Humoral And Cellular Immunity in Convalescent And Vaccinated COVID-19 People With Multiple Sclerosis: Effects Of Disease Modifying Therapies

ABSTRACT

Objectives:

To determine anti-SARS-Cov2 antibodies and T-cell immunity in convalescent people with multiple sclerosis (pwMS) and/or pwMS vaccinated against Covid-19, depending on the disease-modifying therapy, and in comparison to healthy controls (HC).

Multiple sclerosis (Multiple Sclerosis, MS) is an autoimmune disease, and its pathogenesis is related to the disorder of immunity. Under normal circumstances, the immune system attacks pathogens that invade the body and protects the body from infection. But in MS patients, the immune system mistakenly attacks and destroys the myelin sheath in the central nervous system (CNS), causing nerve conduction to be affected, inflammation, and nerve damage. This aggressive immune response may be due to dysregulation of the immune system's self-tolerance. Therefore, regulating and improving the function and balance of the immune system is an important direction for the treatment of multiple sclerosis. From this point of view, we also need to pay attention to our immunity in our daily life. Cistanche has a significant effect on improving immunity. The polysaccharides in the meat can regulate the immune response of the human immune system and improve the stress ability of immune cells. Enhance the bactericidal effect of immune cells.

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Methods:

75 participants were enrolled: Group 1—29 (38.7%) COVID-19 convalescent participants; Group 2—34 (45.3%) COVID-19 vaccinated; Group 3—12 (16.0%) COVID-19 convalescent participants who were later vaccinated against COVID-19. Cellular immunity was evaluated by the determination of several CD4+ and CD8+ cells secreting TNFα, IFNγ, and IL2 after stimulation with SARS-CoV-2 peptides. Results: pwMS treated with ocrelizumab were less likely to develop humoral immunity after COVID-19 recovery or vaccination. No difference was observed in the cellular immunity in all studied parameters between pwMS treated with ocrelizumab compared to HC or pwMS who were treatment naïve or on first-line therapies. These findings were consistent in convalescent, vaccinated, and convalescent+vaccinated participants. COVID-19-vaccinated convalescent pwMS on ocrelizumab compared to COVID-19 convalescent HC who were vaccinated did not show a statistical difference in the rate of seroconversion nor titers of SARS-CoV-2 antibodies. Conclusion: The presence of cellular immunity in pwMS on B-cell depleting therapies is reassuring, as at least partial protection from more severe COVID-19 outcomes can be expected.

1. Introduction

Several studies have indicated an association between B-cell-depleting disease-modifying therapy (DMTs) and a higher probability of a more serious clinical course of COVID-19 (M.P. Sormani et al., 2021; Stastna et al., 2021). Furthermore, more and more data suggest an attenuated humoral response after SARS-COV-2 infection and after COVID-19 vaccination in people with MS (pwMS) who are using ocrelizumab (Habek et al., 2021; Achiron et al., 2021). In light of these data, it is of paramount importance to know how B-cell-depleting agents affect the development of humoral and cellular immunity after the infection, and whether there is an impact on the vaccine response in different populations of pwMS.

The present study aims to determine the development of anti-SARSCov2 antibodies and the development of T-cell mediated immunity in convalescent or/and vaccinated COVID-19 pwMS depending on the DMTs they use and in comparison to healthy controls (HC).

2. Objectives

The primary objective was to investigate the differences in the presence of humoral and cellular immunity in:

a) convalescent COVID-19 pwMS treated with ocrelizumab compared to treatment naïve pwMS or pwMS on 1st line therapies (TN/1st pwMS) and HC.

b) vaccinated COVID-19 pwMS treated with ocrelizumab compared to treatment naïve pwMS or pwMS on 1st line therapies (TN/1st pwMS) and HC.

c) convalescent and vaccinated COVID-19 pwMS treated with ocrelizumab compared to HC.

Fig. 1. Gating strategy to detect SARS-CoV-2 reactive CD4+ and CD8+ T cells after in vitro stimulation for 8 h with surface glycoprotein, matrix, and nucleoprotein overlapping peptide pools. Representative gating of a single live CD4 and CD8 T cells in the upper panel (a), and further enumerated concerning intracellular TNFα, IFNγ, and IL2 (from left to right) cytokine production.

The secondary objectives were to investigate the differences in the titers of SARS-CoV-2 IgG antibodies and absolute values of CD4 and CD8 cells expressing INFγ, TNFα, and IL2 in the same groups as above. Finally, differences in humoral and cellular immunity were investigated between convalescent, vaccinated, and convalescent+vaccinated pwMS on ocrelizumab.

