U.S. patent application number 12/561455 was filed with the patent office on 2010-04-01 for expression and characterization of hiv-1 envelope protein associated with broadly cross reactive neutralizing antibody response.
Invention is credited to Gerald V. Quinnan, JR., Peng Fei Zhang.
Application Number | 20100080818 12/561455 |
Document ID | / |
Family ID | 36781693 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100080818 |
Kind Code |
A1 |
Quinnan, JR.; Gerald V. ; et
al. |
April 1, 2010 |
Expression and Characterization of HIV-1 Envelope Protein
Associated with Broadly Cross Reactive Neutralizing Antibody
Response
Abstract
The present invention relates to HIV-1 envelope proteins from a
donor with non-progressive HIV-1 infection whose serum contains
broadly cross-reactive, primary virus neutralizing antibody. The
invention also relates to isolated or purified proteins and protein
fragments that share certain amino acids at particular positions
with the foregoing HIV-1 proteins.
Inventors: |
Quinnan, JR.; Gerald V.;
(Bethesda, MD) ; Zhang; Peng Fei; (Bethesda,
MD) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Family ID: |
36781693 |
Appl. No.: |
12/561455 |
Filed: |
September 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11473036 |
Jun 23, 2006 |
7608688 |
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12561455 |
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09762261 |
May 29, 2001 |
7090848 |
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PCT/US99/17596 |
Aug 4, 1999 |
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11473036 |
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60095267 |
Aug 4, 1998 |
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Current U.S.
Class: |
424/188.1 ;
424/184.1; 436/501; 530/321; 530/350; 530/387.1 |
Current CPC
Class: |
A61K 39/00 20130101;
A61K 39/12 20130101; C12N 2740/16122 20130101; Y10S 530/826
20130101; A61K 39/21 20130101; A61P 31/18 20180101; C12N 2740/16134
20130101; Y02A 50/30 20180101; C07K 14/005 20130101; Y02A 50/466
20180101 |
Class at
Publication: |
424/188.1 ;
530/350; 424/184.1; 436/501; 530/321; 530/387.1 |
International
Class: |
A61K 39/21 20060101
A61K039/21; G01N 33/566 20060101 G01N033/566; C07K 7/64 20060101
C07K007/64; C07K 16/00 20060101 C07K016/00; A61P 31/18 20060101
A61P031/18 |
Goverment Interests
ACKNOWLEDGMENT OF FEDERAL SUPPORT
[0002] The present invention arose in part from research funded by
the following federal grant monies: NIH AI37436 and AI44339, and
USUHS R087E2
Claims
1. An isolated HIV envelope protein or fragment thereof which, when
administered to a mammal, induces the production of broadly
cross-reactive neutralizing anti-serum against multiple strains of
HIV-1.
2. An isolated HIV envelope protein comprising the amino acid
sequence of SEQ ID NO: 1.
3. An isolated HIV envelope protein or fragment thereof comprising
a proline at a position corresponding to amino acid residue 313, a
methionine at a position corresponding to amino acid residue 314
and a glutamine at a position corresponding to amino acid residue
325 of SEQ ID NO:1.
4. An isolated HIV envelope protein or fragment thereof comprising
a V3 region having the amino acid sequence P M X.sub.1 X.sub.2
X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10 Q,
wherein X.sub.1-X.sub.10 are a natural or non-natural amino
acid.
5. (canceled)
6. An immunogenic composition comprising an isolated HIV-1 envelope
protein or fragment thereof of claim 1 and a pharmaceutically
acceptable carrier.
7. An isolated nucleic acid molecule encoding the HIV-1 envelope
protein or fragment thereof of claim 1.
8. A fusion protein comprising all or a portion of a
microbiological antigen into which the protein of claim 1 has been
inserted.
9. A recombinant delivery vector encoding a fusion protein
comprising all or a portion of a microbiological antigen into which
the protein of claim 1 has been inserted.
10. (canceled)
11. An immunogenic composition comprising the recombinant delivery
vector of claim 9 and a pharmaceutically acceptable carrier.
12. A recombinant delivery vector encoding an attenuated virus
further comprising a nucleotide sequence encoding the protein claim
1.
13. The recombinant delivery vector of claim 12 wherein the
attenuated virus is selected from the group comprising HIV,
encephalitis virus, poliovirus, poxvirus and vaccinia virus.
14. (canceled)
15. An immunogenic composition comprising the recombinant delivery
vector claim 12 and a pharmaceutically acceptable carrier.
16. A method of generating antibodies in a mammal comprising
administering the protein of claim 1 or fragments thereof in an
amount sufficient to induce the production of the antibodies.
17. A method of generating antibodies in a mammal comprising
administering a DNA or mRNA sequence encoding the protein of claim
1 or fragments thereof, in an amount sufficient to induce the
production of the antibodies.
18. The method of claim 17, wherein said DNA is naked DNA.
19. A diagnostic reagent comprising the isolated HIV-1 envelope
protein of claim 1 or fragments thereof.
20. A method of detecting HIV-1 antibodies in a sample comprising
the step of determining whether antibodies in the sample bind to
one or more of the HIV-1 envelope proteins or fragments thereof
claim 1.
21. A cyclic peptide comprising the amino acid sequence of claim
3.
22. An isolated antibody which specifically recognizes the protein
of claim 3.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 11/473,036 (filed Jun. 23, 2006, now U.S. Pat.
No. 7,608,688, issued Oct. 27, 2009) which is a divisional of U.S.
patent application Ser. No. 09/762,261 (filed Feb. 5, 2001, now
U.S. Pat. No. 7,090,848, issued Aug. 15, 2006), which is a U.S.
National Phase Application of International Application
PCT/US99/17596 (filed on Aug. 4, 1999), which claims the benefit of
U.S. Provisional Application 60/095,267 (filed Aug. 4, 1998), all
of which are herein incorporated by reference in their
entirety.
SEQUENCE LISTING SUBMISSION VIA EFS-WEB
[0003] A computer readable text file, entitled
"044508-5001-02-SequenceListing.txt," created on or about Sep. 16,
2009 with a file size of about 20 kb contains the sequence listing
for this application and is hereby incorporated by reference in its
entirety.
TECHNICAL FIELD
[0004] The present invention relates to HIV-1 envelope proteins and
peptides derived from the donor of the Neutralizing Reference Human
Serum (2) which is noted for its capacity to neutralize primary HIV
isolates of varied subtypes.
BACKGROUND OF THE INVENTION
[0005] The development of a successful vaccine against HIV
infection or a vaccine agent capable of preventing HIV disease
progression has been a public health goal for over 15 years. One of
the immune responses that may be required to elicit a protective
immune response against HIV infection is the generation of
antibodies that are virus neutralizing.
[0006] The target of HIV-1 neutralizing antibodies (NA) is the
envelope glycoprotein complex. This complex is a multimeric
structure composed of three or four copies each of the gp120
surface and gp41 transmembrane glycoproteins (Luciw, 1996). There
are a number of neutralization domains on each of the three or four
heterodimeric components of the complex (Thali et al., 1992, 1993;
Zwart et al., 1991; Moore et al., 1993; Trkola et al., 1996; Muster
et al., 1993; Cotropia et al., 1996; Sabri et al., 1996). The amino
acid compositions of the proteins vary substantially from strain to
strain. Some of the neutralization domains are in regions which
tend to vary greatly, while others are in regions which tend to be
highly conserved. The variable neutralization domains include those
in variable (V) regions 1, 2, and 3 of gp120, while the conserved
domains include the primary receptor binding site, and other
epitopes in gp120 and gp41 Amino acid sequence variation is
undoubtedly the explanation for the variation that is seen in
specificity of neutralization sensitivity among virus strains.
However, it has not been possible to classify antigenic subtypes of
HIV-1 based on genetic analyses, and various regions of the
envelope complex even outside of the neutralization domains have
been shown to contribute to antigenic variability (Thali et al.,
1994; Back et al., 1993).
[0007] Recent findings indicate that the neutralization of primary
isolates of HIV may be mediated primarily by antibodies directed
against non-V3 region epitopes expressed on the oligomeric complex
but not on monomeric gp120, while laboratory adapted strains are
more readily neutralized by antibodies directed against V3 (Hioe et
al., 1997; VanCott et al., 1997). The identity of the non-V3
epitopes recognized on primary isolates is not established. The
presence of antibodies which have broadly neutralizing activity
against primary isolates of many subtypes of HIV-1 in sera from
infected people is unusual, but the nature of the envelope proteins
in individuals with such antibodies may be of interest for defining
the epitopes which may be broadly immunogenic in vaccines.
SUMMARY OF THE INVENTION
[0008] The present inventors have cloned and characterized the
envelope genes from the donor of human serum which is noted for its
capacity to neutralize primary HIV isolates of various subtype
(Vujcic, et al. 1995, D'Souza et al., 1991).
[0009] The invention includes an isolated HIV envelope protein or
fragment thereof which, when injected into a mammal, induces the
production of broadly cross-reactive neutralizing anti-serum
against multiple strains of HIV-1.
[0010] The invention further includes an isolated HIV envelope
protein or fragment thereof comprising a proline at a position
corresponding to amino acid residue 313, a methionine at a position
corresponding to amino acid residue 314 and a glutamine at a
position corresponding to amino acid residue 325 of SEQ ID
NO:1.
[0011] In another embodiment, the invention includes an isolated
HIV envelope glycoprotein or fragment thereof comprising an alanine
at a position corresponding to amino acid residue 167 of SEQ ID
NO:1.
[0012] The invention also includes an isolated HIV envelope protein
comprising the amino acid sequence of SEQ ID NO:1 as well as an
isolated nucleic acid molecule encoding the envelope protein, which
was deposited with the American Type Culture Collection (ATCC)
under accession No. PTA-7237 on Dec. 1, 2005 in accordance with the
Budapest Treaty.
[0013] Compositions for eliciting an immune response, such as
vaccines, immunogenic compositions and attenuated viral vaccine
delivery vectors comprising the envelope proteins, peptides and
nucleic acids encoding such proteins and peptides of the invention
are also included. Methods for generating antibodies in a mammal
comprising administering one or more of these proteins, peptides
and nucleic acids, in an amount sufficient to induce the production
of the antibodies, is also included in the invention.
[0014] The invention also comprises a diagnostic reagent comprising
one or more of the isolated HIV-1 envelope proteins and methods for
detecting broadly cross-reactive neutralizing anti-serum against
multiple strains of HIV-1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1: Phylogenetic analysis of the gp120 and gp41
nucleotide coding sequences of clone R2. Alignments were performed
using the Clustial algorithm of Higgins and Sharp in the program
DNA Star (Higgins et al., 1989; Saitou et al., 1987; Myers et al.,
1988). The graphs at the bottom of the two figures indicate the
percent similarity distances represented by the dendograms. Gene
bank accession numbers for the sequences represented are: MW 959,
U08453; MW960, U08454; D747, X65638; BR020, U27401; BR029, U27413;
RU131, U30312; UG975, U27426; AD8, M60472; HXB, K03455; NDK,
M27323; Z2Z6, M22639; UG021, U27399; CM235, L03698; TH022, U09139;
TH006, U08810; UG275, L22951; SF1703, M66533; RW020, U08794; RW00,
U08793; U455, M62320; and Z321, M15896.
