Codon Usage Bias in Hepatitis B Core Antigen, HPV16 L1, and Norovirus VP1 Genes: Implications for Virus‐Like Particle Expression and Vaccine Design

Virus‐like particles from hepatitis B core antigen (HBcAg), human papillomavirus major capsid protein (HPV16 L1), and norovirus VP1 serve as key platforms for chimeric vaccine development. This study analyzed the codon usage patterns of these three genes to clarify their adaptation to human hosts and implications for VLP expression, retrieving 210 HBcAg, 344 HPV16 L1, and 488 norovirus VP1 coding sequences. Nucleotide composition and relative synonymous codon usage (RSCU) analyses revealed that all three genes prefer A/T‐ending codons, with the strongest bias observed in HPV16 L1 and the weakest in norovirus VP1. The effective number of codons (ENC) further indicated limited overall codon bias across these genes. Dinucleotide analysis revealed underrepresentation of CpG across all sequences. Neutrality plot, Parity Rule 2, and ENC‐plot analyses confirmed natural selection, not mutational pressure, as the primary evolutionary force shaping codon usage. Isoacceptor tRNA pool analysis revealed distinct adaptation strategies among the three genes. HBcAg showed broad optimization across multiple amino acids, whereas HPV16 L1 and norovirus VP1 displayed more focused adaptation profiles. The relative codon deoptimization index ranked norovirus VP1 as most human‐adapted, followed by HBcAg and HPV16 L1. This study provides a foundational computational framework for rational mRNA vaccine design, underscoring the need for tailored codon optimization to enhance translational efficiency, ensure proper VLP assembly, and elicit protective immune responses.