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Influenza virus is a highly contagious respiratory pathogen causing between 9.4 and 41 million infections per year in the United States in the last decade. Annual vaccination is recommended by the World Health Organization, with the goal to reduce influenza severity and transmission. Ag-specific single B cell sequencing methodologies have opened up new avenues into the dissection of the Ab response to influenza virus. The improvement of these methodologies is pivotal to reduce the associated costs and optimize the operational workflow and throughput, especially in the context of multiple samples. In this study, PBMCs and serum samples were collected longitudinally from eight influenza vaccinees either vaccinated yearly for four consecutive influenza seasons or once for one season. Following the serological and B cell profiling of their polyclonal Ab response to a panel of historical, recent, and next-generation influenza vaccine hemagglutinin (HA) and virus strains, a single multiplexed Ag-specific single B cell sequencing run allowed to capture HA-specific memory B cells that were analyzed for preferential Ig H chain/L chain pairing, isotype/subclass usage, and the presence of public BCR clonotypes across participants. Binding and functional profiles of representative private and public clonotypes confirmed their HA specificity, and their overall binding and functional activity were consistent with those observed at the polyclonal level. Collectively, this high-resolution and multiplexed Ab repertoire analysis demonstrated the validity of this optimized methodology in capturing Ag-specific BCR clonotypes, even in the context of a rare B cell population, such as in the case of the peripheral Ag-specific memory B cells., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Broadly reactive antibodies that target sequence-diverse antigens are of interest for vaccine design and monoclonal antibody therapeutic development because they can protect against multiple strains of a virus and provide a barrier to evolution of escape mutants. Using LIBRA-seq (linking B cell receptor to antigen specificity through sequencing) data for the B cell repertoire of an individual chronically infected with human immunodeficiency virus type 1 (HIV-1), we identified a lineage of IgG3 antibodies predicted to bind to HIV-1 Envelope (Env) and influenza A Hemagglutinin (HA). Two lineage members, antibodies 2526 and 546, were confirmed to bind to a large panel of diverse antigens, including several strains of HIV-1 Env, influenza HA, coronavirus (CoV) spike, hepatitis C virus (HCV) E protein, Nipah virus (NiV) F protein, and Langya virus (LayV) F protein. We found that both antibodies bind to complex glycans on the antigenic surfaces. Antibody 2526 targets the stem region of influenza HA and the N-terminal domain (NTD) region of SARS-CoV-2 spike. A crystal structure of 2526 Fab bound to mannose revealed the presence of a glycan-binding pocket on the light chain. Antibody 2526 cross-reacted with antigens from multiple pathogens and displayed no signs of autoreactivity. These features distinguish antibody 2526 from previously described glycan-reactive antibodies. Further study of this antibody class may aid in the selection and engineering of broadly reactive antibody therapeutics and can inform the development of effective vaccines with exceptional breadth of pathogen coverage., Competing Interests: M.J.V., A.R.S., and I.S.G. are listed as inventors on patents filed describing the antibodies discovered here. I.S.G. is listed as an inventor on the patent applications for the LIBRA-seq technology. I.S.G. is a co-founder of AbSeek Bio. I.S.G. has served as a consultant for Sanofi. The Georgiev laboratory at VUMC has received unrelated funding from Merck and Takeda Pharmaceuticals. D.J.S has served as a consultant for AstraZeneca AB., (Copyright: © 2024 Vukovich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The continued emergence of deadly human coronaviruses from animal reservoirs highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq), we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryoelectron microscopy (cryo-EM) structure of 54043-5 bound to the prefusion S2 subunit of the severe acute respiratory syndrome coronavirus 2 (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 in vitro, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (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., Competing Interests: Declaration of interests A.R.S. and I.S.G. are co-founders of AbSeek Bio. K.J.K., A.R.S., N.V.J., I.S.G., J.S.M., R.H.C., and J.E.C. are listed as inventors on patents filed describing the antibodies discovered here. R.H.C. is an inventor on patents related to other SARS-CoV-2 antibodies. J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory has received funding support in sponsored research agreements from AstraZeneca, IDBiologics, and Takeda. The Georgiev laboratory at VUMC has received unrelated funding from Takeda Pharmaceuticals., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Viral respiratory diseases affect millions of individuals worldwide each year. Annual vaccinations are recommended by the World Health Organization for some of them, such as influenza and more recently for the coronavirus disease of 2019 (COVID-19) and respiratory syncytial virus, with the goal of reducing disease severity and limiting transmission. In the context of infection and vaccination, it is of primary importance to evaluate the immune response to pathogens to shed light on the mechanisms of protection., (Copyright © 2024 The Cleveland Clinic Foundation. All Rights Reserved.)

