Processing-Optimized Multi-Epitope mRNA Vaccine Enhances HIV-1—Specific CD8+ T Cells 2258806

Abstract Introduction Despite ART, HIV-1 persists in latency and rapidly rebounds after interruption. A functional cure will require potent CD8+ T cells that recognize and eliminate latent reservoirs. mRNA vaccines enable rapid, flexible, multi-epitope design. We tested an mRNA encoding tandem CD8+ epitopes with flanking sequences optimized for antigen processing/MHC-I presentation in mice and human PBMCs and assessed co-stimulation by a TLR8 ligand (TLR8L). Methods We generated mRNAs carrying model epitopes either directly concatenated or appended with flanking regions designed to favor proteasomal generation. HLA-A*02:01 transgenic mice were immunized, and antigen-specific CD8+ T-cell responses and multifunctionality were quantified by ELISpot and flow cytometry. We then established a 10-day in vitro boosting assay using PBMCs from people with HIV-1 cultured with FLT3L and exposed to mRNA encoding tandem HIV-1 epitopes. Epitope-specific CD8+ T-cell frequencies were measured by HLA-tetramers, and cytotoxic potential was assessed by granzyme B and perforin expression. Results In HLA-A*02:01 mice, the flanking-optimized tandem design increased multifunctional CD8+ responses co-producing CD107a/IFN-γ/TNF. In PBMCs from people with HIV-1, the HIV epitope—tandem mRNA increased epitope-specific CD8+ T-cell frequency 3.4-fold versus no vaccine (p = 0.0137); addition of TLR8L further raised this to 4.7-fold (p = 0.0273). The proportion of cells co-expressing granzyme B and perforin rose from 40.8% to 80.5% with TLR8L (p = 0.0137). Conclusion An mRNA vaccine encoding tandem CD8+ T-cell epitopes with optimized flanking sequences elicited quantitatively and qualitatively superior epitope-specific CD8+ responses in HLA-A*02:01 mice and in PBMCs from people with HIV-1. TLR8L further enhanced these responses. This platform supports development of therapeutic mRNA vaccines aimed at strengthening CD8+ T-cell immunity toward a functional cure of HIV-1. Funding Source Japan Society for the Promotion of Science Grant-in-Aid for Early-Career Scientists Topic Categories Vaccines and Immunotherapy (VAC)