Design of Antigen-Specific Antibody CDRH3 Sequences Using AI and Germline-Based Templates.

Antibody-antigen specificity is engendered and refined through a number of complex B cell processes, including germline gene recombination and somatic hypermutation. Here, we present an AI-based technology for de novo generation of antigen-specific antibody CDRH3 sequences using germline-based templates, and validate this technology through the generation of antibodies against SARS-CoV-2. AI-based processes that mimic the outcome, but bypass the complexity of natural antibody generation, can be efficient and effective alternatives to traditional experimental approaches for antibody discovery.

Potent HPIV3-neutralizing IGHV5-51 Antibodies Identified from Multiple Individuals Show L Chain and CDRH3 Promiscuity.

Human parainfluenza virus 3 (HPIV3) is a widespread pathogen causing severe and lethal respiratory illness in at-risk populations. Effective countermeasures are in various stages of development; however, licensed therapeutic and prophylactic options are not available. The fusion glycoprotein (HPIV3 F), responsible for facilitating viral entry into host cells, is a major target of neutralizing Abs that inhibit infection. Although several neutralizing Abs against a small number of HPIV3 F epitopes have been identified to date, relatively little is known about the Ab response to HPIV3 compared with other pathogens, such as influenza virus and SARS-CoV-2. In this study, we aimed to characterize a set of HPIV3-specific Abs identified in multiple individuals for genetic signatures, epitope specificity, neutralization potential, and publicness. We identified 12 potently neutralizing Abs targeting three nonoverlapping epitopes on HPIV3 F. Among these, six Abs identified from two different individuals used Ig heavy variable gene IGHV 5-51, with five of the six Abs targeting the same epitope. However, despite the use of the same H chain variable (VH) gene, these Abs used multiple different L chain variable genes (VL) and diverse H chain CDR 3 (CDRH3) sequences. Together, these results provide further information about the genetic and functional characteristics of HPIV3-neutralizing Abs and suggest the existence of a reproducible VH-dependent Ab response associated with VL and CDRH3 promiscuity. Understanding sites of HPIV3 F vulnerability and the genetic and molecular characteristics of Abs targeting these sites will help guide efforts for effective vaccine and therapeutic development.

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody.

Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

Development of LIBRA-seq for the guinea pig model system as a tool for the evaluation of antibody responses to multivalent HIV-1 vaccines.

Consistent elicitation of serum antibody responses that neutralize diverse clades of HIV-1 remains a primary goal of HIV-1 vaccine research. Prior work has defined key features of soluble HIV-1 Envelope (Env) immunogen cocktails that influence the neutralization breadth and potency of multivalent vaccine-elicited antibody responses including the number of Env strains in the regimen. We designed immunization groups that consisted of different numbers of SOSIP Env strains to be used in a cocktail immunization strategy: the smallest cocktail (group 2) consisted of a set of two Env strains, which were a subset of the three Env strains that made up group 3, which, in turn, were a subset of the six Env strains that made up group 4. Serum neutralizing titers were modestly broader in guinea pigs that were immunized with a cocktail of three Envs compared to cocktails of two and six, suggesting that multivalent Env immunization could provide a benefit but may be detrimental when the cocktail size is too large. We then adapted the LIBRA-seq platform for antibody discovery to be compatible with guinea pigs, and isolated several tier 2 neutralizing monoclonal antibodies. Three antibodies isolated from two separate guinea pigs were similar in their gene usage and CDR3s, establishing evidence for a guinea pig public clonotype elicited through vaccination. Taken together, this work investigated multivalent HIV-1 Env immunization strategies and provides a novel methodology for screening guinea pig B cell receptor antigen specificity at a high-throughput level using LIBRA-seq.IMPORTANCEMultivalent vaccination with soluble Env immunogens is at the forefront of HIV-1 vaccination strategies but little is known about the influence of the number of Env strains included in vaccine cocktails. Our results suggest that adding more strains is sometimes beneficial but may be detrimental when the number of strains is too high. In addition, we adapted the LIBRA-seq platform to be compatible with guinea pig samples and isolated several tier 2 neutralizing monoclonal antibodies, some of which share V and J gene usage and >70% CDR3 identity, thus establishing the existence of public clonotypes in guinea pigs elicited through vaccination.

Dynamics and durability of HIV-1 neutralization are determined by viral replication.

Human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies (nAbs) that prevent infection are the main goal of HIV vaccine discovery. But as no nAb-eliciting vaccines are yet available, only data from HIV-1 neutralizers-persons with HIV-1 who naturally develop broad and potent nAbs-can inform about the dynamics and durability of nAb responses in humans, knowledge which is crucial for the design of future HIV-1 vaccine regimens. To address this, we assessed HIV-1-neutralizing immunoglobulin G (IgG) from 2,354 persons with HIV-1 on or off antiretroviral therapy (ART). Infection with non-clade B viruses, CD4+ T cell counts <200 µl-1, being off ART and a longer time off ART were independent predictors of a more potent and broad neutralization. In longitudinal analyses, we found nAb half-lives of 9.3 and 16.9 years in individuals with no- or low-level viremia, respectively, and 4.0 years in persons who newly initiated ART. Finally, in a potent HIV-1 neutralizer, we identified lower fractions of serum nAbs and of nAb-encoding memory B cells after ART initiation, suggesting that a decreasing neutralizing serum activity after antigen withdrawal is due to lower levels of nAbs. These results collectively show that HIV-1-neutralizing responses can persist for several years, even at low antigen levels, suggesting that an HIV-1 vaccine may elicit a durable nAb response.

