JOANA MARQUES DA CUNHA
Host-parasite interactions in leishmaniasis
In vitro and in vivo study of Leishmania infantum strains isolated from
different human patients
Interações hospedeiro-parasita na leishmaniose
Estudo in vitro e in vivo de estirpes de Leishmania infantum isoladas
de diferentes doentes humanos
Tese de Candidatura ao grau de Doutor em
Patologia e Genética Molecular submetida ao
Instituto de Ciências Biomédicas Abel Salazar e
Faculdade de Medicina da Universidade do Porto
Orientador: Doutora Anabela Cordeiro da Silva
Categoria: Professor Associado com Agregação
Afiliação: Faculdade de Farmácia e Instituto de
Biologia Molecular e Celular, Universidade do
Porto, Portugal
Co-orientador: Doutor Javier Moreno
Categoria: Investigador titular
Afiliação: Centro Colaborador da OMS para a
Leishmaniose, Centro Nacional de Microbiologia,
Instituto de Salud Carlos III, Espanha
The author of this thesis declares that she afforded a major contribution to the conceptual design and technical execution of the work, interpretation of the results and manuscript preparation of the accepted and submitted articles included in this dissertation.
Additionally, she hereby declares that the following original articles/communications were prepared in the scope of this dissertation.
The candidate was supported by a doctoral fellowship (SFRH/BD/48626/2008) given by the “Fundação para a Ciência e a Tecnologia” (FCT; Portugal). The Parasite Disease Group at IBMC - Instituto de Biologia Molecular e Celular (Portugal) and the WHO Collaborating Center for Leishmaniasis at Centro Nacional de Microbiologia, Instituto de Salud Carlos III (Spain) provided the facilities, reagents and logistical supports.
ARTICLES IN INTERNATIONAL PEER-REVIEWED JOURNALS
In the scope of this dissertation
1. Cunha J, Carrillo E, Sánchez C, Cruz I, Moreno J and Cordeiro-da-Silva A: Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis. Parasites and Vectors 2013, 6:122 2. Santarém N and Cunha J, Silvestre R, Silva C, Moreira D, Ouellette M and
Cordeiro-da-Silva A: The impact of distinct culture media in Leishmania infantum biology and infectivity. In press in Parasitology 2013
Participation in other publications in related fields
3. Lima SC, Silvestre R, Cunha J, Barros D, Baltazar MT, Dinis R, Cordeiro-da-Silva A: Crucial CD8+ T lymphocyte cytotoxic role in amphotericin B nanospheres efficacy against experimental visceral leishmaniasis. Submitted to Small 2013 4. Carrillo E, Jimenez MA, Sánchez C, Cunha J, Seva AP, Moreno J: Protein energy
malnutrition weakens the immune response and favors the progression of visceral leishmaniasis in hamsters. Submitted to PLOS Negl Trop Dis 2013
5. Santos AC, Cunha J, Veiga F, Cordeiro-da-Silva A, Ribeiro AJ: Ultrasonication of insulin microgel particles: impact on particle's size and insulin bioactivity. Accepted in Carbohydrate Polymers 2013
6. Resende M, Moreira M, Cunha J, Augusto J, Neves B, Cruz MT, Estaquier J, Cordeiro-da-Silva and Silvestre R: Leishmania-infected MHC-IIhigh dendritic cells polarize CD4+ T cells towards a non-protective T-bet+INFγ+IL10+ phenotype. Journal of immunology 2013, 191(1):262-73
7. Neves BM, Silvestre R, Resende M, Ouaissi A, Cunha J, Tavares J, Loureiro I, Santarem N, Silva AM, Lopes MC et al: Activation of phosphatidylinositol 3-kinase/Akt and impairment of nuclear factor-kappaB: molecular mechanisms behind the arrested maturation/activation state of Leishmania infantum-infected dendritic cells. The American journal of pathology 2010, 177(6):2898-2911.
In the scope of this dissertation
1. Cunha J (presenting author), Carrillo E, Sánchez C, Tavares J, Moreno J and Cordeiro-da-Silva A: Characterization of the virulence of Leishmania infantum isolates from human patients. Immunology 2012, 137, Issue Supplement s1 (Special Issue: Abstracts of the European Congress of Immunology, 5-8 September 2012, Glasgow, Scotland):555.
2. Cunha J (presenting author), Moreno J and Cordeiro-da-Silva A: Comparative study between naturally attenuated and virulent strains of Leishmania infantum: culture conditions and infectiveness. Acta Parasitológica Portuguesa 2010, 17(2; Congresso Português de Parasitologia, 8-10 September 2010, Porto, Portugal):97.
Participation in other publications in related fields
3. Hanniffy S (presenting author), Sánchez C, Cunha J, Carrillo, Cañavate C, Moreno J: Mucosal vaccination using non-pathogenic lactic acid bacteria as a strategy to prevent morbidity and mortality caused by visceral Leishmaniasis European Journal TM&IH Tropical Medicine & International Health 2011, 16 (Supplement 1 - Special Issue: Abstracts of the 7th European Congress on Tropical Medicine and International Health 3-6 October 2011 Barcelona, Spain.):198.
4. Neves B, Silvestre R, Cunha J, Tavares J, Resende M, Ouaissi A, Lopes MC, Cruz MT & Cordeiro-da-Silva A: Immunomodulation of dendritic cells by virulent and attenuated Leishmania infantum strains. European journal of immunology 2009, 39(Supplement 1/09 - Abstracts of the 2nd European Congress of Immunology, September 13-16 2009, Berlim, Germany):365.
In the scope of this dissertation
1. Cunha J (presenting author), Carrillo E, Sánchez C, Tavares, J, Moreno J and Cordeiro-da-Silva A: Characterization of the virulence of Leishmania infantum isolates from human patients. 3rd European Congress of Immunology, September 5-8, 2012, Glasgow, Scotland, Poster 1168
2. Santarém N and Cunha J (presenting authors), Silva C, Moreira D, Silvestre R and Cordeiro-da Silva A: The development of a semi-defined medium for growth of
Leishmania infantum. 3rd I3S Scientific Retreat, May 10-11, 2012, Póvoa de Varzim,
Portugal, Poster 14
3. Cunha J (presenting author), Moreno J, Cordeiro-da-Silva A: In vitro comparative study between naturally attenuated and virulent strains of Leishmania infantum. 2nd I3S Scientific Retreat, May 5-6, 2011, Póvoa de Varzim, Portugal,
Poster 12
4. Cunha J (presenting author), Moreno J, Cordeiro-da-Silva A: Comparative study between naturally attenuated and virulent strains of Leishmania infantum: culture conditions and infectiveness. XIV Congresso Português de Parasitologia 2010, Porto, Portugal, Poster P-31
Participation in other communications in related fields
5. Resende M (presenting author), Moreira D, Cunha J, Neves B, Lima SC, Cruz MT, Cordeiro-da-Silva A & Silvestre R: Infected but not Bystander Dendritic Cells polarize CD4+ T cells towards a non-protective T-bet+ INFγ+ IL10+ phenotype. XXXVII Annual Meeting of the Portuguese Society for Immunology, November 29-30, 2011, Oeiras, Portugal
6. Robalo AL (presenting author), Pereira JA, Resende M, Moreira D, Cunha J, Costa-Lima S, Cordeiro-da-Silva A & Silvestre R: Activation of IL-27 p28 gene transcription on antigen presenting cells infected with Leishmania infantum. XXXVII Annual Meeting of the Portuguese Society for Immunology, November 29-30, 2011, Oeiras, Portugal
7. Hanniffy S (presenting author), Sánchez C, Cunha J, Carrillo, Cañavate C, Moreno J: Mucosal vaccination using non-pathogenic lactic acid bacteria as a strategy to prevent morbidity and mortality caused by visceral Leishmaniasis. 7th European Congress on Tropical Medicine and International Health, October 3-6, 2011, Barcelona, Spain, Poster 1.3-106
8. Resende M, Cunha J, Costa-Lima S, Moreira D, Cordeiro-da-Silva A and Silvestre R (presenting author): Early stages of visceral Leishmania infection. 2nd I3S Scientific Retreat, May 5-6, 2011, Póvoa de Varzim, Portugal, Poster 23
9. Resende M (presenting author), Silvestre R, Neves B, Cunha J, Cruz MT & Cordeiro-da-Silva A: Differential effect of Leishmania infantum on infected and bystander dendritic cells. XXXVI Annual Meeting of the Portuguese Society for Immunology, September 20-11, 2010, Braga, Portugal
10. Neves B, Silvestre R, Cunha J (presenting author), Tavares J, Resende M, Ouaissi A, Lopes MC, Cruz MT & Cordeiro-da-Silva A: Immunomodulation of dendritic cells by virulent and attenuated Leishmania infantum strains. 2nd European Congress of Immunology 2009, Berlin, Germany, Poster PA11/60
I am very grateful to everyone who directly or indirectly made this thesis possible to be developed and concluded. During these more than five years I have worked with amazing people that taught me everything I know in Science, helped me to be more critical about what is known and more curious about the unknown. With all of you I grew up and became the Scientist which I expect to have accurately transposed in this thesis.
