Search Results (148)

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

Background: Toxoplasma gondii (T. gondii) causes severe disease in immunocompromised individuals and pregnant women, underscoring the urgent need for effective vaccines against toxoplasmosis. The dense granule protein 5 (GRA5) of T. gondii plays a key role in parasitic cyst formation. Methods: This study evaluated the protective immune responses induced by a virus-like particle (VLP) vaccine expressing the T. gondii-derived antigen GRA5 in a mouse model challenged with the ME49 strain of T. gondii. GRA5 VLPs were generated using a baculovirus expression system, and VLP formation was confirmed by Western blotting and visualized using transmission electron microscopy. Mice were intranasally immunized with GRA5 VLPs three times at 4-week intervals to induce immune responses, followed by infection with T. gondii ME49. Results: Intranasal immunization with GRA5 VLPs induced parasite-specific IgG antibody responses in the serum and both IgG and IgA antibody responses in the brain. Compared to the non-immunized group, immunized mice exhibited significantly higher levels of germinal center B cells and antibody-secreting cell responses. Moreover, the VLP vaccine suppressed the production of IFN-γ and IL-6 cytokines, leading to a significant reduction in brain inflammation and decreased cyst counts following lethal challenge with T. gondii ME49 infection. Conclusion: These findings suggest that the GRA5 VLP vaccine derived from T. gondii elicits a protective immune response, highlighting its potential as an effective vaccine candidate against toxoplasmosis. Full article

Over the past decade, diseases associated with fowl adenoviruses (FAdVs) have exhibited a new epidemic trend worldwide. The presence of numerous FAdVs serotypes, combined with the virus’s broad host range, positions it as a significant pathogen in the poultry industry. In the current context of intensive poultry production and global trade, co-infections involving multiple FAdVs serotypes, as well as co-infections with FAdVs alongside infectious bursal disease or infectious anemia virus, may occur within the same region or even on the same farm. The frequency of these outbreaks complicates the prevention and control of FAdVs. Therefore, the development of effective, targeted vaccines is essential for providing technical support in the management of FAdVs epidemics. Ongoing vaccine research aims to improve vaccine efficacy and address the challenges posed by emerging FAdVs outbreaks. This review focuses on vaccines developed and studied worldwide for various serotypes of FAdVs in the past decade. It encompasses inactivated vaccines, live attenuated vaccines, e.g., host-adapted attenuated vaccines and gene deletion vaccines, viral vector vaccines, and subunit vaccines (including VLP proteins and chimeric proteins). The current limitations and future development directions of FAdVs vaccine development are also proposed to provide a reference for new-generation vaccines and innovative vaccination strategies against FAdVs, as well as for the rapid development of highly effective vaccines. Full article

Virus-like particles (VLPs) have the potential to become a versatile carrier platform for vaccination against multiple diseases. In the light of short process development timelines and the demand for reliable and robust processes, metabolic modeling of cell culture processes offers great advantages when coupled with a Quality-by-Design (QbD) development approach. A previous work was able to demonstrate the accurate prediction of HEK293F PiggyBac cell concentration as well as VLP titer and metabolite production with a reduced metabolic model. This work presents the reduced metabolic model for a more productive cell line Sleeping Beauty and emphasizes the need for model re-parameterization when the producer cell line changes. The goal of precise prediction for a fed-batch and continuous HEK293 cultivation can, therefore, be achieved. In terms of decision-making for downstream unit operations, a soft sensor for the prediction of main impurities like proteins and DNA was introduced for the first time for the production of lentiviral vectors with several terms describing the release of impurities like DNA and proteins, growth-related protein production, and enzymatic degradation activity associated with cell dissociation in an accurate manner. The additional information can contribute to a more efficient design phase by reducing experimental effort as well as during cultivation with data-based decision-making. With the aid of real-time process data acquisition through process analytical technology (PAT), its predictive power can be enhanced and lead to more reliable processes. Full article

