Enzyme-linked Immunospot Assay Research Articles

Validation of a double-color ELISpot assay of IFN-γ and IL-4 production in human peripheral blood mononuclear cells

The Enzyme-Linked ImmunoSpot (ELISpot) assay detects cytokines secreted during T cell-specific immune responses against pathogens. As this assay has acquired importance in the clinical setting, standard bioanalytical evaluation of this method is required. Here, we describe a formal bioanalytical validation of a double-color ELISpot assay for the evaluation of IFN-γ and IL-4 released by T helper 1 and T helper 2 cells, respectively. As recommended by international guidelines, the parameters assessed were: range and detection limits (limit of detection, LOD; upper and lower limit of quantification, ULOQ and LLOQ), Linearity, Relative Accuracy, Repeatability, Intermediate Precision, Specificity and Robustness. The results obtained in this validation study demonstrate that this assay meets the established acceptability criteria. ELISpot is therefore a reliable technique for measuring T cell-specific immune responses against various antigens of interest.

Aplastic Anemia Patients Have Diverse T Cell Repertoires and Flexible T Cell Responses Similar to Healthy Individuals

Acquired aplastic anemia (AA) is a bone marrow failure disease caused by T cell-mediated autoimmune attack on hematopoietic stem and progenitor cells (HSPCs). Identifying autoreactive T cells in AA has been challenging. Advances in T cell receptor (TCR) sequencing and the discovery of Human Leukocyte Antigen Class I (HLA-I) risk alleles linked to autoantigen presentation in AA have rekindled interest in exploring T cell repertoires in AA. We hypothesized that autoreactive CD8 + T cells in AA may have convergent TCR structures allowing them to recognize AA autoantigens presented by specific HLA-I risk alleles. To test our hypothesis, we analyzed TCR repertoires and HLA-restricted CD8 + T cell reactivity in AA patients. Using TCR Vβ (TRB) sequencing, we analyzed 83 patient samples obtained at AA diagnosis or relapse. High-risk alleles HLA-B*14:02 and B*40:02 were present in 26% and 10% of patients, respectively. HLA-DR*15 was present in 24%. Patient samples were largely sorted bone marrow (BM) CD8 + T lymphocytes. Patient TRB repertoires were compared to those in 709 control samples, largely of unsorted peripheral blood (PB). To account for sampling differences, we downsampled sequences to the minimum number of clones in the matching tissue. Normalized clone size distributions were visualized on rank plots (Fig 1). TRB repertoires in both patients and controls were diverse. Highly expanded clones (>5%) were rare in either group. Patients had 1,415,069 unique CDR3 amino acid sequences (CDR3AA), of which 106,446 (7.5%) were shared by ≥2 patients. Among the 106,446 shared CDR3AA, 95.4% were found in controls, and 4.3% TCRs had reported specificity to exogenous (mostly viral) or endogenous antigens in VDJdb. 4,920 (4.6%) of the shared CDR3AA were detected only in patients (median 2 patients (2-25)). Of these, 190 exhibited TCR convergence in ≥1 patient, such that the same CDR3AA was encoded by ≥2 distinct nucleotide sequences. Clustering 4,920 patient-associated CDR3AA using GLIPH (Grouping of lymphocyte interactions by paratope hotspots) identified 43 TCR antigen convergence groups. CDR3AA in each cluster were not significantly expanded in the majority of patients and were not enriched for the high-risk alleles HLA-B*14:02 or B*40:02. Our data suggest that most CDR3AA in AA are private (unique to individual patients). Shared autoreactive CDR3AA may exist in patients but are likely to also be present in healthy individuals. As the next step towards identifying autoreactive T cells, we focused on AA patients with known HLA-I risk alleles. We tested their CD8 + T cells for reactivity with endogenously expressed antigens by stimulating T cells with hematopoietic cell lines engineered to express single HLA-I alleles matching those of the patients (Fig 2). TRB sequencing showed increased clonality and significant expansion of a subset of clonotypes after T cell stimulation. Interferon γ enzyme-linked immunospot assays confirmed the expanded T cells' responsiveness to antigens presented by HLA alleles used for T cell stimulation. Additionally, these expanded T cells demonstrated variable reactivity to cells expressing other HLA alleles from the same patient. Among the 1% of CDR3AA clonotypes most highly expanded with single HLA-I alleles (63-3000-fold), we identified several that were expanded in an HLA-restricted manner. However, several highly expanded CDR3AA were highly expanded with multiple HLA alleles. Potential reasons for expansion of some CDR3AA after stimulation with cells expressing different HLA alleles include 1) binding of the same CDR3AA to different antigens presented by distinct HLA alleles, 2) expansion in response to the same antigens presented by distinct HLA alleles, or 3) expansion in response to non-HLA antigens. In conclusion, our data show diverse and mostly private TRB repertoires in AA patients. Our in vitro findings suggest that much like T cell responses in non-AA individuals, T cell responses in AA are diverse and exhibit flexibility with respect to activation in response to various endogenous antigens in the context of different self HLA-I alleles. Given the diversity of TCR responses observed in AA patients, the tissue specificity of autoimmune attack and established association with risk HLA-I alleles in AA remain a mystery and likely require HSPC-specific autoantigen expression and efficient autoantigen presentation by individual patients' HLA-I alleles.

