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Leishmania vaccines: from leishmanization to the era of DNA technology

Leishmania vaccines: from leishmanization to the era of DNA technology Volume 2 † Number 1 † March 2009 10.1093/biohorizons/hzp004 ......................................................................................................................................................................................................................................... Review Leishmania vaccines: from leishmanization to the era of DNA technology Naomi Dunning* School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK. * Corresponding author. Tel: þ44 7840358345. Email: naomi_dunning@hotmail.com Supervisor: Dr Selman Ali, School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK. ........................................................................................................................................................................................................................................ Leishmania are obligate intracellular vector-borne parasites that cause significant morbidity and mortality in many countries worldwide. There are several species of the parasite which vary according to geographical location and cause a variety of clinical syndromes ranging from self-limiting cutaneous lesions to potentially fatal infection of the viscera. The disease manifested is dependent on both the species of the parasite and the immune response of the host. Depending on the species of the parasite, resistance to infection is generally associated with a T-helper-1 immune response that activates macrophages to kill intracellular Leishmania in a nitric oxide-dependent manner. Conversely, disease progression is generally associated with a T-helper-2 response that activates humoral immunity. Chemotherapeutic treatments for leishmaniasis exist but are expensive, toxic and ineffective against resistant strains. A vaccine against leishmaniasis is feasible since most individuals that were once infected become resistant to clinical infection when later exposed. However, despite the wealth of information regarding the genetics of the parasite and the experimental immunology of the disease, there is currently no vaccine against Leishmania. A multitude of vaccine strategies have been pursued including the use of killed and genetically modified parasites. Immunization with naked plasmid DNA encoding Leishmania antigens represents a new approach to a Leishmania vaccine and confers several advantages over the more traditional vaccination methods. In order to develop an effective vaccine against leishmaniasis, it is important to understand the mechanisms of the immune response to Leishmania infection. This review discusses such immune mechanisms in detail and also explores several of the Leishmania vaccination strategies employed to date, with particular emphasis on DNA vaccines. Key words: Leishmania, leishmaniasis, immune response, vaccination. ........................................................................................................................................................................................................................................ Introduction Furthermore, available treatments are threatened by drug resistance, which is reviewed by Croft et al. Even with treat- Leishmaniasis has been identified as a category 1 disease by ment, various disease forms can cause lifelong disfigurement the World Health Organisation (WHO) and a rising cause and scarring. Thus, as with all infectious diseases, prevention for concern as an emerging disease with the advent of 1, 2 of leishmaniasis is superior to a cure. A prophylactic vacci- HIV-Leishmania coinfection. Leishmania spp. cause a nation would prove to be the most effective strategy to wide variety of diseases that range in severity from self- control infection and spreading of this group of diseases. healing cutaneous leishmaniasis to fatal disseminated visc- However, despite substantial effort spent in developing a eral leishmaniasis. The disease manifested is determined vaccine, there is currently no licensed vaccine against by both the species of Leishmania and the host immune human leishmaniasis. In order to develop an effective system, although certain species are associated with specific vaccine, it is important to understand the mechanisms of clinical conditions. For example, visceral leishmaniasis the immune response to Leishmania infection so that the usually results from infection with either L. donovani or vaccine can be engineered to induce a protective response L. infantum. rather than one that could result in susceptibility to the Although chemotherapeutic treatments for the leishma- parasite. niases exist, the drugs are costly, limited and toxic. ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 73 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 1. Antagonistic Th and Th responses that confer either resistance or susceptibility to Leishmania. APC, antigen-presenting cell; Th, T-helper cell; IL, 1 2 interleukin; TGF-b, transforming growth factor; TFN-a/b, tumour necrosis factor-a/b; IFN-g, interferon-gamma; Ig, immunoglobulin. receptor-mediated phagocytosis in a process which is The Immune Response to Leishmania crucial for the establishment of infection in the host. The infection primary mechanism for the elimination of Leishmania occurs upon activation of macrophages by IFN-g, secreted The Immune response to Leishmania infection is dependent by NK cells and T-helper-1 cells (Th ), which enables them on both the species of the parasite infecting the host and 1 to kill intracellular Leishmania amastigotes in a nitric the genetics of the host. The severity of disease caused by a oxide (NO)-dependent manner (Fig. 1). Studies have particular species may vary markedly between individuals; demonstrated that the inhibition of nitric oxide production hence, one species of Leishmania can cause more than one from inducible nitric oxide synthase (iNOS) (by clinical syndrome. There are several key components Leishmania) renders macrophages powerless against involved in the immune response to Leishmania. The Leishmania infection. outcome of infection is largely dependent on the ability of Opsonization, the process whereby a pathogen becomes the host to mount a protective T-helper-1 (Th ) response coated with antibody or complement to render it more versus the ability of the parasite to evade and manipulate readily ingested by phagocytic cells, is a protective immune the host’s immune system. response. However, studies have demonstrated that opsoni- Macrophages and effector molecules, dendritic cells (DC), zation of Leishmania major promastigotes with the third T-helper cells (CD4þ T cells), cytotoxic T cells (CD8þ T component of complement (C3) dramatically enhances cells), natural killer (NK) cells and cytokines are all, in one their survival inside macrophages. Providing that the para- way or another, considered to play important roles in the sitized macrophages do not become activated, they provide a immune response to Leishmania infection. safe breeding ground for Leishmania parasites which are Macrophages are phagocytic antigen-presenting cells capable of enhancing macrophage survival after infection. (APC) that play host to Leishmania when promastigotes Therefore, it is evident that macrophages play an important bind to their specific receptors and are internalized by ......................................................................................................................................................................................................................................... 74 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... role in both resistance and susceptibility to Leishmania Macatonia et al. demonstrated that while DC were infection. capable of induction and clonal expansion of T-helper Sanabria et al. emphasized the importance of the inter- cells, they were unable to induce T cell differentiation action between NK cells and DC in the immune response to towards Th or Th without IL-12 and IL-4, respectively. 1 2 Leishmania amazonensis. They found that NK cells pro- This emphasizes the importance of these cytokines in the moted the activation of L. amazonensis infected DC and immune response to Leishmania. Remarkably, the roles of that in turn, the activated DC stimulated NK activation. IL-12 and IL-4 in antagonistic Th and Th responses were 1 2 These findings imply differential roles of DC–NK cross-talk uncovered based upon observations with L. major. at various stages of Leishmania infection and present new The protective role of IL-12 has been demonstrated. In one insight on the interactions between innate immune com- study IL-12-deficient mice, originally derived from a strain ponents during infection with this parasite. genetically resistant to infection with L. major, were suscep- 4 22 Vanloubbeeck and Jones outlined the unique and central tible to infection with this parasite. However, role of DC in pathogen-specific immunity in that they present Vanloubbeeck and Jones found that promoting Th polariz- antigens to CD4þ T cells (T-helper cells) on MHC class II as ation of CD4þ T cells, using IL-12 as an adjuvant, was not well as presenting them to CD8þ T cells (cytotoxic T cells) enough to offer resistance to L. amazonensis. on MHC class I. This is known as cross-priming and is The role of IL-4 in disease progression has been implicated important because both CD8þ T cells and CD4þ T cells in several studies in which the administration of anti-CD4þ are considered to be involved in the immune defence and anti-IL-4 antibodies healed Leishmania infection. 4 23 against Leishmania. DC play an essential role in conferring Furthermore, Kopf et al. demonstrated that the disruption resistance or susceptibility to Leishmania by driving the of the IL-4 gene in susceptible BALB/c mice rendered them differentiation and proliferation of T-helper cells (CD4 þ ) resistant to infection with L. major, a finding that clearly to either T-helper-1 cells (Th ) or T-helper-2 cells (Th ). reveals the effects of this cytokine on disease progression. 