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Accepted Date : 23-Jan-2014 Article type
: Introduction
The path of malaria vaccine development: challenges and perspectives
Charles Arama1,2 & Marita Troye-Blomberg2 From the 1 Malaria Research and Training Center, University of Sciences, Techniques, and Technologies of Bamako (USTTB), Mali; and 2
Department of Molecular Biosciences, The Wenner-Gren Institute
Stockholm University, Stockholm, Sweden
Email: [email protected] Email: [email protected]; [email protected]
Abstract Malaria is a life-threatening disease caused by parasites of the Plasmodium genus. In many parts of the world, the parasites have developed resistance to a number of anti-malarial agents. Key interventions to control malaria include prompt and effective treatment with artemisinin-based combination therapies, use of insecticidal nets by individuals at risk and active research into malaria vaccines. Protection against malaria through vaccination was demonstrated more than 30 years ago when individuals were vaccinated via repeated bites by Plasmodium falciparum-infected and irradiated but still metabolically active mosquitoes. However, vaccination with high doses of irradiated sporozoites injected into humans has long been considered impractical. Yet, following recent success using whole-organism vaccines, the approach has received renewed interest; it was recently reported that repeated injections of irradiated sporozoites increased protection in 80 vaccinated individuals. Other approaches include subunit malaria vaccines, such as the current leading candidate RTS,S (consisting of fusion between a portion of the P. falciparum-derived
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circumsporozoite protein and the hepatitis B surface antigen), which has been demonstrated to induce reasonably good protection. Although results have been encouraging, the level of protection is generally considered to be too low to achieve eradication of malaria. There is great interest in developing new and better formulations and stable delivery systems to improve immunogenicity. In this review we will discuss recent strategies to develop efficient malaria vaccines.
Keywords: adjuvants, malaria, vaccine delivery systems, vaccines.
Introduction Malaria remains one of the most devastating infectious diseases affecting those living in tropical areas of the world, especially young children and pregnant women. Malaria in humans is caused by five protozoan parasite species of the genus Plasmodium: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi [1]. Malaria is a mosquito-borne disease and hence can be controlled in both humans and mosquitoes. Despite major efforts to control the disease, approximately 781 000 deaths occurred in 2009 in children under 5 years of age in sub-Saharan Africa [2]. The control of this life-threatening disease requires a multifaceted approach, including insecticides, chemotherapy and development of cheap, affordable and efficacious malaria vaccines [3]. Because of the large gap in our understanding of the biology of P. falciparum infection, it is difficult to identify the right vaccine candidate among the thousands of potential P. falciparum antigens [4]. This gap can be filled, but only with adequate research support and commitment from experts in all areas of immunobiology of P. falciparum in order to target a variety of potential approaches [5]. Although significant advances have been made in vector control strategies and the effectiveness of artemisinin-based combination therapy, a licensed malaria vaccine is still not available. In addition, current malaria control and elimination programmes are affected by notable heterogeneity of transmission dynamics in endemic areas, including differences in parasite-, vector- and host-related, social and environmental factors. Furthermore, clinical resistance to artemisinin and its derivatives is now well established in P. falciparum populations of western Cambodia [6, 7] and appears to be the greatest threat to global malaria elimination and eradication.
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To date, vaccination is the most effective method of preventing infectious diseases and represents the greatest contribution of immunology to human health. The remarkable success of vaccines against polio, measles, diphtheria, tetanus, rabies and others, and the complete eradication of smallpox in humans, prove the potential of this approach in reducing the global burden of infectious diseases [8]. However, despite this success for some diseases, major logistical and technical challenges remain to be solved in order to develop efficient malaria vaccines that might potentially provide an important tool for use in malaria elimination and eradication programmes.
Recent advances have led to a new era of vaccine development in general and for malaria in particular. Adjuvants and vaccine-delivery systems are becoming increasingly more important for the development of a new generation of vaccines, combining different types of adjuvants into antigen-specific formulations with greater efficacy and improved vaccine formulations [9]. These new approaches offer a wide spectrum of opportunities in malaria vaccine research with direct applications for the near future. The main advantage of vaccine-delivery systems is that they allow co-administration of immunostimulants and more than one antigen into the same system; however, difficulties remain when applying them to human vaccinology. In this review, we will discuss strategies to overcome some of these challenges and suggest several approaches for the development of an effective malaria vaccine.
The life cycle of P. falciparum
The malaria parasite requires mosquitoes and human hosts to complete its life cycle (Fig. 1), and goes through several developmental stages to survive in the human host. During its two-host life cycle, P. falciparum undergoes 10 morphological transitions in five different host tissues [10]. The cycle in humans includes: (i) the pre-erythrocytic stage, which is the first stage of infection in humans with inoculation of sporozoites to infect the hepatocytes; (ii) the erythrocytic stage, which involves asexual reproduction of the parasite in the blood to cause the clinical
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symptoms of the disease; and (iii) the gametocyte stage, which enables sexual reproduction of the parasite in the mosquito and further transmission. The pre-erythrocytic stage begins when an infected female Anopheles mosquito inoculates sporozoites into the skin or into the blood stream of humans during a blood meal. Sporozoites circulate transiently in the bloodstream before invading hepatocytes, in which the asexual cycle takes place. It is estimated that mosquitoes transmit fewer than 100 sporozoites per bite [11, 12]. The sporozoites can remain for up to 6 h at the site of injection [13], and one-third of those leaving the injection site may enter the draining lymph nodes via the lymphatic vessels [14]. The capacity of each of these sporozoites to produce asexual erythrocytic-stage infection is low. In humans, bites of at least five P. falciparum-infected mosquitoes are necessary to ensure that 100% of individuals will become infected [15]. When sporozoites reach the liver parenchyma, they continue to migrate through several hepatocytes before finally infecting one of them. This migration seems to be beneficial for malaria infections in at least two different ways: by activating sporozoites for infection and by increasing the susceptibility of host hepatocytes [16]. After a week, rupture of the merosomes within the lung microvasculature [17] releases thousands of infectious merozoites into the bloodstream where they invade circulating erythrocytes and initiate the clinically important intra-erythrocytic cycle of asexual replication during the following 48 h.
The intra-erythrocytic cycle is responsible for all the clinical symptoms associated with the disease. After 24–32 h, when young parasites mature from rings to the trophozoite stage, infected erythrocytes adhere to endothelial cells in the microcirculation of various organs (known as sequestration) causing cerebral malaria if sequestered in the brain. The trophozoites will eventually mature into schizonts, which finally rupture and release 16–32 daughter merozoites that can invade fresh red blood cells to restart the asexual life cycle. As a survival strategy, blood-stage parasites have been shown to infect and survive and replicate within CD317+ dendritic cells, and small numbers of these cells release parasites that can infect erythrocytes in vivo in a murine model [18].
