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1.  Abortive Intrabronchial Infection of Rhesus Macaques with Varicella-Zoster Virus Provides Partial Protection against Simian Varicella Virus Challenge 
Journal of Virology  2014;89(3):1781-1793.
Varicella-zoster virus (VZV) is a human neurotropic alphaherpesvirus and the etiological agent of varicella (chickenpox) and herpes zoster (HZ, shingles). Previously, inoculation of monkeys via the subcutaneous, intratracheal, intravenous, or oral-nasal-conjunctival routes did not recapitulate all the hallmarks of VZV infection, including varicella, immunity, latency, and reactivation. Intrabronchial inoculation of rhesus macaques (RMs) with simian varicella virus (SVV), a homolog of VZV, recapitulates virologic and immunologic hallmarks of VZV infection in humans. Given that VZV is acquired primarily via the respiratory route, we investigated whether intrabronchial inoculation of RMs with VZV would result in a robust model. Despite the lack of varicella and viral replication in either the lungs or whole blood, all four RMs generated an immune response characterized by the generation of VZV-specific antibodies and T cells. Two of 4 VZV-inoculated RMs were challenged with SVV to determine cross-protection. VZV-immune RMs displayed no varicella rash and had lower SVV viral loads and earlier and stronger humoral and cellular immune responses than controls. In contrast to the results for SVV DNA, no VZV DNA was detected in sensory ganglia at necropsy. In summary, following an abortive VZV infection, RMs developed an adaptive immune response that conferred partial protection against SVV challenge. These data suggest that a replication-incompetent VZV vaccine that does not establish latency may provide sufficient protection against VZV disease and that VZV vaccination of RMs followed by SVV challenge provides a model to evaluate new vaccines and therapeutics against VZV.
IMPORTANCE Although VZV vaccine strain Oka is attenuated, it can cause mild varicella, establish latency, and in rare cases, reactivate to cause herpes zoster (HZ). Moreover, studies suggest that the HZ vaccine (Zostavax) only confers short-lived immunity. The development of more efficacious vaccines would be facilitated by a robust animal model of VZV infection. The data presented in this report show that intrabronchial inoculation of rhesus macaques (RMs) with VZV resulted in an abortive VZV infection. Nevertheless, all animals generated a humoral and cellular immune response that conferred partial cross-protection against simian varicella virus (SVV) challenge. Additionally, VZV DNA was not detected in the sensory ganglia, suggesting that viremia might be required for the establishment of latency. Therefore, VZV vaccination of RMs followed by SVV challenge is a model that will support the development of vaccines that boost protective T cell responses against VZV.
PMCID: PMC4300754  PMID: 25410871
2.  Intrabronchial Infection of Rhesus Macaques with Simian Varicella Virus Results in a Robust Immune Response in the Lungs 
Journal of Virology  2014;88(21):12777-12792.
Varicella-zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (shingles). Primary VZV infection is believed to occur via the inhalation of virus either in respiratory droplets or from shedding varicella lesions or by direct contact with infectious vesicular fluid. However, the ensuing immune response in the lungs remains incompletely understood. We have shown that intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we performed an in-depth analysis of the host immune response to acute SVV infection in the lungs and peripheral blood. We report that acute SVV infection results in a robust innate immune response in the lungs, characterized by the production of inflammatory cytokines, chemokines, and growth factors as well as an increased frequency of plasmacytoid dendritic cells (DCs) that corresponded with alpha interferon (IFN-α) production and a rapid decrease in viral loads in the lungs. This is followed by T and B cell proliferation, antibody production, T cell differentiation, and cytokine production, which correlate with the complete cessation of viral replication. Although terminally differentiated CD8 T cells became the predominant T cell population in bronchoalveolar lavage cells, a higher percentage of CD4 T cells were SVV specific, which suggests a critical role for these cells in the resolution of primary SVV infection in the lungs. Given the homology between SVV and VZV, our data provide insight into the immune response to VZV within the lung.
