in vitro activity of Artemisia infusions against antimalarial drug resistant blood stage parasites

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Transcriptomics of hypnozoites

To date hypnozoite basic biology, mechanistic information regarding hypnozoite formation, persistence and reactivation are not clearly defined. Understanding gene expressions through genetic manipulation-Omics profiling can largely contribute to understanding hypnozoite biology, identifying master regulators that are responsible for hypnozoite mediated quiescent state in the liver. To unravel the mystery, few research teams relentlessly attempted to the approach of preliminary transcriptomes of cultured P. cynomolgi and P. vivax liver-stage parasites utilizing their unique live liver culture protocols and with the advent of transcriptome tools and reagents. It can be noted that in vitro hepatic cultures of relapsing malarial model, infected with sporozoite species (P. vivax and P. cynomolgi) always exhibit two distinct liver parasite populations (non replicating hypnozoite and replicating multinucleated schizont). However to perform the hypnozoite transcriptomics studies, sorting of those liver stage parasites is obligatory which is not easy to manipulate with the conventional culture setup. After subsequent attempts, three hypnozoite transcriptomics has been done so far, findings are available in literature and data are stored in GenBank.

Transcriptome of relapsing sporozoites

It is hypothesized that the decision of hypnozoite formation is pre-committed from the early onset of liver infection and or most probably preordained or pre-committed from mosquito stages -sporozoites. This conceptual issue is still under investigation and really a good clinical research questions for malariologists. Recently a few transcriptomics of relapsing sporozoites were carried out. One group performed a transcriptome of relapsing sporozoites and compared the data set with the proteomic analysis of P. vivax sporozoites118, 119. Ultimately they observed that the transcripts that were identified in sporozoites but were not detectable in protein level. This could be due to the translational repression. However, authors identified a differential profiling of transcripts between sporozoites and asexual blood parasites including hypnozoite enriched liver parasites. Tightly programmed sporozoite transcriptomic profiling defines a broad spectrum of transcriptional activity but is translationally repressed in schizonts and hypnozoites118.
A groundbreaking step will be to eagerly see if single-cell RNA sequencing with relapsing sporozoites may unravel the long cherished scientific questions regarding the sporozoite role in the context of hepatic dormancy and mechanistic insights of stage specific parasite development inside the mosquito vector.

The association of hypnozoite tubulovesicular network with host water- solute channel

To date, very few organelles structures have been identified in hypnozoites. The previous immunofluorescence assay with cell marker antibodies revealed the presence of structures likely mitochondria, cytosol, apicoplast, parasitophorous vacuole membrane (PVM) in hypnozoite in vitro and also in liver sections of humanized mice.
It has been previously shown in rodent parasite P. berghei, host-parasite interaction in Plasmodium hepatic phase is connected with the structural alteration of PVM upon infection where parasite PVM is able to markedly expand towards the host hepatocytes in a dynamic membranous networking (TVN) with clusters, vesicles and tubules. Tubulovesicular network (TVN), mainly a PVM derived network, existed within the cytosol and previously identified in replicating hepatic schizonts120, 121. However recently utilizing high resolution microscopy, an interesting phenomena was observed for the first time that connects the host water solute channel named aquaporin-3 regulating with tubulovesicular network (TVN) of P. vivax hypnozoites.Most surprisingly TVN features were found to be higher in non-replicating hypnozoites compared to schizonts that could implicate and map the differentiation of these two parasite population thorough further critical understanding how such host regulators like aquaporin contributing hypnozoite formation and playing role in its persistence and in its steady metabolomics122.

Hypnozoite re-activation

Nearly 80-90% malaria cases are reported due to hypnozoite mediated relapses. So understanding the relapse following the hypnozoite activation is a high clinical demand. Although the complete relapse picture is neither demonstrated well in vitro nor in vivo to date. in vitro hepatic cultivation of hypnozoites mimic the hypnozoite persistence where non hypnozoite parasite species e.g. schizonts generally egress from day 6 to day 10 post sporozoite infection. Despite this, we could get a hint of hypnozoite activation by consecutively estimating the ratio of hypnozoite versus schizonts at different time points of hepatic cultures. Such hypnozoite activating concept was described in study where a significant number of hypnozoites were decreased from day 6 (67%) to day 10 (19%) cultures indicating activation of hypnozoite123. In addition, molecular characterization reported a high frequency of multiple-clone P. vivax (more than one genetically distinct parasite) carried by individuals where transmission level differed. Consequently the microsatellite genotyping epitomized that relapses might arise from the activation of parasite clones similar to those of the primary infections (e.g. homologous parasites) or genetically distinct clones (heterologous parasites)124. Overall, processes that recapitulate and activate hypnozoite are yet elusive due to lack of proper biological tools although reactivation of hypnozoite is such an enticing hypothesis.

