动物模型在HIV-1杀微生物剂有效性和安全性评价中的应用

HIV/AIDS的流行趋势没有减弱的迹象,人们迫切需要新的预防HIV传播的手段。杀微生物剂旨在通过局部用药于阴道或直肠从而阻止HIV的传播。鉴于目前有大量的杀微生物剂候选物,亟待能够有效评价其有效性及安全性的动物模型。通过比较非灵长类小型动物模型与非人灵长类动物模型在评价HIV杀微生物剂的有效性及安全性上的重要作用,本文总结了评价杀微生物剂有效性及安全性的动物模型的优缺点,同时指出了杀微生物剂研究与发展的方向和建议,希望能够对杀微生物剂的研发有所帮助。     

灵长类动物中TRIMCyp融合基因模式及对逆转录病毒复制的限制作用

TRIM5-CypA融合基因(TRIMCyp)是一种独特的TRIM5基因形式。迄今已发现新大陆猴中包括鹰猴在内的夜猴属所有代表种,以及在北平顶猴、巽他平顶猴、食蟹猴、印度恒河猴和熊猴等旧大陆猴中均存在这种基因融合现象,但在新大陆猴与旧大陆猴中的TRIMCyp融合基因的基因融合模式和表达剪接方式不同。新大陆猴TRIMCyp融合基因是由CypA假基因的cDNA序列通过LINE-1逆转座子介导的逆转座方式插入至TRIM5α基因的第7和第8外显子之间的内含子中形成,而旧大陆猴TRIMCyp融合基因则是由CypA假基因的cDNA序列以相似的逆转座方式插入至TRIM5基因的3’非翻译区(untranslatedregions,UTR)形成。TRIMCyp融合基因在不同灵长类动物中的存在比例、基因型、TRIMCyp融合蛋白的表达以及对逆转录病毒的限制活性均有所差异。鹰猴和平顶猴的TRIMCyp融合基因研究较多,鹰猴TRIMCyp融合蛋白可能以与TRIM5α相似机制限制HIV-1的感染,而平顶猴TRIMCyp融合蛋白则丧失了限制HIV-1的作用。这两个功能截然不同的融合基因为TRIM5α作用机制研究提供了难得的实验材料,也为建立HIV-1感染的新型灵长类动物艾滋病模型奠定了科学依据。该文综述了TRIMCyp融合基因在灵长类动物中的分布、存在形式及其限制逆转录病毒复制的作用机制等方面的研究情况。     

A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice

For prevention of HIV infection many currently licensed anti-HIV drugs and new ones in the pipeline show potential as topically applied microbicides. While macaque models have been the gold standard for in vivo microbicide testing, they are expensive and sufficient numbers are not available. Therefore, a small animal model that facilitates rapid evaluation of potential candidates for their preliminary efficacy is urgently needed in the microbicide field. We previously demonstrated that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and that oral pre-exposure chemo-prophylactic strategies could be tested in this system. Here in these proof-of-concept studies, we extended this system for topical microbicide testing using HIV-1 as the challenge virus. Maraviroc, a clinically approved CCR5 inhibitor drug for HIV treatment, was formulated as a microbicide gel at 5 mM concentration in 2.2% hydroxyl ethyl cellulose. Female RAG-hu mice were challenged vaginally with HIV-1 an hour after intravaginal application of the maraviroc gel. Our results showed that maraviroc gel treated mice were fully protected against vaginal HIV-1 challenge in contrast to placebo gel treated mice which all became infected. These findings highlight the utility of the humanized mouse models for microbicide testing and, together with the recent data from macaque studies, suggest that maraviroc is a promising candidate for future microbicide clinical trials in the field.     

Blood groups of pig-tailed macaques (Macaca nemestrina).

The human-type A-B-O blood groups of 57 pig-tailed macaques (Macaca nemestrina) were determined and the calculated gene frequencies, O = 0.8908, A = 0.0825 and B = 0.0267, gave excellent fit with the hypothesis of inheritance by triple allelic genes. In tests for simian-type blood groups with rhesus, baboon and crab-eating macaque immune antisera, it was shown that the red cells of pig-tailed macaques are polymorphic for several simian-type specificities defined by those cross-reacting sera. Pig-tailed macaques share with other macaque species the complex Drh-graded blood group system, which seems to occupy a special role among red cell antigens of macaques. Normal sera of three female macaques contained spontaneous isoagglutinins which selectively agglutinated the red cells of some pig-tailed as well as stump-tailed macaques.     

