Bidirectional transfer of human cytomegalovirus strains in donor and recipient seropositive lung transplant patients.

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.

Polyomavirus Nephropathy in ABO Blood Group-Incompatible Kidney Transplantation: Torque Teno Virus and Immunosuppressive Burden as an Approximation to the Problem.

Introduction: Earlier reports suggest that patients after ABO-incompatible kidney transplantation (ABOi) are at enhanced risk of developing BK-virus (BKV, also known as BK polyomavirus [BKPyV]) 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 human leukocyte antigen-incompatible (HLAi, i.e. pretransplant donor-specific antibody-positive) and standard-risk transplant recipients. Methods: Our retrospective study screened 2256 consecutive kidney transplantations performed between 2007 and 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- polymerase chain reaction (PCR) and BKV-PCR was carried out at predefined timepoints and ranged from pretransplant until month 24 posttransplantation. TTV loads and immunosuppression were evaluated in the context of BKV-associated complications. Results: ABOi recipients had a higher TTV load compared to HLAi and controls both at month 3 (median 1.5 × 109 vs. 2.4 × 108 vs. 9.1 × 107; P = 0.010) and at month 6 (3.1 × 109 vs. 1.4 × 107 vs. 6.4 × 107; P = 0.014) posttransplantation. 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). Conclusion: Our 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.

A Randomized Phase 2 Trial of Felzartamab in Antibody-Mediated Rejection.

BACKGROUND: Antibody-mediated rejection is a leading cause of kidney-transplant failure. The targeting of CD38 to inhibit graft injury caused by alloantibodies and natural killer (NK) cells may be a therapeutic option. METHODS: In this phase 2, double-blind, randomized, placebo-controlled trial, we assigned patients with antibody-mediated rejection that had occurred at least 180 days after transplantation to receive nine infusions of the CD38 monoclonal antibody felzartamab (at a dose of 16 mg per kilogram of body weight) or placebo for 6 months, followed by a 6-month observation period. The primary outcome was the safety and side-effect profile of felzartamab. Key secondary outcomes were renal-biopsy results at 24 and 52 weeks, donor-specific antibody levels, peripheral NK-cell counts, and donor-derived cell-free DNA levels. RESULTS: A total of 22 patients underwent randomization (11 to receive felzartamab and 11 to receive placebo). The median time from transplantation until trial inclusion was 9 years. Mild or moderate infusion reactions occurred in 8 patients in the felzartamab group. Serious adverse events occurred in 1 patient in the felzartamab group and in 4 patients in the placebo group; graft loss occurred in 1 patient in the placebo group. At week 24, resolution of morphologic antibody-mediated rejection was more frequent with felzartamab (in 9 of 11 patients [82%]) than with placebo (in 2 of 10 patients [20%]), for a difference of 62 percentage points (95% confidence interval [CI], 19 to 100) and a risk ratio of 0.23 (95% confidence interval [CI], 0.06 to 0.83). The median microvascular inflammation score was lower in the felzartamab group than in the placebo group (0 vs. 2.5), for a mean difference of -1.95 (95% CI, -2.97 to -0.92). Also lower was a molecular score reflecting the probability of antibody-mediated rejection (0.17 vs. 0.77) and the level of donor-derived cell-free DNA (0.31% vs. 0.82%). At week 52, the recurrence of antibody-mediated rejection was reported in 3 of 9 patients who had a response to felzartamab, with an increase in molecular activity and biomarker levels toward baseline levels. CONCLUSIONS: Felzartamab had acceptable safety and side-effect profiles in patients with antibody-mediated rejection. (Funded by MorphoSys and Human Immunology Biosciences; ClinicalTrials.gov number, NCT05021484; and EUDRACT number, 2021-000545-40.).

A Multivariant Surrogate Virus Neutralization Test Demonstrates Distinct SARS-CoV-2-Specific Antibody Responses in People Living with HIV after a Fourth Monovalent mRNA Vaccination or an Omicron Breakthrough Infection.

