Plasmablasts In Peripheral Blood Research Articles

#1339 The characteristic of memory B-cell subsets in patients with IgA-nephropathy

Abstract Background and Aims Impaired B-cell development has been postulated to play an important role in the maintaining of autoimmune inflammation what makes memory B-cells a perspective target for a new therapeutic strategy in IgA-nephropathy (IgAN). However, to date there are few and controversial data about the precise role of circulating memory B-cell subsets in clinical progression of IgAN. The aim was to estimate the immunological profile of memory B-cell subpopulations in the peripheral blood of IgAN patients. Method The peripheral blood was obtained from 20 IgAN patients (aged of 32.0 (27.0-36.0) y.o., male/female ratio as 13/7) and 15 donors (aged of 38.0 (30.0-46.0) y.o., male/female ratio as 10/5). The diagnosis was confirmed by morphological examination of the biopsy material. The phenotype of B-cells subsets was determined by a panel of CD38-PE/CD19-ECD/CD27-PC5/IgD-APC antibodies; γδT-lymphocytes—by a panel of TCRγδ-FITC/TCRαβ-PE/HLA-DR-ECD/TCRVδ1-PC7/CD3-APC-A750/TCRVδ2-PB/ CD45-KRO antibodies using flow cytometry method (Cytoflex, Beckman Coulter, USA). The concentration of B-cell activating factor (BAFF) was measured using enzyme-linked immunosorbent assay. Statistical data processing was done in Statistica 10.0 program. Results Based on cell surface markers expression the following B-cells subsets were identified in the peripheral blood: naïve B-cells (CD19+CD27−IgD+), pre-switched B-cells (CD19+CD27+IgD+), switched B-cells (CD19+CD27+IgD−), plasmablasts (CD19+CD27+CD38+) and long-lived plasma cells (CD27hiCD38hi) (Fig. 1). The altered balance in B-cells maturation characterizing by a significant lower percent of pre-switched B-cells and plasmablasts (p < 0.05) as well as a higher trend (>55%) in the naive B-cells (p < 0.01) was established in IgAN patients as compared to healthy donors reflecting the exhaustion of peripheral blood plasmablasts due to constant generation of antibody-producing plasma cells. Moreover, the lower pre-switched B-cells percentage was detected in the blood, the more BAFF production was observed in serum of IgAN patients (R = 0.50; p < 0.05) and the higher proteinuria level was detected (R = 0.47; p < 0.05). The increased total number of γδТ-cells up to 9.42 (6.25-12.44)% in IgAN patients as compared to donors (p = 0.03) was correlated with total memory CD19+CD27+B-cells numbers (R = 0.54; p < 0.05) as well as pre-switched B-cells (R = 0.45; p < 0.05), plasmablasts (R = 0.57; p < 0.01) and long-lived plasma cells (R = 0.51; p < 0.05). A detailed subsets analysis revealed an elevated level of tissue-resident mucosa-associated cells (Vδ1+ and Vδ3+T-lymphocytes) as well as enhanced HLA-DR expression on them (p < 0.05) in combination with decreased normally circulating Vδ2+T-cells numbers (p < 0.01) as compared to healthy donors what characterized Vδ1+ and Vδ3+T-cells subsets as professional antigen presenting cells and indicated their possible role in the priming of antigen specific B-cells. Conclusion Suggesting that most peripheral B-lymphocytes are naïve, the obtained data may reflect a γδT-cells role in migration of pre-switched B-cells from the circulation to mucosa sites for possible further maturation and class-switching what allows us to consider these two parameters as potential useful biomarkers of IgAN disease prognosis in clinical practice.

Highly Cross-Reactive and Protective Influenza A Virus H3N2 Hemagglutinin- and Neuraminidase-Specific Human Monoclonal Antibodies.

