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

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.

Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice.

The ORFs of both native and codon-optimized E7 genes were successfully fused to SPusp45 signal peptide and expressed by a nisin-controlled gene expression system in the NZ9000 strains of Lactococcus lactis. Recombinant strains were confirmed by Western blot analysis. To measure immune responses against the E7 antigen, specific-pathogen-free C57BL/6 mice were inoculated with L lactis harboring pNZ8123-rE7 by oral gavage. Then, specific antibodies and cytokines were measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay, respectively. Oral administration of L lactis strains expressing rE7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4+ T helper and CD8+ T cell precursors. Our outcomes indicated that the HPV-16 E7 specific IL-2- and IFN-γ-secreting T cells in antigen-stimulated splenocytes and intestinal mucosal lymphocytes were significantly higher than the control groups. Our data also demonstrated that mice vaccinated with recombinant L lactis were able to generate potent protective effects against challenge with the E7-expressing tumor cell line (TC-1). Moreover, L lactis containing pNZ8123-HPV16-optiE7 showed strong therapeutic antitumor effects against established tumors in vivo. These findings demonstrate that recombinant L lactis induce both humoral and cellular immune responses in mice and are therefore recommended for therapeutic treatments in humans after oral administration.

Protection against human papillomavirus type 16-induced tumors in C57BL/6 mice by mucosal vaccination with Lactococcus lactis NZ9000 expressing E6 oncoprotein.

Recombinant strains of Lactococcus lactis NZ9000 that express native and codon-optimized E6 protein (fused to the SPusp45 secretion signal) were successfully constructed by using the nisin-controlled gene expression (NICE) system. Expression of the recombinant strains was evaluated by Western blot analysis. Female mice of strain C57BL/6 were immunized orally with recombinant lactococci expressing inducible E6 oncoprotein and the antigen-specific antibody production (IgA and IgG) and cytokines were measured by ELISA and ELISPOT assay, respectively. Our outcomes indicate that the HPV-16 E6 specific IL-2- and IFN-γ-secreting lymphocytes in the antigen-stimulated intestinal mucosal lymphocytes, splenocytes and vaginal lymphocytes were significantly higher than the control groups. We showed that L. lactis having codon-optimized E6 oncogene had better inhibitory effect on tumor growth, better treatment effects on progression of tumor size, and better survival rate in comparison with L. lactis having native E6 oncogene, (P < 0.0001). In conclusion, the rE6 protein displayed by L. lactis can induce humoral and cellular immunity. Taken together, these preclinical results represent a promising step towards the development of recombinant L. lactis as a live oral vector vaccine to treat the HPV-16 associated with cervical cancer.

Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran.

Background & objectives: Diarrhoeagenic Escherichia coli strains are common agents of diarrhoea particularly in developing countries. Food products of animal origin are considered as common carriers of E. coli. This study was undertaken to identify enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) pathotypes in animal-source foods (ASF). Methods: A total of 222 ASF samples were investigated. Based on the culture and biochemical tests, 109 E. coli isolates were identified. Duplex-polymerase chain reaction assay was used to detect ETEC and EPEC. The target genes selected for each category were the lt and st for the ETEC, and eae and bfp for the EPEC isolates. Results: The occurrence of E. coli in dairy and meat products was 45 and 52.5 per cent, respectively. Among the E. coli isolates, two ETEC, one typical EPEC and three atypical EPEC were detected in meat samples, whereas only one typical EPEC and one atypical EPEC were detected in dairy samples. Interpretation & conclusions: Our results showed presence of ETEC and EPEC strains in ASFs. The milk without pasteurization and traditional dairy products produced in unhygienic conditions are most likely the main sources of E. coli pathotypes and other zoonotic pathogens and thus can be considered a potential hazard to the health of the community.

Efficient production and optimization of E7 oncoprotein from Iranian human papillomavirus type 16 in Lactococcus lactis using nisin-controlled gene expression (NICE) system.

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.

Ammonia disinfection of animal feeds --laboratory study.

Animal feeds may be contaminated, accidentally or maliciously, with a number of zoonotic bacteria. Animal infections with these bacterial agents, whether or not they cause animal disease, may lead to human illnesses. Anhydrous ammonia was introduced on farms in developed countries as a high-nitrogen soil amendment, but later found use in enhancing crude protein in low-quality roughage fed to ruminants and in neutralizing mycotoxins in fungus-infested feed grains. Although ammonia has been known to be effective against bacteria in other contexts (e.g., manure, community sewage sludge, seeds for sprouting, and boneless lean beef trimmings), it appears that the antibacterial effect of ammoniating animal feeds had not been tested. In the present study, samples of roughage (wheat straw, corn silage) and concentrates (corn grain, cottonseed) produced as animal feed were contaminated with dried-on zoonotic bacteria (Salmonella Newport in all; Campylobacte jejuni, E. coli O157:H7, Listeria monocytogenes, Yersinia enterocolitica in corn grain only). Disinfection with anhydrous ammonia gas was conducted for 24 h at room temperature ( 25 degrees C). The treatment was least effective in silage because the silage alone showed strong antibacterial activity, which may have been slightly reduced by ammoniation. In the other three feeds, depending on the initial level of contamination, ammonia destruction of >or= 5 log10 cfu/g (99.999%) of the selected contaminant was usually observed.

VacA and cagA genotypes of Helicobacter pylori isolated from raw meat in Isfahan province, Iran.

Foods with animal origins play a substantial role in the transmission of Helicobacter pylori. The present investigation was carried out to study the vacA and cagA genotypes status of H. pylori isolated from various types of meat samples. Two hundred and twenty meat samples were collected and cultured. H. pylori-positive strains were analyzed for the presence of vacA and cagA genotypes. Eleven out of 220 (5.00%) samples were positive for H. pylori. Findings were confirmed by nested PCR. Prevalence of H. pylori in the meat samples of slaughterhouses and butcheries were 72.20% and 27.70%, respectively. The most commonly detected genotypes in the meat samples of slaughterhouses and butcheries were vacAm1a (66.66%) and vacA s1a (37.50%), respectively. The S1am1a was the most commonly detected genotype. Meat sampled from butcheries had the higher prevalence of H. pylori and its genotypes than those of slaughterhouses (p < 0.05). Results showed that meat samples could be the potential sources of virulent strains of H. pylori. Application of sanitary measures in the storage, transportation and sale of meat is essential for reducing the levels of H. pylori cross contamination.

Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363

HPV 16 intratypic sequence variations has been recognized in association with oncogenic potential diverge and geographic distribution. This study aimed to investigate nucleotide modifications and optimization of HPV 16 E7 regions from Iranian infected women. Cervical biopsies from 79/163 HPV 16 positive cancer patients detected in our study were analyzed by PCR in a couple of cloning of a complete ORF of the E7 gene, and sequencing. The most frequently observed variant was C196T in E7 which led to an amino acid change of R66W. In addition, only one common variant T234G was identified from all specimens, but it did not lead to any amino acid change. We also detected nucleotide variations A86G, and C188T in samples. Among 99 codons in E7 gene, 56 codons were improved for Lactococcus lactis subsp. cremoris MG1363 resulting in a reduced G+C content from 43.1% to 34.0%. Also, the AT%, ENC, and CAI values were 66, 20±1.1, and 1.000 instead of 56.90, 60 ±1.1, and 0.406 respectively. Finally we constructed expression vector pNZ8148 encoding optimized E7 oncoprotein of HPV 16. This study declared for the first time, the genetic variations of HPV 16 E7 in IRAN. We conclude that plasmid pNZ8148-HPV 16-opti E7 can be potential vaccine candidates in the future.

Highly Sensitive FRET-Based Fluorescence Immunoassay for Detecting of Aflatoxin B1 Using Magnetic/Silica Core-Shell as a Signal Intensifier.

