Article

Superior Antimalarial Immunity after Vaccination with Late Liver Stage-Arresting Genetically Attenuated Parasites

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

While subunit vaccines have shown partial efficacy in clinical trials, radiation-attenuated sporozoites (RAS) remain the "gold standard" for sterilizing protection against Plasmodium infection in human vaccinees. The variability in immunogenicity and replication introduced by the extensive, random DNA damage necessary to generate RAS could be overcome by genetically attenuated parasites (GAP) designed via gene deletion to arrest at defined points during liver-stage development. Here, we demonstrate the principle that late liver stage-arresting GAP induce larger and broader CD8 T cell responses that provide superior protection in inbred and outbred mice compared to RAS or early-arresting GAP immunizations. Late liver stage-arresting GAP also engender high levels of cross-stage and cross-species protection and complete protection when administered by translationally relevant intradermal or subcutaneous routes. Collectively, our results underscore the potential utility of late liver stage-arresting GAP as broadly protective next-generation live-attenuated malaria vaccines and support their potential as a powerful model for identifying antigens to generate cross-stage protection.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

Supplementary resource (1)

... While the ability of CS-based vaccination to partially limit clinical malaria infection in the field is a major achievement, the modest and rapidly waning efficacy of RTS,S stresses the urgency to develop vaccines with higher and more durable protection. 5 An alternative to subunit vaccines is the use of wholesporozoite (WSp) approaches, based on the generation of immunity against Plasmodium pre-erythrocytic stages following immunisation with infective sporozoites under conditions that prevent the appearance of clinical symptoms, including radiationattenuated sporozoites (RAS), [6][7][8] genetically attenuated parasites (GAP), [9][10][11][12][13] and immunisation with non-attenuated sporozoites in combination with chemoprophylaxis (CPS). [14][15][16] Although CS has been proposed to play an important protective role in WSp vaccines, complete protection following P. yoelii RAS immunization has been shown to occur in transgenic mice that are T-cell tolerant to CS and cannot produce antibodies. ...
... Accordingly, later liver stage-arresting parasites, such as some GAP parasites, and those completing liver stage development, such as the CPS approach, seem to trigger antimalarial immunity superior to that elicited by early-arresting variants. 11,19 Nonetheless, the most advanced WSp approach to human vaccination relies on the intravenous administration of the PfSPZ Vaccine, composed of aseptic, purified, cryopreserved PfRAS. [20][21][22][23] While all current WSp human vaccination strategies rely on the use of Pf sporozoites, alternative WSp vaccines can also be envisaged. ...
... 39 In other studies, mice immunized by irradiated or genetically attenuated (p36p − ) Pb sporozoites were shown to provide partial protection against challenge by P. yoelii (Py) sporozoites, 40 and 100% sterilizing crossspecies protection was observed when mice were immunized with a late liver stage-arresting genetically attenuated Py parasite (Pyabb/f) and challenged with Pb sporozoites. 11 Sedegah et al. 41 reported T cell-mediated protection of mice immunized with attenuated Pb or Py sporozoites against heterologous challenge with Py or Pb, respectively. The requirement for T cells to provide protection against heterologous parasites was consistent with the previous observation that a PyCS CD8 + T cell clone was protective against challenge with the heterologous Pb sporozoites. ...
Article
Full-text available
There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.
... Whole-sporozoite immunization of mice, nonhuman primates, and humans engenders sterilizing pre-erythrocytic immunity (60). Moreover, RC whole-sporozoite vaccines engender superior immunity and engender greater protection at much lower doses than RD sporozoite vaccines (49,61,62). What drives the robust pre-erythrocytic immunity afforded by whole-sporozoite vaccines, and how can these immune responses be further enhanced to improve vaccine efficacy? ...
... RAS immunization drives the production of both central memory CD8 T cells and effector memory CD8 T cells (T EM ). However, immunization with viral vectors bearing a Plasmodium protein generated large numbers of central memory T cells, that did not confer protection (80), in contrast to immunization strategies that induced high levels of T EM that correlated well with protection (61,81). Finally, recent studies with RAS-immunized mice indicate that noncirculating, anti-Plasmodium liver resident memory CD8 T cells (T RM ) are critical to the protection afforded by whole-sporozoite immunization (82)(83)(84). ...
... Immunization via direct i.v. inoculation of wholesporozoites affords greater protection from a sporozoite challenge than s.c. and intradermal inoculation (26,61). Moreover, RC sporozoite immunization generates superior T EM than RD sporozoite immunization (61). ...
Article
Full-text available
A highly efficacious malaria vaccine that prevents disease and breaks the cycle of infection remains an aspirational goal of medicine. Whole parasite vaccines based on the sporozoite forms of the parasite that target the clinically silent pre-erythrocytic stages of infection have emerged as one of the leading candidates. In animal models of malaria, these vaccines elicit potent neutralizing Ab responses against the sporozoite stage and cytotoxic T cells that eliminate parasite-infected hepatocytes. Among whole-sporozoite vaccines, immunization with live, replication-competent whole parasites engenders superior immunity and protection when compared with live replication-deficient sporozoites. As such, the genetic design of replication-competent vaccine strains holds the promise for a potent, broadly protective malaria vaccine. In this report, we will review the advances in whole-sporozoite vaccine development with a particular focus on genetically attenuated parasites both as malaria vaccine candidates and also as valuable tools to interrogate protective immunity against Plasmodium infection.
... In both attenuation models, Plasmodium sporozoites invade hepatocytes within vacuoles, then cease growth completely and do not cause Plasmodium infection of the blood [2,3]. The protection conferred by attenuated sporozoites was confirmed to be mainly mediated by CD8+ T cells targeting LS antigens and not by antigens presented on the surface of migrating sporozoites [17][18][19][20][21]. Recent studies pointed out that when compared to attenuated strains that cease their LS development early, attenuated strains that grow longer in hepatocytes before ceasing growth led to more significant protective immune responses [17]. ...
... In both attenuation models, Plasmodium sporozoites invade hepatocytes within vacuoles, then cease growth completely and do not cause Plasmodium infection of the blood [2,3]. The protection conferred by attenuated sporozoites was confirmed to be mainly mediated by CD8+ T cells targeting LS antigens and not by antigens presented on the surface of migrating sporozoites [17][18][19][20][21]. Recent studies pointed out that when compared to attenuated strains that cease their LS development early, attenuated strains that grow longer in hepatocytes before ceasing growth led to more significant protective immune responses [17]. This indicated that significant exposure to LS antigens can enhance vaccine effectiveness. ...
... Mosquito feeding is conducted by allowing about 150 female mosquitoes to feed on a mouse anesthetized with ketamine/xylazine for 15 min. Salivary gland sporozoite extraction was conducted by dissection of the salivary glands of infected female mosquitoes at day 14 or 15 post mosquito feeding (pmf) in RPMI incomplete medium, as previously described [17,56,57]. Collected salivary glands were mechanically disrupted with a pestle and the salivary gland sporozoites were counted using a hemocytometer. ...
Article
Full-text available
The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%-88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection. Received: 21 November 2019 / Revised: 3 January 2020 / Accepted: 6 January 2020 / Published: 10 January 2020
... Targeted genetic alteration of Plasmodium also generates a homogenous population of attenuated parasites with a distinct genetic identity. The attenuated status of this unique clonal population also does not rely on external technical variables like the radiation dosage (in RAS) or host drug metabolism (in ITV) [89][90][91]. ...
... The deletion of the FabB/F gene involved in fatty acid synthesis resulted in P. yoelii parasites that arrested late in the liver stage [89]. Immunization with Py FabB/FKO elicited a high magnitude of CD8 T cell effector and memory response in both inbred and outbred mice [91]. When compared to the RAS that often arrested early in its liver-stage development, the GAPs that progressed into late-liver-stage engendered higher magnitudes of effector and memory CD8 T cell responses, possibly also directed at a more diverse set of antigens, resulting in better protection from secondary challenges [91]. ...
... Immunization with Py FabB/FKO elicited a high magnitude of CD8 T cell effector and memory response in both inbred and outbred mice [91]. When compared to the RAS that often arrested early in its liver-stage development, the GAPs that progressed into late-liver-stage engendered higher magnitudes of effector and memory CD8 T cell responses, possibly also directed at a more diverse set of antigens, resulting in better protection from secondary challenges [91]. This finding suggested that late-arresting GAPs may be more reliable as a vaccine candidate, compared to either the early-arresting GAPs or RAS vaccines for malaria. ...
Article
Full-text available
Malaria, caused by the protozoan Plasmodium, is a devastating disease with over 200 million new cases reported globally every year. Although immunization is arguably the best strategy to eliminate malaria, despite decades of research in this area we do not have an effective, clinically approved antimalarial vaccine. The current impetus in the field is to develop vaccines directed at the pre-erythrocytic developmental stages of Plasmodium, utilizing novel vaccination platforms. We here review the most promising pre-erythrocytic stage antimalarial vaccine candidates.
... RTS,S/AS0 vaccines reduce episodes of severe malaria and delay the time to new infection but do not offer a durable, high level efficacy (> 50%). Vaccination using whole sporozoites (Plasmodium forms in mosquito salivary glands), though logistically challenging, bears much promise due to exposure of a large repertoire of immunogens to the immune system [3][4][5][6][7][8][9] . Among whole sporozoite vaccines (WSV), Radiation-Attenuated Sporozoites (RAS) has been considered the "gold standard" model for induction of full and sterilizing immunity against malaria 5,10 . ...
... Afterwards, a reverse vaccinology approach based on Plasmodium genomes 11 , bioinformatics tools predicting antigens and/or essential proteins, and improvements in transfection methods for Plasmodium genetic manipulation [12][13][14] has led to the generation of several parasite stage-specific knock-outs exploitable as Genetically Attenuated Parasite (GAP) vaccines 7,15,16 . GAP were designed to overcome the uncertainty associated with random DNA breaks in RAS and minimize the risks associated with radiation, including the production of free radicals. ...
... We show that PbATG8-OE-CVac parasites confer better memory response and longterm protection than WT-FLP-CVac parasites (Figs. 4 and 5). To examine the qualitative difference among the antigen-exposed CD8 + T cells between PbATG8-OE-CVac-and WT-FLP-CVac-immunized mice, we investigated the anti-parasite specific CD8 + T cells population in an approach using the CD8α lo CD11a hi surrogate activation marker 7,27 . T cells expressing low levels of CD8α and high levels of CD11a represent antigenexperienced cells. ...
Preprint
Full-text available
Genetically-attenuated sporozoite vaccines can elicit long-lasting protection against malaria but pose risks of breakthrough infection. Chemoprophylaxis vaccination (CVac) has proven to be the most effective vaccine strategy against malaria. Though CVac with WT sporozoites confers better immunity, the overhanging threat of drug resistance limits its use as a vaccine. Here, we demonstrate that a liver stage-specific mutant of Plasmodium berghei when used as a vaccine under a CVac regimen provides superior long-lasting protection, in both inbred and outbred mice, as compared to WT-CVac. Uniquely, the protection elicited by this mutant is predominantly dependent on a CD8 + T-cell response through an IFN-γ-independent mechanism and is associated with a stable population of antigen-experienced CD8 + T cells. Jointly, our findings support the benefit of liver stage mutants as vaccines over WT, under a CVac protocol. This vaccination strategy is also a powerful model to study the mechanisms of protective immunity and discover new protective antigens.
... RAS, which infect hepatocytes but cannot complete differentiation to merozoites, are capable of eliciting responses in both humans and mice that prevent the parasite from completing liver-stage differentiation and CD8 T cells are major mediators of RAS-induced immunity in mouse models Seder et al., 2013). Genetically attenuated Plasmodium parasites (GAP), tailored to arrest late during hepatocyte infection, induce better protective immunity, and evoke larger Plasmodium-specific CD8 T cell responses than RAS or early arresting GAP without modulating CD8 T cell responses directed at the sporozoite stage (Butler et al., 2011;Vaughan and Kappe, 2017). These data strongly suggest that protective CD8 T cells can be primed not only against sporozoite antigens but also against antigens expressed only after hepatocyte infection. ...
... CD11c + Cells Invade the Liver and Harbor Plasmodium during Liver-Stage Malaria Immunization using late-liver-stage arresting GAP induced superior protective immunity over early arresting GAP (Butler et al., 2011), suggesting that antigens expressed during the liver stage of malaria prime CD8 T cell responses and serve as target antigens for protective immunity. The immunological pathways leading to CD8 T cell responses against Plasmodium liver-stage antigens remain undefined. ...
... Although substantial evidence suggests that CD8 T cell responses against liver-stage Plasmodium antigens occur and contribute to protective immunity (Butler et al., 2011;Vaughan and Kappe, 2017), the cellular and anatomical regulation of this response has remained unknown. In general, antigens presented by MHC-I molecules are derived from intracellular proteins, making them available to be identified by CD8 T cells. ...
