An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.

Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the in silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment antibody sequence-binding datasets may not contain the mutagenic, antigenic, or antibody sequence diversity necessary for deep learning approaches to capture molecular recognition. In part, this is because limited experimental platforms exist for assessing quantitative and simultaneous sequence-function relationships for multiple antibodies. Here we present MAGMA-seq, an integrated technology that combines multiple antigens and multiple antibodies and determines quantitative biophysical parameters using deep sequencing. We demonstrate MAGMA-seq on two pooled libraries comprising mutants of nine different human antibodies spanning light chain gene usage, CDR H3 length, and antigenic targets. We demonstrate the comprehensive mapping of potential antibody development pathways, sequence-binding relationships for multiple antibodies simultaneously, and identification of paratope sequence determinants for binding recognition for broadly neutralizing antibodies (bnAbs). MAGMA-seq enables rapid and scalable antibody engineering of multiple lead candidates because it can measure binding for mutants of many given parental antibodies in a single experiment.

Immunization-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection.

Antigens from viruses or immunizations can persist or are archived in lymph node stromal cells such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration resulted in cross-presentation of archived antigens and boosted memory CD8 + T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8 + T cells specific to the archived antigen from the immunization were significantly higher than memory CD8 + T cells of a different antigen specificity. Finally, the boosted memory CD8 + T cells resulted in increased protection against Listeria monocytogenes expressing the antigen from the immunization, but only for the duration that the antigen was archived. These findings outline an important mechanism by which lymph node stromal cell archived antigens, in addition to bystander activation, can augment memory CD8 + T cell responses during repeated inflammatory insults.

An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.

Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the in silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment antibody sequence-binding datasets may not contain the mutagenic, antigenic, or antibody sequence diversity necessary for deep learning approaches to capture molecular recognition. In part, this is because limited experimental platforms exist for assessing quantitative and simultaneous sequence-function relationships for multiple antibodies. Here we present MAGMA-seq, an integrated technology that combines multiple antigens and multiple antibodies and determines quantitative biophysical parameters using deep sequencing. We demonstrate MAGMA-seq on two pooled libraries comprising mutants of ten different human antibodies spanning light chain gene usage, CDR H3 length, and antigenic targets. We demonstrate the comprehensive mapping of potential antibody development pathways, sequence-binding relationships for multiple antibodies simultaneously, and identification of paratope sequence determinants for binding recognition for broadly neutralizing antibodies (bnAbs). MAGMA-seq enables rapid and scalable antibody engineering of multiple lead candidates because it can measure binding for mutants of many given parental antibodies in a single experiment.

Biochemical and biophysical characterization of natural polyreactivity in antibodies.

To become specialized binders, antibodies undergo a process called affinity maturation to maximize their binding affinity. Despite this process, some antibodies retain low-affinity binding to diverse epitopes in a phenomenon called polyreactivity. Here we seek to understand the molecular basis of this polyreactivity in antibodies. Our results highlight that polyreactive antigen-binding fragments (Fabs) bind their targets with low affinities, comparable to T cell receptor recognition of autologous classical major histocompatibility complex. Extensive mutagenic studies find no singular amino acid residue or biochemical property responsible for polyreactive interaction, suggesting that polyreactive antibodies use multiple strategies for engagement. Finally, our crystal structures and all-atom molecular dynamics simulations of polyreactive Fabs show increased rigidity compared to their monoreactive relatives, forming a neutral and accessible platform for diverse antigens to bind. Together, these data support a cooperative strategy of rigid neutrality in establishing the polyreactive status of an antibody molecule.

Defining and Studying B Cell Receptor and TCR Interactions.

BCRs (Abs) and TCRs (or adaptive immune receptors [AIRs]) are the means by which the adaptive immune system recognizes foreign and self-antigens, playing an integral part in host defense, as well as the emergence of autoimmunity. Importantly, the interaction between AIRs and their cognate Ags defies a simple key-in-lock paradigm and is instead a complex many-to-many mapping between an individual's massively diverse AIR repertoire, and a similarly diverse antigenic space. Understanding how adaptive immunity balances specificity with epitopic coverage is a key challenge for the field, and terms such as broad specificity, cross-reactivity, and polyreactivity remain ill-defined and are used inconsistently. In this Immunology Notes and Resources article, a group of experimental, structural, and computational immunologists define commonly used terms associated with AIR binding, describe methodologies to study these binding modes, as well as highlight the implications of these different binding modes for therapeutic design.

