leishmaniases are a group of complex diseases caused by protozoan parasites of the genus leishmania and transmitted to humans by hematophagous sandflies . there are at least 20 species of the parasite , which vary according to geographical location and cause a variety of clinical manifestations , ranging from self - limiting cutaneous lesions to potentially fatal infection of the viscera [ 2 , 3 ] . it is a disease of tropical and subtropical areas , with more than 12 million cases in 88 countries and 2 million new cases annually including 1.5 million cases of cutaneous leishmaniasis ( cl ) and 0.5 million cases of visceral leishmaniasis ( vl ) . the cutaneous disease is particularly prevalent in afghanistan , algeria , brazil , iran , peru , saudi arabia , and syria , accounting for 90% of the global cl burden . although high - cost chemotherapeutics are available , they show high toxicity and are prone to drug resistance development due to prolonged treatment periods . despite substantial effort spent in developing effective vaccines , a large number of proof - of - principle studies have clearly demonstrated that different vaccine formulations , ranging from killed / live - attenuated parasites to recombinant dna / protein vaccines , can provide significant protection against infection with leishmania spp . in a variety of animal models [ 7 , 8 ] . however , the efficacy of these prophylactic or therapeutic vaccines remains partial , and it is therefore necessary to develop novel and effective vaccines . in this regard , antigen identification represents the most important roadblock in vaccine development against any pathogen , as it is usually achieved through rather empirical , time - consuming , and labour - intensive in vivo and in vitro experiments . efforts have thus been devoted to the development of novel strategies for a more rational and faster identification of antigens among large number of pathogen proteins . in recent development , reverse vaccinology approach can be used to predict those antigens that are most likely to be vaccine candidates using the pathogen genomic sequence . moreover , the genomic information , which contains the sequences of all known and unknown potential antigens of each pathogen , has enabled the prediction and analysis of the entire repertoire of potential cytotoxic t lymphocytes ( ctl ) epitopes using bioinformatics tools . this strategy allows the development of vaccines that were previously difficult or impossible to make and can lead to the discovery of unique antigens that may improve existing vaccines . the recent genomic sequence completion of l. major , l. braziliensis , l. infantum , and l. donovani and the availability of immunoinformatics tools have opened new opportunities for the identification of novel vaccine targets against cl . additionally , the presence of genetically stable but highly conserved antigens among most of the species , including antigens with little or no homology to human proteins , offers hope for the development of a single vaccine for multiple disease indications . as depicted in the literature , effective vaccines must invoke a strong response from both t and b cells ; therefore , ctl epitope mapping is crucial in any vaccine designing strategy . many immunoinformatics algorithms and resources have been available to predict t and b cell immune epitopes for peptide based vaccine design and development . thus , the approach of t cell epitopes prediction and their in vitro / in vivo validations appeared to be a very powerful strategy in rational antigen identification , particularly for a pathogen with a large genome such as leishmania . hence , the current study deals with the analysis of l. major genome ( 33.6  mb ) , considered to express about 8300 proteins , all of which are potential antigens containing effective ctl epitopes with respect to susceptible population . the complete proteome of l. major ( strain friedlin ) , consisting of 8312 protein coding sequences , was extracted from database genedb . we also retrieved five well - characterized control antigens ( csp-401glimvlsfl from plasmodium falciparum , m1 - 58gilgfvftl and np- 265ilrgsvahk from influenza a virus , core-141stlpettvv from hepatitis b virus , and psta1- 41fvvaliplv from mycobacterium tuberculosis ) from database antigendb in order to compare and validate the prediction results . these known antigens have been previously tested and verified in various experimental studies and reported as capable of eliciting ctl responses . initially , the l. major proteome ( 8312 proteins ) was screened for the presence of both signal peptide and gpi anchors using signalp   and dgpi , respectively , and then consensus antigenicity predictions were done using vaxijen   and antigenpro   programs . finally selected candidate antigens were further characterized using tmhmm , scratch protein predictor , and betawrap program . thereafter , these candidate antigens were searched for potential sequence similarity with other closely related species and human and/or mouse proteins , using orthomcl database . furthermore , ctl epitopes prediction was carried out using netctl1.2 [ 25 , 26 ] tool integrating predictions of proteasomal cleavage , tap transport efficiency , and 12 mhc class i supertypes ' binding . finally , optitope ( http://etk.informatik.uni-tuebingen.de/optitope ) was used to determine good vaccine epitopes called the optimal set of epitopes from top scoring naturally processed t cell epitopes , for each population susceptible to cutaneous