BACKGROUND
although the bacille calmette guérin  vaccine for tuberculosis  is the most widely used vaccine worldwide, tb continues to be a tremendous public health problem  <cit> .
a third of the world's population is estimated to be infected with mycobacterium tuberculosis and 2- <dig> million people die of the disease each year  <cit> .
key among the reasons for the unabated spread of tb is the inability of the bcg vaccine to provide adequate protection against pulmonary tb in adults, the most contagious form of tb  <cit> .
developing an improved vaccine for tb, whether a replacement for bcg, a booster to the existing vaccine, or a vaccine specifically directed against latent tb, is of crucial importance in the battle to defeat the disease  <cit> .
while several tb vaccine candidates have demonstrated protective efficacy in animal models and have proceeded to clinical trials in humans  <cit> , even a successful clinical trial cannot guarantee that a vaccine can protect all members of the diverse worldwide human population against all variants of m. tuberculosis.
one promising vaccine candidate is the mtb72f subunit vaccine, a polyprotein composed of the m. tuberculosis proteins pepa and ppe <dig>  <cit> .
the ppe <dig> antigen has been demonstrated to contain at least  <dig> epitopes  <cit>  and the vaccine has been shown to provide protection against tb in cynomolgus monkeys  <cit> ; it is currently under clinical trials in humans.
the peptide sequence of mtb72f, like that of many vaccines, is based on a laboratory strain of the pathogen whose antigens may differ from those found in variable clinical strains in immunologically critical ways  <cit> .
furthermore, the diversity of the human leukocyte antigen  and other immune genes results in variable vaccine efficacy in different individuals even in the absence of pathogen variation  <cit> .
while clinical trials include individuals of many hla genotypes, genotype frequencies vary dramatically in different regions; genotypes that are common in tb-endemic regions may be underrepresented in clinical trial populations.
it is therefore necessary to incorporate information on host and pathogen genetic diversity in vaccine design, development, and testing.
the ppe protein family of m. tuberculosis is a large, 69-member protein family with a currently unknown function  <cit> .
previous work by hebert and colleagues  <cit>  demonstrated that the ppe <dig> protein of mtb72f, but not the pepa protein, had significant variability in a sample of clinical m. tuberculosis isolates.
such variation, however, would be important only if the variation were in regions of the protein that were vital for the human immune response to m. tuberculosis.
to determine whether variation in these proteins might impact the efficacy of mtb72f, we must consider interactions between the m. tuberculosis proteins and the human immune system.
of the many genes involved in the immune response, the major histocompatibility complex  genes - hla in humans - are among the most crucial and yet most variable.
mhc proteins present foreign peptide epitopes from intracellular  and extracellular  pathogens to cd8+ cytotoxic  or cd4+ helper  t cells to initiate the immune response.
the mtb72f vaccine was found to stimulate both cd4+ and cd8+ t cell-mediated responses in a mouse model  <cit> , and the cd4+ t cell response in particular is thought to be essential for preventing m. tuberculosis infection  <cit> .
there are thousands of hla alleles  <cit> , however, and variation in these alleles can significantly impact individual responses to vaccination  <cit> .
recently, several algorithms to predict the affinity of peptide sequences for various class i and class ii mhc alleles have been developed  <cit>  and several have been extensively validated  <cit> .
a consensus approach incorporating three or more programs has been shown to increase the accuracy of mhc class ii epitope predictions  <cit> .
to investigate the impact of host and pathogen variation on tb vaccine efficacy, we have used previously reported protein sequences from clinical isolates of m. tuberculosis  <cit>  and in silico hla epitope prediction programs to assess the protection offered by the mtb72f subunit vaccine against diverse strains of m. tuberculosis in human populations suffering from a high burden of the disease.
this investigation revealed that due to variation in both m. tuberculosis protein sequences and epitope-binding capabilities of different hla alleles, certain human populations with a high burden of tuberculosis may not be optimally protected by the mtb72f vaccine.
RESULTS
comparison of epitopes between the vaccine and clinical strains
to determine the impact of clinical strain variation on the protection offered by the mtb72f vaccine, we examined the conservation of each nonamer sequence predicted to be a binding epitope compared to the conservation of every nonamer sequence in the vaccine.
eight epitope prediction programs were used to determine epitopes predicted to bind .
there was substantial variation in the number of nonamer sequences predicted to act as epitopes for each drb <dig> allele by each prediction program .
however, when a list of all ppe <dig> nonamer cores predicted to bind at least one drb <dig> allele was compiled using the epitope predictions for the five prediction programs that covered the greatest number of drb <dig> alleles, 65% of these nonamers were unconserved.
this percentage was significantly greater than the 60% of total ppe <dig> nonamer sequences that were unconserved .
we also considered individual prediction programs' epitope binding predictions for single drb <dig> alleles.
for  <dig> out of  <dig> individual drb <dig> allele/prediction program combinations, more than 60% of the epitopes predicted to bind were unconserved; for  <dig> of these, 80-100% were unconserved .
no nonamer sequences in the pepa protein were classified as unconserved epitopes.
