An Effective Live Vaccine Strain Of Trypanosoma Cruzi Prevents Chagas Disease In The Mouse Model

Trypanosoma cruzi is the etiologic agent of Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi, which in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation and in intracellular amastigotes participates in generating ROS enhancing parasite growth. We have generated a parasite knock-out mutant of Cyp19 which fails to replicate in cell culture or in mice indicating that lack of Cyp19 is critical for infectivity. Knock-out parasites fail to replicate in or cause clinical disease in immune-decient mice further validating their lack of virulence. Repeated inoculation of knock-out parasites into immuno-competent mice elicits parasite-specic antibodies and T-cell responses. Challenge of immunized mice with wild-type parasites is 100% effective at preventing disease. These results indicate that the knock-out parasite line is a live vaccine candidate for Chagas disease.


Introduction
The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, which is endemic in Mexico, Central and South America 1, 2 . People living in regions within the lower United States, particularly in Arizona, New Mexico and Texas are also at increased risk for acquiring the disease 3 . The disease is transmitted in multiple ways, chie y by the feeding of hematophagous triatomine insects on mammalian hosts 2 . Humans are an incidental hosts and those living rural areas are at greatest risk where poor housing conditions lead to the entrance and feeding of triatomines into houses 4 . Feeding leads to the deposition of fecal material containing infective metacyclic-trypomastigote parasitic forms that end the contamination of mucosae around the feeding sites 3 . Metacyclics bind to and invade host cells wherein they transform into and multiply in the host cell cytosol as intracellular amastigotes. Amastigotes transform thereafter into motile trypomastigotes that exit host cells, which circulate through the body and further infect cells in a variety of target organs. Chronic infection of organs, particularly cardiac myocytes and gastrointestinal smooth muscle, cause the sequelae of chronic Chagas disease 5 . Additional modes of infection are by oral ingestion of parasite infected items, trans-placental maternal-fetal transmission and through transplant of infected blood and tissues to non-infected recipients 6,7,8,9 . Importantly, the global transmigration of chronically infected hosts leads to the diagnosis of Chagas disease in countries that are not endemic for Chagas. Approximately 20-30% of chronically infected individuals develop chronic symptoms, which include Chagas-associated cardiomyopathy, due to chronic in ammation of cardiac tissue due to direct parasite damage and molecular mimicry 10,11 . Chagas disease is the leading cause of heart failure in Latin America. Mega-organ syndromes of the esophagus and large intestine arise from denervation of the gastrointestinal tract due to direct or indirect damage of the smooth muscle 12,13 .
Upon initial infection, there is limited exposure of the immune system to parasite antigens, since the inoculum is small and rapidly becomes intracellular. As parasites replicate intracellularly and then exit host cells in larger numbers immune activation occurs 14 . The immune response to T. cruzi is complex, but involves the induction of both Th1 and Th2 responses. Th-1 responses are signaled by the production of IL-12 leading to IFN-γ and TNF-α and NO production by macrophages. Activated in ammatory cells produce IL-1, -6 and − 18 and are poised to kill intracellular parasites 15,16 . Anti-in ammatory Th2 responses are also activated in order to control over-aggressive immune activation and involve IL-4 and − 10 production 17,18 . Additionally CD8 + lymphocytes and anti-parasite IgG is also produced which limit the increase in parasite load 19 . Chronic smoldering infection ensues after several years due to several factors eventually leading to chronic sequelae of Chagas disease in about one-third of infected patients.
It is unclear why only a subset of infected individuals develops these chronic manifestations, but this is likely due to complex interplay host genetic and parasite factors 20,21,22 . Treatment of Chagas disease is limited to two drugs, nifurtimox and benznidazole, with considerable side effects. There are no vaccines available for prevention of Chagas disease.