1 2 On presentation of Leishmania antigens to CD4þ T cells, Figure 1 demonstrates the antagonistic Th and Th 1 2 the concomitant secretion of IL-12 drives the proliferation responses and the role of each cytokine in response to of (IFN-g secreting) Th cells and NK cells, which activate Leishmania infection. macrophages and inhibit Th responses. Conversely, the Cytotoxic T lymphocytes (CTL) are essential in the secretion of IL-4 during antigen presentation to CD4þ T defence against viruses yet their function with respect to cells drives Th cell development that inhibits Th responses intracellular parasites, such as Leishmania, remains some- 2 1 14 15 and promotes B lymphocyte growth and development what elusive. (Fig. 1). Therefore, it is evident that DC play an essential Experiments conducted using mice deficient in MHC class role in both initiation and regulation of antimicrobial II/CD4þ T cells and mice deficient in MHC class I/CD8þ T immune responses to Leishmania. cells have indicated that CD8þ T cells are not required for The role of Langerhans cells (LC), a specific subset of skin controlling Leishmania infection, as mice deficient in these DC located in the supra-basal epidermal layer, has recently cells were able to control the infection with L. major. This been revised by Ritter et al. Although they were previously was in contrast to MHC class II/CD4þ T cell deficient 17 8 thought to fulfil the same role as other DC, Ritter et al. mice that suffered fatal and uncontrolled infection. Since proposed that LC have a regulatory function and may be Leishmania reside in the parasitophorous vacuole within responsible for the suppression of the inflammatory response macrophages, the route of peptide loading onto MHC class against L. major. This is of great importance as it indicates I and the mechanism of CD8þ T cell activation remain that LC could induce Leishmania-specific immunosupression unclear. Therefore, it is tempting to speculate that these instead of parasite control and therefore has major impli- cells play only a minor role in immunity to Leishmania. cations against the use of LC in vaccine studies. However, activation of cytotoxic T cells may occur as a It has been postulated that the type of immune response to result of cross-priming, a phenomenon which enables DC Leishmania (Th versus Th ) is dependent on the type of to take up extracellular antigens and present them on 1 2 leishmanial antigen presented and recognized by T cells. MHC class I to CD8þ T cells. Therefore, Th responses may be initiated by different anti- The cytokine-producing capability of CTL may play a role gens to Th responses. However, various animal studies in the defence against Leishmania infection as they partici- have implicated that the same parasite epitope may induce pate through IFN-g production that is essential in the host 15 24 aTh response in animals with resolving infection or a Th defence against Leishmania. Muller et al. found that 1 2 response in others susceptible to the disease. CTL were involved in the elimination of L. major as well Many have observed that a Th response to Leishmania as establishment and maintenance of immunity as the inhi- infection, which inhibits inflammatory responses, renders bition of CTL with monoclonal antibodies rendered resistant the host more susceptible to disease and that, conversely, mice susceptible to L. major. Basu et al. demonstrated that 4, 5, 8, 14, 19, 20 resistance is associated with a Th response. CD8þ T cells are vital in a protective response against ......................................................................................................................................................................................................................................... 75 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... L. donovani following hybrid cell vaccination, as the exposed, provides the justification for vaccine development. depletion of these cells resulted in a higher parasite burden Although there is no licensed vaccine against any form of in the spleen and liver. Contrary to these studies, others leishmaniasis for general human use, a vaccine against differ- have shown that CD8þ T cells may contribute towards ent forms should, in theory, be possible. This is considering disease progression as large numbers of these cells have the abundance of genetic and biological information about been isolated in acute phase lesions and in the peripheral the parasite, clinical and experimental immunology of the blood. disease, and the availability of vaccines that offer protection A recent study by Ali et al. (personal communication, in experimental animals against challenge with different School of Science and Technology, NTU) has implicated Leishmania species. The leishmaniases are unique among that L. mexicana are capable of MHC class I downregula- parasitic diseases because a single vaccine could successfully tion. This is a significant finding and may perhaps explain prevent and treat disease and has the potential to protect why the role of CTL in the immune response to against more than one species (disease). To date, there Leishmania has been difficult to establish. have been numerous attempts at developing a successful Currently little is known about the role of cytotoxic T cells vaccine against leishmaniasis and there are several categories in the hosts defence against Leishmania infection. Therefore, of vaccine candidates. further investigation must be undertaken in order to fully Table 1 demonstrates the main categories of Leishmania establish their role. Like viruses, Leishmania are intracellular vaccines in development, according to Khamesipour et al., organisms; therefore, it is feasible that CTL could target and with some specific subcategories and examples of each. eliminate parasitized macrophages in the manner in which Live-Non-attenuated Vaccines they locate and eliminate virus-infected cells. ‘Leishmanization’, the deliberate inoculation of virulent Immune Evasion by Leishmania Leishmania from the exudate of a cutaneous lesion, is the Survival of Leishmania within the mammalian host depends oldest vaccination against cutaneous leishmaniasis and has on their ability to evade and manipulate the anti-leishmanial been practiced for centuries. This is because it has long immune response, thus enabling them to reside long enough been well established that recovery from cutaneous leishma- 19 30 to establish infection. Since Leishmania are intracellular niasis is followed by long lasting immunity to the disease. parasites, once inside the host cell, they evade the humoral Therefore, the parasite could be deliberately introduced to a immune response but become susceptible to attack by cell- concealed area of the body rather than risking multiple mediated immunity. However, despite the presentation of lesions to the face or other exposed regions. This approach Leishmania antigens and the initiation of inflammatory was later developed and live virulent L. major promastigotes responses, Leishmania can reside within the host tissues for were harvested and used in large-scale vaccination trials long periods by exploiting several mechanisms of during the 1970s and 1980s in Israel, Iran and the Soviet 26 27, 29 evasion. There are numerous strategies utilized by Union. Although still practiced in Uzbekistan, the Leishmania to survive within mammalian hosts, some of observation of adverse side effects, including the develop- which are common to other protozoan parasites such as ment of large persistent lesions, psoriasis and immunosup- Toxoplasma gondii and Trypanosoma cruzi. The various pression, led to the discontinuation of Leishmanization in strategies employed by the invading pathogens demonstrate many countries and the focus of vaccine development conse- 28, 29 how Leishmania parasites have evolved to successfully quently shifted towards killed organisms. evade the immune responses designed to target and destroy Nowadays, the development of a new vaccine must them. Such evasion strategies include the modulation of meet several strict criteria where safety, reproducibility macrophage function and the inhibition of antigen presen- and efficacy are of utmost importance. Although tation and T cell stimulation. Investigation of this parasite’s Leishmanization gave a high percentage of successful lesion interaction with the host’s immune system may lead to a development and subsequent immunity to L. major infec- better understanding of how Leishmania infection can be tion, it was neither reproducible nor safe. Furthermore, the resisted or the evasion strategies of these parasites which viability and infectivity of the injected parasites varied and influence susceptibility of the host. Improved understanding organisms without virulence induced delayed-type hypersen- of these areas should assist in the development of an effective sitivity. It is not surprising therefore that Leishmanization prophylactic vaccine, capable of inducing the specific is not recommended by WHO. immune responses required to battle this parasite. Another approach to live-non-attenuated vaccines has been taken by Breton et al. who used a non-pathogenic species, Leishmania tarentolae, to immunize mice. The Vaccination against Leishmaniasis high level of immunological cross-reactivity between Evidence that most individuals that were once infected with species at both the humoral and cellular level provides the Leishmania are resistant to clinical infections, when later rationale for using non-pathogenic Leishmania species to ......................................................................................................................................................................................................................................... 