Jörg Timm

Background: Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is a rare airway disease caused by human papillomavirus (HPV), most commonly HPV6 or HPV11. Affected children often require repeated surgical procedures, and no curative therapy exists. Off-label HPV vaccination has been proposed as an adjunctive treatment, but vaccine-induced immune responses and the relevance of circulating HPV DNA in benign disease remain unclear. Methods: We longitudinally evaluated 2 children with JoRRP who received off-label nonavalent HPV vaccination between 2020 and 2025. Serum HPV-specific binding and neutralizing antibodies were measured before vaccination and after the second and third doses using multiplex serology and a pseudovirion-based neutralization assay. During bronchoscopic follow-up, bronchoalveolar lavage fluid and available biopsy samples were analyzed for HPV DNA. In 1 patient, plasma was additionally tested for circulating HPV DNA by digital polymerase chain reaction. Results: Both children had low or undetectable HPV-specific antibodies at baseline. Vaccination induced strong binding and neutralizing antibody responses against vaccine-included HPV types. During post-vaccination follow-up of 12–29 months after completion of the vaccination series, HPV DNA became undetectable in bronchoalveolar lavage samples, and no new papilloma growth was observed after the last post-vaccination intervention. Circulating HPV DNA was not detected at any time point in the patient tested. Conclusions: Nonavalent HPV vaccination induced robust neutralizing antibody responses in children with JoRRP and was temporally associated with clinical stabilization. Larger studies are needed to clarify clinical benefit and biomarker utility.

Background The 2026 Andes virus (ANDV) outbreak linked to the cruise ship m/v Hondius triggered prolonged diagnostic follow-up of contacts in several countries. ANDV severity, long incubation, and person-to-person spread complicate case ascertainment. The cross-continent introduction of a rare transmissible virus with limited laboratory readiness is a paramount scenario in pandemic preparedness. To build response capacity under the increased regulatory demands of the European in-vitro diagnostic medical devices regulation (IVDR), we conducted an ad-hoc validation of an investigational RT-PCR product for ANDV detection. Methods Members of the EU Reference Laboratory for Vectorborne Viral Pathogens, the HERA network DURABLE, and the German network PAKOP did a rapid survey of hantavirus diagnostic capacity, a sequence-informed review of molecular in-house assays, and a decentralised validation of a strain-specific research-use-only (RUO) RT-PCR product. We tested biobanked patient samples, differential diagnostic materials, and virus cultures; analytical sensitivity was assessed by probit analysis of quantified in-vitro-transcribed RNA. Findings Networks enabled rapid exchange of methods and reference materials, revealing heterogeneous capacity. The RUO assay was evaluated on 302 clinical specimens and 120 cultured materials covering seven Orthohantavirus species and 67 non-hantavirus pathogens. No specificity issues arose; 37 of 38 ANDV samples were positive. Among non-Andes hantaviruses, only a Sin Nombre virus isolate was positive. The 95% limit of detection was 10·65 copies per reaction. Patient-derived material showed concordance with an established in-house RT-PCR. Interpretation Coordinated expert networks can deliver a swift laboratory response and generate ad-hoc validation data for diagnostic products entering regulatory channels. Network-based validation will probably become essential in future European outbreaks under the IVDR.

Background: Human cytomegalovirus (HCMV) drives expansion of adaptive natural killer (NK) cells with Fc receptor-biased effector function. Whether these cells are linked to inflammatory activity and fibrosis in chronic hepatitis B virus (HBV) infection, extend to HBV/hepatitis D virus (HDV) coinfection and are modulated by antiviral therapy is unknown.Methods: We profiled peripheral blood from 44 patients with HBV monoinfection, 12 with HBV/HDV coinfection, and 10 controls by multicolor flow cytometry, stratified by HCMV serostatus and disease stage. NK cell function was assessed by antibody-dependent cellular cytotoxicity (ADCC) and after interleukin-12/-15/-18 stimulation. Adaptive NKG2CposCD57pos NK cells from HCMV seropositive patients with chronic HBV infection without signs of hepatitis (n=3), with chronic hepatitis B (n=4) and from HCMV seropositive controls (n=4) were sorted and the transcriptional profile analysed at the single-cell level.Findings: Expansion of adaptive NK cells was confined to HCMV-seropositive individuals and was further enriched in patients with immune-active hepatitis and cirrhosis, including those with HBV/HDV coinfection, where it correlated with serum HBV DNA levels. Advanced disease was associated with a more differentiated adaptive NK cell phenotype, characterised by reduced Siglec-7 expression and increased granzyme B content. Transcriptional profiling revealed distinct adaptive NK cell states in chronic hepatitis B compared with chronic HBV infection without hepatitis, marked by a cytotoxic terminal effector programme and a greater degree of adaptive differentiation in chronic hepatitis B. In a longitudinal subset, adaptive NK cell frequencies remained stable during nucleos(t)ide analogue therapy despite virological and biochemical improvement, and likewise remained unchanged in HBV/HDV-coinfected patients receiving bulevirtide. Interpretation: Immune-active HBV and HBV/HDV disease amplify an HCMV-imprinted, ADCC-competent NK cell compartment that persists during therapy that may be relevant to immune pathogenesis of HBV infection.

