弓形虫鸡尾酒DNA疫苗的免疫保护作用
|
摘要
弓形虫疫苗研制经历了全虫疫苗、虫体特异组分疫苗、基因工程疫苗、DNA疫苗4个发展过程。但到目前为至,仅有的疫苗是一种活的速殖子S48的减毒株,作为用于羊弓形虫病的商品化疫苗,并没有可用于人的弓形虫疫苗。越来越多的证据表明弓形虫生活期特异的免疫只能诱导局限于该期的保护,较多研究认为多抗原DNA疫苗提供较好的抗弓形虫病保护并且优于单一抗原DNA疫苗。组合多种弓形虫抗原基因的DNA疫苗成为一种主要的研究策略。免疫佐剂的加入可能显著增强原有组合疫苗的免疫效果。当前已有被研究应用为弓形虫DNA疫苗的候选抗原基因主要有:弓形虫膜表面抗原(SAG)、致密颗粒抗原(GRA)、棒状体蛋白(ROP)、微线体蛋白等。在这个思路下,我们构建了包括候选抗原基因GRA1、SAG1、ROP2、AMA1的DNA疫苗,并加入佐剂IL-12的质粒载体,以期取得较好的小鼠受弓形虫感染时的保护效果和免疫效应。
一、弓形虫GRA1、SAG1、ROP2、AMA1鸡尾酒DNA疫苗的构建和鉴定
根据本课题组先期的研究结果,弓形虫DNA疫苗候选基因GRA1和SAG1具有部分的抗弓形虫感染保护作用和免疫效应。他人有关研究认为候选基因ROP2及AMA1也具有这种作用。本研究成功构建了用于组合弓形虫鸡尾酒DNA疫苗的重组质粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1。成功构建用于检测蛋白表达的质粒pVAX1-ROP2His、pVAX1-AMA1His。
将pVAX1-ROP2His、pVAX1-AMA1His转染巨噬细胞后,用Western Blot方法检测到了蛋白的表达。pVAX1-GRA1、pVAX1-SAG1的蛋白表达能力先期本课题组已应用免疫细胞化学染色方法检测验证,本次所构建pVAX1-GRA1、pVAX1-SAG1序列与先期相应重组质粒序列完全一致。将美国Alexander Rakhmilevich博士惠赠的pNGVL3-mIL12重组质粒转染巨噬细胞后,用ELISA方法检测到了蛋白的表达。
二、重组质粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1、pNGVL3-mIL12的免疫效果
不同的鼠系对于弓形虫的敏感性不同,C3H、BALB/c、C57BL/6等小鼠是常用于弓形虫DNA疫苗研究的鼠系,C3H/He (H-2k)是一种对弓形虫感染中等易感的鼠系。本研究选用C3H/He (H-2k)小鼠作为免疫效果观察动物,期望能够观察到一种较长期的免疫保护存活的效果。
早期的弓形虫DNA疫苗研究中往往使用较大剂量的弓形虫进行免疫后小鼠受攻击保护效果的评价,较多的研究使用104及更高剂量的弓形虫RH株速殖子,有研究认为104的剂量较高。这些研究往往都较难以观察到存活时间较长的数据,即可以计算存活率的数据。近期来,弓形虫疫苗研究时,弓形虫的感染攻击剂量已从最早期的105降至104、500直至最新报道的使用50个弓形虫RH株滋养体,最初的延长生存时间变成了存活率指标。
Quan Liu等只用了50个弓形虫RH株的滋养体攻击BALB/c小鼠,BALB/c小鼠又是较C3H小鼠对弓形虫的抵抗力更强的鼠系,我们的研究使用500个弓形虫RH株速殖子的攻击剂量,本研究结果C3H小鼠受保护的存活率不如最近期的Quan Liu等的研究,可能弓形虫攻击剂量仍然较高。
本次研究构建的DNA疫苗pVAX1 - GRA1 + SAG1 + ROP2 + AMA1实验组在C3H小鼠中取得了较好的保护效果,其受弓形虫攻击时生存时间较较少基因pVAX1-ROP2+AMA1组生存时间延长。该种保护又由于IL-12免疫佐剂的应用得到了提高,以弓形虫RH株攻击时观察到了生存时间的较大延长,该实验组10只小鼠受弓形虫攻击时观察到了1只小鼠存活达30天以上。
经鸡尾酒DNA疫苗免疫小鼠血清抗弓形虫(TLA)IgG高于对照组,加免疫佐剂pNGVL3-mIL12组检测到IL-12水平的显著升高,实验组pVAX1- ROP2+AMA1的IFN-γ水平高于pVAX1对照组,更多基因组又较少基因组的IFN-γ水平升高,IL-12佐剂递次诱导了更高IFN-γ水平。实验组与对照组之间的IL-4水平没有显著差异。实验组均较pVAX1对照组淋巴细胞增殖反应更高。pVAX1-GRA1+SAG1+ROP2+AMA1+pNGVL3-mIL12基因免疫组CD4+/CD8+淋巴细胞比值降低。说明该鸡尾酒DNA疫苗诱导了C3H小鼠的特异性免疫应答。
结果表明,该种鸡尾酒DNA疫苗具有良好的免疫保护性,复合免疫的方式增强了免疫效果,这种效果可因免疫佐剂的使用而得到递次提高。
Research of vaccines against Toxoplasma gondii experiences four stages: including whole protozoan body vaccine, parasite-specific composition vaccine, genetic engineering vaccine and DNA vaccine. However, an attenuated T. gondii strain S48 of tachyzoite stage is the only vaccine which has been commercialized for sheep toxoplasmosis and until now there is no vaccine against T. gondii available for human. More and more evidences had showed vaccination with stage-specific antigens can only lead to stage-limited protection and it was also reported multi-antigenic vaccine was more effective than single-antigenic vaccine. Therefore, it has been a trend to develop multi-antigenic vaccines against T. gondii. Moreover, the addition of adjuvant may play an important part in immune protection of the combined multi-antigenic vaccines. At present, the main vaccine candidates are as follows: T. gondii main surface antigens, SAGs; Dense Granule Antigens, GRAs; rhoptry proteins, ROPs; micronemes and so on. In the study, we constructed a combined multi-antigenic DNA vaccine containing GRA1, SAG1, ROP2 and AMA1 which use IL-12 plasmid as an adjuvant to enhance the immune protection.
1. Construction and identification of plasmids GRA1, SAG1, ROP2 and AMA1.
According to our previous study, DNA vaccines GRA1 and SAG1 provide partial immune protection against T. gondii infection. And it is also reported the candidate vaccine genes ROP2 and AMA1 have the same effects. In this study, we constructed recombinant plasmids pVAX1-GRA1, pVAX1-SAG1, pVAX1-ROP2 and pVAX1-AMA1 used for cocktail DNA vaccine, and recombinant expression plasmids pVAX1-ROP2His and pVAX1-AMA1His.