While neutralizing antibodies (nAbs) induced by monovalent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations are primarily directed against the wildtype (WT), subsequent exposure to the Omicron variants may increase the breadth of the antibodies' cross-neutralizing activity. Here, we analyzed the impact of an Omicron breakthrough infection (BTI) or a fourth monovalent mRNA vaccination on nAb profiles in people living with human immunodeficiency virus (PLWH). Using a multivariant surrogate virus neutralization test (sVNT), we quantified nAbs in 36 three-times vaccinated PLWH, of whom 9 acquired a serologically confirmed Omicron BTI, 8 received a fourth vaccine dose, and 19 were neither infected nor additionally vaccinated. While nAbs against WT and Delta increased after the BTI and a fourth vaccination, a significant increase against BA.1, BA.2, and BA.5 was only observed after the BTI. However, there was no significant difference in nAb concentrations between the samples obtained after the BTI and fourth vaccination. In contrast, nAb levels were significantly lower in PLWH, who were neither infected nor additionally vaccinated after three vaccinations. Thus, our study demonstrates the suitability of a multivariant sVNT to assess hybrid humoral immunity after Omicron BTIs in PLWH vaccinated against SARS-CoV-2.

External quality assessments for SARS-CoV-2 genome detection in Austria : A comparison of the first postpandemic round with results from the pandemic era.

BACKGROUND: External quality assessment (EQA) schemes provide objective feedback to participating laboratories about the performance of their analytical systems and information about overall regional analytical performance. The EQAs are particularly important during pandemics as they also assess the reliability of individual test results and show opportunities to improve test strategies. With the end of the COVID-19 pandemic, the testing frequency significantly decreased in Austria. Here, we analyzed whether this decrease had an effect on participation and/or performance in SARS-CoV­2 virus detection EQAs, as compared to the pandemic era. MATERIAL AND METHODS: Identical samples were sent to all participating laboratories, and the EQA provider evaluated the agreement of the reported results with defined targets. The EQA was operated under two schemes with identical samples and therefore we analyzed it as a single EQA round. The performance of testing was reported as true positive ratios, comparing the post-pandemic data to previous rounds. Furthermore, subgroups of participants were analyzed stratified by laboratory type (medical or nonmedical) and the test system format (fully automated or requiring manual steps). RESULTS: While the frequency of false negative results per sample did not change during the 3 years of the pandemic (5.7%, 95% confidence interval [CI] 3.1-8.4%), an average per sample false negative ratio of 4.3% was observed in the first post-pandemic EQA (0%, 1.8%, and 11% for the 3 positive samples included in the test panel, n = 109 test results per sample). In this first post-pandemic EQA medical laboratories (average 0.4% false negative across 3 samples, n = 90) and automated test systems (average 1.2% false negative, n = 261) had lower false negative ratios than nonmedical laboratories (22.8%, n = 19) and manual test systems (16.7%, n = 22). These lower average ratios were due to a low concentration sample, where nonmedical laboratories reported 36.8% and manual test systems 54.5% true positive results. CONCLUSION: Overall ratios of true positive results were below the mean of all results during the pandemic but were similar to the first round of the pandemic. A lower post-pandemic true positive ratio was associated with specific laboratory types and assay formats, particularly for samples with low concentration. The EQAs will continue to monitor the laboratory performance to ensure the same quality of epidemiological data after the pandemic, even if vigilance has decreased.

Virus sequencing performance during the SARS-CoV-2 pandemic: a retrospective analysis of data from multiple rounds of external quality assessment in Austria.

Introduction: A notable feature of the 2019 coronavirus disease (COVID-19) pandemic was the widespread use of whole genome sequencing (WGS) to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Countries around the world relied on sequencing and other forms of variant detection to perform contact tracing and monitor changes in the virus genome, in the hopes that epidemic waves caused by variants would be detected and managed earlier. As sequencing was encouraged and rewarded by the government in Austria, but represented a new technicque for many laboratories, we designed an external quality assessment (EQA) scheme to monitor the accuracy of WGS and assist laboratories in validating their methods. Methods: We implemented SARS-CoV-2 WGS EQAs in Austria and report the results from 7 participants over 5 rounds from February 2021 until June 2023. The participants received sample material, sequenced genomes with routine methods, and provided the sequences as well as information about mutations and lineages. Participants were evaluated on the completeness and accuracy of the submitted sequence and the ability to analyze and interpret sequencing data. Results: The results indicate that performance was excellent with few exceptions, and these exceptions showed improvement over time. We extend our findings to infer that most publicly available sequences are accurate within ≤1 nucleotide, somewhat randomly distributed through the genome. Conclusion: WGS continues to be used for SARS-CoV-2 surveillance, and will likely be instrumental in future outbreak scenarios. We identified hurdles in building next-generation sequencing capacity in diagnostic laboratories. EQAs will help individual laboratories maintain high quality next-generation sequencing output, and strengthen variant monitoring and molecular epidemiology efforts.

