Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA.

Human and simian immunodeficiency virus (HIV and SIV) infections establish lifelong reservoirs of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactivate integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells cotransfected with SIV provirus and plasmids coding for RGNus identified SIV long terminal repeat (LTR), trans-activation response (TAR) element, and ribosome slip site (RSS) regions as the most effective at virus suppression; RGNi targeting these regions inhibited virus production significantly. Multiplex plasmids that coexpressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole-genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations, making these tools a desirable prospect for moving into animal studies. IMPORTANCE Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole-genome sequencing demonstrated effective SIV sequence mutation and inactivation and the absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.

Local immune responses to tuberculin skin challenge in Mycobacterium bovis BCG-vaccinated baboons: a pilot study of younger and older animals.

Individuals over the age of 65 are highly susceptible to infectious diseases, which account for one-third of deaths in this age group. Vaccines are a primary tool to combat infection, yet they are less effective in the elderly population. While many groups have aimed to address this problem by studying vaccine-induced peripheral blood responses in the elderly, work from our lab and others demonstrate that immune responses to vaccination and infectious challenge may differ between tissue sites and the periphery. In this pilot study, we established an in vivo delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and tuberculin skin test in two adult and two aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon tuberculin challenge. We tested short term recall responses (8 weeks post-vaccination) and long term recall responses (25 weeks post-vaccination) by performing skin punch biopsies around the site of tuberculin injection. In short term recall responses, we found increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, in comparison to adult skin. Differences between adult and aged animals normalized in the long term response to tuberculin. In vitro, aged peripheral blood mononuclear cells had increased migration and functional responses to antigen-specific stimulation, suggesting that age-related changes in the tissue in vivo impairs aged immune recall responses to antigenic challenge. These findings highlight the impact of age-associated changes in the local tissue environment in memory recall responses, which may be more broadly applied to the study of other tissues. Moreover, these findings should be considered in future studies aimed at understanding and improving aging immune responses to vaccination and tissue challenge.

Silencing integrated SIV proviral DNA with TAR-specific CRISPR tools.

BACKGROUND: One approach for a functional HIV cure is to prevent transcription from integrated proviral DNA. A critical step in HIV transcription is the Tat protein interaction with the TAR element viral RNA. We tested the strategy of blocking this Tat-TAR interaction in the SIVmac model. METHODS: We designed five CRISPR short guiding RNAs (sgRNAs) targeting the SIVmac TAR element, along with inactive versions of Cas9 (dCas9). These sgRNA constructs were delivered as ribonucleoproteins or plasmid DNA, along with SIV DNA. The constructs were also tested in integrated viral DNA in a cell line chronically infected by SIV. RESULTS: The sgRNAs targeting the coding strand of the TAR element inhibited SIV RNA transcription in association with dCas9-KRAB, but not with dCas9. CONCLUSIONS: Induction of epigenetic modifications may be more effective in inactivating provirus than transcriptional interference and thus may be a better strategy to achieve a functional cure in vivo.

Cross-sectional comparison of health-span phenotypes in young versus geriatric marmosets.

The development of the marmoset as a translational model for healthspan and lifespan studies relies on the characterization of health parameters in young and geriatric marmosets. This cross-sectional study examined health phenotypes in marmosets for five domains of interest for human health and aging: mobility, cognition, metabolism, homeostasis, and immune function. Geriatric marmosets were found to have significant executive function impairment when compared to young animals. While geriatric animals did not show gross abnormalities in mobility and measures of locomotion, their types of movement were altered from young animals. Geriatric marmosets had alterations in cardiac function, with significantly increased mean arterial pressures; metabolism, with significantly lower VO2 ; and suppressed immune function. Further, this study sought to characterize and describe histopathology for both young and geriatric healthy marmosets. Overall this study provides a characterization of health parameters for young and geriatric marmosets which will greatly enhance future aging and interventional testing in marmosets.