After transiently transfected into macrophages cells, the expressions of pVAX1-ROP2His and pVAX1-AMA1His proteins were assayed by Western blot. Our members have previously proved the proteins expressions of pVAX1-GRA1 and pVAX1-SAG1 by immunohistochemistry method. And the sequences of new recombinant plasmids pVAX1-GRA1 and pVAX1-SAG1 we constructed were in consistent with the past ones. After transiently transfected into macrophages cells, the protein expression of pNGVL3-mIL12 was confirmed by ELISA method. pNGVL3-mIL12 recombinant plasmid is the gift of Ph.D Alexander Rakhmilevich.
2. Evaluation of immune protection provided by plasmids pVAX1-GRA1, pVAX1-SAG1, pVAX1-ROP2, pVAX1-AMA1 and pNGVL3-mIL12.
Different strains of inbred mice have different levels of susceptibility to T. gondii caused mortality. The C3H, BALB/c, C57BL/6 etc. were the most frequently used mice for evaluation the effect of DNA vaccines against T. gondii. Because C3H/He (H-2k) are medium susceptible to T. gondii, it is expected to able to evaluate a long-term protection against T. gondii.
In early experiments, mice were often challenged with high dose of T. gondii to determine protective effect of DNA vaccination, such as 104 or more of tachyzoites stage of T. gondii RH strain. And 104 tachyzoites has been considered a too high dose. These researches were always difficult to get ideal data, for instance survival rate. Challenge dose has been decreased from 105 to 104, then 500, finally 50 tachyzoites of T. gondii RH strain as recently reported. And prolongation of the survival time also has been replaced by the index of survival rate.
The C3H mice were challenged in our research with 500 tachyzoites of T. gondii RH strain while BALB/c mice in Quan Liu et al.’s research was challenged 50 trophozoites. And that BALB/c mouse is more resistant to T. gondii than C3H mouse. Compared to Quan Liu et al.’s reports, lower survival rate was obtained in our research. It was maybe an influencing factor that the challenge dose of DNA vaccinated mice in our research was still too high.
In the study, the mice of the group immunized with pVAX1 - GRA1 + SAG1 + ROP2 + AMA1 lived longer than the group immunized with pVAX1 - ROP2 + AMA1, which demonstrated that the first group produced higher immune protection compared to the other. Moreover, the co-immunization with IL-12 adjuvant enhanced the immune protection and prolonged survival time significantly. Even in the experimental group, one mouse among ten survived more than 30 days.
The sera anti-TLA (Toxoplasma lysate antigen) IgG levels of the groups of mice immunized by the cocktail DNA vaccines were higher than the control one’s. The IFN-γlevel of the group pVAX1-ROP2+AMA1 was higher than the pVAX1 control group, more genes group again elevate the IFN-γlevel, and IL-12 adjuvant still more elevate the IFN-γlevel. IL-4 levels of the experimental groups were no significant difference than the control group. The lymphocyte proliferation reactions of all experimental groups were higher than the pVAX1 control group. The ratio of CD4+/CD8+ T lymphocyte of pVAX1-GRA1+SAG1+ROP2+AMA1 + pNGVL3- mIL12 immunized group decreased. These demonstrated that the cocktail DNA vaccine induced specific immune response in C3H mice.
The results confirmed the cocktail DNA vaccine has nice immune protection, combined immunity enhanced immune effect, and the effect was still elevated by the application of immune adjuvant.
一、弓形虫GRA1、SAG1、ROP2、AMA1鸡尾酒DNA疫苗的构建和鉴定
根据本课题组先期的研究结果,弓形虫DNA疫苗候选基因GRA1和SAG1具有部分的抗弓形虫感染保护作用和免疫效应。他人有关研究认为候选基因ROP2及AMA1也具有这种作用。本研究成功构建了用于组合弓形虫鸡尾酒DNA疫苗的重组质粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1。成功构建用于检测蛋白表达的质粒pVAX1-ROP2His、pVAX1-AMA1His。
将pVAX1-ROP2His、pVAX1-AMA1His转染巨噬细胞后,用Western Blot方法检测到了蛋白的表达。pVAX1-GRA1、pVAX1-SAG1的蛋白表达能力先期本课题组已应用免疫细胞化学染色方法检测验证,本次所构建pVAX1-GRA1、pVAX1-SAG1序列与先期相应重组质粒序列完全一致。将美国Alexander Rakhmilevich博士惠赠的pNGVL3-mIL12重组质粒转染巨噬细胞后,用ELISA方法检测到了蛋白的表达。
二、重组质粒pVAX1-GRA1、pVAX1-SAG1、pVAX1-ROP2、pVAX1-AMA1、pNGVL3-mIL12的免疫效果
不同的鼠系对于弓形虫的敏感性不同,C3H、BALB/c、C57BL/6等小鼠是常用于弓形虫DNA疫苗研究的鼠系,C3H/He (H-2k)是一种对弓形虫感染中等易感的鼠系。本研究选用C3H/He (H-2k)小鼠作为免疫效果观察动物,期望能够观察到一种较长期的免疫保护存活的效果。
早期的弓形虫DNA疫苗研究中往往使用较大剂量的弓形虫进行免疫后小鼠受攻击保护效果的评价,较多的研究使用104及更高剂量的弓形虫RH株速殖子,有研究认为104的剂量较高。这些研究往往都较难以观察到存活时间较长的数据,即可以计算存活率的数据。近期来,弓形虫疫苗研究时,弓形虫的感染攻击剂量已从最早期的105降至104、500直至最新报道的使用50个弓形虫RH株滋养体,最初的延长生存时间变成了存活率指标。
Quan Liu等只用了50个弓形虫RH株的滋养体攻击BALB/c小鼠,BALB/c小鼠又是较C3H小鼠对弓形虫的抵抗力更强的鼠系,我们的研究使用500个弓形虫RH株速殖子的攻击剂量,本研究结果C3H小鼠受保护的存活率不如最近期的Quan Liu等的研究,可能弓形虫攻击剂量仍然较高。
本次研究构建的DNA疫苗pVAX1 - GRA1 + SAG1 + ROP2 + AMA1实验组在C3H小鼠中取得了较好的保护效果,其受弓形虫攻击时生存时间较较少基因pVAX1-ROP2+AMA1组生存时间延长。该种保护又由于IL-12免疫佐剂的应用得到了提高,以弓形虫RH株攻击时观察到了生存时间的较大延长,该实验组10只小鼠受弓形虫攻击时观察到了1只小鼠存活达30天以上。
经鸡尾酒DNA疫苗免疫小鼠血清抗弓形虫(TLA)IgG高于对照组,加免疫佐剂pNGVL3-mIL12组检测到IL-12水平的显著升高,实验组pVAX1- ROP2+AMA1的IFN-γ水平高于pVAX1对照组,更多基因组又较少基因组的IFN-γ水平升高,IL-12佐剂递次诱导了更高IFN-γ水平。实验组与对照组之间的IL-4水平没有显著差异。实验组均较pVAX1对照组淋巴细胞增殖反应更高。pVAX1-GRA1+SAG1+ROP2+AMA1+pNGVL3-mIL12基因免疫组CD4+/CD8+淋巴细胞比值降低。说明该鸡尾酒DNA疫苗诱导了C3H小鼠的特异性免疫应答。
结果表明,该种鸡尾酒DNA疫苗具有良好的免疫保护性,复合免疫的方式增强了免疫效果,这种效果可因免疫佐剂的使用而得到递次提高。
Research of vaccines against Toxoplasma gondii experiences four stages: including whole protozoan body vaccine, parasite-specific composition vaccine, genetic engineering vaccine and DNA vaccine. However, an attenuated T. gondii strain S48 of tachyzoite stage is the only vaccine which has been commercialized for sheep toxoplasmosis and until now there is no vaccine against T. gondii available for human. More and more evidences had showed vaccination with stage-specific antigens can only lead to stage-limited protection and it was also reported multi-antigenic vaccine was more effective than single-antigenic vaccine. Therefore, it has been a trend to develop multi-antigenic vaccines against T. gondii. Moreover, the addition of adjuvant may play an important part in immune protection of the combined multi-antigenic vaccines. At present, the main vaccine candidates are as follows: T. gondii main surface antigens, SAGs; Dense Granule Antigens, GRAs; rhoptry proteins, ROPs; micronemes and so on. In the study, we constructed a combined multi-antigenic DNA vaccine containing GRA1, SAG1, ROP2 and AMA1 which use IL-12 plasmid as an adjuvant to enhance the immune protection.
1. Construction and identification of plasmids GRA1, SAG1, ROP2 and AMA1.
According to our previous study, DNA vaccines GRA1 and SAG1 provide partial immune protection against T. gondii infection. And it is also reported the candidate vaccine genes ROP2 and AMA1 have the same effects. In this study, we constructed recombinant plasmids pVAX1-GRA1, pVAX1-SAG1, pVAX1-ROP2 and pVAX1-AMA1 used for cocktail DNA vaccine, and recombinant expression plasmids pVAX1-ROP2His and pVAX1-AMA1His.
After transiently transfected into macrophages cells, the expressions of pVAX1-ROP2His and pVAX1-AMA1His proteins were assayed by Western blot. Our members have previously proved the proteins expressions of pVAX1-GRA1 and pVAX1-SAG1 by immunohistochemistry method. And the sequences of new recombinant plasmids pVAX1-GRA1 and pVAX1-SAG1 we constructed were in consistent with the past ones. After transiently transfected into macrophages cells, the protein expression of pNGVL3-mIL12 was confirmed by ELISA method. pNGVL3-mIL12 recombinant plasmid is the gift of Ph.D Alexander Rakhmilevich.
2. Evaluation of immune protection provided by plasmids pVAX1-GRA1, pVAX1-SAG1, pVAX1-ROP2, pVAX1-AMA1 and pNGVL3-mIL12.
Different strains of inbred mice have different levels of susceptibility to T. gondii caused mortality. The C3H, BALB/c, C57BL/6 etc. were the most frequently used mice for evaluation the effect of DNA vaccines against T. gondii. Because C3H/He (H-2k) are medium susceptible to T. gondii, it is expected to able to evaluate a long-term protection against T. gondii.
In early experiments, mice were often challenged with high dose of T. gondii to determine protective effect of DNA vaccination, such as 104 or more of tachyzoites stage of T. gondii RH strain. And 104 tachyzoites has been considered a too high dose. These researches were always difficult to get ideal data, for instance survival rate. Challenge dose has been decreased from 105 to 104, then 500, finally 50 tachyzoites of T. gondii RH strain as recently reported. And prolongation of the survival time also has been replaced by the index of survival rate.
The C3H mice were challenged in our research with 500 tachyzoites of T. gondii RH strain while BALB/c mice in Quan Liu et al.’s research was challenged 50 trophozoites. And that BALB/c mouse is more resistant to T. gondii than C3H mouse. Compared to Quan Liu et al.’s reports, lower survival rate was obtained in our research. It was maybe an influencing factor that the challenge dose of DNA vaccinated mice in our research was still too high.
In the study, the mice of the group immunized with pVAX1 - GRA1 + SAG1 + ROP2 + AMA1 lived longer than the group immunized with pVAX1 - ROP2 + AMA1, which demonstrated that the first group produced higher immune protection compared to the other. Moreover, the co-immunization with IL-12 adjuvant enhanced the immune protection and prolonged survival time significantly. Even in the experimental group, one mouse among ten survived more than 30 days.
The sera anti-TLA (Toxoplasma lysate antigen) IgG levels of the groups of mice immunized by the cocktail DNA vaccines were higher than the control one’s. The IFN-γlevel of the group pVAX1-ROP2+AMA1 was higher than the pVAX1 control group, more genes group again elevate the IFN-γlevel, and IL-12 adjuvant still more elevate the IFN-γlevel. IL-4 levels of the experimental groups were no significant difference than the control group. The lymphocyte proliferation reactions of all experimental groups were higher than the pVAX1 control group. The ratio of CD4+/CD8+ T lymphocyte of pVAX1-GRA1+SAG1+ROP2+AMA1 + pNGVL3- mIL12 immunized group decreased. These demonstrated that the cocktail DNA vaccine induced specific immune response in C3H mice.
The results confirmed the cocktail DNA vaccine has nice immune protection, combined immunity enhanced immune effect, and the effect was still elevated by the application of immune adjuvant.
引文
[1]詹希美主编.人体寄生虫学[M].北京:人民卫生出版社, 2005: 89.
[2] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet, 2004 Jun 12; 363(9425): 1965-76.
[3] Denkers EY. T lymphocyte-dependent effector mechanisms of immunity to Toxoplasma gondii. Microbes Infect, 1999 Jul; 1(9):699-708.
[4] Yap GS, Sher A. Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function. Immunobiology, 1999 Dec; 201(2):240-7.
