SV40灭活疫苗的制备及其对小鼠免疫的研究

猿猴空泡病毒40(Simian vacuolating virus 40,SV40) 属于乳多空病毒科,是一种DNA肿瘤病毒.亚洲猿类特别是恒河猴是SV40的天然宿主.感染SV40病毒可导致猴体急性病变或呈长期带毒状态,此外能诱使幼鼠产生肿瘤,并能使多种培养细胞发生转化.本研究初步建立了SV40病毒在Vero细胞中的增殖培养方法,并且初步建立了β-丙内脂灭活病毒的方法和纯化工艺.使用SV40病毒灭活疫苗对Balb/c小鼠进行了免疫,结果表明该疫苗具有较好的免疫原性.随后对SV40病毒DNA在免疫小鼠的重要脏器中的整合情况进行了调查,结果表明SV40病毒DNA未在小鼠重要脏器中整合.本研究为SV40病毒灭活疫苗的研制和进一步开展猴体抗SV40感染实验奠定了良好的基础.     

鼠巨细胞病毒 IE-1核酸疫苗和灭活疫苗联合免疫抗病毒感染的研究

巨细胞病毒（Cytomegalovirus,CMV）在人群中感染普遍,对婴幼儿及免疫低下人群中造成严重疾病,目前还没有针对该病毒的商品化疫苗。本研究以BALB/c小鼠为动物模型,探讨鼠巨细胞病毒（Murine cytomega-lovirus,MCMV）IE-1 DNA疫苗和MCMV灭活疫苗联合免疫抗MCMV感染的免疫保护效果。将编码IE-1基因的DNA疫苗（pIE-1）通过肌肉注射辅以电穿孔的方式对小鼠进行初免,再用全病毒灭活疫苗单独或者辅以MF59佐剂进行加强免疫,分别通过ELISA和ELISPOT方法检测到联合免疫策略在免疫组小鼠体内诱导了MC-MV特异性的抗体应答和CTL应答;免疫两周后用3＆#215;LD50致死剂量MCMV感染小鼠,疫苗对小鼠的免疫保护通过检测小鼠存活率、重要器官中的病毒滴度及体重丢失率来评价。结果显示,与单独免疫DNA疫苗或灭活疫苗相比,IE-1 DNA疫苗联合灭活疫苗组能同时在小鼠体内诱导体液免疫和细胞免疫应答,并提供小鼠完全保护;而且MF59辅以灭活疫苗免疫小鼠能增强疫苗的免疫效果。     

SV40病毒的培养、增殖及鉴定

目的：建立SV40病毒在Vero细胞上培养的方法,观察其生长过程,获得SV40病毒,并建立相应的SV40病毒检测方法,为SV40灭活疫苗的制备奠定良好的基础。方法：在Vero细胞上培养和增殖SV40-776株病毒。收获病毒后,应用PCR、免疫荧光以及克隆特异性片段进行测序比较来鉴定SV40病毒。结果：SV40在Vero细胞中增殖很快,并且使细胞出现明显的病变。小规模分离到了SV40病毒颗粒,获得了病毒DNA。不同的鉴定方法均显示出良好的特异性。结论：探讨了SV40病毒病变的基本过程,建立了病毒的培养、增殖和鉴定的方法。     

恒河猴外周血及肾组织SV40病毒基因分析

SV40即猿猴病毒40 (simian virus 40),是DNA肿瘤病毒的原型代表,其基因结构为共价闭合环状双股DNA分子,标准参考株SV40-776含5243个核甘酸。不同分离株bp数略有差异。SV40病毒为强DNA肿瘤病毒,具有使啮齿类动物及人源多种组织培养细胞永生化和转化能力。SV40病毒早期基因编码两个早期非结构蛋白即小T抗原(ST-ag、)和大T抗原(LT-ag),与病毒诱导的肿瘤发生有关。近年来研究表明,从猴体组织新分离的SV40株与实验室参考株SV40-776及SV40-B株相比较,具有明显的遗传异质性,并且SV40遗传变     

