Expression of a conserved synthetic multiepitopic malaria protein CS712 in duckweed: immune response evaluation after oral delivery in BALB/c mice.

BACKGROUND: Malaria remains a significant global health challenge, with hundreds of millions of cases reported annually and limited access to effective and affordable vaccines. Owing to their scalability, safety, and potential for oral administration, plant-based vaccine platforms have emerged as promising alternatives. In this study, we evaluated the immunogenicity of a conserved, synthetic, and multiepitopic chimeric circumsporozoite protein, CS712, derived from Plasmodium vivax and expressed in transgenic Lemna minor, as a candidate antigen for oral malaria vaccine development.

METHODS: The synthetic CS712 gene was cloned into a plant expression vector and introduced into duckweed via Agrobacterium-mediated transformation. Transgenic lines were verified by PCR, and target protein expression was confirmed by Western blot analysis, reaching approximately 0.5% of total soluble protein (TSP) in line L3. BALB/c mice were immunized via three strategies: edible administration, subcutaneous injection, and a prime-boost combination. Immune responses were analyzed by measuring serum IgG, serum IgA, fecal secretory IgA, IgG subclasses, and cytokines, including IFN-γ and IL-5. Splenocyte proliferation was also assessed by flow cytometric cell-cycle analysis.

RESULTS: CS712-expressing duckweed induced both systemic and mucosal immune responses in immunized mice. Edible and prime-boost immunization increased serum IgG and IgA levels as well as fecal secretory IgA responses compared with the non-transgenic control. Notably, serum IgA and fecal s-IgA levels were higher in the edible and prime-boost groups than in the injection group, indicating enhanced IgA-associated responses following oral antigen delivery. IgG subclass analysis and cytokine profiling showed route-dependent immune patterns. The injection group showed the strongest IgG1-dominated and absolute cytokine responses, whereas the edible/oral group showed the highest IgG2a/IgG1 and IFN-γ/IL-5 ratios, suggesting a relatively higher Th1-associated immune balance.

CONCLUSIONS: These findings support the feasibility of using transgenic duckweed as a plant-based platform for producing a multiepitopic malaria vaccine candidate. CS712-expressing duckweed induced systemic antibody responses, mucosal IgA responses, splenocyte proliferation, and cytokine production, supporting its immunogenicity after oral antigen delivery. However, while this study demonstrates immunogenicity, further studies involving dose optimisation, formulation, and challenge experiments are required to determine protective efficacy.