Insights in Chloroquine Action: Perspectives and Implications in Malaria and COVID-19.

Malaria is a threat to human mankind and kills about half a million people every year. On the other hand, COVID-19 resulted in several hundred thousand deaths since December 2019 and remains without an efficient and safe treatment. The antimalarials chloroquine (CQ) and its analog, hydroxychloroquine (HCQ), have been tested for COVID-19 treatment, and several conflicting evidence has been obtained. Therefore, the aim of this review was to summarize the evidence regarding action mechanisms of these compounds against Plasmodium and SARS-CoV-2 infection, together with cytometry applications. CQ and HCQ act on the renin angiotensin system, with possible implications on the cardiorespiratory system. In this context, flow and image cytometry emerge as powerful technologies to investigate the mechanism of therapeutic candidates, as well as for the identification of the immune response and prognostics of disease severity. Data from the large randomized trials support the conclusion that CQ and HCQ do not provide any clinical improvements in disease severity and progression of SARS-CoV-2 patients, as well as they do not present any solid evidence of increased serious side effects. These drugs are safe and effective antimalarials agents, but in SARS-CoV-2 patients, they need further studies in the context of clinical trials. © 2020 International Society for Advancement of Cytometry.

Deciphering host immunity to malaria using systems immunology.

A century of conceptual and technological advances in infectious disease research has changed the face of medicine. However, there remains a lack of effective interventions and a poor understanding of host immunity to the most significant and complex pathogens, including malaria. The development of successful interventions against such intractable diseases requires a comprehensive understanding of host-pathogen immune responses. A major advance of the past decade has been a paradigm switch in thinking from the contemporary reductionist (gene-by-gene or protein-by-protein) view to a more holistic (whole organism) view. Also, a recognition that host-pathogen immunity is composed of complex, dynamic interactions of cellular and molecular components and networks that cannot be represented by any individual component in isolation. Systems immunology integrates the field of immunology with omics technologies and computational sciences to comprehensively interrogate the immune response at a systems level. Herein, we describe the system immunology toolkit and report recent studies deploying systems-level approaches in the context of natural exposure to malaria or controlled human malaria infection. We contribute our perspective on the potential of systems immunity for the rational design and development of effective interventions to improve global public health.

The Promise of a Malaria Vaccine-Are We Closer?

Malaria vaccine development has rapidly advanced in the past decade. The very first phase 3 clinical trial of the RTS,S vaccine was completed with over 15,000 African infants and children, and pilot implementation studies are underway. Next-generation candidate vaccines using novel antigens, platforms, or approaches targeting different and/or multiple stages of the Plasmodium life cycle are being tested. Many candidates, in various stages of development, promise enhanced efficacy of long duration and broad protection against genetically diverse malaria strains, with a few studies under way in target populations in endemic areas. Malaria vaccines together with other interventions promise interruption and eventual elimination of malaria in endemic areas.

Plasmodium co-infection protects against chikungunya virus-induced pathologies.

Co-infection with Plasmodium and chikungunya virus (CHIKV) has been reported in humans, but the impact of co-infection on pathogenesis remains unclear. Here, we show that prior exposure to Plasmodium suppresses CHIKV-associated pathologies in mice. Mechanistically, Plasmodium infection induces IFNγ, which reduces viraemia of a subsequent CHIKV infection and suppresses tissue viral load and joint inflammation. Conversely, concomitant infection with both pathogens limits the peak of joint inflammation with no effect on CHIKV viraemia. Reduced peak joint inflammation is regulated by elevated apoptosis of CD4+ T-cells in the lymph nodes and disrupted CXCR3-mediated CD4+ T-cell migration that abolishes their infiltration into the joints. Virus clearance from tissues is delayed in both infection scenarios, and is associated with a disruption of B cell affinity-maturation in the spleen that reduces CHIKV-neutralizing antibody production.

An expanding toolkit for preclinical pre-erythrocytic malaria vaccine development: bridging traditional mouse malaria models and human trials.

