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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.

Pancreatic islet transplantation efficacy for type 1 diabetes (T1D) management is limited by hypoxia-related graft attrition and need for systemic immunosuppression. To overcome these challenges, we developed the Neovascularized Implantable Cell Homing and Encapsulation (NICHE) device, which integrates direct vascularization for facile mass transfer and localized immunosuppressant delivery for islet rejection prophylaxis. Here, we investigated NICHE efficacy for allogeneic islet transplantation and long-term diabetes reversal in an immunocompetent, male rat model. We demonstrated that allogeneic islets transplanted within pre-vascularized NICHE were engrafted, revascularized, and functional, reverting diabetes in rats for over 150 days. Notably, we confirmed that localized immunosuppression prevented islet rejection without inducing toxicity or systemic immunosuppression. Moreover, for translatability efforts, we showed NICHE biocompatibility and feasibility of deployment as well as short-term allogeneic islet engraftment in an MHC-mismatched nonhuman primate model. In sum, the NICHE holds promise as a viable approach for safe and effective islet transplantation and long-term T1D management.

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.

The focus of present work was to characterize ultra-long acting prodrug of dolutegravir (DTG) and develop biodegradable microparticle formulation. Palmitic acid (PA) conjugated prodrug of DTG was prepared by esterification of hydroxyl group of DTG with the carboxyl group of PA. Physicochemical properties of the prodrug was characterize by MS, NMR, FTIR, SEM, DSC, NIR-CI, pH-solubility, and solid and liquid pH-stability. Comparative solid and liquid stability was performed by storing powder DTG and DTG-Palmitate at 40 °C/75% RH for three months and liquid solution pH 2–8 at room temperature for 24 h, respectively. Pharmacokinetic evaluation was performed in white albino New Zealand rabbits by subcutaneous injection (30 mg/Kg). Poly(lactide-co-glycolide) microparticle formulation was prepared by emulsification-evaporation method and characterized for particle size distribution, shape, drug loading and in-vitro release. MS, NMR, FTIR, SEM, DSC, NIR-CI indicated formation of prodrug. Melting point of the prodrug was lower than DTG but higher than PA. Shape of DTG crystals was irregular while DTG-Palmitate crystals was fine-needle. Solid and liquid stability profiles of the prodrug were similar to DTG. Plasma half-life, area under the curve, and mean-residence time of DTG-Palmitate were 8.8, 2.3 and 14.7 folds of DTG. D90 of DTG and DTG-Palmitate microparticles was 107.1 ± 2.7 and 94.3 ± 3.4 µm, respectively. The in-vitro drug release was almost complete in three weeks from DTG microparticles while it was

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.

CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and inflammatory pathologies, as well as cancer progression. CD36 is known to be expressed by adipocytes and monocytes/macrophages, but its expression by T cells is not clearly established. We found that CD4 and CD8 T cells in adipose tissue and liver of humans, monkeys, and mice upregulated CD36 expression (ranging from ~5–40% CD36+), whereas little to no CD36 was expressed by T cells in blood, spleen, and lymph nodes. CD36 was expressed predominantly by resting CD38-, HLA.DR-, and PD-1- adipose tissue T cells in monkeys, and increased during high-fat feeding in mice. Adipose tissue and liver promote a distinct phenotype in resident T cells characterized by CD36 upregulation.

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.

Pre-exposure prophylaxis (PrEP) with antiretroviral (ARV) drugs are effective at preventing human immunodeficiency virus (HIV) transmission. However, implementation of PrEP presents significant challenges due to poor user adherence, low accessibility to ARVs and multiple routes of HIV exposure. To address these challenges, we developed the nanochannel delivery implant (NDI), a subcutaneously implantable device for sustained and constant delivery of tenofovir alafenamide (TAF) and emtricitabine (FTC) for HIV PrEP. Unlike existing drug delivery platforms with finite depots, the NDI incorporates ports allowing for transcutaneous refilling upon drug exhaustion. NDI-mediated drug delivery in rhesus macaques resulted in sustained release of both TAF and FTC for 83 days, as indicated by concentrations of TAF, FTC and their respectively metabolites in plasma, PBMCs, rectal mononuclear cells and tissues associated with HIV transmission. Notably, clinically relevant preventative levels of tenofovir diphosphate were achieved as early as 3 days after NDI implantation. We also demonstrated the feasibility of transcutaneous drug refilling to extend the duration of PrEP drug delivery in NHPs. Overall, the NDI represents an innovative strategy for long-term HIV PrEP administration in both developed and developing countries.

Recent West African Ebola virus (EBOV) epidemics have led to testing different anti-EBOV vaccines, including a replication-defective adenovirus (RD-Ad) vector (ChAd3-EBOV) and an infectious, replication-competent recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (rVSV-EBOV; also known as rVSV-ZEBOV). While RD-Ads elicit protection, when scaled up to human trials, the level of protection may be much lower than that of vaccines containing viruses that can replicate. Although a replication-competent Ad (RC-Ad) vaccine might generate a level of protection approximating that of rVSV, this infectious vector would also risk causing adenovirus disease. We recently described a "single-cycle" adenovirus (SC-Ad) vector that amplifies antigen genes like RC-Ad, but that avoids the risk of adenovirus infection. Here we have tested an SC-Ad6 vector expressing the glycoprotein (GP) from a 2014 EBOV strain in mice, hamsters, and rhesus macaques. We show that SC-Ad6-EBOV GP induces a high level of serum antibodies in all species and mediates significant protection against pseudo-challenge with rVSV-EBOV expressing luciferase in mice and hamsters. These data suggest that SC-Ad6-EBOV GP may be useful during future EBOV outbreaks.

The establishment of a sylvatic reservoir of Zika virus (ZIKV) in the Americas is dependent on the susceptibility of primates of sufficient population density, the duration and magnitude of viremia, and their exposure to the human mosquito-borne transmission cycle. To assess the susceptibility of squirrel (Saimiri sp.) and owl monkeys (Aotus sp.) to infection, we inoculated four animals of each species with ZIKV from the current epidemic. Viremia in the absence of detectible disease was observed in both species and seroconversion occurred by day 28. ZIKV was detected in the spleen of three owl monkeys: one at 7 days postinoculation (dpi) and two at 14 dpi. This study confirms the susceptibility to ZIKV infection of two Neotropical primate species that live in close proximity to humans in South America, suggesting that they could support a widespread sylvatic ZIKV cycle there. Collectively, establishment of a ZIKV sylvatic transmission cycle in South America would imperil eradication efforts and could provide a mechanism for continued exposure of humans to ZIKV infection and disease.

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.

Long-acting (LA) pre-exposure prophylaxis (PrEP) for HIV prevention is poised to address non-adherence and implementation challenges by alleviating the burden of user-dependent dosing. Due to its potency, tenofovir alafenamide (TAF) is a viable candidate for LA PrEP. However, the inherent hydrolytic instability of TAF presents a challenge for application in LA systems. In this work, we examined the mechanism of TAF hydrolysis in a reservoir-based implant system and characterized TAF degradation kinetics as a function of the solution pH. We determined a pH “stability window” between pH 4.8 – 5.8 in which TAF degradation is substantially mitigated, with minimal degradation at pH 5.3. In a pursuit of a TAF formulation suitable for LA PrEP, we studied trans-urocanic acid (UA) as a buffer excipient. Here we show that UA can maintain the pH of TAF free base (TAFfb) solution inside a surrogate implant model at approximately pH 5.4. Through in vitro analysis, we demonstrated preservation of released TAF purity above 90% for over 9 months. Further, we performed an in vivo assessment of TAFfb-UA formulation in a reservoir-based nanofluidic implant inserted subcutaneously in non-human primates. Preventive levels of tenofovir diphosphate above 100 fmol/106 peripheral blood mononuclear cells were achieved in 2 days and sustained over 35 days. Fluid retrieved from implants after 60 days of implantation showed that UA preserved the aqueous phase in the implant at ~ pH 5.5, effectively counteracting the neutralizing action of interstitial fluids. Moreover, residual TAF in the implants maintained > 98% purity. Overall, TAF-UA represents a viable formulation applicable for LA HIV PrEP.

The current standard for cell encapsulation platforms is enveloping cells in semipermeable membranes that physically isolate transplanted cells from the host while allowing for oxygen and nutrient diffusion. However, long-term viability and function of encapsulated cells are compromised by insufficient oxygen and nutrient supply to the graft. To address this need, a strategy to achieve enhanced vascularization of a 3D-printed, polymeric cell encapsulation platform using platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) is investigated. The study is conducted in rats and, for clinical translation relevance, in nonhuman primates (NHP). Devices filled with PRP, MSCs, or vehicle hydrogel are subcutaneously implanted in rats and NHP and the amount and maturity of penetrating blood vessels assessed via histopathological analysis. In rats, MSCs drive the strongest angiogenic response at early time points, with the highest vessel density and endothelial nitric oxide synthase (eNOS) expression. In NHP, PRP and MSCs result in similar vessel densities but incorporation of PRP ensues higher levels of eNOS expression. Overall, enrichment with PRP and MSCs yields extensive, mature vascularization of subcutaneous cell encapsulation devices. It is postulated that the individual properties of PRP and MSCs can be leveraged in a synergistic approach for maximal vascularization of cell encapsulation platforms.