SARS-CoV-2 antibodies from children exhibit broad neutralization and belong to adult public clonotypes.

From the beginning of the COVID-19 pandemic, children have exhibited different susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, reinfection, and disease compared with adults. Motivated by the established significance of SARS-CoV-2-neutralizing antibodies in adults, here we characterize SARS-CoV-2-specific antibody repertoires in a young cohort of individuals aged from 5 months to 18 years old. Our results show that neutralizing antibodies in children possess similar genetic features compared to antibodies identified in adults, with multiple antibodies from children belonging to previously established public antibody clonotypes in adults. Notably, antibodies from children show potent neutralization of circulating SARS-CoV-2 variants that have cumulatively resulted in resistance to virtually all approved monoclonal antibody therapeutics. Our results show that children can rely on similar SARS-CoV-2 antibody neutralization mechanisms compared to adults and are an underutilized source for the discovery of effective antibody therapeutics to counteract the ever-evolving pandemic.

Negative Binomial Mixture Model for Identification of Noise in Antigen-Specificity Predictions by LIBRA-seq.

Motivation: LIBRA-seq (linking B cell receptor to antigen specificity by sequencing) provides a powerful tool for interrogating the antigen-specific B cell compartment and identifying antibodies against antigen targets of interest. Identification of noise in LIBRA-seq antigen count data is critical for improving antigen binding predictions for downstream applications including antibody discovery and machine learning technologies. Results: In this study, we present a method for denoising LIBRA-seq data by clustering antigen counts into signal and noise components with a negative binomial mixture model. This approach leverages the VRC01 negative control cells included in a recent LIBRA-seq study(Abu-Shmais et al.) to provide a data-driven means for identification of technical noise. We apply this method to a dataset of nine donors representing separate LIBRA-seq experiments and show that our approach provides improved predictions for in vitro antibody-antigen binding when compared to the standard scoring method used in LIBRA-seq, despite variance in data size and noise structure across samples. This development will improve the ability of LIBRA-seq to identify antigen-specific B cells and contribute to providing more reliable datasets for future machine learning based approaches to predicting antibody-antigen binding as the corpus of LIBRA-seq data continues to grow.

Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor.

Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity.

Neutralization fingerprinting technology for characterizing polyclonal antibody responses to dengue vaccines.

Dengue is a major public health threat. There are four dengue virus (DENV) serotypes; therefore, efforts are focused on developing safe and effective tetravalent DENV vaccines. While neutralizing antibodies contribute to protective immunity, there are still important gaps in understanding of immune responses elicited by dengue infection and vaccination. To that end, here, we develop a computational modeling framework based on the concept of antibody-virus neutralization fingerprints in order to characterize samples from clinical studies of TAK-003, a tetravalent vaccine candidate currently in phase 3 trials. Our results suggest a similarity of neutralizing antibody specificities in baseline-seronegative individuals. In contrast, amplification of pre-existing neutralizing antibody specificities is predicted for baseline-seropositive individuals, thus quantifying the role of immunologic imprinting in driving antibody responses to DENV vaccines. The neutralization fingerprinting analysis framework presented here can contribute to understanding dengue immune correlates of protection and help guide further vaccine development and optimization.

Single-cell profiling of the antigen-specific response to BNT162b2 SARS-CoV-2 RNA vaccine.

RNA-based vaccines against SARS-CoV-2 have proven critical to limiting COVID-19 disease severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. Here we identify and characterize antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 using multiple single-cell technologies for in depth analysis of longitudinal samples from a cohort of healthy participants. Mass cytometry and unbiased machine learning pinpoint an expanding, population of antigen-specific memory CD4+ and CD8+ T cells with characteristics of follicular or peripheral helper cells. B cell receptor sequencing suggest progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlate with eventual SARS-CoV-2 IgG, and a participant lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms identify an antigen-specific cellular basis of RNA vaccine-based immunity.

High-Throughput B Cell Epitope Determination by Next-Generation Sequencing.

Development of novel technologies for the discovery of human monoclonal antibodies has proven invaluable in the fight against infectious diseases. Among the diverse antibody repertoires elicited by infection or vaccination, often only rare antibodies targeting specific epitopes of interest are of potential therapeutic value. Current antibody discovery efforts are capable of identifying B cells specific for a given antigen; however, epitope specificity information is usually only obtained after subsequent monoclonal antibody production and characterization. Here we describe LIBRA-seq with epitope mapping, a next-generation sequencing technology that enables residue-level epitope determination for thousands of single B cells simultaneously. By utilizing an antigen panel of point mutants within the HIV-1 Env glycoprotein, we identified and confirmed antibodies targeting multiple sites of vulnerability on Env, including the CD4-binding site and the V3-glycan site. LIBRA-seq with epitope mapping is an efficient tool for high-throughput identification of antibodies against epitopes of interest on a given antigen target.