IMPORTANCE Although primary VZV infection occurs primarily via the respiratory route, the host response in the lungs and its contribution to the cessation of viral replication and establishment of latency remain poorly understood. The difficulty in accessing lung tissue and washes from individuals infected with VZV has hampered efforts to address this knowledge gap. SVV infection of rhesus macaques is an important model of VZV infection of humans; therefore, we utilized this animal model to gain a comprehensive view of the kinetics of the immune response to SVV in the lung and its relationship to the resolution of acute infection in respiratory tissues. These data not only advance our understanding of host immunity to VZV, a critical step in developing new vaccines, but also provide additional insight into immunity to respiratory pathogens.
PMCID: PMC4248928  PMID: 25142604
3.  Chronic Ethanol consumption modulates growth factor release, mucosal cytokine production and microRNA expression in nonhuman primates 
Chronic alcohol consumption has been associated with enhanced susceptibility to both systemic and mucosal infections. However, the exact mechanisms underlying this enhanced susceptibility remain incompletely understood.
Using a nonhuman primate model of ethanol self-administration, we examined the impact of chronic alcohol exposure on immune homeostasis, cytokine and growth factor production in peripheral blood, lung and intestinal mucosa following twelve months of chronic ethanol exposure.
Ethanol exposure inhibited activation-induced production of growth factors HGF, G-CSF and VEGF by peripheral blood mononuclear cells (PBMC). Moreover, ethanol significantly reduced the frequency of colonic Th1 and Th17 cells in a dose-dependent manner. In contrast, we did not observe differences in lymphocyte frequency or soluble factor production in the lung of ethanol-consuming animals. To uncover mechanisms underlying reduced growth factor and Th1/Th17 cytokine production, we compared expression levels of microRNAs in PBMC and intestinal mucosa. Our analysis revealed ethanol-dependent upregulation of distinct microRNAs in affected tissues (miR-181a and miR-221 in PBMC; miR-155 in colon). Moreover, we were able to detect reduced expression of the transcription factors STAT3 and ARNT, which regulate expression of VEGF, G-CSF and HGF and contain targets for these microRNAs. To confirm and extend these observations, PBMC were transfected with either mimics or antagomirs of miR181 and 221and protein levels of the transcription factors and growth factors were determined. Transfection of microRNA mimics led to a reduction in both STAT-3/ARNT as well as VEGF/HGF/G-CSF levels. The opposite outcome was observed when microRNA antagomirs were transfected
Chronic ethanol consumption significantly disrupts both peripheral and mucosal immune homeostasis, and this dysregulation may be mediated by changes in microRNA expression.
PMCID: PMC3984381  PMID: 24329418
Ethanol; Nonhuman Primate; Self-Adminstration; Immunity; microRNA
4.  The Rhesus Rhadinovirus CD200 Homologue Affects Immune Responses and Viral Loads during In Vivo Infection 
Journal of Virology  2014;88(18):10635-10654.
Rhesus macaque rhadinovirus (RRV) is a gammaherpesvirus of rhesus macaque (RM) monkeys that is closely related to human herpesvirus 8 (HHV-8)/Kaposi's Sarcoma-associated herpesvirus (KSHV), and it is capable of inducing diseases in simian immunodeficiency virus (SIV)-infected RM that are similar to those seen in humans coinfected with HIV and HHV-8. Both HHV-8 and RRV encode viral CD200 (vCD200) molecules that are homologues of cellular CD200, a membrane glycoprotein that regulates immune responses and helps maintain immune homeostasis via interactions with the CD200 receptor (CD200R). Though the functions of RRV and HHV-8 vCD200 molecules have been examined in vitro, the precise roles that these viral proteins play during in vivo infection remain unknown. Thus, to address the contributions of RRV vCD200 to immune regulation and disease in vivo, we generated a form of RRV that lacked expression of vCD200 for use in infection studies in RM. Our data indicated that RRV vCD200 expression limits immune responses against RRV at early times postinfection and also impacts viral loads, but it does not appear to have significant effects on disease development. Further, examination of the distribution pattern of CD200R in RM indicated that this receptor is expressed on a majority of cells in peripheral blood mononuclear cells, including B and T cells, suggesting potentially wider regulatory capabilities for both vCD200 and CD200 that are not strictly limited to myeloid lineage cells. In addition, we also demonstrate that RRV infection affects CD200R expression levels in vivo, although vCD200 expression does not play a role in this phenomenon.
