Clinical immunogenicity outcomes from GENEr8-1, a phase 3 study of valoctocogene roxaparvovec, an AAV5-vectored gene therapy for hemophilia A.

Gene transfer therapies utilizing adeno-associated virus (AAV) vectors involve a complex drug design with multiple components that may impact immunogenicity. Valoctocogene roxaparvovec is an AAV serotype 5 (AAV5)-vectored gene therapy for the treatment of hemophilia A that encodes a B-domain-deleted human factor VIII (FVIII) protein controlled by a hepatocyte-selective promoter. Following previous results from the first-in-human phase 1/2 clinical trial, we assessed AAV5-capsid- and transgene-derived FVIII-specific immune responses with 2 years of follow-up data from GENEr8-1, a phase 3, single-arm, open-label study in 134 adult men with severe hemophilia A. No FVIII inhibitors were detected following administration of valoctocogene roxaparvovec. Immune responses were predominantly directed toward the AAV5 capsid, with all participants developing durable anti-AAV5 antibodies. Cellular immune responses specific for the AAV5 capsid were detected in most participants by interferon-γ enzyme-linked immunosorbent spot assay 2 weeks following dose administration and declined or reverted to negative over the first 52 weeks. These responses were weakly correlated with alanine aminotransferase elevations and showed no association with changes in FVIII activity. FVIII-specific cellular immune responses were less frequent and more sporadic compared with those specific for AAV5 and showed no association with safety or efficacy parameters.

Long-chain polyunsaturated lipids associated with responsiveness to anti-PD-1 therapy are colocalized with immune infiltrates in the tumor microenvironment.

The programmed cell death protein-1 (PD-1) is highly expressed on the surface of antigen-specific exhausted T cells and, upon interaction with its ligand PD-L1, can result in inhibition of the immune response. Anti-PD-1 treatment has been shown to extend survival and result in durable responses in several cancers, yet only a subset of patients benefit from this therapy. Despite the implication of metabolic alteration following cancer immunotherapy, mechanistic associations between antitumor responses and metabolic changes remain unclear. Here, we used desorption electrospray ionization mass spectrometry imaging to examine the lipid profiles of tumor tissue from three syngeneic murine models with varying treatment sensitivity at the baseline and at three time points post-anti-PD-1 therapy. These imaging experiments revealed specific alterations in the lipid profiles associated with the degree of response to treatment and allowed us to identify a significant increase of long-chain polyunsaturated lipids within responsive tumors following anti-PD-1 therapy. Immunofluorescence imaging of tumor tissues also demonstrated that the altered lipid profile associated with treatment response is localized to dense regions of tumor immune infiltrates. Overall, these results indicate that effective anti-PD-1 therapy modulates lipid metabolism in tumor immune infiltrates, and we thereby propose that further investigation of the related immune-metabolic pathways may be useful for better understanding success and failure of anti-PD-1 therapy.

Cellular immunity to nucleoproteins (NP) of Crimean-Congo hemorrhagic fever virus (CCHFV) and Hazara Virus (HAZV).

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a globally significant vector-borne pathogen with no internationally-licensed preventative and therapeutic interventions. Hazara virus (HAZV), on the other hand, a related Orthonairovirus, has not been reported as a human pathogen. HAZV has been proposed as a surrogate model for studying CCHFV, bisosafety level 4 (BSL-4) agent. Previously, we investigated the humoral immune responses between NPs of these viruses and in this study, we extended the scrutiny to cellular immune responses elicited by NPs of CCHFV and HAZV. Here, mice were immunized with recombinant CCHFV NP and HAZV NP to evaluate the correlates of cell-mediated immunity (CMI). Delayed-type hypersensitivity (DTH) responses were assessed by challenging immunized mice with CCHFV-rNP or HAZV-rNP on the footpad and lymphocyte proliferation assays (LPAs) were performed by stimulating splenocytes in vitro with CCHFV-rNP or HAZV-rNP to compare cellular immune responses. In all test groups, strong DTH and LPA responses were detected against homologous and heterologous challenging antigens. To assess the cytokine response, an RT-qPCR -specific for cytokine mRNAs was utilized. Interestingly, CCHFV NP stimulated groups exhibited a significantly elevated mRNA level of interleukin 17 A (IL-17) compared to HAZV NP, indicating a notable difference in immune responses. This study presents comparison between CMI elicited by NPs of CCHFV and HAZV and contributes to the understanding of a highly pathogenic virus, particularly in the context of the declaration of CCHFV by World Health Organization's (WHO) as a major viral threat to the world.

