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Soluble 'SOSIP'-stabilized HIV-1 envelope glycoprotein (Env) trimers elicit dominant antibody responses targeting their glycan-free base regions, potentially diminishing neutralizing responses. Previously, using a nonhuman primate model, we demonstrated that priming with fusion peptide (FP)-carrier conjugate immunogens followed by boosting with Env trimers reduced the anti-base response. Further, we demonstrated that longer immunization intervals further reduced anti-base responses and increased neutralization breadth. Here, we demonstrate that long trimer-boosting intervals, but not long FP immunization intervals, reduce the anti-base response. Additionally, we identify that FP priming before trimer immunization enhances antibody avidity to the Env trimer. We also establish that adjuvants Matrix M and Adjuplex further reduce anti-base responses and increase neutralizing titers. FP priming, long trimer-immunization interval, and an appropriate adjuvant can thus reduce anti-base antibody responses and improve Env-directed vaccine outcomes., Competing Interests: C.C., K.X., P.D.K., and J.R.M. are co-inventors on a US Patent Application filed on their behalf by the National Institutes of Health. The other authors declare no conflicts of interest.

Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N -linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers -even those that were stable biochemically- to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response., Competing Interests: NIH has submitted a PCG patent application (PCT/US2023/065009) for Env trimers described in this manuscript on which A.S.O., C.C., T.Z., D.H.R., A.C., R.R., Y.Y., and P.D.K. are co-inventors. The other authors declare no competing interests.

Soluble "SOSIP"-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high-titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the effect on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, here, we quantify the prevalence of trimer-base antibody responses in 49 non-human primates immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. Trimer-base responses account for ∼90% of the overall trimer response in animals immunized with trimer only, ∼70% in animals immunized with a cocktail of SOSIP trimer and FP conjugate, and ∼30% in animals primed with FP conjugates before trimer immunization. Notably, neutralization breadth in FP-conjugate-primed animals correlates inversely with trimer-base responses. Our data provide methods to quantify the prevalence of trimer-base responses and reveal that FP-conjugate priming, either alone or as part of a cocktail, can reduce the trimer-base response and improve the neutralization outcome., Competing Interests: Declaration of interests P.D.K., J.R.M., L.O., Y.T., K.X., and B.Z. are inventors on U.S. Patent Application 62/735,188 filed March 26, 2020, entitled “HIV-1 ENV fusion peptide immunogens and their use.” The other authors declare no competing interests., (Published by Elsevier Inc.)

The HIV fusion peptide (FP) is a promising vaccine target. FP-directed monoclonal antibodies from vaccinated macaques have been identified that neutralize up to ∼60% of HIV strains; these vaccinations, however, have involved ∼1 year with an extended neutralization-eclipse phase without measurable serum neutralization. Here, in 32 macaques, we test seven vaccination regimens, each comprising multiple immunizations of FP-carrier conjugates and HIV envelope (Env) trimers. Comparisons of vaccine regimens reveal FP-carrier conjugates to imprint cross-clade neutralizing responses and a cocktail of FP conjugate and Env trimer to elicit the earliest broad responses. We identify a signature, appearing as early as week 6 and involving the frequency of B cells recognizing both FP and Env trimer, predictive of vaccine-elicited breadth ∼1 year later. Immune monitoring of B cells in response to vaccination can thus enable vaccine insights even in the absence of serum neutralization, here identifying FP imprinting, cocktail approach, and early signature as means to improve FP-directed vaccine responses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization., (Published by Elsevier Inc.)