Search Results (167)

Virus-like particles (VLPs), nanoscale self-assembled structures lacking viral genetic material, have emerged as a versatile platform for vaccines, targeted delivery systems, and gene-editing applications owing to their strong immunogenicity, favorable biosafety profile, and high engineerability. However, the complex architecture of VLPs, their significant size heterogeneity, and the diversity of process- and product-related impurities generated in different expression systems make downstream purification a major bottleneck limiting product quality, yield, and manufacturability. This review systematically discusses advanced downstream purification strategies for VLPs from the perspective of three major objectives: preservation of structure and biological activity, control of product heterogeneity, and assurance of viral safety. First, strategies for maintaining VLP integrity and function are examined, including optimization of solution conditions, adoption of gentle yet efficient separation operations, and integration of process analytical technology (PAT) to reduce process-induced damage. Second, the review summarizes multi-step purification approaches—spanning clarification, ultrafiltration/diafiltration (UF/DF), chromatography, and disassembly/reassembly—to remove host cell proteins, host cell DNA, and product-related impurities while improving particle homogeneity and stability. Third, viral safety is discussed primarily from the perspective of downstream virus clearance under host-dependent risk, with particular attention to orthogonal clearance steps tailored to VLP properties and expression systems such as CHO cells and insect cell–baculovirus platforms. Overall, this review provides a CQA-oriented framework and practical guidance for the development of robust, scalable, and GMP-compliant downstream purification processes for VLP-based products. Full article

Background/Objectives: Epidemiological studies have proven that coxsackievirus B3 (CVB3) is the major virus that causes acute and chronic myocarditis and pancreatitis. Currently, there are no antiviral therapeutic drugs or vaccines that are available for use as clinical therapeutics or vaccines. Subunit polypeptides-based vaccines, especially when combined with adjuvants, represent safe and effective vaccine platforms because they are considered to be better immunogens. The viral capsid protein VP1 of CVB3 is the most immunogenic viral polypeptide, providing opportunities for its use in designing subunit polypeptide vaccines. In the present study, we designed and produced a CVB3 vaccine candidate based on the recombinant expression of the major immunogenic viral protein VP1 of a wild-type CVB3 strain. Methods: We assessed its induced humoral and cellular immune responses and then evaluated its protective immunity against pathogenic CVB3 strain challenges in a Balb/c mouse model. Neutralizing specific antibodies and cytokine interferon gamma (INF-γ) production were determined in the sera of both prime- and prime-boost-immunized mice with the vaccine candidate. Results: Our results demonstrate that the recombinant rVP1 expressed in a eukaryotic insect cell baculovirus vector system elicited cellular and humoral immune responses, protecting Balb/c mice from lethal challenges. Conclusions: Hence, the vaccine produced based on the recombinant expression of VP1 is a promising and potential candidate against natural CVB3 infections. Full article

Synthesized by expressing natural collagen sequences in specific hosts, recombinant collagen exhibits multiple advantages, encompassing a higher content of bioactive domains, enhanced antioxidant activity, the absence of viral pathogens, favorable hydrophilicity, reproducible production, and low immunogenicity. Consequently, it has found extensive use in applications ranging from biomaterials and pharmaceuticals to skincare. This review systematically explores various expression systems for recombinant collagen, including those utilizing Escherichia coli, Pichia pastoris, plants, insect baculovirus, and mammalian cells. It provides a detailed comparison of their differences and commonalities in terms of production efficiency, post-translational modification capability, and cost-effectiveness. Key separation and purification techniques for recombinant collage-notably precipitation, affinity chromatography, ion-exchange chromatography, and gel filtration chromatography are further introduced, with an in-depth analysis of the applicable scenarios and purification outcomes for each method. Finally, the review comprehensively summarizes the characterization methods for both the physicochemical properties and biological functions of recombinant collagen. For physicochemical properties, techniques covered include scanning electron microscopy, micro-differential thermal analysis, circular dichroism spectroscopy, SDS-PAGE, mass spectrometry, and Fourier-transform infrared spectroscopy. For biological functions, the focus is on its roles and the corresponding assessment methods in processes such as cell proliferation, migration, adhesion, and wound healing. Building upon this comprehensive overview, current challenges facing recombinant collagen are identified, and future directions are proposed, emphasizing the need to reduce R&D costs, refine testing methods for cosmetic products, and improve safety evaluation protocols to advance the field. Full article