A strain of Zika virus (ZIKV) of Asian origin associated with birth defects and neurological disorders has emerged and spread through the Americas. ZIKV was first isolated in the blood of nonhuman primates in Africa and has been detected in the blood, saliva, and urine of a few catarrhine species in both Africa and Asia, suggesting that nonhuman primates may serve as both a source and a reservoir of the virus. The recent introduction of ZIKV to human populations in the Americas presents the potential for the virus to spread into nonhuman primate reservoirs. Thus, it is critical to develop efficient and noninvasive detection methods to monitor the spread of the virus in wild nonhuman primate populations. Here, we describe a method for ZIKV detection in noninvasively collected fecal samples of a Neotropical primate. Fecal samples were collected from two captive squirrel monkeys (Saimiri boliviensis boliviensis) that were experimentally infected with ZIKV (Strain Mexico_1_44) and an additional two uninfected squirrel monkeys. Nucleic acids were extracted from these samples, and RT-qPCR was used to assay for the presence of ZIKV using primers flanking a 101 bp region of the NS5 gene. In both ZIKV-inoculated animals, ZIKV was detected 5-11 days post-infection, but was not detected in the uninfected animals. We compare the fecal results to ZIKV detection in serum, saliva, and urine samples from the same individuals. Our results indicate that fecal detection is a cost-effective, noninvasive method for monitoring wild populations of Neotropical primates as possible ZIKV reservoirs.

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.

With nearly 40% of U.S. adults obese, and childhood and adolescent rates rising, obesity and associated comorbidities are serious public health concerns with massive societal costs. Often, lifestyle interventions do not offer sufficient weight loss to improve health, requiring surgery and medications as adjunct management strategies. Here, we present a 4-month case study in which the sustained, low-dose, and constant administration of the thyroid receptor β selective agonist GC-1 (sobetirome) from a novel nanochannel membrane implant was assessed in an obese, pre-diabetic rhesus macaque. Dramatic loss of white adipose tissue in the abdomen from 36 to 18% was observed via magnetic resonance imaging in conjunction with normalized serum insulin and glycemia, with no signs of cardiotoxicity shown. The non-human primate study highlights sustained low-dose delivery of GC-1 from our minimally invasive subcutaneous implant as a valuable approach to induce weight loss and manage obesity and comorbidities, including type 2 diabetes.

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.

Background HIV reservoirs pose major challenges to viral eradication. The main cellular reservoirs include CD4 T cells and macrophages, whereas anatomic reservoirs are thought to be primarily lymphoid tissues. Adipose tissue represents a potentially important non-lymphoid location for HIV replication and persistence because the stromal-vascular-fraction (AT-SVF) contains activated innate and adaptive immune cells that increase in number during infections, obesity, and chronic inflammation. Results Adipose tissue from two groups of SHIV-SF162p3-infected (~4 weeks acute infection) or SIVmac251-infected (~38 weeks chronic infection) rhesus macaques (N = 8 for each group) were studied for immune cell content, viral infectiousness, and metabolic health. The AT-SVF cells from SHIV-infected monkeys contained abundant memory CD4 and CD8 T cells, with fewer NKT cells and macrophages, and no B cells. Proviral DNA (Gag and Env) was readily detectable by nested PCR in AT-SVF cells from multiple adipose depots (subcutaneous and visceral) of acutely infected monkeys, but mostly from visceral fat. More importantly, viral outgrowth assays using input CD4 T cells derived from AT-SVF cells or peripheral blood of chronically infected monkeys resulted in robust replication of infectious virus from both AT-SVF and peripheral blood CD4 T cells. Chronically infected monkeys also experienced adipocyte dysfunction (suppression of major adipogenic genes) and systemic dyslipidemia (decreased serum total cholesterol and free fatty acids, and increased triglycerides), similar to metabolic abnormalities of HIV patients. Conclusions Adipose tissues of SIV-infected rhesus macaques become major compartments for infected immune cells, which in turn induce defects in adipose tissue metabolism. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0260-2) contains supplementary material, which is available to authorized users.

