American Trypanosomiasis

C H A P T E R

69 American Trypanosomiasis 1

Vandanajay Bhatia1, Jian-Jun Wen1, Michele A. Zacks2 and Nisha Jain Garg 3

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA 2 Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA 3 Departments of Microbiology and Immunology and Pathology, Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX, USA

O U T L I N E Historical Background of Chagas Disease Vector and Parasite Vector classification Parasite classification Life cycle of T. cruzi Diagnostic measures Immune Response to T. cruzi Protective immune responses Pathologic immune responses Regulatory immune responses Epidemiology Transmission and geographic distribution Disease burden Potential as a biothreat agent

Pathogenic Mechanisms in Chagas Disease Development Immune-mediated pathology: autoimmunity or parasite persistence Vascular mediators in endothelium Mitochondria dysfunction and oxidative stress-mediated damage Other mechanisms Summary Vaccine Development Against T. cruzi Historical perspective Efforts toward identification of T. cruzi antigenic targets Subunit vaccines in development (over the last 10 years) Prospects for the future Key Issues

Clinical Disease and Control Measures Clinical manifestations of Chagas disease Control measures

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ABSTRACT Trypanosoma cruzi is the etiologic agent of American trypanosomiasis or Chagas disease. It is normally transmitted by the reduviid insect vector, and also by blood transfusion. It is estimated that 16–18 million people are infected in the American continent, with ⬎50,000 deaths reported every year. Acute infection by T. cruzi can be lethal, though the majority exhibit flu-like, nonspecific clinical symptoms. The disease usually evolves into a chronic stage, in which ⬎30% of infected individuals exhibit severe debilitation of the heart. The pathology of Chagas disease presents a complicated and diverse picture in humans. The clinically relevant pathognomonic consequences of human infection by T. cruzi are dilation and hypertrophy of the left ventricle walls and thinning of the apex. The major complications and destructive evolutionary outcomes of chronic infection include ventricular fibrillation, thromboembolism, and congestive heart failure. American trypanosomiasis poses serious public health care and financial concerns. The currently available drugs, though effective against acute infection, are highly toxic and ineffective in arresting or attenuating clinical disease symptoms in chronic patients. No effective vaccines are available. Studies in animal models and human patients have revealed several mechanisms, including parasite persistence, chronic inflammatory responses to self or parasite antigens, and sustained mitochondrial dysfunction and oxidative stress, that contribute to the pathogenic outcome of Chagasic cardiomyopathy. Protective immunity against T. cruzi infection requires the elicitation of Th1 cytokines, lytic antibodies and the concerted activities of macrophages (φs), T helper cells, and cytotoxic T lymphocytes (CTLs). Several antigens, antigen delivery vehicles, and adjuvants have been tested to elicit immune protection to T. cruzi in experimental animals; however, most of these attempts have met with limited success, and the development of an efficacious prophylactic vaccine faces many challenges. There is a critical lack of methods for prevention of infection or treatment of acute infection or chronic disease. This chapter will summarize what is known about the parasite and the current state of knowledge of pathogenesis and protective immunity. We will discuss the research efforts in vaccine development to date and the challenges faced in achieving an efficient prophylactic vaccine against human American trypanosomiasis, as well as the future perspectives.

HISTORICAL BACKGROUND OF CHAGAS DISEASE A detailed history of American trypanosomiasis is presented in a review (Guerra, 1970). The chronicles of colonization of the New World contain many indirect references to Chagas disease, then referred to as Mal de Bicho in Brazil. The first description of the reduviid insect and its blood-sucking habits was made in 1590 by a missionary priest in Tucuman, Argentina. It was ~300 years later when reduviids were shown to be carriers of Trypanosoma cruzi. In 1908, a physician, Carlos Chagas, was commissioned by Oswaldo Cruz, then director of Manguinhos Institute, to the Lassance region of Amazon Basin in Brazil to control a malaria outbreak. During this visit, he noted a large number of reduviid insects, referred to as “barbeiros” or “kissing bug” infesting the poorly constructed rural dwellings. He considered that these insects might be important vector of human disease, and studied them and found flagellates, resembling Crithidia, in their hindgut. Armed with a vector and a possible agent, he allowed the reduviid insects to feed on laboratory animals, and subsequently found flagellated parasites in their blood. The parasite was named, in honor of Oswaldo Cruz, Schyzotrypanum cruzi, and later renamed T. cruzi. Chagas continued his studies and, in 1908, documented the first case of human T. cruzi infection

in a 2-year-old girl. Within a period of less than 2 years, he described salient characteristics of the clinical disease, named Chagas disease after him (Lewinsohn, 1979). Subsequently, Vianna, a colleague of Chagas, made the first histopathological observation of amastigote nests in tissues, and Brunmpt delineated the life cycle of T. cruzi. Cases of T. cruzi infection were reported in Panama, Argentina, and Uruguay from 1931 to 1936. The first case of transfusion-mediated parasite transmission was reported in 1952 in Sao Paulo, Brazil. During the next 10 years, it became evident that the chronic infection is associated with pathologic lesions in the heart, extensively described by Mott and Hagstrom (1965). In 1974, the first observation of anti-heart antibodies in chronically infected patients was made (Cossio et al., 1974), and concurrently, others showed the destruction of heart cells by T. cruzi-sensitized lymphocytes in vitro (Santos-Buch and Teixeira, 1974). These observations initiated the hypothesis of Chagas disease as an autoimmune disease. The earliest studies documenting the immunologic control of T. cruzi showed: the complement-dependent phagocytosis and cytotoxicity to T. cruzi (Kierszenbaum and Budzko, 1973); antibody-dependent, cell-mediated cytotoxicity to bloodform trypomastigotes (Abrahamsohn and Silva, 1977); macrophage activity against T. cruzi (Hoff, 1975); and genetic resistance-versus-susceptibility

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of different inbred mouse strains to T. cruzi (Trischmann et al., 1978). Much has been learned about the biology, pathogenesis, and immunity to T. cruzi since its first description in 1909, and the knowledge related to vaccine development against T. cruzi is summarized here.

VECTOR AND PARASITE Vector Classification The vectors of T. cruzi are insects of the order Hemiptera, family Reduviidae, and subfamily Triatominae. Of the 118 species of triatomines identified, only a few, e.g., Triatoma infestans and Panstrongylus megistus in South America; Rhodnius prolixus in Venezuela, Columbia, and Central America; and T. barberi and T. dimidiata in Mexico, are epidemiologically significant as vectors of T. cruzi. T. sanguisuga is the primary vector of T. cruzi in the southeastern US (Pung et al., 1998; Schofield and Dias, 1999; Guzman-Bracho, 2001) (Fig. 69.1).

Parasite Classification T. cruzi is a hemoflagellate protozoan of the Sarcomastigophora phylum, Mastigophora subphylum, Kinetoplastida order, Trypanosomatidae family, and is characterized by the presence of one flagellum and a

single mitochondrion, which contains the kinetoplast, a specialized DNA-containing organelle. T. cruzi isolates are grouped into two major phylogenetic groups, i.e., I and II, on the basis of zymodemes and several genetic markers. T. cruzi II consists of five related subgroups, IIa–IIe (Brisse et al., 2000). In South America, T. cruzi I is associated with a sylvatic transmission cycle and human disease north of the Amazon basin. T. cruzi II is predominantly distributed in the Southern Cone countries and also associated with a domestic transmission cycle and infection of placental mammals (Barnabe et al., 2001a). In Mexico and the US, the majority of human cases are linked to T. cruzi I (Barnabe et al., 2001b; Bosseno et al., 2002). No direct correlation between Chagas disease severity and parasite lineage has been established.

Life Cycle of T. cruzi T. cruzi evolves during its life cycle into different forms that are morphologically distinct and can also be identified by the relative position of the kinetoplast in relation to the cell nucleus and flagellum emergence (Fig. 69.2) (Tyler et al., 2003). The infective, trypomastigote (15–20 ⫻ 2 μm) exists as a freely swimming, extracellular, nonreplicative form in the bloodstream of mammalian hosts. It may be ingested during a bloodmeal by the triatomine insect vector. In the midgut of the insect, trypomastigotes transform into

(A)

Nu

fg

kt bb um

Human infections Triatoma sanguisuga Triatoma protracta Triatoma gerstaeckeri Triatoma barberi Triatoma dimidiata Rhodinus prolixus Triatoma infestans Panstrongylus megistus

(B)

bb

um Nu

kt fg

(C) kt Nu

bb

FIGURE FIGURE 69.1 Geographical distribution of triatomines of clinical importance and T. cruzi infection in humans.

69.2 Different morphological forms of T. cruzi. (A) Epimastigote, (B) trypomastigote, (C) amastigote. Nu, nucleus; fg, flagellum; um, undulating membrane; kt, kinetoplast; bb, basal body.

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epimastigotes (20 ⫻ 2 μm) that replicate by binary fission, and move to the hindgut and rectum, where they transform into infective metacyclic trypomastigotes. When an infected triatomine takes a bloodmeal and defecates, metacyclic trypomastigotes are released in the feces of the insect and may enter the body of the victim through the puncture wound caused by insect bite, or through mucosal membranes of the mouth, nose, or eyes. Trypomastigotes may also be transmitted by transplacental transmission, blood transfusion, or laboratory accident. There is local inflammation at the entry site where macrophages and/or local tissues are parasitized. Most cells can be parasitized, although there appears to be a tropism for striated muscle, smooth muscle, and neuroectodermal cells. Trypomastigotes escape from the parasitophorous vacuole into the host cell cytoplasm by an unusual mechanism involving transformation of trypomastigotes into spherical, intracellular amastigotes (2–4 μm diameter) that replicate by binary fission within the cytoplasm of the parasitized cells. Within 4–5 days, amastigotes of heavily parasitized cells convert back into trypomastigotes, which are released by cell lysis. The released trypomastigotes may infect other cells or be ingested by the vector to continue the life cycle (Fig. 69.3) (Tyler et al., 2003).

Diagnostic Measures Parasitologic diagnosis of infection by T. cruzi depends upon detection of trypomastigotes by microscopic examination of blood samples. Trypomastigotes

in patients may also be detected by indirect methods, e.g., xenodiagnosis and hemoculture (Chiari, 1992). The polymerase chain reaction (PCR) offers several advantages in the sense that it can be more sensitive, specific, and rapid than the above-mentioned methods. The majority of PCR assays used to detect T. cruzi target the amplification of kinetoplast DNA, ribosomal DNA, or mini-exon sequences (Gonzalez et al., 1994; Gomes et al., 1999; Pizarro et al., 2007). Serological analysis is routinely conducted to determine exposure to T. cruzi. Generally, specific IgM to T. cruzi can be detected 1 week after infection, peaks by day 30, and decreases to undetectable levels in 3–4 months when IgG antibodies reach their maximum level. T. cruzi-specific IgG antibodies persist throughout the life of infected individuals. Anti-T. cruzi antibodies are detected by various methods, including complement fixation, enzyme-linked immunosorbent assay (ELISA), flow cytometry, direct agglutination or indirect hemagglutination, indirect immunoflourescence, and immunoblotting (Ferreira, 1992). T. cruzi-antigen-based serological analysis reagents are commercially available in the endemic countries and used for blood bank screening or seroepidemiological surveys. It is essential to perform multiple tests to confirm seropositivity. In the US, an ELISA assay that uses epimastigote lysate antigens for detection of antibodies to T. cruzi in serum and plasma (OrthoClinical Diagnostics, Raritan, NJ) has been licensed by the Food and Drug Administration, and used by American Red Cross and other blood banks for screening the donors for T. cruzi infection.

Metacyclic trypomastigote

Reduviid insect

Bloodmeal

Skin or mucosa Mammalian host

Trypomastigote in bloodstream

Cell invasion Muscle Cell

Cell lysis Amastigote

FIGURE 69.3 Life cycle of T. cruzi.

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IMMUNE RESPONSE TO T. CRUZI

IMMUNE RESPONSE TO T. CRUZI Protective Immune Responses High parasitemia and wide distribution of T. cruzi in different organs and tissues after initial infection are a result of the parasites’ ability to evade or suppress innate immune mechanisms. The acute phase ends when the host mounts a potent immune response that is largely effective in controlling the parasite. Sterilizing immunity does not exist in T. cruzi infection. The comparison in responses exhibited by susceptible and resistant experimental models has contributed to an understanding of protective immune responses to T. cruzi. Natural killer (NK) cells appear to be the first source of interferon-γ (IFN-γ) that, in turn, augments IL-12 synthesis by φs (Gazzinelli et al., 1992). Surface glycoproteins (mucins) and/or glycophospholipids (GIPLs) of T. cruzi are shown to stimulate the production of multiple cytokines, e.g., IFN-γ, TNF-α, IL-1, and IL-6; and chemokines, e.g., MCP-1, RANTES, and IP-10 by inflammatory φs (Almeida et al., 2000, 2001; Almeida and Gazzinelli, 2001). These cytokines induce φ activation and nitric oxide (NO) production that is important in the killing of T. cruzi (Martins et al., 1998). It is suggested that IFN-γ of NK origin and IL-12 of φ origin skew the differentiation of parasite-specific T helper cells toward a protective Th1 phenotype (Abrahamsohn and Coffman, 1996). Parasite-specific CD4⫹ T cells may assist in the control of T. cruzi through secretion of Th1 cytokines (IFN-γ, IL-2), amplification of the phagocytic activity of macrophages, stimulation of B cell proliferation and antibody production, and differentiation and activation of CD8⫹ T cells (Brener and Gazzinelli, 1997). T. cruzi antigen-specific CD8⫹ T cells are frequently present in infected mice and humans (Wizel et al., 1997, 1998b); and may contribute to T. cruzi control, either by cytolysis of the infected cells or secretion of Th1 cytokines (IFN-γ) that induce trypanocidal activity (DosReis, 1997). A strong lytic antibody response enhances the opsonization, phagocytosis, and complement-dependent killing of the parasites (Krautz et al., 2000). With progression to indeterminate and chronic disease, blood parasitemia is absent, and parasite nests are rarely detectable in cardiac tissue. The presence of parasitespecific antibodies is diagnostic of chronic T. cruzi infection. The mononuclear cells, φs, and IFN-γ-producing CD8⫹ T cells represent the majority of the infiltrate in experimental models (Tarleton, 1996) and human cardiac biopsies (Reis et al., 1993). CD4⫹ T cells are less prominent in the chronic myocardium of infected mice (DosReis, 1997) and human patients (Higuchi et al., 1997). It is likely that immune responses similar

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to those essential for control of acute parasitemia, i.e., substantial antibody response and activation of type 1 CD4⫹ and CD8⫹ T cells, are required to maintain control of T. cruzi during indeterminate and chronic phases of infection and disease. The evidence is provided from the experimental models and natural human infections demonstrating that absence or reduction in any of these immune responses through targeted depletion, immunosuppressive treatments, or infection-induced immunosuppression can result in an exacerbation of parasitemia (Sartori et al., 1995; D’Almeida et al., 1996; Tarleton et al., 1996).

Pathologic Immune Responses Along with their immunoprotective role, CD4⫹ and CD8⫹ T cells are suggested to be involved in immunopathology. During acute infection in mice, CD4⫹ T cells initiate unspecific, polyclonal activation of B cells from spleen and lymph nodes, leading to an increased number of nonspecific, IgG-secreting cells and hypergammaglobulinemia with disease progression (Minoprio et al., 1987, 1989). GIPLs, at least in part, are responsible for triggering of polyclonal B lymphocytes (Bento et al., 1996). CD4⫹ T cells that persist in chronic hearts represent an autoreactive phenotype and are associated with myocyte death, tissue destruction, and increased animal mortality (Soares and Santos, 1999). In humans, most of the initial humoral response is dominated by antibodies to an epitope (Galα1-3Gal) expressed by glycosylphosphatidylinositol (GPI) anchored mucins of T. cruzi and is followed by polyclonal B cell activation and hypergammaglobulinemia with disease progression. CD8⫹ T cells are the major T-cell subset found in the cardiac tissue of both experimental animals and human patients and are thought to be responsible for immunopathology during chronic Chagas disease (Higuchi, 1999; Higuchi et al., 2003).

Regulatory Immune Responses Because T. cruzi infection elicits a strong activation of the immune system, it seems essential that immunoregulatory mechanisms be activated. IL-10 and TGF-β cytokines are important negative modulators of NO synthesis and trypanocidal activity of φs. A lack of IL-10 results in an overwhelming inflammatory response and animal deaths early in infection with T. cruzi (Hunter et al., 1997). Several mechanisms, e.g., regulation of IL-2 expression (Tarleton, 1988), φ activation and NO synthesis (Abrahamsohn and Coffman, 1995), and selective apoptosis of T helper cells (Lopes

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and Dosreis, 1996), may act independently, or in concert, to control the CD4⫹ T cell population in infected mice. Parasite molecules (GIPLs) are shown to block CD8⫹ T cell activation in vitro (DosReis et al., 2002); however, no regulatory mechanisms have been defined that may control CD8⫹ T cell population in vivo. The occurrence of such regulatory mechanisms in human patients, though expected, has not been defined.

EPIDEMIOLOGY Transmission and Geographic Distribution The ancient sylvatic cycle of T. cruzi transmission involves interaction between wild vectors and hosts in different natural ecotypes of American continent. The domestic cycle results from human–vector contact. The reservoir hosts of T. cruzi include ⬎150 mammal species, mainly small animals, that play an important role in maintaining both sylvatic and domestic cycles (WHO, 2002). Humans, followed by dogs, cats, and rodents are important domestic reservoirs. T. cruzi is widespread and endemic in almost all of South and Central America, and Mexico (Fig. 69.1) (Schofield et al., 2006; Cruz-Reyes and Pickering-Lopez, 2006). Several studies have shown the presence of the insect vector as well as infection of domestic dogs and wild animals in the US (Yabsley and Noblet, 2002; Beard et al., 2003; Hall et al., 2007; Hanford et al., 2007). Vectortransmitted human infections have been reported in the southern US (Herwaldt et al., 2000) (Fig. 69.1). Profound economic and social changes have stimulated rural-to-urban migration within the endemic countries and towards the developed countries, e.g., the US and Canada. It is estimated that ⬎300,000 infected individuals live in the city of Sao Paulo; ⬎200,000 each in Rio de Janeiro and Buenos Aires (Dias, 1992b); and ⬎100,000 infected immigrants from Latin America live in the US (Kirchhoff, 1993). These individuals may donate contaminated blood and thereby expose many others to the risk of T. cruzi infection (Dias, 1992a; Leiby et al., 1997). The infectivity risk, defined as the likelihood of infection from receiving an infected transfusion unit, is estimated to be ~20% for T. cruzi (WHO, 2002). Before serologic screening of blood donors for antibody to T. cruzi was implemented, infection rates through blood transfusion ranged from ⬍0.1% to 4% in Argentina, Brazil, Chile, and Uruguay to ⬎10% in Bolivia (Schmunis and Cruz, 2005). Transmission of T. cruzi infection by solid-organ transplantation has been evident in Latin America (Reis et al., 1995; Riarte et al., 1999).

In the US, five cases of T. cruzi transmission by organ transplantation from infected donors have been described (CDC, 2002a, 2006). Three blood banks, situated in California and Arizona states, have conducted a large-scale serosurvey, and documented that 1 in every 4665 blood donations was seropositive for T. cruzi-specific antibodies (CDC, 2002b). This study led to FDA approval of first blood donor screening test for Chagas disease in the US (CDC, 2007). Beginning January 2007, American Red Cross and Blood Systems Inc. have begun screening the blood donations for presence of T. cruzi antibodies and it is expected that all blood-collection establishments in the US will implement screening test for T. cruzi infection in near future. As of July 2007, 458 blood donations were repeat reactive by ELISA and 124 donations were confirmed positive by radioimmunoprecipitation test in the US (AABB Chagas’ Biovigilance Network, http:// www.aabb.org/Content/Programs_and_Services/ Data_Center/Chagas/chagas.htm).

Disease Burden According to World Health Organization estimates from 1998, ⬎120 million people are exposed to risk of T. cruzi infection, ~18 million people are infected with T. cruzi, and ~50,000 children and adults die annually, because of the clinical complications of T. cruziinduced heart disease and lack of effective treatments in endemic countries (WHO, 2002). Approximately 2–5% of fetuses carried by infected mothers are either aborted or born with congenital infection. Revenue loss, in terms of productivity declines due to sickness in prime years and medical costs, has an overwhelming effect on economic growth (WHO, 2002). It is currently estimated that the total annual cost of Chagas disease management in endemic countries is ⬎8 billion US dollars. When considered from a global perspective, Chagas disease represents the third greatest tropical disease burden after malaria and schistosomiasis. American trypanosomiasis is recognized as a potentially emerging infectious disease in the US (Dodd and Leiby, 2004).

Potential as a Biothreat Agent T. cruzi infection poses a minimal risk as a biothreat agent due to a latency period of incubation ranging ~10–30 years prior to development of clinical disease. Chagas disease is, however, an emerging infectious disease in the US and no longer considered a disease of the tropical, developing countries. Since January 2007, the American Red Cross and other blood banks across

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CLINICAL DISEASE AND CONTROL MEASURES

the United States have implemented the donor screening for T. cruzi infection, highlighting the urgent need for clinicians, laboratorians, and public health professionals to understand Chagas disease, its diagnosis, and treatment.

