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Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which can readily mutate to escape acquired immunity. Other regions in the spike S2 subunit, such as the fusion peptide and the stem helix, are highly conserved across sarbecoviruses and recognized by broadly reactive antibodies, providing hope that targeting these epitopes by vaccination could offer protection against both current and emergent viruses. Here we employed computational modeling to design epitope scaffolds that display the fusion peptide and the stem helix epitopes. The engineered proteins bound both mature and germline versions of multiple broad and protective human antibodies with high affinity. Binding specificity was confirmed both biochemically and via high resolution crystal structures. Finally, the epitope scaffolds showed potent engagement of antibodies and memory B-cells from subjects previously exposed to SARS-CoV2, illustrating their potential to elicit antibodies against the fusion peptide and the stem helix by vaccination.

A successful HIV-1 vaccine will require induction of a polyclonal neutralizing antibody (nAb) response, yet vaccine-mediated induction of such a response in primates remains a challenge. We found that a stabilized HIV-1 CH505 envelope (Env) trimer formulated with a Toll-like receptor 7/8 agonist induced potent HIV-1 polyclonal nAbs that correlated with protection from homologous simian-human immunodeficiency virus (SHIV) infection. The serum dilution that neutralized 50% of virus replication (ID 50 titer) required to protect 90% of macaques was 1:364 against the challenge virus grown in primary rhesus CD4 + T cells. Structural analyses of vaccine-induced nAbs demonstrated targeting of the Env CD4 binding site or the N156 glycan and the third variable loop base. Autologous nAb specificities similar to those elicited in macaques by vaccination were isolated from the human living with HIV from which the CH505 Env immunogen was derived. CH505 viral isolates were isolated that mutated the V1 to escape both the infection-induced and vaccine-induced antibodies. These results define the specificities of a vaccine-induced nAb response and the protective titers of HIV-1 vaccine–induced nAbs required to protect nonhuman primates from low-dose mucosal challenge by SHIVs bearing a primary transmitted/founder Env.

SUMMARY HIV-1-infected infants develop broadly neutralizing antibodies (bnAbs) more rapidly than adults, suggesting differences in the neonatal versus adult responses to the HIV-1 envelope (Env). Here, trimeric forms of HIV-1 Env immunogens elicit increased gp120-and gp41-specific antibodies more rapidly in neonatal macaques than adult macaques. Transcriptome analyses of neonatal versus adult immune cells after Env vaccination reveal that neonatal macaques have higher levels of the apoptosis regulator BCL2 in T cells and lower levels of the immunosuppressive interleukin-10 (IL-10) receptor alpha (IL10RA) mRNA transcripts in T cells, B cells, natural killer (NK) cells, and monocytes. In addition, immunized neonatal macaques exhibit increased frequencies of activated blood T follicular helper-like (Tfh) cells compared to adults. Thus, neonatal macaques have transcriptome signatures of decreased immunosuppression and apoptosis compared with adult macaques, providing an immune landscape conducive to early-life immunization prior to sexual debut., In Brief Han et al. demonstrate that infant macaques are capable of responding to HIV vaccines currently studied in human clinical trials. The nature of the neonatal immune system following immunization also suggests a state permissive for generating HIV-1 antibodies. Thus, infant HIV-1 immunization remains a viable strategy to end this pandemic., Graphical abstract

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor binding domain (RBD) and neutralizing antibody epitope presentation affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

Severe acute respiratory syndrome coronaviruses 1 (SARS-CoV) and 2 (SARS-CoV-2), including SARS-CoV-2 variants of concern, can cause deadly infections. The mortality associated with sarbecovirus infection underscores the importance of developing broadly effective countermeasures against them, which could be key in the prevention and mitigation of current and future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV; bat coronaviruses WIV-1 and RsSHC014; and SARS-CoV-2 variants D614G, B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, B.1.617.1, and B.1.617.2 by a receptor binding domain (RBD)–specific human antibody, DH1047. Prophylactic and therapeutic treatment with DH1047 was protective against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B.1.351 infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among sarbecoviruses. Thus, DH1047 is a broadly protective antibody that can prevent infection and mitigate outbreaks caused by SARS-related strains and SARS-CoV-2 variants. Our results also suggest that the conserved RBD epitope bound by DH1047 is a rational target for a universal sarbecovirus vaccine.

