Plasmodium falciparum, with NHP infections, in clinical studies with humans, or using in vitro culture or rodent model systems, others have been dedicated to advancing research on Plasmodium vivax, as well as on phylogenetically related simian species, including Plasmodium cynomolgi, Plasmodium coatneyi, and Plasmodium knowlesi. In-depth study of these four phylogenetically related species over the years has spawned the design of NHP longitudinal infection strategies for gathering information about ongoing infections, which can be related to human infections. These Plasmodium-NHP infection model systems are reviewed here, with emphasis on modern systems biological approaches to studying longitudinal infections, pathogenesis, immunity, and vaccines. Recent discoveries capitalizing on NHP longitudinal infections include an advanced understanding of chronic infections, relapses, anaemia, and immune memory. With quickly emerging new technological advances, more in-depth research and mechanistic discoveries can be anticipated on these and additional critical topics, including hypnozoite biology, antigenic variation, gametocyte transmission, bone marrow dysfunction, and loss of uninfected RBCs. New strategies and insights published by the Malaria Host–Pathogen Interaction Center (MaHPIC) are recapped here along with a vision that stresses the importance of educating future experts well trained in utilizing NHP infection model systems for the pursuit of innovative, effective interventions against malaria.“The Primate Malarias” book has been a uniquely important resource for multiple generations of scientists, since its debut in 1971, and remains pertinent to the present day. Indeed, nonhuman primates (NHPs) have been instrumental for major breakthroughs in basic and pre-clinical research on malaria for over 50 years. Research involving NHPs have provided critical insights and data that have been essential for malaria research on many parasite species, drugs, vaccines, pathogenesis, and transmission, leading to improved clinical care and advancing research goals for malaria control, elimination, and eradication. Whilst most malaria scientists over the decades have been studying  Plasmodium parasites transmitted by Anopheles mosquitoes—has been known since the late nineteenth century [Plasmodium with their ever-changing genetic variation . Once m century ). These iewed in –7). Manyiewed in . Meanwhiiewed in , complemThis article highlights malaria research from early and recent understandings gained from malariotherapy and modern CHMI studies to NHP infection studies and their current utility in support of today’s malaria eradication goals, with emphasis on the value of longitudinal infections and systems biological approaches. This article also pays tribute to the authors of "The Primate Malarias” , 12, as Anopheles mosquito hosts and then be injected into the skin and survive in the liver and blood of humans, NHPs, or other vertebrate hosts. Some parasites must also transform within the blood of the vertebrate host into sexual-stage male and female gametocytes that are infectious for newly biting female Anopheles mosquitoes. The malaria parasite life cycles pose many challenges for both the parasites and hosts in their joint struggle to survive and propagate  comprise between 5000 and 7000 genes [Malaria parasites must successfully develop sequentially in the midgut and salivary glands of female ate Fig. . Throughost RBCs . It is h00 genes –16 versu00 genes .Fig. 1ScPlasmodium infections longitudinally in humans, including the collection of parasitaemia datasets, thereby creating significant knowledge regarding clinical immunity and parasitological features of the disease, albeit in some cases with grave illness and deaths recorded as a result—from Plasmodium falciparum, P. vivax, and Plasmodium malariae infections. Up to 15 malarial paroxysms were observed and monitored in malariotherapy patients. Snounou and Pérignon provide an extraordinary overview of the literature from that time and the numerous retrospective analyses that have followed [From 1917 until 1976, malariotherapy was performed to treat patients with neurosyphilis and progressive paralysis , as malarial infections caused fever, which can activate immune responses (reviewed in ). Over afollowed . Their bfollowed –30, and followed , who volAnopheles mosquitoes, from which enormous parasite-vector knowledge has been gained. Infection with P. vivax was most common. Retrospectively, many overarching conclusions still stand, and these can now be further explored taking into consideration the many since-discovered biological complexities of each parasite species, their Anopheles mosquito vectors, their epidemiology, ecological factors, strain diversity, parasite and host genetics, and the immune systems of either naïve or primed individuals, male or female, young and old  and . patient , now apptibility , 41, thehrocytes , 42–44, hrocytes ).Plasmodium infections from baseline periods to the time of parasitaemia, and helpful for conducting clinical vaccine trials that include sporozoite challenges . In iewed in . CHMI exiewed in , 51, andiewed in –56. The iewed in ). Additiiewed in –61.P. falciparum, and close to a hundred such studies have been performed to date. Following suit, CHMI studies using P. vivax sporozoites have begun to emerge , iniewed in –66. CHMIrozoites –70 and tlunteers  are helplciparum –74, P. vP. vivax , 74–76 amalariae , 78. SucWhile CHMI experiments are generally safe and have demonstrated clear advantages for scientific advancements in understanding malaria, the viewpoints and concerns of participants and their communities and other stakeholders remain important to understand and take into consideration –82.NHP infection models (NHPIMs) have been vital to research on malaria and over 70 human infectious diseases, including many caused by viruses, bacteria and other parasites , 84, andPlasmodium, as elegantly detailed in “The Primate Malarias” [Anopheles mosquito hosts, documenting nuances that distinguish species and strains, and experimental NHP and mosquito host infections. This all helped to set the stage for the use of different host–parasite combinations as model systems for research—whether for drug or vaccine candidate testing, immune response, or pathogenesis studies. The authors described infections of apes and Old World and New World monkeys, the most studied today being macaque (Old World) and Saimiri and Aotus  species. Numerous experiments had been performed to determine whether infections initiated by sporozoites of various species of Plasmodium infect and thrive in the liver and blood, or only the liver, and whether gametocytes were observed. Blood infections were studied including the level of infectivity of the NHP blood to mosquitoes. Numerous publications have followed since, by these authors and others, continuing to shine light on the utility of these model parasite species and the importance of NHP models for malaria research .ussed in , 96, 97)Plasmodium infections in splenectomized animals has also been documented in “The Primate Malarias” [P. falciparum, P. vivax, P. malariae and P. ovale) [Differences in alarias” , 12 and . ovale) –102  ), but th. ovale) .var/SICAvar variant antigen genes and proteins at the surface of Plasmodium-iRBCs, specifically for P. falciparum, P. coatneyi, and P. knowlesi, and likely P. fragile , a subpopulation of P. cynomolgi-iRBCs becomes ‘concealed’ from the circulation [var/SICAvar genes and proteins, compared to others that do not possess comparable genes and proteins. These distinctions are important as they relate to virulence and cytoadhesion and sequestration of iRBCs, or concealment of iRBCs, respectively.Importantly, while splenectomized NHPs have yielded higher parasitaemias of different species for various experimental purposes, and their use has been helpful to ensure the availability of long-term frozen infected RBC (iRBC) stocks for future use, such stocks and splenectomized animals are not advised for immunological and pathology studies seeking to understand the normal course of host–parasite interactions and disease ramifications. Aside from the spleen’s prominent role in removal of damaged RBCs, the spleen has been associated with the expression of parasite virulence factors, namely the iewed in ). Plasmo animals , 105, re animals . Splenic monkeys  and in h monkeys . A studyax genes . Moreoveiewed in ). Intered rhesus . The splculation . In summP. malariae were in fact P. knowlesi zoonoses [P. knowlesi has been documented as a zoonosis throughout most of Southeast Asia and as the main type of human-causing malarial disease in Malaysia [P. cynomolgi has become recognized in the same geographical area as an additional zoonotic parasite of public health concern [P. coatneyi, P. inui, Plasmodium inui-like, and P. simiovale malaria were identified in Malaysia when using molecular methods [Macaca fascicularis and the less-accessible macaque species Macaca nemestrina and Macaca leonina, highlighting their likelihood as well as possible hosts and sources of zoonotic P. knowlesi infections [Macaca arctoides was recently identified as another macaque NHP species that can harbour this simian malaria parasite and be a possible source of zoonotic infections [P. simium, which is genetically and biologically similar to P. vivax [P. brasilianum, which is genetically and biologically similar to P. malariae [P. knowlesi zoonotic infections are the subjects of other papers in this thematic issue.Interestingly, the preface to “The Primate Malarias” noted that this book was timely in 1971 given the recent awareness that monkey malaria parasites can also infect humans , 