U.S. patent application number 12/291533 was filed with the patent office on 2009-07-09 for modifications of hiv env, gag, and pol enhance immunogenicity for genetic immunization.
Invention is credited to Bimal K. Chakrabarti, Yue Huang, Gary J. Nabel, Zengguang Wang.
Application Number | 20090175910 12/291533 |
Document ID | / |
Family ID | 27397438 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090175910 |
Kind Code |
A1 |
Nabel; Gary J. ; et
al. |
July 9, 2009 |
Modifications of HIV Env, Gag, and Pol enhance immunogenicity for
genetic immunization
Abstract
Modified HIV Env, Gag, Pol, or Nef DNA with improved ability to
elicit antibody and CTL responses to HIV antigens have been
identified as prototype immunogens for the treatment and prevention
of HIV infections.
Inventors: |
Nabel; Gary J.; (Washington,
DC) ; Chakrabarti; Bimal K.; (Gaithersburg, MD)
; Huang; Yue; (Bethesda, MD) ; Wang;
Zengguang; (Silver Spring, MD) |
Correspondence
Address: |
KNOBBE, MARTENS, OLSON & BEAR, LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
27397438 |
Appl. No.: |
12/291533 |
Filed: |
November 10, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10359120 |
Feb 4, 2003 |
7470430 |
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12291533 |
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PCT/US01/25721 |
Aug 14, 2001 |
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10359120 |
|
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60279257 |
Mar 28, 2001 |
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60252115 |
Nov 14, 2000 |
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60225097 |
Aug 14, 2000 |
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Current U.S.
Class: |
424/208.1 ;
530/395; 536/23.72 |
Current CPC
Class: |
A61K 39/12 20130101;
C12N 2740/16222 20130101; C07K 2319/00 20130101; C12N 2740/16234
20130101; A61K 2039/54 20130101; C12N 2740/16322 20130101; A61K
2039/53 20130101; A61K 2039/55522 20130101; A61K 39/21 20130101;
A61P 31/18 20180101; C12N 2740/16122 20130101; C07K 14/005
20130101; A61K 2039/545 20130101; A61K 2039/57 20130101; C12N
2740/16134 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/208.1 ;
536/23.72; 530/395 |
International
Class: |
A61K 39/21 20060101
A61K039/21; C07H 21/04 20060101 C07H021/04; C07K 14/16 20060101
C07K014/16 |
Claims
1. A nucleic acid encoding a modified HIV Env glycoprotein wherein
said modified HIV Env glycoprotein has a termination codon at
position 704.
2. The nucleic acid of claim 1, wherein said Env is from an R5
tropic HIV strain.
3. The nucleic acid of claim 1, wherein said Env is from an X4
tropic HIV strain.
4. The nucleic acid of claim 1, wherein the codons in said nucleic
acid have been optimized for expression in humans.
5. The nucleic acid of claim 1, wherein at least one variable
region has been deleted.
6. The nucleic acid of claim 5, wherein said at least one variable
region is the V1 loop.
7. The nucleic acid of claim 6, wherein the V2 loop has been
deleted.
8. The nucleic acid of claim 5, wherein said at least one variable
region is selected from the group consisting of the V1 loop, the V2
loop, the V3 loop and the V4 loop.
9. The nucleic acid of claim 1, wherein said modified HIV Env
glycoprotein is from a lade selected from the group consisting of
Clade A, Clade B and Clade C.
10. The nucleic acid of claim 9, wherein said modified HIV Env
glycoprotein is from Clade C (South African).
11. The nucleic acid of claim 1, wherein said nucleic acid encodes
a modified HIV Env glycoprotein in which the cleavage site, fusion
domain, and at least part of the interspace between the two
heptad-repeats are deleted.
12. The nucleic acid of claim 1, wherein said nucleic acid
comprises the insert in SEQ ID NO: 92 which encodes a modified HIV
Env glycoprotein.
13. The nucleic acid of claim 1, wherein said nucleic acid
comprises a nucleotide sequence at least 95% identical to the
nucleotide sequence of the insert in SEQ ID NO: 92 which encodes a
modified HIV Env glycoprotein.
14. The nucleic acid of claim 1, wherein said nucleic acid encodes
a polypeptide at least 95% identical to the polypeptide encoded by
the insert in SEQ ID NO: 92 which encodes a modified HIV Env
glycoprotein.
15. The nucleic acid of claim 1, wherein said nucleic acid
comprises the insert in SEQ ID NO: 14 which encodes a modified HIV
Env glycoprotein.
16. The nucleic acid of claim 1, wherein said nucleic acid
comprises a nucleotide sequence at least 95% identical to the
nucleotide sequence of the insert in SEQ ID NO: 14 which encodes a
modified HIV Env glycoprotein.
17. The nucleic acid of claim 1, wherein said nucleic acid encodes
a polypeptide at least 95% identical to the polypeptide encoded by
the insert in SEQ ID NO: 14 which encodes a modified HIV Env
glycoprotein.
18. The nucleic acid of claim 1, wherein said nucleic acid
comprises the insert in SEQ ID NO: 96 which encodes a modified HIV
Env glycoprotein.
19. The nucleic acid of claim 1, wherein said nucleic acid
comprises a nucleotide sequence at least 95% identical to the
nucleotide sequence of the insert in SEQ ID NO: 96 which encodes a
modified HIV Env glycoprotein.
20. The nucleic acid of claim 1, wherein said nucleic acid
comprises a nucleotide sequence at least 95% identical to the
nucleotide sequence of the insert in SEQ ID NO: 96 which encodes a
modified HIV Env glycoprotein.
21. The nucleic acid of claim 1, wherein said nucleic acid
comprises the insert which encodes a modified HIV Env glycoprotein
in a sequence selected from the group consisting of SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 50, SEQ ID
NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 954, SEQ ID NO:
55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ
ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ
ID NO: 69, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO:
92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 112, SEQ ID NO: 113,
SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ
ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID
NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:
126, SEQ ID NO: 142, SEQ ID NO: SEQ ID NO: 143, SEQ ID NO: 144, SEQ
ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID
NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:
153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 156 or a
sequence at least 95% identical to the sequence of said insert.
22. The nucleic acid of claim 1, wherein said nucleic acid
comprises a nucleotide sequence encoding a polypeptide at least 95%
identical to the polypeptide encoded by the insert which encodes a
modified HIV Env glycoprotein in a sequence selected from the group
consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID
NO: 14, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,
SEQ ID NO: 954, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID
NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62,
SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID
NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 86, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID
NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:
116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:
120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:
124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 142, SEQ ID NO: SEQ
ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID
NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:
151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:
155, and SEQ ID NO: 156.
23. The nucleic acid of claim 1 further comprising a backbone,
wherein said backbone is a plasmid vector.
24. The nucleic acid of claim 1 further comprising a backbone,
wherein said backbone is an adenoviral vector.
25. A nucleic acid which is at least 95% identical to the nucleic
acid of claim 1, wherein said nucleic acid encodes a modified HIV
Env glycoprotein with a termination codon at position 704.
26. A modified HIV Env glycoprotein encoded by the nucleic acid
molecule of claim 1.
27. A vaccine composition comprising the nucleic acid molecule of
claim 1 and a pharmaceutically acceptable solution in a
prophylactically effective dose.
28. The composition of claim 27 further comprising an adjuvant.
29. A method of generating an antibody or CTL response against
native HIV Env comprising the step of administering the nucleic
acid molecule of claim 1 to a host to generate said antibody or CTL
response against native HIV Env.
30. The method of claim 29, wherein said step is a primary
immunization.
31. The method of claim 29, wherein said step is a boost.
32. The method of claim 29, wherein said step is a primary
immunization with a plasmid vector.
33. The method of claim 29 wherein said step is a primary
immunization with a plasmid vector expressing gp145delCFI and
further comprising a step which is a boost with an adenoviral
vector expressing gp140delCFI.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/359,120, filed Feb. 4, 2003, which is a continuation of
international application number PCT/US01/25721, and claims the
benefit of priority of international application number
PCT/US01/25721 having an international filing date of Aug. 14,
2001, designating the United States of America and published in
English, which claims the benefit of priority of U.S. provisional
patent application No. 60/279,257, filed Mar. 28, 2001, U.S.
provisional patent application No. 60/252,115, filed Nov. 14, 2000,
and U.S. provisional patent application No. 60/225,097, filed Aug.
14, 2000; all of which are hereby expressly incorporated by
reference in their entireties.
SEQUENCE LISTING
[0002] The present application is being filed along with duplicate
copies of a CD-ROM marked "Copy 1" and "Copy 2" containing a
Sequence Listing in electronic format. The duplicate copies of the
CD-ROM each contain a file entitled "NIH206.001 CDV1" created on
Nov. 10, 2008 which is 1,528,044 bytes in size. The information on
these duplicate CD-ROMs is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of molecular
biology. The present invention discloses modified HIV Env, Gag,
Pol, and Nef proteins, related nucleotide sequences, and usage for
genetic immunization.
BACKGROUND OF THE INVENTION
[0004] Protective immunity against human immunodeficiency virus-1
(HIV-1) is likely to require recognition of linear and conformation
epitopes from multiple HIV antigens. Whether these responses can be
elicited more effectively by virion-like structures or fused CTL
epitopes is unknown.
[0005] The immune response to HIV infection in long-term
non-progressors and HIV-exposed sex workers suggests that specific
viral immunity may limit infection and the symptoms of disease. No
single characteristic yet correlates with protective immunity, but
studies in non-human of primates suggest that both humoral and
cellular immunity are required for this response. Depletion of
cytotoxic T cells (CTLs) in chronically-infected macaques enhances
viremia. In humans, higher CTL responses correlate with lower viral
load and stabilization of clinical symptoms. In animal models,
passive transfer of neutralizing antibodies can also contribute to
protection against virus challenge. Neutralizing antibody responses
can also be developed in HIV-infected individuals and are
associated with lower viral loads in long-term non-progressors.
Though this neutralizing antibody response is uncommon, it is
directed largely against the Env protein of the virus.
[0006] In early human vaccine trials, gp120 protein immunogens have
yielded disappointing results: vaccine-induced antibodies have not
been broadly neutralizing and have sometimes enhanced infection in
vitro. Monomeric gp120 loses oligomer-dependent epitopes and does
not include sequences in the ectodomain of the gp41 that become
exposed during virus entry. It is assumed that broadly neutralizing
antibodies bind to native gp120/gp41 complex on the surface of the
virus rather than soluble gp120.
[0007] The development of a cytotoxic T lymphocyte (CTL) response
to viruses is often crucial to the outcome of infections. Lysis of
infected cells prior to the production of progeny virions may limit
virus burst size (Yang, O et al., 1996, J. Virol., 70:5799-5806),
and HIV specific CD8.sup.+ cytotoxic T lymphocytes (CTL) have been
shown to be important in viral clearance and in the control of
initial HIV-1 spread (Borrow, P et al., 1994, J. Virol.,
68:6103-6110; Yang, O et al., 1996, J. Virol. 70:5799-5806). CTL
responses specific to HIV also contribute to reduction in viral
load during acute and asymptomatic infection (Klein, M R, et al.,
1995, J. Exp. Med. 181:1365-1372; Moss, P A H et al., 1995, Proc.
Natl. Acad. Sci. USA, 92:5773-5777) and may be involved in
protection against the establishment of persistent HIV infections
(Rowland-Jones, S L et al., 1993, Lancet, 341:860-861;
Rowland-Jones, S L et. al., 1995, Nat. Med., 1:59-64).
High-frequency CTL responses to HIV-1 correlated with low viral
load and slow disease progression in chronically infected
individuals (Musey, L et al., 1997, N. Engl. J. Med.,
337:1267-1274; Ogg, G S et al., 1998, Science, 279:2103-2106.).
More compelling evidence of an antiviral effect of CD8.sup.+ cells
was demonstrated in controlled studies in macaques, in which
CD8.sup.+ cells were depleted in vivo using a monoclonal antibody.
The viral loads in these animals increased or decreased as the
CD8.sup.+ cells were depleted or reappeared, respectively (Jin, X
et al., 1999, J. Exp. Med., 189:991-998; Schmitz, J E, et al.,
1999, Science, 283:857-860). Therefore, induction of a CTL response
specific to these proteins represents a desirable response in an
HIV-1 vaccine.
[0008] HIV-1 internal structural and enzymatic proteins contain
conserved domains that preserve their functions and thus exhibit
less antigenic diversity that may elicit more effective CTL
responses (Nixon, D F et al., 1988, Nature, 336:484-487.).
Efficient and durable CTL responses require endogenous antigen
synthesis and processing. Current vaccine delivery techniques
include immunization with live, attenuated viruses, inactivated
recombinant virus infection (Letvin, N L, 1998, Science,
280:1875-1880) or plasmid DNA expression vectors. A major obstacle
in the induction of CTL responses with naked DNA or recombinant
virus during development of an HIV vaccine is that the expression
of HIV-1 structural and enzymatic genes is tightly regulated by the
virus itself. The expression of these proteins is heavily dependent
upon the existence of the Rev-responsive element (RRE) of HIV-1 in
recombinant vectors (Cullen, B R, 1992, Microbiol. Rev.,
56:375-394; Felber, B K et al., 1989, Proc Natl Acad Sci USA,
86:1495-1499). Poor expression is caused by the presence of AT rich
inhibitory nucleotide sequences (INS) in the gag, pol and env
genes, which inhibit the nuclear export and efficient expression of
unspliced HIV1 mRNAs. Early studies of DNA vaccination against HIV
in mice required the inclusion of Rev in their expression vectors
(Lu, S et al., 1995, Virology, 209:147-154; Okuda, K et al., 1995,
AIDS Res. Hum. Retroviruses, 11:933-943; Wang, B et al., 1993,
Proc. Natl. Acad. Sci. U.S. A 90:4156-4160), but modification of
INS has been shown to facilitate Rev-independent expression of
HIV-1 Gag (Qiu, J-T et al., 1999, J. Virol., 73:9145-9152; zur
Megede, J et al., 2000, J Virol 74:2628-2635), allowing detectable
humoral and CTL responses against this protein (Qiu, J-T et al.,
1999, J. Virol., 73:9145-9152). These modified HIV-1 Gag genes
produced viral-like particles of the expected density and
morphology and induced an immune response to HIV-1 Gag after DNA
immunization in mice (zur Megede, J et al., 2000, J Virol,
74:2628-2635).
SUMMARY OF THE INVENTION
[0009] Protective immunity against human immunodeficiency virus-1
(HIV-1) is likely to require recognition of linear and conformation
epitopes from multiple HIV antigens, and whether these responses
can be elicited more effectively by virion-like structures or fused
CTL epitopes was previously unknown. Herein is provided a modified
HIV Env with deletions in the cleavage site, fusogenic domain, and
spacing of heptad repeats 1 and 2 to expose the core protein for
optimal antigen presentation and recognition. Additionally we
provide a Gag-Pol or Gag-Pol-Nef fusion protein that is a
polyprotein designed to maximize epitope presentation. The
invention extends the mutation in HIV Env, Gag, Pol, and Nef to any
HIV clade or strain and to related proteins of other viruses.
Different combinations, different orders, and different variations
on a theme are envisioned, the theme being to optimize presentation
of epitopes that generate broad CTL and antibody responses.
[0010] More particularly, we have investigated the effect of
specific mutations in human immunodeficiency virus type 1 (HIV-1)
envelope (Env) on humoral and cellular immune responses after DNA
vaccination. Mice were injected with plasmid expression vectors
encoding HIV Env with modifications of conserved glycosylation
sites or different COOH-terminal mutations intended to mimic a
fusion intermediate. Elimination of conserved glycosylation sites
did not substantially enhance humoral or CTL immunity. In contrast,
a modified gp140 with deletions in the cleavage site, fusogenic
domain and spacing of heptad repeats 1 and 2 enhanced humoral
immunity without reducing the efficacy of the CTL response. Because
of its ability to stimulate the antibody response to native gp160
without affecting cellular immunity, this modified gp140 or a
related derivative is envisioned to be a useful component of an
AIDS vaccine.
[0011] In addition, we have examined the immune response to HIV-1
Gag and Pol after plasmid DNA immunization with Rev-independent
expression vectors encoding various forms of these proteins. Immune
responses were analyzed after vaccination with four expression
vectors, including Gag alone or Gag-Pol, both of which gave rise to
virion-like particles (VLPs), compared to Pol alone or a Gag-Pol
fusion protein that did not form VLPs. The Gag-Pol fusion protein
induced the most broad and potent CTL responses to Gag and Pol in
DNA-vaccinated mice, and this immunogen also readily elicited an
antibody response to HIV-1 Gag and Pol determinants. Through its
ability to induce broad CTL and antibody responses, this Gag-Pol
fusion protein or a related derivative is envisioned to be a useful
component of an AIDS vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0013] FIGS. 1-176. Plasmid Maps. Table 1 provides the description
of FIGS. 1-176, each of which illustrates a map of a plasmid
described herein.
[0014] FIG. 1. Plasmid 2100.
[0015] FIG. 2. Plasmid 2200.
[0016] FIG. 3. Plasmid 2300.
[0017] FIG. 4. Plasmid 2302.
[0018] FIG. 5. Plasmid 2400.
[0019] FIG. 6. Plasmid 2700.
[0020] FIG. 7. Plasmid 2701.
[0021] FIG. 8. Plasmid 2702.
[0022] FIG. 9. Plasmid 2706.
[0023] FIG. 10. Plasmid 2707.
[0024] FIG. 11. Plasmid 2800.
[0025] FIG. 12. Plasmid 2801.
[0026] FIG. 13. Plasmid 2804.
[0027] FIG. 14. Plasmid 2805.
[0028] FIG. 15. Plasmid 2810.
[0029] FIG. 16. Plasmid 2811.
[0030] FIG. 17. Plasmid 2812.
[0031] FIG. 18. Plasmid 2813.
[0032] FIG. 19. Plasmid 2814.
[0033] FIG. 20. Plasmid 2820.
[0034] FIG. 21. Plasmid 2821.
[0035] FIG. 22. Plasmid 2822.
[0036] FIG. 23. Plasmid 2823.
[0037] FIG. 24. Plasmid 2824.
[0038] FIG. 25. Plasmid 2825.
[0039] FIG. 26. Plasmid 2826.
[0040] FIG. 27. Plasmid 2827.
[0041] FIG. 28. Plasmid 2828.
[0042] FIG. 29. Plasmid 2829.
[0043] FIG. 30. Plasmid 2830.
[0044] FIG. 31. Plasmid 2831.
[0045] FIG. 32. Plasmid 2832.
[0046] FIG. 33. Plasmid 2833.
[0047] FIG. 34. Plasmid 2834.
[0048] FIG. 35. Plasmid 2835.
[0049] FIG. 36. Plasmid 2836.
[0050] FIG. 37. Plasmid 2837.
[0051] FIG. 38. Plasmid 2838.
[0052] FIG. 39. Plasmid 2839.
[0053] FIG. 40. Plasmid 2840.
[0054] FIG. 41. Plasmid 2841.
[0055] FIG. 42. Plasmid 2842.
[0056] FIG. 43. Plasmid 2843.
[0057] FIG. 44. Plasmid 2844.
[0058] FIG. 45. Plasmid 2845.
[0059] FIG. 46. Plasmid 2846.
[0060] FIG. 47. Plasmid 2847.
[0061] FIG. 48. Plasmid 2848.
[0062] FIG. 49. Plasmid 2849.
[0063] FIG. 50. Plasmid 2850.
[0064] FIG. 51. Plasmid 2851.
[0065] FIG. 52. Plasmid 2852.
[0066] FIG. 53. Plasmid 2853.
[0067] FIG. 54. Plasmid 2854.
[0068] FIG. 55. Plasmid 2860.
[0069] FIG. 56. Plasmid 2861.
[0070] FIG. 57. Plasmid 2862.
[0071] FIG. 58. Plasmid 2863.
[0072] FIG. 59. Plasmid 2864.
[0073] FIG. 60. Plasmid 2865.
[0074] FIG. 61. Plasmid 2866.
[0075] FIG. 62. Plasmid 2867.
[0076] FIG. 63. Plasmid 2868.
[0077] FIG. 64. Plasmid 2869.
[0078] FIG. 65. Plasmid 2870.
[0079] FIG. 66. Plasmid 2871.
[0080] FIG. 67. Plasmid 2872.
[0081] FIG. 68. Plasmid 2873.
[0082] FIG. 69. Plasmid 2874.
[0083] FIG. 70. Plasmid 2900.
[0084] FIG. 71. Plasmid 3000.
[0085] FIG. 72. Plasmid 3200.
[0086] FIG. 73. Plasmid 3201.
[0087] FIG. 74. Plasmid 3202.
[0088] FIG. 75. Plasmid 3203.
[0089] FIG. 76. Plasmid 3300.
[0090] FIG. 77. Plasmid 3301.
[0091] FIG. 78. Plasmid 3400.
[0092] FIG. 79. Plasmid 3401.
[0093] FIG. 80. Plasmid 3500.
[0094] FIG. 81. Plasmid 3600.
[0095] FIG. 82. Plasmid 3700.
[0096] FIG. 83. Plasmid 3800.
[0097] FIG. 84. Plasmid 5200.
[0098] FIG. 85. Plasmid 5201.
[0099] FIG. 86. Plasmid 5202.
[0100] FIG. 87. Plasmid 5203.
[0101] FIG. 88. Plasmid 5300.
[0102] FIG. 89. Plasmid 5301.
[0103] FIG. 90. Plasmid 5303.
[0104] FIG. 91. Plasmid 5304.
[0105] FIG. 92. Plasmid 5305.
[0106] FIG. 93. Plasmid 5306.
[0107] FIG. 94. Plasmid 5307.
[0108] FIG. 95. Plasmid 5308.
[0109] FIG. 96. Plasmid 5309.
[0110] FIG. 97. Plasmid 5350.
[0111] FIG. 98. Plasmid 5351.
[0112] FIG. 99. Plasmid 5352.
[0113] FIG. 100. Plasmid 5353.
[0114] FIG. 101. Plasmid 5354.
[0115] FIG. 102. Plasmid 5355.
[0116] FIG. 103. Plasmid 5356.
[0117] FIG. 104. Plasmid 5357.
[0118] FIG. 105. Plasmid 5358.
[0119] FIG. 106. Plasmid 5359.
[0120] FIG. 107. Plasmid 5360.
[0121] FIG. 108. Plasmid 5361.
[0122] FIG. 109. Plasmid 5362.
[0123] FIG. 110. Plasmid 5363.
[0124] FIG. 111. Plasmid 5364.
[0125] FIG. 112. Plasmid 5365.
[0126] FIG. 113. Plasmid 5366.
[0127] FIG. 114. Plasmid 5367.
[0128] FIG. 115. Plasmid 5368.
[0129] FIG. 116. Plasmid 5369.
[0130] FIG. 117. Plasmid 5370.
[0131] FIG. 118. Plasmid 5371.
[0132] FIG. 119. Plasmid 5372.
[0133] FIG. 120. Plasmid 5373.
[0134] FIG. 121. Plasmid 5374.
[0135] FIG. 122. Plasmid 5375.
[0136] FIG. 123. Plasmid 5376.
[0137] FIG. 124. Plasmid 5377.
[0138] FIG. 125. Plasmid 5378.
[0139] FIG. 126. Plasmid 5379.
[0140] FIG. 127. Plasmid 5500.
[0141] FIG. 128. Plasmid 5501.
[0142] FIG. 129. Plasmid 5502.
[0143] FIG. 130. Plasmid 5503.
[0144] FIG. 131. Plasmid 5504.
[0145] FIG. 132. Plasmid 5505.
[0146] FIG. 133. Plasmid 5506.
[0147] FIG. 134. Plasmid 5507.
[0148] FIG. 135. Plasmid 5508.
[0149] FIG. 136. Plasmid 5509.
[0150] FIG. 137. Plasmid 5510.
[0151] FIG. 138. Plasmid 5511.
[0152] FIG. 139. Plasmid 5512.
[0153] FIG. 140. Plasmid 5513.
[0154] FIG. 141. Plasmid 5514.
[0155] FIG. 142. Plasmid 5515.
[0156] FIG. 143. Plasmid 5516.
[0157] FIG. 144. Plasmid 5517.
[0158] FIG. 145. Plasmid 5518.
[0159] FIG. 146. Plasmid 5519.
[0160] FIG. 147. Plasmid 5520.
[0161] FIG. 148. Plasmid 5521.
[0162] FIG. 149. Plasmid 5522.
[0163] FIG. 150. Plasmid 5523.
[0164] FIG. 151. Plasmid 5524.
[0165] FIG. 152. Plasmid 5525.
[0166] FIG. 153. Plasmid 5526.
[0167] FIG. 154. Plasmid 5527.
[0168] FIG. 155. Plasmid 5528.
[0169] FIG. 156. Plasmid 5529.
[0170] FIG. 157. Plasmid 3900.
[0171] FIG. 158. Plasmid 3901.
[0172] FIG. 159. Plasmid 4000.
[0173] FIG. 160. Plasmid 4001.
[0174] FIG. 161. Plasmid 4100.
[0175] FIG. 162. Plasmid 4101.
[0176] FIG. 163. Plasmid 4200.
[0177] FIG. 164. Plasmid 4300.
[0178] FIG. 165. Plasmid 4301.
[0179] FIG. 166. Plasmid 4302.
[0180] FIG. 167. Plasmid 4303.
[0181] FIG. 168. Plasmid 4304.
[0182] FIG. 169. Plasmid 4305.
[0183] FIG. 170. Plasmid 4306.
[0184] FIG. 171. Plasmid 4308.
[0185] FIG. 172. Plasmid 4309.
[0186] FIG. 173. Plasmid 4310.
[0187] FIG. 174. Plasmid 4311.
[0188] FIG. 175. Plasmid 4312.
[0189] FIG. 176. Plasmid 4313.
[0190] FIG. 177. Comparison of GP2 with the structures of viral and
cellular membrane fusion proteins. (A) Recombinant Ebola Zaire GP2,
(B) Recombinant Mo-55 from the TM subunit of MoMuLv, (C) Low
pH-treated HA2 from influenza virus, (D) Recombinant,
proteolysis-resistant core of HIV-1 gp41, (E) Recombinant SIV gp41,
NMR structure, (F) Recombinant core coiled segments of the SNARES
syntaxin 1-A, synaptobrevin-II, and SNAP-25B. Weissenhorn et al.,
1998, Molecular Cell, 2, 605-616.
[0191] FIG. 178. Schematic representation of functional domains and
mutations in HIV-1 Env glycoproteins. Full-length envelope
polyprotein, gp160, with the indicated features based on the amino
acid residues of HXB2 is shown (top). Functional domains include
the gp120/gp41 cleavage site (residues 510/511), the fusion domain
(512-527), the two heptad repeats (546-579 and 628-655), the
transmembrane domain (684-705), and the cytoplasmic domain
(706-856). The mutant forms of the envelope proteins are shown
below the structure of gp160. COOH deletions were introduced that
terminate the envelope protein at positions 752, 704, or 680 to
produce gp150, gp145, or gp140, respectively. Two internal
deletions that removed the cleavage site, the fusion domain, and
the region between the two heptad repeats were introduced into
gp160, gp150, gp145, and gp140. A further deletion in the
COOH-terminal region at position 592 removed the second heptad
repeat, the transmembrane domain, and the interspace region to
produce gp128.DELTA.CFI. To disrupt potential glycosylation sites,
asparagine (N) residues at eleven positions (88, 156, 160, 197,
230, 234, 241, 262, 276, 289, and 295) were replaced with aspartic
acid (D) residues in both gp160 and gp150. Versions of both gp160
and gp150 were created with a total of 17 mutated glycosylation
sites by including six additional N to D substitutions at positions
332, 339, 356, 386, 392, and 448.
[0192] FIG. 179. Comparison of the expression of the HIV-1 gp160
with codon-optimized gp160. A. Expression of plasmids encoding
Rev-dependent and Rev-independent codon-modified gp160. Upper
panel: expression of Rev-dependent viral gp160 (left) and
codon-modified gp160 (right) in transfected 293 cells. Lower panel:
comparable expression of .alpha.-actin in these transfected cells.
B. Expression of mutant CXCR4-tropic HIV Env glycoproteins with
COOH-terminal truncations. C and D, respectively. CXCR4-tropic
envelope proteins containing mutant glycosylation sites and mutant
functional domains. The indicated proteins were detected by
immunoblotting as above. Cell lysates produced by transfection with
vector containing no insert were used as controls (first lane in
each panel).
[0193] FIG. 180. Cytotoxicity of full-length gp160 is eliminated by
deletion of the COOH-terminal cytoplasmic domain. Cell rounding and
detachment was not observed in control-transfected 293 cells (A),
in contrast to full-length gp160 (B) and to a lesser extent in
cells transfected with gp150 (C), in contrast to gp145 (D) or gp140
(E).
[0194] FIG. 181. Expression of soluble gp140.DELTA.CFI HIV-1
envelope variant. Immunoprecipitation and Western blot analysis of
supernatants from the indicated transfected cells.
[0195] FIG. 182. Antibody response against HIV-1 envelope proteins
in DNA immunized mice. A. Comparison of the antibody response in
mice immunized with gp140 (.DELTA.CFI) or other Env plasmid
expression vectors. Sera were collected 2 weeks after the last
immunization and used to immunoprecipitate codon-altered gp160 from
lysates of transfected 293 cells. The quantitation of the
immunoprecipitated gp160 was done as described in FIG. 182B. The
average of the normalized data has been presented as a bar diagram.
