Highlights d Electron microscopy polyclonal epitope mapping of immunized and challenged macaques d Diversity of epitopes and specific angles of approach are hallmarks of protection d Neutralization breadth is due in part to fusion peptidespecific antibodies d Cryo-EM analysis of a protected animal details a putatively neutralizing paratope

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several humanderived broadly neutralizing antibodies.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (B GC ) cells that last for at least 6 months. A 186-fold increase in B GC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of B GC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding B GC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells 1 , 2 . Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous B GC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

34The induction of broad and potent immunity by vaccines is the key focus of research efforts 35 aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins 36 have shown promise as vaccine candidates as they can induce potent autologous neutralizing 37 responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated 38 and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better 39 understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of 40 antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-41 epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 42 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and 43 have characteristics akin to several human-derived broadly neutralizing antibodies. 44 (nucleotide level) was 6.4% (range: 2.1%-10.2%) with an average HC complementarity-126 determining region 3 (CDR-H3) length of 15 amino acids (aa) (range: 7-23) ( Table S2). The rh1987 127 KC mAbs utilized HC variable genes from the IGHV3 and IGHV4 families and predominantly used 128 KC V genes from the IGKV1 family (Table S2). All of the rh1987 KC mAbs had a CDR-L3 length of 9 129 aa and their average KC SHM (nucleotide level) was 4.7% (range: 2.6%-6.0%) (Table S2). A single 130 clonal family with 4 members (RM19A) was detected among rh1987 KC mAbs with members 131 isolated from both week 22 and week 25 samples (Table S2). The rh1987 LC mAbs used HC V 132 genes from the IGHV1, IGHV3 and IGHV4 families and LC V genes mainly from the IGLV2 gene 133 family ( Table S2). The rh1987 LC mAbs had an average CDR-L3 length of 10 aa (range: 9-11) with 134 an average LC SHM (nucleotide level) of 3.8% (range: 0.9%-10.6%) (Table S2). Two clonal families 135

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of