Dynamic Evolution of Humoral and T-Cell Specific Immune Response to COVID-19 mRNA Vaccine in Patients with Multiple Sclerosis Followed until the Booster Dose.

This study characterizes antibody and T-cell immune responses over time until the booster dose of COronaVIrus Disease 2019 (COVID-19) vaccines in patients with multiple sclerosis (PwMS) undergoing different disease-modifying treatments (DMTs). We prospectively enrolled 134 PwMS and 99 health care workers (HCWs) having completed the two-dose schedule of a COVID-19 mRNA vaccine within the last 2-4 weeks (T0) and followed them 24 weeks after the first dose (T1) and 4-6 weeks after the booster (T2). PwMS presented a significant reduction in the seroconversion rate and anti-receptor-binding domain (RBD)-Immunoglobulin (IgG) titers from T0 to T1 (p < 0.0001) and a significant increase from T1 to T2 (p < 0.0001). The booster dose in PwMS showed a good improvement in the serologic response, even greater than HCWs, as it promoted a significant five-fold increase of anti-RBD-IgG titers compared with T0 (p < 0.0001). Similarly, the T-cell response showed a significant 1.5- and 3.8-fold increase in PwMS at T2 compared with T0 (p = 0.013) and T1 (p < 0.0001), respectively, without significant modulation in the number of responders. Regardless of the time elapsed since vaccination, most ocrelizumab- (77.3%) and fingolimod-treated patients (93.3%) showed only a T-cell-specific or humoral-specific response, respectively. The booster dose reinforces humoral- and cell-mediated-specific immune responses and highlights specific DMT-induced immune frailties, suggesting the need for specifically tailored strategies for immune-compromised patients to provide primary prophylaxis, early SARS-CoV-2 detection and the timely management of COVID-19 antiviral treatments.

Evaluation of the immunomodulatory effects of interleukin-10 on peripheral blood immune cells of COVID-19 patients: Implication for COVID-19 therapy.

Objective: Several therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform. Methods: Two cohorts were evaluated: in "study population A", plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized "COVID-19 patients" and 29 "NO COVID-19 controls" all unvaccinated. In "study population B", 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis. Results: Baseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in "study population A". Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in "study population B". Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells. Conclusion: This study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.

Longitudinal characterisation of B and T-cell immune responses after the booster dose of COVID-19 mRNA-vaccine in people with multiple sclerosis using different disease-modifying therapies.

BACKGROUND: The decline of humoral response to COVID-19 vaccine led to authorise a booster dose. Here, we characterised the kinetics of B-cell and T-cell immune responses in patients with multiple sclerosis (PwMS) after the booster dose. METHODS: We enrolled 22 PwMS and 40 healthcare workers (HCWs) after 4-6 weeks from the booster dose (T3). Thirty HCWs and 19 PwMS were also recruited 6 months (T2) after the first dose. Antibody response was measured by anti-receptor-binding domain (RBD)-IgG detection, cell-mediated response by an interferon (IFN)-γ release assay (IGRA), Th1 cytokines and T-cell memory profile by flow cytometry. RESULTS: Booster dose increased anti-RBD-IgG titers in fingolimod-treated, cladribine-treated and IFN-ß-treated patients, but not in ocrelizumab-treated patients, although antibody titres were lower than HCWs. A higher number of fingolimod-treated patients seroconverted at T3. Differently, T-cell response evaluated by IGRA remained stable in PwMS independently of therapy. Spike-specific Th1-cytokine response was mainly CD4+ T-cell-mediated, and in PwMS was significantly reduced (p<0.0001) with impaired IL-2 production compared with HCWs at T3. In PwMS, total Th1 and IFN-γ CD4+ T-cell responders to spike protein were increased from T2 to T3.Compared with HCWs, PwMS presented a higher frequency of CD4+ and CD8+ terminally differentiated effector memory cells and of CD4+ effector memory (TEM) cells, independently of the stimulus suggesting the association of this phenotype with MS status. CD4+ and CD8+ TEM cell frequency was further increased at T3 compared with T2. CONCLUSIONS: COVID-19 vaccine booster strengthens humoral and Th1-cell responses and increases TEM cells in PwMS.

