S very low.Molecular Characterization of HIV-1 and Viral Loads (Table 1)Among 11 HIV-1 isolates, 8 were subtype B and 3 were non-B (CRF02_AG, CRF11_cpx and C). All RNA viral loads (VL) were below the threshold of 50 copies/mL. The proviral DNA load ranged from 139 to 3854 DNA copies/million of PBMC. In all samples, the amount of 2-LTR episomic DNA was below the threshold of 10 copies/million PBMC. The Gag sequence of one strain (K) exhibited 5 stop codons. In all cases, tryptophan amino acid was replaced by a stop codon as a result of a switch from TGG (wild) to TAG or TAA.Potential CTL Reactivity against Archived Viral Epitopes According to HLA I AllelesCross-reactivity to Lipo5 lipopeptides. (Table 2) The ANRS HIV-Lipo5 lipopeptides are composed of the Gag 17?5, Gag 253?84, Nef 66?7, Nef 116?45, and Pol 325?55 peptides. On the basis of the different HLA I alleles and the different available sequences of Gag, Nef and Pol obtained 11967625 byDrug Resistance Mutations (DRMs) Analyzed by Ultradeep Pyrosequencing (UDPS) (Table 1)With regard to the frequencies of mutant variants above 1 , 3 proviruses exhibited M184I/V mutations between 4 and 99.6 ,Table 1. Antiretroviral treatment and duration, HIV-1 proviral load, viral subtype, resistance mutations in RT, immunogenetics of patients.PatientsTreatment FTC TDF LPV/rDuration of treatment 4 years JProviral load 817 NA 3854 177 NA 480 2334 NASubtype BRT mutations (UDPS) D67N 0.45 ; K70R 5.90 ; L101I 1.70 NoneHLA A*02:06, *03:01; B*44:02, *51:01 A*02:01, *26:01; B*39:01, *40:A BFTC TDF DRV/r RAL3 years 7 years JB BD67N 0.25 ; M184I 23 NA none 23148522 A*01:01, *02:01; B*08:01, *57:01 A*02:01, 24:02; B*27:05, *51:C D3TC TDF LPV/rFTC TDF ATV/r3 years 8 months JB CRF11_cpxD67N 0.74 ; K219Q 0.20 ; G190E 2.30 NA None A*23:01, *68:02; B*07:02, *81:01 A*01:01, *02:01; B*07:02, *51:E FFTC TDF RPVFTC TDF FPV/r6 yearsBD67N 0.58 ; D67E 0.42 ; M184I 99.60 ; E138G 0.87 ; K219R 20 ; G190E 12 D67N 0.58 ; L101I 0.70 ; K103R 0.60 E138G 0.40 ; K219Q 0,03 M184V 4 ; E138G 23 ; M230L 20 None (NA from aa 219) None A*03:01, *23:01; B*07:02, *35:01 A*02:01; B*40:01, *44:02 A*26:01, *30:02; B*13:03, *18:01 A*23:01, *24:02; B*41:02, *53:01 A*24:02, *33:01; B*14:02, *55:G H I J K3TC d4T LPV/r 3TC TDF LPV/r AZT 3TC LPV/r (NVP) FTC TDF LPV/r FTC TDF EFV9 years 9 years 9 years 5 years 6 years572 458 1490 139B B CRF02_AG C BProviral load expressed as copies/106 PBMC; NA: not available. doi:10.1371/journal.pone.0069029.tToward a New Concept of HIV VaccineFigure 1. Phylogenetic trees of UDPS sequences (Pol RT2) at baseline and at ART success. Title Loaded From File Patients D, B and F according to Table 1. doi:10.1371/journal.pone.0069029.gSanger sequencing, it was not possible to Title Loaded From File obtain exhaustive data although some examples can be given. A. The virus was identified as subtype B HIV-1. With an HLA A*03:01 allele, the patient should recognize the Pol 325?55 epitope (AIFQSSMTK), which is one of the Lipo5 peptides designated from the HXB2 HIV-1 subtype B reference. The archived epitope in the provirus exhibited a substitution (AIFQASMTK) but the presentation with the allele was still excellent (MHC IC50 20.24 versus 12.04). C. The virus was subtype B. With HLA B*08:01, 2 Gag epitopes can be presented according to the HXB2 reference; EIYKRWII with an MHC of 257.38 and GGKKKYKLK with an MHC of 31,060.99 (low affinity). The archived epitopes were identical. E This patient was infected with a CRF11_cpx. With HLA allele B*07:02, 5 epitopes of Nef 