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.

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.).

HLA-E-restricted immune responses are crucial for the control of EBV infections and the prevention of PTLD.

Primary Epstein-Barr virus (EBV) infections may cause infectious mononucleosis (IM), whereas EBV reactivations in solid organ and hematopoietic stem cell transplant recipients are associated with posttransplantation lymphoproliferative disorders (PTLDs). It is still unclear why only a minority of primary EBV-infected individuals develop IM, and why only some patients progress to EBV+PTLD after transplantation. We now investigated whether nonclassic human leukocyte antigen E (HLA-E)-restricted immune responses have a significant impact on the development of EBV diseases in the individual host. On the basis of a large study cohort of 1404 patients and controls as well as on functional natural killer (NK) and CD8+ T-cell analyses, we could demonstrate that the highly expressed HLA-E∗0103/0103 genotype is protective against IM, due to the induction of potent EBV BZLF1-specific HLA-E-restricted CD8+ T-cell responses, which efficiently prevent the in vitro viral dissemination. Furthermore, we provide evidence that the risk of symptomatic EBV reactivations in immunocompetent individuals as well as in immunocompromised transplant recipients depends on variations in the inhibitory NKG2A/LMP-1/HLA-E axis. We show that EBV strains encoding for the specific LMP-1 peptide variants GGDPHLPTL or GGDPPLPTL, presented by HLA-E, elicit strong inhibitory NKG2A+ NK and CD8+ T-cell responses. The presence of EBV strains encoding for both peptides was highly associated with symptomatic EBV reactivations. The further progression to EBV+PTLD was highly associated with the presence of both peptide-encoding EBV strains and the expression of HLA-E∗0103/0103 in the host. Thus, HLA-E-restricted immune responses and the NKG2A/LMP-1/HLA-E axis are novel predictive markers for EBV+PTLD in transplant recipients and should be considered for future EBV vaccine design.

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.

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.

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.

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.

The natural killer cell-associated rs9916629-C allele is a novel genetic risk factor for fatal COVID-19.

The severity of COVID-19 is associated with individual genetic host factors. Among these, genetic polymorphisms affecting natural killer (NK) cell responses, as variations in the HLA-E- (HLA-E*0101/0103), FcγRIIIa- (FcγRIIIa-158-F/V), and NKG2C- (KLRC2wt/del ) receptor, were associated with severe COVID-19. Recently, the rs9916629-C/T genetic polymorphism was identified that indirectly shape the human NK cell repertoire towards highly pro-inflammatory CD56bright NK cells. We investigated whether the rs9916629-C/T variants alone and in comparison to the other risk factors are associated with a fatal course of COVID-19. We included 1042 hospitalized surviving and 159 nonsurviving COVID-19 patients as well as 1000 healthy controls. rs9916629-C/T variants were genotyped by TaqMan assays and were compared between the groups. The patients' age, comorbidities, HLA-E*0101/0103, FcγRIIIa-158-F/V, and KLRC2wt/del variants were also determined. The presence of the rs9916629-C allele was a risk factor for severe and fatal COVID-19 (p < 0.0001), independent of the patients' age or comorbidities. Fatal COVID-19 was more frequent in younger patients (<69.85 years) carrying the FcγRIIIa-158-V/V (p < 0.006) and in older patients expressing the KLRC2del variant (p < 0.003). Thus, patients with the rs9916629-C allele have a significantly increased risk for fatal COVID-19 and identification of the genetic variants may be used as prognostic marker for hospitalized COVID-19 patients.

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.

High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19.

PURPOSE: Host genetic variants in activating natural killer (NK) cell receptors may contribute to differences in severity of COVID-19. NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) responses play, however, a controversial role in SARS-CoV-2 infections. It is unclear whether proinflammatory and cytotoxic SARS-CoV-2-specific ADCC responses limit disease severity or rather contribute to the immunopathogenesis of severe COVID-19. METHODS: Using a genetic association approach and subsequent in vitro antibody-dependent NK cell activation experiments, we investigated whether genetic variants in the FcγRIIIa-encoding FCGR3A gene, resulting in expression of either a low-affinity or high-affinity variant, and individual SARS-CoV-2-specific ADCC response contribute to COVID-19 severity. RESULTS: In our study, we showed that the high-affinity variant of the FcγRIIIa receptor, 158-V/V, is significantly over-represented in hospitalized and deceased patients with COVID-19, whereas the low-affinity FcγRIIIa-158-F/F variant occurs more frequently in patients with mild COVID-19 (P < .0001). Furthermore, functional SARS-CoV-2 antibody-specific NK cell-mediated ADCC assays revealed that significantly higher proinflammatory ADCC responses occur in hospitalized patients with COVID-19, and are especially observed in NK cells expressing the FcγRIIIa-158-V/V variant (P < .0001). CONCLUSION: Our study provides evidence that pronounced SARS-CoV-2-specific NK cell-mediated ADCC responses are influenced by NK cell FcγRIIIa genetic variants and are a hallmark of severe COVID-19.