UIS4 and LISP2 are critical markers of Plasmodium liver stage development: in vitro

Clinically liver residing hypnozoite infection is silent, asymptomatic and undetectable in humans as hypnozoites exist in small numbers. To date, no definitive biomarkers have been detected in humans which made the invasive relapse diagnostic treatment absolutely challenging including the unknown prediction or timing of relapse periodicity.
 UIS4: (Upregulated in infective sporozoite genes 4) mainly positioned to the parasitophorous vacuole membrane, an important organelle between the host and parasite as it separates the parasite from the host cell cytoplasm. The UIS4 encodes a putative transmembrane acidic protein which is transcribed by all malaria species and synteny searching of P. falciparum genome database revealed 74% amino acid sequence identity with P. berghei and P. yoelii.132, 133. However, UIS4 is not critically essential for sporozoite functions such as interaction with vector tissues, motility, gliding. Interestingly UIS4 lacking parasites are able to develop early liver forms that clearly indicates that function of UIS4 is not essential for the conversion of sporozoites to liver trophozoites. To understand whether systematic disruption of UIS4 could lead to blood stage infection. 1000 UIS4 mutant sporozoites were injected in mice who showed no symptoms of infection until 21 days but 59 % of mice were malaria positive when they were infected with high inoculum of 50000 UIS4 mutant sporozoites. The role of UIS4 in blood stage infections remain unknown134. Nowadays, UIS4 is used as a critical marker to identify liver parasites in fixed liver tissue samples both in vitro and in vivo. Hypnozoite possesses a UIS4 prominence in the periphery of parasitophorous vacuole membrane (PVM) and thus possible to detect hypnozoite in culture. As PVM of replicating schizonts can also be marked with anti UIS4 hence UIS4 is not truly hypnozoite specific135.
 LISP 2: Current identification of LISP2 (Liver specific protein 2) facitiliates liver stage Plasmodium studies. Immunolabeling of LISP2 revealed an ability to distinguish non-replicating hypnozoites from early developing liver forms (trophozoites). LIPS2 can be a reliable marker for identification of the reactivating hypnozoites or trophozoites that were derived by the reactivation process of hypnozoite. Interestingly, LIPS2 was found positive to early liver forms/trophozoites/reactivated hypno but donot express in hypnozoite which were positive by UIS4. So LIPS2 showed a differential phenotypic expression in between hypno and reactivated hypno. They confirmed their study in in vitro model of P. cynomolgi and in P. vivax infected humanized mice. Using immunofluorescence assays with anti- PvLISP2, authors observed a major fraction of non-replicating small parasites (hypnozoites) which did not express LISP2 signaling from day 3 to day 18. In contrast trophozoites and mature schizonts clearly expressed LISP2136. Thus LISP2 was proposed as good marker to distinguish dormant hypnozoites from activated hypnozoites that differentiated to developing forms in relapse drug assays.

