A streamlined, automated workflow to screen and triage large numbers of baculoviruses for protein expression.

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.

Baculovirus expression and purification of virion core and envelope proteins of goatpox virus to evaluate their diagnostic potential.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Unseen Weapons: Bacterial Extracellular Vesicles and the Spread of Antibiotic Resistance in Aquatic Environments.

This paper sheds light on the alarming issue of antibiotic resistance (ABR) in aquatic environments, exploring its detrimental effects on ecosystems and public health. It examines the multifaceted role of antibiotic use in aquaculture, agricultural runoff, and industrial waste in fostering the development and dissemination of resistant bacteria. The intricate interplay between various environmental factors, horizontal gene transfer, and bacterial extracellular vesicles (BEVs) in accelerating the spread of ABR is comprehensively discussed. Various BEVs carrying resistance genes like blaCTX-M, tetA, floR, and sul/I, as well as their contribution to the dominance of multidrug-resistant bacteria, are highlighted. The potential of BEVs as both a threat and a tool in combating ABR is explored, with promising strategies like targeted antimicrobial delivery systems and probiotic-derived EVs holding significant promise. This paper underscores the urgency of understanding the intricate interplay between BEVs and ABR in aquatic environments. By unraveling these unseen weapons, we pave the way for developing effective strategies to mitigate the spread of ABR, advocating for a multidisciplinary approach that includes stringent regulations, enhanced wastewater treatment, and the adoption of sustainable practices in aquaculture.

Bacterial extracellular vesicles: biotechnological perspective for enhanced productivity.

Bacterial extracellular vesicles (BEVs) are non-replicative nanostructures released by Gram-negative and Gram-positive bacteria as a survival mechanism and inter- and intraspecific communication mechanism. Due to BEVs physical, biochemical, and biofunctional characteristics, there is interest in producing and using them in developing new therapeutics, vaccines, or delivery systems. However, BEV release is typically low, limiting their application. Here, we provide a biotechnological perspective to enhance BEV production, highlighting current strategies. The strategies include the production of hypervesiculating strains through gene modification, bacteria culture under stress conditions, and artificial vesicles production. We discussed the effect of these production strategies on BEVs types, morphology, composition, and activity. Furthermore, we summarized general aspects of BEV biogenesis, functional capabilities, and applications, framing their current importance and the need to produce them in abundance. This review will expand the knowledge about the range of strategies associated with BEV bioprocesses to increase their productivity and extend their application possibilities.

A seamless connection from the burst sequence to the start codon is essential for polyhedrin hyperexpression in alphabaculoviruses.

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of polyhedrin (POLH), and high-level transcription of the polh gene has been exploited to express foreign proteins in insect cells. While making Bombyx mori nucleopolyhedrovirus (BmNPV) polh mutants using a conventional transfer vector-based method, we noticed that a virus with a short sequence insertion just before the polh start codon produces fewer very small OBs. Detailed analysis of several BmNPV mutants revealed that insertions between the burst sequence and start codon markedly decrease POLH accumulation and polh transcription. We further confirmed this decrease using recombinant viruses expressing a reporter gene driven by the polh promoter. These findings underscore the critical importance of a seamless connection from the burst sequence to the start codon for baculovirus polh hyperexpression.

Impact of dual-baculovirus infection on the Sf9 insect cell transcriptome during rAAV production using single-cell RNA-seq.

The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and virus-like particles. More recently, IC-BEVS has also been used as an alternative to produce recombinant adeno-associated virus (rAAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity (e.g., stochastic infection process and differences in infection kinetics) on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of rAAV of serotype 2 (rAAV2) using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-sequencing (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated with the cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, which was linked to the expression of baculovirus genes as well as to differences in rAAV transgenes (rep, cap and gfp) expression. Noteworthy, at 24 h post-infection, only 29.4% of cells enclosed all three necessary rAAV transgenes to produce packed rAAV2 particles, indicating limitations of the dual-baculovirus system. In addition, the trajectory analysis herein performed highlighted that biological processes such as protein folding, metabolic processes, translation, and stress response have been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight into the rational cell and process engineering toward improved rAAV2 production in IC-BEVS.

Optimization of genetic code expansion in the baculovirus expression vector system (BEVS).

