模拟HIV性传播特点的SIVmac239恒河猴直肠黏膜感染

目的制备SIVmac239恒河猴(Macaca mulatta)细胞适应株病毒,模拟HIV性传播感染特点进行恒河猴直肠黏膜感染研究,探索引起系统性感染的病毒阈值水平与机体病毒、免疫学之间相关性,为我国艾滋病黏膜疫苗等生物制剂有效性评价提供新的模型构建思路。方法参照HIV性传播自然感染剂量范围,选用SIVmac239连续升高的3种剂量直肠黏膜途径感染两只恒河猴,采取多种方法进行病毒血症和免疫反应特点分析。结果两只恒河猴经2×101TCID50和2×102TCID50病毒滴度2次攻击后45d,经检测均未建立系统性感染,病毒特异性免疫反应均为阴性;第3次2×103TCID50病毒滴度攻击后,M296猴表现出典型的系统性感染特点,并诱导特异性免疫反应。结论确认了HIV性传播过程中的病毒剂量效应关系,为预防性生物制剂的猴体有效性评价提供了新的思路。同时,发现SIVmac239Gag区特异性的T细胞免疫反应在病毒控制过程中发挥了关键作用,对于新一代艾滋病黏膜疫苗的抗原选择具有指导性意义。     

表现神经症状的SIVmac251感染猴大脑基底节病毒gp120序列变异分析

目的 研究猴免疫缺陷病毒SIVmac251在中国恒河猴感染传代过程中产生的可能的神经侵袭性和神经嗜性及其分子机制.方法 从静脉感染SIVmac251-155p6N的8只实验猴中出现严重神经症状的1只猴中,监测病毒及免疫指标变化,观察临床症状、猴脑组织病变,单拷贝PCR扩增病毒gp120序列并分析变异及糖基化位点变化情况.结果 感染猴晚期出现明显艾滋病脑病症状,病理切片显示脑组织出现多核巨细胞及神经元变性、坏死.脑基底节分离出单一序列病毒,其氨基酸序列与血浆病毒及感染毒株SlVmac251-155p6序列差异主要位于Gp120的V1和V4区,并且在C1区66位出现一个糖基化位点缺失.结论 SIVmac251在猴体长期传代过程中表现出神经嗜性毒株的特征,对AIDS脑病研究具有重要意义.     

猴免疫缺陷病毒（SIV）感染恒河猴血液学和血液生化的动态变化

目的探讨SIVmac239感染中国恒河猴的血液学和血清生物化学的变化,研究艾滋病的发生机制。方法 20只恒河猴感染SIVmac239后分别在感染前和感染后2、6、9、12、15和18个月在猴空腹状态用盐酸氯胺酮麻醉下静脉釆血。抗凝血用全自动血液细胞分析仪检测血液常规,血清用全自动生化分析仪检测生化指标。结果SIV感染猴红白细胞计数呈逐渐的减少。淋巴细胞计数有较明显减/增的波动。血清酶类ALT和AST没有明显的上升,但在15月时均有所下降P〈0.05～P〈0.01。LDH持续降低,原因未明。CK则逐月上升,至12月时达高峰,后又有所下降。血尿素氮逐月上升,并在6～18个月时P〈0.01。总蛋白量上升,白蛋白逐月降低,球蛋白升高,白蛋白/球蛋白比率倒置。SIV感染猴2个月后总胆固醇、甘油三脂、低密度脂蛋白和高密度脂蛋白均呈上升,至12月时略有下降,但到18月又上升。血糖（GLU）在SIV感染后逐月上升,后期上升更为明显,这可能与胰岛的损害有关。结论 SIVmac239感染猴后,通过SIV侵入相关的器官病变和体液调节失衡导致血液学和血液生物化学产生异常,从而提供有关猴艾滋病的一些基础资料,这将对研究人的艾滋病也许有所帮助。     