leishmaniasis ( figure 1 ) . the tool optitope requires the following input data from the user : ( i ) sequences of known / predicted antigens , ( ii ) a target human population , that is , mhc alleles and corresponding frequency , and ( iii ) the epitope set to be optimized . the input given by the user if it is feasible , optitope will return an optimal set of epitopes along with fraction of immunogenicity contributing to overall immunogenicity . otherwise , program will propose changes to the user 's input that might yield a feasible optimization problem . the information related to mhc alleles frequency in susceptible human populations and geographic areas is retrieved from dbmhc database ( http://www.ncbi.nlm.nih.gov/gv/mhc ) . a good vaccine displays a high overall immunogenicity that means it is capable of inducing potent immunity in a large fraction of the target population including high mutation tolerance as well as a certain degree of allele and antigen coverage . furthermore , the finally selected epitopes should display a high probability of passing through the natural antigen processing pathway . from all possible epitope combinations , the ones with a maximum overall immunogenicity will be called  optimal  ( there may be more than one optimal epitope combination ) . hence , the search for an optimal epitope set for an good vaccine can be considered as an optimization problem : out of a given set of epitopes , choose a subset which , out of all subsets meeting the other input requirements , displays maximum overall immunogenicity i , which can be derived mathematically ( 1 ) as a weighted sum over immunogenicities of epitopes e with respect to the given mhc alleles a : ( 1)i=ee  aapaie , a , where pa is the frequency of allele a in the target population and ie , a measure the immunogenicity of epitope e when bound to allele a ( either predicted or experimentally determined ) . the present study was divided into two major steps : ( i ) we utilized the l. major genome consisting of 8312 protein coding sequences and predicted 25 antigens ( table 1 ) , through successive screening and consensus antigenicity predictions ; ( ii ) we conducted a comprehensive analysis of the epitopes predicted from these 25 candidate antigens ( figure 1 ) . the present strategy is similar to the reverse vaccinology approach adopted by john et al . , for identifying common vaccine candidates from l. major and l. infantum genomes . additionally , singh et al . [ 29 , 30 ] also utilized the similar approach of mhc supertype based epitope identification , as a strategy to mine proteomic data for identification of novel ctl epitopes , in plasmodium falciparum . the previous studies revealed that surface - exposed proteins such as secretory / outer membrane proteins are ideal targets for vaccine development , with respect to those pathogens against which a strong b cell response ( for antibody production ) is critical . however , for vaccine development against those pathogens where t cell response is critical , subcellular localization is not an issue since a t cell response could be directed to any protein target . in addition , gpi anchored proteins are abundantly expressed in the infective and intracellular stages of trypanosoma cruzi ( another kinetoplastid protozoan ) and have been recognized as antigenic targets by both the humoral and cellular immunity . herein , the entire protein repertoire of l. major , consisting of 8312 protein coding sequences , was screened for presence of signal peptides and gpi anchors . out of these , 265 proteins were predicted as gpi anchored proteins , using dgpi tool , and 1798 proteins were found to contain signal peptides / signal anchors , using signalp3.0 tool . however , 151 proteins were predicted to contain both signal peptides / signal anchors and gpi anchors ( data not shown ) . further screening of these 151 proteins , based on consensus antigenicity predictions using vaxijen   and antigenpro   tools , above a predefined threshold of 0.6 , provided a set of 27 antigenic proteins ( data not shown ) . interestingly , three candidate antigens ( genedb i d : lmjf.04.0130 , lmjf.04.0140 , and lmjf.04.0170 ) were found to share a high sequence similarity ( more than 99.6% ) and thus the latter two antigens were excluded from further analysis . finally , 25 candidate antigens were screened for further characterization as vaccine candidates ( table 1 ) . thus , we used solpro tool ( of scratch protein predictor ) to predict the propensity of a protein to be soluble upon overexpression . out of the 25 antigens , 8 ( numbers 3 , 5 , 7 , 12 , 13 , 14 , 16 , and 18 ) were predicted to be soluble upon overexpression while control antigens m1 , core , and psta1 were predicted to be insoluble upon overexpression ( table 1 ) . similarly , proteins with more than one transmembrane ( tm ) region have been found to be difficult to clone , express , and purify . out of 25 predicted antigens , 19 antigens were found to contain less than two ; 5 antigens ( numbers 6 , 11 , 12 , 13 , and 24 ) were found to contain two each while antigen number 1 was found to contain five tm regions . on the other hand , psta1 was found to contain 6 tm regions ( table 1 ) . through literature analysis , it has also been observed that many bacterial and fungal proteins such as toxins , virulence factors , adhesins , and surface proteins have parallel beta - helices which play important role in human infectious disease . therefore , betawrap program   was used to predict the super