SUMMARYThe success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines heralded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticles are superior immunogens compared to trimers alone for priming of broadly neutralizing antibody (bnAb) B cell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs has not been demonstrated. Here we show that mRNAs can encode antigenic Env trimers on ferritin nanoparticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activity in bnAb precursor VH + VL knock-in mice. Next generation sequencing demonstrated acquisition of critical mutations, and monoclonal antibodies that neutralized heterologous HIV-1 isolates were isolated. Thus, mRNA- LNP can encode complex immunogens and are of use in design of germline-targeting and sequential boosting immunogens for HIV-1 vaccine development.

A paradigm shifting study demonstrated that induction of MHC class E and II-restricted CD8+ T cells was associated with the clearance of SIV infection in rhesus macaques. Another recent study highlighted the presence of HIV-1-specific class II-restricted CD8+ T cells in HIV-1 patients who naturally control infection (virus controllers; VCs). However, questions regarding class II-restricted CD8+ T cells ontogeny, distribution across different HIV-1 disease states and their role in viral control remain unclear. In this study, we investigated the distribution and anti-viral properties of HLA-DRB1*0701 and DQB1*0501 class II-restricted CD8+ T cells in different HIV-1 patient cohorts; and whether class II-restricted CD8+ T cells represent a unique T cell subset. We show that memory class II-restricted CD8+ T cell responses were more often detectable in VCs than in chronically infected patients, but not in healthy seronegative donors. We also demonstrate that VC CD8+ T cells inhibit virus replication in both a class I- and class II-dependent manner, and that in two VC patients the class II-restricted CD8+ T cells with an anti-viral gene signature expressed both CD4+ and CD8+ T cell lineage-specific genes. These data demonstrated that anti-viral memory class II-restricted CD8+ T cells with hybrid CD4+ and CD8+ features are present during natural HIV-1 infection.

SUMMARY Antibodies that bind residue K169 in the V2 region of the HIV-1 envelope correlated with reduced risk of infection in the RV144 vaccine trial but were restricted to two ED-motif-encoding light chain genes. Here, we identify an HIV-infected donor with high-titer V2 peptide-binding antibodies and isolate two antibody lineages (CAP228-16H/19F and CAP228–3D) that mediate potent antibody-dependent cell-mediated cytotoxicity (ADCC). Both lineages use the IGHV5–51 heavy chain germline gene, similar to the RV144 antibody CH58, but one lineage (CAP228-16H/19F) uses a light chain without the ED motif. A cocrystal structure of CAP228-16H bound to a V2 peptide identified a IGLV3–21 gene-encoded DDxD motif that is used to bind K169, with a mechanism that allows CAP228-16H to recognize more globally relevant V2 immunotypes. Overall, these data further our understanding of the development of cross-reactive, V2-binding, antiviral antibodies and effectively expand the human light chain repertoire able to respond to RV144-like immunogens., In Brief V2-directed antibodies from the RV144 vaccine trial correlated with reduced HIV-1 infection risk but exhibited restricted light chain gene usage. Here, van Eeden et al. isolate similar antibodies from an HIV-1-infected individual and identify a third V2-reactive light chain gene, increasing the antibody repertoire potentially elicited by vaccination., Graphical Abstract

SARS-CoV in 2003, SARS-CoV-2 in 2019, and SARS-CoV-2 variants of concern (VOC) can cause deadly infections, underlining the importance of developing broadly effective countermeasures against Group 2B Sarbecoviruses, which could be key in the rapid prevention and mitigation of future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV, bat CoVs WIV-1 and RsSHC014, and SARS-CoV-2 variants D614G, B.1.1.7, B.1.429, B1.351 by a receptor-binding domain (RBD)-specific antibody DH1047. Prophylactic and therapeutic treatment with DH1047 demonstrated protection against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B1.351infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among Sarbecoviruses. We conclude that DH1047 is a broadly neutralizing and protective antibody that can prevent infection and mitigate outbreaks caused by SARS-like strains and SARS-CoV-2 variants. Our results argue that the RBD conserved epitope bound by DH1047 is a rational target for pan Group 2B coronavirus vaccines.

SummaryNew SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes.HighlightsCryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion.Adaptation to mink resulted in increased ACE2 binding and spike destabilization.B.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding.E484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation.S protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning.

SUMMARYBetacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

SummarySARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infectionin vitro, while five non-neutralizing NTD antibodies mediated FcγR-independentin vitroinfection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, whilein vitroantibody-enhanced infection does not necessarily herald enhanced infectionin vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

Convergent HIV evolution across species Human immunodeficiency virus (HIV) has a highly diverse envelope protein that it uses to target human cells, and the complexity of the viral envelope has stymied vaccine development. Roark et al. report that the immediate and short-term evolutionary potential of the HIV envelope is constrained because of a number of essential functions, including antibody escape. Consequently, when introduced into humans as HIV or into rhesus macaque monkeys as chimeric simian-human immunodeficiency virus, homologous envelope glycoproteins appear to exhibit conserved patterns of sequence evolution, in some cases eliciting broadly neutralizing antibodies in both hosts. Conserved patterns of envelope variation and homologous B cell responses in humans and monkeys represent examples of convergent evolution that may serve to guide HIV vaccine development. Science , this issue p. eabd2638

The SARS-CoV-2 pandemic that causes COVID-19 respiratory syndrome has caused global public health and economic crises, necessitating rapid development of vaccines and therapeutic countermeasures. The world-wide response to the COVID-19 pandemic has been unprecedented with government, academic, and private partnerships working together to rapidly develop vaccine and antibody countermeasures. Many of the technologies being used are derived from prior government-academic partnerships for response to other emerging infections.

Eliciting protective titers of HIV-1 broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development, but current vaccine strategies have yet to induce bnAbs in humans. Many bnAbs isolated from HIV-1-infected individuals are encoded by immunoglobulin gene rearrangments with infrequent naive B cell precursors and with unusual genetic features that may be subject to host regulatory control. Here, we administer antibodies targeting immune cell regulatory receptors CTLA-4, PD-1 or OX40 along with HIV envelope (Env) vaccines to rhesus macaques and bnAb immunoglobulin knock-in (KI) mice expressing diverse precursors of CD4 binding site HIV-1 bnAbs. CTLA-4 blockade augments HIV-1 Env antibody responses in macaques, and in a bnAb-precursor mouse model, CTLA-4 blocking or OX40 agonist antibodies increase germinal center B and T follicular helper cells and plasma neutralizing antibodies. Thus, modulation of CTLA-4 or OX40 immune checkpoints during vaccination can promote germinal center activity and enhance HIV-1 Env antibody responses., Elucidation of broadly neutralizing antibodies (bnAb) is a goal in HIV vaccine development. Here, Bradley et al. show that administration of CTLA-4 blocking antibody with vaccine antigens increases HIV-1 envelope antibody responses in macaques and a bnAb precursor mouse model.

HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide (“Man9-V3”) for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage (“DH270”), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs—the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen., The V3 region of HIV Env elicits broadly neutralizing antibodies (bnAbs) in patients and represents a potential vaccine antigen. Here, Fera et al. show that the structure of a synthetic V3-glycopeptide closely resembles the conformation in intact HIV Env and identify amino acids in bnAbs that are important for neutralization breadth.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques., Graphical abstract, Convalescent human-derived SARS-CoV-2 RBD and NTD antibodies mediated neutralization as well as infection enhancement in vitro, yet infusion of these antibodies in mice or cynomolgus macaques resulted in suppression of virus replication.

In the RV144 gp120 HIV vaccine trial, decreased transmission risk was correlated with Abs that reacted with a linear epitope at a lysine residue at position 169 (K169) in the HIV-1 envelope (Env) V2 region. The K169 V2 response was restricted to Abs bearing Vλ rearrangements that expressed aspartic acid/glutamic acid in CDR L2. The AE.A244 gp120 in AIDSVAX B/E also bound to the unmutated ancestor of a V2-glycan broadly neutralizing Ab, but this Ab type was not induced in the RV144 trial. In this study, we sought to determine whether immunodominance of the V2 linear epitope could be overcome in the absence of human Vλ rearrangements. We immunized IgH- and Igκ-humanized mice with the AE.A244 gp120 Env. In these mice, the V2 Ab response was focused on a linear epitope that did not include K169. V2 Abs were isolated that used the same human VH gene segment as an RV144 V2 Ab but paired with a mouse λ L chain. Structural characterization of one of these V2 Abs revealed how the linear V2 epitope could be engaged, despite the lack of aspartic acid/glutamic acid encoded in the mouse repertoire. Thus, despite the absence of the human Vλ locus in these humanized mice, the dominance of Vλ pairing with human VH for HIV-1 Env V2 recognition resulted in human VH pairing with mouse λ L chains instead of allowing otherwise subdominant V2-glycan broadly neutralizing Abs to develop.