Mucosal dendritic cells have been implicated in the capture, storage, and transmission of HIV to CD4 + T cells as well as in the promotion of HIV replication in activated CD4 + T cells during the cognate T-cell and DC interaction. We report that HIV induces human genital mucosal epithelial cells to produce thymic stromal lymphopoietin (TSLP) via activation of the NFκB signaling pathway. The TSLP secreted by HIV exposed epithelial cells activated DC, which promoted proliferation and HIV-1 replication of co-cultured autologous CD4 + T cells. In rhesus macaques, we observed dramatic increases in TSLP expression concurrent with an increase in viral replication in the vaginal tissues within the first 2 weeks after vaginal SIV exposure. These data suggest that HIV-mediated TSLP production by mucosal epithelial cells is a critical trigger for DC-mediated amplification of HIV-infection in activated CD4 + T cells. The cross talk between mucosal epithelial cells and DC, mediated by HIV-induced TSLP, may be an important mechanism for the high rate of HIV infection in women through the vaginal mucosa.

Targeted delivery of vaccine candidates to the gastrointestinal (GI) tract holds potential for mucosal immunization, particularly against mucosal pathogens like the human immunodeficiency virus (HIV). Among the different strategies for achieving targeted release in the GI tract, namely the small intestine, pH sensitive enteric coating polymers have been shown to protect solid oral dosage forms from the harsh digestive environment of the stomach and dissolve relatively rapidly in the small intestine by taking advantage of the luminal pH gradient. We developed an enteric polymethacrylate formulation for coating hydroxy-propyl-methyl-cellulose (HPMC) capsules containing lyophilized Adenoviral type 5 (Ad5) vectors expressing HIV-1 gag and a string of six highly-conserved HIV-1 envelope peptides representing broadly cross-reactive CD4(+) and CD8(+) T cell epitopes. Oral immunization of rhesus macaques with these capsules primed antigen-specific mucosal and systemic immune responses and subsequent intranasal delivery of the envelope peptide cocktail using a mutant cholera toxin adjuvant boosted cellular immune responses including, antigen-specific intracellular IFN-gamma-producing CD4(+) and CD8(+) effector memory T cells in the intestine. These results suggest that the combination of oral adenoviral vector priming followed by intranasal protein/peptide boosting may be an effective mucosal HIV vaccination strategy for targeting viral antigens to the GI tract and priming systemic and mucosal immunity.

Vaccine development efforts against HIV-1 have been hindered because of the high mutation rate of the virus, and limitations for direct testing of HIV antigens in animal models to discern the nature of protective immunity. We developed a multivalent vaccine comprised of highly conserved HIV envelope peptide cocktail focused on priming antigen-specific helper T cell and CTL responses. Here we report protection of rhesus macaques against pathogenic SHIV(89.6P) challenge through priming cell-mediated immunity by prophylactic vaccination with the peptide-cocktail delivered by dendritic cells. Compared to monkeys mock-vaccinated or immunized with the peptide cocktail using IFA, vaccination with peptide cocktail-pulsed DC showed significant protection from AIDS-associated mortality and reduction in plasma viremia to undetectable levels.

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

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.

Microgravity has been implicated to play a role in the observed immune dysfunction of astronauts and cosmonauts after either short-term or long-term space travel. These reports, together with studies describing increased levels of microorganisms in the space cabin environment suggest potential risk for in-flight incidences of infectious diseases. In order to understand the mechanism underlying these immune defects, it is important to have a ground-based model that would reliably mimic the effects of microgravity on antigen-specific immune function. We tested the utility of the rotating wall vessel (RWV) technology developed at NASA as a model system because in the RWV the culture medium and the cells rotate synchronously with the vessel, thereby creating simulated microgravity conditions. We compared the RWV to the conventional tissue culture flask (T-flask), for culturing immune precursor cells with cytotoxic T lymphocyte (CTL) activity against synthetic viral peptides. We observed a significant loss of antigen-specific CTL activity in RWV cultures, but not in those from the T-flask, irrespective of the peptide immunogen used for inducing the primary immune response in different mouse strains. Loss of CTL activity in RWV cultures coincided with a significant reduction in CD8+ cells as well as CD4+ cells and DEC205+ dendritic cells, suggesting adverse effects of RWV culturing on both the effector and accessory cells for the loss of antigen-specific CTL function. These results provide a strong parallel to the reported defects in cell-mediated immunity during space travel and strongly support the utility of the RWV technology as an effective ground-based model for identifying key steps in immune cell dysfunction related to microgravity.