T cell-mediated Immune response and correlates of inflammation and their relationship with COVID-19 clinical severity: not an intuitive guess.

BACKGROUND: Predictors of the outcome of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection remain to be fully determined. We evaluated selected viral characteristics and immunological responses that might predict and/or correlate to the clinical outcome of COVID-19. METHODS: For individuals developing divergent clinical outcomes, the magnitude and breadth of T cell-mediated responses were measured within 36 h of symptom onset. Peripheral Blood Mononuclear Cells (PBMCs) were subjected to in vitro stimulation with SARS-CoV-2-based peptides. In addition, SARS-CoV-2 sequences were generated by metagenome, and HLA typing was performed using Luminex technology. FINDINGS: CD4+ T cell activation was negatively correlated with SARS-CoV-2 basal viral load in patients with severe COVID-19 (p = 0·043). The overall cellular immune response, as inferred by the IFN-γ signal, was higher at baseline for patients who progressed to mild disease compared to patients who progressed to severe disease (p = 0·0044). Subjects with milder disease developed higher T cell responses for MHC class I and II-restricted peptides (p = 0·033). INTERPRETATION: Mounting specific cellular immune responses in the first days after symptom onset, as inferred by IFN-γ magnitude in the ELISPOT assay, may efficiently favor a positive outcome. In contrast, progression to severe COVID-19 was accompanied by stronger cellular immune responses, higher CD4 + T cell activation, and a higher number of in silico predicted high-affinity class I HLA alleles.

Molecular assay for detecting MS-H vaccine strain and immune response mechanisms in chickens receiving one or two doses of live MS-H vaccine.

The MS-H vaccine, containing a live strain of Mycoplasma synoviae, is a feasible option for controlling M. synoviae infection in poultry flocks. A comprehensive understanding of vaccinated chickens, including strain differentiation and immune response mechanisms, is required to optimize vaccination strategy. This study aimed to verify the PCR-RFLP molecular assay as a convenient technique for detecting the MS-H vaccine strain and to characterize the immune response mechanisms in experimental layer-type chickens receiving one of three different vaccination programmes; a single dose at either 9 or 12 weeks of age or two doses at both 9 and 12 weeks of age. The PCR-RFLP assay, using restriction enzyme TasI to digest vlhA gene-targeted PCR amplicons, was performed to evaluate vaccine administration by detecting the MS-H vaccine strain in vaccinated chickens and differentiating it from non-vaccine strains such as WVU1853 reference strain and Thai M. synoviae field strains. Results demonstrated that vaccination in layer-type chickens, whether as one or two doses, stimulated immune response mechanisms with no significant advantages of two administrations over a single administration. Serological responses in vaccinated chickens, examined by RPA test and ELISA, were initially detected at 2 weeks post-vaccination, continuously increased, and then remained at the baseline levels from 6 to 9 weeks post-vaccination. Cellular immune responses against both homologous and heterologous antigens, examined by the MTS tetrazolium assay, were similar in the early period post-vaccination, whereas cellular immune response against the homologous MS-H antigen was improved in the late period post-vaccination.

Construction and efficacy testing of DNA vaccines containing HLA-A*02:01-restricted SARS-CoV-2 T-cell epitopes predicted by immunoinformatics.