Article
Plasmodium sporozoites inoculated by mosquitoes migrate to the liver and infect hepatocytes prior to release of merozoites that initiate symptomatic blood-stage malaria. Plasmodium parasites are thought to be restricted to hepatocytes throughout this obligate liver stage of development, and how liver-stage-expressed antigens prime productive CD8 T cell responses remains unknown. We found that a subset of liver-infiltrating monocyte-derived CD11c ⁺ cells co-expressing F4/80, CD103, CD207, and CSF1R acquired parasites during the liver stage of malaria, but only after initial hepatocyte infection. These CD11c ⁺ cells found in the infected liver and liver-draining lymph nodes exhibited transcriptionally and phenotypically enhanced antigen-presentation functions and primed protective CD8 T cell responses against Plasmodium liver-stage-restricted antigens. Our findings highlight a previously unrecognized aspect of Plasmodium biology and uncover the fundamental mechanism by which CD8 T cell responses are primed against liver-stage malaria antigens. There is an urgent need for effective vaccine strategies to protect against malaria. Kurup et al. describe the fundamental mechanisms by which protective CD8 T cell responses are elicited against the liver stage of malaria, possibly helping to refine the rational design of anti-malarial vaccines.
... A CAS-based arrest using the antibiotic azithromycin showed that specifically targeting the Plasmodium apicoplast, a relict non-photosynthetic plastid organelle, resulted in late arrest after complete liver stage maturation (Friesen et al., 2010). Accordingly, two complementary approaches targeting key factors in the Plasmodium apicoplast led to generation of late-arrested GAPs and their testing in vaccine studies (Fig. 3), namely deletion of a fatty acid biosynthesis enzyme (Butler et al., 2011) and a Plasmodium-specific protein of unknown function (Haussig et al., 2011). In both cases potent protection against reinfection was reported and superior protection correlated with extended liver stage maturation. ...
... In both cases potent protection against reinfection was reported and superior protection correlated with extended liver stage maturation. However, safety concerns were reported for ∆PALM whereas safety of fatty acid synthesis II (FASII) enzyme knockout was promising in P. yoelii (Vaughan et al., 2009, Pei et al., 2010, Butler et al., 2011, Haussig et al., 2011, Lindner et al., 2014 but failed to be confirmed in P. berghei, thereby limiting the use of these vaccines candidates (Yu et al., 2008, Annoura et al., 2012, Shears et al., 2017. The additional, unexpected finding of aborted parasite development in mosquitos infected with the corresponding P. falciparum knockouts (Cobbold et al., 2013 essentially eliminated the possibility to develop GAP vaccines by targeted deletion of the FASII pathway. ...
... Comparison studies with GAP-based and RAS vaccines corroborated this notion as GAP immunized animals revealed higher levels of IFN-γ producing T cells (Jobe et al., 2007). Furthermore, increased CD8 + T cell responses were detected in mice immunized with late arresting GAPs (Butler et al., 2011). Expansion of short-lived CD11a hi , CD62 lo , CD44 hi CD8b + cells was observed in late arresting GAP immunized mice. ...
Thesis
Malaria Impfstoffkandidaten, welche Sicherheit und Wirksamkeit gegen prä-erythrozytische Stadien bieten, sind nach wie vor in der Entwicklung. Experimentelle Immunisierungsstudien mit genetisch attenuierten Parasiten (GAP), welche die Entwicklung über das klinisch asymptomatische Leberstadium hinaus verhindern, erwiesen sich als sicher und effizient. ΔSLARP GAP-Sporozoiten arretieren vollständig in der Leber, bieten jedoch keinen langanhaltenden Schutz. Hingegen zeigen Immunisierungen mit ΔP36p/P36 Sporozoiten einen langanhaltenden Schutz, führen jedoch während der Immunisierung gelegentlich zu Blutstadieninfektionen. Diese Studie liefert eine systematische vorklinische Bewertung eines dreifachen KO GAP-Parasiten, durch die Kombination von ΔSLARP und ΔP36p/P36. KO Parasiten arretierten vollständig in vitro und in vivo, aber der zeitnahe Blutinfektionsbeginn nach einer Sporozoiteninfektion in Mäusen zeigte eine verminderte Wirksamkeit des Impfstoffs. Während ein besserer Schutz durch einen späten Leberstadien Entwicklungsstillstand erreicht werden kann, bleiben die zugrundeliegenden molekularen Mechanismen unklar. Eine Vorrausetzung für die Leberzellen Antigenpräsentation ist die Präsenz von parasitären Antigenen im hepatozyten Zytoplasma. Der Proteinexportkomplex PTEX ist in Leberstadien nicht vollständig funktionstüchtig, da das essentielle Hitzeschockprotein 101 (HSP101) nicht exprimiert wird. Um die Rolle von HSP101 für den Leberproteinexport zu klären, wurden transgene HSP101 exprimierende Parasiten erzeugt. Transgene Parasiten weisen in vitro und in vivo schwere Wachstumsstörungen im Leberstadium auf und bieten keinen Impfschutz. Die Ergebnisse legen nahe, dass die Expression von HSP101 streng kontrolliert wird und der Export im frühen Leberstadien nicht wiederhergestellt werden kann. Insgesamt können prä-klinische Studien und die Weiterentwicklung von GAP-basierten Impfstoffkandidaten die laufenden humanen Impfstoffstudien beeinflussen und vorantreiben.
... RTS,S/AS0 vaccines reduce episodes of severe malaria and delay the time to new infection but do not offer a durable, high-level efficacy (>50%). Vaccination using whole sporozoites (Plasmodium forms in mosquito salivary glands), though logistically challenging, bears much promise due to exposure of a large repertoire of immunogens to the immune system [3][4][5][6][7][8][9] . Among whole sporozoite vaccines (WSV), radiation-attenuated sporozoites (RAS) have been considered the "gold standard" model for induction of full and sterilizing immunity against malaria 5,10 . ...
... Afterward, a reverse vaccinology approach based on Plasmodium genomes 11 , bioinformatics tools predicting antigens and/or essential proteins, and improvements in transfection methods for Plasmodium genetic manipulation [12][13][14] has led to the generation of several parasite stage-specific knock-outs exploitable as genetically attenuated parasite (GAP) vaccines 7,15,16 . GAP was designed to overcome the uncertainty associated with random DNA breaks in RAS and minimize the risks associated with radiation, including the production of free radicals. ...
... As opposed to previously reported WSV such as RAS, GAP, and WT-CVac, PbATG8-OE-CVac confer 100% protection at two memory time points (D60 and D80 post-immunization) 7 in a clinically relevant outbred mouse model. The induction of longterm protection elicited by PbATG8-OE-CVac in immunized mice may be due to qualitative and/or quantitative differences (small but nonetheless sufficient) in protective cellular populations. ...
Article
Full-text available
Genetically attenuated sporozoite vaccines can elicit long-lasting protection against malaria but pose risks of breakthrough infection. Chemoprophylaxis vaccination (CVac) has proven to be the most effective vaccine strategy against malaria. Here, we demonstrate that a liver stage-specific autophagy mutant of Plasmodium berghei (ATG8 overexpressor), when used as a live vaccine under a CVac regimen, provides superior long-lasting protection, in both inbred and outbred mice, as compared to WT-CVac. Uniquely, the protection elicited by this mutant is predominantly dependent on a CD8 ⁺ T-cell response through an IFN-γ-independent mechanism and is associated with a stable population of antigen-experienced CD8 ⁺ T cells. Jointly, our findings support the exploitation of liver-stage mutants as vaccines under a CVac protocol. This vaccination strategy is also a powerful model to study the mechanisms of protective immunity and discover new protective antigens.
... In all attenuation methods, Plasmodium sporozoites invade hepatocytes within vacuoles, then cease growth and do not cause Plasmodium infection of the blood 32,37,79,80 . The protection conferred by attenuated sporozoites was confirmed to be mainly mediated by CD8+ T cells targeting LS antigens and not by antigens presented on the surface of migrating sporozoites [81][82][83][84][85] . Recent studies pointed out that when compared to attenuated strains that cease their LS development early, attenuated strains that grow longer in hepatocytes before ceasing growth led to more significant protective immune responses 81 . ...
... The protection conferred by attenuated sporozoites was confirmed to be mainly mediated by CD8+ T cells targeting LS antigens and not by antigens presented on the surface of migrating sporozoites [81][82][83][84][85] . Recent studies pointed out that when compared to attenuated strains that cease their LS development early, attenuated strains that grow longer in hepatocytes before ceasing growth led to more significant protective immune responses 81 . This indicated that significant exposure to LS antigens can enhance vaccine effectiveness. ...
... Mosquito feeding is conducted by allowing about 150 female mosquitoes or less to feed on a mouse anesthetized with ketamine/xylazine for 15 min. Salivary gland sporozoite extraction was conducted by dissection of the salivary glands of infected female mosquitoes at day 14 or 15 post mosquito feeding (pmf) in RPMI incomplete medium, as previously described 81,171,172 . ...
Article
Malaria infects millions of people every year, and despite recent advances in controlling disease spread, it remains a global health concern. Decades of research into both naturally acquired and vaccine mediated immunity have given a broad range of correlates of protection. RTS,S, the only licensed anti-malarial vaccine, has implicated antibodies against the circumsporozoite protein (CSP) as a key correlate. Not to be discounted, CD8+ T cells targeting liver-stage (LS) antigens were associated with protection in attenuated sporozoite vaccination. Clearly there is no panacea for malarial immunity, and a broad range of responses against multiple antigens is crucial. In this work we develop novel synthetic DNA vaccines targeting antigens in multiple Plasmodium pre-erythrocytic life cycle stages, and evaluate the immunity elicited by each in the context of murine models of malaria. To further evaluate protection mediated by Liver stage antigens, we focused on the exported pre-erythrocytic proteins EXP1, PFN, EXP2, ICP, TMP21, and UIS3. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%–88% in non-adjuvanted groups were protected from disease following Plasmodium yoelii challenge. To further evaluate protection mediated by sporozoite antigens, five synDNA vaccines encoding variations of CSP were designed and studied: 3D7, GPI1, ΔGPI, TM, and DD2. ΔGPI generated the most robust immunity, and was the most efficacious in an IV sporozoite challenge. We then compared the immunity generated by ΔGPI vs synDNA mimics for two leading malaria vaccine candidates (RTS,S and R21). They demonstrated similar anti-CSP antibody responses, however ΔGPI induced a more focused T cell response. In an infectious mosquito challenge all three of these constructs generated potent inhibition of liver stage infection, with ΔGPI appearing to also provide the best sterilizing immunity from blood stage parasitemia. Together these studies demonstrated that synDNA vaccines encoding malaria immunogens can provide substantial protection from disease, and highlighted the importance of targeting the pre-erythrocytic life cycle stages to combat malaria.
... Another approach has been possible with the availability of the whole Plasmodium genome sequences (Carlton et al., 2002;Gardner et al., 2002a;Gardner et al., 2002b) and the advancement of gene manipulation technologies (de Koning- Ward et al., 2000;Menard and Nussenzweig, 2000;Thathy and Menard, 2002) which has led to the individualization of genes essential for parasite survival at distinct points during its life cycle. Genetically arrested parasites (GAP) generated by targeted gene deletion(s) designed to arrest at specific points of livers-stage development ( Fig. 12) (Butler et al., 2011;Mueller et al., 2005a;Mueller et al., 2005b;van Dijk et al., 2005) were able to successfully invade the liver but arrest prior to liver stage maturity, generating complete protective immunity against wild type sporozoite challenge in a manner comparable to radiation attenuated sporozoites (Butler et al., 2011;Khan et al., 2012;Mueller et al., 2005b). This immune response is mediated by MHC class I-dependent IFN-γ producing CD8 + T cells (Jobe et al., 2007;Mueller et al., 2007), which results in sterile protracted protection (Jobe et al., 2007;Wang et al., 2009) against sporozoite challenge in rodent models. ...
... Another approach has been possible with the availability of the whole Plasmodium genome sequences (Carlton et al., 2002;Gardner et al., 2002a;Gardner et al., 2002b) and the advancement of gene manipulation technologies (de Koning- Ward et al., 2000;Menard and Nussenzweig, 2000;Thathy and Menard, 2002) which has led to the individualization of genes essential for parasite survival at distinct points during its life cycle. Genetically arrested parasites (GAP) generated by targeted gene deletion(s) designed to arrest at specific points of livers-stage development ( Fig. 12) (Butler et al., 2011;Mueller et al., 2005a;Mueller et al., 2005b;van Dijk et al., 2005) were able to successfully invade the liver but arrest prior to liver stage maturity, generating complete protective immunity against wild type sporozoite challenge in a manner comparable to radiation attenuated sporozoites (Butler et al., 2011;Khan et al., 2012;Mueller et al., 2005b). This immune response is mediated by MHC class I-dependent IFN-γ producing CD8 + T cells (Jobe et al., 2007;Mueller et al., 2007), which results in sterile protracted protection (Jobe et al., 2007;Wang et al., 2009) against sporozoite challenge in rodent models. ...
... Similar results were obtained with clones HRFΔ2 and HRFΔ3 (Supporting Information Fig. S2A and B). As expected, the delayed development of the mutant in the liver greatly decreased its ability to induce CM after sporozoite inoculation, as already reported for other mutants with a developmental block in the liver (Butler et al., 2011) and in humans after RTS,S (repeat and T-cell epitope in the circumsporozoite protein (CSP) of Plasmodium falciparum malaria parasite and a viral envelope protein of the hepatitis B virus, HBsAg) immunization (Heppner et al., 2005). Indeed while 100% C57BL/6 mice developed CM on day 7 or 8 after WT sporozoite inoculation by mosquito bite, only 10% of the mice inoculated with HRFΔ1 sporozoites showed signs of neuropathology (Fig. 1D). ...
