Testing for genuine T cell immune responses to SARS-CoV- 2 in patients with common variable immunodeficiency or B cell depletion

Background: Immunodeficient (ID) patients are among risk groups for severe disease after SARS-CoV2 (CoV2) infection. Despite high population immunity, ID patients remain difficult to medically advice due to their impaired immunity during an evolving CoV2 strains pandemic. Aim: We examined if durable immunisation to CoV2 infection or vaccination is developed. Serology cannot answer this since IgG replacement therapy now contains CoV2 antibodies. Instead, a cellular diagnostic test for patients' CoV2 memory T cells was used Methods: We included 13 ID patients (aged 34-78;7 female) who were vaccinated against CoV2 or previously infected: 5 with common variable immunodeficiency (CVID) and 8 with B cell deficiency (BCD) due to anti-CD20 or BTK inhibition therapy. Peripheral blood mononuclear cells isolated from whole blood were cultured with CoV2 spike protein or peptides for 7 days, then measured for T cell activation markers by flow cytometry. Results: CoV2 spike specific CD4 or CD8 T cells were detectable in 7/8 BCD and all CVID patients. However, only 3/5 CVID and 6/8 BCD patients had both CD4 and CD8 T cell responses. Conclusion: Genuine CoV2 T cell responses are detectable with a cellular diagnostic test in CVID and BCD patients after immunisation. As ID patients are heterogenous, a diagnostic test for memory T cells against CoV2 gives clinicians evidence of a patient's own immune response beyond passive IgG replacement therapy. This aids in consulting advice for infection avoidance strategies and indication for urgent treatment with monoclonal antibodies against CoV2.

Highly specific and reliable in vitro diagnostic analysis of memory T and B lymphocytes in a Swiss cohort of Covid-19 patients

The 2019 novel SARS-CoV-2 pandemic has claimed many lives and disrupted people's quality of life. Diagnostic tests not only confirm past exposure but offer key information to guide patients' healthcare options Current diagnostics for SARS-CoV-2 virus presence or antibodies lack evidence of longer-lasting cellular immunity, partly due to more complex cell culture-based techniques compared to serological tests. We aimed to (1) develop an in vitro flow cytometric diagnostic immunoassay and (2) determine if lasting memory helper CD4 and cytotoxic CD8 T cell activations are distinct between unexposed and convalescent individuals Peripheral blood mononuclear cells (PBMCs) isolated from whole blood of unexposed (n = 10) or convalescent (n = 30) individuals were cultured for 5 days with individual SARS-CoV-2 recombinant proteins or self-designed peptides including the spike, membrane and nucleocapsid proteins. Specific activation of memory T cells and B cells against SARSCoV- 2 antigens were determined by the upregulation of activation markers (CD4 T cells: CD134+ CD137+;CD8 T cells: CD69+ CD137+;CD19 B cells: CD38+). We show both activated T cells and B cells are detectable against all tested antigens, and distinguishable between unexposed and convalescent individuals up to 11 months post-infection. Therefore, with this diagnostic tool, we propose that it would benefit immunocompromised individuals who are unable to mount sustained antibody responses and to study immune correlates of protection, thus enhancing knowledge of the Covid19 disease in a wider range of patient groups.

Allergy testing with SARS-Cov2 mRNA vaccines

Background: The newly approved mRNA-based vaccines BNT162b2 mRNA (Pfizer-BioNTech) and mRNA-1273 (Moderna) may be associated rarely with anaphylaxis. Many uncertainties persist concerning the allergic tests that can be proposed to evaluate the risk of anaphylaxis before vaccination Methods: We report two patients with history of anaphylaxis respectively to paclitaxel and macrogol 3350, who were referred to our allergy clinic to evaluate the risk related to SARS-Cov2 mRNA vaccines. Skin testing included both Pfizer-BioNTech and Moderna vaccines, polysorbate- 80 1% PEG-2000 1%, and trometamol 1% Results: The first case is an 81-year-old woman who was hospitalized in 2018 for an anaphylactic shock attributed to paclitaxel. The second case is a 55-year-old patient who developed generalized pruritus, urticaria dyspnea and dysphonia minutes after starting a preparation of macrogol 3350 for a colonoscopy. Skin prick testing was negative in both patients Intradermal testing (IDT) was positive for both mRNA-based vaccines and polysorbate-80 but not for PEG-2000 and TRIS. Basophil activation tests (BAT) were performed in the first case and were positive for both mRNA vaccines, paclitaxel and to a lesser extent polysorbate-80 and 20 Conclusion: The only shared component between the Pfizer-BioNTech and Moderna vaccines, paclitaxel, polysorbate and macrogol 3350 is PEG, although with different molecular weights. IDT and BAT with mRNA-based vaccines can be useful to evaluate patients with a history of severe anaphylaxis associated with laxatives or parental drugs containing PEG or polysorbate.

12 months follow-up of early phase Covid-19 patients with immune disorders and of members of their family. Cellular and humoral evolution

Aims: Evaluate the lasting immune protection and effects after SARSCoV- 2 infections in healthy individuals and in patients with immune disease Methods: 54 convalescent COVID patients from the 1st wave epidemic and 12 COVID negative close relatives accepted to participate. 29% had an auto-immune disease, 31% allergies and 13% had immune deficiencies Clinical evolution and blood samples were collected monthly for ≥ 12 months. Inflammatory markers and β2microglobulin [β2m] were measured by nephelometry, specific ab anti Spike1 were assayed by immunochromatography and ELISA, neutralizing ab by inhibition ELISA lymphocyte phenotyping by flow cytometry. Lasting memory T CD4+ and T CD8+ and mB CD19+ were determined after in vitro culture with SARS-CoV-2 recombinant proteins or self-designed peptides Results: Along the 12 months of the study, 95% of patients maintained significant antibodies and effective % of neutralising ab (peak: 3rd to 5th month). The persistence of this immunity was corroborated by the presence of circulating memory T CD4+, CD8+ and B CD19+ (month 10 and 12 post COVID). Inflammatory markers returned to pre-infection values within two months after recovery. Elevated β2m, reflecting CD8+ and CD5616+ activation, was observed only during the first month after onset In 12 patients it was again present upon reactivation during the 2nd wave. A decreased or low CD4/CD8 ratio was observed in all for more than 8 months before slowly returning to initial reference Assertion: SARS-CoV-2 immunity and pro-inflammatory profile persist much longer than postulated.