Background: Most immunologically “cold” tumors do not respond durably to checkpoint blockade because tumor antigen (TA) release and presentation are insufficient to prime effective T-cell immunity. While prior work demonstrated synergy between cisplatin and a TLR7/8/9 agonist (CR108) in 4T1 tumors, the underlying mechanism—particularly whether chemotherapy functions as a broad antigen-releasing agent enabling TLR-driven immune amplification—remained undefined. Methods: Using murine models of breast (4T1), melanoma (B16-F10), and colorectal cancer (CT26), we tested multiple chemotherapeutic classes combined with CR108. We quantified intratumoral and systemic soluble TAs, antigen presentation and cross-priming by antigen-presenting cells, tumor-infiltrating lymphocytes, and cytokine production by flow cytometry/ICS. T-cell receptor β (TCRβ) repertoire dynamics in tumor-draining lymph nodes were profiled to assess amplitude and breadth. Tumor microenvironment remodeling was analyzed, and public datasets (e.g., TCGA basal-like breast cancer) were interrogated for expression of genes linked to TA generation/processing and peptide loading. Results: Using cisplatin + CR108 in 4T1 as a benchmark, we demonstrate that diverse chemotherapies—especially platinum agents—broadly increase the repertoire of soluble tumor antigens available for immune recognition. Across regimens, chemotherapy combined with CR108 increased T-cell recognition of candidate TAs and enhanced IFN-γ⁺ CD8⁺ responses, with platinum agents producing the largest expansions in soluble TAs. TCRβ sequencing revealed increased clonal amplitude without loss of repertoire breadth, indicating focused yet diverse antitumor T-cell expansion. Notably, therapeutic efficacy was not predicted by canonical damage-associated molecular pattern (DAMP) signatures but instead correlated with antigen availability and processing capacity. In human basal-like breast cancer, higher expression of genes involved in TA generation and antigen processing/presentation correlated with improved survival. Conclusions: Our findings establish an antigen-centric mechanism underlying chemo–TLR agonist synergy: chemotherapy liberates a broadened repertoire of tumor antigens, which CR108 then leverages via innate immune activation to drive potent, T-cell-mediated antitumor immunity. This framework for rational selection of chemotherapy partners for TLR7/8/9 agonism and support clinical evaluation to convert “cold” tumors into immunologically responsive disease.

Background: Influenza remains a persistent public health challenge due to antigenic drift and shift, necessitating vaccines capable of eliciting broad and durable immunity. Hemagglutinin (HA) antigen serves as the critical target for eliciting protective immune responses against influenza. DNA vaccines offer distinct advantages over conventional platforms, including accelerated development and induction of both humoral and cellular immune responses. Methods: To optimize HA antigen presentation, we designed and systematically compared the immunogenicity and protective efficacy of HA antigen display strategies—bacteriophage T4 fibritin (HA-Foldon) and ferritin-based virus-like particles (HA-Ferritin)—versus monomeric HA DNA vaccines against seasonal influenza viruses. Results: HA-Ferritin showed superior structural stability. All vaccines induced similar HA-specific antibody levels, but HA-Ferritin elicited higher neutralizing antibodies and stronger T cell responses. Upon challenge, HA-Ferritin and HA-Foldon protected mice from weight loss and reduced lung virus loads by 3.27 and 0.76 times, respectively. Monomeric HA provided limited protection, with only 40% survival and minimal viral or pathological reduction. Conclusions: The HA-Ferritin DNA vaccine demonstrated enhanced immunogenicity and protection, supporting structured antigen display as a promising strategy for influenza DNA vaccine development.

Chronic hepatitis B infection remains a significant worldwide health burden, placing persons at risk for hepatocellular cancer and hepatic fibrosis. Chronic hepatitis B virus (CHB) infection is characterized by elevated levels of immunosuppressive regulatory T cells (Tregs), which can inhibit the function of effector T cells and lead to an insufficient immune clearance response against HBV. Theoretically, suppression of Treg cell functionality and percentage could increase anti-HBV reactivity in CHB-infected patients, although this has not yet been explored. We attempted to enhance our previously established anti-CHB protocol utilizing the GM-CSF+IFN-α+rHBVvac regimen (GMI-HBVac) by incorporating mafosfamide (MAF), which has been utilized in anticancer therapy in the past. Intravenous administration of MAF to rAAV8-1.3HBV-infected mice resulted in a dose-dependent reduction of Tregs in the blood, rebounding to pretreatment levels 10 days later. To assess the potential benefit of adding MAF to the anti-CHB protocol, 2 μg/mL MAF was combined with the GMI-HBVac as an anti-Treg treatment in an HBV-infected animal model. When rAAV8-1.3HBV-infected mice were immunized with MAF+GMI-HBVac, peripheral blood Tregs decreased significantly, leading to dendritic cell activation, HBV-specific T cell proliferation, and the upregulation of IFN-gamma-producing CD8⁺T cells. In addition, MAF+GMI-HBVac vaccination stimulated T cell infiltration in HBV-infected livers. These effects may contribute to an enhanced immune response and the clearance of HBV-associated antigens, including serum HBsAg, serum HBcAg, and HBcAg⁺ hepatocytes. Overall, this is the first indication that MAF can act as an adjuvant with GMI-HBVac to deplete Tregs in mice with an established CHB infection. This unique therapeutic vaccine regimen produced a functional cure, as revealed by the remarkable clearance of HBsAg.

Background: The current assessment method of the protective efficacy of adjuvanted vaccines remains slow and labor-intensive, hindered by prolonged immunization protocols and complex assays. Methods: To overcome this bottleneck, we demonstrate that early segregated cellular biomarkers enable rapid prediction of protection, using a respiratory syncytial virus (RSV) pre-fusion F (pre-F) protein model with diverse adjuvants in mice. Results: We identified that germinal center (GC) B cell responses (Days 7 and 9 post-immunization) strongly aligned with protective efficacy, except for Alum, which achieved MF59-level protection despite lower GC responses. Crucially, follicular dendritic cell (FDC) abundance at day 7 universally predicted protection across all adjuvants, including Alum, drastically shortening discovery time and effort from at least 4–6 weeks to within 1 week. Conclusions: FDCs and GC B cells serve as complementary early biomarkers that accurately forecast vaccine efficacy. This approach could potentially reduce the need for prolonged immunization regimens by cellular profiling on days 7–9, offering a modest step toward streamlining adjuvant selection and informing vaccine design.

Background: Inefficient cellular uptake is a significant limitation to the efficacy of DNA vaccines. In this study, we introduce S-Cr9T, a stearyl-modified cell-penetrating peptide (CPP) designed to enhance DNA vaccine delivery by forming stable complexes with plasmid DNA, thereby protecting it from degradation and promoting efficient intracellular uptake. Methods and Results: In vitro studies showed that S-Cr9T significantly improved plasmid stability and transfection efficiency, with optimal performance at an N/P ratio of 0.25. High-content imaging revealed that the S-Cr9T–plasmid complex stably adhered to the cell membrane, leading to enhanced plasmid uptake and transfection. In vivo, S-Cr9T significantly increased antigen expression and triggered a robust immune response, including a threefold increase in IFN-γ secretion and several hundred-fold increases in antibody levels compared to control groups. Conclusions: These findings underscore the potential of S-Cr9T to enhance DNA vaccine efficacy, offering a promising platform for advanced gene therapy and vaccination strategies.

Background: Respiratory syncytial virus (RSV) remains a leading cause of respiratory illness globally, with limited vaccine options, particularly for infants and high-risk populations. This study investigates Cyclosporin A (CsA), traditionally an immunosuppressant, as a novel adjuvant to enhance RSV-specific immunity. Methods: BALB/c mice were subcutaneously immunized with RSV G protein co-administered with varying Cyclosporin A doses, challenged intranasally with RSV, and analyzed for RSV-specific humoral immunity and mechanistic Treg-dependent B-cell responses. Results: We demonstrate that co-administration of CsA with the RSV G protein (G+CsA) dose-dependently boosts RSV-specific IgG and neutralizing antibodies, with selective augmentation of IgG1 and IgG2 subclasses. Mechanistically, G+CsA induces regulatory T cells (Tregs) expressing CD40L and IL-10, which directly promote B-cell activation, proliferation, and plasma cell differentiation. Depletion of Tregs or neutralization of IL-10/CD40L abrogated antibody production, confirming these pathways as critical mediators. Notably, G+CsA-induced Tregs adopt a helper phenotype distinct from conventional Tregs, balancing immune enhancement and homeostasis. Conclusions: CsA demonstrates dual adjuvant properties by enhancing RSV-specific neutralizing IgG titers through Treg-driven B-cell activation, offering a potential strategy to optimize vaccine-induced humoral immunity.