The authors have declared that no competing interests exist.
Conceived and designed the experiments: SZP TC NK GT MLR PB RK DEM JRM BFH JHK MdS NLM RJO DM. Performed the experiments: CW EB RTB XS GT CA MR. Analyzed the data: ACdC RG BK PBG. Contributed reagents/materials/analysis tools: SZP TC NK MLR GT PB AP MdS VN JHK SN SR-N. Wrote the paper: SZP.
The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.
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%
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
Case-control specimens consisted of specimens drawn two weeks after the last immunization from 41 infected vaccinees (cases) and from 205 frequency-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 (Abs) reactive with gp70-V1V2, a scaffolded protein carrying the first and second variable regions of an HIV-1 gp120 envelope glycoprotein fused to murine leukemia virus gp70. Levels of Abs specific for gp70-V1V2 were correlated
The participation of the V2 region of gp120 in the infectious process and the role of V2-specific Abs in protection from infection have been the subject of investigation and controversy for nearly two decades. Although, by definition, “variable” regions vary in amino acid (AA) sequence, many residues in these regions do not vary, or tolerate only conservative changes
Abs specific for V2 occur in only ∼25–40% of HIV-infected individuals
The RV144 clinical vaccine trial was registered with ClinicalTrials.gov; it was assigned NCT00223080 as the registration number. Written informed consent and counseling was conducted as described previously
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, BFH), 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
This assay was previously described
The superscripts denote the following: a “Cyclic V2 (AAs 157–198)” was used in assays in three labs as shown. Throughout this table, blue V2 sequences are identical to the subtype E 92TH023 used in the prime; gray represent scrambled sequences or linkers; brown represents the sequence the central AAs in an extremely polar V2 in subtype A strain QB585.2102M.Ev1v5.C (
This assay was previously described
This method was previously described
V2
Direct binding ELISAs were conducted as previously described
The arrays measured reactivity with 15-mer linear peptides with 12 residue overlaps. Raw peptide microarray data were processed and analyzed as described in
Measurements were conducted with a Biacore T100 as previously described
These assays were performed as previously described
Immune parameters measured two weeks after the last immunizing dose were assessed as correlates of subsequent infection risk using the previously described statistical analysis plan
The lasso model selection procedure
Quantitative | Categorical | ||||||
Assay | Investigator | Institution | Response Rate | OR |
P value | OR |
P value |
|
Nicos Karasavvas | AFRIMS | |||||
Cyclic V2 (AAs 157–198) | 0.47 | NA | NA | 0.82 | 0.63 | ||
Cyclic V2 scrambled mid-region | 0.00 | NA | NA | NA | NA | ||
Cyclic V2 scrambled flanks | 0.91 | 0.76 | 0.10 | 0.84 | 0.66 | ||
|
Mangala Rao | USMHRP | |||||
Cyclic V2 (AAs 157–198) | NA | 0.81 | 0.24 | 0.84 | 0.66 | ||
Cyclic V2 scrambled mid-region | NA | 0.79 | 0.18 | 0.90 | 0.80 | ||
|
Susan Zolla-Pazner | New York University | |||||
Cyclic V2 (AAs 157–198) | 0.95 | 0.82 | 0.26 | 0.65 | 0.31 | ||
Biotin V2 Peptide 6 (AAs 165–178) | 0.76 | 0.95 | 0.80 | 0.85 | 0.76 | ||
gp70-V1V2 | 0.64 | 0.70 | 0.06 | 0.43 | 0.06 | ||
|
Philip Berman | University of California, Santa Cruz | |||||
V2 MN peptide | 0.10 | NA | NA | 0.41 | 0.25 | ||
V2 A244-92TH023 peptide | 0.95 | 0.90 | 0.57 | 0.88 | 0.74 | ||
|
Georgia Tomaras | Duke University | |||||
IgA V2 A244 K178 peptide | 0.06 | NA | NA | 0.79 | 0.77 | ||
IgG V2 A244 K178 peptide | 0.93 | NA | NA | 1.02 | 0.95 | ||
|
David Montefiori | Duke University | |||||
V2 Hotspot analysis | NA | 0.64 | 0.03 | 0.32 | 0.02 |
Estimated 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
Estimated 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 gp70-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.
