SYBR Green Ⅰ实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立

.学者论坛·在动物实验中解决临床难题···························，·······································································……顾玉东(3)基因〕:程     

Single-cycle immunodeficiency viruses provide strategies for uncoupling in vivo expression levels from viral replicative capacity and for mimicking live-attenuated SIV vaccines

To reduce the risks associated with live-attenuated immunodeficiency virus vaccines, single-cycle immunodeficiency viruses (SCIVs) were developed by primer complementation and production of the vaccine in the absence of vif in a vif-independent cell line. After a single intravenous injection of SCIVs into rhesus monkeys, peak viral RNA levels of 10(3) to 10(4) copies/ml plasma were observed, indicating efficient expression of SCIV in the vaccinee. After booster immunizations with SCIVs, SIV-specific humoral and cellular immune responses were observed. Although the vaccine doses used in this pilot study could not protect vaccinees from subsequent intravenous challenge with pathogenic SIVmac239, our results demonstrate that the novel SCIV approach allows us to uncouple in vivo expression levels from the viral replicative capacity facilitating the analysis of the relationship between viral expression levels or viral genes and immune responses induced by SIV.     

实时荧光定量RT-PCR监测恒河猴体内人/猴免疫缺陷病毒载量在传代过程中的变化

目的为分析中国SHIV/猕猴AIDS模型的病毒载量变化趋势,建立一种实时、灵敏、特异的针对人/猴免疫缺陷病毒的定量检测方法。方法体外转录制备RNA标准品,利用TaqManEZRT-PCR试剂盒的反应体系和针对SHIVgag保守区91个碱基的TaqMan探针和引物,建立一步法实时荧光定量RT-PCR。提取126份来自SHIV-CN97001感染恒河猴血浆病毒RNA并定量检测。结果利用梯度稀释的RNA标准品对反应体系进行优化,标准曲线下限达到2×102拷贝/ml,相关性(r>0.99)及重复性(CV=4.14%)均能达到测定要求。病毒载量的检测结果表明SHIV-CN97001在猴体内传代过程中病毒载量有先升后降的趋势,病毒载量通常在接种病毒或感染猴的全血后第14天达到高峰。血浆载量可达到105~106拷贝/ml。结论成功地建立了一步法定量SHIVRNA的实时荧光定量RT-PCR,为SHIV/恒河猴AIDS模型的建立与应用提供了灵敏的病毒载量检测方法。SHIV-CN97001的体内繁殖能力在猴体内传代过程中有所增强。     

Coreceptor Switch of [MLV(SIVagm)] pseudotype vectors by V3-loop exchange

Retroviral vectors derived from murine leukemia virus (MLV) have been pseudotyped with a variant of the envelope glycoprotein (Env) of nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to result in [MLV(SIVagm-wt)] vector particles. The variant env gene encodes a full-length surface envelope glycoprotein (SU) and a C-terminally truncated transmembrane protein (TM). To change the coreceptor usage of this vector from CCR5 to CXCR4, which is predominant on human CD4-positive lymphocytes, the putative V3-loop of SIVagm SU was replaced by that of the T cell tropic HIV-1 variant BH10. The resulting [MLV(SIVagm-X4)] vectors were shown to specifically transduce CD4/CXCR4-positive cell lines, demonstrating the equivalent function in cell entry and choice of coreceptor usage of the V3-loops of SIVagm and HIV-1. These modified vectors were able to transduce primary human lymphocytes and were resistant to neutralization by sera from HIV-1-infected individuals. The [MLV(SIVagm-X4)] pseudotype vector generated is thus a promising candidate vector, e.g., for in vivo gene therapy of HIV-1 infection.     

Analysis of evolutionary conservation in CD1d molecules among primates

The hereditary conservation in the genetically encoded CD1D sequences of various primates was analyzed. Genomic CD1D sequences of 17 rhesus macaques with distinct origins, eight Indian and nine Chinese, were examined and differences of only one or two nucleotides were detected and the consensus sequence of rhesus CD1D was determined. CD1D consensus sequences of three African green monkeys (AGMs) and the rhesus monkeys were then compared to study the evolutionary differences among interspecies. The CD1D consensus sequence determined from AGMs apparently differed by seven nucleotides from the rhesus consensus sequence, and nucleotide difference induced only three amino acid changes within Exon3, corresponding to the alpha2 domain of CD1d having a hydrophobic ligand-binding pocket. Such changes in the alpha2 domain may alter the characteristics of the SIV-derived glycolipid/lipid antigens presented by each CD1d molecule to innate natural killer T cells. In addition, the CD1D genomic sequences of three chimpanzees (chimps) were determined. To our surprise, although Exon2 and Exon3 reflecting antigen-binding alpha1 and alpha2 domains in chimps' CD1D were identical to that in humans except one amino acid, three amino acids within Exon4, reflecting alpha3 domain, were distinct from humans, and one of them was identical to those in rhesus and AGM CD1D. On the basis of the findings, the evolutionary relationship of the CD1d molecules among the various primates and their HIV-1/SIV susceptibility will be discussed.     