Background: Feline panleukopenia virus (FPV) causes acute and frequently fatal disease in cats, underscoring the urgent need for safe, rapidly effective, and scalable vaccines. While virus-like particle (VLP) vaccines are inherently safe and immunogenic, their development is constrained by low yields of recombinant protein in insect cell expression systems. Methods: An optimized baculovirus expression vector system (BEVS) incorporating the hr1-p6.9-p10 transcriptional enhancer and the Ac-ie-01 anti-apoptotic gene was employed to enhance recombinant protein production. VP2 expression levels, viral titers, and hemagglutination activity were quantified using qPCR, SDS-PAGE/Western blotting, transmission electron microscopy (TEM), and functional assays. Immunogenicity and protective efficacy were assessed in both mice and cats through serological analysis, neutralizing antibody detection, and post-challenge clinical monitoring. Results: The optimized BEVS enhanced recombinant protein transcription by 1.5-fold, viral titers by 3.7-fold, and hemagglutination activity by 15-fold. The purified protein self-assembled into uniform 25 nm virus-like particles (VLPs). Immunization elicited earlier responses compared to commercial vaccines. Vaccinated cats maintained normal body temperature, stable leukocyte counts, and minimal viral shedding following FPV challenge. Conclusions: This study validates an enhanced BEVS that effectively overcomes VP2 yield constraints and generates highly immunogenic FPV VLPs. The platform enables rapid-onset protection and offers a scalable strategy for next-generation FPV vaccine development. Full article

Recombinant baculovirus vectors are recognized as effective gene delivery systems for mammalian cells in vitro. However, their application in vivo has been limited due to inactivation by the host’s complement system. We developed a recombinant baculoviral vector derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), incorporating both CMV-IE and polyhedron promoter-driven green fluorescence protein (EGFP) (vAcMBac-CMV-IE-EGFP). We then evaluated the transduction efficiency and safety of vAcMBac-CMV-IE-EGFP at a high multiplicity of infection (MOI) across five distinct cell lines and in Sprague Dawley (SD) rats. In vitro, Sf9, HepG2, and Vero E6 cells showed high transduction rates (95.52 ± 4.86%, 80.53 ± 3.31%, and 80.87 ± 2.50%, respectively), significantly outperforming the other cell types tested, and cell viability remained largely unaffected even at an MOI of 1000. In vivo, EGFP expression was observed in the heart, liver, spleen, lungs, and kidneys of SD rats after tail vein injection. Direct injection of vAcMBac-CMV-IE-EGFP into the rat striatum also resulted in strong EGFP signals in neural tissues. These results demonstrate that a high-MOI baculovirus infection can serve as a remarkably efficient and versatile platform for gene delivery across diverse mammalian cell types as well as in various organs and neural tissues in animal models. This robust method might hold significant promise for future gene therapy applications. Full article

Porcine deltacoronavirus (PDCoV) is a newly discovered porcine intestinal coronavirus that can pose a significant threat to the global commercial swine industry. We established an enzyme-linked immunosorbent assay (ELISA) detection method for the detection of PDCoV antibodies, based on the recombinant nucleocapsid (N) protein expressed using a baculovirus system. The assay was validated using positive and negative serum samples obtained from experimentally immunized rabbits and demonstrated an absence of cross-reactivity with either transmissible gastroenteritis virus (TGEV) or porcine epidemic diarrhea virus (PEDV). The recombinant PDCoV N protein antigen dilution (0.8 μg/mL), sample serum (1:400), and the enzyme-labeled secondary antibody (1:50) were used in this assay. The cut-off value was 0.355, without cross-reactivity including TGEV and PEDV. The ELISA method shows good sensitivity (96.67%), specificity (85.51%), and reproductivity (CV < 10%). We utilized the method to detect PDCoV antibodies in 600 pig serums collected from Zhejiang Province in the last four years (2021–2024). The results showed significant differences in antibody levels between regions and considerable fluctuation in positivity rates across the four-year period. As shown in the results, we developed a sensitive and specific ELISA method for detecting anti-PDCoV N antibodies, which provides a rapid and reliable diagnostic tool for PDCoV surveillance and control. This assay demonstrates significant potential for both epidemiological investigations and commercial applications in swine disease management. Full article