Due to antigenic drift and shift of influenza A viruses (IAV) and the tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to new strains of seasonal IAV and is at risk from viruses with pandemic potential for which limited or no immunity may exist. The genetic drift of H3N2 IAV is specifically pronounced, resulting in two distinct clades since 2014. Here, we demonstrate that immunization with a seasonal inactivated influenza vaccine (IIV) results in increased levels of H3N2 IAV-specific serum antibodies against hemagglutinin (HA) and neuraminidase (NA). Detailed analysis of the H3N2 B cell response indicated expansion of H3N2-specific peripheral blood plasmablasts 7 days after IIV immunization which expressed monoclonal antibodies (MAbs) with broad and potent antiviral activity against many H3N2 IAV strains as well as prophylactic and therapeutic activity in mice. These H3N2-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results demonstrate that IIV-induced H3N2 human MAbs can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IAV H3N2-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza A virus (IAV) infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets within the influenza virus hemagglutinin and neuraminidase proteins. We have demonstrated that seasonal immunization with inactivated influenza vaccine (IIV) stimulates H3N2-specific monoclonal antibodies in humans that are broad and potent in their neutralization of virus in vitro. These antibodies also provide protection from H3N2 IAV in a mouse model of infection. Furthermore, they persist in the bone marrow, where they are expressed by long-lived antibody-producing plasma cells. This significantly demonstrates that seasonal IIV can induce a subset of H3N2-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

The Use of Novel Antibodies to Characterize B cells in Health and Disease

IntroductionCD38, a glycoprotein ectoenzyme found on the surface of majority of hematopoietic cells, can exist in monomeric or multimeric conformations and enzymatic activity correlates with multimerization. During human B cell differentiation, CD38 expression levels are low on naïve and memory B cells, intermediate on transitional cells, and high on antibody-secreting plasmablasts and plasma cells. Conventional anti-CD38 (C-CD38) antibodies, recognize all CD38 formations and are not selective for identification of plasmablasts, thus requiring the use of additional antibodies including CD27. In contrast, a novel sea lamprey derived monoclonal VLRB antibody, tetVLRB MM3 (V-CD38), recognizes an epitope likely located within the catalytic site of active multimeric CD38 and therefore might be more specific to plasmablasts, which express active CD38. ObjectivesCharacterize the B cells recognized by V-CD38 in the peripheral blood of individuals with or without inborn errors of immunity (IEI). MethodsB cell subsets were characterized as described in figure 1 using conventional antibodies to CD3, CD19, IgM, CD27, C-CD38, and by V-CD38 generated by a collaborator. Results were presented as means with standard deviations (SD). Correlations were calculated using the Pearson’s Correlation Coefficient formula. ResultsTo validate our identification of plasmablasts with V-CD38, samples from patients with X-linked agammaglobulinemia lacking B cells, and tonsils rich in plasmablasts were used as negative and positive controls, respectively. Analysis of samples from 16 individuals revealed nearly identical (r = 0.99, p <.00001) proportions of CD27hi plasmablasts recognized by V-CD38 (0.51%+/-0.74%) and C-CD38 (0.49%+/-0.70%), suggesting that all cells expressed multimeric CD38. In contrast, only 45.28% ±16.19% of transitional IgMhighCD27-C-CD38int B cells were also recognized by V-CD38, (2.52%+/–3.62% and 1.15%+/–1.75%, respectively), a finding that was consistent across samples (r = 0.98, p <.00001). Preliminary studies in three IEI patients demonstrated markedly reduced plasmablasts (0.035%+/–0.026%), class-switched memory B cells (1.25% +/–1.52%) and transitional cells (1.25%+/–1.06%) compared to healthy controls (0.59%+/–0.74%, 15.29%+/–8.95%, and 2.81%+/–3.96% respectively). [Display omitted] ConclusionsWhile C-CD38 and V-CD38 antibodies can identify plasmablasts in peripheral blood, V-CD38 allows more selective detection of CD38 on plasmablasts. Some circulating transitional cells also express multimeric CD38. Additional studies will determine whether V-CD38 can better identify patients suffering from abnormal plasmablast formation and impaired production of antibodies.

Analysis of Plasmablasts From Children With Kawasaki Disease Reveals Evidence of a Convergent Antibody Response to a Specific Protein Epitope.

Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. Coronavirus disease 2019 (COVID-19) mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD. We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides. We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common complementarity-determining region 3 (CDR3) motif. These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.

IgE+ plasmablasts predict the onset of clinical allergy.

IgE+ plasmablasts develop following allergen exposure and B cell activation. They secrete IgE and therefore are directly linked to maintain the mechanisms of IgE-mediated allergies. Here, we show that the presence of IgE+ plasmablasts in peripheral blood not only coincides with clinical allergy, but also predicts the upcoming development of clinical disease. Using an equine model of naturally occurring allergy, we compared the timing of allergen exposure, arrival of IgE+ plasmablasts in peripheral blood, and onset of clinical disease. We found that IgE+ plasmablasts predict the development of clinical allergy by at least 3 weeks and can be measured directly by flow cytometry or by IgE secretion following in vitro culture. We also compared the IgE secretion by IgE+ plasmablasts with total plasma IgE concentrations and found that while IgE secretion consistently correlates with clinical allergy, total plasma IgE does not. Together, we describe IgE+ plasmablasts as a reliable and sensitive predictive biomarker of allergic disease development.

Peripheral CD23hi/IgE+ Plasmablasts Secrete IgE and Correlate with Allergic Disease Severity.

Production and secretion of IgE by B cells, plasmablasts, and plasma cells is a central step in the development and maintenance of allergic diseases. IgE can bind to one of its receptors, the low-affinity IgE receptor CD23, which is expressed on activated B cells. As a result, most B cells bind IgE through CD23 on their surface. This makes the identification of IgE producing cells challenging. In this study, we report an approach to clearly identify live IgE+ plasmablasts in peripheral blood for application by both flow cytometry analysis and in vitro assay. These IgE+ plasmablasts readily secrete IgE, upregulate specific mRNA transcripts (BLIMP-1 IRF4, XBP1, CD138, and TACI), and exhibit highly differentiated morphology all consistent with plasmablast differentiation. Most notably, we compared the presence of IgE+ plasmablasts in peripheral blood of allergic and healthy individuals using a horse model of naturally occurring seasonal allergy, Culicoides hypersensitivity. The model allows the comparison of immune cells both during periods of clinical allergy and when in remission and clinically healthy. Allergic horses had significantly higher percentages of IgE+ plasmablasts and IgE secretion while experiencing clinical allergy compared with healthy horses. Allergy severity and IgE secretion were both positively correlated to the frequency of IgE+ plasmablasts in peripheral blood. These results provide strong evidence for the identification and quantification of peripheral IgE-secreting plasmablasts and provide a missing cellular link in the mechanism of IgE secretion and upregulation during allergy.

Peripheral IgE+ plasmablasts as a biomarker of clinical allergy onset and severity

Abstract IgE-mediated allergies depend on the continuous production of allergen-specific IgE to maintain allergic sensitization. While IgE+ plasma cells are the primary source of this IgE, IgE+ plasmablasts can provide a snapshot in peripheral blood of the current response to allergen. We developed a novel approach to characterize IgE+ plasmablasts in peripheral blood and determined the relationship between allergen exposure, IgE+ plasmablasts and clinical disease. We used a unique large animal model of naturally occurring allergy, equine Culicoides hypersensitivity, which allowed all individuals to have similar timing and frequency of allergen exposure and allowed longitudinal collection of samples from the same allergic and healthy individuals over time. We found that peripheral IgE+ plasmablasts spontaneously secrete IgE, express high levels of the IgE receptor CD23 on their surface, and positively correlate with clinical disease severity. In conclusion, IgE+ plasmablasts are critical in the mechanism of allergy through IgE secretion and serve as a quantitative biomarker of allergic disease development and severity. This work was supported by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University; the USDA/NIFA [#2005-01812, #2015-67015-23072, and #2019-67015-29833]; and National Institutes of Health [#1S10RR025502 through Cornell University’s Biotechnology Resource Center].

Increased Frequencies of Switched Memory B Cells and Plasmablasts in Peripheral Blood from Patients with ANCA-Associated Vasculitis.

B cells are thought to play a central role in the pathogenesis of antineutrophil cytoplasmic antibody- (ANCA-) associated vasculitis (AAV). ANCAs have been proposed to cause vasculitis by activating primed neutrophils to damage small blood vessels. We studied a cohort of AAV patients of which a majority were in remission and diagnosed with granulomatosis with polyangiitis (GPA). Using flow cytometry, the frequencies of CD19+ B cells and subsets in peripheral blood from 106 patients with AAV and 134 healthy controls were assessed. B cells were divided into naive, preswitch memory, switched memory, and exhausted memory cells. Naive and switched memory cells were further subdivided into transitional cells and plasmablasts, respectively. In addition, serum concentrations of immunoglobulin A, G, and M were measured and clinical data were retrieved. AAV patients displayed, in relation to healthy controls, a decreased frequency of B cells of lymphocytes (5.1% vs. 8.3%) and total B cell number. For the subsets, a decrease in percentage of transitional B cells (0.7% vs. 4.4%) and expansions of switched memory B cells (22.3% vs. 16.5%) and plasmablasts (0.9% vs. 0.3%) were seen. A higher proportion of B cells was activated (CD95+) in patients (20.6% vs. 10.3%), and immunoglobulin levels were largely unaltered. No differences in B cell frequencies between patients in active disease and remission were observed. Patients in remission with a tendency to relapse had, compared to nonrelapsing patients, decreased frequencies of B cells (3.5% vs. 6.5%) and transitional B cells (0.1% vs. 1.1%) and an increased frequency of activated exhausted memory B cells (30.8% vs. 22.3%). AAV patients exhibit specific changes in frequencies of CD19+ B cells and their subsets in peripheral blood. These alterations could contribute to the autoantibody-driven inflammatory process in AAV.

Evidence of potent humoral immune activity in COVID-19-infected kidney transplant recipients.

Whether kidney transplant recipients are capable of mounting an effective anti‐severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) adaptive immune response despite chronic immunosuppression is unknown and has important implications for therapy. Herein, we analyzed peripheral blood cell surface and intracellular cytokine phenotyping by flow cytometry along with serum antibody testing in 18 kidney transplant recipients with active coronavirus disease 2019 (COVID‐19) infection and 36 matched, transplanted controls without COVID‐19. We observed significantly fewer total lymphocytes and fewer circulating memory CD4+ and CD8+ T cells in the COVID‐19 subjects. We also showed fewer anergic and senescent CD8+ T cells in COVID‐19 individuals, but no differences in exhausted CD8+ T cells, nor in any of these CD4+ T cell subsets between groups. We also observed greater frequencies of activated B cells in the COVID‐19 patients. Sixteen of 18 COVID‐19 subjects tested for anti‐SARS‐CoV‐2 serum antibodies showed positive immunoglobulin M or immunoglobulin G titers. Additional analyses showed no significant correlation among immune phenotypes and degrees of COVID‐19 disease severity. Our findings indicate that immunosuppressed kidney transplant recipients admitted to the hospital with acute COVID‐19 infection can mount SARS‐CoV‐2‐reactive adaptive immune responses. The findings raise the possibility that empiric reductions in immunosuppressive therapy for all kidney transplant recipients with active COVID‐19 may not be required.

Anti-Vibrio cholerae responses in the human periphery and intestine following a live-attenuated vaccine

Abstract Vibrio cholerae is a prototypical mucosal pathogen that causes cholera, a severe diarrheal disease. Cholera affects an estimated 3 to 5 million people annually, resulting in over 100,000 deaths. Infection leads to serotype-specific immunity that can last for up to ten years. Immunity is likely mediated by intestinal antibodies, which primarily target the immunodominant antigens lipopolysaccharide (LPS) and cholera toxin (CT). However, how immunity is generated and maintained is not well understood. Here we have used the live-attenuated cholera vaccine, Vaxchora, to characterize the primary immune response in both the periphery and the human duodenum. Previously, this vaccine showed 80% protective efficacy 90 days after vaccination of naïve volunteers, and induced early antibody responses in levels similar to infection. We developed new technology to identify and characterize V. cholerae-specific plasmablasts and plasma cells by flow cytometry, and found that antigen-specific plasmablasts in peripheral blood quickly expand and upregulate mucosal trafficking markers CCR9 and CCR10. These B cells either develop locally or home to the small intestine, where anti-LPS and anti-CT antibody-secreting cells are present by day 28. Surprisingly, our preliminary data shows that vaccinees may exhibit robust anti-CT responses in the duodenum even when peripheral anti-CT antibody secreting cells are not detected, which provides novel insight into the generation of sustained protection. These data will help us understand not only protective immunity to V. cholerae, but also how the human mucosal plasma cell compartment is developed and maintained.

One step ultra-high-throughput sequencing of the paired antibody VH:VL and TCRβ:α repertoires using cell lysate resistant xenopolymerase in emulsion

Abstract Antibodies and T cell receptors (TCRs) have critical roles in adaptive immunity and thus the determination of their sequences is essential for understanding immune responses in infections or vaccinations, for the discovery of therapeutic antibodies, and for the engineered T cell therapies. Antibodies are composed of variable heavy (VH) and light (VL) chains, and TCRs are composed of variable TCRα/γ and TCRβ/δ chains. Since those chains are encoded by two different transcripts, the determination of functional sequences requires single-cell sequencing. Emulsification of single-cells followed by barcoding of the transcripts or fusion of VH:VL or TCRβ:α transcripts using overlap extension RT-PCR allows high-throughput single-cell sequencing; however, because cell lysate in emulsion inhibits the RT-PCR reaction, these methods require complex microfluidics devices to bypass the inhibition which most of biomedical laboratories cannot access. We discovered recently engineered xenopolymerase, RTX, has an exceptional cell lysate resistance that can overcome this limitation. By employing this unique feature of RTX, we have developed a one-step emulsion-based ultra-high-throughput sequencing method for paired antibody VH:VL and TCRβ:α repertoires not requiring microfluidics or any other specialized equipment. Using this methodology, we obtained high yields (5,000 to &amp;gt;20,000/sample) of paired VH:VL or TCRβ:α clonotypes at low cost. As a demonstration, we applied the methodology to the peripheral blood plasmablasts and T follicular helper (TFH) cells from a seasonal influenza vaccine recipient and discovered high-affinity influenza-specific antibodies and TCRβ:α.

Persistence of HIV-1 Env-Specific Plasmablast Lineages in Plasma Cells after Vaccination in Humans

SummaryInduction of persistent HIV-1 Envelope (Env) specific antibody (Ab) is a primary goal of HIV vaccine strategies; however, it is unclear whether HIV Env immunization in humans induces bone marrow plasma cells, the presumed source of long-lived systemic Ab. To define the features of Env-specific plasma cells after vaccination, samples were obtained from HVTN 105, a phase I trial testing the same gp120 protein immunogen, AIDSVAX B/E, used in RV144, along with a DNA immunogen in various prime and boost strategies. Boosting regimens that included AIDSVAX B/E induced robust peripheral blood plasmablast responses. The Env-specific immunoglobulin repertoire of the plasmablasts is dominated by VH1 gene usage and targeting of the V3 region. Numerous plasmablast-derived immunoglobulin lineages persisted in the bone marrow >8 months after immunization, including in the CD138+ long-lived plasma cell compartment. These findings identify a cellular linkage for the development of sustained Env-specific Abs following vaccination in humans.

A facile technology for the high-throughput sequencing of the paired VH:VL and TCRβ:TCRα repertoires.

Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRb and TCRa) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRb:a repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRb:a clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRb:a.

A Protein Epitope Targeted by the Antibody Response to Kawasaki Disease.

Kawasaki disease (KD) is the leading cause of childhood acquired heart disease in developed nations and can result in coronary artery aneurysms and death. Clinical and epidemiologic features implicate an infectious cause but specific antigenic targets of the disease are unknown. Peripheral blood plasmablasts are normally highly clonally diverse but the antibodies they encode are approximately 70% antigen-specific 1-2 weeks after infection. We isolated single peripheral blood plasmablasts from children with KD 1-3 weeks after onset and prepared 60 monoclonal antibodies (mAbs). We used the mAbs to identify their target antigens and assessed serologic response among KD patients and controls to specific antigen. Thirty-two mAbs from 9 of 11 patients recognize antigen within intracytoplasmic inclusion bodies in ciliated bronchial epithelial cells of fatal cases. Five of these mAbs, from 3 patients with coronary aneurysms, recognize a specific peptide, which blocks binding to inclusion bodies. Sera from 5/8 KD patients day ≥ 8 after illness onset, compared with 0/17 infant controls (P < .01), recognized the KD peptide antigen. These results identify a protein epitope targeted by the antibody response to KD and provide a means to elucidate the pathogenesis of this important worldwide pediatric problem.

Black African and Latino/a identity correlates with increased plasmablasts in MS.

ObjectiveTo determine the influence of self-reported Black African and Latin American identity on peripheral blood antibody-secreting cell (ASC) frequency in the context of relapsing-remitting MS.MethodsIn this cross-sectional study, we recruited 74 subjects with relapsing-remitting MS and 24 age-, and self-reported ethno-ancestral identity-matched healthy donors (HDs) to provide peripheral blood study samples. Subjects with MS were either off therapy at the time of study draw or on monthly natalizumab therapy infusions. Using flow cytometry, we assessed peripheral blood mononuclear cells for antibody-secreting B-cell subsets.ResultsWhen stratified by self-reported ethno-ancestry, we identified significantly elevated frequencies of circulating plasmablasts among individuals with MS identifying as Black African or Latin American relative to those of Caucasian ancestry. Ethno-ancestry–specific differences in ASC frequency were observed only among individuals with MS. By contrast, this differential was not observed among HDs. ASCs linked with poorer MS prognosis and active disease, including IgM+- and class-switched CD138+ subsets, were among those significantly increased.ConclusionThe enhanced peripheral blood plasmablast signature revealed among Black African or Latin American subjects with MS points to distinct underlying mechanisms associated with MS immunopathogenesis. This dysregulation may contribute to the disease disparity experienced by patient populations of Black African or Latin American ethno-ancestry.

1876. A Hepacivirus-Like Protein Is Targeted by the Antibody Response to Kawasaki Disease (KD)

BackgroundClinical and epidemiologic data support a viral cause of KD, but the etiology has eluded 50 years of study. We previously identified virus-like intracytoplasmic inclusion bodies (ICI) in ciliated bronchial epithelium of KD children but not infant controls, but the antigens within the ICI were unknown. At 1–2 weeks following infection, 75% of peripheral blood plasmablasts (PB) specifically target the infectious agent. We cloned the PB response to KD to identify KD-specific antibodies and their target antigens.MethodsWe isolated single PB from children with KD 1–3 weeks after fever onset by flow cytometry, and amplified immunoglobulin VDJ and VJ genes from each PB by RT-PCR. We sequenced the products and made monoclonal antibodies (Mab) from clonally expanded PB in individual patients. Mab were tested for binding to KD tissues and to a viral peptide array containing 29,939 peptides from known B cell epitopes of animal viruses (www.iedb.org).ResultsWe sequenced 1156 PB from 11 KD patients, and identified 44 clonally expanded sets of PB. We prepared 61 Mab from clonally expanded and highly mutated IgA PB, and found that 33/61 bind to KD ICI, 10 strongly and 23 weakly. Of 10 Mab that strongly bind, 2 were VH3-33 (single patient), 2 VH3-23 (single patient), 1 VH3-15, 1 VH3-74, 3 VH1-46 (2 patients), and 1 VH4-59. These Mab CDR3s varied from 11 to 20 aacids, with 4–28 aacid mutations. Mab KD4-2H4 recognized multiple similar peptides from nonstructural protein 4A of hepacivirus C; pt KD4 sera was negative for hepatitis C by fourth-generation ELISA. Amino acid substitution analysis yielded an optimized peptide, and 6 KD Mab recognized this peptide by ELISA. These 6 Mab derived from 3 KD patients, all of whom had coronary aneurysms, and were VH3-74 (n = 1), VH3-33 (n = 2, single patient), VH1-45 (n = 1), and VH3-72 (n = 2, single patient). Strong binding of KD Mab KD4-2H4 and KD6-2B2 to ICI was totally blocked by pre-incubation with optimized peptide. KD but not control sera react with optimized peptide expressed as a glutathione S-transferase fusion protein by western blot.ConclusionChildren with KD make antibodies to a hepacivirus-like protein, and KD ICI contain this protein. These results strongly suggest that a previously unidentified hepacivirus with a respiratory portal of entry is etiologically related to KD.Disclosures All Authors: No reported Disclosures.

Immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase 1 clinical trial

SummaryBackgroundInfluenza viruses cause substantial annual morbidity and mortality globally. Current vaccines protect against influenza only when well matched to the circulating strains. However, antigenic drift can cause considerable mismatches between vaccine and circulating strains, substantially reducing vaccine effectiveness. Moreover, current seasonal vaccines are ineffective against pandemic influenza, and production of a vaccine matched to a newly emerging virus strain takes months. Therefore, there is an unmet medical need for a broadly protective influenza virus vaccine. We aimed to test the ability of chimeric H1 haemagglutinin-based universal influenza virus vaccine candidates to induce broadly cross-reactive antibodies targeting the stalk domain of group 1 haemagglutinin-expressing influenza viruses.MethodsWe did a randomised, observer-blinded, phase 1 study in healthy adults in two centres in the USA. Participants were randomly assigned to one of three prime–boost, chimeric haemagglutinin-based vaccine regimens or one of two placebo groups. The vaccine regimens included a chimeric H8/1, intranasal, live-attenuated vaccine on day 1 followed by a non-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine on day 85; the same regimen but with the inactivated vaccine being adjuvanted with AS03; and an AS03-adjuvanted, chimeric H8/1, intramuscular, inactivated vaccine followed by an AS03-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine. In this planned interim analysis, the primary endpoints of reactogenicity and safety were assessed by blinded study group. We also assessed anti-H1 haemagglutinin stalk, anti-H2, anti-H9, and anti-H18 IgG antibody titres and plasmablast and memory B-cell responses in peripheral blood. This trial is registered with ClinicalTrials.gov, number NCT03300050.FindingsBetween Oct 10, 2017, and Nov 27, 2017, 65 participants were enrolled and randomly assigned. The adjuvanted inactivated vaccine, but not the live-attenuated vaccine, induced a substantial serum IgG antibody response after the prime immunisation, with a seven times increase in anti-H1 stalk antibody titres on day 29. After boost immunisation, all vaccine regimens induced detectable anti-H1 stalk antibody (2·2–5·6 times induction over baseline), cross-reactive serum IgG antibody, and peripheral blood plasmablast responses. An unsolicited adverse event was reported for 29 (48%) of 61 participants. Solicited local adverse events were reported in 12 (48%) of 25 participants following prime vaccination with intramuscular study product or placebo, in 12 (33%) of 36 after prime immunisation with intranasal study product or placebo, and in 18 (32%) of 56 following booster doses of study product or placebo. Solicited systemic adverse events were reported in 14 (56%) of 25 after prime immunisation with intramuscular study product or placebo, in 22 (61%) of 36 after immunisation with intranasal study product or placebo, and in 21 (38%) of 56 after booster doses of study product or placebo. Disaggregated safety data were not available at the time of this interim analysis.InterpretationThe tested chimeric haemagglutinin-based, universal influenza virus vaccine regimens elicited cross-reactive serum IgG antibodies that targeted the conserved haemagglutinin stalk domain. This is the first proof-of-principle study to show that high anti-stalk titres can be induced by a rationally designed vaccine in humans and opens up avenues for further development of universal influenza virus vaccines. On the basis of the blinded study group, the vaccine regimens were tolerable and no safety concerns were observed.FundingBill & Melinda Gates Foundation.

An Efficient Method to Generate Monoclonal Antibodies from Human B Cells.

In the age of personalized medicine, an efficient method to generate monoclonal antibodies (mAbs) is essential for biomedical and immunotherapeutic research. Numerous aspects of basic B-cell biology can be studied at the monoclonal level, includingB-cell development, antibody responses to infection or vaccination, and autoimmune responses. Single-cell B-cell receptor cloning allows for the rapid generation of antigen-specific mAbs in a matter of several weeks. In this chapter, we provide an efficient method to generate mAbs from peripheral blood plasmablasts and memory B cells induced by infection and vaccination. Additionally, we provide a protocol on how to optimize single-cell B-cell sorting for both single-cell B-cell receptor cloning and single-cell RNA-sequencing, for the application of studying B-cell specificity and function (spec-seq). This protocol can be easily adapted for other B-cell populations, B cells in tissues, and B cells from other organisms.

The Epidemiology and Pathogenesis of Kawasaki Disease.

Epidemiologic and clinical features of Kawasaki Disease (KD) strongly support an infectious etiology. KD is worldwide, most prominently in Japan, Korea, and Taiwan, reflecting increased genetic susceptibility among Asian populations. In Hawaii, KD rates are 20-fold higher in Japanese ethnics than in Caucasians, intermediate in other ethnicities. The age distribution of KD, highest in children < 2 yo, lower in those < 6 months, is compatible with infection by a ubiquitous agent resulting in increasing immunity with age and with transplacental immunity, as with some classic viruses. The primarily winter-spring KD seasonality and well-documented Japanese epidemics with wave-like spread also support an infectious trigger. We hypothesize KD pathogenesis involves an RNA virus that usually causes asymptomatic infection but KD in a subset of genetically predisposed children. CD8 T cells, oligoclonal IgA, and upregulation of cytotoxic T cell and interferon pathway genes in the coronaries in fatal KD also support a viral etiology. Cytoplasmic inclusion bodies in ciliated bronchial epithelium identified by monoclonal antibodies made from oligoclonal IgA heavy chains also supports a viral etiology. Recent availability of “second generation” antibodies from KD peripheral blood plasmablasts may identify a specific viral antigen. Thus, we propose an unidentified (“new”) RNA virus infects bronchial epithelium usually causing asymptomatic infection but KD in a subset of genetically predisposed children. The agent persists in inclusion bodies, with intermittent respiratory shedding, entering the bloodstream via macrophages targeting coronaries. Antigen-specific IgA plasma cells and CD8 T cells respond but coronaries can be damaged. IVIG may include antibody against the agent. Post infection, 97–99% of KD patients are immune to the agent, protected against recurrence. The agent can spread either from those with asymptomatic primary infection in winter-spring or from a previously infected contact who intermittently sheds the agent.

Spec-seq unveils transcriptional subpopulations of antibody-secreting cells following influenza vaccination.

Vaccines are among the most effective public health tools for combating certain infectious diseases such as influenza. The role of the humoral immune system in vaccine-induced protection is widely appreciated; however, our understanding of how antibody specificities relate to B cell function remains limited due to the complexity of polyclonal antibody responses. To address this, we developed the Spec-seq framework, which allows for simultaneous monoclonal antibody (mAb) characterization and transcriptional profiling from the same single cell. Here, we present the first application of the Spec-seq framework, which we applied to human plasmablasts after influenza vaccination in order to characterize transcriptional differences governed by B cell receptor (BCR) isotype and vaccine reactivity. Our analysis did not find evidence of long-term transcriptional specialization between plasmablasts of different isotypes. However, we did find enhanced transcriptional similarity between clonally related B cells, as well as distinct transcriptional signatures ascribed by BCR vaccine recognition. These data suggest IgG and IgA vaccine-positive plasmablasts are largely similar, whereas IgA vaccine-negative cells appear to be transcriptionally distinct from conventional, terminally differentiated, antigen-induced peripheral blood plasmablasts.