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine., Competing Interests: Declaration of interests Ted Ross is an inventor on influenza vaccines., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The hepatitis C virus (HCV) is responsible for approximately 50 million infections worldwide. Effective drug treatments while available face access barriers, and vaccine development is hampered by viral hypervariability and immune evasion mechanisms. The CD4+ and CD8+ T-cell responses targeting HCV non-structural (NS) proteins have shown a role in the viral clearance. In this paper, we reviewed the studies exploring the relationship between HCV structural and NS proteins and their effects in contributing to the elicitation of an effective T-cell immune response. The use of different vaccine platforms, such as viral vectors and virus-like particles, underscores their versability and efficacy for vaccine development. Diverse HCV antigens demonstrated immunogenicity, eliciting a robust immune response, positioning them as promising vaccine candidates for protein/peptide-, DNA-, or RNA-based vaccines. Moreover, adjuvant selection plays a pivotal role in modulating the immune response. This review emphasizes the importance of HCV proteins and vaccination strategies in vaccine development. In particular, the NS proteins are the main focus, given their pivotal role in T-cell-mediated immunity and their sequence conservation, making them valuable vaccine targets.

Spike-encoding mRNA vaccines in early 2021 effectively reduced SARS-CoV-2-associated morbidity and mortality. New booster regimens were introduced due to successive waves of distinct viral variants. Therefore, people now have a diverse immune memory resulting from multiple SARS-CoV-2 Ag exposures, from infection to following vaccination. This level of community-wide immunity can induce immunological protection from SARS-CoV-2; however, questions about the trajectory of the adaptive immune responses and long-term immunity with respect to priming and repeated Ag exposure remain poorly explored. In this study, we examined the trajectory of adaptive immune responses following three doses of monovalent Pfizer BNT162b2 mRNA vaccination in immunologically naive and SARS-CoV-2 preimmune individuals without the occurrence of breakthrough infection. The IgG, B cell, and T cell Spike-specific responses were assessed in human blood samples collected at six time points between a moment before vaccination and up to 6 mo after the third immunization. Overall, the impact of repeated Spike exposures had a lower improvement on T cell frequency and longevity compared with IgG responses. Natural infection shaped the responses following the initial vaccination by significantly increasing neutralizing Abs and specific CD4+ T cell subsets (circulating T follicular helper, effector memory, and Th1-producing cells), but it had a small benefit at long-term immunity. At the end of the three-dose vaccination regimen, both SARS-CoV-2-naive and preimmune individuals had similar immune memory quality and quantity. This study provides insights into the durability of mRNA vaccine-induced immunological memory and the effects of preimmunity on long-term responses., (Copyright © 2023 The Authors.)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of interests K.A.P. and I.S.G. are listed as inventors on patents filed describing the antibodies discovered here. A.R.S. and I.S.G. are co-founders of AbSeek Bio. J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines, and is founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics, and AstraZeneca. The Georgiev laboratory at Vanderbilt University Medical Center has received unrelated funding from Takeda Pharmaceuticals., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Notwithstanding the current SARS-CoV-2 pandemic, influenza virus infection still represents a global health concern in terms of hospitalizations and possible pandemic threats. The objective of next-generation influenza vaccines is not only to increase the breadth of response but also to improve the elicitation of an effective and robust immune response, especially in high-risk populations. To achieve this second objective, the administration of adjuvanted influenza vaccines has been considered. In this regard, the monitoring and characterization of the antibody response associated with the administration of adjuvanted vaccines has been evaluated in this study in order to shed light on the kinetic, magnitude and subclass usage of antibody secreting cells (ASCs) as well as of circulating antigen-specific serum antibodies. Specifically, we utilized the DBA/2J mouse model to assess the kinetic, magnitude and IgG subclass usage of the antibody response following an intramuscular (IM) or intraperitoneal (IP) immunization regimen with AddaVax-adjuvanted bivalent H1N1 and H3N2 computationally optimized broadly reactive antigen (COBRA) influenza recombinant hemagglutinins (rHAs). While the serological evaluation revealed a homogeneous kinetic of the antibody response, the detection of the ASCs through a FluoroSpot platform revealed a different magnitude, subclass usage and kinetic of the antigen-specific IgG secreting cells peaking at day 5 and day 9 following the IP and IM immunization, respectively.