Cell-Mediated Immunity (CMI) for SARS-CoV-2 Infection Among the General Population of North India: A Cross-Sectional Analysis From a Sub-sample of a Large Sero-Epidemiological Study.

Background Cell-mediated immunity (CMI), or specifically T-cell-mediated immunity, is proven to remain largely preserved against the variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including Omicron. The persistence of cell-mediated immune response in individuals longitudinally followed up for an extended period remains largely unelucidated. To address this, the current study was planned to study whether the effect of cell-mediated immunity persists after an extended period of convalescence or vaccination. Methods Whole blood specimens of 150 selected participants were collected and tested for Anti-SARS-CoV-2 Interferon-gamma (IFN-γ) response. Ex vivo SARS-CoV-2-specific interferon-gamma Enzyme-linked Immunospot (IFN-γ ELISpot) assay was carried out to determine the levels of virus-specific IFN-γproducing cells in individual samples. Findings Out of all the samples tested for anti-SARS-CoV-2 T-cell-mediated IFN-γ response, 78.4% of samples were positive. The median (interquartile range) spots forming units (SFU) per million levels of SARS-CoV-2-specific IFN-γ producing cells of the vaccinated and diagnosed participants was 336 (138-474) while those who were vaccinated but did not have the disease diagnosis was 18 (0-102); the difference between the groups was statistically significant. Since almost all the participants were vaccinated, a similar pattern of significance was observed when the diagnosedand the never-diagnosed participants were compared, irrespective of their vaccination status. Interpretations Cell-mediated immunity against SARS-CoV-2 persisted, irrespective of age and sex of the participant, for more than six months of previous exposure. Participants who had a history of diagnosed COVID-19 infection had better T-cell response compared to those who had never been diagnosed, in spite of being vaccinated.

Gpibα Caar-T Cells Induce Autoreactive B Cells Elimination to Treat Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is a bleeding disorder caused by autoantibodies against platelet glycoproteins (GPs). A key attempt to treat ITP is to eliminate the autoreactive B cells. Here, we proposed a new strategy for B cell-depletion therapy of ITP, chimeric autoantibody receptors T cells (CAAR-T), which could trap autoreactive B cells and perform specific killing. GPIbα is a significant autoantigen found in ITP patients and indicates a poor response to standard immunosuppressive treatments. We developed GPIbα-based CAARs in this study. Aim: To precisely eliminate the autoreactive B cells to treat ITP. Methods: We constructed various lengths of the native GPIbα ectodomain into the ligand-binding domain of a second-generation CAR structure and expressed the CAAR structure on the HEK293 cell surface to verify its conformational epitope. We immunized BALB/C mice with human platelet lysates to obtain anti-GPIbα hybridomas and performed a binding assay to confirm selected hybridoma antibodies' binding ability to GPIbα CAAR. We performed cytotoxicity assays to assess GPIbα CAAR-T-cell selective cytolysis against anti-GPIbα B cells in vitro and developed a xenograft model to assess the cytolytic efficiency and security of GPIbα CAAR-T cells in vivo. A plasma antibody binding assay was used to test GPIbα CAAR-T cells' ability in reaction with native GPIbα autoantibodies from ITP patients. We applied an enzyme-linked immunospot (ELISpot) assay to verify GPIbα CAAR-T cells' potential in eradicating autoreactive B cells of ITP patients. Results: We successfully expressed GPIbα CAAR structure on the cell surface and proved that the conformational epitope of GPIbα CAAR is consistent with that of the native human platelet GPIbα. The Schematic of GPIbα CAAR structure is presented in Figure 1A. By immunizing BALB/C mice with human platelet lysates, we obtained four anti-GPIbα hybridomas with different CAAR binding sites. Vitro cytotoxicity assays confirmed GPIbα CAAR-T-cell selective cytolysis of anti-GPIbα hybridoma cells. In a hybridoma xenograft model, we verified that the CAAR-T cells could persist and selectively lyse autoreactive B cells and subsequently reduce autoantibody titers without apparent off-target organ toxicity. Through the plasma antibody binding assay, we demonstrated that GPIbα CAAR could react with sera from ITP patients with anti-GPIbα antibodies (Figure 1B). ELISpot analysis showed that GPIbα CAAR-T cells successfully eliminated anti-GPIbα B cells from a patient with refractory ITP, and normal B cells were not destroyed compared to anti-CD19 CAR-T cells. Conclusion: Our study provides a proof of concept for CAAR-T cell therapy to eradicate autoimmune B cells while sparing healthy B cells, which would compensate for the defects of traditional B cell depletion therapy in ITP. GPIbα CAAR-T therapy would be a promising treatment for refractory and relapsed ITP patients.

