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Evaluation of immunological responses to third COVID-19 vaccine among people treated with sphingosine receptor-1 modulators and anti-CD20 therapy

Background : People living with multiple sclerosis (MS) and other disorders treated with immunomodulatory therapies remain concerned about suboptimal responses to coronavirus disease 2019 (COVID-19) vaccines. Important questions persist regarding immunological response to third vaccines, particularly with respect to newer virus variants. The objective of this study is to evaluate humoral and cellular immune responses to a third COVID-19 vaccine dose in people on anti-CD20 therapy and sphingosine 1-phosphate receptor (S1PR) modulators, including Omicron-specific assays. Methods : This is an observational study evaluating immunological responses to third COVID-19 vaccine dose in participants treated with anti-CD20 agents, S1PR modulators, and healthy controls. Neutralizing antibodies against USA-WA1/2020 (WA1) and B.1.1.529 (BA.1) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were measured before and after third vaccine. Groups were compared by one-way ANOVA with Tukey multiple comparisons. Cellular responses to spike peptide pools generated from WA1 and BA.1 were evaluated. Pre-post comparisons were made by Wilcoxon paired t-tests, inter-cohort comparisons by Mann-Whitney t-test. Results : This cohort includes 25 participants on anti-CD20 therapy, 12 on S1PR modulators, and 14 healthy controls. Among those on anti-CD20 therapy, neutralizing antibodies to WA1 were significantly reduced compared to healthy controls (ID50% GM post-vaccination of 8.1 ± 2.8 in anti-CD20 therapy group vs 452.6 ± 8.442 healthy controls, P<0.0001) and neutralizing antibodies to BA.1 were below the threshold of detection nearly universally. However, cellular responses, including to Omicron-specific peptides, were not significantly different from controls. Among those on S1PR modulators, neutralizing antibodies to WA1 were detected in a minority, and only 3/12 had neutralizing antibodies just at the limit of detection to BA.1. Cellular responses to Spike antigen in those on S1PR modulators were reduced by a factor of 100 compared to controls (median 0.0008% vs. 0.08%, p<0.001) and were not significantly “boosted” by a third injection. Conclusions : Participants on anti-CD20 and S1PR modulator therapies had impaired antibody neutralization capacity, particularly to BA.1, even after a third vaccine. T cell responses were not affected by anti-CD20 therapies, but were nearly abrogated by S1PR modulators. These results have clinical implications warranting further study.


Introduction
Many people receiving treatment with immunomodulatory therapies, including those living with multiple sclerosis (MS), remain apprehensive about COVID-19 risk. Among common therapies for MS, there is high concern with anti-CD20 agents and sphingosine 1-phosphate receptor (S1PR) modulators. Anti-CD20 agents deplete circulating B cells and, to a lesser extent, subsets of CD4 and CD8 cells expressing low levels of CD20. 1 In addition to widespread use in MS, anti-CD20 agents are utilized to treat B cell malignancies and a range of immune-mediated neurological and rheumatologic disorders. 2 S1PR modulators functionally block S1PR function, sequestering lymphocytes in the lymph nodes and thymus. 3 In addition to use in MS, the S1PR modulator ozanimod was recently approved for ulcerative colitis and ponesimod is currently in late stage clinical trials for psoriasis and graft-vs.-host disease.
Previous data suggest reduced humoral response to the primary vaccine series related to these treatments [4][5][6] . Limited evidence suggests preserved cellular response to vaccination with anti-CD20 therapies and reduced response with S1PR modulators. 7, 8 Epidemiologic studies have suggested worse clinical outcomes with these therapies. 9 Here we address the most recent significant questions regarding the production of neutralizing antibodies and cellular immune responses before and after third COVID-19 vaccinations in people treated with these immunomodulatory therapies, importantly accounting for the recent Omicron variant.

Sample
All participants were recruited between Mount Sinai's MS Center and Human Immune Monitoring Center. The study was approved by Mount Sinai's Institutional Review Board and all participants provided written informed consent. Eligible participants began treatment with B cell or S1P modulator therapy at least 60 days prior to third vaccine or were healthy volunteers. For this analysis, we included all enrollees who had completed baseline sampling prior to a third injection and a follow-up sample approximately 4 weeks after the third vaccination by early January 2022.

Assessing Humoral and Cellular Immunity
Microneutralization assays against USA-WA1/2020 (WA1, or wild-type) and B.1.1.529 (BA.1, or Omicron variant) SARS-CoV-2 were performed at the Icahn School of Medicine as previously described. 10 Groups were compared by one-way ANOVA with Tukey multiple comparisons. T cell responses were assessed by IFN-gamma ELISPOT using 400,000 peripheral blood mononuclear cells pulsed with various antigens (15-mer overlapping peptide pool covering the entire sequence of Spike antigen (both WT and Omicron variants, from Miltenyi Biotec), N & Orf antigens (Bhardwaj, GenScript), long peptides covering the Spike RBD domain (Biosynthesis), recombinant Spike protein (gift from Drs. Herrera and Garforth, Einstein College of Medicine), and control viral peptide pools, DMSO, and PMA+ionomycine, as described. 11 Spots observed with DMSO (i.e., non-specific reactivity, median number of spots = 3 out of 400,000 PBMCs) were subtracted from spots observed with SARS-CoV-2 antigen pulsed wells. No other costimulation was used in this ex vivo ELISPOT. Responses were considered positive if >25 spots were detected after peptide or protein stimulation, and if they were at least 2x more than the number of spots to DMSO. Pre-post comparisons were made by Wilcoxon paired t-tests, inter-cohort comparisons by Mann-Whitney t-test.