[0016] FIG. 2: Neutralization of clade B viruses and pseudoviruses
by sera from 10 male residents of the Baltimore/Washington, D.C.
area collected from 1985-1990 in the Multicenter AIDS Cohort Study.
The P9 and P10 viruses (P9-V and P10-V) are primary isolates from
two of the serum donors (Quinnan et al., 1998). The neutralization
assays were performed in PM1 cells, as described in the Examples.
Each point represents the results obtained with an individual
serum. The open bars represent the standard deviations about the
geometric means, indicated by the midlines. The numbers above the
results obtained using pseudoviruses indicate the probabilities
obtained from testing the null hypothesis by paired t testing
comparing the individual pseudoviruses to R2.
[0017] FIG. 3 (A) Inhibition of Reference 2-mediated neutralization
of pseudoviruses by synthetic V3 peptides. The neutralization
endpoints for 90% neutralization were calculated as described
previously (Quinnan et al., 1999; Quinnan et al., 1998; Zhang et
al., 1999; Park et al., 1998). Results shown are means of
triplicate determinations. Dose-response effects of R2 linear
17-mer (open square) and cyclic (closed square) (SEQ ID NO:2) and
the 93TH966.8 cyclic (shaded square) (SEQ ID NO:3) V3 peptides on
neutralization of clone R2 pseudovirus. The peptide concentrations
are 3.times.10 raised to the indicated power.
[0018] FIG. 3 (B): Comparative inhibitory effects of peptides on
neutralization of R2 and MN (clone V5) pseudoviruses. All peptides
were tested at 15 .mu.g/ml. The linear peptides (L) corresponded to
the apical sequences of the respective V3 loops. The cyclic
peptides (C) corresponded to the full lengths of the respective V3
regions of the different strains. Neutralization in the absence of
peptide (None), is also shown.
[0019] FIG. 4 (A): Effect of cyclic R2 V3 peptide on neutralization
of pseudoviruses. Fold inhibition of neutralization was calculated
as the ratio of the 50% neutralization titer obtained in the
absence of peptide compared to that obtained in the presence of
cyclic R2 V3 peptide (15 .mu.g/ml). All assays were performed in
triplicate. Neutralization titers were calculated at the midpoints
of the infectivity inhibition curves, since the curves tended to be
most parallel in this region. Similar results were obtained
comparing 90% neutralization endpoints. Peptide inhibition of
neutralization of R2 pseudovirus by sera from MACS donors (donor
numbers 1-10), two assays each, and by Reference 2. Results are
shown for two determinations for each serum from the MACS donors
and for 12 assays of Reference 2 performed during the same time
intervals as the other experiments shown in panels (A) and (B).
[0020] FIG. 4 (B): Peptide inhibition of neutralization of
pseudoviruses expressing MACS patient envelopes (patient numbers 3,
4, 6, 8, 9, and 10) by Reference 2. Results of two or three
separate assays of each pseudovirus are shown.
MODES OF CARRYING OUT THE INVENTION
General Description
[0021] A goal of immunization against HIV is to induce neutralizing
antibody (NA) responses broadly reactive against diverse strains of
virus. The present inventors have studied envelope protein from a
donor with non-progressive HIV-1 infection whose serum contains
broadly cross-reactive, primary virus NA. DNA was extracted from
lymphocytes, which had been collected approximately six and twelve
months prior to the time of collection of the cross reactive serum,
env genes were synthesized by nested PCR, cloned, expressed on
pseudoviruses, and phenotyped in NA assays. Two clones from each
time point had identical V3 region nucleotide sequences, utilized
CCR5 but not CXCR4 for cell entry, and had similar reactivities
with two reference sera. Analysis of the full nucleotide sequence
of one clone demonstrated it to be subtype B, with a predicted
GPGRAF apical V3 sequence, normal predicted glycosylation, and an
intact reading frame. Infectivity assays of R2 pseudovirus in HOS
cells expressing CD4 and various coreceptors demonstrated the
envelope to be CCR5 dependent. R2 pseudovirus was compared to
others expressing env genes of various clades for neutralization by
sera from donors in the United States (presumed or known subtype B
infections), and from individuals infected with subtypes A, C, and
E viruses. Neutralization by the sera from donors in the United
States of pseudoviruses expressing R2 and other clade B envs was
similarly low to moderate, although R2 was uniquely neutralized by
all. R2 was neutralized by sera from people infected with clades
A-F, while other clade B, D, E and G pseudoviruses were neutralized
less often. One highly sensitive clade C pseudovirus was
neutralized by all the sera, although the titers varied more than
250-fold. The results suggest that the epitope(s) which induced the
cross-clade reactive NA in Donor 2 may be expressed on the R2
envelope.
[0022] The present invention relates to HIV-1 envelope proteins
from this donor who had non-progressive HIV-1 infection whose serum
contains broadly cross-reactive, primary virus neutralizing
antibody. The invention also relates to isolated or purified
proteins and protein fragments that share certain amino acids at
particular positions with the foregoing HIV-1 proteins.
SPECIFIC EMBODIMENTS
Proteins and Peptides
[0023] Proteins and peptides of the invention include the full
length envelope protein having the amino acid sequence of Table 3
(SEQ ID NO:1), gp120 having the amino acid sequence corresponding
to gp120 in Table 3 (amino acids: 1-520 of SEQ ID NO:1), gp41
having the amino acid sequence corresponding to gp41 in Table 3
(amino acids 521-866 of SEQ ID NO:1), as well as polypeptides and
peptides corresponding to the V3 domain and other domains such as
V1/V2, C3, V4, C4 and V5. These domains correspond to the following
amino acid residues of SEQ ID NO:1:
TABLE-US-00001 DOMAIN AMINO ACID RESIDUES C1 30-124 V1 125-162 V2
163-201 C2 202-300 V3 301-336 C3 337-387 V4 388-424 C4 425-465 V5
466-509 C5 510-520
[0024] Polypeptides and peptides comprising any single domain may
be of variable length but include the amino acid residues of Table
3 (SEQ ID NO:1) which differ from previously sequenced envelope
proteins. For instance, peptides of the invention which include all
or part of the V3 domain may comprise the sequence: PM X.sub.1
X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9
X.sub.10Q (SEQ ID NO: 5), wherein X.sub.1-X.sub.10 are any natural
or non-natural amino acids (P refers to Proline, M refers to
methionine and Q refers to Glutamine). Non-natural amino acids
include, for example, beta-alanine (beta-Ala), or other omega-amino
acids, such as 3-amino propionic, 2,3-diamino propionic (2,3-diaP),
4-amino butyric and so forth, alpha-aminisobutyric acid (Aib),
sarcosine (Sat), ornithine (Orn), citrulline (Cit), t-butylalanine
(t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle),
phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle),
cysteic acid (Cya) 2-naphthylalanine (2-Nal);
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);
beta-2-thienylalanine (Thi); and methionine sulfoxide (MSO).
Preferably, peptides of the invention are 60%, 70%, 80% or more
preferably, 90% identical to the V3 region of the HIV envelope
protein of Table 3 (SEQ ID NO:1). Accordingly, V3 peptides of the
invention comprise about 13 amino acids but may be 14, 15, 17, 20,
25, 30, 35, 36, 39, 40, 45, 50 or more amino acids in length. In
one embodiment, a V3 peptide of 13 amino acids in length consists
of the sequence PMGPGRAFYTTGQ (amino acids 313-325 of Table 3 (SEQ
ID NO:1).
[0025] In another embodiment of the invention, polypeptides and
peptides comprising all or part of the V1/V2 domain comprise an
amino acid sequence with an alanine residue at a position
corresponding to amino acid 167 Table 3 (SEQ ID NO:1). For
instance, peptides of the invention spanning the V1/V2 domain may
comprise the sequence FNIATSIG (residues 164-171 of SEQ ID NO:1)
and may be about 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more
amino acids in length. As used herein, "at a position corresponding
to" refers to amino acid positions in HIV envelope proteins or
peptides of the invention which are equivalent to a given amino
acid residue in the sequence of Table 1 (SEQ ID NO:1) in the
context of the surrounding residues.
[0026] The peptides of the present invention may be prepared by any
known techniques. Conveniently, the peptides may be prepared using
the solid-phase synthetic technique initially described by
Merrifield (1965), which is incorporated herein by reference. Other
peptide synthesis techniques may be found, for example, in
Bodanszky et al., Peptide Synthesis, 2d ed. (New York, Wiley,
1976).
Nucleic Acids and Recombinant Expression of Peptide or Proteins
[0027] Proteins and peptides of the invention may be prepared by
any available means, including recombinant expression of the
desired protein or peptide in eukaryotic or prokaryotic host cells
(see U.S. Pat. No. 5,696,238). Methods for producing proteins or
peptides of the invention for purification may employ conventional
molecular biology, microbiology, and recombinant DNA techniques
within the ordinary skill level of the art. Such techniques are
explained fully in the literature. See, for example, Maniatis et
al., Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring
Harbor, Cold Spring Harbor Laboratory Press, 1989); Glover, DNA
Cloning: A Practical Approach, Vols. 1-4 (Oxford, IRL Press, 1985);
Gait, Oligonucleotide Synthesis: A Practical Approach (Oxford, IRL
Press, 1984); Hames & Higgins, Nucleic Acid Hybridisation: A
Practical Approach (Oxford, IRL Press, 1985); Freshney, Animal Cell
Culture: A Practical Approach (Oxford, IRL Press, 1992); Perbal, A
Practical Guide To Molecular Cloning (New York, Wiley, 1984).
[0028] The present invention further provides nucleic acid
molecules that encode the proteins or peptides of the invention.
Such nucleic acid molecules can be in an isolated form, or can be
operably linked to expression control elements or vector sequences.
The present invention further provides host cells that contain the
vectors via transformation, transfection, electroporation or any
other art recognized means of introducing a nucleic acid into a
cell.
[0029] As used herein, a "cell line" is a clone of a primary cell
that is capable of stable growth in vitro for many generations.
[0030] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a start codon
at the 5'(amino) terminus and a translation stop codon at the 3'
(carboxy) terminus. A polyadenylation signal and transcription
termination sequence will usually be located 3' to the coding
sequence.
[0031] A "heterologous" region of the DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the gene
will usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genome of the source organism. Another example
of a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., a cDNA where the
genomic coding sequence contains introns, or synthetic sequences
having codons different than the native gene). Allelic variations
or naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
[0032] As used herein, "naked DNA" means nucleic acid molecules
that are free from viral particles, particularly retroviral
particles. This term also means nucleic acid molecules which are
free from facilitator agents including but not limited to the group
comprising: lipids, liposomes, extracellular matrix-active enzymes,
saponins, lectins, estrogenic compounds and steroidal hormones,
hydroxylated lower alkyls, dimethyl sulfoxide (DMSO) and urea.