In Silico Analyses, Experimental Verification and Application in DNA Vaccines of Ebolavirus GP-Derived pan-MHC-II-Restricted Epitopes.

(1) Background and Purpose: Ebola virus (EBOV) is the causative agent of Ebola virus disease (EVD), which causes extremely high mortality and widespread epidemics. The only glycoprotein (GP) on the surface of EBOV particles is the key to mediating viral invasion into host cells. DNA vaccines for EBOV are in development, but their effectiveness is unclear. The lack of immune characteristics resides in antigenic MHC class II reactivity. (2) Methods: We selected MHC-II molecules from four human leukocyte antigen II (HLA-II) superfamilies with 98% population coverage and eight mouse H2-I alleles. IEDB, NetMHCIIpan, SYFPEITHI, and Rankpep were used to screen MHC-II-restricted epitopes with high affinity for EBOV GP. Further immunogenicity and conservation analyses were performed using VaxiJen and BLASTp, respectively. EpiDock was used to simulate molecular docking. Cluster analysis and binding affinity analysis of EBOV GP epitopes and selected MHC-II molecules were performed using data from NetMHCIIpan. The selective GP epitopes were verified by the enzyme-linked immunospot (ELISpot) assay using splenocytes of BALB/c (H2d), C3H, and C57 mice after DNA vaccine pVAX-GPEBO immunization. Subsequently, BALB/c mice were immunized with Protein-GPEBO, plasmid pVAX-GPEBO, and pVAX-LAMP/GPEBO, which encoded EBOV GP. The dominant epitopes of BALB/c (H-2-I-AdEd genotype) mice were verified by the enzyme-linked immunospot (ELISpot) assay. It is also used to evaluate and explore the advantages of pVAX-LAMP/GPEBO and the reasons behind them. (3) Results: Thirty-one HLA-II-restricted and 68 H2-I-restricted selective epitopes were confirmed to have high affinity, immunogenicity, and conservation. Nineteen selective epitopes have cross-species reactivity with good performance in MHC-II molecular docking. The ELISpot results showed that pVAX-GPEBO could induce a cellular immune response to the synthesized selective peptides. The better immunoprotection of the DNA vaccines pVAX-LAMP/GPEBO coincides with the enhancement of the MHC class II response. (4) Conclusions: Promising MHC-II-restricted candidate epitopes of EBOV GP were identified in humans and mice, which is of great significance for the development and evaluation of Ebola vaccines.

Longitudinal Analysis of SARS-CoV-2-Specific Cellular and Humoral Immune Responses and Breakthrough Infection following BNT162b2/BNT162b2/BNT162b2 and ChAdOx1/ChAdOx1/BNT162b2 Vaccination: A Prospective Cohort in Naive Healthcare Workers.

Assessing immune responses post-SARS-CoV-2 vaccination is crucial for optimizing vaccine strategies. This prospective study aims to evaluate immune responses and breakthrough infection in 235 infection-naïve healthcare workers up to 13-15 months after initial vaccination in two vaccine groups (108 BNT/BNT/BNT and 127 ChAd/ChAd/BNT). Immune responses were assessed using the interferon-gamma enzyme-linked immunospot (ELISPOT) assay, total immunoglobulin, and neutralizing activity through surrogate virus neutralization test at nine different time points. Both groups exhibited peak responses one to two months after the second or third dose, followed by gradual declines over six months. Notably, the ChAd group exhibited a gradual increase in ELISPOT results, but their antibody levels declined more rapidly after reaching peak response compared to the BNT group. Six months after the third dose, both groups had substantial cellular responses, with superior humoral responses in the BNT group (p < 0.05). As many as 55 breakthrough infection participants displayed higher neutralization activities against Omicron variants, but similar cellular responses compared to 127 infection-naïve individuals, suggesting cross-immunity. Distinct neutralization classifications (<30%, >80% inhibition) correlated with different ELISPOT results. Our study reveals diverse immune response patterns based on vaccine strategies and breakthrough infections, emphasizing the importance of understanding these dynamics for optimized vaccination decisions.

