Simon Mallal

Background: Multiple class I and II HLA associations have been described in association with nevirapine (NVP) hypersensitivity reaction (HSR) phenotypes. We tested the hypothesis that peptide binding (PB) properties may be shared between these alleles. Methodology: HLA-A,-B-,-C, -DR typing was performed on stored DNA from a retrospective case controlled analysis of NVP HSR (ClinicalTrials.gov NCT00310843) using the 454 FLX platform. Univariate and multivariate analyses stratified for race were performed according to HLA class I/II alleles, HLA supertypes and HLA alleles according to PB (Sidney J, Lund O), Kir ligand groupings and HLA B/C haplotypes for cutaneous and hepatitis phenotypes of NVP HSR. In silico modelling to simulate HLA binding to NVP was performed with the highest ranked candidates. Results: HLA -A,-B,-C and -DR typing resolved to four digits (794 samples (controls =524, cutaneous NVP HSR cases =170, hepatitis NVP HSR cases = 100)). Multivariate analysis of cutaneous NVP HSR in Southeast Asians (SEA) associated DR4 supertype(OR=2.9, p=0.015) and alleles with HLA-35/18 PB properties(OR=6.4, p=0.002). HLA-DRB1*01:02 was associated with hepatitis NVP HSR in Caucasians (OR=2.7,p=0.01) whereas carriage of alleles of the PB B46 were protective (OR=0.3, p=0.04). HLA-C*04:01 was associated with cutaneous NVP HSR, including SJS/TEN across all races (p<0.0001, Mantel-Haenszel test; Caucasians: OR=2.8 [1.3-5.9], p=0.009; African Americans: OR=4.0 [1.4-13.0], p=0.02; SEA: OR=9.0 [3.2-24.9], p<0.0001). However, haplotype analysis of HLA-B/C showed pairing of HLA-C*04:01 with HLA-B alleles with B35 and B18 like PB (HLA-B*35:01,B*35:05,B*35:08,B*53:01,B*18:01,B*18:02, B*44:02,B*4403), and this effect was strongest in SEA where carriage of HLA-C*04:01 when paired with the HLA-B alleles(OR=11.8,p=0.0003) was more strongly associated with cutaneous NVP HSR than HLA-C*04:01 carried alone(OR=4.8,p=0.047). This suggests that risk of cutaneous NVP HSR attributed to carriage of HLA-C*04:01 may be enhanced by HLA-B alleles which are in strong linkage disequilibrium. An in silico and peptide binding model both suggest that NVP non-covalently binds in the F pocket of HLA-B*35:05 and near the B pocket of HLA-C*04:01. In multivariate analyses, Kir ligand groupings Bw4/Bw6 and C1/C2 did not significantly contribute to the modelling of associations with cutaneous hypersensitivity or hepatitis. Conclusions: Cutaneous and hepatitis phenotypes of NVP HSR associate with different HLA-B and DR-alleles respectively that share PB characteristics.The pairing of these HLA-B alleles with HLA-C*04:01 appears important for the development of cutaneous NVP HSR, providing a testable model for the immunopathogenesis of NVP HSR.

Next generation Sequencing (NGS) has enabled high throughput analysis of T cell receptor (TCR) repertoires, which are useful for monitoring antigen- specific T cell responses. Integrating TCR sequencing with expression levels of genes that characterise T cell phenotype at the single cell level allows comprehensive analysis of T cell function and specificity. Single cell TCR sequencing on the Illumina platform enables the identification of paired TCR α/β chains and T cell phenotype from sorted antigen-specific T cell populations. Here we highlight novel techniques available for T cell phenotyping and TCR repertoire analysis.