Booster dose of SARS-CoV-2 messenger RNA vaccines strengthens the specific immune response of patients with rheumatoid arthritis: A prospective multicenter longitudinal study.

OBJECTIVES: To characterize the kinetics of humoral and T-cell responses in rheumatoid arthritis (RA)-patients followed up to 4-6 weeks (T3) after the SARS-CoV-2 vaccine booster dose. METHODS: Health care workers (HCWs, n = 38) and patients with RA (n = 52) completing the messenger RNA vaccination schedule were enrolled at T3. In each cohort, 25 subjects were sampled after 5 weeks (T1) and 6 months (T2) from the first vaccine dose. The humoral response was assessed by measuring anti-receptor-binding domain (RBD) and neutralizing antibodies, the T-cell response by interferon-γ-release assay (IGRA), T cell cytokine production, and B cell phenotype at T3 by flow cytometry. RESULTS: Patients with RA showed a significant reduction of antibody titers from T1 to T2 and a significant increase at T3. T-cell response by IGRA persisted over time in patients with RA, whereas it increased in HCWs. Most patients with RA scored positive for anti-RBD, neutralizing antibody and T-cell responses, although the magnitude was lower than HCWs. The spike-specific-cytokine response was mainly clusters of differentiation (CD)4+ T cells restricted in both cohorts and significantly lower with reduced interleukin-2 response and CD4-antigen-responding naïve T cells in patients with RA. Unswitched memory B cells were reduced in patients with RA compared with HCWs independently of vaccination. CONCLUSION: COVID-19 vaccine booster strengthens the humoral immunity in patients with RA even with a reduced cytokine response.

Coordinated innate and T-cell immune responses in mild COVID-19 patients from household contacts of COVID-19 cases during the first pandemic wave.

Objective: To better define the immunopathogenesis of COVID-19, the present study aims to characterize the early immune responses to SARS-CoV-2 infection in household contacts of COVID-19 cases. In particular, innate, T- and B-cell specific responses were evaluated over time. Methods: Household contacts of COVID-19 cases screened for SARS-CoV-2 infection by nasopharyngeal swab for surveillance purposes were enrolled (T0, n=42). Of these, 28 subjects returned for a follow-up test (T1). The innate response was assessed by detecting a panel of soluble factors by multiplex-technology in plasma samples. Cell-mediated response was evaluated by measuring interferon (IFN)-γ levels by ELISA in plasma harvested from whole-blood stimulated with SARS-CoV-2 peptide pools, including spike (S), nucleocapsid (N) and membrane (M) proteins. The serological response was assessed by quantifying anti-Receptor-Binding-Domain (RBD), anti-Nucleocapsid (N), whole virus indirect immunofluorescence, and neutralizing antibodies. Results: At T0, higher levels of plasmatic IFN-α, IL-1ra, MCP-1 and IP-10, and lower levels of IL-1ß, IL-9, MIP-1ß and RANTES were observed in subjects with positive swab compared to individuals with a negative one (p<0.05). Plasmatic IFN-α was the only cytokine detectable in subjects with positive SARS-CoV-2 swabs with high accuracy for swab score positivity (0.93, p<0.0001). Among subjects with positive swabs, significant negative correlations were found among the RT-PCR cycle threshold values reported for genes S and N and IFN-α or IP-10 levels. At T0, the IFN-γ T-cell specific response was detected in 50% (5/10) of subjects with positive swab, while anti-RBD/anti-N antibodies showed a positivity rate of 10% (1/10). At T1, the IFN-γ T-cell specific response was detected in most of the confirmed-infection subjects (77.8%, 7/9), whereas the serological response was still observed in a minority of them (44.4%, 4/9). Overall, the swab test showed a moderate concordance with the T-cell response (78.6%, k=0.467), and a scarce concordance with the serological one (72.9%, k=0.194). Conclusions: Plasmatic IFN-α and the IFN-γ T-cell specific response appear early even in the absence of seroconversion, and show a greater positivity rate than the serological response in household contacts with positive swab.

Evaluation of the immunomodulatory effects of interleukin-10 on peripheral blood immune cells of COVID-19 patients: Implication for COVID-19 therapy

Objective Several therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform. Methods Two cohorts were evaluated: in “study population A”, plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized “COVID-19 patients” and 29 “NO COVID-19 controls” all unvaccinated. In “study population B”, 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis. Results Baseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in “study population A”. Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in “study population B”. Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells. Conclusion This study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.