An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer.

The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.

Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking.

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.

Sequence and functional characterization of a public HIV-specific antibody clonotype.

Public antibody clonotypes shared among multiple individuals have been identified for several pathogens. However, little is known about the determinants of antibody "publicness". Here, we characterize the sequence and functional properties of antibodies from a public clonotype targeting the CD4 binding site on HIV-1 Env. Our results showed that HIV-1 specificity for the public antibodies studied here, comprising sequences from three individuals, was modulated by the VH, but not VL, germline gene. Non-native pairing of public heavy and light chains from different individuals suggested functional complementation of sequences within this public antibody clonotype. The strength of antigen recognition appeared to be dependent on the specific antibody light chain used, but not on other sequence features such as native-antibody or germline sequence identity. Understanding the determinants of antibody clonotype "publicness" can provide insights into the fundamental rules of host-pathogen interactions at the population level, with implications for clonotype-specific vaccine development.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.

Simultaneous Immunization with Multiple Diverse Immunogens Alters Development of Antigen-Specific Antibody-Mediated Immunity.

Vaccination remains one of the most successful medical interventions in history, significantly decreasing morbidity and mortality associated with, or even eradicating, numerous infectious diseases. Although traditional immunization strategies have recently proven insufficient in the face of many highly mutable and emerging pathogens, modern strategies aim to rationally engineer a single antigen or cocktail of antigens to generate a focused, protective immune response. However, the effect of cocktail vaccination (simultaneous immunization with multiple immunogens) on the antibody response to each individual antigen within the combination, remains largely unstudied. To investigate whether immunization with a cocktail of diverse antigens would result in decreased antibody titer against each unique antigen in the cocktail compared to immunization with each antigen alone, we immunized mice with surface proteins from uropathogenic Escherichia coli, Mycobacterium tuberculosis, and Neisseria meningitides, and monitored the development of antigen-specific IgG antibody responses. We found that antigen-specific endpoint antibody titers were comparable across immunization groups by study conclusion (day 70). Further, we discovered that although cocktail-immunized mice initially elicited more robust antibody responses, the rate of titer development decreases significantly over time compared to single antigen-immunized mice. Investigating the basic properties that govern the development of antigen-specific antibody responses will help inform the design of future combination immunization regimens.

Elicitation of Neutralizing Antibody Responses to HIV-1 Immunization with Nanoparticle Vaccine Platforms.

A leading strategy for developing a prophylactic HIV-1 vaccine is the elicitation of antibodies that can neutralize a large fraction of circulating HIV-1 variants. However, a major challenge that has limited the effectiveness of current vaccine candidates is the extensive global diversity of the HIV-1 envelope protein (Env), the sole target for HIV-neutralizing antibodies. To address this challenge, various strategies incorporating Env diversity into the vaccine formulation have been proposed. Here, we assessed the potential of two such strategies that utilize a nanoparticle-based vaccine platform to elicit broadly neutralizing antibody responses. The nanoparticle immunogens developed here consisted of different formulations of Envs from strains BG505 (clade A) and CZA97 (clade C), attached to the N-termini of bacterial ferritin. Single-antigen nanoparticle cocktails, as well as mosaic nanoparticles bearing both Env trimers, elicited high antibody titers in mice and guinea pigs. Furthermore, serum from guinea pigs immunized with nanoparticle immunogens achieved autologous, and in some cases heterologous, tier 2 neutralization, although significant differences between mosaic and single-antigen nanoparticles were not observed. These results provide insights into the ability of different vaccine strategies for incorporating Env sequence diversity to elicit neutralizing antibodies, with implications for the development of broadly protective HIV-1 vaccines.

Single-Cell Profiling of the Antigen-Specific Response to BNT162b2 SARS-CoV-2 RNA Vaccine.

RNA-based vaccines against SARS-CoV-2 are critical to limiting COVID-19 severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. We used single-cell technologies to identify and characterized antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 in longitudinal samples from a cohort of healthy donors. Mass cytometry and machine learning pinpointed a novel expanding, population of antigen-specific non-canonical memory CD4 + and CD8 + T cells. B cell sequencing suggested progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlated with eventual SARS-CoV-2 IgG and a donor lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms reveal an antigen-specific cellular basis of RNA vaccine-based immunity. ONE SENTENCE SUMMARY: Single-cell profiling reveals the cellular basis of the antigen-specific response to the BNT162b2 SARS-CoV-2 RNA vaccine.

Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking.

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.

Cross-reactive coronavirus antibodies with diverse epitope specificities and Fc effector functions.

The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis-and in some cases trogocytosis-but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies.