疟疾传播阻断疫苗Pys25重组蛋白免疫活性的实验研究

为探讨约氏疟原虫传播阻断疫苗Pys25重组蛋白的免疫效果及其效应,我们以酵母菌表达的Pys25重组蛋白皮下免疫DBA/2小鼠,采用ELISA检测免疫后不同时间小鼠血清中的特异性抗体水平,并通过IFA观察免疫血清对有性阶段虫体发育的阻断效应。初次免疫后2周,小鼠血清中特异性IgG抗体水平开始升高,并于加强免疫后2周达到峰值;感染血液与免疫血清共同培养后,配子体向合子和动合子的发育明显受阻,并且其传播阻断效应与免疫血清呈剂量依赖性。实验表明,酵母菌表达的Pys25重组蛋白具有良好的免疫原性,其免疫血清可显著抑制有性阶段原虫的进一步发育。     

约氏疟原虫丝/苏氨酸磷酸酶5抗血清对有性阶段生长抑制作用的研究

目的:探讨约氏疟原虫丝/苏氨酸磷酸酶5(PyPP5)作为传播阻断疫苗候选抗原的可行性。方法:PCR扩增PyPP5蛋白优势抗原表位,克隆入pET32a (+)载体。IPTG诱导PyPP5重组蛋白(rPyPP5)表达后,纯化并免疫小鼠获取抗血清。体外实验观察抗-PyPP5免疫血清对配子体出丝、动合子形成和转化率的影响。结果:成功制备抗-PyPP5免疫血清。抗-PyPP5免疫血清(1∶5倍稀释)使配子体出丝率、动合子数目和动合子转化率分别下降29.89%(P<0.05)、38.59%(P<0.05)和64.30%(P<0.01)。结论:PyPP5蛋白具有良好的抗原性。抗-PyPP5免疫血清可有效抑制约氏疟原虫传播。     

疟原虫雌性配子体特异性表面蛋白Pys47真核表达及免疫效应评价

P47是特异性表达于疟原虫雌性配子体以及雌性配子表面的蛋白,为了评价其作为传播阻断疫苗候选抗原的免疫效果及其效应,本研究将约氏疟原虫Pys47完整编码区基因序列克隆入真核表达载体pcDNA3.1+,获得核酸质粒,肌肉注射BALB/c小鼠.通过ELISA方法检测免疫小鼠血清滴度,IFA检测疫苗的免疫活性以及免疫血清对有性...     

疟原虫配子体蛋白Pys48核酸疫苗的构建与免疫活性观测

为探讨约氏疟原虫配子体蛋白Pys48核酸疫苗的免疫效果及其效应,以约氏疟原虫Pys48全基因序列克隆和真核表达载体pcDNA3．1为基础获得核酸重组质粒,肌肉注射免疫BALB／c小鼠,ELISA检测小鼠血清中特异性抗体水平,并通过IFA检测疫苗的免疫活性以及免疫血清对有性阶段虫体发育的阻断效应。测序结果和酶切鉴定均证实构建的质粒为Pys48全基因插入质粒。ELISA结果显示疫苗免疫小鼠血清特异性抗体滴度明显高于对照组;IFA结果显示免疫血清能够明显阻断配子体向合子与动合子的发育,并且其传播阻断效应与免疫血清呈剂量依赖性关系。实验表明,真核表达载体pcDNA3．1表达的Pys48核酸疫苗具有良好的免疫原性,其免疫血清可显著抑制有性阶段原虫的进一步发育。     

约氏疟原虫丝/苏氨酸磷酸酶6免疫血清对原虫有性阶段发育抑制作用的研究

为探讨约氏疟原虫丝/苏氨酸磷酸酶6(Plasmodium yoelii Protein Phosphatase 6, PyPP6)作为传播阻断疫苗候选抗原的可行性,采用PCR扩增PP6优势抗原表位并克隆入pET32a(+)载体,诱导PyPP6重组蛋白(rPyPP6)表达与纯化,免疫小鼠获取抗-PyPP6免疫血清(anti-PyPP6)。采用ELISA和Western Blot方法检测anti-PyPP6血清效价和特异性,IFA检测PyPP6蛋白在约氏疟原虫各期定位,体内外实验检测anti-PyPP6血清对雄配子出丝、动合子形成和卵囊发育的影响。结果显示,成功诱导rPyPP6表达,anti-PyPP6血清抗体滴度为1:128000,PyPP6部分定位于约氏疟原虫质膜。与对照组相比,anti-PyPP6免疫血清1∶5倍稀释可显著抑制61.5%配子体出丝,动合子数目和动合子转化率分别降低了75%和19.7%,卵囊形成数量减少了35%。结果表明,PyPP6重组蛋白具有良好的免疫原性和抗原性,抗-PP6免疫血清具有显著的传播阻断效果。     

