Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-β pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.

In chronic hepatitis B virus (HBV) infection, virus-specific T cells are scarce and partially dysfunctional. Therapeutic vaccination is a promising strategy to induce and activate new virus-specific T cells. In long-term or high-level HBV carriers, however, therapeutic vaccination by itself may not suffice to cure HBV. One reason is the impairment of antiviral T cells by immune checkpoints. In this study, we used small-interfering RNA (siRNA) in combination with a heterologous prime-boost therapeutic vaccination scheme (TherVacB) to interfere with a major immune checkpoint, the interaction of programmed death protein-1 (PD-1) and its ligand (PDL-1). In mice persistently replicating HBV after infection with an adeno-associated virus harboring the HBV genome, siRNA targeting PD-L1 resulted in a higher functionality of HBV-specific CD8+ T cells after therapeutic vaccination, and allowed for a more sustained antiviral effect and control of HBV in peripheral blood and in the liver. The antiviral effect was more pronounced if PD-L1 was down-regulated during prime than during boost vaccination. Thus, targeting PD-L1 using siRNA is a promising approach to enhance the efficacy of therapeutic vaccination and finally cure HBV.

While Mpox virus (MPXV) diagnostics were performed in specialized laboratories only, the global emergence of Mpox cases in 2022 revealed the need for a more readily available diagnostic. Automated random‐access platforms with fast nucleic acid extraction and PCR have become established in many laboratories, providing faster and more accessible testing. In this study, we adapted a previously published generic MPXV‐PCR as a lab‐developed test (LDT) on a NeuMoDx Molecular System and isolated MPXV clones from patient materials. To reduce the handling of infectious material, we evaluated a viral lysis buffer (VLB) for sample pretreatment. We further compared the MPXV‐LDT‐PCR to conventional real‐time PCR, determined its sensitivity and specificity using positive swabs, and assessed its performance using external quality assessment samples. Pretreatment of samples with 50% VLB reduced MPXV infectivity by approximately 200‐fold while maintaining PCR sensitivity. The assay demonstrated a sensitivity and specificity of 100% with no cross‐reactivity in the samples tested and performed with a limit of detection of 262 GE/mL. In summary, the assay had a turnaround time of fewer than 2 h and can easily be transferred to other automated PCR platforms, providing a basis for developing rapid assays for upcoming pandemics.

Zusammenfassung Antivirale Medikamente hemmen die Virusvermehrung durch Interaktion mit spezifischen Angriffspunkten im viralen Replikationszyklus. Direkt antivirale Wirkstoffe haben die therapeutischen Möglichkeiten bei chronischen Infektionen mit dem humanen Immundefizienz- (HIV), Hepatitis-B- (HBV) und Hepatitis-C-Virus (HCV) revolutioniert. Fortlaufende pharmakologische Entwicklungen sorgen auch bei Erkrankungen durch Herpesviren für verbesserte therapeutische und prophylaktische Möglichkeiten, was insbesondere immunsupprimierten Patienten zugutekommt. Während Infektionen mit persistierenden Viren wie HIV, HBV oder Herpesviren vom Grundsatz her bislang nicht heilbar sind, können Erreger von akuten Virusinfektionen vollständig eliminiert werden. Akute Infektionen, wie Influenza oder „coronavirus disease 2019“ (COVID-19) weisen durch ihre pathophysiologische Dynamik jedoch schmale therapeutische Zeitfenster für antivirale Therapieansätze auf. Der ideale Therapiezeitpunkt liegt hier unmittelbar nach der Virusexposition, was die Anwendung in der Praxis häufig limitiert. Eine effektive antivirale Prä- oder Postexpositionsprophylaxe hat sich bei Infektionen mit HIV und Influenza A/B etabliert und gewinnt aktuell auch bei Infektionen mit dem „severe acute respiratory syndrome coronavirus 2“ (SARS-CoV‑2) an Bedeutung.

Immunocompromised individuals are at higher risk of developing protracted and severe COVID-19, and understanding individual disease courses and SARS-CoV-2 immune responses in these individuals is of the utmost importance. For more than two years, we followed an immunocompromised individual with a protracted SARS-CoV-2 infection that was eventually cleared in the absence of a humoral neutralizing SARS-CoV-2 antibody response. By conducting an in-depth examination of this individual’s immune response and comparing it to a large cohort of convalescents who spontaneously cleared a SARS-CoV-2 infection, we shed light on the interplay between B- and T-cell immunity and how they interact in clearing SARS-CoV-2 infection.