76 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... Table 1. Examples of vaccines engineered against leishmaniasis. Category Subcategory Principle Example Result ........................................................................................................................................................................................................................................ Live Leishmania Live virulent Vaccination with live, virulent Leishmanization (using L. major) † Life-long protection against (virulent or Leishmania L. major after cure attenuated) Live Vaccination with a live species that Leishmania tarentolae † L. tarentolae induced a non-pathogenic to is non-pathogenic to humans protective immune response humans against L. donovani Knock-out parasites Removal/blocking/replacement of DHFR-TS (enzyme gene) L. major † Survival of knock-out in parasite genes essential for knock-out mice for about 2 months survival without producing a lesion. † Conferred short-term protection against wild type Suicidal cassettes Genetic modification of parasite to L. major strains producing † Enhanced parasite killing induce suicide in response to biologically active † Delayed lesion external signals/to produce granulocyte-macrophage development in susceptible biological substances that activate colony-stimulating factor (GM-CSF) BALB/c mice immune attack against them Killed Leishmania Whole killed A single species of killed Killed single strain of † Significant protection from or fractions parasite promastigotes or more than one L. amazonensis natural infection species † Long lasting Th responses Whole killed Use of whole dead promastigotes Killed L. mexicana/L. braziliensis † Used parasite and and an adjuvant to stimulate promastigotes and BCG immunotherapeutically it adjuvant immune response induced a high cure rate even in severe cases † Cure accompanied by development of Th immune response Fraction of a killed Use of Leishmania antigens and an Fructose Mannose Ligand (FML) † First vaccine against canine parasite and adjuvant to stimulate an immune antigen from surface of parasite and visceral leishmaniasis w 39 adjuvant response saponin adjuvant (Leishmune ) † Potential transmission-blocking vaccine Recombinant DNA vaccination Introduction of bacterial plasmid Multi-antigenic DNA vaccine † Unable to induce and synthetic DNA encoding antigens into host encoding KMPII, TRYP, LACK and protection in dogs against cells in vivo CP63 L. infantum DNA vaccine against parasite † Production of IgG1 and enzyme gamma-glutamylcysteine IgG2a synthetase (g-GCS) † Increased cell-mediated immunity Recombinant Vaccination with recombinant Recombinant hydrophilic acylated † Protection against protein vaccination proteins surface protein B1 (HASPB1) experimental challenge with L. donovani 32 31 immunize against virulent species. Breton et al. believe it parasites to render them avirulent offers a safer, more is a promising approach to vaccination against visceral leish- stable approach to live vaccination. maniasis caused by L. donovani as BALB/c mice were able Live-Attenuated Vaccines to elicit a protective immune response after only a single per- itoneal vaccination. Although the results to this study appear Genetic modification of Leishmania parasites to reduce viru- promising, vaccination with live-non-attenuated parasites is lence, yet maintain immunogenicity, is of current interest in not an appealing prospect and the attenuation of live Leishmania vaccine research. It is an appealing approach as ......................................................................................................................................................................................................................................... 77 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... attenuated parasites closely mimic natural infection that may America have used L. amazonensis autoclaved lysate or a lead to similar immune responses without the danger associ- mixture of native species, whereas L. major is used in most 29 35 ated with infection with live virulent parasites. Due to vaccine studies against Old World leishmaniasis. In advances in molecular biology and the genomic sequencing Venezuela, Convit and his colleagues introduced an auto- of L. major Friedlin, the attenuation of Leishmania parasites claved L. mexicana þ BCG for immunotherapy and/or by removing, blocking or replacing essential genes is a possi- immunochemotherapy, which proved unsuccessful as the 28, 32 bility. Such parasites are referred to as ‘knock-out para- results were either inconclusive or demonstrated low protec- sites’. Leishmania can be engineered without the genes tion induced by the vaccine. Similarly, killed Leishmania required for long-term survival in the host, thus making vaccines failed to confer protection in persons in the 29 29 them safe to immunize with. The first Leishmania knock- Middle East. However, studies in the 1980s revolutionized out was a dihydrofolate reductase thymidylate synthase the use of killed Leishmania as a vaccine by demonstrating gene (DHFR-TS) in L. major. This knock out induced signifi- excellent protection in mice injected intravenously or intra- cant, but temporary, protection in mice when challenged peritoneally (but not subcutaneously) and subsequently with the wild type but gave disappointing results during several formulations of killed vaccines have been devel- 27 27 further studies in monkeys. The production of oped. This emphasizes that the site of administration Leishmania knock-outs can identify essential virulence affects the efficacy of a vaccine; thus, it is therefore important genes. Therefore, as well as providing vaccine candidates to investigate the most effective method of inoculation. In these studies can lead to a better understanding of the Venezuela, autoclaved L. mexicana is currently used immu- parasite. Furthermore, studies using knock-out parasites notherapeutically to treat patients with cutaneous may be key in identifying specific genes that can then leishmaniasis. be applied to DNA vaccines. For example, work by In countries with a rudimentary biotechnology industry 33, 34 Selvapandiyan et al., which highlighted the importance and where a cold-chain distribution of vaccines is not poss- of the centrin proteins for the duplication and cell cycle ible, autoclaving of the killed vaccine is the recommended progression of Leishmania amastigotes, provided the justi- method of sterilization and preservation. However, as fication for the use of the centrin genes as DNA vaccine demonstrated by De Luca et al., autoclaving lowers the candidates by Ali et al. (2008, personal communication). immunogenicity of the parasite by destroying most of the Another approach to attenuating Leishmania parasites is proteins. Therefore, while offering a safer and more stable the addition of suicide cassettes that lead to the death of alternative to live vaccination, vaccination with killed the parasite in response to external stimuli. An example Leishmania does not mimic natural infection and is also of which is the introduction of drug-sensitive genes such as less immunogenic. Saccharomyces cerevisiae cytosine deaminase gene which is sensitive to 5-fluorocytosine. It has been suggested that Vaccines of Leishmania Fractions parasites carrying drug-sensitive cassettes could provide Leishmune , the first vaccine against canine visceral leishma- suitable candidates for Leishmanization as an effective treat- niasis, consists of a purified L. donovani fraction, named ment of non-resolving lesions could be guaranteed. fructose mannose ligand (FML) and a saponin adjuvant. Alternatively, parasites can be modified to produce biological FML has been characterized as a major antigenic complex substances that activate immune attack, such as granulocyte of L. donovani and the main antigen in this complex is macrophage colony stimulating factor (GM-CSF). NH36, an essential enzyme involved in the construction of Attenuated vaccines offer a novel approach to immuniz- 35 w the parasite’s DNA. Leishmune is considered a promising ation against leishmaniasis however, there are fears that the tool for the prevention of canine visceral leishmaniasis and parasite may revert back to a virulent form. Furthermore, furthermore its potential as a transmission-blocking vaccine targeted deletion of essential virulence genes can result in is promising for the control of zoonotic visceral complete destruction of the parasite or mutants that only leishmaniasis. delay lesion development. Such problems are not a concern with the use of killed parasites for vaccine Recombinant Protein Vaccines candidates. Recombinant protein vaccines are produced by genetically Killed Vaccines engineered cells to produce foreign genes that encode anti- Killed Leishmania is an appealing vaccine candidate in terms genic proteins. They are relatively new and can be adminis- of its stable biochemical composition and antigenicity, low tered as purified proteins or as bacteria manufacturing the 37 29 cost and safety. There have been several studies on killed proteins in situ. Various proteins have been tested as poss- Leishmania, with or without adjuvants, as a vaccine. Early ible vaccine candidates including recombinant hydrophilic trials began in Brazil during the 1940s; however, these acylated surface protein B1 (HASPB1) that conferred protec- 29 40 yielded conflicting results. Most killed vaccine studies in tion against experimental challenge with L. donovani. ......................................................................................................................................................................................................................................... 78 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... Novel Vaccine Strategies Therefore, the immune response to the antigen encoded on the plasmid can be geared towards one that will confer resist- To date, there have been many attempts at designing an ance to the parasite. effective vaccine against Leishmaniasis, most of which have DNA vaccines may provide better protection against fallen into the categories previously described. However, in Leishmania than killed or live-attenuated vaccines as they addition to these there have been several novel approaches can induce the expression of Leishmania antigens, which that have also proven to be promising. Such approaches are unaltered in their protein structure and antigenicity. include the use of DC pulsed with Leishmania antigens and 16, 35 Furthermore, bacteria-derived DNA plasmids are naturally vaccines comprised of components of sand fly saliva. immunogenic as their backbones contain unmethylated In light of their crucial role in immunity to Leishmania, cytosine-phosphate-guanosine (CpG) motifs which have recent studies have implicated DC as potential vaccine candi- been shown to readily induce Th cytokine expression and dates serving as vectors for Leishmania antigens. Moll and 42, 43 enhance CD8þ T cell responses. This adjuvant property Berberich observed that DC pulsed ex vivo with L. major is of great use for a Leishmania vaccine as these motifs would antigen induced protection, in otherwise susceptible mice, ensure the induction of cell-mediated immunity which is against live challenge with L. major. known to confer protection against the parasite. Since sand fly saliva is documented to enhance the infec- tivity of Leishmania, vaccines have been engineered against Administration of DNA Vaccines its components. One such component is the SP15 antigen Several methods of DNA vaccine administration have been that was shown to induce significant resistance in mice to tested and it is thought that the method and site of immuniz- challenge with L. major. ation may influence that nature of the immune response eli- cited. Many studies have observed variation in vaccine DNA Vaccination efficacy depending on the site and method of administration The concept of DNA vaccination is relatively new and was despite the use of the same DNAvaccine. To date, successful established by Wolff et al. in 1990 when direct intramuscular DNA vaccination has been achieved using a number of differ- injection of plasmid DNA, encoding reporter genes, resulted ent routes including intramuscular, intravenous, intraepider- 41 –43 in expression of the proteins in myocytes. Their study mal, intraperitoneal, intravaginal, intranasal, intrasplenic, demonstrated that purified recombinant nucleic acids could intrahepatic, subcutaneous and oral. Of these routes, intra- 43, 45 be delivered to cells in vivo to direct protein expression muscular administration is most commonly employed. 