Carroll, W.

Choo, L.

Chopra, A.

Clark, H.

Cooper, D.

Kermode, A.

Leary, S.

Nolan, D.

Strautins, K.

Tschochner, M.

English

Research Repository

August 2013PACTRIMSNovember 2013Multiple Sclerosis Patient Sequence Variation In Epstein-Barr Virus Nuclear Antigen 1 Using 454 FLX technologyTschochner M.1, Leary S.1, Strautins K.1, Clark H.1, Cooper D.1, Chopra A.1, Choo L.1, Carroll W.2,3, Kermode A.1,2,3, Nolan D.1,41Institute for Immunology & Infectious Diseases, Murdoch University, Perth, Western Australia 2Department of Neurology, Sir Charles Gairdner Hospital, Queen Elizabeth II Medical Centre, Western Australia , 3Centre for Neuromuscular and Neurological Disorders, Australian Neuromuscular Research Institute, UWA, Australian Neuromuscular Research Institute, Sir Charles Gairdner Hospital, University of Western Australia, Perth, Western Australia, 4Department of Clinical Immunology, Royal Perth Hospital, Perth, Western AustraliaBackground:Susceptibility to MS is conferred by immune system associated genes, with human leukocyteantigen (HLA) DRB1*1501 identified as a major MS risk allele. Infection with Epstein-Barr Virus(EBV) has also been shown to increase MS risk, and in particular anti-EBV nuclear antigen-1(EBNA-1) antibodies are strongly associated with the disease. Additionally, it has been shownthat EBNA1-specific CD4+ T-cells can cross-recognize MS-associated myelin antigens, and areselectively expanded in MS patients.Acknowledgments:We would like to thank all patients and study participants, all nurses and doctors involved as well as staff at IIID fortheir assistance and input.This study is funded by the McCusker Charitable FoundationOverall, amino acid variability was low and Sanger sequencing results revealed few mixedpositions. Utilizing 454 FLX deep sequencing, quasispecies could be detected, although generallyat a low percentage. In the high coverage samples minorities at ≥10% frequency were detectedin 3.5% (26 positions of 749 positions investigated), increasing to 7.3% for minor speciesdetected at ≥1% (Table 2). At a 1% cut off, 55 of 749 positions contained mixed amino acidsacross all 14 samples. 41 of these occurred in a single sample only but 12 different positions hadmutations which were shared by two samples and 2 positions were shared by three samples(Table 2).Fig 4. Maximum and average reads for 24 MS samples tested on 454 FLX in one lane out of eight.Presenting author contact: m.tschochner@iiid.com.auObjective:To identify MS patient-specific EBNA-1 sequence variation and the potential influence ofminority variants in shaping epitope-specific immune responses.Materials and Methods:MS samples were utilised from the Perth Demyelinating Disease Database (PDDD) (WesternAustralia). DNA was isolated from buffy coats and the C-terminal end of the EBNA-1 gene wasamplified using a novel semi-nested PCR approach with a fully automated setup utilisingBiomek FX robots (Fig 1). First round PCR was performed with the primers EBV109111F-EBV109951R and Expand High Fidelity Taq (Roche) in a 25µl reaction, resulting in a 840bpfragment. A semi-nested PCR was followed using the primer combination EBV109111F-EBV109869R resulting in a final 749 base pair product (Fig 1). Successful PCR samples werepurified using AMPure and Sanger sequenced on an automated 96 capillary ABI 3730XL DNAsequencer, followed by analysis with the ASSIGN V4.0.1.36 software (Conexio Genomics)(Fig 2). Threshold for mixture detection in sanger sequencing has been established to be ~20%.To study sequence minorities in quasispecies present at ≥1%, ultra-deep sequencing wasperformed using the FLX-454 Roche technology for a subset of 23 MS patients and a B95-8control. The same first round PCR products obtained for the sanger sequencing approach wereutilized for the high throughput sequencing approach. Second round primers contained anadditional tag on the 5′ end composing of 11 nucleotides of unique sequence (one each primerpair and sample) to enable pooling of samples in one FLX library. Samples were purified usingAMPure and concentrations measured with Nanodrop. Copy number per sample was calculatedand 1x1011 copies per sample were pooled. After equimolar pooling, the combined sampleswere purified and concentrated to 150.4 ng/µl using a minielute spin column (Qiagen). Onelibrary of all pooled samples was prepared and sequenced with 454 FLX in one lane out of eight(Fig 2) .Fig. 5. Epitope sequence variability in the AEGLRALLARSHVER epitope detected by FLX.grey: difference to reference strain B95-8blue: mixtures detected with FLXDiscussion and Conclusions:This study confirms the feasibility of direct EBNA-1 amplification from patient