Study On The Codon Optimized E7 Gene Of Low Risk Human Papillomaviruses

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 and "low-risk" HPVs, has been linked to cancers of the uterine cervix and benign genital warts respectively.HPV genome is a supercoiled double-stranded circle of DNA , and composed of about 7900 base pairs. The E (early) region of the viral DNA contains seven open-reading frames (E1-E7 ORF), which code for non-structural proteins involved in the DNA replication, transcription and transformation.. The L (late )region contains two ORFs (L1 and L2 ) which code for the viral structural proteins , L1 is the major capsid protein of HPVs.The E6 and E7 genes of "high-risk" HPVs (most notably HPV 16) are the viral transformation genes whose products block p53 and retinoblastoma protein-mediated cell cycle control pathways . HPV-16 E7 is a major oncoprotein which can transform rodent fibroblasts , and in association with E6, immortalize human keratinocytes. The E6 and E7 genes of "low-risk" HPVs demonstrate weaker immortalizing activity in human epithelial cells, combination HPV6 E7 of HPV 16 gene increases the frequency of immortalization threefold over that obtained with HPV 16 gene alone, and also stimulates cell proliferation.HPV E7 protein is also a tumor antigen which contains CD4~+ Th1 and CD8~+ CTL epitopes and continuously expresses on the infected cell surface. Thus, the importance of E7 protein as a target antigen of HPV therapeutic vaccine has been well recognized.The major obstacles that restrict the study of HPV E7 vaccine include the poorer imunogenicity of the protein and weaker immune response to E7 polynucleotide vaccine. In addition, one also worries about the transformation activity of E7 protein. Therefore, the major challenge in the HPV vaccine research field is focusing on the improvements of the imunogenicity of the E7 protein on the basis of non-oncogenicity, and several strategies have been applied to increase the potency of E7 polynucleotide vaccines.The influence of codon usage on the expression of viral gene in their hose cells has been recognized recently. Liu WJ(2002) reported that codon modified HPV 16 E7 DNA vaccine enhanced cytotoxic T-lymphocyte induction and anti-tumor activity.At the present time, the research work of codon modified HPV E7 gene mainly focus on "high-risk" HPV16; however, many problems are still need to be resolved. Such as, whether the codon usage of "low-risk" HPVs is similar to the one of "high-risk" HPVs.? Whether the codon optimization can enhances the expression and immunogenicity of "low-risk" HPVs ? Whether the higher immunogenicity is associated with the expression level of E7 protein ? And how is the mechanism of codon optimization ?We initiated a project to investigate the codon modified E7 gene of "low-risk" HPV6b, and the findings are briefly summarized below.The comparison of codon preference between "low-risk" HPVs and "high-risk" HPVsZhao KN( 2003) first analyed the codon usage bias of 79 HPVs genotypes. The total codon preferences of the 79 HPVs genotypes are significantly different from those of mammalian genes. Apart from the average frequence of codon usage in 79 HPVs genotypes, detailed study in the different type ("low-risk"HPVs and "high-risk" HPVs), and different gene (early gene and late gene)still need to be delineated. Studies on the codon preference between "low-risk" HPVs and"high-risk"HPVs provide a foundation for designing codon modified "low-risk" HPV6b E7 gene.We examined the codon preference of early gene E7 and late gene LI in low-risk HPVs and high-risk HPVs respectively, and comparisons of codon prefernce among HPV 6> HPV 16 andE.coli> yeasU adenovirus > human were also performed . E7 and LI coding sequences, the whole genome of low-risk HPVs and high-risk HPVs were analyzed respectively via CHIPS programs of EMBOSS and the programs of codon usage debats . The usage of HPV preferred codon is generally more than twice as frequently observed in HPV genes as human consensus codon.. The Results showed (1) the Nc value(effective number of codons) of low-risk HPVs E7 and LI gene are 44.8 and 49.5 respectively. The Nc value of high-risk HPVs E7 and LI gene are 53.7 and 44.1 respectively. The Nc value of low-risk HPVs E7 (44.8)is lower than the high-risk HPVs E7 (53.7). (2) the HPV preferred codons in HPV6 and HPV 16 is not consistent with the collective HPV preferred codons in total 79 HPV genotypes . Both HPV6 and HPV 16 display a strong codon usage bias, mainly with T or A at the 3rd position, which is significantly different from those averaged across mammalian genes (3) Thereare 27 ,14 , 21 ,24 codons' frequency of HPV6 evidently disagreeing with E. coli,yeast, adenovirus or human respectively, and 33 ,14 , 30 , 27 codons' frequencyof HPV 16 evidently disagreeing with E. coli , yeast , adenovirus or human respectively.In conclusion : (1) Nc value demonstrated that the frequency of E7' codonof high-risk HPVs is comparatively uniform; whereas the codon uasage bias of low-risk HPVs' E7 is comparatively distinct; indicating that the different type ("low-risk" HPV and "high-risk"HPV) has different codon uasage bias .