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


Generation of attenuated parasites by genetic manipulation and their use as potential vaccines against Chagas disease

Despite the good short-term immunization results achieved with nonreplicating experimental immunogens against T. cruzi40,84—89 and based on the quality and length of the protection achieved, the inoculation and infection by live-attenuated

parasites seem to confer so far the best vaccine effect. These attenuated strains have been shown to yield substantial protection in murine models against a virulent challenge. However, the genetic background and the potential of reversion to a virulent phenotype cannot be foretold, turning them unsuitable for use in human vaccination trials. Nevertheless, the generation of gene transfer experiments as well as the development of new molecular techniques for endogenous gene manipulation has offered the possibility of a better understanding of trypanosomatid genetics and biology. We are now able to identify specific genes in silico, and then, by gene target deletion through homologous recombination, remove them from the parasite genome. This alternative is tempting and promising, since genes related to virulence or persistence could be specifically and permanently deleted or altered. In malaria, the use of genetically modified parasites as experimental vaccines has been well studied.90-92 In different Plasmodium—mouse models, it has been shown that infections with genetically manipulated malaria parasites conferred sterile protection against lethal challenge.93-96 Vaccination approaches based on genetically altered live parasites are also currently under study for Leishmaniasis.97-99 However, in comparison to what has been published for other parasitic organisms, there are relatively few genes which have been genetically manipulated in T. cruzi. The first report of stable transfection of a plasmid vector by homologous recombination in T. cruzi was done in 1993.100 Since then and to our knowledge, there are not many proteins that have been studied in T. cruzi through gene-targeted deletion,101-113 due initially to the lack of more straightforward methods for gene manipulation needed for reverse genetic studies in this organism. However, and until recently, homologous recombination was the only method available for gene suppression or downregulation, since RNA interference has, to date, failed in T. cruzi.114 Recently, advanced and more efficient gene-editing methods have been applied. The CRISPR-Cas9 system was adapted to T. cruzi, demonstrating rapid and efficient downregulation of multiple endogenous genes, including essential genes. This progress holds the promise of developing a variety of genetically defined, mutant T. cruzi strains for immunization.115,116

Most mutants were studied in in vitro systems in order to elucidate metabolic pathways, resistance or susceptibility to different anti-T. cruzi compounds, differentiation of life cycle stages, etc. Only a few T. cruzi mutants have been evaluated in in vivo models and even fewer are the ones evaluated as potential vaccines.117,118

One of the T. cruzi proteins which has been well characterized by reverse genetic studies is the surface glycoprotein GP72. This protein is proposed to be involved in the differentiation of life cycle stages of the parasite119; although its precise role in this process is not completely understood. Parasites carrying a targeted deletion of the gene coding for the GP72 protein were generated (Y-null) and an unexpected altered morphology on the general shape of the parasite was observed as a result of this gene deletion.106 Contrary to that observed for Y wild- type parasites, Y-null parasites could not easily be detected when injected in highly susceptible mouse models.120 PCR reactions carried out in order to determine T. cruzi presence, as well as serological reactions for specific antibodies indicated that Y-null infections were no longer detectable after 90 days postinfection, suggesting that the GP72 protein is essential for sustaining latent infections in immunocompetent animals. Inoculation of Y-null mutant parasites strongly protected adult Swiss mice against a challenge with virulent blood trypo- mastigotes, as shown by a decrease in parasite load in mice preinoculated with the mutant parasites. Even though the protective effect was detectable and significant up to 14 months, the longest interval tested after priming, a weakening of the protection was observed.121 In this sense, and as mentioned before, the duration of the protective effect could be directly related to the antigenic offer at which the immune system is constantly exposed.

In another approach, a monoallelic mutant clone for the calmodulin-ubiquitin (cub) gene (TulCub8) was obtained from the highly virulent Tulahuen strain of T. cruzi. Genetic manipulation of the cub gene resulted in a remarkable reduction in parasite virulence as shown in a murine model; since the mutant clone could only be propagated in mice by means of highly sensitive, hemoculture recovery. Swiss mice were inoculated subcutaneously with doses of TulCub8 epimastigotes and later challenged with virulent wild-type Tulahuen blood trypomastigotes. In this case, a strong protection, based on a reduction in parasite burden in mice preinoculated with TulCub8 epimastigotes, was observed.122

