Dual-expression-site fowl adenovirus 4 (FAdV-4) with high viral titer: a promising viral vector for multivalent poultry vaccines

Enhanced replication and immunogenicity of a genome-modified non-replicating vaccinia virus Tiantan strain for robust low-dose protection in mice

ABSTRACT A non-replicating Tiantan strain-based vaccinia virus (NTV) holds significant application prospects for vaccination and gene therapy and has recently entered clinical trials as a novel and safer vaccine candidate against monkeypox. However, optimization is still required, particularly regarding its production capacity and immunogenicity. In this study, a recombinant virus was constructed by modifying the F1L and C7L genes in a non-replicating viral backbone using CRISPR/Cas9-mediated gene editing and homologous recombination. The resulting construct, designated NTV-ΔF1L-C7L, exhibited significantly enhanced replication in vaccine production cell lines, with viral yields increasing by more than 680-fold in MRC-5 cells compared to those of the parental NTV. Its pathogenicity in mice was significantly reduced, showing more than a 10-fold decrease compared to the pathogenicity of the vaccinia virus Tiantan strain (VTT). Following two intramuscular doses, NTV-ΔF1L-C7L elicited high titers of orthopoxvirus-specific IgG and neutralizing antibodies against vaccinia and monkeypox viruses, along with a robust cellular immune response exhibiting a Th1 bias, which was significantly stronger than that induced by either parental NTV or VTT vaccination. Complete protection (100%) against a lethal challenge with the vaccinia virus Western Reserve strain was achieved in mice immunized with either a low dose (103 PFU) or a single dose (10⁵ PFU) of NTV-ΔF1L-C7L, comparable to that conferred by VTT. These findings demonstrate 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. IMPORTANCE A highly attenuated NTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-ΔF1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-ΔF1L-C7L, demonstrates several significant advantages. Its robust in vitro replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity in vivo , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-ΔF1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox. A highly attenuated NTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-ΔF1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-ΔF1L-C7L, demonstrates several significant advantages. Its robust in vitro replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity in vivo , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-ΔF1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox.

Multi-copy expression of classical swine fever virus E2 in a recombinant pseudorabies virus enhances immunogenicity and supports its potential as a PRV-CSFV bivalent vaccine candidate.

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.

virus-specific immune responses in C57BL/6 mice by immunization with a modified recombinant adenovirus containing the chimeric gene, GcS0.7

Evaluation of a quintuple-gene-deleted PRV vector expressing PEDV S1: safety and immunogenicity in rabbits, mice, and piglets

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.

An orthogonal transcription system enables high-efficiency rescue of measles virus-based vaccine vectors against multiple pathogens

Recombinant measles virus (rMeV) vectors are promising platforms for vaccine development against emerging infectious diseases due to their safety, stability, and potent immunogenicity. However, conventional rMeV rescue systems frequently exhibit low efficiency, thereby constraining their scalability and throughput. In this study, we developed 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. This innovative system facilitated robust cytoplasmic manufacture of both genomic and auxiliary components, eliminating the need for helper virus co-infection (such as modified vaccinia virus) and enhancing rescue efficiency by more than 50-fold relative to traditional rescue approaches. Utilizing this technology, we demonstrated the versatility of the platform by successfully generating six rMeV-based vaccine antigen candidates from influenza virus, Pseudomonas aeruginosa, and Brucella spp. All rescued vaccine candidates exhibited stable transgene expression, sustained replication, and strong antigen production. Immunization studies in golden Syrian hamsters verified that the vaccine candidates elicited high titers of neutralizing and antigen-specific antibodies without any observable adverse effects. These results demonstrate that our orthogonal transcription-based platform facilitates the efficient and safe production of rMeV vectors and provides a proof-of-concept methodological framework for the rapid development of vaccine candidates.

Development and immunological evaluation of a multi-antigen recombinant adenovirus vaccine candidate against PRRSV and PCV2

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.