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Home arrow Economics arrow American Trypanosomiasis Chagas Disease, Second Edition: One Hundred Years of Research


Vaccine development for Chagas disease 32

A.M. Padilla1'*, C.P Brandan2'* and M.A. Basombrio2

''University of Georgia, Athens, GA, United States, 2Universidad Nacional de Salta,

Salta, Argentina

Chapter Outline

Introduction 773

Immune mechanisms associated with protection against Trypanosomacruziinfection 774

DNA vaccination in experimental models of Trypanosoma cruzi infection 778 Vaccination with attenuated parasites (premunition) 780

Basic laboratory studies on premunition against Typanosoma cruzi 780 Field studies on premunition in guinea pigs and dogs 783

Generation of attenuated parasites by genetic manipulation and their use as potential vaccines against Chagas disease 783 Final considerations 788 Acknowledgments 789 References 789


Vaccines have had an indisputable impact on the control of many important human and veterinary diseases and unquestionably have shaped the health landscape of the last generations. Nevertheless the crucial benefits obtained with vaccines for diseases like smallpox and poliomyelitis, many tropical diseases, commonly referred to as neglected diseases, suffer the lack of an effective vaccine. Chagas disease is one of these neglected diseases and it is a major health problem in Latin America countries, especially the poorest ones. The advantages of having a vaccine that prevents Chagas disease have been estimated to be significant not just in terms of public health but also in terms of economic and social development.1 Although some important advances on the comprehension of human immune response to Trypanosoma cruzi infection have been done, the vast majority of our knowledge about immune mechanisms and protective response comes from experimental animal models.

* Both authors contributed equally to this chapter.

American Trypanosomiasis Chagas Disease. DOI:

Copyright © 2017 Elsevier Inc. All rights reserved.

Immune mechanisms associated with protection against Trypanosoma cruzi infection

In recent years increasing knowledge about the immune response associated with Chagas disease has been invaluable for the design and testing of vaccination approaches, although still fundamental questions remain obscure. We need to define the immune response against T. cruzi to better understand the protective mechanisms involved, in order to improve them as well as to identify the weaknesses in the response that allows parasite persistence in the chronic infection.

Trypanosoma cruzi infection is naturally initiated by the invasion of the parasite through mucosa or skin lesions that get in contact with the parasite-containing feces deposited by the insect vector. Once the parasite crosses the skin barrier it encounters the host tissue cells at the site of entry and the normal immune cells that populate that tissue or are recruited there by the lesion. In T. cruzi infection it is possible that the first immune cells to be recruited at the site of entry are also neutrophils as in Leishmania infection2 but probably the majority of the parasites directly infect tissue cells at the site rather than recruited immune cells. This notion is supported by the little parasite migration to surrounding tissues or draining lymph nodes and the evidence of parasite proliferation at the site of infection.3 Therefore, infecting trypomastigotes probably invade host tissue cells at the site of infection (e.g., fibroblasts), transform to cytoplasmic amastigotes forms, and proliferate, with very few parasites spread to some other adjacent tissues or draining lymph nodes.4 It is not well established if the few parasites that get access to the draining lymph nodes right after infection make their way by themselves or are passively transported by immune cells like dendritic cells that migrate to the lymph nodes after acquiring antigen in the periphery. Ultimately, after several rounds of replication inside tissue cells, parasites would be released to gain access to other distant organs through the bloodstream. Thereupon, parasites that arrive to the draining lymph node right after the initial infection do not seem to be effectively presented to trigger an adaptive immune response, which is developed only after the first week postinfection, coincident with the release of parasites that replicate at the site of infection.3,5

Several factors seem to be involved in this delay in the onset of the adaptive immune response during the first days postinfection, including the parasite number and the activation signals provided to the antigen presenting cells that will initiate the adaptive response. This early period seems to be a rather immunologically “silent” one with very few immune mechanisms that reveal the infectious process going on. A vaccination approach that shortens this response time could have a favorable impact controlling the first parasite proliferation at the site of infection. However if during this “silent” period, parasites are not “visible” to the immune system due to an insufficient activation of the antigen presenting cells that display the relevant antigens, the presence already of memory cells from a previous vaccine may not drastically modify this initial response time. On this sense, some evidence suggests that reinfecting parasites may be rapidly controlled by a fully activated immune response maintained by an ongoing infection (premunition), but this

