key: cord-0989173-nv5bv0hz
authors: Iannetta, Marco; Landi, Doriana; Cola, Gaia; Malagnino, Vincenzo; Teti, Elisabetta; Fraboni, Daniela; Buccisano, Francesco; Grelli, Sandro; Coppola, Luigi; Campogiani, Laura; Massimo, Andreoni; Marfia, Girolama Alessandra; Sarmati, Loredana
title: T-cell responses to SARS-CoV-2 in Multiple Sclerosis patients treated with ocrelizumab healed from COVID-19 with absent or low anti-Spike antibody titers
date: 2021-07-21
journal: Mult Scler Relat Disord
DOI: 10.1016/j.msard.2021.103157
sha: 9c57365f0deacd0be3085cb0d52e749cecb23cb9
doc_id: 989173
cord_uid: nv5bv0hz

BACKGROUND: Disease modifying therapies for multiple sclerosis (MS) can impair the specific immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Specifically, it is recognized that ocrelizumab reduces or abrogates anti-SARS-CoV-2 antibody production after natural infection or vaccination, while very little is known about T-cell responses. METHODS: We developed an interferon (IFN)-γ release assay (IGRA) to detect T-cell responses specific to SARS-CoV-2 after overnight stimulation of whole blood with peptide libraries covering the immunodominant sequence domains of the Spike glycoprotein (S) and the Nucleocapsid phosphoprotein (N). RESULTS: Five patients with MS receiving ocrelizumab treatment for at least 1 year and recovered from SARS-CoV-2 infection were enrolled in the study. Despite the absence or the very low concentration of anti-S antibodies, a T-cell response was detectable in all the five MS patients. These results are in accordance with the marked reduction of peripheral B-lymphocyte absolute counts induced by ocrelizumab, that, conversely, did not affect peripheral blood T-lymphocyte subset absolute and relative counts and CD4/CD8 ratio. CONCLUSIONS: The detection of specific T-cell responses to SARS-CoV-2 in patients receiving B-cell depleting therapies represents a useful tool to improve the diagnostic approach in SARS-CoV-2 infection and to accurately assess the immunological response after natural infection or vaccination.

We investigated specific T-cell responses towards severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in patients receiving B-cell depleting treatment with ocrelizumab for multiple sclerosis (MS), who recovered from coronavirus disease-2019 . Little is known about cell-mediated responses in patients receiving B-cell depleting therapies after SARS-CoV-2 natural infection or vaccination (Asplund Högelin et al., 2021) , while several case reports have demonstrated the absence of detectable anti-SARS-CoV-2 antibodies (Ab) (Iannetta et al., 2020; Khayat-Khoei et al., 2021; Smets et al., 2021; Thornton and Harel, 2020) . Recently, Achiron et al. showed poor seroconversion rates after SARS-CoV-2 vaccination in MS patients receiving B-cell depleting treatments (Achiron et al., 2021) . The role of T-cell responses in terms of protection against SARS-CoV-2 infection and re-infection needs to be further elucidated.

Interferon (IFN)- release assay (IGRA) represents a useful tool to identify individuals with specific cellular immunity for SARS-CoV-2 (Murugesan et al., 2020) .

The study was approved by the local Ethic Committees (number 46.20). All patients signed a written informed consent. Anti-Spike Ab specific for SARS-CoV-2 were detected with a commercial electrochemiluminescence immunoassay (Elecsys  Anti-SARS-CoV-2 S, Roche Diagnostics). Peripheral blood lymphocyte subsets were assessed with a lyse no wash standardized protocol, using the BD  Multitest 6-Color TBNK Reagent and trucount tubes (BD Biosciences). T-lymphocyte specific response was assessed with an interferon (IFN)- release assay (IGRA) after overnight stimulation with SARS-CoV-2 peptide libraries. Pools of lyophilized peptides, consisting mainly of 15-mer sequences with 11 amino acids overlap, covering the immunodominant sequence domains of the Spike glycoprotein (S) (GenBank MN908947.3, Protein QHD43416.1) and the Nucleocapsid phosphoprotein (N) (GenBank MN908947.3, Protein QHD43423.2) of SARS-CoV-2 were purchased from Milteny Biotec.