B. Antibody responses in mice immunized with different mutant Env
expression vectors. Antisera from immunized mice were diluted in IP
buffer and 1 .mu.l of each diluted serum was used to
immunoprecipitate codon-altered HIV-1 gp160 from lysates of
transfected 293 cells as described in FIG. 180A. The gels were
scanned and the intensity of the gp160 band was determined by
densitometry using the program Image Quant and presented relative
to the intensity of gp160 immunoprecipitated with positive control
sera (rabbit anti-gp160), which was used to normalize data between
experiments. These data are presented graphically to facilitate
comparison among groups. C. Antibody responses in mice immunized
with gp140 or gp140 (.DELTA.CFI) were determined by
immunoprecipitation and Western blotting. Animals received two
booster doses (100 .mu.g) of the same plasmid, two weeks apart.
Sera (1 .mu.l) collected 2 weeks after the last immunization was
used to immunoprecipitate codon-optimized HIV-1 gp160 from lysates
of transfected 293 cells containing 400 .mu.g of total protein.
Each lane corresponds to the sera from an animal immunized with
either the control vector (lanes 1 and 2), CXCR4-tropic gp140
(lanes 3-6), or plasmid that expresses gp140 with the indicated
mutant functional domains (lanes 7-10). A mouse monoclonal antibody
to gp160 (HIV-1 V3 Monoclonal (IIIB-V3-13), NIH AIDS Reagent
Program) was used as a positive control (lane 11).
[0196] FIG. 183. CTL response against HIV-1 envelope proteins in
DNA immunized mice. The CTL response to CXCR4-tropic Env and
indicated deletion mutants is shown (A). Dependence of CTL activity
on CD8 cells was shown by magnetic bead depletion using the
indicated representative immunogens (B). The CTL responses to
CXCR4-tropic envelope with glycosylation site and .DELTA.CFI
mutations are shown (C and D, respectively). Spleen cells were
isolated from immunized mice two weeks after the final immunization
and stimulated in vitro with irradiated cells expressing gp160 with
addition of hIL2 (5 U/ml) at day 4. The cytolytic activity of the
restimulated spleen cells was tested after 7 days against V3
peptide-pulsed BC10ME cells. Similar findings were observed with
target cells that stably express full-length Env.
[0197] FIG. 184. Schematic representation of HIV-1 Gag-Pol
expression constructs. The protein sequences of Gag (amino acids
1-432) from HXB2 (GenBank accession number K03455) and Pol (amino
acids 3-1003) from NL4-3 (GenBank accession number M19921) were
used to create a synthetic version of hGag-Pol using codons found
in human cells. hGag-Pol.DELTA.FS.DELTA.Pr was made by modification
of the frame shift site (FS) and inactivation of protease. For
hPol, 432 amino acids were deleted from the NH.sub.2-terminal
region of hGag-Pol and addition of an ATG codon. hGag was made by
deletion of 925 amino acids from the COOH-terminal region of
hGag-Pol. hGag-Pol, hGag-Pol.DELTA.FS.DELTA.Pr, hPol and hGag are
expressed from the pNGVL-3 vector backbone.
[0198] FIG. 185. HIV-1 Gag-Pol expression in transfected 293T cells
and stably transfected CT26 and BC10 ME cells. Cell lysates from
293T cells transfected with pCMV .DELTA.R8.2 containing viral
Gag-Pol (vGag-Pol), pNGVL-hGag, hPol, hGag-Pol.DELTA.FS.DELTA.Pr
and hGag-Pol were separated by 4-15% gradient SDS-PAGE, transferred
to nitrocellulose filters, and analyzed by immunoblotting with (A)
human anti HIV-1-IgG, (B) monoclonal anti-p24, and (C) rabbit
anti-RT. (D) Cell lysates from CT26 and BC10ME cells stably
transduced with either hGag or hPol were analyzed with human anti
HIV-1-IgG.
[0199] FIG. 186. Transmission electron microscopy of HIV-1 immature
virus-like particles (VLP) produced by transfected 293T. Cells were
transfected with pNGVL-hGag 48 hours prior to harvesting and fixing
(magnification 25,000.times.).
[0200] FIG. 187. Gag or Pol specific CTL response mediated by CD8
positive cells in immunized mice. Two weeks after mice were
immunized with a control vector, hGag, hPol,
hGag-Pol.DELTA.FS.DELTA.Pr, and hGag-Pol, splenic cells were
harvested and sensitized with naive mouse splenic cells pulsed with
Gag or Pol peptides. One week later, effector cells were tested for
cytolytic activity in a 5-h .sup.51Cr release assay using
.sup.51Cr-labeled BC10ME target cells that were pulsed for 2 hours
with either (A) HIV-1 Gag peptides, or (B) HIV-1 Pol peptides. (C)
CD4+ or CD8+ lymphocytes were depleted from splenic cells of
immunized mice with anti-mouse-CD4+ or CD8+ Dynal beads according
to the manufacturer's instructions.
[0201] FIG. 188. Gag or Pol specific CTL response mediated by CD8
positive cells in immunized mice using stable expressing cell lines
as target cells. Two weeks after immunization in mice, splenic
cells were harvested and sensitized with naive mouse splenic cells
pulsed with Gag or Pol peptides. One week later, effector cells
were tested for cytolytic activity in a 5-h .sup.51Cr release assay
using .sup.51Cr-labeled BC10ME target cells expressing either (A)
HIV-1 Gag or (B) Pol protein.
[0202] FIG. 189. HIV-1 p24 antibody ELISA assays, HIV-1
immunoblotting and immunoprecipitation Western blotting. (A) An
HIV-1 p24 antibody ELISA assay was performed by coating 96-well
plates with 50 .mu.l of purified recombinant HIV-1.sub.IIIB p24
antigen at a concentration of 2 .mu.g/ml in PBS buffer, pH 7.4. (B)
HIV-1 immunoblotting of strips containing HIV-1 proteins were
incubated with pooled mouse sera at a dilution of 1:25. Bands were
visualized using the ECL western blotting detection reagent. (C)
Immunoprecipitation and Western blotting of hPol gene-transfected
293T cell lysates three days after transfection with RIPA buffer.
The pooled mouse serum was diluted with IP buffer. After adding 10
.mu.g of the cell lysate containing HIV-1 Pol protein, the
reactions were incubated overnight on a rotator at 4.degree. C. The
next day, 250 .mu.l of Protein G and A Sepharose beads (10% V/V in
IP buffer) were added, and the reactions were incubated on a
rotator for 2 hours at 4.degree. C. The reactions were washed
4.times. with IP buffer, re-suspended with 30 .mu.l of 1.times.
sample buffer, and then loaded onto SDS-PAGE. The reactions were
transferred to an Immobilon P membrane, and then incubated with
anti HIV-1-IgG. Bands were visualized using the ECL Western
blotting detection reagent.
TABLE-US-00001 TABLE 1 SEQ ID Plasmid Plasmid Name/Description
Plasmid Map Name NO FIG. Env Plasmids 2100 pVR1012x/s R5gp139-Nef
(delta) MHC (delta) CD4/h pVR1012x/s R5gp139-Nef delta MHC delta
CD4/h 1 1 2200 pVR1012x/s R5gp157-Nef (delta) MHC (delta) CD4/h
pVR1012x/s R5gp157-Nef deltaMHC deltaCD4/h 2 2 2300 pVR1012x/s X4
gp139-Nef deltaMHC deltaCD4/h pVR1012x/s X4gp139-Nef delta MHC
delta CD4/h 3 3 2302 pVR1012x/s X4gp130-Nef/h pVR1012x/s
X4gp130-Nef/h 4 4 2400 pVR1012x/s X4gp157-NefDMHCDCD4/h
pVR1012x/sX4gp157-Nef delta MHC delta CD4/h 5 5 2700 pVR1012x/s
X4gp140/h pVR1012x/s X4gp140/h 6 6 2701 pVR1012x/s X4gp140
.DELTA.CFI/h OR pVR1012x/s X4gp140 (del pVR1012x/s X4gp140 (del
F/CL del H IS)/h 7 7 F/CL del H IS)/h 2702 pVR1012x/s X4gp128
.DELTA.CFI/h OR pVR1012x/s X4gp128 (del pVR1012x/s X4gp128 (del
F/CL)/h 8 8 F/CL)/h 2706 pVR1012x/s X4gp145/h pVR1012x/s X4gp145/h
9 9 2707 pVR1012x/s X4gp145 .DELTA.CFI/h OR pVR1012x/s X4gp145 (del
pVR1012x/s X4gp145 (del F/CL del H IS)/h 10 10 F/CL del H IS)/h
2800 pVR1012x/s R5gp140/h pVR1012x/s R5gp140/h 11 11 2801
pVR1012x/s R5gp140 .DELTA.CFI/h OR pVR1012x/sR5gp140 (del
pVR1012x/sR5gp140 (del F/CL del H IS)/h 12 12 F/CL del H IS)/h 2804
pVR1012x/s R5gp145/h pVR1012x/s R5gp145/h 13 13 2805 pVR1012x/s
R5gp145 .DELTA.CFI/h OR pVR1012x/s R5gp145 (del pVR1012x/sR5gp145
(del F/CL del H IS)/h 14 14 F/CL del H IS)/h 2810 pVR1012x/s
R5gp140delC1 (delCFI)/h pVR1012x/sR5gp140delC1 (delCFI)/h 15 15
2811 pVR1012x/s R5gp140delC2 (delCFI)/h pVR1012x/sR5gp140delC2
(delCFI)/h 16 16 2812 pVR1012x/s R5gp140delC3 (delCFI)/h
pVR1012x/sR5gp140delC3 (delCFI)/h 17 17 2813 pVR1012x/s
R5gp140delC4 (delCFI)/h pVR1012x/sR5gp140delC4 (delCFI)/h 18 18
2814 pVR1012x/s R5gp140delC5 (delCFI)/h pVR1012x/sR5gp140delC5
(delCFI)/h 19 19 2820 pVR1012x/s R5gp140 (dCFI)/dV1 pVR1012x/s
R5gp140 (dCFI) dV1/h 20 20 2821 pVR1012x/s R5gp140 (dCFI)/dV2
pVR1012x/s gp140 (dCFI) dV2/h 21 21 2822 pVR1012x/s R5gp140
(dCFI)/dV3 pVR1012x/s gp140 (dCFI) dV3/h 22 22 2823 pVR1012x/s
R5gp140 (dCFI)/dV4 pVR1012x/s R5gp140 (dCFI) dV4/h 23 23 2824
pVR1012x/s R5gp140 (dCFI)/dV12 pVR1012x/s R5gp140 (dCFI) dV12/h 24
24 2825 pVR1012x/s R5gp140 (dCFI)/dV13 pVR1012x/s R5gp140 (dCFI)
dV13/h 25 25 2826 pVR1012x/s R5gp140 (dCFI)/dV14 pVR1012x/s R5gp140
(dCFI) dV14/h 26 26 2827 pVR1012x/s R5gp140 (dCFI)/dv23 pVR1012x/s
R5gp140 (dCFI) dv23/h 27 27 2828 pVR1012x/s R5gp140 (dCFI)/dv24
pVR1012x/s R5gp140 (dCFI) dv24/h 28 28 2829 pVR1012x/s R5gp140
(dCFI)/dv34 pVR1012x/s R5gp140 (dCFI) dv34/h 29 29 2830 pVR1012x/s
R5gp140 (dCFI)/dv123 pVR1012x/s R5gp140 (dCFI) dv123/h 30 30 2831
pVR1012x/s R5gp140 (dCFI)/dv124 pVR1012x/s R5gp140 (dCFI) dv124/h
31 31 2832 pVR1012x/s R5gp140 (dCFI)/dv134 pVR1012x/s R5gp140
(dCFI) dv134/h 32 32 2833 pVR1012x/s R5gp140 (dCFI)/dv234
pVR1012x/s R5gp140 (dCFI) dv234/h 33 33 2834 pVR1012x/s R5gp140
(dCFI)/dV1234 pVR1012x/s R5gp140 (dCFI) dv1234/h 34 34 2835 pAdApt
R5gp140 (dCFI)/dV1 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv1/h 35 35
2836 pAdApt R5gp140 (dCFI)/dV2 pAdApt CMV TbGH (+) R5gp140 (dCFI)
dv2/h 36 36 2837 pAdApt R5gp140 (dCFI)/dV3 pAdApt CMV TbGH (+)
R5gp140 (dCFI) dv3/h 37 37 2838 pAdApt R5gp140 (dCFI)/dV4 pAdApt
CMV TbGH (+) R5gp140 (dCFI) dv4/h 38 38 2839 pAdApt R5gp140
(dCFI)/dV12 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv12/h 39 39 2840
pAdApt R5gp140 (dCFI)/dV13 pAdApt CMV TbGH (+) R5gp140 (dCFI)
dv13/h 40 40 2841 pAdApt R5gp140 (dCFI)/dV14 pAdApt CMV TbGH (+)
R5gp140 (dCFI) dv14/h 41 41 2842 pAdApt R5gp140 (dCFI)/dV23 pAdApt
CMV TbGH (+) R5gp140 (dCFI) dv23/h 42 42 2843 pAdApt R5gp140
(dCFI)/dV24 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv24/h 43 43 2844
pAdApt R5gp140 (dCFI)/dV34 pAdApt CMV TbGH (+) R5gp140 (dCFI)
dv34/h 44 44 2845 pAdApt R5gp140 (dCFI)/dV123 pAdApt CMV TbGH (+)
R5gp140 (dCFI) dv123/h 45 45 2846 pAdApt R5gp140 (dCFI)/dV124
pAdApt CMV TbGH (+) R5gp140 (dCFI) dv124/h 46 46 2847 pAdApt
R5gp140 (dCFI)/dV134 pAdApt CMV TbGH (+) R5gp140 (dCFI) dv134/h 47
47 2848 pAdApt R5gp140 (dCFI)/dV234 pAdApt CMV TbGH (+) R5gp140
(dCFI) dv234/h 48 48 2849 pAdApt R5gp140 (dCFI)/dV1234 pAdApt CMV
TbGH (+) R5gp140 (dCFI) dv1234/h 49 49 2850 pVR1012x/s R5gp145delC1
(delCFI)/h pVR1012x/sR5gp145delC1 (delCFI)/h 50 50 2851 pVR1012x/s
R5gp145delC2 (delCFI)/h pVR1012x/sR5gp145delC2 (delCFI)/h 51 51
2852 pVR1012x/s R5gp145delC3 (delCFI)/h pVR1012x/sR5gp145delC3
(delCFI)/h 52 52 2853 pVR1012x/s R5gp145delC4 (delCFI)/h
pVR1012x/sR5gp145delC4 (delCFI)/h 53 53 2854 pVR1012x/s
R5gp145delC5 (delCFI)/h pVR1012x/sR5gp145delC5 (delCFI)/h 54 54
2860 pVR1012x/s R5gp145 (dCFI)/h/dV1 pVR1012x/s R5 gp145 (dCFI)
dv1/h 55 55 2861 pVR1012x/s R5gp145 (dCFI)/h/dV2 pVR1012x/s R5gp145
(dCFI) dv2/h 56 56 2862 pVR1012x/s R5gp145 (dCFI)/h/dV3 pVR1012x/s
R5gp145 (dCFI) dv3/h 57 57 2863 pVR1012x/s R5gp145 (dCFI)/h/dV4
pVR1012x/s R5gp145 (dCFI) dv4/h 58 58 2864 pVR1012x/s R5gp145
(dCFI)/h/dV12 pVR1012x/s R5gp145 (dCFI) dv12/h 59 59 2865
pVR1012x/s R5gp145 (dCFI)/h/dV13 pVR1012x/s R5gp145 (dCFI) dv13/h
60 60 2866 pVR1012x/s R5gp145 (dCFI)/h/dV14 pVR1012x/s R5gp145
(dCFI) dv14/h 61 61 2867 pVR1012x/s R5gp145 (dCFI)/h/dV23
pVR1012x/s R5gp145 (dCFI) dv23/h 62 62 2868 pVR1012x/s R5gp145
(dCFI)/h/dV24 pVR1012x/s R5gp145 (dCFI) dv24/h 63 63 2869
pVR1012x/s R5gp145 (dCFI)/h/dV34 pVR1012x/s R5gp145 (dCFI) dv34/h
64 64 2870 pVR1012x/s R5gp145 (dCFI)/h/dV134 pVR1012x/s R5gp145
(dCFI) dv134/h 65 65 2871 pVR1012x/s R5gp145 (dCFI)/h/dV234
pVR1012x/s R5gp145 (dCFI) dv234/h 66 66 2872 pVR1012x/s R5gp145
(dCFI) dv123/h pVR1012x/s R5gp145 (dCFI) dv123/h 67 67 2873
pVR1012x/s R5gp145 (dCFI)/h/dV124 pVR1012x/s R5gp145 (dCFI) dv124/h
68 68 2874 pVR1012x/s R5gp145 (dCFI)/h/dV1234 pVR1012x/s R5gp145
(dCFI) dv1234/h 69 69 2900 pVR1012x/s R5gp150/h pVR1012x/s
R5gp150/h 70 70 3000 pVR1012x/s R5gp160/h pVR1012x/sR5gp160/h 71 71
3200 pVR1012x/s X4gp150/h pVR1012x/s X4gp150/h 72 72 3201
pVR1012x/s X4gp150 .DELTA.CFI/h OR pVR1012x/s X4gp150 (del
pVR1012x/s X4gp150 (del F/CL del H IS)/h 73 73 F/CL del H IS)/h
3202 pVR1012x/s X4gp150 .DELTA.gly/h pVR1012x/s X4gp150 delta gly
74 74 3203 pVR1012x/s X4gp150 AB .DELTA.gly/h pVR1012x/s X4gp150 AB
(delta) gly/h 75 75 3300 pVR1012x/s X4gp160/h pVR1012x/s X4gp160/h
76 76 3301 pVR1012x/s X4gp160 .DELTA.CFI/h OR pVR1012x/s X4gp160
(del pVR1012x/s X4gp160 (del F/CL del H IS)/h 77 77 F/CL del H
IS)/h 3400 pVR1012x/s X4gp160 .DELTA.gly/h pVR1012x/s X4gp160 delta
gly 78 78 3401 pVR1012x/s X4gp160 AB .DELTA.gly/h OR pVR1012x/s
pVR1012x/s X4gp160 AB Dgly/h 79 79 X4gp160AB mut .DELTA.gly/h 3500
pVR1012x/s Nef/h pVR1012x/s Nef/h 80 80 3600 pVR1012x/s
NefDMHCDCD4/h pVR1012x/s Nef delta MHC delta CD4/h 81 81 3700
pVR1012x/s NefDCD4/h pVR1012x/s Nef delta CD4/h 82 82 3800
pVR1012x/s NefDMHC/h pVR1012x/s Nef delta MHC/h 83 83 5200 pVR1012
x/s 89.6Pgp128 (del F/CL)/h pVR1012 x/s 89.6Pgp128 (del F/CL)/h 84
84 5201 R5 Clade 89.6P gp140 .DELTA.CFI/h pVR1012x/s 89.6Pgp140
(del F/CL del H IS)/h 85 85 5202 pVR1012 x/s 89.6Pgp145 (del F/CL
del H IS)/h pVR1012 x/s 89.6Pgp145 (del F/CL del H IS)/h 86 86 5203
pVR1012 x/s 89.6Pgp160/h pVR1012 x/s 89.6Pgp160/h 87 87 5300 R5
Clade C gp140 .DELTA.CFI/h OR pVR1012x/s R5 pVR1012x/s CladeC (R5)
gp140 (del F/CL del H IS)/h 88 88 (cladeC) gp140 (del F/CL del H
IS)/h 5301 pVR1012x/s R5 (cladeC) gp145 (del F/CL del H IS)/h
pVR1012x/s CladeC (R5) gp145 (del F/CL del H IS)/h 89 89 5303
pVR1012x/s R5gp145 CladeC (Brazil) delCFI/h pVR1012x/s R5gp145
CladeC (Brazil) delCFI/h 90 90 5304 pVR1012x/s R5 (clade A) gp140
(del F/CL del H IS)/h pVR1012x/s R5gp140CladeA (dCFI)/h 91 91 5305
pVR1012x/s R5 (clade A) gp145 (del F/CL del H IS)/h pVR1012x/s
R5gp145CladeA (dCFI)/h 92 92 5306 pVR1012x/s R5 (clade E) gp140
(del F/CL del H IS)/h pVR1012x/s R5gp140 Clade E (dCFI)/h 93 93
5307 pVR1012x/s R5 (clade E) gp145 (del F/CL del H IS)/h pVR1012x/s
R5 gp145 Clade E (dCFI)/h 94 94 5308 pVR1012x/s R5 (clade C South
African) gp140 (del F/CL del H pVR1012x/s R5gp140 Clade C (SA)
(dCFI)/h 95 95 IS)/h 5309 pVR1012x/s R5 (clade C South African)
gp145 (del F/CL del H pVR1012x/s R5gp145 CladeC (SA) (dCFI)/h 96 96
IS)/h 5350 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV1 pVR1012x/s
R5gp140CladeC (Brazil) dCFIdv1/h 97 97 5351 pVRC1012 (x/s) -gp140
(dCFI) (Brazil C)/dV12 pVR1012x/s R5gp140CladeC (Brazil) (dCFI)
dv12/h 98 98 5352 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV123
pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv123/h 99 99 5353
pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV1234 pVR1012x/s
R5gp140CladeC (Brazil) (dCFI) dv1234/h 100 100 5354 pVRC1012 (x/s)
-gp140 (dCFI) (Brazil C)/dV124 pVR1012x/s R5gp140CladeC (Brazil)
(dCFI) dv124/h 101 101 5355 pVRC1012 (x/s) -gp140 (dCFI) (Brazil
C)/dV13 pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv13/h 102 102
5356 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV134 pVR1012x/s
R5gp140CladeC (Brazil) (dCFI) dv134/h 103 103 5357 pVRC1012 (x/s)
-gp140 (dCFI) (Brazil C)/dV14 pVR1012x/s R5gp140CladeC (Brazil)
(dCFI) dv14/h 104 104 5358 pVRC1012 (x/s) -gp140 (dCFI) (Brazil
C)/dV2 pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv2/h 105 105 5359
pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV23 pVR1012x/s
R5gp140CladeC (Brazil) (dCFI) dv23/h 106 106 5360 pVRC1012 (x/s)
-gp140 (dCFI) (Brazil C)/dV234 pVR1012x/s R5gp140CladeC (Brazil)
(dCFI) dv234/h 107 107 5361 pVRC1012 (x/s) -gp140 (dCFI) (Brazil
C)/dV24 pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv24/h 108 108
5362 pVRC1012 (x/s) -gp140 (dCFI) (Brazil C)/dV3 pVR1012x/s
R5gp140CladeC (Brazil) (dCFI) dv3/h 109 109 5363 pVRC1012 (x/s)
-gp140 (dCFI) (Brazil C)/dV34 pVR1012x/s R5gp140CladeC (Brazil)
(dCFI) dv34/h 110 110 5364 pVRC1012 (x/s) -gp140 (dCFI) (Brazil
C)/dV4 pVR1012x/s R5gp140CladeC (Brazil) (dCFI) dv4/h 111 111 5365
pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV1 pVR1012x/s
R5gp145CladeC (Brazil) (dCFI) dv1/h 112 112 5366 pVRC1012 (x/s)
-gp145 (dCFI) (Brazil C)/dV12 pVR1012x/s R5gp145CladeC (Brazil)
(dCFI) dv12/h 113 113 5367 pVRC1012 (x/s) -gp145 (dCFI) (Brazil
C)/dV123 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv123/h 114 114
5368 pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV1234 pVR1012x/s
R5gp145CladeC (Brazil) (dCFI) dv1234/h 115 115 5369 pVRC1012 (x/s)
-gp145 (dCFI) (Brazil C)/dV124 pVR1012x/s R5gp145CladeC (Brazil)
(dCFI) dv124/h 116 116 5370 pVRC1012 (x/s) -gp145 (dCFI) (Brazil
C)/dV13 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv13/h 117 117
5371 pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV134 pVR1012x/s
R5gp145CladeC (Brazil) (dCFI) dv134/h 118 118 5372 pVRC1012 (x/s)
-gp145 (dCFI) (Brazil C)/dV14 pVR1012x/s R5gp145CladeC (Brazil)
(dCFI) dv14/h 119 119 5373 pVRC1012 (x/s) -gp145 (dCFI) (Brazil
C)/dV2 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv2/h 120 120 5374
pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV23 pVR1012x/s
R5gp145CladeC (Brazil) (dCFI) dv23/h 121 121 5375 pVRC1012 (x/s)
-gp145 (dCFI) (Brazil C)/dV234 pVR1012x/s R5gp145CladeC (Brazil)
(dCFI) dv234/h 122 122 5376 pVRC1012 (x/s) -gp145 (dCFI) (Brazil
C)/dV24 pVR1012x/s R5gp145CladeC (Brazil) (dCFI) dv24/h 123 123
5377 pVRC1012 (x/s) -gp145 (dCFI) (Brazil C)/dV3 pVR1012x/s
R5gp145CladeC (Brazil) (dCFI) dv3/h 124 124 5378 pVRC1012 (x/s)
-gp145 (dCFI) (Brazil C)/dV34 pVR1012x/s R5gp145CladeC (Brazil)
(dCFI) dv34/h 125 125 5379 pVRC1012 (x/s) -gp145 (dCFI) (Brazil
C)/dV4 pVR1012x/s R5gp145CladeC
(Brazil) (dCFI) dv4/h 126 126 5500 pVR1012x/s R5 (SA-C) gp140
(dCFI) dV1/h pVR1012x/s R5 gp140 (dCFI) SA dv1/h 127 127 5501
pVR1012x/s R5 (SA-C) gp140 (dCFI) dV12/h pVR1012x/s R5 gp140 (dCFI)
SA dv12/h 128 128 5502 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV123/h
pVR1012x/s R5 gp140 (dCFI) SA dv123/h 129 129 5503 pVR1012x/s R5
(SA-C) gp140 (dCFI) dV1234/h pVR1012x/s R5 gp140 (dCFI) SA dv1234/h
130 130 5504 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV124/h pVR1012x/s
R5 gp140 (dCFI) SA dv124/h 131 131 5505 pVR1012x/s R5 (SA-C) gp140
(dCFI) dV13/h pVR1012x/s R5 gp140 (dCFI) SA dv13/h 132 132 5506
pVR1012x/s R5 (SA-C) gp140 (dCFI) dV134/h pVR1012x/s R5 gp140
(dCFI) SA dv134/h 133 133 5507 pVR1012x/s R5 (SA-C) gp140 (dCFI)
dV14/h pVR1012x/s R5 gp140 (dCFI) SA dv14/h 134 134 5508 pVR1012x/s
R5 (SA-C) gp140 (dCFI) dV2/h pVR1012x/s R5 gp140 (dCFI) SA dv2/h
135 135 5509 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV23/h pVR1012x/s R5
gp140 (dCFI) SA dv23/h 136 136 5510 pVR1012x/s R5 (SA-C) gp140
(dCFI) dV234/h pVR1012x/s R5 gp140 (dCFI) SA dv234/h 137 137 5511
pVR1012x/s R5 (SA-C) gp140 (dCFI) dV24/h pVR1012x/s R5 gp140 (dCFI)
SA dv24/h 138 138 5512 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV3/h
pVR1012x/s R5 gp140 (dCFI) SA dv3/h 139 139 5513 pVR1012x/s R5
(SA-C) gp140 (dCFI) dV34/h pVR1012x/s R5 gp140 (dCFI) SA dv34/h 140
140 5514 pVR1012x/s R5 (SA-C) gp140 (dCFI) dV4/h pVR1012x/s R5
gp140 (dCFI) SA dv4/h 141 141 5515 pVR1012x/s R5 (SA-C) gp145
(dCFI) dV1/h pVR1012x/s R5 gp145 (dCFI) SA dv1/h 142 142 5516
pVR1012x/s R5 (SA-C) gp145 (dCFI) dV12/h pVR1012x/s R5 gp145 (dCFI)
SA dv12/h 143 143 5517 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV123/h
pVR1012x/s R5 gp145 (dCFI) SA dv123/h 144 144 5518 pVR1012x/s R5
(SA-C) gp145 (dCFI) dV1234/h pVR1012x/s R5 gp145 (dCFI) SA dv1234/h
145 145 5519 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV2/h pVR1012x/s R5
gp145 (dCFI) SA dv2/h 146 146 5520 pVR1012x/s R5 (SA-C) gp145
(dCFI) dV23/h pVR1012x/s R5 gp145 (dCFI) SA dv23/h 147 147 5521
pVR1012x/s R5 (SA-C) gp145 (dCFI) dV234/h pVR1012x/s R5 gp145
(dCFI) SA dv234/h 148 148 5522 pVR1012x/s R5 (SA-C) gp145 (dCFI)
dV24/h pVR1012x/s R5 gp145 (dCFI) SA dv24/h 149 149 5523 pVR1012x/s
R5 (SA-C) gp145 (dCFI) dV3/h pVR1012x/s R5 gp145 (dCFI) SA dv3/h
150 150 5524 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV34/h pVR1012x/s R5
gp145 (dCFI) SA dv34/h 151 151 5525 pVR1012x/s R5 (SA-C) gp145
(dCFI) dV4/h pVR1012x/s R5 gp145 (dCFI) SA dv4/h 152 152 5526
pVR1012x/s R5 (SA-C) gp145 (dCFI) dV13/h pVR1012x/s R5 gp145 (dCFI)
SA dv13/h 153 153 5527 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV134/h
pVR1012x/s R5 gp145 (dCFI) SA dv134/h 154 154 5528 pVR1012x/s R5
(SA-C) gp145 (dCFI) dV124/h pVR1012x/s R5 gp145 (dCFI) SA dv124/h
155 155 5529 pVR1012x/s R5 (SA-C) gp145 (dCFI) dV14/h pVR1012x/s R5
gp145 (dCFI) SA dv14/h 156 156 Gag/Pol plasmids 3900 pVR1012x/s HIV
Gag/h pVR1012x/s Gag/h 157 157 3901 pVR1012x/s SIV Gag/h pVR1012x/s
SIV Gag/h 158 158 4000 pVR1012x/s HIV Gag-Pol .DELTA.FS .DELTA.PR/h
OR pVR1012x/s Gag- pVR1012x/s Gag-PoldeltaFSdeltaPr/h 159 159 Pol/h
4001 pVR1012x/s SIV Gag-Pol/h pVR1012x/s SIV Gag-Pol/h 160 160 4100
pVR1012x/s HIV Pol/h pVR1012x/s Pol/h 161 161 4101 pVR1012x/s SIV
Pol/h pVR1012x/s SIV Pol/h 162 162 4200 pVR1012x/s HIV Gag-Pol/h
pVR1012x/s Gag (fs) Pol/h 163 163 4300 pVR1012x/s HIV Gag-Pol
.DELTA.RT .DELTA.IN/h OR pVR1012 Gag- pVR1012x/s Gag-Pol (fs) RT
(-) IN (-) 164 164 Pol (d delta RT delta IN)/h 4301 pVR1012x/s-Gag
(FS) -Pol-delta RT IN-IRES-R5 gp157-Nef pVR1012x/s-Gag (FS)
-Pol-delta RT IN-IRES-R5 gp157-Nef 165 165 4302 pVR1012x/s HIV
gag-Pol .DELTA.FS .DELTA.PR .DELTA.RT .DELTA.IN/h pVR1012x/s Gag
(delFS) Pol (delta PR delta RT delta IN)/h 166 166 4303 pVR1012x/s
SIV Gag-Pol .DELTA.FS/h OR pVR1012 SIV pVR1012x/s SIV Gag (delFS)
-Pol/h 167 167 Gag (delFS) Pol (delta PR delta RT delta IN)/h 4304
pVR1012 Gag (delFS) Pol delta PR delta RT delta IN/h pVR1012 Gag-C
(delFS) Pol (deltaPR deltaRT deltaIN)/h 168 168 4305 pVR1012 Gag-A
(delFS) Pol (delta PR delta RT delta IN)/h pVR1012 Gag-A (delFS)
Pol (deltaPR delta RT deltaIN)/h 169 169 4306 pVR1012 Gag (delFS)
Pol delta PR delta RT delta IN/Nef/h pVR1012 Gag (delFS) Pol
deltaPR deltaRT deltaIN/Nef/h 170 170 4308 pVR1012 Gag (delFS) Pol
deltaPR deltaRT deltaIN deltaMyr/h pVR1012 Gag (delFS) Pol deltaPR
deltaRT deltaIN delta 171 171 Myr/h 4309 pVR1012 Gag (delFS) Pol
deltaPR deltaRT deltaIN delta pVR1012x/s Gag (delFS) Pol deltaPR
deltaRT deltaIN delta 172 172 Myr/Nef/h Myr/Nef/h 4310 pVR1012 Nef
Gag (del fs) (del Myr) Pol (delta PR delta RT pVR1012 Nef Gag
(delFS) (del Myr) Pol (delta PR delta RT 173 173 delta IN)/h delta
IN)/h 4311 pVR1012 Gag-C (delFS) Pol (delta PR delta RT delta IN)
Nef/h pVR1012 Gag-C (del PS) Pol (delta PR dalta RT delta IN) 174
174 Nef/h 4312 Gag (del fs) (del Myr) Nef Pol
.DELTA.PR.DELTA.RT.DELTA.IN/h pVR1012 Gag (del FS) (del Myr) Nef
Pol (delta PR delta RT 175 175 delta IN)/h 4313 pVR1012 Gag Clade A
(del fs) Pol (.DELTA. PR .DELTA. RT .DELTA. IN)/h pVR1012 Gag-A
(del FS) Pol (delPR delRT delIN) Nef/h 176 176
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0203] The present invention provides modifications of HIV Env,
Gag, Pol, and Nef that enhance immunogenicity for genetic
immunization. Both HIV and SIV are genetically related members of
the lentivirus genus of the Retroviridae family. Lentivirus
isolates from humans are grouped into one of two types, designated
HIV-1 and HIV-2. A classification scheme recognizes nine subtypes
(clades) of HIV-1 (A through 1) and five subtypes of HIV-2 (A
through E). A compendium of HIV and SIV sequence information is
found in a database prepared by Myers et al., Los Alamos, N. Mex.:
Los Alamos National Laboratory.
Nucleic Acid Molecules
[0204] As indicated herein, nucleic acid molecules of the present
invention may be in the form of RNA or in the form of DNA obtained
by cloning or produced synthetically. The DNA may be
double-stranded or single-stranded. Single-stranded DNA or RNA may
be the coding strand, also known as the sense strand, or it may be
the non-coding strand, also referred to as the anti-sense
strand.
[0205] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0206] Nucleic acid molecules of the present invention include DNA
molecules comprising an open reading frame (ORF) of a wild-type HIV
gene; and DNA molecules which comprise a sequence substantially
different from those described above but which, due to the
degeneracy of the genetic code, still encode an ORF of a wild-type
HIV polypeptide. Of course, the genetic code is well known in the
art. Degenerate variants optimized for human codon usage are
preferred.
[0207] In another aspect, the invention provides a nucleic acid
molecule comprising a polynucleotide which hybridizes under
stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above. By "stringent hybridization conditions" is
intended overnight incubation at 42 degree C. in a solution
comprising: 50% formamide, 5 times SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 times
Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1 times SSC at about 65 degree C.
[0208] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide.
[0209] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide. Of
course, a polynucleotide which hybridizes only to a complementary
stretch of T (or U) resides, would not be included in a
polynucleotide of the invention used to hybridize to a portion of a
nucleic acid of the invention, since such a polynucleotide would
hybridize to any nucleic acid molecule containing a poly T (or U)
stretch or the complement thereof (e.g., practically any
double-stranded DNA clone).
[0210] As indicated herein, nucleic acid molecules of the present
invention which encode an HIV polypeptide may include, but are not
limited to those encoding the amino acid sequence of the
full-length polypeptide, by itself, the coding sequence for the
full-length polypeptide and additional sequences, such as those
encoding a leader or secretory sequence, such as a pre-, or pro- or
prepro-protein sequence, the coding sequence of the full-length
polypeptide, with or without the aforementioned additional coding
sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example, ribosome binding
and stability of mRNA; and additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities.
[0211] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the HIV protein. Variants may
occur naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a genome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0212] Such variants include those produced by nucleotide
substitutions, deletions or additions, which may involve one or
more nucleotides. The variants may be altered in coding regions,
non-coding regions, or both. Alterations in the coding regions may
produce conservative or non-conservative amino acid substitutions,
deletions or additions. Especially preferred among these are silent
substitutions, additions and deletions, which do not alter the
properties and activities of the HIV polypeptide or portions
thereof. Also especially preferred in this regard are conservative
substitutions.
[0213] Further embodiments of the invention include nucleic acid
molecules comprising a polynucleotide having a nucleotide sequence
at least 95% identical, and more preferably at least 96%, 97%, 98%
or 99% identical to a nucleotide sequence encoding a polypeptide
having the amino acid sequence of a wild-type HIV polypeptide or a
nucleotide sequence complementary thereto.
[0214] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a HIV polypeptide is intended that the nucleotide sequence
of the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the HIV polypeptide. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These mutations of the reference sequence may
occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0215] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to the
reference nucleotide sequence can be determined conventionally
using known computer programs such as the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive,
Madison, Wis. 53711). Bestfit uses the local homology algorithm of
Smith and Waterman, Advances in Applied Mathematics 2: 482-489
(1981), to find the best segment of homology between two sequences.
When using Bestfit or any other sequence alignment program to
determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present
invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0216] The present application is directed to nucleic acid
molecules at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequences shown herein in the Sequence Listing which
encode a polypeptide having HIV polypeptide activity. By "a
polypeptide having HIV activity" is intended polypeptides
exhibiting HIV activity in a particular biological assay. For
example, Env, Gag, and Pol protein activity can be measured for
changes in immunological character by an appropriate immunological
assay.
[0217] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence
shown herein in the Sequence Listing will encode a polypeptide
"having HIV polypeptide activity." In fact, since degenerate
variants of these nucleotide sequences all encode the same
polypeptide, this will be clear to the skilled artisan even without
performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that
are not degenerate variants, a reasonable number will also encode a
polypeptide having HIV polypeptide activity. This is because the
skilled artisan is fully aware of amino acid substitutions that are
either less likely or not likely to significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second
aliphatic amino acid).
[0218] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that proteins are surprisingly tolerant of amino
acid substitutions.
Polypeptides and Fragments
[0219] The invention further provides a HIV polypeptide having the
amino acid sequence encoded by an open reading frame (ORF) of a
wild-type HIV gene, or a peptide or polypeptide comprising a
portion thereof (e.g., gp120).
[0220] It will be recognized in the art that some amino acid
sequences of the HIV polypeptides can be varied without significant
effect of the structure or function of the protein. If such
differences in sequence are contemplated, it should be remembered
that there will be critical areas on the protein which determine
activity.
[0221] Thus, the invention further includes variations of the HIV
polypeptide which show substantial HIV polypeptide activity or
which include regions of HIV protein such as the protein portions
discussed below. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions. As indicated, guidance
concerning which amino acid changes are likely to be phenotypically
silent can be found in Bowie, J. U., et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990).
[0222] Thus, the fragment, derivative or analog of the polypeptide
of the invention may be (i) one in which one or more of the amino
acid residues are substituted with a conserved or non-conserved
amino acid residue (preferably a conserved amino acid residue) and
such substituted amino acid residue may or may not be one encoded
by the genetic code, or (ii) one in which one or more of the amino
acid residues includes a substituent group, or (iii) one in which
additional amino acids are fused to the mature polypeptide, such as
an IgG Fc fusion region peptide or leader or secretory sequence or
a sequence which is employed for purification of the mature
polypeptide or a proprotein sequence. Such fragments, derivatives
and analogs are deemed to be within the scope of those skilled in
the art from the teachings herein.
[0223] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table A).
TABLE-US-00002 TABLE A Conservative Amino Acid Substitutions
Aromatic Phenylalanine Tryptophan Tyrosine Ionizable: Acidic
Aspartic Acid Glutamic Acid Ionizable: Basic Arginine Histidine
Lysine Nonionizable Polar Asparagine Glutamine Selenocystine Serine
Threonine Nonpolar (Hydrophobic) Alanine Glycine Isoleucine Leucine
Proline Valine Sulfur Containing Cysteine Methionine
[0224] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of amino acid
substitutions for any given HIV polypeptide will not be more than
50, 40, 30, 20, 10, 5 or 3.
[0225] Amino acids in the HIV polypeptides of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as changes in immunological
character.
[0226] The polypeptides of the present invention are conveniently
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present
invention.
[0227] Also intended as an "isolated polypeptide" are polypeptides
that have been purified, partially or substantially, from a
recombinant host cell or a native source. For example, a
recombinantly produced version of the HIV polypeptide can be
substantially purified by the one-step method described in Smith
and Johnson, Gene 67:31-40 (1988).
[0228] The polypeptides of the present invention include a
polypeptide comprising a polypeptide shown herein in the Sequence
Listing; as well as polypeptides which are at least 95% identical,
and more preferably at least 96%, 97%, 98% or 99% identical to
those described above and also include portions of such
polypeptides with at least 30 amino acids and more preferably at
least 50 amino acids.
[0229] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of an
HIV polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the HIV
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0230] As a practical matter, whether any particular polypeptide is
at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown herein in the Sequence Listing can be
determined conventionally using known computer programs such the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0231] The polypeptides of the invention may be produced by any
conventional means. Houghten, R. A. (1985) General method for the
rapid solid-phase synthesis of large numbers of peptides:
specificity of antigen-antibody interaction at the level of
individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135.
This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is
further described in U.S. Pat. No. 4,631,211 to Houghten et al.
(1986).
[0232] The present invention also relates to vectors which include
the nucleic acid molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of HIV polypeptides or fragments thereof by
recombinant techniques.
[0233] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0234] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be
translated.
[0235] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0236] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 available from Pharmacia. Among preferred eukaryotic vectors
are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0237] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0238] The HIV polypeptide can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the
host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may
also include an initial modified methionine residue, in some cases
as a result of host-mediated processes.
Pharmaceutical Formulations, Dosages, and Modes of
Administration
[0239] The compounds of the invention may be administered using
techniques well known to those in the art. Preferably, compounds
are formulated and administered by genetic immunization. Techniques
for formulation and administration may be found in "Remington's
Pharmaceutical Sciences", 18.sup.th ed., 1990, Mack Publishing Co.,
Easton, Pa. Suitable routes may include parenteral delivery, such
as intramuscular, intradermal, subcutaneous, intramedullary
injections, as well as, intrathecal, direct intraventricular,
intravenous, intraperitoneal, intranasal, or intraocular
injections, just to name a few. For injection, the compounds of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer.
[0240] In instances wherein intracellular administration of the
compounds of the invention is preferred, techniques well known to
those of ordinary skill in the art may be utilized. For example,
such compounds may be encapsulated into liposomes, then
administered as described above. Liposomes are spherical lipid
bilayers with aqueous interiors. All molecules present in an
aqueous solution at the time of liposome formation are incorporated
into the aqueous interior. The liposomal contents are both
protected from the external microenvironment and, because liposomes
fuse with cell membranes, are effectively delivered into the cell
cytoplasm.
[0241] Nucleotide sequences of the invention which are to be
intracellularly administered may be expressed in cells of interest,
using techniques well known to those of skill in the art. For
example, expression vectors derived from viruses such as
retroviruses, adenoviruses, adeno-associated viruses, herpes
viruses, vaccinia viruses, polio viruses, or sindbis or other RNA
viruses, or from plasmids may be used for delivery and expression
of such nucleotide sequences into the targeted cell population.
Methods for the construction of such expression vectors are well
known. See, for example, Sambrook et al., 1989, Molecular Cloning,
A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., and Ausubel et al., 1989, Current Protocols In Molecular
Biology, Greene Publishing Associates and Wiley Interscience,
NY.
[0242] The invention extends to the use of a plasmid for primary
immunization (priming) of a host and the subsequent use of a
recombinant virus, such as a retrovirus, adenovirus,
adeno-associated virus, herpes virus, vaccinia virus, polio virus,
or sindbis or other RNA virus, for boosting said host, and vice
versa. For example, the host may be immunized (primed) with a
plasmid by DNA immunization and receive a boost with the
corresponding viral construct, and vice versa. Alternatively, the
host may be immunized (primed) with a plasmid by DNA immunization
and receive a boost with not the corresponding viral construct but
a different viral construct, and vice versa.
[0243] With respect to HIV Env, Gag, and Pol, protein sequences of
the invention may be used as therapeutics or prophylatics (as
subunit vaccines) in the treatment of AIDS or HIV infection. A
therapeutically effective dose refers to that amount of the
compound sufficient to result in amelioration of symptoms or a
prolongation of survival in a patient. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50%
of the population). The dose ratio between toxic and therapeutic
effects is the therapeutic index and it can be expressed as the
ratio LD50/ED50. Compounds which exhibit large therapeutic indices
are preferred. The data obtained from cell culture assays and
animal studies can be used in formulating a range of dosage for use
in humans. The dosage of such compounds lies preferably within a
range of circulating concentrations that includes the ED50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC50 (e.g., the concentration of the test
compound which achieves a half-maximal inhibition of viral
infection relative to the amount of the event in the absence of the
test compound) as determined in cell culture. Such information can
be used to more accurately determine useful doses in humans. Levels
in plasma may be measured, for example, by high performance liquid
chromatography (HPLC).
[0244] The compounds of the invention may, further, serve the role
of a prophylactic vaccine, wherein the host produces antibodies
and/or CTL responses against HIV Env, Gag and Pol, which responses
then preferably serve to neutralize HIV viruses by, for example,
inhibiting further HIV infection. Administration of the compounds
of the invention as a prophylactic vaccine, therefore, would
comprise administering to a host a concentration of compounds
effective in raising an immune response which is sufficient to
elicit antibody and/or CTL responses to HIV Env, Gag, and Pol,
and/or neutralize HIV, by, for example, inhibiting HIV ability to
infect cells. The exact concentration will depend upon the specific
compound to be administered, but may be determined by using
standard techniques for assaying the development of an immune
response which are well known to those of ordinary skill in the
art.
[0245] The compounds may be formulated with a suitable adjuvant in
order to enhance the immunological response. Such adjuvants may
include, but are not limited to mineral gels such as aluminum
hydroxide; surface active substances such as lysolecithin, pluronic
polyols, polyanions; other peptides; oil emulsions; and potentially
useful human adjuvants such as BCG and Corynebacterium parvum.
[0246] Adjuvants suitable for co-administration in accordance with
the present invention should be ones that are potentially safe,
well tolerated and effective in people including QS-21, Detox-PC,
MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B,
Adjumer, PG-026, GSK-1, GcMAF, B-alethine, MPC-026, Adjuvax, CpG
ODN, Betafectin, Alum, and MF59 (see Kim et al., 2000, Vaccine, 18:
597 and references therein).
[0247] Other contemplated adjuvants that may be administered
include lectins, growth factors, cytokines and lymphokines such as
alpha-interferon, gamma-interferon, platelet derived growth factor
(PDGF), gCSF, gMCSF, TNF, epidermal growth factor (EGF), IL-1,
IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12.
[0248] For all such treatments described above, the exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0249] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity, or to organ dysfunctions. Conversely, the
attending physician would also know to adjust treatment to higher
levels if the clinical response were not adequate (precluding
toxicity). The magnitude of an administered dose in the management
of the viral infection of interest will vary with the severity of
the condition to be treated and the route of administration. The
dose and perhaps prime-boost regimen, will also vary according to
the age, weight, and response of the individual patient. A program
comparable to that discussed above may be used in veterinary
medicine.
[0250] The pharmacologically active compounds of this invention can
be processed in accordance with conventional methods of galenic
pharmacy to produce medicinal agents for administration to
patients, e.g., mammals including humans.
[0251] The compounds of this invention can be employed in admixture
with conventional excipients, i.e., pharmaceutically acceptable
organic or inorganic carrier substances suitable for parenteral,
enteral (e.g., oral) or topical application which do not
deleteriously react with the active compounds. Suitable
pharmaceutically acceptable carriers include but are not limited to
water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl
alcohols, polyethylene glycols, gelatine, carbohydrates such as
lactose, amylose or starch, magnesium stearate, talc, silicic acid,
viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, pentaerythritol fatty acid esters, hydroxy
methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical
preparations can be sterilized and if desired mixed with auxiliary
agents, e.g., lubricants, preservatives, stabilizers, wetting
agents, emulsifiers, salts for influencing osmotic pressure,
buffers, coloring, flavoring and/or aromatic substances and the
like which do not deleteriously react with the active compounds.
They can also be combined where desired with other active agents,
e.g., vitamins.
[0252] For parenteral application, which includes intramuscular,
intradermal, subcutaneous, intranasal, intracapsular, intraspinal,
intrasternal, and intravenous injection, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. Formulations for injection may be
presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0253] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules. The
pharmaceutical compositions may be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. A syrup, elixir, or the like can be used
wherein a sweetened vehicle is employed.
[0254] Sustained or directed release compositions can be
formulated, e.g., liposomes or those wherein the active compound is
protected with differentially degradable coatings, e.g., by
microencapsulation, multiple coatings, etc. It is also possible to
freeze dry the new compounds and use the lyophilizates obtained,
for example, for the preparation of products for injection.
[0255] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0256] For topical application, there are employed as non-sprayable
forms, viscous to semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include but
are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, aerosols, etc., which are,
if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives, stabilizers, wetting agents, buffers or salts for
influencing osmotic pressure, etc. For topical application, also
suitable are sprayable aerosol preparations wherein the active
ingredient, preferably in combination with a solid or liquid inert
carrier material, is packaged in a squeeze bottle or in admixture
with a pressurized volatile, normally gaseous propellant, e.g.; a
freon.
[0257] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
Genetic Immunization
[0258] Genetic immunization according to the present invention
elicits an effective immune response without the use of infective
agents or infective vectors. Vaccination techniques which usually
do produce a CTL response do so through the use of an infective
agent. A complete, broad based immune response is not generally
exhibited in individuals immunized with killed, inactivated or
subunit vaccines. The present invention achieves the full
complement of immune responses in a safe manner without the risks
and problems associated with vaccinations that use infectious
agents.
[0259] According to the present invention, DNA or RNA that encodes
a target protein is introduced into the cells of an individual
where it is expressed, thus producing the target protein. The DNA
or RNA is linked to regulatory elements necessary for expression in
the cells of the individual. Regulatory elements for DNA include a
promoter and a polyadenylation signal. In addition, other elements,
such as a Kozak region, may also be included in the genetic
construct.
[0260] The genetic constructs of genetic vaccines comprise a
nucleotide sequence that encodes a target protein operably linked
to regulatory elements needed for gene expression. Accordingly,
incorporation of the DNA or RNA molecule into a living cell results
in the expression of the DNA or RNA encoding the target protein and
thus, production of the target protein.
[0261] When taken up by a cell, the genetic construct which
includes the nucleotide sequence encoding the target protein
operably linked to the regulatory elements may remain present in
the cell as a functioning extrachromosomal molecule or it may
integrate into the cell's chromosomal DNA. DNA may be introduced
into cells where it remains as separate genetic material in the
form of a plasmid. Alternatively, linear DNA which can integrate
into the chromosome may be introduced into the cell. When
introducing DNA into the cell, reagents which promote DNA
integration into chromosomes may be added. DNA sequences which are
useful to promote integration may also be included in the DNA
molecule. Since integration into the chromosomal DNA necessarily
requires manipulation of the chromosome, it is preferred to
maintain the DNA construct as a replicating or non-replicating
extrachromosomal molecule. This reduces the risk of damaging the
cell by splicing into the chromosome without affecting the
effectiveness of the vaccine. Alternatively, RNA may be
administered to the cell. It is also contemplated to provide the
genetic construct as a linear minichromosome including a
centromere, telomeres and an origin of replication.
[0262] The necessary elements of a genetic construct of a genetic
vaccine include a nucleotide sequence that encodes a target protein
and the regulatory elements necessary for expression of that
sequence in the cells of the vaccinated individual. The regulatory
elements are operably linked to the DNA sequence that encodes the
target protein to enable expression.
[0263] The molecule that encodes a target protein is a
protein-encoding molecule which is translated into protein. Such
molecules include DNA or RNA which comprise a nucleotide sequence
that encodes the target protein. These molecules may be cDNA,
genomic DNA, synthesized DNA or a hybrid thereof or an RNA molecule
such as mRNA. Accordingly, as used herein, the terms "DNA
construct", "genetic construct" and "nucleotide sequence" are meant
to refer to both DNA and RNA molecules.
[0264] The regulatory elements necessary for gene expression of a
DNA molecule include: a promoter, an initiation codon, a stop
codon, and a polyadenylation signal. In addition, enhancers are
often required for gene expression. It is necessary that these
elements be operable in the vaccinated individual. Moreover, it is
necessary that these elements be operably linked to the nucleotide
sequence that encodes the target protein such that the nucleotide
sequence can be expressed in the cells of a vaccinated individual
and thus the target protein can be produced.
[0265] Initiation codons and stop codons are generally considered
to be part of a nucleotide sequence that encodes the target
protein. However, it is necessary that these elements are
functional in the vaccinated individual.
[0266] Similarly, promoters and polyadenylation signals used must
be functional within the cells of the vaccinated individual.
[0267] Examples of promoters useful to practice the present
invention, especially in the production of a genetic vaccine for
humans, include but are not limited to promoters from Simian Virus
40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human
Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat
(LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as
the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous
Sarcoma Virus (RSV) as well as promoters from human genes such as
human Actin, human Myosin, human Hemoglobin, human muscle creatine
and human metalothionein.
[0268] Examples of polyadenylation signals useful to practice the
present invention, especially in the production of a genetic
vaccine for humans, include but are not limited to SV40
polyadenylation signals and LTR polyadenylation signals. In
particular, the SV40 polyadenylation signal which is in pCEP4
plasmid (Invitrogen, San Diego Calif.), referred to as the SV40
polyadenylation signal, can be used.
[0269] In addition to the regulatory elements required for DNA
expression, other elements may also be included in the DNA
molecule. Such additional elements include enhancers. The enhancer
may be selected from the group including but not limited to: human
Actin, human Myosin, human Hemoglobin, human muscle creatine and
viral enhancers such as those from CMV, RSV and EBV.
[0270] Genetic constructs can be provided with mammalian origin of
replication in order to maintain the construct extrachromosomally
and produce multiple copies of the construct in the cell. Plasmids
pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the
Epstein Barr virus origin of replication and nuclear antigen EBNA-1
coding region which produces high copy episomal replication without
integration.
[0271] An additional element may be added which serves as a target
for cell destruction if it is desirable to eliminate cells
receiving the genetic construct for any reason. A herpes thymidine
kinase (tk) gene in an expressible form can be included in the
genetic construct. When the construct is introduced into the cell,
tk will be produced. The drug gangcyclovir can be administered to
the individual and that drug will cause the selective killing of
any cell producing tk. Thus, a system can be provided which allows
for the selective destruction of vaccinated cells.
[0272] In order to be a functional genetic construct, the
regulatory elements must be operably linked to the nucleotide
sequence that encodes the target protein. Accordingly, it is
necessary for the initiation and termination codons to be in frame
with the coding sequence.
[0273] Open reading frames (ORFs) encoding the protein of interest
and another or other proteins of interest may be introduced into
the cell on the same vector or on different vectors. ORFs on a
vector may be controlled by separate promoters or by a single
promoter. In the latter arrangement, which gives rise to a
polycistronic message, the ORFs will be separated by translational
stop and start signals. The presence of an internal ribosome entry
site (IRES) site between these ORFs permits the production of the
expression product originating from the second ORF of interest, or
third, etc. by internal initiation of the translation of the
bicistronic or polycistronic mRNA.
[0274] According to the invention, the genetic vaccine may be
administered directly into the individual to be immunized or ex
vivo into removed cells of the individual which are reimplanted
after administration. By either route, the genetic material is
introduced into cells which are present in the body of the
individual. Routes of administration include, but are not limited
to, intramuscular, intraperitoneal, intradermal, subcutaneous,
intravenous, intraarterially, intraoccularly and oral as well as
transdermally or by inhalation or suppository. Preferred routes of
administration include intramuscular, intraperitoneal, intradermal
and subcutaneous injection. Genetic constructs may be administered
by means including, but not limited to, traditional syringes,
needleless injection devices, or microprojectile bombardment gene
guns. Alternatively, the genetic vaccine may be introduced by
various means into cells that are removed from the individual. Such
means include, for example, ex vivo transfection, electroporation,
microinjection and microprojectile bombardment. After the genetic
construct is taken up by the cells, they are reimplanted into the
individual. It is contemplated that otherwise non-immunogenic cells
that have genetic constructs incorporated therein can be implanted
into the individual even if the vaccinated cells were originally
taken from another individual.
[0275] The genetic vaccines according to the present invention
comprise about 1 nanogram to about 1000 micrograms of DNA. In some
preferred embodiments, the vaccines contain about 10 nanograms to
about 800 micrograms of DNA. In some preferred embodiments, the
vaccines contain about 0.1 to about 500 micrograms of DNA. In some
preferred embodiments, the vaccines contain about 1 to about 350
micrograms of DNA. In some preferred embodiments, the vaccines
contain about 25 to about 250 micrograms of DNA. In some preferred
embodiments, the vaccines contain about 100 micrograms DNA.
[0276] The genetic vaccines according to the present invention are
formulated according to the mode of administration to be used. One
having ordinary skill in the art can readily formulate a genetic
vaccine that comprises a genetic construct. In cases where
intramuscular injection is the chosen mode of administration, an
isotonic formulation is preferably used. Generally, additives for
isotonicity can include sodium chloride, dextrose, mannitol,
sorbitol and lactose. In some cases, isotonic solutions such as
phosphate buffered saline are preferred. Stabilizers include
gelatin and albumin. In some embodiments, a vaso-constriction agent
is added to the formulation. The pharmaceutical preparations
according to the present invention are provided sterile and pyrogen
free.
[0277] Genetic constructs may optionally be formulated with one or
more response enhancing agents such as: compounds which enhance
transfection, i.e. transfecting agents; compounds which stimulate
cell division, i.e. replication agents; compounds which stimulate
immune cell migration to the site of administration, i.e.
inflammatory agents; compounds which enhance an immune response,
i.e. adjuvants or compounds having two or more of these
activities.
[0278] In one embodiment, bupivacaine, a well known and
commercially available pharmaceutical compound, is administered
prior to, simultaneously with or subsequent to the genetic
construct. Bupivacaine and the genetic construct may be formulated
in the same composition. Bupivacaine is particularly useful as a
cell stimulating agent in view of its many properties and
activities when administered to tissue. Bupivacaine promotes and
facilitates the uptake of genetic material by the cell. As such, it
is a transfecting agent. Administration of genetic constructs in
conjunction with bupivacaine facilitates entry of the genetic
constructs into cells. Bupivacaine is believed to disrupt or
otherwise render the cell membrane more permeable. Cell division
and replication is stimulated by bupivacaine. Accordingly,
bupivacaine acts as a replicating agent. Administration of
bupivacaine also irritates and damages the tissue. As such, it acts
as an inflammatory agent which elicits migration and chemotaxis of
immune cells to the site of administration. In addition to the
cells normally present at the site of administration, the cells of
the immune system which migrate to the site in response to the
inflammatory agent can come into contact with the administered
genetic material and the bupivacaine. Bupivacaine, acting as a
transfection agent, is available to promote uptake of genetic
material by such cells of the immune system as well.
[0279] In addition to bupivacaine, mepivacaine, lidocaine,
procains, carbocaine, methyl bupivacaine, and other similarly
acting compounds may be used as response enhancing agents. Such
agents acts a cell stimulating agents which promote the uptake of
genetic constructs into the cell and stimulate cell replication as
well as initiate an inflammatory response at the site of
administration.
[0280] Other contemplated response enhancing agents which may
function as transfecting agents and/or replicating agents and/or
inflammatory agents and which may be administered include lectins,
growth factors, cytokines and lymphokines such as alpha-interferon,
gamma-interferon, platelet derived growth factor (PDGF), gCSF,
gMCSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6,
IL-8, IL-10 and IL-12 as well as collagenase, fibroblast growth
factor, estrogen, dexamethasone, saponins, surface active agents
such as immune-stimulating complexes (ISCOMS), Freund's incomplete
adjuvant, LPS analog including monophosphoryl Lipid A (MPL),
muramyl peptides, quinone analogs and vesicles such as squalene and
squalane, hyaluronic acid and hyaluronidase may also be used
administered in conjunction with the genetic construct. In some
embodiments, combinations of these agents are co-administered in
conjunction with the genetic construct. In other embodiments, genes
encoding these agents are included in the same or different genetic
construct(s) for co-expression of the agents.
[0281] With respect to HIV Env, Gag, and Pol nucleotide sequences
of the invention, particularly through genetic immunization, may be
used as therapeutics or prophylatics in the treatment of AIDS or
HIV infection. A therapeutically effective dose refers to that
amount of the compound sufficient to result in amelioration of
symptoms or a prolongation of survival in a patient. Toxicity and
therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD50 (the dose lethal to 50% of
the population) and the ED50 (the dose therapeutically effective in
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD50/ED50. Compounds which exhibit large
therapeutic indices are preferred. The data obtained from cell
culture assays and animal studies can be used in formulating a
range of dosage for use in humans. The dosage of such compounds
lies preferably within a range of circulating concentrations that
includes the ED50 with little or no toxicity. The dosage may vary
within this range depending upon the dosage form employed and the
route of administration utilized. For any compound used in the
method of the invention, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the (e.g., the concentration of
the test compound which achieves a half-maximal inhibition of viral
infection relative to the amount of the event in the absence of the
test compound) as determined in cell culture. Such information can
be used to more accurately determine useful doses in humans. Levels
in plasma may be measured, for example, by high performance liquid
chromatography (HPLC).
[0282] The compounds (for genetic immunization) of the invention
may, further, serve the role of a prophylactic vaccine, wherein the
host produces antibodies and/or CTL responses against HIV Env, Gag,
and Pol which responses then preferably serve to neutralize HIV
viruses by, for example, inhibiting further HIV infection.
Administration of the compounds of the invention as a prophylactic
vaccine, therefore, would comprise administering to a host a
concentration of compounds effective in raising an immune response
which is sufficient to elicit antibody and/or CTL responses to HIV
Env, Gag, and Pol and/or neutralize HIV, by, for example,
inhibiting HIV ability to infect cells. The exact concentration
will depend upon the specific compound to be administered, but may
be determined by using standard techniques for assaying the
development of an immune response which are well known to those of
ordinary skill in the art.
Env
[0283] To improve the immune response to native gp160 and to expose
the core protein for optimal antigen presentation and recognition,
we have analyzed the immune response to modified forms of the
protein. The role of conserved N-linked glycosylation sites has
been studied, and analogues of fusion intermediates have been
developed. Expression vectors with deletions in the cleavage site
(C), the fusion peptide (F), and the interspace (I) between the two
heptad repeats were termed .DELTA.CFI. Plasmid DNA vaccination has
been a useful technology for the development and analysis of
immunogens. This method of vaccination allows appropriate
post-translational modification, proper intracellular trafficking,
and antigen presentation. Direct injection of naked DNA either
intramuscularly or intradermally in rodents induces immune
responses, and the ability to easily modify plasmid expression
vectors to express different forms of HIV envelope proteins enables
rapid and systematic testing of vaccine immunogens. In this
disclosure, we have analyzed the immune response to modified Env
candidates expressed in plasmids with modified codons to improve
gene expression. Both antibody and CTL responses were analyzed
after injection of plasmid DNA into muscle. A modified gp140 DNA
with improved ability to elicit antibody and CTL responses to HIV
Env has now been identified that is envisioned as a prototype
immunogen that can elicit broadly neutralizing antibody responses
to HIV.
Exposing the Core Protein of Viral Membrane Fusion Proteins
[0284] Described herein are modified HIV envelope proteins that
improve the immune response to native gp160 and expose the core
protein for optimal antigen presentation and recognition.
Weissenhorn et al., Molecular Cell, 2, 605-616, 1998 proposes a
core protein as a model for a fusion intermediate of viral
glycoproteins, where the glycoproteins are characterized by a
central triple stranded coiled coil followed by a disulfide-bonded
loop that reverses the chain direction and connects to an .alpha.
helix packed antiparallel to the core helices, as, for example, in
the case of Ebola Zaire GP2, Murine Moloney Leukemia virus (MuMoLv)
55-residue segment of the TM subunit (Mo-55), low-pH-treated
influenza HA2, protease resistant core of HIV gp41, and SIV gp41
(FIG. 177). Thus, the strategy for improving the immune response by
exposing the protease resistant core of HIV gp41 extends to other
viral membrane fusion proteins that are characterized by a central
triple stranded coiled coil followed by a disulfide-bonded loop
that reverses the chain direction and connects to an ax helix
packed antiparallel to the core helices.
[0285] The present approach involves a series of internal mutations
designed to replace the cleavage site (C), the fusion domain (F),
and the interspace (I) between the two heptad repeats all on a
backbone of COOH-terminal truncations to expose the core protein of
the viral membrane fusion protein Env, based on modified gp140 as a
prototype immunogen. By replacement is meant deletions, insertions,
and/or substitutions of amino acid residues. In one embodiment,
deletions are meant (i.e., amino acids are deleted to create the
.DELTA.CFI mutations).
[0286] In this embodiment, the .DELTA.C mutation is intended to
eliminate proteolysis by deleting the gp120/gp41 cleavage site that
links the envelope covalently to the ectodomain by 100%, 99%, 98%,
97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,
84%, 83%, 82%, 81,%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73,%, 72%,
71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%,
58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%,
45%, 44%, 43%, 42, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%,
32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%,
19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, or 1%.
[0287] In this embodiment, the .DELTA.F mutation is intended to
solubilize the molecule by deleting the fusion domain by 100%, 99%,
98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%,
85%, 84%, 83%, 82%, 81,%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73,%,
72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%,
59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%,
46%, 45%, 44%, 43%, 42, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%,
33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%,
20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2%, or 1%.
[0288] In this embodiment, the .DELTA.I mutation is intended to
stabilize oligomer formation by deleting the interspace between the
two heptad repeats by 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,
91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81,%, 80%, 79%,
78%, 77%, 76%, 75%, 74%, 73,%, 72%, 71%, 70%, 69%, 68%, 67%, 66%,
65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%,
52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42, 41%, 40%,
39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%,
26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
[0289] In this embodiment, the COOH-terminal truncation is intended
to reduce toxicity by deleting the cytoplasmic domain by 100%, 99%,
98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%,
85%, 84%, 83%, 82%, 81,%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73,%,
72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%,
59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%,
46%, 45%, 44%, 43%, 42, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%,
33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%,
20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2%, or 1%.
[0290] In this embodiment, optionally, the COOH-terminal truncation
is extended so as to solubilize the molecule by deleting the
transmembrane domain by 100% 99%, 98%, 97%, 96%, 95%, 94%, 93%,
92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81,%, 80%,
79%, 78%, 77%, 76%, 75%, 74%, 73,%, 72%, 71%, 70%, 69%, 68%, 67%,
66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%,
53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42, 41%,
40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%,
27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%,
14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
[0291] Amino acid substitutions may encompass those of a conserved
or non-conserved nature. Presumably, a non-conserved substitution
of a domain would act like a deletion of the domain. Conserved
amino acid substitutions constitute switching one or more amino
acids with amino acids of similar charge, size, and/or
hydrophobicity characteristics. Non-conserved amino acid
substitutions constitute switching one or more amino acids with
amino acids of dissimilar charge, size, and/or hydrophobicity
characteristics. The families of amino acids include the basic
amino acids (lysine, arginine, histidine); the acidic amino acids
(aspartic acid, glutamic acid); the non-polar amino acids (alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan); the uncharged polar amino acids (glycine, asparagine,
glutamine, cysteine, serine, threonine, tyrosine); and the aromatic
amino acids (phenylalanine, tryptophan, and tyrosine). One or more
substitutions may be introduced to achieve the .DELTA.C mutation
intended to eliminate proteolysis by acting like a deletion of the
gp120/gp41 cleavage site to link the envelope covalently to the
ectodomain, the .DELTA.F mutation intended to solubilize the
molecule by acting like a deletion of the fusion domain, the
.DELTA.I mutation intended to stabilize oligomer formation by
acting like a deletion of the interspace between the two heptad
repeats, the COOH-terminal truncation intended to reduce toxicity
by acting like a deletion of the cytoplasmic domain, and,
optionally, the COOH-terminal truncation extended so as to
solubilize the molecule by acting like a deletion of the
transmembrane domain.
[0292] Amino acid insertions may constitute single amino acid
residues or stretches of residues. The insertions may be made at
the carboxy or amino terminal end of a domain, as well as at a
position internal to the domain. Such insertions will generally
range from 2 to 15 amino acids in length. One or more insertions
may be introduced to achieve the .DELTA.C mutation intended to
eliminate proteolysis by acting like a deletion of the gp120/gp41
cleavage site to link the envelope covalently to the ectodomain,
the AF mutation intended to solubilize the molecule by acting like
a deletion of the fusion domain, the .DELTA.I mutation intended to
stabilize oligomer formation by acting like a deletion the
interspace between the two heptad repeats, the COOH-terminal
truncation intended to reduce toxicity by acting like a deletion of
the cytoplasmic domain, and, optionally, the COOH-terminal
truncation extended so as to solubilize the molecule by acting like
a deletion of the transmembrane domain.
[0293] The nucleic acids of the present invention are optionally
DNA, RNA, or mRNA. Most typically, the nucleic acids are provided
by recombinantly making a DNA, which is expressed in a cell as RNA
and/or as mRNA. Given the strategy for making the nucleic acids of
the present invention, one of skill can construct a variety of
clones containing functionally equivalent nucleic acids. Cloning
methodologies to accomplish these ends, and sequencing methods to
verify the sequence of nucleic acids are well known in the art.
Examples of appropriate cloning and sequencing techniques, and
instructions sufficient to direct persons of skill through many
cloning exercises are found in Berger and Kimmel, Guide to
Molecular Cloning Techniques, Methods in Enzymology, volume 152,
Academic Press, Inc., San Diego, Calif. (Berger); Sambrook, et al.
(1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3,
Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.,
(Sambrook); and Current Protocols in Molecular Biology, F. M.
Ausubel, et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(1994 Supplement) (Ausubel). Product information from manufacturers
of biological reagents and experimental equipment also provide
information useful in known biological methods. Such manufacturers
include the SIGMA chemical company (Saint Louis, Mo.), R&D
systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology
(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto,
Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,
Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.
(Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka
Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and
Applied Biosystems (Foster City, Calif.), as well as many other
commercial sources known to one of skill.
[0294] The nucleic acid compositions of this invention, whether
RNA, cDNA, mRNA, genomic DNA, or a hybrid of the various
combinations, are isolated from biological sources or synthesized
in vitro. The nucleic acids of the present invention are present in
transformed or transfected whole cells, in transformed or
transfected cell lysates, or in a partially purified or
substantially pure form.
[0295] In vitro amplification techniques suitable for amplifying
sequences to provide a nucleic acid or for subsequent analysis,
sequencing or subcloning are known. Examples of techniques
sufficient to direct persons of skill through such in vitro
amplification methods, including the polymerase chain reaction
(PCR) the ligase chain reaction (LCR), Q.beta.-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are found in Berger, Sambrook, and Ausubel, as well as
Mullis, et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A
Guide to Methods and Applications (Innis, et al. eds) Academic
Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson
(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)
3:81-94; Kwoh, et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);
Guatelli, et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990);
Lomell, et al., J. Clin. Chem., 35:1826 (1989); Landegren, et al.,
Science, 241:1077-1080 (1988); Van Brunt, Biotechnology, 8:291-294
(1990); Wu and Wallace, Gene, 4:560 (1989); Barringer, et al.,
Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology,
13:563-564 (1995). Improved methods of cloning in vitro amplified
nucleic acids are described in Wallace, et al., U.S. Pat. No.
5,426,039. Improved methods of amplifying large nucleic acids (up
to 40 kb) are summarized in Cheng, et al., Nature, 369:684-685
(1994) and the references therein. One of skill will appreciate
that essentially any RNA can be converted into a double stranded
DNA suitable for restriction digestion, PCR expansion and
sequencing using reverse transcriptase and a polymerase. See,
Ausubel, Sambrook, Innis, and Berger, all supra.
[0296] One of skill will recognize many ways of generating
alterations in a given nucleic acid construct. Such well-known
methods include site-directed mutagenesis, PCR amplification using
degenerate oligonucleotides, exposure of cells containing the
nucleic acid to mutagenic agents or radiation, chemical synthesis
of a desired oligonucleotide (e.g., in conjunction with ligation
and/or cloning to generate large nucleic acids) and other
well-known techniques. See, Giliman and Smith, Gene 8:81-97 (1979),
Roberts, et al., Nature, 328:731-734 (1987) and Sambrook, Innis,
Ausubel, Berger, and Mullis (all supra).
[0297] Most modifications to nucleic acids are evaluated by routine
screening techniques in suitable assays for the desired
characteristic. For instance, changes in the immunological
character of encoded polypeptides can be detected by an appropriate
immunological assay. For instance, changes in the cellular
immunological character of the polypeptide can be detected by an
appropriate antibody or CTL assay. Modifications of other
properties such as nucleic acid hybridization to a complementary
nucleic acid, redox or thermal stability of encoded proteins,
hydrophobicity, susceptibility to proteolysis, or the tendency to
aggregate are all assayed according to standard techniques.
[0298] A wide variety of formats and labels are available and
appropriate for detection of polypeptide sequences. These include
analytic biochemical methods such as spectrophotometry,
radiography, electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, and the like, and various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunoelectrophoresis,
radioimmunoassays (RIAs), enzyme-linked immunosorbent assays
(ELISAs), western blot assays, immunofluorescent assays, and the
like. Several commercially available ELISA assays for the detection
of retroviral components, including Env domains, are available,
allowing one of skill to detect Env in biological samples.
[0299] Similarly, the detection of the nucleic acids of the present
invention proceeds by well known methods such as Southern analysis,
northern analysis, gel electrophoresis, PCR, radiolabeling and
scintillation counting, and affinity chromatography. Many assay
formats are appropriate, including those reviewed in Tijssen (1993)
Laboratory Techniques in biochemistry and molecular
biology--hybridization with nucleic acid probes parts I and II,
Elsevier, N.Y. and Choo (ed) (1994) Methods In Molecular Biology
Volume 33--In Situ Hybridization Protocols, Humana Press Inc., New
Jersey (see also, other books in the Methods in Molecular Biology
series); see especially, Chapter 21 of Choo (id.) "Detection of
Virus Nucleic Acids by Radioactive and Nonisotopic in Situ
Hybridization". Finally, PCR is also routinely used to detect
nucleic acids in biological samples (see, Innis, supra, for a
general description of PCR techniques).
[0300] In one preferred embodiment, antibodies are used to detect
polypeptide sequences. Methods of producing polyclonal and
monoclonal antibodies are known to those of skill in the art, and
many anti-HIV antibodies are available. See, e.g., Coligan (1991)
Current Protocols in Immunology, Wiley/Greene, N.Y.; and Harlow and
Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor
Press, NY; Stites, et al. (eds.) Basic and Clinical Immunology (4th
ed.), Lange Medical Publications, Los Altos, Calif., and references
cited therein; Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.), Academic Press, New York, N.Y.; and Kohler and
Milstein, Nature, 256:495-497 (1975). Other suitable techniques for
antibody preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse, et al., Science,
246:1275-1281 (1989); and Ward, et al., Nature, 341:544-546 (1989).
Specific monoclonal and polyclonal antibodies and antisera will
usually bind with a KD of at least about 0.1 mM, more usually at
least about 1 .mu.M, preferably at least about 0.1 .mu.M or better,
and most typically and preferably, 0.01 .mu.M or better.
Development of HIV Env Vectors
[0301] To develop Env glycoprotein variants that might effectively
induce humoral and cellular immunity, a series of plasmid
expression vectors were generated (FIG. 178). HIV Env is encoded by
nucleic acid sequences that contain RNA structures that limit gene
expression. These vectors were therefore synthesized using codons
found in human genes that allow these structures to be eliminated
without affecting the amino acid sequence. Full-length HIV Env
(gp160) was highly expressed in the absence of HIV accessory
proteins at levels .gtoreq.10-fold higher than Rev-dependent viral
gp 160 in transfected 293 cell by Western blot analysis (FIG.
179A), and the relevant mutant proteins were detected at the
expected apparent molecular weights (FIG. 179B-D). As might be
anticipated, gp160 expressed from the synthetic gene was not
efficiently processed in transfected 293 cells, presumably because
over-expression of gp160 saturates the cellular proteases
responsible for cleavage. (Binley J M et al., 2000, J. Virol,
74:627-643.) Synthetic HIV gp160 induced toxicity in transfected
cells, with cell rounding and detachment evident within 48 hours
(FIG. 180A vs B). This cytotoxicity was reduced by elimination of
the COOH-terminal cytoplasmic domain. Env protein that terminated
at amino acid 752 (gp150) was less cytotoxic than gp160, while the
shorter proteins (gp145 and gp140) produced little or no effect
(FIGS. 180C, D, and E, respectively).
[0302] To alter Env immunogenicity, two different approaches were
explored. First the effects of glycosylation on cellular and
humoral immunity were evaluated by analysis of mutants in which
conserved N-linked glycosylation sites were eliminated by
site-directed mutagenesis. Two sets of mutations were introduced
into both gp160 and gp150 (FIG. 178). The first set included eleven
potential sites (.DELTA.gly11), and the second set included an
additional 6 sites downstream (.DELTA.gly17). Expression studies
showed that the glycosylation mutants were efficiently expressed,
and the glycosylation mutant protein was appropriately reduced in
size compared to wild type gp160 or gp150, consistent with reduced
N-linked glycosylation (FIG. 179C).
[0303] The second approach involved a series of internal deletions
designed to stabilize and expose functional domains of the protein
that might be present in an extended helical structure prior to the
formation of the six-member coiled-coil structure in the hairpin
intermediate. To generate this putative pre-hairpin structure, the
cleavage site was removed to prevent the proteolytic processing of
the envelope and stabilize the protein by linking it covalently to
the gp41 extracellular and/or transmembrane domain. To reduce
toxicity and enhance stability, the fusion peptide domain was
deleted. The heptad repeats in the envelope protein are important
tertiary structure domains involved in the ability of the envelope
protein to form trimers. The sequence between the heptad repeats
was removed to stabilize the formation of trimers and eliminate
formation of the hairpin intermediate. These .DELTA.CFI deletions
were introduced into full-length gp160 and COOH-terminal truncation
mutants. Though cells transfected with vectors encoding
gp140.DELTA.CFI, gp145.DELTA.CFI and gp160 readily expressed these
proteins (FIG. 179D), only gp140.DELTA.CFI, which lacks the
transmembrane domain, was readily detected in the supernatant (FIG.
181), indicating that it can give rise to soluble antigen.
Immunogenicity of Env Mutants after DNA Vaccination
[0304] The ability of these Env proteins to elicit an immune
response was determined in mice by injection with these plasmid DNA
expression vectors. Antibody responses were monitored by the
ability of antisera from injected mice to immunoprecipitate wild
type gp160 from cell lysates by SDS-PAGE and Western blotting (FIG.
182). In some cases, antibody reactivity was also confirmed by
immunofluorescence. To quantitate the antibody response,
immunoprecipitation followed by Western blotting with different
dilutions of immunized mouse sera was tested. The intensity of the
gp160 band was determined by densitometry and standardized relative
to a positive control sera used to normalize data between
experiments. The approximately linear dose response of gp160
intensity with serum dilution allowed quantification of the
anti-gp160 antibody response in mouse sera. Data from immunized
mice showed that none of the wild type Env proteins, neither the
gp160, gp150, gp145, nor gp140 COOH-terminal truncations generated
consistently high antibody responses (FIG. 182A). Of these
proteins, gp140 was somewhat more effective than gp160 in
generating antibody responses but remained low and inconsistent.
Immunization with vectors designed to express glycosylation
deficient envelope proteins also did not improve the humoral
response. In contrast, the .DELTA.CFI mutants in the Env truncation
vectors substantially increased anti-gp160 antibody response (FIG.
182A; gp145, gp140, and gp128). The longer .DELTA.CFI Env proteins
(gp160 and gp150) did not show comparable enhancement of this
response. The gp140 (.DELTA.CFI) provided more consistent and a
greater increase in antibody response than gp128.DELTA.CFI (FIG.
182B). In all cases, these vectors that encoded gp140.DELTA.CFI,
but not wild type gp140, induced antibodies that were reactive to
native gp160 (FIG. 182C).
[0305] To determine whether these modifications of Env adversely
affected CTL responses, spleen cells from immunized mice were
tested for their ability to lyse relevant target cells. All mice
immunized with codon-altered Env vectors, including COOH-terminal
deletion mutations (FIG. 183A) or glycosylation mutants (FIG.
183C), elicited strong CTL responses directed to cell lines pulsed
with HIV Env peptides. These findings were also confirmed using
stably transfected cells that expressed Env. Importantly, gp140
(.DELTA.CFI), which elicited increased antibody responses relative
to the comparable wild typ Env, readily induced CTL responses to
native Env, as did other .DELTA.CFI mutants (FIG. 183D). Addition
of anti-CD8 antibody inhibited cytolytic activity, as did depletion
using magnetic beads coupled with anti-CD8 antibody, thus
confirming a cytotoxic T cell response to these immunogens after
genetic immunization (FIG. 183B). This response was detectable for
at least six months after immunization.
Glycosylation and .DELTA.CFI HIV Env Mutants
[0306] To develop DNA vaccine candidates for HIV, we developed a
series of synthetic genes designed to express HIV Env mutants in
human cells. In the absence of HIV regulatory proteins, these
codon-altered envelope protein genes expressed well in human cells.
Like other DNA vaccines, immunization with these vectors elicited
strong CTL responses in mice, and antibody responses were not
robust in mice immunized with wild type Env expression vectors.
Mutations in highly conserved N-linked glycosylation sites did not
significantly alter humoral or cellular immune response to native
Env. In contrast, a mutant Env with deletions in the cleavage site,
fusion domain, and a region between the heptad repeats elicited a
more potent humoral immune response and retained its ability to
stimulate Env-specific CTL.
[0307] Recent reports suggest that gp160 forms trimers in vivo and
the domain required for trimer formation resides in the ectodomain
of the gp41. Such trimeric forms of HIV envelope protein are likely
to present different epitopes to the immune system compared to
monomeric gp120. In addition to the linear epitopes in the
envelope, this trimeric structure is likely to expose
conformational epitopes important for B cell triggering of a
relevant antibody response. In this regard, gp140 (.DELTA.CFI),
which induced the greatest antibody response, is released in a
soluble form (FIG. 181). In contrast, wild type Env did not elicit
high titer antibody responses. The toxicity of Env in mammalian
cells has been seen and could limit both the amount and duration of
envelope protein expression in vivo that would affect
immunogenicity. The envelope is also heavily glycosylated, and
removal of partial or complete gp120 glycosylation sites has
resulted in higher titers of strain-specific neutralizing antibody
responses to mutant SIVs in monkeys. Though it seemed reasonable
that deglycosylation would reveal epitopes otherwise masked in the
native protein, we did not observe enhanced immune reactivity by
DNA vaccination using different glycosylation site mutants, both in
gp160 and gp150. This difference with the previous study is likely
due to the fact that DNA vaccination rather than viral infection
was utilized for immunization. Though glycosylation mutants are
unlikely to prove helpful with this former method of immunization,
we envision that modification of glycosylation sites will be
effective with other vectors or adjuvants.
[0308] HIV-1 Env is proteolytically cleaved by a cellular
convertase into gp120 and gp41. The gp41 subunit is composed of
cytoplasmic, transmembrane, and ectodomain segments. The role of
the ectodomain of the envelope in membrane fusion, particularly its
hydrophobic glycine-rich fusion peptide, is well established. Two
regions with heptad coiled-coil repeats in the ectodomain of gp41
are involved in viral fusion. Upon fusion, these two alpha helices,
connected via a disulfide-stabilized loop, presumably undergo a
transient conformational change to a fusion active state. These
changes allow the formation of a six-member helical hairpin
intermediate structure that presumably exposes the fusion peptide
at the NH.sub.2-terminus of gp41, allowing fusion to the target
cell membrane. The .DELTA.CFI mutation was intended to eliminate
cleavage of gp140, remove the unstable hydrophobic region and
stabilize oligomer formation. Though detailed structural data is
not yet available on this protein, these mutations apparently
stabilize Env in a conformation that elicits both humoral and
cellular immune responses. For example, the neutralizing epitope in
the ectodomain of gp41 is present in the series of deletions and
truncations of the envelope and gp140 (.DELTA.CFI) is reactive with
the 2F5 neutralizing monoclonal antibody that binds to this
epitope. Importantly, these immunogens also induced CTL responses
to Env. Though gp128 (.DELTA.CFI) induced slightly more potent CTL
activity, gp140 (.DELTA.CFI) was better able to elicit such
responses, both to peptide-pulsed cells and stably transduced
target cells. Thus the enhanced humoral immune response introduced
by this vaccine candidate did not appear to diminish the CTL
response. Taken together, these results indicate that
gp140.DELTA.CFI serves as an improved immunogen that can more
effectively elicit an antibody response against the envelope by DNA
vaccination while preserving its ability to induce a CTL
response.
Gag and Pol
[0309] In this disclosure, we have prepared synthetic HIV-1 B clade
Gag and Pol expression vectors that are based on human (h) codon
usage. These vectors encode hGag-Pol and its derivatives, hGag,
hPol and an hGag-Pol fusion protein. The synthetic Gag-Pol genes
show little nucleotide homology to HIV-1 but are the same in
protein sequence. The modified Gag-Pol genes were subcloned into a
eukaryotic plasmid expression vector for expression and DNA
immunization studies. Synthetic Gag-Pol genes allowed high level
Rev-independent expression of HIV-1 Gag-Pol precursor proteins in
human and mouse cell lines and induced significant cellular and
humoral responses in mice. The Gag-Pol fusion protein induced the
broadest responses to Gag and Pol determinants and thus is
envisioned as a prototype immunogen that maximizes epitope
presentation.
Eliminating the Frame Shift Site to Create Viral Polyproteins
[0310] Described herein are HIV Gag-Pol fusion proteins encoded by
a continuous open reading frame so to improve the immune response
to native Gag and Pol. In some viruses, translational frame
shifting is exploited during protein synthesis. Specific sequences
in the RNA are required for the frame shifting. The viral RNA
sequences cause ribosomal slippage so that viral proteins are
produced in non-equivalent ratios. For example, during translation
in HIV, the ribosomes shift reading frames to synthesize Gag
precursor protein and the gag-pol fusion protein in a 20:1 ratio.
The strategy here is to maximize epitope presentation by
transcribing an immunogen from a continuous open reading frame by
eliminating the frame shift site. Thus, the strategy for improving
the immune response by the use of a HIV Gag-Pol fusion protein
encoded by a continuous open reading frame extends to other viral
proteins that are produced in non-equivalent ratios by virtue of
translational frame shifting.
[0311] The present invention involves HIV Gag-Pol fusion proteins
encoded by a single continuous open reading frame due to mutation
of the frame shift site. The frame shift site is mutated by
deletions, insertions, and/or substitutions of nucleotides to
create a single continuous open reading frame. In one embodiment,
deletions are meant (i.e., nucleotides are deleted to create the
same open reading frame).
[0312] The frame shift site is a mutated frame shift to create a
single continuous open reading frame. For example, a set of similar
retroviral gag-pol frame shift sites are optionally made for a
given fusion protein, for example, by synthesizing different
gag-pol frame shift regions and cloning the sequences
appropriately, or by site-directed mutagenesis of a given frame
shift clone. The efficacy of the frame shift sites are assessed by
measuring the production of the fusion protein. The sequence that
shows the highest level of expression is a "optimized" frame shift
mutation for the set assessed. Alternatively, where a particular
level of expression is desired, a frame shift site from a
particular set of possible frame shift sites which is closest to
the desired activity level is considered to be "optimized."
[0313] Although a full length Gag sequence is preferred for use in
the fusion protein of the present invention, Gag is optionally
deleted of subsequences without negating a polyepitope response.
For example, regions of the matrix protein (p17), regions of the
capsid protein (p24), regions of p2, regions of the nucleocapsid
protein (p7), regions of p1, and regions of p6 can be deleted while
preserving the polyepitope response. Alternatively, regions of the
matrix protein (p17), regions of the capsid protein (p24), regions
of p2, regions of the nucleocapsid protein (p7), regions of p1, and
regions of p6 can be substituted while preserving the polyepitope
response. Alternatively, regions of the matrix protein (p17),
regions of the capsid protein (p24), regions of p2, regions of the
nucleocapsid protein (p7), regions of p1, and regions of p6 can be
interrupted by insertions while preserving the polyepitope
response. Optionally, regions of the matrix protein (p17), regions
of the capsid protein (p24), regions of p2, regions of the
nucleocapsid protein (p7), regions of p1, and regions of p6 can be
mutated to inactivate these proteins.