Hypnozoite biomarkers identified in vivo

Recently a proteomic study revealed the presence of proteins from hypnozoites that circulate in extracellular vesicles. The experimental approach was set up in a humanized mice model that engineered human liver cells inside the mouse liver with a view to mimic human P. vivax infection inside the rodent Thereby authors succeeded to identify 17 different hypnozoite proteins by isolating human derived exosomes from that chimeric mice that might contain host- and parasite-derived proteins and nucleic acids, components of the endocytic pathway and translation machinery and few uncharacterized proteins. These sets of differential proteins can be a predictive marker of latent relapsing infection. However, a detailed investigation is warranted regarding the further biomarker discovery and whether these proteins are precisely specific for Plasmodium vivax liver stage infection rather than P. falciparum137. This informative investigation could largely contribute to developing a non-invasive diagnostic treatment of relapsing malaria.
Recently a clinical cohort study executed in Thailand and Brazil where authors established a panel of serological exposure markers by measuring IgG antibody responses to 342 Plasmodium vivax proteins that capable of accurate prediction of the individuals with recent P. vivax infections that showed strong likelihood of bearing hypnozoites. The unbiased identification was based on patients’ immunogenicity and half-lives of the antibodies that could be targeted by liver stage antimalarials. In general P. vivax blood stage infections induce strong responses of IgG antibodies to a wide range of P. vivax proteins albeit low parasitic burden even after clearance of infection. However, the identified 8 candidate serological markers of exposure can validate their application in especially P. vivax endemic countries. Moreover the authors undertook mathematical modeling for further validation of such serological testing and treatment strategy could reduce P. vivax prevalence by 59–69% and eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy138. Altogether, further investigations of these antibodies are warranted e.g optimization, specificity, purification and testing of these antibodies in other expression systems, antigenicity prediction etc, as there is literature based evidence of cross reactivity of antibody between P. vivax and P. falciparum.

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in vitro and in vivo models facilitates liver stage studies

The first in vitro demonstration of P. vivax and P. falciparum hepatic parasites were shown in a human hepatocytes model146 , 147 . Subsequent attempts were taken to study the Plasmodium liver stage in other cellular models including cell lines117, 123, 148-150 and recently in in vivo humanized models151, 152. Meanwhile, the toolbox of malaria liver stage has evolved. Depsite, hypnozoite activating phenomena was not possible to demonstrate. In fact, studying the entire liver stage of Plasmodium cycle using human parasites under live or laboratory conditions is technically challenging. The use of model organisms, such as the rodent malaria parasite P. berghei is experimentally tractable and the genetic manipulation of this parasite is relatively easy which is already well established. Thus this model allows investigating the EEF development and filling gaps of knowledge that might also be relevant for human Plasmodium species. But genetic makeup of rodent models always does not mimic the human malaria. So as cellular model, cryopreserved primary human hepatocytes are required. Another hypnozoite research limitation is the lack of frequent supply of sporozoites that can be only obtained from relapsing malaria species. Studying P. cynomolgi greatly facilitates in understanding vivax biology and anti-hypnozoite drug screening. Primaquine, the only radical cure drug was also validated with this primate monkey model153. To improve the hepatic infection and for the long term culture mainteance, a sandwich culture was developed. In that approach, simian hepatocytes were seeded into pre-collagen coated plate and the addition of a matrigel cover (a solubilized basement membrane culture substrate, extracted from the Engelbreth-Holm-Swarm mouse sarcoma) greatly facilitated the development of P. cynomolgi exo-erythrocytic forms for over weeks (40 days) providing significant information of hypnozoite reactivation154.

Artemisia infusions: Traditional panacea and future antimalarial drugs discovery aspects

For many decades, the folk medicine either water (infusions or decoctions) or hydro-alcoholic (tinctures) extracts are frequently practiced to benefit from the naturally healing properties offered by plants. Considering the incredible health benefits from such botanical extracts, medicinal plants can offer a wide range of bioactive components (e.g. polyphenols) and can be explored to treat infectious diseases. Artemisia infusions from which the frontline antimalarial artemisinin (ART) is extracted have been traditionally used to treat malaria like symptoms since centuries. Such traditional practices are still included in Chinese pharmacopoeia and also widely popular in a part of Africa subcontinent. The ethnobotanical practice includes the use of whole plant decoction for the treatment of malaria, cough, chill like fever and catarrh. Thus there has been a growing interest in the possible benefits of Artemisia infusions in particular areas where conventional drug therapies are not readily accessible or are too expensive to purchase. In fact the whole flowering plant is documented as anthelmintic, antipyretic, antispasmodic, antiseptic, tonic as well as stomachic260. As mentioned above, A. annua contains artemisinin, which provides a structural chemical base for combinatorial treatment therapy for worldwide antimalarial programs. Moreover research studies also reported that artemisinin is effective for killing human breast cancer cells. Therefore, isolation and characterization of artemisinin has increased the interest in A. annua worldwide. Several ethnobotanical uses in Africa claim that the A. annua tea is also effective against human immunodeficiency virus (HIV). In fact Artemisia infusions are reported effective against human parasitic diseases26.

