Clinical efficacy and safety of switching from eculizumab to ravulizumab in adult patients with aHUS- real-world data.

BACKGROUND: The complement factor 5 (C5)-inhibitor eculizumab has been established as standard-of-care for the treatment of atypical hemolytic uremic syndrome (aHUS). In 2021, the long-acting C5-inhibitor ravulizumab was approved, extending intervals of intravenous treatment from two to eight weeks resulting in improvement of quality of life for patients and lowering direct and indirect therapy associated costs. METHODS: This multicenter, retrospective data analysis of 32 adult patients with aHUS (including 10 kidney transplant recipients) treated with eculizumab for at least three months and switched to ravulizumab aims to evaluate the safety and efficacy of switching medication in the real-world setting. Hematologic parameters, kidney function, concurrent therapy and aHUS associated events were evaluated three months before and until up to 12 months after switching to ravulizumab. RESULTS: Mean age (range) at ravulizumab initiation was 41 years (19-78 years) and 59% of the patients were female. Genetic analysis was available for all patients with 72% showing a pathogenic variant. Median time (range) on eculizumab before switching was 20 months (3-120 months). No new events of TMA or worsening of renal function were reported during up to 12 months of follow-up during ravulizumab treatment. CONCLUSIONS: This is the largest, non-industry derived, multi-center retrospective analysis of adult patients with aHUS switching C5-inhibitor treatment from eculizumab to ravulizumab in the real-world setting. Switching to ravulizumab was safe and efficient resulting in sustained hematological stability and preservation of renal function.

Urinary Biomarkers for Cell Cycle Arrest TIMP-2 and IGFBP7 for Prediction of Graft Function Recovery after Kidney Transplantation.

TIMP-2 and IGFBP7 have been identified and validated for the early detection of renal injury in critically ill patients, but data on recovery of allograft function after kidney transplantation (KTx) are scarce. In a prospective observational multicenter cohort study of renal transplant recipients, urinary [TIMP-2] × [IGFBP7] was evaluated daily from day 1 to 7 after KTx. Different stages of early graft function were defined: immediate graft function (IGF) (decrease ≥ 10% in serum creatinine (s-crea) within 24 h post KTx); slow graft function (SGF) (decrease in s-crea < 10% within 24 h post KTx); and delayed graft function (DGF) (any dialysis needed within the first week after KTx). A total of 186 patients were analyzed. [TIMP-2] × [IGFBP7] was significantly elevated as early as day 1 in patients with DGF compared to SGF and IGF. ROC analysis of [TIMP-2] × [IGFBP7] at day 1 post-transplant for event "Non-DGF" revealed a cut-off value of 0.9 (ng/mL)2/1000 with a sensitivity of 87% and a specificity of 71%. The positive predictive value for non-DGF was 93%. [TIMP-2] × [IGFBP7] measured at day 1 after KTx can predict early recovery of transplant function and is therefore a valuable biomarker for clinical decision making.

Increased SARS-CoV-2 reactive low avidity T cells producing inflammatory cytokines in pediatric post-acute COVID-19 sequelae (PASC).

BACKGROUND: A proportion of the convalescent SARS-CoV-2 pediatric population presents nonspecific symptoms, mental health problems, and a reduction in quality of life similar to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID-19 symptomatic. However, data regarding its clinical manifestation and immune mechanisms are currently scarce. METHODS: In this study, we perform a comprehensive clinical and immunological profiling of 17 convalescent COVID-19 children with post-acute COVID-19 sequelae (PASC) manifestation and 13 convalescent children without PASC manifestation. A detailed medical history, blood and instrumental tests, and physical examination were obtained from all patients. SARS-CoV-2 reactive T-cell response was analyzed via multiparametric flow cytometry and the humoral immunity was addressed via pseudovirus neutralization and ELISA assay. RESULTS: The most common PASC symptoms were shortness of breath/exercise intolerance, paresthesia, smell/taste disturbance, chest pain, dyspnea, headache, and lack of concentration. Blood count and clinical chemistry showed no statistical differences among the study groups. We detected higher frequencies of spike (S) reactive CD4+ and CD8+ T cells among the PASC study group, characterized by TNFα and IFNγ production and low functional avidity. CRP levels are positively correlated with IFNγ producing reactive CD8+ T cells. CONCLUSIONS: Our data might indicate a possible involvement of a persistent cellular inflammatory response triggered by SARS-CoV-2 in the development of the observed sequelae in pediatric PASC. These results may have implications on future therapeutic and prevention strategies.