Baboon CD8 T cells suppress SIVmac infection in CD4 T cells through contact-dependent production of MIP-1α, MIP-1ß, and RANTES.

Simian immunodeficiency virus (SIV) infection in rhesus macaques is often characterized by high viremia and CD4 T cell depletion. By contrast, SIV infection in African nonhuman primate natural hosts is typically nonpathogenic despite active viral replication. Baboons are abundant in Africa and have a geographical distribution that overlaps with natural hosts, but they do not harbor SIVs. Previous work has demonstrated baboons are resistant to chronic SIV infection and/or disease in vivo but the underlying mechanisms remain unknown. Using in vitro SIVmac infections, we sought to identify SIV restriction factors in baboons by comparing observations to the pathogenic rhesus macaque model. SIVmac replicated in baboon PBMC but had delayed kinetics compared to rhesus PBMC. However, SIVmac replication in baboon and rhesus isolated CD4 cells were similar to the kinetics seen for rhesus PBMC, demonstrating intracellular restriction factors do not play a strong role in baboon inhibition of SIVmac replication. Here, we show CD8 T cells contribute to the innate SIV-suppressive activity seen in naïve baboon PBMC. As one mechanism of restriction, we identified higher production of MIP-1α, MIP-1ß, and RANTES by baboon PBMC. Contact between CD4 and CD8 T cells resulted in maximum production of these chemokines and suppression of viral replication, whereas neutralization of CCR5-binding chemokines in baboon PBMC increased viral loads. Our studies indicate baboon natural restriction of SIVmac replication is largely dependent on CD4-extrinsinc mechanisms mediated, in part, by CD8 T cells.

Simian Immunodeficiency Virus Infection of Chimpanzees (Pan troglodytes) Shares Features of Both Pathogenic and Non-pathogenic Lentiviral Infections.

The virus-host relationship in simian immunodeficiency virus (SIV) infected chimpanzees is thought to be different from that found in other SIV infected African primates. However, studies of captive SIVcpz infected chimpanzees are limited. Previously, the natural SIVcpz infection of one chimpanzee, and the experimental infection of six chimpanzees was reported, with limited follow-up. Here, we present a long-term study of these seven animals, with a retrospective re-examination of the early stages of infection. The only clinical signs consistent with AIDS or AIDS associated disease was thrombocytopenia in two cases, associated with the development of anti-platelet antibodies. However, compared to uninfected and HIV-1 infected animals, SIVcpz infected animals had significantly lower levels of peripheral blood CD4+ T-cells. Despite this, levels of T-cell activation in chronic infection were not significantly elevated. In addition, while plasma levels of ß2 microglobulin, neopterin and soluble TNF-related apoptosis inducing ligand (sTRAIL) were elevated in acute infection, these markers returned to near-normal levels in chronic infection, reminiscent of immune activation patterns in 'natural host' species. Furthermore, plasma soluble CD14 was not elevated in chronic infection. However, examination of the secondary lymphoid environment revealed persistent changes to the lymphoid structure, including follicular hyperplasia in SIVcpz infected animals. In addition, both SIV and HIV-1 infected chimpanzees showed increased levels of deposition of collagen and increased levels of Mx1 expression in the T-cell zones of the lymph node. The outcome of SIVcpz infection of captive chimpanzees therefore shares features of both non-pathogenic and pathogenic lentivirus infections.

GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees.

