Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii

The dense granule proteins of Toxoplasma gondii are investigated as possible vaccine candidates against the parasite. The aim of this research was to evaluate the immune responses of sheep injected twice, intramuscularly, with DNA plasmids encoding T. gondii dense granule antigens GRA1, GRA4, GRA6 and GRA7 formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for GRA1, GRA4, GRA6 or GRA7 elicited an immune response after the first and the second injections as indicated by the moderate to high antibody responses. The injection of pGRA7 induced a significant level of anti-GRA7 IgG2 antibody and IFN-γ responses indicating a Th1-like immune response whereas injection with pGRA1, pGRA4 and pGRA6 stimulated a IgG1 type antibody response with a limited, if any, IFN-γ response. The results demonstrate that the intramuscular injection of sheep with a DNA liposome formulated plasmid coding for GRA proteins is an effective system that induces a significant immune response against T. gondii.     

Induction of immune responses in sheep by vaccination with liposome-entrapped DNA complexes encoding Toxoplasma gondii MIC3 gene

Toxoplasma gondii is a parasite that has been extensively studied due to its medical and veterinary importance in terminating pregnancies. Consequently, a satisfactory vaccine is required to control its adverse effects on pregnant animals. The microneme protein, MIC3, is a major adhesion protein that binds to the surface of host cells and parasites, and is therefore a potential vaccine against T. gondii. The viability of MIC3 as a vaccine is investigated in this study. Sheep were injected twice, intramuscularly, with plasmids containing DNA encoding for the mature form of MIC3 protein formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for MIC3 elicited an immune response after the first and second injections as indicated by antibody responses and the production of IFN-gamma. The immune response, as measured by the IgG2 and IgG1 serum levels, was boosted after the injection of the MIC3 DNA vaccine together with high anti-MIC3 antibodies. The results demonstrate that the intramuscular injection of sheep with a plasmid containing DNA coding for MIC3 protein induces a significant and effective immune response against T. gondii.     

Efficacy of DNA vaccination by different routes of immunisation in sheep

DNA vaccination, delivered through various routes, has been used extensively in laboratory animals. Few studies have focused on veterinary species and while results obtained in laboratory animals can often be extrapolated to veterinary species this is not always the case. In this study we have compared the effect of the route of immunisation with DNA on the induction of immune responses and protection of sheep to challenge with live Corynebacterium pseudotuberculosis. Intramuscular injection of plasmid DNA encoding an inactivated form of the phospholipase D (PLD) antigen linked to CTLA4-Ig resulted in the induction of a strong memory response and sterile immunity following challenge in 45% of the animals. In contrast, gene gun delivery or subcutaneous (SC) injection of the DNA vaccine induced comparatively poor responses and insignificant levels of protection. Thus, DNA vaccine efficacy in sheep is strongly influenced by the route of vaccination. Amongst intramuscular vaccinates, protected sheep had significantly elevated IgG2 responses compared to unprotected animals, while both subgroups had equivalent IgG1 levels. This suggests that the presence of IgG2 antibodies and hence a Th1-like response, induced by the DNA vaccine gave rise to protective immunity against C. pseudotuberculosis.     

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

A better understanding of cell-mediated immune responses to classical swine fever virus (CSFV) is essential for the future development of improved vaccines. We analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination. Viral infection induced high T cell responses with high primary and secondary CTL activity correlated with high IFN-gamma production, whereas vaccination with a live vaccine followed by infection mainly induced neutralizing antibody but low cell-mediated responses. Moreover, high IgG1 response was associated with high IFN-gamma response following infection whereas a weak IFN-gamma response was related to a good IgG2 response but a low IgG1 production. These data could reflect Th1/Th2-like balance of immune responses depending upon immunization protocols, which has not yet been described in the pig. T-cell responses to CSFV were evidenced by CSFV-specific CD25 upregulation on CD4-CD8+, but not on CD4+CD8- cells, which further illustrated the importance of CTL responses after infection. Our results indicated that generation of cell-mediated immune responses was much higher following intranasal/oral CSFV infection than after intramuscular vaccination, which implies that the capacity of new CSFV vaccines to induce higher T-cell responses should be considered.     

