Immungenetische Untersuchungen bei Hypersensitivität und HIV-Infektion

Transkript

1 INSTITUT FÜR IMMUNOLOGIE UND GENETIK Immungenetische Untersuchungen bei Hypersensitivität und HIV-Infektion 6. Immundiagnostisches Meeting Dresden, Oktober 2008 Moderne Diagnostik und Therapie immunologisch bedingter Erkrankungen Medizinisches Labor Dr. Bernhard Thiele Hellmut-Hartert-Str. 1 D Kaiserslautern

2 antiretrovirale Therapie (ART)Ä NRTI/NtRTI NNRTI PI Entryinh. AZT (RetrovirÄ) DDI (VidexÄ) D4T (ZeritÄ) ABC (ZiagenÄ) 3TC (EpivirÄ) TDF (VireadÄ) FTC (EmtrivaÄ) DDC (HividÄ) EFV (SustivaÄ) NVP (ViramuneÄ)Ä ETV (Intelence)Ä APV/FPV (Agen.Ä;TelzirÄ) IDV (CrixivanÄ) NFV (ViraceptÄ) SQV (InviraseÄ) LPV (KaletraÄ) ATV (ReyatazÄ) TPV (AptivusÄ) DRV (PrezistaÄ) ENF (FuzeonÄ) MVC (CelsentriÄ)Ä Integraseinh. RAL (InsentressÄ) EVG (GS-9137) RTV (NorvirÄ) slide 2

3 Abacavir Hypersensitivitäts Syndrom slide 3

4 slide 4

5 57.1 AH Markers are Over Represented in the Abacavir Hypersensitive DR7/DQ3 C4A6 B57 DR7/DQ3 C4A6 B57 DR7/DQ3 C4A6 B57 DR7/DQ3 C4A6 B57 DR7/DQ3 C4A6 B57 HIV Negative HIV Positive HIV (+) ABC HIV (+) ABC HIV (+) ABC Controls Controls Controls Hypersensitive Tolerant n = 3212 n = 381 n = 200 n = 18 n = 167 slide 5 Mallal, et al., Lancet :

6 HLA-Loci HLA-A: 489 (390), HLA-B: 830(711), HLA-C: 266(210), HLA-DRB: 545(451) Allele(Proteine)Ä slide 6

7 Interaktion von ABC und HSP 70 Blockade von HSP-70 durch Geldanamycin inhibiert zahlreiche Zytokine Hypothese (Miles)Ä Hsp70 -Polymorphismus ermöglicht die Bindung von ABC oder Metaboliten Die ABC-Bindung setzt ihrerseits gebundene Peptide frei Folge: cytokine storm slide 7

8 Predict-Studie Kontrollarm Start ABC Therapie Auswertbar für klinisch vermutete HSR ITT(EV1) n=847 randomisiert ITT, n=976 ITT(E), n=913 Auswertbar für immunologisch bestätigte HSR ITT(EV2) n=842 ITT, n=1956 Screeningarm ITT, n=980 Start ABC Therapie ITT(E), n=859 Auswertbar für klinisch vermutete HSR ITT(EV1) n=803 Auswertbar für immunologisch bestätigte HSR ITT(EV2) n=802 Ausschluss HLA-B*5701 pos. n=54 Ausschluss HLA-B*5701 pos. n=1 ITT(EV1): Randomisierte Patienten, die min. 41 Tage lang eine Abacavir-haltige Therapie erhielten vor dem Kontrollbesuch zu Woche 6 oder die die Therapie aufgrund eines klinischen Verdachts auf HSR beendeten. ITT(EV2): Patienten aus ITT(EV1) ohne diejenigen, bei denen trotz eines klin. Verdachts auf HSR kein Patch-Test durchgeführt wurde. slide 8 mod. nach Mallal et al.; WESS101

9 ABC Patch-Test slide 9

10 Co-Primäre Endpunkte Klinisch vermutete und immunologisch bestätigte HSR Inzidenz in % slide ,8% (66/847)Ä p<0,0001 3,4% (27/803)Ä Klinische vermutete HSR HSR-Rate in Plazebo-Studien ca. 3% Kontrollarm, kein HLA-B*5701 Screening Prospektives HLA-B*5701 Screening 2,7% (23/842)Ä p<0,0001 0% (0/802)Ä Immunologisch bestätigte HSR mod. nach Mallal et al.; WESS101

