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Alzheimer’s disease is the most common cause of dementia and the only illness among the top 10 causes of death for which there is no disease-modifying therapy. The failure rate of clinical trials is very high, in part due to the premature translation of successful results in transgenic mouse models to patients. Extensive evidence suggests that dysregulation of innate immunity and microglia/macrophages plays a key role in Alzheimer’s disease pathogenesis. Activated resident microglia and peripheral macrophages can display protective or detrimental phenotypes depending on the stimulus and environment. Toll-like receptors (TLRs) are a family of innate immune regulators known to play an important role in governing the phenotypic status of microglia. We have shown in multiple transgenic Alzheimer’s disease mouse models that harnessing innate immunity via TLR9 agonist CpG oligodeoxynucleotides (ODNs) modulates age-related defects associated with immune cells and safely reduces amyloid plaques, oligomeric amyloid-β, tau pathology, and cerebral amyloid angiopathy (CAA) while promoting cognitive benefits. In the current study we have used a non-human primate model of sporadic Alzheimer’s disease pathology that develops extensive CAA—elderly squirrel monkeys. The major complications in current immunotherapeutic trials for Alzheimer’s disease are amyloid-related imaging abnormalities, which are linked to the presence and extent of CAA; hence, the prominence of CAA in elderly squirrel monkeys makes them a valuable model for studying the safety of the CpG ODN-based concept of immunomodulation. We demonstrate that long-term use of Class B CpG ODN 2006 induces a favourable degree of innate immunity stimulation without producing excessive or sustained inflammation, resulting in efficient amelioration of both CAA and tau Alzheimer’s disease-related pathologies in association with behavioural improvements and in the absence of microhaemorrhages in aged elderly squirrel monkeys. CpG ODN 2006 has been well established in numerous human trials for a variety of diseases. The present evidence together with our earlier, extensive preclinical research, validates the beneficial therapeutic outcomes and safety of this innovative immunomodulatory approach, increasing the likelihood of CpG ODN therapeutic efficacy in future clinical trials.

Nonhuman primates are frequently transported to a new location or temporarily relocated within their colony. Both transportation and relocation expose animals to new environments, causing them to undergo a stress response (before adapting). In our NHP colony, the mentioned situations are not infrequent for many reasons, including maintenance. The objective of this study was to determine whether abrupt changes consisting of relocation, housing, separation, and grouping could influence hematological and immunological parameters and thereby functional activity. The current study used squirrel monkeys as a model to investigate the stress-inducing effects of relocation within a facility, while animals acclimated to new situations (physical, housing). A detailed blood analysis revealed significant changes in lymphocytes, triglycerides, total protein, creatinine, and ALT. Flow cytometric analysis of peripheral blood showed reduction in CD3+, CD4+, and CD8+ T cells and monocytes, while B cells and natural killer (NK) cells changed with relocation. Simultaneously, changes in functional activity of immune cells altered proliferative responses and as shown by ELISpot (IFN γ). Though the parameters studied are not affected as severely as those in animals transported by road or air, stress responses induced by intrafacility relocation are significant and worth consideration. Our findings indicate that squirrel monkeys mimic the features seen in humans exposed to social stressors and may serve an important model for understanding the mechanisms of stress-induced immune dysfunction in humans.

HIV vaccine candidates with improved immunogenicity and induction of mucosal T-cell immunity are needed. A prime-boost strategy using a novel HIV glycoprotein 120 DNA vaccine was employed to immunize rhesus macaques. The DNA vaccine encoded a chimeric gp120 protein in fusion with monocyte chemoattractant protein-3, which was hypothesized to improve the ability of antigen-presenting cells to capture viral antigen through chemokine receptor-mediated endocytosis. DNA vaccination induced virus-reactive T cells in peripheral blood, detectable by T cell proliferation, INFgamma ELISPOT and sustained IL-6 production, without humoral responses. With a peptide-cocktail vaccine containing a set of conserved polypeptides of HIV-1 envelope protein, given by nasogastric administration, primed T-cell immunity was significantly boosted. Surprisingly, long-term and peptide-specific mucosal memory T-cell immunity was detected in both vaccinated macaques after one year. Therefore, data from this investigation offer proof-of-principle for potential effectiveness of the prime-boost strategy with a chemokine-fused gp120 DNA and warrant further testing in the nonhuman primate models for developing as a potential HIV vaccine candidate in humans.

