Immunogenic cell death (ICD) provides a promising strategy for cancer treatment by stimulating anti-tumor immunity. However, single ICD inducers often elicit insufficient damage-associated molecular patterns (DAMPs), and the rapid clearance of these DAMPs at the tumor site further prevents sustained immune stimulation. To address these challenges, we developed a polyphenol-based bioadhesive hydrogel antigen reservoir functionalized with dopamine (DA) and loaded with dual ICD inducers. Specifically, thymidine kinase (tk)-deficient oncolytic vaccinia virus (VV, a type II ICD inducer) was combined with oxaliplatin (OXA, a type I ICD inducer), thereby enhancing direct tumor cytotoxicity and synergistically amplifying ICD through distinct mechanisms to promote robust DAMP release. Subsequently, the released DAMPs were captured and retained by the DA-functionalized hyaluronic acid-Pluronic® F127 hydrogel (HADP), forming an antigen reservoir at the tumor site. This synergistic "stimulate-and-retain" strategy not only augments ICD-mediated DAMP release but also prolongs their local availability to sustain immune stimulation. In murine models, the OXA-HADP@VV facilitated dendritic cell (DC) activation, increased IFN-γ [+]CD8[+] T cell infiltration, and reduced regulatory T cells (Tregs), thereby achieving effective tumor suppression and strong anti-recurrence effects. Together, this dual ICD-inducing antigen reservoir provides a versatile platform for improving cancer immunotherapy.

Epidermal growth factor receptor (EGFR) signaling is a major pathway that controls cellular growth, survival, proliferation, and differentiation. Viruses often hijack this pathway to create a favorable environment to support their replication. Vaccinia virus (the prototype member of poxviridae) encodes its own viral growth factor (VGF). VGF is a secreted viral polypeptide with sequence and structural homology to the cellular EGF, which binds to and activates EGFR signaling. Studies from our lab and others have shown that VGF promotes metabolic reprogramming, cell motility, virulence, and spread during VACV infection. However, it remains unclear whether VGF fully mimics EGF signaling or selectively rewires downstream pathways to meet the specific demands of viral replication. To address this question, we used quantitative global phosphoproteomics to systematically compare signaling programs induced by EGF and VGF in primary human foreskin fibroblasts. By analyzing global phosphorylation patterns, kinase activity inference, and motif enrichment, we found that although both peptides engage the same EGFR receptor, EGF and VGF induce distinct downstream phosphorylation and bias downstream kinase activities. Transcriptomic analysis provided a complementary view of the cellular responses to the ligand treatment. Moreover, we found that VGF is more effective at promoting VACV plaque formation than EGF, thereby providing functional support to the VGF-biased EGFR signaling nodes important for viral spread. These findings highlight differences in how viral and cellular growth factors activate EGFR signaling and position poxviral growth factors as powerful tools for examining the fundamental principles of cellular signaling.

Background/Objectives: Recent outbreaks of the mpox virus (MPVX) have highlighted the threat posed by the Orthopoxvirus (OPXV) genus, which includes variola, the causative agent of smallpox. Since routine Vaccinia virus (VACV) vaccination campaigns terminated, the majority of the global population currently lacks immunity against OPXVs. Methods: Using enzyme-linked immunosorbent assays (ELISAs), this study evaluated antibody titers against both VACV proteins and their MPVX orthologs across various age groups. Cross-reactivity was analyzed using 220 serum samples collected from Albanian individuals between November 2023 and January 2024, arranged into five age cohorts. We measured plasma antibody levels against whole VACV, as well as specific surface antigens: L1, B5, A33, and A27 for VACV, and M1, H3, A35, and A29 for MPXV. Results: The data reveal a long-term, gender-independent immunological memory targeting both whole-virus particles and individual viral proteins. Conclusions: Notably, the oldest cohort exhibited higher antibody titers against specific single viral proteins, whereas the younger vaccinated groups showed stronger activity against the whole virus. Additionally, antibodies raised via VACV vaccination recognize MPXV ortholog proteins equally well. This cross-reactivity highlights the broad protective potential of VACV immunization across the entire Orthopoxvirus genus.

