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.

The emergence of SARS-CoV-2 has necessitated the development of versatile vaccines capable of addressing evolving variants. Prime-2-CoV_Beta, a novel Orf virus-based COVID-19 vaccine, was developed to express the SARS-CoV-2 spike and nucleocapsid antigens. This first-in-human, phase I, dose-finding clinical trial was conducted to assess the safety, reactogenicity, and immunogenicity of Prime-2-CoV_Beta as a booster in healthy adults. From June 2022 to June 2023, 60 participants in Germany received varying doses of Prime-2-CoV_Beta. The study demonstrated a favorable safety profile, with no serious adverse events (AEs) reported. All AEs were mild (107) or moderate (10), with the most common symptoms being pain at the injection site, fatigue, and headache. Immunogenicity assessments revealed robust vaccine-induced antigen-specific immune responses. High doses notably elicited significant increases in antibodies against the spike and nucleocapsid proteins as well as neutralizing antibodies against SARS-CoV-2 and its variants. Additionally, the vaccine did not induce ORFV-neutralizing antibodies, indicating the potential for repeated administration. In conclusion, Prime-2-CoV_Beta was safe, well tolerated, and immunogenic, demonstrating potential as a broadly protective vaccine against SARS-CoV-2 and its variants. These promising results support further evaluation of higher doses and additional studies to confirm efficacy and long-term protection. This trial was registered at ClinicalTrials, NCT05389319.

Although dengue virus (DENV) affects almost half of the world’s population there are neither preventive treatments nor any long-lasting and protective vaccines available at this time. The complexity of the protective immune response to DENV is still not fully understood. The most advanced vaccine candidates focus specifically on humoral immune responses and the production of virus-neutralizing antibodies. However, results from several recent studies have revealed the protective role of T cells in the immune response to DENV. Hence, in this study, we generated a novel and potent DENV vaccine candidate based on an Orf virus (ORFV, genus Parapoxvirus) vector platform engineered to encode five highly conserved or cross-reactive DENV human leukocyte antigen (HLA)-A*02- or HLA-B*07-restricted epitopes as minigenes (ORFV-DENV). We showed that ORFV-DENV facilitates the in vitro priming of CD8⁺ T cells from healthy blood donors based on responses to each of the encoded immunogenic peptides. Moreover, we demonstrated that peripheral blood mononuclear cells isolated from clinically confirmed DENV-positive donors stimulated with ORFV-DENV generate cytotoxic T cell responses to at least three of the expressed DENV peptides. Finally, we showed that ORFV-DENV could activate CD8⁺ T cells isolated from donors who had recovered from Zika virus (ZIKV) infection. ZIKV belongs to the same virus family (Flaviviridae) and has epitope sequences that are homologous to those of DENV. We found that highly conserved HLA-B*07-restricted ZIKV and DENV epitopes induced functional CD8⁺ T cell responses in PBMCs isolated from confirmed ZIKV-positive donors. In summary, this proof-of-concept study characterizes a promising new ORFV D1701-VrV-based DENV vaccine candidate that induces broad and functional epitope-specific CD8⁺ T cell responses.

Background: Emerging immune-evasive viral variants threaten the efficacy of current vaccines, underscoring the need for strategies that elicit broad and durable protection. Heterologous prime–boost regimens combining distinct vaccine platforms can enhance humoral and cellular immunity through complementary mechanisms. Methods: Using an intramuscular immunization scheme aligned with clinical vaccination practice, CD-1 mice received homologous or heterologous prime–boost regimens combining a replication-deficient Orf virus (Parapoxvirus orf, ORFV)-based spike vaccine (ORFV-S) with the licensed adjuvanted recombinant protein vaccine VidPrevtyn Beta. Spike-specific humoral and cellular immune responses were assessed. Results: ORFV-S alone induced potent and broad spike-specific IgG responses and achieved the strongest ACE2-binding inhibition across variants of concern. ORFV-S priming followed by VidPrevtyn Beta boosting markedly enhanced the magnitude and cross-variant breadth of antibody responses compared with homologous protein vaccination. Both homologous ORFV-S and heterologous regimens incorporating ORFV-S elicited strong CD4⁺ and CD8⁺ T-cell responses, whereas VidPrevtyn Beta alone induced only modest T-cell activity, demonstrating that ORFV-S effectively complements protein-based vaccines. Conclusions: The ORFV-S vector represents a potent vaccine platform capable of inducing broad humoral and cellular immunity. Its use in heterologous prime–boost combinations enhances both antibody magnitude and breadth beyond homologous protein vaccination, supporting its application in vaccination strategies against evolving viral pathogens.