In the present study, we have quantified the effects of transport, relocation and acclimate/adapt to their new surroundings on female squirrel monkey. These responses are measured in blood samples obtained from squirrel monkeys, at different time points relative to their relocation from their old home to their new home. A group of squirrel monkeys we transported, by truck, for approximately 10 hours. Peripheral blood mononuclear cells (PBMCs) were assayed in order to evaluate the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of cytokines at various time points including immediately before transport, immediately upon arrival, and after approximately 150 days of acclimation. We observed significant changes in T cells and subsets, NK and B cells (CD4+, CD8+, CD4+/CD8+, CD16+, and CD20+). Mitogen specific (e.g. PHA, PWM and LPS) proliferation responses, IFN-γ by ELISPOT assay, and cytokines (IL-2, IL-4 and VEGF) significant changes were observed. Changes seen in the serum chemistry measurements mostly complement those seen in the hematology data. The specific goal was to empirically assess the effects of relocation stress in squirrel monkeys in terms of changes in the numbers and functions of various leukocyte subsets in the blood and the amount of time required for acclimating to their new environment. Such data will help to determine when newly arrived animals become available for use in research studies.

Olive baboons (P. anubis) have provided a useful model of human diseases and conditions, including cardiac, respiratory, and infectious diseases; diabetes; and involving genetics, immunology, aging, and xenotransplantation. The development of a immunologically defined SPF baboons has advanced research further, especially for studies involving the immune system and immunosuppression. In this study, we compare normal immunologic changes of PBMC subsets, and their function in age-matched conventional and SPF baboons. Our results revealed that both groups have comparable numbers of different lymphocyte subsets, but phenotypic differences in central and effector memory T-cell subsets are more pronounced in CD4+ T cells. Despite equal proportions of CD3+ T cells among the conventional and SPF baboons, PBMC from the conventional group showed greater proliferative responses to phytohemagglutinin and pokeweed mitogen and higher numbers of IFNγ-producing cells after stimulation with concanavalin A or pokeweed mitogen, whereas plasma levels of the inflammatory cytokine TNFα were significantly higher in SPF baboons. Exposure of PBMC from conventional baboons to various Toll-like (TLR) ligands, including TLR3, TLR4, and TLR8, yielded increased numbers of IFNγ producing cells, whereas PBMC from SPF baboons stimulated with TLR5 or TLR6 ligand had more IFNγ-producing cells. These findings suggest that although lymphocyte subsets share many phenotypic and functional similarities in conventional and SPF baboons, specific differences in the immune function of lymphocytes could differentially influence the quality and quantity of their innate and adaptive immune responses. These differences should be considered in interpreting experimental outcomes, specifically in studies measuring immunologic endpoints.

One means of stimulating the mammalian innate immune system is via Toll-like receptor 9 (TLR9) being exposed to unmethylated cytosine-phosphate-guanine (CpG) DNA, also known as pathogen-associated molecular patterns (PAMPs) of microbial origin. Synthetic CpG oligodeoxynucleotides (ODNs) with defined CpG motifs possess broad immunostimulatory properties that make CpG ODNs suitable as therapeutic interventions in a variety of human disease conditions, including Alzheimer’s disease (AD). Rodent models are often used to preclinically test the effectiveness of CpG ODN therapeutic agents for AD and other disorders. However, the translatability of findings in such models is limited due to the significant difference of the expression of TLR9 between primates and rodents. The squirrel monkey (SQM), a New World non-human primate (NHP), is known to be phylogenetically proximate to humans, and develops extensive age-dependent cerebral amyloid angiopathy (CAA), a key pathological feature of AD. Hence, this model is currently being used to test AD therapeutics. In the present study, we conducted the first examination of Class C CpG ODN’s immunomodulatory role in elderly SQMs. We documented the effectiveness of CpG ODN to trigger an immune response in an aged cohort whose immune system is senescent. The specific immune response patterns detected here closely resembled CpG ODN-induced immunostimulatory patterns observed in prior human studies. Overall, our findings provide critical data regarding the immunomodulatory potential of CpG ODN in this NHP model, allowing for future translational studies of innate immunity stimulation via TLR9 agonists for diverse indications, including AD therapeutics.