[0314] Likewise, a full length Pol sequence is preferred for use in
the fusion protein of the present invention, and Pol is optionally
deleted of subsequences without negating a polyepitope response.
For example, regions of the protease protein, regions of the
reverse transcriptase protein, and regions of the integrase protein
can be deleted while preserving the polyepitope response.
Alternatively, regions of the protease protein, regions of the
reverse transcriptase protein, and regions of the integrase protein
can be substituted while preserving the polyepitope response.
Alternatively, regions of the protease protein, regions of the
reverse transcriptase protein, and regions of the integrase protein
can be interrupted by insertions while preserving the polyepitope
response. Optionally, regions of the protease protein, regions of
the reverse transcriptase protein, and regions of the integrase
protein can be mutated to inactivate these enzymes.
[0315] In other embodiments, the Gag-Pol fusion proteins are
co-expressed with other proteins, either as fusion proteins or
separately, preferably with Env sequence proteins, such as the
modified HIV Env .DELTA.CFI proteins described herein.
[0316] In another aspect, the invention involves chimeric nucleic
acid molecules. The chimeric nucleic acid molecules of the
invention typically have a retroviral gag nucleic acid sequence and
a retroviral pol nucleic acid sequence in the same open reading
frame due to mutation of the frame shift site. The continuous open
reading frame encodes a fusion protein such as those described
above.
[0317] The chimeric nucleic acids of the present invention are
optionally DNA, RNA, or mRNA. Most typically, the chimeric nucleic
acids are provided by recombinantly making a DNA, which is
expressed in a cell as RNA and/or as mRNA. Given the strategy for
making the chimeric nucleic acids of the present invention, one of
skill can construct a variety of clones containing functionally
equivalent nucleic acids. Cloning methodologies to accomplish these
ends, and sequencing methods to verify the sequence of nucleic
acids are well known in the art. Examples of appropriate cloning
and sequencing techniques, and instructions sufficient to direct
persons of skill through many cloning exercises are found in Berger
and Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology, volume 152, Academic Press, Inc., San Diego, Calif.
(Berger); Sambrook, et al. (1989) Molecular Cloning--A Laboratory
Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor Press, N.Y., (Sambrook); and Current Protocols in
Molecular Biology, F. M. Ausubel, et al., eds., Current Protocols,
a joint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Product
information from manufacturers of biological reagents and
experimental equipment also provide information useful in known
biological methods. Such manufacturers include the SIGMA chemical
company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),
Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH
Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich
Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL
Life Technologies, Inc. (Gaithersberg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City,
Calif.), as well as many other commercial sources known to one of
skill.
[0318] The chimeric nucleic acid compositions of this invention,
whether RNA, cDNA, mRNA, genomic DNA, or a hybrid of the various
combinations, are isolated from biological sources or synthesized
in vitro. The chimeric nucleic acids of the present invention are
present in transformed or transfected whole cells, in transformed
or transfected cell lysates, or in a partially purified or
substantially pure form.
[0319] In vitro amplification techniques suitable for amplifying
sequences to provide a nucleic acid or for subsequent analysis,
sequencing or subcloning are known. Examples of techniques
sufficient to direct persons of skill through such in vitro
amplification methods, including the polymerase chain reaction
(PCR) the ligase chain reaction (LCR), Q.beta.-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are found in Berger, Sambrook, and Ausubel, as well as
Mullis, et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A
Guide to Methods and Applications (Innis, et al. eds) Academic
Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson
(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)
3:81-94; Kwoh, et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);
Guatelli, et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990);
Lomell, et al., J. Clin. Chem., 35:1826 (1989); Landegren, et al.,
Science, 241:1077-1080 (1988); Van Brunt, Biotechnology, 8:291-294
(1990); Wu and Wallace, Gene, 4:560 (1989); Barringer, et al.,
Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology,
13:563-564 (1995). Improved methods of cloning in vitro amplified
nucleic acids are described in Wallace, et al., U.S. Pat. No.
5,426,039. Improved methods of amplifying large nucleic acids (up
to 40 kb) are summarized in Cheng, et al., Nature, 369:684-685
(1994) and the references therein. One of skill will appreciate
that essentially any RNA can be converted into a double stranded
DNA suitable for restriction digestion, PCR expansion and
sequencing using reverse transcriptase and a polymerase. See,
Ausubel, Sambrook, Innis, and Berger, all supra.
[0320] One of skill will recognize many ways of generating
alterations in a given nucleic acid construct. Such well-known
methods include site-directed mutagenesis, PCR amplification using
degenerate oligonucleotides, exposure of cells containing the
nucleic acid to mutagenic agents or radiation, chemical synthesis
of a desired oligonucleotide (e.g., in conjunction with ligation
and/or cloning to generate large nucleic acids) and other
well-known techniques. See, Giliman and Smith, Gene 8:81-97 (1979),
Roberts, et al., Nature, 328:731-734 (1987) and Sambrook, Innis,
Ausubel, Berger, and Mullis (all supra).
[0321] Most modifications to nucleic acids are evaluated by routine
screening techniques in suitable assays for the desired
characteristic. For instance, changes in the immunological
character of encoded polypeptides can be detected by an appropriate
immunological assay. For instance, changes in the cellular
immunological character of the polypeptide can be detected by an
appropriate antibody or CTL assay. Modifications of other
properties such as nucleic acid hybridization to a complementary
nucleic acid, redox or thermal stability of encoded proteins,
hydrophobicity, susceptibility to proteolysis, or the tendency to
aggregate are all assayed according to standard techniques.
[0322] A wide variety of formats and labels are available and
appropriate for detection of fusion protein sequences. These
include analytic biochemical methods such as spectrophotometry,
radiography, electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, and the like, and various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunoelectrophoresis,
radioimmunoassays (RIAs), enzyme-linked immunosorbent assays
(ELISAs), western blot assays, immunofluorescent assays, and the
like. Several commercially available ELISA assays for the detection
of retroviral components, including Env domains, are available,
allowing one of skill to detect Env in biological samples.
[0323] Similarly, the detection of the chimeric nucleic acids of
the present invention proceeds by well known methods such as
Southern analysis, northern analysis, gel electrophoresis, PCR,
radiolabeling and scintillation counting, and affinity
chromatography. Many assay formats are appropriate, including those
reviewed in Tijssen (1993) Laboratory Techniques in biochemistry
and molecular biology--hybridization with nucleic acid probes parts
I and II, Elsevier, N.Y. and Choo (ed) (1994) Methods In Molecular
Biology Volume 33--In Situ Hybridization Protocols, Humana Press
Inc., New Jersey (see also, other books in the Methods in Molecular
Biology series); see especially, Chapter 21 of Choo (id.)
"Detection of Virus Nucleic Acids by Radioactive and Nonisotopic in
Situ Hybridization". Finally, PCR is also routinely used to detect
nucleic acids in biological samples (see, Innis, supra, for a
general description of PCR techniques).
[0324] In one preferred embodiment, antibodies are used to detect
polypeptide sequences. Methods of producing polyclonal and
monoclonal antibodies are known to those of skill in the art, and
many anti-HIV antibodies are available. See, e.g., Coligan (1991)
Current Protocols in Immunology, Wiley/Greene, N.Y.; and Harlow and
Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor
Press, NY; Stites, et al. (eds.) Basic and Clinical Immunology (4th
ed.), Lange Medical Publications, Los Altos, Calif., and references
cited therein; Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.), Academic Press, New York, N.Y.; and Kohler and
Milstein, Nature, 256:495-497 (1975). Other suitable techniques for
antibody preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse, et al., Science,
246:1275-1281 (1989); and Ward, et al., Nature, 341:544-546 (1989).
Specific monoclonal and polyclonal antibodies and antisera will
usually bind with a KD of at least about 0.1 mM, more usually at
least about 1 .mu.M, preferably at least about 0.1 .mu.M or better,
and most typically and preferably, 0.01 .mu.M or better.
Expression of Synthetic HIV-1 Gag and Pol Genes
[0325] Four synthetic HIV-1 Gag-and/or Pol expression vectors,
hGag-Pol, hGag-Pol.DELTA.Fs.DELTA.Pr, hPol and hGag genes were
prepared (FIG. 184). To confirm expression, the synthetic or viral
Gag-Pol genes were transiently transfected into 293T cells, a human
kidney-derived cell line. When cell lysates were analyzed by
immunoblotting with human anti-HIV-1 IgG (FIG. 185A), monoclonal
anti-p24 (FIG. 185B), and rabbit anti-RT (FIG. 185C), Gag p55, Pol
p110 and Gag-Pol p160 precursor proteins were detected in hGag,
hPol, and hGag-Pol fusion plasmids transfected 293T cells, as was
expected. Mature virion proteins, p24 and RTp66, were detected in
the hGag-Pol gene transfected cells (FIGS. 185A, B and C). This
might be a result of the activation of protease inside cells which
was itself a result of the high-level expression of Gag and Gag-Pol
protein. The expression of Gag precursor proteins from
codon-altered vectors was .gtoreq.10-fold higher than viral Gag-Pol
(FIG. 185), determined by quantitative phosphorimaging. The level
of accumulated Gag-Pol fusion protein was 100-fold higher in cells
transfected with hGag-Pol compared to viral Gag-Pol. Virus-like
particles were released from the hGag gene transfected cells (FIG.
186), detected by transmission electron microscopy. Though such
particles were observed at a lower frequency with hGag-Pol, no
particles were seen in cells transfected with
hGag-Pol.DELTA.Fs.DELTA.Pr or hPol vectors. Stable expression of
HIV-1 Gag and Pol proteins from codon-optimized genes in mouse CT26
and BH10ME cells was also observed (FIG. 185D).
Induction of HIV-1 Gag and Pol CTL Responses in Mice by DNA
Vaccination
[0326] To evaluate the cellular immune response to HIV-1 Gag and
Pol proteins, Balb/C female mice were injected intramuscularly with
the eukaryotic expression vector plasmids containing the
codon-optimized genes. Two weeks after the final vaccination,
splenocytes were harvested from the immunized mice and sensitized
with either Gag or Pol peptide-pulsed naive mouse splenocytes. One
week later, CTL responses were analyzed using a 5-hour chromium
release assay.
[0327] CTL responses specific to HIV-1 Gag and/or Pol were first
analyzed using Gag or Pol peptide-pulsed BC10ME cells, or mouse
fibrosarcoma cell lines derived from B/C-N cells. Immunization with
hGag, hGag-Pol.DELTA.FS.DELTA.Pr or hGag-Pol genes induced
comparably strong CTL responses specific to Gag (FIG. 187A);
however, after immunization with hPol, hGag-Pol .DELTA.FS.DELTA.Pr
or hGag-Pol genes, only the fusion protein,
hGag-Pol.DELTA.FS.DELTA.Pr, and hPol to a lesser extent, elicited a
marked CTL response to Pol (FIG. 187B). To confirm that the
specific killing in the CTL assays was induced by CD8.sup.+
cytotoxic T lymphocytes, CD4.sup.+ or CD8.sup.+ cells were depleted
from sensitized splenocytes by Dynal beads (Dynal, Inc., Lake
Success, N.Y.). Depletion of CD8.sup.+ cells abolished the specific
lysis in the hGag-Pol .DELTA.FS.DELTA.Pr gene-immunized mice, while
depletion of CD4.sup.+ had little effect on lysis (FIG. 187C),
suggesting that CD8.sup.+ lymphocytes were responsible for specific
cytotoxicity.
[0328] The responses were further analyzed and confirmed with the
hGag or hPol gene transduced syngeneic CT26 and BC10ME cell lines.
Responses to Gag in the mice immunized with the hGag,
hGag-Pol.DELTA.FS.DELTA.Pr or hGag-Pol genes were similar when
peptide-pulsed cells were used as targets in the CTL assay (FIG.
188A). Mice immunized with the hPol gene generated a specific
response to HIV-1 Pol on BC10ME cell lines stably expressing Pol as
target cells (FIG. 188B). The same results have been observed with
CT26 cell lines. These stably transfected cell lines were therefore
more sensitive as target cells than peptide-pulsed cells in the Pol
CTL assays.
Antibody Response in the Immunized Mice
[0329] Sera from mice immunized with different plasmids was
analyzed with a p24 ELISA. hGag immunized mice demonstrated the
highest p24 antibody titers (FIG. 189A). Unexpectedly, hGag-Pol
virus-like particles elicited the lowest levels of p24 antibody.
Similar results were observed by Western blotting with pooled sera
(FIG. 189B). The HIV-1 Pol specific antibodies were not detected by
a commercially available Western blotting kit (FIG. 189B), but
antibodies to Pol were detected in mice immunized with hPol and
hGag-Pol.DELTA.FS.DELTA.Pr with a more sensitive method, IP/Western
blotting (FIG. 189C). Presumably, this assay is more sensitive and
better able to detect native conformational epitopes. Though such
antibodies were found in mice immunized with Pol and Gag-Pol fusion
proteins in this assay, minimal response was detected in the mice
immunized with hGag-Pol. Though both immunogens elicited similar
Gag responses, the Gag-Pol fusion protein was therefore more
effective in the stimulation of CTL and antibody responses to
Pol.
HIV Gag-Pol Fusion Proteins
[0330] In this disclosure, HIV-1 B-clade Gag and Pol genes were
modified to increase Rev-independent expression of HIV-1 Gag-Pol
proteins. This modification allowed synthesis of HIV Gag and Pol,
as well as fusion proteins at levels 10- to 100-fold higher than
the corresponding Rev-dependent viral gene in the absence of Rev
and RRE elements. These viral proteins were recognized by standard
polyclonal and monoclonal antibodies (FIG. 185), and immature VLP
were produced and released from 293T cells transfected with hGag
(FIG. 186).
[0331] The immune response induced by Rev-independent Gag-Pol was
directed against both Gag and Pol determinants. The hGag, hGag-Pol
fusion and hGag-Pol all induced strong CTL responses specific for
Gag in mice immunized with plasmid DNA, but a significant Pol
response was elicited only in the mice immunized with the
hGag-Pol.DELTA.FS.DELTA.Pr or Pol alone. Because immunization with
hGag-Pol gene failed to induce detectable cellular or humoral
responses to HIV-1 Pol protein, these findings indicate that the
Gag-Pol fusion protein induces a broader range of responses and
allows delivery of an immunogen with a larger number of epitopes in
a single continuous open reading frame. During viral replication,
viral gag-pol produces Gag precursor protein and the gag-pol fusion
protein by frame shifting in a 20:1 ratio (Wilson, W et al., 1988,
Cell, 55:1159-1169). The deletion of a frame shift site in
hGag-Pol.DELTA.FS.DELTA.Pr results in production of only the
Gag-Pol fusion protein. Expression of Gag-Pol proteins alone in
human cells is not adequate to form releasable viral particles
because HIV-1 viral assembly requires Gag precursor proteins (Park,
J and C D Morrow, 1992, J Virol, 66:6304-6313; Smith, A J et al.,
1993, J Virol, 67:2266-2275). The ability of
hGag-Pol.DELTA.FS.DELTA.Pr to elicit strong Gag and Pol specific
CTL responses in mice may be explained by high level expression of
Gag-Pol fusion protein and its retention on the inside of cells,
which could create a new condition not existing during normal viral
replication and could provide sufficient proteins for antigen
presentation. Moreover, the mutation in viral protease prevents
viral protein from intracellular activation and reduced cellular
toxicity. Overexpression of this polyprotein is also likely to
affect its intracellular localization/transport and is envisioned
as improving antigen presentation.
[0332] The Pol gene of HIV-1 is the precursor protein for viral
protease, reverse transcriptase and integrase, which are crucial to
viral replication (Kohl, N E et al., 1988, Proc. Natl. Acad. Sci.
USA, 85:4686-4690.). Retroviral extracellular maturation, resulting
from self-activation of protease after release produces mature RT
and IN which have important functions in reverse transcription and
integration respectively, for HIV-1, HIV-2, and SIV replication.
The catalytic cores of these enzymes have relatively conserved
domains in order to preserve their functions and thus are
envisioned as inducing cross-clade CTL responses. As early as 1988,
CTLs specific for HIV-1 RT were found in blood samples from HIV-1
infected individuals (Hosmalin, A et al., 1990, Proc. Natl. Acad.
Sci. USA, 87:2344-2348; Walker, B D et al., 1988, Science,
240:64-66.). Relatively strong Gag-specific CTL responses have been
shown in numerous non-human primate and human studies, using DNA
vaccines or a live recombinant vector containing viral Gag-Pol
constructs (Evans, T G, 1999, J Infect Dis, 180:290-298; Ferrari,
G, 1997, Blood, 90:2406-2416; Gorse, G J et al., 1999, Vaccine,
18:835-849; Seth, A, 1998, Proc. Natl. Acad. Sci. USA,
95:10112-10116; Seth, A et al., 2000, J Virol, 74:2502-2509), but
fewer Pol-specific CTL responses have been reported. The detection
of significant CTL responses specific to Pol in our disclosure may
be attributed in part to establishment of stable Pol expressing
cell lines, in which codon alteration and inactivation of FS and PR
in the Pol gene allow high level expression of the Pol protein
without cellular toxicity. Though it remains possible that the
hGag-Pol, or a combination of hGag and hPol, may exert similar
effects with appropriate adjuvants or with different prime-boost
regimens, the Rev-independent Gag-Pol fusion protein stimulates
HIV-1 Gag and Pol specific CTL responses in mice and is envisioned
as proving useful in an AIDS vaccine.
Plasmid Descriptions
Env Plasmids
VRC2100
[0333] pVR1012x/s R5gp139-Nef (Delta) MHC (Delta) CD4/h
[0334] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
The envelope protein gene from pX4gp160/h (Nabel lab #1272) was
ligated in frame with the mutant Nef gene from pNefDMHCDCD4/h
(Nabel lab #1278) to produce pX4gp139-NefDMHCDCD4/h. The
envelope-Nef fusion protein expressed from pX4gp139-NefDMHCDCD4/h
contains the first 668 amino acids from the HIV envelope
glycoprotein (gp139) fused to the entire mutant Nef protein. The
truncated envelope polyprotein (gp139) contains the entire SU
protein and a portion of the TM protein including the fusion
domain, but lacking the transmembrane domain and regions important
for oligomer formation. The protein sequence of the Nef protein
from HIV-1 PV22 (GenBank accession number K02083) was used to
create a synthetic version of the Nef gene (Nef/h) using codons
optimized for expression in human cells. To disrupt the ability of
Nef to limit both MHC class I and CD4 expression, point mutations
were introduced into the Nef gene from pNef/h (Nabel lab #1275).
The resulting amino acids substitutions in pNefDMHCDCD4/h are:
P69A, P72A, P75A, P78A, D174A and D175A. X4gp139-NefDMHCDCD4/h is
expressed from the pVR1012x/s (Nabel lab # 1267) vector
backbone.
VRC2200
[0335] pVR1012x/s R5gp157-Nef (Delta) MHC (Delta) CD4/h
[0336] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (Nabel lab number 1272) were replaced with the
corresponding region from the BaL strain of HIV-1 (GeneBank
accession number M68893, again using human preferred codons). The
envelope-Nef fusion protein expressed from pR5gp157-Nef/h contains
the first 820 amino acids from the HIV envelope glycoprotein
(gp157) fused to the entire mutant Nef protein. The gene for gp157
was ligated in frame with the full-length mutant Nef gene from
pNefDMHCDCD4/h (Nabel lab #1278) to produce pR5gp157-NefDMHCDCD4/h.
The protein sequence of the Nef protein from HIV-1 PV22 (GenBank
accession number K02083) was used to create a synthetic version of
the Nef gene (Nef/h) using codons optimized for expression in human
cells. To disrupt the ability of Nef to limit both MHC class I and
CD4 expression, point mutations were introduced into the Nef gene
from pNef/h (Nabel lab #1275). The resulting amino acids
substitutions in pNefDMHCDCD4/h are: P69A, P72A, P75A, P78A, D174A
and D175A. R5gp157-NefDMHCDCD4/h is expressed from the pVR1102x/s
(Nabel lab #1267) vector backbone.
VRC2300
[0337] pVR1012x/s X4gp139-Nef deltaMHC deltaCD4/h
[0338] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
The envelope protein gene from pX4gp160/h (Nabel lab #1272) was
ligated in frame with the mutant Nef gene from pNefDMHC/h (Nabel
lab #1276) to produce pX4gp139-NefDMHC/h. The envelope-Nef fusion
protein expressed from pX4gp139-NefDMHC/h contains the first 668
amino acids from the HIV envelope glycoprotein (gp139) fused to the
entire mutant Nef protein. The truncated envelope polyprotein
(gp139) contains the entire SU protein and a portion of the TM
protein including the fusion domain, but lacking the transmembrane
domain and regions important for oligomer formation. The protein
sequence of the Nef protein from HIV-1 PV22 (GenBank accession
number K02083) was used to create a synthetic version of the Nef
gene (Nef/h) using codons optimized for expression in human cells.
To disrupt the ability of Nef to limit MHC class I expression,
point mutations were introduced into the Nef gene from pNef/h
(Nabel lab #1275). The resulting amino acids substitutions in
pNefDMHC/h are: P69A, P72A, P75A, and P78A. X4gp139-NefDMHC/h is
expressed from the pVR1012x/s (Nabel lab #1267) vector backbone
VRC2302
[0339] pVR1012x/s X4gp130-Nef/h
[0340] For the X4gp130/h (VRC2703) portion, the protein sequence of
the envelope polyprotein (gp160) from HXB2 (X4-tropic, GenBank
accession number K03455) was used to create a synthetic version of
the gene (X4gp160/h) using codons optimized for expression in human
cells. The nucleotide sequence X4gp160/h shows little homology to
the HXB2 gene, but the protein encoded is the same with the
following amino acid substitutions: F53L, N94D, K192S, I215N,
A224T, A346D, and P470L. The full-length X4-tropic version of the
envelope protein from pX4gp160/h (VRC3300) was terminated after the
codon for amino acid 602. The truncated envelope polyprotein
contains the entire SU protein and a portion of the TM protein
including the fusion domain, but lacking the transmembrane domain.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. Regions important for oligomer formation may be
partially functional. This X4 gp130/h (VRC2703) gene was fused to
Nef gene. The Nef protein from HIV-1 PV22 (GenBank accession number
K02083) was used to create the viral Nef gene (pVR1012-Nef)(Nabel
Lab #1093. The nucleotide sequence is homologous to the viral gene,
and the protein encoded is the same. The expression vector backbone
is pVR1012x/s (VRC2000).
VRC2400
[0341] pVR1012x/s X4gp157-NefDMHCDCD4/h
[0342] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
The envelope-Nef fusion protein expressed from
pX4gp157-NefDMHCDCD4/h contains the first 820 amino acids from the
HIV envelope glycoprotein (gp157) fused to the entire mutant Nef
protein. The truncated envelope polyprotein (gp157) contains the
entire SU protein and a portion of the TM protein including the
fusion domain, the transmembrane domain and regions important for
oligomer formation. Heptad(H) 1, Heptad 2 and their Interspace(IS)
are required for oligomerization. The gene for gp157 was ligated in
frame with the full-length mutant Nef gene from pNefDMHCDCD4/h
(VRC3600) to produce pX4gp157-NefDMHCDCD4/h. The protein sequence
of the Nef protein from HIV-1 PV22 (GenBank accession number
K02083) was used to create a synthetic version of the Nef gene
(Nef/h) using codons optimized for expression in human cells. To
disrupt the ability of Nef to limit both MHC class I and CD4
expression, point mutations were introduced into the Nef gene from
pNef/h (VRC3500). The resulting amino acids substitutions in
pNefDMHCDCD4/h are: P69A, P72A, P75A, P78A, D174A and D175A.
X4gp160-NefDMHCDCD4/h is expressed from the pVR1012x/s (VRC2000)
vector backbone.
VRC2700
[0343] pVR1012x/s X4gp140/h
[0344] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
The full-length X4-tropic version of the envelope protein from
pX4gp160/h (VRC3300) was terminated after the codon for amino acid
680. The truncated envelope polyprotein contains the entire SU
protein and a portion of the TM protein including the fusion
domain, but lacking the transmembrane domain. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC2701
[0345] pVR1012x/s X4gp140(del F/CL del H IS)/h
[0346] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. The full-length
X4-tropic version of the envelope protein from pX4gp160/h (VRC3300)
was terminated after the codon for amino acid 680. The truncated
envelope polyprotein contains the entire SU protein and a portion
of the TM protein including the fusion domain, but lacking the
transmembrane domain. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC2702
[0347] pVR1012x/s X4gp128(del F/CL)/h
[0348] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. The full-length
X4-tropic version of the envelope protein from pX4gp160/h (VRC3300)
was terminated after the codon for amino acid 592. The truncated
envelope polyprotein contains the entire SU protein and a portion
of the TM protein including the fusion domain, but lacking the
transmembrane domain. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC2706
[0349] pVR1012x/s X4gp 145/h
[0350] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. The full-length
X4-tropic version of the envelope protein from pX4gp160/h (VRC3300)
was terminated after the codon for amino acid 704. The truncated
envelope polyprotein contains the entire SU protein and a portion
of the TM protein including the fusion domain, but lacking the
transmembrane domain. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC2707
[0351] pVR1012x/s X4gp145(del F/CL del H IS)/h
[0352] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. The full-length
X4-tropic version of the envelope protein from pX4gp160/h (VRC3300)
was terminated after the codon for amino acid 704. The truncated
envelope polyprotein contains the entire SU protein and a portion
of the TM protein including the fusion domain, but lacking the
transmembrane domain. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC2800
[0353] pVR1012x/s R5gp 140/h
[0354] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the transmembrane domain. Regions important for oligomer formation
may be partially functional. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC2801
[0355] pVR1012x/s R5gp140(del F/CL del H IS)/h
[0356] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC2804
[0357] pVR1012x/s R5gp145/h
[0358] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC2805
[0359] pVR1012x/s R5gp145(del F/CL del H IS)/h
[0360] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The Fusion and Cleavage (F/CL) domains, from amino
acids 503-536, have been deleted. The Interspace (IS) between
Heptad (H) 1 and 2, from amino acids 593-620, have been deleted.
The expression vector backbone is pVR1012x/s (VRC2000).
VRC2810
[0361] pVR1012 x/s R5gp140delC1 (delCFI)/h
[0362] Constant domain 1 was deleted from gp140delCFI from amino
acid 33-127 and was replaced with a NheI site.
VRC2811
[0363] pVR1012x/s R5gp140delC2 (delCFI)/h
[0364] Constant domain 2 was deleted from gp140delCFI from amino
acid 199-293, and was replaced with an NheI site
VRC2812
[0365] pVR1012x/s R5gp140delC3 (delCFI)/h
[0366] Constant domain 3 was deleted from gp140delCFI from amino
acid 333-380, and was replaced with an NheI site.
VRC2813
[0367] pVR1012x/s R5gp140delC4 (delCFI)/h
[0368] Constant domain 4 was deleted from gp140delCFI from amino
acid 419-458, and was replaced with an NheI site.
VRC2814
[0369] pVR1012x/s R5gp140delC5 (delCFI)/h
[0370] Constant domain 5 was deleted from gp140delCFI from amino
acid 472-498 and was replaced with an NheI site.
VRC2820
[0371] pVR1012x/s R5gp 140(dCFI)/dV1
[0372] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1 loop (a.a.129 to 154) and transmembrane domain. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The Fusion and Cleavage (F/CL) domains, from amino acids 503-536,
have been deleted. The Interspace (IS) between Heptad (H) 1 and 2,
from amino acids 593-620, have been deleted. Regions important for
oligomer formation may be partially functional. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC2821
[0373] pVR1012x/s R5gp 140(dCFI)/dV2
[0374] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2 loop (a.a.160 to 193) and transmembrane domain. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The Fusion and Cleavage (F/CL) domains, from amino acids 503-536,
have been deleted. The Interspace (IS) between Heptad (H) 1 and 2,
from amino acids 593-620, have been deleted. Regions important for
oligomer formation may be partially functional. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC2822
[0375] pVR1012x/s R5gp140(dCFI)/dV3
[0376] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V3 loop (a.a.299 to 327) and transmembrane domain. Heptad (H)
1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The Fusion and Cleavage (F/CL) domains, from amino
acids 503-536, have been deleted. The Interspace (IS) between
Heptad (H) 1 and 2, from amino acids 593-620, have been deleted.
Regions important for oligomer formation may be partially
functional. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 2823
[0377] pVR1012x/s R5gp 140(dCFI)/dV4
[0378] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V4 loop (a.a.386 to 413) and transmembrane domain. Heptad (H)
1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The Fusion and Cleavage (F/CL) domains, from amino
acids 503-536, have been deleted. The Interspace (IS) between
Heptad (H) 1 and 2, from amino acids 593-620, have been deleted.