<dig> citation numbers are from google scholar
prediction of promiscuous epitopes
as promiscuous epitopes are frequently sought for epitope-based vaccines, we examined such epitopes, defining them as epitopes predicted to bind at least four of the twelve drb <dig> alleles studied and classifying them as conserved or unconserved .
in all, fifteen promiscuous epitopes were predicted, ten from the ppe <dig> protein and five from pepa.
eight of these epitopes, including all of the pepa epitopes, were classified as conserved and the other seven as unconserved.
of note, the three ppe <dig> epitopes predicted to bind the largest numbers of epitopes  were all classified as unconserved.
prediction of drb <dig> allele binding
to find drb <dig> alleles that might be especially well or poorly covered by the mtb72f vaccine, we summed the ppe <dig> and pepa epitope predictions to find the number of total  and conserved  vaccine epitopes predicted to bind each drb <dig> allele.
drb1* <dig> was consistently predicted to bind an extremely large number of epitopes; a median of  <dig> total epitopes  and  <dig> conserved epitopes  were predicted to bind.
by contrast, drb <dig> alleles  <dig>   <dig>  and  <dig> were each predicted to bind a very small number of total or conserved vaccine epitopes.
drb1* <dig> was predicted to bind a median of  <dig> total vaccine epitopes  and four conserved vaccine epitopes  while drb1* <dig> was predicted to bind a median of seven total vaccine epitopes  and three conserved vaccine epitopes .
finally, drb1* <dig> was predicted to bind a median of eight total vaccine epitopes  and five conserved vaccine epitopes .
similar trends were observed when total ppe <dig> epitopes, conserved ppe <dig> epitopes, and pepa epitopes predicted to bind each allele were evaluated separately .
non-tb antigenic regions
because the mtb72f vaccine is a polyprotein composed of a single continuous amino acid chain incorporating the full ppe <dig> protein, the pepa protein separated into two pieces, an n-terminal his tag, and short amino acid insertions between the protein segments, several potential mhc class ii epitopes are found in the vaccine but not in the native m. tuberculosis proteins  <cit> .
if these epitopes bound to drb <dig> proteins, they could misdirect t cells to respond to non-tb epitopes rather than to epitopes found in the pathogen  <cit> .
non-pathogen epitopes make up a relatively high proportion of the epitopes in the mtb72f vaccine compared to other vaccines, such as killed or live attenuated vaccines that do not contain artificial epitopes, and thus these epitopes might misdirect the immune response to mtb72f in a manner not seen with other vaccines.
we therefore evaluated the drb <dig> binding of each potential mhc class ii epitope core found in the vaccine but not in the individual proteins.
the maximum median number of non-tb vaccine epitopes predicted to bind to any of the drb <dig> alleles was two .
of the three drb <dig> epitopes predicted to bind the smallest number of total or conserved vaccine epitopes , two were predicted to bind none of the non-tb epitopes and the third, drb1* <dig>  was predicted to bind a median of one non-tb epitope .
alleles of concern in regions with a high burden of tb
hla drb <dig> alleles of concern were defined as drb <dig> alleles predicted to bind four or fewer conserved epitopes in the mtb72f vaccine.
based on these criteria, we found that the alleles of concern were drb1* <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> .
of these alleles, only four  were predicted to bind as many or more conserved as unconserved epitopes, while the remaining nine were predicted to bind fewer conserved than unconserved epitopes or  to bind no epitopes at all.
five additional alleles  that were not predicted to bind particularly few conserved epitopes nevertheless bound at least as many unconserved as conserved epitopes .
we further examined whether any of these alleles were predicted to bind the non-tb epitopes in the vaccine.
although most of the alleles were not predicted to bind any of these epitopes, drb1* <dig>   <dig>  and  <dig> were each predicted to bind to one non-tb epitope .
1alleles of particular concern  are highlighted with italics.
2n = number of programs able to predict epitope binding to that allele
3nk = none known
4of the study populations included in the allele*frequencies in worldwide populations database  <cit>
populations of concern for reduced vaccine efficacy
based on the alleles of concern determined through epitope binding predictions, we characterized each population in the allele*frequencies database from a tb-endemic country as being of great, moderate, or lesser concern for reduced mtb72f vaccine efficacy.
populations of moderate or great concern were those in which alleles that bound relatively few conserved vaccine epitopes were particularly common .
the populations of moderate concern are the ticuna population of brazil; china's shanxi province and maonan, kazak, bai, lahu, naxi, jino, and yai ethnic minorities throughout the country; a population in delhi, india; the philippines; the venda population of south africa; a population in bangkok, thailand; and vietnam.
the populations of great concern are the kaingang and east amazon indigenous populations of brazil; the xinjiang uyghur autonomous region of china as well as yunnan province's drung, va, lisu, naxi, and nu ethnic minorities; and indonesian java.