3. Materials and methods

3.1. Participants

This single-center, case-control study was performed at University Hospital Center, Zagreb, Croatia, and was approved by the Ethics Committee of the University Hospital Center Zagreb. All pwMS, who came on their regular follow-up visit between July 15th and Aug 15th, 2021, were invited to participate in the study. Inclusion criteria included: 1) recovery from COVID-19 and/or full vaccination against COVID-19 in 12 months before blood sampling, 2) treatment naïve pwMS or pwMS on first-line DMT—interferons, glatiramer acetate, teriflunomide or dimethyl fumarate—or pwMS on ocrelizumab. Exclusion criteria were: 1) all other DMTs except the aforementioned, 2) any other off-label MS therapy, and 3) pwMS who were non-compliant to DMTs. Age and sex-matched convalescent or vaccinated healthy controls (HC) were enrolled as well.

3.2. Humoral immunity

Testing for humoral immunity was performed in the Clinical Institute for Laboratory Diagnostics, University Hospital Center Zagreb, Zagreb, Croatia, using Elecsys® Anti-SARSCoV-2 S assay (Roche Diagnostics Int, Rotkreuz, Switzerland). The assay was performed per the manufacturer’s instructions, using Cobas e 801 analytical unit for immunoassay tests (F. Hoffmann-La Roche Ltd.). Antibody titer ≥0.8 U/mL was considered positive, as recommended by the manufacturer.

3.3. Cellular immunity

Virus-reactive cytokine-producing T cells were enumerated using the human SARS-CoV-2 T Cell Analysis Kit (PBMC, Miltenyi Biotech) following manufacturer protocol, with some modifications.

PBMC were isolated from heparinized whole blood by density gradient centrifugation (Ficoll-Paque Plus from Cytiva). After collection of the PBMC ring, cells were washed twice with RPMI 1640 medium and resuspended in RPMI 1640 media with 10% of human AB serum from healthy, SARS-Cov-2 naive donors with undetectable virus-specific antibodies and undetectable virus-specific T cells. PBMC were stimulated with PepTivator SARS-CoV-2 (Miltenyi Biotec) for complete spike glycoprotein covering the complete protein coding sequence aa 5–1273 (GenBank MN908947.3, Protein QHD43416.1), matrix, and nucleoprotein overlapping peptides, at concentration 1 µg/ml of each for 8 h at 37 ◦C, 5% CO2. Positive control PBMC were stimulated with CytoStim™ whereas negative control PBMC were cultivated in complete medium with 10% DMSO. Brefeldin A at a concentration of 2 µg/ml was added for the last 6 h. Upon harvest, PBMC were labeled with fluorophore-conjugated antibodies for surface markers CD3 APC, CD4 Vio®Bright B515, CD8 VioGreen™, CD14 VioBlue®, and CD20 VioBlue® in the presence of Tandem Signal Enhancer and FcR Blocking Reagent.

After washing in PBS, PBMC was stained with Viobility 405/452 Fixable Dye, and fixed with InsideFix buffer overnight at 4 ◦C. The next day, PBMC was washed with PEB buffer and permeabilized with InsidePerm buffer. For intracellular staining, cells were incubated in a mixture of Tandem Signal Enhancer, FcR Blocking Reagent, and antiIFN-γ PE, anti-TNF-α PE-Vio® 770, and anti-IL-2 PE-Vio® 615 antibodies. Finally, PBMC was resuspended in PEB buffer and analyzed on a 3-laser equipped BD LSR II flow cytometer (BD Biosciences). A live CD3 T cell acquired ranged from 250k-450k per tube.

Data analysis was performed using FlowJo 10.7.1 (TreeStar Inc/ FlowJo LLC). Doublets, debris, and dead cells as well as CD14+ and CD20+ cells were excluded. After predating on live CD3 as well as CD4 and CD8 subtypes, cytokine expression for CD4+ T cells and CD8+ T cells was assessed. Fluorescence minus one (FMO) controls were performed for all fluorescence and used for the gating setting (Fig. 1).

Percentages of cytokine-producing cells were assessed after the deduction of values from unstimulated cells, with the threshold of 0.02%, based on FMO.

3.4. Statistical analysis

Statistical analysis was performed with the IBM SPSS v25 software. The data distribution was tested with the Kolmogorov-Smirnov test. The differences between qualitative variables were tested with the Chi-square test. The differences between the quantitative variables were tested with the parametric independent sample t-test and nonparametric Mann-Whitney test for the comparison between the two groups, and ANOVA test and Kruskal-Wallis test for the comparison between the more than two groups. P-values less than 0.05 were considered significant.