To Professor Anabela I thank the opportunity for accepting me in her lab many years ago where I gave my first steps in Science. Thank you for guiding me in the road that led me to the accomplishment of this work.
To Javier I thank for his kindness when I first arrived to his lab, receiving me as part of his family. It was just a first demonstration of the good heart he has. Thank you for showing me that sometimes I just have to look at things in a more relaxed way.
Eugenia, what a friend and an inspiration you became to me! Thank you for listen to me, think with me and work along me. Your support and open mind were essential. And if we join Carmen “San”, no one can stop us! Carmen, tu alegría es contagiosa! Soy una persona diferente después de haberte conocido.
To Isra, the “master of the Molecular Biology”, thank you for sharing your knowledge (but never your primers or pipettes… ). To Sean, our Scottish “Clark Kent”, for making me feel so well organized . Thanks for your company in some of the working weekends. To Javier Nieto for first introduce me to Madrid. To Carmen Cañavate, Emi, Carmen Chicharro, Maria Flores y las chicas del laboratório. To all of you in the Parasitology Department for the several farewell parties you organized for me. Thank you all for your kindness, especially Espe, Carolina, José and Chus.
To Ramona, “mi abuelita gallega”, thank you for making me feel at home in your home. You truly represented my family in Madrid. Te echaré de menos.
I thank Mariana for the company in the late hours. When it was needed we did it. Thanks for the chocolates and for our nice lunches outside IBMC. Sofia and Diana, thanks for giving me a hand taking care of the cell cultures when I was in vacations. Ricardo, thanks for your expertise in almost every subject. You rock! And Nuno, I still remember that it was you who taught me how to do ELISAS. No one understands your music taste as I do… I thank Joana Tavares for her dedication. Back in the old days you were an example to me.
To my very best friends, Pi e Sarocas, I thank you both for always being there. We grew up, we don’t have the time to be together as before, but I love when me manage to have lunch just us 3 or to do a 6-pack program. I LUV U, girls!
I also thank Joaninha and Bárbara. We are such different 3 Pharmacists… Thanks for presenting me your reality and helping me doing my choices.
I am very grateful to my family that gave me unconditional love and support. You taught me to never give up and to fight for my goals. Mum, thanks for being my mumi. You try your best, and I don’t know if you know, but you are the best! Dad, thank you for always being there for me. I am so proud of being your daughter. I thank you both for raising me and letting me fulfilled whatever dreams I had.
To Sérgio, my beautiful husband, I cannot express how grateful I am for your love, support and patience. I am sorry for making you feel alone when I was gone, or tired of waiting for me in “big experiment days”. Thank you so much for having me. We are now ready to move one step forward in our lives. I love you very much. You’re everything… I also thank to João Leandro for treating me as a daughter. Thanks to your lovely family for being part of my family too. I appreciate your support. You’re great! Thanks to Cristina and João for being part of my life too.
And to all the other members of my family, I thank for your love, support and wise advices.
Finally, I would like to officially thank to FCT for believing in me and financing me with a personal scientific grant and to IBMC and ISCIII for making available all the facilities that made my work possible.
Leishmania spp. are protozoan parasites responsible for a group of clinical manifestations collectively known as leishmaniasis. The parasite is transmitted to the mammalian host by the bite of an infected female phlebotomine sand fly where it exists as promastigote forms. The metacyclic promastigote is the infective stage for the mammalian host but it must rapidly convert into the intracellular amastigote form to survive inside the host’s macrophages.
Leishmaniasis are zoonotic or anthroponotic diseases spread over the Mediterranean, tropic and subtropic regions worldwide. They are considered neglected infectious diseases and the emergence of HIV/Leishmania co-infections in developed countries brought new interest on the disease. Anti-Leishmania drugs are efficient but treatment is highly toxic and associated with elevated cost, not only because of the price of the treatment itself but also due to mandatory hospitalization in most of the cases.
The search for the human vaccine against visceral leishmaniasis, the most severe form of the disease, for long has been attempted but without positive results. However, there are three licensed vaccines for canine leishmaniasis, an important factor in the epidemiology of leishmaniasis in Brazil and Southern Europe, where dogs are the main domestic reservoir for the parasite.
Few years ago our group described the efficacy of the live attenuated sir2 single knockout Leishmania infantum in the protection from the wild type virulent challenge in the murine model of visceral leishmaniasis. With the knowledge that advent from that work and maintaining the interest on the research for a vaccine for visceral leishmaniasis, we wanted to experimentally explore the possibility of the naturally attenuated strains in the protection from a secondary virulent infection.
In some cases, HIV+ patients have been revealing to be infected by Leishmania parasites with rare zymodemes that have never been described in immunocompetent persons or dogs. This fact supports the hypothesis that those strains are only pathogenic in conditions of immunosuppression and, therefore, are less virulent than those found in immunocompetent hosts. Nevertheless, very little is known about the infective, pathogenic, immunogenic and protective capabilities of these naturally attenuated strains in an immunocompetent host. Therefore, experimental confirmation of the non-pathogenicity of these Leishmania attenuated strains would promote its interest as live attenuated vaccines.
To study Leishmania biology a reliable source of parasites is needed. Many are the options available of semi-define and complete define culture media for the in vitro culture of Leishmania promastigotes. However, the choice of the culture medium to use should be
infectivity of the parasites.
This thesis starts with a systematic analysis of the morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity of L. infantum promastigotes when cultivated in different well-established culture media. Indeed, the different media revealed to leave an imprint in the infectivity of the parasite. Furthermore, using a rational approach from the evaluated media, it was developed a simple serum free culture medium that showed to be useful for long-term low-cost maintenance of L. infantum or studies requiring the production of promastigotes in the absence of proteins.
Four L. infantum strains responsible for cutaneous or visceral leishmaniasis in immunocompetent or immunosuppressed patients were studied in the scope of understanding their infective, pathogenic, immunogenic and protective capabilities in an immunocompetent host. To avoid any biased results, the establishment of the culture settings for the four studied strains that allow the generation of similar promastigotes was successfully done. The murine model of visceral leishmaniasis used in this thesis put on evidence the inherent infectivity of each one of the four L. infantum strains and their potential and differential immunomodulatory capacities.
Finally, for the first time it was evaluated the impact of infection-induced immunity on a secondary homologous or heterologous infection with L. infantum strains in the murine model of visceral leishmaniasis. The two most infective strains were used to assess the cellular innate and adaptive immune responses generated 6 weeks after infection and their efficacy in protecting against subsequent challenge. The high infective strain showed partial protection against re-infection due to the expansion of central and effector memory T cell populations and also by the production of IFNγ by both CD4+ and CD8+ T cells and double producers CD4+IFNγ+IL-10+ and CD8+IFNγ+TNFα+. No protection upon virulent challenge was observed when a strain with lower infectivity was used as imprinting, revealing the need of a virulent infection to generate and maintain appropriate immunity. With the work performed within this thesis it was concluded that inherent characteristics of each infective L. infantum strain are responsible for the tropism and memory generation mechanisms, two processes that remarkably influence the outcome and progression of leishmaniasis. These findings contribute to the general acceptance that leishmaniasis is a multifactorial disease with the clinical outcome being highly dependent on the infectivity of the strain and the susceptibility of the host.
tropism, infection-induced immunity, memory, protection.
Leishmania spp. são parasitas protozoários responsáveis por um grupo de manifestações clínicas designadas de leishmaniose. O parasita é transmitido ao mamífero hospedeiro pela picada de uma mosca da areia infetada com forma promastigota. O promastigota metacíclico é a forma infeciosa para o mamífero, mas para sobreviver no interior dos macrófagos do hospedeiro o promastigota tem rapidamente de converter-se na forma amastigota intracelular.
A leishmaniose é uma doença zoonótica e antroponótica dispersa mundialmente pelas regiões mediterrânica, tropicais e subtropicais. Desde sempre tem sido considerada uma doença infeciosa negligenciada, mas a emergência de co-infeções HIV/Leishmania em países desenvolvidos trouxe um novo interesse à doença. Os fármacos anti-Leishmania apesar de eficazes constituem um tratamento altamente tóxico e que está associado a custos elevados, não só pelo preço do tratamento em si mas também devido à hospitalização requerida na maioria dos casos.
A procura da vacina humana para a leishmaniose visceral, a forma mais grave da doença, remonta há várias décadas mas sem nunca alcançar resultados positivos. No entanto, há três vacinas licenciadas para a leishmaniose canina, um fator importante na epidemiologia da leishmaniose no Brasil e sul da Europa onde os cães são o principal reservatório doméstico.
Há alguns anos atrás o nosso grupo descreveu a eficácia de uma estirpe viva de Leishmania infantum geneticamente atenuada pela remoção de um dos alelos do gene sir2 na proteção contra uma infecção com a estirpe selvagem no modelo de leishmaniose visceral murina. Com o conhecimento adquirido nesse trabalho e mantendo o interesse na investigação da vacina para a leishmaniose visceral, quisemos explorar a possibilidade de estirpes naturalmente atenuadas induzirem proteção contra uma infeção virulenta.
Nalguns casos, os doentes HIV+ co-infetados têm demonstrado ser infetados por estirpes de Leishmania com zimodemos pouco frequentes que nunca foram descritos em pessoas imunocompetentes ou em cães. Este facto suporta a hipótese de estas estirpes serem patogénicas apenas em condições de imunossupressão e, por isso, serem menos virulentas do que aquelas encontradas em hospedeiros imunocompetentes. No entanto, pouco se sabe sobre a infetividade, patogenicidade, imunogenicidade e capacidade protetora destas estirpes naturalmente atenuadas num hospedeiro imunocompetente. Por isso, a confirmação experimental da não-patogenicidade destas estirpes atenuadas de Leishmania iria levar ao seu interesse como vacinas vivas atenuadas.
Muitas são as opções disponíveis de meios de cultura semi-definidos e totalmente definidos para a cultura in vitro de promastigotas de Leishmania. No entanto, a escolha do meio de cultura a usar deve ser uma decisão racional uma vez que influencia o crescimento, o desenvolvimento e, consequentemente, a infetividade dos parasitas.
Esta tese inicia-se com a análise sistemática da morfologia, viabilidade, progressão durante o ciclo celular, perfil de metaciclogénese, capacidade de diferenciação em amastigotas axénicos e infetividade de promastigotas de L. infantum em diferentes meios de cultura bem caracterizados na área. Efetivamente, a escolha do meio de cultura revelou-se determinante na infetividade do parasita. Além disto, usando uma abordagem racional a partir dos meios avaliados, desenvolveu-se um meio de cultura simples sem soro que mostrou ser interessante para a manutenção de L. infantum por longos períodos de tempo de forma económica ou para aplicação em estudos que exijam a produção de promastigotas na ausência de proteínas.
Quatro estirpes de L. infantum agentes de leishmaniose cutânea ou visceral em doentes imunocompetentes ou imunodeprimidos foram estudadas no sentido de compreender as suas capacidades de infeção, patogénicas, imunogénicas e protetoras num hospedeiro imunocompetente. Para evitar resultados tendenciosos, o estabelecimento das condições de cultivo para as quatro estirpes em estudo que permitem a geração de promastigotas semelhantes foi conseguido com sucesso. O modelo murino de leishmaniose visceral usado nesta tese evidenciou a infetividade intrínseca de cada uma das quatro estirpes de L. infantum e as suas diferentes capacidades potencialmente imunomodulatórias.
Finalmente, pela primeira vez foi avaliado o impacto da imunidade induzida pela infeção na infeção secundária homóloga ou heteróloga com estirpes de L. infantum no modelo murino de leishmaniose visceral. As duas estirpes com maior infetividade foram usadas para determinar as respostas celulares inata e adaptativa geradas 6 semanas após infeção bem como a sua eficácia na proteção contra uma infeção subsequente. A estirpe com maior infectividade mostrou proteção parcial contra re-infeção devido à expansão das células T de memória central e efetora e também pela produção de IFNγ pelas células T CD4+ e CD8+ e duplas produtoras CD4+IFNγ+IL-10+ e CD8+IFNγ+TNFα+. No entanto, não foi observada proteção quando se usou a estirpe com menor infetividade na primo-infeção, revelando a necessidade de uma infeção virulenta para a geração e manutenção de imunidade adquirida eficaz contra uma re-infeção virulenta.
Com o trabalho realizado nesta tese foi concluído que as características intrínsecas de cada estirpe de L. infantum são responsáveis pelo tropismo e mecanismos de desenvolvimento de memória imunológica, dois processos que influenciam marcadamente a manifestação e a progressão da leishmaniose. Estes dados reforçam a
altamente dependente da capacidade de infeção da estirpe e da suscetibilidade do hospedeiro.
Palavras-chave
Leishmania infantum, leishmaniose visceral, isolados clínicos, meio de cultura, infetividade, tropismo, imunidade induzida pela infeção, memória, proteção.
Leishmania spp. son parásitos protozoarios responsables de un grupo de enfermedades que se conocen de forma conjunta como leishmaniasis. El parásito es trasmitido al hospedador mamífero por la picadura de un flebótomo infectado donde existe en la forma promastigote. El promastigote metacíclico es la forma infecciosa para el mamífero, pero para sobrevivir dentro de los macrófagos hospedadores el promastigote tiene que convertirse rápidamente en la forma amastigote intracelular.
Las leishmaniasis son enfermedades zoonóticas o antroponóticas que se distribuyen por las regiones mediterráneas, tropicales y subtropicales. Se considera una enfermedad infecciosa desatendida, aunque la emergencia de co-infecciones HIV/Leishmania en países desarollados ha generado un nuevo interés por esta enfermedad. Los fármacos anti-Leishmania son eficaces pero el tratamiento es altamente tóxico y está asociado a costes elevados, no sólo por el precio del tratamiento en sí pero también debido a la hospitalización requerida en la mayoría de los casos.