Domains of macaque DC-SIGN essential for capture and transfer of simian immunodeficiency virus

The C-type lectin DC-SIGN mediates the capture and transfer of simian immunodeficiency virus (SIV) from macaque dendritic cells (DCs) to permissive T-cells. To further identify the determinants in macaque DC-SIGN required for capture and transfer of virus, we created mutants containing deletions or point mutations in the extracellular domains, and tested their ability to capture and transmit SIV. We found that SIV bound to the carbohydrate recognition domain (CRD) of macaque DC-SIGN via the envelope protein. In addition, deleting the C-terminal half of the CRD, or mutating amino acids within this region that contact Ca(2+) or mannose, disrupted virion capture activity. However, an N-terminal CRD deletion mutant was capable of binding SIV, indicating that this region was not necessary for binding. Finally, deletion of the neck domain also reduced the capacity for macaque DC-SIGN to capture SIV. Interestingly, ICAM-3, the cellular ligand for DC-SIGN, did not bind to any of the DC-SIGN mutants, including mutants with amino acid changes in the N-terminal region of the CRD. These data suggest that the binding sites for SIV and ICAM-3 may be distinct but overlapping. Together, the data demonstrate the importance of both the neck and the CRD of macaque DC-SIGN for efficient capture of SIV and binding to ICAM-3.     

Use of a Clostridium perfringens vector to express high levels of SIV p27 protein for the development of an oral SIV vaccine

Clostridium perfringens is a normal bacterial flora of the small and large intestines of humans and other animals. The current study investigates the potential use of a noncytotoxic C. perfringens as an oral vaccine vehicle for expression and intestinal delivery of a large amount of SIV antigens. Here we report the construction of a recombinant C. perfringens vaccine vector expressing high levels of SIV p27 during sporulation. Following oral administration of this recombinant C. perfringens vaccine vector to mice, large amounts of intact p27 protein were detected in the terminal ileum where the majority of Peyer's Patches (PPs) are located. Furthermore, dendritic cells (DCs) beneath the mucosal surface in the PPs were able to capture SIV p27 antigen, when PPs were exposed to C. perfringens expressing SIV p27 antigen. In addition, uptake of C. perfringens was able to induce maturation of mouse DCs. These results support the potential use of C. perfringens as an oral SIV/HIV vaccine vector.     

蛋脑啡肽对猴免疫缺陷病毒感染CEM×174细胞kappa阿片受体表达的调节作用(英文)

目的：研究蛋脑啡肽对猴免疫缺陷病毒（ＳＩＶ）感染ＣＥＭ×１７４ 细胞ｋａｐｐａ 阿片受体表达的调节作用。方法：用ＳＩＶ 感染ＣＥＭ×１７４ 细胞并加入不同浓度蛋脑啡肽。在２４ ｈ 提取ＲＮＡ,用ＲＴ－ＰＣＲ 方法扩增ｋａｐｐａ 阿片受体ｍ ＲＮＡ 并且进行定量分析。结果：显示ＳＩＶ 能够抑制ＣＥＭ×１７４ 细胞的生长。在１０－ ７ｍ ｏｌ／Ｌ蛋脑啡肽存在下,ＳＩＶ 对细胞的损害减轻。１０－ ７ｍ ｏｌ／Ｌ和１０－ ６ｍ ｏｌ／Ｌ蛋脑啡肽不能改变正常细胞ｋａｐｐａ 阿片受体的表达,但是在ＳＩＶ 感染组的表达显著增高。结论：实验结果提示蛋脑啡肽能够维持正常淋巴细胞的生长。Ｋａｐｐａ 阿片受体表达的改变可能与蛋脑啡肽对免疫细胞的调节机理有关     

Enhanced Heterosexual Transmission Hypothesis for the Origin of Pandemic HIV-1

HIV-1 M originated from SIVcpz endemic in chimpanzees from southeast Cameroon or neighboring areas, and it started to spread in the early 20th century. Here we examine the factors that may have contributed to simian-to-human transmission, local transmission between humans, and export to a city. The region had intense ape hunting, social disruption, commercial sex work, STDs, and traffic to/from Kinshasa in the period 1899–1923. Injection treatments increased sharply around 1930; however, their frequency among local patients was far lower than among modern groups experiencing parenteral HIV-1 outbreaks. Recent molecular datings of HIV-1 M fit better the period of maximal resource exploitation and trade links than the period of high injection intensity. We conclude that although local parenteral outbreaks might have occurred, these are unlikely to have caused massive transmission. World War I led to additional, and hitherto unrecognized, risks of HIV-1 emergence. We propose an Enhanced Heterosexual Transmission Hypothesis for the origin of HIV-1 M, featuring at the time and place of its origin a coincidence of favorable co-factors (ape hunting, social disruption, STDs, and mobility) for both cross-species transmission and heterosexual spread. Our hypothesis does not exclude a role for parenteral transmission in the initial viral adaptation.     

A critical analysis of the cynomolgus macaque,Macaca fascicularis,as a model to test HIV-1/SIV vaccine efficacy

The use of a number of non-rhesus macaque species, but especially cynomolgus macaques as a model for HIV-1 vaccine development has increased in recent years. Cynomolgus macaques have been used in the United Kingdom, Europe, Canada and Australia as a model for HIV vaccine development for many years. Unlike rhesus macaques, cynomolgus macaques infected with SIV show a pattern of disease pathogenesis that more closely resembles that of human HIV-1 infection, exhibiting lower peak and set-point viral loads and slower progression to disease with more typical AIDS defining illnesses. Several advances have been made recently in the use of the cynomolgus macaque SIV challenge model that allow the demonstration of vaccine efficacy using attenuated viruses and vectors that are both viral and non-viral in origin. This review aims to probe the details of various vaccination trials carried out in cynomolgus macaques in the context of our modern understanding of the highly diverse immunogenetics of this species with a view to understanding the species-specific immune correlates of protection and the efficacy of vectors that have been used to design vaccines.