The two influenza B virus (FLUBV) lineages have continuously diverged from each other since the 1980s, with recent (post-2015) viruses exhibiting accelerated evolutionary rates. Emerging data from human studies and epidemiological models suggest that increased divergence in contemporary viruses may drive differential cross-protection, where infection with Yamagata lineage viruses provides limited immunity against Victoria lineage viruses. Here, we developed animal models to investigate the mechanisms behind asymmetric cross-protection between contemporary FLUBV lineages. Our results show that contemporary Victoria immunity provides robust cross-protection against the Yamagata lineage, whereas Yamagata immunity offers limited protection against the Victoria lineage. This differential cross-protection is driven by Victoria-elicited neuraminidase (NA)-specific antibodies, which show cross-lineage reactivity, unlike those from Yamagata infections. These findings identify a phenomenon in contemporary FLUBV that may help explain the recent disappearance of the Yamagata lineage from circulation, highlighting the crucial role of targeting NA in vaccination strategies to enhance cross-lineage FLUBV protection.

In a phase 1 clinical trial, a chimeric hemagglutinin (cHA) immunogen induced antibody responses against the conserved hemagglutinin (HA) stalk domain as designed. Here, we determined the specificity, function, and subsets of B cells induced by cHA vaccination by pairing single-cell RNA sequencing and B cell receptor repertoire sequencing. We have shown that the cHA-inactivated vaccine with a squalene-based adjuvant induced a robust activated B cell and memory B cell (MBC) phenotype against two broadly neutralizing epitopes in the stalk domain. The overall specificities of the acute plasmablast (PB) and MBC responses clonally overlapped, suggesting B cell convergence to these broadly protective epitopes. At 1 year post immunization, we identified that cHA vaccination reshaped the HA-specific MBC pool to enrich for stalk-binding B cells. Altogether, these data indicate the cHA vaccine induced robust and durable B cell responses against broadly protective epitopes of the HA stalk domain, in line with serological data.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing a context for future vaccine trials and emphasizing the need for further characterization of protective responses toward conserved epitopes.

The COVID-19 pandemic underscores the need to prepare for future emerging coronavriuses (CoVs) by understanding the principles behind effective CoV vaccine design such as protective immunity and antibody responses. To study which epitopes and subdomains contribute to in vivo protection, we utilized the prefusion-stabilized spike protein of MERS-CoV, MERS S-2P, as a vaccine immunogen. Vaccination with MERS S-2P elicited both receptor-binding domain (RBD)- and non-RBD-specific antibodies, including N-terminal domain (NTD)-specific G2-and CDC2-A2-like antibodies. Intriguingly, the immunogen MERS S-2P_ΔRBD, MERS S-2P with the RBDs removed, protects comparably to S1 and S-2P immunogens against MERS-CoV challenge. Moreover, passive transfer studies of polyclonal IgG from MERS S-2P immunized mice depleted of subdomain-specific antibodies demonstrated that non-RBD antibodies protected more than non-NTD antibodies. Altogether, these findings illustrate that in-vivo protection is not solely driven by RBD-specific antibodies and highlights the importance of targeting non-RBD sites in future CoV vaccine designs.

Chimeric hemagglutinins (cHA) appear to be promising for the design and development of universal influenza vaccines. Influenza A group 1 cHAs, cH5/1, cH8/1, and cH11/1, comprising an H1 stem attached to either an H5, H8, or H11 globular head, have been used sequentially as vaccine immunogens in human clinical trials and induced high levels of broadly protective antibodies. Using X-ray crystallography and negative-stain electron microscopy, we determined structures of cH5/1, cH8/1, and cH11/1 HAs in their apo (unliganded) and antibody Fab-bound states. Stem-reactive antibodies 3E1 and 31.b.09 recognize their cognate epitopes in cH5/1, cH8/1, and cH11/1 HAs. However, with cH5/1, the head domains are rotated by 35 to 45° around the threefold axis of the HA trimer compared to native HA with a more splayed-open conformation at the stem base. cH11/1 with 3E1 is structurally more native-like but resembles cH5/1 with 31.b.09, whereas cH8/1 with 31.b.09 exhibited a range of closed-to-open stem configurations with some separation of head and stem domains. Furthermore, all of these group 1 cHAs effectively bound a broad head trimer interface antibody and other broad stem antibodies. Thus, the cHAs exhibit structural plasticity without compromising the stem and head trimer interface epitopes for elicitation of influenza A group 1 cross-reactive antibodies.

H1N1 influenza viruses are responsible for both seasonal and pandemic influenza. The continual antigenic shift and drift of these viruses highlight the urgent need for a universal influenza vaccine to elicit broadly neutralizing antibodies (bnAbs). Identification and characterization of bnAbs elicited in natural infection and immunization to influenza virus hemagglutinin (HA) can provide insights for development of a universal influenza vaccine. Here, we structurally and biophysically characterize four antibodies that bind to a conserved region on the HA membrane-proximal region known as the anchor epitope. Despite some diversity in their VH and VK genes, the antibodies interact with the HA through germline-encoded residues in HCDR2 and LCDR3. Somatic mutations on HCDR3 also contribute hydrophobic interactions with the conserved HA epitope. This convergent binding mode provides extensive neutralization breadth against H1N1 viruses and suggests possible countermeasures against H1N1 viruses.