Autoimmune-mediated connective tissue diseases such as antisynthetase syndrome (ASyS) and systemic sclerosis (SSc) have a high unmet medical need. Here we report on treatment under compassionate use with the CD19×CD3 T cell engager (TCE) blinatumomab and the BCMA×CD3 TCE teclistamab in five patients with treatment-refractory ASyS and in five patients with treatment-refractory SSc, respectively. Induction therapy with blinatumomab or teclistamab reduced target cells in affected muscle and skin, respectively, and decreased autoantibody titers. Blinatumomab induced rapid clinical, serological and histological improvement of myositis and stabilization of interstitial lung disease (ILD) in patients with ASyS. Teclistamab improved skin fibrosis, stabilized ILD and resolved tendon friction rubs in patients with SSc. Inhibition of B cell redifferentiation by maintenance therapy with rituximab (RTX) enabled prolonged disease control, even for patients previously unresponsive to RTX. Treatment was associated with adverse events including cytokine release syndrome (CRS) up to grade 3, in two patients with ASyS and in all patients with SSc. No immune effector cell-associated neurotoxicity syndrome (ICANS) occurred. Respiratory infections treated with antibiotics occurred in six patients. Blinatumomab and teclistamab may offer potential as rescue therapies for patients with treatment-refractory ASyS and SSc.

Hepatitis C virus (HCV) exists as a heterogenous quasispecies, but the phenotypic consequences of viral variability are widely unexplored. Here we identify a replication enhancing domain (ReED) in non-structural protein 5A conferring high replication fitness to clinical isolates. Accumulation of mutations in the ReED mediates high genome replication capacity. In a cohort of liver transplant patients, high replicator variants are exclusively found in individuals with severe disease outcome, suggesting that high viral replication fitness is associated with increased viral pathogenesis. Analysis of large sequence cohorts reveals that overall only 10% of viral genomes show genetic signatures of high replicators, which are enriched in recipients of liver transplantations, patients developing hepatocellular carcinoma and in HIV coinfected individuals. Overall, our data suggests that low replication fitness is a hallmark of HCV, contributing to establishment of persistence, whereas high replicators appear to have an advantage under conditions of immune suppression, thereby enforcing pathogenesis.

Background: The molecular characteristics of hepatitis B virus (HBV) in Africa, including the impact of HIV coinfection, are poorly understood. Methods: We performed whole-genome sequencing (WGS) on biospecimens collected before antiviral therapy in a well-characterized cohort of adults with HBV in Zambia, enriched for HIV coinfection (HBV/HIV). We assessed the frequency of basal core promoter (BCP) and precore variants, substitution frequencies, and the ratio of nonsynonymous to synonymous substitutions (dN/dS ratios), a surrogate for selection pressure. Results: Among 215 participants (median age, 33 years; 36% e antigen [HBeAg] positive, 35% with HBV/HIV), 114 (53.0%) had viral genotype E (gtE), and 101 (47.0%) had genotype A (gtA), subgenotype 1. BCP and precore variants, associated with HBeAg negativity, were more common with increased age, in the absence of HIV, and with gtE. Distinct from gtA, gtE had dN/dS ratios that were increased in the core vs polymerase region. Low dN/dS ratios were observed in HBV/HIV, especially at the lowest CD4 T-cell frequencies. Sequences from acute HBV infection as well as from 5 participants with chronic HBV/HIV who cleared hepatitis B surface antigen early during tenofovir-based antiretroviral therapy showed remarkably low dN/dS ratios. Conclusions: HBV gtE exhibited distinct substitution patterns compared with gtA, and HBV/HIV was associated with reduced HBV sequence diversity, consistent with impaired immune pressure.

Hepatitis C virus (HCV) exists as a heterogenous quasispecies, but the phenotypic consequences of viral variability are widely unexplored. Here we identified a replication enhancing domain (ReED) in nonstructural protein 5A conferring high replication fitness to clinical isolates. Accumulation of mutations in the ReED mediates high genome replication capacity. In a cohort of liver transplant patients, high replicator variants were exclusively found in individuals with severe disease outcome, suggesting that high viral replication fitness is associated with increased viral pathogenesis. Analysis of large sequence cohorts revealed that overall only 10% of viral genomes showed genetic signatures of high replicators, which were enriched in recipients of liver transplantations, patients developing hepatocellular carcinoma and in HIV coinfected individuals. Overall, our data suggests that low replication fitness is a hallmark of HCV, contributing to establishment of persistence, whereas high replicators appear to have an advantage under conditions of immune suppression, thereby enforcing pathogenesis.

Using multi-color flow cytometry analysis, we studied the immunophenotypical differences between leukemic cells from patients with AML/MDS and hematopoietic stem and progenitor cells (HSPCs) from patients in complete remission (CR) following their successful treatment. The panel of markers included CD34, CD38, CD45RA, CD123 as representatives for a hierarchical hematopoietic stem and progenitor cell (HSPC) classification as well as programmed death ligand 1 (PD-L1). Rather than restricting the evaluation on a 2- or 3-dimensional analysis, we applied a t-distributed stochastic neighbor embedding (t-SNE) approach to obtain deeper insight and segregation between leukemic cells and normal HPSCs. For that purpose, we created a t-SNE map, which resulted in the visualization of 27 cell clusters based on their similarity concerning the composition and intensity of antigen expression. Two of these clusters were “leukemia-related” containing a great proportion of CD34⁺/CD38⁻ hematopoietic stem cells (HSCs) or CD34⁺ cells with a strong co-expression of CD45RA/CD123, respectively. CD34⁺ cells within the latter cluster were also highly positive for PD-L1 reflecting their immunosuppressive capacity. Beyond this proof of principle study, the inclusion of additional markers will be helpful to refine the differentiation between normal HSPCs and leukemic cells, particularly in the context of minimal disease detection and antigen-targeted therapeutic interventions. Furthermore, we suggest a protocol for the assignment of new cell ensembles in quantitative terms, via a numerical value, the Pearson coefficient, based on a similarity comparison of the t-SNE pattern with a reference.