重组霍乱毒素B亚基与疟原虫多表位融合蛋白的免疫保护作用研究

对以霍乱毒素B亚基为载体蛋白的重组疟疾多价抗原在小鼠及恒河猴中的免疫原性及对相应疟原虫感染的免疫保护作用进行了研究。结果表明：该抗原免疫小鼠后,对约氏疟子孢子攻击的保护率在50%左右;恒河猴免疫后用1×10⁸食蟹疟裂殖子攻击,对照组2只动物在攻击后4d感染,感染持续30d以上;免疫组2只动物中,两只动物在感染6～7d后完全恢复,且1只推迟3d感染,说明该抗原具有免疫保护作用。     

以恒河猴为模型的DNA疫苗的免疫保护作用研究

研究了以霍乱毒素B亚基（CTB）为载体的重组疟疾多价抗原（AWTE）表位的DNA疫苗在恒河猴中的免疫原性及对相应疟原虫感染的免疫保护作用。结果表明DNA疫苗组免疫2次后即产生了较高水平的细胞免疫和体液免疫,免疫后91天用1.25×10     

性疟原虫DNA疫苗免疫效果的评估

以霍乱毒素Ｂ亚基（ＣＴＢ）为载体,由其基因构建了含有不同时期不同抗原表位的恶性疟原虫的融合基因ＣＴＢ～ＡＷＴＥ、ＣＴＢ～ＮＡＮＰ,前者除含有恶性疟原虫裂殖子表面主要抗原表位杂合多肽基因ＳＰｆ６６外,还含有很强的Ｔ辅助细胞表位ＣＳＴ３和Ｔｃ细胞表位,后者含有子孢子期的Ｂ、Ｔｈ细胞表位。将纯化的质粒免疫Ｂａｌｂ／ｃ纯系小鼠,３次免疫后诱导机体产生了体液免疫和细胞免疫,免疫的小鼠进行疟原虫子孢子攻击实验,保护率为６０％一８０％。将纯化的质粒混合后免疫恒河猴,３次免疫后诱导机体产生了体液免疫和细胞免疫,免疫的恒河猴进行食蟹疟原虫攻击实验,显示了一定的保护作用。     

食蟹猴疟原虫感染恒河猴的长期观察

本项研究证明,食蟹猴疟原虫感染恒河猴后出现的红细胞内期是一个长期的寄生过程,不论血传或子孢子感染,于原虫密度高峰后,均有较长期的低原虫密度阶段。恒河猴均可耐受食蟹猴疟原虫高密度的感染。     

食蟹猴疟原虫B株子孢子感染后供蚊血餐感染时间的初步观察

食蟹猴疟原虫Ｂ株子孢子感染后供蚊血餐感染时间的初步观察宋宗臣第三军区大学寄生虫学教研室重庆６３００３８食蟹猴疟原虫Ｂ株－按蚊－恒河猴系统是疟疾根治药物研究较理想的模型。在使用此模型时,获得好的感染蚊批是重要环节。１９８４年我国引进此株疟原虫后,对子孢...     

A组轮状病毒VP6与霍乱毒素B亚基融合蛋白在大肠杆菌中的表达及生物活性分析

利用霍乱毒素B亚基 (CholeratoxinBsubunit,CTB)的免疫载体作用 ,将轮状病毒相关抗原引入口服免疫体系 ,可激起有效的粘膜免疫反应 ,这里报道了CTB基因与A组轮状病毒地方株T114VP6全基因的融合 ,并在大肠杆菌BL21(DE3 )中进行了融合蛋白的表达。在IPTG诱导下得到分子量为 5 6kD的融合蛋白 ,表达量占菌体蛋白的15 %。分别用抗CT的抗体和抗A组轮状病毒的高价免疫血清进行WesternBlot检测 ,结果证明融合蛋白CTB VP6保留了天然霍乱毒素B亚基及轮状病毒VP6的抗原性。G_M1-ELISA检测表明 ,复性后的融合蛋白具有与神经节苷脂GM1 结合的能力。     

恶性疟原虫子孢子CS抗原融合基因的表达及其生物活性

采用遗传工程技术获得了含有恶性疟原虫子孢子CS抗原融合基因的重组质粒pMC055－CS的工程菌株,能高效分泌CS抗原的融合蛋白至胞外,可达25mg／L．具有霍乱毒素B亚单位（CT－B）和子孢子CS的抗原性．将纯化的融合蛋白免疫C57纯系小鼠,免疫后抗CT－B抗体滴度可达1：3200～6400,抗CS抗体滴度可达1：320～640．免疫小鼠采用约氏疟子孢子腹腔攻击,保护率达34～45％．     

大肠杆菌表达的戊型肝炎病毒ORF2多肽对恒河猴的免疫保护研究

在大肠杆菌中表达的一段戊型肝炎病毒（HEV）结构蛋白NE2,纯化后以弗氏佐剂,按0d,10d,30d的方案10μg/针的剂量免疫3只恒河猴,在第2周抗体阳转,第6周时1只滴度达1∶100 000,另2只滴度1∶20 000,此时以106 PCR滴度的HEV病毒粪悬液攻击。对照组3只均出现血清转氨酶（ALT）升高,抗体阳转,粪便持续排毒1月以上;疫苗组无一发病,未检出非疫苗来源的抗体,其中1只始终未检出粪便排毒,另2只仅出现短暂排毒。以一份NE2免疫后猴血清（滴度1∶20 000）与103 PCR滴度的病毒混匀后感染2只恒河猴,结果对照组2只均持续排毒3周以上,抗体阳转,1只ALT明显升高;而抗体中和组2只猴始终未检出粪便排毒,抗NE2抗体缓慢下降,ALT正常。这些结果表明NE2具有良好的免疫原性和免疫保护性,有可能成为有效的戊肝疫苗。     

恶性疟原虫DNA疫苗免疫效果的评估

以霍乱毒素Ｂ亚基（ＣＴＢ）为载体,由其基因构建了含有不同时期不同抗原表位的恶性疟原虫的融合基因ＣＴＢ～ＡＷＴＥ、ＣＴＢ～ＮＡＮＰ,前者除含有恶性疟原虫裂殖子表面主要抗原表位杂合多肽基因ＳＰｆ６６外,还含有很强的Ｔ辅助细胞表位ＣＳＴ３和Ｔｃ细胞表位,后者含有子孢子期的Ｂ、Ｔｈ细胞表位。将纯化的质粒免疫Ｂａｌｂ．ｃ纯系小鼠,３次免疫后诱导机体产生了体液免疫和细胞免疫,免疫的小鼠进行疟原虫子孢子攻击实验     

恶性疟原虫多抗原表位基因表达载体的构建及其在大豆幼胚中的表达

疟疾是当今最需要研究有效疫苗的主要传染病之一。AWTE基因编码恶性疟原虫多种抗原表位基因 ,CTB基因编码霍乱毒素 B亚基 ,是一种既能引起细胞免疫又能引起体液免疫的免疫载体和佐剂。把 AWTE- CTB融合基因构建到植物表达载体 p BVG- ny2上 ,通过基因枪导入法 ,转化大豆幼胚分生组织。 X- glu染色检测到 GUS基因的表达 ;抗原性分析实验结果表明 ,特异表达的融合蛋白可与 CTB和 AWTE抗体结合 ,具有 CTB抗原性。这个实验结果 ,表明疟疾多抗原表位基因首次在转基因大豆幼胚中得到瞬时表达     

Effect of sequential infection with Plasmodium vivax and P. falciparum in the Aotus trivirgatus monkey.

Aotus trivirgatus monkeys with prior experience with Plasmodium vivax were inoculated with P. falciparum via the bites of infected mosquitoes. The animals with prior malaria had higher parasitemias and significantly higher levels of mosquito infectivity than monkeys with no prior P. vivax experience. Monkeys with a history of P. falciparum that were inoculated with P. vivax had essentially the same parasitemias as those with no prior malaria. However, levels of mosquito infectivity were markedly increased in those monkeys with a history of P. falciparum. The results imply that the introduction of another malaria species into a malarious area may result in higher levels of mosquito infection and more rapid establishment and distribution of that species.     