Interferon-alpha treatment induces complete cytogenetic remission in 25% of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) patients. These remissions are durable unlike remissions induced with other therapies and yet residual leukemia is detectable in most of these patients. Total peripheral blood mononuclear cells (PBMCs) from CML patients in long-term remission following interferon treatment exhibited significantly higher proliferative responses (four- to 15-fold over background) than normals directed against P210 BCR-ABL in extracts of transfected monkey fibroblast cells. Surprisingly, similar enhanced levels of specific proliferative responses were observed with extracts from cells expressing Bcr and/or Abl proteins. In contrast, extracts from vector only or v-Mos-expressing cells had background level responses. Control monkey fibroblast cells lacking BCR-ABL expression failed to induce proliferation over background levels. Normal individuals had no significant responses to Bcr/Abl extracts. On the other hand, peripheral blood mononuclear cells from allogeneic bone marrow transplant CML patients had proliferative responses to cell extracts independent of Bcr-Abl. These data indicate that patients in remission due to alpha-interferon treatment have significantly higher levels of specific cellular immunoreactivity against Bcr/Abl sequences than normal controls, which could play a role in maintaining cytogenetic remission in Ph-positive CML patients.

Due to many physiological and genetic characteristic similarities to humans, squirrel monkeys provide an ideal animal model specifically for studying malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). While squirrel monkeys three years and older are generally considered adult subjects suitable for use in medical research studies, little is known about the functional properties of lymphocytes in relation to the age of these animals, which could significantly impact the quality and quantity of innate and adaptive immune responses. In this study, we investigated differences in the phenotype and function of lymphocytes subsets of young (3-4 years), adult (8-10 years) and aged (16-19 years) squirrel monkeys. In general, animals in all three age groups exhibited comparable numbers of different lymphocyte subsets except for CD20+ B cells that were significantly lower in aged relative to young animals and T cells subsets expressing both CD4 and CD8 (double positive) were significantly higher in aged relative to young animals. With increasing age, phenotypic differences in central and effector memory T cells subsets were observed, that were more pronounced for the CD8+ T cells. Despite equal proportions of CD3+ T cells among the three age groups, responses of peripheral blood mononuclear cells to T cell mitogens PHA and Con A showed lower IFN-γ producing cells in the aged group than that in the young group. Furthermore, aged animals showed significantly higher plasma levels of inflammatory cytokines IL-6, IFN-γ, TNF-α, IL-10 and IL-12. These findings suggest that while the squirrel monkeys in general share phenotypic and functional similarities of lymphocyte subsets with humans in relation to age, specific differences exist in immune function of lymphocytes between young and old animals that could potentially impact experimental outcomes for which the measurement of immunologic endpoints are critical.

Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination.

Recent efforts at understanding the immune response generated against human immunodeficiency virus (HIV) infection have focused on cytotoxic T lymphocyte (CTL)-mediated recognition of HIV antigens. CTLs are a major immune defense mechanism and are necessary for the recovery of many viral infections. We have previously developed a method for screening synthetic peptides for the ability to induce virus-specific major histocompatability complex-restricted CTLs in mice. Using this method, we now report the identification of peptides from the V3 region in gp120 of seven different HIV-1 strains that are capable of inducing a virus-specific CD8+ CTL response in vivo. V3 peptides from MN and SC strains of HIV-1, which are representative of typical strains found in North America and Europe, induced CTLs that exhibited cross-reactivity against a broad range of HIV-1 strains. In addition, immunization of mice with a mixture of these V3 peptides resulted in efficient CTL responses directed against the corresponding HIV-1 strains. These data, together with information in the literature describing the CTL epitope nature of V3 peptide from HIV-1 IIIB in the context of several HLA alleles, indicate the possibility of including V3 synthetic peptides as components of potential vaccines for inducing broadly cross-reactive CTL response against a diverse array of HIV-1 strains.

Objective and design : In this study we used synthetic peptides corresponding to the third variable region (V3) in the envelope protein gp120 of 14 different HIV-1 strains, and tested whether V3-specific T-cell responses are HIV-1 strain-specific or broadly cross-reactive in nine chimpanzees chronically infected with HIVv-1 IIIB . Methods : Peripheral blood mononuclear cells isolated from nine HIV-infected chimpanzees and two uninfected controls were tested, by the [ 3 H]-thymidine incorporation assay, for proliferative responses against phytohemagglutinin, control peptide and V3-loop peptides corresponding to 14 different HIV-1 strains. Serum samples collected from the chimpanzees were analyzed by enzyme-linked immunosorbent assay for antibodies against the V3 peptides. Results : Chimpanzees 100, 139 and 175 exhibited high level of proliferative response directed against the cognate V3 peptide from HIV-1 IIIB and also showed cross-reactivity to V3 peptides from 13, seven and 13 of 13 other HIV-1 strains, respectively. Additionally, five out of nine chimpanzees showed cross-reactive proliferative responses to V3 peptides from at least eight different HIV-1 strains, while significant proliferation to V3 peptides from two or more HIV-1 strains was observed in seven out of nine chimpanzees. On the other hand, four out of nine chimpanzees showed antibody response directed against the cognate V3 peptide from HIV-1 IIIB , and serum from only one chimpanzee (100) showed cross-reactive antibody to six different V3 peptides. Conclusions : Overall, these studies in chimpanzees chronically infected with HIV-1 IIIB indicate that with respect to the immunodominant V3 region, the virus-induced T-cell immunity is directed against a broad spectrum of HIV-1 strains.