La búsqueda de la vacuna humana para la leishmaniasis visceral, la forma más grave de la enfermedad, se remonta a varias décadas pero sin alcanzar nunca resultados positivos completos. En este momento, hay tres vacunas licenciadas para la leishmaniasis canina, lo que constituye una herramienta importante en el control epidemiológico de la leishmaniasis en Brasil y sur de Europa, donde el perro constituye el principal reservorio doméstico del parásito.
Hace algunos años nuestro grupo describió la eficacia de una cepa viva de Leishmania infantum atenuada genéticamente por la deleción de uno de los alelos del gen sir2 en la protección contra un desafío con la cepa salvaje en el modelo murino de leishmaniasis visceral. Con el conocimiento adquirido de ese trabajo y manteniendo el interés en la investigación de una vacuna para la leishmaniasis visceral, hemos querido explorar la posibilidad de utilizar cepas naturalmente atenuadas en la protección contra una segunda infección virulenta.
Se ha confirmado que parte de los enfermos VIH+ coinfectados estaban infectados por cepas de Leishmania con zimodemas poco frecuentes que no habían descritos en personas inmunocompetentes o en perros. Este hecho apoya la hipótesis de estas cepas sean patógenas sólo en condiciones de inmunossupressión y, por eso, sean menos virulentas que aquellas encontradas en hospedadores inmunocompetentes. Sin embargo, se sabe poco sobre la infectividad, patogenicidad, inmunogenicidad y capacidad protectora de estas cepas naturalmente atenuadas en un hospedador inmunocompetente. Por eso, la confirmación experimental de la no-patogenicidad de
atenuadas.
Para estudiar la biología de Leishmania es necesaria una fuente de parásitos de confianza. Muchas son las opciones disponibles de medios de cultivo semi-definidos y totalmente definidos para el cultivo in vitro de promastigotas de Leishmania. Sin embargo, la elección del medio de cultivo a usar debe ser una decisión racional ya que afecta el crecimiento, el desarrollo y, consecuentemente, la infectividade de los parásitos.
Esta tesis se inicia con el análisis sistemático de la morfología, viabilidad, progresión durante el ciclo celular, perfil de metaciclogenesis, capacidad de diferenciación en amastigotes axénicos y infectividad de promastigotes de L. infantum en diferentes medios de cultivo bien caracterizados en este campo. Efectivamente, la elección del medio de cultivo se reveló determinante en la infectividad del parásito. Además, usando un abordaje racional a partir de los medios evaluados, se desarrolló un medio de cultivo simple sin suero que demostró ser particularmente útil para el mantenimiento de L. infantum por periodos largos de tiempo de forma económica o para su aplicación en estudios que exijan la producción de promastigotes en ausencia de proteínas.
Se estudiaron cuatro cepas de L. infantum causantes de leishmaniosis cutánea o visceral en enfermos inmunocompetentes o inmunodeprimidos, con el fin de comprender sus capacidades de infección, patogénicas, inmunogénicas y protectoras en un hospedador inmunocompetente. Para evitar resultados sesgados, se establecieron con éxito condiciones de cultivo para las cuatro cepas en estudio que permitieron la generación de promastigotes similares. El modelo murino de leishmaniasis visceral usado en esta tesis evidenció la infectividad intrínseca de cada una de las cuatro cepas de L. infantum y sus diferentes capacidades potencialmente inmunomoduladoras.
Finalmente, se evaluó por primera vez el impacto de la inmunidad inducida por la infección en la infección secundaria homóloga o heteróloga con cepas de L. infantum en el modelo murino de leishmaniasis visceral. Las dos cepas con mayor infectividade fueron usadas para determinar las respuestas celulares innatas y adaptativas generadas 6 semanas después de infección así como su eficacia en la protección contra un nuevo desafío. La cepa con mayor infectividad mostró protección parcial contra reinfección debido a la expansión de las células T de memoria central y efectora y también por la producción de IFNγ por las células T CD4+ y CD8+ y dobles productoras CD4+IFNγ+IL-10+ y CD8+IFNγ+TNFα+. No se observó protección contra un desafío virulento cuando se usó la cepa con menor infectividad en la primo-infección, revelando la necesidad de una infección virulenta para la generación y mantenimiento de inmunidad adecuada.
Con el trabajo realizado y los resultados obtenidos en esta tesis se puede concluir que las características intrínsecas de cada cepa de L. infantum son responsables del tropismo y
forma decisiva en la aparición y la progresión de la leishmaniasis clínica. Estos datos refuerzan la idea de que la leishmaniasis es una enfermedad multifactorial cuya manifestación clínica es altamente dependiente de la capacidad de infección de la cepa y de la susceptibilidad del hospedador.
Palabras clave
Leishmania infantum, leishmaniasis visceral, aislados clínicos, médio de cultivo, infectividad, tropismo, inmunidad inducida por la infección, memoria, protección.
AUTHOR’S DECLARATION iii
SCIENTIFIC PUBLICATIONS v
Articles in international peer-reviewed journals v
Publications of scientific meetings vi
Poster communications vii
ACKNOWLEDGMENTS ix
SUMMARY xi
RESUMO xiv
RESUMEN xvii
ABBREVIATIONS LIST xxvii
INTRODUCTION 1
THE DISEASE 3
HIV/AIDS and leishmaniasis 5
Canine leishmaniasis 6
LEISHMANIA BIOLOGY AND TRANSMISSION 7
IMMUNOBIOLOGY OF LEISHMANIASIS 8
Parasite’s strategies 8
Host defense 9
MEMORY DEVELOPMENT 10
Memory in leishmaniasis 12
PROPHYLAXIS AND TREATMENT 14
Vaccines 14
Drugs 16
TRANSFERRING LEISHMANIA SP. LIFE CYCLE TO THE LABORATORY 19
Promastigotes cultivation 19
Amastigotes cultivation 21
WHAT’S LEFT TO BE DONE? 23
OBJECTIVES 25
THE IMPACT OF DISTINCT CULTURE MEDIA IN LEISHMANIA INFANTUM BIOLOGY AND
INFECTIVITY 31
Abstract 34
Background 35
Materials and methods 37
Results 41
Discussion 49
Supplemental data 55
References 59
CHARACTERIZATION OF THE BIOLOGY AND INFECTIVITY OF LEISHMANIA INFANTUM
VISCEROTROPIC AND DERMOTROPIC STRAINS ISOLATED FROM HIV+ AND HIV- PATIENTS
IN THE MURINE MODEL OF VISCERAL LEISHMANIASIS 63
Abstract 66
Background 67
Materials and methods 69
Results and Discussion 75
Conclusions 86
Supplemental data 88
References 93
Related unpublished data 98
HIGH INFECTIVE LEISHMANIA INFANTUM STRAIN INDUCES STRONG CENTRAL AND EFFECTOR
MEMORY CD4+ AND CD8+ IMMUNITY REQUIRED FOR PARTIAL PROTECTION AGAINST RE
-INFECTION 99
Abstract 102
Background 103
Materials and methods 106
Results and discussion 109
Conclusions 117
References 118
ON THE IMPORTANCE OF ESTABLISHING THE CULTURE CONDITIONS FOR THE IN VITRO
GROWTH OF LEISHMANIA SP. PARASITES 125
ON THE STRAIN-SPECIFIC CHARACTERISTICS THAT LEAD TO DIFFERENTIAL INFECTIVITY
AND TROPISM 127
ON THE PROTECTIVE ROLE THAT A HIGH INFECTIVE L. INFANTUM STRAIN DISPLAY AGAINST
RE-INFECTION 130
FINAL REMARKS 132
INTRODUCTION
Figure I. Worldwide distribution of VL and CL 4 Figure II. Worldwide distribution of HIV/Leishmania co-infections 6 Figure III. Zoonotic and anthroponotic Leishmania spp. transmission cycles 7
Figure IV. Differentiation progress of CD4+ and CD8+ effector and memory cells 11 Figure V. Leishmania spp. development in the insect vector and in the mammalian host 20
RESULTS
THE IMPACT OF DISTINCT CULTURE MEDIA IN LEISHMANIA INFANTUM BIOLOGY AND INFECTIVITY
Figure 1. Parameters of in vitro development of L. infantum in the different culture media 41
