Specific Binding Characteristics of HLA Alleles Associated with Nevirapine Hypersensitivity Rebecca Pavlos, PhD The Institute for Immunology & Infectious Diseases, Murdoch University, Perth, Western Australia.

Nevirapine Non-nucleoside reverse transcriptase inhibitor (NNRTI) used to treat HIV-1 In triple combination therapy has been shown to suppress viral load Limited by Hypersensitivity in 5% of patients Risk is greater for women with pre-NVP CD4 count >250 cells/mm3 and men with pre-NVP CD4 count >400 cells/mm3.

Complex HLA associations Ethnicity Phenotype Reference HLA-DRB1*01:01 (>25% CD4 T cells) West Australian, Caucasian Hepatic/systemic Martin et al, (2005); Yuan et al, (2011); Keane et al, (2014). HLA-DRB1*01:02 South African Heptatic Phillips et al, (2013). HLA-B*58:01 Hepatic HLA-B*35 Asian, Thai, Indian cADR Yuan et al, (2011); Umapathy et al, (2011). HLA-B*35:01 West Australian Keane et al, (2014). HLA-B*35:05 Thai Rash Chantarangsu et al, (2009) HLA-C*04 All cADRs, All Likanonsakul et al, (2009); Carr et al, (2013); Yuan et al, (2011); Gao et al, (2012). HLA-C*08(-B*14) Sardinian, Japanese, West Australian Hepatic, Rash, Eosinophilia Littera et al, (2006); Gatanga et al, (2007); Keane et al (2014).

Nevirapine HSR Complex HLA Class I and Class II associations (phenotype & ethnic specific) Role for both CD4+ and CD8+ T cells in NVP HSR (Keane et al, 2014, AIDS). Low positive predictive value, many without any known HLA risk alleles

Abacavir van Deurekom et al. Immunogenetics 2015 Aug;67(8):425-36 Illing, et al. 2012 Nature 486:554-832; Ostrov et al. 2012. Proc Natl Acad Sci U S A 109:9959-64; 33; Norcross et al. 2012.. AIDS 26:F21-9

Do similarities in the HLA peptide binding groove explain the HLA-diversity in NVP HSR? Class I. (HLA-A, -B, -C). Pockets A-F van Deurekom et al. Immunogenetics 2015 Aug;67(8):425-36 Class II. (HLA-DRB1). Pockets P1, P4, P6, P7, P9 Murthy et al. Structure. 5 (10): 1385. (1997)

Methods… 5 distinct self-reported ethnicities Samples Case controlled study of cutanous NVP HSR (n=151 cases, n=413 controls) 5 distinct self-reported ethnicities Asian: South-East Asian and Taiwanese; African: African American Caucasian: European and Hispanic Analysis HLA-A,-B-,-C, -DR typing (4 digit) and CYP2B6 genotyping Univariate and multi-variate logistic regression (adjusted for ethnicity) Alleles, supertypes, predicted peptide binding (MHCcluster), binding pocket residues In silico modelling and molecular docking scores for identified candidates.

HLA associations with cutaneous NVP HSR (b)Protective HLA-B B pocket (B62 alleles) (c)Predisposing HLA-B E pocket (B*35:05) (d)Predisposing HLA-DRB1 P4 pocket (DRB1*01 and 04 alleles)

Multivariate modelling was used to examine the overall risk for cutaneous NVP HSR associated with the identified predisposing and protective allele clusters in both whole-cohort and ethnic-specific analysis. Striking similarities in observed relative odds are evident across ancestral groups, despite varying allele frequency distributions.

Conclusions Cutaneous NVP HSR associates with HLA-C alleles having similar peptide binding properties and F pocket structure as HLA-C*04:01. Secondary associations with cutaneous NVP HSR are attributable to Class I and II binding pocket structure; A shared P4 pocket groups HLA-DRB1 risk alleles A characteristic B pocket defines a protective cluster of HLA-B alleles NVP HSR provides a model to explain the basis for complex and multi-allelic associations in drug hypersensitivity

Future work…. Peptide binding ? TCR Peptide elution studies with HLA-C*04:01 and DRB1*01:01 SALs TCR sequencing in HLA characterised patient PBMCs, blister fluid & in affected v normal skin. Peptide binding ? TCR Drug - Nevirapine HLA

Acknowledgements Elizabeth Phillips Simon Mallal Elizabeth McKinnon The Institute for Immunology & Infectious Diseases Murdoch University, Perth WA Elizabeth Phillips Simon Mallal Elizabeth McKinnon Abha Chopra Alec Redwood Silvana Gaudieri David Ostrov University of Florida, USA Jing Yuan Boehringer Ingelheim Pharmaceuticals David Haas Vanderbilt University, USA. Bjoern Peters La Jolla Institute for Allergy and Immunology Soren Buus University of Copenhagen David Koelle University of Washington Mary Carrington Ragon Institute of MGH, MIT & Harvard