Induction of systemic immune responses in sheep by topical application of cholera toxin to skin

Cholera (and related) toxins (CT) when applied topically on unbroken skin induce systemic immune responses in mice, a procedure called transcutaneous immunization (TCI). The current study examined the capacity for TCI to induce systemic immune responses in sheep. Three groups (n=5 per group) were immunized at day 0 (priming) and day 28 (boosting) with 250 microg of CT in water by TCI, with 25 microg of CT in alum by intramuscular injection, or not immunized. Serum samples were taken at days 0, 28, 42, 56 and 70 after immunization for measurement of CT-specific IgG as well as CT-specific IgG1, IgG2, IgA and IgM antibodies by ELISA. After immunization, IgG, IgG1 and IgG2 antibody in immunized groups were significantly higher than in the control group, and boosting further increased these titres. IgG, IgG1 and IgG2 in the injection group were significantly higher than in the TCI group. There was a preponderance of IgG1 antibody, relative to IgG2, in both immunized groups. CT-specific IgA and IgM were detected in both immunized groups. Lymphocyte proliferation to CT was measured at day 90. A CT-specific lymphocyte proliferative response (stimulation index>2) was detected in all sheep from the injection group, in two sheep from the TCI group and in none of the controls. Results demonstrated that TCI induces primary and secondary antibody responses and specific proliferative responses to CT in sheep.     

巨型艾美耳球虫偏菱形样蛋白EmRP免疫原性分析

在前期研究中,从巨型艾美耳球虫(Eimeria maxima)cDNA文库中筛选到了免疫保护性抗原偏菱形样蛋白EmRP,本研究为揭示该抗原的免疫保护机理,进一步检测了其免疫原性。以E.maxima卵囊cDNA为模板,用RT-PCR技术扩增EmRP,并构建真核表达质粒pVAX1-EmRP,将其经腿部肌肉注射2周龄的雏鸡,3周龄加强免疫。分别于首次免疫后和加强免疫后1周,采集血清,用ELISA方法检测血清特异性IgG水平,用荧光定量PCR方法(qPCR)检测细胞因子水平,用流式细胞术检测CD4^+T淋巴细胞和CD8^+T淋巴细胞的比例,分析EmRP诱导的免疫反应。序列分析表明,EmRP含有偏菱形样蛋白超家族成员保守区域,为非跨膜蛋白,分子量约为28.4 kD。真核表达质粒pVAX1-EmRP免疫鸡后,与对照组相比,鸡体IFN-γ、IL-2、IL-10和IL-17等细胞因子转录水平显著提升;CD8^+和CD4^+ T细胞比例显著提高,而血清特异性IgG水平与对照组差异不显著。结果表明,EmRP诱导的免疫保护作用主要是由其诱导的细胞免疫反应实现的,可以作为研制E.maxima新型疫苗的候选抗原。     

Survey of ovine and caprine toxoplasmosis by IFAT and PCR assays in Sardinia, Italy

During the period 1999-2002, we have analyzed 9639 serum samples and 815 aborted samples (670 fetuses and 145 placenta) from 964 ovine and caprine farms distributed over all Sardinia island. After abortion notification, sera collected at random from adult animals were examined to detect simultaneously IgG and IgM antibodies specific to Toxoplasma gondii by indirect immunofluorescence assay, whereas fetuses and placenta were analyzed by a single tube nested PCR assay. Specific IgG antibodies were detected in 2048 (28.4%) sheep and 302 (12.3%) goats, specific IgM antibodies were found in 652 (9%) sheep and 139 (5.6%) goats. From a total of 2471 ovine and 362 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 271 (11.1%) ovine and 23 (6.4%) caprine samples were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. On the one hand, these results indicate that the seroprevalence of T. gondii infection in sheep and goats is relatively high, on the other PCR results demonstrate that T. gondii has a significant role in ovine and caprine abortion. Adequate management might be useful and essential to control the toxoplasmosis in the sheep and goats herds of Sardinia.     

