制备疫苗用W135/Y群脑膜炎球菌菌种的免疫原性及遗传稳定性观察

目的 为保证生产四价流脑(A、C、W135、Y群)结合疫苗质量一致性和可控性,对分离自国内的W135、Y群脑膜炎球菌CMCC(B)29037株和CMCC(B)29028株进行免疫原性及遗传稳定性观察.方法 将W135/Y群脑膜炎球菌工作种子批菌种分别连续传代至30代,并收获3、5、10、15、20、25及30代次菌液,对各代次菌进行免疫原性、抗原性、生化反应、毒性、毒力测定,并将30代次菌发酵培养后提取荚膜多糖进行质量分析.结果 CMCC(B)29037株和CMCC(B)29028株工作种子批菌种诱导小鼠产生总IgG抗体分别为1∶1114和1∶2229,杀菌抗体水平与IgG抗体间差异无统计学意义;试管凝集效价均达到1∶320,生化检定两菌株均发酵葡萄糖、麦芽糖,不发酵果糖、蔗糖、甘露醇和乳糖;两菌株的LD50均＞109,30代次内各代次菌的免疫原性、抗原性、生化反应和毒性均无差异.30代次菌液脑腔毒性测定显示均无病理改变,用第30代次菌生产的W135/Y群脑膜炎球菌荚膜多糖各项检定指标均合格.结论 分离自国内的CMCC(B)29037株和CMCC(B)29028株为脑膜炎球菌W135/Y群菌株,免疫原性、抗原性好,生化反应合格、安全性良好,连续传至30代次仍保持较好的安全性和免疫原性,30代次菌纯化的W135/Y群脑膜炎球菌荚膜多糖质量符合质控要求,可以用作四价流脑结合疫苗生产株.     

宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷型减毒株的生物学特性

ＦＳ５４－２、ＦＳ５４－３、ＦＳ５４－４、ＦＳ５４－５、Ｐｓ５４－６、ＰＳ５４－７及ＦＳ５４－７ｂ等株为本室构建的宋内氏福氏２ａ双价志贺氏菌芳香族氨基酸营养缺陷型减毒株，不但其Ｔｎ１０所携带的四环素抗性已消除，ＦＳ５４－７ｂ株的Ⅰ相大质粒Ｔｎ５所携带的卡那霉素抗性亦已消除。本文为在此基础上，进一步对双价减毒株的侵入力、毒力、免疫原性与免疫保护性及遗传稳定性等进行的研究，证明双价减毒株仍保持了入侵肠上皮细胞的能力，动物实验安全低毒，有很好的免疫原性，对小白鼠有很好的双重免疫保护性。此株显示了良好的应用前景。     

我国鼠疫菌毒力因子的比较与分析

目的 研究自不同生态型和不同鼠疫自然疫源地分离的鼠疫菌4种毒力因子的性状特征。方法 常璺方法检查鼠疫菌毒力因子性状并比较在不同疫源地之间、不同生态型之间的差异。结果 1326株鼠疫菌中仅1株缺失FI、6株缺失PstI,Pgm和WW阳性分别为92.99%、76.47%。结论 自我国不同疫源地、平淡同生态型和不同年代分离的野生型鼠疫菌绝大多数能产生FI和PstI,且不同生态型和不同疫源地分离的菌株这两种毒力因子无明显差别,性状稳定,VW^-和Pgm^-菌株与生态型及疫源地有一定的关系。经长期人工培养传代后。4种毒力因子相比,VW因子较易丧失,该毒力因子具有更不稳定特点。     

痢疾菌的毒力相关抗原及其表型的分析

目的：分析研究该室保存或构建的各类痢疾菌毒株、减毒突变株、菌苗株的毒力及其生物表型的相互关系，探讨与致病有关的成分，为改进现有菌苗和构建新型菌苗提供参考依据。方法：采用刚果红结合试验、接触性溶血试验、HeLa细胞入侵试验及豚鼠角结合膜炎试验，对不同痢疾菌的毒力表型进行检测，并对大质粒及其侵袭相关的基因（4.1kb）进行遗传背景分析。用BA-immunoblot法，对痢疾杆菌和侵袭性大肠杆菌等进行毒力相关抗原的分析。结果：有侵袭毒力的痢疾菌株均与刚果红染料结合、有接触性溶血活性，可侵入HeLa细胞，豚鼠角结合膜试验阳性，并都含有大质粒（120～140MD），与4.1kb侵袭探针杂交也均呈阳性反应；而无毒力的痢疾菌株上述生物表型均为阴性。有毒痢疾菌和侵袭性大肠杆菌均表达4种主要毒力相关抗原Ipa(A,B,C,D），而无毒株不表达这些抗原。痢疾菌的毒力不仅与Ipa有关，而且还与LPS抗原有关。结论：细菌的毒力与生物表型密切有关。并且Ipa与LPS这两类抗原均表达时细菌才有毒力。     

