HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIVmac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIVmac239. The co-crystal structure of a fully glycosylated SIVmac239-gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in ∼83% of SIV strains by glycan N238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility.

The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4β7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a β sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the β sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

Neutralizing antibodies elicited by HIV-1 coevolve with viral envelope proteins (Env) in distinctive patterns, in some cases acquiring substantial breadth. We report that primary HIV-1 envelope proteins-when expressed by simian-human immunodeficiency viruses in rhesus macaques-elicited patterns of Env-antibody coevolution very similar to those in humans, including conserved immunogenetic, structural, and chemical solutions to epitope recognition and precise Env-amino acid substitutions, insertions, and deletions leading to virus persistence. The structure of one rhesus antibody, capable of neutralizing 49% of a 208-strain panel, revealed a V2 apex mode of recognition like that of human broadly neutralizing antibodies (bNAbs) PGT145 and PCT64-35S. Another rhesus antibody bound the CD4 binding site by CD4 mimicry, mirroring human bNAbs 8ANC131, CH235, and VRC01. Virus-antibody coevolution in macaques can thus recapitulate developmental features of human bNAbs, thereby guiding HIV-1 immunogen design.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediates viral entry into cells and is critical for vaccine development against coronavirus disease 2019 (COVID-19). Structural studies have revealed distinct conformations of S, but real-time information that connects these structures is lacking. Here we apply single-molecule fluorescence (Förster) resonance energy transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to human receptor angiotensin-converting enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon exposure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes1-bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to β2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.

Passive transfer of broadly neutralizing antibodies is showing promise in the treatment and prevention of HIV-1. One class of antibodies, the VRC01 class, appears especially promising. To improve VRC01-class antibodies, we combined structure-based design with a matrix-based approach to generate VRC01-class variants that filled an interfacial cavity, used diverse third-complementarity-determining regions, reduced potential steric clashes, or exploited extended contacts to a neighboring protomer within the envelope trimer. On a 208-strain panel, variant VRC01.23LS neutralized 90% of the panel at a geometric mean IC80 less than 1 μg/ml, and in transgenic mice with human neonatal-Fc receptor, the serum half-life of VRC01.23LS was indistinguishable from that of the parent VRC01LS, which has a half-life of 71 d in humans. A cryo-electron microscopy structure of VRC01.23 Fab in complex with BG505 DS-SOSIP.664 Env trimer determined at 3.4-Å resolution confirmed the structural basis for its ~10-fold improved potency relative to VRC01. Another variant, VRC07-523-F54-LS.v3, neutralized 95% of the 208-isolated panel at a geometric mean IC80 of less than 1 μg/ml, with a half-life comparable to that of the parental VRC07-523LS. Our matrix-based structural approach thus enables the engineering of VRC01 variants for HIV-1 therapy and prevention with improved potency, breadth, and pharmacokinetics.

Several influenza antibodies with broad group 2 neutralization have recently been isolated. Here, we analyze the structure, class, and binding of one of these antibodies from an H7N9 vaccine trial, 315-19-1D12. The cryo-EM structure of 315-19-1D12 Fab in complex with the hemagglutinin (HA) trimer revealed the antibody to recognize the helix A region of the HA stem, at the supersite of vulnerability recognized by group 1-specific and by cross-group-neutralizing antibodies. 315-19-1D12 was derived from HV1-2 and KV2-28 genes and appeared to form a new antibody class. Bioinformatic analysis indicated its group 2 neutralization specificity to be a consequence of four key residue positions. We specifically tested the impact of the group 1-specific N33 glycan, which decreased but did not abolish group 2 binding of 315-19-1D12. Overall, this study highlights the recognition of a broad group 2-neutralizing antibody, revealing unexpected diversity in neutralization specificity for antibodies that recognize the HA stem supersite.

