Pig-tailed Macaques Research Articles

Simultaneous measurement of etravirine, maraviroc and raltegravir in pigtail macaque plasma, vaginal secretions and vaginal tissue using a LC-MS/MS assay.

Etravirine (ETR), maraviroc (MVC) and raltegravir (RAL) are promising antiretroviral drugs being used in HIV treatment and may be interesting for prevention applications such as oral or topical pre-exposure prophylaxis. Here we describe a sensitive and accurate method for the simultaneous detection of ETR, MVC and RAL from pigtail macaque plasma, vaginal secretions, and vaginal tissue. This method is characterized by a straightforward precipitation extraction method, a limit of quantification <0.5ngmL(-1) for all three antiretrovirals bolstered by a corresponding internal standard for each drug analyte, and short run time. Quantification is performed using positive ion electrospray triple quadrupole mass spectrometry. This method was validated over clinically relevant ranges for the three ARV drugs in all three matrices: 0.1-100ngmL(-1) for ETR, 0.05-100ngmL(-1) for MVC and 1-100ngmL(-1) for RAL. Our method is accurate and precise, with measured mean inter-assay precision (%CV) and accuracy (% bias) of 5.08% and 1.96%, respectively, while the mean intra-assay precision and accuracy were 3.44% and 1.08%. The overall post-extraction recovery for ETR, MVC and RAL was >94% in all cases. We also show that extracted biological samples are stable after storage at room temperature or 4°C and after three freeze/thaw cycles. This is the first analytical method capable of quantifying ETR, MVC and RAL in biological matrices relevant for pre-clinical testing of oral or topical HIV prevention methods in pigtailed macaques.

761. Targeted Killing of HIV Infected Cells Using CCR5-Disrupted Anti-HIV-CAR T Cells

A cure for HIV remains an important treatment goal for 30 million HIV-infected individuals worldwide. Long term control of HIV following a single treatment will require a mechanism to eradicate the latent reservoir of HIV infected cells that are capable of reactivating. A previous phase II clinical trial using an anti-HIV chimeric antigen receptor (αHIV-CAR) targeting the CD4-binding site on HIV envelope was partially effective. To optimize this approach we have developed a series of CARs based on the scFV of broadly neutralizing HIV antibodies targeting four different structural regions of the HIV envelope: the V1/V2 loop, the V3 loop, the CD4 binding site, and the membrane-proximal external region. αHIV-CAR T cells targeting different epitopes were compared and were able to kill > 80% of HIV-infected cells grown in the presence of ART. However, a limitation of αHIV-CAR T cells is that the αHIV-CAR also serves as a receptor for HIV and allows HIV infection of αHIV-CAR T cells. Therefore we disrupted the major co-receptor required for HIV cell entry, CCR5, using a megaTAL nuclease, as a means of protecting the CAR-expressing cells from HIV infection. We used two strategies for achieving these dual modifications in primary human T cells. For both strategies, the CCR5 megaTAL nuclease was delivered by mRNA electroporation, which has previously been shown to induce a high rate of bi-allelic NHEJ-mediated gene disruption. αHIV-CAR expression cassettes were delivered into the host genome via lentiviral vectors (LV), or were targeted to the megaTAL nuclease cleavage site in CCR5 using an adeno-associated virus (AAV) that included CCR5 homology arms. Both strategies resulted in stable expression of the CAR construct and specific activation of CAR+ T cells in the presence of an HIV+ cell line. In the presence of actively replicating virus, CCR5-megaTAL treated CAR+ T cells out-performed CAR+ T cells generated by LV delivery alone as measured by reduction in HIV capsid protein. To enable testing in non-human primates (NHP), we re-optimized the cell-editing protocol with NHP lymphocytes. Primary T cells from pigtail macaques were successfully transfected with CCR5 megaTAL mRNA and achieved a CCR5 disruption rate of 50%. Cells were subsequently transduced with αHIV-CAR LV resulting in ~70% of manipulated cells with αHIV-CAR. Expansion of the cells in vitro resulted in 60-fold expansion over 8 days. CAR+ NHP cells specifically killed HIV infected human cells demonstrating that NHP-derived αHIV-CAR+ T cells retained killing function. In conclusion, it is feasible to construct αHIV-CAR+ T cells that are protected from HIV infection in human and NHP cells, and warrants further study in vivo.