减毒鼠伤寒沙门菌SL1344ΔsseK1Δasd宿主-载体平衡致死系统的构建及其生物学特性研究

目的:为构建减毒鼠伤寒沙门菌分泌性蛋白K1缺失株平衡致死系统,并将其开发为能够稳定携带外源基因的活疫苗载体。方法:利用重组自杀性质粒(pREΔasd)介导的等位基因交换技术,以减毒鼠伤寒沙门菌株SL1344Δsse K1为亲本株,运用二步法筛选asd基因缺失株SL1344Δsse K1Δasd。将携带有asd基因的无抗性p YA3493质粒电转至上述缺失菌株SL1344Δsse K1Δasd,构建SL1344Δsse K1Δasd(p YA3493)重组菌株。结果:PCR及测序结果表明SL1344Δsse K1Δasd(p YA3493)构建成功。进一步研究表明重组菌株SL1344Δsse K1Δasd(p YA3493)血清型和强毒株SL1344及亲本株SL1344Δsse K1相同,且能稳定遗传缺失后的asd基因;其生化特性和生长速度与强毒株SL1344及亲本株SL1344Δsse K1相比均无明显差异。小鼠毒力试验表明,SL1344Δsse K1Δasd(p YA3493)口服感染6周龄BALB/c小鼠的LD50为5.24×108CFU,毒力较强毒株SL1344约下降至0.048%;免疫保护效力试验显示,运用鼠伤寒沙门菌野生毒株对免疫后第17天的小鼠攻毒有62.5%的保护率,与亲本株SL1344Δsse K1基本一致。结论:以上结果表明,鼠伤寒沙门菌分泌性蛋白K1缺失株宿主-载体平衡致死系统构建成功,且遗传稳定,毒力明显降低,具有较好的免疫原性,为深入研究以鼠伤寒沙门菌为载体的口服多价疫苗奠定基础。     

痢疾双价工程工程株安全保护基础及遗传背景分析

本文通过毒力生物表型、抗原表达及相关基因结构的研究，对痢疾双价工程菌株ＦＳ－１８、ＦＳ－１５Ａ及其回高株ＦＳ－１５Ｖ安全保护的基础及遗传背景进行了分析。认为菌株同时表达福氏及宋内氏Ｏ抗原是其双价保护的。ＦＳ－１８及其亲株４８０２５Ａ质粒缺失２０Ｋｂ左右的侵袭相关基因片段，不表达四种侵袭相关蛋白，丧失侵袭生物表型是其安全与无毒力的分子基础。ＦＳ－１５Ａ与ＦＳ－１５Ｖ侵袭基因完整，但Ｔｎ插入位点的不同     

食品与环境中豚鼠气单胞菌系统发育分析以及毒力和耐药性研究

目的食品与环境中49株豚鼠气单胞菌菌种鉴定、毒力与耐药性检测。方法VITEK、API 20NE生化鉴定,gyrB、rpoD系统发育分析;PCR检测act、alt、ast、lip、ahp、aerA、hlyA、ompA1、fla基因;AST-GN16药敏试验。结果生化鉴定为豚鼠/嗜水气单胞菌株54株,经系统发育分析豚鼠气单胞菌49株,嗜水气单胞菌4株,中国台湾省气单胞菌1株。毒力基因alt、lip、ompA1、fla、act、aerA、hlyA检出率依次为100.00%、100.00%、79.59%、14.29%、2.04%、2.04%、2.04%,未检出ast、ahp;存在4种毒力基因组合,优势组合为alt/lip/ompA1(32/49)。豚鼠气单胞菌氨苄西林、头孢唑林、头孢西丁、阿莫西林/克拉维酸、头孢曲松、复方新诺明、安曲南耐药率依次为79.59%、14.29%、10.20%、6.12%、4.08%、4.08%、2.04%,哌拉西林/他唑巴坦、亚胺培南、阿米卡星、庆大霉素、左氧氟沙星、替加环素、呋喃妥英均敏感;2株多重耐药菌株。结论gyrB、rpoD可有效鉴定豚鼠/嗜水气单胞菌,rpoD可区分近亲缘关系豚鼠与中国台湾省气单胞菌;所有豚鼠气单胞菌均携带2种以上毒力基因,1株环境源菌携带7种毒力基因;青霉素类普遍耐药,产生β-内酰胺酶是最主要耐药机制;未发现碳青霉烯类、氨基糖苷类、氟喹诺酮类、四环素类、呋喃类耐药菌株;食品和生活饮用水存在多重耐药菌。     