Antibody CAP256-VRC26.25 targets the second hypervariable region (V2) at the apex of the HIV envelope (Env) trimer with extraordinary neutralization potency, although less than optimal breadth. To improve breadth, we linked the light chain of CAP256V2LS, an optimized version of CAP256-VRC26.25 currently under clinical evaluation, to the llama nanobody J3, which has broad CD4-binding site-directed neutralization. The J3-linked bispecific antibody exhibited improved breadth and potency over both J3 and CAP256V2LS, indicative of synergistic neutralization. The cryo-EM structure of the bispecific antibody in complex with a prefusion-closed Env trimer revealed simultaneous binding of J3 and CAP256V2LS. We further optimized the pharmacokinetics of the bispecific antibody by reducing the net positive charge of J3. The optimized bispecific antibody, which we named CAP256.J3LS, had a half-life similar to CAP256V2LS in human FcRn knock-in mice and exhibited suitable auto-reactivity, manufacturability, and biophysical risk. CAP256.J3LS neutralized over 97% of a multiclade 208-strain panel (geometric mean concentration for 80% inhibition (IC80) 0.079 μg/ml) and 100% of a 100-virus clade C panel (geometric mean IC80 of 0.05 μg/ml), suggesting its anti-HIV utility especially in regions where clade C dominates.

Current influenza vaccines predominantly induce immunity to the hypervariable hemagglutinin (HA) head, requiring frequent vaccine reformulation. Conversely, the immunosubdominant yet conserved HA stem harbors a supersite that is targeted by broadly neutralizing antibodies (bnAbs), representing a prime target for universal vaccines. Here, we showed that the co-immunization of two HA stem immunogens derived from group 1 and 2 influenza A viruses elicits cross-group protective immunity and neutralizing antibody responses in mice, ferrets, and nonhuman primates (NHPs). Immunized mice were protected from multiple group 1 and 2 viruses, and all animal models showed broad serum-neutralizing activity. A bnAb isolated from an immunized NHP broadly neutralized and protected against diverse viruses, including H5N1 and H7N9. Genetic and structural analyses revealed strong homology between macaque and human bnAbs, illustrating common biophysical constraints for acquiring cross-group specificity. Vaccine elicitation of stem-directed cross-group-protective immunity represents a step toward the development of broadly protective influenza vaccines.

CAP256V2LS, a broadly neutralizing monoclonal antibody (bNAb), is being pursued as a promising drug for HIV-1 prevention. The total level of tyrosine-O-sulfation, a post-translational modification, was known to play a key role for antibody biological activity. More importantly, here wedescribe for the first time the significance of the tyrosine-O-sulfation proteoforms. We developed a hydrophobic interaction chromatography (HIC) method to separate and quantify different sulfation proteoforms, which led to the direct functionality assessment of tyrosine-sulfated species. The fully sulfated (4-SO3) proteoform demonstrated the highest in vitro relative antigen binding potency and neutralization efficiency against a panel of HIV-1 viruses. Interestingly, highly variable levels of 4-SO3 were produced by different clonal CHO cell lines, which helped the bNAb process development towards production of a highly potent CAP256V2LS clinical product with high 4-SO3 proteoform. This study presents powerful insight for any biotherapeutic protein development where sulfation may play an important role in product efficacy.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

The complementary and alternative medicines (CAM) have not been systematically evaluated for the management of HIV/AIDS patients. In a prospective, single-site, open-label, non-randomized, controlled, pilot trial, we evaluated a polyherbal formulation (PHF) for its safety and efficacy in treating subjects with HIV-AIDS.

All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.

The HIV-1 envelope (Env) spike is a trimer of gp120/gp41 heterodimers that mediates viral entry. Binding to CD4 on the host cell membrane is the first essential step for infection but disrupts the native antigenic state of Env, posing a key obstacle to vaccine development. We locked the HIV-1 Env trimer in a pre-fusion configuration, resulting in impaired CD4 binding and enhanced binding to broadly neutralizing antibodies. This design was achieved via structure-guided introduction of neo-disulfide bonds bridging the gp120 inner and outer domains and was successfully applied to soluble trimers and native gp160 from different HIV-1 clades. Crystallization illustrated the structural basis for CD4-binding impairment. Immunization of rabbits with locked trimers from two different clades elicited neutralizing antibodies against tier-2 viruses with a repaired glycan shield regardless of treatment with a functional CD4 mimic. Thus, interdomain stabilization provides a widely applicable template for the design of Env-based HIV-1 vaccines.