Cytomegalovirus antigen-specific T cell immune responses in patients with autoimmune diseases under different cytomegalovirus infection status.

Cytomegalovirus antigen-specific T cell immune responses in patients with autoimmune diseases under different cytomegalovirus infection status.

Optimization of Peripheral Blood Mononuclear Cell Processing for Improved Clinical ELISpot Assay Performance.

Cell and gene therapies have demonstrated impressive therapeutic efficacy in various human diseases. Nevertheless, cellular immune response directed against these therapeutic agents is an obstacle for achieving long-lasting clinical efficacy. Therefore, it is crucial to develop robust assays to accurately monitor cellular immunogenicity towards these therapies. Enzyme-linked immunospot (ELISpot) assay is one of the primarily used methods for measuring cellular immune response in clinical programs, which requires isolation of the peripheral blood mononuclear cells (PBMCs). The quality of this clinical material is one of the most critical factors that impact the robust assessment of cellular immune responses. The optimal blood sample processing conditions, however, remain poorly understood. In this study, we examined the impact of blood sample processing time on the performance characteristics of ELISpot to measure antigen-specific cellular responses. Blood samples that were processed after overnight delay resulted in a loss of ELISpot signals. We subsequently optimized several parameters of sample processing, and successfully recovered ELISpot signals for the blood samples that are processed within 32h. Furthermore, several mitigation strategies were employed that would potentially address the impact of granulocyte contamination on detection of antigen-specific cellular responses. Our investigation provides an extension of sample processing window for clinical studies and is significant for resolving the logistical challenge of whole blood sample shipment for timely PBMC preparation in cell/gene therapy clinical studies.

Immune monitoring of SARS-CoV-2-specific T cell and B cell responses in patients with multiple sclerosis treated with ocrelizumab.

Since the development of the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there has been significant interest in determining the effectiveness of SARS-CoV-2 vaccines in patients under immunomodulatory or immunosuppressive therapies. The aim of this study was to evaluate the impact of ocrelizumab, a monoclonal anti-CD20 antibody, on SARS-CoV-2-specific T cell and B cell responses in patients with relapsing-remitting multiple sclerosis (RRMS). To this end, peripheral blood mononuclear cells (PBMCs) were isolated from n = 23 patients with RRMS. Of these patients, n = 17 were tested before (time point t0) and one month after (time point t1) their first dose of ocrelizumab. In addition, we studied n = 9 RRMS patients that got infected with SARS-CoV-2 over the course of ocrelizumab therapy (time point t2). PBMCs were also isolated from n = 19 age- and gender-matched healthy controls (HCs) after vaccination or infection with SARS-CoV-2, respectively. Interferon-γ (IFN-γ)/interleukin-2 (IL-2) and granzyme B (GzB)/perforin (PFN) double-color enzyme-linked immunospot (ELISPOT) assays or single-color ELISPOT assays were performed to measure SARS-CoV-2 antigen-specific T cell and B cell responses. Anti-viral antibody titers were quantified in the serum by chemiluminescence immunoassay. Our data indicate a significant difference in the SARS-CoV-2 specific IFN-γ (P = 0.0119) and PFN (P = 0.0005) secreting T cell compartment in the MS cohort at t0 compared to HCs. Following the first dose of ocrelizumab treatment, a significant decrease in the number of SARS-CoV-2 spike protein-specific B cells was observed (P = 0.0012). Infection with SARS-CoV-2 in MS patients under ocrelizumab therapy did not significantly alter their existing immune response against the virus. Kaplan-Meier survival analysis suggested that the spike S1 protein-specific immunoglobulin (Ig)G response might be a key parameter for predicting the probability of (re)infection with SARS-CoV-2. Our results call for a critical discussion regarding appropriate vaccination intervals and potential biomarkers for the prediction of (re)infection with SARS-CoV-2 in patients with MS receiving ocrelizumab. DRKS00029110; URL: http://apps.who.int/trialsearch/.

Human herpesvirus 6-specific T-cell immunity in allogeneic hematopoietic stem cell transplant recipients.

