Ruofeng Yan

Vaccination of chickens with a chimeric DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 induces protective immunity against coccidiosis

A fusion DNA vaccine co-expressed Eimeria tenella TA4 and chicken IL-2 (chIL-2) was constructed and its efficacy against E. tenella challenge was observed. TA4 gene of E. tenella and chIL-2 gene were cloned into expression vector pcDNA3.1 and pcDNA4.0c in different forms, producing vaccines pcDNA3.1-TA4-IL-2, pcDNA3.1-TA4 and pcDNA4.0c-IL-2. The expression of aim genes in vivo was detected by RT-PCR and western blot. Animal experiment was carried out to evaluate the immune efficacy of the vaccines. Results indicated these DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. The animal experimental results showed that DNA vaccines could obviously alleviate cecal lesions, body weight loss and increase oocyst decrease ratio. The ACI of pcDNA3.0-TA4-IL-2 group was 192, higher than that of pcDNA3.1-TA4 group. The results suggested that TA4 was an effective candidate antigen for vaccine and co-expression of cytokine with antigen was an alternative method to enhance DNA vaccine immunity.

Efficacy of DNA vaccines carrying Eimeria acervulina lactate dehydrogenase antigen gene against coccidiosis.

The efficacies of DNA vaccines encoding either Eimeria acervulina lactate dehydrogenase (LDH) antigen or a combination of LDH antigen and chicken IL-2 or IFN-gamma were evaluated against chicken coccidiosis. Three vaccine plasmids pVAX-LDH, pVAX-LDH-IFN-gamma and pVAX-LDH-IL-2 were constructed using the eukaryotic expression vector pVAX1. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Average body weight gain, oocyst output, survival rate and lesion scores were measured to evaluate the protective effects of vaccination on challenge infection. The results showed that DNA vaccines could obviously alleviate body weight loss, duodenal lesions, oocyst output and enhance oocyst decrease ratio. Anti-coccidial indexes (ACIs) of pVAX-LDH-IFN-gamma and pVAX-LDH-IL-2 groups were higher than that of other groups. Flow cytometric analysis of T lymphocytes in spleen and cecal tonsil demonstrated that DNA vaccines had significantly increased percentages of CD3(+) T cells compared with pVAX1 alone or TE buffer. The results provided the first proof that DNA vaccine carrying E. acervulina LDH antigen gene induced protective immunity against homologous infection and its effect could be enhanced by co-expression of chicken IL-2 or IFN-gamma.

The optimal immunization procedure of DNA vaccine pcDNA-TA4-IL-2 of Eimeria tenella and its cross-immunity to Eimeria necatrix and Eimeria acervulina.

The immunization procedure of DNA vaccine pcDNA-TA4-IL-2 of Eimeria tenella, including route, dose, time of immunization and age of primary immunization of chicken, was optimized. The stability and the cross-species protection of the vaccine were also analyzed. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and the anti-coccidial index (ACI). Chinese Yellow chickens were randomly distributed into corresponding groups (30/group). The challenged, unchallenged and vector control groups were designed. The results illustrated that 25 microg was the optimal dose and intramuscular injection was the most effective route to induce protective immunity. There were no significant differences of ACIs between boosting and non-boosting groups. Storage time and temperature had little effect on the immunizing efficacy of the vaccine. The vaccine could provide partial cross-protection against the challenge with E. necatrix and E. acervulina, but not with E. maxima.

Construction of DNA vaccines encoding Eimeria acervulina cSZ-2 with chicken IL-2 and IFN-γ and their efficacy against poultry coccidiosis.

The study describes vaccination experiments with highly immunogenic sporozoite E. acervulina cSZ-2 co-administered with chicken IL-2 (chIL-2) and interferon-γ (chIFN-γ) to determine their efficacies against homologue challenge. The entire coding sequence of cSZ2, chIL-2 and chIFN-γ were cloned into eukaryotic expression vector pVAX1, constructing DNA vaccines pVAX1-cSZ2, pVAX1-chIL-2, pVAX1-chIFN-γ, pVAX1-cSZ2-chIL-2 and pVAX1-cSZ2-chIFN-γ. The expression of target genes in vivo was detected by RT-PCR and Western blot. Chicken experiments were carried out by vaccinating chickens two times at dose rate of 100 μg intramuscularly. At 28 days of age, all chickens were inoculated orally with 1×10(5) sporulated oocysts of E. acervulina except the unchallenged control group. Seven days after challenge, all chickens were weighted and slaughtered for duodenum collection. The results indicated that these DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. The findings also demonstrated best synergistic effect of IL-2 with this protein which suggested that co-administration of cytokines with this antigen was a powerful method to enhance immunity by alleviating intestinal lesions, body weight loss and oocyst count imparting partial protection against homologous challenge.

DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice

BackgroundToxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. Currently, the main control strategy depends on the usage of drugs that target the acute stage of the infection, however, drawbacks were encountered while applying this method; therefore, development of an alternative effective method would be important progress. Deoxyribose Phosphate Aldolase (TgDPA) plays an important role supporting cell invasion and providing energy for the parasite.MethodsTgDPA was expressed in Escherichia coli and the purified recombinant protein was used to immunize rats. The antibodies obtained were used to verify in vitro expression of TgDPA. The vector pVAX1 was utilized to formulate a DNA vaccine designated as pTgDPA, which was used to evaluate the immunological changes and the level of protection against challenge with the virulent RH strain of T. gondii.ResultsDNA vaccine, TgDPA revealed that it can induce a strong humoral as well as cellular mediated response in mice. These responses were a contribution of TH1, TH2 and TH17 type of responses. Following challenge, mice immunized with TgDPA showed longer survival rates than did those in control groups.ConclusionsFurther investigation regarding TgDPA is required to shed more light on its immunogenicity and its possible selection as a vaccine candidate.

The protective efficacy of chimeric SO7/IL-2 DNA vaccine against coccidiosis in chickens.

The protective efficacy of recombinant vaccines encoding an Eimeria refractile body antigen SO7 was assessed in broiler chickens following oral infection with Eimeria tenella. The SO7 and chicken IL-2 genes were cloned into the expression vector pVAX1 consecutively to construct DNA vaccines pVAX-SO7 and pVAX-SO7-IL-2. Expression of SO7 and IL-2 gene transcripts and proteins encoded by the plasmid DNAs in vivo was detected by reverse transcription-polymerase chain reaction and Western blot. Chickens were inoculated with 100 μg of plasmids pVAX-SO7 or pVAX-SO7-IL-2, or 200 μg of recombinant SO7 protein or chicken IL-2 protein by leg intramuscular injection. At 28days of age, all chickens except the unchallenged control group were challenged orally with 5×10(4) sporulated oocysts of E. tenella. All chickens were euthanized to determine the effects of immunization on the 7th day post-challenge. The results showed that both DNA vaccines containing the SO7 gene and the recombinant SO7 protein could obviously alleviate body weight loss and cecal lesions compared with unvaccinated and challenged control. These findings also suggested that chicken IL-2 could effectively enhance the immunity of SO7 against E. tenella challenge compared with vaccination using pVAX-SO7 alone.

Immunoproteomic analysis of whole proteins from male and female adult Haemonchus contortus.

Whole proteins of male and female adult Haemonchus contortus were analysed by immunoproteomic techniques. Approximately 662 and 680 spots were detected on proteome maps of male and female nematodes, respectively, stained with Coomassie brilliant blue G-250. There were 609 shared spots. Approximately 193 and 196 spots were recognised on Western blot maps of male and female nematodes, respectively, using antiserum from naturally infected goats as the source of primary antibodies. There were 129 gender-specific spots in male nematodes and 132 in females. Twenty-three shared immunogenic spots were identified by MALDI-TOF or MALDI-TOF-TOF mass spectrometry. These proteins included glutamate dehydrogenase (GDH), homologues of Dim-1, actin, globin-like excretory/secretory protein F6, glutathione S-transferase (GST), ATPase and glyceraldehyde-3-phosphate dehydrogenase. GDH and GST have been identified as immunogenic proteins of H. contortus previously, whereas the other proteins are newly recognised immunogenic proteins in this nematode.

Changes of cytokines and IgG antibody in chickens vaccinated with DNA vaccines encoding Eimeria acervulina lactate dehydrogenase.

