Generation and Characterization of a Bivalent HIV-1 Subtype C gp120 Protein Boost for Proof-of-Concept HIV Vaccine Efficacy Trials in Southern Africa

Abstract

The viral envelope glycoprotein (Env) is the major target for antibody (Ab)-mediated vaccine development against the Human Immunodeficiency Virus type 1 (HIV-1). Although several recombinant Env antigens have been evaluated in clinical trials, only the surface glycoprotein, gp120, (from HIV-1 subtype B, MN, and subtype CRF_01AE, A244) used in the ALVAC prime-AIDSVAX gp120 boost RV144 Phase III HIV vaccine trial was shown to contribute to protective efficacy, although modest and short-lived. Hence, for clinical trials in southern Africa, a bivalent protein boost of HIV-1 subtype C gp120 antigens composed of two complementary gp120s, from the TV1.C (chronic) and 1086.C (transmitted founder) HIV-1 strains, was selected. Stable Chinese Hamster Cell (CHO) cell lines expressing these gp120s were generated, scalable purification methods were developed, and a detailed analytical analysis of the purified proteins was conducted that showed differences and complementarity in the antigenicity, glycan occupancy, and glycan content of the two gp120 molecules. Moreover, mass spectrometry revealed some disulfide heterogeneity in the expressed proteins, particularly in V1V2-C1 region and most prominently in the TV1 gp120 dimers. These dimers not only lacked binding to certain key CD4 binding site (CD4bs) and V1V2 epitope-directed ligands but also elicited reduced Ab responses directed to those epitopes, in contrast to monomeric gp120, following immunization of rabbits. Both monomeric and dimeric gp120s elicited similarly high titer Tier 1 neutralizing Abs as measured in standard virus neutralization assays. These results provide support for clinical evaluations of bivalent preparations of purified monomeric TV1.C and 1086.C gp120 proteins.

Fig 1. Analysis of purified TV1.C gp120 revealed the presence of a dimeric fraction.

Panel A: SDS-PAGE conducted under reduced and non-reduced conditions. Panel B: SEC-HPLC showing non-reduced (upper panel) and reduced (lower panel) conditions. Results confirm the presence of the TV1.C gp120 dimeric form; SEC-HPLC, under reducing, but otherwise native conditions, showed that the reduced gp120 monomer and dimer form a homogenous population. Due to the very low amount of dimeric gp120 protein for 1086.C, comparison of reduced and non-reduced conditions is not shown.

Fig 2. Characterization of potential N-linked glycosylation sites (PNGS).

The glycan compositions (in percentages) of TV1.C (A) and 1086.C (B) glycopeptides were sorted and broadly grouped based on criteria described previously [34]. Here, the glycan profile of each glycopeptide with either single or multiple glycosylation sites were represented by a pair of bars denoting the percentage of the type of glycan: high mannose (red) or processed (blue), according to Env sequence position, as indicated in S2 Table (TV1.C gp120) and 3 (1086.C gp120). (See also S2 and S3 Tables for more detailed information on the absolute occupancies at each site). The asterisks denote sites containing mannose phosphate.

Fig 3. Disulfide bonds identified for TV1.C gp120 monomer and dimer highlighting that the disulfide bonding pattern leading to dimer formation is more complex than what is observed for the monomeric protein.

A similar analysis was not performed for 1086.C gp120 as it was only a very minor species in the preparations.

Fig 4. Binding of purified gp120 proteins to polyclonal sera from 34 HIV+ South African volunteers (filled symbols correspond to TV1.C gp120, open symbols to 1086.C gp120; the line indicates the mean value from triplicate measurements).

All sera bound with high and comparable affinities to both gp120s, with the only exception of SA-C61 which showed higher affinity for TV1.C gp120.

Fig 5. Comparative immunogenicity of TV1.C and 1086.C gp120 monomers and dimers.

Rabbit immune sera collected post-3^rd immunization were evaluated. Serum Ab binding titers (panel A) and avidities (panel B) elicited by monomeric and dimeric fractions of TV1.C and 1086.C gp120s. Strain matched gp120 proteins were used for the binding and avidity analyses. Virus neutralization ID50 titers elicitied by monomeric (solid symbols) and dimeric (open symbols) fractions of TV1.C (panel C) and 1086.C (panel D) against a tier 1 and tier 2 HIV-1 pseudoviruses. No statistically significant differences were observed.

Fig 6. Comparison of epitope-specific binding Abs in rabbit immune sera following immunization with monomeric or dimeric TV1.C and 1086.C gp120s.

V3-specific reactivity was used as a control and, as expected, showed similar binding to monomeric and dimeric gp120s. Monomeric gp120 elicited higher titers of conformational Abs directed against V1V2 and CD4BS epitopes (Ns = not significant; * = p<0.01; ** = p<0.001).