Positive IFN-γ Research Articles

Evaluation of naturally acquired immune responses against novel pre-erythrocytic Plasmodium vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin

BackgroundPlasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity.MethodsTwelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used.ResultsIn those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58–99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9–35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses.ConclusionsThese results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas.

Immunopeptidome mining reveals a novel ERS-induced target in T1D.

Autoreactive CD8+ T cells play a key role in type 1 diabetes (T1D), but the antigen spectrum that activates autoreactive CD8+ T cells remains unclear. Endoplasmic reticulum stress (ERS) has been implicated in β-cell autoantigen generation. Here, we analyzed the major histocompatibility complex class I (MHC-I)-associated immunopeptidome (MIP) of islet β-cells under steady and ERS conditions and found that ERS reshaped the MIP of β-cells and promoted the MHC-I presentation of a panel of conventional self-peptides. Among them, OTUB258-66 showed immunodominance, and the corresponding autoreactive CD8+ T cells were diabetogenic in nonobese diabetic (NOD) mice. High glucose intake upregulated pancreatic OTUB2 expression and amplified the OTUB258-66-specific CD8+ T-cell response in NOD mice. Repeated OTUB258-66 administration significantly reduced the incidence of T1D in NOD mice. Interestingly, peripheral blood mononuclear cells (PBMCs) from patients with T1D, but not from healthy controls, showed a positive IFN-γ response to human OTUB2 peptides. This study provides not only a new explanation for the role of ERS in promoting β-cell-targeted autoimmunity but also a potential target for the prevention and treatment of T1D. The data are available via ProteomeXchange with the identifier PXD041227.

Periodontitis exacerbates pulmonary hypertension by promoting IFNγ+ T cell infiltration in mice

Uncovering the risk factors of pulmonary hypertension and its mechanisms is crucial for the prevention and treatment of the disease. In the current study, we showed that experimental periodontitis, which was established by ligation of molars followed by orally smearing subgingival plaques from patients with periodontitis, exacerbated hypoxia-induced pulmonary hypertension in mice. Mechanistically, periodontitis dysregulated the pulmonary microbiota by promoting ectopic colonization and enrichment of oral bacteria in the lungs, contributing to pulmonary infiltration of interferon gamma positive (IFNγ+) T cells and aggravating the progression of pulmonary hypertension. In addition, we identified Prevotella zoogleoformans as the critical periodontitis-associated bacterium driving the exacerbation of pulmonary hypertension by periodontitis, and the exacerbation was potently ameliorated by both cervical lymph node excision and IFNγ neutralizing antibodies. Our study suggests a proof of concept that the combined prevention and treatment of periodontitis and pulmonary hypertension are necessary.

Identification of novel canonical and cryptic HCMV-specific T-cell epitopes for HLA-A∗03 and HLA-B∗15 via peptide-PRISM

AbstractHuman cytomegalovirus (HCMV) reactivation poses a substantial risk to patients receiving tranplants. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A∗03:01 and HLA-B∗15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes by combining ribosome sequencing (Ribo-seq) and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and Probabilistic Inference of Codon Activities by an EM Algorithm. Furthermore, using machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity, and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as patients with transplants. This resulted in the direct identification of 3 canonical and 1 cryptic HLA-A∗03–restricted immunogenic peptides as well as 5 canonical and 1 cryptic HLA-B∗15–restricted immunogenic peptide, with a specific interferon gamma–positive (IFN-γ+)/CD8+ T-cell response of ≥0.02%. High T-cell responses were detected against 2 HLA-A∗03–restricted and 3 HLA-B∗15–restricted canonical peptides with frequencies of up to 8.77% IFN-γ+/CD8+ T cells in patients after allogeneic stem cell transplantation. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq data sets.

PREVALENCE AND RISK FACTORS OF LATENT TUBERCULOSIS AMONG HEALTHCARE WORKERS IN SANA’A CITY, YEMEN USING WHOLE BLOOD INTERFERON-Γ RELEASE ASSAY