12. Thizoonoses . Since tMalaysia . More re concern , 116 . R methods . Geograpfections , and it fections . New WorP. vivax , and P. malariae , 122, anMany basic biological and clinically relevant questions can be asked in NHP infection experiments (see Box 1). Longitudinal experiments can be designed without the urgent need to treat, and with repeat collection of multiple immunologically relevant tissues, thus capturing data not typically possible in CHMI studies. A holistic view of the immune response is possible with NHPIMs, especially with the Old World macaque models, including the collection and analysis of key tissues in addition to peripheral blood  and integrated analyses of multiple data types during infections and from necropsies. Parenchymal tissues, secondary lymphoid tissues and the lymphatic/humoral circulation all hold relevant information. Over the past few decades, preclinical vaccine trials using NHP models have helped to test the immunogenicity of vaccine constructs, dosing, delivery platforms, and sometimes protection with parasite challenges. Such experiments have been cost-effective in the vaccine development pipeline. Today, these can be perfected to maximize these benefits.P. falciparum, P. vivax, P. malariae and P. ovale) do not develop in the blood of macaques. Simian malaria parasites, with comparable biology to the human malaria parasite species, are therefore relied upon when using these animal models. However, it is worth noting that while P. falciparum blood-stage parasitaemia does not develop in rhesus macaques, rhesus can be infected with P. falciparum up to the liver stage [M. mulatta, P. coatneyi has become recognized as a model for P. falciparum, P. cynomolgi as a model for P. vivax, and P. knowlesi as a model for both, depending on the research question. The history and prospects for using these species were reviewed recently for P. coatneyi [P. cynomolgi [P. knowlesi in general terms [Old World monkey species are valuable for researching the dynamic mechanistic intricacies of malarial naturally acquired and vaccine candidate-induced immunity (NAI and VCI), as well as gametocytogenesis and transmission. They also hold much potential for studying malarial pathogenesis and immune evasion mechanisms, and they are the preferred NHP species for longitudinal systems biological infection experiments. Significantly, the four known human malaria species  has also been shown to be a model for studying malarial pathology including cerebral malaria [Macaca fuscata) have been shown to be an additional viable NHP model for relapsing cynomolgi malaria [The macaque NHP animal models develop a spectrum of malarial disease manifestations that closely resemble the human disease states caused by either human or simian malaria parasites. For example, s tissue –128. Pla anaemia –131, wit anaemia –134. Thuiewed in ). In conenotypes –138, andenotypes , 140. Exoutcomes –144. Pla malaria  and mala malaria . Recentl malaria . JuvenilHistorically, malaria research involving macaque species has been carried out in the United States, the United Kingdom, The Netherlands, China, Thailand, India, Sri Lanka, and other parts of Southeast Asia. Research with these species is currently prominent in The Netherlands, Thailand, Japan, China, and the United States, where different species of macaques are routinely available through domestic breeding programmes or importation. In the United States, National Primate Research Centers (NPRCs), in addition to governmental laboratories, academic institutions, and private businesses, breed and/or may acquire macaque species for malaria research. Macaque species have been generally available as required for malaria research experiments, although at the time of this writing, their availability has been diminished due to their widespread use in SARS-CoV-2/Covid-19 experiments. This situation is likely to reverse, thanks to the development and testing of effective drugs and vaccines to fight the SARS-CoV-2/Covid-19 pandemic. Currently, as a result, severe Covid-19 illness has been subsiding .Aotus and Saimiri have been critical and will remain important for P. falciparum and P. vivax vaccine candidate trials, drug studies, and research into immune responses and pathogenesis caused by these parasite species, as well as basic research involving simian malaria parasite species  that had been adapted to grow in New Word monkeys and stored as frozen iRBC stabilates are highly relevant today. These show individualized characteristics ranging from frequent early relapsing phenotypes  to late-relapsing phenotypes (North Korean strain) [New World monkeys of the genus iewed in , 150). Tmalariae  and P. oarasites . New Worarasites . Neverth include , 152). Achnology –155. Cri strain) –20.Aotus and Saimiri monkey species are currently available from the Keeling Center for Comparative Medicine and Research at The University of Texas MD Anderson Cancer Center, Texas, USA, which has maintained U.S. National Institute of Health (NIH)-supported New World monkey breeding colonies for medical research purposes, including malaria research. Opportunities for malaria research with New World monkey species also exist in Peru, Panama, Brazil, and Colombia.P. coatneyi, P. cynomolgi, and P. knowlesi sporozoites, from which basic biological knowledge has and continues to be gained regarding host infections initiated with each of these species. This research, conducted by the Malaria Host–Pathogen Interaction Center (MaHPIC), has involved longitudinal Plasmodium infections using macaque species, the most common being M. mulatta (rhesus monkeys) and M. fascicularis .Within this article, following an overview of relevant background information, systems biological studies and experimental frameworks are highlighted involving longitudinal NHP infections initiated with Plasmodium parasite species infecting people. NHPIMs cannot recapitulate all health conditions and situations pertinent to humans, but they can begin to take them on, one or a few at a time; tease them apart, deconvolve large datasets, and reveal hidden biological information. It is becoming increasingly possible to gain a multi-dimensional view of the immune system in action, which is critical for determining: (a) what works to the host’s advantage, resulting in reduction or elimination of parasites, inflammation, and pathology, (b) what works to the host’s detriment, resulting in thriving parasites, illness, and pathology, and ultimately, and (c) what interventions may help favour the host. These three areas of inquiry remain at the forefront today.It is well accepted that the host and parasite influence each other, and host–parasite interactions can set off cascades of molecular events and pathways, both locally and systemically, as each thrives to survive. The simian malaria parasites and their NHP model systems continue to provide unique research opportunities for in-depth investigations, aimed to ultimately inform translational solutions to prevent or treat malaria caused by any of the various P. vivax which became recognized as a widespread but important and neglected species [P. falciparum [P. vivax [P. malariae and P. ovale, as well as simian malaria zoonotic species . Tiewed in , 95, 168iewed in ).Plasmodium species and their transmission dynamics, including ‘within-host’ and involving external factors, operating in a multitude of environments. Indeed, the enormous complexity of this overall system has led to new efforts utilizing mathematical and computational modelling strategies to study the problems and trajectories [Anopheles mosquitoes where fertilization occurs and a new brood of parasites forms.Researchers continue to question how to make the next big leap and meet the demands of malaria eradication , 171. Thectories –174. Topectories , with thectories  that traPlasmodium species, NAI, VCI, and malarial disease processes is critical for the development and testing of broadly effective vaccine strategies. Immunological comparisons between malaria naïve and chronically infected individuals are also important, particularly with regards to predicting and understanding the outcomes of candidate vaccine testing in malaria endemic areas with different transmission patterns and intensities, and when a large percentage of individuals may be repeatedly or chronically infected with malaria parasites and have significant levels of NAI against malaria.Vaccination is considered necessary to ultimately prevent infections and reduce (or eliminate) the global burden of malarial disease in humans (reviewed in ). While P. vivax has gained widespread special attention as a predominant malaria parasite species—with raised public health concerns [P. cynomolgi [P. ovale produce dormant parasite-infected cells in the liver that can stay quiescent for weeks, months or years and then become activated to cause relapsing infections in the blood in the absence of new mosquito infections. Consistent with raised attention on this problem, review articles on hypnozoites and relapses have become frequent [Especially in light of the current malaria eradication agenda, concerns , 178–184ynomolgi , 186. Eafrequent , 187–194P. vivax, P. ovale and P. cynomolgi latent parasites, called hypnozoites [P. vivax parasite biomass [The biology of the nozoites  Fig. 1)P. vivax, biomass , and tre biomass , 197. Hy biomass , 198–200P. cynomolgi) and chimpanzees (infected with P. vivax) [P. vivax [P. cynomolgi [P. cynomolgi in vitro blood-stage cultures [P. vivax [P. cynomolgi [P. vivax revealed parasite-derived membraneous networks in both schizonts and hypnozoites and possible functions of aquaporin-3 [P. vivax sporozoites [What causes these species to