38. CCR5 Gene Edited Hematopoietic Stem Cells Engraft in Diverse Anatomical Locales and Undergo SHIV-Dependent Positive Selection in Nonhuman Primates

Background: Gene editing in hematopoietic stem/progenitor cells (HSPCs) has rapidly emerged as a promising therapy for a number of diseases, including human immunodeficiency virus (HIV) infection. We have previously demonstrated the feasibility of this approach in nonhuman primates. Here, we leverage our expertise with gene editing in large animal models to interrogate the clonal persistence, trafficking, and antiviral efficacy of CCR5-edited cells in the pigtailed macaque, M. nemestrina. Our objectives were to map the tissue distribution of HSPC-derived, gene edited progeny, understand how individual gene edited HSPCs persist following autologous transplantation, and develop strategies to select for these cells in vivo.Methods: Zinc Finger Nucleases (ZFNs) are used to target the CCR5 locus in macaque HSPCs. Engraftment and persistence of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured ex vivo and in vivo. Animals are challenged with simian/human immunodeficiency virus (SHIV). Gene edited HSPCs are transplanted either prior to SHIV infection, or in SHIV-infected animals that are treated with combination antiretroviral therapy (cART) in order to approximate a well-suppressed HIV+ patient. Edited cells are measured longitudinally in peripheral blood, bone marrow, gastrointestinal (GI) tract, and lymph nodes, and at necropsy in a panel of 25 tissues, using methods including deep sequencing.Results: We observe up to 14-fold enrichment of CCR5-gene edited memory CD4+ T-cells in SHIV-infected animals, consistent with virus-dependent selection against CCR5 wt memory CD4+ T-cells. Gene edited cells are found in a broad array of anatomical sites, including GI tract and lymph nodes. Spatial and temporal tracking of CCR5 mutations suggests that gene edited cells persist in an analogous fashion to control lentivirus gene-marked cells. Homology directed repair (HDR) pathways can be exploited in macaque CD34+ HSPCs, facilitating knock-in of selectable markers at the disrupted CCR5 locus.Conclusions: Our results in SHIV-infected animals reinforce that this gene editing strategy results in stable engraftment of CCR5-mutated and SHIV-resistant HSPCs and their progeny. Additionally. gene edited CD4+ T-cells undergo positive selection during active infection, further supporting the validity of this approach in the clinic. Moreover, our preliminary ex vivo HDR data suggest that these gene edited cells could be engineered to undergo virus-independent selection.

283. In Vivo Selection of MGMT(P140K) Gene Modified Hematopoietic Cells in the Nonhuman Primate Unmasks a Dormant Pool of Repopulating Clones with Progenitor Cell Ontology

