表达H1N1、H3N2猪流感病毒HA基因重组质粒在小鼠的免疫效力

应用已构建的真核表达质粒pCI-H1-HA、pCAGGS-H1-HA、pCI-H3-HA和pCAGGS-H3-HA作为DNA疫苗，利用BALB／c小鼠进行免疫保护试验，通过测定不同免疫期HI抗体滴度、分析攻毒后BALB／c小鼠体重变化及肺脏病毒含量，评价DNA疫苗的免疫效力。结果表明：构建的DNA疫苗均可诱导小鼠产生免疫力；BALB／c小鼠体重变化统计学分析显示，免疫组与对照组差异极显著（P〈0．01），pCAGGS表达载体构建的DNA疫苗免疫效果优于pCI表达载体构建的DNA疫苗（P〈0．05）。     

Evaluation of immune responses induced by SAG1 and MIC3 vaccine cocktails against Toxoplasma gondii

The aim of this study was to evaluate the immune responses of a SAG1 and MIC3 vaccine cocktail in BALB/c mice. Ninety-six BALB/c mice were randomly divided into eight groups, including three plasmid DNA vaccine groups (pcDNA-MIC3, pcDNA-SAG1, pcDNA-MIC3+pcDNA-SAG1), three recombinant pseudotype baculovirus vaccine groups (BV-G-MIC3, BV-G-SAG1, BV-G-SAG1+BV-G-MIC3) and two control groups (PBS and BV-G-EGFP). All groups were immunized intramuscularly twice at three-week intervals. The production of anti-Toxoplasma gondii lysate antigen (TLA) antibodies, lymphoproliferation, levels of IFN-γ, IL-4 and IL-10 and the survival time were monitored after vaccination. The results showed that immunization of BALB/c mice with MIC3 and SAG1 vaccines stimulated both the cellular and humoral immune responses with the production of anti-T. gondii TLA antibodies. The vaccine cocktails of pcDNA-MIC3+pcDNA-SAG1 or BV-G-SAG1+BV-G-MIC3 induced significantly higher immunogenicity than a single-gene vaccine (P<0.05). Splenocytes from the immunized mice significantly proliferated in response to the TLA and released interferon (IFN)-γ (P<0.05). However, the levels of IL-4 and IL-10 in the sera of the immunized mice were not significantly different from those of the controls (P>0.05). Immunization with the vaccine cocktail (BV-G-SAG1+BV-G-MIC3) in mice significantly prolonged survival (50%; P<0.05) against a lethal challenge of T. gondii (RH tachyzoites), while all mice in the other immunized groups and control groups died within 20 and 4 days post-infection, respectively. Furthermore, the recombinant pseudotype baculovirus vaccines induced better immunogenicity than the plasmid DNA vaccines (P<0.05). These results suggest that an excellent vector-mediated vaccine cocktail strategy might be used to develop a new generation of vaccines against T. gondii infection.     

修饰的ORF5基因增强猪繁殖与呼吸综合征DNA疫苗的体液免疫

为了提高猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因DNA疫苗的免疫效力，将通用型辅助性T淋巴细胞表位(PADRE)插入ORF5的中和表位和覆盖表位间，获得修饰后的ORF5基因ORF5M。在此基础上，进一步构建了ORF5M的真核表达质粒pCl-52M。间接免疫荧光证实体外表达后，免疫BALB／c小鼠，检测免疫后的ELISA抗体和中和抗体，并与未经修饰的ORF5基因真核表达质粒pCl-52进行比较。结果表明，修饰后的DNA疫苗pCl-52M诱导的ELISA抗体和中和抗体均明显优于未经修饰的DNA疫苗pCl-52，是一种具有良好开发前景的PRRS新型疫苗。     

Use of Mycobacterium avium subsp. paratuberculosis specific coding sequences for serodiagnosis of bovine paratuberculosis

In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commercial seroassay (Pourquier test) yielded a sensitivity of 72% for a specificity of only 92%.     

