The pore-forming inflammatory cell death pathway, pyroptosis, was first described in the early 1990s and its role in health and disease has been intensively studied since. The effector molecule GSDMD is cleaved by activated caspases, mainly Caspase 1 or 11 (Caspase 4/5 in humans), downstream of inflammasome formation. In this review, we describe the molecular events related to GSDMD-mediated pore formation. Furthermore, we summarize the so far elucidated ways of SARS-CoV-2 induced NLRP3 inflammasome formation leading to pyroptosis, which strongly contributes to COVID-19 pathology. We also explore the potential of NLRP3 and GSDMD inhibitors as therapeutics to counter excessive inflammation.

The Orf virus (ORFV; Parapoxvirus) strain D1701 with an attenuated phenotype and excellent immunogenic capacity is successfully used for the generation of recombinant vaccines against different viral infections. Adaption for growth in Vero cells was accompanied by additional major genomic changes resulting in ORFV strain variant D1701-V. In this study, restriction enzyme mapping, blot hybridization and DNA sequencing of the deleted region s (A, AT and D) in comparison to the predecessor strain D1701-B revealed the loss of 7 open reading frames (ORF008, ORF101, ORF102, ORF114, ORF115, ORF116, ORF117). The suitability of deletion site D for expression of foreign genes is demonstrated using novel synthetic early promoter eP1 and eP2. Comparison of promoter strength showed that the original vegf-e promoter Pv as well as promoter eP2 display an up to 11-fold stronger expression than promoter eP1, irrespective of the insertion site. Successful integration and expression of the fluorescent marker genes is demonstrated by gene- and insertion-site specific PCR assays, fluorescence microscopy and flow cytometry. For the first time ORFV recombinants are generated simultaneously expressing transgenes in two different insertion loci. That allows production of polyvalent vaccines containing several antigens against one or different pathogens in a single vectored ORFV vaccine.

The potency of viral vector-based vaccines depends on their ability to induce strong transgene-specific immune response without triggering anti-vector immunity. Previously, Orf virus (ORFV, Parapoxvirus) strain D1701-V was reported as a novel vector mediating protection against viral infections. The short-lived ORFV-specific immune response and the absence of virus neutralizing antibodies enables repeated immunizations and enhancement of humoral immune responses against the inserted antigens. However, only limited information exists about the D1701-V induced cellular immunity. In this study we employed major histocompatibility complex (MHC) ligandomics and immunogenicity analysis to identify ORFV-specific epitopes. Using liquid chromatography-tandem mass spectrometry we detected 36 ORFV-derived MHC I peptides, originating from various proteins. Stimulated splenocytes from ORFV-immunized mice did not exhibit specific CD8+ T cell responses against the tested peptides. In contrast, immunization with ovalbumin-expressing ORFV recombinant elicited strong SIINFEKL-specific CD8+ T lymphocyte response. In conclusion, our data indicate that cellular immunity to the ORFV vector is negligible, while strong CD8+ T cell response is induced against the inserted transgene. These results further emphasize the ORFV strain D1701-V as an attractive vector for vaccine development. Moreover, the presented experiments describe prerequisites for the selection of T cell epitopes exploitable for generation of ORFV-based vaccines by reverse genetics.

Background: Orf virus (ORFV)-based vectors are attractive for vaccine development as they enable the induction of potent immune responses against specific transgenes. Nevertheless, the precise mechanisms of immune activation remain unknown. This study therefore aimed to characterize underlying mechanisms in human immune cells.Methods: Peripheral blood mononuclear cells were infected with attenuated ORFV strain D1701-VrV and analyzed for ORFV infection and activation markers. ORFV entry in susceptible cells was examined using established pharmacological inhibitors. Using the THP1-Dual™ reporter cell line, activation of nuclear factor-κB and interferon regulatory factor pathways were simultaneously evaluated. Infection with an ORFV recombinant encoding immunogenic peptides (PepTrio-ORFV) was used to assess the induction of antigen-specific CD8+ T cells.Results: ORFV was found to preferentially target professional antigen-presenting cells (APCs) in vitro, with ORFV uptake mediated primarily by macropinocytosis. ORFV-infected APCs exhibited an activated phenotype, required for subsequent lymphocyte activation. Reporter cells revealed that the stimulator of interferon genes pathway is a prerequisite for ORFV-mediated cellular activation. PepTrio-ORFV efficiently induced antigen-specific CD8+ T cell recall responses in a dose-dependent manner. Further, activation and expansion of naïve antigen-specific CD8+ T cells was observed in response.Discussion: Our findings confirm that ORFV induces a strong antigen-specific immune response dependent on APC uptake and activation. These data support the notion that ORFV D1701-VrV is a promising vector for vaccine development and the design of innovative immunotherapeutic applications.

The Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigates for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by Cottontail Rabbit Papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6, respectively. In two independent experiments we used in total 23 rabbits which immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as control five weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations with the combined four ORFV-CRPV recombinants of rabbits with high tumor burden resulted in significant growth retardation of the tumors as compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden compared to non-immunized rabbits. Tumor growth was significantly reduced after immunization and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors. IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used the Orf Virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing the Cottontail Rabbit Papillomavirus (CRPV) early proteins E1, E2, LE6, and E7, respectively. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieves significant suppression of tumor growth. In total, 84.6 % of all outbred animals benefit from the ORFV-CRPV vaccination showing reduction in tumor size and significant tumor growth inhibition including one animal with complete tumor regression without recurrence.

The Orf virus (ORFV) is a promising vector platform for the generation of vaccines against infectious diseases and cancer, highlighted by its progression to clinical testing phases. One of the critical steps during GMP manufacturing is the clarification of crude harvest because of the enveloped nature and large size of ORFV. This study presents the first description of ORFV clarification process from a HEK suspension batch process. We examined various filter materials, membrane pore sizes, harvest timings, and nuclease treatments. Employing the Ambr® crossflow system for high-throughput, small-volume experiments, we identified polypropylene-based Sartopure® PP3 filters as ideal. These filters, used in two consecutive stages with reducing pore sizes, significantly enhanced ORFV recovery and addressed scalability challenges. Moreover, we demonstrated that the time of harvest and the use of a nuclease play a decisive role to increase ORFV yields. With these findings, we were able to establish an efficient and scalable clarification process of ORFV derived from a suspension production process, essential for advancing ORFV vaccine manufacturing.