Previously published gene modified cell transplantation studies in nonhuman primate models have described several key features of hematopoietic reconstitution (Kim, S. et al., 2014 and Wu, C. et al., 2014). These studies applied either retrovirus integration site analysis (ISA) or DNA barcode sequencing (DBS) to identify hematopoietic clones in bone marrow or peripheral blood cells. However, these distinct methods detected long-term clones at different time points (1 year after transplant by ISA; ~3 months after transplant by DBS), and neither evaluated reconstitution in the context of selective advantage, which is often the case in gene therapy. Here, we tracked tens of thousands of unique clones in 8 pigtail macaques for up to 10 years following myeloablative transplantation with autologous, lentivirus (LV) gene-modified CD34+ cells. Seven animals received cells gene modified with the P140K mutant methylguanine methyltransferase transgene, conferring resistance to O6- benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy. In two animals, MGMT(P140K)-expressing LVs were DNA barcoded, permitting simultaneous ISA and DBS tracking. Gene marking in peripheral blood cells ranged from 2.3% to 66%. Direct comparison of ISA and DBS demonstrated that abundant clones (&gt 1% of sequence reads) are readily detected by both methods, but DBS captures up to 2-fold more lower abundance clones. Before in vivo selection with O6BG/BCNU, we observed a cell dose-dependent, successive pattern of hematopoietic reconstitution by ISA analysis, with short-term clones declining within 100 days after transplantation. Long-term clones were observed as early as 1 month after transplant. In the first year after transplant, persistent clones ranged from 8% to 54% of clones detected at a &gt 1% frequency, and remained stable in the absence of selective pressure. Importantly, when O6BG/BCNU was administered we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving cell doses exceeding 35×106 CD34+ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. Gene ontology analysis of integration loci among early, long-term and dormant clonal populations identified the greatest functional overlap between early and dormant pools. These data suggest that some short-term repopulating clones revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess CD34+ cell numbers results in early dormancy of a large proportion of early repopulating clones. Together, these findings suggest that previous estimates of short- and long-term clonal frequency are an underestimate of true graft repopulation potential.

Socialization in pigtailed macaques (Macaca nemestrina).

In response to new emphasis by regulatory agencies regarding socialization, behavioral management programs are allocating greater resources to maximize socialization opportunities for laboratory primates. Information regarding predictors of compatibility and risk of injury for all laboratory-housed species of macaques are needed to make social introductions and pairings as efficient and safe as possible. This study presents data on 674 pairs of pigtailed macaques (Macaca nemestrina) at the Washington National Primate Research Center over a 7-year period. During pair introduction, behavior was monitored while the degree of tactile contact was gradually increased. Based on observed behavior, pairs were assigned a behavioral introduction score (BIS), rating the quality of their interactions for each day of introduction. Animals deemed compatible, based on the BIS and technologist judgment, were allowed to progress to continuous contact with no staff present. A small proportion of animals deemed compatible at introduction was later separated for subsequent incompatibility or aggression; these proportions were higher in full contact compared to protected contact pairings. Of 674 pairs, 75% were deemed compatible at introduction in protected contact; 86 of these pairs were later transitioned to full contact with 98% compatibility. Predictors of decreased compatibility assessed during protected contact introductions included age (adult pairs were less compatible), the BIS on the last day of introduction, and aggression or injury during the introductory period. Predictors of injuries during the protected contact introduction process included: aggression on the first day of introduction, a negative BIS on the first or last day of introduction, and, surprisingly, the presence of grooming on the first day of introduction. Injuries during both introduction and subsequent pairing in protected contact were rare; however, injury rates increased significantly during full-contact pairing. These findings underscore the necessity of species-specific data to guide decision-making during the social introduction process. Am. J. Primatol. 79:e22556, 2017. © 2016 Wiley Periodicals, Inc.

Conservation of the glycoprotein B homologs of the Kaposi׳s sarcoma-associated herpesvirus (KSHV/HHV8) and old world primate rhadinoviruses of chimpanzees and macaques

The envelope-associated glycoprotein B (gB) is highly conserved within the Herpesviridae and plays a critical role in viral entry. We analyzed the evolutionary conservation of sequence and structural motifs within the Kaposi׳s sarcoma-associated herpesvirus (KSHV) gB and homologs of Old World primate rhadinoviruses belonging to the distinct RV1 and RV2 rhadinovirus lineages. In addition to gB homologs of rhadinoviruses infecting the pig-tailed and rhesus macaques, we cloned and sequenced gB homologs of RV1 and RV2 rhadinoviruses infecting chimpanzees. A structural model of the KSHV gB was determined, and functional motifs and sequence variants were mapped to the model structure. Conserved domains and motifs were identified, including an “RGD” motif that plays a critical role in KSHV binding and entry through the cellular integrin αVβ3. The RGD motif was only detected in RV1 rhadinoviruses suggesting an important difference in cell tropism between the two rhadinovirus lineages.