[5] Golkar M, Shokrgozar MA, Rafati S, et al. Evaluation of protective effect of recombinant dense granule antigens GRA2 and GRA6 formulated in monophosphoryl lipid A (MPL) adjuvant against Toxoplasma chronic infection in mice. Vaccine, 2007 May 22; 25(21):4301-11.
[6] Hughes HP. Toxoplasmosis: the need for improved diagnostic techniques and accurate risk assessment. Curr Top Microbiol Immunol, 1985; 120:105-39.
[7] Zhang J, He S, Jiang H, et al. Evaluation of the immune response induced by multiantigenic DNA vaccine encoding SAG1 and ROP2 of Toxoplasma gondii and the adjuvant properties of murine interleukin-12 plasmid in BALB/c mice. Parasitol Res, 2007 Jul; 101(2):331-8.
[8] Buxton D, Innes EA. A commercial vaccine for ovine toxoplasmosis. Parasitology, 1995; 110 Suppl:S11-6.
[9] Buxton D. Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) in sheep and goats: recent advances. Vet Res, 1998 May-Aug; 29(3-4):289-310.
[10] Ogra PL, Faden HS, Abraham R, et al. Effect of prior immunity on the shedding of virulent revertant virus in feces after oral immunization with live attenuated poliovirus vaccines. J Infect Dis, 1991 Jul; 164(1):191-4.
[11] Fachado A, Rodriguez A, Molina J, et al. Long-term protective immune response elicited by vaccination with an expression genomic library of Toxoplasma gondii.Infect Immun, 2003 Sep; 71(9):5407-11.
[12] Pfefferkorn ER, Pfefferkorn LC. Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants. Exp Parasitol, 1976 Jun; 39(3): 365-76.
[13]Waldeland H, Frenkel JK. Live and killed vaccines against toxoplasmosis in mice. J Parasitol, 1983 Feb; 69(1):60-5.
[14] Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science, 1990 Mar 23; 247(4949 Pt 1):1465-8.
[15]李文妹,陆惠民.弓形虫病核酸疫苗研究进展[J].中国人兽共患病杂志, 2004; 20(5):435-7.
[16] Dautu G, Munyaka B, Carmen G, et al. Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential. Exp Parasitol, 2007 Jul; 116(3):273-82.
[17] Couvreur G, Sadak A, Fortier B, et al. Surface antigens of Toxoplasma gondii. Parasitology, 1988 Aug; 97 ( Pt 1):1-10.
[18] Angus CW, Klivington-Evans D, Dubey JP, et al. Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is immunogenic and protective in rodents. J Infect Dis, 2000 Jan; 181(1):317-24.
[19] Couper KN, Nielsen HV, Petersen E, et al. DNA vaccination with the immunodominant tachyzoite surface antigen (SAG-1) protects against adult acquired Toxoplasma gondii infection but does not prevent maternofoetal transmission. Vaccine, 2003 Jun 20; 21(21-22):2813-20.
[20] Liu Q, Gao S, Jiang L, et al. A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice. Microbes Infect, 2008 Oct; 10(12-13):1355-62.
[21] Capron A, Dessaint JP. Vaccination against parasitic diseases: some alternative concepts for the definition of protective antigens. Ann Inst Pasteur Immunol 1988, Jan-Feb; 139(1):109-17.
[22] Cesbron-Delauw MF, Capron A. Excreted/secreted antigens of Toxoplasma gondii--their origin and role in the host-parasite interaction. Res Immunol, 1993 Jan;144(1):41-4.
[23] Cesbron-Delauw MF. Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitol Today, 1994 Aug; 10(8):293-6.
[24] Cesbron-Delauw MF, Guy B, Torpier G, et al. Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii. Proc Natl Acad Sci U S A, 1989 Oct; 86(19):7537-41.
[25] Scorza T, D'Souza S, Laloup M, et al. A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection. Infect Immun, 2003 Jan; 71(1):309-16.
[26] Vercammen M, Scorza T, Huygen K, et al. DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect Immun, 2000 Jan; 68(1):38-45.
[27] Wang H, Liu Q, Liu K, et al. Immune response induced by recombinant Mycobacterium bovis BCG expressing ROP2 gene of Toxoplasma gondii. Parasitol Int, 2007 Dec; 56(4):263-8.
[28] Chen H, Chen G, Zheng H, et al. Induction of immune responses in mice by vaccination with Liposome-entrapped DNA complexes encoding Toxoplasma gondii SAG1 and ROP1 genes. Chin Med J (Engl), 2003 Oct; 116(10):1561-6.
[29] Dziadek B, Gatkowska J, Brzostek A, et al. Toxoplasma gondii: the immunogenic and protective efficacy of recombinant ROP2 and ROP4 rhoptry proteins in murine experimental toxoplasmosis. Exp Parasitol, 2009 Sep; 123(1):81-9.
[30] Beckers CJ, Dubremetz JF, Mercereau-Puijalon O, et al. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J Cell Biol, 1994 Nov; 127(4):947-61.
[31] Nakaar V, Ngo HM, Aaronson EP, et al. Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii. J Cell Sci, 2003 Jun 1; 116(Pt 11):2311-20.
[32] Echeverria PC, de Miguel N, Costas M, et al. Potent antigen-specific immunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmaniainfantum Hsp83 fusion protein. Vaccine, 2006 May 8; 24(19):4102-10.
[33] Xue M, He S, Cui Y, et al. Evaluation of the immune response elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2 and GRA2 against Toxoplasma gondii. Parasitol Int, 2008 Dec; 57(4):424-9.
[34] Xue M, He S, Zhang J, et al. Comparison of cholera toxin A2/B and murine interleukin-12 as adjuvants of Toxoplasma multi-antigenic SAG1-ROP2 DNA vaccine. Exp Parasitol, 2008 Jul; 119(3):352-7.
[35] Cui YL, He SY, Xue MF, et al. Protective effect of a multiantigenic DNA vaccine against Toxoplasma gondii with co-delivery of IL-12 in mice. Parasite Immunol, 2008 May; 30(5):309-13.
[36] Jongert E, de Craeye S, Dewit J, et al. GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. Parasite Immunol, 2007 Sep; 29(9):445-53.
[37] Lourenco EV, Bernardes ES, Silva NM, et al. Immunization with MIC1 and MIC4 induces protective immunity against Toxoplasma gondii. Microbes Infect, 2006 Apr; 8(5):1244-51.
[38] Ismael AB, Hedhli D, Cerede O, et al. Further analysis of protection induced by the MIC3 DNA vaccine against T. gondii: CD4 and CD8 T cells are the major effectors of the MIC3 DNA vaccine-induced protection, both Lectin-like and EGF-like domains of MIC3 conferred protection. Vaccine, 2009 May 14; 27(22): 2959-66.