汉坦病毒在Vero细胞上的适应传代及疫苗候选株的 …

目的 选育出在Ｖｅｒｏ细胞上繁殖力强、免疫原性和抗原性好的汉滩病及汉城病毒候选株。方法 国内外分离的１６株汉坦病毒（ＨＶ）在Ｖｅｒｏ细胞上进行连续终末稀释传代,研究各株的繁殖特性及传代后病毒滴度及抗原量的变化。结果 经过５次终末稀释传代,选育出Ｌ９９（汉城病毒）和８４ＦＬｉ（汉滩病毒）２株滴度高且稳定的病毒,在Ｖｅｒｏ细胞上具有良好和适应性,７～１０ｄ毒力接近峰值。ＥＬＩＳＡ测抗原量高峰则在１０～     

两株B群流脑菌LOS抗原性的研究

在对我国自７０年代以来从病人和带菌者中分离的９１株Ｂ群脑膜炎奈瑟氏菌（Ｎｍ）进行脂寡糖（ＬＯＳ）免疫型分型、外膜蛋白分型／亚型、多位点酶电泳分型等的基础上，挑选了两株具有代表性的菌株３４０７（Ｂ：１５：Ｐ１．２：Ｌ３，７，９：ＲＦＬＰｂ－２０：克隆群Ｉ）和５４２８５２（Ｂ：ＮＴ：Ｐ１．２：Ｌ３，７，９：ＲＦＬＰｂ－２０：克隆群Ｉ）。提取并纯化了它们的ＬＯＳ，通过ＳＤＳ－ＰＡＧＥ分析发现：两株菌的Ｌ     

宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷…

ＦＳ５４是本室构建的宋内氏福氏２ａ双价志贺氏菌杂交株，具有志贺氏菌的毒力。本研究通过遗传重组，给ＦＳ５４株引入了ａｒｏＤ基因的转座子Ｔｎ１０插入失活性，构建了ＦＳ５４株的芳香族氨基酸营养缺陷型减毒株，并进一步去除了Ｔｎ１０所携带的四环素抗性和宋内氏Ｉ相大质粒上所携带的卡那霉素抗性，得到了宋内氏福氏２ａ双价志贺氏菌芳香族氨基酸营养缺陷型减毒     

Yersinia pestis caf1 variants and the limits of plague vaccine protection

Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.     

连续传代对鼠衣原体生长相关性状及其毒力影响的初步研究

目的探讨体外特殊条件下鼠衣原体(Chlamydia muridarum,Cm)连续传代培养后在感染性、生长发育以及致病性等生物学性状上的变化,为筛选减毒活疫苗,筛查毒力相关基因提供依据。方法将实验室Cm野生株(G0)通过常规细胞培养,在辅助或不辅助培养条件下交替连续传代后,将亲本株G0与获得的传代株G28进行衣原体感染离心依赖性、细胞吸附能力、衣原体感染细胞生长曲线、衣原体形成的噬斑大小以及两种菌株在感染小鼠生殖道后所形成的输卵管病变情况进行检测。结果与亲本株G0相比,传代株G28对感染离心条件的依赖性显著下降(P<0.05);对感染细胞的吸附能力却明显上升(P<0.05);G0与G28在感染细胞32 h内的生长曲线差异并无统计学意义;在感染细胞中所形成的噬斑大小也无明显区别;但体内毒力试验中,G0感染小鼠后致输卵管病变率为87.5%,而G28的输卵管病变率仅为37.5%,两者形成差异有统计学意义。结论Cm传代株G28与亲本株G0相比,在感染能力显著增强的情况下,其致病性却显著降低,为后续鉴定毒力基因,进一步提取基因型单一的减毒株并应用于衣原体减毒活疫苗带来了希望。     

Pathogenicity and Immunogenic Efficacy of a Live Attenuated Plague Vaccine in Vervet Monkeys

