Antibody-secreting Cell Responses Research Articles

Systemic and intestinal porcine epidemic diarrhea virus-specific antibody response and distribution of antibody-secreting cells in experimentally infected conventional pigs

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.

Comparative Analysis of SARS-CoV-2-Specific B Cell and Humoral Responses Elicited by Sputnik V in Naïve and COVID-19-Recovered Vaccine Recipients

Background: The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, several key immunological features of Sputnik V, an adenovirus-based two-component vaccine against SARS-CoV-2, need to be explored in detail. These include the effects of Sputnik V on B cell immunity and its ability to elicit antibody responses that are active against emerging neutralization-resistant SARS-CoV-2 variants. Methods: Our study included a cohort of 22 volunteers who received complete Sputnik V vaccination (two doses 21 days apart), 5 of who had a recent history of mild COVID-19, and 17 constituted a group of SARS-CoV-2 unexposed individuals. The frequencies of receptor-binding domain (RBD)-specific plasmablasts and B memory cells (MBCs), circulating and MBC-derived antibody secreting cells, virus binding, and virus-neutralizing activities of the sera, as well as samples of MBC-derived antibodies were analyzed using flow cytometry, ELISA, enzyme-linked immunosorbent spot (ELISpot), and pseudotyped virus neutralization assay at four time points spanning the period immediately before, during, and after vaccination. Findings: Longitudinal analysis of circulating serum antibodies showed that the anti-RBD IgG levels in naïve vaccine recipients substantially increased after the second vaccine dose (PP=0.0084). In recovered vaccine recipients, RBD-specific MBCs and SARS-CoV-2-specific MBC-derived antibody-secreting cells (ASCs) were already present prior to vaccination and remained stable until day 85. However, in naive vaccine recipients, RBD-specific MBCs and MBC-derived ASCs became detectable after the second dose and by day 85, they reached the levels observed in recovered vaccine recipients. In vitro stimulated MBCs from recovered individuals secreted a significant amount of anti-RBD IgG both on days 28 and 85. These antibodies demonstrated robust neutralization of the Wuhan Spike-pseudotyped lentivirus. In the naïve group, the level of anti-RBD IgG secretion was five- to six-fold reduced compared to that of the recovered group (PInterpretation: B cell and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. All the recovered and most naïve Sputnik V recipients displayed neutralizing antibody responses against the ancestral Wuhan and B.1.351 viruses. Plasmablast, RBD-specific MBCs, SARS-CoV-2-specific MBC-derived ASC responses, and humoral responses were more prominent in the recovered group of vaccinees than in the naïve subgroup, which may be indicative of their higher degree of long-term protection. Funding: This work was supported by the Russian Science Foundation (Project 21-15-00331) and the Russian Fund for Basic Research (20-04-60527).Declaration of Interest: None to declare. Ethical Approval: The study protocol was reviewed and approved by the Medical Ethical Committee of Institute of Immunology (#12-1, December 29, 2020).

Recombinant Live Attenuated Influenza Virus Expressing Conserved G-Protein Domain in a Chimeric Hemagglutinin Molecule Induces G-Specific Antibodies and Confers Protection against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is one of the most important pathogens causing significant morbidity and mortality in infants and the elderly. Live attenuated influenza vaccine (LAIV) is a licensed vaccine platform in humans and it is known to induce broader immune responses. RSV G attachment proteins mediate virus binding to the target cells and they contain a conserved central domain with neutralizing epitopes. Here, we generated recombinant LAIV based on the attenuated A/Puerto Rico/8/1934 virus backbone, expressing an RSV conserved G-domain in a chimeric hemagglutinin (HA) fusion molecule (HA-G). The attenuated phenotypes of chimeric HA-G LAIV were evident by restricted replication in the upper respiratory tract and low temperature growth characteristics. The immunization of mice with chimeric HA-G LAIV induced significant increases in G-protein specific IgG2a (T helper type 1) and IgG antibody-secreting cell responses in lung, bronchioalveolar fluid, bone marrow, and spleens after RSV challenge. Vaccine-enhanced disease that is typically caused by inactivated-RSV vaccination was not observed in chimeric HA-G LAIV as analyzed by lung histopathology. These results in this study suggest a new approach of developing an RSV vaccine candidate while using recombinant LAIV, potentially conferring protection against influenza virus and RSV.