Summary: Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans—a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors. : Most bnAb epitopes on HIV-1 Envelope include host glycans, but previous Env vaccines have not induced glycan-dependent antibodies. Saunders et al. describe here the ontogeny, crystal structure with glycan, and virion Man9GlcNAc2-dependent neutralization for glycan-reactive antibodies induced by envelope vaccination. Keywords: HIV, V3 glycan, vaccination, glycan, long-term immunization

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single−molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env. Structural analysis by single−particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins shows rearrangements in the gp120 topological layer contacts with gp41. Displacement of a conserved tryptophan (W571) from its typical pocket in these Env mutants renders the Env insensitive to CD4 binding. These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design., Here, using cryo-EM and smFRET, Henderson et al. show how tryptophan 571 in the HIV-1 Env acts as a conformational switch during receptor-mediated viral entry and design HIV-1 Env proteins that cannot undergo conformational changes. This has important implications for HIV-1 vaccine design.

INTRODUCTION A major goal of HIV-1 vaccine development is the design of immunogens that induce broadly neutralizing antibodies (bnAbs). However, vaccination of humans has not resulted in the induction of affinity-matured and potent HIV-1 bnAbs. To devise effective vaccine strategies, we previously determined the maturation pathway of select HIV-1 bnAbs from acute infection through neutralizing antibody development. During their evolution, bnAbs acquire an abundance of improbable amino acid substitutions as a result of nucleotide mutations at variable region sequences rarely targeted by activation-induced cytidine deaminase, the enzyme responsible for antibody mutation. A subset of improbable mutations is essential for broad neutralization activity, and their acquisition represents a key roadblock to bnAb development. RATIONALE Current bnAb lineage–based vaccine strategies can initiate bnAb lineage development in animal models but have not specifically elicited the improbable mutations required for neutralization breadth. Induction of bnAbs requires vaccine strategies that specifically engage bnAb precursors and subsequently select for improbable mutations required for broadly neutralizing activity. We hypothesized that vaccination with immunogens that bind with moderate to high affinity to bnAb B cell precursors, and with higher affinity to precursors that have acquired improbable mutations, could initiate bnAb B cell lineages and select for key improbable mutations required for bnAb development. RESULTS We elicited serum neutralizing HIV-1 antibodies in human bnAb precursor knock-in mice and wild-type macaques vaccinated with immunogens designed to select for improbable mutations. We designed two HIV-1 envelope immunogens that bound precursor B cells of either a CD4 binding site or V3-glycan bnAb lineage. In vitro, these immunogens bound more strongly to bnAb precursors once the precursor acquired the desired improbable mutations. Vaccination of macaques with the CD4 binding site–targeting immunogen induced CD4 binding site serum neutralizing antibodies. Antibody sequences elicited in human bnAb precursor knock-in mice encoded functional improbable mutations critical for bnAb development. In bnAb precursor knock-in mice, we isolated a vaccine-elicited monoclonal antibody bearing functional improbable mutations that was capable of neutralizing multiple HIV-1 global isolates. Structures of a bnAb precursor, a bnAb, and the vaccine-elicited antibody revealed the precise roles that acquired improbable mutations played in recognizing the HIV-1 envelope. Thus, our immunogens elicited antibody responses in macaques and knock-in mice that exhibited the mutational patterns, structural characteristics, or neutralization profiles of nascent broadly neutralizing antibodies. CONCLUSION Our study represents a proof of concept for targeted selection of improbable mutations to guide antibody affinity maturation. Moreover, this study demonstrates a rational strategy for sequential immunogen design to circumvent the difficult roadblocks in HIV-1 bnAb induction by vaccination. We show that immunogens should exhibit differences in affinity across antibody maturation stages where improbable mutations are necessary for the desired antibody function. This strategy of selection of specific antibody nucleotides by immunogen design can be applied to B cell lineages targeting other pathogens where guided affinity maturation is needed for a protective antibody response.