约氏疟原虫保护性抗原对鼠疟红内期保护性免疫调节作…

用约氏疟原虫（Ｐ．ｙ）裂殖子抗原免疫ＢＡＬＢ／ｃ小鼠，再用约氏疟原虫非致死株１０＾５ＰＲＢＣ／只攻击。免疫组小鼠血中原虫推迟出现，提早消失，原虫率明显低于未免疫小鼠和佐剂对照小鼠；佐剂对照组仅原虫高峰推迟出现，与易感小鼠无明显差异。抗原免疫小鼠脾脏Ｔ淋巴细胞对抗原特异性和非特异性增殖反应明显高于其它两组，原虫攻击后增殖反应普遍受到抑制，但免疫小鼠比其它两组恢复快，感染后１８天即恢复至正常水平。     

约氏疟原虫可溶性抗原对鼠疟红内期保护性免疫调节作用的研究

用约氏疟原虫（Ｐｌａｓｍｏｄｉｕｍｙｏｅｌｉｙｏｅｌｉ，）可溶性抗原加佐剂免疫小鼠，观察原虫攻击后的保护作用及鼠脾细胞ＣｏｎＡ诱导的ＩＦＮ－γ分泌水平，并设佐剂对照组和空白组。结果显示：免疫组血原虫推迟出现，感染率明显低于其它两组（Ｐ＜００１）。感染时间明显缩短，佐剂对照组原虫率也低于空白组；且与空白组比较，免疫组和佐剂对照组ＩＦＮ－γ高峰出现早，说明ＩＦＮ－γ对抑制血中原虫起一定作用     

约氏疟原虫感染血清对疟原虫配子体感染力作用机制的研究

目的：探讨‘危机血清’介导疟原虫配子体对蛟感染力的作用机制。方法：通过间接免疫荧光实验和直接蚊血食实验,观察感染第5天小鼠血清（D5血清）对配子体感染力的影响。利用ELISA和Griess反应实验,检测D5血清对感染小鼠血清中IFN－γ、TNF－α、IL－4和脾细胞培养上清中NO2^-产生的影响。同时,检测了疟原虫寄生红细胞的提取物（PRBC提取物）诱导NO合成的能力。结果：感染等5天小鼠血液中的配子体向动合子的发育完全发生阻断。D5血清注入感染第3天的小鼠体内后未见立即抑制配子体在蚊体内的发育,但4h后,可促进宿主体内IFN－γ增多,提高NO的表达,并抑制配子体的感染力。并且,PRBC提取物诱导感染小鼠脾细胞NO的合成。结论：危机血清可能通过NO依赖的方式阻断约氏疟原虫媒介昆虫蚊的传播。     

约氏疟原虫保护性抗原对鼠疟红内期保护性免疫调节作用的研究──Ⅱ．53kD抗原细胞增殖反应及血清抗体与保护作用的关系

用约氏疟原虫（Ｐ．ｙ）裂殖子抗原免疫ＥＡＬＢ／ｃ小鼠，再用约氏疟原虫非致死株１０５ＰＲＢＣ／只攻击。免疫组小鼠血中原虫推迟出现，提早消失，原虫率明显低于未免疫小鼠和佐剂对照小鼠；佐剂对照组仅原虫高峰推迟出现，与易感小鼠无明显差异。抗原免疫小鼠脾脏Ｔ淋巴细胞对抗原特异性和非特异性增殖反应明显高于其它两组，原虫攻击后增殖反应普遍受到抑制，但免疫小鼠比其它两组恢复快，感染后１８天即恢复至正常水平。     

约氏疟原虫感染小鼠脾脏B细胞和NK细胞及其细胞因子的检测

为研究约氏疟原虫感染小鼠脾脏不同免疫细胞及其细胞因子的水平变化,将C57BL/6小鼠分为感染组和正常组,分别经小鼠尾静脉注射约氏疟原虫和生理盐水,8d后处死感染组和正常组小鼠并分离脾脏,制备单细胞悬液,然后利用流式细胞术检测小鼠脾脏B细胞和NK细胞及其表达的细胞因子IFN-y、IL-12、IL-10的水平变化情况.结果...     

