Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In , we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family. Here, the authors use cryo-EM to solve the structures of seven potent human antibodies, and demonstrate in vivo protection in a liver burden assay, using chimeric Plasmodium berghei sporozoites expressing Plasmodium falciparum circumsporozoite protein.

Electron microscopy-based polyclonal epitope mapping (EMPEM) can delineate epitope specificities of serum antibodies to a given antigen following vaccination or infection. Here, we present a protocol for the EMPEM method for rapid high-throughput assessment of antibody responses to glycoprotein antigens in vaccination and infection studies. We describe steps for antibody isolation and digestion, antigen complex and purification, and electron microscope imaging. We then detail procedures for processing and analysis of EMPEM data. For complete details on the use and execution of this protocol, please refer to Bianchi et al. (2018). 1

Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4–0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design.

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor–binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Understanding antibody-antigen interactions in a polyclonal immune response in humans and animal models is critical for rational vaccine design. Current approaches typically characterize antibodies that are functionally relevant or highly abundant. Here, we use photo-cross-linking and single-particle electron microscopy to increase antibody detection and unveil epitopes of low-affinity and low-abundance antibodies, leading to a broader structural characterization of polyclonal immune responses. We employed this approach across three different viral glycoproteins and showed increased sensitivity of detection relative to currently used methods. Results were most noticeable in early and late time points of a polyclonal immune response. Additionally, the use of photo-cross-linking revealed intermediate antibody binding states and demonstrated a distinctive way to study antibody binding mechanisms. This technique can be used to structurally characterize the landscape of a polyclonal immune response of patients in vaccination or post-infection studies at early time points, allowing for rapid iterative design of vaccine immunogens.

An effective HIV vaccine likely requires the elicitation of neutralizing antibodies (NAbs) against multiple HIV-1 clades. The recently developed cleavage-independent native flexibly linked (NFL) envelope (Env) trimers exhibit well-ordered conformation and elicit autologous tier 2 NAbs in multiple animal models. Here, we investigated whether the fusion of molecular adjuvant C3d to the Env trimers can improve B- cell germinal center (GC) formation and antibody responses. To generate Env-C3d trimers, we performed a glycine-serine- based (G4S) flexible peptide linker screening and identified a linker range that allowed native folding. A 30–60- amino- acid- long linker facilitates Env-to-C3d association and achieves the secretion of well-ordered trimers and the structural integrity and functional integrity of Env and C3d. The fusion of C3d did not dramatically affect the antigenicity of the Env trimers and enhanced the ability of the Env trimers to engage and activate B cells in vitro. In mice, the fusion of C3d enhanced germinal center formation, the magnitude of Env-specific binding antibodies, and the avidity of the antibodies in the presence of an adjuvant. The Sigma Adjuvant System (SAS) did not affect the trimer integrity in vitro but contributed to altered immunogenicity in vivo, resulting in increased tier 1 neutralization, likely by increased exposure of variable region 3 (V3). Taken together, the results indicate that the fusion of the molecular adjuvant, C3d, to the Env trimers improves antibody responses and could be useful for Env-based vaccines against HIV.

Background Neutralizing antibodies (NAbs) protect against HIV‑1 acquisition in animal models and show promisein treatment of infection. They act by binding to the viral envelope glycoprotein (Env), thereby blocking its recep‑tor interactions and fusogenic function. The potency of neutralization is largely determined by affinity. Less wellexplained is the persistent fraction, the plateau of remaining infectivity at the highest antibody concentrations.Results We observed different persistent fractions for neutralization of pseudovirus derived from two Tier‑2 iso‑lates of HIV‑1, BG505 (Clade A) and B41 (Clade B): it was pronounced for B41 but not BG505 neutralization by NAbPGT151, directed to the interface between the outer and transmembrane subunits of Env, and negligible for eithervirus by NAb PGT145 to an apical epitope. Autologous neutralization by poly‑ and monoclonal NAbs from rabbitsimmunized with soluble native‑like B41 trimer also left substantial persistent fractions. These NAbs largely target acluster of epitopes lining a hole in the dense glycan shield of Env around residue 289. We partially depleted B41‑virionpopulations by incubating them with PGT145‑ or PGT151‑conjugated beads. Each depletion reduced the sensitivityto the depleting NAb and enhanced it to the other. Autologous neutralization by the rabbit NAbs was decreased forPGT145‑depleted and enhanced for PGT151‑depleted B41 pseudovirus. Those changes in sensitivity encompassedboth potency and the persistent fraction. We then compared soluble native‑like BG505 and B41 Env trimers affinity‑purified by each of three NAbs: 2G12, PGT145, or PGT151. Surface plasmon resonance showed differences amongthe fractions in antigenicity, including kinetics and stoichiometry, congruently with the differential neutralization. Thelarge persistent fraction after PGT151 neutralization of B41 was attributable to low stoichiometry, which we explainedstructurally by clashes that the conformational plasticity of B41 Env causes.Conclusion Distinct antigenic forms even of clonal HIV‑1 Env, detectable among soluble native‑like trimer molecules,are distributed over virions and may profoundly mold neutralization of certain isolates by certain NAbs. Affinity puri‑fications with some antibodies may yield immunogens that preferentially expose epitopes for broadly active NAbs,shielding less cross‑reactive ones. NAbs reactive with multiple conformers will together reduce the persistent fractionafter passive and active immunization.

Lassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences among phylogenetically distinct LASV lineages. Despite the sequence diversity of the GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VII, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of the GPC in complex with GP1-A-specific antibodies suggest their neutralization mechanisms. Finally, we present the isolation and characterization of a trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.

A primary objective in malaria vaccine design is the generation of high-quality antibody responses against the circumsporozoite protein of the malaria parasite, Plasmodium falciparum (PfCSP). To enable rational antigen design, we solved a cryo-EM structure of the highly potent anti-PfCSP antibody L9 in complex with recombinant PfCSP. We found that L9 Fab binds multivalently to the minor (NPNV) repeat domain, which is stabilized by a unique set of affinity-matured homotypic, antibody-antibody contacts. Molecular dynamics simulations revealed a critical role of the L9 light chain in integrity of the homotypic interface, which likely impacts PfCSP affinity and protective efficacy. These findings reveal the molecular mechanism of the unique NPNV selectivity of L9 and emphasize the importance of anti-homotypic affinity maturation in protective immunity against P. falciparum. The cryo-EM structure of the highly potent malaria antibody L9 reveals a key role of light-chain derived homotypic interactions in antigen binding and parasite inhibition, enabling antibody engineering and next-generation malaria vaccine design.

Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding. Structural studies of a computationally optimized broadly reactive antigen hemagglutinin in complex with a broadly neutralizing antibody reveal its immunogenic properties and provide insights into flu vaccine design.

SARS-CoV-2 variants evade current monoclonal antibody therapies. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies taking advantage of the avidity and synergy provided by targeting different epitopes. Here we used controlled Fab-arm exchange to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader antibodies that also neutralize SARS-CoV. We demonstrated that the parental antibodies rely on avidity for neutralization using bsAbs containing one irrelevant Fab arm. Using mass photometry to measure the formation of antibody:spike complexes, we determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike, observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent agents to combat circulating and future SARS-like coronaviruses.

Uncleaved prefusion-optimized (UFO) design can stabilize diverse HIV-1 envelope glycoproteins (Envs). Single-component, self-assembling protein nanoparticles (1c-SApNP) can display 8 or 20 native-like Env trimers as vaccine candidates. We characterize the biophysical, structural, and antigenic properties of 1c-SApNPs that present the BG505 UFO trimer with wildtype and modified glycans. For 1c-SApNPs, glycan trimming improves recognition of the CD4 binding site without affecting broadly neutralizing antibodies (bNAbs) to major glycan epitopes. In mice, rabbits, and nonhuman primates, glycan trimming increases the frequency of vaccine responders (FVR) and steers antibody responses away from immunodominant glycan holes and glycan patches. The mechanism of vaccine-induced immunity is examined in mice. Compared with the UFO trimer, the multilayered E2p and I3-01v9 1c-SApNPs show 420 times longer retention in lymph node follicles, 20-32 times greater presentation on follicular dendritic cell dendrites, and up-to-4 times stronger germinal center reactions. These findings can inform future HIV-1 vaccine development. Here the authors present an HIV-1 vaccine strategy that combines Env stabilization, nanoparticle display, and glycan trimming, which improves neutralizing antibody responses, frequency of vaccine responders, and germinal center reactions in animal models.