几种佐剂对肌肉注射猪支原体肺炎活疫苗免疫刺激能力的增强作用研究

为增强现有猪支原体肺炎活疫苗的免疫刺激能力以实现肌肉注射免疫,向现有活疫苗中添加几种不同佐剂,考察其经肌肉免疫后机体的免疫应答情况。结果表明,免疫刺激复合物基质佐剂和左旋咪唑+壳聚糖混合佐剂能有效激发细胞免疫应答和体液免疫应答;左旋咪唑+黄芪多糖混合佐剂能增强疫苗的细胞免疫刺激能力,而对体液免疫无作用;皂角素佐剂未显示明显的免疫增强效果。本试验结果为肌肉注射猪支原体肺炎活疫苗的开发奠定了数据基础。     

蜂胶对猪细小病毒灭活疫苗佐剂作用的试验

68只豚鼠随机均分为4组,1³组分别免疫蜂胶、铝胶和油佐剂PPV疫苗,第4组为空白对照组,注射等量生理盐水。免疫后不同时间点采集血清分析特异性血凝抑制抗体效价评价蜂胶佐剂对免疫豚鼠体液免疫的影响,测定淋巴细胞增殖、IL-2和IL-4含量评价蜂胶佐剂对免疫豚鼠细胞免疫的影响。结果表明,蜂胶、油佐剂和铝胶3种佐剂均能提高豚鼠对PPV灭活疫苗的免疫应答能力,油佐剂提高血清HI抗体效果较好,其次为蜂胶佐剂,再次为铝胶佐剂。蜂胶佐剂促进T淋巴细胞增殖和提高IL-2、IL-4、IL-6和IFN-γ含量效果优于油佐剂和铝胶佐剂。结论:蜂胶能增强PPV灭活疫苗的免疫效果。     

黄芪多糖注射液对猪瘟耐热保护剂活疫苗在兔体内免疫效果的影响

试验旨在研究黄芪多糖(astragalus polysaccharide,APS)注射液作为猪瘟疫苗稀释液及黄芪多糖作为疫苗佐剂提供数据支持。用1%的黄芪多糖注射液、猪瘟疫苗专用稀释液、生理盐水3种不同猪瘟疫苗稀释液稀释兔源猪耐热保护剂活疫苗免疫兔体,比较免疫后不同时间段内血液中的淋巴细胞的活性及血清中的猪瘟抗体、白介素2(interleukin 2,IL-2)、白介素6(interleukin 6,IL-6)、γ干扰素(interferon γ,IFN-γ)、新蝶呤(neopterin,Npt)水平,综合评价3种不同猪瘟疫苗稀释液对兔源猪瘟耐热保护剂活疫苗在兔体内免疫效果的影响。结果表明,黄芪多糖能不同程度的提高抗体水平和多种细胞因子的含量。因此,黄芪多糖注射液可以作为猪瘟疫苗稀释液或改良现有猪瘟疫苗稀释液,黄芪多糖也可以作为猪瘟疫苗佐剂。     

Montanide~(TM) Gel 01 ST水性佐剂对猪乙型脑炎活疫苗免疫小鼠后的早期免疫保护效果的影响

为评估Gel 01 ST佐剂对猪乙型脑炎活疫苗早期免疫保护的影响,本研究以小鼠为动物模型,测定水性疫苗佐剂Montanide~(TM) Gel 01 ST(Gel 01 ST)稀释猪乙型脑炎活疫苗协同免疫小鼠后的攻毒保护情况。以固定病毒含量的不同浓度的Gel 01 ST佐剂与固定佐剂浓度下,不同病毒含量的猪乙型脑炎活疫苗以腹腔免疫小鼠后14 d进行攻毒保护试验,结果显示Gel 01 ST佐剂浓度为10%,病毒含量为10~(7.0)pfu/m L时分别可获得良好的保护效果;以10%Gel 01 ST佐剂稀释猪乙型脑炎活疫苗(病毒含量为10~(7.0)pfu/m L)免疫小鼠,于免疫后不同时间采集血清测定抗体,并于免疫后不同时间进行攻毒保护试验。结果显示Gel 01 ST佐剂稀释的活疫苗免疫小鼠后从第4 d开始其血清抗体水平明显高于无佐剂组(p0.01),且从第7 d开始可提供100%的攻毒保护效力。本研究结果表明,Gel 01 ST佐剂能很好的诱发免疫小鼠的体液免疫应答,增强猪乙型脑炎活疫苗的早期免疫保护。本研究为Gel 01 ST佐剂在动物活疫苗中的应用提供了参考。     