CLINICAL DISEASE AND CONTROL MEASURES Clinical Manifestations of Chagas Disease Infection by T. cruzi elicits nonspecific symptoms, e.g., fatigue, fever, enlarged liver or spleen, swollen lymph glands, and occasionally a characteristic swelling either at the site of entry in the skin, a Chagoma, or in the conjuctiva of eyelid, Romana’s sign. Most acute patients develop heavy parasitism of muscles and some symptoms of cardiac involvement that is usually unrecognized. The clinical manifestations resemble myocarditis, with slight-to-moderate cardiac enlargement. In a small proportion of acute patients (⬍5%), more severe right- and left-sided heart failure may occur with pulmonary and systemic congestion, tachychardia, and other cardiac insufficiencies, occasionally resulting in fatal congestive heart failure. T. cruzi can also invade the gastrointestinal and central nervous systems, but, in general, damage to these systems is uncommon. The signs and symptoms of acute disease resolve spontaneously in 4–8 weeks postinfection, and the majority of patients enter an indeterminate phase of infection. This phase is characterized by very low or undetectable parasitemia, high parasitespecific antibody titers, and lack of clinical symptoms. Between 10 and 20 years after an initial infection, ~40% of infected individuals become symptomatic. The systemic changes during the chronic stage commonly develop in the heart, esophagus, and rectosigmoid colon, and less often in the central nervous system (Kirchhoff et al., 2004). Heart problems in chronic Chagasic patients are evidenced by nonspecific symptoms such as palpitations, dizziness, and syncope. Typically, manifestations of chronic disease are thromboembolism in the brain, limbs, or lungs, congestive heart failure, and sudden death—mainly due to ventricular fibrillation. Once clinical symptoms appear, the prognosis is poor, and fatality occurs within a short period (Rassi et al., 2000). The principal defects in the conduction system present as a mixture of arrythmias, e.g., tachycardia, bradycardia, ventricular fibrillation, and electrical impulse blockages, e.g., right bundle branch and left anterior fasicle block. Myocardial structural defects are mainly cardiomegaly with hypertrophy and dilation of the

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chambers, apical aneurism in left ventricle, and clot formation with thromboembolization (Rossi et al., 2003). The histological examination of sections from both autopsy and biopsy specimens reveal that tissue fibrosis, inflammation, and hypertrophy of cardiac fibers are the chief pathological consequences of Chagas disease. Fibrosis is most pronounced in the left ventricle and apex, and may correspond to increased collagen fiber deposition around the muscle bundles. Inflammation varies in severity and its location may be interstitial and/or diffuse. Echocardiography, serial chest radiographs, and electrocardiography are the most accurate and commonly employed methods for detecting myocardial involvement in Chagasic patients (Rocha et al., 2003). Clinical manifestations of Chagas disease may also be correlated with defective innervation and defective contraction within the myocardium. Pathogenic changes in the parasympathetic branch of the autonomic nervous system are sometimes observed at autopsy. Specifically, a reduction, or in some cases, a complete absence of cells in the neuronal ganglia has been described and may be indicative of a role for impaired heart innervation in cardiac dsyfunction associated with Chagas disease (Marin-Neto, 1998). In some patients, clinical manifestations of Chagas disease include development of a megaesophagus and/or megacolon. Repeated episodes of aspiration pneumonitis are common in patients with severe esophageal dysfunction. Patients with a megacolon suffer from chronic constipation and abdominal pain. Ultrasonography, computed tomography, and magnetic resonance imaging are excellent imaging modalities used to determine the extent of tumor and enlargement, but are very expensive (Mattoso, 1981).

Control Measures Vector Control Three multinational, government-funded programs, initiated in 1991, are underway to control the endemicity of T. cruzi infection in Southern Cone nations of Argentina, Bolivia, Brazil, Chile, Paraguay, and Uruguay (Schofield, 1992; Schofield and Dias, 1999). The primary focus of these programs is the elimination of the domestic insect population through insecticide use, housing improvement, and education. These efforts have been very successful in reducing the domiciliary infestation by triatomines in vast regions of the endemic countries, and subsequently reducing the prevalence of acute infection in young children (Dias and Schofield, 1999; Schofield et al., 2006). The current goal is to eliminate vectorial T. cruzi transmission

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to humans throughout the Southern Cone by 2010. Venezuela and Colombia have also implemented parallel insecticide spray and housing improvement initiatives (WHO, 2002). However, the operational cost to sustain the insecticide spray programs, followed by constant surveillance, is one of the major obstacles in the success of the regional insecticide-based control programs. Insecticide use in the long-term may not be efficacious in blocking T. cruzi transmission, owing to the development of drug resistance by triatomines. Concern remains that reinfestation of homes by secondary sylvatic vectors, e.g., Triatoma sordida, in Brazil and other South American countries, will compromise the long-term efficacy of vector control measures (Moncayo, 2003). Successful elimination of disease as a public health problem through interruption of transmission would not eradicate the parasite due to the zoonotic nature of the disease.

drugs have not yet entered clinical trials possibly due to lack of financial support from the major drug companies for the development of new drugs against Chagas disease (Moncayo and Ortiz Yanine, 2006). Treatment of chronic patients focuses on cardiac management (Rocha et al., 2003) that requires a specialized clinical infrastructure, and thus, is expensive and often beyond the reach of the patient. For example, in Brazil, considering that only 10% of infected individuals would develop chronic disease, the medical costs for the obligatory treatment could reach $250 million. Cardiac transplantation is an option for patients with severe cardiac disease; however, immunosuppression can lead to reactivation of infection with an intensity typical of acute Chagas disease, invasion of transplanted heart by parasites, and heart rejection. No vaccines are currently available against T. cruzi infection.

Blood Supply Screening The problem of transfusion-transmitted Chagas disease was apparent by the 1950s. However, it was only in the 1980s, with the emergence of AIDS that national and regional blood-screening programs were implemented in endemic countries. Among the 18 countries in Latin America for which data are available, the degree of blood bank screening for T. cruzi now reaches 100% of the donors in seven countries, ⱖ99% in three, 75–95% in four, and 25–34% in three (Schmunis and Cruz, 2005). In the US, an ELISA assay that uses epimastigote lysate antigens for detection of antibodies to T. cruzi in serum and plasma (OrthoClinical Diagnostics) has been licensed by the Food and Drug Administration in January 2007, and used by American Red Cross and other blood banks for screening the donors for T. cruzi infection.

PATHOGENIC MECHANISMS IN CHAGAS DISEASE DEVELOPMENT Considerable debate exists regarding the pathomechanisms that are involved in Chagas disease development. No universally accepted model exists to explain the long latency period between infection and disease development, why a subset of individuals in the indeterminate phase progress to chronic disease, or what instigates or perpetuates the damage in the heart that ultimately results in Chagasic disease symptoms. We briefly discuss the pathomechanisms that are described in the literature.

Treatment

Immune-Mediated Pathology: Autoimmunity or Parasite Persistence

Two drugs, benznidazole and nifurtimox, are generally approved for the treatment of T. cruzi infection, and are effective in curing at least 50% of acute or recent cases of infection (Coura, 1996). Both of these drugs have serious and frequent side effects and are not available to many patients either because they are not registered in those countries or drug prices are high. Anti-parasitic drugs are not effective once the patients enter the chronic phase of infection and disease progression (Rodriques Coura and De Castro, 2002). Several other drugs, e.g., posaconazole (Liendo et al., 1998) and thioridazine (Lo Presti et al., 2004; Bustamante et al., 2007) have shown promise in curing T. cruzi infection in murine models, however, these

A significant body of literature suggests that consistent inflammatory immune responses play a role in the development and/or propagation of pathological lesions in Chagasic hearts (Pathologic Immune Responses). In cardiac tissue, antibody- and cytotoxic T lymphocyte (CTL)-mediated responses, as well as the continuous production of cytokines and chemokines, may contribute to the selective destruction of cardiomyocytes, neurons, or endothelial cells, and the development of fibrosis in response to cytopathology (Brener and Gazzinelli, 1997). Two mechanisms, autoimmunity and parasite persistence, are proposed to sustain pathological inflammatory responses in Chagasic hearts (Soares et al., 2001).

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PATHOGENIC MECHANISMS IN CHAGAS DISEASE DEVELOPMENT

The concept of autoimmunity is that polyclonal activation (Minoprio, 2001), molecular mimicry (Iwai et al., 2005), or the presence of cryptic epitopes shared by the host and parasites (Girones et al., 2001b) result in the recognition of self-antigens by the immune response elicited to control the parasite and, subsequently host tissue destruction. A growing number of studies have shown the pathogenic role of autoantibodies present in the serum of chronic mice and patients that react with various antigens in the heart, skeletal muscle, or nervous tissue (reviewed in Kierszenbaum (1999)). These include the β1-adrenoreceptor (Sterin-Borda and Borda, 2000), ribosomal P proteins (Kaplan et al., 1997), cruzipain (Giordanengo et al., 2000), and a novel Cha protein (Girones et al., 2001a). Common antigens between T. cruzi and human myocardial fibers (e.g., myosin) and auto-reactive T cells with specific cytotoxicity against myocardial fibers have been demonstrated in humans (Cunha-Neto et al., 1995; Cunha-Neto and Kalil, 2001). A cardiac myosin-specific autoimmune response and myocarditis can be induced by injecting several doses of subcellular antigens of T. cruzi in animals (Leon et al., 2004). The autoimmune theory, thus, suggests that the humoral and cellular immune reactions, elicited in response to T. cruzi infection, recognize self-antigens, are potentially harmful to the host, and contribute to the development and/or propagation of pathological lesions in Chagasic myocarditis (Leon and Engman, 2003). The lack of evidence that T. cruzi-induced self-reactive antibodies or T cells can induce disease upon transfer into a naïve host precludes us from defining chronic Chagasic cardiomyopathy as an autoimmune disease. It is now recognized that human infection of many years’ duration is associated with a nearly consistent low level of parasitosis (Higuchi et al., 2003). Recent studies using modern techniques, e.g., PCR, immunohistochemistry, and confocal microscopy, have detected parasite DNA or antigens in blood and heart tissue biopsies of experimental animals and chronic human patients (Anez et al., 1999; Mortara et al., 2000; Salomone et al., 2000; Caliari et al., 2002). It is proposed that parasite persistence provides sufficient, consistent antigens that work as a trigger for the hypersensitive response against myocardial fibers, leading to pathologic tissue injury, and, subsequently, to cardiac insufficiency.

Vascular Mediators in Endothelium Yet, clinical-pathological severity is not universally correlated with the presence of parasites (Higuchi et al., 1993) and chronic inflammation (Palomino et al., 2000).

