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.     

The value of primate models for studying human immunodeficiency virus pathogenesis

Abstract: Research on human immunodeficiency virus (HIV) infection is compromised by the obvious limitation in having for study only virus-infected individuals or those exposed to the virus. Steps involved in transmission or pathogenesis require planned experimentation. The identification of animal models of acquired immunodeficiency syndrome (AIDS) has therefore been helpful for evaluating phases of HIV pathogenesis. Of the seven subgenera of lentiviruses now recognized, two share the characteristics with HIV of a T cell tropism and the associated loss of CD4+ cells in the host associated with disease: the feline immunodeficiency virus (FIV) and the simian immunodeficiency virus (SIV) (Table 1). The other animal lentiviruses grow best in macrophages and their infection generally reflects clinical sequellae of infection of this cell type. This review addresses those features of SIV, HIV, and SHIV infections of non-human primates that illustrate the importance of the animal models of AIDS.     

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.     

Determination of a T cell receptor of potent CD8+ T cells against simian immunodeficiency virus infection in Burmese rhesus macaques

Cumulative studies on human immunodeficiency virus (HIV)-infected individuals have shown association of major histocompatibility complex class I (MHC-I) polymorphisms with lower viral load and delayed AIDS progression, suggesting that HIV replication can be controlled by potent CD8⁺ T-cell responses. We have previously established an AIDS model of simian immunodeficiency virus (SIV) infection in Burmese rhesus macaques and found a potent CD8⁺ T cell targeting the Mamu-A1*065:01-restricted Gag_241-249 epitope, which is located in a region corresponding to the HIV Gag_240-249 TW10 epitope restricted by a protective MHC-I allele, HLA-B*57. In the present study, we determined a T cell receptor (TCR) of this Gag_241-249 epitope-specific CD8⁺ T cell. cDNA clones encoding TCR-α and TCR-β chains were obtained from a Gag_241-249-specific CD8⁺ T-cell clone. Coexpression of these TCR-α and TCR-β cDNAs resulted in reconstitution of a functional TCR specifically detected by Gag_241-249 epitope-Mamu-A1*065:01 tetramer. Two of three previously-reported CD8⁺ T-cell escape mutations reduced binding affinity of Gag_241-249 peptide to Mamu-A1*065:01 but the remaining one not. This is consistent with the data obtained by molecular modeling of the epitope-MHC-I complex and TCR. These results would contribute to understanding how viral CD8⁺ T-cell escape mutations are selected under structural constraint of viral proteins.     

SHIV transmission and susceptibility to re-exposure through social contact following vaccination with an HIV synthetic peptide-cocktail: a case study

Abstract: An effective vaccine against human immunodeficiency virus (HIV) should not only protect from infection and development of acquired immunodeficiency syndrome (AIDS), but also prevent potential transmission to naïve partners. We recently reported protection of rhesus macaques from chronic simian‐human immunodeficiency virus (SHIV) infection and AIDS by an HIV envelope peptide‐cocktail vaccine. In the present case study, we observed that one of the vaccinated females, with undetectable circulating virus, when housed in a pair with a naïve male, did not transmit the infection over a 35‐week period of social contact. Subsequent experimental challenge of the male with the same SHIV strain resulted in high‐level infection and transmission to its female cage‐mate. However, the virus was undetectable in the female by 12 weeks without further vaccination, validating the multivalent peptide cocktail vaccine approach in the SHIV‐rhesus model, and suggesting its potential utility as an HIV vaccine strategy for humans.     

Molecular typing of major histocompatibility complex class I alleles in the Indian rhesus macaque which restrict SIV CD8<Superscript>+</Superscript> T cell epitopes

The utility of the rhesus macaque as an animal model in both HIV vaccine development and pathogenesis studies necessitates the development of accurate and efficient major histocompatibility complex (MHC) genotyping technologies. In this paper, we describe the development and application of allele-specific polymerase chain reaction (PCR) amplification for the simultaneous detection of eight MHC class I alleles from the rhesus macaque (Macaca mulatta) of Indian descent. These alleles were selected, as they have been implicated in the restriction of CD8(+) T cell epitopes of simian immunodeficiency virus (SIV). Molecular typing of Mamu-A 01, Mamu-A 02, Mamu-A 08, Mamu-A 11, Mamu-B 01, Mamu-B 03, Mamu-B 04, and Mamu-B 17 was conducted in a high throughput fashion using genomic DNA. Our amplification strategy included a conserved internal control target to minimize false negative results and can be completed in less than 5 h. We have genotyped over 4,000 animals to establish allele frequencies from colonies all over the western hemisphere. The ability to identify MHC-defined rhesus macaques will greatly enhance investigation of the immune responses, which are responsible for the control of viral replication. Furthermore, application of this technically simple and accurate typing method should facilitate selection, utilization, and breeding of rhesus macaques for AIDS virus pathogenesis and vaccine studies.     