In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to develop a mass-spectrometric (MS) method for determining the complete protein sequence of monoclonal antibodies (mAbs). The method was refined on an mAb of a known sequence, a SARS-CoV-1 antireceptor binding domain (RBD) spike monoclonal antibody. The data were searched using Supernovo to generate a complete template-assisted de novo sequence for this and two SARS-CoV-2 mAbs of known sequences resulting in correct sequences for the variable regions and correct distinction of Ile and Leu residues. We then used the method on a set of 25 antihemagglutinin (HA) influenza antibodies of unknown sequences and determined high confidence sequences for >99% of the complementarity determining regions (CDRs). The heavy-chain and light-chain genes were cloned and transfected into cells for recombinant expression followed by affinity purification. The recombinant mAbs displayed binding curves matching the original mAbs with specificity to the HA influenza antigen. Our findings indicate that this methodology results in almost complete antibody sequence coverage with high confidence results for CDR regions on diverse mAb sequences.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes., (Copyright © 2022 by The American Association of Immunologists, Inc.)

In order to longitudinally track SARS-CoV-2 antibody levels after vaccination or infection, we assessed anti-RBD antibody levels in over 1000 people and found no significant decrease in antibody levels during the first 14 months after infection in unvaccinated participants, however, a significant waning of antibody levels was observed following vaccination. Participants who were pre-immune to SARS-CoV-2 prior to vaccination seroconverted to higher antibody levels, which were maintained at higher levels than in previously infected, unvaccinated participants. Older participants exhibited lower level of antibodies after vaccination, but a higher level after infection than younger people. The rate of antibody waning was not affected by pre-immunity or age. Participants who received a third dose of an mRNA vaccine not only increased their antibody levels ~14-fold, but also had ~3 times more antibodies compared to when they received their primary vaccine series. PBMC-derived memory B cells from 13 participants who lost all circulating antibodies were differentiated into antibody secreting cells (ASCs). There was a significant recall of memory B cell ASCs in the absence of serum antibodies in 5-8 of the 10 vaccinated participants, but not in any of the 3 infected participants, suggesting a strong connection between antibody levels and the effectiveness of memory B cell recall.

Competing Interests: The editors declare that they have no conflicts of interest regarding the publication of this special issue.

