Current situation
Vaccine for Chagas disease: reality or utopia?
Isabela Resende Pereira
Laboratory of Hematology – Medical School – UFF
Email: resendeisabela@gmail.com
Joseli Lannes
Laboratory of Biology of Interactions/IOC
Email: lannes@ioc.fiocruz.br, joselilannes@gmail.com
Since the 18th century, when Edward Jenner developed a smallpox vaccine, the use of vaccines has resulted in a reduction in morbidity and mortality from infections throughout the world’s population. Vaccines are indispensable resources for individual and public health. Characteristically, a vaccine is composed of a component of the pathogenic microorganism, and may be in its live attenuated, inactivated form, protein or saccharide fractions, deoxyribonucleic acid (DNA), or carried by genetically modified vectors. Vaccines are expected to prevent or act on the establishment of disease, stimulating the vaccinated organism in antigen recognition and inducing an immune response with antibody production and cellular response, generating immunological memory. Thus, a vaccine to be effective must generate a protective immune response against more than one infecting form of the pathogen (evolutionary forms, strains, lineages, genetic variants) and generate a memory immune response, facilitating the recognition of the pathogen in a specific and faster way in future contacts.
In the context of Chagas disease (CD), vaccine preparations that can prevent the disease have been studied for a long time. The first strategy was used by Emile Brumpt (1913), who showed that infection of animals by Trypanosoma cruzi, the protozoan parasite that causes CD, followed by reinfection by this parasite led to partial protection. In the 1950s, strategies were used to generate avirulent parasites through previous treatment with chemical attenuating agents, radiation, or serial passages in in vitro culture, aiming to preserve (i) the immunogenicity of the preparation, that is, its ability to induce immune response, as well as efficacy, (ii) the ability to induce protection, in addition to being (iii) safe, ie, not inducing disease (Bhatia and collaborators, 2004). In the 1960s, Menezes proposed the use of lyophilized and adjuvanted forms of T. cruzi as a vaccine preparation. However, the protection offered by vaccines with attenuated or killed parasites, as well as those associated with adjuvant, was similar to that offered by immunization with live forms of T. cruzi, when greater survival and lower parasitemia are observed in vaccinated mice (Basombrio and collaborators, 1982; Basombrio and collaborators, 1987; Paiva and collaborators, 1999b). In this sense, in the 1990s, we used the CL-14 strain (non-pathogenic clone originating from the CL strain) of T. cruzi to understand the protective role of components of the vertebrate host’s immune response. We showed that infection of mice with clone CL-14 not only did not induce pathology, it also prevented splenomegaly and polyclonal activation of T-cells, characteristics of T. cruzi infection associated with pathology (Paiva and collaborators, 1999a). On the other hand, exposure of animals to T. cruzi clone CL-14 induced a protective immune response after challenge with infective forms, with reduced parasitemia and increased survival of animals dependent on the activation of CD8+ T-cells. Also, CL-14 vaccination led to reduced CD4+ and CD8+ T-cell hyperactivation after challenge. Taken together, the data suggested that CD8+ T-cell-dependent, less inflammatory and more regulated immune response is associated with disease protection in T. cruzi infection (Paiva and collaborators, 1999b).