discussion
despite the striking variation in the protective efficacy of the bcg vaccine for tb observed among different world populations <cit> , the joint impact of host and pathogen variation on novel tb vaccine candidates has never been characterized.
using epitope prediction programs, we investigated the impact of clinical variations in the mtb72f tb vaccine components, the ppe <dig> and pepa proteins, on epitope binding to alleles of the class ii hla drb <dig> gene.
we identified conserved and unconserved promiscuous epitopes in the ppe <dig> and pepa proteins using the clinical variants described previously  <cit>  and determined that while 60% of potential cd4+ t-cell epitopes in the ppe <dig> protein were unconserved, 65% of the actually predicted t-cell epitopes in this protein were unconserved.
we furthermore found several drb <dig> alleles that bound few vaccine epitopes overall and others that bound predominantly unconserved or non-tb epitopes, allowing us to determine individual genotypes as well as broader populations where the mtb72f vaccine may not offer maximum protection.
our finding that 60% of potential cd4+ t-cell epitopes in ppe <dig> are unconserved is consistent with previous research that has found the ppe gene family to be particularly variable among tuberculosis isolates  <cit> .
the significant increase in the proportion of unconserved t-cell epitopes among the actually predicted epitopes compared to the potential epitopes  likely reflects the selective pressure that the immune system places upon the bacterium to alter antigenic protein regions.
based on the clinical protein sequences previously described  <cit> , no potential epitopes in the pepa protein were classified as unconserved.
although to our knowledge no other studies have compared pepa protein conservation to that of other m. tuberculosis proteins, previous findings that pepa is highly conserved among mycobacterium species  <cit>  suggest that pepa may be a relatively well-conserved protein.
the high conservation of the pepa protein even under selective pressure from the immune response may indicate that little variation in this protein is compatible with protein function, and thus pepa may be a particularly good vaccine target  <cit> .
we also examined the conservation of promiscuous epitopes.
epitope promiscuity is commonly evaluated in the generation of epitope-based vaccines  <cit> , but since they are antigenic, promiscuously binding epitopes should be selected against by a large range of host immune systems.
we would therefore expect to find many unconserved promiscuous epitopes.
this was found to be the case: seven of the ten promiscuous epitopes predicted for ppe <dig> were unconserved.
however, all five of the promiscuous pepa epitopes were conserved, and thus these epitopes could be particularly good candidates for epitope-based vaccines.
although the majority of the promiscuous ppe <dig> epitopes identified were found to be unconserved, it is possible that these epitopes might nevertheless provide protection against the bacterium if they are found in other m. tuberculosis proteins.
we therefore investigated whether these epitopes were found in two proteins  that are closely related to ppe <dig>  this analysis revealed that only three of the promiscuous epitopes were present in all three proteins.
the remaining seven were found only in ppe <dig>
our finding that three of the twelve drb <dig> alleles selected, drb1* <dig>  drb1* <dig>  and drb1* <dig>  were predicted to bind few total and conserved epitopes in the mtb72f vaccine suggests that the vaccine might not be maximally protective in people who have one or more of these alleles.
by contrast, drb1* <dig> was consistently predicted to bind far more total  and conserved  vaccine epitopes than any other drb <dig> allele , suggesting that individuals with this allele might be particularly well protected by the vaccine.
drb1* <dig> is a common allele in the us and belgium, where phase i clinical trials for the mtb72f vaccine were conducted, but unfortunately is rarer in brazil, china, indonesia, and many other countries where tb is endemic according to the allele*frequencies in worldwide populations database  <cit> .
therefore, while the vaccine may be effective in the us and belgium, it may not be equally effective in other regions.
our analysis of all the drb <dig> alleles in the allele*frequencies in worldwide populations database  <cit>  that were found to be one of the top three most common alleles in any population in any of the twenty-two countries designated as tb high-burden countries by the who  <cit>  generated a list of the alleles of concern in high-burden tb countries.
these alleles include drb1* <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  although the epitope predictions for these alleles should be treated with more caution, as predictions for most could be generated by only one or two prediction programs, the importance of these alleles merits their consideration.
while the number of epitopes capable of binding to a particular mhc allele does not necessarily indicate how strong the immune response will be in that mhc background  <cit> , the fact that many of these alleles bind very few conserved epitopes is of concern for two reasons.
first, the analysis conducted here includes only predictions of epitope binding to the mhc molecule and not the cellular processing that must take place before mhc presentation occurs.
because of this processing, many potential antigenic peptides will not be generated in vivo.
for mhc alleles to which few epitopes are predicted to bind, it is possible that none of the potentially binding epitopes will be generated in vivo and thus that no epitopes in the vaccine will be presented on drb <dig> proteins.
furthermore, for some drb <dig> alleles the number of conserved epitopes predicted to bind is much smaller than the number of unconserved epitopes predicted to bind.
in the immune response to a vaccine or pathogen, a single or small number of epitopes is usually immunodominant  <cit>  even though many epitopes could potentially bind to the mhc alleles in question.
