Advancing ORFV-Based Therapeutics to the Clinical Stage

Abstract

The Orf virus (ORFV) is the prototype member of the parapoxvirus family and has long been recognized for its robust immunogenicity, favourable safety profile and its ability to stimulate both cellular and humoural immune responses without inducing significant anti-vector immunity. Despite these inherent advantages, early applications of ORFV-based technologies were limited by challenges in manufacturing scalability and uncertainties regarding clinical safety in humans. However, recent breakthroughs have transformed this therapeutic landscape. A landmark achievement is the development of Prime-2-CoV, an ORFV-based anti-COVID-19 vaccine that has advanced into human clinical trials, providing the first clinical evidence of live ORFV's feasibility, safety and immunogenicity. This milestone, together with the establishment of a good manufacturing practice (GMP)-compliant production process and comprehensive preclinical evaluations, has laid a robust foundation for broader clinical applications of ORFV-based therapeutics. Moreover, the use of ORFV as an oncolytic virus therapy has shown promising results, effectively converting immunologically 'cold' tumours into 'hot' ones, underscoring its versatility as a therapeutic platform. In this review, we critically assess recent advances in ORFV-based therapeutics, with a particular focus on vaccine development and oncolytic virotherapy (OVT). We thoroughly discuss the milestones and impact of the first ORFV-based clinical trial, outline strategies for optimizing the technology and provide insights into overcoming remaining challenges. Collectively, these advancements position ORFV as a highly promising and versatile platform for next-generation prophylactic and therapeutic interventions in both human and veterinary medicine, while also providing a roadmap for future innovations.

Keywords: ORFV; immune stimulation; immunogenic cell death; oncolytic virus; orf virus; parapoxvirus; vaccine; viral vector.

FIGURE 1

Anti‐tumour mechanisms of oncolytic ORFV. Oncolytic ORFV selectively replicates in tumour cells, inducing highly immunogenic cell death through direct lysis as well as the induction of apoptosis, pyroptosis and autophagy, while sparing healthy cells. The release of viral progeny, TAAs and DAMPs or pathogen‐associated molecular patterns (PAMPs) promotes the recruitment and activation of innate immune cells. This leads to the release of pro‐inflammatory cytokines, further enhancing immune activation. As a result, APCs prime a robust humoural and cellular adaptive anti‐tumour response, ultimately transforming the TME from immunologically inert (‘cold’) to highly reactive (‘hot’), facilitating the effective clearance of malignant cells.

FIGURE 2

Mode of action of ORFV D1701‐VrV based vaccines. Upon administration, D1701‐VrV recombinants encoding heterologous antigens (also called transgenes; depicted as yellow and green arrows) under the control of synthetic or native early‐phase promoters are internalized by professional APCs, such as macrophages and DCs, via receptor‐independent macropinocytosis (1). Inside the cytoplasm, the viral core is uncoated, releasing the genome, which is transcribed by viral RNA polymerases and transcription factors prepackaged within the particle (2). This early‐phase transcription includes the encoded transgenes and essential viral factors for sustained expression. Importantly, gene expression remains restricted to the early phase, preventing progression of the poxviral infection cycle to genome replication or late‐phase expression. Expressed transgenes are processed by the proteasome and presented on MHC class I and II molecules, activating CD8+ and CD4+ T cells (3). Concurrently, recognition of the viral double‐stranded DNA genome by the cGAS‐STING pathway (4) triggers innate immune signalling, leading to the activation of interferon regulatory factors (IRF) and NF‐kB (5). This activation drives the release of cytokines, such as IL‐12 and CXCL10, and the upregulation of costimulatory molecules, including CD80, CD86 and OX40L (6). The combination of antigen presentation and APC activation facilitates robust stimulation of adaptive and innate immune responses, including the activation of CD4⁺ and CD8⁺ T cells, B cells and NK cells. This results in potent humoural and cellular immunity against the encoded transgenes.

FIGURE 3

Design of Prime‐2‐CoV as a multi‐antigenic vaccine against SARS‐CoV‐2. (A) Structure of the SARS‐CoV‐2 virion. (B) Illustration of Prime‐2‐CoV, designed to express both the spike and nucleocapsid antigen of SARS‐CoV‐2 from two separate loci within the genome of the D1701‐VrV vector under control of viral early‐phase promoters (P).

FIGURE 4

Milestones of Prime‐2‐CoV to enter clinical trials. Timeline highlighting critical milestones in the development of Prime‐2‐CoV, from the start of the project in response to the Covid‐19 pandemic to the first in human clinical trials, as well as their ultimate impact on D1701‐VrV based therapeutics. Blue borders mark the advances in the pre‐clinical development of the vaccine candidate, red the development of a corresponding GMP compliant production process and green the clinical trials.