U.S. patent application number 15/457505 was filed with the patent office on 2017-07-06 for peptides mimicking hiv-1 viral epitopes in the v2 loop for the gp120 surface envelope glycoprotein.
The applicant listed for this patent is New York University. Invention is credited to Timothy CARDOZO, Xiangpeng KONG, Susan ZOLLA-PAZNER.
Application Number | 20170190764 15/457505 |
Document ID | / |
Family ID | 47880851 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170190764 |
Kind Code |
A1 |
CARDOZO; Timothy ; et
al. |
July 6, 2017 |
PEPTIDES MIMICKING HIV-1 VIRAL EPITOPES IN THE V2 LOOP FOR THE
GP120 SURFACE ENVELOPE GLYCOPROTEIN
Abstract
The present invention relates to an isolated immunogenic peptide
comprising a V2 loop fragment from HIV surface envelope
glycoprotein gp120. This peptide binds specifically with antibodies
in blood of patients vaccinated with a vaccine that has shown
protection from HIV-1 infection, does not react with blood of
matched patients who did not receive the vaccine, and can,
therefore, elicit anti-HIV-1 antibodies which protect against HIV-1
infection. Other aspects of the present invention relate to an
isolated immunogenic polypeptide comprising the peptide inserted
into an immunogenic scaffold protein, a vaccine composition
comprised of the immunogenic peptide and an immunologically or
pharmaceutically acceptable vehicle or excipient as well as methods
of inducing an immune response against HIV-1 and methods of
detecting HIV-1.
Inventors: |
CARDOZO; Timothy; (New York,
NY) ; KONG; Xiangpeng; (New York, NY) ;
ZOLLA-PAZNER; Susan; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New York University |
New York |
NY |
US |
|
|
Family ID: |
47880851 |
Appl. No.: |
15/457505 |
Filed: |
March 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13612300 |
Sep 12, 2012 |
9611294 |
|
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15457505 |
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61533424 |
Sep 12, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2469/20 20130101;
A61K 2039/545 20130101; A61K 2039/6037 20130101; A61P 31/18
20180101; G01N 2333/162 20130101; C12N 2799/023 20130101; C07K
2317/76 20130101; C12N 7/00 20130101; G01N 33/56988 20130101; C07K
2317/34 20130101; A61K 39/21 20130101; C12N 2740/16134 20130101;
C07K 14/245 20130101; G01N 33/6854 20130101; A61K 39/12 20130101;
C07K 14/005 20130101; C07K 2319/55 20130101; C07K 7/08 20130101;
C07K 7/64 20130101; C07K 14/28 20130101; A61K 38/19 20130101; A61K
2039/64 20130101; C07K 16/1063 20130101; C12N 2740/16122
20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; C07K 14/005 20060101 C07K014/005; C07K 7/64 20060101
C07K007/64; C12N 7/00 20060101 C12N007/00 |
Goverment Interests
[0002] The subject matter of this application was made with support
from the United States National Institutes of Health Grant No.
R01-A1084119. The U.S. government has certain rights in the
invention.
Claims
1.-39. (canceled)
40. An isolated antibody raised against an isolated immunogenic
peptide comprising a V2 loop fragment from HIV surface envelope
glycoprotein gp120 which binds specifically with antibodies in
blood of patients vaccinated with a vaccine that has shown
protection from HIV-1 infection, does not react with blood of
matched patients who did not receive the vaccine, and can,
therefore, elicit anti-HIV-1 antibodies which protect against HIV-1
infection.
41. An isolated antibody raised against an isolated immunogenic
peptide comprising a V2 loop fragment from HIV surface envelope
glycoprotein gp120 which binds specifically with antibodies in
blood of patients vaccinated with a vaccine that has shown
protection from HIV-1 infection, does not react with blood of
matched patients who did not receive the vaccine, and can,
therefore, elicit anti-HIV-1 antibodies which protect against HIV-1
infection and an immunogenic scaffold protein, wherein said peptide
is inserted into said scaffold protein, wherein said polypeptide
has a conformation that is recognized by, and bound by, a broadly
neutralizing anti-HIV-1 antibody.
42. An isolated antibody raised against a cyclized form of an
isolated immunogenic peptide comprising a V2 loop fragment from HIV
surface envelope glycoprotein gp120 which binds specifically with
antibodies in blood of patients vaccinated with a vaccine that has
shown protection from HIV-1 infection, does not react with blood of
matched patients who did not receive the vaccine, and can,
therefore, elicit anti-HIV-1 antibodies which protect against HIV-1
infection.
43. (canceled)
Description
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/612,300, filed Sep. 12, 2012, and claims
the benefit of U.S. Provisional Patent Application Ser. No.
61/533,424, filed Sep. 12, 2011, which is hereby incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a peptide mimicking HIV-1
viral epitopes in the V2 loop for the gp120 surface envelope
glycoprotein.
BACKGROUND OF THE INVENTION
[0004] Human Immunodeficiency Virus-1 (HIV-1) infection has been
reported throughout the world in both developed and developing
countries. HIV-2 infection is found predominately in West Africa,
Portugal, and Brazil. At the end of 2008, an estimated 1,178,350
persons aged 13 and older were living with HIV infection in the
United States. Of those, 20% had undiagnosed HIV infections (CDC,
"HIV Surveillance--United States, 1981-2008," MMWR 60(21); 689-693
(2008), which is hereby incorporated by reference in its
entirety).
[0005] The HIV viruses are members of the Retroviridae family and,
more particularly, are classified within the Lentivirinae
subfamily. Like nearly all other viruses, the replication cycles of
members of the Retroviridae family, commonly known as the
retroviruses, include attachment to specific cell receptors, entry
into cells, synthesis of proteins and nucleic acids, assembly of
progeny virus particles (virions), and release of progeny viruses
from the cells. A unique aspect of retrovirus replication is the
conversion of the single-stranded RNA genome into a double-stranded
DNA molecule that must integrate into the genome of the host cell
prior to the synthesis of viral proteins and nucleic acids.
[0006] HIV encodes a number of genes including three structural
genes--gag, pol, and env--that are common to all retroviruses. The
envelope protein of HIV-1 is a glycoprotein of about 160 kd
(gp160). During virus infection of the host cell, gp160 is cleaved
by host cell proteases to form gp120 and the integral membrane
protein, gp41. The gp41 portion is anchored in the membrane bilayer
of virion, while the gp120 segment protrudes into the surrounding
environment. gp120 and gp41 are more covalently associated, and
free gp120 can be released from the surface of virions and infected
cells. The gp120 polypeptide is also instrumental in mediating
entry into the host cell.
[0007] Historically, viral vaccines have been enormously successful
in the prevention of infection by a particular virus. Therefore,
when HIV was first isolated, there was a great amount of optimism
that an HIV vaccine would be developed quickly. However, this
optimism quickly faded, because a number of unforeseen problems
emerged.
[0008] It is widely thought that a successful vaccine should be
able to induce a strong, broadly neutralizing antibody response
against diverse HIV-1. Neutralizing antibodies, by attaching to the
incoming virions, can reduce or even prevent their infectivity for
target cells and prevent the cell-to-cell spread of virus in
tissue. Conventional wisdom suggests that "constant" rather than
"variable" regions of Env would induce the most broadly reactive
antibodies. The failure of the Vaxgen HIV vaccine trial
demonstrated that the sequence-conserved regions of HIV gp120 do
not induce protective neutralizing antibodies, since these regions
were present in the gp120 immunogen used in that study. The failure
of the STEPS HIV vaccine demonstrated that cellular immunity
utilizing non-Env determinants is not protective. Thus, one could
conclude that targeting the sequence-conserved (including non-Env
and the core of Env) regions of the HIV-1 virus for protective
immunity will not work. Thus, there remains a need for envelope
antigens that can elicit an immunological response in a subject
against multiple HIV strains and subtypes, for example when
administered as a vaccine.
[0009] The present invention is directed to overcoming deficiencies
of prior approaches to addressing HIV infection.
SUMMARY OF THE INVENTION
[0010] The present invention relates to an isolated immunogenic
peptide comprising a V2 loop fragment from HIV surface envelope
glycoprotein gp120. This peptide binds specifically with antibodies
in blood of patients vaccinated with a vaccine that has shown
protection from HIV-1 infection, does not react with blood of
matched patients who did not receive the vaccine, and can,
therefore, elicit anti-HIV-1 antibodies which protect against HIV-1
infection.
[0011] Other aspects of the present invention relate to an isolated
immunogenic polypeptide comprising the peptide inserted into an
immunogenic scaffold protein, a vaccine composition comprised of
the immunogenic peptide and an immunologically or pharmaceutically
acceptable vehicle or excipient as well as methods of inducing an
immune response against HIV-1 and methods of detecting HIV-1.
[0012] The RV144 HIV-1 vaccine trial was the first to demonstrate
evidence of protection against HIV-1 infection, with an estimated
vaccine efficacy of 31.2% (Rerks-Ngarm et al., "Vaccination with
ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand," N Engl J
Med. 361:2209-2220 (2009), which is hereby incorporated by
reference in its entirety). This vaccine consisted of four doses of
a recombinant canary pox priming immunogen, ALVAC-HIV (vCP1521),
and two doses of AIDSVAX.RTM. B/E, recombinant HIV-V gp120 proteins
from HIV-1 subtype B and circulating recombinant form 01AE
(CRF01_AE).
[0013] In order to identify correlates of risk of HIV-1 infection
in RV144, two sequential sets of analyses of plasma specimens from
study participants were conducted (Haynes et al., "Immune
Correlates Analysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy
Trial," N Engl J Med. 366:1275-1286 (2012), which is hereby
incorporated by reference in its entirety). The first was a series
of pilot studies in which 32 types of immunologic assays were
performed on sets of plasma and peripheral blood mononuclear cells
from uninfected participants who had received either the placebo or
the vaccine. Results from the pilot studies were used to select
assays for the subsequent case-control study of immune correlates
of infection risk. Assays for the case-control study were chosen if
the results in the pilot studies showed low false positive rates, a
broad dynamic range, low background reactivity, and low specimen
volume requirements (Haynes et al., "Immune Correlates Analysis of
the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial," N Engl J Med.
366:1275-1286 (2012), which is hereby incorporated by reference in
its entirety). Seventeen assay types were selected for the
case-control study, and these generated results for 158 variables.
To preserve maximal statistical power, six were chosen as primary
variables in the case-control study and were analyzed by
multivariate analysis. To expand the search for immune correlates,
all 158 variables were subsequently evaluated by univariate
analyses.
[0014] Case-control specimens consisted of specimens drawn two
weeks after the last immunization from 41 infected vaccinees
(cases) and from 205 matched uninfected vaccinees (controls). Two
of the six primary variables significantly correlated with HIV-1
infection risk in vaccine recipients: 1) the level of plasma IgG
antibodies reactive with gp70-V1V2, a scaffolded protein carrying
the first and second variable regions of the HIV-1 gp120 envelope
glycoprotein fused to murine leukemia virus gp70. Levels of
antibodies specific for gp70-V1V2 were correlated inversely with
the risk of infection; 2) the level of plasma IgA antibodies
reactive with a panel of 14 envelope glycoproteins correlated
directly with risk of infection.
[0015] The participation of the V2 region of gp120 in the
infectious process, and the role of V2 specific antibodies in
protection from infection has been the subject of investigation and
controversy for nearly two decades. Although, by definition,
"variable" regions--like V2--vary in amino acid sequence, many
residues in these regions do not vary, or tolerate only
conservative changes (Zolla-Pazner et al, "Structure-Function
Relationships of HIV-1 Envelope Sequence-Variable Regions Provide a
Paradigm for Vaccine Design," Nat Rev Immunol 10: 527-535 (2010),
which is hereby incorporated by reference in its entirety). These
conserved amino acids can form structural elements that result in
immunologic cross-reactivity between diverse viruses; for example
many V2- and V3-specific antibodies are highly cross-reactive with
diverse HIV-1 envelopes (Gorny et al., "Repertoire of Neutralizing
Human Monoclonal Antibodies Specific For the V3 Domain of HIV-1
gp120," J Immunol. 150: 635-643 (1993); Israel et al., "Prevalence
of a V2 Epitope in Clade B Primary Isolates and its Recognition by
Sera from HIV-1 Infected Individuals," Aids 11: 128-130 (1997);
Krachmarov et al., "Antibodies That are Cross-Reactive for Human
Immunodeficiency Virus Type 1 Clade A and Clade B V3 Domains are
Common in Patient Sera From Cameroon, but Their Neutralization
Activity is Usually Restricted by Epitope Masking," J Virol. 79:
780-790 (2005); Gorny et al., "Functional and Immunochemical
Cross-Reactivity of V2-Specific Monoclonal Antibodies From Human
Immunodeficiency Virus Type 1-Infected Individuals," Virology 427:
198-207 (2012); Nyambi et al., "Immunoreactivity of Intact Virions
of Human Immunodeficiency Virus Type 1 (HIV-1) Reveals the
Existence of Fewer HIV-1 Immunotypes Than Genotypes," J Virol 74:
10670-10680 (2000); Hioe et al., "Anti-V3 Monoclonal Antibodies
Display Broad Neutralizing Activities Against Multiple HIV-1
Subtypes," PLoS ONE 5: e10254 (2010), each of which is hereby
incorporated by reference in its entirety). Moreover, the conserved
structural features are required for these regions to perform
important biologic functions. Thus, for example, conserved elements
within V2 participate in the formation of the bridging sheet (a
constituent of the chemokine receptor binding site (Thali et al.,
"Characterization of Conserved HIV-type 1 gp120 Neutralization
Epitopes Exposed Upon gp120-CD4 Binding," J Virol 67: 3978-3988
(1993); Rizzuto et al., "A Conserved HIV gp120 Glycoprotein
Structure Involved in Chemokine Receptor Binding," Science 280:
1949-1953 (1998); Kwong et al. "Structure of an HIV gp120 Envelope
Glycoprotein in Complex With the CD4 Receptor and a Neutralizing
Human Antibody," Nature 393: 648-659 (1998), each of which is
hereby incorporated by reference in its entirety), and V2 contains
a tripeptide motif in the mid-loop region of V2 that is a putative
.alpha.4.beta.7 integrin binding site (Arthos et al., "HIV-1
Envelope Protein Binds to and Signals Through lintegrin
alpha4beta7, the Gut Mucosal Homing Receptor for Peripheral T
cells," Nat Immunol 9: 301-309 (2008), which is hereby incorporated
by reference in its entirety). Similarly, conserved elements of V3
contribute to its role in binding to the chemokine receptor (Trkola
et al., "CD4-Dependent, Antibody-Sensitive Interactions Between
HIV-1 and its Co-Receptor CCR-5," Nature 384: 184-187 (1996); Hill
et al., "Envelope Glycoproteins From HIV-1, HIV-2 and SIV Can Use
Human CCR5 as a Coreceptor for Viral Entry and Make Direct
CD4-Dependent Interactions With This Chemokine Receptor," J Virol
71: 6296-6304 (1997), each of which is hereby incorporated by
reference in its entirety).
