Citation: The Working Group on Research Priorities for Development of Leishmaniasis Vaccines, Costa CHN, Peters NC, Maruyama SR, de Brito EC Jr, de Miranda Santos IKF (2011) Vaccines for the Leishmaniases: Proposals for a Research Agenda. PLoS Negl Trop Dis 5(3): e943. doi:10.1371/journal.pntd.0000943
Editor: Hechmi Louzir, Institut Pasteur de Tunis, Tunisia
Published: March 29, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: The funders of the Symposium and Working Group that resulted in this policy platform were the Brazilian National Research Council (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq), the Brazilian Ministry of Science and Technology (Ministério de Ciência e Tecnologia), The Oswaldo Cruz Foundation (Fundação Oswaldo Cruz - FIOCRUZ) and the Department of Science and Technology of the Brazilian Ministry of Health (Departamento de Ciência e Tecnologia do Ministério da Saúde - DECIT-MS). None of the authors present financial conflicts of interest with the opinions expressed in this position paper. The funders of the International Symposium for Leishmanisis Vaccines had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The funders of the Symposium and Working Group that resulted in this policy platform were the Brazilian National Research Council (Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq), the Brazilian Ministry of Science and Technology (Ministério de Ciência e Tecnologia - MCT), The Oswaldo Cruz Foundation (Fundação Oswaldo Cruz - FIOCRUZ) and the Department of Science and Technology of the Brazilian Ministry of Health (Departamento de Ciência e Tecnologia do Ministério da Saúde - DECIT-MS). The funders of the International Symposium for Leishmanisis Vaccines had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The International Symposium on Leishmaniasis Vaccines, held in Olinda, Brazil, on March 9–11, 2009, congregated international experts who conduct research on vaccines against the leishmaniases. The questions that were raised during that meeting and the ensuing discussions are compiled in this report and may assist in guiding a research agenda. A group to further discussion on issues raised in this policy platform has been set up at http://groups.google.com/group/leishvaccines-l.
The Impact of the Leishmaniases and Perspectives for Anti-Leishmania Vaccines
The leishmaniases are responsible for the second-highest number of deaths due to parasitic infection globally and are overwhelmingly associated with poverty . They have an estimated prevalence of 12 million humans infected and cause a burden estimated at 2,357,000 disability-adjusted life years . Visceral leishmaniasis (VL) is almost always fatal if not treated, and morbidity caused by the cutaneous leishmaniases (CLs) is also important. Treatments for all forms of leishmaniases are few, toxic, and/or expensive, and, furthermore, drug resistance is on the rise . Canids are significant zoonotic reservoirs for human VL (their role as a reservoir for other species of Leishmania is not fully defined ) and a cause of concern for owners of dogs; they are also important sources of knowledge about the human counterparts of this infection. Leishmaniases are vector-borne diseases, and the impact of global warming on the geographical distribution of parasite-infected sand flies suggests that the leishmaniases may become a widespread and significant problem for public health. Political and socioeconomic changes may have an even more important role than global warming on the changing epidemiology of the leishmaniases, as has been argued for tick-borne diseases in Europe . Indeed, in the last 20 years in Brazil, the epidemiological pattern of VL has changed from a sporadic disease of rural areas to one occurring in peri-urban epidemics that affect all socioeconomic strata, and with a trend towards increasing mortality . The European Centre for Disease Prevention and Control lists the leishmaniases among the ten vector-borne diseases that have the greatest potential to affect European inhabitants . The leishmaniases are, so far, not preventable diseases, and their epidemiological profile is shifting. This current situation demands novel instruments for treatment and control.
The feasibility of controlling the leishmaniases with a vaccine, a likely cost-effective approach for their control in many epidemiological situations, is attested to by the fact that most individuals who were once infected and recovered are resistant to overt clinical manifestations upon re-infections . There are, however, still many challenges to overcome before effective vaccines for prevention of the leishmaniases become a reality. The centuries-old practice of leishmanization, which uses inoculation of live, virulent Leishmania major parasites at cosmetically acceptable sites of the body, affords lifelong protection against CL and represents the only efficacious vaccine for any disease of this complex ; its correlates of protection should be exhaustively explored.
Regarding product profiles, if a live vaccine proves to be the only successful route to controlling the leishmaniases, issues of logistics will certainly arise. However, this aspect has not prevented two other live vaccines to eradicate one disease (smallpox) and almost eradicate another (polio). Product profiles, in turn, directly affect research strategies and clinical development, but should not abort research on useful antigens that otherwise could compose a “second best” product profile. At present there is no consensus about the product profile for vaccines against the leishmaniases, except the minimum requirements for neglected parasitic diseases: safe, effective, and inexpensive.
