Insect Cells Research Articles

Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas

Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host’s immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of Man5GlcNAc2. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn2+, while the addition of EDTA or Cu2+ abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.Graphical abstractIllustration of GnT-I activity. (a) Transfer of GlcNAc to Man5-PA, creating Man5GlcNAc3-PA. (b) Transfer of GlcNAc to MM-PA, creating MGn-PA. (c) Transfer of GlcNAc to GnM-PA, creating GnGn-PA. Blue squares represent N-acetylglucosamine, green cycles depict mannose. Graphic illustration of N-glycans were created using bioRENDER.

The expression system affects the binding affinity between p75NTR and proNGF

ProNGF (nerve growth factor) is a precursor of NGF and a signaling peptide exerting opposite effects on neuronal cells, i.e., apoptotic or neuritogenic. The conflicting biological activity of proNGF depends on the relative levels of two membrane receptors, TrkA and p75NTR. The effect of proNGF depends on the expression levels of these receptor proteins and their affinity to proNGF. Since the affinity of proteins has been studied with various recombinant proteins, it is worth comparing the affinity of these proteins within one experiment with the same method. This study examined the affinity between a recombinant proNGF and p75NTR expressed in common systems: bacterial, insect, and mammalian cells. The extracellular domain of p75NTR expressed in the insect or mammalian systems bound to native mature NGF, with a higher affinity for the insect receptor. The uncleavable proNGF was expressed in the three systems and they showed neuritogenic activity in PC12 cells. These recombinant proteins were used to compare their binding affinity to p75NTR. The insect p75NTR showed a higher binding affinity to proNGF than the mammalian p75NTR. The insect p75NTR bound proNGF from the insect system with the highest affinity, then from the mammalian system, and the lowest from the bacterial system. Conversely, the mammalian p75NTR showed no such preference for proNGF. Because the recombinant proNGF and p75NTR from different expression systems are supposed to have the same amino acid sequences, these differences in the affinity depend likely on their post-translational modifications, most probably on their glycans. Each recombinant proNGF and p75NTR in various expression systems exhibited different mobilities on SDS-PAGE and reactivities with glycosidases and lectins.

Investigation of the insecticidal potential of curcumin derivatives that target the Helicoverpa armigera sterol carrier protein-2

Cotton bollworm (Helicoverpa armigera) is a highly polyphagous, widely prevalent, and persistent Old World insect pest that affects numerous important crops that are directly consumed by people, including tomato, cotton, pigeon pea, chickpea, rice, sorghum, and cowpea. Insects do not synthesize steroids but obtain them from their diet. Inhibition of dietary uptake of steroids by insects is a potentially effective insecticidal mechanism that should not be toxic to humans and other mammals, who synthesize their steroids. Ten curcumin derivatives were tested against H. armigera sterol carrier protein-2 (HaSCP-2) for their potential as insecticidal agents. Curcumin derivatives were initially docked at the binding site of HaSCP-2 to determine their binding affinities and plausible binding modes. The binding modes predominantly show hydrophobic interactions of derivatives with Phe53, Phe110, and Phe89 as core interacting residues in the active site. Validation of in silico results was carried out by performing a fluorescence binding and displacement assay to determine the binding affinities of curcumin derivatives. Among a collection of curcumin derivatives tested, Cur10 showed the lowest IC50 value of 9.64 μM, while Cur07 was 19.86 μM, and Cur06 was 20.79 μM. There was a significant negative correlation between the ability of the curcumin derivatives tested to displace the fluorescent probe from the sterol binding site of HaSCP-2 and to inhibit Sf9 insect cell growth in culture, which is consistent with the curcumin derivatives acting by the novel mechanism of blocking sterol uptake. Then molecular dynamics simulation studies validated the predicted binding modes and the interactions of curcumin derivatives with HaSCP-2 protein. In conclusion, these studies support the potential use of curcumin derivatives as insecticidal agents.

Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro.

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects.

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.

Abstract 3113: Development of HTS enzymatic assays for WRN helicase and exonuclease functions

Abstract WRN, a double strand break repair enzyme with ATP-dependent DNA-helicase and 3’- >5’ exonuclease activities, is under intense focus as a therapeutic target because of its synthetic lethality in tumors with microsatellite instability. To accelerate discovery of WRN inhibitors, we developed and validated homogenous, fluorescence-based high throughput assays for WRN helicase and exonuclease enzymatic activities using the Transcreener HTS Assay platform, which relies on highly selective antibodies to detect nucleotides in a homogenous (mix-and-read) format with far red fluorescent readouts. We produced the WRN helicase domain (aa 500-946, N-terminal 6xHis) in BaV-infected insect cells and optimized detection of its dsDNA-dependent ATPase activity using the Transcreener ADP2 Assay in FP, TR-FRET, and FI detection modes. Optimal reaction conditions included 50 μM ATP (approximate Km) and saturating concentration (100 nM) of a 3’ flapped duplex DNA oligomer; these conditions yielded a good signal with 250 pM WRN, a kcat of 300 min−1, and a Z’ of 0.85. An IC50 of 35 nM was determined for the probe inhibitor NSC-617145, and a 1,280 compound pilot screen (Tocris 2.0 Micro) identified multiple hits that were confirmed in dose response experiments. We produced the WRN 3’-5’ exonuclease domain (aa 1-133-6xHis) in E. coli and used the Transcreener dAMP Exonuclease Assay to allow direct, FP-based detection of dAMP monomers released from a 52 bp dsDNA oligomer with a 5’ overhang. With saturating DNA present (1 μM), the assay produced a good signal with 1 nM enzyme; the Z’ value was 0.89. There are no published inhibitors for the WRN exonuclease, but suramin inhibited with an IC50 of approximately 100 nM. These assays allow sensitive, robust, quantitative detection of WRN ATPase and exonuclease activities using an extensively validated HTS platform; they should be valuable tools for screening and optimizing small molecules targeting specific WRN functions. Citation Format: Ha Pham, Mahbbat Ali, Anibal Ramos Martinez, Robert Lowery. Development of HTS enzymatic assays for WRN helicase and exonuclease functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3113.

