SHIV1157ipd3N4中国恒河猴细胞适应株静脉感染中国恒河猴有效浓度的确定

目的确定SHIV1157ipd3N4静脉途径感染中国恒河猴的有效病毒浓度,明确SHIV1157ipd3N4感染实验猴体内病毒复制和免疫损伤情况。方法 10只正常中国恒河猴分成6组,分别用10倍系列稀释的病毒液1 mL静脉感染,测定血浆病毒载量,CD4＋/CD8＋,CD4＋T淋巴细胞绝对数,分析感染后恒河猴体内病毒复制和免疫损伤情况。结果 5TCID50/mL以上浓度的SHIV1157ipd3N4能通过静脉途径感染中国恒河猴。结论该实验的成功进行为SHIV/中国恒河猴疾病及评价模型的建立奠定了良好的基础,为今后使用此模型评价抗病毒药物或疫苗提供了条件。     

RT-SHIV中国恒河猴适应株细胞水平生物学特性分析

目的体外增值、制备动物感染来源的RT-SHIV病毒中国恒河猴适应株,比较PBMCs和CEMx174两种细胞制备出病毒的差异,同时用TZM-bl、CEMx174、PBMC三种细胞滴定测定病毒TCID50。方法用RT-SHIV病毒静脉感染中国恒河猴,定期采血测定血浆病毒载量,当病毒载量达高峰时采血分离外周血单核淋巴细胞（PBMCs）,与正常恒河猴PBMCs或CEMx174细胞共培养,定期测定培养液中的P24抗原水平,当病毒复制达高峰期时收集培养上清,分装并冻存;测定病毒RNA载量、P24抗原浓度,滴定病毒的TCID50。结果本研究共制备了78 mL PBMCs来源的RT-SHIV病毒和85 mL CEMx174细胞来源的RT-SHIV病毒。RT基因序列和原始序列的相似度为99%,仅在第254和265位的氨基酸发现突变。RT-SHIV（PBMC）和RT-SHIV（CEMx174）病毒载量分别为1.641×108 copies/mL和8.375×108 copies/mL,P24抗原水平分别为20.745 ng/mL和4.28 ng/mL,TZM-bl、CEMx174、PBMC细胞测定病毒的TCID50分别为3.16×105 TCID50/mL和1×104 TCID50/mL,5×102 TCID50/mL和5×105 TCID50/mL,5×102 TCID50/mL和5×103 TCID50/mL。结论 PBMCs细胞来源制备的病毒较CEMx174制备的病毒具有更高的感染性。     

SHIV-KB9感染中国恒河猴动物模型的建立

目的为了进一步确证SHIV-KB9感染中国恒河猴的病毒浓度范围,测试动物对病毒的适应性,明确该动物模型的可重复性。方法实验前采集猴血清并进行血清学检查。选出4只无SIV、STLV、SRV/D和B病毒感染的恒河猴,分别用10倍系列稀释的病毒液静脉感染实验猴,使用流氏细胞术、血常规、病毒分离、DNA-PCR和RT-PCR等方法确定实验猴是否被感染,以及感染后恒河猴体内病毒复制和免疫细胞损伤情况。结果实验猴的血浆病毒载量、病毒分离结果、CD4＋/CD8＋比值和CD4＋T细胞数等证实,4.8×105 copies/mL以上浓度的SHIV-KB9病毒液能成功感染中国恒河猴。结论本研究进一步明确了SHIV-KB9感染中国恒河猴的有效病毒浓度范围,确定了SHIV-KB9病毒感染中国恒河猴的病毒学、免疫学的测定指标,成功的建立了SHIV-KB9/中国恒河猴动物模型。     

体内CD8^＋T细胞剔除对无症状期SHIV感染猴的影响

目的 CD8＋T细胞在一些病毒感染疾病的免疫反应中起着重要的作用,但CD8＋T细胞在HIV无症状期的作用尚不明确,本研究通过体内CD8＋T细胞剔除,研究CD8＋T细胞对SHIV感染猴的影响,进一步了解艾滋病的发病机制。方法选择8只SHIV病毒感染的恒河猴,均处于无症状期,随机分成两组,实验组4只恒河猴在0、3、7 d注射抗CD8＋T抗体cM-T807,不同的时间取外周血、腹股沟淋巴结。流式细胞术测定恒河猴外周血和淋巴结中CD8＋T细胞数目,Real-time RT-PCR法测定实验猴血浆病毒载量,并使用IFN-γElispot方法测定其对猴细胞免疫的影响。结果 CD8＋T细胞敲除后,4只猴的病毒载量都转阳,但反应性不一,HIV-1的靶细胞CD4＋T细胞有轻微下降,后反弹,与病毒载量无相关性;CD8敲除猴的感染情况（血浆病毒载量和CD4细胞）比SHIV病毒急性感染轻,这与ELIPOT结果一致。结论 CD8＋T细胞在HIV无症状期发挥重要的作用,但其作用具有个体差别。     

中国恒河猴Mamu-A*01基因筛查及其对抗原肽p11C的特异性CTL反应

目的 筛查中国恒河猴Mamu-A*01基因,比较中国恒河猴和印度恒河猴的Mamu-A*01基因序列和功能是否相同.方法 PCR方法检测128只中国恒河猴,用特异性引物扩增Mamu-A*01基因,将PCR扩增后的产物克隆测序后与印度恒河猴的Mamu-A*01基因进行同源比对;酶联免疫斑点检测 (ELISPOT) 方法分别检测5只Mamu-A*01基因阳性和5只阴性恒河猴针对SIV、SHIV抗原肽p11C的特异性CTL反应.结果 共筛查出5 只Mamu-A*01基因阳性恒河猴 (3.91%),经测序分析后与印度恒河猴的同源性可达99.1 %.这5只均为SIV/SHIV感染恒河猴,其中四只SIV感染的猴的ELISPOT结果显示针对p11C的高频CTL反应,斑点数在500-1400/106 PBMCs之间,而另1只SHIV感染的恒河猴及5只阴性猴没有斑点出现.结论 中国恒河猴含有Mamu-A*01基因,基因频率有区域性差异,中国恒河猴的Mamu-A*01可提呈特异性抗原肽p11C.     

CXCR4嗜性SHIVKU-1病毒静脉感染中国恒河猴初步研究

目的了解SHIVKU一1静脉途径感染中国恒河猴的感染特点及进展规律。方法两只健康中国恒河猴,静脉感染SHIVKU-1病毒,定期采样检测血浆病毒载量、CD4＋／CD8＋比值、CD4＋T细胞绝对数变化和血清中抗SHIVKU-1特异性IgG抗体水平。多色流式技术分析外周血、腹股沟淋巴结和十二指肠粘膜固有层CD4＋T淋巴细胞记忆细胞亚群变化。结果两只实验猴成功感染SHIVKU-1病毒,一直到感染后3个月均保持稳定水平的病毒载量。外周血CD4＋T淋巴细胞下降明显,CD4＋／CD8＋T细胞比值严重倒置。CD4＋Tcm细胞比例在经历了感染早期的下降后,大幅升高,尤其是外周血和淋巴结。CD4＋Tem则在粘膜固有层中增加明显。结论SHIVKU．1静脉途径成功感染了中国恒河猴,为SHIV／中国恒河猴疾病及评价模型的建立奠定了良好的基础,为今后使用此模型评价抗病毒药物或疫苗提供了条件。     

B亚型SHIV_（SF162p3）中国恒河猴小剂量多次阴道黏膜感染

目的模拟HIV性传播感染特点进行中国恒河猴阴道黏膜小剂量多次感染研究,为我国艾滋病疫苗有效性评价提供新的模型构建思路。方法选用20-30TCID50剂量的SHIVSF162p3病毒阴道黏膜途径感染六只成年雌性中国恒河猴,共感染13次,每次攻毒间隔4～7 d。采取测定血浆病毒载量和外周血CD4＋∶CD8＋。结果 6只中国恒河猴经13次病毒攻击后,经检测均建立系统性感染,血浆病毒载量呈阳性;CD4＋∶CD8＋均有下降。结论成功建立了中国恒河猴阴道黏膜小剂量多次感染模型,为艾滋病研究提供了新的更接近于自然感染状态的模型建立模式。     

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。     

RT-SHIV感染中国恒河猴及体内传代

目的了解RT-SHIV感染中国恒河猴的感染特点,研究RT-SHIV在中国恒河猴中传代特点;建立RT-SHIV中国恒河猴动物模型,为评价HIV-1药物有效性提供动物平台。方法选择4只健康恒河猴,其中两只动物经上肢静脉感染RT-SHIV病毒,感染急性期采取外周血分离CD8-PBMC,扩增病毒,将新制备的病毒静脉感染另外两只中国恒河猴,通过监测血浆病毒载量,CD4＋/CD8＋比值,CD4＋T淋巴细胞和B淋巴细胞的绝对数,了解实验猴的感染状态,同时分析病毒RT基因变异情况。结果 4只动物均获得系统性感染,且传代动物急性期表现更为强烈,RT基因在感染和传代的过程中共观察到3个氨基酸的改变。结论本研究为RT-SHIV中国恒河猴模型的建立提供了基础信息。     

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。     

Molecularly cloned SHIV-1157ipd3N4: a highly replication- competent, mucosally transmissible R5 simian-human immunodeficiency virus encoding HIV clade C Env

Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three of which developed peripheral CD4(+) T-cell depletion. After the first inoculated monkey developed AIDS at week 137 postinoculation, transfer of its infected blood to a na?ve animal induced memory T-cell depletion and thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-1157ipd3, an extra NF-kappaB binding site was engineered into its 3' long terminal repeat, giving rise to SHIV-1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent, mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting HIV-1 clade C Env.     

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models

Background

HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models.

Methodology/Principal Findings

We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an “early,” recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a “late” form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to “late” SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4⁺ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM.

Conclusions/Significance

SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.     

Dynamics of envelope evolution in clade C SHIV-infected pig-tailed macaques during disease progression analyzed by ultra-deep pyrosequencing

Understanding the evolution of the human immunodeficiency virus type 1 (HIV-1) envelope during disease progression can provide tremendous insights for vaccine development, and simian-human immunodeficiency virus (SHIV) infection of non-human primate provides an ideal platform for such studies. A newly developed clade C SHIV, SHIV-1157ipd3N4, which was able to infect rhesus macaques, closely resembled primary HIV-1 in transmission and pathogenesis, was used to infect several pig-tailed macaques. One of the infected animals subsequently progressed to AIDS, whereas one remained a non-progressor. The viral envelope evolution in the infected animals during disease progression was analyzed by a bioinformatics approach using ultra-deep pyrosequencing. Our results showed substantial envelope variations emerging in the progressor animal after the onset of AIDS. These envelope variations impacted the length of the variable loops and charges of different envelope regions. Additionally, multiple mutations were located at the CD4 and CCR5 binding sites, potentially affecting receptor binding affinity, viral fitness and they might be selected at late stages of disease. More importantly, these envelope mutations are not random since they had repeatedly been observed in a rhesus macaque and a human infant infected by either SHIV or HIV-1, respectively, carrying the parental envelope of the infectious molecular clone SHIV-1157ipd3N4. Moreover, similar mutations were also observed from other studies on different clades of envelopes regardless of the host species. These recurring mutations in different envelopes suggest that there may be a common evolutionary pattern and selection pathway for the HIV-1 envelope during disease progression.     

Infectious Simian/Human Immunodeficiency Virus with Human Immunodeficiency Virus Type 1 Subtype C from an African Isolate: Rhesus Macaque Model

Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 56% of all infections in the HIV and AIDS pandemic. It is the predominant subtype in the rapidly expanding epidemic in southern Africa. To develop a relevant model that would facilitate studies of transmission, pathogenesis, and vaccine development for this subtype, we generated SHIV(MJ4), a simian/human immunodeficiency virus (SHIV) chimera based on HIV-1 subtype C. SHIV(MJ4) contains the majority of env, the entire second exon of tat, and a partial sequence of the second exon of rev, all derived from a CCR5-tropic, primary isolate envelope clone from southern Africa. SHIV(MJ4) replicated efficiently in human, rhesus, and pig-tailed macaque peripheral blood mononuclear cells (PBMCs) in vitro but not in CEMx174 cells. To assess in vivo infectivity, SHIV(MJ4) was intravenously inoculated into four rhesus macaques (Macaca mulatta). All four animals became infected as determined through virus isolation, PCR analysis, and viral loads of 10(7) to 10(8) copies of viral RNA per ml of plasma during the primary infection phase. We have established a CCR5-tropic SHIV(MJ4)/rhesus macaque model that may be useful in the studies of HIV-1 subtype C immunology and biology and may also facilitate the evaluation of vaccines to control the spread of HIV-1 subtype C in southern Africa and elsewhere.     

ORIGINAL ARTICLE: AIDS and optic neuritis in a rhesus monkey infected with the R5 clade C SHIV‐1157ipd3N4

A Chinese rhesus macaque infected with the pathogenic CCR5‐tropic clade C simian‐human immunodeficiency virus, SHIV‐1157ipd3N4, had persistent viremia, depletion of CD4⁺ T cells to <200 cells/μl, opportunistic infections, coagulopathy, and gradual development of bilateral blindness. MRI revealed marked thickening of both optic nerves. Histopathological evaluation showed diffuse cellular infiltration at necropsy and a focus of SHIV‐infected cells. This is the first report of CNS pathology following chronic infection with an obligate R5 SHIV.     

Some human immunodeficiency virus type 1 Vpu proteins are able to antagonize macaque BST-2 in vitro and in vivo: Vpu-negative simian-human immunodeficiency viruses are attenuated in vivo

Human immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by targeting BST-2/tetherin, a cellular protein inhibiting virus release. The widely used HIV-1(NL4-3) Vpu functionally inactivates human BST-2 but not murine or monkey BST-2, leading to the notion that Vpu antagonism is species specific. Here we investigated the properties of the CXCR4-tropic simian-human immunodeficiency virus DH12 (SHIV(DH12)) and the CCR5-tropic SHIV(AD8), each of which carries vpu genes derived from different primary HIV-1 isolates. We found that virion release from infected rhesus peripheral blood mononuclear cells was enhanced to various degrees by the Vpu present in both SHIVs. Transfer of the SHIV(DH12) Vpu transmembrane domain to the HIV-1(NL4-3) Vpu conferred antagonizing activity against macaque BST-2. Inactivation of the SHIV(DH12) and SHIV(AD8) vpu genes impaired virus replication in 6 of 8 inoculated rhesus macaques, resulting in lower plasma viral RNA loads, slower losses of CD4(+) T cells, and delayed disease progression. The expanded host range of the SHIV(DH12) Vpu was not due to adaptation during passage in macaques but was an intrinsic property of the parental HIV-1(DH12) Vpu protein. These results demonstrate that the species-specific inhibition of BST-2 by HIV-1(NL4-3) Vpu is not characteristic of all HIV-1 Vpu proteins; some HIV-1 isolates encode a Vpu with a broader host range.