Some viruses including the widespread human cytomegalovirus (HCMV) show a high level of genetic diversity within hosts and across the whole virus population even in relatively conserved genomic regions. To investigate how this diversity is formed, we earlier introduced and analyzed a population model for neutrally evolving viruses that persist in their hosts and are capable of reinfection, mutation and recombination. Based on these results, we fit our model to observed genotype frequencies from Austrian HCMV patients. Despite simply assuming neutral evolution, the model captures the data closely. The inferred parameters are discussed and may give insights about the roles of viral replication, reinfection, mutation and recombination for the evolution of HCMV. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, 443227151 FWF Austrian Science Fund, https://ror.org/013tf3c58, P31503-B26

Torque Teno virus (TTV) is nonpathogenic, highly prevalent, and reflects the immune status of its host. Thus, TTV plasma load was suggested for the guidance of immunosuppression post solid organ transplantation. The present study was designed to determine the kinetics of TTV following changes in calcineurin inhibitor (CNI) dose. A total of 48 adult recipients of a kidney graft transplanted at the Medical University of Vienna between 2018 and 2019 with isolated changes in CNI dose were selected from the prospective TTV-POET trial. TTV plasma load was quantified by in-house PCR. At Day 30 following CNI dose adaptation (median 33% of daily dose) no changes in TTV load were noted. However, at Day 60, following CNI dose reduction a lower TTV load of 6.4 log(10) c/mL (median; interquartile range [IQR] 4.9-8.1) compared with the baseline of 7.1 log(10) c/mL (IQR 5.3-8.9) was noted (p=0.001); there was also a trend toward a higher TTV load following CNI increase (6.6 log(10) c/mL, IQR 4.1-9.7 vs. 5.2 log(10) c/mL, IQR 4.5-6.8; p=0.09). The data suggested that TTV load changes become noticeable only 2 months after CNI dose adaptation, which might be the ideal time point for TTV load monitoring.

IntroductionEarlier reports suggest that patients after ABO-incompatible kidney transplantation (ABOi) are at enhanced risk to develop BK-virus (BKV) nephropathy (BKPyVAN). It remains elusive whether this is a result of more intense immunosuppression or an ABOi-associated ‘intrinsic attribute’. To address this question, we measured Torque-Teno-Virus (TTV) loads as a quantitative proxy for immunosuppressive depth in ABOi recipients and compared them to HLA-incompatible (HLAi, i.e. pre-transplant donor-specific antibody-positive) and standard-risk transplant recipients.MethodsOur retrospective study screened 2256 consecutive kidney transplantations performed between 2007-2020 at the Medical University of Vienna. Out of 629 in-principle eligible transplantations, we were able to include 465 patients: 42 ABOi, 106 HLAi and 317 control recipients. Longitudinal TTV- and BKV-PCR was carried out at pre-defined timepoints and ranged from pre-transplant until month 24 post-transplantation. TTV loads and immunosuppression were evaluated in the context of BKV-associated-complications.ResultsABOi recipients had a higher TTV load compared to HLAi and controls both at month three (median 1.5x109 vs. 2.4x108 vs. 9.1x107, p=0.010) and month six (3.1x109 vs. 1.4x107 vs. 6.4x107, p=0.014) post-transplantation. Tacrolimus exposure was significantly higher in ABOi patients compared to HLAi and control patients (ABOi vs. HLAi: p=0.007, ABOi vs. controls: p<0.0001). Biopsy-proven BKPyVAN was more frequent in ABOi recipients when compared to HLAi- and control-recipients (11.9% vs. 2.8% vs. 4.1%, p=0.046).ConclusionOur data support the assumption that ABOi patients are indeed at higher risk to develop BKPyVAN. A higher TTV load and immunosuppressive burden suggest that intense immunosuppression, rather than an ‘intrinsic attribute’ conferred by ABOi, may contribute to this finding.