Thesis
Recent findings have raised the hypothesis that clinical susceptibility to malaria may be related to allergy-type response. In human infection with P. falciparum, as well as in murine models of malaria, increased levels of histamine have been shown to be associated with disease severity. Histamine releasing factor (HRF), shown to be implicated in the release of pro-inflammatory histamine during late-phase allergy, was demonstrated to be produced by the parasite during mild and severe malaria infections suggesting that Plasmodium HRF may affect host immune responses and contributes to the pathogenesis. The objectives of this work were to evaluate the role of Plasmodium HRF in the development of the immune response and to determine whether its expression is associated with the severity of malaria disease by studying two HRF-deficient (hrfΔ) murine parasites (PbANKA and PbNK65). Infection with PbANKA-hrfΔ sporozoites showed a decrease in the frequency of ECM due to the impairment of the development of the mutant parasites in liver stages as a consequence of the up-regulation of IL-6. Infection with PbNK65-hrfΔ parasites confirmed the importance of HRF in enhancing the virulence of the parasite. Indeed, PbNK65-hrfΔ infection results in parasite clearance leading to a long-lasting protection and immune memory as reflected by an up-regulation of IL-6, a down-regulation of PD-1 expression on T cells and in the enhancement of Ab-mediated phagocytosis. HRF is the first parasite gene which directly modulates the host immune response.
... GAPs confer sterile protection in rodents and data from a recently published Phase I clinical trial testing the safety profile of a firstgeneration early LS-arresting (EA) replication-deficient (RD) P. falciparum GAP showed that GAPs are safe and can engender potent immune responses to sporozoite antigens 6 . Furthermore, in animal models, late LS-arresting (LA), replication-competent (RC) Plasmodium yoelii GAPs afford superior pre-erythrocytic immunity as well as stage-and strain-transcending immunity [7][8][9] as compared to EARD GAPs and RAS. In humans, the superior immunogenicity of RC whole sporozoite vaccines is demonstrated by the observation that in comparison to RAS, ITV requires a fraction of an immunizing sporozoite dose to achieve complete sterilizing protection against controlled human malaria infection 10 . ...
... Replication-competent parasites that unfold their full antigenic potential in the liver are superior to replication-deficient parasites that arrest early in liver infection. LARC GAPs likely constitute the most potent whole sporozoite immunogen given that in mouse models, they afford superior protection as compared to RD RAS and EARD GAPs 7 , and engender stage and speciestranscending protection 7,8 . LARC parasite vaccination also stimulates an innate IFN-1 response and we show here that surprisingly, this negatively impacts the acquisition of protective adaptive immunity, and thus ablation of IFN-1 signaling enhances protection. ...
Article
Full-text available
Immunization with attenuated whole Plasmodium sporozoites constitutes a promising vaccination strategy. Compared to replication-deficient parasites, immunization with replication-competent parasites confers better protection and also induces a type I IFN (IFN-1) response, but whether this IFN-1 response has beneficial or adverse effects on vaccine-induced adaptive immunity is not known. Here, we show that IFN-1 signaling-deficient mice immunized with replication-competent sporozoites exhibit superior protection against infection. This correlates with superior CD8 T cell memory including reduced expression of the exhaustion markers PD-1 and LAG-3 on these cells and increased numbers of memory CD8 T cells in the liver. Moreover, the adoptive transfer of memory CD8 T cells from the livers of previously immunized IFN-1 signaling-deficient mice confers greater protection against liver stage parasites. However, the detrimental role of IFN-1 signaling is not CD8 T cell intrinsic. Together, our data demonstrate that liver stage-engendered IFN-1 signaling impairs hepatic CD8 T cell memory via a CD8 T cell-extrinsic mechanism. Here, Minkah et al. show that, while immunization with replication-competent Plasmodium parasites can confer sterile protection against infection, it also induces a type I interferon response that adversely affects anti-malaria immunity by affecting numbers of protective hepatic CD8 T cells and CD8 T cell function.
... challenge) and heterologous (a different strain) CHMI [5][6][7][8] and against intense natural transmission in Africa 9,10 . Radiation-attenuated PfSPZs are non-replicating parasites and arrest early in the liver stage, as do first-generation genetically attenuated PfSPZs 11,12,13 . ...
... Alternative whole-PfSPZ vaccine approaches are currently being developed, including genetically attenuated parasites that arrest at a specific lifecycle time point. This is hypothetically attractive as it obviates the need for antimalarial drugs, and late liver-stage-arresting parasites enhance sterilizing immunity compared with early-arresting parasites in mice 11 . However, breakthrough infections occurred with some genetically attenuated parasites in the clinic 42 . ...
Article
Full-text available
The global decline in malaria has stalled¹, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))—which kill liver-stage and blood-stage parasites, respectively—and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization (https://clinicaltrials.gov/, NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 10⁴ to 2 × 10⁵ PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains.
... [18][19][20][21] Similar results have been reported after immunisations with late arresting genetically arrested sporozoites in preclinical studies in mice. 22,23 Given that this alternative whole sporozoite vaccine approach ensures full EEF development, the results suggest that the longer the parasites are allowed to develop and mature before arrest, the greater the protection induced by the immunisation strategy. This outcome has been initially interpreted as having more antigens expressed and an increase in antigen biomass during extended parasite development, eliciting a broader range of EEF-specific immune responses needed for protection. ...
... This outcome has been initially interpreted as having more antigens expressed and an increase in antigen biomass during extended parasite development, eliciting a broader range of EEF-specific immune responses needed for protection. 22,23 However, immunogenic proteins expressed in the late EEF, which may increase CD8 + T cell responses, are currently not well defined. ...
Article
Full-text available
Sporozoite antigens are the basis of a number of malaria vaccines being tested, but the contribution of antigens expressed during subsequent liver stage development to pre‐erythrocytic stage immunity is poorly understood. We previously showed that, following immunisation with radiation attenuated sporozoites (RAS), a model epitope embedded in a sporozoite surface protein elicited robust CD8+ T cell responses, whilst the same epitope in a liver stage antigen induced inferior responses. Since RAS arrest early in their development in host hepatocytes, we hypothesised that extending parasite maturation in the liver could considerably improve the epitope‐specific CD8+ T cell response. Here, we employed a late liver stage arrested parasite model, azithromycin prophylaxis alongside live sporozoites, to increase expression of the model epitope until full liver stage maturation. Strikingly, this alternative immunisation strategy, which has been shown to elicit superior protection, failed to improve the resulting epitope‐specific CD8+ T cell responses. Our findings support the notion that liver stage antigens are poorly immunogenic and provide additional caution about prioritising antigens for vaccine development based solely on immunogenicity
... These are vaccines based on the deletion of genes important for merozoite formation [42] or schizogony development [43]. For example, a GAP vaccine based on the deletion of FabB/F, which is involved in the fatty acid synthesis pathway II (FASII) that is essential in late liver-stage development [44], confers better protection than that achieved with RASs, eliciting more intense CD8 + T cell and memory cell responses [45]. Table 1 summarizes these and other candidate GAP vaccines, and relevant features are briefly described here. ...
... Finally, long-lived central memory T cells (TCM) migrate to the draining lymph node of the reinfection site where they proliferate and differentiate into TEFF cells after restimulation to reinforce the primary immune response. [37,45] Trends in Parasitology (continued on next page) ...
Article
The control of diseases caused by protozoan parasites is one of the United Nations' Sustainable Development Goals. In recent years much research effort has gone into developing a new generation of live attenuated vaccines (LAVs) against malaria, Chagas disease and leishmaniasis. However, there is a bottleneck related to their biosafety, production, and distribution that slows downs further development. The success of irradiated or genetically attenuated sporozoites against malaria, added to the first LAV against leishmaniasis to be evaluated in clinical trials, is indicative that the drawbacks of LAVs are gradually being overcome. However, whether persistence of LAVs is a prerequisite for sustained long-term immunity remains to be clarified, and the procedures necessary for clinical evaluation of vaccine candidates need to be standardized.
... The failure of CPS to protect against heterologous parasites is somewhat surprising, given the basic premise that full infection in the liver would increase the magnitude and breadth of T cell responses, as compared with those to RAS 26,75 . One explanation is that the heterologous parasite strains used for CHMI in these CPS studies (NF135.C10 and NF166.C8) are more infectious than the related NF54 and 3D7 strains used in most CHMI studies 73,74 . ...
... A second approach for enhancing the antigenic breadth of sporozoite vaccines compared with RAS is to use GAPs that arrest at a defined point in the late liver stage 71,79 . In mouse models, latearresting GAPs induce a higher frequency of CD8 + T cell responses than RAS vaccination and protect animals at lower immunizing doses than early-arresting parasites 75 . Thus, late-acting GAP vaccines offer the potential advantage of having more robust immunogenicity and breadth than RAS vaccines, but would not require drug treatment as with Cvac. ...
Article
Development of a malaria vaccine remains a critical priority to decrease clinical disease and mortality and facilitate eradication. Accordingly, RTS,S, a protein-subunit vaccine, has completed phase III clinical trials and confers ~30% protection against clinical infection over 4 years. Whole-attenuated-sporozoite and viral-subunit vaccines induce between 20% and 100% protection against controlled human malaria infection, but there is limited published evidence to date for durable, high-level efficacy (>50%) against natural exposure. Importantly, fundamental scientific advances related to the potency, durability, breadth and location of immune responses will be required for improving vaccine efficacy with these and other vaccine approaches. In this Review, we focus on the current understanding of immunological mechanisms of protection from animal models and human vaccine studies, and on how these data should inform the development of next-generation vaccines. Furthermore, we introduce the concept of using passive immunization with monoclonal antibodies as a new approach to prevent and eliminate malaria.
... Late liver stage attenuation can be achieved by genetic engineering of the parasite. Indeed proof-of-concept studies with P. yoelii rodent malaria LARC GAPs, lacking enzymes for type II fatty acid biosynthesis, (e.g., P. yoelii fabb/f -), demonstrated late liver stage arrest and superior protective efficacy in mice when compared with RAS and replication-deficient GAPs (60). Immunizations with P. yoelii fabb/f -LARC GAP not only protected against homologous challenge in both inbred and outbred mice but also conferred protection against a sporozoite challenge using the different rodent malaria species Plasmodium berghei (species-transcending protection) and against lethal blood stage challenge of the same species (stage-transcending protection). ...
... Immunizations with P. yoelii fabb/f -LARC GAP not only protected against homologous challenge in both inbred and outbred mice but also conferred protection against a sporozoite challenge using the different rodent malaria species Plasmodium berghei (species-transcending protection) and against lethal blood stage challenge of the same species (stage-transcending protection). The enhanced protection in P. yoelii fabb/f -LARC GAP-immunized mice was attributed to cytotoxic CD8 + T cells that recognized a broader repertoire of liver stage antigens (60). However, generating the equivalent LARC GAP in P. falciparum failed, as strains lacking enzymes for type II fatty acid biosynthesis did not generate sporozoites, indicating that this pathway is essential for P. falciparum sporozoite formation (37). ...
Article
Full-text available
Whole sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage-arresting replication-competent (LARC) GAPs confer superior protection when compared to early liver stage-arresting replication-deficient (EARD) GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (Pf) has been challenging. Here we report the generation and characterization of an unprecedented Pf LARC GAP generated by targeted gene deletion of the Mei2 gene; Pf mei2-. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for Pf mei2- liver stages in human liver-chimeric mice. However, Pf mei2- liver stages failed to complete development and did not form infectious exo-erythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, Pf mei2- is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against Pf malaria infection.
... Protective epitopes, such as TRAP, are shed during hepatocyte invasion [49] and thus should be accessible to liver APC populations. Vaccination with late-arresting genetically attenuated Plasmodium parasites (GAP) primes CD8 T cell responses with broader specificity than those primed after vaccination with early liver-stage-arresting parasites (RAS), suggesting that unique epitopes are expressed during the development of liver-stage parasites [50,51]. During mouse liver-stage malaria, monocyte-derived CD11c + CSFR1 + F4/80 + APCs infiltrate the liver, capture Plasmodium from infected hepatocytes, and migrate to the liver dLNs to prime naïve CD8 T cells that eventually form protective CD8 Tem and Trm cells [25] ( Figure 1B). ...
Article
Each year over 200 million malaria infections occur, with over 400 000 associated deaths. Vaccines formed with attenuated whole parasites can induce protective memory CD8 T cell responses against liver-stage malaria; however, widespread administration of such vaccines is logistically challenging. Recent scientific findings are delineating how protective memory CD8 T cell populations are primed and maintained and how such cells mediate immunity to liver-stage malaria. Memory CD8 T cell anatomic localization and expression of transcription factors, homing receptors, and signaling molecules appear to play integral roles in protective immunity to liver-stage malaria. Further investigation of how such factors contribute to optimal protective memory CD8 T cell generation and maintenance in humans will inform efforts for improved vaccines.
... Because liver stage is asymptomatic, removal of all liver stages prevents clinical symptoms of malaria and thus is a highly desirable feature of an effective vaccine. Indeed, previous studies have shown that memory CD8 T cells are required for protection against a challenge with a relatively large number of sporozoites (14,15) and that vaccination that induces exclusively memory CD8 T cells of a single specificity can mediate sterilizing protection against a sporozoite challenge (16)(17)(18)(19)(20)(21)(22)(23). Antibodies and CD4 T cells may also contribute to protection in some circumstances, for example, following inoculation of sporozoites by mosquitoes in the skin (24,25). ...
