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Comparative Study
. 2012 Jul 24;109(30):12111-6.
doi: 10.1073/pnas.1204533109. Epub 2012 Jul 5.

HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120

James M Kovacs  1 , Joseph P Nkolola, Hanqin Peng, Ann Cheung, James Perry, Caroline A Miller, Michael S Seaman, Dan H Barouch, Bing Chen
Affiliations
Comparative Study

HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120

James M Kovacs et al. Proc Natl Acad Sci U S A. .

Abstract

HIV-1 envelope glycoprotein is the primary target for HIV-1-specific antibodies. The native HIV-1 envelope spike on the virion surface is a trimer, but trimeric gp140 and monomeric gp120 currently are believed to induce comparable immune responses. Indeed, most studies on the immunogenicity of HIV-1 envelope oligomers have revealed only marginal improvement over monomers. We report here that suitably prepared envelope trimers have nearly all the antigenic properties expected for native viral spikes. These stable, rigorously homogenous trimers have antigenic properties markedly different from those of monomeric gp120s derived from the same sequences, and they induce potent neutralizing antibody responses for a cross-clade set of tier 1 and tier 2 viruses with titers substantially higher than those elicited by the corresponding gp120 monomers. These results, which demonstrate that there are relevant immunologic differences between monomers and high-quality envelope trimers, have important implications for HIV-1 vaccine development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Production of stable and homogenous HIV-1 gp120 and gp140 proteins in 293T cells. (A) His-tagged gp140 proteins derived from the HIV-1 92UG037.8 (clade A) and CZA97012 (clade C) isolates and their corresponding full-length gp120s were purified from supernatants of 293T cells stably transfected with the expression constructs of these proteins. The purified gp120 (Left) and gp140 (Right) proteins were resolved by gel-filtration chromatography on a Superdex 200 and a Superose 6 column, respectively. The molecular weight standards include thyoglobulin (670 kDa), ferritin (440 kDa), γ-globulin (158 kDa), and ovalbumin (44 kDa). Peak fractions were pooled and analyzed by Coomassie-stained SDS/PAGE (Insets). (B) Soluble 4D CD4 was immobilized on a CM-5 chip, and various concentrations (50–1,000 nM) of 92UG037.8 gp120 or 92UG037.8 gp140 were passed over the chip surface. Binding kinetics was evaluated using a 1:1 Langmuir binding model; binding constants are summarized in Table S2. The sensorgrams are shown in black and the fits in green. All injections were carried out in duplicate and gave essentially identical results. Only one of the duplicates is shown.
Fig. 2.
Fig. 2.
Interactions of gp140 and gp120 with CD4 BS antibodies and CD4i antibody. (A) Fab of the CD4 BS antibody VRC01 or b6 was captured on a surface coated with human Fab binder, and varous concentratons(50–1,000 nM) of 92UG037.8 gp120 or 92UG037.8 gp140 were passed over the chip surface. (B) Fab of the CD4i antibody 17b was immobilized on a chip surface using human Fab binder. Then 92UG037.8 gp120 and 92UG037.8 gp140, purified 92UG037.8 gp120-CD4 and 92UG037.8 gp140-CD4 complexes, or purified 92UG037.8 gp120-VRC01 Fab and 92UG037.8 gp140-VRC01 Fab complexes at various concentrations (50–1,000 nM) were passed over the 17b surface. Binding kinetics was evaluated using a 1:1 Langmuir binding model; binding constants are summarized in Table S2. The sensorgrams are shown in black and the fits in green. RU, response units.
Fig. 3.
Fig. 3.
Binding of gp140 and gp120 to bNAbs against the quaternary epitopes. (A) The bNAb PG9 IgG (Left) or PG16 IgG (Right) was captured on a chip surface coated with protein A. Sensorgrams were recorded by passing 92UG037.8 gp140 trimer (1 μM; in red); C97ZA012 gp140 trimer (1 μM; in blue); 92UG037.8 gp120 monomer (1 μM; in dark red); C97ZA012 gp120 monomer (1 μM; in dark blue); or A244 gp120 gD(+) monomer (1 μM; in black) over the antibody surfaces. The apparent differences in response units are caused partly by differences in molecular masses of the flowing analytes. (B) PG9 (Left) or PG16 IgG (Right) was immobilized on the chip surface using human Fab binder. Then various concentrations (50–1,000 nM) of 92UG037.8 gp140 or C97ZA012 gp140 were passed over the antibody surfaces. Binding kinetics was evaluated using a 1:1 Langmuir binding model; binding constants are summarized in Table S2. The sensorgrams are shown in black and the fits in green.
Fig. 4.
Fig. 4.
Immunogenicity of the C97ZA012 trimeric gp140 and monomeric gp120 in guinea pigs. (A) Sera of guinea pigs, vaccinated with either C97ZA012 gp120 or gp140 obtained prevaccination (Pre) and 4 wk after each immunization, were tested in endpoint ELISAs against all 92UG037.8 and C97ZA012 antigens as indicated. Binding antibody responses against 92UG037.8 gp120 monomer (white bars), 92UG037.8 gp140 trimer (black bars), C97ZA012 gp120 monomer (light gray bars), and C97ZA012 gp140 trimer (dark gray bars) are presented as geometric mean titers at each time point ± SD. Horizontal lines indicate background threshold. (B) Guinea pig sera obtained prevaccination (Pre-Bleed), 4 wk after the third vaccination (Post-3×), and 4 wk after the sixth vaccination (Post-6×) were tested against a multiclade panel of tier 1 neutralization-sensitive isolates, including clade A (DJ263.8), clade B (SF162.LS and Bal.26), and cladeC (MW965.26, TV1.21, ZM109F, and ZM197M) HIV-1 Env pseudoviruses and MuLV (negative control) in TZM.bl neutralization assays. NAb titers induced by C97ZA012 gp120 and C97ZA012 gp140 are represented graphically. *P < 0.05; **P < 0.01; unpaired two-tailed t test. Horizontal bars indicate median titers. (C) Sera obtained prevaccination (Pre) and 4 wk after the sixth vaccination (Post-6×) were tested against three tier 2 clade C isolates (Du422, Ce1086_B2, and Ce2010_F5 IMC viruses) in A3R5 neutralization assays. NAb titers induced by C97ZA012 gp120 and C97ZA012 gp140 are represented y graphically. *P < 0.05; unpaired two-tailed t test.
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