32 45 endogenously. DNA vaccines consist of antigenic proteins However, Me´ndeza et al. demonstrated that the dose of encoded on naked plasmid DNA vectors that allow their DNA vaccine required to induce full protection in C57BL/6 3, 5 expression in eukaryotic cells. They represent a promising mice against L. major was five times smaller using the gene approach to vaccine development and have been shown to gun (particle-mediated epidermal delivery) than subcu- generate protective responses against infectious diseases taneous or intramuscular injection. Furthermore, Ali et al. 44, 45 such as leishmaniasis. (personal communication) found that 1 mg of DNA encoding DNA vaccines present a multitude of advantages over other L. mexicana gp63, administered using a gene gun, conferred vaccine strategies and several features have made them an more protection in susceptible BALB/c mice than 100 mgof appealing alternative. Not only are they fast, simple and vaccine administered intramuscularly. These findings clearly cheap to produce on a large scale, they are also temperature demonstrate that DNA vaccine efficacy is higher when admi- stable making storage and transport easier and cheaper as nistered using a gene gun than it is when administered intra- 44, 35 there is no need for a cold-chain of distribution. This is muscularly. This may be because gene gun administration an important advantage for a Leishmania vaccine candidate can directly transfect APC, such as DC, with the plasmid considering that leishmaniasis is a major health concern in DNA. Although myocytes are capable of inducing CTL many developing countries. DNA vaccines provide an extre- responses by presenting peptides (from the encoded antigen) mely flexible method of vaccination as a single plasmid can via MHC class I molecules, unlike APC they are not pro- encode several antigens and multiple plasmids, encoding fessional antigen presenters and do not carry the 45, 46 different antigens, can be delivered in a single adminis- co-stimulatory molecules required to prime T-cells. tration. Therefore, DNA vaccination could potentially Possible Modes of Action of DNA Vaccines provide protection against more than one species. As leishma- niasis can be caused by several different species, this is another Figure 2 illustrates some of the possible immune mechanisms feature that makes DNA vaccination an appealing prospect. induced by gene gun delivery of DNA vaccines. The gene gun Genetic vaccination has been shown to induce both fires DNA-coated gold particles at high speed directly into humoral and cellular immune responses and these responses cells of the epidermis, which include skin cells, LC and can be tailored through modifications of the vector or incor- dermal DC. Upon entry into the cells, the plasmid is trans- poration of cytokine genes with adjuvant properties. ported to the nucleus where the encoded gene is transcribed ......................................................................................................................................................................................................................................... 79 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 2. Possible immune mechanisms induced by particle-mediated epidermal delivery of a Leishmania DNA vaccine (adapted from Encke et al. ). and the protein is subsequently produced, processed into Rodriguez-Cortes et al. found that their multiantigenic peptides by host proteases and then presented in the DNA vaccine encoding KMII, TRYP, LACK and Gp63 did context of MHC class I which then stimulates CD8þ T lym- not protect dogs against L. infantum experimental challenge, phocytes. DC directly transfected with the DNA vaccine despite the hypothesis that an effective immune response was can prime CD8þ T cells by presenting the DNA encoded more likely to be generated following exposure to more than 4 48 antigen in the context of MHC class I. Conversely, there one antigen. Conversely, Carter et al. found that intramus- is evidence to suggest that immature DC can endocytose cular DNA vaccination against the parasite enzyme gamma- soluble proteins and debris from apoptotic transfected cells glutamylcysteine synthetase conferred protection against and express the coded antigen through MHC class I and/ L. donovani in BALB/c mice. The results of these studies 4, 46 or MHC class II after differentiating into mature DC. indicate that multiantigenic vaccines do not necessarily Therefore, DNA vaccination can result in the stimulation confer better protection against Leishmania infection. of both CD4þ T cell and CD8þ T cell populations. The However, these studies used different species of Leishmania unique ability of DC to present extracellular antigens in so it is not possible to directly compare the findings. the context of MHC class I and MCH class II is known as cross-priming, as a result of this phenomenon it is likely Conclusion that DC play a key role in the induction of both humoral and cell-mediated immunity following DNA vaccination. Leishmaniasis is a major cause of morbidity and mortality There have been several studies conducted on potential worldwide. An effective vaccine has the potential to DNA vaccines against Leishmania. Handman et al. control this disease. However, despite great effort, there is demonstrated that DNA vaccines can be used therapeutically currently no licensed vaccine available. To date, there have to treat cutaneous leishmaniasis caused by L. major in both been numerous attempts at developing a successful vaccine genetically resistant C3H/He mice and susceptible BALB/c against leishmaniasis and Table 1 demonstrates only a few mice. This is an important finding, which demonstrates of these. Further work in this field should not only enable that DNA vaccines may have a role to therapeutically cure the improvement of current vaccine strategies but it is disease in both susceptible and resistant individuals. hoped the development of novel methodologies will ......................................................................................................................................................................................................................................... 80 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... 21. Macatonia S, Hsieh C, Murphy K et al. (1993) Dendritic cells and quickly follow suit. Immunization with plasmid DNA encod- macrophages are required for Th1 development of CD4þ T cells from ing Leishmania antigens represents a promising approach to alpha beta TCR transgenic mice: IL-12 substitution for macrophages to vaccination against leishmaniasis in that it has intrinsic adju- stimulate IFN-gamma production is IFN-gamma-dependent. Int Immunol 9: vant properties, induces both humoral and cell-mediated 1119–1128. immune responses and results in long lasting immunity. In 22. Mattner F, Di Padova K, Alber G (1997) Interleukin-12 is indispensable light of its many advantages over other Leishmania vaccine for protective immunity against Leishmania major. Infect Immun 11: 4378–4383. strategies, DNA vaccination could prove to be the best 23. Kopf M, Brombacher F, Khler G et al. (1996) IL-4-deficient Balb/c mice resist approach to both treat and prevent human leishmaniasis. infection with Leishmania major. J Exp Med 184: 1127–1136. 24. Muller I, Pedrazzlni T, Kropf P et al. 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Khamesipour A, Rafati S, Davoudi N et al. (2006) Leishmaniasis vaccine and the implications for vaccine development. Ann N Y Acad Sci 1026: candidates for development: a global overview. Indian J Med Res 123: 267–272. 423–438. 5. Murray H, Berman J, Davies C et al. (2005) Advances in leishmaniasis. Lancet 28. Coler R, Reed S (2005) Second-generation vaccines against leishmaniasis. 366: 1561–1577. Trends Parasitol 21(5): 244–249. 6. Croft S, Sundar S, Fairlamb H (2006) Drug resistance in leishmaniasis. Clin 29. Handman E (2001) Leishmaniasis: current status of vaccine development. Microbiol Rev 19: 111–126. Clin Microbiol Rev 14(2): 229–243. 7. Rodriguez-Cortes A, Ojeda A, Lopez-Fuertez L et al. (2007) Vaccination with 30. Bray R, Modabber F (2000) The History of Leishmaniasis. New York and plasmid DNA encoding KMPII, TRYP, LACK, and GP63 does not protect dogs London: Hodder Arnold Publisher. against Leishmania infantum experimental challenge. Vaccine 25: 31. 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Von Stebut E, Udey M (2004) Requirements for Th1-dependent immunity ment in mice. Am Soc Microbiol 71: 6499–6509. against infection with Leishmania major. Microbes Infect 6: 1102–1109. 37. Giunchetti R et al. (2008) Antigenicity of a whole parasite vaccine as prom- 15. Ruiz J, Becker I (2007) CD8 cytotoxic T cells in cutaneous leishmaniasis. ising candidate against canine leishmaniasis. Res Vet Sci 1: 106–112. Parasite Immunol 29: 671–678. 38. De Luca P, Mayrink W, Alves C et al. (1999) Evaluation of the stability and 16. Moll H, Berberich C (2001) Dendritic cell-based vaccination strategies: induc- immunogenicity of autoclaved and non-autoclaved preparations of a tion of protective immunity against Leishmaniasis. Immunobiology 204: vaccine against American tegumentary Leishmaniasis. Vaccine 17: 659–666. 1179–1185. 17. Ritter U et al. (2004) CD8a- and Langerin-negative dendritic cells, but not 39. Dantas-Torres F (2006) Leishmune vaccine: the newest tool for prevention Langerhans cells, act as principal antigen-presenting cells in leishmaniasis. and control of canine visceral leishmaniosis and its potential as a Eur J Immunol 34: 1542–1550. transmission-blocking vaccine. Vet Parasitol 141: 1–8. 