Bioactivity and efficacy of a hyperimmune bovine colostrum product- Travelan, against shigellosis in a non-Human primate model (Macaca mulatta).

Infectious diarrhea is a World Health Organization public health priority area due to the lack of effective vaccines and an accelerating global antimicrobial resistance crisis. New strategies are urgently needed such as immunoprophylactic for prevention of diarrheal diseases. Hyperimmune bovine colostrum (HBC) is an established and effective prophylactic for infectious diarrhea. The commercial HBC product, Travelan® (Immuron Ltd, Australia) targets multiple strains of enterotoxigenic Escherichia coli (ETEC) is highly effective in preventing diarrhea in human clinical studies. Although Travelan® targets ETEC, preliminary studies suggested cross-reactivity with other Gram-negative enteric pathogens including Shigella and Salmonella species. For this study we selected an invasive diarrheal/dysentery-causing enteric pathogen, Shigella, to evaluate the effectiveness of Travelan®, both in vitro and in vivo. Here we demonstrate broad cross-reactivity of Travelan® with all four Shigella spp. (S. flexneri, S. sonnei, S. dysenteriae and S. boydii) and important virulence factor Shigella antigens. Naïve juvenile rhesus macaques (NJRM) were randomized, 8 dosed with Travelan® and 4 with a placebo intragastrically twice daily over 6 days. All NJRM were challenged with S. flexneri 2a strain 2457T on the 4th day of treatment and monitored for diarrheal symptoms. All placebo-treated NJRM displayed acute dysentery symptoms within 24-36 hours of challenge. Two Travelan®-treated NJRM displayed dysentery symptoms and six animals remained healthy and symptom-free post challenge; resulting in 75% efficacy of prevention of shigellosis (p = 0.014). These results strongly indicate that Travelan® is functionally cross-reactive and an effective prophylactic for shigellosis. This has positive implications for the prophylactic use of Travelan® for protection against both ETEC and Shigella spp. diarrheal infections. Future refinement and expansion of pathogens recognized by HBC including Travelan® could revolutionize current management of gastrointestinal infections and outbreaks in travelers' including military, peacekeepers, humanitarian workers and in populations living in endemic regions of the world.

Impact of sleep deprivation on neurocognition and inflammation in rhesus macaques.

Sleep deprivation in humans is associated with both cognitive impairment and immune dysregulation. An animal model of neuropathogenesis may provide insight to understand the effects of sleep deprivation on the brain. Human neurocognition is more closely mirrored by nonhuman primates (NHP) than other animals. As such, we developed an NHP model to assess the impact of sleep deprivation on neurocognition and markers of systemic immune activation. Six male rhesus macaques underwent three rounds of sleep deprivation (48 h without sleep) at days 0, 14, and 28. We performed domain specific cognitive assessments using the Cambridge Neuropsychological Test Automated Battery (CANTAB) via a touch screen before and after 24 and 48 h of sleep deprivation. Immune activation markers were measured in the blood by multiplex assay and flow cytometry. Although we observed variability in cognitive performance between the three rounds of sleep deprivation, cognitive impairments were identified in all six animals. We noted more cognitive impairments after 48 h than after 24 h of sleep deprivation. Following 48 h of sleep deprivation, elevations in markers of immune activation in the blood were observed in most animals. The observed impairments largely normalized after sleep. The co-occurrence of systemic immune alterations and cognitive impairment establishes this model as useful for studying the impact of sleep deprivation on neurobehavior and immune perturbations in rhesus macaques.

Neurocognitive impact of Zika virus infection in adult rhesus macaques.

