Sequence conservation analysis and in silico human leukocyte antigen-peptide binding predictions for the Mtb72F and M72 tuberculosis candidate vaccine antigens

Table 4 Impact of the alterations introduced for the Mtb72F construction on the peptide binding predictions

Alteration introduced in Mtb72F	Change in the numbers of covered^a alleles for Mtb72F	Explanation
Deletion of the Mtb32A signal sequence.	Loss of 1 covered allele containing 28 predicted epitopes	Epitopes in the Mtb32A signal sequence were predicted for 149 of the 158 alleles assessed. For 148 of the 149 alleles, epitopes were also predicted for the other parts of the protein. Only for 1 allele (DRB1^*16:04), all 28 predicted epitopes were located in the Mtb32A signal sequence, and were thus not predicted for Mtb72F.
Splitting the Mtb32A sequence upstream and downstream of ‘TAAS’ sequence.	No changes in the number of covered alleles.	For each allele with an epitope predicted in this part of the protein there was also an epitope predicted in other parts of the protein. There was an overall loss of 14 predicted epitopes.
Addition of a poly-His tag (MHHHHHH) at the Mtb32A C-terminal end.	No changes in the number of covered alleles.	One epitope (MHHHHHHTAASDNFQ, binding to DRB1^*08:18) was predicted for the Meth-His tag in Mtb72F. There were also other epitopes predicted for this allele.
Addition of 2-amino acid hinge sequences at the junction sites between Mtb32_C and Mtb39A (EF), and between Mtb39A and Mtb32_N (DI).	No changes in the number of covered alleles.	Adding the EF and DI sequences resulted in 13 and 5 additional predicted epitopes, binding to 43 and 31 alleles, respectively. However, the number of alleles with at least one predicted epitope did not change.