肠道病毒71型灭活疫苗免疫恒河猴在攻毒实验中的感染动力学

本文旨在根据前期研究建立的恒河猴感染动物模型,对同期研制的肠道病毒71型(EV71)实验性灭活疫苗免疫动物进行全面的免疫保护性评价。评价指标包括病毒攻击后动物体内病毒载量及病理学变化,根据所得结果进行实验性疫苗免疫后动物在病毒攻击中的感染动力学分析。3个疫苗剂量(20、80、320EU)免疫的恒河猴均出现不同效价的中和抗体,80EU和320EU剂量组在二次免疫后第6周抗体效价达1∶128～1∶256,经104.5CCID50病毒鼻腔攻击后均未检出阳性病毒载量。20EU剂量组中,淋巴器官、中枢神经系统及其他主要脏器均出现比对照组低但仍为阳性的病毒增殖现象。病理学方面,各剂量组免疫恒河猴的中枢神经系统以及肺等器官均未出现相关病理损伤。本实验在确定该疫苗对恒河猴有效保护性的同时,亦为明确EV71灭活疫苗免疫剂量提供了直接的实验依据。     

野生及笼养猕猴SV40T抗原基因检测方法的建立

目的建立猴外周血单核细胞SV40DNA的PCR检测方法,对猕猴SV40T抗原基因进行检测。方法使用PCR方法对分别来自野外（云南宁蒗71只、景东60只）及笼养猕猴（64只）的SV40大T抗原基因进行检测,同时对扩增出的阳性结果进行测序。结果在195份样本中,有4只来自野生猴样本扩增出SV40大T抗原基因（3．1％,4／131）,1只来自笼养猴样本扩增出SV40大T抗原基因（1．6％,1／64）。测序结果显示：猴血样本扩增片段序列与GenBank中的SV40大T抗原C-末端的基因序列片段有15个核苷酸不同（3．3％差异）,与SV40．776标准株的序列基本一致,但SV40—776在nt3020处有一缺口。结论云南野生及笼养猕猴猴群均能检出SV40T抗原基因。因此建立SV40病毒的DNA检测技术,对用于科研及疫苗生产的实验猕猴的病毒学质量控制具有重要意义。     

基因操作原理(续)——第九章 在植物细胞中克隆DNA的可能载体

在原核生物细胞中,克隆外源DNA的技术比较简单,因此,目前在全世界范围内,已有数百家的实验室都能常规地进行这类实验操作。而在真核生物细胞中,克隆外源DNA的工作尚处于相当初步的阶段,这主要是由于缺乏适当的载体。当前唯一可用作真核细胞载体的是猴肾病毒SV40。关于使用SV40作载体的情况,在前一章中已经作过讨论。     

转基因动物研究现状

导言 1980年Gordon等人将疱疹病毒胸苷激酶基因和SV40早期基因启动子重组到质粒pBR322中,再由显微注射植入小鼠受精卵原核,获得第一只转基因小鼠。原理上任何克隆基因都能永久并入哺乳动物胚胎基因组。显微注射后,外源DNA整合进细胞,随后繁衍成种系。因此通过选育能永久建立携带一个或多个新基因的动物谱系。“转基因”(Transgenic)一词应运而生。在此之前,Jaenisch(1976)观察到逆转录病毒(又称逆病毒)感染小鼠胚胎,使单个前病毒DNA插入小鼠染色体DNA。而后含异源基因的重组感染性逆病毒系统的发展导致了用     

PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

两个人生长激素基因在SV40病毒重组体感染的猴细胞中的表达

我们已经建造了带有两个不同的人生长激素(hGH)基因的SV40重组体。用这些重组体感染的猴肾细胞能合成、加工并分泌人生长激素。有着与克隆的人生长激素互补DNA(eDNA)同样编码序列的基因1,共产物从几个标准来看,与垂体hGH没有什么区别。预定编码一个变异蛋白质的基因2,其产物比垂体hGH的免疫反应活性要小,但能有效地与hGH细胞表面受体结合。这些结果表明,基因2有可能表达而产生我们以前未辨别的hGH形式。这些结果显示了在真核细胞中,用基因转移的方法产生成熟的激素是可能的。这些结果也证明了SV40—猴细胞系统可以用于生产和鉴定动物细胞分泌的蛋白质。     

Genetic analysis of simian virus 40 from brains and kidneys of macaque monkeys.

Simian virus 40 (SV40) was isolated from the brains of three rhesus monkeys and the kidneys of two other rhesus monkeys with simian immunodeficiency virus-induced immunodeficiency. A striking feature of these five cases was the tissue specificity of the SV40 replication. SV40 was also isolated from the kidney of a Taiwanese rock macaque with immunodeficiency probably caused by type D retrovirus infection. Multiple full-length clones were derived from all six fresh SV40 isolates, and two separate regions of their genomes were sequenced: the origin (ori)-enhancer region and the coding region for the carboxy terminus of T antigen (T-ag). None of the 23 clones analyzed had two 72-bp enhancer elements as are present in the commonly used laboratory strain 776 of SV40; 22 of these 23 clones were identical in their ori-enhancer sequences, and these had only a single 72-bp enhancer element. We found no evidence for differences in ori-enhancer sequences associated with tissue-specific SV40 replication. The T-ag coding sequence that was analyzed was identical in all clones from kidney. However, significant variation was observed in the carboxy-terminal region of T-ag in SV40 isolated from brain tissues. This sequence variation was located in a region previously reported to be responsible for SV40 host range in cultured cell lines. Thus, SV40 appears to be an opportunistic pathogen in the setting of simian immunodeficiency virus-induced immunodeficiency, similarly to JC virus in human immunodeficiency virus-infected humans, the enhancer sequence organization generally attributed to SV40 is not representative of natural SV40 isolates, and sequence variation near the carboxy terminus of T-ag may play a role in tissue-specific replication of SV40.     

Rearrangement of simian virus 40 regulatory region is not required for induction of progressive multifocal leukoencephalopathy in immunosuppressed rhesus monkeys

Rearrangements of the JC virus (JCV) regulatory region (RR) are consistently found in the brains of patients with progressive multifocal leukoencephalopathy (PML), whereas the archetype RR is present in their kidneys. In addition, the C terminus of the large T antigen (T-Ag) shows greater variability in PML than does the rest of the coding region. To determine whether similar changes in simian virus 40 (SV40) are necessary for disease induction in monkeys, we sequenced the SV40 RR and the C terminus of the T-Ag from the brain of simian/human immunodeficiency virus (SHIV)-infected monkey 18429, which presented spontaneously with an SV40-associated PML-like disease, as well as from the peripheral blood mononuclear cells (PBMC), kidneys, and brains of SV40-seronegative, SHIV-infected monkeys 21289 and 21306, which were inoculated with the 18429 brain SV40 isolate. These animals developed both SV40-associated PML and meningoencephalitis. Thirteen types of SV40 RR were characterized. Compared to the SV40 archetype, we identified RRs with variable deletions in either the origin of replication, the 21-bp repeat elements, or the late promoter, as well as deletions or duplications of the 72-bp enhancer. The archetype was the most prominent RR in the brain of monkey 18429. Shortly after inoculation, a wide range of RRs could be found in the PBMC of monkeys 21289 and 21306. However, the archetype RR became the predominant type in their blood, kidneys, and brains at the time of sacrifice. On the contrary, the T-Ag C termini remained identical in all compartments of the three animals. These results indicate that unlike JCV in humans, rearrangements of SV40 RR are not required for brain disease induction in immunosuppressed monkeys.     

