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Recent advances in single cell RNA sequencing allow users to pool multiple samples into one run and demultiplex in downstream analysis, greatly increasing the experimental efficiency and cost-effectiveness. However, the expensive reagents for cell labeling, limited pooling capacity, non-ideal cell recovery rate and calling accuracy remain great challenges for this approach. To date, there are two major demultiplexing methods, antibody-based cell hashing and Single Nucleotide Polymorphism (SNP)-based genomic signature profiling, and each method has advantages and limitations. Here, we propose a hybrid demultiplexing strategy that increases calling accuracy and cell recovery at the same time. We first develop a computational algorithm that significantly increases calling accuracy of cell hashing. Next, we cluster all single cells based on their SNP profiles. Finally, we integrate results from both methods to make corrections and retrieve cells that are only identifiable in one method but not the other. By testing on several real-world datasets, we demonstrate that this hybrid strategy combines advantages of both methods, resulting in increased cell recovery and calling accuracy at lower cost.HighlightsAn improved algorithm for cell hashing that distinguishes true positive from background for each individual hashtag at higher accuracyThis hybrid strategy increases cell recovery and calling accuracy while lowering experimental costThis hybrid demultiplexing strategy is applicable for single-cell RNA sequencing with different donor species, subjects, and cell populationsDoublet rate is a major determinant of the performance of SNP-based demultiplexing method

SummaryThe rapid evolution of SARS-CoV-2 Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identify S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) and derived from an individual previously infected with SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrates broad cross-neutralization of all dominant variants including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1). Furthermore, it protected hamsters againstin vivochallenges with wildtype, Delta, and BA.1 viruses. Structural analysis reveals that this antibody targets a class 1 epitope via multiple hydrophobic and polar interactions with its CDR-H3, in addition to common class 1 motifs in CDR-H1/CDR-H2. Importantly, this epitope is more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared to diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential, and may inform target-driven vaccine design against future SARS-CoV-2 variants.

Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation, and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme 2 (ACE2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized patients with COVID-19 developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or receptor-binding domain (RBD), to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.

Natural influenza virus infections and seasonal vaccinations often do not confer broadly neutralizing immunity across diverse influenza strains. In addition, the virus is capable of rapid antigenic drift in order to evade pre-existing immunity. The surface glycoproteins, hemagglutinin, and neuraminidase can easily mutate their immunodominant epitopes without impacting fitness. Skewing human antibody repertoires to target more conserved epitopes is thus an expanding area of research: Many groups are attempting to produce universal influenza vaccines that can protect across a wide variety of strains. Achieving this goal will require a detailed understanding of how infection history impacts humoral responses. It will also require the ability to manipulate or enhance B cell selection in order to expand clones that can recognize subdominant but protective epitopes. In this review, we will discuss what immune imprinting means to immunologists and describe efforts to overcome or silence imprinting in order to improve vaccination efficiency.

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.

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.

For development of a long-lasting protective malaria vaccine, it is crucial to understand whether Plasmodium-induced memory B cells (MBCs) or plasma cells develop and stably contribute to protective immunity, or on the contrary the parasite suppresses antibody responses by inducing MBC dysfunction. The expansion of T-bet

Patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme-2 (ACE-2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized COVID-19 patients developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or RBD, to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.

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.

Summary: Multimodal advances in single-cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here, we present LinQ-View, a toolkit designed for multimodal single-cell data visualization and analysis. LinQ-View integrates transcriptional and cell surface protein expression profiling data to reveal more accurate cell heterogeneity and proposes a quantitative metric for cluster purity assessment. Through comparison with existing multimodal methods on multiple public CITE-seq datasets, we demonstrate that LinQ-View efficiently generates accurate cell clusters, especially in CITE-seq data with routine numbers of surface protein features, by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single-cell transcriptional and protein expression data from SARS-CoV-2-infected patients, revealing antigen-specific B cell subsets after infection. Our results suggest LinQ-View could be helpful for multimodal analysis and purity assessment of CITE-seq datasets that target specific cell populations (e.g., B cells). Motivation: Multimodal single-cell sequencing enables multiple aspects for characterizing the dynamics of cell states and developmental processes. Properly integrating information from multiple modalities is a crucial step for interpreting cell heterogeneity. Here, we present LinQ-View, a computational workflow that provides an effective solution for integrating multiple modalities of CITE-seq data for downstream interpretation. LinQ-View balances information from multiple modalities to achieve accurate clustering results and is specialized in handling CITE-seq data with routine numbers of surface protein features.

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 the variants of concern, 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 different variants may have evolved to disrupt the binding of particular neutralizing antibody classes. These results underscore that humans exposed to wildtype (WT) SARS-CoV-2 do 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.

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.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we utilized 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 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., Graphical Abstract, Dugan et al. utilize a multi-antigen bait sorting and single cell sequencing approach to profile B cell immunodominance upon SARS-CoV-2 infection, revealing a dynamic response that evolves toward internal virus proteins over time. Antibodies to internal proteins were non-protective in vivo, suggesting vaccination may generate superior anti-spike immunological memory.

Artificial mutagenesis and chimeric/mosaic protein engineering have laid the foundation for antigenic characterization1 and universal vaccine design2–4 for influenza viruses. However, many methods used for influenza research and vaccine development require sequence editing and protein expression, limiting their applicability and the progress of related research to specialists. Rapid tools allowing even novice influenza researchers to properly analyze and visualize influenza protein sequences with accurate nomenclature are needed to expand the research field. To address this need, we developed Librator, a system for analyzing and designing protein sequences of influenza virus Hemagglutinin (HA) and Neuraminidase (NA). With Librator’s graphical user interface (GUI) and built-in sequence editing functions, biologists can easily analyze influenza sequences and phylogenies, automatically port sequences to visualize structures, then readily mutate target residues and design sequences for antigen probes and chimeric/mosaic proteins efficiently and accurately. This system provides optimized fragment design for Gibson Assembly5 of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible, allowing for significant reagent savings. Use of Librator will significantly facilitate influenza research and vaccine antigen design.

SummaryBroadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is “anchored” to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.

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., 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.

Single cell RNA sequencing is a powerful tool for characterizing the molecular and cellular heterogeneity of immune cells during their development and activation. Multimodal advances in single cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here we present LinQ-View, a toolkit designed for multimodal single cell data visualization and analysis that links transcriptional and cell surface protein expression profiling data. Further, we propose a quantitative metric for cluster purity of CITE-seq data, enabling effective determination of clustering algorithms and their parameters, and finally demonstrate the utility of our toolkit through seamless integration with standard single cell analysis workflows on several public datasets. Through comparison to existing multimodal methods, we demonstrate that LinQ-View generates more accurate cell clusters and is highly efficient, even with massive datasets. LinQ-View is specialized in handling CITE-seq data with routine numbers of surface protein features (e.g. less than 50), by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single cell transcriptional and protein expression data from COVID-19-infected patients and influenza-immunized subjects, revealing antigen-specific B cell subsets and previously unknown T cell subsets post-infection and vaccination.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we utilized single-cell sequencing to profile SARS-CoV-2-reactive B cell subsets in 42 COVID-19 patients. We isolated thousands of B cells in multiple distinct subsets specific to the SARS-CoV-2 spike, endemic coronavirus (HCoV) spikes, nucleoprotein (NP), and open reading frame 8 (ORF8). Spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients 1.5–5 months post-infection. With severe acute infection, we identified substantial populations of endemic HCoV-reactive antibody-secreting cells with highly mutated variable genes, indicative of preexisting immunity. Finally, MBCs exhibited maturation to NP and ORF8 over time relative to spike, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal considerable antibody adaptation to non-neutralizing antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs. Trial Registration Number: This clinical trial was registered at ClinicalTrials.gov with identifier NCT04340050, and clinical information for patients included in the study is detailed in Table S1–S3. Funding: This project was funded in part by the National Institute of Allergy and Infectious Disease (NIAID); National Institutes of Health (NIH) grant numbers U19AI082724 (P.C.W.), U19AI109946 (P.C.W.), U19AI057266 (P.C.W.), the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) grant numbers HHSN272201400005C(P.C.W.). N.W.A. was supported by the Multi-disciplinary Training program in Cancer Research (MTCR) - NIH T32 CA009594. A.J. and R.P.J were supported by federal funds from the NIAID, NIH, and Department of Health and Human Services under Contract HHSN272201700060C. F.K and F.A. were funded by the NIAID CEIRS contractHHSN272201400008C, Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 and the generous support of the JPB foundation, the Open Philanthropy Project (#2020-215611) and other philanthropic donations. Y.K. and P.H.were funded by the Research Program on Emerging and Re-emerging Infectious Disease grant (JP19fk0108113) and the Japan Program for Infectious Diseases Research and Infrastructure (JP20fk0108272) from the Japan Agency for Medical Research and Development (AMED), NIAID CEIRS contract HHSN272201400008C, and CIVIC contract 75N93019C00051. D.F., C.N, Y.D., and P.D.H, were supported by NIAID contracts HHSN272201700060C and 75N93019C00062. M.S.D. and E.S.W. were supported by NIH grants R01 AI157155 and F30 AI152327, respectively. Conflict of Interest: Several antibodies generated from this work are being used by Now Diagnostics in Springdale, AR for the development of a diagnostic test. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. Ethical Approval: All studies were performed with the approval of the University of Chicago institutional review board IRB20-0523 and University of Chicago, University of Wisconsin-Madison, and Washington University in St. Louis institutional biosafety committees. Informed consent was obtained after the research applications and possible consequences of the studies were disclosed to study subjects.

