Antigenicity and immunogenicity of envelope glycoproteins from HIV-1 preseroconversion strains: potential novel vaccine candidates

© 2012 Dr. Sharmila ReddyAn efficacious vaccine is needed to combat the spread of HIV-1 and novel immunogens are required to facilitate this. Immune selection pressure modifies HIV-1 envelope glycoproteins (Envs) during the course of infection to drive escape from neutralizing antibodies (NAbs). However HIV-1 preseroconversion (PSC) strains proliferating in the absence of selection pressure promote Envs featuring reduced glycosylation and shorter variable loops (VLs). These features may potentially favour exposure of neutralization sensitive epitopes and hence PSC Envs as immunogens could elicit NAbs. Blood samples were collected from 38 HIV-1 MSM (men who have sex with men) subjects of the PSC cohort. Based on preliminary env sequence analysis, five samples with fewer potential N-linked glycosylation sites (PNGS) compared to pNL4.3 reference strain were chosen to generate env gp160 clones. These were genotyped for changes in PNGS and length of VLs in comparison to HXB2 (X4) and AD8 (R5) strains and also coreceptor (CoR) usage and changes in binding sites of CD4 and known broadly NAbs (bNAbs) were analyzed. PSC Envs were phenotyped for Env mediated entry, CoR usage, receptor affinity and neutralization sensitivity to CD4 and bNAbs. PSC gp140 clones were constructed and engineered to secrete soluble gp140 trimers and these were assessed for exposure of CD4 binding site (CD4bs) and bNAb epitopes by ELISA and surface plasmon resonance. PSC gp140 DNA and protein immunogens were tested in mice and sera were assessed for binding antibody titres and avidity by ELISA and NAb responses against HIV-1 pseudoviruses by TZM-bl assay. PSC Envs were further tested in rabbits as systemic and mucosal immunogens. A novel lipopeptide R4Pam2Cys adjuvant was primarily used for both systemic and mucosal routes. Montanide and R4Pam2Cys adjuvant combinations for systemic and mucosal routes respectively were also tested. Sera, nasal lavages, cervicovaginal lavages and faecal pellets were evaluated for binding antibody titres and NAb responses against HIV-1 pseudoviruses. Genotypic analysis showed that PSC env clones at intrapatient level were highly identical with fewer PNGS in V1 and V4, shorter VLs in V1, V3 and V4 and fewer changes in binding sites of CD4 and bNAbs. Phenotypic analysis showed that PSC Envs mediated functional entry, were R5 tropic with high efficiency for CCR5 usage and were neutralization sensitive to CD4 and bNAbs b12, 2F5 and 4E10. Antigenicity studies by ELISA and SPR showed exposure of CD4bs and epitopes of bNAbs b12, 2F5 and 447-52D. Immunogenicity studies testing PSC Envs in mice raised high titres of IgG with high avidity, moderate IgA and IgM and bNAbs against clade B and C pseudoviruses. Systemic and mucosal immunization of PSC Envs in rabbits raised effective humoral responses and the group receiving combination of Montanide and R4Pam2Cys for systemic and mucosal routes respectively showed sustained responses. Systemic responses included very high IgG titres with high avidity, low IgA and moderate IgM responses and NAb responses were highly potent but not broad. Mucosal responses included moderate to low IgG, low IgA and low/no IgM responses and NAb responses were broad but the potency varied across the different mucosal compartments. Thus the PSC Envs studied here displayed less glycosylation and shorter VLs compared to HXB2 and AD8 reference strains and exposed bNAb epitopes. When these PSC Envs were tested in mice and rabbit vaccination experiments these antigenic features promoted effective systemic and mucosal humoral responses. Challenge studies in non-human primates will allow further evaluation of the protective potential of PSC Env immunogens