mice , parasites and infections - c57bl/6 ( ly5.2 + ) ( b6 ) mice were 
 purchased from national cancer institute at frederick ( usa ) and were maintained in the 
 university of georgia animal facility in microisolator cages under specific 
 pathogen - free conditions . tissue culture trypomastigotes ( tct ) of the cl strain of 
 t. cruzi were obtained from passage through vero cells . mice were 
 infected intraperitoneally ( i.p . ) with 1,000 brazil strain tct and sacrificed by 
 co2 inhalation at different time points post - infection . mice were 
 reinfected i.p with 10tct of t. cruzi cl strain at 
 different time points post - infection . in some experiments mice were challenged in the 
 hind foot pads with 2.5  10 
 t. cruzi tdtomato trypomastigotes as described previously and the 
 fluorescent intensity as a surrogate of parasite load was measured using a whole animal 
 imaging system ( maestro2 in vivo imaging system cri , usa ) ( canavaci et al . bz treatment - n - benzyl-2-nitroimidazole acetamide ( benznidazole ; 
 rochagan , roche , brazil ) was used as a trypanocidal drug . mice were treated orally with 
 daily doses of bz of 100 mg / kg of body weight for 40 days [ 15 - 55 days post - infection 
 ( dpi ) ] . bz was prepared by pulverisation of one tablet containing 100 mg of the active 
 principle , followed by suspension in distilled water . assessment of treatment efficacy - mice were immunosuppressed with 
 cyclophosphamide ( 200 mg / kg / day ) i.p . at two - three day intervals for a total of four 
 doses . following immunosuppression , blood was collected via tail vein 
 and the number of parasites was quantified using a neubauer haemocytometer . survival was 
 monitored daily . the dna preparation , generation of polymerase chain reaction ( pcr ) 
 standards and detection of parasite tissue load by real - time pcr was carried out as 
 described previously ( cummings et al . skeletal , heart and fat 
 tissues were collected at various time points post - treatment and fixed in 10% buffered 
 formalin . sections ( 5 m ) from paraffin - embedded tissues were stained with haematoxylin 
 and eosin for histopathological analysis . t cell phenotyping - red blood cells ( rbcs ) in single cell suspensions 
 of spleen cells ( sc ) were lysed in a hypotonic ammonium chloride solution and washed in 
 staining buffer { 2% bovine serum albumin , 0.02% azide in phosphate buffered saline ( pbs ) 
 [ pab]}. in some cases , mouse peripheral blood was obtained by retro - orbital venipuncture , 
 collected in na citrate solution and washed in pab . whole blood was incubated with 
 tetramer - pe and the following labelled abs : anti - cd44 fitc , anti - klrg1 pecy7 , anti - cd8 
 efluor 450 , anti - cd127 apc ( ebioscience , usa ) . cells were also stained with anti - cd4 , 
 anti - cd11b and anti - b220 ( caltag - invitrogen laboratories , usa ) for use as an exclusion 
 channel . cells were stained for 45 min at 4c in the dark , washed twice in pab and fixed 
 in 2% formaldehyde . rbcs were lysed in a hypotonic ammonium chloride solution after 
 washing twice in pab . at least 500,000 cells were acquired using a cyan flow cytometer 
 ( dakocytomation , usa ) and analysed with flowjo software ( tree star inc , usa ) . mhc i 
 tetramers tskb20 ( anykftlv / k ) was synthesised at the tetramer core facility 
 ( emory university , usa ) . intracellular cytokine staining - sc from nave , untreated / chronic or 
 treated / cured mice were stimulated with t. cruzi peptides ( 5 m ) at 
 37c for 5 h in the presence of brefeldin a ( golgiplug ; bd pharmingen , usa ) . t. 
 cruzi peptides used in this study were tskb20 ( anykftlv ) and tskb74 
 ( vnydftlv ) ( martin et al . cells were 
 surface stained with anti - cd8 efluor 450 ( ebioscience ) and intracellular cytokine 
 staining was performed with anti - interferon- apc and with a cytofix / ctyoperm kit ( bd 
 biosciences , usa ) in accordance with the manufacturer s instructions . at least 250,000 
 lymphocyte events were acquired and analysed as above . 