Natural killer T (NKT) cells are innate immune cells that are responsible for the first line of antiviral defense, through crosstalk with downstream antigen-presenting cells, natural killer cells, and adaptive immune cells. Previous studies have indicated that NKT cell function is severely impaired in patients with chronic HIV-1 infection. It was reported that alpha-galactosylceramide, a potent agonist antigen for NKT cells, failed to trigger the expansion of NKT cells, or the production of anti-viral cytokines by NKT cells from HIV-1 infected patients in an in vitro assay, in which peripheral blood mononuclear cells (PBMCs) were cultured in the presence of alpha-galactosylceramide. In this study, we stimulated banked peripheral blood mononuclear cells from HIV-1-infected patients with dendritic cells (DC) generated ex vivo and loaded with alpha-galactosylceramide. The results showed that NKT cells were expanded in HIV infected subjects except in patients with advanced AIDS. Expanded NKT cells were capable of producing antiviral cytokines. Our results indicate that NKT cells in HIV infected individuals are potential targets for therapeutic intervention.

NHP are a small, but critical, portion of the animals studied in research laboratories. Many NHP are imported or raised at one facility and subsequently moved to another facility for research purposes. To improve our understanding of the effects of transportation and relocation on the NHP immune system, to minimize potential confounds associated with relocation, and to maximize study validity, we examined the phenotype and function of PBMC in cynomolgus macaques (Macaca fascicularis) that were transported approximately 200 miles by road from one facility to another. We evaluated the phenotype of lymphocyte subsets through flow cytometry, mitogen-specific immune responses of PBMC in vitro, and plasma levels of circulating cytokines before transportation, at approximately 24 h after arrival (day 2), and after 30 d of acclimation. Analyses of blood samples revealed that the CD3+ and CD4+ T-cell counts increased significantly, whereas NK+, NKT, and CD14+ CD16+ nonclassical monocyte subsets were decreased significantly on day 2 after relocation compared with baseline. We also noted significantly increased immune cell function as indicated by mitogen-specific proliferative responses and by IFNγ levels on day 2 compared with baseline. After 30 d of acclimation, peripheral blood CD4+ T-cells and monocyte counts were higher than baseline, whereas B-cell numbers were lower. The mitogen-induced responses to LPS and IFNγ production after stimulation with pokeweed mitogen or phytohemagglutinin remained significantly different from baseline. In conclusion, the effects of transportation and relocation on immune parameters in cynomolgus monkeys are significant and do not fully return to baseline values even after 30 d of acclimation.

Most human immunodeficiency virus type 1 (HIV-1) infections begin at mucosal surfaces. Providing a barrier of protection at these may assist in combating the earliest events in infection. Systemic immunization by intramuscular (i.m.) injection can drive mucosal immune responses, but there are data suggesting that mucosal immunization can better educate these mucosal immune responses. To test this, rhesus macaques were immunized with replicating single-cycle adenovirus (SC-Ad) vaccines expressing clade B HIV-1 gp160 by the intranasal (i.n.) and i.m. routes to compare mucosal and systemic routes of vaccination. SC-Ad vaccines generated significant circulating antibody titers against Env after a single i.m. immunization. Switching the route of second immunization with the same SC-Ad serotype allowed a significant boost in these antibody levels. When these animals were boosted with envelope protein, envelope-binding antibodies were amplified 100-fold, but qualitatively different immune responses were generated. Animals immunized by only the i.m. route had high peripheral T follicular helper (pTfh) cell counts in blood but low Tfh cell counts in lymph nodes. Conversely, animals immunized by the i.n. route had high Tfh cell counts in lymph nodes but low pTfh cell counts in the blood. Animals immunized by only the i.m. route had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity, whereas animals immunized by the mucosal i.n. route had higher ADCC antibody activity. When these Env-immunized animals were challenged rectally with simian-human immunodeficiency virus (SHIV) strain SF162P3 (SHIV(SF162P3)), they all became infected. However, mucosally SC-Ad-immunized animals had lower viral loads in their gastrointestinal tracts. These data suggest that there may be benefits in educating the immune system at mucosal sites during HIV vaccination. IMPORTANCE HIV-1 infections usually start at a mucosal surface after sexual contact. Creating a barrier of protection at these mucosal sites may be a good strategy for to protect against HIV-1 infections. While HIV-1 enters at mucosa, most vaccines are not delivered here. Most are instead injected into the muscle, a site well distant and functionally different than mucosal tissues. This study tested if delivering HIV vaccines at mucosa or in the muscle makes a difference in the quality, quantity, and location of immune responses against the virus. These data suggest that there are indeed advantages to educating the immune system at mucosal sites with an HIV-1 vaccine.

