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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 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., 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.

The present work was aimed at investigating the expression and optimization of a human papillomavirus (HPV) type 16 gene encoding oncoprotein E7 in Lactococcus lactis. We genetically engineered Lactococcus lactis using nisin-controlled gene expression (NICE) system pNZ8148 to express the native and codon optimized E7 oncogenes isolated from Iranian HPV-16. The results of optimizing fermentation showed, the concentration of produced protein was expressively improved by 10 ng/mL nisin after 3.5, and 4 h induction for NZ9000 harboring the codon-optimized, and native E7 respectively. Furthermore the recombinant NZ9000 strains expressed rE7 by maximum value of 4.7 (Codon-optimized), and 1.82 μg/mL (Native) in static flask experiments at initial glucose concentrations of 50 and 75 g/L respectively. The rE7 yield was further enriched in batch fermenter experiments using controlled pH. Thus, the overall production of rE7 under optimized conditions accumulated in the cytoplasm to nearly 33.25 μg/mL by L. lactis NZ9000 containing codon-optimized E7, which was over ∼2.7-fold higher compared to the NZ9000 having native E7 strain (12.01 μg/mL). Accordingly, the maximum biomass production was calculated 4.87, and 1.51 g/L respectively., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory-secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis., (© The American Society of Tropical Medicine and Hygiene.)

Hyalomma anatolicum anatolicum tick is considered as one of the main problem of ruminants' productivity in endemic countries such as parts of Africa, the Middle East and India. The disease is economically important and hence, its control and eradication is a priority. This problem reinforces the need for alternative approach like vaccine to control tick infestations instead of continuous application of acaricide which led to the natural selection of the acaricide-resistant ticks. Therefore, the present study provided evidence for the construction of transformant containing the chromosomally integrated multi-copy expression cassettes of HAO3, its successful and efficient expression in Pichia pastoris yeast and purification of the secreted protein by ultrafiltration (UF) system in a high level yield and purity. The result of antigenicity assay for the rHAO3 protein pointed well toward its capability for the elicitation of antibody response in immunized rabbits. Interestingly, the results indicated that the expressed HAO3 protein reacted well with mid gut antigen (MGAg) and rBm86 (Gavac) antisera in ELISA and western blot assays making it evident that the epitopes present in expressed protein are well recognized by the antibodies against MGAg and rBm86 proteins. Moreover, the presence of cross-reactive epitopes between rHAO3 protein with its native antigen from mid gut cells was also determined., (Copyright © 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)