A comprehensive approach to developing a multi-epitope vaccine against Mycobacterium tuberculosis: from in silico design to in vitro immunization evaluation.

Introduction: The Bacillus Calmette-Guérin (BCG) vaccine, currently used against tuberculosis (TB), exhibits inconsistent efficacy, highlighting the need for more potent TB vaccines. Materials and methods: In this study, we employed reverse vaccinology techniques to develop a promising multi-epitope vaccine (MEV) candidate, called PP13138R, for TB prevention. PP13138R comprises 34 epitopes, including B-cell, cytotoxic T lymphocyte, and helper T lymphocyte epitopes. Using bioinformatics and immunoinformatics tools, we assessed the physicochemical properties, structural features, and immunological characteristics of PP13138R. Results: The vaccine candidate demonstrated excellent antigenicity, immunogenicity, and solubility without any signs of toxicity or sensitization. In silico analyses revealed that PP13138R interacts strongly with Toll-like receptor 2 and 4, stimulating innate and adaptive immune cells to produce abundant antigen-specific antibodies and cytokines. In vitro experiments further supported the efficacy of PP13138R by significantly increasing the population of IFN-γ+ T lymphocytes and the production of IFN-γ, TNF-α, IL-6, and IL-10 cytokines in active tuberculosis patients, latent tuberculosis infection individuals, and healthy controls, revealing the immunological characteristics and compare the immune responses elicited by the PP13138R vaccine across different stages of Mycobacterium tuberculosis infection. Conclusion: These findings highlight the potential of PP13138R as a promising MEV candidate, characterized by favorable antigenicity, immunogenicity, and solubility, without any toxicity or sensitization.

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

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

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.

Development of a rapid neutralization testing system for Rhinovirus C15 based on the enzyme-linked immunospot assay.

Human Rhinoviruses (RVs) are dominant pathogens causing a wide range of respiratory tract diseases, posing a huge threat to public health worldwide. Viruses belonging to the RV-C species are more likely to cause severe illnesses and are strongly associated with asthma onset or exacerbations than RV-A or RV-B. Rapid and sensitive detection of neutralizing antibodies (NAbs) against RV-C can promote the development of vaccines and antiviral drugs and help in the diagnosis of viral infection. In this study, a rapid neutralization testing system for RV-C15, based on an enzyme-linked immunospot assay (Nt-ELISPOT) was developed. A monoclonal antibody (MAb), named 9F9, with high binding efficacy for RV-C15 conjugated to horseradish peroxidase (HRP), was used to detect RV-C15-infected cells at a concentration of 2 µg/ml. The optimal infectious dose of RV-C15 was set at 1 × 104 TCID50/well and the cells were fixed with 0.5% formaldehyde diluted in PBS after incubation for 20 h. Compared with the traditional cytopathic effect (CPE)-based neutralization assay (Nt-CPE), Nt-ELISPOT significantly shortened the detection period and showed good consistency with the detection of neutralizing titers of both sera and NAbs. Using Nt-ELISPOT, three anti-RV-C15 NAbs were obtained with IC50 values of 0.16, 0.27, and 11.8 µg/ml, respectively. Moreover, 64 human serum samples collected from a wide range of age groups were tested for NAb against RV-C15 by Nt-ELISPOT. The total seroprevalence was 48.4% (31/64) and the positive rate was lowest in the group under 6 years old. Thus, the Nt-ELISPOT established in this study can be used as a high-throughput and rapid neutralization assay for the screening of NAbs and for seroepidemiological investigation against RV-C15.

Early immune surveillance to predict cytomegalovirus outcomes after allogeneic hematopoietic stem cell transplantation.