parasite control at the site of infection may be delayed when the previous infection has been resolved leaving resting memory cells that need to be reactivated by antigen presentation. It could be suggested that the delay in the generation of the adaptive response would allow the parasites to reach other tissues like muscle or adipose tissue where immunological or metabolic factors could allow them to chronically persist.6,7 Based on the hypothesis that an insufficient activation of the antigen presenting cells during T. cruzi infection could influence the speed of the origination of the immune response, vaccines based on live-attenuated parasites should incorporate additional immune activating molecules to those originally provided by the parasites. So far some T. cruzi molecules have been shown to activate Toll-like receptors TLR2 and TLR9,8 although these TLR ligands, GPI anchors, and DNA may not be freely available in live parasites initiating the infection which would reduce their activating effect on antigen presenting cells.9 In agreement with this hypothesis, parasites genetically modified to express TLR ligands from other pathogens induce an earlier immune response.10

Once generated, the adaptive immune response is highly efficient in controlling the parasite level, even though this strong response does not reach the total clearance of the parasites. This adaptive immune response is mainly characterized by the presence of specific antibodies, some of which have the capacity to lyse trypomasti- gote forms and are generically called lytic antibodies.11 The cellular branch of this adaptive immune response is characterized by CD41 and CD81 T cells, both of them crucial for parasite control.12,13 Our knowledge about the specific CD81 T cells response notably increased due to the identification of specific parasite epitopes recognized by these immune cells and the application of new immunological techniques.14,15 Among these specific epitopes, the TSKB20 peptide (ANYKFTLV) present in some proteins of the transialidase superfamily has been successfully used to follow the kinetics of the CD81 T cell response by staining with MHC class I complexes containing this peptide. The CD81 T cell response against TSKB20 is one of the highest responses described so far involving approximately 20—30% of the total CD81 T cell population at its peak.14 After the contraction, these CD81 T cells persist in low levels with characteristics predominantly of an effector memory population16 and a smaller subset of cells displaying central memory markers.17 Despite the strong humoral and cellular response mounted against T. cruzi in the acute phase, parasites manage to avoid clearance and persist chronically. The reasons for the incapability of the immune system to completely eliminate parasites are not fully understood and represent an important aspect to be covered for the rationale of developing an effective vaccine. Recently a lower “fitness” of the CD81 T cells generated during T. cruzi infection compared to those generated by a T. cruzi gene-expressing adenovirus vaccine has been suggested as a possible explanation for parasite persistence, however the precise mechanisms required to drive the CD81 T cell response toward a more effective one are not clear.18

Even though a strong CD81 T cell response is elicited against T. cruzi and the relevance of this lymphocyte population for host survival, the precise mechanisms by which these cells control the infection are not completely understood. Depletion or lack of CD81 T cells leads to high susceptibility and mortality of infected mice19 and it has been shown that cytotoxic CD81 T cells from infected animals are able to identify and destroy cells loaded with parasite peptides or parasite infected ones.20 However, it has not been clearly demonstrated yet the importance of these cells to recognize and destroy parasite infected cells in vivo, specially cells known to be chronically infected like muscle fibers or adipocytes, nor that this cytotoxic lysis of infected cells is an effective mechanism to control parasite load in vivo. Furthermore, experiments with perforin-deficient mice yielded contradictory results in terms of susceptibility to the infection.15,21 Therefore, the development of a vaccine that stimulates a strong CD81 T cells response against intracellular parasites is desirable, even when the mechanistic bases of the protection conferred by those cells is still not fully defined.

Other important effector function exerted by CD81 T cells is IFN-gamma production. This cytokine is also produced by CD41 T cells and NK among others cells. IFN-y has been shown crucial in directing the development of naive CD41 T cells toward a Th-1 phenotype as well as activating macrophages. IFN-y is considered a key cytokine involved in the control of T. cruzi because mice deficient in this cytokine are highly susceptible to the infection and succumb in the acute phase. Therefore, it is a generally accepted notion that a vaccine against T. cruzi should induce a response with a Th-1 cytokine profile.22 Regardless of its demonstrated involvement in the resistance to infection and its activation effect on macrophages, there is no clear mechanism linking IFN-y production and parasite control in vivo.

