A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages

Abstract

BackgroundPlasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available.ResultsHere, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite.ConclusionsThe α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Protozoan persister-like cells and drug treatment failure

“…A miniaturized culture system of P. vivax-infected primary human hepatocytes has offered new models to study liver stage infection 28,32 and can be used for high-content imaging-based assays to identify new hypnozoiticidal drugs. Immunodeficient mice, engrafted with human hepatocytes, have enabled the study of P. vivax liver stages 33 , including the elucidation of the hypnozoite maturation process, which enabled the testing of putative hypnozoiticidal drugs, and the identification of UIS4 (upregulated in infectious sporozoites protein 4) as a liver-specific marker for hypnozoites 33,34 However, transcripts that are found only in hypnozoites and not other liver stages were not identified 31 .…”

An adaptable soft-mold embossing process for fabricating optically-accessible, microfeature-based culture systems and application toward liver stage antimalarial compound testing

“…Subcellular analysis of LS parasite morphology within μHEP cultures was performed by fixation with 4% paraformaldehyde followed by two PBS washes and primary antibody staining overnight at 4°C. Primary antibodies were diluted into dilution buffer (formulation above) as follows: 1 : 1000 rabbit anti-upregulated in infectious sporozoites-4 (UIS4) polyclonal antibody purified from immune serum 42 or 1 : 25 000 purified mouse anti-UIS4 recombinant IgG, 43 and 1 : 1000 mouse anti-glucose related protein, binding protein (GRP-BiP) monoclonal IgG. 44 Primary antibody stained cultures were washed thrice with PBS and secondary stained with 2 μg mL −1 Alexafluor 488®-labeled goat anti-rabbit IgG and 2 μg mL −1 Alexafluor 568®-labeled goat anti-mouse IgG as described above, and simultaneously counterstained with Hoechst as described above.…”

8-Aminoquinoline Therapy for Latent Malaria

“…Tremendous progress in systems that may offer that screening capacity has very recently been made, and such systems provide a means of quantifying the synergy of 8-aminoquinolines by any given partner therapy (Table 9) (101,102,(307)(308)(309)(310)(311)(312)(313)(314)(315). Hepatic cellular systems supporting the infection and development of schizonts and hypnozoites may bring the apparently essential involvement of cytochrome P-450 metabolism of the agents under evaluation.…”