Of interest to the etiology of demyelinating autoimmune disease is the potential to aberrantly activate CD4⁺ T cells due to cross-recognition of multiple self-epitopes such as has been suggested for myelin oligodendrocyte glycoprotein epitope 35-55 (MOG_35-55) and neurofilament medium protein epitope 15-35 (NFM_15-35). NFM_15-35 is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG_35-55, a known encephalitogenic Ag. Despite reported cross-reactivity with MOG-specific T cells, the polyclonal response to NFM_15-35 did not expand threshold numbers of MOG_38-49 tetramer-positive T cells. Furthermore, NFM lacked functional synergy with MOG to promote experimental autoimmune encephalomyelitis because NFM-deficient synonymous with knockout mice developed an identical disease course to wild-type mice after challenge with MOG_35-55 Single-cell analysis of encephalitogenic T cells using the peptide:MHC monomer-based two-dimensional micropipette adhesion frequency assay confirmed that NFM was not a critical Ag driving demyelinating disease because NFM_18-30-specific T cells in the CNS were predominantly reactive to MOG_38-49 The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high-affinity, MOG-specific T cells that defined the polyclonal response. Alterations of N-terminal residues outside of the NFM_15-35 core nonamer promoted expansion of high-affinity, MOG_38-49 tetramer-positive T cells and promoted consistent experimental autoimmune encephalomyelitis induction, unlike mice challenged with NFM_15-35 Although NFM_15-35 is immunogenic and cross-reactive with MOG at the polyclonal level, it fails to expand a threshold level of encephalitogenic, high-affinity MOG-specific T cells.

B cells play a vital function in multiple sclerosis (MS) pathogenesis through an array of effector functions. All currently approved MS disease-modifying therapies alter the frequency, phenotype, or homing of B cells in one way or another. The importance of this mechanism of action has been reinforced with the successful development and clinical testing of B-cell-depleting monoclonal antibodies that target the CD20 surface antigen. Ocrelizumab, a humanized anti-CD20 monoclonal antibody, was approved by the Food and Drug Administration (FDA) in March 2017 after pivotal trials showed dramatic reductions in inflammatory disease activity in relapsing MS as well as lessening of disability progression in primary progressive MS. These and other clinical studies place B cells at the center of the inflammatory cascade in MS and provide a launching point for development of therapies that target selective pathogenic B-cell populations.

CD8⁺ T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential autoantigenic targets, prior studies of myelin-reactive CD8⁺ T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8⁺ T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8⁺ T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8⁺ T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8⁺ T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8⁺ T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8⁺ T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20⁺ CD8⁺ T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8⁺ T cells in MS, indicates these cells may be attractive targets in MS therapy.

Objective

To examine humoral and cellular response in multiple sclerosis patients on anti-CD20 therapy after third BNT162b2 mRNA SARS-CoV-2 vaccination.

Methods

A prospective longitudinal study design from first throughout third vaccination in Danish and American MS centers. All participants were treated with ocrelizumab. Antibody (Ab) levels were assessed before and after third vaccination using SARS-CoV-2 IgG II Quant assay (Abbott Laboratories). B- and T-lymphocytes enumeration was done with BD Multitest™6-color TBNK reagent. Spike-specific T-cell responses were measured through PBMC stimulation with spike peptide pools (JPT Peptide Technologies).

Results

We found that 14.0%, 37.7%, and 33.3% were seropositive after first, second and third vaccination. The median Ab-levels were 74.2 BAU/mL (range: 8.5-2427) after second vaccination, as well as 43.7 BAU/ml (range: 7.8-366.1) and 31.3 BAU/mL (range: 7.9-507.0) before and after third vaccination, respectively. No difference was found in levels after second and third vaccination (p = 0.1475). Seropositivity dropped to 25.0% of participants before the third vaccination, a relative reduction of 33.3% (p = 0.0020). No difference was found between frequencies of spike reactive CD4⁺and CD8⁺ T-cells after second (0.65 ± 0.08% and 0.95 ± 0.20%, respectively) and third vaccination (0.99 ± 0.22% and 1.3 ± 0.34%, respectively).

Conclusion

In this longitudinal cohort we found no significant increased humoral or cellular response with administration of a third SARS-CoV-2 mRNA vaccination. These findings suggest the need for clinical strategies to include allowance of B cell reconstitution before repeat vaccination and/or provision of pre-exposure prophylactic monoclonal antibodies.