Haemobartonellosis in squirrel monkeys (Saimiri sciureus): antagonism between Haemobartonella sp. and experimental Plasmodium falciparum malaria

A hemotropic parasite of the genus Haemo bartonella (rickettsial parasite of the Family Anaplasmataceae) is responsible for latent asymptomatic infection in colony-born Saimiri monkeys. Indeed, many of these animals develop a patent Haemobartonella infection following splenectomy. Such patent parasitism is characterized by an intense Haemobartonella parasitemia which peaks between days 12 and 14 after removal of the spleen and then decreases to become undetectable between days 25 and 30. During the resolving phase of parasitemia, a moderate anemia associated with monocytosis and erythrophagocytosis is observed. In certain Saimiri monkeys, Haemobartonella parasitemia remains latent following removal of the spleen. This indicates that the spleen plays a role but is not necessary to maintain latent Haemobartonella parasitism. It also suggests the existence of heterogeneity in the host immune reactivity to the parasite. Latent or patent haemobartonellosis might raise a problem when Saimiri monkeys are used as experimental hosts of Plasmodium falciparum asexual blood stages, as already noticed with "rodent malaria." Thus we investigated the relationship between Haemobartonella and P. falci parum in splenectomized monkeys. When animals harboring latent Haemobartonella sp. were infected with P. falciparum, the former remained latent and exerted no influence on the course of the P. falciparum parasitemia. In constrast, when P. falciparum was initiated in animals which were in the process of developing patent haemobarto nellosis, the course of the former was protracted and either the animal resisted longer, or it self-cleared the P. falciparum infection. Conversely, patent haemobartonellosis was delayed when splenectomy was performed at different times after initiation of P. falciparum infection in intact monkeys. Our results do not allow us to draw conclusions as to the mechanism(s) of the antagonism between the two parasites, but they emphasize the need to monitor the presence of Haemobartonella when splenectomized Saimiri monkeys are used as experimentals hosts for P. falciparum parasitism.     

The putative haemobartonella that influences Plasmodium falciparum parasitaemia in squirrel monkeys is a haemotrophic mycoplasma

Splenectomised squirrel monkeys (Saimiri sciureus) are increasingly being used as an experimental host for human malaria studies, notably for the assessment of candidate vaccines against Plasmodium falciparum blood-stage infection. Recently, S. sciureus monkeys in our primate-breeding colony were reported to be asymptomatic carriers of a putative Haemobartonella species. Patent haemobartonella infection is frequently activated following splenectomy, and may interfere with studies on the course of P. falciparum parasitaemia in these animals. Here, we show by 16S rRNA gene sequence analysis that this wall-less bacterium is not a rickettsia but, instead, is a haemotrophic mycoplasma. Haemotrophic mycoplasmas are a newly identified group of mycoplasmas that parasitise the surfaces of erythrocytes of a wide variety of vertebrate hosts.     

Azithromycin: antimalarial profile against blood- and sporozoite-induced infections in mice and monkeys

The spectrum of antimalarial activity of the new macrolide antibiotic azithromycin was evaluated against blood- and sporozoite-induced infections with a chloroquine-resistant strain of Plasmodium yoelii nigeriensis (N-67) in Swiss mice and with simian parasite Plasmodium cynomolgi B in rhesus monkeys. Against experimental rodent malaria, a 70 mg/kg/day dose showed curative blood-schizontocidal activity in a four-dose regimen administered orally from day 0 to day 3 or from day 2 to day 5 to mice harboring established infection. The curative response was also obtained with a 40 mg/kg/day dose administered in an extended seven-dose (days 0-6) regimen. Azithromycin was also effective in the causal prophylactic test, since a 50 mg/kg dose from day -1 to day +2 protected mice against P. y. nigeriensis (N-67) sporozoite challenge. In comparison, erythromycin did not show either of the above activities up to a 405 mg/kg/day dose in identical regimens. Comparison of the ED(90) values showed that azithromycin was 31-fold more effective than erythromycin as a blood schizontocide. In the simian model, trophozoite-induced infections of P. cynomolgi B were cured with 25 mg/kg/day azithromycin administered for 7 days. In the causal prophylactic test, the prepatent period was significantly extended in monkeys challenged with P. cynomolgi B sporozoites, presumably because of the growth inhibition of preerythrocytic schizonts in hepatocytes. Azithromycin did not exhibit any hypnozoitocidal (dormant exoerythrocytic stages) activity at 25 mg/kg/day in a seven-dose regimen.     

