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

Amir Hossein Mohseni,Sedigheh Taghinezhad-S,Vadood Razavilar,Hossein Keyvani

doi:10.1186/s12896-019-0499-5

Abstract

BackgroundWe 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.ResultsThe 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 flask experiments, while the corresponding strains gave a maximum yield of 35.49 and 14.24 μg/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-γ-secreting T cells were detected in antigen-stimulated splenocytes and intestinal mucosal lymphocytes, when compared to the control groups.ConclusionsWe 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.

Highlights

We aimed at constructing Lactococcus lactis strains expressing Human papillomavirus (HPV) type 16 (HPV-16) recombinant E7 oncoprotein and examining its overproduction ability followed by optimizing batch and fed-batch fermentations
According to SignalP 4.1, we obtained a discrimination score (D-score) of 0.640 at the position of 27–28 amino acid residues, which was above the default cut-off point of 0.450, suggesting that there is a signal peptide at the N-terminus of recombinant HPV type 16 (HPV-16) E7 protein (Fig. 1a)
The results indicated that the maximum values of recombinant E7 (rE7) concentrations were achieved by the end of the fermentation, while being still considerably higher compared to our previous batch production of intracellular rE7 oncoprotein (1.8916–2.6350 with 95% confidence interval (CI) of difference, P = 0.0001 for E7 and, 1.9321–2.4746 with 95% CI of difference, P < 0.0001 for optiE7)

Summary

Introduction

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. Several studies have examined the use of bacteria (such as Salmonella and Mycobacterium spp) as E7 antigen delivery vehicles to elicit an immune response (2019) 19:8 against HPV-16 [8] These organisms could recover their pathogenic potential and are not totally safe for use in humans, especially in older people, children, and immunosuppressed patients [9]. Lactic acid bacteria (LAB) are promising candidates for in vivo delivery of antigens because of their long and safe association with humans and their food These dairy microorganisms have been used for centuries in the fermentation and preservation of numerous foods and are considered to be safe microorganisms with a GRAS (Generally Recognized As Safe) status [10, 11]. Oral administration with genetic engineered L. lactis has proved to elicit both systemic and mucosal immunity [8, 15]

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Discussion

Conclusion