Aquaporin-4 (AQP4)-specific Th17 cells are thought to have a central role in neuromyelitis optica (NMO) pathogenesis. When modeling NMO, only AQP4-reactive Th17 cells from AQP4-deficient (AQP4^-/-), but not wild-type (WT) mice, caused CNS autoimmunity in recipient WT mice, indicating that a tightly regulated mechanism normally ensures tolerance to AQP4. Here, we found that pathogenic AQP4 T cell epitopes bind MHC II with exceptionally high affinity. Examination of T cell receptor (TCR) α/β usage revealed that AQP4-specific T cells from AQP4^-/- mice employed a distinct TCR repertoire and exhibited clonal expansion. Selective thymic AQP4 deficiency did not fully restore AQP4-reactive T cells, demonstrating that thymic negative selection alone did not account for AQP4-specific tolerance in WT mice. Indeed, AQP4-specific Th17 cells caused paralysis in recipient WT or B cell-deficient mice, which was followed by complete recovery that was associated with apoptosis of donor T cells. However, donor AQP4-reactive T cells survived and caused persistent paralysis in recipient mice deficient in both T and B cells or mice lacking T cells only. Thus, AQP4 CNS autoimmunity was limited by T cell-dependent deletion of AQP4-reactive T cells. In contrast, myelin oligodendrocyte glycoprotein (MOG)-specific T cells survived and caused sustained disease in WT mice. These findings underscore the importance of peripheral T cell deletional tolerance to AQP4, which may be relevant to understanding the balance of AQP4-reactive T cells in health and in NMO. T cell tolerance to AQP4, expressed in multiple tissues, is distinct from tolerance to MOG, an autoantigen restricted in its expression.

The wide spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with phenotypes impacting transmission and antibody sensitivity necessitates investigation of immune responses to different spike protein versions. Here, we compare neutralization of variants of concern, including B.1.617.2 (delta) and B.1.1.529 (omicron), in sera from individuals exposed to variant infection, vaccination, or both. We demonstrate that neutralizing antibody responses are strongest against variants sharing certain spike mutations with the immunizing exposure, and exposure to multiple spike variants increases breadth of variant cross-neutralization. These findings contribute to understanding relationships between exposures and antibody responses and may inform booster vaccination strategies.

Background

Our study investigated the rate of breakthrough SARS-CoV-2 infection and clinical outcomes in a cohort of multiple sclerosis (MS) patients who were treated with the anti-CD20 monoclonal antibody (Ab), ocrelizumab, before first, second and third BNT162b2 mRNA vaccinations. To correlate clinical outcomes with the humoral and cellular response.

Methods

The study was a prospective non-randomised controlled multicentre trial observational study. Participants with a diagnosis of MS who were treated for at least 12 months with ocrelizumab prior to the first BNT162b2 mRNA vaccination were prospectively followed up from January 2021 to June 2022.

Results

Out of 54 participants, 32 (59.3%) developed a positive SARS-CoV-2 PCR test in the study period. Mild infection was observed in all infected participants. After the third vaccination, the non-infected participants had higher mean Ab levels compared to the infected participants (54.3 binding antibody unit (BAU)/mL vs 26.5 BAU/mL, p=0.030). The difference in reactivity between spike-specific CD4⁺ and CD8⁺ T lymphocytes in the two groups was not significant.

Conclusion and relevance

The study results demonstrate rates of 59% in breakthrough infections after the third SARS-CoV-2 mRNA vaccination in ocrelizumab-treated patients with MS, without resulting in critical disease courses. These findings suggest the need for continuous development of prophylactic treatments when proved important in the protection of severe breakthrough infection.

Background and objectives

Prospective, deeply phenotyped research cohorts monitoring individuals with chronic neurologic conditions, such as multiple sclerosis (MS), depend on continued participant engagement. The COVID-19 pandemic restricted in-clinic research activities, threatening this longitudinal engagement, but also forced adoption of televideo-enabled care. This offered a natural experiment in which to analyze key dimensions of remote research: (1) comparison of remote vs in-clinic visit costs from multiple perspectives and (2) comparison of the remote with in-clinic measures in cross-sectional and longitudinal disability evaluations.

Methods

Between March 2020 and December 2021, 207 MS cohort participants underwent hybrid in-clinic and virtual research visits; 96 contributed 100 "matched visits," that is, in-clinic (Neurostatus-Expanded Disability Status Scale [NS-EDSS]) and remote (televideo-enabled EDSS [tele-EDSS]; electronic patient-reported EDSS [ePR-EDSS]) evaluations. Clinical, demographic, and socioeconomic characteristics of participants were collected.

Results

The costs of remote visits were lower than in-clinic visits for research investigators (facilities, personnel, parking, participant compensation) but also for participants (travel, caregiver time) and carbon footprint (p < 0.05 for each). Median cohort EDSS was similar between the 3 modalities (NS-EDSS: 2, tele-EDSS: 1.5, ePR-EDSS: 2, range 0.6.5); the remote evaluations were each noninferior to the NS-EDSS within ±0.5 EDSS point (TOST for noninferiority, p < 0.01 for each). Furthermore, year to year, the % of participants with worsening/stable/improved EDSS scores was similar, whether each annual evaluation used NS-EDSS or whether it switched from NS-EDSS to tele-EDSS.

Discussion

Altogether, the current findings suggest that remote evaluations can reduce the costs of research participation for patients, while providing a reasonable evaluation of disability trajectory longitudinally. This could inform the design of remote research that is more inclusive of diverse participants.