Circulating Ab-levels are known to decrease from month two to six, also in healthy individuals (Levin?et?al., 2021). in levels after second and third vaccination (p?=?0.1475). Seropositivity dropped to 25.0% of participants before the third vaccination, a relative reduction of 33.3% (p?=?0.0020). No difference was found between frequencies of spike reactive CD4+and CD8+ T–cells after second (0.65??0.08% and 0.95??0.20%, respectively) and third vaccination (0.99??0.22% and 1.3??0.34%, respectively). Conclusion In this longitudinal cohort we found no significant increased humoral or cellular response with administration of a third SARS-CoV-2 mRNA vaccination. These findings suggest the need for clinical strategies to include allowance of B cell reconstitution before repeat vaccination and/or provision of pre-exposure prophylactic monoclonal antibodies. Keywords: Multiple sclerosis, Anti-CD20, Ocrelizumab, HDAC10 Antibody response, BNT162b2, Booster vaccine, mRNA vaccine, SARS-CoV-2 Abbreviations: MS, multiple sclerosis; Ab or Abs, antibody or antibodies; RBD, 25-hydroxy Cholesterol receptor binding domain; AIM, Activation-induced marker; BAU/mL, binding antibodies unit per milliliter; Vx, Visit x; PBMCs, peripheral blood mononuclear cells 1.?Introduction The ongoing Covid-19 pandemic raises concerns about its effects on the most vulnerable patients. Anti-CD20 medications such as ocrelizumab, rituximab, and ofatumumab are widely used to treat multiple sclerosis (MS), blood cancers, and autoimmune diseases. Anti-CD20 targets B-lymphocytes, thus leading to cell lysis, and thereby reduction of disease activity in both relapsing and progressive MS (Krumbholz?et?al., 2012). Furthermore, compared to other disease modifying therapies, anti-CD20 treatment is associated with more severe complications to SARS-CoV-2 infection (MP?Sormani et?al., 2021) e.g., higher rates of hospitalizations and severe disease course (Salter?et?al., 2021). A COVID-19-specific strategy for patients at specific risk in Denmark has therefore been to offer early re-vaccination to anti-CD20 treated patients. It has previously been shown by our and other investigators that patients on B-cell depleting treatments have significantly reduced humoral immunity after COVID-19 vaccines compared to healthy controls (Novak?et?al., 2021; Sabatino?et?al., 2022). Several studies confirm that vaccination, in general, generates a decreased humoral response in anti-CD20 treated patients (Achiron?et?al., 2021; Bar-Or?et?al., 2020; Hua?et?al., 2014; Ammitzb?ll?et?al., 2021). Data indicate that higher levels of B-cells at the time of vaccination and longer intervals between anti-CD20 treatments improve the response to vaccination (Disanto et?al., 2021). However, extending dosage interval 25-hydroxy Cholesterol is currently considered an off-label treatment, and the effect on relapse risk, while appearing to date to not be substantial, is still unknown (Ammitzb?ll?et?al., 2021; Killestein?et?al., 2020; Nguyen?et?al., 2017; MP?Sormani et?al., 2021). One study has advocated that vaccination should occur one month before the next treatment infusion (Day?et?al., 2020). Still, the timing of treatment with anti-CD20 infusion and subject vaccination is still being debated (Novak?et?al., 2021; Rico?et?al., 2021). The initial two vaccinations can lead to successful seroconversion in a subset of anti-CD20 patients (Novak?et?al., 2021). Accordingly, the Danish National Board of Health, European Medicines 25-hydroxy Cholesterol Agency (EMA), and the U.S. Food and Drug Administration (FDA) have all recommended or authorized an additional third vaccine for these patients. The primary goal of this study is to determine whether additional mRNA SARS-CoV-2 vaccination can increase levels of specific SARS-CoV-2 spike receptor binding domain (RBD) antibodies (Abs) generated in MS patients treated with anti-CD20 therapy (ocrelizumab). We also assessed whether a third vaccine dose can increase T cell responses and the proportion of seropositive individuals among these participants. To address this question, we examined frequencies of spike-reactive T cells and levels of SARS-CoV-2 Abs before and after a third SARS-CoV-2 vaccination in a large cohort of anti-CD20-treated MS-patients from two international MS centers. 2.?Method 2.1. Study population and design In this observational study, we included prospectively adult participants (18 years or older) with confirmed MS (2017 McDonald Criteria) on ocrelizumab (anti-CD20) therapy. All participants had received two doses of mRNA SARS-CoV-2 vaccination and were enrolled prior to a third booster vaccine of the mRNA SARS-CoV-2 vaccine. Results from first and second vaccination were already published in a recent paper (Novak?et?al., 2021). No other immunosuppressive treatment beyond infusion-related methylprednisolone was given to the participants during this study. Patients were included from two Danish MS clinics (Esbjerg, Viborg) and the University of California, centre for MS and Neuroinflammation, in San Francisco (USA). 25-hydroxy Cholesterol All participants followed standard clinical practice by their treating neurologist and interval of time between second and third vaccination was not standardised (Baden?et?al., 2021; Polack?et?al., 2020). 2.2. Sample collection Blood samples were collected at two time points: 0C7 days before the third booster (V4) vaccination and two to four weeks after the third booster vaccination.