Breadth of neutralizing antibodies elicited by stable, homogeneous clade A and clade C HIV-1 gp140 envelope trimers in guinea pigs

Abstract

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.

FIG. 1.

Biochemical analysis of the clade C trimer. (A) The purified CZA97.012 (clade C) gp140 trimer was resolved by gel filtration chromatography on Superpose 6 columns. The apparent molecular mass was calculated by using the standards thyroglobulin (670 kDa), ferritin (440 kDa), and catalase (232 kDa). (Inset) Peak fractions were pooled and analyzed by SDS-PAGE. (B) The clade C trimer was treated with various concentrations (lanes 1 to 7, 0, 0.05, 0.25, 0.5, 1, 2, and 5 mM, respectively) of ethylene glycol bis(succinimidylsuccinate). Cross-linked products were analyzed by SDS-PAGE by using a 4% gel (lane 8, the gp140 trimer under nonreducing conditions). The molecular standard was cross-linked phosphorylase b (Sigma). Similar analyses were previously reported for the purified clade A 92UG037.8 gp140 trimer (17).

FIG. 2.

ELISA titers against gp140 in guinea pig sera. Sera obtained 4 weeks after each immunization were tested in endpoint ELISAs against the clade A and clade C trimer antigens in the clade A (A) and clade C (B) vaccinated guinea pigs. Data are presented as geometric mean titers at each time point ± standard deviations. The horizontal line indicates the background threshold.

FIG. 3.

Summary of NAb titers against tier 2 viruses. NAb ID₅₀ titers against six clade A (red), clade B (blue), and clade C (green) tier 2 primary isolates are summarized for each guinea pig. (A) Preimmunization. (B) Postimmunization. The horizontal line indicates a titer of 60 as the threshold for positivity.

FIG. 4.

Sample NAb data for purified serum IgG against the tier 2 clade C virus ZM109F.PB4. Serial dilutions of purified IgG from clade A (A) or clade C (B) trimer-immunized guinea pigs and naïve control animals were tested for NAb activity against the tier 2 clade C virus ZM109F.PB4.

FIG. 5.

Variable loop peptide inhibition experiments. (A) Titrations of purified IgG obtained from the sera of representative animals immunized with the clade A (guinea pig 5) or clade C (guinea pig 10) trimer were preincubated with linear V3 loop or scrambled peptides at 20 μg/ml and then tested in NAb assays against the SF162.LS (tier 1-B), ZM109F.PB4 (tier 2-C), and 6535.3 (tier 2-B) viruses. The data shown in the graph are for a single IgG concentration of 500 μg/ml. (B) Sequence alignment of the 92UG037.8 and CZA97012 V1, V2, and V3 peptide loops. Amino acid residues highlighted in gray indicate sequence homology.

FIG. 6.

ELISA titers against gp140 following heterologous prime/boost vaccination regimens. Sera were obtained 4 weeks after immunization with the clade A (A and C) or the clade C (B and D) trimer in the context of DNA/protein (A and B) or DNA/rAd26 (C and D) groups. Antibody titers were assessed by ELISA. Graphs show geometric mean titers for each time point ± standard deviations. Immunizations 1, 2, and 3 reflect DNA priming. 3* indicates ELISA titers after 3 immunizations but prior to boosting. 4 or 4-6 indicates rAd26 or protein boosting, respectively. The horizontal line indicates the background threshold.