Velmurugu, Y. | bioRxiv Science

bioRxiv2025

Persistent Classical and Atypical Memory B Cells Underlie Heterogeneous Vaccine Responses in Ocrelizumab-Treated Multiple Sclerosis

Background and ObjectivesPatients with multiple sclerosis (pwMS) treated with ocrelizumab (OCR), a B-cell-depleting therapy, exhibit heterogeneous humoral responses to SARS-CoV-2 mRNA vaccination. The mechanisms underlying this variability remain incompletely understood. We performed a longitudinal analysis of B-cell subset dynamics and antigen-specific T cell responses in OCR-treated pwMS and healthy controls to determine how immune cell composition, timing of OCR infusion, and lymphocyte dynamics influence humoral response outcomes. MethodsBased on post-vaccination anti-Spike IgG titers measured by multiplex bead immunoassay, pwMS were categorized as super-responders (SR), responders (R), or non-responders (NR). A 35-marker spectral flow cytometry panel was used to characterize T- and B-cell subsets longitudinally, at baseline and following stimulation with a SARS-CoV-2 peptide pool. ResultsCD4+ and CD8+ T cell populations were preserved across OCR-treated pwMS, and SARS-CoV-2-specific T cells remained detectable for more than 6 months after vaccination. In contrast, residual B-cell subset composition distinguished responders from non-responders. DN2-like B cells (CD19+CD27-IgD-T-bet+CD11c+CXCR5-) persisted despite repeated OCR infusions and were enriched in SR compared with NR. Repletion of mature naive B cells in peripheral blood correlated with time since last OCR infusion and with stronger humoral immune responses. ConclusionsB-cell subsets that resist OCR-mediated depletion may contribute to vaccine responsiveness in OCR-treated pwMS. Altered repletion kinetics of naive B cell subsets in non-responders suggest that specific B-cell populations may serve as predictive biomarkers of vaccine-induced humoral immunity, despite preserved T-cell responses.

immunology↗

bioRxiv2017

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine experiments and simulations to examine the folding/unfolding pathways of the VPK pseudoknot, a variant of the Mouse Mammary Tumor Virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK, and laser temperature-jump approaches serve as local probes allowing us to monitor the order of assembly of VPK with two helices with different intrinsic stabilities. The experiments and molecular simulations show that at 50 mM KCl the dominant folding pathway populates only the more stable partially folded hairpin. As the salt concentration is increased a parallel folding pathway emerges, involving the less stable hairpin structure as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. The findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. Our study not only unambiguously demonstrates that PK folds by parallel pathways, but also establishes that quantitative description of RNA self-assembly requires a synergistic combination of experiments and simulations.\n\nSIGNIFICANCEThe assembly mechanism of RNA, vital to describing their functions, depends on both the sequence and the metal ion concentration. How the latter influences the folding trajectories remains an important unsolved problem. Here, we examine the folding pathways of an RNA pseudoknot (PK) with key functional roles in transcription and translation, using a combination of experiments and simulations. We demonstrate that the PK, consisting of two helices with differing stabilities, folds by parallel pathways. Surprisingly, the flux between them is modulated by monovalent salt concentration. Our work shows that the order of assembly of PKs is determined by the relative stability of the helices, implying that the folding landscape can be controlled by sequence and ion concentration.

biophysics↗