Antibody-secreting Cell Responses Research Articles

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3–6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

A First in Human Clinical Trial Assessing the Safety and Immunogenicity of Two Intradermally Delivered Enterotoxigenic Escherichia coli CFA/I Fimbrial Tip Adhesin Antigens with and without Heat-Labile Enterotoxin with Mutation LT(R192G).

Enterotoxigenic E. coli (ETEC) is a leading cause of diarrhea in travelers as well as for children living in low- to middle-income countries. ETEC adhere to intestinal epithelium via colonization factors (CFs). CFA/I, a common CF, is composed of a polymeric stalk and a tip-localized minor adhesive subunit, CfaE. Vaccine delivery by the transcutaneous immunization of dscCfaE was safe but was poorly immunogenic in a phase 1 trial when administered to volunteers with LTR(192G) and mLT. To potentially enhance the immunogenicity of CfaE while still delivering via a cutaneous route, we evaluated the safety and immunogenicity of two CfaE constructs administered intradermally (ID) with or without mLT. CfaE was evaluated as a donor strand-complemented construct (dscCfaE) and as a chimeric construct (Chimera) in which dscCfaE replaces the A1 domain of the cholera toxin A subunit and assembles non-covalently with the pentamer of heat-labile toxin B (LTB). Subjects received three ID vaccinations three weeks apart with either dscCfaE (1, 5, and 25 µg) or Chimera (2.6 and 12.9 µg) with and without 0.1 µg of mLT. Subjects were monitored for local and systemic adverse events. Immunogenicity was evaluated by serum and antibody-secreting cell (ASC) responses. The vaccine was well-tolerated with predominantly mild and moderate local vaccine site reactions characterized by erythema, induration and post-inflammatory hyperpigmentation. High rates of serologic and ASC responses were seen across study groups with the most robust responses observed in subjects receiving 25 µg of dscCfaE with 0.1 mcg of LT(R192G). Both ETEC adhesin vaccine prototypes were safe and immunogenic when co-administered with mLT by the ID route. The observed immune responses induced with the high dose of dscCfaE and mLT warrant further assessment in a controlled human infection model.

GMP manufacture of Shigella flexneri 2a Artificial Invaplex (InvaplexAR) and evaluation in a Phase 1 Open-label, dose escalating study administered intranasally to healthy, adult volunteers

Shigella species cause severe disease among travelers to, and children living in, endemic countries. Although significant efforts have been made to improve sanitation, increased antibiotic resistance and other factors suggest an effective vaccine is a critical need. Artificial Invaplex (InvaplexAR) is a subunit vaccine approach complexing Shigella LPS with invasion plasmid antigens. In pre-clinical studies, the InvaplexAR vaccine demonstrated increased immunogenicity as compared to the first generation product and was subsequently manufactured under cGMP for clinical testing in a first-in-human Phase 1 study. The primary objective of this study was the safety of S. flexneri 2a InvaplexAR given by intranasal (IN) immunization (without adjuvant) in a single-center, open-label, dose-escalating Phase 1 trial and secondarily to assess immunogenicity to identify a dose of InvaplexAR for subsequent clinical evaluations. Subjects received three IN immunizations of InvaplexAR, two weeks apart, in increasing dose cohorts (10 µg, 50 µg, 250 µg, and 500 μg). Adverse events were monitored using symptom surveillance, memory aids, and targeted physical exams. Samples were collected throughout the study to investigate vaccine-induced systemic and mucosal immune responses. There were no adverse events that met vaccination-stopping criteria. The majority (96%) of vaccine-related adverse events were mild in severity (most commonly nasal congestion, rhinorrhea, and post-nasal drip). Vaccination with InvaplexAR induced anti-LPS serum IgG responses and anti-Invaplex IgA and IgG antibody secreting cell (ASC) responses at vaccine doses ≥250 µg. Additionally, mucosal immune responses and functional antibody responses were seen from the serum bactericidal assay measurements. Notably, the responder rates and the kinetics of ASCs and antibody lymphocyte secretion (ALS) were similar, suggesting that either assay may be employed to identify IgG and IgA secreting cells. Further studies with InvaplexAR will evaluate alternative immunization routes, vaccination schedules and formulations to further optimize immunogenicity. (Clinical Trial Registry Number NCT02445963).

