U.S. patent application number 11/975643 was filed with the patent office on 2011-06-30 for mva expressing modified hiv envelope, gag, and pol genes.
Invention is credited to Patricia L. Earl, Leigh Anne Eller, Matthew Edward Harris, Bernard Moss, Thomas C. VanCott, Linda Wyatt.
Application Number | 20110159036 11/975643 |
Document ID | / |
Family ID | 33131868 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110159036 |
Kind Code |
A1 |
Moss; Bernard ; et
al. |
June 30, 2011 |
MVA expressing modified HIV envelope, GAG, and POL genes
Abstract
The invention provides modified virus Ankara (MVA), a
replication-deficient strain of vaccinia virus, expressing human
immunodeficiency virus (HIV) env, gag, and pol genes.
Inventors: |
Moss; Bernard; (Bethesda,
MD) ; Earl; Patricia L.; (Chevy Chase, MD) ;
Wyatt; Linda; (Rockville, MD) ; Eller; Leigh
Anne; (Kampala, UG) ; VanCott; Thomas C.;
(Brookeville, MD) ; Harris; Matthew Edward;
(Poway, CA) |
Family ID: |
33131868 |
Appl. No.: |
11/975643 |
Filed: |
October 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11238155 |
Sep 28, 2005 |
7303754 |
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11975643 |
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PCT/US2004/009906 |
Mar 29, 2004 |
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11238155 |
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60459175 |
Mar 28, 2003 |
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Current U.S.
Class: |
424/205.1 ;
424/93.2; 435/235.1; 435/320.1 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2710/24143 20130101; C12N 2740/16122 20130101; C12N 2830/15
20130101; C12N 2740/16222 20130101; C12N 2830/00 20130101; A61K
2039/53 20130101; C12N 15/86 20130101; A61K 2039/57 20130101; C12N
2830/60 20130101; A61K 2039/5256 20130101 |
Class at
Publication: |
424/205.1 ;
424/93.2; 435/320.1; 435/235.1 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 35/76 20060101 A61K035/76; C12N 15/63 20060101
C12N015/63; C12N 7/04 20060101 C12N007/04 |
Claims
1. A pharmaceutical composition comprising a recombinant MVA virus
expressing an HIV env, gag, and pol gene or modified gene thereof
for production of an HIV Env, Gag, and Pol antigen by expression
from said recombinant MVA virus, wherein said HIV env gene is
modified to encode an HIV Env protein composed of gp120 and the
membrane-spanning and ectodomain of gp41 but lacking part or all of
the cytoplasmic domain of gp41, and a pharmaceutically acceptable
carrier, wherein said HIV env, gag, and pol genes are isolatable
from an individual infected with Ugandan clade D isolate
99UGA03349, 99UGA07412, or 98UG57128.
2. The pharmaceutical composition of claim 1 comprising 99UGA03349
gagpol in Appendix 1 or sequence having at least about 90%, 95% or
99.9% identity thereto.
3. The pharmaceutical composition of claim 1 comprising 99UGA07412
gagpol in Appendix 1 or sequence having at least about 90%, 95% or
99.9% identity thereto.
4. The pharmaceutical composition of claim 1 comprising 99UGA03349
envelope in Appendix 1 or sequence having at least about 90%, 95%
or 99.9% identity thereto.
5. The pharmaceutical composition of claim 1 comprising 99UGA07412
envelope in Appendix 1 or sequence having at least about 90%, 95%
or 99.9% identity thereto.
6. The pharmaceutical composition of claim 1 comprising 98UG57128
envelope in Appendix 1 or sequence having at least about 90%, 95%
or 99.9% identity thereto.
7. The pharmaceutical composition of claim 1, wherein said
recombinant MVA virus is MVA/UGD-1 defined as comprising 99UGA07412
gagpol in Appendix 1 or sequence having at least about 90%, 95% or
99.9% identity thereto, and 99UGA07412 envelope in Appendix 1 or
sequence having at least about 90%, 95% or 99.9% identity
thereto.
8. The pharmaceutical composition of claim 1, wherein said
recombinant MVA virus is MVA/UGD-2 defined as comprising 99UGA03349
gagpol in Appendix 1 or sequence having at least about 90%, 95% or
99.9% identity thereto, and 98UG57128 envelope in Appendix 1 or
sequence having at least about 90%, 95% or 99.9% identity
thereto.
9. The pharmaceutical composition of claim 1, wherein said
recombinant MVA virus is MVA/UGD-3 defined as comprising 99UGA07412
gagpol in Appendix 1 or sequence having at least about 90%, 95% or
99.9% identity thereto, and 99UGA03349 envelope in Appendix 1 or
sequence having at least about 90%, 95% or 99.9% identity
thereto.
10. The pharmaceutical composition of claim 1, wherein said
recombinant MVA virus is MVA/UGD-4 defined as comprising 99UGA03349
gagpol in Appendix 1 or sequence having at least about 90%, 95% or
99.9% identity thereto, and 99UGA07412 envelope in Appendix 1 or
sequence having at least about 90%, 95% or 99.9% identity
thereto.
11. The pharmaceutical composition of claim 1, wherein said
recombinant MVA virus is MVA/UGD-5 defined as comprising 99UGA03349
gagpol in Appendix 1 or sequence having at least about 90%, 95% or
99.9% identity thereto, and 98UG57128 envelope in Appendix 1 or
sequence having at least about 90%, 95% or 99.9% identity
thereto.
12. The pharmaceutical composition of claim 1 wherein said
recombinant MVA virus additionally expresses an additional HIV gene
or modified gene thereof for production of an HIV antigen by
expression from said recombinant MVA virus, wherein said additional
HIV gene is a member selected from the group consisting of vif,
vpr, tat, rev, vpu, and nef.
13. An MVA shuttle plasmid comprising pLAS-1 of Appendix 2 or
sequence having at least about 90%, 95% or 99.9% identity thereto,
or pLAS-2 of Appendix 2 or sequence having at least about 90%, 95%
or 99.9% identity thereto.
14. A method of making a recombinant MVA virus comprising preparing
the MVA shuttle plasmid of claim 13 and combining said MVA shuttle
plasmid with a MVA virus to produce said recombinant MVA virus, and
isolating said recombinant MVA virus.
15. A method of boosting a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of claim 1, whereby a
CD8.sup.+ T cell immune response to the antigen previously primed
in the primate is boosted.
16. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of claim 1, whereby a
CD8.sup.+ T cell immune response to the antigen in the primate is
induced.
17. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a priming composition comprising
nucleic acid encoding said antigen and then provision in the
primate of a boosting composition which comprises claim 1, whereby
a CD8.sup.+ T cell immune response to the antigen is induced.
18. The method of claim 15, wherein the primate is a human.
19. The method of claim 15, wherein administration of the
recombinant MVA virus is by needleless injection.
20. The method of claim 15, wherein the priming composition
comprises plasmid DNA encoding said antigen.
21. MVA 1974/NIH Clone 1.
22. A pharmaceutical composition comprising a recombinant MVA virus
expressing an HIV env, gag, and pol gene or modified gene thereof
for production of an HIV Env, Gag, and Pol antigen by expression
from said recombinant MVA virus, wherein said HIV env gene is
modified to encode an HIV Env protein composed of gp120 and the
membrane-spanning and ectodomain of gp41 but lacking part or all of
the cytoplasmic domain of gp41, and a pharmaceutically acceptable
carrier, wherein said HIV env, gag, and pol genes are isolatable
from an individual infected with Kenyan clade A isolate
00KE-KER2008, 00KE-KNH1144, or 00KE-KNH1207.
23. The pharmaceutical composition of claim 22 comprising
00KE-KNH2008 gagpol in Appendix 3 or sequence having at least about
90%, 95% or 99.9% identity thereto.
24. The pharmaceutical composition of claim 22 comprising
00KE-KNH1144 envelope in Appendix 3 or sequence having at least
about 90%, 95% or 99.9% identity thereto.
25. The pharmaceutical composition of claim 22 comprising
00KE-KNH1207 envelope in Appendix 3 or sequence having at least
about 90%, 95% or 99.9% identity thereto.
26. The pharmaceutical composition of claim 22, wherein said
recombinant MVA virus is MVA/KEA-1 defined as comprising
00KE-KNH2008 gagpol in Appendix 3 or sequence having at least about
90%, 95% or 99.9% identity thereto, and 00KE-KNH1144 envelope in
Appendix 3 or sequence having at least about 90%, 95% or 99.9%
identity thereto.
27. The pharmaceutical composition of claim 22, wherein said
recombinant MVA virus is MVA/KEA-2 defined as comprising
00KE-KNH2008 gagpol in Appendix 3 or sequence having at least about
90%, 95% or 99.9% identity thereto, and 00KE-KNH1207 envelope in
Appendix 3 or sequence having at least about 90%, 95% or 99.9%
identity thereto.
28. The pharmaceutical composition of claim 22, wherein said
recombinant MVA virus is MVA/KEA-3 defined as comprising
00KE-KNH2008 gagpol in Appendix 3 or sequence having at least about
90%, 95% or 99.9% identity thereto, and 00KE-KNH1144 envelope in
Appendix 3 or sequence having at least about 90%, 95% or 99.9%
identity thereto.
29. The pharmaceutical composition of claim 22, wherein said
recombinant MVA virus is MVA/KEA-4 defined as comprising
00KE-KNH2008 gagpol in Appendix 3 or sequence having at least about
90%, 95% or 99.9% identity thereto, and 00KE-KNH1144 envelope in
Appendix 3 or sequence having at least about 90%, 95% or 99.9%
identity thereto.
30. The pharmaceutical composition of claim 22, wherein said
recombinant MVA virus is MVA/KEA-5 defined as comprising
00KE-KNH2008 gagpol in Appendix 3 or sequence having at least about
90%, 95% or 99.9% identity thereto, and 00KE-KNH1144 envelope in
Appendix 3 or sequence having at least about 90%, 95% or 99.9%
identity thereto.
31. The pharmaceutical composition of claim 22 wherein said
recombinant MVA virus additionally expresses an additional HIV gene
or modified gene thereof for production of an HIV antigen by
expression from said recombinant MVA virus, wherein said additional
HIV gene is a member selected from the group consisting of vif,
vpr, tat, rev, vpu, and nef.
32. A method of boosting a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of claim 22, whereby a
CD8.sup.+ T cell immune response to the antigen previously primed
in the primate i2s boosted.
33. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of claim 22, whereby a
CD8.sup.+ T cell immune response to the antigen in the primate is
induced.
34. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a priming composition comprising
nucleic acid encoding said antigen and then provision in the
primate of a boosting composition which comprises claim 22, whereby
a CD8.sup.+ T cell immune response to the antigen is induced.
35. The method of claim 32, wherein the primate is a human.
36. The method of claim 32, wherein administration of the
recombinant MVA virus is by needleless injection.
37. The method of claim 32, wherein the priming composition
comprises plasmid DNA encoding said antigen.
38. A pharmaceutical composition comprising a recombinant MVA virus
expressing an HIV env, gag, and pol gene or modified gene thereof
for production of an HIV Env, Gag, and Pol antigen by expression
from said recombinant MVA virus, wherein said HIV env gene is
modified to encode an HIV Env protein composed of gp120 and the
membrane-spanning and ectodomain of gp41 but lacking part or all of
the cytoplasmic domain of gp41, and a pharmaceutically acceptable
carrier, wherein said HIV env, gag, and pol genes are isolatable
from an individual infected with Tanzanian clade C isolate
00TZA-246 or 00TZA-125.
39. The pharmaceutical composition of claim 38 comprising 00TZA-246
gagpol in Appendix 4 or sequence having at least about 90%, 95% or
99.9% identity thereto.
40. The pharmaceutical composition of claim 38 comprising 00TZA-125
envelope in Appendix 4 or sequence having at least about 90%, 95%
or 99.9% identity thereto.
41. The pharmaceutical composition of claim 38, wherein said
recombinant MVA virus is MVA/TZC-1 defined as comprising 00TZA-246
gagpol in Appendix 4 or sequence having at least about 90%, 95% or
99.9% identity thereto, and 00TZA-125 envelope in Appendix 4 or
sequence having at least about 90%, 95% or 99.9% identity
thereto.
42. The pharmaceutical composition of claim 38 wherein said
recombinant MVA virus additionally expresses an additional HIV gene
or modified gene thereof for production of an HIV antigen by
expression from said recombinant MVA virus, wherein said additional
HIV gene is a member selected from the group consisting of vif,
vpr, tat, rev, vpu, and nef.
43. A method of boosting a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of claim 38, whereby a
CD8.sup.+ T cell immune response to the antigen previously primed
in the primate is boosted.
44. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of claim 38, whereby a
CD8.sup.+ T cell immune response to the antigen in the primate is
induced.
45. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a priming composition comprising
nucleic acid encoding said antigen and then provision in the
primate of a boosting composition which comprises claim 38, whereby
a CD8.sup.+ T cell immune response to the antigen is induced.
46. The method of claim 43, wherein the primate is a human.
47. The method of claim 43, wherein administration of the
recombinant MVA virus is by needleless injection.
48. The method of claim 43, wherein the priming composition
comprises plasmid DNA encoding said antigen.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/238,155, filed Sep. 28, 2005, which is continuation of
International Patent Application No. PCT/US2004/009906, filed Mar.
29, 2004 designating the U.S. and published in English on Oct. 14,
2004 as WO 2004/087201, which claims the benefit of U.S.
Provisional Patent Application No. 60/459,175, filed Mar. 28, 2003,
all of with are hereby expressly incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The invention provides modified vaccinia Ankara (MVA), a
replication-deficient strain of vaccinia virus, expressing human
immunodeficiency virus (HIV) env, gag, and pol genes.
BACKGROUND OF THE INVENTION
[0003] Cellular immunity plays an important role in the control of
immunodeficiency virus infections (P. J. Goulder et al. 1999 AIDS
13:S121). Recently, a DNA vaccine designed to enhance cellular
immunity by cytokine augmentation successfully contained a highly
virulent immunodeficiency virus challenge (D. H. Barouch et al.
2000 Science 290:486). Another promising approach to raising
cellular immunity is DNA priming followed by recombinant poxvirus
boosters (H. L. Robinson et al. 2000 AIDS Rev 2:105). This
heterologous prime/boost regimen induces 10- to 100-fold higher
frequencies of T cells than priming and boosting with DNA or
recombinant poxvirus vaccines alone. Previously, investigators
showed that boosting a DNA-primed response with a poxvirus was
superior to boosting with DNA or protein for the control of a
non-pathogenic immunodeficiency virus (H. L. Robinson et al. 1999
Nat Med 5:526). There is a need for the control of a pathogenic
immunodeficiency virus.
SUMMARY OF THE INVENTION
[0004] Here we report that DNA priming followed by a recombinant
modified vaccinia Ankara (rMVA) booster has controlled a highly
pathogenic immunodeficiency virus challenge in a rhesus macaque
model. Both the DNA and rMVA components of the vaccine expressed
multiple immunodeficiency virus proteins. Two DNA inoculations at 0
and 8 weeks and a single rMVA booster at 24 weeks effectively
controlled an intrarectal challenge administered seven months after
the booster. These findings are envisioned as indicating that a
relatively simple multiprotein DNA/MVA vaccine can help to control
the acquired immune deficiency syndrome (AIDS) epidemic. We also
report that inoculations of rMVA induce good immune responses even
without DNA priming.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] 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.
[0006] FIG. 1. Phylogenetic relationships of HIV-1 and HIV-2 based
on identity of pol gene sequences. SIV.sub.cpz, and SIV.sub.smm are
subhuman primate lentiviruses recovered from a chimpanzee and sooty
mangabey monkey, respectively.
[0007] FIG. 2. Phylogenetic relationships of HIV-1 groups M, N and
O with four different SIV.sub.cpz, isolates based on full-length
pol gene sequences. The bar indicates a genetic distance of 0.1
(10% nucleotide divergence) and the asterisk positions group N
HIV-1 isolates based on env sequences.
[0008] FIG. 3. Tropic and biologic properties of HIV-1
isolates.
[0009] FIG. 4. HIV-encoded proteins. The location of the HIV genes,
the sizes of primary translation products (in some cases
polyproteins), and the processed mature viral proteins are
indicated.
[0010] FIG. 5. Schematic representation of a mature HIV-1
virion.
[0011] FIG. 6. Linear representation of the HIV-1 Env glycoprotein.
The arrow indicates the site of gp160 cleavage to gp120 and gp41.
In gp120, cross-hatched areas represent variable domains (V.sub.1
to V.sub.5) and open boxes depict conserved sequences (C.sub.1 to
C.sub.5). In the gp41 ectodomain, several domains are indicated:
the N-terminal fusion peptide, and the two ectodomain helices (N-
and C-helix). The membrane-spanning domain is represented by a
black box. In the gp41 cytoplasmic domain, the Tyr-X-X-Leu (YXXL)
endocytosis motif (SEQ ID NO: 9) and two predicted helical domains
(helix-1 and -2) are shown. Amino acid numbers are indicated.
[0012] FIG. 7. Temporal frequencies of Gag-specific T cells. (A)
Gag-specific CD8 T cell responses raised by DNA priming and rMVA
booster immunizations. The schematic presents mean Gag-CM9-tetramer
data generated in the high-dose i.d. DNA-immunized animals. (B)
Gag-specific IFN-.gamma. ELISPOTs in A*01 (open bars) and non-A*01
(filled bars) macaques at various times before challenge and at two
weeks after challenge. Three pools of 10 to 13 Gag peptides
(22-mers overlapping by 12) were used for the analyses. The numbers
above data bars represent the arithmetic mean.+-.SD for the
ELISPOTs within each group. The numbers at the top of the graphs
designate individual animals. *, data not available; #, <20
ELISPOTs per 1.times.10.sup.6 peripheral blood mononuclear cells
(PBMC). Temporal data for Gag-CM9-Mamu-A*01 tetramer-specific T
cells can be found in FIG. 12.
[0013] FIG. 8. Temporal viral loads, CD4 counts, and survival after
challenge of vaccinated and control animals. (A) Geometric mean
viral loads and (B) geometric mean CD4 counts. (C) Survival curve
for vaccinated and control animals. The dotted line represents all
24 vaccinated animals. (D) Viral loads and (E) CD4 counts for
individual animals in the vaccine and control groups. The key to
animal numbers is presented in (E). Assays for the first 12 weeks
after challenge had a detection level of 1000 copies of RNA per
milliliter of plasma. Animals with loads below 1000 were scored
with a load of 500. For weeks 16 and 20, the detection level was
300 copies of RNA per milliliter. Animals with levels of virus
below 300 were scored at 300.
[0014] FIG. 9. Postchallenge T cell responses in vaccine and
control groups. (A) Temporal tetramer.sup.+ cells (dashed line) and
viral loads (solid line). (B) Intracellular cytokine assays for
IFN-.gamma. production in response to stimulation with the Gag-CM9
peptide at two weeks after challenge. This ex vivo assay allows
evaluation of the functional status of the peak postchallenge
tetramer.sup.+ cells displayed in FIG. 7A. (C) Proliferation assay
at 12 weeks after challenge. Gag-Pol-Env (open bars) and Gag-Pol
(hatched bars) produced by transient transfections were used for
stimulation. Supernatants from mock-transfected cultures served as
control antigen. Stimulation indices are the growth of cultures in
the presence of viral antigens divided by the growth of cultures in
the presence of mock antigen.
[0015] FIG. 10. Lymph node histomorphology at 12 weeks after
challenge. (A) Typical lymph node from a vaccinated macaque showing
evidence of follicular hyperplasia characterized by the presence of
numerous secondary follicles with expanded germinal centers and
discrete dark and light zones. (B) Typical lymph node from an
infected control animal showing follicular depletion and
paracortical lymphocellular atrophy. (C) A representative lymph
node from an age-matched, uninfected macaque displaying nonreactive
germinal centers. (D) The percentage of the total lymph node area
occupied by germinal centers was measured to give a non-specific
indicator of follicular hyperplasia. Data for uninfected controls
are for four age-matched rhesus macaques.
[0016] FIG. 11. Temporal antibody responses. Micrograms of total
Gag (A) or Env (B) antibody were determined with ELISAs. The titers
of neutralizing antibody for SHIV-89.6 (C) and SHIV-89.6P (D) were
determined with MT-2 cell killing and neutral red staining (D. C.
Montefiori et al. 1988 J Clin Microbiol 26:231). Titers are the
reciprocal of the serum dilution giving 50% neutralization of the
indicated viruses grown in human PBMC. Symbols for animals are the
same as in FIG. 8.
[0017] FIG. 12. Gag-CM9-Mamu-A*01 tetramer-specific T cells in
Mamu-A*01 vaccinated and control macaques at various times before
challenge and at two weeks after challenge. The number at the upper
right corner of each plot represents the frequency of
tetramer-specific CD8 T cells as a % of total CD8 T cells. The
numbers above each column of FACS data designate individual
animals.
[0018] FIG. 13. Map of plasmid transfer vector pLW-48.
[0019] FIG. 14 A-I. Sequences of plasmid transfer vector pLW-48
(SEQ ID NO: 1), Psyn II promoter (which controls ADA envelope
expression) (SEQ ID NO: 2), ADA envelope truncated (SEQ ID NO: 3),
PmH5 promoter (which controls HXB2 gag pol expression) (SEQ ID NO:
4), and HXB2 gag pol (with safety mutations, .DELTA. integrase)
(SEQ ID NO: 5).
[0020] FIG. 15. Plasmid transfer vector pLW-48 and making MVA
recombinant virus MVA/HIV 48.
[0021] FIG. 16. A clade B gag pol.
[0022] FIG. 17. Sequence of new Psyn II promoter (SEQ ID NO:
2).
[0023] FIG. 18. pLAS-1 and pLAS-2.
[0024] FIG. 19. pLAS-1/UGDgag.
[0025] FIG. 20. pLAS-2/UGDenv.
[0026] FIG. 21. pLAS-2/UGDrev env.
[0027] FIG. 22. Schematic for recombinant MVA production.
[0028] FIG. 23. Overview of making recombinant MVA/UGD viruses.
[0029] FIG. 24. Immunoprecipitation analysis.
[0030] FIG. 25. Functional analysis of expressed proteins. A,
Virus-like partikle assay. B. Env fusion assay.
[0031] FIG. 26. MVA/UGD induced HIV env- and gag-specific antibody
responses. A. HIV p24-specofoc serum IgG responses. B. HIV
env-specific serum IgG responses. C. MVA/UGD induced HIV env- and
gag-specific antibody responses (study 1).
[0032] FIG. 27. MVA/UGD induced gag-specific intracellular
IFN-.gamma. production.
[0033] FIG. 28A. MVA/UGD induced gag-specific IFN-.gamma.
ELISPOT.
[0034] FIG. 28B. MVA/UGD induced pol-specific IFN-.gamma.
ELISPOT.
[0035] FIG. 29. MVA/UGD induced gag-specific tetramer staining.
[0036] FIG. 30. MVA/UGD induced gag-specific antibody responses
(study 2).
[0037] FIGS. 31A & B. MVA/UGD induced gag- and pol-specific
intracellular IFN-.gamma. production (study 2).
[0038] FIGS. 32 A, B, & C. MVA/UGD induced gag- and
pol-specific ELISPOT (study 2).
[0039] FIG. 33. MVA/UGD induced gag-specific tetramer staining
(study 2).
[0040] FIG. 34. MVA/UGD induced gag-specific cytotoxic T cell
killing.
[0041] FIG. 35. Immunoprecipitation analysis of cell lysates
(MVA/KEA).
[0042] FIG. 36. Gag particle assay (MVA/KEA).
[0043] FIG. 37. Fusion assay (MVA/KEA).
[0044] FIG. 38. MVA/KEA induced HIV-1 env-specific antibody
responses.
[0045] FIG. 39. MVA/KEA induced gag-specific intracellular
production.
[0046] FIG. 40. MVA/KEA induced gag-specific tetramer staining.
[0047] FIG. 41. MVA/KEA induced gag-specific IFN-.gamma.
ELISPOT.
[0048] FIG. 42. Immunoprecipitation analysis of cell lysates
(MVA/TZC).
[0049] FIG. 43. Fusion assay (MVA/TZC).
BRIEF DESCRIPTION OF THE APPENDICES
[0050] Appendix 1. DNA sequences of gagpol and env genes from
Ugandan HIV-1 clade D isolates (SEQ ID NOs: 51, 52, 53, 54,
55).
[0051] Appendix 2. DNA sequences of MVA shuttle plasmids, pLAS-1
and pLAS-2 (SEQ ID NOs: 56, 57).
[0052] Appendix 3. DNA sequences of gagpol and env genes from
Kenyan HIV-1 clade A isolates (SEQ ID NOs: 58, 59, 60).
[0053] Appendix 4. DNA sequences of gagpol and env genes from
Tanzanian HIV-1 clade C isolates (SEQ ID NOs: 61, 62).
[0054] Appendix 5. American Type Culture Collection, Budapest
Treaty Deposit Form BP/1, questions 1-8, regarding MVA 1974/NIH
Clone 1.
[0055] Appendix 6. American Tissue Type Collection, Additional
Information Required When Depositing A Virus for Patent Purposes,
regarding MVA 1974/NIH Clone 1.
Deposit of Microorganism
[0056] The following microorganism has been deposited in accordance
with the terms of the Budapest Treaty with the American Type
Culture Collection (ATCC), Manassas, Va., on the date
indicated:
TABLE-US-00001 Microorganism Accession No. Date MVA 1974/NIH Clone
1 PTA-5095 Mar. 27, 2003
[0057] MVA 1974/NIH Clone 1 was deposited as ATCC Accession No.
PTA-5095 on Mar. 27, 2003 with the American Type Culture Collection
(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, USA. This
deposit was made under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure and the Regulations thereunder
(Budapest Treaty). This assures maintenance of a viable culture of
the deposit for 30 years from date of deposit. The deposit will be
made available by ATCC under the terms of the Budapest Treaty, and
subject to an agreement between Applicant and ATCC which assures
permanent and unrestricted availability of the progeny of the
culture of the deposit to the public upon issuance of the pertinent
U.S. patent or upon laying open to the public of any U.S. or
foreign patent application, whichever comes first, and assures
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 USC .sctn.122 and the Commissioner's rules pursuant
thereto (including 37 CFR .sctn.1.14). Availability of the
deposited strain is not to be construed as a license to practice
the invention in contravention of the rights granted under the
authority of any government in accordance with its patent laws.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Recombinant MVA Virus
[0058] Vaccinia virus, a member of the genus Orthopoxvirus in the
family of Poxyiridae, was used as live vaccine to immunize against
the human smallpox disease. Successful worldwide vaccination with
vaccinia virus culminated in the eradication of variola virus, the
causative agent of the smallpox ("The global eradication of
smallpox. Final report of the global commission for the
certification of smallpox eradication". History of Public Health,
No. 4, Geneva: World Health Organization, 1980). Since that WHO
declaration, vaccination has been universally discontinued except
for people at high risk of poxvirus infections (e.g. laboratory
workers).
[0059] More recently, vaccinia viruses have also been used to
engineer viral vectors for recombinant gene expression and for the
potential use as recombinant live vaccines (Mackett, M. et al. 1982
PNAS USA 79:7415-7419; Smith, G. L. et al. 1984 Biotech Genet Engin
Rev 2:383-407). This entails DNA sequences (genes) which code for
foreign antigens being introduced, with the aid of DNA
recombination techniques, into the genome of the vaccinia viruses.
If the gene is integrated at a site in the viral DNA which is
non-essential for the life cycle of the virus, it is possible for
the newly produced recombinant vaccinia virus to be infectious,
that is to say able to infect foreign cells and thus to express the
integrated DNA sequence (EP Patent Applications No. 83,286 and No.
110,385). The recombinant vaccinia viruses prepared in this way can
be used, on the one hand, as live vaccines for the prophylaxis of
infectious diseases, on the other hand, for the preparation of
heterologous proteins in eukaryotic cells.
[0060] For vector applications health risks would be lessened by
the use of a highly attenuated vaccinia virus strain. Several such
strains of vaccinia virus were especially developed to avoid
undesired side effects of smallpox vaccination. Thus, the modified
vaccinia Ankara (MVA) has been generated by long-term serial
passages of the Ankara strain of vaccinia virus (CVA) on chicken
embryo fibroblasts (for review see Mayr, A. et al. 1975 Infection
3:6-14; Swiss Patent No. 568,392). The MVA virus is publicly
available from American Type Culture Collection as ATCC No.
VR-1508. MVA is distinguished by its great attenuation, that is to
say by diminished virulence and ability to replicate in primate
cells while maintaining good immunogenicity. The MVA virus has been
analyzed to determine alterations in the genome relative to the
parental CVA strain. Six major deletions of genomic DNA (deletion
I, II, III, IV, V, and VI) totaling 31,000 base pairs have been
identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). The
resulting MVA virus became severely host cell restricted to avian
cells.
[0061] Furthermore, MVA is characterized by its extreme
attenuation. When tested in a variety of animal models, MVA was
proven to be avirulent even in immunosuppressed animals. More
importantly, the excellent properties of the MVA strain have been
demonstrated in extensive clinical trials (Mayr A. et al. 1978
Zentralbl Bakteriol [B] 167:375-390; Stickl et al. 1974 Dtsch Med
Wschr 99:2386-2392). During these studies in over 120,000 humans,
including high-risk patients, no side effects were associated with
the use of MVA vaccine.
[0062] MVA replication in human cells was found to be blocked late
in infection preventing the assembly to mature infectious virions.
Nevertheless, MVA was able to express viral and recombinant genes
at high levels even in non-permissive cells and was proposed to
serve as an efficient and exceptionally safe gene expression vector
(Sutter, G. and Moss, B. 1992 PNAS USA 89:10847-10851).
Additionally, novel vaccinia vector vaccines were established on
the basis of MVA having foreign DNA sequences inserted at the site
of deletion III within the MVA genome (Sutter, G. et al. 1994
Vaccine 12:1032-1040).
[0063] The recombinant MVA vaccinia viruses can be prepared as set
out hereinafter. A DNA-construct which contains a DNA-sequence
which codes for a foreign polypeptide flanked by MVA DNA sequences
adjacent to a naturally occurring deletion, e.g. deletion III, or
other non-essential sites, within the MVA genome, is introduced
into cells infected with MVA, to allow homologous recombination.
Once the DNA-construct has been introduced into the eukaryotic cell
and the foreign DNA has recombined with the viral DNA, it is
possible to isolate the desired recombinant vaccinia virus in a
manner known per se, preferably with the aid of a marker. The
DNA-construct to be inserted can be linear or circular. A plasmid
or polymerase chain reaction product is preferred. The
DNA-construct contains sequences flanking the left and the right
side of a naturally occurring deletion, e.g. deletion III, within
the MVA genome. The foreign DNA sequence is inserted between the
sequences flanking the naturally occurring deletion. For the
expression of a DNA sequence or gene, it is necessary for
regulatory sequences, which are required for the transcription of
the gene, to be present on the DNA. Such regulatory sequences
(called promoters) are known to those skilled in the art, and
include for example those of the vaccinia 11 kDa gene as are
described in EP-A-198,328, and those of the 7.5 kDa gene
(EP-A-110,385). The DNA-construct can be introduced into the MVA
infected cells by transfection, for example by means of calcium
phosphate precipitation (Graham et al. 1973 Virol 52:456-467;
Wigler et al. 1979 Cell 16:777-785), by means of electroporation
(Neumann et al. 1982 EMBO J. 1:841-845), by microinjection
(Graessmann et al. 1983 Meth Enzymol 101:482-492), by means of
liposomes (Straubinger et al. 1983 Meth Enzymol 101:512-527), by
means of spheroplasts (Schaffner 1980 PNAS USA 77:2163-2167) or by
other methods known to those skilled in the art.
HIVs and Their Replication
[0064] The etiological agent of acquired immune deficiency syndrome
(AIDS) is recognized to be a retrovirus exhibiting characteristics
typical of the lentivirus genus, referred to as human
immunodeficiency virus (HIV). The phylogenetic relationships of the
human lentiviruses are shown in FIG. 1. HIV-2 is more closely
related to SIV.sub.smm, a virus isolated from sooty mangabey
monkeys in the wild, than to HIV-1. It is currently believed that
HIV-2 represents a zoonotic transmission of SIV.sub.smm to man. A
series of lentiviral isolates from captive chimpanzees, designated
SIV.sub.cpz, are close genetic relatives of HIV-1.
[0065] The earliest phylogenetic analyses of HIV-1 isolates focused
on samples from Europe/North America and Africa; discrete clusters
of viruses were identified from these two areas of the world.
Distinct genetic subtypes or clades of HIV-1 were subsequently
defined and classified into three groups: M (major); O (outlier);
and N (non-M or O) (FIG. 2). The M group of HIV-1, which includes
over 95% of the global virus isolates, consists of at least eight
discrete clades (A, B, C, D, F, G, H, and J), based on the sequence
of complete viral genomes. Members of HIV-1 group O have been
recovered from individuals living in Cameroon, Gabon, and
Equatorial Guinea; their genomes share less than 50% identity in
nucleotide sequence with group M viruses. The more recently
discovered group N HIV-I strains have been identified in infected
Cameroonians, fail to react serologically in standard whole-virus
enzyme-linked immunosorbent assay (ELISA), yet are readily
detectable by conventional Western blot analysis.
[0066] Most current knowledge about HIV-1 genetic variation comes
from studies of group M viruses of diverse geographic origin. Data
collected during the past decade indicate that the HIV-1 population
present within an infected individual can vary from 6% to 10% in
nucleotide sequence. HIV-1 isolates within a clade may exhibit
nucleotide distances of 15% in gag and up to 30% in gp120 coding
sequences. Interclade genetic variation may range between 30% and
40% depending on the gene analyzed.
[0067] All of the HIV-1 group M subtypes can be found in Africa.
Clade A viruses are genetically the most divergent and were the
most common HIV-1 subtype in Africa early in the epidemic. With the
rapid spread of HIV-1 to southern Africa during the mid to late
1990s, clade C viruses have become the dominant subtype and now
account for 48% of HIV-1 infections worldwide. Clade B viruses, the
most intensively studied HIV-1 subtype, remain the most prevalent
isolates in Europe and North America.
[0068] High rates of genetic recombination are a hallmark of
retroviruses. It was initially believed that simultaneous
infections by genetically diverse virus strains were not likely to
be established in individuals at risk for HIV-1. By 1995, however,
it became apparent that a significant fraction of the HIV-1 group M
global diversity included interclade viral recombinants. It is now
appreciated that HIV-1 recombinants will be found in geographic
areas such as Africa, South America, and Southeast Asia, where
multiple HIV-1 subtypes coexist and may account for more than 10%
of circulating HIV-1 strains. Molecularly, the genomes of these
recombinant viruses resemble patchwork mosaics, with juxtaposed
diverse HIV-1 subtype segments, reflecting the multiple crossover
events contributing to their generation. Most HIV-1 recombinants
have arisen in Africa and a majority contains segments originally
derived from clade A viruses. In Thailand, for example, the
composition of the predominant circulating strain consists of a
clade A gag plus pol gene segment and a clade E env gene. Because
the clade E env gene in That HIV-1 strains is closely related to
the clade E env present in virus isolates from the Central African
Republic, it is believed that the original recombination event
occurred in Africa, with the subsequent introduction of a
descendent virus into Thailand. Interestingly, no full-length HIV-1
subtype E isolate (i.e., with subtype E gag, pol, and env genes)
has been reported to date.
[0069] The discovery that .alpha. and .beta. chemokine receptors
function as coreceptors for virus fusion and entry into susceptible
CD4.sup.+ cells has led to a revised classification scheme for
HIV-1 (FIG. 3). Isolates can now be grouped on the basis of
chemokine receptor utilization in fusion assays in which HIV-1
gp120 and CD4.sup.+ coreceptor proteins are expressed in separate
cells. As indicated in FIG. 3, HIV-1 isolates using the CXCR4
receptor (now designated X4 viruses) are usually T cell line
(TCL)-tropic syncytium inducing (SI) strains, whereas those
exclusively utilizing the CCR5 receptor (R5 viruses) are
predominantly macrophage (M)-tropic and non-syncytium inducing
(NSI). The dual-tropic R5/X4 strains, which may comprise the
majority of patient isolates and exhibit a continuum of tropic
phenotypes, are frequently SI.
[0070] As is the case for all replication-competent retroviruses,
the three primary HIV-1 translation products, all encoding
structural proteins, are initially synthesized as polyprotein
precursors, which are subsequently processed by viral or cellular
proteases into mature particle-associated proteins (FIG. 4). The
55-kd Gag precursor Pr55.sup.Gag is cleaved into the matrix (MA),
capsid (CA), nucleocapsid (NC), and p6 proteins. Autocatalysis of
the 160-kd Gag-Pol polyprotein, Pr160.sup.Gag-Pol, gives rise to
the protease (PR), the heterodimeric reverse transcriptase (RT),
and the integrase (IN) proteins, whereas proteolytic digestion by a
cellular enzyme(s) converts the glycosylated 160-kd Env precursor
gp160 to the gp120 surface (SU) and gp41 transmembrane (TM)
cleavage products. The remaining six HIV-1-encoded proteins (Vif,
Vpr, Tat, Rev, Vpu, and Nef) are the primary translation products
of spliced mRNAs.
Gag
[0071] The Gag proteins of HIV, like those of other retroviruses,
are necessary and sufficient for the formation of noninfectious,
virus-like particles. Retroviral Gag proteins are generally
synthesized as polyprotein precursors; the HIV-1 Gag precursor has
been named, based on its apparent molecular mass, Pr55.sup.Gag. As
noted previously, the mRNA for Pr55.sup.Gag is the unspliced 9.2-kb
transcript (FIG. 4) that requires Rev for its expression in the
cytoplasm. When the pol ORF is present, the viral protease (PR)
cleaves Pr55.sup.Gag during or shortly after budding from the cell
to generate the mature Gag proteins p17 (MA), p24 (CA), p7 (NC),
and p6 (see FIG. 4). In the virion, MA is localized immediately
inside the lipid bilayer of the viral envelope, CA forms the outer
portion of the cone-shaped core structure in the center of the
particle, and NC is present in the core in a ribonucleoprotein
complex with the viral RNA genome (FIG. 5).
[0072] The HIV Pr55.sup.Gag precursor oligomerizes following its
translation and is targeted to the plasma membrane, where particles
of sufficient size and density to be visible by EM are assembled.
Formation of virus-like particles by Pr55.sup.Gag is a
self-assembly process, with critical Gag-Gag interactions taking
place between multiple domains along the Gag precursor. The
assembly of virus-like particles does not require the participation
of genomic RNA (although the presence of nucleic acid appears to be
essential), pol-encoded enzymes, or Env glycoproteins, but the
production of infectious virions requires the encapsidation of the
viral RNA genome and the incorporation of the Env glycoproteins and
the Gag-Pol polyprotein precursor Pr160.sup.Gag-Pol.
Pol
[0073] Downstream of gag lies the most highly conserved region of
the HIV genome, the pol gene, which encodes three enzymes: PR, RT,
and IN (see FIG. 4). RT and IN are required, respectively, for
reverse transcription of the viral RNA genome to a double-stranded
DNA copy, and for the integration of the viral DNA into the host
cell chromosome. PR plays a critical role late in the life cycle by
mediating the production of mature, infectious virions. The pol
gene products are derived by enzymatic cleavage of a 160-kd Gag-Pol
fusion protein, referred to as Pr160.sup.Gag-Pol. This fusion
protein is produced by ribosomal frameshifting during translation
of Pr55.sup.Gag (see FIG. 4). The frame-shifting mechanism for
Gag-Pol expression, also utilized by many other retroviruses,
ensures that the poi-derived proteins are expressed at a low level,
approximately 5% to 10% that of Gag. Like Pr55.sup.Gag, the
N-terminus of Pr160.sup.Gag-Pol is myristylated and targeted to the
plasma membrane.