Background and aims: Tuberculosis (TB) is a critical problem of public health. Diagnosis and screening of latent Mycobacterium tuberculosis infections (LTBI) are essential to control their spread. The goal of the study was to clarify the prevalence and risk factors for LTBI using IGRA among healthcare works (HCWs) in Sana’a city, Yemen. Methods: The cross-sectional study was performed in three chosen tertiary hospitals in Sana'a city. Healthcare workers who participated in this survey were asked to complete a questionnaire and donate 5 ml of blood to perform the IGRA. A total of 180 healthcare workers were involved in this study; aged 20-60 years, 45.6% males and 54.4% females. Blood samples were collected and then tested by Interferon-gamma (IFN-γ) release assays that measure the presence of specific M. tuberculosis reactive T-cells sensitized by a previous infection with M. tuberculosis using the Quanti FERON-TB Gold In Tube assay (QFT-GIT). The data were evaluated for the positive association of interferon-gamma. Results: The overall positivity rate IFN-γ of our healthcare workers was 20%, and for females it was 28.6%, with an associated odds ratio equal to 3.7, CI=1.5-8.7, P=0.001, while for males it was only 9.7%. Positive rates IFN-γ increased with increasing age, as a high IFN-γ positive rate occurred in the 50-60-year age group was 50%, with OR=5.5, CI=2.2-13.6, P &lt; 0.001. There was a significant association between IFN-γ positive latent tuberculosis and unimmunized childhood BCG (OR=2.2, CI=1.03-4.7, P=0.03), diabetics (OR=4.2, CI=1.5-12, P=0.003), smoking (OR=2.7, CI=1.2-6.1, P=0.009) and contacting cases with active TB (OR=4.1, CI=1.4-12.2, P=0.007). Conclusion: It was found that the rate of LTBI among HCWs is high in Sana'a, central Yemen. Factors at both the institutional and individual level can influence the prevalence of LTBI among HCWs. Since contact with active TB patients has been identified as a risk factor for LTBI, more effective TB infection control measures are necessary in health care facilities and congregate settings and to create awareness about TB risk factors in hospitals and as an occupational hazard. Peer Review History: Received: 5 September 2022; Revised: 17 October; Accepted: 6 November, Available online: 15 November 2022 Academic Editor: Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, amaka_mgbahurike@yahoo.com Received file: Reviewer's Comments: Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.5/10 Reviewers: Dr. Bilge Ahsen KARA, Ankara Gazi Mustafa Kemal Hospital, Turkey, ahsndkyc@gmail.com Dr. Wadhah Hassan Ali Edrees, Hajja University, Yemen, edress2020@gmail.com Similar Articles: DETERMINATION OF RIFAMPICIN MONO-RESISTANCE MYCOBACTERIUM TUBERCULOSIS IN THE NATIONAL TUBERCULOSIS CONTROL PROGRAMME IN SANA'A CITY-YEMEN: A SIGNIFICANT PHENOMENON IN WAR REGION WITH HIGH PREVALENCE TUBERCLOISIS

Differences in Humoral and Cellular Response to COVID-19 Vaccine Against Different Variants over Time in Chronic Lymphocytic Leukemia

Background Patients with chronic lymphocytic leukemia (CLL) are at an increased risk of adverse clinical outcomes following COVID-19 infection, due to impaired humoral and cellular response to infection and vaccination. Most of the data available focus on humoral responses to SARS-CoV-2 vaccination or T cell responses following 2-dose vaccine series. However, the effect of emerging variants of concern (VoC) on T cell recognition in this population has not been previously reported. Aim of the study To evaluate functional humoral response and functional SARS-CoV-2 spike-specific T cell response in CLL patients after the second, the third dose, and 12-months since the first dose. To determine the effect of treatment on immune outcomes. To investigate T cell response to Omicron (BA.2) and ancestral strains, and the impact of Omicron-defining mutations on total spike-specific functional response. Methods CLL patients from Mayo Clinic, Rochester who submitted a blood sample soon after the second and the third dose, and 12-months after the first dose of an mRNA-based vaccination were included. The samples collected following a documented SARS-CoV-2 infection were excluded. Spike-specific T cell responses were quantified using IFN-γ ELISpot assay (R&D Systems). PBMCs were stimulated with a peptide pool spanning the complete sequence of ancestral SARS-CoV-2 spike and two 68-peptide pools: the BA.2 mutation pool selectively covering the mutated spike regions, and reference pool containing the wild-type sequences for the same regions (Peptivator pools, Miltenyi Biotec). The results were considered positive if at least two of the following three were met: average spot count > 5 SFUs/2x10^5; mean spot count >3-fold higher than in negative control; and statistically significant difference (paired t-test<0.5) between Ag-specific and negative control wells.For evaluation of humoral response, we utilized an assay measuring antibody capable of blocking spike receptor binding domain (RBD)-ACE2 (cPASS GenScript). Statistical analysis was performed using t-test and Mann-Whitney test for continuous variables; Chi-square (or Fisher's Exact test) was used for comparing categorical variables. The Mayo Clinic IRB approved the study. Results Thirty patients with CLL were included in the study. Eight patients received a fourth vaccine dose before the 12-months blood draw. The rate of positivity for receptor blocking activity was significantly higher in the untreated group after the 2ndand 3rd dose (0% vs. 75%, 38.5% vs. 93.3%, p>0.05). This difference was not observed for the cellular response to spike-specific ancestral in all the three timepoints. At 12 months 54.16 % of the patients had positive IFN-γ ELISpot response, with no significant difference between treated and untreated (58.3% vs. 50%, p=1.0), and patients with and without a fourth dose (62.5% vs. 50%, p=0.67). Among treated CLL patients, seven (70%) and five (41.6%) patients without neutralizing antibodies had detectable cellular response after the 2nd and 3rd dose, respectively. On average, less than 25% of the total spike-specific T cell reactivity was directed against the epitopes in the regions mutated in BA.2 subvariant. Moreover, we did not detect statistically significant differences in IFN-γspot counts between the mutational and reference 68-peptide pools at any timepoint (median spots T1: 1.62 vs. 2.33, p=0.4; T2: 5 vs. 4.33, p=0.31; T3: 5 vs. 6, p=0.49). Conclusion Treatment status impacts humoral, but no cellular response to SARS-CoV-2 vaccination CLL patients. However, the overall rate of positive cellular response one year after the first dose is low. This, combined with no boosting of cellular response upon the 4th dose, reflects an impaired response even after additional doses. Suboptimal cellular immunity may put CLL patients at higher risk of severe COVID-infection. We did not observe significant difference in the cellular response between ancestral and Omicron BA.2 strains, and only the smaller fraction of T cell response was directed against regions harboring BA.2-specific mutations. These findings confirm the greater observed cross-reactivity to VoC with cellular compared to humoral immune responses. They also suggest significant cellular immunity may remain in the face of Omicron circulation, at least among patients who have response to the vaccine. Given the small sample size, our findings need to be confirmed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expression of miRNA-29a-3p and IFN-γ as biomarkers in active and latent pulmonary tuberculosis.