Avian influenza is an economically significant disease affecting poultry worldwide and is caused by influenza A viruses that can range from low to highly pathogenic strains. These viruses primarily target the respiratory, digestive, and nervous systems of birds, leading to severe outbreaks that threaten poultry production and pose zoonotic risks. The ectodomain of the avian influenza virus (AIV) matrix protein 2 (M2e), known for its high conservation across influenza strains, has emerged as a promising candidate for developing a universal influenza vaccine in a mouse model. However, the efficacy of such expression against poultry AIVs remains limited. The objective of this study was to evaluate the immunogenicity of nodavirus-like particles displaying the M2e proteins. In this study, three synthetic heterologous M2e genes originated from AIV strains H5N1, H9N2 and H5N2 were fused with the nodavirus capsid protein (NVC) of the giant freshwater prawn Macrobrachium rosenbergii (NVC-3xAvM2e) prior to immunogenicity characterisations in chickens. The expression vector pTRcHis-TARNA2 carrying the NVC-3xAvM2e gene cassette was introduced into E. coli TOP-10 cells. The recombinant proteins were purified, inoculated into one-week-old specific pathogen-free chickens subcutaneously and analysed. The recombinant protein NVC-3xAvM2e formed virus-like particles (VLPs) of approximately 25 nm in diameter when observed under a transmission electron microscope. Dynamic light scattering (DLS) analysis revealed that the VLPs have a polydispersity index (PDI) of 0.198. A direct ELISA upon animal experiments showed that M2e-specific antibodies were significantly increased in vaccinated chickens after the booster, with H5N1 M2e peptides having the highest mean absorbance value when compared with those of H9N2 and H5N2. A challenge study using low pathogenic AIV (LPAI) strain A/chicken/Malaysia/UPM994/2018 (H9N2) at 10^6.5 EID₅₀ showed significant viral load in the lung and cloaca, but not in the oropharyngeal of vaccinated animals when compared with the unvaccinated control group. Collectively, this study suggests that nodavirus-like particles displaying three heterologous M2e have the potential to provide protection against LPAI H9N2 in chickens, though the vaccine’s efficacy and cross-protection across different haemagglutinin (HA) subtypes should be further evaluated. Full article

Background/Objectives: Feline panleukopenia, caused by FPV, is a highly contagious disease in cats. Current vaccines face challenges including complex production, high cost, and safety risks. Developing safer, more efficient alternatives is crucial. This study aimed to produce FPV virus-like particles (VLPs) using a recombinant baculovirus system expressing the VP2 gene and evaluate their immunogenicity and protective efficacy in cats. Methods: Sf9 insect cells were infected with recombinant baculovirus to express VP2 protein. The VP2 protein was purified using ultrafiltration and size-exclusion chromatography (SEC). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) confirmed the assembly of VLPs. Twenty healthy cats were randomly divided into four groups; three groups received different doses (5 μg, 15 μg, and 45 μg) of FPV VLP vaccine, while the fourth group served as the control group immunized with PBS. Blood samples were collected on day 21 to measure hemagglutination inhibition (HI) and virus-neutralizing (VN) antibody responses. Cats in the 15 μg dose group were challenged with virulent FPV strain 708 on day 21, and clinical signs and white blood cell counts were monitored for 10 days. Results: Immunized cats exhibited significantly higher HI and VN antibody titers compared to controls. After challenge, vaccinated cats showed no clinical signs of disease, and their white blood cell counts remained stable. In contrast, control cats developed severe symptoms and experienced significant leukopenia. Conclusions: The FPV VLP vaccine generated in this study are highly immunogenic and provide effective protection against virulent FPV challenge, demonstrating their potential as a safer vaccine candidate for feline panleukopenia. Full article

Foot-and-mouth disease virus (FMDV) continues to pose a significant threat to livestock health and the global agricultural economy, particularly in endemic regions of Asia, Africa, and the Middle East. Current vaccines based on chemically inactivated FMDV present several challenges, including biosafety risks, high production costs, and limited effectiveness against emerging viral variants. To overcome these limitations, we developed virus-like particle (VLP) vaccines targeting FMDV serotypes O, A, and Asia1 using a recombinant Escherichia coli expression system. The resulting VLPs self-assembled into 25–30 nm particles with native-like morphology and antigenic properties, as confirmed by transmission electron microscopy, SDS-PAGE, and Western blot analysis. Immunogenicity was evaluated in mice and pigs using ELISA and virus neutralization tests (VNT), and protective efficacy was assessed through viral challenge studies. All VLPs induced strong serotype-specific antibody responses, with ELISA PI values exceeding 50% and significantly increased VNT titers after booster immunization. In mice, PD₅₀ values were 73.5 (A-type), 32.0 (O-type), and 55.7 (Asia1-type); in pigs, PD₅₀ values reached 10.6 (O-type) and 22.6 (Asia1-type). Notably, the vaccines induced robust immune responses even at lower antigen doses, suggesting the feasibility of dose-sparing formulations. These findings demonstrate that FMDV VLPs produced in E. coli are highly immunogenic and capable of eliciting protective immunity, highlighting their promise as safe, scalable, and cost-effective alternatives to conventional inactivated FMD vaccines. Full article