Epizootic hemorrhagic disease (EHD) is an important livestock disease caused by Epizootic hemorrhagic disease virus (EHDV). The recent incursion and wide distribution of EHDV in Europe have increased the need for effective vaccine candidates. Background/Objectives: The VP2 protein of EHDV forms the outer capsid layer of the virion and is essential for viral assembly and host cell entry. Owing to its antigenic properties, VP2 represents a major target for vaccine development. However, the recombinant production of VP2 is limited by low stability and poor yields, representing a significant barrier for the generation of safe and effective subunit vaccines. Methods: To overcome these limitations, the VP2 protein from EHDV serotype 8 (EHDV-8) was rationally engineered with targeted modifications at both the amino and carboxyl termini of its coding sequence. Recombinant expression was performed using a baculovirus vector-mediated system in Trichoplusia ni pupae (CrisBio^® technology), employed as living biofactories. Results: The engineering of VP2 resulted in up to a tenfold increase in protein yields compared with the wild-type sequence, while maintaining the trimeric structural integrity of the recombinant protein. Both wild-type and engineered VP2 protein variants were formulated and used to immunize IFNAR(−/−) mice, a model susceptible to EHDV infection. Both engineered and wild-type VP2 formulations elicited comparable neutralizing antibody responses in vaccinated animals. Furthermore, immunization with either formulation conferred full protection against lethal EHDV-8 challenge. Conclusions: In this work, we demonstrated that the rational engineering of the VP2 protein significantly improved recombinant expression yields in a baculovirus-based system without compromising structural integrity or immunogenicity. These findings additionally demonstrate the feasibility of producing high-quality VP2 antigens in T. ni pupae using CrisBio^® technology and support their potential application in the development of subunit vaccines against EHDV. Full article

Piscine orthoreovirus genotype 1 (PRV-1) is an emerging viral pathogen in salmon aquaculture that causes Heart and Skeletal Muscle Inflammation (HSMI), with high prevalence in salmon-producing countries such as Chile. A significant obstacle in PRV-1 vaccine development is the inability to culture the virus in vitro, which limits the scalability and production of traditional inactivated or DNA-based vaccine strategies. This study describes the development of a novel virus-like particle (VLP)-based vaccine against PRV-1. Recombinant VLP were produced by co-expressing the six structural proteins of PRV-1 (λ1, λ2, μ1, σ1, σ2, σ3) using a baculovirus-based expression system in insect cells. In addition, to enable differentiating infected from vaccinated animals (DIVA) strategies, the σ1 protein was modified by adding of a cmyc epitope tag. The results demonstrated that the native VLP vaccine (VLP6n) significantly reduced viral loads in Atlantic salmon challenged with PRV-1. Moreover, in rainbow trout, the cmyc-tagged VLP-like vaccine (VLP6c) elicited a specific antibody response against the cmyc epitope, allowing differentiation between vaccinated and naturally infected fish. Overall, this VLP-based vaccine platform represents a promising strategy for controlling PRV-1 prevalence in salmon-producing counties, supporting the implementation of serological surveillance programs. Full article

Background: Seasonal influenza vaccines must be reformulated annually due to the high genetic variability and antigenic drift of circulating influenza viruses. The annual update, guided by World Health Organization (WHO) recommendations, results in significant challenges, including compressed production time periods, elevated manufacturing costs, and global distribution pressures. Moreover, mismatches between vaccine strains and circulating viruses can severely reduce protective efficacy, underscoring the urgent need for broadly protective and long-lasting influenza vaccines. Methods: In this study, we developed an adjuvanted trivalent recombinant influenza virus-like particle vaccine (a-RIV) using the baculovirus–insect cell expression system and formulated it with an AS01-like adjuvant. The vaccine comprises full-length hemagglutinin (HA) proteins from WHO-recommended seasonal influenza strains: A/H1N1 (AH1), A/H3N2 (AH3), and B/Victoria (B/vic) lineages. The purified HA proteins were subsequently formulated with a liposomal adjuvant to enhance the immunogenicity. Results: In mouse immunization studies, the a-RIV vaccine elicited significantly stronger humoral and cellular immune responses than the licensed recombinant vaccine Flublok and the conventional inactivated influenza vaccine (IIV). High levels of functional anti-HA antibodies and antigen-specific T cell responses persisted for at least six months post-vaccination. Moreover, a-RIV induced broadly reactive antibodies capable of cross-binding to heterologous AH1 and AH3 influenza strains. Conclusions: Our data demonstrate that the a-RIV elicits enhanced, durable, and broadly cross-reactive immune responses against multiple influenza subtypes. These findings support the potential of adjuvanted recombinant HA-based vaccine as a promising candidate for the development of next-generation influenza vaccine. Full article

African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, febrile, highly contagious, and lethal disease that poses a severe threat to the global pig farming industry. Currently, no globally recognized, safe, and effective commercial ASF vaccine has been developed, making vaccination a crucial strategy for outbreak control. The ASFV structural proteins p72, p30, and p54 are key targets for vaccine development. In this study, we developed a novel baculovirus vector-based system for surface display of a recombinant protein comprising epitopes from p72, p30, and p54. Upon infection, the recombinant protein was expressed and anchored on the plasma membrane of Sf-9 cells. Purified virus analysis revealed that the Bac-recombinant protein enhanced gene delivery and transgene expression in mammalian cells compared to the Bac-Wild Type (Bac-WT). In a murine model, the Bac-recombinant protein induced significantly higher IFN-γ and IL-4 levels than Bac-p30 and the negative control. However, further evaluation in swine models is required to confirm its protective potential against ASFV. Furthermore, it also elicited a robust antibody response, generating high-titer Bac-recombinant protein-specific antibodies. Therefore, these findings suggest that the ASFV Bac-recombinant protein is a promising candidate for a vector-based vaccine. Full article

Background/Objectives: Virus-like particle (VLP) vaccines based on human papillomavirus (HPV) L1 proteins have high efficacy for preventing cervical cancer and other HPV-associated diseases. The production yields of commercial HPV VLPs remain suboptimal. We aimed to improve HPV VLP production efficiency using a hyper-expression vector system for the expression of L1 proteins of four major HPV serotypes—HPV 6, 11, 16, and 18. Methods: HPV L1 proteins were expressed in Trichoplusia ni (Hi5) insect cells via a hyper-expression baculovirus vector system. Following cell lysis using a microfluidizer, VLPs were purified through a two-step chromatographic process. Particle morphology was characterized using transmission electron microscopy and dynamic light scattering. Immunogenicity was evaluated using a murine model; mice received three intramuscular injections of the purified quadrivalent VLPs. The resulting IgG and neutralizing antibody responses were compared with those elicited by the commercial quadrivalent vaccine, Gardasil. Results: The L1 proteins from HPV 6, 11, 16, and 18 were successfully expressed at high levels in Hi5 cells, forming uniformly sized VLPs with hydrodynamic diameters of 50–60 nm. The average production yield of the quadrivalent VLPs exceeded 40 mg/L, an improvement over conventional yields. The candidate VLPs elicited strong HPV-specific IgG and neutralizing antibody responses in mice, comparable to those induced by Gardasil. Conclusions: The hyper-expression baculovirus vector system enables high-yield production of HPV L1 VLPs with desirable structural and immunogenic properties. This approach holds promise for the cost-effective and scalable manufacturing of next-generation HPV VLP vaccines, facilitating broader global access to HPV immunization. Full article

The use of viruses as natural pathogens of pest insects is a promising approach in biocontrol. The main drawback of this approach is its relatively slow mode of action, which could be addressed through genetic modifications of the strains used. In this article, we propose the use of the commercially available Bac-to-Bac system for infecting pest insects with a recombinant virus and assessing the impact of various molecules on their viability. Using the laboratory-friendly model organism Galleria mellonella and baculovirus carrying the eGFP gene, we demonstrated a 100% infection rate, with a consistent and stable spread of the viruses throughout insect bodies and a gradual increase in recombinant protein expression. Notably, at day 5 post-infection the insects remained viable and active. This approach enables the identification of pathogenic effects caused by the virus-induced expression of other molecules, such as toxins. Full article