Immunotherapies, including oncolytic viruses (OV), are promising therapies that can enhance anti-tumor immune responses. However, preclinical success of immunotherapies in mouse models has not always translated to clinical benefit in cancer patients. This study compared preclinical efficacy and mechanism of action for ASP9801, a vaccinia virus expressing IL-7 and IL-12, using mouse models of colorectal cancer (CRC) in vivo and in human organotypic tumor slice models ex vivo. The murine surrogate for ASP9801 significantly reduced tumor volumes in treated and abscopal tumors in two different CRC models in vivo (MC38 and RO100). Treatment efficacy was accentuated when combined with anti-PD1 treatment, and single-cell RNA sequencing analysis revealed depletion of tumor cells and increased T cell infiltration and activation in both treated and abscopal tumors. However, human tissue analysis ex vivo (E-slices) using PDX models and patient samples showed that ASP9801 is not effective in CRC, consistent with clinical trial results. On the other hand, ASP9801 was highly effective in GBM, indicating indication-specific efficacy of ASP9801, and how E-slice assays can be used to identify treatment-sensitive indications. This study demonstrates the superiority of E-slices over mouse models for predicting clinical response and its utility in planning clinical trials.

Single-reaction multiplex nucleic acid testing (NATs) must both co-amplify multiple targets and distinguish the resulting amplicons within the product mixture. Most conventional assays achieve this distinction using target-specific reporters or other external encoding elements. Here, we introduce a primer-programmed multiplexing strategy in which primer design generates target-associated amplicons with predefined lengths that are read electrically with nanopores. In a proof-of-concept implementation, one-pot recombinase polymerase amplification (RPA) generated four amplicons with predefined product lengths from Monkeypox (Mpox), Cowpox, Vaccinia, and human RNase P. The resulting amplicons were analyzed with glass nanopores, and individual translocation events were classified by a deep neural network (DNN) based on a six-dimensional feature set derived from nanopore recordings. Event-level decoding achieved >95% accuracy, >94.7% sensitivity, and >98.3% specificity, and aggregation of classified events yielded sample-level calls with >93.3% sensitivity and >93.9% specificity in blinded samples, with qualitative calls matching polymerase chain reaction (PCR) results across the 9 PCR-benchmarked samples. These results demonstrate a probe-free multiplexing architecture in which target identity is programmed through primer-defined amplicon design and decoded from the resulting multidimensional nanopore response using a DNN, eliminating the need for target-specific reporter chemistry and opening new opportunities for all-electrical multiplex nucleic acid testing based on unmodified amplification products.

Hepatocellular carcinoma (HCC) develops in a chronically injured and immunologically specialized organ that is both the treatment target and a major determinant of viral clearance and toxicity. Oncolytic viruses (OVs) can couple tumor-selective infection with immunogenic lysis, innate sensing, antigen presentation, and adaptive immune recruitment, but the clinical consequences of these processes depend on viral platform, delivery route, liver-resident cell handling, spatial disease distribution, and hepatic reserve. This review synthesizes HCC OV biology across four linked levels: platform-specific tumor-cell susceptibility; replication and interferon restriction; regulation by Kupffer cells, liver sinusoidal endothelial cells, dendritic-cell subsets, hepatic stellate cells, fibroblasts, and resident lymphoid populations; and spatial pharmacodynamics across injected tumors, non-injected intrahepatic lesions, cirrhotic parenchyma, and extrahepatic disease. We compare adenovirus, vaccinia virus, herpes simplex virus, and candidate RNA viruses using route- and assay-matched criteria and interpret clinical evidence according to study design, biological context, and permissible inference. Early Pexa-Vec studies established biological activity, whereas PHOCUS defined a negative boundary for Pexa-Vec followed by sorafenib in an unselected first-line population. H101 plus nivolumab and VG161 support early feasibility and pharmacodynamic investigation, while integrated human pharmacodynamic and diseased-liver HCC-model reovirus data highlight schedule-dependent immune effects and potential off-tumor liver inflammation. VIRAL-HCC is used only as a non-scored organizational matrix for prospective trial design and biomarker interpretation. Future development should link tumor exposure and productive infection or payload expression to compartment-resolved immune effects, clinically relevant tumor control, and preservation of hepatic reserve.