Discovery of durable memory B cell (MBC) subsets against neutralizing viral epitopes is critical for determining immune correlates of protection from SARS-CoV-2 infection. Here, we identified functionally distinct SARS-CoV-2-reactive B cell subsets by profiling the repertoire of convalescent COVID-19 patients using a high-throughput B cell sorting and sequencing platform. Utilizing barcoded SARS-CoV-2 antigen baits, we isolated thousands of B cells that segregated into discrete functional subsets specific for the spike, nucleocapsid protein (NP), and open reading frame (ORF) proteins 7a and 8. Spike-specific B cells were enriched in canonical MBC clusters, and monoclonal antibodies (mAbs) from these cells were potently neutralizing. By contrast, B cells specific to ORF8 and NP were enriched in naïve and innate-like clusters, and mAbs against these targets were exclusively non-neutralizing. Finally, we identified that B cell specificity, subset distribution, and affinity maturation were impacted by clinical features such as age, sex, and symptom duration. Together, our data provide a comprehensive tool for evaluating B cell immunity to SARS-CoV-2 infection or vaccination and highlight the complexity of the human B cell response to SARS-CoV-2.

Relapsing malaria caused by Plasmodium vivax is a neglected tropical disease and an important cause of malaria worldwide. Vaccines to prevent clinical disease and mosquito transmission of vivax malaria are needed to overcome the distinct challenges of this important public health problem. In this vaccine immunogenicity study in mice, we examined key variables of responses to a P. vivax Duffy binding protein vaccine, a leading candidate to prevent the disease-causing blood-stages. Significant sex-dependent differences were observed in B cell (CD80+) and T cell (CD8+) central memory subsets, resulting in significant differences in functional immunogenicity and durability of anti-DBP protective efficacy. These significant sex-dependent differences in inbred mice were in the CD73+CD80+ memory B cell, H2KhiCD38hi/lo, and effector memory subsets. This study highlights sex and immune genes as critical variables that can impact host responses to P. vivax antigens and must be taken into consideration when designing clinical vaccine studies.

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.

Broadly neutralizing antibodies are critical for protection against drifted and novel influenza viruses. Here, we reveal humans have pre-existing immunity against two broadly neutralizing epitopes of the HA head: the lateral patch and receptor-binding site (RBS). Monoclonal antibodies generated against these epitopes are broadly neutralizing against 40+ years of H1N1 evolution and provide potent protection in vivo. First exposure to the novel 2009 pandemic H1N1 virus recalls memory B cells that target the conserved RBS and lateral patch epitopes. We identified unique repertoire, structural, and binding features of lateral patch and RBS binding antibodies including a lateral patch binding motif shared across subjects. Together, our data indicate that the conserved RBS and lateral patch epitopes are readily targeted by the human memory B cell pool and that universal vaccine strategies should aim to drive antibodies against both conserved head and stalk domain epitopes.

Background: The Plasmodium vivax merozoite surface protein 1 paralog (PvMSP1P-19) is a glycosylphosphatidylinositol (GPI)-anchored blood-stage protein that is expressed on the merozoite surface. It is proposed as a blood-stage vaccine candidate against P. vivax because of its ability to induce immune responses upon natural P. vivax exposure and in immunized animals. This study aimed to demonstrate the presence of inhibitory antibodies and memory B cell responses to the PvMSP1P-19 antigen during acute P. vivax infection and after recovery from infection. Methods: To evaluate the antibody responses to PvMSP1P-19 during and after recovery from P. vivax infection, heparinized blood was collected from P. vivax-infected patients and recovered subjects to detect the total IgG response. The seropositive samples were defined into high and low responders, according to their optical density (OD) values obtained from ELISA. High responders were the subjects who had OD values above the OD of antisera from non-exposed controls plus 4x standard deviations, whereas low responders were the subjects who had OD values less than OD of antisera from non-exposed controls plus 4x standard deviations. The plasma from high and low responders were taken for testing the inhibitory activity against PvMSP1P-19-erythrocyte binding by in vitro EBIA. The sustainability of PvMSP1P-19-specific memory B cell responses after recovery from infection was analysed by ELISPOT. Results: The anti-PvMSP1P-19 antibody levels were significantly higher in acutely infected P. vivax patients compared to healthy controls (P

BackgroundRhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes.Plasmodium vivaxrhoptry proteins have been shown to induce humoral immunity during natural infections. Therefore, these proteins may be potential novel vaccine candidates. However, there is a lack of data on the duration of antibody and memory B cell (MBC) responses. Here, the longitudinal analysis of antibody and MBC responses to theP. vivaxrhoptry proteins PvRALP1-Ecto and PvRhopH2 were monitored and analysed in individuals to determine their persistence.MethodsThirty-nine samples fromP. vivax-infected subjects (age 18–60 years) were recruited to explore the frequency and persistence of antibody and MBC responses against rhoptry proteins (PvRALP1-Ecto and PvRhopH2) using both cross-sectional and longitudinal cohort study designs. Antibody levels were determined by ELISA during clinical malaria, and at 3, 9 and 12 months post-infection. The frequency of MBC sub-sets and presence of rhoptry-specific MBCs in subjects 18 months after treatment were detected by flow cytometry and ELISPOT assay.ResultsThe seroprevalence of antibodies against PvRALP1-Ecto and PvRhopH2 proteins was found to be high during acute infection, with IgG1, IgG2 and IgG3 sub-classes predominant. However, these anti-rhoptry responses were short-lived and significantly decreased at 9 months post-infection. To relate the durability of these antibody responses to MBC persistence at post-infection, 18-month post-infection peripheral blood mononuclear cells (PBMCs) samples were taken to detect rhoptry-specific MBCs and frequency of MBC sub-sets, and correlate with antibody responses. These late post-infection samples revealed that rhoptry-specific MBCs were present in about 70% of total subjects. However, the persistence of specific MBCs was not correlated with antibody responses as the majority of malaria subjects who were positive for PvRALP1-Ecto- or PvRhopH2-specific MBCs were seronegative for the rhoptry antigens. The frequencies of classical MBCs were increased after infection, whereas those of activated and atypical MBCs were decreased, indicating that MBC responses could switch from activated or atypical MBCs to classical MBCs after parasite clearance, and were maintained in blood circulating at post-infection.ConclusionThe study showed that rhoptry antigens induced the development and persistence of MBC responses inP. vivax-infected subjects who lived in a region of low malaria transmission, which were not related to the longevity of antibody responses.

Additional file 1. PvRALP1-Ecto and PvRhopH2 specific MBC response by ELISPOT assay.

The major challenge in designing a protective Duffy binding protein region II (DBPII)-based vaccine against blood-stage vivax malaria is the high number of polymorphisms in critical residues targeted by binding-inhibitory antibodies. Here, longevity of antibody and memory B cell response (MBCs) to DBL-TH variants, DBL-TH2, -TH4, -TH5, -TH6 and -TH9 were analyzed in P. vivax-exposed individuals living in a low malaria transmission area of southern Thailand. Antibody to DBL-TH variants were significantly detected during P. vivax infection and it was persisted for up to 9 months post-infection. However, DBL-TH-specific MBC responses were stably maintained longer than antibody response, at least 3 years post-infection in the absence of re-infection. Phenotyping of B cell subsets showed the expansion of activated and atypical MBCs during acute and recovery phase of infection. While the persistence of DBL-TH-specific MBCs was found in individuals who had activated and atypical MBC expansion, anti-DBL-TH antibody responses was rapidly declined in plasma. The data suggested that these two MBCs were triggered by P. vivax infection, its expansion and stability may have impact on antibody responses. Our results provided evidence for ability of DBPII variant antigens in induction of long-lasting MBCs among individuals who were living in low malaria endemicity.

Background Plasmodium vivax is the most geographically widespread malaria species and codominates with Plasmodium falciparum, the deadliest form of the malaria parasite. For the last few years, the number of vivax malaria cases has increased, but vivax malaria is still considered a neglected disease. During the blood stages of their life cycle, P. vivax parasites export several hundred proteins into host red blood cells. Some of these exported proteins have been discovered and studied for use as a blood-stage malaria vaccine. The P. vivax glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (PvGAMA) was identified in previous study, which plays an important role in parasite invasion. To support the hypothesis that PvGAMA can induce an immune response in natural exposure, the antibody responses and cellular immunity against this antigen was demonstrated during and post-infection. Methods The recombinant protein PvGAMA was expressed and purified by wheat germ cell-free (WGCF) system. The analysis of humoral and cellular immune responses to the PvGAMA antigen during infection and post-infection with the P. vivax parasite were done by enzyme-linked immunosorbent assay (ELISA) techniques. Results During P. vivax infection, 95% of patients showed significant antibody responses to PvGAMA antigen. The cytophilic IgG1 and IgG3 isotypes were the major isotypes produced in response to PvGAMA. A cross-sectional study of anti-PvGAMA responses during and post-infection with P. vivax found that the majority of individuals, approximately 54% of patients, were shown to maintain a positive anti-PvGAMA titre at 3 months post-infection, and some patients had the ability to maintain an antibody response for up to 12 months post-infection. Moreover, PvGAMA had the ability to stimulate a cellular immune response that was characterized by the production of the cytokines IL-2, IFN-γ and IL-10. The levels of the cytokines IFN-γ and IL-10 were significantly increased in PvGAMA-stimulated lymphocyte cultures. Conclusions Taken together, PvGAMA had potential to induce an immune response both humoral and cellular immunity in naturally acquired P. vivax infection individuals during infection and post-infection. Therefore, PvGAMA could be as a vaccine candidate to stimulate immune response against P. vivax infection.

Background Thirty-one glycosylphosphatidylinositol (GPI)-anchored proteins of Plasmodium vivax, merozoite surface protein 1 (MSP1), MSP1 paralogue, MSP4, MSP5, MSP8, and MSP10 have been reported from homologs of Plasmodium falciparum by gene annotation with bioinformatics tools. These GPI-anchored proteins contain two epidermal growth factor (EGF)-like domains at its C-terminus. Here, P. vivax merozoite surface protein 8 (PvMSP8) are considered as potential targets of protective immunity. Methods Recombinant PvMSP8 (rPvMSP8) was expressed, purified, and used for the assessment of humoral and cellular immune responses in P. vivax-infected patients and immune mice. Moreover, the target epitope of ant-PvMSP8 antibodies and subcellular localization of PvMSP8 was also determined. Results The rPvMSP8 was successfully expressed and purified as soluble form as ~55 kDa. PvMSP8 was localized to the outer circle of pigments associated with the food vacuole. The rPvMSP8 protein had a high antigenicity (73.2% in sensitivity and 96.2% in specificity) in patients infected with P. vivax. IgG2 antibody subtype was the predominantly responses to this antigen. Antibody response to PvMSP8 increased up to day 7 and after that slightly decreased within a month. The longevity of anti-PvMSP8 antibody was stably sustained up to 12-year recovery patient samples. Most anti-PvMSP8 antibodies recognized two epitopes that were located outside the C-terminal EGF-like domain. The cellular immune response in P. vivax-exposed individuals produced high levels of IFN-γ and IL-10 upon PvMSP8 antigen stimulation in vitro. Conclusions All data in this study suggest that PvMSP8 antigen has a potential to induce both humoral and cellular immune responses in patients with P. vivax infection. The subcellular localization of PvMSP8 confirmed that it was associated with the parasite food vacuole in blood-stage parasites. A further characterization of this protein will be useful for blood stage P. vivax vaccine development. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1837-5) contains supplementary material, which is available to authorized users.

Additional file 1: Figure S1. Cytokine levels in supernatant of 72 hr splenocyte cultures of rPbMSP8-immunized BABL/C mice stimulated with PvMSP8 in vitro. (A) The levels (O.D. 450 nm) of splenocytes from mice immunized with rPvMSP8 after stimulation with 2.5 Îźg/mL of rPvMSP8. The asterisks indicate significant differences on the graph. (B) The levels of IFN-g, TNF-a, IL-2, IL-4, IL-10 cytokines from rPvMSP8-immunized mouse splenocytes stimulated with PvMSP8. Table S1. Peptide sequence information of overlapped 18-mer from the C terminus of PvMSP8. Table S2. Prevalence (% positive), 95% confidence intervals, and mean fluorescence intensity of IgG responses to rPvMSP8 in patient and healthy individual serum samples. Table S3. Prevalence, 95% confidence intervals, and normalized mean fluorescence intensity of IgG responses to rPvMSP8 in 1-month follow-up and archival sera samples.

Rhoptry-associated membrane antigen (RAMA) is an abundant glycophosphatidylinositol (GPI)-anchored protein that is embedded within the lipid bilayer and is implicated in parasite invasion. Antibody responses against rhoptry proteins are produced by individuals living in a malaria-endemic area, suggesting the immunogenicity of Plasmodium vivax RAMA (PvRAMA) for induction of immune responses during P. vivax infection. To determine whether PvRAMA contributes to the acquisition of immunity to malaria and could be a rational candidate for a vaccine, the presence of memory T cells and the stability of the antibody response against PvRAMA were evaluated in P. vivax-exposed individuals. The immunogenicity of PvRAMA for the induction of T cell responses was evaluated by in vitro stimulation of peripheral blood mononuclear cells (PBMCs). High levels of interferon (IFN)-γ and interleukin (IL)-10 cytokines were detected in the culture supernatant of PBMCs, and the CD4+ T cells predominantly produced IL-10 cytokine. The levels of total anti-PvRAMA immunoglobulin G (IgG) antibody were significantly elevated, and these antibodies persisted over the 12 months of the study. Interestingly, IgG1, IgG2 and IgG3 were the major antibody subtypes in the response to PvRAMA. The frequency of IgG3 in specific to PvRAMA antigen maintained over 12 months. These data could explain the immunogenicity of PvRAMA antigen in induction of both cell-mediated and antibody-mediated immunity in natural P. vivax infection, in which IFN-γ helps antibody class switching toward the IgG1, IgG2 and IgG3 isotypes and IL-10 supports PvRAMA-specific antibody production.

There is an error in the affiliation for the ninth author. Eun-Taek Hun is not affiliated with #3 but with #2 Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, Republic of Korea.

Background Plasmodium vivax merozoite surface protein-1 paralog (PvMSP1P) is a glycosylphosphatidylinositol-anchored protein expressed on the merozoite surface. This molecule is a target of natural immunity, as high anti-MSP1P-19 antibody levels were detected during P. vivax infection and the antibody inhibited PvMSP1P-erythrocyte binding. Recombinant PvMSP1P antigen results in production of a significant Th1 cytokine response in immunized mice. The present study was performed to characterize natural cellular immunity against PvMSP1P-19 and PvDBP region II in acute and recovery P. vivax infection. Methods Peripheral blood mononuclear cells (PBMCs) from acute and recovery P. vivax infection were obtained for lymphocyte proliferation assay upon PvMSP1P-19 and PvDBP region II antigen stimulation. The culture supernatant was examined for the presence of the cytokines IL-2, TNF, IFN-γ and IL-10 by enzyme-linked immunosorbent assay (ELISA). To determine whether Th1 or Th2 have a memory response against PvMSP1P-19 and PvDBPII protein antigen, PBMCs from subjects who had recovered from P. vivax infection 8–10 weeks prior to the study were obtained for lymphocyte proliferation assay. Cytokine-producing cells were analysed by flow cytometry. Results IL-2 was detected at high levels in lymphocyte cultures from acutely infected P. vivax patients upon PvMSP1P-19 stimulation. Analysis of the Th1 or Th2 memory response in PBMC cultures from subjects who had recovered from P. vivax infection showed significantly elevated levels of PvMSP1P-19 and PvDBPII-specific IFN-γ-producing cells (P