Leishmaniasis: Recombinant DNA Vaccination and Different Approaches for Vaccine Development

Review: Clinical Trial Outcomes

Leishmaniasis: recombinant DNA vaccination and different approaches for vaccine development

Clin. Invest. (2013) 3(11), 1023–1044

Designing an ideal vaccine against leishmaniasis using a suitable Tahereh Taheri* & Sima Rafati candidate antigen and appropriate antigen delivery system to induce the Molecular Immunology & Vaccine Research accurate type of immune response is a process still under investigation. Laboratory, Pasteur Institute of Iran, Pasteur There are some different strategies applied for vaccination against Avenue No. 69, Tehran, 13164, Iran leishmaniasis including conventional and modern (genetically-modified *Author for correspondence: Tel.: +98 21 6649 6560 vaccines) approaches. The type of induced immune response for each Fax: +98 21 6649 6560 vaccine depends on the type of vaccine and route of administration, E-mail: [email protected] the amount and nature of the antigen/s, as well as adjuvant (if used). Recombinant DNA technology or genetic engineering has accelerated the identification of new types of prophylaxis approaches using crude lysates to single or multi-proteins, epitopes and DNA vaccines that are capable of creating less or more partial protection. At present, a few limited vaccine formulations are licensed in Brazil and Europe just for canines. Vaccine development for humans and even for canines is under serious study. In this review, we focus on current and suggested vaccination approaches based on recombinant DNA strategies against leishmaniasis.

Keywords: cocktail vaccines • DNA vaccine • leishmaniasis • prime–boost vaccination • recombinant DNA vaccines

Infection by Leishmania protozoan parasites causes leishmaniasis, which is associated with relatively high rates of morbidity and mortality worldwide. It is still a global challenge and serious health problem for humans and some animals in many endemic countries [1] . There are different clinical forms of leishmaniasis including cutaneous leishmaniasis (CL), mucocutaneous leishamaniasis and visceral leishmaniasis (VL). It is known that the clinical manifestations of leishmaniasis are dependent on the interactions between the parasite species and host immune system [2]. Leishmaniasis creates more serious health problems in people suffering from other infectious diseases, such as HIV, and even noninfectious diseases, such as cancer. According to a WHO report, leishmaniasis is endemic in 98 countries on five continents [3]. The estimated incidence is 0.2–0.4 million VL cases and 0.7–1.2 million CL cases [3]. This disease is one of the most important neglected diseases and is ranked the second in mortality rate and the fourth in morbidity rate among the tropical infections [4]. Due to infection of internal organs, symptoms of VL are more severe than CL. More than 95% of global VL cases occur in six countries: India, Bangladesh, Sudan, South Sudan, Brazil and Ethiopia. CL is more widely distributed in the Americas, the Mediterranean basin, and western Asia from the Middle East to central Asia. The ten countries with the highest estimated case counts are Afghani- stan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, Sudan, Costa Rica and Peru [3]. The people living in endemic areas, war zones or suffering from poverty, especially in third-world countries, account for the majority of the high-risk population who

need an executive control program for prevention of causative agents for VL, but humans are the only known leishmaniasis and other infectious diseases. hosts for L. donovani; while L. infantum is primarily a Annually, the emergence rate of new cases of leish- zoonotic disease and canine species are the main ani- maniasis is widely increasing, because there is no suc- mal reservoir [8]. Reservoir animals are usually used as cessful program to control the disease. There are several animal models for experimental studies. L. major and factors that complicate the control of leishmaniasis, such L. tropica are causative agents for the cutaneous form of as lack of an effective vaccine or safe therapy, resistance disease. For many years, BALB/c mice have been used to current antileishmanials, probable transmission as the best model for CL caused by L. major. However, through tourists and change in geographic distribution some scientists believe that due to high susceptibility of the insect habitats due to global warming [5]. of BALB/c mice to the cutaneous form of leishmani- In general, Leishmania has two different vertebrate asis, they are not a suitable host to test the protective hosts – humans and animals. In addition, the dimorphic effect of antigens [12] . The animal reservoir for L. tropica life cycle in different species of parasite make Leishma- is still unknown and debatable. Furthermore, human nia a very complex microorganism. Usually, the infec- heterogeneity is another major obstacle. The severity tion is transmitted from animal to animal/human by and symptoms of disease are not the same in differ- the bite of an infected female sandfly. However, some ent infected individuals. However, there are still many other rates of transmission are reported, such as human known and unknown factors resulting in the lack of a to human transmission of Leishmania, as is the case good strategy to prevent this disease. There are some with Leishmania donovani [4,6] and Leishmania tropica. major questions that should be considered in order to L. tropica infection has been described as anthropo- select the best strategy, as demonstrated in Figure 1. zoonosis, but some cases of canine infection have been Due to the mentioned reasons, vaccine development reported [6]. Actually, cell cycles of all species are not for leishmaniasis is a more difficult task than for other completely characterized. The human is generally con- organisms such as bacteria and viruses [10,13] . sidered as a second (or accidental) host [7] for some Leish- To design a potential vaccine, three main fac- mania species (e.g., Leishmania major and Leishmania tors should be considered: identification of a suitable infantum) and is the unique host for some other species, antigen/s; using appropriate antigen delivery route and such as L. donovani. a potential adjuvant. Nowadays, advances in molecular Post-kala-azar dermal leishmaniasis is a second out- biology knowledge help us to identify some potential come of L. donovani infection in some individuals, virulence factors for prophylactic or diagnostic use. which may occur either during or after treatment [8]. Recombinant DNA technology allows scientists to Hence, vaccination of both humans and animal reser- identify and transfer different genes to prokaryotic voirs, especially dogs for VL, are the two main rational and/or eukaryotic hosts such as bacteria, yeast and even main missions for controlling the disease. Induction of Leishmania parasites to express protein (transiently or stable immunological memory in humans after natural permanent) and release it in different conditions, such infection shows that antiparasitic vaccine development as secretory form or inclusion bodies. Most of these anti- is possible. So far, various forms of vaccines have been gens have conferred effective immune responses when developed and a few formulated vaccines are licensed for delivered as a vaccine in different animal models. In pets (Leishmune® vaccine [since 2004 in Brazil], Leish- clinical trials, however, the results showed weaker pro- Tec ® [since 2008 in Brazil], CaniLeish® [since 2011 in tection when the same antigens or vaccines are used. Europe]), although there is still no licensed (approved) Development of molecular tools also allows manipu- commercial vaccine for human use [1,9] . lation of parasite genome, disruption and insertion of The existence of several infectious species of Leish- interested (target) genes to create new lines of parasites mania and different forms of the disease has made it as live vaccines. difficult to design a vaccine against Leishmania and In this review, we will first provide a short history more knowledge of the vaccine, further investigations of vaccines, followed by new approaches to vaccine and experiences are required. Another problematic issue development for leishmaniasis. in vaccine development is differences in known virulence factors between different species [10] . For example, Immune response of host against leishmania L. major in the old world and Leishmania mexicana/ During leishmaniasis a collection of immune cells Leishmania amazonensis in the new world are CL- and immunomodulators, cytokines and antibodies are causing species; however lipophosphoglycan (LPG) is involved in combating the parasite. Result of this fight a potent virulence factor for L. major but not for L. mex- is either removing the parasite and establishment of icana [11] . In addition, reservoirs of all Leishmania species a long-lasting immunity or survival of parasite in the are not the same. L. donovani and L. infantum are both macrophages and disease progression. However, this

1024 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes depends on the type of parasite, activation of CD4+ and CD8+ T cells and secreted cytokines. Immunity to …antigen/s should be used? leishmaniasis is mediated by both arms of the immune …strategy provides long-term immunity? systems: innate (by neutrophils, macrophages, natural Which… …adjuvant should be used for the highest effect? = Vaccine killer [NK] and dendritic cells) and adaptive (T cells) …animals model are closer to human immunity? responses [14] . …route of administration is suitable? Neutrophils are the first line of host immune defense and primary effector cells of the innate immune sys- Figure 1. Major questions that should be considered in order to select tem against entrance of Leishmania and induction of the best strategy for vaccine design. inflammatory reactions [15–17] . After infection, these cells produce higher levels of IL-8 that act as mediators vaccinated mice [23–25]. Therefore, a safe and effective to recruit more neutrophils. vaccine should be able to increase significant levels of + Progression of disease is associated with inability of CD8 cytotoxic T lymphocytes [26]. neutrophils to kill the parasite. NK cells are another component of the innate immune response and the Short history of traditional & first-generation primary source of IFN-g and IL-12. In addition, vaccines against leishmaniasis IFN-g, TNF and IL-12 are among other stimulator Up to now, several attempts have been done with dif- molecules for NK cells that migrate to the infection ferent vaccination approaches in human and animals, site [18]. The second group of cells involved in immu- especially canines, including traditional live vaccines nity are monocytes/macrophages. In leishmaniasis, (leishmanization) and whole killed vaccines. Each the host immune system produces IL-12, which is approach has some benefits and risks (Table 1). One of essential for cellular immunity through macrophages the important advantages of live vaccines is generation and dendritic cells. and maintenance of the immunological memory during T-cell responses are very critical to protect against Leishmania infection [27], although it has some ethical intracellular pathogens like Leishmania, which induce issues for routine clinical use. Recently, live vaccines both innate and adaptive immune responses [19] . have been classified into two main groups: conventional Two major, distinct CD4+ T-cells, Th1 and Th2 are live vaccine and genetically attenuated live vaccines. known. Many studies have shown that recovery and There is another new approach where nonpathogenic protection against most intracellular pathogens, such as species of Leishmania are used. Leishmanization and leishmanial infection, directly depends on induction of live attenuated vaccines are examples of conventional a Th1 type of immune response, while Th2 responses vaccine. are associated with nonprotective or nonhealing prog- nosis. Some cytokines, such as IFN-g, TNF-a and ■■Leishmanization (using live virulent IL-12, are hallmarks of the Th1 response, while IL-4, promastigotes) IL-5 and IL-10 are markers to identify Th2 response For many years, leishmanization as a vaccination [20]. Most studies on vaccination against Leishmania method from the first-generation of live vaccines was the have been done in the murine model. In mice mod- only way to control cutaneous form of disease. In this els, development of an immune response depends on method, some covered parts of the body are inoculated genetic background of animals and Leishmania species. with live virulent promastigotes. In spite of its dangers Infection course and clinical signs in C57BL/6 mice are (Table 1), it is still used in humans only in urgent condi- very mild because parasite replication is controlled effi- tions in endemic areas such as Uzbekistan, Afghanistan, ciently, whereas BALB/c mice develop a severe course Iraq and Iran [28]. Leishmanization was used during of disease [17,21] . In BALB/c mice, nonhealing and pro- the Iran–Iraq war but after that time it stopped due to gressive infection is associated with lack or lower level probable dangers [29]. of IFN-g production and also increased production of IL-4, IL-5 and IL-10, and the level of IL-10 is critical ■■Live inactivated vaccines in vaccine failure or success [22]. Live attenuated vaccines are derived from a wild-type The other important issue in vaccination against parasite grown in natural conditions, followed by some leishmaniasis is maintenance of immune memory that modifications to attenuate or weaken the parasites, usu- is responsible for long-lasting immunity provided by ally by repeated culturing, chemical reagents, heat or T-memory cells. Several studies have shown that CD8+ radiation. These parasites are avirulent and cannot T cells are responsible for controlling leishmaniasis generate disease but retain the ability to grow, repli- and inducing adaptive immunity, since depletion of cate, trigger the immune system and revert to virulent CD8+ T cells abolishes the generated protection in the form. Within traditional methods, live vaccines were