American Trypanosomiasis is an important neglected reemerging tropical parasitism, infecting about 8 million people worldwide. Its agent, Trypanosoma cruzi, exhibits multiple mechanisms to evade the host immune response and infect host cells. An important immune evasion strategy of T. cruzi infective stages is its capacity to inhibit the complement system activation on the parasite surface, avoiding opsonizing, immune stimulating and lytic effects. Eight male M. fascicularis between 8 and 10 years of age of Chinese origin were infected through oral administration of T. cruzi-infected triatomine bugs and followed over 6 weeks. The purpose of this study was to define cellular immunological responses in T. Cruzi challenged M. fascicularis. We measured immunofluorescent staining and multifunctional T. cruzi antigen specific T-cell responses in experimental infected M. fascicularis. Significant increases in the CD4+ T-cell population and polyfunctional T cell responses were observed in all experimental-infected animals compared with the uninfected time points. We observed a decrease in absolute number of lymphocytes, T cells and T cell subsets, in early phase of infection but recovered in later period. T. cruzi antigen specific IFN-g and IL-12p40 ELISPOT showed no significant infection specific responses during the study period. Circulating cytokines measurement in plasma revealed a significant increase IFN-g and IL-1ra during infection period. In conclusion, our present findings provide the first evidence demonstrating the Trypanosoma cruzi infection by oral route.

Synthetic CpG oligodeoxynucleotides (ODNs) with defined CpG motifs and immunomodulatory properties signal through an endosomal membrane-based type of pattern recognition receptor (PRR), the Toll-like receptor 9 (TLR9), that has been identified in dendritic cells (DCs), B cells, and other human immune cell types. CpG-ODNs influence several signaling pathways, leading to cytokine production in many mammalian species that make CpG ODNs suitable as therapeutic interventions in a variety of human disease conditions. The squirrel monkey (SQM), a well-established New World non-human primate (NHP) model is known to be used for studying various diseases such as Alzheimer’s disease (AD), malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). In the present study, we generated the first report of immunoregulatory activity of class C CpG-ODN in aged squirrel monkey. CpG-ODN exhibits very potent effects on squirrel monkey immune cells by inducing IFN-γ/IFN-α from peripheral blood mononuclear cells and demonstrates accelerated kinetics of activity. In addition, class C CpG-ODN specifically activates pDCs to undergo maturation and secrete cytokines, including high levels of IFN-α. To best to our knowledge, this is the first documentation describing immune status in squirrel monkeys immunized with C class CpG-ODN by concentrating on lymphocyte surface antigen expression and function. Overall, our findings provide critical data regarding the immunomodulatory potential of CpG-ODN in this NHP model allowing for future therapeutic trials of innate immunity stimulation via TLR9 agonists for diverse indications including AD therapeutics.