for mhc alleles that are predicted to bind many fewer conserved than unconserved epitopes, it is likely that the immune response to the vaccine would be dominated by responses to unconserved epitopes and therefore would not be optimally protective against all m. tuberculosis strains.
most  of the alleles of concern were predicted to bind at least as many unconserved as conserved epitopes, and one was predicted to bind no conserved or unconserved epitopes at all.
only five of the  <dig> alleles predicted to bind more than four conserved epitopes were predicted to bind at least as many unconserved as conserved epitopes.
individuals with these alleles - drb1* <dig>   <dig>   <dig>   <dig>  and  <dig> - might also be less efficiently protected by the mtb72f vaccine.
because mtb72f is a polyprotein containing several non-tb potential epitopes at the junction of the ppe <dig> and pepa proteins and at the n-terminus of the polyprotein, we investigated whether epitopes present in the mtb72f vaccine but not in the native ppe <dig> and pepa proteins might misdirect the immune response upon immunization.
fortunately, most of the alleles of concern noted above did not bind any of the non-tb epitopes in the vaccine, but drb1* <dig>   <dig>   <dig>  and  <dig> were each predicted to bind to a median of one non-tb epitope.
this finding is of particular concern for drb1* <dig>  a common allele in south africa, because it was not predicted to bind any of the protective epitopes  in the vaccine.
the drb1-mediated immune response to mtb72f in people with this allele might thus be misdirected against epitopes that are found in the vaccine but not in tb and thus this portion of the immune response would not be protective.
as south africa is one of the sites for the phase ii clinical trials of the mtb72f vaccine, it would be beneficial to collect immunological data from vaccine recipients in order to determine whether the vaccine is effective in persons with the drb1* <dig> allele.
while this analysis should be useful for assessing population coverage of the mtb72f vaccine, it is important to recognize the limitations inherent in the use of epitope prediction programs.
no epitope prediction program is perfectly accurate, and there is substantial disagreement among the epitopes predicted by each program  <cit> .
we increased the accuracy of our analysis by using multiple prediction programs for each drb <dig> allele when possible, but there are nevertheless likely to be differences between the predicted and actual epitopes for many alleles.
however, as we obtained good agreement among programs as far as which alleles were predicted to bind relatively many or few vaccine epitopes, the lists of alleles and populations of concern would not likely be substantially changed by improvements in the prediction programs or experimental confirmation.
although we compared epitope predictions across eight different programs, these programs are not perfectly comparable.
several of the programs, such as vaxign, provided information on peptide affinity predictions only for epitopes predicted to bind each allele.
as we could not obtain affinity information for peptides predicted not to bind, we were unable to evaluate the binding threshold to which the prediction program was automatically set.
furthermore, the binding cutoffs used by several of the programs likely differ from the ic <dig> ≤  <dig> nm cutoff that we imposed on programs generating ic <dig> predictions.
however, as the number of epitopes predicted by programs with non-ic50-based cutoffs generally though not always fell within the range of programs that did use the ic <dig> cutoff, it is unlikely that the differing thresholds among programs severely skewed the results.
finally, it should be noted that the hla allele frequencies in different populations  <cit>  that were utilized to generate the list of alleles of concern were in some cases generated through only a few small studies.
it is likely that further study of hla genotypes would alter our predictions of which populations might be more or less effectively protected by the mtb72f vaccine.
CONCLUSIONS
we conclude that the mtb72f vaccine may be less protective in certain populations that suffer from a high burden of tb and thus that additional protective measures may be needed in these populations in addition to this promising vaccine candidate.
while the findings from this in silico analysis should be verified with immunological studies, this analysis complements in vitro and in vivo experimentation by vastly expanding the range of host and pathogen factors that can be examined and incorporating an analysis of many more genotypes than a laboratory or even clinical study could include.
furthermore, this type of analysis can be used to aid rational selection of populations in which to conduct clinical trials for vaccines.
this method of vaccine evaluation could also be usefully extended to other subunit vaccine candidates for tb, as well as vaccine candidates for hiv and other diseases, and might provide a means of comparing different vaccines to select the best vaccine to use in a given population.
however, continued improvements to epitope prediction software, refinement of programs to predict pre-presentation epitope processing, and further study of the hla genotypes of world populations could help to increase the accuracy and utility of such vaccine evaluations.