The third T. cruzi mutant tested in protection assays was the T. cruzi clone L16, a Lyt1~/2 null mutant carrying a biallelic deletion of the gene. The infective and protective behavior of the biallelic knockout clone L16 in a murine model was analyzed by Zago et al.123 A significant reduction in the infective capacity of clone L16 was observed in adult Swiss mice, determined by fresh blood mounts, spleen index, and tissue parasite load. Furthermore, a considerable reduction in the muscle inflammatory response elicited by the L16 clone as compared to wild-type parasites was detected. However, a latent and persistent infection with this mutant clone was shown by positive T. cruzi DNA detection in blood samples until 12 months postinfection. Long-term protection was also observed in Swiss mice inoculated with L16 epimastigotes. Fourteen months later, these animals were still strongly protected against a challenge with Tulahuen wild-type blood trypomastigotes as shown by a reduction in the parasite load in blood.123

Another set of genes involved in amastigote energy metabolism is the one encoding the putative enoyl-coenzyme A hydratase/isomerase proteins (ECH1 and ECH2). Mutants carrying only one copy of the echl gene and none of the ech2 genes failed to establish persistent infections in mice. However, oral gavage of ech mutants in mice induced a systemic muscle tissue infection and a potent T. cruzi specific CD81 T cell response.124 Protection conferred by these mutant parasites was obtained after three doses of 105 trypomastigotes administered 2 weeks apart by oral route. Challenge was performed in the footpad using fluorescent trypomasti- gotes125 45 days after the last immunization dose. A high number of activated CD81 T cells in peripheral blood of ech mutant vaccinated group was detected and it was in concordance with a strong protective response, indicating thus T cell proliferation could be a good indicator of the effectiveness of the vaccine.

Since the molecular basis of attenuation in the wild-type TCC strain of T. cruzi is still unknown, attempts to introduce targeted gene deletions into this clone, as a

safety mechanism against eventual reversion to the virulent phenotype were carried out. A TCC monoallelic mutant clone for the dihydrofolate reductase— thymidylate synthase gene (dhfr-ts) was generated.112 In trypanosomatids, dihydrofolate reductase—thymidylate synthase (dhfr-ts) is a single-copy gene encoding an important enzyme involved in the thymidine biosynthesis pathway and, therefore, in the DNA synthesis. Besides showing some delay in growth rate in axenic cultures, TCC dhfr-ts+/~ parasites also showed a striking attenuation in in vivo models. A remarkably low percentage of T. cruzi specific CD81 T cell was detected in mice inoculated with TCC dhfr-ts+/~ parasites. This is not ideal, since the generation of genetically attenuated parasites capable of inducing a strong CD81 T cell response is desirable. However, these mutant parasites retained their protective effect against a virulent T. cruzi challenge in different mouse strains. Even more, these mutant parasites retained their protective effect against a virulent challenge with virulent fluorescent trypomastigote parasites even after a year postinoculation. In all experiments performed, the protection induced by mutant parasites did not differ from that obtained with wild-type parasites, suggesting that the deletion of one dhfr-ts allele did not alter the protective capacity of the original wild-type live immunogen.112

The TCC strain was also genetically manipulated at the crt locus113 since mutant parasites with a monoallelic deletion of the TcCRT gene were generated. T. cruzi Calreticulin is a calcium-binding chaperone involved in the quality control of endoplasmic reticulum nascent proteins. After being translocated to the flagellum pocket, TcCRT inhibits the activation of the classical and lectin complement pathways evading the lytic action of the complement in the host blood system. TcCRT mutant epimastigotes parasites have a high susceptibility to the lytic action of the complement system, compared with the wild-type strain and displayed a stable loss of virulence.113 Mice receiving a prime and boost immunization regimen of 105 mutant or wild-type TCC metacyclic tripomastigotes were protected against a virulent challenge with a T. cruzi field isolate126 after 120 days of the last immunization dose. Mice immunized with TcCRT mutant and TCC wild-type parasites displayed, after challenge, a significantly lowered parasite density in peripheral blood. Necropsies at day 60 postinfection revealed that mice immunized with mutant parasites showed reduced inflammatory response in heart and muscle tissues, compared to controls. Also, the spleen index was significantly reduced in mutant and wild- type TCC-immunized mice.127