Malaria remains a significant public health burden with 214 million new infections and over 400,000 deaths in 2015. Elucidating relevant Plasmodium parasite biology can lead to the identification of novel ways to control and ultimately eliminate the parasite within geographic areas. Particularly, the development of an effective vaccine that targets the clinically silent pre-erythrocytic stages of infection would significantly augment existing malaria elimination tools by preventing both the onset of blood-stage infection/disease as well as spread of the parasite through mosquito transmission. In this Perspective, we discuss the role of small animal models in pre-erythrocytic stage vaccine development, highlighting how human liver-chimeric and human immune system mice are emerging as valuable components of these efforts.

Novel approaches to whole sporozoite vaccination against malaria.

The parasitic disease malaria threatens more than 3 billion people worldwide, resulting in more than 200 million clinical cases and almost 600,000 deaths annually. Vaccines remain crucial for prevention and ultimately eradication of infectious diseases and, for malaria, whole sporozoite based immunization has been shown to be the most effective in experimental settings. In addition to immunization with radiation-attenuated sporozoites, chemoprophylaxis and sporozoites (CPS) is a highly efficient strategy to induce sterile protection in humans. Genetically attenuated parasites (GAP) have demonstrated significant protection in rodent studies, and are now being advanced into clinical testing. This review describes the existing pre-clinical and clinical data on CPS and GAP, discusses recent developments and examines how to transform these immunization approaches into vaccine candidates for clinical development.

The impact of lipid-based nutrient supplementation on anti-malarial antibodies in pregnant women in a randomized controlled trial.

BACKGROUND: Malaria and undernutrition frequently coexist, especially in pregnant women and young children. Nutrient supplementation of these vulnerable groups might reduce their susceptibility to malaria by improving immunity. METHODS: Antibody immunity to antigens expressed by a placental-binding parasite isolate, a non-placental binding parasite isolate, merozoites and schizonts at enrolment (before 20 gestation weeks) and at 36 gestation weeks were measured in 1,009 Malawian pregnant women receiving a daily lipid-based nutrient supplement, multiple micronutrients or iron and folic acid, who were participants in a randomized clinical trial assessing the effects of nutrient supplementation on pregnancy outcomes and child development (registration ID: NCT01239693). RESULTS: Antibodies to placental-binding isolates significantly increased while antibodies to most merozoite antigens declined over pregnancy. Overall, after adjustment for covariates, the type of supplementation did not influence antibody levels at 36 gestation weeks or the rate of change in antibody levels from enrolment to 36 weeks. A negative association between maternal body mass index and opsonizing antibodies to placental-binding antigens (coefficient (95% CI) -1.04 (-1.84, -0.24), was observed. Similarly, women with higher socioeconomic status had significantly lower IgG and opsonizing antibodies to placental-binding antigens. Neither of these associations was significantly influenced by the supplementation type. CONCLUSIONS: In the current cohort nutrient supplementation did not affect anti-malarial antibody responses, but poor and undernourished mothers should be a priority group in future trials.

An alternative paradigm for the role of antimalarial plants in Africa.

Most investigations into the antimalarial activity of African plants are centered on finding an indigenous equivalent to artemisinin, the compound from which current frontline antimalarial drugs are synthesized. As a consequence, the standard practice in ethnopharmacological research is to use in vitro assays to identify compounds that inhibit parasites at nanomolar concentrations. This approach fails to take into consideration the high probability of acquisition of resistance to parasiticidal compounds since parasite populations are placed under direct selection for genetic that confers a survival advantage. Bearing in mind Africa's long exposure to malaria and extensive ethnobotanical experimentation with both therapies and diet, it is more likely that compounds not readily overcome by Plasmodium parasites would have been retained in the pharmacopeia and cuisine. Such compounds are characterized by acting primarily on the host rather than directly targeting the parasite and thus cannot be adequately explored in vitro. If Africa's long history with malaria has in fact produced effective plant therapies, their scientific elucidation will require a major emphasis on in vivo investigation.

