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SummaryCongenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM responses contribute to ZIKV immunity in pregnancy, mediating neutralization up to three months post symptoms. From a ZIKV-infected pregnant woman, we established a B cell line secreting a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets a novel envelope dimer epitope within Domain II. The arrangement of this epitope on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not achievable by IgG antibodies. DH1017.IgM protected against lethal ZIKV challenge in mice. Our findings identify a unique role of antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunoprophylactic, particularly during pregnancy.Key pointsPlasma IgM contributes to early ZIKV neutralization during pregnancyUltrapotent neutralization by pentameric DH1017.IgM mAb depends on isotypeDH1017.IgM can engage all binding sites concurrently through different angles of approachDH1017.IgM protects mice against lethal ZIKV challenge

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

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

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth., A subset of HIV-infected individuals can develop broadly neutralizing antibodies. Boyd and colleagues studied such HIV-infected individuals and found significant perturbations in their antibody repertoires, including increased frequencies of B cells expressing antibodies with features associated with autoreactivity.

In the RV144 HIV-1 phase III trial, vaccine efficacy directly correlated with the magnitude of the variable region 2–specific (V2-specific) IgG antibody response, and in the presence of low plasma IgA levels, with the magnitude of plasma antibody-dependent cellular cytotoxicity. Reenrollment of RV144 vaccinees in the RV305 trial offered the opportunity to define the function, maturation, and persistence of vaccine-induced V2-specific and other mAb responses after boosting. We show that the RV144 vaccine regimen induced persistent V2 and other HIV-1 envelope–specific memory B cell clonal lineages that could be identified throughout the approximately 11-year vaccination period. Subsequent boosts increased somatic hypermutation, a critical requirement for antibody affinity maturation. Characterization of 22 vaccine-induced V2-specific mAbs with epitope specificities distinct from previously characterized RV144 V2-specific mAbs CH58 and CH59 found increased in vitro antibody-mediated effector functions. Thus, when inducing non-neutralizing antibodies, one method by which to improve HIV-1 vaccine efficacy may be through late boosting to diversify the V2-specific response to increase the breadth of antibody-mediated anti–HIV-1 effector functions.

Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion.

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.

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy. IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV + ) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases the propensity of Env to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC.

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.

Immunoglobulin A (IgA) antibodies exist in monomeric, dimeric, and secretory forms. Dimerization of IgA depends on a 15-kD polypeptide termed "joining (J) chain," which is also part of the binding site for an epithelial glycoprotein called "secretory component (SC)," whether this after apical cleavage on secretory epithelia is ligand bound in secretory IgA (SIgA) or in a free form. Uncleaved membrane SC, also called the "polymeric Ig receptor," is thus crucial for transcytotic export of SIgA to mucosal surfaces, where it interacts with and modulates commensal bacteria and mediates protective immune responses against exogenous pathogens. To evaluate different forms of IgA, we have produced mouse monoclonal antibodies (MAbs) against human J-chain and free SC. We found that J-chain MAb 9A8 and SC MAb 9H7 identified human dimeric IgA and SIgA in enzyme-linked immunoassay and western blot analysis, as well as functioning in immunohistochemistry to identify cytoplasmic IgA of intestinal lamina propria plasmablasts/plasma cells and crypt epithelium of distal human intestine. Finally, we demonstrated that SC MAb 9H7 cross-reacted with rhesus macaque SIgA. These novel reagents should be of use in the study of the biology of various forms of IgA in humans and SIgA in macaques, as well as in monitoring the production and/or isolation of these forms of IgA.

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.

Sequences of rearranged Ig light chain variable region genes of human anti-alpha3(IV)NC1 collagen monoclonal antibody 2D6. Sequence of mAb 2D6 light chain V-J region and sequences of the closest corresponding germline variable region gene segments identified in the IMGT/V-QUEST reference databases, with CDR-IMGT delineated according to the IMGT unique numbering for V-DOMAIN.

Sequences of rearranged Ig heavy chain variable region genes of human anti-alpha3(IV)NC1 collagen monoclonal antibody 2D6. Sequence of mAb 2D6 heavy chain V-D-J region and sequences of the closest corresponding germline variable region gene segments identified in the IMGT/V-QUEST reference databases, with CDR-IMGT delineated according to the IMGT unique numbering for V-DOMAIN.

Human monoclonal antibody CH58 isolated from an RV144 vaccinee binds at Lys169 of the HIV-1 Env gp120 V2 region, a site of vaccine-induced immune pressure. CH58 neutralizes HIV-1 CRF_01 AE strain 92TH023 and mediates ADCC against CD4 + T cell targets infected with CRF_01 AE tier 2 virus. CH58 and other antibodies that bind to a gp120 V2 epitope have a second light chain complementarity determining region (LCDR2) bearing a glutamic acid, aspartic acid (ED) motif involved in forming salt bridges with polar, basic side amino acid side chains in V2. In an effort to learn how V2 responses develop, we determined the crystal structures of the CH58-UA antibody unliganded and bound to V2 peptide. The structures showed an LCDR2 structurally pre-conformed from germline to interact with V2 residue Lys169. LCDR3 was subject to conformational selection through the affinity maturation process. Kinetic analyses demonstrate that only a few contacts were responsible for a 2000-fold increase in KD through maturation, and this effect was predominantly due to an improvement in off-rate. This study shows that preconformation and preconfiguration can work in concert to produce antibodies with desired immunogenic properties., Graphical abstract, Highlights • With only 2-3% mutation from germline, the HIV-1 antibody CH58 developed neutralizing and ADCC capabilities. • The LCDR2 Glu–Asp motif of the RV144 antibody CH58 is pre-conformed from germline to interact with the gp120 V2 loop. • Affinity and neutralization gains resulted from tuning local interactions rather than gross sequence or structure changes. • Structural analyses show the second light chain complementarity determining region Glu–Asp motif of the CH58 antibody isolated from an RV144 vaccinee is optimally pre-conformed from germline to interact with the gp120 V2 loop. The increased binding affinity and neutralization capacity of the mature antibody compared to its germline precursor were achieved with only 2–3% mutation from germline, and the fact that these gains appeared to be a result of the tuning of local interactions rather than gross sequential or conformational changes provides hope that a rational immunogen design for HIV-1 treatment may become a reality.

Background Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. Results The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. Conclusions These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0078-8) contains supplementary material, which is available to authorized users.