18. Moreno J (2007) Changing views on Langerhans cell functions in 40. Stager S, Smith D, Kaye P (2000) Immunization with a recombinant stage- Leishmaniasis. Trends Parasitol 23(3): 86–88. regulated surface protein from Leishmania donovani induces protection 19. Bogdan C, Rollinghoff M (1998) The immune response to Leishmania: against visceral Leishmaniasis. J Immunol 165: 7064–7071. mechanisms of parasite control and evasion. Int J Parasitol 28: 121–134. 41. Wolff J, Malone R, Williams P et al. (1990) Direct gene transfer into mouse 20. Alexander J, Bryson K (2005) T helper (h)1/Th2 and Leishmania: paradox muscle in vivo. Science 247(4949): 1465–1468. rather than paradigm. Immunol Lett 99: 17–23. ......................................................................................................................................................................................................................................... 81 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... 42. Ivory C, Chadee K (2004) DNA vaccines: designing strategies against parasitic 46. Donnelly J, Liu M, Ulmer J (2000) Antigen presentation and DNA vaccines. infections. Genet Vaccines Ther 2: 17. Am J Respir Crit Care Med 162: S190–S193. 43. Garmory H, Brown K, Titball R (2003) DNA vaccines: improving expression of 47. Handman E, Noormohammadib E, Curtisa J et al. (2000) Therapy of antigens. Genet Vaccines Ther 1: 2. murine cutaneous leishmaniasis by DNA vaccination. Vaccine 18: 3011–3017. 44. Encke J, Putlitza J, Wandsa J (1999) DNA Vaccines. Intervirology 42: 117–124. 48. Carter K, Henriquez F, Campbell S et al. (2007) DNA vaccination against the parasite enzyme gamma-glutamylcysteine synthetase 45. Me´ndeza S, Belkaid Y, Seder R et al. (2002) Optimization of DNA vaccination confers protection against Leishmania donovani infection. Vaccine 25: against cutaneous leishmaniasis. Vaccine 20: 3702–3708. 4502–4509. ........................................................................................................................................................................................................................................ Submitted on 30 September 2008; accepted on 21 January 2009; advance access publication 17 February 2009 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Leishmania vaccines: from leishmanization to the era of DNA technology

Bioscience Horizons , Volume 2 (1) – Mar 17, 2009

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Volume 2 † Number 1 † March 2009 10.1093/biohorizons/hzp004 ......................................................................................................................................................................................................................................... Review Leishmania vaccines: from leishmanization to the era of DNA technology Naomi Dunning* School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK. * Corresponding author. Tel: þ44 7840358345. Email: naomi_dunning@hotmail.com Supervisor: Dr Selman Ali, School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK. ........................................................................................................................................................................................................................................ Leishmania are obligate intracellular vector-borne parasites that cause significant morbidity and mortality in many countries worldwide. There are several species of the parasite which vary according to geographical location and cause a variety of clinical syndromes ranging from self-limiting cutaneous lesions to potentially fatal infection of the viscera. The disease manifested is dependent on both the species of the parasite and the immune response of the host. Depending on the species of the parasite, resistance to infection is generally associated with a T-helper-1 immune response that activates macrophages to kill intracellular Leishmania in a nitric oxide-dependent manner. Conversely, disease progression is generally associated with a T-helper-2 response that activates humoral immunity. Chemotherapeutic treatments for leishmaniasis exist but are expensive, toxic and ineffective against resistant strains. A vaccine against leishmaniasis is feasible since most individuals that were once infected become resistant to clinical infection when later exposed. However, despite the wealth of information regarding the genetics of the parasite and the experimental immunology of the disease, there is currently no vaccine against Leishmania. A multitude of vaccine strategies have been pursued including the use of killed and genetically modified parasites. Immunization with naked plasmid DNA encoding Leishmania antigens represents a new approach to a Leishmania vaccine and confers several advantages over the more traditional vaccination methods. In order to develop an effective vaccine against leishmaniasis, it is important to understand the mechanisms of the immune response to Leishmania infection. This review discusses such immune mechanisms in detail and also explores several of the Leishmania vaccination strategies employed to date, with particular emphasis on DNA vaccines. Key words: Leishmania, leishmaniasis, immune response, vaccination. ........................................................................................................................................................................................................................................ Introduction Furthermore, available treatments are threatened by drug resistance, which is reviewed by Croft et al. Even with treat- Leishmaniasis has been identified as a category 1 disease by ment, various disease forms can cause lifelong disfigurement the World Health Organisation (WHO) and a rising cause and scarring. Thus, as with all infectious diseases, prevention for concern as an emerging disease with the advent of 1, 2 of leishmaniasis is superior to a cure. A prophylactic vacci- HIV-Leishmania coinfection. Leishmania spp. cause a nation would prove to be the most effective strategy to wide variety of diseases that range in severity from self- control infection and spreading of this group of diseases. healing cutaneous leishmaniasis to fatal disseminated visc- However, despite substantial effort spent in developing a eral leishmaniasis. The disease manifested is determined vaccine, there is currently no licensed vaccine against by both the species of Leishmania and the host immune human leishmaniasis. In order to develop an effective system, although certain species are associated with specific vaccine, it is important to understand the mechanisms of clinical conditions. For example, visceral leishmaniasis the immune response to Leishmania infection so that the usually results from infection with either L. donovani or vaccine can be engineered to induce a protective response L. infantum. rather than one that could result in susceptibility to the Although chemotherapeutic treatments for the leishma- parasite. niases exist, the drugs are costly, limited and toxic. ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 73 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 1. Antagonistic Th and Th responses that confer either resistance or susceptibility to Leishmania. APC, antigen-presenting cell; Th, T-helper cell; IL, 1 2 interleukin; TGF-b, transforming growth factor; TFN-a/b, tumour necrosis factor-a/b; IFN-g, interferon-gamma; Ig, immunoglobulin. receptor-mediated phagocytosis in a process which is The Immune Response to Leishmania crucial for the establishment of infection in the host. The infection primary mechanism for the elimination of Leishmania occurs upon activation of macrophages by IFN-g, secreted The Immune response to Leishmania infection is dependent by NK cells and T-helper-1 cells (Th ), which enables them on both the species of the parasite infecting the host and 1 to kill intracellular Leishmania amastigotes in a nitric the genetics of the host. The severity of disease caused by a oxide (NO)-dependent manner (Fig. 1). Studies have particular species may vary markedly between individuals; demonstrated that the inhibition of nitric oxide production hence, one species of Leishmania can cause more than one from inducible nitric oxide synthase (iNOS) (by clinical syndrome. There are several key components Leishmania) renders macrophages powerless against involved in the immune response to Leishmania. The Leishmania infection. outcome of infection is largely dependent on the ability of Opsonization, the process whereby a pathogen becomes the host to mount a protective T-helper-1 (Th ) response coated with antibody or complement to render it more versus the ability of the parasite to evade and manipulate readily ingested by phagocytic cells, is a protective immune the host’s immune system. response. However, studies have demonstrated that opsoni- Macrophages and effector molecules, dendritic cells (DC), zation of Leishmania major promastigotes with the third T-helper cells (CD4þ T cells), cytotoxic T cells (CD8þ T component of complement (C3) dramatically enhances cells), natural killer (NK) cells and cytokines are all, in one their survival inside macrophages. Providing that the para- way or another, considered to play important roles in the sitized macrophages do not become activated, they provide a immune response to Leishmania infection. safe breeding ground for Leishmania parasites which are Macrophages are phagocytic antigen-presenting cells capable of enhancing macrophage survival after infection. (APC) that play host to Leishmania when promastigotes Therefore, it is evident that macrophages play an important bind to their specific receptors and are internalized by ......................................................................................................................................................................................................................................... 74 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... role in both resistance and susceptibility to Leishmania Macatonia et al. demonstrated that while DC were infection. capable of induction and clonal expansion of T-helper Sanabria et al. emphasized the importance of the inter- cells, they were unable to induce T cell differentiation action between NK cells and DC in the immune response to towards Th or Th without IL-12 and IL-4, respectively. 