The virulence of T. cruzi is dependent on a myriad of protein-, lipid-and glycan-based virulence factors, which lead to the binding, invasion and intracellular replication of parasites in both phagocytic and nonphagocytic cells 23,24 . A number of years ago our laboratory identi ed a secreted cis-trans peptidyl-prolyl isomerase, termed cyclophilin 19, which was expressed by insect stage T. cruzi and promoted the resistance of parasites to killing by insect anti-microbial peptides as well as induction of parasite calcineurin leading to partial transformation of parasites into metacyclic forms and enhancement of infectivity 25 . Since then we have found that cyclophilin 19 is expressed by and secreted by all parasite stages including intracellular amastigotes, where is promotes the reactive oxygen species important for intracellular parasite growth and survival (Pedroso, et al, unpublished). Cyclophilins are a highly conserved family of enzymes, which play a myriad of roles in biology, mainly acting as protein chaperones by isomerization of proline residues within protein substrates 26,27 . Aberrant expression of cyclophilins, particularly the closest human homologue to cyclophilin 19, cyclophilin A, is associated with enhanced invasiveness of certain malignant tumors 28 and the induction of cardiac in ammation 29 . The binding and isomerization of proline within the cell signaling ligand, CD147 by extracellular cyclophilin A, leading to its activation, is mechanistically implicated in these phenotypes 30,31 .
The genome sequence of T. cruzi indicated that cyclophilin 19 is encoded by a single-copy gene, so in order to determine the role of cyclophilin 19 in parasite virulence and pathogenesis, we employed a double-allelic homologous recombination gene knock-out strategy to remove cyclophilin 19 genes from parasites. Double knock-out parasite mutants with depleted cyclophilin 19 were unable to infect host cells in vitro or mice in vivo. Repeated infection of mice with these mutants led to increasing levels of parasite speci c T-and B-cell responses that were 100% effective at preventing death of acute Chagas disease in mice indicating this may be an effective vaccine strain for prevention of Chagas disease. The mutant strain did not replicate in immune-de cient mice strains and dexamethasone treatment of mice infected with the mutant strain did not develop clinical disease or the emergence of parasites, indicating that the vaccine strain is safe in immune-suppressed host conditions. Lastly, a single dose of this attenuated parasite line led to complete protection of high dose challenge with highly virulent wild type parasites.
Overall, this data is proof of principle that this mutant line is an effective and safe live vaccine strain against Chagas disease.
Genomic DNA Isolation. Epimastigotes from T. cruzi wild type (WT) and cyp19 knock out (KO) were collected from the culture and washed twice with PBS. Genomic total DNA was extracted using DNeasy Blood & Tissue Kits (Qiagen) according to the described manufacturer's protocol. PCR was performed to amplify various targets to clone into plasmids and to con rm the presence and absence of genes.
Transfection, selection and cloning of T. cruzi. A total of 1 × 10 7 early log phase of T. cruzi epimastigotes was used to transfect with 50 µg of linearized Neo/Hyg plasmid using the electroporator (Harvard Apparatus BTX, Holliston, MA). Transfected parasites were maintained for 24 h in LDNT medium alone.
The selection pressure was created with 200 µg/ml of G418 and 200 µg/ml of Hygromycin B for transfectants with neomycin phosphotransferase and hygromycin phosphotransferase gene cassette, respectively. Parasites were selected for 4-5 weeks post-transfection and considered fully selected when all the parasites with no resistant marker gene cassette were dead. Individual clones were obtained by limiting dilution into a 96-well plate. Individual cloned cells were further analyzed for the absence of cyp19 gene.
Nuclear genomic sequencing and analysis. The libraries were sequenced using a PE75 protocol (75-bp reads from each paired end) on a HiSeq2000 at the UW Sequencing Northwest Genomics Center. After deindexing, we obtained between 21.2M (JM255) and 44.1M (JM256) reads per library. Aligned all reads from the 7 WGS libraries against the 43 chromosomes of the TcBrA4 genome from TriTrypDBv46 using Geneious assembler containing the following loci: 1) Cyp19: A 21,328-bp fragment from PRFA01000019 (reverse complemented) containing three genes on either side of C4B63_19g183), which is the orthologue of TcCLB.506925.300. This locus is on Chr39-S in CL Brener; 2) Cyp11: A 12,966-bp fragment from PRFA01000149 containing 4-5 genes on either side of a paralogue (C4B63_149g20) of TcCLB.506925.300. This locus is on Chr22-S in CL Brener. The alignments were manually examined and the reads-per-kilobase-per-million (RPKM) calculated for each of the contig in order to compare gene copy numbers. Depending on the library ~ 10-20% of the reads did not align. Chromosomal number was estimated in each sample using two different approaches: 1) Normalization of the mean read coverage for each chromosome by dividing by the median of all 43 chromosomes and multiply by 2; 2) Normalization of the RPKM (from Geneious) for each gene by dividing by the median for all genes and multiplying by 2 to generate the "copy number" of each gene, allowing to calculate the copy number mean for all genes on each chromosome. Buffer Blue (National Diagnostics, Atlanta, GA), and boiled for 5 min. Proteins were separated in 15% polyacrylamide gels and transferred to a polyvinylidene di uoride (PVDF) or nitrocellulose membranes.