[39] Alexander J, Jebbari H, Bluethmann H, et al. Immunological control of Toxoplasma gondii and appropriate vaccine design. Curr Top Microbiol Immunol, 1996; 219:183-95.
[40] Innes EA, Vermeulen AN. Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora. Parasitology, 2006; 133 Suppl:S145-68.
[41] Liu S, Shi L, Cheng YB, et al. Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice. Parasitol Res, 2009 Jul; 105(1):267-74.
[42] Fachado A, Rodriguez A, Angel SO, et al. Protective effect of a naked DNAvaccine cocktail against lethal toxoplasmosis in mice. Vaccine, 2003 Mar 28; 21(13-14): 1327-35.
[43] Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol, 2005 Jul 15; 175(2):633-9.
[44] Denkers EY, Gazzinelli RT. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin Microbiol Rev, 1998 Oct; 11(4):569-88.
[45] Denkers EY, Del Rio L, Bennouna S. Neutrophil production of IL-12 and other cytokines during microbial infection. Chem Immunol Allergy, 2003; 83:95-114.
[46] He XW, Wang F, Jiang L, et al. Induction of mucosal and systemic immune response by single-dose oral immunization with biodegradable microparticles containing DNA encoding HBsAg. J Gen Virol, 2005 Mar; 86(Pt 3):601-10.
[47] Shah MA, Yan R, Xu L, et al. A recombinant DNA vaccine encoding Eimeria acervulina cSZ-2 induces immunity against experimental E. tenella infection. Vet Parasitol, 2010 Apr; 169(1-2):185-9.
[48] Yang Y, Zhang Z, Yang J, et al. Oral vaccination with Ts87 DNA vaccine delivered by attenuated Salmonella typhimurium elicits a protective immune response against Trichinella spiralis larval challenge. Vaccine, 2010 Mar; 28(15): 2735-42.
[49] Lin J, Lin X, Yang GH, et al. Toxoplasma gondii: Expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages. Exp Parasitol, 2010 Feb; 125: 165–171.
[50] Saavedra R, de Meuter F, Decourt JL, et al. Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli. J Immunol, 1991 Sep 15; 147(6):1975-82.
[51] Liu MM, Yuan ZG, Peng GH, et al. Toxoplasma gondii microneme protein 8 (MIC8) is a potential vaccine candidate against toxoplasmosis. Parasitol Res, 2010 Apr; 106(5): 1079-84.
[52] Donahue CG, Carruthers VB, Gilk SD, et al. The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels. Mol Biochem Parasitol, 2000 Nov;111(1):15-30.
[53] Bunnell BA, Morgan RA. Gene therapy for infectious diseases. Clin Microbiol Rev, 1998 Jan; 11(1):42-56.
[54]李德才. Th细胞在机体免疫应答中的作用[J].中华现代临床医学杂志, 2009; 7(4): 318-321.
[55]吴春风,李淑红.弓形虫病免疫学研究现状[J].国外医学寄生虫病分册, 2004, 3月; 31(2): 62-65.
[56] Gurunathan S, Wu CY, Freidag BL, et al. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol, 2000 Aug; 12(4):442-7.
[57]谭岩,方艳秋,杨贵贞. IL-6对IFN诱导的小鼠腹腔巨噬细胞抗弓形虫作用的影响[J].上海免疫学杂志, 1999; 19(1): 39-41.
[58] Wang H, He S, Yao Y, et al. Toxoplasma gondii: protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice. Exp Parasitol, 2009 Jul; 122(3):226-32.
[59] Fang R, Feng H, Nie H, et al. Construction and immunogenicity of pseudotype baculovirus expressing Toxoplasma gondii SAG1 protein in BALB/c mice model. Vaccine, 2010 Feb; 28(7): 1803-7.
[60] Subauste CS, Remington JS. Role of gamma interferon in Toxoplasma gondii infection. Eur J Clin Microbiol Infect Dis, 1991 Feb; 10(2):58-67.
[61]何维主编.医学免疫学[M].北京:人民卫生出版社, 2006.
[62] Mevelec MN, Bout D, Desolme B, et al. Evaluation of protective effect of DNA vaccination with genes encoding antigens GRA4 and SAG1 associated with GM-CSF plasmid, against acute, chronical and congenital toxoplasmosis in mice. Vaccine, 2005 Aug 22; 23(36):4489-99.
[63] Liu Q, Shang L, Jin H, et al. The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18. Res Vet Sci, 2010 Aug; 89(1): 93-7.
[64]史霖,刘珊,程雁斌等.弓形虫多表位DNA疫苗的构建及其免疫保护作用[J].细胞与分子免疫学杂志, 2008; 24 (7): 689-691.
[1]詹希美.人体寄生虫学[M].北京:人民卫生出版社, 2005.
[2] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet, 2004 Jun 12; 363(9425): 1965-76.
[3] Golkar M, Shokrgozar MA, Rafati S, et al. Evaluation of protective effect of recombinant dense granule antigens GRA2 and GRA6 formulated in monophosphoryl lipid A (MPL) adjuvant against Toxoplasma chronic infection in mice. Vaccine, 2007 May 22; 25(21):4301-11.
[4] Zhang J, He S, Jiang H, et al. Evaluation of the immune response induced bymultiantigenic DNA vaccine encoding SAG1 and ROP2 of Toxoplasma gondii and the adjuvant properties of murine interleukin-12 plasmid in BALB/c mice. Parasitol Res, 2007 Jul; 101(2):331-8.
[5] Tan TG, Mui E, Cong H, et al. Identification of T. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans. Vaccine, 2010 Mar; 28(23): 3977-89.
[6]李文妹,陆惠民.弓形虫病核酸疫苗研究进展[J].中国人兽共患病杂志2004; 20(5):435-7.
[7] Couvreur G, Sadak A, Fortier B, et al. Surface antigens of Toxoplasma gondii. Parasitology, 1988 Aug; 97 ( Pt 1):1-10.
[8] Fang R, Feng H, Nie H, et al. Construction and immunogenicity of pseudotype baculovirus expressing Toxoplasma gondii SAG1 protein in BALB/c mice model. Vaccine, 2010 Feb; 28(7): 1803-7.
[9] Cesbron-Delauw MF, Capron A. Excreted/secreted antigens of Toxoplasma gondii--their origin and role in the host-parasite interaction. Res Immunol, 1993 Jan; 144(1):41-4.
[10] Cesbron-Delauw MF. Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitol Today, 1994 Aug; 10(8):293-6.
[11] Scorza T, D'Souza S, Laloup M, et al. A GRA1 DNA vaccine primes cytolytic CD8+ T cells to control acute Toxoplasma gondii infection. Infect Immun, 2003 Jan; 71(1):309-16.