Diagnosis and management of latent tuberculosis (TB) infections are one of the challenges of eradicating pulmonary TB. A critical aspect of controlling pulmonary TB spread is early diagnosis. One TB biological marker type under evaluation is microRNAs (miRNAs). Mycobacterium tuberculosis infection causes epigenetic changes. The upregulation of miRNA-29a-3p suppresses the immune response by post-transcriptionally inhibiting interferon (INF)-γ expression in T cells, increasing susceptibility to pulmonary TB. This study aimed to assess miRNA-29a-3p expression as a biomarker of active and latent pulmonary TB infection. This case-control study included 50 individuals with active TB, 33 household contacts with a positive IFN-γ release assay (IGRA), and 30 healthy controls. An enzyme-linked immunosorbent assay-based IGRA was used to determine latent pulmonary TB infection in household contacts. Quantitative real-time PCR was used to quantify miRNA-29a-3p expression. Data analysis used analyses of variance and receiver operating characteristic (ROC) curves. miRNA-29a-3p expression differed significantly between active TB, latent TB, and healthy participants (controls; p = <0.001. ROC curve analysis showed that miRNA-29a-3p expression had 86% sensitivity and 73% specificity with an area under the ROC curve (AUC) of 0.763 (95% confidence interval [CI]: 0.668-0.858). The miRNA-29a-3p ROC curve had 84.8% sensitivity and 70% specificity with an AUC of 0.808 (95% CI: 0.698-0.919) for latent TB. Additionally, miRNA-29a-3p expression was significantly correlated with active (p<0.0001) and latent (p<0.0001) pulmonary TB. However, miRNA-29a-3p expression was not significantly correlated with INF-γ levels in patients with active (R=0.005; p=0.62) and latent (R=0.010; p=0.38) pulmonary TB or healthy controls (R=0.060; p=0.19). miRNA-29a-3p expression was increased in patients with active and latent pulmonary TB. Therefore, miRNA-29a-3p represents a potential biomarker for latent and active pulmonary TB. However, IFN-γ levels were not correlated with miR-29a-3p expression.

COVID-19 Vaccination Responses with Different Vaccine Platforms in Patients with Inborn Errors of Immunity.

Patients with inborn errors of immunity (IEI) in Argentina were encouraged to receive licensed Sputnik, AstraZeneca, Sinopharm, Moderna, and Pfizer vaccines, even though most of the data of humoral and cellular responses combination on available vaccines comes from trials conducted in healthy individuals. We aimed to evaluate the safety and immunogenicity of the different vaccines in IEI patients in Argentina. The study cohort included adults and pediatric IEI patients (n = 118) and age-matched healthy controls (HC) (n = 37). B cell response was evaluated by measuring IgG anti-spike/receptor binding domain (S/RBD) and anti-nucleocapsid(N) antibodies by ELISA. Neutralization antibodies were also assessed with an alpha-S protein-expressing pseudo-virus assay. The T cell response was analyzed by IFN-γ secretion on S- or N-stimulated PBMC by ELISPOT and the frequency of S-specific circulating T follicular-helper cells (TFH) was evaluated by flow cytometry.No moderate/severe vaccine-associated adverse events were observed. Anti-S/RBD titers showed significant differences in both pediatric and adult IEI patients versus the age-matched HC cohort (p < 0.05). Neutralizing antibodies were also significantly lower in the patient cohort than in age-matched HC (p < 0.01). Positive S-specific IFN-γ response was observed in 84.5% of IEI patients and 82.1% presented S-specific TFH cells. Moderna vaccines, which were mainly administered in the pediatric population, elicited a stronger humoral response in IEI patients, both in antibody titer and neutralization capacity, but the cellular immune response was similar between vaccine platforms. No difference in humoral response was observed between vaccinated patients with and without previous SARS-CoV-2 infection.In conclusion, COVID-19 vaccines showed safety in IEI patients and, although immunogenicity was lower than HC, they showed specific anti-S/RBD IgG, neutralizing antibody titers, and T cell-dependent cellular immunity with IFN-γ secreting cells. These findings may guide the recommendation for a vaccination with all the available vaccines in IEI patients to prevent COVID-19 disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-022-01382-7.