Characterization of an anti-lymphocyte function-associated antigen-1 antibody in a simian immunodeficiency virus-pig-tailed macaque (Macaca nemestrina) model

The simian immunodeficiency virus (SIV)/pig-tailed macaque (Macaca nemestrina) model of acquired immune deficiency syndrome (AIDS) is a powerful system in which to study cell adhesion molecules and retroviral pathogenesis in vivo. Preliminary experiments were conducted to examine the role of lymphocyte function-associated antigen 1 (LFA-1) in early SIV infection in vivo by using an LFA-1 monoclonal antibody (MHM.23) specific to human LFA-1. In vitro studies revealed that at concentrations of > or = 20 microg/ml, MHM.23 blocked LFA-1-mediated adhesion and T-cell activation (>90%) of pig-tailed macaque peripheral blood mononuclear cells (PBMCs). In addition, SIVmac239 infection of macaque cells was inhibited in a dose-dependant manner by MHM.23. Administration of MHM.23 to pig-tailed macaques inhibited LFA-1-ICAM-1-mediated activity in vivo and maintained binding on macaque cells for < or = 4 d. Our in vitro studies indicated that at an MHM.23 concentration of 20 microg/ml, macaque PBMCs were completely saturated. Our in vivo studies determined that 5 mg/kg MHM.23 intravenously every 24 h was required to maintain saturating levels and inhibit LFA-1-ICAM-1 function in pig-tailed macaques.     

Inoculation of Macaca fascicularis with simian immunodeficiency virus, SIVmne immunologic, serologic, and pathologic changes

Previous studies had tested the susceptibility of two macaque species, Macaca nemestrina and M. mulatta, to infection with the primate lymphotropic lentivirus SIVmne. In this report we describe the results obtained after infecting eleven M. fascicularis with SIVmne. Six of the animals had previously been immunized with a recombinant vaccinia virus expressing the envelope gene of HIV-1. All eleven animals became seropositive. To date ten animals have died 43 to 155 weeks post infection of an AIDS-like disease.     

SIV of stump-tailed macaque (SIVstm) is a divergent Asian isolate.

Analysis of molecularly cloned DNAs of SIVs isolated from Asian rhesus macaque (Macaca mulatta; SIVmac) and pig-tailed macaque (Macaca nemestrina; SIVmne) has indicated a high degree of sequence homology between these viruses. Thus SIVmac and SIVmne might have originated from the same or very closely related viruses. We have cloned and sequenced a PCR-amplified segment containing the LTR sequences of SIV originating from a stump-tailed macaque (Macaca arctoides; SIVstm). Comparative sequence analysis indicates that SIVstm belongs to the SIV/HIV-2 group; however, it is genetically distinct from the other members.     

Pneumatocele in a pig-tailed macaque (Macaca nemestrina)

Radiographic examination of a pig-tailed macaque (Macaca nemestrina) with pneumonia revealed a large pneumatocele. The pneumatocele, a thin-walled, partially fluid filled radiolucent area, occupied approximately one-third of the left thorax. Rapid resolution of the pneumatocele accompanied antimicrobial treatment of the pneumonia and coincided with clinical improvement. Severe pulmonary acariasis was found at postmortem 15 months later.     

Pigtailed macaque model refinement: topical microbicide safety in the presence of coitus

Background Inclusion of sexual activity in the macaque model for topical microbicide safety evaluation would more closely mimic human use of topical microbicides and provide a more rigorous safety assessment. Methods Initially, male–female partners were monitored in cohousing arrangements to determine whether macaques would copulate ad libitum. The logistics of performing vaginal examinations before and after coital visits were analyzed and optimized. Findings from cervicovaginal examinations conducted before and after sexual activity were compared. Results Coital activity was reliably observed in the majority of cohousing sessions, representing all phases of the menstrual cycle. Female macaques were trained to be restrained while fully alert for pre‐coital vaginal sampling. Post‐coital examinations occur under general sedation. Post‐coital examinations reveal alterations to tissues, microbiology, and pH compared with pre‐coital visits. Conclusions This work clearly demonstrates that it is feasible to incorporate sexual activity in the macaque model for topical microbicide safety assessment.     

Variable prevalence and functional diversity of the antiretroviral restriction factor TRIMCyp in Macaca fascicularis

