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HIV monoclonal antibodies for viral reservoir eradication strategies will likely need to recognize reactivated infected cells and potently drive Fc-mediated innate effector cell activity. We systematically characterize a library of 185 HIV-envelope-specific antibodies derived from 15 spontaneous HIV controllers (HCs) that selectively exhibit robust serum Fc functionality and compared them to broadly neutralizing antibodies (bNAbs) in clinical development. Within the 10 antibodies with the broadest cell-recognition capability, seven originated from HCs and three were bNAbs. V3-loop-targeting antibodies are enriched among the top cell binders, suggesting the V3-loop may be selectively exposed and accessible on the cell surface. Fc functionality is more variable across antibodies, which is likely influenced by distinct binding topology and corresponding Fc accessibility, highlighting not only the importance of target-cell recognition but also the need to optimize for Fc-mediated elimination. Ultimately, our results demonstrate that this comprehensive selection process can identify monoclonal antibodies poised to eliminate infected cells., Competing Interests: Declaration of interests G.A. has a financial interest (founder) in SeromYx, a company developing platform technology that describes the antibody immune response. G.A.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. The other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies., Competing Interests: Declaration of interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is the founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from and IDBiologics and AstraZeneca. Vanderbilt University has applied for a patent that is related to antibodies discussed in this work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Artificial glycan holes on recombinant Env-based vaccines occur when a potential N-linked glycosylation site (PNGS) is under-occupied, but not on their viral counterparts. Native-like SOSIP trimers, including clinical candidates, contain such holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To eliminate glycan holes and mimic the glycosylation of native BG505 Env, we replace all 12 NxS sequons on BG505 SOSIP with NxT. All PNGS, except N133 and N160, are nearly fully occupied. Occupancy of the N133 site is increased by changing N133 to NxS, whereas occupancy of the N160 site is restored by reverting the nearby N156 sequon to NxS. Hence, PNGS in close proximity, such as in the N133-N137 and N156-N160 pairs, affect each other's occupancy. We further apply this approach to improve the occupancy of several Env strains. Increasing glycan occupancy should reduce off-target immune responses to vaccine antigens., Competing Interests: Declaration of interests The International AIDS Vaccine Initiative (IAVI) has previously filed a patent relating to the BG505 SOSIP.664 trimer: U.S. provisional application 61/772,739, entitled “HIV-1 Envelope Glycoprotein,” with J.P.M., A.B.W., and R.W.S. among the co-inventors, but no patents have been filed on any work described here., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Structural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstitute a full-length Env clone into a nanodisc, complex it with a membrane-proximal external region (MPER) targeting antibody 10E8, and structurally define the full quaternary epitope of 10E8 consisting of lipid, MPER, and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapt the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with a peptidisc scaffold., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The antibody ADI-15878 was previously identified from a human survivor of Ebola virus Makona variant (EBOV/Mak) infection. This mAb demonstrated potent neutralizing activity against all known ebolaviruses and provided protection in rodent and ferret models against three ebolavirus species. Here, we describe the unliganded crystal structure of ADI-15878 as well as the cryo-EM structures of ADI-15878 in complex with the EBOV/Mak and Bundibugyo virus (BDBV) glycoproteins (GPs). ADI-15878 binds through an induced-fit mechanism by targeting highly conserved residues in the internal fusion loop (IFL), bridging across GP protomers via the heptad repeat 1 (HR1) region. Our structures provide a more complete description of the ebolavirus immunogenic landscape, as well as a molecular basis for how rare but potent antibodies target conserved filoviral fusion machinery., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Broadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)