Boosting of vaginal HSV-2-specific B and T cell responses by intravaginal therapeutic immunization results in diminished recurrent HSV-2 disease.

Boosting herpes simplex virus (HSV)-specific immunity in the genital tissues of HSV-positive individuals to increase control of HSV-2 recurrent disease and virus shedding is an important goal of therapeutic immunization and would impact HSV-2 transmission. Experimental therapeutic HSV-2 vaccines delivered by a parenteral route have resulted in decreased recurrent disease in experimental animals. We used a guinea pig model of HSV-2 infection to test if HSV-specific antibody and cell-mediated responses in the vaginal mucosa would be more effectively increased by intravaginal (Ivag) therapeutic immunization compared to parenteral immunization. Therapeutic immunization with HSV glycoproteins and CpG adjuvant increased glycoprotein-specific IgG titers in vaginal secretions and serum to comparable levels in Ivag- and intramuscular (IM)-immunized animals. However, the mean numbers of HSV glycoprotein-specific antibody secreting cells (ASCs) and IFN-γ SCs were greater in Ivag-immunized animals demonstrating superior boosting of immunity in the vaginal mucosa compared to parenteral immunization. Therapeutic Ivag immunization also resulted in a significant decrease in the cumulative mean lesion days compared to IM immunization. There was no difference in the incidence or magnitude of HSV-2 shedding in either therapeutic immunization group compared to control-treated animals. Collectively, these data demonstrated that Ivag therapeutic immunization was superior compared to parenteral immunization to boost HSV-2 antigen-specific ASC and IFN-γ SC responses in the vagina and control recurrent HSV-2 disease. These results suggest that novel antigen delivery methods providing controlled release of optimized antigen/adjuvant combinations in the vaginal mucosa would be an effective approach for therapeutic HSV vaccines. IMPORTANCE HSV-2 replicates in skin cells before it infects sensory nerve cells where it establishes a lifelong but mostly silent infection. HSV-2 occasionally reactivates, producing new virus which is released back at the skin surface and may be transmitted to new individuals. Some HSV-specific immune cells reside at the skin site of the HSV-2 infection that can quickly activate and clear new virus. Immunizing people already infected with HSV-2 to boost their skin-resident immune cells and rapidly control the new HSV-2 infection is logical, but we do not know the best way to administer the vaccine to achieve this goal. In this study, a therapeutic vaccine given intravaginally resulted in significantly better protection against HSV-2 disease than immunization with the same vaccine by a conventional route. Immunization by the intravaginal route resulted in greater stimulation of vaginal-resident, virus-specific cells that produced antibody and produced immune molecules to rapidly clear virus.

Virus-Like Particles Assembled Using Respiratory Syncytial Virus Matrix Protein Elicit Protective Immunity in Mice.

Heterologous virus-like particle (VLP) assembly involving influenza or the Newcastle disease virus matrix protein (M) has been extensively used to explore the efficacies of VLP vaccines against the respiratory syncytial virus (RSV). Here, we attempted to generate homologous RSV VLPs by expressing the pre-fusion (pre-F) or the glycoprotein (G) on the RSV M protein and evaluated their protective efficacy in mice. We generated VLPs using the baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Recombinant baculoviruses expressing the RSV pre-F, G, and M antigens were inoculated into Sf9 cells, and particles were self-assembled. Mice were immunized with either pre-F or G-expressing VLPs, and immune parameters were assessed to determine protection. Our findings show that successful VLP assembly can be achieved by utilizing recombinant baculoviruses expressing the RSV pre-F or G proteins with the native matrix protein. Mice immunized with either pre-F or the G antigen-expressing VLPs elicited robust serum-mediated virus neutralization. VLP immunization evoked Th1-biased RSV-specific antibody responses in the sera of mice. Following challenge infection with the RSV A2 strain, immunized mice experienced lesser eosinophil and IL-4 accumulation in the lungs, though a substantial increase in TNF-α secretion was observed from CD4+ T cells. Interestingly, splenic antibody-secreting cell responses were substantially enhanced against RSV F antigen, but not against the RSV G antigen following immunization and challenge infection. Immunizing mice with the VLPs significantly inhibited pulmonary histopathology development, as indicated by the diminished inflammatory immune cell influx and mucin secretion. Combined, these vaccine-induced immune responses contributed to successfully inhibiting the RSV replication in the lungs of mice and demonstrated that RSV VLP assembly using insect cell-derived homologous RSV matrix protein is a feasible approach.