samples. Patientderived sequences were divergent from the B95-8 reference strain and clustered in four closegroups. FLX sequencing technology could detect additional low-level sequence variants but theywere generally rare, consistent with known limited dsDNA viral sequence variation. Of note,four samples could be sequenced using 454 FLX which could not be sequenced with Sangersequencing technology. However, these samples preferentially amplified chromosomal DNA withlow EBV reads, suggesting low EBV viral load in these samples.Analysing sequence variation in previously described EBNA-1 epitopes demonstrated concordantresults between Sanger and FLX 454 sequencing techniques, with limited evidence of viralquasispecies. The frequent detection of sequences that are divergent from B95-8 referencestrain suggests that studies investigating epitope-specific immune responses can be usefullyguided by autologous patient sequence information.In conclusion, low-level sequence variation was identified by sensitive FLX technology but wasgenerally rare. It remains to be determined if these minority species affect EBV-specific immuneresponses or disease outcomes.For FLX data analysis all homopolymers not present in the majority consensus sequence of thepatient sample have been excluded. All minorities present at <1% were not taken intoconsideration and insertion deletions were also excluded.14 samples tested with 454 FLX hadhigh coverage of the C-terminus withaverage reads between 80-1632. 10samples had low coverage and orreads (average reads <15) (Fig 4).Four samples (38585, 38695, 38939,38593) which could be amplified butnot Sanger sequenced weresuccessfully sequenced using FLX.Detailed analysis showed highamount of contigs representingchromosomal DNA but all showedalso low frequency EBV reads ofunique sequence.Table 2. Mixed amino acid positions for 14 samples with high coverage tested on 454 FLX. Minorities present at 1%, 2%, 5% and 10% cut off.Results:EBV could successfully be amplified in 72samples without previous culture to enrichfor EBV episomes. EBV Sanger sequencesclustered in four main groups. Interestingly,most of the samples did not cluster wellwith the EBV reference strain B59-8 (Fig.3).Genotyping of position 487 of EBNA-1identified the strain 487-threonine (n=52)and 487-alanine (n=14) as most prevalent,with alanine also present in the B95-8reference strain (Table 1).Fig. 3: Phylogenetic tree of C-terminal EBNA-1 patient sequences and the B95-8 reference sequence.Epitope coding sequence variability was directly compared using 14 samples sequenced withSanger and 454 FLX sequencing technology for three previously described epitopes: a putative Bcell epitope GRRPFFHPVGE (aa 401-411), and two HLA-DRB1*1501 epitopes (AEGLRALLARSHVER(aa 482-496) and MVFLQTHIFAEVLKD (aa 563-477). In our cohort, the most frequent variantswere identified as PPPGRRPFFHPVGEAD, AEGLRTLLARCHVER (Fig. 5) and IV/AFLQTHIFAEGLK.Binding scores (NetMHCIIpan) of patient derived epitope sequences revealed that FLXtechnology identified additional low frequency variants with HLA-DRB1*1501 restriction.Table 1. Genotyping amino acid position 487 of the EBNA-1 gene in 72 samples.Both sequencing methodswere concordant in detectingsequence variation potentiallyrelevant to epitope recognitionand presentation, with only aminority of sequences sharingidentity with the B95-8reference strain.Fig 1. Semi-nested, C-terminal EBNA-1 PCR. Numbers reflect the position of the primers in the B95-8 reference sequence.Fig 2. Workflow comparison sanger and 454 FLX sequencingcodons B95-8 gca gaa ggt tta aga gct ctc ctg gct agg agt cac gta gaa aggaa  B95-8 A E G L R A L L A R S H V E R samplesFLX - - - - - - - - - - - - - - -Sanger - - - - - - - - - - - - - - - 2FLX - - - - - GTT - - - - - - - - -Sanger - - - - - GTT - - - - - - - - - 1FLX - - - - AAA - - - - - - - - - -Sanger - - - - AAA - - - - - - - - - - 1FLX - - - - - RCT - - - - WGT - - - -Sanger - - - - - - - - - - - - - - - 1FLX - - GGY - - ACT - - - - TGT - - - -Sanger - - - - - ACT - - - - TGT - - - - 1FLX - - - - - RCT - - - - WGT - - - -Sanger - - - - - ACT - - - - TGT - - n.a. - 1FLX - - - - - ACT - - - - TGT - - - -Sanger - - - - - ACT - - - - TGT - - - - 7Samples with mutation in same position1 2 3total mutationsminorities 1% 41 12 2 55minorities 2% 21 13 1 35minorities 5% 23 6 1 30minorities 10% 20 6 0 26

Multiple Sclerosis Patient Sequence Variation In Epstein-Barr Virus Nuclear Antigen 1

https://researchrepository.murdoch.edu.au/id/eprint/36779/1/MS%20poster.pdf

Multiple Sclerosis Patient Sequence Variation In Epstein-Barr Virus Nuclear Antigen 1

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