Regions important for oligomer formation may be partially
functional. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 2824
[0379] pVR1012x/s R5gp 140(dCFI)/dV12
[0380] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2 loops (a.a.129 to 154, and a.a.160 to 193) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2825
[0381] pVR1012x/s R5gp 140(dCFI)/dV13
[0382] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V3 loops (a.a.129 to 154, and a.a.299 to 327) and
transmembrane domain. Heptad (H) 1, Heptad 2 and their Interspace
(IS) are required for oligomerization. The Fusion and Cleavage
(F/CL) domains, from amino acids 503-536, have been deleted. The
Interspace (IS) between Heptad (H) 1 and 2, from amino acids
593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2826
[0383] pVR1012x/s R5gp 140(dCFI)/dV14
[0384] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V4 loops (a.a.129 to 154, and a.a.386 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2827
[0385] pVR1012x/s R5gp140(dCFI)/dV23
[0386] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2, V3 loops (a.a.160 to 193, and a.a.299 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2828
[0387] pVR1012x/s R5gp140(dCFI)/dV24
[0388] The protein sequence of the envelope polyprotein (gp 160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2, V4 loops (a.a.160 to 193, and a.a.386 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2829
[0389] pVR1012x/s R5gp 140(dCFI)/dV34
[0390] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V3, V4 loops (a.a.299 to 327, and a.a.386 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2830
[0391] pVR1012x/s R5gp 140(dCFI)/dV123
[0392] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2, V3 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.299
to 327) and transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2831
[0393] pVR1012x/s R5gp 140(dCFI)/dV124
[0394] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2, V4 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.386
to 413) and transmembrane domain. Heptad (H) 1, Heptad 2 and their
Interspace (IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2832
[0395] pVR1012x/s R5gp140(dCFI)/dV134
[0396] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V3, V4 loops (a.a. 129 to 154, a.a.299 to 327, and a.a.386
to 413) and transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2833
[0397] pVR1012x/s R5gp 140(dCFI)/dV234
[0398] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2, V3, V4 loops (a.a. 160 to 193, a.a.299 to 327, and a.a.386
to 413) and transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC 2834
[0399] pVR1012x/s R5gp 140(dCFI)/dV1234
[0400] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2, V3, V4 loops (a.a. 129 to 154, a.a. 160 to 193, a.a.299
to 327, and a.a.386 to 413) and transmembrane domain. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The Fusion and Cleavage (F/CL) domains, from amino acids 503-536,
have been deleted. The Interspace (IS) between Heptad (H) 1 and 2,
from amino acids 593-620, have been deleted. Regions important for
oligomer formation may be partially functional. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC 2835
[0401] pAdApt R5gp140(dCFI)/dV1
[0402] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1 loop (a.a.129 to 154) and transmembrane domain. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The Fusion and Cleavage (F/CL) domains, from amino acids 503-536,
have been deleted. The Interspace (IS) between Heptad (H) 1 and 2,
from amino acids 593-620, have been deleted. Regions important for
oligomer formation may be partially functional. The expression
vector backbone is pAdApt.
VRC 2836
[0403] pAdApt R5gp140(dCFI)/dV2
[0404] The protein sequence of the envelope polyprotein (gp 160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2 loop (a.a. 160 to 193) and transmembrane domain. Heptad(H)
1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The Fusion and Cleavage (F/CL) domains, from amino
acids 503-536, have been deleted. The Interspace (IS) between
Heptad (H) 1 and 2, from amino acids 593-620, have been deleted.
Regions important for oligomer formation may be partially
functional. The expression vector backbone is AdApt.
VRC 2837
[0405] pAdApt R5gp140(dCFI)/dV3
[0406] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V3 loop (a.a.299 to 327) and transmembrane domain. Heptad (H)
1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The Fusion and Cleavage (F/CL) domains, from amino
acids 503-536, have been deleted. The Interspace (IS) between
Heptad (H) 1 and 2, from amino acids 593-620, have been deleted.
Regions important for oligomer formation may be partially
functional. The expression vector backbone is pAdApt.
VRC 2838
[0407] pAdApt R5gp140(dCFI)/dV4
[0408] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V4 loop (a.a.386 to 413) and transmembrane domain. Heptad (H)
1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The Fusion and Cleavage (F/CL) domains, from amino
acids 503-536, have been deleted. The Interspace (IS) between
Heptad (H) 1 and 2, from amino acids 593-620, have been deleted.
Regions important for oligomer formation may be partially
functional. The expression vector backbone is pAdApt.
VRC 2839
[0409] pAdApt R5gp 140(dCFI)/dV12
[0410] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2 loops (a.a.129 to 154, and a.a.160 to 193) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2840
[0411] pAdApt R5gp 140(dCFI)/dV13
[0412] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V3 loops (a.a.129 to 154, and a.a.299 to 327) and
transmembrane domain. Heptad (H) 1, Heptad 2 and their Interspace
(IS) are required for oligomerization. The Fusion and Cleavage
(F/CL) domains, from amino acids 503-536, have been deleted. The
Interspace (IS) between Heptad (H) 1 and 2, from amino acids
593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2841
[0413] pAdApt R5gp 140(dCFI)/dV14
[0414] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V4 loops (a.a.129 to 154, and a.a.386 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2842
[0415] pAdApt R5gp140(dCFI)/dV23
[0416] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2, V3 loops (a.a.160 to 193, and a.a.299 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2843
[0417] pAdApt R5gp140(dCFI)/dV24
[0418] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2, V4 loops (a.a.160 to 193, and a.a.386 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2844
[0419] pAdApt R5gp140(dCFI)/dV34
[0420] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V3, V4 loops (a.a.299 to 327, and a.a.386 to 413) and
transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2845
[0421] pAdApt R5gp 140(dCFI)/dV123
[0422] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2, V3 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.299
to 327) and transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt
VRC 2846
[0423] pAdApt R5gp140(dCFI)/dV124
[0424] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2, V4 loops (a.a. 129 to 154, a.a.160 to 193, and a.a.386
to 413) and transmembrane domain. Heptad (H) 1, Heptad 2 and their
Interspace (IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2847
[0425] pAdApt R5gp 140(dCFI)/dV134
[0426] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V3, V4 loops (a.a. 129 to 154, a.a.299 to 327, and a.a.386
to 413) and transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2848
[0427] pAdApt R5gp 140(dCFI)/dV234
[0428] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V2, V3, V4 loops (a.a. 160 to 193, a.a.299 to 327, and a.a.386
to 413) and transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pAdApt.
VRC 2849
[0429] pAdApt R5gp 140(dCFI)/dV1234
[0430] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 680. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the V1, V2, V3, V4 loops (a.a. 129 to 154, a.a. 160 to 193, a.a.299
to 327, and a.a.386 to 413) and transmembrane domain. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The Fusion and Cleavage (F/CL) domains, from amino acids 503-536,
have been deleted. The Interspace (IS) between Heptad (H) 1 and 2,
from amino acids 593-620, have been deleted. Regions important for
oligomer formation may be partially functional. The expression
vector backbone is pAdApt.
VRC2850
[0431] pVR1012x/s R5gp145delC1 (delCFI)/h
[0432] Constant domain 1 was deleted from gp145delCFI from amino
acid 33-127 and was replaced with an Nhe I site.
VRC2851
[0433] pVR1012 x/s R5gp145delC2 (delCFI)/h
[0434] Constant domain 2 was deleted from gp145dCFI from amino acid
199-293 and was replaced with an Nhe I site.
VRC2852
[0435] pVR1012x/s R5gp145delC3 (delCFI)/h
[0436] Constant domain 3 was deleted from gp145delCFI from amino
acid 333-380 and was replaced with an Nhe I site.
VRC2853
[0437] pVR1012 x/s R5gp145delC4 (delCFI)/h
[0438] Constant domain 4 was deleted from gp145delCFI from amino
acid 419-458 and was replaced with an Nhe I site.
VRC2854
[0439] pVR1012 x/s R5gp145delC5 (delCFI)/h
[0440] Constant domain 5 was deleted from gp145delCFI from amino
acid 472-498 and was replaced with an Nhe I site.
VRC 2860
[0441] pVR1012x/s R5gp145(dCFI)/h/dV1
[0442] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The V1 loop (a.a. 129-154) and Fusion and Cleavage
(F/CL) domains (a.a. 503-536) have been deleted. Also, the
Interspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have been
deleted. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 2861
[0443] pVR1012x/s R5gp145(dCFI)/h/dV2
[0444] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The V2 loop (a.a. 160-193) and Fusion and Cleavage
(F/CL) domains (a.a. 503-536) have been deleted. Also, the
Interspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have been
deleted. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 2862
[0445] pVR1012x/s R5gp145(dCFI)/h/dV3
[0446] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The V3 loop (a.a. 299-327) and Fusion and Cleavage
(F/CL) domains (a.a. 503-536) have been deleted. Also, the
Interspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have been
deleted. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 2863
[0447] pVR1012x/s R5gp145(dCFI)/h/dV4
[0448] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V4 loop (a.a. 386-413) and Fusion and Cleavage
(F/CL) domains (a.a. 503-536) have been deleted. Also, the
Interspace (IS) between Heptad (H) 1 and 2 (a.a.593-620) have been
deleted. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 2864
[0449] pVR10I2x/s R5gp145(dCFI)/h/dV12
[0450] The protein sequence of the envelope polyprotein (gp 160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The V1, V2 loops (a.a. 129-154 and 160-193) and
Fusion and Cleavage (F/CL) domains (a.a. 503-536) have been
deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR0102x/s (VRC2000).
VRC 2865
[0451] pVR1012x/s R5gp145(dCFI)/h/dV13
[0452] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V1, V3 loops (a.a. 129-154 and 299-327) and
Fusion and Cleavage (F/CL) domains (a.a. 503-536) have been
deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2866
[0453] pVR1012x/s R5gp145(dCFI)/h/dV14
[0454] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V1, V4 loops (a.a. 129-154 and 386-413) and
Fusion and Cleavage (F/CL) domains (a.a. 503-536) have been
deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2867
[0455] pVR1012x/s R5gp145(dCFI)/h/dV23
[0456] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad(H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V2, V3 loops (a.a. 160-193 and 299-327) and
Fusion and Cleavage (F/CL) domains (a.a. 503-536) have been
deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2868
[0457] pVR1012x/s R5gp145(dCFI)/h/dV24
[0458] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad(H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V2, V4 loops (a.a. 160-193 and 386-413) and
Fusion and Cleavage (F/CL) domains (a.a. 503-536) have been
deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2869
[0459] pVR1012x/s R5gp 145(dCFI)/h/dV34
[0460] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V3, V4 loops (a.a. 299-327 and 386-413) and
Fusion and Cleavage (F/CL) domains (a.a. 503-536) have been
deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2870
[0461] pVR1012x/s R5gp145(dCFI)/h/dV134
[0462] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V1, V3, V4 loops (a.a. 129-154, 299-327 and
386-413) and Fusion and Cleavage (F/CL) domains (a.a. 503-536) have
been deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2871
[0463] pVR1012x/s R5gp 145(dCFI)/h/dV234
[0464] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The V2, V3, V4 loops (a.a. 160-193, 299-327 and
386-413) and Fusion and Cleavage (F/CL) domains (a.a. 503-536) have
been deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2872
[0465] pVR1012x/s R5gp145(dCFI)dv123/h
[0466] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V1, V2, V4 loops (a.a. 129-154, 160-193 and
386-413) and Fusion and Cleavage (F/CL) domains (a.a. 503-536) have
been deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2873
[0467] pVR1012x/s R5gp 145(dCFI)/h/dV124
[0468] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V1, V2, V4 loops (a.a. 129-154, 160-193 and
386-413) and Fusion and Cleavage (F/CL) domains (a.a. 503-536) have
been deleted. Also, the Interspace (IS) between Heptad (H) 1 and 2
(a.a.593-620) have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 2874
[0469] pVR1012x/s R5gp 145(dCFI)/h/dV1234
[0470] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an
R5-tropic version of the envelope protein (R5gp160/h), the region
encoding HIV-1 envelope polyprotein amino acids 275 to 361 from
X4gp160/h (VRC3300) were replaced with the corresponding region
from the BaL strain of HIV-1 (GeneBank accession number M68893,
again using human preferred codons). The full-length R5-tropic
version of the envelope protein gene from pR5gp160/h (VRC3000) was
terminated after the codon for amino acid 704. The truncated
envelope polyprotein (gp145) contains the entire SU protein and a
portion of the TM protein including the fusion domain, the
transmembrane domain, and regions important for oligomer formation.
Heptad (H) 1, Heptad 2 and their Interspace (IS) are required for
oligomerization. The V1, V2, V3, V4 loops (a.a. 129-154, 160-193,
299-327 and 386-413) and Fusion and Cleavage (F/CL) domains (a.a.
503-536) have been deleted. Also, the Interspace (IS) between
Heptad (H) 1 and 2 (a.a.593-620) have been deleted. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC2900
[0471] pVR1012x/s R5gp150/h
[0472] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). The full-length
R5-tropic version of the envelope protein gene from pR5gp160/h
(VRC3000) was terminated after the codon for amino acid 752. The
truncated envelope polyprotein (gp150) contains the entire SU
protein and a portion of the TM protein including the fusion
domain, the transmembrane domain, and regions important for
oligomer formation. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC3000
[0473] pVR1012x/s R5gp 160/h
[0474] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
produce an R5-tropic version of the envelope protein (R5gp160/h),
the region encoding HIV-1 envelope polyprotein amino acids 275 to
361 from X4gp160/h (VRC3300) were replaced with the corresponding
region from the BaL strain of HIV-1 (GeneBank accession number
M68893, again using human preferred codons). Full length SU and TM
proteins are expressed from the pVR1012x/s (VRC2000) vector
backbone.
VRC3200
[0475] pVR1012x/s X4gp150/h
[0476] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
The full-length X4-tropic version of the envelope protein gene from
pX4gp160/h (VRC3300) was terminated after the codon for amino acid
752. The truncated envelope polyprotein (gp150) contains the entire
SU protein and a portion of the TM protein including the fusion
domain, the transmembrane domain, and regions important for
oligomer formation. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC3201
[0477] pVR1012x/s X4gp150(del F/CL del H IS)/h
[0478] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
The full-length X4-tropic version of the envelope protein gene from
pX4gp160/h (VRC3300) was terminated after the codon for amino acid
752. The truncated envelope polyprotein (gp150) contains the entire
SU protein and a portion of the TM protein including the fusion
domain, the transmembrane domain, and regions important for
oligomer formation. Heptad(H) 1, Heptad 2 and their Interspace(IS)
are required for oligomerization. The Fusion and Cleavage (F/CL)
domains, from amino acids 503-536, have been deleted. The
Interspace (IS) between Heptad (H) 1 and 2, from amino acids
593-620, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC3202
[0479] pVR1012x/s X4gp150 .DELTA.gly/h.
[0480] Eukaryotic vector with humanized codons expressing the HIV
envelope glycoprotein gp150 from HXB2, X4 tropic mutated in the
Glycosylation sites. VRC3202 pVR1012x/s X4gp150Dgly/h The protein
sequence of the envelope polyprotein (gp160) from HXB2 (X4-tropic,
GenBank accession number K03455) was used to create a synthetic
version of the gene (X4gp160/h) using codons optimized for
expression in human cells. The nucleotide sequence X4gp160/h shows
little homology to the HXB2 gene, but the protein encoded is the
same with the following amino acid substitutions: F53L, N94D,
K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To disrupt
potential glycoslylation sites in the HIV-1 envelope proteins,
point mutations were introduced into the full-length X4-tropic
version of the envelope protein gene from pX4gp160/h (VRC3300). The
resulting amino acids substitutions in X4gp160Dgly/h are: N88D,
N156D, N160D, N197E, N230D, N234D, N241D, N276D, L288V, N289D,
S291T, N295D, N332D, N339D, N356D, N386D, and N448D. The
full-length X4-tropic version of the envelope protein gene from
pX4gp160/h (VRC3300) was terminated after the codon for amino acid
752. Heptad(H) 1, Heptad 2 and their Interspace(IS) are required
for oligomerization. Full length SU and TM proteins are expressed
from the pVR1012x/s (VRC2000) vector backbone.
VRC3203
[0481] pVR1012x/s X4gp 150 AB .DELTA.gly/h
[0482] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The AB designation means
the amino acids from 1-307 (XbaI to EcoR1). The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
disrupt potential glycoslylation sites in the HIV-1 envelope
proteins, point mutations were introduced into the full-length
X4-tropic version of the envelope protein gene from pX4gp160/h
(VRC3300). The resulting amino acids substitutions in X4gp160Dgly/h
are: N88D, N156D, N160D, N197E, N230D, N234D, N241D, N276D, L288V,
N289D, S291T, and N295D. The full-length X4-tropic version of the
envelope protein gene from pX4gp160/h (VRC3300) was terminated
after the codon for amino acid 752. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. Full length SU and
TM proteins are expressed from the pVR1012x/s (VRC2000) vector
backbone.
VRC3300
[0483] pVR1012x/s X4gp 160/h
[0484] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. Full length SU and TM proteins are expressed from
the pVR1012x/s (VRC2000) vector backbone.
VRC3301
[0485] pVR1012x/s X4gp160(del F/CL del H IS)/h
[0486] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T.
Full length SU and TM proteins are Expressed. Heptad(H) 1, Heptad 2
and their Interspace(IS) are required for oligomerization. The
Fusion and Cleavage (F/CL) domains, from amino acids 503-536, have
been deleted. The Interspace (IS) between Heptad (H) 1 and 2, from
amino acids 593-620, have been deleted. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC3400
[0487] pVR1012x/s X4gp160.DELTA.gly/h
[0488] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
disrupt potential glycoslylation sites in the HIV-1 envelope
proteins, point mutations were introduced into the full-length
X4-tropic version of the envelope protein gene from pX4gp160/h
(VRC3300). The resulting amino acids substitutions in X4gp160Dgly/h
are: N88D, N156D, N160D, N197E, N230D, N234D, N241D, N276D, L288V,
N289D, S291T, N295D, N332D, N339D, N356D, N386D, and N448D.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. Full length SU and TM proteins are expressed from
the pVR1012x/s (VRC2000) vector backbone.
VRC3401
[0489] pVR1012x/s X4gp 160AB mut .DELTA.gly/h
[0490] The protein sequence of the envelope polyprotein (gp160)
from HXB2 (X4-tropic, GenBank accession number K03455) was used to
create a synthetic version of the gene (X4gp160/h) using codons
optimized for expression in human cells. The AB designation means
the amino acids from 1-307 (XbaI to EcoR1). The nucleotide sequence
X4gp160/h shows little homology to the HXB2 gene, but the protein
encoded is the same with the following amino acid substitutions:
F53L, N94D, K192S, I215N, A224T, A346D, P470L, T7231, and S745T. To
disrupt potential glycoslylation sites in the HIV-1 envelope
proteins, point mutations were introduced into the full-length
X4-tropic version of the envelope protein gene from pX4gp160/h
(VRC3300). The resulting amino acids substitutions in X4gp160Dgly/h
are: N88D, N156D, N160D, N197E, N230D, N234D, N241D, N276D, L288V,
N289D, S291T, and N295D. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. Full length SU and
TM proteins are expressed from the pVR1012x/s (VRC2000) vector
backbone.
VRC3500
[0491] pVR1012x/s Nef/h
[0492] The protein sequence of the Nef protein from HIV-1 PV22
(GenBank accession number K02083) was used to create a synthetic
version of the Nef gene (Nef/h) using codons optimized for
expression in human cells. The nucleotide sequence Nef/h shows
little homology to the viral gene, but the protein encoded is the
same. Nef/h is expressed from the pVR1012x/s (VRC2000) vector
backbone.
VRC3600
[0493] pVR1012x/s NefDMHCDCD4/h
[0494] The protein sequence of the Nef protein from HIV-1 PV22
(GenBank accession number K02083) was used to create a synthetic
version of the Nef gene (Nef/h) using codons optimized for
expression in human cells. To disrupt the ability of Nef to limit
both MHC class I and CD4 expression point mutations were introduced
into the Nef gene from pNef/h (VRC3500). The resulting amino acids
substitutions in pNefDMHCDCD4/h are: P69A, P72A, P75A, P78A, D174A
and D175A. pNefDMHCDCD4/h is expressed from the pVR1012x/s
(VRC2000) vector backbone.
VRC3700
[0495] pVR1012x/s NefDCD4/h
[0496] The protein sequence of the Nef protein from HIV-1 PV22
(GenBank accession number K02083) was used to create a synthetic
version o f the Nef gene (Nef/h) using codons optimized for
expression in human cells. To disrupt the ability of Nef to limit
CD4 expression, point mutations were introduced into the Nef gene
from pNef/h (VRC3500). The resulting amino acids substitutions in
pNefDCD/h are: D174A and D175A. pNefDCD4/h is expressed from the
pVR1012x/s (VRC2000) vector backbone. VRC3700 pVR1012x/s NefDCD4/h
The protein sequence of the Nef protein from HIV-1 PV22 (GenBank
accession number K02083) was used to create a synthetic version of
the Nef gene (Nef/h) using codons optimized for expression in human
cells. To disrupt the ability of Nef to limit CD4 expression, point
mutations were introduced into the Nef gene from pNef/h (VRC3500).
The resulting amino acids substitutions in pNefDCD/h are: D174A and
D175A. pNefDCD4/h is expressed from the pVR1012x/s (VRC2000) vector
backbone.
VRC3800
[0497] pVR1012x/s NefDMHC/h
[0498] The protein sequence of the Nef protein from HIV-1 PV22
(GenBank accession number K02083) was used to create a synthetic
version of the Nef gene (Nef/h) using codons optimized for
expression in human cells. To disrupt the ability of Nef to limit
MHC class I expression, point mutations were introduced into the
Nef gene from pNef/h (VRC3500). The resulting amino acids
substitutions in pNefDMHC/h are: P69A, P72A, P75A, and P78A.
pNefDMHC/h is expressed from the pVR1012x/s (VRC2000) vector
backbone.
VRC5200
[0499] pVR1012x/s 89.6 Pgp128(del F/CL)/h
[0500] The protein sequence of the envelope polyprotein (gp160)
from 89.6P (Dual-tropic, GenBank accession number
u89134/LOCUS:SIU89134) was used to create a synthetic version of
the gene (89.6 Pgp160/h) using codons optimized for expression in
human cells. The nucleotide sequence 89.6 Pgp160/h shows little
homology to the 89.6P gene, but the protein encoded is the same.
The full-length 89.6P, dual-tropic version of the envelope protein
gene from 89.6P gp160/h (VRC3000) was terminated after the codon
for amino acid 596. The truncated envelope polyprotein contains the
entire SU protein and a portion of the TM protein including the
fusion domain, but lacking the transmembrane domain. The Fusion and
Cleavage (F/CL) domains, from amino acids 508-541, have been
deleted. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC5201
[0501] pVR1012x/s 89.6 Pgp140(del F/CL del H IS/h
[0502] The protein sequence of the envelope polyprotein (gp160)
from 89.6P (dual-tropic, GenBank accession number u89134/locus
SIU89134) was used to create a synthetic version of the gene
(Dualtropic gp160/h) using codons optimized for expression in human
cells. The nucleotide sequence dualtropic gp160/h shows little
homology to the 89.6P gene, but the protein encoded is the same.
The full-length 89.6P, dual-tropic version of the envelope protein
gene was terminated after the codon for amino acid 683. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 508-541, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 597-625, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC5202
[0503] pVR1012x/s 89.6 Pgp145(del F/CL del H IS/h
[0504] The protein sequence of the envelope polyprotein (gp160)
from 89.6P (dual-tropic, GenBank accession number u89134/locus
SIU89134) was used to create a synthetic version of the gene
(Dualtropic gp 160/h) using codons optimized for expression in
human cells. The nucleotide sequence dualtropic gp160/h shows
little homology to the 89.6P gene, but the protein encoded is the
same. The full-length 89.6P, dual-tropic version of the envelope
protein gene was terminated after the codon for amino acid 709. The
truncated envelope polyprotein contains the entire SU protein and a
portion of the TM protein including the fusion domain, but lacking
the transmembrane domain. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 508-541, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 597-625, have been deleted. Regions important for oligomer
formation may be partially functional. The expression vector
backbone is pVR1012x/s (VRC2000).
VRC5203
[0505] pVR1012x/s 89.6 Pgp160/h
[0506] The protein sequence of the envelope polyprotein (gp160)
from 89.6P (Dual-tropic, GenBank accession number U89134/LOCUS:
SIU89134) was used to create a synthetic version of the gene (dual
tropic gp160/h) using codons optimized for expression in human
cells. The nucleotide sequence 89.6P gp160/h shows little homology
to the 89.6P gene, but the protein encoded is the same. Full length
SU and TM proteins are expressed from the pVR1012x/s (VRC2000)
vector backbone
VRC5300
[0507] pVR1012x/s R5(clade C)gp140(del F/CL del H IS)/h
[0508] The protein sequence of the envelope polyprotein (gp160)
from 92br025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (R5gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
R5gp160/h shows little homology to the 92br025 gene, but the
protein encoded is the same. The full-length R5-tropic version of
the envelope protein was synthesized by Operon under the name:
kongene. The XbaI (18 nt up-stream from ATG) to BglII (1376 nt
down-stream from ATG) fragment which contains polylinker at the 5'
end, Kozak sequence and ATG was cloned into the XbaI to BglII sites
of VRC2701 pVR1012x/s X4gp140(del F/CL del H IS)/h backbone.
Therefore, the gene is R5 (clade C) gp160/h up to the BglII site
(1376 nt from ATG) and the rest of the gene after BglII site is
VRC2701 pVR1012x/s X4gp140(del F/CL del H IS)/h. The truncated
envelope polyprotein contains the entire SU protein and a portion
of the TM protein including the fusion domain, but lacking the
transmembrane domain. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000). The full-length X4-tropic version of the
envelope protein from pX4gp160/h (VRC3300) was terminated after the
codon for amino acid 680.
VRC5301
[0509] pVR1012x/s R5(clade C)gp145(del F/CL del H IS)/h
[0510] The protein sequence of the envelope polyprotein (gp160)
from 92br025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (R5gp160/h) using codons
optimized for expression in human cells. The nucleotide sequence
R5gp160/h shows little homology to the 92br025 gene, but the
protein encoded is the same. The full-length R5-tropic version of
the envelope protein was synthesized by Operon under the name:
kongene. The XbaI (18 nt up-stream from ATG) to BglII (1376 nt
down-stream from ATG) fragment which contains polylinker at the 5'
end, Kozak sequence and ATG was cloned into the XbaI to BglII sites
of VRC2701 pVR1012x/s X4gp140(del F/CL del H IS)/h backbone.
Therefore, the gene is R5 (clade C) gp160/h up to the BglII site
(1376 nt from ATG) and the rest of the gene after BglII site is
VRC2707 pVR1012x/s X4gp145(del F/CL del H IS)/h. The truncated
envelope polyprotein contains the entire SU protein and a portion
of the TM protein including the fusion domain, but lacking the
transmembrane domain. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 503-536, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 593-620, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000). The full-length X4-tropic version of the
envelope protein from pX4gp160/h (VRC3300) was terminated after the
codon for amino acid 704.
VRC5303
[0511] pVR1012x/s R5gp145CladeC(Brazil) delCFI/h
[0512] Authentic Clade C unlike 5301 which is a hybrid between C
and B.
VRC 5304
[0513] pVR1012x/s R5(clade A)gp 140(del F/CL del H IS)/h
[0514] The protein sequence of the envelope polyprotein (gp160)
from 92rw020 (R5-tropic, GenBank accession number U51283) was used
to create a synthetic version of the gene (Clade-A gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence R5gp145delCFI shows little homology to the
92rw020 gene, but the protein encoded is the same. The XbaI (18 nt
up-stream from ATG) to BamH1 (1837 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains the entire SU
protein and the TM domain, but lacking the Fusion domain and
Cytoplasmic domain. Regions important for oligomer formation may be
partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 486-519, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 576-604, have been deleted. The expression vector backbone is
pVR1102x/s (VRC2000).
VRC 5305
[0515] pVR1012x/s R5(clade A)gp145(del F/CL del H IS)/h
[0516] The protein sequence of the envelope polyprotein (gp160)
from 92rw020 (R5-tropic, GenBank accession number U51283) was used
to create a synthetic version of the gene (Clade-A gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence R5gp145delCFI shows little homology to the
92rw020 gene, but the protein encoded is the same. The XbaI (18 nt
up-stream from ATG) to BamH1 (1912 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains the entire SU
protein and the TM domain, but lacking the Fusion domain and
Cytoplasmic domain. Regions important for oligomer formation may be
partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 486-519, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 576-604, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5306
[0517] pVR1012x/s R5(clade E)gp140(del F/CL del H IS)/h
[0518] The protein sequence of the envelope polyprotein
(gp140delCFI) from 93th966.8 (R5-tropic, GenBank accession number
U08456) was used to create a synthetic version of the gene (Clade-C
gp140delCFI) using codons optimized for expression in human cells.
The nucleotide sequence R5gp140delCFI shows little homology to the
gene 93th966.8, but the protein encoded is the same. The XbaI (18
nt up-stream from ATG) to BamH1 (1856 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains the entire SU
protein and the TM domain, but lacking the Fusion domain and
Cytoplasmic domain. Regions important for oligomer formation may be
partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 497-530, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 588-613, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5307
[0519] pVR1012x/s R5(clade E)gp145(del F/CL del H IS)/h
[0520] The protein sequence of the envelope polyprotein
(gp145delCFI) from 93th966.8 (R5-tropic, GenBank accession number
U08456) was used to create a synthetic version of the gene (Clade-C
gp145delCFI) using codons optimized for expression in human cells.
