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Bacterial vaginosis (BV) is a polymicrobial infection of the female reproductive tract. BV is characterized by replacement of health-associated Lactobacillus species by diverse anerobic bacteria, including the well-known Gardnerella vaginalis. Prevotella timonensis, and Prevotella bivia are anerobes that are found in a significant number of BV patients, but their contributions to the disease process remain to be determined. Defining characteristics of anerobic overgrowth in BV are adherence to the mucosal surface and the increased activity of mucin-degrading enzymes such as sialidases in vaginal secretions. We demonstrate that P. timonensis, but not P. bivia, strongly adheres to vaginal and endocervical cells to a similar level as G. vaginalis but did not elicit a comparable proinflammatory epithelial response. The P. timonensis genome uniquely encodes a large set of mucus-degrading enzymes, including four putative fucosidases and two putative sialidases, PtNanH1 and PtNanH2. Enzyme assays demonstrated that fucosidase and sialidase activities in P. timonensis cell-bound and secreted fractions were significantly higher than for other vaginal anerobes. In infection assays, P. timonensis efficiently removed fucose and α2,3- and α2,6-linked sialic acid moieties from the epithelial glycocalyx. Recombinantly expressed P. timonensis NanH1 and NanH2 cleaved α2,3 and α2,6-linked sialic acids from the epithelial surface, and sialic acid removal by P. timonensis could be blocked using inhibitors. This study demonstrates that P. timonensis has distinct virulence-related properties that include initial adhesion and a high capacity for mucin degradation at the vaginal epithelial mucosal surface. Our results underline the importance of understanding the role of different anerobic bacteria in BV. IMPORTANCE: Bacterial vaginosis (BV) is a common vaginal infection that affects a significant proportion of women and is associated with reduced fertility and increased risk of secondary infections. Gardnerella vaginalis is the most well-known BV-associated bacterium, but Prevotella species including P. timonensis and P. bivia may also play an important role. We showed that, similar to G. vaginalis, P. timonensis adhered well to the vaginal epithelium, suggesting that both bacteria could be important in the first stage of infection. Compared to the other bacteria, P. timonensis was unique in efficiently removing the protective mucin sugars that cover the vaginal epithelium. These results underscore that vaginal bacteria play different roles in the initiation and development of BV.
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Objectives: High-magnitude CD8+ T cell responses are associated with mild COVID-19 disease; however, the underlying characteristics that define CD8+ T cell-mediated protection are not well understood. The antigenic breadth and the immunodominance hierarchies of epitope-specific CD8+ T cells remain largely unexplored and are essential for the development of next-generation broad-protective vaccines. This study identified a broad spectrum of conserved SARS-CoV-2 CD8+ T cell epitopes and defined their respective immunodominance and phenotypic profiles following SARS-CoV-2 infection. Methods: CD8+ T cells from 51 convalescent COVID-19 donors were analysed for their ability to recognise 133 predicted and previously described SARS-CoV-2-derived peptides restricted by 11 common HLA class I allotypes using heterotetramer combinatorial coding, which combined with phenotypic markers allowed in-depth ex vivo profiling of CD8+ T cell responses at quantitative and phenotypic levels. Results: A comprehensive panel of 49 mostly conserved SARS-CoV-2-specific CD8+ T cell epitopes, including five newly identified low-magnitude epitopes, was established. We confirmed the immunodominance of HLA-A*01:01/ORF1ab1637-1646 and B*07:02/N105-113 and identified B*35:01/N325-333 as a third epitope with immunodominant features. The magnitude of subdominant epitope responses, including A*03:01/N361-369 and A*02:01/S269-277, depended on the donors' HLA-I context. All epitopes expressed prevalent memory phenotypes, with the highest memory frequencies in severe COVID-19 donors. Conclusion: SARS-CoV-2 infection induces a predominant CD8+ T memory response directed against a broad spectrum of conserved SARS-CoV-2 epitopes, which likely contributes to long-term protection against severe disease. The observed immunodominance hierarchy emphasises the importance of T cell epitopes derived from nonspike proteins to the overall protective and cross-reactive immune response, which could aid future vaccine strategies.
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Background: Patients affected by different types of autoimmune diseases, including common conditions such as multiple sclerosis (MS) and rheumatoid arthritis (RA), are often treated with immunosuppressants to suppress disease activity. It is not fully understood how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and cellular immunity induced by infection and/or upon vaccination is affected by immunosuppressants. Methods: The dynamics of cellular immune reactivation upon vaccination of SARS-CoV-2 experienced MS patients treated with the humanized anti-CD20 monoclonal antibody ocrelizumab (OCR) and RA patients treated with methotrexate (MTX) monotherapy were analyzed at great depth via high-dimensional flow cytometry of whole blood samples upon vaccination with the SARS-CoV-2 mRNA-1273 (Moderna) vaccine. Longitudinal B and T cell immune responses were compared to SARS-CoV-2 experienced healthy controls (HCs) before and 7 days after the first and second vaccination. Results: OCR-treated MS patients exhibit a preserved recall response of CD8+ T central memory cells following first vaccination compared to HCs and a similar CD4+ circulating T follicular helper 1 and T helper 1 dynamics, whereas humoral and B cell responses were strongly impaired resulting in absence of SARS-CoV-2-specific humoral immunity. MTX treatment significantly delayed antibody levels and B reactivation following the first vaccination, including sustained inhibition of overall reactivation marker dynamics of the responding CD4+ and CD8+ T cells. Conclusions: Together, these findings indicate that SARS-CoV-2 experienced MS-OCR patients may still benefit from vaccination by inducing a broad CD8+ T cell response which has been associated with milder disease outcome. The delayed vaccine-induced IgG kinetics in RA-MTX patients indicate an increased risk after the first vaccination, which might require additional shielding or alternative strategies such as treatment interruptions in vulnerable patients. Funding: This research project was supported by ZonMw (The Netherlands Organization for Health Research and Development, #10430072010007), the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (#792532 and #860003), the European Commission (SUPPORT-E, #101015756) and by PPOC (#20_21 L2506), the NHMRC Leadership Investigator Grant (#1173871).