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Table 1. Advantages and disadvantages of conventional and modern vaccination strategies. Type of vaccine Advantages Disadvantages Live vaccines Single dose, long-term immunity Not safe (leishmanization) Stimulates immune system naturally Has side effects Produces strong immunity Uses whole pathogen Elicits both humoral and cellular immune response Generation of unfavorable immune response Multiplication in the host Induces innate and adaptive immune responses Induces CD4+ and CD8+ T cells Killed vaccines No risk and safer than live vaccines Less powerful than live vaccines Inexpensive Requires multiple doses to have significant No amplification in the host reactogenicity Increased risk of allergic reactions DNA vaccines Safe to use, no adjuvant needed Low potency in humans Stable at room temperature and shipment is Often weak immunogenicity inexpensive Easy for manipulation and production Elicits antigen-specific immune responses Induces both CD4+ and CD8+ T cells Elicits both humoral and cellular immune response Due to CpG motifs, are immunomodulators Prime antigen-specific memory T cells Recombinant No risk Needs cold chain for transportation vaccines No interference with the maternal immunity and/or Expensive, no biological activity other vaccines No native conformation Induces strong immune response Needs adjuvant to enhance immunogenicity Contamination with bacteria substances Large-scale preparation is hard Needs a large amount of antigen to stimulate immunity Prime–boost Long-lasting response Needs cold chain for transportation vaccine Antigen-specific memory T cells Live genetically Stable Risk of reversion to virulent state modified vaccines Safer than general live vaccines Needs cold chain for transportation (avirulent) Stimulate immune system as in natural infection Unknown memory formation and duration Multiplication in the host Stimulates a weaker immune system response Generation of unfavorable immune response Live non- Safe to use Unknown memory formation and duration pathogenic Life-long immunity Need cold chain for transportation vaccines No reversion to virulent state (avirulent) Stimulate immune system similar to natural infection Multiplication in the host

highly successful. These vaccines induce T-cell-medi- as HIV-positive patients due to the damage in their ated immune responses through intracellular processing immune system. and presentation of antigens with MHC class I and II antigens, which is critical to induce protection against ■■Killed parasite vaccine intracellular pathogens [19] . The great advantage of this Since 1930, whole killed vaccines have been used as an type of vaccine is the eliciting of a strong cellular and effective approach in both therapeutic and prophylactic humoral response like a natural infection in disease, vaccines [8]. Killed vaccines with or without adjuvant but milder than wild-type lines, and conferring life- are considered as an old method in the first-generation long immunity. In spite of this advantage, this type category of vaccines. Nevertheless, remarkable results of of vaccine has many drawbacks (Table 1). They need vaccination of dogs with killed parasite vaccines show a booster shot to maintain immunity in individuals. that it seems, in spite of many risks, still one of the best Moreover, not all people can receive live vaccines, such strategies for vaccination [30,31]. Advantage of this group

1026 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes of vaccines is induction of both CD4+ and CD8+ T-cell a single antigen, due to possible synergistic effect of responses, which require both endogenous as well as immune response. Therefore, these vaccines are safer exogenous antigen processing and presentation of anti- than traditional vaccines. Another attractive aspect of gens with MHC class I and II molecules [19] . In addi- modern vaccination is incorporation of immunologi- tion, due to stability of their biochemical composition cally stimulating proteins to increase the release of spe- and antigenicity, lower cost and higher safety compared cific cytokines, though in some cases they have some with live vaccines, scientists pay more attention to these limitations or disadvantages (Table 1). vaccines [30]. However, killed vaccines cannot stimulate Many antigens of different Leishmania species have the immune response as efficiently as live vaccines, so been tested as recombinant proteins and DNA vaccine. they need a booster to have a significant effect. These However, none of them could elicit a perfect long-term vaccines could increase the risk of allergic reactions due protection and just generated a partial protection with to large amounts of antigen involved (Table 1). different degree of immunity. Hence, researchers are In Uzbekistan, to decrease the danger of using live using improved strategies to increase the duration of vaccines, a mixture of live virulent L. major with killed immune response in host, such as cocktail vaccines and parasites has been used as a prophylactic vaccine [29]. In heterologous prime–boost vaccination. Venezuela, autoclaved killed L. mexicana is used to treat Importantly, other factors such as the type of patients with CL in specific condition[29] . This vaccine selected animal with respect to high susceptibility/ for CL has shown various results in different countries. semisusceptibility/resistance to infection, the age of the However, in Phase III of clinical trials in Colombia animal, passage number of parasite in culture and type [32,33], Ecuador [32,34] and Brazil [32], they showed low of infection are very critical. Lack of suitable animal efficacies against L. amazonensis. Likewise, these kind model for some species, like L. infantum and L. tropica, of vaccines were not protective against L. major and do not allow precise studies with some antigens [14] . L. donovani in Iran and Sudan, respectively [32]. ■■Recombinant protein vaccines (second- Modern vaccination approaches against generation vaccines) leishmaniasis In recent decades, a large numbers of antigens from Recently, DNA sequence determination of the human different strains of Leishmania have been identified and genome as well as Leishmania provides new opportunities characterized. Some of these are considered to be an to identify novel approaches for vaccinology. Recombi- attractive vaccine candidate due to their critical role nant vaccines consist of recombinant protein vaccines, in entry and survival of the parasite. Among them, recombinant DNA vaccines, genetically modified live fucose/mannose ligand (FML; Leishmune vaccine) vaccines, recombinant viral-based vaccines, epitope- and A2 are successful examples from recombinant based or peptide vaccines and recombinant subunit vac- protein vaccines that achieved license for vaccination cines that have been developed through recombinant in dogs [5]. DNA techniques. Table 1 shows a summary of advantages In spite of their limitations, proteins induce strong and disadvantages of different forms of vaccines. immune responses but shorter duration for protection Before emergence of recombinant vaccines, crude against Leishmania infection. The recombinant pro- Leishmania antigens were used to immunize vulner- teins have no interference with the maternal immunity able individuals. Following development of recombi- and/or other vaccines. These vaccines are safe due to nant DNA technology, different parasite components, their inability to replicate in the host; hence, a large especially essential genes for parasite survival, were amount of antigen is needed to stimulate immunity. focused on and their induced immune response in host Preparation of recombinant protein vaccines is very has been characterized. In another approach, genetically difficult and expensive since it needs purification sys- attenuated live vaccines were made by knocking out tems and adjuvants or carriers to enhance immuno- of some main genes. Designing recombinant vaccines genicity [35]. The purified proteins are often associ- against Leishmania is based on fundamental informa- ated with bacterial component after purification. In tion about genes involved in structure, metabolism and addition, these vaccines have no biological activity and virulence. The main advantages of the recombinant vac- native conformation, but some of the tested antigens cine technology are using just a single or a number of showed a better ability to stimulate the immune system proteins (mixing or fusing together) from one or more (Table 1). species of an organism instead of the whole parasite cell As mentioned above, one of the major drawbacks antigens, so there is no risk of disease due to adverse of recombinant proteins is essentiality of using an virulent properties. There are reports suggesting that adjuvant because the sole protein induces only Th2 the use of several antigens is more effective than using response, while, coadministration of this type of

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vaccines with an adjuvant shifts the immune response ■■Recombinant protein vaccines versus DNA toward Th1. For example, although rLACK alone was vaccines not protective against L. major, rLACK plus rIL-12 There are a lot of debates over protein and DNA vac- as adjuvant was shown to induce partial protection in cination and their induction of natural immunity. Both BALB/c mice [23]. In contrast, a similar immunization DNA and protein vaccines have some advantages and program (rLACK+rIL-12) could not generate protec- limitations, as summarized in Table 1. There is a long list tion against L. amazonensis infection [36]. This shows of candidate leishmanial molecules that were adminis- the species specificity of selected antigens in inducing trated as recombinant protein or DNA-based vaccines immune response. to evaluate prevention or reduction effect against CL and VL models with different adjuvants and routes of ■■DNA vaccines (third-generation vaccines) vaccination. However, none of them conferred complete Parallel to development of protein vaccine, DNA vac- long-term protection against leishmaniasis and often cines encoding one or more immunogenic proteins have generated just partial protection. been introduced as a successful delivery system. In 1990, Several reports with different antigens from different for the first time, RNA and DNA expression vectors laboratories have confirmed that vaccination with DNA containing reporter genes were introduced for injection is more potent and induces a stronger immune response into mouse skeletal muscle in vivo [37]. DNA vaccination than recombinant protein alone or a combination of holds considerable promise for vaccination against dif- DNA and protein (prime–boost) [23,41–47]. However, ferent diseases in which Th1 responses and cell-mediated there are some examples of failed DNA vaccinations immunity are responsible for generating protection, such using the TRYP gene alone [26] and g-GCLC [48]. as with leishmaniasis. During the last two decades, DNA Many factors, such as nature [13], conformation [49] vaccines have been developed against infectious micro- and dose of antigen, nature of used adjuvant, frequency organisms because this approach of immunization elic- and route of vaccine administration [13] and the time its both humoral and cellular immune response against interval between prime–boost immunizations are very native forms of protein, since antigens are produced in important and could have varying degress of influence the host natural conformation due to post-translational on a protective level. Low immunogenicity of DNA modifications of the expressed proteins inside the cells. vaccines is the main drawback of these vaccines when In addition, lower quantities of DNA should be used to compared with protein vaccines [40]. To solve this induce protection. Of course, different elements could problem, some strategies have been developed, such influence the magnitude of immune response generated, as cocktail vaccines, prime–boost vaccines and using including immunogenicity of the desired expressed gene novel adjuvant and delivery systems as good alternatives and synthetic oligodeoxynucleotides. The CpG motifs to increase the efficacy. present in plasmid DNA are immunomodulators and by acting as an adjuvant switch the immune response Some candidate molecules for vaccination toward Th1 [38]. In addition, immunization with DNA Many antigenic molecules as vaccine candidates have alone activates TLR9, whcih leads to high IL-12 produc- been prepared and tested by several groups against tion and finally a predominant Th1 immune response Leishmania infections and induced different levels of [39]. Another property of DNA vaccines is being more protection. Some of them were shown to induce protec- stable in shipping and transition. They are also cheaper tion against more than one Leishmania species. In addi- than recombinant protein vaccines due to not need- tion, coadministration of different antigens in the same ing any adjuvant and purification system or refolding regime dramatically increased the potency of vaccine. procedures (Table 1). Leish-111f is a single polyprotein antigen composed In addition, a single multivalent DNA vaccine, in of three fused components, L. major thiol-specific which a single plasmid would carry more than one antioxidant (TSA), L. major stress-inducible protein 1 gene encoding protective proteins, would be more cost- and Leishmania braziliensis elongation initiation fac- effective and easier to produce. On the other hand, tor [50–52]. These antigens are present in both amasti- naked DNA has a short lifetime within the cell since gote and the promastigote forms and are highly con- they get degraded by the enzymes. Some alternative served among different Leishmania spp. This was the methods such as coating DNA with nano- or micro- or first candidate for a subunit vaccine that was tested gold-particles protect plasmids from degradation and in Phase I and II clinical trials [45]. It has been shown increase phagocytic uptake by professional antigen- that combined recombinant Leish111f with monophos- presenting cell [40]. A major drawback of DNA vaccines phoryl lipid A-stable emulsion or IL-12 could protect is their very low immunogenicity in humans when various animals against L. major, L. amazonensis and compared with animal models. L. infantum infection [51,53–55]. Furthermore, Leish-111f