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This leads to the suggestion that other mechanisms must be involved. The hypothesis that vasculopathy contributes to Chagasic heart disease has developed from the observations indicating that both human and experimental infections cause a reduction in blood flow and an intense vasculitis. Bradykinin, a potent vasodilator, and endothelin 1 (ET-1), and thromboxane A2 (TXA2), two potent vasoconstrictors, have been identified in Chagasic hearts. Kinins convey the vasodilating response through their interaction with vascular receptors and lessen the detrimental effects of ischemia (Yang et al., 2001). Despite increased kinin expression, T. cruzi-mediated decrease in coronary flow was not normalized in infected rats, suggesting impaired kinin receptor function. Instead, activation of kinin receptors on cardiovascular cells was shown to contribute to an increased infectivity by T. cruzi (Scharfstein et al., 2000; Todorov et al., 2003). TXA2 and ET-1 may potentiate disease severity by promoting microvascular pathology, e.g., vasospasm, ischemia, and microthrombi (Petkova et al., 2001). Vascular mediators, in conjunction with cytokines induced by T. cruzi infection, may be significant to the development of microcirculatory abnormalities that are well documented in Chagasic hearts (reviewed in Mukherjee et al. (2003)).

Mitochondria Dysfunction and Oxidative Stress-Mediated Damage Our studies suggest that Chagasic myocardium may be pre-disposed to sustained oxidative stress associated with mitochondrial dysfunction (reviewed in Garg (2005) and Zacks et al. (2005)). Cardiac tissue is extraordinarily dependent upon oxidative phosphorylation for energy to perform its functions. Accordingly, in cardiomyocytes, mitochondria are abundant. Mitochondria are the main producers of reactive oxygen species (ROS) in the heart and brain. Under normal conditions, NADH-ubiquinone oxidoreductase (CI) and ubiquinol-cytochrome c reductase (CIII) complexes of the respiratory chain release 2–4% of electrons to oxygen, resulting in superoxide (O2⫺) formation in mitochondria that is then converted to highly toxic hydroxyl ion (OH⫺) and H2O2 by various reactions (Ide et al., 1999; Chen et al., 2003). An alteration in CI and CIII activities may result in increased ROS production in mitochondria (Ide et al., 1999; Chen et al., 2003), and is found in the myocardia of T. cruziinfected mice (Vyatkina et al., 2004; Wen and Garg, 2004) and peripheral blood of seropositive Chagasic subjects (Wen et al., 2006b). Manganese superoxide dismutase (MnSOD) is the major O2⫺ scavenger in mitochondria, and its decreased activity was noted during

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acute-to-chronic disease development in infected murine myocardium (Wen et al., 2004). It was, therefore, proposed that a functional decline in the respiratory chain, increased ROS generation, coupled with an inability to efficiently scavenge the mitochondrial free radicals, predisposes Chagasic hearts to sustained ROS during infection and disease development. At low levels, ROS induces cytokine and chemokine production (Rahman and MacNee, 2000), thus providing a potential mechanistic link between mitochondrial ROS generation and acute and/or chronic inflammation in Chagasic cardiomyopathy. The cytotoxicity of sustained oxidative stress is related to ROS’ ability to oxidize cell constituents, including proteins, lipids, and DNA, which lead to deterioration of cellular structure and function and, ultimately, to cell death (Tsutsui, 2001; Martindale and Holbrook, 2002). The sustained occurrence of oxidative damage, as evidenced by consistent presence of protein carbonylation and lipids peroxidation products, was shown in the myocardium of an experimental model of Chagas disease (Wen et al., 2004) and in peripheral blood of human Chagasic subjects (Wen et al., 2006b; de Oliveira et al., 2007). Oxidative damage during Chagas disease may not only be due to increased ROS formation but also exacerbated by inefficient glutathione antioxidant capacity (glutathione peroxidase-GSH-glutathione reductase), as is noted in infected mice (Wen et al., 2004) and human subjects (Perez-Fuentes et al., 2003). Subsequently, recent studies have suggested that combinatorial anti-parasite/ antioxidant therapies would be useful in controlling Chagas disease pathology. Treatment of experimental animals with phenylbutylnitrone antioxidant (Wen et al., 2006a) and human Chagasic patients with vitamin C and vitamin E antioxidants (Macao et al., 2007) was shown to be effective in reducing the oxidative insult–associated pathology in Chagas disease. In summary, mitochondria are targets of a variety of endogenous and exogenous insults, including inflammatory mediators, elicited by T. cruzi infection. Depending upon the extent and duration of mitochondrial dysfunction, oxidative stress may initiate or contribute to destruction of heart tissue and consequent dysfunction through sustenance of inflammatory responses and oxidative damage of the cardiac components.

Other Mechanisms Less well explored is the potential for the T. cruzi strain or clonal variation to account for different clinical outcomes (Tibayrenc, 1998). Human genetic differences

may also contribute to the dichotomy between indeterminate and chronic disease manifestations (WilliamsBlangero et al., 2003), although no conclusive evidence for genetic differences is present to date.

Summary We conclude that the contributions of parasite persistence, autoimmunity, vascular mediators, mitochondria dysfunction, oxidative stress, and potentially, parasite strain or clonal variation and host genetics are not mutually exclusive and could contribute in part and/or act at distinct time points during T. cruzi infection to initiate and sustain the observed, multifaceted cardiac pathology. The sum of these factors may also determine the degree of pathophysiology the infection may evoke and the severity of chronic disease. An important implication of these studies is that preventing infection or controlling the acute parasite load below a threshold level would be effective in decreasing the tissue damage imposed by multiple pathogenic mechanisms and lead to decreased disease severity. Obviously, these observations provided an impetus for vaccine development against T. cruzi, which is also favored by the fact that the effector mechanisms capable of controlling parasite burden were delineated.

VACCINE DEVELOPMENT AGAINST T. CRUZI Given the current knowledge about the status of protective immunity from T. cruzi (see section Protective Immune Responses), a successful vaccine against the parasite is envisioned to elicit a long-term, lytic antibody response, type 1 cytokines, and CTLs. There are two stages of the parasite against which vaccines can be designed. Vaccines against trypomastigotes as they enter host cells following the bite of an infected triatomine, or the burst of an infected cell, will prevent the initiation or persistence of infection, and limit the parasitemia. Vaccines against intracellular replicative amastigotes would arrest the propagation of parasite in a host and prevent the parasite from entering the blood. Both types of vaccines would arrest or attenuate disease development in humans and the reservoir mammalian host. In addition, vaccines against either stage of the parasite would prevent triatomine infection and, thus, interrupt or reduce parasite transmission in both human and reservoir populations, as well as in insects.

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TABLE 69.1 Traditional vaccination approaches against T. cruzi

Historical Perspective Early efforts in developing prophylactic vaccines against T. cruzi tested killed parasite preparations as immunogens. Examples include T. cruzi killed by: (i) chemical treatment, e.g., thimerosal, formalin, glutaraldehyde; (ii) physical methods, e.g., pulverization, freezing and thawing, sonication, or pressure disruption; and (iii) irradiation. These antigenic preparations were tested for vaccine potential in various animal models, including mice, guinea pigs, dogs, and monkeys. Vaccination with chemically treated T. cruzi failed to provide any protection from lethal challenge infection. Other vaccination approaches generated a degree of resistance to T. cruzi; immunized animals controlled acute parasitemia and survived (70–100%) acute infection (Table 69.1). Similar efforts utilizing subcellular fractions, e.g., flagellar, soluble, or membranes, as vaccines also demonstrated an elicitation of partially protective immunity. For example, immunization with crude flagellar extracts enhanced resistance to T. cruzi, evidenced by a 90% survival rate of mice from challenge infection (Ruiz et al., 1986). Immunization of mice with T. cruzi soluble extract elicited IFN-γ and prevented death from an otherwise lethal infection (Garcia et al., 2000). Most of these approaches utilized the epimastigote, the insect stage of the parasite, that was later identified as expressing different antigens from those found in the infective and intracellular stages of T. cruzi. An absence of immunogenic proteins in epimastigotes, or a loss of protective epitopes during inactivation and fractionation, was believed to be the cause for the limited success met in these attempts in vaccine development. The next series of efforts tested live vaccines, consisting of T. cruzi strains attenuated by treatment with drugs or pharmacological agents, or by serial passage in cultures, for their potency in experimental animals (mice and dogs). These vaccines were largely effective in controlling subsequent infections by virulent strains. Vaccinated animals exhibited decreased parasitemia and increased survival rates compared to unimmunized animals (Table 69.1). The danger of reversion of the attenuated strains to a virulent form and the likelihood of increased virulence of attenuated strains in immunocompromised individuals rendered these vaccines impractical. These studies, however, showed that a prophylactic vaccine capable of eliciting protective immunity with a minimal risk of biological reversion to virulent phenotype would be useful in controlling T. cruzi infection and disease, and provided a foundation for the identification of target antigens of the immune responses and the development of subunit vaccines.

Vaccination strategy

% Survival (dpia)

T. cruzi treated with Thimerosal

0

Formalin Glutaraldehyde

0 100 (60)

Pulverization Freezing and thawing Sonication

100 (10–15) 70 (120) 0

Pressure disruption

80–100 (120)

Irradiation

80–100

T. cruzi subcellular fractions Flagellar 90 (60) Soluble 85 (150) Membranes

Not detected

Live T. cruzi attenuated by Drug treatment Trypaflavine Actinomycin D

100(30) 100 (13)

Bayer 7602

100

Primaquine

100

L-furaltodone

100 (11)

Serial passage in culture

100 (13–77)

References

Kagan and Norman (1961) Hauschka (1949) Basso et al. (2007) Rego (1959) Basombrio (1990) Seneca and Peer (1966) Gonzalez Cappa et al. (1974) Okanla et al. (1982)

Ruiz et al. (1986) Garcia et al. (2000) Ruiz et al. (1985)

Collier (1931) Fernandes et al. (1966) Hauschka et al. (1950) Pizzi and Prager (1952) Brener and Chiari (1967) Menezes (1968, 1969)

100 (30) a

Experimental animals were observed for survival for n days postinfection.

Efforts Toward Identification of T. cruzi Antigenic Targets The antigenic targets capable of eliciting the desired immune responses (see Protective Immune Response) at a level that confers protection from infection are the best choice for subunit vaccine development. Early studies employed random approaches, e.g., screening of expression libraries with immune sera from infected animals or human patients, screening with antigen-specific monoclonal antibodies, and amplification of the antigen-encoding genes (Ruiz et al., 1990; Malchiodi et al., 1993; Low and Tarleton, 1997) for the identification of putative vaccine candidates. These approaches, though successful in characterization of a variety of T. cruzi proteins, often led to the discovery of antigens capable of eliciting an antibody response only.

