Humoral immunity to SARS-CoV-2 mRNA vaccination in multiple sclerosis: the relevance of time since last rituximab infusion and first experience from sporadic revaccinations.

INTRODUCTION: The effect of disease-modifying therapies (DMT) on vaccine responses is largely unknown. Understanding the development of protective immunity is of paramount importance to fight the COVID-19 pandemic. OBJECTIVE: To characterise humoral immunity after mRNA-COVID-19 vaccination of people with multiple sclerosis (pwMS). METHODS: All pwMS in Norway fully vaccinated against SARS-CoV-2 were invited to a national screening study. Humoral immunity was assessed by measuring anti-SARS-CoV-2 SPIKE RBD IgG response 3-12 weeks after full vaccination, and compared with healthy subjects. RESULTS: 528 pwMS and 627 healthy subjects were included. Reduced humoral immunity (anti-SARS-CoV-2 IgG <70 arbitrary units) was present in 82% and 80% of all pwMS treated with fingolimod and rituximab, respectively, while patients treated with other DMT showed similar rates as healthy subjects and untreated pwMS. We found a significant correlation between time since the last rituximab dose and the development of humoral immunity. Revaccination in two seronegative patients induced a weak antibody response. CONCLUSIONS: Patients treated with fingolimod or rituximab should be informed about the risk of reduced humoral immunity and vaccinations should be timed carefully in rituximab patients. Our results identify the need for studies regarding the durability of vaccine responses, the role of cellular immunity and revaccinations.

Absolute two-point quantification of proteins using dimethylated proteotypic peptides.

Absolute targeted proteomics typically employs known amounts of synthetic stable isotopically labeled peptides which are mixed with the analyte and analysed by LC-MS to determine the concentration of proteins. In order to obtain more data, we evaluated the use of two different stable isotopes of the same peptide as spike-in for absolute quantification. For this purpose, peptide labeling by reductive amination was applied, which is a mild reaction for dimethylation of amine groups with very high yield. Three different forms can be generated with e.g., light and heavy labels for spike-in peptides, and medium label for endogenous peptides. The method was studied with peptides of apolipoprotein A-I, apolipoprotein B-100, and leucine-rich alpha-2-glycoprotein without and with serum. In serum, the endogenous protein concentrations were measured across four orders of magnitude by the two-point quantification method. Less than 20% of coefficient of variation (CV) values and strong correlation with R2 of 0.99 across three analytical replicates was observed. Most importantly, the two-point quantification method allows an internal quality control of the spike-in peptide as strong deviations in ratios calculated between the first and second reference indicate a methodical error. Because of the significant lower costs than synthetically stable isotopically labeled peptides, this approach might be particularly interesting for the absolute quantification of multiple proteins.

Quantitative phosphoproteome analysis of cisplatin-induced apoptosis in Jurkat T cells.

Protein phosphorylation is one of the most important post-translational modifications (PTMs) due to its vital role in cellular functions and signaling pathways. Protein phosphorylation is also known to be involved in the regulation of apoptosis. Previously, we have performed a SILAC-based analysis of tyrosine phosphorylated peptides of cisplatin-induced apoptotic Jurkat T cells. Here, we analyzed the global phosphorylation profile by enrichment of serine/threonine/tyrosine phosphorylated peptides using TiO2 beads. More than 7000 phosphopeptides of more than 2500 phosphoproteins were identified in four biological replicates. Using two different normalized collision energy (NCE) values for fragmentation by higher-energy collisional dissociation (HCD) revealed complementary results. HCD with NCE 25 accounted for 31% and NCE 35 for 12% uniquely identified phosphopeptides, whereas 57% were found at both NCEs. Different peptide lengths and amino acid compositions were observed at different NCE. A phosphopeptide database was generated out of the results obtained using the Swiss-Prot protein database in order to find differences in regulation of specific phosphorylated sites within multiphosphorylated proteins. Several members of the MAPK signaling pathway were found to be upregulated in apoptotic compared to control cells. Changes of phosphorylation of the transcription factors JUN and ATF2 during apoptosis was confirmed by Western blotting.