1028 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes vaccine has potential application in both prevention and BALB/c mice and can stimulate Th1 response against treatment [56]. L. major in two regimens, DNA/DNA and prime–boost Leishmania homologue of receptors for activated (DNA/protein) [70]. C-kinase (LACK) is another attractive target that CPs are other vaccine candidates that belong to the has been successfully used as an experimental vaccine group of papain-like enzymes and are expressed in the against leishmaniasis. So far, this antigen is used fre- amastigote form [2,71]. CPs are among the important quently in vaccine studies against both CL and VL. Dif- virulence factors that have several critical functions in ferent studies have shown that LACK antigen is a pro- establishment of infection [72,73]. There are different tective antigen against L. major [23] and L. amazonensis families of the CP group, including CPA, CPB and CPC [36] infection, but it did not confer significant protection in different species of Leishmania, which are charac- against L. donovani infection [57]. terized and the relation between their expression and Gp63 (leishmanolysin) is a zinc-metallo protease parasite virulence is described [2]. Several early reports membrane surface glycoprotein expressed in promas- have highlighted the role of CPs in pathogenicity, invad- tigotes of different species of Leishmania. It has been ing the host cells, replication, and finally exiting the successfully used as a candidate vaccine with differ- infected cells to establish further infections of L. major ent formulations in mouse models, and is a promising [24,74], L. mexicana [75,76], L. chagasi [77], L. tropica [78] vaccine candidates against leishmaniasis. and L. (L.) amazonensis [79] amastigote in mammalian FML is an antigenic glycoprotein complex from the host. Type I enzymes (CPB) are encoded by multi-copy promastigote form of L. donovani. This is the main anti- genes (19 copies) and contain an unusual C-terminal gen in Leishmune vaccine, the first commercial vaccine extension about 110 amino acids, which is absent in against VL. In several studies, immunization potential other CPs of the papain superfamily [80]. The C-termi- of FML was evaluated with different adjuvants, such as nal extension has been postulated to be highly immu- saponin and aluminium, and it demonstrated the ability nogenic and may play a role in the diversion of the host to create strong protection and enhanced production immune response [77,81–84]. of IgG in mice [58] and dogs [5,59,60]. In addition, the The acidic ribosomal P0 protein of L. infantum is a FML vaccine induced a significant long-lasting protec- structural component of the large ribosome subunit. tive effect against canine kala-azar in the field [61,62]. Vaccination based on the ribosomal P0 protein-DNA Protection against canine kala-azar was also investigated or rLiP0 protein plus CpG oligodeoxynucleotides pro- in naturally exposed dogs vaccinated with the FML vac- tected C57BL/6 mice from dermal pathology, accompa- cine [59–60,63] and showed 95% seropositivity to FML nied by production of IFN-g and reduced parasite load, and 100% intradermal reaction to L. donovani lysate but was not able to prevent the progressive disease in 7 months after vaccination. BALB/c mice, despite the induced Th1 immunological The nucleoside hydrolase (NH36) antigen is a 36-kDa response in both models [12] . surface glycoprotein complex and is the main antigen of A2 was identified for the first time inL. donovani and is the FML complex, which is identified inL. donovani, one of the excellent candidate antigens against VL identi- L. chagasi, L. amazonensis and L. major [64,65]. fied so far and tested in different species ofL. donovani, LPG is a dominant surface molecule expressed in L. infantum and L. chagasi [85]. A2 genes are conserved all Leishmania species and is associated with several in species of the L. donovani complex [36]. Attention to processes in promastigotes, such as parasite survival, this antigen is due to its amastigote-specific expression in interaction with host cell receptors, inhibition of midgut L. donovani. It is required for survival of VL-causing para- proteases, attachment and entry into the host macro- sites in visceral organs, but it is present only in a truncated phages and sand flies. Moreover, it also plays an impor- form in L. major [86] and is absent in Leishmania taren- tant role in parasite resistance to complement molecules, tolae [87], which is a member of nonpathogenic species. manipulation of signal transduction pathways and gene Immunization of mice with A2, in recombinant protein expression in macrophages, and resistance to oxidative or DNA vaccination form, leads to protection against ® stress. Altogether, through all the mentioned pathways, L. donovani infection [88,89]. At present, LeishTec is one this protein has a pivotal role in initiation and estab- of the commercially available vaccines on the market that lishment of a durable infection [66–69]. In addition, it is based on A2 antigen. This vaccine is a recombinant has been observed that LPG3 helps to synthesize the A2-antigen of Leishmania amastigotes plus saponin as LPG, as the most important surface molecule in pro adjuvant [85]. mastigotes [66,67]. LPG3 is the Leishmania homolog Finally, kinetoplastid membrane protein-11 is a of the mammalian endoplasmic reticulum chaperone highly conserved membrane protein with high epitope GRP94, a member of HSP90 family that is involved density. This hydrophobic protein has been described in assembly of LPG. LPG3 is highly immunogenic in to be associated with LPG. The protectivity effects of

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this antigen as a DNA or protein alone or combined introduced as a prime–boost vaccination that is more with some adjutants have been shown protection against potent than DNA or protein alone [46]. two prevalent forms of leishmaniasis, CL and VL [90,91] . Prime–boost vaccination Cocktail vaccines Early studies using different antigens with different con- So far, various leishmanial antigens have been ditons simultaneously suggested that induced protection tested separately in order to induce protection with after prime–boost vaccination is more stable and power- long-term immunity in animal models (Table 2) ful. A number of scientists have compared the potency [12,23,24,36,41,43,44,47,48,51,53,54,70,74,88,89,92–106]. Although and efficacy of different strategies using DNA or pro- the responses are variable, one of the limitations on using tein alone (homologous prime–boost) and heterologous a single antigen is their weak immune response. Thus, prime–boost (HPB)-based vaccination. Although there in order to achieve higher levels of protection, cocktail are different reports, most of them confirmed that the vaccines in form of combined recombinant proteins or DNA-prime/boost-protein approach due to primary genetically fused or even coadministrated with sand fly expression of antigen within cells is able to stimulate salivary molecules as a novel source of antigens have robust cellular and humoral responses. Of course, as been suggested [107] . There are several examples, such as mentioned above, type of antigens, rout of immuniza- KSAC (which is a genetically fused protein comprised tion and the type of adjuvant are critical factors that of KMP-1, SMT, A2 and CPB [108]) and recombinant should be considered. antigens (L. major stress-inducible protein 1 and TSA) plus IL-12 [101] . Due to the high antigenic diversity of ■■Homologous prime–boost vaccination the Leishmania parasites, using more than one antigen Homologous prime–boost strategy is a repeated vac- could increase the chance of vaccine usability on a wide cination program using the same antigens (boost- range of species [96]. ing) [112] . Usually, protein or DNA vaccines (DNA/ The efficacy of DNA vaccinations as a cocktail of DNA or protein/protein) are applied for two or three the two distinct plasmids encoding CPA and CPB was steps of vaccination (or revaccination). One of the evaluated by their capability to induce protection and a problems using this strategy is induction of antivec- specific immune response toLeishmania infection in the tor immunity encountered when using the same vec- BALB/c mice as model. This vaccine induced a long- tor. Evaluation of protective efficacy and comparison lasting protective response in immunized mice, whereas between these two regimes revealed that in some cases the separate injection of cysteine protease genes is not DNA/DNA strategy is more potent than recombinant protective [74]. The next study showed no significant dif- protein alone or combination with DNA in order to ferences between the levels of induced protection in vac- control infection [23,41,42,44]. cination with bicistronic plasmid (encoding CPA and CPB separately) and monocistronic plasmid (encoding ■■HPB vaccination CPA/B hybrid protein) [102] . Cocktail vaccines have sev- As discussed above, while both recombinant protein eral advantages. Production of a vaccine composed of and DNA vaccines are powerful approaches to induce a several antigens separately is more difficult to standard- potent immune response, in many cases the generated ize and also more expensive to produce than a single- immune responses are weak. In order to enhance the product vaccine [103] . In addition, multivalent vaccines protective response, a HPB vaccination strategy encod- containing a broader range of protective epitopes that ing the same antigen is introduced as an alternative would cover a wide range of MHC types in a heteroge- approach instead of homologous boosts. In this method, neous outbreed population, such as humans and dogs a particular antigen is administered in two rounds using [102], elicited a protective response stronger than when more than one delivery method and two different vec- individually used [93,105]. This type of antigen delivery tors [112,113]. Usually, DNA vaccines are used for priming can induce cross-protection against different species of and recombinant proteins or live vectors are used for Leishmania. boosting. The first example of this approach was DNA The recombinant protein Q (a genetic fusion of five vaccine for the priming and virus-based vaccine (attenu- intracellular antigenic fragments, from the L. infan- ated avian poxvirus) for boosting; but now, different tum acidic ribosomal proteins Lip2a, Lip2b, P0 and microorganisms could be used as delivery systems such histone H2A) when mixed with BCG or CpG will be as viruses, bacteria and parasites. more protective than when administered alone against The major proof of using this approach is that it does a L. infantum infection in mice [109] and dogs [110,111]. not need any adjuvant. Due to carrying stimulatory Recently, a cocktail vaccine containing four candidate unmethylated CpG motifs in their backbone, DNA antigens, KMPII, TRYP, LACK and PAPLE22, was plasmids induce cellular immunity via expression of

1030 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes

[41] [41] [74] [74] [95] [93] [23] [23] [23] [92] [97] [94] [24] [24] [36] [96] Ref. / IL-5 g g, , ↓ IL-10 , , g g g / IL-5 , ↑ IL-5, g g , ↓ Il-4, g , ↓ IL-5 , ↓ IL-5 g g ⁄IL-4,↓ Parasite load g T-cells-induced protection T-cells-induced ↑ Lower level of IFN- Results ↑ IgG1, ↓ Swelling size, protectionNo response,↑ Th1 ↓ Swelling size, ↑ High production of IFN- ↑ IgG2a, Partial protection No protection, ↓ IFN- Partial protection, ↓ Footpad swelling, ↑ IFN- ↑ Protective response, ↓ IgG2a, ↑ IFN- ↑ Protection level, ↓ parasite load, response, ↑ Th1 ↑ IgG2a/IgG1, ↑ IFN-γ/IL-5 Protective response, ↑ IgG2a, ↑ Control of disease progression, ↓ Parasite burden, ↑ IFN- CD8 protectionNo Protective response, ↑ IgG2a, ↑ Control of disease progression, ↓ Parasite burden, ↑ IFN- No protection, ↑ Parasite load protection,↓ 81% ↑ IgG2a, ↑ IFN- Protection,↑ IFN- protection,Significant ↓ lesion size Partial protection, ↑ IFN- ↑ IgG, ↑ IgG2a, ↓ IgG1 ↓ 66% parasite burden, ↑ IgG2a, ↑ IFN- ↓ Parasite burden, ↑ IgG2a/IgG1 Th1 response,↑ Th1 ↑ IgG2a/IgG1, ↓ Parasite load BALB/c Model BALB/c BALB/c BALB/c C57BL/6 BALB/c BALB/C BALB/C BALB/C BALB/C BALB/C BALB/C BALB/c Canine BALB/C BALB/c + Montanide 720 + . – Adjuvant – 407 Poloxamer 407 Poloxamer SLN SLN – – rIL-12 rIL-12 CpG – ODN CpG – Saponin CpG ODN CpG

Leishmania amazonensis mexicana major major major major major major major major major major major major chagasi chagasi major L. Challenge strain L. L. L. L. L. L. L. L. Leishmania L. L. L. L. Leishmania L. L. chagasi donovani major major major major major major major major major major major chagasi major Leishmania major Antigen origin L. L. L. L. L. L. L. L. Leishmania L. L. L. Leishmania L. L. -CTE DNA DNA Single Ag cpa Table 2. Candidate antigens for immunization against immunization for antigens Candidate 2. Table Protein or DNA cpb rCPA Rcpb CPB Protein/protein CPA/CPB/CPC DNA/DNA LACK DNA rLACK rLACK rLACK rSPase SPase DNA Gp63 Liposomal A2 DNA rA2 Liposomal Liposomal rLmSTI1 Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type 18 E6 protein human papillomavirus; LDU: Leishman–Donovan units; LN: Lipid nanoparticles; 18 E6 protein Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type ODN: Oligodeoxynucleotide; SLN: Solid lipid nanoparticles.

future science group Clin. Invest. (2013) 3(11) 1031 Review: Clinical Trial Outcomes Taheri & Rafati

[12] [12] [12] [47] [99] [88] [48] [43] [43] [36] [96] [44] [44] Ref. [89,98] mexicana chagasi, mexicana , ↑ IgG2a , ↑ IgG2a g g g, g g, g, 70% parasite, ↓ 70% load IL-12, ↓ IL-4 , ↑ IL-12, ↓ IL-4 , ↑ IL-12, and, Th1 Th2 response and -4 , ↑ IL-2 g g g g chagasi Protection, ↑ IFN- ↓ parasite load, and ↓ IL-4 -10 Protection, ↑ IFN- reduction65% in LDU Partial protection 30% of mice Protection, response, ↑ Th1 ↑ IgG2a/IgG1, ↑ IFN- Partial protection, response,↑ Th1 ↑ IgG2a/IgG1, ↑ IFN- response, Protection, Th1 ↓ parasite load, ↑ IFN- Partial protection, ↑ IgG2a, ↑ IFN- Finally progressive disease response, Protection, Th1 ↓ parasite load, ↑ IFN- reactions,↓ DTH ↓ Ab, ↓ parasitem load, ↓ IL-10, ↑ IFN- protectionNo Significant protection against L. Partial protection against L. Results Protection, ↑ IFN- ↑ titer Ab, and ↑ Th1 Th2 response Partial protection ↓ 88% parasite load of L. parasite↓ 65% load of L. Protect, ↑ IFN- Delay in lesion growth, ↑ Ab titers BALB/c BALB/c BALB/c BALB/c BALB/c C57BL/6 BALB/C C57BL/6 Canine BALB/c BALB/c Model C57B/6 BALB/c BALB/C (cont.). rIL-12 pCDNA3 – ODN CpG ODN CpG ODN CpG ODN CpG ODN CpG – – Saponin Adjuvant Heat killed Propianzibactrium acnes – Nonionic surfactant vesicles

Leishmania amazonensis donovani major donovani donovani major major major chagasi chagasi amazonensis chagasi mexicana donovani chagasi mexicana mexicana L. L. L. L. L. L. L. L. L. L. L. L. L. Challenge strain L. L. L. L. infantum donovani donovani major donovani donovani infantum infantum donovani donovani donovani donovani donovani donovani L. L. L. L. L. Leishmania L. L. L. L. L. Antigen origin L. L. L. GCS g Single Ag rA2 Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type 18 E6 protein human papillomavirus; LDU: Leishman–Donovan units; LN: Lipid nanoparticles; 18 E6 protein Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type ODN: Oligodeoxynucleotide; SLN: Solid lipid nanoparticles. Table 2. Candidate antigens for immunization against immunization for antigens Candidate 2. Table A2 DNA Gp63 DNA Gp63 DNA/DNA rgp63 rLiP0 rLiP0 LiP0 DNA NH DNA NH DNA rNH rLd Protein or DNA rA2 NH DNA