Mechanisms of Protective Immunity
In animal models, immunity against Leishmania has historically been cast within the Th1/Th2 paradigm. Cellular immunity is considered to be the key mediator of resistance by means of IFN-γ, which upregulates the production of nitric oxide, leading to oxidative burst in phagocytes that harbor these intracellular pathogens. However, evidence of Th2 and T regulatory responses in resistant mice, and progressive disease in resistant strains that have defects in Th1 differentiation but do not default to a Th2 pathway, has led to a modification of this paradigm to state that a Th2 response does not promote disease, but the absence of Th1 immunity does . In addition, the discovery of IL-10-mediated regulation of protective IFN-γ-producing cells has shown that a highly regulated Th1 response can also lead to susceptibility . The Th1 immune response can also be associated with immunopathology; for example, development of lesions in human tegumentary leishmaniasis is due to activation of type 1 immune responses. Moreover, individuals infected with L. braziliensis but who do not develop disease produce less type 1 cytokines than patients with cutaneous lesions . Studies must be done to determine whether such individuals did not develop disease because they had small Th1 immune responses or because they received a low dose of antigen.
The precise role of antibodies in conferring resistance to infections with Leishmania is controversial and needs to be reappraised. The role of antibodies in tuberculosis, caused by an intracellular pathogen, was also controversial. However, recent work shows that antibodies participate in important effector mechanisms in the response against tuberculosis . Antibodies also regulate the outcome of immune responses through different types of Fc receptors . Animals lacking B cells are resistant to infections with Leishmania , and the elevated levels of IgG seen in human and canine VL suggest that antibodies are not relevant for protection in the absence of an appropriate cellular response. More recent work, however, supports a role of antibodies in protection against Leishmania. Natural antibodies in normal human sera have been shown to very rapidly kill infective culture promastigotes by the classical pathway of complement . Sera from dogs vaccinated against L. infantum with LiESAp antigen killed promastigotes and amastigotes and inhibited the in vitro infectivity of promastigotes for canine macrophages . Mice lacking IgG1 are more resistant to L. mexicana infection and mount a strong IgG2a/c antibody response . L. major taken up via different phagocytic receptors produces different outcomes of infection: mice infected with IgG-opsonized parasites showed enhanced protective immunity as well as increased numbers of L. major–infected lesional dendritic cells, leading to production of IL-12 and priming of Th1 and Tc1 cells and efficient parasite killing by lesional macrophages . C57BL/6 mice co-infected with L. amazonensis and L. major develop chronic disease and produce less antigen-specific antibodies compared to similarly co-infected C3HeB/FeJ mice, which heal . These and other results indicate that the subclass, specificity, and, possibly, affinity of anti-Leishmania antibodies may determine outcome of infection.
Kedzierski and colleagues  caution against extrapolating results obtained with the murine models of immunity to humans: there are still no unambiguous correlates of protection, and the induction of a putatively protective IFN-γ response via vaccines will not be sufficient to induce protection if levels of IL-10 are disproportionately elevated. They propose that testing for IL-10 is as important as testing for IFN-γ to determine whether a vaccine has induced protective responses. So far the leishmanin (Montenegro) skin test is the most informative and practical immunological surrogate marker used in clinical trials . With a complementary rationale Campos-Neto  has shown that disease-associated Th2 antigens of Leishmania can be protective if a Th1 response to them is generated before infection. Moreover, the immune response to a given antigen (e.g., LACK) can be protective (Th1) or exacerbating (Th2), depending on the way the antigen is introduced  or on the genetic background of the mouse. More recently, Nylén and Akuffo  proposed a strategy to search for the different biomarkers of outcomes of infection with Leishmania parasites in human subjects. These biomarkers may be essential not only for elucidating details of protective immunity, but for determining the efficacy of vaccines and for assisting in screening of novel antigens. This same approach is now also guiding research on AIDS vaccines . The focus on individuals with infection but no clinical manifestations is logical because while natural immunity against Leishmania may not prevent infection, the fact that the majority of infections do not result in disease indicates that efficacious immune mechanisms exist. This situation is seen in dogs , and more emphasis should be given to canine immunology. This natural protective response should be the gold standard, and the development and efficacy of vaccines should be considered within the context of this standard.
Persistence of Parasites: Role in Immunity Vaccine Development
Leishmanization is unacceptable for many individuals and regulatory agencies because it can be accompanied by minor complications and is potentially problematic in immunocompromised recipients; however, co-injection of live parasites with CpG oligodeoxynucleotides attenuates the severity of disease following leishmanization in mice . Vaccination with live parasites that harbor suicide cassettes, making them susceptible to treatment with antibiotics, was tested successfully in mice as a means to provide a safe live challenge in clinical trial, but it has not been pursued further as a vaccine . Leishmanization has not been tested against disease caused by species of Leishmania other than L. major; furthermore, the degree of cross-protection provided has not been sufficiently evaluated. It is noteworthy that epidemiological evidence indicates that individuals from Sudan with history of CL have lower incidence of VL . Okwor and Uzonna  argue that vaccination with live-parasite-based vaccines for CL would induce effector and central memory cells. The challenge will be to achieve attenuation of live parasites without losing efficacy. In this regard, observations in animal models indicate that antigen persistence may be as important as the specific protein or parasite component employed in a vaccine .
Vector Saliva and Parasite Proteo-Phosphoglycans: Role in Immunity and in Vaccine Development
Salivary antigens of the vector are important additional components of a vaccine . This aspect has received little attention in spite of the fact that salivary proteins from the vector are also delivered to the host during natural transmission of the pathogen, and, in at least some cases, these proteins are immunogenic or immunomodulatory for the host , . The rationales behind including salivary antigens of the sand fly vector in vaccines against the leishmaniases are 2-fold and are not necessarily mutually exclusive. If some of these salivary proteins are immunosuppressants, they may also compromise induction of protective immunity with Leishmania-derived antigens. Thus, a vaccine may neutralize this immunosuppressant activity. Conversely, several salivary proteins are potent immunogens and induce lymphocytic infiltration and production of IFN-γ and IL-12; this reaction may set the local stage for a protective anti-Leishmania immune response. Sand fly bites induce hypersensitivity reactions in many hosts, and the problem of how to target sand fly salivary proteins while avoiding these reactions also needs to be addressed. Rogers and colleagues  argue that parasite-secreted proteo-phosphoglycans, rather than vector saliva, are responsible for enhanced disease following sand-fly-transmitted versus needle infection and are viable vaccine targets. Proteo-phosphoglycans are regurgitated by phebotomines into the host, where they activate host macrophages through the alternative arginase pathway. Arginase synthesizes polyamines, which are essential growth factors for Leishmania . The neglect of the role of the vector in vaccine development is reflected by the fact that the efficacy of all but two ,  experimental vaccines has been tested by needle, rather than infected sand fly challenge (see the Table S1). Kedzierski and colleagues  call attention to the influence of wound repair triggered by the bite of the vector, a little-studied aspect of resistance to leishmaniasis.
Antigens for Anti-Leishmaniasis Vaccines: State of the Art
Most of the 25 vaccines licensed for use in humans are effective because they drive responses to many different targets on the pathogens . Therefore, the capacity to respond to multiple antigens may be an essential requisite of an efficacious vaccine. This observation brings up the issue of antigen discovery. As seen in Table S1, 34 subunits of Leishmania antigens are available, but they have not been evaluated as a set of more than three components. Given the complexity of the eukaryotic Leishmania parasite, the fact that the response observed following live vaccination or healed infection in humans is poly-specific and that immunization with a combination of the antigens LmST11 and TSA, a polyprotein vaccine, apparently confers the best degree of protection so far in nonhuman primates  suggests that an increase in the known repertoire of immunogenic antigens for this parasite is necessary. The new sequencing technologies should facilitate this task. Table S1 is a starting point, a database of vaccine candidates similar to that created for malaria . A comparative analysis of the genomes of Plasmodium vivax, P. falciparum, P. yoelii, and P. knowlesi  revealed novel gene families and invasion pathways in P. vivax. A similar advance with more genomes of Leishmania parasites could reveal alternative mechanisms of parasitism and, thus, new targets for vaccines. Another case in point is L. tarentolae, a lizard pathogen that is nonpathogenic to humans. It expresses a cysteine protease B, lipophosphoglycan LPG3, and the leishmanolysin GP63, which are virulence genes of Leishmania species pathogenic for humans. On the other hand, the A2 gene, expressed by the L. donovani complex and which promotes visceralization, is absent in L. tarentolae . L. tarentolae could play the same role in leishmaniasis control that vaccinia virus played in the eradication of smallpox, where a species nonpathogenic for humans induced protective immunity against a related species of pathogen. Finally, pathoantigens should be identified to better understand the pathological process, but also to preclude these molecules during selection of vaccine candidates and, if they are essential for protective responses, to research strategies to render them nonpathogenic.