Vaccines play essential roles in the fight against the COVID-19 pandemic. The development and assessment of COVID-19 vaccines have generally focused on the induction and boosting of neutralizing antibodies targeting the SARS-CoV-2 spike (S) protein. Due to rapid and continuous variation in the S protein, such vaccines need to be regularly updated to match newly emerged dominant variants. T-cell vaccines that target MHC I- or II-restricted epitopes in both structural and non-structural viral proteins have the potential to induce broadly cross-protective and long-lasting responses. In this work, the entire proteome encoded by SARS-CoV-2 (Wuhan-hu-1) is subjected to immunoinformatics-based prediction of HLA-A*02:01-restricted epitopes. The immunogenicity of the predicted epitopes is evaluated using peripheral blood mononuclear cells from convalescent Wuhan-hu-1-infected patients. Furthermore, predicted epitopes that are conserved across major SARS-CoV-2 lineages and variants are used to construct DNA vaccines expressing multi-epitope polypeptides. Most importantly, two DNA vaccine constructs induce epitope-specific CD8 + T-cell responses in a mouse model of HLA-A*02:01 restriction and protect immunized mice from challenge with Wuhan-hu-1 virus after hACE2 transduction. These data provide candidate T-cell epitopes useful for the development of T-cell vaccines against SARS-CoV-2 and demonstrate a strategy for quick T-cell vaccine candidate development applicable to other emerging pathogens.

The Structural Characterization of a Polysaccharide from the Dried Root of Salvia miltiorrhiza and Its Use as a Vaccine Adjuvant to Induce Humoral and Cellular Immune Responses.

In order to supplement the research gap concerning Salvia miltiorrhiza polysaccharide extracted from Danshen in NMR analysis, and to clarify its immune enhancement effect as an adjuvant, we isolated and purified SMPD-2, which is composed of nine monosaccharides such as Ara, Gal, and Glc from Danshen. Its weight average molecular weight was 37.30 ± 0.096 KDa. The main chain was mainly composed of →4)-α-D-Galp-(1→, →3,6)-ß-D-Glcp-(1→ and a small amount of α-L-Araf-(1→. After the subcutaneous injection of SMPD-2 as an adjuvant to OVA in mice, we found that it enhanced the immune response by activating DCs from lymph nodes, increasing OVA-specific antibody secretion, stimulating spleen lymphocyte activation, and showing good biosafety. In conclusion, SMPD-2 could be a promising candidate for an adjuvant.

Factors Associated With Vaccine-Induced T-Cell Immune Responses Against Severe Acute Respiratory Syndrome Coronavirus 2 in Kidney Transplant Recipients.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important prophylactic measure in kidney transplant recipients (KTRs), but the immune response is often impaired. Here, we examined the T-cell immune response against SARS-CoV-2 in 148 KTRs after 3 or 4 vaccine doses, including 35 KTRs with subsequent SARS-CoV-2 infection. The frequency of spike-specific T cells was lower in KTRs than in immunocompetent controls and was correlated with the level of spike-specific antibodies. Positive predictors for detection of vaccine-induced T cells were detection of spike-specific antibodies, heterologous immunization with messenger RNA and a vector vaccine, and longer time after transplantation. In vaccinated KTRs with subsequent SARS-CoV-2 infection, the T-cell response was greatly enhanced and was significantly higher than in vaccinated KTRs without SARS-CoV-2 infection. Overall, the data show a correlation between impaired humoral and T-cell immunity to SARS-CoV-2 vaccination and provide evidence for greater robustness of hybrid immunity in KTRs.

Alpha-galactosylceramide as adjuvant induces protective cell-mediated immunity against Leishmania mexicana infection in vaccinated BALB/c mice.

Adjuvants represent a promising strategy to improve vaccine effectiveness against infectious diseases such as leishmaniasis. Vaccination with the invariant natural killer T cell ligand α-galactosylceramide (αGalCer) has been used successfully as adjuvant, generating a Th1-biased immunomodulation. This glycolipid enhances experimental vaccination platforms against intracellular parasites including Plasmodium yoelii and Mycobacterium tuberculosis. In the present study, we assessed the protective immunity induced by a single-dose intraperitoneal injection of αGalCer (2 µg) co-administrated with a lysate antigen of amastigotes (100 µg) against Leishmania mexicana infection in BALB/c mice. The prophylactic vaccination led to 5.0-fold reduction of parasite load at the infection site, compared to non-vaccinated mice. A predominant pro-inflammatory response was observed in challenged vaccinated mice, represented by a 1.9 and 2.8-fold-increase of IL-1ß and IFN-γ producing cells, respectively, in the lesions, and by 23.7-fold-increase of IFN-γ production in supernatants of restimulated splenocytes, all compared to control groups. The co-administration of αGalCer also stimulated the maturation of splenic dendritic cells and modulated a Th1-skewed immune response, with high amounts of IFN-γ production in serum. Furthermore, peritoneal cells of αGalCer-immunized mice exhibited an elevated expression of Ly6G and MHCII. These findings indicate that αGalCer improves protection against cutaneous leishmaniasis, supporting evidence for its potential use as adjuvant in Leishmania-vaccines.