1032 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes [51] [74] [74] [48] [48] [48] [54] [101] [103] [102] [100] [104] Ref. [53–54] T cells, + , ↓ IL-4 g / IL-5, g , g , ↑ IL-2 IL-5, ↑ IgG2a, , ↓ IL-5, g g g , ↑ IgG2a, ↓ IL-4 g TNF, ↓ IgG ↑ TNF, , ↑ IL-2, T cells, ↑ IFN- g + No protectionNo ↑ Ab titers, ↑ IL-4 protection Transient ↓ Lesion size, ↑ Ab titers ↑ IgG2a/IgG1, ↓ parasite burden High level protection, immunity for 3 months, ↑ IFN- Protection, ↓ lesion size, ↑ IFN- ↓ Parasite loads of 91.7%, ↑ CD4 Protection: ↓ parasite loads of 99.6%, ↑ CD4 ↑ Humoral and cellular, ↓ lesion size Induce excellent protection reduction71% of infection, long lasting protection, ↑ IFN- phenotype,Th1 ↑ IFN- reduction81% of infection Partial protection response,Th1 ↑ IFN- Significant protection, ↑ IFN- Results Protection Delay in lesion growth ↑ Ab titers ↑ IFN- partial protection BALB/C BALB/C BALB/c BALB/c BALB/c, C57BL/6C57BL/10 Hamster BALB/c C57BL/6 BALB/c monkey Rhesus BALB/c BALB/c BALB/c BALB/c Model BALB/C (cont.). – – MPL-SE or Ribi 529-SE IL-12 or MPL-SE MPL MPL-SE MPL MPL-SE – IL-12 – – 407 Poloxamer SLN Adjuvant Nonionic surfactant vesicles

Leishmania mexicana major major major infantum infantum major major major major major major major L. L. L. L. L. L. L. L. L. L. L. L. Challenge strain L. braziliansis donovani donovani major major braziliansis major braziliansis major braziliansis major major major major major major donovani L. L. L. Leishmania L. L. L. L. L. L. L. L. L. L. L. L. Antigen origin L. -CTE GCS + cpb + cpb GCS GCS g g g Single Ag rLd Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type 18 E6 protein human papillomavirus; LDU: Leishman–Donovan units; LN: Lipid nanoparticles; 18 E6 protein Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type ODN: Oligodeoxynucleotide; SLN: Solid lipid nanoparticles. Table 2. Candidate antigens for immunization against immunization for antigens Candidate 2. Table DNA Ld DNA Ld Cocktail vaccine rLeish-111f rLeish-111f rLeish-111f rLeish-111f TSA/LmSTI1 DNA rTSA/LmSTI1 cpa DNA, monocistronic plasmid cpa DNA, bicistronic plasmid Hybrid rCPA/CPB CPA/CPB DNA/DNA Protein or DNA

future science group Clin. Invest. (2013) 3(11) 1033 Review: Clinical Trial Outcomes Taheri & Rafati

cytokines related to Th1 response. Addition of CpG [70] [88] [36] [96] [105] [106] Ref. to vaccines enhances and increases the long-lasting , , protection and can efficiently shift the Th1/Th2 mixed g g response towards protective Th1-biased response [44]. IFN-

IFN- Currently, there are four different regimes of HPB against leishmanial infection as described below (Table 3) [22,26,41,44,46,70,74,83,84,90,97,105,114–125].

g DNA/protein strategy lesion size, ↑ lesions size, ↑ size, lesions So far, different candidate leishmanial antigens have been tested by this strategy (Table 3). Recently, in an interesting study, three vaccination strategies have been compared using kinetoplastid membrane protein-11, TRYP, LACK, and PAPLE22 vaccine candidate anti- /IL-5, ↓ lesion/IL-5, size, ↓ parasite burden g

IL-5, ↓ parasite, ↓ IL-5, load in LN gens against VL in the hamster model. The compari- g son between naked DNA, raw insect-derived recom- Significant protection, response, ↑ Th1 IFN- Results High level of protection, 80% reduction in LDU, response, Th1 ↑ IFN- No protection, ↑ parasite load Significant protection, ↓ and↑ IL-4 -10 Significant protection, ↑ IgG2a, ↑ IFN- ↓ responses, Protective parasite burden ↓ parasite ↓ IL-10, in spleen binant protein and heterologous prime–boost strategies showed that the prime–boost strategy and raw insect-derived recombinant proteins elicited a stronger cell-mediated immune response and parasitological protection against L. infantum than naked DNA. This is while the recombinant proteins derived from Th1 response Th1 Model BALB/c BALB/c BALB/C BALB/c BALB/c baculovirus-infected insect cells was not protective [46].

DNA/virus strategy In parallel to using DNA as a vehicle for vaccination, recombinant viral vectors were developed as powerful vaccine delivery systems. A major advantage of this approach is that it is suitable for use in humans [126] . Using recombinant viral vectors to deliver leishmanial

(cont.). antigens or peptides [127] is another new approach to – Adjuvant – – – Montanide 720 ODN CpG vaccination. Vaccinia virus Ankara (MVA), recombinant Vaccinia virus, attenuated adenoviruses and attenuated pox viruses like fowl pox are some examples

Leishmania from modified viruses that are safe to use. These vectors lose replication properties after long serial passages into cells like chicken embryo fibroblasts, which is used for MVA [128] . However some inconsistent reports are published. As summarized in Table 3, immunization braziliensis donovani amazonensis amazonensis chagasi major major braziliensis L. Challenge strain L. L. L. L. L. L. L. of BALB/c mice with DNA-LACK/MVA-LACK was successful and induced protection against L. major [128]; however, another group could not prove this vaccination regimen [22]. infantum DNA/bacteria strategy Live-attenuated Salmonella enterica serovar Typhimurium is another optimal vaccine vehicle for boosting strategy, donovani amazonensis donovani major major Leishmania chagasi Antigen origin L. HPV L. L. L. L. since it stimulates both humoral and cellular immune responses. The main advantage of these systems is possibility for oral delivery. Vaccination with DNA-Sal- monella as prime–boost regimen, while DNA encoded LACK antigen, expressing LACK antigen enhanced IFN-g production and protection against L. major Cocktail vaccine H2A, H2B, H3, H4 DNA Table 2. Candidate antigens for immunization against immunization for antigens Candidate 2. Table Protein or DNA A2 DNA + E6 rA2 + rLACK A2 + NH DNA NT + LPG3 DNA rLmL5 or rLmL3 Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type 18 E6 protein human papillomavirus; LDU: Leishman–Donovan units; LN: Lipid nanoparticles; 18 E6 protein Ab: Antibody; Ag: Antigen; CTE: C-terminal extension; DTH: delayed-type hypersensitivity; E6: Type ODN: Oligodeoxynucleotide; SLN: Solid lipid nanoparticles. challenge in susceptible BALB/c mice [114] .

1034 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes

Table 3. Examples of prime/boost vaccination against different species of Leishmania. Ag prime/boost Source of Ag Challenge strain Adjuvant Animal model Results Ref. DNA/protein ORFF Leishmania donovani L. donovani – BALB/C ↓ Parasite load (75%-80%) [116] DNA/protein ↓ (not induce the typical T-helper response), ↓ IgG2a, ↓ IFN-g rA2 Leishmania chagasi L. chagasi Saponin Canine Partial protection [97] H2A, H2B, H3, H4 Leishmania infantum L. braziliensis CpG BALB/c Strong immune response, [105] 2 x DNA/protein chagasi ↓ IFN-g, ↓ IL-5, ↓ parasite load in LN, ↓ inhibition of disease CPA + CPB Leishmania major L. major IFA BALB/c ↓ Th1 response, ↓ swelling [74] DNA/protein size, ↓ high production of IFN-g, ↓ IgG2a, partial protection CPA + CPB L. infantum L. infantum CpG ODN or BALB/c Th1 response [84] DNA/protein Montanide 720 CPA + CPB L. infantum L. infantum CpG ODN or Canine Protection [83] DNA/protein Montanide 720 CTE (CPB) L. infantum L. infantum CpG ODN or BALB/c Th1/Th2 response [117] DNA/protein Montanide 720 CPC L. infantum L. infantum CpG ODN or BALB/c Significant protection, [118] DNA/protein Montanide 720 ↓ IgG2a/IgG1, ↓ NO, ↓ IFN-g, ↓ parasite burden SPase L. major L. major CpG ODN + BALB/c ↓ 70% parasite burden [41] DNA/protein Montanide 720 ↓ IgG2a, ↓ IFN-g, ↓ IL-5 gp63 L. donovani L. donovani CpG BALB/c Induce Th1 responses, [44] DNA/protein ↑ IFN-g, ↑ IL-12, ↑ NO, ↓ IgG2a/IgG1, ↓ IL-4, IL-10, ↓ parasite load LPG3 L. major L. major Montanide 720 BALB/c Significant protection, [70] DNA/protein ↓ IgG2a, ↓ IFN-g/IL-5, ↓ lesion size, ↓ parasite burden in spleen KMPII, TRYP, L. imfantum L. infantum – Hamster ↓ Parasite load, ↓ NO [46] LACK, PAPLE22 3 x DNA/ 2 x protein DNA/virus LACK L. infantum L. infantum – Canine 60% protection, ↑ IL-4, [119] DNA/rVV ↑ IFN-g and ↑ IL-12 mRNA LACK L. infantum L. infantum – BALB/c High levels of protection, [120] DNA/WR or DNA/ ↓ parasite burden, ↑ IFN-g, MVA ↑ TNF LACK L. infantum L. major – BALB/c Long lasting protection, [128] DNA/MVA ↑ IFN-g and TNF-a secreting CD8+ T cells, ↓ lesion size by 65–92% LACK L. infantum L. infantum – Canine ↓ VL symptoms [121] DNA/MVA ↓ T-cell activation, ↓ Ab Ag: Antigen; CTE: C-terminal extension; IFA: Incomplete Freund’s adjuvant; SLN: Lipid nanoparticles; MVA: Vaccinia virus Ankara; ODN: Oligodeoxynucleotide; ORFF: Open-reading frame gene from the LD1 locus; rVV: Recombinant Vaccinia virus; VL: Visceral leishmaniasis; WR: Vaccina Western Reserve virus.

future science group Clin. Invest. (2013) 3(11) 1035 Review: Clinical Trial Outcomes Taheri & Rafati

Table 3. Examples of prime/boost vaccination against different species of Leishmania (cont.). Ag prime/boost Source of Ag Challenge strain Adjuvant Animal model Results Ref. DNA/virus LACK L. infantum L. major – BALB/c ↑ IFN-g, ↑ TNF-a and [122] DNA ↑ IL-2, ↑ CD8+ and CD4+ T cell P36/LACK L. infantum L. major – BALB/c ↓ lesion size, ↑IFN-g, [123] DNA/rVV ↓ parasite load (1000 ×) Influenza A LACK L. major – BALB/c ↑ IFN-g producing CD4+ [124] viruses single peptide T cells, ↓ IL-4, IL-5, IL-10 and IL-13 LACK L. major L. major – BALB/c Not protective, Th2 response, [22] DNA/MVA IgG2a/IgG1 = 0.04 TRYP L. major L. major – BALB/c Protective, Th1 response DNA/MVA IgG2a/IgG1 = 0.91 TRYP Leishmania (Viannia) L. (V.) a-GalCer BALB/c Induce protection but no [26] DNA/MVA panamensis panamensis LPS, CpG protection Pam3CSK4 MALP-2 TRYP L. infantum L. infantum – Out bred Th1 response, ↑ IgG2, ↑ IFN-g [125] DNA/MVA canine KMP-11 L. donovani L. donovani – Mice, hamster ↑ Activation CD4+ and [90] DNA/rVV CD8+ T cells, ↑ high-quality T cells ↑ IFN-g, IL-2 and TNF-a DNA/bacteria LACK DNA- L. major L. major – BALB/c Enhance protection, ↑ IFN-g, [120] Salmonella/ ↓ IL-4 and IL-10, ↑ IgG2a, protein ↓ IgG1 DNA/Leishmania A2-CPA-CPB-CTE L. infantum L. infantum SLN BALB/c Th1 response, protection [115] DNA/L. tarentolae ↑ IFN-g, ↓ IL-10, ↑ IFN-g/ IL-10, ↑ NO, ↓ parasite burden Ag: Antigen; CTE: C-terminal extension; IFA: Incomplete Freund’s adjuvant; SLN: Lipid nanoparticles; MVA: Vaccinia virus Ankara; ODN: Oligodeoxynucleotide; ORFF: Open-reading frame gene from the LD1 locus; rVV: Recombinant Vaccinia virus; VL: Visceral leishmaniasis; WR: Vaccina Western Reserve virus.