The retroviral restriction factor TRIMCyp, derived from the TRIM5 gene, blocks replication at a postentry step. TRIMCyp has so far been found in four species of Asian macaques, Macaca fascicularis, M. mulatta, M. nemestrina, and M. leonina. M. fascicularis is commonly used as a model for AIDS research, but TRIMCyp has not been analyzed in detail in this species. We analyzed the prevalence of TRIMCyp in samples from Indonesia, Indochina, the Philippines, and Mauritius. We found that TRIMCyp is present at a higher frequency in Indonesian than in Indochinese M. fascicularis macaques and is also present in samples from the Philippines. TRIMCyp is absent in Mauritian M. fascicularis macaques. We then analyzed the restriction specificity of TRIMCyp derived from three animals of Indonesian origin. One allele, like the prototypic TRIMCyp alleles described for M. mulatta and M. nemestrina, restricts human immunodeficiency virus type 2 (HIV-2) and feline immunodeficiency virus (FIV) but not HIV-1. The others restrict HIV-1 and FIV but not HIV-2. Mutagenesis studies confirmed that polymorphisms at amino acid residues 369 and 446 in TRIMCyp (or residues 66 and 143 in the cyclophilin A [CypA] domain) confer restriction specificity. Additionally, we identified a polymorphism in the coiled-coil domain that appears to affect TRIMCyp expression or stability. Taken together, these data show that M. fascicularis has the most diverse array of TRIM5 restriction factors described for any primate species to date. These findings are relevant to our understanding of the evolution of retroviral restriction factors and the use of M. fascicularis models in AIDS research.     

Inoculation of baboons and macaques with simian immunodeficiency virus/Mne, a primate lentivirus closely related to human immunodeficiency virus type 2

A primate lymphotropic lentivirus was isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma. This isolate, originally designated M. nemestrina immunodeficiency virus (MnIV) and now classified as simian immunodeficiency virus (SIV/Mne), was inoculated intravenously into three juvenile rhesus monkeys (Macaca mulatta), three juvenile pig-tailed macaques (M. nemestrina), and two juvenile baboons (Papio cynocephalus). All six macaques became viremic by 3 weeks after inoculation, whereas neither of the baboons developed viremia. One pig-tailed macaque died at 15 weeks with suppurative peritonitis secondary to ulcerative, necrotizing colitis. Immunologic abnormalities included a marked decrease in CD4+ peripheral blood lymphocytes. Although five macaques mounted an antibody response to SIV/Mne, the animal that died at 15 weeks remained antibody negative. Three other macaques (two rhesus and one pig-tailed) died 66 to 87 weeks after inoculation after exhibiting progressive weight loss, anemia, and diarrhea. Histopathologic findings at necropsy included various manifestations of immune deficiency, nephropathy, subacute encephalitis, pancreatitis, adenocarcinoma, and lymphoid atrophy. SIV/Mne could be readily isolated from the spleens and lymph nodes of all necropsied macaques, and from the cerebrospinal fluid, brains, bone marrow, livers, and pancreas of some of the animals. SIV antigens were localized by avidin-biotin immunohistochemistry to pancreatic islet cells and to bone marrow endothelial cells. The data suggest that African baboons may be resistant to infection by SIV/Mne, whereas Asian macaques are susceptible to infection with this pathogenic primate lentivirus.     

VivaGel (SPL7013 Gel): a candidate dendrimer--microbicide for the prevention of HIV and HSV infection

Microbicides are compounds that applied vaginally or rectally, protect the user from sexually transmitted infections. Although no commercial product is yet available, many candidates are under development. A leading candidate, VivaGel (SPL7013 Gel) is the product of nanotechnology. The active ingredient is SPL7013, a dendrimer that was designed specifically with HIV and HSV antiviral activity and human safety in mind. SPL7013 has demonstrated efficacy against human immunodeficiency virus and herpes simplex virus in in vitro and animal models. VivaGel appears to be well tolerated in both animals and humans. This review summarizes the studies of VivaGel and its active ingredient, SPL7013.     

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: epidemiologic studies

At the University of Washington Regional Primate Research Center, a simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in 82 macaques since 1976, including 77 pigtailed macaques (Macaca nemestrina), two long-tailed macaques (M. fascicularis), one Japanese macaque (M. fuscata) and two rhesus macaques (M. mulatta). The syndrome is characterized by immunodeficiency accompanied by a fibroproliferative lesion, primarily affects young monkeys (1-3 years) and has a high case fatality rate. Based on the occurrence of RF in colony-born and non-colony-born monkeys, the minimum incubation period for natural exposure is believed to be about 9 months. The incidence of RF was 0.9% in M. nemestrina, 0.1% in M. fascicularis, 1.0% in M. fuscata and 0.4% in M. mulatta. There were no significant differences in the incidence of RF by sex or seasonality. Epidemiologic studies were focused on 42 juvenile M. nemestrina that developed RF between January 1980 and June 1983, and the results were compared with 42 age- and sex-matched controls. The incidence of RF was 5.7% in monkeys 12-24 months old and 3.4% in monkeys 24-36 months old, but less than 1.0% in age groups of under 1 year and over 3 years. No significant associations were found for housing history, parentage, generations or ancestral origins. Epidemiologic information and preliminary viral studies suggest a type D retrovirus may be the causative agent in RF and SAIDS. RF associated with SAIDS appears to be an excellent model for Kaposi's sarcoma associated with human AIDS.     