Background: There is no method of predicting human cytomegalovirus (HCMV) outcomes in allogeneic hematopoietic stem cell transplant recipients clinically, leading in some cases to excessive or insufficient antiviral therapy. We evaluated the early immune response of recipients with disparate HCMV outcomes. Methods: The HCMV outcomes of recipients were determined by long-term monitoring of HCMV DNA levels posttransplant. HCMV IgG and IgM concentrations at 1 week before and 1 week after transplantation, absolute lymphocyte counts, and HCMV-specific IFN-γ secreting cells at 1 month posttransplant were evaluated based on HCMV outcome. Results: All recipients were negative for HCMV IgM. Significant differences between recipients with and without HCMV reactivation were observed in pre- and post-transplant HCMV IgG antibody levels, absolute lymphocyte counts, and HCMV-specific IFN-γ secreting cells (P < 0.05). HCMV IgG antibody levels significantly increased after transplantation in recipients with HCMV reactivation (P = 0.032), but not in those without reactivation. Multivariate analysis revealed that except for the absolute lymphocyte count these biomarkers were related to HCMV reactivation, independent of other clinical factors. In time-to-event analyses, lower levels of these biomarkers were associated with an increased 150-day cumulative incidence of HCMV reactivation (log-rank P < 0.05). In recipients with HCMV reactivation, the duration of HCMV DNAemia had negative correlation with HCMV-specific IFN-γ-secreting cells (P = 0.015, r = -0.372). The relationships between the peak HCMV DNA load and absolute lymphocyte count and HCMV-specific IFN-γ-secreting cells followed the same trends (P = 0.026, r = -0.181 and P = 0.010, r = -0.317). Conclusions: HCMV IgG, absolute lymphocyte count, and HCMV-specific IFN-γ secreting cells represent the humoral and cellular immune response. Early monitoring of these immune markers could enable prediction of HCMV outcomes posttransplant and assessment of the severity of HCMV DNAemia.

Development of a rapid neutralization assay for the detection of neutralizing antibodies against coxsackievirus B1.

Coxsackievirus B1 (CVB1) is a major pathogen that causes viral myocarditis and aseptic meningitis and is implicated as a cause of type 1 diabetes mellitus. The rapid detection of neutralizing antibodies can help in the prevention and diagnosis of viral infection. The traditional cytopathic effect (CPE)-based neutralization assay (Nt-CPE) is time-consuming and labor-intensive. In this study, an efficient neutralization test based on an enzyme-linked immunospot assay and a monoclonal antibody 2E6 against CVB1 (Nt-Elispot) was developed. In this optimal Nt-Elispot, a multiplicity of infection (MOI) of 1 per well was set as the infection dose, and an incubation time of 18 hours was selected as the checkpoint. Compared with Nt-CPE, Nt-Elispot significantly shortened the detection period and displayed a good correlation with it. This established CVB1 Nt-Elispot could be applied to efficiently screen neutralizing antibodies and evaluate the level of NAb against CVB1 in large cohorts.

Is Fibromyalgia Associated with Borrelia-specific T Lymphocytes?

BACKGROUND: Although fibromyalgia is a common cause of chronic musculoskeletal pain, its aetiology and pathophysiology are uncertain. It has recently been suggested that fibromyalgia symptomatology represents a T lymphocyte-mediated immune response to pathogens, which are known risk factors for autoimmune diseases. One major suggested candidate pathogen is the bacterial genus Borrelia. However, to date, this hypothesis has not been tested. OBJECTIVE: The aim was to carry out the first test of this hypothesis by comparing Borrelia-specific T lymphocyte reactivity in fibromyalgia patients and matched controls. METHODS: The enzyme-linked immunospot assay was used to detect T-lymphocyte reactivity to Borrelia burgdorferi sensu stricto (full antigen), outer surface protein (Osp) A from Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, native OspC plus decorin binding protein A recombinant and lymphocyte function antigen-1 (shared epitope) in 27 patients who fulfilled the revised diagnostic criteria for fibromyalgia of the American College of Rheumatology and in 26 control subjects. The assays were carried out blind to the group status of the participants. RESULTS: The two groups did not differ by age, sex or ethnicity. They did not differ significantly in respect of T lymphocyte reactivity to Borrelia burgdorferi sensu stricto (full antigen) (p = 0.847), Osp mix (p = 0.709) or lymphocyte function antigen-1 (p = 0.367). CONCLUSION: This novel controlled study provides no evidence of an association between fibromyalgia and Borrelia-specific T lymphocytes.