Reference methods in antiviral drug discovery, the plaque reduction assay (PRA) and viral yield reduction, are labor-intensive and low-throughput, whereas tetrazolium-based metabolic assays (MTT) may be unsuitable for viruses that sustain host-cell metabolism during productive infection, including poxviruses. We validated a digital densitometric assay (DDA), which quantifies antiviral and cytotoxic effects in crystal violet-stained monolayers using the open-source software Fiji/ImageJ, against three reference methods, with vaccinia virus strain Western Reserve in Vero cells. Across 38 fungal extracts, DDA agreed substantially with PRA for antiviral activity (Lin's concordance correlation coefficient, CCC = 0.869; 95% limits of agreement -31.5 to +21.1 percentage points) and moderately with MTT for cytotoxicity (CCC = 0.677) and was more precise between replicates (median coefficient of variation 2.5% versus 5.5%). For the test compound FAC22, the half-maximal cytotoxic concentration was 238.3 μM by DDA and 46.4 μM by MTT, a five-fold difference between readouts; the half-maximal effective concentration was 8.83 μM by viral yield reduction and 11.9 μM by DDA over its non-cytotoxic range. In infected cultures MTT separated virus and cell controls by only 1.08 to 1.39-fold, precluding estimation of an effective concentration, against 1.8 to 2.3-fold for DDA. DDA met conventional robustness criteria on the screening plate (Z' = 0.818 antiviral, 0.861 cytotoxicity), comparable to PRA (Z' = 0.91), cut reagent consumption by 80 to 90%, and shortened analysis from 20 min to under 1 min per plate. DDA is accurate, resource-efficient and scalable for poxvirus screening, and applicable to other cytopathic viruses pending validation.

Vaccinia-related kinase 1 (VRK1) is a nuclear serine/threonine kinase that contributes to genome stability through the phosphorylation of diverse substrates. VRK1 is frequently upregulated in multiple malignancies. Although its function as a chromatin kinase has been extensively characterized, its potential role in RNA processing remains largely unexplored. Using label-free quantitative phosphoproteomics, we identified the splicing scaffold protein serine/arginine repetitive matrix protein 2 (SRRM2) as a high-confidence VRK1-dependent phosphorylation target. We show that VRK1-dependent SRRM2 phosphorylation is required for efficient alternative splicing of DNA damage response (DDR)-related transcripts and contributes to cellular resistance to genotoxic stress. Collectively, this study reveals that VRK1 supports DDR-related splicing by regulating SRRM2 phosphorylation, providing a novel molecular mechanism by which VRK1 contributes to the DNA damage response.

Tumors often exhibit immunosuppressive microenvironments (TMEs) and present as immunologically "cold." Oncolytic viruses (OVs), particularly engineered vaccinia viruses (oncoVVs), are an encouraging therapy that directly lyses cancer cells and reprograms the TME. In this study, we evaluated the therapeutic potential of an oncolytic vaccinia virus expressing human C-type lectin domain family 2 member A (oncoVV-CLEC2A) in melanoma and ovarian cancer cells. OncoVV-CLEC2A elevated reactive oxygen species (ROS) levels, suppressed Nrf2-mediated antioxidant responses, and triggered autophagy. These changes promoted cytokine production (IL-6, IFN-α/β/γ, TNF-α) via activation of AP-1 and IRF3/7. In a B16-F10 melanoma model, oncoVV-CLEC2A increased serum levels of IFN-γ, accompanied by augmented infiltration of CD8[+] T cells and activation of NK cells. Taken together, oncoVV-CLEC2A reshapes the TME by intensifying oxidative stress and autophagic activity, effectively turning "cold" tumors "hot." Notably, the mutual reinforcement between ROS production and autophagy activation not only enhances viral replication but also triggers cytokine-mediated immunity.