阳离子脂质体包裹CaG对猪瘟猪肺疫猪丹毒三联疫苗免疫效应的影响

对3组分别注射猪瘟猪肺疫猪丹毒三联疫苗（简称三联疫苗），CpG十三联疫苗，阳离子脂质体包裹CpG十三联疫苗的小鼠细胞和体液免疫反应进行了比较分析。结果显示，阳离子脂质体包裹CpG十三联疫苗免疫小鼠脾细胞增殖反应明显增强，白细胞介素-2（IL-2）诱生活性和免疫细胞数量，血液IgG含量较仅注射三联疫苗的小鼠显著增加。说明阳离子脂质体包裹CpG能增强小鼠对三联疫苗的体液和细胞免疫应答反应。使疫苗的免疫原性增强。     

黄芪多糖对猪瘟疫苗体液免疫和细胞免疫的影响

为查明黄芪多糖对猪瘟疫苗体液免疫IgG抗体与猪白细胞介素-4(IL-4)、猪干扰素-γ(IFN-γ)、猪肿瘤坏死因子α(TNF-α)三种细胞免疫因子的影响,选用黄芪多糖注射液直接稀释高效猪瘟活疫苗(细胞源)的方法进行了免疫试验。证实了黄芪多糖直接稀释猪瘟活疫苗不但可以提高猪瘟疫苗IgG抗体水平,还可以提高猪干扰素-γ(IFN-γ)、猪肿瘤坏死因子α(TNF-α)浓度,降低猪白细胞介素-4(IL-4)浓度,证明在使用猪瘟疫苗免疫防控中添加黄芪多糖可增强猪群免疫力,为控制猪瘟流行提供了依据。     

牛新孢子虫NcSAG1基因工程亚单位疫苗的免疫试验

为了解新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗对实验动物的免疫效果,本试验提取延边黄牛新孢子虫野毒株基因组DNA,用PCR技术扩增新孢子虫NcSAG1表面蛋白基因,并在大肠杆菌中进行原核表达,将Western-blot鉴定具有免疫活性的重组蛋白与弗氏佐剂混合制备NcSAG1基因工程亚单位疫苗,免疫BALB/c小鼠,应用间接ELISA方法测定体液免疫水平,应用流式细胞技术测定细胞免疫水平,以此评价疫苗对实验动物的免疫应答反应。结果显示,制备的NcSAG1基因工程亚单位疫苗免疫BALB/c小鼠后,在三免后第3天时,检测抗体的OD450nm值达0.688;CD4+/CD8+值达3.650,均显著高于重组蛋白免疫组和PBS对照组,说明制备的基因工程亚单位疫苗能够提高实验动物的体液免疫和细胞免疫水平。本试验为牛新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗的深入研究奠定了基础。     

纳米铝胶佐剂增强猪细小病毒灭活疫苗猪体免疫的效果

将20头35日龄断奶仔猪随机分为4组,将制备的纳米铝胶佐剂猪细小病毒(Porcine parvovirus,PPV)灭活疫苗肌肉注射免疫接种,并以常规铝胶佐剂PPV灭活疫苗组、市售PPV油乳剂灭活疫苗组为疫苗对照,生理盐水组做阴性对照,应用HI和ELISA检测接种猪的体液免疫水平,流式细胞仪检测免疫猪的细胞免疫水平。体液免疫检测结果显示,PPV纳米铝胶佐剂疫苗组能显著提高机体产生抗体的速度,在首免后第2周产生有效保护抗体水平显著高于油乳剂疫苗和常规铝胶佐剂疫苗组(P0.05),在第3周其抗体水平达到峰值;细胞免疫检测结果显示,3种疫苗均能诱导机体产生细胞免疫应答,但各组之间差异不显著(P0.05)。结果表明,以纳米铝胶为佐剂制备的猪细小病毒灭活疫苗,能显著提高机体的免疫水平,并较早刺激机体产生抗体,具有较好的免疫保护效果。     