The production of recombinant proteins containing unnatural amino acids, commonly known as genetic code expansion (GCE), represents a breakthrough in protein engineering that allows for the creation of proteins having novel designed properties. The naturally occurring orthogonal pyrrolysine tRNA/aminoacyl-tRNApyl synthetase pair (tRNApyl/PylRS) found in Methanosarcinaceae species has provided a rich platform for protein engineers to build a library of amino acid derivatives suitable for the introduction of novel chemical functionalities. While reports of the production of such recombinant proteins utilizing the tRNApyl/PylRS pair, or mutants thereof, is commonplace in Escherichia coli and mammalian cell expression systems, there has only been a single such report of GCE in the other stalwart of recombinant protein production, the baculovirus expression vector system (BEVS). However, that report formulates protein production within the designs of the MultiBac expression system [1]. The current study frames protein production within the strategies of the more commonplace Bac-to-Bac system of recombinant baculovirus production, via the development of novel baculovirus transfer vectors that harbor the tRNApyl/PylRS pair. The production of recombinant proteins harboring an unnatural amino acid(s) was examined using both an in cis and an in trans arrangement of the tRNApyl/PylRS pair relative to the target protein ORF i.e. the latter resides, respectively, on either the same vector as the tRNApyl/PylRS pair, or on a separate vector and deployed in a viral co-infection experiment. Aspects of the transfer vector designs and the viral infection conditions were investigated.

A sensitive assay for scrutiny of Autographa californica multiple nucleopolyhedrovirus genes using CRISPR-Cas9.

Baculoviruses have very large genomes and previous studies have demonstrated improvements in recombinant protein production and genome stability through the removal of some nonessential sequences. However, recombinant baculovirus expression vectors (rBEVs) in widespread use remain virtually unmodified. Traditional approaches for generating knockout viruses (KOVs) require several experimental steps to remove the target gene prior to the generation of the virus. In order to optimize rBEV genomes by removing nonessential sequences, more efficient techniques for establishing and evaluating KOVs are required. Here, we have developed a sensitive assay utilizing CRISPR-Cas9-mediated gene targeting to examine the phenotypic impact of disruption of endogenous Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes. For validation, 13 AcMNPV genes were targeted for disruption and evaluated for the production of GFP and progeny virus - traits that are essential for their use as vectors for recombinant protein production. The assay involves transfection of sgRNA into a Cas9-expressing Sf9 cell line followed by infection with a baculovirus vector carrying the gfp gene under the p10 or p6.9 promoters. This assay represents an efficient strategy for scrutinizing AcMNPV gene function through targeted disruption, and represents a valuable tool for developing an optimized rBEV genome. KEY POINTS: [Formula: see text] A method to scrutinize the essentiality of baculovirus genes was developed. [Formula: see text] The method uses Sf9-Cas9 cells, a targeting plasmid carrying a sgRNA, and a rBEV-GFP. [Formula: see text] The method allows scrutiny by only needing to modify the targeting sgRNA plasmid.

Distribution and Location of BEVs in Different Genotypes of Bananas Reveal the Coevolution of BSVs and Bananas.

Members of the family Caulimoviridae contain abundant endogenous pararetroviral sequences (EPRVs) integrated into the host genome. Banana streak virus (BSV), a member of the genus Badnavirus in this family, has two distinct badnaviral integrated sequences, endogenous BSV (eBSV) and banana endogenous badnavirus sequences (BEVs). BEVs are distributed widely across the genomes of different genotypes of bananas. To clarify the distribution and location of BEVs in different genotypes of bananas and their coevolutionary relationship with bananas and BSVs, BEVs and BSVs were identified in 102 collected banana samples, and a total of 327 BEVs were obtained and categorized into 26 BEVs species with different detection rates. However, the majority of BEVs were found in Clade II, and a few were clustered in Clade I. Additionally, BEVs and BSVs shared five common conserved motifs. However, BEVs had two unique amino acids, methionine and lysine, which differed from BSVs. BEVs were distributed unequally on most of chromosomes and formed hotspots. Interestingly, a colinear relationship of BEVs was found between AA and BB, as well as AA and SS genotypes of bananas. Notably, the chromosome integration time of different BEVs varied. Based on our findings, we propose that the coevolution of bananas and BSVs is driven by BSV Driving Force (BDF), a complex interaction between BSVs, eBSVs, and BEVs. This study provides the first clarification of the relationship between BEVs and the coevolution of BSVs and bananas in China.