Immunogenicity, efficacy and safety of COVID-19 vaccines: an update of data published by 31 December 2021.

The unprecedented coronavirus disease 2019 (COVID-19) pandemic has caused a disaster for public health in the last 2 years, without any sign of an ending. Various vaccines were developed rapidly as soon as the outbreak occurred. Clinical trials demonstrated the reactogenicity, immunogenicity and protection efficacy in humans, and some of the vaccines have been approved for clinical use. However, waves of infections such as the recently circulating Omicron variant still occur. Newly emerging variants, especially the variants of concern, and waning humoral responses pose serious challenges to the control of the COVID-19 pandemic. Previously, we summarized the humoral and cellular immunity, safety profiles and protection efficacy of COVID-19 vaccines with clinical data published by 21 May 2021. In this review, we summarize and update the published clinical data of COVID-19 vaccines and candidates up to 31 December 2021.

Cross-reactive immune responses to monkeypox virus induced by MVA vaccination in mice.

Mpox (monkeypox) infection cases increased recently in non-Mpox outbreak areas, potentially causing an international threat. The desire to defend against a potential outbreak has led to renewed efforts to develop Mpox vaccines. In this report, mice were immunized with various doses of modified vaccinia virus Ankara (MVA) to evaluate the cross-reactive immune response of MVA immunization against protective antigens of the current monkeypox virus. We demonstrated that MVA induced specific antibodies against protective antigens (A29, A35, B6, M1, H3, and I1), mediating the neutralization abilities against the MVA and the monkeypox virus (MPXV). Moreover, recombinant protective antigens of the MPXV elicited cross-binding and cross-neutralizing activities for MVA. Hence, the MVA induced cross-reactive immune responses, which may guide future efforts to develop vaccines against the recent MPXV. Notably, compared to the other protective antigens, the predominant A29 and M1 antigens mediated higher cross-neutralizing immune responses against the MVA, which could serve as antigen targets for novel orthologous orthopoxvirus vaccine.

Humoral and cellular immune responses after three or four doses of BNT162b2 in patients with hematological malignancies.

OBJECTIVES: Initial responses to coronavirus disease 2019 vaccination are impaired in patients with hematological malignancies. We investigated immune responses after three or four doses of BNT162b2 in patients with myeloid and lymphoid malignancies compared to controls, and identified risk factors for humoral and cellular nonresponse 1 year after first vaccination. METHODS: In 407 hematological patients (45 myeloid, 362 lymphoid) and 98 matched controls, we measured immunoglobulin G (IgG) and neutralizing antibodies specific for the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at baseline, 3 weeks, 2, 6, and 12 months, and interferon-γ release at 12 months. RESULTS: In patients with lymphoid malignancies, SARS-CoV-2 receptor-binding domain IgG concentration and mean neutralizing capacity was lower than in controls at all time points. A diagnosis of chronic lymphocytic B-cell leukemia (CLL) or lymphoma was associated with humoral nonresponse at 12 months compared to having multiple myeloma/amyloidosis (p < .001 and p = .013). Compared to controls, patients with lymphoid malignancies had increased risk of cellular nonresponse. A lymphoma diagnosis was associated with lower risk of cellular nonresponse compared to patients with multiple myeloma/amyloidosis, while patients with CLL had comparable response rates to patients with multiple myeloma/amyloidosis (p = .037 and p = .280). CONCLUSIONS: In conclusion, long-term humoral and cellular immune responses to BNT162b2 were impaired in patients with lymphoid malignancies.