The nucleotide sequence R5gp145delCFI shows little homology to the
gene 93th966.8, but the protein encoded is the same. The XbaI (18
nt up-stream from ATG) to BamH1 (1928 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains the entire SU
protein and the TM domain, but lacking the Fusion domain and
Cytoplasmic domain. Regions important for oligomer formation may be
partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The Fusion and
Cleavage (F/CL) domains, from amino acids 497-530, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 588-613, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5308
[0521] pVR1012x/s R5(clade C South African)gp140(del F/CL del H
IS)/h
[0522] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1833 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein contains
the entire SU protein and the TM domain, but lacking the Fusion
domain and Cytoplasmic domain. Regions important for oligomer
formation may be partially functional. Heptad(H) 1, Heptad 2 and
their Interspace(IS) are required for oligomerization. The Fusion
and Cleavage (F/CL) domains, from amino acids 487-520, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 577-605, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5309
[0523] pVR1012x/s R5(clade C South African)gp145(del F/CL del H
IS)/h
[0524] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein contains
the entire SU protein and the TM domain, but lacking the Fusion
domain and Cytoplasmic domain. Regions important for oligomer
formation may be partially functional. Heptad(H) 1, Heptad 2 and
their Interspace(IS) are required for oligomerization. The Fusion
and Cleavage (F/CL) domains, from amino acids 487-520, have been
deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino
acids 577-605, have been deleted. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5350
[0525] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV1
[0526] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1 loop(a.a.133-148) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembrane
domain, and cytoplasmic domain in the TM protein. Regions important
for oligomer formation may be partially functional. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The expression vector backbone is pVR1012x/s (VRC2000).
VRC 5351
[0527] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV12
[0528] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2 loops (a.a.133-191) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembrane
domain, and cytoplasmic domain in the TM protein. Regions important
for oligomer formation may be partially functional. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The expression vector backbone is pVR1012x/s (VRC2000).
VRC 5352
[0529] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV123
[0530] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2, V3 loops (a.a.130-191, 330-358) of the SU protein. It
also lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529),
the Interspace (IS) between Heptad (H) 1 and 2(a.a.586-612),
transmembrane domain, and cytoplasmic domain in the TM protein.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5353
[0531] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV1234
[0532] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2, V3, V4 loops (a.a.130-191, 330-358, 384-408) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), transmembrane domain, and cytoplasmic domain in the TM
protein. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5354
[0533] pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV124
[0534] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2, V4 loops (a.a. 130-191, 384-408) of the SU protein. It
also lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529),
the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),
transmembrane domain, and cytoplasmic domain in the TM protein.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5355
[0535] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV13
[0536] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V3 loops (a.a.133-148, 330-358) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),
transmembrane domain, and cytoplasmic domain in the TM protein.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5356
[0537] pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV134
[0538] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V3, V4 loops (a.a.130-148, 330-358, 384-408) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), transmembrane domain, and cytoplasmic domain in the TM
protein. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5357
[0539] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV14
[0540] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V4 loops (a.a. 130-148, 384-408) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),
transmembrane domain, and cytoplasmic domain in the TM protein.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5358
[0541] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV2
[0542] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V2 loop(a.a.154-191) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembrane
domain, and cytoplasmic domain in the TM protein. Regions important
for oligomer formation may be partially functional. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The expression vector backbone is pVR1012x/s (VRC2000).
VRC 5359
[0543] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV23
[0544] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140 (delCFI) shows little
homology to the gene 92BR025, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1910 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein contains
deletion in the V2, V3 loops (a.a. 154-191, 330-358) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), transmembrane domain, and cytoplasmic domain in the TM
protein. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5360
[0545] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV234
[0546] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V2, V3, V4 loops (a.a.154-191, 330-358, 384-408) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), transmembrane domain, and cytoplasmic domain in the TM
protein. Regions important for oligomer formation may be partially
functional: Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5361
[0547] pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV24
[0548] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V2, V4 loops (a.a. 154-191, 384-408) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),
transmembrane domain, and cytoplasmic domain in the TM protein.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5362
[0549] pVRC1012(x/s)-gp 140(dCFI)(Brazil C)/dV3
[0550] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V3 loop(a.a.330-358) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembrane
domain, and cytoplasmic domain in the TM protein. Regions important
for oligomer formation may be partially functional. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The expression vector backbone is pVR1012x/s (VRC2000).
VRC 5363
[0551] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV34
[0552] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V3, V4 loops (a.a.330-358, 384-408) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612),
transmembrane domain, and cytoplasmic domain in the TM protein.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5364
[0553] pVRC1012(x/s)-gp140(dCFI)(Brazil C)/dV4
[0554] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp140delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp140(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V4 loop(a.a.384-408) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), transmembrane
domain, and cytoplasmic domain in the TM protein. Regions important
for oligomer formation may be partially functional. Heptad(H) 1,
Heptad 2 and their Interspace(IS) are required for oligomerization.
The expression vector backbone is pVR1012x/s (VRC2000).
VRC 5365
[0555] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV1
[0556] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1 loop(a.a.133-148) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), and Cytoplasmic
domain in the TM protein. Regions important for oligomer formation
may be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC 5366
[0557] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV12
[0558] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2 loops (a.a.133-191) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), and Cytoplasmic
domain in the TM protein. Regions important for oligomer formation
may be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC 5367
[0559] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV123
[0560] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2, V3 loops (a.a. 133-191, 330-358) of the SU protein. It
also lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529),
the Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), and
Cytoplasmic domain in the TM protein. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC 5368
[0561] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV1234
[0562] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2, V3, V4 loops (a.a. 133-191,330-358, 384-408) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), and Cytoplasmic domain in the TM protein. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 5369
[0563] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV124
[0564] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V2, V4 loops (a.a. 133-148,154-191, 384-408) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), and Cytoplasmic domain in the TM protein. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 5370
[0565] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV13
[0566] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V3 loops (a.a.133-148, 330-358) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), and
Cytoplasmic domain in the TM protein. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC 5371
[0567] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV134
[0568] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V3, V4 loops (a.a. 133-148,330-358, 384-408) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), and Cytoplasmic domain in the TM protein. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 5372
[0569] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV14
[0570] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V1, V4 loops (a.a.133-148, 384-408) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), and
Cytoplasmic domain in the TM protein. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC 5373
[0571] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV2
[0572] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V2 loop (a.a.154-191) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), and Cytoplasmic
domain in the TM protein. Regions important for oligomer formation
may be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC 5374
[0573] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV23
[0574] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V2, V3 loops (a.a.154-191, 330-358) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), and
Cytoplasmic domain in the TM protein. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC 5375
[0575] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV234
[0576] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V2, V3, V4 loops (a.a. 154-191,330-358, 384-408) of the SU
protein. It also lacks the Fusion and Cleavage (F/CL) domains(a.a.
496-529), the Interspace (IS) between Heptad (H) 1 and 2(a.a.
586-612), and Cytoplasmic domain in the TM protein. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC 5376
[0577] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV24
[0578] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V2, V4 loops (a.a.154-191, 384-408) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), and
Cytoplasmic domain in the TM protein. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC 5377
[0579] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV3
[0580] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V3 loop (a.a.330-358) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), and Cytoplasmic
domain in the TM protein. Regions important for oligomer formation
may be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC 5378
[0581] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV34
[0582] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V3, V4 loops (a.a.330-358, 384-408) of the SU protein. It also
lacks the Fusion and Cleavage (F/CL) domains(a.a. 496-529), the
Interspace (IS) between Heptad (H) 1 and 2(a.a. 586-612), and
Cytoplasmic domain in the TM protein. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC 5379
[0583] PVRC1012(x/s)-gp145(dCFI)(Brazil C)/dV4
[0584] The protein sequence of the envelope polyprotein (gp160)
from 92BR025 (R5-tropic, GenBank accession number U52953) was used
to create a synthetic version of the gene (Brazil-C gp145delCFI)
using codons optimized for expression in human cells. The
nucleotide sequence of Brazil-C gp145(delCFI) shows little homology
to the gene 92BR025, but the protein encoded is the same. The XbaI
(18 nt up-stream from ATG) to BamH1 (1910 nt down-stream from ATG)
fragment which contains polylinker at the 5' end, Kozak sequence
and ATG was cloned into the XbaI to BamH1 sites of pVR1012x/s
backbone. The truncated envelope polyprotein contains deletion in
the V4 loop (a.a.384-408) of the SU protein. It also lacks the
Fusion and Cleavage (F/CL) domains(a.a. 496-529), the Interspace
(IS) between Heptad (H) 1 and 2(a.a. 586-612), and Cytoplasmic
domain in the TM protein. Regions important for oligomer formation
may be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC5500
[0585] pVR1012x/s R5(SA-C)gp 140(dCFI) dV1/h
[0586] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1 loop (a.a.136-150), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5501
[0587] pVR1012x/s R5(SA-C)gp140(dCFI) dV12/h
[0588] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V2 loops (a.a.136-194), the Fusion and Cleavage (F/CL) domains
(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC 5502
[0589] pVR1012x/s R5(SA-C)gp140(dCFI) dV23/h
[0590] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V2, V3 loops (a.a. 136-194, 297-325), the Fusion and Cleavage
(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad
(H) 1 and 2 (a.a.577-605), the transmembrane domain and the
intracellular region. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC5503
[0591] pVR1012x/s R5(SA-C)gp 140(dCFI)dV1234/h
[0592] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V2, V3, V4 loops (a.a.136-194,297-325, 385-399), the Fusion and
Cleavage (F/CL) domains (a.a. 487-520), the Interspace (IS) between
Heptad (H) 1 and 2 (a.a.577-605), the transmembrane domain and the
intracellular region. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC5504
[0593] pVR1012x/s R5(SA-C)gp 140(dCFI)dV124/h
[0594] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1102x/s backbone. The truncated envelope polyprotein lacks the
V1, V2, V4 loops (a.a. 136-194, 385-399), the Fusion and Cleavage
(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad
(H) 1 and 2 (a.a.577-605), the transmembrane domain and the
intracellular region. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC550S
[0595] pVR1012x/s R5(SA-C)gp 140(dCFI)dV13/h
[0596] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V3 loops (a.a.136-150, 297-325), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5506
[0597] pVR1012x/s R5(SA-C)gp140(dCFI)dV134/h
[0598] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V3, V4 loops (a.a. 136-150, 297-325, 385-399), the Fusion and
Cleavage (F/CL) domains (a.a. 487-520), the Interspace (IS) between
Heptad (H) 1 and 2 (a.a.577-605), the transmembrane domain and the
intracellular region. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC5507
[0599] pVR1012x/s R5(SA-C)gp140(dCFI)dV14/h
[0600] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V4 loops (a.a.136-150, 385-399), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5508
[0601] pVR1012x/s R5(SA-C)gp 140(dCFI)dV2/h
[0602] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2 loop (a.a.156-194), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1102x/s (VRC2000).
VRC5509
[0603] pVR1012x/s R5(SA-C)gp140(dCFI)dV23/h
[0604] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2, V3 loops (a.a.156-194, 297-325), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5510
[0605] pVR1012x/s R5(SA-C)gp 140(dCFI)dV234/h
[0606] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2, V3, V4 loops (a.a. 156-194, 297-325, 385-399), the Fusion and
Cleavage (F/CL) domains (a.a. 487-520), the Interspace (IS) between
Heptad (H) 1 and 2 (a.a.577-605), the transmembrane domain and the
intracellular region. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC5511
[0607] pVR1012x/s R5(SA-C)gp140(dCFI)dV24/h
[0608] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2, V4 loops (a.a.156-194, 385-399), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5512
[0609] pVR1012x/s R5(SA-C)gp 140(dCFI)dV3/h
[0610] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V3 loop (a.a.297-325), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5513
[0611] pVR1012x/s R5(SA-C)gp 140(dCFI)dV34/h
[0612] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V3, V4 loops (a.a. 297-325, 385-399), the Fusion and Cleavage
(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad
(H) 1 and 2 (a.a.577-605), the transmembrane domain and the
intracellular region. Regions important for oligomer formation may
be partially functional. Heptad(H) 1, Heptad 2 and their
Interspace(IS) are required for oligomerization. The expression
vector backbone is pVR1012x/s (VRC2000).
VRC5514
[0613] pVR1012x/s R5(SA-C)gp140(dCFI)dV4/h
[0614] The protein sequence of the envelope polyprotein
(gp140delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp140delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp140delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V4 loop (a.a 385-399), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), the transmembrane domain and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5515
[0615] pVR1012x/s R5(SA-C)gp 145(dCFI)dV1/h
[0616] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1 loop (a.a.136-150), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), and the intracellular region. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC5516
[0617] pVR1012x/s R5(SA-C)gp145(dCFI)dV12/h
[0618] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V2 loops (a.a.136-194), the Fusion and Cleavage (F/CL) domains
(a.a. 487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), and the intracellular region. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC5517
[0619] pVR1012x/s R5(SA-C)gp 145(dCFI)dV123/h
[0620] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V2, V3 loops (a.a.136-194, 297-325), the Fusion and Cleavage
(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad
(H) 1 and 2 (a.a.577-605), and the intracellular region. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC5518
[0621] pVR1012x/s R5(SA-C)gp 145(dCFI)dV1234/h
[0622] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V2, V3, V4 loops (a.a.136-194, 297-325, 385-399), the Fusion
and Cleavage (F/CL) domains (a.a. 487-520), the Interspace (IS)
between Heptad (H) 1 and 2 (a.a.577-605), and the intracellular
region. Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5519
[0623] pVR1012x/s R5(SA-C)gp 145(dCFI)dV2/h
[0624] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2 loop (a.a.156-194), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), and the intracellular region. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC5520
[0625] pVR1012x/s R5(SA-C)gp 145(dCFI)dV23/h
[0626] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2, V3 loops (a.a.156-194, 297-325), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), and the intracellular region. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC5521
[0627] pVR1012x/s R5(SA-C)gp145(dCFI)dV234/h
[0628] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2, V3, V4 loops (a.a.156-194, 297-325, 385-399), the Fusion and
Cleavage (F/CL) domains (a.a. 487-520), the Interspace (IS) between
Heptad (H) 1 and 2 (a.a.577-605), and the intracellular region.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5522
[0629] pVR1012x/s R5(SA-C)gp 145(dCFI)dV24/h
[0630] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V2, V4 loops (a.a.156-194, 385-399), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), and the intracellular region. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC5523
[0631] pVR1012x/s R5(SA-C)gp145(dCFI)dV3/h
[0632] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V3 loop (a.a.297-325), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), and the intracellular region. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC5524
[0633] pVR1012x/s R5(SA-C)gp145(dCFI)dV34/h
[0634] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V3, V4 loops (a.a. 297-325, 385-399), the Fusion and Cleavage
(F/CL) domains (a.a. 487-520), the Interspace (IS) between Heptad
(H) 1 and 2 (a.a.577-605), and the intracellular region. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC5525
[0635] pVR1012x/s R5(SA-C)gp 145(dCFI)dV4/h
[0636] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V4 loop (a.a.385-399), the Fusion and Cleavage (F/CL) domains (a.a.
487-520), the Interspace (IS) between Heptad (H) 1 and 2
(a.a.577-605), and the intracellular region. Regions important for
oligomer formation may be partially functional. Heptad(H) 1, Heptad
2 and their Interspace(IS) are required for oligomerization. The
expression vector backbone is pVR1012x/s (VRC2000).
VRC5526
[0637] pVR1012x/s R5(SA-C)gp 145(dCFI)dV13/h
[0638] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V3 loops (a.a.136-150, 297-325), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the Interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), and the intracellular region. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
VRC5527
[0639] pVR1012x/s R5(SA-C)gp145(dCFI)dV134/h
[0640] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V3, V4 loops (a.a.136-150, 297-325, 385-399), the Fusion and
Cleavage (F/CL) domains (a.a. 487-520), the Interspace (IS) between
Heptad (H) 1 and 2 (a.a.577-605), and the intracellular region.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5528
[0641] pVR1012x/s R5(SA-C)gp145(dCFI)dV124/h
[0642] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V2, V4 loops (a.a.136-150, 156-194, 385-399), the Fusion and
Cleavage (F/CL) domains (a.a. 487-520), the Interspace (IS) between
Heptad (H) 1 and 2 (a.a.577-605), and the intracellular region.
Regions important for oligomer formation may be partially
functional. Heptad(H) 1, Heptad 2 and their Interspace(IS) are
required for oligomerization. The expression vector backbone is
pVR1012x/s (VRC2000).
VRC5529
[0643] pVR1012x/s R5(SA-C)gp 145(dCFI)dV14/h
[0644] The protein sequence of the envelope polyprotein
(gp145delCFI) from 97ZA012 (R5-tropic, GenBank accession number
AF286227) was used to create a synthetic version of the gene
(Clade-C gp145delCFI) using codons optimized for expression in
human cells. The nucleotide sequence R5gp145delCFI shows little
homology to the gene 97ZA012, but the protein encoded is the same.
The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream
from ATG) fragment which contains polylinker at the 5' end, Kozak
sequence and ATG was cloned into the XbaI to BamH1 sites of
pVR1012x/s backbone. The truncated envelope polyprotein lacks the
V1, V4 loops (a.a.136-150, 385-399), the Fusion and Cleavage (F/CL)
domains (a.a. 487-520), the interspace (IS) between Heptad (H) 1
and 2 (a.a.577-605), and the intracellular region. Regions
important for oligomer formation may be partially functional.
Heptad(H) 1, Heptad 2 and their Interspace(IS) are required for
oligomerization. The expression vector backbone is pVR1012x/s
(VRC2000).
Gag-Pol Plasmids
VRC3900
[0645] pVR1012x/s Gag/h
[0646] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HXB2 (GenBank accession number K03455) was used
to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HXB2 gene, but
the protein encoded is the same. The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein to produce
pGag(fs)Pol/h (VRC4200). The protein sequence of the pol
polyprotein (amino acids 3-1003) from NL4-3 (GenBank accession
number M19921) was used to create a synthetic version of the pol
gene using codons optimized for expression in human cells. To
produce a gene that expresses all of the gag proteins, the region
encoding pol amino acids 77-1003 were deleted from Gag(fs)Pol/h to
produce Gag/h. This construct also encodes most of the protease (98
amino acids) gene encoding amino acids 3-77. Gag/h is expressed
from the pVR1012x/s vector backbone.
VRC3901
[0647] pVR1012x/s SIV Gag/h
[0648] The protein sequence of the gag polyprotein (amino acid from
1-550) SIVmac239 (GenBank accession number M33262) was used to
create a synthetic version of the gag gene using codons optimized
for expression in human cells. The nucleotide sequence of the
synthetic gag gene shows little homology to the SIVMac239 gene, but
the protein encoded is the same.
VRC4000
[0649] pVR1012x/s Gag-Pol/h
[0650] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HXB2 (GenBank accession number K03455) was used
to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HXB2 gene, but
the protein encoded is the same. The synthetic gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein. The protein
sequence of the pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the pol gene (Pol/h) using codons optimized for
expression in human cells. Gag-Pol/h is expressed from the
pVR1012x/s vector backbone.
VRC4001
[0651] pVR1012x/s SIVGag-Pol/h
[0652] Eukaryotic vector with humanized codons expressing the
Gag-pol gene of SIVmac239. The Sal 1-XbaI fragment of SIV
gag(VRC3901) was inserted into Sal 1-XbaI of SIV Pol(VRC4101) to
create VRC4001.
VRC4100
[0653] pVR1012x/s Pol/h
[0654] The protein sequence of the pol polyprotein (amino acids
3-1003) from NL4-3 (GenBank accession number M19921) was used to
create a synthetic version of the pol gene using codons optimized
for expression in human cells. To initiate translation at the
beginning of Pol, a methionine codon was added to the 5'-end of the
synthetic polymerase gene to create the Pol/h gene. Pol/h is
expressed from the pVR1012x/s (VRC2000) vector backbone.
VRC4101
[0655] pVR1102x/s SIV Pol/h
[0656] The protein sequence of the pol polyprotein (amino acids
3-1017) from SIVmac239 (GenBank accession number M19921) was used
to create a synthetic version of the pol gene using codons
optimized for expression in human cells. To initiate translation at
the beginning of Pol, a methionine codon was added to the 5'-end of
the synthetic polymerase gene to create the Pol/h gene. The
Protease (Pr) mutation is at pol amino acid 123 and is AAG->GGA
or amino acids R->G. Reverse transcriptase(RT) mutation is at aa
352(GAC to CAT D(D to H)-RT) and Integrase mutation is at aa
788(GAC to GGC(D to A)-IN). Pol/h is expressed from the pVR1012x/s
(VRC2000) vector backbone.
VRC4200
[0657] pVR1012x/s Gag(fs)Pol/h
[0658] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HXB2 (GenBank accession number K03455) was used
to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HXB2 gene, but
the protein encoded is the same. The synthetic gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein. The protein
sequence of the pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the pol gene (Pol/h) using codons optimized for
expression in human cells. To create the possibility for
translational frameshifting as means to express the gag-pol
polyprotein, the synthetic coding region for the last four amino
acids of the NC protein through the rest of gag plus an additional
3 amino acids from pol were replaced with the corresponding viral
sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3
(GenBank accession number M19921). The substitution of viral for
synthetic sequences both introduces the sites required for
frameshifting and restores the ability to express all gag proteins,
including p1 and p6. Gag(fs)Pol/h is expressed from the pVR1012x/s
vector backbone.
VRC4300
[0659] pVR1012 Gag-Pol(d delta RT delta IN)/h
[0660] Eukaryotic vector with humanized codons expressing the Gag
and the frame shifted Pol genes of HIV HXB2 subtype B with
deletions in Reverse transcriptase, and Integrase regions. The
protein sequence of the gag polyprotein (Pr55, amino acids 1-432)
from HXB2 (GenBank accession number K03455) was used to create a
synthetic version of the gag gene using codons optimized for
expression in human cells. The nucleotide sequence of the synthetic
gag gene shows little homology to the HXB2 gene, but the protein
encoded is the same. The synthetic gag gene contains all of the
mature Gag proteins except for the last two that are normally
cleaved from the carboxy-terminus of the gag polyprotein, p1 and p6
(amino acids 433-500). The synthetic gag gene was ligated in frame
with sequences encoding the pol polyprotein. The protein sequence
of the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBank
accession number M19921) was used to create a synthetic version of
the pol gene (Pol/h) using codons optimized for expression in human
cells. To create the possibility for translational frameshifting as
means to express the gag-pol polyprotein, the synthetic coding
region for the last four amino acids of the NC protein through the
rest of gag plus an additional 3 amino acids from pol were replaced
with the corresponding viral sequences (nucleotides 2074-2302 on
the HXB2 genome) from NL4-3 (GenBank accession number M19921). The
substitution of viral for synthetic sequences both introduces the
sites required for frameshifting and restores the ability to
express all gag proteins, including p1 and p6 The Reverse
Transcriptase (RT) mutation is at gag-pol amino acid 771 and is
GAC->CAC or amino acids D->H. The Integrase (IN) mutation is
at gag-pol amino acid 1209 and is ACT->CAT or amino
acids-D->A. Note: This vector is not in the pVR1012x/s
backbone.
VRC4301
[0661] pVR1012x/s-Gag(FS)-Pol-delta RT IN-IRES-R5gp157-Nef
[0662] For the Gag(FS)Pol delta RT delta IN/h portion, VRC4302
pVR1012x/s Gag(fs)Pol(delta PR delta/h was used for the protein
sequence of the gag polyprotein (Pr55, amino acids 1-432) from HXB2
(GenBank accession number K03455) was used to create a synthetic
version of the gag gene using codons optimized for expression in
human cells. The nucleotide sequence of the synthetic gag gene
shows little homology to the HXB2 gene, but the protein encoded is
the same. The synthetic gag gene contains all of the mature Gag
proteins except for the last two that are normally cleaved from the
carboxy-terminus of the gag polyprotein, p1 and p6 (amino acids
433-500). The synthetic gag gene was ligated in frame with
sequences encoding the pol polyprotein. The protein sequence of the
pol polyprotein (amino acids 3-1003) from NL4-3 (GenBank accession
number M19921) was used to create a synthetic version of the pol
gene (Pol/h) using codons optimized for expression in human cells.
To create the possibility for translational frameshifting as means
to express the gag-pol polyprotein, the synthetic coding region for
the last four amino acids of the NC protein through the rest of gag
plus an additional 3 amino acids from pol were replaced with the
corresponding viral sequences (nucleotides 2074-2302 on the HXB2
genome) from NL4-3 (GenBank accession number M19921). The
substitution of viral for synthetic sequences both introduces the
sites required for frameshifting and restores the ability to
express all gag proteins, including p1 and p6. Gag(fs)Pol/h is
expressed from the pVR1012x/s vector backbone. The Reverse
Transcriptase (RT) mutation is at gag-pol amino acid 771 and is
GAC->CAC or amino acids D->H. The Integrase (IN) mutation is
at gag-pol amino acid 1209 and is ACT->CAT or amino acids
D->A. This gene has been fused to an Internal Ribosomal Entry
Site (IRES) and then fused to the R5gp157-Nef from VRC2200
pVR1012x/s R5gp157-NefDMHCDCD4/h in which the protein sequence of
the envelope polyprotein (gp160) from HXB2 (X4-tropic, GenBank
accession number K03455) was used to create a synthetic version of
the gene (X4gp160/h) using codons optimized for expression in human
cells. The nucleotide sequence X4gp160/h shows little homology to
the HXB2 gene, but the protein encoded is the same with the
following amino acid substitutions: F53L, N94D, K192S, I215N,
A224T, A346D, P470L, T7231, and S745T. To produce an R5-tropic
version of the envelope protein (R5gp160/h), the region encoding
HIV-1 envelope polyprotein amino acids 275 to 361 from X4gp160/h
(VRC3300) were replaced with the corresponding region from the BaL
strain of HIV-1 (GeneBank accession number M68893, again using
human preferred codons). The envelope-Nef fusion protein expressed
from pR5gp157-Nef/h contains the first 820 amino acids from the HIV
envelope glycoprotein (gp 157) fused to the entire mutant Nef
protein. The gene for gp157 was ligated in frame with the
full-length mutant Nef gene from pNefDMHCDCD4/h (VRC3600) to
produce pR5gp157-NefDMHCDCD4/h. The protein sequence of the Nef
protein from HIV-1 PV22 (GenBank accession number K02083) was used
to create a synthetic version of the Nef gene (Nef/h) using codons
optimized for expression in human cells. To disrupt the ability of
Nef to limit both MHC class I and CD4 expression, point mutations
were introduced into the Nef gene from pNef/h (VRC3500). The
resulting amino acids substitutions in pNefDMHCDCD4/h are: P69A,
P72A, P75A, P78A, D174A and D175A. R5gp157-NefDMHCDCD4/h is
expressed from the pVR1012x/s (VRC2000) vector backbone.
VRC4302
[0663] pVR1012 Gag(delFS)Pol(delta PR delta RT delta IN)/h
[0664] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HXB2 (GenBank accession number K03455) was used
to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HXB2 gene, but
the protein encoded is the same. The synthetic gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein. The protein
sequence of the pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the pol gene (Pol/h) using codons optimized for
expression in human cells. To create the possibility for
translational frameshifting as means to express the gag-pol
polyprotein, the synthetic coding region for the last four amino
acids of the NC protein through the rest of gag plus an additional
3 amino acids from pol were replaced with the corresponding viral
sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3
(GenBank accession number M19921). The substitution of viral for
synthetic sequences both introduces the sites required for
frameshifting and restores the ability to express all gag proteins,
including p1 and p6. The deleted Frame Shifted (delFS) has 5 T
nucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h
is expressed from the pVR1012x/s vector backbone. The Protease (PR)
mutation is at gag-pol amino acid 553 and is AGG->GGC or amino
acids R->G. The Reverse Transcriptase (RT) mutation is at
gag-pol amino acid 771 and is GAC->CAC or amino acids D->H.
The Integrase (IN) mutation is at gag-pol amino acid 1209 and is
ACT->CAT or amino acids D->A. Note: This vector is not in the
pVR1012x/s backbone.
VRC4303
[0665] pVR1012SIV Gag(delFS)Pol(delta PR delta RT delta IN)/h
[0666] Eukaryotic vector with humanized codons expressing the Gag
and the frame-shift-deleted Pol genes SIVmac239 with deletions in
the Protease, Reverse transcriptase, and Integrase regions. The 5
of Ts from 3188 to 3192 of SIV gag-pol(VRC4001) were deleted to
create VRC4303.
VRC4304
[0667] pVR1012 Gag (delFS)Pol delta PR delta RT delta IN/h
[0668] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HIV-1 C clade(GenBank accession number U52953)
was used to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HIV-1 gene, but
the protein encoded is the same. The synthetic gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein. The protein
sequence of the pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the pol gene (Pol/h) using codons optimized for
expression in human cells. To create the possibility for
translational frameshifting as means to express the gag-pol
polyprotein, the synthetic coding region for the last four amino
acids of the NC protein through the rest of gag plus an additional
3 amino acids from pol were replaced with the corresponding viral
sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3
(GenBank accession number M19921). The substitution of viral for
synthetic sequences both introduces the sites required for
frameshifting and restores the ability to express all gag proteins,
including p1 and p6. The deleted Frame Shifted (delFS) has 5 T
nucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h
is expressed from the pVR1012x/s vector backbone. The Protease (PR)
mutation is at gag-pol amino acid 553 and is AGG->GGC or amino
acids R->G. The Reverse Transcriptase (RT) mutation is at
gag-pol amino acid 771 and is GAC->CAC or amino acids D->H.
The Integrase (IN) mutation is at gag-pol amino acid 1209 and is
ACT->CAT or amino acids D->A. Note: This vector is not in the
pVR1012x/s backbone.
VRC4305
[0669] pVR1012 Gag-A(delFS)Pol(delta PR delta RT delta IN)/h
[0670] Eukaryotic vector with humanized-codons expressing the Gag
and the frame shifted Pol genes of HIV HIV-1A clade with deletions
in the Protease, Reverse transcriptase, and Integrase regions.