Endotoxin-Induced Physiological and Psychological Sickness Responses in Healthy Humans: Insights into the Post-Acute Phase.

INTRODUCTION: Experimental endotoxemia is a translational model of systemic inflammation that has contributed significantly to our current understanding of sickness behavior and inflammation-associated depression. Previous studies using this model revealed a strong association between cytokine levels, endocrine changes, and psychological sickness symptoms during the acute phase of inflammation. The objective of this randomized, double-blind, placebo-controlled crossover study was to gain insight into potential post-acute physiological and psychological consequences of endotoxin administration that may either persist or newly emerge between 24 and 72 h after injection. The main focus was on associations between serum levels of C-reactive protein (CRP) and affective symptoms as well as alterations in diurnal cortisol profile, the two key features of inflammation-associated depression. METHODS: Healthy male volunteers (N = 18) received an injection of either endotoxin (0.8 ng/kg) or placebo on two separate but otherwise identical study days, 7 days apart. Blood and saliva samples were collected during acute and post-acute phases after injection to measure blood inflammatory markers (interleukin [IL]-6, IL-1 receptor antagonist [ra], CRP) and salivary cortisol levels. In addition, participants completed a comprehensive battery of questionnaires to assess physical and psychological sickness symptoms. RESULTS: Endotoxin treatment induced a short-time rise in plasma IL-6 and a longer increase in IL-1ra. The increase in serum CRP was delayed compared to cytokines, peaking at 24 h and gradually decreasing until 72 h after injection. The inflammatory response was accompanied by bodily and psychological sickness symptoms which occurred only in the acute phase, whereas none of the symptoms persisted or recurred in the post-acute phase. Salivary cortisol levels were significantly increased during the acute phase and exhibited pronounced circadian changes. However, no significant differences in diurnal cortisol profiles were observed between placebo and endotoxin conditions on the days after treatment. CONCLUSION: Our findings suggest that CRP, which is elevated in patients with inflammation-associated depression, does not appear to be responsible for depressive symptomatology. Moreover, a single inflammatory episode is not sufficient to alter diurnal cortisol profiles, as observed in inflammation-associated depression. In addition, the absence of persistent lipopolysaccharide-induced psychological and physiological changes beyond the acute phase further supports the safety of endotoxin administration in humans.

Fading SARS-CoV-2 humoral VOC cross-reactivity and sustained cellular immunity in convalescent children and adolescents.

Cross-reactive cellular and humoral immunity can substantially contribute to antiviral defense against SARS-CoV-2 variants of concern (VOC). While the adult SARS-CoV-2 cellular and humoral immunity and its cross-recognition potential against VOC is broadly analyzed, similar data regarding the pediatric population are missing. In this study, we perform an analysis of the humoral and cellular SARS-CoV-2 response immune of 32 convalescent COVID-19 children (children), 27 convalescent vaccinated adults(C + V+) and 7 unvaccinated convalescent adults (C + V-). Similarly to adults, a significant reduction of cross-reactive neutralizing capacity against delta and omicron VOC was observed 6 months after SARS-CoV-2 infection. While SAR-CoV-2 neutralizing capacity was comparable among children and C + V- against all VOC, children demonstrated as expected an inferior humoral response when compared to C + V+. Nevertheless, children generated SARS-CoV-2 reactive T cells with broad cross-recognition potential. When compared to V + C+, children presented even comparable frequencies of WT-reactive CD4 + and CD8 + T cells with high avidity and functionality. Taking into consideration the limitations of study - unknown disease onset for 53% of the asymptomatic pediatric subjects, serological detection of SARS-CoV-2 infection-, our results suggest that following SARS-CoV-2 infection children generate a humoral SARS-CoV-2 response with neutralizing potential comparable to unvaccinated COVID-19 convalescent adults as well a sustained SARS-CoV-2 cellular response cross-reactive to VOC.

SARS-CoV-2-specific humoral and cellular immunity in two renal transplants and two hemodialysis patients treated with convalescent plasma.

When patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) they can face two specific problems: virus-specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten recovery, is contraindicated. Antiviral treatment with convalescent plasma (CP) could be an alternative treatment option. In this case report, we present two kidney transplant recipients and two hemodialysis patients who were infected with SARS-CoV-2 and received CP. Antibodies against the receptor-binding domain in the S1 subunit of the SARS-CoV-2 spike protein were determined sequentially by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and neutralization assay and specific cellular responses by interferon-gamma ELISpot. Before treatment, in both kidney transplant recipients and one hemodialysis patient antibodies were undetectable by ELISA (ratio < 1.1), corresponding to low neutralizing antibody titers (≤1:40). ELISpot responses in the four patients were either weak or absent. After CP treatment, we observed an increase of SARS-CoV-2-specific antibodies (IgG ratio and neutralization titer) and of specific cellular responses. After intermittent clinical improvement, one kidney transplant recipient again developed typical symptoms on Day 12 after treatment and received a second cycle of CP treatment. Altogether, three patients clinically improved and could be discharged from the hospital. However, one 83-year-old multimorbid patient deceased. Our data suggest that the success of CP therapy may only be temporary in patients with chronic kidney disease; which requires close monitoring of viral load and antiviral immunity and possibly an adaptation of the treatment regimen.

Failure of first meningococcal vaccination in patients with atypical haemolytic uraemic syndrome treated with eculizumab.

BACKGROUND: The C5 complement inhibitor eculizumab is a first-line treatment in atypical haemolytic uraemic syndrome (aHUS). Therapy with eculizumab is associated with a highly increased risk for meningococcal infection. Therefore, vaccination is highly recommended before beginning treatment. Efficacy of quadrivalent meningococcal vaccines (MenACWY) in patients treated with the C5 complement inhibitor eculizumab in aHUS has not yet been determined. METHODS: Patients with aHUS received one dose of a MenACWY conjugate vaccine before eculizumab treatment commenced. Bactericidal titres against meningococcal serogroups A, C, W and Y were determined using baby rabbit complement in 25 patients. RESULTS: Full immune response to meningococcal vaccination was detected in five patients (20%), while seven patients (28%) showed no immune response in any of the tested serogroups. The remaining 13 patients showed incomplete immune response with proof of protective antibody titres for one to three serogroups without perceptible preference for any serogroup. Bactericidal titres after re-vaccination were available for 17 patients. Nine patients with incomplete immune response after first vaccinations showed protective antibody titres for all serogroups after re-vaccination. Kidney function had improved in >50% of patients at the time of re-vaccination compared with the time of first vaccination and immunosuppressive therapy was only applied to re-vaccinated patients following kidney transplantation. CONCLUSIONS: Immunogenicity of first quadrivalent meninongococcal vaccination is insufficient in patients with aHUS. Booster response is promising, but incomplete. Therefore, establishing antibiotic prophylaxes seems pivotal.

Prevalence of active hepatitis E virus infection and efficacy of ribavirin treatment in renal allograft recipients.

BACKGROUND: Hepatitis E virus (HEV) genotype 3 infection frequently progresses to chronic disease with persisting HEV viremia in immunocompromised patients. Here, we evaluated the prevalence of HEV infection in renal allograft recipients and investigated the efficacy and tolerability of ribavirin monotherapy. METHODS: A total of 947 recipients on average 8.7 years post transplant were screened for anti-HEV IgG, IgM and HEV-RNA. Sixteen HEV-viremic renal allograft recipients were treated with ribavirin for 12 weeks. HEV-RNA concentration, laboratory and clinical parameters were assessed at baseline, during therapy and 12 weeks after treatment cessation. HEV-genotyping was performed in all HEV-viremic patients. RESULTS: Past HEV infection was detected serologically in 18% of the renal allograft recipients. Ongoing HEV replication was found in 16 recipients (all genotype 3). Unanimously, distinct HEV sequences were revealed in all HEV-viremic patients. At the start of ribavirin treatment, median HEV-RNA viral load was 4.3 × 106 (8000-5.0 × 106 ) IU/mL. Ninety-four percentage of HEV-infected allograft recipients showed a sustained virological response 12 weeks after treatment cessation. Ribavirin treatment was associated with rapid decrease in liver enzymes and rare occurrence of anemia. CONCLUSIONS: Prevalence of active HEV infection is important in renal transplant patients without signs of nosocomial infection. Ribavirin treatment was safe and effective.

Randomized, open-label, comparative phase IV study on the bioavailability of Ciclosporin Pro (Teva) versus Sandimmun® Optoral (Novartis) under fasting versus fed conditions in patients with stable renal transplants.