11 Sekundäre Endpunkte Spezifität, Sensitivität, PPV und NPV des HLA-B*5701 Screenings für immunologisch bestätigte HSR Immunologisch bestätigte HSR Keine immunologisch bestätigte HSR Gesamt HLA-B*5701 positiv HLA-B*5701 negativ Gesamt Prävalenz HLA-B*5701: 5,7% Spezifität: 794/819 = 96,9% 95% KI (95,5%, 98,0%)Ä Sensitivität: 23/23 = 100% 95% KI (85,2%, 100,0%)Ä PPV*: 23/48 = 47,9% 95% KI (33,3%, 62,8%)Ä NPV**: 794/794 = 100% 95% KI (99,5%, 100,0%)Ä * positiver prädiktiver Wert ** negativer prädiktiver Wert mod. nach Mallal et al.; WESS101 slide 11

12 Implikation des prospektiven HLA-B*5701 Screenings * 100 ABC-naive Patienten 94 HLA-B*5701 negativ 6 HLA-B*5701 positiv sehr geringes Risiko einer HSR klinische Überwachung weiterhin erforderlich 2x wahrscheinlich keine HSR 4x wahrscheinlich klin. Diag. HSR * basierend auf den Ergebnissen von PREDICT-1 mod. nach Mallal et al.; WESS101 slide 12

13 HLA-B*5701 Prävalenz mod. nach Phillips et al.; HIV/AIDS; 43:103-5 slide 13

14 HLA B*5701 Phänotypfrequenz eigene Daten HLA B*5701 6,57 % HLA B*5703 0,65 % HLA B57 neg 92,78 % n=609 slide 14

15 Richtlinien Die Testung auf Vorliegen von HLA- B*5701 vor geplanter Therapie mit ABC ist richtlinienkonform. DHHS Guidelines for the Use of Antiretroviral Agents in HIV-1- Infected Adults and Adolescents European AIDS Clinical Society (EACS) Guidelines for the Clinical Management and Treatment of HIV Infected Adults in Europe slide 15

16 INSTITUT FÜR IMMUNOLOGIE UND GENETIK The Koblenz HIV-superbug Unterzeile zum Titel Medizinisches Labor Dr. Bernhard Thiele Hellmut-Hartert-Str. 1 D Kaiserslautern

17 slide 17

18 Institut fär Immunologie und Genetik Serokonversion?? Home-brew blot, PEI, PD Dr. Schnierle Å HIV Blot 2.2 (MP Biomedicals Asia Pacific Pte. Ltd.) p24 ++ gp160/ Å Chiron RIBA HIV-1/HIV-2 SIA, B.Nr.1176/06 (Chiron Corp.) gp slide 18

19 Mutationsprofil Pat. Nr. 611, S9R ART: naiv VL: cop/ml Protease: V 3 I, L 10 I, Q 18 H, S 37 N, R 41 K/R, M 46 I, K 55 R, R 57 K, I 62 V, L 63 H/P, C 67 C/Y, G 73 T, I 84 V, L 90 M, I 93 L, C 95 F Reverse Transkriptase: E 6 D, T 39 A, M 41 L, S 68 G, L 74 I/L/V, E 122 K, D 123 E, D 177 E, Y 181 C/F/I/N/S/Y, T 200 A, Q 207 E, L 210 W, R 211 K, L 214 F, T 215 C/D/G/N/S/Y, V 245 K, R 277 K, I 293 V, E 297 E/G, L 301 I/L, E 312 A, I 326 V, Q 334 L Subtyp B slide 19

20 Interpretation NRTIs slide 20

21 Interpretation PIs slide 21

22 Interpretation NNRTIs slide 22

23 Interpretation Korezeptor slide 23

24 Therapieempfehlung TDF+FTC, AZT, DRV/r, ENV (T20, Fuzeon)Ä slide 24

25 Mutationsprofil Pat. Nr. 611, S9R ART: naiv VL: cop/ml Protease: V 3 I, L 10 I, Q 18 H, S 37 N, R 41 K/R, M 46 I, K 55 R, R 57 K, I 62 V, L 63 H/P, C 67 C/Y, G 73 T, I 84 V, L 90 M, I 93 L, C 95 F Reverse Transkriptase: E 6 D, T 39 A, M 41 L, S 68 G, L 74 I/L/V, E 122 K, D 123 E, D 177 E, Y 181 C/F/I/N/S/Y, T 200 A, Q 207 E, L 210 W, R 211 K, L 214 F, T 215 C/D/G/N/S/Y, V 245 K, R 277 K, I 293 V, E 297 E/G, L 301 I/L, E 312 A, I 326 V, Q 334 L Subtyp B slide 25

