Dataset Information

Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis.

ABSTRACT: BACKGROUND:We aimed at constructing Lactococcus lactis strains expressing HPV-16 recombinant E7 (rE7) oncoprotein and examining its overproduction ability followed by optimizing batch and fed-batch fermentations. Thereafter, in order to assess the immunogenicity of recombinant L. lactis cells, C57BL/6 mice were immunized by oral gavage. RESULTS:The results suggested that recombinant strains harboring optiE7 and E7 genes produced a maximum of 4.84 and 1.91??g/mL of rE7 in static flask experiments, while the corresponding strains gave a maximum yield of 35.49 and 14.24??g/mL in batch experiments, respectively. Fed-batch study indicated that the concentration of rE7 protein significantly increased after feeding yeast extract plus GM17 medium. The rE7 production of the best performing strains was 2.09- and 1.48-fold higher than that of the strains during the batch fermentation. Furthermore, biomass levels were 1.98- and 1.92-fold higher than those in batch cultivation. Oral immunization of C57BL/6 mice with recombinant L. lactis produced significant specific IgG and IgA antibody responses in serum and vaginal fluids, respectively. Our outcomes suggest that vaccination with L. lactis expressing rE7 can generate significant protective effects against E7-expressing cell line. Also, our study provides evidence that the presence of large amounts of E7-specific CD4+ T helper and CD8+ T cell precursors was stimulated. Significantly higher frequencies of HPV-16 E7 specific IL-2- and IFN-?-secreting T cells were detected in antigen-stimulated splenocytes and intestinal mucosal lymphocytes, when compared to the control groups. CONCLUSIONS:We conclude that optimization of culture conditions along with recombinant protein expression can highly stimulate both specific humoral and cell-mediated immune responses in mice after oral immunization. These promising results represent a step towards fast-tracking a vaccine against HPV-16-associated cervical cancer.

SUBMITTER: Mohseni AH

PROVIDER: S-EPMC6346575 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis.

Mohseni Amir Hossein AH Taghinezhad-S Sedigheh S Keyvani Hossein H Razavilar Vadood V

BMC biotechnology 20190124 1

<h4>Background</h4>We aimed at constructing Lactococcus lactis strains expressing HPV-16 recombinant E7 (rE7) oncoprotein and examining its overproduction ability followed by optimizing batch and fed-batch fermentations. Thereafter, in order to assess the immunogenicity of recombinant L. lactis cells, C57BL/6 mice were immunized by oral gavage.<h4>Results</h4>The results suggested that recombinant strains harboring optiE7 and E7 genes produced a maximum of 4.84 and 1.91 μg/mL of rE7 in static fl ...[more]

PMID: 30678667

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Project description:High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.

Dataset Information

Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis.

Publications

Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis.

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