key: cord-1046092-b019pyd2
authors: Maniscalco, Giorgia Teresa; Ferrara, Anne Lise; Liotti, Antonietta; Manzo, Valentino; Di Battista, Maria Elena; Salvatore, Simona; Graziano, Daniela; Viola, Assunta; Amato, Gerardino; Moreggia, Ornella; Di Giulio Cesare, Daniele; Alfieri, Gennaro; Di Iorio, Walter; Della Rocca, Gennaro; Andreone, Vincenzo; De Rosa, Veronica
title: Long term persistence of SARS-CoV-2 humoral response in multiple sclerosis subjects
date: 2022-04-12
journal: Mult Scler Relat Disord
DOI: 10.1016/j.msard.2022.103800
sha: 29280f1b34a138462f8d197a042ce60e0888a65a
doc_id: 1046092
cord_uid: b019pyd2

BACKGROUND & OBJECTIVES: The persistence of the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 pandemic, partly due to the appearance of highly infectious variants, has made booster vaccinations necessary for vulnerable groups. Here, we present data regarding the decline of the SARS-CoV-2 BNT162b2 mRNA vaccine-induced humoral immune response in a monocentric cohort of MS patients. METHODS: 96 MS patients undergoing eight different DMTs, all without previous SARS-CoV-2 infection, were evaluated for anti-Spike IgG levels, 21 days (T1) and 5–6 months (T2) after the second SARS-CoV-2 BNT162b2 mRNA vaccine dose. The anti-Spike IgG titre from MS subjects was compared with 21 age- and sex-matched healthy controls (HC). RESULTS: When compared with SARS-CoV-2 IgG levels at T2 in HC, we observed comparable levels in interferon-β 1a-, dimethyl fumarate-, teriflunomide- and natalizumab-treated MS subjects, but an impaired humoral response in MS subjects undergoing glatiramer acetate-, cladribine-, fingolimod- and ocrelizumab-treatments. Moreover, comparison between SARS-CoV-2 IgG Spike titre at T1 and T2 revealed a faster decline of the humoral response in patients undergoing dimethyl fumarate-, interferon-β 1a- and glatiramer acetate-therapies, while those receiving teriflunomide and natalizumab showed higher persistence compared to healthy controls. CONCLUSION: The prominent decline in humoral response in MS subjects undergoing dimethyl fumarate-, interferon-β 1a- and glatiramer acetate-therapies should be considered when formulating booster regimens as these subjects would benefit of early booster vaccinations.

Vaccination against the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has become a global priority as coronavirus disease 2019 (COVID-19) presents a severe or lifethreatening disease course up to 5-10% of cases [1] . Among all the available vaccines, BNT162b2 mRNA has been shown to induce high levels of the anti-SARS-CoV-2 Spike-receptor binding domain (RBD) IgG neutralizing antibodies (NAbs) that strongly correlate with clinical immune protection from COVID-19 in healthy subjects [2] [3] [4] . Furthermore, NAbs also induce a good level of seroprotection in Multiple Sclerosis (MS) patients, although with different effectiveness depending on the disease-modifying therapy (DMT). Indeed, while MS patients treated with immunosuppressive therapies such as cladribine (CLAD), fingolimod (FTY) and ocrelizumab (OCRE) exhibited an impaired secretion of SARS-CoV-2 anti-Spike IgGs [5] [6] [7] , an increased humoral vaccine response has been reported in interferon- 1a (IFN)-treated MS subjects [7] . In light of the current discussion regarding SARS-CoV-2 booster vaccinations, several studies have evaluated the duration of anti-SARS-CoV-2 immune protection in healthy individuals [8, 9] ; however clinical data suggest an attenuated short-term humoral response to SARS-CoV-2 vaccines in patients with MS receiving DMTs [5, 10] .

Here we presented a prospective study aimed at assessing the six-months persistence of the humoral response in a monocentric cohort of MS patients treated with eight different DMTs compared with healthy subjects, all receiving BNT162b2 mRNA vaccine and without previous SARS-CoV-2 infection.

This is a prospective monocentric study to evaluate the kinetic of SARS-CoV-2 IgG Spike titre 5-6 months after the second dose of BNT162b2 vaccine in MS subjects undergoing vaccination at the Multiple Sclerosis Centre of the Cardarelli Hospital (Naples, Italy) from March to November 2021.

