The Thai Phase III HIV Type 1 Vaccine trial (RV144) regimen induces antibodies that target conserved regions within the V2 loop of gp120

Abstract

The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200-3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100-200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169-184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100-800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100-3200) and 3570 (range: 200-12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.

FIG. 1.

Graphic representation of the cyclic V2 loop and alignment of V2 loop amino acid sequences. (A) Amino acid sequence of the cyclic (Cyc) V2 loop of the HIV-1 CRF01_AE 92TH023 strain. The flanks and mid-region are labeled. (B) Alignment of V2 loop amino acid sequences. Sequences that vary from 92TH023 are boxed and the first (157) and last (196) amino acids of the V2 are shown on top of the alignment. Numbering is based on the HXB2 strain. Cyclic V2 peptides were synthesized based on clone 92TH023. Peptides for microarray analysis were based on consensus (Con) sequences and peptides 1–6 represent linear N-linked biotinylated peptides.

FIG. 2.

IgG antibody responses to recombinant HIV-1 Env proteins in RV144 vaccinees and placebo recipients. (A) Antibody responses to recombinant Env proteins gp120 MN, A244, and 92TH023 at visits 1, 8, and 9 determined by ELISA using plasma samples from set Z at a dilution of 1:100. Absorbance at 405 nm is shown on the y-axis. (B) Antibody responses to the same recombinant proteins and visits in placebo recipients. Dotted line indicates the cutoff for the assay in the absence of the capturing protein. Each sample was run in triplicate and data represent the average of two independent experiments. Bars represent the standard error of the mean.

FIG. 3.

Evaluation of antibody responses to overlapping peptides of V2 by peptide microarray analysis. Microarray peptide analysis was done using plasma from 80 HIV-1-uninfected vaccine recipients and 20 placebo recipients (set A). (A) Reactivity of plasma with peptides from seven HIV-1 Group M subtypes (M consensus, A, B, C, D, CRF01, and CRF02). (B) Overall reactivity of RV144 plasma samples to the V2 loop. Peptide positions are shown on the x-axis and percent responses are shown on the y-axis. The peptide positions are aligned with the amino acid sequence of HXB2 protein and the sequence of 92TH023 V2 loop is shown for comparison.

FIG. 4.

Antibody responses to cyclic V2 loop peptides by ELISA and Biacore. (A) Antibody responses at visits 1 and 8 to cyclic peptides Cyc V2, Cyc V2 Scr Fl, and Cyc V2 Scr MR by ELISA. (B) Antibody responses to Cyc V2, Cyc V2 Scr Fl, and Cyc V2 Scr MR by ELISA using 20 infected and four uninfected subjects. (C) Antibody responses to shorter cyclic V2 peptides by Biacore. In Biacore, plasma samples were used at a 1:50 dilution and the values are reported as response units. A representative experiment of three independent experiments is shown. ELISA and Biacore analyses were performed using the identical plasma samples (set Z). In ELISAs, plasma samples were used at a dilution of 1:100 and responses were considered positive if they exceeded 2.5 times the background (absence of capturing peptide) and are indicated by the dotted line. Each sample was tested in triplicate and the results represent the average of at least two independent experiments. Absorbance at 405 nm is shown on the y-axis.

FIG. 5.

Antibody responses to cyclic V2 loop decline over time. (A) Antibody responses to Cyc V2 peptide at visits 1, 8, and 9 by ELISA. Responses were considered positive if they exceeded 2.5 times the background (absence of capturing peptide) and are indicated by the dotted line. (B) Antibody responses to Cyc V2 peptide at visits 1, 8, and 9 by Biacore. Plasma samples were used at a 1:50 dilution and the values are reported as response units. A representative experiment of three independent experiments is shown. ELISA and Biacore analyses were done using plasma sample set Z.

FIG. 6.

Antibody responses to linear V2 loop peptides decline over time. (A) Antibody responses to linear biotinylated peptides 1, 2, 3, 4, and 6 at visits 1, 8, and 9 by ELISA using plasma sample set C. Absorbance at 405 nm is shown on the y-axis. (B) ELISA using the same peptides and plasma of 20 placebos, set C. Plasma was used at a dilution of 1:100 and peptide aa sequences are shown in the right upper corner of (B). Samples were run in duplicate and are the average of two experiments.

FIG. 7.

Antibody responses to cyclic V3 peptide in vaccinated uninfected subjects, HIV-1-infected and HIV-uninfected. Antibody responses to Cyc V3 peptide (strain 92TH023) using plasma samples from RV144 visits 1 and 8 (set Z) and from 20 HIV-1-infected and four HIV-1-uninfected subjects. Plasma samples were used at a starting dilution of 1:100. Cutoff values are indicated by the dotted line. Panels represent the average of at least two independent experiments.

FIG. 8.

Comparison of antibody responses to V2 and V3 loops in RV144 vaccinees and HIV-1 naturally infected subjects. (A) Comparison of antibody responses to gp120 V2 and V3 loops from HIV-1-infected individuals. (B) Antibody responses to gp120 V2 and V3 loops in RV 144 vaccinated individuals. Plasma dilutions were at 1:100 and absorbance at 405 nm is shown on the y-axis. Cutoff values are indicated by the dotted line.