IMPORTANCE Cellular CD200 and its receptor, CD200R, compose a pathway that is important in regulating immune responses and is known to play a role in a variety of human diseases. A number of pathogens have been found to modulate the CD200-CD200R pathway during infection, including human herpesvirus 8 (HHV-8), the causative agent of Kaposi's sarcoma and B cell neoplasms in AIDS patients, and a closely related primate virus, rhesus macaque rhadinovirus (RRV), which infects and induces disease in rhesus macaque monkeys. HHV-8 and RRV encode homologues of CD200, termed vCD200, which are thought to play a role in preventing immune responses against these viruses. However, neither molecule has been studied in an in vivo model of infection to address their actual contributions to immunoregulation and disease. Here we report findings from our studies in which we analyzed the properties of a mutant form of RRV that lacks vCD200 expression in infected rhesus macaques.
PMCID: PMC4178886  PMID: 24991004
5.  Chikungunya Viruses That Escape Monoclonal Antibody Therapy Are Clinically Attenuated, Stable, and Not Purified in Mosquitoes 
Journal of Virology  2014;88(15):8213-8226.
Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1−/− mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle.
IMPORTANCE Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.
PMCID: PMC4135940  PMID: 24829346
6.  Could moderate alcohol intake be recommended to improve vaccine responses? 
Expert review of vaccines  2014;13(7):817-819.
The impacts of alcohol consumption on human health are complex and modulated by several factors such as patterns and amounts of drinking, genetics, the organ system studied, as well as the sex and the age of the user. There is strong evidence that chronic ethanol abuse is associated with increased morbidity and mortality, immunosuppression, and increased susceptibility to both bacterial and viral infections. In contrast, moderate alcohol consumption exerts positive effects including decreased mortality, and improved cardiovascular disease and insulin sensitivity. Interestingly, accumulating evidence also supports an immune boosting effect of moderate alcohol. In this editorial, we summarize the findings that support a positive effect of moderate alcohol on host immunity. We also discuss the limitations of the previous data and emphasize the importance of additional studies to uncover mechanisms for these immune-stimulating effects in order to extend these benefits to vulnerable segments of the population who cannot consume alcohol.
PMCID: PMC4245072  PMID: 24872009
Alcohol; vaccine; immunity; inflammation; macaque
7.  Pathophysiologic and Transcriptomic Analyses of Viscerotropic Yellow Fever in a Rhesus Macaque Model 
Infection with yellow fever virus (YFV), an explosively replicating flavivirus, results in viral hemorrhagic disease characterized by cardiovascular shock and multi-organ failure. Unvaccinated populations experience 20 to 50% fatality. Few studies have examined the pathophysiological changes that occur in humans during YFV infection due to the sporadic nature and remote locations of outbreaks. Rhesus macaques are highly susceptible to YFV infection, providing a robust animal model to investigate host-pathogen interactions. In this study, we characterized disease progression as well as alterations in immune system homeostasis, cytokine production and gene expression in rhesus macaques infected with the virulent YFV strain DakH1279 (YFV-DakH1279). Following infection, YFV-DakH1279 replicated to high titers resulting in viscerotropic disease with ∼72% mortality. Data presented in this manuscript demonstrate for the first time that lethal YFV infection results in profound lymphopenia that precedes the hallmark changes in liver enzymes and that although tissue damage was noted in liver, kidneys, and lymphoid tissues, viral antigen was only detected in the liver. These observations suggest that additional tissue damage could be due to indirect effects of viral replication. Indeed, circulating levels of several cytokines peaked shortly before euthanasia. Our study also includes the first description of YFV-DakH1279-induced changes in gene expression within peripheral blood mononuclear cells 3 days post-infection prior to any clinical signs. These data show that infection with wild type YFV-DakH1279 or live-attenuated vaccine strain YFV-17D, resulted in 765 and 46 differentially expressed genes (DEGs), respectively. DEGs detected after YFV-17D infection were mostly associated with innate immunity, whereas YFV-DakH1279 infection resulted in dysregulation of genes associated with the development of immune response, ion metabolism, and apoptosis. Therefore, WT-YFV infection is associated with significant changes in gene expression that are detectable before the onset of clinical symptoms and may influence disease progression and outcome of infection.
