Post-Vaccination Coronavirus Disease 2019: A Case-Control Study and Genomic Analysis of 119 Breakthrough Infections in Partially Vaccinated Individuals.

BACKGROUND: Post-vaccination infections challenge the control of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: We matched 119 cases of post-vaccination severe acute respiratory syndrome coronavirus 2 infection with BNT162b2 mRNA or ChAdOx1 nCOV-19 to 476 unvaccinated patients with COVID-19 (September 2020-March 2021) according to age and sex. Differences in 60-day all-cause mortality, hospital admission, and hospital length of stay were evaluated. Phylogenetic, single-nucleotide polymorphism (SNP), and minority variant allele (MVA) full-genome sequencing analysis was performed. RESULTS: Overall, 116 of 119 cases developed COVID-19 post-first vaccination dose (median, 14 days). Thirteen of 119 (10.9%) cases and 158 of 476 (33.2%) controls died (P < .001), corresponding to the 4.5 number needed to treat (NNT). Multivariably, vaccination was associated with a 69.3% (95% confidence interval [CI]: 45.8 to 82.6) relative risk (RR) reduction in mortality. Similar results were seen in subgroup analysis for patients with infection onset ≥14 days after first vaccination and across vaccine subgroups. Hospital admissions (odds ratio, 0.80; 95% CI: .51 to 1.28) and length of stay (-1.89 days; 95% CI: -4.57 to 0.78) were lower for cases, while cycle threshold values were higher (30.8 vs 28.8, P = .053). B.1.1.7 was the predominant lineage in cases (100 of 108, 92.6%) and controls (341 of 446, 76.5%). Genomic analysis identified 1 post-vaccination case that harbored the E484K vaccine-escape mutation (B.1.525 lineage). CONCLUSIONS: Previous vaccination reduces mortality when B.1.1.7 is the predominant lineage. No significant lineage-specific genomic changes during phylogenetic, SNP, and MVA analysis were detected.

Diagnostic accuracy of Abbott Architect Assay as a screening tool for human T-cell leukaemia virus type-1 and type-2 infection in a London teaching hospital with a large solid organ transplant centre.

AIM: In the United Kingdom, organ donors/recipients are screened for evidence of human T-cell leukaemia virus type-1 and type-2 (HTLV-1/2) infections. Since the United Kingdom is a low prevalence country for HTLV infections, a screening assay with high sensitivity and specificity is required. Samples with repeat reactivity on antibody testing are sent to a reference lab for confirmatory serological and molecular testing. In the case of donor screen, this leads to delays in the release of organs and can result in wastage. We aim to assess whether a signal/cut-off (S/CO) ratio higher than the manufacturer's recommendation of 1.0 in the Abbott Architect antibody assay is a reliable measure of HTLV-1/2 infection. METHODS: We conducted a 5 year retrospective analysis of 7245 patients from which 11 766 samples were tested on the Abbott Architect rHTLV I/II assay. Reactive samples (S/CO >1) were referred for confirmatory serological and molecular detection (Western Blot and proviral DNA) at UK Health Security Agency, (formerly PHE, Colindale), the national reference laboratory. Electronic, protected laboratory and hospital patient databases were employed to collate data. RESULTS: A total of 45 patients had initially reactive samples. 42.2% (n = 19/45) had an S/CO ratio > 20, with HTLV infection confirmed in n = 18/19 and indeterminate confirmatory results in n = 1/19. No samples with an S/CO ratio <4 (48.9%, n = 22/45) or 4-20 (8.9%, n = 4/45) had positive confirmatory results on subsequent confirmatory testing. CONCLUSION: Samples with an S/CO >20 likely represent a true HTLV-1/2 infection. Reactive samples with an S/CO <4 were unlikely to confirm for HTLV infections. Interpretation of these ratios can assist clinicians in the assessment of low reactive samples and reiterates the need for faster access to confirmatory testing.

High Viral Diversity and Mixed Infections in Cerebral Spinal Fluid From Cases of Varicella Zoster Virus Encephalitis.

