Aug 2026· Veterinary Research· Vol 57· 0 citations· 42 references
Medicine
TL;DR
The recombinant virus maintained wild-type-comparable titers while carrying 3606 bp of total foreign sequences, highlighting its dual-site advantage and potential as a multivalent poultry vaccine vector.
Abstract
Fowl adenovirus serotype 4 (FAdV-4) is a nonenveloped double-stranded DNA virus with a 43–45 kb genome, making it a promising viral vector for developing multivalent poultry vaccines. However, limited genomic studies have hindered the design of strategies for generating recombinant FAdV-4 capable of expressing multiple foreign proteins while maintaining high viral titers. In this study, three deletion mutants (rD14AB, rD2042, rD19A) were generated to assess their roles in viral replication and pathogenicity. Only rD2042 showed impaired replication and virulence compared with wild-type FAdV-4. Foreign gene expression assays demonstrated that the D14AB site had a maximum insertion capacity of 2349 bp, and an SV40 poly(A) signal was required for efficient protein expression and viral propagation at this locus. In contrast, the D19A site tolerated insertions of up to 1257 bp in the absence of this signal. Using the insertion strategy optimized for wild-type CH_JS_2017, we modified the previously reported nonpathogenic rON1 strain (harboring the Hexon gene from the attenuated ON1 strain) to co-express mCherry, eGFP, and mTagBFP2 at the two independent sites. SPF chicken trials verified the safety of the engineered virus at immunization age and the genetic stability of exogenous genes. The recombinant virus maintained wild-type-comparable titers while carrying 3606 bp of total foreign sequences, highlighting its dual-site advantage and potential as a multivalent poultry vaccine vector.
It is demonstrated that NTV-ΔF1L-C7L combines high safety with enhanced replication and immunogenicity, supporting its value as a novel vaccine vector and its potential application in controlling the current global monkeypox outbreak.
Jiao Ren, Shi-Yuan Liu, Hang Yuan et al.· Journal of Virology· 0 citations
Classical swine fever virus (CSFV) and pseudorabies virus (PRV) remain significant threats to the swine industry. Although recombinant PRV vectors represent promising platforms for bivalent vaccine development, their application is frequently limited by insufficient expression of heterologous antigens, which may compromise protective efficacy. To address this limitation, we employed a multi-copy expression strategy to enhance CSFV E2 protein levels in a PRV-based vector. Using CRISPR/Cas9-mediated gene editing, we constructed recombinant PRVs expressing one, two, or three copies of the E2 gene by sequential insertion into the gE/gI, tk, and gG loci. In vitro validation demonstrated increased detectable E2 expression in the multi-copy recombinant viruses. Meanwhile, the recombinant viruses maintained virion morphology and replication kinetics comparable to those of the parental strain PRV-GX. Immunogenicity studies in rabbits showed that PRV-3CE2 elicited stronger E2-specific humoral responses and E2-associated cytokine recall responses than PRV-2CE2, while both recombinant viruses induced detectable neutralizing activity. In challenge experiments, although vaccination did not completely prevent febrile responses following CSFV challenge, both PRV-2CE2 and PRV-3CE2 reduced CSFV RNA loads in blood compared with the mock group, suggesting partial protective efficacy. Additionally, both PRV-2CE2 and PRV-3CE2 provided complete protection against lethal PRV challenge in rabbits. These findings suggest that multi-copy E2 expression enhances E2-associated immunogenicity and supports further optimization and evaluation of PRV-3CE2 as a PRV-CSFV bivalent vaccine candidate.
Xianfei Shang, Hui Zhao, Yufeng He et al.· Veterinary Microbiology· 0 citations
The CAG hybrid promoter improved not only the expression level, but also the immunogenicity of the fusion protein, and may thus provide a promising strategy for HTNV vaccine research.
LI Kai, Pu-yuan Li, Xing-an Wu et al.· 0 citations
Pseudorabies virus (PRV) and Porcine epidemic diarrhea virus (PEDV) are currently co-circulation among swine herd with multiple variant strains, which cause severe economic losses to the global swine industry. In the present study, a recombinant PRV-based vaccine candidate was constructed backbone of a PRV variant via deletion of five genes (gI, gE, US2, US9, and TK) and insertion of the S1 gene derived from a PEDV G2b strain, and the resulting recombinant virus was generated as rPRV-Δ5-S1. The safety of the quintuple-gene-deleted PRV vector (rPRV-Δ5) was firstly evaluated in rabbits and compared with the previously generated triple-gene-deleted vector (rPRV-Δ3). Rabbits inoculated with rPRV-Δ5 exhibited milder clinical manifestations, lower viral loads in tissues, and fewer histopathological lesions, indicating an improved safety profile. Subsequent immunization trials in mice demonstrated that rPRV-Δ5-S1 induced PEDV S1-specific antibodies and PEDV-neutralizing antibodies, which were higher than those observed in the commercial inactivated PEDV vaccine group. Furthermore, rPRV-Δ5-S1 elicited higher neutralizing antibody titers against PRV variants than the Bartha-K61 vaccine and provided complete protection against lethal challenge with the PRV variant. To further validate the immunogenicity and protective efficacy of rPRV-Δ5-S1, challenge experiments were conducted in piglets. The results showed that piglets immunized with rPRV-Δ5-S1 developed detectable antibody responses against both PRV and PEDV. After challenge with the PEDV G2b variant, rPRV-Δ5-S1-immunized piglets exhibited reduced diarrhea severity, decreased viral shedding, alleviated intestinal lesions, and improved weight gain compared with control animals. Collectively, these findings demonstrate that the quintuple-gene-deleted PRV vector exhibits an improved safety profile and can serve as an effective platform for heterologous antigen delivery. The recombinant virus rPRV-Δ5-S1 can induced immune responses against both PRV and PEDV in multiple animal models, which supports its potential as a bivalent vaccine candidate against these two economically critical swine pathogens.
Junda Li, Jiadeng Jiang, Ruhai Guo et al.· Frontiers in Microbiology· 0 citations
A modular, helper-virus-free and high-efficiency rescue platform based on an orthogonal transcription system utilizing orthogonal promoters and engineered RNA polymerases fused to an mRNA capping enzyme is developed and provides a proof-of-concept methodological framework for the rapid development of vaccine candidates.
Zihan Ma, Weijun Wang, Qiuli Lou et al.· Synthetic and Systems Biotec...· 0 citations
In this study, a recombinant adenovirus vaccine candidate constructed and evaluated to addressed the widespread clinical problem of co-infections of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus Type 2 (PCV2). Epidemiological analysis revealed a PRRSV-positive rate of 40.8%, with prevalent strains belonging to Lineage 1 and Lineage 8. Analysis of key amino acid sites in the GP5 protein indicated the presence of highly virulent strain characteristics and widespread wild-type strain variation. Based on these findings and previous studies, we selected multiple PRRSV proteins (GP2–GP5, M, N) from multiple strains and the validated PCV2 Cap protein fused with dominant T/B cell epitopes, and successfully constructed four recombinant adenoviruses using a 2A peptide-linked strategy. These constructs stably expressed the target antigens in HEK293 cells and demonstrated good passage stability. Animal experiments showed that piglets immunized with the recombinant adenoviruses developed high levels of PRRSV- and PCV2-specific antibodies and neutralizing antibodies, and exhibited lymphocyte proliferation and secretion of cytokines including IFN-γ, IL-2, and IL-4, indicating the vaccine simultaneously elicited both humoral and cellular immune responses. Following PRRSV challenge, immunized groups, particularly the multi-antigen combined immunization group, demonstrated significant clinical protection. This study provides a promising vaccine candidate and a theoretical foundation for the coordinated control of PRRSV and PCV2.
Yuwan Li, Jun-Zhi Ji, K. Wang et al.· BMC Veterinary Research· 0 citations
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