Objectives Results of earlier external quality assessment (EQA) rounds suggested remarkable differences in the sensitivity of SARS-CoV PCR assays. Although the test systems are intended to detect SARS-CoV-2 in individual samples, screening is often applied to sample pools to increase efficiency and decrease costs. However, it is unknown to what extent these tests actually meet the manufacturer's specifications for sensitivity and how they perform when testing sample pools. Methods The sensitivity of assays in routine use was evaluated with a panel of positive samples in a round of a SARS-CoV-2 virus genome detection EQA scheme. The panel consisted of samples at or near the lower limit of detection ("weakly positive"). Laboratories that routinely test sample pools were asked to also analyze the pooled EQA samples according to their usual pool size and dilution method. Results All participants could detect a highly positive patient-derived sample (>106 copies/mL). Most (96%) of the test systems could detect at least 1,000 copies/mL, meeting the minimum acceptable benchmark, and many (94%) detected the vRNA in a sample with lower concentration (500 copies/mL). The false negative ratio increased to 16 and 26% for samples with 100 and 50 copies/mL, respectively. Conclusions The performance of most assays met or exceeded their specification on sensitivity. If assays are to be used to analyze sample pools, the sensitivity of the assay and the number of pooled samples must be balanced.

ABSTRACT Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell–cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion. IMPORTANCE Human cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.

Glycoproteins in the viral envelope of human cytomegalovirus (HCMV) orchestrate virion tethering, receptor recognition and fusion with cellular membranes. The glycoprotein gB acts as fusion protein. The gHgL complexes gHgLgO and gHgLpUL(128,130,131A) define the HCMV cell tropism. Studies with HCMV lacking gO had indicated that gO, independently of binding to its cellular receptor PDGFR alpha, plays an important second role in infection. Here, we identified a gO mutation which abolished virus particle infectivity by preventing the interaction of gHgLgO with host cell heparan sulfate proteoglycans (HSPGs). We could not only show that gHgLgO - HSPG interactions are a genuine second role of gHgLgO, but also that gHgLgO is the main player in determining the infectivity of HCMV virus particles. This challenges long-accepted textbook knowledge on the role of gB and gMgN complexes in virion tethering. Additionally, it adds the gHgLgO complex to the antigens of interest for future HCMV vaccines or treatments. ### Competing Interest Statement The authors have declared no competing interest.

Donor and recipient human cytomegalovirus (HCMV) seropositive (D+R+) lung transplant recipients (LTRs) often harbor multiple strains of HCMV, likely due to transmitted donor (D) strains and reactivated recipient (R) strains. To date, the extent and timely occurrence of each likely source in shaping the post-transplantation (post-Tx) strain population is unknown. Here, we deciphered the D and R origin of the post-Tx HCMV strain composition in blood, bronchoalveolar lavage (BAL), and CD45+ BAL cell subsets. We investigated either D and/or R formalin-fixed paraffin-embedded blocks or fresh D lung tissue from four D+R+ LTRs obtained before transplantation. HCMV strains were characterized by short amplicon deep sequencing. In two LTRs, we show that the transplanted lung is reseeded by R strains within the first 6 months after transplantation, likely by infiltrating CD14+ CD163+/- alveolar macrophages. In three LTRs, we demonstrate both rapid D-strain dissemination and persistence in the transplanted lung for >1 year post-Tx. Broad inter-host diversity contrasts with intra-host genotype sequence stability upon transmission, during follow-up and across compartments. In D+R+ LTRs, HCMV strains of both, D and R origin can emerge first and dominate long-term in subsequent episodes of infection, indicating replication of both sources despite pre-existing immunity.

The ubiquitous Torque teno virus (TTV) establishes a chronically persistent infection in the human host. TTV has not been associated with any apparent disease, but, as part of the human virome, it may confer a regulatory imprint on the human immune system with as yet unclear consequences. However, so far, only few studies have characterized the TTV-specific immune responses or the overall immunological imprints by TTV. Here, we reveal that TTV infection leads to a highly exhausted TTV-specific CD8+ T-cell response, hallmarked by decreased IFN-γ production and the expression of the inhibitory NKG2A-receptor. On a functional level, we identified a panel of highly polymorphic TTV-encoded peptides that lead to an expansion of regulatory NKG2A+ natural killer, NKG2A+CD4+, and NKG2A+CD8+ T cells via the stabilization of the non-classical HLA-E molecule. Our results thus demonstrate that TTV leads to a distinct imprint on the human immune system that may further regulate overall human immune responses in infectious, autoimmune, and malignant diseases.

