Antibody sequence determinants of viral antigen specificity.

Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, resulting in the generation of diverse, antigen-specific antibody repertoires. A paramount feature of antibodies that enables their critical contributions in counteracting recurrent and novel pathogens, and consequently fostering their utility as valuable targets for therapeutic and vaccine development, is the exquisite specificity displayed against their target antigens. Yet, there is still limited understanding of the determinants of antibody-antigen specificity, particularly as a function of antibody sequence. In recent years, experimental characterization of antibody repertoires has led to novel insights into fundamental properties of antibody sequences but has been largely decoupled from at-scale antigen specificity analysis. Here, using the LIBRA-seq technology, we generated a large data set mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against 20 viral antigens representing diverse pathogens of biomedical significance. Analysis uncovered virus-specific patterns in variable gene usage, gene pairing, somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals, including the presence of public antibody clonotypes. Notably, our results showed that, for B-cell receptors originating from different individuals but leveraging an identical combination of heavy and light chain variable genes, there is a specific CDRH3 identity threshold above which B cells appear to exclusively share the same antigen specificity. This finding provides a quantifiable measure of the relationship between antibody sequence and antigen specificity and further defines experimentally grounded criteria for defining public antibody clonality.IMPORTANCEThe B-cell compartment of the humoral immune system plays a critical role in the generation of antibodies upon new and repeated pathogen exposure. This study provides an unprecedented level of detail on the molecular characteristics of antibody repertoires that are specific to each of the different target pathogens studied here and provides empirical evidence in support of a 70% CDRH3 amino acid identity threshold in pairs of B cells encoded by identical IGHV:IGL(K)V genes, as a means of defining public clonality and therefore predicting B-cell antigen specificity in different individuals. This is of exceptional importance when leveraging public clonality as a method to annotate B-cell receptor data otherwise lacking antigen specificity information. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

Phase 1 dose-escalation trial evaluating a group 2 influenza hemagglutinin stabilized stem nanoparticle vaccine.

The relative conservation of the influenza hemagglutinin (HA) stem compared to that of the immunodominant HA head makes the HA stem an attractive target for broadly protective influenza vaccines. Here we report the first-in-human, dose-escalation, open-label trial (NCT04579250) evaluating an unadjuvanted group 2 stabilized stem ferritin nanoparticle vaccine based on the H10 A/Jiangxi-Donghu/346/2013 influenza HA, H10ssF, in healthy adults. Participants received a single 20 mcg dose (n = 3) or two 60 mcg doses 16 weeks apart (n = 22). Vaccination with H10ssF was safe and well tolerated with only mild systemic and local reactogenicity reported. No serious adverse events occurred. Vaccination significantly increased homologous H10 HA stem binding and neutralizing antibodies at 2 weeks after both first and second vaccinations, and these responses remained above baseline at 40 weeks. Heterologous H3 and H7 binding antibodies also significantly increased after each vaccination and remained elevated throughout the study. These data indicate that the group 2 HA stem nanoparticle vaccine is safe and induces stem-directed binding and neutralizing antibodies.

Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase 1 clinical trial investigating a ferritin nanoparticle vaccine displaying H2 hemagglutinin (HA) in H2-naive and H2-exposed adults enabled us to perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited. We temporally map the epitopes targeted by serum antibodies after vaccine prime and boost, revealing that previous H2 exposure results in higher responses to the variable HA head domain. In contrast, initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryoelectron microscopy and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the HA head, including the receptor-binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses post-vaccination.

Enterovirus virus-like-particle and inactivated poliovirus vaccines do not elicit substantive cross-reactive antibody responses.

Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.

Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses.

Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.

Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase.

Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.

C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Combinatorial immune refocusing within the influenza hemagglutinin RBD improves cross-neutralizing antibody responses.

The receptor-binding domain (RBD) of influenza virus hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies. Here, we evaluate the ability of several immunofocusing techniques to enhance the functional breadth of vaccine-elicited immune responses against the HA RBD. We present a series of "trihead" nanoparticle immunogens that display native-like closed trimeric RBDs from the HAs of several H1N1 influenza viruses. The series includes hyperglycosylated and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the receptor-binding site periphery. Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicit higher hemagglutination inhibition (HAI) and neutralizing activity than the corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation. By contrast, mosaic nanoparticle display and antigen hypervariation do not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Our results yield important insights into antibody responses against the RBD and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.

Antigen spacing on protein nanoparticles influences antibody responses to vaccination.

