Search Results (28)

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

Tuberculosis (TB), a human and animal disease caused by Mycobacterium tuberculosis (M.tb), has the highest global mortality rate after coronavirus disease 2019 (COVID-19) and poses a major public health threat globally. Since 1890, vaccine candidates for various forms of TB have been developed for different age groups, but these vaccine candidates have not provided intended protection in adolescents and adults in clinical trials. To help prevent and control the spread of TB, the development of a safe and effective TB vaccine is imperative. The MTB39A protein and the molecular adjuvant MTB32C protein were expressed by an insect-baculovirus expression system, and the recombinant baculovirus surface-displayed particles were evaluated for their immunogenicity in BALB/c mice and calves. The results showed that the rvAc-71CA/rvAc-MTB39A recombinant baculovirus surface-displayed particles exhibited good immunogenicity in mice and calves and could be further developed as potential candidates. Full article

The development of effective vaccines necessitates a delicate balance between maximizing immunogenicity and minimizing safety concerns. Subunit vaccines, while generally considered safe, often fail to elicit robust and durable immune responses. Nanotechnology presents a promising approach to address this dilemma, enabling subunit antigens to mimic critical aspects of native pathogens, such as nanoscale dimensions, geometry, and highly repetitive antigen display. Various expression systems, including Escherichia coli (E. coli), yeast, baculovirus/insect cells, and Chinese hamster ovary (CHO) cells, have been explored for the production of nanoparticle vaccines. Among these, E. coli stands out due to its cost-effectiveness, scalability, rapid production cycle, and high yields. However, the E. coli manufacturing platform faces challenges related to its unfavorable redox environment for disulfide bond formation, lack of post-translational modifications, and difficulties in achieving proper protein folding. This review focuses on molecular and protein engineering strategies to enhance protein solubility in E. coli and facilitate the in vitro reassembly of virus-like particles (VLPs). We also discuss approaches for antigen display on nanocarrier surfaces and methods to stabilize these carriers. These bioengineering approaches, in combination with advanced nanocarrier design, hold significant potential for developing highly effective and affordable E. coli-derived nanovaccines, paving the way for improved protection against a wide range of infectious diseases. Full article

The enormous effects of avian influenza on poultry production and the possible health risks to humans have drawn much attention to this disease. The H9N2 subtype of avian influenza virus is widely prevalent among poultry, posing a direct threat to humans through infection or by contributing internal genes to various zoonotic strains of avian influenza. Despite the widespread use of H9N2 subtype vaccines, outbreaks of the virus persist due to the rapid antigenic drift and shifts in the influenza virus. As a result, it is critical to develop a broader spectrum of H9N2 subtype avian influenza vaccines and evaluate their effectiveness. In this study, a recombinant baculovirus expressing the broad-spectrum HA protein was obtained via bioinformatics analysis and a baculovirus expression system (BES). This recombinant hemagglutinin (HA) protein displayed cross-reactivity to positive sera against several subbranch H9 subtype AIVs. An adjuvant and purified HA protein were then used to create an rHA vaccine candidate. Evaluation of the vaccine demonstrated that subcutaneous immunization of the neck with the rHA vaccine candidate stimulated a robust immune response, providing complete clinical protection against various H9N2 virus challenges. Additionally, virus shedding was more effectively inhibited by rHA than by the commercial vaccine. Thus, our findings illustrate the efficacy of the rHA vaccine candidate in shielding chickens against the H9N2 virus challenge, underscoring its potential as an alternative to conventional vaccines. Full article

Influenza viruses can cause highly infectious respiratory diseases, posing noteworthy epidemic and pandemic threats. Vaccination is the most cost-effective intervention to prevent influenza and its complications. However, reliance on embryonic chicken eggs for commercial influenza vaccine production presents potential risks, including reductions in efficacy due to HA gene mutations and supply delays due to scalability challenges. Thus, alternative platforms are needed urgently to replace egg-based methods and efficiently meet the increasing demand for vaccines. In this study, we employed a baculovirus expression vector system to engineer HA, NA, and M1 genes from seasonal influenza strains A/H1N1, A/H3N2, B/Yamagata, and B/Victoria, generating virus-like particle (VLP) vaccine antigens, H1N1-VLP, H3N2-VLP, Yamagata-VLP, and Victoria-VLP. We then assessed their functional and antigenic characteristics, including hemagglutination assay, protein composition, morphology, stability, and immunogenicity. We found that recombinant VLPs displayed functional activity, resembling influenza virions in morphology and size while maintaining structural integrity. Comparative immunogenicity assessments in mice showed that our quadrivalent VLPs were consistent in inducing hemagglutination inhibition and neutralizing antibody titers against homologous viruses compared to both commercial recombinant HA and egg-based vaccines (Vaxigrip). The findings highlight insect cell-based VLP vaccines as promising candidates for quadrivalent seasonal influenza vaccines. Further studies are worth conducting. Full article

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio^® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio^®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio^® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines. Full article

To simultaneously express and improve expression levels of multiple viral proteins of a porcine reproductive and respiratory syndrome virus (PRRSV), polycistronic baculovirus surface display vectors were constructed and characterized. We engineered polycistronic baculovirus surface display vectors, namely, pBacDual Display EGFP(BacDD)-2GP2-2GP4 and pBacDD-4GP5N34A/N51A (mtGP5), which simultaneously express and display the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP4-gp64TM-CTD, and His-tagged mtGP5-gp64TM-CTD fusion proteins of PRRSV on cell membrane of Sf-9 cells. Specific pathogen-free (SPF) pigs were administered intramuscularly in 2 doses at 21 and 35 days of age with genetic recombinant baculoviruses-infected cells. Our results revealed a high level of ELISA-specific antibodies, neutralizing antibodies, IL-4, and IFN-γ in SPF pigs immunized with the developed PRRSV subunit vaccine. To further assess the co-expression efficiency of different gene combinations, pBacDD-GP2-GP3-2GP4 and pBacDD-2mtGP5-2M constructs were designed for the co-expression of the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP3-gp64TM-CTD, and His-tagged GP4-gp64TM-CTD proteins as well as the ectodomain of His-tagged mtGP5-gp64TM-CTD and His-tagged M-gp64TM-CTD fusion proteins of PRRSV. To develop an ELISA assay for detecting antibodies against PRRSV proteins, the sequences encoding the ectodomain of the GP2, GP3, GP4, mtGP5, and M of PRRSV were amplified and subcloned into the pET32a vector and expressed in E. coli. In this work, the optimum conditions for expressing PRRSV proteins were evaluated, and the results suggested that 4 × 10⁵ of Sf-9 cells supplemented with 7% fetal bovine serum and infected with the recombinant baculoviruses at an MOI of 20 for three days showed a higher expression levels of the protein. Taken together, the polycistronic baculovirus surface display system is a useful tool to increase expression levels of viral proteins and to simultaneously express multiple viral proteins of PRRSV for the preparation of subunit vaccines. Full article

Baculoviridae is a large family of arthropod-infective viruses. Recombinant baculoviruses have many applications, the best known is as a system for large scale protein production in combination with insect cell cultures. More recently recombinant baculoviruses have been utilized for the display of proteins of interest with applications in medicine. In the present review we analyze the different strategies for the display of proteins and peptides on the surface of recombinant baculoviruses and provide some examples of the different proteins displayed. We analyze briefly the commercially available systems for recombinant baculovirus production and display and discuss the future of this emerging and powerful technology. Full article

Virus-like particles (VLPs) are versatile vaccine carriers for conferring broad protection against influenza by enabling high-level display of multiple hemagglutinin (HA) strains within the same particle construct. The insect cell-baculovirus expression vector system (IC-BEVS) is amongst the most suitable platforms for VLP expression; however, productivities vary greatly with particle complexity (i.e., valency) and the HA strain(s) to be expressed. Understanding the metabolic signatures of insect cells producing different HA-VLPs could help dissect the factors contributing to such fluctuations. In this study, the metabolic traces of insect cells during production of HA-VLPs with different valences and comprising HA strains from different groups/subtypes were assessed using targeted metabolic analysis and metabolic flux analysis. A total of 27 different HA-VLP variants were initially expressed, with titers varying from 32 to 512 HA titer/mL. Metabolic analysis of cells during the production of a subset of HA-VLPs distinct for each category (i.e., group 1 vs. 2, monovalent vs. multivalent) revealed that (i) expression of group-2 VLPs is more challenging than for group-1 ones; (ii) higher metabolic rates are not correlated with higher VLP expression; and (iii) specific metabolites (besides glucose and glutamine) are critical for central carbon metabolism during VLPs expression, e.g., asparagine, serine, glycine, and leucine. Principal component analysis of specific production/consumption rates suggests that HA group/subtype, rather than VLP valency, is the driving factor leading to differences during influenza HA-VLPs production. Nonetheless, no apparent correlation between a given metabolic footprint and expression of specific HA variant and/or VLP design could be derived. Overall, this work gives insights on the metabolic profile of insect High Five cells during the production of different HA-VLPs variants and highlights the importance of understanding the metabolic mechanisms that may play a role on this system’s productivity. Full article