Measuring Variant-Specific Neutralizing Antibody Profiles after Bivalent SARS-CoV-2 Vaccinations Using a Multivariant Surrogate Virus Neutralization Microarray.

The capability of antibodies to neutralize different SARS-CoV-2 variants varies among individuals depending on the previous exposure to wild-type or Omicron-specific immunogens by mono- or bivalent vaccinations or infections. Such profiles of neutralizing antibodies (nAbs) usually have to be assessed via laborious live-virus neutralization tests (NTs). We therefore analyzed whether a novel multivariant surrogate-virus neutralization test (sVNT) (adapted from a commercial microarray) that quantifies the antibody-mediated inhibition between the receptor angiotensin-converting enzyme 2 (ACE2) and variant-specific receptor-binding domains (RBDs) can assess the neutralizing activity against the SARS-CoV-2 wild-type, and Delta Omicron BA.1, BA.2, and BA.5 subvariants after a booster with Omicron-adapted bivalent vaccines in a manner similar to live-virus NTs. Indeed, by using the live-virus NTs as a reference, we found a significant correlation between the variant-specific NT titers and levels of ACE2-RBD binding inhibition (p < 0.0001, r ≤ 0.78 respectively). Furthermore, the sVNTs identified higher inhibition values against BA.5 and BA.1 in individuals vaccinated with Omicron-adapted vaccines than in those with monovalent wild-type vaccines. Our data thus demonstrate the ability of sVNTs to detect variant-specific nAbs following a booster with bivalent vaccines.

Natural killer cell functional genetics and donor-specific antibody-triggered microvascular inflammation.

Antibody-mediated rejection (ABMR) is a leading cause of graft failure. Emerging evidence suggests a significant contribution of natural killer (NK) cells to microvascular inflammation (MVI). We investigated the influence of genetically determined NK cell functionality on ABMR development and activity. The study included 86 kidney transplant recipients subjected to systematic biopsies triggered by donor-specific antibody detection. We performed killer immunoglobulin-like receptor typing to predict missing self and genotyped polymorphisms determining NK cell functionality (FCGR3AV/F158 [rs396991], KLRC2wt/del, KLRK1HNK/LNK [rs1049174], rs9916629-C/T). Fifty patients had ABMR with considerable MVI and elevated NK cell transcripts. Missing self was not related to MVI. Only KLRC2wt/wt showed an association (MVI score: 2 [median; interquartile range: 0-3] vs 0 [0-1] in KLRC2wt/del recipients; P = .001) and remained significant in a proportional odds multivariable model (odds ratio, 7.84; 95% confidence interval, 2.37-30.47; P = .001). A sum score incorporating all polymorphisms and missing self did not outperform a score including only KLRC2 and FCGR3A variants, which were predictive in univariable analysis. NK cell genetics did not affect graft functional decline and survival. In conclusion, a functional KLRC2 polymorphism emerged as an independent determinant of ABMR activity, without a considerable contribution of missing self and other NK cell gene polymorphisms.

Ineffective control of Epstein-Barr-virus-induced autoimmunity increases the risk for multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the CNS. Epstein-Barr virus (EBV) contributes to the MS pathogenesis because high levels of EBV EBNA386-405-specific antibodies cross react with the CNS-derived GlialCAM370-389. However, it is unclear why only some individuals with such high autoreactive antibody titers develop MS. Here, we show that autoreactive cells are eliminated by distinct immune responses, which are determined by genetic variations of the host, as well as of the infecting EBV and human cytomegalovirus (HCMV). We demonstrate that potent cytotoxic NKG2C+ and NKG2D+ natural killer (NK) cells and distinct EBV-specific T cell responses kill autoreactive GlialCAM370-389-specific cells. Furthermore, immune evasion of these autoreactive cells was induced by EBV-variant-specific upregulation of the immunomodulatory HLA-E. These defined virus and host genetic pre-dispositions are associated with an up to 260-fold increased risk of MS. Our findings thus allow the early identification of patients at risk for MS and suggest additional therapeutic options against MS.

