the ethics committee of gois federal university approved this study under protocol # 
 020/2008 .
recombinant mycobacterium smegmatis production : m. 
 smegmatis mc 155 ( kindly provided by dr .
luciana leite of the 
 butantan institute , brazil ) was grown in middlebrook 7h9 broth ( himedia , mumbai , india ) 
 supplemented with 0.05% of tween 80 at 37c for three days .
cells were washed in 10% 
 glycerol , and electrocompetent cells were aliquoted in 100 l volumes in 
 cryotubes at 80c until use .
the recombinant plasmid containing the gene of the fusion protein cmx constructed in our 
 lab ( pla71/ cmx )   and the empty plasmid ( pla71 , 
 kindly provided by dr .
brigitte gicquel , from pasteur institute , france ) were introduced 
 into m. smegmatis mc 155 ( mc - cmx and 
 mc - pla71 , respectively ) by electroporation .
construction of the recombinant m. smegmatis - cmx ( mc - cmx ) was 
 described by junqueira - kipnis et al . .
briefly , the dna sequence containing the immunodominant epitopes from ag85c 
 and mpt51 and the entire hspx gene , described previously by de sousa et al . 
 , was used as template for pcr amplification 
 using a set of primers to create flanking restriction enzyme sites and facilitate posterior 
 cloning .
the product of this amplification was cloned into pgem - t easy vector ( promega , 
 madison , wis , u.s.a . ) .
the recombinant pgem - t easy vector containing the fused gene ( cmx ) 
 was digested with kpni and noti for elution of the cmx gene .
the eluted cmx / kpni / noti gene 
 was ligated to the mycobacteria / e .
coli shuttle vector pla71 digested with 
 the same enzymes , the size of which was 12,248 pb , and as a selective marker , pla71 contains 
 a kanamycin resistance gene .
the cmx gene was 
 inserted downstream of the -lactamase gene ( blaf * ) promoter .
this promoter codes the 32 
 amino acids of the mature -lactamase signal sequence and the first 5 amino acids of the 
 mature protein for expression and export of cmx protein .
pla71/cmx and pla71 vectors were transformed into 
 mc and screened on media with kanamycin , to produce the recombinant vaccine 
 mc - cmx and mc - pla71 control vaccines , respectively . animals and experimental design : thirty nelore calves that ranged in age 
 from 10 to 12 months and were negative for the tuberculin skin test ( from the goiano federal 
 institute  uruta campus ) were separated into three groups randomly .
group 1 received 1.0 
 ml ( 1  10 cfu / ml ) of the recombinant 
 vaccine ( m. smegmatis mc - cmx ) ; group 2 received 1.0 
 ml ( 1  10 cfu / ml ) of the empty vector 
 vaccine ( m. smegmatis mc - pla71 ) ; and group 3 received 1.0 
 ml of pbs .
prior to each immunization , blood collection was performed 
 such that there were blood samples corresponding to the basal level , the levels at 
 21 
 days after the first and second immunizations and the level at 30 days after 
 the last immunization . in order to test the maintenance of memory immune responses ,
the animals were revaccinated 
 a fourth time at 200 days after the third vaccination .
blood was collected from the tail artery / vein without an 
 anti - coagulant .
sera were obtained after centrifugation at 2,500 g for 10 min at 4c and 
 stored at 20c until use .
blood was also collected with heparin as an anti - coagulant for cytometric analysis . 
 peripheral blood mononuclear cell ( pbmc )
was obtained after centrifugation at 2,500 g for 15 
 min at 4c , treatment with erythrocytes lysis solution and resuspension in rpmi 1640 media 
 ( gibco , carlsbad , ca , u.s.a . ) supplemented with 24 mm of sodium bicarbonate , 
 10% heat - inactivated fetal calf serum , 2 mm of l - glutamine ( sigma - aldrich , st 
 louis , mo , u.s.a . ) , 1% of penicillin - streptomycin ( sigma - aldrich ) , 1 mm sodium 
 pyruvate ( sigma - aldrich ) and nonessential amino acids 
 ( sigma - aldrich ) .
immunoenzymatic assay ( elisa ) : ninety - six - well polystyrene plates ( santa 
 cruz biotechnology , dallas , tx , u.s.a . )
were coated with the lysates of mc - cmx 
 vaccine or purified recombinant protein ( rcmx , rag85c , rmpt51 or rhspx , 10 
 g / ml ) diluted in 15 mm carbonate / bicarbonate buffer ( ph 
 9.8 ) .
after 18 hr of incubation at 4c , the plates were blocked with carbonate / bicarbonate 
 buffer containing 1% gelatin for 2 hr at 37c .
serum , diluted ( 1:320 ) in pbs containing 0.1% 
 gelatin , was added and incubated for 2 hr at 37c .
extensive washing with pbs tween ( 0.05% ) 
 was performed followed by addition of an anti - bovine igg conjugate ( jackson immunoresearch 
 laboratories , inc . , london , u.k . )
diluted in pbs 0.1% gelatin ( 1:10,000 ) and incubation for 
 1 hr at 37c .
after additional washing with pbs tween ( 0.05% ) , citrate buffer ( ph 5.0 ) 
 containing o - phenylenediamine ( opd ) and hydrogen peroxide was added .
samples were analyzed in a multiskan 
 plus ( thermo scientific , walthan , ma , u.s.a . ) elisa reader at 492 nm . flow cytometry : flow cytometry analysis was performed after stimulation of 
 10 pbmcs with rcmx ( 10 g / ml ) or 
 phytohemagglutinin ( pha 1 g / ml ) for 6 hr at 37c in 5% 
 co2 .
cells were further incubated with monensin ( ebioscience , , san diego , ca , 
 u.s.a . ) for 4 hr at 37c in 5% co2 .
cell staining was performed with antibodies 
 conjugated with surface membrane or intracellular markers , pe - cd8 , percp - ifn- and apc - cd4 , 
 for 30 min , washed with pbs containing 0.1% sodium azide , fixed and permeabilized with perm 
 fix / perm wash ( bd pharmingen , sanjose , ca , u.s.a . ) .
cell selection after flow cytometry 
 acquisition of 50,000 events ( bd biosciences , san diego , ca , u.s.a .
facscanto ii ) was based 
 on size ( fsc ) and granularity ( ssc ) to gate lymphocytes using bd facsdiva software .
histopathological analysis : ninety days after the booster immunization , 
 superficial cervical lymph node biopsies were performed at the site of vaccine injections . 

biopsy samples were conditioned in 10% buffered formalin , posteriorly submitted to routine 
 processing and stained with hematoxylin eosin ( he )   and fite s acid fast staining .
single cervical tuberculin skin test : all animals were submitted to 
 tuberculin skin testing prior to beginning the experiments and after the end of all 
 immunizations . the test was performed by intradermally injecting 0.1 ml of 
 m. bovis standard ppd ( 0.1 mg of m. bovis strain an5 ) in 
 the mid - cervical area .
the animals were examined for a hypersensitivity response 72 hr post 
 injection . statistical analysis : the results were analyzed using the microsoft excel 
 ( version 14.3.4 , 2011 ) and graphpad ( prism 4.0 ) software . differences between groups were 
 analyzed using anova test .
individual groups were compared by the nonparametric 
 kruskal - wallis test followed by dunn s test .
results with a p - value of less than 0.05% were 
 considered statistically significant .
in order to verify if mc - cmx was able to induce an immune humoral response in 
 bovine , an elisa was developed to detect the presence of vaccine - specific antibodies 
 ( fig .
( a ) serum levels of 
 anti - mc - cmx antibodies among bovine immunized with mc - cmx , 
 mc and pbs .
( b ) serum levels of anti - rcmx antibodies among bovine 
 immunized with mccmx , mc and pbs .
animals were immunized 
 with 1  10 cfu / ml of each vaccine subcutaneously .
sera 
 were obtained at different time points following vaccination and analyzed by elisa . 

each time point represents the mean and standard error of the mean of the optical 
 density from 10 animals per group .
* statistically significant difference between 
 the mc - cmx and pbs groups ( p<0.05 ) . *
* statistically 
 significant difference between the mc and pbs groups 
 ( p<0.05 ) . ) .
a progressive increase in igg antibody levels was observed as 
 immunizations were performed , and there was a significant difference in 
 mc - cmx - specific antibody levels at the latter time points of immunizations 
 compared with at the initial time points , as well as compared with the control groups 
 immunized with mc or pbs .
an elisa was also performed to detect the presence of 
 rcmx - specific antibodies ( fig .
1b ) , and the 
 results were similar to those of the first test .
a progressive increase in igg antibodiy 
 levels occurred , with higher titers being observed at latter time points of immunization . 

