Study of the biology of the sporozoite in the invasion of the vector salivary glands and human hepatocytes in vivax malaria

Malaria is still one of the most important infectious diseases worldwide today. In Brazil, P. vivax is responsible for 83% of cases of the disease, making it the most prevalent species of plasmodium in the country. Transmission of malaria to humans occurs through inoculation of the sporozoite stage by mosquitoes of the genus Anopheles. For this transmission mechanism to be successful, the fixation of sporozoites in the basal lamina of the salivary glands, invasion of glandular cells, followed by migration to the vector’s secretory cavity are essential. Several studies have revealed that several sporozoite proteins are important for salivary gland invasion. Recently, it was found that Roptria neck protein 2 (RON-2) is located in the rhoptry of P. berghei sporozoites (rodent plasmodium), playing an important role in the invasion of salivary glands, in addition to possibly playing an important role in the infection of hepatocytes, an essential step to begin the disease cycle in humans. It is also known that for this invasion of hepatocytes to be successful, it requires signaling by two secondary messengers responsible for sporozoite motility: cGMP-dependent protein kinase (PKG) and calcium-dependent protein kinase 4 (CDPK4). Thus, after invasion of hepatocytes, sporozoites can differentiate and begin the replication process, transforming into a hepatic schizont or the latency stage of the parasite, hypnozoites. Few drugs have the ability to eliminate these hepatic stages of the cycle; one of them being primaquine and, more recently, its potential substitute, tafenoquine, which is already being used in some health units in Brazil, for the treatment of vivax malaria. However, both drugs can be toxic to individuals deficient in the G6PD enzyme, which leads to the search for new drugs for this group, as well as pregnant women, where treatment must be exclusively with chloroquine. To this end, after consent via ICF, blood from patients positive for P. vivax will be collected at FMT-HVD, Manaus, Amazonas. These will be used to artificially feed the species An. darlingi for the production of sporozoites, which will be used: to verify the expression of the PvRON-2 protein in P. vivax sporozoites (1) through IFI, where they will be incubated with different concentrations of polyclonal antibodies obtained after immunization of animals against PvRON2 and Western Blot, where the extracted protein content will also be incubated with the antibody and finally mass spectrometry to verify the intensity of the band. To evaluate the role of the PvRON-2 protein in the invasion of P. vivax sporozoites into the salivary glands (2) and hepatocytes (3), invasion assays will be performed in the presence and absence of the anti-PvRon-2 antibody. Finally, to select compounds with a blocking action on the production of hepatic schizonts and hypnozoites, promising drugs with schizonticidal/hypnozoiticidal action (inhibitors of the CDPK4 protein) will be tested by adding sporozoites to hepatocyte cultures. Therefore, this study aims to elucidate new targets for vaccine production as well as select new candidate compounds for radical cures for P. vivax, fundamental tools for controlling and eliminating the disease.

Project details

Basic Science
P. vivax

Jan 2023

Brazil