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Some parasites inside the erythrocytes differentiate into male and female gametocytes. It remains poorly understood which factors stimulate gametogenesis [19]. However, it has been suggested that it is influenced by environmental factors, drugs or innate immune factors [20, 21]. Without treatment, most patients with P. falciparum malaria will develop gametocytaemia within 10 to 40 days after the onset of parasitaemia [22]. Upon ingestion by a feeding female mosquito, the male and female gametes undergo fertilization in the mosquito midgut to form a zygote and subsequently a motile ookinete. Okinetes penetrate the midgut epithelial cells and rest between the midgut epithelium and the basal lamina to form oocysts. The oocysts undergo a complex asexual development stage, which eventually generates infective sporozoites that can be introduced into the human host at the next blood meal via the mosquito saliva, thereby ensuring the continuation of the parasite life cycle.
Vaccines against malaria
The purpose of vaccination strategies is to induce protective memory immune responses in advance of infection, to provide protection in the case of encountering the disease-causing agent again. Malaria vaccine development is an active research area with enormous challenges. As the parasite proceeds from a sporozoite through the liver stage to the replicating cycle of the blood stage, it undergoes morphological changes and displays antigenic variations. This allows the parasite to evade the protective immune responses of the host. As a result, the acquisition of long-term sterile immunity, which is often associated with recovery from many other infectious diseases, is not observed in malaria-infected individuals. Despite these difficulties, the most convincing evidence that vaccination against malaria is feasible has come from experimental studies in rodents, monkeys and human subjects in which attenuated sporozoites induced sterile protective immunity. The main malaria vaccines currently in clinical trials are summarised in Table 1. An ideal malaria vaccine requires three essential features: (i) multiple components that will induce an effective immune response to the different stages of the malaria infection (sporozoites, infected hepatocytes and asexual and sexual stages); (ii) multiple epitopes that are restricted to presentation by different major
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histocompatibility complex (MHC) molecules in order to overcome genetic diversity and antigenic variation; and (iii) multi-immunogenicity inducing more than one type of immune response, including cell-mediated and humoral components. Such a multicomponent vaccine should increase the probability of a sustainable and effective host response [23]. Three types of vaccine candidate targeting different stages in the life cycle of the malaria parasite have been intensively investigated: (i) transmission-blocking vaccines (TBVs); (ii) pre-erythrocytic vaccines; and (iii) blood-stage vaccines (Fig 2). TBVs
TBVs target antigens on gametes, zygotes and ookinetes in order to prevent parasite development in the mosquito midgut. The aim of these vaccines is to induce antibodies against the sexual-stage antigens to block ookinete-to-oocyst transition to stop the subsequent generation of infectious sporozoites [24]. Of note, TBVs do not protect the recipient from contracting malaria, but could be helpful in preventing the spread of the disease. These vaccines are intended to protect entire/selected communities from infection. The leading vaccine candidates in this group include the P. falciparum ookinete surface antigens Pfs25 [25] and Pfs28 and their P. vivax homologues Pvs25 and Pvs28 [26, 27]. In order to improve the immunogenicity, Pfs25 was expressed as a recombinant protein that was chemically cross-linked to ExoProtein A and delivered as a nanoparticle. This enhanced the immunogenicity of the vaccine in mice, and it is currently undergoing Phase I trials in humans [28]. Because the antigens are never naturally presented to the human immune system, one of the potential limitations of the TBV approach is that the absence of natural boosting following immunization might limit efficacy [29]. Nevertheless, TBVs could be important tools for a malaria elimination and eradication programme, for prevention of transmission of the disease [30].
Pre-erythrocytic vaccines Research on pre-erythrocytic vaccines has quickly progressed during this last decade, and a number of new approaches are in the pipeline that could contribute to improve the second generation of malaria vaccines. Liver-stage vaccines are designed to prevent malaria in the
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human host. However, because of the high rate of replication of sporozoites, a single parasite may be sufficient for the infection to proceed to the blood stage. The liver stage of P. falciparum is an attractive therapeutic target for the development of both anti-malarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage [31]. However, an efficient liver stage vaccine must be 100% effective in order to protect humans with no natural immunity. Such vaccines include those containing whole killed sporozoites and those based on antigenic portions of the circumsporozoite proteins [32]. A landmark finding that set the standards for immunological protection against malaria infection was established by immunisation with irradiated sporozoites [33, 34]. To date, the most advanced pre-erythrocytic vaccine candidate is RTS,S, which consists of a truncated circumsporozoite protein (CSp) of P. falciparum directly fused to the hepatitis B surface (S) antigen. This vaccine has shown 30–50% protection in human field trials in Africa [35, 36]. As protection was relatively short lasting, how to improve the efficacy by manipulating the immune response of the host is a current challenge. The mechanism by which RTS,S confers protection against blood-stage disease remains poorly understood. It seems that RTS,S induces protection against clinical malaria by temporarily reducing the number of merozoites emerging from the liver. This may allow prolonged exposure to subclinical levels of asexual blood-stage parasites, therefore boosting the naturally acquired blood-stage immunity [37]. A novel antigen, the cell-traversal protein for ookinetes and sporozoites (CelTOS), has been identified as an essential protein for the traversal of the malaria parasite in both mammalian and the insect hosts [38]. In a murine model, CelTOS is a potentially interesting pre-erythrocytic vaccine candidate, which induces complete sterile protection against sporozoite challenge [39].
The case for a whole sporozoite vaccine originated from the early finding that mice could be protected by inoculation of P. berghei-irradiated sporozoites by intravenous injection [33]. Today, there is renewed interest in the whole-organism vaccine as a result of a recent highly successful human trial using experimental sporozoite inoculation under chloroquine prophylaxis [40]. In this trial, four of the six volunteers mounted sterile protection against P. falciparum rechallenges 2 years later [41]. Sporozoites attenuated by targeted gene disruption are being evaluated as whole-parasite vaccines in which the favourite candidates are genetically attenuated late liver stage-arresting parasites [42]. This type of vaccine induces high levels of cross-stage and cross-species protection and complete protection when administered This article is protected by copyright. All rights reserved.
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by intradermal or subcutaneous routes [43]. However, the results have not been reproduced. For instance, cross-stage protection is not proven in human infection, with recent data suggesting a complete lack of protection against blood-stage challenge and indicating that chemoprophylaxis with P. falciparum sporozoiteinduced protection is mediated by immunity against pre-erythrocytic stages [44]. Genetically attenuated vaccines can avoid the potentially problematic irradiation step in the manufacturing process, but other challenges of manufacturing and delivering a viable cryopreserved whole parasite vaccine remain [45]. However, the results of a recent vaccine trial showed that these challenges could be overcome [46]. With regard to this encouraging finding, the next step will be the implementation of such a trial in malaria endemic areas with diverse transmission settings. The fact that whole sporozoite vaccines induce better protection than subunit vaccines [47, 48] suggests that antigen combination strategies are necessary. Therefore, further research into other potential malaria vaccine antigens and strategies for their delivery is essential.
Blood-stage vaccines
Blood-stage vaccines are designed to elicit anti-invasion and anti-disease responses [49]. The underlying principle of these strategies is that if a vaccine could block the invasion of erythrocytes by merozoites, it would prevent malarial disease. At present, several blood-stage antigens are in clinical trials: apical membrane antigen 1 (AMA1) [50], erythrocyte-binding antigen-175 (EBA-175) [51], glutamate-rich protein (GLURP) [52, 53], merozoite surface protein (MSP) 1 [54], MSP2 [55] and MSP3 [56, 57] and serine repeat antigen 5 (SERA5) [58, 59]. All these antigens are highly expressed on the surface of merozoites. Of note, AMA1 and MSP1 did not demonstrate efficacy in African children [50, 54], probably due to the highly polymorphic nature of the vaccine structures [60]. Efforts to enhance the vaccine efficacy of AMA1 and MSP1 with novel adjuvants [61, 62], using viral vector prime-boost strategies [63], or by combining AMA1 and MSP1 [64] have been investigated. The extensive genetic diversity of the parasite and the selective pressure exerted by the host’s immune response are major factors to be considered in the development of effective blood-stage vaccines [65]. How to address genetic polymorphism constitutes an important issue to be explored with regard to this
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group of vaccines. It has been suggested that efforts would be emphasised on antigens or constructs inducing cross-reactive immune responses, which could cover genetic diversity. With increasing research in this field, new antigens with great potential as bloodstage vaccine candidates have been discovered. For example, P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) has been shown to induce inhibitory antibodies that are effective across common PfRH5 genetic variants [66, 67]. In addition, rhoptry-associated leucine zipper-like protein 1 (RALP1), which plays an important role during merozoite invasion into erythrocytes, was recognized by malaria-immune serum samples from Mali and Thailand, suggesting the potential of this protein as a blood-stage vaccine candidate [68]. These candidates showed strong immunogenicity; therefore it is time to proceed to Phase I/IIa human trials and, if successful, they could be combined with vaccines targeting other stages of the P. falciparum life cycle.
Adjuvants and delivery systems for malaria vaccines
A range of adjuvant formulations and viral/bacterial vectors is available for use with different malaria vaccine candidates. An ideal adjuvant is a component that enhances the potency, longevity and quality of specific immune responses to antigens, with minimal toxicity to the recipient. Adjuvants have been divided into two main groups according to their component sources, physiochemical properties or mechanisms of action: (i) immunostimulants such as Toll-like receptor (TLR) ligands, cytokines, saponins and bacterial exotoxins that act directly on the immune system to increase the response to antigens; and (ii) vehicles such as mineral salts, emulsions, liposomes, virosomes and biodegradable polymer microspheres that present vaccine antigens [69, 70]. Effective protection against different pathogens requires distinct types of immune responses and therefore the role of adjuvants is of great importance. Indeed, the nature of the adjuvant can determine the specific type of immune response, which may bias towards cytotoxic T lymphocyte (CTL) responses, antibody responses or particular types of T-helper (Th) cell responses. Although adjuvants have been used since the early vaccination trials, their mechanism of action is only now becoming clear. The adjuvants that have been
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approved for human clinical trials or tested in malaria vaccines include alum, saponins [Quil-A, immunostimulating complexes (ISCOMs), QS-21, AS02 and AS01], montanides (ISA51, ISA720), the oil-in-water emulsion MF59™, the monophosphoryl lipid A (MPL®), virus-like particle, virosomes and cholera toxin [70]. Today, increasingly more immunostimulatory oligonucleotides (CpG motifs), imidazoquinolines (imiquimod and resiquimod) and other TLR ligands are used as adjuvants due to their ability to induce Th1 type immunity and CTL responses [72]. Moreover, some adjuvants such as virosomes, liposomes and ISCOMs seem to promote priming of CD8+ T cells and antigen cross-presentation [73].
Bacillus Calmette-Guérin as a potential malaria vaccine delivery system
Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been used for a century as a vaccine against tuberculosis (TB). The long-lasting adjuvant and intrinsic immunostimulatory properties of BCG form the basis of its registration as a treatment for bladder carcinoma [74, 75]. BCG contains ligands for TLR2 and TLR4 and is the gold standard for vaccination against TB Previous studies have shown that vaccination with recombinant BCG-based vaccine modalities results in strong humoral and cell-mediated responses, elicited by the host to a variety of foreign antigens inserted into and expressed by the recombinant vector [77]. Therefore, we assessed the immunogenicity of a recombinant BCG-expressing circumsporozoite protein (BCG-CS) as a malaria vaccine candidate by performing in vitro and in vivo studies in BALB/c mice. We demonstrated that BCG-CS is highly effective in activating innate immune responses and priming the adaptive immune system [78]. In addition, by using heterologous prime–boost strategies, we showed that priming with a replication-defective human adenovirus serotype 35 (Ad35) vector encoding circumsporozoite protein (Ad35-CS), followed by BCG-CS boosting, enhanced the quality of antibody responses, and significantly increased the numbers of long-lived plasma cells and interferon-γ-producing cells in response to CSp peptides [79]. These findings highlight that BCG is a promising vaccine-delivery system and provide a rationale for its use in malaria vaccine development. In general, multiantigen vaccines are better than single-antigen vaccines, because broad immune coverage inhibits immune escape of viruses and parasites. Furthermore, a novel strategy is to formulate multi-adjuvanted vaccines, as adjuvants trigger the innate This article is protected by copyright. All rights reserved.
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immune response, which regulates the quality and magnitude of adaptive immunity [80, 81]. The rationale behind this approach is that such vaccines would trigger multiple signalling pathways and elicit distinct and synergistic immune responses. In this regard, the inclusion of TLR4 and TLR7/8 agonists in malaria vaccines produced mixed effects on the diversity and breadth of antibody responses [82]. In addition, this combination may activate follicular dendritic cells within germinal centres that play a crucial role during somatic hypermutation and the affinity maturation process [83]. As more compelling data emerge, hopefully a better understanding of the mechanism of action of novel adjuvants will lead to the development of efficacious multi-adjuvanted malaria vaccines.
Concluding remarks It is clear that further advances are still required for malaria vaccine development, based on empirical approaches and basic research, to identify new target antigens and provide improved understanding of how different adjuvants will affect the balance and durability of effector, memory and regulatory responses. By taking advantage of new tools and strategies, this will speed up the development of a new generation of malaria vaccines that are highly efficacious. Conflict of interest statement The authors have no conflicts of interest to declare. Acknowledgements We acknowledge the contribution of Prof. Kai Matuschewski at the Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany, for critical reading and feedback on the manuscript.
