Comparable Antigenicity and Immunogenicity of Oligomeric Forms of a Novel, Acute HIV-1 Subtype C gp145 Envelope for Use in Preclinical and Clinical Vaccine Research

Abstract

Eliciting broadly reactive functional antibodies remains a challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development that is complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C, and novel antigenic properties have been described for subtype C Env proteins. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an infected person in the acute phase (Fiebig stage I/II) was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane-proximal external region (MPER), extended by a polylysine tail. Soluble gp145 was enriched for trimers that yielded the expected "fan blade" motifs when visualized by cryoelectron microscopy. CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16, and VRC01 HIV-1 neutralizing monoclonal antibodies (MAbs), as well as the V1/V2-specific PGT121, 697, 2158, and 2297 MAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimers, and larger multimers elicited similar levels of cross-subtype binding and neutralizing antibodies to tier 1 and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus but did inhibit the homologous infectious molecular clone in a peripheral blood mononuclear cell (PBMC) assay. This Env is currently in good manufacturing practice (GMP) production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine.

Importance: At present, the product pipeline for HIV vaccines is insufficient and is limited by inadequate capacity to produce large quantities of vaccine to standards required for human clinical trials. Such products are required to evaluate critical questions of vaccine formulation, route, dosing, and schedule, as well as to establish vaccine efficacy. The gp145 Env protein presented in this study forms physical trimers, binds to many of the well-characterized broad neutralizing MAbs that target conserved Env epitopes, and induce cross-subtype neutralizing antibodies as measured in both cell line and primary cell assays. This subtype C Env gp145 protein is currently undergoing good manufacturing practice production for use as a reagent for preclinical studies and for human clinical research. This product will serve as a reagent for comparative studies and may represent a next-generation candidate HIV immunogen.

FIG 1

Purified gp145 was fractionated and analyzed by BN-PAGE (A; lane 1, unfractionated gp145; lane 2, higher-order oligomer; lane 3, trimer; lane 4 dimer; lane 5, molecular mass marker) and SDS-PAGE under nonreducing conditions (B; lane 1, reduced unfractionated gp145; lane 2, unfractionated gp145; lane 3, cross-linked unfractionated gp145; lane 4, cross-linked higher-order oligomers; lane 5, cross-linked trimer; lane 6, cross-linked dimer). EGS-cross-linked phosphorylase b was run in lane 7 as a protein molecular mass marker. The positions and natures of the deduced oligomeric species are indicated by the arrows at the left. (C) Averaged, top-down views of gp145 monomer (part 1), dimer (part 2), trimer (part 3), and oligomer (part 4), as visualized by cryo-EM.

FIG 2

CO6980v0c22 gp145 binding to V2-specific antibodies and α4β7. (A) HIV V2-specific antibodies were tested by ELISA for binding to CO6980v0c22 gp145. MAbs 697, 2158, and 2297 bound (black symbols), while MAbs 1357, 1361, 1393, and 830A and nonspecific MAb 1418 did not bind (open symbols). (B) CO6980v0c22 gp145 binding to α4β7 was determined by using flow cytometry to measure the binding of biotinylated gp145 to RPMI 8866 cells. gp145 binding (blue peak) was observed as a shift from the cell control without gp145 (shaded area) and was inhibited by the incubation of RPMI 8866 cells with α4 antibody HP2/1 before the addition of gp145 (red peak). A single representative experiment is shown. OD, optical density.

FIG 3

Binding antibody titers elicited by vaccination with CO6980v0c22 gp145. (A) Geometric mean binding antibody titers were determined with week 10 sera collected 2 weeks after the final immunization in study 1. Immunogenicity of unfractionated gp145 was evaluated in four different adjuvant formulations: aluminum hydroxide plus gp145 (solid light blue), L(DMPG/MPLA) plus gp145 (blue bars with black dots), L(DMPG/MPLA plus gp145) (hatched bars), and L(PIP/MPLA plus gp145) (solid dark blue bars). Binding antibody responses to the following four Env antigens were tested: IIIB gp41 (subtype B), CO6980v0c22 gp145 (subtype C), C.ZA.1197MB gp120 (subtype C), and CN54 gp140 (subtype C). The data are the geometric mean endpoint titers of individual serum samples from four rabbits in each group assayed in triplicate and the standard error of the mean (SEM). (B and C) Week 0 and 10 sera from nine rabbits. One rabbit in group 5, L(DMPG/MPLA) without gp145; four rabbits in group 1; and four rabbits in group 3 were tested for antibodies binding to the gp70 V1V2 scaffolded antigen by ELISA (B) and a V3 peptide by BLI (C). (D) Study 2 binding antibody responses to the following four Env antigens: cyclic V2 from Con C (subtype C) and CO6980v0c22 (subtype C), C.ZA.1197MB gp120 (subtype C), and CN54 gp140 (subtype C). The bars indicate the responses of animals immunized with unfractionated (solid pink bar) or fractionated gp145 (dimer, red bar with black dots; trimer, hatched bar; higher-order oligomer, solid dark red bar). Antigen for all groups was formulated by mixing gp145 with aluminum hydroxide. The data are the mean endpoint titers of individual serum samples from four rabbits in each group assayed in triplicate and the SEM.

FIG 4

Detection of NAbs elicited by vaccination with CO6980v0c22 gp145. NAbs were detected by both the TZM-bl (A and C) and PBMC (B and D) neutralization assay platforms with viruses from subtypes B (SF162, tier 1A; BZ167, tier 1B; BaL, tier 1B), C (CO6980, tier 2; GS015, tier 1A; ETH2220, tier 1B; MW965, tier 1A), and CRF01_AE (CM235, tier 2; NI1046, tier 2). For panels A and B, week 10 sera were screened at a dilution of 1:40 (TZM-bl) or 1:50 (PBMC); percent neutralization was calculated as the reduction of infection observed in the postimmunization sera compared to the preimmunization sera. The dashed line at 50% represents the negative cutoff for neutralization. The box plots represent the mean percent neutralization and SEM of the four individual rabbits in each group. Individual values were calculated as the mean of two independent experiments. In the graphs in panels C and D, the group mean ID₅₀s from study 2 are presented and the box plots are as described above.