Defining HPV-specific B cell responses in patients with head and neck cancer.

SUMMARYTumours can harbour significant numbers of B cells and plasma cells, however, little is known about the antigen specificity of intra-tumoral B cells1–8. Here we show that human papillomavirus (HPV)-specific B cell responses are detectable in HPV-positive head and neck cancers, with active production of HPV-specific IgG antibodies in situ. HPV-specific antibody secreting cells (ASCs) were readily detectable (~0.7-25% of IgG+ ASCs) in the tumour microenvironment (TME) with minimal bystander recruitment of influenza-specific cells, suggesting a localised and antigen-specific ASC response. HPV-specific ASC responses, which correlated with plasma IgG titres, were directed against the HPV proteins E2, E6, and E7, with E2-specific responses tending to be the most dominant. Using intra-tumoral B cells and plasma cells, we generated several HPV-specific human monoclonal antibodies, which exhibited a high degree of somatic hypermutation, consistent with chronic antigen exposure. scRNAseq analyses revealed the presence of activated B cells, germinal centre B cells, and ASCs within the TME. ASCs and B cells were preferentially localised in the tumour stroma, with well-formed clusters of activated B cells indicating ongoing germinal centre reactions. Overall, we show that antigen-specific activated and germinal centre B cells as well as plasma cells can be found in the TME. Our findings provide a better understanding of humoral immune responses in human cancer and suggest that tumour-infiltrating B cells could be harnessed for the development of novel therapeutic agents.

The Effects of Imprinting and Repeated Seasonal Influenza Vaccination on Adaptive Immunity after Influenza Vaccination.

(1) Background: The influenza virus continues to cause significant annual morbidity and mortality. The overall efficacy of seasonal influenza vaccination is suboptimal, which is partly due to host immune factors. The effects of imprinting and repeated seasonal influenza vaccination were investigated to assess for immune factors and mechanisms that impact influenza vaccine responses. (2) Methods: Twenty participants were enrolled into a prospective pilot study based on birth cohort and seasonal influenza immunization history. Immunologic parameters were assessed over a six-month period after the seasonal influenza vaccine was administered. (3) Results: There was no significant imprinting effect, as measured by hemagglutination inhibition (HAI) fold change, HAI geometric mean titer (GMT) for Day 29 or Day 180 post-vaccination and antigen- specific antibody-secreting cells (ASC) for Day 8 post-vaccination. Individuals who had minimal prior seasonal influenza vaccination had a higher magnitude ASC response and a higher HAI fold change post-vaccination than individuals who were repeatedly vaccinated. (4) Conclusions: Repeated seasonal influenza vaccination resulted in a decreased fold change of the immune response, although individuals in this cohort tended to have high HAI titers at baseline that persisted after vaccination. Imprinting effects were not observed in this cohort. These host immune factors should be considered in the development of universal influenza vaccines. ClinicalTrials.gov Identifier: NCT03686514.

A first in human clinical trial assessing the safety and immunogenicity of transcutaneously delivered enterotoxigenic Escherichia coli fimbrial tip adhesin with heat-labile enterotoxin with mutation R192G

