Biology subjects

Martin, A. L.

Publications and source records attributed to Martin, A. L..

4 recordsLinked to original sources

bioRxiv2026

Circular RNA vaccine performance is determined by RNA quality and epitranscriptomic tuning rather than innate activation

Circular RNA (circRNA) is an emerging vaccine modality that is proposed to improve stability, reduce reactogenicity and extend antigen expression compared with linear mRNA. However, the relative contributions to vaccine performance of its covalently closed structure, its purity, and of nucleotide modifications remain poorly described. Here, we systematically dissected these parameters in vivo across two antigen systems. We identified RNA quality as a major determinant of circRNA reactogenicity, with differences in innate immune activation tracking with the presence of residual RNA species in less refined preparations. In contrast, highly purified circRNA exhibited markedly reduced reactogenicity compared with linear mRNA, independent of nucleotide modification. Despite these differences, circRNA and mRNA vaccines elicited comparable antibody titres and T cell responses, indicating that reduced innate activation does not enhance adaptive immune magnitude. Notably, incorporating N6-methyladenosine (m6A) did not affect reactogenicity or antigen expression but selectively enhanced antibody quality, increasing binding affinity and neutralisation capacity. CircRNA vaccination also altered the anatomical distribution of germinal centre responses, reducing splenic antigen-specific germinal centre B cells while preserving lymph node responses. Together, these findings show that circRNA vaccine performance is governed by RNA preparation quality and epitranscriptomic tuning rather than innate activation alone.

immunology↗

bioRxiv2026

Engineering antigenic breadth against SARS-CoV-2 by pairing divergent RBDs within a single mRNA immunogen

Vaccines capable of eliciting broadly neutralising antibodies (bnAbs) are a major goal for pandemic preparedness. A persistent challenge across vaccine Wields is how to deliberately recruit the rare B cell clones that recognise conserved epitopes shared across diverse viral variants. BnAbs have been known to frequently emerge through extensive somatic hypermutation during afWinity maturation, here we describe an alternative, structure-driven mechanism for bnAb selection. We designed an mRNA vaccine in which two antigenically distinct SARS-CoV-2 variants (Omicron and Delta; O-{Delta}) receptor binding domains (RBDs) are physically fused on a single polypeptide. This design is predicted to favour B cell antigen receptors capable of engaging conserved epitopes on both RBDs with enhanced avidity. A matched non-divergent tandem RBD (Delta-Delta; {Delta}-{Delta}) served as a control. The divergent (O-{Delta}) immunogen was robustly expressed and retained high-afWinity ACE2 binding. In mice, immunisation elicited potent antibody responses and increased the frequency of antigen-speciWic cross-reactive B cells, recognising Delta, Omicron, and the 2002 pandemic strain SARS-CoV RBDs. Using multicolour RBD tetramers and single-cell B cell receptor sequencing, we show that breadth arises via two distinct pathways. The divergent vaccine preferentially enriches clonally distinct cross-reactive B cells (not present within non-cross-reactive B cell pools) with low levels of somatic hypermutation (SHM), consistent with selection of germline-biased precursors. In contrast, the matched control vaccine yields cross-reactivity primarily within existing clonal lineages (clonal overlap between cross-reactive and non-cross-reactive cells) and at higher mutational burdens, consistent with afWinity-maturation-driven acquisition of breadth. Together, these Windings demonstrate that antigen structure can bias B cell selection towards cross-reactive speciWicities without requiring extensive SHM. This work establishes a simple, modular antigen-design principle in which juxtaposing appropriately divergent antigens on a single scaffold promotes the enrichment of bnAb-prone B cells, providing a scalable strategy for vaccine development against rapidly evolving pathogens.

immunology↗

bioRxiv2024

Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections.