Somatic mutations within antibody variable and framework regions (FWR) can alter thermostability and structural flexibility, but their impact on functional potency is unclear. Here we study thermostability and use molecular dynamics (MD) simulations to assess the role of FWR mutations during maturation of HIV-1 broadly neutralizing antibodies (bnAbs). The tested bnAbs show lower thermostability than their unmutated ancestor antibodies. FWR mutations in the Fab elbow region are frequently observed in HIV-1 bnAbs and MD simulations show that such FWR mutations alter interdomain flexibility in two HIV-1 bnAbs. In a CD4-binding site lineage, reversion mutations result in a loss of neutralization potency in an early intermediate and affinity-matured bnAb against autologous and heterologous Tier-2 viruses, respectively. Elbow region reversion mutations in a glycan-V3 bnAb modestly reduces potency against an autologous virus isolate. Thus, selection of mutations in the Fab elbow region impacts interdomain conformational flexibility and paratope plasticity during bnAb development., Somatic mutations within antibody framework regions (FWR) can alter structural flexibility, but their role in maturation of broadly neutralizing antibodies (bnAbs) is unclear. Here the authors show how FWR mutations impact interdomain conformational flexibility and paratope plasticity during bnAb development.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells., Graphical abstract, Structural and functional analyses identify a category of glycan-reactive antibodies in macaques and humans that are marked by the dimerization of the antigen-binding fragment. These antibodies are involved in HIV neutralization and also recognize the S2 protein of SARS-CoV-2.

Summary Elicitation of VRC01-class broadly neutralizing antibodies (bnAbs) is an appealing approach for a preventative HIV-1 vaccine. Despite extensive investigations, strategies to induce VRC01-class bnAbs and overcome the barrier posed by the envelope N276 glycan have not been successful. Here, we inferred a high-probability unmutated common ancestor (UCA) of the VRC01 lineage and reconstructed the stages of lineage maturation. Env immunogens designed on reverted VRC01-class bnAbs bound to VRC01 UCA with affinity sufficient to activate naive B cells. Early mutations defined maturation pathways toward limited or broad neutralization, suggesting that focusing the immune response is likely required to steer B cell maturation toward the development of neutralization breadth. Finally, VRC01 lineage bnAbs with long CDR H3s overcame the HIV-1 N276 glycan barrier without shortening their CDR L1, revealing a solution for broad neutralization in which the heavy chain, not CDR L1, is the determinant to accommodate the N276 glycan., Graphical Abstract, Highlights • A high-probability VRC01 lineage UCA was inferred and CDRH3 evolution defined • Env immunogens bind to VRC01 UCA with affinity sufficient to activate naive B cells • Early mutations defined maturation pathways toward limited or broad neutralization • Antibodies with long CDRH3s achieved neutralization breadth without shortening CDRL1s, Understanding how HIV-1 VRC01-class broadly neutralizing antibodies overcome maturation barriers is key for vaccine development. Bonsignori et al. inferred the unmutated common ancestor of the VRC01 lineage, reconstructed the stages of lineage maturation, and identified multiple solutions adopted by evolving B cells to overcome the N276 glycan barrier and achieve broad neutralization.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env+B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env− upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env+ (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools., An efficient HIV-1 vaccine will likely depend on eliciting broadly neutralizing antibodies (bnAb). Here the authors analyze the B cell repertoire in macaques and knock-in mice in response to sequential immunization with Env variants that induce a bnAb targeting the CD4-binding site of Env in a HIV-1 infected individual.

Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. Antibody 10E8, reactive with the distal portion of the membrane-proximal external region (MPER) of HIV-1 gp41, is broadly neutralizing. However, the ontogeny of distal MPER antibodies and the relationship of memory B cell to plasma bnAbs are poorly understood. HIV-1–specific memory B cell flow sorting and proteomic identification of anti-MPER plasma antibodies from an HIV-1–infected individual were used to isolate broadly neutralizing distal MPER bnAbs of the same B cell clonal lineage. Structural analysis demonstrated that antibodies from memory B cells and plasma recognized the envelope gp41 bnAb epitope in a distinct orientation compared with other distal MPER bnAbs. The unmutated common ancestor of this distal MPER bnAb was autoreactive, suggesting lineage immune tolerance control. Construction of chimeric antibodies of memory B cell and plasma antibodies yielded a bnAb that potently neutralized most HIV-1 strains.