[12] Saavedra R, Becerril MA, Dubeaux C, et al. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii. Infect Immun, 1996 Sep; 64(9):3858-62.
[13] Martin V, Supanitsky A, Echeverria PC, et al. Recombinant GRA4 or ROP2 protein combined with alum or the gra4 gene provides partial protection in chronic murine models of toxoplasmosis. Clin Diagn Lab Immunol, 2004 Jul; 11(4):704-10.
[14] Echeverria PC, de Miguel N, Costas M, et al. Potent antigen-specific immunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmania infantum Hsp83 fusion protein. Vaccine, 2006 May 8; 24(19):4102-10.
[15] Dautu G, Munyaka B, Carmen G, et al. Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential. Exp Parasitol, 2007 Jul; 116(3):273-82.
[16] Cui YL, He SY, Xue MF, et al. Protective effect of a multiantigenic DNA vaccine against Toxoplasma gondii with co-delivery of IL-12 in mice. Parasite Immunol, 2008 May; 30(5):309-13.
[17] Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol, 2005 Jul 15; 175(2):633-9.
[18] Denkers EY, Gazzinelli RT. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin Microbiol Rev, 1998 Oct; 11(4):569-88.
[19] Denkers EY, Del Rio L, Bennouna S. Neutrophil production of IL-12 and other cytokines during microbial infection. Chem Immunol Allergy, 2003; 83:95-114.
[20] Xue M, He S, Cui Y, et al. Evaluation of the immune response elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2 and GRA2 against Toxoplasma gondii. Parasitol Int, 2008 Dec; 57(4):424-9.
[21] Moire N, Dion S, Lebrun M, et al. Mic1-3KO tachyzoite a live attenuated vaccine candidate against toxoplasmosis derived from a type I strain shows features of type II strain. Exp Parasitol, 2009 Oct; 123(2):111-7.
[22] Saeij JP, Boyle JP, Coller S, et al. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science, 2006 Dec 15; 314(5806):1780-3.
[23] Taylor S, Barragan A, Su C, et al. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science, 2006 Dec 15; 314(5806):1776-80.
[24] Vercammen M, Scorza T, Huygen K, et al. DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect Immun, 2000 Jan; 68(1):38-45.
[25] Blackwell JM, Roberts CW, Alexander J. Influence of genes within the MHC on mortality and brain cyst development in mice infected with Toxoplasma gondii: kinetics of immune regulation in BALB H-2 congenic mice. Parasite Immunol, 1993 Jun; 15(6):317-24.
[26] Nielsen HV, Lauemoller SL, Christiansen L, et al. Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene. Infect Immun, 1999 Dec; 67(12):6358-63.
[27] Jongert E, de Craeye S, Dewit J, et al. GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. Parasite Immunol, 2007 Sep; 29(9):445-53.
[28] Machado AV, Caetano BC, Barbosa RP, et al. Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity. Vaccine, 2010 Apr 19; 28(18):3247-56.
[29] Gurunathan S, Wu CY, Freidag BL, et al. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol, 2000 Aug; 12(4):442-7.
[2] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet, 2004 Jun 12; 363(9425): 1965-76.
[3] Denkers EY. T lymphocyte-dependent effector mechanisms of immunity to Toxoplasma gondii. Microbes Infect, 1999 Jul; 1(9):699-708.
[4] Yap GS, Sher A. Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function. Immunobiology, 1999 Dec; 201(2):240-7.
[5] Golkar M, Shokrgozar MA, Rafati S, et al. Evaluation of protective effect of recombinant dense granule antigens GRA2 and GRA6 formulated in monophosphoryl lipid A (MPL) adjuvant against Toxoplasma chronic infection in mice. Vaccine, 2007 May 22; 25(21):4301-11.
[6] Hughes HP. Toxoplasmosis: the need for improved diagnostic techniques and accurate risk assessment. Curr Top Microbiol Immunol, 1985; 120:105-39.
[7] Zhang J, He S, Jiang H, et al. Evaluation of the immune response induced by multiantigenic DNA vaccine encoding SAG1 and ROP2 of Toxoplasma gondii and the adjuvant properties of murine interleukin-12 plasmid in BALB/c mice. Parasitol Res, 2007 Jul; 101(2):331-8.
[8] Buxton D, Innes EA. A commercial vaccine for ovine toxoplasmosis. Parasitology, 1995; 110 Suppl:S11-6.
[9] Buxton D. Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) in sheep and goats: recent advances. Vet Res, 1998 May-Aug; 29(3-4):289-310.
[10] Ogra PL, Faden HS, Abraham R, et al. Effect of prior immunity on the shedding of virulent revertant virus in feces after oral immunization with live attenuated poliovirus vaccines. J Infect Dis, 1991 Jul; 164(1):191-4.
[11] Fachado A, Rodriguez A, Molina J, et al. Long-term protective immune response elicited by vaccination with an expression genomic library of Toxoplasma gondii.Infect Immun, 2003 Sep; 71(9):5407-11.
[12] Pfefferkorn ER, Pfefferkorn LC. Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants. Exp Parasitol, 1976 Jun; 39(3): 365-76.
[13]Waldeland H, Frenkel JK. Live and killed vaccines against toxoplasmosis in mice. J Parasitol, 1983 Feb; 69(1):60-5.
[14] Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science, 1990 Mar 23; 247(4949 Pt 1):1465-8.
[15]李文妹,陆惠民.弓形虫病核酸疫苗研究进展[J].中国人兽共患病杂志, 2004; 20(5):435-7.
[16] Dautu G, Munyaka B, Carmen G, et al. Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential. Exp Parasitol, 2007 Jul; 116(3):273-82.
[17] Couvreur G, Sadak A, Fortier B, et al. Surface antigens of Toxoplasma gondii. Parasitology, 1988 Aug; 97 ( Pt 1):1-10.
[18] Angus CW, Klivington-Evans D, Dubey JP, et al. Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is immunogenic and protective in rodents. J Infect Dis, 2000 Jan; 181(1):317-24.
[19] Couper KN, Nielsen HV, Petersen E, et al. DNA vaccination with the immunodominant tachyzoite surface antigen (SAG-1) protects against adult acquired Toxoplasma gondii infection but does not prevent maternofoetal transmission. Vaccine, 2003 Jun 20; 21(21-22):2813-20.
[20] Liu Q, Gao S, Jiang L, et al. A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice. Microbes Infect, 2008 Oct; 10(12-13):1355-62.
[21] Capron A, Dessaint JP. Vaccination against parasitic diseases: some alternative concepts for the definition of protective antigens. Ann Inst Pasteur Immunol 1988, Jan-Feb; 139(1):109-17.