Antibody and skin-test responses of sheep vaccinated against Johne's Disease

Current vaccines against Mycobacterium avium subsp. paratuberculosis (MAP, Johne's Disease) may cause animals to react positively when tested for Mycobacterium bovis (Bovis). Therefore, the effects of vaccination on MAP serum Ab and skin-test responses to MAP and Bovis PPD were compared in 25 ewes vaccinated against MAP with 24 control ewes in an infected flock 3 years post-vaccination. MAP-specific Ab levels were higher (P<0.001) in vaccinated ewes than in control ewes. All increases in skinfold-thickness from 0 to 48h were greater (P<0.0001) than zero while increases in skinfold-thickness from 48 to 72h were greater (P<0.05) than zero for Johnin but not for Bovis PPD. The Vaccine x PPD x Time interaction for skinfold-thickness was significant (P<0.001) with greater increases to Johnin than to Bovis, but with much greater increases in vaccinated ewes. These data suggest that administration of vaccines against MAP developed from whole organisms increase the likelihood that animals will be classified as "responders" to a Bovis screening test and negative by the follow-up comparative cervical tuberculin test, but they also show that vaccination initiates both humoral and cell-mediated MAP-specific responses.     

DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza

The influenza virus vaccines that are commercially-available for humans, horses and pigs in the United States are inactivated, whole-virus or subunit vaccines. While these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. DNA vaccines are a novel alternative to conventional vaccination strategies, and offer many of the potential benefits of live virus vaccines without their risks. In particular, because immunogens are synthesized de novo within DNA transfected cells, antigen can be presented by MHC class I and II molecules, resulting in stimulation of both humoral and cellular immune responses. Influenza virus has been used extensively as a model pathogen in DNA vaccine studies in mice, chickens, ferrets, pigs, horses and non-human primates, and clinical trials of DNA-based influenza virus vaccines are underway in humans. Our studies have focused on gene gun delivery of DNA vaccines against equine and swine influenza viruses in mice, ponies and pigs, including studies employing co-administration of interleukin-6 DNA as an approach for modulating and adjuvanting influenza virus hemagglutinin-specific immune responses. The results indicate that gene gun administration of plasmids encoding hemagglutinin genes from influenza viruses is an effective method for priming and/or inducing virus-specific immune responses, and for providing partial to complete protection from challenge infection in mice, horses and pigs. In addition, studies of interleukin-6 DNA co-administration in mice clearly demonstrate the potential for this approach to enhance vaccine efficacy and protection.     

Gene immunization of mice with plasmid DNA expressing rabies virus glycoprotein

Gene immunization can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses. We constructed plasmid vectors expressing the full-length Vnukovo-32 rabies virus glycoprotein G under the control of CMV IE promoter and enhancer, adenovirus tripartite leader sequences and poly A signal of SV40. The gene vaccines were evaluated for the ability to elicit neutralizing antibodies and to protect BALB/c mice against lethal rabies virus challenge. First, mice were injected intramuscularly (i.m.) into the left hind leg and by the intradermoplantar (i.d.p.) route with equal amounts of plasmid DNA (0.25-0.1 mg). Two weeks later, immunization was boosted with an additional dose of the DNA. The immunized mice were challenged by intracerebral (i.c.) inoculation of CVS-27 (10-50 LD50) rabies virus. All mice produced anti-rabies virus neutralizing antibodies with a titre of > or = 1:45 after immunization with 0.1-0.4 mg of DNA. In challenge experiments, 83 to 91.6% protection was observed. These results confirm that a DNA vaccine could be a simple and effective solution for preventing the spread of rabies.     

