Pig-tailed Macaques Research Articles

Uterine overdistention induces preterm labor mediated by inflammation: observations in pregnant women and nonhuman primates

Uterine overdistention is thought to induce preterm labor in women with twin and multiple pregnancies, but the pathophysiology remains unclear. We investigated for the first time the pathogenesis of preterm birth associated with rapid uterine distention in a pregnant nonhuman primate model. A nonhuman primate model of uterine overdistention was created using preterm chronically catheterized pregnant pigtail macaques (Macaca nemestrina) by inflation of intraamniotic balloons (N = 6), which were compared to saline controls (N = 5). Cesarean delivery was performed due to preterm labor or at experimental end. Microarray, quantitative reverse transcriptase polymerase chain reaction, Luminex (Austin, TX), and enzyme-linked immunosorbent assay were used to measure messenger RNA (mRNA) and/or protein levels from monkey (amniotic fluid, myometrium, maternal plasma) and human (amniocytes, amnion, myometrium) tissues. Statistical analysis employed analysis of covariance and Wilcoxon rank sum. Biomechanical forces were calculated using the law of Laplace. Preterm labor occurred in 3 of 6 animals after balloon inflation and correlated with greater balloon volume and uterine wall stress. Significant elevations of inflammatory cytokines and prostaglandins occurred following uterine overdistention in an "inflammatory pulse" that correlated with preterm labor (interleukin [IL]-1β, tumor necrosis factor [TNF]-α, IL-6, IL-8, CCL2, prostaglandin E2, prostaglandin F2α, all P < .05). A similar inflammatory response was observed in amniocytes in vitro following mechanical stretch (IL1β, IL6, and IL8 mRNA multiple time points, P < .05), in amnion of women with polyhydramnios (IL6 and TNF mRNA, P < .05) and in amnion (TNF-α) and myometrium of women with twins in early labor (IL6, IL8, CCL2, all P < .05). Genes differentially expressed in the nonhuman primate after balloon inflation and in women with polyhydramnios and twins are involved in tissue remodeling and muscle growth. Uterine overdistention by inflation of an intraamniotic balloon is associated with an inflammatory pulse that precedes and correlates with preterm labor. Our results indicate that inflammation is an early event after a mechanical stress on the uterus and leads to preterm labor when the stress is sufficiently great. Further, we find evidence of uterine tissue remodeling and muscle growth as a common, perhaps compensatory, response to uterine distension.

Efficacy of topical tenofovir against transmission of a tenofovir-resistant SHIV in macaques.

BackgroundTopically delivered tenofovir (TFV) from intravaginal rings, tablets, or gels is being evaluated for HIV prevention. We previously demonstrated that TFV delivered vaginally by gel protected macaques from vaginal infection with SHIV. Here we investigated efficacy of the TFV gel against vaginal transmission of a TFV-resistant SHIV containing the K65R mutation (SHIV162P3K65R) and its relationship to drug levels in vaginal tissues.ResultsSHIV162P3K65R shows approximately a 5-fold reduction in susceptibility to TFV compared to wild-type SHIV. Efficacy was evaluated in pig-tailed macaques exposed vaginally twice-weekly (up to 10 weeks) to SHIV162P3K65R 30 min after receiving placebo (n = 6) or 1% TFV (n = 6) gel. Four of the six controls were infected after a median of 5 exposures. In contrast, five of six macaques that received TFV gel remained uninfected after 20 vaginal SHIV162P3K65R exposures, resulting in an estimated efficacy of 75%. The mean intracellular TFV-diphosphate (TFV-DP) concentrations in vaginal lymphocytes 4 h after a single gel dose were found to be high (1,631 fmol/106 cells, range 492–3,847) and within the in vitro IC75 range (1,206 fmol/106 cells) for SHIV162P3K65R.ConclusionBoth the modest resistance conferred by K65R and the high TFV-DP exposure in vaginal lymphocytes, likely explain the observed protection. The findings in this model do not predict complete loss of protection by topical TFV against vaginal exposure to HIV-1K65R viruses and provide a tissue drug target for high efficacy. These data will facilitate the development of TFV delivery platforms that have high activity on both wild-type and TFV-resistant viruses.