Human herpesvirus 6 (HHV-6) can reactivate after allogeneic hematopoietic stem cell transplant (allo-HSCT) and may lead to severe symptoms. HHV-6-specific immune responses after HSCT are largely unexplored. We conducted a prospective observational study on 208 consecutive adult patients who received allo-HSCT to investigate HHV-6 reactivations and specific immune responses. Interferon gamma-producing HHV-6-specific T cells were quantified using enzyme-linked immunospot assay (ELISpot). HHV-6 reactivation occurred in 63% of patients, at a median of 25 days from allo-HSCT. Only 40% of these presented a clinically relevant infection, defined by the presence of classical HHV-6 end-organ diseases (EODs), based on European Conference on Infections in Leukaemia (ECIL) guidelines, and other possible HHV6-related EODs. Using multivariate analysis, we identified risk factors for HHV-6 reactivation: previous allo-HSCT, posttransplant cyclophosphamide (PT-Cy), and time-dependent steroids introduction. The use of PT-Cy and steroids were associated with clinically relevant infections, whereas higher CD3+ cell counts seemed to be protective. Interestingly, circulating HHV-6-specific T cells were significantly higher in patients with reactivated virus. Moreover, HHV-6-specific T-cell responses, quantified at >4 days after the first viremia detection, predicted clinically relevant infections (P< .0001), with higher specificity (93%) and sensitivity (79%) than polyclonal CD3+ cells per μL. Overall survival and transplant-related mortality were not affected by time-dependent HHV-6 reactivation, whereas a significant association was observed between clinically relevant infections and acute graft-versus-host disease. These results shed light on the role of HHV-6 in allo-HSCT and may affect HHV-6 monitoring and treatment.

A Subunit Vaccine Candidate Composed of Mpox Virus A29L, M1R, A35R, and B6R Elicits Robust Immune Response in Mice.

With no specific antiviral drugs and preventive vaccines against Mpox virus (MPXV), the epidemic has led to the declaration of a Public Health Emergency of International Concern. As a developmental direction for new vaccines, studies of subunit vaccines based upon MPXV antigen proteins are lacking. In this study, A29L, M1R, A35R, and B6R of MPXV were expressed and purified from a prokaryotic system. The four MPXV antigen proteins in combination were mixed with aluminum hydroxide or CpG7909 as adjuvant, and subsequently used to inoculate mice. The results of enzyme-linked immunosorbent assay (ELISA), flow cytometry analyses, and enzyme-linked immunospot (ELISPOT) assays indicated that A29L, M1R, A35R, and B6R elicited high-level antigen-specific antibodies and CD4+ T cells-based cellular immune response in mice. Moreover, the results of virus neutralization assays suggested that sera from the mice immunized with four proteins elicited high neutralizing activities against the vaccinia virus. Notably, the results of ELISA, ELISPOT, and virus neutralization assays also showed that the CpG7909 adjuvant was more effective in inducing an immune response compared with the aluminum adjuvant. In summary, this study offers valuable insights for further studies of subunit vaccine candidates for the prevention of MPXV and other orthomyxoviruses.

MTHFD2 ablation in T cells protects against heart transplant rejection by perturbing IRF4/PD-1 pathway through the metabolic-epigenetic nexus

One-carbon metabolism supports the activation, proliferation, and function of multiple immune cells. However, researchers have not clearly determined whether and how one-carbon metabolic enzymes contribute to heart transplant rejection. We investigated the dynamic metabolic adaptation in grafts during heart transplant rejection by conducting transcriptomics, metabolomics and single-cell RNA sequencing studies of cardiac tissue from human and mouse heart transplant recipients. We also assessed the expression of the one-carbon metabolic enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in cardiac grafts by immunofluorescence and flow cytometry assays. Then we constructed a murine heart transplant model with T cell-specific Mthfd2 knockout mice, analyzed T cells function by flow cytometry assays and enzyme-linked immunospot assays, and studied the mechanism by Cleavage Under Targets and Tagmentation assays. Finally, we studied the effect of a pharmacological inhibitor of MTHFD2 in humanized skin transplant model. We revealed that the one-carbon metabolism enzyme MTHFD2 was a hallmark of alloreactive T cells and was linked to T cell proliferation and function after exposure to alloantigen. And, Mthfd2 ablation prevented murine heart transplant rejection. Mechanistically, we found Mthfd2 ablation affected the interferon regulatory factor 4/programmed death-1 pathway through a metabolic-epigenetic mechanism involving H3K4me3. Furthermore, we found that inhibiting MTHFD2 attenuated human allograft rejection in a humanized skin transplant model. These data show that the one-carbon metabolic enzyme MTHFD2 serves as a metabolic checkpoint of alloreactive T cells and suggest that it may be a potential therapeutic target for heart transplant rejection.