[0016] Antibodies specific for V2 occur in only .about.25-40% of
HIV-infected individuals (Israel et al., "Prevalence of a V2
Epitope in Clade B Primary Isolates and its Recognition by Sera
from HIV-1 Infected Individuals," Aids 11: 128-130 (1997); Kayman
et al., "Presentation of Native Epitopes in the V1/V2 and V3
Regions of Human Immunodeficiency Virus Type 1 gp120 by Fusion
Glycoproteins Containing Isolated gp120 Domains," J Virol. 68:
400-410 (1994), each of which is hereby incorporated by reference
in its entirety). Interestingly, the cross-reactivity of these
antibodies does not require extensive mutation from the VH germ
line since V2-specific monoclonal antibodies from HIV-infected
individuals display a mean 6.2% mutation frequency from germ line
(Gorny et al., "Functional and Immunochemical Cross-Reactivity of
V2-Specific Monoclonal Antibodies From Human Immunodeficiency Virus
Type 1-Infected Individuals," Virology 427: 198-207 (2012), which
is hereby incorporated by reference in its entirety) which is
comparable to a mean 6.8% mutation frequency found in Abs from
normal individuals (Tiller et al., "Autoreactivity in Human IgG+
Memory B Cells," Immunity 26: 205-213 (2007), which is hereby
incorporated by reference in its entirety). Thus, because a large
body of data suggests that V2 may be a site of HIV-1 vulnerability,
and because a strong antibody response to gp70-V1V2 was correlated
with reduced infection in the RV144 clinical vaccine trial, a
thorough analysis of all V2 antibody assays used in the RV144
immune correlates study was undertaken, as disclosed here.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the ELISA reactivity of human anti-V2
monoclonal antibodies 697-D and 2158 with AIDSVAX subtype E and
AIDSVAX subtype B gp120 immunogens. The dashed line represents
twice background levels.
[0018] FIG. 2 shows ELISA reactivity with gp70-V1V2 of plasma
specimens used in the pilot studies of the RV144 clinical vaccine
trial (Set C). The results from one of three experiments are shown.
The open and filled blue diamonds depict negative responses at
weeks 0 and 26, respectively. The open and closed red circles
depict positive responses at weeks 0 and 26, respectively. Each
vertical line connects a single patient's specimen drawn at Week 0
and Week 26. The specimens are ordered by the difference in
reactivity between the Week 0 and Week 26 specimens, with the
biggest increasers on the right. Plasma were tested at a final
dilution of 1:100, and a positive response was defined as being
>0.276, the cut-off OD value which was defined as the mean+3
standard deviations based on values derived from vaccinees at week
0 (the pre-immunization time point).
[0019] FIG. 3 shows Spearman rank correlations between the V2
assays run in the case-control study.
[0020] FIG. 4 shows ELISA reactivity of anti-V2 IgG antibodies in
plasma from vaccine recipients in the RV144 pilot study (Set L)
against three different V2 antigens (gp70-V1V2, V2 cyclic peptide
from clade E strain 92TH023, and the K178 V2 peptide) run in
parallel in the same assay at a plasma dilution of 1:50. Data shown
are from one of two experiments. Plasma from placebo recipients
were negative. Statistical comparisons between groups for positive
responders were performed using Student's t-test.
[0021] FIGS. 5A-B show boxplots showing ELISA reactivity of V2
peptides with plasma (diluted 1:100) from 80 vaccinees' specimens
from the pilot study (Set C). The distributions of the reactivities
are shown where the left edge of each box equals the 25th
percentile; the vertical line in each box is the 50th percentile,
and the right edge of each box equals the 75th percentile. The
boxplots were prepared using the scientific graphing program,
GraphPad Prism, with "whiskers" showing the minimum and maximum
responses. FIG. 5A shows four 21-mer N-terminal biotinylated
peptides (Peptides 1-4 (SEQ ID NOS: 3 to 6)) were selected on the
basis of a bioinformatics analysis of V2 sequences from the LANL
HIV Database. FIG. 5B shows a second peptide panel designed upon
inspection of the amino acids in Peptides 1-4 (SEQ ID NOS: 3 to 6)
in FIG. 5A revealed amino acids that distinguish Peptides 1 (SEQ ID
NO: 3) and 3 (SEQ ID NO: 5) from 2 (SEQ ID NO: 4) and 4 (SEQ ID NO:
6); these appear at positions 165, 169, 172, and 174. To maximally
enhance the availability of the epitopes on the peptides used in
the fine mapping of the V2 antibodies, a spacer of three glycines
was inserted between the biotin tag at the N-terminus of the
peptide and the V2 sequences.
[0022] FIG. 6 shows a ribbon diagram of the backbone fold of the
V1V2 domain bound to the CDR H3 loop of monoclonal antibody PG9
(transparent stippled spheres are the atoms of the PG9 CDR H3). The
ribbon backbones of amino acids 165-176 are identical to Peptide 6
(SEQ ID NO: 7), is labeled C. The strands are labeled A-D according
to the convention established recently (McLellan et al., "Structure
of HIV-1 gp120 V1/V2 Domain With Broadly Neutralizing Antibody
PG9," Nature 480:336-343 (2011), which is hereby incorporated by
reference in its entirety).
[0023] FIG. 7 shows estimated odds ratios (ORs) and 95% confidence
intervals for each of the V2 assays. Data are derived from the
categorical analyses shown in Table 2. Estimated ORs compare
subgroups defined by high vs. low responses except for comparisons
for analyses of IgA V2 A244 K178 and V2 MN which compare positive
vs. negative responses. For evaluation of biotinylated V2 peptide 6
(SEQ ID NO: 7), comparison is between high and negative
responses.
[0024] FIGS. 8A-B show microarray analysis of the V2 antibody
response in plasma from vaccinees in the case-control study. FIG.
8A shows an aggregate response across all sub-types averaged across
vaccinees as a function of HxB2 positions. An individual aggregate
response is computed using a sliding window mean statistic of 9
amino acids, i.e., peptides with HxB2 positions of 9 contiguous
amino acids averaged together. In FIG. 8B, the actual sequence of
each of the overlapping 15-mers (SEQ ID NOS: 17 to 65) spanning V2
positions 160-183 is shown. The seven sets of peptides represent
the consensus V2 regions of HIV-1 Group M and of subtypes A, B, C,
D, CRF01_AE and CRF02_AG. Peptides are shaded according to their
average response across all vaccinees, with a scale of 0 (white) to
1.8 (black). The V2 sequence of HxB2 is shown above the graph with
the corresponding HxB2 numbering, and this numbering is also shown
on the x-axis. The bold arrow indicates that the estimated
aggregate response is highest when centered at position 173, though
peptides centered on position 170, i.e., with the V2 peptide
spanning residues 163-177 have the highest response. All responses
are calculated using normalized intensities and by subtracting the
intensities of baseline pre-bleeds.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention relates to an isolated immunogenic
peptide comprising a V2 loop fragment from HIV surface envelope
glycoprotein gp120. This peptide binds specifically with antibodies
in blood of patients vaccinated with a vaccine that has shown
protection from HIV-1 infection, does not react with blood of
matched patients who did not receive the vaccine, and can,
therefore, elicit anti-HIV-1 antibodies which protect against HIV-1
infection.
[0026] In accordance with this aspect of the present invention,
suitable isolated immunogenic peptides include peptides of the
amino acid sequence
X.sub.1X.sub.2DX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7YX.sub.8X.sub.9X.sub.10-
X.sub.11X.sub.12 (SEQ ID NO: 1), where X.sub.1 is L, V, I, M, F, W
or A; X.sub.2 is any amino acid; X.sub.3 is R, K or H; X.sub.4 is
K, D, E, R, H, S, T, C, N, Q, Y, A, V or M; X.sub.5 is K, D, E, R,
H, S, T, C, N, Q, Y or A; X.sub.6 is K, D, E, R, H, S, T, C, N, Q,
Y or A; X.sub.7 is L, V, I, M, F, W, A or E; X.sub.8 is K, D, E, R,
H, S, T, C, N, Q, Y or A; X.sub.9 is L, V, I, M, F, W or A;
X.sub.10 is F or T, X.sub.11 is K, D, E, R, H, S, T, C, N, Q, Y or
A; X.sub.12 is K, D, E, R, H, S, T, C, N, Q, Y or A. Examples of
specific immunogenic peptides in accordance with the present
invention include the following amino acid sequences:
LRDKKQRVYSLFYK (SEQ ID NO: 7), IRDKKQRVYSLFYK (SEQ ID NO: 11),
LRDKVQRVYSLFYK (SEQ ID NO: 12), LRDKKQREYSLFYK (SEQ ID NO: 13),
LRDKKQRVYALFYK (SEQ ID NO: 14), or LQNKKQQVYSLFYQ (SEQ ID NO:
15).
[0027] Another aspect of the present invention is an isolated
immunogenic peptide including the amino acid sequence of SEQ ID NO:
2. In accordance with this aspect of the present invention,
suitable isolated immunogenic peptides include peptides of the
amino acid sequence of SEQ ID NO: 1 with X.sub.11 as any amino acid
other than Y. In particular, SEQ ID NO: 2 has the following amino
acid sequence:
X.sub.1X.sub.2DX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7YX.sub.8X.sub.9X.sub.10-
X.sub.11X.sub.12, where X.sub.1 is L, V, I, M, F, W or A; X.sub.2
is any amino acid; X.sub.3 is R, K or H; X.sub.4 is K, D, E, R, H,
S, T, C, N, Q, Y, A, V or M; X.sub.5 is K, D, E, R, H, S, T, C, N,
Q, Y or A; X.sub.6 is K, D, E, R, H, S, T, C, N, Q, Y or A; X.sub.7
is L, V, I, M, F, W, A or E; X.sub.8 is K, D, E, R, H, S, T, C, N,
Q, Y or A; X.sub.9 is L, V, I, M, F, W or A; X.sub.10 is F or T,
X.sub.11 is any amino acid other than Y; X.sub.12 is K, D, E, R, H,
S, T, C, N, Q, Y or A.
[0028] In another aspect of the present invention, the isolated
immunogenic peptide comprises the amino acid sequence of
LRDKMQKVYALTYK (SEQ ID NO: 16). This is a sequence that does not
occur in nature and has a mutation, relative to the amino acid
sequence of SEQ ID: 1, which requires Y at position X.sub.11. This
destroys a cathepsin protease cleavage site.
[0029] The present invention also relates to an isolated
immunogenic polypeptide of the present invention comprising the
isolated immunogenic peptide described above and an immunogenic
scaffold protein. The polypeptide has a conformation that is
recognized by, and bound by, a broadly neutralizing anti-HIV-1
antibody.
[0030] As used herein, a "broadly neutralizing" antibody or
antibody response is an antibody or response that results in
binding and neutralization of at least one group of heterologous
HIV-1 viruses that are members of a different subtype or clade than
that of the source of the immunizing antigen. The scaffold protein
can be one that is highly immunogenic and capable of enhancing the
immunogenicity of any heterologous sequences fused to/inserted in
it. Suitable scaffold proteins include, without limitation, a
cholera toxin and an enterotoxin.
[0031] In one embodiment of the present invention, the scaffold
protein is cholera toxin subunit B (CTB). CTB is highly immunogenic
and has been used in fusion constructs to enhance immunogenicity of
its fusion partner polypeptide or peptide (McKenzie et al.,
"Cholera Toxin B Subunit as a Carrier to Stimulate a Mucosal Immune
Response," J Immunol. 133:1818-1824 (1984); Czerkinsky et al.,
"Oral Administration of a Streptococcal Antigen Coupled to Cholera
Toxin B Subunit Evokes Strong Antibody Responses in Salivary Glands
and Extramucosal Tissues," Infect Immun. 57:1072-1077 (1989), each
of which is hereby incorporated by reference in its entirety). CTB
has also been described as a mucosal adjuvant for vaccines (Areas
et al., "Expression and Characterization of Cholera Toxin
B-Pneumococcal Surface Adhesin A Fusion Protein in Escherichia
Coli: Ability of CTB-PsaA to Induce Humoral Immune Response in
Mice," Biochem Biophys Res Commun. 321:192-196 (2004), which is
hereby incorporated by reference in its entirety).
[0032] In another embodiment of the present invention, the scaffold
protein is an E. coli enterotoxin, preferably heat labile
entertoxin. This protein is also highly immunogenic and has been
used in fusion constructs to enhance immunogenicity of its fusion
partner polypeptide or peptide (Lipscombe et al., "Intranasal
Immunization Using the B Subunit of the Escherichia Coli Heat
Labile Toxin Fused to an Epitope of the Bordetella Pertussis P.69
Antigen," Mol Microbiol. 5:1385-1392 (1991), which is hereby
incorporated by reference in its entirety).