M2e合成肽流感通用疫苗的研究

M2蛋白是甲型流感病毒的跨膜蛋白,其优点为不易变异,且N端24个氨基酸组成的胞外区(M2e)十分保守,适合作为通用流感疫苗的靶标.因此本实验将4个拷贝的M2e通过赖氨酸与通用Th细胞表位偶联获得M2e分支肽(M2e-MAP),添加弗氏佐剂制备成合成肽疫苗免疫小鼠,评价其免疫原性及异源攻毒保护效力.ELISA实验结果表明,该疫苗能刺激机体产生高水平的抗M2e特异性IgG抗体；细胞因子IL-4的检测进一步说明了其能诱导产生高的体液免疫应答水平,ELISPOT实验从IL-4和IFN-γ两个方面分别说明了该疫苗不仅可以诱导高的体液免疫应答,而且能刺激机体产生高水平的细胞免疫应答.对免疫小鼠攻H9N2,通过肺病毒分离滴定和肺组织病理切片等方法检测攻毒保护效果,结果均表明,M2e分支肽疫苗能对异源病毒的攻击产生较好的保护效力.上述结果为流感通用疫苗的研究提供了一种参考思路.     

蓝耳病疫苗对猪瘟弱毒疫苗免疫效果的影响

为了探明猪蓝耳病阳性场猪蓝病TJM-F92株弱毒疫苗是否对猪瘟疫苗免疫效果存在影响,按照不同的免疫模式将试验动物分为7组,在注射疫苗前(第0周)、注射疫苗后(第2,4,6周)采集血样,通过酶联免疫吸附试验(ELISA)检测各组试验猪猪瘟、蓝耳病抗体水平,用流式细胞术检测各组猪的淋巴细胞亚群水平,从体液免疫和细胞免疫两个方面分别验证猪蓝耳病阳性场猪蓝耳病TJM-F92株弱毒疫苗对猪瘟疫苗的免疫效果是否有干扰作用。结果表明:猪蓝耳病阳性猪场蓝耳病弱毒疫苗对猪瘟疫苗体液免疫和细胞免疫应答干扰作用不明显(P0.05),说明在蓝耳病阳性猪场猪蓝耳病TJM-F92株弱毒疫苗可以和猪瘟疫苗同时免疫。     

Preparation of Adjuvant Diluent for Live Vaccine against Classical Swine Fever and Evaluation of its Immune Effects

The aim of this study was to develop a novel oil-in-water (O/W) emulsion as adjuvant diluents (AD) for live vaccine against classical swine fever (CSF) that could effectively enhance the immune effect of vaccine.The AD was prepared by high-pressure homogenization technique.Formulations and preparation parameters were optimized with response surface design.Its stability, particle size, polydispersity (PDI) and Zeta potential were characterized.The humoral immune response and cellular immune response of the AD were evaluated with BALB/c mice by intramuscular injection.The particle size of the AD prepared by optimized formulation and parameters was 100.4 nm, PDI was 0.147, and Zeta potential was —28.7 mV.The experiment results showed that the AD had good stability.The AD was inoculated combined with live vaccine against CSF into BALB/c mice by intramuscular injection.The results showed that the live vaccine against CSF specific immune responses could be evoked in mice by co-inoculation with AD and vaccine.The cellular immune response levels in co-inoculated groups were significantly higher than control group (P<0.05), with obvious phenomena of higher levels of IFN-γ, IL-6 and IL-4 in serum.The result revealed that cellular immune capability significantly improved with the AD.The results strongly revealed that cellular immune capability significantly improved by introducing AD for effective immune-adjuvant for live vaccine against CSF.     

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.     