Background: Varicella zoster virus (VZV) may cause encephalitis, both with and without rash. Here we investigate whether viruses recovered from the central nervous system (CNS; encephalitis or meningitis) differ genetically from those recovered from non-CNS samples. Methods: Enrichment-based deep sequencing of 45 VZV genomes from cerebral spinal fluid (CSF), plasma, bronchoalveolar lavage (BAL), and vesicles was carried out with samples collected from 34 patients with and without VZV infection of the CNS. Results: Viral sequences from multiple sites in the same patient were identical at the consensus level. Virus from vesicle fluid and CSF in cases of meningitis showed low-level diversity. By contrast, plasma, BAL, and encephalitis had higher numbers of variant alleles. Two CSF-encephalitis samples had high genetic diversity, with variant frequency patterns typical of mixed infections with different clades. Conclusions: Low viral genetic diversity in vesicle fluid is compatible with previous observations that VZV skin lesions arise from single or low numbers of virions. A similar result was observed in VZV from cases of VZV meningitis, a generally self-limiting infection. CSF from cases of encephalitis had higher diversity with evidence for mixed clade infections in 2 cases. We hypothesize that reactivation from multiple neurons may contribute to the pathogenesis of VZV encephalitis.

Transmission of Hepatitis B Core Antibody and Galactomannan Enzyme Immunoassay Positivity via Immunoglobulin Products: A Comprehensive Analysis.

BACKGROUND: Therapeutic immunoglobulins are used as replacement or immunomodulatory therapy, but can transmit clinically important molecules. We investigated hepatitis B virus (HBV) antibodies and galactomannan enzyme immunoassay (GM-EIA) positivity. Detection of HBV core antibody may prompt antiviral prophylaxis when commencing therapy such as rituximab; a positive GM-EIA result prompts investigation or treatment for invasive fungal disease. METHODS: We performed a cross-sectional analysis of HBV serology in 80 patients established (>6 months) on immunoglobulin therapy; prospective analysis of HBV serology in 16 patients commencing intravenous immunoglobulin (IVIG); and pre- and post-infusion analysis of GM-EIA in 37 patients receiving IVIG. RESULTS: Pre-IVIG, 9 of 80 patients tested positive for HBV surface antibody and 1 of 80 tested equivocal for HBV core antibody. On IVIG, 79 of 79 tested positive for surface antibody, 37 of 80 tested positive for core antibody, and 10 of 80 tested equivocal for core antibody. There were significant differences by product, but among patients receiving products that appear to transmit core antibody, negative results correlated with lower surface antibody titers and longer time since infusion, suggesting a simple concentration effect. There was a progressive increase with each infusion in the percentage of patients testing positive for HBV core antibody among patients newly commencing IVIG. Some patients "seroreverted" to negative during therapy. Certain IVIG products tested positive for GM-EIA and there were rises in index values in corresponding patient samples from pre- to post-infusion. Overall, 5 of 37 patient samples pre-infusion and 15 of 37 samples post-infusion tested positive for GM-EIA. CONCLUSIONS: HBV antibodies and GM-EIA positivity are common in patients receiving IVIG and confound diagnostic results.

Recombination of Globally Circulating Varicella-Zoster Virus.

UNLABELLED: Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpes virus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE: Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

The prevalence of occult hepatitis B virus (HBV) infection in a large multi-ethnic haemodialysis cohort.

BACKGROUND: Haemodialysis patients are at increased risk of exposure to blood borne viruses. To reduce transmission in the UK, all haemodialysis patients are regularly screened, and if susceptible to Hepatitis B virus (HBV) infection, vaccinated. METHODS: This retrospective study was undertaken to determine the HBV immune status in a large dialysis cohort and the prevalence of occult HBV infection, defined as the presence of anti-HBcore antibody (anti-HBcAb) and HBV DNA without detectable HB surface antigen (HBsAg). Information on HBV status was retrieved from haemodialysis patients under the care of The Royal Free Hospital, London, UK between 2009-2010. Available sera from 138 of 161 anti-HBcAb positive/HBsAg negative individuals were anonymised and tested for HBV DNA by a real time quantitative PCR. RESULTS: 15 (2%) of 793 patients had chronic HBV infection (HBsAg positive). 161 (20%) were anti-HBcAb positive but HBsAg negative suggesting past infection. 335 (54%) of the remaining 617 patients were considered immune following vaccination (anti-HBsAb > 10 IU/L). Three (2.2%) of the 138 anti-HBcAb positive, HBsAg negative patients had detectable HBV DNA (3, 5 and 9 IU/ml). Standard liver function tests were normal in these patients. CONCLUSIONS: In a large multi-ethnic London haemodialysis cohort, 20% patients had evidence of past HBV infection. Despite this, the prevalence of occult HBV was found to be low and the very low levels of HBV DNA detected are unlikely to pose a nosocomial transmission risk in the presence of robust vaccination and infection control measures.

Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease.

Epstein-Barr virus (EBV) is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV-specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV-associated non-haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre-terminal. Thus, this third party donor-derived EBV-specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes.

Serological cross reactivity to CMV and EBV causes problems in the diagnosis of acute hepatitis E virus infection.

Hepatitis E virus (HEV) infection is an important public health concern as a major cause of enterically-transmitted hepatitis worldwide. The detectable window of viraemia is narrow, and HEV IgM and IgG rise simultaneously in acute infection. Furthermore, previous investigators have shown HEV IgM false positive reactions occur against EBV, CMV and potentially hepatitis A. A retrospective analysis of HEV serology testing was performed at a London tertiary referral hospital over a 3-year period. A thousand four hundred and twenty three serum samples were tested for HEV serology, with 33 samples HEV IgM positive and 28 HEV IgM equivocal. One hundred and eleven samples were HEV IgG positive but IgM negative suggesting past infection. No patients with HEV IgM positivity had false positive reactions against hepatitis A. A high degree of EBV and CMV cross reactivity was noted, with 33.3% and 24.2% of HEV IgM positive samples also testing positive for EBV and CMV IgM, respectively. HEV RNA was detected in four HEV IgM positive samples, indicating true positivity, although three demonstrated cross reactivity against EBV. Only 13.3% of samples with positive HEV IgM were HEV PCR positive, highlighting a low positive predictive value of serology testing. Overall a high level of HEV, EBV and CMV IgM cross reactivity was demonstrated, indicating that serology is unreliable in the diagnosis of acute viral hepatitis. It is concluded that that the diagnosis of viral hepatitis should be based on clinical features, raised transaminases, serology, and confirmatory PCR testing.

Interleukin 28B gene polymorphisms and Epstein-Barr virus-associated lymphoproliferative diseases.

OBJECTIVES: Single-nucleotide polymorphisms (SNPs) near the interleukin (IL) 28B gene encoding a type III interferon (IFN-λ) are the most important genetic predictors of treatment response to hepatitis C virus (HCV). This retrospective study was undertaken to determine any association between IL28B SNPs and the development of viraemia in Epstein-Barr virus (EBV)-driven acute infectious mononucleosis (IM) and post-transplant lymphoproliferative disease (PTLD). METHODS: Genomic DNA extracted from plasma from 45 EBV seropositive controls and 46 acute IM, 23 non-PTLD (transplant) and 21 PTLD patients was tested by PCR for 2 SNPs within IL28B. EBV DNA levels were tested in IM and PTLD samples by a real-time quantitative PCR. RESULTS: No significant differences were seen in SNP frequencies at rs12979860 and rs8099917 in IM and PTLD patients compared to EBV seropositive controls and transplant patients. EBV DNA levels were lower in IM and PTLD patients with CC (a favourable genotype in HCV) at rs12979860 compared to non-CC genotypes (p = 0.055). Acute IM patients with CC had significantly lower levels of EBV DNA in plasma compared to those with non-CC genotypes (p = 0.011). CONCLUSIONS: Genotype CC may influence anti-viral responses of IFN-λ, thereby allowing better control of EBV viraemia during lymphoproliferation, particularly in IM.

Characteristics of Epstein-Barr viraemia in adult liver transplant patients: a retrospective cohort study.

Therapeutic immunosuppression following solid organ transplantation increases the risk of Epstein-Barr (EBV) viraemia, which is implicated in post-transplant lymphoproliferative disease (PTLD). We retrospectively analysed the incidence of EBV viraemia and clinical outcomes in 98 liver transplant recipients. Patients underwent EBV DNA monitoring by whole-blood PCR: EBV levels were correlated with clinical parameters and outcomes for a median of 249 days. 67% patients developed EBV viraemia (EBV DNA ≥100 copies/ml) and 30% had sustained viraemia. There was a trend towards higher hazard ratios for viraemia with exposure to aciclovir (HR 1.57, P = 0.12) or in recipients of a poorly HLA-matched graft (HR 1.62, P = 0.10). These associations became significant in the subgroup with >90 days surveillance; HR 2.54 (P = 0.0015) for aciclovir and HR 1.99 (P = 0.03) for poorly matched grafts. The converse was true with ganciclovir (HR 0.56 P = 0.13). Viraemia was more prolonged in men (median duration 7 days vs 1; P = 0.01) and in those with lower UKELD scores (11 days vs 1 day; P = 0.001) but shortened with ganciclovir exposure (P = 0.06). Younger patients were more likely to have high peak viral loads (P = 0.07). No clinical signs or symptoms or adverse outcomes were associated with EBV reactivation.

Opt-out HIV screening in adults attending the Emergency Department of a London teaching hospital.

BACKGROUND: Opt-out Emergency Department blood borne virus (EDBBV) screening was introduced at the Royal Free Hospital under the NHSEI (NHS England and NHS Improvement) programme to expand opt-out testing in local authority areas with high HIV prevalence. This initiative was part of the "Toward Zero" policy towards ending HIV transmission in England by 2030. METHODS: All patients attending the Royal Free Hospital Emergency Department (ED) aged 16 and over were screened for blood borne viruses (HIV/HBV/HCV) unless they opted out. We looked at HIV data from patients seen in ED between the initiation of EDBBV testing on the 12th of April and 12th of August 2022. Hepatitis B and C data was reviewed in a separate study. OUTCOME: A total of 12,208 samples from 10,641 patients were screened for HIV. Amongst these samples there were 88 which were positive, giving a seroprevalence of 0.84 %. There were 48 patients who were already known to local HIV services, 35 were known to HIV services outside of our Trust and 5 were new diagnoses. CONCLUSION: Our results confirmed our local HIV prevalence to be very high, as per the UK Health Security Agency and supports the need for HIV testing. Opt-out ED BBV screening has been a highly effective method for identifying people living with HIV who are unaware of their status.

Opt-out testing for hepatitis B and C infections in adults attending the emergency department of a large London teaching hospital.

BACKGROUND: The National Health Service (NHS) in England commissioned opt-out testing in London Emergency Departments (ED) in April 2022 to allow early identification and management of hepatitis B (HBV) and hepatitis C virus (HCV) infection in patients unaware of their infection status. METHODS: All adults over the age of 16 undergoing blood tests in the ED at the Royal Free Hospital were tested for HBV surface antigen and anti-HCV IgG unless they opted out. Data was collected between the 12th of April and 22nd of August 2022. OUTCOME: Of 11,215 patients tested for HCV, 164 patients were found to be anti-HCV IgG positive, giving a seroprevalence rate of 1.46 %. 52 of the anti-HCV IgG positive patients did not have any previous HCV serology result. 23 of the anti-HCV IgG positive patients were also HCV RNA positive giving an RNA seroprevalence of 0.21 %, and 17 of those were new diagnoses of HCV viraemia. For HBV testing, 82 (0.73 %) out of 11,192 patients tested were found to be HBsAg positive, including one patient who presented acutely with a positive HBV core IgM. 39 of the HBsAg positive patients were previously unknown to us; of these, 9 had an HBV viral load of more than 2000 IU/mL, including 3 patients with positive HBV e antigen and one patient with hepatitis D virus co-infection. CONCLUSION: Opt-out screening of HBV and HCV in ED is effective at identifying patients with previously undiagnosed viral hepatitis infection and providing an opportunity to engage them in specialist care.

Cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant in transplant recipients: a phase 2 randomised placebo-controlled trial.

BACKGROUND: Cytomegalovirus end-organ disease can be prevented by giving ganciclovir when viraemia is detected in allograft recipients. Values of viral load correlate with development of end-organ disease and are moderated by pre-existing natural immunity. Our aim was to determine whether vaccine-induced immunity could do likewise. METHODS: We undertook a phase-2 randomised placebo controlled trial in adults awaiting kidney or liver transplantation at the Royal Free Hospital, London, UK. Exclusion criteria were pregnancy, receipt of blood products (except albumin) in the previous 3 months, and simultaneous multiorgan transplantation. 70 patients seronegative and 70 seropositive for cytomegalovirus were randomly assigned from a scratch-off randomisation code in a 1:1 ratio to receive either cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant or placebo, each given at baseline, 1 month and 6 months later. If a patient was transplanted, no further vaccinations were given and serial blood samples were tested for cytomegalovirus DNA by real-time quantitative PCR (rtqPCR). Any patient with one blood sample containing more than 3000 cytomegalovirus genomes per mL received ganciclovir until two consecutive undetectable cytomegalovirus DNA measurements. Safety and immunogenicity were coprimary endpoints and were assessed by intention to treat in patients who received at least one dose of vaccine or placebo. This trial is registered with ClinicalTrials.gov, NCT00299260. FINDINGS: 67 patients received vaccine and 73 placebo, all of whom were evaluable. Glycoprotein-B antibody titres were significantly increased in both seronegative (geometric mean titre 12,537 (95% CI 6593-23,840) versus 86 (63-118) in recipients of placebo recipients; p<0.0001) and seropositive (118,395; 64,503-217,272) versus 24,682 (17,909-34,017); p<0.0001) recipients of vaccine. In those who developed viraemia after transplantation, glycoprotein-B antibody titres correlated inversely with duration of viraemia (p=0.0022). In the seronegative patients with seropositive donors, the duration of viraemia (p=0.0480) and number of days of ganciclovir treatment (p=0.0287) were reduced in vaccine recipients. INTERPRETATION: Although cytomegalovirus disease occurs in the context of suppressed cell-mediated immunity post-transplantation, humoral immunity has a role in reduction of cytomegalovirus viraemia. Vaccines containing cytomegalovirus glycoprotein B merit further assessment in transplant recipients. FUNDING: National Institute of Allergy and Infectious Diseases, Grant R01AI051355 and Wellcome Trust, Grant 078332. SPONSOR: University College London (UCL).

Soluble CD30: a serum marker for Epstein-Barr virus-associated lymphoproliferative diseases.

The soluble form of CD30 (sCD30), a member of tumor necrosis factor receptor superfamily, has been used as a marker of disease activity in various lymphomas. Epstein-Barr virus (EBV) is a potent stimulator of CD30 expression. The study aims to evaluate whether sCD30 can be used as a diagnostic marker for EBV-associated infectious mononucleosis (IM) and post-transplant lymphoproliferative disease (PTLD). Plasma from EBV seropositive healthy controls (N = 90), acute IM patients (n = 90), non-PTLD heart/lung transplant recipients (N = 30) and EBV-positive PTLD patients (N = 23) was tested for sCD30 using a commercially available ELISA kit. EBV DNA was tested by real time quantitative polymerase chain reaction assay. Significantly higher sCD30 levels were observed in acute IM patients (median 242.9 ng/ml) compared to EBV seropositive controls (median 15.7 ng/ml; P < 0.0001). These levels were highest in IM patients within 14 days of onset of illness. PTLD patients had significantly higher sCD30 levels (median 94 ng/ml) than healthy controls (P < 0.0001) and transplant patients (median 27 ng/ml; P = 0.0007). EBV DNA was detected mostly in acute IM and PTLD patients. In both cases there was a significant correlation between sCD30 and EBV DNA levels in plasma (P < 0.0001). This study demonstrates that sCD30 and EBV DNA levels can be used as potential markers for diagnosis of IM and PTLD.

Epitope specificity and clonality of EBV-specific CTLs used to treat posttransplant lymphoproliferative disease.

In a recent phase II clinical trial using banked allogeneic CTL lines to treat EBV-associated posttransplant lymphoproliferative disease, a response rate of 52% was recorded 6 mo posttreatment. Tumor response was associated with an increase in both CTL/recipient HLA matches and CD4(+) T cells within the infused CTL lines. The present study was undertaken to correlate tumor response with CTL specificity. The majority of CTL lines infused recognized EBV-encoded nuclear Ag-3 proteins, but CTL protein specificity itself did not correlate with tumor response. Specificity in conjunction with donor/recipient functional HLA matching as opposed to HLA matching alone, however, was important for tumor response. CTL receptor TCR beta-chain variable gene subfamilies were polyclonal, with no preferential use of a particular family. However, tumor response was improved in those receiving CTL lines with polyclonal vs clonal distribution for subfamilies 2, 3, and 9. Interestingly, in five of six tumors (five Hodgkin's-like and one Burkitt's-like posttransplant lymphoproliferative disease) with restricted viral gene expression a complete response was recorded, although in some cases the tumor cells did not express the proteins recognized by the infused CTL. Thus CTL were advantageous when functionally HLA matched but for certain tumor types complete responses occurred in the absence of detectable specific CTL/tumor recognition. We suggest that either the allogenic CTL contained small, undetectable, EBV-specific, HLA-matched T cell populations or perhaps they stimulated nonspecific inflammatory responses in vivo, which were beneficial for tumor regression. These observations should be considered when designing and implementing CTL therapies.

Whole genome sequencing of Herpes Simplex Virus 1 directly from human cerebrospinal fluid reveals selective constraints in neurotropic viruses.

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70 per cent of the global population. Clinical manifestations are largely restricted to recurrent epidermal vesicles. However, HSV-1 also leads to encephalitis, the infection of the brain parenchyma, with high associated rates of mortality and morbidity. In this study, we performed target enrichment followed by direct sequencing of HSV-1 genomes, using target enrichment methods on the cerebrospinal fluid (CSF) of clinical encephalitis patients and from skin swabs of epidermal vesicles on non-encephalopathic patients. Phylogenetic analysis revealed high inter-host diversity and little population structure. In contrast, samples from different lesions in the same patient clustered with similar patterns of allelic variants. Comparison of consensus genome sequences shows HSV-1 has been freely recombining, except for distinct islands of linkage disequilibrium (LD). This suggests functional constraints prevent recombination between certain genes, notably those encoding pairs of interacting proteins. Distinct LD patterns characterised subsets of viruses recovered from CSF and skin lesions, which may reflect different evolutionary constraints in different body compartments. Functions of genes under differential constraint related to immunity or tropism and provide new hypotheses on tissue-specific mechanisms of viral infection and latency.

Cytolytic mechanisms and T-cell receptor Vbeta usage by ex vivo generated Epstein-Barr virus-specific cytotoxic T lymphocytes.

Ex-vivo-generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable beta-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0.0001) and ethylene glycol-bis tetraacetic acid (P < 0.0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-gamma and TNF-alpha. Granzyme B, perforin and Fas ligand were detected in CD8(+) and CD4(+) cells in all CTL; however, a greater proportion of CD8(+) than CD4(+) T cells expressed granzyme B (P < 0.0001) and more granzyme B was detected in CD8(+) T cells than in CD4(+) T cells (P = 0.001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.

Cytomegalovirus viral load parameters associated with earlier initiation of pre-emptive therapy after solid organ transplantation.

BACKGROUND: Human cytomegalovirus (HCMV) can be managed by monitoring HCMV DNA in the blood and giving valganciclovir when viral load exceeds a defined value. We hypothesised that such pre-emptive therapy should occur earlier than the standard 3000 genomes/ml (2520 IU/ml) when a seropositive donor transmitted virus to a seronegative recipient (D+R-) following solid organ transplantation (SOT). METHODS: Our local protocol was changed so that D+R- SOT patients commenced valganciclovir once the viral load exceeded 200 genomes/ml; 168 IU/ml (new protocol). The decision point remained at 3000 genomes/ml (old protocol) for the other two patient subgroups (D+R+, D-R+). Virological outcomes were assessed three years later, when 74 D+R- patients treated under the old protocol could be compared with 67 treated afterwards. The primary outcomes were changes in peak viral load, duration of viraemia and duration of treatment in the D+R- group. The secondary outcome was the proportion of D+R- patients who developed subsequent viraemia episodes. FINDINGS: In the D+R- patients, the median values of peak viral load (30,774 to 11,135 genomes/ml, p<0.0215) were significantly reduced on the new protocol compared to the old, but the duration of viraemia and duration of treatment were not. Early treatment increased subsequent episodes of viraemia from 33/58 (57%) to 36/49 (73%) of patients (p< 0.0743) with a significant increase (p = 0.0072) in those episodes that required treatment (16/58; 27% versus 26/49; 53%). Median peak viral load increased significantly (2,103 to 3,934 genomes/ml, p<0.0249) in the D+R+ but not in the D-R+ patient subgroups. There was no change in duration of viraemia or duration of treatment for any patient subgroup. INTERPRETATION: Pre-emptive therapy initiated at the first sign of viraemia post-transplant significantly reduced the peak viral load but increased later episodes of viraemia, consistent with the hypothesis of reduced antigenic stimulation of the immune system.

Respiratory Infections and Antibiotic Usage in Common Variable Immunodeficiency.

BACKGROUND: Patients with common variable immunodeficiency (CVID) suffer frequent respiratory tract infections despite immunoglobulin replacement and are prescribed significant quantities of antibiotics. The clinical and microbiological nature of these exacerbations, the symptomatic triggers to take antibiotics, and the response to treatment have not been previously investigated. OBJECTIVES: To describe the nature, frequency, treatment, and clinical course of respiratory tract exacerbations in patients with CVID and to describe pathogens isolated during respiratory tract exacerbations. METHODS: We performed a prospective diary card exercise in 69 patients with CVID recruited from a primary immunodeficiency clinic in the United Kingdom, generating 6210 days of symptom data. We collected microbiology (sputum microscopy and culture, atypical bacterial PCR, and mycobacterial culture) and virology (nasopharyngeal swab multiplex PCR) samples from symptomatic patients with CVID. RESULTS: There were 170 symptomatic exacerbations and 76 exacerbations treated by antibiotics. The strongest symptomatic predictors for commencing antibiotics were cough, shortness of breath, and purulent sputum. There was a median delay of 5 days from the onset of symptoms to commencing antibiotics. Episodes characterized by purulent sputum responded more quickly to antibiotics, whereas sore throat and upper respiratory tract symptoms responded less quickly. A pathogenic virus was isolated in 56% of respiratory exacerbations and a potentially pathogenic bacteria in 33%. CONCLUSIONS: Patients with CVID delay and avoid treatment of symptomatic respiratory exacerbations, which could result in structural lung damage. However, viruses are commonly represented and illnesses dominated by upper respiratory tract symptoms respond poorly to antibiotics, suggesting that antibiotic usage could be better targeted.