Article
Full-text available
Malaria, a disease caused by parasites of the Plasmodium genus, begins when Plasmodium-infected mosquitoes inject malaria sporozoites while searching for blood. Sporozoites migrate from the skin via blood to the liver, infect hepatocytes, and form liver stages which in mice 48 h later escape into blood and cause clinical malaria. Vaccine-induced activated or memory CD8 T cells are capable of locating and eliminating all liver stages in 48 h, thus preventing the blood-stage disease. However, the rules of how CD8 T cells are able to locate all liver stages within a relatively short time period remains poorly understood. We recently reported formation of clusters consisting of variable numbers of activated CD8 T cells around Plasmodium yoelii (Py)-infected hepatocytes. Using a combination of experimental data and mathematical models we now provide additional insights into mechanisms of formation of these clusters. First, we show that a model in which cluster formation is driven exclusively by T-cell-extrinsic factors, such as variability in “attractiveness” of different liver stages, cannot explain distribution of cluster sizes in different experimental conditions. In contrast, the model in which cluster formation is driven by the positive feedback loop (i.e., larger clusters attract more CD8 T cells) can accurately explain the available data. Second, while both Py-specific CD8 T cells and T cells of irrelevant specificity (non-specific CD8 T cells) are attracted to the clusters, we found no evidence that non-specific CD8 T cells play a role in cluster formation. Third and finally, mathematical modeling suggested that formation of clusters occurs rapidly, within few hours after adoptive transfer of CD8 T cells, thus illustrating high efficiency of CD8 T cells in locating their targets in complex peripheral organs, such as the liver. Taken together, our analysis provides novel insights into and attempts to discriminate between alternative mechanisms driving the formation of clusters of antigen-specific CD8 T cells in the liver.
... One late-blocked FASII mutant, the P. yoelii FABB/F À GAP, was shown to induce broader CD8 + T cell responses than RAS as well as some degree of crossstage and cross-species (P. berghei) protection [10]. Therefore, late liver-stage development may enhance protective efficacy by allowing the presentation of a larger set of antigens to the immune system. ...
Chapter
Vaccines that target the preerythrocytic phase of malaria hold great promise as elimination tools since they are the sole vaccines that can achieve sterile protection against a challenge. This chapter focuses on preerythrocytic stage vaccines based on live attenuated parasites. It first summarizes the main conclusions that have emerged from studies in rodents, which compared various parasite attenuation methods, and then presents the vaccination regimens that are currently being tested in humans.
... Another approach uses gene deletion to create genetically attenuated parasites (GAP), which arrest at a defined developmental stage in the liver (Mueller et al., 2005). Immunisation experiments in mice showed an improved protection in comparison to radiation attenuated sporozoites (Butler et al., 2011). ...
Thesis
Malaria in humans is most often caused by either Plasmodium falciparum or P. vivax. Research efforts have mostly focused on P. falciparum, despite P. vivax being the most widespread Plasmodium species. This parasite species differs from P. falciparum in some key aspects like its ability to form dormant liver forms, an early appearance of gametocytes in the blood stream and its strict tropism to reticulocytes. Although P. vivax and P. falciparum invade different developmental stages of red blood cells, both express a highly abundant protein on the surface of their merozoites, the merozoite surface protein 1 (MSP-1). Initially expressed as a precursor protein, MSP-1 undergoes proteolytic processing and is cleaved into four processing fragments called p83, p30, p38 and p42. These fragments remain non-covalently associated and are attached to the surface of the merozoite via a GPI anchor at the C-terminal fragment p42. MSP-1 is considered a promising vaccine candidate and P. falciparum MSP-1D is currently tested as a vaccine in a clinical trial. However, no similar vaccine was developed for P. vivax yet. The establishment of a protein production process for P. vivax MSP-1 (PvMSP-1) was an essential part of this study. Two different methods were developed, which both use individually expressed fusion proteins to assemble the full length MSP-1 protein. One fusion protein consists of the fragments p83 and p30, the other includes p38 and p42. In the first approach, the two halves are refolded together and purified by ion exchange chromatography and size exclusion chromatography. This protocol was also applied to produce P. berghei MSP-1 and test whether the assembly of a hybrid protein consisting of one P. falciparum half and one P. vivax half is possible. In the second approach contaminants are first removed from the individual fusion proteins by high resolution ion exchange chromatography before reconstitution of MSP-1. Both techniques result in a pure MSP-1 preparation, but the second approach results in a higher yield. The produced recombinant proteins were structurally characterised using a combination of bioinformatical, biophysical and biochemical methods. All recombinant proteins were cleaved by P. falciparum SUB1, indicating a high conservatism of the cleavage motifs. CD spectroscopy showed a mostly alpha-helical structure and only partial unfolding of the protein at high temperatures. Interestingly, this unfolding increased when MSP-1D was processed by SUB1 before performing thermal denaturation. This suggests an increased stability of the MSP-1 heterodimer compared to the complex consisting of four processing fragments. Prediction of the secondary structure revealed unstructured regions in all analysed MSP-1 proteins. The function of these regions remains unknown, but these sites could be responsible for interactions with other proteins. Cross-linking of MSP-1 paired with mass spectrometry uncovered a previously unknown interaction between the p83 and p42 processing fragment, leading to an updated model of MSP-1 with a globular instead of asymmetrical shape. This project provides two production processes for recombinant P. vivax MSP-1, which is now available for further immunological studies. The structural information acquired with this and other MSP-1 proteins can be used as a base to further elucidate its function and three-dimensional structure.
... Building upon this success will necessitate identifying new pre-erythrocytic stage targets and expanding the repertoire of antigens to include late liver stage proteins. 7 In this study we focus on the final phase of the Plasmodium pre-erythrocytic stage, the merosomes, characterizing their proteome to obtain a better understanding of their biology and provide data to inform target selection for future interventions. ...
Article
The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for antimalarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique data set will allow us to explore key questions about the biology of merosomes and hepatic merozoites.
... Current GAP in clinical development include early-arresting liver stage parasites that arrest at a similar developmental stage as RAS (<24 h) (8,9). More recent P. yoelii pre-clinical GAP studies have engineered late-arresting liver stage GAP (2-3 days) (10)(11)(12). Whole parasites that arrest later in liver stage development (late-arresting GAP) or progress to the blood stage (CPS), provide a larger biomass and broader repertoire of immunogens, permitting lower SPZ doses for protection. For example, clinical CPS studies showed volunteers required 10-to 100-fold fewer parasites than RAS to achieve complete protection against malaria challenge, and pre-clinical late-arresting GAP studies showed that at least 10-fold fewer parasites could provide sterilizing protection and also protect against blood stage challenge (12,13). ...
Article
Full-text available
Each year malaria kills hundreds of thousands of people and infects hundreds of millions of people despite current control measures. An effective malaria vaccine will likely be necessary to aid in malaria eradication. Vaccination using whole sporozoites provides an increased repertoire of immunogens compared to subunit vaccines across at least two life cycle stages of the parasite, the extracellular sporozoite, and intracellular liver stage. Three potential whole sporozoite vaccine approaches are under development and include genetically attenuated parasites, radiation attenuated sporozoites, and wild-type sporozoites administered in combination with chemoprophylaxis. Pre-clinical and clinical studies have demonstrated whole sporozoite vaccine immunogenicity, including humoral and cellular immunity and a range of vaccine efficacy that depends on the pre-exposure of vaccinated individuals. While whole sporozoite vaccines can provide protection against malaria in some cases, more recent studies in malaria-endemic regions demonstrate the need for improvements. Moreover, challenges remain in manufacturing large quantities of sporozoites for vaccine commercialization. A promising solution to the whole sporozoite manufacturing challenge is in vitro culturing methodology, which has been described for several Plasmodium species, including the major disease-causing human malaria parasite, Plasmodium falciparum. Here, we review whole sporozoite vaccine immunogenicity and in vitro culturing platforms for sporozoite production.
... Live genetically-attenuated parasites lacking three genes (p52-/p36-/sap1-; "PfGAP3KO") arrest early in liver-stage development, and were safe with no observed breakthrough infection following administration into human subjects by infective mosquito bites (16). Compared to irradiated sporozoite vaccine and early liver stage-arresting genetically-attenuated parasites, second generation genetically-attenuated parasites that arrest late liver stages have shown to demonstrate superior antimalarial immunity following vaccination in mice by having a greater antigen repertoire (40,41). These candidate vaccines could also have greater efficacy in humans, but this remains to be demonstrated. ...
Article
Full-text available
Despite continuous efforts, the century-old goal of eradicating malaria still remains. Multiple control interventions need to be in place simultaneously to achieve this goal. In addition to effective control measures, drug therapies and insecticides, vaccines are critical to reduce mortality and morbidity. Hence, there are numerous studies investigating various malaria vaccine candidates. Most of the malaria vaccine candidates are subunit vaccines. However, they have shown limited efficacy in Phase II and III studies. To date, only whole parasite formulations have been shown to induce sterile immunity in human. In this article, we review and discuss the recent developments in vaccination with sporozoites and the mechanisms of protection involved.
... The recent development of the Clustered, Randomly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to improve the efficiency of genome editing. This has been observed for both Plasmodium falciparum, the human-infectious species responsible for the majority of cases and deaths, and Plasmodium yoelii, a rodentinfectious species typically favored for rapid genetic manipulation, the ability to interrogate the entire life cycle, and similarities with P. falciparum in mosquito stage development and host-pathogen interactions (12)(13)(14)(15)(16). Importantly, CRISPR-based gene editing doesn't rely on integration of a selection cassette into the genome, and the edited genome can therefore be free of drug resistance/fluorescent markers and scar sequences. ...
Article
Full-text available
Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causal parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite's growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene of interest to understand its importance and function. However, these approaches can be limited by the availability of selectable markers and the time required to generate transgenic parasites. Moreover, there also is a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for that utilizes a ribozyme–guide–ribozyme (RGR) single guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free parasites with homology arms as short as 80–100 bp. Additionally, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and desired locus replacement editing events, whereas the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding catalytically dead SpCas9 (dSpCas9) to the region upstream of a gene of interest, resulting in a position-dependent, but strand-independent reduction in gene expression. This robust and flexible system facilitates efficient genetic characterizations of rodent-infectious species.
... Heterologous protection was dependent on CD8 + T-cells whereas antibodies from immunized mice only recognized homologous, but not heterologous, sporozoites. In another study, 100% of mice immunized with genetically attenuated P. yoelii sporozoites were protected against P. berghei sporozoite challenge (53). The authors suggested that late-liver stage arresting sporozoites elicited a broadly protective CD8 + T-cell response. ...
Article
Full-text available
The infection dynamics between different species of Plasmodium that infect the same human host can both suppress and exacerbate disease. This could arise from inter-parasite interactions, such as competition, from immune regulation, or both. The occurrence of protective, cross-species (heterologous) immunity is an unlikely event, especially considering that strain-transcending immunity within a species is only partial despite lifelong exposure to that species. Here we review the literature in humans and animal models to identify the contexts where heterologous immunity can arise, and which antigens may be involved. From the perspective of vaccine design, understanding the mechanisms by which exposure to an antigen from one species can elicit a protective response to another species offers an alternative strategy to conventional approaches that focus on immunodominant antigens within a single species. The underlying hypothesis is that certain epitopes are conserved across evolution, in sequence or in structure, and shared in antigens from different species. Vaccines that focus on conserved epitopes may overcome the challenges posed by polymorphic immunodominant antigens; but to uncover these epitopes requires approaches that consider the evolutionary history of protein families across species. The key question for vaccinologists will be whether vaccines that express these epitopes can elicit immune responses that are functional and contribute to protection against Plasmodium parasites.
... Here we provide the first proteome of this life cycle stage using the Plasmodium berghei rodent malaria model. success will necessitate identifying new pre-erythrocytic stage targets and expanding the repertoire of antigens to include late liver stage proteins (7). In this study we focus on the final phase of the Plasmodium pre-erythrocytic stage, the merosomes, characterizing their proteome to obtain a better understanding of their biology and provide data to inform target selection for future interventions. ...
Preprint
The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for anti-malarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique dataset will allow us to explore key questions about the biology of merosomes and hepatic merozoites. Highlights First proteome of the merosome stage of malaria parasites Quantitative detection of 1188 parasite proteins across 3 biological replicates Comparison to blood stage proteomes identifies shared and unique proteins Discovery of cleaved PEXEL motifs highlights liver stage protein export In Brief The merosome stage that links malaria liver and blood stage infection is poorly understood. Here we provide the first proteome of this life cycle stage using the Plasmodium berghei rodent malaria model. Graphical Abstract
... Sporozoite antigen-specific CD8 + T cell responses are generated in the skindraining lymph nodes at the site of inoculation, primarily by the uptake and cross-presentation of sporozoites by CD8 + CD11c + DCs 157,158 . However, the observation that late-liver-stage-arresting genetically attenuated Plasmodium parasites (GAPs) elicited better protective CD8 + T cell responses than early-liver-stage-arresting GAPs or radiation-attenuated Plasmodium sporozoites (RAS) suggested that the developmental progression of Plasmodium in infected hepatocytes had a decisive role in generating better, perhaps antigenically broader CD8 + T cell responses 159 . Recent findings from our laboratory identified a class of monocyte-derived CD11b + CSF1R + CD207 + F4/80 + CD11c + APCs in the liver that acquired Plasmodium following hepatocyte infection, to prime CD8 + T cell responses against liver-stage specific antigens in the liver-draining lymph nodes 160 . ...