Cerebrospinal Fluid Biomarkers of Simian Immunodeficiency Virus Encephalitis : CSF Biomarkers of SIV Encephalitis.

Antiretroviral therapy has led to increased survival of HIV-infected patients but also increased prevalence of HIV-associated neurocognitive disorders. We previously identified YKL40 as a potential cerebrospinal fluid (CSF) biomarker of lentiviral central nervous system (CNS) disease in HIV-infected patients and in the macaque model of HIV encephalitis. The aim of this study was to define the specificity and sensitivity along with the predictive value of YKL40 as a biomarker of encephalitis and to assess its relationship to CSF viral load. CSF YKL40 and SIV RNA concentrations were analyzed over the course of infection in 19 SIV-infected pigtailed macaques and statistical analyses were performed to evaluate the relationship to encephalitis. Using these relationships, CSF alterations of 31 neuroimmune markers were studied pre-infection, during acute and asymptomatic infection, at the onset of encephalitis, and at necropsy. YKL40 CSF concentrations above 1122ng/ml were found to be a specific and sensitive biomarker for the presence of encephalitis and were highly correlated with CSF viral load. Macaques that developed encephalitis had evidence of chronic CNS immune activation during early, asymptomatic, and end stages of infection. At the onset of encephalitis, CSF demonstrated a rise of neuroimmune markers associated with macrophage recruitment, activation and interferon response. CSF YKL40 concentration and viral load are valuable biomarkers to define the onset of encephalitis. Chronic CNS immune activation precedes the development of encephalitis while some responses suggest protection from CNS lentiviral disease.

Viral seroprevalence in northern pig-tailed macaques (Macaca leonina) derived from Ho Chi Minh City, Vietnam.

Non-human primates are natural virus reservoirs, whether wild or domestic. In this study, we determined the seroprevalence of common viruses by ELISA in a northern pig-tailed macaque (Macaca leonina) colony derived from Ho Chi Minh City, Vietnam. A total of 20 types of virus which are commonly selected as target microorganisms for specific-pathogen-free colonies, or which have zoonotic potential were included in this study. The results showed only 2 in 90 northern pig-tailed macaques were seronegative for all the detected viruses, and at least 16 out of the total 20 types of virus tested were prevalent in this colony, so these macaques were commonly infected by various viruses. These macaques should be carefully assessed for viral seroprevalence in order to prevent zoonotic diseases from being transferred to human beings.

Persistence of mammals in a selectively logged forest in Malaysian Borneo

Many tropical mammals with important functional roles in forest ecosystems are threatened with extinction, yet how they respond to increasingly prevalent habitat disturbance, such as selective logging, is not well understood. We deployed 43 motion-triggered camera traps in 2013 and 2014 in unlogged forest and in a forest logged three decades previously in Malaysian Borneo. We used camera trap photographs to assess whether selective logging influenced the local abundance of medium to large-bodied ground-dwelling mammals. We focused our study on six locally common species: sambar deer, (Rusa unicolor), yellow muntjac (Muntiacus atherodes), chevrotains (Tragulus spp.), banded civet (Hemigalus derbyanus), Malay civet (Viverra tangalunga), and pig-tailed macaque (Macaca nemestrina). Occupancy models were used to estimate local abundance. None of the mammals in our study exhibited decreased abundance in logged forest, although yellow muntjac and pig-tailed macaque were associated with canopy height. Contiguous forest connects these two areas, allowing animal movement between them, potentially explaining the lack of responses. The elapsed time since logging may have further influenced our findings. The effects of logging on mammal local abundance may no longer detectable after 30 years. Overall, our results demonstrate the importance of regenerating forest as habitat for medium- to large-sized mammal species.

Accurate measurement of female genital tract fluid dilution in cervicovaginal lavage samples.