Protease
[0074] Early pulse-chase studies performed with avian retroviruses
clearly indicated that retroviral Gag proteins are initially
synthesized as polyprotein precursors that are cleaved to generate
smaller products. Subsequent studies demonstrated that the
processing function is provided by a viral rather than a cellular
enzyme, and that proteolytic digestion of the Gag and Gag-Pol
precursors is essential for virus infectivity. Sequence analysis of
retroviral PRs indicated that they are related to cellular
"aspartic" proteases such as pepsin and renin. Like these cellular
enzymes, retroviral PRs use two apposed Asp residues at the active
site to coordinate a water molecule that catalyzes the hydrolysis
of a peptide bond in the target protein. Unlike the cellular
aspartic proteases, which function as pseudodimers (using two folds
within the same molecule to generate the active site), retroviral
PRs function as true dimers. X-ray crystallographic data from HIV-1
PR indicate that the two monomers are held together in part by a
four-stranded antiparallel .beta.-sheet derived from both N- and
C-terminal ends of each monomer. The substrate-binding site is
located within a cleft formed between the two monomers. Like their
cellular homologs, the HIV PR dimer contains flexible "flaps" that
overhang the binding site and may stabilize the substrate within
the cleft; the active-site Asp residues lie in the center of the
dimer. Interestingly, although some limited amino acid homology is
observed surrounding active-site residues, the primary sequences of
retroviral PRs are highly divergent, yet their structures are
remarkably similar.
Reverse Transcriptase
[0075] By definition, retroviruses possess the ability to convert
their single-stranded RNA genomes into double-stranded DNA during
the early stages of the infection, process. The enzyme that
catalyzes this reaction is RT, in conjunction with its associated
RNaseH activity. Retroviral RTs have three enzymatic activities:
(a) RNA-directed DNA polymerization (for minus-strand DNA
synthesis), (b) RNaseH activity (for the degradation of the tRNA
primer and genomic RNA present in DNA-RNA hybrid intermediates),
and (c) DNA-directed DNA polymerization (for second- or plus-strand
DNA synthesis).
[0076] The mature HIV-1 RT holoenzyme is a heterodimer of 66 and 51
kd subunits. The 51-kd subunit (p51) is derived from the 66-kd
(p66) subunit by proteolytic removal of the C-terminal 15-kd RNaseH
domain of p66 by PR (see FIG. 4). The crystal structure of HIV-1 RT
reveals a highly asymmetric folding in which the orientations of
the p66 and p51 subunits differ substantially. The p66 subunit can
be visualized as a right hand, with the polymerase active site
within the palm, and a deep template-binding cleft formed by the
palm, fingers, and thumb subdomains. The polymerase domain is
linked to RNaseH by the connection subdomain. The active site,
located in the palm, contains three critical Asp residues (110,
185, and 186) in close proximity, and two coordinated Mg.sup.2+
ions. Mutation of these Asp residues abolishes RT polymerizing
activity. The orientation of the three active-site Asp residues is
similar to that observed in other DNA polymerases (e.g., the Klenow
fragment of E. coli DNA polI). The p51 subunit appears to be rigid
and does not form a polymerizing cleft; Asp 110, 185, and 186 of
this subunit are buried within the molecule. Approximately 18 base
pairs of the primer-template duplex lie in the nucleic acid binding
cleft, stretching from the polymerase active site to the RNaseH
domain.
[0077] In the RT-primer-template-dNTP structure, the presence of a
dideoxynucleotide at the 3' end of the primer allows visualization
of the catalytic complex trapped just prior to attack on the
incoming dNTP. Comparison with previously obtained structures
suggests a model whereby the fingers close in to trap the template
and dNTP prior to nucleophilic attack of the 3'-OH of the primer on
the incoming dNTP. After the addition of the incoming dNTP to the
growing chain, it has been proposed that the fingers adopt a more
open configuration, thereby releasing the pyrophosphate and
enabling RT to bind the next dNTP. The structure of the HIV-1
RNaseH has also been determined by x-ray crystallography; this
domain displays a global folding similar to that of E. coli
RNaseH.
Integrase
[0078] A distinguishing feature of retrovirus replication is the
insertion of a DNA copy of the viral genome into the host cell
chromosome following reverse transcription. The integrated viral
DNA (the provirus) serves as the template for the synthesis of
viral RNAs and is maintained as part of the host cell genome for
the lifetime of the infected cell. Retroviral mutants deficient in
the ability to integrate generally fail to establish a productive
infection.
[0079] The integration of viral DNA is catalyzed by integrase, a
32-kd protein generated by PR-mediated cleavage of the C-terminal
portion of the HIV-1 Gag-Pol polyprotein (see FIG. 4).
[0080] Retroviral IN proteins are composed of three structurally
and functionally distinct domains: an N-terminal,
zinc-finger-containing domain, a core domain, and a relatively
nonconserved C-terminal domain. Because of its low solubility, it
has not yet been possible to crystallize the entire 288-amino-acid
HIV-1 IN protein. However, the structure of all three domains has
been solved independently by x-ray crystallography or NMR methods.
The crystal structure of the core domain of the avian sarcoma virus
IN has also been determined. The N-terminal domain (residues 1 to
55), whose structure was solved by NMR spectroscopy, is composed of
four helices with a zinc coordinated by amino acids His-12, His-16,
Cys-40, and Cys-43. The structure of the N-terminal domain is
reminiscent of helical DNA binding proteins that contain a
so-called helix-turn-helix motif; however, in the HIV-1 structure
this motif contributes to dimer formation. Initially, poor
solubility hampered efforts to solve the structure of the core
domain. However, attempts at crystallography were successful when
it was observed that a Phe-to-Lys change at IN residue 185 greatly
increased solubility without disrupting in vitro catalytic
activity. Each monomer of the HIV-1 IN core domain (IN residues 50
to 212) is composed of a five-stranded .beta.-sheet flanked by
helices; this structure bears striking resemblance to other
polynucleotidyl transferases including RNaseH and the bacteriophage
MuA transposase. Three highly conserved residues are found in
analogous positions in other polynucleotidyl transferases; in HIV-1
IN these are Asp-64, Asp-116 and Glu-152, the so-called D,D-35-E
motif. Mutations at these positions block HIV IN function both in
vivo and in vitro. The close proximity of these three amino acids
in the crystal structure of both avian sarcoma virus and HIV-1 core
domains supports the hypothesis that these residues play a central
role in catalysis of the polynucleotidyl transfer reaction that is
at the heart of the integration process. The C-terminal domain,
whose structure has been solved by NMR methods, adopts a
five-stranded n-barrel folding topology reminiscent of a Src
homology 3 (SH3) domain. Recently, the x-ray structures of SIV and
Rous sarcoma virus IN protein fragments encompassing both the core
and C-terminal domains have been solved.
Env
[0081] The HIV Env glycoproteins play a major role in the virus
life cycle. They contain the determinants that interact with the
CD4 receptor and coreceptor, and they catalyze the fusion reaction
between the lipid bilayer of the viral envelope and the host cell
plasma membrane. In addition, the HIV Env glycoproteins contain
epitopes that elicit immune responses that are important from both
diagnostic and vaccine development perspectives.
[0082] The HIV Env glycoprotein is synthesized from the singly
spliced 4.3-kb Vpu/Env bicistronic mRNA (see FIG. 4); translation
occurs on ribosomes associated with the rough endoplasmic reticulum
(ER). The 160-kd polyprotein precursor (gp160) is an integral
membrane protein that is anchored to cell membranes by a
hydrophobic stop-transfer signal in the domain destined to be the
mature TM Env glycoprotein, gp41 (FIG. 6). The gp160 is
cotranslationally glycosylated, forms disulfide bonds, and
undergoes oligomerization in the ER. The predominant oligomeric
form appears to be a trimer, although dimers and tetramers are also
observed. The gp160 is transported to the Golgi, where, like other
retroviral envelope precursor proteins, it is proteolytically
cleaved by cellular enzymes to the mature SU glycoprotein gp120 and
TM glycoprotein gp41 (see FIG. 6). The cellular enzyme responsible
for cleavage of retroviral Env precursors following a highly
conserved Lys/Arg-X-Lys/Arg-Arg motif is furin or a furin-like
protease, although other enzymes may also catalyze gp160
processing. Cleavage of gp160 is required for Env-induced fusion
activity and virus infectivity. Subsequent to gp160 cleavage, gp120
and gp41 form a noncovalent association that is critical for
transport of the Env complex from the Golgi to the cell surface.
The gp120-gp41 interaction is fairly weak, and a substantial amount
of gp120 is shed from the surface of Env-expressing cells.
[0083] The HIV Env glycoprotein complex, in particular the SU
(gp120) domain, is very heavily glycosylated; approximately half
the molecular mass of gp160 is composed of oligosaccharide side
chains. During transport of Env from its site of synthesis in the
ER to the plasma membrane, many of the side chains are modified by
the addition of complex sugars. The numerous oligosaccharide side
chains form what could be imagined as a sugar cloud obscuring much
of gp120 from host immune recognition. As shown in FIG. 6, gp120
contains interspersed conserved (C.sub.1 to C.sub.5) and variable
(V.sub.1 to V.sub.5) domains. The Cys residues present in the
gp120s of different isolates are highly conserved and form
disulfide bonds that link the first four variable regions in large
loops.
[0084] A primary function of viral Env glycoproteins is to promote
a membrane fusion reaction between the lipid bilayers of the viral
envelope and host cell membranes. This membrane fusion event
enables the viral core to gain entry into the host cell cytoplasm.
A number of regions in both gp120 and gp41 have been implicated,
directly or indirectly, in Env-mediated membrane fusion. Studies of
the HA.sub.2 hemagglutinin protein of the orthomyxoviruses and the
F protein of the paramyxoviruses indicated that a highly
hydrophobic domain at the N-terminus of these proteins, referred to
as the fusion peptide, plays a critical role in membrane fusion.
Mutational analyses demonstrated that an analogous domain was
located at the N-terminus of the HIV-1, HIV-2, and SIV TM
glycoproteins (see FIG. 6). Nonhydrophobic substitutions within
this region of gp41 greatly reduced or blocked syncytium formation
and resulted in the production of noninfectious progeny
virions.
[0085] C-terminal to the gp41 fusion peptide are two amphipathic
helical domains (see FIG. 6) which play a central role in membrane
fusion. Mutations in the N-terminal helix (referred to as the
N-helix), which contains a Leu zipper-like heptad repeat motif,
impair infectivity and membrane fusion activity, and peptides
derived from these sequences exhibit potent antiviral activity in
culture. The structure of the ectodomain of HIV-1 and SIV gp41, the
two helical motifs in particular, has been the focus of structural
analyses in recent years. Structures were determined by x-ray
crystallography or NMR spectroscopy either for fusion proteins
containing the helical domains, a mixture of peptides derived from
the N- and C-helices, or in the case of the SIV structure, the
intact gp41 ectodomain sequence from residue 27 to 149. These
studies obtained fundamentally similar trimeric structures, in
which the two helical domains pack in an antiparallel fashion to
generate a six-helix bundle. The N-helices form a coiled-coil in
the center of the bundle, with the C-helices packing into
hydrophobic grooves on the outside.
[0086] In the steps leading to membrane fusion CD4 binding induces
conformation changes in Env that facilitate coreceptor binding.
Following the formation of a ternary gp120/CD4/coreceptor complex,
gp41 adopts a hypothetical conformation that allows the fusion
peptide to insert into the target lipid bilayer. The formation of
the gp41 six-helix bundle (which involves antiparallel interactions
between the gp41 N- and C-helices) brings the viral and cellular
membranes together and membrane fusion takes place.
Use of Recombinant MVA Virus To Boost CD+8 Cell Immune Response
[0087] The present invention relates to generation of a CD8.sup.+ T
cell immune response against an antigen and also eliciting an
antibody response. More particularly, the present invention relates
to "prime and boost" immunization regimes in which the immune
response induced by administration of a priming composition is
boosted by administration of a boosting composition. The present
invention is based on inventors' experimental demonstration that
effective boosting can be achieved using modified vaccinia Ankara
(MVA) vectors, following priming with any of a variety of different
types of priming compositions including recombinant MVA itself.
[0088] A major protective component of the immune response against
a number of pathogens is mediated by T lymphocytes of the CD8.sup.+
type, also known as cytotoxic T lymphocytes (CTL). An important
function of CD8.sup.+ cells is secretion of gamma interferon
(IFN.gamma.), and this provides a measure of CD8.sup.+ T cell
immune response. A second component of the immune response is
antibody directed to the proteins of the pathogen.
[0089] The present invention employs MVA which, as the experiments
described below show, has been found to be an effective means for
providing a boost to a CD8.sup.+ T cell immune response primed to
antigen using any of a variety of different priming compositions
and also eliciting an antibody response.
[0090] Remarkably, the experimental work described below
demonstrates that use of embodiments of the present invention
allows for recombinant MVA virus expressing an HIV antigen to boost
a CD8.sup.+ T cell immune response primed by a DNA vaccine and also
eliciting an antibody response. The MVA was found to induce a
CD8.sup.+ T cell response after intradermal, intramuscular or
mucosal immunization. Recombinant MVA has also been shown to prime
an immune response that is boosted by one or more inoculations of
recombinant MVA.
[0091] Non-human primates immunized with plasmid DNA and boosted
with the MVA were effectively protected against intramucosal
challenge with live virus. Advantageously, the inventors found that
a vaccination regime used intradermal, intramuscular or mucosal
immunization for both prime and boost can be employed, constituting
a general immunization regime suitable for inducing CD8.sup.+ T
cells and also eliciting an antibody response, e.g. in humans.
[0092] The present invention in various aspects and embodiments
employs an MVA vector encoding an HIV antigen for boosting a
CD8.sup.+ T cell immune response to the antigen primed by previous
administration of nucleic acid encoding the antigen and also
eliciting an antibody response.
[0093] A general aspect of the present invention provides for the
use of an MVA vector for boosting a CD8.sup.+ T cell immune
response to an HIV antigen and also eliciting an antibody
response.
[0094] One aspect of the present invention provides a method of
boosting a CD8.sup.+ T cell immune response to an HIV antigen in an
individual, and also eliciting an antibody response, the method
including provision in the individual of an MVA vector including
nucleic acid encoding the antigen operably linked to regulatory
sequences for production of antigen in the individual by expression
from the nucleic acid, whereby a CD8.sup.+ T cell immune response
to the antigen previously primed in the individual is boosted.
[0095] An immune response to an HIV antigen may be primed by
immunization with plasmid DNA or by infection with an infectious
agent.
[0096] A further aspect of the invention provides a method of
inducing a CD8.sup.+ T cell immune response to an HIV antigen in an
individual, and also eliciting an antibody response, the method
comprising administering to the individual a priming composition
comprising nucleic acid encoding the antigen and then administering
a boosting composition which comprises an MVA vector including
nucleic acid encoding the antigen operably linked to regulatory
sequences for production of antigen in the individual by expression
from the nucleic acid.
[0097] A further aspect provides for use of an MVA vector, as
disclosed, in the manufacture of a medicament for administration to
a mammal to boost a CD8.sup.+ T cell immune response to an HIV
antigen, and also eliciting an antibody response. Such a medicament
is generally for administration following prior administration of a
priming composition comprising nucleic acid encoding the
antigen.
[0098] The priming composition may comprise any viral vector, such
as a vaccinia virus vector such as a replication-deficient strain
such as modified vaccinia Ankara (MVA) or NYVAC (Tartaglia et al.
1992 Virology 118:217-232), an avipox vector such as fowlpox or
canarypox, e.g. the strain known as ALVAC (Paoletti et al. 1994 Dev
Biol Stand 82:65-69), or an adenovirus vector or a vesicular
stomatitis virus vector or an alphavirus vector.
[0099] The priming composition may comprise DNA encoding the
antigen, such DNA preferably being in the form of a circular
plasmid that is not capable of replicating in mammalian cells. Any
selectable marker should not be resistance to an antibiotic used
clinically, so for example Kanamycin resistance is preferred to
Ampicillin resistance. Antigen expression should be driven by a
promoter which is active in mammalian cells, for instance the
cytomegalovirus immediate early (CMV IE) promoter.
[0100] In particular embodiments of the various aspects of the
present invention, administration of a priming composition is
followed by boosting with a boosting composition, or first and
second boosting compositions, the first and second boosting
compositions being the same or different from one another. Still
further boosting compositions may be employed without departing
from the present invention. In one embodiment, a triple
immunization regime employs DNA, then adenovirus as a first
boosting composition, then MVA as a second boosting composition,
optionally followed by a further (third) boosting composition or
subsequent boosting administration of one or other or both of the
same or different vectors. Another option is DNA then MVA then
adenovirus, optionally followed by subsequent boosting
administration of one or other or both of the same or different
vectors.
[0101] The antigen to be encoded in respective priming and boosting
compositions (however many boosting compositions are employed) need
not be identical, but should share at least one CD8.sup.+ T cell
epitope. The antigen may correspond to a complete antigen, or a
fragment thereof. Peptide epitopes or artificial strings of
epitopes may be employed, more efficiently cutting out unnecessary
protein sequence in the antigen and encoding sequence in the vector
or vectors. One or more additional epitopes may be included, for
instance epitopes which are recognized by T helper cells,
especially epitopes recognized in individuals of different HLA
types.
[0102] An HIV antigen of the invention to be encoded by a
recombinant MVA virus includes polypeptides having immunogenic
activity elicited by an amino acid sequence of an HIV Env, Gag,
Pol, Vif, Vpr, Tat, Rev, Vpu, or Nef amino acid sequence as at
least one CD8.sup.+ T cell epitope. This amino acid sequence
substantially corresponds to at least one 10-900 amino acid
fragment and/or consensus sequence of a known HIV Env or Pol; or at
least one 10-450 amino acid fragment and/or consensus sequence of a
known HIV Gag; or at least one 10-100 amino acid fragment and/or
consensus sequence of a known HIV Vif, Vpr, Tat, Rev, Vpu, or
Nef.
[0103] Although a full length Env precursor sequence is presented
for use in the present invention, Env is optionally deleted of
subsequences. For example, regions of the gp120 surface and gp41
transmembrane cleavage products can be deleted.
[0104] Although a full length Gag precursor sequence is presented
for use in the present invention, Gag is optionally deleted of
subsequences. For example, regions of the matrix protein (p17),
regions of the capsid protein (p24), regions of the nucleocapsid
protein (p7), and regions of p6 (the C-terminal peptide of the Gag
polyprotein) can be deleted.
[0105] Although a full length Pol precursor sequence is presented
for use in the present invention, Pol is optionally deleted of
subsequences. For example, regions of the protease protein (p10),
regions of the reverse transcriptase protein (p66/p51), and regions
of the integrase protein (p32) can be deleted.
[0106] Such an HIV Env, Gag, or Pol can have overall identity of at
least 50% to a known Env, Gag, or Pol protein amino acid sequence,
such as 50-99% identity, or any range or value therein, while
eliciting an immunogenic response against at least one strain of an
HIV.
[0107] Percent identify can be determined, for example, by
comparing sequence information using the GAP computer program,
version 6.0, available from the University of Wisconsin Genetics
Computer Group (UWGCG). The GAP program utilizes the alignment
method of Needleman and Wunsch (J Mol Biol 1970 48:443), as revised
by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP
program defines identity as the number of aligned symbols (i.e.,
nucleotides or amino acids) which are identical, divided by the
total number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include: (1) a
unitary comparison matrix (containing a value of 1 for identities
and 0 for non-identities) and the weighted comparison matrix of
Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by
Schwartz and Dayhoff (eds., Atlas of Protein Sequence and
Structure, National Biomedical Research Foundation, Washington,
D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an
additional 0.10 penalty for each symbol in each gap; and (3) no
penalty for end gaps.
[0108] In a preferred embodiment, an Env of the present invention
is a variant form of at least one HIV envelope protein. Preferably,
the Env is composed of gp120 and the membrane-spanning and
ectodomain of gp41 but lacks part or all of the cytoplasmic domain
of gp41.
[0109] Known HIV sequences are readily available from commercial
and institutional HIV sequence databases, such as GENBANK, or as
published compilations, such as Myers et al. eds., Human
Retroviruses and AIDS, A Compilation and Analysis of Nucleic Acid
and Amino Acid Sequences, Vol. I and II, Theoretical Biology and
Biophysics, Los Alamos, N. Mex. (1993), or using http:// for
hiv-web.lan1.gov/.
[0110] Substitutions or insertions of an HIV Env, Gag, or Pol to
obtain an additional HIV Env, Gag, or Pol, encoded by a nucleic
acid for use in a recombinant MVA virus of the present invention,
can include substitutions or insertions of at least one amino acid
residue (e.g., 1-25 amino acids). Alternatively, at least one amino
acid (e.g., 1-25 amino acids) can be deleted from an HIV Env, Gag,
or Pol sequence. Preferably, such substitutions, insertions or
deletions are identified based on safety features, expression
levels, immunogenicity and compatibility with high replication
rates of MVA.
[0111] Amino acid sequence variations in an HIV Env, Gag, or Pol of
the present invention can be prepared e.g., by mutations in the
DNA. Such HIV Env, Gag, or Pol include, for example, deletions,
insertions or substitutions of nucleotides coding for different
amino acid residues within the amino acid sequence. Obviously,
mutations that will be made in nucleic acid encoding an HIV Env,
Gag, or Pol must not place the sequence out of reading frame and
preferably will not create complementary domains that could produce
secondary mRNA structures.
[0112] HIV Env, Gag, or Pol-encoding nucleic acid of the present
invention can also be prepared by amplification or site-directed
mutagenesis of nucleotides in DNA or RNA encoding an HIV Env, Gag,
or Pol and thereafter synthesizing or reverse transcribing the
encoding DNA to produce DNA or RNA encoding an HIV Env, Gag, or
Pol, based on the teaching and guidance presented herein.
[0113] Recombinant MVA viruses expressing HIV Env, Gag, or Pol of
the present invention, include a finite set of HIV Env, Gag, or
Pol-encoding sequences as substitution nucleotides that can be
routinely obtained by one of ordinary skill in the art, without
undue experimentation, based on the teachings and guidance
presented herein. For a detailed description of protein chemistry
and structure, see Schulz, G. E. et al., 1978 Principles of Protein
Structure, Springer-Verlag, New York, N.Y., and Creighton, T. E.,
1983 Proteins: Structure and Molecular Properties, W. H. Freeman
& Co., San Francisco, Calif. For a presentation of nucleotide
sequence substitutions, such as codon preferences, see Ausubel et
al. eds. Current Protocols in Molecular Biology, Greene Publishing
Assoc., New York, N.Y. 1994 at .sctn..sctn. A.1.1-A.1.24, and
Sambrook, J. et al. 1989 Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. at Appendices C and D.
[0114] Thus, one of ordinary skill in the art, given the teachings
and guidance presented herein, will know how to substitute other
amino acid residues in other positions of an HIV env, gag, or pol
DNA or RNA to obtain alternative HIV Env, Gag, or Pol, including
substitutional, deletional or insertional variants.
[0115] Within the MVA vector, regulatory sequences for expression
of the encoded antigen will include a natural, modified or
synthetic poxvirus promoter. By "promoter" is meant a sequence of
nucleotides from which transcription may be initiated of DNA
operably linked downstream (i.e. in the 3' direction on the sense
strand of double-stranded DNA). "Operably linked" means joined as
part of the same nucleic acid molecule, suitably positioned and
oriented for transcription to be initiated from the promoter. DNA
operably linked to a promoter is "under transcriptional initiation
regulation" of the promoter. Other regulatory sequences including
terminator fragments, polyadenylation sequences, marker genes and
other sequences may be included as appropriate, in accordance with
the knowledge and practice of the ordinary person skilled in the
art: see, for example, Moss, B. (2001). Poxyiridae: the viruses and
their replication. In Fields Virology, D. M. Knipe, and P. M.
Howley, eds. (Philadelphia, Lippincott Williams & Wilkins), pp.
2849-2883. Many known techniques and protocols for manipulation of
nucleic acid, for example in preparation of nucleic acid
constructs, mutagenesis, sequencing, introduction of DNA into cells
and gene expression, and analysis of proteins, are described in
detail in Current Protocols in Molecular Biology, 1998 Ausubel et
al. eds., John Wiley & Sons.
[0116] Promoters for use in aspects and embodiments of the present
invention must be compatible with poxvirus expression systems and
include natural, modified and synthetic sequences.
[0117] Either or both of the priming and boosting compositions may
include an adjuvant, such as granulocyte macrophage-colony
stimulating factor (GM-CSF) or encoding nucleic acid therefor.
[0118] Administration of the boosting composition is generally
about 1 to 6 months after administration of the priming
composition, preferably about 1 to 3 months.
[0119] Preferably, administration of priming composition, boosting
composition, or both priming and boosting compositions, is
intradermal, intramuscular or mucosal immunization.
[0120] Administration of MVA vaccines may be achieved by using a
needle to inject a suspension of the virus. An alternative is the
use of a needleless injection device to administer a virus
suspension (using, e.g., BIOJECTOR.TM. needleless injector) or a
resuspended freeze-dried powder containing the vaccine, providing
for manufacturing individually prepared doses that do not need cold
storage. This would be a great advantage for a vaccine that is
needed in rural areas of Africa.
[0121] MVA is a virus with an excellent safety record in human
immunizations. The generation of recombinant viruses can be
accomplished simply, and they can be manufactured reproducibly in
large quantities. Intradermal, intramuscular or mucosal
administration of recombinant MVA virus is therefore highly
suitable for prophylactic or therapeutic vaccination of humans
against AIDS which can be controlled by a CD8.sup.+ T cell
response.
[0122] The individual may have AIDS such that delivery of the
antigen and generation of a CD8.sup.+ T cell immune response to the
antigen is of benefit or has a therapeutically beneficial
effect.
[0123] Most likely, administration will have prophylactic aim to
generate an immune response against HIV or AIDS before infection or
development of symptoms.
[0124] Components to be administered in accordance with the present
invention may be formulated in pharmaceutical compositions. These
compositions may comprise a pharmaceutically acceptable excipient,
carrier, buffer, stabilizer or other materials well known to those
skilled in the art. Such materials should be non-toxic and should
not interfere with the efficacy of the active ingredient. The
precise nature of the carrier or other material may depend on the
route of administration, e.g. intravenous, cutaneous or
subcutaneous, nasal, intramuscular, intraperitoneal routes.
[0125] As noted, administration is preferably intradermal,
intramuscular or mucosal.
[0126] Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0127] For intravenous, cutaneous, subcutaneous, intramuscular or
mucosal injection, or injection at the site of affliction, the
active ingredient will be in the form of a parenterally acceptable
aqueous solution which is pyrogen-free and has suitable pH,
isotonicity and stability. Those of relevant skill in the art are
well able to prepare suitable solutions using, for example,
isotonic vehicles such as Sodium Chloride Injection, Ringer's
Injection, Lactated Ringer's Injection. Preservatives, stabilizers,
buffers, antioxidants and/or other additives may be included as
required.
[0128] A slow-release formulation may be employed.
[0129] Following production of MVA particles and optional
formulation of such particles into compositions, the particles may
be administered to an individual, particularly human or other
primate. Administration may be to another mammal, e.g. rodent such
as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig,
horse, cow, donkey, dog or cat.
[0130] Administration is preferably in a "prophylactically
effective amount" or a "therapeutically effective amount" (as the
case may be, although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual. The actual
amount administered, and rate and time-course of administration,
will depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage etc, is within
the responsibility of general practitioners and other medical
doctors, or in a veterinary context a veterinarian, and typically
takes account of the disorder to be treated, the condition of the
individual patient, the site of delivery, the method of
administration and other factors known to practitioners. Examples
of the techniques and protocols mentioned above can be found in
Remington's Pharmaceutical Sciences, 16th edition, 1980, Osol, A.
(ed.).
[0131] In one preferred regimen, DNA is administered at a dose of
250 .mu.g to 2.5 mg/injection, followed by MVA at a dose of
10.sup.6 to 10.sup.9 infectious virus particles/injection.
[0132] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated.
[0133] Delivery to a non-human mammal need not be for a therapeutic
purpose, but may be for use in an experimental context, for
instance in investigation of mechanisms of immune responses to an
antigen of interest, e.g. protection against HIV or AIDS.
[0134] Further aspects and embodiments of the present invention
will be apparent to those of ordinary skill in the art, in view of
the above disclosure and following experimental exemplification,
included by way of illustration and not limitation, and with
reference to the attached figures.
Example 1
Control of a Mucosal Challenge and Prevention of AIDS by a
Multiprotein DNA/MVA Vaccine
[0135] Here we tested DNA priming and poxvirus boosting for the
ability to protect against a highly pathogenic mucosal challenge.
The 89.6 chimera of simian and human immunodeficiency viruses
(SHIV-89.6) was used for the construction of immunogens and its
highly pathogenic derivative, SHIV-89.6P, for challenge (G. B.
Karlsson et al. 1997 J Virol 71:4218). SHIV-89.6 and SHIV-89.6P do
not generate cross-neutralizing antibody (D. C. Montefiori et al.
1998 J Virol 72:3427) and allowed us to address the ability of
vaccine-raised T cells and non-neutralizing antibodies to control
an immunodeficiency virus challenge. Modified vaccinia Ankara (MVA)
was used for the construction of the recombinant poxvirus. MVA has
been highly effective at boosting DNA-primed CD8 T cells and enjoys
the safety feature of not replicating efficiently in human or
monkey cells (H. L. Robinson et al. 2000 AIDS Reviews 2:105).
[0136] To ensure a broad immune response both the DNA and
recombinant MVA (rMVA) components of the vaccine expressed multiple
immunodeficiency virus proteins. The DNA prime (DNA/89.6) expressed
simian immunodeficiency virus (SIV) Gag, Pol, Vif, Vpx, and Vpr and
human immunodeficiency virus-1 (HIV-1) Env, Tat, and Rev from a
single transcript (R. J. Gorelick et al. 1999 Virology 253:259; M.
M. Sauter et al. 1996 J Cell Biol 132:795).
[0137] Molecularly cloned SHIV-89.6 sequences were cloned into the
vector pGA2, using ClaI and RsrII sites. This cloning deleted both
long terminal repeats (LTRs) and nef. The SHIV-89.6 sequences also
were internally mutated for a 12-base pair region encoding the
first four amino acids of the second zinc finger in nucleocapsid.
This mutation renders SHIV viruses noninfectious (R. J. Gorelick et
al. 1999 Virology 253:259). A mutation in gp41 converted the
tyrosine at position 710 to cysteine to achieve better expression
of Env on the plasma membrane of DNA-expressing cells (M. M. Sauter
et al. 1996 J Cell Biol 132:795). pGA2 uses the CMV immediate early
promoter without intron A and the bovine growth hormone
polyadenylation sequence to express vaccine inserts. Vaccine DNA
was produced by Althea (San Diego, Calif.). In transient
transfections of 293T cells, DNA/89.6 produced about 300 ng of Gag
and 85 ng of Env per 1.times.10.sup.6 cells.
[0138] The rMVA booster (MVA/89.6) expressed SIV Gag, Pol, and
HIV-1 Env under the control of vaccinia virus early/late
promoters.
[0139] The MVA double recombinant virus expressed both the HIV 89.6
Env and the SIV 239 Gag-Pol, which were inserted into deletion II
and deletion III of MVA, respectively. The 89.6 Env protein was
truncated for the COOH-terminal 115 amino acids of gp41. The
modified H5 promoter controlled the expression of both foreign
genes.
[0140] Vaccination was accomplished by priming with DNA at 0 and 8
weeks and boosting with rMVA at 24 weeks (FIG. 7A).
[0141] I.d. and i.m. DNA immunizations were delivered in
phosphate-buffered saline (PBS) with a needleless jet injector
(Bioject, Portland, Oreg.) to deliver five i.d. 100-.mu.l
injections to each outer thigh for the 2.5-mg dose of DNA or one
i.d. 100-.mu.l injection to the right outer thigh for the
250-.mu..mu.g dose of plasmid. I.m. deliveries of DNA were done
with one 0.5-ml injection of DNA in PBS to each outer thigh for the
2.5-mg dose and one 100-.mu.l injection to the right outer thigh
for the 250-.mu.g dose. 1.times.10.sup.8 pfu of MVA/89.6 was
administered both i.d. and i.m. with a needle. One 100-.mu.l dose
was delivered to each outer thigh for the i.d. dose and one
500-.mu.l dose to each outer thigh for the i.m dose. Control
animals received 2.5 mg of the pGA2 vector without vaccine insert
with the Bioject device to deliver five 100-.mu.l doses i.d. to
each outer thigh. The control MVA booster immunization consisted of
2.times.10.sup.8 pfu of MVA without an insert delivered i.d. and
i.m. as described for MVA/89.6.
[0142] Four groups of six rhesus macaques each were primed with
either 2.5 mg (high-dose) or 250 .mu.g (low-dose) of DNA by
intradermal (i.d.) or intramuscular (i.m.) routes using a
needleless jet injection device (Bioject, Portland, Oreg.) (T. M.
Allen et al. 2000 J Immunol 164:4968).
[0143] Young adult rhesus macaques from the Yerkes breeding colony
were cared for under guidelines established by the Animal Welfare
Act and the NIH "Guide for the Care and Use of Laboratory Animals"
with protocols approved by the Emory University Institutional
Animal Care and Use Committee. Macaques were typed for the
Mamu-A*01 allele with polymerase chain reaction (PCR) analyses (M.
A. Egan et al. 2000 J Virol 74:7485; I. Ourmanov et al. 2000 J
Virol 74:2740). Two or more animals containing at least one
Mamu-A*01 allele were assigned to each group. Animal numbers are as
follows: 1, RBr-5*; 2, RIm-5*; 3, RQf-5*; 4, RZe-5; 5, ROm-5; 6,
RDm-5; 7, RAj-5*; 8, RJi-5*; 9, RAl-5*; 10, RDe-5*; 11, RAi-5; 12,
RPr-5; 13, RKw-4*; 14, RWz-5*; 15, RGo-5; 16, RLp-4; 17, RWd-6; 18,
RAt-5; 19, RPb-5*; 20, RIi-5*; 21, RIq-5; 22, RSp-4; 23, RSn-5; 24,
RGd-6; 25, RMb-5*; 26, RGy-5*; 27, RUs-4; and 28, RPm-5. Animals
with the A*01 allele are indicated with asterisks.
[0144] Gene gun deliveries of DNA were not used because these had
primed non-protective immune responses in a 1996-98 trial (H. L.
Robinson et al. 1999 Nat Med 5:526). The MVA/89.6 booster
immunization (2.times.10.sup.8 plaque-forming units, pfu) was
injected with a needle both i.d. and i.m. A control group included
two mock immunized animals and two naive animals. The challenge was
given at 7 months after the rMVA booster to test for the generation
of long-term immunity. Because most HIV-1 infections are
transmitted across mucosal surfaces, an intrarectal challenge was
administered.
[0145] DNA priming followed by rMVA boosting generated high
frequencies of virus-specific T cells that peaked at one week
following the rMVA booster (FIG. 7). The frequencies of T cells
recognizing the Gag-CM9 epitope were assessed by means of Mamu-A*01
tetramers, and the frequencies of T cells recognizing epitopes
throughout Gag were assessed with pools of overlapping peptides and
an enzyme-linked immunospot (ELISPOT) assay (C. A. Power et al.
1999 J Immunol Methods 227:99).
[0146] For tetramer analyses, about 1.times.10.sup.6 peripheral
blood mononuclear cells (PBMC) were surface-stained with antibodies
to CD3 conjugated to fluorescein isothiocyanate (FITC) (FN-18;
Biosource International, Camarillo, Calif.), CD8 conjugated to
peridinin chlorophyl protein (PerCP) (SK1; Becton Dickinson, San
Jose, Calif.), and Gag-CM9 (CTPYDINQM)-Mamu-A*01 tetramer (SEQ ID
NO: 6) conjugated to allophycocyanin (APC), in a volume of 100
.mu.l at 8.degree. to 10.degree. C. for 30 min. Cells were washed
twice with cold PBS containing 2% fetal bovine serum (FBS), fixed
with 1% paraformaldehyde in PBS, and analyzed within 24 hrs on a
FACScaliber (Becton Dickinson, San Jose, Calif.). Cells were
initially gated on lymphocyte populations with forward scatter and
side scatter and then on CD3 cells. The CD3 cells were then
analyzed for CD8 and tetramer-binding cells. About 150,000
lymphocytes were acquired for each sample. Data were analyzed using
FloJo software (Tree Star, San Carlos, Calif.).
[0147] For interferon-.gamma. (IFN-.gamma.) ELISPOTs, MULTISCREEN
96 well filtration plates (Millipore Inc. Bedford, Mass.) were
coated overnight with antibody to human IFN-.gamma. (Clone B27,
Pharmingen, San Diego, Calif.) at a concentration of 2 .mu.g/ml in
sodium bicarbonate buffer (pH 9.6) at 8.degree. to 10.degree. C.
Plates were washed two times with RPMI medium and then blocked for
1 hour with complete medium (RPMI containing 10% FBS) at 37.degree.
C. Plates were washed five more times with plain RPMI medium, and
cells were seeded in duplicate in 100 .mu.l complete medium at
numbers ranging from 2.times.10.sup.4 to 5.times.10.sup.5 cells per
well. Peptide pools were added to each well to a final
concentration of 2 .mu.g/ml of each peptide in a volume of 100
.mu.l in complete medium. Cells were cultured at 37.degree. C. for
about 36 hrs under 5% CO.sub.2. Plates were washed six times with
wash buffer (PBS with 0.05% TWEEN.RTM.-20) and then incubated with
1 .mu.g of biotinylated antibody to human IFN-.gamma. per
milliliter (clone 7-86-1; Diapharma Group, West Chester, Ohio)
diluted in wash buffer containing 2% FBS. Plates were incubated for
2 hrs at 37.degree. C. and washed six times with wash buffer.
Avidin-horseradish peroxidase (Vector Laboratories, Burlingame,
Calif.) was added to each well and incubated for 30 to 60 min at
37.degree. C. Plates were washed six times with wash buffer and
spots were developed using stable DAB as substrate (Research
Genetics, Huntsville, Ala.). Spots were counted with a stereo
dissecting microscope. An ovalbumin peptide (SIINFEKL) (SEQ ID NO:
7) was included as a control in each analysis. Background spots for
the ovalbumin peptide were generally <5 for 5.times.10.sup.5
PBMCs. This background when normalized for 1.times.10.sup.6 PBMC
was <10. Only ELISPOT counts of twice the background
(.gtoreq.20) were considered significant. The frequencies of
ELISPOTs are approximate because different dilutions of cells have
different efficiencies of spot formation in the absence of feeder
cells (C. A. Power et al. 1999 J Immunol Methods 227: 99). The same
dilution of cells was used for all animals at a given time point,
but different dilutions were used to detect memory and acute
responses.