Prime-boost immunization with HA/C3d DNA followed by a recombinant pseudorabies virus boost enhanced protective immunity against H3N2 swine influenza virus in mice

DNA and recombinant virus vaccines against swine influenza virus (SIV) have been pursued with promising results, but induce poor immunogenicity. This study evaluated the effects of a vaccine regimen in mice including priming with three DNA vaccines expressing soluble HA (sHA), complete HA (tmHA), or sHA fused with three copies murine C3d (sHA-mC3d3) and boosting with recombinant pseudorabies virus expressing HA (rPRV-HA). Immune responses were monitored by ELISA, HI assays, and virus neutralization. Protective efficacy was evaluated by virus isolation from lungs, distribution in tissues, and pathology following challenge with H3N2 SIV. Priming with sHA-mC3d3 and boosting with rPRV-HA induced higher levels of HA-specific antibodies and yielded the most effective protection. This finding implied that priming with a DNA vaccine expressing C3d fused with antigen and boosting with a recombinant vector vaccine is an effective way to induce protective humoral immunity and prevent some infectious diseases.     

马流感病毒血凝素基因甲病毒复制子重组表达质粒的构建及其免疫效力评价

为评价马流感病毒(EIV)HA基因核酸免疫效果,本研究以甲病毒复制子载体pSFV1CS分别构建了表达EIV H3N8亚型的美洲型和欧洲型HA基因的重组真核表达质粒。并将其转染293T细胞,经间接免疫荧光鉴定表明HA基因获得表达;以重组质粒免疫的BALB/c鼠能够检测到特异性抗体产生,而且HI抗体水平持续升高,同时小鼠体内IFN-γ、IL-4分泌水平也有所升高。攻毒后小鼠表现轻度临床症状,但病毒分离和RT-PCR均未检测到病毒。上述结果表明,该重组质粒pSFV1CS-EIV-HA具有良好的免疫原性并且可以诱导免疫动物产生较高免疫应答的能力。     

Evaluation of the immunogenicity of Mycobacterium avium subsp. paratuberculosis (MAP) stress-associated recombinant proteins

The aim of this study was to assess the humoral immune responses in MAP-infected and uninfected sheep against 27 MAP stress-associated recombinant proteins that were regulated in in vitro models of physiological stress. These include evaluation of 5 proteins, which were previously reported by Gumber et al. (2009b), using serum samples from sheep with a wide range of disease stages. For purification of recombinant his-tagged proteins expressed as an insoluble protein, on-column refolding purification was applied as well as one-step denaturing purification. All purifications together resulted in a total of 48 recombinant antigen preparations. In antibody ELISA tests, 23 of these, representing 18 MAP proteins, showed significant differences in responses between infected and uninfected sheep. Recombinant antigen preparations MAP2281c, MAP3555 (refolded form), and MAP0711c (refolded form) when incorporated in an ELISA, had similar sensitivity to a commercial antibody ELISA test at the cutpoint of 90% specificity, and showed relatively high values in receiver operating characteristic (ROC) curve analysis. However, as some of the sera from uninfected sheep also reacted to recombinant antigens, further development of the assays is necessary prior to practical application. Compared to the commercial antibody ELISA, MAP0593c, MAP2281c, MAP2411, MAP3555, and MAP3200 detected more infected sheep with a lower grade of lesion, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected sheep at an early stage of infection. This is consistent with the hypothesis of latency or dormancy in subclinical mycobacterial infection.     

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both T_h1 and T_h2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases.     

禽分枝杆菌副结核亚种hsp65与鹅副黏病毒HN融合基因真核表达载体的构建与表达

为探索禽分枝杆菌副结核亚种（MAP）的热休克蛋白65（hsp65）对鹅副黏病毒（GPMV）血凝素神经氨酸酶（HN）的分子佐剂作用及构建GPMVDNA疫苗,针对GPMV的HN基因和MAP的hsp65基因设计引物,PCR克隆扩增两个基因,分别将二者先后与真核表达载体pVAXl连接,构建了pVAXl-HN、pVAXl-hsp65-HN,并通过PCR、酶切和序列鉴定所获重组质粒;应用脂质体法将其转染至Marc-145细胞,RT-PCR和间接免疫荧光试验证实hsp65和HN在Marc-145细胞中获得了表达。本研究为制备新型GPMVDNA疫苗及探索hsp65在DNA疫苗中的应用奠定了基础。     