All human subjects were enrolled after obtaining informed consent. The study was approved by the Institutional Review Board of the Cardarelli Hospital. We enrolled 96 MS and 21 healthy controls receiving the two doses of BNT162b2 vaccine according to the recommendations of Italian Authority of Health, all without previous SARS-CoV-2 infection. MS subjects were vaccinated according to specific timing; more in detail, MS subjects treated with IFN, GA, TERI, DMF, FTY and NAT were vaccinated without any interruption of immunomodulatory treatment, while CLADand OCRE-treated MS subjects were vaccinated at least 1 or 3 months respectively after the last therapeutic administration, according to the recommendations of Italian Authority of Health. Blood samples were collected at 9:00 AM into heparinized Vacutainers (BD Biosciences) and processed within the following 4h. Demographic and clinical characteristics of the study cohort are shown in Table 1 . Inclusion criteria were patients aged between 18-65 years, diagnosed with multiple sclerosis treated with DMTs for at least 6 months. Exclusion criteria were previous SARS-CoV-2 infection (antibody screening), any relapse and/or steroid use in the last 30 days before enrolment.

Healthy subjects were matched for age and sex and had no history of inflammation, endocrine or autoimmune disease. The ethnic distribution among the groups was comparable, with all participants being Caucasian.

Quantitative determination of antibodies to the SARS-CoV-2 Spike protein was carried out by Roche Elecsys® Anti-SARS-CoV-2 S assay (Roche Diagnostics International Ltd, Rotkreuz, Switzerland). The assay was performed using a recombinant protein representing the RBD of the S antigen leading to a double-antigen sandwich assay complex which favors detection of high affinity antibodies against SARS-CoV-2 (range between 0.4 to 250 U/mL), resulting in a sensitivity of 98.8 % (95 % CI: 98.1 -99.3 %).

The study was conducted according the Good Clinical Practice guidelines and the ethical principles of the Declaration of Helsinki. Investigators obtained ethic committee approval for the study protocol and amendments by the local Ethic Committee of A.O.R.N. A. Cardarelli/Santobono-Pausilipon (protocol number 2821). All subjects give written informed consent to participate to the study.

Descriptive analyses were presented as mean (± standard error of the mean), median and interquartile range (IQR). Categorial variables were described as frequency and percentage. A Shapiro-wilk test was performed to assess the normal distribution of data. In case of not-normal distribution appropriate non-parametric tests were performed (Wilcoxon test). The fold persistence of SARS-CoV-2 Spike IgG titre was calculated as the percentage ratio between IgG titre at T2 and T1, relative to HC (100 %). P-value less of 0.05 indicated significance. A multilinear regression model was used to compare the antibody levels across subjects treated with differerent DMTs after adjusting for age, sex, EDSS levels, disease duration, DMT duration and antibody levels in the prebooster samples. Data analyses were performed using Graphpad Prism (version 8).

Data were collected from March 2021 to November 2021. In this prospective monocentric study, we excluded subjects previously infected with SARS-CoV-2, through the measurement of nucleocapsid-specific antibodies. After assessment, 96 MS patients and 21 HC were evaluated for anti-Spike IgG levels, 21 days (T1) and 5-6 months (T2) after the second SARS-CoV-2 BNT162b2 mRNA vaccine dose. The demographic and clinical characteristics are reported in Table 1 . In the MS group, 57 were female (59.4%) and 39 male (40.6%) and the mean age was 40.7 ± 10.5 (mean ± SD) years. In the control group (HC), 11 subjects were females (52.4%) and 10 males (47.6%), 

We previously reported that IFN-treated MS subjects showed a significant increase of anti-Spike

IgG levels compared to HC, while humoral response was profoundly affected in MS subjects undergoing CLAD, FTY and OCRE, 21 days after the second BNT162b2 mRNA vaccine dose [6] .