Article
Immunity to malaria has been linked to the availability and function of helper CD4 ⁺ T cells, cytotoxic CD8 ⁺ T cells and γδ T cells that can respond to both the asymptomatic liver stage and the symptomatic blood stage of Plasmodium sp. infection. These T cell responses are also thought to be modulated by regulatory T cells. However, the precise mechanisms governing the development and function of Plasmodium-specific T cells and their capacity to form tissue-resident and long-lived memory populations are less well understood. The field has arrived at a point where the push for vaccines that exploit T cell-mediated immunity to malaria has made it imperative to define and reconcile the mechanisms that regulate the development and functions of Plasmodium-specific T cells. Here, we review our current understanding of the mechanisms by which T cell subsets orchestrate host resistance to Plasmodium infection on the basis of observational and mechanistic studies in humans, non-human primates and rodent models. We also examine the potential of new experimental strategies and human infection systems to inform a new generation of approaches to harness T cell responses against malaria. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
... An aligned rank transformation was performed on non-parametric data CD49d to examine differences in antigen-experienced CD4 + T cells between WT and Icos -/mice. This approach has been confirmed for viral infection [33] and other models of rodent malaria [34][35][36]. In the absence of CQ, there was a similar percentage and number of CD49d + CD11a + CD4 + T cells in the spleen of WT and Icos -/mice (S1 Fig). ...
Article
Full-text available
The co-stimulatory molecule ICOS is associated with the induction and regulation of T helper cell responses, including the differentiation of follicular helper T (Tfh) cells and the formation and maintenance of memory T cells. However, the role of ICOS signaling in secondary immune responses is largely unexplored. Here we show that memory T cell formation and maintenance are influenced by persistent infection with P. chabaudi chabaudi AS infection, as memory T cell numbers decline in wild-type and Icos-/- mice after drug-clearance. Following drug-clearance Icos-/- mice display a relapsing parasitemia that occurs more frequently and with higher peaks compared to wild-type mice after re-challenge. The secondary immune response in Icos-/- mice is characterized by significant impairment in the expansion of effector cells with a Tfh-like phenotype, which is associated with a diminished and delayed parasite-specific Ab response and the absence of germinal centers. Similarly, the administration of an anti-ICOSL antagonizing antibody to wild-type mice before and after reinfection with P. c. chabaudi AS leads to an early defect in Tfh cell expansion and parasite-specific antibody production, confirming a need for ICOS-ICOSL interactions to promote memory B cell responses. Furthermore, adoptive transfer of central memory T (TCM) cells from wild-type and Icos-/- mice into tcrb-/- mice to directly evaluate the ability of TCM cells to give rise to Tfh cells revealed that TCM cells from wild-type mice acquire a mixed Th1- and Tfh-like phenotype after P. c. chabaudi AS infection. While TCM cells from Icos-/- mice expand and display markers of activation to a similar degree as their WT counterparts, they displayed a reduced capacity to upregulate markers indicative of a Tfh cell phenotype, resulting in a diminished humoral response. Together these findings verify that ICOS signaling in memory T cells plays an integral role in promoting T cell effector responses during secondary infection with P. c. chabaudi AS.
... Early arrest of liver stage development has been shown to offer long lasting and sterile protection in rodent models and this can be achieved by deleting various genes which play a critical role in completion of liver stage development, such as: (i) UIS3 (Mueller et al., 2005b), (Kublin et al., 2017) liver stage genes which were administered by infective mosquito bites and conferred protection with and without breakthrough infections respectively in human subjects. To enhance further the efficacy of GAP vaccines, second generation genetically attenuated parasites (next generation), such as fabb/f- (Butler et al., 2011) and plasmei2-/LISP2- (Vaughan et al., 2018) have been developed and arrest during late stage liver development allowing the host to be exposed to a greater antigen repertoire, thereby conferring superior protection in rodent models. One major hurdle facing the further development of GAP vaccines is the difficulty in generating late arresting P. falciparum next generation GAPs. ...
Thesis
Full-text available
Malaria is still a disease of global health significance. Its causative agent, Plasmodium, has a complex lifecycle alternating between the female Anopheles mosquito and the vertebrate host. The blood stage of the infection is thought to cause the clinical symptoms of the disease and is also targeted by the humoral immune response of the host. Natural acquired immunity to malaria is largely mediated by immunoglobulins and is reflected both by a lower prevalence of infection with age and lower rates of disease. Previous studies have generally focussed on understanding the protective role of naturally acquired functional antibodies targeting the mature infected erythrocyte (mIE) and highly immunogenic merozoite. However, the ring-infected erythrocyte (rIE), has hardly been explored to date in the context of naturally acquired protective immunity. As it is the stage of the infection that is predominantly found in the peripheral circulation, its immunological significance is uncertain. This thesis was aimed at contributing to this knowledge gap by investigating the role and significance of rIEs in protective immunity. Firstly, to investigate the immunogenicity of rIEs, I tested whether they were recognised by malaria-immune antibodies using immunofluorescence and flow cytometry. I confirmed that malaria-immune antibodies do bind to rIEs using both laboratory-adapted (lab) and field isolates. This binding was strongly correlated with that directed against mIEs and merozoites, both of which have been shown to be important targets of protective immunity. Malaria-immune antibody binding to rIEs had no direct inhibitory effect on parasite survival and maturation. In separate assays, I tested whether rIEs were retained in the spleen independently of antibody using a microsphiltration assay. As has been previously reported, rIEs were retained in the spleen and I demonstrated for the first time that the retention rate was comparable between lab and field parasite isolates of the same age, suggesting that this may be a mechanism of parasite clearance that contributes to the low parasite densities observed in malaria endemic areas. Secondly, to investigate the likely physiological relevance of antibody binding to rIEs during P. falciparum infection, I tested whether the in vitro opsonic phagocytosis of rIEs predicted the outcome of infection in a controlled human malaria infection (CHMI) study. I established the flow cytometry based opsonic phagocytosis assay (OPA) of rIEs in our laboratory. I showed for the first time, to the best of my knowledge, that opsonic phagocytosis of rIEs was significantly associated with the outcome of infection (lower parasite densities and the need for treatment) in the CHMI study. Interestingly, phagocytosis of uninfected erythrocytes (uEs) in the ring culture was higher in untreated as compared to the treated individuals, suggesting a potential contribution to anaemia, as others have reported. In addition, I developed competition OPAs and used stage-specific spent media, to demonstrate that merozoite specific antibodies mediated the observed opsonic phagocytosis of the ring culture cells and uEs incubated in spent media respectively. Finally, to investigate the specific parasite antigens on the surface of ring culture cells and uEs incubated in spent media, I employed surface trypsinization, followed by mass spectrometry of supernatants. This is the first surface proteomics study to be conducted on rIEs and in this single study I was able to identify parasite proteins on the surface of rIEs and uEs that had previously been identified singly, in a piecemeal fashion (EBA140, EBA175, RAP1, RAP2, RhopH1, RhopH2, RhopH3 and MCP1), and ENO which I showed for the first time. These proteins have been speculated to be transferred by the merozoite to the surface of the newly invaded erythrocyte during invasion and have also been shown to be correlates of protective immunity, with the exception of MCP1, whose role in parasite clearance and protective immunity has not yet been explored. This thesis shows for the first time that antibody mediated clearance of rIEs in vitro, is correlated with the outcome of P. falciparum infection in a CHMI study and was mediated by anti-merozoite antibodies. This study also demonstrates that there are shared parasite targets between merozoites and rIEs. I conclude that rIEs are not immunologically inert and are targeted by antibodies leading to parasite clearance through mechanisms such as phagocytosis. This activity was significantly associated with lower parasite densities and resistance against clinical symptoms in a human malaria challenge study suggesting that it contributes to protective immunity. The enhanced phagocytosis of uEs in protected individuals suggest a potential detrimental impact of anaemia that requires further investigation.
... In endemic areas where reinfection is frequent, immunity enhancement via IAP inhibitors could be naturally boosted with re-exposure to malaria parasites and continued drug administration. This could eventually drive robust immunity to pre-erythrocytic stages and potentially blood stages if the arrested EEFs are sufficiently mature (Butler et al., 2011). The feasibility of such an approach is underscored by the tolerability and safety of some monovalent IAP antagonists that can be administered daily for protracted periods. ...
Article
Full-text available
Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite.
... Another element potentially shaping the immunogenicity of RPL6 and TRAP is their contrasting expression patterns. Prolonged antigen expression has been postulated to favor protection via specific CD8 + T cells (Butler et al., 2011;Speake et al., 2016). TRAP is highly expressed by sporozoites, but not by liver-stage parasites (Amino et al., 2006), which may limit the amount of antigen that infected hepatocytes can display throughout liver-stage development for recognition by liver T RM cells. ...
Article
Liver-resident memory CD8+ T (TRM) cells remain in and constantly patrol the liver to elicit rapid immunity upon antigen encounter and can mediate efficient protection against liver-stage Plasmodium infection. This finding has prompted the development of immunization strategies where T cells are activated in the spleen and then trapped in the liver to form TRM cells. Here, we identify PbRPL6120-127, a H2-Kb-restricted epitope from the putative 60S ribosomal protein L6 (RPL6) of Plasmodium berghei ANKA, as an optimal antigen for endogenous liver TRM cell generation and protection against malaria. A single dose vaccination targeting RPL6 provided effective and prolonged sterilizing immunity against high dose sporozoite challenges. Expressed throughout the parasite life cycle, across Plasmodium species, and highly conserved, RPL6 exhibits strong translation potential as a vaccine candidate. This is further advocated by the identification of a broadly conserved, immunogenic HLA-A∗02:01-restricted epitope in P. falciparum RPL6.
... As an alternative to radiation-based attenuation, genetic modification generates a homogeneous formulation of PfSPZ, which stop development in the liver at a well-defined point (10). In rodent models, immunization with genetically attenuated malaria SPZ can induce similar, or even greater, protective immunity compared to radiation-attenuated malaria SPZ (11). The intrinsic and irreversible nature of the genetic attenuation greatly reduces safety risks during manufacturing of PfSPZ. ...
Article
Immunization with attenuated Plasmodium sporozoites can induce protection against malaria infection, as shown by Plasmodium falciparum (Pf) sporozoites attenuated by radiation in multiple clinical trials. As alternative attenuation strategy with a more homogeneous population of Pf sporozoites (PfSPZ), genetically engineered Plasmodium berghei sporozoites (SPZ) lacking the genes b9 and slarp induced sterile protection against malaria in mice. Consequently, PfSPZ-GA1 Vaccine, a Pf identical double knockout (Pf∆b9∆slarp), was generated as a genetically attenuated malaria parasite vaccine and tested for safety, immunogenicity, and preliminary efficacy in malaria-naïve Dutch volunteers. Dose-escalation immunizations up to 9.0 × 105 PfSPZ of PfSPZ-GA1 Vaccine were well tolerated without breakthrough blood-stage infection. Subsequently, groups of volunteers were immunized three times by direct venous inoculation with cryopreserved PfSPZ-GA1 Vaccine (9.0 × 105 or 4.5 × 105 PfSPZ, N = 13 each), PfSPZ Vaccine (radiation-attenuated PfSPZ, 4.5 × 105 PfSPZ, N = 13), or normal saline placebo at 8-week intervals, followed by exposure to mosquito bite controlled human malaria infection (CHMI). After CHMI, 3 of 25 volunteers from both PfSPZ-GA1 groups were sterilely protected, and the remaining 17 of 22 showed a patency ≥9 days (median patency in controls, 7 days; range, 7 to 9). All volunteers in the PfSPZ Vaccine control group developed parasitemia (median patency, 9 days; range, 7 to 12). Immunized groups exhibited a significant, dose-related increase in anti-Pf circumsporozoite protein (CSP) antibodies and Pf-specific interferon-γ (IFN-γ)-producing T cells. Although no definite conclusion can be drawn on the potential strength of protective efficacy of PfSPZ-GA1 Vaccine, the favorable safety profile and induced immune responses by PfSPZ-GA1 Vaccine warrant further clinical evaluation.
... Taking advantage of pathogen stage conversion for immunization strategies is one way to attack the parasite at its developmental bottleneck (Kreutzfeld et al., 2017). Studies have shown that superior immunity can be achieved by a developmental arrest shortly before liver-to-blood stage conversion, as a larger and broader array of antigens is presented compared to early arresting GAPs (Butler et al., 2011;Haussig et al., 2011). Therefore, we performed immunizations with azithromycin attenuated late arresting liver stages and challenged vaccinated animals to explore whether restoring HSP101 in liver stages can elicit superior immune responses. ...
Article
Full-text available
Host cell remodeling is critical for successful Plasmodium replication inside erythrocytes and achieved by targeted export of parasite-encoded proteins. In contrast, during liver infection the malarial parasite appears to avoid protein export, perhaps to limit exposure of parasite antigens by infected liver cells. HSP101, the force-generating ATPase of the protein translocon of exported proteins (PTEX) is the only component that is switched off during early liver infection. Here, we generated transgenic Plasmodium berghei parasite lines that restore liver stage expression of HSP101. HSP101 expression in infected hepatocytes was achieved by swapping the endogenous promoter with the ptex150 promoter and by inserting an additional copy under the control of the elongation one alpha (ef1α) promoter. Both promoters drive constitutive and, hence, also pre-erythrocytic expression. Transgenic parasites were able to complete the life cycle, but failed to export PEXEL-proteins in early liver stages. Our results suggest that PTEX-dependent early liver stage export cannot be restored by addition of HSP101, indicative of alternative export complexes or other functions of the PTEX core complex during liver infection.
... Keitany et al. 2014b;Tarun et al. 2007;Moita et al. 2021). In agreement with this, further interrogation of the T cell responses in animals and human subjects revealed the persistent induction of IFN-γ, TNF-α, NO and abundance of sporozoite-specific central and effector memory CD8 + T cells, and IFN-γ producing CD8 + T cells (Belnoue et al. 2004;Butler et al. 2011;Trimnell et al. 2009;Tarun et al. 2007). Moreover, in vitro studies further demonstrated that the treatment of sporozoite-infected hepatocytes with IFN-γ showed eradication of parasites (P. ...
Article
Malaria represents one of the major life-threatening diseases that poses a huge socio-economic impact, worldwide. Chemoprophylaxis vaccination using a relatively low number of wild-type infectious sporozoites represents an attractive and effective vaccine strategy against malaria. However, the role of immune responses to pre-erythrocytic versus blood-stage parasites in protection against different antimalarial drugs remains unclear. Here, in the present study, we explored the immune responses against the repetitive inoculation of live Plasmodium yoelii (P. yoelii) sporozoites in an experimental Swiss mouse model under antimalarial drug lumefantrine chemoprophylaxis (CPS-LMF). We monitored the liver stage parasitic load, pro/anti-inflammatory cytokines expression, and erythrocytic stage patency, following repetitive cycles of sporozoites inoculations. It was found that repetitive sporozoites inoculation under CPS-LMF results in delayed blood-stage infection during the fourth sporozoites challenge, while sterile protection was produced in mice following the fifth cycle of sporozoites challenge. Intriguingly, we observed a significant up-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and iNOS response and down-regulation of anti-inflammatory cytokines (IL-4, IL-10 and TGF-β) in the liver HMNC (hepatic mononuclear cells) and spleen cells after 4th and 5th cycle of sporozoites challenge in the CPS-LMF mice. Meanwhile, we also noticed that the liver stage parasites load under CPS-LMF immunization has gradually reduced after 2nd, 3rd, 4th and 5th sporozoites challenge. Overall, our study suggests that chemoprophylaxis vaccination under LMF drug cover develops strong immune responses and confer superior long-lasting protection against P. yoelii sporozoites. Furthermore, this vaccination strategy can be used to study the protective and stage-specific immunity against new protective antigens. Supplementary information: The online version contains supplementary material available at 10.1007/s13205-021-03022-0.
... Pb.ANKA, P. berghei ANKA; Py.N67, P. y. nigeriensis N67; Py.YM, P. y. yoelii YM; Pc.AS, P. chabaudi AS. studied. Late liver stage-arresting and replication-competent (LARC) of genetically-attenuated sporozoites have been shown to provide good cross-stage and cross-species protection in mice (Butler et al., 2011;Vaughan et al., 2018). However, a recent study using P. yoelii LARC sporozoites showed that the liver stageengendered IFN-I signaling impaired hepatic CD8 + T cell responses, which is critical for liver stage protection. ...
Article
Full-text available
Type I interferons (IFN-Is) are important cytokines playing critical roles in various infections, autoimmune diseases, and cancer. Studies have also shown that IFN-Is exhibit ‘conflicting’ roles in malaria parasite infections. Malaria parasites have a complex life cycle with multiple developing stages in two hosts. Both the liver and blood stages of malaria parasites in a vertebrate host stimulate IFN-I responses. IFN-Is have been shown to inhibit liver and blood stage development, to suppress T cell activation and adaptive immune response, and to promote production of proinflammatory cytokines and chemokines in animal models. Different parasite species or strains trigger distinct IFN-I responses. For example, a Plasmodium yoelii strain can stimulate a strong IFN-I response during early infection, whereas its isogenetic strain does not. Host genetic background also greatly influences IFN-I production during malaria infections. Consequently, the effects of IFN-Is on parasitemia and disease symptoms are highly variable depending on the combination of parasite and host species or strains. Toll-like receptor (TLR) 7, TLR9, melanoma differentiation-associated protein 5 (MDA5), and cyclic GMP-AMP synthase (cGAS) coupled with stimulator of interferon genes (STING) are the major receptors for recognizing parasite nucleic acids (RNA/DNA) to trigger IFN-I responses. IFN-I levels in vivo are tightly regulated, and various novel molecules have been identified to regulate IFN-I responses during malaria infections. Here we review the major findings and progress in ligand recognition, signaling pathways, functions, and regulation of IFN-I responses during malaria infections.
... The poor protection by overirradiated sporozoites suggests that heat is not the only factor that reduces Plasmodium immunogenicity. The precise requirements for the generation of optimal CD8 + T-cell-mediated protective immunity to liver-stage Plasmodium parasites are not well defined, but there is evidence that GAP vaccination induces higher numbers of memory CD8 + T cells than RAS vaccination [36], suggesting that metabolism and the ability of the parasites to invade and persist within hepatocytes for longer periods of time may be important. This is also relevant in the context of HKS as the heat-killed parasites are not metabolically active and cannot migrate to and invade the liver. ...
Article
Full-text available
Malaria remains a major cause of mortality in the world, and an efficient vaccine is the best chance of reducing the disease burden. Vaccination strategies for the liver stage of disease that utilise injection of living radiation‐attenuated sporozoites (RAS) confer sterile immunity, which is mediated by CD8⁺ memory T cells, with liver‐resident memory T cells (TRM) being particularly important. We have previously described a TCR transgenic mouse, termed PbT‐I, where all CD8⁺ T cells recognize a specific peptide from Plasmodium. PbT‐I form liver TRM cells upon RAS injection and are capable of protecting mice against challenge infection. Here we utilize this transgenic system to examine whether non‐living sporozoites, killed by heat treatment (HKS), could trigger the development of Plasmodium‐specific liver TRM cells. We found that HKS vaccination induced the formation of memory CD8⁺ T cells in the spleen and liver, and importantly, liver TRM cells were fewer in number than that induced by RAS. Crucially, we showed the number of TRM cells was significantly higher when HKS were combined with the glycolipid α‐galactosylceramide as an adjuvant. In the future, this work could lead to development of an anti‐malaria vaccination strategy that does not require live sporozoites, providing greater utility. This article is protected by copyright. All rights reserved
... Additional regulations, specific to each national jurisdiction, apply to the use and potential release of genetically modified parasite pathogens. This includes genetically modified malaria parasites that have been used in clinical trials 56,57 (ANZCTR ID: ACTRN12617000824369). The inadvertent release of genetically modified parasites into local malaria vectors could lead to unforeseen or additional negative effects beyond that caused by the release of a wild type organism. ...
Article
Malaria clinical studies entailing the experimental infection of healthy volunteers with Plasmodium parasites by bites from infected mosquitos, injection of cryopreserved sporozoites, or injection of blood-stage parasites provide valuable information for vaccine and drug development. Success of these studies depends on maintaining safety. In this mini-review, we discuss the safety risks and associated mitigation strategies of these three types of experimental malaria infection. We aimed to inform researchers and regulators who are currently involved in or are planning to establish experimental malaria infection studies in endemic or non-endemic settings.
... Both CSP and TRAP subunit-based vaccines (RTS,S and ME-TRAP, respectively) have successfully elicited sterile immunity against malaria infection in humans; however, both have demonstrated only modest efficacy (27). The protection elicited using subunit-based vaccines is low compared to the protection seen with RAS, suggesting the key protective antigens have not been identified and/or the highly protective RAS immunity is generated in response to multiple antigens expressed in the liver stage (16,(27)(28)(29). In either case, discovery of novel antigens and their inclusion in multi-antigen/ subunit vaccines is needed to increase effectiveness of a liver stage malaria vaccine. ...
Article
Full-text available
Immunization with radiation-attenuated sporozoites (RAS) has been shown to protect against malaria infection, primarily through CD8 T cell responses, but protection is limited based on parasite strain. Therefore, while CD8 T cells are an ideal effector population target for liver stage malaria vaccine development strategies, such strategies must incorporate conserved epitopes that cover a large range of class I human leukocyte antigen (HLA) supertypes to elicit cross-strain immunity across the target population. This approach requires identifying and characterizing a wide range of CD8 T cell epitopes for incorporation into a vaccine such that coverage across a large range of class I HLA alleles is attained. Accordingly, we devised an experimental framework to identify CD8 T cell epitopes from novel and minimally characterized antigens found at the pre-erythrocytic stage of parasite development. Through in silico analysis we selected conserved P. falciparum proteins, using P. vivax orthologues to establish stringent conservation parameters, predicted to have a high number of T cell epitopes across a set of six class I HLA alleles representative of major supertypes. Using the decision framework, five proteins were selected based on the density and number of predicted epitopes. Selected epitopes were synthesized as peptides and evaluated for binding to the class I HLA alleles in vitro to verify in silico binding predictions, and subsequently for stimulation of human T cells using the Modular IMmune In-vitro Construct (MIMIC ® ) technology to verify immunogenicity. By combining the in silico tools with the ex vivo high throughput MIMIC platform, we identified 15 novel CD8 T cell epitopes capable of stimulating an immune response in alleles across the class I HLA panel. We recommend these epitopes should be evaluated in appropriate in vivo humanized immune system models to determine their protective efficacy for potential inclusion in future vaccines.
Article
Effective malaria vaccines are urgently needed. Now, clinical evidence indicates that a vaccination approach that uses live parasites growing in the liver can generate high levels of immune protection from infection. Clinical evidence points to an effective vaccination approach.
Article
Radiation attenuated sporozoite (RAS), a whole parasite vaccine approach provides sterile protection against malaria. However, RAS immunization does not confer protection for long, and that has been correlated with the waning parasite-induced memory CD8⁺ T cell responses. Interestingly, an intermittent infectious (wild-type) sporozoite challenge to the RAS vaccinated mice lengthened the protection period from 6 to 18 months. Herein, we have studied the changes that infectious sporozoite brought in RAS-induced memory CD8⁺ T cells for conferring lengthened protection. We observed that the infectious sporozoite challenge has boosted the frequency of foreign antigen-experienced memory CD8⁺ T cells. In those CD8⁺ T cells, it has reduced the Annexin-V reactivity, raised Bcl-2 expression, and also more cells undergone homeostatic proliferation (Ki-67⁺). It has also scaled down the frequency of Nur77 and CX3CR1 high expressing cells in those memory CD8⁺ T cell populations which we further correlated with better survival signals. This article is protected by copyright. All rights reserved.
Preprint
Functional characterization of genes in Plasmodium parasites often relies on genetic manipulations to disrupt or modify a gene-of-interest. However, these approaches are limited by the time required to generate transgenic parasites for P. falciparum and the availability of a single drug selectable marker for P. yoelii . In both cases, there remains a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a SpCas9-based gene editing system for Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) sgRNA expression strategy. Using this system with P. yoelii , we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free and scar-free parasites with homology arms as short as 80-100bp. Moreover, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and the desired locus replacement editing events, while the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding dCas9 to targets in the gene control region of a gene-of-interest, resulting in a modest reduction in gene expression. This robust and flexible system should open the door for in-depth and efficient genetic characterizations in both rodent- and human-infectious Plasmodium species. Importance Plasmodium parasites, the causative agent of malaria, still pose an enormous threat to public health worldwide. Gaining additional insight into the biology of the parasite is essential for generating an effective vaccine and identifying novel drug targets. To this end, CRISPR/Cas9 tools have been developed to more efficiently interrogate the Plasmodium genome than is possible with conventional reverse genetics approaches. Here, we describe CRISPR-RGR as an addition to the CRISPR/Cas9 toolbox for the rodent-infectious Plasmodium parasites. By using multiple ribozyme-flanked single guide RNAs expressed from RNA polymerase II promoters, transgenic parasites can be rapidly generated as designed without leaving selectable markers. Moreover, CRISPR-RGR can be adapted for use as a CRISPR interference (CRISPRi) system to alter gene expression without genome modification. Together, CRISPR-RGR for gene editing and CRISPRi application can hasten investigations into the biology and vulnerabilities of the malaria parasite.
