Antigen persistence and TLR stimulation contribute to induction of a durable HIV-1-specific neutralizing antibody response

Abstract

HIV-1 Env glycoprotein (Env) immunogenicity is limited in part by structural instability and extensive glycan shielding and is likely the greatest obstacle to an HIV-1 vaccine. Stabilized Env trimers can elicit serum neutralizing antibodies, but the response is short-lived. Here we use Newcastle Disease Virus-like particle (NDV-VLP) platform to present stabilized versions of HIV-1 Env at high valency and in the context of varied conformational stability, adjuvants, dose, and antigen persistence. Influenza virus hemagglutinin, or SARS-CoV2 Spike-bearing VLPs rapidly induce neutralizing antibodies, in contrast, they were not induced by those bearing Env. A replicating adenovirus type 4 expressing Env rapidly induces autologous neutralizing antibodies. However, durable neutralizing antibodies are induced only when multiple features of a replicating virus infection are combined, with the largest impact from dose and escalating dose. In summary, we show here immunogenicity of HIV-1 Env could be improved by reproducing features of virus infection.

Fig. 1. Chimeric NDV VLP production and characterization.

A Schematics of chimeric NDV F expressing HIV-1 Envelope (1086 C, 1086 C SOSIP, 1086 C NFL-TD), Influenza HA or SARS-CoV2 spike proteins designed for this study. B Western blots of lysates of A549 cells transfected with the indicated chimeric NDV F chimeric proteins. C Detection of indicated chimeric NDV F and NP proteins in purified VLPs by western blot. D The ratio of indicated chimeric NDV F protein chimeras to NDV nucleoprotein measured by western blot (1086 C constructs, n = 7; H5 and CoV2, n = 6). The samples derive from the same experiment, and the blots were processed in parallel. E Quantification of protein spikes expressed on the VLP surface performed by negative stain electron microscopy. F Representative EM images of VLP variants. The scale bars correspond to 50 nm.

Fig. 2. Expression of native-like trimers on producer cells and NDV VLPs.

A Binding of HIV-1 Envelope monoclonal antibodies to chimeric NDV F/1086 C protein on transfected A549 cells by flow cytometry. B Summary of HIV-1 Envelope monoclonal antibodies binding to chimeric NDV F/1086 C proteins on transfected, or Ad4 FDE3 Env150 infected A549 cells. MFI is normalized to VRC01. C Binding of VRC01 and PGT145 to stabilized 1086 C NFL-TD and SOSIP proteins on chimeric NDV F VLPs by flow virometry.

Fig. 3. Immunogenicity of high valency chimeric NDV VLP variants in rabbits.

A–D Immunogenicity of 150 μg intramuscular (IM) dose administered NDV VLP variant expressing influenza virus HA (A), SARS-CoV2 Spike (B), SOSIP stabilized HIV-1 envelope protein (C), or NFL-TD stabilized HIV-1 envelope protein (D). E Immunogenicity of replication-competent Ad4 FDE3 Env150 recombinant (10¹¹, IM) expressing non-stabilized 1086 C HIV-1 Envelope protein. Serum HIV-1 neutralizing antibody titers (C–E) of each rabbit (n = 6) are shown in red squares (SF162) and blue dots (1086 C). F Serum neutralization breadth (ID₅₀) of rabbits (n = 6) immunized with Ad4 FDE3 Env150 at the indicated weeks against pseudoviruses of HIV-1 Env SF162, 1086 C and SIV Env mac256. Blue arrows indicate the week of IM-administered immunization. Serum neutralizing antibody titers (ID₅₀) of each rabbit (n = 6) are shown in dots, and the median value for each time point is connected with a solid line.

Fig. 4. Impact of TLR stimulation on immunogenicity of VLP variants.

A–F Immunogenicity of 150 μg intramuscular (IM) dose of chimeric NDV VLP variant expressing the indicated protein with AS01 adjuvant (A, B) or TLR agonist encapsidated RNA40 (C–F). Animals immunized with constructs without RNA40 are shown in gray. G Heatmap of serum neutralizing activity (ID₅₀) against a panel of 10 HIV-1 Env pseudoviruses from clades (A–C), and SIVmac256. Blue arrows indicate the week of the IM-administered immunization. Serum neutralizing antibody titers (ID₅₀) of each rabbit (n = 6) are shown in dots, and the median value for each time point is connected with a solid line.

Fig. 5. Impact of dose on immunogenicity of VLP variants.

A–D Immunogenicity of TLR agonist RNA40 encapsidated chimeric NDV VLP variants expressing the indicated stabilized HIV-1 envelope protein with or without AS01 adjuvant and IM administered at high doses (500 μg). E Heatmap of neutralization activity (ID₅₀) in rabbit serum against a panel of 10 HIV-1 Env pseudoviruses from clades (A–C), and SIVmac256. Blue arrows indicate the week of IM-administered immunization. Serum neutralizing antibody titers (ID₅₀) of each rabbit (n = 6) are shown in dots, and the median value for each time point is connected with a solid line.

Fig. 6. Impact of dose escalation on immunogenicity of VLP variants.

A, B Immunogenicity of TLR agonist RNA40 encapsidated chimeric NDV VLP variants expressing the indicated HIV-1 envelope formulated with AS01 adjuvant given at an escalating dose of 500 μg. The immunogen was split into 7 IM immunizations in a dose-escalation manner. Four separate weeks of dose-escalation IM immunizations are indicated by the blue histogram. Serum neutralizing antibody titers of each rabbit are shown in dots, and the median value for each time point is connected with a solid line. C Heatmap of rabbit serum (Week 28) neutralization activity (ID₅₀) against a panel of 10 HIV-1 Env pseudoviruses from clades (A–C) and SIVmac256.

Fig. 7. Binding assay and EMPEM.

A Sera from week 28 from rabbits (n = 6) immunized with the indicated vaccine regimen was assayed against the indicated stabilized trimer protein. The area under the curve (AUC) of serial dilutions is displayed. B Negative-stain EMPEM 3D reconstruction of polyclonal antibody fragment antigen-binding (pFab) from rabbit 142 (RB142) at week 31 in complex with 1086 C NFL trimer (left). The map is segmented and colored by component. Docking of an Env gp140 protomer model into the map reveals that the polyclonal antibodies bind near the V3 and V1 regions of gp120, near N301 (middle and right panels). Relevant potential N-linked glycosylation sites in 1086 C NFL are labeled in green, and those commonly present in other HIV-1 genotypes but not in 1086 C are labeled in red.