Failure of FIV-infected cats to control Toxoplasma gondii correlates with reduced IL2, IL6, and IL12 and elevated IL10 expression by lymph node T cells

Increased susceptibility to intracellular pathogens in HIV-infected individuals and FIV-infected cats is attributed to a defective T-helper 1 (Th1) immune response. However, little is known about specific cytokine responses to secondary pathogens. To address this question, control and FIV-infected cats were challenged with Toxoplasma gondii, and lymph node cells analyzed for cytokine mRNA expression. Twenty-four weeks post-FIV infection, prior to T. gondii challenge, IL2 and IL12 mRNAs were depressed, whereas IL10 and IFNgamma mRNAs were increased in CD4+ and CD8+ subsets. Following T. gondii challenge, control cats showed increased expression of IL2, IFNgamma, IL10, IL12, and IL6 mRNAs. In contrast, IL2, IL6, IFNgamma, and IL12 mRNAs were suppressed in FIV-T. gondii co-infected cats, whereas IL10 remained at the high prechallenge levels. IFNgamma and IL10 mRNAs were produced by both CD4+ and CD8+ cells in FIV-T. gondii cats. Elevated IL10 may suppress a Th1 cytokine response to T. gondii challenge.     

基因枪与肌肉注射猪繁殖与呼吸综合征病毒ORF5基因疫苗诱导小鼠体液及细胞免疫的比较研究

猪繁殖与呼吸综合征病毒ORF5基因疫苗（pcDNA—PRRSV—ORF5）以不同免疫途径（基因枪和肌肉注射）免疫BALB／c小鼠，以PBS和空载质粒pcDNA3．1（＋）为对照，采用流式细胞仪（FACS）、淋巴细胞增殖试验（MTT法）及间接ELISA试验分别对小鼠外周血中CD4^＋、CD8^＋T淋巴细胞数、T淋巴细胞的转化功能及小鼠血清中特异性PRRSV血清抗体IgG动态变化进行了检测。结果表明，pcDNA—PRRSV-ORF5基因疫苗接种小鼠后外周血对ConA有明显的反应性，试验组与对照组比较差异极显著（P〈0．01）或显著（P〈0．05），CD4^＋T淋巴细胞数在免疫后7d高于对照组（P〈0．01），CD8^＋T淋巴细胞在免疫后28d高于对照组，不同途径基因疫苗接种小鼠后均诱导小鼠产生PRRSV特异性IgG。在诱导细胞免疫方面，基因枪和肌肉注射各组间无明显差异；在诱导体液免疫方面，基因枪法优于肌肉注射。研究表明制备的pcDNA—PRRSV—ORF5基因疫苗免疫小鼠能够诱导其机体产生良好的体液和细胞免疫应答，基因枪法较肌肉注射更能诱导体液免疫应答的产生。     

The role of colorstrum on the occurrence of immunoglobulin G subclasses and antibody production in neonatal goats.

Quantitative determinations of IgG1 and IgG2, in one group of colostrum-fed and one group of colostrum-deprived neonatal goats revealed that the occurrence of the IgG1 subclass preceeded that of the IgG2 in both cases. In the colostrum-fed animals the IgG2 appeared, on an average, in the fourth week of life whereas in the colostrum-deprived animals the IgG2 was detected as early as three weeks after birth. At the age of twelve weeks the mean concentrations for IgG, and IgG2 were higher in the animals deprived of colostrum. The immune response to human gamma globulin was studied in colostrum-fed and colostrum-deprived neonatal goats which were immunized at birth and again after four and eight weeks. Following the first two antigen administrations a significantly higher response was obtained in the colostrum-fed neonates. However, the third injection determined a similar response in both groups. A marked suppressive effect on the immune response was observed in colostrum-fed neonatal goats when specific antibodies were present in the colostrum after preimmunization of the mothers with human gamma globulin.     

Comparison of antibody and cell-mediated immune responses in horses following feeding of a novel dietary antigen, ovalbumin, and rotavirus.

Adult ponies which were fed ovalbumin (OVA) daily for 2 weeks had significantly greater serum anti-OVA IgG (P = 0.001) and antigen specific lymphocyte responses (P = 0.031) after intramuscular injection with OVA given with saponin than control ponies which had not been fed the antigen. This suggests that, despite the lack of evidence of B- or T-cell activation in peripheral blood during the period of OVA feeding, the animals were primed for an active secondary immune response. Adult ponies were challenged with equine rotavirus, strain H-2, but no statistically significant differences were found in serum IgG-associated antibody responses or antigen-specific lymphocyte responses between the rotavirus-challenged group and the control group, either following rotavirus challenge or intramuscular injection of rotavirus antigen given with saponin. Our findings, that feeding the non-replicating protein antigen OVA appeared to prime for an increased immune response rather than inducing oral tolerance, may be of relevance to future studies on the way the equine gastrointestinal tract handles usually harmless antigens.     