[0148] Gag-CM9 tetramer analyses were restricted to macaques that
expressed the Mamu-A*01 histocompatibility type, whereas ELISPOT
responses did not depend on a specific histocompatibility type. As
expected, the DNA immunizations raised low levels of memory cells
that expanded to high frequencies within 1 week of the rMVA booster
(FIGS. 7 and 12). In Mamu-A*01 macaques, CD8 cells specific to the
Gag-CM9 epitope expanded to frequencies as high as 19% of total CD8
T cells (FIG. 12). This peak of specific cells underwent a 10- to
100-fold contraction into the DNA/MVA memory pool (FIGS. 7A and
12). ELISPOTs for three pools of Gag peptides also underwent a
major expansion (frequencies up to 4000 spots for 1.times.10.sup.6
PBMC) before contracting from 5- to 20-fold into the DNA/MVA memory
response (FIG. 7B). The frequencies of ELISPOTs were the same in
macaques with and without the A*01 histocompatibility type
(P>0.2).
[0149] Simple linear regression was used to estimate correlations
between postbooster and postchallenge ELISPOT responses, between
memory and postchallenge ELISPOT responses, and between
logarithmically transformed viral loads and ELISPOT frequencies.
Comparisons between vaccine and control groups and A*01 and non
A*01 macaques were performed by means of two-sample t tests with
logarithmically transformed viral load and ELISPOT responses.
Two-way analyses of variance were used to examine the effects of
dose and route of administration on peak DNA/MVA ELISPOTs, on
memory DNA/MVA ELISPOTs, and on logarithmically transformed Gag
antibody data.
[0150] At both peak and memory phases of the vaccine response, the
rank order for the height of the ELISPOTs in the vaccine groups was
2.5 mg i.d.>2.5 mg i.m.>250 .mu.g i.d.>250 .mu.g i.m.
(FIG. 7B). The IFN-.gamma. ELISPOTs included both CD4 and CD8
cells. Gag-CM9-specific CD8 cells had good lytic activity after
restimulation with peptide.
[0151] The highly pathogenic SHIV-89.6P challenge was administered
intrarectally at 7 months after the rMVA booster, when
vaccine-raised T cells were in memory (FIG. 7).
[0152] The challenge stock (5.7.times.10.sup.9 copies of viral RNA
per milliliter) was produced by one intravenous followed by one
intrarectal passage in rhesus macaques of the original SHIV-89.6P
stock (G. B. Karlsson et al. 1997 J Virol 71:4218). Lymphoid cells
were harvested from the intrarectally infected animal at peak
viremia, CD8-depleted, and mitogen-stimulated for stock production.
Before intrarectal challenge, fasted animals were anesthetized
(ketamine, 10 mg/kg) and placed on their stomach with the pelvic
region slightly elevated. A feeding tube (8Fr (2.7 mm).times.16
inches (41 cm); Sherwood Medical, St. Louis, Mo.) was inserted into
the rectum for a distance of 15 to 20 cm. Following insertion of
the feeding tube, a syringe containing 20 intrarectal infectious
doses in 2 ml of RPMI-1640 plus 10% FBS was attached to the tube
and the inoculum was slowly injected into the rectum. After
delivery of the inoculum, the feeding tube was flushed with 3.0 ml
of RPMI without FBS and then slowly withdrawn. Animals were left in
place, with pelvic regions slightly elevated, for a period of ten
minutes after the challenge.
[0153] The challenge infected all of the vaccinated and control
animals (FIG. 8). However, by 2 weeks after challenge, titers of
plasma viral RNA were at least 10-fold lower in the vaccine groups
(geometric means of 1.times.10.sup.7 to 5.times.10.sup.7) than in
the control animals (geometric mean of 4.times.10.sup.8) (FIG. 8A)
(S. Staprans et al. in: Viral Genome Methods K. Adolph, ed. CRC
Press, Boca Raton, Fla., 1996 pp. 167-184; R. Hofmann-Lehmann et
al. 2000 AIDS Res Hum Retroviruses 16:1247).
[0154] For the determination of SHIV copy number, viral RNA from
150 .mu.l of ACD anticoagulated plasma was directly extracted with
the QIAamp Viral RNA kit (Qiagen), eluted in 60 .mu.l of AVE
buffer, and frozen at -80.degree. C. until SHIV RNA quantitation
was performed. Five microliters of purified plasma RNA was
reverse-transcribed in a final 20-.mu.l volume containing 50 mM
KCl, 10 mM Tris-HCl (pH 8.3), 4 mM MgCl.sub.2, 1 mM each
deoxynucleotide triphosphate (dNTP), 2.5 .mu.M random hexamers, 20
units MultiScribe RT, and 8 units ribonuclease inhibitor. Reactions
were incubated at 25.degree. C. for 10 min, followed by incubation
at 42.degree. C. for 20 min, and inactivation of reverse
transcriptase at 99.degree. C. for 5 min. The reaction mix was
adjusted to a final volume of 50 .mu.l containing 50 mM KCl, 10 mM
Tris-HCl (pH 8.3), 4 mM MgCl.sub.2, 0.4 mM each dNTP, 0.2 .mu.M
forward primer, 0.2 .mu.M reverse primer, 0.1 .mu.M probe, and 5
units AmpliTaq Gold DNA polymerase (all reagents from PerkinElmer
Applied Biosystems, Foster City, Calif.). The primer sequences
within a conserved portion of the SIV gag gene are the same as
those described previously (S. Staprans et al. in: Viral Genome
Methods K. Adolph, ed. CRC Press, Boca Raton, Fla., 1996 pp.
167-184). A PerkinElmer Applied Biosystems 7700 Sequence Detection
System was used with the PCR profile: 95.degree. C. for 10 min,
followed by 40 cycles at 93.degree. C. for 30 s, and 59.5.degree.
C. for 1 min. PCR product accumulation was monitored with the 7700
sequence detector and a probe to an internal conserved gag gene
sequence: 6FAM-CTGTCTGCGTCATTTGGTGC-Tamra (SEQ ID NO: 8), where FAM
and Tamra denote the reporter and quencher dyes. SHIV RNA copy
number was determined by comparison with an external standard curve
consisting of virion-derived SIVmac239 RNA quantified by the SIV
bDNA method (Bayer Diagnostics, Emeryville, Calif.). All specimens
were extracted and amplified in duplicate, with the mean result
reported. With a 0.15-ml plasma input, the assay has a sensitivity
of 10.sup.3 RNA copies per milliliter of plasma and a linear
dynamic range of 10.sup.3 to 10.sup.8 RNA copies (R.sup.2=0.995).
The intraassay coefficient of variation was <20% for samples
containing >10.sup.4 SHIV RNA copies per milliliter, and <25%
for samples containing 10.sup.3 to 10.sup.4 SHIV RNA copies per
milliliter. To more accurately quantitate low SHIV RNA copy number
in vaccinated animals at weeks 16 and 20, we made the following
modifications to increase the sensitivity of the SHIV RNA assay:
(i) Virions from <1 ml of plasma were concentrated by
centrifugation at 23,000 g at 10.degree. C. for 150 min before
viral RNA extraction, and (ii) a one-step reverse transcriptase PCR
method was used (R. Hofmann-Lehmann et al. 2000 AIDS Res Hum
Retroviruses 16:1247). These changes provided a reliable
quantification limit of 300 SHIV RNA copies per milliliter, and
gave SHIV RNA values that were highly correlated to those obtained
by the first method used (r=0.91, P<0.0001).
[0155] By 8 weeks after challenge, both high-dose DNA-primed groups
and the low-dose i.d. DNA-primed group had reduced their geometric
mean loads to about 1000 copies of viral RNA per milliliter. At
this time, the low-dose i.m. DNA-primed group had a geometric mean
of 6.times.10.sup.3 copies of viral RNA and the nonvaccinated
controls had a geometric mean of 2.times.10.sup.6. By 20 weeks
after challenge, even the low-dose i.m. group had reduced its
geometric mean copies of viral RNA to 1000. Among the 24 vaccinated
animals, only one animal, animal number 22 in the low-dose i.m.
group, had intermittent viral loads above 1.times.10.sup.4 copies
per milliliter (FIG. 8D).
[0156] By 5 weeks after challenge, all of the nonvaccinated
controls had undergone a profound depletion of CD4 cells (FIG. 8B).
All of the vaccinated animals maintained their CD4 cells, with the
exception of animal 22 in the low dose i.m. group (see above),
which underwent a slow CD4 decline (FIG. 8E). By 23 weeks after
challenge, three of the four control animals had succumbed to AIDS
(FIG. 8C). These animals had variable degrees of enterocolitis with
diarrhea, cryptosporidiosis, colicystitis, enteric campylobacter
infection, splenomegaly, lymphadenopathy, and SIV-associated giant
cell pneumonia. In contrast, all 24 vaccinated animals maintained
their health.
[0157] Containment of the viral challenge was associated with a
burst of antiviral T cells (FIGS. 7 and 9A). At one week after
challenge, the frequency of tetramer.sup.+ cells in the peripheral
blood had decreased, potentially reflecting the recruitment of
specific T cells to the site of infection (FIG. 9A). However, by
two weeks after challenge, tetramer.sup.+ cells in the peripheral
blood had expanded to frequencies as high as, or higher than, after
the rMVA booster (FIGS. 7 and 9A). The majority of the
tetramer.sup.+ cells produced IFN-.gamma. in response to a 6-hour
peptide stimulation (FIG. 9B) (S. L. Waldrop et al. 1997 J Clin
Invest 99:1739) and did not have the "stunned" IFN-.gamma. negative
phenotype sometimes observed in viral infections (F. Lechner et at
2000 J Exp Med 191:1499).
[0158] For intracellular cytokine assays, about 1.times.10.sup.6
PBMC were stimulated for 1 hour at 37.degree. C. in 5 ml
polypropylene tubes with 100 .mu.g of Gag-CM9 peptide (CTPYDINQM)
(SEQ ID NO: 6) per milliliter in a volume of 100 .mu.l RPMI
containing 0.1% bovine serum albumin (BSA) and 1 .mu.g of antibody
to human CD28 and 1 .mu.g of antibody to human CD49d (Pharmingen,
San Diego, Calif.) per milliliter. Then, 900 .mu.l of RPMI
containing 10% FBS and monensin (10 .mu.g/ml) was added, and the
cells were cultured for an additional 5 hrs at 37.degree. C. at an
angle of 5.degree. under 5% CO.sub.2. Cells were surface stained
with antibodies to CD8 conjugated to PerCP (clone SK1, Becton
Dickinson) at 8.degree. to 10.degree. C. for 30 min, washed twice
with cold PBS containing 2% FBS, and fixed and permeabilized with
CYTOFIX/CYTOPERM.TM. solution (Pharmingen). Cells were then
incubated with antibodies to human CD3 (clone FN-18; Biosource
International, Camarillo, Calif.) and IFN-.gamma. (Clone B27;
Pharmingen) conjugated to FITC and phycoerythrin, respectively, in
Perm wash solution (Pharmingen) for 30 min at 4.degree. C. Cells
were washed twice with Perm wash, once with plain PBS, and
resuspended in 1% paraformaldehyde in PBS. About 150,000
lymphocytes were acquired on the FACScaliber and analyzed with
FloJo software.
[0159] The postchallenge burst of T cells contracted concomitant
with the decline of the viral load. By 12 weeks after challenge,
virus-specific T cells were present at about one-tenth of their
peak height (FIGS. 7A and 9A). In contrast to the vigorous
secondary response in the vaccinated animals, the naive animals
mounted a modest primary response (FIGS. 7B and 9A). Tetramer.sup.+
cells peaked at less than 1% of total CD8 cells (FIG. 9A), and
IFN-.gamma.-producing ELISPOTs were present at a mean frequency of
about 300 as opposed to the much higher frequencies of 1000 to 6000
in the vaccine groups (FIG. 7B) (P<0.05).
[0160] The tetramer.sup.+ cells in the control group, like those in
the vaccine group, produced IFN-.gamma. after peptide stimulation
(FIG. 9B). By 12 weeks after challenge, three of the four controls
had undetectable levels of IFN-.gamma.-producing ELISPOTs. This
rapid loss of antiviral T cells in the presence of high viral loads
may reflect the lack of CD4 help.
[0161] T cell proliferative responses demonstrated that
virus-specific CD4 cells had survived the challenge and were
available to support the antiviral immune response (FIG. 9C).
[0162] About 0.2 million PBMC were stimulated in triplicate for 5
days with the indicated antigen in 200 .mu.l of RPMI at 37.degree.
C. under 5% CO.sub.2. Supernatants from 293T cells transfected with
DNA expressing either SHIV-89.6 Gag and Pol or SHIV-89.6 Gag, Pol
and Env were used directly as antigens (final concentration of
.about.0.5 .mu.g of p27 Gag per milliliter). Supernatants from mock
DNA (vector alone)-transfected cells served as negative controls.
On day six, cells were pulsed with 1 .mu.Ci of tritiated thymidine
per well for 16 to 20 hours. Cells were harvested with an automated
cell harvester (TOMTEC, Harvester 96, Model 1010, Hamden, Conn.)
and counted with a Wallac 1450 MICROBETA Scintillation counter
(Gaithersburg, Md.). Stimulation indices are the counts of
tritiated thymidine incorporated in PBMC stimulated with 89.6
antigens divided by the counts of tritiated thymidine incorporated
by the same PBMC stimulated with mock antigen.
[0163] At 12 weeks after challenge, mean stimulation indices for
Gag-Pol-Env or Gag-Pol proteins ranged from 35 to 14 in the vaccine
groups but were undetectable in the control group. Consistent with
the proliferation assays, intracellular cytokine assays
demonstrated the presence of virus-specific CD4 cells in vaccinated
but not control animals. The overall rank order of the vaccine
groups for the magnitude of the proliferative response was 2.5 mg
i.d.>2.5 mg i.m.>250 .mu.g i.d.>250 .mu.g i.m.
[0164] At 12 weeks after challenge, lymph nodes from the vaccinated
animals were morphologically intact and responding to the
infection, whereas those from the infected controls had been
functionally destroyed (FIG. 10). Nodes from vaccinated animals
contained large numbers of reactive secondary follicles with
expanded germinal centers and discrete dark and light zones (FIG.
10A). By contrast, lymph nodes from the non-vaccinated control
animals showed follicular and paracortical depletion (FIG. 10B),
while those from unvaccinated and unchallenged animals displayed
normal numbers of minimally reactive germinal centers (FIG. 10C).
Germinal centers occupied <0.05% of total lymph node area in the
infected controls, 2% of the lymph node area in the uninfected
controls, and up to 18% of the lymph node area in the vaccinated
groups (FIG. 10D). More vigorous immune reactivity in the low-dose
than the high-dose DNA-primed animals was suggested by more
extensive germinal centers in the low dose group (FIG. 10D). At 12
weeks after challenge, in situ hybridization for viral RNA revealed
rare virus-expressing cells in lymph nodes from 3 of the 24
vaccinated macaques, whereas virus-expressing cells were readily
detected in lymph nodes from each of the infected control animals.
In the controls, which had undergone a profound depletion in CD4 T
cells, the cytomorphology of infected lymph node cells was
consistent with a macrophage phenotype.
[0165] The prime/boost strategy raised low levels of antibody to
Gag and undetectable levels of antibody to Env (FIG. 11).
Postchallenge, antibodies to both Env and Gag underwent anamnestic
responses with total Gag antibody reaching heights approaching 1
mg/ml and total Env antibody reaching heights of up to 100
.mu.g/ml.
[0166] Enzyme-linked immunosorbent assays (ELISAs) for total
antibody to Gag used bacterially produced SIV gag p27 to coat wells
(2 .mu.g per milliliter in bicarbonate buffer). ELISAs for antibody
to Env antibody used 89.6 Env produced in transiently transfected
293T cells and captured with sheep antibody against Env (catalog
number 6205; International Enzymes, Fairbrook Calif.). Standard
curves for Gag and Env ELISAs were produced with serum from a
SHIV-89.6-infected macaque with known amounts of immunoglobulin G
(IgG) specific for Gag or Env. Bound antibody was detected with
peroxidase-conjugated goat antibody to macaque IgG (catalog
#YNGMOIGGFCP; Accurate Chemical, Westbury, N.Y.) and TMB substrate
(Catalog #T3405; Sigma, St. Louis, Mo.). Sera were assayed at
threefold dilutions in duplicate wells. Dilutions of test sera were
performed in whey buffer (4% whey and 0.1% TWEEN.RTM. 20 in
1.times.PBS). Blocking buffer consisted of whey buffer plus 0.5%
nonfat dry milk. Reactions were stopped with 2M H.sub.2SO.sub.4 and
the optical density read at 450 nm. Standard curves were fitted and
sample concentrations were interpolated as .mu.g of antibody per ml
of serum using SOFTmax 2.3 software (Molecular Devices, Sunnyvale,
Calif.).
[0167] By 2 weeks after challenge, neutralizing antibodies for the
89.6 immunogen, but not the SHIV-89.6P challenge, were present in
the high-dose DNA-primed groups (geometric mean titers of 352 in
the i.d. and 303 in the i.m. groups) (FIG. 11C) (D. C. Montefiori
et al. 1988 J Clin Microbiol 26:231). By 5 weeks after challenge,
neutralizing antibody to 89.6P had been generated (geometric mean
titers of 200 in the high-dose i.d. and 126 in the high-dose i.m.
group) (FIG. 11D) and neutralizing antibody to 89.6 had started to
decline. By 16 to 20 weeks after challenge, antibodies to Gag and
Env had fallen in most animals.
[0168] Our results demonstrate that a multiprotein DNA/MVA vaccine
can raise a memory immune response capable of controlling a highly
virulent mucosal immunodeficiency virus challenge. Our levels of
viral control were more favorable than have been achieved using
only DNA (M. A. Egan et al. 2000 J Virol 74:7485) or rMVA vaccines
(I. Ourmanov et al. 2000 J Virol 74:2740) and were comparable to
those obtained for DNA immunizations adjuvanted with interleukin-2
(D. H. Barouch et al. 2000 Science 290:486). All of these previous
studies have used more than three vaccine inoculations, none have
used mucosal challenges, and most have challenged at peak effector
responses and not allowed a prolonged post vaccination period to
test for "long term" efficacy.
[0169] The dose of DNA had statistically significant effects on
both cellular and humoral responses (P<0.05), whereas the route
of DNA administration affected only humoral responses. Intradermal
DNA delivery was about 10 times more effective than i.m.
inoculations for generating antibody to Gag (P=0.02). Neither route
nor dose of DNA appeared to have a significant effect on
protection. At 20 weeks after challenge, the high-dose DNA-primed
animals had slightly lower geometric mean levels of viral RNA
(7.times.10.sup.2 and 5.times.10.sup.2) than the low-dose
DNA-primed animals (9.times.10.sup.2 and 1.times.10.sup.3).
[0170] The DNA/MVA vaccine controlled the infection, rapidly
reducing viral loads to near or below 1000 copies of viral RNA per
milliliter of blood. Containment, rather than prevention of
infection, affords the opportunity to establish a chronic infection
(H. L. Robinson et al. 1999 Nat Med 5:526). By rapidly reducing
viral loads, a multiprotein DNA/MVA vaccine will extend the
prospect for long-term non-progression and limit HIV transmission
(J. W. Mellors et al. 1996 Science 272:1167; T. C. Quinn et al.
2000 N Engl J Med 342:921).
Example 2
MVA Expressing Modified HIV Env, Gag, and Pol Genes
[0171] This disclosure describes the construction of a modified
vaccinia Ankara (MVA) recombinant virus, MVA/HIV clade B
recombinant virus expressing the HIV strain ADA env and the HXB2
gag pol (MVA/HIV ADA env+HXB2 gag pol). For amplification, the lab
name of MVA/HIV 48 will be used, which denotes the plasmid from
which the construct comes.
[0172] The HIV gag-pol genes were derived from the Clade B
infectious HXB2 virus. The gag-pol gene was truncated so that most
of the integrase coding sequences were removed and amino acids 185,
266, and 478 were mutated to inactivate reverse transcriptase,
inhibit strand transfer activity, and inhibit the RNaseH activity,
respectively. The Clade B CCR5 tropic envelope gene was derived
from the primary ADA isolate; TTTTTNT (SEQ ID NO: 14) sequences
were mutated without changing coding capacity to prevent premature
transcription termination and the cytoplasmic tail was truncated in
order to improve surface expression, immunogenicity, and stability
of the MVA vector. The HIV genes were inserted into a plasmid
transfer vector so that gag-pol gene was regulated by the modified
H5 early/late vaccinia virus promoter and the env gene was
regulated by the newly designed early/late Psyn II promoter to
provide similar high levels of expression. A self-deleting GUS
reporter gene was included to allow detection and isolation of the
recombinant virus. The HIV genes were flanked by MVA sequences to
allow homologous recombination into the deletion 3 site so that the
recombinant MVA would remain TK positive for stability and high
expression in resting cells. The recombinant MVA was isolated and
shown to express abundant amounts of gag-pol-env and to process
gag. Production of HIV-like particles was demonstrated by
centrifugation and by electron microscopy. The presence of env in
the HIV-like particles was demonstrated by immunoelectron
microscopy.
TABLE-US-00002 Table of Sequences Description SEQ ID NO FIG. NO
pLW-48 map N/A 13 pLW-48 sequence 1 14 Psyn II promoter 2 14 ADA
envelope truncated 3 14 PmH5 promoter 4 14 HXB2 gag pol 5 14
Plasmid Transfer Vector
[0173] The plasmid transfer vector used to make the MVA recombinant
virus, pLW-48, (FIG. 15) by homologous recombination was
constructed as follows:
[0174] 1. From the commercially obtained plasmid, pGem-4Z
(Promega), flanking areas on either side of deletion III,
designated flank 1 and flank 2, containing 926 and 520 base pairs
respectively, were amplified by PCR from the MVA stains of vaccinia
virus. Within these flanks, a promoter, the mH5, which had been
modified from the originally published sequence by changing two
bases that had been shown by previously published work to increase
the expression of the cloned gene, was added.
[0175] 2. A clade B gag pol (FIG. 16) was truncated so that the
integrase was removed and was cloned into the plasmid so that it
was controlled by the mH5 promoter. This gene contained the
complete HXB2 sequence of the gag. The pol gene has reverse
transcriptase safety mutations in amino acid 185 within the active
site of RT, in amino acid 266 which inhibits strand transfer
activity, and at amino acid 478 which inhibits the RNaseH activity.
In addition, the integrase gene was deleted past EcoRI site.
[0176] 3. A direct repeat of 280 basepairs, corresponding to the
last 280 base pairs of MVA flank 1, was added after flank 1.
[0177] 4. The p11 promoter and GUS reporter gene were added between
the two direct repeats of flank 1 so that this screening marker
could initially be used for obtaining the recombinant virus, yet
deleted out in the final recombinant virus (Scheiflinger, F. et al.
1998 Arch Virol 143:467-474; Carroll, M. W. and B. Moss 1995
BioTechniques 19:352-355).
[0178] 5. A new promoter, Psyn II, was designed to allow for
increased expression of the ADA env. The sequence of this new
early/late promoter is given in FIG. 17.
[0179] 6. A truncated version of the ADA envelope with a silent
5TNT mutation was obtained by PCR and inserted in the plasmid under
the control of the Psyn II promoter. The envelope was truncated in
the cytoplasmic tail of the gp41 gene, deleting 115 amino acids of
the cytoplasmic tail. This truncation was shown to increase the
amount of envelope protein on the surface of infected cells and
enhance immunogenicity of the envelope protein in mice, and
stability of the recombinant virus in tissue culture.
Recombinant MVA Construction
[0180] 1. MVA virus, which may be obtained from ATCC Number
VR-1508, was plaque purified three times by terminal dilutions in
chicken embryo fibroblasts (CEF), which were made from 9 day old
SPF Premium SPAFAS fertile chicken eggs, distributed by B and E
Eggs, Stevens, Pa.
[0181] 2. Secondary CEF cells were infected at an MOI of 0.05 of
MVA and transfected with 2 .mu.g of pLW-48, the plasmid described
above. Following a two-day incubation at 37.degree. C., the virus
was harvested, frozen and thawed 3.times., and plated out on CEF
plates.
[0182] 3. At 4 days, those foci of infection that stained blue
after addition of X-gluc substrate, indicating that recombination
had occurred between the plasmid and the infecting virus, were
picked and inoculated on CEF plates. Again, those foci that stained
blue were picked.
[0183] 4. These GUS containing foci were plated out in triplicate
and analyzed for GUS staining (which we wanted to now delete) and
ADA envelope expression. Individual foci were picked from the 3rd
replicate plates of those samples that had about equal numbers of
mixed populations of GUS staining and nonstaining foci as well as
mostly envelope staining foci.
[0184] 5. These foci were again plated out in triplicate, and
analyzed the same way. After 5 passages, a virus was derived which
expressed the envelope protein but which had deleted the GUS gene
because of the double repeat. By immunostaining, this virus also
expressed the gag pol protein.
Characterization of MVA Recombinant Virus, MVA/HIV 48
[0185] 1. Aliquots of MVA/HIV 48 infected cell lysates were
analyzed by radioimmunoprecipitation and immunostaining with
monoclonal antibodies for expression of both the envelope and gag
pol protein. In both of these tests, each of these proteins was
detected.
[0186] 2. The recombinant virus was shown to produce gag particles
in the supernatant of infected cells by pelleting the
.sup.35S-labeled particles on a 20% sucrose cushion.
[0187] 3. Gag particles were also visualized both outside and
budding from cells as well as within vacuoles of cells in the
electron microscope in thin sections. These gag particles had
envelope protein on their surface.
[0188] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer, and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
SUMMARY
[0189] In summary, we have made a recombinant MVA virus, MVA/HIV
48, which has high expression of the ADA truncated envelope and the
HXB2 gag pol. The MVA recombinant virus is made using a transiently
expressed GUS marker that is deleted in the final virus. High
expression of the ADA envelope is possible because of a new hybrid
early/late promoter, Psyn II. In addition, the envelope has been
truncated because we have shown truncation of the envelope enhances
the amount of protein on the surface of the infected cells, and
hence enhances immunogenicity; stability of the recombinant is also
enhanced. The MVA recombinant makes gag particles which have been
shown by pelleting the particles through sucrose and analyzing by
PAGE. Gag particles with envelope protein on the surface have also
been visualized in the electron microscope.
Example 3
Additional Modified or Synthetic Promoters Designed for Gene
Expression in MVA Or Other Poxviruses
[0190] Additional modified or synthetic promoters were designed for
gene expression in MVA or other poxviruses. Promoters were modified
to allow expression at early and late times after infection and to
reduce possibility of homologous recombination between identical
sequences when multiple promoters are used in same MVA vector.
Promoters are placed upstream of protein coding sequence.
TABLE-US-00003 m7.5 promoter (SEQ ID NO: 10):
CGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAA
TTTCTCGTAAAAATTGAAAAACTATTCTAATTTATTGCACGGT Psyn II promoter (SEQ
ID NO: 2): TAAAAAATGAAAAAATATTCTAATTTATAGGACGGTTTTGATTTTCTTT
TTTTCTATGCTATAAATAATAAATA Psyn III promoter (SEQ ID NO: 11):
TAAAAATTGAAAAAATATTCTAATTTATAGGACGGTTTTGATTTTCTTTT
TTTCTATACTATAAATAATAAATA Psyn IV promoter (SEQ ID NO: 12):
TAAAAATTGAAAAACTATTCTAATTTATAGGACGGTTTTGATTTTCTTTT
TTTCTATACTATAAATAATAAATA Psyn V promoter (SEQ ID NO: 13):
AAAAAATGATAAAGTAGGTTCAGTTTTATTGCTGGTTTAAAATCACGCT
TTCGAGTAAAAACTACGAATATAAAT
Example 4
Tables A-F
[0191] Table A: MVA/48 Immunization--Guinea Pigs.
[0192] Groups of guinea pigs were immunized at days 0 and 30 with
1.times.10.sup.8 infectious units of MVA/48 by either the
intramuscular (IM) or intradermal (ID) route. As a control another
group was immunized IM with the same dose of non-recombinant MVA.
Sera taken before as well as after each immunization were analyzed
for neutralizing activity against HIV-1-MN. Titers are the
reciprocal serum dilution at which 50% of MT-2 cells were protected
from virus-induced killing. Significant neutralizing activity was
observed in all animals after the second immunization with MVA/48
(day 49).
[0193] Table B: Frequencies of HIV-1 gag-Specific T Cells Following
Immunization of Mice with MVA/48.
[0194] Groups of Balb/c mice were immunized at days 0 and 21 with
1.times.10.sup.7 infectious units of MVA/48 by one of three routes:
intraperitoneal (IP), intradermal (ID), or intramuscular (IM). A
control group was immunized with non-recombinant MVA. At 5 weeks
after the last immunization, splenocytes were prepared and
stimulated in vitro with an immunodominant peptide from HIV-1 p24
for 7 days. The cells were then mixed either with peptide-pulsed
P815 cells or with soluble peptide. Gamma interferon-producing
cells were enumerated in an ELISPOT assay. A value of >500 was
assigned to wells containing too many spots to count. Strong T cell
responses have been reported in mice immunized IP with other
viruses. In this experiment, IP immunization of mice with MVA/48
elicited very strong HIV-1 gag-specific T cell responses.
[0195] Table C: DNA Prime and MVA/48 Boost--Total ELISPOTs Per
Animal.
[0196] Ten rhesus macaques were primed (weeks 0 and 8) with a DNA
vaccine expressing HIV-1 antigens including Ada envelope and HXB2
gagpol. At week 24 the animals were boosted intramuscularly with
1.times.10.sup.8 infectious units of MVA/48. Fresh peripheral blood
mononuclear cells (PBMC) were analyzed for production of gamma
interferon in an ELISPOT assay as follows: PBMC were incubated for
30-36 hours in the presence of pools of overlapping peptides
corresponding to the individual HIV-1 antigens in the vaccines. The
total number of gamma interferon-producing cells from each animal
is shown in the table. T cell responses to DNA vaccination were
limited (weeks 2-20). However, boosting with MVA/48 resulted in
very strong HIV-1-specific T cell responses in all animals (week
25).
[0197] Table D: Antibody Response Following Immunization of
Macaques with MVA/SHIV KB9.
[0198] Groups of rhesus macaques were immunized with
2.times.10.sup.8 infectious units of MVA/SHIV-KB9 at weeks 0 and 4
by one of several routes: Tonsilar, intradermal (ID), or
intramuscular (IM). Another group was immunized with
non-recombinant MVA using the same routes. Serum samples from 2
weeks after the second immunization were analyzed for binding to
KB9 envelope protein by ELISA and for neutralization of SHIV-89.6P
and SHIV-89.6. In the ELISA assay, soluble KB9 envelope protein was
captured in 96 well plates using an antibody to the C-terminus of
gp120. Serial dilutions of sera were analyzed and used to determine
the endpoint titers. Neutralization of SHIV-89.6P and SHIV-89.6 was
determined in an MT-2 cell assay. Titers are the reciprocal serum
dilution at which 50% of the cells were protected from
virus-induced killing. In in vitro neutralization assays,
SHIV-89.6P and SHIV-89.6 are heterologous, i.e. sera from animals
infected with one of the viruses do not neutralize the other virus.
Thus, two immunizations with MVA/SHIV-KB9 elicited good ELISA
binding antibodies in all animals and neutralizing antibodies to
the homologous virus (SHIV-89.6P) in some animals. In addition,
heterologous neutralizing antibodies were observed in a subset of
animals.
[0199] Table E: Frequencies of Gag CM-9-Specific CD3/CD8 T Cells
Following Immunization of Macaques with MVA/SHIV-KB9.
[0200] Groups of MamuA*01 positive rhesus macaques were immunized
with 2.times.10.sup.8 infectious units of MVA/SHIV-KB9 at weeks 0
and 4 by one of several routes: tonsilar, intradermal (ID), or
intramuscular IM). Another group was immunized with non-recombinant
MVA. The frequencies of CD3+/CD8+ T cells that bound tetrameric
complex containing the SIV gag-specific peptide CM9 were determined
by flow cytometry at various times after each immunization. Time
intervals were as follows: 1a, 1b, and 1d were one, two, and four
weeks after the first immunization, respectively; 2a, 2b, 2c, and
2d were one, two, three, and twelve weeks after the second
immunization, respectively. Values above background are shown in
bold face. Strong SIV gag-specific responses were observed after a
single immunization with MVA/SHIV-KB9 in all immunized animals.
Boosting was observed in most animals following the second
immunization. In addition, measurable tetramer binding was still
found twelve weeks after the second immunization.
[0201] Table F: Frequencies of Specific T Cells Following
Immunization of Macaques with MVA/SHIV KB9.
[0202] Groups of macaques were immunized with MVA/SHIV-KB9 as
described above. MVA/SHIV-KB9 expresses 5 genes from the chimeric
virus, SHIV-89.6P: envelope, gag, polymerase, tat, and nef. Thus,
the frequencies of T cells specific for each of the 5 antigens were
analyzed using pools of peptides corresponding to each individual
protein. Fresh PBMC were stimulated with pools of peptides for
30-36 hours in vitro. Gamma interferon-producing cells were
enumerated in an ELISPOT assay. The total number of cells specific
for each antigen is given as "total # spots". In addition, the
number of responding animals and average # of spots per group is
shown. PBMC were analyzed at one week after the first immunization
(1a) and one week after the second immunization (2a). Another group
of 7 animals was immunized with non-recombinant MVA. In these
animals, no spots above background levels were detected. Thus, a
single immunization with MVA/SHIV-KB9 elicited strong SHIV-specific
T cell responses in all animals. Gag and envelope responses were
the strongest; most animals had responses to gag, all animals had
responses to envelope. The Elispot responses were also observed
after the second immunization with MVA/SHIV-KB9, albeit at lower
levels. At both times, the rank order of responses was:
tonsilar>ID>IM. We show good immune response to nef and some
immune response to tat.
TABLE-US-00004 TABLE A MVA/48 immunization - guinea pigs HIV-MN
neutralizing antibody - reciprocal titer Day 4 day 3 day MVA day
MVA day Animal # Group Route 0 #1 30 #2 49 885 MVA I.M. <20 I.M.
31 I.M. 24 891 '' '' <20 '' 85 '' <20 882 MVA/48 I.M. <20
I.M. <20 I.M. 5,524 883 '' '' <20 '' 68 '' 691 886 '' ''
<20 '' <20 '' 4,249 890 '' '' <20 '' 180 '' 89 879 MVA/48
I.D. <20 I.D. <20 I.D. 817 881 '' '' <20 '' <20 '' 234
888 '' '' <20 '' 24 '' 112 889 '' '' <20 '' 22 '' 376
TABLE-US-00005 TABLE B Frequencies of HIV-gag-specific T cells
following immunization of mice with MVA/48 Group P815 cells + gag
peptide gag peptide no stimulation MVA control 0 2 0 4 1 2 MVA/48
(IP) >500 >500 >500 >500 8 8 MVA/48 (ID) 12 5 49 33 4 2
MVA/48 (IM) 22 18 66 49 12 8
TABLE-US-00006 TABLE C DNA prime and MVA/48 boost. Total ELISPOTS
per Animal WEEKS Animal # -2 2 6 10 .sup.2 14 .sup.2 20 .sup.2 25
.sup.2 I. RLW 4 731* < 47 43 50 3905 RVl 5 997* < < < 8
205 Roa .sup. < .sup.1 < 1 < < < 245 RHc < <
< < < < 535 Ryl < < < < < < 4130 RQk
< 46 < < < < 630 RDr < < < 14 < <
1965 RZc < 5 < 58 < < 925 RSf < 118 < < <
20 5570 Ras < 69 < < < < 1435 Total 9 1966 1 119 43
78 19545 Geo Mean 4.5 105.3 1.0 33.7 43.0 20.0 1147.7 DNA primes
were at 0 and 8 weeks and MVA/48 boost was at 24 weeks .sup.1 <
= Background (2x the number of ELISPOTs in the unstimulated control
+ 10) .sup.2 Costimulatory antibodies were added to the ELISPOT
incubations *Animals from this bleed date exhibited higher than
usual ELISPOTs.
TABLE-US-00007 TABLE D Antibody response following immunization of
macaques with MVA/SHIV KB9 SHIV- KB9 env SHIV- SHIV-89.6 89.6P
ELISA KB9 env elisa 89.6 SHIV-89.6P # pos # pos Animal # Route
titer average std dev. Nab titer Nab titer animals animals 598
tonsil 25,600 31,086 20,383 <20 <20 3 2 601 '' 51,200 <20
<20 606 '' 25,600 <20 <20 642 '' 51,200 75 31 646 ''
51,200 61 48 653 '' 6,400 <20 <20 654 '' 6,400 22 <20 602
i.d. 25,600 18,800 15,341 38 <20 2 4 604 '' 12,800 <20 262
608 '' 3,200 20 66 637 '' 12,800 <20 35 638 '' 51,200 <20
<20 645 '' 25,600 <20 <20 647 '' 12,800 32 162 650 ''
6,400 <20 <20 599 i.m. 6,400 17,000 16,516 <20 <20 0 3
600 '' 6,400 <20 29 609 '' 6,400 <20 <20 639 '' 51,200
<20 85 640 '' 12,800 <20 <20 641 '' 25,600 <20 41 649
'' 1,600 <20 <20 651 '' 25,600 20 <20 603 Control <100
<100 <20 <20 0 0 605 '' <100 <20 <20 607 ''
<100 <20 <20 643 '' <100 <20 <20 644 '' <100
<20 <20 648 '' <100 <20 <20 652 '' <100 <20
<20
TABLE-US-00008 TABLE E Frequencies of gag CM9-specific CD3/CD8 T
cells following immunization of macaques with MVA/SHIV KB9 pre-
Animal # Route Virus bleed 1a 1b 1d 2a 2b 2c 2d 598 Tonsil MVA/KB9
0.018 0.41 0.79 0.25 2.64 1.13 0.51 0.21 601 '' '' 0.071 0.34 0.38
0.27 0.83 0.7 0.36 0.039 646 '' '' 0.022 0.68 0.76 0.43 1.12 0.91
0.53 0.15 653 '' '' 0.041 0.69 0.85 0.53 0.68 0.49 0.47 0.3 648 ''
MVA 0.033 0.039 0.022 0.058 0.033 0.013 602 i.d. MVA/KB9 0.019 0.17
0.92 0.5 0.95 0.59 0.5 0.2 604 '' '' 0.013 0.11 0.38 0.32 0.44 0.38
0.19 0.25 650 '' '' 0.095 0.17 0.6 0.23 2.87 1.12 0.9 0.16 647 ''
'' 0.032 0.22 0.38 0.14 0.84 0.91 0.34 0.17 652 '' MVA 0.041 0.038
0.059 0.025 0.022 0.026 0.055 599 i.m. MVA/KB9 0.081 0.31 0.082
0.12 0.054 0.11 600 '' '' 0.034 0.15 0.41 0.17 0.29 0.27 0.16 0.049
649 '' '' 0.00486 0.35 1.34 0.56 2.42 0.77 0.69 0.22 651 '' ''
0.049 0.12 0.69 0.25 1.01 0.32 0.24 0.22 603 '' MVA 0.024 0.087
0.073 0.082 0.027 0.17
TABLE-US-00009 TABLE F Frequencies of specific T cells following
immunization of macaques with MVA/SHIV KB9 Gag specific Tat
specific Nef specific Env specific Total # Total average # total
average # total average # Total Average # Study responding # #
responding # # responding # # Responding # # Responding groups
animals spots spots animals spots spots animals spots spots animals
spots spots animals tonsil 4/6 1325 221 0/6 0 0 3/6 195 33 6/6 8760
1460 6/6 1a tonsil 5/6 1405 234 0/6 0 0 1/6 560 93 6/6 4485 748 6/6
2a i.d. 7/7 1335 191 0/7 0 0 2/7 215 31 7/7 7320 1046 7/7 1a i.d.