however , no response was observed to the individual recombinant antigens ( rag85c , rmpt51 or 
 rhspx , data not shown ) .
( a ) serum levels of 
 anti - mc - cmx antibodies among bovine immunized with mc - cmx , 
 mc and pbs .
( b ) serum levels of anti - rcmx antibodies among bovine 
 immunized with mccmx , mc and pbs .
animals were immunized 
 with 1  10 cfu / ml of each vaccine subcutaneously .
sera 
 were obtained at different time points following vaccination and analyzed by elisa . 

each time point represents the mean and standard error of the mean of the optical 
 density from 10 animals per group .
* statistically significant difference between 
 the mc - cmx and pbs groups ( p<0.05 ) . *
* statistically 
 significant difference between the mc and pbs groups 
 ( p<0.05 ) . to evaluate
if vaccination altered the number of cd4 t cells in the peripheral 
 blood , pbmcs were analyzed , and the total number of cd4 t cells from each animal 
 was obtained .
cattle were revaccinated 200 days 
 after the third immunization , and pbmcs were obtained .
( a ) total cd4 t 
 cells from the vaccinated group were quantified by flow cytometry .
the dots represent 
 the number of cd4 t cell / ml from each animal .
( b ) pbmcs from 
 mc - cmx - vaccinated animals were stimulated in vitro with 
 medium , pha or cmx , and the percentage of cd4ifn--positive t cells was 
 analyzed .
no 
 significant statistical difference was observed . ) . since it is well known that the cellular immune response to btb is crucial for 
 protection , after the booster vaccination to induce reactivation of memory cells , 
 ifn--producing cd4 t lymphocyte populations were quantified .
stimulation of 
 pbmcs from mc - cmx - vaccinated bovine with recombinant rcmx did not reveal an 
 increase in the amount of ifn--producing cd4 t lymphocytes , regardless of the 
 animal immunization status ( fig .
cattle were revaccinated 200 days 
 after the third immunization , and pbmcs were obtained .
( a ) total cd4 t 
 cells from the vaccinated group were quantified by flow cytometry .
the dots represent 
 the number of cd4 t cell / ml from each animal .
( b ) pbmcs from 
 mc - cmx - vaccinated animals were stimulated in vitro with 
 medium , pha or cmx , and the percentage of cd4ifn--positive t cells was 
 analyzed .
no 
 significant statistical difference was observed . after the second immunization , animals that received the mc - cmx vaccine 
 presented accentuated lymphadenomegaly in superficial cervical lymph nodes .
neither necrosis nor 
 inflammatory infiltrate or granuloma formation was found within the analyzed biopsy samples 
 ( fig . 3fig .
3.histopathological analysis of a superficial cervical lymph node from the 
 mc - cmx vaccinated group .
the lymph node from a representative animal was 
 sectioned and stained with hematoxylin eosin ( h&e ) ( a , c and d ) and fite s acid 
 fast stain ( b ) 90 days after the last immunization of the group .
note the absence of 
 inflammatory infiltrate or granuloma formation or necrosis ( a ) .
fite s acid fast 
 staining revealed no presence of acid - fast bacilli ( b ) .
the structure of a normal 
 lymph node with a germinal center , medular region and blood vessels can be seen ( c and 
 d ) . ) .
histopathological analysis of a superficial cervical lymph node from the 
 mc - cmx vaccinated group .
the lymph node from a representative animal was 
 sectioned and stained with hematoxylin eosin ( h&e ) ( a , c and d ) and fite s acid 
 fast stain ( b ) 90 days after the last immunization of the group .
note the absence of 
 inflammatory infiltrate or granuloma formation or necrosis ( a ) .
fite s acid fast 
 staining revealed no presence of acid - fast bacilli ( b ) .
the structure of a normal 
 lymph node with a germinal center , medular region and blood vessels can be seen ( c and 
 d ) .
all animals remained negative for the tuberculin skin test until the end of the 
 experimental period .
in the present study , we investigated the capacity of a recombinant vaccine , 
 mc - cmx , to induce specific igg antibodies in bovine .
the cervical lymph nodes of mc - cmx - vaccinated 
 animals presented follicular hyperplasia characteristic of cellular proliferation .
the 
 animals remained negative in the tuberculin skin test throughout the duration of the 
 study . in recent years
, several approaches have been made to increase the protective efficacy of 
 bcg , in addition to the search of new immunogenic antigens from mtb as well as from other 
 mycobacteria , in order to develop improved vaccines .
an effective vaccine against tb or btb 
 must be capable of preventing infection and establishing a long - lasting protection . 