With the evolution of methods and techniques that allowed more refined biochemical and molecular studies, it became possible to select proteins from parasite fractions, as well as immunogenic epitopes contained in a given protein and test their ability to generate an immune response and protection to the challenge with T. cruzi. Some molecular candidates have excelled in inducing a protective immune response, such as cruzipain proteins, present in amastigote and trypomastigote forms, surface proteins of trypomastigote forms of the trans-sialidase (TS) family, paraflagellar rod protein, among others (Cazorla and collaborators, 2009). In the last 20 years, numerous groups have been testing in different protocols and experimental models the use of recombinant proteins, DNA vaccines or vaccines having recombinant viruses as vectors, expressing epitopes or genes of the TS family in order to obtain a protective immune response against T. cruzi infection (Garg and Tarleton, 2002; De Alencar and collaborators, 2009; Boscardin and collaborators, 2003; Vasconcelos and collaborators, 2004; Machado and collaborators, 2006). The amastigote surface protein (ASP)-2, important for the establishment of chronic infection (Boscardin and collaborators, 2003; Vasconcelos and collaborators, 2004), and TS, an enzyme of the trypomastigote forms that catalyzes the transfer of acid sialic acid from host glycoproteins to receptor molecules on the parasite’s membrane (Schenkman et al 1994), belong to the same gene family and have been described as immunodominant proteins (Low and collaborators, 1998; Myahira and collaborators, 2005; Araujo and collaborators , 2005). Prophylactic administration of vaccine preparations containing ASP2 and TS elicited a humoral and cellular immune response, in addition to reducing parasitemia and increasing survival of mice vaccinated and challenged with the Y – DTU II strain of T. cruzi (Machado and collaborators, 2006; Haolla and collaborators, 2009, de Alencar and collaborators, 2009; Barbosa and collaborators, 2013). Vaccination with DNA of plasmids containing genes encoding cruzipain proteins (Schnapp and collaborators, 2002) and trypomastigote surface antigen-1 (TSA-1) (Wizel and collaborators, 1998) resulted in induction of partial immunity, without leading to immunity capable of preventing infection. Another strategy used was the heterologous protocol using plasmid DNA in the prime (induction) and the recombinant human non-replicating adenovirus type 5 (rAd5) carrying sequences of ASP2 in the boost (boost). This vaccine proposal stimulated a protective immune response, associated with an increase in the frequency of T. cruzi-specific memory CD8+ T-cells (Rigato and collaborators, 2011). Importantly, the prophylactic administration of vaccine preparations containing ASP2 and TS elicited a humoral and cellular immune response, in addition to reducing parasitemia and increasing the survival of vaccinated mice, with a reduction in the percentage of animals with electrical alterations in the chronic phase of infection (Machado and collaborators, 2006; Haolla and collaborators, 2009, de Alencar and collaborators, 2009; Barbosa and collaborators, 2013). In addition to the classic proposal for immunoprophylactic use, we proposed the use of vaccine as a therapeutic strategy with the objective of stimulating protective immunity and preventing the progression of the cardiac form of CD. In this study, we used the strategy of the homologous prime-boost protocol with rAd5 carrying sequences encoding ASP2 and TS from T. cruzi (rAdVax). This protocol preserved specific immunity mediated by interferon gamma (IFNγ) and decreased the frequency of potentially cytotoxic (perforin-expressing) CD8+ T-cells. In addition, vaccination with rAdVax reversed electrical changes, reduced histopathological changes such as cardiac fibrosis, and increased the survival of animals infected with different strains of T. cruzi (CL – DTU VI, Colombian – DTU I). Furthermore, the therapeutic vaccine administered to animals chronically infected with the Colombian strain of T. cruzi, which presented electrical alterations compatible with chronic chagasic cardiomyopathy (CCC) and a systemic inflammatory profile, resulted in reversal of cardiac and immune alterations (Figures 1 and 2), suggesting that this is a potential vaccine candidate to be used in the treatment of the cardiac form of CD (Pereira and collaborators, 2015).The protective immune response in T. cruzi infection mainly involves immunological mechanisms that result in the production of specific antibodies to parasite antigens and the immune response involving cells with cytotoxic activity, that is, capable of killing infected cells or expressing parasite antigens, especially cytotoxic T lymphocytes (Figure 1).
Aiming to develop a recombinant vaccine against Chagas disease that would stimulate these protective mechanisms, researchers from Fiocruz and the Interdisciplinary Center for Gene Therapy at the Federal University of São Paulo got together to build recombinant adenoviruses containing T. cruzi antigens. The antigens chosen were trans-sialidase (TS, Figure 2) and amastigote surface protein 2 (ASP-2, Figure 3).
References:
Aparicio-Burgos JE, Zepeda-Escobar JA, de Oca-Jimenez RM, Estrada-Franco JG, Barbabosa-Pliego A, Ochoa-García L, Alejandre-Aguilar R, Rivas N, Peñuelas-Rivas G, Val-Arreola M, Gupta S, Salazar-García F, Garg NJ, Vázquez-Chagoyán JC. Immune protection against Trypanosoma cruzi induced by TcVac4 in a canine model. PLoS Negl Trop Dis. 9: e0003625, 2015.