go dormant in hepatocytes—seemingly unaffected by the immune system; and what causes the dormant infected cells to reactivate and produce broods of thriving infectious parasites once again capable of causing blood-stage infections? While hypnozoites were discovered almost 40 years ago—in rhesus macaques (infected with . vivax) –203, theP. vivax , 205 andynomolgi –208. Robcultures  have alscultures , 211. HeP. vivax , 212–214ynomolgi . Recentlaporin-3 . This tyaporin-3 , 218. Toaporin-3 , and celaporin-3 , 221 arerozoites  and hostrozoites .P. cynomolgi and P. vivax sporozoites to initiate hepatocyte culture infections. Notably the first hepatocyte culture system to show results involved M. fascicularis and P. cynomolgi. P. cynomolgi blood-stage parasites were grown in macaques to generate gametocytes to feed mosquitoes to develop sporozoites to infect M. fascicularis hepatocyte cultures [P. cynomolgi or P. vivax gametocytes from infected NHP blood for mosquito feeds, and conducting ex vivo experiments with liver tissue samples.NHP models have become an important if not critical resource for enabling ongoing research with hepatocyte cultures. First, they have been vital for establishing gametocytaemia and feeding mosquitoes to generate cultures . More inP. vivax clinical cases declines in endemic areas [P. vivax clinical cases continue to decline, as hoped—logistical challenges will be compounded that include having sufficient availability of patient infected-blood donors, mosquito insectary operations, and experts for establishing, infecting, and analyzing the data coming from hepatocyte cultures. Meanwhile, there is much yet to be learned about the biology of hypnozoites to effectively identify targets for drug intervention, and multi-omic approaches that can distinguish host and parasite targets will be essential [Importantly, as the number of ic areas , the demic areas , 224. Thssential , 223. Wissential , may oneP. vivax malaria and potentially P. ovale—for which less data is available [P. vivax relapses are responsible for over 50% and potentially as high as 95% of blood-stage infections caused by these species [Plasmodium parasites, and recurrent infections. Studying relapsing malaria, caused by the activation of dormant P. vivax hypnozoites, is especially challenging, impractical—and arguably not possible—in human populations, where a main challenge is the difficulty of distinguishing relapse infections from new mosquito-borne infections. There are a few praise-worthy exceptions on the feasibility questions. Two major studies used molecular methods including whole genome sequencing and protein microarray analyses to distinguish new infections from relapses [bona fide relapses [Relapses are the ‘strong suit’ of vailable –228. P.  species , 229. Yerelapses , 231. Otrelapses , 233.P. vivax relapses can also be studied via controlled experimental infections of New World monkeys, thus overcoming the challenge of discerning relapses from new infections in human studies. Monkey-adapted isolates of P. vivax can be capitalized upon that exhibit different relapse patterns .iewed in ). Moreovr humans –117. Crimacaques –137 and macaques , 139 andquitoes , which has domains known to adhere to various endothelial receptors [Plasmodium species for which all developmental iRBC forms circulate. Questions remain regarding what other functions the adhesive PfEMP-1 and related proteins may serve for the parasites [Cytoadhesion of iewed in , 248). Tproteins –254, preeceptors , 256. Wharasites , 277.P. vivax and P. cynomolgi to account for non-circulating iRBCs that do not express EMP-1 related proteins [Plasmodium species, the circulating iRBCs represented in blood smear parasitaemias, do not necessarily represent the entire parasite load, or parasite burden in infected individuals . FP. falciparum gametocytes have been found in the bone marrow, where their maturation takes place [P. vivax gametocytes [P. vivax iRBCs in humans [Aotus and Saimiri monkey NHP infection studies have demonstrated and quantified the burden of P. vivax iRBCs in these tissues relative to others [P. cynomolgi infected M. mulatta, examined to better understand P. vivax infections and anaemia, a computational model was used and concluded that a subpopulation of iRBCs was in fact concealed in the tissues [In the early stages of infection, es place –261, andetocytes . Surprisn humans –265, pren humans ). Aotus o others , 266. Fu tissues . As withPlasmodium knowlesi iRBCs have been identified in the tissues of experimentally infected M. fascicularis and M. mulatta [P. knowlesi parasitization, tissues of the gastrointestinal tract stood out, consistent with gastrointestinal complications in patients with P. knowlesi [P. knowlesi infection of baboons has also resulted in the localization of parasites in vital organs, including the brain and placenta [ mulatta , 277. Inknowlesi , 277. P.placenta , 146.P. falciparum RTS,S candidate vaccine  was approved for widespread use in Africa after showing roughly 30% efficacy in preventing severe malaria in children after four sequential injections [Throughout the 1980s, once a few malaria parasite genes had been characterized, a significant R & D focus developed towards a vaccine mimicking immunogenic regions of the parasite’s immunodominant circumsporozoite protein (CSP), hypothesizing that such a vaccine could prevent sporozoite infection of hepatocytes (reviewed in ). Howeveiewed in , 269–282P. vivax , 284. Rejections , 286. Thjections , 288. Thjections , 289–293Plasmodium species and zoonotic infections. Uncounted lessons have been learned during the roughly 30 years of RTS,S development, testing, delivery/administration, and monitoring phases. While sterilizing immunity—killing all parasites—has yet to be reached in large clinical trials, it must remain the goal as malaria vaccinology work continues alongside research to understand each parasite species’ biology, diversity, antigenic variation, and evasion strategies, as well as host immune responses, including those that are protective and those that may be detrimental and cause pathology. Moreover, differences in responsiveness between malaria naïve volunteers and people living in different malaria endemic areas must be considered . UP. falciparum vaccines [P. vivax Duffy Binding Protein (PvDBP) as a one-shot key prospect immunogen to prevent entry of P. vivax merozoites into Duffy glycoprotein-positive host RBCs  end of the merozoite and is known to specifically attach to the cognate RBC receptor prior to invading the host RBC [P. vivax can also invade Duffy negative RBCs by utilizing alternate invasion mechanisms . Thiewed in  as a rechost RBC , 302–304iewed in , 306).P. falciparum vaccines [P. vivax research has been progressing at a competitive pace, even prior to the genome sequencing era, greatly aided by the use of both Aotus and Saimiri monkey infections and short-term in vitro blood-stage cultures permitting studies on P. vivax from human infected blood samples [P. vivax parasites for genetic and biological analyses  [A. nancymaae monkeys to clinical vaccine trials in humans [P. vivax proteins have been investigated as prospective vaccine candidates in field immunogenicity studies [P. vivax infections in S. boliviensis and A. nancymaae monkeys, thereby pointing to roles in typical or alternative invasion pathways [Discovery of candidate vaccine proteins and testing them continues, predominantly to develop vaccines , 308. No samples , 310. Oviewed in , 20, 311P. vivax –314, as  decades . From a iewed in , 311). Tiewed in , 316–320P. vivax , 322 (reP. vivax ). Importues (Rh) –326 and ues (Rh)  has progn humans –331. Sev studies , 333, anpathways . IdentifP. falciparum vaccine candidate antigens [An overarching challenge in the malaria vaccine development pipeline has been to express recombinant proteins or make peptides that can be representative of high-priority target proteins, structurally and antigenically, and on a population scale. While ongoing studies continue to establish a basis for new directions for malaria vaccine development, they are just the beginning. For example, a recent study assessed the global diversity and population structure of high priority antigens . This anantigens , 335, anantigens –338. TheP. falciparum sporozoite immunogens have shown homologous protection in malaria naïve individuals [Plasmodium infections and malaria. By contrast, P. falciparum sporozoite immunizations along with prophylactic drugs have shown homologous and heterologous efficacy and have been suggested for use by travelers to Africa and potentially widespread use in Africa [P. vivax sporozoite vaccines has yet to be reported. This research has been hampered by the unmet challenge of reliably generating sporozoite immunogens in the absence of blood-stage culture systems that can reliably produce infectious gametocytes for infecting Anopheles mosquitoes. A few studies with sporozoite immunizations concurrent with blood-stage drug cover have been carried out with P. knowlesi [P. cynomolgi in M. mulatta . Thus, n Africa –345. Comknowlesi , 346 andmulatta  responses [P. vivax immunogenicity field studies, raising questions on the origin and role of IgM compared to IgG [In brief, innate immune signaling pathways are activated by parasite-derived materials such as DNA, RNA, glycosylphosphatidylinositol (GPI), and haemozoin, otherwise known as Pathogen Associated Molecular Patterns (PAMPs) or Danger Associated Molecular Patterns (DAMPs) (reviewed in –352). Suesponses –363. Lond to IgG , 365. Ald to IgG .Plasmodium [CHMI experiments have served as an alternative to traditional field studies by attempting to explain the immune response to malaria