The membranes were blocked with 5% skimmed milk containing Tween 20 (0.5%) for 1 h. The membranes were probed with primary antibodies at 4°C overnight followed by the incubation with the corresponding secondary antibodies labeled with horseradish-peroxidase for 90 min. The bound antibodies were detected by enhanced chemiluminescence reagents (Millipore, Burlington, MA, USA). Visualization of the transferred protein was done with FluoroChem HD2 (Protein Simple, CA, USA).
Histopathology. Heart tissues from mice were xed in 10% buffered formalin. Tissue sections (5 µm) were stained with Haematoxylin-Eosin (H&E) and examined under light microscope for parasites. Images were taken using a LEICA DMi1 using the LAS V4.12 software.
Explant culture. Explants of organs from T. cruzi-infected mice were cultured in LDNT medium. Brie y, organs were collected and excised into smaller pieces and put into 25 cm 2 non-vented ask containing 10 ml LDNT medium. Cultures were incubated at 26°C and examined twice a week for any growth of parasites for up to 8 weeks.
Determination of IgG2a in blood serum by antibody ELISA. For analysis of IgG2a, T. cruzi antigen was prepared by lysis of whole cells through rapid freeze-thaw technique. 96-well microplates were coated with T. cruzi antigen (5 µg/ml) at 4°C overnight using antigen coating buffer (phosphate-buffered saline, pH 9.0). Total IgG2a antibodies against T. cruzi were measured from blood of uninfected and infected mice. The antibody concentrations were measured at a 405 nm wavelength using SpectraMax microplate reader and data were analyzed by Softmax Pro Software (Molecular Devices LLC, Sunnyvale, CA, USA).
T-cell proliferation and cytokine determination. Splenocytes were harvested from wild type and STAT1 or STAT4 knock out mice. The cells were plated at a concentration of 5 ⋅ 10 6 cells /ml in RPMI 1640 medium supplemented with 10% FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin, and 1% HEPES. After stimulation of cells with 20 µg/ml T. cruzi freeze-thaw whole -cell antigen for 72 h, supernatants were collected, and the production of cytokines were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Cytokines were analyzed according to the manufacture's protocol using capture and detection antibody (BioLegend, San Diego, CA, USA). Cytokine concentrations were measured at a 405 nm wavelength using SpectraMax microplate reader and data were analyzed by Softmax Pro Software (Molecular Devices LLC, Sunnyvale, CA, USA).
Complement-mediated killing assay. Normal human serum was purchased from Sigma-Aldrich. The stationary-growth phase of T. cruzi culture was collected and washed twice with phosphate buffered saline. The cells were mixed with normal human serum at a nal 30% concentration, and incubated at 37°C for 30 min with intermittent shaking in between. The cells were centrifuged at 1500 rpm for 5 min and kept at room temperature for 60 min so metacyclic parasites emerge from the pellet into the supernatant. The supernatant was collected, washed ve times with phosphate buffered saline and the number of metacyclics were counted using improved Neubauer counting chambers.
Scanning Electron Microscopy. Cells were xed and processed for SEM at the Campus Microscopy and Imaging Facility (CMIF), The Ohio State University, OH, USA. SEM images were obtained with a FEI Nova NanoSEM 400 Scanning Electron Microscope equipped with secondary and low-vacuum detector with a eld-emission gun (FEG) electron source.
In vitro infections. All in vitro infections were performed in H9C2 and RAW cells. Host cells were plated at a concentration of 1 ⋅ 10 5 cells/well in 12-well plates containing DMEM medium containing 10% HIFBS. Infection of H9C2 cells was performed using serum selected metacyclic-trypomastigotes and incubated at 37°C. After 24 h, the cells were washed with PBS in order to remove any extracellular trypomastigotes.