(2) The codon preferences of different HPV type have its individuality. The codonusage bias of HPV are significantly different from those averaged across mammalian genes, suggesting that codon usage and DNA base composition could be one of the determinants of the rate of HPV gene expression within mammalian cells. (3) Codon preference of HPV was close to that of the yeast system and deviated from E. coli, Adenovirus and human. The results indicatedthat yeast system should be more suitable for HPV' gene expression.Knowledge of codon usage pattern in low-risk HPV6 and high-risk HPV 16 respectively may therefore contribute the development of DNA vaccines and improve understanding of the evolution and pathogenesis of HPVs. The effects of codon optimization on the expression and immunogenicity of HPV6b E7 proteinThe comparison of codon preference between "low-risk" HPVs and "high-risk" HPVs revealed that the codon usage of HPV E7 gene does not completely match with their hosts, indicating that codon usage might play an important role in the interactions between viruses and their hosts.The purpose of this proect is to investigate whether codon modification can enhance the expression of HPV6b E7 gene and immunogenicity of HPV6b E7 DNA vaccine.To do so, the full length E7 gene of HPV6b were modified to substitute human preferred codon for rarely used codon , and three mutations were introduced into the pRB binding site of HPV6b E7 to eliminate its transformation potential. The codon optimized (humanized) and mutated E7 gene (named HPV6b hu-mE7) were cloned into the Kpnl and EcoRI sites of the pcDNA3 mammalian expression vector, and the expresssion of the HPV6bhu-mE7 gene and the immunogenicity of HPV6bhu-mE7 DNA vaccine were compared with the HPV 6b wild-type E7 gene(named HPV6b wt-E7).To compare the expression of codon modified HPV6b E7 gene (pcDNA3/hu-mE7) and wild-type HPV6b E7 gene (pcDNA3/wt-E7 ) , Cos-1 cells were transfected with mammalian expression construct pcDNA3/hu-mE7and pcDNA3/wt-E7. E7 protein was determined 44 -48h after transfection via immunofluorescence. Of cells transfected with pcDNA3/hu-mE7 , 30-40%produced E7 protein within the cell nucleolus. Only occasional cells ( about 5%) transfected with pcDNA3/wt-E7 produced sufficient E7 protein to be recognized by immunofluorescence . The enhanced expression of E7 protein with pcDNA3/hu-mE7 was also confirmed via FACS analysis again.To examine in vivo immune responses induced by the wild-type and codon modified HPV6b E7 expression constructs, female C57BL/6 mice were immunized intramuscularly, with the pcDNA3/hu-mE7 and pcDNA3/wt-E7 respectively. The results showed that the codon modified HPV6b E7 gene induced the stronger BFN-gammma ratios than did the HPV6b wt-E7 gene. HPV6b E7-specific antibody was detected in vaccinated mice by ELISA and there was statistically significant difference in the response to the 6b E7 antigen beteewen the codon modified E7 gene groups and the wild type E7 gene groups.Our data confirm that codon optimisation enhances the expression of HPV 6b E7 protein in mammalian cells and the immunogenicity of DNA vaccine encoding the HPV 6b E7 gene. Codon optimized HPV6b E7 DNA vaccine can induce an E7-specific CMI and IgG antibody immune response. As found by others for HIV proteins and for HPV LI proteins , altering codon usage in an 6bE7 DNA vaccine toward mammalian codon usage results in better expression of protein in vitro and an improved immune response in vivo. The enhanced immune response by codon modification is associated with improved expression of E7 protein. These results suggest that the optimizing codon usage contribute to the enhanced gene expression and immunogenicity of HPV6b E7 DNA vaccine.The study on the mechanism of codon optimization and the enhanced expression of HPV proteinsThe studies have demonstrated that codon usage appears to be the major determinant of HPV gene expression, but very little is known about themoclecular mechanisms .To study how the codon optimisation enhances the expression of HPVprotein in mammalian cells , we first compared the mRNA and protein expressionlevels of codon optimized HPV6b E7 gene and wild type HPV6b E7 gene,subsequently we investigated whether expression of a single tRNA gene( human(tRNAser (CGA)) can assist expression HPV gene in cultured mammalian cells.The mammalian Cos-1 and WISH cells were transfected with recombinant pcDNA3/hu-mE7 and pcDNA3/wt-E7 plasmids respectively. The 6bE7 mRNA expression levels of the two plasmids were analysed by RT-PCR. The result of semi-quantitative RT-PCR showed that the cell transfected with pcDNA3 /hu-mE7 and pcDNA3/wt-E7 both gave rise to the specific amplified fragment of 190 bps,. Statistical analysis of the mRNA expression levels of cultures transfected with two plasmids indicated that there was no evident difference between codon optimized HPV6b E7 gene and wild type HPV6b E7 gene ( p>0.05). The same results had also been obtained in the study on the mRNA expression levels of the codon optimized BPV LI gene and wild type BPV LI gene. The results suggested that the posttranscriptional mechanisms may account for the low expression of HPV protein.To disclose the factors that might be involved in the low expression of HPV protein at the posttranscriptional level , we investigate whether expression of a single tRNA gene ( human (tRNAser (CGA) ) can assist expression HPV 6b E7 gene in cultured mammalian cells.The recombinant eukaryotic expression construct expressing human (tRNAser (CGA) was generated. The Cos-1 cells were transfected with (tRNAser (CGA) gene (pSV2neo tRNAser (CGA) ) together with either wild type 6bE7 gene (pcDNA3/wt-E7) or codon optimized 6bE7 gene( pcDNA3/hu-mE7) , and the E7 protein were analyzed by immunofluorescence . The result showed that the E7 protein was increased in the cells co-transfected with the wild type E7 and tRNAser (CGA)gene compared to the cultures transfeced with E7 gene alone , indicating that tRNAser (CGA) can assist expression HPV6b E7 gene in cultured...