Influenza remains one of the most contagious infectious diseases. Approximately, 25 to 50 million people suffer from influenza-like illness in the United States annually, leading to almost 1 million hospitalizations. Globally, the World Health Organization (WHO) estimates 250,000 to 500,000 mortalities associated with secondary respiratory complications due to influenza virus infection every year. Currently, seasonal vaccination represents the best countermeasure to prevent influenza virus spread and transmission in the general population. However, presently licensed influenza vaccines are about 60% effective on average, and their effectiveness varies from season to season and among age groups, as well as between different influenza subtypes within a single season. The hemagglutination inhibition (HAI) assay represents the gold standard method for measuring the functional antibody response elicited following standard-of-care vaccination, along with evaluating the efficacy of under-development influenza vaccines in both animal models and clinical trial settings. However, using the classical HAI approach, it is not possible to dissect the complexities of variable epitope recognition within a polyclonal antibody response. In this paper, we describe a straightforward competitive HAI-based method using a combination of influenza virus and recombinant hemagglutinin (HA) proteins to dissect the HAI functional activity of HA-specific antibody populations in a single assay format. IMPORTANCE The hemagglutination inhibition (HAI) assay is a well-established and reproducible method that quantifies functional antibody activity against influenza viruses and, in particular, the capability of an antibody formulation to inhibit the binding of hemagglutinin (HA) to sialic acid. However, the HAI assay does not provide full insights on the breadth and epitope recognition of the antibody formulation, especially in the context of polyclonal sera, where multiple antibody specificities contribute to the overall observed functional activity. In this report we introduce the use of Y98F point-mutated recombinant HA (HAΔSA) proteins, which lack sialic acid binding activity, in the context of the HAI assay as a means to absorb out certain HA-directed (i.e., strain-specific or cross-reactive) antibody populations. This modification to the classical HAI assay, referred to as the competitive HAI assay, represents a new tool to dissect the magnitude and breadth of polyclonal antibodies elicited through vaccination or natural infection.

Influenza is a highly contagious viral respiratory disease that affects million of people worldwide each year. Annual vaccination is recommended by the World Health Organization with the goal of reducing influenza severity and limiting transmission through elicitation of antibodies targeting the hemagglutinin (HA) glycoprotein. The antibody response elicited by current seasonal influenza virus vaccines is predominantly strain-specific, but pre-existing influenza virus immunity can greatly impact the serological antibody response to vaccination. However, it remains unclear how B cell memory is shaped by recurrent annual vaccination over the course of multiple seasons, especially in high-risk elderly populations. Here, we systematically profiled the B cell response in young adult (18-34 year old) and elderly (65+ year old) vaccine recipients that received annual split inactivated influenza virus vaccination for 3 consecutive seasons. Specifically, the antibody serological and memory B-cell compartments were profiled for reactivity against current and historical influenza A virus strains. Moreover, multiparametric analysis and antibody landscape profiling revealed a transient increase in strain-specific antibodies in the elderly, but with an impaired recall response of pre-existing memory B-cells, plasmablast (PB) differentiation and long-lasting serological changes. This study thoroughly profiles and compares the immune response to recurrent influenza virus vaccination in young and elderly participants unveiling the pitfalls of current influenza virus vaccines in high-risk populations., Competing Interests: GAK is currently affiliated with Cellular Technology Limited (CTL) but his contributions to this manuscript preceded his current position at CTL, and CTL had no involvement in this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H 2 O 2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN - ) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1 -/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1β, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN - is generated by LPO using DUOX1-derived H 2 O 2 and inactivates several influenza strains in vitro. We also show that OSCN - diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H 2 O 2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN - in an H 2 O 2 -dependent manner in vitro. OSCN - does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN - , and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections., Competing Interests: The authors declare no competing interest.

Feline morbillivirus (FeMV) was identified for the first time in stray cats in 2012 in Hong Kong and, since its discovery, it was reported in domestic cats worldwide. Although a potential association between FeMV infection and tubulointerstitial nephritis (TIN) has been suggested, this has not been proven, and the subject remains controversial. TIN is the most frequent histopathological finding in the context of feline chronic kidney disease (CKD), which is one of the major clinical pathologies in feline medicine. FeMV research has mainly focused on defining the epidemiology, the role of FeMV in the development of CKD, and its in vitro tropism, but the pathogenicity of FeMV is still not clear, partly due to its distinctive biological characteristics, as well as to a lack of a cell culture system for its rapid isolation. In this review, we summarize the current knowledge of FeMV infection, including genetic diversity of FeMV strains, epidemiology, pathogenicity, and clinicopathological findings observed in naturally infected cats.

Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016-2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches., Competing Interests: Some of the authors are affiliated with Atreca, Inc., but the company provided no funding for the study and no competing interests exist. The commercial affiliation of those authors does not alter our adherence to PLoS ONE policies on sharing data and materials.

SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad hoc parameter combinations is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains that are poised to host (several) epitopes and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes, and those with weaker coupling could themselves be prone to antibody recognition.

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

Influenza is a highly contagious viral respiratory disease that affects millions of people worldwide each year. Annual vaccination is recommended by the World Health Organization to reduce influenza severity and limit transmission through elicitation of antibodies targeting mainly the hemagglutinin glycoprotein of the influenza virus. Antibodies elicited by current seasonal influenza vaccines are predominantly strain-specific. However, continuous antigenic drift by circulating influenza viruses facilitates escape from pre-existing antibodies requiring frequent reformulation of the seasonal influenza vaccine. Traditionally, immunological responses to influenza vaccination have been largely focused on IgG antibodies, with almost complete disregard of other isotypes. In this report, young adults (18-34 years old) and elderly (65-85 years old) subjects were administered the split inactivated influenza vaccine for 3 consecutive seasons and their serological IgA and IgG responses were profiled. Moreover, correlation analysis showed a positive relationship between vaccine-induced IgA antibody titers and traditional immunological endpoints, exposing vaccine-induced IgA antibodies as an important novel immune correlate during influenza vaccination., (Copyright © 2020 Abreu, Clutter, Attari, Sautto and Ross.)

Influenza is a highly contagious viral pathogen with more than 200,000 cases reported in the United States during the 2017-2018 season. Annual vaccination is recommended by the World Health Organization, with the goal to reduce influenza severity and transmission. Currently available vaccines are about 60% effective, and vaccine effectiveness varies from season to season, as well as between different influenza subtypes within a single season. Immunological imprinting from early-life influenza infection can prominently shape the immune response to subsequent infections. Here, the impact of preexisting B cell memory in the response to quadrivalent influenza vaccine was assessed using blood samples collected from healthy subjects (18-85 years old) prior to and 21-28 days following influenza vaccination. Influenza vaccination increased both HA-specific antibodies and memory B cell frequency. Despite no apparent differences in antigenicity between vaccine components, most individuals were biased toward one of the vaccine strains. Specifically, responses to H3N2 were reduced in magnitude relative to the other vaccine components. Overall, this study unveils a potentially new mechanism underlying differential vaccine effectiveness against distinct influenza subtypes.

Computationally optimized broadly reactive Ags (COBRA) targeting H1 elicit a broad cross-reactive and cross-neutralizing Ab response against multiple H1N1 viral strains. To assess B cell breadth, Mus musculus (BALB/c) Ab-secreting cells elicited by a candidate COBRA hemagglutinin (HA) (termed P1) were compared with Ab-secreting cells elicited by historical H1N1 vaccine strains. In addition, to evaluate the Ab response elicited by P1 HA at increased resolution, a panel of P1 HA-specific B cell hybridomas was generated following immunization of mice with COBRA P1 and the corresponding purified mAbs were characterized for Ag specificity and neutralization activity. Both head- and stem-directed mAbs were elicited by the P1 HA Ag, with some mAbs endowed with Ab-dependent cell-mediated cytotoxicity activity. P1 HA-elicited mAbs exhibited a wide breadth of HA recognition, ranging from narrowly reactive to broadly reactive mAbs. Interestingly, we identified a P1 HA-elicited mAb (1F8) exhibiting broad hemagglutination inhibition activity against both seasonal and pandemic H1N1 influenza strains. Furthermore, mAb 1F8 recognized an overlapping, but distinct, epitope compared with other narrowly hemagglutination inhibition-positive mAbs elicited by the P1 or wild-type HA Ags. Finally, P1 HA-elicited mAbs were encoded by distinct H chain variable and L chain variable gene segment rearrangements and possessed unique CDR3 sequences. Collectively, the functional characterization of P1 HA-elicited mAbs sheds further insights into the underlying mechanism(s) of expanded Ab breadth elicited by a COBRA HA-based immunogen and advances efforts toward design and implementation of a more broadly protective influenza vaccine., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Each year millions of people are infected by influenza viruses, and this causes a substantial economic and health burden on our society. Influenza epidemics and pandemics are attributable to the ongoing evolution of influenza viruses through antigenic drift and shift, respectively. One of the reasons for the continuous circulation of influenza viruses in the human population is the incomplete protection conferred by currently available seasonal influenza vaccines against possible arising drifted or shifted influenza strains. Recently, tremendous efforts have been focused on the development of a more effective broadly reactive or universal influenza vaccine. The main objective of underdevelopment vaccines is to protect the human population not only from currently circulating virus strains but also from possible future variants without the need for their continuous update. Different approaches have been developed to reach this goal and elicit an effective and cross-protective immune response. Among these, consensus-based prophylactic approaches to effectively prevent influenza infections are the major focus of this review.