consequently , it is essential that a robust induction of effector and memory cells take 
 place and that a significant level of antibodies is raised following vaccination .
it is known that antibodies play an important role 
 in mycobacterial infections , as they can induce microbicidal activities of macrophages and 
 attract monocytes , neutrophils and nk cells to the site of infection , in addition to 
 activating dendritic cells and macrophages , enabling efficient antigen processing and 
 presentation by these cells [ 19 , 25 , 39 , 41 ] . in this study , a significant increase in specific igg levels against mc - cmx and 
 rcmx was observed in mc - cmx - vaccinated animals ( fig .
1 ) . despite the observation of an initial response among animals 
 vaccinated with m. smegmatis ( mc ) alone
, there was a 
 significant increase in the antibody level in animals receiving mc - cmx after the 
 second time point of vaccination ( p<0.05 ) .
this cross - reaction observed 
 among the control groups could be explained by the presence of ag85c and mpt51 analog 
 proteins in m. smegmatis   and by 
 the constant exposure of animals to environmental mycobacteria as well .
bovines vaccinated 
 with mc - cmx could not produce igg antibodies against individual ag85c , mpt51 or 
 hspx recombinant antigens when tested in an indirect elisa ( data not shown ) , reflecting the 
 immunogenicity of the rcmx protein in the bovine model .
similar results were observed when 
 mice were immunized with mc - cmx or rcmx [ 15 , 24 ] . a possible explanation for these 
 observations could involve the tertiary structure of the secreted fusion protein produced by 
 the recombinant m. smegmatis vaccine hiding or exposing unique epitopes not 
 present in the single antigens .
to circumvent the absence of response observed to the 
 individual wild - type antigens , further engineering of the recombinant fusion protein is 
 needed , such as replacing the hinge regions with bigger and/or different amino acid 
 sequences to allow independence of each epitope exposition and folding .
similarly , we have shown that rcmx , a recombinant fusion protein , induces production of 
 igg1 and igg2a in mice when used as a subunit vaccine in addition to the ability of this 
 recombinant protein to detect high levels of specific igg and igm in the sera of active tb 
 individuals .
these results corroborate with 
 immunogenic potential of the recombinant protein in the context of the vaccine .
a recent 
 report showed the ability of a recombinant m. smegmatis vaccine expressing 
 a fusion protein comprised of the antigens esat- 6 and cfp-10 from mtb to induce a high 
 titer of antibodies in the mouse .
thus , the 
 fusion protein cmx can be considered a candidate to be used in the production of recombinant 
 or subunit vaccines , as they presented antigenicity and immunogenicity in different study 
 host models .
further development of the fusion protein should be performed before this 
 vaccine is considered a candidate to protect btb .
the essential role of cell - mediated immune response in the protection against tb has been 
 well characterized [ 13 , 30 , 34 ] .
, as they 
 produce ifn- and tnf- , which are key cytokines in the immune response against 
 mycobacterial infections .
in addition , 
 cd8 t lymphocytes also contribute to a long - lasting protection against tb 
 [ 28 , 31 , 
 33 ] , as they can use a diverse repertoire of 
 mechanisms against infected cells , especially phagocytes , the main target of mycobacteria , 
 and non - phagocytic cells that can also be infected by this pathogen . here , we show that there was no significant increase in specific 
 cd4 ifn--positive t cells ( fig .
this phenomena could be explained by a ) the majority of the effector/ 
 memory t cell being localized in the draining lymph nodes instead of in peripheral blood 
 [ 26 , 35 ] , b ) 
 30 days after the booster possibly being too long for the persistence of this population of 
 cells in the periphery [ 26 , 39 ] and c ) cmx not having been presented by bovine antigen - presenting 
 cells .
it is well known that mycobacteria can be drained into the lymph node by infected resident 
 macrophages .
consequently , we sought to understand 
 if the observed lymphadenomegaly in superficial cervical lymph nodes was an inflammatory 
 process caused by m. smegmatis proliferation .
the histopathological 
 analysis revealed neither necrosis nor inflammatory infiltrate or granuloma formation within 
 the analyzed biopsy samples .