 although the bacille calmette guérin vaccine for tuberculosis is the most widely used vaccine worldwide, tb continues to be a tremendous public health problem. a third of the world's population is estimated to be infected with mycobacterium tuberculosis and 2- million people die of the disease each year. key among the reasons for the unabated spread of tb is the inability of the bcg vaccine to provide adequate protection against pulmonary tb in adults, the most contagious form of tb. developing an improved vaccine for tb, whether a replacement for bcg, a booster to the existing vaccine, or a vaccine specifically directed against latent tb, is of crucial importance in the battle to defeat the disease. while several tb vaccine candidates have demonstrated protective efficacy in animal models and have proceeded to clinical trials in humans, even a successful clinical trial cannot guarantee that a vaccine can protect all members of the diverse worldwide human population against all variants of m. tuberculosis. one promising vaccine candidate is the mtb72f subunit vaccine, a polyprotein composed of the m. tuberculosis proteins pepa and ppe. the ppe antigen has been demonstrated to contain at least epitopes and the vaccine has been shown to provide protection against tb in cynomolgus monkeys; it is currently under clinical trials in humans. the peptide sequence of mtb72f, like that of many vaccines, is based on a laboratory strain of the pathogen whose antigens may differ from those found in variable clinical strains in immunologically critical ways. furthermore, the diversity of the human leukocyte antigen and other immune genes results in variable vaccine efficacy in different individuals even in the absence of pathogen variation. while clinical trials include individuals of many hla genotypes, genotype frequencies vary dramatically in different regions; genotypes that are common in tb-endemic regions may be underrepresented in clinical trial populations. it is therefore necessary to incorporate information on host and pathogen genetic diversity in vaccine design, development, and testing. the ppe protein family of m. tuberculosis is a large, 69-member protein family with a currently unknown function. previous work by hebert and colleagues demonstrated that the ppe protein of mtb72f, but not the pepa protein, had significant variability in a sample of clinical m. tuberculosis isolates. such variation, however, would be important only if the variation were in regions of the protein that were vital for the human immune response to m. tuberculosis. to determine whether variation in these proteins might impact the efficacy of mtb72f, we must consider interactions between the m. tuberculosis proteins and the human immune system. of the many genes involved in the immune response, the major histocompatibility complex genes - hla in humans - are among the most crucial and yet most variable. mhc proteins present foreign peptide epitopes from intracellular and extracellular pathogens to cd8+ cytotoxic or cd4+ helper t cells to initiate the immune response. the mtb72f vaccine was found to stimulate both cd4+ and cd8+ t cell-mediated responses in a mouse model, and the cd4+ t cell response in particular is thought to be essential for preventing m. tuberculosis infection. there are thousands of hla alleles, however, and variation in these alleles can significantly impact individual responses to vaccination. recently, several algorithms to predict the affinity of peptide sequences for various class i and class ii mhc alleles have been developed and several have been extensively validated. a consensus approach incorporating three or more programs has been shown to increase the accuracy of mhc class ii epitope predictions. to investigate the impact of host and pathogen variation on tb vaccine efficacy, we have used previously reported protein sequences from clinical isolates of m. tuberculosis and in silico hla epitope prediction programs to assess the protection offered by the mtb72f subunit vaccine against diverse strains of m. tuberculosis in human populations suffering from a high burden of the disease. this investigation revealed that due to variation in both m. tuberculosis protein sequences and epitope-binding capabilities of different hla alleles, certain human populations with a high burden of tuberculosis may not be optimally protected by the mtb72f vaccine.  comparison of epitopes between the vaccine and clinical strains to determine the impact of clinical strain variation on the protection offered by the mtb72f vaccine, we examined the conservation of each nonamer sequence predicted to be a binding epitope compared to the conservation of every nonamer sequence in the vaccine. eight epitope prediction programs were used to determine epitopes predicted to bind. there was substantial variation in the number of nonamer sequences predicted to act as epitopes for each drb allele by each prediction program. however, when a list of all ppe nonamer cores predicted to bind at least one drb allele was compiled using the epitope predictions for the five prediction programs that covered the greatest number of drb alleles, 65% of these nonamers were unconserved. this percentage was significantly greater than the 60% of total ppe nonamer sequences that were unconserved. we also considered individual prediction programs' epitope binding predictions for single drb alleles. for out of individual drb allele/prediction program combinations, more than 60% of the epitopes predicted to bind were unconserved; for of these, 80-100% were unconserved. no nonamer sequences in the pepa protein were classified as unconserved epitopes. citation numbers are from google scholar prediction of promiscuous epitopes as promiscuous epitopes are frequently sought for epitope-based vaccines, we examined such epitopes, defining them as epitopes predicted to bind at least four of the twelve drb alleles studied and classifying them as conserved or unconserved. in all, fifteen promiscuous epitopes were predicted, ten from the ppe protein and five from pepa. eight of these epitopes, including all of the pepa epitopes, were classified as conserved and the other seven as unconserved. of note, the three ppe epitopes predicted to bind the largest numbers of epitopes were all classified as unconserved. prediction of drb allele binding to find drb alleles that might be especially well or poorly covered by the mtb72f vaccine, we summed the ppe and pepa epitope predictions to find the number of total and conserved vaccine epitopes predicted to bind each drb allele. drb1* was consistently predicted to bind an extremely large number of epitopes; a median of total epitopes and conserved epitopes were predicted to bind. by contrast, drb alleles and were each predicted to bind a very small number of total or conserved vaccine epitopes. drb1* was predicted to bind a median of total vaccine epitopes and four conserved vaccine epitopes while drb1* was predicted to bind a median of seven total vaccine epitopes and three conserved vaccine epitopes. finally, drb1* was predicted to bind a median of eight total vaccine epitopes and five conserved vaccine epitopes. similar trends were observed when total ppe epitopes, conserved ppe epitopes, and pepa epitopes predicted to bind each allele were evaluated separately. non-tb antigenic regions because the mtb72f vaccine is a polyprotein composed of a single continuous amino acid chain incorporating the full ppe protein, the pepa protein separated into two pieces, an n-terminal his tag, and short amino acid insertions between the protein segments, several potential mhc class ii epitopes are found in the vaccine but not in the native m. tuberculosis proteins. if these epitopes bound to drb proteins, they could misdirect t cells to respond to non-tb epitopes rather than to epitopes found in the pathogen. non-pathogen epitopes make up a relatively high proportion of the epitopes in the mtb72f vaccine compared to other vaccines, such as killed or live attenuated vaccines that do not contain artificial epitopes, and thus these epitopes might misdirect the immune response to mtb72f in a manner not seen with other vaccines. we therefore evaluated the drb binding of each potential mhc class ii epitope core found in the vaccine but not in the individual proteins. the maximum median number of non-tb vaccine epitopes predicted to bind to any of the drb alleles was two. of the three drb epitopes predicted to bind the smallest number of total or conserved vaccine epitopes, two were predicted to bind none of the non-tb epitopes and the third, drb1* was predicted to bind a median of one non-tb epitope. alleles of concern in regions with a high burden of tb hla drb alleles of concern were defined as drb alleles predicted to bind four or fewer conserved epitopes in the mtb72f vaccine. based on these criteria, we found that the alleles of concern were drb1* and. of these alleles, only four were predicted to bind as many or more conserved as unconserved epitopes, while the remaining nine were predicted to bind fewer conserved than unconserved epitopes or to bind no epitopes at all. five additional alleles that were not predicted to bind particularly few conserved epitopes nevertheless bound at least as many unconserved as conserved epitopes. we further examined whether any of these alleles were predicted to bind the non-tb epitopes in the vaccine. although most of the alleles were not predicted to bind any of these epitopes, drb1* and were each predicted to bind to one non-tb epitope. 1alleles of particular concern are highlighted with italics. 2n = number of programs able to predict epitope binding to that allele 3nk = none known 4of the study populations included in the allele*frequencies in worldwide populations database populations of concern for reduced vaccine efficacy based on the alleles of concern determined through epitope binding predictions, we characterized each population in the allele*frequencies database from a tb-endemic country as being of great, moderate, or lesser concern for reduced mtb72f vaccine efficacy. populations of moderate or great concern were those in which alleles that bound relatively few conserved vaccine epitopes were particularly common. the populations of moderate concern are the ticuna population of brazil; china's shanxi province and maonan, kazak, bai, lahu, naxi, jino, and yai ethnic minorities throughout the country; a population in delhi, india; the philippines; the venda population of south africa; a population in bangkok, thailand; and vietnam. the populations of great concern are the kaingang and east amazon indigenous populations of brazil; the xinjiang uyghur autonomous region of china as well as yunnan province's drung, va, lisu, naxi, and nu ethnic minorities; and indonesian java. discussion despite the striking variation in the protective efficacy of the bcg vaccine for tb observed among different world populations, the joint impact of host and pathogen variation on novel tb vaccine candidates has never been characterized. using epitope prediction programs, we investigated the impact of clinical variations in the mtb72f tb vaccine components, the ppe and pepa proteins, on epitope binding to alleles of the class ii hla drb gene. we identified conserved and unconserved promiscuous epitopes in the ppe and pepa proteins using the clinical variants described previously and determined that while 60% of potential cd4+ t-cell epitopes in the ppe protein were unconserved, 65% of the actually predicted t-cell epitopes in this protein were unconserved. we furthermore found several drb alleles that bound few vaccine epitopes overall and others that bound predominantly unconserved or non-tb epitopes, allowing us to determine individual genotypes as well as broader populations where the mtb72f vaccine may not offer maximum protection. our finding that 60% of potential cd4+ t-cell epitopes in ppe are unconserved is consistent with previous research that has found the ppe gene family to be particularly variable among tuberculosis isolates. the significant increase in the proportion of unconserved t-cell epitopes among the actually predicted epitopes compared to the potential epitopes likely reflects the selective pressure that the immune system places upon the bacterium to alter antigenic protein regions. based on the clinical protein sequences previously described, no potential epitopes in the pepa protein were classified as unconserved. although to our knowledge no other studies have compared pepa protein conservation to that of other m. tuberculosis proteins, previous findings that pepa is highly conserved among mycobacterium species suggest that pepa may be a relatively well-conserved protein. the high conservation of the pepa protein even under selective pressure from the immune response may indicate that little variation in this protein is compatible with protein function, and thus