The aim of this study was to investigate the changes of cytokines and specific serum IgG in chickens following vaccination with DNA vaccines encoding either Eimeria acervulina (E. acervulina) lactate dehydrogenase (LDH) antigen or LDH and chicken IL-2 or IFN-γ. Two-week-old chickens were randomly divided into five groups. Experimental group of chickens were immunized with DNA vaccines while control group of chickens were injected with pVAX1 plasmid alone or sterile water. All immunizations were boosted 2 weeks later. The LDH-specific IgG antibody response was measured at weeks 1-6 post-second immunization. The result showed that the antibody titers in chickens vaccinated with DNA vaccines were significantly different from those of the control groups 1 week after the second immunization (P<0.05) and reached the maximum values 3 weeks post-second immunization. The systemic and local cytokine mRNA expression was determined by quantitative RT-PCR 7 days post-second immunization. The specific IgG antibody levels against LDH of all chickens vaccinated with vaccines were increased compared to those of sterile water (H(2)O) and plasmid (pVAX1) control chickens 1-6 weeks post-second immunization (P<0.05). The mRNA levels of IFN-γ, IL-2, TNFSF15, IL-17D as well as TGF-β4 in both spleen and cecal tonsil were also increased in experimental chickens. In contrast, the only significant change of IL-4 mRNA level was observed in spleen of chickens immunized with pVAX-LDH-IL-2 compared with pVAX-LDH and control groups (P<0.05). These results suggested that DNA vaccines could increase the IgG antibody level and induce the expressions of cytokines.

Transcriptional and proteomic analysis reveal recombinant galectins of Haemonchus contortus down-regulated functions of goat PBMC and modulation of several signaling cascades in vitro

UNLABELLED In this study, a combined proteomic and transcriptomic analysis was performed to understand the mechanisms underlying the immunomodulation induced by recombinant galectins of Haemonchus contortus (rHco-gal-m/f) on goat peripheral blood mononuclear cells (PBMC). We demonstrated that rHco-gal-m/f could be distinguished by antisera from goats experimentally infected with H. contortus and bound to the surface of goat PBMC. Following rHco-gal-m/f exposure, 16 differentially expressed proteins were identified, which function in biological processes such as stimulus response, biological regulation and localization. According to Gene Ontology Annotation, 15 proteins (93.8%) had binding activity and 9 proteins (56.3%) had catalytic activity. A series of transcriptomic analyses were performed subsequently to assess the expression change of certain pathway members. The integrated results of proteomic and transcriptomic analysis suggested that the activation of VEGF pathway, free radical producing pathway, NFκB pathway and ubiquitin-proteasome pathway was inhibited following exposure to rHco-gal-m/f, while the TLR pathway and CASPASE pathway were activated. Cytokine production and T cell differentiation were also influenced. Cell migration assays and ELISA were performed and the results were in accordance with the change of the proteins and genes. The protein and gene profiles determined here identified several mechanisms underlying the rHco-gal-m/f-induced immunomodulation of goat PBMC. BIOLOGICAL SIGNIFICANCE This research provided insight into the interactive relationship between parasitic nematode galectins and host PBMC. It also shed new lights on the understanding of molecular mechanisms of helminthic immune evasion.

Vaccination of goats with glyceraldehyde-3-phosphate dehydrogenase DNA vaccine induced partial protection against Haemonchus contortus.

Owing to its critical functions in worm physiology, glyceraldehyde-3-phosphate dehydrogenase in Haemonchus contortus (HcGAPDH) is potential candidates for vaccine to control haemonchosis. In this study, DNA vaccine expressing HcGAPDH antigen was tested for protection against experimental H. contortus infections in goats. Fifteen goats (9-10 months of age) were allocated into three trial groups. Group 1 was vaccinated with HcGAPDH DNA vaccine twice on days 0 and 14, and then challenged with 5000 infective H. contortus L3 (third stage larvae) on days 28. Group 2 was an unvaccinated positive control group challenged with H. contortus L3 on days 28. Group 3 was an unvaccinated negative control group that was not challenged with L3. By the method of RT-PCR and Western-blot, transcription and expression of HcGAPDH DNA vaccine were identified at local injection sites post immunizations, respectively. After immunization with the DNA vaccine, significantly high levels of serum IgG, serum IgA, mucosal IgA, CD4(+) T lymphocytes and B lymphocytes were generated. Also, increased numbers of blood eosinophils and decreased haemoglobin level after challenge were observed in the vaccinated group. Meanwhile, cumulative mean faecal worm egg counts and worm burdens in vaccinated group were reduced by 34.9% and 37.73%, respectively. In brief, recombinant HcGAPDH DNA vaccine induced partial immune response against H. contortus infection in goats.