Female Antibodies Go the Distance against Influenza Viruses.

Females have long been described to generate superior humoral immune responses relative to those in males. In the article by Ursin et al. (R. L. Ursin, S. Dhakal, H. Liu, S. Jayaraman, et al., mBio 13:e01839-22, 2022, https://doi.org/10.1128/mbio.01839-22), the authors showed that female mice generated a more robust, broadly reactive, and protective humoral immune response against influenza viruses in comparison to their male counterparts. Female mice demonstrated more efficient germinal center responses, including increased class switching and affinity maturation. Therefore, sex plays an important role in acquisition of protection against influenza viruses by modulating the generation of protective B cell responses. In this commentary, we dive into how this study builds on our understanding of how females generate superior antibody responses against influenza viruses and how this informs vaccine design.

Librator: a platform for the optimized analysis, design, and expression of mutable influenza viral antigens.

Artificial mutagenesis and protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used in this process require manual sequence editing and protein expression, limiting their efficiency and utility in high-throughput applications. More streamlined in silico tools allowing researchers to properly analyze and visualize influenza viral protein sequences with accurate nomenclature are necessary to improve antigen design and productivity. To address this need, we developed Librator, a system for analyzing and designing custom protein sequences of influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Within Librator's graphical interface, users can easily interrogate viral sequences and phylogenies, visualize antigen structures and conservation, mutate target residues and design custom antigens. Librator also provides optimized fragment design for Gibson Assembly of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible across related subtypes, thereby promoting reagent savings. Finally, the program facilitates single-cell immune profiling, epitope mapping of monoclonal antibodies and mosaic protein design. Using Librator-based antigen construction, we demonstrate that antigenicity can be readily transferred between HA molecules of H3, but not H1, lineage viruses. Altogether, Librator is a valuable tool for analyzing influenza virus HA and NA proteins and provides an efficient resource for optimizing recombinant influenza antigen synthesis.

Broadly neutralizing antibodies target a haemagglutinin anchor epitope.

Broadly neutralizing antibodies that target epitopes of haemagglutinin on the influenza virus have the potential to provide near universal protection against influenza virus infection1. However, viral mutants that escape broadly neutralizing antibodies have been reported2,3. The identification of broadly neutralizing antibody classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design. Here we report a distinct class of broadly neutralizing antibodies that target a discrete membrane-proximal anchor epitope of the haemagglutinin stalk domain. Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with H2 and H5 viruses that are a pandemic threat. Antibodies that target this anchor epitope utilize a highly restricted repertoire, which encodes two public binding motifs that make extensive contacts with conserved residues in the fusion peptide. Moreover, anchor epitope-targeting B cells are common in the human memory B cell repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric haemagglutinin vaccine4,5, which is a potential universal influenza virus vaccine. To maximize protection against seasonal and pandemic influenza viruses, vaccines should aim to boost this previously untapped source of broadly neutralizing antibodies that are widespread in the human memory B cell pool.

Cross-Neutralization of Emerging SARS-CoV-2 Variants of Concern by Antibodies Targeting Distinct Epitopes on Spike.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have arisen that exhibit increased viral transmissibility and partial evasion of immunity induced by natural infection and vaccination. To address the specific antibody targets that were affected by recent viral variants, we generated 43 monoclonal antibodies (mAbs) from 10 convalescent donors that bound three distinct domains of the SARS-CoV-2 spike. Viral variants harboring mutations at K417, E484, and N501 could escape most of the highly potent antibodies against the receptor binding domain (RBD). Despite this, we identified 12 neutralizing mAbs against three distinct regions of the spike protein that neutralize SARS-CoV-2 and variants of concern (VOCs), including B.1.1.7 (alpha), P.1 (gamma), and B.1.617.2 (delta). Notably, antibodies targeting distinct epitopes could neutralize discrete variants, suggesting that different variants may have evolved to disrupt the binding of particular neutralizing antibody classes. These results underscore that humans exposed to the first pandemic wave of prototype SARS-CoV-2 possess neutralizing antibodies against current variants and that it is critical to induce antibodies targeting multiple distinct epitopes of the spike that can neutralize emerging variants of concern. IMPORTANCE We describe the binding and neutralization properties of a new set of human monoclonal antibodies derived from memory B cells of 10 coronavirus disease 2019 (COVID-19) convalescent donors in the first pandemic wave of prototype SARS-CoV-2. There were 12 antibodies targeting distinct epitopes on spike, including two sites on the RBD and one on the N-terminal domain (NTD), that displayed cross-neutralization of VOCs, for which distinct antibody targets could neutralize discrete variants. This work underlines that natural infection by SARS-CoV-2 induces effective cross-neutralization against only some VOCs and supports the need for COVID-19 vaccination for robust induction of neutralizing antibodies targeting multiple epitopes of the spike protein to combat the current SARS-CoV-2 VOCs and any others that might emerge in the future.

