Construction of Recombinant Baculoviruses.

The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Evaluation of recombinant baculovirus clearance during rAAV production in Sf9 cells using a newly developed fluorescent-TCID50 assay.

Introduction: Recombinant adeno-associated virus (rAAV) vectors provide a safe and efficient means for in vivo gene delivery, although its large-scale production remains challenging. Featuring high manufacturing speed, flexible product design, and inherent safety and scalability, the baculovirus/Sf9 cell system offers a practical solution to the production of rAAV vectors in large quantities and high purity. Nonetheless, removal and inactivation of recombinant baculoviruses during downstream purification of rAAV vectors remain critical prior to clinical application. Methods: The present study utilized a newly developed fluorescent-TCID50 (F-TCID50) assay to determine the infectious titer of recombinant baculovirus (rBV) stock after baculovirus removal and inactivation, and to evaluate the impact of various reagents and solutions on rBV infectivity. Results and discussion: The results showed that a combination of sodium lauryl sulfate (SLS) and Triton X-100 lysis, AAVx affinity chromatography, low pH hold (pH3.0), CsCl ultracentrifugation, and NFR filtration led to effective removal and/or inactivation of recombinant baculoviruses, and achieved a log reduction value (LRV) of more than 18.9 for the entire AAV purification process. In summary, this study establishes a standard protocol for downstream baculovirus removal and inactivation and a reliable F-TCID50 assay to detect rBV infectivity, which can be widely applied in AAV manufacturing using the baculovirus system.

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Virus-Like Particles Assembled Using Respiratory Syncytial Virus Matrix Protein Elicit Protective Immunity in Mice.

Purpose: Heterologous virus-like particle (VLP) assembly involving influenza or the Newcastle disease virus matrix protein (M) has been extensively used to explore the efficacies of VLP vaccines against the respiratory syncytial virus (RSV). Here, we attempted to generate homologous RSV VLPs by expressing the pre-fusion (pre-F) or the glycoprotein (G) on the RSV M protein and evaluated their protective efficacy in mice. Methods: We generated VLPs using the baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Recombinant baculoviruses expressing the RSV pre-F, G, and M antigens were inoculated into Sf9 cells, and particles were self-assembled. Mice were immunized with either pre-F or G-expressing VLPs, and immune parameters were assessed to determine protection. Results: Our findings show that successful VLP assembly can be achieved by utilizing recombinant baculoviruses expressing the RSV pre-F or G proteins with the native matrix protein. Mice immunized with either pre-F or the G antigen-expressing VLPs elicited robust serum-mediated virus neutralization. VLP immunization evoked Th1-biased RSV-specific antibody responses in the sera of mice. Following challenge infection with the RSV A2 strain, immunized mice experienced lesser eosinophil and IL-4 accumulation in the lungs, though a substantial increase in TNF-α secretion was observed from CD4+ T cells. Interestingly, splenic antibody-secreting cell responses were substantially enhanced against RSV F antigen, but not against the RSV G antigen following immunization and challenge infection. Immunizing mice with the VLPs significantly inhibited pulmonary histopathology development, as indicated by the diminished inflammatory immune cell influx and mucin secretion. Conclusion: Combined, these vaccine-induced immune responses contributed to successfully inhibiting the RSV replication in the lungs of mice and demonstrated that RSV VLP assembly using insect cell-derived homologous RSV matrix protein is a feasible approach.

Study on the Protective Immunity Induced by Pseudotyped Baculovirus Expressing the E Protein of Tembusu Virus in Ducklings.

The Duck Tembusu virus (DTMUV), a pathogenic flavivirus, has been causing significant economic losses in the Chinese poultry industry since 2010. This virus can severely decrease egg production and inhibit the growth of laying ducks and ducklings. While many vaccines have been developed to prevent DTMUV infection, fresh outbreaks continue to occur, as few effective vaccines are available. The E glycoprotein of DTMUV is the primary target for inducing protective immunity in the natural host. Therefore, we conducted an investigation and successfully developed a recombinant baculovirus containing the DTMUV E gene. Ducklings were then vaccinated with the purified protein derived from this virus as a potential vaccine candidate. Our findings demonstrated that the E glycoprotein of DTMUV was highly expressed in Sf9 cells. The vaccination of ducklings with the recombinant baculovirus Bac-E resulted in the induction of strong humoral and cellular immune responses. Most significantly, we observed that the vaccine provided 100% protective immunity against lethal challenges with the DTMUV YY5 strain.

Zika virus-like particles (VLPs) produced in insect cells.

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Enhanced virulence of genetically engineered Autographa californica nucleopolyhedrovirus owing to accelerated viral DNA replication aided by inserted ascovirus genes.