Baboons (papio spp.) have served as a successful model of human disease such as cardiac and respiratory, infectious, diabetes, genetics, immunology, aging, and xenotransplantation. The development of an immunologically defined specific-pathogen free (SPF) baboon model has further advanced research, especially with studies involving the immune system and immunosuppression. In this study, we compare normal immunological changes of peripheral blood mononuclear cell (PBMC) subsets, and their function in age-matched conventional and SPF baboons. Our results demonstrate that both groups have comparable numbers of different lymphocyte subsets, but there are phenotypic differences in central and effector memory T cells subsets that are more pronounced in the CD4+ T cells. Despite equal proportions of CD3+ T cells among the conventional and SPF baboon groups, PBMC show higher proliferative responses to mitogens PHA and PWM and higher IFN-γ producing cells to Con A and PWM in the conventional group. Plasma levels of the inflammatory cytokine TNF-α were significantly higher in SPF baboons. Exposure of PBMC from conventional baboons to various Toll like ligands (TLR ligands) TLR-3, TLR-4 and TLR-8 show higher IFN-γ producing cells while PBMC from SPF baboons stimulated with TLR-5 and TLR-6 ligand show higher IFN-γ producing cells. These findings suggest that while the lymphocyte subsets in conventional and SPF baboons share many phenotypic and functional similarities, specific differences exist in immune function of lymphocytes which could impact the quality and quantity of innate and adaptive immune responses. These differences should be considered for better experimental outcomes, specifically in studies measuring immunological endpoints.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus with devastating outcomes seen recently in the Americas due to the association of maternal ZIKV infection with fetal microcephaly and other fetal malformations not previously associated with flavivirus infections. Here, we have developed the olive baboon (Papio anubis) as a nonhuman primate (NHP) translational model for the study of ZIKV pathogenesis and associated disease outcomes to contrast and compare with humans and other major NHPs, such as macaques. Following subcutaneous inoculation of adult male and nonpregnant female baboons, viremia was detected at 3 and 4 days postinfection (dpi) with the concordant presentation of a visible rash and conjunctivitis, similar to human ZIKV infection. Furthermore, virus was detected in the mucosa and cerebrospinal fluid. A robust ZIKV-specific IgM and IgG antibody response was also observed in all the animals. These data show striking similarity between humans and the olive baboon following infection with ZIKV, suggesting our model is a suitable translational NHP model to study ZIKV pathogenesis and potential therapeutics.IMPORTANCE ZIKV was first identified in 1947 in a sentinel rhesus monkey in Uganda and subsequently spread to Southeast Asia. Until 2007, only a small number of cases were reported, and ZIKV infection was relatively minor until the South Pacific and Brazilian outbreaks, where more severe outcomes were reported. Here, we present the baboon as a nonhuman primate model for contrast and comparison with other published animal models of ZIKV, such as the mouse and macaque species. Baboons breed year round and are not currently a primary nonhuman primate species used in biomedical research, making them more readily available for studies other than human immunodeficiency virus studies, which many macaque species are designated for. This, taken together with the similarities baboons have with humans, such as immunology, reproduction, genetics, and size, makes the baboon an attractive NHP model for ZIKV studies in comparison to other nonhuman primates.

Cellular immune responses were tested to determine the effect of fenbendazole on the function of lymphocytes from Bolivian squirrel monkeys (Samiri boliviensis boliviensis). Giardia-infected squirrel monkeys were treated with commercially available fenbendazole (FBZ)-medicated monkey chow. Immune responses were compared between historical controls (Giardia naive, untreated with FBZ (control animals)) and Giardia-infected, FBZ-treated squirrel monkeys (study animals). Peripheral blood lymphocytes from study monkeys had significantly lower stimulation indices compared to control animals when cultured in vitro with concanavalin A (Con A) (p

Chimpanzees are the closest phylogenetic relatives to humans, sharing more than 98% genetic sequence identity. These genetic similarities prompted the belief that chimpanzees can serve as an ideal model for human disease conditions and vaccine development. However, in light of the recent NIH decision to phase out biomedical research in chimpanzees and retire NIH-supported chimpanzees, data from the present study will continue to provide value for the care of aged and sick chimpanzees located in zoos, sanctuaries, and primate centers. Surprisingly little information has been published regarding the normal chimpanzee immune system, and most extant studies have been based on small numbers of animals. In the current study, we provide a better understanding of the chimpanzee immune system with regard to age and sex. We examined immune parameters of chimpanzees (n = 94; 51 female, 43 male; age, 6 to 47 y) by using flow cytometry, immune function analysis, and cytokine analysis. Because lymphocytes are key mediators of cellular immune responses, particularly to intracellular pathogens such as viruses, we surveyed the phenotypic and functional attributes of T and B lymphocytes in this healthy and age-stratified population of chimpanzees. We noted a significantly higher percentage of CD16+T cells in male compared with female chimpanzees but no significant changes in percentages of CD3+, CD4+, CD8+, or CD4+CD8+ T cells with age or sex. In addition, aging was associated with decreased proliferative responses to mitogens in both sexes. Sex-specific differences also were present in the percentage of NK cells but not in their cytotoxic activity and in circulating cytokine levels in plasma. Going forward, the data presented here regarding immune cell changes associated with aging in healthy chimpanzees will serve to enhance the care of geriatric and ill animals.

Owl monkeys (Aotus nancymaae) are New World NHP that serve an important role in vaccine development and as a model for human disease conditions such as malaria. Despite the past contributions of this animal model, limited information is available about the phenotype and functional properties of peripheral blood lymphocytes in reference to sex and age. Using a panel of human antibodies and a set of standardized human immune assays, we identified and characterized various peripheral blood lymphocyte subsets, evaluated the immune functions of T cells, and analyzed cytokines relative to sex and age in healthy owl monkeys. We noted age- and sex-dependent changes in CD28+ (an essential T cell costimulatory molecule) and CD95+ (an apoptotic surface marker) T cells and various levels of cytokines in the plasma. In immune assays of freshly isolated peripheral blood mononuclear cells, IFNγ and perforin responses were significantly higher in female than in male monkeys and in young adults than in juvenile and geriatric groups, despite similar lymphocyte (particularly T cell) populations in these groups. Our current findings may be useful in exploring Aotus monkeys as a model system for the study of aging, susceptibility to infectious diseases, and age-associated differences in vaccine efficacy, and other challenges particular to pediatric and geriatric patients.

Studies in nonhuman primates (NHP) for prospective immune cell monitoring subsequent to infection and/or vaccination usually rely on periodic sampling of the blood samples with only occasional collections of biopsies from mucosal tissues because of safety concerns and practical constraints. Here we present evidence in support of cytobrush sampling of oral, rectal, and genital mucosal tissues as a minimally invasive approach for the phenotypic analyses of different T cells subsets de novo as well as prospectively after intranasal immunization in rhesus macaques. Significant percentages of viable lymphocytes were obtained consistently from both naive and chronically SIV-infected rhesus macaques. The percentages of CD3+ T cells in the blood were significantly higher compared to those in the mucosal tissues analyzed in the naive animals, while in the SIV+ animals the CD3+ T cells were significantly elevated in the rectal tissues, relative to all other sites analyzed. In the naive, but not SIV+ macaques, the rectal and vaginal mucosal tissues, compared to oral mucosa and blood, showed higher diversity and percentages of CD4+ T cells expressing the HIV entry co-receptor CCR5 and mucosal specific adhesion (CD103) as well as activation (HLA-DR) and proliferation (Ki67) markers. Sequential daily cytobrush sampling from the oral, rectal, and genital mucosal tissues was performed in SIV+ animals from an ongoing study where they were administered intranasal immunization with adenoviral vectored vaccines incorporating the green fluorescent protein (GFP) reporter gene. We detected a transient increase in GFP+ CD4 T cells in only oral mucosa suggesting limited mucosal trafficking. In general, CD4+ and CD8+ T cells expressing Ki67 transiently increased in all mucosal tissues, but those expressing the CCR5, HLA-DR, and CD103 markers exhibited minor changes. We propose the minimally invasive cytobrush sampling as a practical approach for effective and prospective immune monitoring of the oral-genital mucosal tissues in NHP.

Studies in nonhuman primates (NHP) for prospective immune cell monitoring subsequent to infection and/or vaccination usually rely on periodic sampling of the blood samples with only occasional collections of biopsies from mucosal tissues because of safety concerns and practical constraints. Here we present evidence in support of cytobrush sampling of oral, rectal, and genital mucosal tissues as a minimally invasive approach for the phenotypic analyses of different T cells subsets de novo as well as prospectively after intranasal immunization in rhesus macaques. Significant percentages of viable lymphocytes were obtained consistently from both naïve and chronically SIV-infected rhesus macaques. The percentages of CD3+ T cells in the blood were significantly higher compared to those in the mucosal tissues analyzed in the naïve animals, while in the SIV+ animals the CD3+ T cells were significantly elevated in the rectal tissues, relative to all other sites analyzed. In the naïve, but not SIV+ macaques, the rectal and vaginal mucosal tissues, compared to oral mucosa and blood, showed higher diversity and percentages of CD4+ T cells expressing the HIV entry co-receptor CCR5 and mucosal specific adhesion (CD103) as well as activation (HLA-DR) and proliferation (Ki67) markers. Sequential daily cytobrush sampling from the oral, rectal, and genital mucosal tissues was performed in SIV+ animals from an ongoing study where they were administered intranasal immunization with adenoviral vectored vaccines incorporating the green fluorescent protein (GFP) reporter gene. We detected a transient increase in GFP+ CD4 T cells in only oral mucosa suggesting limited mucosal trafficking. In general, CD4+ and CD8+ T cells expressing Ki67 transiently increased in all mucosal tissues, but those expressing the CCR5, HLA-DR, and CD103 markers exhibited minor changes. We propose the minimally invasive cytobrush sampling as a practical approach for effective and prospective immune monitoring of the oral-genital mucosal tissues in NHP.