Experimental validation of immunogenic SARS-CoV-2 T cell epitopes identified by artificial intelligence

During the COVID-19 pandemic we utilized an AI-driven T cell epitope prediction tool, the NEC Immune Profiler (NIP) to scrutinize and predict regions of T cell immunogenicity (hotspots) from the entire SARS-CoV-2 viral proteome. These immunogenic regions offer potential for the development of universally protective T cell vaccine candidates. Here, we validated and characterized T cell responses to a set of minimal epitopes from these AI-identified universal hotspots. Utilizing a flow cytometry-based T cell activation-induced marker (AIM) assay, we identified 59 validated screening hits, of which 56% (33 peptides) have not been previously reported. Notably, we found that most of these novel epitopes were derived from the non-spike regions of SARS-CoV-2 (Orf1ab, Orf3a, and E). In addition, ex vivo stimulation with NIP-predicted peptides from the spike protein elicited CD8+ T cell response in PBMC isolated from most vaccinated donors. Our data confirm the predictive accuracy of AI platforms modelling bona fide immunogenicity and provide a novel framework for the evaluation of vaccine-induced T cell responses

Immune complexes, innate immunity, and NETosis in ChAdOx1 vaccine-induced thrombocytopenia

Aims - We recently reported five cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) 7–10 days after receiving the first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine against corona virus disease 2019 (COVID-19). We aimed to investigate the pathogenic immunological responses operating in these patients. Methods and results - We assessed circulating inflammatory markers by immune assays and immune cell phenotyping by flow cytometry analyses and performed immunoprecipitation with anti-platelet factor (PF)4 antibody in plasma samples followed by mass spectrometry from all five patients. A thrombus was retrieved from the sinus sagittal superior of one patient and analysed by immunohistochemistry and flow cytometry. Precipitated immune complexes revealed multiple innate immune pathway triggers for platelet and leucocyte activation. Plasma contained increased levels of innate immune response cytokines and markers of systemic inflammation, extensive degranulation of neutrophils, and tissue and endothelial damage. Blood analyses showed activation of neutrophils and increased levels of circulating H3Cit, dsDNA, and myeloperoxidase–DNA complex. The thrombus had extensive infiltration of neutrophils, formation of neutrophil extracellular traps (NETs), and IgG deposits. Conclusions - The results show that anti-PF4/polyanion IgG-mediated thrombus formation in VITT patients is accompanied by a massive innate immune activation and particularly the fulminant activation of neutrophils including NETosis. These results provide novel data on the immune response in this rare adenoviral vector-induced VITT

The persistence of anti-Spike antibodies following two SARS-CoV-2 vaccine doses in patients on immunosuppressive therapy compared to healthy controls—a prospective cohort study

The durability of vaccine-induced humoral immunity against SARS-CoV-2 in patients with immune mediated inflammatory diseases (IMIDs) on immunosuppressive therapy is not known. The aim of this study was to compare the persistence of anti-Spike antibodies following two-dose SARS-CoV-2 vaccination between IMID patients and healthy controls and to identify factors associated with antibody decline.publishedVersio

Humoral immunity to SARS-CoV-2 mRNA vaccination in multiple sclerosis: the relevance of time since last rituximab infusion and first experience from sporadic revaccinations

Introduction The effect of disease-modifying therapies (DMT) on vaccine responses is largely unknown. Understanding the development of protective immunity is of paramount importance to fight the COVID-19 pandemic. Objective To characterise humoral immunity after mRNA-COVID-19 vaccination of people with multiple sclerosis (pwMS). Methods All pwMS in Norway fully vaccinated against SARS-CoV-2 were invited to a national screening study. Humoral immunity was assessed by measuring anti-SARS-CoV-2 SPIKE RBD IgG response 3–12 weeks after full vaccination, and compared with healthy subjects. Results 528 pwMS and 627 healthy subjects were included. Reduced humoral immunity (anti-SARS-CoV-2 IgG <70 arbitrary units) was present in 82% and 80% of all pwMS treated with fingolimod and rituximab, respectively, while patients treated with other DMT showed similar rates as healthy subjects and untreated pwMS. We found a significant correlation between time since the last rituximab dose and the development of humoral immunity. Revaccination in two seronegative patients induced a weak antibody response. Conclusions Patients treated with fingolimod or rituximab should be informed about the risk of reduced humoral immunity and vaccinations should be timed carefully in rituximab patients. Our results identify the need for studies regarding the durability of vaccine responses, the role of cellular immunity and revaccinations. This article is made freely available for use in accordance with BMJ’s website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by BMJ. You may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained.publishedVersio

Association of variable number of tandem repeats in the coding region of the FAM46A gene, FAM46A rs11040 SNP and BAG6 rs3117582 SNP with susceptibility to tuberculosis

