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The transmission of Plasmodium spp. sporozoites to the mammalian host is the first step in the initiation of the mosquito-borne disease known as malaria. The exact route of transmission from the bloodstream to the liver is still not clearly elucidated, and identification of the host glycan structures bound by the sporozoites may inform as to which host cells are involved. Here, we provide a comprehensive analysis of the glycan structures that sporozoites from the human pathogen, P. falciparum, and the rodent pathogen, P. yoelii, recognize and bind. Glycan array analysis was used to profile the glycans bound by the sporozoites, and the binding affinities of these sporozoite-glycan interactions were then determined by surface plasmon resonance. Data showed that the different Plasmodium spp. bind different classes of glycans. P. falciparum was observed to bind to glycans with terminal N-acetylgalactosamine (GalNAc) or Galactose (Gal) linked to a GalNAc, and the highest-affinity observed was with the GalNAc monosaccharide (12.5 nM). P. yoelii bound glycosaminoglycans, mannosyl glycans, Gal linked to N-acetylglucosamine structures, and the αGal epitope. The highest-affinity interaction for P. yoelii was with the αGal epitope (31.4 nM). This is the first study to identify the key host glycan structures recognized by human and rodent Plasmodium spp. sporozoites. An understanding of how Plasmodium sporozoites interact with the specific glycan structures identified here may provide further insight into this infectious disease that could help direct the design of an effective therapeutic.

Introduction: Despite decades of research into the development of a vaccine to combat the malaria parasite, a highly efficacious malaria vaccine is not yet available. Different whole parasite-based vaccine approaches, including deliberate Plasmodium infection and drug cure (IDC), have been evaluated in pre-clinical and early phase clinical trials. The advantage of whole parasite vaccines is that they induce immune responses against multiple parasite antigens, thus lowering the impact of antigenic diversity. Deliberate Plasmodium IDC, as a vaccine approach, involves administering infectious, live parasites in combination with an anti-malarial drug, which controls the infection and enables induction of protective immune responses.

Contains fulltext : 238206.pdf (Publisher’s version ) (Open Access) Introduction: An effective vaccine against malaria forms a global health priority. Both naturally acquired immunity and sterile protection induced by irradiated sporozoite immunization were described decades ago. Still no vaccine exists that sufficiently protects children in endemic areas. Identifying immunological correlates of vaccine efficacy can inform rational vaccine design and potentially accelerate clinical development.Areas covered: We discuss recent research on immunological correlates of malaria vaccine efficacy, including: insights from state-of-the-art omics platforms and systems vaccinology analyses; functional anti-parasitic assays; pre-immunization predictors of vaccine efficacy; and comparison of correlates of vaccine efficacy against controlled human malaria infections (CHMI) and against naturally acquired infections.Expert Opinion: Effective vaccination may be achievable without necessarily understanding immunological correlates, but the relatively disappointing efficacy of malaria vaccine candidates in target populations is concerning. Hypothesis-generating omics and systems vaccinology analyses, alongside assessment of pre-immunization correlates, have the potential to bring about paradigm-shifts in malaria vaccinology. Functional assays may represent in vivo effector mechanisms, but have scarcely been formally assessed as correlates. Crucially, evidence is still meager that correlates of vaccine efficacy against CHMI correspond with those against naturally acquired infections in target populations. Finally, the diversity of immunological assays and efficacy endpoints across malaria vaccine trials remains a major confounder.

Babesiosis is a tick-borne disease caused by intraerythrocytic Babesia parasites. It is a well-known illness in companion animals and livestock, resulting in substantial economic losses in the cattle industry. Babesiosis is also recognized as an emerging zoonosis of humans in many countries worldwide. There is no vaccine against human babesiosis. Currently, preventive measures are focused on vector avoidance. Although not always effective, treatment includes antimicrobial therapy and exchange transfusion. In this review, we discuss the host's immune response to the parasite, vaccines being used to prevent babesiosis in animals, and lessons from malaria vaccine development efforts to inform the development of a human babesiosis vaccine. An effective human vaccine would be a significant advance towards curtailing this rapidly emerging disease.