SIVmac239感染恒河猴急性期回肠派氏淋巴结中淋巴细胞亚群的变化

目的分析SIVmac239感染早期中国恒河猴回肠派氏淋巴结淋巴细胞数量及亚群的变化,探讨这些变化与疾病进展的可能关系。方法以静脉注射SIVmac239制备恒河猴AIDS模型,对回肠派氏淋巴结进行CD4和CD8免疫组化标记,分离Peyer’s集合淋巴结淋巴细胞,分别标记CD3、CD4、CD8、CD28、CD95单克隆抗体,以流式细胞仪检测T细胞及其亚群的表达情况。结果 SIVmac239感染急性期中国恒河猴Peyer淋巴结中CD4＋/CD8＋比值持续下降,记忆性细胞比例升高,但Peyer淋巴结形态及CD4＋T细胞数量未见明显变化,CD8＋T细胞从第5天开始持续升高。结论 SIVmac239感染急性期,中国恒河猴回肠派氏淋巴结形态及CD4＋T细胞数量基本维持,向记忆性细胞的转化增加,但是CD4＋/CD8＋比值下降。     

猴免疫缺陷病毒（SIV）在艾滋病模型恒河猴组织中的分布和载量测定

目的研究当艾滋病恒河猴模型的血浆病毒载量处于低水平或阴性时,猴免疫缺陷病毒（simian immunodeficiency viruses,SIV）在宿主组织中的分布情况。方法SIVmac251感染恒河猴10只,定期检测其血浆载量,感染病毒平均高峰时间第14天时,活检取淋巴结。选取感染18个月后病毒载量最低水平和阴性的2只艾滋病猴（SAIDS）,经安死术后取淋巴结、脾、肝、肺、肾、脑等组织,用原位杂交和实时荧光定量PCR的方法检测病毒在组织中的分布和组织中的病毒载量。结果感染后14d,10只猴血浆病毒载量达到10^7copies/mL,淋巴结组织病毒载量为10^5-10^8copies/g,原位杂交方法在腹股沟淋巴结中检测到强阳性斑点。感染后第18个月的2只猴,血浆病毒载量下降并维持不高于10^2copies/mL水平或阴性,但组织分布不尽相同,在肠系膜淋巴结、肾上腺、海马回、空肠、脾脏等组织中检测到10^5-10^6copies/g的病毒载量,于一只猴的脑积液中检测到10^3copies/mL的病毒载量。用原位杂交的方法在肠系膜淋巴结和空肠中检测到强阳性斑点,其它组织中未检测到阳性斑点。结论实验证实SAIDS猴在血浆病毒载量低甚至阴性时,病毒在不同组织中仍有分布,有些组织中甚至出现高病毒载量,提示在制备SIV/SAIDS模型中,尤其在药物筛选和疫苗评价时,应考虑组织病毒载量指标的测定和药物、疫苗对组织病毒的治疗清除作用的评价。     

ELISPOT方法检测SIVmac239感染猴特异性细胞免疫水平

目的为了完善现有的SIV／恒河猴模型,掌握恒河猴被SIV感染后体内细胞免疫应答状态,为评价HIV疫苗提供方法和数据上的参考,我们测定了SIV感染猴体内病毒特异性的细胞免疫水平。方法实验前选出4只无SIV、sTLV、SRV／D和B病毒感染的恒河猴,用SIVmac239病毒液静脉感染实验猴,使用RT-PCR、流氏细胞术和ELISPOT等方法,监测SIVmac239病毒在恒河猴体内复制情况、感染猴的外周免疫损伤情况和细胞免疫情况,持续测定一年。结果实验结果显示IFN-γ ELISPOT方法能有效的评估实验猴的细胞免疫情况,IFN—YELISPOT结果和CD4＋T细胞数无相关性,与血浆病毒载量稍有相关。结论本实验明确了SIVmac239感染中国恒河猴体内CTL的基本趋势和范围,了解了外周血病毒载量、外周免疫损伤与细胞免疫状况之间的联系,完善了SIV／SAIDS模型评价指标,为使用此模型评价抗病毒药物或疫苗提供了基础条件。     

葡萄糖代谢异常导致SIVmac239感染北平顶猴急性期体重变化

目的探讨艾滋病急性期炎症因子、脂类代谢及葡萄糖代谢对体重的影响。方法检测SIVmac239感染北平顶猴急性期炎症因子(TNF-α、IL-6)、脂类代谢(胆固醇、三酰甘油、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇)及葡萄糖代谢参数(胰岛素、胰岛素抵抗指数),比较体重增加组和体重减少组SIV感染北平顶猴之间的差异,运用Pearson相关分析探讨胰岛素抵抗与血浆病毒载量的相关性。结果在SIVmac239感染北平顶猴急性期,炎症因子和血脂四项在体重增加组与体重减少组之间无明显差异;体重增加组胰岛素水平明显下降,体重减少组胰岛素水平明显升高,并出现胰岛素抵抗表现;Pearson分析显示,感染后第5周及第11周胰岛素抵抗指数变化与血浆病毒载量呈正相关。结论 SIVmac239感染急性期,葡萄糖代谢异常可能是导致北平顶猴体重变化的主要原因。     

