Researchers found more than one type of T. cruzi parasite strain in the Chilean highlands; they also found that the different strains had higher or lower parasitemia levels. Each T. cruzi strain displayed a unique characteristic. González and colleagues (1995:131) hypothesize that tissue tropism of individual T. cruzi strains and geographic distribution of different strains and their source (sylvatic or domestic) may play a role in the wide variety of clinical signs encountered in Chagas’ disease (Rassi 1977). Conclusions from the Andean northern of Chile study are as follows:
Finally, the parasite sample studied here from humans resembles the main ones circulating in humans of other endemic areas of Chile as described before (Solari et al. 1992; Hendriksson et al. 1993; Mufioz et al. 1994), in spite of the fact that other T. cruzi populations are also transmitted by the insect vectors and their poor infective capacity in the murine experimental models. This observation probably is explained by a special adaptation of the Zymodeme 30 parasite type in human hosts from the presumably mixed infective T. cruzi populations circulating in nature. This observation and the early adaptation of T. cruzi to humans in the [Andean] highlands and other endemic areas of Chile as well, compared to other countries, could explain the more benign character of Chagas’ disease in this geographic area. The biochemical characterization shows that several T. cruzi subpopulations exist in the endemic area of Chile, but it remains to be demonstrated whether the clinical evolution of this parasitism on humans varies depending upon the infective strain involved (Gonzalez et al. 1995:132-33)
APPENDIX 3
Immunization against T. cruzi
Immunization against trypanosomes needs to be targeted at the surface membrane of the parasite, which presents a number of considerations. American trypanosomes, such as T. cruzi, differ from African trypanosomes and do not employ the dramatic surface modulation seen with African trypanosomes, such as T. bruceigambiense and T. b. rhodesiense, that cause sleeping sickness. T. cruzi’s evasive strategies are to share antigens with host cells and to alter host antigens so that when host antibodies attack the parasites they simultaneously attack host cells (Avila 1994, Brener 1994). This is an evasive function of the surface membrane.
A most vital part of any parasite is its surface, which is the site of nutrient acquisition, the site of dealing with host immunity, and the site where it protects itself from biotic and abiotic components of the environment. The surface membrane of T. cruzi as well as similar protozoa exhibits a wide variety of housekeeping functions. For example, it is responsible for ion balance, nutrient transport, and resisting the physical and chemical perils offered by the vinchuca’s gut. The parasite’s surface is also a key participator in the adherence to and penetration of host cells. Moreover, it is this organelle that deals effectively with the host’s immune response.
The more that is known about T. cruzi’s surface, the more we can learn about how it penetrates cells, what it eats, and how it eats. Consequently, extensive research is being done concerning the biochemistry of T. cruzi’s surface, especially on how it interacts with the host’s immune system.
T. cruzi has a very complex life cycle, which is composed of various stages that pass through a multitude of microenvironments within its mammalian and insect hosts. These microenvironments present many hostile elements which must be overcome since they are essential to the parasite as home or transportation and provide the protozoan with the space and nutrients to survive and reproduce. The capabilities of the organism cannot be assumed to be the same in each environment, since the environments differ so dramatically. If T. cruzi alters its surface to deal with environments, it also consequently alters the basis for attack by the host immune system. T. cruzi’s basic survival strategy is alteration of its surface as it passes through various stages within the vector insectfrom trypomastigotes in the foregut, to epimastigotes in the midgut, and to metacyclic trypomastigotes in the hindgut, which are passed in the feces and deposited on the skin of the animal/human host, and, within the host, from metacyclics in the blood, to amastigote forms in tissues, to trypomastigotes circulating in the blood (see Figure 7).
In comparison, African trypanosomes pass through only two stages, trypomastigote and epimastigote. Within a vertebrate host, African trypanosomes multiply as trypomastigotes in the blood and lymph, whereas T. cruzi multiplies intracellularly as amastigotes. African trypanosomes survive by continually varying their coats and presenting new antigens, thereby exhausting the immune system and setting the stage for secondary infections.
African trypanosomes change their surface coat to defeat the immune system, but American trypanosomes have taken up an intracellular existence and interacted with several broad modulations of vertebrate immune function to evade immunity in more subtle but equally effective ways in terms of its survival. T. cruzi doesn’t undergo a dramatic alteration of the glycoprotein coat as do African trypanosomes, but it does have many different strains (See Appendix 2: Strains of T. cruzi). In Bolivia well over a hundred strains of T. cruzi have been identified which have different surface architecture. T. cruzi uses its surface architecture as an immunoprophylactic. For example, once T. cruzi trypomastigotes have penetrated host cells, surface components protect it from lysosomal enzymes and products ofoxidative burst.
The adaptive successes of African and American trypanosomes lie with their surface coat, which is quite able to outsmart immune systems of the host. Generally speaking, the outer surface of any parasite is one of the most important organs in its symbiotic relationship because it provides an interface between the parasite and its vertebrate and invertebrate hosts. A large measure of the success of trypanosomes lies in their ability to modulate their outer surface in response to attack from host antibodies and immune cells and to hostile components from the environment they encounter in the insect gut and in the cells, fluids, and tissues of a vertebrate host. If prophylactic vaccinations are developed against T. cruzi and the African trypanosomes, they will probably have to somehow alter or incapacitate the outer surface of the parasite.