BACKGROUND: The influence of pre- or postprandial administration on pharmacokinetics of cyclosporine is supposed to be less in gel-based formulations than in microemulsions. This study was designed to investigate the influence of a high-fat meal on the pharmacokinetic profile of the two cyclosporine containing formulations Ciclosporin Pro (gel-based emulsion) and Sandimmun®Optoral (microemulsion) in renal transplant recipients. METHODS: A randomized, open-label, repeated-measurement, comparative phase IV trial was conducted with two sequence groups for nutrition condition (fasting→fed, fed→fasting) and two treatment phases (Sandimmun® Optoral → Ciclosporin Pro), each covering both nutrition conditions. Primary pharmacokinetic variable of interest was the reduction of bioavailability due to high-fat food compared to fasting conditions measured by the difference D of ln-transformed bioavailability variables (AUCSS, τ, Css, max, und Css, min). RESULTS: A nutrition effect was found for both study medications with respect to the parameters AUCSS, τ and CSS, max, but not to CSS, min. The reduction of bioavailability caused by high-fat food was not significantly different for Sandimmun®Optoral and Ciclosporin Pro. CONCLUSIONS: An effect of high-fat breakfast prior to the morning dose on AUCSS, τ and CSS, max was found for Sandimmun® Optoral and for Ciclosporin Pro. Trough level monitoring did not capture ingestion-related variability. Conversion to Ciclosporin Pro seems to be safe with regard to intra-individual pharmacokinetic variability. TRIAL REGISTRATION: EudraCT No. 2009-011354-18 (29th April 2019).

The single nucleotide polymorphism rs4986790 (c.896A>G) in the gene TLR4 as a protective factor in corona virus disease 2019 (COVID-19).

Background and aims: Several factors, such as hypertension and diabetes mellitus, are known to influence the course of coronavirus disease 2019 (COVID-19). However, there is currently little information on genetic markers that influence the severity of COVID-19. In this study, we specifically investigated the single nucleotide polymorphism (SNP) rs4986790 in the TLR4 gene to identify a universal marker for preclinical prediction of COVID-19 disease progression. Methods: We analyzed the influence of demographics, pre-existing conditions, inflammatory parameters at the time of hospitalization, and TLR4 rs4986790 genotype on the outcome of COVID-19 in a comprehensive cohort (N = 1570). We performed multivariable analysis to investigate the impact of each factor. Results: We confirmed that younger patient age and absence of pre-existing conditions were protective factors against disease progression. Furthermore, when comparing patients with mild SARS-CoV-2 infection with patients who required hospitalization or intensive care or even died due to COVID-19, the AG/GG genotype of TLR4 rs4986790 was found to be a protective factor against COVID-19 disease progression (OR: 0.51, 95% CI: 0.34 - 0.77, p = 0.001). In addition, we demonstrated that low levels of interleukin-6 (IL-6) and procalcitonin (PCT) had a favorable effect on COVID-19 disease severity. In the subsequent multivariable analysis, we confirmed the absence of cardiovascular disease, low levels of IL-6 and PCT, and TLR4 rs4986790 AG/GG genotypes as independent predictors of potential hospitalization and reduction of severe or fatal disease course. Conclusion: In this study, we identified an additional genetic factor that may serve as an invariant predictor of COVID-19 outcome. The TLR4 rs4986790 AG/GG genotype reduced by half the risk of COVID-19 patients requiring hospitalization, intensive care or to have a fatal outcome. In addition, we were able to confirm the influence of previously known factors such as pre-existing conditions and inflammatory markers upon the onset of disease on the course of COVID-19. Based on these observations, we hereby provide another prognostic biomarker that could be used in routine diagnostics as a predictive factor for the severity of COVID-19 prior to SARS-CoV-2 infection.

Low avidity circulating SARS-CoV-2 reactive CD8+ T cells with proinflammatory TEMRA phenotype are associated with post-acute sequelae of COVID-19.