In 2022, a global Mpox outbreak occurred, and the World Health Organization (WHO) declared it a Public Health Emergency of International Concern (PHEIC). Although the outbreak was quickly constrained, new cases of infection associated with mutated virions continue to be documented worldwide. In recent global health crises, there is an urgent need to develop rapid, cost-effective diagnostic assays. While nucleic acid assays (e.g., PCR and CRISPR-based methods) represent the current gold standard for Mpox virus (MPXV) detection, point-of-care (POC) assays for Mpox remain limited. Here, we developed a molecularly imprinted polymer (MIP)-based MPXV sensor targeting A29L, a surface protein involved in host cell binding. The MIP sensor exhibits a remarkable detection limit of 7.7 pg/mL (0.54 pM). Moreover, we demonstrated that the MIP sensor detected A29L in artificial interstitial fluid (AIF) with a detection limit of 12.4 pg/mL (0.86 pM), supporting sensitive detection in a lesion-relevant model matrix. The MIP sensor preferentially responded to A29L over MPXV B6R, vaccinia virus A27L, and biointerferents and retained its response after 30 days. The results indicate that the MIP-based MPXV sensor has strong potential for clinical Mpox detection and could serve as a valuable diagnostic platform in future pandemics.

The noncanonical nuclear factor κB (NF-κB) pathway regulates adaptive immune responses and antiviral innate immunity. Orthopoxviruses, including the human pathogen variola virus and zoonotic viruses such as vaccinia virus and monkeypox virus, have evolved multiple strategies to suppress host antiviral signaling. Our previous work using in vitro immune cell models demonstrated that the mouse-specific orthopoxvirus ectromelia virus (ECTV) inhibits the noncanonical NF-κB signaling pathway. Here, we investigated the impact of ECTV on noncanonical NF-κB signaling in human epithelial HeLa cells. Tumor necrosis factor superfamily ligands were used to activate or modulate noncanonical NF-κB signaling. Using fluorescence microscopy and immunoblot analyses, we found that ECTV reduced LIGHT (TNFSF14)- and lymphotoxin α1β2 (LTα1β2)-induced nuclear accumulation of the NF-κB subunits RelB and p52. ECTV infection alone increased whole-cell RelB and NF-κB2/p100 levels but did not significantly alter nuclear RelB levels. In TNF-α-stimulated cells, ECTV reduced levels of cellular inhibitor of apoptosis protein 2 (cIAP2), whereas in LTα1β2-stimulated cells, ECTV reduced levels of TNF receptor-associated factor 3 (TRAF3). RT-qPCR analysis revealed that ECTV upregulated RELB, NFKB2, and CHUK expression. Viral infection also increased expression of TNFAIP3 and OTUD7B, genes encoding regulators of noncanonical NF-κB signaling. Moreover, infected cells exhibited increased expression of selected RelB-responsive genes, including FBRS, CCR10, and TNFRSF6B. Together, these findings indicate that ECTV modulates components associated with noncanonical NF-κB signaling in epithelial cells in vitro, without evidence of constitutive pathway activation.