Vaccines are recognized worldwide as one of the most important tools for combating infectious diseases. Despite the tremendous value conferred by currently available vaccines toward public health, the implementation of additional vaccine platforms is also of key importance. In fact, currently available vaccines possess shortcomings, such as inefficient triggering of a cell-mediated immune response and the lack of protective mucosal immunity. In this regard, recent work has been focused on vaccine delivery systems, as an alternative to injectable vaccines, to increase antigen stability and improve overall immunogenicity. In particular, novel strategies based on edible or intradermal vaccine formulations have been demonstrated to trigger both a systemic and mucosal immune response. These novel vaccination delivery systems offer several advantages over the injectable preparations including self-administration, reduced cost, stability, and elimination of a cold chain. In this review, the latest findings and accomplishments regarding edible and intradermal vaccines are described in the context of the system used for immunogen expression, their molecular features and capacity to induce a protective systemic and mucosal response.

Influenza viruses represent a threat to the world population. The currently available standard of care influenza vaccines are offered for each influenza season to prevent infection and spread of influenza viruses. Current vaccine formulations rely on using wild-type Ags, including the hemagglutinin (HA) and neuraminidase (NA) proteins as the primary immune targets of the vaccine. However, vaccine effectiveness varies from season to season, ranging from 10 to 75% depending on season and on age group studied. To improve rates of vaccine effectiveness, a new generation of computationally optimized broadly reactive Ags (COBRA)-based vaccines have been developed as a next-generation influenza vaccine. In this report, mice were intranasally, i.p., or i.m. primed with reassortant influenza viruses expressing different H1N1 COBRA HA proteins. These mice were subsequently boosted i.p. or i.m. with the same viruses. Sera collected from mice that were intranasally infected and i.p. boosted with COBRA-based viruses had broad anti-HA IgG binding, hemagglutination inhibition, and neutralizing activity against a panel of seasonal and pandemic H1N1 viruses. Mice immunized with viruses expressing a seasonal or pandemic H1N1 HA protein had antisera that recognized fewer viruses in the panel. Overall, COBRA-based HA proteins displayed on the surface of a virus elicited a breadth of Abs that recognized and neutralized historical H1N1 strains as well as more contemporary H1N1 viruses., (Copyright © 2018 The Authors.)

Influenza virus infection is an ongoing health and economic burden causing epidemics with pandemic potential, affecting 5-30% of the global population annually, and is responsible for millions of hospitalizations and thousands of deaths each year. Annual influenza vaccination is the primary prophylactic countermeasure aimed at limiting influenza burden. However, the effectiveness of current influenza vaccines are limited because they only confer protective immunity when there is antigenic similarity between the selected vaccine strains and circulating influenza isolates. The major targets of the antibody response against influenza virus are the surface glycoprotein antigens hemagglutinin (HA) and neuraminidase (NA). Hypervariability of the amino acid sequences encoding HA and NA is largely responsible for epidemic and pandemic influenza outbreaks, and are the consequence of antigenic drift or shift, respectively. For this reason, if an antigenic mismatch exists between the current vaccine and circulating influenza isolates, vaccinated people may not be afforded complete protection. There is currently an unmet need to develop an effective "broadly-reactive" or "universal" influenza vaccine capable of conferring protection against both seasonal and newly emerging pre-pandemic strains. A number of novel influenza vaccine approaches are currently under evaluation. One approach is the elicitation of an immune response against the "Achille's heel" of the virus, i.e. conserved viral proteins or protein regions shared amongst seasonal and pre-pandemic strains. Alternatively, other approaches aim toward eliciting a broader immune response capable of conferring protection against the diversity of currently circulating seasonal influenza strains.In this review, the most promising under-development universal vaccine approaches are discussed with an emphasis on those targeting the HA glycoprotein. In particular, their strengths and potential short-comings are discussed. Ultimately, the upcoming clinical evaluation of these universal vaccine approaches will be fundamental to determine their effectiveness against preventing influenza virus infection and/or reducing transmission and disease severity.

