Advancing Key Gaps in the Knowledge of Plasmodium vivax Cryptic Infections Using Humanized Mouse Models and Organs-on-Chips

Abstract

Plasmodium vivax is the most widely distributed human malaria parasite representing 36.3% of disease burden in the South-East Asia region and the most predominant species in the region of the Americas. Recent estimates indicate that 3.3 billion of people are under risk of infection with circa 7 million clinical cases reported each year. This burden is certainly underestimated as the vast majority of chronic infections are asymptomatic. For centuries, it has been widely accepted that the only source of cryptic parasites is the liver dormant stages known as hypnozoites. However, recent evidence indicates that niches outside the liver, in particular in the spleen and the bone marrow, can represent a major source of cryptic chronic erythrocytic infections. The origin of such chronic infections is highly controversial as many key knowledge gaps remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Due to ethical and technical considerations, working with the liver, bone marrow and spleen from natural infections is very difficult. Recent advances in the development of humanized mouse models and organs-on-a-chip models, offer novel technological frontiers to study human diseases, vaccine validation and drug discovery. Here, we review current data of these frontier technologies in malaria, highlighting major challenges ahead to study P. vivax cryptic niches, which perpetuate transmission and burden.

Keywords: Key gaps in the knowledge; Plasmodium vivax; humanized mouse; models; organs-on-a-chip.

Figure 1

Life cycle of Plasmodium vivax highlighting new cryptic erythrocytic stages. During a blood meal, malaria-infected mosquitos inject sporozoites which after reaching the bloodstream enter hepatocytes initiating the pre-erythrocytic cycle. Within the liver, P. vivax either differentiates (i) into a dormant stage called a hypnozoite which, upon reactivation, causes clinical relapses, or (ii) into tissue schizonts, which after thousands of mitotic replications in membranous sacks known as merosomes, release merozoites into the bloodstream initiating the erythrocytic cycle. In this cycle, P. vivax merozoites predominantly, if not exclusively, invade reticulocytes starting asexual blood stage differentiation of rings, trophozoites, schizonts and egress to invade new red blood cells. This cyclical developmental process takes about 48 h. In addition, P. vivax produces specific proteins to create caveola–vesicle complexes that appear as profuse speckling in Giemsa-stained blood smears, known as Schüfnner’s dots. Moreover, some P. vivax parasites can differentiate into mature gametocytes before a clinical infection and illness develops, thus having the advantage of continued transmission to the insect vector before the appearance of clinical symptoms and subsequent treatment. Remarkably, presence of parasites in the spleen and the bone marrow represents novel cryptic erythrocytic infections that need to be incorporated in the life cycle of this species (boxed). Infections of these organs can be either directly from invasion of merozoites into the reticulocyte-rich bone marrow and spleen, or by infected-reticulocytes in peripheral blood. Circulating gametocytes are rounded shape and on uptake in the blood meal of anopheles mosquitoes begin the sexual cycle. This includes release of the male and female gametes, fertilisation, and formation of a motile ookinete that crosses the midgut epithelium. Differentiation into a new replicative form known as the oocyst, release of sporozoites, migration, and invasion of the salivary glands ends this complex life cycle in which the parasite undergoes more than ten stages of cellular differentiation and invades at least four types of cells within two different hosts. (Created with BioRender.com by Carmen Fernandez-Becerra).

Figure 2

Plasmodium falciparum and Plasmodium vivax life cycle together with the currently available immunodeficient mouse models to study the different parasite stages. Models available to study liver stages are Alb-uPA, FRG (N) huHep, TK-NOG and AFC8 model (this last one has not already been tested for Plasmodium infections). Models to study blood stages are FRG (N) huHep model with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection) administration; NSG, NOG and NRG models engrafted with human HSCs and treated with exogenous human cytokines or directly administered with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection); NSGW41 (or NBSGW) and MISTRG models engrafted with human HSC (this last one has not already been tested for Plasmodium infections). Models to study sexual stages are DRAG model engrafted with human HSC, FRG (N) huHep model with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection) administration, NSG, NOG and NRG models engrafted with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection), NSG model engrafted with human HSC, treated with Clo-Lip and NIMP-R14 to deplete remaining murine neutrophils. *Clo-Lip treatment improves huRBCs engraftment/development. Human hepatocytes (huhep); human red blood cells (huRBC); human reticulocytes (huRetics); hematopoietic stem cells (HSC); Clodronate liposomes (Clo-Lip); Plasmodium falciparum (Pf); Plasmodium vivax (Pv); albumin-urokinase-type plasminogen activator (Alb-uPA); Knock-out (KO); FAH, Rag2^-/- and IL2rγ^null (FRG); thymidine kinase NOG (TK-NOG); NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NGS); NOD.Cg-Prkdc^scid Il2rg^tm1Sug/JicTac (NOG); NOD.Cg-Rag1^tm1Mom Il2rg^tm1Wjl/SzJ (NRG); C;129S4-Rag2^tm1.1Flv Csf ^tm1(CSF1)FlvCsf2/Il3^{tm1.1(CSF2,IL3)Flv} Thpo^{tm1.1(TPO)Flv} Il2^gtm1.1Flv Tg(SIRPA)1Flv/J (MISTRG); NOD.Cg-Kit^W−41J Prkdcscid Il2rg^tm1Wjl/ WaskJ (NSGW41); HLA-DR4 Rag^-/- IL2rg^-/- NOD (DRAG). (Created with Inkscape by Iris Aparici-Herraiz).

Figure 3

Organs-on-a-chip for cryptic infections in malaria research. Microfluidic system connecting the minimal functional units of a liver, bone marrow and spleen-on-a-chip on a PDMS support with controlled perfusion rate. Different molecular-design hydrogels can be used for tissue dimensionality, i.e., matrigel, alginate, fibrin/collagen. The small circles show the presence of malaria parasites and extracellular vesicles; the big circles show 3D cultures with illustrative examples of cell types present in each specific organ (Created with BioRender.com by Nuria Sima).