26 Mutationsprofil Index Patient ART: TDF, ABC, FPV/r VL: cop/ml Protease: V3I, L10I, Q18HQ, S37N, M46I, K57KR, I62V, L63P, A71AV, G73T, V77IM, P79N, I84V, I85IV, L90M, I93L Reverse Transkriptase: 6D, 41L, 43Q, 118IV, 122K, 123E, 177E, 200A, 207E, 210W, 211K, 214F, 215Y, 245K, 277K, 293V, 312A, 326V, 331K Subtyp B slide 26

27 Mutationsprofil Pat. Nr. 611, S9R4 Protease: V 3 I, L 10 I, Q 18 H, S 37 N, R 41 K/R, M 46 I, K 55 R, R 57 K, I 62 V, L 63 H/P, C 67 C/Y, G 73 T, I 84 V, L 90 M, I 93 L, C 95 F Reverse Transkriptase: E 6 D, T 39 A, M 41 L, S 68 G, L 74 I/L/V, E 122 K, D 123 E, D 177 E, Y 181 C/F/I/N/S/Y, T 200 A, Q 207 E, L 210 W, R 211 K, L 214 F, T 215 C/D/G/N/S/Y, V 245 K, R 277 K, I 293 V, E 297 E/G, L 301 I/L, E 312 A, I 326 V, Q 334 L L 74 I/L/V: Y 181 C: ddi oder ABC NVP oder EFV slide 27

28 Therapiehistorie Indexpatient 01/1993: Videx (ddi)ä 01/1995: Retrovir (AZT)Ä 04/1995: Retrovir, Hivid (AZT, ddc)ä 01/1996: Retrovir, Epivir (AZT, 3TC)Ä 10/1996: Zerit, Epivir (d4t, 3TC)Ä 01/1998: Zerit, Videx, Crixivan (d4t, ddi, IDV 08/1999: Ziagen, Sustiva Viracept (ABC, EFV, NFV)Ä 08/2000: Retrovir, Epivir, Crixivan (AZT, 3TC, IDV/r)Ä 12/2004: Viread, Ziagen, Telzir (TDF, ABC, APV/r)Ä... VL: 11/1999: cop/ml 02/2000: cop/ml 05/2000: cop/ml Mgl. Infektionszeitraum slide 28

29 HLA Typisierung Index Patient ART: ddi, FTC, TPV/r VL: cop/ml Subtyp B HLA B*1501/3501 slide 29

30 HLA Typisierung Pat. Nr. 611, S9R ART: TDF, FTC, AZT, DRV/r, ENF VL: <50 cop/ml Subtyp B HLA B*3501/3502 slide 30

31 slide 31

32 Problem definition Do transmitted resistance mutations correlate with certain HLA types indicating escape from the immune surveillance? slide 32

33 Role of HLA in HIV-1 disease progression HLA types which are associated with a high viremia and fast progression of disease Ä fast progression: HLA-A1-B8-DR3, B37 Ä slow progression: HLA-B14/Cw8, B57, B27, A32 slide 33

34 Institut fär Immunologie und Genetik Patients and Methods Å Å Å Å 103 therapy-naive patients (RESINA) with documented drug resistance mutations in protease or / and reverse transcriptase Determination of HLA-A and ÇB type by sequencing (n=75) Frequencies of HLA-types were correlated with mutations in protease and reverse transcriptase Significance of correlation determined using FisherÉs exact probability test slide 34 Schweitzer et al., XVII IHDRW, Sitges 2009