恒河猴艾滋病模型中枢神经系统及淋巴结病变的电镜观察

目的观察恒河猴艾滋病模型不同感染阶段中枢神经系统及淋巴结的超微结构改变,探讨艾滋病的发病机制和病理改变的发展过程。方法 15只恒河猴,1只为正常对照,其余14只静脉注射感染SIVmac239猴艾滋病病毒,分别于感染后1周,2周,1个月,2个月,12个月,18个月取腹股沟淋巴结及下丘脑组织进行透射电镜检查。结果感染后1周即可出现淋巴结内淋巴细胞病变,表现为线粒体肿胀,嵴溶解,出现自噬体等。感染后2周及1个月时淋巴细胞病变主要表现为线粒体肿胀,嵴溶解;感染2个月时淋巴细胞细胞核形态出现明显改变。感染后12个月淋巴结内多数淋巴细胞出现病变,细胞肿胀,细胞器正常形态大部分消失;部分细胞出现溶解性坏死的特征。至感染后期（18个月时）,淋巴结内局部淋巴细胞稀疏,细胞核肿胀或形态不规则。中枢神经系统的病变在感染后1周出现,表现为神经纤维肿胀,感染后2周出现神经元的病变,表现为神经元线粒体肿胀,粗面内质网脱颗粒,尼氏体消失;神经纤维内出现空泡。感染后12个月及18个月时,神经元和神经纤维的病变加重。结论在SIV感染早期,淋巴结内淋巴细胞及中枢神经系统即可出现病变,维持一段相对稳定的时间后,至疾病后期,病变加重。中枢神经系统与淋巴结的超微结构病变发展规律有一定的相似性。     

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。     

SIVmac239病毒经不同途径感染恒河猴急性期CD4~＋T细胞数量与其细胞表面CD95表达量的变化关系

目的研究SIVmac239病毒分别通过直肠（rectal infection：IR）及静脉（intravenous infection：IV）感染恒河猴,在感染急性期外周血CD4＋T细胞数量与其细胞表面的死亡受体（CD95）表达量之间的变化关系。方法将SIVmac239经直肠及静脉途径各感染14只恒河猴。监测病毒载量、CD4＋T淋巴细胞数量及CD4＋/CD8＋比值的变化,从而明确感染。同时,在感染急性期内9个时间点采集静脉血,并利用流式细胞术分析CD4＋T细胞表面CD95的表达量。结果静脉组恒河猴CD95表达量于第2天开始升高,第7天达到最高,同时CD4＋T淋巴细胞数降到最低。直肠组恒河猴也于感染后第2天开始升高,但第10天才达到最高值,CD4＋T淋巴细胞数于第17天降到最低。同静脉组相比,直肠组CD95表达量增高及CD4＋T细胞数降低均较晚出现,且其CD4＋T细胞数量降至最低晚于CD95表达量达到峰值一周后出现。结论 SIVmac239经直肠及静脉感染恒河猴后,伴随CD4＋T细胞表面CD95表达的升高,CD4＋T细胞数量逐渐下降。但不同感染途径对CD4＋T细胞表面CD95的表达量与CD4＋T淋巴细胞数的变化不尽相同,这可能由于不同感染途径造成CD95在感染急性期CD4＋T细胞数量降低中发挥的作用不同。     

Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research,two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4 T lymphocyte in peripheral blood,plasma viral loads,proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection,proviral DNA had been detected in PBMCs,and infectious SIVmac239 virus had been isolated from PBMCs. At the same period,the numbers of CD4 T lymphocytes were significantly decreased,and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover,antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.     