The plates were re-incubated in complete medium at 37°C 1-2 weeks. The formation of amastigotes and trypomastigotes were observed up to 2-3 weeks under light inverted microscope.
Animal infections. In order to examine the infectivity of T. cruzi wild type (WT) and Cyp19 −/− DKO (double allele knock out) mutant parasites, AJ, STAT1 −/− (BALB/c), and STAT4 −/− (BALB/c) mice (Jackson Laboratories, Bar Harbor, ME, USA) were infected with 1 ⋅ 10 5 serum-selected trypomastigotes in 100 µl PBS through intraperitoneal route. Mice were examined daily for clinical symptoms to determine overall survival. All of the mice were examined for parasitemia by collecting blood from the tail vein twice a week. The survival days was counted from the day of inoculation until mice died. The statistically signi cant difference in survival between WT and DKO was calculated by Kaplan Meier survival analysis.
Immunization and challenge infection in mice. Mouse immunization was performed with serum-selected Cyp19 knock out trypomastigotes in AJ mice. Mice were immunized intraperitoneally four times with 1 ⋅ 10 5 trypomastigotes at 0, 4, 8, and 14 weeks. At 18 week, mice were challenged with 1 ⋅ 10 5 T. cruzi wildtype metacyclic trypomastigotes. Control AJ mice were infected with 1⋅10 5 serum-selected T. cruzi wild type trypomastigotes. All of the control mice died after 3 weeks of infection whereas immunized mice survived without developing any clinical illness (speci cally weight loss, huddling behavior, decreased motility and shaking). All of the immunized and challenged mice were harvested at 34 week to evaluate further including explant culture.
Statistical Analysis. All data are expressed as mean ± SD. All statistical analyses were done in GraphPad Prism software. A student t test was used to determine statistical signi cance of differences among the groups. A P value of < 0.05 was considered signi cant and indicated with asterisk.

Results
Generation of a Cyp19 knock-out parasite line. Since the published genome sequence of T. cruzi (CL Brener) 32 indicated that cyclophilin 19 is expressed from a single copy gene 33,34 we opted to create a Cyp19 null mutant using double allelic gene replacement (Fig. 1A). Plasmid constructs were engineered to contain 5'-and 3-UTRs of Cyp19 anking sequences surrounding drug resistance genes for neomycinand hygromycin phospho-transferases (NEO and HYG, respectively). These were ampli ed by PCR, cloned into pET15b plasmid, linearized and transfected separately into Brazil strain epimastigotes and selected using neomycin and hygromycin, respectively. Drug resistant parasites were cloned using limited dilution and examined for appropriate chromosomal integration of drug resistant constructs using PCR. A second allelic ablation was performed from a Neo-resistant clone with another round of transfection using a HYG-resistance marker. After double drug selection and limited dilution cloning, parasites were analyzed for appropriate integration by PCR for integration of transgenes, Cyp19 and anking sequences (Fig. 1B). Western blot analysis of doubly resistant parasites con rmed loss of Cyp19 protein expression (Fig. 1C).