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The diploid genome (106–110 Mb) of T. cruzi, distributed in 35–40 chromosomal bands, contains ~22,500 proteins encoded by genes (El-Sayed et al., 2005). The complexity of the T. cruzi genome has necessitated the development of logical strategies to determine which parasite proteins are the likely choice for immune activation. Garg et al. (Garg et al., 1997) employed a transfection system and expressed chicken ovalbumin (OVA), the classical immunogen, in different cellular compartments of T. cruzi. Antigen-presenting cells infected with T. cruzi expressing OVA as a GPIanchored surface protein or as a secreted protein were recognized and lysed by the OVA-specific CTLs in vitro. Also, parasites expressing GPI-anchored or secreted OVA elicited the expansion of OVA-specific CTLs and antibodies in infected mice (Garg et al., 1997; Kumar and Tarleton, 2001). It was inferred that during the process of replication and/or differentiation, T. cruzi releases GPI-anchored and secretory proteins in host cell cytoplasm where they are degraded by proteosome enzymes. The resulting peptides that transported to endoplasmic reticulum are associated with MHC class I molecules, and displayed on the surface of infected cells, where they can be recognized by circulating CD8⫹ T cells. On extracellular trypomastigotes and amastigotes, these same proteins expressed in a membrane-associated form, are engulfed and processed by phagocytic cells, displayed in association with MHC class II molecules, and can be recognized by CD4⫹ T cells that provide help for the activation and proliferation of CD8⫹ T and B cells. These studies concluded that GPI-anchored proteins, abundantly expressed in infective and intracellular stages of T. cruzi, and the secreted proteins are the most likely source of peptides for immune activation. Accordingly, during the last 10 years, several surface proteins of T. cruzi have been identified, and their immunogenic potential examined. The extracellular and intracellular life cycle stages of T. cruzi adapt to sudden changes in the environment and survive in generally unfavorable conditions by changing the composition of surface glycoproteins. Most (but not all) of these surface proteins are attached to the plasma membrane by GPI anchor, their expression is developmentally regulated, and contain stage-specific modifications that ultimately reflect their functional importance in the life cycle and provide plausible targets for vaccine development. We briefly describe the abundantly expressed and other surface proteins of T. cruzi and evidence that they are recognized as antigenic targets of immune responses elicited in infected experimental animals and humans.

Abundantly Expressed Surface Antigens Many of the GPI-anchored surface proteins are encoded by genes belonging to large families, e.g., trans-sialidase (TS) super family (737 genes), mucins (662 genes), mucin-associated surface proteins (MASPs, 944 genes), and glycoprotein 63s (GP63s, 174 genes) (El-Sayed et al., 2005). These gene families are largely T. cruzi-specific and account for ⬎18% of the total protein-encoding genome. Trans-sialidases are a heterogenous group of GPIanchored proteins, a majority of which are expressed in the trypomastigote stage. Most TS family members contain a conserved sialidase super family motif (VTVxNVxLYNR) but are not enzymatically active. The enzymatically active TS variants consist of 12-amino-acids long shed acute phase antigen (SAPA) repeats, are expressed in the trypomastigote stage, and catalyze the sialylation of parasite mucins through transfer of sialic acid from the host cell surface (Schenkman et al., 1994). This step is necessary for T. cruzi attachment and invasion of host cells, survival in and escape from the parasitophorous vacuole, and differentiation into a replicative amastigote form (Hall, 1993; Schenkman et al., 1994; Tan and Andrews, 2002). TC85, another member of the TS super family, binds to laminin and cytokeratin in vitro, and is hypothesized to facilitate movement of T. cruzi through the extracellular matrix, allowing it to traverse the tissues to invade other organs (Magdesian et al., 2001). The substantial diversity of the TS gene family has likely developed in response to host immune pressure. Accordingly, several of the TS family members are identified to be targets of humoral and cell-mediated immune responses in Chagasic patients and experimental animals. A trypomastigote surface antigen, TSA1 (85 kDa), was the first T. cruzi protein shown to be the target of CD8⫹ T lymphocytes in infected mice (Wizel et al., 1997) and humans (Wizel et al., 1998b). TSA1 peptide-specific CD8⫹ T cell lines were cytotoxic against peptide-sensitized or infected cells, secreted IFN-γ and TNF-α, and were able, upon adoptive transfer, to confer substantial protection against challenge infection in mice (Wizel et al., 1997). Antibodies to enzymatically active TS (120–200 kDa) have been detected in the sera of Chagasic patients and experimental animals infected with T. cruzi (Pereira-Chioccola et al., 1994). The TS catalytic domain was identified to contain epitopes recognized by IFN-γ-producing type 1 cells and antibodies in Chagasic patients (Ribeirao et al., 2000). Amastigote stage-specific proteins of TS family, i.e., ASP1 (Santos et al., 1997), ASP2 (Low and Tarleton, 1997), and ASP9 (Boscardin et al., 2003) have been identified, although their precise role is presently

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unknown. ASP1 (78 kDa) and ASP2 (83 kDa) were shown to be the targets of in vivo (mice)-generated CTLs that were parasite- and peptide-specific, MHCrestricted, and CD8-dependent (Low et al., 1998). Human circulating CTLs specific for ASP1- and ASP2derived peptides were detected in HLA-A2⫹ T. cruziinfected patients (Wizel et al., 1998b). Mucins contain a large proportion (85%) of Oglycosidic-linked carbohydrates. Mucins of 35–50 kDa are present in insect stage epimastigotes and metacyclic trypomastigotes, whereas larger (80–200 kDa) mucin proteins are expressed in bloodform trypomastigotes. The large heterogeneity of mucins is due to the presence of different core proteins along with the complexity and extent of oligosaccharide side chains (Schenkman et al., 1993). Mucins serve as acceptors of sialic acid in a trans-sialylation reaction catalyzed by TS and are essential for parasite protection and survival in a mammalian host (Schenkman et al., 1993; Almeida et al., 1999). T. cruzi mucins, such as GP35/50 and SSP3, expressed in metacyclic and trypomastigote stages, respectively, have been ascribed an analogous function in parasite ligand–host receptor binding (Schenkman et al., 1993). The GPI component of mucins is a powerful inducer of polyclonal B cells, inflammatory cytokines, and macrophages (AcostaSerrano et al., 2001). The differences in the structure, composition, and abundance of the mucin proteins may contribute to infectivity, virulence, and tissue tropism of T. cruzi or constitute an immune-evasion mechanism, but this has not yet been experimentally proven. MASP family members consist of chimeric domains, i.e., an N- or C-terminal conserved domain combined with the N- or C-terminal domain of mucin or the C-terminal domain from the TS super family (El-Sayed et al., 2005). The mechanism of generation of such chimeric MASPs, their functions in parasite life cycle or immunogenic potential are unknown. GP63 family members were recently identified in T. cruzi. These are homologs of Leishmania major surface protease, GP63, and may serve analogous function as a surface protease (Cuevas et al., 2003). The immunologic potential of T. cruzi GP63s has not been examined to date. Other Immunogenic Proteins Complement regulatory protein (CRP), the 160 kDa surface antigen of trypomastigotes was named so because of its ability to protect T. cruzi from complement-mediated lysis (Norris et al., 1989). Antibodies to CRP have been detected in sera of human Chagasic patients (Norris et al., 1994).

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Cruzipain, the major lysosomal cysteine proteinase (60 kDa) of T. cruzi, is encoded by multiple polymorphic tandemly organized genes located on different chromosomes (Campetella et al., 1992). Cruzipain consists of a catalytic moiety with high homology to cathepsins S and L, and a C-terminal domain, characteristic of type I cysteine proteinases of trypanosomatids (Cazzulo et al., 2001). The proteolytic function of cruzipain is essential for parasite differentiation and invasion of mammalian cells (Cazzulo et al., 2001). Irreversible inhibitors of cruzipain blocked parasite development, exhibited trypanocidal activity, and are currently examined for their therapeutic utility against T. cruzi (Santos et al., 2005). Cruzipain-derived epitopes were recognized by antibodies and IFN-γproducing, CD8⫹ immune cells in Chagasic patients, signifying its immunogenicity (Fonseca et al., 2005). FCaBP, a 24 kDa flagellar calcium-binding protein, is expressed in all life cycle stages of T. cruzi. The N-terminal 24 amino acids, modified by palmitoylation and myristoylation, facilitate the flagellar localization of FCaBP (Godsel and Engman, 1999). Immune sera from T. cruzi-infected experimental animals and humans exhibit highly specific, sensitive reactivity with FCaBP (Godsel et al., 1995). FCaBP-specific CD8⫹ T-cell are generated in mice infected with T. cruzi (Fralish and Tarleton, 2003). GP90 (90 kDa) and GP82 (82 kDa), expressed on a plasma membrane of metacyclic trypomastigotes, interact with distinct host cell receptors. Attachment of the GP82-host cell receptor triggers a Ca2⫹ response in the target cell as well as in the parasite, an event that enables parasites to gain entry to the host cell (Yoshida et al., 2000). Binding of GP90 to its receptor failed to induce a Ca2⫹ response in parasites as well as host cells (Ruiz et al., 1998). Reduction in GP90 expression on the parasite surface resulted in increased infectivity, leading to a suggestion that GP90 may modulate parasite invasion by altering GP82-host receptor interactions (Malaga and Yoshida, 2001). GP82-specific antibodies cross-reacted with heterologous antigens (GP90 and GP30/55), while antisera to GP90 reacted only with the homologous antigen (Yoshida et al., 1993). GP90 is specifically recognized by antisera from Chagasic, but not leishmaniasis, patients (Schechter et al., 1983), thus, suggesting its diagnostic importance. Immunization with GP82 or GP90 stimulated T. cruzi-specific CD4⫹ T cell proliferation in lymph nodes of mice, signifying immunogenicity of the two glycoproteins (Yoshida et al., 1993). A 11 kDa, cytoskeleton-associated, kinetoplastid membrane protein, KMP11, is highly conserved among trypanosomes (Thomas et al., 2000), though the biological role it plays remains unclear. The immunologic role

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of KMP11 was evidenced by the detection of KMP11specific antibodies in sera of chronically infected patients (Trujillo et al., 1999). Spleen cells from KMP11-immunized C57BL/6A2/Kb transgenic mice, when incubated with an immunodominant A2-specific peptide from KMP11, lysed antigen, or peptide-pulsed JurkatA2/Kb cells, suggesting the presence of a human CTL epitope in KMP11 (Maranon et al., 2001). LYT1 (61 kDa), a secreted antigen, is expressed in all parasite life cycle stages. Genetic deletion studies showed that LYT1 is not required for viability of epimastigotes, but is essential for parasite development in mammalian cells. LYT1-deficient mutants exhibited accelerated in vitro development, reduced infectivity, and diminished hemolytic activity that was restored upon complementation with episomally expressed LYT1 (Manning-Cela et al., 2001). LYT1-derived H-2Kb peptides were presented in association with MHC I molecules. Antigen-specific CTLs generated in T. cruzi-infected mice showed high levels of lytic activity against peptide-pulsed target cells, thus signifying the immunogenicity of LYT1 (Fralish and Tarleton, 2003). Paraflagellar rod proteins (PFR1-4, MW ~70 kDa; PFR5-6, 78–86 kDa) are highly conserved among different T. cruzi strains of clinical importance (Fouts et al., 1998; Clark et al., 2005). PFRs constitute a major structural component of flagellum and are critical for cell motility (Fouts et al., 1998; Clark et al., 2005). PFRs are highly immunogenic. PFR-specific CD8⫹ T cells were elicited in infected mice and showed high levels of lytic activity against peptide-pulsed target cells and secreted IFN-γ in response to parasiteinfected target cells (Wrightsman et al., 2002). PFRs are recognized by sera antibodies from indeterminate and chronic Chagasic patients (Michailowsky et al., 2003). Incubation of recombinant PFRs with PBMCs of Chagasic patients induced proliferation of antigen-specific, IFN-γ producing CD4⫹ and CD8⫹ lymphocytes (Michailowsky et al., 2003). TC52, a 52 kDa trypanothione: glutathione disulfide thioltransferase enzyme, is essential for maintaining the intracellular thiol-disulphide redox balance in T. cruzi (Moutiez et al., 1997). T. cruzi heterozygous mutants deleted of one TC52 allele exhibited low virulence and caused an attenuated form of Chagas disease in experimental animals. Deletion of both TC52 alleles was lethal, indicating the importance of TC52 in parasite survival (Garzon et al., 2003). This enzyme is specific for the parasite (Moutiez et al., 1997) and targeted for therapeutics development. While TC52 was found to be immunologically silent in acutely infected animals, the immunomodulatory role of TC52 was suggested by the fact that recombinant TC52, in

synergy with IFN-γ, stimulated gene expression for iNOS and IL-12, and NO production in φs (FernandezGomez et al., 1998). A TC52-specific immune response appeared late in T. cruzi infection and may play a role in the modulation of its biological function(s) (Fernandez-Gomez et al., 1998). The continuing progress in T. cruzi genomics, proteomics, and bioinformatics, followed by wet-lab testing of several genes in experimental models, is likely to further expand the pool of antigenic targets of T. cruzi.

Subunit Vaccines in Development (over the Last 10 Years) A Brief Overview Armed with data on protective immune mechanisms and their antigenic targets, researchers interested in vaccine development against T. cruzi were required to identify effective antigen delivery systems to elicit potent immune responses to the vaccine candidates. Initial studies utilized a protein immunization approach to test the vaccine potential of T. cruzi antigens. Most of these studies were designed such that the protective efficacy of putative vaccine candidates was determined based upon survival following a lethal challenge infection. Efforts were made to enhance the protective efficacy of protein vaccines by codelivery of adjuvants, use of alternative routes of antigen delivery, and by increasing the amount or the number of doses. In subsequent studies, a DNA immunization approach was favored due to the ease of construction and production of the vectors, the stability of DNA, and the ability to enhance the immune response by the codelivery of genes encoding cytokines. Most importantly, this method of antigen delivery has proved to be efficient in eliciting antibodies, Th1 cytokines, and CD8⫹ T cell immune responses to encoded antigens. Successful induction of humoral and/or cellular immune responses to the plasmid-encoded antigens using various routes of gene delivery has been shown to provide partial or complete protection against numerous infectious agents (reviewed in Donnelly et al. (2005)). T. cruzi proteins identified as targets of specific antibodies and CTLs in infected mice and humans (see section Efforts Toward Identification of T. cruzi Antigenic Targets) were tested as DNA vaccines in experimental models. Considering that complete genes were incorporated into the DNA vectors, epitopes capable of being presented by many MHC alleles were expected to be present. Accordingly, several of the genes were tested and afforded protective immunity in multiple mouse strains.

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In parallel with the efforts toward identification of vaccine candidates, adjuvants were tested to enhance or skew the immune responses toward desirable Th1 type. Use of adjuvants to increase protective immunity against T. cruzi dates back to 1965 (Menezes, 1965) when saponin, a derivative from the bark of Quillaja saponaria (Quil A), was injected to enhance the protective efficacy of immunogens. Co-inoculation of saponin with freeze-thaw-inactivated parasites stimulated a Th1 type immune response, and slightly increased protection from challenge infection (Johnson et al., 1963). Recent studies have examined the utility of IL-12, GM-CSF, CD40, HSP70, and CpG oligodeoxynucleotides in enhancing the Th1 responses to defined antigen vaccines. GM-CSF was chosen as a genetic adjuvant because it is a potent cytokine capable of enhancing the antigen presentation capability of antigen-presenting cells, such as dendritic cells. In addition, it facilitates B- and T-cell-mediated immunity (Warren and Weiner, 2000). IL-12 is a key cytokine involved in CD8⫹ T cell activation and proliferation, and in directing the immune responses to type 1 (Pan et al., 1999). Similarly, other adjuvants were chosen for their ability to skew the immune response to a protective Th1 type through different mechanisms. We discuss the efforts to date focused on testing and enhancing the protective efficacy of subunit vaccines against T. cruzi (Table 69.2). Subunit Protein Vaccines Initial studies examined the vaccine potential of GPI-proteins of T. cruzi, selected for their abundant expression and immunogenicity (see section Efforts Toward Identification of T. cruzi Antigenic Targets). GP90 Though recognized as an antigenic target in infected mice and humans, GP90 proved to be poorly immunogenic as a vaccine. Specific antibody and cellmediated immunity were potentiated when GP90 was codelivered with saponin adjuvant, and these immune responses were effective against a range of T. cruzi strains. Further, the levels of acute tissue damage, as measured by production of auto antitissue immunoglobulins, were significantly reduced in immunized mice and marmosets (Scott et al., 1985). All animals, immunized and nonimmunized, remained xenodiagnosis-positive (tested at 60 weeks postinfection), which suggested sterile immunity was not achieved. GP82 Mice immunized with a C-terminal peptide of GP82 (224–516 amino acids) plus alum elicited specific antibodies that were not protective; these antibodies

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lacked agglutinating or complement-dependent cytotoxicity and failed to neutralize parasite infectivity. The substantial resistance exhibited by immunized mice to acute infection was correlated with intense proliferation in the spleen of IFN-γ-producing CD4⫹ T cells (Santori et al., 1996). GP56 Immunization of mice with purified GP56, followed by challenge infection, provided increased survival by only 4 days compared to those not immunized before infection. Considering an epimastigote stage protein that is not expressed by infective trypomastigote and intracellular stages, limited protection afforded by GP56 was not surprising (Harth et al., 1994). Cruzipain Cruzipain-immunized mice exhibited delayed death upon lethal challenge infection (Laderach et al., 1996). Efforts to enhance the protective efficacy of cruzipain utilized codelivery of IL12 (plus neutralizing anti-IL-4 antibody) (Schnapp et al., 2002) or CpGODN (Frank et al., 2003), selected for their capacity to drive immunity toward a Th1 bias. Immunization of mice by either of these vaccines elicited high specific antibody titers. Spleen cells from immunized mice, when stimulated in vitro with antigen or infected φs, strongly proliferated and produced high levels of IL-2 and/or IFN-γ cytokines. All immunized mice exhibited substantial resistance to T. cruzi, evidenced by low parasitemia and 80% survival to acute infection. While cruzipain-specific Th1 responses were associated with protective immunity to T. cruzi in vitro and in vivo, adoptively transferred cruzipain-specific T cells failed to confer protection against challenge infection in mice, indicating that additional immune mechanisms were important to cruzipain-specific immunity. Recombinant attenuated Salmonella typhimurium serovar expressing cruzipain served to elicit immunity to mucosal T. cruzi infection in mice. PFR The protective efficacy of PFR proteins has been tested in detail. First, PFRs purified from T. cruzi were shown to protect mice against lethal challenge infection that was markedly governed by an antigen delivery route. The subcutaneous route of antigen delivery elicited protective immunity, while intraperitoneal injections elicited a strong, nonprotective antibody response (Wrightsman et al., 1995). Similar observations were made with recombinant PFRs. The subcutaneous, but not intraperitoneal, administration of PFR1 and PFR2 (individually or in combination) with Freund or alum adjuvant provided significant protection in 100% and 83% mice, respectively, against

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TABLE 69.2 Subunit vaccines against T. cruzi Antigen

Adjuvant used

Experimental model mouse (T. cruzi) strain

% Survival (dpia)

References

60 (100)c Not detectedc 40 (12) 67–80 (60–100)c

Scott et al. (1985) Santori et al. (1996) Harth et al. (1994) Laderach et al. (1996), Schnapp et al. (2002), Frank et al. (2003) Miller et al. (1996), Wrightsman and Manning (2000), Wrightsman et al. (2002), Luhrs et al. (2003) Ouaissi et al. (2002) Sepulveda et al. (2000) Araujo et al. (2005)

Protein vaccine GP90 GP82 GP56 Cruzipainb

Saponin Alum Freund adjuvant IL-12, CpGODN

PFR1, PFR2b

Alum, Freund, IL-12

CBA, Marmoset (Y) Balb/c (CL) Swiss-Webster (Y) C3H/HeN (RA) Balb/c (Tulahuen) C57BL/6, Balb/c (Peru)

TC52 CRP ASP2

Alum, Bordtella pertusis Freund adjuvant Alum, CpGODN

Balb/c (Y) Balb/c (Y) A/Sn (Y)

62 (120)c 10 (40)e 53.3 (60)d

DNA vaccine CRP TSA1b

None IL-12 ⫹ GM-CSF

100 (40)c 60 (140)d

ASP1b ASP2b ASP2 ASP9 TSb TSSAb

IL-12 ⫹ GM-CSF IL-12 ⫹ GM-CSF None None None IL-12, RANK-L

KMP11 LYT1 FCaBP TCβ3 PFR2 PFR3 ASP1 ⫹ ASP2 ⫹ TSA1 TS family members Mucin family members ASP2 ⫹ TSA1

HSP-70 IL-12 ⫹ GM-CSF IL-12 ⫹ GM-CSF IL-12 ⫹ GM-CSF HSP70 HSP70 IL-12 ⫹ GM-CSF None None None

Balb/c (Y) Balb/c, C3H/HeSnJ, C57BL/6 (Brazil) C3H/HeSnJ, C57BL/6 (Brazil) C3H/HeSnJ, C57BL/6 (Brazil) A/Sn (Y) Balb/c (Y) Balb/c (Y) Balb/c, C3H/HeJ, C57BL/6, B6 (Tulahuen) Balb/c (Y) C57BL/6 (Brazil) C57BL/6 (Brazil) C57BL/6 (Brazil) Balb/c (Y) Balb/c (Y) C3H/HeSnJ, C57BL/6 (Brazil) C57BL/6 (Brazil) C57BL/6 (Brazil) A/Sn (Y)

83–100 (30–60)c

50 (70)c 80 (75) 0 (75) 0 (75) 100 (35)c,f 100 (35)c 83 (140)c 75 (75) 25 (75) 86 (60)c,f

