Simultaneous Induction of Multiple Classes of Broadly Neutralizing Antibody Precursors via Combination Germline-Targeting Immunization

Inducing broadly neutralizing antibodies (bnAbs) against HIV remains a key challenge in vaccine development. Germline targeting immunogens have successfully primed bnAb B cell lineages to individual HIV envelope epitopes in humans and non-human primates. However, eliciting consistent breadth will require the induction of multiple bnAb classes. We investigated whether immunization with a combination of germline-targeting immunogens could concurrently prime multiple bnAb lineages in non-human primates. Animals were immunized with three immunogens, targeting distinct epitopes: the V3-glycan/N332 supersite, the V2 Apex region and the membrane-proximal external region (MPER), either individually or in combinations of two or all three. Triple combination immunization transiently reduced V2 Apex and V3-glycan responses, but by 8 weeks post-boost bnAb-precursor lineages were observed to all three epitopes. Similar somatic hypermutation was observed across groups, indicative of permissive germinal center responses. These findings support combination germline-targeting immunization as a viable strategy to prime multiple bnAb lineages simultaneously. Overall design: N332-GT5, Apex-GT6 and 10E8-GT12 -specific B cells from PBMCs were isolated by fluorescence-activated cell sorting (FACS) and analyzed by scRNA-seq, including VDJ and GEX libraries, using the 10X Genomics platform. Samples were multiplexed using TotalSeq™-C hashtag antibodies (BioLegend).