Araujo AF, de Alencar BC, Vasconcelos JR, Hiyane MI, Marinho CR, Penido ML, Boscardin SB, Hoft DF, Gazzinelli RT, Rodrigues MM. CD8+-T-cell-dependent control of Trypanosoma cruzi infection in a highly susceptible mouse strain after immunization with recombinant proteins based on amastigote surface protein 2. Infect Immun. 73(9):6017–6025, 2005.
Barbosa RP, Filho BG, Dos Santos LI, Junior PA, Marques PE, Pereira RV, Cara CD, Bruña-Romero O, Rodrigues MM, Gazzinelli RT, Machado AV. Vaccination using recombinants influenza and adenoviruses encoding amastigote surface protein-2 are highly effective on protection against Trypanosoma cruzi infection. PLoSOne 8:e61795, 2013.
Basombrio MA, Arredes H. Long-term immunological response induced by attenuated Trypanosoma cruzi in mice. Journal of Parasitology. 73: 236–238, 1987.
Basombrio MA, Besuschio S, Cossio PM. Side effects of immunization with live attenuated Trypanosoma cruzi in mice and rabbits. Infection and Immunity 36: 342–350, 1982.
Basso B, Cervetta L, Moretti E, Carlier Y, Truyens C. Acute Trypanosoma cruzi infection: IL-12, IL-18, TNF, sTNFR and NO in T. rangeli-vaccinated mice. Vaccine. 22: 1868–1872, 2004.
Basso B, Moretti E, Fretes R.Vaccination with epimastigotes of diferente strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection. Mem Inst Oswaldo Cruz. 103: 370–374, 2008.
Bhatia V, Sinha M, Luxon B, Garg N. Utility of the Trypanosoma cruzi sequence database for identification of potential vaccine candidates by in silico and in vitro screening. Infect Immun. 72: 6245–6254, 2004.
Boscardin SB, Kinoshita SS, Fujimura AE, Rodrigues MM. Immunization with Cdna expressed by amastigotes of Trypanosoma cruzi elicits protective immune response against experimental infection. Infect Immun. 71: 2744–2757, 2003.
Brumpt E. “Immunité partielle dans les infections à Trypanosoma cruzi”, transmission de ce trypanosome par Cimex rotundatus. Rôle régulateur des hotes intermédiaires. Passage à travers la peau. Bulletin de la Societé de Pathologie Exotique 6:172–176, 1913.
Cazorla SI, Frank FM, Malchiodi EL. Vaccination approaches against Trypanosoma cruzi infection. Expert Rev Vaccines. 8: 921–935, 2009.
De Alencar BC, Persechini PM, Haolla FA, de Oliveira G, Silverio JC, etal. Perforina and gamma interferon expression. Are required for CD4+ and CD8+T-cell-dependent protective immunity against a human parasite,Trypanosoma cruzi, elicited by heterologous plasmid DNA prime-recombinant adenovirus 5 boost vaccination. Infect Immun 77: 4383–4395, 2009.
De Souza W. Trypanosoma cruzi–Host Cell Interaction Front Immunol. 5: 339, 2014.
Garg N, Tarleton RL. Genetic immunization elicits antigen-specific protective immune responses and decreases disease severity in Trypanosoma cruzi infection. Infect Immun. 70: 5547–5555, 2002.
Gupta S, Garg NJ. A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term Protective T Cell Immunity against Trypanosoma cruzi. PLoS Pathog. 11: e1004828, 2015.
Haolla FA, Claser C, de Alencar BC, Tzelepis F, de Vasconcelos JR, de Oliveira G, Silvério JC, Machado AV, Lannes-Vieira J, Bruna-Romero O, Gazzinelli RT, dos Santos RR, Soares MB, Rodrigues MM. Strain-specific protective immunity following vaccination against experimental Trypanosoma cruzi infection. Vaccine. 27: 5644–5653, 2009.
Higuchi MD, Ries MM, Aiello VD, Benvenuti LA, Gutierrez PS, Bellotti G, Pileggi F. Association of an increase in CD8+ T cells with the presence of Trypanosoma cruzi antigens in chronic, human, chagasic myocarditis. Am J Trop Med Hyg 56: 485–489, 1997.