Short Communication: Practical Experience with Analysis and Design of Repeat Low-Dose SHIVSF162P3 Exposure Studies in Female Pigtail Macaques with Varying Susceptibility During Menstrual Cycling.

Vaginal SHIVSF162P3 acquisition in pigtail macaques (Macaca nemestrina) is dependent on time point during the menstrual cycle. Susceptibility is higher around menstruation and lower at ovulation in mid cycle. This complicates the design of repeat low-dose (RLD) SHIV exposure studies because virus challenges given during low susceptibility periods have lower chances to infect. To account for fluctuating susceptibility, we analyzed menstrual cycles rather than exposures until infection following virus challenges. We first reanalyzed infection data of 41 macaques receiving placebo or no treatment during once (n=18) or twice (n=23) weekly virus exposures. The same number of cycles was required for infection with either challenge frequency, while it took a median four or six challenges for once or twice weekly exposures, respectively. More virus challenges to infection likely reflect frequent unsuccessful exposures in frequently exposed animals. When reanalyzing two previously reported biomedical HIV intervention studies, we found 1% tenofovir gel was 74% or 86% efficacious based on cycles or exposures (p=0.019 or p=0.003, respectively, Fisher's exact test), while 1% raltegravir gel was 84% or 89 % efficacious, respectively (p=0.047 or p=0.031). Evaluating the number of menstrual cycles rather than exposures until infection can account for varying susceptibility during the menstrual cycle. Our observations have implications for future study designs such as planning the frequency of virus exposures. Menstrual cycle analysis may also avoid potential overestimation of efficacy against vaginal challenges during low susceptibility periods in the cycle that are unlikely to cause infection.

Analysis of putative mucosal SHIV susceptibility factors during repeated DMPA treatments in pigtail macaques.

Depot-medroxyprogesterone acetate (DMPA) has been associated in some studies with increased HIV susceptibility in women. We used a pigtail macaque model to document the effects of repeated DMPA treatments and their potential contribution to increased SHIV susceptibility. Nine pigtails were administered 2.5, 1.5, or 0.5 mg/kg DMPA in study weeks one and four. Menstrual cycling, vaginal epithelial thickness, and other SHIV susceptibility factors were monitored for a mean of 24 study weeks. All DMPA treatments suppressed menstrual cycling and increased vaginal pH. The vaginal epithelium thinned naturally during baseline menstrual cycles (from mean of 351 to 161 μm in late-luteal phase). Following DMPA, the non-nucleated layer was temporarily absent. Two weeks post-second DMPA injection, mean epithelial thickness was 53, 45, and 167 μm for the descending doses, respectively. All animals showed temporal vaginal epithelial thinning with loss of the non-nucleated layer, and vaginal pH changes post-DMPA injections.

Repeated Vaginal SHIV Challenges in Macaques Receiving Oral or Topical Preexposure Prophylaxis Induce Virus-Specific T-Cell Responses.

Preexposure prophylaxis (PrEP) for HIV prevention is a novel biomedical prevention method. We have previously modeled PrEP during rectal SHIV exposures in macaques and identified that Simian/Human Immunodeficiency Virus chimera (SHIV)-specific T-cell responses were induced in the presence of antiretroviral drugs, an observation previously termed T-cell chemo-vaccination. This report expands those initial findings by examining a larger group of macaques that were given oral or topical PrEP during repeated vaginal virus exposure. Thirty-six female pigtail macaques received up to 20 repeat low-dose vaginal inoculations with wild-type (WT) SHIVSF162P3 (n = 24) or a clonal derivative with the tenofovir (TFV) K65R drug-resistant mutation (n = 12). PrEP consisted of oral Truvada (n = 6, WT), TFV vaginal gel (n = 6, K65R), or TFV intravaginal ring (n = 6, WT). The remaining animals were PrEP-inexperienced controls (n = 12, WT; n = 6, K65R). SHIV-specific T cells were identified and characterized using interferon γ Enzyme-Linked ImmunoSpot (ELISPOT) and multiparameter flow cytometry. Of 9 animals that were on PrEP and remained uninfected during WT SHIV vaginal challenges, 8 (88.9%) developed virus-specific T-cell responses. T cells were in CD4 and CD8 compartments, reached up to 4900 interferon γ-producing cells per million peripheral blood mononuclear cells, and primarily pol directed. In contrast, the replication-impaired K65R virus did not induce detectable T-cell responses, likely reflecting the need for adequate replication. Virus-specific T-cell responses occur frequently in oral or topical PrEP-protected pigtail macaques after vaginal exposure to WT SHIV virus. The contribution of such immune responses to protection from infection during and after PrEP warrants further investigation.