BACKGROUND & AIMS: Direct-acting antiviral agents suppress hepatitis B virus (HBV) load, but they require life-long use. Stimulation of the innate immune system could increase its ability to control the virus and have long-lasting effects after a finite regimen. We investigated the effects of immune activation with GS-9620--a potent and selective orally active small molecule agonist of Toll-like receptor 7--in chimpanzees with chronic HBV infection. METHODS: GS-9620 was administered to chimpanzees every other day (3 times each week) for 4 weeks at 1 mg/kg and, after a 1-week rest, for 4 weeks at 2 mg/kg. We measured viral load in plasma and liver samples, the pharmacokinetics of GS-9620, and the following pharmacodynamics parameters: interferon-stimulated gene expression, cytokine and chemokine levels, lymphocyte and natural killer cell activation, and viral antigen expression. Clinical pathology parameters were monitored to determine the safety and tolerability of GS-9620. RESULTS: Short-term oral administration of GS-9620 provided long-term suppression of serum and liver HBV DNA. The mean maximum reduction of viral DNA was 2.2 logs, which occurred within 1 week of the end of GS-9620 administration; reductions of >1 log persisted for months. Serum levels of HBV surface antigen and HBV e antigen, and numbers of HBV antigen-positive hepatocytes, were reduced as hepatocyte apoptosis increased. GS-9620 administration induced production of interferon-α and other cytokines and chemokines, and activated interferon-stimulated genes, natural killer cells, and lymphocyte subsets. CONCLUSIONS: The small molecule GS-9620 activates Toll-like receptor 7 signaling in immune cells of chimpanzees to induce clearance of HBV-infected cells. This reagent might be developed for treatment of patients with chronic HBV infection.

Impact of mucosal inflammation on oral simian immunodeficiency virus transmission.

Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus (HIV). There is epidemiological evidence that genital mucosal inflammation leads to enhanced HIV type 1 (HIV-1) transmission. The objective of this study was to assess the influence of periodontal inflammation on oral HIV transmission using a nonhuman primate model of teeth ligature-induced periodontitis. Simian immunodeficiency virus (SIV) was nontraumatically applied to the gingiva after moderate gingivitis was identified through clinical and immunologic analyses (presence of inflammatory cytokines). Overall oral SIV infection rates were similar in the gingivitis-induced and control groups (5 infections following 12 SIV administrations for each), although more macaques were infected with multiple viral variants in the gingivitis group. SIV infection also affected the levels of antiviral and inflammatory cytokines in the gingival crevicular fluid, and a synergistic effect was observed, with alpha interferon and interferon-inducible protein 10 undergoing significant elevations following SIV infection in macaques with gingivitis compared to controls. These increases in antiviral and inflammatory immune modulators in the SIV-infected gingivitis macaques could also be observed in blood plasma, although the effects at both compartments were generally restricted to the acute phase of the infection. In conclusion, while moderate gingivitis was not associated with increased susceptibility to oral SIV infection, it resulted in elevated levels of cytokines in the oral mucosa and plasma of the SIV-infected macaques. These findings suggest a synergy between mucosal inflammation and SIV infection, creating an immune milieu that impacts the early stages of the SIV infection with potential implications for long-term pathogenesis.

Characterization of γδT cells in naïve and HIV-infected chimpanzees and their responses to T-cell activators in vitro.

BACKGROUND: γδT cells are effector cells that eliminate cancer and virus-infected cells. Chimpanzees are an endangered species that can naturally and experimentally be infected with SIV and HIV, respectively, but no information about the functionality of γδT cells during chronic lentiviral infection is currently available. METHODS: Healthy and HIV-infected chimpanzee γδT cells were characterized by flow cytometry. γδT subsets were studied after stimulation with T-cell activators, and the release of cytokines was analyzed by Luminex assay. RESULTS: γδT-cell subsets, Vδ1 and Vδ2Vγ9, showed different patterns in the expression of CD4, CD195, CD159a, and CD159c. Stimulation of γδT cells resulted in increased levels of CD4 and HLA-DR, which is more pronounced in Vδ1 T cells. Distinct cytokine patterns were found between healthy and HIV-infected chimpanzees. CONCLUSIONS: Analyses of major chimpanzee γδT subsets show similarities to human γδT cells and suggest different functionality and roles in their immune response against HIV infection.

Molecular Approaches for the Validation of the Baboon as a Nonhuman Primate Model for the Study of Zika Virus Infection.