Infant BCG vaccination and risk of pulmonary and extrapulmonary tuberculosis throughout the life course: a systematic review and individual participant data meta-analysis

BCG vaccines are given to more than 100 million children every year, but there is considerable debate regarding the effectiveness of BCG vaccination in preventing tuberculosis and death, particularly among older children and adults. We therefore aimed to investigate the age-specific impact of infant BCG vaccination on tuberculosis (pulmonary and extrapulmonary) development and mortality. In this systematic review and individual participant data meta-analysis, we searched MEDLINE, Web of Science, BIOSIS, and Embase without language restrictions for case-contact cohort studies of tuberculosis contacts published between Jan 1, 1998, and April 7, 2018. Search terms included "mycobacterium tuberculosis", "TB", "tuberculosis", and "contact". We excluded cohort studies that did not provide information on BCG vaccination or were done in countries that did not recommend BCG vaccination at birth. Individual-level participant data for a prespecified list of variables, including the characteristics of the exposed participant (contact), the index case, and the environment, were requested from authors of all eligible studies. Our primary outcome was a composite of prevalent (diagnosed at or within 90 days of baseline) and incident (diagnosed more than 90 days after baseline) tuberculosis in contacts exposed to tuberculosis. Secondary outcomes were pulmonary tuberculosis, extrapulmonary tuberculosis, and mortality. We derived adjusted odds ratios (aORs) using mixed-effects, binary, multivariable logistic regression analyses with study-level random effects, adjusting for the variable of interest, baseline age, sex, previous tuberculosis, and whether data were collected prospectively or retrospectively. We stratified our results by contact age and Mycobacterium tuberculosis infection status. This study is registered with PROSPERO, CRD42020180512. We identified 14 927 original records from our database searches. We included participant-level data from 26 cohort studies done in 17 countries in our meta-analysis. Among 68 552 participants, 1782 (2·6%) developed tuberculosis (1309 [2·6%] of 49 686 BCG-vaccinated participants vs 473 [2·5%] of 18 866 unvaccinated participants). The overall effectiveness of BCG vaccination against all tuberculosis was 18% (aOR 0·82, 95% CI 0·74-0·91). When stratified by age, BCG vaccination only significantly protected against all tuberculosis in children younger than 5 years (aOR 0·63, 95% CI 0·49-0·81). Among contacts with a positive tuberculin skin test or IFNγ release assay, BCG vaccination significantly protected against tuberculosis among all participants (aOR 0·81, 95% CI 0·69-0·96), participants younger than 5 years (0·68, 0·47-0·97), and participants aged 5-9 years (0·62, 0·38-0·99). There was no protective effect among those with negative tests, unless they were younger than 5 years (0·54, 0·32-0·90). 14 cohorts reported on whether tuberculosis was pulmonary or extrapulmonary (n=57 421). BCG vaccination significantly protected against pulmonary tuberculosis among all participants (916 [2·2%] in 41 119 vaccinated participants vs 334 [2·1%] in 16 161 unvaccinated participants; aOR 0·81, 0·70-0·94) but not against extrapulmonary tuberculosis (106 [0·3%] in 40 318 vaccinated participants vs 38 [0·2%] in 15 865 unvaccinated participants; 0·96, 0·65-1·41). In the four studies with mortality data, BCG vaccination was significantly protective against death (0·25, 0·13-0·49). Our results suggest that BCG vaccination at birth is effective at preventing tuberculosis in young children but is ineffective in adolescents and adults. Immunoprotection therefore needs to be boosted in older populations. National Institutes of Health.

Anti-PD-L1 Antibody Enhances T Cell Immune Responses and Reduces Resistance of Breast Cancer Cells to Radiotherapy.