Author Summary
Yellow fever virus causes ∼200,000 infections and 30,000 deaths annually in Africa and South America. Although this is an important human pathogen, the basis of yellow fever disease severity remains poorly understood. Rhesus macaques are susceptible to yellow fever and develop similar symptoms as severe as those observed in humans. In this study, we characterized disease progression in this model and observed a profound loss of lymphocytes that preceded the appearance of serum markers of virus-induced liver pathology. This change might provide an early indicator of fatal yellow fever. In addition, we also identified significant changes in gene expression in white blood cells that occur before any measurable disease symptoms and these genetic signatures may provide future targets for antiviral therapeutics and better diagnostics.
PMCID: PMC4238990  PMID: 25412185
8.  Age-related dysregulation of CD8+ T-cell memory specific for a persistent virus is independent of viral replication 
The immune system devotes substantial resources to the life-long control of persistent pathogens, which were hypothesized to play an important role in immune aging. Specifically, the presence of latent herpesviruses has been correlated with immune exhaustion and shorter lifespan in octogenarian humans. But neither the causality nor the mechanistic link(s) were established, and the relative roles of persistent antigenic stimulation and of virus-independent homeostatic disturbances in T-cell aging remain unresolved. We longitudinally analyzed expansion, contraction and long-term maintenance of CD8+ T-cells responding to localized infection with a latent virus, HSV-1. Young mice exhibited the expected expansion and contraction of HSV-1-specific cells and the stable maintenance of memory T-cells into advanced adulthood. However, upon entry into senescence, many (>40%) animals exhibited an accumulation in Ag-specific cells (memory inflation) which in some animals was comparable to that observed in acute infection. Inflation occurred to the same extent in control mice and mice continuously treated with the anti-HSV drug famciclovir, which inhibits viral replication and was able to reduce expression of the gB protein; this was also seen in mice infected with an acute virus. The inflating cells largely maintained Ag-specific function, and exhibited typical central memory phenotype, with no signs of Ag-specific activation. They exhibited increased expression of CD122 and CD127, akin to the antigen-independent T-cell clonal expansions (TCE) found in old specific pathogen free (SPF) laboratory mice. This collectively suggests that in this model the inflating cells may be selected for high responsiveness to environmental cytokines largely in an antigen-independent manner.
PMCID: PMC4161215  PMID: 18354208
9.  Genome-Wide Analysis of T Cell Responses during Acute and Latent Simian Varicella Virus Infections in Rhesus Macaques 
Journal of Virology  2013;87(21):11751-11761.
Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.
PMCID: PMC3807353  PMID: 23986583
10.  Age and Immune Status of Rhesus Macaques Impact Simian Varicella Virus Gene Expression in Sensory Ganglia 
Journal of Virology  2013;87(15):8294-8306.
Simian varicella virus (SVV) infection of rhesus macaques (RMs) recapitulates the hallmarks of varicella-zoster virus (VZV) infection of humans, including the establishment of latency within the sensory ganglia. Various factors, including age and immune fitness, influence the outcome of primary VZV infection, as well as reactivation resulting in herpes zoster (HZ). To increase our understanding of the role of lymphocyte subsets in the establishment of viral latency, we analyzed the latent SVV transcriptome in juvenile RMs depleted of CD4 T, CD8 T, or CD20 B lymphocytes during acute infection. We have previously shown that SVV latency in sensory ganglia of nondepleted juvenile RMs is associated with a limited transcriptional profile. In contrast, CD4 depletion during primary infection resulted in the failure to establish a characteristic latent viral transcription profile in sensory ganglia, where we detected 68 out of 69 SVV-encoded open reading frames (ORFs). CD-depleted RMs displayed a latent transcriptional profile that included additional viral transcripts within the core region of the genome not detected in control RMs. The latent transcriptome of CD20-depleted RMs was comparable to the latent transcription in the sensory ganglia of control RMs. Lastly, we investigated the impact of age on the establishment of SVV latency. SVV gene expression was more active in ganglia from two aged RMs than in ganglia from juvenile RMs, with 25 of 69 SVV transcripts detected. Therefore, immune fitness at the time of infection modulates the establishment and/or maintenance of SVV latency.