Clinical Significance of Insulin Peptide-specific Interferon-γ-related Immune Responses in Ketosis-prone Type 2 Diabetes.

CONTEXT: Unprovoked A-ß+ ketosis-prone type 2 diabetes (KPD) is characterized by the sudden onset of diabetic ketosis/ketoacidosis (DK/DKA) without precipitating factors, negative anti-islet autoantibodies ("A-"), and preservation of ß-cell function ("ß+") after recovery from DKA. Although this phenotype often appears with acute hyperglycemia and DK/DKA just like acute-onset type 1 diabetes (AT1D), the involvement of anti-islet immune responses remains unknown. OBJECTIVE: We sought to clarify the immunological role of insulin-associated molecules in unprovoked A-ß+ KPD. METHODS: In this cross-sectional study, blood samples from 75 participants (42 with AT1D and 33 with KPD) were evaluated for interferon (IFN)-γ-secreting peripheral blood mononuclear cells (PBMCs) reactive to 4 insulin B-chain amino acid 9-23-related peptides (B:9-23rPep) using an enzyme-linked immunospot (ELISpot) assay. RESULTS: Overall, 36.4% (12/33) of KPD participants showed positive IFN-γ ELISpot assay results; the positivity rate in KPD was similar to that in AT1D (38.1%; 16/42) and statistically significantly higher than the previously reported rate in type 2 diabetes (8%; 2/25; P < .0167). Moreover, B:9-23rPep-specific IFN-γ-producing PBMC frequency was negatively correlated with age and ad lib serum C-peptide levels in all KPD participants and positively correlated with glycated hemoglobin A1c level in KPD participants with positive IFN-γ ELISpot results. CONCLUSION: These findings suggest the involvement of B:9-23rPep-specific IFN-γ-related immunoreactivity in the pathophysiology of some unprovoked A-ß+ KPD. Moreover, increased immunoreactivity may reflect transiently decreased ß-cell function and increased disease activity at the onset of DK/DKA, thereby playing a key role in DK/DKA development in this KPD phenotype.

Integrative Analysis of HTNV Glycoprotein Derived MHC II Epitopes by In Silico Prediction and Experimental Validation.

Hantaan virus (HTNV), the causative pathogen of hemorrhagic fever with renal syndrome (HFRS), is a negative RNA virus belonging to the Orthohantaviridae family. HTNV envelope glycoprotein (GP), encoded by the genomic medium segment, is immunogenic and is therefore a promising vaccine candidate. Major histocompatibility complex class I (MHC-I) epitopes derived from HTNV has been extensively studied, but little is known of MHC-II epitopes. In silico predictions based on four databases indicated that the full-length HTNV GP has 1121 15-mer epitopes, of which 289 had a high score for binding to the human and murine MHC-II superfamily. It found that epitope ILTVLKFIANIFHTS could potentially bind most MHC-II molecules covering human and murine haplotypes. Dominant epitopes were validated by enzyme-linked immunospot assay of splenocytes from immunized mice; 6 of 10 epitopes supported the predictions including TATYSIVGPANAKVP, TKTLVIGQCIYTITS, FSLLPGVAHSIAVEL, CETYKELKAHGVSCP, CGLYLDRLKPVGSAY, and NLGENPCKIGLQTSS. Conservation analysis of dominant epitopes revealed host-virus interactions without geographic stratification, thus meeting the requirements of candidate vaccines for large-population prophylaxis. These findings provide insight into hantavirus antigenicity and suggest that vaccines targeting MHC-II could provide immune protection in large population to complement symptomatic therapies for the treatment of HFRS.

Warfarin-induced Stevens-Johnson syndrome with severe liver injury.

Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a life-threatening mucocutaneous disease that is predominantly drug-induced. Warfarin is the most commonly used drug for long-term anti-coagulant therapy; however, warfarin-induced SJS/TEN is seldom reported. In this study, we presented the case of a 61-year-old man who developed SJS after receiving multiple-drug therapy following aortic valve replacement surgery. The patient was diagnosed with drug-induced liver injury (DILI) based on significantly abnormal liver function test results. Warfarin was identified as the culprit drug using the algorithm of drug causality for epidermal necrolysis (ALDEN) score, enzyme-linked immunospot (ELISPOT) assay, and Roussel Uclaf Causality Assessment Method (RUCAM). After warfarin discontinuation and corticosteroid therapy, the lesions and liver function test findings improved. Human leukocyte antigen typing was conducted to detect the risk allele. To our knowledge, this is the first reported case of warfarin-induced SJS/TEN with DILI. This case suggests that commonly used and safe pharmaceutical agents such as warfarin can potentially cause serious adverse events, including SJS/TEN and DILI. The application of ALDEN, the ELISPOT assay, and RUCAM could be useful in identifying culprit drugs.

The Role of In Vivo and Ex Vivo Diagnostic Tools in Severe Delayed Immune-Mediated Adverse Antibiotic Drug Reactions.

BACKGROUND: The use of in vivo and ex vivo diagnostic tools for delayed immune-mediated adverse drug reactions is currently ill defined. OBJECTIVE: To determine whether the combination of skin testing and/or IFN-γ enzyme-linked immunoSpot assay (ELISpot) can aid diagnosis of these allergy phenotypes. METHODS: Patients with antibiotic-associated severe delayed immune-mediated adverse drug reaction hypersensitivity, including Stevens-Johnson syndrome and toxic epidermal necrolysis, drug reaction with eosinophilia and systemic symptoms (DRESS), acute generalized exanthematous pustulosis, generalized bullous fixed drug eruption, and severe maculopapular exanthema, were prospectively recruited. In vivo testing was completed to the implicated drug(s), and ex vivo testing was performed with the patient's PBMCs stimulated with the relevant antibiotic concentrations for IFN-γ release ELISpot measurement. RESULTS: Eighty-one patients met the inclusion criteria, with DRESS (42; 51.9%) accounting for most cases. Among the 63 (78%) who had an ELISpot assay performed, 34 (54%) were positive to at least 1 implicated antibiotic (median spot-forming units/million cells, 99.5; interquartile range, 68-187), with glycopeptide being a strong predictor of positivity (adjusted odds ratio, 6.11; 95% CI, 1.74-21.42). In combination (in vivo and ex vivo), 51 (63%) of those tested were positive to an implicated antibiotic. For DRESS and severe maculopapular exanthema associated with penicillins and cephalosporins, this combination confirmed the culprit agent in 11 of the 12 cases and in 6 of 7 for DRESS associated with glycopeptides. CONCLUSIONS: This study demonstrates that using in vivo in combination with ex vivo testing can enhance the diagnostic approach in these severe phenotypes by assisting with the identification of possible culprit antibiotics.

Usefulness of BK virus-specific interferon-γ enzyme-linked immunospot assay for predicting the outcome of BK virus infection in kidney transplant recipients.

BACKGROUND/AIMS: To investigate if BK virus (BKV)-specific T cell immunity measured by an interferon-γ enzyme-linked immunospot (ELISPOT) assay can predict the outcome of BK virus infection in kidney transplant recipients (KTRs). METHODS: We included 68 KTRs with different viremia status (no viremia [n = 17], BK viremia [n = 27], and cleared viremia [n = 24]) and 44 healthy controls (HCs). The BK viremia group was divided into controller (< 3 months) and noncontroller (> 3 months) according to sustained duration of BKV infection. We compared BKV-ELISPOT results against five BKV peptides (large tumor antigen [LT], St, VP1-3). RESULTS: BKV-ELISPOT results were higher in three KTRs groups with different BKV infection status than the HCs group (p < 0.05). In KTR groups, they were higher in cleared viremia group than no viremia or BK viremia group. Within the BK viremia group, controller group had higher LT-ELISPOT results compared to noncontroller group (p = 0.032). Also, KTRs without BK virus-associated nephropathy (BKVN) had higher LT, St, VP1, and VP2-ELISPOT results than those with BKVN (p < 0.05). CONCLUSION: BKV-ELISPOT assay may be effective in predicting clinical outcomes of BKV infection in terms of clearance of BK virus and development of BKVN.