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea among travelers and pediatric populations worldwide. The tip-localized adhesin of colonization factor antigen (CFA)/I fimbriae was engineered as a donor strand complemented variant (dscCfaE) and delivered via transcutaneous immunization. Preclinical vaccine testing demonstrated safety, immunogenicity and efficacy. A series of open-label dose-escalating phase 1 studies evaluated a 3-dose (days 0, 21, 42) regimen via a transcutaneous skin patch. A total of forty-six subjects were enrolled into one of four vaccine dose levels (10, 50, 250, or 1250 µg) co-administered with single-mutant heat-labile enterotoxin (LTR(192G)). At the 50 µg dose level, ten subjects received the dscCfaE vaccine without LT(R192G). The vaccine was well tolerated with mild local vaccine site reactions characterized by an erythematous papular rash and pruritus, which were less frequent and reactive in the group not receiving LT(R192G). The frequency of responses to dscCfaE were moderate, whereas anti-toxin responses (serum IgA/IgG) ranged from 75 to 100% across groups that received LT(R192G). Antigen-specific antibody-secreting cell responses were elicited at all dose levels, but were generally low. Follow-on studies will optimize construct and route of delivery and assess efficacy in an ETEC challenge study.

Efficacy of Heterologous Prime-Boost Vaccination with H3N2 Influenza Viruses in Pre-Immune Individuals: Studies in the Pig Model.

In a previous study in influenza-naïve pigs, heterologous prime-boost vaccination with monovalent, adjuvanted whole inactivated vaccines (WIV) based on the European swine influenza A virus (SwIAV) strain, A/swine/Gent/172/2008 (G08), followed by the US SwIAV strain, A/swine/Pennsylvania/A01076777/2010 (PA10), was shown to induce broadly cross-reactive hemagglutination inhibition (HI) antibodies against 12 out of 15 antigenically distinct H3N2 influenza strains. Here, we used the pig model to examine the efficacy of that particular heterologous prime-boost vaccination regimen, in individuals with pre-existing infection-immunity. Pigs were first inoculated intranasally with the human H3N2 strain, A/Nanchang/933/1995. Seven weeks later, they were vaccinated intramuscularly with G08 followed by PA10 or vice versa. We examined serum antibody responses against the hemagglutinin and neuraminidase, and antibody-secreting cell (ASC) responses in peripheral blood, draining lymph nodes, and nasal mucosa (NMC), in ELISPOT assays. Vaccination induced up to 10-fold higher HI antibody titers than in naïve pigs, with broader cross-reactivity, and protection against challenge with an antigenically distant H3N2 strain. It also boosted ASC responses in lymph nodes and NMC. Our results show that intramuscular administration of WIV can lead to enhanced antibody responses and cross-reactivity in pre-immune subjects, and recall of ASC responses in lymph nodes and NMC.

Interferon gamma signaling regulates the generation of influenza-specific memory B cells

Abstract Memory B cells(Bmem), which can rapidly differentiate into antibody secreting cells (ASC) upon secondary challenge, are key mediators of humoral immunity. However, the signals that govern the formation and function of these cells remain poorly understood. To date, most Bmem studies have been performed with alum-adjuvanted proteins that induce type 2 immune responses. This differs significantly from the type I and II interferon (IFN) response generated after viral infection. Using a murine model of influenza, we showed that Bmem cell intrinsic expression of the IFNγ-inducible transcription factor (TF) T-bet was required for Bmem secondary ASC responses following viral challenge. Here, we demonstrate that B cell-specific deletion of the IFNγR and the IFNg-induced TF STAT1 also affects the flu-specific Bmem compartment and greatly diminishes Bmem recall responses. We demonstrate that T-bet is expressed by a subset of all flu-specific Bmem, including those expressing IgG1 and IgM receptors. Therefore, we hypothesized that T-bet expression could be used to subset the Bmem cells into those that did or did not receive prior IFNg and STAT1 programming signals. Sorting Bmem from flu-infected mice, we show that Bmem cells with the highest levels of T-bet differentiate quickly and robustly following ex vivo stimulation. By contrast, those Bmem expressing lower levels of T-bet differentiate with delayed kinetics. Taken together, these data support the notion that B cell intrinsic IFNγR signaling, mediated by STAT1, drives formation of a distinct population of T-bet expressing effector memory B cells that are programmed to rapidly differentiate following re-exposure to antigen.