African trypanosomes have coats of glycoprotein, which are capable of producing thousands of antigenic variations. After African metacyclic trypomastigotes have infected a person, the humoral immune system responds by producing antibodies primarily of two classes, IgM and IgG. The IgM class is the first of these defense proteins produced in response to infection. They destroy by agglutination and lysis all antigenically identical organisms within a given population of parasites. Some trypomastigotes escape because they have different surface antigens, however, and they quickly reproduce until there is another attack by a new variety of IgM antibodies. Another group survives with antigenic variations on their surface coats, and another IgM antibody contingent rushes out to kill them. Eventually, the parasites win this battle because their possibility of variation and survival is greater than the strength of the host’s immune system, continually weakened by the stress of the attacks. Moreover, continual lysing of antigens releases toxic substances into the victim’s body. Every wave of antibodies quickly becomes useless because the trypanosomes have selected new coats with new antigens which evade the previous antibodies (Katz, Despommier, and Gwadz 1988). In short, African trypanosomes display a “moving target,” a continual variation of antigenic coatings, so that just as the host mounts an antibody response to one, another type proliferates (Schmidt and Roberts 1989).
Glycoproteins on T. cruzi’s Surface
In the parasite Trypanosoma cruzi, mucin-like glycoproteins play an important role in the organism’s interaction with the surface of the mammalian cell during the invasion process (Di Noia, Sánchez, and Frasch 1995). Mucins are highly glycosylated proteins expressed by most secretory epithelial tissues in vertebrates; but recent research has shown that these geneencoding molecules have been detected in Leishmania major (Murray and Spithill 1991) and in Trypanosoma cruzi (Reyes, Pollevick, and Frasch 1994). These mucin-like genes have a defined basic structure and sequence, which allows their
inclusion in a gene family.
Trypanosoma cruzi has a family of putative mucin genes whose organization resembles the ones present in mammalian cells (Di Noia, Sánchez, and Frasch 1995). Different parasite isolates have different sets of genes, as defined by their central domain. Much work has been done on the biochemical and functional characterization of mucin-like surface glycoconjugates (Schenkman et al. 1994). These heavily O-glycosylated molecules are Thr-, Ser-, and Pro-rich and are attached to a membrane by a glycophosphatidylinositol anchor (Schenkman et al. 1993). Mucins in T. cruzi are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase. These molecules are involved in the cell-invasion process, probably mediating adhesion of the parasite to the mammalian cell surface (Ruiz et al. 1993, Yoshida et al. 1989). A putative mucin gene in T. cruzi has been identified (Reyes et al. 1994), having a small size and encoding five repeat units with the consensus sequence T8KP2. In a later study, Di Noia, Sànchez, and Frasch (1995) establish that T. cruzi does in fact have a putative mucin gene family resembling the one present in vertebrate cells. Their members have a Thr/Ser/Pro-rich central domain, which might or might not be organized in repetitive units, and highly conserved non-repetitive flanking domains.
In earlier studies (e.g., Snary 1985:144-48), the following glycoprotein or antigens have been found on T. cruzi’s surface. Lipopeptido phosphoglycan (LPPG) is a complex surface component found on the membrane of epimastigotes. This large complex molecule includes three glycoproteins with a molecular weight (MW) of 37,000, 31,000, and 24,000 (referred to as GP-37, GP-31, and GP-24, respectively). Indirect evidence indicates that LPPG may also be found on amastigotes, because antibodies against LPPG have been found in the serum of patients with Chagas’ disease. Generally, epimastigotes are thought to occur only in insects and not in the vertebrate host, although this conclusion is not definitive. The presence of LPPG antibodies in hosts apparently indicates that this glycoprotein is also found on trypanosomes in the host. It is not clear why the current reasoning is that it is found on amastigotes alone and not also on trypomastigotes.
The function of LPPG in T. cruzi is not known, but it is postulated that it provides some protection for free-living protozoa from the macroenvironment. Epimastigotes face an environment in the insect’s gut with many hostile components. LPPG, then, may be part of the baggage still carried by T. cruzi from among the characteristics displayed by free-living ancestors of the parasite. Once the ancestors of American trypanosomes gave up their free-living life-style, perhaps they kept LPPG because it came in handy inside the insect’s gut. In other words, it was a pre-adaptive feature allowing them to settle in what would otherwise be a hostile environment.
Glycoprotein (GP-90) is another cell-surface antigen found on all three life-cycle forms-trypomastigote, epimastigote, and amastigote. One parasite-promoting function of GP-90 is that it interferes with complement-mediated lysis of the parasite. Antibodies to GP-90 are found in all patients, and these antibodies do not cross-react with leishmaniasis antigens. Consequently, GP90 shows great promise as a diagnostic tool to differentiate between Chagas’ disease and leishmaniasis in Bolivia, where patients are subject to infection from T. cruzi and from Leishmania sp. and where diagnostic testing frequently does not discriminate between them.