additionally , no acid - fast bacilli were detected within the 
 analyzed tissues ( fig .
the observed 
 lymphadenomegaly was probably due to cellular proliferation as a result of a lymphocytic 
 activation .
it is possible that the observed hyperplasia was strictly correlated with higher 
 levels of antibodies induced against cmx , although activation of cd4 or 
 cd8 t cells was not observed .
previous studies stated that bovine have a dichotomous response to mycobacteria , that is , 
 alternating high levels of antibody production with cellular immune response [ 35 , 38 , 50 ] . in line with this , we can infer that a humoral 
 immune response was elicited in response to the fusion protein cmx and that a cellular 
 response was directed only toward m. smegmatis , and thus , it was not 
 observed in our assays .
an inflammatory response or the presence of bacteria was not 
 observed in the lymphoid tissue ( fig .
furthermore , the maintenance of a negative tst status among animals indicates that the 
 vaccine did not elicit a cross - reaction with the test .
explanations for pitfalls in human and animal tb vaccines are encountered throughout the 
 literature .
some studies affirm that previous contact with environmental mycobacteria can 
 reduce the immune response induced by tb vaccines .
different animal model studies have 
 already proven the interference of sensitization to other mycobacteria in the immune 
 response against tuberculosis [ 7 , 14 , 30 ] .
a study 
 with cattle demonstrated that , prior to bcg immunization , bovines exhibited high levels of 
 ifn- in response to ppd from mycobacterium avium , and these animals showed 
 an ifn- response to ppd from m. bovis for only one week after vaccination 
 with bcg ; on the other hand , groups that received a newly derived attenuated m. 
 bovis strain maintained this response for two weeks .
failure in bcg vaccination among animal models is consistent with 
 studies in humans , where it has been shown that previous contact with other mycobacteria 
 could block multiplication of the vaccine and induction of an immune response .
regarding 
 humans , a study with populations from malawi and the united kingdom showed that people from 
 malawi had lower levels of ifn- one year after bcg vaccination , which was attributed to a 
 preexposure to environmental mycobacteria .
the development of new therapies against infectious diseases that affect human populations 
 is a constant challenge for the scientific community . in regard to tb ,
a zoonosis causing a 
 significant death toll in humans and important economic losses worldwide , these challenges 
 are even greater .
an 
 important limitation of this study was the necessity to validate the real potential of 
 mc - cmx as a vaccine in a protective study in bovines challenged with 
 m. bovis .
this pitfall does not diminish the importance of our findings , 
 as we showed for the first time the bovine immune response to a recombinant fusion protein 
 used as a btb vaccine . in conclusion
, this work shows that the mc - cmx vaccine was able to induce a 
 humoral immune response and did not induce cross - reactivity in the tst , indicating that it 
 can potentially be further tested as a vaccine for bovine tuberculosis .
 abstractthis study aimed to evaluate the immunogenicity of a recombinant 
 mycobacterium smegmatis vaccine expressing the cmx fusion protein 
 composed of immunodominant epitopes ag85c , mpt51 and hspx of mycobacterium 
 tuberculosis , which are important mycobacteria virulence factors . 
 a group of 
 nelore heifers that were 10 to 12 months of age and negative for the tuberculin skin test 
 ( tst ) were immunized with four doses of the recombinant vaccine mc2-cmx 
 ( m. smegmatis - ag85c - mpt51-hspx ) during a period of one 
 year . before each immunization 
 , blood was collected to obtain sera for antibody analysis . 
 
 serological analysis demonstrated that mc2-cmx was able to induce a humoral 
 response with increased levels of specific igg antibodies against cmx , despite minimum 
 antibody levels being detected for individual ag85c , mpt51 or hspx recombinant antigens . 
 
 however , there was no significant increase in specific cd4 + ifn--positive t 
 cells . 
 lymphadenomegaly was observed in superficial cervical lymph nodes adjacent to the 
 site of vaccination among mc2-cmx - vaccinated bovines , and the histopathological 
 analysis demonstrated follicular hyperplasia without inflammatory infiltrate or granuloma 
 formation . 
 animals remained negative for the tst until the end of the experiments , showing 
 no cross - reactivity with the recombinant vaccine and tuberculin proteins . 
 we discuss the 
 potential of mc2-cmx to induce an immune response in cattle . 
MATERIALS AND METHODS
RESULTS
DISCUSSION