Single cell transcriptomics of in vivo differentiating antibody-secreting cells reveals a divergent activated B cell program dependent on IRF4

Abstract The generation of antibody-secreting cells (ASC) requires reprogramming the B cell transcriptome, epigenome, and metabolic potential to support the processes necessary to synthesize and secrete antibodies. These molecular changes have largely been studied at defined stages representing naive B cells, activated B cells, and ASC. To further refine when ASC fate decisions occur, we adoptively transferred CTV labeled B cells and monitored differentiation to both type I and II T cell independent antigens. At the peak of ASC responses, transferred cells were phenotyped by flow cytometry and isolated for single-cell RNA-seq. These data captured a continuum of responding B cells representing all stages of differentiation, with the largest heterogeneity observed among actB. Computationally ordering cells along differentiation trajectories, revealed divergent actB differentiation paths, with one branch leading to ASC formation that was dependent on IRF4. Moreover, cells that followed this trajectory downregulated the surface marker L-selectin and could be separated from cells that followed the alternative non-ASC differentiation branch. Indeed, isolation of actB by L-selectin status confirmed that L-selectin negative cells were ten times more likely to form ASC in culture. In summary these data provide insights into the cell division kinetics, molecular heterogeneity, and differentiation paths that lead to ASC formation.

Virus-like particle vaccine displaying Toxoplasma gondii apical membrane antigen 1 induces protection against T. gondii ME49 infection in mice

Toxoplasmosis is an intracellular parasitic disease caused by the protozoa Toxoplasma gondii, which affects about half of the world's population. In spite of the strenuous endeavors, a T. gondii vaccine for clinical use remains unreported to date. In the present study, we generated virus-like particles (VLPs) containing T. gondii apical membrane antigen 1 (AMA1) and assessed its efficacy in a murine model. VLPs were characterized using western blot and TEM. T. gondii-specific IgG and IgA antibody responses in sera, germinal center B cell responses in spleen, brain cyst counts and their sizes were determined. Elevated T. gondii-specific IgG and IgA antibody responses were observed from the sera of AMA1 VLP-immunized mice. Immunization with AMA1 VLPs enhanced T. gondii-specific antibody-secreting cell responses and germinal center B cell responses upon antigen stimulation. Brain tissue analysis revealed that AMA1 VLP-immunization reduced cyst formation and its size compared to control. Also, VLP-immunized mice were less susceptible to body weight loss and displayed enhanced survival rate compared to the control group. Our results demonstrated that the immune response induced by T. gondii AMA1 VLPs confer partial protection against T. gondii infection and provides important insight into potential T. gondii vaccine design strategy.

Flavivirus-Mediating B Cell Differentiation Into Antibody-Secreting Cells in Humans Is Associated With the Activation of the Tryptophan Metabolism.

Patients infected with the Dengue virus (DENV) often present with a massive generation of DENV-specific antibody-secreting cells (ASCs) in the blood. In some cases, these ASCs represent more than 50% of the circulating B cells, a higher magnitude than those induced by other infections, vaccinations, and plasma cell lymphomas. However, it remains unclear how the DENV infection elicits this colossal response. To address this issue, we utilised an in vitro strategy to induce human PBMCs of healthy individuals incubated with DENV particles (DENV4 TVP/360) to differentiate into ASCs. As controls, PBMCs were incubated with a mitogen cocktail or supernatants of uninfected C6/36 cells (mock). The ASC phenotype and function were increasingly detected in the DENV and mitogen-cultured PBMCs as compared to mock-treated cells. In contrast to the in vivo condition, secreted IgG derived from the PBMC-DENV culture was not DENV-specific. Lower ASC numbers were observed when inactivated viral particles or purified B cells were added to the cultures. The physical contact was essential between B cells and the remaining PBMCs for the DENV-mediated ASC response. Considering the evidence for the activation of the tryptophan metabolism detected in the serum of Dengue patients, we assessed its relevance in the DENV-mediated ASC differentiation. For this, tryptophan and its respective metabolites were quantified in the supernatants of cell cultures through mass spectrophotometry. Tryptophan depletion and kynurenine accumulation were found in the supernatants of PBMC-DENV cultures, which presented enhanced detection of indoleamine 2,3-dioxygenase 1 and 2 transcripts as compared to controls. In PBMC-DENV cultures, tryptophan and kynurenine levels strongly correlated to the respective ASC numbers, while the kynurenine levels were directly proportional to the secreted IgG titers. Contrastingly, PBMCs incubated with Zika or attenuated Yellow Fever viruses showed no correlation between their kynurenine concentrations and ASC numbers. Therefore, our data revealed the existence of distinct pathways for the DENV-mediated ASC differentiation and suggest the involvement of the tryptophan metabolism in this cellular process triggered by flavivirus infections.