During an epidemic, individual test results form the basis of epidemiological indicators such as case numbers or incidence. Therefore, the accuracy of measures derived from these indicators depends on the reliability of individual results. In the COVID-19 pandemic, monitoring and evaluating the performance of the unprecedented number of testing facilities in operation, and novel testing systems in use, was urgently needed. External quality assessment (EQA) schemes are unique sources of data reporting on testing performance, and their providers are recognised contacts and support for test facilities (for technical-analytical topics) and health authorities (for planning the monitoring of infection diagnostics). To identify information provided by SARS-CoV-2 genome detection EQA schemes that is relevant for public health microbiology, we reviewed the current literature published in PubMed between January, 2020, and July, 2022. We derived recommendations for EQA providers and their schemes for best practices to monitor pathogen-detection performance in future epidemics. We also showed laboratories, test facilities, and health authorities the information and benefits they can derive from EQA data, and from the non-EQA services of their providers.

Background: Torque Teno virus (TTV) is non-pathogenic, highly prevalent and reflects the immune status of its host. TTV plasma load was suggested for risk stratification of graft rejection and infection post kidneytransplantation, for which most studies applied an in-house PCR. Recently, a commercial PCR was CEcertified for clinical use. The present study was designed to assess the performance of TTV load as quantified by the commercial PCR in the prediction of graft rejection and infection. Methods: Patients and events were selected from the prospective TTV-POET trial, including 683 consecutive adult recipients of a kidney-graft transplanted at the Medical University Vienna, 2016-2020. TTV was quantified in plasma drawn in Months 4-12 post-transplant by in-house and commercial PCR and associated with consecutive infections and graft rejections until Month 12 post-transplantation.Results: A total of 342 samples from 314 patients with 85 biopsies (rejection, n = 18) and 79 infectious events were assessed. The two PCRs were highly associated (estimate 0.91, 95%CI 0.89-0.93), with a mean difference of 1.38 log10 copies/mL (95%CI 1.46-1.30). The risk of rejection decreased by 25% with every log10 increase in TTV load as quantified by commercial PCR (RR 0.75, 95%CI 0.67-0.85), and the risk of infection increased by 6% (RR 1.06, 95%CI 1.00-1.12). Conclusion: These data support the value of TTV quantification by commercial PCR for the risk stratification of graft rejection and infection in the first year post kidney-transplantation. The test performance determined within this study may serve to design clinical trials and subsequently, support application in clinical routine.

Background: The aim of external quality assessment (EQA) schemes is to provide an evaluation of the analytical performance of laboratories and test systems in a near-to-real-life setting. This monitoring service not only provides confidential feedback to participant laboratories, but also serves as a control measure for the epidemiological assessment of the regional incidence during a pandemic. Particularly, when regulations for diagnostic procedures, test systems and facilities are suspended in times of a pandemic, such independent assessments of the analytical quality are essential.Methods: In this study, a total of 6260 qualitative PCR test results reported by 191 laboratories for 67 samples in 14 rounds of EQA schemes performed on SARS-CoV-2 pathogen detection between May 2020 and March 2023 were retrospectively analysed. The analysis particularly aimed to assess false-negativity rates in samples in relation to different virus loads and laboratory types, as well as the changes in performance over time.Findings: During three years and 14 EQA rounds, a total of 93·7% of PCR test results were true positive, 5·2% were false negative, and 1·1% were inconclusive. Detection rates decreased with in samples with lower virus concentration. In samples with < 100000 copies/mL (Ct >30), the correct positive detection rate was 91·5%, while the false negative rate was 7·1% (1·4% results were inconclusive). A total 0·6% of test results obtained from samples negative for SARS-CoV-2 RNA were false positive, with 2·9% inconclusive. Notably the overall test performance did not change significantly over the three years. The odds of a nonmedical laboratory reporting a false negative result were 2·7 times the odds of a medical laboratory.Interpretation: High rates of false negativity in all PCR analyses evaluated in comprehensive, multiple and repeated EQA schemes outline a clear path for improvement in the future.Funding: None.Declaration of Interest: The authors declare no competing interests.Ethical Approval: Ethical approval is not required for our work as we do not include any patient data but only report on results of external quality assessment schemes.

