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A modular, multi-purpose, and cost-effective electrochemical biosensor based on a five-stranded four-way junction (5S-4WJ) system was developed for SARS-CoV-2 (genes S and N) and Influenza A virus (gene M) detection. The 5S-4WJ structure consists of an electrode-immobilized universal stem-loop (USL) strand, two auxiliary DNA strands, and a universal methylene blue redox strand (UMeB). This design allows for the detection of specific nucleic acid sequences using square wave voltammetry (SWV). The sequence-specific auxiliary DNA strands (m and f) ensure selectivity of the biosensor for target recognition utilizing the same USL and UMeB components. An important feature of this biosensor is the ability to reuse the USL-modified electrodes to detect the same or alternative targets in new samples. This is accomplished by a simple procedure involving rinsing the electrodes with water to disrupt the 5S-4WJ structure and subsequent re-hybridization of the USL strand with the appropriate set of strands for a new analysis. The biosensor exhibited minimal loss in signal after rehybridization, demonstrating its potential as a viable multiplex assay for both current and future pathogens, with a low limit of quantification (LOQ) of as low as 17 pM.

Protective immunity following vaccination is sustained by long-lived antibody-secreting cells and resting memory B cells (MBCs). Responses to two-dose SARS-CoV-2 mRNA-1273 vaccination are evaluated longitudinally by multimodal single-cell analysis in three infection-naïve individuals. Integrated surface protein, transcriptomics, and B cell receptor (BCR) repertoire analysis of sorted plasmablasts and spike + (S-2P + ) and S-2P - B cells reveal clonal expansion and accumulating mutations among S-2P + cells. These cells are enriched in a cluster of immunoglobulin G-expressing MBCs and evolve along a bifurcated trajectory rooted in CXCR3 + MBCs. One branch leads to CD11c + atypical MBCs while the other develops from CD71 + activated precursors to resting MBCs, the dominant population at month 6. Among 12 evolving S-2P + clones, several are populated with plasmablasts at early timepoints as well as CD71 + activated and resting MBCs at later timepoints, and display intra- and/or inter-cohort BCR convergence. These relationships suggest a coordinated and predictable evolution of SARS-CoV-2 vaccine-generated MBCs., Competing Interests: Declaration of interests S.H.K. receives consulting fees from Peraton. K.B.H. receives consulting fees from Prellis Biologics., (Published by Elsevier Inc.)

SARS-CoV-2 mRNA booster vaccines provide protection from severe disease, eliciting strong immunity that is further boosted by previous infection. However, it is unclear whether these immune responses are affected by the interval between infection and vaccination. Over a 2-month period, we evaluated antibody and B cell responses to a third-dose mRNA vaccine in 66 individuals with different infection histories. Uninfected and post-boost but not previously infected individuals mounted robust ancestral and variant spike-binding and neutralizing antibodies and memory B cells. Spike-specific B cell responses from recent infection (<180 days) were elevated at pre-boost but comparatively less so at 60 days post-boost compared with uninfected individuals, and these differences were linked to baseline frequencies of CD27 lo B cells. Day 60 to baseline ratio of BCR signaling measured by phosphorylation of Syk was inversely correlated to days between infection and vaccination. Thus, B cell responses to booster vaccines are impeded by recent infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)