Circulating serum HBsAg level is a biomarker for HBV-specific T and B cell responses in chronic hepatitis B patients

Chronic hepatitis B (CHB) infection functional cure is defined as sustained loss of HBsAg and several therapeutic strategies are in clinical development designed to pharmacologically reduce serum HBsAg, break immune tolerance, and increase functional cure rates. However, little is known about pre-treatment HBsAg levels as an indicator of HBV immune potential. Here, we compared the phenotypes and HBV-specific response of lymphocytes in CHB patients stratified by serum HBsAg levels <500 (HBslo) or >50,000 IU/ml (HBshi) using immunological assays (flow cytometry, ICS, ELISPOT). HBshi patients had significantly higher expression of inhibitory PD-1 on CD4+ T cells, particularly among TEMRA subset, and higher FcRL5 expression on B cells. Upon HBcAg(core) or HBsAg(env)-stimulation, 85% and 60% of HBslo patients had IFNγ+TNFα+ and IFNγ+ IL2+ CD4+ T cell responses respectively, in comparison to 33% and 13% of HBshi patients. Checkpoint blockade with αPD-1 improved HBV-specific CD4+ T cell function only in HBslo patients. HBsAg-specific antibody-secreting cells (ASCs) response was not different between these groups, yet αPD-1 treatment resulted in significantly higher fold change in ASCs among patients with HBsAg <100 IU/ml compared to patients with HBsAg >5,000 IU/ml. Thus, serum HBsAg correlates with inhibitory receptor expression, HBV-specific CD4+ T cell responses, and augmentation by checkpoint blockade.

Virus-like particles containing multiple antigenic proteins of Toxoplasma gondii induce memory T cell and B cell responses.

Although the efforts to develop vaccine against Toxoplasma gondii infection were made for decades, there is currently no licensed vaccine available for humans. Upon discovering a number of T or B cell epitope regions from T. gondii IMC, ROP18 and MIC8, multi-antigen VLPs or combination VLPs were generated. Mice immunized with multi-antigen VLPs or combination VLPs were challenge infected with T. gondii (ME49). T. gondii-specific IgG, IgG isotypes and IgA antibody responses, memory T and B cell responses and protection were evaluated. All the mice survived upon T. gondii challenge infection by multi-antigen VLPs vaccination. Vaccinated mice elicited higher levels of parasite-specific IgG and IgG2a antibody responses in sera, IgA antibody responses in feces, CD4+ and CD8+ T cell responses, and cytokines (IFN-γ, IL-10) responses compared to combination VLPs. In particular, the multi-antigen VLPs vaccination showed significantly higher levels of antibody secreting cell (ASC) responses, CD4+ and CD8+ effector memory T cells, and memory B cells than combination VLPs. Multi-antigen VLPs vaccination showed significant reduction of brain cyst counts and size, and all mice survived. Prediction and analysis of epitopes have indicated that IMC, ROP18 and MIC8 showed partially overlapping epitopes for T and B cells. Our results indicated that antibody responses, memory T and B cells induced by multi-antigen VLPs vaccination might contribute to the complete protection upon T. gondii (ME49) challenge infection.

