Microalgae *Chlamydomonas reinhardtii* successfully produces HPV16 E6 and E7 vaccine antigens

Globally, cervical cancer remains a critical health challenge, particularly in low- and middle-income countries where access to effective prevention and treatment is limited. Persistent infection with high-risk human papillomavirus (HPV), specifically HPV16, is the primary driver of malignant transformation, largely due to the continuous expression of the viral oncoproteins E6 and E7. These tumor-specific antigens are highly promising targets for the development of therapeutic vaccines. However, current production systems for recombinant HPV antigens often lack the scalability and affordability required to meet global demand, highlighting a significant gap in vaccine development.

Researchers established Chlamydomonas reinhardtii as a microalgal platform for producing HPV16 E6 and E7 proteins. Codon-optimized sequences encoding HPV16 E6 (519bp) and HPV16 E7 (481bp) were integrated into the nuclear genome of C. reinhardtii using electroporation-mediated transformation. Following transformation, successful protein expression was analyzed by SDS-PAGE and immunoblotting to detect recombinant proteins at their predicted molecular weights. The identity and integrity of the recombinant HPV antigens were definitively confirmed using LC-MS/MS analysis.

Protein analysis by SDS-PAGE and immunoblotting successfully detected recombinant E6 at approximately 18kDa and E7 at approximately 11kDa, consistent with their predicted molecular weights, confirming successful translation within the microalgal system. This initial detection provided strong evidence of expression. Critically, LC-MS/MS analysis subsequently identified multiple high-confidence tryptic peptides corresponding to both HPV16 E6 and HPV16 E7 antigens. This mass spectrometry-based validation achieved sequence coverage ranging from 22-35%, providing definitive confirmation of the recombinant protein identity and integrity. These findings collectively demonstrate the robust capability of C. reinhardtii to produce clinically relevant HPV16 antigens.

LC-MS/MS analysis definitively identified multiple high-confidence tryptic peptides for both HPV16 E6 and E7, with sequence coverage of 22-35%, confirming successful recombinant protein production.