A Synthetic E7 Gene of Human Papillomavirus Type 16 That Yields Enhanced Expression of the Protein in Mammalian Cells and Is Useful for DNA Immunization Studies

Abstract

A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20-to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G ؉ C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system.Human papillomaviruses (HPVs) are small double-stranded-DNA viruses that infect stratified epithelia and cause benign and malignant proliferative lesions. A subset of HPVs with mucosal tropism, the so-called "high-risk" HPVs, has been linked to cancers of the uterine cervix (55), which account for about 11% of the global cancer incidence in women. Of these, more than 90% contain HPV DNA, most notably HPV type 16 (HPV-16) and HPV-18, integrated into the host cell genome, where they express two viral early genes, E6 and E7, whose products block p53 and retinoblastoma protein-mediated cell cycle control pathways (38,56). Indeed, the tumorigenic phenotype of HPV relies on continuous expression of the E6 and E7 genes (9). E7 is an oncoprotein which can transform rodent fibroblasts (24), cooperate with activated ras to transform primary cells (34), and, in association with E6, immortalize keratinocytes (10,19,30).Progression from detectable HPV infection to invasive cancer occurs in less than 1% of cases and usually takes more than 2 decades, indicating that additional factors are involved in the process of carcinogenesis. Several lines of evidence suggest that such factors may act by intensifying HPV early gene expression, as illustrated in the following examples. First, disruption or mutation of the viral E2 gene, which is known to repress the HPV early promoter and hence expression of E6 and E7, is observed in cervical carcinogenesis (7,36,42,47,52). Moreover, the site of chromosomal integration of the viral DNA influences expression of viral early genes (48). Second, viral load appears to be a determinant for the development of cervical carcinoma ...

The efficacy of a DNA vaccine containing inserted and replicated regions of the E7 gene for treatment of HPV-16 induced tumors

“…Comparatively, an advantage offered by the E7IR vaccine strategy is that the entire coding region is intact and thus there are no HLA restrictions that require consideration in wide-spread vaccine application. In addition, duplication of the gene sequence should cause duplication of all inherent CTL epitopes and the E7IR construct was designed with codon modifications that favor translation in mice, a strategy that has proved effective in enhancing translation of the E7 gene [10,29]. Mechanistically, enhanced expression of CTL epitopes is probably accomplished by increased protein production as the western blot in figure 2b would suggest.…”

“…Several different approaches have been examined including the attachment of ubiquitin to DNA constructs [6,7], targeting of tumor antigens to centrosomal compartments to enhance MHC class I presentation [8], the attachment to constructs of calreticulin to target HPV antigens to the MHC I pathway [9], optimization of codon usage for enhanced expression and presentation of HPV oncogenes [6,10,11] , utilization of modified genes to decrease the transformation capacity [12][13][14] or enhancement of intercellular spreading in order to increase the number of cells expressing antigen [15]. All of these approaches have demonstrated increased antigen presentation and superior tumor eradication in mouse models.…”

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

“…This is accomplished by modification of codon composition to affect translational efficiency. Findings from several studies suggest that viral codon usage modified to resemble human gene codon usage can lead to an increased expression of the viral protein, as has been observed in the L1 and L2 capsid proteins of bovine papillomavirus type 1 as well as HPV-16 E7 protein [11,12]. The reported increase in HPV-16 E7 protein expression has also been shown to enhance the immunogenicity of E7-specific DNA vaccines.…”

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene

“…This is even true for viral genes from viruses that infect humans as the natural host. 25,26 One would assume that such viral genes would already be humanized due to the co-evolution with the human host. However, the virus may have for certain reasons evolved not to have all or certain genes codon optimized since this might help the virus to avoid the immune response.…”