We have previously reported the induction of MHC class I-restricted, CD8+ cytotoxic T lymphocytes (CTLs) specific to human immunodeficiency virus type 1 (HIV-1) in mice by a 15-amino acid peptide (R15K) from the V3 loop in gp120. We now present evidence showing that CTL activity induced by R15K was stable for 8-10 weeks after a single injection and that as little as 20 micrograms peptide was sufficient for efficient CTL induction in vivo. While induction of CTLs was efficient with R15K emulsified in either complete or incomplete Freund's adjuvant, only a low-level CTL response was observed in mice immunized with R15K in either alum or saline. We analyzed a series of carrier-free synthetic peptides ranging in length from 8 to 24 amino acids from the V3 loop region and observed that peptide R10I consisting of 10 amino acids from the middle portion of R15K was more efficient for CTL induction. Additionally, lymph node cells from mice immunized with 24 and 15 amino acid peptides (N24G and R15K, respectively) when restimulated in vitro with R10I exhibited greater HIV-1 env-specific CTL activity than when either of the longer peptides was used for restimulation. A peptide consisting of only 8 amino acids (R8K) was sufficient neither for inducing primary CTLs nor for in vitro restimulation of lymph node CTL precursors. These results establish that a carrier-free 10-amino acid synthetic peptide from the V3 loop region in HIV-1 gp120 has the optimal sequence for efficient induction of HIV env-specific CTLs in mice.

The antioxidant and antimicrobial efficacy of grape seed extract (GSE) was studied in restructured mutton slices (RMS) under aerobic and vacuum packaging conditions during refrigerated storage. The RMS treated with grape seed extract (GSE) had significantly (P0.05) lower thiobarbituric acid reactive substance (TBARS) values and free fatty acids (FFA) % compared to control (C) and butylated hydroxy anisole (BHA) treated RMS during storage at 4±1°C. Addition of GSE significantly (P0.05) reduced the total psychrophilic and coliform counts in RMS during refrigerated storage. The GSE treated mutton slices recorded significantly (P0.05) superior scores of color, flavor, juiciness and overall palatability than C and BHA treated RMS. The TBARS values, FFA % and microbial counts increased significantly (P0.05) during storage. It can be concluded that GSE has excellent antioxidant and antimicrobial properties compared to control and BHA treated RMS during refrigerated storage under aerobic and vacuum conditions.

In HIV infection there is a paucity of literature about the degree of immune dysfunction to potentially correlate and/or predict disease progression relative to CD4(+) T cells count or viral load. We assessed functional characteristics of memory T cells subsets as potential prognostic markers for changing viral loads and/or disease progression using the SHIV-infected rhesus macaque model. Relative to long-term non-progressors with low/undetectable viral loads, those with chronic plasma viremia, but clinically healthy, exhibited significantly lower numbers and functional impairment of CD4(+) T cells, but not CD8(+) T cells, in terms of IL-2 production by central memory subset in response to PMA and ionomycine (PMA+I) stimulation. Highly viremic animals showed impaired cytokine-production by all T cells subsets. These results suggest that functional impairment of CD4(+) T cells in general, and of central memory subset in particular, may be a potential indicator/predictor of chronic infection with immune dysfunction, which could be assayed relatively easily using non-specific PMA+I stimulation.

We have previously identified several synthetic peptides from conserved regions of the human immunodeficiency virus (HIV) envelope protein gp160 that have the capacity to induce broadly reactive T cell responses against gp160 in mice of several major histocompatibility complex (MHC) haplotypes. In the present investigation three rhesus monkeys were immunized with a mixture of eight synthetic peptides that are capable of inducing T cell activity in mice. Peripheral blood mononuclear cells (PBMCs) from these monkeys were monitored every 2 weeks for a period of 34 weeks for proliferative responses against individual peptides and recombinant gp160. Peripheral blood mononuclear cells from all three rhesus monkeys showed good proliferative responses with peptides 104 (aa 45-55), 111 (aa 118-130), and 63 (aa 519-543), whereas weak responses were observed with peptides 113 (aa 204-216) and 116 (aa 240-252). Two of the three rhesus monkey-derived PBMC preparations also showed good proliferative responses with peptide 61 (aa 586-598). Significant responses were not observed with peptides 105 (aa 48-61) and R15K (aa 315-329) in any of the monkeys immunized. However, PBMCs from all three monkeys showed significantly high proliferative responses with recombinant gp160, the HIV-1 envelope protein precursor. These results demonstrate that mixtures of synthetic peptides from HIV env gene product can prime gp160-specific T cell responses in rhesus monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and anti-Ad T cell responses. Also, HD vectors have the benefit of being packaged by all subgroup C serotype helper viruses. We found that HD serotypes 1, 2, 5, and 6 induce anti-HIV responses equivalently. By using these HD serotypes in heterologous succession we showed that HD vectors can be used to significantly boost anti-HIV immune responses in mice and in FG-Ad5-immune macaques. Since HD vectors have been show to have an increased safety profile, do not possess any Ad genes, can be packaged by multiple serotype helper viruses, and elicit strong anti-HIV immune responses, they warrant further investigation as alternatives to FG vectors as gene-based vaccines.

Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-gamma-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activity against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV(KU2). Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-gamma production, higher levels of vaccine-specific IFN-gamma producing CD4(+) cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.

The E6 and E7 oncoproteins of the high-risk HPV type16 represent ideal targets for HPV vaccine development, they being consistently expressed in cervical cancer lesions. Since HPV-16 is primarily transmitted through genital mucosal route, mucosal immune responses constitute an essential feature for vaccination strategies against HPV-associated lesions. We present here evidence showing that mucosal immunization of mice by the intranasal route with a mixture of peptides E7(44-62) and E6(43-57) from the E7 and E6 oncoproteins of HPV-16, respectively, using a mutant cholera toxin adjuvant (CT-2*), primed strong antigen-specific cellular immune responses in systemic and mucosal tissues. Significant levels of IFN-gamma production by both CD4 and CD8 cells were observed along with CTL responses that were effective against both peptide-pulsed targets as well as syngeneic tumor cells (TC-1) expressing the cognate E6 and E7 proteins. Furthermore, mice immunized with the peptide mixture and CT-2* effectively resisted TC-1 tumor challenge. These results together with our earlier observations that T cell responses to these peptides correlate with recurrence-free survival in women after ablative treatment for HPV-associated cervical intraepithelial neoplasia, support the potential of these E6 and E7 peptides for inclusion in vaccine formulations.

The close similarities in hematopoietic and immune systems of humans and rhesus macaques (Macaca mulatta) make them the desired nonhuman primate animal model for developing vaccines against infectious diseases relevant to humans. The best example is the simian immunodeficiency virus infection in macaques as a model for AIDS, resulting from infection of humans by HIV-1. Vaccine efficacy against viruses depends on priming cell-mediated immunity by the use of sensitive assays that can accurately detect even small levels of antigen-specific T-cell responses that may otherwise be missed easily. With this in mind, we developed the dendritic cell enzyme-linked immunospot (DC-ELISPOT) protocol by incorporating antigen-pulsed DCs to stimulate lymphocytes, as opposed to the conventional ELISPOT assay, which cultures mixtures of various low-level populations of indigent antigen-presenting cells and responding lymphocytes with antigens. In rhesus macaques immunized with an HIV envelope peptide cocktail vaccine, the DC-ELISPOT protocol enabled more accurate enumeration of the cellular immune responses, as antigen-specific interferon-gamma-producing cells, with up to 18-fold increase in detection sensitivity compared with conventional ELISPOT and elimination of false negative results. The increased sensitivity of DC-ELISOT protocol is further validated in tests determining recall antigen-specific responses in human volunteers after tuberculin skin testing.

Vaccination with peptide antigens is an effective strategy against mucosal viral infections. We tested a two-codon mutant of cholera toxin (CT-2*) lacking ADP-ribosylating activity and toxicity as a mucosal adjuvant for T cell epitope peptides for intranasal immunization of mice. Efficient induction of helper and cytotoxic T lymphocyte responses associated with TH1 cytokine production were observed in the systemic and mucosal compartments including nasal, gut, and vaginal associated lymphoid tissues. Single or multiple dosing with the peptide antigen and CT-2* induced strong memory immunity without tolerance. These results demonstrate CT-2* as a suitable mucosal adjuvant for priming antigen-specific cellular immune responses.