Background/Objectives: Avian influenza represents a major threat to both animal and public health. Our group has tracked avian influenza viruses circulating in wild birds in Peru during the last 20 years. While most of these viruses are low-pathogenic avian influenza strains, some exhibit genetic changes that significantly diverge from common circulating viruses. We selected a highly divergent hemagglutinin H2 gene from a genetically characterized avian influenza virus to develop a recombinant protein using a baculovirus system. Methods: We administered 5 µg and 20 µg doses of the recombinant H2 protein (rH2) into 3-week-old chickens using an abdominal cavity inoculation model to evaluate the activation of innate immune responses. Chickens were euthanized at 24 and 72 h post inoculation and an abdominal lavage was performed to harvest the abdominal cavity content. Results: Infiltrating cells were counted and their cell viability was measured using an Annexin V/PI staining. At 24 h, a large proportion of infiltrating leukocytes were identified as heterophils, monocyte/macrophages and lymphocytes. These proportions changed at 72 h, with a decrease in heterophils and increase in monocyte and lymphocyte pools. We observed strong cellular activity in abdominal leukocytes at 24 h, with a decline in activation levels at 72 h. Cytokine expression suggested a tightly regulated immune response during the 72 h period, while a more sustained response was observed at the 20 µg dose. Antibody levels demonstrated the capacity of the rH2 protein to induce long-term responses. Conclusions: These results revealed that the baculovirus-expressed rH2 protein induces a controlled immune activation, a long-term immune response, holding promise as a potential vaccine candidate for animal health. Full article

Bovine Norovirus (BNoV) is a member of the enterovirus family that can cause gastroenteritis in calves. This virus poses a significant risk to calf growth and development as well as to the long-term sustainability of the cattle industry in China and elsewhere. No specific treatment or vaccine is currently available; thus, the development of a safe and effective vaccine is paramount. Here, we describe a strategy to assemble BNoV virus-like particles (VLPs) using the insect baculovirus expression system (BEV) to express the major structural protein, VP1, and demonstrate their potentiality as vaccines. The results showed that the BNoV-VLP self-assembled into complete spherical particles with a diameter of approximately 40 nm. When it was immunized in mice, the levels of specific IgG and IgA antibodies peaked at weeks 6 and 7 post-immunization, respectively, with maximum titers of 1:25,600 and 1:200. Moreover, we observed a significant increase in the CD4⁺/CD8⁺ T-cell ratio in splenic lymphocytes of immunized mice (p < 0.05), accompanied by a significant increase in TNF-α⁺CD4⁺ T-cells and TNF-α⁺CD8⁺ T-cells (p < 0.05). These results demonstrate that BNoV-VLPs are promising vaccine candidates for providing immunoprotection in the future. These studies support the significant practical implications of using a scientific basis for the development of a BNoV-VLP vaccine. Full article

Background/Objectives: Porcine circovirus type 2 (PCV2) infection causes porcine circovirus disease (PCVD), a global immunosuppressive disease in pigs. Its clinical manifestations include post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS), which cause significant economic losses to the swine industry. The Cap protein, which is the major protective antigen of PCV2, can self-assemble to form virus-like particles (VLPs) in the insect baculovirus expression system. Few studies have compared the expression of Cap proteins in different baculovirus expression systems. Methods: In this study, we compared two commonly commercialized baculovirus construction systems with the Cap protein expression in various insect cells. Results: The results demonstrate that the flashBAC system expressed the Cap protein at higher levels than the Bac-to-Bac system. Notably, when expressing four copies of the Cap protein, the flashBAC system achieved the highest protein yield in High Five cells, where it reached 432 μg/mL at 5 days post-infection (dpi) with 27 °C cultivation. Animal experiments confirmed that the purified Cap protein effectively induced specific antibody production in mice and swine. Conclusions: This study provides critical data for optimizing the production of the PCV2 Cap protein, which is of great significance for reducing the production cost of PCV2 vaccines and improving the industrial production efficiency. Full article