[22] Cesbron-Delauw MF, Capron A. Excreted/secreted antigens of Toxoplasma gondii--their origin and role in the host-parasite interaction. Res Immunol, 1993 Jan;144(1):41-4.
[23] Cesbron-Delauw MF. Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitol Today, 1994 Aug; 10(8):293-6.
[24] Cesbron-Delauw MF, Guy B, Torpier G, et al. Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii. Proc Natl Acad Sci U S A, 1989 Oct; 86(19):7537-41.
[25] Scorza T, D'Souza S, Laloup M, et al. A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection. Infect Immun, 2003 Jan; 71(1):309-16.
[26] Vercammen M, Scorza T, Huygen K, et al. DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect Immun, 2000 Jan; 68(1):38-45.
[27] Wang H, Liu Q, Liu K, et al. Immune response induced by recombinant Mycobacterium bovis BCG expressing ROP2 gene of Toxoplasma gondii. Parasitol Int, 2007 Dec; 56(4):263-8.
[28] Chen H, Chen G, Zheng H, et al. Induction of immune responses in mice by vaccination with Liposome-entrapped DNA complexes encoding Toxoplasma gondii SAG1 and ROP1 genes. Chin Med J (Engl), 2003 Oct; 116(10):1561-6.
[29] Dziadek B, Gatkowska J, Brzostek A, et al. Toxoplasma gondii: the immunogenic and protective efficacy of recombinant ROP2 and ROP4 rhoptry proteins in murine experimental toxoplasmosis. Exp Parasitol, 2009 Sep; 123(1):81-9.
[30] Beckers CJ, Dubremetz JF, Mercereau-Puijalon O, et al. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J Cell Biol, 1994 Nov; 127(4):947-61.
[31] Nakaar V, Ngo HM, Aaronson EP, et al. Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii. J Cell Sci, 2003 Jun 1; 116(Pt 11):2311-20.
[32] Echeverria PC, de Miguel N, Costas M, et al. Potent antigen-specific immunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmaniainfantum Hsp83 fusion protein. Vaccine, 2006 May 8; 24(19):4102-10.
[33] Xue M, He S, Cui Y, et al. Evaluation of the immune response elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2 and GRA2 against Toxoplasma gondii. Parasitol Int, 2008 Dec; 57(4):424-9.
[34] Xue M, He S, Zhang J, et al. Comparison of cholera toxin A2/B and murine interleukin-12 as adjuvants of Toxoplasma multi-antigenic SAG1-ROP2 DNA vaccine. Exp Parasitol, 2008 Jul; 119(3):352-7.
[35] Cui YL, He SY, Xue MF, et al. Protective effect of a multiantigenic DNA vaccine against Toxoplasma gondii with co-delivery of IL-12 in mice. Parasite Immunol, 2008 May; 30(5):309-13.
[36] Jongert E, de Craeye S, Dewit J, et al. GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. Parasite Immunol, 2007 Sep; 29(9):445-53.
[37] Lourenco EV, Bernardes ES, Silva NM, et al. Immunization with MIC1 and MIC4 induces protective immunity against Toxoplasma gondii. Microbes Infect, 2006 Apr; 8(5):1244-51.
[38] Ismael AB, Hedhli D, Cerede O, et al. Further analysis of protection induced by the MIC3 DNA vaccine against T. gondii: CD4 and CD8 T cells are the major effectors of the MIC3 DNA vaccine-induced protection, both Lectin-like and EGF-like domains of MIC3 conferred protection. Vaccine, 2009 May 14; 27(22): 2959-66.
[39] Alexander J, Jebbari H, Bluethmann H, et al. Immunological control of Toxoplasma gondii and appropriate vaccine design. Curr Top Microbiol Immunol, 1996; 219:183-95.
[40] Innes EA, Vermeulen AN. Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora. Parasitology, 2006; 133 Suppl:S145-68.
[41] Liu S, Shi L, Cheng YB, et al. Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice. Parasitol Res, 2009 Jul; 105(1):267-74.
[42] Fachado A, Rodriguez A, Angel SO, et al. Protective effect of a naked DNAvaccine cocktail against lethal toxoplasmosis in mice. Vaccine, 2003 Mar 28; 21(13-14): 1327-35.
[43] Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol, 2005 Jul 15; 175(2):633-9.
[44] Denkers EY, Gazzinelli RT. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin Microbiol Rev, 1998 Oct; 11(4):569-88.
[45] Denkers EY, Del Rio L, Bennouna S. Neutrophil production of IL-12 and other cytokines during microbial infection. Chem Immunol Allergy, 2003; 83:95-114.
[46] He XW, Wang F, Jiang L, et al. Induction of mucosal and systemic immune response by single-dose oral immunization with biodegradable microparticles containing DNA encoding HBsAg. J Gen Virol, 2005 Mar; 86(Pt 3):601-10.
[47] Shah MA, Yan R, Xu L, et al. A recombinant DNA vaccine encoding Eimeria acervulina cSZ-2 induces immunity against experimental E. tenella infection. Vet Parasitol, 2010 Apr; 169(1-2):185-9.
[48] Yang Y, Zhang Z, Yang J, et al. Oral vaccination with Ts87 DNA vaccine delivered by attenuated Salmonella typhimurium elicits a protective immune response against Trichinella spiralis larval challenge. Vaccine, 2010 Mar; 28(15): 2735-42.
[49] Lin J, Lin X, Yang GH, et al. Toxoplasma gondii: Expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages. Exp Parasitol, 2010 Feb; 125: 165–171.
[50] Saavedra R, de Meuter F, Decourt JL, et al. Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli. J Immunol, 1991 Sep 15; 147(6):1975-82.
[51] Liu MM, Yuan ZG, Peng GH, et al. Toxoplasma gondii microneme protein 8 (MIC8) is a potential vaccine candidate against toxoplasmosis. Parasitol Res, 2010 Apr; 106(5): 1079-84.
[52] Donahue CG, Carruthers VB, Gilk SD, et al. The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels. Mol Biochem Parasitol, 2000 Nov;111(1):15-30.
[53] Bunnell BA, Morgan RA. Gene therapy for infectious diseases. Clin Microbiol Rev, 1998 Jan; 11(1):42-56.
[54]李德才. Th细胞在机体免疫应答中的作用[J].中华现代临床医学杂志, 2009; 7(4): 318-321.
[55]吴春风,李淑红.弓形虫病免疫学研究现状[J].国外医学寄生虫病分册, 2004, 3月; 31(2): 62-65.
[56] Gurunathan S, Wu CY, Freidag BL, et al. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol, 2000 Aug; 12(4):442-7.