Figure 2. Dominant morphology of logarithmic and stationary-phase L. infantum 42
Figure 3. In vitro and in vivo virulence of promastigotes grown in different culture media 43 Figure 4. Relative expression of metacyclogenesis-related genes in L. infantum
promastigotes grown in the different media
44
Figure 5. Development of a protein-free medium for the growth of L. infantum
promastigotes
45
Figure 6. Biology of L. infantum promastigotes in cRPMI 47
Figure 7. Overtime virulence loss 48
Figure S1. Adjustment of initial inoculum for SDM and Schneider media 55
Figure S2. Histogram of cell cycle analysis of parasites in different media 55 Figure S3. Cell cycle analysis of parasites in different media 56 Figure S4. Dot plot of bone marrow-derived macrophages infected with CFSE-labeled
parasites
56
Figure S5. Relative gene expression of metacyclogenesis-related genes of promastigotes
cultivated in the different media
57
Figure S6. Dominant promastigote morphology during the process of development of the protein-free media
57
Figure S7. Growth curves of L. infantum in cRPMI after 4 or 20 in vitro passages 58 Figure S8. Influence of different FCS lots on the growth of L. infantum cultivated in RPMI 58
IN THE MURINE MODEL OF VISCERAL LEISHMANIASIS
Figure 1. Molecular characterization of L. infantum isolates 75 Figure 2. Growth of L. infantum in different culture medium 77 Figure 3. Indirect measurement of metacyclogenesis 78 Figure 4. In vitro differential infectivity of L. infantum strains 79 Figure 5. Quantitative distribution of L. infantum in BALB/c mice 2 and 6 weeks after
infection 81
Figure 6. Cell populations in spleens of naive and Leishmania-infected mice in the acute
and chronic phases 84
Figure 7. Leishmania-specific humoral response 85 Additional figure 1. Validation of the qPCR methodology for quantification of the parasite
loads in murine tissues
89
Additional figure 2. K26 gene alignment 90
Additional figure 3. Cell cycle analysis 91
Additional figure 4. Variation of the transcription of metacyclogenesis-dependent genes 92
Additional figure 5. Organ weight 2 and 6 weeks post-infection 92 Figure VI. RFLP patterns of mixed infections 98
HIGH INFECTIVE LEISHMANIA INFANTUM STRAIN INDUCES STRONG CENTRAL AND EFFECTOR
MEMORY CD4+ AND CD8+IMMUNITY REQUIRED FOR PARTIAL PROTECTION AGAINST RE-INFECTION
Figure 1. Parasite load after infection and challenge with L. infantum strains
with different infectivity 109
Figure 2. Splenic cellular populations after infection and challenge with highly and low
infective L. infantum strain 111
Figure 3. Expression of CCR2 on splenic macrophages, neutrophils and dendritic cells 112
Figure 4. T cell memory repertoire 114
INTRODUCTION
Table I. Main disease manifestations of leishmaniasis and the Leishmania species
frequently associated with the disease 4
RESULTS
THE IMPACT OF DISTINCT CULTURE MEDIA IN LEISHMANIA INFANTUM BIOLOGY AND INFECTIVITY
Table S1. Comparative cost of the culture media used in the study 58
CHARACTERIZATION OF THE BIOLOGY AND INFECTIVITY OF LEISHMANIA INFANTUM
VISCEROTROPIC AND DERMOTROPIC STRAINS ISOLATED FROM HIV+ AND HIV- PATIENTS IN THE MURINE MODEL OF VISCERAL LEISHMANIASIS
Table 1. Estimated overall parasite load of L. infantum-infected BALB/c mice 2 and 6
AIDS Acquired Immunodeficiency Syndrome BMMo Bone marrow-derived macrophages CCR CC chemokine receptor CD Cluster of differentiation Challg Challenge
CL Cutaneous leishmaniasis
DCs Dendritic cells
FBS Fetal bovine serum FCS Fetal calf serum
HIV Human immunodeficiency virus IL Interleukin
IFNγ Interferon-γ
ITS Ribosomal internal transcribed spacer IVDUs Intravenous drug users
MCL Mucocutaneous leishmaniasis MLEE Multilocus enzyme electrophoresis MLMT Multilocus microsatellite typing
NK Natural killer
NNN Novy-McNeal-Nicolle medium PCR Polymerase chain reaction
PKDL Post kala-azar dermal leishmaniasis
qPCR Quantitative real time polymerase chain reaction Re-Inf Re-infection
RFLP Restriction fragment length polymorphism
RT-PCR Reverse transcriptase - polymerase chain reaction
SHERP Small Hydrophilic Endoplasmic Reticulum-associated Protein TCM Central memory T cells
TEM Effector memory T cells TGFβ Transforming growth factor-β Th Helper T lymphocyte
TNFα Tumor necrosis factor-α
VL Visceral leishmaniasis
Introduction
The disease
Leishmaniasis designate a group of parasitic diseases caused by the infection of the Leishmania spp. protozoa in humans and several domestic and sylvatic animals. In humans, four main clinical forms of the disease can be identified according to the clinical manifestations:
Cutaneous leishmaniasis (CL) comprises cutaneous lesions that are more or less difficult to heal (depending on the species involved) and leave visible scars when cured, many times in exposed areas like the face or the arms which can be socially stigmatizing [1].
Mucocutaneous leishmaniasis (MCL) affects the mucous layers of the nose, mouth, throat and surrounding tissues, many times leading to mutilating lesions, which can lead to social exclusion. In contrast to CL, treatment is always required because the disease can be life-threatening [1].
Visceral leishmaniasis (VL), also known as kala-azar, is the most severe form of the disease, since it affects the spleen, liver and bone marrow, producing hepatosplenomegalia, weight loss, pancytopenia, hypergammaglobulinemia and intermittent low-grade fever. Is fatal if untreated due to severe cachexia and bleeding (owing to thrombocytopenia that installs with time) [2].
Post kala-azar dermal leishmaniasis (PKDL) is a manifestation that can appear weeks to years after cure of VL by L. donovani infection [1]. It consists of skin papules and hypopigmented macules dispersed through the skin. It is frequent in African and Indian patients but its emergence is not well understood [2].
Up to date, 30 species of Leishmania were described, 20 of them are pathogenic for the humans [3]. In Table I are depicted the main species that affect humans and the related disease manifestation.
Table I. Main disease manifestations of leishmaniasis and the Leishmania species frequently associated with the disease
Main type of the disease Species
Old World, subgenus Leishmania
Visceral leishmaniasis Leishmania donovani and Leishmania infantum
Cutaneous leishmaniasis Leishmania major, Leishmania tropica and Leishmania aethiopica Post kala‐azar dermal leishmaniasis Leishmania donovani New World, subgenus Leishmania Visceral leishmaniasis Leishmania infantum Cutaneous leishmaniasis Leishmania infantum, Leishmania mexicana, Leishmania pifanoi and Leishmania amazonensis New World, subgenus Viannia Cutaneous leishmaniasis Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis and Leishmania peruviana
Mucocutaneous leishmaniasis Leishmania braziliensis and Leishmania panamensis
Adapted from [4]
Leishmaniasis is endemic in 98 countries and 3 territories ranging the Mediterranean Basin, the Middle East, the Indian sub-continent, and the tropical regions from America and Africa [5] (Figure I). The last WHO report on the epidemiology of leishmaniasis estimates that every year 0.7 to 1.2 million new cases of CL are mounted and 0.2 to 0.4 million people develop VL which, in turn, is responsible for 20 000 to 40 000 deaths [5]. Nevertheless, in endemic countries most of the L. infantum- or L. donovani- infected people are asymptomatic carriers or self-healers [6, 7].