DNA vaccines for veterinary medicine

DNA vaccination represents one of the most recent novel approaches to vaccine development. Experimentally, DNA vaccines induce a broad range of long lasting immune responses including humoral and cell-mediated immunity against infectious diseases in humans and animals. Furthermore, DNA vaccines are potentially useful for the treatment of autoimmune diseases or cancer. However, most information on the efficacy of DNA vaccines has been generated in mice and studies in larger animals are limited. In this review, the potential application of DNA vaccines in livestock and pet animals are discussed. The principle of this new technology, its potency and future perspectives for use in veterinary medicine will be outlined.     

Immune responses of immunocompetent and immunocompromised mice experimentally infected with Bartonella henselae

The aim of this study is to understand host immune responses in immunocompetent and immunocompromised mice against Bartonella henselae infection. BALB/c and nude (BALB/c nu/nu) mice were inoculated intraperitoneally with 10(8) colony forming units of B. henselae (Houston-1 strain). Blood, brain, liver, spleen, kidney and bone marrow samples were collected 0, 3, 7, 14, 21 and 28 days after infection and submitted to bacteriological, serological and genetical examinations. B. henselae was isolated only from the liver 3 days after infection. DNA of the inoculums was detected by polymerase chain reaction from blood, liver, and spleen samples collected from BALB/c and blood from nude mice 3 and 7 days after infection. No bacterial DNA was detected from both BALB/c and nude mice thereafter during 4 weeks observation periods. These results indicate that the T-cell may not participate in the effective elimination of the organisms from mice. In addition, western blot analysis revealed that the antigens of 27.3- and 31.5-kDa reacted with IgM antibodies from the blood of BALB/c and nude mice after 3 days of infection, suggesting that these antigens were recognized by thymus-independent mechanism. Furthermore the antigens were detected from the culture-supernatants of B. henselae, indicating that these antigens were secreted from the organisms.     

密码子优化的PRRSV GP5基因DNA疫苗在鼠体内免疫效力的评价

为了提高表达含有猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）GP5基因DNA疫苗的死疫效应,本研究通过人工合成的方法将GP5基因的密码子改造为猪体嗜好的密码子,并将其作为免疫原基因,连同野生型GP5基因,分别插入表达载体pIRES1neo中,构建重组质粒pIR-optiGP5和pIR-GP5.将这两种质粒分别转染293T细胞,转染后48 h,采用间接免疫荧光和Western blot方法检测GP5基因的体外表达情况,结果两种检测方法显示重组质粒pIR-optiGP5的蛋白表达量明显多于pIR-GP5。为了评价DNA免疫质粒pIR-optiGP5的免疫效果,选取30只6～8周龄雌性BALB/c小鼠,将pIRES1neo、pIR-GP5、pIR-optiGP5分别以100μg/只剂量,进行肌肉多点注射,共免疫3次,每次间隔2周,同时,利用间接免疫荧光技术、流式细胞技术、淋巴细胞特异性增殖试验等方法检测其体液免疫和细胞免疫水平。结果显示：DNA免疫质粒pIR-optiGP5在二免后即可检测到荧光抗体,而pIR-GP5在三免后才能检测到;且发现用pIR-optiGP5质粒免疫小鼠,其CD4^＋、CD8^＋ T淋巴细胞百分数及特异性刺激指数均高于pIR-GP5免疫小鼠,推测经过密码子优化的GP5基因其蛋白在小鼠体内得到了较高水平的表达,并诱导了较强的免疫应答,这为进一步研究和设计有效的PRRSV DNA疫苗提供了新的思路。     

Immune response of BALB/c mouse immunized with recombinant MSPs proteins of Anaplasma marginale binding to immunostimulant complex (ISCOM)

Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60-105kDa), rMSP1b (100kDa), rMSP4 (47kDa), and rMSP5 (29kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.     