 in vivo cytotoxicity assay - sc from nave mice were incubated either 
 with the t. cruzi peptide tskb20 ( anykftlv ) , tskb74 ( vnydftlv ) or with 
 no peptide for 1 h at 37c . cells were washed twice with pbs and incubated with 2.5 m 
 cfse ( cfse ) for tskb20 peptide - loaded cells , 1.0 m cfse 
 ( cfse ) , for tskb74 peptide - loaded cells or 0.25 m cfse 
 ( cfsel ) for unpulsed cells for 3 min at room temperature . the cfse was 
 quenched with fbs and the cfseand cfse cells were combined 
 and transferred intravenously into nave , untreated / chronic and treated / cured mice . scs 
 were harvested after 16 h and cfse , cfseand cfselow 
 cells were detected by flow cytometry . the percentage of specific killing was 
 determined using the formula : { 1- [ ( % cfsenave/%cfse 
 nave)/(%cfse chronic/%cfse chronic ) ] } 100% . ethics - all animal protocols were approved by the university of 
 georgia institutional animal care and use committee . effective vaccines faithfully mimic the immune protection afforded by survival of a 
 full - fledged infection . since complete spontaneous clearance of t. 
 cruzi infection is rare , we simulated the immunity induced by resolution of 
 infection by curing established infections using bz treatment . our previous studies have 
 established the conditions for achieving and methods to monitor parasitological cure 
 using this approach ( bustamante et al . mice were infected with 10 trypomastigotes and bz 
 treatment was initiated on day 15 post - treatment and then challenge infection was 
 delivered ~150 days after the completion of drug treatment ( fig . mice were then immunosuppressed ~45 days after challenge 
 to reveal persistence of infection as assessed by parasites in blood and tissues . previous studies have established this immunosuppression protocol as a standard for 
 detecting persistent infection and for determining the curative potential of candidate 
 drugs ( bustamante et al . 1b , treated but not re - challenged mice were free of infection , 
 demonstrating again the ability of this bz treatment regimen to achieve parasitological 
 cure . however both persistently infected ( untreated ) and infected / cured mice ( treated ) 
 exhibited parasites in the blood and in tissues following re - challenge ( fig . thus , mice cured of t. 
 cruzi infection are not resistant to reinfection with a low dose of 
 t. cruzi . mice infected for 15 days with 
 103 trypomastigotes of the t. cruzi cl strain were left 
 untreated or were treated with a 40 day course of benznidazole ( bz ) at 100 
 mg / kg / day . mice were there challenge infected with the same strain / dose of 
 t. cruzi or not challenged and then 45 days later ( day 265 
 of the experiment ) immunosuppressed with cyclophosphamide as previously 
 described ( bustamante et al . 2008 ) ; b : detection of parasites in blood or 
 muscle tissue ( c ) two weeks after immunosuppression indicates the failure of 
 infection / cure protocol to protect mice from challenge infection . treated but 
 not - re - challenged mice had no detectable parasitaemia following 
 immunosuppression ( b ) indicating the ability of the bz treatment to provide 
 parasitological cure ; c : histological sections of the skeletal muscle at 280 
 days post - infection ( dpi ) . presence of absence of tissue parasites was 
 confirmed by quantitative polymerase chain reaction ( data not shown ) . bar = 200 
 m . 
  to follow the reinfection process at the infection site more closely , we assessed 
 initial protection using fluorescent protein - expressing parasites delivered into the 
 footpads of single or multiple dose  immunised  as previously described ( collins et al . 2a ) . as predicted , a single round of infection and cure provided 
 essentially no protection based on the footpad infection model ( fig . in contrast , similar challenge infection in continuously 
 infected mice ( not bz - treated group ) resulted in more rapid control of the footpad 
 challenge relative to either noninfected or infected / cured mice . red arrows 
 indicate points at which a subset of mice was immunosuppressed to assess 
 infection status ( b , d , f ) . parasite load at the site of the infection assessed 
 by quantification of the fluorescent signal from mice primarily infected with 1 
  103 cl wild - type parasites that underwent one , two or three cured infections 
 and their untreated counterparts submitted to challenged in the hind foot pads 
 with 2.5  105trypanosoma cruzi tdtomato trypomastigotes ( c , 
 e , g ) . parasitaemias in mice described in b , d and f at ~15 days after 
 administration of the immunosuppressant cyclophosphamide ; bz : benznidazole ; 
 dpi : days post - infection . we then asked if boosting of the immune protection could be achieved by multiple rounds 
 of infection and cure . attainment of parasitological cure after each round of infection 
 and treatment was confirmed by detection of predominantly cd127-expressing , t central 
 memory phenotype t. cruzi - specific t cells in the blood of mice ( fig . continuously infected mice maintained a 
 relatively stable and low level of cd127-expressing t cells specific for the 
 immunodominant tskb20 t. cruzi trans - sialidase ( ts)-derived epitope 
 whereas drug - cured mice exhibited a decrease in the overall number of tskb20-specific t 
 cells and an increasing proportion of cd127 expression , indicative of the absence of 
 continuous antigen stimulation in these cured mice ( bustamante et al . multiple rounds of infection and cure increased 
 the ability of mice to control initial parasite growth in the footpad , with three rounds 
 of infection and cure providing a level of protection nearly equivalent to that afforded 
 by continuous infection ( fig . 3:parasite - specific memory t cell responses in mice undergoing multiple 
 rounds of infection and cure . a : expression of the memory maintenance marker 
 cd127 in blood on cd8 + tskb20-tetramer+ t cells from infected mice that 
 underwent three cured infections and their untreated counterparts ; b : the 
 mhc - peptide tetramer of the immunodominant tskb20/kb epitope was used to detect 
 trypanosoma cruzi - specific cd8 + t cells in the blood of 
 mice described above ; bz : benznidazole ; dpi : days post - infection . even nonvaccinated mice ultimately control t. cruzi at the initial 
 infection site in the footpad resulting in the absence of detectable parasites by 
 immunofluorescence at > 2 weeks post - infection ( fig . 