Protective mucosal and systemic immunity induced by virus-like particles expressing Toxoplasma gondii cyst wall protein.

Toxoplasma gondii host cellular invasion factors such as the rhoptry proteins, micronemal antigens, or other subcellular compartment proteins have shown limited vaccine efficacies. T. gondii cyst wall protein (CST1) as a cyst persistence factor is critical for cyst wall integrity and bradyzoite persistence. Here, we generated influenza virus-like particles (VLPs) expressing the T. gondii CST1 and evaluated the mucosal as well as systemic immunities induced by VLPs. Intranasal immunization with the VLPs induced parasite-specific IgG and IgA antibody responses in sera and intestines. VLP immunization showed higher levels of germinal center B cell response and antibody-secreting cell (ASC) response upon challenge infection, indicating memory B cell response was induced. VLP-immunized mice showed a significant reduction of cyst counts and lower levels of pro-inflammatory cytokines (IFN-γ, IL-6) production in the brain upon T. gondii ME49 challenge infection compared to unimmunized control. Thus, VLP immunization protected mice from the lethal dose challenge infection with T. gondii ME49 and did not incur bodyweight loss. These results indicated that T. gondii CST1 containing VLPs can induce mucosal and systemic immunity and also suggest its developmental potential as an effective vaccine candidate against T. gondii infection.

A transcriptionally distinct subset of influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans

Seasonal influenza vaccination elicits hemagglutinin (HA)-specific memory B (Bmem) cells, and although multiple Bmem cell populations have been characterized, considerable heterogeneity exists. We found that HA-specific human Bmem cells differed in the expression of surface marker FcRL5 and transcriptional factor T-bet. FcRL5+T-bet+ Bmem cells were transcriptionally similar to effector-like memory cells, while T-betnegFcRL5neg Bmem cells exhibited stem-like central memory properties. FcRL5+ Bmem cells did not express plasma-cell-commitment factors but did express transcriptional, epigenetic, metabolic, and functional programs that poised these cells for antibody production. Accordingly, HA+ T-bet+ Bmem cells at day 7 post-vaccination expressed intracellular immunoglobulin, and tonsil-derived FcRL5+ Bmem cells differentiated more rapidly into antibody-secreting cells (ASCs) invitro. The T-bet+ Bmem cell response positively correlated with long-lived humoral immunity, and clonotypes from T-bet+ Bmem cells were represented in the secondary ASC response to repeat vaccination, suggesting that this effector-like population predicts influenza vaccine durability and recall potential.

Use of a MAIT-Activating Ligand, 5-OP-RU, as a Mucosal Adjuvant in a Murine Model of Vibrio cholerae O1 Vaccination.

Background:Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in the mucosa with capacity for B-cell help. We hypothesize that targeting MAIT cells, using a MAIT-activating ligand as an adjuvant, could improve mucosal vaccine responses to bacterial pathogens such as Vibrio cholerae.Methods:We utilized murine models of V. cholerae vaccination to test the adjuvant potential of the MAIT-activating ligand, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). We measured V. cholerae-specific antibody and antibody-secreting cell responses and used flow cytometry to examine MAIT-cell and B-cell phenotype, in blood, bronchoalveolar lavage fluid (BALF), and mucosal tissues, following intranasal vaccination with live V. cholerae O1 or a V. cholerae O1 polysaccharide conjugate vaccine.Results:We report significant expansion of MAIT cells in the lungs (P < 0.001) and BALF (P < 0.001) of 5-OP-RU treated mice, and higher mucosal (BALF, P = 0.045) but not systemic (serum, P = 0.21) V. cholerae O-specific-polysaccharide IgG responses in our conjugate vaccine model when adjuvanted with low-dose 5-OP-RU. In contrast, despite significant MAIT cell expansion, no significant differences in V. cholerae-specific humoral responses were found in our live V. cholerae vaccination model.Conclusions:Using a murine model, we demonstrate the potential, as well as the limitations, of targeting MAIT cells to improve antibody responses to mucosal cholera vaccines. Our study highlights the need for future research optimizing MAIT-cell targeting for improving mucosal vaccines.