Elite Control, Gut CD4 T Cell Sparing, and Enhanced Mucosal T Cell Responses in Macaca nemestrina Infected by a Simian Immunodeficiency Virus Lacking a gp41 Trafficking Motif.

Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques (Macaca mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4(+) T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques (Macaca nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4(+) T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of <15 to 50 RNA copies/ml. CD4(+) T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4(+) T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8(+) cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

8th IAS Conference on HIV Pathogenesis, Treatment & Prevention 19–22 July 2015, Vancouver, Canada

IntroductionSensitive assays are needed for detection of residual HIV in patients with undetectable plasma viral loads to determine if eradication strategies are effective. The gold standard quantitative viral outgrowth assay (QVOA) underestimates the magnitude of the viral reservoir, while sensitive PCR-based assays lack the ability to distinguish replication competent from defective virus. We sought to determine whether xenograft of leukocytes from HIV-1 infected patients with undetectable plasma viral loads into severely immunocompromised mice would result in viral amplification and measurable viral loads within the aberrant murine host.MethodsWe evaluated whether xenograft of 1) peripheral blood mononuclear cells (PBMCs) from five HIV-1+ patients on suppressive antiretroviral therapy (ART), 2) PBMCs or purified resting CD4+ T cells from 5 HIV-1+ elite suppressors (ES), or 3) PBMCs from a Simian Immunodeficiency Virus (SIV)+ pigtailed macaque on suppressive ART, all with undetectable plasma viral loads, into NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice resulted in viral amplification in the mouse. Successful xenograft of mice was confirmed by flow cytometry. Human CD8+ T cells were depleted in humanized mice with depleting antibody, and CD4+ T cells were activated in a subset of mice with activating anti-CD3. Plasma viral loads in xenografted mice were quantified using qRT-PCR, and compared to plasma viral load and QVOA results from the human or macaque donor.ResultsWith this murine viral outgrowth assay (MVOA), we amplified HIV-1 from all 10 HIV+ subjects with undetectable plasma viral load, including an ES from whom we were unable to recover virus by QVOA. We detected HIV in mice an average of 20 days after xenograft with PBMCs from patients on suppressive ART, and an average of 28 days after xenograft with PBMCs or resting CD4+ T cells from ES. For two of the mice xenografted with CD4+ T cells from ES, we detected HIV only after activation with anti-CD3. We similarly detected SIV in macaquized mice by seven days post-xenograft.ConclusionsThe MVOA has the potential to serve as a powerful tool to identify residual HIV-1 in patients with undetectable viral loads, such as those who have undergone promising cure therapies.

Critical Role for the Adenosine Pathway in Controlling Simian Immunodeficiency Virus-Related Immune Activation and Inflammation in Gut Mucosal Tissues.

The role of the adenosine (ADO) pathway in human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV) infection remains unclear. We compared SIVsab-induced changes of markers related to ADO production (CD39 and CD73) and breakdown (CD26 and adenosine deaminase) on T cells from blood, lymph nodes, and intestine collected from pigtailed macaques (PTMs) and African green monkeys (AGMs) that experience different SIVsab infection outcomes. We also measured ADO and inosine (INO) levels in tissues by mass spectrometry. Finally, we assessed the suppressive effect of ADO on proinflammatory cytokine production after T cell receptor stimulation. The baseline level of both CD39 and CD73 coexpression on regulatory T cells and ADO levels were higher in AGMs than in PTMs. Conversely, high INO levels associated with dramatic increases in CD26 expression and adenosine deaminase activity were observed in PTMs during chronic SIV infection. Immune activation and inflammation markers in the gut and periphery inversely correlated with ADO and directly correlated with INO. Ex vivo administration of ADO significantly suppressed proinflammatory cytokine production by T cells in both species. In conclusion, the opposite dynamics of ADO pathway-related markers and contrasting ADO/INO levels in species with divergent proinflammatory responses to SIV infection support a key role of ADO in controlling immune activation/inflammation in nonprogressive SIV infections. Changes in ADO levels predominately occurred in the gut, suggesting that the ADO pathway may be involved in sparing natural hosts of SIVs from developing SIV-related gut dysfunction. Focusing studies of the ADO pathway on mucosal sites of viral replication is warranted. The mechanisms responsible for the severe gut dysfunction characteristic of progressive HIV and SIV infection in humans and macaques are not completely elucidated. We report that ADO may play a key role in controlling immune activation/inflammation in nonprogressive SIV infections by limiting SIV-related gut inflammation. Conversely, in progressive SIV infection, significant degradation of ADO occurs, possibly due to an early increase of ADO deaminase complexing protein 2 (CD26) and adenosine deaminase. Our study supports therapeutic interventions to offset alterations of this pathway during progressive HIV/SIV infections. These potential approaches to control chronic immune activation and inflammation during pathogenic SIV infection may prevent HIV disease progression.

Analysis of the complete mitochondrial genome and characterization of diverse NUMTs of Macaca leonina

As a non-human primate, the pig-tailed macaque has received wide attention because it can be infected by HIV-1. In this study, we determined the complete mtDNA sequence of the northern pig-tailed macaque (Macaca leonina). Unexpectedly, during the amplification of the mtDNA control region (D-loop region) we observed several D-loop-like sequences, which were NUMTs (nuclear mitochondrial sequences) and a total of 14 D-loop-like NUMT haplotypes were later identified in five individuals. The neighbor-joining tree and estimated divergence time based on these D-loop-like NUMT sequences of M. leonina provide some insights into the understanding of the evolutionary history of NUMTs. D-loop-like haplotypes G and H, which also exist in the nuclear genome of mulatta, appear to have been translocated into the nuclear genome before the divergence of M. mulatta and M. leonina. The other D-loop-like NUMT haplotypes were translocated into the nuclear genome of M. leonina after the divergence of the two species. Later sequence conversion was predicted to occur among these 14 D-loop-like NUMT haplotypes. The overall structure of the mtDNA of M. leonina was found to be similar to that seen in other mammalian mitochondrial genomes. Phylogenetic analysis based on the maximum likelihood method shows M. leonina clustered with Macaca silenus among the analyzed mammalian species.

Cataloguing of Potential HIV Susceptibility Factors during the Menstrual Cycle of Pig-Tailed Macaques by Using a Systems Biology Approach.

Our earlier studies with pig-tailed macaques demonstrated various simian-human immunodeficiency virus (SHIV) susceptibilities during the menstrual cycle, likely caused by cyclic variations in immune responses in the female genital tract. There is concern that high-dose, long-lasting, injectable progestin-based contraception could mimic the high-progesterone luteal phase and predispose women to human immunodeficiency type 1 (HIV-1) acquisition and transmission. In this study, we adopted a systems biology approach employing proteomics (tandem mass spectrometry), transcriptomics (RNA microarray hybridization), and other specific protein assays (enzyme-linked immunosorbent assays and multiplex chemokine and cytokine measurements) to characterize the effects of hormonal changes on the expression of innate factors and secreted proteins in the macaque vagina. Several antiviral factors and pathways (including acute-phase response signaling and complement system) were overexpressed in the follicular phase. Conversely, during the luteal phase there were factors overexpressed (including moesins, syndecans, and integrins, among others) that could play direct or indirect roles in enhancing HIV-1 infection. Thus, our study showed that specific pathways and proteins or genes might work in tandem to regulate innate immunity, thus fostering further investigation and future design of approaches to help counter HIV-1 acquisition in the female genital tract. HIV infection in women is poorly understood. High levels of the hormone progesterone may make women more vulnerable to infection. This could be the case during the menstrual cycle, when using hormone-based birth control, or during pregnancy. The biological basis for increased HIV vulnerability is not known. We used an animal model with high risk for infection during periods of high progesterone. Genital secretions and tissues during the menstrual cycle were studied. Our goal was to identify biological factors upregulated at high progesterone levels, and we indeed show an upregulation of genes and proteins which enhance the ability of HIV to infect when progesterone is high. In contrast, during low-progesterone periods, we found more HIV inhibitory factors. This study contributes to our understanding of mechanisms that may regulate HIV infection in females under hormonal influences. Such knowledge is needed for the development of novel prevention strategies.

Species-specific differences in the expression and regulation of α4β7 integrin in various nonhuman primates.

Among nonhuman primates, SIV-infected Asian pigtailed macaques (PM) are relatively more susceptible to infection and disease progression than SIV-infected rhesus macaques (RM). In addition, SIV-infected African natural hosts such as the sooty mangabeys (SM) are resistant to disease. The mechanisms associated with such species-related variable clinical outcomes remain ill-defined but hold the potential to provide insights into the underlying mechanisms surrounding HIV pathogenesis. Recent findings indicate that the expression of the heterodimeric gut homing integrin α4β7 can influence both susceptibility and disease progression in RM. It was reasoned that differences in the frequencies/surface densities of α4β7-expressing lymphocytes might contribute to the differences in the clinical outcome of SIV infection among NHPs. In this article, we report that CD4(+) T cells from PM constitutively express significantly higher levels of α4β7 than RM or SM. Retinoic acid, a key regulator of α4β7 expression, was paradoxically found at higher levels in the plasma of SM versus RM or PM. We also observed pairing of β7 with αE (αEβ7) on CD4(+) T cells in the peripheral blood of SM, but not PM or RM. Finally, the differential mean density of expression of α4β7 in RM versus SM versus PM was predominantly dictated by species-specific sequence differences at the level of the β7 promoters, as determined by in vitro reporter/promoter construct transfection studies. We propose that differences in the regulation and expression of α4β7 may explain, in part, the differences in susceptibility and SIV disease progression in these NHP models.

Fear reactions to snakes in naïve mouse lemurs and pig-tailed macaques

Primates have been predated on by snakes throughout their evolution and as a result, antipredator responses accompanied by signs of fear are often witnessed in the wild. In captivity, however, the fear of snakes is less clear, as experiments with naïve nonhuman primates have given inconsistent results. In this study, we present evidence that naïve mouse lemurs (Microcebus murinus) and putatively naïve pig-tailed macaques (Macaca nemestrina) do exhibit fear of snakes, even though the apparent reactions are mild. In an experiment with control- or snake-odoured boxes, mouse lemurs clearly avoided feeding in the latter. When the latency of touching rubber models was measured, pig-tailed macaques took longer to touch a toy snake compared with a toy lizard. Our findings that fear of snakes is shown by naïve individuals support the hypothesis that it is innate in primates.

Spider monkeys (Ateles geoffroyi) are less sensitive to the odor of aliphatic ketones than to the odor of other classes of aliphatic compounds