The number of experimental vaccines developed in the last 30 years that target the different forms of leishmaniasis surpasses 100. These experimental vaccines have undergone various stages of evaluation and comprise antigens consisting of whole live, attenuated, genetically modified, or killed parasites, or one or more subunits or fusion proteins. They employ different adjuvants, delivery systems, and vectors. Finally, they have been evaluated in five different animal models as well as in humans. Table S1 (an interactive version of this table is available at http://www.leishvaccines.net) and Figures 1–3 summarize all of these efforts, and the numbers they depict attest to the fact that vaccinology is still an imprecise science and can be bewildering to authorities and planners.
Data are derived from Table S1, which contains a summary of all vaccines to date (both experimental and in clinical use). Black bars represent CL: L. major, L. mexicana, L. tropica, L. amazonensis, and L. braziliensis; gray bars represent VL: L. donovani, L. chagasi, and L. infantum.
Data are derived from Table S1, which contains a summary of all vaccines to date (both experimental and in clinical use). Bar graphs display the number of trials for each Leishmania species and the type of antigens that have been tested.
The main questions raised during debates at the International Symposium on Leishmaniasis Vaccines are summarized in Table 1. Except when specifically noted, the questions addressed issues concerning all of the leishmaniases. They addressed live attenuated or genetically modified (GM) parasites, the role of vectors, further antigen discovery, and elucidation of protective immunity for formulation of vaccines, models and clinical trials, funding policies, and dissemination of information on all these issues, on which the development of vaccines for the leishmaniases depends.
Funding agencies must be persuaded that the profile of the leishmaniases is shifting and that a concerted action is needed with new scientific approaches. A strategic plan is needed to attain truly efficacious vaccines against the leishmaniases. The first bottleneck to overcome is caused by scientific gaps; therefore, a call for grant proposals should focus on a few scientific questions and research priorities in vaccinology for VL and CL that are potentially very informative. Proposals should ideally be executed as a network in order to maximize existing information generated by individual groups and to generate new insights. The research priorities could be validated through consultations with participants in the discussion group that the Working Group has set up (http://groups.google.com.br/group/leishvaccines-l) during a fixed period of time, six months. In principle these research priorities might address the following:
- The role of parasite persistence in protective immunity and live versus killed/subunit vaccines in order to define (i) a product profile for VL and CL and (ii) whether it is valid to improve leishmanization for CL;
- The role of immunity to vector saliva in immunity against different species of Leishmania and in composition of vaccines;
- The significance and immunological profile of the hosts who are able to maintain subclinical infections and their role in obtaining biomarkers of resistance and protective immunity;
- Further application of genomic approaches for (i) identifying virulence factors in different species of Leishmania and (ii) antigen discovery;
- Re-formulation of a previously tested subunit vaccines that include novel antigens, including antigens of the 30 subunits previously identified by different groups (Table S1), and/or new adjuvants.
Conceptual and technological advances in immunology, parasitology, genetics, genomics, and bioinformatics have increased our capacity to address the research priorities with a more effective systemic approach to the vector–Leishmania–host interface. However, these advances do not affect the constraints that spring from the placement of political will and personal scientific convictions. The size of this task and the seriousness of the problem represented by these neglected diseases warrant a large collaborative effort. At least 30 vaccine subunits (listed in the Table S1) have been tested with relative success by several different groups. If a multicomponent vaccine is a sine qua non condition for achieving an effective vaccine, many of these antigens will probably be part of the composition. In this case, the issue of intellectual property becomes an significant barrier for attracting companies willing to develop, manufacture, and market a vaccine. The funding agencies and grantees must devise a solution that is fair for all interested parties, including the patients.
An example in science where competing groups (with large financial interests at stake) eventually united and then achieved a common goal is seen in the cloning of the gene causing cystic fibrosis. In contrast to the the situation for patients with neglected leishmaniases, patients with cystic fibrosis and their families count on the active Cystic Fibrosis Foundation, incorporated in 1955, which created a network of research centers and a research program in 1980, the same time that efforts began for developing subunit vaccines for Leishmania. In less than nine years, in the pre-genomics era, the defective cystic fibrosis gene and its protein product were described. Presently, more than 30 drugs that modulate the cystic fibrosis gene product itself are in the pipeline, including phase 3 trials. Individuals at risk for developing leishmaniasis need a similar voice and concerted action.