Two antigens on zygotes and ookinetes of Plasmodium yoelii and Plasmodium berghei that are distinct targets of transmission-blocking immunity.

We have developed transmission-blocking monoclonal antibodies (MAbs) against Plasmodium yoelii 21-kDa (Pys21) and 28-kDa (Pys25) ookinete surface proteins. These MAbs block infectivity of P. yoelii to Anopheles stephensi. One MAb, 14, cross-reacted by Western blotting with a 28-kDa surface protein (Pbs25) of P. berghei ookinetes and blocked oocyst development, as assayed by direct mosquito feeds on passively immunized P. berghei-infected mice. In total, we have identified two ookinete surface proteins in P. yoelii, one of which is also present in P. berghei. The transmission-blocking activity of the anti-Pys25 MAb 4 was complete and more potent than that of the anti-Pys21 MAb 2. Moreover, Fab fragments of MAb 4 had transmission-blocking activity in mice. In comparison, Fab fragments of MAb 2 did not have detectable transmission-blocking effect, although F(ab')2 did. Furthermore, MAb 2 and MAb 4 appeared to block the in vitro formation and development of zygotes as well.     

Plasmodium falciparum: natural and experimental transmission-blocking immunity

Fc-dependent regulation of humoral immune responses was investigated by immunization of BALB/c mice with immune complexes. These complexes were composed of DNP- and PC-conjugated KLH or Ficoll, and monoclonal T15 idiotype-positive anti-PC antibodies of different isotypes but indistinguishable V-region properties. Since the response to DNP was analyzed, effects due to masking of antigenic determinants by anti-PC antibodies are excluded. The responses to free and complexed antigens showed significant differences in the proportions of DNP-specific IgM, IgG1, IgG2 and IgG3 antibodies. Complexes containing the T-dependent carrier KLH elicited serum antibodies to DNP with significantly decreased IgM levels, irrespective of the isotype in the complex. Under these conditions, however, DNP-specific IgG classes were augmented to various extents, depending on the isotype in the complex. In the case of the T-independent carrier Ficoll, only complexes with IgM or IgG3 suppressed the IgM response. Moreover, immunization with complexes composed of IgM or IgG2a led to a significant decrease of DNP-specific IgG1. In contrast to changes induced by antibodies bound to the T-dependent antigen, immunization with T-independent complexes did not enhance the production of any of the immunoglobulin isotypes.     

Malaria transmission-blocking immunity induced by natural infections of Plasmodium vivax in humans.

Immunity to malarial infections in human populations is known to affect the development of the asexual blood stages of the parasites in the human host and to be capable of conferring significant protection against morbidity and mortality due to the disease. In this study we show that during acute infection with Plasmodium vivax malaria, one of the two main malarial pathogens of humans, most individuals also develop immunity that suppresses the infectivity of the sexual stages of the parasite to mosquitoes. The immunity is antibody mediated and is directed against the parasites in the mosquito midgut shortly after ingestion of blood by a mosquito. This immunity could be expected to have significant effects on the natural transmission of P. vivax malaria.     