35 Mutations in RT Frequency of HLA-B*44 Occurrence [%] 45,0% 40,0% 35,0% 30,0% 25,0% 20,0% 15,0% 10,0% 5,0% 0,0% 41,7% 9,3% 5,8% Frequencies of HLA-types in subgroups compared to the whole group as well as literature values Subgroup (K 103 Whole Group R) n = 6; P = Literature Value Frequency of HLA-B*07 Frequency of HLA-B*40 Frequency of HLA-B*07 + HLA-B*40 Occurrence [%] 25,0% 20,0% 15,0% 10,0% 5,0% 0,0% 20,6% Subgroup (V 118 I) 10,7% Whole Group 12,2% Literature Value Occurrence [%] 14,0% 12,0% 10,0% 8,0% 6,0% 4,0% 2,0% 0,0% 11,8% Subgroup (V 118 I) 8,0% Whole Group Occurrence [%] 20,0% 18,0% 16,0% 14,0% 12,0% 10,0% 8,0% 6,0% 4,0% 2,0% 0,0% 17,6% Subgroup (V 118 I) 5,3% Whole Group n = 17; P = n = 17; P = n = 17; P = ,7% Literature Value 0,8% Literature Value slide 35 Schweitzer et al., XVII IHDRW, Sitges 2009

36 Mutations in RT Frequency of HLA-B*44 Occurrence [%] 45,0% 40,0% 35,0% 30,0% 25,0% 20,0% 15,0% 10,0% 5,0% 0,0% 40,0% 9,3% 5,8% n = 5; P = Frequency of HLA-A*11 Subgroup (L 210 F) Whole Group Literature Value 60,0% 50,0% 50,0% Occurrence [%] 40,0% 30,0% 20,0% 10,0% 8,0% 6,2% n = 2; P = ,0% Subgroup (V 75 I) Whole Group Literature Value slide 36 Schweitzer et al., XVII IHDRW, Sitges 2009

37 Mutations in PR Occurrence [%] 40,0% 35,0% 30,0% 25,0% 20,0% 15,0% 10,0% 5,0% 0,0% 37,5% Subgroup (L 33 F) Frequency of HLA-A*01 12,7% Whole Group 15,2% Literature Value for Caucasian Population 5,7% Literature Value for African Population Occurrence [%] 30,0% 25,0% 20,0% 15,0% 10,0% 5,0% 0,0% 25,0% Subgroup (L 33 F) Frequency of HLA-B*35 14,7% Whole Group n = 4; P = n = 4; P = ,7% 8,5% Literature Value for Caucasian Population Literature Value for African Population Frequency of HLA-A*01 + HLA-B*35 Occurrence [%] 60,0% 50,0% 40,0% 30,0% 20,0% 10,0% 0,0% 50,0% Subgroup (L 33 F) 5,3% Whole Group n = 4; P = ,5% 0,5% Literature Value for Caucasian Population Literature Value for African Population slide 37 Schweitzer et al., XVII IHDRW, Sitges 2009

38 Mutations in PR Frequency of HLA-A*03 Frequency of HLA-B*35 Occurrence [%] 45,0% 40,0% 35,0% 30,0% 25,0% 20,0% 15,0% 10,0% 5,0% 0,0% 40,0% Subgroup (M 46 I / L) 14,0% 13,4% Whole Group Literature Value Occurrence [%] 45,0% 40,0% 35,0% 30,0% 25,0% 20,0% 15,0% 10,0% 5,0% 0,0% 40,0% Subgroup (M 46 I / L) 14,7% Whole Group n = 5; P = n = 5; P = ,7% Literature Value Frequency of HLA-A*03 + HLA-B*35 Occurrence [%] 70,0% 60,0% 50,0% 40,0% 30,0% 20,0% 10,0% 0,0% 60,0% 12,0% Subgroup (M 46 I Whole Group / L) n = 5; P = ,3% Literature Value slide 38 Schweitzer et al., XVII IHDRW, Sitges 2009

39 Transmission of resistance Resistance Mutation = CTL escape mutation? Statistical analysis of correlations between drug resistance associated mutations and HLA class I molecules from RESINA (n=73 Therapy Naive Patients) Frequency in genotype Mutation HLA-A / -B Subgroup Whole group Lit. Value P value K103R B* % 9.3 % 5.8 % V118I B*07 + B* % 5.3 % 0.8 % L210F B*44 40 % 9.3 % 5.8 % V75I A*11 50 % 8 % 6.2 % L33F A*01 + B*35 50% 5.3 % 1.5 % Caucasians 0.5 % Africans M46I/L A*03 40 % 14 % 13.4 % M46I/L B*35 40 % 14.7 % 9.7 % M46I/L A*03 + B*35 60% 12 % 1.3 % Drug resistance mutations can be assigned to specific HLA alleles slide 39 Schweitzer et al., XVII IHDRW, Sitges 2009

