P.N.P.G. against the fusion peptide. Subject terms:Protein vaccines, Antibodies == Introduction == Recent efforts in vaccine design for the HIV-1 virus have focused on developing neutralizing adaptive immune responses to the HIV-1 Env glycoprotein via sequential immunization13. Studies of broadly neutralizing antibodies (bNAbs) isolated from Edasalonexent HIV + human donors have informed immunogen design efforts for various epitopes on the Env trimer, including the V3-glycan patch4,5, the fusion peptide (FP)6,7, and the CD4-binding site8,9. In some cases, on-target antibody responses are accompanied by off-target responses in which antibodies are made against undesired epitopes on the Env trimer including Edasalonexent the bottom or base epitope10and/or a minimally-glycosylated region (glycan hole)11. These antibodies target immunodominant but non-neutralizing epitopes and therefore do not contribute meaningfully to a neutralizing antibody response. We previously described the design and characterization of RC1, a BG505 BCLX SOSIP.66412-based engineered immunogen targeting the V3-glycan patch on the gp120 subunit of Env trimer4. We showed that RC1 and/or RC1-4fill (modified from RC1 to include additional potential N-linked glycosylation sites; PNGSs) that had been multimerized on virus-like particles (VLPs) elicited antibodies that recognized the V3-glycan patch in wild-type mice, rabbits, and non-human primates (NHPs)4. We subsequently boosted a subset of RC1-4fill-primed NHPs, isolated single Env-specific B cells, and derived antibody sequences from which monoclonal antibodies (mAbs) were produced13. Here, we describe a single-particle cryo-EM structure of a BG505 Env trimer bound to a monoclonal antibody (Ab1245) isolated from a rhesus macaque after a sequential immunization protocol that included multimerized HIV-1 SOSIP Envs derived from different clades. Ab1245 binds to an epitope overlapping with the FP-targeting bNAb VRC346at the interface of the Env gp41 and gp120 subunits, but unlike VRC34, Ab1245 displaces the FP and fusion peptide-proximal region (FPPR). In addition, Ab1245 contains a methionine residue that structurally mimics Met530gp41, a key residue for the stability of the Env trimer, by engaging the tryptophan clasp formed by three gp41 tryptophan residues14,15. Despite inducing FP rearrangement and overlap with the neutralizing VRC34 epitope, Ab1245 did not neutralize BG505 or other viral strains, perhaps because of its sub-stoichiometric binding to Env trimer. These previously unseen features of gp120-gp41 interface antibodies demonstrate that HIV-1 Env can elicit non-neutralizing antibodies that block a neutralizing epitope, inform immunogen design protocols to prevent elicitation of similar antibodies, and provide potential mechanistic insight into HIV-1 Env-mediated fusion of the host and viral membranes. == Results and discussion == == Sequential immunization after RC1-4-fill priming elicited Ab1245, a non-V3-targeting antibody == We previously described a V3-glycan patch targeting immunogen, RC1, which was modified from a designed V3 immunogen, 11MUTB16, by removing the N-linked glycan attached to gp120 residue N156gp1204. Both RC1 and 11MUTB were derived from clade A BG505 SOSIP.664 native-like Env trimers12. RC1-4fill and 11MUTB-4fill were modified Edasalonexent from RC1 and 11MUTB, respectively, to reduce antibody responses to off-target epitopes11,1719by inserting PNGSs to add glycans to residues 230gp120, 241gp120, 289gp120, and 344gp1204. In addition, to enhance avidity effects and limit Edasalonexent antibody access to the Env trimer base, we multimerized immunogens on VLPs using the SpyTag-SpyCatcher system20,21. Four NHPs primed with RC1-4fill-VLPs4were boosted sequentially with (i) VLPS coupled with 11MUTB-4fill16(clade A), (ii) VLPs coupled with B41 SOSIP (clade B), and (iii) VLPs coupled with a mixture of AMC011 and DU422 SOSIPs (clades B and C) over the course of 9 months22(Fig.1a). == Fig. 1. Characterization of Ab1245 elicited in macaques by sequential immunization. == aSequential immunization protocol for the macaque that produced Ab1245 over the course of 40 weeks.bSequence alignment of Ab1245 heavy.