[0033] An important factor for the immunogenic property of CTB and
heat labile enterotoxin is their binding to GM1 ganglioside, which
is present on the surface of mucosal cells. This results in its
propensity to induce mucosal immunity and is highly desirable for
an HIV immunogen or vaccine, because infection commonly occurs via
a mucosal route. In addition, the availability of structural
information of these proteins allows protein design that avoids or
minimizes disruption of the GM1 binding site, thereby preserving
the inherent immunogenicity of these polypeptides.
[0034] In accordance with this aspect of the present invention, the
immunogenic peptide can be inserted directly into the scaffold's
tertiary structure. This yields a polypeptide in which an
exceptionally high fraction of the molecular surface presents V2
epitopes that are recognized by broadly-reactive neutralizing
anti-gp120 antibodies and can elicit anti-HIV-1 antibody responses
that preferably are broadly-reactive and neutralize the virus.
Molecular modeling is used to test in-silico, whether various
insertion positions in the scaffold and different loop lengths
result in loop conformations that present the epitopes.
Specifically, there are two approaches. Firstly, the scaffold is
scanned for amino-acid positions that can be superimposed on the
termini of the loop as observed in the V2/antibody complex. When
superposition within small tolerances (<0.5 .ANG. root mean
square deviation (RMSD) for the terminal residues is achieved, the
model is evaluated for the absence of clashes with the scaffold
structure. Secondly, the loop is inserted in a random conformation
and subjected to conformational sampling. Low energy conformations
generated during sampling are compared to the desired V2
conformation as observed in the V2/antibody complex. Sampling is
over a restricted energy range. When the construct is such that
conformations within 1.0 .ANG. backbone RMSD of the desired V2
conformation are identified in the simulation, a model of the
immunogen-antibody complex is built to ensure that the scaffold
does not interfere with the V2 loop/antibody binding.
[0035] The isolated immunogenic peptides described above may also
exist in a cyclized form. These cyclic peptides can be synthesized
and include two cysteine residues that bond via a disulfide linkage
forming the cyclic peptide. Alternatively, the peptide may be
cyclized by chemical means without relying upon disulfide bonding
of two cysteine residues, for example, by introduction of a
linker.
[0036] The cyclic peptide compositions of the present invention may
be synthesized using ordinary skill in the art of organic synthesis
and peptide synthesis. Methods for restricting the secondary
structure of peptides and proteins are highly desirable for the
rational design of therapeutically useful
conformationally-restricted (or "locked") pharmacophores. The
purely chemical approaches for restricting secondary structure
often require extensive multistep syntheses (Olson, G. L., J. Am.
Chem. Soc. 112:323 (1990)). An alternative approach involves
installing covalent bridges in peptides. However, due to the
sensitivity of the peptide backbone and side chains, this method
necessitates careful protection/deprotection strategies.
[0037] The general guiding principles determining the design of
useful cyclic peptides are well-known in the art and are dictated
by the need to maintain the antibody reactivity and immunogenicity
of the V2 peptide, particularly for induction of broadly reactive,
neutralizing antibodies while enhancing its stability as well as
the ability to be inserted into a desired scaffold protein without
disrupting the "function" of the latter, i.e., immunogenicity and
other binding characteristics of the scaffold such as the binding
of recombinant V2-CTB to the glycolipid targets of CTB. In addition
to testing a cyclic peptide serologically, it may be analyzed more
extensively by structural (biophysical) techniques, such as NMR
spectroscopy or X-ray crystallographic methods, in solution or when
bound to a characterizing broadly-reactive neutralizing monoclonal
antibody such as 447-52D (see publication WO04/069863, which is
hereby incorporated by reference in its entirety)
[0038] The one or more peptides in the present invention can be
synthesized by solid phase or solution phase peptide synthesis,
recombinant expression, or can be obtained from natural sources.
Automatic peptide synthesizers are commercially available from
numerous suppliers, such as Applied Biosystems, Foster City, Calif.
Standard techniques of chemical peptide synthesis are well known in
the art (see e.g., SYNTHETIC PEPTIDES: A USERS GUIDE 93-210
(Gregory A. Grant ed., 1992), which is hereby incorporated by
reference in its entirety). Peptide production via recombinant
expression can be carried out using bacteria, such as E. coli,
yeast, insect cells or mammalian cells and expression systems.
Procedures for recombinant protein/peptide expression are well
known in the art and are described by Sambrook et al., Molecular
Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed., 1989),
which is hereby incorporated by reference in its entirety.
[0039] Recombinantly expressed peptides can be purified using any
one of several methods readily known in the art, including ion
exchange chromatography, hydrophobic interaction chromatography,
affinity chromatography, gel filtration, and reverse phase
chromatography. The peptide is preferably produced in purified form
(preferably at least about 80% to 85% pure, more preferably at
least about 90% or 95% pure) by conventional techniques. Depending
on whether the recombinant host cell is made to secrete the peptide
into growth medium (see U.S. Pat. No. 6,596,509 to Bauer et al.,
which is hereby incorporated by reference in its entirety), the
peptide can be isolated and purified by centrifugation (to separate
cellular components from supernatant containing the secreted
peptide) followed by sequential ammonium sulfate precipitation of
the supernatant. The fraction containing the peptide is subjected
to gel filtration in an appropriately sized dextran or
polyacrylamide column to separate the peptides from other proteins.
If necessary, the peptide fraction may be further purified by
HPLC.
[0040] Another aspect of the present invention is directed to an
immunogenic vaccine composition comprising the linear or cyclized
isolated immunogenic peptides or polypeptides described above, and
an immunologically and pharmaceutically acceptable vehicle or
excipient.
[0041] Suitable vehicles and excipients are described in
REMINGTON'S PHARMACEUTICAL SCIENCE (19th ed., 1995), which is
hereby incorporated by reference in its entirety. The incorporation
of such immunologically and pharmaceutically acceptable components
depends on the intended mode of administration and therapeutic
application of the pharmaceutical composition. Typically, however,
the vaccine composition will include a pharmaceutically-acceptable,
non-toxic carrier or diluent, which are defined as vehicles
commonly used to formulate pharmaceutical compositions for animal
or human administration. The diluent is selected so as not to
affect the biological activity of the composition. Exemplary
carriers or diluents include distilled water, physiological
phosphate-buffered saline, Ringer's solutions, dextrose solution,
and Hank's solution.
[0042] Vaccine compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized sepharose, agarose, cellulose),
polymeric amino acids, amino acid copolymers, and lipid aggregates
(such as oil droplets or liposomes).
[0043] The vaccine composition of the present invention may also be
supplemented with an immunostimulatory cytokine. Preferred
cytokines are GM-CSF (granulocyte-macrophage colony stimulating
factor), interleukin 1, interleukin 2, interleukin 12, interleukin
18, or interferon-gamma.
[0044] The vaccine composition of the present invention can further
contain an adjuvant. One class of preferred adjuvants is aluminum
salts, such as aluminum hydroxide, aluminum phosphate, or aluminum
sulfate. Such adjuvants can be used with or without other specific
immunostimulating agents such as MPL or 3-DMP, QS-21, flagellin,
polymeric or monomeric amino acids such as polyglutamic acid or
polylysine, or pluronic polyols. Oil-in-water emulsion formulations
are also suitable adjuvants that can be used with or without other
specific immunostimulating agents such as muramyl peptides (e.g.,
N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-
-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy
propylamide (DTP-DPP) Theramide.RTM., or other bacterial cell wall
components). A suitable oil-in-water emulsion is MF59.RTM.
(containing 5% Squalene, 0.5% Tween.RTM. 80, and 0.5% Span.RTM. 85
(optionally containing various amounts of MTP-PE) formulated into
submicron particles using a microfluidizer such as Model 110Y
Microfluidizer.RTM. (Microfluidics, Newton Mass.) as described in
WO90/14837 to Van Nest et al., which is hereby incorporated by
reference in its entirety. Other suitable oil-in-water emulsions
include SAF (containing 10% Squalene, 0.4% Tween.RTM. 80, 5%
Pluronic.RTM.-blocked polymer L121, and thr-MDP, either
microfluidized into a submicron emulsion or vortexed to generate a
larger particle size emulsion) and Ribiadjuvant system (RAS;
containing 2% squalene, 0.2% Tween.RTM. 80, and one or more
bacterial cell wall components). Another class of suitable
adjuvants are saponin adjuvants, such as Stimulon.RTM. (QS-21) or
particles generated therefrom such as ISCOMs (immunostimulating
complexes) and ISCOMATRIX.TM.. Other suitable adjuvants include
incomplete or complete Freund's Adjuvant (IFA). Such adjuvants are
generally available from commercial sources.
[0045] Another aspect of the present invention relates to a method
of inducing a broadly neutralizing antibody response against a V2
epitope of HIV-1 gp120 in a subject. This method comprises
administering to the subject the immunogenic peptide, cyclized
peptide, or polypeptides, described above, under conditions
effective to induce, in the subject, a neutralizing antibody
response against the V2 epitope of the HIV-1 gp120. In a preferred
embodiment of this aspect, the selected subject is HIV-1
positive.
[0046] In accordance with this aspect of the present invention, a
neutralizing antibody response is an antibody or response that
results in binding and neutralization of at least one group of
heterologous HIV-1 viruses that are members of a different subtype
or clade than that of the source of the immunizing antigen. Such a
response is an active response induced by administration of the
immunogenic peptide or polypeptide and represents a means for
vaccination against HIV-1.
[0047] An MT-2 assay can be performed to measure neutralizing
antibody responses. Antibody-mediated neutralization of selected
strains or isolates of HIV-1 can be measured in an MT-2
cell-killing assay (Montefiori et al., "Evaluation of Antiviral
Drugs and Neutralizing Antibodies to Human Immunodeficiency Virus
By a Rapid and Sensitive Microtiter Infection Assay," J Clin
Microbiol. 26(2):2310-235 (1988), which is hereby incorporated by
reference in its entirety). HIV-1.sub.IIIB and HIV-1.sub.MN induce
the formation of syncytia in MT-2 cells. The inhibition of the
formation of syncytia by the sera shows the activity of
neutralizing antibodies present within the sera, induced by
vaccination. Immunized test and control sera can be exposed to
virus-infected cells (e.g. cells of the MT-2 cell line).
Neutralization can be measured by any method that determines viable
cells, such as staining, e.g., with Finter's neutral red.
Percentage protection can be determined by calculating the
difference in absorption between test wells (cells+virus) and
dividing this result by the difference in absorption between cell
control wells (cells only) and virus control wells (virus only).
Neutralizing titers may be expressed, for example, as the
reciprocal of the plasma dilution required to protect at least 50%
of cells from virus-induced killing.
[0048] Another aspect of the present invention relates to a method
of inducing a protective and non-neutralizing antibody response
against a V2 epitope of HIV-1 gp120 in a subject. This method
comprises administering to the subject the immunogenic peptide,
cyclized peptide, or polypeptides, as described above, under
conditions effective to induce, in the subject, a protective,
non-neutralizing antibody response against the V2 epitope of the
HIV-1 gp120. In a preferred embodiment of this aspect, the selected
subject is HIV-1 positive.
[0049] In accordance with this aspect of the present invention,
non-neutralizing antibodies will not impair virus entry into cells.
However, a non-neutralizing antibody response will trigger
antibody-dependent cell-mediated viral inhibition (ADCVI), which
may be effective against HIV-1 (Asmal et al., "Antibody Dependent
Cell Mediated Viral Inhibition Emerges After Simian
Immunodeficiency Virus SIVmac251 Infection of Rhesus Monkeys
Coincident With gp140-Binding Antibodies and is Effective Against
Neutralization Resistant Viruses," J Virol. 85(11) 5465-5475
(2011), which is hereby incorporated by reference in its
entirety).
[0050] ADCVI can be measured by infecting polybrene-treated
CEM.NKR-CCR5 cells (National Institutes of Health AIDS Research and
Reference Program) with a clinical strain of HIV-1
(HIV-1.sub.92US657). The addition of effector cells from healthy
donors and sera from vaccine subjects to the infected target cells
can lead to ADCVI. Levels of p24, as determined by ELISA can
determine the presence of ADCVI (Forthal et al., "Recombinant gp120
Vaccine-Induced Antibodies Inhibit Clinical Strains of HIV-1 in the
Presence of Fc Receptor Bearing Effector Cells and Correlate
Inversely with HIV Infection Rate," J of Immunol. 178(10):
6596-6603 (2007), which is hereby incorporated by reference in its
entirety).
[0051] Another aspect of the present invention relates to a method
of inducing protective antibodies against a V2 epitope of HIV-1
gp120 in a subject. This method comprises administering to the
subject the immunogenic peptide, cyclized peptide, or polypeptide,
as described above, under conditions effective to induce, in the
subject, a protective antibody response against the V2 epitope of
the HIV-1 gp120. In one embodiment, the selected subject is HIV-1
positive.
[0052] The presence of a protective humoral immunological response
can be determined and monitored by testing a biological sample
(e.g., blood, plasma, serum, urine, saliva, feces, CSF or lymph
fluid) from the subject for the presence of antibodies directed to
the immunogenic component of the administered pharmaceutical
composition.
[0053] The immunization protocol preferably includes at least one
priming dose, followed by one or multiple boosting doses
administered over time. An exemplary range for an immunogenically
effective amount of the present immunogenic polypeptides is about 5
to about 500 .mu.g/kg body weight. A preferred range is about
10-100 .mu.g/kg.
[0054] Compositions of the present invention can be administered by
parenteral, topical, intravenous, oral, subcutaneous,
intraperitoneal, intranasal, or intramuscular means. The most
typical route of administration for compositions formulated to
induce an immune response is subcutaneous, although others can be
equally effective. The next most common is intramuscular injection.
This type of injection is most typically performed in the arm or
leg muscles. Intravenous injections as well as intraperitoneal
injections, intra-arterial, intracranial, or intradermal injections
are also effective in generating an immune response.
[0055] The compositions of the present invention may be formulated
for parenteral administration. Solutions or suspensions of the
agent can be prepared in water suitably mixed with a surfactant
such as hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof in
oils. Illustrative oils are those of petroleum, animal, vegetable,
or synthetic origin, for example, peanut oil, soybean oil, or
mineral oil. In general, water, saline, aqueous dextrose and
related sugar solution, and glycols, such as propylene glycol or
polyethylene glycol, are preferred liquid carriers, particularly
for injectable solutions. Under ordinary conditions of storage and
use, these preparations contain a preservative to prevent the
growth of microorganisms.