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59. Palacpac NM, Ntege E, Yeka A et al. Phase 1b randomized trial and follow-up study in Uganda of the blood-stage malaria vaccine candidate BK-SE36. PLoS One 2013; 8: e64073. 60. Takala SL, Coulibaly D, Thera MA et al. Extreme polymorphism in a vaccine antigen and risk of clinical malaria: implications for vaccine development. Sci Transl Med 2009; 1: 2ra5. 61. Ellis RD, Martin LB, Shaffer D et al. Phase 1 trial of the Plasmodium falciparum blood stage vaccine MSP1(42)-C1/Alhydrogel with and without CPG 7909 in malaria naive adults. PLoS One 2010; 5: e8787. 62. Sagara I, Ellis RD, Dicko A et al. A randomized and controlled Phase 1 study of the safety and immunogenicity of the AMA1-C1/Alhydrogel + CPG 7909 vaccine for Plasmodium falciparum malaria in semi-immune Malian adults. Vaccine 2009; 27: 7292-8. 63. Hill AV, Reyes-Sandoval A, O'Hara G et al. Prime-boost vectored malaria vaccines: progress and prospects. Hum Vaccin 2010; 6: 78-83. 64. Malkin E, Hu J, Li Z et al. A phase 1 trial of PfCP2.9: an AMA1/MSP1 chimeric recombinant protein vaccine for Plasmodium falciparum malaria. Vaccine 2008; 26: 6864-73. 65. Takala SL, Plowe CV. Genetic diversity and malaria vaccine design, testing and efficacy: preventing and overcoming 'vaccine resistant malaria'. Parasite Immunol 2009; 31: 560-73. 66. Bustamante LY, Bartholdson SJ, Crosnier C et al. A full-length recombinant Plasmodium falciparum PfRH5 protein induces inhibitory antibodies that are effective across common PfRH5 genetic variants. Vaccine 2013; 31: 373-9. 67. Douglas AD, Williams AR, Illingworth JJ et al. The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody. Nat Commun 2011; 2: 601. 68. Ito D, Hasegawa T, Miura K et al. RALP1 is a rhoptry-neck erythrocyte-binding protein of Plasmodium falciparum merozoite and a potential blood-stage vaccine candidate antigen. Infect Immun 2013;. 69. Coler RN, Carter D, Friede M, Reed SG. Adjuvants for malaria vaccines. Parasite Immunol 2009; 31: 520-8. 70. Bruder JT, Angov E, Limbach KJ, Richie TL. Molecular vaccines for malaria. Hum Vaccin 2010; 6: 54-77. 71. Mbow ML, De Gregorio E, Valiante NM, Rappuoli R. New adjuvants for human vaccines. Curr Opin Immunol 2010; 22: 411-6. 72. Bevan MJ. Helping the CD8(+) T-cell response. Nat Rev Immunol 2004; 4: 595-602. 73. Reed SG, Bertholet S, Coler RN, Friede M. New horizons in adjuvants for vaccine development. Trends Immunol 2009; 30: 23-32.
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74. Alexandroff AB, Jackson AM, O'Donnell MA, James K. BCG immunotherapy of bladder cancer: 20 years on. Lancet 1999; 353: 1689-94. 75. Kawai K, Miyazaki J, Joraku A, Nishiyama H, Akaza H. Bacillus Calmette-Guerin (BCG) immunotherapy for bladder cancer: current understanding and perspectives on engineered BCG vaccine. Cancer Sci 2013; 104: 22-7. 76. Heldwein KA, Liang MD, Andresen TK et al. TLR2 and TLR4 serve distinct roles in the host immune response against Mycobacterium bovis BCG. J Leukoc Biol 2003; 74: 277-86. 77. Matsumoto S, Yukitake H, Kanbara H, Yamada T. Recombinant Mycobacterium bovis bacillus Calmette-Guerin secreting merozoite surface protein 1 (MSP1) induces protection against rodent malaria parasite infection depending on MSP1-stimulated interferon gamma and parasite-specific antibodies. J Exp Med 1998; 188: 845-54. 78. Arama C, Waseem S, Fernandez C et al. A recombinant Bacille Calmette-Guerin construct expressing the Plasmodium falciparum circumsporozoite protein enhances dendritic cell activation and primes for circumsporozoite-specific memory cells in BALB/c mice. Vaccine 2012; 30: 5578-84. 79. Arama C, Assefaw-Redda Y, Rodriguez A et al. Heterologous prime-boost regimen adenovector 35-circumsporozoite protein vaccine/recombinant Bacillus Calmette-Guerin expressing the Plasmodium falciparum circumsporozoite induces enhanced long-term memory immunity in BALB/c mice. Vaccine 2012; 30: 4040-5. 80. Mount A, Koernig S, Silva A, Drane D, Maraskovsky E, Morelli AB. Combination of adjuvants: the future of vaccine design. Expert Rev Vaccines 2013; 12: 733-46. 81. Mutwiri G, Gerdts V, van Drunen Littel-van den Hurk,S. et al. Combination adjuvants: the next generation of adjuvants?. Expert Rev Vaccines 2011; 10: 95-107. 82. Wiley SR, Raman VS, Desbien A et al. Targeting TLRs expands the antibody repertoire in response to a malaria vaccine. Sci Transl Med 2011; 3: 93ra69. 83. Garin A, Meyer-Hermann M, Contie M et al. Toll-like receptor 4 signaling by follicular dendritic cells is pivotal for germinal center onset and affinity maturation. Immunity 2010; 33: 84-95.
Figure and table legends Fig. 1 The life cycle of Plasmodium falciparum. The life cycle of P. falciparum in humans consists of the pre-erythrocytic stage, the asexual blood stage and the gametocyte stage. This life cycle involves humans and female Anopheles mosquitoes. Each stage can be characterised by the expression of stage-specific proteins that are targets of host immune
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responses. The potential immune mechanisms induced at each stage are shown. RBC, red blood cell; NK, natural killer; ROS, reactive oxygen species. Fig. 2 The development of malaria vaccines. Target sites in the malaria life cycle that could be interrupted by vaccines. Three types of vaccine candidates are shown: pre-erythrocytic, blood-stage and transmission-blocking vaccines.
Table 1 The main malaria vaccines that are currently undergoing clinical trials. RTS,S, circumsporozoite (CS) protein fused with hepatitis B surface antigen; PfCelTOS, Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites; CSVAC, vaccine of chimpanzee adenovirus 63 (ChAD63) expressing CS and modified vaccinia virus Ankara (MVA) virus expressing CS; PfSPZ, P. falciparum sporozoite vaccine; SE36, vaccine based on the N-terminal domain of serine repeat antigen 5 (SERA5) of P. falciparum; NMRC-M3V-AdPfCA, human adenovirus 5-vectored P. falciparum vaccine encoding CSP and AMA1; EBA 175.R2, vaccine consisting of erythrocyte-binding antigen 175; GMZ2, fusion protein of P. falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP); METRAP, multiple epitope string with thrombospondin-related adhesion protein; FMP2.1, apical membrane antigen 1 (AMA1) candidate malaria vaccine. Table adapted from the World Health Organisation: (http://www.who.int/vaccine_research/links/Rainbow/en/index.html).