Infection with malaria parasites continues to be a major global public health issue. While current control measures have enabled a significant decrease in morbidity and mortality over the last 20 years, additional tools will be required if we are to progress toward malaria parasite eradication. Malaria vaccine research has focused on the development of subunit vaccines; however, more recently, interest in whole-parasite vaccines has reignited. Whole-parasite vaccines enable the presentation of a broad repertoire of antigens to the immune system, which limits the impact of antigenic polymorphism and genetic restriction of the immune response. We previously reported that whole-parasite vaccines can be prepared using chemically attenuated parasites within intact red blood cells or using killed parasites in liposomes, although liposomes were less immunogenic than attenuated parasites. If they could be frozen or freeze-dried and be made more immunogenic, liposomal vaccines would be ideal for vaccine deployment in areas where malaria is endemic. Here, we develop and evaluate a Plasmodium yoelii liposomal vaccine with enhanced immunogenicity and efficacy due to incorporation of TLR4 agonist, 3D(6-acyl) PHAD, and mannose to target the liposome to antigen-presenting cells. Following vaccination, mice were protected, and strong cellular immune responses were induced, characterized by parasite-specific splenocyte proliferation and a mixed Th1/Th2/Th17 cytokine response. Parasite-specific antibodies were induced, predominantly of the IgG1 subclass. CD4+ T cells and gamma interferon were critical components of the protective immune response. This study represents an important development toward evaluation of this whole-parasite blood-stage vaccine in a phase I clinical trial.

Malaria is a devastating parasitic disease for which a vaccine is urgently needed. Seminal studies in the 1940s demonstrated that a whole parasite malaria vaccine could induce protective immunity i...

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

Background The pathogenesis of malaria in pregnancy (MiP) involves accumulation of P. falciparum-infected red blood cells (pRBCs) in the placenta, contributing to poor pregnancy outcomes. Parasite accumulation is primarily mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). Magnitude of IgG to pRBCs has been associated with reduced risk of MiP in some studies, but associations have been inconsistent. Further, antibody effector mechanisms are poorly understood, and the role of antibody complement interactions is unknown. Methods Studying a longitudinal cohort of pregnant women (n=302) from a malaria-endemic province in Papua New Guinea (PNG), we measured the ability of antibodies to fix and activate complement using placental binding pRBCs and PfEMP1 recombinant domains. We determined antibody-mediated complement inhibition of pRBC binding to the placental receptor, chondroitin sulfate A (CSA), and associations with protection against placental parasitemia. Results Some women acquired antibodies that effectively promoted complement fixation on placental-binding pRBCs. Complement fixation correlated with IgG1 and IgG3 antibodies, which dominated the response. There was, however, limited evidence for membrane attack complex activity or pRBC lysis or killing. Importantly, a higher magnitude of complement fixing antibodies was prospectively associated with reduced odds of placental infection at delivery. Using genetically modified P. falciparum and recombinant PfEMP1 domains, we found that complement-fixing antibodies primarily targeted a specific variant of PfEMP1 (known as VAR2CSA). Furthermore, complement enhanced the ability of antibodies to inhibit pRBC binding to CSA, which was primarily mediated by complement C1q protein. Conclusions These findings provide new insights into mechanisms mediating immunity to MiP and reveal potential new strategies for developing malaria vaccines that harness antibody-complement interactions.

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BackgroundThe pathogenesis of malaria in pregnancy (MiP) involves accumulation of P. falciparum-infected red blood cells (pRBCs) in the placenta, contributing to poor pregnancy outcomes. Parasite accumulation is primarily mediated by P. falciparum erythrocyte membrane 1 (PfEMP1). Magnitude of IgG to pRBCs has been associated with reduced risk of MiP in some studies, but associations have been inconsistent. Further, antibody effector mechanisms are poorly understood, and the role of antibody complement interactions is unknown.MethodsStudying a longitudinal cohort of pregnant women (n=302) from a malaria-endemic province in Papua New Guinea (PNG), we measured the ability of antibodies to fix and activate complement using placental binding pRBCs and PfEMP1 recombinant domains. We determined antibody-mediated complement inhibition of pRBC binding to the placental receptor, chondroitin sulfate A (CSA) and associations with protection against placental parasitaemia.ResultsSome women acquired antibodies that effectively promoted complement fixation on placental-binding pRBCs. Complement fixation correlated with IgG1 and IgG3 antibodies, which dominated the response. There was, however, limited evidence for membrane attack complex activity or pRBC lysis or killing. Importantly, a higher magnitude of complement fixing antibodies was prospectively associated with reduced odds of placental infection at delivery. Using genetically-modified P. falciparum and recombinant PfEMP1 domains, we found that complement-fixing antibodies primarily targeted a specific variant of PfEMP1 (known as VAR2CSA). Furthermore, complement enhanced the ability of antibodies to inhibit pRBC binding to CSA, which was primarily mediated by complement C1q protein.ConclusionThese findings provide new insights into mechanisms mediating immunity to MiP and reveal potential new strategies for developing malaria vaccines that harness antibody-complement interactions.