[0033] As used herein, a "nucleic acid molecule" refers to the
polymeric form of deoxyribonucleotides (adenine, guanine, thymine,
and/or cytosine) in either its single stranded form, or in
double-stranded helix as well as RNA. This term refers only to the
primary and secondary structure of the molecule and is not limited
to any particular tertiary form. In discussing the structure of
particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the nontranscribed
strand of DNA (e.g., the strand having a sequence homologous to the
mRNA). Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0034] As used herein, a "promoter sequence" is a DNA regulatory
region capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. For
purposes of defining the present invention, the promoter sequence
is bounded (inclusively) at its 3' terminus by the transcription
initiation site and extends upstream (5' direction) to include the
minimum number of bases or elements necessary to initiate
transcription at levels detectable above background. Within the
promoter sequence will be found a transcription initiation site, as
well as protein binding domains responsible for the binding of RNA
polymerase. Eukaryotic promoters will often, but not always,
contain "TATA" boxes and "CAT" boxes.
[0035] As used herein, a "replicon" is any genetic element (e.g.,
plasmid, chromosome, virus) that functions as an autonomous unit of
DNA replication in vivo; i.e., capable of replication under its own
control.
[0036] A "signal sequence" can be included before the coding
sequence or the native 29 amino acid signal sequence from the
envelope protein of Table 3 may be used. This sequence encodes a
signal peptide, N-terminal to the polypeptide, that communicates to
the host cell to direct the polypeptide to the cell surface or
secrete the polypeptide into the media. This signal peptide is
clipped off by the host cell before the protein leaves the cell.
Signal sequences can be found associated with a variety of proteins
native to prokaryotes and eukaryotes. For instance, alpha-factor, a
native yeast protein, is secreted from yeast, and its signal
sequence can be attached to heterologous proteins to be secreted
into the media (See U.S. Pat. No. 4,546,082, and EP 0116201).
Further, the alpha-factor and its analogs have been found to
secrete heterologous proteins from a variety of yeast, such as
Saccharomyces and Kluyveromyces, (EP 88312306.9; EP 0324274
publication, and EP 0301669). An example for use in mammalian cells
is the tPA signal used for expressing Factor VIIIc light chain.
[0037] As used herein, DNA sequences are "substantially homologous"
when at least about 85% (preferably at least about 90% and most
preferably at least about 95%) of the nucleotides match over the
defined length of the DNA sequences. Sequences that are
substantially homologous can be identified in a Southern
hybridization experiment under, for example, stringent conditions
as defined for that particular system. Defining appropriate
hybridization conditions is within the skill of the art. See, for
example, Maniatis et al., supra.
[0038] A cell has been "transformed" by exogenous or heterologous
DNA when such DNA as been introduced inside the cell. The
transforming DNA may or may not be integrated (covalently linked)
into chromosomal DNA making up the genome of the cell. In
prokaryotes, for example, the transforming DNA may be maintained on
an episomal element such as a plasmid or viral vector. With respect
to eukaryotic cells, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that it
is inherited by daughter cells through chromosome replication. This
stability is demonstrated by the ability of the eukaryotic cell to
establish cell lines or clones comprised of a population of
daughter cells containing the transforming DNA.
[0039] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then translated
into the protein encoded by the coding sequence.
[0040] As used herein, a "vector" is a replicon, such as plasmid,
phage or cosmid, to which another DNA segment may be attached so as
to bring about the replication of the attached segment.
[0041] Vectors are used to simplify manipulation of the DNA which
encodes the HIV proteins or peptides, either for preparation of
large quantities of DNA for further processing (cloning vectors) or
for expression of the HIV proteins of peptides (expression
vectors). Vectors comprise plasmids, viruses (including phage), and
integrated DNA fragments, i.e., fragments that are integrated into
the host genome by recombination. Cloning vectors need not contain
expression control sequences. However, control sequences in an
expression vector include transcriptional and translational control
sequences such as a transcriptional promoter, a sequence encoding
suitable ribosome binding sites, and sequences which control
termination of transcription and translation. The expression vector
should preferably include a selection gene to facilitate the stable
expression of HIV gene and/or to identify transformants. However,
the selection gene for maintaining expression can be supplied by a
separate vector in cotransformation systems using eukaryotic host
cells.
[0042] Suitable vectors generally will contain replicon (origins of
replication, for use in non-integrative vectors) and control
sequences which are derived from species compatible with the
intended expression host. By the term "replicable" vector as used
herein, it is intended to encompass vectors containing such
replicons as well as vectors which are replicated by integration
into the host genome. Transformed host cells are cells which have
been transformed or transfected with vectors containing HIV peptide
or protein encoding DNA. The expressed HIV proteins or peptides may
be secreted into the culture supernatant, under the control of
suitable processing signals in the expressed peptide, e.g.
homologous or heterologous signal sequences.
[0043] Expression vectors for host cells ordinarily include an
origin of replication, a promoter located upstream from the HIV
protein or peptide coding sequence, together with a ribosome
binding site, a polyadenylation site, and a transcriptional
termination sequence. Those of ordinary skill will appreciate that
certain of these sequences are not required for expression in
certain hosts. An expression vector for use with microbes need only
contain an origin of replication recognized by the host, a promoter
which will function in the host, and a selection gene.
[0044] Commonly used promoters are derived from polyoma, bovine
papilloma virus, CMV (cytomegalovirus, either murine or human),
Rouse sarcoma virus, adenovirus, and simian virus 40 (SV40). Other
control sequences (e.g., terminator, polyA, enhancer, or
amplification sequences) can also be used.
[0045] An expression vector is constructed so that the HIV protein
or peptide coding sequence is located in the vector with the
appropriate regulatory sequences, the positioning and orientation
of the coding sequence with respect to the control sequences being
such that the coding sequence is transcribed and translated under
the "control" of the control sequences (i.e., RNA polymerase which
binds to the DNA molecule at the control sequences transcribes the
coding sequence). The control sequences may be ligated to the
coding sequence prior to insertion into a vector, such as the
cloning vectors described above. Alternatively, the coding sequence
can be cloned directly into an expression vector which already
contains the control sequences and an appropriate restriction site.
If the selected host cell is a mammalian cell, the control
sequences can be heterologous or homologous to the HIV coding
sequence, and the coding sequence can either be genomic DNA
containing introns or cDNA.
[0046] Higher eukaryotic cell cultures may be used to express the
proteins of the present invention, whether from vertebrate or
invertebrate cells, including insects, and the procedures of
propagation thereof are known. See, for example, Kruse &
Patterson, Tissue Culture (New York, Academic Press, 1973).
[0047] Suitable host cells for expressing HIV proteins or peptides
in higher eukaryotes include: monkey kidney CVI line transformed by
SV40 (COS-7, ATCC CRL1651); baby hamster kidney cells (BHK, ATCC
CRL10); Chinese hamster ovary-cells-DHFR (Urlaub & Chasin,
1980); mouse Sertoli cells (Mather, 1980); monkey kidney cells (CVI
ATCC CCL70); African green monkey kidney cells (VERO76, ATCC
CRL1587); human cervical carcinoma cells (HeLa, ATCC CCL2); canine
kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL3A,
ATCC CRL1442); human lung cells (W138, ATCC CCL75); human liver
cells (HepG2, HB8065); mouse mammary tumor (MMT 060652, ATCC
CCL51); rat hepatoma cells (Baumann et al., 1980) and TRI cells
(Mather et al., 1982).
[0048] It will be appreciated that when expressed in mammalian
tissue, the recombinant HIV gene products may have higher molecular
weights than expected due to glycosylation. It is therefore
intended that partially or completely glycosylated forms of HIV
preproteins or peptides having molecular weights somewhat different
from 160, 120 or 41 kD are within the scope of this invention.
[0049] Other preferred expression vectors are those for use in
eukaryotic systems. An exemplary eukaryotic expression system is
that employing vaccinia virus, which is well-known in the art. See,
for example, Macket et al. (1984); Glover, supra; and WO 86/07593.
Yeast expression vectors are known in the art. See, for example,
U.S. Pat. Nos. 4,446,235; 4,443,539; 4,430,428; and EP 103409; EP
100561; EP 96491.
[0050] Another preferred expression system is vector pHSI, which
transforms Chinese hamster ovary cells (see WO 87/02062). Mammalian
tissue may be cotransformed with DNA encoding a selectable marker
such as dihydrofolate reductase (DHFR) or thymidine kinase and DNA
encoding the HIV protein or peptide. If wild type DHFR gene is
employed, it is preferable to select a host cell which is deficient
in DHFR, thus permitting the use of the DHFR coding sequence as
marker for successful transfection in hgt medium, which lacks
hypoxanthine, glycine, and thymidine. An appropriate host cell in
this case is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity, prepared and propagated as described by Urlaub &
Chasin, (1980).
[0051] Depending on the expression system and host selected, HIV
proteins or peptides are produced by growing host cells transformed
by an exogenous or heterologous DNA construct, such as an
expression vector described above under conditions whereby the HIV
protein is expressed. The HIV protein or peptide is then isolated
from the host cells and purified. If the expression system secretes
the protein or peptide into the growth media, the protein can be
purified directly from cell-free media. The selection of the
appropriate growth conditions and initial crude recovery methods
are within the skill of the art.
[0052] Once a coding sequence for an HIV protein or peptide of the
invention has been prepared or isolated, it can be cloned into any
suitable vector and thereby maintained in a composition of cells
which is substantially free of cells that do not contain an HIV
coding sequence. Numerous cloning vectors are known to those of
skill in the art. Examples of recombinant DNA vectors for cloning
and host cells which they can transform include the various
bacteriophage lambda vectors (E. coli), pBR322 (E. coli), pACYC177
(E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative
bacteria), pLAFRI (gram-negative bacteria), pME290 (non-E. coli
gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis),
pBD9 (Bacillus), pIJ61 (Streptomyces). pUC6 (Streptomyces),
actinophage, fC31 (Streptomyces). YIpS (Saccharomyces), YCp19
(Saccharomyces), and bovine papilloma virus (mammalian cells). See
generally, Glover, supra; T. Maniatis et al., supra; and Perbal,
supra.
Fusion Proteins
[0053] HIV envelope fusion proteins and methods for making such
proteins have been previously described (U.S. Pat. No. 5,885,580).
It is now a relatively straight forward technology to prepare cells
expressing a foreign gene. Such cells act as hosts and may include,
for the fusion proteins of the present invention, yeasts, fungi,
insect cells, plants cells or animals cells. Expression vectors for
many of these host cells have been isolated and characterized, and
are used as starting materials in the construction, through
conventional recombinant DNA techniques, of vectors having a
foreign DNA insert of interest. Any DNA is foreign if it does not
naturally derive from the host cells used to express the DNA
insert. The foreign DNA insert may be expressed on extrachromosomal
plasmids or after integration in whole or in part in the host cell
chromosome(s), or may actually exist in the host cell as a
combination of more than one molecular form. The choice of host
cell and expression vector for the expression of a desired foreign
DNA largely depends on availability of the host cell and how
fastidious it is, whether the host cell will support the
replication of the expression vector, and other factors readily
appreciated by those of ordinary skill in the art.