Broadly neutralizing antibodies (bNAbs) represent a promising alternative to antiretroviral drugs for HIV-1 prevention and treatment. Selected antibodies to the CD4-binding site bolster envelope trimer binding via quaternary contacts. Here, we rationally engraft a new paratope, i.e., the extended heavy-chain framework region 3 (FR3) loop of VRC03, which mediates quaternary interaction, onto several potent bNAbs, enabling them to reach an adjacent gp120 protomer. The interactive quaternary surface is delineated by solving the crystal structure of two FR3 loop-chimeric antibodies. Chimerization enhances the neutralizing activity of several potent bNAbs against a majority of global HIV-1 strains. Compared to unmodified antibodies, chimeric antibodies display lower autoreactivity and prolonged in vivo half-life in huFcRn mice and rhesus macaques. Thus, paratope engraftment may be used to expand the epitope repertory of natural antibodies, improving their functionality for disease prevention and treatment.

Human noroviruses are non-enveloped, single-strand RNA viruses that cause pandemic outbreaks of acute gastroenteritis. A bivalent vaccine containing GI.1 and GII.4 virus-like particles (VLPs) has been shown to be safe and highly immunogenic, but its efficacy and durability have been limited. Here, we show that norovirus GI.1 VLPs are unstable and contain a substantial fraction of dissociated VLP components. Broadly reactive, non-neutralizing antibodies isolated from vaccinated donors bound to the dissociated components, but not to the intact VLPs. Engineering of interprotomer disulfide bonds within the shell domain prevented disassembly of the VLPs, while preserving antibody accessibility to blockade epitopes. Without adjuvant, mice immunized with stabilized GI.1 VLPs developed faster blockade antibody titers compared to immunization with wild-type GI.1 VLPs. In addition, immunization with stabilized particles focused immune responses toward surface-exposed epitopes and away from occluded epitopes. Overall, disulfide-stabilized norovirus GI.1 VLPs elicited improved responses over the non-disulfide-stabilized version, suggesting their promise as candidate vaccines.

Antibodies targeting the V1V2 apex of the HIV-1 envelope (Env) trimer comprise one of the most commonly elicited categories of broadly neutralizing antibodies. Structures of these antibodies indicate diverse modes of Env recognition typified by antibodies of the PG9 class and the PGT145 class. The mode of recognition, however, has been unclear for the most potent of the V1V2 apex-targeting antibodies, CAP256-VRC26.25 (named for donor-lineage.clone and referred to hereafter as VRC26.25). Here, we determine the cryoelectron microscopy structure at 3.7 Å resolution of the antigen-binding fragment of VRC26.25 in complex with the Env trimer thought to have initiated the lineage. The 36-residue protruding loop of VRC26.25 displays recognition incorporating both strand-C interactions similar to the PG9 class and V1V2 apex insertion similar to the PGT145 class. Structural elements of separate antibody classes can thus intermingle to form a "combined" class, which in this case yields an antibody of extraordinary potency.

Understanding maturation pathways of broadly neutralizing antibodies (bnAbs) against HIV-1 can be highly informative for HIV-1 vaccine development. A lineage of J038 bnAbs is now obtained from a long-term SHIV-infected macaque. J038 neutralizes 54% of global circulating HIV-1 strains. Its binding induces a unique "up" conformation for one of the V2 loops in the trimeric envelope glycoprotein and is heavily dependent on glycan, which provides nearly half of the binding surface. Their unmutated common ancestor neutralizes the autologous virus. Continuous maturation enhances neutralization potency and breadth of J038 lineage antibodies via expanding antibody-Env contact areas surrounding the core region contacted by germline-encoded residues. Developmental details and recognition features of J038 lineage antibodies revealed here provide a new pathway for elicitation and maturation of V2-targeting bnAbs.