Dapsone hypersensitivity syndrome.

Dapsone hypersensitivity syndrome.

Comparison of Four T-cell Assays and Two Binding Antibody Assays in SARS-CoV-2 Vaccinees With or Without Omicron Breakthrough Infection.

Several T-cell response assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are available; however, their comparability and correlations with antibody responses remain unclear. We compared four SARS-CoV-2 T-cell response assays and two anti-SARS-CoV-2 spike antibody assays. We enrolled 89 participants who had received a booster dose of the BNT162b2 vaccine after two doses of the ChAdOx1 or BNT162b2 vaccine. Fifty-six participants without breakthrough infection (BI) (ChAdOx1/BNT162b2 group: N=27; BNT162b2 group: N=29) and 33 with BI were included. We evaluated two whole-blood interferon-gamma release assays (IGRAs) (QuantiFERON and Euroimmun), T-SPOT.COVID, an in-house enzyme-linked immunospot (ELISPOT) assay (targeting the spike and nucleocapsid peptides of wild-type and Omicron SARS-CoV-2), Abbott IgG II Quant, and Elecsys Anti-S, using Mann-Whitney U, Wilcoxon signed-rank, and Spearman's correlation tests. The correlations between the IGRAs and between the ELISPOT assays (ρ=0.60-0.70) were stronger than those between the IGRAs and ELISPOT assays (ρ=0.33-0.57). T-SPOT.COVID showed a strong correlation with Omicron ELISPOT (ρ=0.70). The anti-spike antibody assays showed moderate correlations with T-SPOT.COVID, Euroimmun IGRA, and ELISPOT (ρ=0.43-0.62). Correlations tended to be higher in the BI than in the noninfected group, indicating that infection induces a stronger immune response. T-cell response assays show moderate to strong correlations, particularly when using the same platform. T-SPOT.COVID exhibits potential for estimating immune responses to the Omicron variant. To accurately define SARS-CoV-2 immune status, both T-cell and B-cell response measurements are necessary.

The recombinant truncated envelope protein of West Nile virus adjuvanted with Alum/CpG induces potent humoral and T cell immunity in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus distributed globally for decades and can cause disease in humans and animals. So far, no WNV vaccine has been licensed for human use. Therefore, the development of novel candidate vaccines and the improvement of vaccination strategies is imperative. As the WNV envelope (E) glycoprotein plays an important role in mediating viral binding to cellular receptors and virus-cell membrane fusion, it is a critical target for the host humoral response. Here, we prepared a recombinant truncated envelope protein of WNV (rWNV-80E) and developed a WNV subunit vaccine formulation with a combination of aluminum hydroxide (alum) and a synthetic oligonucleotide CpG as adjuvants. C57BL/6 mice were immunized twice intramuscularly at 28-day intervals with 5 µg purified rWNV-80E adjuvanted with Alum/CpG. WNV E-specific IgG was detected by enzyme-linked immunosorbent assay and neutralizing antibodies (nAbs) was detected using single-round infectious particles of WNV. Furthermore, T cell immunity was detected by enzyme-linked immunospot assay and intracellular cytokine staining assay. Notably, rWNV-80E was highly immunogenic and elicited potent humoral and cell immunity, as evidenced by significant levels of IFN-γ and TNF-α secretion in the T cells of mice. In summary, the Alum/CpG-adjuvanted rWNV-80E subunit vaccine elicited potent and balanced B- and T-cell immunity in mice, and therefore it is a promising candidate vaccine that warrants further investigation for use in human or veterinary applications.