Immunogenicity and protective efficacy of two recombinant pseudorabies viruses expressing Toxoplasma gondii SAG1 and MIC3 proteins

Toxoplasma gondii is one of the most common parasitic pathogens in humans and warm-blooded animals, causing toxoplasmosis. One of the efficient ways to control this disease is immunization. In this study, two recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV-SAG1) and TgMIC3 (rPRV-MIC3) based on the PRV vaccine strain were developed by homologous recombination and used for immunizing BALB/c mice. Ninety BALB/c mice were randomly divided into five groups including four experimental groups (inoculated twice in 4 weeks interval with PRV TK-/gG-/EGFP+, rPRV-SAG1, rPRV-MIC3, rPRV-SAG1+rPRV-MIC3, respectively) and one control group (inoculated with medium). All mice vaccinated with rPRV developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-γ and IL-2 production. These results demonstrated that rPRV could induce significant humoral and cellular Th1 immune responses. Moreover, rPRV immunization induced partial protection against a lethal challenge with T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. The mice immunized with the rPRV-SAG1 and rPRV-MIC3 cocktail could develop higher T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge. These results suggested that expression of protective antigens of T. gondii in PRV is a novel approach towards the development of a vaccine against both animal pseudorabies and toxoplasmosis.     

Immune response and protective efficacy against homologous challenge in BALB/c mice vaccinated with DNA vaccine encoding Toxoplasma gondii actin depolymerizing factor gene

A DNA vaccine (pVAX1-TgADF) encoding Toxoplasma gondii actin depolymerizing factor (ADF) gene was constructed and the immune response and protective efficacy of this vaccine against homologous challenge in BALB/c mice were evaluated. High titers of specific antibody and increases in the percentage of CD4(+) and CD8(+) T lymphocyte cells were observed from BALB/c mice vaccinated with pVAX1-TgADF (P<0.05), when PBS group was used as control. The survival time of BALB/c mice in pVAX1-TgADF group was longer than those in control groups. The numbers of brain cysts in the experimental BALB/c mice immunized with pVAX1-TgADF reduced significantly compared with those in PBS group (P<0.05), and the rate of reduction could reach to around 42.8%. These results suggested that the DNA vaccine pVAX1-TgADF could generate specific humoral and cellular immune responses, prolong survival times, and reduce brain cysts load against T. gondii infection in BALB/c mice.     

Interferon-gamma and interleukin-2 release by lymphocytes derived from the blood, mesenteric lymph nodes and intestines of normal sheep and those affected with paratuberculosis (Johne's disease).

This study sought to determine if T-cell cytokine responses to mycobacterial infections in sheep were similar to those in other species and if such responses correlated with prevailing gut pathology. Lymphocytes were isolated from the blood (PBL), mesenteric lymph nodes (MLN) and ileal lamina propria (LPL) of control sheep and of sheep with clinical Johne's disease due to infection with Mycobacterium avium ssp. paratuberculosis (M.a. paratuberculosis). These animals had previously been categorised into two groups exhibiting either the 'tuberculoid' (paucibacillary) form of lesion or the 'lepromatous' (multibacillary) form. Lymphocytes were examined for their capacity, following stimulation with johnin-PPD, to release interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) characteristic of the Th1 subset of MHC Class II-restricted CD4+ (helper) T-cells in other species. The expression of the two cytokines appeared related to the type of histological lesion observed. Antigen-stimulated lymphocytes from the tuberculoid group exhibited greater release of IFN-gamma and IL-2 than lymphocytes from the lepromatous group suggesting a Th1-type of response in the former in which expression of IFN-gamma by PBL showed a significant positive correlation with that expressed by MLN and LPL. Lymphocytes from animals with lepromatous lesions released lesser mycobacterium-induced IFN-gamma and IL-2 indicating a diminished role for a Th1 subset in this group of sheep. Differences in cytokine expression were much more apparent with lymphocytes which were derived from MLN.     

Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.     

