Immunogenicity of a duckweed-expressed multivariant circumsporozoite antigen administered orally to 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 chimeric circumsporozoite protein (CS712) derived from Plasmodium vivax , expressed in transgenic Lemna minor , as a candidate oral malaria vaccine. Methods The CS712 gene was subsequently cloned and inserted into a plant expression vector and successfully introduced into duckweed via Agrobacterium -mediated transformation. The transgenic lines were verified via PCR, and target protein expression was confirmed via Western blot analysis; the protein reached concentrations of 0.4% of the total soluble protein. BALB/c mice were immunized via three different routes: oral administration, subcutaneous injection, and a prime–boost combination. Immune responses were analyzed by quantifying total IgG, IgA, and IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3) and cytokine levels (IL-5 and IFN-γ). Splenocyte proliferation was also assessed via flow cytometry. Results Both the oral and prime-boost immunization strategies elicited robust humoral and mucosal immune responses, as evidenced by significant increases in serum IgG and IgA as well as fecal IgA. Analysis of the IgG subclass distribution and cytokine profiles revealed the activation of both Th1- and Th2-type immune pathways. Conclusions These findings support the feasibility of using transgenic duckweed as a plant-based bioreactor for oral vaccine candidate production. The capacity of CS712-expressing duckweed to induce both mucosal and systemic immunity underscores its potential as a cost-effective and scalable platform for developing oral malaria vaccines targeting P. vivax .