Molecular and immunological characterization of the calcyclin binding protein in rodent malaria parasite.

Malaria remains as a global life-threatening disorder due to the emergence of resistance against standard antimalarials. Consequently, there is a serious need to better understand the biology of the malaria parasite in order to determine appropriate targets for new interventions. Calcyclin binding protein (CacyBP) is a multi-functional and multi-ligand protein that is not well characterized in malaria disease. In this study, we have cloned CacyBP from rodent species Plasmodium yoelii nigeriensis and purified the recombinant protein to carry out its detailed molecular, biophysical and immunological characterization. Molecular characterization indicates that PyCacyBP is a ∼27 kDa protein in parasite lysate and exists in monomer and dimer forms. Bioinformatic analysis of CacyBP showed significant sequence and structural similarities between rodent and human malaria parasites. CacyBP is expressed in all blood stages of P. yoelii nigeriensis parasite. In silico studies proposed the immunogenic potential of CacyBP. The rPyCacyBP immunized mice exhibited elevated levels of IgG1, IgG2a, IgG2b and IgG3 in their serum. Notably, cellular immune response in splenocytes from immunized mice showed increased expression of pro-inflammatory cytokines such as IL-12, IFN-γ and TNF-α. This CacyBP exhibited pro-inflammatory immune response in rodent host. These finding revealed that CacyBP may have the potential to boost the host immunity for protection against malaria infection. The present study provides basis for further exploration of the biological function of CacyBP in malaria parasite.

Genotoxicity mechanism of food preservative propionic acid in the in vivo Drosophila model: gut damage, oxidative stress, cellular immune response and DNA damage.

Propionic acid is a short-chain fatty acid that is the main fermentation product of the enteric microbiome. It is found naturally and added to foods as a preservative and evaluated by health authorities as safe for use in foods. However, propionic acid has been reported in the literature to be associated with both health and disease. The purpose of this work is to better understand how propionic acid affects Drosophila melanogaster by examining some of the effects of this compound on the D. melanogaster hemocytes. D. melanogaster was chosen as a suitable in vivo model to detect potential risks of propionic acid (at five concentrations ranging from 0.1 to 10 mM) used as a food preservative. Toxicity, cellular immune response, intracellular oxidative stress (reactive oxygen species, ROS), gut damage, and DNA damage (via Comet assay) were the end-points evaluated. Significant genotoxic effects were detected in selected cell targets in a concentration dependent manner, especially at two highest concentrations (5 and 10 mM) of propionic acid. This study is the first study reporting genotoxicity data in the hemocytes of Drosophila larvae, emphasizing the importance of D. melanogaster as a model organism in investigating the different biological effects caused by the ingested food preservative product.

Krüppel-like factor 5 regulates wound repair and the innate immune response in human airway epithelial cells.

A complex network of transcription factors regulates genes involved in establishing and maintaining key biological properties of the human airway epithelium. However, detailed knowledge of the contributing factors is incomplete. Here we characterize the role of Krüppel-like factor 5 (KLF5), in controlling essential pathways of epithelial cell identity and function in the human lung. RNA-seq following siRNA-mediated depletion of KLF5 in the Calu-3 lung epithelial cell line identified significant enrichment of genes encoding chemokines and cytokines involved in the proinflammatory response and also components of the junctional complexes mediating cell adhesion. To determine direct gene targets of KLF5, we defined the cistrome of KLF5 using ChIP-seq in both Calu-3 and 16HBE14o- lung epithelial cell lines. Occupancy site concordance analysis revealed that KLF5 colocalized with the active histone modification H3K27ac and also with binding sites for the transcription factor CCAAT enhancer-binding protein beta (C/EBPß). Depletion of KLF5 increased both the expression and secretion of cytokines including IL-1ß, a response that was enhanced following exposure to Pseudomonas aeruginosa lipopolysaccharide. Calu-3 cells exhibited faster rates of repair after KLF5 depletion compared with negative controls in wound scratch assays. Similarly, CRISPR-mediated KLF5-null 16HBE14o- cells also showed enhanced wound closure. These data reveal a pivotal role for KLF5 in coordinating epithelial functions relevant to human lung disease.

