CRPV genomes with synonymous codon optimizations in the CRPV E7 gene show phenotypic differences in growth and altered immunity upon E7 vaccination

Abstract

Papillomaviruses use rare codons relative to their hosts. Recent studies have demonstrated that synonymous codon changes in viral genes can lead to increased protein production when the codons are matched to those of cells in which the protein is being expressed. We theorized that the immunogenicity of the virus would be enhanced by matching codons of selected viral genes to those of the host. We report here that synonymous codon changes in the E7 oncogene are tolerated in the context of the cottontail rabbit papillomavirus (CRPV) genome. Papilloma growth rates differ depending upon the changes made indicating that synonymous codons are not necessarily neutral. Immunization with wild type E7 DNA yielded significant protection from subsequent challenge by both wild type and codon-modified genomes. The reduction in growth was most dramatic with the genome containing the greatest number of synonymous codon changes.

Figure 1. Location of codon changes in H. CRPV E7.

Each of the codon changes introduced into H. CRPV E7 is noted. The changes were cumulative. For example E7/14 contains all the changes in E7/8 plus 6 unique to itself; E7/18 contains the 14 in E7/14 plus 4 unique to itself; E7/22 contains all of the changes noted. Upper lines represent wild type E7 sequence and lower lines, the codon-modified sequences with changes noted in bold. Single letter notations are used for the amino acids. The numbers above selected amino acids identify the genome containing the codon modification of that amino acid.

Figure 2. Immunoprecipitation of E7 proteins.

Protein from transiently transfected cells was immunoprecipitated, run out on a PAGE gel and detected using a monoclonal antibody to E7, MAb E7-1. Increasing amounts of protein were detected with increasing numbers of codon changes.

Figure 3. FSCAN analysis of cells transfected with E7 expression constructs.

Expression of E7 protein was detected in immortalized rabbit cells transfected with expression constructs of the E7 genes and subjected to FSCAN analysis. All analyses were done in duplicate. E7/18 and E7/22 showed the highest expression levels.( p<0.05, unpaired student t test), when compared to the control.

Figure 4. Analysis of expression of EGFP, used as a control for transfection efficiency.

Expression of EGFP was detected in all cells cotransfected with E7 genes and an EGFP expression construct. All transfections were done in duplicate. The amount of protein expressed declined with increasing numbers of optimized codons in the E7 genes suggesting down regulation due to expression of E7 (p<0.05, unpaired student t test).

Figure 5. Northern blot analysis of cells transiently transfected with E7 expression constructs.

RNA from transiently transfected cells was blotted to Hybond N⁺ membrane and probed for E7 message using a mixture of all E7 genes for the probe. Message was standardized to beta actin control. Elevated E7 RNA signals were obtained for all codon-modified genes; E7/8 gave the highest signal.

Figure 6. Quantitative RT PCR analysis on total RNA from cells transiently transfected with wild type E7 or codon-modified E7 expression constructs demonstrated that E7/22 message was present at about four times the numbers of copies of the other genes.

RNA was isolated from transiently transfected cells and subjected to QRT- PCR. Standard curves were run with each reaction and quantitation of message was done using these curves. E7/22 message was present at levels four fold higher than the other genes.

Figure 7. An Actinomycin D time course demonstrated that message stability increased with increasing numbers of codon changes.

RNA was isolated from transiently transfected cells at different time points from 0 to six hours post treatment. RNA stability increased with increasing numbers of codon changes.

Figure 8. Sizes of papillomas differed among the different genotypes.

Growth curves of papillomas following infection with wild type and codon-optimized genomes. E7/8 and E7/14 papillomas grew at about wild type rate; E7/18 papillomas grew statistically faster than E7/22 papillomas (P<0. 001, unpaired student's t test); E7/18 papillomas grew statistically faster at later time points than wild type papillomas (P<0.03 unpaired student's t test).

Figure 9. Results of vaccination study.

Animals were vaccinated with either wild type E7 (◯,▵,□) or empty vector (•,▴,▪) and were then challenged with each of the CRPV genomes including wild type. Vaccination resulted in a reduction in size of all papillomas. Volumes of wild type, E7/8, E7/14 and E7/18 papillomas were reduced 30–50%, and volumes of E7/22 papillomas were reduced 75% (see Table 1). Figure shows results for wild type (◯,•), E7/18 (▵,▴) and E7/22 (□,▪) papillomas.