Scope of anti-hypnozoite compounds fishing from natural resources

Preliminary screening of chosen plants or fractions is generally performed in in vitro models against rodent parasites. When results were obtained, they could be screened on human malaria especially against relapsing malaria. Surprisingly screening of plant derived extracts or metabolites against liver stage of relapsing malaria is totally unprecendented in literature database PubMed after a thorough search of the relevant scientific databases under these key words ‘plant metabolites P. vivax’, natural products, marine products P. vivax.etc. Yet a few plant derived compounds (morphinan) and secondary metabolites (triterpenoids, isolated from Momordica balsamina) were tested against rodent Plasmodium liver stage266, 267 . True anti-relapsing compound is very scarce in the current antimalarial pipeline. Only a bonafide radical cure drug (a drug able to kill the dormant hypnozoites inside the liver during the blood stage infection and treatment) can leverage the gap. Drug screening with natural resources largely conducted in Plasmodium blood stages but recently anti-hypnozoite drug screening has gained momentum and expanded well mainly due to the development of robust hepatic culture models that facilitate culture more than a week. Hence from that perspective, an idea to further explore natural resources for a specific effect (most strikingly for an anti hypnozoite effect) without any clue is not rational.

Table of contents :

Abstract
Chapter 1 General Introduction
2.1 Life Cycle
2.2 Geographical distribution of Plasmodium vivax
2.3 Rising reports of Plasmodium vivax in sub-Saharan Africa: Barrier of malaria elimination agenda
3.1 Discovery of pre-erythrocytic malaria stage
3.2 Unique biology of P. vivax
3.3 Hypnozoite discovery and its distinct features
3.4 Reactivation of hypnozoites and relapse pattern
3.5 Mathematical concept of reactivation kinetics
4.1 8 aminoquinolines, the only anti-relapsing therapies for vivax malaria and limitations
4.2 Proposed mode primaquine action
5.1 Sporozoite: factors contributing hepatocyte permissiveness
5.2 Role of hepatocyte surface proteins in liver infection
5.3 Liver Host molecules accelerate Plasmodium exo-erythrocytic development
6.1 Few research cues of hypnozoites are mentioned below-
6.2 Transcriptomics of hypnozoites
6.3 Transcriptome of relapsing sporozoites
6.4 The association of hypnozoite tubulovesicular network with host water- solute channel .
6.5 Hypnozoite re-activation
6.6 Relapse Pattern
7.1 UIS4 and LISP2 are critical markers of Plasmodium liver stage development: in vitro
7.2 Hypnozoite biomarkers identified in vivo
8.1 in vitro and in vivo models facilitates liver stage studies
8.2 Hepatic models for hypnozoite research: in vitro
8.3 Humanized mice in vivo
8.4 Hepatic organoid model convenient to study relapsing malaria
8.5 Development of reporter lines facilitate understanding hypnozoite features
8.6 Challenges associated with anti–hypnozoite drug discovery
9.1 Drugs identified active against malaria liver stage
9.2 Quantitative high throughput screening facilitates liver stage drug discovery
10.1 Previous drug screening failed to find a radical cure drug
10.3 Necessity of anti-hypnozoite drugs
10.4. Inability of current malaria diagnostic tools to detect hypnozoite mediated relapses from recrudescence and reinfection
10.5 Reactivating drugs: New avenue for relapsing therapy
11.1 Historical perspective
11.3 Scope of anti-hypnozoite compounds fishing from natural resources
11.4 Other natural products
COVID19: can provoke Plasmodium vivax relapse ?
Chapter 2 Supplementation with a cocktail of methyl paraben and penicillin-streptomycin in mosquito diet prevents microbial contamination in hepatic Plasmodium cult MP-PS effect on microbial burden in salivary gland extracts
Chapter 3 Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites
(ARTICLE 1 : Manuscript under revision in BIORXIV )
Chapter 4 Artemisia infusions perturbs the apicoplast biogenesis of liver resident Plasmodium parasites including hypnozoites
Introduction
Chapter 5 in vitro activity of Artemisia infusions against antimalarial drug resistant blood stage parasites
Abstract
Result and Discussion
General Discussion
CONCLUSION & PERSPECTIVE
ANNEX
I. Extracts derived from Artemisia infusions show potent inhibitory activity against rodent malaria species
List of abréviations
RESUME

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