VRC4305 pVR1012 Gag(-AdelFS)Pol(delta PR delta RT delta IN)/h The
protein sequence of the gag polyprotein (Pr55, amino acids 1-432)
from HIV-1 A clade (GenBank accession number AF004885) was used to
create a synthetic version of the gag gene using codons optimized
for expression in human cells. The nucleotide sequence of the
synthetic gag gene shows little homology to the HIV-1 gene, but the
protein encoded is the same. The synthetic gag gene contains all of
the mature Gag proteins except for the last two that are normally
cleaved from the carboxy-terminus of the gag polyprotein, p1 and p6
(amino acids 433-500). The synthetic gag gene was ligated in frame
with sequences encoding the pol polyprotein. The protein sequence
of the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBank
accession number M19921) was used to create a synthetic version of
the pol gene (Pol/h) using codons optimized for expression in human
cells. To create the possibility for translational frameshifting as
means to express the gag-pol polyprotein, the synthetic coding
region for the last four amino acids of the NC protein through the
rest of gag plus an additional 3 amino acids from pol were replaced
with the corresponding viral sequences (nucleotides 2074-2302 on
the HXB2 genome) from NL4-3 (GenBank accession number M19921). The
substitution of viral for synthetic sequences both introduces the
sites required for frameshifting and restores the ability to
express all gag proteins, including p1 and p6. The deleted Frame
Shifted (delFS) has 5 T nucleotides deleted between the Gag and Pol
sequences. Gag(fs)Pol/h is expressed from the pVR1012x/s vector
backbone. The Protease (PR) mutation is at gag-pol amino acid 553
and is AGG->GGC or amino acids R->G. The Reverse
Transcriptase (RT) mutation is at gag-pol amino acid 771 and is
GAC->CAC or amino acids D->H. The Integrase (IN) mutation is
at gag-pol amino acid 1209 and is ACT->CAT or amino acids
D->A. Note: This vector is not in the pVR1012x/s backbone.
pVRC4306 pVR1012 Gag(delFS)Pol delta PR delta RT delta IN/Nef/h
[0671] The protein sequence of the Gag polyprotein (Pr55, amino
acids 1-432) from HXB2 (GenBank accession number K03455) was used
to create a synthetic version of the Gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic Gag gene shows little homology to the HXB2 gene, but
the protein encoded is the same. The synthetic Gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the Gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic Gag gene was ligated
in frame with sequences encoding the Pol polyprotein. The protein
sequence of the Pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the Pol gene (Pol/h) using codons optimized for
expression in human cells. To create the possibility for
translational frameshifting as means to express the Gag-Pol
polyprotein, the synthetic coding region for the last four amino
acids of the NC protein through the rest of Gag plus an additional
3 amino acids from Pol were replaced with the corresponding viral
sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3
(GenBank accession number M19921). The substitution of viral for
synthetic sequences both introduces the sites required for
frameshifting and restores the ability to express all Gag proteins,
including p1 and p6. The deleted Frame Shifted (delFS) has 5 T
nucleotides deleted between the Gag and Pol sequences. The Protease
(PR) mutation is at Gag-Pol amino acid 553 and is AGG->GGC or
amino acids R->G. The Reverse Transcriptase (RT) mutation is at
Gag-Pol amino acid 771 and is GAC->CAC or amino acids D->H.
The Integrase (IN) mutation is at Gag-Pol amino acid 1209 and is
ACT->CAT or amino acids D->A. The Nef/h gene was fused to
downstream of Pol gene of Gag(delFS)Pol(delta PR delta RT delta
IN). No loss or extra-amino acid was created by the fusion between
Nef and Pol. The ATG of Nef was preserved. The protein sequence of
the Nef protein from HIV-1 PV22 (GenBank accession number K02083)
was used to create a synthetic version of the Nef gene (Nef/h)
using codons optimized for expression in human cells. The
nucleotide sequence Nef/h shows little homology to the viral gene,
but the protein encoded is the same. Note: This vector is not in
the pVR1012x/s backbone.
VRC 4308
VRC4302-myr
[0672] pVR1012 Gag (delFS) Pol deltaPR deltaRT deltaIN
deltaMyr/h
[0673] The myristylation site was deleted from pVRC4302. The
protein sequence of the gag polyprotein (Pr55, amino acids 1-432)
from HXB2 (GenBank accession number K03455) was used to create a
synthetic version of the gag gene using codons optimized for
expression in human cells. The nucleotide sequence of the synthetic
gag gene shows little homology to the HXB2 gene, but the protein
encoded is the same. The synthetic gag gene contains all of the
mature Gag proteins except for the last two that are normally
cleaved from the carboxy-terminus of the gag polyprotein, p1 and p6
(amino acids 433-500). The synthetic gag gene was ligated in frame
with sequences encoding the pol polyprotein. The protein sequence
of the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBank
accession number M19921) was used to create a synthetic version of
the pol gene (Pol/h) using codons optimized for expression in human
cells. To create the possibility for translational frameshifting as
means to express the gag-pol polyprotein, the synthetic coding
region for the last four amino acids of the NC protein through the
rest of gag plus an additional 3 amino acids from pol were replaced
with the corresponding viral sequences (nucleotides 2074-2302 on
the HXB2 genome) from NL4-3 (GenBank accession number M19921). The
substitution of viral for synthetic sequences both introduces the
sites required for frameshifting and restores the ability to
express all gag proteins, including p1 and p6. The deleted Frame
Shifted (delFS) has 5 T nucleotides deleted between the Gag and Pol
sequences. Gag(fs)Pol/h is expressed from the pVR1012x/s vector
backbone. The Protease (PR) mutation is at gag-pol amino acid 553
and is AGG->GGC or amino acids R->G. The Reverse
Transcriptase (RT) mutation is at gag-pol amino acid 771 and is
GAC->CAC or amino acids D->H. The Integrase (IN) mutation is
at gag-pol amino acid 1209 and is ACT->CAT or amino acids
D->A. The myristylation site was deleted. Note: This vector is
not in the pVR1012x/s backbone.
VRC 4309
[0674] pVR1012 Gag (delFS) Pol deltaPR deltaRT deltaIN delta
Myr/Nef/h
[0675] The myristylation site was deleted from pVRC4306. The
protein sequence of the gag polyprotein (Pr55, amino acids 1-432)
from HXB2 (GenBank accession number K03455) was used to create a
synthetic version of the gag gene using codons optimized for
expression in human cells. The nucleotide sequence of the synthetic
gag gene shows little homology to the HXB2 gene, but the protein
encoded is the same. The synthetic gag gene contains all of the
mature Gag proteins except for the last two that are normally
cleaved from the carboxy-terminus of the gag polyprotein, p1 and p6
(amino acids 433-500). The synthetic gag gene was ligated in frame
with sequences encoding the pol polyprotein. The protein sequence
of the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBank
accession number M19921) was used to create a synthetic version of
the pol gene (Pol/h) using codons optimized for expression in human
cells. To create the possibility for translational frameshifting as
means to express the gag-pol polyprotein, the synthetic coding
region for the last four amino acids of the NC protein through the
rest of gag plus an additional 3 amino acids from pol were replaced
with the corresponding viral sequences (nucleotides 2074-2302 on
the HXB2 genome) from NL4-3 (GenBank accession number M19921). The
substitution of viral for synthetic sequences both introduces the
sites required for frameshifting and restores the ability to
express all gag proteins, including p1 and p6. The deleted Frame
Shifted (delFS) has 5 T nucleotides deleted between the Gag and Pol
sequences. Gag(fs)Pol/h is expressed from the pVR1012x/s vector
backbone. The Protease (PR) mutation is at gag-pol amino acid 553
and is AGG->GGC or amino acids R->G. The Reverse
Transcriptase (RT) mutation is at gag-pol amino acid 771 and is
GAC->CAC or amino acids D->H. The Integrase (IN) mutation is
at gag-pol amino acid 1209 and is ACT->CAT or amino acids
D->A. The stop codon TAG was removed and synthetic B clade Nef
(Genbank access number) was fusion to the 3' end of pol by PCR.
Note: This vector is not in the pVR1012x/s backbone.
VRC 4310
[0676] pVR1012Nef Gag (del fs) (del Myr) Pol (delta PR delta RT
delta IN)/h
[0677] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HXB2 (GenBank accession number K03455) was used
to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HXB2 gene, but
the protein encoded is the same. The synthetic gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein. The protein
sequence of the pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the pol gene (Pol/h) using codons optimized for
expression in human cells. To create the possibility for
translational frameshifting as means to express the gag-pol
polyprotein, the synthetic coding region for the last four amino
acids of the NC protein through the rest of gag plus an additional
3 amino acids from pol were replaced with the corresponding viral
sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3
(GenBank accession number M19921). The substitution of viral for
synthetic sequences both introduces the sites required for
frameshifting and restores the ability to express all gag proteins,
including p1 and p6. The deleted Frame Shifted (delFS) has 5 T
nucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h
is expressed from the pVR1012x/s vector backbone. The Protease (PR)
mutation is at gag-pol amino acid 553 and is AGG->GGC or amino
acids R->G. The Reverse Transcriptase (RT) mutation is at
gag-pol amino acid 771 and is GAC->CAC or amino acids D->H.
The Integrase (IN) mutation is at gag-pol amino acid 1209 and is
ACT->CAT or amino acids D->A. The stop codon TAG of synthetic
B clade Nef (Genbank access number) gene was removed and was fused
to the 5' end of gene by PCR. Note: This vector is not in the
pVR1012x/s backbone.
VRC 4311
[0678] pVR1012 Gag-C(delFS)Pol(delta PR delta RT delta IN)
Nef/h
VRC4304+Clade C Nef
[0679] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HIV-1 C clade(GenBank accession number U52953)
was used to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HIV-1 gene, but
the protein encoded is the same. The synthetic gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein. The protein
sequence of the pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the pol gene (Pol/h) using codons optimized for
expression in human cells. To create the possibility for
translational frameshifting as means to express the gag-pol
polyprotein, the synthetic coding region for the last four amino
acids of the NC protein through the rest of gag plus an additional
3 amino acids from pol were replaced with the corresponding viral
sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3
(GenBank accession number M19921). The substitution of viral for
synthetic sequences both introduces the sites required for
frameshifting and restores the ability to express all gag proteins,
including p1 and p6. The deleted Frame Shifted (delFS) has 5 T
nucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h
is expressed from the pVR1012x/s vector backbone. The Protease (PR)
mutation is at gag-pol amino acid 553 and is AGG->GGC or amino
acids R->G. The Reverse Transcriptase (RT) mutation is at
gag-pol amino acid 771 and is GAC->CAC or amino acids D->H.
The Integrase (IN) mutation is at gag-pol amino acid 1209 and is
ACT->CAT or amino acids D->A. The stop codon TAG was removed
and synthetic C clade Nef (Genbank accession number: U52953) was
fusion to the 3' end of pol by PCR. Note: This vector is not in the
pVR1012x/s backbone.
VRC 4312
Gag (del fs) (del Myr) Nef Pol .DELTA.PR.DELTA.RT.DELTA.IN/h
VRC-Myr-gag-Dnef-Dpol
[0680] (Eukaryotic Vector with Humanized Codons Expressing the Gag,
Truncated Nef, and Truncated Pol Proteins of HIV Subtype B.Ns.)
[0681] This construct was derived from VRC4302. The protein
sequence of the gag polyprotein (Pr55, amino acids 1-432) from HXB2
(GenBank accession number K03455) was used to create a synthetic
version of the gag gene using codons optimized for expression in
human cells. The nucleotide sequence of the synthetic gag gene
shows little homology to the HXB2 gene, but the protein encoded is
the same. The synthetic gag gene contains all of the mature Gag
proteins except for the last two that are normally cleaved from the
carboxy-terminus of the gag polyprotein, p1 and p6 (amino acids
433-500). The synthetic gag gene was ligated in frame with
sequences encoding synthetic nef gene that 51 aa were deleted from
5'. 77 aa were deleted from 5' of pol polyprotein, and ligated with
3' of nef in which tag stop codon was deleted. The protein sequence
of the pol polyprotein (amino acids 3-78-1003) from NL4-3 (GenBank
accession number M19921) was used to create a synthetic version of
the pol gene (Pol/h) using codons optimized for expression in human
cells. To create the possibility for translational frameshifting as
means to express the gag-pol polyprotein, the synthetic coding
region for the last four amino acids of the NC protein through the
rest of gag plus an additional 3 amino acids from pol were replaced
with the corresponding viral sequences (nucleotides 2074-2302 on
the HXB2 genome) from NL4-3 (GenBank accession number M19921). The
substitution of viral for synthetic sequences both introduces the
sites required for frameshifting and restores the ability to
express all gag proteins, including p1 and p6. The deleted Frame
Shifted (delFS) has 5 T nucleotides deleted between the Gag and Pol
sequences. Gag(fs)Pol/h is expressed from the pVR1102x/s vector
backbone. The Protease (PR) mutation is at gag-pol amino acid 553
and is AGG->GGC or amino acids R->G. The Reverse
Transcriptase (RT) mutation is at gag-pol amino acid 771 and is
GAC->CAC or amino acids D->H. The Integrase (IN) mutation is
at gag-pol amino acid 1209 and is ACT->CAT or amino acids
D->A. Note: This vector is not in the pVR012x/s backbone.
VRC 4313
[0682] pVR1012 Gag Clade A (del fs)Pol(.DELTA. PR .DELTA. RT
.DELTA. IN)/h
VRC4305+Clade A Nef
[0683] The protein sequence of the gag polyprotein (Pr55, amino
acids 1-432) from HIV-1 A clade (GenBank accession number AF004885)
was used to create a synthetic version of the gag gene using codons
optimized for expression in human cells. The nucleotide sequence of
the synthetic gag gene shows little homology to the HIV-1 gene, but
the protein encoded is the same. The synthetic gag gene contains
all of the mature Gag proteins except for the last two that are
normally cleaved from the carboxy-terminus of the gag polyprotein,
p1 and p6 (amino acids 433-500). The synthetic gag gene was ligated
in frame with sequences encoding the pol polyprotein. The protein
sequence of the pol polyprotein (amino acids 3-1003) from NL4-3
(GenBank accession number M19921) was used to create a synthetic
version of the pol gene (Pol/h) using codons optimized for
expression in human cells. To create the possibility for
translational frameshifting as means to express the gag-pol
polyprotein, the synthetic coding region for the last four amino
acids of the NC protein through the rest of gag plus an additional
3 amino acids from pol were replaced with the corresponding viral
sequences (nucleotides 2074-2302 on the HXB2 genome) from NL4-3
(GenBank accession number M19921). The substitution of viral for
synthetic sequences both introduces the sites required for
frameshifting and restores the ability to express all gag proteins,
including p1 and p6. The deleted Frame Shifted (delFS) has 5 T
nucleotides deleted between the Gag and Pol sequences. Gag(fs)Pol/h
is expressed from the pVR1012x/s vector backbone. The Protease (PR)
mutation is at gag-pol amino acid 553 and is AGG->GGC or amino
acids R->G. The Reverse Transcriptase (RT) mutation is at
gag-pol amino acid 771 and is GAC->CAC or amino acids D->H.
The Integrase (IN) mutation is at gag-pol amino acid
[0684] 1209 and is ACT->CAT or amino acids D->A. The stop
codon TAG was removed and synthetic A clade Nef (Genbank accession
number:AF069670) was fused to the 3' end of pol by PCR. Note: This
vector is not in the pVR1012x/s backbone.
EXAMPLES
Env
Immunogens
[0685] Plasmids expressing the CXCR4-tropic HIV-1 HXB2 Env were
made synthetically with sequences designed to disrupt viral RNA
structures that limit protein expression by using codons typically
found in human cells. Briefly, the synthetic Env gene of HXB2
(GenBank accession number K03455) was generated in three fragments
by assembling the overlapping synthetic oligonucleotides using PCR
amplification. To produce a CCR5-tropic version of the HIV-1
envelope, the region encoding amino acids 275 to 361 of HXB2
(CXCR4-tropic) gp160 was replaced with CCR5-tropic HIV-1 BaL
sequence (GenBank accession number M68893), which includes the V3
loop. Glycosylation mutants were generated by site-directed
mutagenesis to replace asparagine with glutamic acid residues at
seventeen conserved glycosylation sites between amino acids 88 and
448. To express truncation mutant Env proteins, stop codons were
introduced after positions 752, 704, 680 or 592 to produce gp150,
gp145, gp140, or gp128, respectively. The Env protein was further
changed by deleting amino acids 503 to 537 and 593 to 619, which
removes the cleavage site sequence, the fusion domain, and a part
of the spacer between the two heptad repeats. The structures of the
synthetic HIV envelope genes are shown (FIG. 178). The cDNAs were
cloned in the expression vector pNGVL or pVR1012 under the control
of the cytomegalovirus immediate-early enhancer, promoter, and
first intron. The protein sequence was identical to HXB2 Env except
for the following amino acid substitutions: F53L, N94D, K192S,
I215N, A224T, A346D, P470L, T7231, and S745T.
Expression of Envelope Proteins in Transfected Cells
[0686] 293 cells (10.sup.6) were plated in 60 mm dishes. Cells were
transfected on the following day with 2 .mu.g of plasmid using
calcium phosphate. Cells were harvested 48 hours after transfection
and lysed in buffer containing 50 mM HEPES pH 7.0, 250 mM NaCl and
0.5% NP40. The protein concentration in the lysates was determined
using the Bradford reagent (Bio-Rad). Proteins (25 .mu.g) in
lysates were separated by 7.5% SDS-PAGE and transferred to
Immobilon-P membrane (Millipore, Bedford, Mass.). Env was detected
by immunoprecipitation followed by Western blotting using
polyclonal antibody against gp160 (Intracel, Rockville, Md.).
Cell Surface Expression of Envelope by FACS Analysis
[0687] 293 cells were harvested 48 hours after transfection and
washed twice with phosphate-buffered saline (PBS) containing 1%
bovine serum albumin and incubated for 30 minutes on ice with
polyclonal immunoglobulin from an HIV-1 infected patient. The
secondary antibody against human IgG conjugated with FITC (Jackson
Immuno Research) was added, incubated for 30 minutes on ice, washed
3 times with PBS, and analyzed by flow cytometry (FACScan). The
median fluorescent intensity values were derived using Cell Quant
software.
DNA Injection in Mice
[0688] Six week old, female BALB/c mice were injected
intramuscularly with 100 .mu.g of purified plasmid DNA suspended in
200 .mu.l of normal saline. For each plasmid DNA, a group of 4 mice
was injected three times at intervals of two weeks. The mice were
bled two weeks after the last injection, sera collected and stored
at 4.degree. C.
Quantitation of the Antibody Response
[0689] Immunoprecipitation and Western blotting was used to detect
the antibodies that bind to native envelope proteins. Sera from
immunized mice were used to immunoprecipitate gp160 from cell
lysates of gp160-transfected 293 cells. Indicated dilutions were
used to immunoprecipitate gp160 from the cell lysate (400 .mu.g).
Immunocomplexes were separated by 7.5% SDS-PAGE and analyzed by
immunoblotting using the polyclonal antibody against gp 160.
Analysis of CTL Response
[0690] Spleens were removed aseptically and gently homogenized to a
single cell suspension, washed, and resuspended to a final
concentration of 5.times.10.sup.7 cells/ml. Cells were incubated
for 7 days in presence of IL-2 (10 U/ml) and either irradiated
peptide-pulsed splenocytes from naive mice or an irradiated stable
cell line expressing the full length gp160 BC-env/rev. Three types
of target cells were used: peptide-pulsed P815 cells (ATCC TIB64),
BC10ME cells stably expressing gp160, and BC10ME cells pulsed with
peptides derived from gp160 sequence. Target cells were labeled
with .sup.51Cr for 90 minutes and washed three times with RPMI-1640
with 10% FBS, 2 mM glutamine, 5.times.10.sup.- .sup.5M
.beta.-mercaptoethanol and fungisone, 250units/ml, and resuspended
in this media. Cytolytic activity was determined in triplicate
samples using all different target cell dilutions in a 5-hour
.sup.51Cr-release assay.
Gag and Pol
Development of Synthetic HIV-1 Gag-Pol Expression Vectors
[0691] The protein sequences of Gag (amino acids 1-432) from HXB2
(GenBank accession number K03455) and Pol (amino acids 3-1003) from
NL4-3 (GenBank accession number M19921) were reverse translated by
the GCG Package (Genetic Computer Group, Inc., Madison, Wis.) using
codons expected for human cells. A 226-bp fragment spanning the
frame shift site and the overlapping region of the two reading
frames from NL4-3 were retained to allow expression of Gag and
Gag-Pol precursor polyproteins in the same construct. 86
oligonucleotides covering 4325 DNA base pairs with 5' SalI and 3'
EcoRI sites were purchased from GIBCO Life Technologies. Each of
the oligonucleotides was 75 base-pairs with 25 nt of overlap. The
codon optimized Gag-Pol gene (hGag-Pol) was assembled by PCR with
Pwo (Boehringer Mannheim) and Turbo Pfu (Stratagene) high fidelity
DNA polymerase. The PCR conditions were optimized with a PCR
optimization kit (Stratagene) on a gradient Robocycler
(Stratagene). Full-length synthetic Gag-Pol gene was cloned into
the Sal I and blunted Bgl II site of the mammalian expression
vector pNGVL-3 (Xu, Ling et al., 1998, Nature Medicine, 4:37-42)
and confirmed by DNA sequencing. Three additional constructs were
derived from the hGag-Pol gene. 5 Thymidines (Ts) in the frame
shift site (FS) of the hGag-Pol gene were deleted (.DELTA.FS) and
the protease was inactivated by replacing AGG in protease to
GGC(R42G) to create hGag-Pol.DELTA.FS.DELTA.Pr. 432 amino acids of
the NH2-terminal of hGag-Pol gene were deleted and an ATG start
codon was added to create the hPol gene. 925 amino acids of the
COOH-terminal hGag-Pol were deleted to create the hGag gene.
hGagPol.DELTA.FS.DELTA.Pr, hPol and hGag genes were expressed in
the pNGVL-3 plasmid, derived by insertion of a polylinker into
pVR1012. The plasmid expressing viral Gag-Pol, pCMV.DELTA.8.2 was a
kind gift.
Transient Transfection and Analysis of Expression
[0692] 293T cells were maintained in Dulbecco's modified Eagle
medium (DMEM; GIBCO-BRL), supplemented with 10% fetal bovine serum
(FBS). Plasmid DNAs were purified with double cesium chloride
sedimentation gradients. Approximately 3.times.10.sup.6 293T cells
were placed in a 10-cm dish one day before transfection. 10 .mu.g
of pCMVdR8.2 plasmid (containing the viral gag-pol gene), or 5 pg
of pVR1012s (containing the codon altered genes), were used to
transfect 293T cells, using the calcium phosphate method. Three
days after transfection, cell lysates were prepared with RIPA
buffer (Boehringer Mannheim, Indianapolis, Ind.) and separated by
4-15% gradient SDS-polyacrylamide gel electrophoresis (PAGE), then
transferred onto an Immobilon P membrane (Millipore). Membranes
were then incubated with anti HIV-1-IgG (AIDS Research and
Reference Reagent Program), monoclonal anti-p24 (ICN), or rabbit
anti-RT (Intracel, Rockville, Md.). Bands were visualized using the
ECL Western blotting detection reagent (Amersham Pharmacia Biotech,
Piscataway, N.J.), as described by the manufacturer. Expression
levels were determined using a phosphorimager.
Generation of Stably Transfected Cell Lines
[0693] hGag and hPol genes were individually subcloned into the Xho
I and EcoRI sites of a retroviral vector, pPGS-CITE-Neo. Three
plasmid systems were used to produce recombinant retroviruses
containing the hGag or hPol genes. 48 hours after transfection, the
supernatants were collected to transduce CT26 and BC10ME which are
syngeneic to Balb/C mice, and selected in 0.8 mg/ml of G418 two
days after infection. The positive clones were screened and
confirmed by Western blotting and maintained in 10% FCS
supplemented RPMI (GIBCO-BRL) with 0.5 mg/ml G418.
DNA Vaccination of Mice
[0694] Female BALB/C mice, 6-8 weeks old, were used for
immunogenicity studies. For DNA vaccination, mice were immunized
with 100 .mu.l (0.5 .mu.g/ml DNA and 0.9% NaCl) in the quadriceps
muscle of each hind leg every two weeks for a total of 4
injections.
CTL Assay
[0695] Animals were sacrificed after the last immunization and
spleens removed from both naive and immunized mice using aseptic
techniques. Splenic lymphocytes were harvested and the chromium
release CTL assay was performed in triplicate as previously
described. The peptides used for sensitizing cells are as follows:
Two peptide mixtures from the Gag protein P17(88-115) and
p24(62-76). Seven peptide mixtures from the Pol protein: 1)
P66(175-189), 2) P66(179-193), 3) P66(183-197), 4) P66(187-201), 5)
P66(223-237), 6) P66(227-241), 7) P66(367-381).
Measurement of Antibody Responses
[0696] Anti-p24 ELISA assays
[0697] The anti-p24 ELISA assay was performed in Immunlon
ninety-six-well plates (Dynet Technologies Inc., Chantilly, Va.).
The plates were coated with 50 .mu.l of purified recombinant
HIV-11IIB p24 antigen (Intracel) at a concentration of 2 .mu.g/ml
in PBS buffer, pH 7.4 (GIBCO) with 0.05% sodium azide. Plates were
washed 3.times. in PBS containing 3% BSA and 0.05% Tween20
(blocking buffer) and incubated for 2 hours. Mouse sera were
serially diluted from 1:100 to 1:12,800 in blocking buffer, added
to the p24-coated plates and incubated overnight at 4.degree. C.
Plates were then washed four times with PBS (0.05% Tween 20), and
incubated with goat anti-mouse IgG (1:10,000 dilution, Sigma) for 2
hours at room temperature. Plates were washed four times, and then
pNPP alkaline phosphatase substrate (75 .mu.l; Sigma, St. Louis,
Mo.) was added to each well. The reaction was stopped after one
hour by addition of 0.5 N NaOH (25 .mu.l). The plates were read on
an ELISA reader at 405 nm, and titers were calculated at a cutoff
optical density of 0.4.
HIV-1 immunoblotting
[0698] The strips containing HIV-1 proteins (Immunectics Inc.,
Cambridge, Mass.) were incubated with pooled mouse sera at a
dilution of 1:25. Purified human anti-HIV IgG (AIDS Research and
Reference Reagent Program, Rockville, Md.) was used as a positive
control. Bands were visualized using the ECL Western blotting
detection reagent (Amersham Pharmacia Biotech, Piscataway,
N.J.).
Immunoprecipitation and Western Blotting
[0699] Three days after transfection, hPol gene-transfected 293T
cell lysates were prepared with RIPA buffer. The pooled mouse sera
were diluted with immunoprecipitation (IP) buffer (100 mM KCl, 2.5
mM MgCl.sub.2, 20 mM HEPES, pH 7.9, 0.1% NP-40, 1 mM DTT and
proteinase inhibitors). After adding 10 Hg of the cell lysate
containing the HIV-1 Pol protein, the reactions were incubated
overnight on a rotator at 4.degree. C. 250 .mu.l of Protein G and A
Sepharose beads (10% V/V in IP buffer) were then added, and the
reactions were incubated for 2 hours on a rotator at 4.degree. C.
The beads were washed four times with IP buffer, resuspended in 30
.mu.l of 1.times. sample buffer and loaded onto SDS-PAGE. After
transfer onto an Immobilon P membrane (Millipore), membranes were
incubated with anti HIV-1-IgG (AIDS Research and Reference Reagent
Program). Bands were visualized using the ECL Western blotting
detection reagent (Amersham Pharmacia Biotech).
Prime/Boost Vaccination Strategy
[0700] Groups of guinea pigs (4/group) were immunized (primed) with
the vectors listed below, 3 times at 2 week intervals. Two weeks
after the third DNA immunization, blood was collected for immune
analysis. 2-3 days after the blood collection, the animals received
a boost with a corresponding adenoviral construct, AdApt (Sullivan,
N. et al., 2000, Nature, 408:605-608). Blood was again collected 2
weeks after the adenoviral boost. Blood was tested for neutralizing
antibodies and (using an ELISA) the presence of antibody against
the envelope. The groups are summarized below.
TABLE-US-00003 Prime Adenoviral Boost gp140delCFI (R5) Adv
gp140delCFI (R5) gp145delCFI (R5) Adv gp145delCFI (R5) gp145delCFI
(R5) Adv gp140delCFI (R5) gp140delCFI (89.6P) Adv gp140delCFI
(89.6P) gp145delCFI (89.6P) Adv gp145delCFI (89.6P) gp145delCFI
(89.6P) Adv gp140delCFI (89.6P) Adv gp140delCFI (R5) Adv
gp140delCFI (R5) Adv gp145delCFI (R5) Adv gp145delCFI (R5) Adv
gp145delCFI (R5) Adv gp140delCFI (R5) Adv gp140delCFI (89.6P) Adv
gp140delCFI (89.6P) Adv gp145delCFI (89.6P) Adv gp145delCFI (89.6P)
Adv gp145delCFI (89.6P) Adv gp140delCFI (89.6P)
Results
[0701] The level of antibody in the sera after adenoviral boost was
8- to 10-fold higher than the level of antibody present after 3
injections of DNA alone, in almost all of the cases. Neutralizing
antibodies were detected in the sera of animals immunized with
either gp145delCFI DNA or gp140delCFI DNA followed by
Advgp140delCFI.
[0702] Neutralization was also observed in animals immunized with
gp145delCFI DNA followed by Adv gp145delCFI, and in animals both
primed and boosted with adenovirus.
[0703] Optimal neutralization was seen when animals were primed
with gp145delCFI DNA followed by adenovirus expressing gp140delCFI
in both lade B (R5-Bal strain) and 89.6P (Clade B, dual tropic
strain).
[0704] All neutralizing activity was able to be blocked with the V3
peptide of the corresponding strains.
[0705] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
figures, tables, and appendices, as well as patents, applications
and publications referred to above are hereby incorporated by
reference.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090175910A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090175910A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References