Immune escape is a frequent occurrence, which limits the duration of antitumor immune responses to radiotherapy. Here, we aimed to ascertain the roles and underlying mechanisms of programmed death ligand 1 (PD-L1) in tolerance of breast cancer (BC) to radiotherapy. We first quantified microRNA-21 (miR-21) and PD-L1 expression in BC tissues and cells, followed by identification of the interactions between miR-21, PD-L1, and programmed cell death protein 4 (PDCD4). miR-21 knock-in mice were used to construct tumor-bearing models, which were then treated with anti-PD-L1 antibody and irradiation, followed by measurement of tumor growth and tumor immune escape. Finally, we evaluated the synergistic effects of radiotherapy and anti-PD-L1 antibody in vivo. The results showed increased miR-21 expression in BC tissues and cells, which was positively correlated with PD-L1 expression. The treatment with radiotherapy or anti-PD-L1 antibody in the miR-21 knock-in mice diminished tumor weight and volume, along with decreased CD3+CD8+ positive cells, serum IL-2 and IFN-γ levels, and lower PD-L1 expression, but augmented apoptosis of T and BC cells. Moreover, miR-21 significantly augmented PD-L1 expression via PI3K/Akt pathway activation by targeting PDCD4 in BC cells. Thus, radiotherapy and anti-PD-L1 antibody synergistically accelerated the therapeutic effect against BC in mice, thereby implicating a close interplay between radiotherapy, T cells, and the miR-21/PDCD4/PI3K/Akt/PD-L1 axis.

The Clinical and Immunological Activity Depending on the Presence of Interferon γ in Primary Sjögren's Syndrome-A Pilot Study.

The upregulation of IFN pathways and their stimulated genes is associated with primary Sjögren’s syndrome (pSS). The recent studies also indicate the involvement of interferon γ (IFNγ) in the pathogenesis of pSS. The study aimed to assess the clinical and immunological activity depending on the concentration of IFNγ in the peripheral blood in pSS patients. Methods: The study group consisted of patients over 18 years of age with a confirmed diagnosis of pSS. Based on the collected data, disease activity was assessed using the EULAR Sjögren’s syndrome disease activity index (ESSDAI) and the EULAR Sjögren’s syndrome patient reported index (ESSPRI). Results: Among 40 pSS patients, 33 (82%) showed increased levels of IFNγ. The group with positive IFNγ was younger (43 years) than the group with negative IFNγ (57 years) (p < 0.05). In the positive IFNγ group, the time to diagnosis was shorter (p < 0.05). There was a difference in ESSDAI among patients with and without IFNγ (p < 0.05). There were no differences between the groups in ESSPRI and the presence of cryoglobulins, specific anti-SSA, and anti-SSB antibodies and in C3 and C4 hypocomplementemia. RF occurred in both groups with a similar frequency (p = 0.6), but in patients with IFNγ presence, significantly higher RF titers were observed (34.9 vs. 10.5; p < 0.05). Conclusion: In the group of patients with positive IFNγ, the mean value of RF and ESSDAI was higher. This group was also younger than patients with pSS without IFNγ.

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

Unprovoked A-β+ 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 β-cell function ("β+") 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. We sought to clarify the immunological role of insulin-associated molecules in unprovoked A-β+ KPD. 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. 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. These findings suggest the involvement of B:9-23rPep-specific IFN-γ-related immunoreactivity in the pathophysiology of some unprovoked A-β+ KPD. Moreover, increased immunoreactivity may reflect transiently decreased β-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.

Long-Term Course of Humoral and Cellular Immune Responses in Outpatients After SARS-CoV-2 Infection.

Characterization of the naturally acquired B and T cell immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for the development of public health and vaccination strategies to manage the burden of COVID-19 disease. We conducted a prospective, cross-sectional analysis in COVID-19 recovered patients at various time points over a 10-month period in order to investigate how circulating antibody levels and interferon-gamma (IFN-γ) release by peripheral blood cells change over time following natural infection. From March 2020 till January 2021, we enrolled 412 adults mostly with mild or moderate disease course. At each study visit, subjects donated peripheral blood for testing of anti-SARS-CoV-2 IgG antibodies and IFN-γ release after SARS-CoV-2 S-protein stimulation. Anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies were positive in 316 of 412 (76.7%) and borderline in 31 of 412 (7.5%) patients. Our confirmation assay for the presence of neutralizing antibodies was positive in 215 of 412 (52.2%) and borderline in 88 of 412 (21.4%) patients. Likewise, in 274 of 412 (66.5%) positive IFN-γ release and IgG antibodies were detected. With respect to time after infection, both IgG antibody levels and IFN-γ concentrations decreased by about half within 300 days. Statistically, production of IgG and IFN-γ were closely associated, but on an individual basis, we observed patients with high-antibody titres but low IFN-γ levels and vice versa. Our data suggest that immunological reaction is acquired in most individuals after natural infection with SARS-CoV-2 and is sustained in the majority of patients for at least 10 months after infection after a mild or moderate disease course. Since, so far, no robust marker for protection against COVID-19 exists, we recommend utilizing both, IgG and IFN-γ release for an individual assessment of the immunity status.