Diagnostic value of combined islet antigen-reactive T cells and autoantibodies assays for type 1 diabetes mellitus.

AIMS/INTRODUCTION: Type 1 diabetes mellitus is a T cell-mediated autoimmune disease. However, the determination of the autoimmune status of type 1 diabetes mellitus relies on islet autoantibodies (Abs), as T-cell assay is not routinely carried out. This study aimed to investigate the diagnostic value of combined assay of islet antigen-specific T cells and Abs in type 1 diabetes mellitus patients. MATERIALS AND METHODS: A total of 54 patients with type 1 diabetes mellitus and 56 healthy controls were enrolled. Abs against glutamic acid decarboxylase (GAD), islet antigen-2 and zinc transporter 8 were detected by radioligand assay. Interferon-γ-secreting T cells responding to glutamic acid decarboxylase 65 and C-peptide (CP) were measured by enzyme-linked immunospot. RESULTS: The positive rate for T-cell responses was significantly higher in patients with type 1 diabetes mellitus than that in controls (P < 0.001). The combined positive rate of Abs and T-cell assay was significantly higher than that of Abs assay alone (85.2% vs 64.8%, P = 0.015). A significant difference in fasting CP level was found between the T+ and T- groups (0.07 ± 0.05 vs 0.11 ± 0.09 nmol/L, P = 0.033). Furthermore, levels of fasting CP and postprandial CP were both lower in the Ab- T+ group than the Ab- T- group (fasting CP 0.06 ± 0.05 vs 0.16 ± 0.12 nmol/L, P = 0.041; postprandial CP 0.12 ± 0.13 vs 0.27 ± 0.12 nmol/L, P = 0.024). CONCLUSIONS: Enzyme-linked immunospot assays in combination with Abs detection could improve the diagnostic sensitivity of autoimmune diabetes.

[Comparison of islet autoantigen-specific T cell response detected by direct enzyme-linked immunospot (ELISPOT) assay and accelerated co-cultured dendritic cells (acDCs) assay].

Objective: To investigate the effect of enzyme-linked immunospot (ELISPOT) on accelerated co-cultured dendritic cells (acDCs) and direct detection of islet full-length antigen-specific T cell response in peripheral blood of patients with type 1 diabetes mellitus (T1DM). Methods: Sixteen patients with T1DM[9 males, 7 females, mean age(28.5±9.4)years] and 12 age-and sex-matched healthy controls were selected in the Department of Metabolism and Endocrinology, the Second Xiangya Hospital between March 2012 and August 2014. The numbers of IFN-γ secreting CD4(+)T cells responding to glutamic acid decarboxylase 65 (GAD(65)), C-peptide (CP) and insulin (INS) were detected by ELISPOT-acDCs and ELISPOT-direct assays, respectively. The positive rate of islet autoantigen and associated antigen reactive T cells under different detection assays were compared. Results: The positive rate for GAD(65), INS, and CP antigen reactive T cells detected by ELISPOT-acDCs was 1/16, 6/16 and 4/16, respectively, and T cells positive for INS in T1DM patients were higher than that in the controls (0/12) (P=0.024). Combining GAD(65), CP and INS-ELISPOT-acDCs detection, the positive rate for CD4(+) T cells in T1DM patients was higher than that in the controls (9/16 vs 1/12, P=0.016). The positive rate for GAD(65), INS, and CP antigen reactive T cells detected by ELISPOT-direct detection was 2/16, 1/16 and 7/16, respectively, and T cells positive for CP was higher than that in the controls (1/12), but the difference was not statistically significant (P=0.088). Likewise, the positive rate for CD4(+) T cells was higher in T1DM patients than that in the controls by combined GAD(65), CP and INS-ELISPOT-direct detection (8/16 vs 1/12, P=0.039). Compared with the ELISPOT-direct assay, the positive rate of INS antigen specific T cell response detected by ELISPOT-acDCs was higher (P=0.041). No statistical differences of other antigens were found between the two groups (all P>0.05). Conclusions: Both multiple islet antigens-combined CD4(+)-ELISPOT-acDCs and direct assays could provide diagnostic value of cellular immunology for T1DM patients. The ELISPOT-acDCs assay is superior to the ELISPOT-direct assay in the detection of INS antigen-specific T cell response.