Squirrel monkeys support replication of BK virus more efficiently than simian virus 40: an animal model for human BK virus infection

We performed experiments to test the suitability of squirrel monkeys (Saimiri sciureus) as an experimental model for BK virus (BKV) and simian virus 40 (SV40) infection. Four squirrel monkeys received intravenous inoculation with BKV Gardner strain, and six squirrel monkeys received intravenous inoculation with SV40 777 strain. Eight of 10 monkeys received immunosuppression therapy, namely, cyclophosphamide subcutaneously either before or both before and after viral inoculation. The presence of viral infection was assessed by quantitative real-time PCR amplification of viral DNA from blood, urine, and 10 tissues. We found that squirrel monkeys were susceptible to infection with BKV, with high viral copy number detected in blood and viral genome detected in all tissues examined. BKV genome was detected in urine from only one monkey, while three monkeys manifested focal interstitial nephritis. BKV T antigen was expressed in renal peritubular capillary endothelial cells. By contrast, SV40 was detected at very low copy numbers in only a few tissues and was not detected in blood. We conclude that the squirrel monkey is a suitable animal for studies of experimental BKV infection and may facilitate studies of viral entry, pathogenesis, and therapy.     

Infection of newborn Taiwan monkeys (Macaca cyclopis) with human adenovirus type 12 and simian virus 40.

For testing biological response of newborn Taiwan monkeys to infection of human adenovirus type 12 (Ad12) and simian virus 40 (SV40), 40 newborn Taiwan monkeys were inoculated with Ad12, Ad12, plus SV40, Ad12 supplemented with Ad12-induced hamster tumor tissue (Ad12 tumor) or control specimens (HeLa or African green monkey kidney cell lysate). Among them 26 survived including 8 newborn monkeys inoculated with control specimens. The survivors were observed for 4 years but no tumor was produced. The increase of body weight and intake of calories and protein in each test group during the first 12 wk were similar to those of corresponding control groups. Intrapulmonary inoculation of 108.2TCD50 of Ad12 with additional subcutaneous dose of Ad12 (108.8TCD50), Ad12 plus SV40 (108TCD50) or Ad12 plus Ad12 tumor killed 78% of newborn monkeys (7 of 9) in 18 days. The newborn could stand subcutaneous inoculation of SV40 (108TCD50) with 1 dose of Ad12 (108.8TCD50) or 3 doses of Ad12 (108.5, 108.5 and 108.2TCD50) at 24-hr intervals. When 108.8TCD50 or more Ad12 were inoculated, the virus could be isolated as late as 44 and 26 days from rectal and throat swab specimens respectively. The Ad12 neutralizing antibody in baby monkeys inoculated with multiple doses of Ad12 persisted, in low titer, longer than those injected with single high doses of Ad12, but anti-Ad12 T (tumor) antibody disappeared by 35 wk in both groups. Although SV40 antibody response was better than Ad12 antibody response in baby Taiwan monkeys, pre-infection of SV40 did not potentiate the production of anti-Ad12 T antibody.     

A serological survey of simian virus 40 in monkeys

A serological survey of simian virus 40 (SV 40) was conducted by an immune adherence hemagglutination (IAHA) test in breeding monkeys. Of a total of 356 monkeys tested, 168 (47.2%) were seropositive. All 168 seropositive monkeys were detected from 224 monkeys which were bred or kept in Japan for a long time. In contrast, none of the 132 monkeys which were newly imported from Southeast Asia was seropositive. If a comparison was made in the same breeding place, the positive rate of 80.4% (111/138) of Japanese monkeys was significantly (P less than 0.01) higher than the 59.5% (25/42) among rhesus monkeys. The positive rate and the IAHA titers were higher in older age group (greater than 5 years) but similar in male and female. These results indicated that SV 40 was highly prevalent among breeding monkeys in Japan.     