The role of adaptive SARS-CoV-2 specific immunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although a growing population of convalescent COVID-19 patients with manifestation of PASC is observed. We analyzed the SARS-CoV-2-specific immune response, via pseudovirus neutralizing assay and multiparametric flow cytometry in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. Although frequencies of SARS-CoV-2-reactive CD4+ T cells were similar between the studied cohorts, a stronger SARS-CoV-2 reactive CD8+ T cell response, characterized by IFNγ production and predominant TEMRA phenotype but low functional TCR avidity was detected in PASC patients compared to controls. Of interest, high avidity SARS-CoV-2-reactive CD4+ and CD8+ T cells were comparable between the groups demonstrating sufficient cellular antiviral response in PASC. In line with the cellular immunity, neutralizing capacity in PASC patients was not inferior compared to controls. In conclusion, our data suggest that PASC may be driven by an inflammatory response triggered by an expanded population of low avidity SARS-CoV-2 reactive pro-inflammatory CD8+ T cells. These pro-inflammatory T cells with TEMRA phenotype are known to be activated by a low or even without TCR stimulation and lead to a tissue damage. Further studies including animal models are required for a better understanding of underlying immunopathogensis. Summary: A CD8+ driven persistent inflammatory response triggered by SARS-CoV-2 may be responsible for the observed sequelae in PASC patients.

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients.

Introduction: Triple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets. Materials and methods: To follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3' UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival. Results: sHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3' UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy response. Conclusion: The results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets.

Peripheral HLA-G/ILT-2 immune checkpoint axis in acute and convalescent COVID-19 patients.

The immunosuppressive non-classical human leukocyte antigen-G (HLA-G) can elicits pro-viral activities by down-modulating immune responses. We analysed soluble forms of HLA-G, IL-6 and IL-10 as well as on immune effector cell expression of HLA-G and its cognate ILT-2 receptor in peripheral blood obtained from hospitalised and convalescent COVID-19 patients. Compared with convalescents (N = 202), circulating soluble HLA-G levels (total and vesicular-bound molecules) were significantly increased in hospitalised patients (N = 93) irrespective of the disease severity. During COVID-19, IL-6 and IL-10 levels were also elevated. Regarding the immune checkpoint expression of HLA-G/ILT-2 on peripheral immune effector cells, the frequencies of membrane-bound HLA-G on CD3+ and CD14+ cells were almost identical in patients during and post COVID-19, while the frequency of ILT-2 receptor on CD3+ and CD14+ cells was increased during acute infection. A multi-parametric correlation analysis of soluble HLA-G forms with IL-6, IL-10, activation markers CD25 and CD154, HLA-G, and ILT-2 expression on immune cells revealed a strong positive correlation of soluble HLA-G forms with membrane-bound HLA-G molecules on CD3+/CD14+ cells only in convalescents. During COVID-19, only vesicular-bound HLA-G were positively correlated with the activation marker CD25 on T cells. Thus, our data suggest that the elevated levels of soluble HLA-G in COVID-19 are due to increased expression in organ tissues other than circulating immune effector cells. The concomitant increased expression of soluble HLA-G and ILT-2 receptor frequencies supports the concept that the immune checkpoint HLA-G/ILT-2 plays a role in the immune-pathogenesis of COVID-19.

Immune responses in COVID-19 patients during breakthrough infection with SARS-CoV-2 variants Delta, Omicron-BA.1 and Omicron-BA.5.

Background: Breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are increasingly observed in vaccinated individuals. Immune responses towards SARS-CoV-2 variants, particularly Omicron-BA.5, are poorly understood. We investigated the humoral and cellular immune responses of hospitalized COVID-19 patients during Delta and Omicron infection waves. Methods: The corresponding SARS-CoV-2 variant of the respective patients were identified by whole genome sequencing. Humoral immune responses were analyzed by ELISA and a cell culture-based neutralization assay against SARS-CoV-2 D614G isolate (wildtype), Alpha, Delta (AY.43) and Omicron (BA.1 and BA.5). Cellular immunity was evaluated with an IFN-γ ELISpot assay. Results: On a cellular level, patients showed a minor IFN-γ response after stimulating PBMCs with mutated regions of SARS-CoV-2 variants. Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced. Double-vaccinated patients with Delta breakthrough infection showed a significantly increased neutralizing antibody response against Delta compared to double-vaccinated uninfected controls (median complete neutralization titer (NT100) 640 versus 80, p<0.05). Omicron-BA.1 infection increased neutralization titers against BA.1 in double-vaccinated patients (median NT100 of 160 in patients versus 20 in controls, p=0.07) and patients that received booster vaccination (median NT100 of 50 in patients versus 20 in controls, p=0.68). For boosted patients with BA.5 breakthrough infection, we found no enhancing effect on humoral immunity against SARS-CoV-2 variants. Conclusion: Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced in SARS-CoV-2 breakthrough infections. Delta and Omicron-BA.1 but not Omicron-BA.5 infections boosted the humoral immunity in double-vaccinated patients and patients with booster vaccination. Despite BA.5 breakthrough infection, those patients may still be vulnerable for reinfections with BA.5 or other newly emerging variants of concern.