Figure I - Worldwide distribution of VL and CL
The relation of leishmaniasis with poverty catalogues it as a neglected tropical disease. In fact, 72 of the endemic countries are developing nations with a burden of 90% of the VL, CL and MCL [9]. In these regions, the majority of the population lives in rural areas, where higher densities of sand flies are found, and are malnourished, a condition that leads to immunosuppression. In addition, HIV concomitant infection is frequent, contributing to a severe state of immunodeficiency [10].
However, leishmaniasis is nowadays an important issue in developed countries due to coinfection cases with HIV where Leishmania arises as an opportunistic infectious agent, the third of the parasitic infections, after Toxoplasma gondii and Cryptosporidium spp. [10]. Indeed, 90% of the reported HIV/Leishmania cases are from Southern European countries, namely Spain, Portugal, Italy and France [11].
HIV/AIDS AND LEISHMANIASIS
HIV/Leishmania co-infections correspond to 2-9% of all the VL cases in endemic countries (Figure II) [12]. The close geographical overlap of Leishmania and HIV infections promote the concomitant infection of both pathogens. In fact, HIV infection increases in 100-2320 times the risk of developing VL in the endemic regions. Intravenous drug users (IVDUs) are an important group in the epidemiology of HIV/Leishmania co-infections. Indeed, IVDUs have contributed for 76% (between 1990 and 1998) to 67% (between 2000 and 2006) of the HIV/Leishmania co-infected cases [12]. Also, 10% of the overall HIV infections are transmitted by the use of intravenous drugs; in some regions of Eastern Europe and Central Asia this number rises to 80% [13]. However, data mainly from the Southern Europe revealed that since the introduction of the HAART regimes routinely the number of co-infection cases has dropped [12].
Both diseases present synergistic detrimental effects on the cellular immune response because they infect similar target cells (macrophages and dendritic cells). Leishmania induces the secretion of tumor necrosis factor-α (TNFα) and IL-1α which upregulate HIV gene expression by NF-κB, while HIV-dependent immunosuppression promotes Leishmania multiplication [14]. VL promotes the progression of HIV infection leading to the clinical manifestation of AIDS, whereas HIV+ patients present repetitive relapses of leishmaniasis and, frequently, amastigotes are found in unusual locations, such as the lungs, tonsils or intestinal tract [12].
Figure II. Worldwide distribution of HIV/Leishmania co-infections
Adapted from [14].
CANINE LEISHMANIASIS
In the veterinary field, dogs are a main concern, not only because of the disease itself, but also because of their importance as reservoir for human transmission. Canine leishmaniasis (CanL) is endemic in 70 countries of the same affected regions as human leishmaniasis, including also the United States of America. In non-endemic countries, however, imported sick dogs constitute an important veterinary and public health problem [15]. CanL is associated to L. infantum infection and, being a systemic disease, clinical manifestations are unspecific. Nonetheless, the existence of skin ulcers, onychogryphosis and marked cachexia are common visible signs in sick dogs. Renal disease is frequently the only symptom in dogs with CanL and chronic renal failure is often the cause of death [15].
Leishmania biology and transmission
Leishmania spp. are trypanosomatid parasites with a digenetic life cycle that is required for the perpetuation of the species. The promastigote is a flagellated motile form that develops in the sand fly vector (Phlebotomus spp. in the Old World or Lutzomyia spp. in the New World). In the digestive tract of the insect, promastigotes multiply and evolve through sequential stages until they differentiate in the final infective form, the metacyclic promastigote [16, 17]. Upon a blood meal on a susceptible mammalian host, metacyclic promastigotes are deposited in the skin and rapidly captured by neutrophils [18], dendritic cells (DCs) [19] and macrophages [20]. To survive inside these cells, a dramatic differentiation step occurs and promastigotes develop into amastigotes, non-motile intracellular forms which are able to resist and multiply inside the aggressive milieu of the phagocytes phagolysosomes [21]. Intracellular amastigotes are spread throughout the organism reaching distant locations from bitten sites, whether residing in the skin or being capable to enter the viscera and bone marrow.
Leishmania spp. can be transmitted by zoonotic or anthroponotic cycles. L. major [22,23] and L. infantum are usually of zoonotic transmission, with dogs as the main reservoir, while L. donovani and L. tropica are generally of anthroponotic origin [24]. A segregation of the different clinical manifestations related with its reservoir host can, then, be made as follows: zoonotic VL (by L. infantum), zoonotic CL (by L. major), anthroponotic VL (by L. donovani) and anthroponotic CL (by L. tropica). In addition to these natural cycles, an important alternative artificial anthroponotic cycle has been revealed more than one decade ago with the analysis of blood remnants in syringes discarded by IVDUs [25] (Figure III).
Immunobiology of leishmaniasis
PARASITE’S STRATEGIES
The diversity of clinical manifestations expressed in leishmaniasis derives from a complex interaction between the parasite and the host’s immune system. In fact, the outcome of the disease is strongly influenced not only by inherent characteristics of the infective strain but also by the fitness of the immune system in generating a protective response.
Leishmania spp. are masters at disguising their entry into the host as they downregulate the activation signals that otherwise would prompt an effective immune response. Promastigote antigens from the surface glycocalix like gp63 protease (leishmanolysin) or the lipophosphoglycans (LPG) are known to provide specific resistance to complement-mediated lysis, the first attack to pathogens that reach the blood, and to participate in the silent entry of the parasite into the phagocytes [27]. On their hand, amastigotes released from disrupted macrophages use opsonization with IgG’s Fcγ moiety to enter in new macrophages without triggering their activation through ligation to Fcγ receptor [27]. A more elegant approach relies on the “Trojan horse” strategy, where Leishmania takes advantage of the rapid arrival of neutrophils to the inoculation site and uses them to blindly enter the macrophages as they phagocyte apoptotic infected neutrophils [28]. However, this mechanism has not yet been demonstrated to occur in vivo and in situ studies failed to prove it, though the involvement of neutrophils in the early steps of infection is undoubtable [4].
Once inside the phagocyte, Leishmania is able to downregulate the co-stimulatory molecules CD40 and CD86 on DCs [29] and CD40 and MHC class II on macrophages [30] that are needed for the proper activation of T cells. Also, it induces the establishment of a more favorable anti-inflammatory milieu by increasing 10 and diminishing 12, IL-6 and TNFα secretion by those cells [29, 30]. This immunomodulation arrests the maturation process that is usually driven by the interaction of a pathogen-associated molecular pattern (PAMP) with Toll-like receptors (TLRs) and interferes with the cross-talking between innate and acquired immune responses, allowing the parasite to favorably infect the spleen, liver, lymph nodes and bone marrow in VL [27].
One of the hallmarks in leishmaniasis immunobiology is the hypergammaglobulinemia due to polyclonal expansion of B cells. In CL a clear segregation of IgG2/IgG1 antibodies in the dichotomy of Th1/ Th2 responses is related to protection/progression of the disease [31, 32]. In VL this is not the case and, in general terms, a strong humoral response is detrimental and increases the severity of the disease [33, 34]. However, the appearance of IgG2 after vaccination is usually considered a good marker of protection [35].