Background The aim of external quality assessment (EQA) schemes is to evaluate the analytical performance of laboratories and test systems in a near-to-real-life setting. This monitoring service provides feedback to participant laboratories and serves as a control measure for the epidemiological assessment of the regional incidence of a pathogen, particularly during epidemics. Using data from EQA schemes implemented as a result of the intensive effort to monitor SARS-CoV-2 infections in Austria, we aimed to identify factors that explained the variation in laboratory performance for SARS-CoV-2 detection over the course of the COVID-19 pandemic. Methods For this observational study, we retrospectively analysed 6308 reverse transcriptase quantitative PCR (RT-qPCR) test results reported by 191 laboratories on 71 samples during 14 rounds of three SARS-CoV-2 pathogen detection EQA schemes in Austria between May 18, 2020, and Feb 20, 2023. We calculated the overall rates of false and true-negative, false and true-positive, and inconclusive results. We then assessed laboratory performance by estimating the sensitivity by testing whether significant variation in the odds of obtaining a true-positive result could be explained by virus concentration, laboratory type, or assay format. We also assessed whether laboratory performance changed over time. Findings 4371 (937%) of 4663 qPCR test results were true-positive, 241 (52%) were false-negative, and 51 (11%) were inconclusive. The mean per-sample sensitivity was 997% in samples with high virus concentrations (1383 [994%] true-positive, three [02%] false-negative, and five [04%] inconclusive results for 1391 tests in which the sample cycle threshold was <= 32), whereas detection rates were lower in samples with low virus concentrations (mean per-sample sensitivity 925%; 2988 [913%] true-positive, 238 [73%] false-negative, and 46 [14%] inconclusive results for 3272 tests in which the cycle threshold was >32). Of the 1645 results expected to be negative, 1561 (949%) were correctly reported as negative, 10 (06%) were incorrectly reported as positive, and 74 (45%) were reported as inconclusive. Notably, the overall performance of the tests did not change significantly over time. The odds of reporting a correct result were 294 (95% CI 175-496) times higher for a medical laboratory than for a non-medical laboratory, and 460 (291-741) times greater for automated test systems than for manual test systems. Automated test systems within medical laboratories had the highest sensitivity when compared with systems requiring manual intervention in both medical and non-medical laboratories. Interpretation High rates of false-negativity in all PCR analyses evaluated in comprehensive, multiple, and repeated EQA schemes outline a clear path for improvement in the future. The performance of some laboratories (eg, non-medical laboratories or those using non-automated test systems) should receive additional scrutiny-for example, by requiring additional EQA schemes for certification or accreditation-if the aggregated data from EQA rounds suggest lower sensitivity than that recorded by others. This strategy will provide assurances that epidemiological data as a whole are reliable when testing on such a large scale. Although performance did not improve over time, we cannot exclude extenuating circumstances-such as shortages and weakened supply chains-that could have prevented laboratories from seeking alternative methods to improve performance.

The caseA 32-year-old female Caucasian patient presented to the emergency department in January 2020 with diarrhea, fever, cough and progressing leg edema, after returning from Turkey.Patient history revealed relapsing remitting multiple sclerosis (MS) since 2003 with various immunosuppressive treatments.In 2013 she received rituximab (2 × 375 mg/m 2 body surface, two weeks apart), and no further MS relapses recurred thereafter (EDSS 2.0).Initial chest CT scans showed extensive bilateral lung infiltrations and pleural effusions, while laboratory results revealed microcytic, hypochromic anemia (Hb 9.9 g/dL), elevated acute phase parameters (leukocytosis 16.6 G/L, C-reactive protein 307.7 mg/L), hypoalbuminemia (1.84 g/dL) and proteinuria (spot urine protein/creatinine ratio 1777 mg/g, normal serum creatinine).Empiric treatment with cefuroxime, azithromycin and loop diuretics improved most symptoms,

