Longitudinal increase of humoral responses after four SARS-CoV-2 vaccinations and infection in MS patients on fingolimod

Background: Humoral responses after SARS-CoV-2 vaccination are greatly impaired in multiple sclerosis (MS) patients on fingolimod. Effects of repeated vaccination and infections on long-term responses are unclear. Methods: Prospective study in 60 MS patients on fingolimod measuring humoral responses after up to four vaccinations and 8 months after fourth vaccination. Results: Anti-WH1 antibody titers increased with each additional vaccination. At long-term follow-up titers increased further and most patients developed new humoral responses against the BA.1 omicron variant. Conclusion: Repeated SARS-CoV-2 vaccinations boost humoral immunity and, probably together with SARS-CoV-2 infections, induce humoral responses on the long-term in almost all patients.


Introduction
2][3] Based on this decreased response, MS patients on fingolimod are offered additional SARS-CoV-2 vaccinations in many countries, albeit with little effect on the humoral immune response after a third vaccination and no effect after a fourth vaccination. 1,2However, the number of patients who have experienced a SARS-CoV-2 infection has increased considerably over time, providing an additional boost in immune responses.The combined effect of repeated vaccination and infections on long-term humoral responses in patients on fingolimod is unknown.
In this study, we longitudinally assessed humoral responses after a maximum of four SARS-CoV-2 vaccinations and at long-term follow-up in MS patients on fingolimod.

Methods
This is a substudy of an ongoing prospective observational multicenter cohort study on immunity against SARS-CoV-2 in various immune-mediated inflammatory diseases (Target-to-B!(T2B); Trial NL8900; Dutch Trial register).The medical ethical committee of the Amsterdam UMC, location AMC (2020.194)approved the study and participants provided written informed consent.
In this study, we included patients with MS, who received at least one SARS-CoV-2 mRNA vaccine, used fingolimod during primary immunization, and with at least one serum sample after any vaccination available for SARS-CoV-2 antibody measurement.Clinical data were retrieved from the medical files.Data on antigen test or polymerase chain reaction proven SARS-CoV-2 infections and vaccinations were collected by digital questionnaires.Samples were obtained by self-performed finger prick at home or by venipuncture in the hospital at 28 days after first, second, third, and booster vaccination.This third

Longitudinal increase of humoral responses after four SARS-CoV-2 vaccinations and infection in MS patients on fingolimod
vaccination was offered to patients on fingolimod as an additional early booster to complement primary immunization ahead of the regular booster vaccination.As additional vaccinations, participants were vaccinated with mRNA vaccines.Approximately 8 months after booster vaccination, from September to November 2022, a long-term follow-up sample was obtained in a subgroup of participants who donated blood through venipuncture during primary immunization.Healthy controls were included when a longterm follow-up sample and at least one sample after vaccination were available.SARS-CoV-2 antibodies against Wuhan-Hu-1 (WH1) and Omicron BA.1 receptor-binding domain (RBD) were measured at Sanquin laboratory using an IgGspecific quantitative enzyme-linked immunosorbent assay (ELISA). 4The cut-off for a positive ELISA was 4.0 arbitrary units per ml (AU/mL), representing 99% specificity in pre-outbreak samples. 4,5 assessed the median antibody titer and proportion of seropositive participants at each time point, and in patients with paired samples at sequential vaccinations, we compared changes in titers and seropositivity from second to third vaccination, from third to booster, and from booster to long-term follow-up using the Wilcoxon signed-rank test and McNemar's test, respectively.The number of patients with SARS-CoV-2 infections was too low to allow comparison between patients with or without infection.Data analysis was performed in R version 4.3.0(R foundation for Statistical Computing, Vienna, Austria).

Results
Sixty patients and 43 healthy controls were included in this study (Supplemental Figure 1).Participant characteristics are summarized in Table 1.Three patients switched to other disease modifying therapies (DMT) after primary immunization (Supplemental Table 1).Not all samples were available for all time points (Supplemental Table 3).Three patients experienced a SARS-CoV-2 infection before primary immunization.At successive sampling time points, the cumulative proportion of samples from patients with a previous SARS-CoV-2 infection increased from 1/44 (2.3%) after first vaccination, 2/52 (3.8%) after second vaccination, 5/55 (9.1%) after third vaccination, 6/33 (18.2%) after booster vaccination, and 11/15 (73.3%) at long-term follow-up.

Discussion
We observed a gradual increase in antibody titers following repeated vaccinations and (breakthrough) infections in MS patients on fingolimod, and approximately 18 months after the start of the pandemic, most patients had a well measurable humoral response.Moreover, we show that the antibody repertoire can broaden over time and that new, in this case anti-BA.1,responses can develop.Together, these findings show that MS patients on fingolimod eventually develop and maintain a humoral response, although it should be noted that antibody titers remain considerably lower compared to titers that have been found in controls or patients on other immunosuppressants. 6her studies on the effect of repeated vaccination in MS patients on fingolimod differed in findings on seropositivity, possibly due to small groups.König et al. 1 reported seropositivity in 72% (21/29) patients after third vaccination compared to 55% (16/29) after primary immunization.In another cohort, 33% (7/21) of patients were seropositive after primary immunization, and 45% (13/29) were seropositive after third vaccination. 2This is lower than the seropositivity proportions in our cohort, which are similar to the findings from König et al.Due to a low number of prior infections, we were unable to separate the individual effects of repeated vaccinations and infections.Arguably, this is less important as the prevalence of SARS-CoV-2 infections has increased considerably over time.At long-term follow-up, antibody titers remained high, either from the booster vaccination, infections, or a combination of both.To our knowledge, we are the first to report on the humoral response after up to four SARS-CoV-2 vaccinations and at long-term follow-up, in a fairly large group of patients on fingolimod.
Strengths of this study are the longitudinal followup, measurement of two different types of antibody responses and a relatively large cohort.Limitations are the combination of vaccinations and intercurrent infections making the observed humoral response less generalizable, only mRNA vaccines as additional vaccinations, the absence of cellular immunity measurements and monitoring subclinical infections, and only a small group at long-term follow-up making findings on this timepoint less generalizable.
In conclusion, in our study, almost all MS patients on fingolimod develop an adapting humoral response due to repeated vaccinations and infections at longterm follow-up.grant 10430072010007) for the funding of the study and the T2B partners, including the patient groups and Health~Holland for the support in this study.This collaboration project is financed by the PPP.Allowance made available by Top Sector Life Sciences & Health to Samenwerkende Gezondheidsfondsen (SGF) under project number LSHM18055-SGF to stimulate publicprivate partnerships and co-financing by health foundations that are part of the SGF.The authors also thank E.P. Moll van Charante, J.A. Bogaards, and R.A. Scholte for their guidance in the data safety monitoring board.

Data Availability
Data will be made available upon reasonable request.
FU: follow-up; IQR: interquartile range; MS: multiple sclerosis; SD: standard deviation.*Additionalvaccinations were either BNT162b2 (Pfizer-BioNTech) or CX-024414 (Moderna) independent of which vaccine was received in primary immunization, an early booster (third vaccination) was offered to patients on fingolimod before the regular booster.Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/ or publication of this article: FE and TK report (governmental) grants from ZonMw to study immune response after SARS-CoV-2 vaccination in autoimmune diseases.FE also reports grants from Prinses Beatrix Spierfonds, CSL Behring, Kedrion, Terumo BCT, Grifols, Takeda Pharmaceutical Company, and GBS-CIDP Foundation; consulting fees from UCB