BACKGROUND: Recently, some new nanobiosensors using different nanoparticles or microarray systems for detection of mycotoxins have been designed . However, rapid, sensitive and early detection of aflatoxicosis would be very helpful to distinguish high-risk persons. OBJECTIVES: We report a highly sensitive competitive immunoassay using magnetic/silica core shell as a signal intensifier for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from Cd/Te quantum dots (antiaflatoxin B1 antibody immobilized on the surface of Cd/Te quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The specific immune-reaction between the anti-aflatoxin B1 antibody on the QDs and the labeledaflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photo-excitation of the QDs. Using magnetic/silica core shell to intensify the obtained signal is the novelty of this study. MATERIALS AND METHODS: Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and tellurium in the presence of sodium borohydride under nitrogen atmosphere. Magnetic nanoparticles were synthesized using FeSO4 and FeCl3 (1:2 molar ratio) and ammonia as an oxidizing agent under nitrogen atmosphere. The prepared magnetic nanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Nanoparticles synthesis and monodispersity confirmed by TEM. Immobilization of Cd/Te QDs to antibodies and labeling of aflatoxin B1-albumin by Rho 123 were performed by EDC/NHS reaction in reaction mixture buffer, pH 6, at room temperature. RESULTS: By using the magnetic/silica core shell sensitivity of the system changed from 2×10-11 in our previous study to 2×10-12 in this work. The feasibility of the method established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the decreased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spiked samples, over the range of 0.01-0.06 µmol.mL-1. CONCLUSIONS: This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require multiple separation steps and excessive washing.

Antioxidant and antimicrobial carboxymethyl cellulose films containing Zataria multiflora essential oil.

The present study describes the physical, antioxidant, and antimicrobial properties of biodegradable films prepared by incorporating different concentrations (1, 2, and 3% v/v) of Zataria multiflora Boiss (avishan-e shirazi) essential oil (ZEO) into carboxymethyl cellulose (CMC) film. The films' tensile strength, elongation at break, water-vapor permeability, optical characteristics, microstructure, and antimicrobial and antioxidant properties were investigated. The results indicated that the film containing 1% ZEO had the highest tensile strength and elongation at break. The control film showed the lowest water-vapor permeability. The resulting optical data showed that the control films were transparent in appearance; transparency was significantly reduced by an increase in ZEO concentration. Solubility in water decreased with increased ZEO. Films with ZEO, especially at higher concentrations, were more effective against all tested bacteria than the control film. Those films incorporating essential oil revealed antioxidant properties as well; this effect was greatly improved when the proportion of ZEO was increased. The results indicated that the antioxidant and antibacterial activity of ZEO is retained when it is used in CMC film. These properties with some good physical characteristics suggest applications for ZEO-incorporated film in a wide range of food products.

Interactive Effect of Temperature, Atmosphere and Storage Time on the Probability of Colony Formation on Blood Agar by Four Listeria Species.

The probability (P) of one Listeria cell and the cells needed to initiate colony formation on sheep blood agar plates as affected by atmospheric conditions (AC), storage temperature (T), time (t), and Listeria species was evaluated. The factorial design experiments included Listeria monocytogenes (2 strains), Listeria seeligeri , Listeria ivanovii , and Listeria innocua , as test organisms, storage of the plates at 4, 8, 20, and 30°C under air (A), modified atmosphere (MA) of 5% O2, + 10% CO2 + 85% N2, 100% CO2 (CO2), vacuum (V), and candle jar (CJ) for 7, 14, 21, 42, and 56 d. Statistical analysis indicated the significant effect of AC (p<0.0004), T (p<0.0001), t (p<0.0001) but not of species (p>0.71). None of the interactions with atmospheric conditions were significant (AC × species, AC × t, AC × T, all with p>0.46). Pairwise comparison of the P's for each of the AC's indicated that 100% CO2 was significantly more inhibitory to growth initiation than any other AC (p<0.004). No difference among the other AC's was shown. The effect of CO2 on delaying growth of Listeria was enhanced with decreasing storage T. Thus, under CO2, ≤6 cells of L. monocytogenes formed a colony within 7 d at 20°C and 42 d at 4°C. L. ivanovii was the most sensitive to CO2 and required 4.7 × 104 cells to form a colony after 42 d at 4°C. CO2 (100%) extended the lag phase at ≤8°C and decreased the rate of growth of the test organisms at 4°C but not at higher temperatures.