Abacavir (ABC) binds non-covalently to the floor of the peptide-binding groove of HLA-B*57:01, altering the chemistry and shape of the antigen binding cleft. This allows previously untolerized self-peptides to be presented by HLA-B*57:01 which upon T-cell receptor binding initiates a CD8+ T-cell response and a clinical hypersensitivity reaction(HSR). Endoplasmic reticulum aminopeptidases (ERAPs) trim peptides for MHC Class I presentation, influencing the degree and specificity of the CD8+ T-cell response. Genetic variation within ERAP adds to the positive predictive value (PPV) of the HLA class I risk allele in autoimmune diseases such as HLA-B27 positive ankylosing spondylitis. Considering the altered peptide repertoire mechanism of ABC HSR we hypothesize that variation in ERAP may help explain why 45% carrying HLA-B*57:01 can tolerate ABC. SNPs within ERAP1 (rs27044, rs17482078, rs30187, rs27434, rs2287987) were examined in HLA-B*57:01+ ABC HSR patch test positive (PT+)[n=53] and HLA-B*57:01+ ABC tolerant[n=22] with sequence-based typing. Rs2248374, a tag SNP for functional ERAP2 haplotypes was also examined. Haplotype A is tagged by the (A) allele, while haplotype B is tagged by rs2248374(G). Fisher exact tests and multiple logistic regressions were used to compare genotypes between the ABC HSR PT+ and tolerant groups. HLA-B*57:01+ ABC tolerance was associated with rs27434(GG) (18/22(82%) vs 24/53(45%) in ABC HSR PT+, p=0.005). This SNP maps to the active site within ERAP1 (AA356). For an HLA-B*57:01 positive population the estimated PPV for rs27434 genotypes for ABC HSR PT+ is AA(100%), AG(77%) and GG(40%). A missense mutation within the domain junction (rs30187(C)) important in conformation change of ERAP1 (AA528), was overrepresented in HLA-B*57:01+ ABC tolerant individuals (p=0.04). Analysis indicated linkage between rs27044 and rs30187, rs17482078 and rs2287987, and between rs30187 and rs27434 (all p < 0.0001). In a multivariable model with rs27434(GG), the ERAP2 SNP (rs2248372(G)) that tags haplotype B which is characterized by a truncated protein, was decreased in tolerant individuals (p = 0.005). ERAP and particularly ERAP1 variants are important in the development of ABC HSR. ERAP activity may influence the repertoire of peptides presented by HLA-B*57:01 or influence early changes in immunodominant epitope selection. This provides a potential pathogenic mechanism for the development of ABC HSR or ABC tolerance in HLA-B*57:01 carriers.

Background: HIV cure is limited by persistence of long lived latently infected CD4+ T cells. Latently infected cell lines are widely used in vitro to study HIV latency. We identified and tested the stability of HIV integration sites in latently infected cell lines, using a newly developed high throughput method. Method: To determine assay sensitivity/efficiency, genomic DNA of seven latent HIV cell lines (20 cells each) were isolated and mixed with genomic DNA from one million HIV negative PBMCs. Additionally, the latently infected cell lines ACH-2, U1 and J1.1 containing replication competent HIV and J-Lat 8.4, 9.2, 10.6 and 15.4 cell lines that contain a single replication deficient HIV were passed. DNA, isolated after passage 0, 2, 4, 6 and 8 was enzymatically cut to random sized fragments. These were end-repaired and a linker was ligated to the fragments. The fragments were subjected to LTR based nested PCR with barcoded nested primers and prepared for Miseq sequencing. Chromosomal alignment was determined using the Blat-UCSC Genome Browser (GRCH38/hg38). Results: The efficiency was 35% and detected one HIV integration site in 50,000 uninfected PBMCs. J-Lat cell lines showed single integration sites. ACH-2, U1 and J1.1 demonstrated multiple distinct HIV integration sites per 150,000 cells (74, 42 and 93 respectively). J1.1, which is reported to have a single integrated copy per cell, demonstrated two major integration sites in equal frequency. ACH-2 cells, when passaged, demonstrated a 2-fold increase in unique HIV integration sites found across the human genome. Conclusion: Cell lines latently infected with replication competent HIV demonstrated multiple unique HIV integration sites indicating these cell lines are not clonal. Furthermore, the increase and change in sites of HIV integration observed in ACH-2 cells over time is suggestive of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency.