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.
Hepatitis A virus (HAV) is an hepatotropic human picornavirus that is associated only with acute infection. Its pathogenesis is not well understood because there are few studies in animal models ...using modern methodologies. We characterized HAV infections in three chimpanzees, quantifying viral RNA by quantitative RT-PCR and examining critical aspects of the innate immune response including intrahepatic IFN-stimulated gene expression. We compared these infection profiles with similar studies of chimpanzees infected with hepatitis C virus (HCV), an hepatotropic flavivirus that frequently causes persistent infection. Surprisingly, HAV-infected animals exhibited very limited induction of type I IFN-stimulated genes in the liver compared with chimpanzees with acute resolving HCV infection, despite similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Minimal IFN-stimulated gene 15 and IFIT1 responses peaked 1-2 wk after HAV challenge and then subsided despite continuing high hepatic viral RNA. An acute inflammatory response at 3-4 wk correlated with the appearance of virus-specific antibodies and apoptosis and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months, remaining present long after clearance from serum and feces and revealing dramatic differences in the kinetics of clearance in the three compartments. Viral RNA was detected in the liver for significantly longer (35 to >48 wk) than HCV RNA in animals with acute resolving HCV infection (10-20 wk). Collectively, these findings indicate that HAV is far stealthier than HCV early in the course of acute resolving infection. HAV infections represent a distinctly different paradigm in virus-host interactions within the liver.
Theta-defensins (θ-defensins) are macrocyclic antimicrobial peptides expressed in leukocytes of Old World monkeys. The peptides are broad spectrum microbicides in vitro and numerous θ-defensin ...isoforms have been identified in granulocytes of rhesus macaques and Olive baboons. Several mammalian α- and β-defensins, genetically related to θ-defensins, have proinflammatory and immune-activating properties that bridge innate and acquired immunity. In the current study we analyzed the immunoregulatory properties of rhesus θ-defensins 1-5 (RTDs 1-5). RTD-1, the most abundant θ-defensin in macaques, reduced the levels of TNF, IL-1α, IL-1β, IL-6, and IL-8 secreted by blood leukocytes stimulated by several TLR agonists. RTDs 1-5 suppressed levels of soluble TNF released by bacteria- or LPS-stimulated blood leukocytes and THP-1 monocytes. Despite their highly conserved conformation and amino acid sequences, the anti-TNF activities of RTDs 1-5 varied by as much as 10-fold. Systemically administered RTD-1 was non-toxic for BALB/c mice, and escalating intravenous doses were well tolerated and non-immunogenic in adult chimpanzees. The peptide was highly stable in serum and plasma. Single dose administration of RTD-1 at 5 mg/kg significantly improved survival of BALB/c mice with E. coli peritonitis and cecal ligation-and-puncture induced polymicrobial sepsis. Peptide treatment reduced serum levels of several inflammatory cytokines/chemokines in bacteremic animals. Collectively, these results indicate that the anti-inflammatory properties of θ-defensins in vitro and in vivo are mediated by the suppression of numerous proinflammatory cytokines and blockade of TNF release may be a primary effect.
Hepatitis A virus (HAV) infection typically resolves within 4-7 wk but symptomatic relapse occurs in up to 20% of cases. Immune mechanisms that terminate acute HAV infection, and prevent a relapse of ...virus replication and liver disease, are unknown. Here, patterns of T cell immunity, virus replication, and hepatocellular injury were studied in two HAV-infected chimpanzees. HAV-specific CD8(+) T cells were either not detected in the blood or failed to display effector function until after viremia and hepatitis began to subside. The function of CD8(+) T cells improved slowly as the cells acquired a memory phenotype but was largely restricted to production of IFN-γ. In contrast, CD4(+) T cells produced multiple cytokines when viremia first declined. Moreover, only CD4(+) T cells responded during a transient resurgence of fecal HAV shedding. This helper response then contracted slowly over several months as HAV genomes were eliminated from liver. The findings indicate a dominant role for CD4(+) T cells in the termination of HAV infection and, possibly, surveillance of an intrahepatic reservoir of HAV genomes that decays slowly. Rapid contraction or failure to sustain such a CD4(+) T cell response after resolution of symptoms could increase the risk of relapsing hepatitis A.
Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey ...adenovirus (TMAdV), as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus) at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus). Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.
Quantifying cortisol concentration in hair is a non-invasive biomarker of long-term hypothalamic-pituitary-adrenal (HPA) activation, and thus can provide important information on laboratory animal ...health. Marmosets (Callithrix jacchus) and capuchins (Cebus apella) are New World primates increasingly used in biomedical and neuroscience research, yet published hair cortisol concentrations for these species are limited. Review of the existing published hair cortisol values from marmosets reveals highly discrepant values and the use of variable techniques for hair collection, processing, and cortisol extraction. In this investigation we utilized a well-established, standardized protocol to extract and quantify cortisol from marmoset (n = 12) and capuchin (n = 4) hair. Shaved hair samples were collected from the upper thigh during scheduled exams and analyzed via methanol extraction and enzyme immunoassay. In marmosets, hair cortisol concentration ranged from 2,710 to 6,267 pg/mg and averaged 4,070 ± 304 pg/mg. In capuchins, hair cortisol concentration ranged from 621 to 2,089 pg/mg and averaged 1,092 ± 338 pg/mg. Hair cortisol concentration was significantly different between marmosets and capuchins, with marmosets having higher concentrations than capuchins. The incorporation of hair cortisol analysis into research protocols provides a non-invasive measure of HPA axis activity over time, which offers insight into animal health. Utilization of standard protocols across laboratories is essential to obtaining valid measurements and allowing for valuable future cross-species comparisons.
Objective This study assesses in a baboon model the hemodynamics and human leukocyte antigen immunogenicity of chronically implanted bioengineered (decellularized with collagen conditioning ...treatments) human and baboon heart valve scaffolds. Methods Fourteen baboons underwent pulmonary valve replacement, 8 with decellularized and conditioned (bioengineered) pulmonary valves derived from allogeneic (N = 3) or xenogeneic (human) (N = 5) hearts; for comparison, 6 baboons received clinically relevant reference cryopreserved or porcine valved conduits. Panel-reactive serum antibodies (human leukocyte antigen class I and II), complement fixing antibodies (C1q binding), and C-reactive protein titers were measured serially until elective sacrifice at 10 or 26 weeks. Serial transesophageal echocardiograms measured valve function and geometry. Differences were analyzed with Kruskal–Wallis and Wilcoxon rank-sum tests. Results All animals survived and thrived, exhibiting excellent immediate implanted valve function by transesophageal echocardiograms. Over time, reference valves developed a smaller effective orifice area index (median, 0.84 cm2 /m2 ; range, 1.22 cm2 /m2 ), whereas all bioengineered valves remained normal (effective orifice area index median, 2.45 cm2 /m2 ; range, 1.35 cm2 /m2 ; P = .005). None of the bioengineered valves developed elevated peak transvalvular gradients: 5.5 (6.0) mm Hg versus 12.5 (23.0) mm Hg ( P = .003). Cryopreserved valves provoked the most intense antibody responses. Two of 5 human bioengineered and 2 of 3 baboon bioengineered valves did not provoke any class I antibodies. Bioengineered human (but not baboon) scaffolds provoked class II antibodies. C1q+ antibodies developed in 4 recipients. Conclusions Valve dysfunction correlated with markers for more intense inflammatory provocation. The tested bioengineering methods reduced antigenicity of both human and baboon valves. Bioengineered replacement valves from both species were hemodynamically equivalent to native valves.
Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika ...syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV's protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.
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