Another useful glycoprotein, GP-72 (MW 72,000) is specific for epimastigote and metacyclic trypomastigote (infective parasites) insect stages, but it is not found on blood trypomastigotes or intracellular amastigotes. GP-72 is very useful for isolating different strains (zymodemes) of T. cruzi, because each strain shows a different concentration ofGP-72the higher the concentration of GP-72, the more pathogenic the strain. Moreover, different strains of the parasite cause different clinical syndromes: some strains tend to megavisceralize and cause megasyndromes; others might concentrate in the heart and nerves.
Antigenic Targets for Immunizations
Glycoprotein (GP) antigens found on T. cruzi’s coat are considered as antigenic targets for immunizations. Because GP-90 is found in all stages of T. cruzi’s life cycle, it may be a candidate target of a vaccine against the parasite. Experimentally, acutely infected chagasic mice were vaccinated with such an antibody and did not die; however, they remained infected. Although this vaccine may be suitable to curtail the ravages of acute infection in infants, it is less than adequate for adults who are suffering with chronic infections.
Because GP-72 is found on the surface of infective trypomastigotes, it would be a good antigen to target in the development of a vaccine. The function of GP-72 is demonstrated by the fact that if GP-72 is stripped from the surface of epimastigotes, they are unable to transform into metacyclic trypomastigotes. GP-72 is essential for this transformation process.
Entomologically, GP-72 is valuable for the making of maps plotted according to geographical areas where various strains (zymodemes) of T. cruzi are found. The pathology of Chagas’ disease is related to zymodemes. From the different regions of Bolivia, triatomines are collected, epimastigotes extracted from infected bugs, and GP-72 measured. This provides a guide to the prevalent strains and pathologies in the departments of Bolivia. Glycoprotein GP-25 is useful in immunodiagnosis and appears on epimastigotes and trypomastigotes.
Another possibility for vaccine involves GP-85, which is thought to be a glycoprotein that allows the parasite to attach itself to the host’s cell membranes. If a vaccine can destroy GP-85, the parasite cannot penetrate and attach itself to cells.
With any of these possibilities, a major problem is that antibodies specific to T. cruzi antigens also cross-react with human host cells. Therefore, even if it worked in mice, there is the possibility that a vaccine may aggravate the situation by inducing antibodies that cross-react with host cell antigens. Autoimmunity is considered to play a central role in the pathology of Chagas’ disease.
Conclusions from research on various biochemical coatings on surfaces of T. cruzi indicate that immunoprophylaxis does not appear to be possible for the following reasons (Snary 1985:144-48): scientists have not found a vaccine that induces sterile immunity, and, furthermore, in regard to experimental vaccines, scientists have not established that those tested would not also induce autoantibody production in the host.
The conclusion is that Trypanosoma cruzi has evolved to fit an intricately complex niche in the organic world. The biology of this organism is complex, involving intimate interactions with two different hosts which include a variety of different life-cycle stages that exhibit major differences in structural and functional biochemistry. The organism’s survival involves highly successful evasive strategies against the human immune system as well as the extraordinary ability to survive in a variety of hostile environments inside insects and humans and to make use of the complex relationship between insects and mammals in the reproduction and transmission of the species.
Prospects for Immunizations
The facts that most of the pathology from Chagas’ disease relates to the immune response and that living organisms are necessary for acquired partial immunity present slim possibilities for developing a vaccine against Chagas’ disease. To produce a vaccine for Chagas’ disease, the following requirements are necessary:
1) Any vaccine should not induce active infection; therefore, immunization with living T. cruzi is out of the question. Reasons include the facts that even weaker strains could elicit autoimmune pathology and that weaker strains are not found, only less virulent strains. Moreover, the risk would be much too great to be undertaken by international health organizations.
2) Any vaccine must confer total and sterile protection. Vaccines that have been tried in mice employing either live and attenuated (weakened) parasites, killed intact organisms, or cell homogenates were able only to delay mortality. These vaccines produce partial protection from acute infections and do not provide protection from chronic infections, which in some cases are a more horrible way to die. Nonetheless, partial protection may be important to prevent the high incidenc
e of deaths of children from acute infections.
3) Any vaccine cannot induce an autoimmune response, so the vaccine has to be very specific and exclusionary in targeting only parasite antigens and not those that the parasite shares with the host. Any misdirection could lead to creating EVI antibodies that are in themselves sources of pathology in the host. Moreover, the targeted parasite antigen must be essential to the parasite and on its surface throughout its trypomastigote and amastigote forms. This implies highly complicated research and very involved testing of vaccines, with the result most likely being high-cost vaccine production that will be unaffordable to people in endemic areas.
If and when a suitable antigen is found and vaccines are developed with it, there are most likely some strains of T. cruzi to which the chosen antigen may be ineffective in inducing protective immunity. Even if all strains are affected, there will be mutant individuals which the vaccine will not affect, leading to the evolution of resistant strains of the parasite. These “super” T. cruzi will continue on.
The Kiss of Death Page 24