66?7 should be recog.S very low.Molecular Characterization of HIV-1 and Viral Loads (Table 1)Among 11 HIV-1 isolates, 8 were subtype B and 3 were non-B (CRF02_AG, CRF11_cpx and C). All RNA viral loads (VL) were below the threshold of 50 copies/mL. The proviral DNA load ranged from 139 to 3854 DNA copies/million of PBMC. In all samples, the amount of 2-LTR episomic DNA was below the threshold of 10 copies/million PBMC. The Gag sequence of one strain (K) exhibited 5 stop codons. In all cases, tryptophan amino acid was replaced by a stop codon as a result of a switch from TGG (wild) to TAG or TAA.Potential CTL Reactivity against Archived Viral Epitopes According to HLA I AllelesCross-reactivity to Lipo5 lipopeptides. (Table 2) The ANRS HIV-Lipo5 lipopeptides are composed of the Gag 17?5, Gag 253?84, Nef 66?7, Nef 116?45, and Pol 325?55 peptides. On the basis of the different HLA I alleles and the different available sequences of Gag, Nef and Pol obtained 11967625 byDrug Resistance Mutations (DRMs) Analyzed by Ultradeep Pyrosequencing (UDPS) (Table 1)With regard to the frequencies of mutant variants above 1 , 3 proviruses exhibited M184I/V mutations between 4 and 99.6 ,Table 1. Antiretroviral treatment and duration, HIV-1 proviral load, viral subtype, resistance mutations in RT, immunogenetics of patients.PatientsTreatment FTC TDF LPV/rDuration of treatment 4 years JProviral load 817 NA 3854 177 NA 480 2334 NASubtype BRT mutations (UDPS) D67N 0.45 ; K70R 5.90 ; L101I 1.70 NoneHLA A*02:06, *03:01; B*44:02, *51:01 A*02:01, *26:01; B*39:01, *40:A BFTC TDF DRV/r RAL3 years 7 years JB BD67N 0.25 ; M184I 23 NA none 23148522 A*01:01, *02:01; B*08:01, *57:01 A*02:01, 24:02; B*27:05, *51:C D3TC TDF LPV/rFTC TDF ATV/r3 years 8 months JB CRF11_cpxD67N 0.74 ; K219Q 0.20 ; G190E 2.30 NA None A*23:01, *68:02; B*07:02, *81:01 A*01:01, *02:01; B*07:02, *51:E FFTC TDF RPVFTC TDF FPV/r6 yearsBD67N 0.58 ; D67E 0.42 ; M184I 99.60 ; E138G 0.87 ; K219R 20 ; G190E 12 D67N 0.58 ; L101I 0.70 ; K103R 0.60 E138G 0.40 ; K219Q 0,03 M184V 4 ; E138G 23 ; M230L 20 None (NA from aa 219) None A*03:01, *23:01; B*07:02, *35:01 A*02:01; B*40:01, *44:02 A*26:01, *30:02; B*13:03, *18:01 A*23:01, *24:02; B*41:02, *53:01 A*24:02, *33:01; B*14:02, *55:G H I J K3TC d4T LPV/r 3TC TDF LPV/r AZT 3TC LPV/r (NVP) FTC TDF LPV/r FTC TDF EFV9 years 9 years 9 years 5 years 6 years572 458 1490 139B B CRF02_AG C BProviral load expressed as copies/106 PBMC; NA: not available. doi:10.1371/journal.pone.0069029.tToward a New Concept of HIV VaccineFigure 1. Phylogenetic trees of UDPS sequences (Pol RT2) at baseline and at ART success. Patients D, B and F according to Table 1. doi:10.1371/journal.pone.0069029.gSanger sequencing, it was not possible to obtain exhaustive data although some examples can be given. A. The virus was identified as subtype B HIV-1. With an HLA A*03:01 allele, the patient should recognize the Pol 325?55 epitope (AIFQSSMTK), which is one of the Lipo5 peptides designated from the HXB2 HIV-1 subtype B reference. The archived epitope in the provirus exhibited a substitution (AIFQASMTK) but the presentation with the allele was still excellent (MHC IC50 20.24 versus 12.04). C. The virus was subtype B. With HLA B*08:01, 2 Gag epitopes can be presented according to the HXB2 reference; EIYKRWII with an MHC of 257.38 and GGKKKYKLK with an MHC of 31,060.99 (low affinity). The archived epitopes were identical. E This patient was infected with a CRF11_cpx. With HLA allele B*07:02, 5 epitopes of Nef 66?7 should be recog.
