Modification of HPV type 16 E6 and E7 genes, and analysis of stability and immunogenicity of the modified proteins / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To select the mutants of HPV type 16 E6 and E7 genes suitable for construction of vaccine for treatment of cervical cancer.</p><p><b>METHODS</b>E6 and E7 genes were modified by site-directed mutagenesis. Several recombinant vaccina viruses were constructed by inserting the E6 or E7 mutants into the genome of vaccina virus Tiantan strain and employed to study their antigenicity.</p><p><b>RESULTS</b>Western blot assay showed that the E6 ?mutant? with substitution of Gly for Leu at amino acid site 50 and E7 mutant with substitution of Gly for Cys-24 and Glu-26 had no effect on their stability and antigenicity, but change of the Cys at position 91 of E7 dramatically reduced its stability and antigencity. Conclusion The results confirmed that the Zinc-finger structure at the E7 C-terminal? plays an important role in the integrity and stability of E7 protein.</p>

Construction and identification of the replication-deficient recombinant vaccinia virus co-expressing HPV type 16 L1 and L2 proteins / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate an HPV16 prophylactic vaccine candidate for cervical cancer.</p><p><b>METHODS</b>HPV16 major capsid protein L1 gene and minor capsid protein L2 gene were amplified using PCR. These genes were mutated by PCR site-directed mutagenesis for removal of sequence motifs (TTTTTNT) which would cause transcription termination when expressed from a vaccinia virus early promoter, then inserted into a vaccinia virus expression vector. A strain replication-deficient recombinant vaccinia virus containing the mutant sequences was obtained through a homologous recombination and identified.</p><p><b>RESULTS</b>The nucleotide sequence remained the correct amino acid sequence of the L1 and L2 proteins after mutated. Full-length L1 and L2 proteins were generated in cells infected with the recombinant virus. The virus strain propagated at very low titer or could not reproduce in some kinds of cell derived from different human tissues.</p><p><b>CONCLUSIONS</b>The authors have generated a strain replication-deficient recombinant vaccinia virus expressing HPV16 L1 plus L2 proteins as an HPV16 prophylactic vaccine candidate for cervical cancer.</p>

Construction of recombinant vaccinia virus co-expressing mutant E6 plus E7 proteins and detection of its immunogenicity and antitumor response / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate a candidate HPV16 vaccine for experimental and therapeutical use for cervical cancer.</p><p><b>METHODS</b>The mutants of HPV16 early E6 and E7 genes were inserted into a vaccinia virus expression vector. A strain of recombinant vaccinia virus was constructed through homologous recombination.</p><p><b>RESULTS</b>Showed that the mutant E6 and E7 genes were located at TK gene region of vaccinia virus Tiantan strain in a head to head orientation under the control of early/late promoters, H6 and 7.5K respectively. Studies in mice indicated that VmE6E7 could elicit specific antibodies against E6 and E7, and retarded or prevented tumor development in a proportion of C57 BL/6 mice challenged by syngeneic HPV16E6 and E7 transformed tumor cells.</p><p><b>CONCLUSIONS</b>The success in constructing VmE6E7 provides a basis for the further development of HPV16 therapeutic vaccine.</p>