Table 1 The main malaria vaccines that are currently undergoing clinical trials
Phase Phase Phase Phase Phase Trial registration 1a 1b 2a 2b 3 number
Trial sponsor
RTS,S/AS01E
x
x
x
RTS,S-AS01 delayed fractional third dose
x
x
x
GlaxoSmithKline, Belgium GlaxoSmithKline, Belgium
PfCelTOS FMP012
x
Vaccines
Pre-erythrocytic x
x
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NCT00866619 NCT01857869
NCT01540474
US Army Medical Research and Materiel Command
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CSVAC
x
NCT01450280
University of Oxford, UK GlaxoSmithKline, Belgium
Adenovirus (Ad35) vectored CS and RTS,SAS01 in heterologous prime-boost regimen
x
x
ChAd63/MVA (CS; METRAP)
x
x
x
NCT01364883
University of Oxford, UK
PfSPZ
x
x
x
NCT01001650
Sanaria Inc., USA
NCT01366534
Blood-stage ChAd63.AMA1/MVA. x AMA1 +Al/CPG7909 x AMA1-C1Alhydrogel+CPG 7909
NCT01142765 x
BSAM-2-Alhydrogel + CPG 7909
x
x
EBA 175.R2
x
x
SE36
x
x
ChAd63/MVA AMA1
x
x
x
FMP2.1-AS01B (AMA1 3D7)
x
x
x
NMRC.M3V.Ad.PfCA x
x
x
x
x
x
MSP3 [181-276]
University of Oxford, UK NCT00414336 National Institute of Allergy and Infectious Diseases (NIAID) NCT00889616 National Institute of Allergy and Infectious Diseases (NIAID) NCT01026246 National Institute of Allergy and Infectious Diseases (NIAID) ISRCTN71619711 Research Foundation for Microbial Diseases of Osaka University, Japan NCT01142765 University of Oxford, UK NCT00385047 US Army Medical Research and Materiel Command
x
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NCT00392015
US Army Medical Research and Materiel Command
NCT00652275
African Malaria Network Trust (AMANET)
x
Accepted Article
GMZ2
Transmissionblocking Pfs25-EPA
x
x
x
x
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NCT00424944
African Malaria Network Trust (AMANET)
NCT01434381
National Institute of Allergy and Infectious Diseases (NIAID)
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Accepted Date : 23-Jan-2014 Article type
: Introduction
The path of malaria vaccine development: challenges and perspectives
Charles Arama1,2 & Marita Troye-Blomberg2 From the 1 Malaria Research and Training Center, University of Sciences, Techniques, and Technologies of Bamako (USTTB), Mali; and 2
Department of Molecular Biosciences, The Wenner-Gren Institute
Stockholm University, Stockholm, Sweden
Email: [email protected] Email: [email protected]; [email protected]
Abstract Malaria is a life-threatening disease caused by parasites of the Plasmodium genus. In many parts of the world, the parasites have developed resistance to a number of anti-malarial agents. Key interventions to control malaria include prompt and effective treatment with artemisinin-based combination therapies, use of insecticidal nets by individuals at risk and active research into malaria vaccines. Protection against malaria through vaccination was demonstrated more than 30 years ago when individuals were vaccinated via repeated bites by Plasmodium falciparum-infected and irradiated but still metabolically active mosquitoes. However, vaccination with high doses of irradiated sporozoites injected into humans has long been considered impractical. Yet, following recent success using whole-organism vaccines, the approach has received renewed interest; it was recently reported that repeated injections of irradiated sporozoites increased protection in 80 vaccinated individuals. Other approaches include subunit malaria vaccines, such as the current leading candidate RTS,S (consisting of fusion between a portion of the P. falciparum-derived
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circumsporozoite protein and the hepatitis B surface antigen), which has been demonstrated to induce reasonably good protection. Although results have been encouraging, the level of protection is generally considered to be too low to achieve eradication of malaria. There is great interest in developing new and better formulations and stable delivery systems to improve immunogenicity. In this review we will discuss recent strategies to develop efficient malaria vaccines.
Keywords: adjuvants, malaria, vaccine delivery systems, vaccines.
Introduction Malaria remains one of the most devastating infectious diseases affecting those living in tropical areas of the world, especially young children and pregnant women. Malaria in humans is caused by five protozoan parasite species of the genus Plasmodium: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi [1]. Malaria is a mosquito-borne disease and hence can be controlled in both humans and mosquitoes. Despite major efforts to control the disease, approximately 781 000 deaths occurred in 2009 in children under 5 years of age in sub-Saharan Africa [2]. The control of this life-threatening disease requires a multifaceted approach, including insecticides, chemotherapy and development of cheap, affordable and efficacious malaria vaccines [3]. Because of the large gap in our understanding of the biology of P. falciparum infection, it is difficult to identify the right vaccine candidate among the thousands of potential P. falciparum antigens [4]. This gap can be filled, but only with adequate research support and commitment from experts in all areas of immunobiology of P. falciparum in order to target a variety of potential approaches [5]. Although significant advances have been made in vector control strategies and the effectiveness of artemisinin-based combination therapy, a licensed malaria vaccine is still not available. In addition, current malaria control and elimination programmes are affected by notable heterogeneity of transmission dynamics in endemic areas, including differences in parasite-, vector- and host-related, social and environmental factors. Furthermore, clinical resistance to artemisinin and its derivatives is now well established in P. falciparum populations of western Cambodia [6, 7] and appears to be the greatest threat to global malaria elimination and eradication.
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To date, vaccination is the most effective method of preventing infectious diseases and represents the greatest contribution of immunology to human health. The remarkable success of vaccines against polio, measles, diphtheria, tetanus, rabies and others, and the complete eradication of smallpox in humans, prove the potential of this approach in reducing the global burden of infectious diseases [8]. However, despite this success for some diseases, major logistical and technical challenges remain to be solved in order to develop efficient malaria vaccines that might potentially provide an important tool for use in malaria elimination and eradication programmes.
Recent advances have led to a new era of vaccine development in general and for malaria in particular. Adjuvants and vaccine-delivery systems are becoming increasingly more important for the development of a new generation of vaccines, combining different types of adjuvants into antigen-specific formulations with greater efficacy and improved vaccine formulations [9]. These new approaches offer a wide spectrum of opportunities in malaria vaccine research with direct applications for the near future. The main advantage of vaccine-delivery systems is that they allow co-administration of immunostimulants and more than one antigen into the same system; however, difficulties remain when applying them to human vaccinology. In this review, we will discuss strategies to overcome some of these challenges and suggest several approaches for the development of an effective malaria vaccine.
The life cycle of P. falciparum
The malaria parasite requires mosquitoes and human hosts to complete its life cycle (Fig. 1), and goes through several developmental stages to survive in the human host. During its two-host life cycle, P. falciparum undergoes 10 morphological transitions in five different host tissues [10]. The cycle in humans includes: (i) the pre-erythrocytic stage, which is the first stage of infection in humans with inoculation of sporozoites to infect the hepatocytes; (ii) the erythrocytic stage, which involves asexual reproduction of the parasite in the blood to cause the clinical
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symptoms of the disease; and (iii) the gametocyte stage, which enables sexual reproduction of the parasite in the mosquito and further transmission. The pre-erythrocytic stage begins when an infected female Anopheles mosquito inoculates sporozoites into the skin or into the blood stream of humans during a blood meal. Sporozoites circulate transiently in the bloodstream before invading hepatocytes, in which the asexual cycle takes place. It is estimated that mosquitoes transmit fewer than 100 sporozoites per bite [11, 12]. The sporozoites can remain for up to 6 h at the site of injection [13], and one-third of those leaving the injection site may enter the draining lymph nodes via the lymphatic vessels [14]. The capacity of each of these sporozoites to produce asexual erythrocytic-stage infection is low. In humans, bites of at least five P. falciparum-infected mosquitoes are necessary to ensure that 100% of individuals will become infected [15]. When sporozoites reach the liver parenchyma, they continue to migrate through several hepatocytes before finally infecting one of them. This migration seems to be beneficial for malaria infections in at least two different ways: by activating sporozoites for infection and by increasing the susceptibility of host hepatocytes [16]. After a week, rupture of the merosomes within the lung microvasculature [17] releases thousands of infectious merozoites into the bloodstream where they invade circulating erythrocytes and initiate the clinically important intra-erythrocytic cycle of asexual replication during the following 48 h.
The intra-erythrocytic cycle is responsible for all the clinical symptoms associated with the disease. After 24–32 h, when young parasites mature from rings to the trophozoite stage, infected erythrocytes adhere to endothelial cells in the microcirculation of various organs (known as sequestration) causing cerebral malaria if sequestered in the brain. The trophozoites will eventually mature into schizonts, which finally rupture and release 16–32 daughter merozoites that can invade fresh red blood cells to restart the asexual life cycle. As a survival strategy, blood-stage parasites have been shown to infect and survive and replicate within CD317+ dendritic cells, and small numbers of these cells release parasites that can infect erythrocytes in vivo in a murine model [18].
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Some parasites inside the erythrocytes differentiate into male and female gametocytes. It remains poorly understood which factors stimulate gametogenesis [19]. However, it has been suggested that it is influenced by environmental factors, drugs or innate immune factors [20, 21]. Without treatment, most patients with P. falciparum malaria will develop gametocytaemia within 10 to 40 days after the onset of parasitaemia [22]. Upon ingestion by a feeding female mosquito, the male and female gametes undergo fertilization in the mosquito midgut to form a zygote and subsequently a motile ookinete. Okinetes penetrate the midgut epithelial cells and rest between the midgut epithelium and the basal lamina to form oocysts. The oocysts undergo a complex asexual development stage, which eventually generates infective sporozoites that can be introduced into the human host at the next blood meal via the mosquito saliva, thereby ensuring the continuation of the parasite life cycle.
Vaccines against malaria
The purpose of vaccination strategies is to induce protective memory immune responses in advance of infection, to provide protection in the case of encountering the disease-causing agent again. Malaria vaccine development is an active research area with enormous challenges. As the parasite proceeds from a sporozoite through the liver stage to the replicating cycle of the blood stage, it undergoes morphological changes and displays antigenic variations. This allows the parasite to evade the protective immune responses of the host. As a result, the acquisition of long-term sterile immunity, which is often associated with recovery from many other infectious diseases, is not observed in malaria-infected individuals. Despite these difficulties, the most convincing evidence that vaccination against malaria is feasible has come from experimental studies in rodents, monkeys and human subjects in which attenuated sporozoites induced sterile protective immunity. The main malaria vaccines currently in clinical trials are summarised in Table 1. An ideal malaria vaccine requires three essential features: (i) multiple components that will induce an effective immune response to the different stages of the malaria infection (sporozoites, infected hepatocytes and asexual and sexual stages); (ii) multiple epitopes that are restricted to presentation by different major
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histocompatibility complex (MHC) molecules in order to overcome genetic diversity and antigenic variation; and (iii) multi-immunogenicity inducing more than one type of immune response, including cell-mediated and humoral components. Such a multicomponent vaccine should increase the probability of a sustainable and effective host response [23]. Three types of vaccine candidate targeting different stages in the life cycle of the malaria parasite have been intensively investigated: (i) transmission-blocking vaccines (TBVs); (ii) pre-erythrocytic vaccines; and (iii) blood-stage vaccines (Fig 2). TBVs
TBVs target antigens on gametes, zygotes and ookinetes in order to prevent parasite development in the mosquito midgut. The aim of these vaccines is to induce antibodies against the sexual-stage antigens to block ookinete-to-oocyst transition to stop the subsequent generation of infectious sporozoites [24]. Of note, TBVs do not protect the recipient from contracting malaria, but could be helpful in preventing the spread of the disease. These vaccines are intended to protect entire/selected communities from infection. The leading vaccine candidates in this group include the P. falciparum ookinete surface antigens Pfs25 [25] and Pfs28 and their P. vivax homologues Pvs25 and Pvs28 [26, 27]. In order to improve the immunogenicity, Pfs25 was expressed as a recombinant protein that was chemically cross-linked to ExoProtein A and delivered as a nanoparticle. This enhanced the immunogenicity of the vaccine in mice, and it is currently undergoing Phase I trials in humans [28]. Because the antigens are never naturally presented to the human immune system, one of the potential limitations of the TBV approach is that the absence of natural boosting following immunization might limit efficacy [29]. Nevertheless, TBVs could be important tools for a malaria elimination and eradication programme, for prevention of transmission of the disease [30].
Pre-erythrocytic vaccines Research on pre-erythrocytic vaccines has quickly progressed during this last decade, and a number of new approaches are in the pipeline that could contribute to improve the second generation of malaria vaccines. Liver-stage vaccines are designed to prevent malaria in the
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human host. However, because of the high rate of replication of sporozoites, a single parasite may be sufficient for the infection to proceed to the blood stage. The liver stage of P. falciparum is an attractive therapeutic target for the development of both anti-malarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage [31]. However, an efficient liver stage vaccine must be 100% effective in order to protect humans with no natural immunity. Such vaccines include those containing whole killed sporozoites and those based on antigenic portions of the circumsporozoite proteins [32]. A landmark finding that set the standards for immunological protection against malaria infection was established by immunisation with irradiated sporozoites [33, 34]. To date, the most advanced pre-erythrocytic vaccine candidate is RTS,S, which consists of a truncated circumsporozoite protein (CSp) of P. falciparum directly fused to the hepatitis B surface (S) antigen. This vaccine has shown 30–50% protection in human field trials in Africa [35, 36]. As protection was relatively short lasting, how to improve the efficacy by manipulating the immune response of the host is a current challenge. The mechanism by which RTS,S confers protection against blood-stage disease remains poorly understood. It seems that RTS,S induces protection against clinical malaria by temporarily reducing the number of merozoites emerging from the liver. This may allow prolonged exposure to subclinical levels of asexual blood-stage parasites, therefore boosting the naturally acquired blood-stage immunity [37]. A novel antigen, the cell-traversal protein for ookinetes and sporozoites (CelTOS), has been identified as an essential protein for the traversal of the malaria parasite in both mammalian and the insect hosts [38]. In a murine model, CelTOS is a potentially interesting pre-erythrocytic vaccine candidate, which induces complete sterile protection against sporozoite challenge [39].
The case for a whole sporozoite vaccine originated from the early finding that mice could be protected by inoculation of P. berghei-irradiated sporozoites by intravenous injection [33]. Today, there is renewed interest in the whole-organism vaccine as a result of a recent highly successful human trial using experimental sporozoite inoculation under chloroquine prophylaxis [40]. In this trial, four of the six volunteers mounted sterile protection against P. falciparum rechallenges 2 years later [41]. Sporozoites attenuated by targeted gene disruption are being evaluated as whole-parasite vaccines in which the favourite candidates are genetically attenuated late liver stage-arresting parasites [42]. This type of vaccine induces high levels of cross-stage and cross-species protection and complete protection when administered This article is protected by copyright. All rights reserved.
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by intradermal or subcutaneous routes [43]. However, the results have not been reproduced. For instance, cross-stage protection is not proven in human infection, with recent data suggesting a complete lack of protection against blood-stage challenge and indicating that chemoprophylaxis with P. falciparum sporozoiteinduced protection is mediated by immunity against pre-erythrocytic stages [44]. Genetically attenuated vaccines can avoid the potentially problematic irradiation step in the manufacturing process, but other challenges of manufacturing and delivering a viable cryopreserved whole parasite vaccine remain [45]. However, the results of a recent vaccine trial showed that these challenges could be overcome [46]. With regard to this encouraging finding, the next step will be the implementation of such a trial in malaria endemic areas with diverse transmission settings. The fact that whole sporozoite vaccines induce better protection than subunit vaccines [47, 48] suggests that antigen combination strategies are necessary. Therefore, further research into other potential malaria vaccine antigens and strategies for their delivery is essential.
Blood-stage vaccines
Blood-stage vaccines are designed to elicit anti-invasion and anti-disease responses [49]. The underlying principle of these strategies is that if a vaccine could block the invasion of erythrocytes by merozoites, it would prevent malarial disease. At present, several blood-stage antigens are in clinical trials: apical membrane antigen 1 (AMA1) [50], erythrocyte-binding antigen-175 (EBA-175) [51], glutamate-rich protein (GLURP) [52, 53], merozoite surface protein (MSP) 1 [54], MSP2 [55] and MSP3 [56, 57] and serine repeat antigen 5 (SERA5) [58, 59]. All these antigens are highly expressed on the surface of merozoites. Of note, AMA1 and MSP1 did not demonstrate efficacy in African children [50, 54], probably due to the highly polymorphic nature of the vaccine structures [60]. Efforts to enhance the vaccine efficacy of AMA1 and MSP1 with novel adjuvants [61, 62], using viral vector prime-boost strategies [63], or by combining AMA1 and MSP1 [64] have been investigated. The extensive genetic diversity of the parasite and the selective pressure exerted by the host’s immune response are major factors to be considered in the development of effective blood-stage vaccines [65]. How to address genetic polymorphism constitutes an important issue to be explored with regard to this
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group of vaccines. It has been suggested that efforts would be emphasised on antigens or constructs inducing cross-reactive immune responses, which could cover genetic diversity. With increasing research in this field, new antigens with great potential as bloodstage vaccine candidates have been discovered. For example, P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) has been shown to induce inhibitory antibodies that are effective across common PfRH5 genetic variants [66, 67]. In addition, rhoptry-associated leucine zipper-like protein 1 (RALP1), which plays an important role during merozoite invasion into erythrocytes, was recognized by malaria-immune serum samples from Mali and Thailand, suggesting the potential of this protein as a blood-stage vaccine candidate [68]. These candidates showed strong immunogenicity; therefore it is time to proceed to Phase I/IIa human trials and, if successful, they could be combined with vaccines targeting other stages of the P. falciparum life cycle.
Adjuvants and delivery systems for malaria vaccines
A range of adjuvant formulations and viral/bacterial vectors is available for use with different malaria vaccine candidates. An ideal adjuvant is a component that enhances the potency, longevity and quality of specific immune responses to antigens, with minimal toxicity to the recipient. Adjuvants have been divided into two main groups according to their component sources, physiochemical properties or mechanisms of action: (i) immunostimulants such as Toll-like receptor (TLR) ligands, cytokines, saponins and bacterial exotoxins that act directly on the immune system to increase the response to antigens; and (ii) vehicles such as mineral salts, emulsions, liposomes, virosomes and biodegradable polymer microspheres that present vaccine antigens [69, 70]. Effective protection against different pathogens requires distinct types of immune responses and therefore the role of adjuvants is of great importance. Indeed, the nature of the adjuvant can determine the specific type of immune response, which may bias towards cytotoxic T lymphocyte (CTL) responses, antibody responses or particular types of T-helper (Th) cell responses. Although adjuvants have been used since the early vaccination trials, their mechanism of action is only now becoming clear. The adjuvants that have been
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approved for human clinical trials or tested in malaria vaccines include alum, saponins [Quil-A, immunostimulating complexes (ISCOMs), QS-21, AS02 and AS01], montanides (ISA51, ISA720), the oil-in-water emulsion MF59™, the monophosphoryl lipid A (MPL®), virus-like particle, virosomes and cholera toxin [70]. Today, increasingly more immunostimulatory oligonucleotides (CpG motifs), imidazoquinolines (imiquimod and resiquimod) and other TLR ligands are used as adjuvants due to their ability to induce Th1 type immunity and CTL responses [72]. Moreover, some adjuvants such as virosomes, liposomes and ISCOMs seem to promote priming of CD8+ T cells and antigen cross-presentation [73].