DNA/parasite strategy Recently, the ability of recombinant L. tarentolae to More recently, a new strategy using recombinant live induce protective immunity against leishmaniasis in mice nonpathogenic Leishmania species carrying antigens model was evaluated using heterologous prime–boost derived from pathogenic species are introduced as vec- immunization techniques, priming with plasmid DNA tors to express that protein endogenously, present it followed by a boost with the recombinant parasite [115,129] . to the immune system and elicit immune response In another study, BALB/c mice were immunized [115,129–131]. L. tarentolae (isolated from lizard) is an with a heterologous prime–boost regimen using DNA/ example of the genus Leishmania that could not gen- live parasite carrying a trifusion gene (A2-CPA-CPB- erate any signs of leishmaniasis in human, hence it CTE) against L. infantum challenge. For priming, the is known as nonpathogenic for human [130] . Using trifusion gene was formulated with cationic solid lipid Leishmania species has some advantages, such as its nanoparticles acting as a delivery system. The protec- ability to grow in cell-free media condition within tive immunity was associated with a Th1-type immune a cheap/easy media, with a mammalian-type N-gly- response with high levels of IFN-g prior and after chal- cosylation pattern [132] . Previous studies have shown lenge and with lower levels of IL-10 production and that L. tarentolae can be used as a live vaccine against parasite burden after challenge. Moreover, this immun L. donovani and elicits a protective Th1 immune ization elicited high IgG1 and IgG2a humoral immune response [130] . responses [115] .

1036 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes

Nonpathogenic organism-based live vaccines localization of the antigen has more influence on the In spite of high challenges of live immunization, scien- generated immune response [134] . tists still believe that live vaccines are the most powerful known vaccine to date. As summarized in Table 1, Genetically attenuated live vaccines live vaccines induce T-cell mediated immune responses Immunization using live parasites is stronger in induc- and mimic natural infection through correct processing ing the immune system and eliciting powerful cytokines and presentation of antigens in association with MHC or antibodies than other vaccines, due to stability of the class I and II antigens [19] . In vaccination with live para- immune response and higher antigenicity [30]. Therefore, sites, both CD4+ and CD8+ T cells are induced to create scientists are trying to generate genetically manipulated protective immune responses and long-term immunity, strains to stimulate the immune system without replica- hence, induce appropriate inflammatory and regulatory tion of parasite within the host and manifestation of the immune responses in the host animals [19] . However, the disease. In order to identify the parasites with the least risk of reversion of the organism to its virulent state is virulence potential, essential gene/s for parasite growth the main obstacle when using these vaccines. Another and infectivity in amastigote form of the parasite are tar- approach to reach the best safe condition is using live geted for disruption. DHFR-TS was the first gene that was vaccines through heterologous gene expression in a live targeted as an essential metabolic gene from L. major’s nonpathogenic microorganism such as L. tarentolae [130] . genome to obtain genetically attenuated parasite vaccine This parasite does not survive in mice [130] . Table 4 shows [137] . After that, many target genes in Leishmania were a list of nonpathogenic microorganisms used as carrier to disrupted and the knock-out strains were evaluated for express heterologous proteins [115,124,129–131,133–135]. infectivity potential and protectivity. However, the most Recently, with development of transfection techniques, important concern about safety and the possibility of a number of genes (virulence or reporter genes) were trans- reversion to virulence state [14] remained to be solved. fected in L. tarentolae episomally or integrated into rRNA For example lpg2-mutant L. mexicana maintain their abil- locus of the genome through homologous recombination ity to cause disease in the absence of the lpg2 gene [138] to obtain knock-in recombinant parasites [115,133,136]. through an unknown compensatory mechanism [139] . The A2 gene, which is expressed specifically by the Consequently, to decrease infectivity potential of live L. donovani complex and promotes visceralization, is attenuated parasite vaccines, it is likely that more than absent in L. tarentolae [87]. In 2010, effectiveness of one gene should be disrupted [14] . A2-recombinant L. tarentolae as live vaccine was stud- ied with two administration systems, intravenous and Saliva-based vaccines intraperitoneal injection. The results demonstrated that Leishmania parasite is transmitted by different species of a single intraperitoneal vaccination of BALB/c mice infected female sand fly such asLutzomyia (new world) with recombinant L. tarentolae expressing A2 antigens and Phlebotomus (old world) species. In a natural path- derived from L. infantum elicited a strong cell prolif- way of infection, parasites are delivered into the skin of eration after challenging with L. infantum that was the host while accompanied by secretory saliva that helps accompanied by reduced levels of IL-5 production after induce protective immunity. Sand fly saliva contains challenge, leading to a potent Th1 immune response. immunogenic or immunomodulatory molecules, which In contrast, intravenous injection elicited a Th2 type are potential targets for development of vaccines to control response, characterized by higher levels of IL-5 and high Leishmania infection [140] . humoral immune response, resulting in a less efficient Many studies have shown that different sand fly pro- protection [133] . Moreover, vaccination of mice with live/ teins from different species allow progression of infec- live parasite carrying trifusion gene (A2-CPA-CPB- tion, increase in parasite numbers in the animal host CTE) against L. infantum showed the same protective [141,142], and development of protection against Leish- effect as of heterologous DNA/live injection [115] . mania infection. After identifying the effectiveness of In another study, three recombinant Lactococcus lactis the salivary gland of the sand fly and its components strains were generated to express A2 and used as live vac- in 1998, many researchers have focused on further cines in order to induce specific immune responses against characterization of salivary proteins and used salivary L. donovani infection in BALB/c mice. L. lactis expressing gland homogenates in their experiments to stimulate Leishmania A2 protein at different subcellular locations the biological milieu of natural transmission. Different (cytoplasm, secreted outside the cell and anchored to the salivary proteins from Phlebotomus papatasi, the vector of cell wall) were tested as live bacterial vaccines against VL L. major, have been characterized [143]. The role of these caused by L. donovani infection in BALB/c mice. Com- proteins in immunization are different; for example: parison between three different patterns of intracellular SP44 caused exacerbation while SP15 led to protection expression of the cell demonstrated that the subcellular against L. major infection [143,144]. Some of the salivary

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Table 4. Nonpathogenic vectors used as live vaccine candidate against Leishmania. Deliver host Antigen Infection agent Immunization method Animal model Results Ref. Toxoplasma KMP-11 Lactococcus ip. BALB/c Protection. Ultimately not [135] gondii mutant major control Lactococcus L. tarentolae Lactococcus ip. BALB/c ↑ Activates the DC cell [130] tarentolae donovani maturation, ↑ IFN-g ↑ T-cell responses L. tarentolae HIV-1 Gag HIV-1 ip. BALB/c ↓ 75% virus replication, [131] protein ↓ memory specific CD4+ T lymphocytes, ↑ antibodies titers L. tarentolae A2 Lactococcus ip. BALB/c ↑ IFN-g, ↓ IL-5 [133] infantum iv. L. tarentolae HPV type 16 TC-1 tumor cells sc. C57BL/6 ↑ IgG2a , ↑ IFN-g, [129] E7 gene ↓ Tumor size L. tarentolae A2-CPA-CPB- L. infantum sc. in footpad BALB/c ↑ Th1 response, ↑ IFN-g, [115] CTE ↓ IL-10, ↑ NO, ↑ IgG1, ↑ IgG2a, ↓ Parasite burden Lactococcus A2 L. donovani sc. BALB/c ↑ Expression of A2 anchored to [134] lactis the cell wall, ↑ serum antibodies Influenza A LACK L. major ip. BALB/c ↑ Th1 response, ↑ IFN-g [124] viruses single peptide ↑ CD4+ T cell, ↓ Lesion size ↓ Parasite burden DC: Dendritic cell; HPV: Human papillomavirus; ip.: Intraperitoneal; iv.: Intravenous; sc.: Subcutaneous.

proteins could be potential targets for Leishmania infec- [64]. In 1993, FML was shown to be the most potent tion in animal models and vaccine development, either inhibitor of both promastigote and amastigote internal- alone or in combination with other antigens. However, ization, and to be present on the parasite surface during this potency is variable in different animals. For example, the vertebrate–host cycle. FML antigenic fraction was a protective effect of PpSP15 is confirmed in mice [143], but potent immunogen in rabbits [148] . The protective effect not in Rhesus monkeys [140] . In addition, immunization of this vaccine has been confirmed in different projects of mice with LPG combined with salivary gland lysates in Phase I–III trials on small laboratory animals (mouse failed to generate protection [145] . and hamster) and dogs in Brazil [64]. In Brazil, in Phase III So far, two sand fly salivary proteins, maxadi- trials it was shown that the number of deaths and symp- lan from Lutzomyia longipalpis and PpSP15 from toms derived from VL in dogs has decreased more than P. papatasi [143,144], have been characterized as protective 92% with a long-lasting protection, followed by reduced molecules against leishmaniasis. The maxadilan protein transmission of disease to humans [61,147]. can exacerbate the infection with L. major, but vaccina- Leish-Tec ® is the second commercial vaccine against tion against maxadilan could protect the mice against VL in dogs that was licensed in Brazil in 2008 [1] . This infection with L. major [146] . vaccine contains recombinant A2 antigens from VL- causing Leishmania species that caused VL, plus saponin The first licensed leishmania vaccines (or first as an adjuvant [85]. leishmaniasis vaccines approved) The third vaccine licensed in Europe in 2011 is ® To date, different vaccination strategies have failed to be CaniLeish [1] . It belongs to the second-generation of effective in human and animals. However, there are some vaccines and is within the excreted–secreted proteins of available licensed vaccines that are limited to be used only the supernatant of cultures of L. infantum plus QA21 against VL in dogs. (highly purified fraction of theQuilaja saponaria saponin) Leishmune vaccine contains recombinant FML antigen as adjuvant (LiESP/QA-21) [5,9,149]. isolated from L. donovani with saponin as adjuvant, which was the first licensed vaccine against canine visceral leish- Future perspective maniasis in Brazil since 2004 [1,59,61,147]. FML contains an During the past few years, vaccine study has undergone antigenic marker called NH36 (nucleoside hydrolase), great progression. Some vaccines developed are accept- which is a potential candidate for diagnosis and vaccine able for vaccination in animal models (mice and dogs)

1038 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes but could not help in the elimination and/or control of to act properly. The harmony between innate and adap- leishmnaiasis in humans or even dogs. The majority of tive immune response is highly important, although still known antigens have been checked in different modalities more knowledge is needed for better shaping the vaccine as a vaccine and shown contrastable protection. development. The main challenge is the difficulty in creating substantial long-term immunity in the host. Selection of Acknowledgments vaccine candidates is still problematic, although derived The authors thank A Mizbani for critically reading and editing the proteins from sand fly saliva could open a new therapeutic manuscript. avenue for the development of effective vaccine. The other main obstacle is to know and have deeper Financial & competing interested disclosure understanding of the host’s genetic background, virulence This project was supported by Pasteur Institute of Iran (Grant factors and specific components of sand fly vectors for number 653 to TT) and National Science Foundation of Iran designing a preventable strategy against leishmaniasis. (grant number 91053539 to SR). The authors have no other We need to know more information on how immune relevant affiliations or financial involvement with any organiza- systems react with non pathogenic Leishmania such as tion or entity with a financial interest in or financial conflict with L. tarentolae in comparison with pathogenic strains. We the subject matter or materials discussed in the manuscript. No need to know and establish different criteria to increase writing assistance was utilized in the production of this the immunogenicity of live attenuated/nonpathogenic manuscript. strains using different adjuvants. This strategy is highly important in HPB vaccination. One of the main crucial Ethical conduct of research steps is how the innate immunity is stimulated and in The authors state that they have obtained appropriate institutional fact how innate immunity can create a milieu for stron- review board approval or have followed the principles outlined in the ger and durable adaptive immune responses. It is highly Declaration of Helsinki for all human or animal experimental inves- important to manipulate the activation of T-regulatory tigations. In addition, for investigations involving human subjects, cells in order to control as well as helping other T cells informed consent has been obtained from the participants involved.

Executive summary Immunology focusing ■■Vaccine delivery systems are critical for elicited immune response and protection provided by specific antigens. ■■The CD8+ T cells, due to their essential role in mediating host defense, should be considered as an important target for vaccine development against Leishmania parasites. ■■Proper stimulation of innate immune responses are important for having proper milieu for adaptive immune response against Leishmania parasite infection. Prime–boost strategy ■■Administration of cocktail antigens through a suitable heterologous prime–boost system seems to be the best approach to create high-level protection. ■■Prime–boost strategy using DNA and parasite could present a powerful vaccine approach against leishmaniasis. Live vaccination ■■Proteins expressed by live nonpathogenic Leishmania are biologically active and close to the native protein, hence they could be the most powerful approach for vaccination against leishmaniasis. ■■Genetically attenuated or nonpathogenic Leishmania have gained further attention as an alternative in eukaryotic delivery systems. ■■The species-specific genes and virulence factors should be determined.

References Papers of special note have been highlighted as: 2 Silva-Almeida Basp M, Ribeiro-Guimarães predicting the future from past and present n of interest ML, Alves CR. Proteinases as virulence experience. J. Biomed. Res. 27(2), 85–102 n n of considerable interest factors in Leishmania spp. infection in (2013). mammals 5, 160 (2012). 1 Palatnik-de-Sousa CB, Day MJ. One health: . Parasit. Vectors 5 Palatnik-DE-Sousa CB. Vaccines for canine the global challenge of epidemic and endemic 3 Alvar J, Velez ID, Bern C et al. Leishmaniasis leishmaniasis. Front. Immunol. 3, 69 (2012). leishmaniasis. Parasit. Vectors 4, 197 (2011). worldwide and global estimates of its 6 Banuls AL, Hide M, Prugnolle F. Leishmania incidence. PLoS One 7(5), e35671 (2012). n n Outlines different ways to control and the leishmaniases: a parasite genetic leishmaniasis with WHO recommendations 4 Mutiso JM, Macharia JC, Kiio MN, update and advances in taxonomy, and global problems for research, diagnosis, Ichagichu JM, Rikoi H, Gicheru MM. epidemiology and pathogenicity in humans. therapy and vaccination. Development of Leishmania vaccines: Adv. Parasitol. 64, 1–109 (2007).

future science group Clin. Invest. (2013) 3(11) 1039 Review: Clinical Trial Outcomes Taheri & Rafati

7 Basyoni MM. Leishmania vaccines updates. 19 Innes EA, Bartley PM, Rocchi M et al. overview. Indian J. Med. Res. 123(3), PUJ 5(1), 1–10 (2012). Developing vaccines to control protozoan 423–438 (2006). 8 Evans KJ, Kedzierski L. Development of parasites in ruminants: dead or alive? Vet. 30 Giunchetti RC, Reis AB, Da Silveira-Lemos D vaccines against visceral leishmaniasis. Parasitol. 180, 155–163 (2011). et al. Antigenicity of a whole parasite vaccine as J. Trop. Med. 2012, 892817 (2012). 20 Alexander J, Bryson K. T helper (h)1/Th2 and promising candidate against canine leishmania: paradox rather than paradigm. leishmaniasis. Res. Vet. Sci. 85(1), 106–112 n Systematic review on different approaches Immunol. Lett. 99(1), 17–23 (2005). (2008). for Leishmania vaccination and summarization of advances in vaccination 21 Ritter U, Mattner J, Rocha JS, Bogdan C, 31 Bruhn KW, Birnbaum R, Haskell J. Killed but strategies against visceral leishmaniasis. Korner H. The control of Leishmania metabolically active Leishmania infantum as a (Leishmania) major by TNF in vivo is novel whole-cell vaccine for visceral 9 Moreno J, Vouldoukis I, Martin V, dependent on the parasite strain. Microbes leishmaniasis. Clin. Vaccine Immunol. 19, 490– McGahie D, Cuisinier AM, Gueguen S. Use Infect. 6(6), 559–565 (2004). 498 (2012). of a LiESP/QA-21 Vaccine (CaniLeish) stimulates an appropriate Th1-dominated 22 Stober CB, Lange UG, Roberts MT, 32 Noazin S, Modabber F, Khamesipour A et al. cell-mediated immune response in dogs. Alcami A, Blackwell JM. IL-10 from First generation leishmaniasis vaccines: a review PLoS Negl. Trop. Dis. 6, e1683 (2012). regulatory T cells determines vaccine efficacy of field efficacy trials. Vaccine 26, 6759–6767 in murine Leishmania major infection. (2008). 10 Kedzierski L. Leishmaniasis. Hum. Vaccin. J. Immunol. 175(4), 2517–2524 (2005). 7(11), 1204–1214 (2011). 33 Velez ID, Gilchrist K, Arbelaez MP et al. 23 Gurunathan S, Sacks DL, Brown DR et al. Failure of a killed Leishmania amazonensis 11 Garami A, Mehlert A, Ilg T. Glycosylation Vaccination with DNA encoding the vaccine against American cutaneous defects and virulence phenotypes of immunodominant LACK parasite antigen leishmaniasis in Colombia. Trans. R. Soc. Trop. Leishmania mexicana phosphomannomutase confers protective immunity to mice infected Med. Hyg. 99, 593–598 (2005). and dolicholphosphate-mannose synthase with Leishmania major. J. Exp. Med. 186(7), gene deletion mutants. Mol. Cell. Biol. 34 Armijos RX, Weigel MM, Calvopina M, 1137–1147 (1997). 21(23), 8168–8183 (2001). Hidalgo A, Cevallos W, Correa J. Safety, 24 Rafati S, Kariminia A, Seyde-Eslami S, immunogenecity, and efficacy of an autoclaved 12 Iborra S, Carrion J, Anderson C, Alonso C, Narimani M, Taheri T, Lebbatard M. Leishmania amazonensis vaccine plus BCG Sacks D, Soto M. Vaccination with the Recombinant cysteine proteinases-based adjuvant against new world cutaneous Leishmania infantum acidic ribosomal P0 vaccines against Leishmania major in BALB/c leishmaniasis. Vaccine 22, 1320–1326 (2004). protein plus CpG oligodeoxynucleotides mice: the partial protection relies on induces protection against cutaneous 35 Fernandez-Robledo JA, Vasta GR. Production interferon gamma producing CD8(+) T leishmaniasis in C57BL/6 mice but does not of recombinant proteins from protozoan lymphocyte activation. Vaccine 20(19–20), prevent progressive disease in BALB/c mice. parasites. Trends Parasitol. 26(5), 244–254 2439–2447 (2002). Infect. Immun. 73(9), 5842–5852 (2005). (2010). 25 Stager S, Rafati S. CD8(+) T cells in 13 Kubar J, Fragaki K. Recombinant DNA- 36 Coelho EA, Tavares CA, Carvalho FA et al. leishmania infections: friends or foes? Front. derived Leishmania proteins: from the Immune responses induced by the Leishmania Immunol. 3, 5 (2012). laboratory to the field. Lancet Infect. Dis. 5, (Leishmania) donovani A2 antigen, but not by + 107–114 (2005). n Description of important role of CD8 the LACK antigen, are protective against T-cells in human cutaneous and visceral experimental Leishmania (Leishmania) 14 Selvapandiyan A, Dey R, Gannavaram S et amazonensis infection. Infect. Immun. 71(7), al. Immunity to visceral leishmaniasis using leishmaniasis and its mechanism in cure or 3988–3994 (2003). genetically defined live-attenuated parasites. resistance to leishmaniasis. J. Trop. Med. 2012,631460 (2012). 26 Jayakumar A, Castilho TM, Park E, 37 Wolff JA, Malone RW, Williams P et al. Direct gene transfer into mouse muscle 15 Laskay T, Van Zandbergen G, Solbach W. Goldsmith-Pestana K, Blackwell JM, in vivo. Science 247(4949Pt 1), 1465–1468 Neutrophil granulocytes – Trojan horses for McMahon-Pratt D. TLR1/2 activation during (1990). Leishmania major and other intracellular heterologous prime–boost vaccination (DNA- microbes? Trends Microbiol. 11(5), 210–214 MVA) enhances CD8+ T-Cell responses 38 Bode C, Zhao G, Steinhagen F, Kinjo T, (2003). providing protection against Leishmania Klinman ADM. CpG DNA as a vaccine (Viannia). PLoS Negl. Trop. Dis. 5(6), e1204 adjuvant. Expert Rev. Vaccines 10(4), 499–511 16 Laskay T, Van Zandbergen G, Solbach W. (2011). (2011). Neutrophil granulocytes as host cells and transport vehicles for intracellular 27 Raman VS, Duthie MS, Fox CB, 39 Gurunathan S, Klinman DM, Seder RA. pathogens: apoptosis as infection-promoting Matlashewski G, Reed SG. Adjuvants for DNA vaccines: immunology, application, and factor. Immunobiology 213(3–4), 183–191 Leishmania vaccines: from models to clinical optimization. Annu. Rev. Immunol. 18, (2008). application. Front. Immunol. 3, 1–15 (2012). 927–974 (2000). 17 Ritter U, Frischknecht F, Van Zandbergen 28 Okwor I, Uzonna J. Persistent parasites and 40 Li L, Saade F, Petrovsky N. The future of G. Are neutrophils important host cells for immunologic memory in cutaneous human DNA vaccines. J. Biotechnol. 162, Leishmania parasites? Trends Parasitol. leishmaniasis: implications for vaccine designs 171–182 (2012). and vaccination strategies. Immunol. Res. 25(11), 505–510 (2009). n Extensive review on current state-of-the-art 41(2), 123–136 (2008). 18 Bogdan C. Natural killer cells in knowledge in human DNA vaccines experimental and human leishmaniasis. 29 Khamesipour A, Rafati S, Davoudi N, including its advantages and limitations, Front. Cell. Infect. Microbiol. 2, 69 (2012). Maboudi F, Modabber F. Leishmaniasis delivery systems and systems approaches to vaccine candidates for development: a global DNA vaccine design.

1040 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes

41 Rafati S, Ghaemimanesh F, Zahedifard F. novel Leishmania major recombinant protein 61 Borja-Cabrera GP, Correia Pontes NN, Da Comparison of potential protection induced encoded by members of a multicopy gene Silva VO et al. Long lasting protection against by three vaccination strategies (DNA/DNA, family. Infect. Immun. 66(7), 3279–3289 canine kala-azar using the FML-QuilA protein/protein and DNA/protein) against (1998). saponin vaccine in an endemic area of Brazil Leishmania major infection using Signal 51 Campos-Neto A, Porrozzi R, Greeson K et al. (Sao Goncalo do Amarante, RN). Vaccine Peptidase type I in BALB/c mice. Vaccine Protection against cutaneous leishmaniasis 20(27–28), 3277–3284 (2002). 24(16), 3290–3297 (2006). induced by recombinant antigens in murine 62 Palatnik-De-Sousa CB, De Figueiredo Barbosa 42 Iborra S, Soto M, Carrion J et al. The and nonhuman primate models of the human A, Oliveira SM et al. FML vaccine against Leishmania infantum acidic ribosomal protein disease. Infect. Immun. 69, 4103–4108 canine visceral leishmaniasis: from second- P0 administered as a DNA vaccine confers (2001). generation to synthetic vaccine. Expert Rev. protective immunity to Leishmania major 52 Skeiky YAW, Kennedy M, Kaufman D et al. Vaccines 7(6), 833–851 (2008). infection in BALB/c mice. Infect. Immun. LeIF: a recombinant Leishmania protein that 63 Da Silva VO, Borja-Cabrera GP, Correia 71(11), 6562–6572 (2003). induces an IL-12-mediated Th1 cytokine Pontes NN et al. A Phase III trial of efficacy of 43 Aguilar-Be I, Da Silva Zardo R, Paraguai De profile. J. Immunol. 161, 6171–6179 (1998). the FML-vaccine against canine kala-azar in Souza E et al. Cross-protective efficacy of a 53 Coler RN, Skeiky YA, Bernards K et al. an endemic area of Brazil (Sao Goncalo do prophylactic Leishmania donovani DNA Immunization with a polyprotein vaccine Amaranto, RN). Vaccine 19(9–10), 1082–1092 vaccine against visceral and cutaneous murine consisting of the T-cell antigens thiol-specific (2000). leishmaniasis. Infect. Immun. 73(2), 812–819 antioxidant, Leishmania major stress- 64 Santana DM, Borja-Cabrera GP, Paraguai De (2005). inducible protein 1, and Leishmania Souza E, Sturm NR, Palatnik De Sousa CB, 44 Mazumder S, Maji M, Das A, Ali N. Potency, elongation initiation factor protects against Campbell DA. Nucleoside hydrolase from efficacy and durability of DNA/DNA, DNA/ leishmaniasis. Infect. Immun. 70(8), Leishmania (L.) donovani is an antigen protein and protein/protein based vaccination 4215–4225 (2002). diagnostic for visceral leishmaniasis. Mol. using gp63 against Leishmania donovani in 54 Coler RN, Goto Y, Bogatzki L, Raman V, Biochem. Parasitol. 120(2), 315–319 (2002). BALB/c Mice. PLoS One 6(2), e14644 (2011). Reed SG. Leish-111f, a recombinant 65 Shi W, Schramm VL, Almo SC. Nucleoside 45 Nagill R, Kaur S. Vaccine candidates for polyprotein vaccine that protects against Hydrolase from Leishmania major. J. Biol. leishmaniasis: a review. Int. visceral Leishmaniasis by elicitation of CD4+ Chem. 274, 21114–21120 (1999). Immunopharmacol. 11, 1464–1488 (2011). T-cells. Infect. Immun. 75(9), 4648–4654 66 Beverley SM, Turco SJ. Lipophosphoglycan (2007). n Describes leishmaniasis and current (LPG) and the identification of virulence genes candidate approaches. Almost all antigens 55 Fujiwara RT, Vale AM, Al AE. in the prozoan parasite Leishmania. Trends with different regimes that are used for Immunogenicity in dogs of three Microbiol. 6, 35–40 (1998). vaccine strategy have been outlined. recombinant antigens (TSA, LeIF and 67 Descoteaux A, Avila HA, Zhang K, Turco SJ, LmSTI1) potential vaccine candidates for 46 Todoli F, Rodriguez-Cortes A, Nunez Mdel Beverley SM. Leishmania LPG3 encodes a canine visceral leishmaniasis. Vet. Res. 36, C et al. Head-to-head comparison of three GRP94 homolog required for phosphoglycan 827–838 (2005). vaccination strategies based on DNA and raw synthesis implicated in parasite virulence but insect-derived recombinant proteins against 56 Reed SG, Coler RN, Campos-Neto A. not viability. EMBO J. 21(17), 4458–4469 Leishmania. PLoS One 7(12), e51181 (2012). Development of a leishmaniasis vaccine: the (2002). importance of MPL. Expert Rev. Vaccines 47 Walker PS, Scharton-Kersten T, Rowton ED 68 Ilg T. Lipophosphoglycan is not required for 2(2), 239–252 (2003). et al. Genetic immunization with infection of macrophages or mice by glycoprotein 63 cDNA results in a helper T 57 Dumonteil E, Maria Jesus RS, Javier EO, Leishmania mexicana. EMBO J. 19(9), cell type 1 immune response and protection Maria Del Rosario GM. DNA vaccines 1953–1962 (2000). in a murine model of leishmaniasis. Hum. induce partial protection against Leishmania 69 Kamhawi S. Phlebotomine sand flies and Gene Ther. 9(13), 1899–1907 (1998). mexicana. Vaccine 21(17–18), 2161–2168 Leishmania parasites: friends or foes? Trends (2003). 48 Campbell SA, Alawa J, Doro B et al. Parasitol. 22(9), 439–445 (2006). Comparative assessment of a DNA and 58 Santos WR, Aguiar IA, Paraguai de Souza E, 70 Abdian N, Gholami E, Zahedifard F, protein Leishmania donovani gamma de Lima VM, Palatnik M, Palatnik-de- Safaee N, Rafati S. Evaluation of DNA/DNA glutamyl cysteine synthetase vaccine to cross- Sousa CB. Immunotherapy against murine and prime–boost vaccination using LPG3 protect against murine cutaneous experimental visceral leishmaniasis with the against Leishmania major infection in leishmaniasis caused by L. major or FML-vaccine. Vaccine 21, 4668–4676 susceptible BALB/c mice and its antigenic L. mexicana infection. Vaccine 30(7), (2003). properties in human leishmaniasis. Exp. 1357–1363 (2012). 59 Parra LE, Borja-Cabrera GP, Santos FN, Parasitol. 127(3), 627–636 (2011). 49 Sjolander A, Baldwin TM, Curtis JM, Souza LO, Palatnik-De-Sousa CB, Menz I. 71 Pral EM, Bijovsky AT, Balanco JM, Alfieri SC. Bengtsson KL, Handman E. Vaccination Safety trial using the Leishmune vaccine Leishmania mexicana: proteinase activities and with recombinant parasite surface antigen 2 against canine visceral leishmaniasis in megasomes in axenically cultivated amastigote- from Leishmania major induces a Th1 type of Brazil. Vaccine 25(12), 2180–2186 (2007). like forms. Exp. Parasitol. 77(1), 62–73 (1993). immune response but does not protect against 60 Santos FN, Borja-Cabrera GP, Miyashiro LM 72 Donnelly S, Dalton JP, Robinson MW. How infection. Vaccine 16(20), 2077–2084 (1998). et al. Immunotherapy against experimental pathogen-derived cysteine proteases modulate 50 Webb JR, Campos-Neto A, Ovendale PJ et al. canine visceral leishmaniasis with the saponin host immune responses. Adv. Exp. Med. Biol. Human and murine immune responses to a enriched-Leishmune vaccine. Vaccine 25(33), 712, 192–207 (2011). 6176–6190 (2007).

future science group Clin. Invest. (2013) 3(11) 1041 Review: Clinical Trial Outcomes Taheri & Rafati

73 McKerrow JH, Caffrey C, Kelly B, Loke P, protective immunity in murine visceral liposome enhances immune response and Sajid M. Proteases in parasitic diseases. Annu. leishmaniasis. Vaccine 24(12), 2169–2175 protection against leishmaniasis in immunized Rev. Pathol. 1, 497–536 (2006). (2006). BALB/c mice. Clin. Vaccine Immunol. 15(4), 74 Rafati S, Salmanian AH, Taheri T, Vafa M, 85 Fernandes AP, Costa MMS, Coelho EAF et al. 668–674 (2008). Fasel N. A protective cocktail vaccine against Protective immunity against challenge with 95 Jaafari MR, Badiee A, Khamesipour A et al. murine cutaneous leishmaniasis with DNA Leishmania (Leishmania) chagasi in beagle dogs The role of CpG ODN in enhancement of encoding cysteine proteinases of Leishmania vaccinated with recombinant A2 protein. immune response and protection in BALB/c major. Vaccine 19(25–26), 3369–3375 (2001). Vaccine 26, 5888–5895 (2008). mice immunized with recombinant major 75 Denise H, McNeil K, Brooks DR, 86 Zhang WW, Mendez S, Ghosh A et al. surface glycoprotein of Leishmania (rgp63) Alexander J, Coombs GH, Mottram JC. Comparison of the A2 gene locus in encapsulated in cationic liposome. Vaccine Expression of multiple CPB genes encoding Leishmania donovani and Leishmania major and 25(32), 6107–6117 (2007). cysteine proteases is required for Leishmania its control over cutaneous infection. J. Biol. 96 Zanin FHC, Coelho EAF, Tavares CAP et al. mexicana virulence in vivo. Infect. Immun. Chem. 278(37), 35508–35515 (2003). Evaluation of immune responses and protection 71(6), 3190–3195 (2003). 87 Azizi H, Hassani K, Taslimi Y, Najafabadi HS, induced by A2 and nucleoside hydrolase (NH) 76 Alexander J, Coombs GH, Mottram JC. Papadopoulou B, Rafati S. Searching for DNA vaccines against Leishmania chagasi and Leishmania mexicana cysteine proteinase- virulence factors in the non-pathogenic parasite Leishmania amazonensis experimental deficient mutants have attenuated virulence to humans Leishmania tarentolae. Parasitology infections. Microbes Infect. 9(9), 1070–1077 for mice and potentiate a Th1 response. 136(7), 723–735 (2009). (2007). J. Immunol. 161(12), 6794–6801 (1998). 88 Ghosh A, Labrecque S, Matlashewski G. 97 Fernandes AP, Costa MMS, Coelho EAF et al. 77 Mundodi V, Kucknoor AS, Gedamu L. Role Protection against Leishmania donovani Protective immunity against challenge with of Leishmania (Leishmania) chagasi amastigote infection by DNA vaccination: increased DNA Leishmania (Leishmania) chagasi in beagle dogs cysteine protease in intracellular parasite vaccination efficiency through inhibiting the vaccinated with recombinant A2 protein. survival: studies by gene disruption and cellular p53 response. Vaccine 19(23–24), Vaccine 26(46), 5888–5895 (2008). antisense mRNA inhibition. BMC Mol. Biol. 3169–3178 (2001). 98 Ghosh A, Zhang WW, Matlashewski G. 6, 3 (2005). 89 Ghosh A, Zhang WW, Matlashewski G. Immunization with A2 protein results in a 78 Mahmoudzadeh-Niknam H, McKerrow J. Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response Leishmania tropica: cysteine proteases are mixed Th1/Th2 and a humoral response which which protects mice against Leishmania essential for growth and pathogenicity. Exp. protects mice against Leishmania donovani donovani infections. Vaccine 20(1–2), 59–66 Parasitol. 106(3–4), 158–163 (2004). infections. Vaccine 20(1–2), 59–66 (2001). (2002). 79 De Souza Leao S, Lang T, Prina E, Hellio R, 90 Guha R, Das S, Ghosh J et al. Heterologous 99 Borja-Cabrera GP, Santos FB, Picillo E, Antoine JC. Intracellular Leishmania priming-boosting with DNA and vaccinia virus Gravino AE, Manna L, Palatnik-De-Sousa CB. amazonensis amastigotes internalize and expressing kinetoplastid membrane protein-11 Nucleoside hydrolase DNA vaccine against degrade MHC class II molecules of their host induces potent cellular immune response and canine visceral leishmaniasis. Procedia Vaccinol. cells. J. Cell Sci. 108, 3219–3231 (1995). confers protection against infection with 1, 104–109 (2009). 80 Mottram JC, North MJ, Barry JD, Coombs antimony resistant and sensitive strains of 100 Skeiky YAW, Coler RN, Brannon M et al. GH. A cysteine proteinase cDNA from Leishmania (Leishmania) donovani. Vaccine Protective efficacy of a tandemly linked, multi- Trypanosoma brucei predicts an enzyme with an 31(15), 1905–1915 (2013). subunit recombinant leishmanial vaccine ® unusual C-terminal extension. FEBS Lett. 91 Bhaumik S, Basu R, Sen S, Naskar K, Roy S. (Leish-111f ) formulated in MPL adjuvant. 258(2), 211–215 (1989). KMP-11 DNA immunization significantly Vaccine 20, 3292–3303 (2002). 81 Mottram JC, Robertson CD, Coombs GH, protects against L. donovani infection but 101 Campos-Neto A, Webb JR, Greeson K, Barry JD. A developmentally regulated cysteine requires exogenous IL-12 as an adjuvant for Coler RN, Skeiky YA, Reed SG. Vaccination proteinase gene of Leishmania mexicana. Mol. comparable protection against L. major. with plasmid DNA encoding TSA/LmSTI1 Microbiol. 6(14), 1925–1932 (1992). Vaccine 27(9), 1306–1316 (2009). leishmanial fusion proteins confers protection against Leishmania major infection in 82 Mottram JC, Frame MJ, Brooks DR et al. 92 Doroud D, Zahedifard F, Vatanara A, susceptible BALB/c mice. Infect. Immun. The multiple cpb cysteine proteinase genes Najafabadi AR, Rafati S. Cysteine proteinase 70(6), 2828–2836 (2002). of Leishmania mexicana encode isoenzymes type I, encapsulated in solid lipid nanoparticles that differ in their stage regulation and induces substantial protection against 102 Zadeh-Vakili A, Taheri T, Taslimi Y, Doustdari substrate preferences. J. Biol. Chem. Leishmania major infection in C57BL/6 mice. F, Salmanian AH, Rafati1 S. Bivalent DNA 272(22), 14285–14293(1997). Parasite Immunol. 33(6), 335–348 (2011). vaccination with genes encoding Leishmania major cysteine proteinases type I and II protects 83 Rafati S, Nakhaee A, Taheri T et al. Protective 93 Doroud D, Zahedifard F, Vatanara A et al. mice against infectious challenge. Iranian vaccination against experimental canine Delivery of a cocktail DNA vaccine encoding J. Biotechnol. 2(1), 35–43 (2004). visceral leishmaniasis using a combination of cysteine proteinases type I, II and III with solid DNA and protein immunization with cysteine lipid nanoparticles potentiate protective 103 Zadeh-Vakili A, Taheri T, Taslimi Y, Doustdari proteinases type I and II of L. infantum. immunity against Leishmania major infection. F, Salmanian AH, Rafati S. Immunization Vaccine 23(28), 3716–3725 (2005). J. Control. Release 153(2), 154–162 (2011). with the hybrid protein vaccine, consisting of Leishmania major cysteine proteinases Type I 84 Rafati S, Zahedifard F, Nazgouee F. Prime– 94 Badiee A, Jaafari MR, Samiei A, Soroush D, (CPB) and Type II (CPA), partially protects boost vaccination using cysteine proteinases Khamesipour A. Coencapsulation of CpG against leishmaniasis. Vaccine 22(15–16), type I and II of Leishmania infantum confers oligodeoxynucleotides with recombinant Leishmania major stress-inducible protein 1 in 1930–1940 (2004).