4/7 755 108 0/7 0 0 1/7 55 8 7/7 2700 386 7/7 2a i.m. 7/7 925 132
1/7 60 9 3/7 180 26 7/7 5490 784 7/7 1a i.m. 4/7 250 36 0/7 0 0 0/7
0 0 6/7 2205 315 6/7 2a
Example 5
Construction and Pre-clinical Immunogenicity of a Recombinant MVA
Vaccine Expressing Subtype D HIV-1 Env, Gag and Pol for Use in
Uganda
[0203] Recombinant MVA vaccines have been successful in generating
SIV and SHIV specific humoral and CD8 T cell responses in non-human
primates and, alone or in combination with DNA vaccines, have
provided protection in rhesus macaques from disease after
pathogenic SHIV challenge. An overall program goal is to conduct
clinical vaccine trials in Africa using vaccines that induce both
neutralizing antibody and CD8 T cell specific responses and that
are based upon representative full-length HIV-1 sequences isolated
from the target vaccine cohorts. The predominant incident and
prevalent HIV-1 subtype in Uganda is subtype D. Several R5 subtype
D HIV-1 strains were selected and used to prepare recombinant MVA
vaccines expressing env, gag, protease and RT. Initially, multiple
env and gag/pol clones from 3 pure Ugandan subtype D isolates were
selected. These sequences were separately cloned into pCR2.1 and
tested for expression levels in-vitro by immunoprecipitation, and
for envelope function as assessed by envelope-mediated fusion with
CD4 and CCR5 or CXCR4 expressing cells. Based on these in-vitro
analyses, several R5 subtype D env and gag/pol sequences were
selected and cloned into MVA shuttle plasmids containing GFP and
the modified H5 promoter for sequential cloning into deletions II
and III, respectively, of MVA. The parent MVA used was a 1974 stock
chosen to eliminate FDA concerns regarding potential BSE
contamination. Several recombinant MVA (rMVA-UGD) expressing
subtype D env and gag/pol were prepared in primary CEF cells using
gamma-irradiated FBS from a USDA approved source and selected using
GFP expression. These rMVA-UGD were further plaque-purified and
amplified to titers sufficient for in-vivo immunogenicity studies.
Pre-clinical humoral and cellular immunogenicity of the various
rMVA-UGD were then assessed in BALB/c mice.
MVA Expressing Altered HIV-1 Envelope, Gag, and Polymerase Genes
from Ugandan Clade D
[0204] This example describes the construction of 5 recombinant
Modified Vaccinia Virus Ankara (MVA) viruses expressing envelope
(env) and gagpol genes from HIV-1 clade D isolates from Uganda.
Sequences from Ugandan Clade D
[0205] Env and gagpol genes from three Ugandan clade D HIV-1
isolates were used:
TABLE-US-00010 HIV-1 Isolate name GenBank Accession # Lab
designation (LVD) 99UGA03349 AF484518 B 99UGA07412 AF484477 C
98UG57128 AF484502 D
[0206] Env and gagpol genes were PCR amplified from Ugandan HIV-1
clade D isolates by short term co-cultures on normal human PBMC
(Harris et al. 2002 AIDS Research and Human Retroviruses 18:1281)
using the oligonucleotides shown in Table G and cloned into
pCR2.1-TOPO (Invitrogen). (HIV-I infected individuals contain a
population or quasi-species of related but distinct viruses. Upon
co-culture, multiple viruses can emerge such that the sequences of
individual amplified genes from the co-culture may differ from the
sequence of the full genome.) The resulting amplified env genes
have a C-terminal deletion of 115 amino acids that was previously
shown to enhance expression and yield a more stable recombinant
virus. The resulting gagpol genes have a deletion of the entire
integrase and Rnase H portions of the genes. Within both the env
and gagpol genes, several mutations were made by site-directed
mutagenesis (Quik Change from Stratagene). In the env genes, silent
mutations were made to eliminate two naturally occurring vaccinia
virus early transcription termination signals (TTTTTNT, SEQ ID NO:
14) (Earl et. al 1990 J. Virol 64:2451). In the pol genes, two
mutations were made in separate locations in the active site of
reverse transcriptase to abolish enzymatic activity (Larder et. al
1987 Nature 327:716)(see Tables H(i) & (ii) for details on
changes made to env and gagpol genes).
[0207] PCR2.1-TOPO plasmids containing the amplified genes were
first characterized with respect to the orientation of the gene.
Clones in which the gene was oriented properly with respect to the
T7 promoter were chosen and protein expression was analyzed as
previously described (Earl et al. 1997 J Virol 71:2674). Briefly,
BS-C-1 cells were infected with vTF7-3 (Fuerst et al. 1986 PNAS USA
83:8122), a recombinant vaccinia virus expressing T7 RNA
polymerase, transfected with a plasmid, and metabolically labeled.
Cell lysates were subjected to immunoprecipitation with serum
pooled from several HIV-1 clade D-infected individuals. Proteins
were analyzed by SDS-polyacrylamide gel electrophoresis and
visualized by autoradiography. One env and one gagpol DNA clone
from each clade D isolate was chosen for construction of
recombinant MVA viruses. DNA sequencing was performed to confirm
the integrity of each gene. Sequences are presented in Appendix
1.
Cloning into Shuttle Plasmids
[0208] Two MVA shuttle plasmids, pLAS-1 and pLAS-2 (FIG. 18), were
used for construction of recombinant MVA viruses. DNA sequences of
both plasmids are presented in Appendix 2. In both plasmids, the
foreign gene is driven by the modified H5 promoter. In addition,
both plasmids contain a cassette with the gene for green
fluorescent protein (GFP) driven by the vaccinia p11 promoter. This
cassette is flanked by direct repeats that will readily recombine
to eliminate GFP during virus propagation. Thus, GFP is used as a
positive screening marker in early rounds of plaque purification,
and as a negative screening marker in final recombinant virus
selection (FIG. 22). MVA flanking sequences in pLAS-1 and pLAS-2
direct recombination into deletion III (Del III) and deletion II
(Del II) of MVA, respectively.
[0209] Gagpol genes from 2 isolates (99UGA03349 and 99UGA07412)
were cloned separately into pLAS-1 for insertion into Del III of
MVA. The plasmids were named pLAS-1/UGD/Bgag and pLAS-1/UGD/Cgag
(FIG. 19 & Table I). When the env gene is cloned into the NotI
restriction site, a short open reading frame precedes the env open
reading frame. This open reading frame is out of frame with env and
terminates at approximately nucleotide 75 in the env gene.
[0210] Env genes from three isolates (99UGA03349, 99UGA07412,
98UG57128) were cloned separately into MVA shuttle plasmid pLAS-2,
for insertion into Del II of MVA. Plasmids were named
pLAS-2/UGD/Benv, pLAS-2/UGD/Cenv, and pLAS-2/UGD/Denv (FIG. 20
& Table I). When the env gene is cloned into the NotI
restriction site, a short open reading frame precedes the env open
reading frame. This open reading frame is out of frame with env and
terminates at approximately nucleotide 75 in the env gene.
[0211] Foreign genes in pLAS-1 and pLAS-2 recombine into the
vaccinia genome in the same orientation as the surrounding vaccinia
genes. To test the effect of reversing the orientation of the env
gene on the level of gene expression and stability of viruses, two
of the env genes and their promoters were excised from pLAS-2 with
restriction endonucleases BspE1 and EcoRV; sticky ends were filled
in with Klenow enzyme; and the fragments were then reinserted into
pLAS-2 the opposite orientation (FIG. 21). Plasmids were named
pLAS-2/UGD/revCenv and pLAS-2/UGD/revDenv (Table I).
Recombinant MVA Construction
[0212] Parent MVA: MVA 1974/NIH Clone 1 was used as the parent for
all recombinant viruses. It was derived from a stock of MVA at
passage 572, prepared on Feb. 22, 1974 in the laboratory of A.Mayr
in Germany. After receipt in the Laboratory of Viral Diseases, this
stock was passaged a total of 6 times in chicken embryo fibroblast
(CEF) cells, including 3 clonal purifications. Amplification was
performed on the final, clonally purified virus. All CEF cells were
derived from specific pathogen-free (SPAFAS) eggs.
[0213] Recombinant viruses expressing gagpol: CEF cells were
infected with MVA 1974/NIH Clone 1 and transfected with either
pLAS-1/UGD/Bgag or pLAS-1/UGD/Cgag for insertion into Del III. Two
to three rounds of plaque purification were performed based on GFP
expression. Further rounds of plaque purification were performed by
picking plaques based on lack of GFP expression and concomitant
positive gag expression as measured by immunostaining using a
monoclonal antibody to HIV-1 p24 (183-H12-5C; obtained from the NIH
AIDS Research and Reference Reagent Program) (FIG. 22). Recombinant
gagpol-expressing viruses were amplified and characterized for gag
expression by immunoprecipitation as described above. The two
viruses were named MVA/UGD/Bgag and MVA/UGD/Cgag. These viruses
were then used as the parent in making gagpol/env recombinant
viruses (see below).
[0214] Recombinant viruses expressing gagpol and env: Recombinant
viruses, MVA/UGD/Bgag and MVA/UGD/Cgag were used as parent viruses
for insertion of env genes. Thus, CEF cells were infected with
either MVA/UGD/Bgag or MVA/UGD/Cgag and subsequently transfected
with one of the pLAS-2-env-containing plasmids described above
(FIG. 23 & Table I). As above, the first two rounds of plaque
purification were performed based on GFP expression. In subsequent
rounds of purification, plaques were selected based on loss of GFP
expression and positive gag and env expression as measured by
immunostaining in duplicate cultures (FIG. 22). A total of 5
gagpol/env-expressing viruses (MVA/UGD-1 through -5) were amplified
and characterized (Table J).
Characterization of Recombinant MVA/UGD Viruses
[0215] The 5 MVA/UGD viruses have been characterized for gene
expression and function. Immunoprecipitation of env and gag
proteins is shown in FIG. 24. BS-C-1 cells were infected with
individual recombinant viruses at a multiplicity of infection of
10, metabolically labeled, and lysates were subjected to
immunoprecipitation with a pool of sera from HIV-1 clade D infected
individuals. Viruses expressing gagpol only (MVA/UGD/Bgag and Cgag)
were included, as was non-recombinant MVA as a negative control and
MVA/CMDR as a positive control. The latter virus expresses
gagpol/env from a Clade E HIV-1 isolate. All viruses produced high
levels of gag protein and efficient processing into p24 was
observed. In addition, all env-expressing viruses produced high
levels of env protein (gp160).
[0216] FIG. 25 demonstrates that the gag and env proteins produced
by the MVA/UGD viruses are functional. Virus-like particles were
obtained by centrifugation of the supernatant of infected cells
through a sucrose cushion (Karacostas et al. 1993 Virology
193:661). Pelleted material was then separated by
SDS-polyacrylamide gel electrophoresis and analyzed by
autoradiography (Panel A). As seen, p55 and p24 gag proteins were
found in the pellet indicating that virus-like particles were
formed. Panel B shows results of an assay in which env-expressing
cells (infected with MVA/UGD virus) were mixed with cells
expressing CD4 and co-receptor (X4 or R5) (Nussbaum, Broder, &
Berger 1994 J Virol 68:5411). Fusion was measured by
beta-galactosidase activity in cell lysates. As shown, all five
MVA/UGD viruses induced fusion with CD4/R5-expressing cells.
Immunogenicity of Recombinant MVA/UGD Viruses (Study 1)
[0217] Groups of Balb/c mice were immunized with individual MVA/UGD
viruses, non-recombinant MVA (negative control), or MVA/CMDR
(positive control--expressing clade E gagpol/env) at weeks 0 and 3.
The dose was 10.sup.7 infectious units per immunization and the
route was intraperitoneal. Humoral and cell mediated responses were
measured and are shown in FIGS. 26-28.
[0218] Antibody responses after 2 immunizations are shown in FIG.
26. Reciprocal endpoint ELISA titers to p24 at various times after
immunization are shown in Panel A. All UGD viruses elicited
gag-specific antibodies after 2 immunizations. Env-specific
responses are shown in Panel B. In this experiment, pooled sera
from groups of mice were used to immunoprecipitate metabolically
labeled, autologous gp160 proteins. As seen, sera from mice
immunized with MVA/UGD-1, -3, and -4 reacted with gp160 (the other
viruses were not tested in this assay). Reciprocal endpoint titers
to gp140 env at various times after immunization are shown in Panel
C. All UGD viruses elicited env-specific antibodies after 2
immunizations.
[0219] T cell responses were measured with several assays. First,
gag and pol peptide-specific intracellular interferon gamma
(IFN-.gamma.) responses were measured by intracellular cytokine
staining. Splenocytes were collected 3 weeks after immunization,
stimulated in-vitro for 7 days, and then cultured overnight with
peptide-pulsed P815 cells. Brefeldin A was added to prevent
secretion of INF-.gamma.. CD3 positive, CD8 positive, IFN-.gamma.
positive cells were enumerated by flow cytometry. Analyses were
performed after one and two immunizations. Both gag- and
pol-specific responses were observed after two immunizations (FIG.
27) (samples from animals immunized with MVA/UGD-2, and 5 were not
assayed). Second, gag- and pol-specific INF-.gamma.responses were
measured by ELISPOT (FIG. 28 A & B). Briefly, splenocytes from
immunized mice were mixed with gag or pol peptide-pulsed P815 cells
in 96-well nitrocellulose plates coated with
anti-IFN-.gamma.antibody. After overnight incubation, spots were
visualized by sequential incubation with anti-IFN-.gamma. biotin
antibody, straptavidin-HRP, and AEC substrate. Spots were
enumerated using a Zeiss ELISPOT reader. Gag peptide-specific
responses were found after one immunization and were boosted in
most groups after the second immunization. Pol peptide-specific
responses were found in several groups after two immunizations.
Third, gag peptide-specific responses were measured by tetramer
staining (H-2Kd gag LAI tetramer: AMQMLKETI, SEQ ID NO: 63) (FIG.
29). Splenocytes were stimulated in vitro with either gag peptide
or MVA/CM240gagpol, a recombinant virus expressing a clade E
gagpol. CD3 positive, CD8 positive, tetramer positive cells were
enumerated by flow cytometry. Positive tetramer staining was
observed with cells from several groups of mice.
Immunogenicity of Recombinant MVA/UGD Viruses (Study 2)
[0220] A second mouse immunogenicity study was performed to confirm
the humoral and cellular immunogenicity of MVA/UGD-3 and MVA/UGD-4.
BALB/c mice (10 per group) were administered intraperitoneal
immunizations of 10.sup.7 infectious units of MVA at weeks 0 and 3.
Five mice per group were sacrificed two weeks after the 1.sup.st
and 2.sup.nd immunizations and spleens were removed for assessment
of cellular immunogenicity. Sera were collected from each mouse at
weeks -1, 0, 1, 2, 3, 4 and 5. Splenocytes and sera were pooled
together by group. HIV gag-specific serum IgG responses were
detected from all MVA/UGD-immunized groups after the 2.sup.nd
immunization (FIG. 30). These gag-specific responses were
predominantly of subclass IgG2a for both MVA/UGD-3 and MVA/UGD-4
demonstrating a Th1-type response (Table Q).
[0221] HIV-specific cell-mediated immunity was assessed by four
separate assays: (1) intracellular IFN-.gamma. staining by flow
cytometry (ICS), (2) IFN-.gamma. secretion by ELISPOT, (3)
gag-peptide specific tetramer staining and (4) cytotoxic T
lymphocyte (CTL) killing. (1) ICS: Splenocytes were collected two
weeks after the 1.sup.st immunization, stimulated for 7 days with
MVA-infected P815 cells and then incubated overnight with P815
cells pulsed with a gag or pol peptide previously shown to be
target of CD8 T cells in BALB/c mice (Casimiro et al. 2002 J Virol
76:185). Brefeldin A was included in the overnight incubation to
prevent cytokine secretion. Both HIV gag- and pol-specific
responses were detected for the MVA/UGD-immunized, but not control
immunized, mice as evidenced by the production of intracellular
INF-.gamma. after peptide stimulation (FIG. 31A). For example, 8.5%
and 5% of splenocyte lymphocytes from MVA/UGD-4-immunized mice were
positive for gag and pol, respectively. Similar results were
obtained for the MVA/UGD immunized mice after the 2.sup.nd
immunization (FIG. 31B). (2) IFN-.gamma. ELISPOT: HIV gag-specific
IFN-.gamma. responses were detected by ELISPOT without prior
in-vitro stimulation after both the 1.sup.st (FIG. 32A) and
2.sup.nd immunization (FIG. 32B) with a boost detected after the
2.sup.nd immunization. HIV gag-specific responses were stronger
than the pol-specific responses. HIV pol-specific responses were
detectable after a 7-day in-vitro stimulation with P815 cells
pulsed with pol peptide (FIG. 32C). (3) Tetramer staining: HIV gag
peptide-specific responses were measured by tetramer staining
(H-2Kd gag LAI tetramer: AMQMLKETI, SEQ ID NO: 63) (FIG. 33).
Splenocytes were stained pre or post a 7-day in-vitro stimulation
with P815 cells either pulsed with gag peptide or infected with
MVA/CM240gag/pol, a recombinant virus expressing a subtype E
gagpol. CD3 positive/CD8 positive/tetramer positive cells were
enumerated by flow cytometry. Positive tetramer staining was
observed for all of the MVA/UGD immunized groups both pre-IVS and
post-IVS with MVA-infected P815 cells. (4) CTL: Splenocytes removed
2 weeks after the 1.sup.st immunization were stimulated in-vitro
with MVA/CME (a recombinant MVA expressing env and gagpol from a
subtype E HIV-1 isolate) infected P815 cells for 7 days and tested
for the ability to lyse P815 cells pulsed with gag peptide.
Splenocytes from all MVA/UGD, but not MVA control, immunized mice
efficiently lysed peptide-pulsed P815 cells at E:T ratios of 20:1
(FIG. 34).
Example 6
Construction and Pre-Clinical Immunogenicity of a Recombinant MVA
Vaccine Expressing Subtype A HIV-1 Env, Gag and Pol for Use in
Kenya
[0222] As part of the overall program goal to conduct clinical
vaccine trials in Africa, HIV-1 sequences from Kenya were selected.
The predominant incident and prevalent HIV-1 subtype in Kenya is
subtype A. Several R5 subtype A HIV-1 strains were selected and
used to prepare recombinant MVA vaccines expressing env and gagpol
(gag, protease and RT). One gagpol and two env clones from pure
Kenyan subtype A isolates were selected.
TABLE-US-00011 HIV-1 Isolate name GenBank Accession # Publication
00KE-KER2008 AF457052 AIDS 16: 1809 (2002) 00KE-KNH1144 AF457066 ''
00KE-KNH1207 AF457068 ''
[0223] All the steps described in EXAMPLE 5 for construction of
subtype D recombinant MVA viruses were followed for the selected
subtype A clones. These include:
[0224] PCR amplification of truncated genes and cloning into
pCR2.1.
[0225] Testing for in vitro expression.
[0226] Testing for env and gag function.
[0227] Site directed mutagenesis in env to eliminate vaccinia virus
early transcription termination sites.
[0228] Site directed mutagenesis in pol to inactivate enzymatic
activity.
[0229] Cloning into MVA shuttle plasmids pLAS-1 (gagpol) and pLAS-2
(env).
[0230] Recombination of gagpol into MVA 1974/NIH Clone 1 using
primary CEF cells.
[0231] Recombination of env into the recombinant virus expressing
gag to produce a single virus expressing both gagpol and env.
[0232] Recombination of env into MVA 1974/NIH Clone 1 to produce a
virus expressing env only.
[0233] In addition to the mutations described in EXAMPLE 5 and
utilized with the Ugandan subtype D env genes, two other mutations
were introduced into one of the Kenyan clade A env genes (KNH1144).
First, the tyr at position 717 was mutated using site directed
mutagenesis to either ala or ser. This mutation has been shown to
increase cell surface expression of env proteins (Rowell et. al.
1995 J Immunol 155:473; LaBranche et al. 1995 J Virol 69:5217).
Second, the env protein was further truncated at the C-terminus
just prior to the transmembrane domain yielding a soluble, secreted
form of the protein. Published studies have shown that immunization
with this form of the env protein results in enhanced antibody
production as compared to membrane bound env.
[0234] The specifics of deletions and mutations in env and gagpol
genes for KEA isolates are given in Table K. Plasmids and viruses
expressing KEA env and gagpol genes are given in Table L and M.
Characterization of Recombinant MVA/KEA Viruses
[0235] The MVA/KEA viruses were characterized for gene expression
and function. Immunoprecipitation of env and gag proteins is shown
in FIG. 35. BSC-1 cells were infected with individual recombinant
viruses at a multiplicity of infection of 10, metabolically
labeled, and lysates were subjected to immunoprecipitation with a
pool of antibodies including: monoclonal antibody T24 (env),
monoclonal antibody 183-H12-5C (gag), and pooled HIV-1+ sera.
MVA/UGD and WR/vEJW-1 were included as positive controls for env
and gag expression, respectively. All MVA/KEA viruses express high
levels of env and/or gag, as expected.
[0236] Virus-like particles were obtained by centrifugation of the
supernatant of infected cells through a sucrose cushion (Karacostas
et al. 1993 Virology 193:661). Gag p24 protein was found in the
pelleted material indicating the formation of virus-like particles
(FIG. 36).
[0237] A cell-cell fusion assay was used to assess the function of
expressed, membrane bound env. In this assay env-expressing cells
(infected with MVA/KEA virus) were mixed with cells expressing CD4
and co-receptor (X4 or R5) (Nussbaum, et al. 1994 J Virol 68:5411).
Fusion was measured by beta-galactosidase activity in cell lysates.
All viruses expressing membrane bound env induced fusion with
CD4/R5-expressing cells (FIG. 37).
Immunogenicity of Env and Gag in Mice
[0238] The 5 recombinant MVA viruses expressing env, gag and pol
from Kenyan subtype A HIV-1 isolates (MVA/KEA-1 through MVA/KEA-5),
the 3 viruses expressing subtype A env alone (MVA/KEA-6 through
MVA/KEA-8) and the MVA expressing subtype A gag/pol (MVA/KEA-9)
were evaluated in an in-vivo mouse immunogenicity study designed to
measure the humoral and cellular immunogenicity of these vaccines.
BALB/c mice (10 per group) were administered intraperitoneal
immunizations of 10.sup.7 infectious units of individual MVA/KEA
viruses at weeks 0 and 3. Five mice per group were sacrificed two
weeks after the 1.sup.st and 2.sup.nd immunizations and spleens
were removed for assessment of cellular immunogenicity. Sera were
collected from each mouse at weeks -1, 0, 1, 2, 3, 4 and 5.
Splenocytes and sera were pooled together by group. HIV
env-specific serum IgG responses were detected from all
MVA/KEA-immunized groups after the 2.sup.nd immunization (FIG. 38).
While env-specific responses were detected in all groups except for
the control group, they were strongest in MVA/KEA-3, 4, 5, 6 and
8.
[0239] HIV-specific cell-mediated immunity was assessed by three
assays: (1) intracellular IFN-.gamma. staining by flow cytometry
(ICS), (2) IFN-.gamma. secretion by ELISPOT, and (3) gag-peptide
specific tetramer staining. (1) ICS: Splenocytes were collected two
weeks after the 2.sup.nd immunization, stimulated for 7 days with
MVA-infected P815 cells and then incubated overnight with P815
cells pulsed with a gag or pol peptide previously shown to be
target of CD8 T cells in BALB/c mice (Casimiro et al. 2002 J Virol
76:185). Brefeldin A was included during the overnight incubation
to prevent cytokine secretion. HIV gag-specific responses were
detected for each of the groups immunized with MVA/KEA viruses
expressing env, gag and pol or gag and pol, but not control
immunized, mice as evidenced by the production of intracellular
IFN-.gamma. after peptide stimulation (FIG. 39). Splenocytes
positive for HIV gag ranged from 7% to 22% for the
MVA/KEA-immunized mice. (2) IFN-.gamma. ELISPOT: HIV gag-specific
IFN-.gamma. responses from splenocytes without prior in-vitro
stimulation were detected in all groups receiving MVA/KEA viruses
expressing gag after the 1.sup.st immunization (FIG. 40). (3)
Tetramer staining: HIV gag peptide-specific responses were measured
by tetramer staining (H-2Kd gag LAI tetramer: AMQMLKETI, SEQ ID NO:
63) (FIG. 41). Splenocytes were stained pre-IVS. CD3 positive CD8
positive, tetramer positive cells were enumerated by flow
cytometry. Similar to the above ICS and IFN-.gamma. ELISPOT
results, positive tetramer staining was observed for all of the
groups immunized with MVA-KEA expressing gag.
II. EXAMPLE 7
Construction of a Recombinant MVA Vaccine Expressing Subtype C
HIV-1 Env, Gag, and Pol for Use in Tanzania
[0240] As part of the overall program goal to conduct clinical
vaccine trials in Africa, HIV-1 sequences from Tanzania were
selected. The predominant incident and prevalent HIV-1 subtype in
Tanzania is subtype C. Several R5 subtype C HIV-1 strains were
selected and used to prepare recombinant MVA vaccines expressing
env, gag, protease and RT. One gagpol and one env clone from pure
Tanzanian subtype C isolates were selected.
TABLE-US-00012 HIV-1 Isolate name GenBank Accession # Publication
00TZA-125 AY253304 AIDS Res & Hum Retrov, in press 00TZA-246
AY253308 AIDS Res & Hum Retrov, in press
[0241] Steps described in EXAMPLE 5 were followed for the selected
subtype C clones. These include:
[0242] PCR amplification of truncated genes and cloning into
pCR2.1.
[0243] Testing for in vitro expression.
[0244] Testing for env and gag function.
[0245] Site directed mutagenesis in env to eliminate vaccinia virus
early transcription termination sites.
[0246] Site directed mutagenesis in pol to inactivate enzymatic
activity.
[0247] Cloning into MVA shuttle plasmids pLAS-1 (gagpol) and pLAS-2
(env).
[0248] Recombination of gagpol into MVA 1974/NIH Clone 1 using
primary CEF cells.
[0249] Recombination of env into the recombinant virus expressing
gag to produce a single virus expressing both gagpol and env.
[0250] Recombination of env into MVA 1974/NIH Clone 1 to produce a
virus expressing env only.
[0251] The specifics of deletions and mutations in env and gagpol
genes for TZC isolates is given in Table N. Plasmids and viruses
expressing TZC env and gagpol genes are given in Table O and P.
Characterization of Recombinant MVA/TZA Viruses
[0252] The KEA viruses were characterized for gene expression.
Immunoprecipitation of env and gag proteins is shown in FIG. 42.
BSC-1 cells were infected with individual recombinant viruses at a
multiplicity of infection of 10, metabolically labeled, and lysates
were subjected to immunoprecipitation with a pool of sera from
HIV-1 infected individuals. MVA/CMDR and MVA were included as
positive and negative controls, respectively. The MVA/KEA virus
expresses high levels of env and gag.
[0253] Virus-like particles were obtained by centrifugation of the
supernatant of infected cells through a sucrose cushion (Karacostas
et al. 1993 Virology 193:661). Gag p24 protein was found in the
pelleted material indicating the formation of virus-like particles
(FIG. 43).
Example 8
Stability of Expression of HIV-1 Genes in Recombinant MVA
Viruses
[0254] The stability of the inserted env and gagpol genes in
recombinant viruses from each of the subtypes was tested after
serial passage in CEF cells. Viruses were grown in CEF cells using
a procedure that mimics that used for expansion of virus for large
scale vaccine production, i.e. infection at low multiplicity,
growth for 3 days at 37 C, harvesting of virus from cell lysates.
After repeated passage, the virus stocks were tested for stability
of the inserts using a 3-day immunostaining protocol. In this
protocol, CEF cell monolayers were infected at a low multiplicity
allowing for visualization of individual viral foci. After 3 days,
the monolayers were fixed and then stained with monoclonal
antibodies specific for either env or gag. Staining and
non-staining foci were enumerated and results are shown in Table R.
Very few non-staining foci were detected after 10-11 passages of
each virus indicating that the inserted genes are stable after
repeated passage in culture.