IntroductionPregnant women have an increased risk of P. falciparum infection, which is associated with low birth weight and preterm delivery. VAR2CSA, a variant surface antigen expressed on the parasitized erythrocyte surface, enables sequestration in the placenta. Few studies have prospectively examined relationships between antibody responses during pregnancy and subsequent adverse birth outcomes, and there are limited data outside Africa.MethodsLevels of IgG against VAR2CSA domains (DBL3; DBL5) and a VAR2CSA-expressing placental-binding P. falciparum isolate (PfCS2-IE) were measured in 301 women enrolled at their first visit to antenatal care which occurred mid-pregnancy (median = 26 weeks, lower and upper quartiles = 22, 28). Associations between antibody levels at enrolment and placental infection, birthweight and estimated gestational age at delivery were assessed by linear and logistic regression with adjustment for confounders. For all outcomes, effect modification by gravidity and peripheral blood P. falciparum infection at enrolment was assessed.ResultsAmong women who had acquired P. falciparum infection at enrolment, those with higher levels of VAR2CSA antibodies (75th percentile) had infants with higher mean birthweight (estimates varied from +35g to +149g depending on antibody response) and reduced adjusted odds of placental infection (aOR estimates varied from 0.17 to 0.80), relative to women with lower levels (25th percentile) of VAR2CSA antibodies. However, among women who had not acquired an infection at enrolment, higher VAR2CSA antibodies were associated with increased odds of placental infection (aOR estimates varied from 1.10 to 2.24).ConclusionsWhen infected by mid-pregnancy, a better immune response to VAR2CSA-expressing parasites may contribute to protecting against adverse pregnancy outcomes.

Objectives A major COVID‐19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor‐binding domain (RBD) and angiotensin‐converting enzyme 2 (ACE2). These vaccines will also induce T‐cell responses. However, concerns were raised that aberrant vaccine‐induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology. Methods We procured a series of overlapping 20‐amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID‐19 convalescent patients. Identified epitopes were conjugated to diphtheria‐toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2. Results Seven putative vaccine epitopes were identified. Memory B‐cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. Conclusion COVID‐19 convalescent patients have SARS‐CoV‐2‐specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope‐specific antibodies synergistically block RBD–ACE2 interaction., We identified SARS‐COV‐2 receptor‐binding domain (RBD)‐specific antibodies in convalescent patients, specificities of which could be defined with short peptides. These peptides when used as immunogens, induced neutralising antibodies which synergistically blocked RBD and ACE 2 interaction. Our approach will aid the design of a vaccine containing minimal antigenic material, thus improving the vaccine's safety profile.

The apicoplast, a relic plastid found in most Apicomplexan parasites, is a notable drug target. Certain antibiotics elicit a delayed death phenotype by targeting this organelle. Here, we review apicoplast-targeting drugs and their targets, particularly those that cause delayed death, and highlight its potential uses in malaria vaccine development.

Background Low birth weight (LBW) and preterm birth (PTB) are major contributors to infant mortality and chronic childhood morbidity. Understanding factors that contribute to or protect against these adverse birth outcomes is an important global health priority. Anaemia and iron deficiency are common in malaria-endemic regions, but there are concerns regarding the value of iron supplementation among pregnant women in malaria-endemic areas due to reports that iron supplementation may increase the risk of malaria. There is a lack of evidence on the impact of iron deficiency on pregnancy outcomes in malaria-endemic regions. Methods We determined iron deficiency in a cohort of 279 pregnant women in a malaria-endemic area of Papua New Guinea. Associations with birth weight, LBW and PTB were estimated using linear and logistic regression. A causal model using sequential mediation analyses was constructed to assess the association between iron deficiency and LBW, either independently or mediated through malaria and/or anaemia. Results Iron deficiency in pregnant women was common (71% at enrolment) and associated with higher mean birth weights (230 g; 95% confidence interval, CI 118, 514; p

Background: Overcoming antigenic diversity is a key challenge in the development of effective Plasmodium falciparum malaria vaccines. Strategies that promote the generation of antibodies targeting conserved epitopes of vaccine antigens may provide protection against diverse parasites strains. Understanding differences between vaccine-induced and naturally acquired immunity is important to achieving this goal. Methods: We analyzed antibodies generated in a phase 1 human vaccine trial, MSP2-C1, which included 2 allelic forms of MSP2, an abundant vaccine antigen on the merozoite surface. Vaccine-induced responses were assessed for functional activity against multiple parasite strains, and cross-reactivity of antibodies was determined using competition ELISA and epitope mapping approaches. Results: Vaccination induced cytophilic antibody responses with strain-transcending opsonic phagocytosis and complement-fixing function. In contrast to antibodies acquired via natural infection, vaccine-induced antibodies were directed towards conserved epitopes at the C-terminus of MSP2, whereas naturally acquired antibodies mainly targeted polymorphic epitopes. Functional activity of C-terminal–targeted antibodies was confirmed using monoclonal antibodies that promoted opsonic phagocytosis against multiple parasite strains. Conclusion: Vaccination generated markedly different responses to polymorphic antigens than naturally acquired immunity and targeted conserved functional epitopes. Induction of antibodies targeting conserved regions of malaria antigens provides a promising vaccine strategy to overcome antigenic diversity for developing effective malaria vaccines.

