Training rhesus macaques to take daily oral antiretroviral therapy for preclinical evaluation of HIV prevention and treatment strategies.

BACKGROUND: Macaque models of simian or simian/human immunodeficiency virus (SIV or SHIV) infection are critical for the evaluation of antiretroviral (ARV)-based HIV treatment and prevention strategies. However, modelling human oral ARV administration is logistically challenging and fraught by limited adherence. Here, we developed a protocol for administering daily oral doses of ARVs to macaques with a high rate of compliance. METHODS: Parameters of positive reinforcement training (PRT), behavioral responses and optimal drug delivery foods were defined in 7 male rhesus macaques (Macaca mulatta). Animals were trained to sit in a specified cage location prior to receiving ARVs, emtricitabine (FTC) and tenofovir alafenamide (TAF), in a blended food mixture, which was followed immediately with a juice chaser. Consistency of daily oral adherence was evaluated in 4 trained macaques receiving clinically equivalent doses of FTC and TAF (20 and 1.5 mg/kg, respectively) in a short-term (1 month) and an extended (6 month) trial. Adherence was monitored using medication diaries and by quantifying intracellular FTC-triphosphate (FTC-TP) and tenofovir-diphosphate (TFV-DP) concentrations in peripheral mononuclear blood cells (PBMCs). RESULTS: Trained macaques quickly and consistently took daily oral ARVs for 1 month with an average 99.8% observed adherence. Intracellular concentrations of TFV-DP (median = 845.8 fmol/million cells [range, 620.8-1031.3]) and FTC-TP (median = 367.0 fmol/million cells [range, 289.5-413.5) in PBMCs were consistent with high adherence. Extended treatment with select subjects yielded similar observations for three months (99.5% adherence, 352/356 complete doses taken), although a sudden drop in adherence was observed after splenic biopsy surgery. CONCLUSIONS: We demonstrate that trained macaques reliably adhere to a daily oral ARV regimen, although unexpected adherence issues are possible. Our approach, using clinical doses of oral FTC and TAF daily, further refines macaque models of HIV treatment and prevention by mimicking the human route and timing of ARV administration.

Development of an Occupational Risk Assessment Tool for Laboratory Animal Facilities.

Introduction: Laboratory animal facilities aim to provide excellence in animal care and welfare and support scientific research. Critical to these goals is to ensure a safe work environment for personnel comprising veterinary and animal care, laboratory research, and maintenance staff. Objective: Thus, performing occupational risk assessments allows for evaluation of risks from identified hazards associated with a variety of tasks ongoing in laboratory animal facilities. Methods: Herein, we present the development of an occupational risk assessment tool purposed to capture the dynamics of work performed in laboratory animal facilities, calculate and prioritize identified risks associated with procedures and processes, and inform and evaluate risk mitigations. Results: We also discuss a risk assessment for refining sharps use in nonhuman primate husbandry and care to demonstrate the utility of this tool to improve occupational safety in our animal facility. Conclusion: This tool and framework evolve into a holistic occupational risk management system that identifies, evaluates, and mitigates occupational risks; determines risk acceptability; consistently ensures communication and consultation with frontline personnel, stakeholders, senior leadership, and subject matter experts in biosafety, science, and animal care and welfare; and continuously strives to improve and enhance the operations of laboratory animal facilities.

Monoclonal Antibodies as Prophylactic and Therapeutic Agents Against Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for considerable epidemics worldwide and recently emerged in the Americas in 2013. CHIKV may cause long-lasting arthralgia after acute infection. With currently no licensed vaccines or antivirals, the design of effective therapies to prevent or treat CHIKV infection is of utmost importance and will be facilitated by increased understanding of the dynamics of chikungunya. In this article, monoclonal antibodies against CHIKV as viable prophylactic and therapeutic agents will be discussed.

A natural Anopheles-associated Penicillium chrysogenum enhances mosquito susceptibility to Plasmodium infection.

Whereas studies have extensively examined the ability of bacteria to influence Plasmodium infection in the mosquito, the tripartite interactions between non-entomopathogenic fungi, mosquitoes, and Plasmodium parasites remain largely uncharacterized. Here we report the isolation of a common mosquito-associated ascomycete fungus, Penicillium chrysogenum, from the midgut of field-caught Anopheles mosquitoes. Although the presence of Pe. chrysogenum in the Anopheles gambiae midgut does not affect mosquito survival, it renders the mosquito significantly more susceptible to Plasmodium infection through a secreted heat-stable factor. We further provide evidence that the mechanism of the fungus-mediated modulation of mosquito susceptibility to Plasmodium involves an upregulation of the insect's ornithine decarboxylase gene, which sequesters arginine for polyamine biosynthesis. Arginine plays an important role in the mosquito's anti-Plasmodium defense as a substrate of nitric oxide production, and its availability therefore has a direct impact on the mosquito's susceptibility to the parasite. While this type of immunomodulatory mechanism has already been demonstrated in other host-pathogen interaction systems, this is the first report of a mosquito-associated fungus that can suppress the mosquito's innate immune system in a way that would favor Plasmodium infection and possibly malaria transmission.

The Anopheles innate immune system in the defense against malaria infection.

The multifaceted innate immune system of insects is capable of fighting infection by a variety of pathogens including those causing human malaria. Malaria transmission by the Anopheles mosquito depends on the Plasmodium parasite's successful completion of its lifecycle in the insect vector, a process that involves interactions with several tissues and cell types as well as with the mosquito's innate immune system. This review will discuss our current understanding of the Anopheles mosquito's innate immune responses against the malaria parasite Plasmodium and the influence of the insect's intestinal microbiota on parasite infection.

Caudal is a negative regulator of the Anopheles IMD pathway that controls resistance to Plasmodium falciparum infection.

Malaria parasite transmission depends upon the successful development of Plasmodium in its Anopheles mosquito vector. The mosquito's innate immune system constitutes a major bottleneck for parasite population growth. We show here that in Anopheles gambiae, the midgut-specific transcription factor Caudal acts as a negative regulator in the Imd pathway-mediated immune response against the human malaria parasite Plasmodium falciparum. Caudal also modulates the mosquito midgut bacterial flora. RNAi-mediated silencing of Caudal enhanced the mosquito's resistance to bacterial infections and increased the transcriptional abundance of key immune effector genes. Interestingly, Caudal's silencing resulted in an increased lifespan of the mosquito, while it impaired reproductive fitness with respect to egg laying and hatching.

Low- and high-tech approaches to control Plasmodium parasite transmission by anopheles mosquitoes.

Current efforts have proven inadequate to stop the transmission of Plasmodium parasites, and hence the spread of malaria, by Anopheles mosquitoes. Therefore, a novel arsenal of strategies for inhibiting Plasmodium infection of mosquitoes is urgently needed. In this paper, we summarize research on two approaches to malaria control, a low-tech strategy based on parasite inhibition by the mosquito's natural microflora, and a high-tech strategy using genetic modification of mosquitoes that renders them resistant to infection and discuss advantages and disadvantages for both approaches.

Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae.

Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development before invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.

Depletion of mitochondrial acyl carrier protein in bloodstream-form Trypanosoma brucei causes a kinetoplast segregation defect.

Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.