Diagnostic usefulness of the cytomegalovirus (CMV)-specific T cell-based assay for predicting CMV infection after kidney transplant.

BACKGROUND/AIMS: We evaluated the usefulness in kidney transplant (KT) candidates of cytomegalovirus (CMV)-specific enzyme-linked immunospot (ELISPOT) assays for predicting the development of post-transplant CMV infections. METHODS: All adult recipients admitted for living-donor KT between March 2014 and March 2015 were prospectively enrolled except donor CMV-seropositive and recipient seronegative (D+/R-) recipients. All the enrolled patients underwent CMV-specific ELISPOT assays before transplant, and a researcher blinded to the results of these assays examined the patients for CMV infection at least 6 months post-transplant. RESULTS: Of 133 KT recipients, 44 (33%) developed CMV infections. When we used the cut-off determined by receiver operator characteristic curve, 16 of the 34 patients (47%) with negative pp65-specific ELISPOT results (< 11 spots/200,000 cells) developed CMV infections, whereas 28 of the 99 patients (39%) with positive pp65-specific ELISPOT results at baseline (≥ 11 spots/200,000 cells) developed CMV infections after KT (p = 0.02). Based on the multivariable Cox regression model, negative pp65-specific ELISPOT assay results was an independent risk factor for CMV infection (adjusted hazard ratio [AHR], 1.87; 95% confidence interval [CI], 1.01 to 3.46; p = 0.047) as well as age (AHR, 1.05; 95% CI, 1.01 to 1.08; p = 0.007). CONCLUSION: Pre-transplant CMV-specific ELISPOT assay appears to predict the development of CMV infections after KT in recipients at moderate risk such as CMV-seropositive recipients (Clinical Trial Registration Number NCT02025335).

The establishment of CMV antigen-specific enzyme-linked immunospot assay among Chinese: A pilot study.

BACKGROUND: The prevalence of cytomegalovirus in China is high and the infection could result in disastrous consequences. Cellular immunity is the main mechanism for virus controlling. We explored the performance of cytomegalovirus antigen-specific enzyme-linked immunospot assay in patients with different infection states in endemic area. METHODS: Twenty-eight patients with reliable results were included in statistical analysis. Peripheral blood mononuclear cells were extracted and were stimulated by cytomegalovirus phosphoprotein 65 or immediate early protein-1 antigen respectively. Spot forming cells (SFCs) were counted to evaluate the cellular immune response elicited by antigens. RESULTS: Spots could be clearly displayed, and evenly dispersed with a clean background. The numbers of SFCs were 0 [0-0], 426 [210-601] and 230 [57-513] for uninfected individuals (n = 7), latently infected patients (n = 11) and actively infected patients (n = 10) respectively, which were statistically different. The number of SFCs stimulated by phosphoprotein 65 was significantly higher than that by immediate early protein-1. CONCLUSIONS: The number of SFCs was significantly different among patients with different infection state. The stimulatory effect of phosphoprotein 65 was better than that of immediate early protein-1.

The effect of pneumococcal immunization on total and antigen-specific B cells in patients with severe chronic kidney disease.