Induction of systemic, mucosal and memory antibody responses targeting Vibrio cholerae O1 O-specific polysaccharide (OSP) in adults following oral vaccination with an oral killed whole cell cholera vaccine in Bangladesh.

BackgroundOral cholera vaccine (OCV) containing killed Vibrio cholerae O1 and O139 organisms (Bivalent-OCV; Biv-OCV) are playing a central role in global cholera control strategies. OCV is currently administered in a 2-dose regimen (day 0 and 14). There is a growing body of evidence that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae mediate protection against cholera. There are limited data on anti-OSP responses in recipients of Biv-OCV. We assessed serum antibody responses against O1 OSP, as well as antibody secreting cell (ASC) responses (a surrogate marker for mucosal immunity) and memory B cell responses in blood of adult recipients of Biv-OCV in Dhaka, Bangladesh.Methodology/Principal findingsWe enrolled 30 healthy adults in this study and administered two doses of OCV (Shanchol) at days 0 and 14. Blood samples were collected before vaccination (day 0) and 7 days after each vaccination (day 7 and day 21), as well as on day 44. Serum responses were largely IgA with minimal IgG and IgM responses in this population. There was no appreciable boosting following day 14 vaccination. There were significant anti-OSP IgA ASC responses on day 7 following the first vaccination, but none after the second immunization. Anti-OSP IgA memory B cell responses were detectable 30 days after completion of the vaccination series, with no evident induction of IgG memory responses. In this population, anti-Ogawa OSP responses were more prominent than anti-Inaba responses, perhaps reflecting impact of previous exposure. Serum anti-OSP responses returned to baseline within 30 days of completing the vaccine series.ConclusionOur results call into question the utility of the 2-dose regimen separated by 14 days in adults in cholera endemic areas, and also suggest that Biv-OCV-induced immune responses targeting OSP are largely IgA in this highly endemic cholera area. Studies in children in cholera-endemic areas need to be performed. Protective efficacy that extends for more than a month after vaccination presumably is mediated by direct mucosal immune response which is not assessed in this study. Our results suggest a single dose of OCV in adults in a cholera endemic zone may be sufficient to mediate at least short-term protection.

Recombinant live attenuated influenza virus expressing G protein fragment in a chimeric hemagglutinin molecule induces G-specific antibodies and confers protection against respiratory syncytial virus

Abstract Respiratory syncytial virus (RSV) is one of the most important pathogens associated with significant morbidity and mortality in infants and the elderly. Live attenuated influenza vaccine (LAIV) is a licensed platform for vaccination in humans and known to induce broader immune responses locally and systemically. RSV G attachment proteins mediate virus binding to the target cells and contain a conserved central domain with neutralizing epitopes. Here, we generated recombinant LAIV based on the attenuated A/PR8/34 virus backbone, expressing a RSV G conserved domain in a chimeric hemagglutinin (HA) fusion molecule (HA-G). The attenuated phenotypes of chimeric HA-G LAIV were evident by restricted replication in the upper respiratory (nose) but not in the lower respiratory track lung tissues of the mice. Chimeric HA-G LAIV grew better at low temperature 33□. Prime and boost intranasal vaccination of mice with HA-G LAIV induced G-protein specific IgG and IgG2a (T helper type 1) isotype antibodies. Immunization of mice with chimeric HA-G LAIV showed significant increases in G-protein specific IgG and antibody-secreting cell responses in lung, bronchioalveolar fluid, bone marrow, and spleens after challenge. Vaccine-enhanced disease typically caused by inactivated-RSV vaccination was not observed in chimeric HA-G LAIV as analyzed by lung histopathology and infiltrating innate and adaptive immune cells after RSV challenge. Immunity of chimeric HA-G LAIV against influenza virus was not compromised. These results in this study suggest a novel approach of developing RSV vaccine candidates using LAIV, potentially dual vaccines conferring protection against both pathogens.

