Antigen-specific Plasmablasts Research Articles

Increasing human monoclonal antibody cloning efficiency with a whole-cell modified immunoglobulin-capture assay (mICA).

Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, Streptococcus pneumoniae and Neisseria meningitidis. Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods - representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned via mICA versus ~53% cloned via the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilization of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approaches such as reverse vaccinology 2.0 (RV 2.0) for bacterial vaccine antigen discovery.

Capturing the Functional Antibody Repertoire from Multiple B-cell Subsets Following Antigen Stimulation.

Abstract Antigen stimulation of mature B-cells induces activated B-cells followed by differentiation to either memory B-cells or plasmablasts. Each subset has a unique phenotype and potentially unique immunoglobulin repertoire that can be mined for therapeutic antibody candidates. As new targets for therapeutic antibodies have become more challenging, primary B-cell technologies have arisen to meet this challenge, but, their use has typically been limited to one of the B-cell subsets. We sought to develop a platform that could be used generally to interrogate all of the B-cell subsets (i.e., activated, memory and plasmablasts) that are potential sources of useful antibodies. Using fluorescence activated cell sorting (FACS), we have identified and sorted antigen-specific activated and memory B-cells by staining for CD19, surface IgG, and fluorochrome-tagged antigen. Antigen-specific plasmablasts have been identified and sorted after encapsulation of cells in microdroplets, capture of secreted IgG, CD19/CD138 staining, and fluorochrome-tagged antigen staining. Additionally, hybridomas have been evaluated using either protocol. The sorted cells have been sequenced by next generation sequencing (NGS) of paired heavy and light chains. With this platform ~10,000–20,000 antigen specific B-cells from a population of a few million viable lymphocytes have been sorted in 1–2 hours. From the sorted population, 100 – 700 unique paired clonotypes have been identified using this platform. The platform enables the capture of immunoglobulin repertoires from multiple B-cell subsets that is comprehensive, flexible, and cost-effective.

Divergent age-related humoral correlates of protection against respiratory syncytial virus infection in older and young adults: a pilot, controlled, human infection challenge model

Respiratory viral infections are typically more severe in older adults. Older adults are more vulnerable to infection and do not respond effectively to vaccines due to a combination of immunosenescence, so-called inflamm-ageing, and accumulation of comorbidities. Although age-related changes in immune responses have been described, the causes of this enhanced respiratory disease in older adults remain poorly understood. We therefore performed volunteer challenge with respiratory syncytial virus (RSV) in groups of younger and older adult volunteers. The aim of this study was to establish the safety and tolerability of this model and define age-related clinical, virological, and immunological outcomes. In this human infection challenge pilot study, adults aged 18-55 years and 60-75 years were assessed for enrolment using protocol-defined inclusion and exclusion criteria. Symptoms were documented by self-completed diaries and viral load determined by quantitative PCR of nasal lavage. Peripheral blood B cell frequencies were measured by enzyme-linked immunospot and antibodies against pre-fusion and post-fusion, NP, and G proteins in the blood and upper respiratory tract were measured. The study was registered with ClinicalTrials.gov, NCT03728413. 381 adults aged 60-75 years (older cohort) and 19 adults aged 18-55 years (young cohort) were assessed for enrolment using protocol-defined inclusion and exclusion criteria between Nov 12, 2018, and Feb 26, 2020. 12 healthy volunteers aged 60-75 years and 21 aged 18-55 years were inoculated intranasally with RSV Memphis-37. Nine (67%) of the 12 older volunteers became infected, developing mild-to-moderate upper respiratory tract symptoms that resolved without serious adverse events or sequelae. Viral load peaked on day 6 post-inoculation and symptoms peaked between days 6 and 8. Increases in circulating IgG-positive and IgA-positive antigen-specific plasmablasts, serum neutralising antibodies, and pre-F specific IgG were similar younger and older adults. However, in contrast to young participants, secretory IgA titres in older volunteers failed to increase during infection and, unlike serum IgG, did not correlate with protection. Better understanding of age-related differences in clinical outcomes and immune correlates of protection can overcome reduction in vaccine efficacy with advancing age. We identify correlates of protection in older adults, revealing previously unrecognised factors which might have implications for targeted vaccine discovery and drug development in this vulnerable group. Medical Research Council and GlaxoSmithKline EMINENT Consortium.