[0056] Vaccine formulations suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0057] When it is desirable to deliver the vaccine of the present
invention systemically, it may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing,
and/or dispersing agents.
[0058] Intraperitoneal or intrathecal administration of the agents
of the present invention can also be achieved using infusion pump
devices such as those described by Medtronic, Northridge, Calif.
Such devices allow continuous infusion of desired compounds
avoiding multiple injections and multiple manipulations.
[0059] In addition to the formulations described previously, the
agents may also be formulated as a depot preparation. Such long
acting formulations may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0060] The present invention is further directed to an isolated
antibody raised against the immunogenic peptide or polypeptide of
the present invention. The isolated antibody of the present
invention encompasses any immunoglobulin molecule that specifically
binds the V2 epitope of HIV-1 gp120. As used herein, the term
"antibody" is meant to include intact immunoglobulins derived from
natural sources or from recombinant sources, as well as
immunoreactive portions (i.e., antigen binding portions) of intact
immunoglobulins. The antibodies of the present invention may exist
in a variety of forms including, for example, polyclonal
antibodies, monoclonal antibodies, intracellular antibodies
("intrabodies"), antibody fragments (e.g. Fv, Fab and F(ab)2), as
well as single chain antibodies (scFv), chimeric antibodies and
humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A
LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999);
Houston et al., "Protein Engineering of Antibody Binding Sites:
Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv
Analogue Produced in Escherichia coli," Proc Natl Acad Sci USA
85:5879-5883 (1988); Bird et al, "Single-Chain Antigen-Binding
Proteins," Science 242:423-426 (1988), each of which is hereby
incorporated by reference in its entirety).
[0061] Antibodies of the present invention may also be synthetic
antibodies. A synthetic antibody is an antibody which is generated
using recombinant DNA technology, such as, for example, an antibody
expressed by a bacteriophage. Alternatively, the synthetic antibody
is generated by the synthesis of a DNA molecule encoding and
expressing the antibody of the present invention or the synthesis
of an amino acid specifying the antibody, where the DNA or amino
acid sequence has been obtained using synthetic DNA or amino acid
sequence technology which is available and well known in the
art.
[0062] Methods for monoclonal antibody production may be carried
out using the techniques described herein or other well-known in
the art (MONOCLONAL ANTIBODIES--PRODUCTION, ENGINEERING AND
CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds.,
1995), which is hereby incorporated by reference in its entirety).
Generally, the process involves obtaining immune cells
(lymphocytes) from the spleen of a mammal which has been previously
immunized with the antigen of interest (i.e., a polymerized first
or second peptide or fusion peptide) either in vivo or in
vitro.
[0063] The antibody-secreting lymphocytes are then fused with
myeloma cells or transformed cells, which are capable of
replicating indefinitely in cell culture, thereby producing an
immortal, immunoglobulin-secreting cell line. Fusion with mammalian
myeloma cells or other fusion partners capable of replicating
indefinitely in cell culture is achieved by standard and well-known
techniques, for example, by using polyethylene glycol (PEG) or
other fusing agents (Milstein and Kohler, "Derivation of Specific
Antibody-Producing Tissue Culture and Tumor Lines by Cell Fusion,"
Eur J Immunol 6:511 (1976), which is hereby incorporated by
reference in its entirety). The immortal cell line, which is
preferably murine, but may also be derived from cells of other
mammalian species, is selected to be deficient in enzymes necessary
for the utilization of certain nutrients, to be capable of rapid
growth, and have good fusion capability. The resulting fused cells,
or hybridomas, are cultured, and the resulting colonies screened
for the production of the desired monoclonal antibodies. Colonies
producing such antibodies are cloned, and grown either in vivo or
in vitro to produce large quantities of antibody.
[0064] Alternatively monoclonal antibodies can be made using
recombinant DNA methods as described in U.S. Pat. No. 4,816,567 to
Cabilly et al, which is hereby incorporated by reference in its
entirety. The polynucleotides encoding a monoclonal antibody are
isolated from mature B-cells or hybridoma cells, for example, by
RT-PCR using oligonucleotide primers that specifically amplify the
genes encoding the heavy and light chains of the antibody. The
isolated polynucleotides encoding the heavy and light chains are
then cloned into suitable expression vectors, which when
transfected into host cells such as E. coli cells, simian COS
cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, monoclonal antibodies
are generated by the host cells. Also, recombinant monoclonal
antibodies or fragments thereof of the desired species can be
isolated from phage display libraries (McCafferty et al., "Phage
Antibodies: Filamentous Phage Displaying Antibody Variable
Domains," Nature 348:552-554 (1990); Clackson et al., "Making
Antibody Fragments using Phage Display Libraries," Nature
352:624-628 (1991); and Marks et al., "By-Passing Immunization.
Human Antibodies from V-Gene Libraries Displayed on Phage," J. Mol.
Biol. 222:581-597 (1991), which are hereby incorporated by
reference in their entirety).
[0065] The polynucleotide(s) encoding a monoclonal antibody can
further be modified using recombinant DNA technology to generate
alternative antibodies. For example, the constant domains of the
light and heavy chains of a mouse monoclonal antibody can be
substituted for those regions of a human antibody to generate a
chimeric antibody. Alternatively, the constant domains of the light
and heavy chains of a mouse monoclonal antibody can be substituted
for a non-immunoglobulin polypeptide to generate a fusion antibody.
In other embodiments, the constant regions are truncated or removed
to generate the desired antibody fragment of a monoclonal antibody.
Furthermore, site-directed or high-density mutagenesis of the
variable region can be used to optimize specificity and affinity of
a monoclonal antibody.
[0066] The monoclonal antibody of the present invention can be a
humanized antibody. Humanized antibodies are antibodies that
contain minimal sequences from non-human (e.g., murine) antibodies
within the variable regions. Such antibodies are used
therapeutically to reduce antigenicity and human anti-mouse
antibody responses when administered to a human subject. In
practice, humanized antibodies are typically human antibodies with
minimal to no non-human sequences. A human antibody is an antibody
produced by a human or an antibody having an amino acid sequence
corresponding to an antibody produced by a human.
[0067] Procedures for raising polyclonal antibodies are also well
known. Typically, such antibodies can be raised by administering
the peptide or polypeptide containing the epitope of interest (i.e.
polymerized first or second peptides or fusion peptides)
subcutaneously to rabbits (e.g. New Zealand white rabbits), goats,
sheep, swine or donkeys which have been bled to obtain pre-immune
serum. The antigens can be injected in combination with an
adjuvant. The rabbits are bled approximately every two weeks after
the first injection and periodically boosted with the same antigen
three times every six weeks. Polyclonal antibodies are recovered
from the serum by affinity chromatography using the corresponding
antigen to capture the antibody. This and other procedures for
raising polyclonal antibodies are disclosed in Ed Harlow and David
Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor
Laboratory Press, 1988), which is hereby incorporated by reference
in its entirety.
[0068] In addition to whole antibodies, the present invention
encompasses binding portions of such antibodies. Such binding
portions include the monovalent Fab fragments, Fv fragments (e.g.,
single-chain antibody, scFv), and single variable V.sub.H and
V.sub.L domains, and the bivalent F(ab').sub.2 fragments, Bis-scFv,
diabodies, triabodies, minibodies, etc. These antibody fragments
can be made by conventional procedures, such as proteolytic
fragmentation procedures, as described in James Goding, MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press, 1983)
and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold
Spring Harbor Laboratory, 1988), which are hereby incorporated by
reference in their entirety, or other methods known in the art.
[0069] It may further be desirable, especially in the case of
antibody fragments, to modify the antibody in order to increase its
serum half-life. This can be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment by mutation of the appropriate region in the
antibody fragment or by incorporating the epitope into a peptide
tag that is then fused to the antibody fragment at either end or in
the middle (e.g., by DNA or peptide synthesis).
[0070] Antibody mimics are also suitable for use in accordance with
the present invention. A number of antibody mimics are known in the
art including, without limitation, those known as monobodies, which
are derived from the tenth human fibronectin type III domain
(10Fn3) (Koide et al., "The Fibronectin Type III Domain as a
Scaffold for Novel Binding Proteins," J Mol Biol 284:1141-1151
(1998); Koide et al., "Probing Protein Conformational Changes in
Living Cells by Using Designer Binding Proteins: Application to the
Estrogen Receptor," Proc Natl Acad Sci USA 99:1253-1258 (2002),
each of which is hereby incorporated by reference in its entirety);
and those known as affibodies, which are derived from the stable
alpha-helical bacterial receptor domain Z of staphylococcal protein
A (Nord et al., "Binding Proteins Selected from Combinatorial
Libraries of an alpha-helical Bacterial Receptor Domain," Nature
Biotechnol 15(8):772-777 (1997), which is hereby incorporated by
reference in its entirety).
[0071] The present invention is further directed to a method of
detecting whether a subject is infected with HIV-1. This method
includes providing a sample from the subject. The sample is
contacted with the immunogenic peptide described above under
conditions effective to cause an immunogenic reaction between
antibodies in the sample and the immunogenic peptide. Any subject,
where the contacting results in the immunogenic reaction, is
identified as being infected with HIV-1. The diagnosis of HIV-1 is
based on the detection of V2-specific antibodies in the subject.
The presence of antibodies reactive with the V2-specific peptides
can be detected using standard electrophoretic and immunodiagnostic
techniques, including immunoassays such as competition, direct
reaction, or sandwich type assays. Such assays include, but are not
limited to: western blots; agglutination tests; enzyme-labeled and
mediated immunoassays, such as ELISAs; biotin/avidin type assays;
radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc.
The reactions generally involve using labels such as fluorescent,
chemiluminescent, radioactive, or enzymatic labels or dye
molecules, or other methods for detecting the formation of a
complex between the antigen and the antibody.
EXAMPLES
[0072] The following examples are provided to illustrate
embodiments of the present invention but they are by no means
intended to limit its scope.
Materials and Methods for Examples 1-4
[0073] Specimens.
[0074] The initial pilot studies were performed using various sets
of plasma from the RV144 participants which were selected randomly,
evenly balanced for men and women, and derived from participants at
visit 1 (pre-bleed), visit 8 (week 26 after the first immunization
[two weeks after the last immunization]), and visit 9 (52 weeks).
The pilot studies were performed with plasma sets C (SZP, PB), A
and L (GT, BH), and Z (MR, NK). Subsequently, case-control plasma
specimens, described above, were tested for the primary and
secondary variables selected as described above and in Haynes et
al., "Immune Correlates Analysis of the ALVAC-AIDSVAX HIV-1 Vaccine
Efficacy Trial," N Engl J Med. 366:1275-1286 (2012), which is
hereby incorporated by reference in its entirety.
[0075] Written informed consent and counseling was conducted as
described previously (Rerks-Ngarm et al., "Vaccination with ALVAC
and AIDSVAX to Prevent HIV-1 Infection in Thailand," N Engl J Med.
361:2209-2220 (2009); Haynes et al., "Immune Correlates Analysis of
the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial," N Engl J Med.
366:1275-1286 (2012), each of which is hereby incorporated by
reference in its entirety), and the protocol was reviewed by the
ethics committees of the Thai Ministry of Public Health, the Royal
Thai Army, Mahidol University, and the Human Subjects Research
Review Board of the U.S. Army Medical Research and Materiel
Command. It was also independently reviewed and endorsed by the
World Health Organization and the Joint United Nations Program on
HIV/AIDS and by the AIDS Vaccine Research Working Group of the
National Institute of Allergy and Infectious Diseases at the
National Institutes of Health. The manufacturers were full trial
collaborators and were a part of the Phase III trial steering
committee.
[0076] ELISA for Cyclic Peptides and Recombinant gp120 (NK).
[0077] This assay was previously described (Haynes et al., "Immune
Correlates Analysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy
Trial," N Engl J Med. 366:1275-1286 (2012), which is hereby
incorporated by reference in its entirety). Briefly, U-bottom ELISA
plates were coated with either 1 .mu.g/ml of cyclic peptide (Table
1) or with 3 .mu.g/ml of AIDSVAX recombinant gp120 immunogen A244
or MN.
