Boosting with AIDSVAX B/E Enhances Env Constant Region 1 and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and Potency

Abstract

Induction of protective antibodies is a critical goal of HIV-1 vaccine development. One strategy is to induce nonneutralizing antibodies (NNAbs) that kill virus-infected cells, as these antibody specificities have been implicated in slowing HIV-1 disease progression and in protection. HIV-1 Env constant region 1 and 2 (C1C2) monoclonal antibodies (MAbs) frequently mediate potent antibody-dependent cellular cytotoxicity (ADCC), making them an important vaccine target. Here, we explore the effect of delayed and repetitive boosting of RV144 vaccine recipients with AIDSVAX B/E on the C1C2-specific MAb repertoire. It was found that boosting increased clonal lineage-specific ADCC breadth and potency. A ligand crystal structure of a vaccine-induced broad and potent ADCC-mediating C1C2-specific MAb showed that it bound a highly conserved Env gp120 epitope. Thus, boosting to affinity mature these types of IgG C1C2-specific antibody responses may be one method by which to make an improved HIV vaccine with higher efficacy than that seen in the RV144 trial.IMPORTANCE Over one million people become infected with HIV-1 each year, making the development of an efficacious HIV-1 vaccine an important unmet medical need. The RV144 human HIV-1 vaccine regimen is the only HIV-1 clinical trial to date to demonstrate vaccine efficacy. An area of focus has been on identifying ways by which to improve upon RV144 vaccine efficacy. The RV305 HIV-1 vaccine regimen was a follow-up boost of RV144 vaccine recipients that occurred 6 to 8 years after the conclusion of RV144. Our study focused on the effect of delayed boosting in humans on the vaccine-induced Env constant region 1 and 2 (C1C2)-specific antibody repertoire. It was found that boosting with an HIV-1 Env vaccine increased C1C2-specific antibody-dependent cellular cytotoxicity potency and breadth.

Keywords: HIV vaccine; antibody function.

FIG 1

Identification of RV305 C1C2-specific MAbs. (A) The C1C2-specific MAbs A32 and C11 were assayed by ELISA for reactivity with full-length AE.A244gp120 or AE.A244g120Δ11. An A32-specific mutant protein was designed (AE.A244g120Δ11 F53S H72L V75A E106K D107H S110A Q114L) to identify A32-like MAb responses. 19B was used as a positive control and CH65 as a negative control. (B) RV305 nonneutralizing MAbs were assayed for A32 blocking by ELISA. (C) RV305 nonneutralizing A32-blockable MAb heavy- and light-chain gene sequence mutation frequencies were analyzed by Cloanalyst (13) and compared to previously published RV144 heavy- and light-chain gene sequence mutation frequencies (percent nucleotide) (12). Statistical significance was determined using a Wilcoxon rank sum test. Red bar represents the means. (D) RV305 nonneutralizing A32-blockable MAbs were assayed by ELISA for binding to AE.A244g120Δ11 and AE.A244g120Δ11 F53S H72L V75A E106K D107H S110A Q114L. Data are expressed as percent binding of the mutant protein relative to the wild type. Shown are the means with standard deviations from two independent experiments.

FIG 2

RV305 boosting increased the apparent affinity and antibody-dependent cellular cytotoxicity breadth and potency of the C1C2-specific RV144-derived DH677 memory B cell clonal lineage. DH677.1 was isolated by AE.A244gp120Δ11-specific single-cell sorting of PBMCs collected from a vaccinee 2 weeks after the final boost in the RV144 vaccine trial. DH677.2, DH677.3, and DH677.4 were isolated by AE.A244gp120Δ11-specific single-cell sorting of PBMCs collected from the same vaccinee after the second AIDSVAX B/E (RV305 group II) boost given in RV305 (∼7 years later). The intermediate ancestor one (IA1), intermediate ancestor two (IA2), intermediate ancestor three (IA3), and unmutated common ancestor (UCA) were inferred using Cloanalyst (13). The MAbs were recombinantly expressed and assayed by surface plasmon resonance for binding to the AIDSVAX B/E proteins AE.A244g120 full length, AE.A244g120Δ11, and B.MNg120Δ11. Shown are the antibody apparent affinity measurements (K_d), expressed in nanomolars. NB, no detectable binding. MAbs were also assayed for ADCC against AE.C235-, B.WITO-, C.TV-1-, C.MW965-, C.1086C-, C.DU151-, and C.DU422-infected CEM.NKR_CCR5 cells. An ADCC score (see Materials and Methods) was used to account for ADCC breadth and potency.

FIG 3

Comparison of the two copies of the DH677.3 Fab-gp120_93TH057 core_e-M48U1 complex and the two Fab copies in the apo Fab structure from the asymmetric unit of crystals. (A) The root mean square deviation (RMSD) between complex copies is 0.946 Å for main-chain residues. (B) The RMSD between the Fab copies in the apo Fab structure is 0.540 Å for main-chain residues. (C) Comparison of the free and bound DH677.3 Fab. The α-carbon backbone diagram of superposition of the structures of DH677.3 Fab alone (dark cyan, heavy chain; light cyan, light chain) and N5-i5 Fab bound to CD4-triggered gp120 (dark brown, heavy chain; light brown, light chain). The average RMSD between free and bound Fabs is 0.818 Å for main-chain residues.