Whole nuclear genome sequencing of wild-type and Cyp19 knock-out strains. In order to verify the removal of Cyp19 genes and the appropriate integration of drug markers we performed whole genome sequencing of several knock-out clones (denoted as SKO-NEO and -HYG, DKO-D0, -D11, -D12 in Figs S1-S4) and two WT parental strains. DKO-D0 was parasite line used in the vaccination studies in this paper. DKO lines D11 and D12 were recloned from the DKO-DO cloned line more than one year after continued growth in culture. The WT strains included parental WT strain used for the knock-out (termed WT-att), and an additional virulent WT strain (termed WT-vir) derived from WT-2 by multi-passage through immunede cient STAT 4 −/− mice. Recently the genome sequence of Brazil strain A4 32 was published which we used as a reference to compare of the genome sequences of our WT strains and the SKO and DKO mutant strains. This analysis con rmed the absence of two Cyp19 genes with NEO and HYG as expected (Fig. S1). Unexpectedly, we found that there was a preexisting trisomy of the central portion of chromosome 1, which contains another copy of the Cyp19 gene, in both the WT strains (shown for WT-att in Fig. S1 and for both in Fig. S2 and S3), indicating that the Cyp19 trisomy pre-existed in both WT parasite lines before transfection and is not as a result of compensation for loss of two Cyp19 gene copies. There is considerable aneuploidy in between the cell lines and there are increased copy number of several putative cyclophilin-like genes in the DKO lines in chromosomes 17 and 22 (Fig. S4). It is unclear whether these changes are a result of functional compensation for loss of two of the three Cyp19 genes in the DKO. The overall genomic features of the DKO lines is highly stable inasmuch as DKO-DO and DKO-D11, -12 are similar even after one year of continuous growth in culture (without drug pressure) without any repair of Cyp19 alleles by the remaining intact gene copy. Nevertheless, the loss of two Cyp19 genes results in attenuation of pathogenesis of in the DKO strain (see sections below). We have subsequently reanalyzed the DKO clones by Western blotting for the expression of Cyp19 from the remaining gene Cyp19 and have found intermittent expression of low levels of Cyp19 in from the DKO lines (not shown). Sequence analysis indicates that this additional copy is it identical to the other Cyp19 copies. We do not yet understand the nature of the ability of the variable expression of Cyp19 from these cells. However, we have not seen a change in the biologic phenotype (see sections below) related to Cyp19 deletion. We have attempted to remove the remaining Cyp19 copy using both a third round of targeted integration of a third drug-resistance marker and using CRISPR/Cas9 technology and both have been unsuccessful (not shown and Pedroso, et al, unpublished). We surmise that complete removal of Cyp19 from T. cruzi is not achievable, as Cyp19 is an essential gene for parasite survival.
Parasite growth and infection studies in vitro. Epimastigotes of the Cyp19 null mutants (DKOs) grew slower and reached peak densities that are signi cantly lower than their wild-type controls ( Fig. 2A). The proportion of metacyclic forms that arose spontaneously in culture appeared earlier in the course of growth in culture and are signi cantly more abundant than wild-type parasites, reaching approximately 85-90% of the population ( Fig. 2A). Single knock-out (SKO) parasites reached approximately half the peak density of WT parasites and produced about 35-50% metacyclic forms during growth in culture ( Fig. 2A). The metacyclics of Cyp19 null parasites are resistant to complement mediated killing using normal human serum, as those of wild-type parasites (not shown) and had morphology similar to wildtype metacyclic forms (Fig. 2B). We suspect that diminished Cyp19 expression in the knock-out parasite lines leads to faster differentiation of epimastigotes into metacyclic trypomastigotes resulting in the higher proportion of metacyclics in the overall culture. Since metacyclic parasites do not replicate, this limit results in a reduced overall density of parasites in the culture.
In order to test the ability of SKO and DKO parasite lines to infect we used a rat heart myoblast (RHMs) cell line (H9C2), our prototype line for in vitro assessment of parasite infection, and incubated them with isolated metacyclic stages the DKO, SKO and WT parasite lines. Infection of RHMs with WT parasites results in the development of intracellular amastigotes by 2-3 days post-infection that increase in number, eventually transforming into motile tissue-culture tryomastiogotes (TCTs) which then exit host cells (Fig. 2C, upper panels). The course of infection of RHMs with SKO parasites is similar to that of WT parasites, although they appear to grow slower intra-cytoplasmically, but eventually reach similar densities and transform into TCTs that exit the cell (Fig. 3C, middle panels). Infection of RHMs with DKO parasites results in the production of scant amastigotes, which fail to replicate intracellularly and eventually degenerate (Fig. 3C, lower panels). Infections of RHMs with DKO parasites fail to give rise to any demonstrable TCTs. We have not observed amastigote or TCTs forms produced over prolonged observations of RHMs cultures incubated with DKO parasites for 1-2 months. We have repeated infectivity studies with the DKO line at least 10 times with the same results. Additional DKO clones, D-11 and D-12, obtained by recloning the DKO line after one year on continuous culture, were repeatedly tested for their ability to infect and replicate in H9C2 cells. Neither parasite line was able to infect these cells or produce TCTs (not shown) as we have seen with the original DKO parasite line. We compared the ability of WT and DKO parasites to cause productive infection in phagocytic RAW cells (Fig. 3). As expected infection with WT metacyclics led to the development of amastigotes and formation and release of extracellular trypomastigotes (Fig. 3 lower panel). In contrast, infection of cells with DKO metacyclics did not lead to a productive infection with either the development of replicating intracellular amastigote or the production of extracellular trypomastigotes.