An ion chromatographic method with conductivity detection for the precise and accurate analysis of lithium ions in phosphate-buffered saline, used as a cervicovaginal lavage (CVL) fluid, was developed and validated. The lithium ion dilution factor during the CVL is used to calculate the volume of cervicovaginal fluid (CVF) collected. Initial CVL Li(+) concentrations of 1mM and 10mM were evaluated. The method is robust, practical, and afforded an accurate measurement (5% of the measurement, or better) at 24μL of vaginal fluid simulant collected per mL of CVL fluid, as low as 5μLmL(-1) using 10mM Li(+) with a measurement accuracy of 6.7%. Ion chromatograms of real-world CVL samples collected in vivo from common animal models (sheep and pig-tailed macaque) and a human volunteer demonstrate that the analysis is interference-free. The method is readily transferrable and should enable the accurate measurement of CVF volume collected during CVLs benefitting a broad range of research disciplines, including pharmacokinetic, pharmacodynamic, metabolomic, and microbiome studies.

High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo

The low fidelity of HIV replication facilitates immune and drug escape. Some reverse transcriptase (RT) inhibitor drug-resistance mutations increase RT fidelity in biochemical assays but their effect during viral replication is unclear. We investigated the effect of RT mutations K65R, Q151N and V148I on SIV replication and fidelity in vitro, along with SIV replication in pigtailed macaques. SIVmac239-K65R and SIVmac239-V148I viruses had reduced replication capacity compared to wild-type SIVmac239. Direct virus competition assays demonstrated a rank order of wild-type>K65R>V148I mutants in terms of viral fitness. In single round in vitro-replication assays, SIVmac239-K65R demonstrated significantly higher fidelity than wild-type, and rapidly reverted to wild-type following infection of macaques. In contrast, SIVmac239-Q151N was replication incompetent in vitro and in pigtailed macaques. Thus, we showed that RT mutants, and specifically the common K65R drug-resistance mutation, had impaired replication capacity and higher fidelity. These results have implications for the pathogenesis of drug-resistant HIV.

Clinicopathologic, Immunohistochemical, and Ultrastructural Findings of a Fatal Case of Middle East Respiratory Syndrome Coronavirus Infection in the United Arab Emirates, April 2014

Middle East respiratory syndrome coronavirus (MERS-CoV) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal MERS-CoV infection is unknown. We describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of MERS-CoV in the world, which was related to a hospital outbreak in the United Arab Emirates in April 2014. The main histopathologic finding in the lungs was diffuse alveolar damage. Evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. Double staining immunoassays that used anti–MERS-CoV antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of MERS-CoV antigen; double immunostaining with dipeptidyl peptidase 4 showed colocalization in scattered pneumocytes and syncytial cells. No evidence of extrapulmonary MERS-CoV antigens were detected, including the kidney. These results provide critical insights into the pathogenesis of MERS-CoV in humans.

Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque.

This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and NRs in macaque tissues were usually comparable to those of human tissues. In addition, menopause did not significantly alter the ectocervical mRNA levels of CYP1A1, CYP1B1, or NRs.

Cloning, sequencing, and polymorphism analysis of novel classical MHC class I alleles in northern pig-tailed macaques (Macaca leonina).

The northern pig-tailed macaque (Macaca leonina) has been confirmed to be an independent species from the pig-tailed macaque group of Old World monkey. We have previously reported that the northern pig-tailed macaques were also susceptible to HIV-1. Here, to make this animal a potential HIV/AIDS model and to discover the mechanism of virus control, we attempted to assess the role of major histocompatibility complex (MHC) class I-restricted immune responses to HIV-1 infection, which was associated with viral replication and disease progression. As an initial step, we first cloned and characterized the classical MHC class I gene of northern pig-tailed macaques. In this study, we identified 39 MHC class I alleles including 17 MHC-A and 22 MHC-B alleles. Out of these identified alleles, 30 were novel and 9 were identical to alleles previously reported from other macaque species. The MHC-A and MHC-B loci were both duplicates as rhesus macaques and southern pig-tailed macaques. In addition, we also detected the patterns of positive selection in northern pig-tailed macaques and revealed the existence of balance selection with 20 positive selection sites in the peptide binding region. The analysis of B and F peptide binding pockets in northern and southern pig-tailed macaques and rhesus macaques suggested that they were likely to share a few common peptides to present. Thus, this study provides important MHC immunogenetics information and adds values to northern pig-tailed macaques as a promising HIV/AIDS model.