 2 ) . to determine if the multiply infection / cured mice additionally were able 
 to clear infection throughout the body ( not just at the site of initial infection ) , mice 
 were immunosuppressed and then reassessed for parasites in the blood and tissues using 
 microscopy ( fig . as expected both the 
 continuously infected / challenged and the nave / challenged mice had detectable 
 parasitaemias following immunosuppression . however even mice submitted to three rounds 
 of infection and cure failed to prevent the establishment of the infection following 
 footpad challenge . the failure to prevent reinfection following one or two rounds of 
 infection / cure is also evident from the fact that mice challenged with a low dose 
 ( 10 ) of t. cruzi i.p . show very low levels of cd127 
 expression on t. cruzi - specific t cells , indicative of a persistent 
 antigen presence ( fig . although immunity induced by cure of an initial infection is generally the standard 
 against which vaccines are measured , there are multiple reasons why the infection / cure 
 protocol used herein failed to provide sterilising immunity . to exclude the possibility 
 that immune effectors might not be generated , or are lost after bz - induced cure , we 
 monitored effector functions in t. cruzi - specific t cells during the 
 infection cure cycle . the boost in the frequency of t. cruzi - specific t 
 cells in cured mice following each round of reinfection is indicative of sustained and 
 competent t cell memory following infection cure ( fig . although the overall frequency of t. cruzi - specific 
 cd8 t cells decreases , as expected following infection cure ( bustamante et al . 2008 ) ( figs 3b , 4a ) , the quality of 
 these memory t cells is evidenced by their in vivo cytolytic activity ( fig . 4b ) and ex vivo cytokine production in response 
 to t. cruzi epitopes ( fig . thus the failure of the infection cure protocol to provide protection is not due to the 
 absence of an inducible effector t cell population that could be recalled upon 
 challenge . 4:functional effector t cell responses in trypanosoma 
 cruzi - infected and cured mice in the late stages of the infection . a : 
 frequency of tskb20/kb+ t cells in the spleens of untreated or treated mice 615 
 days post - infection ( dpi ) ( 560 days post - treatment ) ; b : in vivo cytotoxic t 
 lymphocyte activity on tskb20 and tskb74-pulsed targets 16 h after transfer to 
 nave , untreated or treated t. cruzi - infected mice ( 615 dpi ) . numbers above the peaks are 
 percentage of specific lysis calculated as described in the material and 
 methods ; c : intracellular cytokine staining for interferon ( ifn)- production 
 by cd8 + t cells with or without stimulation with tskb20 peptide in 
 representative mice at 400 dpi . numbers represent the percentage of cd8 + t 
 cells producing ifn-. 
  the argument could be made that the immunity induced by the infection / cure protocol used 
 here induces an insufficient and/or poorly targeted immune response . the enhanced 
 protection conveyed by active infection relative to cured infection evident in the 
 footpad challenge model might suggest that higher number of parasite - specific t cells , 
 effector t cells , rather than memory t cells or effector mechanisms not measured in 
 these assays , may be required for optimal infection control . 2002 , 2007 ) that superinfection ( a  new  infection in an 
 already infected host ) is not uncommon in the case of t. cruzi , 
 indicating that simply having more effector t cells ( as is the case during an active 
 infection ) rather than memory t cells ( in cured infection ) is not sufficient to prevent 
 establishment of  new  infections . thus , immunity induced by prior infection , whether 
 cured or not , is insufficient to prevent reinfection . we believe that the results of several recent studies shed some light on the failure of 
 active infection and the infection / cure protocol used here , as well as other vaccine 
 protocols in t. cruzi and may be informative with respect to defining 
 the overall likelihood of developing competent prophylactic vaccines for t. 