[0054] The foreign DNA insert of interest comprises any DNA
sequence coding for fusion proteins including any synthetic
sequence with this coding capacity or any such cloned sequence or
combination thereof. For example, fusion proteins coded and
expressed by an entirely recombinant DNA sequence is encompassed by
this invention but not to the exclusion of fusion proteins peptides
obtained by other techniques.
[0055] Vectors useful for constructing eukaryotic expression
systems for the production of fusion proteins comprise the fusion
protein's DNA sequence, operatively linked thereto with appropriate
transcriptional activation DNA sequences, such as a promoter and/or
operator. Other typical features may include appropriate ribosome
binding sites, termination codons, enhancers, terminators, or
replicon elements. These additional features can be inserted into
the vector at the appropriate site or sites by conventional
splicing techniques such as restriction endonuclease digestion and
ligation.
[0056] Yeast expression systems, which are the preferred variety of
recombinant eukaryotic expression system, generally employ
Saccharomyces cerevisiae as the species of choice for expressing
recombinant proteins. Other species of the genus Saccharomyces are
suitable for recombinant yeast expression system, and include but
are not limited to carlsbergensis, uvarum, rouxii, montanus,
kluyveri, elongisporus, norbensis, oviformis, and diastaticus.
Saccharomyces cerevisiae and similar yeasts possess well known
promoters useful in the construction of expression systems active
in yeast, including but not limited to GAP, GAL10, ADH2, PHO5, and
alpha mating factor.
[0057] Yeast vectors useful for constructing recombinant yeast
expression systems for expressing fusion proteins include, but are
not limited to, shuttle vectors, cosmid plasmids, chimeric
plasmids, and those having sequences derived from two micron circle
plasmids. Insertion of the appropriate DNA sequence coding for
fusion proteins into these vectors will, in principle, result in a
useful recombinant yeast expression system for fusion proteins
where the modified vector is inserted into the appropriate host
cell, by transformation or other means. Recombinant mammalian
expression system are another means of producing the fusion
proteins for the vaccines/immunogens of this invention. In general,
a host mammalian cell can be any cell that has been efficiently
cloned in cell culture. However, it is apparent to those skilled in
the art that mammalian expression options can be extended to
include organ culture and transgenic animals. Host mammalian cells
useful for the purpose of constructing a recombinant mammalian
expression system include, but are not limited to, Vero cells,
NIH3T3, GH3, COS, murine C127 or mouse L cells. Mammalian
expression vectors can be based on virus vectors, plasmid vectors
which may have SV40, BPV or other viral replicons, or vectors
without a replicon for animal cells. Detailed discussions on
mammalian expression vectors can be found in the treatises of
Glover, DNA Cloning: A Practical Approach, Vols. 1-4 (Oxford, IRL
Press, 1985).
[0058] Fusion proteins may possess additional and desirable
structural modifications not shared with the same organically
synthesized peptide, such as adenylation, carboxylation,
glycosylation, hydroxylation, methylation, phosphorylation or
myristylation. These added features may be chosen or preferred as
the case may be, by the appropriate choice of recombinant
expression system. On the other hand, fusion proteins may have its
sequence extended by the principles and practice of organic
synthesis.
Vaccines and Immunogenic Compositions
[0059] When used in vaccine or immunogenic compositions, the
proteins or peptides of the present invention may be used as
"subunit" vaccines or immunogens. Such vaccines or immunogens offer
significant advantages over traditional vaccines in terms of safety
and cost of production; however, subunit vaccines are often less
immunogenic than whole-virus vaccines, and it is possible that
adjuvants with significant immunostimulatory capabilities may be
required in order to reach their full potential.
[0060] Currently, adjuvants approved for human use in the United
States include aluminum salts (alum). These adjuvants have been
useful for some vaccines including hepatitis B, diphtheria, polio,
rabies, and influenza. Other useful adjuvants include Complete
Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA),
Muramyl dipeptide (MDP) (see Ellouz et al., 1974), synthetic
analogues of MDP (reviewed in Chedid et al., 1978),
N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-d]palmitoy-
l-s-glycero-3-(hydroxyphosphoryloxy)]ethylamide (MTP-PE) and
compositions containing a metabolizable oil and an emulsifying
agent, wherein the oil and emulsifying agent are present in the
form of an oil-in-water emulsion having oil droplets substantially
all of which are less than one micron in diameter (see EP
0399843).
[0061] The formulation of a vaccine or immunogenic compositions of
the invention will employ an effective amount of the protein or
peptide antigen. That is, there will be included an amount of
antigen which, in combination with the adjuvant, will cause the
subject to produce a specific and sufficient immunological response
so as to impart protection to the subject from subsequent exposure
to an HIV virus. When used as an immunogenic composition, the
formulation will contain an amount of antigen which, in combination
with the adjuvant, will cause the subject to produce specific
antibodies which may be used for diagnostic or therapeutic
purposes.
[0062] The vaccine compositions of the invention may be useful for
the prevention or therapy of HIV-1 infection. While all animals
that can be afflicted with HIV-1 can be treated in this manner, the
invention, of course, is particularly directed to the preventive
and therapeutic use of the vaccines of the invention in man. Often,
more than one administration may be required to bring about the
desired prophylactic or therapeutic effect; the exact protocol
(dosage and frequency) can be established by standard clinical
procedures.
[0063] The vaccine compositions are administered in any
conventional manner which will introduce the vaccine into the
animal, usually by injection. For oral administration the vaccine
composition can be administered in a form similar to those used for
the oral administration of other proteinaceous materials. As
discussed above, the precise amounts and formulations for use in
either prevention or therapy can vary depending on the
circumstances of the inherent purity and activity of the antigen,
any additional ingredients or carriers, the method of
administration and the like.
[0064] By way of non-limiting illustration, the vaccine dosages
administered will typically be, with respect to the gp120 antigen,
a minimum of about 0.1 mg/dose, more typically a minimum of about 1
mg/dose, and often a minimum of about 10 mg/dose. The maximum
dosages are typically not as critical. Usually, however, the dosage
will be no more than 500 mg/dose, often no more than 250 mg/dose.
These dosages can be suspended in any appropriate pharmaceutical
vehicle or carrier in sufficient volume to carry the dosage.
Generally, the final volume, including carriers, adjuvants, and the
like, typically will be at least 0.1 ml, more typically at least
about 0.2 ml. The upper limit is governed by the practicality of
the amount to be administered, generally no more than about 0.5 ml
to about 1.0 ml.
[0065] Peptides of the invention corresponding to domains of the
envelope protein such as V3 may be constructed or formulated into
compounds or compositions comprising multimers of the same domain
or multimers of different domains. For instance, peptides
corresponding to the V3 domain may be circularized by oxidation of
the cysteine residues to form multimers containing 1, 2, 3, 4 or
more individual peptide epitopes. The circularized form may be
obtained by oxidizing the cysteine residues to form disulfide bonds
by standard oxidation procedures such as air oxidization.
[0066] Synthesized peptides of the invention may also be
circularized in order to mimic the geometry of those portions as
they occur in the envelope protein. Circularization may be
facilitated by disulfide bridges between existing cysteine
residues. Cysteine residues may also be included in positions on
the peptide which flank the portions of the peptide which are
derived from the envelope protein. Alternatively, cysteine residues
within the portion of a peptide derived from the envelope protein
may be deleted and/or conservatively substituted to eliminate the
formation of disulfide bridges involving such residues. Other means
of circularizing peptides are also well known. The peptides may be
circularized by means of covalent bonds, such as amide bonds,
between amino acid residues of the peptide such as those at or near
the amino and carboxy termini (see U.S. Pat. No. 4,683,136).
[0067] In an alternative format, vaccine or immunogenic
compositions may be prepared as vaccine vectors which express the
HIV protein or peptide of the invention in the host animal. Any
available vaccine vector may be used, including live Venezuelan
Equine Encephalitis virus (see U.S. Pat. No. 5,643,576), poliovirus
(see U.S. Pat. No. 5,639,649), pox virus (see U.S. Pat. No.
5,770,211) and vaccina virus (see U.S. Pat. Nos. 4,603,112 and
5,762,938). Alternatively, naked nucleic acid encoding a protein or
peptide of the invention may be administered directly to effect
expression of the antigen (see U.S. Pat. No. 5,739,118).
Diagnostic Reagents
[0068] The HIV protein or peptide compositions of the present
invention may be used as diagnostic reagents in immunoassays to
detect anti-HIV antibodies, particularly anti-gp120 antibodies.
Many HIV immunoassay formats are available. Thus, the following
discussion is only illustrative, not inclusive. See generally,
however, U.S. Pat. Nos. 4,743,678; 4,661,445; and 4,753,873 and EP
0161150 and EP 0216191.
[0069] Immunoassay protocols may be based, for example, upon
composition, direct reaction, or sandwich-type assays. Protocols
may also, for example, be heterogeneous and use solid supports, or
may be homogeneous and involve immune reactions in solution. Most
assays involved the use of labeled antibody or polypeptide. The
labels may be, for example, fluorescent, chemiluminescent,
radioactive, or dye molecules. Assays which amplify the signals
from the probe are also known, examples of such assays are those
which utilize biotin and avidin, and enzyme-labeled and mediated
immunoassays, such as ELISA assays.
[0070] Typically, an immunoassay for anti-HIV antibody will involve
selecting and preparing the test sample, such as a biological
sample, and then incubating it with an HIV protein or peptide
composition of the present invention under conditions that allow
antigen-antibody complexes to form. Such conditions are well known
in the art. In a heterogeneous format, the protein or peptide is
bound to a solid support to facilitate separation of the sample
from the polypeptide after incubation. Examples of solid supports
that can be used are nitrocellulose, in membrane or microtiter well
form, polyvinylchloride, in sheets or microtiter wells, polystyrene
latex, in beads or microtiter plates, polyvinlyidine fluoride,
diazotized paper, nylon membranes, activated beads, and Protein A
beads. Most preferably, Dynatech, Immulon.RTM. microtiter plates or
0.25 inch polystyrene beads are used in the heterogeneous format.
The solid support is typically washed after separating it from the
test sample.
[0071] In homogeneous format, on the other hand, the test sample is
incubated with the HIV protein or peptide in solution, under
conditions that will precipitate any antigen-antibody complexes
that are formed, as is known in the art. The precipitated complexes
are then separated from the test sample, for example, by
centrifugation. The complexes formed comprising anti-HIV antibody
are then detected by any number of techniques. Depending on the
format, the complexes can be detected with labeled anti-xenogenic
Ig or, if a competitive format is used, by measuring the amount of
bound, labeled competing antibody. These and other formats are well
known in the art.
[0072] Diagnostic probes useful in such assays of the invention
include antibodies to the HIV-1 envelope protein. The antibodies to
may be either monoclonal or polyclonal, produced using standard
techniques well known in the art (See Harlow & Lane,
Antibodies: A Laboratory Manual, (Cold Spring Harbor, Cold Spring
Harbor Laboratory Press, 1988). They can be used to detect HIV-1
envelope protein by specifically binding to the protein and
subsequent detection of the antibody-protein complex by ELISA,
Western blot or the like. The HIV-1 envelope protein used to elicit
these antibodies can be any of the variants discussed above.