Antibiotic and Antiinflammatory Therapy Transiently Reduces Inflammation and Hypercoagulation in Acutely SIV-Infected Pigtailed Macaques.

Increased chronic immune activation and inflammation are hallmarks of HIV/SIV infection and are highly correlated with progression to AIDS and development of non-AIDS comorbidities, such as hypercoagulability and cardiovascular disease. Intestinal dysfunction resulting in microbial translocation has been proposed as a lead cause of systemic immune activation and hypercoagulability in HIV/SIV infection. Our goal was to assess the biological and clinical impact of a therapeutic strategy designed to reduce microbial translocation through reduction of the microbial content of the intestine (Rifaximin-RFX) and of gut inflammation (Sulfasalazine-SFZ). RFX is an intraluminal antibiotic that was successfully used in patients with hepatic encephalopathy. SFZ is an antiinflammatory drug successfully used in patients with mild to moderate inflammatory bowel disease. Both these clinical conditions are associated with increased microbial translocation, similar to HIV-infected patients. Treatment was administered for 90 days to five acutely SIV-infected pigtailed macaques (PTMs) starting at the time of infection; seven untreated SIVsab-infected PTMs were used as controls. RFX+SFZ were also administered for 90 days to three chronically SIVsab-infected PTMs. RFX+SFZ administration during acute SIVsab infection of PTMs resulted in: significantly lower microbial translocation, lower systemic immune activation, lower viral replication, better preservation of mucosal CD4+ T cells and significantly lower levels of hypercoagulation biomarkers. This effect was clear during the first 40 days of treatment and was lost during the last stages of treatment. Administration of RFX+SFZ to chronically SIVsab–infected PTMs had no discernible effect on infection. Our data thus indicate that early RFX+SFZ administration transiently improves the natural history of acute and postacute SIV infection, but has no effect during chronic infection.

Central nervous system-specific consequences of simian immunodeficiency virus Gag escape from major histocompatibility complex class I-mediated control.

In the fourth decade of the HIV epidemic, the relationship between host immunity and HIV central nervous system (CNS) disease remains incompletely understood. Using a simian immunodeficiency virus (SIV)/macaque model, we examined CNS outcomes in pigtailed macaques expressing the MHC class I allele Mane-A1*084:01 which confers resistance to SIV-induced CNS disease and induces the prototypic viral escape mutation Gag K165R. Insertion of gag K165R into the neurovirulent clone SIV/17E-Fr reduced viral replication in vitro compared to SIV/17E-Fr. We also found lower cerebrospinal fluid (CSF), but not plasma, viral loads in macaques inoculated with SIV/17E-Fr K165R versus those inoculated with wildtype. Although escape mutation K165R was genotypically stable in plasma, it rapidly reverted to wildtype Gag KP9 in both CSF and in microglia cultures. We induced robust Gag KP9-specific CTL tetramer responses by vaccinating Mane-A*084:01-positive pigtailed macaques with a Gag KP9 virus-like particle (VLP) vaccine. Upon SIV/17E-Fr challenge, vaccinated animals had lower SIV RNA in CSF compared to unvaccinated controls, but showed no difference in plasma viral loads. These data clearly demonstrate that viral fitness in the CNS is distinct from the periphery and underscores the necessity of understanding the consequences of viral escape in CNS disease with the advent of new therapeutic vaccination strategies.

