Endogenous Specialized Proresolving Mediator Profiles in a Novel Experimental Model of Lymphatic Obstruction and Intestinal Inflammation in African Green Monkeys.

Changes in the intestinal lymphatic vascular system, such as lymphatic obstruction, are characteristic features of inflammatory bowel diseases. The lymphatic vasculature forms a conduit to enable resolution of inflammation; this process is driven by specialized endogenous proresolving mediators (SPMs). To evaluate contributions of lymphatic obstruction to intestinal inflammation and to study profiles of SPMs, we generated a novel animal model of lymphatic obstruction using African green monkeys. Follow-up studies were performed at 7, 21, and 61 days. Inflammation was determined by histology. Luminex assays were performed to evaluate chemokine and cytokine levels. In addition, lipid mediator metabololipidomic profiling was performed to identify SPMs. After 7 days, lymphatic obstruction resulted in a localized inflammatory state, paralleled by an increase in inflammatory chemokines and cytokines, which were found to be up-regulated after 7 days but returned to baseline after 21 and 61 days. At the same time, a distinct pattern of SPMs was profiled, with an increase for D-series resolvins, protectins, maresins, and lipoxins at 61 days. These results indicate that intestinal lymphatic obstruction can lead to an acute inflammatory state, accompanied by an increase in proinflammatory mediators, followed by a phase of resolution, paralleled by an increase and decrease of respective SPMs.

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1).

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8⁺ T cells in early viral evolution.

The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8(+) T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8(+) T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8(+) T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.

Selection-driven immune escape is not a significant factor in the failure of CD4 T cell responses in persistent hepatitis C virus infection.

UNLABELLED: Immune escape driven by selection pressure from virus-specific CD8 T cells has been demonstrated in both chimpanzees and humans infected with the hepatitis C virus (HCV). Although escape mutations have also been characterized in major histocompatibility complex (MHC) class II-restricted HCV epitopes, it is unknown whether selection-driven immune escape by CD4 T cell epitopes is a significant factor in the failure of these responses or contributes to persistent infection. To address this issue, evolution of MHC class I- and class II-restricted HCV epitopes was compared in four chimpanzees persistently infected with the virus for more than 10 years. We identified an amino acid change in a CD4 epitope of the HCV NS3 protein in one of the chimpanzees 3 years after infection. This mutation resulted in diminished activation, cytokine production (interferon-gamma and interleukin-2), and proliferation by an epitope-specific CD4 T cell line. We expanded our analysis to determine if mutations were common in multiple CD4 versus CD8 T cell epitopes in the four chronically infected animals. Whereas we observed mutations in over 75% of CD8 T cell epitopes analyzed in this study, only 18% of CD4 T cell epitopes analyzed showed amino acid changes. The frequency of changes in class II epitopes was not different from flanking regions, so CD4 T cells rarely exert selection pressure against the HCV genome. CONCLUSION: Apparent mutational escape can occur in MHC class II-restricted epitopes, but this is uncommon when compared with class I-restricted epitopes in the same individual. This indicates that other mechanisms for silencing CD4 T cells are dominant in persistent HCV infections.

Variable patterns of programmed death-1 expression on fully functional memory T cells after spontaneous resolution of hepatitis C virus infection.

The inhibitory receptor programmed death-1 (PD-1) is present on CD8(+) T cells in chronic hepatitis C virus (HCV), but expression patterns in spontaneously resolving infections are incompletely characterized. Here we report that PD-1 was usually absent on memory CD8(+) T cells from chimpanzees with resolved infections, but sustained low-level expression was sometimes observed in the absence of apparent virus replication. PD-1-positive memory T cells expanded and displayed antiviral activity upon reinfection with HCV, indicating conserved function. This animal model should facilitate studies of whether PD-1 differentially influences effector and memory T-cell function in resolved versus persistent human infections.

The Final (Oral Ebola) Vaccine Trial on Captive Chimpanzees?

Could new oral vaccine technologies protect endangered wildlife against a rising tide of infectious disease? We used captive chimpanzees to test oral delivery of a rabies virus (RABV) vectored vaccine against Ebola virus (EBOV), a major threat to wild chimpanzees and gorillas. EBOV GP and RABV GP-specific antibody titers increased exponentially during the trial, with rates of increase for six orally vaccinated chimpanzees very similar to four intramuscularly vaccinated controls. Chimpanzee sera also showed robust neutralizing activity against RABV and pseudo-typed EBOV. Vaccination did not induce serious health complications. Blood chemistry, hematologic, and body mass correlates of psychological stress suggested that, although sedation induced acute stress, experimental housing conditions did not induce traumatic levels of chronic stress. Acute behavioral and physiological responses to sedation were strongly correlated with immune responses to vaccination. These results suggest that oral vaccination holds great promise as a tool for the conservation of apes and other endangered tropical wildlife. They also imply that vaccine and drug trials on other captive species need to better account for the effects of stress on immune response.

Age, rank, and personality effects on the cortisol sedation stress response in young chimpanzees.

Primates and other mammals exhibit a glucocorticoid response to somatic and psychosocial stressors. The pattern and degree of response varies both within and between species, but the factors affecting within-species variability have rarely been considered. Here we describe the 90-min serum cortisol response of 14 juvenile and adolescent chimpanzees to the stress of sedation with ketamine hydrochloride. We show age differences in both baseline cortisol and time of peak cortisol, with younger individuals (ages 4-5 years) showing higher baseline levels and an earlier peak response than older individuals (ages 8-9 years). However, we found no sex or rank-related differences in any of the sedation measures: baseline cortisol, cortisol peak time, peak cortisol level, or cortisol change from baseline. We also examined the relationship between these sedation stress measures and behavioral style score, and found that individuals scoring high in the "mellow" behavioral style component showed a greater stress response than individuals scoring lower in this component. Future studies should consider the role of individual differences in age and personality in the cortisol response to stress.

A disposable vaginal speculum for the chimpanzee (Pan troglodytes).

The unanticipated underutilization of chimpanzees (Pan troglodytes) by the National Institutes of Health (NIH) for current NIH-supported research has prompted the application of a variety of contraceptive forms. Among the many methods attempted, the intrauterine device (IUD) has been described as having an efficacy similar to that described for humans. In addition, the device is both a financially sound and practical solution to prevention of overpopulation, while minimizing disruption of normal cyclic hormonal fluctuations. However, variations in the size and shape of the sexual skin of the female chimpanzee and the depth of the vaginal vault have posed physical constraints on the use of instrumentation developed for humans for assisting with insertion of the device. In addition the literature is lacking in specific methodology for pelvic examination in chimpanzees. Previously, the University of Louisiana at Lafayette New Iberia Research Center used a 3-ml plastic syringe case as a vaginal speculum to accommodate requirements of an approved research protocol in African green monkeys (Chlorocebus aethiops). Similarly, a simple and effective disposable vaginal speculum was developed for the chimpanzee. The closed tip of a plastic syringe case of appropriate size (20 to 60 ml) was removed to provide an open tube; momentary heating smoothed the cut edge. This simple speculum allowed for sufficient visualization of the cervix for assessment and assistance in the insertion of the IUD. Variously sized speculums were prepared to accommodate differences in animal size. This simple and effective speculum was clean, disposable, and inexpensive.

Immunogenicity of attenuated vesicular stomatitis virus vectors expressing HIV type 1 Env and SIV Gag proteins: comparison of intranasal and intramuscular vaccination routes.

An experimental AIDS vaccine based on attenuated, recombinant vesicular stomatitis virus (rVSV), when administered by a combination of parenteral and mucosal routes, has proven effective at preventing AIDS in a rhesus macaque model (Rose NF, et al.: Cell 2001;106:539-549). In an effort to determine the optimal route of vaccine administration we evaluated the ability of rVSV-based vaccine vectors expressing HIV-1 Env and SIV Gag proteins, when given either intramuscularly (i.m.) or intranasally (i.n.), to elicit antigen-specific cellular and humoral immune responses, and to protect from a subsequent vaginal challenge with simian-human immunodeficiency virus (SHIV89.6P). Our results demonstrate that macaques vaccinated by the i.n. route developed significantly higher antigen-specific cellular immune responses as determined by MHC class I tetramer staining, IFN-gamma ELISPOT, and cytotoxic T cell assays. However, systemic and mucosal humoral immune responses did not vary significantly with the route of vaccine administration. Given the importance of cell-mediated immune responses in slowing AIDS progression, intranasal delivery of a VSV-based AIDS vaccine may be an optimal as well as practical route for vaccination and should be considered in design of clinical trials.

Legg-Calvé-Perthes disease in a rhesus macaque (Macaca mulatta).

A juvenile rhesus macaque presented with atrophy of the musculature of its left leg. Physical examination localized the problem to the coxofemoral joint. Radiography revealed changes consistent with Legg-Calvé-Perthes (LCP) disease. Femoral head ostectomy was performed, and the femoral head was submitted for histologic examination, results of which confirmed a diagnosis of LCP.

An Atypical Case of Mycobacterium bovis in a Cynomolgus Macaque (Macaca fascicularis) Imported From the Philippines.

An incidental case of Mycobacterium bovis was confirmed in a cynomolgus macaque from a shipment of 100 animals imported to the University of Southwestern Louisiana New Iberia Research Center from the Philippines. The macaque was euthanized 4 weeks into the quarantine period (October 1997) for failure to thrive and suspected melioidosis. Approximately 6 months later, on 9 March 1998, culture and antigenic probes from kidney tissues identified the etiologic agent as M. bovis. Five remaining cohort animals were euthanized after we obtained the results from the index case. Kidney tissues from two of the cohort animals were positive for M. avium. All animals had negative intradermal skin tests prior to euthanasia. All three animals positive for Mycobacterium were sero-negative for retroviruses. The unusual presentation of this case, coupled with the inability to detect disease by standard means, serves to emphasize the importance of follow-up examination and culture of tissues obtained from imported non-human primates.

Semen evaluation for verification of azoospermia after vasectomy in chimpanzees (Pan troglodytes).

Standards for the reproductive management of captive chimpanzees stipulate that chimpanzees admitted into the National Chimpanzee Sanctuary System must undergo vasectomy followed by laboratory confirmation of azoospermia. In light of the observations of ourselves and others, we questioned whether azoospermia is a necessary indicator of successful vasectomy. Therefore, the objectives of the present study were to assess how much time is required between vasectomy and semen evaluation for azoospermia to be reached and to determine the percentage of vasectomized chimpanzees that actually are azoospermic. The study population comprised 39 adult male chimpanzees that underwent vasectomy and subsequent semen examination at 0.5 to 24 mo afterward. Overall, spermatozoa were found in the semen of at least 1 chimpanzee in almost every month in which animals were evaluated. Of the animals evaluated repeatedly after vasectomy, 20% had no sperm at any examination, 60% were azoospermic then positive during at least 1 subsequent examination, 13.3% were positive at least once and then azoospermic, and 6.7% were positive at every examination. After 0.5 mo postvasectomy, all sperm observed were nonmotile. The results suggest that azoospermia is not a necessary indicator of successful vasectomy.