Antibodies are also produced from peptide sequences of HIV-1
envelope proteins using standard techniques in the art (Harlow
& Lane, supra). Fragments of the monoclonals or the polyclonal
antisera which contain the immunologically significant portion can
also be prepared.
[0073] The following working examples specifically point out
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the disclosure.
Other generic configurations will be apparent to one skilled in the
art. All references, including U.S. or foreign patents, referred to
in this application are herein incorporated by reference in their
entirety.
EXAMPLES
[0074] The following methods were used in the Examples:
Reference Serum Donor Envelope Gene Cloning
[0075] The donor of the HIV-1 Neutralizing Serum (2) (Reference 2),
available in the NIH AIDS Research and Reference Reagent Program
(Catalog Number: 1983) is a participant in a long term cohort study
at the Clinical Center, NIH (Vujcic et al., 1995). The blood used
to prepare Reference 2 had been collected in the Spring of 1989.
Peripheral blood mononuclear cells that had been cryopreserved from
donations obtained approximately six months and one year prior to
the time of Reference 2 collections were used as sources of DNA for
env gene cloning. The cells had not been stored to maintain
viability. DNA was extracted using phenol/chloroform from
approximately 1-3.times.10.sup.6 cells from each donation (Quinnan
et al., 1998). The DNA was used as template in a nested polymerase
chain reaction, similar to that described previously, except rTth
was used as the DNA polymerase, following the manufacturer's
instructions (Barnes, 1992; Cariello et al., 1991). The DNA was
cloned into the expression vector pSV7d, as previously described
(Quinnan et al., 1998; Stuve et al., 1987).
Other env Gene Clones and Virus Pools
[0076] The following HIV-1 env clones in the expression vector pSV3
were obtained from the AIDS Research and Reference Reagent Program,
93MW965.26 (clade C), 92RWO20.5 (clade A), 93TH966.8 (clade E),
92UG975.10 (clade G) (Gao et al., 1994). The production of env
clones from the molecular virus clones NL43, AD8, and SF162 has
been previously described (Quinnan et al., 1998; Adachi et al.,
1986; Theodore et al., 1996; Englund et al., 1995). env gene of the
Z2Z6 strain was cloned similarly, using molecular virus clone
plasmid as template in polymerase chain reaction, and cloning the
genes into the plasmid pSV7d (Seth et al., 1993). The production of
primary isolate env clones from participants in the Multicenter
AIDS Cohort Study, designated here P9 and P10, has also been
previously described (Quinnan et al., 1998). P9 and P10-virus pools
were prepared by single subpassages of the cell culture media from
primary cultures in PHA blasts (Quinnan et al., 1998). The use of
molecular virus clones for preparation of virus pools of NL43 in H9
cells, and NL (SF 162) and AD8, in PHA blasts, has also been
previously described (Quinnan et al., 1998).
Cell Cultures
[0077] The H9 cell line was obtained from Robert Gallo (Mann et
al., 1989). The Molt 3 cell line was obtained from the American
Type Culture Collection, Rockville, Md. (ATCC). (Daniel et al.,
1988) The HOS cell lines expressing CD4 and various coreceptors for
HIV-1 were obtained from the NIH AIDS Research and Reference
Reagent Program, as was the PM1 cell line (Deng et al., 1996;
Landau et al., 1992; Lusso et al., 1995). The 293T cell line was
obtained from the ATCC, with permission from the Rockefeller
Institute (Liou et al., 1994). The H9, Molt3 and PM1 cell cultures
were maintained in RPMI-1640 medium supplemented with 10% fetal
bovine serum and antibiotics (Gibco). The HOS and 293T cells were
maintained in Dulbecco's Minimal Essential Medium (Gibco), with
similar supplements, except that the HOS cell medium was
supplemented with puromycin for maintenance of plasmid stability.
Cryopreserved human peripheral blood lymphocytes were stimulated
with PHA and used for virus infections (Quinnan et al., 1998;
Mascola et al., 1994).
Reverse Transcriptase Assay
[0078] Reverse transcriptase activity was assayed as previously
described (Park et al., 1998).
Virus Neutralization Assays
[0079] The virus NL43 was used in neutralization assays which
employed Molt3 cells as target cells and used giant cell formation
for endpoint determination, as previously described (Vujcic et al.,
1995). The amounts of virus used were sufficient to result in the
formation of 30-50 giant cells per well (Vujcic et al., 1995;
Lennette, 1964). The viruses, NL (SF162) and AD8, P9 and P10 were
tested for neutralization in PHA stimulated human lymphoblasts in
the presence of IL-2 (Quinnan et al., 1998; Mascola et al., 1994).
In the latter assays ten percent of the cell suspension was removed
each week, fifty percent of the medium was changed each week, and
medium was sampled twice weekly from each well for reverse
transcriptase assay. The reverse transcriptase assays were
performed on the test samples from the first sampling date at which
the non-neutralized control wells had reverse transcriptase
activity about 10-20.times. background, generally on day 14 or 17
of the assay. The neutralization endpoint was considered to be the
highest dilution of serum at which reverse transcriptase activity
was reduced at least fifty percent. The Reference Neutralizing Sera
1 and 2 and the Negative Reference Serum were used as positive and
negative controls (NIH AIDS Research and Reference Reagent
Program)
Pseudovirus Construction and Assays of Pseudoviruses for
Infectivity and Neutralization
[0080] Pseudoviruses were constructed and assayed using methods
similar to those described previously (Quinnan et al., 1998; Deng
et al., 1996; Park et al., 1998). pSV7d-env plasmid DNA and
pNL43.luc+.E-R- were cotransfected into 70 to 80% confluent 293T
cell cultures using the calcium phosphate/Hepes buffer technique,
following manufacturer's instructions (Promega, Madison, Wis.), in
24 well plastic tissue culture trays or 25 cm.sup.2 flasks (Quinnan
et al., 1998; Deng et al., 1996; Park et al., 1998). After 24 hours
the medium was replaced with medium containing one mM sodium
butyrate (Quinnan et al., 1998; Park et al., 1998). Two days after
transfection medium was harvested, passed through a 45 .mu.m
sterile filter (Millipore Corp, Bedford, Mass.), supplemented with
an additional 20% fetal bovine serum and stored at -80.degree.
C.
[0081] Pseudovirus infectivity was assayed in PM1 or HOS-CD4 cells
expressing various co-receptors. Transfection supernatants were
serially diluted and inoculated into cells in 96 well plates, 50
.mu.l per well. Assays were routinely performed in triplicate. The
cultures were incubated for four days, centrifuged at 400.times.g
for ten minutes if PM1 cells were used, and medium removed by
aspiration. The cells were washed twice with phosphate buffered
saline, lysed with 25 .mu.l cell culture lysing reagent according
to the manufacturer's instructions (Promega, Madison, Wis.); the
cells were then tritiated into the medium, and 10 .mu.l of the
suspensions were transferred to wells of 96 well luminometer
plates. Substrate was added in 100 .mu.l volumes automatically, and
the luminescence read using a MicroLumatPlus luminometer (EG&G
Berthold, Hercules, Calif.). Mock PV controls were used in each
assay consisting of media harvested from 293T cell cultures
transfected with pSV7d (without an env insert) and pNL43.Luc.E-R-,
and processed in the same way as cultures for PV preparation.
Infectivity endpoints were determined by a modified Reed Munch
method; an individual well was considered positive if the
luminescence was at least 10-fold greater than the mock control,
and the endpoint was considered to be the highest dilution at which
the calculated frequency of positivity was .gtoreq.50% (Quinnan et
al., 1998; Park et al., 1998; Lennette, 1964). Luminescence
resulting from infection with minimally diluted samples was
generally about 10,000-fold greater than background.
[0082] Neutralization tests were performed using PM1 or HOS-CD4
cells. Aliquots of 25 .mu.l of two-fold serial serum dilutions were
mixed with equal volumes of diluted PV in wells of 96 well plates.
The PV dilutions were selected so as to expect luminescence in the
presence of non-neutralizing serum of about 100-fold of background.
Assays were performed in triplicate. The virus serum mixtures were
incubated for sixty minutes at 40.degree. C., after which 150 .mu.l
aliquots of PM1 cell suspensions were added, which each contained
1.5.times.10.sup.4 cells, or the suspensions were transferred to
wells containing HOS-CD4 cells. The assays were then processed
similarly to the infectivity assays. The neutralization endpoints
were calculated by a modified Reed-Munch method in which the
endpoint was considered to be the highest serum dilution calculated
to have a frequency of .gtoreq.50% for reducing luminescence by
.gtoreq.90% compared to the non-neutralized control. PV titrations
were conducted in duplicate in parallel with each neutralization
assay.
Nucleic Acid Sequencing
[0083] Nucleotide sequence analysis was performed using the
di-deoxy cycle sequencing technique and AmpliTaq FS DNA polymerase,
according to manufacturer's directions (Perkin Elmer Applied
Biosystems, Foster City, Calif.). After the sequencing reaction the
DNA was purified using Centriflex Gel Filtration Cartridges
(Advanced Genetic Technologies, Gaithersberg, Md.). Sequencing gels
were run and analyzed using an Applied Biosystems Prism, Model 377
DNA Sequencer. Sequencing was performed on both strands. Sequence
alignment was performed using the Editseq SEQMAN, and Megalign
programs in DNA Star according to the method of Higgins and Sharp
(1989).
Example 1
Comparability of Clones Isolated from Different Time Points
[0084] From the samples of patient cells from each of the two time
points, env clones were recovered which encoded proteins which were
capable of mediating pseudovirus entry into target cells. Two such
clones from each time point were further characterized. As shown in
Table 1, the envelopes of all four clones mediated infection for
PM1 cells and were neutralized comparably by References 1 and 2.
Pseudoviruses carrying envelopes corresponding to each clone were
also tested for infectivity for HOS-CD4 cells expressing either
CXCR4 or CCR5, and all four were infectious only for the cells
expressing CCR5, as shown in Table 2. Nucleotide sequences
including the V3 regions were analyzed for each clone, with more
than 300 bases assigned for each, and no differences between the
clones were found (results not shown). Based on the absence of
demonstration of differences in these assays, a single clone from
the March sample was selected for use in subsequent assays, and is
designated R2, hereafter.
Example 2
Clone R2 Genotype and Host Range Phenotype
[0085] The complete nucleotide sequence of the env gene clone R2
was determined and found to have an open reading frame of 2598
bases (Genbank Accession Number: AF 128126) (SEQ ID NO: 24). The
amino acid sequence deduced from this sequence is shown in Table 3
(SEQ ID NO: 1). There are thirty predicted glycosylation sites,
compared to twenty-nine in the consensus clade B sequence; four
consensus glycosylation sites are lacking in R2, including those at
residues 146, 215, 270 and 368 (numbering according to the Human
Retroviruses and AIDS Database clade B consensus sequence), in the
V2, C2, C2 and V4 regions of gp120, respectively (Myers et al.,
1993). The consensus glycosylation sequences at residues 215 and
270 are highly and moderately variable, respectively.