A survey of antigens, adjuvants, delivery systems, and models employed for developing vaccines for the VLs and CLs.
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The authors thank the Brazilian National Research Council (Conselho Nacional de Desenvolvimento Científico e Tecnológico), the Brazilian Ministry of Science and Technology (Ministério de Ciência e Tecnologia), the Oswaldo Cruz Foundation (Fundação Oswaldo Cruz), and the Department of Science and Technology of the Brazilian Ministry of Health (Departamento de Ciência e Tecnologia do Ministério da Saúde) for supporting the International Symposium on Leishmaniasis Vaccines. The authors also thank Dr. Farrokh Modabber and anonymous reviewers for valuable insights that improved the manuscript.
The members of the Working Group on Research Priorities for Development of Leishmaniasis Vaccines are: Nahid Ali, Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, Kolkata, India, firstname.lastname@example.org; Eldo Cardoso de Brito, Jr., Institute of Tropical Diseases Natan Portella, Federal University of Piauí, Teresina, Brazil, email@example.com; Cláudia Brodskyn, Research Center Gonçalo Moniz, Fiocruz, Salvador, Brazil, firstname.lastname@example.org; Antônio Campos-Neto, The Forsyth Institute, Boston, Massachusetts, United States of America, email@example.com; Edgar M. Carvalho, Service of Immunology, University Hospital Professor Edgard Santos, Federal University of Bahia, Salvador, Brazil, firstname.lastname@example.org; Kwang Poo Chang, Chicago Medical School, Rosalind Franklin University, Chicago, Illinois, United States of America, KwangPoo.Chang@rosalindfranklin.edu; Carlos Henrique Nery Costa, Institute of Tropical Diseases Natan Portella, Federal University of Piauí, Teresina, Brazil, email@example.com; Ana Paula Fernandes, Department of Clinical and Toxicological Analysis, School of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Brazil, firstname.lastname@example.org; Ricardo Fujiwara, Laboratory of Immunology and Genomics of Parasites, Department of Parasitology, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, Brazil, email@example.com; Ricardo Gazzinelli, René Rachou Institute, Fiocruz, and Department of Biochemistry and Immunology, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, Brazil, firstname.lastname@example.org; Hiro Goto, Tropical Diseases Institute of São Paulo and School of Medicine, University of São Paulo (Instituto de Medicina Tropical de São Paulo e Faculdade de Medicina, Universidade de São Paulo), São Paulo, Brazil, email@example.com; Gabriel Grimaldi, Laboratory of Research in Leishmaniasis, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil, firstname.lastname@example.org; Paul Kaye, Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, York, United Kingdom, email@example.com; Lukasz Kedzierski, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, firstname.lastname@example.org; Ali Khamesipour, Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran, email@example.com; Carla Maia, Institute of Hygiene and Tropical Medicine (Instituto de Higiene e Medicina Tropical), Universidade Nova de Lisboa, Lisbon, Portugal, firstname.lastname@example.org; Sandra Regina Maruyama, Department of Biochemistry and Immunology, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, Brazil, email@example.com; W. Robert McMaster, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, firstname.lastname@example.org; Sérgio C. F. Mendonça, Oswaldo Cruz Institute, Rio de Janeiro, Brazil, email@example.com; Hira Lal Nakhasi, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland, United States of America, firstname.lastname@example.org; Nathan C. Peters, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, NPeters@niaid.nih.gov; Franco Piazza, Infectious Disease Research Institute, Seattle, Washington, United States of America, email@example.com; Rupert Quinnell, Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom, R.J.Quinnell@leeds.ac.uk; Alexandre Barbosa Reis, Federal University of Ouro Preto and Research Center René Rachou, Ouro Preto, Brazil, firstname.lastname@example.org; Isabel K. Ferreira de Miranda Santos, Department of Biochemistry and Immunology, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, Brazil, email@example.com; Gabriela Santos-Gomes, Hygiene and Tropical Institute (Instituto de Higiene e Medicina Tropical), New University of Lisbon, Lisbon, Portugal, firstname.lastname@example.org; Jeffrey Shaw, Department of Parasitology, Institute of Biomedical Research, University of São Paulo, São Paulo, Brazil, email@example.com; Jesus Valenzuela, Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America, firstname.lastname@example.org; Peter Walden, Department of Dermatology and Allergy, Charité—Universitätsmedizin Berlin, Humboldt University, Berlin, Germany, email@example.com; Guilherme Werneck, National School of Public Health Sérgio Arouca, Fiocruz, Rio de Janeiro, Brazil, firstname.lastname@example.org.
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