Torque Teno Virus quantification for monitoring of immunomodulation with biologic compounds in the treatment of rheumatoid arthritis.

OBJECTIVES: RA patients who fail to respond to MTX can receive biologic dMARDs (bDMARDs). The Torque Teno Virus (TTV) is a potential novel candidate for monitoring of immunosuppression. We explore TTV in these patients and its association with clinical response to bDMARDs. METHODS: The BioBio Study is a multicentre randomized open-label trial, including RA patients with insufficient response to MTX. Patients were randomized to either TNFi (infliximab, INF), anti-IL-6 (tocilizumab, TCZ), CTLA4-Ig (abatacept, ABA) or anti-CD20 (rituximab, RTX) in addition to MTX. PCR was used to quantify TTV in the peripheral blood. RESULTS: TTV was measured in 95 patients (INF, n = 23; TCZ, n = 22; ABA, n = 27; RTX; n = 23). TTV increased by a median of 4.5 × 104 copies/ml [c/ml; interquartile range (IQR) 0-7.5 × 105] after 3 months. TTV levels at month 3 were associated with the Simplified Disease Activity Index (SDAI) (P = 0.03) and the Clinical Disease Activity Index (CDAI) response (P = 0.026) at month 6. A TTV cut-off level of 1.2 × 106 c/ml at month 3 had a positive likelihood ratio of 2.7 for prediction of an 85% reduction in SDAI at month 6. CONCLUSION: Our data suggest that TTV levels increase upon TNF, CD20 and costimulation blockade and are associated with the clinical response to bDMARDs in RA patients. TRIAL REGISTRATION: ClinicalTrials.gov; https://clinicaltrials.gov; NCT01638715.

Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes.

BACKGROUND: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. METHODS: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus. RESULTS: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1 , and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. CONCLUSION: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2-neutralizing antibodies conferring sterilizing immunity.

Results of a SARS-CoV-2 virus genome detection external quality assessment round focusing on sensitivity of assays and pooling of samples.

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.

Inadequate design of mutation detection panels prevents interpretation of variants of concern: results of an external quality assessment for SARS-CoV-2 variant detection.

OBJECTIVES: Mutation-specific PCR assays have quickly found their way into laboratory diagnostics due to their capacity to be a fast, easy to implement and high-throughput method for the detection of known SARS-CoV-2 variants of concern (VoCs). However, little is known about the performance of such assays in routine laboratory analysis. METHODS: The results reported in a recent round of an external quality assessment (EQA) scheme for SARS-CoV-2 mutation-specific PCR were retrospectively analyzed. For the determination of individual variant-specific sequences as well as for the interpretation results for certain virus variants, correct, incorrect, and unreported results were evaluated, and their possible causes were investigated. RESULTS: A total of 34 laboratories participated in this study. For five samples containing the VoC Alpha + E484K, Beta, Gamma, Delta, or B.1.1.318 (as a variant of interest), 848 results for SARS-2-CoV mutation detection were reported, 824 (97.2%, range per sample 88-100%) of which were correct. Melting curve assays gave 99% correct results, real-time RT-qPCR 94%, microarray-based assays 100%, and MALDI-TOF MS 96%. A total of 122/167 (73%) reported results for SARS-CoV-2 variant determination were correct. Of the 45 inconclusive or incorrect results, 33 (73%) were due to inadequate selection of targets that did not allow identification of contemporary VoC, 11 (24%) were due to incorrect results, and one (3%) was due to correct results of mutation-specific PCR. CONCLUSIONS: Careful and up-to-date selection of the targets used in mutation-specific PCR is essential for successful detection of current SARS-CoV-2 variants.

Assessment of S1-, S2-, and NCP-Specific IgM, IgA, and IgG Antibody Kinetics in Acute SARS-CoV-2 Infection by a Microarray and Twelve Other Immunoassays.

In this study, we comprehensively analyzed multispecific antibody kinetics of different immunoglobulins in hospitalized patients with acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Three hundred fifty-four blood samples longitudinally obtained from 81 IgG-seroconverting progressed coronavirus disease 2019 (CoVID-19) patients were quantified for spike 1 (S1), S2, and nucleocapsid protein (NCP)-specific IgM, IgA, IgG, and total Ig antibodies using a microarray, 11 different enzyme-linked immunosorbent assays (ELISAs)/chemiluminescence immunoassays (CLIAs), and 1 rapid test by seven manufacturers. The assays' specificity was assessed in 130 non-CoVID-19 pneumonia patients. Using the microarray, NCP-specific IgA and IgG antibodies continuously displayed higher detection rates during acute CoVID-19 than S1- and S2-specific ones. S1-specific IgG antibodies, however, reached higher peak values. Until the 26th day post-symptom onset, all patients developed IgG responses against S1, S2, and NCP. Although detection rates by ELISAs/CLIAs generally resembled those of the microarray, corresponding to the target antigen, sensitivities and specificities varied among all tests. Notably, patients with more severe CoVID-19 displayed higher IgG and IgA levels, but this difference was mainly observed with S1-specific immunoassays. In patients with high SARS-CoV-2 levels in the lower respiratory tract, we observed high detection rates of IgG and total Ig immunoassays with a particular rise of S1-specific IgG antibodies when viral concentrations in the tracheal aspirate subsequently declined over time. In summary, our study demonstrates that differences in sensitivity among commercial immunoassays during acute SARS-CoV-2 infection are only partly related to the target antigen. Importantly, our data indicate that NCP-specific IgA and IgG antibodies are detected earlier, while higher S1-specific IgA antibody levels occur in severely ill patients.

Influence of Human Cytomegalovirus Glycoprotein O Polymorphism on the Inhibitory Effect of Soluble Forms of Trimer- and Pentamer-Specific Entry Receptors.

Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO trimer and gH/gL/UL128-131 pentamer, are important for cell-free HCMV entry. While soluble NRP2-Fc (sNRP2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble platelet-derived growth factor receptor α-Fc (sPDGFRα-Fc) interacts with gO, thereby inhibiting infection of all cell types. Since gO is the most variable subunit, we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, and GT5 sequences in the bacterial artificial chromosome (BAC) TB40-BAC4-luc background (where luc is luciferase). All mutants were tested for fibroblast and epithelial cell infectivity, for virion content of gB, gH, and gO, and for infection inhibition by sPDGFRα-Fc and sNRP2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in gB and gH levels in mutant virions. All gO GT mutants except recombinant gO GT1c/3 displayed a nearly complete inhibition at 1.25 µg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%), and all experienced complete inhibition on fibroblasts (≥99%). While gO GT replacement did not influence sNRP2-Fc inhibition at 1.25 µg/ml on epithelial cells (97% to 99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblast inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope of >1.0), sNRP2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0, suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2 in a cell type-dependent manner.IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism, as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the amounts of trimer in virions and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information for the future development of anti-HCMV therapeutic compounds based on sPDGFRα.

Deletion of the NKG2C receptor encoding KLRC2 gene and HLA-E variants are risk factors for severe COVID-19.

PURPOSE: Host genetic variants may contribute to severity of COVID-19. NKG2C+ NK cells are potent antiviral effector cells, potentially limiting the extent of SARS-CoV-2 infections. NKG2C is an activating NK cell receptor encoded by the KLRC2 gene, which binds to HLA-E on infected cells leading to NK cell activation. Heterozygous or homozygous KLRC2 deletion (KLRC2del) may naturally occur and is associated with a significantly lower or absent NKG2C expression level. In addition, HLA-E*0101/0103 genetic variants occur, caused by a single-nucleotide polymorphism. We therefore investigated whether the severity of COVID-19 is associated with these genetic variants. METHODS: We investigated the distribution of KLRC2 deletion and HLA-E*0101/0103 allelic variants in a study cohort of 361 patients with either mild (N = 92) or severe (N = 269) COVID-19. RESULTS: Especially the KLRC2del, and at a lower degree the HLA-E*0101, allele were significantly overrepresented in hospitalized patients (p = 0.0006 and p = 0.01), particularly in patients requiring intensive care (p < 0.0001 and p = 0.01), compared with patients with mild symptoms. Both genetic variants were independent risk factors for severe COVID-19. CONCLUSION: Our data show that these genetic variants in the NKG2C/HLA-E axis have a significant impact on the development of severe SARS-CoV-2 infections, and may help to identify patients at high-risk for severe COVID-19.

Diagnosis of COVID-19 using multiple antibody assays in two cases with negative PCR results from nasopharyngeal swabs.

We report of two cases of progressed COVID-19 with negative PCR tests from nasopharyngeal swabs, in whom diagnosis was made by different antibody assays, including a lateral flow rapid test and multiple commercial ELISAs, finally confirmed by comprehensive serological assays. These cases highlight that commercial ELISAs and even rapid tests might significantly aid the diagnosis of COVID-19, particularly, if a combination of serological assays is used with a specific clinical question, in severely ill patients after seroconversion and when comprehensive serological methods are used for confirmation.

Variability of cycle threshold values in an external quality assessment scheme for detection of the SARS-CoV-2 virus genome by RT-PCR.

OBJECTIVES: The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. METHODS: Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. RESULTS: A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. CONCLUSIONS: In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.

The versatility of external quality assessment for the surveillance of laboratory and in vitro diagnostic performance: SARS-CoV-2 viral genome detection in Austria.

OBJECTIVES: External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. METHODS: Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. RESULTS: A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (Ct ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of Ct values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. CONCLUSIONS: Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.