HLA-B - (GFID) eV

INSTITUT FÜR IMMUNOLOGIE UND GENETIK 

Medizinisches Labor Kaiserslautern 

Immungenetische Untersuchungen bei 

Hypersensitivität und HIV-Infektion 

6. Immundiagnostisches Meeting Dresden, 7. - 9. Oktober 2008 

Moderne Diagnostik und Therapie 

immunologisch bedingter Erkrankungen 

Medizinisches Labor 

Dr. Bernhard Thiele 

Hellmut-Hartert-Str. 1 

D-67655 Kaiserslautern

Institut für Immunologie und Genetik 


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



Abacavir Hypersensitivitäts Syndrom 

slide 3



slide 4



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 2002 359:727-732



HLA-Loci 

HLA-A: 489 (390), HLA-B: 830(711), HLA-C: 266(210), 

HLA-DRB: 545(451) 

Allele(Proteine)Ä 

slide 6 

http://www.ebi.ac.uk/imgt/hla/stats.html


Medizinisches Labor Kaiserslautern 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



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, n=1956 

Screeningarm 

ITT, n=980 

Start ABC 

Therapie 

ITT(E), n=859 

Auswertbar für klinisch vermutete HSR 


Auswertbar für immunologisch bestätigte HSR 


Ausschluss 

HLA-B*5701 

pos. n=54 

Ausschluss 


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



ABC Patch-Test 

slide 9



Co-Primäre Endpunkte 

Klinisch vermutete und immunologisch bestätigte HSR 

Inzidenz in % 

slide 10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

7,8% 

(66/847)Ä 

p



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 23 25 48 

HLA-B*5701 negativ 0 794 794 

Gesamt 23 819 842 

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



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



HLA-B*5701 Prävalenz 

mod. nach Phillips et al.; HIV/AIDS; 43:103-5 

slide 13



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



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

INSTITUT FÜR IMMUNOLOGIE UND GENETIK 


„The Koblenz HIV-superbug“ 

Unterzeile zum Titel 

Medizinisches Labor 

Dr. Bernhard Thiele 

Hellmut-Hartert-Str. 1 

D-67655 Kaiserslautern



slide 17

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 ++ 

gp160/120 ++ 

Å Chiron RIBA HIV-1/HIV-2 SIA, B.Nr.1176/06 

(Chiron Corp.) 

gp41 +++ 

slide 18



Mutationsprofil Pat. Nr. 611, S9R4 

23.11.2006 

ART: naiv 

VL: 8.800 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



Interpretation NRTIs 

slide 20



Interpretation PIs 

slide 21



Interpretation NNRTIs 

slide 22



Interpretation Korezeptor 

slide 23



Therapieempfehlung 

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

slide 24




23.11.2006 

ART: naiv 




C 95 F 





Subtyp B 

slide 25



Mutationsprofil Index Patient 

24.06.2005 

ART: TDF, ABC, FPV/r 


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






C 95 F 





L 74 I/L/V: 

Y 181 C: 

ddI oder ABC 

NVP oder EFV 

slide 27



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: 7.300 cop/mL 

02/2000: 90.000 cop/mL 

05/2000: 84.000 cop/mL 

Mgl. Infektionszeitraum 

slide 28



HLA Typisierung Index Patient 

07.2007 

ART: ddI, FTC, TPV/r 


Subtyp B 

HLA B*1501/3501 

slide 29



HLA Typisierung Pat. Nr. 611, S9R4 

08.2007 

ART: TDF, FTC, AZT, DRV/r, ENF 

VL:



slide 31



Problem definition 

Do transmitted resistance mutations 

correlate with certain HLA types indicating 

escape from the immune surveillance 

slide 32



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



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



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 = 0.0018 

Literature Value 



Frequency of HLA-B*07 + HLA-B*40 


25,0% 

20,0% 

15,0% 

10,0% 

5,0% 

0,0% 

20,6% 

Subgroup (V 118 

I) 

10,7% 

Whole Group 

12,2% 



14,0% 

12,0% 

10,0% 

8,0% 

6,0% 

4,0% 

2,0% 

0,0% 

11,8% 


I) 