PMCID: PMC3719833  PMID: 23698305
11.  Improving immunity in the elderly: current and future lessons from nonhuman primate models 
Age  2011;34(5):1157-1168.
The immune system must overcome daily challenges from pathogens to protect the body from infection. The success of the immune response to infection relies on the ability to sense and evaluate microbial threats and organize their elimination, while limiting damage to host tissues. This delicate balance is achieved through coordinated action of the innate and adaptive arms of the immune system. Aging results in several structural and functional changes in the immune system, often described under the umbrella term “immune senescence”. Age-related changes affect both the innate and adaptive arms of the immune system and are believed to result in increased susceptibility and severity of infectious diseases, which is further exacerbated by reduced vaccine efficacy in the elderly. Therefore, multiple strategies to improve immune function in the aged are being investigated. Traditionally, studies on immune senescence are conducted using inbred specific pathogen free (SPF) rodents. This animal model has provided invaluable insight into the mechanisms of aging. However, the limited genetic heterogeneity and the SPF status of this model restrict the successful transfer of immunological discoveries between murine models and the clinical setting. More recently, nonhuman primates (NHPs) have emerged as a leading translational model to investigate immune senescence and to test interventions aimed at delaying/reversing age-related changes in immune function. In this article, we review and summarize advances in immuno-restorative approaches investigated in the NHP model system and discuss where the NHP model can support the development of novel therapeutics.
PMCID: PMC3448983  PMID: 22180097
Immune senescence; Nonhuman primates; Cytokine; Caloric restriction; Hormones
12.  Overview of aging research using nonhuman primate models 
Age  2011;34(5):1047-1049.
PMCID: PMC3448985  PMID: 22207280
13.  Neutralizing Polyclonal IgG Present during Acute Infection Prevents Rapid Disease Onset in Simian-Human Immunodeficiency Virus SHIVSF162P3-Infected Infant Rhesus Macaques 
Journal of Virology  2013;87(19):10447-10459.
Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIVSF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression.
PMCID: PMC3807376  PMID: 23885083
14.  Bacterial artificial chromosome derived simian varicella virus is pathogenic in vivo 
Virology Journal  2013;10:278.
Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that infects humans and results in chickenpox and herpes zoster. A number of VZV genes remain functionally uncharacterized and since VZV is an obligate human pathogen, rigorous evaluation of VZV mutants in vivo remains challenging. Simian varicella virus (SVV) is homologous to VZV and SVV infection of rhesus macaques (RM) closely mimics VZV infection of humans. Recently the SVV genome was cloned as a bacterial artificial chromosome (BAC) and BAC-derived SVV displayed similar replication kinetics as wild-type (WT) SVV in vitro.
RMs were infected with BAC-derived SVV or WT SVV at 4x105 PFU intrabronchially (N=8, 4 per group, sex and age matched). We collected whole blood (PBMC) and bronchoalveolar lavage (BAL) at various days post-infection (dpi) and sensory ganglia during latent infection (>84 dpi) at necropsy and compared disease progression, viral replication, immune response and the establishment of latency.
Viral replication kinetics and magnitude in bronchoalveolar lavage cells and whole blood as well as rash severity and duration were similar in RMs infected with SVV BAC or WT SVV. Moreover, SVV-specific B and T cell responses were comparable between BAC and WT-infected animals. Lastly, we measured viral DNA in sensory ganglia from both cohorts of infected RMs during latent infection.
SVV BAC is as pathogenic and immunogenic as WT SVV in vivo. Thus, the SVV BAC genetic system combined with the rhesus macaque animal model can further our understanding of viral ORFs important for VZV pathogenesis and the development of second-generation vaccines.
PMCID: PMC3846606  PMID: 24010815
Herpesvirus; Simian varicella virus; Rhesus macaque; Bacterial artificial chromosome; Pathogenesis
15.  Attenuation of the Adaptive Immune Response in Rhesus Macaques Infected with Simian Varicella Virus Lacking Open Reading Frame 61 
Journal of Virology  2013;87(4):2151-2163.
Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Infection of rhesus macaques (RM) with the homologous simian varicella virus (SVV) recapitulates hallmarks of VZV infection. We have shown that an antisense transcript of SVV open reading frame 61 (ORF61), a viral transactivator, was detected most frequently in latently infected RM sensory ganglia. In this study, we compared disease progression, viral replication, immune response, and the establishment of latency following intrabronchial infection with a recombinant SVV lacking ORF61 (SVVΔORF61) to those following infection with wild-type (WT) SVV. Varicella severity and viral latency within sensory ganglia were comparable in RMs infected with SVVΔORF61 and WT SVV. In contrast, viral loads, B and T cell responses, and plasma inflammatory cytokine levels were decreased in RMs infected with SVVΔORF61. To investigate the mechanisms underlying the reduced adaptive immune response, we compared acute SVV gene expression, frequency and proliferation of dendritic cell (DC) subsets, and the expression of innate antiviral genes in bronchoalveolar lavage (BAL) samples. The abundance of SVV transcripts in all kinetic classes was significantly decreased in RMs infected with SVVΔORF61. In addition, we detected a higher frequency and proliferation of plasmacytoid dendritic cells in BAL fluid at 3 days postinfection in RMs infected with SVVΔORF61, which was accompanied by a slight increase in type I interferon gene expression. Taken together, our data suggest that ORF61 plays an important role in orchestrating viral gene expression in vivo and interferes with the host antiviral interferon response.
PMCID: PMC3571457  PMID: 23221560
16.  Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity 
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.
Author Summary
Chikungunya virus (CHIKV) is a re-emerging Alphavirus that has caused recent massive outbreaks in the Indian Ocean region. In addition, outbreaks have been documented in Europe and elsewhere in the world, initiated by infected travelers returning to their homelands. The recent outbreak strains possess extended vector range and as such, raise the potential of CHIKV outbreaks in the Southeastern parts of the United States. In this study, we examined CHIKV immunity in adult and aged Rhesus macaques following infection with two different CHIKV strains (recent outbreak strain CHIKV-LR and a West African Strain CHIKV-37997). CHIKV-LR causes persistent infection in the aged animals and replicates, on average, to higher levels than CHIKV-37997. Irrespective of the viral strain used, aged animals had delayed and/or reduced immunity compared to adult animals. Our data support the clinical findings of CHIKV susceptibility in vulnerable populations including the aged and provide mechanistic evidence that an effective immune response directed against the virus is required for preventing persistent CHIKV infection.
PMCID: PMC3723534  PMID: 23936572
17.  Autophagic Killing Effects against Mycobacterium tuberculosis by Alveolar Macrophages from Young and Aged Rhesus Macaques 
PLoS ONE  2013;8(6):e66985.
Non-human primates, notably rhesus macaques (Macaca mulatta, RM), provide a robust experimental model to investigate the immune response to and effective control of Mycobacterium tuberculosis infections. Changes in the function of immune cells and immunosenescence may contribute to the increased susceptibility of the elderly to tuberculosis. The goal of this study was to examine the impact of age on M. tuberculosis host-pathogen interactions following infection of primary alveolar macrophages derived from young and aged rhesus macaques. Of specific interest to us was whether the mycobactericidal capacity of autophagic macrophages was reduced in older animals since decreased autophagosome formation and autophagolysosomal fusion has been observed in other cells types of aged animals. Our data demonstrate that alveolar macrophages from old RM are as competent as those from young animals for autophagic clearance of M. tuberculosis infection and controlling mycobacterial replication. While our data do not reveal significant differences between alveolar macrophage responses to M. tuberculosis by young and old animals, these studies are the first to functionally characterize autophagic clearance of M. tuberculosis by alveolar macrophages from RM.
PMCID: PMC3688994  PMID: 23825603
18.  Targeting oxidized LDL improves insulin sensitivity and immune cell function in obese Rhesus macaques★ 
Molecular Metabolism  2013;2(3):256-269.
Oxidation of LDL (oxLDL) is a crucial step in the development of cardiovascular disease. Treatment with antibodies directed against oxLDL can reduce atherosclerosis in rodent models through unknown mechanisms. We demonstrate that through a novel mechanism of immune complex formation and Fc-γ receptor (FcγR) engagement, antibodies targeting oxLDL (MLDL1278a) are anti-inflammatory on innate immune cells via modulation of Syk, p38 MAPK phosphorylation and NFκB activity. Subsequent administration of MLDL1278a in diet-induced obese (DIO) nonhuman primates (NHP) resulted in a significant decrease in pro-inflammatory cytokines and improved overall immune cell function. Importantly, MLDL1278a treatment improved insulin sensitivity independent of body weight change. This study demonstrates a novel mechanism by which an anti-oxLDL antibody improves immune function and insulin sensitivity independent of internalization of oxLDL. This identifies MLDL1278a as a potential therapy for reducing vascular inflammation in diabetic conditions.
PMCID: PMC3773831  PMID: 24049738
Nonhuman primate; Obesity; Atherosclerosis; ox-LDL; Inflammation; Diabetes
19.  A longitudinal analysis of circulating stress-related proteins and chronic ethanol self-administration in cynomolgus macaques 
Alcoholics have alterations in endocrine and immune function and increased susceptibility to stress-related disorders. A longitudinal analysis of chronic ethanol intake on homeostatic mechanisms is, however, incompletely characterized in primates.
Plasma proteins (n = 60; Luminex) and hormones (adrenocorticotropic hormone, ACTH; cortisol) were repeatedly measured in adult male cynomolgus monkeys (Macaca fascicularis, n = 10) during a 32-month experimental protocol at baseline, during induction of water and ethanol (4% w/v in water) self-administration, after 4 months and after 12 months of 22-h daily concurrent access to ethanol and water.
Significant changes were observed in ACTH, cortisol and 45/60 plasma proteins: a majority (28/45) were suppressed as a function of ethanol self-administration, eight proteins were elevated and nine showed biphasic changes. Cortisol and ACTH were greatest during induction, and correlations between these hormones and plasma proteins varied across the experiment. Pathway analyses implicated nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) as possible mediators of ethanol-induced effects on immune-related proteins in primates.
Chronic ethanol consumption in primates leads to an allostatic state of physiological compromise with respect to circulating immune- and stress-related proteins in NF-κB- and STAT/JAK-related pathways in correlation with altered endocrine activity.
PMCID: PMC3324628  PMID: 22141444
non-human primates; ethanol; Luminex; adrenocorticotropic hormone; cortisol
20.  Animal Models of Varicella Zoster Virus Infection 
Pathogens  2013;2(2):364-382.
Primary infection with varicella zoster virus (VZV) results in varicella (chickenpox) followed by the establishment of latency in sensory ganglia. Declining T cell immunity due to aging or immune suppressive treatments can lead to VZV reactivation and the development of herpes zoster (HZ, shingles). HZ is often associated with significant morbidity and occasionally mortality in elderly and immune compromised patients. There are currently two FDA-approved vaccines for the prevention of VZV: Varivax® (for varicella) and Zostavax® (for HZ). Both vaccines contain the live-attenuated Oka strain of VZV. Although highly immunogenic, a two-dose regimen is required to achieve a 99% seroconversion rate. Zostavax vaccination reduces the incidence of HZ by 51% within a 3-year period, but a significant reduction in vaccine-induced immunity is observed within the first year after vaccination. Developing more efficacious vaccines and therapeutics requires a better understanding of the host response to VZV. These studies have been hampered by the scarcity of animal models that recapitulate all aspects of VZV infections in humans. In this review, we describe different animal models of VZV infection as well as an alternative animal model that leverages the infection of Old World macaques with the highly related simian varicella virus (SVV) and discuss their contributions to our understanding of pathogenesis and immunity during VZV infection.
PMCID: PMC4235715  PMID: 25437040
varicella zoster virus; guinea pigs; SCID-humanized mouse model; non-human primates; simian varicella virus
21.  Cytomegalovirus CC Chemokine Promotes Immune Cell Migration 
Journal of Virology  2012;86(21):11833-11844.
Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4+ T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.
PMCID: PMC3486311  PMID: 22915808
22.  Increased Viral Loads and Exacerbated Innate Host Responses in Aged Macaques Infected with the 2009 Pandemic H1N1 Influenza A Virus 
Journal of Virology  2012;86(20):11115-11127.
In contrast to seasonal influenza virus infections, which typically cause significant morbidity and mortality in the elderly, the 2009 H1N1 virus caused severe infection in young adults. This phenomenon was attributed to the presence of cross-protective antibodies acquired by older individuals during previous exposures to H1N1 viruses. However, this hypothesis could not be empirically tested. To address this question, we compared viral replication and the development of the immune response in naïve young adult and aged female rhesus macaques infected with A/California/04/2009 H1N1 (CA04) virus. We show higher viral loads in the bronchoalveolar lavage (BAL) fluid and nasal and ocular swabs in aged animals, suggesting increased viral replication in both the lower and upper respiratory tracts. T cell proliferation was higher in the BAL fluid but delayed and reduced in peripheral blood in aged animals. This delay in proliferation correlated with a reduced frequency of effector CD4 T cells in old animals. Aged animals also mobilized inflammatory cytokines to higher levels in the BAL fluid. Finally, we compared changes in gene expression using microarray analysis of BAL fluid samples. Our analyses revealed that the largest difference in host response between aged and young adult animals was detected at day 4 postinfection, with a significantly higher induction of genes associated with inflammation and the innate immune response in aged animals. Overall, our data suggest that, in the absence of preexisting antibodies, CA04 infection in aged macaques is associated with changes in innate and adaptive immune responses that were shown to correlate with increased disease severity in other respiratory disease models.
PMCID: PMC3457171  PMID: 22855494
23.  Measures of Anxiety, Amygdala Volumes, and Hippocampal Scopolamine phMRI Response in Elderly Female Rhesus Macaques 
Neuropharmacology  2011;62(1):385-390.
In nonhuman primates, anxiety levels are typically assessed by observing social hierarchies or behavior in an intruder task. As measures of anxiety might influence performance on a particular cognitive task, it is important to analyze these measures in the same room as used for the cognitive task. As we use a playroom for the spatial maze test, we classified elderly female rhesus macaques (Macaca mulatta) monkeys, as bold or reserved monkeys based on the time spent in specific areas of this room. Based on their exploratory behavior in the playroom, bold monkeys were defined as animals that spent 20% more time in the unprotected areas of the room than in the protected areas, whereas reserved monkeys spent a comparable amount of time in both areas. MRI analyses showed that reserved monkeys had a smaller amygdala compared to bold monkeys but there were no group differences in hippocampal volumes. In addition, the amount of time spent in the corners of the room was negatively correlated with the right and total amygdala size. Finally, reserved monkeys showed a lower phMRI response to the muscarinic receptor antagonist scopolamine compared to the bold monkeys. Thus, in elderly female nonhuman primates measures of anxiety are associated with structural amygdala differences and hippocampal muscarinic receptor function.
PMCID: PMC3195963  PMID: 21867720
anxiety; nonhuman primate; amygdala; hippocampus; scopolamine; phMRI
24.  Simian Varicella Virus Gene Expression During Acute and Latent Infection of Rhesus Macaques 
Journal of Neurovirology  2011;17(6):600-612.
Varicella zoster virus (VZV) is a neurotropic α-herpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Reactivation of VZV results in herpes zoster and other neurological complications. Our understanding of the VZV transcriptome during acute and latent infection in immune competent individuals remains incomplete. Infection of rhesus macaques with the homologous simian varicella virus (SVV) recapitulates the hallmarks of VZV infection. We therefore characterized the SVV transcriptome by quantitative real-time reverse transcriptase PCR (RT-qPCR) during acute infection in bronchial alveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC), and during latency in sensory ganglia obtained from the same rhesus macaques. During acute infection, all known SVV open reading frames (ORFs) were detected and the most abundantly expressed ORFs are involved in virus replication and assembly such as the transcriptional activator ORF 63 and the structural proteins ORF 41 and ORF 49. In contrast, latent SVV gene expression is highly restricted. ORF 61, a viral transactivator and latency-associated transcript, is the most prevalent transcript detected in sensory ganglia. We also detected ORFs A, B, 4, 10, 63, 64, 65, 66 and 68 though significantly less frequently than ORF 61. This comprehensive analysis has revealed genes that potentially play a role in the establishment and/or maintenance of SVV latency.
PMCID: PMC3286235  PMID: 22052378
Herpesvirus; simian varicella virus; gene expression; latency; rhesus macaque

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