Apidermins (APDs) are known as structural cuticular proteins in insects, but their additional roles are poorly understood. In this study, we characterized the honeybee, Apis mellifera, APD 2 (AmAPD 2), which displays activity suggesting antimicrobial properties. In A. mellifera worker bees, the AmAPD 2 gene is transcribed in the epidermis, hypopharyngeal glands, and fat body, and induced upon microbial ingestion. Particularly in the epidermis of A. mellifera worker bees, the AmAPD 2 gene showed high expression and responded strongly to microbial challenge. Using a recombinant AmAPD 2 peptide, which was produced in baculovirus-infected insect cells, we showed that AmAPD 2 is heat-stable and binds to live bacteria and fungi as well as carbohydrates of microbial cell wall molecules. This binding action ultimately induced structural damage to microbial cell walls, which resulted in microbicidal activity. These findings demonstrate the antimicrobial role of AmAPD 2 in honeybees. Full article

Adaptive laboratory evolution has been used to improve production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) in insect cells. However, little is known about the underlying biological mechanisms promoting higher HA-VLP expression in such adapted cell lines. In this article, we present a study of gene expression patterns associated with high-producer insect High Five cells adapted to neutral pH, in comparison to non-adapted cells, during expression of influenza HA-VLPs. RNA-seq shows a decrease in the amount of reads mapping to host cell genomes along infection, and an increase in those mapping to baculovirus and transgenes. A total of 1742 host cell genes were found differentially expressed between adapted and non-adapted cells throughout infection, 474 of those being either up- or down-regulated at both time points evaluated (12 and 24 h post-infection). Interestingly, while host cell genes were found up- and down-regulated in an approximately 1:1 ratio, all differentially expressed baculovirus genes were found to be down-regulated in infected adapted cells. Pathway analysis of differentially expressed genes revealed enrichment of ribosome biosynthesis and carbohydrate, amino acid, and lipid metabolism. In addition, oxidative phosphorylation and protein folding, sorting and degradation pathways were also found to be overrepresented. These findings contribute to our knowledge of biological mechanisms of insect cells during baculovirus-mediated transient expression and will assist the identification of potential engineering targets to increase recombinant protein production in the future. Full article

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods. Full article

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines. Full article

Varicella–zoster virus (VZV) is the causative agent of varicella and herpes zoster (HZ) and can pose a significant challenge to human health globally. The initial VZV infection—more common in children—causes a self-limiting chicken pox. However, in later life, the latent VZV can become reactivated in these patients, causing HZ and postherpetic neuralgia (PHN), a serious and painful complication. VZV glycoprotein E (gE) has been developed into a licensed subunit vaccine against HZ (Shingrix). However, its efficacy relies on the concomitant delivery of a robust adjuvant (AS01B). Here, we sought to create a new immunogen for vaccine design by displaying the VZV–gE on the baculovirus surface (Bac–gE). Correct localization and display of gE on the engineered baculovirus was verified by flow cytometry and immune electron microscopy. We show that Bac–gE provides excellent antigenicity against VZV and induces not only stronger gE-specific CD4⁺ and CD8⁺ T cell responses but also higher levels of VZV–specific neutralizing antibodies as compared with other vaccine strategies in mice. Collectively, we show that the baculovirus display of VZV–gE confers ideal humoral and cellular immune responses required for HZ vaccine development, paving the way for a baculovirus-based vaccine design. Full article

Here, rabbits were immunized with a virus-like particle (VLP) vaccine prepared by expressing 239 amino acids of the swine hepatitis E virus (HEV)-3 capsid protein using a baculovirus system. Thirty specific-pathogen-free rabbits were divided into five groups (negative and positive control and 10, 50, and 100 μg VLP-vaccinated). Positive control group rabbits showed viremia and fecal viral shedding, whereas rabbits vaccinated with 10 μg VLP showed transient fecal viral shedding, and rabbits vaccinated with 50 and 100 μg VLP did not show viremia or fecal viral shedding. Serum anti-HEV antibody titers increased in a dose-dependent manner. Anti-HEV antibody titers were significantly higher (p < 0.05) in 100 μg VLP-vaccinated rabbits than in the negative control rabbits at week 4. Anti-HEV antibody titers were significantly higher in 50 and 10 μg VLP-vaccinated rabbits than in the negative control rabbits at weeks 8 and 11, respectively. Serum IFN-γ and IL-12 levels were significantly higher (p < 0.01) in rabbits vaccinated with 50 and 100 μg VLP than in the negative control rabbits at weeks 4 and 6. Liver tissues of 50 and 100 μg VLP-vaccinated rabbits displayed significantly less (p < 0.05) fibrosis than those of the positive control rabbits. The prepared VLP vaccine demonstrated dose-dependent immunogenicity sufficient for inducing anti-HEV antibody production, thus protecting rabbits against swine HEV-3. Full article