V1V2-specific complement activating serum IgG as a correlate of reduced HIV-1 infection risk in RV144

Abstract

Non-neutralizing IgG to the V1V2 loop of HIV-1 gp120 correlates with a decreased risk of HIV-1 infection but the mechanism of protection remains unknown. This V1V2 IgG correlate was identified in RV144 Thai trial vaccine recipients, who were primed with a canarypox vector expressing membrane-bound gp120 (vCP1521) and boosted with vCP1521 plus a mixture gp120 proteins from clade B and clade CRF01_AE (B/E gp120). We sought to determine whether the mechanism of vaccine protection might involve antibody-dependent complement activation. Complement activation was measured as a function of complement component C3d deposition on V1V2-coated beads in the presence of RV144 sera. Variable levels of complement activation were detected two weeks post final boosting in RV144, which is when the V1V2 IgG correlate was identified. The magnitude of complement activation correlated with V1V2-specific serum IgG and was stronger and more common in RV144 than in HIV-1 infected individuals and two related HIV-1 vaccine trials, VAX003 and VAX004, where no protection was seen. After adjusting for gp120 IgA, V1V2 IgG, gender, and risk score, complement activation by case-control plasmas from RV144 correlated inversely with a reduced risk of HIV-1 infection, with odds ratio for positive versus negative response to TH023-V1V2 0.42 (95% CI 0.18 to 0.99, p = 0.048) and to A244-V1V2 0.49 (95% CI 0.21 to 1.10, p = 0.085). These results suggest that complement activity may have contributed in part to modest protection against the acquisition of HIV-1 infection seen in the RV144 trial.

Fig 1. V1V2 sequences in the gp70 scaffolds.

Shown is an amino acid sequence alignment of the V1V2 and flanking regions used as gp70-V1V2 scaffolds (with the exception of GNE8) to assess complement-activating antibodies. The hotspot peptide region (aa 165–178) targeted by V2 antibodies in RV144 [6, 7] is underlined. Amino acids in this hotspot that differ from TH023.6 and A244 are shown in boldface type. Lysine (K) at position 169 and valine (V) at position 172 are indicated by a solid diamond. The sequence for GNE8 used in VAX003 is shown as a reference but was not made as a gp70-V1V2 to assess complement activation.

Fig 2. V1V2-specific complement activating antibodies in vaccine recipients and HIV-1-infected individuals.

Samples obtained post-immunization or post-infection were assayed with gp70 and gp70-V1V2 scaffolds in the presence of either NHS as a source of complement (black open circles) or C3-deficient human serum (gray circles). Pre-immunization samples from vaccine recipients were assayed in the presence of normal human serum (black solid circles). Magnitude of complement activation is expressed as mean fluorescence intensity (MFI) of C3d detection for RV144 (A), VAX003 (B), VAX004 (C) and HIV-1-infected individuals (D). Note that post-immunization samples in VAX004 were not assayed in the presence of C3-deficient human serum. E. Percent of positive reactions in RV144, VAX003, VAX004 (n = 24 each) and CRF01_AE HIV-1-infected individuals (n = 9). Values >2x the highest negative control value were considered positive (values above the horizontal lines). Pre-immune sera from vaccine recipients assayed in the presence of NHS served as negative controls for RV144, VAX003 and VAX004. Assays in the presence of C3-deficient human serum served as negative controls for HIV-1-infected individuals.

Fig 3. Complement activation requires a critical threshold level of V1V2-specific IgG.

MFI values for complement activation by RV144 post-immune plasmas (visit 8) assayed in the presence of NHS were adjusted for non-specific activity by subtracting the MFI of corresponding pre-immune samples (visit 1) assayed in NHS (both MFI values in Fig 2A). The adjusted MFI values are plotted against the corresponding MFI values for V1V2-specific IgG in post-immune samples (after subtraction of non-specific IgG binding activity in pre-immune samples). Shown are results for individual antigens (A-D) and aggregate results (E) pooling over the four antigens for gp70-V1V2 of 92TH023, A244, MN and Ce1086. (F) Adjusted MFI values are plotted against corresponding neutralization titers against 92TH023 (black) and MN (grey).

Fig 4. RV144 case-control assays for V1V2-specific complement activating antibodies.

Plasma obtained 2 weeks post final boosting (week 26) from 41 vaccine recipients (cases) who acquired HIV-1 infection after week 26, and from an additional 205 vaccine recipients (controls) who had not acquired infection by the end of the trial (month 42) were assayed for complement activation against V1V2 of the three vaccine strains (92TH023, A244 and MN). Week 26 plasma from 20 placebo recipients who acquired HIV-1 infection after week 26, and 20 placebo recipients who remained uninfected at the end of the trial were included as negative controls. Readouts are shown as box plots of reactivity with each of the 3 V1V2-scaffold antigens with plasma from the 286 case-control specimens. Box plots show the 25th percentile (lower edge of the box), 50th percentile (horizontal line in the box), and 75th percentile (upper edge of the box). Participants are stratified according to HIV-1 infection status and treatment assignment. Gender and immune response categories are indicated by the color and shape of the points. Negative, Medium, and High are defined are defined in the Methods section and are divided by the gray shaded horizontal bands.

Fig 5. Correlation between RV144 case-control readouts for three V1V2-specific complement activating antibody specificities (92TH023, A244, MN) and primary IgA and IgG V1V2 readouts.

The log-transformed MFI or OD values are displayed in pairwise scatter plots below the diagonal or as a histogram on the diagonal. For each pair of readouts the Spearman correlation is displayed above the diagonal. Scatter plots include a blue loess smooth line. The primary IgA and IgG V1V2 primary variables were studied in the original case-control analysis [2].

Fig 6. Association of complement activation with the primary ADCC variable in RV144.

Shown are associations between complement activation and ADCC activity against CM2544, MN.3 and TH023.