Isabela Resende Pereira
Laboratory of Hematology – Medical School – UFF
Laboratory of Biology of Interactions/IOC
Email: email@example.com, firstname.lastname@example.org
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 et al., 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 et al., 1982; Basombrio et al., 1987; Paiva et al., 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 et al., 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 et al., 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 et al., 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 et al., 2009; Boscardin et al., 2003; Vasconcelos et al., 2004; Machado et al., 2006). The amastigote surface protein (ASP)-2, important for the establishment of chronic infection (Boscardin et al., 2003; Vasconcelos et al., 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 et al., 1998; Myahira et al., 2005; Araujo et al. , 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 et al., 2006; Haolla et al., 2009, de Alencar et al., 2009; Barbosa et al., 2013). Vaccination with DNA of plasmids containing genes encoding cruzipain proteins (Schnapp et al., 2002) and trypomastigote surface antigen-1 (TSA-1) (Wizel et al., 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 et al., 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 et al., 2006; Haolla et al., 2009, de Alencar et al., 2009; Barbosa et al., 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 et al., 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).
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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
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:
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.
Preventive vaccine for Chagas’ disease
University of Texas
Email: email@example.comUnder construction.