Immune response in mice and cattle after immunization with a Boophilus microplus DNA vaccine containing bm86 gene

Plasmid pBMC2 encoding antigen Bm86 from a Colombian strain of cattle tick Boophilus microplus, was used for DNA-mediated immunization of BALB/c mice, employing doses of 10 and 50microg, delivered by intradermic and intramuscular routes. Anti-Bm86 antibody levels were significantly higher compared to control mice treated with PBS. In the evaluation of immunoglobulin isotypes, significant levels of IgG2a and IgG2b were observed in mice immunized with 50microg of pBMC2. Measurement of interleukine (IL) levels (IL-4, IL-5, IL-12(p40)) and interferon-gamma (IFN-gamma) in the sera of mice immunized with pBMC2 indicated high levels of IL-4 and IL-5, although there were also significant levels of IFN-gamma. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN-gamma secretion. In all trials, mice injected intramuscularly with 50microg of pBMC2 presented the highest immune response. Moreover, cattle immunized with this DNA vaccine showed antibody production significantly different to the negative control. In conclusion, these results suggest the potential of DNA immunization with pBMC2 to induce humoral and cellular immune responses against B. microplus.     

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.     

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.     

Assessment in mice of vapA-DNA vaccination against Rhodococcus equi infection

There is a need to produce a vaccine against Rhodococcus equi pneumonia in foals in which immunity against infection is largely based on a type 1, cell-mediated, immune response. The VapA protein of the virulence plasmid of R. equi is highly immunogenic. To assess the potential of vapA-DNA to produce immunity, C57BL/6 and BALB/c mice were immunized with a DNA vaccine constructed from vapA incorporated into pcDNA3.1. The plasmid construct expressed VapA in a COS-7 cell line. Intramuscular immunization of mice resulted in enhanced clearance of R. equi from the liver of intravenously challenged mice compared to non-immunized controls. This effect was more marked when pORF-IL-12, a plasmid expressing murine IL12, was included with the vaccine. Antibody developed to VapA, with an IgG2a response being more marked in mice immunized with pcDNA-vapA than in non-immunized or in mice immunized with the mixed vapA and IL-12 plasmid constructs. In conclusion, this study has shown for the first time that DNA immunization with vapA enhances the immune responses of mice against R. equi infection, that the IgG subisotype response is consistent with a type 1-based immune response, and that this can be enhanced by injection of the IL-12 gene.     

The adjuvant effect of a single dose of interleukin-12 on murine immune responses to live or killed Brucella abortus strain RB51.

This study was designed to determine if a single 0.5 microg administration of recombinant murine interleukin-12 (IL-12) would influence immune responses of mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). Mice were vaccinated intraperitoneally with 5 x 10(8) cfu of live or gamma-irradiated SRB51 bacteria alone, or in combination with 0.5 microg of IL-12. Control mice received saline or 0.5 microg of IL-12. Serologic responses and spleen weights after vaccination were greater in mice vaccinated with live SRB51 when compared to mice receiving killed SRB51 or control treatments. Administration of a single dose of IL-12 as a vaccine adjuvant did not influence immune responses, clearance of live SRB51, or resistance against B. abortus strain 2308 (S2308) challenge. The results of this study suggest that a single administration of 0.5 microg of IL-12 at the time of vaccination does not have significant adjuvant effects on vaccine-induced immune responses against live or killed Brucella.     

法氏囊提取液对新城疫活疫苗免疫效力的影响

本文对鸡法氏囊提取液与ND活疫苗的使用方法进行了分析。通过血凝抑制抗体滴度的测定结果表明,预先接种鸡法氏囊提取液时,经肌肉注射、口服和点眼滴鼻接种方式均能提高ND疫苗免疫鸡的抗体产生能力,且经肌肉注射法最佳。但是,在整个试验期间,法氏囊提取液只能增强鸡群对ND活疫苗的早期免疫应答,而法氏囊提取液与ND活疫苗同时使用时更能刺激免疫鸡群的早期抗体产生水平。     

Simultaneous vaccination of piglets against foot-and-mouth disease and classical swine fever

Six-week-old piglets, born of unvaccinated sows, were vaccinated against foot-and-mouth disease (FMD) with a trivalent, inactivated vaccine containing an adjuvant or vaccinated against classical swine fever (CSF) with a live attenuated vaccine or against both diseases simultaneously at two different sites. The antibody response to the FMD vaccine was not significantly influenced by the simultaneous vaccination against CSF. FMD vaccine administered simultaneously with the CSF vaccine produced a significantly higher antibody response to CSF than occurred with CSF vaccination only.