HOST DEFENSE
The primary strategy that the host presents to fight against Leishmania is non-specific and relies on innate mechanisms of the immune response. As mentioned above, complement-mediated lysis is the first trap that promastigotes must circumvent to succeed in infection and, in fact, “the messers become the messies” since Leishmania cleave the C3b factor transforming it in a C3b1-like molecule that is used by the parasite to enter unnoticeably in macrophages [27]. Also, Leishmania takes advantage of the C5a cleaved fragment since it has a negative impact on the TLR4-induced synthesis of IL-12 family of cytokines [27]. Even so, activation of DCs upon contact with Leishmania still occurs in those cells that did not engulf parasites - the so called bystander cells - leading to the secretion of IL-12 [33,37].
IL-12 is considered a key cytokine in the early development of the effective immune response due to its requirement for the activation of NK cells and T lymphocytes [38]. Activation of these cells leads to the secretion of interferon-γ (IFNγ), another commander cytokine.
Both in mice as in humans, macrophages are classically activated by IFNγ. This leads to the transcription of inducible nitric oxide synthase (iNOS) and phagocyte NADPH oxidase (phox) that produce nitric oxide (NO) and reactive oxygen species (ROS), respectively, specimens generally considered indispensable for macrophage-direct killing of Leishmania [27]. Macrophages activated by IL-12-driven IFNγ secretion by Th1 lymphocytes - M1 macrophages - also produce TNFα, IL-1β and IL-6, pro-inflammatory cytokines that favor the protective response against Leishmania infection. These macrophages are, then, both effectors and inducers of the Th1 polarized immune response [39]. Nevertheless, the strong Th1 pro-inflammatory response must be balanced with the secretion of IL-10 and transforming growth factor-β (TGFβ) to avoid immunopathology through excessive tissue damage [40].
However, depending on the Leishmania species involved and the fitness of the host’s immune system, an alternative activation of the macrophages, through IL-4 and IL-13 secretion by Th2 lymphocytes, can command an immune response favoring the progression of the infection. These so-called M2 macrophages upregulate arginase 1 which promotes the biosynthesis of polyamines that Leishmania can take up and use for proliferation [40].
Memory development
Effector CD4+ and CD8+ T cells that were activated by the recognition of Leishmania antigens on the cognate T cell receptor (TCR) and expanded to respond to infection will face a massive contraction on their numbers of about 90% after the elimination of the parasite, leaving a subset of experienced cells that constitute the memory pool. Memory cells are long-lived cells that rapidly expand in response to a secondary challenge with the priming antigen [41]. They form a heterogeneous pool with distinct abilities in proliferation, migration and cytokine production that can be easily identified by the differential expression of some surface molecules, usually CD44 and CD62L in mice [42] or CD45RA/RO and CD62L or CCR7 in humans [43].
CD44 is the most widely marker for selecting antigen-exposed T cells as, after it has been upregulated on activated lymphocytes, its expression is sustained on effector and memory murine cells. Through the interaction with its major ligand, the hyaluronic acid, CD44 regulates cell adhesion and migration, critical phenomena for the recruitment and function of effector and memory cells. Besides increasing T cell activation and promoting T cell survival, CD44 also contributes to the regulation of the contraction phase that takes place after the elimination of the pathogen and the maintenance of tolerance [44]. In humans, CD45RA is expressed in naïve cells, whereas memory cells upregulate CD45RO in response to antigen priming [45].
CD62L (L-selectin) is a transmembrane protein with cell adhesion properties and receptor signaling functions. Both in mice and in humans, it is expressed in the surface of the majority of circulating leukocytes contributing to their regulated recruitment to the lymph nodes and inflamed tissues [46, 47]. In the lymph nodes, CD62L+ T cells become activated by the interaction with antigen presenting cells, which leads to its cleavage. CD62L shedding from the surface of activated T cells allows them to re-enter circulation and be directed to target sites where they are needed to employ their effector functions [48]. The surface expression of CD62L allows, then, and in coordination with CD44 expression to differentiate the two main memory populations, both in CD4+ and in CD8+ T cells: central memory T cells (TCM) are CD44hiCD62L+ while effector memory T cells (TEM) are CD44hiCD62L- [42].
The CCR7 surface expression is another segregation marker for effector and central memory cells. Although it is usually synchronized with CD62L, some heterogeneity on its expression has been found in murine memory cells, making CD62L a more reliable indicator of memory populations in mice [48].
The divergent expression of CD62L translates into different trafficking and tissue predominance of TCM and TEM cells, as well as cytokine production and proliferative
capacities. TCM cells are found principally in the lymph nodes and spleen where they secrete high amounts of IL-2 and exhibit high proliferation. TEM cells, on the contrary, are found mainly in the blood, peripheral organs and also in the spleen, show low proliferative capacity and strong effector function due to immediate ability of IFNγ secretion and accumulation of granzyme B and perforine in granules characteristic of CD8+ T cells [42, 48].
Antigen-specific TCM cells develop in the presence of the pathogen, though after its elimination they endure and are the ones that support the rapid response mounted upon challenge [47, 49]. On the contrary, TEM cells are strong effectors but are eliminated in the absence of the antigen [47, 49]; when needed to face a challenge they are provided by the TCM experienced pool [50]. This is in accordance with the linear model of differentiation observed in mice, humans and non-human primates in which cells progressively gain function from TCM to TEM to terminal effectors [48], as shown in Figure IV. In this figure the relation of the memory phenotype and the cytokines these cells produce is well discriminated.
In CD4+ T cells TNFα is the most common cytokine to be detected when a Th1 milieu is developed [51]. Also IL-2 is found very often in conjunction with TNFα and, although it has no major effector function, IL-2 promotes lifelong memory cells (even in the absence of TNFα). These highly sustainable cells can further secrete IFNγ upon another activation signal, eventually becoming short-lived terminal effector IFNγ single producers cells if the antigen persists (Figure IV-a) [51]. CD8+ T cells follow a linear progression similar to the CD4+ T cells trajectory, though TEM cells are able to regain IL-2 expression and revert to TCM state that concomitantly produce IFNγ, TNFα and IL-2. The fixed lineage model is also depicted in Figure IV-b showing that TEM cells can be directly originated from effector cytotoxic CD8+ T cells [51]. The desirable memory phenotype is, then, to have multifunctional effector cells that concomitantly produce IFNγ and TNFα along with IL-2 that enhances the expansion of the memory pools, contributing to a better effector response [51].
MEMORY IN LEISHMANIASIS
Memory cells were demonstrated to have great importance in the control of leishmaniasis, with distinct roles described for TCM and TEM cells. Zaph et al. have shown that in mice both TCM and TEM CD4+ cells require parasite presence to be developed, though maintenance of TCM is independent of antigen persistence [49]. This achievement, however, seems highly dependent on the initial overall T cell response, since in some immunization experiments that used low dose of parasites protection was lost after the elimination of the parasites, possibly due to insufficient expansion of the TCM pool [52]. Adoptive transfer of TCM from L. major-infected mice to naïve animals conferred protection upon a challenge. When facing the antigen, TCM expanded in the lymph nodes, acquired effector functions, including CD62L downregulation which allowed their migration to the infection site and effective protection [49].
Nevertheless, concomitant immunity, i.e. efficient protection upon a challenge due to the long-term and simultaneous persistence of the pathogen, seems to be a hallmark in leishmaniasis [53]. Studies using mice models have shown that a small numbers of parasites restricted to the inoculation site, without causing clinical manifestations, are essential for protection from a virulent challenge [52]. In fact, this is the concept behind the leishmanization strategy applied in humans, discussed below, where a small amount of Leishmania virulent parasites are inoculated in a hidden location of the skin with the objective of protection from a real challenge [54].