Genetic engineering technology is an ideal method to improve insecticidal efficiency by combining the advantages of different pathogenic microorganisms. Thus, six ascovirus genes were introduced into the genomic DNA of Autographa californica nucleopolyhedrovirus (AcMNPV) to possibly transfer the intrinsically valuable insecticidal properties from ascovirus to baculovirus. The viral budded virus (BV) production and viral DNA replication ability of AcMNPV-111 and AcMNPV-165 were significantly stronger than that of AcMNPV-Egfp (used as the wild-type virus in this study), whereas AcMNPV-33 had reduced ones. AcMNPV-111 and AcMNPV-165 also exhibited excellent insecticidal efficiency in the in vivo bioassays: AcMNPV-111 showed a 24.1% decrease in the LT50 value and AcMNPV-165 exhibited a 56.3% decrease in the LD50 value compared with AcMNPV-Egfp against the 3rd instar of Spodoptera exigua larvae, respectively. Furthermore, the size of the occlusion bodies (OBs) of AcMNPV-33, AcMNPV-111, and AcMNPV-165 were significantly increased compared to that of AcMNPV-Egfp. AcMNPV-111 and AcMNPV-165 had stable virulence against the 2nd to 4th instars tested larvae and higher OB yield than AcMNPV-Egfp in the 3rd and 4th instar larvae. Correlation and regression analyses indicated that it is better to use 5 OBs/larva virus to infect the 2nd instar larvae to produce AcMNPV-111 and 50 OBs/larva virus to infect the 3rd instar larvae to produce AcMNPV-165. The results of this study obtained recombinant viruses with enhanced virulence and exhibited a diversity of ascovirus gene function based on the baculovirus platform, which provided a novel strategy for the improvement of baculovirus as a biological insecticide.

Sf9 Cells Metabolism and Viability When Coinfected with Two Monocistronic Baculoviruses to Produce Rabies Virus-like Particles.

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at different multiplicities of infection (MOI). Schott flasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and glutamate in a broad MOI (pfu/cell) range of BVG (0.15-12.5) and BVM (0.1-5.0) using SF900 III serum free culture medium. Death phase initiation and the specific death rate depend on BV MOI. The wave pattern of nutrient/metabolite profiles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5-4.5) and BVM (1.0-3.0) for maximum protein expression was defined. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifically, for progressing in a rabies VLP vaccine development.

Zika virus-like particles (VLPs) produced in insect cells

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins’ conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Sf9 cell metabolism throughout the recombinant Baculovirus and rabies virus-like particles production in two culture systems

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to fnally obtain rabies VLP in two culture systems: Schott fask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specifc rates were quantifed over the exponential growth phase and infection stage. The highest uptake specifc rate was observed for glucose (42.5× 10–12 mmol cell/h) in SF and for glutamine (30.8× 10–12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10–10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The fndings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Performance comparison of recombinant Baculovirus and rabies virus-like particles production using two culture platforms

This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM’s multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Oxygen uptake and transfer rates throughout production of recombinant baculovirus and rabies virus-like particles

The technologies used in rabies vaccines manufacturing for human use are based on the inactivated virus platform. An alternative to traditional vaccines is virus-like particles (VLPs). This work aimed to characterize the oxygen uptake and transfer rate parameters throughout recombinant baculovirus (rBV) and rabies VLPs production using Sf9 cells in stirred tank bioreactor (STB) for a better bioprocess understanding and scalability. Four runs in a bench STB were performed: cell culture without infection; cells infected singly with rBV bearing rabies virus glycoprotein (rBVG, multiplicity of infection, MOI=0.1 pfu/cell) and matrix protein (rBVM, MOI=0.1 pfu/cell), and coinfected with BVG and BVM at MOI of 3 and 2 pfu/cell, respectively. The specific oxygen uptake rate () and volumetric oxygen transfer coefficient () were monitored throughout the reactions, as well as viable cell concentration, viability, rBV titers, and protein concentration. According to the results herein, the aeration and agitation systems in a bioreactor at a higher scale could be designed using the criterium for scale-up of constant , without oxygen facilities. Besides, rabies VLPs volumetric yield of 2.8 mg/L with a typical size (55–68 nm) was obtained. These findings suggest a promising bioprocess for rabies VLPs at a commercial scale.

Study of the heterologous gene expression characteristics of a new clone of a cell line derived from Papilio xuthus and its serum-free adaptation.

The baculovirus expression vector system using insect cells as a bioreactor has been used for in vitro expression of recombinant proteins and plays an important role in the fields of biology, agronomy, and medicine. Screening suitable host cell lines is an important part of the construction of insect cell baculovirus expression systems. In previous research, we used a single-cell cloning process with the Papilio xuthus cell line RIRI-PX1 and obtained the monoclonal cell line RIRI-PX1-C31. In this study, we compared the basic biological and recombinant protein expression characteristics of RIRI-PX1-C31 and its parent cell line RIRI-PX1 and found that the expression of recombinant ß-galactosidase in RIRI-PX1-C31 was significantly higher than that in the parental cell line. Further serum-free adaptation studies confirmed that RIRI-PX1-C31 can adapt to the growth environment of Express Five Serum-free medium and that its expression level of recombinant ß-galactosidase was significantly higher than that before adaptation.