Chimeric Human Papillomavirus-16 Virus-like Particles Presenting HIV-1 P18I10 Peptide: Expression, Purification, Bio-Physical Properties and Immunogenicity in BALB/c Mice.

Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG (p = 0.6409) was observed as Gardasil anti-HPV vaccine-immunized mice. Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1.

Blood plasma derived extracellular vesicles (BEVs): particle purification liquid chromatography (PPLC) and proteomic analysis reveals BEVs as a potential minimally invasive tool for predicting response to breast cancer treatment.

PURPOSE: Circulating blood plasma derived extracellular vesicles (BEVs) containing proteins hold promise for their use as minimally invasive biomarkers for predicting response to cancer therapy. The main goal of this study was to establish the efficiency and utility of the particle purification liquid chromatography (PPLC) BEV isolation method and evaluate the role of BEVs in predicting breast cancer (BC) patient response to neoadjuvant chemotherapy (NAC). METHODS: PPLC isolation was used to separate BEVs from non-EV contaminants and characterize BEVs from 17 BC patients scheduled to receive NAC. Using LC-MS/MS, we compared the proteome of PPLC-isolated BEVs from patients (n = 7) that achieved a pathological complete response (pCR) after NAC (responders [R]) to patients (n = 10) who did not achieve pCR (non-responders [NR]). Luminal MCF7 and basaloid MDA-MB-231 BC cells were treated with isolated BEVs and evaluated for metabolic activity by MTT assay. RESULTS: NR had elevated BEV concentrations and negative zeta potential (ζ-potential) prior to receipt of NAC. Eight proteins were enriched in BEVs of NR. GP1BA (CD42b), PECAM-1 (CD31), CAPN1, HSPB1 (HSP27), and ANXA5 were validated using western blot. MTT assay revealed BEVs from R and NR patients increased metabolic activity of MCF7 and MDA-MB-231 BC cells and the magnitude was highest in MCF7s treated with NR BEVs. CONCLUSION: PPLC-based EV isolation provides a preanalytical separation process for BEVs devoid of most contaminants. Our findings suggest that PPLC-isolated BEVs and the five associated proteins may be established as predictors of chemoresistance, and thus serve to identify NR to spare them the toxic effects of NAC.

A review of alternative promoters for optimal recombinant protein expression in baculovirus-infected insect cells.

Generating recombinant proteins in insect cells has been made possible via the use of the Baculovirus Expression Vector System (BEVS). Despite the success of many proteins via this platform, some targets remain a challenge due to issues such as cytopathic effects, the unpredictable nature of co-infection and co-expressions, and baculovirus genome instability. Many promoters have been assayed for the purpose of expressing diverse proteins in insect cells, and yet there remains a lack of implementation of those results when reviewing the landscape of commercially available baculovirus vectors. In advancing the platform to produce a greater variety of proteins and complexes, the development of such constructs cannot be avoided. A better understanding of viral gene regulation and promoter options including viral, synthetic, and insect-derived promoters will be beneficial to researchers looking to utilize BEVS by recruiting these intricate mechanisms of gene regulation for heterologous gene expression. Here we summarize some of the developments that could be utilized to improve the expression of recombinant proteins and multi-protein complexes in insect cells.

Solutions against emerging infectious and noninfectious human diseases through the application of baculovirus technologies.

Baculoviruses are insect pathogens widely used as biotechnological tools in different fields of life sciences and technologies. The particular biology of these entities (biosafety viruses 1; large circular double-stranded DNA genomes, infective per se; generally of narrow host range on insect larvae; many of the latter being pests in agriculture) and the availability of molecular-biology procedures (e.g., genetic engineering to edit their genomes) and cellular resources (availability of cell lines that grow under in vitro culture conditions) have enabled the application of baculoviruses as active ingredients in pest control, as systems for the expression of recombinant proteins (Baculovirus Expression Vector Systems-BEVS) and as viral vectors for gene delivery in mammals or to display antigenic proteins (Baculoviruses applied on mammals-BacMam). Accordingly, BEVS and BacMam technologies have been introduced in academia because of their availability as commercial systems and ease of use and have also reached the human pharmaceutical industry, as incomparable tools in the development of biological products such as diagnostic kits, vaccines, protein therapies, and-though still in the conceptual stage involving animal models-gene therapies. Among all the baculovirus species, the Autographa californica multiple nucleopolyhedrovirus has been the most highly exploited in the above utilities for the human-biotechnology field. This review highlights the main achievements (in their different stages of development) of the use of BEVS and BacMam technologies for the generation of products for infectious and noninfectious human diseases. KEY POINTS: • Baculoviruses can assist as biotechnological tools in human health problems. • Vaccines and diagnosis reagents produced in the baculovirus platform are described. • The use of recombinant baculovirus for gene therapy-based treatment is reviewed.