Immunogen design approaches aim to control the specificity and quality of antibody responses elicited by next-generation vaccines. Here, we use computational protein design to generate a nanoparticle vaccine platform based on the receptor-binding domain (RBD) of influenza hemagglutinin (HA) that enables precise control of antigen conformation and spacing. HA RBDs are presented as either monomers or native-like closed trimers that are connected to the underlying nanoparticle by a rigid linker that is modularly extended to precisely control antigen spacing. Nanoparticle immunogens with decreased spacing between trimeric RBDs elicit antibodies with improved hemagglutination inhibition and neutralization potency as well as binding breadth across diverse H1 HAs. Our "trihead" nanoparticle immunogen platform provides insights into anti-HA immunity, establishes antigen spacing as an important parameter in structure-based vaccine design, and embodies several design features that could be used in next-generation vaccines against influenza and other viruses.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase I clinical trial investigating a ferritin nanoparticle displaying H2 hemagglutinin in H2-naïve and H2-exposed adults. Therefore, we could perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited after H2 vaccination. We temporally map the epitopes targeted by serum antibodies after first and second vaccinations and show previous H2 exposure results in higher responses to the variable head domain of hemagglutinin while initial responses in H2-naïve participants are dominated by antibodies targeting conserved epitopes. We use cryo-EM and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the hemagglutinin head including the receptor binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses.

Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera.

Multivalent antigen display is a fast-growing area of interest toward broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor binding domain. However, targeting conserved non-RBD epitopes could limit the potential for antigenic escape. To explore new potential targets, we engineered protein nanoparticles displaying coronavirus prefusion-stabilized spike (CoV_S-2P) trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in female mice. Monotypic SARS-1 nanoparticles elicit cross-neutralizing antibodies against MERS-CoV and protect against MERS-CoV challenge. MERS and SARS nanoparticles elicit S1-focused antibodies, revealing a conserved site on the S N-terminal domain. Moreover, mosaic nanoparticles co-displaying distinct CoV_S-2P trimers elicit antibody responses to distant cross-group antigens and protect male and female mice against MERS-CoV challenge. Our findings will inform further efforts toward the development of pan-coronavirus vaccines.

Corrigendum: Impact of adjuvant: trivalent vaccine with quadrivalent-like protection against heterologous Yamagata-lineage influenza B virus.

[This corrects the article DOI: 10.3389/fimmu.2022.1002286.].

Refined semi-lethal aerosol H5N1 influenza model in cynomolgus macaques for evaluation of medical countermeasures.

Highly pathogenic avian influenza A H5N1 viruses cause high mortality in humans and have pandemic potential. Effective vaccines and treatments against this threat are urgently needed. Here, we have refined our previously established model of lethal H5N1 infection in cynomolgus macaques. An inhaled aerosol virus dose of 5.1 log10 plaque-forming unit (pfu) induced a strong febrile response and acute respiratory disease, with four out of six macaques succumbing after challenge. Vaccination with three doses of adjuvanted seasonal quadrivalent influenza vaccine elicited low but detectable neutralizing antibody to H5N1. All six vaccinated macaques survived four times the 50% lethal dose of aerosolized H5N1, while four of six unvaccinated controls succumbed to disease. Although vaccination did not protect against severe influenza, vaccinees had reduced respiratory dysfunction and lower viral load in airways compared to controls. We anticipate that our macaque model will play a vital role in evaluating vaccines and antivirals against influenza pandemics.

Designed nanoparticles elicit cross-reactive antibody responses to conserved influenza virus hemagglutinin stem epitopes.

Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that tandem copies of an alpha-helical fragment from the conserved stem region (helix-A) can be displayed on the protruding spikes structures of a capsid scaffold. The stem region of HA on these designed chimeric nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, mice vaccinated with H1-nanoparticles were protected from lethal challenge with H1N1 influenza virus. By using a nanoparticle library approach with this helix-A nanoparticle design, we show that this vaccine nanoparticle construct design could be applicable to different influenza HA subtypes. Importantly, antibodies elicited by H1, H5, and H7 nanoparticles demonstrated homosubtypic and heterosubtypic cross-reactivity binding to different HA subtypes. Also, helix-A nanoparticle immunizations were used to isolate mouse monoclonal antibodies that demonstrated heterosubtypic cross-reactivity and provided protection to mice from viral challenge via passive-transfer. This tandem helix-A nanoparticle construct represents a novel design to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes.

Combinatorial immune refocusing within the influenza hemagglutinin head elicits cross-neutralizing antibody responses.

The head domain of influenza hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies during infection and vaccination. Here we evaluated a series of immunogens that combined several immunofocusing techniques for their ability to enhance the functional breadth of vaccine-elicited immune responses. We designed a series of "trihead" nanoparticle immunogens that display native-like closed trimeric heads from the HAs of several H1N1 influenza viruses, including hyperglycosylated variants and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the periphery of the receptor binding site (RBS). Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicited higher HAI and neutralizing activity against vaccine-matched and -mismatched H1 viruses than corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation, indicating that both of these engineering strategies contributed to improved immunogenicity. By contrast, mosaic nanoparticle display and antigen hypervariation did not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Serum competition assays and electron microscopy polyclonal epitope mapping revealed that the trihead immunogens, especially when hyperglycosylated, elicited a high proportion of antibodies targeting the RBS, as well as cross-reactive antibodies targeting a conserved epitope on the side of the head. Our results yield important insights into antibody responses against the HA head and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.

Antigen spacing on protein nanoparticles influences antibody responses to vaccination.

