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.
Abstract
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).
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.
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).
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.
Japan Society for the Promotion of Science Grant-in-Aid for Early-Career Scientists
Vaccines and Immunotherapy (VAC)
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mRNA vaccines have transformed vaccinology in recent years, shortening timelines and enabling precise response to infectious diseases. One key modification that made this platform so successful is the replacement of uridine (U) with N1-methylpseudouridine (m¹ψ), which improved antigen expression and tolerability. The use of adjuvants for mRNA platforms represents a promising approach to increase immunogenicity of these vaccines, lowering the doses and offering more potent CD8+ T cell priming. However, successful adjuvant strategies remain largely undefined.
Here, we developed a novel and structurally defined double-stranded RNA (dsRNA) and evaluated its immunogenic potential to induce immune responses against infectious diseases using an mRNA encoding influenza hemagglutinin (HA) as a model. C57BL/6 mice were immunized with 3 doses of HA m¹ψ mRNA-LNP (HA-LNP) alone or in co-administered with dsRNA-LNP using intramuscular-prime/ intravenous-boost administration. Peripheral blood mononuclear cells (PBMCs) were collected after each dose and stimulated ex vivo with HA peptides for intracellular cytokine staining (ICS) or ELISpot. Sera was collected 21 days after immunization for humoral analyses.
Co-administration of HA-LNP and dsRNA-LNP induces more robust humoral responses with ∼3-fold higher specific IgG-antibody titers and increased neutralizing ability compared to HA-LNP alone. Evaluation of cellular arm showed that dsRNA-LNP potentializes the cellular immune responses with enhanced frequency of IFNγ production by CD4+ and CD8+ subsets, T cell activation (CD69+), CD8+ T cell polyfunctionality (IFNγ/TNFα), and higher cytotoxic profile (Granzyme B+/ Perforin+).
Overall, the dsRNA-LNP acts as a potent adjuvant for nucleoside-modified mRNA vaccination, inducing a strong immune activation, representing a potent platform for eliciting balanced, durable antiviral immunity.
This work was supported by the National Institute of Allergy and Infectious Disease (NIAID) and National Cancer Institute (NCI) of the US National Institutes of Health (NIH) under award numbers R01AI153064 (NP) and R01CA283736 (NP and CGR), respectively.
Vaccines and Immunotherapy (VAC)
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Cal Poly Pomona Project Hatchery
Immune Response Regulation: Cellular Mechanisms (IRC)
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