TABLE-US-00001 TABLE 1 V2-Related Reagents and Assays Used in the
Pilot and Case-Controlled Analysis Used for Pilot Case- Assay and
Reagents/Lab PI Study control ELISA/Karasavva .sup.aCyclic V2 (AAs
157-198) + + (SEQ ID NO: 66) Cyclic scrambled V2 crown
QVLFKDIHKIVKPLYA + + (SEQ ID NO: 69) Cyclic scrambled V2 flanks
CENLTDKMFTSR SESRLDETNYNISC + + (SEQ ID NO: 70) ELISA/Zolla-Pazner
.sup.bPeptide 1: Most polar V2 LRDKKQRVYSLFYKLDVVQIN + sequence
(AAs165-185) (SEQ ID NO: 3) Peptide 2 Most common 40
IRDKVQKEYALFYKLDVVPID + AA V2 sequence (SEQ ID NO: 4) Peptide 3
Most polar 40 LRDKKQQVYSLFYRLDIEKIN + AA V2 sequence (SEQ ID NO: 5)
Peptide 4 Consensus 40 AA IRDKKQKEYALFYKLDVVPID + V2 sequence (SEQ
ID NO: 6) Peptide 6: First 14 AA of LRDKKQRVYSLFYK + + Peptide 1
(AAs 165-178) (SEQ ID NO: 7) Peptide 6G: First 14 AA of
GGGLRDKKQRVYSLFYK + Peptide 1 with linker (SEQ ID NO: 10) Peptide
7: Central 14-mer KQRVYSLFYKLDVV + of peptide 1 (SEQ ID NO: 8)
Peptide 8: C-term 13-mer YSLFYKLDVVQIN + Peptide 1 (SEQ ID NO: 9)
Peptide 17: L165I Mutant GGGIRDKKQRVYSLFYK + of Peptide 6 (SEQ ID
NO: 11) Peptide 18: K169V Mutant GGGLRDKVQRVYSLFYK + of Peptide 6
(SEQ ID NO: 12) Peptide 19: V172E Mutant GGGLRDKKQREYSLFYK + of
Peptide 6 (SEQ ID NO: 13) Peptide 20: S174A Mutant of Peptide 6
GGGLRDKKQRVYALFYK + (SEQ ID NO: 14) .sup.aCyclic V2 (AAs 157-198)
(SEQ ID NO: 66) gp70-V1V2 [from subtype + + B Case A2] (SEQ ID NO:
71) ELISA/Berman V2 A244-92TH023 peptide + + (SEQ ID NO: 72) V2 MN
peptide + + (SEQ ID NO: 73) ELISA/Tomaras V2 peptide K178 KK KKK +
(SEQ ID NO: 74) SPR/Rao Cyclic V2 scrambled crown QVLFKDIHKIVKPLYA
+ + (SEQ ID NO: 69) .sup.aCyclic V2 (AAs 157-198) + + (SEQ ID NO:
66) Luminex/Tomaras IgG binding to biotiny- KK KKK + + latedV2
peptide K178 (SEQ ID NO: 74) IgA binding to biotiny- KK KKK + +
latedV2 peptide K178 (SEQ ID NO: 74) *Hotspot/Montefiori + +
.sup.a"Cyclic V2 (amino acids 157-198)" was used in assays in three
labs as shown. Throughout this table, bold italicized V2 sequences
are identical to the subtype E 92TH023 used in the prime;
underlined sequences represent scrambled sequences or linkers;
italicized sequences represent the sequence the central amino
acidss in an extremely polar V2 in subtype A strain
QB585.2102M.Ev1v5.C with individual mutations shown in bold; plain
black represents sequences chosen for particular properties, as
described bold underlined sequences represent the V1V2 from subtype
B Case A2 (Pinter et al., "Potent Neutralization of Primary HIV-1
Isolates By Antibodies Directed Against Epitopes Present in the
V1N2 Domain of HIV-1 gp120," Vaccine 16: 1803-1811(1998), which is
hereby incorporated by reference in its entirety) and the central
23-mer of V2 from subtype B strain MN. .sup.bAll peptides were
biotinylated at the N-terminus with the exception of peptide K178
and peptide V2 A244-92TH023 which were biotinylated at the
C-terminus. *Multiple V2 peptides from various strains (see Table 1
and Karasavvas et al., "The Thai Phase Iii Clinical Trial (RV144)
Induces the Generation of Antibodies that Target a Conserved Region
Within the V2 Loop of gp120; The Thai Phase Iii Clinical Trial
(RV144) Induces the Generation of Antibodies that Target a
Conserved Region Within the V2 Loop of gp120", Abstract OA07.08 LB;
Bangkok, Thailand pp. OA07.08 LB (2011), which is hereby
incorporated by reference in its entirety).
[0078] After washing, two-fold serial dilutions of plasma at an
initial dilution of 1:100 or, alternatively, anti-V2 human
monoclonal antibodies 2158 or 697-D (Gorny et al., "Human Anti-V2
Monoclonal Antibody That Neutralizes Primary But Not Laboratory
Isolates of HIV-1," J Virol. 68:8312-8320 (1994); Pinter at al.,
"The V1/V2 Domain of gp120 is a Global Regulator of Sensitivity of
Primary Human Immunodeficiency Virus Type 1 Isolates to
Neutralization by Antibodies Commonly Induced Upon Infection," J
Virol. 78:5205-5215 (2004), each of which is hereby incorporated by
reference in its entirety) were used at concentrations of 0.002-10
.mu.g/ml. Color was developed with HRP-conjugated goat anti-human
IgG and substrate, and read A405 nm. The background value was
determined from wells that did not contain recombinant proteins or
peptides.
[0079] Biotinylated Linear Peptide ELISAs (SZP).
[0080] This assay was previously described (Haynes et al., "Immune
Correlates Analysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy
Trial," N Engl J Med. 366:1275-1286 (2012), which is hereby
incorporated by reference in its entirety). Briefly, StreptaWell
ELISA plates (Roche) were coated with 1 .mu.g/ml of one of several
N-terminus biotinylated linear V2 peptides (Table 1); the plates
were washed and incubated with RV144 plasma specimens diluted 1:100
in RPMI medium containing 15% fetal bovine sera. Alkaline
phosphatase (AP)-conjugated goat anti-human IgG and diethanolamine
substrate were used to develop color which was read at A405 nm. At
each step, every well contained 50 al; specimens were run in
duplicate in each experiment, and three experiments were
performed.
[0081] Binding ELISA with gp70-V1V2 (SZP).
[0082] This method was previously described (Haynes et al., "Immune
Correlates Analysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy
Trial," N Engl J Med. 366:1275-1286 (2012), which is hereby
incorporated by reference in its entirety). Briefly, plates were
coated with 1 .mu.g/ml gp70-V1V2 (Table 1 and Pinter et al.,
"Potent Neutralization of Primary HIV-1 Isolates by Antibodies
Directed Against Epitopes Present in the V1/V2 Domain of HIV-1
gp120," Vaccine 16:1803-1811 (1998), which is hereby incorporated
by reference in its entirety), washed, and then incubated for 1.5 h
at 37.degree. C. with RV144 plasma diluted 1:100 in RPMI containing
15% fetal bovine sera. After further washing, bound antibodies were
visualized using AP-conjugated goat anti-human IgG and
diethanolamine substrate, and read at 405 nm. At each step, every
well contained 50 al; specimens were run in duplicate in each
experiment, and three experiments were performed.
[0083] V2 Linear Peptide ELISA (PB).
[0084] These assays were performed as previously described (Gilbert
et al., "Correlation Between Immunologic Responses to a Recombinant
Glycoprotein 120 Vaccine and Incidence of HIV-1 Infection in a
Phase 3 HIV-1 Preventative Vaccine Trial," J Infect Dis.
191:666-677 (2005), which is hereby incorporated by reference in
its entirety). Briefly, plates were coated with 0.5 .mu.g/well of
peptide (Table 1) and incubated overnight at 4.degree. C.
Three-fold dilutions of test sera were run in duplicate using a
starting dilution of 1:30. HRP-labeled anti-human IgG and substrate
(OPD) were used to develop color.
[0085] ELISAs of Linear and Cyclic Peptides and gp70-V1V2 (GT).
[0086] Direct binding ELISAs were conducted as previously described
(Haynes et al., "Immune Correlates Analysis of the ALVAC-AIDSVAX
HIV-1 Vaccine Efficacy Trial," N Engl J Med. 366:1275-1286 (2012),
which is hereby incorporated by reference in its entirety) in
384-well ELISA plates coated with 2 .mu.g/ml of linear or cyclic V2
peptides or gp70-V1V2 and incubated with three-fold serial
dilutions of plasma at a starting dilution of 1:50, followed by
washing and incubation with 10 .mu.l of HRP-conjugated goat
anti-human Ig secondary antibody and substrate (SureBlue
Reserve.TM.). Plates were read at 450 nm.
[0087] Overlapping Peptide Microarray Assay (DM). The arrays
measured reactivity with 15-mer peptides with 12 residue overlaps.
Raw peptide microarray data were processed and analyzed as
described in Tomaras et al., "Initial B-cell Responses to
Transmitted Human Immunodeficiency Virus Type 1: Virion-Binding
Immunoglobulin M (IgM) and IgG Antibodies Followed by Plasma
Anti-gp41 Antibodies With Ineffective Control of Initial Viremia,"
J Virol. 82:12449-12463 (2008), which is hereby incorporated by
reference in its entirety. Peptide sequences were provided by LANL
to cover the entire gp160 HIV Env from six HIV-1 Group M subtypes
(A, B, C, D, CRF01_AE and CRF02_AG) for a total of 1423 peptides.
The specific peptides were determined by LANL's method for
generating the mosaic peptide set (Ngo et al., "Identification and
Testing of Control Peptides for Antigen Microarrays," Journal of
Immunological Methods. 343:68-78 (2009), which is hereby
incorporated by reference in its entirety) and were manufactured by
JPT Peptide Technologies (Berlin, Germany).
[0088] Surface Plasmon Resonance (MR).
[0089] Measurements were conducted with a Biacore.RTM. T100 as
previously described (Haynes et al., "Immune Correlates Analysis of
the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial," N Engl J Med.
366:1275-1286 (2012), which is hereby incorporated by reference in
its entirety). Briefly, lysozyme (reference surface) and
streptavidin (for peptide capture) were immobilized onto CM5 chips.
Biotinylated V2 peptides (1 .mu.M) (Table 1) were manually injected
over the streptavidin-coated chip surface. Heat-inactivated plasma
samples diluted 1:50 were injected over the chip surface followed
by a dissociation period, after which a 50 nM solution of
affinity-purified .gamma.-chain-specific sheep anti-human IgG was
passed over the peptide coated-Ig bound surface. Non-specific
binding was subtracted and data analysis was performed using
BIAevaluation.TM. 4.1 software. Case-control samples were run in
triplicate.
[0090] IgG and IgA Binding Multiplex Assays (GT). These assays were
performed as previously described (Haynes et al., "Immune
Correlates Analysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy
Trial," N Engl J Med. 366:1275-1286 (2012); Tomaras et al.,
"Initial B-cell Responses to Transmitted Human Immunodeficiency
Virus Type 1: Virion-Binding Immunoglobulin M (IgM) and IgG
Antibodies Followed by Plasma Anti-gp41 Antibodies With Ineffective
Control of Initial Viremia," J Virol. 82:12449-12463 (2008), each
of which is hereby incorporated by reference in its entirety) using
peptide K178 which represents a linear portion of V2 from
immunogens A244 and 92TH023 (Table 1). HIV-specific antibody
isotypes were detected with goat anti-human IgA and mouse
anti-human IgG.
[0091] Statistical Analyses.
[0092] Immune biomarkers measured two weeks after the last
immunizing dose were assessed as correlates of subsequent infection
risk using the previously described statistical analysis plan
(Haynes et al., "Immune Correlates Analysis of the ALVAC-AIDSVAX
HIV-1 Vaccine Efficacy Trial," N Engl J Med. 366:1275-1286 (2012),
which is hereby incorporated by reference in its entirety).
Briefly, for each immune biomarker, logistic regression accounting
for the sampling design was used to estimate the odds ratio (OR) of
infection, controlling for gender and baseline behavioral risk. The
OR was estimated both for the immune biomarker as a categorical
variable and for variables with greater than 50% of vaccinees
exhibiting a positive response, as a quantitative variable (scaled
to have a SD=1). For the categorical analysis, if the positive
response rate is less than 50%, then the OR compares positive vs.
negative responders. If the positive response rate is 50-85%, then
the OR compares high vs. negative where high is above the median of
the positive responders. For positive response rates>85%, the OR
compares high vs. low where high and low are the upper and bottom
tertiles of the response for vaccine recipients. The statistical
analysis plan was finalized before data analysis and is described
in detail in Haynes et al., "Immune Correlates Analysis of the
ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial," N Engl J Med.
366:1275-1286 (2012), which is hereby incorporated by reference in
its entirety.
[0093] The lasso model selection procedure (Friedman et al.,
"Regularization Paths for Generalized Linear Models Via Coordinate
Descent," J Stat Softw. 33:1-22 (2010), which is hereby
incorporated by reference in its entirety) as implemented in the R
software package was used to assess the ability of 12 V2 variables
from Table 2 to predict infection when included in a multivariate
logistic model adjusting for gender and behavioral risk score.
TABLE-US-00002 TABLE 2 Response Rate and Odds Ratios (ORs)
Calculated From the Case Control Specimens Tested With 13 V2
Variables. Quantitative Categorical Inves- P P Assay tigator
Institution OR.sup.1,2 value OR.sup.1,3 value V2 cyclic Nicos
AFRIMS peptides-ELISA Karasavvas Cyclic V2 NA NA 0.82 0.63 (AAs
157-198) Cyclic V2 NA NA NA NA scrambled crown Cyclic V2 0.76 0.10
0.84 0.66 scrambled flanks V2 cyclic Mangala USMHRP peptides-SPR
Rao Cyclic V2 0.79 0.18 0.90 0.80 scrambled crown Cyclic V2 0.81
0.24 0.84 0.66 (AAs 157-198) V2 reagents- Susan New York ELISA
Zolla- University Pazner Cyclic V2 0.82 0.26 0.65 0.31 (AAs
157-198) Biotin V2 Peptide 0.95 0.80 0.85 0.76 6 (AAs 165-178)
gp70-V1V2 0.70 0.06 0.43 0.06 V2 linear Philip University
peptides-ELISA Berman of California, Santa Cruz V2 MN peptide NA NA
0.41 0.25 V2 A244-92TH023 0.90 0.57 0.88 0.74 peptide IgA and IgG
Abs Georgia Duke vs. V2 peptide- Tomaras University Luminex IgA V2
A244 NA NA 0.79 0.77 K178 peptide IgG V2 A244 NA NA 1.02 0.95 K178
peptide Peptide David Duke Microarray Montefiori University V2
Hotspot 0.64 0.03 0.32 0.02 analysis .sup.1Estimated odds ratios
are computed using a logistic regression model accounting for the
sampling design and adjusting for gender and behavioral risk score,
as described in Haynes et al., "Immune Correlates Analysis of the
ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial," N Engl J Med.
366:1275-1286 (2012), which is hereby incorporated by reference in
its entirety. .sup.2Estimated odds ratio per one standard deviation
increment in the immune biomarker; not available (NA) if response
rates, when applicable, are less than 50%. For example, the OR of
0.70 (ELISA binding to gp7O-V1V2) means that for every higher SD
value, the rate of infection is reduced by 30%, while the OR of
0.43 means that vaccinees with responses in the upper third had an
infection rate 57% lower than vaccinees with responses in the lower
third. .sup.3Estimated odds ratios comparing subgroups defined by
high vs. low responses except for two (IgA V2 A244 K178 and V2 MN)
which compare positive vs. negative response and one (biotin V2
peptide 6) which compares high vs. negative; not available (NA) for
Cyclic V2 scrambled crown (ELISA) which has no positive
responses.
[0094] The cyclic V2 scrambled crown variable was excluded, because
it had no positive responses. Two of the variables with low
response rates, IgA V2 A244 K178 peptide and V2 MN peptide, were
dichotomized as 1 for response and 0 for non-response, while the
remaining ten variables were included on a quantitative scale. The
best parsimonious model was chosen based on the average area under
the receiver operating characteristic curve derived from 1,000
10-fold cross-validation splits.
Example 1--Antigenicity of the Boosting Immunogens Used
[0095] In RV144, the V2 sequence in the recombinant ALVAC priming
immunogen derived from subtype E strain 92TH023 was:
TABLE-US-00003 (SEQ ID NO: 66)
.sup.157CSFNMTTELRDKKQKVHALFYKLDIVPIEDNTSS.SEYRLINC.sup.198
The V2 sequence in the protein boosting gp120 immunogen AIDSVAX E
(strain A244) was:
TABLE-US-00004 (SEQ ID NO: 67)
.sup.157CSFNMTTELRDKKQKVHALFYKLDIVPIEDNNDS.SEYRLINC.sup.198
The V2 sequence of the protein boosting gp120 immunogen AIDVAX B
(strain MN) was:
TABLE-US-00005 (SEQ ID NO: 68)
.sup.157CSFNITTSIGDKMQKEYALLYKLDIEPI.DN.DSTS.YRLISC.sup.198
Insertion of periods in the sequences allows for alignment.
Numbering shown and used throughout this report is that assigned to
strain HxB2 (Ratner et al., "Complete Nucleotide Sequences of
Functional Clones of the AIDS Virus," AIDS Res Hum Retroviruses
3:57-69 (1987), which is hereby incorporated by reference in its
entirety).
[0096] The antigenic reactivity of the V2 region in AIDSVAX B and E
was assessed using human anti-V2 monoclonal antibodies 697D and
2158 (Gorny et al., "Human Anti-V2 Monoclonal Antibody That
Neutralizes Primary But Not Laboratory Isolates of HIV-1," J Virol.
68:8312-8320 (1994); Pinter at al., "The V1/V2 Domain of gp120 is a
Global Regulator of Sensitivity of Primary Human Immunodeficiency
Virus Type 1 Isolates to Neutralization by Antibodies Commonly
Induced Upon Infection," J Virol. 78:5205-5215 (2004), each of
which is hereby incorporated by reference in its entirety). As
shown in FIG. 1, the titration curves for each of these monoclonal
antibodies with the two boosting immunogens could be superimposed,
with half-maximal binding achieved at 0.0057 and 0.0055 .mu.g/ml of
monoclonal antibody 697D, and 0.0041 and 0.0039 .mu.g/ml of
monoclonal antibody 2158 vs. AIDSVAX A244 and AIDSVAX MN,
respectively. This analysis suggests that, with respect to the
highly conformational V2 epitopes recognized by these monoclonal
antibodies (Gorny et al., "Functional and Immunochemical
Cross-Reactivity of V2-specific Monoclonal Antibodies from Human
Immunodeficiency Virus Type 1-infected Individuals," Virology 427:
198-207 (2012); Pinter et al., "The V1/V2 Domain of gp120 is a
Global Regulator of Sensitivity of Primary Human Immunodeficiency
Virus Type 1 Isolates to Neutralization by Antibodies Commonly
Induced Upon Infection," J Virol. 78: 5205-5215 (2004); Gorny,
"Production of Human Monoclonal Antibodies Via Fusion of
Epstein-Barr Virus-Transformed Lymphocytes with Heteromyeloma," In:
Celis, editor. In: Cell Biology: A Laboratory Handbook: Academic
Press 276-281 (1994), each of which is hereby incorporated by
reference in its entirety), the antigenicity of the A244 and MN
gp120 immunogens are similar. Notably, these two monoclonal
antibodies also bind to gp70-V1V2 (Gorny et al., "Functional and
Immunochemical Cross-Reactivity of V2-specific Monoclonal
Antibodies from Human Immunodeficiency Virus Type 1-infected
Individuals," Virology 427: 198-207 (2012); Karasavvas et al., "The
Thai Phase Iii Clinical Trial (RV144) Induces the Generation of
Antibodies that Target a Conserved Region Within the V2 Loop of
gp120; The Thai Phase Iii Clinical Trial (RV144) Induces the
Generation of Antibodies that Target a Conserved Region Within the
V2 Loop of gp120," Abstract OA07.08 LB; Bangkok, Thailand. pp.
OA07.08 LB (2011), each of which is hereby incorporated by
reference in its entirety).
Example 2--The V2 Antibody Response in RV144 can be Detected with
Both Linear and V1V2-Scaffolded Antigens
[0097] A gp70-V1V2 scaffolded protein carrying the V1 and V2 loops
from a clade B strain, case A2, was previously described (Pinter et
al., "Potent Neutralization of Primary HIV-1 Isolates by Antibodies
Directed Against Epitopes Present in the V1/V2 Domain of HIV-1
gp120," Vaccine 16:1803-1811 (1998), which is hereby incorporated
by reference in its entirety). When Set C plasma specimens (from 20
placebo and 80 vaccine recipients) were tested in the pilot studies
at a dilution of 1:100, none of the specimens from the placebo
recipients contained detectable antibodies to gp70-V1V2. In
contrast, the plasma of 67 of 80 (84%) vaccine recipients contained
antibodies reactive with this reagent (FIG. 2). Moreover, the
dynamic range of the assay was large, covering an optical density
range from the cut-off, 0.276 OD units, to 1.918. A relatively poor
correlation was found between this assay and other assays that
measured various V2 variables, suggesting that the antibody
response measured with gp70-V1V2 represents a unique "immunologic
space" (FIG. 3).
[0098] The frequency of V2 responses detected with pilot study
specimens derived from vaccinees varied with the assay used,
ranging from 6% for IgA antibodies reactive with a linear V2
peptide (K178) when measured by Luminex (see Table 1 and Haynes et
al., "Immune Correlates Analysis of the ALVAC-AIDSVAX HIV-1 Vaccine
Efficacy Trial," N Engl J Med. 366:1275-1286 (2012), which is
hereby incorporated by reference in its entirety) to 97% for IgG
antibodies reactive with an A244 (subtype E) cyclic V2 peptide (see
Table 1 and Karasavvas et al., "The Thai Phase Iii Clinical Trial
(RV144) Induces the Generation of Antibodies that Target a
Conserved Region Within the V2 Loop of gp120; The Thai Phase Iii
Clinical Trial (RV144) Induces the Generation of Antibodies that
Target a Conserved Region Within the V2 Loop of gp120", Abstract
OA07.08 LB; Bangkok, Thailand. pp. OA07.08 LB (2011), which is
hereby incorporated by reference in its entirety).
[0099] When reactivity to various V2 reagents were compared in
parallel by ELISA, the response to the K178 peptide was
significantly stronger than that to gp70-V1V2 or to cyclic V2
peptide (amino acids 157-198), as shown in FIG. 4. Thus, the RV144
vaccine induced antibodies that reacted to both scaffolded-V1V2 and
to linear V2 peptides, but the response to the latter appears to be
stronger.
Example 3--Delineation of the Linear V2 Epitopes Recognized by
Plasma Antibodies from Vaccinees
[0100] For fine mapping of linear V2 epitopes recognized by
antibodies in the plasma of RV144 vaccinees, four 21-mer peptides
(Peptides 1-4 (SEQ ID NOS: 3 to 6) in Table 1 and FIG. 5A) were
selected on the basis of a bioinformatics analysis of V2 sequences
from the LANL HIV Database. Peptide 1 (SEQ ID NO: 3) was derived
from the V2 of a strain with the highest number of polar amino
acids (subtype A strain QB585.2102M.Ev1v5.C from Kenya); this V2
was 38 amino acids in length. Since V2 is most frequently 40 amino
acids in length (Zolla-Pazner et al., "Structure-Function
Relationships of HIV-1 Envelope Sequence-Variable Regions Provide a
Paradigm for Vaccine Design. Nat Rev Immunol. 10:527-535 (2010),
which is hereby incorporated by reference in its entirety), further
analyses identified sequences from viruses containing V2 regions 40
amino acids long: Peptide 2 (SEQ ID NO: 4) represents the central
21 amino acids of V2 in the most common naturally occurring
sequence (derived from subtype B strain 878v3_2475). Peptide 3 (SEQ
ID NO: 5) is the V2 with the highest number of polar amino acids
(from subtype A strain 01TZA341). Peptide 4 (SEQ ID NO: 6) is the
consensus V2 sequence among all viruses with V2 regions of 40 amino
acids.
[0101] As illustrated by the ELISA data (FIG. 5A), Peptide 1 (SEQ
ID NO: 3) was the most reactive with plasma from the RV144
vaccinees: 61% of plasma showed positive reactivity, i.e., had OD
values above the cut-off which was based on the mean+3 SD of
control plasma (from placebo recipients). Peptide 3 (SEQ ID NO: 5)
was also reactive (49% positive), while Peptides 2 (SEQ ID NO: 4)
and 4 (SEQ ID NO: 6) were poorly reactive (0% and 7% positive,
respectively). Three overlapping peptides (Peptides 6-8 (SEQ ID
NOS: 7 to 9)) were synthesized based on the sequence of the most
strongly reactive Peptide 1 (SEQ ID NO: 3). Results with the
overlapping peptides showed that the epitope maps to the 14
residues in Peptide 6 (SEQ ID NO: 7) containing amino acids 165-178
(FIG. 5A). The residues in this V2 region form the outer C strand
of the 3-sheet folded domain of VV2 (FIG. 6) (McLellan et al.,
"Structure of HIV-1 gp120 V1/V2 Domain With Broadly Neutralizing
Antibody PG9," Nature 480:336-343 (2011), which is hereby
incorporated by reference in its entirety).
[0102] To distinguish the amino acids that play a critical role in
determining anti-V2 antibody reactivity, a further series of
peptides was designed. Results with these peptides indicated that
L165I (Peptide 17 (SEQ ID NO: 11)) and S174A (Peptide 20 (SEQ ID
NO: 14)) replacements had little effect on reactivity (FIG. 5B). In
contrast, the K169V (Peptide 18 (SEQ ID NO: 12)) and the V172E
(Peptide 19 (SEQ ID NO: 13)) replacements profoundly reduced the
reactivity of the plasma, indicating that these two residues are
critical for binding of V2 peptides by the vaccine-induced
antibodies. These data acquire enhanced importance in light of
results showing that (a) sieve analysis showed that a residue other
than K at position 169 was more frequent in breakthrough viruses in
vaccine recipients (Rolland et al., "Sequence Analysis of HIV-1
Breakthrough Infections in the RV144 Trial. Characterization of
Breakthrough Viruses," Sequence Analysis of HIV-1 Breakthrough
Infections in the RV144 Trial, Abstract S07.02 S07.02 (2011) and
Edlefsen et al., "Sieve Analysis of RV144," Abstract S07.04 S07.04
(2011), each of which is hereby incorporated by reference in its
entirety), as well as (b) Gln (E) strongly predominates in subtype
E whereas valine (V) slightly predominates at position 172 in
subtype B. Notably, the poor response to Peptide 19 (SEQ ID NO:
13), in which the V172E mutation appears, suggests that the V2
antibody response was induced by the subtype E (A244) gp120
boosting immunogen rather than the subtype B (MN) immunogen.
Example 4--Comparison of V2 Assays Run in the Case-Control
Study
[0103] Based on the pilot studies, six assay types were chosen for
measuring 13 variables with case-control specimens (Table 2). One
of these (ELISA binding to gp70-V1V2) was chosen as a primary
variable. The secondary variables provided by the 12 additional V2
assays were run in exploratory analyses with case-control
specimens. The primary and secondary variables were chosen to
represent assays whose results did not correlate with one another.
The heat map in FIG. 3 represents the Spearman rank correlations
between the V2 assays, and demonstrates that the primary variable,
binding of IgG antibodies to gp70-V1V2, correlated only weakly with
ELISA binding to cyclic V2 (amino acids 157-198) (Spearman
correlation: 0.5-0.6). Analysis of the microarray "hotspot" data
was not performed until after completion of the analysis of the
initial pilot and case-control studies. Interestingly, the V2
hotspot variable correlates poorly with all of the other V2
variables.
[0104] For the univariate analysis, as in the multivariate analysis
(Haynes et al., "Immune Correlates Analysis of the ALVAC-AIDSVAX
HIV-1 Vaccine Efficacy Trial," N Engl J Med. 366:1275-1286 (2012),
which is hereby incorporated by reference in its entirety),
statistical significance was approached or achieved with the
primary variable of ELISA binding to gp70-V1V2 (p=0.06 and p=0.02,
respectively). With one of the secondary variables (V2 hotspot),
significance was achieved (p=0.03 quantitative, and p=0.02
categorical, Table 2). The ORs calculated for each V2 variable are
shown in FIG. 7 and Table 2. The univariate ORs for all V2
variables were <1.02, compatible with the hypothesis that V2
antibodies played a role in reducing infection. When binding
antibodies were assessed by peptide microarray analysis using
linear overlapping peptides covering the entire V2 region of seven
major genetic subtypes, the lowest and most statistically
significant OR was achieved (FIG. 7 and Table 2). The results are
shown in FIG. 8 for V2 residues 160-183. Most of the remaining
C-terminal portion of V2 is poorly immunogenic (Zolla-Pazner et
al., "Structure-Function Relationships of HIV-1 Envelope
Sequence-Variable Regions Provide a Paradigm for Vaccine Design,"
Nat Rev Immunol. 10:527-535 (2010), which is hereby incorporated by
reference in its entirety), and similarly, there was little or no
reactivity with peptides in the V1 region. The aggregate response
(FIG. 8A), shows that the V2 response is centered around residue
K.sup.173. The peptides with the strongest reactivity encompass
residues 163-177 (FIG. 8B), which matches the results from the
independent ELISA data described above. It is also noteworthy that
the weakest reactivity in the microarray analysis was detected with
the subtype B subset of V2 peptides, confirming the poor reactivity
with Peptide 19 (SEQ ID NO: 13) (FIG. 5B) which represents a
canonical subtype B V2 containing a Glu (E) at position 172.
