PLLA–ZnO nanowire nano-vaccine orchestrates balanced Th1/Th2 immunity and confers prophylactic antitumor protection

Abstract

Cancer vaccines require both cellular and humoral immunity for optimal therapeutic efficacy. We previously demonstrated that carcinoembryonic antigen (CEA)-coated poly- l -lactic acid-zinc oxide (PLLA-ZnO) nanocomposites induce strong cellular immunity and therapeutic antitumor effects. However, the humoral immune response and prophylactic potential remained unexplored. Female C57BL/6 mice (n = 6/group) were immunized subcutaneously with CEA-coated PLLA-ZnO nanocomposites at two-week intervals. ELISA measured serum antibody responses (IgG1 and IgG2c) at Days 14 and 28 post-immunizations. Flow cytometry assessed IFNγ-producing T cells, and intracellular cytokine staining (ICS) quantified TNF-α, IL-2, and IFN-γ in splenocytes of tumor-bearing mice. Lymphoid cell profiling by multi-parameter flow cytometry characterized immune remodeling in the spleen and tumor microenvironment. Prophylactic efficacy was evaluated in a tumor challenge model using MC38/CEA cells. PLLA-ZnO/CEA immunization induced robust and balanced Th1/Th2 humoral immunity, with both IgG1 and IgG2c antibodies reaching high levels after the second immunization (OD₄₅₀ ~ 1.10 and ~ 1.55, respectively). Critically, PLLA-ZnO/CEA elicited significantly higher IgG2c responses than the traditional Alum adjuvant at day 28 (1.55 vs. 1.05 OD₄₅₀, p < 0.001), demonstrating superior Th1 immunity. The IgG1/IgG2c ratio for PLLA-ZnO/CEA (0.72) indicated balanced immunity, contrasting sharply with Alum’s Th2 bias (ratio 1.33). In vitro studies confirmed substantial Type I interferon production and CCR7+ dendritic cell migration. Flow cytometry demonstrated robust IFNγ+ CD4+ and CD8+ T cell responses upon antigen restimulation. In a prophylactic tumor model, PLLA-ZnO/CEA vaccination significantly reduced tumor growth by 65–70% compared to PBS controls (p < 0.001), with sustained protection throughout the observation period. Vaccinated tumor-bearing mice exhibited robust antigen-specific polyfunctional T-cell responses (TNF-α, IL-2, and IFN-γ in both CD4+ and CD8+ splenocytes) and significant expansion of splenic CD8+ T cells and tumor-infiltrating NK cells, indicating active immune remodeling within the tumor microenvironment. CEA-coated PLLA-ZnO nanocomposites elicit balanced Th1/Th2 humoral immunity alongside robust cellular responses, providing significant prophylactic protection against tumor growth. These findings, together with characterization of CEA antigen immobilization and dendritic cell uptake, complement our previous demonstration of therapeutic efficacy and establish PLLA-ZnO/CEA as a versatile, mechanistically characterized vaccine platform superior to traditional adjuvants for both preventive and therapeutic cancer immunotherapy.