Based on our prior studies in mouse, monkey, chimpanzee, and human experimental systems, we identified six peptides encoded by highly conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope gene that selectively induce cellular immune responses in the absence of anti-viral antibody production. We tested a cocktail of the six peptides as a prototype vaccine for protection from simian human immunodeficiency virus (SHIV) infection and acquired immunodeficiency syndrome (AIDS) in a rhesus monkey model. Three monkeys were vaccinated with the peptide cocktail in Freund’s adjuvant followed by autologous dendritic cells (DC) pulsed with these peptides. All the vaccinated animals exhibited significant induction of T-cell proliferation and cytotoxic T lymphocytes (CTL) responses, but no neutralizing antibodies. Two control mock-vaccinated monkeys showed no specific immune responses. Upon challenge with the pathogenic SHIV KU-2 , both the control and vaccinated monkeys were infected, but efficient clearance of virus-infected cells was observed in all the three vaccinated animals within 14 weeks. These animals also experienced a boosting of antiviral cellular immune responses after infection, and maintained antigen-specific IFN-γ-producing cells in circulation beyond 42 weeks post-challenge. In contrast, the two mock-vaccinated monkeys had low to undetectable cellular immune responses and maintained significant levels of viral-infected cells and infectious virus in circulation. Further, in both the control monkeys plasma viremia was detectable beyond 38 weeks post-challenge indicating chronic phase infection. In one control monkey, the CD4 + cells dropped to very low levels by 2 weeks post-challenge and became undetectable by week 39 coinciding with high plasma viremia and AIDS, which included cachexia and ataxia. These results serve as proof of principle for the effectiveness of the HIV envelope peptide cocktail vaccine against chronic infection and AIDS, and support the development of multivalent peptide-based vaccine as a viable strategy to induce cell-mediated immunity (CMI) for protection against HIV and AIDS in humans.

Induction of cytotoxic T lymphocyte (CTL) responses is an important defense mechanism against infectious agents, specifically viruses. In the present investigation we employed a mouse assay system we previously developed, for rapid induction of CTLs by synthetic peptides from E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16). In particular, we compared the efficiency of CTL induction by HPV-16 peptides synthesized as linear monomers with those containing a dipalmitoyl-lysine-glycine-glycine (P2-KGG) moiety at the amino-terminus. Our results identified a 15-amino-acid peptide from E6(Q15L, aa 43-57) to be capable of inducing CTLs in vivo and addition of the lipid tail significantly increased CTL induction over that seen with the linear form of the peptide. Further, we identified a shorter peptide, V1OC, with 9 of 10 amino acids overlapping with Q15L peptide (aa 49-58) to be capable of inducing CTLs against both V1OC and Q15L. In case of E7 protein, our results demonstrated usefulness of P2-KGG moiety for enhanced CTL induction by previously identified CTL epitope peptides Q19D (aa 44-62) and R9F (aa 49-57). CTLs induced by both the E6 and E7 peptides were MHC class I-restricted and exhibited strict allele specificity and CD8+ phenotype. Our results showing enhanced cell-mediated immune responses with lipid-tailed forms of peptides add strength to the concept of a synthetic peptide-based vaccine for prophylaxis and therapy of HPV-associated cervical cancer.

Our previous reports established that immunization of mice in the footpad with a 15-amino acid synthetic peptide (R15K) from the V3 loop region in the envelope protein gp120 of human immunodeficiency virus type 1 (HIV-1) resulted in rapid induction of major histocompatability complex (MHC) class I-restricted, CD8+ HIV-1 envelope-specific cytotoxic T lymphocytes (CTLs) in the proximal popliteal lymph node. While efficient CTL activity could be assayed in lymph node cells for 8 to 10 weeks after a single injection, spleen cells from these mice showed low to negligible levels of specific CTLs at 4 to 8 weeks postimmunization. We tested immunizing mice with a noncovalent mixture of a helper T cell (Th) activity-inducing peptide and R15K and observed efficient induction of R15K-specific CTL response that could be assayed up to 8 weeks postimmunization in cells obtained from both lymph node and spleen. Efficient CTL priming was observed when Th peptides from either of two different conserved regions in the HIV env were mixed with R15K, containing a dipalmityl-lysine-glycine-glycine moiety at the amino terminus. These data confirm reports in literature describing requirement of Th activity for efficient priming of CTL response in vivo. Additionally, these studies strongly suggest the possibility of formulating potential vaccine candidates consisting of mixtures of synthetic peptides capable of inducing Th and CTL responses in the context of multiple MHC haplotypes.