Interestingly, the reactivity with the V2 peptide representing the
sequence of vaccine strain MN (subtype B) also includes E.sup.172.
Only 24 of 246 vaccinees' specimens reacted with the V2 MN peptide,
again confirming the poor reactivity with the subtype B V2;
however, strikingly, none of these 24 vaccinees were infected,
resulting in an OR for positive vs. negative responders of 0.41
(Table 2 and FIG. 7). The 0.25 p-value reflects the low power of
these data due to the very few positive responders and could also
be due to poor sensitivity of the assay since the results were
reported as endpoint titers after a starting dilution of 1:30.
Discussion of Examples 1-4
[0105] In this study, the results achieved with the entire panel of
V2 assays used in the RV144 pilot and case-control studies were
probed in order to understand more fully the nature of the V2
antibody response and why the high response to epitopes in this
region is associated with a lower rate of infection in vaccinees.
The data presented include all of the data describing the V2
antibodies induced by the vaccine and available from both the pilot
and the case-control specimens.
[0106] These studies document at least two types of V2 antibodies
induced by the RV144 vaccine: antibodies reactive with a scaffolded
V1V2 protein, gp70-V1V2, and antibodies specific for linear V2
peptides. Studies with human monoclonal antibodies suggest that
these may be non-overlapping antibody populations since monoclonal
antibodies such as 697D and 2158 react with conformational V1V2
epitope(s) carried by gp70-V1V2 but not with linear peptides (Gorny
et al., "Functional and Immunochemical Cross-Reactivity of
V2-Specific Monoclonal Antibodies From Human Immunodeficiency Virus
Type 1-Infected Individuals," Virology 427:198-207 (2012); Gorny et
al., "Human Anti-V2 Monoclonal Antibody That Neutralizes Primary
but Not Laboratory Isolates of HIV-1," J Virol. 68:8312-8320
(1994), each of which is hereby incorporated by reference in its
entirety), while monoclonal antibodies such as CH58 and CH59 react
with linear V2 peptides but not with gp70-V1V2. The primary
variable that correlated with reduced risk of infection measured
antibody activity in ELISA with gp70-V1V2. This reagent retains a
conformation presented in vivo during infection since it is
recognized by antibodies in sera of infected individuals and was
used for the selection of two monoclonal antibody-producing
hybridomas from the cells of HIV-infected individuals which are
broadly cross-reactive with diverse envelopes and neutralize
several Tier 1 pseudoviruses (Gorny et al., "Functional and
Immunochemical Cross-Reactivity of V2-Specific Monoclonal
Antibodies From Human Immunodeficiency Virus Type 1-Infected
Individuals," Virology 427:198-207 (2012); Pinter et al., "The
V1/V2 Domain of gp120 is a Global Regulator of Sensitivity of
Primary Human Immunodeficiency Virus Type I Isolates to
Neutralization by Antibodies Commonly Induced Upon Infection," J
Virol. 78:5205-5215 (2004), each of which is hereby incorporated by
reference in its entirety).
[0107] The reactivity of vaccinees' antibodies with overlapping V2
peptides also correlated with reduced risk of infection (Table 2),
generating the hypothesis that antibodies to linear V2 epitopes
were also involved in reducing the rate of HIV infection. The
observation that none of the vaccinees who produced antibodies
reactive with the linear subtype B MN V2 peptide were infected with
HIV during the trial is intriguing, although the low power of the
result reduces confidence in the significance of this observation.
The data generated with various linear V2 peptides indicate that:
(a) the dominant immunogenic linear V2 epitope in the RV144 vaccine
encompasses residues 165 to 178; (b) the V2 antibodies were induced
primarily by subtype E A244 rather than the subtype B MN gp120
boost; (c) the V2 antibodies were cross-reactive with V2 peptides
derived from several subtypes, (d) the dominant linear V2 epitope
was located in the C .beta.-strand of the V1V2 complex (FIG. 6 and
McLellan et al., "Structure of HIV-1 gp120 V1/V2 Domain With
Broadly Neutralizing Antibody PG9" Nature 480: 336-343 (2011),
which is hereby incorporated by reference in its entirety), (e)
residues K.sup.169 and V.sup.172, were critical for the binding of
vaccinees' plasma antibodies to V2 peptides, and (f) the V2 epitope
includes the lysine at position 169 which was identified by sieve
analysis to be mismatched in breakthrough infections (Rolland et
al., "Sequence Analysis of HIV-1 Breakthrough Infections in the
RV144 Trial. Characterization of Breakthrough Viruses," Sequence
Analysis of HIV-1 Breakthrough Infections in the RV144 Trial,
Abstract S07.02 S07.02 (2011), which is hereby incorporated by
reference in its entirety).
[0108] It is noteworthy that the single primary variable showing an
inverse correlate of infection risk in the RV144 case-control study
was antibody reactivity with gp70-V1V2 which contains the V1V2
sequence of case A2, a subtype B strain (Pinter et al., "Potent
Neutralization of Primary HIV-1 Isolates by Antibodies Directed
Against Epitopes Present in the V1/V2 Domain of HIV-1 gp120,"
Vaccine 16:1803-1811 (1998), which is hereby incorporated by
reference in its entirety) which carries both the V169 and the E172
residues that reduce reactivity of vaccinees' antibodies with V2
peptides. Notably, however, as shown above, vaccinees' plasma do
react with subtype B-derived linear V2 peptides, though with less
potent and less frequent reactivity than with V2 peptides from
other subtypes. The data with gp70-V1V2 and the linear peptides may
suggest that the effective antibody populations are those which are
broadly cross-reactive, targeting conformational and linear
epitopes shared by diverse HIV-1 subtypes. Indeed, these data,
together with bioinformatics data on V2 (Zolla-Pazner et al.,
"Structure-Function Relationships of HIV-1 Envelope
Sequence-Variable Regions Provide a Paradigm for Vaccine Design,"
Nat Rev Immunol. 10:527-535 (2010), which is hereby incorporated by
reference in its entirety) and studies of the preferential gene
usage of VH families by V2-specific monoclonal antibodies (Gorny et
al., "Functional and Immunochemical Cross-Reactivity of V2-Specific
Monoclonal Antibodies From Human Immunodeficiency Virus Type
1-Infected Individuals," Virology 427:198-207 (2012), which is
hereby incorporated by reference in its entirety), support the
presence of conserved and immunologically cross-reactive elements
in the V2 loop. The role of shared structures and antigenic
determinants in the variable loops of the envelope in inducing
potentially protective antibody responses is also suggested by the
involvement of the V2 and V3 loops as components of the epitopes
recognized by the class of potently neutralizing antibodies that
target quaternary epitopes and proteoglycans on the envelope spike
(Gorny et al., "Identification of a New Quaternary Neutralizing
Epitope on Human Immunodeficiency Virus Type I Virus Particles," J
Virol. 79:5232-5237 (2005); Walker et al., "Broad and Potent
Neutralizing Antibodies From an African Donor Reveal a New HIV-1
Vaccine Target," Science 326:285-289 (2009); Changela et al.,
"Crystal Structure of Human Antibody 2909 Reveals Conserved
Features of Quaternary-Specific Antibodies that Potentially
Neutralize HIV-1," J Virol. 85:2524-2535 (2011); Spurrier et al.,
"Structural Analysis and Computational Modeling of Human and
Macaque Monoclonal Antibodies Provide a Model for the Quaternary
Neutralizing Epitope of HIV-1 gp120," Structure 19:691-699 (2011);
Wu et al., "Immunotypes of a Quaternary Structure of the HIV-1
Envelope Affect Viral Vulnerability to Neutralizing Antibodies," J
Virol. 85:4578-4585 (2011); Bonsignori et al., "Analysis of a
Clonal Lineage of HIV-1 Envelope V2/V3 Comformational
Epitope-Specific Broadly Neutralizing Antibodies and Their Inferred
Unmutated Common Ancestors," Journal of Virology 85:9998-10009
(2011); Walker at al., "Broad Neutralization Coverage of HIV by
Multiple Highly Potent Antibodies," Nature 477:466-470 (2011);
Pejchal et al., "A Potent and Broad Neutralizing Antibody
Recognizes and Penetrates the HIV Glycan Shield," Science
334:1097-1103 (2011), each of which is hereby incorporated by
reference in its entirety).
[0109] The explanation for the strong induction of V2 antibodies by
the A244 subtype E gp120 immunogen compared to the weak response
induced by the MN subtype B gp120 despite the similar antigenicity
of the two proteins has several possible explanations. It may be
due to a proteolytic cleavage site in the V2 loop of MN; a
cathepsin D cleavage site (QKEYALL (SEQ ID NO: 75)) exists in the
V2 of MN (Yu et al., "Protease Cleavage Sites in HIV-1 gp120
Recognized by Antigen Processing Enzymes are Conserved and Located
at Receptor Binding Sites," J Virol. 84:1513-1526 (2010), which is
hereby incorporated by reference in its entirety), while this site
is absent from the V2 of A244 (QKVHALF (SEQ ID NO: 76)). The
importance of proteoloysis by lysosomal enzymes on antigen
presentation and induction of immune responses to gp120 was
previously described (Chien et al., "Human Immunodeficiency Virus
Type I Evades T-helper Responses by Expoiting Antibodies that
Suppress Antigen Processing," J Virol. 78:7645-7652 (2004), which
is hereby incorporated by reference in its entirety), providing a
theoretical explanation for understanding the differential antibody
responses to these two gp120 immunogens. Alternatively, the
immunogenicity of the V2 in the MN gp120 boosting immunogen may be
less than that of the A244 immunogen, and/or the subtype E rather
than the subtype B V2 region is the greater similarity of the
AIDSVAX subtype E gp120 protein boost to the subtype E Env used in
the prime. To address these issues, further studies of V2 responses
with specimens from other vaccine trials, e.g., VAX003 and VAX004,
are underway, along with assays using additional peptides,
proteoglycans, and epitope-scaffolded proteins.
[0110] Finally, the mechanisms by which anti-V2 antibodies may
reduce HIV infection have yet to be understood. As noted, anti-V2
monoclonal antibodies can neutralize many Tier 1 pseudoviruses in
the TZM.bl assay (Gorny et al., "Functional and Immunochemical
Cross-Reactivity of V2-Specific Monoclonal Antibodies From Human
Immunodeficiency Virus Type 1-Infected Individuals," Virology
427:198-207 (2012), which is hereby incorporated by reference in
its entirety). It is possible that they mediate broader
neutralizing activity than is detected in this particular assay.
Plasma samples from RV144 neutralized some Tier 1 viruses in the
TZM-bl assay and in a more sensitive assay with A3R5 cells; however
no neutralization of Tier 2 viruses was detected in either assay.
Since V2 can be detected on the surface of virions (Nyambi et al.,
"Conserved and Exposed Epitopes on Intact, Native, Primary Human
Immunodeficiency Virus Type I Virions of Group M," J Virol.
74:7096-7107 (2000), which is hereby incorporated by reference in
its entirety) and infected cells (Zolla-Pazner et al., "Serotyping
of Primary Human Immunodeficiency Virus Type I Isolates From
Diverse Geographic Locations by Flow Cytometry," J Virol.
69:3807-3815 (1995), which is hereby incorporated by reference in
its entirety), these antibodies may also mediate various other
anti-viral functions such as ADCC, ADCVI, virolysis, virus
opsonization, virus aggregation, etc. Along with current studies of
the potential biologic functions of V2 antibodies, assessment is
on-going to test several hypotheses, including those that postulate
that anti-V2 antibodies prevent conformational changes in the
envelope necessary for binding to CCR5, and that these antibodies
may, or may not, prevent binding of the envelope to 4037.
Interestingly, after vaccination of non-human primates with Ad26
and MVA containing SIVsm543 inserts, a low dose intra-rectal
heterologous SIVmac251 challenge identified a potential V2
correlate of protection (Barouch et al., "Vaccine Protection
Against Acquisition of Neutralization-Resistant SIV Challenges in
Rhesus Monkeys," Nature 482:89-93 (2012), which is hereby
incorporated by reference in its entirety). While the relevance of
the SIV model to ALVAC-HIV and AIDSVAX B/E responses in humans may
be unclear, the presence of this analogous protective response
after vaccination, in addition to the results of the RV144 immune
correlates analysis, may provide a means to illuminate the
postulated mechanism for reducing the risk of infection.
[0111] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow.
