Plasmodium vivax malaria is a neglected tropical disease, despite being more geographically widespread than any other form of malaria.
Plasmodium vivax malaria is a neglected tropical disease, despite being more geographically widespread than any other form of malaria , and causes 132–391 million clinical infections each year . Compared to P. falciparum, P. vivax has a broader temperature tolerance and an earlier onset of gametocyte development, enabling the parasites to spread through diverse climates  and making them more difficult to control and eliminate . Currently, there is no vaccine available for P. vivax, though several preventative medications have been shown to be effective . The epidemiology of P. vivax malaria is further complicated by the pathogen’s unique ability to form dormant-stage hypnozoites in the host liver cells, giving rise to recurrent relapse infections from weeks/months to years later . Relapse infections have substantially impacted progress in malaria control, especially in countries that are approaching elimination .
Plasmodium vivax was previously thought to be rare or absent in Africa because people of African descent often lack the expression of a Duffy blood group antigen, known as the Duffy antigen–chemokine receptor (DARC). It is believed that the fixation of the Duffy negativity trait, and the rarity of P. vivax infection in Africa supports that Duffy-negative individuals are refractory to P. vivax. Unlike P. falciparum, which utilizes multiple erythrocyte receptors for invasion and has merozoite proteins with overlapping and redundant receptor-binding functions, invasion of erythrocyte by P. vivax merozoites exclusively relies on the interaction between PvDBP and DARC expressed on the surface of erythrocytes and reticulocytes. DARC is a glycoprotein on the surface of red blood cells (RBCs) that allows P. vivax to bind and invade human erythrocytes at the cysteine-rich region II of Duffy Binding Protein 1 (DBP1) . However, recent studies have reported several cases of P. vivax in Duffy-negative people in different parts of Africa where Duffy-negative populations are predominant . It is apparent that Duffy-negative individuals are no longer resistant to P. vivax malaria . This phenomenon raises important questions of how P. vivax invades erythrocytes of Duffy-negative individuals. To date, only a single P. vivax ligand protein PvDBP1 has been studied in great detail . It has been hypothesized that either mutations in PvDBP1 provided a new pathway of entry, or a low expression of DARC in Duffy-negative individuals binds readily with parasites that contain high copies of PvDBP1 . Recent studies have shown that, despite several mutational differences observed in PvDBP1 between Duffy-positive and Duffy-negative infections, none of them bind to Duffy-negative erythrocytes , implying that an alternative parasite ligand is being used.
The investigation of erythrocyte invasion mechanisms in P. vivax could be complicated by the genetic characteristics and epidemiology of P. vivax in Duffy-negative individuals. P. vivax has a significantly higher nucleotide diversity at the genome level, compared to P. falciparum . Such a contrast could be attributed to frequent gene flow via human movement, intense transmission, and variation in host susceptibility . Genes associated with erythrocyte binding, such as Duffy binding protein (PvDBP), erythrocyte binding protein (PvEBP), reticulocyte binding protein (PvRBP), merozoite surface protein (PvMSP), apical membrane antigen 1 (PvAMA1), and tryptophan-rich antigen genes (PvTRAg) families, are highly diverse in P. vivax from Africa and Southeast Asia . These genes have been shown to play a role in reticulocyte invasion  and patient antigenicity  and provide explanations to high levels of selection detected at the genome levels in P. vivax from South Korea , Kyrgyz Republic , New Guinea , and Thailand . Proteins such as RBP, TRAg, anchored micronemal antigen (GAMA), and Rhoptry neck protein (RON) have been suggested to play a role in red cell invasion, especially in low-density infections . Unfortunately, studies that investigated erythrocyte invasion pathways are scattered with no definitive evidence and systematic approaches to clarify the exact role of these target genes. Due to a lack of reliable and logistical long-term in vitro methods , P. vivax remains a parasite for which it is difficult to effectively study the molecular mechanisms and biology in detail, beyond genetic characterizations.
Recent findings of P. vivax cases in Duffy-negative individuals suggest that some lineages may have evolved to use ligands other than Duffy for erythrocyte invasion . This significantly increases the risk of P. vivax infection in the African populations and may eventually become a new cause of epidemics and severe disease across Africa. To establish how the phenomenon of P. vivax infection of Duffy-negative individuals has evolved and identify potential vaccine candidates to target it, it is important to understand how this parasite invades Duffy-negative erythrocytes and, hence, causes malaria. The investigations of P. vivax at the cellular and molecular levels have been restricted by the lack of a continuous in vitro culturing of live parasites. With the advancement in P. vivax genome sequencing technology, coupled with the ability to mature ex vivo isolates, it is now possible to obtain high-quality transcriptomes of the blood stages. However, there is still a lack of viable methods to indefinitely culture P. vivax, due to the need for young reticulocytes to sustain long-term culture. Strategies to overcoming this problem have been proposed but remain impractical due to a large initial and continuous investment of labor and infrastructure . The successes of short-term culture utilizing young reticulocytes from placental blood  and indefinite culture in Saimiri boliviensis and Aotus nancymae monkeys  shed light on pathogenesis in humans and potential ligands for invasion ; however, several unanswered questions remain.