Profiling CD8+ Tcell epitopes of COVID-19 convalescents reveals reduced cellular immune responses to SARS-CoV-2 variants.

Cellular immunity is important in determining the disease severity of COVID-19 patients. However, current understanding of SARS-CoV-2 epitopes mediating cellular immunity is limited. Here we apply T-Scan, a recently developed method, to identify CD8+ T cell epitopes from COVID-19 patients of four major HLA-A alleles. Several identified epitopes are conserved across human coronaviruses, which might mediate pre-existing cellular immunity to SARS-CoV-2. In addition, we identify and validate four epitopes that were mutated in the newly circulating variants, including the Delta variant. The mutations significantly reduce T cell responses to the epitope peptides in convalescent and vaccinated samples. We further determine the crystal structure of HLA-A∗02:01/HLA-A∗24:02 in complex with the epitope KIA_S/NYN_S, respectively, which reveals the importance of K417 and L452 of the spike protein for binding to HLA. Our data suggest that evading cellular immunity might contribute to the increased transmissibility and disease severity associated with the new SARS-CoV-2 variants.

A peptide-based vaccine ACP derived from antigens of Mycobacterium tuberculosis induced Th1 response but failed to enhance the protective efficacy of BCG in mice

BackgroundTuberculosis (TB) is a global infectious disease, but there is no ideal vaccine against TB except the Bacille Calmette-Guérin (BCG) vaccine. MethodsHerein, 25 candidate peptides were predicted from four antigens of Mycobacterium tuberculosis based on their high-affinity binding capacity for the human leukocyte antigen (HLA) DRB1∗0101. Three T-helper 1 (Th1) immunodominant peptides (Ag85B12-26, CFP2112-26, and PPE18149-163) were identified by ELISPOT assays in the humanized C57BL/6 mice. They resulted in a novel Th1 peptide-based vaccine ACP named by the first letter of the three peptides. In addition, the protective efficacy was evaluated in humanized or wild-type C57BL/6 mice and the humoral and cellular immune responses were confirmed in vitro. ResultsCompared with the PBS group, the ACP vaccinated mice showed slight decreases in colony-forming units (CFUs) and pathological lesions. However, when using it as a booster, the ACP vaccine did not significantly enhance the protective efficacy of BCG in humanized or wild-type mice. Interestingly, we found that ACP vaccination significantly increased the number of interferon-γ positive (IFN-γ+) T lymphocytes and the levels of IFN-γ cytokines as well as antibodies. Furthermore, the IL-2 level was significantly higher in humanized mice prime-boosted with BCG and ACP. ConclusionsOur results suggested that ACP vaccination could stimulate higher levels of cytokines and antibodies but failed to improve the protective efficacy of BCG in mice, indicating that the secretion level of IFN-γ may not be positively correlated with the protection efficiency of the vaccine. These findings provided important information on the feasibility of a peptide vaccine as a booster for enhancing the protective efficacy of BCG.

Tuberculose pulmonaire sous traitement par immunothérapie type anti-PD1

Immunotherapy is now a standard of care in oncology. There is a need to improve our knowledge about immune-related adverse events, especially infectious diseases. We describe the case of a 49-year old male who received anti-PD1 therapy, to treat metastatic melanoma with pulmonary metastasis. After 3 cycles of nivolumab, computed tomography scanning showed a decrease of the pulmonary metastasis in the upper left lobe, but revealed new pulmonary lesions such as tree-in-bud and a lung cavity in the same lobe. This was diagnosed as pulmonary tuberculosis with no antibiotic resistance identified. The patient continued the immunotherapy and was initiated onto a standard anti-tuberculosis therapy. In the absence of an initial positive IFN-γ release assay (Quantiferon) test, but as there might have been a history of primary infection during childhood, a reactivation of tuberculosis was considered to be likely. This is the ninth case of tuberculosis infection under immunotherapy and it underlines the need to consider infection risks in patients undergoing immunotherapy. An INF-γ release assay screening test should be considered an essential part of the pre-treatment work-up.

Co-Regulation of Immune Checkpoint PD-L1 with Interferon-Gamma Signaling is Associated with a Survival Benefit in Renal Cell Cancer.