Although there is some contradiction about the importance of the CD41 T cells in the development of the CD81 T cell response during T. cruzi infection,5,23 their role in the control of the parasite is clearly demonstrated by the high susceptibility of mice defective in CD41 T cells which do not survive the acute phase of the infection.24 The helper functions of the CD41 lymphocyte subset in the maturation of the antibody producing B lymphocytes as well as orchestrating the cytokine profile make them a fundamental branch of the immune response. However, relatively little is known about their priming characteristics during T. cruzi infection or other antiparasitic features that would be advantageous to develop during a vaccination protocol. Recently, a new subset of noncirculating memory cells called T resident memory cells has shown promising features as a first line of defense in skin and mucosa, which are common infection routes for many pathogens including T. cruzi. The capacity of these cells induced by vaccination to confer protection has been demonstrated in experimental infections with the closely related parasite Leishmania major.25

As mentioned before, despite the strong immune response against T. cruzi mounted in the acute phase, parasites survive and settle down to a chronic infection. How do parasites avoid the complete clearance by the immune system? How can we boost or modify this response by prophylactic vaccination to block the progression of the infection and prevent the persistence of the parasites?

One of the mechanisms suggested to participate in the chronic persistence of parasites is the apparent dysfunction of the CD81 T cells infiltrating the infected muscle tissue. As demonstrated by Leavey and Tarleton,6 these cells have a lower capacity to produce IFN-y after restimulation in vitro than their counterparts isolated from spleen. However, this dysfunction does not seem to be induced by T regulatory cells or TGF-(3.2627 Although chronic infecting parasites cannot be efficiently removed by the immune system, they can successfully be eliminated by the administration of the trypanomicidal drug Benznidazole.28 After clearance of the parasites, the CD81 T cells populations specific against parasite epitopes change their phenotype from effector to central memory cells. This change in the phenotype agrees with the current opinion that the pathogen persistence continuously stimulates the cells, turning them antigen addictive and avoiding the development of a central memory population. Therefore, the expression of central memory markers (e.g., CD62L and CD127) in the specific CD81 T cell population has been proposed as an indirect evidence of the efficacy of the drug treatment in this mouse model of T. cruzi infection. Unfortunately, the cure by drug treatment does not provide sterile immunity and cured mice displaying specific central memory CD81 T cells are able to better control a subsequent infection but unable to completely eliminate the totality of the reinfecting parasites.29 This poses a considerable challenge for the development of a vaccine against T. cruzi infection that provides sterile immunity, since such a vaccine should stimulate and maintained protective mechanisms that are not obtained after the cure of a natural infection. Regarding this matter, the immune response elicited and maintained by a current infection is usually strong enough to provide protection against a second infection. So an ideal vaccine should elicit and keep a response as strong as the one produced by a virulent infection but lacking the pathogenic effects produced by persistent parasites. So far some experimental vaccines with attenuated live parasites have been shown to provide a strong protection against a subsequent reinfection with more virulent parasites, although the parasites from the secondary infection are rarely completely cleared. Also it is highly probably that the maintenance of the protective effect is associated with the persistence of the vaccinating parasites. Moreover, commonly the strength of the immune response originated by the attenuated live parasites used as a vaccine is lower than a response induced by fully virulent parasites.

Here is another paradox of the immune response in T. cruzi infection: How is the immune response maintained in the chronic phase so strong as to provide protection against a reinfection but at the same time so inefficient to clear the chronic parasites from the previous infection? How can we boost or redirect the chronic immune response to recognize and eliminate the persistent parasites? Can we design a therapeutic vaccine to modify the already established immune response?

These specific responses persist during the chronic phase and the CD81 T cells specific against TSKB20 do not seem to suffer exhaustion, a common phenomenon seen in other chronic infections which results in the loss of the effector functions of the cells and their final deletion.30 This characteristic of the chronic CD81 T cell response in T. cruzi opens the possibility for a therapeutic vaccine that could boost the immune response to achieve the complete clearance of the chronic parasites. However, choosing the right antigens against which to restimulate the response and turn the chronic parasites “targetable,” as well as modifying the already established immunodominance hierarchy, could be a complicated task. Currently a consortium of academic and industrial partners is working on the development of a therapeutic vaccine based on two recombinant T. cruzi antigens,31 although the capacity of this approach to prevent or delay the onset of Chagasic pathology in human patients is under debate.

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