FIG 4

Crystal structure of the DH677.3 Fab-gp120_93TH057 core_e-M48U1 complex. (A) The overall structure of the complex is shown as a ribbon diagram (left) with the molecular surface displayed over the Fab molecule (middle), colored based on electrostatic charge: red, negative; blue, positive. The gp120 outer domain is gray and the inner domain colored to indicate inner domain mobile layers 1 (yellow), 2 (cyan), and 3 (light orange) and the 7-stranded β-sandwich (magenta). Complementary determining regions (CDRs) are in the following colors: CDR H1, light blue; CDR H2, dark green; CDR H3, black; CRL1, light green; CDR L2, brown; CDRL3, blue. A blow-up view shows the network of hydrogen (H) bonds formed at the Fab-gp120 interface. H bonds contributed by side-chain and main-chain atoms of gp120 residues are colored magenta and blue, respectively. (B) Fab buried surface area (BSA) and gp120 residues forming DH677.3 epitope are shaded in blue according to BSA (antibody) and percent conservation of gp120 residues (Env). gp120 main-chain (blue) and side-chain (red) hydrogen bonds (H) and salt bridges (S) are shown above the residue. (C) The DH677.3 Fab-gp120_93TH057 core_e interface. CDRs are shown as ribbons (left) and balls and sticks of residues contributing the binding (right) over the gp120 core. The molecular surface of gp120 is colored as described for panel A (left) and by electrostatic potential (right).

FIG 5

DH677.3 heavy- and light-chain contact residues. MAb side-chain (+) and main-chain (−) contact residues, colored green for hydrophobic, blue for hydrophilic, and black for both, as determined by a 5-Å cutoff value over the corresponding sequence. CDRs are colored as described for Fig. 1, and buried surface residues as determined by PISA are shaded.

FIG 6

Recognition of HIV-1 Env by DH677.3 and other cluster A MAbs. (A) The overlay of DH677.3 and cluster A MAbs A32 and N12-i3 (C11-like) bound to the gp120 core. Crystal structures of the gp120 antigen in complex with the Fab of DH677.3, A32 (PDB code 4YC2), and N12-i3 (PDB code 5W4L), superimposed based on gp120. The d1 and d2 domains of the target cell receptor CD4 were added to replace peptide mimetic M48U1 of the DH677.3 Fab-gp120_93TH057 core_e-M48U1 complex. Molecular surfaces are displayed over Fab molecules and colored in lighter and darker shades of brown, blue, and green for the heavy and light chains of DH677.3, A32, and N12-i3, respectively. A blow-up view shows details of the DH677.3 interaction with the 8-stranded β-sandwich of the gp120 inner domain. The 8th strand (colored in blue) formed by the 11 N-terminal residues of gp120 in the N12-i3 bound conformation (PDB entry 5W4L) was modeled into the DH677.3 Fab-gp120_93TH057 core_e-M48U1 complex. CDR H1 and H2 of DH677.3 are colored light blue and dark green, respectively. (B and C) Comparison of DH677.3, A32, and N12-i3 epitope footprints. (B) The DH677.3 epitope footprint (shown in red) is plotted on the gp120 surface with layers colored, as described for Fig. 1, with the A32 and N12-i2 epitope footprints shown in black. (C) DH677.3, A32, and N12-i3 gp120 contact residues are mapped onto the gp120 sequence. Side-chain (+) and main-chain (−) contact residues are colored green for hydrophobic, blue for hydrophilic, and black for both, as determined by a 5-Å cutoff value over the corresponding sequence. Buried surface residues, as determined by PISA, are shaded. The DH677.3 epitope footprint overlays with the epitopes of both A32 and N12-i3.

FIG 7

Gating strategy for infected cell elimination assay. Cells are gated on forward scatter and side scatter (FSC and SSC, respectively), followed by distinguishing viable cells using NFL-1 (a dead cell marker) and TFL-4 (target cell marker). Target cells are then gated on CD4 and p24. Mock-infected cells are used to determine positioning of the gate for p24⁺. The proportion of p24⁺, p24⁺ CD4⁺, and p24⁺ CD4⁻ target cells in the presence of effectors only, effectors plus negative control, or effectors plus sample was determined.

FIG 8

RV305-derived C1C2-specific MAb DH677.3 is significantly better than A32 at mediating antibody-dependent cellular cytotoxicity against CD4 downmodulated infectious molecular clone (IMC)-infected cells. Cells were infected with clade B and clade C full-length IMC that do not contain a reporter gene. Surface CD4 expression was analyzed by flow cytometry, and p24 expression was measured in live/viable, all p24⁺ (A), p24⁺ CD4⁺ (B), and p24⁺ CD4⁻ (C) IMC-infected cell populations. Data are shown with the means and standard deviations.