Prospects for malaria elimination in non-Amazonian regions of Latin America.

Latin America contributes 1-1.2 million clinical malaria cases to the global malaria burden of about 300 million per year. In 21 malaria endemic countries, the population at risk in this region represents less than 10% of the total population exposed worldwide. Factors such as rapid deforestation, inadequate agricultural practices, climate change, political instability, and both increasing parasite drug resistance and vector resistance to insecticides contribute to malaria transmission. Recently, several malaria endemic countries have experienced a significant reduction in numbers of malaria cases. This is most likely due to actions taken by National Malaria Control Programs (NMCP) with the support from international funding agencies. We describe here the research strategies and activities to be undertaken by the Centro Latino Americano de Investigación en Malaria (CLAIM), a new research center established for the non-Amazonian region of Latin America by the National Institute of Allergy and Infectious Diseases (NIAID). Throughout a network of countries in the region, initially including Colombia, Guatemala, Panama, and Peru, CLAIM will address major gaps in our understanding of changing malaria epidemiology, vector biology and control, and clinical malaria mainly due to Plasmodium vivax. In close partnership with NMCPs, CLAIM seeks to conduct research on how and why malaria is decreasing in many countries of the region as a basis for developing and implementing new strategies that will accelerate malaria elimination.

Ajuga remota Benth.: from ethnopharmacology to phytomedical perspective in the treatment of malaria.

Treatment and control of malaria have become more difficult with the spread of drug-resistant parasites and insecticide-resistant mosquito vectors. In the search for new antimalarial drugs, ethnopharmacological sources should merit more attention. Establishing the safety of traditional herbal medicines, along with identifying their active principles, are essential steps in the production of a properly standardized and accessible herbal medicine. Phytochemical characterization could also serve as a base for the development of new chemical compounds. The genus of Ajuga belongs to the family Lamiaceae and contains at least 301 species. Many of these plants have been used in traditional medicine. Ajuga remota in particular is traditionally used as a herbal remedy for fever and infections, and is prescribed for malaria by 66% of the Kenyan herbalists. A large number of compounds have already been isolated from A. remota, including ergosterol-5,8-endoperoxide (6), ajugarin-I (1), 8-O-acetylharpagide (5) and several phytoecdysteroids. In vitro pharmacological studies have been conducted on constituents of A. remota of which some of them displayed a concentration-dependent inhibition of chloroquine-sensitive and -resistant Plasmodium falciparum and Mycobacterium tuberculosis. Inhibition of parasitaemia was demonstrated in mouse models with P. berghei, supporting the traditional use of the plant against malaria. In this state-of-the-art review, A. remota as a possible therapeutic tool for malaria is discussed.

Host-mediated regulation of superinfection in malaria.

In regions of high rates of malaria transmission, mosquitoes repeatedly transmit liver-tropic Plasmodium sporozoites to individuals who already have blood-stage parasitemia. This manifests itself in semi-immune children (who have been exposed since birth to Plasmodium infection and as such show low levels of peripheral parasitemia but can still be infected) older than 5 years of age by concurrent carriage of different parasite genotypes at low asymptomatic parasitemias. Superinfection presents an increased risk of hyperparasitemia and death in less immune individuals but counterintuitively is not frequently observed in the young. Here we show in a mouse model that ongoing blood-stage infections, above a minimum threshold, impair the growth of subsequently inoculated sporozoites such that they become growth arrested in liver hepatocytes and fail to develop into blood-stage parasites. Inhibition of the liver-stage infection is mediated by the host iron regulatory hormone hepcidin, whose synthesis we found to be stimulated by blood-stage parasites in a density-dependent manner. We mathematically modeled this phenomenon and show how density-dependent protection against liver-stage malaria can shape the epidemiological patterns of age-related risk and the complexity of malaria infections seen in young children. The interaction between these two Plasmodium stages and host iron metabolism has relevance for the global efforts to reduce malaria transmission and for evaluation of iron supplementation programs in malaria-endemic regions.