parasites over time in malaria naïve and non-malaria naïve individuals. For example, one noteworthy observation is that epigenetic imprinting occurs in innate immune cells after exposure to blood-stage parasites in vivo . This isasmodium , 369, anasmodium . It remaA hallmark of acute malaria is an array of inflammatory processes that can cause fever and other symptoms and signs of illness, which result in possible mild to severe damage to the body’s tissues and organs. Inflammatory processes can aid a host in its anti-malarial fight and recovery, but they can also overwhelm the host . NumerouPlasmodium infections are asymptomatic with chronic long-lasting infections that provide a blood-borne reservoir of parasites for transmission to Anopheles mosquitoes and propagation of the disease [The majority of human  disease , 375. Un disease –379. The disease ). Recent disease , 380–382 disease .Future studies using NHPIMs can help advance this critical line of research by determining from which B cell tissue compartments these cells originate, the extent of their persistence, to what cytokines they respond, and to the extent they help or hinder the development of immunological memory responses against the parasites . DelineaPlasmodium infections causing immunosuppression, including the influence of regulatory T cells, B cell populations, and antigen presenting cells [In 2013, Mueller and colleagues reviewed known information relating to ng cells . This tong cells . As immuDecades of immune response studies converge on similar conclusions with regards to the need to understand much more about the intricate host–parasite relationships and immune responses that allow for sustained parasitism , 46–48. P. coatneyi when modelling P. falciparum infection [P. cynomolgi modelling P. vivax infection [or are restricted to achieve) a reduction in parasitaemia and illness over elimination of all parasites, therewith supporting the retention of sexual stage gametocytes and prospects for transmission to mosquitoes.Normal physiology is affected by malarial infections and tissues can become damaged; in severe cases organ damage may be irreparable. These facts are also evidenced from rare cases of irreversible severe illness caused by nfection  and P. cnfection . Key quenfection , 399. Thnfection , 400) annfection ). SpecifSystems-based studies of the immune response and cellular dynamics during longitudinal infections will continue to be informative to help address long-standing questions and arrive at new interventions. While clinical immunity can be generated, resulting in asymptomatic infections, chronic sub-patent blood-stage infections remain the norm in endemic areas. This situation naturally serves the parasite well, allowing for transmission of gametocyte progeny to mosquitoes, but remains a health concern for infected individuals as well as the population at large that presents targets for future bites by mosquitoes and propagation of the disease. If natural inoculation of the parasite itself does not lead to sterilizing immunity, what are the chances that a subunit or whole parasite vaccine would? The often-presumed answer is that sterilizing immunity is unlikely, or an extremely elusive goal , regardlA complete understanding of malaria parasite species, their biology, host–parasite interactions, and population dynamics that are essential for their survival, is still lacking. In 1971, at the time of the publication of “The Primate Malarias”, host–parasite interaction(s) was not common terminology , 405. ThP. vivax CHMI trial to define specific immune responses [P. coatneyi and P. falciparum [P. knowlesi infections in M. mulatta and M. fascicularis, which highlight mechanisms of resilience in the latter species [Whole blood transcriptomics has been a starting point for numerous functional genomics projects. Transcriptomic models—especially across multiple time points from in vivo infection—gain multi-dimensional complexity when epigenetic interactions and time-dependent biological and immunological phenomena or the impact of the microbiota or co-infections are considered as well esponses , and by lciparum . Gupta e species , 413. Sy species . Recentl species . Baselin species . Pre-imm species ). These  species  raise th species , 294). T species , 417.The predictive capabilities, based on mathematical models and the integration of diverse datasets and metadata, support the identification of signature biomarker profiles that may be prognostic or diagnostic, validation experiments that may include perturbations, and in silico screening and testing of candidate drugs and vaccines. Traditional ELISPOT tests, ELISAs, isotype analyses, protein microarrays, and various functional assays  continue to serve a purpose, to validate findings and develop new hypotheses and research directions to delve deeper into the biology of specific cell types and immune networks. Meanwhile, bioinformatic analysis of large and multi-omic datasets, dynamic modelling and spatial imaging technologies are becoming critical for advancing such research, whether on single cells or tissue samples . FThe integration of multiple-omic and other data types is a key goal for designing quasi-realistic computational models for biological processes. In the simplest case, these are static representations of networks that show which components interact with each other. Dynamic modelling raises the bar further by also addressing questions pertaining to the constant changes in biological systems, such as a host cell or organism, whether these occur within seconds, minutes, a day or longer , 419. GeP. falciparum and three rodent malaria parasite species were tackled first, at the turn of the century [Laverania malaria parasite species [P. falciparum causing the most cases of malaria throughout Sub-Saharan Africa, including severe cases and death, and because P. falciparum and corresponding rodent species are recognized as being most widely amenable to research work in laboratories around the world. However, the in vitro culture environment is very different from the in vivo host environment, with all its complex organ physiology, biology, biochemistry, and immune system, and rodent models—while valuable—do not suffice to reveal the host–parasite interactions that occur in humans or NHPs [P. falciparum genomes and robust culture systems, functional genomics studies have become plentiful for P. falciparum for the different parasite development stages throughout the parasite’s life cycle [Genomic and post-genomic technologies have greatly advanced knowledge of malaria parasites of a number of species in various stages of their life cycles. The genome sequences of  century , 423, fo species  plus a f species . This sp or NHPs , 427. Pafe cycle , 428.Plasmodium vivax and P. knowlesi genome sequences were published in 2008 [P. vivax [P. knowlesi [Plasmodium vivax transcriptomes have since become available representing sporozoites [P. knowlesi, importantly showing differences in the parasite’s gene expression between the in vivo NHP environment and in vitro cultures [M. mulatta and M. fascicularis host responses [Plasmodium knowlesi ex vivo stage-specific blood-stage proteomes are under analysis, helping to flesh out the biology of this species . Plasmodium cynomolgi genome sequences were first published in 2010 [P. coatneyi genome sequence was published in 2016 [P. ovale and P. malariae [M. mulatta, M. fascicularis and Aotus and Saimiri species) genome sequences have also been published [Anopheles vector species and ongoing advancements for studying Plasmodium genomics data [ in 2008 , 430, foP. vivax –433 and knowlesi –437 genorozoites , 439, sprozoites , 213 androzoites –448, androzoites –454. Likcultures  as well esponses . Plasmod in 2010 , followe in 2010  and tran in 2010 , 214. Fi in 2016  and in 2malariae , 456. Huublished –463. So,ics data .Plasmodium proteins remain undefined, but expedited progress with biological discoveries are likely given the immense amounts of genomic and functional -omic data that continues to be developed and analysed. Technological tools and capabilities have advanced considerably to obtain genomic sequences with improved quality and much more readily from unknown parasite strains, as well as -omic information on host tissues and single cells [Still and all, the biological functions of most le cells , and whele cells . Today, The malaria research field and its vision for what’s important and possible have advanced considerably with the recent benefits of CHMI and systems biological and immunological approaches, generating new levels of understanding and numerous new hypotheses. Still today, underlying biological and molecular mechanisms of NAI, VCI and pathogenesis are largely unknown, leaving a hazy picture of the immune response under various scenarios with many gaps and open questions , 47. HowNumerous investigations for decades have relied upon rodent malaria models to delve into immune response and pathogenesis questions . ANHP infection models (NHPIMs) allow for the in-depth investigation of both broad overarching and specific hypotheses, providing opportunities to enhance what can be achieved using rodents and through clinical studies with humans. Ideally, research using NHPs will continue to complement or validate studies involving rodents, where putative mechanisms can be investigated in greater depth with the benefit of genetic tools and much larger experimental cohorts. Still, rodents can develop exceedingly high parasitaemias, well over 10% in most cases, which undoubtedly must affect immune responses. Such a high level of parasitaemia is rarely seen in human infections or carefully monitored and controlled NHP