TABLE-US-00013 TABLE G Oligonucleotides used for PCR amplification
of env and gagpol genes Clade D Ugandan HIV- envelope 1 isolates 5'
primer 3' primer 99UGA03349 GCGCCCCGGGTCGACGCGGCCGCGCCATGAG
GCGCCCCGGGCGGCCGCAGAAAAATTAGCCTTG AGTGAGGGGGATACAGAGGAAC (SEQ ID
NO: CTCTCCACCTTCTTCTTCTATTCC (SEQ ID NO: 15) 16) 99UGA07412
GCGCCCCGGGTCGACGCGGCCGCGCCATGAG GCGCCCCGGGCGGCCGCAGAAAAATTAGCCTTG
AGTGAGGGAGACAGTGAGGAATTAT (SEQ ID CTCTCCACCTTCTTCTTCTATTCC (SEQ ID
NO: NO: 17) 18) 98UG57128 GCGCCCCGGGTCGACGCGGCCGCGCCATGAG
GCGCCCCGGGCGGCCGCAGAAAAATTAGCCTTG AGTGAGGGGGATAGAGAGGAATTAT (SEQ ID
CTCTCCACCTTCTCCTTC (SEQ ID NO: 20) NO: 19) Clade D Ugandan HIV-
gagpol 1 isolates 5' primer 3' primer 99UGA03349
GCGCCCCGGGGCCATGGGTGCGAGAGCGTCA GCGCCCCGGGAGAAAAATTAGAAGGTTTCTGCT
GTATTAAGC (SEQ ID NO: 21) CCTACTATGGGTTCCT (SEQ ID NO: 22)
99UGA07412 GCGCCCCGGGGCCATGGGTGCGAGAGCGTCA
GCGCCCCGGGAGAAAAATTAGAAAGTTTCTGCT GTGTTAAGT (SEQ ID NO: 23)
CCTACTATGGGTTCCT (SEQ ID NO: 24)
TABLE-US-00014 TABLE H Deletions/mutations in env genes envelope
C-terminal truncation Clade D # of Ugandan amino acid last TTTTTNT
silent mutations HIV-1 DNA sequence at C- # of last sequence at C-
amino amino acid amino acid isolates terminus nucleotide terminus
acid nucleotide # nucleotide # 99UGA0334 GGAATAGAAGAAGAAG 2178
GIEEEGGEQG 726 phe to phe 168 phe to phe 374 GTGGAGAGCAAGGC (SEQ ID
NO: 26) T to C 504 T to C 1122 (SEQ ID NO: 25) 99UGA07412
GGAATAGAAGAAGAAG 2211 GIEEEGGEQG 737 phe to phe 173 phe to phe 384
GTGGAGAGCAAGGC (SEQ ID NO: 28) T to C 519 T to C 1152 (SEQ ID NO:
27) 98UG57128 GGAACAGAAGGAGAA 2262 GTEGEGGEQG 754 phe to phe 182
phe to phe 391 GGTGGAGAGCAAGGC (SEQ ID NO: 30) T to C 546 T to C
1173 (SEQ ID NO: 29) Deletions/mutations in gagpol genes gagpol
C-terminal truncation Clade D # of pol Ugandan amino acid last RT
active site mutations HIV-1 DNA sequence at C- # of last sequence
at C- amino amino acid amino acid isolates terminus nucleotide
terminus acid nucleotide # nucleotide # 99UGA03349 AAGGAACCCATAG
3065 KEPIVGAETF 1022 asp to glu 692 asp to his 767 TAGGAGCAGAAAC
(SEQ ID NO: T to G 2075 GAT to CAC 2298 & 2300 CTTC (SEQ ID NO:
32) 31) 99UGA07412 AAGGAACCCATAG 3077 KEPIVGAETF 1026 asp to glu
696 asp to his 771 TAGGAGCAGAAAC (SEQ ID NO: T to G 2087 GAT to CAC
2310 & 2312 TTTC (SEQ ID NO: 34) 33)
TABLE-US-00015 TABLE I UGD Plasmids Direction Insertion in rela-
parent site tion to Plasmid plasmid HIV isolate in MVA vaccinia
pLAS-1/UGD/Bgag pLAS-1 99UGA03349 Del III same pLAS-1/UGD/Cgag
pLAS-1 99UGA07412 Del III same pLAS-2/UGD/Benv pLAS-2 99UGA03349
Del II same pLAS-2/UGD/Cenv pLAS-2 99UGA07412 Del II same
pLAS-2/UGD/Denv pLAS-2 98UG57128 Del II same pLAS-2/UGD/revCenv
pLAS-2 99UGA07412 Del II reverse pLAS-2/UGD/revDenv pLAS-2
98UG57128 Del II reverse
TABLE-US-00016 TABLE J UGD Viruses gagpol Env Direction Direction
in rela- in rela- gag/pol tion to env tion to Virus HIV isolate
vaccinia HIV isolate vaccinia MVA/UGD-1 99UGA07412 same 99UGA07412
Same MVA/UGD-2 99UGA03349 same 98UG57128 Reverse MVA/UGD-3
99UGA07412 same 99UGA03349 Same MVA/UGD-4 99UGA03349 same
99UGA07412 Reverse MVA/UGD-5 99UGA03349 same 98UG57128 Same
MVA/UGD- 99UGA03349 same -- -- Bgag MVA/UGD- 99UGA07412 same -- --
Cgag
TABLE-US-00017 TABLE K Deletions/mutations in env genes (KEA)
envelope C-terminal truncation TTTTTNT Clade A amino acid # of last
mutations Tyrosine mutations Kenyan HIV-1 DNA sequence at # of last
sequence at amino amino acid amino acid isolates C-terminus
nucleotide C-terminus acid nucleotide # nucleotide # 00KE-KNH1207
AGAATCGAAGG 2209 GRIEGEGGE 736 phe to phe 378 AGAAGGTGGAG QD T to C
1134 AGCAAGAC (SEQ ID (SEQ ID NO: 35) NO: 36) 00KE-KNH1144
AGAATCGAAGG 2239 GRIEGEGGE 746 phe to phe 389 AGAAGGTGGAG QD T to C
1167 AGCAAGAC (SEQ ID (SEQ ID NO: 37) NO: 38) 00KE-KNH1144
AGAATCGAAGG 2239 GRIEGEGGE 746 tyr to ala 717 AGAAGGTGGAG QD TAC to
GCG 2149 to 2151 AGCAAGAC (SEQ ID (SEQ ID NO: 39) NO: 40)
00KE-KNH1144 AGAATCGAAGG 2239 GRIEGEGGE 746 tyr to ser 717
AGAAGGTGGAG QD TAC to AGC 2149 to 2151 AGCAAGAC (SEQ ID (SEQ ID NO:
41) NO: 42) 00KE-KNH1144 GACATATCAAAT 2064 DISNWLWY 688
TGGCTGTGGTAT IR ATAAGA (SEQ ID (SEQ ID NO: 43) NO: 44)
Mutations/deletions in gagpol genes (KEA) gagpol C-terminal
truncation # of pol last RT active site mutations amino #amino acid
#amino acid # Clade A DNA # of last amino acid acid # in pol in pol
Kenyan HIV-1 sequence at nucleotide sequence at in amino acid
nucleotide # amino acid nucleotide # isolates C-terminus in gagpol
C-terminus pol nucleotide in gagpol nucleotide in gagpol#
00KE-KER2008 GACCCCATAG 3074 KDPIAGA 595 asp to glu 265 asp to his
340 CAGGAGCAGA ETF (SEQ T to G 2084 GAT to CAC 2307 to 2309 GACTTTC
ID NO: 46) (SEQ ID NO: 45)
TABLE-US-00018 TABLE L KEA Plasmids Direction Insertion in rela-
parent site tion to Plasmid plasmid HIV isolate in MVA vaccinia
pLAS-l/KER2008gag pLAS-1 00KE-KER2008 Del III same
pLAS-2/KNH1144env pLAS-2 00KE-KNH1144 Del II same pLAS-2/KNH1207env
pLAS-2 00KE-KNH1207 Del II same pLAS-2/KNH1144gp140 pLAS-2
00KE-KNH1144 Del II same pLAS-2/KNH1144(Y/A) pLAS-2 00KE-KNH1144
Del II same pLAS-2/KNH1144(Y/S) pLAS-2 00KE-KNH1144 Del II same
TABLE-US-00019 TABLE M KEA Viruses gag/pol env Direction Direction
in rela- in rela- gag/pol tion to env tion to Virus HIV isolate
vaccinia HIV isolate vaccinia MVA/KEA-1 00KE-KER2008 same
00KE-KNH1144 same MVA/KEA-2 00KE-KER2008 same 00KE-KNH1207 same
MVA/KEA-3 00KE-KER2008 same 00KE-KNH1144 same (gp140) MVA/KEA-4
00KE-KER2008 same 00KE-KNH1144 same (Y/A) MVA/KEA-5 00KE-KER2008
same 00KE-KNH1144 same (Y/S) MVA/KEA-6 -- 00KE-KNH1144 same
MVA/KEA-7 -- 00KE-KNH1207 same MVA/KEA-8 -- 00KE-KNH1144 same
(gp140) MVA/KEA-9 00KE-KER2008 same --
TABLE-US-00020 TABLE N Mutations/deletions in env genes (TZC)
envelope Clade C TTTTTNT Tanzanian C-terminal truncation silent
mutations HIV-1 # of last amino acid sequence # of last amino acid
isolates DNA sequence at C-terminus nucleotide at C-terminus amino
acid nucleotide # 00TZA-125 GGAATCGAAGAAGAAGGTGGA 2223 GIEEEGGEQD
741 phe to phe 396 GAGCAAGAC (SEQ ID NO: 47) (SEQ ID NO: 48) T to C
1161 Mutations/deletions in gagpol (TZC) gagpol C-terminal
truncation pol # of RT active site mutations Clade C amino acid
last amino acid amino acid Tanzanian # of last sequence at amino
amino # in pol # in pol HIV-1 DNA sequence at nucleotide C-terminus
acid in acid nucleotide amino acid nucleotide isolates C-terminus
of gagpol of pol pol nucleotide # in gagpol nucleotide # in gagpol
00TZA-246 AAAGAACCCATA 3083 KEPIVGAETF 601 asp to glu 271 asp to
his 346 GTAGGAGCAGAA (SEQ ID NO: T to G 2093 GAT to CAC 2316 &
2318 ACTTTCT (SEQ ID 50) NO: 49)
TABLE-US-00021 TABLE O TZC Plasmids Direction Insertion in rela-
parent site tion to Plasmid plasmid HIV isolate in MVA vaccinia
pLAS-1/ pLAS-1 00TZA-246 Del III same TZC246gag pLAS-2/ pLAS-2
00TZA-125 Del II same TZC125env
TABLE-US-00022 TABLE P TZC Viruses gag/pol env Direction Direction
in rela- in rela- gag/pol tion to env tion to Virus HIV isolate
vaccinia HIV isolate vaccinia MVA/TZC-1 00TZA-246 same 00TZA-125
same MVA/TZC-2 00TZA-246 same -- MVA/TZC-3 -- 00TZA-125 same
TABLE-US-00023 TABLE Q HIV-1 gag IgG2a/IgG1 ratios Ratio Ratio
Ratio Group Week 1 G2a/G1 Week 2 G2a/G1 Week 3 G2a/G1 MVA/CMDR G1
34,100 2.5 34,100 2.5 29,900 2 G2a 85,300 85,300 59,700 MVA control
G1 <100 -- <100 -- <100 -- G2a <100 <100 <100
MVA/UGD-4 G1 34,100 1.6 25,600 1.6 19,200 1.8 G2a 55,500 41,600
35,200 MVA/UGD-3 G1 3,200 3.3 3,200 5.3 3,200 5.3 G2a 10,600 17,100
17,100 MVA/UGD-1 G1 9,600 0.7 4,800 1.3 4,800 1.3 G2a 6,400 6,400
6,400 MVA/UGD G1 4,800 6.2 3,200 9.3 2,400 12.4 gag G2a 29,900
29,900 29,900
TABLE-US-00024 TABLE R Stability of inserts in rMVA viruses Gag Env
non- non- total staining total staining Virus foci # foci # % foci
# foci # % After 10 270 3 1.1 270 3 1.1 passages MVA/UGD-4 After 11
passages MVA/KEA-1 360 2 0.56 310 0 0 MVA/KEA-2 230 3 1.3 280 0 0
MVA/KEA-3 340 0 0 340 0 0 MVA/KEA-4 210 0 0 210 0 0 MVA/KEA-5 220 1
0.45 210 0 0 After 11 600 5 0.83 800 1 0.13 passages MVA/TZC
TABLE-US-00025 APPENDIX 1 DNA sequences of gagpol and env genes
from Ugandan HIV-1 clade D isolates: 99UGA03349 gagpol (SEQ ID NO:
51):
ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATGAATGGGAAAAAATTCGGTTACG
GCCAGGGGGAAACAAAAAATATAGATTAAAACATTTAGTATGGGCAAGCAGGGAGCTAGAACGAT
TTGCACTTAATCCTGGTCTTTTAGAAACATCAGAAGGCTGTAGACAAATAATAGAACAGCTACAAC
CATCTATTCAGACAGGATCAGAGGAACTTAAATCATTACATAATACAGTAGTAACCCTCTATTGTG
TACATGAAAGGATAAAGGTAGCAGATACCAAGGAAGCTTTAGATAAGATAAAGGAAGAACAAACC
AAAAGTAAGAAAAAAGCACAGCAAGCAACAGCTGACAGCAGCCAGGTCAGCCAAAATTATCCTAT
AGTACAAAACCTACAGGGGCAAATGGTACACCAGTCCTTATCACCTAGGACTTTGAATGCATGGGT
AAAAGTAATAGAAGAGAAGGCTTTCAGCCCAGAAGTAATACCCATGTTTTCAGCATTATCAGAAG
GAGCCACCCCAACAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAA
ATGTTAAAAGAGACTATCAATGAGGAAGCTGCAGAATGGGATAGGCTACATCCAGTGCCTGCAGG
GCCTGTTGCACCAGGCCAAATGAGAGAACCAAGGGGAAGTGATATAGCAGGAACTACCAGTACCC
TTCAGGAACAAATAGGATGGATGACAAGCAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGA
TGGATAATCCTAGGATTAAATAAAATAGTAAGAATGTATAGCCCTGTCAGCATTTTGGACATAAGA
CAAGGACCAAAGGAACCCTTTAGAGACTATGTAGATCGGTTCTATAAAACTCTACGAGCCGAGCA
AGCTTCACAGGATGTAAAAAATTGGATGACTGAAACCTTGTTAGTCCAAAATGCGAATCCAGATTG
TAAAACTATCTTAAAAGCATTGGGACCAGCGGCTACATTAGAAGAAATGATGACAGCATGTCAGG
GAGTGGGGGGACCCAGTCATAAAGCAAGAGTTTTGGCTGAGGCAATGAGCCAAGCATCAAACACA
AATGCTGTTATAATGATGCAGAGGGGCAATTTCAAGGGCAAGAAAATCATTAAGTGTTTCAACTGT
GGCAAAGAAGGACACCTAGCAAAAAATTGTAGGGCTCCTAGGAAAAGAGGCTGTTGGAAATGTGG
AAAGGAAGGGCACCAAATGAAAGATTGTAATGAAAGACAGGCTAATTTTTTAGGGAGAATTTGGC
CTTCCCACAAGGGGAGGCCAGGGAATTTCCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGCA
GAGAGCTTCGGGTTTGGGGAAGAGATAACACCCTCCCAGAAACAGGAGGGGAAAGAGGAGCTGT
ATCCTTCAGCCTCCCTCAAATCACTCTTTGGCAACGACCCCTAGTCACAATAAAAATAGGGGGACA
GCTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTAGTAGAAGAAATGAATTTGCCAG
GAAAATGGAAACCAAAAATGATAGGGGGAATTGGGGGCTTTATCAAAGTAAGACAGTATGATCAA
ATACTCGTAGAAATCTATGGATATAAGGCTACAGGTACAGTATTAGTAGGACCTACACCTGTCAAC
ATAATTGGAAGAAATTTGTTGACTCAGATTGGTTGCACTTTAAATTTTCCAATTAGTCCTATTGAAA
CTGTACCAGTAAAATTAAAGTCAGGGATGGATGGTCCAAGAGTTAAACAATGGCCATTGACAGAA
GAGAAAATAAAAGCACTAATAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAACTTTCAAGAAT
TGGACCTGAAAATCCATACAATACTCCAATATTTGCCATAAAGAAAAAAGACAGTACTAAGTGGA
GAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAACTAGGA
ATACCACATCCTGCAGGGCTAAAAAAGAAAAAATCAGTAACAGTACTGGAGGTGGGTGATGCATA
TTTTTCAGTTCCCTTATATGAAGACTTTAGAAAATACACTGCATTCACCATACCTAGTATAAACAAT
GAGACACCAGGAATTAGATATCAGTACAATGTGCTTCCACAAGGATGGAAAGGATCACCGGCAAT
ATTCCAAAGTAGCATGACAAAAATTTTAGAACCTTTTAGAAAACAAAATCCAGAAGTGGTTATCTA
CCAATACATGCACGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAATAAAAATAG
AGGAATTAAGGGGACACCTATTGAAGTGGGGATTTACCACACCAGACAAAAATCATCAGAAGGAA
CCTCCATTTCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAAAACTG
CCAGAAAAAGAAAGCTGGACTGTCAATGATCTGCAGAAGTTAGTGGGGAAATTAAATTGGGCAAG
TCAAATTTATTCAGGAATTAAAGTAAGACAATTATGCAAATGCCTTAGGGGAACCAAAGCACTGAC
AGAAGTAGTACCACTGACAGAAGAAGCAGAATTAGAACTGGCAGAAAACAGGGAACTTCTAAAAG
AAACAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAACAAGGG
CAAGACCAATGGACATATCAAATTTATCAAGAACAATATAAAAATTTGAAAACAGGAAAGTATGC
AAAGAGGAGGAGTACCCACACTAATGATGTAAAACAATTAACAGAGGCAGTGCAAAAAATAGCCC
AAGAATGTATAGTGATATGGGGAAAGACTCCTAAATTCAGACTACCCATACAAAAGGAAACATGG
GAAACATGGTGGACAGAGTATTGGCAGGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCT
CCCTTGGTTAAATTATGGTACCAGTTAGAGAAGGAACCCATAGTAGGAGCAGAAACCTTCTAA
99UGA07412 gagpol (SEQ ID NO: 52):
ATGGGTGCGAGAGCGTCAGTGTTAAGTGGGGGAAAATTAGATGAATGGGAAAGAATTCGGTTACG
GCCAGGGGGAAACAAAAGATATAAACTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAGCGA
TTTGCACTTAATCCTGGCCTTTTAGAAACATCAGAAGGCTGTAAACAAATATTGGGACAGCTACAA
CCAGCTATTCAGACAGGATCAGAAGAACTTAAATCATTATATAATACAGTAGCAACCCTCTATTGT
GTACATGAGAGGCTAAAGGTAACAGACACCAAGGAAGCTTTAGACAAAATAGAGGAAGAACAAA
CCAAAAGTAAGAAAAAAGCACAGCAAGCAACAGCTGACACAAAAAACAGCAGCCAGGTCAGCCA
AAATTATCCTATAGTACAAAACCTACAGGGGCAAATGGTACACCAGGCTATATCACCTAGAACGTT
GAACGCATGGGTAAAAGTAATAGAGGAGAAGGCTTTCAGCCCAGAAGTAATACCCATGTTTTCAG
CATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAG
GCAGCCATGCAGATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGGTTACATCC
AGTACATGCAGGGCCTATTGCACCAGGACAAATGAGAGAACCAACAGGAAGTGATATAGCAGGAA
CTACTAGTACCCTTCAGGAACAAATAGGATGGATGACCAGCAATCCACCTATCCCAGTAGGAGAA
ATCTATAAAAGATGGATAATCCTAGGATTAAATAAAATAGTAAGGATGTATAGCCCTGTCAGTATT
TTGGACATAAAACAAGGGCCAAAGGAACCCTTTAGAGACTATGTAGATCGGTTCTATAAAACTCTA
AGGGCCGAGCAAGCTTCACAGGAGGTAAAAGGTTGGATGACCGAAACCTTGTTGGTCCAAAATGC
AAACCCAGATTGTAAAACCATCTTAAAAGCATTGGGACCAGCGGCTACATTAGAAGAAATGATGA
CAGCATGTCAGGGAGTGGGGGGACCCGGTCATAAAGCAAGAGTTTTGGCTGAGGCAATGAGTCAA
GTCTCAACAAATACTGCTATAATGATGCAGAGAGGCAATTTTAAGGGCCCAAAGAAAAGCATTAA
GTGTTTTAACTGTGGCAAAGAAGGTCACACAGCAAAAAACTGTAGAGCTCCTAGGAAAAGGGGCT
GTTGGAAATGTGGAAGGGAAGGACATCAAATGAAAGATTGCACTGAAAGACAGGCTAATTTTTTA
GGGAAAATTTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTCCTTCAGAACAGACCAGAGCCAAC
AGCCCCACCAGAAGAAAGCTTCGGGTTTGGGGAAGAGATAACACCCTCTCAGAAACAGGAGAAGA
AGGACAAGGAGCTGTATCCTGTAGCTTCCCTCAAATCACTCTTTGGCAACGACCCCTTGTCACAAT
AAAGATAGGGGGACAGCTAAAGGAAGCTCTACTAGATACAGGAGCAGATGATACAGTATTAGAAG
AAATAAATTTGCCAGGAAAATGGAAACCAAAAATGATAGGGGGAATTGGAGGCTTTATCAAAGTA
AGACAGTATGAGCAAATACTTGTAGAAATCTGTGGACAGAAAGCTATAGGTACAGTATTAGTAGG
GCCTACACCTGTCAACATAATTGGAAGAAATTTGTTGACTCAGATTGGTTGCACTTTAAATTTTCC
AATTAGCCCTATTGAAACTGTACCAGTAAAATTAAAGCCAGGGATGGACGGTCCAAAAGTTAAAC
AATGGCCATTGACAGAAGAAAAGGTAAAAGCACTAATAGAAATTTGTACAGAAATGGAAAAGGAA
GGAAAAATTTCAAGAATTGGACCTGAAAATCCATACAATACTCCAATATTTGCCATAAAGAAAAA
GGACAGTACTAAGTGGAGAAAATTAGTAGATTTCAGGGAACTTAATAAGAGAACTCAAGACTTCT
GGGAAGTTCAACTAGGAATACCACATCCTGCGGGGCTAAAAAAGAAAAAATCAGTAACAGTACTG
GAGGTGGGTGATGCATATTTTTCAGTTCCCTTATATGAAGATTTTAGAAAATATACTGCATTCACC
ATACCTAGTATAAACAATGAAACACCAGGAATTAGATATCAGTACAATGTGCTTCCACAAGGGTG
GAAAGGATCACCAGCAATATTCCAAAGTAGCATGACAAAAATCTTAGAACCTTTTAGAAAACAAA
ATCCAGAAATGGTTATCTATCAATACATGCACGATTTGTATGTAGGATCTGACTTAGAAATAGGGC
AGCATAGAATAAAAATAGAAGAATTAAGGGGACACCTGTTGAAGTGGGGATTTACCACACCAGAC
AAAAAGCATCAGAAAGAACCTCCATTTCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACA
GTACAGTCTATAAAACTGCCAGAACAAGAAAGCTGGACTGTCAATGATATACAGAAGTTAGTGGG
AAAATTAAATTGGGCAAGCCAGATTTATCCAGGAATTAAGGTAAGACAATTATGCAAATGCATTA
GGGGTACCAAAGCACTGACAGAAGTAGTACCACTGACAGAAGAAGCAGAATTAGAACTGGCAGAA
AACAGGGAAATTCTAAGAGAACCAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGC
AGAGATACAGAAACAAGGGCAAGACCAGTGGACATACCAAATTTATCAAGAACAATATAAAAATC
TGAAAACAGGAAAGTATGCAAAAGTGAGGGGTACCCACACTAATGATGTAAAACAATTAACAGAG
GCAGTACAAAAAATAACCCAAGAATGTATAGTGATATGGGGAAAGCCTCCTAAATTTAGACTACC
CATACAAAAAGAAACATGGGAAATATGGTGGACAGAGTATTGGCAGGCCACCTGGATTCCTGAGT
GGGAGTTTGTCAATACCCCTCCTTTAGTTAAATTATGGTACCAATTAGAGAAGGAACCCATAGTAG
GAGCAGAAACTTTCTAA 99UGA03349 envelope (SEQ ID NO: 53):
ATGAGAGTGAGGGGGATACAGAGGAACTATCAAAACTTGTGGAGATGGGGCACCTTGCTCCTTGG
GATGTTGATGATATGTAAGGCTACAGAACAGTTGTGGGTCACAGTTTACTATGGGGTACCTGTGTG
GAAAGAAGCAACCACTACTCTATTTTGTGCATCAGATGCTAAATCATATAAAGAAGAAGCACATA
ATATCTGGGCTACACATGCCTGTGTACCAACAGACCCCAACCCACGAGAATTAATAATAGAAAATG
TCACAGAAAACTTTAACATGTGGAAAAATAACATGGTGGAGCAGATGCATGAGGATATAATCAGT
TTATGGGATCAAAGCCTAAAACCATGTGTAAAATTAACCCCACTCTGTGTCACTTTAAACTGCACT
GAATGGAGGAAGAATAACACTATCAATGCCACCAGAATAGAAATGAAAAACTGCTCTTTCAATCT
AACCACAGAAATAAGAGATAGGAAAAAGCAAGTGCATGCACTTTTCTATAAACTTGATGTGGTAC
CAATAGATGATAATAATAGTACTAATACCAGCTATAGGTTAATAAATTGTAATACCTCAGCCATTA
CACAGGCGTGTCCAAAGGTAACCTTTGAGCCAATTCCCATACATTATTGTGCCCCAGCTGGATATG
CGATTCTAAAATGTAACAATAAGAAGTTCAATGGGACAGGTCCATGCGATAATGTCAGTACAGTA
CAGTGTACACATGGAATTAGGCCAGTAGTATCCACTCAATTGTTGTTGAATGGCAGTCTAGCAGAA
GAAGACATAATAATTAGATCTGAGAATCTCACAAATAATGCTAAAATCATAATAGTACAGCTTAAT
GAGTCTGTAACAATTAATTGCACAAGGCCCTACAACAATACAAGAAGAGGTGTACATATAGGACC
AGGGCGAGCATACTATACAACAGACATAATAGGAGATATAAGACAAGCACATTGTAACATTAGTG
GAGCAGAATGGAATAAGACTTTACATCGGGTAGCTAAAAAATTAAGAGACCTATTTAAAAAGACA
ACAATAATTTTTAAACCGTCCTCCGGAGGGGACCCAGAAATTACAACACACAGCTTTAATTGTAGA
GGGGAATTCTTCTACTGCAATACAACAAGACTGTTTAATAGCATATGGGGAAATAATAGTACAGG
AGTTGATGAGAGTATAACACTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGGGAGTAG
GAAAAGCAATGTATGCCCCTCCCATTGAAGGACTAATCAGCTGCTCATCAAATATTACAGGATTAC
TGTTGACAAGAGATGGTGGTGGAAGTAACAGTAGTCAGAATGAGACCTTCAGACCTGGAGGGGGA
GATATGAGAGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAGAATTGAACCATTAGG
TCTAGCACCCTCCAAGGCAAAAAGAAGAGTAGTAGAAAGAGAGAAAAGAGCAATAGGACTAGGA
GCTATGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACGATGGGCGCAGCGTCACTGACGCTGAC
GGTACAGGCCAGACAGCTATTGTCTGGTATAGTGCAACAGCAAAACAATTTGCTGAAGGCTATAG
AGGCGCAACAGCACCTGTTGCAACTCACAGTCTGGGGCGTTAAACAGCTCCAGGCAAGAGTCCTG
GCTGTGGAAAGCTACCTAAGGGATCAACAGCTCCTAGGAATTTGGGGTTGCTCTGGAAAACACATT
TGCACCACCAATGTGCCCTGGAACTCTAGCTGGAGTAATAAAACTCTAAAATCAATTTGGGATAAC
ATGACCTGGATGGAGTGGGAAAGAGAAATTGACAATTACACAGGGATAATATACAATTTACTTGA
AGAATCGCAAACCCAGCAAGAAAGAAATGAACAAGACCTATTGAAATTGGACCAATGGGCAAGTT
TGTGGAATTGGTTTAGCATAACAAAATGGCTGTGGTATATAAAAATATTTATAATGATAGTAGGAG
GCTTGATAGGCTTAAGGATAGTTTTTGCTGTGCTTTCTATAGTAAATAGAGTTAGGCAGGGATATT
CACCTCTGTCGTTTCAGACCCTCCTCCCAGCCCCGCGGGGACCCGACAGGCCCGAAGGAATAGAAG
AAGAAGGTGGAGAGCAAGGCTAA 99UGA07412 envelope (SEQ IS NO: 54):
ATGAGAGTGAGGGAGACAGTGAGGAATTATCAGCACTTGTGGAGATGGGGCATCATGCTCCTTGG
GATGTTAATGATATGTAGTGCTGCAGACCAGCTGTGGGTCACAGTGTATTATGGGGTACCTGTGTG
GAAAGAAGCAACCACTACTCTATTTTGTGCATCAGATGCTAAAGCACATAAAGCAGAGGCACATA
ATATCTGGGCTACACATGCCTGTGTACCAACAGACCCCAATCCACGAGAAATAATACTAGGAAATG
TCACAGAAAACTTTAACATGTGGAAGAATAACATGGTAGAGCAGATGCATGAGGATATAATCAGT
TTATGGGATCAAAGTCTAAAACCATGTGTAAAATTAACCCCACTCTGTGTTACTTTAAACTGCACT
ACATATTGGAATGGAACTTTACAGGGGAATGAAACTAAAGGGAAGAATAGAAGTGACATAATGAC
ATGCTCTTTCAATATAACCACAGAAATAAGAGGTAGAAAGAAGCAAGAAACTGCACTTTTCTATAA
ACTTGATGTGGTACCACTAGAGGATAAGGATAGTAATAAGACTACCAACTATAGCAGCTATAGATT
AATAAATTGCAATACCTCAGTCGTGACACAGGCGTGTCCAAAAGTAACCTTTGAGCCAATTCCCAT
ACATTATTGTGCCCCAGCTGGATTTGCGATTCTGAAATGTAATAATAAGACGTTCAATGGAACGGG
TCCATGCAAAAATGTCAGCACAGTACAGTGTACACATGGAATTAGGCCAGTAGTGTCAACTCAACT
GTTGTTGAATGGCAGTCTAGCAGAAGAAGAGATAATAATTAGATCTGAAAATATCACAAATAATG
CAAAAACCATAATAGTACAGCTTAATGAGTCTGTAACAATTGATTGCATAAGGCCCAACAACAATA
CAAGAAAAAGTATACGCATAGGACCAGGGCAAGCACTCTATACAACAGACATAATAGGGAATATA
AGACAAGCACATTGTAATGTTAGTAAAGTAAAATGGGGAAGAATGTTAAAAAGGGTAGCTGAAAA
ATTAAAAGACCTTCTTAACCAGACAAAGAACATAACTTTTGAACCATCCTCAGGAGGGGACCCAGA
AATTACAACACACAGCTTTAATTGTGGAGGGGAATTCTTCTACTGCAATACATCAGGACTATTTAA
TGGGAGTCTGCTTAATGAGCAGTTTAATGAGACATCAAATGATACTCTCACACTCCAATGCAGAAT
AAAACAAATTATAAACATGTGGCAAGGAGTAGGAAAAGCAATGTATGCCCCTCCCATTGCAGGAC
CAATCAGCTGTTCATCAAATATTACAGGACTATTGTTGACAAGAGATGGTGGTAATACTGGTAATG
ATTCAGAGATCTTCAGACCTGGAGGGGGAGATATGAGAGACAATTGGAGAAGTGAATTATACAAA
TATAAAGTAGTAAGAATTGAACCAATGGGTCTAGCACCCACCAGGGCAAAAAGAAGAGTGGTGGA
AAGAGAAAAAAGAGCAATAGGACTGGGAGCTATGTTCCTTGGGTTCTTGGGAGCGGCAGGAAGCA
CGATGGGCGCAGCGTCACTGACGCTGACGGTACAGGCCAGACAGTTATTGTCTGGTATAGTGCAA
CAGCAAAACAATTTGCTGAGAGCTATAGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGG
CATTAAACAGCTCCAGGCAAGAGTCCTGGCTATGGAAAGCTACCTAAAGGATCAACAGCTCCTAG
GAATTTGGGGTTGCTCTGGAAAACACATTTGCACCACTACTGTGCCCTGGAACTCTACCTGGAGTA
ATAGATCTGTAGAGGAGATTTGGAATAATATGACCTGGATGCAGTGGGAAAGAGAAATTGAGAAT
TACACAGGTTTAATATACACCTTAATTGAAGAATCGCAAACCCAGCAAGAAAAGAATGAACAAGA
ACTATTGCAATTGGATAAATGGGCAAGTTTGTGGAATTGGTTTAGTATAACAAAATGGCTGTGGTA
TATAAAAATATTCATAATGATAGTAGGAGGCTTAATAGGTTTAAGAATAGTTTTTGCTGTGCTTTC
TTTAGTAAATAGAGTTAGGCAGGGATATTCACCTCTGTCTTTTCAGACCCTCCTCCCAGCCCCGAG
GGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGCAAGGCTAA 98UG57128
envelope (SEQ ID NO: 55):
ATGAGAGTGAGGGGGATAGAGAGGAATTATCAGCACTTATGGTGGAGATGGGGCACCATGCTCCT
TGGGATATTGATGATATGTAGTGCTGCAGAACAATTGTGGGTCACAGTTTATTATGGGGTACCTGT
GTGGAAAGAAGCAACCACTACTCTATTTTGTGCATCAGATGCTAAAGCATATAAAGCAGAGGCAC
ACAATATCTGGGCTACACATGCCTGTGTACCAACAGACCCCAACCCACAAGAAATAGTACTAGAA
AATGTCACAGAAAACTTTAACATGTGGAAAAATAGCATGGTGGAGCAGATGCATGAGGATGTAAT
CAGTTTATGGGATCAAAGCCTAAAACCATGTGTAAAATTAACCCCACTCTGTGTCACTTTAAACTG
CACTAATGCCACTGCCACTAATGCCACTGCCACTAGTCAAAATAGCACTGATGGTAGTAATAAAAC
TGTTAACACAGACACAGGAATGAAAAACTGCTCTTTCAATGTAACCACAGATCTAAAAGATAAGA
AGAGGCAAGACTATGCACTTTTCTATAAACTTGATGTGGTACGAATAGATGATAAGAATACCAATG
GTACTAATACCAACTATAGATTAATAAATTGTAATACCTCAGCCATTACACAAGCGTGTCCAAAGA
TAACCTTTGAGCCAATTCCCATACATTATTGTGCCCCAGCTGGATATGCGATTCTAAAATGTAATA
ATAAGACATTCAATGGGACGGGTCCATGCAAAAACGTCAGCACAGTACAGTGTACACATGGGATT
AGGCCAGTAGTGTCAACTCAACTGTTGTTGAATGGCAGTCTAGCAGAGGAAGAGATAGTAATTAG
ATCTGAAAACCTCACAAATAATGCTAAAATTATAATAGTACAGCTTAATGAAGCTGTAACAATTAA
TTGCACAAGACCCTCCAACAATACAAGACGAAGTGTACATATAGGACCAGGGCAAGCAATCTATT
CAACAGGACAAATAATAGGAGATATAAGAAAAGCACATTGTAATATTAGTAGAAAAGAATGGAAT
AGCACCTTACAACAGGTAACTAAAAAATTAGGAAGCCTGTTTAACACAACAAAAATAATTTTTAAT
GCATCCTCGGGAGGGGACCCAGAAATTACAACACACAGCTTTAATTGTAACGGGGAATTCTTCTAC
TGCAATACAGCAGGACTGTTTAATAGTACATGGAACAGGACAAATAGTGAATGGATAAATAGTAA
ATGGACAAATAAGACAGAAGATGTAAATATCACACTTCAATGCAGAATAAAACAAATTATAAACA
TGTGGCAGGGAGTAGGAAAAGCAATGTATGCCCCTCCCGTTAGTGGAATAATCCGATGTTCATCAA
ATATTACAGGACTGTTGCTGACAAGAGATGGTGGTGGTGCAGATAATAATAGGCAGAATGAGACC
TTCAGACCTGGGGGAGGAGATATGAGAGACAATTGGAGAAGTGAATTATACAAATATAAAGTAGT
AAGAATTGAACCACTAGGTATAGCACCCACCAAGGCAAGGAGAAGAGTGGTGGAAAGAGAAAAA
AGAGCAATAGGACTGGGAGCCTTGTTCCTTGGGTTCTTGGGAACAGCAGGAAGCACGATGGGCGC
AGTGTCAATGACGCTGACGGTACAGGCCAGACAAGTATTGTCTGGTATAGTGCAACAGCAAAACA
ATCTGCTGAGGGCTATAGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATTAAACAGC
TCCAGGCAAGAATCCTGGCTGTGGAAAGCTACCTAAAGGATCAACAGCTCCTAGGAATTTGGGGT
TGCTCTGGAAAACACATTTGCACCACTAATGTGCCCTGGAACTCTAGCTGGAGTAATAAATCTCTA
AATTATATTTGGAATAACATGACCTGGATGGAGTGGGAAAAGGAAATTGACAATTACACAGAATT
AATATACAGCTTAATTGAAGTATCGCAAATCCAGCAAGAAAAGAATGAACAAGAACTATTGAAAT
TGGACAGTTGGGCAAGTTTGTGGAATTGGTTTAGCATAACAAAATGGCTGTGGTATATAAAAATAT
TCATAATGATAGTAGGAGGCTTGATAGGCTTAAGAATAGTTTTTGCTGTGCTTTCTTTAGTAAATA
GAGTTAGGCAGGGATACTCACCTCTGTCGTTTCAGACCCTTATCCCAGCCTCGAGGGGACCCGACA
GGCCCGAAGGAACAGAAGGAGAAGGTGGAGAGCAAGGCTAA
TABLE-US-00026 APPENDIX 2 DNA sequences of MVA shuttle plasmids:
pLAS-1 (SEQ ID NO: 56):
GAATTCGTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGTAAAACATGG
CGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAACCATGTTGTGAAAAAGTCAAGAACG
TTCACATTGGCGGACAATCTAAAAACAATACAGTGATTGCAGATTTGCCATATATGGATAATGCGG
TATCCGATGTATGCAATTCACTGTATAAAAAGAATGTATCAAGAATATCCAGATTTGCTAATTTGA
TAAAGATAGATGACGATGACAAGACTCCTACTGGTGTATATAATTATTTTAAACCTAAAGATGCCA
TTCCTGTTATTATATCCATAGGAAAGGATAGAGATGTTTGTGAACTATTAATCTCATCTGATAAAG
CGTGTGCGTGTATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGGATGTTTCCTTTTTTAC
CAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATTTCTCTATCGATGCGGCACCTCTCTTAAGA
AGTGTAACCGATAATAATGTTATTATATCTAGACACCAGCGTCTACATGACGAGCTTCCGAGTTCC
AATTGGTTCAAGTTTTACATAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTGTTGATG
GATCTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAGCAAAAATATATTAGACA
ATACTACAATTAACGATGAGTGTAGATGCTGTTATTTTGAACCACAGATTAGGATTCTTGATAGAG
ATGAGATGCTCAATGGATCATCGTGTGATATGAACAGACATTGTATTATGATGAATTTACCTGATG
TAGGCGAATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGG
TGGCTGGGTACCAGGCGCGCCTTTCATTTTGTTTTTTTCTATGCTATAAATGGTGAGCAAGGGCGA
GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT
TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGC
ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGC
TTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC
GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTT
CGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACA
TCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAG
AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGC
CGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCT
GAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGT
TCGTGACCGCCGCCGGGATCACTCTCGGCATGCACGAGCTGTACAAGTAAGAGCTCGGTTGTTGAT
GGATCTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAGCAAAAATATATTAGAC
AATACTACAATTAACGATGAGTGTAGATGCTGTTATTTTGAACCACAGATTAGGATTCTTGATAGA
GATGAGATGCTCAATGGATCATCGTGTGATATGAACAGACATTGTATTATGATGAATTTACCTGAT
GTAGGCGAATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCG
GTGGCTGGCGGCCCGCTCGAGGCCGCTGGTACCCAACCTAAAAATTGAAAATAAATACAAAGGTT
CTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCCCGGGGATCCTCTAGAGT
CGACCTGCAGGGAAAGTTTTATAGGTAGTTGATAGAACAAAATACATAATTTTGTAAAAATAAATC
ACTTTTTATACTAATATGACACGATTACCAATACTTTTGTTACTAATATCATTAGTATACGCTACAC
CTTTTCCTCAGACATCTAAAAAAATAGGTGATGATGCAACTTTATCATGTAATCGAAATAATACAA
ATGACTACGTTGTTATGAGTGCTTGGTATAAGGAGCCCAATTCCATTATTCTTTTAGCTGCTAAAA
GCGACGTCTTGTATTTTGATAATTATACCAAGGATAAAATATCTTACGACTCTCCATACGATGATCT
AGTTACAACTATCACAATTAAATCATTGACTGCTAGAGATGCCGGTACTTATGTATGTGCATTCTTT
ATGACATCGCCTACAAATGACACTGATAAAGTAGATTATGAAGAATACTCCACAGAGTTGATTGTA
AATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTACACATTCACCAGAAACTAGT
TAAGCTTGTCTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGT
GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTG
GGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCGAGTCGGG
AAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG
GGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTAT
CAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG
TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCGATAG
GCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAG
GACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC
CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG
TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA
GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATC
GCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT
TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA
AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGC
GGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTG
ATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA
TTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGT
ATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC
TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC
TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA
GCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCAT
CCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT
TGTTGGCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGT
TCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGT
CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT
AATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCAT
TCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGC
CACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGA
TCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT
TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA
GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG
GTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC
GCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATA
AAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGAC
ACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGT
CAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGAT
TGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCA
TCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGC
TATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTT
TCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGGATTTAGGTGACACTATA pLAS-2
(SEQ ID NO: 57):
CCTCCTGAAAAACTGGAATTTAATACACCATTTGTGTTCATCATCAGACATGATATTACTGGATTTA
TATTGTTTATGGGTAAGGTAGAATCTCCTTAATATGGGTACGGTGTAAGGAATCATTATTTTATTT
ATATTGATGGGTACGTGAAATCTGAATTTTCTTAATAAATATTATTTTTATTAAATGTGTATATGTT
GTTTTGCGATAGCCATGTATCTACTAATCAGATCTATTAGAGATATTATTAATTCTGGTGCAATATG
ACAAAAATTATACACTAATTAGCGTCTCGTTTCAGACATGGATCTGTCACGAATTAATACTTGGAA
GTCTAAGCAGCTGAAAAGCTTTCTCTCTAGCAAAGATGCATTTAAGGCGGATGTCCATGGACATAG
TGCCTTGTATTATGCAATAGCTGATAATAACGTGCGTCTAGTATGTACGTTGTTGAACGCTGGAGC
ATTGAAAAATCTTCTAGAGAATGAATTTCCATTACATCAGGCAGCCACATTGGAAGATACCAAAAT
AGTAAAGATTTTGCTATTCAGTGGACTGGATGATTCGAGGTACCAGGCGCGCCCTTTCATTTTGTT
TTTTTCTATGCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCA
CCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCC
TCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG
ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACG
GCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG
AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG
CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCC
ACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGAC
GGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAAC
GAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGCAC
GAGCTGTACAAGTAAGAGCTCGCTTTCTCTCTAGCAAAGATGCATTTAAGGCGGATGTCCATGGAC
ATAGTGCCTTGTATTATGCAATAGCTGATAATAACGTGCGTCTAGTATGTACGTTGTTGAACGCTG
GAGCATTGAAAAATCTTCTAGAGAATGAATTTCCATTACATCAGGCAGCCACATTGGAAGATACCA
AAATAGTAAAGATTTTGCTATTCAGTGGACTGGATGATTCTCCGGATGGTACCCAACCTAAAAATT
GAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAAG
CCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGC
AGAATTCGGCTTGGGGGGCTGCAGGTGGATGCGATCATGACGTCCTCTGCAATGGATAACAATGA