Opportunistic infections in non-human primates exposed to immunomodulatory biotherapeutics: considerations and case examples.

Immunomodulatory biotherapeutics are most often evaluated for safety preclinically by way of repeat dose toxicity studies in non-human primates. Since immunomodulation is expected with this class of therapeutics, and since non-human primates share many opportunistic or latent infectious agents with humans, non-human primates in these toxicity studies may present with opportunistic or recrudescent infections that would be of concern if they occurred clinically in humans. In such instances, it is suggested that non-clinical safety assessment scientists consider a series of key questions that aim to clarify the relationship of the findings to the biotherapeutic under study and the expected predictivity of the findings to the human clinical setting. In this review, relevant case examples are considered comprising (i) gammaherpesviruses-mediated B-lymphocyte proliferation associated with a T-lymphocyte depleting fusion protein; (ii) increased plasmodial hemoparasite burdens associated with a monoclonal antibody inhibitory to T-lymphocyte trafficking and macrophage function, and (iii) the expected predictivity of non-human primate models for the occurrence of encephalic polyomavirus infections.

Development of self-assembled nanoceramic carrier construct(s) for vaccine delivery.

Hydroxyapatite (HA) has been extensively investigated as scaffolds for tissue engineering, as drug delivery agents, as non-viral gene carriers, as prosthetic coatings, and composites. Recent studies in our laboratory demonstrated the immunoadjuvant properties of HA when administered with malarial merozoite surface protein-1(19) (MSP-1(19)). HA nanoceramic carrier was prepared by co-precipitation method that comprises of sintering and spray-drying technique. Prepared systems were characterized for crystallinity, size, shape, and antigen loading efficiency. Small size and large surface area of prepared HA demonstrated good adsorption efficiency of immunogens. Prepared nanoceramic formulations also showed slower in vitro antigen release and slower biodegrability behavior, which may lead to a prolonged exposure to antigen-presenting cells and lymphocytes. Furthermore, addition of mannose in nanoceramic formulation may additionally lead to increased stability and immunological reactions. Immunization with MSP-1(19) in nanoceramic-based adjuvant systems induced a vigorous immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. In addition considerable amount of IFN-g and IL-2 was observed in spleen cells of mice immunized with nanoceramic-based vaccines. On the contrary, mice immunized with MSP-1(19) alone or with alum did not exhibit a significant cytotoxic response. The antibody responses to vaccine co-administered with HA was a mixed Th1/Th2 compared to the Th2-biased response obtained with alum. The prepared HA nanoparticles exhibit physicochemical properties that appear promising to make them a suitable immunoadjuvant to be used as antigen carriers for immunopotentiation.

In vivo induction of the nitric oxide pathway in hepatocytes after injection with irradiated malaria sporozoites, malaria blood parasites or adjuvants.

The mechanisms responsible for malarial immunity induced by repetitive injections of X-irradiated sporozoites have not been fully established. We demonstrate here that a single injection of irradiated sporozoites induced, as soon as 24 h after, a non-permissive state to hepatocyte reinfection with sporozoites in vitro. The same effect was observed when malarial blood forms, irradiated promastigotes of Leishmania infantum, adjuvants (muramyl dipeptide, poly acidylic uridylic) or interferon-gamma was injected. Activation of the nitric oxide (NO) pathway in the hepatocyte by these factors was found to be responsible for hepatocyte refractory status. Additionally, this metabolic pathway is involved in protection given by repeated injections of irradiated sporozoites since protection could be reversed by treating mice at the time of sporozoite challenge with a competitive inhibitor (NG-monomethyl-L-arginine) of the NO pathway. These results suggest that, in view of an antisporozoite vaccine, further studies are needed to find out how to activate specifically a long-lasting nonspecific immune response.

Role of non-human primates in malaria vaccine development: memorandum from a WHO meeting.