Most broadly neutralizing antibodies (BNAbs) elicited in response to HIV-1 infection are extraordinarily mutated. One goal of HIV-1 vaccine development is to induce antibodies that are similar to the most potent and broad BNAbs isolated from infected subjects. The most effective BNAbs have very high mutation frequencies, indicative of the long periods of continual activation necessary to acquire the BNAb phenotype through affinity maturation. Understanding the mutational patterns that define the maturation pathways in BNAb development is critical to vaccine design efforts to recapitulate through vaccination the successful routes to neutralization breadth and potency that have occurred in natural infection. Studying the mutational changes that occur during affinity maturation, however, requires accurate partitioning of sequence data into B-cell clones and identification of the starting point of a B-cell clonal lineage, the initial V(D)J rearrangement. Here, we describe the statistical framework we have used to perform these tasks. Through the recent advancement of these and similar computational methods, many HIV-1 ancestral antibodies have been inferred, synthesized and their structures determined. This has allowed, for the first time, the investigation of the structural mechanisms underlying the affinity maturation process in HIV-1 antibody development. Here, we review what has been learned from this atomic-level structural characterization of affinity maturation in HIV-1 antibodies and the implications for vaccine design.

A preventive HIV-1 vaccine should induce HIV-1-specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.

Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

A goal for an HIV-1 vaccine is to overcome virus variability by inducing broadly neutralizing antibodies (bnAbs). One key target of bnAbs is the glycan-polypeptide at the base of the envelope (Env) third variable loop (V3). We have designed and synthesized a homogeneous minimal immunogen with high-mannose glycans reflective of a native Env V3-glycan bnAb epitope (Man9-V3). V3-glycan bnAbs bound to Man9-V3 glycopeptide and native-like gp140 trimers with similar affinities. Fluorophore-labeled Man9-V3 glycopeptides bound to bnAb memory B cells and were able to be used to isolate a V3-glycan bnAb from an HIV-1–infected individual. In rhesus macaques, immunization with Man9-V3 induced V3-glycan-targeted antibodies. Thus, the Man9-V3 glycopeptide closely mimics an HIV-1 V3-glycan bnAb epitope and can be used to isolate V3-glycan bnAbs.

Development of an HIV vaccine is a global priority. A major roadblock to a vaccine is an inability to induce protective broadly neutralizing antibodies (bnAbs). HIV gp41 bnAbs have characteristics that predispose them to be controlled by tolerance. We used gp41 2F5 bnAb germline knock-in mice and macaques vaccinated with immunogens reactive with germline precursors to activate neutralizing antibodies. In germline knock-in mice, bnAb precursors were deleted, with remaining anergic B cells capable of being activated by germline-binding immunogens to make gp41-reactive immunoglobulin M (IgM). Immunized macaques made B cell clonal lineages targeted to the 2F5 bnAb epitope, but 2F5-like antibodies were either deleted or did not attain sufficient affinity for gp41-lipid complexes to achieve the neutralization potency of 2F5. Structural analysis of members of a vaccine-induced antibody lineage revealed that heavy chain complementarity-determining region 3 (HCDR3) hydrophobicity was important for neutralization. Thus, gp41 bnAbs are controlled by immune tolerance, requiring vaccination strategies to transiently circumvent tolerance controls.

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Proteins often undergo conformational changes when binding to each other. A major fraction of backbone conformational changes involves motion on the protein surface, particularly in loops. Proper accounting for the motion of protein surface loops represents a challenge for protein-protein docking algorithms. A first step in addressing this challenge is to distinguish protein surface loops that are likely to undergo backbone conformational changes upon protein-protein binding (mobile loops) from those that are not (stationary loops). In this study we developed a machine learning strategy based on support vector machines (SVM). Our SVM uses three features of loop residues in the unbound protein structures — Ramachandran angles, crystallographic B-factors and relative accessible surface area — to distinguish mobile loops from stationary ones. This method yields an average prediction accuracy of 75.3% compared with a random prediction accuracy of 50%, and an average of 0.79 area under the receiver operating characteristic (ROC) curve, using 4-fold cross-validation. Testing the method on an independent dataset, we obtained a prediction accuracy of 70.5%. Finally, we applied the method to 11 complexes that involve members from the Ras superfamily and achieved prediction accuracy of 92.8% for the Ras superfamily proteins and 74.4% for their binding partners.

An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.

SummaryMonoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

SummaryIn HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.