Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
Plasmid encoding papillomavirus Type 16 (HPV16) DNA constructed with codon optimization improved the immunogenicity against HPV infection
Human papillomavirus Type 16 (HPV16) infections can cause neoplasia, which is thought to be closely associated with the development of cervical cancers. In the study, we attempted to construct a DNA plasmid encoding a HPV16 capsid protein (L1) and a HPV16 oncoprotein (E7), which was capable of preventing HPV16 infection and eliminating HPV16-infected cells. A plasmid, L1E7hpSCA1, encoding the L1 and E7 genes with the codon usage optimized for mammalian cell expression, was constructed. Mutations were introduced into the E7 gene sequence for reducing its oncogenicity. C57BL/6 mice were intramuscularly immunized at tibialis anterior (TA) muscles with the newly constructed L1E7hpSCA1 plasmid. The immune responses induced by the L1E7hpSCA1 plasmid (with codon optimization) and a control L1E7pSCA1 plasmid (without codon optimization) were compared. It is shown that the L1E7hpSCA1 was able to induce much stronger immune responses than the L1E7pSCA1. Sera obtained from immunized animals were found to contain anti-HPV16 antibodies as detected by ELISA and hemagglutination inhibition (HAI) assays. Cytotoxicity and interferon-gamma assays showed that spleenocytes from immunized animals were able to recognize and lyze E7 expressing tumor TC-1 cells. Moreover, the growth of E7 expressing tumor mass was inhibited in vaccinated mice. In vivo tumor protection test indicated that tumor formation was prevented in the experimental animals (67%) after vaccination with L1E7hpSCA1, while for the control group injected with L1E7pSCA1 only and the animal group injected with pSCA1 only, tumor formation was observed in all experimental animals. Our results suggest that the L1E7h gene (with codon optimization) is more effective against HPV16 than the L1E7 gene (without codon optimization). The L1E7hpSCA1 plasmid was able to provide protection against E7 expressing tumor, and it might have the potential to be a vaccine candidate for HPV prevention.
Vaccine. 23(5): 629-38.
To induce higher protein expression level and to improve the immunogenicity against HPV infection, the recoded genes E7h and L1h were constructed. E7 sequence obtained from Genebank(K02718) was altered to optimized the codon usage for mammalian cells using overlapping PCR with Pfu polymerase and six primers, which can be found in the article. The codon were adapted according to the codon usage for Homo sapiens from Genebank. Mutagenesis of E7 gene was simultaneously introduced in the codons at position 24 and 26 so that the glycines became translated instead of cysteine 24 and glutamic acid 26. L1hpBK-CMV was used as a template for producing L1h synthetic gene. As expected, a DNA plasmid, L1E7hpSCA1, encoding codon-optimized HPV 16 L1 and E7 genes, elicited a higher immune response than the codon-unoptimized L1E7pSCA1.