pepa may be a particularly good vaccine target. we also examined the conservation of promiscuous epitopes. epitope promiscuity is commonly evaluated in the generation of epitope-based vaccines, but since they are antigenic, promiscuously binding epitopes should be selected against by a large range of host immune systems. we would therefore expect to find many unconserved promiscuous epitopes. this was found to be the case: seven of the ten promiscuous epitopes predicted for ppe were unconserved. however, all five of the promiscuous pepa epitopes were conserved, and thus these epitopes could be particularly good candidates for epitope-based vaccines. although the majority of the promiscuous ppe epitopes identified were found to be unconserved, it is possible that these epitopes might nevertheless provide protection against the bacterium if they are found in other m. tuberculosis proteins. we therefore investigated whether these epitopes were found in two proteins that are closely related to ppe this analysis revealed that only three of the promiscuous epitopes were present in all three proteins. the remaining seven were found only in ppe our finding that three of the twelve drb alleles selected, drb1* drb1* and drb1* were predicted to bind few total and conserved epitopes in the mtb72f vaccine suggests that the vaccine might not be maximally protective in people who have one or more of these alleles. by contrast, drb1* was consistently predicted to bind far more total and conserved vaccine epitopes than any other drb allele, suggesting that individuals with this allele might be particularly well protected by the vaccine. drb1* is a common allele in the us and belgium, where phase i clinical trials for the mtb72f vaccine were conducted, but unfortunately is rarer in brazil, china, indonesia, and many other countries where tb is endemic according to the allele*frequencies in worldwide populations database. therefore, while the vaccine may be effective in the us and belgium, it may not be equally effective in other regions. our analysis of all the drb alleles in the allele*frequencies in worldwide populations database that were found to be one of the top three most common alleles in any population in any of the twenty-two countries designated as tb high-burden countries by the who generated a list of the alleles of concern in high-burden tb countries. these alleles include drb1* and although the epitope predictions for these alleles should be treated with more caution, as predictions for most could be generated by only one or two prediction programs, the importance of these alleles merits their consideration. while the number of epitopes capable of binding to a particular mhc allele does not necessarily indicate how strong the immune response will be in that mhc background, the fact that many of these alleles bind very few conserved epitopes is of concern for two reasons. first, the analysis conducted here includes only predictions of epitope binding to the mhc molecule and not the cellular processing that must take place before mhc presentation occurs. because of this processing, many potential antigenic peptides will not be generated in vivo. for mhc alleles to which few epitopes are predicted to bind, it is possible that none of the potentially binding epitopes will be generated in vivo and thus that no epitopes in the vaccine will be presented on drb proteins. furthermore, for some drb alleles the number of conserved epitopes predicted to bind is much smaller than the number of unconserved epitopes predicted to bind. in the immune response to a vaccine or pathogen, a single or small number of epitopes is usually immunodominant even though many epitopes could potentially bind to the mhc alleles in question. for mhc alleles that are predicted to bind many fewer conserved than unconserved epitopes, it is likely that the immune response to the vaccine would be dominated by responses to unconserved epitopes and therefore would not be optimally protective against all m. tuberculosis strains. most of the alleles of concern were predicted to bind at least as many unconserved as conserved epitopes, and one was predicted to bind no conserved or unconserved epitopes at all. only five of the alleles predicted to bind more than four conserved epitopes were predicted to bind at least as many unconserved as conserved epitopes. individuals with these alleles - drb1* and - might also be less efficiently protected by the mtb72f vaccine. because mtb72f is a polyprotein containing several non-tb potential epitopes at the junction of the ppe and pepa proteins and at the n-terminus of the polyprotein, we investigated whether epitopes present in the mtb72f vaccine but not in the native ppe and pepa proteins might misdirect the immune response upon immunization. fortunately, most of the alleles of concern noted above did not bind any of the non-tb epitopes in the vaccine, but drb1* and were each predicted to bind to a median of one non-tb epitope. this finding is of particular concern for drb1* a common allele in south africa, because it was not predicted to bind any of the protective epitopes in the vaccine. the drb1-mediated immune response to mtb72f in people with this allele might thus be misdirected against epitopes that are found in the vaccine but not in tb and thus this portion of the immune response would not be protective. as south africa is one of the sites for the phase ii clinical trials of the mtb72f vaccine, it would be beneficial to collect immunological data from vaccine recipients in order to determine whether the vaccine is effective in persons with the drb1* allele. while this analysis should be useful for assessing population coverage of the mtb72f vaccine, it is important to recognize the limitations inherent in the use of epitope prediction programs. no epitope prediction program is perfectly accurate, and there is substantial disagreement among the epitopes predicted by each program. we increased the accuracy of our analysis by using multiple prediction programs for each drb allele when possible, but there are nevertheless likely to be differences between the predicted and actual epitopes for many alleles. however, as we obtained good agreement among programs as far as which alleles were predicted to bind relatively many or few vaccine epitopes, the lists of alleles and populations of concern would not likely be substantially changed by improvements in the prediction programs or experimental