Membrane Protein Production in Insect Cells.

Membrane proteins are an essential part of the machinery of life. They connect the interior and exterior of cells, play an important role in cell signaling and are responsible for the influx and efflux of nutrients and metabolites. For their structural and functional analysis high yields of correctly folded and modified protein are needed. Insect cells, such as Sf9 cells, have been one of the major expression hosts for eukaryotic membrane proteins in structural investigations during the last decade, as they are easier to handle than mammalian cells and provide more natural posttranslational modifications than microbial systems. Here we describe general techniques for establishing and maintaining insect cell cultures, the generation and amplification of recombinant baculovirus stocks using the flashBAC™ or Bac-to-Bac™ systems, membrane protein production, as well as the production of membrane preparations for extraction and purification experiments.

Combined Subcutaneous-Intranasal Immunization With Epitope-Based Antigens Elicits Binding and Neutralizing Antibody Responses in Serum and Mucosae Against PRRSV-2 and SARS-CoV-2.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.

Performance Comparison of Recombinant Baculovirus and Rabies Virus-like Particles production Using Two Culture Platforms.

This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM's multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Chemical composition, antioxidant, antimicrobial and antiviral activities of the leaf extracts of Syzygium myrtifolium.

This study was conducted to evaluate the chemical composition and biological activities of the leaf extracts of Syzygium myrtifolium Walp. (Myrtaceae). The results indicate that the leaf extracts of S. myrtifolium contain various classes of phytochemicals (alkaloids, anthraquinones, flavonoids, phenolics, saponins, tannins and triterpenoids) and possess antioxidant, antibacterial, antifungal and antiviral activities. Ethyl acetate, ethanol, methanol, and water extracts exhibited significantly higher (p < 0.05) oxygen radical absorbance capacity and ferric-reducing antioxidant power than the hexane and chloroform extracts. However, all extracts exhibited stronger inhibitory activity against four tested species of yeasts (minimal inhibitory concentration: 0.02-0.31 mg mL-1) than against six tested species of bacteria (minimal inhibitory concentration: 0.16-1.25 mg mL-1). The ethanolic extract offered the highest protection of Vero cells (viability > 70 %) from the cytopathic effect caused by the Chikungunya virus while the ethyl acetate extract showed significant replication inhibitory activity against the virus (p < 0.001) using the replicon-enhanced green fluorescent protein reporter system.

Sf9 Cells metabolism and viability when coinfected with two monocistronic baculoviruses to produce rabies virus‑like particles

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at diferent multiplicities of infection (MOI). Schott fasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and lutamate in a broad MOI (pfu/cell) range of BVG (0.15–12.5) and BVM (0.1–5.0) using SF900 III serum free culture medium. Death phase initiation and the specifc death rate depend on BV MOI. The wave pattern of nutrient/metabolite profles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5–4.5) and BVM (1.0–3.0) for maximum protein expression was defned. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifcally, for progressing in a rabies VLP vaccine development.

Mucosal Administration of Recombinant Baculovirus Displaying Toxoplasma gondii ROP4 Confers Protection Against T. gondii Challenge Infection in Mice.

Pathogens require physical contact with the mucosal surface of the host organism to initiate infection and as such, vaccines eliciting both mucosal and systemic immune responses would be promising. Studies involving the use of recombinant baculoviruses (rBVs) as mucosal vaccines are severely lacking despite their inherently safe nature, especially against pathogens of global importance such as Toxoplasma gondii. Here, we generated rBVs displaying T. gondii rhoptry protein 4 (ROP4) and evaluated their protective efficacy in BALB/c mice following immunization via intranasal (IN) and oral routes. IN immunization with the ROP4-expressing rBVs elicited higher levels of parasite-specific IgA antibody responses compared to oral immunization. Upon challenge infection with a lethal dose of T. gondii ME49, IN immunization elicited significantly higher parasite-specific antibody responses in the mucosal tissues such as intestines, feces, vaginal samples, and brain than oral immunization. Marked increases in IgG and IgA antibody-secreting cell (ASC) responses were observed from intranasally immunized mice. IN immunization elicited significantly enhanced induction of CD4+, CD8+ T cells, and germinal center B (GC B) cell responses from secondary lymphoid organs while limiting the production of the inflammatory cytokines IFN-γ and IL-6 in the brain, all of which contributed to protecting mice against T. gondii lethal challenge infection. Our findings suggest that IN delivery of ROP4 rBVs induced better mucosal and systemic immunity against the lethal T. gondii challenge infection compared to oral immunization.