Accuracy of QuantiFERON SARS-CoV-2 research use only assay and characterization of the CD4+ and CD8+ T cell-SARS-CoV-2 response: comparison with a homemade interferon-γ release assay.

OBJECTIVES: In this study, we aimed to characterize the SARS-CoV-2-specific T cell response detected by the QuantiFERON SARS-CoV-2 research use only assay in terms of accuracy and T cell subsets involved compared with a homemade interferon (IFN)-γ release assay (IGRA). METHODS: We evaluated T cell response by the standardized QuantiFERON SARS-CoV-2 tubes (antigen [Ag]1 and Ag2) and a homemade IGRA quantifying IFN-γ response to SARS-CoV-2 spike peptides (homemade-IGRA-SPIKE test). We evaluated the T cell subsets mediating the specific response using flow cytometry. RESULTS: We prospectively enrolled 66 individuals: COVID-19 or post-COVID-19 subjects and NO-COVID-19-vaccinated subjects, including healthy donors and immunocompromised subjects. The standardized kit detected 62.1% (41/66) of T cell responders. Ag2 tube showed a higher IFN-γ quantitative and qualitative response. Ag1 tube response was mainly mediated by CD4+ T cells; Ag2 tube response was mediated by CD4+ and CD8+ T cells. The homemade-IGRA-SPIKE test detected a higher number of responders (52/66, 78.8%) than the QuantiFERON SARS-CoV-2 assay (P = 0.056). The response was found in both T cell subsets, although a higher magnitude and response rate was observed in the CD4+ T cell subset. CONCLUSION: The QuantiFERON SARS-CoV-2 response is mediated by CD4+ and CD8+ T cells. A lower number of responders is found compared with the homemade-IGRA-SPIKE test, likely because of the different peptide composition.

Coordinated innate and T-cell immune responses in mild COVID-19 patients from household contacts of COVID-19 cases during the first pandemic wave

Objective To better define the immunopathogenesis of COVID-19, the present study aims to characterize the early immune responses to SARS-CoV-2 infection in household contacts of COVID-19 cases. In particular, innate, T- and B-cell specific responses were evaluated over time. Methods Household contacts of COVID-19 cases screened for SARS−CoV−2 infection by nasopharyngeal swab for surveillance purposes were enrolled (T0, n=42). Of these, 28 subjects returned for a follow-up test (T1). The innate response was assessed by detecting a panel of soluble factors by multiplex-technology in plasma samples. Cell-mediated response was evaluated by measuring interferon (IFN)-γ levels by ELISA in plasma harvested from whole-blood stimulated with SARS−CoV−2 peptide pools, including spike (S), nucleocapsid (N) and membrane (M) proteins. The serological response was assessed by quantifying anti-Receptor-Binding-Domain (RBD), anti-Nucleocapsid (N), whole virus indirect immunofluorescence, and neutralizing antibodies. Results At T0, higher levels of plasmatic IFN-α, IL-1ra, MCP-1 and IP-10, and lower levels of IL-1β, IL-9, MIP-1β and RANTES were observed in subjects with positive swab compared to individuals with a negative one (p<0.05). Plasmatic IFN-α was the only cytokine detectable in subjects with positive SARS-CoV-2 swabs with high accuracy for swab score positivity (0.93, p<0.0001). Among subjects with positive swabs, significant negative correlations were found among the RT-PCR cycle threshold values reported for genes S and N and IFN-α or IP-10 levels. At T0, the IFN-γ T-cell specific response was detected in 50% (5/10) of subjects with positive swab, while anti-RBD/anti-N antibodies showed a positivity rate of 10% (1/10). At T1, the IFN-γ T-cell specific response was detected in most of the confirmed-infection subjects (77.8%, 7/9), whereas the serological response was still observed in a minority of them (44.4%, 4/9). Overall, the swab test showed a moderate concordance with the T-cell response (78.6%, k=0.467), and a scarce concordance with the serological one (72.9%, k=0.194). Conclusions Plasmatic IFN-α and the IFN-γ T-cell specific response appear early even in the absence of seroconversion, and show a greater positivity rate than the serological response in household contacts with positive swab.