Studies on Nondefective Adenovirus-Simian Virus 40 Hybrid Viruses I. A Newly Characterized Simian Virus 40 Antigen Induced by the Ad2+ND1 Virus

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), does not induce heat-labile SV40 T antigen but does induce a previously uncharacterized heat-stable SV40 antigen-the SV40 "U" antigen. This antigen is detectable by both immunofluorescence and complement fixation by using sera from hamsters with SV40 tumors. Sera from hamsters bearing SV40 tumors can be divided into two groups, those that react with both SV40 T and U antigens (T(+)U(+) sera) and those that react with SV40 T antigen only (T(+)U(-) sera). SV40 U-specific sera from monkeys immunized with Ad2(+)ND(1)-infected cells do not react with SV40 T antigen by immunofluorescence but do react with an antigen in the nucleus of SV40-transformed cells and with an early, cytosine arabinoside-resistant antigen present in the nucleus of SV40-infected cells. A heat-stable SV40 antigen detectable by complement fixation with T(+)U(+) hamster sera is present in extracts of SV40-induced hamster tumors and in cell packs of SV40-infected or -transformed cells. SV40 U-antigen synthesis by Ad2(+)ND(1) virus is partially sensitive to inhibitors of deoxyribonucleic acid synthesis, whereas U-antigen synthesis by SV40 virus is an early cytosine arabinoside-resistant event. As an early SV40 antigen differing from SV40 T antigen, U antigen may play a role in malignant transformation mediated by SV40.     

Airborne Stability of Simian Virus 40

The influence of relative humidity on the airborne survival of simian virus 40 (SV40) was studied by allowing virus aerosols to age in rotating drums at 21 or 32 C and at a relative humidity (RH) value ranging from 22 to 88%. Airborne SV40 virus was stable at every RH tested at 21 C, but aerosols maintained at 32 C were inactivated within 60 min at mid-range RH values. The unusual stability at 21 C over a broad RH range indicates that potentially biohazardous situations may occur under laboratory conditions if this virus becomes accidentally airborne.     

Inactivation of T Antigen-Forming Capacities of Simian Virus 40 and Adenovirus 12 by Ultraviolet Irradiation

Methods to measure T antigen-forming capacities of simian virus 40 (SV40) and adenovirus 12 (Ad12) were investigated, and a method to measure the capacity in terms of T antigen-forming units was employed by the use of cytosine arabinoside. Plaque-forming units and T antigen-forming units of SV40, SV40 deoxyribonucleic acid, or Ad12 were inactivated by ultraviolet (UV) irradiation at the same rate, roughly following a single-hit curve. T-antigen formation by UV-irradiated SV40 and Ad12 was enhanced in cells multiply infected and in cells in a growing state. These observations showed that it was difficult or impossible to estimate the size of the gene for T antigen by UV inactivation.     

Transformation of primary rat kidney cells by fragments of simian virus 40 DNA.

Linear simian virus 40 (SV40) DNA molecules of genome length and DNA fragments smaller than genome length when prepared with restriction endonucleases and tested for transforming activity on primary cultures of baby rat kidney cells. The linear molecules of genome length (prepared with endonucleases R-EcoRI, R-BamHI, and R-HpaII or R-HapII), a 74% fragment (EcoRI/HpaII or HapII-A), and a 59% fragment (BamHI/HapII-A) could all transform rat kidney cells with the same efficiency as circular SV40 DNA. All transformed lines tested contained the SV40-specific T-antigen in 90 to 100% of the cells, which was taken as evidence that the transformation was SV40 specific. The DNA fragments with transforming activity contained the entire early region of SV40 DNA. Endo R-HpaI, which introduced one break in the early region, apparently inactivated the transforming capacity of SV40 DNA, since no transformation was observed with any of the three HpaI fragments tested. Attempts were made to rescue infectious virus from some of the transformed lines by fusion with permissive BSC-1 cells. Infectious virus was only recovered from the cells transformed by circular form I DNA. No infectious virus could be isolated from any of the other types of transformed cells.