Oncolytic virotherapy has emerged as a promising cancer treatment that combines selective tumor destruction with potent stimulation of antitumor immunity. Unlike conventional therapies, oncolytic viruses (OVs) preferentially infect and replicate within malignant cells while sparing normal tissues, leading to direct tumor lysis and the release of tumor-associated antigens, pathogen-associated molecular patterns, and damage-associated molecular patterns that activate both innate and adaptive immune responses. Advances in molecular virology and genetic engineering have transformed naturally occurring viruses into programmable therapeutic platforms capable of delivering immunomodulatory payloads that enhance antitumor efficacy and remodel the tumor microenvironment. Multiple DNA- and RNA-based viral platforms, including herpes simplex virus, adenovirus, vaccinia virus, reovirus, measles virus, Newcastle disease virus, vesicular stomatitis virus, and coxsackievirus, are currently under clinical investigation, with regulatory approval of talimogene laherparepvec (T-VEC) and G47Δ providing important proof of clinical feasibility. Increasing evidence indicates that OVs can convert immunologically "cold" tumors into "hot" tumors, thereby improving responsiveness to immune checkpoint inhibitors and other therapeutic modalities. Consequently, oncolytic virotherapy has become an attractive component of combination strategies involving immunotherapy, chemotherapy, radiotherapy, targeted therapies, and cellular therapies. Nevertheless, several challenges continue to limit its broader clinical application, including antiviral immune clearance, inefficient systemic delivery, restricted intratumoral spread, tumor heterogeneity, manufacturing complexity, and the lack of robust predictive biomarkers. This review provides a comprehensive overview of the biological foundations of oncolytic virotherapy, viral engineering strategies, mechanisms of tumor selectivity, direct oncolysis, immunogenic cell death, and immune remodeling of the tumor microenvironment. It further summarizes the major oncolytic virus platforms, recent preclinical and clinical advances, emerging combination strategies, current translational challenges, and future directions expected to advance precision oncolytic virotherapy and expand its role in modern cancer treatment.

During poxvirus morphogenesis, the external scaffold defines the shape and size of immature virions. However, its structural characterization has been hindered by the pleomorphic nature of authentic virus particles. Here, we present single-particle cryo-electron microscopy and cryo-electron tomography structures of two distinct in vitro assemblies of the vaccinia virus scaffold protein D13 that recapitulate oligomeric states previously observed in situ. These structures reveal a dramatic transition from rod-like oligomers, representing a pre-assembly state, to a fully formed scaffold, triggered by the docking of the N-terminal peptide of the viral membrane protein A17 into the base cavity of D13. We propose that this interaction destabilizes the pre-assembly conformation and initiates scaffold formation upon viral membrane recruitment, ultimately leading to the formation of immature virions. Our findings provide structural insights into the early stages of poxvirus morphogenesis and establish a mechanistic link between conformational changes in D13 and scaffold assembly.

The rapid development of effective COVID-19 vaccines was enabled by the parallel assessment of multiple candidates early in the pandemic, including candidates based on the Modified Vaccinia virus Ankara (MVA) vector platform. This Phase 1 trial (NCT04569383) evaluated the MVA-based vaccine candidate MVA-SARS-2-S, expressing the full-length native SARS-CoV-2 spike glycoprotein (Wuhan-HU-1). Between October 2020 and May 2021, 30 healthy, SARS-CoV-2-naive adults received two doses of either 10[7] or 10[8] international units (IU) of MVA-SARS-2-S 28 days apart, with 6-month follow-up. A subgroup (n = 12) received two BNT162b2 booster vaccinations. Safety and reactogenicity were primary objectives; secondary and exploratory objectives included S1 seroconversion rates, S-domain-specific antibodies, SARS-CoV-2-neutralizing antibodies, and spike-specific T cell responses. MVA-SARS-2-S demonstrated a good safety profile. No severe or serious adverse events occurred. Immunogenicity was poor: S1-specific seroconversion occurred in 0/15 (10[7] IU group) and 3/15 (10[8] IU group) participants. Binding antibodies were predominantly directed against the S2 subunit, with peak median anti-S2 fold changes of 6.0 and 5.2 in the 10[7] IU and 10[8] IU groups, respectively. Neutralizing antibodies were detected in one participant (10[8] IU group); spike-specific T cell responses were observed in 5/15 (10[7] IU group) and 2/13 (10[8] IU group) participants. Heterologous BNT162b2 vaccination induced S1 seroconversion and detectable neutralizing antibody and T cell responses in all boosted participants, without safety concerns. In conclusion, while MVA-SARS-2-S was well tolerated and did not adversely affect subsequent BNT162b2 boosting, the vaccine candidate elicited insufficient immunogenicity, underscoring the need for optimized antigen design.