40 CTL response / escape HIV HIV peptides TCR MHC I CTL CD4+cell HIV mutant peptides CTL X slide 40

41 Institut fär Immunologie und Genetik Procedure of analysis Å Å Å Å Å Statistical analysis of HIV-resistance associated mutations and HLA alleles Epitope identification with 15 long overlapping peptides Design of peptides representing optimal epitopes Epitope confirmation with ELISPOT Parallel re-evaluation of statistically derived results in patients from a different cohort slide 41 Schweitzer et al., XVII IHDRW, Sitges 2009

42 Institut fär Immunologie und Genetik Epitope mapping: V75I (RT)Ñ Putative epitopes were predicted using ELF. ( ELISPOT (biological assay)ä Pat EA HLA A11, A11, B35, B62, Cw3, Cw4 LVDFRELNKRTQD FW n.p. KWRKLVDFRELNKRT n.p. KDSTKWRKLVDFREL n.p. AIKKKDSTKWRKLVD n.p. SFU/ cells slide 42 Schweitzer et al., XVII IHDRW, Sitges 2009

43 Results of the epitope mapping Peptides comprising following amino acid HLA allele shared by investigated patients Number of Patients recognizing peptides 33 (PR)Ä 46 (PR)Ä 46 (PR)Ä 75 (RT)Ä 103 (RT)Ä 118 (RT)Ä HLA-A*01 HLA-A*03 HLA-B*35 HLA-A*11 HLA-B*44 HLA-B*07 4 out of out of 28 4 out of 10 4 out of 9 6 out of 22 7 out of 17 Epitopes at positions 46, 75, 118 were detected from >40% of the tested patients slide 43 Schweitzer et al., XVII IHDRW, Sitges 2009

44 Institut fär Immunologie und Genetik CTL recognition: L210 (RT)Ñ B*44 TKIEELRQHLLRWGLTTPDKK Sequence-wt: GQHRTKIEELRQHLLRWGFTTPD L210F: EELRQHLFRW L210W: EELRQHLWRW Pat ZS (L210) HLA A24, A24, B44, B13, Cw5, Cw6 Pat. BF, (L210F), HLA: A02, A02, B44, B F W-- EELRQHLLRW n.p. SFU/ cells F W-- EELRQHLLRW n. P. ls e c /1 U F S slide 44 Schweitzer et al., XVII IHDRW, Sitges 2009

45 Institut fär Immunologie und Genetik Summary Å Å Å Statistical analysis revealed correlations between HLA class I alleles und the occurrence of drug resistance mutations in treatment-naöve patients peptides comprising these amino acid positions were recognized by CD8+ T cells 210F acts as escape mutation in B44+ patients Ä selection for this mutation may occur in B44+ patients The HLA-system provides the selective pressure for the occurrence and maintenance of certain transmitted drug resistance mutations in treatment-naöve patients. slide 45 Schweitzer et al., XVII IHDRW, Sitges 2009

46 Institut fär Immunologie und Genetik Conclusions Å Å Å The results of this study contribute to the understanding of the relationship between HIV infection and immune response The knowledge about HLA restricted HIV drug resistance mutations might be helpful in designing new therapy strategies The results of this study indicate that the prevalence of transmitted drug resistance could effectively be higher than determined by several nationwide survey programs slide 46 Schweitzer et al., XVII IHDRW, Sitges 2009

47 Acknowledgements F Schweitzer SM Mueller M Däumer R Klein R Kaiser M Oette K Roomp T Lengauer T Harrer University of Maastricht, Maastricht, The Netherlands; University of Cologne, Cologne, Germany; University Hospital Erlangen, Erlangen, Germany Institute for Immunology and Genetics, Kaiserslautern, Germany Institute for Immunology and Genetics, Kaiserslautern, Germany University of Cologne, Cologne, Germany University Hospital Düsseldorf, Düsseldorf, Germany; Krankenhaus der Augustinerinnen, Cologne, Germany Max Planck Institute for Informatics, Saarbrücken, Germany Max Planck Institute for Informatics, Saarbrücken, Germany University Hospital Erlangen, Erlangen, Germany slide 47