 cruzi infection . for the immune system to prevent the establishment of 
 t. cruzi infection , the potentially protective response must either 
 absolutely prevent parasites from infecting host cells , thus halting the replication of 
 t. cruzi before it can begin or must rapidly identify and kill 
 infected cells before the initial round of parasite replication can be completed ( and 
 the resulting expanded population of parasites spreads throughout the body ) . with respect to the preventing host cell entry , the passive transfer of high - titre 
 antibodies can reduce parasite loads ( franchin et al . 1997 ) , presumably through the ability to target extracellular trypomastigotes 
 for destruction by complement or phagocytic cells , or to block the ability of these 
 parasites to invade host cells . however this approach has not been demonstrated to 
 actually prevent infection and the antibodies produced during the course of acute and 
 chronic infections , although contributing to immune control of the infection ( kumar & tarleton 1998 ) clearly lack the ability 
 to prevent the constant reinfection of host cells that maintains the infection . so the 
 ability to generate a targeted antibody response sufficient to block 100% of invading 
 parasites seems questionable - certainly no previously tested method has . investigation of the early events in the generation of parasite - specific t cell 
 responses in t. cruzi have provided some insights into how the nave as 
 well as the primed immune system deals with t. cruzi . despite the 
 strength and the focus of the cd8 cells generated in t. 
 cruzi infection , this response is nevertheless extremely slow in developing 
 ( padilla et al . this delay is evident 
 in the initiation of proliferation and development of effector function in 
 parasite - specific t cells , as well as in the recruitment of leukocytes into the draining 
 lymph nodes . these observations suggest initially t. cruzi is infection 
 occurs  under the radar  , failing to trigger significant recognition by pathogen sensors 
 on with innate and adaptive immune responses depend . we have attributed this clandestine 
 infection process by t. cruzi to two factors : ( i ) the mechanism of cell 
 invasion by t. cruzi that occurs without significant host cell damage 
 and thus with little release of damage associated molecular patterns ( damps ) and ( ii ) 
 the absence of significant pathogen - associated molecular patterns ( pamps ) in the 
 invading t. cruzi trypomastigotes . these conclusions are supported by 
 the observation of the delayed generation of responses , the timing of which follows 
 closely the destruction of host cells ( with the consequent release of damps ) during the 
 exit of parasites at four - five dpi cruzi t cell responses can be accelerated and enhanced by supplying bona 
 fide pamps with the infection , either by administration of ligands for tlr2 and tlr9 
 coincident with infection ( padilla et al . 2009 ) 
 or by endogenous expression of classical bacterial pamps by t. cruzi 
 ( kurup & tarleton 2013 ) . the insufficiency of t. cruzi in triggering of innate immune sensors 
 not only delays the initiation of responses in newly infected hosts , but also has a 
 long - lasting impact on the ultimate course of t. cruzi infection . 
 infections with t. cruzi lines expressing salmonella 
 flagellin , a well - studied bacterial pamp , control the infection better , exhibit 
 much - reduced pathogen load and evidence of cure in some cases ( kurup & tarleton 2013 ) . the implications of these results for 
 the potential of vaccines to prevent t. cruzi infection are 
 substantial . first , if t. cruzi can avoid eliciting an inflammatory 
 response - at least until after completion of the first round of replication in and 
 release from host cells - then no level of pre - existing t cell immunity ( i.e. , 
 vaccine - induced immunity ) will be able to prevent establishment of the infection in a 
 newly infected host or quickly extinguish that infection once it is established . site in an infected host 
 ( e.g. a site lacking primed effector cells ) parasite proliferation would also be 
 expected to proceed there unfettered until the inflammatory signals generated by the 
 destruction of the host cell recruit these effector cells beginning four - five days later 
 ( upon completion of the replication cycle ) . 2 ; for the one and 
 two - time infected / cured mice , as well as early infection in the continuously infected 
 mice , there is essentially no control of parasite load detected in the infection site 
 until after day 4 of infection . a second potential complicating factor in immunity to t. cruzi and 
 vaccine development is the targets of these immune responses . the major focus of both 
 cellular and humoral immune responses in t. cruzi infection are 
 proteins encoded by large gene families . with respect to cd8 t cell 
 responses , epitopes encoded by the ts family of genes are strongly immunodominant ( martin et al . the high variability ( the ts 
 family is composed of > 3,000 individual genes / gene fragments in the t. cruzi 
 cl brener genome ) and the strain - specific variation and recombinational 
 potential of these genes ( p weatherly et al . , unpublished observations ) makes the ts 
 proteins moving targets for immune responses and thus a relatively poor choice as 
 vaccine targets . furthermore , we have recently reported that ts epitopes are not 
 effectively presented by infected host cells until 24 - 48 h after host cell infection . in 
 contrast , other nonvariant but sub - dominant immune targets from flagellar proteins are 
 detected by t cells within 6 h after host cell invasion ( kurup & tarleton 2014 ) . thus the normal t cell response to t. 