Kinetics of the B- and T-Cell Immune Responses After 6 Months From SARS-CoV-2 mRNA Vaccination in Patients With Rheumatoid Arthritis.

Objective: To assess the kinetics of the humoral and cell-mediated responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in rheumatoid arthritis (RA) patients treated with different immunosuppressive therapies. Methods: Following vaccine completed schedule, health care workers (HCWs, n = 49) and RA patients (n = 35) were enrolled at 5 weeks (T1) and 6 months (T6) after the first dose of BNT162b2-mRNA vaccination. Serological response was assessed by quantifying anti-receptor-binding domain (RBD)-specific immunoglobulin G (IgG) and SARS-CoV-2 neutralizing antibodies, while cell-mediated response was assessed by a whole-blood test quantifying the interferon (IFN)-γ response to spike peptides. B-cell phenotype and IFN-γ-specific T-cell responses were evaluated by flow cytometry. Results: After 6 months, anti-RBD antibodies were still detectable in 91.4% of RA patients, although we observed a significant reduction of the titer in patients under Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4)-Ig [median: 16.4 binding antibody units (BAU)/ml, interquartile range (IQR): 11.3-44.3, p < 0.0001] or tumor necrosis factor (TNF)-α inhibitors (median: 26.5 BAU/ml, IQR: 14.9-108.8, p = 0.0034) compared to controls (median: 152.7 BAU/ml, IQR: 89.3-260.3). All peripheral memory B-cell (MBC) subpopulations, in particular, the switched IgG+ MBCs (CD19+CD27+IgD-IgM-IgG+), were significantly reduced in RA subjects under CTLA-4-Ig compared to those in HCWs (p = 0.0012). In RA patients, a significantly reduced anti-RBD IgG titer was observed at T6 vs. T1, mainly in those treated with CTLA-4-Ig (p = 0.002), interleukin (IL)-6 inhibitors (p = 0.015), and disease-modifying antirheumatic drugs (DMARDs) ± corticosteroids (CCSs) (p = 0.015). In contrast, a weak nonsignificant reduction of the T-cell response was reported at T6 vs. T1. T-cell response was found in 65.7% of the RA patients at T6, with lower significant magnitude in patients under CTLA-4-Ig compared to HCWs (p < 0.0001). The SARS-CoV-2 IFN-γ-S-specific T-cell response was mainly detected in the CD4+ T-cell compartment. Conclusions: In this study, in RA patients after 6 months from COVID-19 vaccination, we show the kinetics, waning, and impairment of the humoral and, to a less extent, of the T-cell response. Similarly, a reduction of the specific response was also observed in the controls. Therefore, based on these results, a booster dose of the vaccine is crucial to increase the specific immune response regardless of the immunosuppressive therapy.

Humoral- and T-Cell-Specific Immune Responses to SARS-CoV-2 mRNA Vaccination in Patients With MS Using Different Disease-Modifying Therapies.

