Optimization of Plasmodium vivax infection of colonized Amazonian Anopheles darlingi.

Obtaining Plasmodium vivax sporozoites is essential for in vitro culture of liver stage parasites, not only to understand fundamental aspects of parasite biology, but also for drug and vaccine development. A major impediment to establish high-throughput in vitro P. vivax liver stage assays for drug development is obtaining sufficient numbers of sporozoites. To do so, female anopheline mosquitoes have to be fed on blood from P. vivax-infected patients through an artificial membrane-feeding system, which in turns requires a well-established Anopheles colony. In this study we established conditions to provide a robust supply of P. vivax sporozoites. Adding a combination of serum replacement and antibiotics to the membrane-feeding protocol was found to best improve sporozoite production. A simple centrifugation method appears to be a possible tool for rapidly obtaining purified sporozoites with a minimal loss of yield. However, this method needs to be better defined since sporozoite viability and hepatocyte infection were not evaluated.

Developing Plasmodium vivax Resources for Liver Stage Study in the Peruvian Amazon Region.

To develop new drugs and vaccines for malaria elimination, it will be necessary to discover biological interventions, including small molecules that act against Plasmodium vivax exoerythrocytic forms. However, a robust in vitro culture system for P. vivax is still lacking. Thus, to study exoerythrocytic forms, researchers must have simultaneous access to fresh, temperature-controlled patient blood samples, as well as an anopheline mosquito colony. In addition, researchers must rely on native mosquito species to avoid introducing a potentially dangerous invasive species into a malaria-endemic region. Here, we report an in vitro culture system carried out on site in a malaria-endemic region for liver stage parasites of P. vivax sporozoites obtained from An. darlingi, the main malaria vector in the Americas. P. vivax sporozoites were obtained by dissection of salivary glands from infected An. darlingi mosquitoes and purified by Accudenz density gradient centrifugation. HC04 liver cells were exposed to P. vivax sporozoites and cultured up to 9 days. To overcome low P. vivax patient parasitemias, potentially lower mosquito vectorial capacity, and humid, nonsterile environmental conditions, a new antibiotic cocktail was included in tissue culture to prevent contamination. Culturing conditions supported exoerythrocytic (EEF) P. vivax liver stage growth up to 9 days and allowed for maturation into intrahepatocyte merosomes. Some of the identified small forms were resistant to atovaquone (1 µM) but sensitive to the phosphatidylinositol 4-kinase inhibitor, KDU691 (1 µM). This study reports a field-accessible EEF production process for drug discovery in a malaria-endemic site in which viable P. vivax sporozoites are used for drug studies using hepatocyte infection. Our data demonstrate that the development of meaningful, field-based resources for P. vivax liver stage drug screening and liver stage human malaria experimentation in the Amazon region is feasible.

Continuous Supply of Plasmodium vivax Sporozoites from Colonized Anopheles darlingi in the Peruvian Amazon.

In vitro culture of Plasmodium vivax liver stages underlies key understandings of the fundamental biology of this parasite, particularly the latent, hyponozoite stage, toward drug and vaccine development. Here, we report systematic production of Plasmodium vivax sporozoites in colonized Anopheles darlingi mosquitoes in the Peruvian Amazon. Human subject-derived P. vivax-infected blood was fed to Anopheles darlingi females using standard membrane feedings assays. Optimizing A. darlingi infection and sporozoite production included replacement of infected patient donor serum with naïve donor serum, comparing anticoagulants in processing blood samples, and addition of penicillin-streptomycin and ATP to infectious blood meals. Replacement of donor serum by naïve serum in the P. vivax donor blood increased oocysts in the mosquito midgut, and heparin, as anticoagulant, was associated with the highest sporozoite yields. Maintaining blood-fed mosquitoes on penicillin-streptomycin in sugar significantly extended mosquito survival which enabled greater sporozoite yield. In this study, we have shown that a robust P. vivax sporozoite production is feasible in a malaria-endemic setting where infected subjects and a stable A. darlingi colony are brought together, with optimized laboratory conditions.