A comparison of DNA vaccines expressing the 45W, 18k and 16k host-protective antigens of Taenia ovis in mice and sheep

The immunogenicity of DNA vaccines encoding three different Taenia ovis host-protective antigens was compared in mice and sheep. DNA vaccines encoding the 45W, 18k and 16k antigens of T. ovis were constructed. The ability of DNA vaccines encoding the 45W and 18k genes to express antigen was confirmed by Western blotting of transfected Cos-7 cells. BALB/c mice were vaccinated intramuscularly with 45W, 18k or 16k DNA vaccines and the humoral immune response analysed by ELISA. DNA vaccines expressing 45W, 18k or 16k antigen were immunogenic in mice and generated significant titres of antigen-specific antibody. Intramuscular vaccination of outbred sheep with the T. ovis DNA vaccines generated significantly lower titres of 45W-specific antibody and failed to generate 18k or 16k-specific antibody. The findings of this study show that each of the three T. ovis host-protective antigens are amenable to delivery via DNA vaccines, and that the parameters governing the efficacy of DNA vaccines in sheep require further investigation.     

Protection of mice against Chlamydophila abortus infection with a bacteriophage-mediated DNA vaccine expressing the major outer membrane protein

A bacteriophage-delivered DNA vaccine against Chlamydophila abortus was constructed by cloning a eukaryotic cassette containing the ompA gene (which expresses the Major Outer Membrane Protein) into a bacteriophage lambda vector. Four groups, each of 20 BALB/c mice were inoculated separately with the phage vaccine, a conventional DNA vaccine based on the same ompA expression cassette, a live attenuated vaccine (strain 1B) or the empty phage vector. The phage and DNA vaccines and empty phage vector were administered intramuscularly on days 0, 14 and 28; the attenuated vaccine was given once on day 0. Half the animals in each group were challenged on day 42 by intraperitoneal injection of live C. abortus and sacrificed on day 49. Phage-vaccinated mice developed moderate antibody levels against C. abortus and yielded higher levels of IFN-γ and IL-2 compared with the attenuated live vaccine group. Clearance of chlamydiae from spleens was significantly better in the attenuated vaccine group compared with the phage vaccine group, while both groups were significantly superior to the DNA vaccine and control groups (p<0.01). Although levels of protection in the mouse model were lower in phage-vaccinated animals, than in 1B vaccinated animals, phage vaccines offer several other advantages, such as easier handling and safety, potentially cheaper production and no chance of reversion to virulence. Although these are preliminary results in a model system, it is possible that with further optimisation immunization with phage vaccines may provide a novel way to improve protection against C. abortus infection and trials in large animals are currently being initiated.     

捻转血矛线虫Hc38基因DNA疫苗对绵羊免疫保护性效果评价

为了研究基因疫苗对绵羊的免疫保护效果,本研究构建了捻转血矛线虫(H.contortus)Hc38基因DNA疫苗.将H.contortus Hc38基因保守结构域克隆到真核表达载体pcDNA3.1中,免疫鼠8d后用RT-PCR检测到该疫苗在鼠肌肉组织中进行了转录.将纯化的DNA疫苗免疫绵羊后,用western blot和ELISA方法检测疫苗在绵羊体内的翻译和诱导IgG的产生.二免后2周用10 000条H.contortus第3期幼虫攻击实验动物,检测绵羊粪便虫卵排出、成虫数量等免疫保护性指标.该H.contortus Hc38 DNA疫苗与对照组比较,免疫组绵羊排出虫卵减少66.6%、成虫减少33.1%.特别值得注意的是免疫组的静脉注射方式产生抗体最高,相应羊的虫卵数和成虫数低.本实验证明Hc38基因DNA疫苗对绵羊虫卵及成虫发育具有明显的抑制作用.