Abha Chopra

Objectives. Inclusion body myositis (IBM) is a progressive inflammatory and degenerative disease of the skeletal muscles that affects individuals over the age of 45 and leads to a gradual loss of mobility. It has been widely reported that a subgroup of 33 to 72% of IBM patients produce self-reactive antibodies that bind cytosolic 5’-nucleotidase 1A (NT5C1A) within the muscles and possibly exacerbate the disease severity. A genetic association between immune-related genes with IBM has been described. This study aimed to deepen our knowledge about the human leukocyte antigen (HLA) genes that alter the risk to develop IBM and to investigate whether specific HLA alleles may contribute to the occurrence of NT5C1A-directed antibodies.

Methods. In this study, we used Illumina next-generation sequencing to resolve the high resolution HLA haplotype of 102 Caucasian IBM patients from the Western Australian cohort. We then compared the frequency and carriage of the identified alleles within the IBM cohort to reference databases of Caucasian cohorts. We additionally compared the HLA allele carriage within the genotypes of anti-NT5C1A-positive and-negative patients within our IBM cohort.

Results. Our results confirmed the previously identified association risk of the 8.1 MHC ancestral haplotype with IBM. We also lifted ambiguities and clarified the identity of the risk-associated alleles that have been previously reported at a lower level of resolution. Additionally, we identified previously unreported risk allele associations with IBM. Furthermore, our analysis validated previously reported protective HLA alleles, and also revealed reduced carriage frequency of additional alleles, suggesting their protective role in the disease. Lastly, our study revealed two alleles, which carriage are associated with anti-NT5C1A antibody production.

Conclusions. Our findings refine and expand on the knowledge of the HLA genetic background of IBM. Stratifying patients based on their HLA genotype provides a genetic basis for new therapeutic intervention strategies early in the disease process to slow down symptom development.

Background: Understanding the spread of HIV during HIV prevention trials has high potential to inform future intervention programming. Here, we provide a first characterisation of the residual age- and gender-specific transmission dynamics during the HPTN 071 (PopART) trial using orthogonal methods, and investigate the potential impact of suppressing transmissions from inferred source populations.

Methods: First, epidemic predictions were made using an individual-based model (IBM) calibrated on trial data. Model parameters of the sexual network were derived from surveys on sexual behaviour. Second, model predictions were tested against phylogenetic estimates obtained with phyloscanner from viral short-read next-generation sequencing (NGS) data from three trial communities in Zambia.

Results: Phylogenetic analysis identified 180 probable transmission pairs and the direction of transmission between them. 62% of these transmissions were from men to women, the same as was predicted by the IBM. The phylogenetic analysis predicted men to be 5.4 years than women in male-to-female transmission (IBM predicted 5.5 years), and 3.9 years older in female-to-male transmissions (the IBM predicted 2.9 years). The age distribution of transmitters agreed with modelling predictions, more closely after adjusting for sampling bias (figure 1). According to both the model and phylogenetics analysis, onwards transmissions peaked in 25-29 year old (y.o.) men and 20-24 y.o. women. Modelling the prevention of all transmissions from 25-29 y.o. men and 20-24 y.o. women reduced cumulative incidence over the trial period (mid-2014 to 2018) by 20% and 19% respectively.

Conclusions: Our results validate predictions of a mathematical model using phylogenetic data. These results support observations that there is a significant contribution of young people to HIV transmission in sub-Saharan Africa, especially 25 to 29 y.o. men. These results highlight that if universal testing-and-treatment (UTT) does not reach young people, and 25 to 29 y.o. men in particular, then the effect of UTT on reducing incidence may be limited.

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.

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.