Bacillus Calmette-Guérin as a potential malaria vaccine delivery system
Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been used for a century as a vaccine against tuberculosis (TB). The long-lasting adjuvant and intrinsic immunostimulatory properties of BCG form the basis of its registration as a treatment for bladder carcinoma [74, 75]. BCG contains ligands for TLR2 and TLR4 and is the gold standard for vaccination against TB Previous studies have shown that vaccination with recombinant BCG-based vaccine modalities results in strong humoral and cell-mediated responses, elicited by the host to a variety of foreign antigens inserted into and expressed by the recombinant vector [77]. Therefore, we assessed the immunogenicity of a recombinant BCG-expressing circumsporozoite protein (BCG-CS) as a malaria vaccine candidate by performing in vitro and in vivo studies in BALB/c mice. We demonstrated that BCG-CS is highly effective in activating innate immune responses and priming the adaptive immune system [78]. In addition, by using heterologous prime–boost strategies, we showed that priming with a replication-defective human adenovirus serotype 35 (Ad35) vector encoding circumsporozoite protein (Ad35-CS), followed by BCG-CS boosting, enhanced the quality of antibody responses, and significantly increased the numbers of long-lived plasma cells and interferon-γ-producing cells in response to CSp peptides [79]. These findings highlight that BCG is a promising vaccine-delivery system and provide a rationale for its use in malaria vaccine development. In general, multiantigen vaccines are better than single-antigen vaccines, because broad immune coverage inhibits immune escape of viruses and parasites. Furthermore, a novel strategy is to formulate multi-adjuvanted vaccines, as adjuvants trigger the innate This article is protected by copyright. All rights reserved.
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immune response, which regulates the quality and magnitude of adaptive immunity [80, 81]. The rationale behind this approach is that such vaccines would trigger multiple signalling pathways and elicit distinct and synergistic immune responses. In this regard, the inclusion of TLR4 and TLR7/8 agonists in malaria vaccines produced mixed effects on the diversity and breadth of antibody responses [82]. In addition, this combination may activate follicular dendritic cells within germinal centres that play a crucial role during somatic hypermutation and the affinity maturation process [83]. As more compelling data emerge, hopefully a better understanding of the mechanism of action of novel adjuvants will lead to the development of efficacious multi-adjuvanted malaria vaccines.
Concluding remarks It is clear that further advances are still required for malaria vaccine development, based on empirical approaches and basic research, to identify new target antigens and provide improved understanding of how different adjuvants will affect the balance and durability of effector, memory and regulatory responses. By taking advantage of new tools and strategies, this will speed up the development of a new generation of malaria vaccines that are highly efficacious. Conflict of interest statement The authors have no conflicts of interest to declare. Acknowledgements We acknowledge the contribution of Prof. Kai Matuschewski at the Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany, for critical reading and feedback on the manuscript.
References 1. Singh B, Kim Sung L, Matusop A et al. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 2004; 363: 1017-24. 2. World Health Organization. World Malaria Report 2012 2012;.
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Figure and table legends Fig. 1 The life cycle of Plasmodium falciparum. The life cycle of P. falciparum in humans consists of the pre-erythrocytic stage, the asexual blood stage and the gametocyte stage. This life cycle involves humans and female Anopheles mosquitoes. Each stage can be characterised by the expression of stage-specific proteins that are targets of host immune
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responses. The potential immune mechanisms induced at each stage are shown. RBC, red blood cell; NK, natural killer; ROS, reactive oxygen species. Fig. 2 The development of malaria vaccines. Target sites in the malaria life cycle that could be interrupted by vaccines. Three types of vaccine candidates are shown: pre-erythrocytic, blood-stage and transmission-blocking vaccines.
Table 1 The main malaria vaccines that are currently undergoing clinical trials. RTS,S, circumsporozoite (CS) protein fused with hepatitis B surface antigen; PfCelTOS, Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites; CSVAC, vaccine of chimpanzee adenovirus 63 (ChAD63) expressing CS and modified vaccinia virus Ankara (MVA) virus expressing CS; PfSPZ, P. falciparum sporozoite vaccine; SE36, vaccine based on the N-terminal domain of serine repeat antigen 5 (SERA5) of P. falciparum; NMRC-M3V-AdPfCA, human adenovirus 5-vectored P. falciparum vaccine encoding CSP and AMA1; EBA 175.R2, vaccine consisting of erythrocyte-binding antigen 175; GMZ2, fusion protein of P. falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP); METRAP, multiple epitope string with thrombospondin-related adhesion protein; FMP2.1, apical membrane antigen 1 (AMA1) candidate malaria vaccine. Table adapted from the World Health Organisation: (http://www.who.int/vaccine_research/links/Rainbow/en/index.html).
Table 1 The main malaria vaccines that are currently undergoing clinical trials
Phase Phase Phase Phase Phase Trial registration 1a 1b 2a 2b 3 number
Trial sponsor
RTS,S/AS01E
x
x
x
RTS,S-AS01 delayed fractional third dose
x
x
x
GlaxoSmithKline, Belgium GlaxoSmithKline, Belgium
PfCelTOS FMP012
x
Vaccines
Pre-erythrocytic x
x
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NCT00866619 NCT01857869
NCT01540474
US Army Medical Research and Materiel Command
Accepted Article
CSVAC
x
NCT01450280
University of Oxford, UK GlaxoSmithKline, Belgium
Adenovirus (Ad35) vectored CS and RTS,SAS01 in heterologous prime-boost regimen
x
x
ChAd63/MVA (CS; METRAP)
x
x
x
NCT01364883
University of Oxford, UK
PfSPZ
x
x
x
NCT01001650
Sanaria Inc., USA
NCT01366534
Blood-stage ChAd63.AMA1/MVA. x AMA1 +Al/CPG7909 x AMA1-C1Alhydrogel+CPG 7909
NCT01142765 x
BSAM-2-Alhydrogel + CPG 7909
x
x
EBA 175.R2
x
x
SE36
x
x
ChAd63/MVA AMA1
x
x
x
FMP2.1-AS01B (AMA1 3D7)
x
x
x
NMRC.M3V.Ad.PfCA x
x
x
x
x
x
MSP3 [181-276]
University of Oxford, UK NCT00414336 National Institute of Allergy and Infectious Diseases (NIAID) NCT00889616 National Institute of Allergy and Infectious Diseases (NIAID) NCT01026246 National Institute of Allergy and Infectious Diseases (NIAID) ISRCTN71619711 Research Foundation for Microbial Diseases of Osaka University, Japan NCT01142765 University of Oxford, UK NCT00385047 US Army Medical Research and Materiel Command
x
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NCT00392015
US Army Medical Research and Materiel Command
NCT00652275
African Malaria Network Trust (AMANET)
x
Accepted Article
GMZ2
Transmissionblocking Pfs25-EPA
x
x
x
x
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NCT00424944
African Malaria Network Trust (AMANET)
NCT01434381
National Institute of Allergy and Infectious Diseases (NIAID)
Accepted Article This article is protected by copyright. All rights reserved.