8,0% 

Whole Group 


20,0% 

18,0% 

16,0% 

14,0% 

12,0% 

10,0% 

8,0% 

6,0% 

4,0% 

2,0% 

0,0% 

17,6% 


I) 

5,3% 

Whole Group 

n = 17; P = 0.0531 n = 17; P = 0.4700 

n = 17; P = 0.0344 

6,7% 


0,8% 


slide 35 




Mutations in RT 



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 = 0.0074 

Frequency of HLA-A*11 

Subgroup (L 210 

F) 

Whole Group 


60,0% 

50,0% 

50,0% 


40,0% 

30,0% 

20,0% 

10,0% 

8,0% 

6,2% 

n = 2; P = 0.0323 

0,0% 

Subgroup (V 75 I) Whole Group Literature Value 

slide 36 




Mutations in PR 


40,0% 

35,0% 

30,0% 

25,0% 

20,0% 

15,0% 

10,0% 

5,0% 

0,0% 

37,5% 

Subgroup (L 

33 F) 


12,7% 

Whole Group 

15,2% 

Literature 

Value for 

Caucasian 

Population 

5,7% 

Literature 

Value for 

African 

Population 


30,0% 

25,0% 

20,0% 

15,0% 

10,0% 

5,0% 

0,0% 

25,0% 

Subgroup (L 

33 F) 


14,7% 

Whole Group 

n = 4; P = 0.0643 n = 4; P = 0.6044 

9,7% 8,5% 

Literature 

Value for 

Caucasian 

Population 

Literature 

Value for 

African 

Population 

Frequency of HLA-A*01 + HLA-B*35 


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 = 0.0125 

1,5% 0,5% 

Literature 

Value for 

Caucasian 

Population 

Literature 

Value for 

African 

Population 

slide 37 




Mutations in PR 




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 



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 = 0.0344 n = 5; P = 0.0406 

9,7% 


Frequency of HLA-A*03 + HLA-B*35 


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 = 0.0109 

1,3% 


slide 38 




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*44 41.7 % 9.3 % 5.8 % 0.0018 

V118I B*07 + B*40 17.6 % 5.3 % 0.8 % 0.0344 

L210F B*44 40 % 9.3 % 5.8 % 0.0074 

V75I A*11 50 % 8 % 6.2 % 0.0323 

L33F A*01 + B*35 50% 5.3 % 

1.5 % 

Caucasians 

0.5 % 

Africans 

0.0125 

M46I/L A*03 40 % 14 % 13.4 % 0.0344 

M46I/L B*35 40 % 14.7 % 9.7 % 0.0406 

M46I/L A*03 + B*35 60% 12 % 1.3 % 0.0109 

Drug resistance mutations can be assigned to specific HLA alleles 

slide 39 




CTL response / escape 

HIV 

HIV peptides 

TCR 

MHC I 

CTL 

CD4+cell 

HIV mutant peptides 

CTL 

X 

slide 40



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 




Epitope mapping: V75I (RT)Ñ 

Putative epitopes were predicted using ELF. 

(http://www.hiv.lanl.gov/content/sequence/ELF/epitope_analyzer.html)Ä 

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/100 000 cells 

0 500 1000 

slide 42 




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 






4 out of 28 

17 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 




CTL recognition: L210 (RT)Ñ 

B*44 

TKIEELRQHLLRWGLTTPDKK 

200 210 220 

Sequence-wt: GQHRTKIEELRQHLLRWGFTTPD 

L210F: EELRQHLFRW 

L210W: EELRQHLWRW 

Pat ZS (L210) HLA A24, A24, B44, B13, Cw5, Cw6 

Pat. BF, (L210F), HLA: A02, A02, B44, B44 

-------F-- 

-------W-- 

EELRQHLLRW 

n.P. 

SFU/100 000 cells 

-------F-- 

-------W-- 

EELRQHLLRW 

n. P. 

ls 

e 

c 

0 

0 

0 

/1 

U 

F 

S 

0 500 1000 1500 

0 20 40 

slide 44 




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 




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 




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

HLA-B - (GFID) eV

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