Analysis of humoral immune responses in rhesus macaques vaccinated with attenuated SIVmac239Δnef and challenged with pathogenic SIVmac251

Background To determine the correlation between protection and humoral immune response against simian immunodeficiency virus (SIVmac251), 11 macaques were immunized with live‐attenuated SIVmac239Δnef either intravenously or via the tonsils and exposed to SIVmac251 after either 6 or 15 months along with unvaccinated controls. Results Independent of the route of vaccine application, viremia was significantly reduced in vaccinees compared with controls 2 weeks post‐challenge. Concomitantly, viremia correlated inversely with SIV‐specific IgG, complement‐mediated lysis and neutralizing antibodies and these parameters seemed to contribute to reduced viremia. During chronic infection, six monkeys controlled viremia in the circulation (two or fewer infectious units per 10⁶ PBMCs) and showed no signs of trapping in lymphatic tissues (Appendix S1). Conclusions As no significant differences were observed throughout the study, with respect to the humoral immune response and viremia control, between the two vaccinated cohorts, mucosal immunization strategies are recommended due to more simplified application.     

Vaccine protection by a triple deletion mutant of simian immunodeficiency virus.

Twelve rhesus monkeys were vaccinated with SIVmac316 delta nef (lacking nef sequences), and 12 were vaccinated with SIVmac239 delta3 (lacking nef, vpr, and upstream sequences in U3). SIVmac316 and SIVmac239 differ by only eight amino acids in the envelope; these changes render SIVmac316 highly competent for replication in macrophages. Seventeen of the animals developed persistent infections with the vaccine viruses. Seven of the 24 vaccinated animals, however, developed infections that were apparently transient in nature. Six of these seven yielded virus from peripheral blood when tested at weeks 2 and/or 3, three of the seven had transient antibody responses, but none of the seven had persisting antibody responses. The 24 monkeys were challenged in groups of four with 10 rhesus monkey infectious doses of wild-type, pathogenic SIVmac251 at weeks 8, 20, and 79 following receipt of vaccine. None of the seven with apparently transient infections with vaccine virus were protected upon subsequent challenge. Analysis of cell-associated viral loads, CD4+ cell counts, and viral gene sequences present in peripheral blood in the remainder of the monkeys following challenge allowed a number of conclusions. (i) There was a trend toward increased protection with length of time of vaccination. (ii) Solid vaccine protection was achieved by 79 weeks with the highly attenuated SIV239 delta3. (iii) Solid long-term protection was achieved in at least two animals in the absence of complete sterilizing immunity. (iv) Genetic backbone appeared to influence protective capacity; animals vaccinated with SIV239 delta3 were better protected than animals receiving SIV316 delta nef. This better protection correlated with increased levels of the replicating vaccine strain. (v) The titer of virus-neutralizing activity in serum on the day of challenge correlated with protection when measured against a primary stock of SIVmac251 but not when measured against a laboratory-passaged stock. The level of binding antibodies to whole virus by enzyme-linked immunosorbent assay also correlated with protection.     

Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac.

To identify viral determinants of simian immunodeficiency virus (SIV) virulence, two pairs of reciprocal recombinants constructed from a pathogenic (SIVmac239) and a nonpathogenic (SIVmac1A11) molecular clone of SIV were tested in rhesus macaques. A large 6.2-kb fragment containing gag, pol, env, and the regulatory genes from each of the cloned (parental) viruses was exchanged to produce one pair of recombinant viruses (designated SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11gag-env/239 to indicate the genetic origins of the 5'/internal/3' regions, respectively, of the virus). A smaller 1.4-kb fragment containing the external env domain of each of the parental viruses was exchanged to create the second pair (SIVmac1A11/239env/1A11 and SIVmac239/1A11env/239) of recombinant viruses. Each of the two parental and four recombinant viruses was inoculated intravenously into four rhesus macaques, and all 24 animals were viremic by 4 weeks postinoculation (p.i.). Virus could not be isolated from peripheral blood mononuclear cells (PBMC) of any animals infected with SIVmac1A11 after 6 weeks p.i. but was consistently isolated from all macaques inoculated with SIVmac239 for 92 weeks p.i. Virus isolation was variable from animals infected with recombinant viruses; SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11env/239 were isolated most frequently. Animals inoculated with SIVmac239 had 10 to 100 times more virus-infected PBMC than those infected with recombinant viruses. Three animals infected with SIVmac239 died with simian AIDS (SAIDS) during the 2-year observation period after inoculation, and the fourth SIVmac239-infected animal had clinical signs of SAIDS. Two animals infected with recombinant viruses died with SAIDS; one was infected with SIVmac239/1A11gag-env/239, and the other was infected with SIVmac1A11/239gag-env/1A11. The remaining 18 macaques remained healthy by 2 years p.i., and 13 were aviremic. One year after inoculation, peripheral lymph nodes of some of these healthy, aviremic animals harbored infected cells. All animals seroconverted within the first few weeks of infection, and the magnitude of antibody response to SIV was proportional to the levels and duration of viremia. Virus-suppressive PBMC were detected within 2 to 4 weeks p.i. in all animals but tended to decline as viremia disappeared. There was no association of levels of cell-mediated virus-suppressive activity and either virus load or disease progression. Taken together, these results indicate that differences in more than one region of the viral genome are responsible for the lack of virulence of SIVmac1A11.     

