Plasmodium cynomolgi Berok Growth Inhibition Assay by Thiol-reactive Probe Based Flow Cytometric Measurement.

The relapsing malaria species, Plasmodium vivax, is the most widely distributed and difficult-to-treat cause of human malaria. The merozoites of P. vivax preferentially invade ephemeral human CD71+ reticulocytes (nascent reticulocytes), thereby limiting the development of a robust continuous culture in vitro. Fortunately, P. vivax's sister species, P. cynomolgi Berok, can be cultured continuously, providing the ability to screen novel therapeutics drug and vaccine candidates in a reliable and high-throughput manner. Based on well-established growth inhibition activity (GIA) assays against P. falciparum and P. knowlesi, this protocol adopts the current flow cytometry assay methodology and investigates P. vivax inhibitory antibodies using the P. cynomolgi Berok invasion model based on the thiol-reactivity and DNA abundance of viable parasites in macaque erythrocytes. Established GIA assays screen antibodies at either a single concentration or high/low dose concentrations to provide quick insights for prioritizing potential antibodies capable of specifically interrupting parasite ligand and host receptor binding with minimal concentrations. Hence, this protocol expands on the existing GIA assay by using serially diluted antibodies and generating a dose-response curve to better quantify the inhibitory efficacy amongst selected vaccine candidates.

A Chimeric Plasmodium vivax Merozoite Surface Protein Antibody Recognizes and Blocks Erythrocytic P. cynomolgi Berok Merozoites In Vitro.

Research on erythrocytic Plasmodium vivax merozoite antigens is critical for identifying potential vaccine candidates in reducing P. vivax disease. However, many P. vivax studies are constrained by its inability to undergo long-term culture in vitro Conserved across all Plasmodium spp., merozoite surface proteins are essential for invasion into erythrocytes and highly expressed on erythrocytic merozoites, thus making it an ideal vaccine candidate. In clinical trials, the P. vivax merozoite surface protein 1 (PvMSP1-19) vaccine candidate alone has shown to have limited immunogenicity in patients; hence, we incorporate the highly conserved and immunogenic C terminus of both P. vivax merozoite surface protein 8 (PvMSP8) and PvMSP1-19 to develop a multicomponent chimeric protein rPvMSP8+1 for immunization of mice. The resulted chimeric rPvMSP8+1 antibody was shown to recognize native protein MSP8 and MSP1-19 of mature P. vivax schizonts. In the immunized mice, an elevated antibody response was observed in the rPvMSP8+1-immunized group compared to that immunized with single-antigen components. In addition, we examined the growth inhibition of these antibodies against Plasmodium cynomolgi (Berok strain) parasites, which is phylogenetically close to P. vivax and sustains long-term culture in vitro Similarly, the chimeric anti-rPvMSP8+1 antibodies recognize P. cynomolgi MSP8 and MSP1-19 on mature schizonts and showed strong inhibition in vitro via growth inhibition assay. This study provides support for a new multiantigen-based paradigm rPvMSP8+1 to explore potential chimeric vaccine candidates against P. vivax malaria using sister species P. cynomolgi.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

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.

Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection.

OBJECTIVE: HCV infection affects millions of people worldwide, and many patients develop chronic infection leading to liver cancers. For decades, the lack of a small animal model that can recapitulate HCV infection, its immunopathogenesis and disease progression has impeded the development of an effective vaccine and therapeutics. We aim to provide a humanised mouse model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. DESIGN: Recently, we have established human liver cells with a matched human immune system in NOD-scid Il2rg(-/-) (NSG) mice (HIL mice). These mice are infected with HCV by intravenous injection, and the pathologies are investigated. RESULTS: In this study, we demonstrate that HIL mouse is capable of supporting HCV infection and can present some of the clinical symptoms found in HCV-infected patients including hepatitis, robust virus-specific human immune cell and cytokine responses as well as liver fibrosis and cirrhosis. Similar to results obtained from the analysis of patient samples, the human immune cells, particularly T cells and macrophages, play critical roles during the HCV-associated liver disease development in the HIL mice. Furthermore, our model is demonstrated to be able to reproduce the therapeutic effects of human interferon alpha 2a antiviral treatment. CONCLUSIONS: The HIL mouse provides a model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. It could also serve as a platform for antifibrosis and immune-modulatory drug testing.