Comparison of anti-nucleocapsid antibody assays for the detection of SARS-CoV-2 Omicron vaccine breakthroughs after various intervals since the infection.

Antibody assays with the nucleocapsid (NC) protein as the target antigen can identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections when polymerase chain reaction (PCR) analyses are unavailable. Regarding the kinetics of NC-specific antibodies, vaccine breakthroughs with Omicron subvariants may differ from infections with the ancestral wild-type virus. Therefore, we evaluated which assays have the highest sensitivity for detecting NC-specific antibodies after various intervals since breakthrough infections with an Omicron subvariant. The study included 279 samples from vaccinated subjects who experienced PCR-confirmed Omicron breakthrough infections between 21 and 266 days before sampling. The samples were comparatively assessed with the Elecsys® Anti-SARS-CoV-2 N (Roche), the Anti-SARS-CoV-2-NCP-ELISA (Euroimmun), the recomLine SARS-CoV-2 IgG (Mikrogen), and the SARS-CoV-2 ViraChip IgG assays (Viramed). In the whole cohort, the Elecsys® Anti-SARS-CoV-2 N assay displayed the highest sensitivity (93%, p < 0.0001), followed by the recomLine SARS-CoV-2 IgG assay (70%), the SARS-CoV-2 ViraChip IgG assay (41%) and the Anti-SARS-CoV-2-NCP-ELISA (35%). Although measured antibody levels and time-dependent sensitivities differed, the extent of the antibody decrease was similar among all assays. As demonstrated by this study, manufacturer-dependent differences in the sensitivities of NC-specific antibody assays should be considered when serology is applied to link previous SARS-CoV-2 infections with potential post-COVID sequelae.

Performance of SARS-CoV-2 nucleic acid amplification testing in Austria as measured by external quality assessment schemes during 3 years of the COVID-19 pandemic: an observational retrospective study.

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 (93·7%) of 4663 qPCR test results were true-positive, 241 (5·2%) were false-negative, and 51 (1·1%) were inconclusive. The mean per-sample sensitivity was 99·7% in samples with high virus concentrations (1383 [99·4%] true-positive, three [0·2%] false-negative, and five [0·4%] 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 92·5%; 2988 [91·3%] true-positive, 238 [7·3%] false-negative, and 46 [1·4%] inconclusive results for 3272 tests in which the cycle threshold was >32). Of the 1645 results expected to be negative, 1561 (94·9%) were correctly reported as negative, 10 (0·6%) were incorrectly reported as positive, and 74 (4·5%) 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 2·94 (95% CI 1·75-4·96) times higher for a medical laboratory than for a non-medical laboratory, and 4·60 (2·91-7·41) 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. FUNDING: None.

Effect of monovalent COVID-19 vaccines on viral interference between SARS-CoV-2 and several DNA viruses in patients with long-COVID syndrome.

Epstein-Barr virus (EBV) reactivation may be involved in long-COVID symptoms, but reactivation of other viruses as a factor has received less attention. Here we evaluated the reactivation of parvovirus-B19 and several members of the Herpesviridae family (DNA viruses) in patients with long-COVID syndrome. We hypothesized that monovalent COVID-19 vaccines inhibit viral interference between SARS-CoV-2 and several DNA viruses in patients with long-COVID syndrome, thereby reducing clinical symptoms. Clinical and laboratory data for 252 consecutive patients with PCR-verified past SARS-CoV-2 infection and long-COVID syndrome (155 vaccinated and 97 non-vaccinated) were recorded during April 2021-May 2022 (median 243 days post-COVID-19 infection). DNA virus-related IgG and IgM titers were compared between vaccinated and non-vaccinated long-COVID patients and with age- and sex-matched non-infected, unvaccinated (pan-negative for spike-antibody) controls. Vaccination with monovalent COVID-19 vaccines was associated with significantly less frequent fatigue and multiorgan symptoms (p < 0.001), significantly less cumulative DNA virus-related IgM positivity, significantly lower levels of plasma IgG subfractions 2 and 4, and significantly lower quantitative cytomegalovirus IgG and IgM and EBV IgM titers. These results indicate that anti-SARS-CoV-2 vaccination may interrupt viral cross-talk in patients with long-COVID syndrome (ClinicalTrials.gov Identifier: NCT05398952).

Association of torque teno virus viremia with liver fibrosis in the first year after liver transplantation.