Sepulveda et al. (2000) Wizel et al. (1998a), Garg and Tarleton (2002) Garg and Tarleton (2002) Garg and Tarleton (2002) Vasconcelos et al. (2004) Boscardin et al. (2003) Costa et al. (1998) Katae et al. (2002), Miyahira et al. (2003) Planelles et al. (2001) Fralish and Tarleton (2003) Fralish and Tarleton (2003) Fralish and Tarleton (2003) Morell et al. (2006) Morell et al. (2006) Garg and Tarleton (2002) Fralish and Tarleton (2003) Fralish and Tarleton (2003) Vasconcelos et al. (2004)

⬍60 (140)d 80 (140)c,f 63 (60)d,f 100 (60)c 100 (50)c,f 80–100 (40)c

Immunotherapeutic DNA vaccine TSA1b None TC24b None TSA1 None TSA1 None TSA1 None ASP9 None TS None TSA1 None Tc52 None Tc24 None

Balb/c (H4), acute model Balb/c (H4), acute model CD1 (H1), chronic model ICR (H1), acute model ICR (H1), chronic model ICR (H1), acute model ICR (H1), acute model ICR (H1), acute model ICR (H1), acute model ICR (H1), acute model

70 (45)c,f 100 (45)c,f 100 (140)f Not detectedc,f Not detectedc,f 50 (50)e 50 (50)e 70 (50)c,f 75 (50)c 85 (50)c,f

Dumonteil et al. (2004) Dumonteil et al. (2004) Dumonteil et al. (2004) Zapata-Estrella et al. (2006) Zapata-Estrella et al. (2006) Sanchez-Burgos et al. (2007) Sanchez-Burgos et al. (2007) Sanchez-Burgos et al. (2007) Sanchez-Burgos et al. (2007) Sanchez-Burgos et al. (2007)

Recombinant virus vaccine Ad-TSSA/MVA-TSSA rAD-ASP2 rAD-TS rAD-ASP2 ⫹ rAD-TS

C57BL/6 (Tulahuen) Balb/c (Y) Balb/c (Y) Balb/c (Y)

100 (50)c 80 (160)c 50 (160)d 100 (160)c

Miyahira et al. (2005) Machado et al. (2006) Machado et al. (2006) Machado et al. (2006)

MVA-RANK-L None None None

a

Experimental animals were observed for survival for n days postinfection. These antigens were shown to provide variable degree of protection in different mouse strains (data presented is from the animal model that exhibited best protection). c Upon challenge infection, immunized animals exhibited very low (ⱕ10%) parasitemia as detected in unimmunized/infected animals (data presented is from the animal model that exhibited best protection). d Upon challenge infection, immunized animals exhibited moderate (~50%) parasitemia as detected in unimmunized/infected animals (data presented is from the animal model that exhibited best protection). e Upon challenge infection, immunized animals exhibited similar parasitemia as detected in unimmunized/infected animals (data presented is from the animal model that exhibited best protection). f Immunization with these antigens was effective in decreasing the severity of chronic disease, evaluated by histopathological analysis of cardiac tissue biopsies. b

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a lethal T. cruzi challenge. PFRs alone or delivered with other adjuvants (QS-21, Ribi-700, IL-12), irrespective of the route of antigen delivery, failed to control T. cruzi infection (Miller et al., 1996). Protection afforded by PFRs plus Freunds or alum adjuvants was associated with high levels of IFN-γ and IL-2 and low levels of IL-4 production in immunized mice. Using knockout mice depleted in specific immune functions, it was shown that PFR-induced protective immunity was MHC I- and CD8⫹ T cell-dependent, and B cell responses were not necessary for resistance to T. cruzi infection (Miller et al., 1997). The function of CD4⫹ T cells in PFR-immunized mice was associated with parasite clearance, as was also evidenced by the fact that these CD4⫹ T cells released IFN-γ and stimulated NO production in infected φs, essential for killing of T. cruzi (Miller et al., 1997). Others showed that protection against T. cruzi in PFR-immunized mice was provided by induction of a highly polarized type 1 cytokine profile (Luhrs et al., 2003). TC52 TC52 recombinant protein–elicited protection from infection in immunized mice was associated with TC52-mediated: (i) alleviation of immunosuppression otherwise presented by acute T. cruzi infection, and (ii) enhanced maturation of dendritic cells that play a central role in initiation of Th1 immunity (Ouaissi et al., 2002). The activation/maturation of dendritic cells from immunized mice was evidenced by increased CD83 and CD86 expression, inflammatory chemokines (IL-8, MCP-1, and MIP-1α) production, and presentation of potent co-stimulatory properties. Codelivery of alum or Bordtella pertusis adjuvants enhanced the protective efficacy of TC52 in immunized/infected mice. CRP Immunization of mice with either the protein or DNA CRP vaccine elicited a Th1 type T cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with CRP-encoding plasmid produced antibodies that exhibited complement-dependent cytotoxicity to parasites and were protected against a lethal challenge infection with T. cruzi; thus suggesting the superiority of DNA immunization over protein immunization with recombinant CRP (Sepulveda et al., 2000). ASP2 Protective efficacy of ASP2 was reported recently. Immunization of A/Sn mice with ASP2 recombinant protein alone or with ASP2 DNA followed by ASP-2 recombinant protein resulted in 53% and 75 % survival, respectively (Araujo et al., 2005). Six ASP2-recombinant peptides, representing different segments of the ASP2 protein were tested

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for their efficacy as immunogen with alum and CPGODN 1826 adjuvants. ASP2 peptide consisting of the CD8⫹ epitope provided 100% survival from challenge infection, indicating the importance of a CD8⫹ T cell response in eliciting protection against T. cruzi infection (Araujo et al., 2005).

Subunit DNA Vaccines Trans-Sialidase Genes The vaccine potential of several members of the TS gene family has been examined by the authors and others. TSA1, to our knowledge, was the first subunit DNA vaccine tested against T. cruzi. Intramuscular immunization of TSA1 in C57BL/6 (H-2b) and Balb/c (H-2d) mice generated efficient antiparasite antibody responses and primed CTLs that lysed antigen-presenting cells in an antigenspecific, CD8⫹ T cell-dependent manner. When challenged with T. cruzi, immunized mice (64% C57BL/6 and 89% Balb/c) survived lethal infection (Wizel et al., 1998a). We have evaluated the protective efficacy of ASP1 and ASP2 DNA vaccines. C57BL/6 mice immunized with ASP1- or ASP2-encoding plasmids elicited protective levels of immune responses, evidenced by decreased parasitemia and 50% and 80% survival rates, respectively, from lethal infection (Garg and Tarleton, 2002). Codelivery of IL-12- and GM-CSF-encoding plasmids with ASP1- or ASP2-expression vectors resulted in splenomegaly, an indicator of the increase in lymphocyte activation and enhanced immune reactivity. In addition, an increase in the induction of immune responses, i.e., antigen-specific CTL activity, parasite-specific humoral immune responses, and secretion of type 1 cytokines (IFN-γ), correlated with improved resistance to challenge infection, was observed following co-administration of cytokines with T. cruzi antigen-encoding plasmids. Importantly, mice immunized with ASP2 plus cytokine adjuvants exhibited better control of chronic inflammation and pathological lesions. This was the first observation to demonstrate that vaccination strategies capable of reducing the parasite burden below a threshold level will be successful in controlling the severity of chronic disease and provided a foundation for testing the vaccine potential of other T. cruzi antigenic targets as DNA vaccines. Others have reported the protective efficacy of ASP2 in A/Sn mice, which are highly susceptible to T. cruzi. Mice immunized with ASP2-encoding plasmid exhibited moderate parasitemia and minimal inflammatory foci in the heart and striated muscle. Subsequently, 63% of the immunized mice survived

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as compared to controls that succumbed to challenge infection. Protective immunity was associated with increased secretion of IFN-γ by spleen cells and the presence of ASP-2-specific CD8⫹ T cells (Vasconcelos et al., 2004). The immunogenic potential of a TS gene encoding the enzymatically active trans-sialidase was demonstrated by Costa et al. (1998). Mice immunized with TS-expressing plasmid were protected from challenge infection. The protective efficacy achieved by a DNA–DNA TS vaccine was not increased by DNArecombinant protein vaccination approach (Vasconcelos et al., 2003). Along with the protection afforded by T-cell-mediated immune responses, a strong antibody response that was elicited in immunized mice contributed to protection from infection through inhibition of the enzymatic activity of the native enzyme. It was proposed that inhibition of TS activity prevented the sialylation of parasite surface mucins, a step essential for attachment, invasion, and survival of T. cruzi (details in section Abundantly Expressed Surface Antigens). TSSA, another TS surface antigen, when delivered as a DNA vaccine in mice, elicited CD4⫹ and CD8⫹ T cell-dependent protective immunity against T. cruzi. Codelivery of IL-12 DNA improved the antigenspecific CD8⫹ T cell activity and vaccine efficacy of TSSA, as evidenced by the efficient control of parasitemia and tissue inflammation, and increased survival from infection in immunized mice (Katae et al., 2002). RANK-L, the ligand to receptor activator of NFκB, is implicated in CD40-CD40L-independent T cell priming by dendritic cells. Similar to IL-12, the coadministration of RANK-L-encoding gene enhanced the CD8⫹ T cell-dependent vaccine efficacy of TSSA in mice (Miyahira et al., 2003). KMP11, nonimmunogenic itself, was expressed in fusion with heat shock protein 70 (HSP70) that has adjuvant properties (Qazi et al., 2005). Murine immunization with KMP11-HSP70 elicited antigenspecific, long-lasting IgG2a antibody response and CD8⫹ CTL lytic activity, and subsequently, better control of parasitemia and 50% survival from lethal infection (Planelles et al., 2001). A LYT1-encoding gene delivered with IL-12 and GM-CSF elicited antigen-specific CD8⫹ CTLs and resulted in an 80% survival rate following lethal challenge infection (Fralish and Tarleton, 2003). PFR2 and PFR3 were expressed in fusion with HSP70 for its adjuvant properties, and delivered to chronically infected mice. Immunization with PFR2HSP70 and PFR3-HSP70 induced high levels of PFRspecific IgG2a antibodies. However, only PFR2-HSP70 elicited a statistically significant CTL response that

was sufficient to provide protection against T. cruzi infection (Morell et al., 2006). Not all of the antigenic targets were found to be useful as DNA vaccine candidates. For example, genes encoding FCaBP, TCβ3, mucins, and CCL4/MIP-1β failed to provide any protection from T. cruzi infection (Katae et al., 2002; Fralish and Tarleton, 2003; Roffe et al., 2006). It is interesting that FCaBP and TCβ3 were recognized by CTLs in infected mice and elicited a cell-mediated immune response in mice when delivered as a DNA vaccine, implying that mere elicitation of CD8⫹ T cell responses by an antigen is not indicative of its vaccine potential. Similarly, all of the genes that provided protection in one mouse strain were not protective in other inbred strains of mice. Examples include ASP1, ASP2, TSA1 that were tested individually or in combination (Garg and Tarleton, 1998), and TSSA (Katae et al., 2002). All of these genes failed to alter susceptibility of C3H mice to T. cruzi infection, suggesting host genetic restriction may also contribute to inefficacy of vaccine candidates. Multicomponent Subunit Vaccines Considering that an increase in the level and diversity of T. cruzi-specific immune responses could enhance the protective capacity of DNA vaccines, some investigators tested immunization with multiple genes encoding members of the TS or mucin families. The ability of the mixed genes to elicit protective immune responses depended upon: (i) the amount of a given plasmid sufficient to elicit protective responses, and (ii) the total amount of DNA that can be injected without toxicity. Various dilutions of TS family members (ASP1, ASP2, and TSA1) (0.001–33 μg/mouse) were tested and showed that codelivery of as little as 1 ng of each gene (plus IL-12 and GM-CSF adjuvants) resulted in the activation of a substantial, antigen-specific CTL response, while 10 ng of each DNA was needed to induce moderate levels of a parasite-specific antibody response (Garg and Tarleton, 1998). The level of resistance to T. cruzi infection correlated with the amount of DNA delivered, the maximal protection being obtained with 1 μg of each vaccine DNA plus cytokines. An inhibitory effect on the elicitation of antigen-specific immune responses was not observed when mice were immunized with the mixture of plasmids, indicating that multiple genes with or without cytokine adjuvants can be used in developing immunization strategies for control of T. cruzi infection. The level of protection from T. cruzi infection induced in mice immunized with mixture of TS family members (Garg and Tarleton, 2002; Fralish and Tarleton, 2003) was, however, not significantly better than that induced in mice immunized with individual