Strain-specific neutralizing determinant in the transmembrane protein of simian immunodeficiency virus.

Monoclonal antibody SF8/5E11, which recognizes the transmembrane protein (TMP) of simian immunodeficiency virus of macaque monkeys (SIVmac), displayed strict strain specificity. It reacted with cloned and uncloned SIVmac251 but not with cloned SIVmac142 and SIVmac239 on immunoblots. This monoclonal antibody neutralized infection by cloned, cell-free SIVmac251 and inhibited formation of syncytia by cloned SIVmac251-infected cells; these activities were specific to cloned SIVmac251 and did not occur with the other viruses. Site-specific mutagenesis was used to show that TMP amino acids 106 to 110 (Asp-Trp-Asn-Asn-Asp) determined the strain specificity of the monoclonal antibody. This strain-specific neutralizing determinant is located within a variable region of SIVmac and human immunodeficiency virus type 2 (HIV-2) which includes conserved, clustered sites for N-linked glycosylation. The determinant corresponds exactly to a variable, weak neutralizing epitope in HIV-1 TMP which also includes conserved, clustered sites for N-linked glycosylation. Thus, the location of at least one neutralizing epitope appears to be common to both SIVmac and HIV-1. Our results suggest a role for this determinant in the viral entry process. Genetic variation was observed in this neutralizing determinant following infection of a rhesus monkey with molecularly cloned SIVmac239; variant forms of the strain-specific, neutralizing determinant accumulated during persistent infection in vivo. Selective pressure from the host immune response in vivo may result in sequence variation in this neutralizing determinant.     

Establishment of AIDS animal model with SIVmac239 infected Chinese rhesus monkey

In the present research, two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID₅₀ of SIVmac239. The changes in the numbers of CD4⁺ T lymphocyte in peripheral blood, plasma viral loads, proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection, proviral DNA had been detected in PBMCs, and infectious SIVmac239 virus had been isolated from PBMCs. At the same period, the numbers of CD4⁺ T lymphocytes were significantly decreased, and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover, antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches. These authors contributed equally to this work.     

Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac.

Infection of macaque monkeys with simian immunodeficiency virus (SIV) is probably the best animal model currently available for studying acquired immunodeficiency syndrome. In this report, we describe three infectious molecular clones of SIVmac and one of human immunodeficiency virus type 2 (HIV-2) and their use in the study of cell and species specificity, animal infection, and the relationship of gene sequence to function. Replication of the cloned viruses in different cell lines varied dramatically. Some human CD4+ cell lines (HUT 78 and MT-4) supported the replication of SIVmac and HIV-2, while others (CEM and Jurkat-T) supported the replication of HIV-2 but not SIVmac. Growth of cloned virus in macaque lymphocytes in vitro was predictive of macaque infection in vivo. Macaque lymphocytes supported the replication of SIVmac239 and SIVmac251 but not SIVmac142 or HIV-2ROD. Using virus recovery and antibody response as criteria for infection, macaques that received cloned SIVmac251 and SIVmac239 became infected, while macaques receiving cloned SIVmac142 and HIV-2ROD did not become infected. Nucleotide sequences from the envelope region of all four cloned viruses demonstrated that there is considerable flexibility in the location of the translational termination (stop) signal. These infectious molecular clones will be very useful for future studies directed at the molecular basis for persistence, pathogenicity, tropism, and cell and species specificity.     

Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus.

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.