Project description:W614A-3S peptide is a modified 3S motif of the HIV-gp41 (mutation W614A). We previously detected the presence of natural neutralizing antibodies directed against W614A-3S peptide (NAbs) in long-term non-progressor HIV+ patients. Here, we compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in inducing broadly-NAbs (bNAbs). Rabbit and mouse models were used to screen the induction of bNAbs following 4 immunizations. SQE was more efficient than Alum formulation in inducing W614A-3S-specific bNAbs with up to 67-93% of HIV strains neutralized. We then analyzed the quality of peptide-specific murine B cells by single-cell gene expression by quantitative Reverse Transcription-PCR and single-cell V(D)J sequencing. We found more frequent germinal center B cells in SQE than in Alum, albeit with a different gene expression profile. The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations. All sixteen BCR sequences which were cloned were specific of peptide. Adjuvant formulations of W614A-3S-peptide-conjugated immunogen impact the quantity and quality of B cell immune responses at both the gene expression level and BCR sequence. We used a single-cell quantitative Reverse Transcription-PCR (qRT-PCR) approach to compare between the two formulations the quality of W614A-3S-specific B cell populations isolated from draining lymph nodes, one week after 2nd and 3rd immunizations (W3 and W5, respectively).
Project description:Selenoproteins are defined as proteins that contain selenocysteine (Sec, U), playing crucial roles in living organisms. The incorporation of Sec involves a unique mechanism that primarily includes the UGA codon, the SECIS element, and its binding protein SBP2. Currently, the 25 known human selenoproteins were largely discovered through predictions of stem-looped SECIS structures. However, the exploration of yet-to-be-defined selenoproteins and SECIS elements has been hindered by the accuracy and depth of these predictions. In this study, we focus on SBP2, the core SECIS-binding protein, to analyze all its binding RNAs using RNA immunoprecipitation sequencing (RIP-Seq) technology. Based on this data, we have constructed a robust selenoprotein repository known as the 3S-DB (SBP2-bound RNA sequencing-supported selenoprotein database, SSS-DB or 3S-DB). This 3S-DB contains 1,333 RNA sequences, including all those known selenoprotein RNAs, that can bind to SBP2 with potential SECIS function. Importantly, we validate that the 3' UTRs of PDF and ATP5MJ exhibit SECIS activity. In summary, the established 3S-DB highlights the mRNA that SBP2 can directly bind to, potentially recruiting the entire Sec insertion machinery to generate new selenoproteins. Our results provide a crucial resource for uncovering novel selenoproteins and previously unidentified SECIS elements.
Project description:We compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in BCR repertoire of peptide-specific B cells after 4 immunizations (at week 11 after the first intramuscular injection). The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations.