B19-Specific CD8⁺ T Cells Persist at High Levels for Many Months after Acute Infection

<div><p>(A) Representative A24 FYT tetramer staining of individual O3\′s PBMCs. Plots are gated on live CD8⁺ lymphocytes stained directly ex vivo. Percentages shown are those of tetramer-positive CD8⁺ T cells. Time points indicated refer to the number of months after first symptoms reported. Symptoms in this individual lasted 5 wk.</p> <p>(B) Frequency of B19-specific responses over time for six acutely infected individuals in the Oxford cohort (O1–O5) and five remotely infected individuals (OR1–OR5). In one case, two epitopes were studied.</p> <p>(C) Frequency of B19-specific responses over time for five acutely infected individuals in the Stockholm cohort (S1–S5). In two cases, two epitopes were studied.</p></div

Representative Ex Vivo Phenotyping of B19-Specific CD8⁺ T Cell Populations over Time

<div><p>Percentages shown are the frequency of marker-positive cells amongst multimer-positive cells (plots gated on live CD8⁺ lymphocytes ex vivo).</p> <p>(A) Patient S2 B40 TEA pentamer staining at 4 mo (left) and 21 mo (right), showing perforin and CD62L levels.</p> <p>(B) Patient O3 A24 FYT tetramer staining at 2 mo (left) and 20 mo (right), showing CD27 and CD28 staining.</p> <p>(C) Patient O5 A2 GLC tetramer staining at 4 mo (left) and 18 mo (right), showing CD38 and CD57 staining.</p></div

Acutely infected individuals Maintain Activated Mature Effector Cells after Resolution of Acute Infection

<p>Remotely infected individuals show a less mature/activated phenotype but express only low levels of CCR7 and CD62L (Oxford cohort). The y-axis show the frequency of marker-positive cells amongst tetramer-positive CD8⁺ cells, while the x-axis show the number of months after symptom onset. Data are shown in perforin, CD38, CD57, CD27, CD28, and CCR7: data are derived from six acutely infected individuals in the Oxford cohort (O1–O3, O5, and O6; insufficient cells available for O4) and five remotely infected individuals (OR1–OR5).</p

Longitudinal Phenotype Analysis of B19-Specific CD8⁺ T Cell Responses in Acutely Infected Individuals in Stockholm Cohort

<div><p>(A) Blood from patient S1 was stained with the two tetramers A2 LLH and A2 GLC.</p> <p>(B) Blood from patient S2 was stained with tetramers A2 GLC and the Pro5 B40 TEA pentamer. The top panels show the frequency of tetramer-positive cells over time for the two different responses in each individual (data equivalent to those in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020343#pmed-0020343-g001" target="_blank">Figure 1</a>C). The subsequent panels show the frequency of B19-specific CD8⁺ cells positive for perforin, CD38, CD57, and CD62L in both patients.</p></div

B19-Specific CD8⁺ T Cells Secrete IFNγ Ex Vivo, Proliferate, and Show Cytolytic Function In Vitro

<div><p>(A) Left panel shows that PBMCs from acutely infected patient O1 secrete IFNγ ex vivo after 18 h of FYTPLADQF peptide stimulation. Negative control (zero spots) and two peptide-stimulated wells from an ELISpot plate are shown. Numbers represent IFNγ-secreting cells per 250,000 PBMCs. Right panel shows A24 FYT tetramer staining of PBMCs at same time point, displaying the number of tetramer-positive cells expressed as a percentage of CD8⁺ T cells.</p> <p>(B) Tetramer staining of patient S2\′s PBMCs ex vivo (left) and after short-term TEADVQQWL peptide stimulation in vitro (right).</p> <p>(C) Ex vivo IFNγ ELISpot results for remotely infected individual OR3. Mean and standard deviations of triplicates are shown. Cells were stimulated for 18 h with no peptide, GLCPHCINV, or TEADVQQWL.</p> <p>(D) ⁵¹Cr release assay using HLA-A2-restricted GLCPHCINV-specific CTLs from individual OR1. PBMCs were stimulated for 14 d with GLCPHCINV peptide and cytolysis was tested against HLA-A*0201-transfected LBL.721.220 target cells at various effector-to-target-cell (E:T) ratios.</p></div

Hepatitis B virus (HBV): serological markers of infection and immunity in a cohort of South African children

Children age 6-60 months were recruited as part of the Co-infection in South-African Children (COSAC) study, in Kimberley, South Africa. HIV-negative participants (n=174) were recruited through the Kimberley Respiratory Cohort (KReC). These children were admitted to hospital with a clinical diagnosis of respiratory tract infection between July 2014 and August 2016. HIV-positive children (n=136) were recruited primarily from HIV outpatient clinics, (recruited between September 2009 and July 2016). Children had research blood samples taken at the same time as routine clinical blood tests. We tested all samples for markers of hepatitis B virus (HBV) infection and immunity. HBsAg testing was carried out in Kimberley Hospital, South Africa using the Magnetic parcel chemiluminometric immunoassay (MPCI; Advia Centaur platform). Confirmatory HBsAg testing, together with anti-HBs and anti-HBc testing, was carried out by the microbiology department at Oxford University Hospitals (OUH) NHS Foundation Trust, Oxford, UK (Architect i2000). Ethics approval for this study was obtained from the Ethics Committee of the Faculty of Health Science, University of the Free State, Bloemfontein, South Africa (HIV Study Ref: ETOVS Nr 08/09 and COSAC Study Ref: ECUFS NR 80/2014). Written consent for enrollment into the study was obtained from the child’s parent or guardian

Oxford Screening of CSF and Respiratory Samples ('OSCAR'): Supplementary resources for a project using Next Generation Sequencing (NGS) for identification of viruses from clinical laboratory samples

<p>This is a file set that describes the methods used in a small pilot study to investigate 'next generation sequencing' (NGS) derived from an Illumina platform, applied to clinical diagnostic samples following routine testing in a UK microbiology laboratory.</p><p>We briefly summarise the benefits and challenges of an NGS approach to diagnostics, and conclude with some potential methodological improvements.</p><p>The file set includes anonymised metadata for the samples tested (10 respiratory and 10 CSF).</p><p>Results have been made available through a separate DOI: 10.6084/m9.figshare.5712091 and the entire genomic metadata have been submitted to European Nucleotide Archive (ENA); (primary accession PRJEB22949). </p><p>This study was approved through the UK integrated research application system (REC reference 14/LO/1077).</p

Oxford Screening of CSF and Respiratory Samples ('OSCAR'): interactive data visualisation using Krona to display results from a pilot project using Next Generation Sequencing (NGS) for identification of viruses from clinical laboratory samples

This is an html file that allows visualisation of metagenomic dataset from a pilot project to screen CSF and respiratory samples using Illumina HiSeq after completion of routine diagnostic testing in a UK microbiology laboratory.<div><br></div><div>Methods and underlying metadata are available through a separate DOI (<a rel="noreferrer noopener" target="_blank">10.6084/m9.figshare.5670007). The entire metagenomic dataset has been uploaded to the European Nucleotide Archive (</a>primary accession PRJEB22949). <a rel="noreferrer noopener" target="_blank">A subset of these data have been used for a project to identify human herpesvirus 6 (HHV-6) in clinical samples; this manuscript is available on BioRxiv (</a>https://doi.org/10.1101/236083).</div><div><div><div><div><br></div></div><div>This study was approved through the UK integrated research application system (REC reference 14/LO/1077).</div></div></div

HCV+ Subjects 14.08.18.xlsx

We performed a retrospective study of HCV infection in a UK teaching hospital to evaluate and compare the performance of different laboratory tests, to describe the population with active HCV infection and to determine the proportion of these individuals who access clinical review and treatment. Microbiology records for all HCV screening tests performed at Oxford University Hospitals within two defined time-intervals (Group 1 - 18 months; Jan 2013 - June 2014, Group 2 - 15 months; Jan 2015 - March 2016) were studied . For those testing positive we collected follow-up testing data. We recorded patient age, sex, and the location from which the sample was sent. We used an analytical tool to estimate ethnicity applying Onolytics software for all patients whom a full name was part of the electronic record. Ethics approval was not required, as this study was undertaken as a departmental quality improvement exercise within microbiology using anonymised patient data, and completed the audit cycle for previously approved audit projects. Data for Onolytics analysis were handled separately and were subject to a confidential disclosure agreement drawn up by University of Oxford Research Services (February 2016). <div><div><br><div><br></div><div><br></div><div><br></div><div><br></div><div><br></div><div><br></div><div><br></div><div><br></div><div><br></div><div><br></div><div><br></div></div></div

Relationship between HBV status and markers of HIV disease in HIV-positive women from South Africa and Botswana.

<p>Panels (A) and (B): South Africa (Durban + Kimberley cohorts pooled); Panels (C) and (D): Botswana (Gaborone). Left-hand column (panels (A) and (C)) shows CD4+ T cell counts; right-hand column (panels (B) and (D)) shows HIV-1 RNA viral load. In each case, box represents median and 25/75^th centiles, whiskers 5-95^th centiles. P values by Mann Whitney U test.</p