The regulation of the effector responses prone to Leishmania elimination to avoid the development of the disease but still leaving a restricted parasite population that maintains the lifelong memory is done by the CD4+CD25+FoxP3+ regulatory T cells [53]. However, these cells are also responsible for the reactivation of the persistent parasites in mice [55], so a tight balance between effector and regulatory T cells must be achieved in order to retrieve efficient recall responses.
Prophylaxis and treatment
VACCINES
Until date, the only successful, long-lasting strategy for human immunization against leishmaniasis was the leishmanization process. It consists on the inoculation of live virulent parasites in a hidden area of the skin of healthy people with the purpose of development of immunity when challenged by a natural infection. Leishmanization showed 100% protection when used as prophylaxis for cutaneous leishmaniasis (CL) throughout the ex-Soviet Union, Asia, and the Middle East [56]. Due to risk of complications in healthy people and difficult standardization of the live L. major inoculum, this procedure was mostly abandoned. However, this is still a current practice in Uzbekistan [56] and a few years ago it was reported to be applied in the evaluation of the efficacy of new vaccines [57].
A “natural” form of leishmanization may be the reason why in Sri Lanka so many cases of CL by L. donovani are reported while VL is rare [8]. McCall et al. have recently reproduced this scenario in the BALB/c model, immunizing the mice subcutaneously with a dermotropic L. donovani strain from Sri Lanka followed by intravenous challenge with a viscerotropic autochthonous strain, and indeed, partial protection was obtained in the liver of the infected mice [58]. The authors attributed the ability of the cutaneous strain to protect against the challenge with the visceral strain to a probable great similarity between the two L. donovani strains, to justify the opposing phenotype observed by others [59]. Also, an epidemiological study in Sudan indicated that only individuals previously negative for leishmanin (Montenegro skin test) developed VL, thus, though without scientific evidences, the leishmanin-positive individuals that were possibly formerly infected with L. major were protected against the visceral disease [60].
First generation vaccines comprise killed parasites and live attenuated parasites. They were primarily developed to overcome one of the major concerns related to leishmanization: the risk of disease development in immunocompetent persons and the total improperness for immunosuppressed patients for this same reason.
With more or less success, some examples of killed vaccines include L. braziliensis crude antigens tested in dogs [61] and trivalent (L. braziliensis + L. guayanensis + L. amazonensis) phenol-killed whole Leishmania promastigotes with bacille Calmette-Guérin (BCG) as adjuvant in Ecuadorian children [62]. According to a meta-analysis conducted in 2009 by Noazin et.al. to evaluate the efficacy of the clinical trials performed with whole killed parasites in endemic areas since 1970s, with the exception of this latter in Ecuador,
none of the other eight clinical trials (based on autoclaved L. major with BCG tested against CL in the Old World and L. amazonensis or multivalent preparations inactivated with merthiolate used against CL in the New World) showed significant protection against natural infection [63]. A new option was tested recently: a killed but metabolically active (KBMA) L. infantum. This vaccine showed partial protection in spleen and liver of BALB/c mice 2 and 8 weeks after challenge triggering a mixed Th1/Th2 response but the authors claim that improving results could be obtained by adding TLR agonists and Th1 adjuvants [64].
For the live attenuated parasites many are the works reported whether using physical, chemical or genetic manipulation for reducing the virulence of the strains or even naturally attenuated strains, like the non-pathogenic L. tarentolae [65]. Some of the most successful vaccine candidates for VL based on genetically altered live parasites were L donovani biopterin transporter gene knockout (KO) (BT1−/−) [66], L donovani replication deficient centrin gene KO (Cen−/−) [67], L donovani cytochrome c oxidase complex component p27 gene KO (Ldp27−/−) [68], L. infantum silent information regulatory 2 single KO (SIR2+/−) [69] and L. tarentolae expressing L. donovani A2 antigen [70]. Despite showing hopeful efficiency in murine models, the promising candidates that were tested in human and canine diseases failed to protect (reviewed in [71]).
A different approach relies on recombinant proteins, polyproteins, DNA vaccines, liposomal formulations and dendritic cell vaccine delivery systems [56]; these constitute the second generation vaccines. A variety of antigens have been tested, though only few in the scope of VL. These include rgp63, rHASPB1, rA2 and the polyprotein rLeish-111f in the group of the recombinant proteins; LiESA, FML and amastigote P8 as purified antigens; LACK and KMP-11 in DNA vaccines (all reviewed in detail in [72]).
For VL, the best vaccine candidate so far tested is the Leish-111f Leishmania recombinant antigen combined with MPL-SE adjuvant. After having proved to protect in murine CL [73] and VL [74] it has also demonstrated to be safe and well tolerated in humans [75]. Clinical trials in dogs have resulted in disparate conclusions about the efficacy of the vaccine in the prophylaxis of CanL [76, 77], though survival of infected dogs was increased after vaccination and treatment with glucantime [78].
In canine vaccinology, however, authorized vaccine options are available. Leishmune® was the first vaccine licensed for the prevention of CanL but is authorized only in Brazil. It consists of L. donovani purified fuccose-mannose ligand (FML antigen) in combination with a saponin adjuvant. Clinical trials have showed that Leishmune® reduces the risk of infection but also prevents disease progression in already infected dogs, though the
manufacturer does not recommend the vaccine as immunotherapy. A transmission-blocking activity was also attributed to this vaccine, making it highly appealing for the control of the zoonosis [79].
Some years later, Leish-Tec® was released, also only in Brazil. The recombinant A2 protein is the antigen that constitutes the vaccine along with saponin adjuvant. Protection was found to be related to high levels of IgG and IgG2 anti-A2 antibodies, without the presence of IgG1, and high amounts of IFNγ with low levels of IL-10 [80].
Recently, a new vaccine, CaniLeish®, the only authorized in Europe, has entered the market for the prophylaxis of CanL. The manufacturer claims that vaccinated dogs reduce the risk of developing the disease in 4 fold compared to non-vaccinated animals [81]. The use of L. infantum excreted/secreted proteins associated to QA-21 adjuvant (LiESP/QA-21) leads to the increase of IgG2 specific antibodies, stronger Leishmania-specific lymphoproliferation with increased IFNγ-producing T cell population that is able to activate a significant leishmanicidal macrophage ability in vitro due to NO production [35].
DRUGS
According to WHO guidelines, treatment should be given only after confirmation of the disease and following national and regional recommendations. In some cases supportive treatment, like rehydration, nutritional supplementation or blood transfusions might be needed before starting the therapy to improve prognosis [24].
Currently six drugs are available for the treatment of VL: pentavalent antimonials (sodium stibogluconate and meglumine antimoniate), the antifungal amphotericin B (AmpB in the deoxycholate form and the lipid formulation), the anticancer drug miltefosine and the antibiotics paramomycin and pentamidine. Pentavalent antimonials are the first line treatment, followed by AmpB and miltefosine [24]. The treatment is usually prolonged and requires parenteral (intravenous or intramuscular) administration, with the exception of miltefosine that is available in oral formulation. All six drugs present high toxicity which increases the treatment cost due to the necessary monitoring to control the adverse reactions.
Adverse reactions of pentavalent antimonials include nausea and vomiting, headache, myalgia and arthralgia, pancreatitis, cardiotoxicity, pancytopenia and peripheral neuropathy. Following AmpB administration it is frequent to observe thrombophlebitis of the injected vein, high fever, rigor and chills; hypokalemia and myocarditis are less common but serious, making hospitalization mandatory during AmpB treatment. Lipid formulations of AmpB allow the reduction of some side-effects though maintaining the