ACCTAAAGTACTAGAAATGGTATATGATGCTACAATTTTACCCGAAGGTAGTAGCATGGATTGTAT
AAACAGACACATCAATATGTGTATACAACGCACCTATAGTTCTAGTATAATTGCCATATTGGATAG
ATTCCTAATGATGAACAAGGATGAACTAAATAATACACAGTGTCATATAATTAAAGAATTTATGAC
ATACGAACAAATGGCGATTGACCATTATGGAGAATATGTAAACGCTATTCTATATCAAATTCGTAA
AAGACCTAATCAACATCACACCATTAATCTGTTTAAAAAAATAAAAAGAACCCGGTATGACACTTT
TAAAGTGGATCCCGTAGAATTCGTAAAAAAAGTTATCGGATTTGTATCTATCTTGAACAAATATAA
ACCGGTTTATAGTTACGTCCTGTACGAGAACGTCCTGTACGATGAGTTCAAATGTTTCATTGACTA
CGTGGAAACTAAGTATTTCTAAAATTAATGATGCATTAATTTTTGTATTGATTCTCAATCCTAAAAA
CTAAAATATGAATAAGTATTAAACATAGCGGTGTACTAATTGATTTAACATAAAAAATAGTTGTTA
ACTAATCATGAGGACTCTACTTATTAGATATATTCTTTGGAGAAATGACAACGATCAAACCGGGCA
TGCAAGCTTGTCTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCT
GTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC
TGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCGAGTCG
GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT
TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT
ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA
TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCGAT
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC
AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT
GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC
TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTT
CAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA
TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA
GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT
GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA
GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTT
TGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGA
GATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA
GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA
TCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG
GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTAT
CAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCC
ATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAAC
GTTGTTGGCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG
GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCG
GTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGC
ATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC
ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGC
GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAG
GATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCT
TTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAAT
AAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCA
GGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC
GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTA
TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTG
ACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCC
GTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAG
ATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCG
CATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTC
GCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGT
TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGGATTTAGGTGACACTATAGAATACG
AATTC
TABLE-US-00027 APPENDIX 3 DNA sequences of gagpol and env genes
from Kenyan HIV-1 clade A isolates: KER2008 gagpol (SEQ ID NO: 58):
ATGGGTGCGAGAGCGTCAGTATTAAGTGGGGGAAAATTAGATGCATGGGAGAAAATTCGGTTAAG
GCCAGGGGGAAAGAAAAAATATAGACTGAAACACTTAGTATGGGCAAGCAGGGAGCTGGAAAAA
TTCGTACTTAACCCTAGCCTTTTAGAAACTTCAGAAGGATGTCAGCAAATAATGAACCAAATACAA
CCAGCTCTTCAGACAGGAACAGAAGAACTTAGATCATTATTTAATGCAGTAGCAACCCTCTATTGT
GTACATCAACGGATAGAGGTAAAAGACACCAAGGAAGCTTTAGATAAAGTAGAGGAAATACAAAA
CAAGAGCAAGCAAAAGACACAACAGGCAGCAGCTGATACAGGAAACAACAGCAAGGTCAGCCAT
AATTACCCTATAGTGCAAAATGCACAAGGGCAAATGATACATCAGTCCTTATCACCAAGGACTTTG
AATGCATGGGTAAAGGTAATAGAAGAAAGGGGTTTCAGCCCAGAAGTAATACCCATGTTCTCAGC
ATTATCAGAAGGAGCCATCCCACAAGATTTAAATATGATGCTGAACATAGTGGGGGGACACCAGG
CAGCTATGCAAATGTTAAAAGAAACTATCAATGAGGAAGCTGCAGAATGGGACAGGTTACATCCA
GCACAGGCAGGGCCTATTCCACCAGGCCAGATAAGAGACCCAAGGGGAAGTGACATAGCAGGAAC
TACTAGTACCCCTCAGGAACAAATAACATGGATGACAAACAACCCACCTATCCCAGTGGGAGACA
TCTATAAAAGATGGATAATCCTAGGATTAAATAAAATAGTAAGAATGTATAGCCCTGTTAGCATTT
TAGATATAAAACAGGGGCCAAAAGAACCCTTCAGAGACTATGTAGATAGGTTCTTTAAAGTTCTCA
GAGCCGAACAAGCTACACAGGAAGTAAAAGGCTGGATGACAGAGACCCTGCTGGTTCAAAATGCA
AATCCAGATTGTAAGTCCATTTTAAGAGCATTAGGAACAGGGGCTACATTAGAAGAAATGATGAC
AGCATGTCAGGGAGTGGGAGGACCCGGCCATAAAGCAAGGGTTTTAGCTGAGGCAATGAGTCAAG
CACAACAGGCAAATGTAATGATGCAGAGGGGCAGCTTTAAGGGGCAGAAAAGAATTAAGTGCTTC
AACTGTGGCAAAGAGGGACACCTAGCCAGAAATTGCAGAGCCCCTAGGAAAAAAGGCTGTTGGAA
GTGTGGGAAAGAAGGACACCAAATGAAAGATTGCAATGAGAGACAGGCTAATTTTTTAGGGAAAA
TTTGGCCTTCCAGCAAGGGGAGGCCAGGAAATTTTCCCCAGAGCAGACCGGAGCCAACAGCCCCA
CCAGCAGAGATCTTTGGGATGGGGGAAGAGATAACCTCCCCTCCGAAGCAGGAGCAGAAAGAGAG
GGAACAAACCCCACCCTTTGTTTCCCTCAAATCACTCTTTGGCAACGACCCGTTGTCACAGTAAAA
GTAGGAGGAGAAATGAGAGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGATAT
AAATTTGCCAGGAAAATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAGGTAAAAC
AATATGATCAGGTATCTATAGAAATTTGTGGAAAAAAGGCTATAGGTACGGTATTAGTAGGACCT
ACACCTGTCAACATAATTGGAAGAAATATGTTGACTCAGATTGGTTGTACCTTAAATTTTCCAATT
AGTCCTATTGAGACTGTACCAGTAACATTAAAGCCAGGAATGGATGGCCCAAGGGTTAAACAATG
GCCATTGACAGAAGAGAAAATAAAAGCATTGACAGAAATTTGTAAAGAGATGGAAAAGGAAGGA
AAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAATATTTGCAATAAAGAAAAAAGAT
AGCACTAAATGGAGGAAATTAGTAGATTTCAGAGAGCTCAATAAAAGAACACAAGACTTTTGGGA
AGTTCAATTAGGGATACCGCATCCAGCGGGCCTAAAAAAGAAAAAATCAGTAACAGTACTAGAGG
TGGGGGATGCATATTTTTCAGTTCCCCTAGATAAAAACTTTAGAAAGTATACTGCATTTACCATAC
CTAGTTTAAATAATGAAACACCAGGAATCAGGTATCAGTACAATGTGCTTCCACAAGGATGGAAA
GGATCACCAGCAATATTCCAGTGCAGTATGACAAAAATCTTAGAGCCCTTTAGATCAAAAAATCCA
GAAATAATTATCTATCAATACATGCACGACTTGTATGTAGGATCAGATTTAGAAATAGGGCAGCAT
AGAGCAAAAATAGAAGAATTAAGAGCTCATCTACTGAGCTGGGGATTTACTACACCAGACAAAAA
GCATCAGAAAGAACCTCCATTCCTTTGGATGGGATATGAGCTCCATCCTGACAAGTGGACAGTCCA
GCCTATAGAGCTGCCAGAAAAAGAAAGCTGGACTGTCAATGATATACAGAAATTAGTGGGAAAAC
TAAATTGGGCCAGTCAAATTTATCCAGGAATTAAAGTAAAGCAATTGTGTAAACTTCTCAGGGGAG
CCAAAGCCCTAACAGATATAGTAACACTGACTGAGGAAGCAGAATTAGAATTAGCAGAGAACAGG
GAGATTCTAAAAGACCCTGTGCATGGGGTATATTATGACCCATCAAAAGACTTAATAGCAGAAAT
ACAGAAACAAGGGCAAGACCAATGGACATACCAAATTTATCAGGAGCCATTTAAAAATCTAAAAA
CAGGAAAATATGCAAGAAAAAGGTCTGCTCACACTAATGATGTAAGACAATTAGCAGAAGTAGTG
CAGAAAGTGGTCATGGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAACTACCCATACA
AAAAGAGACATGGGAGACATGGTGGATGGACTATTGGCAAGCTACCTGGATTCCTGAGTGGGAGT
TTGTCAATACCCCTCCCCTAGTAAAATTATGGTACCAGTTAGAGAAAGACCCCATAGCAGGAGCAG
AGACTTTCTAA KNH1144 envelope (SEQ ID NO: 59):
ATGAGAGTGATGGGGATACAGATGAATTGTCAGCACTTATTGAGATGGGGAACTATGATCTTGGG
ATTGATAATAATCTGTAATGCTGTAAACAGCAACTTGTGGGTTACTGTCTATTATGGGGTACCTGT
GTGGAAAGATGCAGAGACCACCTTATTTTGTGCATCAGATGCTAAAGCATATAAAACAGAAAAGC
ATAATGTCTGGGCTACACATGCCTGTGTGCCCACAGACCCCAACCCACAAGAAATACCTTTGGAAA
ATGTGACAGAAGAGTTTAACATGTGGAAAAATAAAATGGTAGAACAAATGCATACAGATATAATC
AGTCTATGGGACCAAAGCCTACAGCCATGTGTAAAGTTAACCCCTCTCTGCATTACTTTAAACTGT
ACAGATGTTACTAATGTTACAGATGTTAGTGGTACGAGGGGCAACATCACCATCATGAAAGAGAT
GGAGGGAGAAATAAAAAACTGTTCTTTCAATATGACCACAGAAATAAGGGATAAGAAACAGAAAG
TATATTCACTCTTTTATAGACTTGATGTAGTACCAATAAATCAGGGTAATAGTAGTAGTAAAAACA
GTAGTGAGTATAGATTAATAAGTTGTAATACCTCAGCCATTACACAAGCTTGCCCAAAGGTAAGCT
TTGAGCCAATTCCCATACATTATTGTGCCCCAGCTGGTTTTGCGATCCTGAAGTGTAGGGATAAGG
AGTTCAATGGAACAGGGGAATGCAAGAATGTCAGCACAGTCCAATGCACACATGGAATCAAGCCA
GTAGTATCAACTCAACTACTGTTAAATGGCAGTCTAGCAGAAGAAAAGGTAAAAATCAGAACTGA
AAATATCACAAACAATGCCAAAACTATAGTAGTACAACTTGTCGAGCCTGTGAGAATTAATTGTAC
TAGACCTAATAACAATACAAGAGAGAGTGTGCGTATAGGGCCAGGACAAGCATTCTTTGCAACAG
GTGACATAATAGGGGATATAAGACAAGCACATTGTAATGTCAGTAGATCACAATGGAATAAGACT
TTACAACAGGTAGCTGAACAATTAAGAGAACACTTTAAAAACAAAACAATAATATTTAACAGTTCC
TCAGGAGGGGATCTAGAAATCACAACACATAGTTTCAATTGTGGAGGAGAATTCTTCTATTGTAAT
ACATCAGGTCTGTTCAATAGCACCTGGAATACCAGCATGTCAGGGTCAAGTAACACGGAGACAAA
TGACACTATAACTCTCCAATGCAGAATAAAGCAAATTATAAATATGTGGCAGAGAACAGGACAAG
CAATATATGCCCCTCCCATCCAGGGAGTGATAAGGTGTGAATCAAACATCACAGGACTACTGTTAA
CAAGAGATGGTGGGGAGGAGAAGAACAGTACAAATGAAATCTTCAGACCTGGAGGAGGAGATAT
GAGGGACAACTGGAGAAGTGAATTATATAAGTATAAAGTAGTAAAAATTGAACCACTAGGAGTAG
CACCCACCAGGGCAAGGAGAAGAGTGGTGGGAAGAGAAAAAAGAGCAGTTGGAATAGGAGCTGT
TTTCCTTGGGTTCTTAGGAGCAGCAGGAAGCACTATGGGCGCGGCGTCAATAACGCTGACGGTACA
GGCCAGGCAATTATTGTCTGGCATAGTGCAGCAGCAGAGCAATTTGCTGAGGGCTATAGAGGCTC
AACAACATATGTTGAAACTCACGGTCTGGGGCATTAAACAGCTCCAGGCAAGAGTCCTTGCTGTGG
AAAGATACCTAAGGGATCAACAGCTCCTAGGAATTTGGGGCTGCTCTGGAAAACTCATCTGCACCA
CTAATGTGCCCTGGAACTCTAGTTGGAGTAATAAATCTCAGGATGAAATATGGAACAACATGACCT
GGCTGCAATGGGATAAAGAAATTAGCAATTACATAAACCTAATATATAGTCTAATTGAAGAATCG
CAAAACCAGCAGGAAAAGAATGAACAAGACTTATTGGCATTGGGCAAGTGGGCAAATCTGTGGAC
TTGGTTTGACATATCAAATTGGCTGTGGTATATAAGAATATTTATAATGATAGTAGGAGGCTTAAT
AGGATTAAGAATAGTTTTTGCTGTGCTTGCTGTAATAAAGAGAGTTAGGCAGGGATACTCACCTGT
GTCATTTCAGATCCATGCCCCAAACCCAGGGGGTCTCGACAGGCCCGGAAGAATCGAAGGAGAAG
GTGGAGAGCAAGACTAA KNH1207 envelope (SEQ ID NO: 60)
ATGAGAGTGATGGGGATACAGATGAATTGTCAAAGCTTGTGGAGATGGGGAACTATGATCTTGGG
AATGTTAATGATTTGTAGTGTTGCAGGAAACTTGTGGGTTACTGTCTACTATGGGGTACCTGTGTG
GAAAGAGGCAGACACCACCTTATTTTGTGTATCAAATGCTAGAGCATATGATACAGAAGTGCATA
ATGTCTGGGCTACACATGCCTGTGTACCTACGGACCCCAACCCACAAGAAATAGATTTGGAGAATG
TGACAGAAGAGTTTAACATGTGGAAAAATAACATGGTAGAGCAAATGCATACAGATATAATTAGT
CTATGGGACCAAAGCCTAAAACCATGTGTAAAGTTAACCCCTCTCTGCGTTACTTTAGATTGTGGC
TATAATGTAACCAACTTGAATTTCACCAGTAACATGAAAGGAGACATAACAAACTGCTCTTACAAT
ATGACCACAGAAATAAGGGATAGGAAACAGAAAGTGTATTCACTTTTCTATAGGCTTGATATAGTA
CCAATTAATGAAGAAAAGAATAATAGCAGGGAGACTAGTCCGTATAGATTAATAAATTGTAATAC
CTCAGCCATTACACAAGCTTGTCCTAAGGTATCTTTTGAACCAATTCCCATACATTATTGTGCCCCA
GCCGGTTTTGCGATTCTAAAATGTAAGGATGCAGAGTTCAATGGAACAGGGCCATGCAAGAATGT
CAGCACAGTACAATGTACACATGGAATCAGGCCAGTAATATCAACTCAACTGCTGTTAAATGGCAG
TTTAGCAGAGAATGGGACAAAGATTAGATCTGAAAATATCACAAACAATGCCAAAACCATAATAG
TACAACTTAACGAGACCGTACAAATTAATTGTACCAGACCTAGCAACAATACAAGAAAAAGTGTA
CGTATAGGACCAGGACAAGCATTCTATACAACAGGTGATATAACAGGGGATATAAGACAAGCATA
TTGTAATGTCAGTAGACAAGAATGGGAACAAGCATTAAAAGGGGTAGTTATACAATTAAGAAAAC
ACTTTAACAAAACAATAATCTTTAACAGTTCCTCAGGAGGGGATTTAGAAATTACAACACATAGTT
TTAATTGTGGAGGAGAATTCTTCTATTGTGATACATCAGGCCTGTTTAATAGCACCTGGAACACGA
ACACCACCGAGCCAAACAACACAACGTCAAATGGCACTATCATTCTCCAATGCAGAATAAAGCAA
ATTATAAATCTGTGGCAGAGAACCGGACAAGCAATGTATGCCCCTCCCATCCAAGGGGTAATAAG
GTGTGATTCCAACATTACAGGACTACTATTAACAAGAGATGGTGGAGTAGTTGATAGTATAAATGA
AACCGAAATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAGT
ATAAAGTAGTAAAAATTGAACCACTAGGAGTAGCACCCACCGGGGCAAAGAGAAGAGTGGTGGA
GAGAGAAAAAAGAGCAGTTGGCATAGGAGCTGTATTCATTGGGTTCTTAGGAGCAGCAGGAAGCA
CTATGGGCGCGGCGTCAATAACGCTGACGGTACAGGCCAGACAATTATTGTCTGGCATAGTGCAA
CAGCAAAGCAATTTGCTGAGGGCTATAGAGGCTCAACAGCATATGTTGAGACTCACGGTCTGGGG
CATTAAGCAGCTCCAGGCAAGAGTCCTGGCTGTGGAAAGATACCTAAGGGATCAACAGCTCCTAG
GAATTTGGGGCTGCTCTGGAAAACTCATCTGCACCACTAATGTGCCCTGGAACTCTAGTTGGAGTA
ATAAATCTCAGGAGGAAATATGGGGTAACATGACCTGGCTGCAATGGGATAAAGAAATTAGCAAT
TACACACAAACAATATATAACCTACTTGAAGAATCGCAGAACCAGCAGGAAAAGAATGAACAAGA
CTTATTGGCATTGGACAAGTGGGCAAATTTGCGGACTTGGTTTGACATAACAAATTGGCTGTGGTA
TATAAAAATGTTTATAATGATAGTAGGAGGCTTAATAGGATTAAGAATAGTTTTTGCTGTGCTTTC
TGTAATAAATAGAGTTAGGCAGGGATACTCACCTCTGTCGTTTCAGACCCATATCCCGAGCCCAAG
GGGTCTCGATAGGCCCGGAAGAATCGAAGGAGAAGGTGGAGAGCAAGACTAA
TABLE-US-00028 APPENDIX 4 DNA sequences of gagpol and env genes
from Tanzanian HIV-1 clade C isolates: TZA-246 gagpol (SEQ ID NO:
61):
ATGGGTGCGAGAGCGTCAATATTAAGAGGGGGAAAATTAGATCGATGGGAAAAAATTAGGTTAAG
GCCAGGGGGAAAGAAAAGCTATATGATAAAACACTTAGTATGGGCAAGCAGGGAGCTGGAAAGA
TTTGCACTTAACCCTAGCCTTTTAGAGACATCAGAAGGCTGTAAACAAATAATGAAACAGCTACAA
CCAGCTCTTCAGACAGGAACAGAAGAACTTAAATCATTATTCAATGCAATAGCAGTTCTCTATTGT
GTACATGAAGGGATAGATGTAAAAGACACCAAGGAAGCCTTAGACAAGATAGAGGAAGAACAGA
ACAAAAGTCAGCAAAAAACACAGCAGGCAGAAGCAGCTGGCGGAAAAGTCAGTCAAAATTATCCT
ATAGTGCAGAATCTCCAAGGACAAATGGTACACCAGTCCATATCACCTAGAACTTTGAATGCATGG
GTAAAAGTAATAGAGGAAAAGGCTTTTAGCCCAGAGGTAATACCCATGTTTACAGCATTATCAGA
AGGAGCCACCCCACAAGATTTAAACACCATGCTAAATACAGTGGGGGGACATCAAGCAGCCATGC
AAATGTTAAAAGATACCATCAATGAGGAGGCTGCAGAATGGGATAGGATACATCCAGTACATGCA
GGGCCTACTGCACCAGGCCAAATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTAC
CCTTCAGGAACAAATAGCATGGATGACAGCTAACCCACCTGTTCCAGTGGGAGAAATCTACAAAA
GATGGATAATACTGGGTTTAAATAAAATAGTAAGAATGTATAGCCCTGTCAGCATTTTGGACATAA
AACAAGGGCCAAAGGAACCCTTTAGAGACTATGTAGATCGGTTCTTTAAAACTTTAAGAGCTGAAC
AGGCTACACAAGATGTAAAAAATTGGATGACAGACACCTTGTTGGTCCAAAATGCGAACCCAGAT
TGTAAGACCATTTTAAGAGCATTAGGACCAGGGGCTACATTAGAAGAAATGATGACAGCATGTCA
AGGAGTGGGAGGACCTGGCCACAAAGCCAGAGTTTTGGCTGAGGCAATGAGCCAAGCAAACACAC
ACATAATGATGCAGAGAAGCAATTTTAAAGGCTCTAAAAGAATTGTTAAATGTTTCAACTGTGGCA
AGGAAGGGCACATAGCCAGAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAA
GGAAGGACACCAAATGAAAGACTGTACTGAGAGGCAGGCTAATTTTTTAGGGAAAATTTGGCCTT
CCCACAAGGGGAGGCCAGGGAATTTCCTTCAGAACAGGTCAGAGCCAACAGCCCCACCAACGAAC
AGGCCAGAGCCAACAGCTCCACCAGCAGAGAGCTTCAGGTTCGAGGAAGCAACCCCTGCTCCGAA
GCAGGAGCTGAAAGACAGGGAACCTTTAATTTCCCTCAAATCACTCTTTGGCAGCGACCCCTCGTC
TCAATAAAAGTAGGGGGTCAAACAAAGGAGGCTCTTTTAGACACAGGAGCAGATGATACAGTATT
AGAAGAAATAAATTTGCCAGGAAAATGGAAACCCAAAATGATAGGAGGAATTGGAGGTTTTATCA
AAGTAAGACAGTATGATCAGATAGTTATAGAAATTTGTGGAAAAAAGGCTATAGGTACAGTATTA
GTAGGACCCACCCCTGTCAACATAATTGGAAGAAATATGTTGACTCAGCTTGGATGCACACTAAAT
TTTCCAATTAGTCCTATTGAAACTGTACCAGTAAAGTTAAAGCCAGGAATGGATGGCCCAAAGGTT
AAACAATGGCCATTGACAGAAGAAAAAATAAAGGCATTAACAGCAATTTGTGAAGAAATGGAGAA
GGAAGGAAAAATTACAAAGATTGGGCCTGAAAATCCATATAACACTCCAGTATTTGCCATAAAAA
AGAAGGACAGTACTAAGTGGAGAAAATTAGTAGATTTCAGGGAACGCAATAAAAGAACTCAAGAT
TTTTGGGAAGTTCAATTAGGCATACCACACCCAGCAGGGTTAAAAAAGAAAAAATCAGTGACAGT
ACTGGAGGTGGGGGATGCATACTTCTCAGTTCCTTTAGATGAAGGCTTCAGGAAATATACTGCATT
CACCATACCTAGTATAAACAATGAAACACCAGGAATTAGATATCAATACAATGTGCTTCCACAGGG
ATGGAAAGGATCACCAGCAATATTCCAGAGTAGCATGACAAAAATCTTAGAGCCCTTTAGAGCAC
AAAATCCAGAAATAGTCATCTATCAATATATGCACGACTTATATGTAGGATCTGACTTAGAAATAG
GGCAACATAGAGCAAAAATAGAGGAATTAAGAGAACATCTATTAAAGTGGGGATTTACCACACCA
GACAAGAAACATCAGAAAGAACCCCCATTTCTTTGGATGGGGTATGAACTCCATCCTGACAAATGG
ACAGTACAGCCTATAACGCTGCCAGAAAAGGAAAGCTGGACTGTCAATGATATACAGAAGTTAGT
GGGAAAACTAAACTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAACTGTATAAACTCC
TTAGGGGAGCCAAAGCACTAACAGACATAGTACCACTAACTGAAGAGGCAGAATTAGAATTGGCA
GAGAACAGGGAAATTCTAAAAGAACCAGTACATGGGGTATATTATGACCCATCAAAAGACTTGAT
AGCTGAAATACAGAAACAAGGGCATGACCAATGGACATATCAAATTTACCAAGAACCATTCAAAA
ATCTGAAAACAGGGAAGTATGCAAAAATGAGGAGTGCCCACACTAATGATGTAAAACAATTAACA
GAGGCAGTGCAAAAAATAGCCATGGAAGGCATAGTAATATGGGGAAAGACTCCTAAATTTAGACT
GCCCATTCAAAAGGAAACATGGGAAACATGGTGGACAGACTATTGGCAAGCCACCTGGATTCCTG
AGTGGGAGTTTGTTAATACCCCTCCCCTAGTAAAATTATGGTACCAGCTGGAGAAAGAACCCATAG
TAGGAGCAGAAACTTTC TZA-125 envelope (SEQ ID NO: 62):
ATGAGAGTGAAGGGGATATTGAGGAATTGGCAACACAGGTGGATATGGATCTGGATCATCTTAGG
CTTTTGGATGCTAATGATTTGTAATGGGAACTTGTGGGTCACTGTCTACTATGGGGTACCTGTGTG
GAAAGAAGCAAATGCTCCTCTATTTTGTGCATCAGATGCTAAAGCATATGAGAAAGAAGTGCATA
ATGTCTGGGCTACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAACTAGACTTGGTAAATG
TAACAGAAAATTTTAACATGTGGAAAAATGACATGGTAGATCAGATGCATGAGGATATAATCAGT
TTATGGGATGAAAGCCTAAAGCCATGTGTAAAGTTGACCCCACTCTGTGTCACTCTAAACTGTACT
AATGCTAATATTAATAATGATACTGTTGCTAATAGTGGTACTTTTAAGGTTGATAATAGTAGTAAT
GTAGTAAAAAATTGCTCTTTCAATATAACCACAGAAATAAGAGATAAGAAGAAAAAAGAATATTC
ATTGTTTTATAGACTTGATATATTACCACTTGATAACTCTAGTGAGTCTAAGAACTATAGTGAGTAT
GTATTAATAAATTGTAATGCCTCAACCGTAACACAAGCCTGTCCAAAGGTCTCTTTTGACCCAATT
CCTATACATTATTGTGCTCCAGCTGGTTATGCGATTCTAAAGTGTAAAGATAAGACATTCAATGGA
ACAGGACCATGCAGTAATGTCAGCACAGTACTATGTACACATGGAATTAAGCCAGTGGTATCAACT
CAATTACTGTTAAATGGTAGCCTAGCAGAAGAAGGGATAGTAATTAGATCTGAAAATCTGACAAA
CAATGCCAAAACAACAATAGTACAGCTTAATGAACCTGTAGAAATTATGTGTGTAAGACCCGGCA
ATAATACAAGAAAAAGTGTGAGGATAGGACCAGGACAAACATTCTATGCAACAGGAGGCATAATA
GGAGATATAAGACAAGCACATTGTAACATTAGTAGAAGTGATTGGAATAAAACTTTACAAGAGGT
AGGTAAAAAATTACGAGAATACTTCCACAATAAAACAATAAGATTTAAACCGGCGGTCGTAGGAG
GGGACCTGGAAATTACAACACATAGCTTTAATTGTAGAGGAGAATTCTTCTATTGCAATACATCAG
AACTGTTTACAGGTGAATATAATGGTACTGAGTATAAGAATACTTCAAATTCAAATCCTAACATCA
CACTCCCATGTAGAATAAAACAATTTGTAAACATGTGGCAGAGGGTAGGACGAGCAATGTATGCC
CCTCCTATTGAAGGAAACATAACATGTAACTCAAGTATCACAGGACTACTATTGACATGGGATGGA
GGAAACAATACTAATGGCACAGAGACATTTAGACCTGGAGGAGGAGATATGAGGGATAATTGGAG
AAGTGAATTATATAAATATAAAGTGGTAGAAATTAAACCATTAGGAATAGCACCCACTAGTGCAA
AAAGGAGAGTGGTGGAGAGAGAGAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTA
GGAGCAGCAGGAAGCACTATGGGCGCAGCATCAATAACGCTGACGGTACAGGCCAGACAATTATT
GTCTGGTATAGTGCAACAGCAAAGCAATTTGCTGAGGGCCATAGAGGCGCAACAGCATATGTTGC
AACTCACAGTCTGGGGCATTAAACAGCTCCAGACAAGAGTCCTGGCTATAGAAAGATACCTAAAG
GATCAACAGCTCCTAGGGATTTGGGGCTGCTCTGGAAAACTCATCTGCACCACTGCTGTGCCTTGG
AACACTAGTTGGAGTAATAAAACTGAACAGGACATTTGGAATCTAACCTGGATGCAGTGGGATAG
AGAAGTTAGTAATTACACAGACATAATATACAGGTTGCTTGAAGACTCACAAATCCAGCAGGAAA
ACAATGAAAAGGATTTACTAGCATTGGACAGTTGGAAAAATCTGTGGAATTGGTTTGACATAACA
AATTGGTTGTGGTATATAAGAACATTCATAATGATAGTAGGAGGCTTGATAGGCTTAAGGATAATT
TTTGCTGTAATTTCTATAGTGAATAGAGTTAGGCAGGGATACTCACCTTTGTCATTTCAGACCCTTA
CCCCAACCCCGAGGGGACCAGAAAGGCTCGGAGGAATCGAAGAAGAAGGTGGAGAGCAAGACTAA
[0255] 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, referenced to above, are hereby incorporated by
reference.
Sequence CWU 1
1
63112225DNAArtificial SequencePlasmid pLW-48 1gaattcgttg gtggtcgcca
tggatggtgt tattgtatac tgtctaaacg cgttagtaaa 60acatggcgag gaaataaatc
atataaaaaa tgatttcatg attaaaccat gttgtgaaaa 120agtcaagaac
gttcacattg gcggacaatc taaaaacaat acagtgattg cagatttgcc
180atatatggat aatgcggtat ccgatgtatg caattcactg tataaaaaga
atgtatcaag 240aatatccaga tttgctaatt tgataaagat agatgacgat
gacaagactc ctactggtgt 300atataattat tttaaaccta aagatgccat
tcctgttatt atatccatag gaaaggatag 360agatgtttgt gaactattaa
tctcatctga taaagcgtgt gcgtgtatag agttaaattc 420atataaagta
gccattcttc ccatggatgt ttcctttttt accaaaggaa atgcatcatt
480gattattctc ctgtttgatt tctctatcga tgcggcacct ctcttaagaa
gtgtaaccga 540taataatgtt attatatcta gacaccagcg tctacatgac
gagcttccga gttccaattg 600gttcaagttt tacataagta taaagtccga
ctattgttct atattatata tggttgttga 660tggatctgtg atgcatgcaa
tagctgataa tagaacttac gcaaatatta gcaaaaatat 720attagacaat
actacaatta acgatgagtg tagatgctgt tattttgaac cacagattag
780gattcttgat agagatgaga tgctcaatgg atcatcgtgt gatatgaaca
gacattgtat 840tatgatgaat ttacctgatg taggcgaatt tggatctagt
atgttgggga aatatgaacc 900tgacatgatt aagattgctc tttcggtggc
tgggtaccag gcgcgccttt cattttgttt 960ttttctatgc tataaatggt
acgtcctgta gaaaccccaa cccgtgaaat caaaaaactc 1020gacggcctgt
gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg
1080gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag gcagttttaa
cgatcagttc 1140gccgatgcag atattcgtaa ttatgcgggc aacgtctggt
atcagcgcga agtctttata 1200ccgaaaggtt gggcaggcca gcgtatcgtg
ctgcgtttcg atgcggtcac tcattacggc 1260aaagtgtggg tcaataatca
ggaagtgatg gagcatcagg gcggctatac gccatttgaa 1320gccgatgtca
cgccgtatgt tattgccggg aaaagtgtac gtatcaccgt ttgtgtgaac
1380aacgaactga actggcagac tatcccgccg ggaatggtga ttaccgacga
aaacggcaag 1440aaaaagcagt cttacttcca tgatttcttt aactatgccg
gaatccatcg cagcgtaatg 1500ctctacacca cgccgaacac ctgggtggac
gatatcaccg tggtgacgca tgtcgcgcaa 1560gactgtaacc acgcgtctgt
tgactggcag gtggtggcca atggtgatgt cagcgttgaa 1620ctgcgtgatg
cggatcaaca ggtggttgca actggacaag gcactagcgg gactttgcaa
1680gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc tctatgaact
gtgcgtcaca 1740gccaaaagcc agacagagtg tgatatctac ccgcttcgcg
tcggcatccg gtcagtggca 1800gtgaagggcg aacagttcct gattaaccac
aaaccgttct actttactgg ctttggtcgt 1860catgaagatg cggacttgcg
tggcaaagga ttcgataacg tgctgatggt gcacgaccac 1920gcattaatgg
actggattgg ggccaactcc taccgtacct cgcattaccc ttacgctgaa
1980gagatgctcg actgggcaga tgaacatggc atcgtggtga ttgatgaaac
tgctgctgtc 2040ggctttaacc tctctttagg cattggtttc gaagcgggca
acaagccgaa agaactgtac 2100agcgaagagg cagtcaacgg ggaaactcag
caagcgcact tacaggcgat taaagagctg 2160atagcgcgtg acaaaaacca
cccaagcgtg gtgatgtgga gtattgccaa cgaaccggat 2220acccgtccgc
aaggtgcacg ggaatatttc gcgccactgg cggaagcaac gcgtaaactc
2280gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca
caccgatacc 2340atcagcgatc tctttgatgt gctgtgcctg aaccgttatt
acggatggta tgtccaaagc 2400ggcgatttgg aaacggcaga gaaggtactg
gaaaaagaac ttctggcctg gcaggagaaa 2460ctgcatcagc cgattatcat
caccgaatac ggcgtggata cgttagccgg gctgcactca 2520atgtacaccg
acatgtggag tgaagagtat cagtgtgcat ggctggatat gtatcaccgc
2580gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc
cgattttgcg 2640acctcgcaag gcatattgcg cgttggcggt aacaagaaag
ggatcttcac tcgcgaccgc 2700aaaccgaagt cggcggcttt tctgctgcaa
aaacgctgga ctggcatgaa cttcggtgaa 2760aaaccgcagc agggaggcaa
acaatgagag ctcggttgtt gatggatctg tgatgcatgc 2820aatagctgat
aatagaactt acgcaaatat tagcaaaaat atattagaca atactacaat
2880taacgatgag tgtagatgct gttattttga accacagatt aggattcttg
atagagatga 2940gatgctcaat ggatcatcgt gtgatatgaa cagacattgt
attatgatga atttacctga 3000tgtaggcgaa tttggatcta gtatgttggg
gaaatatgaa cctgacatga ttaagattgc 3060tctttcggtg gctggcggcc
cgctcgagta aaaaatgaaa aaatattcta atttatagga 3120cggttttgat
tttctttttt tctatgctat aaataataaa tagcggccgc accatgaaag
3180tgaaggggat caggaagaat tatcagcact tgtggaaatg gggcatcatg
ctccttggga 3240tgttgatgat ctgtagtgct gtagaaaatt tgtgggtcac
agtttattat ggggtacctg 3300tgtggaaaga agcaaccacc actctatttt
gtgcatcaga tgctaaagca tatgatacag 3360aggtacataa tgtttgggcc
acacatgcct gtgtacccac agaccccaac ccacaagaag 3420tagtattgga
aaatgtgaca gaaaatttta acatgtggaa aaataacatg gtagaacaga
3480tgcatgagga tataatcagt ttatgggatc aaagcctaaa gccatgtgta
aaattaaccc 3540cactctgtgt tactttaaat tgcactgatt tgaggaatgt
tactaatatc aataatagta 3600gtgagggaat gagaggagaa ataaaaaact
gctctttcaa tatcaccaca agcataagag 3660ataaggtgaa gaaagactat
gcacttttct atagacttga tgtagtacca atagataatg 3720ataatactag
ctataggttg ataaattgta atacctcaac cattacacag gcctgtccaa
3780aggtatcctt tgagccaatt cccatacatt attgtacccc ggctggtttt
gcgattctaa 3840agtgtaaaga caagaagttc aatggaacag ggccatgtaa
aaatgtcagc acagtacaat 3900gtacacatgg aattaggcca gtagtgtcaa
ctcaactgct gttaaatggc agtctagcag 3960aagaagaggt agtaattaga
tctagtaatt tcacagacaa tgcaaaaaac ataatagtac 4020agttgaaaga
atctgtagaa attaattgta caagacccaa caacaataca aggaaaagta
4080tacatatagg accaggaaga gcattttata caacaggaga aataatagga
gatataagac 4140aagcacattg caacattagt agaacaaaat ggaataacac
tttaaatcaa atagctacaa 4200aattaaaaga acaatttggg aataataaaa
caatagtctt taatcaatcc tcaggagggg 4260acccagaaat tgtaatgcac
agttttaatt gtggagggga attcttctac tgtaattcaa 4320cacaactgtt
taatagtact tggaatttta atggtacttg gaatttaaca caatcgaatg
4380gtactgaagg aaatgacact atcacactcc catgtagaat aaaacaaatt
ataaatatgt 4440ggcaggaagt aggaaaagca atgtatgccc ctcccatcag
aggacaaatt agatgctcat 4500caaatattac agggctaata ttaacaagag
atggtggaac taacagtagt gggtccgaga 4560tcttcagacc tgggggagga
gatatgaggg acaattggag aagtgaatta tataaatata 4620aagtagtaaa
aattgaacca ttaggagtag cacccaccaa ggcaaaaaga agagtggtgc
4680agagagaaaa aagagcagtg ggaacgatag gagctatgtt ccttgggttc
ttgggagcag 4740caggaagcac tatgggcgca gcgtcaataa cgctgacggt
acaggccaga ctattattgt 4800ctggtatagt gcaacagcag aacaatttgc
tgagggctat tgaggcgcaa cagcatctgt 4860tgcaactcac agtctggggc
atcaagcagc tccaggcaag agtcctggct gtggaaagat 4920acctaaggga
tcaacagctc ctagggattt ggggttgctc tggaaaactc atctgcacca
4980ctgctgtgcc ttggaatgct agttggagta ataaaactct ggatatgatt
tgggataaca 5040tgacctggat ggagtgggaa agagaaatcg aaaattacac
aggcttaata tacaccttaa 5100ttgaggaatc gcagaaccaa caagaaaaga
atgaacaaga cttattagca ttagataagt 5160gggcaagttt gtggaattgg
tttgacatat caaattggct gtggtatgta aaaatcttca 5220taatgatagt
aggaggcttg ataggtttaa gaatagtttt tactgtactt tctatagtaa
5280atagagttag gcagggatac tcaccattgt catttcagac ccacctccca
gccccgaggg 5340gacccgacag gcccgaagga atcgaagaag aaggtggaga
cagagactaa tttttatgcg 5400gccgctggta cccaacctaa aaattgaaaa
taaatacaaa ggttcttgag ggttgtgtta 5460aattgaaagc gagaaataat
cataaataag cccggggatc ctctagagtc gacaccatgg 5520gtgcgagagc
gtcagtatta agcgggggag aattagatcg atgggaaaaa attcggttaa
5580ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
agggagctag 5640aacgattcgc agttaatcct ggcctgttag aaacatcaga
aggctgtaga caaatactgg 5700gacagctaca accatccctt cagacaggat
cagaagaact tagatcatta tataatacag 5760tagcaaccct ctattgtgtg
catcaaagga tagagataaa agacaccaag gaagctttag 5820acaagataga
ggaagagcaa aacaaaagta agaaaaaagc acagcaagca gcagctgaca
5880caggacacag caatcaggtc agccaaaatt accctatagt gcagaacatc
caggggcaaa 5940tggtacatca ggccatatca cctagaactt taaatgcatg
ggtaaaagta gtagaagaga 6000aggctttcag cccagaagtg atacccatgt
tttcagcatt atcagaagga gccaccccac 6060aagatttaaa caccatgcta
aacacagtgg ggggacatca agcagccatg caaatgttaa 6120aagagaccat
caatgaggaa gctgcagaat gggatagagt gcatccagtg catgcagggc
6180ctattgcacc aggccagatg agagaaccaa ggggaagtga catagcagga
actactagta 6240cccttcagga acaaatagga tggatgacaa ataatccacc
tatcccagta ggagaaattt 6300ataaaagatg gataatcctg ggattaaata
aaatagtaag aatgtatagc cctaccagca 6360ttctggacat aagacaagga
ccaaaagaac cctttagaga ctatgtagac cggttctata 6420aaactctaag
agccgagcaa gcttcacagg aggtaaaaaa ttggatgaca gaaaccttgt
6480tggtccaaaa tgcgaaccca gattgtaaga ctattttaaa agcattggga
ccagcggcta 6540cactagaaga aatgatgaca gcatgtcagg gagtaggagg
acccggccat aaggcaagag 6600ttttggctga agcaatgagc caagtaacaa
attcagctac cataatgatg cagagaggca 6660attttaggaa ccaaagaaag
attgttaagt gtttcaattg tggcaaagaa gggcacacag 6720ccagaaattg
cagggcccct aggaaaaagg gctgttggaa atgtggaaag gaaggacacc
6780aaatgaaaga ttgtactgag agacaggcta attttttagg gaagatctgg
ccttcctaca 6840agggaaggcc agggaatttt cttcagagca gaccagagcc
aacagcccca ccagaagaga 6900gcttcaggtc tggggtagag acaacaactc
cccctcagaa gcaggagccg atagacaagg 6960aactgtatcc tttaacttcc
ctcagatcac tctttggcaa cgacccctcg tcacaataaa 7020gatagggggg
caactaaagg aagctctatt agatacagga gcagatgata cagtattaga
7080agaaatgagt ttgccaggaa gatggaaacc aaaaatgata gggggaattg
gaggttttat 7140caaagtaaga cagtatgatc agatactcat agaaatctgt
ggacataaag ctataggtac 7200agtattagta ggacctacac ctgtcaacat
aattggaaga aatctgttga ctcagattgg 7260ttgcacttta aattttccca
ttagccctat tgagactgta ccagtaaaat taaagccagg 7320aatggatggc
ccaaaagtta aacaatggcc attgacagaa gaaaaaataa aagcattagt
7380agaaatttgt acagaaatgg aaaaggaagg gaaaatttca aaaattgggc
ctgagaatcc 7440atacaatact ccagtatttg ccataaagaa aaaagacagt
actaaatgga ggaaattagt 7500agatttcaga gaacttaata agagaactca
agacttctgg gaagttcaat taggaatacc 7560acatcccgca gggttaaaaa
agaaaaaatc agtaacagta ctggatgtgg gtgatgcata 7620tttttcagtt
cccttagatg aagacttcag gaagtatact gcatttacca tacctagtat
7680aaacaatgag acaccaggga ttagatatca gtacaatgtg cttccacagg
gatggaaagg 7740atcaccagca atattccaaa gtagcatgac aaaaatctta
gagcctttta aaaaacaaaa 7800tccagacata gttatctatc aatacatgaa
cgatttgtat gtaggatctg acttagaaat 7860agggcagcat agaacaaaaa
tagaggagct gagacaacat ctgttgaggt ggggacttac 7920cacaccagac
aaaaaacatc agaaagaacc tccattcctt tggatgggtt atgaactcca
7980tcctgataaa tggacagtac agcctatagt gctgccagaa aaagacagct
ggactgtcaa 8040tgacatacag aagttagtgg ggaaattgaa taccgcaagt
cagatttacc cagggattaa 8100agtaaggcaa ttatgtaaac tccttagagg
aaccaaagca ctaacagaag taataccact 8160aacagaagaa gcagagctag
aactggcaga aaacagagag attctaaaag aaccagtaca 8220tggagtgtat
tatgacccat caaaagactt aatagcagaa atacagaagc aggggcaagg
8280ccaatggaca tatcaaattt atcaagagcc atttaaaaat ctgaaaacag
gaaaatatgc 8340aagaatgagg ggtgcccaca ctaatgatgt aaaacaatta
acagaggcag tgcaaaaaat 8400aaccacagaa agcatagtaa tatggggaaa
gactcctaaa tttaaactac ccatacaaaa 8460ggaaacatgg gaaacatggt
ggacagagta ttggcaagcc acctggattc ctgagtggga 8520gtttgttaat
acccctcctt tagtgaaatt atggtaccag ttagagaaag aacccatagt
8580aggagcagaa accttctatg tagatggggc agctaacagg gagactaaat
taggaaaagc 8640aggatatgtt actaacaaag gaagacaaaa ggttgtcccc
ctaactaaca caacaaatca 8700gaaaactcag ttacaagcaa tttatctagc
tttgcaggat tcaggattag aagtaaacat 8760agtaacagac tcacaatatg
cattaggaat cattcaagca caaccagata aaagtgaatc 8820agagttagtc
aatcaaataa tagagcagtt aataaaaaag gaaaaggtct atctggcatg
8880ggtaccagca cacaaaggaa ttggaggaaa tgaacaagta gataaattag
tcagtgctgg 8940aatcaggaaa atactatttt tagatggaat agataaggcc
caagatgaac attagttttt 9000atgtcgacct gcagggaaag ttttataggt
agttgataga acaaaataca taattttgta 9060aaaataaatc actttttata
ctaatatgac acgattacca atacttttgt tactaatatc 9120attagtatac
gctacacctt ttcctcagac atctaaaaaa ataggtgatg atgcaacttt
9180atcatgtaat cgaaataata caaatgacta cgttgttatg agtgcttggt
ataaggagcc 9240caattccatt attcttttag ctgctaaaag cgacgtcttg
tattttgata attataccaa 9300ggataaaata tcttacgact ctccatacga
tgatctagtt acaactatca caattaaatc 9360attgactgct agagatgccg
gtacttatgt atgtgcattc tttatgacat cgcctacaaa 9420tgacactgat
aaagtagatt atgaagaata ctccacagag ttgattgtaa atacagatag
9480tgaatcgact atagacataa tactatctgg atctacacat tcaccagaaa
ctagttaagc 9540ttgtctccct atagtgagtc gtattagagc ttggcgtaat
catggtcata gctgtttcct 9600gtgtgaaatt gttatccgct cacaattcca
cacaacatac gagccggaag cataaagtgt 9660aaagcctggg gtgcctaatg
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 9720gctttcgagt
cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
9780agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
gctgcgctcg 9840gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
cggtaatacg gttatccaca 9900gaatcagggg ataacgcagg aaagaacatg
tgagcaaaag gccagcaaaa ggccaggaac 9960cgtaaaaagg ccgcgttgct