infections. Moreover, relapse biology and associated immune responses currently cannot be studied using rodent malarial infection models , aIn many cases, immune cell subsets between humans and macaques are comparable. Peripheral blood B cell subsets and T-cells can be phenotyped and classified similarly between humans and macaques , 475. OtP. vivax [The time is opportune to maximize NHPIM efforts, building upon experience gained from longitudinal NHP experiments by many investigators over many decades (described above), and recently by the MaHPIC, described below. Incidentally, the MaHPIC was established soon after the publication of a review article, titled “Acquired Immunity to Malaria”, where the authors, Doolan, Dobaño and Baird wrote: “One likely starting point for more firmly establishing age-dependent, strain-transcending immunity in malaria and for beginning to sort through the myriad possible mechanisms at work may be a nonhuman primate model” . SimilarP. vivax .” As notP. vivax . Also, LP. vivax , also inP. vivax . Silva-FP. vivax .Anopheles mosquitoes to obtain infectious sporozoites —and infecting them through direct blood meal feedings on NHPs.Between 1999 and 2019, the Yerkes NPRC (YNPRC) at Emory University in Atlanta, Georgia  became recognized as the main NPRC (in the USA) with malaria as a major area of research emphasis, and with a recent history of systemic longitudinal malarial infection studies and an on-site insectary facility. Other academic institutions offer the possibility for NHP studies, or mosquito work, but rarely are these animal and mosquito resources found together in one location, so that NHPs can become experimentally infected via mosquito bites or the inoculation of freshly dissected sporozoites from the salivary glands of mosquitoes. The Yerkes Insectary for Malaria Research (YIFMR) was designed in 2015 and opened in 2017, in response to the Centers for Disease Control and Prevention  plans to eliminate malaria research projects that involved NHPs. Thus, there was the need to fill this gap, building upon the CDC’s renowned basic infection and evolutionary studies using a variety of primate malaria species and NHP hosts to document host–parasite compatibilities, vaccine trials, and the routine production of gametocytes for infecting female iewed in , 12). UpComparable setups have since been established at two other academic institutions in the United States: the University of Georgia  with New World and Old World monkey resources and the Oregon NPRC  with Old World monkey resources. The expansion of such research is important, especially now while there is potential for in-depth exploration of the life cycle, immunity and pathogenesis using systems biology, immunology, and vaccinology approaches.Malaria is so complex that systemic analyses have become critical, and these require large transdisciplinary teams that encompass many scientific disciplines. The Malaria Host–Pathogen Interaction Center (MaHPIC) is an example of such a large team effort, which has been focused primarily on NHPIMs and using systems approaches to develop and integrate multi-omic, clinical and parasitological datasets from longitudinal infections. As of the time of this writing, no comparable research program has been noted in the literature, and as discussed above, these studies complement CHMI investigations.Plasmodium infections in multiple host species, which showed either severe or resilient outcomes .The MaHPIC was established in 2012 at the YNPRC of Emory University  and condPlasmodium pathogens as model systems will provide insights into mechanisms as well as indicators for human malarial disease conditions”. The MaHPIC’s work has been built upon three underlying scientific premises: (1) that the host environment is critical for studying malaria parasite biology, infections, immunity, pathogenesis, and pathology, (2) that studying the dynamics of longitudinal infections is critical, and (3) that the integration and modelling of parasitological, clinical, and multi-omic datasets will reveal novel host–parasite interactions, pathways, and networks.The MaHPIC team put forth the overarching hypothesis that “Nonhuman primate host interactions with P. coatneyi (Hackeri strain) as a model of P. falciparum, P. cynomolgi (M/B and Ceylon strains) as a model of P. vivax, and P. knowlesi  to better understand malaria caused by this zoonotic species in its natural (human and Macaca fascicularis) and the traditional experimental (M. mulatta) hosts. Several infection experiments were also carried out with P. vivax in A. nancymaae and S. boliviensis monkeys. Each major longitudinal NHP infection was initiated with sporozoites and allowed to progress for over one month, and in many instances three or more months, when repeat homologous or heterologous sporozoite-initiated infections, relapsing malaria, or recrudescing malaria and chronic disease were being explored. Typically, capillary blood samples were obtained daily as warranted for parasitaemia readings and reticulocyte count analyses, blood chemistry readings using an iSTAT System (Abbot Labs), and targeted and/or untargeted plasma-based metabolomics experiments. Designated time points for venous blood draw samples were determined and selectively utilized as required to generate various large-scale-omic datasets , cytokine analyses, serology, adaptive and innate immune profiles, complete blood count haematology measurements, and the enumeration of haemozoin-containing leukocytes. The details of sample collections, methods, and data generated are being compiled elsewhere , in addition to related details already reported with MaHPIC’s public database depositions and in the team’s original research publications . This bM. mulatta to P. cynomolgi sporozoite immunizations in the face of blood-stage drug cover and the outcome from challenging the animals with infectious sporozoites .The MaHPIC’s original mandate did not include investigations of the immune response in depth, but the team increasingly built the foundation for doing so given the immune response’s prime relevance to infection and disease. At the start of the MaHPIC, a few basic innate and adaptive immune profiling flow cytometry panels were included in the project’s experimental design, to enable initial comparative analyses of the dynamics of cell types that are activated in the course of infection by different malaria parasite species. The group’s foray into malarial immunity and pathogenesis gained increasing attention, and the design of major infection experiments shifted to address specific outstanding biological, immunobiological, and pathogenesis questions of importance to the field of malaria research. As a result, NIAID also awarded the team supplementary funding to design and implement a systems vaccinology trial experiment, specifically to examine the immune response in P. cynomolgi, P. coatneyi or P. knowlesi. Special flow cytometry panels were developed to track and evaluate specific cell types in A. nancymaae and S. boliviensis involved in the immune response to P. vivax. As a result, numerous fluorescently labelled antibodies were identified, tested, and validated in MaHPIC experiments .In total, the MaHPIC successfully optimized an extensive number of flow cytometry panels and gating strategies to track B cells, T cells, monocytes, dendritic cells, neutrophils, and NK cells, as well as cell activation, co-stimulation and trafficking functional markers when relevant. RBC flow cytometry panels and procedures were also developed to monitor erythrocyte progenitor cells, different stages of RBC development from nucleated RBCs to reticulocytes and normocytes, and to distinguish uninfected and infected RBCs . 45 cytoIterative longitudinal infection experiments were executed by the MaHPIC. A few representative examples are summarized here to demonstrate the types and results of these experiments. The experimental numbers reflect those incorporated in the group’s Laboratory Information Management System.M. mulatta with 100 P. coatneyi sporozoites [M. mulatta developed severe anaemia, coagulopathy, renal impairment, and a generalized metabolic dysfunction comparable to what is often seen in severe human cases. Major turnover of erythrocytes was demonstrated and attributed to dysfunctional bone marrow. Disease severity turned out to be less once the animals were semi-immune and re-infected [The MaHPIC’s initially designed longitudinal infection experiment , involved repeat infections of four malaria naïve rozoites . This warozoites . Then, iinfected .P. coatneyi develop chronic infections after receiving subcurative artemether treatment and that they recapitulate the decrease in haemoglobin levels characteristic of persistent infection in humans [P. knowlesi showed evidence of a similar phenotype, but without the administration of subcurative anti-malarial treatment (see E07 below) [M. mulatta that were comparable to those observed in human P. falciparum cases. E03 data are still being analysed by the MaHPIC team, for instance, with respect to the integration of transcriptomic and immunological data. Of particular importance has been an analysis of the development of specific antibody responses over time, as chronic infections are established, and the dynamic production and function of different isotypes. A main question relates to how these antibody responses may result in a balance of protective responses with the killing of infected RBCs and adverse responses that result in the elimination of a majority of uninfected bystander RBCs, as determined by Fonseca et al. using a mathematical model [Specifically, E03  showed tn humans , 488. A 7 below) . Both si7 below) . Furtheral