BACKGROUND: While the 23-valent pneumococcal polysaccharide vaccine (PPV23) is routinely used in Canada and some other countries to prevent pneumococcal infection in adults with chronic kidney disease (CKD), patients develop a suboptimal antibody response to PPV23 due to their immune dysfunction. The 13-valent pneumococcal conjugate vaccine (PCV13) has superior immunogenicity in some categories of immunocompromised adults; however, its effect on the immune response in CKD patients has only been addressed by two recent studies with conflicting results. The effect of PPV23 or PCV13 on B cells in these patients has not been previously studied. We studied the absolute numbers and proportions of B cells and subpopulations in two groups of adult patients with severe CKD pre- and 7 days post-immunization with PCV13: pneumococcal vaccine naïve and previously immunized with PPV23 (over one year ago). RESULTS: PPV23 immunized patients had significantly lower proportions and absolute numbers of class switched memory (CD19 + CD27 + IgM-), as well as lower absolute numbers of IgM memory (CD19 + CD27 + IgM+) and class switched B cells (CD19 + CD27-IgM-) compared to PPV23 naïve patients. Following PCV13 immunization, the differences in absolute numbers of B-cell subpopulations between groups remained significant. The PPV23 immunized group had higher proportions of CD5- B cells along with lower proportions and absolute numbers of CD5+ B cells compared to PPV23 naïve patients both pre- and post-immunization with PCV13. However, previous PPV23 immunization did not have a noticeable effect on the numbers of total IgG or serotype 6B and 14 specific antibody-secreting cells detected 7 days post-immunization with PCV13. Nevertheless, fold increase in anti-serotype 14 IgG concentrations 28 days post-PCV13 was greater in PPV23 naïve than in previously immunized patients. CONCLUSIONS: The results suggest that immunization with PPV23 may result in long-term changes in B-cell subpopulations such as increased prevalence of CD5- B cells and decreased prevalence of class switched memory B cells in the peripheral blood. Because previous immunization with PPV23 in patients with CKD is associated with a significant decrease in the total class switched memory B cells in response to subsequent immunization with PCV13, this may reduce PCV13 immunogenicity in the setting of PPV23 followed by PCV13. TRIAL REGISTRATION: Registered February 24, 2015 at ClinicalTrials.gov (NCT02370069).

Significance of peripheral mononuclear cells producing interferon-γ in response to insulin B:9-23-related peptides in subtypes of type 1 diabetes.

Type 1 diabetes is largely caused by ß-cell destruction through anti-islet autoimmunity. Reportedly, interferon (IFN)-γ-secreting peripheral blood mononuclear cells (PBMCs) specific to four insulin B-chain amino acid 9-23-related peptides (B:9-23rPep) were increased in type 1 diabetes participants. This study aimed to investigate the PBMC frequencies in subtypes of type 1 diabetes using enzyme-linked immunospot assay. In this cross-sectional study, peripheral blood samples were obtained from 148 participants including 72 with acute-onset type 1 diabetes (AT1D), 51 with slowly progressive insulin-dependent diabetes mellitus (SPIDDM), and 25 with type 2 diabetes. The frequency of B:9-23rPep-specific IFN-γ-producing PBMCs was significantly higher in AT1D participants than in SPIDDM and type 2 diabetes participants. Meanwhile, a significant inverse correlation was observed between the PMBC frequencies and insulin secretion capacity in SPIDDM participants. These findings suggest that the increased peripheral B:9-23rPep-specific IFN-γ immunoreactivity reflects decreased functional ß-cell mass and greater disease activity of type 1 diabetes.

[P(0) protein promotes the expression of INF-γ and IL-10 in peripheral blood of n-hexane neuropathy patients].

Objective: To study the changes of monocyte cytokines in peripheral blood of n-hexane neuropathy patients induced by P(0) protein, and to explore the role of autoimmunity in n-hexane neuropathy patients. Methods: In May 2018, 5 patients with peripheral neuropathy diagnosed as n-hexane poisoning were selected as case group in Shenzhen Prevention and Treatment Center for Occupational Disease in 2017. 6 workers exposure to n-hexane and 6 workers without n-hexane exposure were selected as contact group and control group. Peripheral blood mononuclear cells(PBMC) were isolated from venous blood. Results: The number of spots produced by INF-γ and IL-10 increased after stimulation with P(0) protein in case group, and the positive rate was significantly higher than control group and the contact group. Conclusion: Autoimmunity induced by P(0) protein may be involved in the occurrence of myelin sheath damage in n-hexane neuropathy patients.