To our knowledge, the studies mentioned above are the only ones involving knockout T. cruzi clones in protection assays. These studies included control groups where mice preinoculated with the “wild-type,” nonengineered T. cruzi strain were challenged with virulent parasites. Inhibition of acute phase parasitemia was as strong as with the mutants, but this “protection” (or premunition) was obtained at the expense of a previous infection with heavy parasite load and development of disease. These control groups thus showed that the mutated parasites retained the immunogenic effects of a mild infection, sparing the untoward effects of a previous acute and chronic phase disease. However, the immune bases of the protection elicited by these genetically modified parasites, as well as the presence of

inflammatory infiltrates or amastigote nests in target organs after challenge was not always fully evaluated. However, these results reinforce the possibility of generating transgenic experimental vaccines that combine the immunogenicity of live vaccines and a genetically supported built-in safety modification, currently absent in naturally attenuated parasites.

Also attractive is the generation of mutant parasites expressing foreign genes such as specific immune factors. CD40 is a cell surface receptor belonging to the TNF family and its ligand, CD40L, is a costimulatory protein belonging to the same family. The CD40/CD40L complex not only presents immunomodulatory properties but is also involved in the humoral and cellular response.128 It has been previously shown that T. cruzi parasitemia and mortality rate could significantly be reduced in infected mice by the inoculation of CD40L-transfected fibroblasts together with T. cruzi parasites.129 Mice infected with transgenic T. cruzi parasites carrying a plasmid encoding the gene CD40L were able to better control infection than those infected with wild-type T. cruzi ones. IFN-y production and lymphocyte proliferation was observed by immunization with CD40L-parasites. Even more important is the protective capacity conferred by these parasites. A very low or even null level of parasites was detected in the group of mice first infected with the transgenic parasites, suggesting that the infection by these parasites is able to induce protection against a subsequent virulent infection.130

In summary, T. cruzi is an organism where gene-targeted deletion and gene silencing have so far not been as successfully applied as in bacteria or even as in other trypanosomatids, such as Trypanosoma brucei and Leishmania. Studies where T. cruzi mutants were compared with wild-type parasites using infectivity measurements, almost invariably revealed a change toward attenuation. Sometimes this change has been profound and sometimes partial. Therefore, targeted deletion of specific genes can be conceived as a potential procedure to generate clones able to develop in culture, but less efficient to invade and persist in vivo, providing a potential for mass production coupled with a built-in safety device against reversion to the virulent phenotype. The use of T. cruzi attenuated parasites is very appealing since the infection obtained thereof is much alike to a natural infection and may consequently lead to the establishment of a protective immune response. However, genome manipulation could lead to a loss of the protective immunity, either because such genetically modified parasites no longer express antigen epitopes essentials for triggering a good immune response or because they are not able to persist long enough to fully activate the immune system. In this sense, a wide spectrum of specific individual genes or a combination of them, as well as different T. cruzi strains should be evaluated.

Considering the recent improvements in genetic manipulation, a live- genetically attenuated parasite vaccine applicable to dogs and other mammals, which act as natural reservoirs, and with capacity to reduce the intensity and spread of the disease seems to be a possible and realistic achievement. An important factor to be considered is the presence of antibiotic-resistant genes in the genome of these “vaccine” parasites and the possible implications that this could bring. Drug resistance genes are the common mechanism to select genetically modified parasites; nonetheless the release of drug-resistant parasites to the environment implies an undesirable and potential risk. Hence, one of the main objectives for these mutant parasites is to make them incapable of persistence in the host and completely unable to be spread or transmitted to the insect vectors. Another approach to tackle the drug resistance problem is a new strategy exploited in the generation of Leishmania mutant parasites, in which after the target deletion, the drug-resistant gene is removed from the parasite genome.131 Furthermore, the generation of T. cruzi parasites genetically modified in order to “commit suicide” as a response to external stimulus is also an appealing alternative.132 In this approach, genetically modified parasites could be inoculated and allowed to infect the mice until a fully immune response is developed before being induced to die. This technology has been well documented in other parasitic organisms such as Leishmania.133,134 Based on this, genetically modified T. cruzi parasites could be achieved and safely used as live vaccines.

The recent elucidation of the trypanosomatids genomes and the identification of new targets for genetic manipulation open the possibility of generating a wide variety of mutant parasites that could be evaluated as potential vaccines against Chagas disease. The generation of parasites carrying more than one gene deletion or a combination of gene deletions and/or expression of foreign genes could also be feasible. The strategy of superimposing attenuating mutations into already naturally attenuated and protective strains might provide additional mechanisms against reversion to virulent phenotypes.

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