A live attenuated Yersinia pestis (Pasteurella pestis) vaccine strain designated EV51f, which had been passaged through guinea pigs previously treated with ferrous sulfate, was shown to be pathogenic for African green vervet monkeys (Cercopithecus aethips pygerythrus), but not for guinea pigs. The bacilli multiplied in the monkeys, as shown by positive blood cultures, caused an elevation of white cell counts and rectal temperatures, and resulted in death of 26% (13/50) of animals. Postmortem findings of these animals were typical of bubonic-septicemic plague. This vaccine did not cause deaths in 50 guinea pigs even in doses up to 100 million viable bacilli inoculated subcutaneously. It is suggested that the virulence of an attenuated Y. pestis strain which does not produce pigment on a defined medium containing hemin, but possesses all other known virulence determinants, is dependent on the availability of iron in vivo. The serological response of the monkeys as determined by the hemagglutinating and mouse protective antibodies was high one month after vaccination and also in guinea pigs, as shown by virulent challenge. This antibody level declined in monkeys over a period of nearly 6 months, and a decline in immunity was confirmed by virulent challenge which resulted in the death of 30% of vaccinated monkeys. The level of immunity in monkeys did not appear to be related to the dose of vaccine.     

Production in Vero cells of an inactivated rabies vaccine from strain FRV/K for animal and human use

A new concentrated and purified rabies vaccine was produced in Vero cells. Two rabies virus strains, the fixed rabies virus Pasteur (FRV) and Pittman Moore (PM) were adapted to Vero cells by 20 cycles of alternating passages in the brain of weaning mice. Intracerebral (i.c.) inoculation of weaning mice was followed then by 17 and 20 serial passages in Vero cells of RFV and PM strains, respectively. The adapted strains designated as FRV/K and PM/K gave titres of 10(6) +/- 1.5 log (LD50/ml for i.c. inoculated mice) in several harvests taken from one infected cell culture. Pooled harvests were concentrated 20-fold by ultrafiltration and were tested as animal vaccine after inactivation with beta-propiolactone (BPL). Another vaccine preparation destined for human use, in addition to concentration and inactivation, was also purified by gel filtration. Control tests revealed that the antigenic content of different strain FRV/K harvests was very high in comparison with that of strain PM/K and the reference tissue culture vaccine (RIV, Netherland). In sheep the antibody response induced by the FRV/K strain was very high; serum neutralizing index (NI) higher than 4 was reached 40 days after the second vaccine dose, whereas the vaccine preparation from strain PM/K gave NI of 2.3 and the reference vaccine NI of 3.8, respectively. Safety tests in rabbits and guinea pigs showed neither pyrogenicity nor toxicity.     

Evaluation of live attenuated plague vaccines in Praomys (Mastomys) natalensis.

A live attenuated Yersinia pestis vaccine designated EV76-51f, which had previously been shown to be pathogenic in vervet monkeys but not in guinea pigs, was tested in the multimammate mouse Praomys (Mastomys) natalensis. Doses of 10(6) viable organisms inoculated subcutaneously as either a lyophilized suspension or an agar-grown culture resulted in vaccination fatalities in Praomys but not in white mice. Hemagglutinating antibodies to the fraction 1 antigen were not stimulated by doses lower than 10(4) viable organisms. Agar-grown cultures of the vaccine gave better protection against a virulent Y. pestis challenge than did a lyophilized suspension. All Praomys vaccinated with a dose of 10(6) agar-grown EV76-51f protected against a virulent challenge, whereas even doses up to 10(8) lyophilized bacilli failed to give complete protection. The pathogenicity of a live attenuated plague vaccine derived from the Y. pestis EV76 vaccine strain can be detected in Praomys (Mastomys) natalensis, a rodent species highly susceptible to plague. This animal species may therefore be valuable for the testing of live attenuated plague vaccines before they are tested in costly nonhuman primates.     

Development of in vitro correlate assays of immunity to infection with Yersinia pestis

Pneumonic plague is a severe, rapidly progressing disease for which there is no effective vaccine. Since the efficacy of new vaccines cannot be tested in humans, it is essential to develop in vitro surrogate assays that are valid predictors of immunity. The F1 capsule antigen stimulates a protective immune response to most strains of Yersinia pestis. However, strains of Y. pestis that are F1- but still virulent have been isolated, and an in vitro assay, the results which can predict protection against both F1+ and F1- strains, is needed. The virulence antigen (V) is an essential virulence factor of Y. pestis and stimulates protective antibodies. We investigated potential correlates of plague immunity that are based on anti-V antibody-mediated neutralization of Yersinia-induced macrophage cytotoxicity. The neutralizing activity of sera from mice vaccinated with an F1-V fusion candidate vaccine was determined. The decrease in the level of the apoptosis-specific enzyme caspase-3 significantly predicted survival in one- and two-dose vaccination experiments. Sera from F1-V-vaccinated nonhuman primates were evaluated with macrophage assays based on caspase-3 and on other markers manifested at the different stages in cell death. Using murine- and human-derived macrophages in microscopic and fluorescence-activated-cell-sorting-based live/dead staining assays of terminal necrosis, we demonstrated a strong association between in vitro neutralization of macrophage cytotoxicity induced by serum-treated Yersinia and in vivo protection against lethal infection. These results provide a strong base for the development of reliable in vitro correlate bioassays that are predictive of protective immunity to plague.     