IntroductionTorque teno virus (TTV) replication is controlled by immune status, mirroring a degree of immunosuppression after solid organ transplantation. TTV viraemia (TTVv) was associated with acute cellular rejection and infection within the first year after liver transplantation (LT). Long-term data on TTV after LT and correlation with graft injury from protocol biopsies are limited. MethodsOne hundred plasma samples paired with graft biopsies from a prospective single-center biorepository were analyzed.ResultsThe median time post-LT was 23 months (range, 2–298). TTVv was detectable in 97%. TTVv decreased over time after LT and showed a significant decline from year 1 to later time points. Hence, TTVv correlated negatively with histologic liver fibrosis (liver allograft fibrosis and Ishak scores) and positively with the overall immunosuppression degree quantified by an immunosuppression score in the first year after LT. There was no association with dosages or trough levels of single immunosuppressants. The pharmacodynamic marker TTVv did not correlate with pharmacokinetic assessments of immunosuppression degree [calcineurin inhibitor (CNI) trough levels or immunosuppressant dosages]—our clinical gold standards to guide immunosuppressive therapy. TTVv was independently associated with histologically proven liver fibrosis after LT in the first year after LT in multivariate analysisDiscussionThe independent association of histological graft fibrosis with lower TTVv in year 1 underscores that a pharmacodynamic marker would be preferable to individualize immunosuppression after LT. However, a high variability of TTVv at the low immunosuppression doses given after the first year precludes TTV as a clinically useful marker after LT in the long-term liver transplant recipients.

Background: The detection of SARS-CoV-2 vRNA in clinical samples has relied almost exclusively on RT-qPCR as the gold standard test. Published results from various external quality assessments ("ring trials") worldwide have shown that there is still a large variability in results reported for the same samples. As reference standards of SARS-CoV-2 RNA are available, we tested whether using standard curves to convert Ct values into copies/mL (cp/mL) improved harmonization.Methods: Nine laboratories using 23 test systems (15 of which were unique) prepared standard dilution curves to convert Ct values of 13 SARS-CoV-2 positive samples to cp/mL (hereafter IU/mL). The samples were provided in three rounds of a virus genome detection external quality assessment (EQA) scheme. We tested the precision and accuracy of results reported in IU/mL, and attempted to identify the sources of variability.Results: Reporting results as IU/mL improved the precision of the estimated concentrations of all samples compared to reporting Ct values, although some inaccuracy remained. Variance analysis showed that nearly all variability in data was explained by individual test systems within individual laboratories. When controlling for this effect, there was no significant difference between all other factors tested (test systems, EQA rounds, sample material).Conclusions: Converting results to copies/mL improved precision across laboratory test systems. However, it seems the results are still very specific to test systems within laboratories. Further efforts could be made to improve accuracy and achieve full harmonization across diagnostic laboratories.

Background The virome of lung transplant recipients (LTRs) under immunosuppressive therapy is dominated by non-pathogenic Anelloviridae and further includes several pathogenic viruses such as Herpesviruses or respiratory viruses. It is unclear whether the donor-derived virome in the transplanted lung influences recipient virome dynamics in other body compartments and if so, to which degree. Likewise, it is unknown whether dependencies exist among virus populations that mutually shape viral loads and kinetics. Results To address these questions, we characterized viral communities in airways and plasma of 49 LTRs and analyzed their abundance patterns in a data modeling approach. We found distinct viral clusters that were specific for body compartments and displayed independent dynamics. These clusters robustly gathered specific viral species across the patient cohort. In the lung, viral cluster abundance associated with time after transplantation and we detected mutual exclusion of viral species within the same human host. In plasma, viral cluster dynamics were associated with the indication for transplantation lacking significant short-time changes. Interestingly, pathogenic viruses in the plasma co-occurred specifically with Alpha torque virus genogroup 4 and Gamma torque virus strains suggesting shared functional or ecological requirements. Conclusions In summary, the detailed analysis of virome dynamics after lung transplantation revealed host, body compartment, and time-specific dependency patterns among viruses. Furthermore, our results suggested genetic adaptation to the host microenvironment at the level of the virome and support the hypothesis of functional complementarity between Anellovirus groups and other persistent viruses.

Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 10 4 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Potent immunosuppressive drugs have been introduced into clinical care for solid organ transplant recipients. It is now time to guide these drugs on an individual level to optimize their efficacy. An ideal tool simultaneously detects overimmunosuppression and underimmunosuppression, is highly standardized, and is straightforward to implement into routine. Randomized controlled interventional trials are crucial to demonstrate clinical value. To date, proposed assays have mainly focused on the prediction of rejection and were based on the assessment of few immune compartments. Recently, novel tools have been introduced based on a more integrated approach to characterize the immune function and cover a broader spectrum of the immune system. In this respect, the quantification of the plasma load of a highly prevalent and apathogenic virus that might reflect the immune function of its host has been proposed: the torque teno virus (TTV). Although TTV control is driven by T cells, other major immune compartments might contribute to the hosts' response. A standardized in-house polymerase chain reaction and a conformité européenne-certified commercially available polymerase chain reaction are available for TTV quantification. TTV load is associated with rejection and infection in solid organ transplant recipients, and cutoff values for risk stratification of such events have been proposed for lung and kidney transplantation. Test performance of TTV load does not allow for the diagnosis of rejection and infection but is able to define at-risk patients. Hitherto TTV load has not been used in interventional settings, but two interventional randomized controlled trials are currently testing the safety and efficacy of TTV-guided immunosuppression.