Background We report findings from an experimental medicine study of rationally designed prefusion stabilised native-like HIV envelope glycoprotein (Env) immunogens, representative of global circulating strains, delivered by sequential intramuscular injection. Methods Healthy adult volunteers were enrolled into one of five groups (A to E) each receiving a different schedule of one of two consensus Env immunogens (ConM SOSIP, ConS UFO, either unmodified or stabilised by chemical cross-linking, followed by a boost with two mosaic Env immunogens (Mos3.1 and Mos3.2). All immunogens were co-formulated with liposomal Monophosphoryl-Lipid A (MPLA) adjuvant, and volunteers were followed up for 28 days post final Mosaic booster injection. Participants gave written informed consent to join the study. The study is registered on ClinicalTrials.gov ID NCT03816137. Findings Fifty-one participants (men n = 23 and women n = 28) aged 18–55 were enrolled. The seroconversion rate against Env was 100% with all participants having measurable anti-Env IgG antibodies after their second injection and throughout the study. Neutralisation was detected against the ConM pseudovirus in sera of those who had received both ConM and ConS immunogens. However, this activity was limited in breadth and was neither boosted nor broadened in those receiving the Mos3.1 and Mos3.2 immunogens. Neutralising antibody function correlated with binding to V1/V3 and V5 epitopes and peaked after the third injection. Interpretation Rationally designed prefusion-stabilised native-like Env trimers are robustly immunogenic in a prime-boost schedule. When given alone they are insufficient to induce neutralising antibody titres of significant breadth, but they represent potentially valuable polishing immunogens after germline-targeting. Funding European Aids Vaccine initiative (EAVI2020) received funding from EU Horizon 2020, grant number 681137. Structural studies were supported by the Bill and Melinda Gates Foundation (INV-002916).

Vaccination strategies against HIV-1 aim to elicit broadly neutralizing antibodies (bnAbs) using prime-boost regimens with HIV envelope (Env) immunogens. Epitope mapping has shown that early antibody responses are directed to easily accessible nonneutralizing epitopes on Env instead of bnAb epitopes. Autologously neutralizing antibody responses appear upon boosting, once immunodominant epitopes are saturated. Here, we use electron microscopy–based polyclonal epitope mapping (EMPEM) to elucidate how repeated immunization with HIV Env SOSIP immunogens results in the generation of Ab2α anti-idiotypic antibodies in rabbits and rhesus macaques. We present the structures of six anti–immune complex antibodies and find that they target idiotopes composed of framework regions of antibodies bound to Env. Examination of cryo–electron microscopy density enabled prediction of sequences for an anti–immune complex antibody, the paratope of which is enriched with aromatic amino acids. This work sheds light on current vaccine development efforts for HIV, as well as for other pathogens in which repeated exposure to antigen is required.

Prevention of severe COVID‐19 disease by SARS‐CoV‐2 in high‐risk patients, such as immuno‐compromised individuals, can be achieved by administration of antibody prophylaxis, but producing antibodies can be costly. Plant expression platforms allow substantial lower production costs compared to traditional bio‐manufacturing platforms depending on mammalian cells in bioreactors. In this study, we describe the expression, production and purification of the originally human COVA2‐15 antibody in plants. Our plant‐produced mAbs demonstrated comparable neutralizing activity with COVA2‐15 produced in mammalian cells. Furthermore, they exhibited similar capacity to prevent SARS‐CoV‐2 infection in a hamster model. To further enhance these biosimilars, we performed three glyco‐ and protein engineering techniques. First, to increase antibody half‐life, we introduced YTE‐mutation in the Fc tail; second, optimization of N‐linked glycosylation by the addition of a C‐terminal ER‐retention motif (HDEL), and finally; production of mAb in plant production lines lacking β‐1,2‐xylosyltransferase and α‐1,3‐fucosyltransferase activities (FX‐KO). These engineered biosimilars exhibited optimized glycosylation, enhanced phagocytosis and NK cell activation capacity compared to conventional plant‐produced S15 and M15 biosimilars, in some cases outperforming mammalian cell produced COVA2‐15. These engineered antibodies hold great potential for enhancing in vivo efficacy of mAb treatment against COVID‐19 and provide a platform for the development of antibodies against other emerging viruses in a cost‐effective manner.