Multilineage polyclonal engraftment of Cal-1 gene-modified cells and in vivo selection after SHIV infection in a nonhuman primate model of AIDS

We have focused on gene therapy approaches to induce functional cure/remission of HIV-1 infection. Here, we evaluated the safety and efficacy of the clinical grade anti-HIV lentiviral vector, Cal-1, in pigtailed macaques (Macaca nemestrina). Cal-1 animals exhibit robust levels of gene marking in myeloid and lymphoid lineages without measurable adverse events, suggesting that Cal-1 transduction and autologous transplantation of hematopoietic stem cells are safe, and lead to long-term, multilineage engraftment following myeloablative conditioning. Ex vivo, CD4+ cells from transplanted animals undergo positive selection in the presence of simian/human immunodeficiency virus (SHIV). In vivo, Cal-1 gene-marked cells are evident in the peripheral blood and in HIV-relevant tissue sites such as the gastrointestinal tract. Positive selection for gene-marked cells is observed in blood and tissues following SHIV challenge, leading to maintenance of peripheral blood CD4+ T-cell counts in a normal range. Analysis of Cal-1 lentivirus integration sites confirms polyclonal engraftment of gene-marked cells. Following infection, a polyclonal, SHIV-resistant clonal repertoire is established. These findings offer strong preclinical evidence for safety and efficacy of Cal-1, present a new method for tracking protected cells over the course of virus-mediated selective pressure in vivo, and reveal previously unobserved dynamics of virus-dependent T-cell selection.

Plasmodium knowlesi: an update

There were only four species of Plasmodium that were thought to cause malaria in humans until a large number of human infections by Plasmodium knowlesi, a malaria parasite typically found in long-tailed and pig-tailed macaques, were reported in 2004 in Malaysian Borneo. Since then, cases of knowlesi malaria have been reported throughout South-east Asia and also in travellers returning from the region. This article describes the molecular, entomological and epidemiological data which indicate that P. knowlesi is an ancient parasite that is primarily zoonotic, and there are three highly divergent sub-populations. It also describes the detection methods for P. knowlesi, which is morphologicaly similar to P. malariae, and the clinical features and treatment of this malaria parasite that is potentially fatal.

Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques.

Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10(8) PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.

Changes in the primate trade in indonesian wildlife markets over a 25-year period: Fewer apes and langurs, more macaques, and slow lorises.

Indonesia has amongst the highest primate species richness, and many species are included on the country's protected species list, partially to prevent over-exploitation. Nevertheless traders continue to sell primates in open wildlife markets especially on the islands of Java and Bali. We surveyed 13 wildlife markets in 2012-2014 and combined our results with previous surveys from 1990-2009 into a 122-survey dataset with 2,424 records of 17 species. These data showed that the diversity of species in trade decreased over time, shifting from rare rainforest-dwelling primates traded alongside more widespread species that are not confined to forest to the latter type only. In the 1990s and early 2000s orangutans, gibbons and langurs were commonly traded alongside macaques and slow lorises but in the last decade macaques and slow lorises comprised the bulk of the trade. In 2012-2014 we monitored six wildlife markets in Jakarta, Bandung and Garut (all on Java), and Denpasar (Bali). During 51 surveys we recorded 1,272 primates of eight species. Traders offered long-tailed macaque (total 1,007 individuals) and three species of slow loris (228 individuals) in five of the six markets, whereas they traded ebony langurs (18 individuals), and pig-tailed macaques (14 individuals) mostly in Jakarta. Pramuka and Jatinegara markets, both in Jakarta, stood out as important hubs for the primate trade, with a clear shift in importance over time from the former to the latter. Slow lorises, orangutans, gibbons and some langurs are protected under Indonesian law, which prohibits all trade in them; of these protected species, only the slow lorises remained common in trade throughout the 25-year period. Trade in non-protected macaques and langurs is subject to strict regulations-which market traders did not follow-making all the market trade in primates that we observed illegal. Trade poses a substantial threat to Indonesian primates, and without enforcement, the sheer volume of trade may mean that species of Least Concern or Near Threatened may rapidly decline. Am. J. Primatol. 79:e22517, 2017. © 2015 Wiley Periodicals, Inc.