Passive immunization against transmissible gastroenteritis virus in piglets by ingestion of milk of sows inoculated with attenuated virus.

Pregnant sows were inoculated with the attenuated strain, TO--163, of swine transmissible gastroenteritis virus. Suckling piglets born from them received challenge inoculation with the virulent virus at 3 days after birth, and examined for ability to prevent infection and the immunoglobulin (Ig) classes of antibody in milk. A pregnant sow was inoculated intramuscularly with a dose of 10(8.0) TCID50 and intranasally with a dose of 10(9.3) TCID50 of attenuated virus. Piglets born from it suffered from diarrhea after challenge inoculation, but none of them died eventually. Their dam was also affected with diarrhea for 4 to 7 days after challenge inoculation of them. Another pregnant sow was inoculated twice with 10(9.3) TCID50 of attenuated virus, first by the intramuscular and secondly by the intranasal route. Of nine piglets born from it, one excreted soft feces after challenge inoculation, but all survived to grow normally. Their dam manifested no clinical symptoms at all after challenge inoculation of them. The higher the titer of virus inoculated into pregnant sows, the higher the neutralizing antibody titer in serum and milk of the sows after farrowing. The puerperal sow which had received two doses of 10(9.3) TCID50 each of attenuated virus by the intramuscular and intranasal route, respectively, presented the highest neutralizing antibody titer of all the inoculated sows. This titer was 2,048 in serum and 14,183 in colostrum immediately after farrowing. In that sow IgG was the main class of immunoglobulins in neutralizing antibody in milk. Even the IgA antibody titer of that sow was higher than that of any other sow which had been administered with virus of low titer. It was 392 and 19 3 and 9 days, respectively, after farrowing.     

Studies on the Santa Lucia (El Salvador) strain of Plasmodium falciparum in Aotus trivirgatus monkeys.

The Santa Lucia strain of Plasmodium falciparum was isolated from El Salvador, Central America, and established in Aotus trivirgatus monkeys. Transmission from monkey to monkey via the bites of infected Anopheles freeborni, A. maculatus, and A, albimanus mosquitoes was obtained in 20 of 27 attempts. Prepatent periods in the monkeys ranged from 17 to 46 days with a mean of 24.3 days. Parasitemias and mortality were higher following sporozoite inoculation into animals which had been previously infected with P. vivax than in those with no previous malaria experience. Monkeys previously infected with P. vivax and P. cynomolgi had lower maximum parasitemias than those previously infected with P. vivax only.     

DNA prime-protein boost immunization in monkeys: efficacy of a novel construct containing functional domains of Plasmodium cynomolgi CS and TRAP

We report the efficacy of a bimodal immunization regimen that involved priming with naked DNA (multiple doses) followed by a booster with recombinant protein in rhesus monkeys with a chimeric construct containing the N-terminus of thrombospondin-related adhesive protein and the C-terminus of circumsporozoite protein of Plasmodium cynomolgi. The vaccinated animals developed high titer antibodies against the chimeric antigen, the two components of the hybrid and the native proteins of sporozoites. The peripheral blood mononuclear cells isolated from the vaccinated animals had significant in vitro T cell proliferation activity when stimulated with the recombinant chimeric protein. Furthermore, following challenge with 1000 P. cynomolgi sporozoites, the peak and total parasitemia were significantly lower in vaccinated animals than in the control animals.     

Intranasal H5N1 Vaccines,Adjuvanted with Chitosan Derivatives,Protect Ferrets against Highly Pathogenic Influenza Intranasal and Intratracheal Challenge

We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model.Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings.In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals.This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.