[0086] Genotypic analyses conducted included evaluation of the
gp120 and gp41 nucleotide coding sequences in comparison to those
of a number of strains of clades A through G, as shown in FIG. 1
(Saitou et al., 1987; Myers & Miller, 1988). Both coding
regions were more closely related to clade B than non-clade B
sequences. Comparative analyses of regions of the predicted gp120
and gp41 amino acid sequences were also performed (results not
shown). The regions analyzed included: each constant and variable
region of gp120; the proximal gp41 ectodomain including the leucine
zipper region; the part of gp41 extending from the end of the
leucine zipper to the second cysteine; the remaining gp41
ectodomain, and the transmembrane region; and the cytoplasmic
region. R2 consistently related more closely with the clade B
sequences than the others.
Example 3
Comparative Sensitivity of R2 and Other Clade B Viruses and
Pseudoviruses to Neutralization by Sera from Individuals with Clade
B Infections
[0087] The neutralization of R2 pseudovirus was compared to other
clade B viruses and pseudoviruses as shown in FIG. 2. Of the five
virus-pseudovirus comparisons made (P9, P10, NL43, AD8 and SF162 V
and PV), there were no significant differences in the
neutralization of matched viruses and pseudoviruses by paired t
test (statistical results not shown). Each of the pseudovirus
preparations was neutralized by seven, eight, or nine of the sera
tested, and the geometric mean titers ranged from 1:13.9 to 1:56,
while the R2-PV was neutralized by all ten of the sera tested, with
a geometric mean titer of 1:73.5. Although the neutralization
titers of each of the different sera against R2 and the other
pseudoviruses were frequently within four-fold, the neutralization
of R2-PV was significantly greater by paired t test than four of
the other PV preparations.
Example 4
Comparative Neutralization of Pseudoviruses Expressing R2 and Other
Envelopes of Diverse Subtypes by Sera from Diverse Subtype
Infections
[0088] The results of comparative neutralization testing using sera
from individuals infected with HIV-I strains of subtypes A, C and
E, and the Reference 1 and 2, and one Thai clade B serum are shown
in Table 4. Reference 2 neutralized the pseudovirus expressing the
homologous R2 envelope at the modest titer of 1:64 in the
experiment shown and within two-fold of this titer in many other
experiments. It neutralized the other seven pseudoviruses tested at
low to moderate titers, as well. The R2 pseudovirus was neutralized
by seventeen of twenty-four sera, including sera from people
infected with each of the clades A-F. The other two clade B
pseudoviruses were neutralized less frequently and were also
neutralized infrequently by the clade E sera. The frequency of
neutralization by sera from individuals infected with different
clades was not significantly skewed for any of the other four
pseudoviruses. Clade A, C, D and G pseudoviruses were neutralized
by eight, seventeen, six and three of the seventeen sera tested,
respectively. The clade C pseudovirus was substantially more
sensitive to neutralization, in general than the others tested. The
clade E pseudovirus was neutralized by five of five clade D sera
and seven of eight clade E sera but only one of the sera from
people infected by other clades.
Example 5
Synthetic Peptides Generated from V3 Amino Acid Sequences from R2
Strain
[0089] R2 strain V3 peptides were synthesized using an automated
ABI synthesizer and FMOC chemistry (Zeng et al., 1997). The
sequences of these peptides were KSIPMGPGRAFYTTGQI (SEQ ID NO:2)
and CSRPNNNTRKSIPMGPGRAFYTTGQIIGDIRQAHC (SEQ ID NO:3). The mutant
R2 (313-4 PM/HI, 325 Q/D) V3 peptide was prepared similarly. Strain
93TH966.8 V3 peptide, sequence: CTRPSNNTRTSTTIGPGQVFYRTGDITGNIRKAYC
(SEQ ID NO:4) was synthesized using the same methods. The peptides
were purified using C18, acetonitrile-in-water gradient
chromatography with a Waters High Performance Liquid Chromatograph.
Sequences of the purified peptides were verified using an ABI
automated sequencer. Peptides were lyophilized and stored at
4-8.degree. C. Preparation of a linear MN strain V3 peptide has
been described previously (Carrow et al., 1991). Cyclic MN strain
35-mer peptide was obtained from the AIDS Research and Reference
Reagent Program (Catalog #1841) provided by Catasti et al.,
(1996).
[0090] The R2V3 35-mer was insoluble in water, while all other
peptides tested were soluble in water to at least 10 mg/ml. To
obtain cyclic peptides, solutions of the R2 and R2 (313-4 PM/HI,
325 Q/D) V3 35-mers in dimethylsulfoxide (DMSO), 10 mg/ml, were
diluted 1:10 in water at room temperature or 37.degree. C. and the
pH was adjusted to 8.5 with ammonium hydroxide. These solutions
were aerated by bubbling air through the solutions for periods
>1 hour. Following aeration, the pH was adjusted to 7.4 using
hydrochloric acid. A portion of the R235-mer peptide precipitated
during these procedures. To obtain an approximate quantitation of
the amount of R2 V3 35-mer that remained in solution, the turbidity
of the suspension was determined at 480 nm wavelength visible light
using a spectrophotometer. The spectrophotometer was blanked with a
solution of 10 percent DMSO in water, and a standard curve was
produced using slurries of known amounts of the 35-mer peptide
suspended in water. The amount of precipitate estimated by
turbidity was subtracted from the amount of peptide added at the
beginning of the preparation procedure to estimate the amount
remaining in solution. The solubility of the oxidized R235-mer
peptide in 10 percent DMSO solution at pH=7.4 was estimated to be
300-350 .mu.g/ml when processed at room temperature, or 850-900
.mu.g/ml when processed at 37.degree. C. Peptides were sterilized
by passage through 0.22.mu. pore size filters prior to use.
Example 6
Peptide Blocking of Neutralizing Antibody Activity Against Clone R2
Pseudovirus
[0091] The neutralization blocking effects of synthetic V3 peptides
were examined to test the contribution of V3-anti-V3 interactions
in the neutralizing cross reactivities of Reference 2 and clone R2.
The blocking effects of peptides on neutralizing activity of
Reference 2 against clone R2 pseudovirus are shown in FIG. 3A.
Usually, the linear 17-mer peptide had no inhibitory effect on
neutralization, as shown. In only one of several experiments
two-fold reduction of neutralization was observed in the presence
of 17-mer peptide. Concentration-dependent inhibitory effects of
the cyclic 35-mer R2 V3 peptide on neutralization of clone R2
pseudovirus by Reference 2 was observed in the experiment shown and
in numerous other similar experiments. Maximum effect was observed
at approximately 15 .mu.g/ml. No inhibitory effect was observed
using a cyclic peptide homologous to the V3 region of the HIV-1
93TH966.8 strain.
[0092] The comparative effects of the R2 and MN strain V3 peptides
on neutralization of the R2 and MN strain pseudoviruses are shown
in FIG. 3B. The results shown are representative of two additional
experiments. Only the cyclic R2 V3 peptide produced consistent
blocking of R2 pseudovirus neutralization. The linear R2 and MN,
and the cyclic MN peptides did not block R2 neutralization in two
experiments and blocked only two-fold in a third experiment. In
contrast, the MN cyclic and linear peptides consistently inhibited
MN strain neutralization eight- to sixteen-fold in these
experiments, and the R2 peptides had consistent two-fold inhibitory
effects on neutralization of the MN strain. These effects of MN
peptides on MN strain neutralization are consistent with previous
reports (Carrow et al., 1991; Park et al., 1999).
Example 7
Cyclic R2 V3 Peptide Inhibition of Neutralization of R2
Pseudoviruses by Sera from MACS Patients
[0093] Inhibition of heterologous serum neutralization of R2
pseudovirus by cyclic R2 V3 peptide was evaluated to determine if
cross reactivity of these sera with R2 included effects of anti-V3
antibodies. The comparative neutralization titers of sera from ten
patients from the MACS against clone R2 pseudovirus in the presence
and absence of cyclic R2 V3 peptide are shown in FIG. 4A (Quinnan
et al., 1998). These sera have been described previously, and have
been shown to neutralize primary HIV-1 enveloped pseudoviruses
cross reactively, but to a lesser extent than Reference 2 (Zhang et
al., 1999). Each serum was tested twice. Seven of the sera appeared
to be inhibited at least two-fold in one or both experiments. The
geometric mean inhibitory effect of all the tests was 1.9-fold. The
results of twelve tests conducted at the same times as those tests
shown in FIGS. 4A and 4B are shown for Reference 2; the geometric
mean inhibitory effect was 3.56.
Example 8
Cyclic R2 V3 Peptide Inhibition of Reference 2 Neutralization of
Pseudoviruses Expressing Envelopes from the MACS Patients
[0094] Inhibition of Reference 2 neutralization of pseudoviruses
expressing heterologous envelopes by cyclic R2 V3 peptide was
evaluated to determine whether anti-V3 antibody contributed to the
neutralizing cross reactivity of Reference 2. The results of these
experiments are shown in FIG. 4B. Each pseudovirus was tested two
or three times. The peptide appeared to exert a two-fold inhibitory
effect in one, two, or three of the experiments using each of the
six pseudoviruses. The geometric mean inhibitory effect was
1.6-fold.
Example 9
Induction of Cross-Reactive Neutralizing Antibodies in Mice
Following Immunization with Recombinant Delivery Vectors Encoding
HIV-1 Envelope Proteins
[0095] The DNA clone encoding the R2 envelope was introduced into
an expression vector which can be used to express the envelope
protein complex in vivo for immunization. The recombinant delivery
vector expressing the R2 envelope clone was been administered to
mice, both in its full length, encoding both gp120 and gp41, or in
a truncated form. The truncated form is secreted by cells which
express gp140. Both the full-length and truncated form of these
constructs induced neutralizing antibodies in mice. The mice which
received the gp 140 construct, which includes the V3 region, have
developed neutralizing antibodies which neutralize at least three
different strains of HIV-1, including the R2 strain, a macrophage
tropic laboratory strain known as SF162, and a primary strain which
is not laboratory adapted. The amount of cross-reactivity observed
exceeds that induced by most or all other HIV immunogens that have
been tested as single agents.
TABLE-US-00002 TABLE 1 Comparative Neutralization of Pseudoviruses
Expressing Multiple Envelope Clones From Donor 2 Neutralization
Titer Against Clone Serum 10.1 10.2 3.1 3.2 Reference 1 1:32 1:64
1:32 1:64 Reference 2 1:128 1:128 1:128 1:128
TABLE-US-00003 TABLE 2 Coreceptor Dependency of R2 Pseudovirus
Entry Into HOS-CD4 Cells Infectivity Titer In Pseudo- In HOS-CD4
Cells Expressing PM1 virus CCR1 CCR2b CCR3 CCR4 CCR5 CXCR4 Cells R2
<1:4 <1:4 <1:4 <1:4 1:64 <1:4 1:32 P9 <1:4
<1:4 <1:4 <1:4 1:256 <1:4 1:8 NL4-3 <1:4 <1:4
<1:4 <1:4 1:32 >1:256 1:8 AD8 <1:4 <1:4 <1:4
<1:4 1:256 <1:4 1:32
TABLE-US-00004 TABLE 3 Inferred Amino Acid Sequence of the R2
Envelope Clone from Donor 2. ##STR00001## .sup.aAmino Acid residues
are identified by standard single letter designations. Predicted
N-linked glycosylation sites are indicated by shading and
boding.