Here, the anti-malarial activity of two gold(I) phosphine compounds auranofin and [Au(d2pype)2]Cl (where d2pype is 1,2-bis(di-2-pyridylphosphino)ethane), were examined to inform their use as potential drugs and malaria parasite-attenuating agents. In vitro, the gold compounds were active against Plasmodium falciparum and P. knowlesi as well as the rodent parasite P. chabaudi AS. Attenuation of the parasite was observed when mice were inoculated with P. chabaudi AS infected red blood cells treated in vitro with [Au(d2pype)2]Cl (1 or 2 μM) or auranofin (2 μM) for 2 or 3 h. Quantitative PCR data showed persistence of low levels of parasite DNA up to 8 days post inoculation. In some experiments, there was microscopically detectable parastiemia following inoculation which subsequently cleared. Following 1 or 3 doses of gold compound-treated parasitized red blood cells (pRBCs), protection was not observed when these mice were subsequently challenged with wild type P. chabaudi AS. In experiments where microscopically detectable parasites were observed following in vivo inoculation, mice were subsequently fully protected against a challenge infection with wildtype parasites. In an infect-and-treat rodent model, the gold compounds were unable to inhibit P. chabaudi AS growth in vivo when administered orally. Gold compounds act via the inhibition of antioxidant systems which are critical in the pathogen's survival from attack by the host oxidants. In vitro, they directly inhibit the parasite thioredoxin reductase, hence the observed suppressive activity. On the other hand, in vivo, the gold compounds may not be readily available for absorption and thus pharmacokinetic studies will be required to further examine drug bioavailability following administration. With structural differences in redox mechanisms of P. falciparum and the human host being identified, gold compounds can be better designed to more efficiently target and selectively inhibit the parasite.

After many decades of research, an effective vaccine for malaria is still not available. Most research efforts have focused on identifying a key target antigen and then using powerful adjuvants to generate specific antibodies that can block parasites from entering host cells (hepatocytes, red blood cells). However, the inability to generate sufficiently potent antibody responses has led to significant disappointment with current vaccine programs. An additional challenge for sub-unit vaccines is that key vaccine antigens are highly polymorphic. These challenges have spurred radically different approaches to malaria vaccine development. Many of these involve the use of "whole parasites"-either extracted from mosquitoes or cultured. With these, every parasite molecule for that particular strain is included in the vaccine. This strategy is showing great promise following several clinical trials with irradiated sporozoites. However, a whole-parasite approach to a blood stage vaccine has not advanced as quickly. This article outlines the history, the different approaches that are being taken and the challenges associated with whole parasite blood stage vaccines and discusses recent exciting developments as these vaccines now move into the clinic.

The development of a blood-stage malaria vaccine has largely focused on the subunit approach. However, the limited success of this strategy, mainly due to antigenic polymorphism and the failure to maintain potent parasite-specific immune responses, indicates that other approaches must be considered. Whole parasite (WP) vaccines offer many advantages over sub-units; they represent every antigen on the organism, thus limiting the effects of antigenic polymorphism, and similarly they compensate for individual Immune-Response (Ir) gene-regulated non-responsiveness to any particular antigen. From a development perspective, they negate the need to identify and compare the relative efficacies of individual candidate antigens. WP vaccines induce protective immunity that is largely cell-mediated. However, WP blood-stage vaccines present a number of challenges for the development pathway. Key issues are cryopreservation and storage and the possible induction of antibodies against red blood cell surface antigens, even if the parasites are grown in blood group O, Rh negative blood. Here, we used a novel adaptation of an immunomagnetic method from STEMCELL™ Technologies to remove the red cell membranes from human red blood cells parasitized with P. falciparum. We then used these antigens to construct liposomes which were modified to present mannose on their membrane to target the liposome to antigen presenting cells. We then compared the immunogenicity of freshly prepared and lyophilized liposome vaccines. Following vaccination of mice, liposomes induced significantly lower antibody responses to human red cells but potent strain- and species-transcending cell-mediated immune responses to parasite antigens. These data support transitioning the P. falciparum liposomal vaccine into clinical studies.

In the development of vaccines, the ability to initiate both innate and subsequent adaptive immune responses need to be considered. Live attenuated vaccines achieve this naturally, while inactivated and sub-unit vaccines generally require additional help provided through delivery systems and/or adjuvants. Liposomes present an attractive adjuvant/delivery system for antigens. Here, we review the key aspects of immunity against Plasmodium parasites, liposome design considerations and their current application in the development of a malaria vaccine.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

Plasmodium falciparum is the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-grade P. falciparum parasites (NF54, 7G8, and 3D7B) and ex vivo -derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants for in vitro characterization. All parasites were infective in vivo . However, infectivity of NF54 was dramatically reduced. In vitro characterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggesting P. falciparum erythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence.