We analyzed for association between the Family with sequence similarity 46, member A (FAM46A) gene (located on chromosome 6q14.1), BCL2-Associated Athanogene 6 (BAG6) gene (located on chromosome 6p21.3) and tuberculosis in Croatian Caucasian. We genotyped the FAM46A rs11040 SNP, FAM46A VNTR and BAG6 rs3117582 polymorphisms in a case-control study with 257 tuberculosis patients and 493 healthy individuals in a Croatian Caucasian population. We found that genotype FAM46A 3/3 (three VNTR repeats homozygote) was associated with susceptibility to tuberculosis (p<0.0015, Pcorr.<0.029, Odds ratio = 2.42, 95% Confidence Interval = 1.34–4.3). This association suggests that the protein domain encoded by the VNTR might be important for the function of the FAM46A protein, which, in turn, could be relevant in developing tuberculosis. In addition, we found that FAM46A rs11040 SNP:FAM46A VNTR:BAG6 haplotype 132 (G-3-C) is associated with susceptibility to tuberculosis (p<0.012, pcorr.<0.024, Odds ratio 3.45, 95% Confidence Interval = 1.26–9.74). This may suggests that the interaction between the FAM46A and BAG6 proteins may be involved in tuberculosis etiology. We found also that infection of human macrophages with heat-killed M. tuberculosis (H37Rv) led to over-expression of FAM46A (VNTR 3/4) transcript. This is the first study to show associations between the FAM46A gene VNTR polymorphisms, FAM46A rs11040 SNP:FAM46A VNTR:BAG6 haplotypes and any disease

Cryopreservation of primary B cells minimally influences their signaling responses

Phospho flow is a powerful approach to detect cell signaling aberrations, identify biomarkers and assess pharmacodynamics, and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here, B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions, fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes

A national intercalated medical student research program–student perceptions, satisfaction, and factors associated with pursuing a PhD

Background To counteract a decreasing number of physician-scientists, a national intercalated Medical Student Research Programme (MSRP) was launched in Norway in 2002. We aimed to assess whether the students’ favourable perceptions and satisfaction with the program had prevailed since the inception in 2002 and until 2015, and to identify factors associated with pursuing a PhD. Methods The study was an incorporation of data from two previous national evaluations of the MSRP performed in 2007 and 2015. We used electronic questionnaires to explore demographic characteristics, area and type of research, student satisfaction, and future scientific goals. In 2007, questionnaires were sent to all 208 students, and 183 (88%) replied. In 2015, the corresponding numbers were 279, and 240 (86%). Categorical data were analysed using either Kruskal-Wallis or Pearson’s chi square test. Differences between sample means were assessed with Student`s t-test while logistic regression was used to test associations between selected covariates and the students’ ambitions to pursue a PhD degree. Results Overall, the student satisfaction was 79%. However, more students in 2015 received less regular and less supervision time and expressed a need for more of it. Seventy-seven per cent expressed an ambition to pursue a PhD. Students were more likely to have a PhD ambition if they were satisfied with the program, had a supervisor with high expectations for them, or had already published some of their results. At both time points, students (86% vs. 89%) responded that the MSRP had a positive impact on their regular curriculum achievements. Conclusions The high degree of satisfaction with the national MSRP among undergraduate students has prevailed since the inception in 2002. By far, the program has also met its goal to increase the number of aspiring physician-scientists. However, to maintain that goal over time, adequate and personal supervision is a prerequisite

A national intercalated medical student research program–student perceptions, satisfaction, and factors associated with pursuing a PhD

Background: To counteract a decreasing number of physician-scientists, a national intercalated Medical Student Research Programme (MSRP) was launched in Norway in 2002. We aimed to assess whether the students’ favourable perceptions and satisfaction with the program had prevailed since the inception in 2002 and until 2015, and to identify factors associated with pursuing a PhD. Methods: The study was an incorporation of data from two previous national evaluations of the MSRP performed in 2007 and 2015. We used electronic questionnaires to explore demographic characteristics, area and type of research, student satisfaction, and future scientific goals. In 2007, questionnaires were sent to all 208 students, and 183 (88%) replied. In 2015, the corresponding numbers were 279, and 240 (86%). Categorical data were analysed using either Kruskal-Wallis or Pearson’s chi square test. Differences between sample means were assessed with Student`s t-test while logistic regression was used to test associations between selected covariates and the students’ ambitions to pursue a PhD degree. Results: Overall, the student satisfaction was 79%. However, more students in 2015 received less regular and less supervision time and expressed a need for more of it. Seventy-seven per cent expressed an ambition to pursue a PhD. Students were more likely to have a PhD ambition if they were satisfied with the program, had a supervisor with high expectations for them, or had already published some of their results. At both time points, students (86% vs. 89%) responded that the MSRP had a positive impact on their regular curriculum achievements. Conclusions: The high degree of satisfaction with the national MSRP among undergraduate students has prevailed since the inception in 2002. By far, the program has also met its goal to increase the number of aspiring physician-scientists. However, to maintain that goal over time, adequate and personal supervision is a prerequisite