Nonhuman primates (NHP) are particularly important for modeling infections with viruses that do not naturally replicate in rodent cells. Zika virus (ZIKV) has been responsible for sporadic epidemics, but in 2015 a disseminated outbreak of ZIKV resulted in the World Health Organization declaring it a global health emergency. Since the advent of this last epidemic, several NHP species, including the baboon, have been utilized for modeling and understanding the complications of ZIKV infection in humans; several health issues related to the outcome of infection have not been resolved yet and require further investigation. This study was designed to validate, in baboons, the molecular signatures that have previously been identified in ZIKV-infected humans and macaque models. We performed a comprehensive molecular analysis of baboons during acute ZIKV infection, including flow cytometry, cytokine, immunological, and transcriptomic analyses. We show here that, similar to most human cases, ZIKV infection of male baboons tends to be subclinical, but is associated with a rapid and transient antiviral interferon-based response signature that induces a detectable humoral and cell-mediated immune response. This immunity against the virus protects animals from challenge with a divergent ZIKV strain, as evidenced by undetectable viremia but clear anamnestic responses. These results provide additional support for the use of baboons as an alternative animal model to macaques and validate omic techniques that could help identify the molecular basis of complications associated with ZIKV infections in humans.

Maintenance or emergence of chronic phase secondary cytotoxic T lymphocyte responses after loss of acute phase immunodominant responses does not protect SIV-infected rhesus macaques from disease progression.

The simian immunodeficiency virus- (SIV-) infected rhesus macaque is the preferred animal model for vaccine development, but the correlates of protection in this model are not completely understood. In this paper, we document the cytotoxic T lymphocyte (CTL) response to SIV and its effects on viral evolution in an effort to identify events associated with disease progression regardless of MHC allele expression. We observed the evolution of epitopes targeted by CTLs in a group of macaques that included long-term nonprogressing (LTNP), slowly progressing (SP), normally progressing (NP), and rapidly progressing (RP) animals. Collectively, our data (1) identify novel CTL epitopes from an SP animal that are not restricted by known protective alleles, (2) illustrate that, in this small study, RP and NP animals accrue more mutations in CTL epitopes than in SP or LTNP macaques, and (3) demonstrate that the loss of CTL responses to immunodominant epitopes is associated with viral replication increases, which are not controlled by secondary CTL responses. These findings provide further evidence for the critical role of the primary cell-mediated immune responses in the control of retroviral infections.

Publisher Correction: Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16-18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.

Novel application of nonhuman primate tethering system for evaluation of acute phase SIVmac251 infection in rhesus macaques (Macaca mulatta).

Infection of rhesus macaques with simian immunodeficiency virus (SIV) is the preferred animal model for the development and testing of human immunodeficiency virus (HIV) vaccines, and animals protected from SIV challenge by live attenuated vaccines are an invaluable tool for determining immune correlates of protection. The acute phase of SIV infection, in which immune responses are most critical for slowing disease progression, occurs within the first 4 weeks of exposure. The small window of time available for observing critical immune responses makes obtaining adequate blood samples with sufficient frequency difficult. This study is the first to apply a previously reported nonhuman primate (NHP) tether system to study viral immunology. The use of the tether allows for frequent blood sampling without using restraints or sedation, thereby reducing the potentially confounding physiological changes induced by stress. We performed comparative analysis of acute phase immune responses in vaccinated and unvaccinated animals challenged with SIV-mac251. Our results demonstrate live attenuated vaccine-induced protection, which is associated with small increases in the cytotoxic T-cell (CTL) response to immunodominant epitopes, but not with increases in antibody titers. Additionally, vaccination was shown to establish a pool of antigen-specific CD8+ memory cells available for expansion after challenge. The confirmatory nature of these data indicates the validity of using the tether system for evaluation of acute phase anti-SIV responses and can be applied to the study of immune responses in other viral infections in which frequent sampling in small windows of time would be useful.