Jones EM, Colley DG, Tostes S, Lopes ER, Vnencak-Jones CL, McCurley TL. Amplification of a Trypanosoma cruzi DNA sequence from inflammatory lesions in human chagasic cardiomyopathy. Am J Trop Med Hyg 48: 348–357, 1993.
Jones K, Versteeg L , Damania A, Keegan B, et al. Vaccine-linked chemotherapy improves benznidazole efficacy for acute Chagas disease. Infect Immun. IAI.00876-817, 2018.
Kierszenbaum F. Where do we stand on the autoimmunity hypothesis of Chagas disease? Trends Parasitol. 21: 513–516, 2005.
Low HP, Santos MA, Wizel B, Tarleton RL. Amastigote surface proteins of Trypanosoma cruzi are targets for CD8+ CTL. J Immunol. 160: 1817–1823, 1998.
Machado AV, Cardoso JE, Claser C, Rodrigues MM, Gazzinelli RT, Bruna-Romero O. Long-term protective immunity induced against Trypanosoma cruzi infection after vaccination with recombinant adenoviruses encoding amastigote surface protein-2 and trans-sialidase. Hum Gene Ther. 17: 898–908, 2006.
Menezes H. The use of adjuvants in the vaccination of mice with lyophilized Trypanosoma cruzi. Hospital (Rio J). 68(6):1341-1346,1965.
Miyahira Y, Takashima Y, Kobayashi S, Matsumoto Y, Takeuchi T, Ohyanagi-Hara M, Yoshida A, Ohwada A, Akiba H, Yagita H, Okumura K, Ogawa H. Immune responses against a single CD8+-T-cell epitope induced by virus vector vaccination can successfully control Trypanosoma cruzi infection. Infect Immun. 73: 7356–7365, 2005.
Paiva CN, Castelo-Branco MT, Rocha JA, Lannes-Vieira J, Gattass CR. Trypanosoma cruzi: lack of T cell abnormalities in mice vaccinated with live trypomastigotes. Parasitol Res. 85(12):1012–1017, 1999.
Paiva CN, Castelo-Branco MTL, Lannes-Vieira J, and Gattass CR. Trypanosoma cruzi: Protective Response of Vaccinated Mice Is Mediated by CD8+ Cells, Prevents Signs of Polyclonal T Lymphocyte Activation, and Allows Restoration of a Resting Immune State after Challenge. Experimental Parasitology. 91: 7–19, 1999.
Pereira IR, Vilar-Pereira G, Marques V, da Silva AA, Caetano B, Moreira OC, et al. A Human Type 5 Adenovirus-Based Trypanosoma cruzi Therapeutic Vaccine Re-programs Immune Response and Reverses Chronic Cardiomyopathy. PLoS Pathog 11: e1004594, 2015.
Quijano-Hernández IA, Castro-Barcena A, Vázquez-Chagoyán JC, Bolio-González ME, Ortega-López J, Dumonteil E. Preventive and therapeutic DNA vaccination partially protect dogs against an infectious challenge with Trypanosoma cruzi. Vaccine. 31: 2246–2252, 2013.
Reis DD, Jones EM, Tostes S Jr, Lopes ER, Gazzinelli G, Colley DG, McCurley TL 1993. Characterization of inflammatory infiltrates in chronic chagasic myocardial lesions: presence of tumor necrosis factor-alpha+ cells and dominance of granzyme A+, CD8+ lymphocytes. Am J Trop Med Hyg 48: 637–644, 1993.
Rigato PO, de Alencar BC, de Vasconcelos JR, Dominguez MR, Araújo AF, Machado AV, Gazzinelli RT, Bruna-Romero O, Rodrigues MM. Heterologous plasmid DNA prime-recombinant human adenovírus 5 boost vaccination generates a stable pool of protective long-lived CD8(+) T effector memory cells specific for a human parasite Trypanosoma cruzi. Infect Immun 79: 2120–2130, 2011.
Schenkman S, Eichinger D, Pereira ME, Nussenzweig V. Structural and functional properties of Trypanosoma trans-sialidase. Annu Rev Microbiol 48: 499–523, 1994.
Schnapp AR, Eickhoff CS, Sizemore D, Curtiss R, 3rd, Hoft DF. Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice. Infect Immun. 70: 5065–5074, 2002.
Tarleton RL. New approaches in vaccine development for parasitic infections. Cell Microbiol. 7: 1379–1386, 2005.
Vasconcelos JRC, Hiyane MI, Marinho CRF, Claser C, et al. Protective Immunity Against Trypanosoma cruzi Infection in a Highly Susceptible Mouse Strain After Vaccination with Genes Encoding the Amastigote Surface Protein-2 and Trans-Sialidase. Human Gene Therapy, 15:878–886, 2004.
Wizel B, Garg N, Tarleton RL. Vaccination with trypomastigote surface antigen 1encoding plasmid DNA confers protection against lethal Trypanosoma cruzi infection. Infect Immun. 66: 5073–5081, 1998.
Zingales B. Trypanosoma cruzi genetic diversity: Something new for something known about Chagas disease manifestations, serodiagnosis and drug sensitivity. Acta Trop. 184:38–52, 2018.
Background
Background: Critical requirements for a Chagas’ disease vaccine
Erney Plessmann Camargo
President of the National Council for Scientific and Technological Development (CNPq) and member of the Superior Council of CAPES, Brasília, DF, Brazil
Email: erney@usp.br
Just 4 years after Carlos Chagas’ discoveries, Emile Brumpt published the first tests on the induction of an “Immunité partielle dans les infections à Trypanosoma cruzi”. This work by Brumpt contained a statement that would mark ninety-odd years in the history of the search for a vaccine against Chagas’ disease: the protection induced against experimental infections by T. cruzi would always be partial, never accompanied by sterile immunity.
Let’s go through the history of this “immunité partielle” remembering four basic requirements for a prophylactic or preventive anti-Chagas vaccine:
- A prophylactic vaccine should provide total protection, that is, sterile immunity to primary infections. As Zigman Brener and I had already warned: “A vaccine which merely attenuates the acute phase of the infection – a possibly acceptable procedure for other infectious disease – would be of questionable value in Chagas’ disease.” This is because, in the pathogenesis of the disease, the involvement of target organs is a chronic sequel of the long course of the disease, not an exclusive event of the acute phase. A few surviving acute-phase trypanosomes could lead to chronic-phase lesions. Hence the requirement of sterile immunity for a prophylactic vaccine against Chagas. Not to mention the fact that non-sterile immunities do not eliminate the source of infection. Notwithstanding this basic principle, most vaccine attempts have only taken into account mortality rates in the acute phase, without worrying about residual parasitemias and chronic disease in vaccinated animals.
- 2. A vaccine should provide protection against all strains of cruzi capable of infecting humans. Since Brumpt, strains of T. cruzi of greater or lesser virulence were known. Today we know more about the T. cruzi strains that circulate in nature and we know that they can be grouped into zymodemes or genotypes that exhibit virulence and distinct antigens. Potential vaccines should at least be tested against the known and epidemiologically important genotypes of T. cruzi and should be shown to be effective against all.
- A vaccine could not induce autoimmune disease, a postulate that derived from evidence and beliefs that Chagas’ disease has a large autoimmune component and that T. cruzi fractions and antigens are capable of inducing pathological manifestations of chronic disease in the absence of infections. Although the importance of this autoimmunity is questionable (see Kierszenbaum’s review, just as a precaution this possibility should be examined.
- 4. Finally, as the vaccine was intended for humans, it had to be developed and tested in experimental models that mimicked the human pathogenesis and immune response. Unfortunately, the model universally adopted was the murine model, which has proved to be a model. Just as an example, mice do not recognize alpha-galactopyranosyl epitopes on the surface of trypanosomes as antigenic. These epitopes are responsible for the induction of lytic antibodies in human infections. Thus, an effective vaccine against T. cruzi infection in mice may not mean a vaccine against Chagas’ disease. Despite this, few alternative experimental models were tested. Occasional, albeit promising, incursions were made in the rabbit and especially dog models, which were not followed up. The monkey was also little explored and, attention, Muniz, in 1947 showed that Rhesus monkeys vaccinated with dead forms of typanosomes developed “hyperergic” myocarditis, which in itself shows that different models can lead to absolutely dissonant observations. Surprisingly, the dog, a simple and accessible model, which besides the monkey (primates), is the only one that is infected by T. cruzi in nature and that exhibits a pathology in many aspects similar to that of man, has been little tested.
