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

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

Broadly neutralizing antibodies target a haemagglutinin anchor epitope.

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

A hybrid demultiplexing strategy that improves performance and robustness of cell hashing.

Cell hashing, a nucleotide barcode-based method that allows users to pool multiple samples and demultiplex in downstream analysis, has gained widespread popularity in single-cell sequencing due to its compatibility, simplicity, and cost-effectiveness. Despite these advantages, the performance of this method remains unsatisfactory under certain circumstances, especially in experiments that have imbalanced sample sizes or use many hashtag antibodies. Here, we introduce a hybrid demultiplexing strategy that increases accuracy and cell recovery in multi-sample single-cell experiments. This approach correlates the results of cell hashing and genetic variant clustering, enabling precise and efficient cell identity determination without additional experimental costs or efforts. In addition, we developed HTOreader, a demultiplexing tool for cell hashing that improves the accuracy of cut-off calling by avoiding the dominance of negative signals in experiments with many hashtags or imbalanced sample sizes. When compared to existing methods using real-world datasets, this hybrid approach and HTOreader consistently generate reliable results with increased accuracy and cell recovery.

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

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

Imprinting, immunodominance, and other impediments to generating broad influenza immunity.

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.

The persistence of naturally acquired antibodies and memory B cells specific to rhoptry proteins of Plasmodium vivax in patients from areas of low malaria transmission.

BACKGROUND: Rhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes. Plasmodium vivax rhoptry 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 the P. vivax rhoptry proteins PvRALP1-Ecto and PvRhopH2 were monitored and analysed in individuals to determine their persistence. METHODS: Thirty-nine samples from P. 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. RESULTS: The 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. CONCLUSION: The study showed that rhoptry antigens induced the development and persistence of MBC responses in P. vivax-infected subjects who lived in a region of low malaria transmission, which were not related to the longevity of antibody responses.

The acquisition of long-lived memory B cell responses to merozoite surface protein-8 in individuals with Plasmodium vivax infection.

BACKGROUND: The ability of a malaria antigen to induce effective, long-lasting immune responses is important for the development of a protective malaria vaccine. Plasmodium vivax merozoite surface protein-8 (PvMSP8) has been shown to be immunogenic in natural P. vivax infections and produces both cell-mediated and antibody-mediated immunity. Thus, PvMSP8 has been proposed as a vaccine candidate following fusion with other merozoite antigens in blood stage vaccine design. Here, the long-term responses of antibodies and memory B cells (MBCs) specific to PvMSP8 in individuals were monitored in a longitudinal cohort study. METHODS: Both cross-sectional surveys and cohort studies were utilized to explore the persistence of antibody and MBC responses to PvMSP8. Antibody titers were detected in individuals with acute disease and those who recovered from an infection for 4 years. The dominant peptide epitope of PvMSP8 recognized by naturally acquired antibodies was examined to observe the durability of the post-infection antibody response. PvMSP8-specific MBCs were also in subjects 4 years post-infection using an enzyme-linked immunospot assay. RESULTS: The prevalence of antibodies to PvMSP8 was high during and after infection. The antibody levels in individual responders were monitored for up to 12 months post-infection and showed that most patients maintained their seropositive response. Interestingly, the anti-PvMSP8 antibody responses stably persisted in some patients who had recovered from an infection for 4 years. Positive PvMSP8-specific MBCs were also detected at 4 years post-infection. However, analysis in these individuals showed no correlation with the presence or titer of circulating antibody. CONCLUSION: PvMSP8 had the ability to induce a long-term humoral immune response. The antibodies and MBCs specific for this antigen developed and persisted in subjects who acquired a natural P. vivax infection. Inclusion of the PvMSP8 antigen in blood stage vaccine design should be considered.

Immunogenicity of glycosylphosphatidylinositol-anchored micronemal antigen in natural Plasmodium vivax exposure.

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.

Immunogenicity of the Plasmodium vivax merozoite surface protein 1 paralog in the induction of naturally acquired antibody and memory B cell responses.

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 4× standard deviations, whereas low responders were the subjects who had OD values less than OD of antisera from non-exposed controls plus 4× 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 <  0.0001). Monitoring of the anti-PvMSP1P-19 antibody titre showed that the antibody was maintained for up to 9 months after recovery. Almost all high-responder groups strongly inhibited PvMSP1P-19 binding to erythrocytes, whereas no inhibition was shown in most low-responder samples. Interestingly, the inhibitory activity of the antibodies in some individuals from high-responder samples were stable for at least 12 months. The longevity of the antibody response was associated with the presence of PvMSP1P-19-specific memory B cells at 9 months after recovery from infection. CONCLUSIONS: The PvMSP1P-19 antigen has immunogenicity during the induction of the antibody response, in which both the levels and inhibitory activity are maintained after the patient recovered from P. vivax infection. The maintenance of the antibody response was associated with the response of PvMSP1P-19-specific memory B cells. Therefore, the PvMSP1P-19 antigen should also be considered as a reliable vaccine candidate to develop a blood-stage vaccine against P. vivax.

Naturally acquired humoral and cellular immune responses to Plasmodium vivax merozoite surface protein 8 in patients with 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.

Naturally-acquired cellular immune response against Plasmodium vivax merozoite surface protein-1 paralog antigen.

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  <  0.05). Interestingly, the response of IFN-γ-producing cells in PvMSP1P stimulation was fourfold greater in recovered subjects than that in acute-infection patients. CD4(+) T cells were the major cell phenotype involved in the response to PvMSP1P-19 and PvDBPII antigen. CONCLUSIONS: PvMSP1P-19 strongly induces a specific cellular immune response for protection against P. vivax compared with PvDBPII as the antigen induces activation of IFN-γ-producing effector cells following natural P. vivax exposure. Upon stimulation, PvMSP1P-19 has the potential to activate the recall response of Th1 effector memory cells that play a role in killing the parasite.

Lack of memory recall in human CD4 T cells elicited by the first encounter with SARS-CoV-2.

The studies reported here focus on the impact of pre-existing CD4 T cell immunity on the first encounter with SARS-CoV-2. They leverage PBMC samples from plasma donors collected after a first SARS-CoV-2 infection, prior to vaccine availability and compared to samples collected prior to the emergence of SARS-CoV-2. Analysis of CD4 T cell specificity across the entire SARS-CoV-2 proteome revealed that the recognition of SARS-CoV-2-derived epitopes by CD4 memory cells prior to the pandemic are enriched for reactivity toward non-structural proteins conserved across endemic CoV strains. However, CD4 T cells after primary infection with SARS-CoV-2 focus on epitopes from structural proteins. We observed little evidence for preferential recall to epitopes conserved between SARS-CoV-2 and seasonal CoV, a finding confirmed through use of selectively curated conserved and SARS-unique peptides. Our data suggest that SARS-CoV-2 CD4 T cells elicited by the first infection are primarily established from the naive CD4 T cell pool.

Epitopes recognition of SARS-CoV-2 nucleocapsid RNA binding domain by human monoclonal antibodies.

Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NPRBD) binds RNA and the C-terminal domain is responsible for dimerization. After infection, NP is highly expressed and triggers robust host immune response. The anti-NP antibodies are not protective and not neutralizing but can effectively detect viral proliferation soon after infection. Two structures of SARS-CoV-2 NPRBD were determined providing a continuous model from residue 48 to 173, including RNA binding region and key epitopes. Five structures of NPRBD complexes with human mAbs were isolated using an antigen-bait sorting. Complexes revealed a distinct complement-determining regions and unique sets of epitope recognition. This may assist in the early detection of pathogens and designing peptide-based vaccines. Mutations that significantly increase viral load were mapped on developed, full length NP model, likely impacting interactions with host proteins and viral RNA.

A hybrid demultiplexing strategy that improves performance and robustness of cell hashing.

Recent advances in single cell RNA sequencing allow users to pool multiple samples and demultiplex in downstream analysis, which greatly increase experimental efficiency and cost-effectiveness. Among all the demultiplexing methods, nucleotide barcode-based cell hashing has gained widespread popularity due to its compatibility and simplicity. Despite these advantages, certain issues of this technic remain to be solved, such as challenges in distinguishing true positive from background, high reagent cost for samples with large cell numbers, and unpredictable false negative and false doublet rates. Here, we propose a hybrid demultiplexing strategy that increases calling accuracy and cell recovery of cell hashing without adding experimental cost. In this approach, we computationally cluster all single cells based on their natural genetic variations and assign donor identity by finding the dominant hashtag in each genotype cluster. This hybrid strategy assigns donor identity to any cell that is identified as singlet by either genotype clustering or cell hashing, which allows us to demultiplex most majority of cells even if only a small fraction of cells are labeled with hashtags. When comparing its performance with cell hashing on multiple real-world datasets, this hybrid approach consistently generates reliable demultiplexing results with increased cell recovery and accuracy.

Site of vulnerability on SARS-CoV-2 spike induces broadly protective antibody against antigenically distinct Omicron subvariants.

The rapid evolution of the severe acute respiratory syndrome coronavirus 2 (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 identified S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) that was derived from an individual previously infected with WT SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrated 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/XBB). Furthermore, S728-1157 protected hamsters against in vivo challenges with WT, Delta, and BA.1 viruses. Structural analysis showed that this antibody targets a class 1/RBS-A epitope in the receptor binding domain via multiple hydrophobic and polar interactions with its heavy chain complementarity determining region 3 (CDR-H3), in addition to common motifs in CDR-H1/CDR-H2 of class 1/RBS-A antibodies. Importantly, this epitope was more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared with diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential and may inform target-driven vaccine designs against future SARS-CoV-2 variants.

Interferon-γ signal drives differentiation of T-bethi atypical memory B cells into plasma cells following Plasmodium vivax infection.

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-bethi atypical MBCs is described in chronic Plasmodium falciparum-exposed individuals. However, it remains unclear whether accumulation of T-bethi atypical MBCs is indicative of a protective role or rather an impaired function of the immune system in malaria. Here, the phenotypic and functional features of T-bethi atypical MBCs were studied in P. vivax patients living in an area of low malaria transmission. During P. vivax infection, the patients produced a twofold higher frequency of T-bethi atypical MBCs compared to malaria non-exposed individuals. This distinct atypical MBC subset had a switched IgG phenotype with overexpression of activation markers and FcRL5, and decreased Syk phosphorylation upon BCR stimulation. Post-infection, expansion of T-bethi IgG+ atypical MBCs was maintained for at least 3 months. Further studies of the contribution of T-bethi atypical MBC function to humoral immunity showed that synergizing IFN-γ with TLR7/8 and IL-21 signals was required for their differentiation into plasma cells and antibody secretion.

Cross-reactive inhibitory antibody and memory B cell responses to variant strains of Duffy binding protein II at post-Plasmodium vivax infection.

Duffy binding protein region II (DBPII) is considered a strong potential vaccine candidate of blood-stage P. vivax. However, the highly polymorphic nature of this protein often misdirects immune responses, leading them to be strain-specific. Details of cross-reactive humoral immunity to DBPII variants have therefore become an important focus for the development of broadly protective vaccines. Here, cross-reactive humoral immunity against a panel of Thai DBPII variants (DBL-THs) was demonstrated in immunized BALB/c mice and P. vivax patients, by in vitro erythrocyte-binding inhibition assay. Sera from immunized animals showed both strain-transcending (anti-DBL-TH2 and -TH4) and strain-specific (anti-DBL-TH5, -TH6 and -TH9) binding to DBL-TH variants. Using anti-DBL-TH sera at 50% inhibitory concentration (IC50) of the homologous strain, anti-DBL-TH2 sera showed cross inhibition to heterologous DBL-TH strains, whereas anti-DBL-TH5 sera exhibited only strain-specific inhibition. In P. vivax patients, 6 of 15 subjects produced and maintained cross-reactive anti-DBL-TH inhibitory antibodies through the 1-year post-infection timepoint. Cross-reactive memory B cell (MBC) responses to DBL-TH variants were analyzed in subjects recovered from P. vivax infection (RC). The plasma samples from 5 RC subjects showed broad inhibition. However, MBC-derived antibodies of these patients did not reveal cross-inhibition. Altogether, broadly anti-DBP variant inhibitory antibodies developed and persisted in P. vivax infections. However, the presence of cross-reactive anti-DBL-TH inhibitory function post-infection was not related with MBC responses to these variants. More detailed investigation of long-lasting, broadly protective antibodies to DBPII will guide the design of vivax malaria vaccines.

Severe COVID-19 induces autoantibodies against angiotensin II that correlate with blood pressure dysregulation and disease severity.

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.

Variable immunogenicity of a vivax malaria blood-stage vaccine candidate.

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.

Cross neutralization of emerging SARS-CoV-2 variants of concern by antibodies targeting distinct epitopes on spike.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have arisen that exhibit increased viral transmissibility and partial evasion of immunity induced by natural infection and vaccination. To address the specific antibody targets that were affected by recent viral variants, we generated 43 monoclonal antibodies (mAbs) from 10 convalescent donors that bound three distinct domains of the SARS-CoV-2 spike. Viral variants harboring mutations at K417, E484 and N501 could escape most of the highly potent antibodies against the receptor binding domain (RBD). Despite this, we identified 12 neutralizing mAbs against three distinct regions of the spike protein that neutralize SARS-CoV-2 and 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.