The elicitation of cross-neutralizing or broadly neutralizing Abs (bNAbs) to diverse HIV strains by Env vaccination remains a high priority for a broadly efficacious vaccine. The elicitation of bNAbs against conserved Env determinants remains elusive; however, the recent isolation of bNAbs to the fusion peptide and other sites of vulnerability demark promising leads in this process. Using N-glycan deleted NFL trimer-liposome priming and heterologous boosting/restoration, cross-neutralizing responses in rabbits were elicited with isolation of a CD4 binding site (CD4bs)-directed bNAb, E70, and 1C2 (87% breadth) directed toward the gp120:gp41 interface, as delineated by high resolution cryoEM (Dubrovskaya et al., Immunity 2019). More recently, we have also elicited cross-neutralizing responses in guinea pigs using this approach with novel, full length stabilized ?MIF? trimers as well as autologous tier 2 neutralizing responses in wild type mice following mRNA lipid nanoparticle (LNP) vaccination. Accordingly, the major objective of Project 1 will be to leverage these initial promising small animal results to elicit bNAbs in non-human primates (NHPs) using an ?epitope-targeted? approach against the CD4bs and the gp120:gp41 trimer interface. As both sites are conserved Env protein determinants ringed by glycans, the N-glycan deletion priming and restoration regimen will be further optimized. The NFL trimers will be modified to enhance presentation of the targeted sites, while improving trimer stability and homogeneity by tail-anchoring on covalently coupled trimer-liposomes, the cell surface from mRNA, or with a heterologous trimer motif (MIF). The three presentation platforms will be cross-compared for effectiveness and translatability. Further, based on studies indicating human infants and adolescents more readily develop bNAbs compared to HIV-infected adults, immunization responses will be compared between juvenile and adult macaques. As a secondary objective, the same regimens will be tested in guinea pigs to cross-validate the animal models. Guided approaches monitoring ?real-time? serum IgG responses by EM polyclonal epitope mapping (EMPEM; Core C) as well as rapid monoclonal Ab (mAb) isolation (Project 2/VRC) will be utilized to inform boosting from a select, diverse panel of structure-based, stabilized and homogeneous NFLs, iterative redesign and subsequent experiments. All NFL trimers will be produced in Project 1 for the entire P01, validated by biophysical methods, including DSC, EM and crystallography (Core C). Following elicitation of Env serum responses, isolated mAbs will be screened to confirm elicitation and neutralization specificity. By these integrated processes and comprehensive analysis, we will elicit and preferentially drive neutralizing antibodies to cross-neutralizing sites in primates in anticipation of human testing in the clinic.
The major objective of Project 1 in this HIVRAD proposal is to translate the promising elicitation of broadly neutralizing antibodies (bNAbs) following vaccination of the novel NFL trimers in small animal models in NHPs, along with small animal cross-validation. We will use the exciting new technique of serum-derived Fabs binding to the trimer by negative stain EM (EMPEM, Core C), coupled with glycan-deleted trimer priming, to improve the efficiency by ?real-time? analytical guidance of each Env trimer boost. From animals displaying the presence of bNAbs in their anti-sera, we will isolate (Project 2) and map both neutralizing and off-target non-neutralizing mAbs (VRC), which, once integrated for immunogen redesign in Project 1, will be a significant step forward toward the goal of generating an effective HIV-1 vaccine that would have a substantial impact on improving human public health.