Worldwide survey of AIDS vaccine challenge studies in nonhuman primates: vaccines associated with active and passive immune protection from live virus challenge.

An AIDS Vaccine Surveillance System (AVSS) was designed and implemented to track the rapidly growing international database supporting the development of promising AIDS vaccines. Both preclinical nonhuman primate (NHP) and clinical human trials are tracked by the AVSS. This report presents summary data generated from the AVSS on the NHP AIDS vaccine/live virus challenge studies only. Summary data on more than 100 preclinical HIV/SIV vaccines are presented within the framework of 1) 13 arbitrary Vaccine Types, 2) studies grouped by animal model (i.e., chimpanzee/HIV-1, and macaque/SIV, HIV-2), and 3) immunization approach (i.e., active and passive). Systematic and timely presentations of these summary data, both here and in future reports, aim to promote a clearer understanding of both earlier and more recent preclinical AIDS vaccine developments.     

Identification of SIV Nef CD8 T cell epitopes restricted by a MHC class I haplotype associated with lower viral loads in a macaque AIDS model

Virus-specific CD8⁺ T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag_206–216 and Gag_241–249 epitope-specific CD8⁺ T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8⁺ T-cell responses induced in all the 90-120-Ia⁺ macaques on SIV replication remains unknown. Here, we identified three CD8⁺ T-cell epitopes, Nef_9–19, Nef_89–97, and Nef_193–203, associated with 90-120-Ia. Nef_9–19 and Nef_193–203 epitope-specific CD8⁺ T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8⁺ T cells, indicating that these CD8⁺ T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia.     

Peptide immunization restimulates the memory CD4 T cell response but fails to induce cytotoxic T lymphocytes in cynomolgus monkeys

A potential strategy to induce peptide specific CTL in vivo was investigated. A synthetic vaccine consisting of an SIV-derived, HLA-A2.1-binding CTL epitope and a tetanus toxin-derived T helper epitope was evaluated for its capacity to induce peptide-specific CTL in monkeys. Thirteen animals were immunized and boosted twice with 150 μg of CTL plus 250 μg of the T helper peptide (p30). Peripheral blood mononuclear cells (PBMC) were regularly analysed for cytotoxic and proliferative responses before, between, and after the immunizations, and the serum was tested for anti-peptide antibodies. No unequivocal induction of SIV peptide-specific CTL in any of the monkeys was observed. However, a wide pattern of mild and transient side reactions were observed, ranging from local redness at the injection site to generalized exanthema, myalgias, arthralgias, and fever. The side-effects were related to the T helper epitope, as they were similar to the side-effects experienced after tetanus immunization, correlated to the magnitude of the p30-specific in vitro proliferative response, and occurred only if p30 was co-injected. No antibody against the SIV-derived peptides nor against p30 was detectable in the serum after repeated immunizations. The data suggest that the CTL peptide, at the concentration used in this study, failed to induce a cytotoxic immune response in vivo, although the T helper peptide seems to be capable of restimulating the specific memory T cells.     

Assessing genetic-based therapies for AIDS using the simian immunodeficiency virus

Abstract: A plasmid encoding the full-length infectious molecular proviral clone of SIV_mac239 was generated. Virus derived from cells transfected with this clone replicated to high levels and was cytopathic for some transformed human CD4⁺ cell lines and primary rhesus macaque peripheral blood mononuclear cells. Since replication of SIV requires the functional expression of the viral encoded rev protein, transient co-transfection studies were initiated with the infectious proviral clone and a well-characterized trans-dominant negative HIV-1 rev mutant.     

Age-dependent changes in T cell homeostasis and SIV load in sooty mangabeys

Sooty mangabeys ( Cercocebus atys ) showed age-dependent changes in T cell regeneration. Younger animals had a high percentage of CD4+ CD45RA+ T cells and a high concentration of T cell receptor excisional circles (TRECs) in peripheral blood, which indicated active thymopoiesis. In contrast, older animals had an increased T cell turnover, which suggested that most T cell production occurred in the periphery. In addition, the number of peripheral CD4+ T cells naturally decreased with age. Non-pathogenic SIVsm infection did not significantly change the T cell proliferation rate or the TREC concentration, though it did cause a moderate loss of peripheral CD4+ T cells. The viral load correlated negatively with age, which could be accounted for by the reduced availability of CD4+ target cells in older mangabeys. Thus, the number of susceptible target cells may be a limiting factor in natural SIV infection.     