Infection studies on vivo. In order to test the effect of the loss of Cyp19 on parasite virulence in animals we used immunocompetent AJ mice (males 4-6 weeks of age) infected with puri ed metacyclics of the either WT or DKO parasites (10 5  DKO parasites induce anti-T. cruzi immunity which protects against acute Chagas disease. Because DKO parasites do not infect cells in vitro or mice, we tested the ability of repeated infections of DKO parasites to produce an anti-T. cruzi immune responses. Repeated inoculation of animals with WT produces parasite-speci c IgG2a levels to rise reaching a peak at near the time of death (Fig. 6, left panel).
Repeated IP inoculation of immunocompetent AJ mice with 10 5 parasites also did not result in clinical disease or death over multiple experiments. However, the level of parasite-speci c IgG2a increasingly rose with repeated injection of DKOs, indicating the development of B-cell anti-parasite immunity. Splenic cells harvested from DKO "immunized" animals produced high levels of IFN-γ and lower levels of IL-4 and IL-10 when stimulated with parasite antigen indicating the development of Th1-cell immunity (Fig. 6B). We tested whether the anti-parasite immunity developed by repeated injection was able to protect mice from development of disease or death from infection by WT parasites (Fig. 7). Infection of mice with WT parasites, as expected, resulted in the onset of clinical infection and death between 3-4 weeks postinfection ( Fig. 7A and B). Challenge of mice previously "immunized" with repeated injections of DKO parasites with WT parasites did not develop clinical disease and are completely healthy up to 34 weeks post-challenge (Fig. 7B). We found no evidence of parasite infection in DKO immunized WT challenged animals (Fig. 7D). We have repeated this experiment 3 times with the same results. In order to test whether latent infection existed in DKO-immunized WT challenged animals we treated animals with dexamethasone approximately one year post-infection. After 60 days of dexamethasone treatment, we observed no clinical infection (Fig. 7B) or the presence of parasites in explantation or histopathologic analyses (not shown).
A single immunization with DKO parasites is su cient for protective immunity. Our experiments up to this point indicated that four doses of DKO parasites given over a span of 4.5 months induced effective protective immunity to WT challenge. We tested whether a single dose of the DKO vaccine could elicit protection (Fig. 8). DKO administered as described previously and animals were challenged 6 months after immunization. Observation of immunized animals over 6 months post-infection resulted in no evidence of clinical infection (Fig. 8B) whereas, as expected, 100% of non-immunized animals died within 21-25 days post challenge ( Fig. 8A and B). Explantation and histopathologic analyses indicated the presence of parasites only in the non-immunized challenged animals ( Fig. 8C and D).

Discussion
This report describes the engineering of Trypanosoma cruzi in the genes encoding cyclophilin 19, an enzyme which catalyzes cis-trans peptidyl-prolyl isomerization of proteins (PPIases). Our previous studies of this enzyme fortuitously found that it was secreted by epimastigotes and important in the neutralization of anti-parasiticidal proline-containing insect anti-microbial peptides 25 . As a group, PPIases are expressed by organisms from all Kingdoms where they serve myriad functions including acting as a protein chaperone (folding of nascent proteins and re-folding of aggregated proteins), in ROS production and scavenging 37 , gene expression and RISC complex formation and miRNA regulation 38,39 .
Cyp19 is the only T. cruzi PPIase that has been reported to be secreted 25,34,40,41 , which portends a role in interacting with extracellular substrates. We found that Cyp19 is expressed in and released extracellularly by all life-stages of the parasite (Pedroso, et al, unpublished), suggesting in each parasite stage it may function to engage and modify different host target proteins. In fact, our recent data indicated that secreted Cyp19 induces the intracellular ROS production in host cells which is critical for amastigote replication (Pedroso, et al, unpublished).