[57]谭岩,方艳秋,杨贵贞. IL-6对IFN诱导的小鼠腹腔巨噬细胞抗弓形虫作用的影响[J].上海免疫学杂志, 1999; 19(1): 39-41.
[58] Wang H, He S, Yao Y, et al. Toxoplasma gondii: protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice. Exp Parasitol, 2009 Jul; 122(3):226-32.
[59] Fang R, Feng H, Nie H, et al. Construction and immunogenicity of pseudotype baculovirus expressing Toxoplasma gondii SAG1 protein in BALB/c mice model. Vaccine, 2010 Feb; 28(7): 1803-7.
[60] Subauste CS, Remington JS. Role of gamma interferon in Toxoplasma gondii infection. Eur J Clin Microbiol Infect Dis, 1991 Feb; 10(2):58-67.
[61]何维主编.医学免疫学[M].北京:人民卫生出版社, 2006.
[62] Mevelec MN, Bout D, Desolme B, et al. Evaluation of protective effect of DNA vaccination with genes encoding antigens GRA4 and SAG1 associated with GM-CSF plasmid, against acute, chronical and congenital toxoplasmosis in mice. Vaccine, 2005 Aug 22; 23(36):4489-99.
[63] Liu Q, Shang L, Jin H, et al. The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18. Res Vet Sci, 2010 Aug; 89(1): 93-7.
[64]史霖,刘珊,程雁斌等.弓形虫多表位DNA疫苗的构建及其免疫保护作用[J].细胞与分子免疫学杂志, 2008; 24 (7): 689-691.
[1]詹希美.人体寄生虫学[M].北京:人民卫生出版社, 2005.
[2] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet, 2004 Jun 12; 363(9425): 1965-76.
[3] Golkar M, Shokrgozar MA, Rafati S, et al. Evaluation of protective effect of recombinant dense granule antigens GRA2 and GRA6 formulated in monophosphoryl lipid A (MPL) adjuvant against Toxoplasma chronic infection in mice. Vaccine, 2007 May 22; 25(21):4301-11.
[4] Zhang J, He S, Jiang H, et al. Evaluation of the immune response induced bymultiantigenic DNA vaccine encoding SAG1 and ROP2 of Toxoplasma gondii and the adjuvant properties of murine interleukin-12 plasmid in BALB/c mice. Parasitol Res, 2007 Jul; 101(2):331-8.
[5] Tan TG, Mui E, Cong H, et al. Identification of T. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans. Vaccine, 2010 Mar; 28(23): 3977-89.
[6]李文妹,陆惠民.弓形虫病核酸疫苗研究进展[J].中国人兽共患病杂志2004; 20(5):435-7.
[7] Couvreur G, Sadak A, Fortier B, et al. Surface antigens of Toxoplasma gondii. Parasitology, 1988 Aug; 97 ( Pt 1):1-10.
[8] Fang R, Feng H, Nie H, et al. Construction and immunogenicity of pseudotype baculovirus expressing Toxoplasma gondii SAG1 protein in BALB/c mice model. Vaccine, 2010 Feb; 28(7): 1803-7.
[9] Cesbron-Delauw MF, Capron A. Excreted/secreted antigens of Toxoplasma gondii--their origin and role in the host-parasite interaction. Res Immunol, 1993 Jan; 144(1):41-4.
[10] Cesbron-Delauw MF. Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitol Today, 1994 Aug; 10(8):293-6.
[11] Scorza T, D'Souza S, Laloup M, et al. A GRA1 DNA vaccine primes cytolytic CD8+ T cells to control acute Toxoplasma gondii infection. Infect Immun, 2003 Jan; 71(1):309-16.
[12] Saavedra R, Becerril MA, Dubeaux C, et al. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii. Infect Immun, 1996 Sep; 64(9):3858-62.
[13] Martin V, Supanitsky A, Echeverria PC, et al. Recombinant GRA4 or ROP2 protein combined with alum or the gra4 gene provides partial protection in chronic murine models of toxoplasmosis. Clin Diagn Lab Immunol, 2004 Jul; 11(4):704-10.
[14] Echeverria PC, de Miguel N, Costas M, et al. Potent antigen-specific immunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmania infantum Hsp83 fusion protein. Vaccine, 2006 May 8; 24(19):4102-10.
[15] Dautu G, Munyaka B, Carmen G, et al. Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential. Exp Parasitol, 2007 Jul; 116(3):273-82.
[16] Cui YL, He SY, Xue MF, et al. Protective effect of a multiantigenic DNA vaccine against Toxoplasma gondii with co-delivery of IL-12 in mice. Parasite Immunol, 2008 May; 30(5):309-13.
[17] Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol, 2005 Jul 15; 175(2):633-9.
[18] Denkers EY, Gazzinelli RT. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin Microbiol Rev, 1998 Oct; 11(4):569-88.
[19] Denkers EY, Del Rio L, Bennouna S. Neutrophil production of IL-12 and other cytokines during microbial infection. Chem Immunol Allergy, 2003; 83:95-114.
[20] Xue M, He S, Cui Y, et al. Evaluation of the immune response elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2 and GRA2 against Toxoplasma gondii. Parasitol Int, 2008 Dec; 57(4):424-9.
[21] Moire N, Dion S, Lebrun M, et al. Mic1-3KO tachyzoite a live attenuated vaccine candidate against toxoplasmosis derived from a type I strain shows features of type II strain. Exp Parasitol, 2009 Oct; 123(2):111-7.
[22] Saeij JP, Boyle JP, Coller S, et al. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science, 2006 Dec 15; 314(5806):1780-3.
[23] Taylor S, Barragan A, Su C, et al. A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science, 2006 Dec 15; 314(5806):1776-80.
[24] Vercammen M, Scorza T, Huygen K, et al. DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect Immun, 2000 Jan; 68(1):38-45.
[25] Blackwell JM, Roberts CW, Alexander J. Influence of genes within the MHC on mortality and brain cyst development in mice infected with Toxoplasma gondii: kinetics of immune regulation in BALB H-2 congenic mice. Parasite Immunol, 1993 Jun; 15(6):317-24.
[26] Nielsen HV, Lauemoller SL, Christiansen L, et al. Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene. Infect Immun, 1999 Dec; 67(12):6358-63.
[27] Jongert E, de Craeye S, Dewit J, et al. GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. Parasite Immunol, 2007 Sep; 29(9):445-53.
[28] Machado AV, Caetano BC, Barbosa RP, et al. Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity. Vaccine, 2010 Apr 19; 28(18):3247-56.
[29] Gurunathan S, Wu CY, Freidag BL, et al. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol, 2000 Aug; 12(4):442-7.