Preprint
Plasmodium falciparum ( Pf ) malaria causes high rates of morbidity and mortality and lacks a sufficiently effective vaccine. Clinical immunity develops in residents of malaria endemic regions which confers reduced clinical symptoms during infection and protection against severe disease. We hypothesized that understanding the immune mechanisms of clinical immunity could inform vaccine design to improve efficacy. We compared the peripheral blood cellular and humoral immune responses during a mild episode of Pf malaria infection. Participants were classified as either clinically susceptible or clinically protected, based on the number of recurrent clinical infections over an 18-month longitudinal study in a malaria endemic region in Malawi. Susceptible participants had three or more recurrent clinical episodes while clinically immune individuals had one or none. Protected participants exhibited higher plasma immunoglobulin G (IgG) breadth and titers against Pf antigens, and greater antibody (Ab)-dependent Pf opsonization compared to susceptible participants. Using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses, we identified expanded memory CD4 ⁺ T cell clones sharing identical T cell receptor clonotypes in the blood of protected participants during malaria infection. These cells express a strong cytolytic T helper 1 effector program with transcripts encoding granzymes (A, B, H, M), granulysin, NKG7 and the Zeb2 master transcriptional regulator of terminally differentiated effector T cells. Memory CD4 ⁺ T cells expressing Zeb2 ⁺ were CD39 hi TIGIT hi and expressed multiple chemotactic and checkpoint inhibitory receptors, although the cellular levels of several of these receptors were reduced in protected compared to susceptible individuals. We propose that clonally expanded Zeb2 ⁺ cytolytic memory CD4 ⁺ Th1 cells could represent essential contributors to clinical immunity against Pf malaria. One Sentence Summary A population of cytolytic memory CD4 ⁺ T cells is clonally expanded in patients with Plasmodium falciparum malaria and has reduced chemotactic and inhibitory receptor expression in patients with naturally acquired clinical malaria immunity.
Article
Introduction Tissue-resident memory T cells (TRM cells) are powerful mediators of protracted adaptive immunity to infection in peripheral organs. Harnessing TRM cells through vaccination hence promises unprecedented potential for protection against infection. A paramount example of this is malaria, a major infectious disease for which immunity through traditional vaccination strategies remains challenging. Liver TRM cells appear to be highly protective against malaria, and recent developments in our knowledge of the biology of these cells have defined promising, novel strategies for their induction. Areas covered Here, we describe the path that led to the discovery of TRM cells and discuss the importance of liver TRM cells in immunity against Plasmodium spp. infection; we summarize current knowledge on TRM cell biology and discuss the current state and potential of TRM-based vaccination against malaria. Expert opinion TRM based vaccination has emerged as a promising means to achieve efficient protection against malaria. Recent advances provide a solid basis for continuing the development of this area of research. Deeper understanding of the mechanisms that mediate TRM formation and maintenance and identification of immunogenic and protective target epitopes suitable for human vaccination remain the main challenges for translation of these discoveries.
Preprint
Full-text available
Malaria, a disease caused by parasites of the Plasmodium genus, begins when Plasmodium-infected mosquitoes inject malaria sporozoites while searching for blood. Sporozoites migrate from the skin via blood to the liver, infect hepatocytes, and form liver stages which in mice 48 hours later escape into blood and cause clinical malaria. Vaccine-induced activated or memory CD8 T cells are capable of locating and eliminating all liver stages in 48 hours, thus preventing the blood-stage disease. However, the rules of how CD8 T cells are able to locate all liver stages within a relatively short time period remains poorly understood. We recently reported formation of clusters consisting of variable numbers of activated CD8 T cells around Plasmodium yoelii (Py)-infected hepatocytes. Using a combination of experimental data and mathematical models we now provide additional insights into mechanisms of formation of these clusters. First, we show that a model in which cluster formation is driven exclusively by T-cell-extrinsic factors, such as variability in "attractiveness" of different Py-infected cells, cannot explain distribution of cluster sizes in different experimental conditions. In contrast, the model in which cluster formation is driven by the positive feedback loop (i.e., larger clusters attract more T cells) can accurately explain the available data. Second, while both Py-specific CD8 T cells and T cells of irrelevant specificity (non-specific T cells) are attracted to the clusters, we found no evidence that non-specific T cells play a role in cluster formation. Third and finally, mathematical modeling suggested that formation of clusters occurs rapidly, within few hours after adoptive transfer of T cells, thus illustrating high efficiency of T cells in locating their targets in complex peripheral organs such as the liver. Taken together, our analysis provides novel insights into and discriminates between alternative mechanisms driving the formation of clusters of antigen-specific CD8 T cells in the liver.
Article
Introduction A safe and effective vaccine will likely be necessary for the control or eradication of malaria which kills 400,000 annually. Our most advanced vaccine candidate to date is RTS,S which is based on the Plasmodium falciparum circumsporozoite protein (PfCSP) of the malaria parasite. However, protection by RTS,S is incomplete and short-lived. Areas covered Here we summarize results from recent clinical trials of RTS,S and critically evaluate recent studies that aim to understand the correlates of protective immunity and why vaccine-induced protection is short-lived. In particular, recent systems serology studies have highlighted a key role for the necessity of inducing functional antibodies. In-depth analyses of immune responses to CSP in both mouse models and vaccinated humans have also highlighted difficulties in generating the maintaining high-quality antibody responses. Finally, in recent years biophysical and structural studies of antibody binding to PfCSP have led to a better understanding of how highly potent antibodies can block infection, which can inform vaccine design. Expert Opinion We highlight how both structure-guided vaccine design and a better understanding of the immune response to PfCSP can inform a second generation of PfCSP-based vaccines stimulating a broader range of protective targets within PfCSP.
Article
Full-text available
Radiation‐attenuated sporozoites induce sterilizing immunity and remain the 'gold standard' for malaria vaccine development. Despite practical challenges in translating these whole sporozoite vaccines to large‐scale intervention programmes, they have provided an excellent platform to dissect the immune responses to malaria pre‐erythrocytic (PE) stages, comprising both sporozoites and exoerythrocytic forms. Investigations in rodent models have provided insights that led to the clinical translation of various vaccine candidates—including RTS,S/AS01, the most advanced candidate currently in a trial implementation programme in three African countries. With advances in immunology, transcriptomics and proteomics, and application of lessons from past failures, an effective, long‐lasting and wide‐scale malaria PE vaccine remains feasible. This review underscores the progress in PE vaccine development, focusing on our understanding of host‐parasite immunological crosstalk in the tissue environments of the skin and the liver. We highlight possible gaps in the current knowledge of PE immunity that can impact future malaria vaccine development efforts.
Chapter
Full-text available
Vaccination was developed by Edward Jenner in 1796. Since then, vaccination and vaccine development research has been a hotspot of research in the scientific community. Various ways of vaccine development are successfully employed in mass production of vaccines. One of the most successful ways to generate vaccines is the method of virulence attenuation in pathogens. The attenuated strains of viruses, bacteria, and parasites are used as vaccines which elicit robust immune response and confers protection against virulent pathogens. This chapter brings together the most common and efficient ways of generating live attenuated vaccine strains in viruses, bacteria, and parasites.
Article
Full-text available
Human malaria vaccine trials have revealed vaccine efficacy but improvement is still needed. In this study, we aimed to re-evaluate vaccination with blood-stage naturally attenuated parasites, as a whole-organism vaccine model against cross-strain and cross-species malaria, to establish a better vaccination strategy. C57BL/6 mice controlled blood-stage Plasmodium yoelii 17XNL (PyNL) within 1 month of infection, while mice with a variety of immunodeficiencies demonstrated different susceptibilities to PyNL, including succumbing to hyperparasitemia. However, after recovery, survivors had complete protection against a challenge with the lethal strain PyL. Unlike cross-strain protection, PyNL-recovered mice failed to induce sterile immunity against Plasmodium berghei ANKA, although prolonged survival was observed in some vaccinated mice. Splenomegaly is a typical characteristic of malaria; the splenic structure became reorganized to prioritize extra-medullary hematopoiesis and to eliminate parasites. We also found that the peritoneal lymph node was enlarged, containing activated/memory phenotype cells that did not confer protection against PyL challenge. Hemozoins remained in the spleen several months after PyNL infection. Generation of an attenuated human blood-stage parasite expressing proteins from multiple species of malaria would greatly improve anti-malaria vaccination.
Article
Malaria is a global health scourge for which a highly effective vaccine remains frustratingly elusive. Recent identification of an endogenous malaria antigen that stimulates robust TRM-mediated immunity in mice by Valencia-Hernandez et al. strengthens the case for prime-and-trap malaria vaccines and will greatly aid further investigations of cellular antimalarial immunity.
Article
While the liver and blood stages of the Plasmodium life cycle are commonly regarded as two separate fields of malaria research, several studies have pointed towards the existence of a bidirectional cross-talk, where one stage of mammalian infection may impact the establishment and progression of the other. Despite the constraints in experimentally addressing concurrent liver and blood stage Plasmodium infections, animal models and clinical studies have unveiled a plethora of molecular interactions between the two. Here, we review the current knowledge on the reciprocal influence of hepatic and erythrocytic infection by malaria parasites, and discuss its impacts on immunity, pathology and vaccination against this deadly disease.
Article
Full-text available
Malaria is a mosquito-borne disease that is transmitted by inoculation of the Plasmodium parasite sporozoite stage. Sporozoites invade hepatocytes, transform into liver stages, and subsequent liver-stage development ultimately results in release of pathogenic merozoites. Liver stages of the parasite are a prime target for malaria vaccines because they can be completely eliminated by sterilizing immune responses, thereby preventing malarial infection. Using expression profiling, we previously identified genes that are only expressed in the pre-erythrocytic stages of the parasite. Here, we show by reverse genetics that one identified gene, UIS3 (upregulated in infective sporozoites gene 3), is essential for early liver-stage development. uis3-deficient sporozoites infect hepatocytes but are unable to establish blood-stage infections in vivo, and thus do not lead to disease. Immunization with uis3-deficient sporozoites confers complete protection against infectious sporozoite challenge in a rodent malaria model. This protection is sustained and stage specific. Our findings demonstrate that a safe and effective, genetically attenuated whole-organism malaria vaccine is possible.
Article
Full-text available
Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages – liver stages – of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans.
Article
Full-text available
Intravenous injection of mice with attenuated Plasmodium berghei sporozoites induces sterile immunity to challenge with viable sporozoites. Non-intravenous routes have been reported to yield poor immunity. Because intravenous immunization has been considered to be unacceptable for large scale vaccination of humans, assessment was made of the results of intradermal immunization of mice with Plasmodium yoelii, a rodent malaria parasite whose infectivity resembles that of human malaria. Mice were immunized with two injections of isolated, radiation-attenuated P. yoelii sporozoites, either by intravenous (IV) or intradermal (ID) inoculation. In an attempt to enhance protective immunogenicity of ID-injections, one group of experimental mice received topical application of an adjuvant, Imiquimod, while another group had their injections accompanied by local "tape-stripping" of the skin, a procedure known to disrupt the stratum corneum and activate local immunocytes. Challenge of immunized and non-immunized control mice was by bite of sporozoite-infected mosquitoes. Degree of protection among the various groups of mice was determined by microscopic examination of stained blood smears. Statistical significance of protection was determined by a one-way ANOVA followed by Tukey's post hoc test. Two intravenous immunizations produced 94% protection to mosquito bite challenge; intradermal immunization produced 78% protection, while intradermal immunization accompanied by "tape-stripping" produced 94% protection. There were no statistically significant differences in degree of protective immunity between immunizations done by intravenous versus intradermal injection. The use of a sub-microlitre syringe for intradermal injections yielded excellent protective immunity. ID-immunization with large numbers of radiation-attenuated P. yoelii sporozoites led to levels of protective immunity comparable to those achieved by IV-immunization. It remains to be determined whether an adjuvant treatment can be found to substantially reduce the numbers of attenuated sporozoites required to achieve a strong protective immunity with as few doses as possible for possible extension to immunization of humans.
Article
Full-text available
Malaria caused by Plasmodium falciparum remains a major public health threat, especially among children and pregnant women in Africa. An effective malaria vaccine would be a valuable tool to reduce the disease burden and could contribute to elimination of malaria in some regions of the world. Current malaria vaccine candidates are directed against human and mosquito stages of the parasite life cycle, but thus far, relatively few proteins have been studied for potential vaccine development. The most advanced vaccine candidate, RTS,S, conferred partial protection against malaria in phase II clinical trials and is currently being evaluated in a phase III trial in Africa. New vaccine targets need to be identified to improve the chances of developing a highly effective malaria vaccine. A better understanding of the mechanisms of naturally acquired immunity to malaria may lead to insights for vaccine development.
Article
Full-text available
The RTS,S/AS02(D) vaccine has been shown to have a promising safety profile, to be immunogenic and to confer protection against malaria in children and infants. We did a randomized, controlled, phase I/IIb trial of RTS,S/AS02(D) given at 10, 14 and 18 weeks of age staggered with routine immunization vaccines in 214 Mozambican infants. The study was double-blind until the young child completed 6 months of follow-up over which period vaccine efficacy against new Plasmodium falciparum infections was estimated at 65.9% (95% CI 42.6-79.8, p<0.0001). We now report safety, immunogenicity and estimated efficacy against clinical malaria up to 14 months after study start. Vaccine efficacy was assessed using Cox regression models. The frequency of serious adverse events was 32.7% in the RTS,S/AS02(D) and 31.8% in the control group. The geometric mean titers of anti-circumsporozoite antibodies declined from 199.9 to 7.3 EU/mL from one to 12 months post dose three of RTS,S/AS02(D), remaining 15-fold higher than in the control group. Vaccine efficacy against clinical malaria was 33% (95% CI: -4.3-56.9, p = 0.076) over 14 months of follow-up. The hazard rate of disease per 2-fold increase in anti-CS titters was reduced by 84% (95% CI 35.1-88.2, p = 0.003). The RTS,S/AS02(D) malaria vaccine administered to young infants has a good safety profile and remains efficacious over 14 months. A strong association between anti-CS antibodies and risk of clinical malaria has been described for the first time. The results also suggest a decrease of both anti-CS antibodies and vaccine efficacy over time. ClinicalTrials.gov NCT00197028.