The double-knock-out (DKO) mutant parasite line with diminished Cyp19 expression is viable in culture, but has a diminished growth rate, and is defective in its ability to reach a peak density of that of wild type parasites, suggesting a defect in replication as epimastigotes. The mutant line differentiates rapidly into metacyclic trypomastigotes, further suggesting a defect in normal cell cycle control. We surmise that the rapid conversion of epimastigotes to metacyclic-forms at early points in cultivation of cells contributes to the inability of these cells to reach higher densities, since metacyclic forms do not undergo replication. The replication of low numbers or epimastigotes prior to their differentiation into metacyclic form is the key to their continued survival in culture. The DKO metacyclics are resistant to complement-mediated killing by normal human serum and are morphologically similar to those of wild-type parasites, suggesting the decreased Cyp19 expression does not affect these properties. When used to infect mammalian host cells, metacyclic parasites enter cells at a reduced rate and form scant amastigotes-like structures, which degenerate and disappear from host cells. DKO parasites do not lead to productive infection and fail to give rise to cell-derived trypomastigotes (TCTs Splenocytes harvested from these animals produce high levels of IFN-γ, IL-12 and low levels of IL-4 and IL-10 indicative of a Th-1 response, which has been reported to advantageous to the clearance of parasites 15,16 . Challenge of "DKO-immunized" with high-dose virulent wild-type Brazil strain T. cruzi metacyclics failed to develop clinical signs of infection disease and had 100% survival whereas infected non-immunized animals had 100% mortality between 21-30 days post-infection. This indicates the anti-T. cruzi immunity elicited by the DKO-parasite immunization was 100% effective at protecting animals from acute Chagas disease. Surprisingly, a single immunization with the DKO line was effective at providing completely protection to wild-type parasite challenge 6 months after inoculation. This indicates that the immune response elicited by the DKO vaccine strain is highly immunogenic and long-lasting. An advantage of the DKO mutant is that it provides exposure of the immune response to a large array of metacyclic stage antigens which may give rise to greater immunity than would subunit vaccines composed of a single of few antigens. In addition, the using the whole parasite might serve as a natural adjuvant, naturally bolstering the immune response. We have not found any deleterious effects from administration of the DKO vaccine strain to animals. This is a concern since the development of host immunity to certain parasite antigens can serve to generate autoimmunity through molecular mimicry and this could lead to tissue in ammation 42 . Our safety studies included using STAT-1 and STAT-4 de cient mouse strains to test the DKO. The mutant parasite line was unable to infect these mutant mouse strains, indicating that even in immune-de cient mice the DKO line is unable to replicate. Induction of immunosuppression using dexamethasone, in immunocompetent mice infected with DKO parasites did not result in recrudesced infection, indicating that the DKO strain does not cause smoldering latent infection. Further, dexamethasone treatment of immunized, WT-challenged animals did not result in emergence of latent infection. This is highly important since it suggests that the immunity induced by the DKO vaccine results in sterile immunity not allowing wild-type parasites to reach or growth within target tissues.
Currently, there are no approved vaccines for the prevention or treatment of Chagas disease. There are several reports of Chagas vaccines in development including those based on recombinant protein subunits 43,44,45 , naked plasmid DNA-protein expressing constructs 46 , peptides 47 , adenoviral and Salmonella vector systems for expression of parasite proteins and several live attenuated T. cruzi vaccines and a live T. rangeli vaccine 48,49,50,51 . Several mutant parasite lines have been tested as live vaccine strains; either those attenuated by long-term in vitro cultivation 52 or those engineered with speci c genetic defects resulting in diminished growth in culture and reduced virulence in animals but are able to provoke anti-T. cruzi immunity and partially reduce the magnitude of infection of animals when challenged with the parental wild-type strain 53,54,55 .
Our results demonstrate that the DKO mutant described here is highly effective live parasite vaccine for prevention of acute Chagas disease in the mouse model. It is completely effective in a single dose and it provides sterile immunity and is safe in immune-de cient hosts. Other potential roles of this strain including using this as a heterologous antigen delivery system. Further studies with this strain include its ability to cross-protect against infection by other T. cruzi strains, the length of protective immunity.