Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.
Animals ; Hepacivirus ; genetics ; immunology ; Mice ; Plasmids ; genetics ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

Mice and 3-day-old chickens were orally inoculated with the recombinant attenuated Salmonella typhimurium strain ZJ111 carrying pcDNA3-F expression plasmid encoding the fusion protein of Newcastle disease virus (NDV). The results showed that ZJ111/pcDNA3-F was relatively safe. The recombinant plasmid pcDNA3-F was stable within the host stain ZJ111 in vitro and in vivo as shown by restriction enzyme analysis and PCR identification of the F gene. In an experimental vaccination study, 3-day-old chickens were orally immunized with ZJ111/pcDNA3-F with a dose of 108 cfu per chicken and boosted two weeks later. At week 4 post boosting, all chickens were challenged with a lethal dose of a virulent NDV strain F48 E9. The results showed that oral vaccination with ZJ111/pcDNA3-F induced stronger humoral and cellular immune responses than intramuscular immunization with naked pcDNA3-F plasmid. It also exhibited higher protection rate than the latter (66.7% vs 50%). This study indicates that the DNA vaccine using attenuated Salmonella typhimurium as delivery carrier had good safety, stability and immunogenicity and exhibited good potential of low cost and convenience for poultry disease control.
Animals ; Chickens ; Immunity, Cellular ; immunology ; Immunity, Humoral ; immunology ; Mice ; Newcastle Disease ; immunology ; virology ; Plasmids ; Polymerase Chain Reaction ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; adverse effects ; genetics ; immunology

OBJECTIVETo increase the immune effect of gene vaccine, T7 RNA polymerase was used to establish a system of cytoplasmic expression.

METHODS(1) The plasmid pT7 EMCVP1, including T7 promoter sequence, 5'-untranslated sequence of encephalomyocarditis (EMC) virus, VP1 sequence of coxsackievirus B3 (CVB3), was cotransfected with the plasmid pAR 3132, which codes for the T7 RNA polymerase, into HeLa cells and murine peritoneal macrophages. (2) The plasmid pT7 EMCVP1 and pAR 3132 were respectively transformed into the attenuated Salmonella typhimurium SL 7207. The two kinds of transformed bacteria were coinfected into murine peritoneal macrophages.

RESULTS(1) The target antigen VP1 in the cytoplasm was about 2-4-fold higher than that of pcDNA3 VP1 singly transfected. (2) After the murine peritoneal macrophages were coinfected by two kinds of transformed bacteria, the target antigen VP1 could also be detected.

CONCLUSIONThe pT7 EMCVP1 and pAR 3132 could be expressed in the cytoplasm of HeLa cells and murine peritoneal macrophages and the amount of the antigen VP1 increased remarkably as compared with that of pcDNA3 VP1 singly transfected.

Animals ; Bacteriophage T7 ; genetics ; Capsid Proteins ; genetics ; metabolism ; Cytoplasm ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enterovirus B, Human ; genetics ; Gene Expression ; HeLa Cells ; Humans ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Salmonella typhimurium ; genetics ; Transfection

To examine if polyprotein gene (VP2/VP4/VP3) of Infectious Bursal Disease Virus (IBDV) could be delivered into mammalian cells and expressed using attenuated Salmonella typhimurium as vector. The IBDV polyprotein gene was amplified by RT-PCR and inserted in to pCI, an eukaryotic expression plasmid. The resulting recombinant pCI-VP2/VP4/VP3 was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam- and phoP-), which was then use to transfect the Vero cells. Gene specific RT-PCR revealed that VP2/VP4/VP3 was transcribed into mRNA in the Vero cells. Indirect immunofluorscence assay, SDS-PAGE and Western-blot analysis showed that VP2/VP4/VP3 was expressed and the product was immuno-reactive with anti-IBDV serum. This work provides essential precondition for developing a new oral DNA vaccine against IBDV.
Animals ; Cercopithecus aethiops ; Electroporation ; Genetic Vectors ; genetics ; Infectious bursal disease virus ; genetics ; metabolism ; Polyproteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Salmonella typhimurium ; genetics ; metabolism ; Transfection ; Vero Cells ; Viral Proteins ; genetics

To construct a non-resistance and attenuated Salmonella typhimurium strain which expresses conservative region of adhesin(AB) of Helicobacter pylori(Hp). The AB gene was amplified by PCR and inserted into the expression vector pYA248 containing asd gene and was introduceded into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain by twice transformations, which is a balanced lethal recombinant. Bridged ELISA method was used to measure AB expressed in sonicate and culture supernatant. According to Meacock's way and growth curve, stability of the recombinant is evaluated. Semi-lethal capacity test was used to evaluate the safty of recombinant. Results showed S. typhimurium X4072(pYA248-AB) was constructed successfully, recombinant X4072(pYA248- AB) content of supernatant serum was higher than that of thallus lytic liquor confirmed by bridged ELISA, and after recombinant pYA248- AB cultured 100 generation without selection pressure, all the recombinant germ selected randomly can grow, and the AB antigen was positive by ELISA detection. The growth curve of the recombinant germ showed that the growth state of X4072(pYA248) and X4072(pYA248- AB) were coincidence on the whole, and the survival rate of C57BL/6 was still 100%, 30 days after taking X4072(pYA248- AB) 1.0 x 10(10)cfu. orally. Non-resistance S. typhimurium X4072(pYA248- AB) was constructed successfully. The recombinant plasmid was stable indicated by in vitro experiment. And the recombinant strain was safe confirmed by animal experiment. This live vaccine strain is worthy to be considered as a new live oral vaccine candidate against Hp infection.
Adhesins, Bacterial ; genetics ; metabolism ; Animals ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; Helicobacter pylori ; genetics ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; Polymerase Chain Reaction ; Salmonella typhimurium ; genetics ; growth & development ; metabolism

OBJECTIVETo prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection.