Human cytomegalovirus (CMV) utilizes different glycoproteins to enter into fibroblast and epithelial cells. A trimer of glycoproteins H, L, and O (gH/gL/gO) is required for entry into all cells, whereas a pentamer of gH/gL/UL128/UL130/UL131A is selectively required for infection of epithelial, endothelial, and some myeloid-lineage cells, but not of fibroblasts. Both complexes are of considerable interest for vaccine and immunotherapeutic development but present a conundrum: gH/gL-specific antibodies have moderate potency yet neutralize CMV entry into all cell types, whereas pentamer-specific antibodies are more potent but do not block fibroblast infection. Which cell types and neutralizing activities are important for protective efficacy in vivo remain unclear. Here, we present evidence that certain CMV strains have evolved polymorphisms in gO to evade trimer-specific neutralizing antibodies. Using luciferase-tagged variants of strain TB40/E in which the native gO is replaced by gOs from other strains, we tested the effects of gO polymorphisms on neutralization by monoclonal antibodies (mAbs) targeting four independent epitopes in gH/gL that are common to both trimer and pentamer. Neutralization of fibroblast entry by three mAbs displayed a range of potencies that depended on the gO type, a fourth mAb failed to neutralize fibroblast entry regardless of the gO type, while neutralization of epithelial cell entry by all four mAbs was potent and independent of the gO type. Thus, specific polymorphisms in gO protect the virus from mAb neutralization in the context of fibroblast but not epithelial cell entry. No influence of gO type was observed for protection against CMV hyperimmune globulin or CMV-seropositive human sera, suggesting that antibodies targeting protected gH/gL epitopes represent a minority of the polyclonal neutralizing repertoire induced by natural infection.

Abstract BACKGROUND AND AIMS The nonpathogenic and ubiquitous Torque Teno virus (TTV) plasma load is associated with immunosuppression in solid organ transplant recipients. An optimal TTV range has been defined for risk stratification of graft rejection and infection in the first year post kidney transplant applying an in-house PCR. Recently, a commercial PCR—the TTV R-GENE® kit—has been CE certified for clinical use. The present study was designed to validate and refine the optimal TTV range applying the TTV R-GENE® kit. METHOD Patients and events were selected from the prospective TTV-POET trial, including all 628 consecutive adult recipients of a kidney allograft transplanted at the Medical University of Vienna, between January 2016 and July 2020. Patients were followed for 12 months post-transplant or until drop-out. TTV was quantified longitudinally by the TTV R-GENE® kit. The primary outcome was biopsy proven graft rejection and the secondary end-point was infection. RESULTS A total of 78 patients with 85 graft biopsies (rejection, n = 18) and 274 patients with 80 infectious events following TTV quantification after reaching steady state in month 3 post-transplantation were selected. TTV was quantified at a median of 17 days before rejection was diagnosed and 63 days before onset of infection. The risk for rejection decreased by 25% with every log level increase in TTV load [RR 0.75, 95% confidence interval (95% CI) 0.67–0.85; < 0.001]. For TTV loads < 5log10 c/mL a high specificity of 90% for rejection was calculated. The risk for an infection increased by 6% with every log level increase of TTV load (RR 1.06, IQR 1.00–0.12; = 0.047). For TTV loads > 7log10 c/mL a high specificity of 91% for infection was calculated. Multivariate modelling revealed an independent associated between TTV and rejection and infection, respectively. CONCLUSION These data support the value of TTV quantified by TTV R-GENE® for risk stratification of graft rejection and infection in the first year post kidney transplantation. The optimal TTV range defined within this study will be applied in an interventional randomized controlled trial to assess the safety of TTV-guided immunosuppression: the TTVguideIT trial.