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.

Combining RNA in situ hybridization and spectral flow cytometry to investigate the leukocyte glycocalyx in autoimmunity

Abstract The glycocalyx surrounding leukocytes has been studied for its role in immunomodulation and its potential as a therapeutic target. 4-methylumbelliferone (4MU) is a small molecule that inhibits production of the glycocalyx’s primary polysaccharide component hyaluronan (HA), by targeting the HA synthase (HAS). Oral treatment with 4MU has proved efficacious in preventing the development and progression of autoimmunity, though the mechanism is contested. We have previously shown that 4MU strongly inhibits T-cell mediated autoimmunity, and report here that it also attenuates formation of antigen-specific plasmablasts and subsequent autoantibody production. This suggests that the HA glycocalyx plays a role in promoting the humoral component of autoimmunity. The HAS proteins are deeply membrane embedded, preventing the use of traditional flow cytometry antibodies to define expression levels across leukocyte compartments. This limitation has made it difficult to identify cellular targets of 4MU. Alternative measurements must be used to study the HA glycocalyx. Surface HA content of a cell can be measured with labelled HA binding protein (HABP), though this does not explain whether HA was synthesized or scavenged with HA-binding receptors. RNA measurement can also be used to look at transcript levels of the HAS proteins. Here, we combine RNA in situ hybridization, fluorescently labeled HA binding protein (HABP), and conventional flow cytometry antibodies to define the HA glycocalyx on leukocytes during autoimmunity. Utilizing Spectral Flow Cytometry, we can assess parameters in numbers comparable to Mass Cytometry (CyTOF) to identify which cells are most likely targeted by 4MU to prevent antibody formation during autoimmunity.

Tfh cell response in influenza vaccines in humans: what is visible and what is invisible.

Elucidating the immune mechanism by which seasonal influenza vaccines induce a protective immune response is of great importance to gain insights into the design of next-generation vaccines conferring more effective and long-lasting immune protection. Recent studies have established that T follicular helper (Tfh) cells play a major role for the generation of antibody response following influenza vaccination. Yet, the evidence is gained largely through the analysis of blood samples, and our knowledge on the role of Tfh cells in influenza vaccination is still largely limited to the generation of antigen-specific plasmablasts. Recently, influenza vaccination was shown to induce the expansion of two types of memory B cells in addition to plasmablasts. It is plausible that activated Tfh cells that remain in the lymph nodes after vaccination, a cell population missed in the analysis of blood samples, might also contribute to the diversification of memory B cell repertoire. However, current evidence shows no increase of somatic hypermutation of the expanded memory B cell clones, suggesting that this mechanism is not efficiently active in current influenza vaccines.

Specific and cross-reactive immune response to oral Salmonella Typhi Ty21a and parenteral Vi capsular polysaccharide typhoid vaccines administered concomitantly

BackgroundSince protective efficacy of the current typhoid vaccines—oral whole-cell Salmonella Typhi Ty21a and parenteral Vi-capsular polysaccharide preparation—is not optimal, and no vaccines are available against paratyphoid or non-typhoidal Salmonella (NTS) serotypes, new approaches deserve to be explored. The immunological mechanisms elicited by the two typhoid vaccines are mainly targeted against different structures. We studied whether these vaccines would enhance S. Typhi-specific immune response and cross-reactivity against other Salmonellae, if administered concomitantly. Materials and methodsVolunteers were immunized simultaneously with Ty21a and Vi vaccines (Ty21a+Vi group) or with either of the two singly (Ty21a and Vi groups). All volunteers were investigated for circulating specific and cross-reactive plasmablasts, identified by ELISPOT as IgA, IgG or IgM antibody-secreting cells (ASC) reactive with S. Typhi, S. Paratyphi A/B/C, or selected NTS serotypes (S. Enteritidis, S. Typhimurium). ResultsIn the Ty21a+Vi group, no specific or cross-reactive plasmablasts were detected before vaccination. After vaccination, the number of S. Typhi-specific plasmablasts (878 ASC/106 PBMC, 95%CI 554–1201) proved higher than in the Ty21a (339 ASC/106 PBMC; p<0.001) and Vi (149 ASC/106 PBMC; p<0.001) groups. Likewise, cross-reactive responses in the Ty21a+Vi group were higher than in the Ty21a and Vi groups (Ty21a+Vi vs Ty21a: ASC against S. Paratyphi A/B, S. Enteritidis and S. Typhimurium p<0.05, against S. Paratyphi C p<0.01; Ty21a+Vi vs Vi: against S. Paratyphi C not significant, others p<0.0001). A gut-directed homing profile was seen among O antigen-specific and a systemic one among Vi antigen-specific plasmablasts. ConclusionsConcomitant administration of Ty21a and Vi vaccines is well tolerated and induces an additive immune response to the two vaccines. Thus it enhances the magnitude of both typhoid-specific plasmablast responses and those cross-reacting with paratyphoid and most important NTS serotypes. The data encourage concomitant use of Ty21 and Vi vaccines for those at risk.

Ongoing activation of autoantigen-specific B cells in primary biliary cirrhosis.

The serologic hallmark of primary biliary cirrhosis (PBC), the antimitochondrial response to the E2 component of the pyruvate dehydrogenase complex (PDC-E2), has unique features, including continuous high titers of immunoglobulin M (IgM) and IgG reactivity throughout all stages of disease, capable not only of target enzyme inhibition, but also crossreactive with chemical xenobiotics that share molecular homology with the inner lipoyl domain of PDC-E2; such chemicals have been proposed as potential etiological agents. We used flow cytometry and enzyme-linked immunospot assay (ELISPOT) to examine B-cell subsets in 59 subjects, including 28 with PBC, 13 with primary sclerosing cholangitis (PSC), and 18 healthy controls. Strikingly, in PBC, although there were no significant differences in B-cell phenotype subpopulations, 10% of the total IgG and IgA plasmablast population and 23% of the IgM plasmablast population were uniquely reactive with PDC-E2, detected in the CXCR7+ CCR10low plasmablast population. In contrast, plasmablast reactivity to a control antigen, tetanus toxoid, was minimal and similar in all groups. Additionally, we isolated plasmablast-derived polyclonal antibodies and compared reactivity with plasma-derived antibodies and noted a distinct noncirculating tissue source of xenobiotic crossreacting antibodies. The high levels of autoantigen specific peripheral plasmablasts indicate recent activation of naive or memory B cells and a continuous and robust activation. The presence of CXCR7+ CCR10low PDC-E2-specific ASCs suggests a mechanistic basis for the migration of circulating antigen specific plasmablasts to the mucosal epithelial ligands CXCL12 and CCL28. Our findings suggest a sustained rigorous B-cell response in PBC, likely activated and perpetuated by cognate autoantigen.

In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells.

The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/beige mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.

An In Vivo Human-Plasmablast Enrichment Technique Allows Rapid Identification of Therapeutic Influenza A Antibodies

Recent advances enabling the cloning of human immunoglobulin G genes have proven effective fordiscovering monoclonal antibodies with therapeutic potential. However, these antibody-discovery methods are often arduous and identify only a few candidates from numerous antibody-secreting plasma cells or plasmablasts. We describe an invivo enrichment technique that identifies broadly neutralizing human antibodies with high frequency. For this technique, human peripheral blood mononuclear cells from vaccinated donors are activated and enriched in an antigen-specific manner for the production of numerous antigen-specific plasmablasts. Using this technology, we identified four broadly neutralizing influenza A antibodies by screening only 840 human antibodies. Two of these antibodies neutralize every influenza A human isolate tested and perform better than the current anti-influenza A therapeutic, oseltamivir, in treating severe influenza infection in mice and ferrets. Furthermore, these antibodies elicit robust invivo synergism when combined with oseltamivir, thus highlighting treatment strategies that could benefit influenza-infected patients.

Pathogen-Specific Circulating Plasmablasts in Patients with Pneumonia

Lower respiratory tract infections (LRTI) are the leading cause of death world-wide, with Streptococcus pneumoniae (Pnc) as the most prevalent pathogen. Local immune mechanisms appear central to protection against the disease, yet they are poorly characterized. Infections at other, non-respiratory mucosal sites are associated with a transient circulation of mucosa-originating lymphocytes from the mucosal site to blood and back to the mucosa. The present study explored whether pathogen-specific plasmablasts appear in the circulation also in patients with infection of the lower respiratory tract. 16 patients with bacteremic Pnc pneumonia and 14 healthy volunteers were explored for circulating plasmablasts secreting antibodies against their own pathogenic Pnc strain isolated in blood cultures (patients) or against several pathogenic strains from pneumonia patients (14 controls) or a mixture of nine different purified pneumococcal polysaccharides (8 controls). Both patients and volunteers were studied for all plasmablasts. The cells were identified with ELISPOT as Pnc-specific antibody-secreting cells (ASC) and as all immunoglobulin-secreting cells (ISC). High numbers of circulating Pnc-specific ASC were found in the acute phase of the disease in all patients with pneumonia (median 97 ASC/106 PBMC), but in none of the controls. IgG isotype predominated in 9/16 patients. The numbers of ISC were significantly higher in the patients than in the healthy controls, yet Pnc-specific ASC only accounted for 0.7% of all the patients' ISC.The present study is the first to show that antigen-specific plasmablasts appear in the circulation in pneumonia, suggesting that pulmonary lypmhocytes recirculate in humans. Assessing these cells provides a novel tool for studying immune response to antigens encountered at the LRT.

Persistence of Diarrheal Pathogens Is Associated with Continued Recruitment of Plasmablasts in the Circulation

Intestinal antigen encounter leads to recirculation of antigen-specific plasmablasts via lymphatics and blood back to the intestine. Investigating these gut-originating cells in blood provides a less invasive tool for studying intestinal immune responses, with the limitation that the cells disappear from the circulation in two weeks. No data exist on situations where pathogens persist in the intestine. Patients with Salmonella, Yersinia, or Campylobacter gastroenteritis and volunteers receiving an oral typhoid vaccine were assayed for plasmablasts specific to each subject's own pathogen/antigen weekly until the response faded. In vaccinees, plasmablasts disappeared in two weeks. In gastroenteritis, the response faded 2-3 and 3–7 weeks after the last positive Salmonella or Yersinia stool culture. Even in symptomless patients, pathogens persisting in the intestine keep seeding plasmablasts into the circulation. Assaying these cells might offer a powerful tool for research into diseases in which persisting microbes have a potential pathogenetic significance.

Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response

AbstractMaintenance of protective humoral immunity depends on the generation and survival of antibody-secreting cells. The bone marrow provides niches for long-term survival of plasma cells generated in the course of systemic immune responses in secondary lymphoid organs. Here, we have analyzed migratory human plasma blasts and plasma cells after secondary vaccination with tetanus toxin. On days 6 and 7 after immunization, CD19+/CD27high/intracellular immunoglobulin Ghigh (IgGhigh)/HLA-DRhigh/CD38high/CD20–/CD95+ tetanus toxin–specific antibody-secreting plasma blasts were released in large numbers from the secondary lymphoid organs into the blood. These cells show chemotactic responsiveness toward ligands for CXCR3 and CXCR4, probably guiding them to the bone marrow or inflamed tissue. At the same time, a population of CD19+/CD27high/intracellular IgGhigh/HLA-DRlow/CD38+/CD20–/CD95+ cells appeared in the blood in large numbers. These cells, with the phenotype of long-lived plasma cells, secreted antibodies of unknown specificity, not tetanus toxoid. The appearance of these plasma cells in the blood indicates successful competition for survival niches in the bone marrow between newly generated plasma blasts and resident plasma cells as a fundamental mechanism for the establishment of humoral memory and its plasticity.