We aimed at evaluating the persistence of the SARS-CoV-2 IgG titre 5-6 months after the second vaccine dose (T2), in our monocentric cohort of MS subjects undergoing different DMTs, respect to age-and sex-matched HC. We found that SARS-CoV Table 2 ). The reduction was not significant in TERI-, CLA-and OCRE-treated MS groups, while subjects under FTY showed a slight -albeit irrelevant -increase of anti-Spike IgG at T2 compared to their T1 levels ( Figure 1B e Table 2 ). Finally, in those groups of DMT-treated MS subjects having shown a significant antibody production after the second SARS-CoV-2 BNT162b2 mRNA vaccine dose, we evaluated the persistence of the humoral response at 6 months, expressed as the percentage ratio between IgG titre at T2 and T1, relative to HC (Figure 1C) . When compared to healthy controls, we observed a faster decline of anti-Spike IgG levels in patients undergoing DMF-(75.7 ± 11.5 %), IFN-(62.4 ± 6.7 %) and GA-(54.3 ± 11.3 %) therapies, while those receiving TERI and NAT show an increased persistence (130.9 ± 34.4 % and 127.2 ± 32.6%, respectively), still not reaching the statistical significance ( Figure 1C) . We performed linear regression analyses to rule out that individual factors or clinical variables (age, sex, EDSS, DMT duration, time to last therapeutic administration) could affect the SARS-CoV-2 vaccine humoral response at T2, but we did not find any significant correlation between these parameters and IgG-Spike titre in the different DMT groups (data not shown). Our data highlight how DMTs could differentially promote the persistence of protective SARS-CoV-2 humoral response in MS subjects.

Our study showed that antibody responses to SARS-CoV-2 BNT162b2 mRNA vaccine are broadly distributed and declined substantially in most individuals over time. Antibodies are key immune effectors that confer protection against pathogens [7] . The longevity of the antibody response to SARS-CoV-2 vaccine in multiple sclerosis (MS) subjects are still not well defined. In this context, decisions regarding initiation or continuation of specific disease modifying therapy (DMT) have to consider the potential relevance to the pandemic. Understanding the possible distinctive effects of each therapeutic agent on the immune response to the vaccine is essential during this special time. However, based on their anti-Spike IgG levels at T1, we evaluated the antibody persistence in those DMT-groups exhibiting an adequate humoral response after vaccination. We observed that, when compared to HC, TERI-and NAT-treated MS subjects showed an increased persistence of anti-Spike specific IgG antibody response to SARS-CoV-2 BNT162b2 mRNA vaccine after 5-6 months, while DMF-, IFN-and GA-therapies affected the retention of the humoral response overtime. An important implication of our data is the possible existence of an efficient SARS-CoV-2 vaccine ""holder"" phenotype, defined as individuals who experience relatively sustained anti-SARS-CoV-2 IgG production. This seems to be the case of teriflunomide, which selectively inhibits dihydro-orotate dehydrogenase (DHODH), an important mitochondrial enzyme in the de novo pyrimidine synthesis pathway. The downstream effect is the reduced proliferation of rapidly dividing cells, including activated T and B lymphocytes. It has been reported that TERI-treated MS patients developed effective immunity to seasonal influenza after vaccination. In addition, there is emerging evidence suggesting a direct antiviral effect for teriflunomide and other DHODH inhibitors against a range of viruses such as Theiler"s, respiratory syncytial, Ebola, cytomegalovirus, Epstein-Barr, and picornavirus [8] . Moreover, our data are in line with the successful development of anti-SARS-CoV-2 antibodies described in patients under teriflunomide after COVID-19 infection [9] . Regarding natalizumab, it acts as an antagonist to alpha-4 integrin on leukocyte surface, blocking their interaction with vascular cell adhesion molecules and preventing leukocyte migration to the CNS. Data from influenza vaccine studies strongly support its treatment under the COVID-19 pandemic; however, some concerns have been raised regarding the potential increase of viral shedding due to the reduction of lymphocyte trafficking in the lungs [10] . Since our data unveiled a slight increase in both T1 and T2 anti-Spike IgG levels compared to HC and a good humoral persistence, this could suggest a delayed booster timing for NAT-treated MS subjects. The same holds true also for TERI-treated MS subjects which, despite a lower antibody production at T1, retained an excellent humoral reservoir at T2. According to the literature, our study shows that different humoral persistence could be observed among immuno-modulating DMTs [5, 10, 16] ; this offers new evidence that, if confirmed in a larger cohort of patients, should be considered when formulating booster regimens in MS subjects. 

DMF: Dimethyl fumarate; DMTs: Disease modifying therapies; EDSS: Expanded disability status scale; FTY: Fingolimod; GA: Glatiramer acetate; HC: Healthy controls; IgG: Immunoglobulin G; INF: Interferon β 1a; IQR: Interquartile range; MS: Multiple sclerosis; NAT: Natalizumab; OCRE: Ocrelizumab; PPMS: Primary progressive multiple sclerosis; RRMS: Relapsing remitting multiple sclerosis; SARS-CoV-2: severe acute respiratory syndrome coronavirus-2

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