Background/Objectives: Foot-and-mouth disease virus (FMDV) poses a continuous threat to livestock health and agricultural economies. Current vaccines require high biosafety standards and are costly to produce. While novel vaccine technologies have been explored, most fail to meet industrial scalability, cost-efficiency, or multiserotype flexibility required for effective FMD control. This study aimed to evaluate the feasibility of using a high-cell density transient gene expression (TGE) system in CHO cells for the production of FMDV virus-like particles (VLPs) as a recombinant vaccine platform. Methods: VLP expression was optimized by adjusting cDNA and polyethyleneimine (PEI) concentrations. Expression yields were compared at 24 and 48 h post-transfection to determine optimal harvest timing. We further tested the system’s capacity to express different serotypes and chimeric constructs, incorporating VP1 sequences from various FMDV strains. Immunogenicity was evaluated in swine using VLPs from the A2001 Argentina strain as a model. Results: Optimal VLP expression was achieved at 24 h post-transfection. Chimeric constructs incorporating heterologous VP1 regions were successfully expressed. Immunized pigs developed protective antibody titers as measured by a virus neutralization test (VNT, log₁₀ titer 1.43) and liquid-phase blocking ELISA (LPBE, titer 2.20) at 28 days post-vaccination (dpv). Titers remained above protective thresholds up to 60 dpv with a single dose. A booster at 28 dpv further elevated titers to levels comparable to those induced by the inactivated vaccine. Conclusions: Our results demonstrate the feasibility of using CHO cell-based TGE for producing immunogenic FMDV VLPs. This platform shows promise for scalable, cost-effective, and biosafe development of recombinant FMD vaccines. Full article

Insect cell lines are a cornerstone of recombinant protein production, providing a versatile platform for biopharmaceutical and research applications. In the early 20th century, scientists first attempted to culture insect cells in vitro, developing continuous cell lines to produce the first insect cell-derived recombinant protein, IFN-β. Initial successes, along with advancements in the use of insect cells for recombinant protein manufacturing, primarily relied on baculovirus expression vector systems (BEVSs), which enable heterologous gene expression in infected cells. Today, growing attention is focused on baculovirus-free systems based on the transfection of insect cells with plasmid DNA. This approach simplifies the final product purification process and facilitates the development of stable monoclonal cell lines that produce recombinant proteins or protein complexes, particularly virus-like particles (VLPs). Thanks to advancements in genetic engineering and the application of adaptive laboratory evolution (ALE) methods, significant strides have been made in overcoming many limitations associated with insect cell BEVSs, ultimately enhancing the reliability, yield, and quality of the biomanufacturing process. Our manuscript discusses the history of developing insect cell lines, presents various recombinant protein production systems utilizing these cells, and summarizes modifications aimed at improving insect cell lines for recombinant protein biomanufacturing. Finally, we explore their implications in pharmaceutical production, particularly on Nuvaxovid^®/Covovax, which is the latest approved vaccine developed using insect cell BEVSs for protection against SARS-CoV-2. Full article

Background/Objectives: N-Myristoyltransferase inhibitors (NMTi) represent a novel antiviral strategy against mammarenaviruses such as Lassa and Junin viruses. The Z matrix protein inhibits viral ribonucleoprotein (vRNP) activity in a dose-dependent manner. Here, we investigated whether Z-mediated vRNP inhibition depends on Z myristoylation or oligomerization. Methods: We used HEK293T cells transfected with wild-type (WT) or G2A-mutated Z constructs in LCMV minigenome (MG) assays. Cells were treated with the NMTi IMP-1088 and the proteasome inhibitor MG132. Z protein expression, vRNP activity, and VLP production were analyzed by immunofluorescence, western blotting, and colocalization analyses. Results: IMP-1088 treatment led to proteasome-mediated degradation of Z, reducing its inhibition of vRNP activity, which was restored by MG132. The non-myristoylated Z G2A mutant retained vRNP inhibitory activity but showed impaired oligomerization and budding capacity. These findings demonstrate that Z-mediated vRNP inhibition is independent of myristoylation and oligomerization. Conclusions: Z myristoylation and oligomerization are not required for its inhibitory vRNP activity. Targeting Z myristoylation with NMTi impairs virus assembly and budding without affecting Z-mediated inhibition of vRNP activity, supporting the development of NMTi as a promising broad-spectrum antiviral strategy against mammarenaviruses. Full article

Background: Biologics is an exciting and growing area of medicine. Within the larger field of biologics, the use of viral vectors and virus-like particles (VLPs) is increasingly common, making it crucial to develop innovative and practical unit operations for the related purification process. Objective: Some scientists and engineers propose that membrane-based downstream virus purification (MVP) platforms would allow for more scalable and cost-effective production of these critical particles. However, the so-cial, political, and ethical implications of these advancements remain largely unex-plored. This paper aims to explore various pivotal facets of MVP technology govern-ance and regulations within the U.S. context, including (1) government policy ar-rangements related to the implementation of the technologies, (2) stakeholder atti-tudes, policy preferences, and behaviors, and (3) the fundamental factors that shape these attitudes, policy preferences, and behaviors. Methods: In doing so, we analyze publicly available federal and state government documents pertaining to biomanu-facturing, healthcare, and legislative attempts. Additionally, we will perform a stake-holder analysis on relevant industries, healthcare service providers, and recipients. Conclusions: Our goal is to outline the socio-political, ethical, and regulatory factors pertaining to the regulation and governance of these technologies. Full article

Reverse genetics systems for influenza C virus encounter challenges due to the inefficient production of infectious virus particles. In the present study, we explored the underlying cause by assessing the efficiency of generating influenza C virus-like particles (C-VLPs) containing specific virus RNA (vRNA) segments. Using 293T cells transfected with plasmids encoding GFP- and DsRed2-vRNAs (each flanked by the non-coding regions of Segments 5 and 6, respectively), along with plasmids expressing virus proteins, we observed that C-VLPs containing a single vRNA segment were generated efficiently. However, the simultaneous packaging of two vRNA segments into a single C-VLP was less frequent, as demonstrated by flow cytometry and reverse-transcription PCR analyses. Statistical evaluations revealed a decreased efficiency of incorporating multiple vRNA segments into single particles, which likely contributes to the reduced production of infectious virus particles in reverse genetics systems. These findings highlight a critical limitation in the vRNA incorporation mechanism of influenza C virus, contrasting with that of influenza A virus. Hence, further studies are required to elucidate specific vRNA packaging signals and optimize vRNA expression levels to improve the production of infectious influenza C virus particles. Full article

Orthohantaviruses (also known as hantaviruses) are pathogens that cause two distinct, yet related forms of severe human disease: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases pose a significant threat to global public health due to their high case fatality rates, which can range from 1% to 50%. In recent years, an increasing number of countries and regions have reported human cases, underscoring the urgent need for improved understanding, prevention, and treatment strategies. Given the severity of these diseases and the lack of specific post-exposure antiviral treatments, preventive measures are critical. For several decades, substantial efforts have been dedicated to developing orthohantavirus vaccines, leading to significant advancements. The first large-scale deployment involved inactivated vaccines, which played a crucial role in reducing HFRS incidence in South Korea and China. Subunit vaccines, viral vector vaccines, and virus-like particle (VLP) vaccines have also been extensively researched. Nucleic acid vaccines, including both mRNA and DNA vaccines, hold the greatest potential for future development due to their rapid design and production cycles, ability to elicit robust immune responses, ease of storage and transportation, and adaptable production platforms. Ongoing advancements in computer technology and artificial intelligence promise to further enhance the development of more effective orthohantavirus vaccines. Full article

Enzymes play an essential role in many different industries; however, their operating conditions are limited due to the loss of enzyme activity in the presence of proteases and at temperatures significantly above physiological conditions. One way to improve the stability of these enzymes against high temperatures and proteases is to encapsulate them in protective shells or virus-like particles. This work presents a streamlined, three-step, cell-free protein synthesis (CFPS) procedure that enables rapid in vitro enzyme production, targeted encapsulation in protective virus-like particles (VLPs), and facile purification using a 6× His-tag fused to the VLP coat protein. This process is performed in under 12 h and overcomes several limitations of enzyme encapsulation, such as the control of packing density, speed, and complexity of the process. Here, we encapsulate the enzyme Candida antarctica lipase B in the VLP from the bacteriophage Qβ, while in the presence of a linking RNA aptamer. The encapsulated enzymes largely retained their activity in comparison to the free enzymes. Additionally, when subjected to 90 °C temperatures or 5 h incubation with proteases, the encapsulated enzymes maintained their activity, whereas the free enzymes lost their activity. In this work, we also demonstrate control over packing density by achieving packing densities of 4.7 and 6.5 enzymes per VLP based off the concentration of enzyme added to the encapsulation step. Full article

Hand, foot, and mouth disease (HFMD) represents an emerging health concern whose main causative agents are Coxsackievirus A6 (CVA6) and enterovirus A71 (EV71). The lack of a CVA6 vaccine and the rise of new HFMD-causing strains due to the containment of established HFMD-causing viruses necessitates the search for alternative vaccine technologies, including virus-like particle (VLP) vaccine candidates. While studies have demonstrated that production of enterovirus-like particles in various organisms can be achieved by expression of the viral P1 structural proteins and the 3CD protease, optimization based on the interplay between the three most commonly altered infection parameters (multiplicity of infection (MOI), viable cell density at the time of infection (VCD), and the infection period) is often not investigated. To address this challenge, we have performed Design of Experiments (DoE) to optimize the production of CVA6 and EV71 VLPs. Our results indicate that CVA6 VLP production peaks at high MOI, high VCD, and long infection periods. Our subsequent downstream purification processes yielded 38 mg and 158 mg of purified CVA6 and EV71 VLPs from 1 L crude harvest, respectively. This translates into thousands of potential vaccine doses and highlights the economic potential of enterovirus-like particles for vaccine purposes. Full article