1 2 Leishmania amazonensis. They found that NK cells pro- This emphasizes the importance of these cytokines in the moted the activation of L. amazonensis infected DC and immune response to Leishmania. Remarkably, the roles of that in turn, the activated DC stimulated NK activation. IL-12 and IL-4 in antagonistic Th and Th responses were 1 2 These findings imply differential roles of DC–NK cross-talk uncovered based upon observations with L. major. at various stages of Leishmania infection and present new The protective role of IL-12 has been demonstrated. In one insight on the interactions between innate immune com- study IL-12-deficient mice, originally derived from a strain ponents during infection with this parasite. genetically resistant to infection with L. major, were suscep- 4 22 Vanloubbeeck and Jones outlined the unique and central tible to infection with this parasite. However, role of DC in pathogen-specific immunity in that they present Vanloubbeeck and Jones found that promoting Th polariz- antigens to CD4þ T cells (T-helper cells) on MHC class II as ation of CD4þ T cells, using IL-12 as an adjuvant, was not well as presenting them to CD8þ T cells (cytotoxic T cells) enough to offer resistance to L. amazonensis. on MHC class I. This is known as cross-priming and is The role of IL-4 in disease progression has been implicated important because both CD8þ T cells and CD4þ T cells in several studies in which the administration of anti-CD4þ are considered to be involved in the immune defence and anti-IL-4 antibodies healed Leishmania infection. 4 23 against Leishmania. DC play an essential role in conferring Furthermore, Kopf et al. demonstrated that the disruption resistance or susceptibility to Leishmania by driving the of the IL-4 gene in susceptible BALB/c mice rendered them differentiation and proliferation of T-helper cells (CD4 þ ) resistant to infection with L. major, a finding that clearly to either T-helper-1 cells (Th ) or T-helper-2 cells (Th ). reveals the effects of this cytokine on disease progression. 1 2 On presentation of Leishmania antigens to CD4þ T cells, Figure 1 demonstrates the antagonistic Th and Th 1 2 the concomitant secretion of IL-12 drives the proliferation responses and the role of each cytokine in response to of (IFN-g secreting) Th cells and NK cells, which activate Leishmania infection. macrophages and inhibit Th responses. Conversely, the Cytotoxic T lymphocytes (CTL) are essential in the secretion of IL-4 during antigen presentation to CD4þ T defence against viruses yet their function with respect to cells drives Th cell development that inhibits Th responses intracellular parasites, such as Leishmania, remains some- 2 1 14 15 and promotes B lymphocyte growth and development what elusive. (Fig. 1). Therefore, it is evident that DC play an essential Experiments conducted using mice deficient in MHC class role in both initiation and regulation of antimicrobial II/CD4þ T cells and mice deficient in MHC class I/CD8þ T immune responses to Leishmania. cells have indicated that CD8þ T cells are not required for The role of Langerhans cells (LC), a specific subset of skin controlling Leishmania infection, as mice deficient in these DC located in the supra-basal epidermal layer, has recently cells were able to control the infection with L. major. This been revised by Ritter et al. Although they were previously was in contrast to MHC class II/CD4þ T cell deficient 17 8 thought to fulfil the same role as other DC, Ritter et al. mice that suffered fatal and uncontrolled infection. Since proposed that LC have a regulatory function and may be Leishmania reside in the parasitophorous vacuole within responsible for the suppression of the inflammatory response macrophages, the route of peptide loading onto MHC class against L. major. This is of great importance as it indicates I and the mechanism of CD8þ T cell activation remain that LC could induce Leishmania-specific immunosupression unclear. Therefore, it is tempting to speculate that these instead of parasite control and therefore has major impli- cells play only a minor role in immunity to Leishmania. cations against the use of LC in vaccine studies. However, activation of cytotoxic T cells may occur as a It has been postulated that the type of immune response to result of cross-priming, a phenomenon which enables DC Leishmania (Th versus Th ) is dependent on the type of to take up extracellular antigens and present them on 1 2 leishmanial antigen presented and recognized by T cells. MHC class I to CD8þ T cells. Therefore, Th responses may be initiated by different anti- The cytokine-producing capability of CTL may play a role gens to Th responses. However, various animal studies in the defence against Leishmania infection as they partici- have implicated that the same parasite epitope may induce pate through IFN-g production that is essential in the host 15 24 aTh response in animals with resolving infection or a Th defence against Leishmania. Muller et al. found that 1 2 response in others susceptible to the disease. CTL were involved in the elimination of L. major as well Many have observed that a Th response to Leishmania as establishment and maintenance of immunity as the inhi- infection, which inhibits inflammatory responses, renders bition of CTL with monoclonal antibodies rendered resistant the host more susceptible to disease and that, conversely, mice susceptible to L. major. Basu et al. demonstrated that 4, 5, 8, 14, 19, 20 resistance is associated with a Th response. CD8þ T cells are vital in a protective response against ......................................................................................................................................................................................................................................... 75 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... L. donovani following hybrid cell vaccination, as the exposed, provides the justification for vaccine development. depletion of these cells resulted in a higher parasite burden Although there is no licensed vaccine against any form of in the spleen and liver. Contrary to these studies, others leishmaniasis for general human use, a vaccine against differ- have shown that CD8þ T cells may contribute towards ent forms should, in theory, be possible. This is considering disease progression as large numbers of these cells have the abundance of genetic and biological information about been isolated in acute phase lesions and in the peripheral the parasite, clinical and experimental immunology of the blood. disease, and the availability of vaccines that offer protection A recent study by Ali et al. (personal communication, in experimental animals against challenge with different School of Science and Technology, NTU) has implicated Leishmania species. The leishmaniases are unique among that L. mexicana are capable of MHC class I downregula- parasitic diseases because a single vaccine could successfully tion. This is a significant finding and may perhaps explain prevent and treat disease and has the potential to protect why the role of CTL in the immune response to against more than one species (disease). To date, there Leishmania has been difficult to establish. have been numerous attempts at developing a successful Currently little is known about the role of cytotoxic T cells vaccine against leishmaniasis and there are several categories in the hosts defence against Leishmania infection. Therefore, of vaccine candidates. further investigation must be undertaken in order to fully Table 1 demonstrates the main categories of Leishmania establish their role. Like viruses, Leishmania are intracellular vaccines in development, according to Khamesipour et al., organisms; therefore, it is feasible that CTL could target and with some specific subcategories and examples of each. eliminate parasitized macrophages in the manner in which Live-Non-attenuated Vaccines they locate and eliminate virus-infected cells. ‘Leishmanization’, the deliberate inoculation of virulent Immune Evasion by Leishmania Leishmania from the exudate of a cutaneous lesion, is the Survival of Leishmania within the mammalian host depends oldest vaccination against cutaneous leishmaniasis and has on their ability to evade and manipulate the anti-leishmanial been practiced for centuries. This is because it has long immune response, thus enabling them to reside long enough been well established that recovery from cutaneous leishma- 19 30 to establish infection. Since Leishmania are intracellular niasis is followed by long lasting immunity to the disease. parasites, once inside the host cell, they evade the humoral Therefore, the parasite could be deliberately introduced to a immune response but become susceptible to attack by cell- concealed area of the body rather than risking multiple mediated immunity. However, despite the presentation of lesions to the face or other exposed regions. This approach Leishmania antigens and the initiation of inflammatory was later developed and live virulent L. major promastigotes responses, Leishmania can reside within the host tissues for were harvested and used in large-scale vaccination trials long periods by exploiting several mechanisms of during the 1970s and 1980s in Israel, Iran and the Soviet 26 27, 29 evasion. There are numerous strategies utilized by Union. Although still practiced in Uzbekistan, the Leishmania to survive within mammalian hosts, some of observation of adverse side effects, including the develop- which are common to other protozoan parasites such as ment of large persistent lesions, psoriasis and immunosup- Toxoplasma gondii and Trypanosoma cruzi. The various pression, led to the discontinuation of Leishmanization in strategies employed by the invading pathogens demonstrate many countries and the focus of vaccine development conse- 28, 29 how Leishmania parasites have evolved to successfully quently shifted towards killed organisms. evade the immune responses designed to target and destroy Nowadays, the development of a new vaccine must them. Such evasion strategies include the modulation of meet several strict criteria where safety, reproducibility macrophage function and the inhibition of antigen presen- and efficacy are of utmost importance. Although tation and T cell stimulation. Investigation of this parasite’s Leishmanization gave a high percentage of successful lesion interaction with the host’s immune system may lead to a development and subsequent immunity to L. major infec- better understanding of how Leishmania infection can be tion, it was neither reproducible nor safe. Furthermore, the resisted or the evasion strategies of these parasites which viability and infectivity of the injected parasites varied and influence susceptibility of the host. Improved understanding organisms without virulence induced delayed-type hypersen- of these areas should assist in the development of an effective sitivity. It is not surprising therefore that Leishmanization prophylactic vaccine, capable of inducing the specific is not recommended by WHO. immune responses required to battle this parasite. Another approach to live-non-attenuated vaccines has been taken by Breton et al. who used a non-pathogenic species, Leishmania tarentolae, to immunize mice. The Vaccination against Leishmaniasis high level of immunological cross-reactivity between Evidence that most individuals that were once infected with species at both the humoral and cellular level provides the Leishmania are resistant to clinical infections, when later rationale for using non-pathogenic Leishmania species to ......................................................................................................................................................................................................................................... 