BackgroundProgrammed death ligand (PD-L1)-based immune checkpoint blockade therapy for metastatic renal cell carcinoma (RCC) achieves significant response rates in a subgroup of patients. The relevance of PD-L1 gene regulation for disease outcome is not clear.ObjectiveTo evaluate PD-L1 expression and its dependence on interferon-γ (IFN-γ) in RCC cell lines and tissues in relation to disease outcome.Methods and PatientsRegulation of PD-L1-mRNA and PD-L1 protein was studied in cell lines from clear cell RCC (ccRCC) and papillary RCC (pRCC) by quantitative RT-PCR and Western-blot analysis. PD-L1-mRNA correlation and gene-set enrichment analysis (GSEA) of the IFN-γ pathway were conducted with RNA-Seq from ccRCC, pRCC, and skin cutaneous melanoma (SKCM) tissue. In addition, patient overall survival (OS) and disease-free survival (DFS) (cBioPortal for Cancer Genomics) were considered.ResultsIn ccRCC-like cell lines, PD-L1 was induced by canonical IFN-γ signaling, whereas in a pRCC-like cell line, PD-L1 was refractory towards IFN-γ signaling. In ccRCC and SKCM tissues, GSEA revealed significant IFN-γ pathway activation in tissue samples with high PD-L1-mRNA levels. This was not observed in pRCC tissue. ccRCC and SKMC patients with low PD-L1-mRNA levels had significantly shorter OS and DFS than those with high PD-L1-mRNA levels. In pRCC patients, no significant difference in OS and DFS with regard to PD-L1-mRNA tissue levels was obvious.ConclusionsThe findings suggest that ccRCC and pRCC differ with respect to PD-L1 regulation by IFN-γ-signaling. High PD-L1-mRNA levels in tumor tissues with a positive IFN-γ signature favorably affect OS and DFS.Electronic supplementary materialThe online version of this article (10.1007/s11523-020-00728-8) contains supplementary material, which is available to authorized users.

Optimizing the Design of Latent Tuberculosis Treatment Trials: Insights from Mathematical Modeling.

Rationale: Noninferiority trials of treatment for latent tuberculosis infection (LTBI) are challenging because of imperfect LTBI diagnostic tests.Objectives: To assess the effect on study outcomes of different enrollment strategies for a noninferiority trial of LTBI treatment.Methods: We mathematically simulated a two-arm randomized clinical trial of LTBI in which the experimental therapy was 50% efficacious and the control was 80% efficacious, with an absolute 0.75% noninferiority margin. Five enrollment strategies were assessed: 1) enroll based on no LTBI diagnostic test; 2) enroll based on a positive tuberculin skin test (TST); 3) enroll based on a positive IFN-γ release assay (IGRA); 4) enroll based on either a positive TST or IGRA; and 5) enroll regardless of test result, assuming 70% had negative TSTs, 20% positive TSTs, and 10% unknown results.Measurements and Main Results: Under most LTBI prevalence assumptions, enrolling based on a positive IGRA was least likely to result in falsely declaring noninferiority of the experimental regimen. Enrolling based on no test or regardless of test result led to falsely declaring noninferiority unless LTBI prevalence in the underlying population was higher than 45%. Enrolling based on a mix of TST and IGRA substantially reduced the likelihood of falsely declaring noninferiority over enrolling based on TST alone, even if as many as 70% of participants were enrolled based on positive TST.Conclusions: Noninferiority trials of LTBI should enroll based on the most specific diagnostic tests available (i.e., IGRAs) to avoid misclassifying inferior treatment regimens as noninferior.

Hepatic interferon γ and tumor necrosis factor a expression in infants with neonatal cholestasis and cytomegalovirus infection.

Aim of the studyTo determine the hepatic interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) levels in infants with neonatal cholestasis (NC) and associated cytomegalovirus (CMV) infection.Material and methodsThis study was conducted in 21 infants with NC over a period of 6 months from June 2017 to December 2017 to determine the hepatic IFN-γ and TNF-α levels in infants with NC and associated CMV infection.ResultsIFN-γ levels were positive in 16 (80%), low positive in 3 (16%) and negative in 1 (5%) patients. High positive and positive TNF-α levels were seen in 9 (56.3%) patients with positive liver CMV PCR and low positive levels were seen in 7 (43.7%) patients with positive liver CMV PCR (odds ratio [OR] = 2.6). Positive IFN-γ was present in 13 (81.3%) patients with positive liver CMV PCR and low positive or negative IFN-γ was seen in 3 (18.7%) patients with positive liver CMV PCR (OR = 2.2). Six (60%) patients with positive or high positive TNF-α levels in liver tissue had biliary atresia (BA) whereas 7 (77.7%) with low positive TNF-α levels had non-BA neonatal hepatitis (OR = 5.25). Six (37.5%) patients with positive IFN-γ had BA whereas 2 (50%) patients with low positive or negative IFN-γ had BA (OR = 0.6).ConclusionsThere is high prevalence of CMV in liver tissues in patients with NC and elevated TNF-α and IFN-γ levels are seen in these patients. Elevated TNF-α is also seen in patients with BA. The association of elevated TNF-α, BA and CMV infection needs to be evaluated further.