Data Availability Statement
Anonymized data not published within this article will be made available by request from any qualified investigator, as permitted by institutional regulations. Table 1 describes 25 participants on B cell therapy, 12 on S1PR modulators, and 14 controls. Figure 1 illustrates neutralizing antibody titers. Few on B-cell therapies mounted detectable neutralizing antibodies to WA1 (n=4, 2 just above the limit of detection (LoD)), with some increasing after the third vaccination (n=7, 4 just above the LoD). None had detectable neutralizing antibodies to BA.1 before the third vaccination, and only one converted after third vaccination. Among patients on S1PR modulators, three participants had neutralizing antibodies to BA.1 just at the level of detection post third injection. This contrasts with healthy controls, where a third vaccination resulted in detectable neutralizing antibodies to WA1 (ID50% GM of 59.25 ± 6.536 pre-vs 452.6 ± 8.442 post-vaccination, P<0.01) and BA.1 (ID50% GM of 6.13 ± 1.418 pre-vs 75.71 ± 3.405 post-vaccination, P<0.0001) nearly universally. Although there was more variability among those on B cell therapies compared to controls, overall T cell responses were not significantly different between B cell participants and controls. B cell participants showed significantly increased responses to WA1 and BA.1 peptides after a third injection (median 0.04% increase, p=0.008 by Wilcoxon paired test). Responses to WT Spike peptide pool were significantly greater in controls and B cell participants compared to S1PR participants (median 300-450 spots vs. 3 spots out of 400,000 PBMC, p<0.0001). Among S1PR participants, responses were not significantly different before and after third injection (median 0.004% increase). These results were confirmed with the Omicron peptide pool, RBD peptide pool, and Spike full-length protein, though these were generally less reactive in controls as well. Responses to the peptide pool generated from nucleocapsid (N) and open reading frame (ORF) proteins, which reflect naturally occurring immunity rather than vaccine-induced immunity, were not significantly different in MS participants compared to controls. Interestingly, responses to CEFT (cytomegalovirus, Epstein-Barr virus, influenza A virus, Clostridium tetani) peptide pools were significantly lower in B cell participants' responses compared to controls. 6 Participants on both therapies demonstrated significantly reduced production of neutralizing antibodies, particularly to BA.1, as compared to controls. Cellular immune responses to vaccination were similar among those on B cell therapies to controls and increased after a third vaccination. However, S1P participants demonstrated less robust cellular responses. Driving the lack of significant increase among S1P participants is that there were very few "converts" after the third vaccination. That is, the cellular response increased with a third vaccination among those who had a measurable response to their first two injections, but did not increase for patients who never responded to the first two injections. This observation has significant consequences for expected responses to additional vaccines among those on S1PR modulators.

Discussion
Timing of therapy initiation may play a role in vaccine response. The lone B cell participant with a high level of neutralizing antibodies to BA.1 after the third vaccination received the first vaccinations prior to starting therapy, though notably two others who started therapy after the first vaccinations did not develop neutralizing antibodies to BA.1 after the third vaccine. Of three S1P participants with neutralizing antibodies to BA.1 just at LOD, two received their first injections prior to starting therapy. These are important preliminary observations that will be relevant to those with new diagnoses or needing to change therapy going forward.
Strengths of our study include the presence of a control group and the study of S1PR modulators in addition to B cell therapy. We have assessed the levels of neutralizing antibodies, which reflect the effectiveness of the humoral immune response and represent a mechanistic correlate of protection against SARS-CoV-2, 12, 13 and measured cellular immunity. We have also studied Omicron-specific assays, which more accurately reflect the current circulating strains.
Our study is limited by its small size. The cohort includes only one participant over age 65 because of the timing of enrollment (many of those over 65 had already received a third vaccine when enrollment began). We did not quantify the relative contribution of CD8 vs. CD4 T cell responses, though target antigen formulations used in our assays suggest that the third dose is better at increasing CD8 than CD4 T cells. 14 Study design did not permit correlation to clinical outcomes.
Future directions include assessing durability of vaccine responses over time, studying the impact of natural infection with new variants, and most importantly, correlation with clinical outcomes. For those on S1PR modulators, future studies could consider brief cessation of therapy to aim for an improved response to additional injection, weighing potential benefits and risks of such a strategy.