Monoclonal neutralizing antibodies (mAbs) are considered an important prophylactic against SARS-CoV-2 infection in at-risk populations and a strategy to counteract future sarbecovirus-induced disease. However, most mAbs isolated so far neutralize only a few sarbecovirus strains. Therefore, there is a growing interest in bispecific antibodies (bsAbs) which can simultaneously target different spike epitopes and thereby increase neutralizing breadth and prevent viral escape. Here, we generate and characterize a panel of 30 novel broadly reactive bsAbs using an efficient controlled Fab-arm exchange protocol. We specifically combine some of the broadest mAbs described so far, which target conserved epitopes on the receptor binding domain (RBD). Several bsAbs show superior cross-binding and neutralization compared to the parental mAbs and cocktails against sarbecoviruses from diverse clades, including recent SARS-CoV-2 variants. BsAbs which include mAb COVA2–02 are among the most potent and broad combinations. As a result, we study the unknown epitope of COVA2–02 and show that this mAb targets a distinct conserved region at the base of the RBD, which could be of interest when designing next-generation bsAb constructs to contribute to a better pandemic preparedness.

Clade 2.3.4.4b H5N1 is causing an unprecedented outbreak in dairy cows in the United States. To understand if recent H5N1 viruses are changing their receptor use, we screened recombinant hemagglutinin (HA) from historical and recent 2.3.4.4b H5N1 viruses for binding to distinct glycans bearing terminal sialic acids using a glycan microarray. We find that H5 from A/Texas/37/2024, an isolate from the dairy cow outbreak, has increased binding breadth to core glycans bearing terminal α2,3 sialic acids, the avian receptor, compared to historical and recent 2.3.4.4b H5N1 viruses. We do not observe any binding to α2,6 sialic acids, the receptor used by human seasonal influenza viruses. Using molecular dynamics and a cryo-EM structure of A/Texas/37/2024 H5, we show A/Texas/37/2024 H5 is more flexible within the receptor-binding site compared to a 2.3.4.4b H5 from 2022. We identify a single mutation outside of the receptor binding site, T199I, is responsible for increased binding breadth, as it increases receptor binding site flexibility. Together, these data show recent H5N1 viruses are evolving increased receptor binding breadth which could impact the host range and cell types infected with H5N1.

Antibodies targeting epitopes through germline-encoded motifs can be found in different individuals. While these public antibodies are often beneficial, they also pose hurdles for subdominant antibodies to emerge. Here, we use transgenic mice that reproduce the human IGHV1-69∗01 germline-encoded antibody response to the conserved stem epitope on group 1 hemagglutinin (HA) of influenza A virus to show that this germline-endowed response can be overridden by a subdominant yet cross-group reactive public antibody response. Immunization with a non-cognate group 2 HA stem enriched B cells harboring the IGHD3-9 gene, thereby switching from IGHV1-69- to IGHD3-9-encoded motif-dependent epitope recognition. These IGHD3-9 antibodies bound, neutralized, and conferred cross-group protection in mice against influenza A viruses. A cryoelectron microscopy (cryo-EM) structure of an IGHD3-9 antibody resembled the human broadly neutralizing antibody FI6v3, which uses IGHD3-9. Together, our findings offer insights into vaccine regimens that engage an immunoglobulin repertoire with broader cross-reactivity to influenza A viruses.

Malaria pathology is driven by the accumulation of Plasmodium falciparum-infected erythrocytes in microvessels1. This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis2. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins, as well as parasite sequestration in bioengineered 3D human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies are likely to represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria. Two broadly reactive and inhibitory human monoclonal antibodies against the malaria parasite Plasmodium falciparum have been characterized, providing insights into immunity, prevention and treatment of severe malaria.

HIV-1 infection leads to chronic disease requiring life-long treatment and therefore alternative therapeutics, a cure and/or a protective vaccine are needed. Antibody-mediated effector functions could have a role in the fight against HIV-1. However, the properties underlying the potential beneficial effects of antibodies during HIV-1 infection are poorly understood. To identify a specific profile of antibody features associated with delayed disease progression, we studied antibody polyfunctionality during untreated HIV-1 infection in the well-documented Amsterdam Cohort Studies. Serum samples were analyzed from untreated individuals with HIV-1 at approximately 6 months (n = 166) and 3 years (n = 382) post-seroconversion (post-SC). A Luminex antibody Fc array was used to profile 15 different Fc features for serum antibodies against 20 different HIV-1 envelope glycoprotein antigens and the resulting data was also compared with data on neutralization breadth. We found that high HIV-1 specific IgG1 levels and low IgG2 and IgG4 levels at 3 years post-SC were associated with delayed disease progression. Moreover, delayed disease progression was associated with a broad and polyfunctional antibody response. Specifically, the capacity to interact with all Fc γ receptors (FcγRs) and C1q, and in particular with FcγRIIa, correlated positively with delayed disease progression. There were strong correlations between antibody Fc features and neutralization breadth and several antibody features that were associated with delayed disease progression were also associated with the development of broad and potent antibody neutralization. In summary, we identified a strong association between broad, polyfunctional antibodies and delayed disease progression. These findings contribute new information for the fight against HIV-1, especially for new antibody-based therapy and cure strategies.