HLA-E-restricted Hantaan virus-specific CD8+ T cell responses enhance the control of infection in hemorrhagic fever with renal syndrome

Infection with the Hantaan virus (HTNV) may result in severe hemorrhagic fever with renal syndrome (HFRS). The functions of HLA-E-restricted CD8+ T lymphocytes in virus control and vaccine development have recently received increased attention. The purpose of this research is to discover HLA-E-restricted CD8+ T cell epitopes on HTNV as well as the features of these epitope-specific CD8+ T cells in HFRS patients. To anticipate HLA-E-restricted HTNV epitopes, the NetMHCpan servers were utilized. The K562/HLA-E cell binding test and the enzyme-linked immunospot assay were used to confirm epitope binding to HLA-E. The number and features of HLA-E-restricted epitope-specific CD8+ T lymphocytes in HFRS patients were investigated using tetramer staining, intracellular cytokine labeling, proliferation, and cytotoxicity assays. Six HTNV-derived HLA-E-restricted CD8+ T cell epitopes were found in this study. In mild/moderate HFRS patients, the frequency of HLA-E-restricted epitope-specific CD8+ T cells was greater than in severe/critical patients. CD38+HLA-DR+ HLA-E-restricted CD8+ T cells were identified. Meanwhile, CD45RA+CCR7− effector memory-re-expressing CD45RA T cells with early and intermediate maturation and differentiation characteristics predominated. Notably, CD8+ T cells from milder HFRS patients produced more interferon-γ, interleukin-2, and granzyme B, had a stronger proliferative potential, and were inversely linked with the amount of plasma HTNV virus load. Furthermore, HLA-E-restricted epitope-specific CD8+ T cells demonstrated improved cytotoxic activity in vitro during the acute stage of HFRS. Taken together, the findings demonstrate the protective effects of HLA-E-restricted CD8+ T cells during HTNV infection, suggesting that HLA-E-targeted vaccines against HTNV might be developed for HLA-diverse populations.

Identification of two novel T cell epitopes on the E2 protein of classical swine fever virus C-strain

C-strain, also known as the HCLV strain, is a well-known live attenuated vaccine against classical swine fever (CSF), a devastating disease caused by classical swine fever virus (CSFV). Vaccination with C-strain induces a rapid onset of protection, which is associated with virus-specific gamma interferon (IFN-γ)-secreting CD8+ T cell responses. The E2 protein of CSFV is a major protective antigen. However, the T cell epitopes on the E2 protein remain largely unknown. In this study, eight overlapping nonapeptides of the E2 protein were predicted and synthesized to screen for potential T cell epitopes on the CSFV C-strain E2 protein. Molecular docking was performed on the candidate epitopes with the swine leukocyte antigen-1*0401. The analysis obtained two highly conserved T cell epitopes, 90STEEMGDDF98 and 331ATDRHSDYF339, which were further identified by enzyme-linked immunospot assay. Interestingly, the mutants deleting or substituting the epitopes are nonviable. Further analysis demonstrated that 90STEEMGDDF98 is crucial for the E2 homodimerization, while CSFV infection is significantly inhibited by the 331ATDRHSDYF339 peptide treatment. The two novel T cell epitopes can be used to design new vaccines that are able to provide rapid-onset protection.

Humoral and cellular BNT162b2 mRNA-based booster vaccine-induced immunity in patients with multiple myeloma and persistence of neutralising antibodies: results of a prospective single-centre cohort study.

Currently available messenger ribonucleic acid (mRNA)-based vaccines against coronavirus disease (COVID-19) have been shown to be effective even in highly immunocompromised hosts, including patients with multiple myeloma. However, vaccination failure can be observed in all patient groups. This prospective study longitudinally assessed the humoral and cellular responses to a third booster dose of BNT162b2 mRNA-based vaccine in patients with myeloma (n = 59) and healthy controls (n = 22) by measuring the levels of anti-spike (S) antibodies (electro-chemiluminescence immunoassay) including neutralising antibodies and specific T-cells (enzyme-linked immunospot assay) following booster administration. The third booster dose showed a high immunogenicity on the serological level among the patients with multiple myeloma (median anti-S level = 41 binding antibody units [BAUs]/ml pre-booster vs 3902 BAU/ml post-booster, p <0.001; increase in the median neutralising antibody level from 19.8% to 97%, p <0.0001). Four of five (80%) patients with a complete lack of any serological response (anti-S immunoglobulin level <0.8 BAU/ml) after two vaccine doses developed detectable anti-S antibodies after booster vaccination (median anti-S level = 88 BAU/ml post-booster). T-cell responses were largely preserved among the patients with multiple myeloma with no difference from the healthy controls following baseline vaccination (median spot-forming units [SFU]/106of peripheral blood mononuclear cells = 193 vs 175, p = 0.711); these responses were augmented significantly after booster administration among the patients with multiple myeloma (median SFU/106of peripheral blood mononuclear cells = 235 vs 443, p <0.001). However, the vaccination responses remained highly heterogeneous and diminished over time, with insufficient serological responses occurring even after booster vaccination in a few patients irrespective of the treatment intensity. Our data demonstrate improvements in humoral and cellular immunity following booster vaccination and support the assessment of the humoral vaccine response in patients with multiple myeloma until a threshold for protection against severe COVID-19 is validated. This strategy can allow the identification of patients who might benefit from additional protective measures (e.g. pre-exposure prophylaxis via passive immunisation).

