Long-lasting neutralizing antibodies and T cell response after the third dose of mRNA anti-SARS-CoV-2 vaccine in multiple sclerosis

Abstract

Background and objectives: Long lasting immune response to anti-SARS-CoV-2 vaccination in people with Multiple Sclerosis (pwMS) is still largely unexplored. Our study aimed at evaluating the persistence of the elicited amount of neutralizing antibodies (Ab), their activity and T cell response after three doses of anti-SARS-CoV-2 vaccine in pwMS.

Methods: We performed a prospective observational study in pwMS undergoing SARS-CoV-2 mRNA vaccinations. Anti-Region Binding Domain (anti-RBD) of the spike (S) protein immunoglobulin G (IgG) titers were measured by ELISA. The neutralization efficacy of collected sera was measured by SARS-CoV-2 pseudovirion-based neutralization assay. The frequency of Spike-specific IFNγ-producing CD4+ and CD8+ T cells was measured by stimulating Peripheral Blood Mononuclear Cells (PBMCs) with a pool of peptides covering the complete protein coding sequence of the SARS-CoV-2 S.

Results: Blood samples from 70 pwMS (11 untreated pwMS, 11 under dimethyl fumarate, 9 under interferon-γ, 6 under alemtuzumab, 8 under cladribine, 12 under fingolimod and 13 under ocrelizumab) and 24 healthy donors were collected before and up to six months after three vaccine doses. Overall, anti-SARS-CoV-2 mRNA vaccine elicited comparable levels of anti-RBD IgGs, neutralizing activity and anti-S T cell response both in untreated, treated pwMS and HD that last six months after vaccination. An exception was represented by ocrelizumab-treated pwMS that showed reduced levels of IgGs (p<0.0001) and a neutralizing activity under the limit of detection (p<0.001) compared to untreated pwMS. Considering the occurrence of a SARS-CoV-2 infection after vaccination, the Ab neutralizing efficacy (p=0.04), as well as CD4+ (p=0.016) and CD8+ (p=0.04) S-specific T cells, increased in treated COVID+ pwMS compared to uninfected treated pwMS at 6 months after vaccination.

Discussion: Our follow-up provides a detailed evaluation of Ab, especially in terms of neutralizing activity, and T cell responses after anti-SARS-CoV-2 vaccination in MS context, over time, considering a wide number of therapies, and eventually breakthrough infection. Altogether, our observations highlight the vaccine response data to current protocols in pwMS and underline the necessity to carefully follow-up anti-CD20- treated patients for higher risk of breakthrough infections. Our study may provide useful information to refine future vaccination strategies in pwMS.

Keywords: COVID-19; T-cell response; anti-SARS-CoV-2 vaccination; disease modifying therapies; multiple sclerosis; neutralizing antibodies.

Figure 1

Kinetics of anti-RBD IgG levels in pwMS and HD. IgG titers have been compared between HD and untreated and treated pwMS (A); then stratified by the occurrence of a natural infection after three doses of vaccine (B) or by specific therapy (C). Anti-RBD IgGs have been quantified at three time points: immediately before vaccination (P0), one (P1) and six months (P6) after the third dose of vaccine. Dotted line corresponds to the cut-off threshold of 1.4 IU/ml (log10). (A) Statistic was performed by one-way ANOVA with Bonferroni correction for multiple comparisons. Asterisks correspond to p-value thresholds of one-way ANOVA with Bonferroni correction for multiple comparisons: * indicates HD at P1 vs. each other group at P1 (**p<0,002) (B) Subjects under interferon have been excluded from the analysis because no subjects under interferon experienced natural infection after vaccination. Statistic was assessed by the two-tailed unpaired t-test. (C) Statistic was performed by one-way ANOVA with Bonferroni correction for multiple comparisons. Different symbols have been used for different comparisons within each group: * indicates NT at P1 vs. each other group at P1 (***p<0.001); § indicates NT at P6 vs. each other group at P6(§§§p<0.001). HD, healthy donors; NT, not treated; T, treated, DMF, dimethyl fumarate; IFN, Interferon; COVID +, anti-N positive subjects; COVID -, anti-N negative subjects.

Figure 2

SARS-CoV-2 pseudovirion production and characterization. In (A, B) the SARS-CoV-2 pseudovirion production is reported. (A) Uninfected and untreated 293T cells (mock control). (B) 293T cells transfected with pcDNA3.1.S2 and infected with G*ΔG-VSV-luc observed under inverted microscope at 48h. The arrow highlights the observed syncytia. In (C) SARS-CoV-2 pseudovirion characterization by means of WB analysis is reported, showing the incorporation of the SARS-CoV-2 spike on the VSV virions.

Figure 3

Neutralizing activity of serum samples from pwMS and HD. Neutralization titer has been compared between HD and pwMS stratified by therapy (A) or by the occurrence of a natural infection after three doses of vaccine (B). Neutralization assay were performed at two time points: one (P1) and six months (P6) after the third vaccination dose. Dotted line corresponds to the cut-off threshold of 1.3 NT₅₀ (log10). (A) Statistical significance was assessed by one-way ANOVA with Bonferroni correction for multiple comparisons. Different symbols were used for different comparisons within each group: * indicates NT at P1 vs. each other group at P1 (***p<0,001); § indicates NT at P6 vs. each other group at P6 (§§§ p<0.001). (B) Statistical significance was assessed by two-tailed unpaired t-test within each group (* p<0.03). Subjects under Interferon were excluded from the analysis because no subjects under interferon experienced natural infection after vaccination. All the results are presented as the mean values of two independent experiments. NT₅₀ PBNA, neutralizing titer 50 calculated with pseudovirion based neutralization assay; HD, healthy donors; NT, not treated; T, treated; DMF, dimethyl fumarate; IFN, Interferon; COVID +, anti-N positive subjects; COVID -, anti-N negative subjects.

Figure 4

Correlation plots between anti-RDB IgG levels and neutralizing titer 50 in HD (A) and pwMS (B). Plots have been generated using values from all groups. Pearson correlation coefficients were computed to assess the linear relationship between Anti-RDB IgG levels and NT₅₀.

Figure 5

Frequency of Spike specific CD4+ and CD8+ T cells in PBMCs of pwMS and HD. Percentage of Spike specific CD4+ and CD8+ T cells has been obtained by in-vitro restimulation of PBMCs with Spike peptides, followed by intracellular staining for IFNγ. The percentage of Spike specific CD4+ (A, B) or CD8+ T (C, D) cells was obtained by subtracting values of unstimulated cells. Obtained percentage were compared between HD and pwMS stratified by therapy (A, C) or by the occurrence of a natural infection after three doses of vaccine (B, D). Statistics were assessed by two-tailed unpaired t-test (*p<0.03). HD, healthy donors; NT, not treated; T, treated; DMF, dimethyl fumarate; IFN, Interferon; COVID +, anti-N positive subjects; COVID -, anti-N negative subjects.