4. Results

Altogether, 75 participants were enrolled: 29 (38.7%) COVID-19 convalescent participants – group 1, 34 (45.3%) COVID-19 vaccinated - group 2, and 12 (16.0%) COVID-19 convalescent participants who were later vaccinated against COVID-19 – group 3. The demographic characteristics of the cohort are presented in Table 1.

Results regarding the presence or absence of humoral and cellular immunity across groups are presented in Table 1. To summarize the results, the absence of humoral immunity was more frequent in ocrelizumab-treated pwMS in convalescent and vaccinated groups compared to HC and TN/1st pwMS. No difference was observed in Group 3. On the contrary, there was no difference in cellular immunity between ocrelizumab-treated pwMS, TN/1st pwMS, and HC in all three groups (Table 1).

A similar observation was made while interpreting data presented as absolute values.

In convalescent groups, the titer of SARS-CoV2 IgG was significantly lower in pwMS on ocrelizumab, compared to TN/1st pwMS (0.21 (0 − 250) vs. 87.60 (5.25–250), p = 0.006) (Fig. 2). In the vaccinated group, the titer of SARS-CoV2 IgG was significantly lower in pwMS on ocrelizumab, compared to healthy controls and TN/1st pwMS (0.42 (0–250) vs. 250 (143–250) vs. 250 (37.5–250), < 0.001; respectively), (Fig. 2). In Group 3, there was no statistically significant difference in titer of SARS-CoV2 IgG between the pwMS on ocrelizumab and healthy controls (p = 0.106).

In convalescent groups, the titer of SARS-CoV2 IgG was significantly lower in pwMS on ocrelizumab, compared to TN/1st pwMS (0.21 (0 − 250) vs. 87.60 (5.25–250), p = 0.006) (Fig. 2). In the vaccinated group, the titer of SARS-CoV2 IgG was significantly lower in pwMS on ocrelizumab, compared to healthy controls and TN/1st pwMS (0.42 (0–250) vs. 250 (143–250) vs. 250 (37.5–250), < 0.001; respectively), (Fig. 2). In Group 3, there was no statistically significant difference in titer of SARS-CoV2 IgG between the pwMS on ocrelizumab and healthy controls (p = 0.106).

Finally, no differences were observed in the presence of humoral or cellular immunity between convalescent, vaccinated, and convalescent+vaccinated pwMS on ocrelizumab (all p>0.05).

5. Discussion

The results of our study have shown that although a significant proportion of pwMS on B-cell depleting therapy do not mount a humoral response after recovery from COVID-19 or vaccination against COVID-19, they can mount sufficient cellular immunity against COVID-19. Specifically, no difference was observed in both CD4 or CD8-positive INFγ, TNFα, and IL2 cells in pwMS on B-cell depleting therapy compared to healthy controls or treatment naïve pwMS and/or pwMS on 1st line therapies.

Our findings may have implications in mitigating the risk associated with the treatment of pwMS with B-cell-depleting therapy.

First, results from the MSBase registry, with the largest cohort of pwMS with COVID-19, had demonstrated consistent associations of rituximab with increased risk of hospitalization, ICU admission, and requiring artificial ventilation, and ocrelizumab with hospitalization and ICU admission. (Simpson-Yap et al., 5) Further, several other studies have identified that pwMS who are receiving ocrelizumab is not able to mount a protective humoral response. (Habek et al., 2021; Achiron et al 9) Recent data also suggest that the development of a humoral immune response remains rare in pwMS using B-cell depleting therapies, even after the third dose of the homologous SARS-CoV-2 mRNA vaccine. (Achtnichts et al., 2021) 

Studies have shown a significant correlation between time since the last dose of B-cell depleting therapies and the development of humoral immunity after vaccination, with rituximab having longer intervals (mean=386 days) than ocrelizumab (mean=129 days). (Konig ¨ et al., 20; M.P. Sormani et al., 2021) It has been suggested that, in selected clinically and radiologically stable pwMS, delaying the cycle of B-cell depleting therapies by 3 to 6 months before vaccination could increase the probability of developing appropriate humoral responses. (Disanto et al., 2021) All these data indicate that pwMS treated with B-cell-depleting therapies should be informed about the risk of more severe COVID-19 outcomes and the risk of reduced humoral immunity after vaccinations if they are already on therapy.

