Th strands using an Applied Biosystems 3500xls Dx MC-LR site Genetic Analyzer and primers from the second PCR. The sequences of the study are available in GenBank under accession numbers KC899326 C899346.RT Ultra-deep PyrosequencingRT UDPS was performed using the Roche GS Junior equipment. Amplicons previously obtained, purified and quantitated were pooled at equimolar concentrations. Clonal amplification on beads (EmPCR) was performed using the 454 Life Science reagents that enable bidirectional sequencing, composed of 30 cycles of PCR amplification. DNA-containing beads were recovered and UDPS was performed on the GS Junior sequencer (454 Life Sciences; Roche). UDPS generated a median of 11.000 68181-17-9 site sequence reads per sample. These reads were analyzed using the Amplicon Variant Analyzer software, 454, Roche. The UDPS results of the study are available in GenBank under accession number SRA073324.Toward a New Concept of HIV VaccineTable 4. Primers used for Gag, Nef and Pol amplification.sequence 59-39 GAG First PCR 1 amplicon Primer 59 Primer 39 First PCR 2nd amplicon Primer 59 Primer 39 Second PCR 1st amplicon Primer 59 Primer 39 Second PCR 2nd amplicon Primer 59 Primer 39 Second PCR 3rd amplicon Primer 59 Primer 39 Second PCR 4th amplicon Primer 59 Primer 39 NEF First PCR Primer 59 Primer 39 Second PCR Primer 59 Primer 39 POL First PCR Primer 59 Primer 39 Second PCR 1st amplicon Primer 59 Primer 39 Second PCR 2nd amplicon Primer 59 Primer 39 Second PCR 3rd amplicon Primer 59 18204824 Primer 39 doi:10.1371/journal.pone.0069029.t004 CTRGATGTGGGTGATGCA CNYTATAGGCTGTACTGTCC GAAAATCCATACAATACTCCAGTATTTGC CTATGCTGCCCTATTTCTAAGTCAGAT TTGGTTGCACTTTAAATTTTCCCATTAGCCCTATT CTTTCCATCCCTGTGGAAGCACATT AGTAGGACCTACACCTGTCA CTGTTAGTGCTTTGGTTCCTCT CCTAGAAGAATAAGACAGGGCTTGGAAAG ACGCCTCCCTGGAAAGTCCCCAGCGG GCCACAGCCATAGCAGTAGCTGAGGGG CCAGTACAGGCAAAAAGCAGCTGCTTATA TCAGAGCAGACCAGAGCCAACAGCCCCA AATGCTTTTATTTTTTCTTCTGTCAATGGC ATAATCCACCTATCCCAGTAGGAGAAATT AGGGGTCGTTGCCAAAGA CACCTAGAACTTTAAATGCATGGGT TTTGGTCCTTGTCTTATGTCCAGA GACTAGCGGAGGCTAGAA GTTCTAGGTGATATGGCCTGATG ATAATCCACCTATCCCAGTAGGAGAAATT ATGCTTTTATTTTTTCTTCTGTCAATGGC GACTAGCGGAGGCTAGAA TTTGGTCCTTGTCTTATGTCCAGAstHXB2 genome position764?81 1635?1544?572 2621?764?81 1219?1232?256 1635?1544?572 2264?2136?163 2621?8673?699 9511?8754?782 9443?2480?499 3399?2530?564 2988?2706?734 3119?2874?891 3284?HLA Class I TypingGenomic DNA was extracted from the frozen white blood cell pellets and quantitated as described above. Intermediate-to-high resolution was performed by reverse Polymerase Chain ReactionSequence Specific Oligonucleotide (PCR-SSO) hybridization using the LuminexH flow beads LabTypeH assay (InGen, Chilly-Mazarin, France) for the A and B loci. Allelic ambiguities were solved with PCR-Sequence Specific Primer (SSP) amplification using Olerup assays (BioNoBis, Montfort L’Amaury, France). The manufacturers’ recommendations were strictly followed. Allele assignment was performed by comparison with the official nomenclature and validated by the WHO committee for HLA system factors.Immune Recognition ToolsThe viral epitopes considered for the study were those from the Los Alamos database. Recognition between the HLA groove and the peptides or their variants was predicted using the immune epitope database (www.immuneepitope.org). We evaluated the affinity of the epitopes for the MHC molecules with the MHC IC50 (nM) value. Small values are associated with better binders. A value of 500 nM is often used as the threshold