Effective delivery of STING agonist using exosomes suppresses tumor growth and enhances antitumor immunity.

The Stimulator of Interferon Genes (STING) pathway is implicated in the innate immune response and is important in both oncogenesis and cancer treatment. Specifically, activation of the cytosolic DNA sensor STING in antigen-presenting cells (APCs) induces a type I interferon response and cytokine production that facilitates antitumor immune therapy. However, use of STING agonists (STINGa) as a cancer therapeutic has been limited by unfavorable pharmacological properties and targeting inefficiency due to rapid clearance and limited uptake into the cytosol. Exosomes, a class of extracellular vesicles shed by all cells are under consideration for their use as effective carriers of drugs owing to their innate ability to be taken up by cells and their biocompatibility for optimal drug biodistribution. Therefore, we engineered exosomes to deliver the STING agonist cyclic GMP-AMP (iExoSTINGa), to exploit their favorable pharmacokinetics and pharmacodynamics. Selective targeting of the STING pathway in APCs with iExoSTINGa was associated with superior potency compared with STINGa alone in suppressing B16F10 tumor growth. Moreover, iExoSTINGa showed superior uptake of STINGa into dendritic cells compared with STINGa alone, which led to increased accumulation of activated CD8+ T-cells and an antitumor immune response. Our study highlights the potential of exosomes in general, and iExoSTINGa specifically, in enhancing cancer therapy outcomes.

Immunological characteristics of MAV/06 strain of varicella-zoster virus vaccine in an animal model.

BACKGROUND: Varicella-zoster virus (VZV) is a pathogen that causes chickenpox and shingles in humans. Different types of the varicella vaccines derived from the Oka and MAV/06 strains are commercially available worldwide. Although the MAV/06 vaccine was introduced in 1990s, little was known about immunological characteristics. RESULTS: Here, we evaluated B and T cell immune response in animals inoculated with the Oka and MAV/06 vaccines as well as a new formulation of the MAV/06 vaccine. A variety of test methods were applied to evaluate T and B cell immune response. Plaque reduction neutralization test (PRNT) and fluorescent antibody to membrane antigen (FAMA) assay were conducted to measure the MAV/06 vaccine-induced antibody activity against various VZVs. Glycoprotein enzyme-linked immunosorbent assay (gpELISA) was used to compare the degree of the antibody responses induced by the two available commercial VZV vaccines and the MAV/06 vaccine. Interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assays and cytokine bead array (CBA) assays were conducted to investigate T cell immune responses. Antibodies induced by MAV/06 vaccination showed immunogenicity against a variety of varicella-zoster virus and cross-reactivity among the virus clades. CONCLUSIONS: It is indicating the similarity of the antibody responses induced by commercial varicella vaccines and the MAV/06 vaccine. Moreover, VZV-specific T cell immune response from MAV/06 vaccination was increased via Th1 cell response. MAV/06 varicella vaccine induced both humoral and cellular immune response via Th1 cell mediated response.

Immunogenicity and reactogenicity of homologous mRNA-based and vector-based SARS-CoV-2 vaccine regimens in patients receiving maintenance dialysis.

Patients receiving maintenance dialysis (MD) are vulnerable to COVID-19-related morbidity and mortality. Currently, data on SARS-CoV-2-specific cellular and humoral immunity post-vaccination in this population are scarce. We conducted a prospective single-center study exploring the specific cellular (interferon-γ and interleukin-2 ELISpot assays) and humoral immune responses (dot plot array and chemiluminescent microparticle immunoassay [CMIA]) at 4 weeks and 6 weeks following a single dose or a complete homologous dual dose SARS-CoV-2 vaccine regimen in 60 MD patients (six with a history of COVID-19). Our results show that MD patients exhibit a high seroconversion rate (91.7%) but the anti-spike IgG antibodies (CMIA) tend to wane rapidly after full immunization. Only 51.7% of the patients developed T cell immune response. High anti-spike IgG antibodies may predict a better cellular immunity. While patients with prior COVID-19 showed the best response after one, SARS-CoV-2-naïve patients may benefit from a third vaccine injection.