Robust immune responses are observed after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2-experienced individuals.

The use of coronavirus disease 2019 (COVID-19) vaccines will play the major role in helping to end the pandemic that has killed millions worldwide. COVID-19 vaccines have resulted in robust humoral responses and protective efficacy in human trials, but efficacy trials excluded individuals with a prior diagnosis of COVID-19. As a result, little is known about how immune responses induced by mRNA vaccines differ in individuals who recovered from COVID-19. Here, we evaluated longitudinal immune responses to two-dose BNT162b2 mRNA vaccination in 15 adults who had experienced COVID-19, compared to 21 adults who did not have prior COVID-19. Consistent with prior studies of mRNA vaccines, we observed robust cytotoxic CD8+ T cell responses in both cohorts after the second dose. Furthermore, SARS-CoV-2–naive individuals had progressive increases in humoral and antigen-specific antibody-secreting cell (ASC) responses after each dose of vaccine, whereas SARS-CoV-2–experienced individuals demonstrated strong humoral and antigen-specific ASC responses to the first dose but these responses were not further enhanced after the second dose of the vaccine at the time points studied. Together, these data highlight the relevance of immunological history for understanding vaccine immune responses and may have implications for personalizing mRNA vaccination regimens used to prevent COVID-19, including for the deployment of booster shots.

Single-Cell Technologies for the Study of Antibody-Secreting Cells.

Antibody-secreting cells (ASC), plasmablasts and plasma cells, are terminally differentiated B cells responsible for large-scale production and secretion of antibodies. ASC are derived from activated B cells, which may differentiate extrafollicularly or form germinal center (GC) reactions within secondary lymphoid organs. ASC therefore consist of short-lived, poorly matured plasmablasts that generally secrete lower-affinity antibodies, or long-lived, highly matured plasma cells that generally secrete higher-affinity antibodies. The ASC population is responsible for producing an immediate humoral B cell response, the polyclonal antibody repertoire, as well as in parallel building effective humoral memory and immunity, or potentially driving pathology in the case of autoimmunity. ASC are phenotypically and transcriptionally distinct from other B cells and further distinguishable by morphology, varied lifespans, and anatomical localization. Single cell analyses are required to interrogate the functional and transcriptional diversity of ASC and their secreted antibody repertoire and understand the contribution of individual ASC responses to the polyclonal humoral response. Here we summarize the current and emerging functional and molecular techniques for high-throughput characterization of ASC with single cell resolution, including flow and mass cytometry, spot-based and microfluidic-based assays, focusing on functional approaches of the secreted antibodies: specificity, affinity, and secretion rate.

Vectored antibody gene delivery restores host B and Tcell control of persistent viral infection.

Passive antibody therapy and vectored antibody gene delivery (VAGD) in particular offer an innovative approach to combat persistent viral diseases. Here, we exploit a small animal model to investigate synergies of VAGD with the host's endogenous immune defense for treating chronic viral infection. An adeno-associated virus (AAV) vector delivering the lymphocytic choriomeningitis virus (LCMV)-neutralizing antibody KL25 (AAV-KL25) establishes protective antibody titers for >200days. When therapeutically administered to chronically infected immunocompetent wild-type mice, AAV-KL25 affords sustained viral load control. In contrast, viral mutational escape thwarts therapeutic AAV-KL25 effects when mice are unable to mount LCMV-specific antibody responses or lack CD8+ Tcells. VAGD augments antiviral germinal center B cell and antibody-secreting cell responses and reduces inhibitory receptor expression on antiviral CD8+ Tcells. These results indicate that VAGD fortifies host immune defense and synergizes with B cell and CD8 Tcell responses to restore immune control of chronic viral infection.

