Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites.

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.

Elevated Detection of Dual Antibody B Cells Identifies Lupus Patients With B Cell-Reactive VH4-34 Autoantibodies.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of "B cells that are naïve IgD+" (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Neutralizing antibody: a savior in the Covid-19 disease.

Coronavirus outbreak was declared a pandemic by World Health Organization (WHO) in March 2020. The pandemic has led to a devastating loss of life. It has shown us how infectious diseases can cause human existence at stake, and community health is important. The spike protein is the most immunogenic component of the virus. Most vaccine development strategies have focused on the receptor-binding domain (RBD) in the spike protein because it is the most specific target site that recognizes and interacts with human lung cells. Neutralizing antibodies are generated by the humoral immune system and reduce the viral load by binding to spike protein components. Neutralizing antibodies are the proteins secreted by plasma cells and serve as an important part of the defense mechanism. In the recent Covid-19 infection, neutralizing antibodies can be utilized for both diagnostic such as immune surveillance and therapeutic tools such as plasma therapy. So far, many monoclonal antibodies are in the clinical trial phase, and few of them are already in use. In this review, we have discussed details about neutralizing antibodies and their role in combating Covid-19 disease.

Respuesta vacunal frente a SARS-CoV-2 en trabajadores de un hospital / Vaccine response to SARS-CoV-2 in hospital workers

Introducción: Evaluar la inmunidad de los trabajadores de un hospital tras haber completado la vacunación Pfizer-BionTech, y su relación con factores individuales. También describir los efectos adversos de la vacuna. Método: Estudio transversal de una muestra de los trabajadores del Hospital General Universitario de Castellón vacunados con dos dosis en enero y febrero de 2021. Un mes después se detectaron: anticuerpos IgG frente a la proteína N (IgG-NP), de IgM frente a la proteína S (IgM-S) y detección cuantitativa de IgG frente a la proteína S (IgG-Quant). Se utilizó un cuestionario para recoger datos demográficos, factores de riesgo y efectos secundarios. En el análisis estadístico se utilizaron modelos de regresión múltiple. Resultados: La participación fue del 96,8% (275/284). Presentaron IgG-Quant el 99,6%, 14,9% IgM-S, y 4,4% IgG-NP. El nivel ajustado de IgG-Quant aumentó significativamente con la obesidad, en no fumadores y con positividad IgM-S y/o IgG-NP. La prevalencia de IgM-S era mayor en varones, y se asociaba con los mismos factores que la IgG-Quant. De los infectados por COVID-19, el 42,9% no presentaron IgG-NP. Un 86,5% sufrió algún efecto secundario que se asoció a tener IgG-NP, mayores niveles de IgG-Quant, y fue más frecuente en jóvenes y mujeres. Conclusiones: Todos los participantes desarrollaron inmunidad humoral excepto uno. Tuvieron mayores niveles de anticuerpos los que habían padecido la COVID-19. Un porcentaje alto desarrolló efectos secundarios leves, más frecuentes en los que habían padecido la enfermedad (AU)

Introduction: The aim of this study was to measure anti-SARS-CoV-2 immunity of hospital workers after a completed 2-dose Pfizer-BionTech vaccination, and to examine factors potentially associated with immunity status. Side effects of the vaccine were also studied. Method: This was a cross-sectional study of a representative sample of General University Hospital of Castellon workers, vaccinated with two doses in January and February 2021. We measured IgG antibodies against protein N (IgG-NP), IgM against protein S (IgM-S), and quantitative levles of IgG against protein S (IgG-Quant) one month after the last dose. We obtained information on demographic, risk factors, and vaccine side effects via a self-completed questionnaire. For the statistical analysis we used multiple regression models. Results: Two hundred seventy-five workers participated (96.8%, 275/284). Positive IgG-Quant, IgM-S, and IgG-NP were 99.6%, 14.9% and 4.4%, respectively. Adjusted IgG-Quant levels increased significantly with obesity, nonsmoking status, positive IgM-S, and/or IgG-NP. The prevalence of IgM-S was higher in males, and associated with the same factors as those for IgG-Quant. Among those with a history of COVD-19 infection, 42.9% did not have IgG-NP. Overall 86.5% of participants had side effects, which were associated with positive IgG-NP, high IgG-Quant levels, younger age, and being female. Conclusions: All but one participant developed immunity. Those who had suffered from COVID-19 infection had higher antibody levels. A high proportion of participants had mild secondary effects, especially those with previous COVID-19 infection (AU)

Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases.