Malaria is a disease caused by a protozoan of the Plasmodium genus and results in 0.5–0.7 million deaths per year. Increasing drug resistance of the parasite and insecticide resistance of mosquitoes necessitate alternative control measures. Numerous vaccine candidates have been identified but none have been able to induce robust, long-lived protection when evaluated in malaria endemic regions. Rodent studies have demonstrated that chemically attenuated blood stage parasites can persist at sub-patent levels and induce homologous and heterologous protection against malaria. Parasite-specific cellular responses were detected, with protection dependent on CD4+ T cells. To investigate this vaccine approach for Plasmodium falciparum, we characterised the persistence and immunogenicity of chemically attenuated P. falciparum FVO strain parasites (CAPs) in non-splenectomised Aotus nancymaae monkeys following administration of a single dose. Control monkeys received either normal red blood cells or wild-type parasites followed by drug treatment. Chemical attenuation was performed using tafuramycin A, which irreversibly binds to DNA. CAPs were detected in the peripheral blood for up to 2 days following inoculation as determined by thick blood smears, and for up to 8 days as determined by quantitative PCR. Parasite-specific IgG was not detected in monkeys that received CAPs; however, in vitro parasite-specific T cell proliferation was observed. Following challenge, the CAP monkeys developed an infection; however, one CAP monkey and the infection and drug-cure monkeys showed partial or complete resistance. These experiments lay the groundwork for further assessment of CAPs as a potential vaccine against malaria.

The development of a vaccine is essential for the elimination of malaria. However, despite many years of effort, a successful vaccine has not been achieved. Most subunit vaccine candidates tested in clinical trials have provided limited efficacy, and thus attenuated whole-parasite vaccines are now receiving close scrutiny. Here, we test chemically attenuated Plasmodium yoelii 17X and demonstrate significant protection following homologous and heterologous blood-stage challenge. Protection against blood-stage infection persisted for at least 9 months. Activation of both CD4 + and CD8 + T cells was shown after vaccination; however, in vivo studies demonstrated a pivotal role for both CD4 + T cells and B cells since the absence of either cell type led to loss of vaccine-induced protection. In spite of significant activation of circulating CD8 + T cells, liver-stage immunity was not evident. Neither did vaccine-induced CD8 + T cells contribute to blood-stage protection; rather, these cells contributed to pathogenesis, since all vaccinated mice depleted of both CD4 + and CD8 + T cells survived a challenge infection. This study provides critical insight into whole-parasite vaccine-induced immunity and strong support for testing whole-parasite vaccines in humans.

SUMMARYNaturally acquired immunity to the blood-stage of the malaria parasite develops slowly in areas of high endemicity, but is not sterilizing. It manifests as a reduction in parasite density and clinical symptoms. Immunity as a result of blood-stage vaccination has not yet been achieved in humans, although there are many animal models where vaccination has been successful. The development of a blood-stage vaccine has been complicated by a number of factors including limited knowledge of human-parasite interactions and which antigens and immune responses are critical for protection. Opinion is divided as to whether this vaccine should aim to accelerate the acquisition of responses acquired following natural exposure, or whether it should induce a different response. Animal and experimental human models suggest that cell-mediated immune responses can control parasite growth, but these responses can also contribute to significant immunopathology if unregulated. They are largely ignored in most blood-stage malaria vaccine development strategies. Here, we discuss key observations relating to cell-mediated immune responses in the context of experimental human systems and field studies involving naturally exposed individuals and how this may inform the development of a blood-stage malaria vaccine.

Naturally acquired immunity to malaria is robust and protective against all strains of the same species of Plasmodium. This develops as a result of repeated natural infection, taking several years to develop., Naturally acquired immunity to malaria is robust and protective against all strains of the same species of Plasmodium. This develops as a result of repeated natural infection, taking several years to develop. Evidence suggests that apoptosis of immune lymphocytes due to uncontrolled parasite growth contributes to the slow acquisition of immunity. To hasten and augment the development of natural immunity, we studied controlled infection immunization (CII) using low-dose exposure to different parasite species (Plasmodium chabaudi, P. yoelii, or P. falciparum) in two rodent systems (BALB/c and C57BL/6 mice) and in human volunteers, with drug therapy commencing at the time of initiation of infection. CIIs with infected erythrocytes and in conjunction with doxycycline or azithromycin, which are delayed death drugs targeting the parasite’s apicoplast, allowed extended exposure to parasites at low levels. In turn, this induced strong protection against homologous challenge in all immunized mice. We show that P. chabaudi/P. yoelii infection initiated at the commencement of doxycycline therapy leads to cellular or antibody-mediated protective immune responses in mice, with a broad Th1 cytokine response providing the best correlate of protection against homologous and heterologous species of Plasmodium. P. falciparum CII with doxycycline was additionally tested in a pilot clinical study (n = 4) and was found to be well tolerated and immunogenic, with immunological studies primarily detecting increased cell-associated immune responses. Furthermore, we report that a single dose of the longer-acting drug, azithromycin, given to mice (n = 5) as a single subcutaneous treatment at the initiation of infection controlled P. yoelii infection and protected all mice against subsequent challenge.

Background The continuing morbidity and mortality associated with infection with malaria parasites highlights the urgent need for a vaccine. The efficacy of sub-unit vaccines tested in clinical trials in malaria-endemic areas has thus far been disappointing, sparking renewed interest in the whole parasite vaccine approach. We previously showed that a chemically attenuated whole parasite asexual blood-stage vaccine induced CD4+ T cell-dependent protection against challenge with homologous and heterologous parasites in rodent models of malaria. Methods In this current study, we evaluated the immunogenicity and safety of chemically attenuated asexual blood-stage Plasmodium falciparum (Pf) parasites in eight malaria-naïve human volunteers. Study participants received a single dose of 3 × 107 Pf pRBC that had been treated in vitro with the cyclopropylpyrolloindole analogue, tafuramycin-A. Results We demonstrate that Pf asexual blood-stage parasites that are completely attenuated are immunogenic, safe and well tolerated in malaria-naïve volunteers. Following vaccination with a single dose, species and strain transcending Plasmodium-specific T cell responses were induced in recipients. This included induction of Plasmodium-specific lymphoproliferative responses, T cells secreting the parasiticidal cytokines, IFN-γ and TNF, and CD3+CD45RO+ memory T cells. Pf-specific IgG was not detected. Conclusions This is the first clinical study evaluating a whole parasite blood-stage malaria vaccine. Following administration of a single dose of completely attenuated Pf asexual blood-stage parasites, Plasmodium-specific T cell responses were induced while Pf-specific antibodies were not detected. These results support further evaluation of this chemically attenuated vaccine in humans. Trial registration Trial registration: ACTRN12614000228684. Registered 4 March 2014.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1β, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation., MIF is a cytokine best known for its modulatory effect on expression of proinflammatory cytokines. Here the authors show that MIF facilitates the NLRP3–vimentin interaction, resulting in Nlrp3 inflammasome activation.

Objectives: Blood stage malaria parasites attenuated with seco‐cyclopropyl pyrrolo indole (CPI) analogues induce robust immunity in mice to homologous and heterologous malaria parasites and are being considered for the development of a human vaccine. However, it is not understood how attenuated parasites induce immunity. We showed that following vaccination, parasite DNA persisted in blood for several months, raising the possibility that ongoing immune stimulation may be critical. However, parasites were not seen microscopically beyond 24 h postvaccination. We aimed to provide a mechanistic understanding of immune induction. Methods: Mice were vaccinated with chemically attenuated Plasmodium chabaudi parasites. PCR and adoptive transfer studies were used to determine the presence of parasites and antigen in vivo. In other experiments, Plasmodium falciparum parasitised red blood cells were attenuated in vitro and RNA and antigen expression studied. Results: We show that blood transferred from vaccinated mice into naive mice activates T cells and induces complete protective immunity in the recipient mice strongly suggesting that there is persistence of parasite antigen postvaccination. This is supported by the presence of parasite RNA in vaccinated mice and both RNA and antigen expression in P. falciparum cultures treated with CPI drugs in vitro. In addition, drugs that block parasite growth also prevent the induction of immunity in vaccinated mice, indicating that some growth of attenuated parasites is required for immune induction. Conclusions: Attenuated parasites persist at submicroscopic levels in the blood of mice postvaccination with the ability to activate T cells and induce ongoing protective immune responses.

Controlled human malaria infection (CHMI) entails deliberate infection with malaria parasites either by mosquito bite or by direct injection of sporozoites or parasitized erythrocytes. When required, the resulting blood-stage infection is curtailed by the administration of antimalarial drugs. Inducing a malaria infection via inoculation with infected blood was first used as a treatment (malariotherapy) for neurosyphilis in Europe and the United States in the early 1900s. More recently, CHMI has been applied to the fields of malaria vaccine and drug development, where it is used to evaluate products in well-controlled early-phase proof-of-concept clinical studies, thus facilitating progression of only the most promising candidates for further evaluation in areas where malaria is endemic. Controlled infections have also been used to immunize against malaria infection. Historically, CHMI studies have been restricted by the need for access to insectaries housing infected mosquitoes or suitable malaria-infected individuals. Evaluation of vaccine and drug candidates has been constrained in these studies by the availability of a limited number of Plasmodium falciparum isolates. Recent advances have included cryopreservation of sporozoites, the manufacture of well-characterized and genetically distinct cultured malaria cell banks for blood-stage infection, and the availability of Plasmodium vivax -specific reagents. These advances will help to accelerate malaria vaccine and drug development by making the reagents for CHMI more widely accessible and also enabling a more rigorous evaluation with multiple parasite strains and species. Here we discuss the different applications of CHMI, recent advances in the use of CHMI, and ongoing challenges for consideration.