Neuraminidase in Virus-like Particles Contributes to the Protection against High Dose of Avian Influenza Virus Challenge Infection.

Neuraminidase is an important target for influenza vaccination. In this study, we generated avian influenza VLPs, expressing hemagglutinin (HA), neuraminidase (NA), HA and NA co-expressed (HANA), to evaluate the protective role of NA against a high (10LD50) and low (2LD50) dose of avian influenza virus challenge infections. A single immunization with HANA VLPs elicited the highest level of virus-specific IgG, IgG1, and IgG2a responses from the sera post-vaccination and the lungs post-challenge-infection. Potent antibody-secreting cell responses were observed from the spleens and lungs of HANA-VLP-immunized mice post-challenge-infection. HANA VLPs induced the highest CD4+ T cell, CD8+ T cell, and germinal center B cells, while strongly limiting inflammatory cytokine production in the lungs compared to other VLP immunization groups. In correlation with these findings, the lowest bodyweight losses and lung virus titers were observed from HANA VLP immunization, and all of the immunized mice survived irrespective of the challenge dose. Contrastingly, VLPs expressing either HA or NA alone failed to elicit complete protection. These results indicated that NA in VLPs played a critical role in inducing protection against a high dose of the challenge infection.

Mucosal Administration of Recombinant Baculovirus Displaying Toxoplasma gondii ROP4 Confers Protection Against T. gondii Challenge Infection in Mice.

Pathogens require physical contact with the mucosal surface of the host organism to initiate infection and as such, vaccines eliciting both mucosal and systemic immune responses would be promising. Studies involving the use of recombinant baculoviruses (rBVs) as mucosal vaccines are severely lacking despite their inherently safe nature, especially against pathogens of global importance such as Toxoplasma gondii. Here, we generated rBVs displaying T. gondii rhoptry protein 4 (ROP4) and evaluated their protective efficacy in BALB/c mice following immunization via intranasal (IN) and oral routes. IN immunization with the ROP4-expressing rBVs elicited higher levels of parasite-specific IgA antibody responses compared to oral immunization. Upon challenge infection with a lethal dose of T. gondii ME49, IN immunization elicited significantly higher parasite-specific antibody responses in the mucosal tissues such as intestines, feces, vaginal samples, and brain than oral immunization. Marked increases in IgG and IgA antibody-secreting cell (ASC) responses were observed from intranasally immunized mice. IN immunization elicited significantly enhanced induction of CD4+, CD8+ T cells, and germinal center B (GC B) cell responses from secondary lymphoid organs while limiting the production of the inflammatory cytokines IFN-γ and IL-6 in the brain, all of which contributed to protecting mice against T. gondii lethal challenge infection. Our findings suggest that IN delivery of ROP4 rBVs induced better mucosal and systemic immunity against the lethal T. gondii challenge infection compared to oral immunization.

An enzyme-linked immunospot assay to evaluate Pneumovax response when on intravenous immunoglobulin