A new in vitro model for stem cell differentiation and interaction

Development involves an interplay between various cell types from their birth to their disappearance by differentiation, migration, or death. Analyzing these interactions provides insights into their roles during the formation of a new organism. As a study tool for these interactions, we have created a model based on embryoid bodies (EBs) generated from mouse embryonic stem (mES) cells, which can be used to visualize the differentiation of mES cells into specific cell types while at the same time allowing controlled removal of this same cell population using an enzyme–prodrug approach. Cell-specific expression of Cre induces a switch of EGFP expression to LacZ. Furthermore, it leads to the expression of nitroreductase (NTR), which in combination with the prodrug CB1954 induces apoptosis. Here, we validate this model by showing expression of LacZ and NTR after Cre-mediated recombination. Additionally we show, as an example, that we can target the endothelial cells in EBs using the Tie-2 promoter driving Cre. Ablating Cre-expressing cells by adding CB1954 to the culture led to an abrogated vascular formation. This system can easily be adapted to determine the fate and interaction of many different cell types provided that there is a cell-type-specific promoter available.     

Toward developing a genome-wide microsatellite marker set for linkage analysis in the rhesus macaque (Macaca mulatta): identification of 76 polymorphic markers

Linkage analysis can be problematic in humans because of the lack of large, multigenerational pedigrees and the difficulties in obtaining phenotypic data on all family members. In contrast, large, captive colonies of rhesus macaque are a potentially valuable resource for linkage studies because detailed phenotypic and genealogical data are kept, inbreeding is avoided, and DNA samples can usually be obtained. Microsatellite marker sets for genome-wide screening are available in a number of species, but not for the rhesus macaque. We tested primers to 400 human microsatellite markers from a genome-wide mapping set using DNA from nine unrelated female rhesus macaques. We found that 76 (19%) of the primers amplified a polymorphic product using the standard protocols for human DNA. The average heterozygosity of the markers in humans was 0.80, compared to 0.65 in the rhesus macaques. This study provides preliminary data, which could be used toward the development of a linkage mapping set in this species. There would be a need, however, to confirm the Mendelian inheritance of the markers.     

Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro

In this study, we investigated whether a type of retroviral interference might be one mechanism that mediates the powerful protection induced by live attenuated SIVC8. Our results show that retroviral interference could be demonstrated between SIV and SHIV-HXBc2 in human T-cell lines chronically infected with either SIVC8 or SIVJ5. Lymphocytes from macaques infected with live attenuated SIVC8 were significantly less sensitive (P < 0.05) to in vitro infection by virulent SIVJ5 and SHIV-HXBc2 than were lymphocytes from naive controls. However, this significant difference in the sensitivity of lymphocytes to virus infection was not observed for more efficiently replicating viruses such as SHIVSF33 and SIVsm3. Virus growth was significantly enhanced (P < 0.01) by depletion of CD8+ T-cells, suggesting a role for these cells in the control of SIV replication, both in vitro and in vivo. We found that levels of the beta-chemokines regulated upon activation, normal T-cell expressed and secreted, macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta did not correlate with inhibition of virus replication. Taken together, our findings do not support the hypothesis that retroviral interference is the mechanism by which live attenuated SIVC8 induces protection.     

Teeth-derived stem cells: A source for cell therapy

Cell therapy is one of the important therapeutic approaches in the treatment of many diseases such as cancer, degenerative diseases, and cardiovascular diseases. Among various cell types, which could be used as cell therapies, stem cell therapy has emerged as powerful tools in the treatment of several diseases. Multipotent stem cells are one of the main classes of stem cells that could originate from different parts of the body such as bone marrow, adipose, placenta, and tooth. Among several types of multipotent stem cells, tooth-derived stem cells (TDSCs) are associated with special properties such as accessible, easy isolation, and low invasive, which have introduced them as a good source for using in the treatment of several diseases such as neural injuries, liver fibrosis, and Cohrn’s disease. Here, we provided an overview of TDSCs particular stem cells from human exfoliated deciduous teeth and clinical application of them. Moreover, we highlighted molecular mechanisms involved in the regulation of dental stem cells fate.     

A panel of 20 highly variable microsatellite polymorphisms in rhesus macaques (Macaca mulatta) selected for pedigree or population genetic analysis

This paper reports 20 new microsatellite loci that are highly polymorphic in rhesus macaques (Macaca mulatta). We screened known human microsatellite loci to identify markers that are polymorphic in rhesus macaques, and then selected specific loci that show substantial levels of heterozygosity and robust, reliable amplification. The 20 loci reported here were chosen to include one highly informative microsatellite from each rhesus monkey autosomal chromosome. Fourteen of the 20 polymorphisms are tetranucleotide repeats, and all can be analyzed using standard PCR and electrophoresis procedures. These new rhesus markers have an average of 15.5 alleles per locus and average heterozygosity of 0.83. This panel of DNA polymorphisms will be useful for a variety of different genetic analyses, including pedigree testing, paternity analysis, and population genetic studies. Many of these loci are also likely to be informative in other closely related Old World monkey species.