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family members (Table 69.2). Attempts to enhance the protective efficacy of TS family members by a DNA prime-protein boost approach were also not successful (Vasconcelos et al., 2003). No protection was observed in mice immunized with a pool of genes encoding mucin family members (Fralish and Tarleton, 2003). The findings of either no or little induction of protective immunity with a cocktail of antigen-encoding vectors was attributed to the fact that genes of large families may express shared epitopes that do not present any protective benefits in inbred mice. It was anticipated that the potential synergistic immunologic benefit of a combination of epitopes from multiple genes would induce a higher frequency of immune effectors in heterogeneous host populations and provide effective immunity against diverse parasite strains, both of which would likely be verified in future studies.

Genome-Based Vaccines By using the sequence database of T. cruzi (El-Sayed et al., 2005), there is the potential to conduct a large-scale, unbiased screening of the T. cruzi genome for the identification of genes of interest. Sophisticated bioinformatics programs are designed to evaluate gene functions on the basis of homologies to genes characterized in other organisms and the presence of motifs predictive of targeting, cellular localization, surface expression, and functional characteristics of the gene product. Such programs have the ability to circumvent the time-consuming, laborious experimental techniques and allow us to directly proceed from sequence information to antigenic target identification and vaccine design. Web-based bioinformatics tools coupled with an experimental strategy have been employed for the identification of putative genes encoding GPI-anchored or secreted proteins in a T. cruzi-expressed sequence tag (EST) database. Molecular and biochemical characterization of eight of the sequences selected by this approach demonstrated that the encoded proteins were conserved in the genome of T. cruzi strains of clinical importance and expressed as surface proteins during different developmental stages of the parasite. When delivered as a DNA vaccine in mice, the selected antigens elicited a trypanolytic antibody response that was in agreement with the intensity of the surface expression of these proteins in infective and intracellular stages of the parasite (Bhatia et al., 2004). This study validated the hypothesis that a T. cruzi sequence database committed to appropriate screening strategies would be an efficient resource for the identification of potential vaccine candidates.

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Therapeutic Vaccines No approved postexposure immunoprophylactic or therapeutic treatment exists for T. cruzi. Several studies have shown improved survival and preservation of cardiac structure in acutely or chronically infected mice treated with TSA1-, Tc52-, and TC24-encoding genes (Dumonteil et al., 2004; Sanchez-Burgos et al., 2007) that was associated with vaccine-induced rapid increase in the number of CD4⫹ and CD8⫹ T cells (Zapata-Estrella et al., 2006). Conversely, treatment of acutely infected mice with ASP2-, and TS-encoding plasmids was not effective in controlling parasetimia or prolonging survival (Sanchez-Burgos et al., 2007; Dumonteil, 2007).

Recombinant Virus Vaccines Replication-deficient recombinant human viruses have an unprecedented ability to induce strong Th1 type immune response (Rocha et al., 2004). Several studies have been performed to evaluate the ability of the recombinant viruses encoding T. cruzi genes in inducing a long-lasting and protective immunity against T. cruzi infection in experimental models. Priming of immune response in mice with adenovirus encoding a single CD8⫹-T-cell epitope derived from TSSA antigen, followed by boosting with vaccinia virus encoding the same CD8⫹-T-cell epitope along with vaccinia virus encoding RANK-L as adjuvant provided significant protection against lethal T. cruzi infection (Miyahira et al., 2005) (Table 69.2). Further, recombinant adenoviruses encoding ASP2 and TS antigens, alone or in combination, elicited strong antibody and T-cell responses and provided high level of protection against lethal T. cruzi challenge in mice (Machado et al., 2006). These studies have opened another arena for developing and testing the potential of T. cruzi vaccines.

Summary Taken together, the above studies established the immunogenic potential of several of the parasite surface antigens and provided evidence for the usefulness of a DNA immunization approach in eliciting protective immune responses against T. cruzi. Unfortunately, all of the tested antigen(s) that conferred protection for a short term (2–4 weeks after vaccination) failed to confer long-term immunity. Further, all of the defined antigens tested so far, individually or in combination, with or without adjuvants, failed to produce sterile immunity and/or prevent death from infection in 100% of the vaccinated animals. It is surmised that

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the immune responses elicited by the tested vaccine candidates were either below the threshold required for generation of sterile immunity or were not rapid enough in their kinetics to control T. cruzi efficiently during the acute phase of infection. The ability of parasites to utilize multiple immune-evasion mechanisms may also contribute to T. cruzi survival in the immunized host.

PROSPECTS FOR THE FUTURE Studies in experimental models have delineated the effector mechanisms that are essential to provide resistance to T. cruzi infection. Several defined vaccine candidates are known to elicit partially protective immunity against T. cruzi. A systemic approach to vaccine development against T. cruzi in future studies would require further characterization of protective immune responses, identification of new antigenic targets of these protective responses, development of efficient antigen delivery systems, and use of adjuvants and vaccination regimens to enhance the protective responses to known vaccine candidates. Given the complexity of the T. cruzi genome, multiple life cycle stages in the mammalian host, and strain variations, it is essential that substantial efforts are employed in selecting the appropriate vaccine candidates. Microarray analysis has emerged as a powerful technology to assess differential gene expression in various stages or strains of pathogens, and identification of stage-specific virulence genes that could be potent drug or vaccine targets (Rathod et al., 2002; Grifantini et al., 2002). The development and free availability of complete genome arrays will facilitate the efforts of the research community in identifying stage-specific novel genes that may also be conserved across various lineages of T. cruzi and serve as potential vaccine candidates. Development of computational strategies for efficient screening of the T. cruzi EST database has proven to be useful in identifying novel vaccine candidates (Bhatia et al., 2004) and we anticipate that complete sequencing and annotation of T. cruzi genome (El-Sayed et al., 2005) would facilitate the unbiased identification and testing of additional vaccine candidates in the next 5 years. Once the selected antigens are shown to be effective in eliciting protective immunity in mice, testing and optimization of the gene-mixes consisting of protective candidates that synergize in their antigenic activity would facilitate formulation of the multivalent vaccines capable of providing maximally protective immunity against T. cruzi. Further development of small (mice) and

large animal models (dogs) would be essential to conduct the field studies and adequately assess the protective efficacy of vaccines in providing short- and long-term immunity before a clinical trial in humans can be envisioned. Given the parasites’ ability to evade immune detection and survive long-term in an immuno-competent host, it is unlikely that anti-T. cruzi vaccines would be effective in preventing infection or in providing sterile immunity. It is, however, likely that vaccines capable of eliciting immune responses that are sufficient to keep the parasite burden below a threshold level would be effective in eliminating the parasite-mediated pathology and tissue injury and thus arrest the progression of Chagas disease severity.

KEY ISSUES ●

●

●

●

●

●

●

●

Several studies have shown that control of acute parasitemia and tissue parasite burden is an effective approach in arresting the development and progression of disease. Some success with the first-generation vaccines, including fractionated or inactivated parasites, led the identification of effector immune mechanisms against T. cruzi. The use of new technologies and experimental systems has allowed a better understanding of the mechanisms underlying the protective immune response against T. cruzi. Secreted and GPI-anchored proteins of T. cruzi are recognized as the best choice for the development of subunit vaccines. The DNA vaccination approach has proved to be promising in eliciting both humoral and cellular immune responses and testing and identification of vaccine candidates against T. cruzi. Completion of T. cruzi genome sequencing and development of a variety of bioinformatics and computational tools would allow the global screening of the genome for the identification of novel vaccine candidates. The complexity of the parasite life cycle, expression of developmentally regulated proteins in different morphological stages, and multifaceted manifestations of Chagas disease caused by different T. cruzi strains necessitates the identification of multiple antigenic targets. The complexity of the protective immune responses constituted by lytic antibodies, CTLs, and type 1 cytokines complicates the selection of protective vaccine candidates.

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ACKNOWLEDGMENTS ●

●

●

●

The expression of immune-suppressive membrane antigens (SAPA (Gorelik et al., 1998), AGC10 (Kierszenbaum et al., 1999)) and the parasite’s ability to utilize multiple immune-evasion mechanisms (Sztein and Kierszenbaum, 1993; Ouaissi et al., 1995, 2001) may limit the immunoprotective capacity of anti-T. cruzi vaccines. The lack of interest of large pharmaceutical companies in developing vaccines against tropical diseases requires a major commitment from the health care agencies of developed and endemic countries. Concerted efforts are needed in testing the vaccine potential of known and novel antigenic targets, followed by the design of a multicomponent vaccine cocktail that is effective in eliciting protective immunity in multiple hosts against different parasite strains. Evaluation of route and mode of delivery, immunization dose and regimens, and the role of adjuvants in eliciting maximal protective immunity in experimental hosts would provide an impetus for future developments.

ACKNOWLEDGMENTS The work in N. J. Garg’s laboratory has been supported in part by grants from the American Heart Association, John Sealy Memorial Endowment Fund for Biomedical Research, American Health Assistance Foundation, and National Institutes of Health. V. Bhatia was an awardee of a Postdoctoral Fellowship from the Sealy Center of Vaccine Development. M. A. Zacks was awarded the James W. McLaughlin Predoctoral Fellowship and a Postdoctoral Fellowship in the Emerging Infectious Disease Program at the US Centers for Disease Control and Prevention. Our thanks are due to Ms. Mardelle Susman for proofreading and editing of the manuscript.

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