TABLE-US-00005 TABLE 4 Neutralization of Pseudoviruses Expressing
Envelopes of Various Clades by Sera from People Infected with
Various Clades of HIV-1 NA Titer Against Pseudovirus (Clade).sup.a
R2 P9 P10 RW020 MW965 Z2Z6 TH966 UG975 Clade Serum.sup.b (B) (B)
(B) (A) (C) (D) (E) (G) B Ref 1 32 16 32 <10 256 10 <8 <10
Ref 2 64 32 64 10 128 40 8 10 WR8465 20 .sup. NT.sup.c 80 <10
640 10 <10 10 A 37570 320 160 20 80 2560 <10 <10 <10
35374 40 <10 <10 <10 640 <10 <10 <10 35837 40 20
<10 80 2560 <10 <10 <10 C 5107 40 10 <10 10 1280
<10 <10 <10 5708 10 <10 <10 <10 320 <10 <10
<10 5218 80 <10 <10 <10 1280 <10 <10 <10 D
UG9240 <10 NT NT NT NT NT 20 NT UG9370 <10 NT NT NT NT NT 10
NT UG9386 <10 NT NT NT NT NT 10 NT UG93097 10 NT NT NT NT NT 10
NT UG94118 10 NT NT NT NT NT 20 NT E WR5659 10 <10 <10 <10
20 <10 40 <10 WR5901 <10 <10 <10 40 320 10 40 10
WR8177 <10 <10 <10 40 640 10 80 <10 WR8657 <10
<10 10 10 640 <10 80 <10 WR8593 <10 <10 <10
<10 160 10 40 <10 1008 <10 <10 <10 <10 10 <10
<10 <10 1053 20 <10 <10 <10 40 <10 20 <10 1062
20 10 <10 10 320 <10 20 <10 F BR9318 <10 NT NT NT NT NT
<10 NT BR93019 10 NT NT NT NT NT <10 NT BR93020 20 NT NT NT
NT NT <10 NT BR93029 10 NT NT NT NT NT <10 NT
.sup.aNeutralization titers are the dilutions at which 90%
inhibition of luminescence was observed. .sup.bSera were the
Reference Neutralizing Human Serum 1 and 2, or were provided by Dr.
J. Mascola, HIVNET, or the UNAIDS Program, as described in the
text. .sup.cNT = not tested.
TABLE-US-00006 TABLE 5 Comparison of V3 Region Amino Acid Sequences
of Clone R2 with Phenetic Subgroup Consensus Sequences 1 Through 13
and Clade A Through E Consensus Sequences..sup.a Clone, Subgroup or
Clade V3 Region Amino Acid Sequence R2
NNTR.KSIPMGPGRAFYTTGQIIGDIRQAHC PHENETIC 1 (SEQ ID NO: 6)
----.---HI----------D---------- PHENETIC 2 (SEQ ID NO: 7)
----.---SI-------A--E---------- PHENETIC 3 (SEQ ID NO: 8)
----.---SI-------A--K---------- PHENETIC 4 (SEQ ID NO: 9)
----.---RI---Q---A--D---------- PHENETIC 5 (SEQ ID NO: 10)
----.---HI-------A--K---------- PHENETIC 6 (SEQ ID NO: 11)
K--RRR-H.I---------K----------- PHENETIC 7 (SEQ ID NO: 12)
----.T--TI---QV--R--K---------- PHENETIC 8 (SEQ ID NO: 13)
KKM-.T-ARI----V-HK--K---S-TK-Y- PHENETIC 9 (SEQ ID NO: 14)
----.Q-THI---Q-L---.D---K------ PHENETIC 10 (SEQ ID NO: 15)
----.QGTHI-----Y---.N---------- PHENETIC 11 (SEQ ID NO: 16)
----.QRTSI-Q-QAL---.E-R------A- PHENETIC 12 (SEQ ID NO: 17)
D-IKIQRT-I-Q-Q-L---RITGYI.G---- PHENETIC 13 (SEQ ID NO: 18)
Q-K-.QGT-I-L-Q-L---R.-K----K--- CLADE A (SEQ ID NO: 19)
----.--VHI---Q---A--D---------- CLADE B (SEQ ID NO: 20)
----.---HI----------E---------- CLADE C (SEQ ID NO: 21)
----.---RI---QT-YA--D---------- CLADE D (SEQ ID NO: 22)
----.QRTHI---Q-L---.R---------- CLADE E (SEQ ID NO: 23)
----.T--TI---QV--R--D------K-Y- .sup.aDashes indicate residues at
which the individual sequences are identical to R2. The periods
indicate sites of insertions or deletions.
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Sequence CWU 1
1
241866PRTHuman immunodeficiency virus type 1R2 strain envelope
protein (gp 160) 1Met Arg Val Lys Gly Ile Arg Arg Asn Tyr Gln His
Trp Trp Gly Trp1 5 10 15Gly Thr Met Leu Leu Gly Leu Leu Met Ile Cys
Ser Ala Thr Glu Lys 20 25 30Leu Trp Val Thr Val Tyr Tyr Gly Val Pro
Val Trp Lys Glu Ala Thr 35 40 45Thr Thr Leu Phe Cys Ala Ser Asp Ala
Lys Ala Tyr Asp Thr Glu Ala 50 55 60His Asn Val Trp Ala Thr His Ala
Cys Val Pro Thr Asp Pro Asn Pro65 70 75 80Gln Glu Val Glu Leu Val
Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 90 95Asn Asn Met Val Glu
Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110Gln Ser Leu
Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125Asn
Cys Thr Asp Leu Arg Asn Thr Thr Asn Thr Asn Asn Ser Thr Asp 130 135
140Asn Asn Asn Ser Asn Ser Glu Gly Thr Ile Lys Gly Gly Glu Met
Lys145 150 155 160Asn Cys Ser Phe Asn Ile Ala Thr Ser Ile Gly Asp
Lys Met Gln Lys 165 170 175Glu Tyr Ala Leu Leu Tyr Lys Leu Asp Ile
Glu Pro Ile Asp Asn Asp 180 185 190Asn Thr Ser Tyr Arg Leu Ile Ser
Cys Asn Thr Ser Val Ile Thr Gln 195 200 205Ala Cys Pro Lys Ile Ser
Phe Glu Pro Ile Pro Ile His Tyr Cys Ala 210 215 220Pro Ala Gly Phe
Ala Ile Leu Lys Cys Asn Asp Lys Lys Phe Ser Gly225 230 235 240Lys
Gly Ser Cys Lys Asn Val Ser Thr Val Gln Cys Thr His Gly Ile 245 250
255Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu
260 265 270Glu Glu Val Val Ile Arg Ser Glu Asn Phe Thr Asn Asn Ala
Lys Thr 275 280 285Ile Ile Val Gln Leu Arg Glu Pro Val Lys Ile Asn
Cys Ser Arg Pro 290 295 300Asn Asn Asn Thr Arg Lys Ser Ile Pro Met
Gly Pro Gly Arg Ala Phe305 310 315 320Tyr Thr Thr Gly Gln Ile Ile
Gly Asp Ile Arg Gln Ala His Cys Asn 325 330 335Ile Ser Lys Thr Asn
Trp Thr Asn Ala Leu Lys Gln Val Val Glu Lys 340 345 350Leu Gly Glu
Gln Phe Asn Lys Thr Lys Ile Val Phe Thr Asn Ser Ser 355 360 365Gly
Gly Asp Pro Glu Ile Val Thr His Ser Phe Asn Cys Ala Gly Glu 370 375
380Phe Phe Tyr Cys Asn Thr Thr Gln Leu Phe Asp Ser Ile Trp Asn
Ser385 390 395 400Glu Asn Gly Thr Trp Asn Ile Thr Arg Gly Leu Asn
Asn Thr Gly Arg 405 410 415Asn Asp Thr Ile Thr Leu Pro Cys Arg Ile
Lys Gln Ile Ile Asn Arg 420 425 430Trp Gln Glu Val Gly Lys Ala Met
Tyr Ala Pro Pro Ile Lys Gly Asn 435 440 445Ile Ser Cys Ser Ser Asn
Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly 450 455 460Gly Lys Asp Asp
Asn Ser Arg Asp Gly Asn Glu Thr Phe Arg Pro Gly465 470 475 480Gly
Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys 485 490
495Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg
500 505 510Arg Val Val Gln Arg Glu Glu Arg Ala Val Gly Leu Gly Ala
Met Phe 515 520 525Phe Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly
Ala Ala Ser Val 530 535 540Thr Leu Thr Val Gln Ala Arg Gln Leu Leu
Ser Gly Ile Val Gln Gln545 550 555 560Gln Ser Asn Leu Leu Arg Ala
Ile Glu Ala Gln Gln His Leu Leu Gln 565 570 575Leu Thr Val Trp Gly
Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val 580 585 590Glu Arg Tyr
Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser 595 600 605Gly
Lys Leu Ile Cys Thr Thr Thr Val Pro Trp Asn Ala Ser Trp Ser 610 615
620Lys Asn Lys Thr Leu Glu Ala Ile Trp Asn Asn Met Thr Trp Met
Gln625 630 635 640Trp Asp Lys Glu Ile Asp Asn Tyr Thr Ser Leu Ile
Tyr Ser Leu Ile 645 650 655Glu Glu Ser Pro Ile Gln Gln Glu Lys Asn
Glu Gln Glu Leu Leu Glu 660 665 670Leu Asp Lys Trp Ala Asn Leu Trp
Asn Trp Phe Asp Ile Ser Asn Trp 675 680 685Leu Trp Tyr Ile Lys Ile
Phe Ile Met Ile Val Gly Gly Leu Val Gly 690 695 700Leu Arg Ile Val
Phe Val Val Leu Ser Ile Val Asn Arg Val Arg Gln705 710 715 720Gly
Tyr Ser Pro Leu Ser Phe Gln Thr Arg Leu Pro Ala Pro Arg Gly 725 730
735Pro Asp Arg Pro Glu Glu Ile Glu Glu Glu Gly Gly Asp Arg Asp Arg
740 745 750Asp Arg Ser Gly Leu Leu Val Asp Gly Phe Leu Thr Leu Ile
Trp Val 755 760 765Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr His Arg
Leu Arg Asp Leu 770 775 780Leu Leu Ile Val Thr Arg Ile Val Glu Leu
Leu Gly Arg Arg Gly Trp785 790 795 800Glu Ile Leu Lys Tyr Trp Trp
Asn Leu Leu Gln Tyr Trp Ser Gln Glu 805 810 815Leu Lys Asn Ser Ala
Val Ser Leu Phe Asn Ala Thr Ala Ile Ala Val 820 825 830Ala Glu Gly
Thr Asp Arg Val Ile Gln Val Leu Gln Arg Val Gly Arg 835 840 845Ala
Leu Leu His Ile Pro Thr Arg Ile Arg Gln Gly Leu Glu Arg Ala 850 855
860Leu Leu865217PRTHuman immunodeficiency virus type 1segment of R2
strain V3 domain 2Lys Ser Ile Pro Met Gly Pro Gly Arg Ala Phe Tyr
Thr Thr Gly Gln1 5 10 15Ile335PRTHuman immunodeficiency virus type
1R2 strain V3 domain 3Cys Ser Arg Pro Asn Asn Asn Thr Arg Lys Ser