BACKGROUND: Zika virus (ZIKV) is a mosquito-transmitted flavivirus that affects many regions of the world. Infection, in utero, causes microcephaly and later developmental and neurologic impairments. The impact of ZIKV infection on neurocognition in adults has not been well described. The objective of the study was to assess the neurocognitive impact of ZIKV infection in adult rhesus macaques. METHODS: Neurocognitive assessments were performed using the Cambridge Neuropsychological Test Automated Battery (CANTAB) via a touch screen and modified Brinkman Board before and after subcutaneous ZIKV inoculation. Immune activation markers were measured in the blood and cerebral spinal fluid (CSF) by multiplex assay and flow cytometry. RESULTS: All animals (N = 8) had detectable ZIKV RNA in plasma at day 1 post-inoculation (PI) that peaked at day 2 PI (median 5.9, IQR 5.6-6.2 log10 genome equivalents/mL). In all eight animals, ZIKV RNA became undetectable in plasma by day 14 PI, but persisted in lymphoid tissues. ZIKV RNA was not detected in the CSF supernatant at days 4, 8, 14 and 28 PI but was detected in the brain of 2 animals at days 8 and 28 PI. Elevations in markers of immune activation in the blood and CSF were accompanied by a reduction in accuracy and reaction speed on the CANTAB in the majority of animals. CONCLUSIONS: The co-occurrence of systemic and CSF immune perturbations and neurocognitive impairment establishes this model as useful for studying the impact of neuroinflammation on neurobehavior in rhesus macaques, as it pertains to ZIKV infection and potentially other pathogens.

Chronic Wound Infection Model of Acinetobacter baumannii in Outbred Mice.

INTRODUCTION: We established a murine wound infection model with doxycycline treatment against multidrug-resistant Acinetobacter baumannii (AB5075) in Institute of Cancer Research (ICR) outbred mice. METHODS: Using three groups of neutropenic ICR mice, two full-thickness dorsal dermal wounds (6 mm diameter) were made on each mouse. In two groups, wounds were inoculated with 7.0 × 104 colony-forming units of AB5075. Of these two groups, one received a 6-day regimen of doxycycline while the other was sham treated with phosphate-buffered saline as placebo control. Another uninfected/untreated group served as a control. Wound closure, clinical symptoms, bacterial burden in wound beds and organs, and wound histology were investigated. RESULTS: Doxycycline-treated wounds completely healed by day 21, but untreated, infected wounds failed to heal. Compared to controls, wound infections without treatment resulted in significant reductions in body weight and higher bacterial loads in wound beds, lung, liver, and spleen by day 7. Histological evaluation of wounds on day 21 revealed ulcerated epidermis, muscle necrosis, and bacterial presence in untreated wounds, while wounds treated with doxycycline presented intact epidermis. CONCLUSIONS: Compared to the previously developed BALB/c dermal wound model, this study demonstrates that the mouse strain selected impacts wound severity and resolution. Furthermore, this mouse model accommodates two dorsal wounds rather than only one. These variations offer investigators increased versatility when designing future studies of wound infection. In conclusion, ICR mice are a viable option as a model of dermal wound infection. They accommodate two simultaneous dorsal wounds, and upon infection, these wounds follow a different pattern of resolution compared to BALB/c mice.

TLR7 agonist, N6-LS and PGT121 delayed viral rebound in SHIV-infected macaques after antiretroviral therapy interruption.

Toll-like receptor 7 (TLR7) agonist and PGT121 (broadly neutralizing antibody, bnAb) administration previously delayed viral rebound and induced SHIV remission. We evaluated the impact of GS-986 (TLR7 agonist) and dual bnAbs on viral rebound after antiretroviral therapy (ART) interruption. Rhesus macaques inoculated with SHIV-1157ipd3N4 were initiated on daily suppressive ART from Day 14 post SHIV inoculation. Active arm animals (n = 8) received GS-986, N6-LS and PGT121 after plasma viral suppression, starting from week 14. GS-986 induced immune activation and SHIV-specific T cell responses but not viral expression in all the active arm animals. After ART interruption, median time to viral rebound was 6 weeks in the active and 3 weeks in the control arm (p = 0.024). In this animal model, the administration of the combination of GS-986 and dual bnAbs was associated with a modest delay in viral rebound. This strategy should be further evaluated to better understand the underlying mechanisms for the induction of virus-specific immune responses and delay in viral rebound.

Hepatic spheroids used as an in vitro model to study malaria relapse.

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.

Strict tropism for CD71+/CD234+ human reticulocytes limits the zoonotic potential of Plasmodium cynomolgi.

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P vivax These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.

Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll-like receptor 4 antagonist.

Shigella dysenteriae causes the most severe of all infectious diarrhoeas and colitis. We infected rhesus macaques orally and also treated them orally with a small and non-absorbable polypropyletherimine dendrimer glucosamine that is a Toll-like receptor-4 (TLR4) antagonist. Antibiotics were not given for this life-threatening infection. Six days later, the clinical score for diarrhoea, mucus and blood was 54% lower, colon interleukin-8 and interleukin-6 were both 77% lower, and colon neutrophil infiltration was 75% less. Strikingly, vasculitis did not occur and tissue fibrin thrombi were reduced by 67%. There was no clinical toxicity or adverse effect of dendrimer glucosamine on systemic immunity. This is the first report in non-human primates of the therapeutic efficacy of a small and orally bioavailable TLR antagonist in severe infection. Our results show that an oral TLR4 antagonist can enable controlled resolution of the infection-related-inflammatory response and can also prevent neutrophil-mediated gut wall necrosis in severe infectious diarrhoeas.

Pharmacokinetics and pharmacodynamics of (+)-primaquine and (-)-primaquine enantiomers in rhesus macaques (Macaca mulatta).

Primaquine (PQ) remains the sole available drug to prevent relapse of Plasmodium vivax malaria more than 60 years after licensure. While this drug was administered as a racemic mixture, prior studies suggested a pharmacodynamic advantage based on differential antirelapse activity and/or toxicities of its enantiomers. Oral primaquine enantiomers prepared using a novel, easily scalable method were given for 7 days to healthy rhesus macaques in a dose-rising fashion to evaluate their effects on the blood, liver, and kidneys. The enantiomers were then administered to Plasmodium cynomolgi-infected rhesus macaques at doses of 1.3 and 0.6 mg/kg of body weight/day in combination with chloroquine. The (-)-PQ enantiomer had higher clearance and apparent volume of distribution than did (+)-PQ and was more extensively converted to the carboxy metabolite. There is evidence for differential oxidative stress with a concentration-dependent rise in methemoglobin (MetHgb) with increasing doses of (+)-PQ greater than that seen for (-)-PQ. There was a marked, reversible hepatotoxicity in 2 of 3 animals dosed with (-)-PQ at 4.5 mg/kg. (-)-PQ in combination with chloroquine was successful in preventing P. cynomolgi disease relapse at doses of 0.6 and 1.3 mg/kg/day, while 1 of 2 animals receiving (+)-PQ at 0.6 mg/kg/day relapsed. While (-)-PQ was also associated with hepatotoxicity at higher doses as seen previously, this has not been identified as a clinical concern in humans during >60 years of use. Limited evidence for increased MetHgb generation with the (+) form in the rhesus macaque model suggests that it may be possible to improve the therapeutic window for hematologic toxicity in the clinic by separating primaquine into its enantiomers.

Absolute bioavailability of cis-mirincamycin and trans-mirincamycin in healthy rhesus monkeys and ex vivo antimalarial activity against Plasmodium falciparum.

The pharmacokinetics, oral bioavailability, and ex vivo antimalarial activity of mirincamycin isomers in a healthy rhesus monkey model were assessed to support lead optimization of novel nonhemolytic drugs for radical cure and causal prophylaxis of malaria. Fourteen male rhesus monkeys were randomized to four groups, which included cis and trans isomers by the oral and intravenous routes, with vehicle-only controls for each dosing route. Concentration-time data were collected for 7 days and were analyzed by noncompartmental analysis. cis-Mirincamycin had an absolute oral bioavailability of 13.6%, which was slightly higher than that of trans-mirincamycin (11.7%), but this difference was not statistically significant. There was a statistically significant difference between the area under the concentration-time curve from zero to 48 h (AUC(0-48)) of cis-mirincamycin and that of trans-mirincamycin after oral dosing. When cultured in vitro with the W2 clone of Plasmodium falciparum, the 50% inhibitory concentrations for cis-mirincamycin, trans-mirincamycin, and dihydroartemisinin were 11,300, 12,300, and 2.30 nM, respectively. However, when dosed primate plasma was cultured ex vivo against the W2 clone, both isomers had much greater relative potencies than their in vitro activities relative to results for dihydroartemisinin, an increase of approximately 100-fold for the cis isomer and 150-fold for the trans isomer. Further, oral ex vivo activity was significantly higher than intravenous activity for both isomers, particularly during the first 90 min following dosing, suggesting the first-pass formation of one or more metabolites with blood-stage antimalarial activity. Identification of the metabolic pathways and metabolites may help to further delineate the properties of this class of drugs with previously demonstrated liver-stage antimalarial activity.

Radical curative efficacy of tafenoquine combination regimens in Plasmodium cynomolgi-infected Rhesus monkeys (Macaca mulatta).

BACKGROUND: Tafenoquine is an 8-aminoquinoline being developed for radical cure (blood and liver stage elimination) of Plasmodium vivax. During monotherapy treatment, the compound exhibits slow parasite and fever clearance times, and toxicity in glucose-6-phosphate dehydrogenase (G6PD) deficiency is a concern. Combination with other antimalarials may mitigate these concerns. METHODS: In 2005, the radical curative efficacy of tafenoquine combinations was investigated in Plasmodium cynomolgi-infected naïve Indian-origin Rhesus monkeys. In the first cohort, groups of two monkeys were treated with a three-day regimen of tafenoquine at different doses alone and in combination with a three-day chloroquine regimen to determine the minimum curative dose (MCD). In the second cohort, the radical curative efficacy of a single-day regimen of tafenoquine-mefloquine was compared to that of two three-day regimens comprising tafenoquine at its MCD with chloroquine or artemether-lumefantrine in groups of six monkeys. In a final cohort, the efficacy of the MCD of tafenoquine against hypnozoites alone and in combination with chloroquine was investigated in groups of six monkeys after quinine pre-treatment to eliminate asexual parasites. Plasma tafenoquine, chloroquine and desethylchloroquine concentrations were determined by LC-MS in order to compare doses of the drugs to those used clinically in humans. RESULTS: The total MCD of tafenoquine required in combination regimens for radical cure was ten-fold lower (1.8 mg/kg versus 18 mg/kg) than for monotherapy. This regimen (1.8 mg/kg) was equally efficacious as monotherapy or in combination with chloroquine after quinine pre-treatment to eliminate asexual stages. The same dose of (1.8 mg/kg) was radically curative in combination with artemether-lumefantrine. Tafenoquine was also radically curative when combined with mefloquine. The MCD of tafenoquine monotherapy for radical cure (18 mg/kg) appears to be biologically equivalent to a 600-1200 mg dose in humans. At its MCD in combination with blood schizonticidal drugs (1.8 mg/kg), the maximum observed plasma concentrations were substantially lower than (20-84 versus 550-1,100 ng/ml) after administration of 1, 200 mg in clinical studies. CONCLUSIONS: Ten-fold lower clinical doses of tafenoquine than used in prior studies may be effective against P. vivax hypnozoites if the drug is deployed in combination with effective blood-schizonticidal drugs.

Mycobacterium tuberculosis infection in a closed colony of rhesus macaques (Macaca mulatta).

Mycobacterium infections in nonhuman primates can devastate the colonies and place human handlers at risk. Despite conservative measures to prevent exposure, infections occur even in closed colonies. Here we describe a recent case of M. tuberculosis within a closed colony of rhesus macaques at our Thailand facility and the procedures instituted to prevent subsequent infections. Investigation of the outbreak did not confirm the source of the infection, but even with intensive occupational safety measures in place, human contact remains the most likely possibility.