While mature asexual P. vivax and its transmissive gametocytes occur in peripheral blood, histological analyses of P. vivax in Aotus and Saimiri monkeys have shown immature gametocytes and few asexual schizonts present in the parenchyma of bone marrow . Asexual schizonts appear to be more concentrated in the sinusoids of the liver , suggesting that bone marrow could be a critical reservoir for P. vivax gametocyte development and proliferation. Indeed, the bone marrow reservoirs may suggest that microscopic detection is not ideal for active case detection and treatment of P. vivax until bone marrow samples are accessible. As P. vivax requires reticulocytes for growth , the general low proportion of reticulocytes (that make up only 1% of the total number of host erythrocytes) may explain low parasite loads in symptomatic patients  and a lack of observable schizonts in blood circulation . Additionally, pathological analyses of S. boliviensis tissues showed that P. vivax infections also affect the lungs and kidneys, both of which had mononuclear infiltrates, higher macrophage levels, alveolar wall thickening, collagen deposition, and type II pneumocyte hyperplasia . The level of tissue damage is parasite-load dependent and determined by the amount of by-product, namely hemozoin, being produced . These findings may imply a large number of asymptomatic P. vivax carriers in the general populations. It is well-known that P. vivax has the ability to relapse from dormant liver-stage hypnozoites, from weeks to years after the clearance of the primary blood-stage infection, and this is a major obstacle to its control and elimination . The liver and bone marrow have been shown to be major parasite reservoirs for P. vivax hypnozoites in Saimiri monkey models , but mechanisms of hypnozoite development remain largely unknown and are difficult to study due to a lack of long-term in vitro culture. Moreover, relapse varies systemically by geographic region and/or seasonal changes in the environment . In regions where P. vivax transmission is intense and stable, relapse is common and enhances local transmission , whereas, in Africa, P. vivax transmission is relatively low and usually seasonal and unstable . The rate of relapse is largely unknown. There is, as of yet, no information on the frequency and clinical impacts of relapse in Duffy-negative P. vivax infections, nor reliable biomarkers for relapse detection, due to limited technologies and substantial knowledge gaps in the biology of P. vivax hypnozoites and relapse. Future investigations employing a longitudinal study design that monitors the dynamics and consequences of relapse infections in both Duffy-positive and Duffy-negative individuals will offer deep insights into the epidemiology and biology of P. vivax infections.
Our current knowledge of the molecular mechanisms of erythrocyte invasion in several Plasmodium species offers a reference model on candidate invasion ligands in P. vivax. Plasmodium falciparum invades a wide range of red blood cells, from young reticulocytes to mature normocytes. One of the main binding protein ligands is the erythrocyte binding ligand (EBL) family, which includes multiple members, such as EBA-175, EBA-140, EBL-1, and EBA-181. EBA-175 binds to the sialic acid–containing structure on human erythrocyte receptor glycophorin A (GpA) during invasion . The role of the EBA-175 protein has been shown to be critical for erythrocyte invasion, as antibodies raised against EBA-175 prevent binding to GpA in vitro . EBA-175 triggers changes in the erythrocyte membrane , and the shedding of EBA-175 causes uninfected red blood cells to cluster or form rosette, which allows for immune evasion . The host immune responses may explain the polymorphisms and diversifying selection observed in EBA-175 . Other ligands, such as EBA-140 and EBL-1, are known to bind to glycophorin C (GpC)  and glycophorin B (GpB), respectively, on the erythrocytes. Unlike GpA and GpC, the GpB exhibits high levels of polymorphisms, particularly in people of African ancestry, suggesting that a strong selective pressure may have provided an evolutionary advantage to parasite invasion . For example, the S-s-U- and Dantu GpB phenotypes both showed moderate protection against invasion; however, this does not hold true for all GpB phenotypes . To the best of our knowledge, the specific receptor for EBA-181 is chymotrypsin-sensitive, trypsin-resistant, and neuraminidase-sensitive to erythrocytic treatment , although it remains to be identified.