The domestic ferret ( Mustela putorius furo ) is a commonly used animal model for the study of influenza virus infection and vaccination. Recently, our group has developed murine mAbs with specificity for the κ (Igκ) and λ (Igλ) L chains of ferret Ig. These mAbs were used to quantify the abundance of Igκ and Igλ in serum and to evaluate L chain usage of the Ab response against the hemagglutinin (HA) protein elicited by influenza infection. After influenza A infection of immunologically naive ferrets with various H1N1 or H3N2 strains, the acute Ab response against HA exhibited an inherent bias toward λ L chain usage. In contrast, secondary infection of H1N1 preimmune ferrets with an antigenically distinct H1N1 virus elicited a recall response against the original HA that was no longer biased toward Igλ and possessed differential specificity. Moreover, sequential infection of ferrets with H1N1 influenza viruses elicited an Igκ-biased Ab response directed against the HA globular head and stem regions. Furthermore, sequential infection of ferrets with viral vectors expressing chimeric HA, aimed at boosting Ab reactivity against the HA stem region, also elicited an Igκ-biased response. Collectively, these findings suggest that ferret B cells expressing an Igκ or Igλ BCR possess differential specificities, and highlight the utility of our recently developed mAbs for studying the immune response to influenza virus infection and vaccination in the ferret model., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The present study focused on inhibition of HSV-1 and -2 replication and pathogenesis in vitro and in vivo, through the selective targeting of the envelope glycoprotein D. Firstly, a human monoclonal antibody (Hu-mAb#33) was identified that could neutralise both HSV-1 and -2 at nM concentrations, including clinical isolates from patients affected by different clinical manifestations and featuring different susceptibility to acyclovir in vitro. Secondly, the potency of inhibition of both infection by cell-free viruses and cell-to-cell virus transmission was also assessed. Finally, mice receiving a single systemic injection of Hu-mAb#33 were protected from death and severe clinical manifestations following both ocular and vaginal HSV-1 and -2 lethal challenge. These results pave the way for further studies reassessing the importance of HSV entry as a novel target for therapeutic intervention and inhibition of cell-to-cell virus transmission., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The design of vaccine strategies and the development of drugs targeting the early stages of Hepatitis C virus (HCV) infection are hampered by the lack of structural information about its surface glycoproteins E1 and E2, the two constituents of HCV entry machinery. Despite the recent crystal resolution of limited versions of both proteins in truncated form, a complete picture of the E1E2 complex is still missing. Here we combined deep computational analysis of E1E2 secondary, tertiary and quaternary structure with functional and immunological mutational analysis across E1E2 in order to propose an in silico model for the ectodomain of the E1E2 heterodimer. Our model describes E1-E2 ectodomain dimerization interfaces, provides a structural explanation of E1 and E2 immunogenicity and sheds light on the molecular processes and disulfide bridges isomerization underlying the conformational changes required for fusion. Comprehensive alanine mutational analysis across 553 residues of E1E2 also resulted in identifying the epitope maps of diverse mAbs and the disulfide connectivity underlying E1E2 native conformation. The predicted structure unveils E1 and E2 structures in complex, thus representing a step towards the rational design of immunogens and drugs inhibiting HCV entry.