T-bet Transcription Factor Promotes Antibody-Secreting Cell Differentiation by Limiting the Inflammatory Effects of IFN-γ on B Cells

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

Evaluation of the safety and immunogenicity of the oral inactivated multivalent enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-blind, randomized, placebo-controlled Phase I trial using electrochemiluminescence and ELISA assays for immunogenicity analyses

The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology.Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA.ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87–100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62–93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens.The promising results in adults supported testing ETVAX in descending age groups of children.ClinicalTrials.gov Identifier: NCT02531802.

Immune Correlates of Protection Induced by Virus-Like Particles Containing 2009 H1N1 Pandemic Influenza HA, NA or M1 Proteins

ABSTRACTInfluenza virus-like particle (VLPs) vaccines are a promising alternative to conventional egg-based vaccines. Evaluation of vaccine efficacy induced by HA-M1 VLPs, NA-M1 VLPs or M1 VLPs against virus challenge infection would provide important insight into vaccine design strategy. In this study, we generated VLPs containing hemagglutinin (HA), neuraminidase (NA) or M1 proteins derived from the A/California/04/09. Mice were immunized intramuscularly with HA-M1, NA-M1 or M1 VLPs and protective immunity was evaluated by assessing lung virus loads against low (5LD50) or high (100LD50) lethal dose of homologous virus challenges. High levels of virus-specific serum IgG antibody responses were induced in mice after HA-M1 VLPs immunization, whereas low or no IgG antibody responses were detected from immunization with NA-M1 VLPs or M1 VLPs, independently. Mice that were immunized with HA-M1 VLPs showed below the limit of detection on lung virus loads against low dose (5LD50) of challenge and significant reduction against high dose (100LD50) of challenge infection. Mice that were immunized with NA-M1 or M1 VLPs also displayed reduced lung viral loads compared to naïve control. In vitro cultures of cells from mouse spleen and bone marrow revealed that HA-M1 VLPs and NA-M1 VLPs induced higher levels of antibody-secreting cell (ASC) responses compared to naïve control, whereas M1 VLPs showed no ASC responses. HA-M1, NA-M1 or M1 VLPs immunization demonstrated varying degree of protection with respect to body weight changes and survival rates, which are consistent with the levels of antibody responses in sera and ASC responses from spleen and bone marrow.

The Art and Science of Diagnosing Mycoplasma pneumoniae Infection.

Mycoplasma pneumoniae causes a significant burden of disease in children as both upper and lower respiratory tract infections (URTIs and LRTIs). A positive pharyngeal polymerase chain reaction (PCR) or serology for M. pneumoniae can be found in 4–39% of children hospitalized with community-acquired

Antibody secreting B cells and plasma antibody response to rotavirus vaccination in infants from Kolkata India

BackgroundAssessing immune response after rotavirus vaccination consists in measuring serum or plasma IgA and IgG antibodies, but these assays provide very little information about the mucosal immune response. Thus the development of assays for detection of mucosal immune response following rotavirus vaccination is essential. We evaluate to assess circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immune responses to rotavirus vaccine. Methods372 subjects, aged 6 weeks, were enrolled in the study. All the subjects were assigned to receive two doses of Rotarix® vaccine. Using a micro-modified whole blood-based ELISPOT assay, circulating rotavirus type-specific IgA- and IgG-ASCs, including gut homing β7+ ASCs, were enumerated on week 6 before the first dose of Rotarix vaccination at 7 weeks of age and week 18 after the second vaccination at 17 weeks of age. Plasma samples collected before vaccination, and after two doses of Rotarix® vaccination were tested for plasma rotavirus IgA titers. ResultsTwo doses of Rotarix® provided to induce sero-protective titer of ≥ 20 Units in 35% of subjects. Total blood IgA- ASC responses were detected in 26.4% of subjects who were non-responder before vaccination. Among responders, 47% of the subjects also have sero-protective plasma IgA titers. DiscussionOur results suggest that virus-specific blood gut homing ASCs were detected and provide insight into mucosal immune response after rotavirus vaccination. Further studies are needed to evaluate the duration of such immune responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the rotavirus immunization program.