A multivalent clade C HIV-1 Env trimer cocktail elicits a higher magnitude of neutralizing antibodies than any individual component

Abstract

The sequence diversity of human immunodeficiency virus type 1 (HIV-1) presents a formidable challenge to the generation of an HIV-1 vaccine. One strategy to address such sequence diversity and to improve the magnitude of neutralizing antibodies (NAbs) is to utilize multivalent mixtures of HIV-1 envelope (Env) immunogens. Here we report the generation and characterization of three novel, acute clade C HIV-1 Env gp140 trimers (459C, 405C, and 939C), each with unique antigenic properties. Among the single trimers tested, 459C elicited the most potent NAb responses in vaccinated guinea pigs. We evaluated the immunogenicity of various mixtures of clade C Env trimers and found that a quadrivalent cocktail of clade C trimers elicited a greater magnitude of NAbs against a panel of tier 1A and 1B viruses than any single clade C trimer alone, demonstrating that the mixture had an advantage over all individual components of the cocktail. These data suggest that vaccination with a mixture of clade C Env trimers represents a promising strategy to augment vaccine-elicited NAb responses.

Importance: It is currently not known how to generate potent NAbs to the diverse circulating HIV-1 Envs by vaccination. One strategy to address this diversity is to utilize mixtures of different soluble HIV-1 envelope proteins. In this study, we generated and characterized three distinct, novel, acute clade C soluble trimers. We vaccinated guinea pigs with single trimers as well as mixtures of trimers, and we found that a mixture of four trimers elicited a greater magnitude of NAbs than any single trimer within the mixture. The results of this study suggest that further development of Env trimer cocktails is warranted.

FIG 1

Acute clade C HIV-1 Env gp140 trimer expression, stability, and homogeneity. (A) Expression levels of novel, acute gp140 envelope protein sequences. Supernatant collected from 293T cells transiently transfected with HIV-1 Env gp140 sequences was assessed for protein expression by Western blotting. (B) Coomassie-stained SDS-PAGE gel of pooled peaks of acute, clade C trimers after a single freeze/thaw cycle or incubation at 4°C for 2 weeks. Trimers are as follows for both SDS-PAGE gels: lanes 1, C97ZA012; lanes 2, 405C; lanes 3, 459C; lanes 4, 939C gp140. (C) Gel filtration chromatography traces of 459C, 405C, and 939C gp140 trimers as run on a Superose 6 column. Molecular mass standards for traces include thyoglobin (670 kDa), ferritin (440 kDa), and γ-globin (158 kDa).

FIG 2

Maximum-likelihood trees and sequence alignments of clade C gp140 sequences. (A) Phylogenetic tree comparing each of the four clade C vaccine envelope (Env) sequences to 489 clade C sequences sampled from the year 2004. Colors indicate the country of origin for each sequence according to the key provided (ZA, South Africa; MW, Malawi; ZM, Zambia; TZ, Tanzania; CN, China; CY, Cyprus; BR, Brazil; GB, Great Britain; X, all other countries sampled; ES, Spain; IN, India; Ccon, consensus C; FR, France; US, United States; ZW, Zimbabwe). (B) Phylogenetic tree comparing each of the four clade C vaccine Env sequences to 506 clade C sequences from South Africa starting from the year 2000 (00). Years of origin are shown in shades of gray according to the key provided. For panels A and B, vaccine Env strains are highlighted in red, the consensus clade C Env sequence is shown in cyan, and the HXB2 sequence (outgroup) is indicated in dark blue. The scale bar indicates phylogenetic distance, with the bar length corresponding to 0.01 genetic change, or nucleotide substitution, per site. (C) Alignment of CD4 binding site contact residues for clade C immunogens, (D) alignment of PG9 contact residues for clade C immunogens, (E) alignment of V3 loop and C-terminal glycan contact residues for clade C immunogens. For panels C, D, and E, sequence alignments were compared to a consensus C sequence and are aligned using HXB2 numbering, with ranking of sequence centrality denoted with red numbers, 1 being most central and 4 being least central. pos, positions.