model .P. cynomolgi-rhesus macaque model of relapsing malaria to delve into specific immunobiological questions and begin the process of using systems biological approaches to understand relapse biology and anaemia as it relates to P. vivax in humans [P. cynomolgi M/B strain sporozoites that had been freshly isolated from Anopheles mosquito salivary glands [These four iterative experiments adopted the n humans . The couy glands , 140. Thy glands  and E25 y glands .P. vivax patients and confirmed a similar mechanism in humans [Based on data from E04, Tang et al. used Weighted Gene Coexpression Network Analysis (WGCNA) to integrate flow cytometry, RNA-sequencing, and systemic cytokine measurements . Despiten humans . After dn humans , the mecn humans .P. cynomolgi-antigen-specific ELISAs, and opsonic phagocytosis assays to support the functionality of P. cynomolgi antigen-specific antibodies. Together, the various data types and validation experiments demonstrated that immunity after the primary parasitaemia and a bout of clinical illness was associated with a rapid expansion of memory B cells (MBCs) in a recall response and the production of anti-parasite IgG1 that was able to mediate clearance of parasitized RBCs. Antibodies were also detected that specifically reacted with uninfected RBCs, consistent with them being a causal factor in the observed anaemia. Specific B-cell subpopulations and immunoglobulin isotypes were identified among the peripheral blood mononuclear cells. As in humans, the B cell subpopulations identified included: naive (IgD+CD27−), unswitched memory (USM: IgD+CD27+), switched memory (SM: IgD−CD27+), and double-negative B cells (DN: IgD−CD27−). The USM and SM B cells dramatically and rapidly expanded during relapses, including IgG+SM B cells, and IgM+SM B cells though fewer in number; in contrast, only IgG+SM B cells increased during the homologous reinfections, however, IgM recognizing both infected and uninfected RBCs was significantly increased. Overall, the reduction of infected RBCs in the peripheral blood by phagocytosis coincided with specific MBC and antibody responses and the lack of clinically detectable illness in both relapses and homologous reinfections. Having generated insights into the B-cell dynamics involved in the generation of strain-specific immune memory, E23 and E24 informed new possible directions to understand B-cell dynamics and functions in future experiments.Systems analyses are showing that macaques are particularly well-suited for investigating changes in the bone marrow and possible relationships to systemic changes impacting not only anaemia, but also the development of long-lived serological immunity. E23 and E24 demonstrated that—as with humans —clinical immunity can form after a single sporozoite-initiated blood-stage infection and prevent illness; this was the case both during E23 relapses and E24 homologous strain reinfections . For thiP. cynomolgi, were inoculated a few months later with 2,000 freshly isolated P. cynomolgi sporozoites of the Ceylon strain; the analysis and integration of the results from this project are in progress, alongside validation experiments . For this series of experiments, rectal swabs were also acquired and microbiome and multi-omic integrative analyses are underway to understand host–parasite–microbiome relationships .Experiment 25 was subsequently performed to test the hypothesis that protective recall responses projected (and then observed) for E24 would not function with a heterologous challenge. The six rhesus monkeys that had been infected in E23 and then re-infected in E24 with the M/B strain of P. cynomolgi mirror clinical manifestations and immunity results observed in humans with P. vivax . In reciewed in . In futuAnopheles mosquitoes [P. vivax and P. falciparum infections .P. knowlesi infections that had been initiated with Malayan strain sporozoites in M. fascicularis of Mauritius origin and demonstrated that these natural macaque hosts developed clinical illness [M. mulatta [P. coatneyi or P. cynomolgi [P. knowlesi infected M. mulatta and M. fascicularis, analysed temporally, show dramatic differences at the onset of the acute infection that seem to support the recovery of M. fascicularis, whereas M. mulatta continues to show inflammatory and anti-inflammatory processes [M. fascicularis responded to the parasite as early as 3 days after inoculation with sporozoites, unlike M. mulatta, showing early cytokine signaling along with interferon responses while the parasites were still in the liver. Among other findings, Gupta and colleagues also report pre-infection differences in neutrophils and naïve CD4+T cells that lead to differences in Ca2+ homeostasis, which ultimately balances inflammation and cell proliferation during the log phase of parasitaemia expansion [P. knowlesi infection experiments also incorporated unique telemetry measurements generated from implanted devices that continuously tracked temperature, blood pressure, heart rate and activity levels .Peterson et al. delved into the pathogenesis of  illness . However mulatta —did not ynomolgi , 140. Onrocesses . Perhapsxpansion . These PP. coatneyi, P. cynomolgi, or P. knowlesi replicates the hemoglobin kinetics of humans with P. falciparum or P. vivax [The MaHPIC established that the development of anaemia in rhesus macaques infected with P. vivax , 277. DuP. vivax . Indeed,P. vivax , 495. ThPlasmodium infection regardless of the infecting species. In one example, Tang et al. interpreted peripheral blood transcriptomics data from E03 and E04 by use of a dynamic model of purine metabolism and showed how gene expression associated with purine metabolism from each of these experiments was reflected in specific downstream alterations in metabolomic signatures [Data from E03 and E04 were also studied together to determine what common changes may occur in the host due to gnatures . Specifignatures . Other egnatures , 483.In 2019 many celebrated the 50th anniversary of the first landing of humans on the moon, and another USA-led trip to the moon was projected for 2024. A year later, the SARS-CoV-2/Covid-19 pandemic struck hard and inspired the development, testing, approval, production and distribution of multiple SARS-CoV-2/Covid-19 vaccines in a matter of months  and suchMalaria research interests and agendas have become all-encompassing with consideration of today’s malaria eradication goals. The pursuit of effective vaccines continues alongside epidemiological tracking of parasites, species and infections, and focused attention on the need to detect and treat relapse and chronic asymptomatic cases of malaria. Improved diagnostics, drugs and issues relating to insecticide availability and resistance are also at the forefront. New efforts include rapid species-specific detection of infections using non-invasive procedures , 505, thAs a result of the recent SARS-CoV-2/Covid-19 pandemic, communities and their malaria control/elimination/eradication agendas have been disrupted in countless unexpected ways  and manyToday with the benefits of cellular imaging, -omic technologies, and computational methods, scientists can visualize biological functions and dynamics, in essence beginning to gain traction on understanding complex in vivo dynamical systems. The advent of systems biological approaches offers hope that it is possible to capitalize on NHP model systems hand-in-hand with CHMI studies. Notwithstanding, despite advances recognized in this review, the use of NHPs for malaria research has arguably been quite limited to date, especially compared to other infectious diseases of widespread importance. Any reluctance is unfortunate because clear evidence is mounting that NHPs can be an effective tool to tease out the details of NAI, VCI and pathogenesis in controlled longitudinal infection studies. As mechanistic depth is required to address many hypotheses regarding the molecular basis of host–parasite interactions, necessitating rigorous sampling of blood, bone marrow, or other tissues, NHPs may be the preferred host of the future.P. vivax primary infections, relapses, and repeat infections using robust Old World macaque-P. cynomolgi infection models [P. vivax in the New World monkeys [P. coatneyi and -P. knowlesi infection models are excellent for studying acute and chronic immune responses and pathogenesis, as well as antigenic variation based on the large Schizont Infected Cell Agglutination variant antigens (SICAvar) multigene family that corresponds to the var gene family in P. falciparum [P. coatneyi, P. cynomolgi and P. knowlesi in rhesus monkeys and P. knowlesi in baboons , and fuComparative studies with different parasite species, strains, and co-infections, possibly involving animals of different age groups and sexes , could also be fruitful toward understanding NAI and VCI in these populations. Such studies may also lead to insights supporting the development of vaccines that provide species- or strain-transcending immunity, comparable to multiple natural exposures in malaria endemic areas to homologous or heterologous parasites that can result in the building of NAI, with a lack of symptomology. While this level of immunity would be a major achievement toward eradication, the problem of asymptomatic infections and chronicity must also be addressed. Otherwise, transmission will remain a likely fact, and the chances for eradication will be much reduced. Interventions focused on NAI goals must include both anti-disease and anti-parasite immunity, as well as the elimination of infectious gametocytes.Plasmodium and their functional attributes in NHP infections. It will be informative to assess these at several time points during infections, when possible, from sequential biopsies on individual animals, or from a cohort of animals with staggered terminal analyses, such that the cell types within different tissues could be examined. These studies can be performed, for example, during the liver stage and acute and chronic stages of infections, post treatment, and during relapses in the case of P. vivax or P. cynomolgi. With such analyses, a holistic picture of the dynamics of infection, immunity and disease will take shape. Similarly, future iterations of the types of experiments carried out by the MaHPIC will permit the pursuit of hypothesis-driven questions that will gradually become more specific. Thus, within the limits imposed by ethical regulations, more information will be gained from a specific period of an infection or from a specific tissue or cell type and targeted studies will yield insights into the living tissue architecture and the parasite’s environment.A caveat for studying the cellular dynamics within the immune system while relying solely on peripheral blood or bone marrow is that not all immune cell types of interest, or their specific activated phenotypes, are necessarily circulating in the blood or present in the bone marrow. Moreover, a subset of iRBCs can become sequestered or concealed in various tissues and organs , 277, soPlasmodium, may not be in the realm of realistic possibilities—today. However, with greater fundamental understanding of the workings of the immune system and host–parasite interactions, strategic, fortuitous—or serendipitous—discoveries will pave the way forward in ways unbeknown today. All things considered, from the hundreds of millions of Plasmodium infections and clinical cases of malaria that still occur annually in close to 100 countries [The ever-elusive efficacious malaria vaccine, which can be counted on for complete protection against one or all species, strains and variants of ountries , and theountries .”Fifty years ago, the authors of “The Primate Malarias” demonstrated much curiosity and dedication, thereby consciously or not paving the way for decades of malaria research involving NHPs to follow their lead. Once again, the malaria research field is at an important juncture, and the question is whether society has the determination to utilize all available resources and the cornucopia of amazing new technologies to advance our understanding of malaria, immunity, and pathogenesis for the benefit of translational research that will ultimately benefit communities affected by this disease worldwide. The catch phrase “use them or lose them” needs to be taken to heart when it comes to NHP model systems. The discussion of advances based on NHP models described here is intended to inspire curiosity and the pursuit of new avenues toward knowledge regarding the multi-faceted system that encompasses malaria. The necessary tools are emerging. They are powerful and unprecedented, but they will only be efficacious if they are used and relentlessly refined and improved by new cohorts of well-trained malariologists. This article’s goal—while honoring leaders from the past—is thus a call to action. The longitudinal infection experiments performed by the MaHPIC are an example of the benefits reaped from decades of prior research with NHPs. It was timely in 2012 for the MaHPIC team to begin developing the systems biological underpinnings for longitudinal NHP infection research, and their work added new dimensions of understanding to key aspects of the disease, such as relapses, chronic infections, anaemia, and immune memory. However, much is yet to be learned, venturing into uncharted territory. If technologies continue to advance as expected, and the tenacity for new research using NHPIMs remains strong, insights into malaria in these models will continue to provide a multi-dimensional holistic view of parasitism at the cellular, tissue and organ levels, and spawn a wider, and at the same time sharper focus onto possible targets for interventions and disease eradication. Terabytes of data from MaHPIC experiments have been deposited in the NIAID-supported Bioinformatics Resource Centers, PlasmoDB , 276 and


Plasmodium vivax to malaria elimination in Cambodia. In Papua New Guinea, these studies document the complex host–vector–parasite interactions that are underlying both the stunning reductions in malaria burden from 2006 to 2014 and the significant resurgence in transmission in 2016 to 2018. Here we describe the novel analytical, surveillance, molecular, and immunological tools that are being applied in our ongoing Asia-Pacific ICEMR research program.Gaining an in-depth understanding of malaria transmission requires integrated, multifaceted research approaches. The Asia-Pacific International Center of Excellence in Malaria Research (ICEMR) is applying specifically developed molecular and immunological assays, in-depth entomological assessments, and advanced statistical and mathematical modeling approaches to a rich series of longitudinal cohort and cross-sectional studies in Papua New Guinea and Cambodia. This is revealing both the essential contribution of forest-based transmission and the particular challenges posed by  Since then, progress has been mixed, with several countries reaching elimination and others reducing local transmission to very low levels. In other countries, progress has slowed or reversed, even before the COVID-19 pandemic put additional stresses on local public health systems.In 2014 and 2015, after a decade of significant progress in reducing malaria transmission, 21 Asia-Pacific countries signed up to the joint goal of regional malaria elimination by 2030 and established the Asia-Pacific Leaders Malaria Alliance  distribution rounds, the prevalence of Plasmodium sp. infections and number of confirmed malaria cases were reduced by 50% to 90% between 2008–2009  and 2014 (MIS: 0.9%).–P. vivax and P. falciparum are comparable in PNG, P. falciparum still accounts for a clear majority of clinical cases.Cambodia and Papua New Guinea (PNG) are two countries that exemplify the Asia-Pacific malaria elimination challenges. After the confirmation of artemisinin resistant P. falciparum and P. vivax in PNG and Cambodia, the Asia-Pacific and previous South-West Pacific International Center of Excellence in Malaria Research (ICEMR) have been conducting coordinated sets of epidemiological and entomological studies in three sites  to prevent blood-stage replication.P .falciparum clinical immunity, we found that antibodies to merozoites fix and activate complement to inhibit invasion and lyse merozoites, and complement-fixing antibodies were more strongly correlated with protection than growth-inhibitory antibodies.,P. falciparum–infected erythrocytes also contribute to immunity, especially in young children in our study populations, and antibodies predominantly target P. falciparum erythrocyte membrane protein 1.–In studies of P. vivax presents additional challenges, one of which is the severely limited capacity to propagate parasites in vitro. To get around this, we have optimized assays to quantify antibody complement-fixation and FcγR-mediated functions using arrays of P. vivax recombinant antigens.–Studying functional immunity to P. falciparum and P. vivax is predominantly associated with prevention of clinical illness, rather than prevention of infection.–,–Importantly, we and others have found that acquired immunity against ,However, the role of immunity in maintaining asymptomatic infections in populations with low malaria transmission such as in Cambodia is less clear because the current paradigm is that effective acquired immunity requires substantial malaria exposure to develop and requires regular exposure to be maintained. Some populations have transitioned from high to low transmission intensity. Therefore, ongoing protective immunity may be partly provided by established memory from prior exposures. However, there are other populations where low transmission has been established for an extended time, with lower magnitude of acquired Abs, yet low-density asymptomatic infections are still commonly observed.P. falciparum,P. vivax  to track clones in longitudinal cohort studies; 2) SNP barcodes: for P. falciparum  and P. vivaxTo map how changes in transmission affect parasite populations and investigate whether the genetic composition of parasite population may contribute to high asymptomatic carriage, the Asia Pacific ICEMR Genomics Core has developed genotyping assays for malaria parasites using amplicon sequencing. These include 1) short single nucleotide polymorphism (SNP) microhaplotype assays for P. falciparum and P. vivax have demonstrated high levels of diversity in PNG parasite populations, but low diversity in Cambodia, which reflects the lower transmission intensity and a largely clonal multidrug resistant P. falciparum population in Cambodia. A lower level of parasite diversity may enable protective immunity to be acquired more quickly, and studies are ongoing to understand this. In PNG ICEMR sites in Madang and East Sepik, parasite diversity was measured using 10 microsatellite markers but did not reveal any substantial changes, even with a large reduction in transmission intensity. However, an increase in multilocus disequilibrium, a measure of parasite relatedness, was observed suggesting this parameter may be a marker of transmission reduction. Contrary to expectations, P. falciparum population structure present at the first ICEMR timepoint in 2005–2006 was lost after LLINs were introduced and was attributed to increased human migration between the two ICEMR sites, whereas P. vivax remained unstructured both pre- and post-LLIN.P. falciparum lineages