Despite a century of research focused on the development and implementation of effective control strategies, infection with the malaria parasite continues to result in significant morbidity and mortality worldwide. An effective malaria vaccine is considered by many to be the definitive solution. Yet, after decades of research, we are still without a vaccine that is capable of inducing robust, long lasting protection in naturally exposed individuals. Extensive sub-unit vaccine development focused on the blood stage of the malaria parasite has thus far yielded disappointing results. There is now a renewed focus on whole parasite vaccine strategies, particularly as they may overcome some of the inherent weaknesses deemed to be associated with the sub-unit approach. This review discusses the whole parasite vaccine strategy focusing on the blood stage of the malaria parasite, with an emphasis on recent advances and challenges in the development of killed and live attenuated vaccines.

Individuals in areas of Plasmodium falciparum endemicity develop immunity to malaria after repeated exposure. Knowledge of the acquisition and nature of protective immune responses to P. falciparum is presently limited, particularly for young children. We examined antibodies (IgM, IgG, and IgG subclasses) to merozoite antigens and their relationship to the prospective risk of malaria in children 1 to 4 years of age in a region of malaria endemicity in Papua New Guinea. IgG, IgG1, and IgG3 responses generally increased with age, were higher in children with active infection, and reflected geographic heterogeneity in malaria transmission. Antigenic properties, rather than host factors, appeared to be the main determinant of the type of IgG subclass produced. High antibody levels were not associated with protection from malaria; in contrast, they were typically associated with an increased risk of malaria. Adjustment for malaria exposure, using a novel molecular measure of the force of infection by P. falciparum , accounted for much of the increased risk, suggesting that the antibodies were markers of higher exposure to P. falciparum . Comparisons between antibodies in this cohort of young children and in a longitudinal cohort of older children suggested that the lack of protective association was explained by lower antibody levels among young children and that there is a threshold level of antibodies required for protection from malaria. Our results suggest that in populations with low immunity, such as young children, antibodies to merozoite antigens may act as biomarkers of malaria exposure and that, with increasing exposure and responses of higher magnitude, antibodies may act as biomarkers of protective immunity.

During gestational malaria, Plasmodium falciparum-infected erythrocytes can sequester within the placenta, contributing to poor pregnancy outcomes, especially low birth weight. In children and non-pregnant adults, pigmented leukocytes may serve as markers of sequestered parasite burden and predict clinical outcomes. Here, we investigated circulating pigmented leukocyte numbers as predictors of clinical outcomes in pregnant women presenting with malaria at enrolment. The number of circulating pigmented neutrophils at enrolment negatively correlated with birth weight (Rho=-25, P=.04), suggesting these cells may have a pathogenic role in, and could serve as prognostic markers for, malaria-associated low birth weight.

Introduction During pregnancy, immunoglobulin G (IgG) is transferred from the mother to the fetus, providing protection from disease in early infancy. Plasmodium falciparum infections may reduce maternofetal antibody transfer efficiency, but mechanisms remain unclear. Methods Mother-cord paired serum samples collected at delivery from Papua New Guinea (PNG) and the Thailand-Myanmar Border Area (TMBA) were tested for IgG1 and IgG3 to four P. falciparum antigens and measles antigen, as well as total serum IgG. Multivariable linear regression was conducted to assess the association of peripheral P. falciparum infection during pregnancy or placental P. falciparum infection assessed at delivery with maternofetal antibody transfer efficiency. Path analysis assessed the extent to which associations between P. falciparum infection and antibody transfer were mediated by gestational age at delivery or levels of maternal total serum IgG. Results Maternofetal antibody transfer efficiency of IgG1 and IgG3 was lower in PNG compared to TMBA (mean difference in cord antibody levels (controlling for maternal antibody levels) ranged from -0.88 to 0.09, median of -0.20 log2 units). Placental P. falciparum infections were associated with substantially lower maternofetal antibody transfer efficiency in PNG primigravid women (mean difference in cord antibody levels (controlling for maternal antibody levels) ranged from -0.62 to -0.10, median of -0.36 log2 units), but not multigravid women. The lower antibody transfer efficiency amongst primigravid women with placental infection was only partially mediated by gestational age at delivery (proportion indirect effect ranged from 0% to 18%), whereas no mediation effects of maternal total serum IgG were observed. Discussion Primigravid women may be at risk of impaired maternofetal antibody transport with placental P. falciparum infection. Direct effects of P. falciparum on the placenta, rather than earlier gestational age and elevated serum IgG, are likely responsible for the majority of the reduction in maternofetal antibody transfer efficiency with placental infection.