Multiple antigen stimulating cellular therapy (MASCT)-I for metastatic urothelial carcinoma (mUC): A multicenter, phase I study (MASCT-I-1001).

e16572 Background: Immune checkpoint inhibitors (ICIs) showed unsatisfactory efficacy in mUC. This study aimed to evaluate the role of MASCT-I alone, or plus camrelizumab or chemotherapy in patients (pts) with mUC. Methods: Eligible pts were enrolled in five groups (G): MASCT-I alone as salvage treatment in pts who failed standard treatment (G1), or as maintenance therapy following first-line platinum based-chemotherapy (G2); MASCT-I plus camrelizumab in pts who had progressed after first-line chemotherapy (G3) or ICI (G4), and MASCT-I plus platinum-based chemotherapy as first-line treatment (G5). Pts in G2 were allowed to be enrolled in G3 when disease progressed. The primary endpoint was safety. The secondary endpoints included progression-free survival (PFS) and overall survival (OS). Dynamic biomarker analysis of specific immune responses against 15 antigens was performed by enzyme-linked immunospot assays (ELISPOT) in peripheral blood mononuclear cells from pts. Results: Thirty-nine pts were included from February 16th, 2017 to March 3rd, 2022, with two, 18, nine, four and six pts in G1, 2, 3, 4 and 5, respectively. The pts were 37-72 years old, and 69.2% were male. The main MASCT-I-related adverse events (AEs) included grade 1/2 flushing, pruritus, rash, muscle cramp, fever, and arthralgia. Two pts experienced serious AEs related to both MASCT-I and camrelizumab, including grade 3 rash, adrenocortical insufficiency, and pleural effusion in one pt, and grade 3 elevated transaminases in another. No MASCT-I-related death occurred. No new safety signal related to chemotherapy or camrelizumab was reported. Thirty-six out of 39 pts had at least one response evaluation, with one complete response, six partial response and 13 stable diseases. The median PFS and OS for all pts was 2.3 months and 15.5 months, respectively. The median PFS and OS for pts in G2 was 3.0 months and 39.3 months, respectively. Pts in G2 presented significantly superior OS compared with pts in other groups ( P&lt; 0.001). In nine pts enrolled in G3 after progression in G2, the OS was significantly longer than other nine pts in G3 (median OS: not reached vs 15.4 months, hazard ratio [HR] = 0.14, 95% confidence interval [CI]: 0.03-0.61, P= 0.034). Among 24 pts with available ELISPOT analysis, six were defined as responders, which showed a significant prolonged PFS and OS compared with the non-responders (median PFS: 10.1 vs 4.5 months, HR = 0.33, 95% CI: 0.13-0.81, P&lt; 0.005; median OS: not reached vs 13.5 months, HR = 0.19, 95% CI: 0.05-0.70, P&lt; 0.005). Conclusions: Both MASCT-I alone or combined with immunotherapy or chemotherapy showed manageable safety profiles. Maintenance of MASCT-I after first-line chemotherapy presented inspiring survival benefit. Synergistic effects between MASCT-I and ICIs may exist. Potential candidates for MASCT-I treatment may be identified by ELISPOT analysis. Clinical trial information: NCT03034304 .

Interferon-gamma FlowSpot assay for the measurement of the T-cell response to cytomegalovirus

ObjectivesWe describe a new method, FlowSpot, to assess CMV-specific T-cell response by quantification of interferon-gamma (IFN-γ). CMV-specific, T-cell-released IFN-γ was captured by flow beads and measured via flow cytometry. In the present study, we used FlowSpot to assess CMV-specific T-cell response in healthy individuals. The FlowSpot results were compared with those of serological analysis and enzyme-linked immunospot (ELISpot) assay. MethodsExperimental results and parameter analysis were investigated by using serological, ELISpot, and FlowSpot assays. ResultsThe levels of IFN-γ, which is released from CMV-specific T-cells, were measured, and the results and parameter analysis showed a good correlation between FlowSpot and ELISpot. However, FlowSpot was more sensitive and better reflected the strength of IFN-γ secretion than did ELISpot. ConclusionsCompared to ELISpot, FlowSpot has a high sensitivity and is cost and time effective. Thus, this method can be used in wider clinical and scientific applications.