remained while P. vivax rapidly diversified (unpublished). This difference in results for the different marker panels is likely to be due to the SNPs more accurately characterizing parasite relatedness, and therefore changes in population structure. ICEMR studies are now utilizing these barcodes in PNG and Cambodia (Asia Pacific ICEMR), in addition to Mali (West Africa ICEMR). Whole genome sequencing is also being conducted in the three countries to validate SNP barcodes and confirm patterns observed.AmpSeq markers for both kelch13 C580Y mutations associated with artemisinin were discovered for the first time in PNG by genotyping P. falciparum isolates collected from Wewak, East Sepik Province in 2016.kelch 13 C580Y emerging in the local parasite population, it was not clear whether it emerged in PNG, West Papua, or another nearby location. We recently developed a multiplex amplicon sequencing assay known as “DResCode” that targets well-known drug resistance genes  in addition to a PNG genetic background that is associated with the kelch13 C580Y mutation. Using this assay as well as a real-time quantitative PCR assay for rapid detection of kelch13 C580Y mutations, ICEMR samples are being studied in a wider surveillance effort to confirm whether the kelch13 C580Y mutation had spread to other parts of PNG.Using ICEMR samples to monitor the evolution of antimalarial drug resistance mutations is a major goal of the program. Recently, Although the ICEMR site of Maprik is only 50 km from Wewak, where the first mutations were detected, no mutations were found among more than 300 samples collected in 2016. Genotyping of more than 600 samples collected from eight locations in PNG between 2015 and 2018, identified only a single C580Y mutant in Lae, Morobe Province.Anopheles mosquitoes on forest fringes and in forest sites.–Anopheles vectors in villages from 6 pm to 6 am. To obtain a better knowledge of mosquito behavior in the Cambodian forest setting, we used odor-baited double nets traps to assess Anopheles behavior and host preferences over a 24-hour period in different ecological settings.Anopheles vectors were captured during daytime, when people are active and not protected by a bed net, highlighting the importance of daytime biting to sustaining residual transmission in this area. Infectious mosquitoes (sporozoite positive in heads and thoraces) were collected in both human- and cow-baited double net traps, and key Anopheles vectors  exhibited mainly generalist or zoophilic host preferences. These observations suggest that many of the Anopheles mosquito populations are largely maintained by feeding on animals, and that, in contrast to African malaria vectors, the notion of highly preferential human feeding may be inaccurate in the Greater Mekong Subregion. Knowing that opportunistic feeding is very common could inform the design of new vector control tools to control malaria infection levels.Despite that spending extended periods of time in the forest is one of the main malaria risk factors in the Greater Mekong Subregion, few studies have collected We continue to investigate mosquito ecology by designing collection based on data acquired from land cover analyses and by conducting human mobility studies to better comprehend the transmission patterns. Our ICEMR is particularly interested in determining more finely the vector–human contact patterns in time and space. To do so, we assessed the evolution of the land cover over 30 years and showed a high deforestation rate with wooded areas decreasing from 91% to 47% in our study site.An. punctulatus group, exhibiting opportunistic host-seeking behavior and a preference for outdoor biting. In addition, An. farauti, the dominant coastal vector species in PNG, is very active shortly after sunset. The effect of LLINs on these outdoor, zoophilic vector populations remains unclear. Our ICEMR has contributed significantly to the detailed understanding of these important vector ecology-related factors sustaining malaria transmission. By conducting targeted human landing catch studies indoors, outdoors, and in social gathering places, we quantified where transmission occurs. In addition, barrier screen studies revealed important host-seeking behaviors.In the southwest Pacific, malaria transmission is known to be peri-domestic, and control relies heavily on the mass distribution of LLINs. Although there is no pyrethroid resistance in the Anopheline populations in PNG,Although malaria prevalence in high-burden provinces in PNG is high on average, it is still characterized by a striking degree of spatiotemporal heterogeneity, resulting from human and mosquito-related factors. Our longitudinal mosquito collections revealed striking species-specific seasonality patterns in some settings, indicating that different species maintain malaria transmission through the wet and dry seasons, respectively (unpublished data). We also observed high differences in Anopheline biting frequency in villages that are just a few kilometers apart, indicating that high variability in local mosquito abundance is the main cause of transmission heterogeneity in PNG. A particular focus of our ICEMR lies on investigating when and where humans are most vulnerable to Anopheline biting and which population groups are most at risk. By matching human DNA collected in cross-sectional surveys with DNA obtained from blood-fed mosquitoes, our studies indicated that, as in other settings, younger males are at particular risk to receive mosquito bites, which is another parallel to the Cambodian setting, although the underlying reasons based on human occupational or behavioral patterns are different.–P. falciparum and P. vivax infections in both settings but also demonstrated the possibility of transmission from asymptomatic P. vivax infections. Further studies to determine the contribution of symptomatic versus asymptomatic infections to local transmission are ongoing.Last but not least, we have established mosquito-to-human infection studies in PNG and Cambodia to investigate the contribution of different population groups  to malaria transmission.By closely coordinating our entomology work with epidemiological studies, we are able to gain a holistic understanding of malaria transmission in both PNG and Cambodian communities.P. falciparum and P. vivax.,Cross-sectional and longitudinal studies implemented in PNG and Cambodia provide a rich understanding of the epidemiology of P. vivax transmission was calibrated to malaria prevalence data collected after the introduction of insecticide treated nets.P. vivax cases if bed net distributions were stopped—a prediction consistent with most epidemiologists’ intuition. However, the model provided additional insight, predicting a rebound of malaria cases to levels even higher than before interventions. This is because children born during an era of high bed net coverage grow up with limited malaria exposure and consequently have low levels of immunity. If bed net distribution stops, this cohort is extremely vulnerable to new malaria cases. These findings from mathematical models add to the evidence base needed to justify continuing bed net distribution.In PNG, a model of P. vivax prevalence were observed (23%), and infections were distributed across men and women of all ages. Outside the forest, P. vivax prevalence was lower (3%) with significant clustering of infections in men aged 16 to 50 years, consistent with a pattern of occupational exposure where working-age men are exposed to malaria during forest visits. This is in contrast to the “free-mixing” assumption of many malaria transmission models, where any mosquito can bite any human. These epidemiological findings have initiated theoretical model development to account for the structured interactions between humans and mosquitoes.In Cambodia, age and gender stratified analysis of data from cross-sectional surveys revealed important covariates for malaria risk.The Asia-Pacific ICEMR’s ability to bring together long-term epidemiological and entomological follow-up with state-of-the-art laboratory assays and analytical tools has provided unique insights into the complex host–parasite–vector interactions that underlie the different trajectories and challenges of malaria control and elimination in PNG and Cambodia.P. vivax infections. This will require both the implementation of effective radical cure with Primaquine and the development of novel interventions, such as serological testing and treatment (PvSeroTAT) that allow identifying and targeting potential hypnozoite carriers. In PNG, the ICEMR not only provided a detailed understanding of the key factors that have driven both the decrease in transmission between 2010 and 2014 but also for the resurgence after 2015. By linking the continuous monitoring of the key threats of increasing drug resistance and variations in vector control efficacy, our studies are uniquely placed to support the PNG national malaria control program in policy and implementation. Studies of the targets and mechanisms of malaria immunity, including diversity of key target antigens, will inform the development of vaccines for P. falciparum and P. vivax to help achieve long-term control and elimination of malaria in the Asia-Pacific region, and elsewhere.Our Cambodian results strengthen the hypothesis that forests are the main risk areas for human malaria transmission and highlight the importance of daytime biting behavior as a potential source for transmission. Approaches targeted at risk groups based only on forest proximity may be more cost-effective for the national malaria control program. Our results also highlight the importance of specific control efforts aimed at asymptomatic