Sequence CWU 1
1
76114PRTArtificial Sequenceconsensus peptide from V2 loop of HIV
gp120 1Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa 1 5
10 214PRTArtificial Sequenceconsensus peptide from V2 loop of HIV
gp120 2Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa 1 5
10 321PRTArtificial Sequencepeptide of most polar V2 sequence from
HIV gp120 3Leu Arg Asp Lys Lys Gln Arg Val Tyr Ser Leu Phe Tyr Lys
Leu Asp 1 5 10 15 Val Val Gln Ile Asn 20 421PRTArtificial
Sequencepeptide of most common 40 amino acid V2 sequence from HIV
gp120 4Ile Arg Asp Lys Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu
Asp 1 5 10 15 Val Val Pro Ile Asp 20 521PRTArtificial
Sequencepeptide of most polar 40 amino acid V2 sequence from HIV
gp120 5Leu Arg Asp Lys Lys Gln Gln Val Tyr Ser Leu Phe Tyr Arg Leu
Asp 1 5 10 15 Ile Glu Lys Ile Asn 20 621PRTArtificial
Sequenceconsensus peptide of 40 amino acid V2 sequence from HIV
gp120 6Ile Arg Asp Lys Lys Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu
Asp 1 5 10 15 Val Val Pro Ile Asp 20 714PRTArtificial
Sequencepeptide of first 14 amino acids of SEQ ID NO 3 7Leu Arg Asp
Lys Lys Gln Arg Val Tyr Ser Leu Phe Tyr Lys 1 5 10 814PRTArtificial
Sequencepeptide of central 14-mer of SEQ ID NO 3 8Lys Gln Arg Val
Tyr Ser Leu Phe Tyr Lys Leu Asp Val Val 1 5 10 913PRTArtificial
Sequencepeptide of C-terminal 13-mer of SEQ ID NO 3 9Tyr Ser Leu
Phe Tyr Lys Leu Asp Val Val Gln Ile Asn 1 5 10 1017PRTArtificial
Sequencepeptide of SEQ ID NO 7 with N-terminal glycines 10Gly Gly
Gly Leu Arg Asp Lys Lys Gln Arg Val Tyr Ser Leu Phe Tyr 1 5 10 15
Lys 1117PRTArtificial Sequencepeptide of SEQ ID NO 7 with L165I
mutation 11Gly Gly Gly Ile Arg Asp Lys Lys Gln Arg Val Tyr Ser Leu
Phe Tyr 1 5 10 15 Lys 1217PRTArtificial Sequencepeptide of SEQ ID
NO 7 with K169V mutation 12Gly Gly Gly Leu Arg Asp Lys Val Gln Arg
Val Tyr Ser Leu Phe Tyr 1 5 10 15 Lys 1317PRTArtificial
Sequencepeptide of SEQ ID NO 7 with V172E mutation 13Gly Gly Gly
Leu Arg Asp Lys Lys Gln Arg Glu Tyr Ser Leu Phe Tyr 1 5 10 15 Lys
1417PRTArtificial Sequencepeptide of SEQ ID NO 7 with S174A
mutation 14Gly Gly Gly Leu Arg Asp Lys Lys Gln Arg Val Tyr Ala Leu
Phe Tyr 1 5 10 15 Lys 1514PRTArtificial Sequencepeptide from V2
loop of HIV gp120 15Leu Gln Asn Lys Lys Gln Gln Val Tyr Ser Leu Phe
Tyr Gln 1 5 10 1614PRTArtificial Sequencepeptide of SEQ ID NO 1
which requires Y at position 11 16Leu Arg Asp Lys Met Gln Lys Val
Tyr Ala Leu Thr Tyr Lys 1 5 10 176PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 Group M 17Arg Leu Asp Val Val Pro 1 5
189PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
Group M 18Leu Phe Tyr Arg Leu Asp Val Val Pro 1 5 1912PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 Group M 19Val Tyr
Ala Leu Phe Tyr Arg Leu Asp Val Val Pro 1 5 10 2015PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 Group M 20Lys Gln
Lys Val Tyr Ala Leu Phe Tyr Arg Leu Asp Val Val Pro 1 5 10 15
2115PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
Group M 21Arg Asp Lys Lys Gln Lys Val Tyr Ala Leu Phe Tyr Arg Leu
Asp 1 5 10 15 2215PRTArtificial Sequencepeptide of consensus V2
region of HIV-1 Group M 22Thr Glu Ile Arg Asp Lys Lys Gln Lys Val
Tyr Ala Leu Phe Tyr 1 5 10 15 2315PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 Group M 23Asn Met Thr Thr Glu Ile Arg
Asp Lys Lys Gln Lys Val Tyr Ala 1 5 10 15 246PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype A 24Arg Leu
Asp Val Val Gln 1 5 259PRTArtificial Sequencepeptide of consensus
V2 region of HIV-1 subtype A 25Leu Phe Tyr Arg Leu Asp Val Val Gln
1 5 2612PRTArtificial Sequencepeptide of consensus V2 region of
HIV-1 subtype A 26Val Tyr Ser Leu Phe Tyr Arg Leu Asp Val Val Gln 1
5 10 2715PRTArtificial Sequencepeptide of consensus V2 region of
HIV-1 subtype A 27Lys Gln Lys Val Tyr Ser Leu Phe Tyr Arg Leu Asp
Val Val Gln 1 5 10 15 2815PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype A 28Arg Asp Lys Lys Gln Lys
Val Tyr Ser Leu Phe Tyr Arg Leu Asp 1 5 10 15 2915PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype A 29Thr Glu
Leu Arg Asp Lys Lys Gln Lys Val Tyr Ser Leu Phe Tyr 1 5 10 15
3015PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype A 30Asn Met Thr Thr Glu Leu Arg Asp Lys Lys Gln Lys Val Tyr
Ser 1 5 10 15 316PRTArtificial Sequencepeptide of consensus V2
region of HIV-1 subtype B 31Lys Leu Asp Val Val Pro 1 5
329PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype B 32Leu Phe Tyr Lys Leu Asp Val Val Pro 1 5
3312PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype B 33Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro 1 5 10
3415PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype B 34Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val
Pro 1 5 10 15 3515PRTArtificial Sequencepeptide of consensus V2
region of HIV-1 subtype B 35Arg Asp Lys Val Gln Lys Glu Tyr Ala Leu
Phe Tyr Lys Leu Asp 1 5 10 15 3615PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype B 36Thr Ser Ile Arg Asp Lys
Val Gln Lys Glu Tyr Ala Leu Phe Tyr 1 5 10 15 3715PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype B 37Asn Ile
Thr Thr Ser Ile Arg Asp Lys Val Gln Lys Glu Tyr Ala 1 5 10 15
386PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype C 38Arg Leu Asp Ile Val Pro 1 5 399PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype C 39Leu Phe
Tyr Arg Leu Asp Ile Val Pro 1 5 4012PRTArtificial Sequencepeptide
of consensus V2 region of HIV-1 subtype C 40Val Tyr Ala Leu Phe Tyr
Arg Leu Asp Ile Val Pro 1 5 10 4115PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype C 41Lys Gln Lys Val Tyr Ala
Leu Phe Tyr Arg Leu Asp Ile Val Pro 1 5 10 15 4215PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype C 42Arg Asp
Lys Lys Gln Lys Val Tyr Ala Leu Phe Tyr Arg Leu Asp 1 5 10 15
4315PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype C 43Thr Glu Ile Arg Asp Lys Lys Gln Lys Val Tyr Ala Leu Phe
Tyr 1 5 10 15 4415PRTArtificial Sequencepeptide of consensus V2
region of HIV-1 subtype C 44Asn Thr Thr Thr Glu Ile Arg Asp Lys Lys
Gln Lys Val Tyr Ala 1 5 10 15 456PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype D 45Lys Leu Asp Val Val Pro 1
5 469PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype D 46Leu Phe Tyr Lys Leu Asp Val Val Pro 1 5
4712PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype D 47Val Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro 1 5 10
4815PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype D 48Lys Lys Gln Val Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val
Pro 1 5 10 15 4915PRTArtificial Sequencepeptide of consensus V2
region of HIV-1 subtype D 49Arg Asp Lys Lys Lys Gln Val Tyr Ala Leu
Phe Tyr Lys Leu Asp 1 5 10 15 5015PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype D 50Thr Glu Val Arg Asp Lys
Lys Lys Gln Val Tyr Ala Leu Phe Tyr 1 5 10 15 5115PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype D 51Asn Ile
Thr Thr Glu Val Arg Asp Lys Lys Lys Gln Val Tyr Ala 1 5 10 15
526PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype CRF01_AE 52Lys Leu Asp Ile Val Gln 1 5 539PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype CRF01_AE
53Leu Phe Tyr Lys Leu Asp Ile Val Gln 1 5 5412PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype CRF01_AE
54Val Tyr Ala Leu Phe Tyr Lys Leu Asp Ile Val Gln 1 5 10
5515PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype CRF01_AE 55Lys Gln Lys Val Tyr Ala Leu Phe Tyr Lys Leu Asp
Ile Val Gln 1 5 10 15 5615PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype CRF01_AE 56Arg Asp Lys Lys Gln
Lys Val Tyr Ala Leu Phe Tyr Lys Leu Asp 1 5 10 15 5715PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype CRF01_AE
57Thr Glu Leu Arg Asp Lys Lys Gln Lys Val Tyr Ala Leu Phe Tyr 1 5
10 15 5815PRTArtificial Sequencepeptide of consensus V2 region of
HIV-1 subtype CRF01_AE 58Asn Met Thr Thr Glu Leu Arg Asp Lys Lys
Gln Lys Val Tyr Ala 1 5 10 15 596PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype CRF02_AG 59Arg Leu Asp Val Val
Gln 1 5 609PRTArtificial Sequencepeptide of consensus V2 region of
HIV-1 subtype CRF02_AG 60Leu Phe Tyr Arg Leu Asp Val Val Gln 1 5
6112PRTArtificial Sequencepeptide of consensus V2 region of HIV-1
subtype CRF02_AG 61Val Tyr Ala Leu Phe Tyr Arg Leu Asp Val Val Gln
1 5 10 6215PRTArtificial Sequencepeptide of consensus V2 region of
HIV-1 subtype CRF02_AG 62Lys Gln Lys Val Tyr Ala Leu Phe Tyr Arg
Leu Asp Val Val Gln 1 5 10 15 6315PRTArtificial Sequencepeptide of
consensus V2 region of HIV-1 subtype CRF02_AG 63Arg Asp Lys Lys Gln
Lys Val Tyr Ala Leu Phe Tyr Arg Leu Asp 1 5 10 15 6415PRTArtificial
Sequencepeptide of consensus V2 region of HIV-1 subtype CRF02_AG
64Thr Glu Leu Arg Asp Lys Lys Gln Lys Val Tyr Ala Leu Phe Tyr 1 5
10 15 6515PRTArtificial Sequencepeptide of consensus V2 region of
HIV-1 subtype CRF02_AG 65Asn Met Thr Thr Glu Leu Arg Asp Lys Lys
Gln Lys Val Tyr Ala 1 5 10 15 6642PRTArtificial Sequencepeptide V2
sequence in the recombinant ALVAC priming immunogen 66Cys Ser Phe
Asn Met Thr Thr Glu Leu Arg Asp Lys Lys Gln Lys Val 1 5 10 15 His
Ala Leu Phe Tyr Lys Leu Asp Ile Val Pro Ile Glu Asp Asn Thr 20 25
30 Ser Ser Ser Glu Tyr Arg Leu Ile Asn Cys 35 40 6742PRTArtificial
Sequencepeptide V2 sequence in the protein boosting gp120 immunogen
AIDSVAX E 67Cys Ser Phe Asn Met Thr Thr Glu Leu Arg Asp Lys Lys Gln
Lys Val 1 5 10 15 His Ala Leu Phe Tyr Lys Leu Asp Ile Val Pro Ile
Glu Asp Asn Asn 20 25 30 Asp Ser Ser Glu Tyr Arg Leu Ile Asn Cys 35
40 6840PRTArtificial Sequencepeptide V2 sequence in the protein
boosting gp120 immunogen AIDSVAX B 68Cys Ser Phe Asn Ile Thr Thr
Ser Ile Gly Asp Lys Met Gln Lys Glu 1 5 10 15 Tyr Ala Leu Leu Tyr
Lys Leu Asp Ile Glu Pro Ile Asp Asn Asp Ser 20 25 30 Thr Ser Tyr
Arg Leu Ile Ser Cys 35 40 6942PRTArtificial Sequencepeptide of
cyclic V2 scrambled crown 69Cys Ser Phe Asn Met Thr Thr Glu Leu Arg
Asp Lys Gln Val Leu Phe 1 5 10 15 Lys Asp Ile His Lys Ile Val Lys
Pro Leu Tyr Ala Glu Asp Asn Thr 20 25 30 Ser Ser Ser Glu Tyr Arg
Leu Ile Asn Cys 35 40 7042PRTArtificial Sequencepeptide of cyclic
V2 scrambled flanks 70Cys Glu Asn Leu Thr Asp Lys Met Phe Thr Ser
Arg Lys Gln Lys Val 1 5 10 15 His Ala Leu Phe Tyr Lys Leu Asp Ile
Val Pro Ile Ser Glu Ser Arg 20 25 30 Leu Asp Glu Thr Asn Tyr Asn
Ile Ser Cys 35 40 7182PRTArtificial Sequencepeptide of gp70-VIV2
from subtype B case A2 71Cys Val Thr Leu Asn Cys Ile Asp Leu Arg
Asn Ala Thr Asn Ala Thr 1 5 10 15 Ser Asn Ser Asn Thr Thr Asn Thr
Thr Ser Ser Ser Gly Gly Leu Met 20 25 30 Met Glu Gln Gly Glu Ile
Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser 35 40 45 Ile Arg Asp Lys
Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp 50 55 60 Ile Val
Pro Ile Asp Asn Pro Lys Asn Ser Thr Asn Tyr Arg Leu Ile 65 70 75 80
Ser Cys 7219PRTArtificial Sequencepeptide of V2 A244-92TH023 72Asn
Met Thr Thr Glu Leu Arg Asp Lys Lys Gln Lys Val His Ala Leu 1 5 10
15 Phe Tyr Lys 7323PRTArtificial Sequencepeptide of V2 MN 73Ile Thr
Thr Ser Ile Gly Asp Lys Met Gln Lys Glu Tyr Ala Leu Leu 1 5 10 15
Tyr Lys Leu Asp Ile Glu Pro 20 7421PRTArtificial Sequencepeptide of
V2 K178 74Lys Lys Lys Val His Ala Leu Phe Tyr Lys Leu Asp Ile Val
Pro Ile 1 5 10 15 Glu Asp Lys Lys Lys 20 757PRTArtificial
Sequencepeptide of cathepsin D cleavage site from V2 of MN 75Gln
Lys Glu Tyr Ala Leu Leu 1 5 767PRTArtificial Sequencepeptide of V2
of A244 76Gln Lys Val His Ala Leu Phe 1 5
* * * * *