Given these premises, the story of the search for a vaccine against Chagas’ disease would be a coming and going through the labyrinth of Brumpt’s fateful “immunité partielle”.
What was first tried in Chagas, following a practice common to vaccine attempts in other diseases, was to induce protection with previous immunizations, either with sub-lethal doses of T. cruzi or with cruzi strains of alleged attenuated virulence. These attempts began with Brumpt’s seminal work and continued into the late 1970s. In these 60-odd years, immunizations took place with several strains of T. cruzi, in addition to other species of trypanosomes and, later, with trypanosomatids parasites of insects. All without success and all limited to the measurement of mortality in the post-challenge phase with a virulent strain of T. cruzi.
Several vaccination attempts have also been made with live cultural forms of T. cruzi, inactivated by radiation or chemical multiplication blocking agents, or cultural forms killed by the most varied fixatives and antiseptics. The results obtained were always discouraging when, after several vaccination schedules, the animals were challenged with infective doses of T. cruzi. The reduction in mortality was never accompanied by sterile immunity, and post-challenge parasitemias were always positive.
There were numerous vaccine trials with cellular sub-fractions, the most notorious of them with flagellar fractions. Although, in some cases, mortality rates were zero after challenge with infective forms, when properly investigated, parasitemias never were zero.
A step forward was taken when purified antigens began to replace cell fractions in vaccine attempts. Not because the results were better than precedents in the direction of sterile immunity. But because rational research for antigens, particularly surface antigens, replaced the empirical search with the rationalist search for a vaccine. At the same time, efforts were made to better understand the molecular organization of the surface of T. cruzi and its antigens, especially trans-sialidase (TS), an enzyme unique to T. cruzi. The association of the definition of the antigen with immunological studies allowed significant advances in the understanding of the immune response to T. cruzi, especially in the clarification of the role of CD8+ cells in the assembly of an organ-protective defense in chagasic infection.
Despite these advances in our knowledge of the chagasic disease pathogenesis, the available and prospective experimental vaccines were not able to overcome the stigma of “immunité partielle”.
In recent years, new perspectives have opened up with DNA vaccines, with the work developed by the group of Rodrigues et al. showing that they simply consist of inserting T. cruzi genes into plasmids and injecting them into animals to be vaccinated. The genes preliminarily chosen were surface genes and among them, transialidase. The first results did not escape the stigma of “immunité partielle”. Recently, the choice of a T1-type immunity-stimulating plasmid (with the production of pro-inflammatory cytokines, such as interferon-gamma) carrying an association of two surface genes, including transialidase, provided zero mortality rates after the challenge between mice vaccinated for six months of observation, although parasitemias persisted in most animals.
In any case, the prospects are optimistic, thanks to the properties of DNA vaccines to permanently produce vaccine antigens, sometimes for the life (at least) of the mice. In this way, even if the infection is not completely overcome (sterile immunity), the constantly elicited defenses can keep the infection under control, reducing chronic tissue and organ damage. This fact, which still needs to be better investigated, opens up possibilities for the use of DNA vaccines as curative vaccines.
Another interesting perspective concerns the protection of mucous membranes, particularly as oral infections are becoming more frequent (or more frequently recorded), notably in the Amazon. In this sense, some works developed by Hoft et al. already point to the induction of mucosal immunity against T. cruzi antigens.
However, without being pessimistic, it should always be remembered that the murine model serves the mouse and may not be suitable for humans. It should also be remembered that different strains of T. cruzi express different epitopes of transialidases and possibly other antigens.
Finally, there is a final problem that has not even been addressed in terms of vaccine logistics. How to epidemiologically verify the effectiveness of a vaccine against Chagas’ disease? Which population to use in the vaccine test phase? Considering that Chagas’ disease is of chronic-late expression, for how many years should the test population be observed for the release of the vaccine? What population would a vaccine against Chagas’ disease in Brazil target?Fortunately, these are just problems, not insurmountable obstacles, and science always ends up finding solutions to its problems. However, in the meantime, let’s not even think about neglecting vector control services and blood banks.
Prophylactic Vaccine
Preventive vaccine for Chagas’ disease
Rosa Maldonado
University of Texas
Email: ramaldonado@utep.eduUnder construction.