Aliphatic ketones are widely present in body-borne and food odors of primates. Therefore, we used an operant conditioning paradigm and determined olfactory detection thresholds in four spider monkeys for a homologous series of aliphatic 2-ketones (2-butanone to 2-nonanone) and two of their isomers (3- and 4-heptanone). We found that, with the exception of the two shortest-chained ketones, all animals detected concentrations <1ppm (parts per million), and with five odorants individual animals even reached threshold values <0.1ppm. Further, we found a significant correlation between olfactory sensitivity of the spider monkeys and carbon chain length of the 2-ketones which can best be described as a U-shaped function. In contrast, no significant correlation was found between olfactory sensitivity and position of the functional carbonyl group. Across-odorant and across-species comparisons revealed the following: spider monkeys are significantly less sensitive to the odors of aliphatic ketones than to the odor of other classes of aliphatic compounds (1-alcohols, n-aldehydes, n-acetic esters, and n-carboxylic acids) sharing the same carbon length. Spider monkeys do not differ significantly in their olfactory sensitivity for aliphatic ketones from squirrel monkeys and pigtail macaques, but are significantly less sensitive to these odorants compared to human subjects and mice. These findings support the notion that neuroanatomical and genetic properties do not allow for reliable predictions with regard to a species’ olfactory sensitivity. Further, we conclude that the frequency of occurrence of a class of odorants in a species’ chemical environment does not allow for reliable predictions of the species’ olfactory sensitivity.

Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species.

Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system.

The networked animal.

The article discusses the shaping of animal behavior and specifically how they relate to one another in social networks which play a large part in mating, the transmission of disease, and teaching survival skills. The authors comment on the behavior of the primate pig-tailed macaques, Central America's manakins, and bottlenose dolphins.

Dynamic vaginal microbiota in pigtail macaques is associated with menstrual cycle and inflammation (MUC9P.753)

Abstract A challenge in prevention of mucosal HIV transmission is incomplete understanding of correlates of vaginal transmission, including mucosal inflammation. Vaginal SIV/SHIV transmission in pigtail macaques (Macaca nemestrina) is an excellent model for HIV transmission, however little is known about how genital microbes may influence inflammation and transmission. Broad-range 16S rRNA gene PCR and pyrosequencing was performed on vaginal swabs to assess vaginal microbiome in healthy pigtail macaques. Bacteria were identified to the species or genus level. Luminex was used to measure cytokine and chemokine levels in vaginal swabs. Pigtail macaques had diverse bacterial communities at all phases of the menstrual cycle, with a range of dominant bacteria. Several species were identified that are commonly found in bacterial vaginosis (BV) in humans, and three animals exhibitied Lactobacillus-dominance. Longitudinal sampling demonstrated that the vaginal microbiome is dynamic and possibly influenced by the menstrual cycle. Animals with Lactobacillus-dominant vaginal microbiota had decreased IL-8 (p=0.009) and IL-1Ra (p=0.044). In conclusion, the macaque vaginal microbiota is diverse, dynamic, and can resemble the human vaginal microbiota, including BV-associated bacteria as well as Lactobacillus spp, which is associated with decreased innate inflammation. These data provide a foundation for understanding how the vaginal microbiome may impact risk of HIV/SIV transmission.

242. Leukocyte Telomere Length Is Maintained Following Transplant and Chemoselection in Macaques with Polyclonal Engraftment of Gene Modified Blood Cells

Hematopoietic stem cell (HSC) gene therapy relies on the ability to achieve stable therapeutic levels of gene-modified HSC engraftment. The selectable variant methylguanine methyltransferase gene, P140K, has demonstrated effective selection of gene modified cells in vivo after administration of O6-benzylguanine (O6BG) and non-myeloablative bis-chloroethylnitrosurea (BCNU) in several pre-clinical models. However, the biological consequences of chemoselective pressure on HSC are unknown. It is known that HSC transplantation causes leukocyte telomere shortening, likely due to the proliferative demand on engrafted blood progenitor cells required for hematopoietic reconstitution following myeloablative conditioning. Additionally, telomere shortening is associated with chromosomal instability preceding malignant evolution in blood cells. We and others demonstrated stable telomere length in rhesus and pigtail macaques following myeloablative transplantation of autologous lentivirus gene modified HSCs. However, the selective pressure placed on P140K-modified HSC after O6BG/BCNU treatment may contribute to telomere attrition. To test this hypothesis, we performed a longitudinal assessment of telomere length on peripheral blood leukocytes using a quantitative PCR method in five pigtail macaques. Macaques received myeloablative total body irradiation (1020cGy) followed by infusion of autologous HSC gene-modified with either a gammaretrovirus or a lentivirus encoding the P140K transgene. In all animals, O6BG/BCNU effectively increased the contribution of gene-modified cells in the blood. However, in two animals (J02370 and M02426), chemoselection was associated with a loss in total leukocyte telomere length. We sorted modified and non-modified cells in J02370 based on a fluorescent marker, and found that gene-modified leukocytes in animal J02370 displayed significantly shorter telomeres than the non-modified leukocytes. To determine the relative number of gene-modified clones contributing to this phenomenon, we investigated gene-modified clonal contribution over time in each of the five animals. Telomere shortening in animals J02370 and M02426 correlated temporally with emergence of clonal dominance in vivo, whereas animals displaying stable leukocyte telomere length maintained clonal diversity in vivo. In both J02370 and M02426, shorter total leukocyte telomere length was stably maintained, as were the respective dominant clones, over time (up to 1,700 days). Telomere shortening in these animals could be attributed to either increased cell division during clonal outgrowth, or to clonal selection of a progenitor that originally began with shorter telomeres. Importantly, our data suggest that chemoselective pressure on transduced HSC does not impact telomere length in the setting of polyclonal hematopoiesis. While clonality may exert a negative effect on telomere length in the nonhuman primate, this level of telomere attrition is not associated with malignant transformation or bone marrow failure.

684. Long-Term, Multilineage Engraftment of Zinc Finger Nuclease-Edited Hematopoietic Stem Cells in Nonhuman Primates

Background: Nuclease-mediated gene editing in hematopoietic stem cells (HSCs) holds great promise for diseases including HIV infection and hemoglobinopathies, but little information is available regarding the feasibility of this approach in large animal models. To better evaluate the function of HSCs following gene editing, we have engineered cells with disrupted CCR5 alleles and assessed engraftment following autologous transplant in the pigtailed macaque, M. nemestrina. Disrupted CCR5 alleles in this model should directly protect against infection with simian/human immunodeficiency virus (SHIV). We are evaluating the extent to which CCR5-disrupted cell progeny engraft in macaques, and testing whether these cells impede infection by SHIV.Methods: Zinc Finger Nucleases (ZFNs) are used to target the CCR5 locus in macaque HSCs. 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 SHIV virus containing an HIV envelope; to approximate the status of an HIV+ patient, three-drug combination antiretroviral therapy (cART) is initiated following viral set point. Animals reach undetectable levels of plasma viremia prior to autologous transplant with gene-edited cells.Results: CCR5 targeting experiments yield up to 60% gene disruption in CD34+ cells ex vivo, translating to approximately 5% steady state bulk disruption in vivo. Gene-disrupted cells demonstrate long-term, multilineage engraftment in macaques, including comparable levels of disruption in CD3+, CD20+, CD14+, and granulocyte subsets. We also observe biallelic disruption of CCR5 in colony forming assays. Importantly, this approach is equally feasible in SHIV-naive and in SHIV-infected, cART-suppressed animals. During robust SHIV replication, our preliminary data suggest that CCR5-deleted cells undergo positive selection in vivo.Conclusions: This is the first demonstration of successful long-term multilineage engraftment of ZFN-edited HSCs in a NHP transplantation model. The CCR5 gene editing/HSCT strategy presented here results in robust levels of target gene disruption in vivo, yet does not impair HSC engraftment or differentiation. CCR5-deleted cells can undergo positive selection following challenge with SHIV. Our model enables the evaluation of novel therapeutic approaches in a number of infectious and genetic diseases including HIV infection. Specifically, these results are applicable not only in the context of acute HIV exposure, but also in the clinically relevant setting of pre-existing latent HIV infection.

Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection.

CD8(+) T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8(+) T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8(+) T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8(+) T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8(+) T cells reach 0.5% of total CD8(+) T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8(+) T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8(+) T cells.

Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates

Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34(+) cells expressing the mC46 anti-HIV fusion protein. We show that SHIV(+), ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34(+) cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV(+), ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4(+) T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4(+)CCR5(+)T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial.