Development of platforms to dissect interactions between the transcriptional networks of chronic viral hepatitis B and Plasmodium parasite co-infections in the liver

Abstract

espite significant progress in the last decade, malaria remains an important and difficult global health challenge, with over 438,000 deaths in the last year alone. Five species of the Plasmodium parasite, transmitted by the female Anopheles mosquito vector, cause malaria in humans. Most regions affected by malaria are also highly co-endemic with hepatitis B virus (HBV) infections, which affect over 240 million people worldwide. Malaria parasites and HBV both infect the liver, specifically at the hepatocyte level which serves as the obligate site for all human Plasmodium liver stage development and the unique host reservoir for HBV. Despite their high co-endemicity, little is known how these hepatotropic pathogens interact at the transcriptomic level or collectively influence host response and disease severity. Research efforts have been thwarted by a lack of reliable models that physiologically recapitulate and support hepatotropic infection in vivo. To this end, the Ploss Lab has developed the novel human liver chimeric FAH-/- NOD-Rag1-/-/IL-2Rγnull (FNRG) mouse model to study co-infections of the different human Plasmodium species and HBV. Given the tremendous logistical challenges with obtaining malaria parasites, we first sought to develop the scientific infrastructure for infecting FNRG mice with Plasmodium species. Here, we demonstrate established pipelines and procedures for dissecting mosquito salivary glands to obtain Plasmodium falciparum sporozoites, for infecting mice via live feeding of Plasmodium vivax-infected mosquitoes in Iquitos, Peru, and for quantifying relative parasite abundance of Plasmodium malariae and Plasmodium ovale in coinfected patients in Blantyre, Malawi. Furthermore, we demonstrate that the FNRG model is capable of supporting high levels of persistent HBV infection, which is a requisite for subsequent analysis of hepatotropic co-infections. In addition, to successfully visualize infection, we have developed indirect immunofluorescence assays for Plasmodium and HBV antigens and applied the fluorescent lipophilic dye DiR for tracing infection in vivo. Lastly, we have optimized a laser capture microdissection protocol to extract small numbers of cells for subsequent RNA quantification. These platforms, encompassing the diagnosis of malaria patients, generation of mosquitoes, development of sporozoites, infection of mice, quantification and visualization of viremia and parasitemia, and isolation of infected cells, demonstrate the vast potential of this framework toward the ultimate aim of characterizing the transcriptomic networks of malaria and HBV co-infections in the human liver.