between bind.Th strands using an Applied Biosystems 3500xls Dx Genetic Analyzer and primers from the second PCR. The sequences of the study are available in GenBank under accession numbers KC899326 C899346.RT Ultra-deep PyrosequencingRT UDPS was performed using the Roche GS Junior equipment. Amplicons previously obtained, purified and quantitated were pooled at equimolar concentrations. Clonal amplification on beads (EmPCR) was performed using the 454 Life Science reagents that enable bidirectional sequencing, composed of 30 cycles of PCR amplification. DNA-containing beads were recovered and UDPS was performed on the GS Junior sequencer (454 Life Sciences; Roche). UDPS generated a median of 11.000 sequence reads per sample. These reads were analyzed using the Amplicon Variant Analyzer software, 454, Roche. The UDPS results of the study are available in GenBank under accession number SRA073324.Toward a New Concept of HIV VaccineTable 4. Primers used for Gag, Nef and Pol amplification.sequence 59-39 GAG First PCR 1 amplicon Primer 59 Primer 39 First PCR 2nd amplicon Primer 59 Primer 39 Second PCR 1st amplicon Primer 59 Primer 39 Second PCR 2nd amplicon Primer 59 Primer 39 Second PCR 3rd amplicon Primer 59 Primer 39 Second PCR 4th amplicon Primer 59 Primer 39 NEF First PCR Primer 59 Primer 39 Second PCR Primer 59 Primer 39 POL First PCR Primer 59 Primer 39 Second PCR 1st amplicon Primer 59 Primer 39 Second PCR 2nd amplicon Primer 59 Primer 39 Second PCR 3rd amplicon Primer 59 18204824 Primer 39 doi:10.1371/journal.pone.0069029.t004 CTRGATGTGGGTGATGCA CNYTATAGGCTGTACTGTCC GAAAATCCATACAATACTCCAGTATTTGC CTATGCTGCCCTATTTCTAAGTCAGAT TTGGTTGCACTTTAAATTTTCCCATTAGCCCTATT CTTTCCATCCCTGTGGAAGCACATT AGTAGGACCTACACCTGTCA CTGTTAGTGCTTTGGTTCCTCT CCTAGAAGAATAAGACAGGGCTTGGAAAG ACGCCTCCCTGGAAAGTCCCCAGCGG GCCACAGCCATAGCAGTAGCTGAGGGG CCAGTACAGGCAAAAAGCAGCTGCTTATA TCAGAGCAGACCAGAGCCAACAGCCCCA AATGCTTTTATTTTTTCTTCTGTCAATGGC ATAATCCACCTATCCCAGTAGGAGAAATT AGGGGTCGTTGCCAAAGA CACCTAGAACTTTAAATGCATGGGT TTTGGTCCTTGTCTTATGTCCAGA GACTAGCGGAGGCTAGAA GTTCTAGGTGATATGGCCTGATG ATAATCCACCTATCCCAGTAGGAGAAATT ATGCTTTTATTTTTTCTTCTGTCAATGGC GACTAGCGGAGGCTAGAA TTTGGTCCTTGTCTTATGTCCAGAstHXB2 genome position764?81 1635?1544?572 2621?764?81 1219?1232?256 1635?1544?572 2264?2136?163 2621?8673?699 9511?8754?782 9443?2480?499 3399?2530?564 2988?2706?734 3119?2874?891 3284?HLA Class I TypingGenomic DNA was extracted from the frozen white blood cell pellets and quantitated as described above. Intermediate-to-high resolution was performed by reverse Polymerase Chain ReactionSequence Specific Oligonucleotide (PCR-SSO) hybridization using the LuminexH flow beads LabTypeH assay (InGen, Chilly-Mazarin, France) for the A and B loci. Allelic ambiguities were solved with PCR-Sequence Specific Primer (SSP) amplification using Olerup assays (BioNoBis, Montfort L’Amaury, France). The manufacturers’ recommendations were strictly followed. Allele assignment was performed by comparison with the official nomenclature and validated by the WHO committee for HLA system factors.Immune Recognition ToolsThe viral epitopes considered for the study were those from the Los Alamos database. Recognition between the HLA groove and the peptides or their variants was predicted using the immune epitope database (www.immuneepitope.org). We evaluated the affinity of the epitopes for the MHC molecules with the MHC IC50 (nM) value. Small values are associated with better binders. A value of 500 nM is often used as the threshold between bind.