Introduction: Torque 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. Methods: One hundred plasma samples paired with graft biopsies from a prospective single-center biorepository were analyzed. Results: The 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 analysis. Discussion: The 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.

Inhibitory NKG2A+ and absent activating NKG2C+ NK cell responses are associated with the development of EBV+ lymphomas.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, which infects over 90% of the adult human population worldwide. After primary infections, EBV is recurrently reactivating in most adult individuals. It is, however, unclear, why these EBV reactivations progress to EBV+ Hodgkin (EBV+HL) or non-Hodgkin lymphomas (EBV+nHL) only in a minority of EBV-infected individuals. The EBV LMP-1 protein encodes for a highly polymorphic peptide, which upregulates the immunomodulatory HLA-E in EBV-infected cells, thereby stimulating the inhibitory NKG2A-, but also the activating NKG2C-receptor on natural killer (NK) cells. Using a genetic-association approach and functional NK cell analyses, we now investigated, whether these HLA-E-restricted immune responses impact the development of EBV+HL and EBV+nHL. Therefore, we recruited a study cohort of 63 EBV+HL and EBV+nHL patients and 192 controls with confirmed EBV reactivations, but without lymphomas. Here, we demonstrate that in EBV+ lymphoma patients exclusively the high-affine LMP-1 GGDPHLPTL peptide variant-encoding EBV-strains reactivate. In EBV+HL and EBV+nHL patients, the high-expressing HLA-E*0103/0103 genetic variant was significantly overrepresented. Combined, the LMP-1 GGDPHLPTL and HLA-E*0103/0103 variants efficiently inhibited NKG2A+ NK cells, thereby facilitating the in vitro spread of EBV-infected tumor cells. In addition, EBV+HL and EBV+nHL patients, showed impaired pro-inflammatory NKG2C+ NK cell responses, which accelerated the in vitro EBV-infected tumor cells spread. In contrast, the blocking of NKG2A by monoclonal antibodies (Monalizumab) resulted in efficient control of EBV-infected tumor cell growth, especially by NKG2A+NKG2C+ NK cells. Thus, the HLA-E/LMP-1/NKG2A pathway and individual NKG2C+ NK cell responses are associated with the progression toward EBV+ lymphomas.

A Multivariant Surrogate Neutralization Assay Identifies Variant-Specific Neutralizing Antibody Profiles in Primary SARS-CoV-2 Omicron Infection.

Primary infection with the Omicron variant of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) can be serologically identified with distinct profiles of neutralizing antibodies (nAbs), as indicated by high titers against the Omicron variant and low titers against the ancestral wild-type (WT). Here, we evaluated whether a novel surrogate virus neutralization assay (sVNT) that simultaneously quantifies the binding inhibition of angiotensin-converting enzyme 2 (ACE2) to the proteins of the WT- and Omicron-specific receptor-binding domains (RBDs) can identify nAb profiles after primary Omicron infection with accuracy similar to that of variant-specific live-virus neutralization tests (NTs). Therefore, we comparatively tested 205 samples from individuals after primary infection with the Omicron variant and the WT, and vaccinated subjects with or without Omicron breakthrough infections. Indeed, variant-specific RBD-ACE2 binding inhibition levels significantly correlated with respective NT titers (p < 0.0001, Spearman's r = 0.92 and r = 0.80 for WT and Omicron, respectively). In addition, samples from individuals after primary Omicron infection were securely identified with the sVNT according to their distinctive nAb profiles (area under the curve = 0.99; sensitivity: 97.2%; specificity: 97.84%). Thus, when laborious live-virus NTs are not feasible, the novel sVNT we evaluated in this study may serve as an acceptable substitute for the serological identification of individuals with primary Omicron infection.

Association between human genetic variants affecting the host NK cell response and the development of herpes simplex virus type 1 encephalitis.