Enhanced replication of HIV-1 in vivo in pigtailed macaques (Macaca nemestrina)

Non-human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV-1) and animals that are readily available for research. HIV-1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for enhanced viral replication was first found in one of the group 2 animals, and in both group 3 animals. Observations that underlie this conclusion are sustained viral recovery from peripheral blood mononuclear cells (PBMCs), increased and accelerated production of antiviral antibodies, and the ability to detect plasma viral ribonucleic acid (RNA) months after infection. There was no evidence of CD4 depletion in any of the animals during the follow-up period. These data suggest that a useful non-human primate model for AIDS can be attained in pigtailed macaques ( M. nemestrina ).     

Enhanced replication of HIV-1 in vivo in pigtailed macaques (Macaca nemestrina)

Non-human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV-1) and animals that are readily available for research. HIV-1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for enhanced viral replication was first found in one of the group 2 animals, and in both group 3 animals. Observations that underlie this conclusion are sustained viral recovery from peripheral blood mononuclear cells (PBMCs), increased and accelerated production of antiviral antibodies, and the ability to detect plasma viral ribonucleic acid (RNA) months after infection. There was no evidence of CD4 depletion in any of the animals during the follow-up period. These data suggest that a useful non-human primate model for AIDS can be attained in pigtailed macaques (M. nemestrina).     

Characterization of pig-tailed macaque classical MHC class I genes: implications for MHC evolution and antigen presentation in macaques

MHC-dependent CD8(+) T cell responses have been associated with control of viral replication and slower disease progression during lentiviral infections. Pig-tailed macaques (Macaca nemestrina) and rhesus monkeys (Macaca mulatta), two nonhuman primate species commonly used to model HIV infection, can exhibit distinct clinical courses after infection with different primate lentiviruses. As an initial step in assessing the role of MHC class I restricted immune responses to these infections, we have cloned and characterized classical MHC class I genes of pig-tailed macaques and have identified 19 MHC class I alleles (Mane) orthologous to rhesus macaque MHC-A, -B, and -I genes. Both Mane-A and Mane-B loci were found to be duplicated, and no MHC-C locus was detected. Pig-tailed and rhesus macaque MHC-A alleles form two groups, as defined by 14 polymorphisms affecting mainly their B peptide-binding pockets. Furthermore, an analysis of multiple pig-tailed monkeys revealed the existence of three MHC-A haplotypes. The distribution of these haplotypes in various Old World monkeys provides new insights about MHC-A evolution in nonhuman primates. An examination of B and F peptide-binding pockets in rhesus and pig-tailed macaques suggests that their MHC-B molecules present few common peptides to their respective CTLs.     

Analysis of immunoglobulin,complements and CRP levels in serum of captive northern pig-tailed macaques (Macaca leonina)

The northern pig-tailed macaque(NPM,Macaca leonina) has become a widely used animal model in biomedical research. In this study, we measured serum immunoglobulin IgG, IgM, IgA, complement C3, C4 and CRP levels in 3-11 year old captive northern pig-tailed macaques using HITACHI 7600-20 automated chemistry analyzer in order to determine the influences of age and gender on these items. The results showed that serum IgA, IgM, C3 and C4 levels were not correlated with age(P0.05), while serum IgG levels increased progressively with age(r=0.202;P=0.045). Serum IgG, IgA, IgM and C3 levels were higher in females than in males(P0.05). Moreover, serum C3 concentration was both positively and strongly correlated with that of C4(r=0.700; P0.0001). This study provides basic serum immunoglobulin and complement data of captive northern pig-tailed macaques, which may prove useful for future breeding efforts and biomedical research.     

Phylogenetic relationships among the macaques: evidence from the nuclear locus NRAMP1.

The macaques, genus Macaca, represent one of the most successful radiations within the Order Primates, with a geographical distribution that ranks second in size only to that of humans among extant primates. Although the number of macaque species recognized depends on the classification scheme used, most authors currently follow the classifications of either Fooden or Delson, both of whom recognize 19 extant macaque species. These two classifications differ in their placement of macaque species into more inclusive taxa (i.e., species groups). While researchers have attempted to use mitochondrial DNA (mtDNA) to resolve these phylogenetic relationships, different studies have generated conflicting conclusions. Consequently, we screened nuclear DNA sequences of a large number of macaques to determine if such data provide greater insight into the phylogenetic relationships among macaques. The data generated from the comparison of two (noncoding) introns within the natural resistance-associated macrophage protein 1 (NRAMP1) gene generally agree with the classification scheme of Delson. However, the data also support several novel observations. Specifically, the NRAMP1 data demonstrate that M. silenus and M. nemestrina lack nuclear genetic variation, while M. assamensis and M. radiata exhibit the greatest amount of genetic variation. In addition, these data suggest that M. fascicularis may not be as "primitive" (with respect to other members of the fascicularis group) as the mtDNA based data suggest.