ggcgtttttc gataggctcc gcccccctga cgagcatcac 10020aaaaatcgac
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
10080tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
taccggatac 10140ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
atagctcacg ctgtaggtat 10200ctcagttcgg tgtaggtcgt tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag 10260cccgaccgct gcgccttatc
cggtaactat cgtcttgagt ccaacccggt aagacacgac 10320ttatcgccac
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
10380gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
agtatttggt 10440atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
ttggtagctc ttgatccggc 10500aaacaaacca ccgctggtag cggtggtttt
tttgtttgca agcagcagat tacgcgcaga 10560aaaaaaggat ctcaagaaga
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 10620gaaaactcac
gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
10680cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
aacttggtct 10740gacagttacc aatgcttaat cagtgaggca cctatctcag
cgatctgtct atttcgttca 10800tccatagttg cctgactccc cgtcgtgtag
ataactacga tacgggaggg cttaccatct 10860ggccccagtg ctgcaatgat
accgcgagac ccacgctcac cggctccaga tttatcagca 10920ataaaccagc
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
10980atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
taatagtttg 11040cgcaacgttg ttggcattgc tacaggcatc gtggtgtcac
gctcgtcgtt tggtatggct 11100tcattcagct ccggttccca acgatcaagg
cgagttacat gatcccccat gttgtgcaaa 11160aaagcggtta gctccttcgg
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 11220tcactcatgg
ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
11280ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg 11340agttgctctt gcccggcgtc aatacgggat aataccgcgc
cacatagcag aactttaaaa 11400gtgctcatca ttggaaaacg ttcttcgggg
cgaaaactct caaggatctt accgctgttg 11460agatccagtt cgatgtaacc
cactcgtgca cccaactgat cttcagcatc ttttactttc 11520accagcgttt
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
11580gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
aagcatttat 11640cagggttatt gtctcatgag cggatacata tttgaatgta
tttagaaaaa taaacaaata 11700ggggttccgc gcacatttcc ccgaaaagtg
ccacctgacg tctaagaaac cattattatc 11760atgacattaa cctataaaaa
taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt 11820gatgacggtg
aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
11880gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
cgggtgtcgg 11940ggctggctta actatgcggc atcagagcag attgtactga
gagtgcacca tatgcggtgt 12000gaaataccgc acagatgcgt aaggagaaaa
taccgcatca ggcgccattc gccattcagg 12060ctgcgcaact gttgggaagg
gcgatcggtg cgggcctctt cgctattacg ccagctggcg 12120aaagggggat
gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
12180cgttgtaaaa cgacggccag tgaattggat ttaggtgaca ctata
12225274DNAArtificial SequencePsyn II promoter 2taaaaaatga
aaaaatattc taatttatag gacggttttg attttctttt tttctatgct 60ataaataata
aata 7432214DNAArtificial SequenceADA envelope truncated
3atgaaagtga aggggatcag gaagaattat cagcacttgt ggaaatgggg catcatgctc
60cttgggatgt tgatgatctg tagtgctgta gaaaatttgt gggtcacagt ttattatggg
120gtacctgtgt ggaaagaagc aaccaccact ctattttgtg catcagatgc
taaagcatat 180gatacagagg tacataatgt ttgggccaca catgcctgtg
tacccacaga ccccaaccca 240caagaagtag tattggaaaa tgtgacagaa
aattttaaca tgtggaaaaa taacatggta 300gaacagatgc atgaggatat
aatcagttta tgggatcaaa gcctaaagcc atgtgtaaaa 360ttaaccccac
tctgtgttac tttaaattgc actgatttga ggaatgttac taatatcaat
420aatagtagtg agggaatgag aggagaaata aaaaactgct ctttcaatat
caccacaagc 480ataagagata aggtgaagaa agactatgca cttttctata
gacttgatgt agtaccaata 540gataatgata atactagcta taggttgata
aattgtaata cctcaaccat tacacaggcc 600tgtccaaagg tatcctttga
gccaattccc atacattatt gtaccccggc tggttttgcg 660attctaaagt
gtaaagacaa gaagttcaat ggaacagggc catgtaaaaa tgtcagcaca
720gtacaatgta cacatggaat taggccagta gtgtcaactc aactgctgtt
aaatggcagt 780ctagcagaag aagaggtagt aattagatct agtaatttca
cagacaatgc aaaaaacata 840atagtacagt tgaaagaatc tgtagaaatt
aattgtacaa gacccaacaa caatacaagg 900aaaagtatac atataggacc
aggaagagca ttttatacaa caggagaaat aataggagat 960ataagacaag
cacattgcaa cattagtaga acaaaatgga ataacacttt aaatcaaata
1020gctacaaaat taaaagaaca atttgggaat aataaaacaa tagtctttaa
tcaatcctca 1080ggaggggacc cagaaattgt aatgcacagt tttaattgtg
gaggggaatt cttctactgt 1140aattcaacac aactgtttaa tagtacttgg
aattttaatg gtacttggaa tttaacacaa 1200tcgaatggta ctgaaggaaa
tgacactatc acactcccat gtagaataaa acaaattata 1260aatatgtggc
aggaagtagg aaaagcaatg tatgcccctc ccatcagagg acaaattaga
1320tgctcatcaa atattacagg gctaatatta acaagagatg gtggaactaa
cagtagtggg 1380tccgagatct tcagacctgg gggaggagat atgagggaca
attggagaag tgaattatat 1440aaatataaag tagtaaaaat tgaaccatta
ggagtagcac ccaccaaggc aaaaagaaga 1500gtggtgcaga gagaaaaaag
agcagtggga acgataggag ctatgttcct tgggttcttg 1560ggagcagcag
gaagcactat gggcgcagcg tcaataacgc tgacggtaca ggccagacta
1620ttattgtctg gtatagtgca acagcagaac aatttgctga gggctattga
ggcgcaacag 1680catctgttgc aactcacagt ctggggcatc aagcagctcc
aggcaagagt cctggctgtg 1740gaaagatacc taagggatca acagctccta
gggatttggg gttgctctgg aaaactcatc 1800tgcaccactg ctgtgccttg
gaatgctagt tggagtaata aaactctgga tatgatttgg 1860gataacatga
cctggatgga gtgggaaaga gaaatcgaaa attacacagg cttaatatac
1920accttaattg aggaatcgca gaaccaacaa gaaaagaatg aacaagactt
attagcatta 1980gataagtggg caagtttgtg gaattggttt gacatatcaa
attggctgtg gtatgtaaaa 2040atcttcataa tgatagtagg aggcttgata
ggtttaagaa tagtttttac tgtactttct 2100atagtaaata gagttaggca
gggatactca ccattgtcat ttcagaccca cctcccagcc 2160ccgaggggac
ccgacaggcc cgaaggaatc gaagaagaag gtggagacag agac
2214470DNAArtificial SequencePmH5 promoter 4aaaaattgaa aataaataca
aaggttcttg agggttgtgt taaattgaaa gcgagaaata 60atcataaata
7053479DNAArtificial SequenceHXB2 gag pol 5atgggtgcga gagcgtcagt
attaagcggg ggagaattag atcgatggga aaaaattcgg 60ttaaggccag ggggaaagaa
aaaatataaa ttaaaacata tagtatgggc aagcagggag 120ctagaacgat
tcgcagttaa
tcctggcctg ttagaaacat cagaaggctg tagacaaata 180ctgggacagc
tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
240acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
caaggaagct 300ttagacaaga tagaggaaga gcaaaacaaa agtaagaaaa
aagcacagca agcagcagct 360gacacaggac acagcaatca ggtcagccaa
aattacccta tagtgcagaa catccagggg 420caaatggtac atcaggccat
atcacctaga actttaaatg catgggtaaa agtagtagaa 480gagaaggctt
tcagcccaga agtgataccc atgttttcag cattatcaga aggagccacc
540ccacaagatt taaacaccat gctaaacaca gtggggggac atcaagcagc
catgcaaatg 600ttaaaagaga ccatcaatga ggaagctgca gaatgggata
gagtgcatcc agtgcatgca 660gggcctattg caccaggcca gatgagagaa
ccaaggggaa gtgacatagc aggaactact 720agtacccttc aggaacaaat
aggatggatg acaaataatc cacctatccc agtaggagaa 780atttataaaa
gatggataat cctgggatta aataaaatag taagaatgta tagccctacc
840agcattctgg acataagaca aggaccaaaa gaacccttta gagactatgt
agaccggttc 900tataaaactc taagagccga gcaagcttca caggaggtaa
aaaattggat gacagaaacc 960ttgttggtcc aaaatgcgaa cccagattgt
aagactattt taaaagcatt gggaccagcg 1020gctacactag aagaaatgat
gacagcatgt cagggagtag gaggacccgg ccataaggca 1080agagttttgg
ctgaagcaat gagccaagta acaaattcag ctaccataat gatgcagaga
1140ggcaatttta ggaaccaaag aaagattgtt aagtgtttca attgtggcaa
agaagggcac 1200acagccagaa attgcagggc ccctaggaaa aagggctgtt
ggaaatgtgg aaaggaagga 1260caccaaatga aagattgtac tgagagacag
gctaattttt tagggaagat ctggccttcc 1320tacaagggaa ggccagggaa
ttttcttcag agcagaccag agccaacagc cccaccagaa 1380gagagcttca
ggtctggggt agagacaaca actccccctc agaagcagga gccgatagac
1440aaggaactgt atcctttaac ttccctcaga tcactctttg gcaacgaccc
ctcgtcacaa 1500taaagatagg ggggcaacta aaggaagctc tattagatac
aggagcagat gatacagtat 1560tagaagaaat gagtttgcca ggaagatgga
aaccaaaaat gataggggga attggaggtt 1620ttatcaaagt aagacagtat
gatcagatac tcatagaaat ctgtggacat aaagctatag 1680gtacagtatt
agtaggacct acacctgtca acataattgg aagaaatctg ttgactcaga
1740ttggttgcac tttaaatttt cccattagcc ctattgagac tgtaccagta
aaattaaagc 1800caggaatgga tggcccaaaa gttaaacaat ggccattgac
agaagaaaaa ataaaagcat 1860tagtagaaat ttgtacagaa atggaaaagg
aagggaaaat ttcaaaaatt gggcctgaga 1920atccatacaa tactccagta
tttgccataa agaaaaaaga cagtactaaa tggaggaaat 1980tagtagattt
cagagaactt aataagagaa ctcaagactt ctgggaagtt caattaggaa
2040taccacatcc cgcagggtta aaaaagaaaa aatcagtaac agtactggat
gtgggtgatg 2100catatttttc agttccctta gatgaagact tcaggaagta
tactgcattt accataccta 2160gtataaacaa tgagacacca gggattagat
atcagtacaa tgtgcttcca cagggatgga 2220aaggatcacc agcaatattc
caaagtagca tgacaaaaat cttagagcct tttaaaaaac 2280aaaatccaga
catagttatc tatcaataca tgaacgattt gtatgtagga tctgacttag
2340aaatagggca gcatagaaca aaaatagagg agctgagaca acatctgttg
aggtggggac 2400ttaccacacc agacaaaaaa catcagaaag aacctccatt
cctttggatg ggttatgaac 2460tccatcctga taaatggaca gtacagccta
tagtgctgcc agaaaaagac agctggactg 2520tcaatgacat acagaagtta
gtggggaaat tgaataccgc aagtcagatt tacccaggga 2580ttaaagtaag
gcaattatgt aaactcctta gaggaaccaa agcactaaca gaagtaatac
2640cactaacaga agaagcagag ctagaactgg cagaaaacag agagattcta
aaagaaccag 2700tacatggagt gtattatgac ccatcaaaag acttaatagc
agaaatacag aagcaggggc 2760aaggccaatg gacatatcaa atttatcaag
agccatttaa aaatctgaaa acaggaaaat 2820atgcaagaat gaggggtgcc
cacactaatg atgtaaaaca attaacagag gcagtgcaaa 2880aaataaccac
agaaagcata gtaatatggg gaaagactcc taaatttaaa ctacccatac
2940aaaaggaaac atgggaaaca tggtggacag agtattggca agccacctgg
attcctgagt 3000gggagtttgt taatacccct cctttagtga aattatggta
ccagttagag aaagaaccca 3060tagtaggagc agaaaccttc tatgtagatg
gggcagctaa cagggagact aaattaggaa 3120aagcaggata tgttactaac
aaaggaagac aaaaggttgt ccccctaact aacacaacaa 3180atcagaaaac
tcagttacaa gcaatttatc tagctttgca ggattcagga ttagaagtaa
3240acatagtaac agactcacaa tatgcattag gaatcattca agcacaacca
gataaaagtg 3300aatcagagtt agtcaatcaa ataatagagc agttaataaa
aaaggaaaag gtctatctgg 3360catgggtacc agcacacaaa ggaattggag
gaaatgaaca agtagataaa ttagtcagtg 3420ctggaatcag gaaaatacta
tttttagatg gaatagataa ggcccaagat gaacattag 347969PRTArtificial
SequenceGag-CM9 peptide 6Cys Thr Pro Tyr Asp Ile Asn Gln Met1
578PRTArtificial Sequenceovalbumin peptide 7Ser Ile Ile Asn Phe Glu
Lys Leu1 5820DNAArtificial Sequencesynthetic probe 8ctgtctgcgt
catttggtgc 2094PRTArtificial Sequenceendocytosis motif 9Tyr Xaa Xaa
Leu11093DNAArtificial Sequencem7.5 promoter 10cgctttttat agtaagtttt
tcacccataa ataataaata caataattaa tttctcgtaa 60aaattgaaaa actattctaa
tttattgcac ggt 931174DNAArtificial SequencePsyn III promoter
11taaaaattga aaaaatattc taatttatag gacggttttg attttctttt tttctatact
60ataaataata aata 741274DNAArtificial SequencePsyn IV promoter
12taaaaattga aaaactattc taatttatag gacggttttg attttctttt tttctatact
60ataaataata aata 741375DNAArtificial SequencePsyn V promoter
13aaaaaatgat aaagtaggtt cagttttatt gctggtttaa aatcacgctt tcgagtaaaa
60actacgaata taaat 75147DNAArtificial Sequenceearly transcription
termination signal 14tttttnt 71553DNAArtificial Sequence5' primer
15gcgccccggg tcgacgcggc cgcgccatga gagtgagggg gatacagagg aac
531657DNAArtificial Sequence3' primer 16gcgccccggg cggccgcaga
aaaattagcc ttgctctcca ccttcttctt ctattcc 571756DNAArtificial
Sequence5' primer 17gcgccccggg tcgacgcggc cgcgccatga gagtgaggga
gacagtgagg aattat 561857DNAArtificial Sequence3' primer
18gcgccccggg cggccgcaga aaaattagcc ttgctctcca ccttcttctt ctattcc
571956DNAArtificial Sequence5' primer 19gcgccccggg tcgacgcggc
cgcgccatga gagtgagggg gatagagagg aattat 562051DNAArtificial
Sequence3' primer 20gcgccccggg cggccgcaga aaaattagcc ttgctctcca
ccttctcctt c 512140DNAArtificial Sequence5' primer 21gcgccccggg
gccatgggtg cgagagcgtc agtattaagc 402249DNAArtificial Sequence3'
primer 22gcgccccggg agaaaaatta gaaggtttct gctcctacta tgggttcct
492340DNAArtificial Sequence5' primer 23gcgccccggg gccatgggtg
cgagagcgtc agtgttaagt 402449DNAArtificial Sequence3' primer
24gcgccccggg agaaaaatta gaaagtttct gctcctacta tgggttcct
492530DNAArtificial SequenceHIV env C-terminal truncation sequence
25ggaatagaag aagaaggtgg agagcaaggc 302610PRTArtificial SequenceHIV
env C-terminal truncation sequence 26Gly Ile Glu Glu Glu Gly Gly
Glu Gln Gly1 5 102730DNAArtificial SequenceHIV env C-terminal
truncation sequence 27ggaatagaag aagaaggtgg agagcaaggc
302810PRTArtificial SequenceHIV env C-terminal truncation sequence
28Gly Ile Glu Glu Glu Gly Gly Glu Gln Gly1 5 102930DNAArtificial
SequenceHIV env C-terminal truncation sequence 29ggaacagaag
gagaaggtgg agagcaaggc 303010PRTArtificial SequenceHIV env
C-terminal truncation sequence 30Gly Thr Glu Gly Glu Gly Gly Glu
Gln Gly1 5 103130DNAArtificial SequenceHIV gagpol C-terminal
truncation sequence 31aaggaaccca tagtaggagc agaaaccttc
303210PRTArtificial SequenceHIV gagpol C-terminal truncation
sequence 32Lys Glu Pro Ile Val Gly Ala Glu Thr Phe1 5
103330DNAArtificial SequenceHIV gagpol C-terminal truncation
sequence 33aaggaaccca tagtaggagc agaaactttc 303410PRTArtificial
SequenceHIV gagpol C-terminal truncation sequence 34Lys Glu Pro Ile
Val Gly Ala Glu Thr Phe1 5 103530DNAArtificial SequenceHIV envelope
C-terminal truncation sequence 35agaatcgaag gagaaggtgg agagcaagac
303611PRTArtificial SequenceHIV envelope C-terminal truncation
sequence 36Gly Arg Ile Glu Gly Glu Gly Gly Glu Gln Asp1 5
103730DNAArtificial SequenceHIV envelope C-terminal truncation
sequence 37agaatcgaag gagaaggtgg agagcaagac 303811PRTArtificial
SequenceHIV envelope C-terminal truncation sequence 38Gly Arg Ile
Glu Gly Glu Gly Gly Glu Gln Asp1 5 103930DNAArtificial SequenceHIV
envelope C-terminal truncation sequence 39agaatcgaag gagaaggtgg
agagcaagac 304011PRTArtificial SequenceHIV envelope C-terminal
truncation sequence 40Gly Arg Ile Glu Gly Glu Gly Gly Glu Gln Asp1
5 104130DNAArtificial SequenceHIV envelope C-terminal truncation
sequence 41agaatcgaag gagaaggtgg agagcaagac 304211PRTArtificial
SequenceHIV envelope C-terminal truncation sequence 42Gly Arg Ile
Glu Gly Glu Gly Gly Glu Gln Asp1 5 104330DNAArtificial SequenceHIV
envelope C-terminal truncation sequence 43gacatatcaa attggctgtg
gtatataaga 304410PRTArtificial SequenceHIV envelope C-terminal
truncation sequence 44Asp Ile Ser Asn Trp Leu Trp Tyr Ile Arg1 5
104527DNAArtificial SequenceHIV gagpol C-terminal truncation
sequence 45gaccccatag caggagcaga gactttc 274610PRTArtificial
SequenceHIV gagpol C-terminal truncation sequence 46Lys Asp Pro Ile
Ala Gly Ala Glu Thr Phe1 5 104730DNAArtificial SequenceHIV envelope
C-terminal truncation sequence 47ggaatcgaag aagaaggtgg agagcaagac
304810PRTArtificial SequenceHIV envelope C-terminal truncation
sequence 48Gly Ile Glu Glu Glu Gly Gly Glu Gln Asp1 5
104931DNAArtificial SequenceHIV gagpol C-terminal truncation
sequence 49aaagaaccca tagtaggagc agaaactttc t 315010PRTArtificial
SequenceHIV gagpol C-terminal truncation sequence 50Lys Glu Pro Ile
Val Gly Ala Glu Thr Phe1 5 10513068DNAHIV-1 51atgggtgcga gagcgtcagt
attaagcggg ggaaaattag atgaatggga aaaaattcgg 60ttacggccag ggggaaacaa
aaaatataga ttaaaacatt tagtatgggc aagcagggag 120ctagaacgat
ttgcacttaa tcctggtctt ttagaaacat cagaaggctg tagacaaata
180atagaacagc tacaaccatc tattcagaca ggatcagagg aacttaaatc
attacataat 240acagtagtaa ccctctattg tgtacatgaa aggataaagg
tagcagatac caaggaagct 300ttagataaga taaaggaaga acaaaccaaa
agtaagaaaa aagcacagca agcaacagct 360gacagcagcc aggtcagcca
aaattatcct atagtacaaa acctacaggg gcaaatggta 420caccagtcct
tatcacctag gactttgaat gcatgggtaa aagtaataga agagaaggct
480ttcagcccag aagtaatacc catgttttca gcattatcag aaggagccac
cccaacagat 540ttaaacacca tgctaaacac agtgggggga catcaagcag
ccatgcaaat gttaaaagag 600actatcaatg aggaagctgc agaatgggat
aggctacatc cagtgcctgc agggcctgtt 660gcaccaggcc aaatgagaga
accaagggga agtgatatag caggaactac cagtaccctt 720caggaacaaa
taggatggat gacaagcaat ccacctatcc cagtaggaga aatctataaa
780agatggataa tcctaggatt aaataaaata gtaagaatgt atagccctgt
cagcattttg 840gacataagac aaggaccaaa ggaacccttt agagactatg
tagatcggtt ctataaaact 900ctacgagccg agcaagcttc acaggatgta
aaaaattgga tgactgaaac cttgttagtc 960caaaatgcga atccagattg
taaaactatc ttaaaagcat tgggaccagc ggctacatta 1020gaagaaatga
tgacagcatg tcagggagtg gggggaccca gtcataaagc aagagttttg
1080gctgaggcaa tgagccaagc atcaaacaca aatgctgtta taatgatgca
gaggggcaat 1140ttcaagggca agaaaatcat taagtgtttc aactgtggca
aagaaggaca cctagcaaaa 1200aattgtaggg ctcctaggaa aagaggctgt
tggaaatgtg gaaaggaagg gcaccaaatg 1260aaagattgta atgaaagaca
ggctaatttt ttagggagaa tttggccttc ccacaagggg 1320aggccaggga
atttccttca gagcagacca gagccaacag ccccaccagc agagagcttc
1380gggtttgggg aagagataac accctcccag aaacaggagg ggaaagagga
gctgtatcct 1440tcagcctccc tcaaatcact ctttggcaac gacccctagt
cacaataaaa atagggggac 1500agctaaagga agctctatta gatacaggag
cagatgatac agtagtagaa gaaatgaatt 1560tgccaggaaa atggaaacca
aaaatgatag ggggaattgg gggctttatc aaagtaagac 1620agtatgatca
aatactcgta gaaatctatg gatataaggc tacaggtaca gtattagtag
1680gacctacacc tgtcaacata attggaagaa atttgttgac tcagattggt
tgcactttaa 1740attttccaat tagtcctatt gaaactgtac cagtaaaatt
aaagtcaggg atggatggtc 1800caagagttaa acaatggcca ttgacagaag
agaaaataaa agcactaata gaaatttgta 1860cagaaatgga aaaggaagga
aaactttcaa gaattggacc tgaaaatcca tacaatactc 1920caatatttgc
cataaagaaa aaagacagta ctaagtggag aaaattagta gatttcagag
1980aacttaataa gagaactcaa gatttctggg aagttcaact aggaatacca
catcctgcag 2040ggctaaaaaa gaaaaaatca gtaacagtac tggaggtggg
tgatgcatat ttttcagttc 2100ccttatatga agactttaga aaatacactg
cattcaccat acctagtata aacaatgaga 2160caccaggaat tagatatcag
tacaatgtgc ttccacaagg atggaaagga tcaccggcaa 2220tattccaaag
tagcatgaca aaaattttag aaccttttag aaaacaaaat ccagaagtgg
2280ttatctacca atacatgcac gatttgtatg taggatctga cttagaaata
gggcagcata 2340gaataaaaat agaggaatta aggggacacc tattgaagtg
gggatttacc acaccagaca 2400aaaatcatca gaaggaacct ccatttcttt
ggatgggtta tgaactccat cctgataaat 2460ggacagtaca gcctataaaa
ctgccagaaa aagaaagctg gactgtcaat gatctgcaga 2520agttagtggg
gaaattaaat tgggcaagtc aaatttattc aggaattaaa gtaagacaat
2580tatgcaaatg ccttagggga accaaagcac tgacagaagt agtaccactg
acagaagaag 2640cagaattaga actggcagaa aacagggaac ttctaaaaga
aacagtacat ggagtgtatt 2700atgacccatc aaaagactta atagcagaaa
tacagaaaca agggcaagac caatggacat 2760atcaaattta tcaagaacaa
tataaaaatt tgaaaacagg aaagtatgca aagaggagga 2820gtacccacac
taatgatgta aaacaattaa cagaggcagt gcaaaaaata gcccaagaat
2880gtatagtgat atggggaaag actcctaaat tcagactacc catacaaaag
gaaacatggg 2940aaacatggtg gacagagtat tggcaggcca cctggattcc
tgagtgggag tttgtcaata 3000cccctccctt ggttaaatta tggtaccagt
tagagaagga acccatagta ggagcagaaa 3060ccttctaa 3068523080DNAHIV-1
52atgggtgcga gagcgtcagt gttaagtggg ggaaaattag atgaatggga aagaattcgg
60ttacggccag ggggaaacaa aagatataaa ctaaaacata tagtatgggc aagcagggag
120ctagagcgat ttgcacttaa tcctggcctt ttagaaacat cagaaggctg
taaacaaata 180ttgggacagc tacaaccagc tattcagaca ggatcagaag
aacttaaatc attatataat 240acagtagcaa ccctctattg tgtacatgag
aggctaaagg taacagacac caaggaagct 300ttagacaaaa tagaggaaga
acaaaccaaa agtaagaaaa aagcacagca agcaacagct 360gacacaaaaa
acagcagcca ggtcagccaa aattatccta tagtacaaaa cctacagggg
420caaatggtac accaggctat atcacctaga acgttgaacg catgggtaaa
agtaatagag 480gagaaggctt tcagcccaga agtaataccc atgttttcag
cattatcaga aggagccacc 540ccacaagatt taaacaccat gctaaacaca
gtggggggac atcaggcagc catgcagatg 600ttaaaagaga ccatcaatga
ggaagctgca gaatgggata ggttacatcc agtacatgca 660gggcctattg
caccaggaca aatgagagaa ccaacaggaa gtgatatagc aggaactact
720agtacccttc aggaacaaat aggatggatg accagcaatc cacctatccc
agtaggagaa 780atctataaaa gatggataat cctaggatta aataaaatag
taaggatgta tagccctgtc 840agtattttgg acataaaaca agggccaaag
gaacccttta gagactatgt agatcggttc 900tataaaactc taagggccga
gcaagcttca caggaggtaa aaggttggat gaccgaaacc 960ttgttggtcc
aaaatgcaaa cccagattgt aaaaccatct taaaagcatt gggaccagcg
1020gctacattag aagaaatgat gacagcatgt cagggagtgg ggggacccgg
tcataaagca 1080agagttttgg ctgaggcaat gagtcaagtc tcaacaaata
ctgctataat gatgcagaga 1140ggcaatttta agggcccaaa gaaaagcatt
aagtgtttta actgtggcaa agaaggtcac 1200acagcaaaaa actgtagagc
tcctaggaaa aggggctgtt ggaaatgtgg aagggaagga 1260catcaaatga
aagattgcac tgaaagacag gctaattttt tagggaaaat ttggccttcc
1320cacaagggaa ggccagggaa tttccttcag aacagaccag agccaacagc
cccaccagaa 1380gaaagcttcg ggtttgggga agagataaca ccctctcaga
aacaggagaa gaaggacaag 1440gagctgtatc ctgtagcttc cctcaaatca
ctctttggca acgacccctt gtcacaataa 1500agataggggg acagctaaag
gaagctctac tagatacagg agcagatgat acagtattag 1560aagaaataaa
tttgccagga aaatggaaac caaaaatgat agggggaatt ggaggcttta
1620tcaaagtaag acagtatgag caaatacttg tagaaatctg tggacagaaa
gctataggta 1680cagtattagt agggcctaca cctgtcaaca taattggaag
aaatttgttg actcagattg 1740gttgcacttt aaattttcca attagcccta
ttgaaactgt accagtaaaa ttaaagccag 1800ggatggacgg tccaaaagtt
aaacaatggc cattgacaga agaaaaggta aaagcactaa 1860tagaaatttg
tacagaaatg gaaaaggaag gaaaaatttc aagaattgga cctgaaaatc
1920catacaatac tccaatattt gccataaaga aaaaggacag tactaagtgg
agaaaattag 1980tagatttcag ggaacttaat aagagaactc aagacttctg
ggaagttcaa ctaggaatac 2040cacatcctgc ggggctaaaa aagaaaaaat
cagtaacagt actggaggtg ggtgatgcat 2100atttttcagt tcccttatat
gaagatttta gaaaatatac tgcattcacc atacctagta 2160taaacaatga
aacaccagga attagatatc agtacaatgt gcttccacaa gggtggaaag
2220gatcaccagc aatattccaa agtagcatga caaaaatctt agaacctttt
agaaaacaaa 2280atccagaaat ggttatctat caatacatgc acgatttgta
tgtaggatct gacttagaaa 2340tagggcagca tagaataaaa atagaagaat
taaggggaca cctgttgaag tggggattta 2400ccacaccaga caaaaagcat
cagaaagaac ctccatttct ttggatgggt tatgaactcc 2460atcctgataa
atggacagta cagtctataa aactgccaga acaagaaagc tggactgtca
2520atgatataca gaagttagtg ggaaaattaa attgggcaag ccagatttat
ccaggaatta 2580aggtaagaca attatgcaaa tgcattaggg gtaccaaagc
actgacagaa gtagtaccac 2640tgacagaaga agcagaatta gaactggcag
aaaacaggga aattctaaga gaaccagtac 2700atggagtgta ttatgaccca
tcaaaagact taatagcaga gatacagaaa caagggcaag 2760accagtggac
ataccaaatt tatcaagaac aatataaaaa tctgaaaaca ggaaagtatg
2820caaaagtgag gggtacccac actaatgatg taaaacaatt aacagaggca
gtacaaaaaa 2880taacccaaga atgtatagtg atatggggaa agcctcctaa
atttagacta cccatacaaa 2940aagaaacatg ggaaatatgg tggacagagt
attggcaggc cacctggatt cctgagtggg 3000agtttgtcaa tacccctcct
ttagttaaat tatggtacca attagagaag gaacccatag 3060taggagcaga
aactttctaa 3080532181DNAHIV-1 53atgagagtga gggggataca gaggaactat
caaaacttgt ggagatgggg caccttgctc 60cttgggatgt tgatgatatg taaggctaca
gaacagttgt gggtcacagt ttactatggg 120gtacctgtgt ggaaagaagc
aaccactact ctattttgtg
catcagatgc taaatcatat 180aaagaagaag cacataatat ctgggctaca
catgcctgtg taccaacaga ccccaaccca 240cgagaattaa taatagaaaa
tgtcacagaa aactttaaca tgtggaaaaa taacatggtg 300gagcagatgc
atgaggatat aatcagttta tgggatcaaa gcctaaaacc atgtgtaaaa
360ttaaccccac tctgtgtcac tttaaactgc actgaatgga ggaagaataa
cactatcaat 420gccaccagaa tagaaatgaa aaactgctct ttcaatctaa
ccacagaaat aagagatagg 480aaaaagcaag tgcatgcact tttctataaa
cttgatgtgg taccaataga tgataataat 540agtactaata ccagctatag
gttaataaat tgtaatacct cagccattac acaggcgtgt 600ccaaaggtaa
cctttgagcc aattcccata cattattgtg ccccagctgg atatgcgatt
660ctaaaatgta acaataagaa gttcaatggg acaggtccat gcgataatgt
cagtacagta 720cagtgtacac atggaattag gccagtagta tccactcaat
tgttgttgaa tggcagtcta 780gcagaagaag acataataat tagatctgag
aatctcacaa ataatgctaa aatcataata 840gtacagctta atgagtctgt
aacaattaat tgcacaaggc cctacaacaa tacaagaaga 900ggtgtacata
taggaccagg gcgagcatac tatacaacag acataatagg agatataaga
960caagcacatt gtaacattag tggagcagaa tggaataaga ctttacatcg
ggtagctaaa 1020aaattaagag acctatttaa aaagacaaca ataattttta
aaccgtcctc cggaggggac 1080ccagaaatta caacacacag ctttaattgt
agaggggaat tcttctactg caatacaaca 1140agactgttta atagcatatg
gggaaataat agtacaggag ttgatgagag tataacactc 1200ccatgcagaa
taaaacaaat tataaacatg tggcagggag taggaaaagc aatgtatgcc
1260cctcccattg aaggactaat cagctgctca tcaaatatta caggattact
gttgacaaga 1320gatggtggtg gaagtaacag tagtcagaat gagaccttca
gacctggagg gggagatatg 1380agagacaatt ggagaagtga attatataaa
tataaagtag taagaattga accattaggt 1440ctagcaccct ccaaggcaaa
aagaagagta gtagaaagag agaaaagagc aataggacta 1500ggagctatgt
tccttgggtt cttgggagca gcaggaagca cgatgggcgc agcgtcactg
1560acgctgacgg tacaggccag acagctattg tctggtatag tgcaacagca
aaacaatttg 1620ctgaaggcta tagaggcgca acagcacctg ttgcaactca
cagtctgggg cgttaaacag 1680ctccaggcaa gagtcctggc tgtggaaagc
tacctaaggg atcaacagct cctaggaatt 1740tggggttgct ctggaaaaca
catttgcacc accaatgtgc cctggaactc tagctggagt 1800aataaaactc
taaaatcaat ttgggataac atgacctgga tggagtggga aagagaaatt
1860gacaattaca cagggataat atacaattta cttgaagaat cgcaaaccca
gcaagaaaga 1920aatgaacaag acctattgaa attggaccaa tgggcaagtt
tgtggaattg gtttagcata 1980acaaaatggc tgtggtatat aaaaatattt
ataatgatag taggaggctt gataggctta 2040aggatagttt ttgctgtgct
ttctatagta aatagagtta ggcagggata ttcacctctg 2100tcgtttcaga
ccctcctccc agccccgcgg ggacccgaca ggcccgaagg aatagaagaa
2160gaaggtggag agcaaggcta a 2181542214DNAHIV-1 54atgagagtga
gggagacagt gaggaattat cagcacttgt ggagatgggg catcatgctc 60cttgggatgt
taatgatatg tagtgctgca gaccagctgt gggtcacagt gtattatggg
120gtacctgtgt ggaaagaagc aaccactact ctattttgtg catcagatgc
taaagcacat 180aaagcagagg cacataatat ctgggctaca catgcctgtg
taccaacaga ccccaatcca 240cgagaaataa tactaggaaa tgtcacagaa
aactttaaca tgtggaagaa taacatggta 300gagcagatgc atgaggatat
aatcagttta tgggatcaaa gtctaaaacc atgtgtaaaa 360ttaaccccac
tctgtgttac tttaaactgc actacatatt ggaatggaac tttacagggg
420aatgaaacta aagggaagaa tagaagtgac ataatgacat gctctttcaa
tataaccaca 480gaaataagag gtagaaagaa gcaagaaact gcacttttct
ataaacttga tgtggtacca 540ctagaggata aggatagtaa taagactacc
aactatagca gctatagatt aataaattgc 600aatacctcag tcgtgacaca
ggcgtgtcca aaagtaacct ttgagccaat tcccatacat 660tattgtgccc
cagctggatt tgcgattctg aaatgtaata ataagacgtt caatggaacg
720ggtccatgca aaaatgtcag cacagtacag tgtacacatg gaattaggcc
agtagtgtca 780actcaactgt tgttgaatgg cagtctagca gaagaagaga
taataattag atctgaaaat 840atcacaaata atgcaaaaac cataatagta
cagcttaatg agtctgtaac aattgattgc 900ataaggccca acaacaatac
aagaaaaagt atacgcatag gaccagggca agcactctat 960acaacagaca
taatagggaa tataagacaa gcacattgta atgttagtaa agtaaaatgg
1020ggaagaatgt taaaaagggt agctgaaaaa ttaaaagacc ttcttaacca
gacaaagaac 1080ataacttttg aaccatcctc aggaggggac ccagaaatta
caacacacag ctttaattgt 1140ggaggggaat tcttctactg caatacatca
ggactattta atgggagtct gcttaatgag 1200cagtttaatg agacatcaaa
tgatactctc acactccaat gcagaataaa acaaattata 1260aacatgtggc
aaggagtagg aaaagcaatg tatgcccctc ccattgcagg accaatcagc
1320tgttcatcaa atattacagg actattgttg acaagagatg gtggtaatac
tggtaatgat 1380tcagagatct tcagacctgg agggggagat atgagagaca
attggagaag tgaattatac 1440aaatataaag tagtaagaat tgaaccaatg
ggtctagcac ccaccagggc aaaaagaaga 1500gtggtggaaa gagaaaaaag
agcaatagga ctgggagcta tgttccttgg gttcttggga 1560gcggcaggaa
gcacgatggg cgcagcgtca ctgacgctga cggtacaggc cagacagtta
1620ttgtctggta tagtgcaaca gcaaaacaat ttgctgagag ctatagaggc
gcaacagcat 1680ctgttgcaac tcacagtctg gggcattaaa cagctccagg
caagagtcct ggctatggaa 1740agctacctaa aggatcaaca gctcctagga
atttggggtt gctctggaaa acacatttgc 1800accactactg tgccctggaa
ctctacctgg agtaatagat ctgtagagga gatttggaat 1860aatatgacct
ggatgcagtg ggaaagagaa attgagaatt acacaggttt aatatacacc
1920ttaattgaag aatcgcaaac ccagcaagaa aagaatgaac aagaactatt
gcaattggat 1980aaatgggcaa gtttgtggaa ttggtttagt ataacaaaat
ggctgtggta tataaaaata 2040ttcataatga tagtaggagg cttaataggt
ttaagaatag tttttgctgt gctttcttta 2100gtaaatagag ttaggcaggg
atattcacct ctgtcttttc agaccctcct cccagccccg 2160aggggacccg
acaggcccga aggaatagaa gaagaaggtg gagagcaagg ctaa 2214552265DNAHIV-1
55atgagagtga gggggataga gaggaattat cagcacttat ggtggagatg gggcaccatg
60ctccttggga tattgatgat atgtagtgct gcagaacaat tgtgggtcac agtttattat
120ggggtacctg tgtggaaaga agcaaccact actctatttt gtgcatcaga
tgctaaagca 180tataaagcag aggcacacaa tatctgggct acacatgcct
gtgtaccaac agaccccaac 240ccacaagaaa tagtactaga aaatgtcaca
gaaaacttta acatgtggaa aaatagcatg 300gtggagcaga tgcatgagga
tgtaatcagt ttatgggatc aaagcctaaa accatgtgta 360aaattaaccc
cactctgtgt cactttaaac tgcactaatg ccactgccac taatgccact
420gccactagtc aaaatagcac tgatggtagt aataaaactg ttaacacaga
cacaggaatg 480aaaaactgct ctttcaatgt aaccacagat ctaaaagata
agaagaggca agactatgca 540cttttctata aacttgatgt ggtacgaata
gatgataaga ataccaatgg tactaatacc 600aactatagat taataaattg
taatacctca gccattacac aagcgtgtcc aaagataacc 660tttgagccaa
ttcccataca ttattgtgcc ccagctggat atgcgattct aaaatgtaat
720aataagacat tcaatgggac gggtccatgc aaaaacgtca gcacagtaca
gtgtacacat 780gggattaggc cagtagtgtc aactcaactg ttgttgaatg
gcagtctagc agaggaagag 840atagtaatta gatctgaaaa cctcacaaat
aatgctaaaa ttataatagt acagcttaat 900gaagctgtaa caattaattg
cacaagaccc tccaacaata caagacgaag tgtacatata 960ggaccagggc
aagcaatcta ttcaacagga caaataatag gagatataag aaaagcacat
1020tgtaatatta gtagaaaaga atggaatagc accttacaac aggtaactaa
aaaattagga 1080agcctgttta acacaacaaa aataattttt aatgcatcct
cgggagggga cccagaaatt 1140acaacacaca gctttaattg taacggggaa
ttcttctact gcaatacagc aggactgttt 1200aatagtacat ggaacaggac
aaatagtgaa tggataaata gtaaatggac aaataagaca 1260gaagatgtaa
atatcacact tcaatgcaga ataaaacaaa ttataaacat gtggcaggga
1320gtaggaaaag caatgtatgc ccctcccgtt agtggaataa tccgatgttc
atcaaatatt 1380acaggactgt tgctgacaag agatggtggt ggtgcagata
ataataggca gaatgagacc 1440ttcagacctg ggggaggaga tatgagagac
aattggagaa gtgaattata caaatataaa 1500gtagtaagaa ttgaaccact
aggtatagca cccaccaagg caaggagaag agtggtggaa 1560agagaaaaaa
gagcaatagg actgggagcc ttgttccttg ggttcttggg aacagcagga
1620agcacgatgg gcgcagtgtc aatgacgctg acggtacagg ccagacaagt
attgtctggt 1680atagtgcaac agcaaaacaa tctgctgagg gctatagagg
cgcaacagca tctgttgcaa 1740ctcacagtct ggggcattaa acagctccag
gcaagaatcc tggctgtgga aagctaccta 1800aaggatcaac agctcctagg
aatttggggt tgctctggaa aacacatttg caccactaat 1860gtgccctgga
actctagctg gagtaataaa tctctaaatt atatttggaa taacatgacc
1920tggatggagt gggaaaagga aattgacaat tacacagaat taatatacag
cttaattgaa 1980gtatcgcaaa tccagcaaga aaagaatgaa caagaactat
tgaaattgga cagttgggca 2040agtttgtgga attggtttag cataacaaaa
tggctgtggt atataaaaat attcataatg 2100atagtaggag gcttgatagg
cttaagaata gtttttgctg tgctttcttt agtaaataga 2160gttaggcagg
gatactcacc tctgtcgttt cagaccctta tcccagcctc gaggggaccc
2220gacaggcccg aaggaacaga aggagaaggt ggagagcaag gctaa
2265565326DNAArtificial SequenceMVA shuttle plasmid pLAS-1
56gaattcgttg gtggtcgcca tggatggtgt tattgtatac tgtctaaacg cgttagtaaa
60acatggcgag gaaataaatc atataaaaaa tgatttcatg attaaaccat gttgtgaaaa
120agtcaagaac gttcacattg gcggacaatc taaaaacaat acagtgattg
cagatttgcc 180atatatggat aatgcggtat ccgatgtatg caattcactg
tataaaaaga atgtatcaag 240aatatccaga tttgctaatt tgataaagat
agatgacgat gacaagactc ctactggtgt 300atataattat tttaaaccta
aagatgccat tcctgttatt atatccatag gaaaggatag 360agatgtttgt
gaactattaa tctcatctga taaagcgtgt gcgtgtatag agttaaattc
420atataaagta gccattcttc ccatggatgt ttcctttttt accaaaggaa
atgcatcatt 480gattattctc ctgtttgatt tctctatcga tgcggcacct
ctcttaagaa gtgtaaccga 540taataatgtt attatatcta gacaccagcg
tctacatgac gagcttccga gttccaattg 600gttcaagttt tacataagta
taaagtccga ctattgttct atattatata tggttgttga 660tggatctgtg
atgcatgcaa tagctgataa tagaacttac gcaaatatta gcaaaaatat
720attagacaat actacaatta acgatgagtg tagatgctgt tattttgaac
cacagattag 780gattcttgat agagatgaga tgctcaatgg atcatcgtgt
gatatgaaca gacattgtat 840tatgatgaat ttacctgatg taggcgaatt
tggatctagt atgttgggga aatatgaacc 900tgacatgatt aagattgctc
tttcggtggc tgggtaccag gcgcgccttt cattttgttt 960ttttctatgc
tataaatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
1020ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
cgagggcgag 1080ggcgatgcca cctacggcaa gctgaccctg aagttcatct
gcaccaccgg caagctgccc 1140gtgccctggc ccaccctcgt gaccaccctg
acctacggcg tgcagtgctt cagccgctac 1200cccgaccaca tgaagcagca
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 1260gagcgcacca
tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
1320gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
ggaggacggc 1380aacatcctgg ggcacaagct ggagtacaac tacaacagcc
acaacgtcta tatcatggcc 1440gacaagcaga agaacggcat caaggtgaac
ttcaagatcc gccacaacat cgaggacggc 1500agcgtgcagc tcgccgacca
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 1560ctgcccgaca
accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
1620cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
cggcatgcac 1680gagctgtaca agtaagagct cggttgttga tggatctgtg
atgcatgcaa tagctgataa 1740tagaacttac gcaaatatta gcaaaaatat
attagacaat actacaatta acgatgagtg 1800tagatgctgt tattttgaac
cacagattag gattcttgat agagatgaga tgctcaatgg 1860atcatcgtgt
gatatgaaca gacattgtat tatgatgaat ttacctgatg taggcgaatt
1920tggatctagt atgttgggga aatatgaacc tgacatgatt aagattgctc
tttcggtggc 1980tggcggcccg ctcgaggccg ctggtaccca acctaaaaat
tgaaaataaa tacaaaggtt 2040cttgagggtt gtgttaaatt gaaagcgaga
aataatcata aataagcccg gggatcctct 2100agagtcgacc tgcagggaaa
gttttatagg tagttgatag aacaaaatac ataattttgt 2160aaaaataaat
cactttttat actaatatga cacgattacc aatacttttg ttactaatat
2220cattagtata cgctacacct tttcctcaga catctaaaaa aataggtgat
gatgcaactt 2280tatcatgtaa tcgaaataat acaaatgact acgttgttat
gagtgcttgg tataaggagc 2340ccaattccat tattctttta gctgctaaaa
gcgacgtctt gtattttgat aattatacca 2400aggataaaat atcttacgac
tctccatacg atgatctagt tacaactatc acaattaaat 2460cattgactgc
tagagatgcc ggtacttatg tatgtgcatt ctttatgaca tcgcctacaa
2520atgacactga taaagtagat tatgaagaat actccacaga gttgattgta
aatacagata 2580gtgaatcgac tatagacata atactatctg gatctacaca
ttcaccagaa actagttaag 2640cttgtctccc tatagtgagt cgtattagag
cttggcgtaa tcatggtcat agctgtttcc 2700tgtgtgaaat tgttatccgc
tcacaattcc acacaacata cgagccggaa gcataaagtg 2760taaagcctgg
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
2820cgctttcgag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
aacgcgcggg 2880gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
ctcactgact cgctgcgctc 2940ggtcgttcgg ctgcggcgag cggtatcagc
tcactcaaag gcggtaatac ggttatccac 3000agaatcaggg gataacgcag
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 3060ccgtaaaaag
gccgcgttgc tggcgttttt cgataggctc cgcccccctg acgagcatca
3120caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
gataccaggc 3180gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
accctgccgc ttaccggata 3240cctgtccgcc tttctccctt cgggaagcgt
ggcgctttct catagctcac gctgtaggta 3300tctcagttcg gtgtaggtcg
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 3360gcccgaccgc
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
3420cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
atgtaggcgg 3480tgctacagag ttcttgaagt ggtggcctaa ctacggctac
actagaagga cagtatttgg 3540tatctgcgct ctgctgaagc cagttacctt
cggaaaaaga gttggtagct cttgatccgg 3600caaacaaacc accgctggta
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 3660aaaaaaagga
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
3720cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
tcacctagat 3780ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
atatatgagt aaacttggtc 3840tgacagttac caatgcttaa tcagtgaggc
acctatctca gcgatctgtc tatttcgttc 3900atccatagtt gcctgactcc
ccgtcgtgta gataactacg atacgggagg gcttaccatc 3960tggccccagt
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
4020aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
tatccgcctc 4080catccagtct attaattgtt gccgggaagc tagagtaagt
agttcgccag ttaatagttt 4140gcgcaacgtt gttggcattg ctacaggcat
cgtggtgtca cgctcgtcgt ttggtatggc 4200ttcattcagc tccggttccc
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 4260aaaagcggtt
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
4320atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
ccgtaagatg 4380cttttctgtg actggtgagt actcaaccaa gtcattctga
gaatagtgta tgcggcgacc 4440gagttgctct tgcccggcgt caatacggga
taataccgcg ccacatagca gaactttaaa 4500agtgctcatc attggaaaac
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 4560gagatccagt
tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
4620caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
agggaataag 4680ggcgacacgg aaatgttgaa tactcatact cttccttttt
caatattatt gaagcattta 4740tcagggttat tgtctcatga gcggatacat
atttgaatgt atttagaaaa ataaacaaat 4800aggggttccg cgcacatttc
cccgaaaagt gccacctgac gtctaagaaa ccattattat 4860catgacatta
acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
4920tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
cttgtctgta 4980agcggatgcc gggagcagac aagcccgtca gggcgcgtca
gcgggtgttg gcgggtgtcg 5040gggctggctt aactatgcgg catcagagca
gattgtactg agagtgcacc atatgcggtg 5100tgaaataccg cacagatgcg
taaggagaaa ataccgcatc aggcgccatt cgccattcag 5160gctgcgcaac
tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
5220gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
cccagtcacg 5280acgttgtaaa acgacggcca gtgaattgga tttaggtgac actata
5326575143DNAMVA shuttle plasmid pLAS-2 57cctcctgaaa aactggaatt
taatacacca tttgtgttca tcatcagaca tgatattact 60ggatttatat tgtttatggg
taaggtagaa tctccttaat atgggtacgg tgtaaggaat 120cattatttta
tttatattga tgggtacgtg aaatctgaat tttcttaata aatattattt
180ttattaaatg tgtatatgtt gttttgcgat agccatgtat ctactaatca
gatctattag 240agatattatt aattctggtg caatatgaca aaaattatac
actaattagc gtctcgtttc 300agacatggat ctgtcacgaa ttaatacttg
gaagtctaag cagctgaaaa gctttctctc 360tagcaaagat gcatttaagg
cggatgtcca tggacatagt gccttgtatt atgcaatagc 420tgataataac
gtgcgtctag tatgtacgtt gttgaacgct ggagcattga aaaatcttct
480agagaatgaa tttccattac atcaggcagc cacattggaa gataccaaaa
tagtaaagat 540tttgctattc agtggactgg atgattcgag gtaccaggcg
cgccctttca ttttgttttt 600ttctatgcta taaatggtga gcaagggcga
ggagctgttc accggggtgg tgcccatcct 660ggtcgagctg gacggcgacg
taaacggcca caagttcagc gtgtccggcg agggcgaggg 720cgatgccacc
tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
780gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
gccgctaccc 840cgaccacatg aagcagcacg acttcttcaa gtccgccatg
cccgaaggct acgtccagga 900gcgcaccatc ttcttcaagg acgacggcaa
ctacaagacc cgcgccgagg tgaagttcga 960gggcgacacc ctggtgaacc
gcatcgagct gaagggcatc gacttcaagg aggacggcaa 1020catcctgggg
cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
1080caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
aggacggcag 1140cgtgcagctc gccgaccact accagcagaa cacccccatc
ggcgacggcc ccgtgctgct 1200gcccgacaac cactacctga gcacccagtc
cgccctgagc aaagacccca acgagaagcg 1260cgatcacatg gtcctgctgg
agttcgtgac cgccgccggg atcactctcg gcatgcacga 1320gctgtacaag
taagagctcg ctttctctct agcaaagatg catttaaggc ggatgtccat
1380ggacatagtg ccttgtatta tgcaatagct gataataacg tgcgtctagt
atgtacgttg 1440ttgaacgctg gagcattgaa aaatcttcta gagaatgaat
ttccattaca tcaggcagcc 1500acattggaag ataccaaaat agtaaagatt
ttgctattca gtggactgga tgattctccg 1560gatggtaccc aacctaaaaa
ttgaaaataa atacaaaggt tcttgagggt tgtgttaaat 1620tgaaagcgag
aaataatcat aaataagccc ggggatcctc tagagtcgac ctgcaggcat
1680gctcgagcgg ccgccagtgt gatggatatc tgcagaattc ggcttggggg
gctgcaggtg 1740gatgcgatca tgacgtcctc tgcaatggat aacaatgaac
ctaaagtact agaaatggta 1800tatgatgcta caattttacc cgaaggtagt
agcatggatt gtataaacag acacatcaat 1860atgtgtatac aacgcaccta
tagttctagt ataattgcca tattggatag attcctaatg 1920atgaacaagg
atgaactaaa taatacacag tgtcatataa ttaaagaatt tatgacatac
1980gaacaaatgg cgattgacca ttatggagaa tatgtaaacg ctattctata
tcaaattcgt 2040aaaagaccta atcaacatca caccattaat ctgtttaaaa
aaataaaaag aacccggtat 2100gacactttta aagtggatcc cgtagaattc
gtaaaaaaag ttatcggatt tgtatctatc 2160ttgaacaaat ataaaccggt
ttatagttac gtcctgtacg agaacgtcct gtacgatgag 2220ttcaaatgtt
tcattgacta cgtggaaact aagtatttct aaaattaatg atgcattaat
2280ttttgtattg attctcaatc ctaaaaacta aaatatgaat aagtattaaa
catagcggtg 2340tactaattga tttaacataa aaaatagttg ttaactaatc
atgaggactc tacttattag 2400atatattctt tggagaaatg acaacgatca
aaccgggcat gcaagcttgt ctccctatag 2460tgagtcgtat tagagcttgg
cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 2520tccgctcaca
attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
2580ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
tcgagtcggg 2640aaacctgtcg tgccagctgc attaatgaat cggccaacgc
gcggggagag gcggtttgcg 2700tattgggcgc tcttccgctt cctcgctcac
tgactcgctg cgctcggtcg ttcggctgcg 2760gcgagcggta tcagctcact
caaaggcggt aatacggtta tccacagaat caggggataa 2820cgcaggaaag
aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
2880gttgctggcg tttttcgata ggctccgccc ccctgacgag catcacaaaa
atcgacgctc 2940aagtcagagg tggcgaaacc cgacaggact ataaagatac
caggcgtttc cccctggaag 3000ctccctcgtg cgctctcctg ttccgaccct
gccgcttacc
ggatacctgt ccgcctttct 3060cccttcggga agcgtggcgc tttctcatag
ctcacgctgt aggtatctca gttcggtgta 3120ggtcgttcgc tccaagctgg
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 3180cttatccggt
aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
3240agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
cagagttctt 3300gaagtggtgg cctaactacg gctacactag aaggacagta
tttggtatct gcgctctgct 3360gaagccagtt accttcggaa aaagagttgg
tagctcttga tccggcaaac aaaccaccgc 3420tggtagcggt ggtttttttg
tttgcaagca gcagattacg cgcagaaaaa aaggatctca 3480agaagatcct
ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
3540agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
taaattaaaa 3600atgaagtttt aaatcaatct aaagtatata tgagtaaact
tggtctgaca gttaccaatg 3660cttaatcagt gaggcaccta tctcagcgat
ctgtctattt cgttcatcca tagttgcctg 3720actccccgtc gtgtagataa
ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3780aatgataccg
cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
3840cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
agtctattaa 3900ttgttgccgg gaagctagag taagtagttc gccagttaat
agtttgcgca acgttgttgg 3960cattgctaca ggcatcgtgg tgtcacgctc
gtcgtttggt atggcttcat tcagctccgg 4020ttcccaacga tcaaggcgag
ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 4080cttcggtcct
ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
4140ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
ctgtgactgg 4200tgagtactca accaagtcat tctgagaata gtgtatgcgg
cgaccgagtt gctcttgccc 4260ggcgtcaata cgggataata ccgcgccaca
tagcagaact ttaaaagtgc tcatcattgg 4320aaaacgttct tcggggcgaa
aactctcaag gatcttaccg ctgttgagat ccagttcgat 4380gtaacccact
cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
4440gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
cacggaaatg 4500ttgaatactc atactcttcc tttttcaata ttattgaagc
atttatcagg gttattgtct 4560catgagcgga tacatatttg aatgtattta
gaaaaataaa caaatagggg ttccgcgcac 4620atttccccga aaagtgccac
ctgacgtcta agaaaccatt attatcatga cattaaccta 4680taaaaatagg
cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
4740cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
atgccgggag 4800cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
tgtcggggct ggcttaacta 4860tgcggcatca gagcagattg tactgagagt
gcaccatatg cggtgtgaaa taccgcacag 4920atgcgtaagg agaaaatacc
gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg 4980ggaagggcga
tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
5040tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
gtaaaacgac 5100ggccagtgaa ttggatttag gtgacactat agaatacgaa ttc
5143583077DNAHIV-1 clade A 58atgggtgcga gagcgtcagt attaagtggg
ggaaaattag atgcatggga gaaaattcgg 60ttaaggccag ggggaaagaa aaaatataga
ctgaaacact tagtatgggc aagcagggag 120ctggaaaaat tcgtacttaa
ccctagcctt ttagaaactt cagaaggatg tcagcaaata 180atgaaccaaa
tacaaccagc tcttcagaca ggaacagaag aacttagatc attatttaat
240gcagtagcaa ccctctattg tgtacatcaa cggatagagg taaaagacac
caaggaagct 300ttagataaag tagaggaaat acaaaacaag agcaagcaaa
agacacaaca ggcagcagct 360gatacaggaa acaacagcaa ggtcagccat
aattacccta tagtgcaaaa tgcacaaggg 420caaatgatac atcagtcctt
atcaccaagg actttgaatg catgggtaaa ggtaatagaa 480gaaaggggtt
tcagcccaga agtaataccc atgttctcag cattatcaga aggagccatc
540ccacaagatt taaatatgat gctgaacata gtggggggac accaggcagc
tatgcaaatg 600ttaaaagaaa ctatcaatga ggaagctgca gaatgggaca
ggttacatcc agcacaggca 660gggcctattc caccaggcca gataagagac
ccaaggggaa gtgacatagc aggaactact 720agtacccctc aggaacaaat
aacatggatg acaaacaacc cacctatccc agtgggagac 780atctataaaa
gatggataat cctaggatta aataaaatag taagaatgta tagccctgtt
840agcattttag atataaaaca ggggccaaaa gaacccttca gagactatgt
agataggttc 900tttaaagttc tcagagccga acaagctaca caggaagtaa
aaggctggat gacagagacc 960ctgctggttc aaaatgcaaa tccagattgt
aagtccattt taagagcatt aggaacaggg 1020gctacattag aagaaatgat
gacagcatgt cagggagtgg gaggacccgg ccataaagca 1080agggttttag
ctgaggcaat gagtcaagca caacaggcaa atgtaatgat gcagaggggc
1140agctttaagg ggcagaaaag aattaagtgc ttcaactgtg gcaaagaggg
acacctagcc 1200agaaattgca gagcccctag gaaaaaaggc tgttggaagt
gtgggaaaga aggacaccaa 1260atgaaagatt gcaatgagag acaggctaat
tttttaggga aaatttggcc ttccagcaag 1320gggaggccag gaaattttcc
ccagagcaga ccggagccaa cagccccacc agcagagatc 1380tttgggatgg
gggaagagat aacctcccct ccgaagcagg agcagaaaga gagggaacaa
1440accccaccct ttgtttccct caaatcactc tttggcaacg acccgttgtc
acagtaaaag 1500taggaggaga aatgagagaa gctctattag atacaggagc
agatgataca gtattagaag 1560atataaattt gccaggaaaa tggaaaccaa
aaatgatagg gggaattgga ggttttatca 1620aggtaaaaca atatgatcag
gtatctatag aaatttgtgg aaaaaaggct ataggtacgg 1680tattagtagg
acctacacct gtcaacataa ttggaagaaa tatgttgact cagattggtt
1740gtaccttaaa ttttccaatt agtcctattg agactgtacc agtaacatta
aagccaggaa 1800tggatggccc aagggttaaa caatggccat tgacagaaga
gaaaataaaa gcattgacag 1860aaatttgtaa agagatggaa aaggaaggaa
aaatttcaaa aattgggcct gaaaatccat 1920acaatactcc aatatttgca
ataaagaaaa aagatagcac taaatggagg aaattagtag 1980atttcagaga
gctcaataaa agaacacaag acttttggga agttcaatta gggataccgc
2040atccagcggg cctaaaaaag aaaaaatcag taacagtact agaggtgggg
gatgcatatt 2100tttcagttcc cctagataaa aactttagaa agtatactgc
atttaccata cctagtttaa 2160ataatgaaac accaggaatc aggtatcagt
acaatgtgct tccacaagga tggaaaggat 2220caccagcaat attccagtgc
agtatgacaa aaatcttaga gccctttaga tcaaaaaatc 2280cagaaataat
tatctatcaa tacatgcacg acttgtatgt aggatcagat ttagaaatag
2340ggcagcatag agcaaaaata gaagaattaa gagctcatct actgagctgg
ggatttacta 2400caccagacaa aaagcatcag aaagaacctc cattcctttg
gatgggatat gagctccatc 2460ctgacaagtg gacagtccag cctatagagc
tgccagaaaa agaaagctgg actgtcaatg 2520atatacagaa attagtggga
aaactaaatt gggccagtca aatttatcca ggaattaaag 2580taaagcaatt
gtgtaaactt ctcaggggag ccaaagccct aacagatata gtaacactga
2640ctgaggaagc agaattagaa ttagcagaga acagggagat tctaaaagac
cctgtgcatg 2700gggtatatta tgacccatca aaagacttaa tagcagaaat
acagaaacaa gggcaagacc 2760aatggacata ccaaatttat caggagccat
ttaaaaatct aaaaacagga aaatatgcaa 2820gaaaaaggtc tgctcacact
aatgatgtaa gacaattagc agaagtagtg cagaaagtgg 2880tcatggaaag
catagtaata tggggaaaga ctcctaaatt taaactaccc atacaaaaag
2940agacatggga gacatggtgg atggactatt ggcaagctac ctggattcct
gagtgggagt 3000ttgtcaatac ccctccccta gtaaaattat ggtaccagtt
agagaaagac cccatagcag 3060gagcagagac tttctaa 3077592241DNAHIV-1
Clade A 59atgagagtga tggggataca gatgaattgt cagcacttat tgagatgggg
aactatgatc 60ttgggattga taataatctg taatgctgta aacagcaact tgtgggttac
tgtctattat 120ggggtacctg tgtggaaaga tgcagagacc accttatttt
gtgcatcaga tgctaaagca 180tataaaacag aaaagcataa tgtctgggct
acacatgcct gtgtgcccac agaccccaac 240ccacaagaaa tacctttgga
aaatgtgaca gaagagttta acatgtggaa aaataaaatg 300gtagaacaaa
tgcatacaga tataatcagt ctatgggacc aaagcctaca gccatgtgta
360aagttaaccc ctctctgcat tactttaaac tgtacagatg ttactaatgt
tacagatgtt 420agtggtacga ggggcaacat caccatcatg aaagagatgg
agggagaaat aaaaaactgt 480tctttcaata tgaccacaga aataagggat
aagaaacaga aagtatattc actcttttat 540agacttgatg tagtaccaat
aaatcagggt aatagtagta gtaaaaacag tagtgagtat 600agattaataa
gttgtaatac ctcagccatt acacaagctt gcccaaaggt aagctttgag
660ccaattccca tacattattg tgccccagct ggttttgcga tcctgaagtg
tagggataag 720gagttcaatg gaacagggga atgcaagaat gtcagcacag
tccaatgcac acatggaatc 780aagccagtag tatcaactca actactgtta
aatggcagtc tagcagaaga aaaggtaaaa 840atcagaactg aaaatatcac
aaacaatgcc aaaactatag tagtacaact tgtcgagcct 900gtgagaatta
attgtactag acctaataac aatacaagag agagtgtgcg tatagggcca
960ggacaagcat tctttgcaac aggtgacata ataggggata taagacaagc
acattgtaat 1020gtcagtagat cacaatggaa taagacttta caacaggtag
ctgaacaatt aagagaacac 1080tttaaaaaca aaacaataat atttaacagt
tcctcaggag gggatctaga aatcacaaca 1140catagtttca attgtggagg
agaattcttc tattgtaata catcaggtct gttcaatagc 1200acctggaata
ccagcatgtc agggtcaagt aacacggaga caaatgacac tataactctc
1260caatgcagaa taaagcaaat tataaatatg tggcagagaa caggacaagc
aatatatgcc 1320cctcccatcc agggagtgat aaggtgtgaa tcaaacatca
caggactact gttaacaaga 1380gatggtgggg aggagaagaa cagtacaaat
gaaatcttca gacctggagg aggagatatg 1440agggacaact ggagaagtga
attatataag tataaagtag taaaaattga accactagga 1500gtagcaccca
ccagggcaag gagaagagtg gtgggaagag aaaaaagagc agttggaata
1560ggagctgttt tccttgggtt cttaggagca gcaggaagca ctatgggcgc
ggcgtcaata 1620acgctgacgg tacaggccag gcaattattg tctggcatag
tgcagcagca gagcaatttg 1680ctgagggcta tagaggctca acaacatatg
ttgaaactca cggtctgggg cattaaacag 1740ctccaggcaa gagtccttgc
tgtggaaaga tacctaaggg atcaacagct cctaggaatt 1800tggggctgct
ctggaaaact catctgcacc actaatgtgc cctggaactc tagttggagt
1860aataaatctc aggatgaaat atggaacaac atgacctggc tgcaatggga
taaagaaatt 1920agcaattaca taaacctaat atatagtcta attgaagaat
cgcaaaacca gcaggaaaag 1980aatgaacaag acttattggc attgggcaag
tgggcaaatc tgtggacttg gtttgacata 2040tcaaattggc tgtggtatat
aagaatattt ataatgatag taggaggctt aataggatta 2100agaatagttt
ttgctgtgct tgctgtaata aagagagtta ggcagggata ctcacctgtg
2160tcatttcaga tccatgcccc aaacccaggg ggtctcgaca ggcccggaag
aatcgaagga 2220gaaggtggag agcaagacta a 2241602211DNAHIV-1 Clade A
60atgagagtga tggggataca gatgaattgt caaagcttgt ggagatgggg aactatgatc
60ttgggaatgt taatgatttg tagtgttgca ggaaacttgt gggttactgt ctactatggg
120gtacctgtgt ggaaagaggc agacaccacc ttattttgtg tatcaaatgc
tagagcatat 180gatacagaag tgcataatgt ctgggctaca catgcctgtg
tacctacgga ccccaaccca 240caagaaatag atttggagaa tgtgacagaa
gagtttaaca tgtggaaaaa taacatggta 300gagcaaatgc atacagatat
aattagtcta tgggaccaaa gcctaaaacc atgtgtaaag 360ttaacccctc
tctgcgttac tttagattgt ggctataatg taaccaactt gaatttcacc
420agtaacatga aaggagacat aacaaactgc tcttacaata tgaccacaga
aataagggat 480aggaaacaga aagtgtattc acttttctat aggcttgata
tagtaccaat taatgaagaa 540aagaataata gcagggagac tagtccgtat
agattaataa attgtaatac ctcagccatt 600acacaagctt gtcctaaggt
atcttttgaa ccaattccca tacattattg tgccccagcc 660ggttttgcga
ttctaaaatg taaggatgca gagttcaatg gaacagggcc atgcaagaat
720gtcagcacag tacaatgtac acatggaatc aggccagtaa tatcaactca
actgctgtta 780aatggcagtt tagcagagaa tgggacaaag attagatctg
aaaatatcac aaacaatgcc 840aaaaccataa tagtacaact taacgagacc
gtacaaatta attgtaccag acctagcaac 900aatacaagaa aaagtgtacg
tataggacca ggacaagcat tctatacaac aggtgatata 960acaggggata
taagacaagc atattgtaat gtcagtagac aagaatggga acaagcatta
1020aaaggggtag ttatacaatt aagaaaacac tttaacaaaa caataatctt
taacagttcc 1080tcaggagggg atttagaaat tacaacacat agttttaatt
gtggaggaga attcttctat 1140tgtgatacat caggcctgtt taatagcacc
tggaacacga acaccaccga gccaaacaac 1200acaacgtcaa atggcactat
cattctccaa tgcagaataa agcaaattat aaatctgtgg 1260cagagaaccg
gacaagcaat gtatgcccct cccatccaag gggtaataag gtgtgattcc
1320aacattacag gactactatt aacaagagat ggtggagtag ttgatagtat
aaatgaaacc 1380gaaatcttca gacctggagg aggagatatg agggacaatt
ggagaagtga attatataag 1440tataaagtag taaaaattga accactagga
gtagcaccca ccggggcaaa gagaagagtg 1500gtggagagag aaaaaagagc
agttggcata ggagctgtat tcattgggtt cttaggagca 1560gcaggaagca
ctatgggcgc ggcgtcaata acgctgacgg tacaggccag acaattattg
1620tctggcatag tgcaacagca aagcaatttg ctgagggcta tagaggctca
acagcatatg 1680ttgagactca cggtctgggg cattaagcag ctccaggcaa
gagtcctggc tgtggaaaga 1740tacctaaggg atcaacagct cctaggaatt
tggggctgct ctggaaaact catctgcacc 1800actaatgtgc cctggaactc
tagttggagt aataaatctc aggaggaaat atggggtaac 1860atgacctggc
tgcaatggga taaagaaatt agcaattaca cacaaacaat atataaccta
1920cttgaagaat cgcagaacca gcaggaaaag aatgaacaag acttattggc
attggacaag 1980tgggcaaatt tgcggacttg gtttgacata acaaattggc
tgtggtatat aaaaatgttt 2040ataatgatag taggaggctt aataggatta
agaatagttt ttgctgtgct ttctgtaata 2100aatagagtta ggcagggata
ctcacctctg tcgtttcaga cccatatccc gagcccaagg 2160ggtctcgata
ggcccggaag aatcgaagga gaaggtggag agcaagacta a 2211613083DNAHIV-1
Clade C 61atgggtgcga gagcgtcaat attaagaggg ggaaaattag atcgatggga
aaaaattagg 60ttaaggccag ggggaaagaa aagctatatg ataaaacact tagtatgggc
aagcagggag 120ctggaaagat ttgcacttaa ccctagcctt ttagagacat
cagaaggctg taaacaaata 180atgaaacagc tacaaccagc tcttcagaca
ggaacagaag aacttaaatc attattcaat 240gcaatagcag ttctctattg
tgtacatgaa gggatagatg taaaagacac caaggaagcc 300ttagacaaga
tagaggaaga acagaacaaa agtcagcaaa aaacacagca ggcagaagca
360gctggcggaa aagtcagtca aaattatcct atagtgcaga atctccaagg
acaaatggta 420caccagtcca tatcacctag aactttgaat gcatgggtaa
aagtaataga ggaaaaggct 480tttagcccag aggtaatacc catgtttaca
gcattatcag aaggagccac cccacaagat 540ttaaacacca tgctaaatac
agtgggggga catcaagcag ccatgcaaat gttaaaagat 600accatcaatg
aggaggctgc agaatgggat aggatacatc cagtacatgc agggcctact
660gcaccaggcc aaatgagaga accaagggga agtgacatag caggaactac
tagtaccctt 720caggaacaaa tagcatggat gacagctaac ccacctgttc
cagtgggaga aatctacaaa 780agatggataa tactgggttt aaataaaata
gtaagaatgt atagccctgt cagcattttg 840gacataaaac aagggccaaa
ggaacccttt agagactatg tagatcggtt ctttaaaact 900ttaagagctg
aacaggctac acaagatgta aaaaattgga tgacagacac cttgttggtc
960caaaatgcga acccagattg taagaccatt ttaagagcat taggaccagg
ggctacatta 1020gaagaaatga tgacagcatg tcaaggagtg ggaggacctg
gccacaaagc cagagttttg 1080gctgaggcaa tgagccaagc aaacacacac
ataatgatgc agagaagcaa ttttaaaggc 1140tctaaaagaa ttgttaaatg
tttcaactgt ggcaaggaag ggcacatagc cagaaattgc 1200agggccccta
ggaaaaaggg ctgttggaaa tgtggaaagg aaggacacca aatgaaagac
1260tgtactgaga ggcaggctaa ttttttaggg aaaatttggc cttcccacaa
ggggaggcca 1320gggaatttcc ttcagaacag gtcagagcca acagccccac
caacgaacag gccagagcca 1380acagctccac cagcagagag cttcaggttc
gaggaagcaa cccctgctcc gaagcaggag 1440ctgaaagaca gggaaccttt
aatttccctc aaatcactct ttggcagcga cccctcgtct 1500caataaaagt
agggggtcaa acaaaggagg ctcttttaga cacaggagca gatgatacag
1560tattagaaga aataaatttg ccaggaaaat ggaaacccaa aatgatagga
ggaattggag 1620gttttatcaa agtaagacag tatgatcaga tagttataga
aatttgtgga aaaaaggcta 1680taggtacagt attagtagga cccacccctg
tcaacataat tggaagaaat atgttgactc 1740agcttggatg cacactaaat
tttccaatta gtcctattga aactgtacca gtaaagttaa 1800agccaggaat
ggatggccca aaggttaaac aatggccatt gacagaagaa aaaataaagg
1860cattaacagc aatttgtgaa gaaatggaga aggaaggaaa aattacaaag
attgggcctg 1920aaaatccata taacactcca gtatttgcca taaaaaagaa
ggacagtact aagtggagaa 1980aattagtaga tttcagggaa cgcaataaaa
gaactcaaga tttttgggaa gttcaattag 2040gcataccaca cccagcaggg
ttaaaaaaga aaaaatcagt gacagtactg gaggtggggg 2100atgcatactt
ctcagttcct ttagatgaag gcttcaggaa atatactgca ttcaccatac
2160ctagtataaa caatgaaaca ccaggaatta gatatcaata caatgtgctt
ccacagggat 2220ggaaaggatc accagcaata ttccagagta gcatgacaaa
aatcttagag ccctttagag 2280cacaaaatcc agaaatagtc atctatcaat
atatgcacga cttatatgta ggatctgact 2340tagaaatagg gcaacataga
gcaaaaatag aggaattaag agaacatcta ttaaagtggg 2400gatttaccac
accagacaag aaacatcaga aagaaccccc atttctttgg atggggtatg
2460aactccatcc tgacaaatgg acagtacagc ctataacgct gccagaaaag
gaaagctgga 2520ctgtcaatga tatacagaag ttagtgggaa aactaaactg
ggcaagtcag atttatgcag 2580ggattaaagt aaggcaactg tataaactcc
ttaggggagc caaagcacta acagacatag 2640taccactaac tgaagaggca
gaattagaat tggcagagaa cagggaaatt ctaaaagaac 2700cagtacatgg
ggtatattat gacccatcaa aagacttgat agctgaaata cagaaacaag
2760ggcatgacca atggacatat caaatttacc aagaaccatt caaaaatctg
aaaacaggga 2820agtatgcaaa aatgaggagt gcccacacta atgatgtaaa
acaattaaca gaggcagtgc 2880aaaaaatagc catggaaggc atagtaatat
ggggaaagac tcctaaattt agactgccca 2940ttcaaaagga aacatgggaa
acatggtgga cagactattg gcaagccacc tggattcctg 3000agtgggagtt
tgttaatacc cctcccctag taaaattatg gtaccagctg gagaaagaac
3060ccatagtagg agcagaaact ttc 3083622226DNAHIV-1 Clade C
62atgagagtga aggggatatt gaggaattgg caacacaggt ggatatggat ctggatcatc
60ttaggctttt ggatgctaat gatttgtaat gggaacttgt gggtcactgt ctactatggg
120gtacctgtgt ggaaagaagc aaatgctcct ctattttgtg catcagatgc
taaagcatat 180gagaaagaag tgcataatgt ctgggctaca catgcctgtg
tacccacaga ccccaaccca 240caagaactag acttggtaaa tgtaacagaa
aattttaaca tgtggaaaaa tgacatggta 300gatcagatgc atgaggatat
aatcagttta tgggatgaaa gcctaaagcc atgtgtaaag 360ttgaccccac
tctgtgtcac tctaaactgt actaatgcta atattaataa tgatactgtt
420gctaatagtg gtacttttaa ggttgataat agtagtaatg tagtaaaaaa
ttgctctttc 480aatataacca cagaaataag agataagaag aaaaaagaat
attcattgtt ttatagactt 540gatatattac cacttgataa ctctagtgag
tctaagaact atagtgagta tgtattaata 600aattgtaatg cctcaaccgt
aacacaagcc tgtccaaagg tctcttttga cccaattcct 660atacattatt
gtgctccagc tggttatgcg attctaaagt gtaaagataa gacattcaat
720ggaacaggac catgcagtaa tgtcagcaca gtactatgta cacatggaat
taagccagtg 780gtatcaactc aattactgtt aaatggtagc ctagcagaag
aagggatagt aattagatct 840gaaaatctga caaacaatgc caaaacaaca
atagtacagc ttaatgaacc tgtagaaatt 900atgtgtgtaa gacccggcaa
taatacaaga aaaagtgtga ggataggacc aggacaaaca 960ttctatgcaa
caggaggcat aataggagat ataagacaag cacattgtaa cattagtaga
1020agtgattgga ataaaacttt acaagaggta ggtaaaaaat tacgagaata
cttccacaat 1080aaaacaataa gatttaaacc ggcggtcgta ggaggggacc
tggaaattac aacacatagc 1140tttaattgta gaggagaatt cttctattgc
aatacatcag aactgtttac aggtgaatat 1200aatggtactg agtataagaa
tacttcaaat tcaaatccta acatcacact cccatgtaga 1260ataaaacaat
ttgtaaacat gtggcagagg gtaggacgag caatgtatgc ccctcctatt
1320gaaggaaaca taacatgtaa ctcaagtatc acaggactac tattgacatg
ggatggagga 1380aacaatacta atggcacaga gacatttaga cctggaggag
gagatatgag ggataattgg 1440agaagtgaat tatataaata taaagtggta
gaaattaaac cattaggaat agcacccact 1500agtgcaaaaa ggagagtggt
ggagagagag aaaagagcag tgggaatagg agctttgttc 1560cttgggttct
taggagcagc aggaagcact atgggcgcag catcaataac gctgacggta
1620caggccagac aattattgtc tggtatagtg caacagcaaa gcaatttgct
gagggccata 1680gaggcgcaac agcatatgtt gcaactcaca gtctggggca
ttaaacagct ccagacaaga 1740gtcctggcta tagaaagata cctaaaggat
caacagctcc tagggatttg gggctgctct 1800ggaaaactca tctgcaccac
tgctgtgcct tggaacacta gttggagtaa taaaactgaa 1860caggacattt
ggaatctaac ctggatgcag tgggatagag aagttagtaa ttacacagac
1920ataatataca ggttgcttga agactcacaa atccagcagg aaaacaatga
aaaggattta 1980ctagcattgg acagttggaa aaatctgtgg aattggtttg
acataacaaa ttggttgtgg 2040tatataagaa cattcataat gatagtagga
ggcttgatag
gcttaaggat aatttttgct 2100gtaatttcta tagtgaatag agttaggcag
ggatactcac ctttgtcatt tcagaccctt 2160accccaaccc cgaggggacc
agaaaggctc ggaggaatcg aagaagaagg tggagagcaa 2220gactaa
2226639PRTArtificial Sequencesynthetic peptide 63Ala Met Gln Met
Leu Lys Glu Thr Ile1 5
* * * * *
References