Herpes simplex virus encephalitis (HSE) is a rare complication of herpes simplex virus type 1 (HSV-1) infection or reactivation. It is so far unclear why only few patients develop HSE. As natural killer (NK) cells provide an important defense against HSV-1, we investigated whether there is an association between distinct human genetic variants associated with the host NK cell response and HSE. Forty-nine adult patients with confirmed HSE and 247 matched controls were analyzed for the distribution of the following genotypes: CD16A (FcγRIIIA) V/F and IGHG1 G1m3/17, both influencing antibody-dependent cellular cytotoxicity; HLA-E*0101/*0103, associated with NK cell activation; and SLFN13 rs9916629C/T  associated with NK cell response. Homozygous HLA-E*0101:0101 and HLA-E*0103:0103 variants as well as the rs9916629CC genotype were overrepresented in HSE patients compared to controls (p ≤ 0.001). Notably, cooccurrence of the homozygous HLA-E*0101 and rs9916629CC genotypes was present in 19% of patients but totally absent in controls (p ≤ 0.0001). Distribution of CD16A and IGHG1 variants did not differ between patients and controls. Our data show that the rare combination of HLA-E*0101:0101 and rs9916629CC is significantly associated with HSE. Possibly, these genetic variations could be useful as clinical markers predicting HSE prognosis and helping to adapt the treatment of HSE in the individual patient.

Design of external quality assessment schemes and definition of the roles of their providers in future epidemics.

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.

Prediction of humoral and cellular immune response to COVID-19 mRNA vaccination by TTV load in kidney transplant recipients and hemodialysis patients.

BACKGROUND: Immunosuppressed individuals such as kidney transplant recipients (KTR) and hemodialysis patients (DP) show impaired immune responses to COVID-19 vaccination. Plasma Torque Teno Virus (TTV) DNA load is used as surrogate for the individual degree of immunosuppression. We now assessed the association of TTV load at time of COVID-19 vaccination with humoral and cellular immune response rates to vaccination in KTR, DP, and healthy medical personnel (MP). METHODS: A total of 100 KTR, 115 DP and 54 MP were included. All were SARS-CoV-2 seronegative at the time of vaccination with either BNT162b2 or mRNA-1273. Plasma TTV loads were assessed at the time of first vaccination. After two-dose vaccination, seroconversion (de novo detection of SARS-CoV-2 S1-IgA and/or IgG) was determined. In addition, cellular responses as assessed by interferon γ release and neutralizing antibodies were assessed in a subset of participants. ROC analyses were performed to define TTV load cut-offs predicting specific immune responses to vaccination. RESULTS: Plasma TTV loads at the time of first vaccination were negatively associated with seroconversion after two-dose vaccination in KTR (OR 0.87, 95% CI 0.76-0.99). TTV loads were significantly lower in KTR who developed humoral and cellular immune responses to vaccination compared to non-responders (p = 0.0411 and 0.0030, respectively). Of patients with TTV loads above 106 copies/ml, none developed cellular immune responses against SARS-CoV-2, and only 2 of 17 (12%) seroconverted in response to vaccination. CONCLUSION: Plasma TTV loads at the time of first vaccination in immunosuppressed individuals may be useful to predict individual vaccine-specific immune responses.

Three rounds of a national external quality assessment reveal a link between disharmonic anti-SARS-CoV-2 antibody quantifications and the infection stage.

OBJECTIVES: The WHO's standardized measuring unit, "binding antibody units per milliliter (BAU/mL)," should allow the harmonization of quantitative results by different commercial Anti-SARS-CoV-2 immunoassays. However, multiple studies demonstrate inter-assay discrepancies. The antigenic changes of the Omicron variant affect the performance of Spike-specific immunoassays. This study evaluated the variation of quantitative Anti-SARS-CoV-2-Spike antibody measurements among 46, 50, and 44 laboratories in three rounds of a national external quality assessment (EQA) prior to and after the emergence of the Omicron variant in a diagnostic near-to-real-life setting. METHODS: We analyzed results reported by the EQA participant laboratories from single and sequential samples from SARS-CoV-2 convalescent, acutely infected, and vaccinated individuals, including samples obtained after primary and breakthrough infections with the Omicron variant. RESULTS: The three immunoassays most commonly used by the participants displayed a low intra-assay and inter-laboratory variation with excellent reproducibility using identical samples sent to the participants in duplicates. In contrast, the inter-assay variation was very high with all samples. Notably, the ratios of BAU/mL levels quantified by different immunoassays were not equal among all samples but differed between vaccination, past, and acute infection, including primary infection with the Omicron variant. The antibody kinetics measured in vaccinated individuals strongly depended on the applied immunoassay. CONCLUSIONS: Measured BAU/mL levels are only inter-changeable among different laboratories when the same assay was used for their assessment. Highly variable ratios of BAU/mL quantifications among different immunoassays and infection stages argue against the usage of universal inter-assay conversion factors.