METHODSUsing genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected.

RESULTSThe sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed.

CONCLUSIONThe attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; administration & dosage ; immunology ; Female ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Helicobacter Infections ; immunology ; prevention & control ; Helicobacter pylori ; enzymology ; genetics ; immunology ; Immunoglobulin G ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Mice, Inbred BALB C ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Urease ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; genetics ; immunology ; Weight Loss

OBJECTIVETo evaluate the feasibility of oral cytokine gene therapy against tumor using live attenuated Salmonella as a vector.

METHODSA live attenuated AraA- autotrophic mutant of Salmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1 and pCMVmIL-12 administered orally in BALB/c and C57BL/6 mice. After 6 weeks, the mice were challenged with 4T1 or Lewis tumor cells, respectively, and flow cytometry and confocal microscopy were used to detect the expression of green fluorescence protein (GFP) in the tissues. PCR and ELISA were performed to detect the integration and expression of mIL-12 gene, and the survival time of the mice was also recorded.

RESULTSGFP expression and mIL-12 gene integration could be detected in the liver, spleen, intestinal, kidney and tumor tissues of the mice. The serum level of mIFN-gamma, mIL-12 increased significantly in mice with oral mIL-12 administration (P<0.05), which resulted in the survival prolongation of the mice as compared with the control mice (P<0.05).

CONCLUSIONOral gene therapy using live attenuated Salmonella can be potentially a simple, effective and above all, safe means for tumor treatment.

Administration, Oral ; Animals ; Carcinoma, Lewis Lung ; therapy ; Flow Cytometry ; Genetic Therapy ; methods ; Genetic Vectors ; administration & dosage ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Interleukin-12 ; genetics ; metabolism ; Mammary Neoplasms, Animal ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Microscopy, Confocal ; Neoplasms, Experimental ; therapy ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; immunology

OBJECTIVETo investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L(-1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder.

CONCLUSIONLack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites.

Animals ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Genes, Bacterial ; genetics ; Kidney ; enzymology ; Liver ; enzymology ; Male ; Microsomes, Liver ; drug effects ; enzymology ; Mutagenicity Tests ; Phthalic Acids ; pharmacokinetics ; toxicity ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Salmonella typhimurium ; genetics ; Urinary Bladder ; enzymology ; beta-Galactosidase ; metabolism

Th-2-biased immune responses are known to play a key role in the pathogenesis of atopic dermatitis. In particular, the macrophage-derived chemokine CCL22 is directly implicated in Th-2-associated skin inflammatory reactions, and its levels are significantly elevated in serum and are correlated with disease severity in atopic dermatitis. In this study, we tested the development of genetic therapeutic options to treat atopic dermatitis using bacteria expressing miRNA. We constructed a recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) for the in vivo knockdown of CCL22. The CCL22 gene was downregulated with CCL22 miRNA in activated lymphocytes. In mice with a cutaneous disease similar to atopic dermatitis, interleukin-4 was inhibited and interferon-gamma was induced after treatments with ST-miRCCL22. Furthermore, CCL22 levels were suppressed in the atopic mice treated with ST-miRCCL22. These results suggest that ST-miRCCL22 may be an effective genetic agent for treating atopic dermatitis.
Animals ; Cell Line ; *Chemokine CCL22/genetics ; Cytokines/blood ; Dermatitis, Atopic/pathology ; Disease Models, Animal ; Female ; Gene Expression Regulation/drug effects ; Gene Silencing ; Immunoglobulin E/blood ; Mice ; *MicroRNAs/genetics/pharmacology ; *Organisms, Genetically Modified/genetics ; *Salmonella typhimurium/genetics/metabolism ; Skin/*drug effects/pathology

OBJECTIVETo study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMV-beta as report gene.

METHODSMurine peritoneal macrophages were infected with SL7207(pCMV-beta) in vitro, then the expression of the beta-gal were detected by X-gal staining or RT-PCR. After mice were orally immunized with SL7207(pCMV-beta), the expression of beta-gal in the lymphoid tissue were tested by RT-PCR, humoral responses were tested by ELISA, splenic lymphocyte proliferation were tested by 3H-TdR incorporation and cytotoxic T lymphocyte reaction were tested by JAM test.

RESULTSThe results indicated that the plasmid pCMV-beta could be delivered by SL7207 into the nucleus of the murine macrophages efficiently and expressed well in vitro; after mice received oral immunizations with attenuated S.typhimurium SL7207 harboring plasmid pCMV-beta mice, the expression of beta-gal could be detected in the spleen, mesenteric lymph nodes and Peyers patches of the mice. Furthermore, the experiments demonstrated that specific humoral immune responses and cell-mediated immune responses were successfully induced in these immunized mice. Compared with the naked DNA vaccination, SL7207 (pCMV-beta) oral immunization were more efficient in inducing cellular immune responses.

CONCLUSIONSAttenuated Salmonella typhimurium SL7207 could be used as DNA delivery vehicle for oral immunization, which have the ability to deliver the antigen-encoding DNA specifically to APC directly for inducing the specific immune response being dominant with cellular immune response.

Animals ; Bacterial Vaccines ; immunology ; Cell Proliferation ; Cytotoxicity, Immunologic ; Female ; Genes, Reporter ; Genetic Vectors ; Macrophages, Peritoneal ; metabolism ; microbiology ; Mice ; Mice, Inbred BALB C ; Mutation ; Salmonella typhimurium ; genetics ; T-Lymphocytes, Cytotoxic ; cytology ; Transfection ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; beta-Galactosidase ; genetics ; immunology ; metabolism