A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening.

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

Biotechnological applications of occlusion bodies of Baculoviruses.

The ability of Baculoviruses to hyper-express very late genes as polyhedrin, the major component of occlusion bodies (OBs) or polyhedra, has allowed the evolution of a system of great utility for biotechnology. The main function of polyhedra in nature is to protect Baculovirus in the environment. The possibility of incorporating foreign proteins into the crystal by fusing them to polyhedrin (POLH) opened novel potential biotechnological uses. In this review, we summarize different applications of Baculovirus chimeric OBs. Basically, the improvement of protein expression and purification with POLH as a fusion partner; the use of recombinant polyhedra as immunogens and antigens, and the incorporation of proteins into polyhedra to improve Baculoviruses as bioinsecticides. The results obtained in each area and the future trends in these topics are also discussed.

Overexpression of c-Myc enhances recombinant protein production in High Five cells after baculovirus infection.

Due to their numerous advantages, baculovirus expression vector systems (BEVS) have been widely used to express recombinant proteins for different purposes. Different strategies have been adopted to increase recombinant protein production. In this study, we transiently or stably expressed mouse c-Myc in High Five cells using a commercial pIB/V5 vector. Under the control of the OpIE2 promoter, this vector could enhance recombinant protein production. We found that transient expression of c-Myc in High Five cells improved recombinant protein production. Furthermore, we established two stable cell lines, High Five-c-Myc #1 and High Five-c-Myc #2, that stably expressed mouse c-Myc. We further found that the expression level of the recombinant protein was increased in these stable cell lines compared to control cell lines. These data indicate that overexpressing c-Myc in cells is a promising way to improve recombinant protein production in BEVS.

Production of human type II collagen using an efficient baculovirus-silkworm multigene expression system.

Human type II collagen is a macromolecular protein found throughout the human body. The baculovirus expression vector system is one of the most ideal systems for the routine production and display of recombinant eukaryotic proteins in insect, larvae, and mammalian cells. We use this system to express a full-length gene, human type II collagen cDNA (4257 bp), in cultured Spodoptera frugiperda 9 cells (Sf9), Bombyx mori cells, and silkworm larvae. In this study, the expression of human type II collagen gene in both insect cells and silkworm larvae was purified by nickel column chromatography, leading to 300-kDa bands in SDS-PAGE and western blotting indicative of collagen α-chains organized in a triple-helical structure. About 1 mg/larva human type II collagen is purified from silkworm skin, which shows a putative large scale of collagen production way. An activity assay of recombinant human type II collagen expressed by silkworm larvae demonstrated that the recombinant protein has considerable bioactive properties. Scanning electron microscopy of purified proteins clearly reveals randomly distributed and pitted structures. We conclude that the baculovirus-silkworm multigene expression system can be used as an efficient platform for express active human type II collagen and other complicated eukaryotic proteins.

Fundamentals of Baculovirus Expression and Applications.

In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology.

Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system.

Expression of recombinant proteins by BEVS in a macula-like virus-free silkworm cell line.

We previously established the first Bombyx mori macula-like virus (BmMLV)-free cell line (BmVF cells) from a B. mori embryo. In this study, we evaluated the expression of recombinant proteins in BmVF cells using a B. mori nucleopolyhedrovirus (BmNPV)-derived expression vector. Our results showed that BmVF cells are susceptible to BmNPV, and both the promoter activity of the polyhedrin gene and the post-translated modifications of a recombinant protein are equivalent between BmMLV-negative BmVF and -positive BmN4 cells. These findings indicate that persistent infection with BmMLV has no discernible effect on BmNPV-mediated protein production in B. mori cells.