BACKGROUND AND OBJECTIVES: To evaluate the immune-specific response after full severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination of patients with multiple sclerosis (MS) treated with different disease-modifying drugs by the detection of both serologic and T-cell responses. METHODS: Healthcare workers (HCWs) and patients with MS, having completed the 2-dose schedule of an mRNA-based vaccine against SARS-CoV-2 in the past 2-4 weeks, were enrolled from 2 parallel prospective studies conducted in Rome, Italy, at the National Institute for Infectious diseases Spallanzani-IRCSS and San Camillo Forlanini Hospital. Serologic response was evaluated by quantifying the region-binding domain (RBD) and neutralizing antibodies. Cell-mediated response was analyzed by a whole-blood test quantifying interferon (IFN)-γ response to spike peptides. Cells responding to spike stimulation were identified by fluorescence-activated cell sorting analysis. RESULTS: We prospectively enrolled 186 vaccinated individuals: 78 HCWs and 108 patients with MS. Twenty-eight patients with MS were treated with IFN-ß, 35 with fingolimod, 20 with cladribine, and 25 with ocrelizumab. A lower anti-RBD antibody response rate was found in patients treated with ocrelizumab (40%, p < 0.0001) and fingolimod (85.7%, p = 0.0023) compared to HCWs and patients treated with cladribine or IFN-ß. Anti-RBD antibody median titer was lower in patients treated with ocrelizumab (p < 0.0001), fingolimod (p < 0.0001), and cladribine (p = 0.010) compared to HCWs and IFN-ß-treated patients. Serum neutralizing activity was present in all the HCWs tested and in only a minority of the fingolimod-treated patients (16.6%). T-cell-specific response was detected in the majority of patients with MS (62%), albeit with significantly lower IFN-γ levels compared to HCWs. The lowest frequency of T-cell response was found in fingolimod-treated patients (14.3%). T-cell-specific response correlated with lymphocyte count and anti-RBD antibody titer (ρ = 0.554, p < 0.0001 and ρ = 0.255, p = 0.0078 respectively). IFN-γ T-cell response was mediated by both CD4+ and CD8+ T cells. DISCUSSION: mRNA vaccines induce both humoral and cell-mediated specific immune responses against spike peptides in all HCWs and in the majority of patients with MS. These results carry relevant implications for managing vaccinations, suggesting promoting vaccination in all treated patients with MS. CLASSIFICATION OF EVIDENCE: This study provides Class III data that SARS-CoV-2 mRNA vaccination induces both humoral and cell-mediated specific immune responses against viral spike proteins in a majority of patients with MS.

ImmunosuppressiveTherapies Differently Modulate Humoral- and T-Cell-Specific Responses to COVID-19 mRNA Vaccine in Rheumatoid Arthritis Patients.

Objective: To assess in rheumatoid arthritis (RA) patients, treated with different immunosuppressive therapies, the induction of SARS-CoV-2-specific immune response after vaccination in terms of anti-region-binding-domain (RBD)-antibody- and T-cell-specific responses against spike, and the vaccine safety in terms of clinical impact on disease activity. Methods: Health care workers (HCWs) and RA patients, having completed the BNT162b2-mRNA vaccination in the last 2 weeks, were enrolled. Serological response was evaluated by quantifying anti-RBD antibodies, while the cell-mediated response was evaluated by a whole-blood test quantifying the interferon (IFN)-γ-response to spike peptides. FACS analysis was performed to identify the cells responding to spike stimulation. RA disease activity was evaluated by clinical examination through the DAS28crp, and local and/or systemic clinical adverse events were registered. In RA patients, the ongoing therapeutic regimen was modified during the vaccination period according to the American College of Rheumatology indications. Results: We prospectively enrolled 167 HCWs and 35 RA patients. Anti-RBD-antibodies were detected in almost all patients (34/35, 97%), although the titer was significantly reduced in patients under CTLA-4-inhibitors (median: 465 BAU/mL, IQR: 103-1189, p<0.001) or IL-6-inhibitors (median: 492 BAU/mL, IQR: 161-1007, p<0.001) compared to HCWs (median: 2351 BAU/mL, IQR: 1389-3748). T-cell-specific response scored positive in most of RA patients [24/35, (69%)] with significantly lower IFN-γ levels in patients under biological therapy such as IL-6-inhibitors (median: 33.2 pg/mL, IQR: 6.1-73.9, p<0.001), CTLA-4-inhibitors (median: 10.9 pg/mL, IQR: 3.7-36.7, p<0.001), and TNF-α-inhibitors (median: 89.6 pg/mL, IQR: 17.8-224, p=0.002) compared to HCWs (median: 343 pg/mL, IQR: 188-756). A significant correlation between the anti-RBD-antibody titer and spike-IFN-γ-specific T-cell response was found in RA patients (rho=0.432, p=0.009). IFN-γ T-cell response was mediated by CD4+ and CD8+ T cells. Finally, no significant increase in disease activity was found in RA patients following vaccination. Conclusion: This study showed for the first time that antibody-specific and whole-blood spike-specific T-cell responses induced by the COVID-19 mRNA-vaccine were present in the majority of RA patients, who underwent a strategy of temporary suspension of immunosuppressive treatment during vaccine administration. However, the magnitude of specific responses was dependent on the immunosuppressive therapy administered. In RA patients, BNT162b2 vaccine was safe and disease activity remained stable.