Ile Pro Met Gly Pro1 5 10 15Gly Arg Ala Phe Tyr Thr Thr Gly Gln Ile
Ile Gly Asp Ile Arg Gln 20 25 30Ala His Cys 35435PRTHuman
immunodeficiency virus type 1V3 domain of strain 93TH966.8 4Cys Thr
Arg Pro Ser Asn Asn Thr Arg Thr Ser Thr Thr Ile Gly Pro1 5 10 15Gly
Gln Val Phe Tyr Arg Thr Gly Asp Ile Thr Gly Asn Ile Arg Lys 20 25
30Ala Tyr Cys 35513PRTArtificial SequenceDescription of Artificial
Sequence derivatives of segment of V3 domain in R2 strain 5Pro Met
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln1 5 10630PRTHuman
immunodeficiency virus type 1sequence of Phenetic 1 in V3 region
6Asn Asn Thr Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr1 5
10 15Thr Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln Ala His Cys 20 25
30730PRTHuman immunodeficiency virus type 1sequence of Phenetic 2
in V3 region 7Asn Asn Thr Arg Lys Ser Ile Ser Ile Gly Pro Gly Arg
Ala Phe Tyr1 5 10 15Ala Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln Ala
His Cys 20 25 30830PRTHuman immunodeficiency virus type 1sequence
of Phenetic 3 in V3 region 8Asn Asn Thr Arg Lys Ser Ile Ser Ile Gly
Pro Gly Arg Ala Phe Tyr1 5 10 15Ala Thr Gly Lys Ile Ile Gly Asp Ile
Arg Gln Ala His Cys 20 25 30930PRTHuman immunodeficiency virus type
1sequence of Phenetic 4 in V3 region 9Asn Asn Thr Arg Lys Ser Ile
Arg Ile Gly Pro Gly Gln Ala Phe Tyr1 5 10 15Ala Thr Gly Asp Ile Ile
Gly Asp Ile Arg Gln Ala His Cys 20 25 301030PRTHuman
immunodeficiency virus type 1sequence of Phenetic 5 in V3 region
10Asn Asn Thr Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr1
5 10 15Ala Thr Gly Lys Ile Ile Gly Asp Ile Arg Gln Ala His Cys 20
25 301130PRTHuman immunodeficiency virus type 1sequence of Phenetic
6 in V3 region 11Lys Asn Thr Arg Arg Arg Ser His Ile Gly Pro Gly
Arg Ala Phe Tyr1 5 10 15Thr Thr Lys Gln Ile Ile Gly Asp Ile Arg Gln
Ala His Cys 20 25 301230PRTHuman immunodeficiency virussequence of
Phenetic 7 in V3 region 12Asn Asn Thr Arg Thr Ser Ile Thr Ile Gly
Pro Gly Gln Val Phe Tyr1 5 10 15Arg Thr Gly Lys Ile Ile Gly Asp Ile
Arg Gln Ala His Cys 20 25 301330PRTHuman immunodeficiency virus
type 1sequence of Phenetic 8 in V3 region 13Lys Lys Met Arg Thr Ser
Ala Arg Ile Gly Pro Gly Arg Val Phe His1 5 10 15Lys Thr Gly Asp Ile
Ile Gly Ser Ile Thr Lys Ala Tyr Cys 20 25 301429PRTHuman
immunodeficiency virus type 1sequence of Phenetic 9 in V3 region
14Asn Asn Thr Arg Gln Ser Thr His Ile Gly Pro Gly Gln Ala Leu Tyr1
5 10 15Thr Thr Asp Ile Ile Gly Lys Ile Arg Gln Ala His Cys 20
251529PRTHuman immunodeficiency virus type 1sequence of Phenetic 10
in V3 region 15Asn Asn Thr Arg Gln Gly Thr His Ile Gly Pro Gly Arg
Ala Tyr Tyr1 5 10 15Thr Thr Asn Ile Ile Gly Asp Ile Arg Gln Ala His
Cys 20 251629PRTHuman immunodeficiency virussequence of Phenetic 11
in V3 region 16Asn Asn Thr Arg Gln Arg Thr Ser Ile Gly Gln Gly Gln
Ala Leu Tyr1 5 10 15Thr Thr Glu Ile Arg Gly Asp Ile Arg Gln Ala Ala
Cys 20 251730PRTHuman immunodeficiency virus type 1sequence of
Phenetic 12 in V3 region 17Asp Asn Ile Lys Ile Gln Arg Thr Pro Ile
Gly Gln Gly Gln Ala Leu1 5 10 15Tyr Thr Thr Arg Ile Thr Gly Tyr Ile
Gly Gln Ala His Cys 20 25 301829PRTHuman immunodeficiency virus
type 1sequence of Phenetic 13 in V3 region 18Gln Asn Lys Arg Gln
Gly Thr Pro Ile Gly Leu Gly Gln Ala Leu Tyr1 5 10 15Thr Thr Arg Ile
Lys Gly Asp Ile Arg Lys Ala His Cys 20 251930PRTHuman
immunodeficiency virus type 1sequence of Clade A in V3 region 19Asn
Asn Thr Arg Lys Ser Val His Ile Gly Pro Gly Gln Ala Phe Tyr1 5 10
15Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln Ala His Cys 20 25
302030PRTHuman immunodeficiency virus type 1sequence of Clade B in
V3 region 20Asn Asn Thr Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala
Phe Tyr1 5 10 15Thr Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln Ala His
Cys 20 25 302130PRTHuman immunodeficiency virus type 1sequence of
Clade C in V3 region 21Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro
Gly Gln Thr Phe Tyr1 5 10 15Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg
Gln Ala His Cys 20 25 302229PRTHuman immunodeficiency virus type
1sequence of Clade D in V3 region 22Asn Asn Thr Arg Gln Arg Thr His
Ile Gly Pro Gly Gln Ala Leu Tyr1 5 10 15Thr Thr Arg Ile Ile Gly Asp
Ile Arg Gln Ala His Cys 20 252330PRTHuman immunodeficiency virus
type 1sequence of Clade E in V3 region 23Asn Asn Thr Arg Thr Ser
Ile Thr Ile Gly Pro Gly Gln Val Phe Tyr1 5 10 15Arg Thr Gly Asp Ile
Ile Gly Asp Ile Arg Lys Ala Tyr Cys 20 25 30242601DNAHuman
immunodeficiency virus type 1 24atgagagtga aggggatcag gaggaattat
cagcactggt ggggatgggg cacgatgctc 60cttgggttat taatgatctg tagtgctaca
gaaaaattgt gggtcacagt ctattatggg 120gtacctgtgt ggaaagaagc
aaccaccact ctattttgtg catcagatgc caaagcatat 180gatacagagg
cacataatgt ttgggccaca catgcctgtg tacccacaga ccccaaccca
240caagaagtag aattggtaaa tgtgacagaa aattttaaca tgtggaaaaa
taacatggta 300gaacagatgc atgaggatat aatcagttta tgggatcaaa
gcctaaagcc atgcgtaaaa 360ttaaccccac tctgtgttac tttaaattgc
actgatttga ggaatactac taataccaat 420aatagtactg ataataacaa
tagtaatagc gagggaacaa taaagggagg agaaatgaaa 480aactgctctt
tcaatatcgc cacaagcata ggagataaga tgcagaaaga atatgcactt
540ctttataaac ttgatataga accaatagat aatgataata ccagctatag
gttgataagt 600tgtaatacct cagtcattac acaagcttgt ccaaagatat
cctttgagcc aattcccata 660cactattgtg ccccggctgg ttttgcgatt
ctaaagtgta acgataaaaa gttcagtgga 720aaaggatcat gtaaaaatgt
cagcacagta caatgtacac atggaattag gccagtagta 780tcaactcaac
tgctgttaaa tggcagtcta gcagaagaag aggtagtaat tagatctgag
840aatttcacaa acaatgctaa aaccataata gtacagctga gagaacctgt
aaaaattaat 900tgttcaagac ccaacaacaa tacaagaaaa agtataccta
tgggaccagg gagagcattt 960tatacaacag gacaaataat aggagatata
agacaagcac attgtaatat tagtaaaaca 1020aattggacta acgctttaaa
acaggtagtt gaaaaactag gggaacaatt caacaagaca 1080aaaatagtct
ttacgaactc ctcaggaggg gacccagaaa ttgtaacgca cagttttaat
1140tgtgcagggg aatttttcta ctgtaataca acacaactgt ttgatagtat
ttggaatagt 1200gagaatggta cttggaatat tactaggggg ttaaataaca
ctggaagaaa tgacacaatc 1260acactcccat gcaggataaa acaaattata
aacaggtggc aggaagtagg aaaagcaatg 1320tatgcccctc ccatcaaagg
aaacattagc tgttcatcaa atattacagg gctgctatta 1380acaagagatg
gtggtaagga tgataatagc agggacggga acgagacctt cagacctgga
1440ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
agtaaaaatt 1500gaaccattag gagtagcacc caccaaggca aagagaagag
tggtgcaaag agaagaaaga 1560gcagtgggac taggagctat gttcattggg
ttcttgggag cagcaggaag cactatgggc 1620gcagcgtcag tgacgctgac
ggtacaggcc aggcaattat tgtctggtat agtgcaacag 1680cagagcaatt
tgctgagagc tattgaggcg caacagcatc tgttgcaact cacagtctgg
1740ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
ggatcaacag 1800ctcctaggga tttggggttg ctctggaaaa ctcatttgca
ccactactgt gccttggaat 1860gctagttgga gtaagaataa aactctggaa
gctatttgga ataacatgac ctggatgcag 1920tgggacaaag agattgacaa
ttacacaagc ttaatatact ccttaattga agaatcgcag 1980atccaacaag
aaaagaatga acaagaatta ttggaattag ataaatgggc aaatctgtgg
2040aattggtttg acatatcaaa ctggctgtgg tatataaaaa tattcataat
gatagtagga 2100ggcttggtag gtttaaggat agtttttgtt gtactttcta
tagtgaatag agttaggcag 2160ggatactcac cattatcgtt tcagacccgc
ctcccagccc cgaggggacc cgacaggccc 2220gaagaaatcg aagaagaagg
tggagacaga gacagagaca gatccgggct cttagtggat 2280ggattcttaa
cacttatctg ggtcgacctg cggagcctgt gcctcttcag ctaccaccgc
2340ttgagagact tactcttgat tgtgacgagg attgtggaac ttctgggacg
cagggggtgg 2400gaaatcctca aatattggtg gaatctcctg cagtattgga
gtcaggaact aaagaatagt 2460gctgttagct tgttcaacgc caccgccata
gcagtagctg agggaacaga tagggttata 2520gaagtattac aaagagttgg
tagagctttg ctccacatac ctacaagaat aagacagggc 2580ttggaaaggg
ctttgctata a 2601
* * * * *