Characterization of the immune response to Mycoplasma bovis lung infection

To better understand the interaction between Mycoplasma bovis and its bovine host, we have characterized the immune response generated during an experimental lung infection with M. bovis. Proliferation ([3H]-thymidine blastogenesis) and Th1/Th2 cytokine production were used to monitor peripheral cellular immune responses. Flow cytometry analysis was used to determine T-cell subset activity by CD25 expression. Humoral immune response was monitored by the identification of antigen-specific IgG1 and IgG2 isotypes over time. Herein, we show that M. bovis antigen stimulates activation of CD4+ and CD8+ cells in vitro in a manner consistent with memory, and that gammadelta-T cells are activated by antigen in a manner consistent with innate immunity. In addition, the percentage of cells producing IFN-gamma during recall response is equal to that of IL-4 producing cells. Serological analysis shows M. bovis stimulates increased production of antigen-specific IgG1 while very little IgG2 is produced. We therefore submit that experimental lung infection of cattle with M. bovis results in a Th2-skewed immune response.     

Haemonchus contortus (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response

Selection of resistant animals and host immunization have been proposed as alternative methods for the control of gastrointestinal nematode parasites. However, a better knowledge of the mechanisms involved in protective immunity against these parasites is required for the development of optimal strategies. In this study, 3 month old INRA 401 lambs (n = 81) were allocated into three groups: uninfected control, challenged either once (primary-infected animals) or twice (previously-infected animals) with 10,000 Haemonchus contortus L3. Uninfected control and challenged animals were sequentially sacrificed at 3, 7, 15 and 28 days post challenge. In both challenged groups, a clear Th2-oriented immune response was observed in the abomasal lymph node and mucosa. IL-4 and IL-13 mRNA over-expression, recruitment of eosinophils, mast cells and globule leukocytes and production of specific systemic IgG and mucosal IgA were observed earlier in previously-infected animals than in primary-infected ones. At 28 days post infection, no differences between intensities of these responses were observed between the challenged groups. Worm establishment rates were similar in previously-infected and primary-infected lambs. However, reductions of worm development, female fecundity and fecal egg output were observed in previously-infected sheep. We conclude that H. contortus infection in young INRA 401 lambs induced an unequivocal Th2 immune response resulting in the regulation of worm egg production without affecting their establishment.     

基因重排狂犬病病毒疫苗株免疫效果的初步研究

试验探究了基因重排狂犬病病毒(RV)弱毒疫苗株能否刺激小鼠产生RV抗体免疫球蛋白G(IgG),比较了其不同免疫方式、不同免疫剂量对小鼠抗体水平的影响及安全性评估。选取28只体重(20±3)g、35日龄左右的昆明系小白鼠,随机分为7组:第1组为低剂量口服组:复合佐剂50μL+病毒液50μL;第2组为中等剂量口服组:复合佐剂150μL+病毒液150μL;第3组为高剂量口服组:复合佐剂300μL+病毒液300μL;第4组为低剂量注射组:复合佐剂50μL+病毒液50μL;第5组为中等剂量注射组:复合佐剂150μL+病毒液150μL;第6组为高剂量注射组:复合佐剂300μL+病毒液300μL;第7组为口服对照组:复合佐剂300μL+SRV9亲本毒株300μL,分别于0、7、14d进行免疫,采用ELISA定量检测试剂盒对各免疫组小鼠的血清IgG水平进行检测,免疫期结束后取各免疫组小鼠的肝脏、肾脏、肺脏、脑组织制作病理组织切片对疫苗的安全性进行评估。结果显示,各免疫组均产生了RV抗体IgG;肌内注射免疫产生的抗体速度较口服免疫快,但肌内注射免疫会导致小鼠发病死亡;600μL口服免疫组产生的抗体水平显著高于100、300μL口服免疫组及亲本毒株SRV9对照组(P<0.05),说明适当加大口服免疫剂量会使口服免疫组产生的抗体水平增加,并且不会引起不良反应。病理组织学检查发现,各口服免疫组小鼠的肝脏、肾脏、肺脏、脑组织的形态结构较为正常,未发生病变,表明基因重排RV疫苗株口服免疫副作用小、安全性较好。本试验结果为研制新型口服疫苗开辟了新的方向。