Background Four studies previously indicated that the effect of malaria infection during pregnancy on the risk of low birthweight (LBW, In a meta-analysis using individual participant data, Jordan Cates and colleagues explore potential interactions between malaria and nutrition during pregnancy and their associations with low birthweight., Author summary Why was this study done? More than 125 million pregnant women are at risk of malaria in pregnancy annually, producing detrimental effects on maternal, newborn, and infant health. Maternal undernutrition is estimated to be responsible for 800,000 newborn deaths annually. Prior evidence from 4 small studies indicated that the harmful impact of malaria on fetal growth and birthweight (BW) may depend upon the macronutrient nutritional status of the mother. If malaria and maternal undernutrition have synergistic negative impacts on pregnancy outcomes, interventions targeted to high-risk women might provide substantial public benefit. The present study provides a robust assessment of potential malaria–nutrition interactions in pregnancy and overcomes size and methodological limitations of earlier exploratory studies. What did the researchers do and find? We present a large, pooled analysis of individual participant data from 13 studies conducted in sub-Saharan Africa and the Western Pacific investigating the interaction between maternal malaria infection and malnutrition on the risk of low birthweight (LBW) and reduced mean BW. The findings suggest that women who are both infected with malaria and malnourished are at greater risk of LBW than their uninfected, well-nourished counterparts. However, the study found no conclusive evidence of interaction between the 2, i.e., the impact of malaria on BW was independent of the macronutrient nutritional status of the mother. Subgroup analyses did find that studies conducted just in Africa had slight evidence of interaction, but this was not consistent throughout all analyses. What do these findings mean? Although there was no overall evidence of malaria–nutrition interactions, more than 1 in 3 pregnant women suffered from malaria and/or undernutrition, emphasizing the importance of joint approaches to decrease maternal malaria and improve nutrition to minimize adverse pregnancy outcomes.

Malaria vaccine development has been dominated by the subunit approach; however, many subunit vaccine candidates have had limited efficacy in settings of malaria endemicity. As our search for an efficacious malaria vaccine continues, the development of a whole-organism vaccine is now receiving much scrutiny. One strategy currently being explored in the development of a whole-organism vaccine involves chemical attenuation of the malaria parasite. In vivo and in vitro chemical attenuation of both liver-stage and blood-stage Plasmodium parasites has been investigated. Here, we discuss both approaches of chemical attenuation in the development of a whole-organism vaccine against malaria.

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity.

Despite decades of effort, an effective malaria vaccine has not yet been developed. Continued issues with the efficacy of the subunit vaccine approach have led to a renewed interest in the use of the whole-parasite vaccine approach for malaria. This approach maximizes the number of antigens presented to the immune system; antigenic diversity will thus have limited impact on efficacy. In this chapter we discuss the use of whole blood-stage parasites in the development of a malaria vaccine. This approach is being developed using killed or attenuated parasites. Attenuation methods include γ-irradiation, genetic deletion/disruption, or chemical treatment of parasites. We discuss these strategies and review the immune responses induced following vaccination. This is an exciting field; however, many challenges remain including issues relating to the use of red blood cells in the vaccine and suitable methods for vaccine storage.

Malaria remains one of the most prevalent and lethal human infectious diseases worldwide. A comprehensive characterization of antibody responses to blood stage malaria is essential to support the development of future vaccines, sero-diagnostic tests, and sero-surveillance methods. We constructed a proteome array containing 4441 recombinant proteins expressed by the blood stages of the two most common human malaria parasites, P. falciparum (Pf) and P. vivax (Pv), and used this array to screen sera of Papua New Guinea children infected with Pf, Pv, or both (Pf/Pv) that were either symptomatic (febrile), or asymptomatic but had parasitemia detectable via microscopy or PCR. We hypothesized that asymptomatic children would develop antigen-specific antibody profiles associated with antidisease immunity, as compared with symptomatic children. The sera from these children recognized hundreds of the arrayed recombinant Pf and Pv proteins. In general, responses in asymptomatic children were highest in those with high parasitemia, suggesting that antibody levels are associated with parasite burden. In contrast, symptomatic children carried fewer antibodies than asymptomatic children with infections detectable by microscopy, particularly in Pv and Pf/Pv groups, suggesting that antibody production may be impaired during symptomatic infections. We used machine-learning algorithms to investigate the relationship between antibody responses and symptoms, and we identified antibody responses to sets of Plasmodium proteins that could predict clinical status of the donors. Several of these antibody responses were identified by multiple comparisons, including those against members of the serine enriched repeat antigen family and merozoite protein 4. Interestingly, both P. falciparum serine enriched repeat antigen-5 and merozoite protein 4 have been previously investigated for use in vaccines. This machine learning approach, never previously applied to proteome arrays, can be used to generate a list of potential seroprotective and/or diagnostic antigens candidates that can be further evaluated in longitudinal studies.