confirmation. although we compared epitope predictions across eight different programs, these programs are not perfectly comparable. several of the programs, such as vaxign, provided information on peptide affinity predictions only for epitopes predicted to bind each allele. as we could not obtain affinity information for peptides predicted not to bind, we were unable to evaluate the binding threshold to which the prediction program was automatically set. furthermore, the binding cutoffs used by several of the programs likely differ from the ic ≤ nm cutoff that we imposed on programs generating ic predictions. however, as the number of epitopes predicted by programs with non-ic50-based cutoffs generally though not always fell within the range of programs that did use the ic cutoff, it is unlikely that the differing thresholds among programs severely skewed the results. finally, it should be noted that the hla allele frequencies in different populations that were utilized to generate the list of alleles of concern were in some cases generated through only a few small studies. it is likely that further study of hla genotypes would alter our predictions of which populations might be more or less effectively protected by the mtb72f vaccine.  we conclude that the mtb72f vaccine may be less protective in certain populations that suffer from a high burden of tb and thus that additional protective measures may be needed in these populations in addition to this promising vaccine candidate. while the findings from this in silico analysis should be verified with immunological studies, this analysis complements in vitro and in vivo experimentation by vastly expanding the range of host and pathogen factors that can be examined and incorporating an analysis of many more genotypes than a laboratory or even clinical study could include. furthermore, this type of analysis can be used to aid rational selection of populations in which to conduct clinical trials for vaccines. this method of vaccine evaluation could also be usefully extended to other subunit vaccine candidates for tb, as well as vaccine candidates for hiv and other diseases, and might provide a means of comparing different vaccines to select the best vaccine to use in a given population. however, continued improvements to epitope prediction software, refinement of programs to predict pre-presentation epitope processing, and further study of the hla genotypes of world populations could help to increase the accuracy and utility of such vaccine evaluations. 
due to variation in both m. tuberculosis protein sequences and epitope-binding capabilities of different hla alleles, certain human populations with a high burden of tuberculosis may not be optimally protected by the mtb72f vaccine. we evaluated the binding of epitopes in the vaccine to different alleles of the human drb class ii mhc protein using freely available epitope prediction programs and compared protein sequences from clinical isolates to the sequences included in the mtb72f vaccine. this analysis predicted that the mtb72f vaccine would be less effective for several drb genotypes, due either to limited vaccine epitope binding to the drb proteins or to binding primarily by unconserved ppe epitopes. although the mtb72f vaccine candidate has shown promise in animal and clinical trials thus far, it may not be optimally effective in some genotypic backgrounds. the efficacy of the mtb72f vaccine should be further examined in these particular populations to determine whether additional protective measures might be necessary for these regions. previous work by hebert and colleagues demonstrated that the ppe protein of mtb72f had significant variability in a sample of clinical m. tuberculosis isolates. the present study assesses mtb72f's predicted efficacy in people with different drb genotypes to predict whether the vaccine will protect against diverse clinical strains of m. tuberculosis in a diverse host population. the mtb72f subunit vaccine for tuberculosis, currently in clinical trials, is hoped to provide improved protection compared to the current bcg vaccine. it is not clear, however, whether mtb72f would be equally protective in the different human populations suffering from a high burden of tuberculosis. furthermore, we found that these less-protective drb alleles are found at a very high frequency in several populations with a high burden of tuberculosis.
 the mtb72f subunit vaccine for tuberculosis, currently in clinical trials, is hoped to provide improved protection compared to the current bcg vaccine. it is not clear, however, whether mtb72f would be equally protective in the different human populations suffering from a high burden of tuberculosis. previous work by hebert and colleagues demonstrated that the ppe protein of mtb72f had significant variability in a sample of clinical m. tuberculosis isolates. however, whether this variation might impact the efficacy of mtb72f in the context of the microbial and host immune system interactions remained to be determined. the present study assesses mtb72f's predicted efficacy in people with different drb genotypes to predict whether the vaccine will protect against diverse clinical strains of m. tuberculosis in a diverse host population.  we evaluated the binding of epitopes in the vaccine to different alleles of the human drb class ii mhc protein using freely available epitope prediction programs and compared protein sequences from clinical isolates to the sequences included in the mtb72f vaccine. this analysis predicted that the mtb72f vaccine would be less effective for several drb genotypes, due either to limited vaccine epitope binding to the drb proteins or to binding primarily by unconserved ppe epitopes. furthermore, we found that these less-protective drb alleles are found at a very high frequency in several populations with a high burden of tuberculosis.  although the mtb72f vaccine candidate has shown promise in animal and clinical trials thus far, it may not be optimally effective in some genotypic backgrounds. due to variation in both m. tuberculosis protein sequences and epitope-binding capabilities of different hla alleles, certain human populations with a high burden of tuberculosis may not be optimally protected by the mtb72f vaccine. the efficacy of the mtb72f vaccine should be further examined in these particular populations to determine whether additional protective measures might be necessary for these regions. 