Briefly, 500µl of fresh heparinized blood were incubated overnight at 37°C and 5% CO 2 , after stimulation with PepTivator  S1, S, and N (3 conditions for each peptide library plus 1 condition with pooled peptides) at a final concentration of 1µg/ml. For each patient a negative and positive (phytohemagglutinin 5µg/ml) condition was also included. After 18 hours, supernatants were collected and stored at -80°C. IFN- production was assessed with a commercial enzyme linked immunosorbent assay (ELISA) kit (Human IFN- DuoSet ELISA,

and T-lymphocyte specific responses were assessed on the same day.

Five patients (4 males, median age 33 years, range 27-50) with a relapsing remitting (RR) MS, receiving ocrelizumab treatment, who experienced SARS-CoV-2 infection, were enrolled in this study. All patients had been exposed to ocrelizumab for at least 1 year.

Median expanded disability status scale (EDSS) was 0.5 (range 0-3). SARS-CoV-2 infection was diagnosed through SARS-CoV-2 RNA molecular detection on nasopharyngeal swab.

Two patients needed hospitalization because of COVID-19 pneumonia, and were classified as severe. Patients were sampled after a median of 89 days from COVID-19 onset (range: 66-176) and 89 days from the last ocrelizumab infusion (range: . Clinical data are reported in table 1.

Anti-S Ab were detectable only in two subjects (OCRE1: 0.54 U/ml and OCRE5: 155.6 U/ml) at low concentrations. Flow cytometry analysis of peripheral blood showed a reduction in CD19+ B-lymphocyte absolute and relative counts (median: 1 cell/µl, range 0-15; median 0.04%, range 0-0.96; respectively). Conversely, CD4+ and CD8+ T-cell absolute and relative counts were within normal ranges, and CD4/CD8 ratio was above 1 for all the subjects (table   1) .

Whole blood stimulation showed detectable levels of IFN- upon PepTivator  S1 and N stimulation in all patients with the exception of subject OCRE3. After stimulation with PepTivator  S, IFN- was undetectable in supernatants of 2 subjects (OCRE1 and OCRE3).

Interestingly, after pooling together the three peptide libraries (S1, S and N), an IFN- production was detectable in all the subjects, including subject OCRE3 (figure 1).

Results from three healthy controls are reported in supplementary table 1, showing the consistency of the stimulation.

In this case collection of 5 MS patients under ocrelizumab treatment who recovered from COVID-19, despite the absence of anti-S antibody production, a specific T-cell response was One limitation of our study is represented by the lack of pre-infection samples to evaluate SARS-CoV-2 specific T-cell responses prior to COVID-19 disease. It has been shown that in some SARS-CoV-2 unexposed donors T-cells display significant reactivity to SARS-CoV-2 antigen peptide pools, due to cross-reactivity with other coronaviruses (Sette and Crotty, 2020) . antibodies; Anti-Spike Ab limit of detection: 0.40 U/ml For each subject, 6 conditions were set up: unstimulated (NS); stimulation with PepTivator  SARS-CoV-2 Prot_S1 [1µg/ml] (S1); stimulation with PepTivator  SARS-CoV-2 Prot_S [1µg/ml] (S); stimulation with PepTivator  SARS-CoV-2 Prot_N [1µg/ml] (N); stimulation with pooled peptides, S1 + S + N [1µg/ml] (P); stimulation with phytohemagglutinin 5µg/ml (PHA). After overnight stimulation, IFN- concentration (ng/ml) in supernatants was assessed with a commercial ELISA KIT. Each sample was tested in duplicate. A 7-point standard curve was included in each ELISA session. IFN- response was defined as peptide or PHA stimulated condition minus unstimulated condition.

Humoral immune response to COVID-19 mRNA vaccine in patients with multiple sclerosis treated with high-efficacy disease-modifying therapies

Development of Humoral and Cellular Immunological Memory Against SARS-CoV-2 Despite B-Cell Depleting Treatment in Multiple Sclerosis (SSRN Scholarly Paper No. ID 3796531)

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