BackgroundTesting for common variable immunodeficiency (CVID) requires evaluation of specific antibody responses to vaccines. Current practice of evaluating pneumococcal serotype-specific immunoglobulin (Ig)G levels after Pneumovax (P23) has several limitations and is not accurate for patients already on immunoglobulin replacement. In contrast, the enzyme-linked immunospot (ELISPOT) assay can be interpreted in patients on immunoglobulin replacement as ex vivo measurement of specific antibody-secreting cells (ASCs). ObjectiveTo optimize and test an ELISPOT assay to evaluate vaccination response to P23 and compare with P23 serotype-specific IgG for patients on intravenous immunoglobulin (IVIG). MethodsWe prospectively enrolled a total of 15 adults: normal controls (n = 8), patients with CVID on IVIG replacement (n = 2), patients on IVIG replacement for recurrent infections who did not meet diagnostic criteria for CVID, considered IgG deficiency (n = 2), and patients without immunodeficiency on high-dose IVIG for other diagnosis (n = 3). We measured P23 serotype-specific IgG before and 4 weeks after P23 and ELISPOT ASCs before and 1 week after P23 (with B-cell subpopulation analysis by flow cytometry in patients on IVIG). ResultsNormal controls had a vaccination response by P23 serotype-specific IgG, whereas patients on IVIG did not. Except for true patients with CVID on IVIG, a P23 ELISPOT ASC response was found in normal controls (highest) and most patients on IVIG for recurrent infections or other diagnosis. ConclusionOur pilot study suggests that an optimized ELISPOT protocol has utility to evaluate the P23-specific antibody response after vaccination. Our ELISPOT assay seemed reliable for patients on IVIG and may help differentiate true patients with CVID from those with a less stringent diagnosis while on IVIG.

Viremia controls Env-specific antibody-secreting cell responses in simian immunodeficiency virus infected macaques pre and post-antiretroviral therapy.

The aim of this study was to investigate the kinetics of Env (gp140)-specific antibody-secreting cells (ASCs) during acute and early chronic simian immunodeficiency virus (SIV) infection, and prior to and postantiretroviral therapy (ART) in rhesus macaques. At week 0, rhesus macaques were inoculated intravenously with SIVmac239 and the viral loads were allowed to develop. Daily ART was initiated at week 5 post infection until week 18, though the animals were monitored until week 28 for the following parameters: enumeration of SIV gp140-specific ASCs by ELISPOT; quantification of viremia and SIV gp140-specific IgG titres through qRT-PCR and ELISA, respectively; estimation of monocytes, follicular helper T cells (Tfh) and memory B cell frequencies using polychromatic flow cytometry. Direct correlations were consistently found between blood SIV gp140-specific ASC responses and viremia or SIV Env-specific IgG titres. In contrast, SIV gp140-specific ASC responses showed inverse correlations with the percentage of total memory B cells in the blood. In lymph nodes, the magnitude of the SIV gp140-specific ASC responses also followed the viral load kinetics. In contrast, the number of SIV gp140-specific ASCs presented did not correlate with frequencies of circulating activated monocyte (CD14+CD16+) or Tfh cells. Blood and/or lymph node viral loads may regulate the onset and magnitude of SIV gp140-specific ASCs during SIV infection and following ART in rhesus macaques.

Cellular and Humoral Immune Responses in Covid-19 and Immunotherapeutic Approaches.

Coronavirus disease 2019 (Covid-19), caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can range in severity from asymptomatic to severe/critical disease. SARS-CoV-2 uses angiotensin-converting enzyme 2 to infect cells leading to a strong inflammatory response, which is most profound in patients who progress to severe Covid-19. Recent studies have begun to unravel some of the differences in the innate and adaptive immune response to SARS-CoV-2 in patients with different degrees of disease severity. These studies have attributed the severe form of Covid-19 to a dysfunctional innate immune response, such as a delayed and/or deficient type I interferon response, coupled with an exaggerated and/or a dysfunctional adaptive immunity. Differences in T-cell (including CD4+ T-cells, CD8+ T-cells, T follicular helper cells, γδ-T-cells, and regulatory T-cells) and B-cell (transitional cells, double-negative 2 cells, antibody-secreting cells) responses have been identified in patients with severe disease compared to mild cases. Moreover, differences in the kinetic/titer of neutralizing antibody responses have been described in severe disease, which may be confounded by antibody-dependent enhancement. Importantly, the presence of preexisting autoantibodies against type I interferon has been described as a major cause of severe/critical disease. Additionally, priorVaccine and multiple vaccine exposure, trained innate immunity, cross-reactive immunity, and serological immune imprinting may all contribute towards disease severity and outcome. Several therapeutic and preventative approaches have been under intense investigations; these include vaccines (three of which have passed Phase 3 clinical trials), therapeutic antibodies, and immunosuppressants.

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.