Article
Full-text available
Radiation-attenuated Plasmodium sporozoites (RAS) are the only vaccine shown to induce sterilizing protection against malaria in both humans and rodents. Importantly, these "whole-parasite" vaccines are currently under evaluation in human clinical trials. Studies with inbred mice reveal that RAS-induced CD8 T cells targeting liver-stage parasites are critical for protection. However, the paucity of defined T cell epitopes for these parasites has precluded precise understanding of the specific characteristics of RAS-induced protective CD8 T cell responses. Thus, it is not known whether quantitative or qualitative differences in RAS-induced CD8 T cell responses underlie the relative resistance or susceptibility of immune inbred mice to sporozoite challenge. Moreover, whether extraordinarily large CD8 T cell responses are generated and required for protection following RAS immunization, as has been described for CD8 T cell responses following single-antigen subunit vaccination, remains unknown. Here, we used surrogate T cell activation markers to identify and track whole-parasite, RAS-vaccine-induced effector and memory CD8 T cell responses. Our data show that the differential susceptibility of RAS-immune inbred mouse strains to Plasmodium berghei or P. yoelii sporozoite challenge does not result from host- or parasite-specific decreases in the CD8 T cell response. Moreover, the surrogate activation marker approach allowed us for the first time to evaluate CD8 T cell responses and protective immunity following RAS-immunization in outbred hosts. Importantly, we show that compared to a protective subunit vaccine that elicits a CD8 T cell response to a single epitope, diversifying the targeted antigens through whole-parasite RAS immunization only minimally, if at all, reduced the numerical requirements for memory CD8 T cell-mediated protection. Thus, our studies reveal that extremely high frequencies of RAS-induced memory CD8 T cells are required, but may not suffice, for sterilizing anti-Plasmodial immunity. These data provide new insights into protective CD8 T cell responses elicited by RAS-immunization in genetically diverse hosts, information with relevance to developing attenuated whole-parasite vaccines.
Article
Full-text available
The global research community must take up the challenge to work toward the eradication of malaria. In the past, malaria research has focused on drugs and vaccines that target the blood stage of infection, and mainly on the most deadly species, Plasmodium falciparum, all of which is justified by the need to prevent and treat the disease. This work remains critically important today. However, an increased research focus is now being placed on potential interventions that aim to kill the parasite stages transmitted to and by the mosquito vector because they may represent more vulnerable targets to stop the spread of malaria. Here, we highlight some of the research into malaria parasite biology that has the potential to provide new intervention targets for antimalarial drugs and vaccines.
Article
Full-text available
Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.
Article
Full-text available
Malaria results in >1,000,000 deaths per year worldwide. Although no licensed vaccine exists, much effort is currently focused on subunit vaccines that elicit CD8 T cell responses directed against Plasmodium parasite liver stage Ags. Multiple immune-effector molecules play a role in antimicrobial immunity mediated by memory CD8 T cells, including IFN-gamma, perforin, TRAIL, Fas ligand, and TNF-alpha. However, it is not known which pathways are required for memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. In this study, we used a novel immunization strategy to generate memory CD8 T cells in the BALB/c mouse model of P. berghei or P. yoelii sporozoite infection to examine the role of immune-effector molecules in resistance to the liver stage infection. Our studies reveal that endogenous memory CD8 T cell-mediated protection against both parasite species is, in part, dependent on IFN-gamma, whereas perforin was only critical in protection against P. yoelii. We further show that neutralization of TNF-alpha in immunized mice markedly reduces memory CD8 T cell-mediated protection against both parasite species. Thus, our studies identify IFN-gamma and TNF-alpha as important components of the noncytolytic pathways that underlie memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. Our studies also show that the effector pathways that memory CD8 T cells use to eliminate liver stage infection are, in part, Plasmodium species specific.
Article
Full-text available
The difficulty of inducing protective immunity through antibodies against sporozoites led to efforts to assess vectored vaccines as a means of inducing protective T-cell immunity against the malaria liver-stage parasite. Although DNA vectored vaccines used alone were poorly immunogenic and not protective, high levels of parasite clearance in the liver has been achieved with viral vectored vaccines used in heterologous prime-boost regimes. Such vectored vaccination regimes represent one of only two approaches that have induced repeatable partial efficacy in human P. falciparum subunit vaccine trials. Interestingly, vectors expressing the TRAP antigen have been consistently been more immunogenic and protective than vectors expressing the circumsporozoite protein in human trials. However, sterile protection requires induction of very potent T-cell responses that are currently only achievable with heterologous prime-boost regimes. Recently, simian adenoviruses have been assessed as priming agents in Adenovirus-MVA regimes in both phase I and phase IIa trials in the UK, based on very promising pre-clinical results showing better immunogenicity and efficacy than previous prime-boost regimes. The same vectors are also being assessed clinically expressing blood-stage antigens, attempting to induce both protective antibodies and T cells as recently demonstrated in murine efficacy studies. These viral vectors now provide a major option for inclusion in a high efficacy multi-stage malaria vaccine that should achieve deployable levels of efficacy in endemic settings.
Article
Full-text available
Determining the magnitude and kinetics, together with the phenotypic and functional characteristics of responding CD8 T cells, is critical for understanding the regulation of adaptive immunity as well as in evaluating vaccine candidates. Recent technical advances have allowed tracking of some CD8 T cells responding to infection, and a body of information now exists describing phenotypic changes that occur in CD8 T cells of known Ag-specificity during their activation, expansion, and memory generation in inbred mice. In this study, we demonstrate that Ag but not inflammation-driven changes in expression of CD11a and CD8alpha can be used to distinguish naive from Ag-experienced (effector and memory) CD8 T cells after infection or vaccination. Interestingly and in contrast to inbred mice, tracking polyclonal CD8 T cell responses with this approach after bacterial and viral infections revealed substantial discordance in the magnitude and kinetics of CD8 T cell responses in outbred hosts. These data reveal limitations to the use of inbred mouse strains as preclinical models at vaccine development and suggest the same dose of infection or vaccination can lead to substantial differences in the magnitude and timing of Ag-specific CD8 expansion as well in differences in protective memory CD8 T cell numbers in outbred individuals. This concept has direct relevance to development of vaccines in outbred humans.
Article
Full-text available
Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.
Article
Full-text available
Immunization with radiation attenuated Plasmodium sporozoites (RAS) elicits sterile protective immunity against sporozoite challenge in murine models and in humans. Similarly to RAS, the genetically attenuated sporozoites (GAPs) named uis3(-), uis4(-) and P36p(-) have arrested growth during the liver stage development, and generate a powerful protective immune response in mice. We compared the protective mechanisms in P. yoelii RAS, uis3(-) and uis4(-) in BALB/c mice. In RAS and GAPs, sterile immunity is only achieved after one or more booster injections. There were no differences in the immune responses to the circumsporozoite protein (CSP) generated by RAS and GAPs. To evaluate the role of non-CSP T-cell antigens we immunized antibody deficient, CSP-transgenic BALB/c mice, that are T cell tolerant to CSP, with P. yoelii RAS or with uis3(-) or uis4(-) GAPs, and challenged them with wild type sporozoites. In every instance the parasite liver stage burden was approximately 3 logs higher in antibody deficient CSP transgenic mice as compared to antibody deficient mice alone. We conclude that CSP is a powerful protective antigen in both RAS and GAPs viz., uis3(-) and uis4(-) and that the protective mechanisms are similar independently of the method of sporozoite attenuation.
Article
Full-text available
Protecting against both liver and blood stages of infection is a long-sought goal of malaria vaccine design. Recently, we described the use of replication-defective viral vaccine vectors expressing the malaria antigen merozoite surface protein-1 (MSP-1) as an antimalarial vaccine strategy that elicits potent and protective antibody responses against blood-stage parasites. Here, we show that vaccine-induced MSP-1-specific CD4(+) T cells provide essential help for protective B cell responses, and CD8(+) T cells mediate significant antiparasitic activity against liver-stage parasites. Enhanced survival is subsequently seen in immunized mice following challenge with sporozoites, which mimics the natural route of infection more closely than when using infected red blood cells. This effect is evident both in the presence and absence of protective antibodies and is associated with decreased parasite burden in the liver followed by enhanced induction of the cytokine IFN-gamma in the serum. Multistage immunity against malaria can thus be achieved by using viral vectors recombinant for MSP-1.
Article
Full-text available
Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.
Article
Full-text available
Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4(+) and CD8(+) T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-gamma, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.
Article
Full-text available
As a consequence of gene cloning and DNA sequencing several gene families are emerging in the field of cell-cell recognition. These include immunoglobulins, integrins, certain extracellular glycoproteins and a family of functionally unrelated proteins which include factor B. We report here the cloning and sequencing of a gene from Plasmodium falciparum, coding for a protein we call thrombospondin related anonymous protein (TRAP), which shares certain sequence motifs common to other well-characterized proteins. The most significant homology is based around the sequence Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), present in three copies in region I of thrombospondin (TSP), six copies in properdin (P) and one copy in all the circumsporozoite (CS) proteins sequenced so far. TRAP also shares with certain extracellular glycoproteins, including TSP, the cell-recognition signal Arg-Gly-Asp (RGD), which has been shown to be crucial in the interaction of several extracellular glycoproteins with members of the integrin superfamily. Unlike the CS protein, TRAP is expressed during the erythrocytic stage of the parasite life cycle.
Article
Full-text available
It has been reported previously that immunization with recombinant protein containing the two epidermal growth factor (EGF)-like modules from merozoite surface protein 1 (MSP-1) of Plasmodium yoelii (strain YM) protects mice against a lethal blood-stage challenge with the same parasite strain. Since MSP-1 is expressed in both liver- and blood-stage schizonts and on the surface of merozoites, we evaluated the effectiveness of immunization with recombinant proteins containing either the individual or the two combined EGF-like modules in producing a protective response against a sporozoite challenge. The recombinant protein expressing the combined EGF-like modules of the YM strain protected mice against a homologous sporozoite challenge, and sterile protection, as defined by the absence of detectable blood-stage parasites, was observed in the majority of the mice. In contrast, mice immunized with recombinant P. yoelii YM MSP-1 were not protected against a heterologous challenge with sporozoites from strain 265 BY of P. yoelii. The lack of protection may be explained by differences identified in the amino acid sequences of MSP-1 for the two strains. A recombinant protein containing the two EGF-like modules of MSP-1 from P. yoelii 265 BY was produced and used to immunize mice. These mice were protected against a homologous challenge with sporozoites of P. yoelii 265 BY. The results suggest that a recombinant MSP-1 has potential as a vaccine against malaria, but its efficacy may be limited by sequence polymorphism and selection of variants.
Article
Full-text available
Sterile protective immunity against challenge with Plasmodium spp. sporozoites can be induced in multiple model systems and humans by immunization with radiation-attenuated Plasmodium spp. sporozoites. The infected hepatocyte has been established as the primary target of this protection, but the underlying mechanisms have not been completely defined. Abs, CD8+ T cells, CD4+ T cells, cytokines (including IFN-gamma and IL-12), and NO have all been implicated as critical effectors. Here, we have investigated the mechanisms of protective immunity induced by immunization with different vaccine delivery systems (irradiated sporozoites, plasmid DNA, synthetic peptide/adjuvant, and multiple Ag peptide) in genetically distinct inbred strains, genetically modified mice, and outbred mice. We establish that there is a marked diversity of T cell-dependent immune responses that mediate sterile protective immunity against liver-stage malaria. Furthermore, we demonstrate that distinct mechanisms of protection are induced in different strains of inbred mice by a single method of immunization, and in the same strain by different methods of immunization. These data underscore the complexity of the murine host response to a parasitic infection and suggest that an outbred human population may behave similarly. Data nevertheless suggest that a pre-erythrocytic-stage vaccine should be designed to induce CD8+ T cell- and IFN-gamma-mediated immune responses and that IFN-gamma responses may represent an in vitro correlate of pre-erythrocytic-stage protective immunity.
Article
Full-text available
Screening of a Plasmodium falciparum genomic expression library for antigens expressed at the pre-erythrocytic stages resulted in the isolation of a recombinant phage (DG249) whose insert corresponded to regions II and III of a 175-kDa erythrocyte-binding antigen (EBA-175). EBA-175 is a parasite ligand implicated in red blood cell invasion. Reverse-transcriptase polymerase chain reaction, indirect immunofluorescent antibody test, and Western blot analysis confirmed that EBA-175 is expressed not only in blood-stage parasites but also in infected hepatocytes and on the sporozoite surface. The presence of EBA-175 on pre-erythrocytic parasites enhances the vaccine potential of this antigen by adding another target to the immune responses elicited by immunization
Article
Full-text available
During 1989–1999, 11 volunteers were immunized by the bites of 1001–2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2–9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23–42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.
Article
Full-text available
How and when memory T cells form during an immune response are long-standing questions. To better understand memory CD8 T cell development, a time course of gene expression and functional changes in antigen-specific T cells during viral infection was evaluated. The expression of many genes continued to change after viral clearance in accordance with changes in CD8 T cell functional properties. Even though memory cell precursors were present at the peak of the immune response, these cells did not display hallmark functional traits of memory T cells. However, these cells gradually acquired the memory cell qualities of self-renewal and rapid recall to antigen suggesting the model that antigen-specific CD8 T cells progressively differentiate into memory cells following viral infection.