Experimental Zika Virus Inoculation in a New World Monkey Model Reproduces Key Features of the Human Infection.

A monkey model of Zika virus (ZIKV) infection is urgently needed to better understand transmission and pathogenesis, given its proven association with fetal brain defects in pregnant women and acute neurological illness. Here we experimentally infected 4 male marmosets with ZIKV (prototype 1947 African strain) and monitored them clinically with sampling of various body fluids and tissues for nearly 3 months. We show that the course of acute infection with ZIKV in these New World monkeys resembles the human illness in many respects, including (1) lack of apparent clinical symptoms in most cases, (2) persistence of the virus in body fluids such as semen and saliva for longer periods of time than in serum, and (3) generation of neutralizing antibodies as well as an antiviral immunological host response. Importantly, ZIKV-infected saliva samples (in addition to serum) were found to be infectious, suggesting potential capacity for viral transmission by the oral route. Re-challenge of a previously infected marmoset with a contemporary outbreak strain SPH2015 from Brazil resulted in continued protection against infection, no viral shedding, and boosting of the immune response. Given the key similarities to human infection, a marmoset model of ZIKV infection may be useful for testing of new drugs and vaccines.

Three weekly courses of betamethasone administered to pregnant baboons at 0.6, 0.65, and 0.7 of gestation alter fetal and maternal lymphocyte populations at 0.95 of gestation.

The hypothalamic-pituitary-adrenal (HPA) axis plays a major role in the communication between the immune and neuroendocrine systems. Glucocorticoids are potent immunomodulatory hormones. In the present study, we evaluated the effect of three weekly courses of betamethasone, administered to pregnant baboons at 0.6, 0.65, and 0.7 of gestation, on maternal hematological parameters during treatment, maternal and fetal hematological parameters and lymphocyte populations at 0.95 of gestation, and fetal lymphoid organs and placental structure. Each weekly betamethasone course resulted in decreased granulocytes and increased lymphocytes and monocytes in maternal circulation (by percentage, p < 0.05). The percentage and absolute number of CD8+ T-cells in the maternal circulation were lower and CD4+ T-cells higher (p < 0.05) in treated pregnant animals at 0.95 gestation. The percentage of proliferating CD3- CD8+ cells was lower in blood obtained from the fetal heart of betamethasone-treated animals. In the betamethasone group, the number of CD8+ T-cells and NK cells were elevated and the number of T and CD4+ T-cells were reduced in fetal heart blood compared with the umbilical vein blood. The number of placental macrophages (CD68+ cells) per visual field in betamethasone-treated and control animals were not different. Taken together, our data show that betamethasone treatment of pregnant females with no indication of preterm labor affects some components of the fetal and maternal immune system, altering the maternal CD4+/CD8+ ratio and absolute number of fetal NK cell and maternal CD8+ T-cell.

Increases in NKG2C Expression on T Cells and Higher Levels of Circulating CD8+ B Cells Are Associated with Sterilizing Immunity Provided by a Live Attenuated SIV Vaccine.

Vaccines based on live attenuated viruses are highly effective immunogens in the simian immunodeficiency virus (SIV)/rhesus macaque animal model and offer the possibility of studying correlates of protection against infection with virulent virus. We utilized a tether system for studying, in naive macaques and animals vaccinated with a live-attenuated vaccine, the acute events after challenge with pathogenic SIV. This approach allowed for the frequent sampling of small blood volumes without sedation or restraining of the animals, thus reducing the confounding effect of sampling stress. Before challenge, vaccinated animals presented significantly higher levels of proliferating and activated B cells than naive macaques, which were manifested by high expression of CD8 on B cells. After SIV challenge, the only changes observed in protected vaccinated macaques were significant increases in expression of the NK marker NKG2C on CD4 and CD8 T cells. We also identified that infection of naive macaques with SIV resulted in a transient peak of expression of CD20 on CD8 T cells and a constant rise in the number of B cells expressing CD8. Finally, analysis of a larger cohort of vaccinated animals identified that, even when circulating levels of vaccine virus are below the limit of detection, live attenuated vaccines induce systemic increases of IP-10 and perforin. These studies indicate that components of both the innate and adaptive immune systems of animals inoculated with a live-attenuated SIV vaccine respond to and control infection with virulent virus. Persistence of the vaccine virus in tissues may explain the elevated cytokine and B-cell activation levels. In addition, our report underpins the utility of the tether system for the intensive study of acute immune responses to viral infections.

Phenotypic changes associated with advancing gestation in maternal and fetal baboon lymphocytes.

Baboons are very similar to humans in ontogeny, reproductive physiology, and placentation, and thus serve as an excellent nonhuman primate model for use in both normative and perturbation studies of pregnancy that cannot be performed on pregnant women. However, little is known about the changes induced by normal pregnancy in the maternal and fetal baboon in lymphocyte subset composition, and lymphocyte activation and proliferation. We performed multicolor flow cytometry analysis of peripheral venous blood samples obtained from pregnant baboons at mid-gestation (0.5 G), and from matched fetal heart (FH) and umbilical cord (UC) blood at the end of gestation (0.95 G). Compared with their mothers at 0.95 G, fetal lymphocytes had higher percentages of B and CD4+ T cells, and lower numbers of NK and CD8+ T cells. When comparing pregnant baboons at 0.5 and 0.95 G, we also found that pregnancy induces immune stimulation, measured as higher activation without proliferation of CD8+ T and NK cells in the maternal circulation. Our study adds new data to support the notion of pregnancy-induced immune activation and strengthens the value of the baboon as a nonhuman primate model for studies pertinent to human reproductive physiology, pathology, and vaccination.

Establishment of a neonatal rhesus macaque model to study Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) is the causative agent of human tuberculosis (TB) with an estimated 8.8 million new TB cases and 1.4 million deaths annually. Tuberculosis is the leading cause of death in AIDS patients worldwide but very little is known about early TB infection or TB/HIV co-infection in infants. A clinically relevant newborn animal model to study TB infection is urgently needed. We have successfully established an aerosol newborn/infant model in neonatal nonhuman primates (NHPs) that mimics clinical and bacteriological characteristics of Mtb infection as seen in human newborns/infants. Further, this model will allow the establishment of a TB coinfection model of pediatric AIDS. Aerosol versus intra broncho-alveolar Mtb infection was studied. Interestingly, 42 days post infection specific lesions were detected suggestive of the classic Ghon focus in human children. Concurrently, specific cellular immune responses developed 4-6 weeks after Mtb infection. Using the enzyme-linked immunospot (ELISPOT) assays, we found that IL-12 production correlated with early Mtb infection lesions seen by routine thoracic radiographs. Overall, this work represents the first example of early Mtb infection of newborn macaques. This study gives us a unique opportunity to further characterize immunopathogenesis and establish a TB/SIV co-infection model for pediatric AIDS.

Evaluation of RepliVAX WN, a single-cycle flavivirus vaccine, in a non-human primate model of West Nile virus infection.

West Nile virus (WNV) causes serious neurologic disease, but no licensed vaccines are available to prevent this disease in humans. We have developed RepliVAX WN, a single-cycle flavivirus with an expected safety profile superior to other types of live-attenuated viral vaccines. In this report we describe studies examining RepliVAX WN safety, potency, and efficacy in a non-human primate model of WNV infection. A single immunization of four rhesus macaques with RepliVAX WN was safe and elicited detectable neutralizing antibody titers and IgM and IgG responses, and IgG titers were increased in two animals that received a second immunization. After challenge with WNV, three of four immunized animals were completely protected from viremia, and the remaining animal showed minimal viremia on one day. In contrast, the unvaccinated animal developed viremia that lasted six days. These results demonstrate the efficacy and safety of RepliVAX WN in this primate model of WNV infection.