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Single-cell transcriptome profiling and the use of AID deficient mice reveal that B cell activation combined with antibody class switch recombination and somatic hypermutation do not benefit the control of experimental trypanosomosis.

Salivarian trypanosomes are extracellular protozoan parasites causing infections in a wide range of mammalian hosts, with Trypanosoma evansi having the widest geographic distribution, reaching territories far outside Africa and occasionally even Europe. Besides causing the animal diseases, T. evansi can cause atypical Human Trypanosomosis. The success of this parasite is attributed to its capacity to evade and disable the mammalian defense response. To unravel the latter, we applied here for the first time a scRNA-seq analysis on splenocytes from trypanosome infected mice, at two time points during infection, i.e. just after control of the first parasitemia peak (day 14) and a late chronic time point during infection (day 42). This analysis was combined with flow cytometry and ELISA, revealing that T. evansi induces prompt activation of splenic IgM+CD1d+ Marginal Zone and IgMIntIgD+ Follicular B cells, coinciding with an increase in plasma IgG2c Ab levels. Despite the absence of follicles, a rapid accumulation of Aicda+ GC-like B cells followed first parasitemia peak clearance, accompanied by the occurrence of Xbp1+ expressing CD138+ plasma B cells and Tbx21+ atypical CD11c+ memory B cells. Ablation of immature CD93+ bone marrow and Vpreb3+Ly6d+Ighm+ expressing transitional spleen B cells prevented mature peripheral B cell replenishment. Interestingly, AID-/- mice that lack the capacity to mount anti-parasite IgG responses, exhibited a superior defense level against T. evansi infections. Here, elevated natural IgMs were able to exert in vivo and in vitro trypanocidal activity. Hence, we conclude that in immune competent mice, trypanosomosis associated B cell activation and switched IgG production is rapidly induced by T. evansi, facilitating an escape from the detrimental natural IgM killing activity, and resulting in increased host susceptibility. This unique role of IgM and its anti-trypanosome activity are discussed in the context of the dilemma this causes for the future development of anti-trypanosome vaccines.

Selective functional antibody transfer into the breastmilk after SARS-CoV-2 infection.

Antibody transfer via breastmilk represents an evolutionary strategy to boost immunity in early life. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies have been observed in the breastmilk, the functional quality of these antibodies remains unclear. Here, we apply systems serology to characterize SARS-CoV-2-specific antibodies in maternal serum and breastmilk to compare the functional characteristics of antibodies in these fluids. Distinct SARS-CoV-2-specific antibody responses are observed in the serum and breastmilk of lactating individuals previously infected with SARS-CoV-2, with a more dominant transfer of immunoglobulin A (IgA) and IgM into breastmilk. Although IgGs are present in breastmilk, they are functionally attenuated. We observe preferential transfer of antibodies capable of eliciting neutrophil phagocytosis and neutralization compared to other functions, pointing to selective transfer of certain functional antibodies to breastmilk. These data highlight the preferential transfer of SARS-CoV-2-specific IgA and IgM to breastmilk, accompanied by select IgG subpopulations, positioned to create a non-pathologic but protective barrier against coronavirus disease 2019 (COVID-19).

Diagnostic Vaccination in Clinical Practice.

Testing the antibody response to vaccination (diagnostic vaccination) is crucial in the clinical evaluation of primary immunodeficiency diseases. Guidelines from the American Academy of Allergy, Asthma & Immunology (AAAAI) provide detailed recommendations for diagnostic vaccination with pure pneumococcal polysaccharide vaccines (PPV). However, the degree of compliance with these guidelines and the utility of the guidelines in actual practice are undescribed. To address this, we systematically evaluated diagnostic vaccination in adult patients with suspected primary immunodeficiency diseases in a single tertiary center from 2011 to 2016 (n = 229). We found that full compliance with the AAAAI guidelines was achieved for only 39 patients (17%), suggesting that the guidelines are not easy to follow. Worse, interpretation according to the guidelines was heavily influenced by which serotype-specific antibodies that were used for the evaluation. We found that the arbitrary choices of serotype-specific antibodies could change the fraction of patients deemed to have 'adequate immunity' by a factor of four, exposing an inherent flaw in the guidelines. The flaw relates to dichotomous principles for data interpretation under the AAAAI guidelines. We therefore propose a revised protocol for diagnostic vaccination limited to PPV vaccination, subsequent antibody measurements, and data interpretation using Z-scores. The Z-score compiles multiple individual antibody levels, adjusted for different weighting, into one single continuous variable for each patient. In contrast to interpretation according to the AAAAI guidelines, the Z-scores were robust to variations in the choice of serotype-specific antibodies used for interpretation. Moreover, Z-scores revealed reduced immunity after vaccination in the patients with recurrent pneumonia (a typical symptom of antibody deficiency) compared with control patients. Assessment according to the AAAAI guidelines failed to detect this difference. We conclude that our simplified protocol and interpretation with Z-scores provides more robust clinical results and may enhance the value of diagnostic vaccination.

