The donors recognized four peptides of the 23 20-mer peptides in DENV-1, five peptides of the 35 20-mer peptides of DENV-2, five peptides of the 35 peptides of the DENV-3 and five peptides of the 28 20-mer peptides of DENV-4 (Table 2). All dengue immune donors responded to the peptides of at least two DENV serotypes. Two donors responded to peptides of all four DENV serotypes. The number of healthy donors responding to at least two peptides of the four DENV serotypes in the cultured ELISPOT assays is shown in Table 3. Eight of 20 (40%) of the individuals responded

to at least two peptides of DENV-4 and responses to at least two peptides of other serotypes ranged from 30 to 50% (Table 3). The frequency RAD001 cost of cultured ELISPOT responses to each of these peptides is shown in Fig. 1. These peptides had <15% homology between the four DENV serotypes except for 30% homology for four peptides (DENV-1 peptide with DENV-1 pep-11, DENV-2 pep-33, DENV-4 pep-12, DENV-2 pep-11, DENV-3 pep-11. DENV-2 peptide 17 with DENV-3 pep-21, DENV-3 pep-11 with DENV-4 pep-19). Of the 19 conserved and non-cross-reactive regions identified from the four DENV serotypes, two peptides were from the envelope region,

one peptide from the DENV-2 was from the NS1 region, six peptides were from the NS2A region, two peptides from the NS2B region, one peptide of learn more DENV-1 was from the NS3 region, four peptides were from the NS4A region and three peptides were from the NS5 region (Table 2). Of the six peptides identified which were from the NS2A Tenofovir region, one peptide each was from DENV-2 and DENV-3, two peptides from DENV-4 and two of the peptides were from DENV-1. Three of six of these peptides were from the region represented by amino acids (aa) 99–133, and two of six peptides were from the region represented by

aa 184–216. One peptide from DENV-4 was from the aa 135–148. Variants of all the peptides are shown in supplementary Table S1 and are based on NCBI Virus Variation website data. In the current study we have used the most common sequence, which accounted for >90% of the detected variation in the majority of cases. The three peptides, from aa 99 to 133, were again found to be highly conserved. Of these three peptides, peptide 28 of DENV-3 (RENLLLGVGLAMATTLQLPE), which was the most frequently recognized peptide among all donors (nine of 20), had two changes in the amino acids in only two sequences. In these two variants, threonine in position 14 is replaced by alanine and arginine in position 17 was replaced by methionine. Peptide 10 of DENV-4 (AMTTTLSIPHDLMELIDGIS) had the amino acid leucine in position 6 replaced by isoleucine in some sequences. Although we also used this sequence in our peptide matrix, we did not detect any responses to the sequence with the altered amino acid.