76 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... Table 1. Examples of vaccines engineered against leishmaniasis. Category Subcategory Principle Example Result ........................................................................................................................................................................................................................................ Live Leishmania Live virulent Vaccination with live, virulent Leishmanization (using L. major) † Life-long protection against (virulent or Leishmania L. major after cure attenuated) Live Vaccination with a live species that Leishmania tarentolae † L. tarentolae induced a non-pathogenic to is non-pathogenic to humans protective immune response humans against L. donovani Knock-out parasites Removal/blocking/replacement of DHFR-TS (enzyme gene) L. major † Survival of knock-out in parasite genes essential for knock-out mice for about 2 months survival without producing a lesion. † Conferred short-term protection against wild type Suicidal cassettes Genetic modification of parasite to L. major strains producing † Enhanced parasite killing induce suicide in response to biologically active † Delayed lesion external signals/to produce granulocyte-macrophage development in susceptible biological substances that activate colony-stimulating factor (GM-CSF) BALB/c mice immune attack against them Killed Leishmania Whole killed A single species of killed Killed single strain of † Significant protection from or fractions parasite promastigotes or more than one L. amazonensis natural infection species † Long lasting Th responses Whole killed Use of whole dead promastigotes Killed L. mexicana/L. braziliensis † Used parasite and and an adjuvant to stimulate promastigotes and BCG immunotherapeutically it adjuvant immune response induced a high cure rate even in severe cases † Cure accompanied by development of Th immune response Fraction of a killed Use of Leishmania antigens and an Fructose Mannose Ligand (FML) † First vaccine against canine parasite and adjuvant to stimulate an immune antigen from surface of parasite and visceral leishmaniasis w 39 adjuvant response saponin adjuvant (Leishmune ) † Potential transmission-blocking vaccine Recombinant DNA vaccination Introduction of bacterial plasmid Multi-antigenic DNA vaccine † Unable to induce and synthetic DNA encoding antigens into host encoding KMPII, TRYP, LACK and protection in dogs against cells in vivo CP63 L. infantum DNA vaccine against parasite † Production of IgG1 and enzyme gamma-glutamylcysteine IgG2a synthetase (g-GCS) † Increased cell-mediated immunity Recombinant Vaccination with recombinant Recombinant hydrophilic acylated † Protection against protein vaccination proteins surface protein B1 (HASPB1) experimental challenge with L. donovani 32 31 immunize against virulent species. Breton et al. believe it parasites to render them avirulent offers a safer, more is a promising approach to vaccination against visceral leish- stable approach to live vaccination. maniasis caused by L. donovani as BALB/c mice were able Live-Attenuated Vaccines to elicit a protective immune response after only a single per- itoneal vaccination. Although the results to this study appear Genetic modification of Leishmania parasites to reduce viru- promising, vaccination with live-non-attenuated parasites is lence, yet maintain immunogenicity, is of current interest in not an appealing prospect and the attenuation of live Leishmania vaccine research. It is an appealing approach as ......................................................................................................................................................................................................................................... 77 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... attenuated parasites closely mimic natural infection that may America have used L. amazonensis autoclaved lysate or a lead to similar immune responses without the danger associ- mixture of native species, whereas L. major is used in most 29 35 ated with infection with live virulent parasites. Due to vaccine studies against Old World leishmaniasis. In advances in molecular biology and the genomic sequencing Venezuela, Convit and his colleagues introduced an auto- of L. major Friedlin, the attenuation of Leishmania parasites claved L. mexicana þ BCG for immunotherapy and/or by removing, blocking or replacing essential genes is a possi- immunochemotherapy, which proved unsuccessful as the 28, 32 bility. Such parasites are referred to as ‘knock-out para- results were either inconclusive or demonstrated low protec- sites’. Leishmania can be engineered without the genes tion induced by the vaccine. Similarly, killed Leishmania required for long-term survival in the host, thus making vaccines failed to confer protection in persons in the 29 29 them safe to immunize with. The first Leishmania knock- Middle East. However, studies in the 1980s revolutionized out was a dihydrofolate reductase thymidylate synthase the use of killed Leishmania as a vaccine by demonstrating gene (DHFR-TS) in L. major. This knock out induced signifi- excellent protection in mice injected intravenously or intra- cant, but temporary, protection in mice when challenged peritoneally (but not subcutaneously) and subsequently with the wild type but gave disappointing results during several formulations of killed vaccines have been devel- 27 27 further studies in monkeys. The production of oped. This emphasizes that the site of administration Leishmania knock-outs can identify essential virulence affects the efficacy of a vaccine; thus, it is therefore important genes. Therefore, as well as providing vaccine candidates to investigate the most effective method of inoculation. In these studies can lead to a better understanding of the Venezuela, autoclaved L. mexicana is currently used immu- parasite. Furthermore, studies using knock-out parasites notherapeutically to treat patients with cutaneous may be key in identifying specific genes that can then leishmaniasis. be applied to DNA vaccines. For example, work by In countries with a rudimentary biotechnology industry 33, 34 Selvapandiyan et al., which highlighted the importance and where a cold-chain distribution of vaccines is not poss- of the centrin proteins for the duplication and cell cycle ible, autoclaving of the killed vaccine is the recommended progression of Leishmania amastigotes, provided the justi- method of sterilization and preservation. However, as fication for the use of the centrin genes as DNA vaccine demonstrated by De Luca et al., autoclaving lowers the candidates by Ali et al. (2008, personal communication). immunogenicity of the parasite by destroying most of the Another approach to attenuating Leishmania parasites is proteins. Therefore, while offering a safer and more stable the addition of suicide cassettes that lead to the death of alternative to live vaccination, vaccination with killed the parasite in response to external stimuli. An example Leishmania does not mimic natural infection and is also of which is the introduction of drug-sensitive genes such as less immunogenic. Saccharomyces cerevisiae cytosine deaminase gene which is sensitive to 5-fluorocytosine. It has been suggested that Vaccines of Leishmania Fractions parasites carrying drug-sensitive cassettes could provide Leishmune , the first vaccine against canine visceral leishma- suitable candidates for Leishmanization as an effective treat- niasis, consists of a purified L. donovani fraction, named ment of non-resolving lesions could be guaranteed. fructose mannose ligand (FML) and a saponin adjuvant. Alternatively, parasites can be modified to produce biological FML has been characterized as a major antigenic complex substances that activate immune attack, such as granulocyte of L. donovani and the main antigen in this complex is macrophage colony stimulating factor (GM-CSF). NH36, an essential enzyme involved in the construction of Attenuated vaccines offer a novel approach to immuniz- 35 w the parasite’s DNA. Leishmune is considered a promising ation against leishmaniasis however, there are fears that the tool for the prevention of canine visceral leishmaniasis and parasite may revert back to a virulent form. Furthermore, furthermore its potential as a transmission-blocking vaccine targeted deletion of essential virulence genes can result in is promising for the control of zoonotic visceral complete destruction of the parasite or mutants that only leishmaniasis. delay lesion development. Such problems are not a concern with the use of killed parasites for vaccine Recombinant Protein Vaccines candidates. Recombinant protein vaccines are produced by genetically Killed Vaccines engineered cells to produce foreign genes that encode anti- Killed Leishmania is an appealing vaccine candidate in terms genic proteins. They are relatively new and can be adminis- of its stable biochemical composition and antigenicity, low tered as purified proteins or as bacteria manufacturing the 37 29 cost and safety. There have been several studies on killed proteins in situ. Various proteins have been tested as poss- Leishmania, with or without adjuvants, as a vaccine. Early ible vaccine candidates including recombinant hydrophilic trials began in Brazil during the 1940s; however, these acylated surface protein B1 (HASPB1) that conferred protec- 29 40 yielded conflicting results. Most killed vaccine studies in tion against experimental challenge with L. donovani. ......................................................................................................................................................................................................................................... 