Construction and characterization of an attenuated purine auxotroph in a Francisella tularensis live vaccine strain

Francisella tularensis is a facultative intracellular pathogen and is the etiological agent of tularemia. It is capable of escaping from the phagosome, replicating to high numbers in the cytosol, and inducing apoptosis in macrophages of a variety of hosts. F. tularensis has received significant attention recently due to its potential use as a bioweapon. Currently, there is no licensed vaccine against F. tularensis, although a partially protective live vaccine strain (LVS) that is attenuated in humans but remains fully virulent for mice was previously developed. An F. tularensis LVS mutant deleted in the purMCD purine biosynthetic locus was constructed and partially characterized by using an allelic exchange strategy. The F. tularensis LVS delta purMCD mutant was auxotrophic for purines when grown in defined medium and exhibited significant attenuation in virulence when assayed in murine macrophages in vitro or in BALB/c mice. Growth and virulence defects were complemented by the addition of the purine precursor hypoxanthine or by introduction of purMCDN in trans. The F. tularensis LVS delta purMCD mutant escaped from the phagosome but failed to replicate in the cytosol or induce apoptotic and cytopathic responses in infected cells. Importantly, mice vaccinated with a low dose of the F. tularensis LVS delta purMCD mutant were fully protected against subsequent lethal challenge with the LVS parental strain. Collectively, these results suggest that F. tularensis mutants deleted in the purMCD biosynthetic locus exhibit characteristics that may warrant further investigation of their use as potential live vaccine candidates.     

A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity.     

New findings in live, attenuated hepatitis A vaccine development

Strain CR326F of hepatitis A virus, derived from a fecal specimen of Costa Rican patient 033-03, was passed 15 times in fetal rhesus monkey kidney (FRhK6) cell cultures plus eight times in human diploid lung (MRC5) cell cultures to yield variant F and 16 times in MRC5 cell cultures to yield variant F'. Both variants were purified by limit dilution passages. Virulence for marmosets was assessed at six different passage levels, including variants F and F'. There was a gradual loss of virulence with in vitro passage. Variant F retained slight virulence for marmosets; variant F' showed no evidence of virulence. Both variants induced hepatitis A antibody in most marmosets that received them, and the animals were immune to infection when challenged. Variants F and F' were also assessed in chimpanzees. As in marmosets, F retained slight virulence but F' did not. Experimental vaccines made from variants F and F' were then inoculated parenterally into adult human volunteers. A portion of recipients of variant F showed brief, low-order enzyme elevations; none was seen in recipients of F', although their occurrence could not be totally ruled out. As in the animal models, F' appeared more attenuated than F. Most persons developed hepatitis A antibody, indicating the feasibility of developing a live, attenuated hepatitis A vaccine for human beings.     

Isolation and characterization of mutant strains of Bordetella bronchiseptica lacking dermonecrotic toxin-producing ability.

Mutant strains of Bordetella bronchiseptica, named B-42, B-76, B-84, and B-119, were obtained after serial passages of a parent strain, L3, on Bordet-Gengou agar plates containing 20% horse blood and 200 micrograms of nalidixic acid per ml (BGN-20 agar plates) at 42 degrees C. Mutant strains completely lacked dermonecrotic toxin-producing ability, and lethal activity of the strains for mice was apparently reduced compared with that of strain L3. Mutant strains were able to grow at 42 degrees C, and the strains were nalidixic acid resistant. The mutant strains showed domed (Dom+) colony morphology with smooth texture (Scs+) and no production of zone of hemolysis (Hly-), but the agglutinability of these strains to antiserum prepared with Dom+ Scs+ Hly+ organisms of strain L3 was the same as that of strain L3. When strain B-42 was inoculated intramuscularly or intranasally into guinea pigs, all the animals survived without manifesting clinical signs and produced a high-level of serum agglutination antibodies against strain L3. These inoculated animals were protected against intranasal challenge with strain L3. These properties of mutant strains are hereditarily stable after 50 subcultures on BGN-20 agar plates or 20 passages in mice. These data suggest that the mutant strains lacking dermonecrotic toxin-producing ability can be used as a live attenuated vaccine against swine atrophic rhinitis.