Immunogen design approaches aim to control the specificity and quality of antibody responses to enable the creation of next-generation vaccines with improved potency and breadth. However, our understanding of the relationship between immunogen structure and immunogenicity is limited. Here we use computational protein design to generate a self-assembling nanoparticle vaccine platform based on the head domain of influenza hemagglutinin (HA) that enables precise control of antigen conformation, flexibility, and spacing on the nanoparticle exterior. Domain-based HA head antigens were presented either as monomers or in a native-like closed trimeric conformation that prevents exposure of trimer interface epitopes. These antigens were connected to the underlying nanoparticle by a rigid linker that was modularly extended to precisely control antigen spacing. We found that nanoparticle immunogens with decreased spacing between closed trimeric head antigens elicited antibodies with improved hemagglutination inhibition (HAI) and neutralization potency as well as binding breadth across diverse HAs within a subtype. Our "trihead" nanoparticle immunogen platform thus enables new insights into anti-HA immunity, establishes antigen spacing as an important parameter in structure-based vaccine design, and embodies several design features that could be used to generate next-generation vaccines against influenza and other viruses.

EV-D68 virus-like particle vaccines elicit cross-clade neutralizing antibodies that inhibit infection and block dissemination.

Enterovirus D68 (EV-D68) causes severe respiratory illness in children and can result in a debilitating paralytic disease known as acute flaccid myelitis. No treatment or vaccine for EV-D68 infection is available. Here, we demonstrate that virus-like particle (VLP) vaccines elicit a protective neutralizing antibody against homologous and heterologous EV-D68 subclades. VLP based on a B1 subclade 2014 outbreak strain elicited comparable B1 EV-D68 neutralizing activity as an inactivated viral particle vaccine in mice. Both immunogens elicited weaker cross-neutralization against heterologous viruses. A B3 VLP vaccine elicited more robust neutralization of B3 subclade viruses with improved cross-neutralization. A balanced CD4+ T helper response was achieved using a carbomer-based adjuvant, Adjuplex. Nonhuman primates immunized with this B3 VLP Adjuplex formulation generated robust neutralizing antibodies against homologous and heterologous subclade viruses. Our results suggest that both vaccine strain and adjuvant selection are critical elements for improving the breadth of protective immunity against EV-D68.

An influenza H1 hemagglutinin stem-only immunogen elicits a broadly cross-reactive B cell response in humans.

Current yearly seasonal influenza vaccines primarily induce an antibody response directed against the immunodominant but continually diversifying hemagglutinin (HA) head region. These antibody responses provide protection against the vaccinating strain but little cross-protection against other influenza strains or subtypes. To focus the immune response on subdominant but more conserved epitopes on the HA stem that might protect against a broad range of influenza strains, we developed a stabilized H1 stem immunogen lacking the immunodominant head displayed on a ferritin nanoparticle (H1ssF). Here, we evaluated the B cell response to H1ssF in healthy adults ages 18 to 70 in a phase 1 clinical trial (NCT03814720). We observed both a strong plasmablast response and sustained elicitation of cross-reactive HA stem-specific memory B cells after vaccination with H1ssF in individuals of all ages. The B cell response was focused on two conserved epitopes on the H1 stem, with a highly restricted immunoglobulin repertoire unique to each epitope. On average, two-thirds of the B cell and serological antibody response recognized a central epitope on the H1 stem and exhibited broad neutralization across group 1 influenza virus subtypes. The remaining third recognized an epitope near the viral membrane anchor and was largely limited to H1 strains. Together, we demonstrate that an H1 HA immunogen lacking the immunodominant HA head produces a robust and broadly neutralizing HA stem-directed B cell response.

An influenza hemagglutinin stem nanoparticle vaccine induces cross-group 1 neutralizing antibodies in healthy adults.

Influenza vaccines could be improved by platforms inducing cross-reactive immunity. Immunodominance of the influenza hemagglutinin (HA) head in currently licensed vaccines impedes induction of cross-reactive neutralizing stem-directed antibodies. A vaccine without the variable HA head domain has the potential to focus the immune response on the conserved HA stem. This first-in-human dose-escalation open-label phase 1 clinical trial (NCT03814720) tested an HA stabilized stem ferritin nanoparticle vaccine (H1ssF) based on the H1 HA stem of A/New Caledonia/20/1999. Fifty-two healthy adults aged 18 to 70 years old enrolled to receive either 20 µg of H1ssF once (n = 5) or 60 µg of H1ssF twice (n = 47) with a prime-boost interval of 16 weeks. Thirty-five (74%) 60-µg dose participants received the boost, whereas 11 (23%) boost vaccinations were missed because of public health restrictions in the early stages of the COVID-19 pandemic. The primary objective of this trial was to evaluate the safety and tolerability of H1ssF, and the secondary objective was to evaluate antibody responses after vaccination. H1ssF was safe and well tolerated, with mild solicited local and systemic reactogenicity. The most common symptoms included pain or tenderness at the injection site (n = 10, 19%), headache (n = 10, 19%), and malaise (n = 6, 12%). We found that H1ssF elicited cross-reactive neutralizing antibodies against the conserved HA stem of group 1 influenza viruses, despite previous H1 subtype head-specific immunity. These responses were durable, with neutralizing antibodies observed more than 1 year after vaccination. Our results support this platform as a step forward in the development of a universal influenza vaccine.