The domestication of cattle provided Propionibacteriaceae the opportunity to adapt to human skin. These bacteria constitute a distinct genus (Cutibacterium), and a single species within that genus (C. acnes) dominates 25% of human skin. C. acnes protects humans from pathogen colonization, but it can also infect indwelling medical devices inserted through human skin. Proteins that help Cutibacteria live on our skin may also act as virulence factors during an opportunistic infection, like a shoulder periprosthetic joint infection (PJI). To better understand the evolution of this commensal and opportunistic pathogen, we sought to extensively characterize one of these proteins, RoxP. This secreted protein is only found in the Cutibacterium genus, helps C. acnes grow in oxic environments, and is required for C. acnes to colonize human skin. Structure-based sequence analysis of twenty-one RoxP orthologs (71-100% identity to C. acnes strain KPA171202 RoxP_1) revealed a high-degree of molecular surface conservation and helped identify a potential heme-binding interface. Biophysical evaluation of a subset of seven RoxP orthologs (71-100% identity) demonstrated that heme-binding is conserved. Computational modeling of these orthologs suggests that RoxP heme-binding is mediated by an invariant molecular surface composed of a surface-exposed tryptophan (W66), adjacent cationic pocket, and nearby potential heme axial ligands. Further, these orthologs were found to undergo heme-dependent oligomerization. To further probe the role of this protein in C. acnes biology, we developed four monoclonal anti-RoxP antibodies, assessed the binding of those antibodies to a subset of ten RoxP orthologs (71-100% identity), developed an anti-RoxP sandwich ELISA (sELISA) with sub-nanogram sensitivity, and adapted that sELISA to quantitate RoxP in human biofluids that can be infected by C. acnes (serum, synovial fluid, cerebrospinal fluid). This study expands our understanding of how an environmental bacterium evolved to live on humans, and the assays developed in this work can now be used to identify this organism when it gains access to sterile sites to cause opportunistic infections. Author SummaryThe longer humans live, the more they require internal "replacement parts," like prosthetic joints. Increased placement of these and other medical devices has increased their complications, which frequently are infections caused by microbes that live on humans. One of these microbes is Cutibacterium acnes, which dominates 25% of human skin. It appears that when humans domesticated cattle, a C. acnes ancestor adapted from living in cows to living on people. One of these adaptations was RoxP, a protein only found in Cutibacterium and carried by all C. acnes. Here, we describe our extensive characterization of RoxP. We found that distantly related RoxP conserve high stability at the low pH found on human skin. They also conserve the ability to bind heme, a source of iron used by microbes when they infect humans. As a part of this work, we developed tests that measure RoxP to identify C. acnes growth. In a clinic or hospital, these tests could allow a doctor to rapidly identify C. acnes infections, which would improve patient outcomes and lower healthcare costs. This work has helped us better understand how C. acnes adapted to live on humans and to identify C. acnes infections of medical devices.

microbiology↗

bioRxiv2023

Mouse Embryonic Stem Cell Pluripotency Factors Regulate RNA Methylation

The pluripotency of embryonic stem cells (ESCs) is actively promoted by a diverse set of factors, including leukemia inhibitory factor (LIF), glycogen synthase kinase-3 (Gsk-3) and mitogen-activated protein kinase kinase (MEK) inhibitors, ascorbic acid, and -ketoglutarate. Strikingly, several of these factors intersect with the post-transcriptional methylation of RNA (m6A), which has also been shown to play a role in ESC pluripotency. Therefore, we explored the possibility that these factors converge on this biochemical pathway to promote the retention of ESC pluripotency. Mouse ESCs were treated with various combinations of small molecules, and the relative levels of m6A RNA were measured, as well as the expression of genes marking naive and primed ESCs. The most surprising result was the discovery that replacing glucose with high levels of fructose pushed ESCs to a more naive state and reduced m6A RNA abundance. Our results suggest a correlation between molecules previously shown to promote the retention of ESC pluripotency and m6A RNA levels, strengthening a molecular connection between reduced m6A RNA and the pluripotent state, and provides a foundation for future mechanistic studies on the role of m6A and ESC pluripotency.

molecular biology↗