Identification of Novel T-Cell Epitopes on Infectious Bronchitis Virus N Protein and Development of a Multi-epitope Vaccine.

Cellular immune responses play a key role in the control of viral infection. The nucleocapsid (N) protein of infectious bronchitis virus (IBV) is a major immunogenic protein that can induce protective immunity. To screen for potential T-cell epitopes on IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. Four T-cell epitope peptides were identified by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot), intracellular cytokine staining, and carboxyfluorescein succinimidyl ester (CFSE) lymphocyte proliferation assays; among them, three peptides (N211-230, N271-290, and N381-400) were cytotoxic T lymphocyte (CTL) epitopes, and one peptide (N261-280) was a dual-specific T-cell epitope, which can be recognized by both CD8+ and CD4+ T cells. Multi-epitope gene transcription cassettes comprising four neutralizing epitope domains and four T-cell epitope peptides were synthesized and inserted into the genome of Newcastle disease virus strain La Sota between the P and M genes. Recombinant IBV multi-epitope vaccine candidate rLa Sota/SBNT was generated via reverse genetics, and its immune protection efficacy was evaluated in specific-pathogen-free chickens. Our results show that rLa Sota/SBNT induced IBV-specific neutralizing antibody and T-cell responses and provided significant protection against homologous and heterologous IBV challenge. Thus, the T-cell epitope peptides identified in this study could be good candidates for IBV vaccine development, and recombinant Newcastle disease virus-expressing IBV multi-epitope genes represent a safe and effective vaccine candidate for controlling infectious bronchitis. IMPORTANCE T-cell-mediated immune responses are critical for the elimination of IBV-infected cells. To screen conserved T-cell epitopes in the IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. By combining IFN-γ ELISpot, intracellular cytokine staining, and CFSE lymphocyte proliferation assays, we identified three CTL epitopes and one dual-specific T-cell epitope. The value of T-cell epitope peptides identified in the N protein was further verified by the design of an IBV multi-epitope vaccine. Results show that IBV multi-epitope vaccine candidate rLa Sota/SBNT provided cross protection against challenges with a QX-like or a TW-like IBV strain. So, T-cell-mediated immune responses play an important role in the control of viral infection, and conserved T-cell epitopes serve as promising candidates for use in multi-epitope vaccine construction. Our results provide a new perspective for the development of a safer and more effective IBV vaccine.

Anti-SARS-CoV-2 T-stem cell memory persists in ocrelizumab-treated MS patients.

BACKGROUND: Development of long-lasting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) T-cell responses in persons with multiple sclerosis (pwMS) treated with ocrelizumab is questioned. OBJECTIVE: Investigate antiviral T-cell responses after infection with SARS-CoV-2 in ocrelizumab-treated pwMS. Control groups included ocrelizumab-treated pwMS without SARS-CoV-2 infection, and non-MS individuals with and without SARS-CoV-2 infection. METHODS: Peripheral blood mononuclear cells were stimulated with SARS-CoV-2 peptide pools and T-cell reactivity was assessed by ELISPOT for interferon (IFN)-γ detection, and by multiparametric fluorescence-activated cell sorting (FACS) analyses for assessment and characterization of T-cell activation. RESULTS: ELISPOT assay against the spike and the N protein of SARS-CoV-2 displayed specific T-cell reactivity in 28/29 (96%) pwMS treated with ocrelizumab and infected by SARS-CoV-2, similar to infected persons without MS. This reactivity was present 1 year after infection and independent from the time of ocrelizumab infusion. FACS analysis following stimulation with SARS-CoV-2 peptide pools showed the presence of activation-induced markers (AIMs) in both CD4+ and CD8+ T-cell subsets in 96% and 92% of these individuals, respectively. Within naïve AIM+ CD4+ and CD8+ T-cells, we detected T memory stem cells, suggesting the acquisition of long-term memory. CONCLUSIONS: B-cell depletion using ocrelizumab does not impair the development of long-lasting anti-SARS-CoV-2 T-cell responses.