This raises the question of whether COVID-19 convalescent patients or patients who have been vaccinated against COVID-19 and do not develop an antibody response, will have adequate immunity from subsequent SARS-Cov-2 infections. Although data on reinfection or infection after vaccination in pwMS on B-cell-depleting therapies is currently lacking, several studies have emphasized T-cell immunity as an important factor in the protection against SARS-CoV-2. The first study compared B cell and T cell responses longitudinally in 20 pwMS on anti-CD20 antibody monotherapy with 10 HC after BNT162b2 or mRNA-1273 mRNA vaccination. In this study, all pwMS treated with aCD20 therapy generated antigen-specific CD4 and CD8 T cell responses after vaccination. (Apostolidis et al., 14) 

Moreover, several subsequent studies confirmed that pwMS who were treated with ocrelizumab generated comparable SARS-CoV-2-specific T-cell responses with healthy controls and/or pwMS on other MS therapies. (Brill et al., 23; Sabatino et al., 14; Gadani et al., 16; N. Madelon et al., 2021)

The results of our study further expand the knowledge on T-cell immunity, not only on vaccination but also on COVID-19 convalescent pwMS. We have found no difference in any studied parameter of T-cell immunity in COVID-19 convalescent pwMS regardless of DMT and HC. Furthermore, COVID-19 convalescent pwMS on ocrelizumab who were vaccinated compared to COVID-19 convalescent HC who were vaccinated do not show statistically different rates of seroconversion or titers of SARS-CoV-2 antibodies. This finding is in line with a previous study showing that humoral response to mRNA vaccine is significantly influenced by previous SARS-CoV-2 infection. (Pitzalis et al., 2021)

With the rapid spread of the SARS-CoV-2 variant Omicron, the question raised whether T-cell responses to the Omicron variant are conserved in pwMS using B-cell depleting therapies after COVID-19 mRNA vaccination. Preliminary, non-peer-reviewed data, suggest that in pwMS using B-cell depleting therapies the vaccine-induced T-cell responses are little affected by the mutations carried by Omicron, and a third vaccine dose improves cytotoxic T-cell responses. (N. Madelon et al., 2021)

Limitations of this study are the small number of participants and the lack of longitudinal data on both humoral and cellular immunity in the studied sample of participants.

In conclusion, the presence of cellular immunity in pwMS on B-cell-depleting therapies is reassuring, as at least partial protection from more severe COVID-19 outcomes can be expected. Our data on COVID-19 convalescent persons, who are later vaccinated against COVID-19, support the use of booster SARS-CoV-2 vaccinations in patients receiving B-cell depleting therapies.

Authors’ contributions

Study concept and design: Habek. In vitro experimental setup and flow cytometric acquisition: Cveti´c, Bendelja. Flow cytometry data analysis: Savi´c Mlakar, Bendelja. Acquisition of data: Habek, Rogi´c, Adamec, Barun, Gabeli´c, Krbot Skori´c. Analysis and interpretation of data: Habek, Cveti´c, Savi´c Mlakar, Bendelja, Rogi´c, Adamec, Barun, Gabeli´c, Krbot Skori´c. Drafting of the manuscript: Habek. Critical revision of the manuscript for important intellectual content: Habek, Cveti´c, Savi´c Mlakar, Bendelja, Rogi´c, Adamec, Barun, Gabeli´c, Krbot Skori´c. Administrative, technical, and material support: Habek, Cveti´c, Savi´c Mlakar, Bendelja, Rogi´c, Adamec, Barun, Gabeli´c, Krbot Skori´c.

Declaration of Competing Interest

MH: Participated as a clinical investigator and/or received consultation and/or speaker fees from Biogen, Sanofi Genzyme, Merck, Bayer, Novartis, Pliva/Teva, Roche, Alvogen, Actelion, Alexion Pharmaceuticals, TG Pharmaceuticals.

ZC: ˇ Reports no conflict of interest. 

ASM: Reports no conflict of interest. 

KB: Reports no conflict of interest. 

DR: Reports no conflict of interest.

IA: Participated as a clinical investigator and/or received consultation and/or speaker fees from Biogen, Sanofi Genzyme, Merck, Bayer, Novartis, Pliva/Teva, Roche, Alvogen, Actelion, Alexion Pharmaceuticals, TG Pharmaceuticals.

BB: Participated as a clinical investigator and/or received consultation and/or speaker fees from Biogen, Sanofi Genzyme, Merck, Bayer, Novartis, Pliva/Teva, Roche, Alvogen, Actelion, Alexion Pharmaceuticals.

TG: Participated as a clinical investigator and/or received consultation and/or speaker fees from Biogen, Sanofi Genzyme, Merck, Bayer,

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

No funding was received for this study.

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