DC Vaccination Induces Antigen Specific Immune Responses in AML Patients: A 1-Year Interim Assessment

Acute myeloid leukemia (AML) is a lethal hematological malignancy characterized by high rates of relapse (50%). Studies of immune responses in AML patients led to identification of two leukemia-associated antigens (Wilms' tumor protein 1 (WT1) and preferentially expressed antigen of melanoma (PRAME) for which cellular and humoral immune responses were detected. Based on expression profiles in AML and normal tissues, WT1 and PRAME were selected as target antigens for a DC vaccine, aiming to elicit adaptive and innate immune responses in AML patients. The primary objective of the trial (EudraCT No.: 2014-003520-44) is to evaluate safety and feasibility of active immunotherapy with antigen-loaded autologous DCs. The secondary objectives are to assess DC vaccination-induced T cell responses against WT1 and PRAME and to evaluate the clinical outcome over a period of 2 years or until disease progression/relapse. WT1-positive AML patients, with or without PRAME positivity were included, if they had a morphological remission with or without hematological recovery (CRi) after induction chemotherapy and were not eligible for allogeneic hematopoietic stem cell transplantation. DCs were administered intradermally (2.5x106 WT1 plus 2.5x106 PRAME DCs) on a weekly basis for 4 consecutive weeks, followed by administration on week 6 and then every 4 weeks until 2 years, tumor progression or patient withdrawal. This report focusses on the immune-monitoring data collected during the first year of treatment. Multiple parameters were evaluated, including WT1- and PRAME specific T cell responses in peripheral blood (IFN-g ELISPOT), the recurrence of WT1- and/or PRAME-positive AML blasts in bone marrow (qRT-PCR) and peripheral blood (flow cytometry), and occurrence of AML-specific mutations in bone marrow (NGS). DCs were assessed for expression of WT1 and PRAME and costimulatory molecules by flow cytometry, while secretion of IL10 and IL12 was evaluated by double-color ELISPOT. Twenty CR/CRi AML patients were vaccinated, and vaccination was well tolerated, and no signs of autoimmunity were observed. 12/20 patients remained stable throughout the first year of treatment, while 8/20 relapsed. 2/8 relapsed patients died due to progression of disease during the first year. In patients in stable remission, 7/12 patients had no detectable IFN-γ ELISPOT response in peripheral blood, 3/12 mounted a response and 2/12 were not evaluable due to high background production of IFN-γ. Positive IFN-γ ELISPOT responses were observed within 1-30 weeks of treatment and associated with a decrease over time in levels of WT1 (3/3) and/or PRAME mRNA (1/3) expression. Except for 2 patients who remained MRD-negative throughout the first year of treatment, ELISPOT-negative patients in remission (5/7) exhibited low, relatively stable levels of target antigens. Stable remissions were associated with one (3/12) or no (8/12) AML-related mutations while one patient was not evaluable. AML relapses were observed within 50 days in 4 patients, within 51-100 days in 1 patient, and after more than 150 days in 3 patients. Overall, 6/8 relapsing patients exhibited positive IFN-γ ELISPOT responses, while 2 patients relapsing in &lt;50 days failed to respond and one patient relapsing on day 77 exhibited an IFN-γ response at baseline. In all other relapsing patients, IFN-γ ELISPOT responses were detected within 6-30 weeks after vaccination, in association with rapid and progressive increases in levels of WT1 and/or PRAME. AML-related gene mutations were detected in 6/8 relapses, while one patient was not evaluable, and one had no mutation; In 4 of the relapsing patients, 3 to 6 mutations were detected, while in 2/8 a single mutation was observed. In conclusion, IFN-γ responses to WT1 and/or PRAME were detected in peripheral blood of 75% (6/8) of relapsing patients, but only 25% (3/12) of the patients in remission. It is tempting to speculate that detection of IFN-γ response in peripheral blood is linked to the concomitant presence of AML blasts in the periphery of relapsing patients. Stable or decreasing levels of WT1 and/or PRAME mRNA in bone marrow of most patients in remission is compatible with the hypothesis that a local response against AML antigens may be ongoing, despite a negative IFN-γ response in peripheral blood. Monitoring of the patients currently in remission may shed some light on the role of DC vaccination on the prevention of AML recurrence. Disclosures Eckl: Medigene Immunotherapies GmbH: Employment. Raffegerst:Medigene Immunotherapies GmbH: Employment. Schnorfeil:Medigene Immunotherapies GmbH: Employment. Prinz-Schulz:Medigene Immunotherapies GmbH: Employment. Fingerhut:Medigene Immunotherapies GmbH: Employment. Bigalke:BioNTech Innovative Manufacturing Services GmbH: Employment. Pinkernell:Medigene AG: Employment. Schendel:Medigene AG: Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Tafuri:Medigene Immunotherapies GmbH: Employment.