78 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... Novel Vaccine Strategies Therefore, the immune response to the antigen encoded on the plasmid can be geared towards one that will confer resist- To date, there have been many attempts at designing an ance to the parasite. effective vaccine against Leishmaniasis, most of which have DNA vaccines may provide better protection against fallen into the categories previously described. However, in Leishmania than killed or live-attenuated vaccines as they addition to these there have been several novel approaches can induce the expression of Leishmania antigens, which that have also proven to be promising. Such approaches are unaltered in their protein structure and antigenicity. include the use of DC pulsed with Leishmania antigens and 16, 35 Furthermore, bacteria-derived DNA plasmids are naturally vaccines comprised of components of sand fly saliva. immunogenic as their backbones contain unmethylated In light of their crucial role in immunity to Leishmania, cytosine-phosphate-guanosine (CpG) motifs which have recent studies have implicated DC as potential vaccine candi- been shown to readily induce Th cytokine expression and dates serving as vectors for Leishmania antigens. Moll and 42, 43 enhance CD8þ T cell responses. This adjuvant property Berberich observed that DC pulsed ex vivo with L. major is of great use for a Leishmania vaccine as these motifs would antigen induced protection, in otherwise susceptible mice, ensure the induction of cell-mediated immunity which is against live challenge with L. major. known to confer protection against the parasite. Since sand fly saliva is documented to enhance the infec- tivity of Leishmania, vaccines have been engineered against Administration of DNA Vaccines its components. One such component is the SP15 antigen Several methods of DNA vaccine administration have been that was shown to induce significant resistance in mice to tested and it is thought that the method and site of immuniz- challenge with L. major. ation may influence that nature of the immune response eli- cited. Many studies have observed variation in vaccine DNA Vaccination efficacy depending on the site and method of administration The concept of DNA vaccination is relatively new and was despite the use of the same DNAvaccine. To date, successful established by Wolff et al. in 1990 when direct intramuscular DNA vaccination has been achieved using a number of differ- injection of plasmid DNA, encoding reporter genes, resulted ent routes including intramuscular, intravenous, intraepider- 41 –43 in expression of the proteins in myocytes. Their study mal, intraperitoneal, intravaginal, intranasal, intrasplenic, demonstrated that purified recombinant nucleic acids could intrahepatic, subcutaneous and oral. Of these routes, intra- 43, 45 be delivered to cells in vivo to direct protein expression muscular administration is most commonly employed. 32 45 endogenously. DNA vaccines consist of antigenic proteins However, Me´ndeza et al. demonstrated that the dose of encoded on naked plasmid DNA vectors that allow their DNA vaccine required to induce full protection in C57BL/6 3, 5 expression in eukaryotic cells. They represent a promising mice against L. major was five times smaller using the gene approach to vaccine development and have been shown to gun (particle-mediated epidermal delivery) than subcu- generate protective responses against infectious diseases taneous or intramuscular injection. Furthermore, Ali et al. 44, 45 such as leishmaniasis. (personal communication) found that 1 mg of DNA encoding DNA vaccines present a multitude of advantages over other L. mexicana gp63, administered using a gene gun, conferred vaccine strategies and several features have made them an more protection in susceptible BALB/c mice than 100 mgof appealing alternative. Not only are they fast, simple and vaccine administered intramuscularly. These findings clearly cheap to produce on a large scale, they are also temperature demonstrate that DNA vaccine efficacy is higher when admi- stable making storage and transport easier and cheaper as nistered using a gene gun than it is when administered intra- 44, 35 there is no need for a cold-chain of distribution. This is muscularly. This may be because gene gun administration an important advantage for a Leishmania vaccine candidate can directly transfect APC, such as DC, with the plasmid considering that leishmaniasis is a major health concern in DNA. Although myocytes are capable of inducing CTL many developing countries. DNA vaccines provide an extre- responses by presenting peptides (from the encoded antigen) mely flexible method of vaccination as a single plasmid can via MHC class I molecules, unlike APC they are not pro- encode several antigens and multiple plasmids, encoding fessional antigen presenters and do not carry the 45, 46 different antigens, can be delivered in a single adminis- co-stimulatory molecules required to prime T-cells. tration. Therefore, DNA vaccination could potentially Possible Modes of Action of DNA Vaccines provide protection against more than one species. As leishma- niasis can be caused by several different species, this is another Figure 2 illustrates some of the possible immune mechanisms feature that makes DNA vaccination an appealing prospect. induced by gene gun delivery of DNA vaccines. The gene gun Genetic vaccination has been shown to induce both fires DNA-coated gold particles at high speed directly into humoral and cellular immune responses and these responses cells of the epidermis, which include skin cells, LC and can be tailored through modifications of the vector or incor- dermal DC. Upon entry into the cells, the plasmid is trans- poration of cytokine genes with adjuvant properties. ported to the nucleus where the encoded gene is transcribed ......................................................................................................................................................................................................................................... 79 Review Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 2. Possible immune mechanisms induced by particle-mediated epidermal delivery of a Leishmania DNA vaccine (adapted from Encke et al. ). and the protein is subsequently produced, processed into Rodriguez-Cortes et al. found that their multiantigenic peptides by host proteases and then presented in the DNA vaccine encoding KMII, TRYP, LACK and Gp63 did context of MHC class I which then stimulates CD8þ T lym- not protect dogs against L. infantum experimental challenge, phocytes. DC directly transfected with the DNA vaccine despite the hypothesis that an effective immune response was can prime CD8þ T cells by presenting the DNA encoded more likely to be generated following exposure to more than 4 48 antigen in the context of MHC class I. Conversely, there one antigen. Conversely, Carter et al. found that intramus- is evidence to suggest that immature DC can endocytose cular DNA vaccination against the parasite enzyme gamma- soluble proteins and debris from apoptotic transfected cells glutamylcysteine synthetase conferred protection against and express the coded antigen through MHC class I and/ L. donovani in BALB/c mice. The results of these studies 4, 46 or MHC class II after differentiating into mature DC. indicate that multiantigenic vaccines do not necessarily Therefore, DNA vaccination can result in the stimulation confer better protection against Leishmania infection. of both CD4þ T cell and CD8þ T cell populations. The However, these studies used different species of Leishmania unique ability of DC to present extracellular antigens in so it is not possible to directly compare the findings. the context of MHC class I and MCH class II is known as cross-priming, as a result of this phenomenon it is likely Conclusion that DC play a key role in the induction of both humoral and cell-mediated immunity following DNA vaccination. Leishmaniasis is a major cause of morbidity and mortality There have been several studies conducted on potential worldwide. An effective vaccine has the potential to DNA vaccines against Leishmania. Handman et al. control this disease. However, despite great effort, there is demonstrated that DNA vaccines can be used therapeutically currently no licensed vaccine available. To date, there have to treat cutaneous leishmaniasis caused by L. major in both been numerous attempts at developing a successful vaccine genetically resistant C3H/He mice and susceptible BALB/c against leishmaniasis and Table 1 demonstrates only a few mice. This is an important finding, which demonstrates of these. Further work in this field should not only enable that DNA vaccines may have a role to therapeutically cure the improvement of current vaccine strategies but it is disease in both susceptible and resistant individuals. hoped the development of novel methodologies will ......................................................................................................................................................................................................................................... 80 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Review ......................................................................................................................................................................................................................................... 21. Macatonia S, Hsieh C, Murphy K et al. (1993) Dendritic cells and quickly follow suit. 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Submitted on 30 September 2008; accepted on 21 January 2009; advance access publication 17 February 2009 .........................................................................................................................................................................................................................................

Journal

Bioscience HorizonsOxford University Press

Published: Mar 17, 2009

Keywords: Key words Leishmania leishmaniasis immune response vaccination

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