This Memorandum discusses the coordination and standardization of malaria vaccine research in non-human primates to encourage optimum use of the available animals in experiments that are fully justified both scientifically and ethically. The requirements for experimentation in non-human primates, the availability of suitable animals for malaria vaccine studies, and the criteria for testing candidate vaccines are considered. The policy and legislation relevant to the use of non-human primates in biomedical research are also briefly discussed. The Memorandum concludes with eight recommendations.

[Lipoproteins and malaria. I. Immunogenicity of serum lipoproteins in mice infected with Plasmodium chabaudi]. / Lipoprotéines et malaria: I. Immunogénicité des lipoprotéines sériques chez la souris infectée par Plasmodium chabaudi.

Anti-lipoprotein antibodies (anti-Lp Ab) have been investigated during the course of acute infection with P. chabaudi in Swiss mice using a radio-immunoprecipitation assay with (125)-iodine radiolabelled lipoproteins from normal or infected mice. Antibodies were detected 11 days after the beginning of infection; however, the highest lipoprotein precipitations were observed with purified (125)-I labelled lipoproteins from day-5 or day-7 infected mice. P. chabaudi infected mice were treated with chloroquine at various intervals after the beginning of infection and anti-Lp Ab were assayed on day 13. Anti-Lp Ab were not observed in mice treated before day 7 but were present in all mice treated after day 7. Anti-Lp Ab were not detected in mice infected with P. yoelii 17 X. Injection of purified lipoproteins from day-7 P. chabaudi infected mice (Lp day-7) to normal uninfected mice did not induce an antibody response to lipoproteins but anti-Lp Ab were observed when the same injection was performed in P. yoelii infected mice. Moreover, anti-Lp Ab were detected in uninfected mice injected concomitantly with Lp day-7 and hematin extracted from malarial pigment. Our results suggest that anti-Lp Ab observed in P. chabaudi infected mice are mainly against modified lipoproteins produced during infection and that the induction of the antibody response against lipoproteins requires an adjuvant effect such as the hematin which is released during infection.

A comparison of saponin with other adjuvants for the potentiation of protective immunity by a killed Plasmodium yoelii vaccine in the mouse.

The protective immunity conferred by subcutaneous injection of outbred CD-1 mice with a killed Plasmodium yoelii (YM strain) vaccine was strongly potentiated by saponin. By adjusting the dose of antigen, the number of immunizations and the number of living parasites in the challenge infection, conditions were defined where antigen alone was non-protective but 100% protection was obtained by the addition of saponin. Inbred BALB/c, CBA/CA and C57 B1 mice were much less responsive than the CD-1 mice. The following adjuvants were compared with saponin: mineral oil emulsions (Freund's incomplete and complete adjuvants); A1(OH)3(Alhydrogel); bacteria and synthetic bacterial derivatives (Bordetella pertussis, Corynebacterium parvum and muramyl dipeptide); surface active materials (digitonin, vitamin A, Arquad 18, dimethyldioctadecyl ammonium bromide, and the polyene antibiotics, Nystatin and Amphotericin B). None of these adjuvants were as effective as saponin, although FCA, A1(OH)3 and C. parvum augmented immunity considerably. The possible reasons for the efficacy of saponin as an adjuvant for protozoal vaccines are discussed. The P. yoelli/mouse system provides a sensitive and rapid screening assay for comparison of potential adjuvants suitable for use with a malaria vaccine.

Immunity to Plasmodium yoelii and Babesia microti: modulation by the CBA/N X-chromosome.

CBA/N mice carry an X-linked recessive defect expressed in cells of the B cell lineage. The major deficiency in these mice is an almost complete inability to respond to certain thymus-independent antigens, such as pneumococcal polysaccharide type III (S III). We have examined the responses of mice carrying the CBA/N X-chromosome to the malaria parasite Plasmodium yoelii and the piroplasm Babesia microti. We have found that the duration and severity of these infections is increased in mice carrying the CBA/N X-chromosome and that this is associated with a markedly defective IgM antibody response to the parasitized red cell and a failure to produce autoantibodies to bromelain-treated mouse RBCs. An autoimmune response directed at modified determinants on the red cell membrane may be one of the factors involved in the control of these infections.