Estimated 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 mid-region (ELISA) which has no positive responses.
In RV144, the V2 sequence in the recombinant ALVAC priming immunogen derived from subtype E strain 92TH023 was:
157
The V2 sequence in the protein boosting gp120 immunogen AIDSVAX E (strain A244) was:
The V2 sequence of the protein boosting gp120 immunogen AIDVAX B (strain MN) was:
Insertion of periods in the sequences allows for alignment. Numbering shown and used throughout this report is that assigned to strain HxB2
The antigenic reactivity of the V2 region in AIDSVAX B and E was assessed using human anti-V2 mAbs 697D and 2158
The pink dashed line represents twice background levels.
A gp70-V1V2 scaffolded protein carrying the V1 and V2 variable regions from a subtype B strain, case A2, was previously described
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 >0.283, 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).
The frequency of V2 responses detected with pilot study specimens derived from vaccinees varied with the assay used, ranging from 6% for IgA Abs reactive with a linear V2 peptide (K178) when measured by Luminex (see
For fine mapping of linear V2 epitopes recognized by Abs in the plasma of RV144 vaccinees, four 21-mer peptides (Peptides 1–4 in
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 Prism, with “whiskers” showing the minimum and maximum responses. Panel A: Four 21-mer N-terminal biotinylated peptides (Peptides 1–4) were selected on the basis of a bioinformatics analysis of V2 sequences from the LANL HIV Database (see text). Panel B: A second peptide panel designed upon inspection of the AAs in Peptides 1–4 in Panel A revealed AAs that distinguish Peptides 1 and 3 from 2 and 4; 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 Abs, a spacer of three glycines was inserted between the biotin tag at the N-terminus of the peptide and the V2 sequences.
As illustrated by the ELISA data (
The ribbon backbone of AAs 165–176, which are identical to Peptide 6 (in
To distinguish the AAs that play a critical role in determining anti-V2 Ab reactivity, a further series of peptides was designed.
Based on the pilot studies, six assay types were chosen for measuring 13 variables with case-control specimens (
For the univariate analysis, as in the multivariate analysis
Data are derived from the categorical analyses shown in
The upper panel shows an aggregate response across all sub-types averaged across vaccinees as a function of the V2 sequence in subtype B strain HxB2. An individual aggregate response is computed using a sliding window mean statistic of 9 AAs, i.e., peptides with HxB2 positions of 9 contiguous AAs averaged together. In the lower portion of the figure, the actual sequence of each of the overlapping 15-mers 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 colored according to their average response across all vaccinees, with a scale of 0 (white) to 1.76 (red). All responses are calculated using normalized intensities and by subtracting the intensities of baseline pre-bleeds.
In this study, we have probed the results achieved with the entire panel of V2 assays used in the RV144 pilot and case-control studies in order to understand more fully the nature of the V2 Ab 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
Our studies document at least two types of V2 Abs induced by the RV144 vaccine: Abs reactive with a scaffolded V1V2 protein, gp70-V1V2, and Abs specific for linear and cyclic V2 peptides. Studies with human mAbs suggest that these may be non-overlapping Ab populations since human mAbs such as 697D and 2158 react with conformational V1V2 epitope(s) carried by glycosylated gp70-V1V2 containing a subtype B sequence, but not with linear or cyclic (non-glycosylated) peptides
The reactivity of vaccinees' Abs with overlapping V2 peptides also correlated with reduced risk of infection (
It is noteworthy that the single primary variable showing an inverse correlate of infection risk in the RV144 case-control study was Ab reactivity with gp70-V1V2 which contains the V1V2 sequence of case A2, a subtype B strain
The explanation for the strong induction of V2 Abs 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 may be due to the fact that the V2 domain of A244 rgp120 is more immunogenic than that of MN rgp120, and that it usually takes three immunizations with protein, given at appropriate intervals, to elicit good seroconversion to the V2 domain
Finally, the mechanisms by which anti-V2 Abs may reduce HIV infection have yet to be understood. As noted, anti-V2 mAbs can neutralize many Tier 1 pseudoviruses in the TZM.bl assay
We acknowledge, in particular, Ellen Turk for her technical expertise in the acquisition of the peptide microarray data.