 cruzi is fixated on a set of molecules that are highly variable and that are 
 not expressed until late in the cycle of host cell infection - a poor  choice  for the 
 effective elimination of infected host cells . collectively , the data presented in these studies and previously reported suggest the 
 following scenario of events in t. cruzi infection . infection of nave , 
 previously infected or persistently infected hosts , or in new sites within an infected 
 host , occurs with minimal triggering of innate immune sensors , thus allowing the 
 establishment of t. cruzi in these hosts / sites . t cell responses are 
 generated and local inflammation is induced only after completion of a round of parasite 
 replication and host cell destruction , which releases damps and possibly pamps . cruzi t cells that are normally induced in the infection 
 target primarily highly variant proteins that are not effectively presented on host 
 cells until late in the host cell infection process . thus t cells that are induced by 
 infection or vaccination are both not initially recruited to sites of infection and when 
 they ultimately get to these sites , they do not have the specificities that allow them 
 to rapidly identify infected host cells early in the infection process . vaccination 
 could be used to redirect t cell responses to nonvariant antigens , such as those encoded 
 in flagellar proteins - this process has been shown to have some merit ( kurup & tarleton 2014 ) . however it is difficult 
 to see how a vaccination can overcome the issue of the relatively silent invasion 
 process used by t. cruzi . the best one might be able to hope for in an 
 anti - t . cruzi vaccine is to enhance the rate of parasite clearance 
 and reduce the time required to and frequency of cure of the infection - but not prevent 
 the infection . while this would be an improvement , whether that is sufficient to justify 
 a vaccine development program is uncertain . the studies discussed here present the possibility that development of an effective 
 prophylactic vaccine for t. cruzi may not be feasible . the incredible 
 impact of vaccines in curbing deaths due to infectious agents notwithstanding , it may 
 just be that there are infections that will allude protection by prophylactic 
 vaccination and chagas disease may be among these . others have promoted the idea of therapeutic vaccines 
 ( dumonteil et al . 2012 ) . however these would 
 likely suffer from the same problems as described here for prophylactic vaccines , as 
 well as the concern of their utility given the availability of therapeutic drugs that 
 can be highly effective , despite their variability and toxicities . because t. 
 cruzi infection is zoonotic and the fact that companion animals contribute 
 significantly to its transmission to humans , transmission blocking vaccines that reduce 
 pathogen load ( without having to necessary eliminate the infection ) have some promise . lastly , related to the effectiveness of drugs to cure t. cruzi 
 infection - as done in this study - it is important to note that hosts cured of 
 t. cruzi infection are susceptible to reinfection . so the use and 
 development of better methods of transmission control will continue to be a necessity if 
 the impact of t. cruzi infection in humans is to be reduced .
prevention of trypanosoma cruzi infection in mammals likely depends 
 on either prevention of the invading trypomastigotes from infecting host cells or the 
 rapid recognition and killing of the newly infected cells by t. 
 cruzi - specific t cells . 
 we show here that multiple rounds of infection 
 and cure ( by drug therapy ) fails to protect mice from reinfection , despite the 
 generation of potent t cell responses . 
 this disappointing result is similar to that 
 obtained with many other vaccine protocols used in attempts to protect animals 
 from t. cruzi infection . 
 we have previously shown that immune 
 recognition of t. cruzi infection is significantly delayed both at 
 the systemic level and at the level of the infected host cell . 
 the systemic delay 
 appears to be the result of a stealth infection process that fails to trigger 
 substantial innate recognition mechanisms while the delay at the cellular level is 
 related to the immunodominance of highly variable gene family proteins , in particular 
 those of the trans - sialidase family . 
 here we discuss how these previous studies and 
 the new findings herein impact our thoughts on the potential of prophylactic 
 vaccination to serve a productive role in the prevention of t. cruzi 
 infection and chagas disease .