Bridging the B Cell Gap: Novel Technologies to Study Antigen-Specific Human B Cell Responses.

The generation of high affinity antibodies is a crucial aspect of immunity induced by vaccination or infection. Investigation into the B cells that produce these antibodies grants key insights into the effectiveness of novel immunogens to induce a lasting protective response against endemic or pandemic pathogens, such as influenza viruses, human immunodeficiency virus, or severe acute respiratory syndrome coronavirus-2. However, humoral immunity has largely been studied at the serological level, limiting our knowledge on the specificity and function of B cells recruited to respond to pathogens. In this review, we cover a number of recent innovations in the field that have increased our ability to connect B cell function to the B cell repertoire and antigen specificity. Moreover, we will highlight recent advances in the development of both ex vivo and in vivo models to study human B cell responses. Together, the technologies highlighted in this review can be used to help design and validate new vaccine designs and platforms.

B Cell Responses against Influenza Viruses: Short-Lived Humoral Immunity against a Life-Long Threat.

Antibodies are critical for providing protection against influenza virus infections. However, protective humoral immunity against influenza viruses is limited by the antigenic drift and shift of the major surface glycoproteins, hemagglutinin and neuraminidase. Importantly, people are exposed to influenza viruses throughout their life and tend to reuse memory B cells from prior exposure to generate antibodies against new variants. Despite this, people tend to recall memory B cells against constantly evolving variable epitopes or non-protective antigens, as opposed to recalling them against broadly neutralizing epitopes of hemagglutinin. In this review, we discuss the factors that impact the generation and recall of memory B cells against distinct viral antigens, as well as the immunological limitations preventing broadly neutralizing antibody responses. Lastly, we discuss how next-generation vaccine platforms can potentially overcome these obstacles to generate robust and long-lived protection against influenza A viruses.

An Egg-Derived Sulfated N-Acetyllactosamine Glycan Is an Antigenic Decoy of Influenza Virus Vaccines.

Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess different glycosylation patterns than viruses circulating among humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived glycan that is an antigenic decoy, with egg-binding MAbs reacting with a sulfated N-acetyllactosamine (LacNAc). Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken-derived cells, suggesting that only egg-grown vaccines can induce antiegg antibodies. Notably, antibodies against the egg antigen utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and had a simple heavy-chain complementarity-determining region 3. By analyzing a public data set of influenza virus vaccine-induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.

First exposure to the pandemic H1N1 virus induced broadly neutralizing antibodies targeting hemagglutinin head epitopes.

Broadly neutralizing antibodies are critical for protection against both drifted and shifted influenza viruses. Here, we reveal that first exposure to the 2009 pandemic H1N1 influenza virus recalls memory B cells that are specific to the conserved receptor-binding site (RBS) or lateral patch epitopes of the hemagglutinin (HA) head domain. Monoclonal antibodies (mAbs) generated against these epitopes are broadly neutralizing against H1N1 viruses spanning 40 years of viral evolution and provide potent protection in vivo. Lateral patch-targeting antibodies demonstrated near universal binding to H1 viruses, and RBS-binding antibodies commonly cross-reacted with H3N2 viruses and influenza B viruses. Lateral patch-targeting mAbs were restricted to expressing the variable heavy-chain gene VH3-23 with or without the variable kappa-chain gene VK1-33 and often had a Y-x-R motif within the heavy-chain complementarity determining region 3 to make key contacts with HA. Moreover, lateral patch antibodies that used both VH3-23 and VK1-33 maintained neutralizing capability with recent pH1N1 strains that acquired mutations near the lateral patch. RBS-binding mAbs used a diverse repertoire but targeted the RBS epitope similarly and made extensive contacts with the major antigenic site Sb. Together, our data indicate that RBS- and lateral patch-targeting clones are abundant within the human memory B cell pool, and universal vaccine strategies should aim to drive antibodies against both conserved head and stalk epitopes.

Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.

Hemolysis-associated phosphatidylserine exposure promotes polyclonal plasmablast differentiation.

Antimalarial antibody responses are essential for mediating the clearance of Plasmodium parasite-infected RBCs from infected hosts. However, the rapid appearance of large numbers of plasmablasts in Plasmodium-infected hosts can suppress the development and function of durable humoral immunity. Here, we identify that the formation of plasmablast populations in Plasmodium-infected mice is mechanistically linked to both hemolysis-induced exposure of phosphatidylserine on damaged RBCs and inflammatory cues. We also show that virus and Trypanosoma infections known to trigger hemolytic anemia and high-grade inflammation also induce exuberant plasmablast responses. The induction of hemolysis or administration of RBC membrane ghosts increases plasmablast differentiation. The phosphatidylserine receptor Axl is critical for optimal plasmablast formation, and blocking phosphatidylserine limits plasmablast expansions and reduces Plasmodium parasite burden in vivo. Our findings support that strategies aimed at modulating polyclonal B cell activation and phosphatidylserine exposure may improve immune responses against Plasmodium parasites and potentially other infectious diseases that are associated with anemia.

Influenza hemagglutinin-specific IgA Fc-effector functionality is restricted to stalk epitopes.

In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.

Extrafollicular CD4 T cell-derived IL-10 functions rapidly and transiently to support anti-Plasmodium humoral immunity.

Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti-Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria.

SARS-CoV-2 Infection Severity Is Linked to Superior Humoral Immunity against the Spike.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity of the antibody response mounted against this novel virus is not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and nonstructural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.IMPORTANCE With the ongoing pandemic, it is critical to understand how natural immunity against SARS-CoV-2 and COVID-19 develops. We have identified that subjects with more severe COVID-19 disease mount a more robust and neutralizing antibody response against SARS-CoV-2 spike protein. Subjects who mounted a larger response against the spike also mounted antibody responses against other viral antigens, including the nucleocapsid protein and ORF8. Additionally, this study reveals that subjects with more severe disease mount a larger memory B cell response against the spike. These data suggest that subjects with more severe COVID-19 disease are likely better protected from reinfection with SARS-CoV-2.

Preexisting immunity shapes distinct antibody landscapes after influenza virus infection and vaccination in humans.

Humans are repeatedly exposed to variants of influenza virus throughout their lifetime. As a result, preexisting influenza-specific memory B cells can dominate the response after infection or vaccination. Memory B cells recalled by adulthood exposure are largely reactive to conserved viral epitopes present in childhood strains, posing unclear consequences on the ability of B cells to adapt to and neutralize newly emerged strains. We sought to investigate the impact of preexisting immunity on generation of protective antibody responses to conserved viral epitopes upon influenza virus infection and vaccination in humans. We accomplished this by characterizing monoclonal antibodies (mAbs) from plasmablasts, which are predominantly derived from preexisting memory B cells. We found that, whereas some influenza infection-induced mAbs bound conserved and neutralizing epitopes on the hemagglutinin (HA) stalk domain or neuraminidase, most of the mAbs elicited by infection targeted non-neutralizing epitopes on nucleoprotein and other unknown antigens. Furthermore, most infection-induced mAbs had equal or stronger affinity to childhood strains, indicating recall of memory B cells from childhood exposures. Vaccination-induced mAbs were similarly induced from past exposures and exhibited substantial breadth of viral binding, although, in contrast to infection-induced mAbs, they targeted neutralizing HA head epitopes. Last, cocktails of infection-induced mAbs displayed reduced protective ability in mice compared to vaccination-induced mAbs. These findings reveal that both preexisting immunity and exposure type shape protective antibody responses to conserved influenza virus epitopes in humans. Natural infection largely recalls cross-reactive memory B cells against non-neutralizing epitopes, whereas vaccination harnesses preexisting immunity to target protective HA epitopes.