secondary structural motif in primary amino acid sequences of 25 antigens . a total 9 candidate antigens ( numbers 26 , 9 , 11 , 13 , and 24 ) , heterologous immunity may exist to cross - reactive epitopes in other strains of the same organism . thus , we identified the potential orthologs in the available leishmania genomes annotations using orthomcl database   through blastp homology prediction program . all the selected 25 candidate antigens showed orthologs in other related species , namely , l. braziliensis , l. infantum , and l. mexicana except antigen number 11 . one of the greatest barriers in vaccine development is the possibility that a particular vaccine may cross - react between host and parasite antigens . thus , vaccine candidates showing sequence similarity with the hosts ( e.g. , human or mouse ) proteins are likely to cause autoimmunity in the host and should be discarded to avoid potential autoimmunity . out of 25 antigens , 3 ( numbers 3 , 9 , and 18 ) showed orthologs in human as well as mouse . for elicitation of t cell responses , the subcellular location and function of target protein are less important than the presence of appropriate mhc binding epitopes in the protein sequences . in past 15 years , significant efforts have been made toward generation of procedures / algorithms for accurate prediction of mhc binding affinity and t cell epitopes . utilizing the clustering method , majority of hla molecules have been classified in relatively few hla supertypes on the basis of their peptide binding specificities [ 36 , 37 ] . one approach to identifying targets of ctl responses in an antigen is based on prediction of high affinity mhc class i restricted t cell epitopes using computerized algorithms . it is also demonstrated that peptides that possess in vitro binding affinity ( ic50 ) values of  500  nm are more immunogenic in vivo . thus , in the current study , the immunogenicity screening was limited to the predicted peptides that were able to bind hla class i supertypes , with binding affinities ( ic50 ) 500  nm . furthermore , it is important to consider whether each mhc binding peptide is being correctly processed from the native antigen and subsequently displayed on the surface of antigen presenting cells . at present , it is possible to predict the naturally processed peptides using netctl algorithm above a combined epitope processing score of 0.75 , which includes predictions of proteasomal cleavage , tap binding , and hla binding . thus , in order to identify candidate cd8 + t cell epitopes , 25 candidate antigens selected from l. major were screened using netctl . the tool netctl1.2 provides a comprehensive prediction about epitopes binding to the 12 different hla class i supertypes : hla - a1 , hla - a2 , hla - a3 , hla - a24 , and hla - a26 and hla - b7 , hla - b8 , hla - b27 , hla - b39 , hla - b44 , hla - b58 , and hla - b62 . a total of 3756 putative ctl epitopes were predicted , including 1373 hla - a ( 230-a1 , 429-a2 , 269-a3 , 207-a24 , and 238-a26 ) and 2383 hla - b ( 542-b7 , 283-b8 , 318-b27 , 288-b39 , 157-b44 , 304-b58 , and 491-b62 ) supertype binding peptides ( figure 2 ) . predictions for the 5 control antigens showed that csp had 28-hla - a , 40-hla - b , m1 had 33-hla - a , 55-hla - b , core had 21-hla - a , 48-hla - b , psta1 had 83-hla - a , 104-hla - b , and np had 59-hla - a , 126-hla - b restricted ctl epitopes . apart from this , their experimentally validated ctl epitopes were also predicted by netctl ranked in top five . from the analysis , antigen number 1 was found to have largest number of predicted ctl epitopes for hla - a and hla - b supertypes while antigens numbers 4 , 16 , 19 , and 21 had higher number of ctl epitopes for hla - a supertypes and antigens numbers 13 , 16 , 19 , and 21 had higher number of ctl epitopes for hla - b supertypes in comparison to the control antigens . overall , test antigen number 1 showed highest number of supertype epitopes and was thus predicted as best antigen . from among the predicted ctl epitopes , the epitopes which displayed the top processing score for the respective mhc supertypes are presented in tables 2 and 3 . these 624 potential ctl epitopes were also checked for their potential similarity with human proteins , using human protein reference database ( http://www.hprd.org/ ) . however , none of the epitopes were found similar to any human proteins . mhc is highly polymorphic ; hence each individual possesses a set of mhc molecules of differing specificities ; that is , different patients typically bind different repertoires of peptides . thus , a crucial step in the design of effective peptide based vaccine is the selection of the good epitopes set which yields the best immune response in a given population or individual . furthermore , the frequency of an mhc allele to occur within the target human population directly affects the allele 's contribution to the overall immunogenicity . sette et al . have also demonstrated a correlation between immunogenicity and mhc class i binding affinity . it is therefore reasonable to use mhc class i binding affinity prediction methods for calculation of the overall immunogenicity . hence , the present study employed optitope ( using bimas method ) to determine the optimal set of epitopes from the selected epitopes , which calculate the best immune response in the susceptible target populations of cutaneous leishmaniasis , namely , southwest asia , north africa , and south america . initially , the top scoring epitope sets predicted using netctl from each of the 25 candidate antigens were tried to optimize by optitope and screened the bimas based hla nonbinders ( negative ) for the target populations . out of these , 10 epitope sets ( from antigen numbers 1 , 2 , 4 , 5 , 6 , 8 , 15 , 21 , 22 , and 25 ) that were predicted hla binders ( positive ) are clubbed together to form a set of 120 candidate epitopes . however , when this combined epitope set was further optimized for the three different target populations , no optimization solution was obtained for any population . therefore , epitope set of antigen number 21 was randomly excluded from the combined set and got the optimized results with the 108 combined epitopes set ( derived from the 9 positive antigens ) for the different target populations . for the target population of southwest asia , out of the 108 candidate epitopes set , optitope selected a subset of 60 epitopes restricted by the 19 mhc class i alleles covering 96.58 % human population . the most immunogenic epitope , 127slwsllagv , from antigen number 1 , was found to bind with allele hla - a0201 , contributing 7% , while the least immunogenic epitope , 246kssalahkl , from antigen number 25 , contributed < 1% to the overall immunogenicity ( table 4 ) . similarly , for the target population of north africa , out of the 108 candidate epitopes ' set , optitope selected a subset of 45 epitopes restricted by the 13 mhc class i alleles covering 88.48 % human population . here again , the epitope 127slwsllagv , from antigen number 1 , was most immunogenic , covered the allele hla - a0201 , and contributes 9% , and the least immunogenic epitope , 4llprlflaf , from antigen number 8 , contributed < 1% to the overall immunogenicity ( table 5 ) . also , for the target population of south america , out of the 108 candidate epitopes ' set , optitope selected a subset of 34 epitopes restricted by the 9 mhc class i alleles covering 98.97 % human population . for a third time , the epitope 127slwsllagv , from antigen number 1 , which binds to the allele hla - a0201 , was predicted most immunogenic and contributed 10% , while the least immunogenic epitope , 127simslqiry , from antigen number 8 , was found to contribute < 1% to the overall immunogenicity ( table 6 ) . thus , overall , it was found that 6 antigens ( numbers 1 , 4 , 13 , 16 , 19 , and 21 ) had larger number of predicted ctl epitopes as compared to control antigens which could be tested in vivo for validation . similarly , the epitope 127slwsllagv , from antigen number 1 , binds to hla - a0201 molecule and was predicted as most immunogenic for all the three populations ' susceptibility to leishmaniasis [ 42 , 43 ] . hence , these antigens / peptides may be considered as suitable candidates for vaccine and diagnostics design . further , these protective epitopes conform to the anchor - based mhc binding motifs ' concept used for t cell epitope identification by many researchers such as sette et al .   and rtzschke et al . the current study aimed to mine l. major genome for antigens selection and characterization as vaccine components based on criteria such as presence of transmembrane domains and orthologs analysis . furthermore , the immunogenic epitopes predicted from these antigens can be analyzed for hla - supertype binding and optimization of good vaccine epitopes against susceptible human populations to l. major infection . in light of the results obtained , it can be concluded that the combined use of reverse vaccinology and immunoinformatics along with in vitro / in vivo validation strategies has emerged as the most promising approach in designing successful vaccine against tropical diseases . in future , it would be helpful to use modeling and simulation system where critical experiments may be performed in a computer in order to predict the effects of experimental modifications on the immune system and thus offer a criterion for the selection of the most likely meaningful experimental tests to be conducted in vivo or in vitro .
despite the wealth of information regarding genetics of the causative parasite and experimental immunology of the cutaneous leishmaniasis , there is currently no licensed vaccine against it . in the current study , 
 a two - level data mining strategy was employed , to screen the leishmania major genome for promising vaccine candidates . 
 first , we screened a set of 25 potential antigens from 8312 protein coding sequences , based on presence of signal peptides , gpi anchors , and consensus antigenicity predictions . 
 second , we conducted a comprehensive immunogenic analysis of the 25 antigens based on epitopes predicted by netctl tool . 
 interestingly , results revealed that candidate antigen number 1 ( lmjf.03.0550 ) had greater number of potential t cell epitopes , as compared to five well - characterized control antigens ( csp - plasmodium falciparum , m1 and np - influenza a virus , core protein - hepatitis b virus , and psta1-mycobacterium tuberculosis ) . in order to determine an optimal set of epitopes among the highest scoring predicted epitopes , 
 the optitope tool was employed for populations susceptible to cutaneous leishmaniasis . 
 the epitope ( 127slwsllagv ) from antigen number 1 , found to bind with the most prevalent allele hla - a0201 ( 25% frequency in southwest asia ) , was predicted as most immunogenic for all the target populations . 
 thus , our study reasserts the potential of genome - wide screening of pathogen antigens and epitopes , for identification of promising vaccine candidates .