1042 www.future-science.com future science group Recombinant DNA vaccination & different approaches for vaccine development Review: Clinical Trial Outcomes

104 Doroud D, Zahedifard F, Vatanara A et al. in mice. Infect. Immun. 72(8), 4924–4928 123 Gonzalo RM, Real GD, Rodriguez JR et al. A C-terminal domain deletion enhances the (2004). heterologous prime–boost regime using DNA protective activity of cpa/cpb loaded solid 115 Saljoughian N, Taheri T, Zahedifard F et al. and recombinant vaccinia virus expressing the lipid nanoparticles against Leishmania major Development of novel prime–boost strategies Leishmania infantum P36/LACK antigen in BALB/c mice. PLoS Negl. Trop. Dis. 5(7), based on a tri-gene fusion recombinant L. protects BALB/c mice from cutaneous e1236 (2011). tarentolae vaccine against experimental murine leishmaniasis. Vaccine 20, 1226–1231 (2002). 105 Carneiro MW, Santos DM, Fukutani KF visceral leishmaniasis. PLoS Negl. Trop. Dis. 124 Kedzierska K, Curtis JM, Valkenburg SA et al. et al. Vaccination with L. infantum chagasi 7(4), e2174 (2013). Induction of protective CD4+ T cell-mediated nucleosomal histones confers protection immunity by a Leishmania peptide delivered in n n Novel study introducing a new prime–boost against new world cutaneous leishmaniasis recombinant influenza viruses. PLoS One 7(3), vaccine regimen using DNA cocktail in caused by leishmania braziliensis. 7(12), e33161 (2012). priming and recombinant Leishmania e52296 (2012). tarentolae expressing of Leishmanial antigens 125 Carson C, Antoniou M, Ruiz-Argüello MBN 106 Ramírez L, Santos DM, Souza AP et al. as fusion gene. et al. A prime/boost DNA/modified vaccinia Evaluation of immune responses and analysis virus Ankara vaccine expressing recombinant 116 Tewary P, Jain M, Sahani MH, Saxena S, of the effect of vaccination of the Leishmania Leishmania DNA encoding TRYP is safe and Madhubala R. A heterologous prime–boost major recombinant ribosomal proteins L3 or immunogenic in outbred dogs, the reservoir of vaccination regimen using ORFF DNA and L5 in two different murine models of zoonotic visceral leishmaniasis. Vaccine 27, recombinant ORFF protein confers protective cutaneous leishmaniasis. Vaccine 31(9), 1080–1086 (2009). immunity against experimental visceral 1312–1319 (2013). leishmaniasis. J. Infect. Dis. 191(12), 126 Gilbert SC. Clinical development of modified 107 Collin N, Gomes R, Teixeira C et al. Sand fly 2130–2137 (2005). vaccinia virus ankara vaccines. Vaccine salivary proteins induce strong cellular 31(39), 4241–4246 (2013). 117 Rafati S, Zahedifard F, Azari MK, Taslimi Y, immunity in a natural reservoir of visceral Taheri T. Leishmania infantum: prime boost 127 Maroof A, Brown N, Smith B et al. leishmaniasis with adverse consequences for vaccination with C-terminal extension of Therapeutic vaccination with recombinant leishmania. PLoS Pathog. 5(5), e1000441 cysteine proteinase type I displays both type 1 adenovirus reduces splenic parasite burden in (2009). and 2 immune signatures in BALB/c mice. experimental visceral leishmaniasis. J. Infect. 108 Goto Y, Bhatia A, Raman VS et al. KSAC, the Exp. Parasitol. 118(3), 393–401 (2008). Dis. 205(5), 853–863 (2012). first defined polyprotein vaccine candidate for 118 Khoshgoo N, Zahedifard F, Azizi H, 128 Pérez-Jiménez E, Kochan G, Gherardi MM, visceral leishmaniasis. Clin. Vaccine Immunol. Taslimi Y, Alonso MJ, Rafati S. Cysteine Esteban M. MVA-LACK as a safe and 18, 1118–1124 (2011). proteinase type III is protective against efficient vector for vaccination against 109 Parody N, Soto M, Requena JM, Alonso C. Leishmania infantum infection in BALB/c mice leishmaniasis. Microbes Infect. 8, 810–822 Adjuvant guided polarization of the immune and highly antigenic in visceral leishmaniasis (2006). humoral response against a protective individuals. Vaccine 26, 5822–5829 (2008). 129 Salehi M, Taheri T, Mohit E et al. multicomponent antigenic protein (Q) from 119 Ramiro MJ, Zarate JJ, Hanke T et al. Recombinant Leishmania tarentolae encoding Leishmania infantum . A CpG + Q mix Protection in dogs against visceral the HPV type 16 E7 gene in tumor mice protects Balb/c mice from infection. Parasite leishmaniasis caused by Leishmania infantum is model. Immunotherapy 4(11), 1107–1120 Immunol. 26(6–7), 283–93 (2004). achieved by immunization with a heterologous (2012). 110 Carcelén J, Iniesta V, Fernández-Cotrina J prime–boost regime using DNA and vaccinia 130 Breton M, Tremblay MJ, Ouellette M, et al. The Chimerical Multi-Component Q recombinant vectors expressing LACK. Vaccine Papadopoulou B. Live nonpathogenic protein from Leishmania in the absence of 21(19–20), 2474–2484 (2003). parasitic vector as a candidate vaccine against adjuvant protects dogs against an experimental 120 Dondji B, Pérez-Jimenez E, Goldsmith-Pestana visceral leishmaniasis. Infect. Immun. 73(10), Leishmania infantum infection. Vaccine 27, K, Esteban M, McMahon-Pratt D. 6372–6382 (2005). 5964-5973 (2009). Heterologous prime–boost vaccination with 131 Breton M, Zhao C, Ouellette M, 111 Molano I, Alonso MG, Miron C et al. A the LACK antigen protects against murine Tremblay MJ, Papadopoulou B. A Leishmania infantum multi-component visceral leishmaniasis. Infect. Immun. 73, recombinant non-pathogenic Leishmania antigenic protein mixed with live BCG confers 5286–5289 (2005). vaccine expressing human immunodeficiency protection to dogs experimentally infected 121 Ramos I, Alonso A, Marcen JM et al. virus 1 (HIV-1) Gag elicits cell-mediated with L. infantum. Vet. Immunol. Heterologous prime–boost vaccination with a immunity in mice and decreases HIV-1 Immunopathol. 92, 1–13 (2003). non-replicative vaccinia recombinant vector replication in human tonsillar tissue following 112 Woodland DL. Jump-starting the immune expressing LACK confers protection against exposure to HIV-1 infection. J. Gen. Virol. system: prime–boosting comes of age. Trends canine visceral leishmaniasis with a 88(Pt 1), 217–225 (2007). Immunol. 25, 98–104 (2004). predominant Th1-specific immune response. 132 Niimi T. Recombinant protein production in 113 Ivory C, Chadee K. DNA vaccines: designing Vaccine 26(3), 333–344 (2008). the eukaryotic protozoan parasite Leishmania strategies against parasitic infections. Genet. 122 Sánchez-Sampedro L, GóMez CE, Mejías- tarentolae: a review. Methods Mol. Biol. 824, Vaccines Ther. 2(1), 17 (2004). Pérez E, Sorzano COS, Esteban M. High 307–315 (2012). 114 Lange UG, Mastroeni P, Blackwell JM, Stober quality long-term CD4+ and CD8+ effector 133 Mizbani A, Taheri T, Zahedifard F et al. CB. DNA-Salmonella enterica serovar memory populations stimulated by DNA- Recombinant Leishmania tarentolae Typhimurium primer-booster vaccination biases LACK/MVA-LACK regimen in Leishmania expressing the A2 virulence gene as a novel towards T-helper 1 responses and enhances major BALB/c model of infection. PLoS One candidate vaccine against visceral protection against Leishmania major infection 7(6), e38859 (2012). leishmaniasis. Vaccine 28(1), 53–62 (2010).

future science group Clin. Invest. (2013) 3(11) 1043 Review: Clinical Trial Outcomes Taheri & Rafati

134 Yam KK, Hugentobler F, Pouliot P et al. 139 Uzonna JE, Spath GF, Beverley SM, Scott P. 145 Karanja R, Ingonga J, Mwangi M et al. Generation and evaluation of A2-expressing Vaccination with phosphoglycan-deficient Immunization with a combination of Lactococcus lactis live vaccines against Leishmania major protects highly susceptible Leishmania major lipophosphoglycan (LPG) Leishmania donovani in BALB/c mice. mice from virulent challenge without inducing and phlebotomus duboscqi salivary gland J. Med. Microbiol. 60, 1248–1260 (2011). a strong Th1 response. J. Immunol. 172(6), lysates (SGLs) abrogates protective effect of 3793–3797 (2004). LPG against L. major in BALB/c mice. Afr. 135 Ramı́Rez JR, Gilchrist K, Robledo S et al. Attenuated Toxoplasma gondii ts-4 mutants 140 Coutinho-Abreu IV, Ramalho-Ortigao M. J. Health Sci. 18, 1–5, (2011). engineered to express the Leishmania Ecological genomics of sand fly salivary gland 146 Morris RV, Shoemaker CB, David JR, antigen KMP-11 elicit a specific immune genes: an overview. J. Vector Ecol. 36(Suppl. 1), Lanzaro GC, Titus RG. Sandfly maxadilan response in BALB/c mice. Vaccine 20(3–4), S58–S63 (2011). exacerbates infection with Leishmania major 455–461 (2001). 141 Theodos CM, Ribeiro JM, Titus RG. Analysis and vaccinating against it protects against L. 136 Bolhassani A, Taheri T, Taslimi Y et al. of enhancing effect of sand fly saliva on major infection. J. Immunol. 167, 5226–5230 Fluorescent Leishmania species: Leishmania infection in mice. Infect. Immun. (2001). development of stable GFP expression and 59(5), 1592–1598 (1991). 147 Nogueira FS, Moreira MA, Borja-Cabrera GP its application for in vitro and in vivo 142 Belkaid Y, Kamhawi S, Modi G et al. et al. Leishmune vaccine blocks the studies. Exp. Parasitol. 127(3), 637–645 Development of a natural model of cutaneous transmission of canine visceral leishmaniasis: (2011). leishmaniasis: powerful effects of vector saliva absence of Leishmania parasites in blood, skin 137 Titus RG, Gueiros-Filho FJ, De Freitas LA, and saliva preexposure on the long-term and lymph nodes of vaccinated exposed dogs. Beverley SM. Development of a safe live outcome of Leishmania major infection in the Vaccine 23(40), 4805–4810 (2005). Leishmania vaccine line by gene mouse ear dermis. J. Exp. Med. 188(10), 148 Palatnik-De-Sousa CB, Dutra HS, Borojevic replacement. Proc. Natl Acad. Sci. USA 1941–1953 (1998). R. Leishmania donovani surface 92(22), 10267–10271 (1995). 143 Valenzuela JG, Belkaid Y, Garfield MK et al. glycoconjugate GP36 is the major 138 Spaeth GF, Lye LF, Segawa H, Turco SJ, Toward a defined anti-Leishmania vaccine immunogen component of the fucose- Beverley ASM. Identification of a targeting vector antigens: characterization of a mannose ligand (FML). Acta Trop. 53(1), compensatory mutant (lpg2REV) of protective salivary protein. J. Exp. Med. 194(3), 59–72 (1993). Leishmania major able to survive as 331–342 (2001). 149 Lemesre JL, Holzmuller P, Gonc Alves RB et amastigotes within macrophages without 144 Oliveira F, Lawyer PG, Kamhawi S, Valenzuela al. Long-lasting protection against canine LPG2-dependent glycoconjugates and its JG. Immunity to distinct sand fly salivary visceral leishmaniasis using the LiESAp-MDP significance to virulence and immunization proteins primes the anti-Leishmania immune vaccine in endemic areas of France: double- strategies. Infect. Immun. 72, 3622–3627 response towards protection or exacerbation of blind randomised efficacy field trial. Vaccine (2004). disease. PLoS Negl. Trop. Dis. 2, e226 (2008). 25, 4223–4234 (2007).

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