Effect of HLA-G5 Immune Checkpoint Molecule on the Expression of ILT-2, CD27, and CD38 in Splenic B cells.

The human leukocyte antigen G (HLA-G) is an immune checkpoint molecule with a complex network of interactions with several inhibitory receptors. Although the effect of HLA-G on T cells and NK cells is well studied, the effect of HLA-G on B cells is still largely elusive. B cells are of particular interest in the context of the HLA-G-ILT-2 interaction because the ILT-2 receptor is constitutively expressed on most B cells, whereas it is only present on some subsets of T and NK cells. To characterize the effect of HLA-G5 molecules on B cells, we studied splenic B cells derived from cytomegalovirus (CMV) sero-positive donors after CMV stimulation with antigens in the presence and absence of soluble HLA-G5. In the presence of HLA-G5, increased expression of the ITIM-bearing Ig-like transcript (ILT-2) was observed on B cells, but its expression was not affected by stimulation with CMV antigens. Moreover, it became evident that HLA-G5 exposure resulted in a decreased expression of CD27 and CD38 and, accordingly, in lower proportions of CD19+CD27+CD38+ and higher proportions of CD19+CD27-CD38- B cells. Taken together, our in vitro findings demonstrate that soluble HLA-G5 suppresses markers of B cell activation, suggesting that HLA-G5 has an impact on splenic B cell differentiation and activation. Based on these results, further investigation regarding the role of HLA-G as a prognostic factor and a potential therapeutic agent with respect to B cell function appears reasonable.

A Multipathogen Bile Sample-based PCR Assay Can Guide Empirical Antimicrobial Strategies in Cholestatic Liver Diseases.

Background and objectives: Polymerase chain reaction (PCR) techniques provide rapid detection of pathogens. This pilot study evaluated the diagnostic utility and clinical impact of multiplex real-time PCR (mRT-PCR, SeptiFast) vs. conventional microbial culture (CMC) in bile samples of patients with chronic cholestatic liver diseases (cCLDs), endoscopic retrograde cholangio-pancreatography (ERCP), and peri-interventional-antimicrobial-prophylaxis (pAP). Methods: We prospectively collected bile samples from 26 patients for microbiological analysis by CMC and mRT-PCR. Concordance of the results of both methods was determined by Krippendorff's alpha (α) for inter-rater reliability and the Jaccard index of similarity. Results: mRT-PCRbile and CMCbile results were concordant for only Candida albicans (α=0.8406; Jaccard index=0.8181). mRT-PCRbile detected pathogens in 8/8 cases (100%), CMCbile in 7/8 (87.5%), and CMCblood in 5/8 (62.5%) with clinical signs of infection. mRT-PCRbile, CMCbile, and CMCblood had identical detection results in 3/8 (37.5%) with clinical signs of infection (two Klebsiella spp. and one Enterococcus faecium). The total pathogen count was significantly higher with mRT-PCRbile than with CMCbile (62 vs. 31; χ2=30.031, p<0.001). However, pathogens detected by mRT-PCRbile were more often susceptible to pAP according to the patient infection/colonization history (PI/CH) and surveillance data for antibiotic resistance in our clinic (DARC). Pathogens identified by mRT-PCRbile and resistant to pAP by PI/CH and DARC were likely to be clinically relevant. Conclusions: mRT-PCR in conjunction with CMCs for bile analysis increased diagnostic sensitivity and may benefit infection management in patients with cholestatic diseases. Implementation of mRT-PCR in a bile sample-based diagnostic routine can support more rapid and targeted use of antimicrobial agents in cCLD-patients undergoing ERCP and reduce the rate/length of unnecessary administration of broad-spectrum antibiotics.

COVID-19 in Elderly, Immunocompromised or Diabetic Patients-From Immune Monitoring to Clinical Management in the Hospital.

The novel, highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic of acute respiratory illness worldwide and remains a huge threat to the healthcare system's capacity to respond to COVID-19. Elderly and immunocompromised patients are at increased risk for a severe course of COVID-19. These high-risk groups have been identified as developing diminished humoral and cellular immune responses. Notably, SARS-CoV-2 RNA remains detectable in nasopharyngeal swabs of these patients for a prolonged period of time. These factors complicate the clinical management of these vulnerable patient groups. To date, there are no well-defined guidelines for an appropriate duration of isolation for elderly and immunocompromised patients, especially in hospitals or nursing homes. The aim of the present study was to characterize at-risk patient cohorts capable of producing a replication-competent virus over an extended period after symptomatic COVID-19, and to investigate the humoral and cellular immune responses and infectivity to provide a better basis for future clinical management. In our cohort, the rate of positive viral cultures and the sensitivity of SARS-CoV-2 antigen tests correlated with higher viral loads. Elderly patients and patients with diabetes mellitus had adequate cellular and humoral immune responses to SARS-CoV-2 infection, while immunocompromised patients had reduced humoral and cellular immune responses. Our patient cohort was hospitalized for longer compared with previously published cohorts. Longer hospitalization was associated with a high number of nosocomial infections, representing a potential hazard for additional complications to patients. Most importantly, regardless of positive SARS-CoV-2 RNA detection, no virus was culturable beyond a cycle threshold (ct) value of 33 in the majority of samples. Our data clearly indicate that elderly and diabetic patients develop a robust immune response to SARS-CoV-2 and may be safely de-isolated at a ct value of more than 35.

Superior humoral immunity in vaccinated SARS-CoV-2 convalescence as compared to SARS-COV-2 infection or vaccination.

Emerging variants of concern (VOC) raise obstacles in shaping vaccination strategies and ending the pandemic. Vaccinated SARS-CoV-2 convalescence shapes the current immune dynamics. We analyzed the SARS-CoV-2 VOC-specific cellular and humoral response of 57 adults: 42 convalescent mRNA vaccinated patients (C+V+), 8 uninfected mRNA vaccinated (C-V+) and 7 unvaccinated convalescent individuals (C+V-). While C+V+ demonstrated a superior humoral SARS-CoV-2 response against all analyzed VOC (alpha, delta, omicron) compared to C-V+ and C+V-, SARS-CoV-2 reactive CD4+ and CD8+ T cells, which can cross-recognize the alpha, delta and omicron VOC after infection and/or vaccination were observed in all there groups without significant differences between the groups. We observed a preserved cross-reactive C+V+ and C-V+ T cell memory. An inferior humoral response but preserved cross-reactive T cell memory in C+V- compared to C+V+ was observed, as well as an inferior humoral response but preserved cross-reactive T cell memory in C+V- compared to C-V+. Adaptive immunity generated after SARS-CoV-2 infection and vaccination leads to superior humoral immune response against VOC compared to isolated infection or vaccination. Despite the apparent loss of neutralization potential caused by viral evolution, a preserved SARS-CoV-2 reactive T cell response with a robust potential for cross-recognition of the alpha, delta and omicron VOC was detected in all studied cohorts. Our results may have implications on current vaccination strategies.

Cellular and Humoral Immunity against Different SARS-CoV-2 Variants Is Detectable but Reduced in Vaccinated Kidney Transplant Patients.

In kidney transplant (KTX) patients, immune responses after booster vaccination against SARS-CoV-2 are inadequately examined. We analyzed these patients a median of four months after a third/fourth vaccination and compared them to healthy controls. Cellular responses were analyzed by interferon-gamma (IFN-γ) and interleukin-2 (IL-2) ELISpot assays. Neutralizing antibody titers were assessed against SARS-CoV-2 D614G (wild type) and the variants alpha, delta, and omicron by a cell culture-based neutralization assay. Humoral immunity was also determined by a competitive fluorescence assay, using 11 different variants of SARS-CoV-2. Antibody ratios were measured by ELISA. KTX patients showed significantly lower SARS-CoV-2-specific IFN-γ responses after booster vaccination than healthy controls. However, SARS-CoV-2-specific IL-2 responses were comparable to the T cell responses of healthy controls. Cell culture-based neutralizing antibody titers were 1.3-fold higher in healthy controls for D614G, alpha, and delta, and 7.8-fold higher for omicron (p < 0.01). Healthy controls had approximately 2-fold higher concentrations of potential neutralizing antibodies against all 11 variants than KTX patients. However, more than 60% of the KTX patients displayed antibodies to variants of SARS-CoV-2. Thus, KTX patients should be partly protected, due to neutralizing antibodies to variants of SARS-CoV-2 or by cross-reactive T cells, especially those producing IL-2.