SARS-CoV-2 Antibody Isotypes in Systemic Lupus Erythematosus Patients Prior to Vaccination: Associations With Disease Activity, Antinuclear Antibodies, and Immunomodulatory Drugs During the First Year of the Pandemic.

Objectives: Impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic on individuals with arthritis has been highlighted whereas data on other rheumatic diseases, e.g., systemic lupus erythematosus (SLE), are scarce. Similarly to SLE, severe SARS-CoV-2 infection includes risks for thromboembolism, an unbalanced type I interferon response, and complement activation. Herein, SARS-CoV-2 antibodies in longitudinal samples collected prior to vaccination were analyzed and compared with SLE progression and antinuclear antibody (ANA) levels. Methods: One hundred patients (83 women) with established SLE and a regular visit to the rheumatologist (March 2020 to January 2021) were included. All subjects donated blood and had done likewise prior to the pandemic. SARS-CoV-2 antibody isotypes (IgG, IgA, IgM) to the cell receptor-binding S1-spike outer envelope protein were detected by ELISA, and their neutralizing capacity was investigated. IgG-ANA were measured by multiplex technology. Results: During the pandemic, 4% had PCR-confirmed infection but 36% showed SARS-CoV-2 antibodies of ≥1 isotype; IgA was the most common (30%), followed by IgM (9%) and IgG (8%). The antibodies had low neutralizing capacity and were detected also in prepandemic samples. Plasma albumin (p = 0.04) and anti-dsDNA (p = 0.003) levels were lower in patients with SARS-CoV-2 antibodies. Blood group, BMI, smoking habits, complement proteins, daily glucocorticoid dose, use of hydroxychloroquine, or self-reported coronavirus disease 2019 (COVID-19) symptoms (except fever, >38.5°C) did not associate with SARS-CoV-2 antibodies. Conclusion: Our data from early 2021 indicate that a large proportion of Swedish SLE patients had serological signs of exposure to SARS-CoV-2 but apparently with a minor impact on the SLE course. Use of steroids and hydroxychloroquine showed no distinct effects, and self-reported COVID-19-related symptoms correlated poorly with all antibody isotypes.

Integrated longitudinal immunophenotypic, transcriptional and repertoire analyses delineate immune responses in COVID-19 patients.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Dynamic Characteristic Analysis of Antibodies in Patients With COVID-19: A 13-Month Study.

There is a worldwide pandemic of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; yet our understanding remains limited on the characteristic of antibodies, especially for dynamic long-term tracking. Sequential serum samples were collected up to 416 days post onset of symptoms (POS) from 102 patients who were hospitalized with coronavirus disease 2019 (COVID-19). Immunoglobulin (Ig)G, IgM, and IgA levels targeting SARS-CoV-2 spike 1 receptor-binding domain (S1-RBD), spike 2 extracellular domain (S2-ECD), and nucleocapsid protein (N) were quantified as well as neutralizing activity. We were pleasantly surprised to find that the antibody remained detective and effective for more than a year POS. We also found the varied reactions of different antibodies as time passed: N-IgA rose most rapidly in the early stage of infection, while S2-IgG was present at a high level in the long time of observation. This study described the long traceable antibody response of the COVID-19 and offered hints about targets to screen for postinfectious immunity and for vaccination development of SARS-CoV-2.

Immunoglobulin subtype-coated bacteria are correlated with the disease activity of inflammatory bowel disease.

Immune response involving various immunoglobulin (Ig) isotypes and subtypes to microbiome is involved in the pathogenesis and disease activity of inflammatory bowel diseases (IBDs). To clarify the presence of Ig-coated bacteria in the intestine and its association with disease activity in ulcerative colitis (UC) and Crohn's disease (CD), we extracted and classified Ig-coated bacteria from fecal samples of 42 patients with IBD and 12 healthy controls (HCs) using flow cytometry and 16S ribosomal RNA sequence analysis. The percentage of bacteria coated with IgA and IgM was higher in patients with IBD than in HCs, and IgG-coated bacteria were found only in patients with IBD. Moreover, the percentages of bacteria coated with IgG1, IgG2, IgG3, and IgM in UC samples and IgG3, IgG4, and IgM in CD samples were correlated with disease activities. The proportions of Bacteroides ovatus and Streptococcus increased during the active phase of CD. Hence, the detailed analysis of Ig-coated bacteria and Ig subtypes using flow cytometry could aid in developing useful indicators of disease activity and identifying more disease-related bacteria, which could become novel treatment targets for IBDs.

The IgE Blocking Activity Induced by Dermatophagoides pteronyssinus Subcutaneous Immunotherapy Does Not Correlate with Specific IgA but with IgG4 in both Serum and Saliva.

BACKGROUND: The role of salivary-specific IgG4 and IgA in subcutaneous immunotherapy (SCIT) is not well defined. We aimed to investigate the change of IgG4 and IgA in both serum and saliva and their correlations with IgE-blocking-factor (IgE-BF) during SCIT. METHOD: 307 Dermatophagoides pteronyssinus (DP) allergic rhinitis and/or asthma patients were recruited for this study. 286 patients received DP-SCIT for 1 year. Twenty-one patients received only symptomatic treatment. DP-, Der p 1-, and Der p 2-specific IgE in serum, specific-IgG4 and Der p 2-specific IgA1 and IgA2 in both serum and saliva were measured at timepoints 0, 4, and 12 months during DP-SCIT. Correlation between salivary and serological IgG4, IgA, and their correlation with DP-specific IgE-BF measured in serum was evaluated. RESULTS: During DP-SCIT, the allergen-specific IgG4 in both saliva and serum increased and correlated significantly, the correlation becomes stronger over the treatment time. DP-specific IgE-BF significantly correlated with DP-specific IgG4 in serum (p < 0.0001) at different timepoints and in saliva at 12 months of SCIT (p < 0.01). No change in Der p 2-specific IgA during DP-SCIT was observed, and the IgA in serum did not correlate with IgA in saliva. There was no correlation between DP IgE-BF and Der p 2-specific IgA in serum or saliva. The control group did not exhibit significant changes in any antibody level measured. CONCLUSION: The IgE blocking activity induced by DP-SCIT treatment correlated with specific IgG4 and not IgA. The IgG4 in saliva correlates with serum IgG4 and can be an alternative immunological marker beyond 1 year of SCIT treatment.

Functional Homology for Antibody-Dependent Phagocytosis Across Humans and Rhesus Macaques.

Analyses of human clinical HIV-1 vaccine trials and preclinical vaccine studies performed in rhesus macaque (RM) models have identified associations between non-neutralizing Fc Receptor (FcR)-dependent antibody effector functions and reduced risk of infection. Specifically, antibody-dependent phagocytosis (ADP) has emerged as a common correlate of reduced infection risk in multiple RM studies and the human HVTN505 trial. This recurrent finding suggests that antibody responses with the capability to mediate ADP are most likely a desirable component of vaccine responses aimed at protecting against HIV-1 acquisition. As use of RM models is essential for development of the next generation of candidate HIV-1 vaccines, there is a need to determine how effectively ADP activity observed in RMs translates to activity in humans. In this study we compared ADP activity of human and RM monocytes and polymorphonuclear leukocytes (PMN) to bridge this gap in knowledge. We observed considerable variability in the magnitude of monocyte and PMN ADP activity across individual humans and RM that was not dependent on FcR alleles, and only modestly impacted by cell-surface levels of FcRs. Importantly, we found that for both human and RM phagocytes, ADP activity of antibodies targeting the CD4 binding site was greatest when mediated by human IgG3, followed by RM and human IgG1. These results demonstrate that there is functional homology between antibody and FcRs from these two species for ADP. We also used novel RM IgG1 monoclonal antibodies engineered with elongated hinge regions to show that hinge elongation augments RM ADP activity. The RM IgGs with engineered hinge regions can achieve ADP activity comparable to that observed with human IgG3. These novel modified antibodies will have utility in passive immunization studies aimed at defining the role of IgG3 and ADP in protection from virus challenge or control of disease in RM models. Our results contribute to a better translation of human and macaque antibody and FcR biology, and may help to improve testing accuracy and evaluations of future active and passive prevention strategies.

Comparative Analysis of Antigen-Specific Anti-SARS-CoV-2 Antibody Isotypes in COVID-19 Patients.

Serological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Abs in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect Abs against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various Ag-specific Ab isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via PCR test. We developed IgG, IgM, and IgA measurement assays for each Ag, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full-length S protein, S trimer, and nucleocapsid (N) domain, based on ELISA. The assays of the S protein for all isotypes showed high specificity, whereas the assays for all isotypes against N protein showed lower specificity. The sensitivity of all Ag-specific Ab isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 d postsymptom onset. The best correlation with virus-neutralizing activity was found for IgG against RBD, and levels of IgG against RBD in sera from four patients with severe COVID-19 increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa.

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Impact of Immunoglobulin Isotype and Epitope on the Functional Properties of Vibrio cholerae O-Specific Polysaccharide-Specific Monoclonal Antibodies.

Vibrio cholerae causes the severe diarrheal disease cholera. Clinical disease and current oral cholera vaccines generate antibody responses associated with protection. Immunity is thought to be largely mediated by lipopolysaccharide (LPS)-specific antibodies, primarily targeting the O-antigen. However, the properties and protective mechanism of functionally relevant antibodies have not been well defined. We previously reported on the early B cell response to cholera in a cohort of Bangladeshi patients, from which we characterized a panel of human monoclonal antibodies (MAbs) isolated from acutely induced plasmablasts. All antibodies in that previous study were expressed in an IgG1 backbone irrespective of their original isotype. To clearly determine the impact of affinity, immunoglobulin isotype and subclass on the functional properties of these MAbs, we re-engineered a subset of low- and high-affinity antibodies in different isotype and subclass immunoglobulin backbones and characterized the impact of these changes on binding, vibriocidal, agglutination, and motility inhibition activity. While the high-affinity antibodies bound similarly to O-antigen, irrespective of isotype, the low-affinity antibodies displayed significant avidity differences. Interestingly, despite exhibiting lower binding properties, variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies, suggesting that how the MAb binds to the O-antigen may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.IMPORTANCE Immunity to the severe diarrheal disease cholera is largely mediated by lipopolysaccharide (LPS)-specific antibodies. However, the properties and protective mechanisms of functionally relevant antibodies have not been well defined. Here, we have engineered low and high-affinity LPS-specific antibodies in different immunoglobulin backbones in order to assess the impact of affinity, immunoglobulin isotype, and subclass on binding, vibriocidal, agglutination, and motility inhibition functional properties. Importantly, we found that affinity did not directly dictate functional potency since variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies. This suggests that how the antibody binds sterically may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.

Determinants of Serum Immunoglobulin Levels: A Systematic Review and Meta-Analysis.

Background: An up-to-date overview of determinants of serum immunoglobulins in adults is pivotal for clinical practice and research, but currently lacking. We therefore performed a systematic review and meta-analysis to identify determinants of serum immunoglobulin levels. Methods: Embase, Web of Science, Medline, Cochrane, and Google Scholar were searched from inception to July 11th, 2019 for articles reporting on determinants of serum immunoglobulin A, G or M (IgA, IgG or IgM) in adult humans. Random and fixed effect models were applied to obtain pooled mean differences (MDs) and 95% confidence intervals (CIs) for the association of age and sex with serum immunoglobulins. Results: We retrieved 117 articles reporting on determinants of serum immunoglobulins, of which 28 could be meta-analyzed. Older compared to younger individuals had higher IgA (MD: 0.38; CI: 0.18 - 0.58), but lower IgM levels (MD: -0.40; 95%: -0.66 - -0.14). Men had higher IgA (MD: 0.22; CI: 0.03 - 0.42), but lower IgM levels (MD: -0.21; CI: -0.32 - -0.10) than women. Age and sex did not influence IgG. Caucasian ethnicity was associated with lower IgA, IgG, and IgM. Smoking and corticosteroid use were associated with lower IgG. Positive associations were reported of probiotics with IgG, alcohol with IgA, hypertension with IgA and IgG, and acute psychological stress with IgA, IgG, and IgM. Conclusions: Older age and male sex are associated with higher IgA, but lower IgM, and urge investigation of age- and sex-specific reference ranges of immunoglobulins. Other identified determinants were ethnicity, diet, lifestyle and cardio-metabolic factors.

Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects.

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.