Objective: The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2)., Design: Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc)., Results: In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes., Conclusions: Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Memory B cells (B mem ) provide the second wall of adaptive humoral host defense upon specific antigen rechallenge when the first wall, consisting of preformed antibodies originating from a preceding antibody response, fails. This is the case, as recently experienced with SARS-CoV-2 infections and previously with seasonal influenza, when levels of neutralizing antibodies decline or when variant viruses arise that evade such. While in these instances, reinfection can occur, in both scenarios, the rapid engagement of preexisting B mem into the recall response can still confer immune protection. B mem are known to play a critical role in host defense, yet their assessment has not become part of the standard immune monitoring repertoire. Here we describe a new generation of B cell ELISPOT/FluoroSpot (collectively ImmunoSpot ® ) approaches suited to dissect, at single-cell resolution, the B mem repertoire ex vivo, revealing its immunoglobulin class/subclass utilization, and its affinity distribution for the original, and for variant viruses/antigens. Because such comprehensive B cell ImmunoSpot ® tests can be performed with minimal cell material, are scalable, and robust, they promise to be well-suited for routine immune monitoring., (© 2024. The Author(s).)

The affinity distribution of the antigen-specific memory B cell (B mem ) repertoire in the body is a critical variable that defines an individual's ability to rapidly generate high-affinity protective antibody specificities. Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here, we introduce two variants of the B cell ImmunoSpot ® assay that are suitable for simultaneously assessing the affinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using relatively little labor and with high-throughput capacity. First, we experimentally validated that both ImmunoSpot ® assay variants are suitable for establishing functional affinity hierarchies using B cell hybridoma lines as model antibody-secreting cells (ASC), each producing mAb with known affinity for a defined antigen. We then leveraged both ImmunoSpot ® variants for characterizing the affinity distribution of SARS-CoV-2 Spike-specific ASC in PBMC following COVID-19 mRNA vaccination. Such ImmunoSpot ® assays promise to offer tremendous value for future B cell immune monitoring efforts, owing to their ease of implementation, applicability to essentially any antigenic system, economy of PBMC utilization, high-throughput capacity, and suitability for regulated testing., (© 2024. The Author(s).)

Viral infections and opportunistic fungal pathogens represent a major menace for immunocompromised patients. Despite the availability of antifungal and antiviral drugs, mortality in these patients remains high, underlining the need of novel therapeutic options based on completely different strategies. This review describes the potential of several T-cell-based therapeutic approaches in the prophylaxis and treatment of infectious diseases with a particular focus on persistent viral infections and opportunistic fungal infections, as these mostly affect immunocompromised patients.

The lack of structural information on hepatitis C virus (HCV) surface proteins has so far hampered the development of effective vaccines. Recently, two crystallographic structures have described the core portion (E2c) of E2 surface glycoprotein, the primary mediator of HCV entry. Despite the importance of these studies, the E2 overall structure is still unknown and, most importantly, several biochemical and functional studies are in disagreement with E2c structures. Here, the main literature will be discussed and an alternative disulfide bridge pattern will be proposed, based on unpublished human monoclonal antibody reactivity. A modeling strategy aiming at recapitulating the available structural and functional studies of E2 will also be proposed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) is the major cause of chronic liver disease worldwide. There is evidence that neutralizing anti-HCV antibodies may find potential applications in novel prophylactic and therapeutic strategies. This paper describes the very high neutralization activity and unique biological features of two broadly cross-reactive and cross-neutralizing anti-HCV human monoclonal IgG1 derived from human monoclonal recombinant Fab fragments.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease and the most common indication for liver transplantation. Current therapies are ineffective in a relevant percentage of patients raising the urgent medical need to develop adequate therapies for this infection. Broadly neutralizing human monoclonal antibodies (mAbs) directed against the HCV E2 glycoprotein (HCV/E2), the major target of the neutralizing humoral immune response, are considered as a possible novel therapeutic strategy for this infection. In the last few years, several anti-HCV/E2 human mAbs have been described in literature to be possibly used for therapeutic or prophylactic purposes. In this review, we illustrate the best candidates for an anti-HCV mAb-based therapy, considering their cross-neutralization profiles and their ability to overcome possible viral escape mechanisms.

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.