Another important binding protein family of P. falciparum is the reticulocyte-binding homologue (PfRh) that includes PfRh1, PfRh2a, PfRh2b, PfRh4, and PfRh5. PfRh1 binds to an unidentified receptor “Y”, which has been characterized to be trypsin- and chymotrypsin-resistant and neuraminidase-sensitive . PfRh1 is necessary for sialic acid–dependent invasion of human red blood cells . Antibodies raised against PfRh1 have been shown to block invasion by inhibiting calcium signaling in the merozoite . PfRh2a and PfRh2b are identical for much of the N-terminus region, but each has a unique 500 C-terminus region  and differential expressions in various P. falciparum lines, including deletions, such as a deletion of PfRh2b in P. falciparum D10 . The loss of PfRh2b does not appear to impact invasion or growth of the parasites and suggests compensatory mechanisms for the loss of PfRh2b . PfRh2a binds to more than one receptor on erythrocytes, but these receptors have yet to be identified . PfRh2b has been shown to be involved in merozoite calcium signaling . It binds to an unknown receptor “Z” on erythrocytes, which is neuraminidase- and trypsin-resistant and chymotrypsin-sensitive . PfRh4 has been shown to have sialic acid–independent binding activity with the complement receptor type I (CR1) on erythrocytes . The PfRh5 complex is composed of PfRh5, Ripr, CyRPA, and Pf113, which collectively promote successful merozoite invasion of erythrocytes by binding to basigin (BSG, CD147) . A BSG variant on erythrocytes, known as Oka-, has been shown to reduce merozoite binding affinities and invasion efficiencies . This variant was reported so far only from people of Japanese ancestry . Previous knockout or double-knockout experiments have indicated that the EBL and PfRh gene families work cooperatively or can functionally compensate for the loss of each other . For example, a loss of EBA-175 can activate PfRh4 . When EBA-181 expression was disrupted, PfRH2b was no longer functional . When EBA-181 and EBA-140 genes were disrupted, the parasite deleted the PfRh2b gene . Further study is needed to gain a deeper understanding of how they may work synergistically to promote invasion and immune evasion.
Until recently, P. knowlesi was considered primarily a simian malaria that infects Macaca fascicularis, Macaca nemestrina, and Presbystis melalophos . P. knowlesi is now confirmed to cause malarial infections in humans . P. knowlesi has been shown to use different ligands to invade macaques and human erythrocytes . Two gene families, DBL and RBP, are responsible for erythrocyte binding. The DBL gene family comprises PkDBP-α, PkDBP-β, and PkDBP-γ. In humans, the parasite ligand responsible for erythrocyte invasion is PkDBP-α, which binds to the DARC receptor. The other two Duffy-binding proteins, PkDBP-β and PkDBP-γ, bind only to macaque but not human erythrocytes . The normocyte-binding protein Xa (NBPXa) is required for binding in human erythrocytes, but it is not necessary for invasion of Macaca mulatta erythrocytes . Variation in PkNBPXa has been shown to be linked with parasite virulence and severity of disease . The receptors for NBPXa and NBPXb necessary for invasion for either human or M. mulatta erythrocytes have yet to be identified . Unlike P. vivax, both P. falciparum and P. knowlesi can be maintained in long-term culture, making them ideal systems for studying invasion mechanisms .
P. cynomolgi is a vivax-like simian malaria that shares many genomic and phenotypic characteristics with P. vivax and has been often used as a reference model of P. vivax . Two gene families, erythrocyte binding-like (EBL) and reticulocyte binding-like (RBL), are responsible for erythrocyte binding and invasion in P. cynomolgi . The EBL gene family encodes PcyDBP-1 and PcyDBP-2, similar to PkDBP, which binds to the complementary DARC receptor on Duffy-positive erythrocytes. PcyDBP-1 is an ortholog for PkDBP-α, while PcyDBP-2 has no known orthologs with other Plasmodium DBPs . Previous studies have shown no variation in gene copy number of either PcyDBP-1 or PcyDBP-2 among P. cynomolgi laboratory strains . Studies of field isolates of both P. cynomolgi and P. knowlesi have shown that PcyDBP-1 exhibits high levels of nucleotide diversity, as compared to PcyDBP2 or PkDBPs . The RBL gene family is composed of PcyRBP1, PcyRBP1a, PcyRBP1b, PcyRBP2a, PcyRBP2b, PcyRBP2c, PcyRBP2d, PcyRBP2e, PcyRBP2f, and PcyRBP3, most of which are responsible for mediating parasite invasion into reticulocytes . Functional studies of PcyRBPs are further complicated, as different strains of P. cynomolgi have a different set of RBL genes. For example, PcyRBP2a is present in the P. cynomolgi B and P. cynomolgi Cambodian strains but absent in the P. cynomolgi Berok strain. Similarly, PcyRBP1b is present in the P. cynomolgi Berok and Gombak strain but absent in the P. cynomolgi B, Cambodian and Rossan strain . While it is possible that the loss of PcyRBP1b can be compensated by the presence of PcyRBP2a , the relative role of PcyRBP1b and PcyRBP2a in RBC invasion requires further investigations .