FIG 3

Presentation of CD4 and CD4i epitopes by acute, clade C trimers. (A) Soluble two-domain CD4 was irreversibly coupled to a CM5 chip, and 459C, 405C, or 939C gp140 was flowed over the chip at concentrations of 62.5 to 1,000 nM. (B to D) Protein A was irreversibly coupled to a CM5 chip, and (B) 17b IgG was captured. HIV-1 Env 459C, 405C, or 939C gp140 was flowed over the bound IgG at a concentration of 1,000 nM in the presence or absence of CD4 bound to the immunogen. 17b binding alone is indicated in red, and CD4 coupled to trimer binding to 17b IgG is shown in blue. VRC01 IgG (C) and 3BNC117 IgG (D) were captured, and HIV-1 Env 459C, 405C, and 939C gp140 trimers were flowed over the bound IgG at concentrations of 62.5 to 1,000 nM. Sensorgrams are presented in black and kinetic fits in green. RU, response units.

FIG 4

Presentation of V3 and glycan-dependent epitopes by acute, clade C trimers. For all experiments, protein A was irreversibly coupled to a CM5 chip and IgGs were captured. HIV-1 Env 459C, 405C, and 939C gp140 trimers were flowed over bound PGT126 IgG (A), PGT121 IgG (B), and 10-1074 IgG (C) at concentrations of 62.5 to 1,000 nM. Sensorgrams are presented in black and kinetic fits in green. RU, response units.

FIG 5

Presentation of V1/V2, glycan-dependent, quaternary-preferring epitopes by acute, clade C trimers and monomers. For all experiments, protein A was irreversibly coupled to a CM5 chip and IgGs were captured. 459C, 405C, and 939C trimers and monomers were flowed over bound PGT145 IgG at concentrations of 62.5 to 1,000 nM. Sensorgrams are presented in black and kinetic fits in green. RU, response units.

FIG 6

Binding antibody titers from guinea pigs vaccinated with clade C trimers. (A) Vaccination scheme for all vaccinated guinea pigs. Animals were vaccinated at weeks 0, 4, and 8 and bled at weeks 0, 4, 8, and 12. (B) Binding antibody titers from guinea pig sera against gp140 antigens after vaccination with clade C trimeric immunogen. Sera were tested in endpoint ELISAs against a panel of trimeric antigens in guinea pigs vaccinated with HIV-1 Env C97ZA012 (n = 14 animals), 459C (n = 10), 405C (n = 5), and 939C (n = 5) gp140 trimeric protein immunogens. 2C mixture (n = 5), C97ZA012+459C gp140; 3C mixture (n = 5), C97ZA012+459C+405C gp140; 4C mixture (n = 10), C97ZA012+405C+459C+939C gp140. Colors correspond to coating proteins as listed. The horizontal dotted line indicates background, and error bars indicate standard deviations.

FIG 7

Magnitude of neutralizing antibody titers after vaccination with single clade C or multivalent vaccination regimens. Guinea pig sera obtained prevaccination (pre) and 4 weeks after the third vaccination (post) were tested against a multiclade panel of tier 1 clade C, clade B, and clade A neutralization-sensitive isolates in the TZM.bl neutralization assay. Horizontal bars indication median titers, and the dotted black line indicates the limit of detection for the assay. The x-axis immunogen names refer to the vaccination regimen. C97 is HIV-1 Env C97ZA012 gp140, 2C includes HIV-1 Env C97ZA012+459C gp140, 3C includes HIV-1 Env C97ZA012+459C+405C gp140, and 4C includes HIV-1 Env C97ZA012+459C+405C+939C gp140 trimeric immunogens.

FIG 8

Comparison of titers of neutralizing antibodies elicited by vaccination regimens by clade C trimers as measured by the TZM.bl neutralization assay. (A) Geometric means over test pseudovirions for each animal. Each point represents a geometric mean response per animal for all pseudovirions, compared with the entire pseudovirion panel (top) and tier 1B pseudovirions only (bottom). Boxes show median and interquartile ranges, and vaccines are indicated with colors according to the key. (B) Geometric means of background subtracted neutralizing antibody ID₅₀ titers by vaccination group at week 12 stratified by test pseudovirion (1 point per vaccination group per test pseudovirion). Each vaccination group is indicated in a color according to the key and connected by a single line.