Early nonspecific immune responses and immunity to blood-stage nonlethal Plasmodium yoelii malaria

The early role of natural killer cells and gamma delta T cells in the development of protective immunity to the blood stage of nonlethal Plasmodium yoelii infection was studied. Splenic cytokine levels were measured 24 h after infection of natural killer cell-depleted immunodeficient and littermate mice or transiently T-cell-depleted normal mice. Splenic gamma interferon levels were significantly increased above background in immunodeficient and littermate mice 24 h after infection. Depletion of natural killer cells resulted in markedly depressed gamma interferon levels and poor control of parasitemia, particularly in severe combined immunodeficient mice. In the littermates, gamma interferon levels were partially reduced, but parasitemias were resolved normally. However, in athymic mice, natural killer cell depletion had no effect on gamma interferon production. Levels of tumor necrosis factor alpha were increased in all animals 24 h after infection, and responses were not affected by natural killer cell depletion. However, in T-cell-depleted animals, both gamma interferon and tumor necrosis factor alpha levels were decreased 24 h after infection, and depleted mice were unable to control their parasitemia. These results suggest that the early production of both cytokines is important in the early control of parasitemia and that both natural killer and gamma delta T cells contribute equally towards their production. The data also suggest that the subsequent resolution of infection requires early production of gamma interferon, which might act by switching on the appropriate T-helper-cell subsets and other essential parasitotoxic effector mechanisms.     

约氏疟原虫BY265株减毒子孢子诱导小鼠保护性免疫的研究

目的探讨约氏疟原虫BY265株减毒子孢子免疫能否诱导小鼠产生完全保护性免疫及其效应分子。方法确定减毒子孢子合适辐照剂量并免疫小鼠后,间接免疫荧光和ELISPOT分别检测抗环子孢子蛋白(CSP)抗体滴度和CSP特异的CD8+T细胞分泌IFN-γ及其抵御子孢子的攻击感染情况。结果免疫小鼠的外周血能检测到子孢子CSP特异的抗体(1∶400)和产生IFN-γ的CSP特异CD8+T细胞;与对照小鼠相比,1000个子孢子尾静脉注射攻击减毒子孢子免疫小鼠后,直到14d也检测不到红内期疟原虫。结论经辐照减毒子孢子免疫后的小鼠对野生株子孢子产生了完全保护性免疫,为红外期疫苗的研究提供基础与理论依据。     

Effect of CpG oligodeoxynucleotides on the immunogenicity of Pfs25, a Plasmodium falciparum transmission-blocking vaccine antigen

Antibodies directed against Pfs25, a protein present on the surface of zygotes and ookinetes of Plasmodium falciparum, completely block pathogen transmission. We evaluated the immunomodulatory effect of CpG oligodeoxynucleotides (ODN) on the immunogenicity of recombinant Pfs25 (rPfs25) formulated in alum (Al). Immunization of mice with rPfs25 plus CpG ODN improved both the antibody titer (a 30-fold-higher antibody response than that with rPfs25-Al alone) and avidity. Coadministration of CpG ODN dramatically enhanced the titer of immunoglobulin G2A (IgG2a) compared to the titer of the IgG1-dominant response caused by rPfs25-Al alone, and the sera from the CpG ODN-coadministered group completely blocked the transmission of P. falciparum parasites to mosquitoes, as determined by membrane feeding assays. However, transmission-blocking experiments revealed that blocking efficacy was dependent on high-titer antibody levels, independent of isotypes. These results suggest that CpG ODN can be used as an adjuvant to enhance the immunogenicity of rPfs25 as a malaria transmission-blocking vaccine.     

Murine model for assessment of Plasmodium falciparum transmission-blocking vaccine using transgenic Plasmodium berghei parasites expressing the target antigen Pfs25

Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmission-blocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmission-blocking antibodies is typically assessed using an assay known as the membrane feeding assay (MFA). It is an in vitro method that involves mixing antibodies with cultured P. falciparum gametocytes and feeding them to mosquitoes through an artificial membrane followed by assessment of infection in the mosquitoes. We genetically modified Plasmodium berghei to express Pfs25 and demonstrated that the transgenic parasites (TrPfs25Pb) are susceptible to anti-Pfs25 antibodies during mosquito-stage development. The asexual growth kinetics and mosquito infectivity of TrPfs25Pb were comparable to those of wild-type parasites, and TrPfs25Pb displayed Pfs25 on the surface of ookinetes. Immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi. Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These studies describe development of an animal malaria model alternative to the in vitro MFA and show that the model can facilitate P. falciparum transmission-blocking vaccine evaluation based on the target antigen Pfs25. We believe that an animal model to test transmission-blocking vaccines would be superior to the MFA, since there may be additional immune factors that synergize the transmission-blocking activity of antibodies in vivo.