Abstract 5783: Receptor tyrosine kinase Tyro3 interaction with ligands and inhibitors

Abstract TAM receptors (Tyro3, Axl, Mer) are a sub-family of receptor tyrosine kinases that regulate cell survival and homeostasis primarily through their role in clearance of apoptotic cells. While several receptor tyrosine kinases (RTKs) are targeted in various types of cancer therapies, interest in Tyro3 as a potential target in breast cancers has only recently grown. Determining the structure of Tyro3 with and without tyrosine kinase inhibitors (TKIs) bound will provide important missing information to aid in the design of treatments that can be combined with existing cancer treatments, as dual treatment is often necessary. I have designed constructs that will be used for crystallization of the Tyro3 kinase domains. I mapped the full portion of the kinase (called "Small"), an extended kinase ("Big"), and a smaller catalytic domain ("Cat"). The kinases were made through a molecular cloning method (Gibson Assembly), sequence verified, and expressed in insect cells for large scale purification. Ni-NTA and gel filtration will be used, followed by crystallization and soaking with inhibitors. Citation Format: Keisha Nicole Hardeman, Chrystal Starbird. Receptor tyrosine kinase Tyro3 interaction with ligands and inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5783.

Abstract 3114: Development of HTS enzymatic assays for RNA helicases: DDX3, DDX5, DDX17, RIG-1 and MDA5

Abstract RNA helicases are a broad class of enzymes that bind and modify RNA in an ATP-dependent manner and play diverse roles in RNA metabolism, regulation of gene expression, and innate immunity. Several RNA helicases, especially in the dead box helicase (DDX) family, have been shown to be dysregulated in cancer, and there have been limited efforts to develop small molecule inhibitors. To accelerate development of selective helicase inhibitors while avoiding off target effects, especially with helicases that promote tumor immunity, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP2 assay for homogenous detection of RNA-dependent ATPase activity with a far-red fluorescence polarization (FP) readout. Here we describe development of assays for DDX3, DDX17 and DDX5, which have all been implicated in cancer, and the Rig-1-like receptor (RLR) helicases, RIG-1 and MDA5, which trigger expression of type I interferons important for tumor immunity. We produced the recombinant enzymes using BaV-infected insect cells, as full-length polypeptides, or in some cases truncated to remove disordered N-terminal domains. We determined RNA substrate and cofactor requirements and kinetic parameters with the purified enzymes and optimized the assays for initial velocity detection of RNA-dependent ATPase activity with a Z’ value greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays. We validated each of the helicase ATPase assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming hits in dose response mode with the enzymatic assay and with thermal shift assays to confirm target binding. These assays will enable screening and hit-to-lead/SAR for DDX helicases that drive tumorigenesis as well as selectivity profiling to avoid off target effects with RLR helicases that promote tumor immunity. Citation Format: Mahbbat Ali, Ha Pham, Anibal Ramos Martinez, Robert Lowery. Development of HTS enzymatic assays for RNA helicases: DDX3, DDX5, DDX17, RIG-1 and MDA5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3114.

Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production.

Lab-grown insect meat – Chemical and biological insights – A comprehensive review

Abstract Lab-grown insect meat is a promising alternative to traditional livestock for sustainable food production. This review paper aims to provide a comprehensive overview of the current state of knowledge regarding lab-grown insect meat, emphasizing key aspects such as life cycle assessment, insect cell culture history, technological advancements, and bio-robotics in insect cell culture. Comparisons and challenges between insect and mammalian/avian cell culture methodologies are presented. The nutritional content of edible insects (proximate, amino acid, mineral, and vitamin content) and the potential health benefits of consuming insect meat are discussed. The paper also explores embryonic and adult myogenesis processes in insect cells, as well as the significance of insect body fat and muscle cells in culture. Applications of insect cell culture in various fields, such as food production and pharmaceutical development, are presented. Moreover, the potential occurrence of mutations in lab-grown insect cells is examined. Lastly, the review addresses the drawbacks and limitations of insect labriculture, discussing factors such as scalability, cost-efficiency, and public acceptance. Overall, this comprehensive review provides essential insights into the chemical and biological aspects of lab-grown insect meat, paving the way for further research and development in this emerging field. This article is the first review article reporting the chemical and biological insights of lab-grown insect meat.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3–48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

Biocontrol Potential of Sodin 5, Type 1 Ribosome-Inactivating Protein from Salsola soda L. Seeds.

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as 'agretti', is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 µg/mL (1.4 µM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 µg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome-inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.

Biochemical characterization of cardiac α-actin mutations A21V and D26N implicated in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α-actin are the cause in 1%-5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM-linked cardiac α-actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α-actin variants and wild-type protein revealed improper N-terminal processing in the Spodoptera frugiperda (Sf-9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies. Therefore, we produced N-terminal deletion mutants lacking the N-terminal cysteine (ΔC2). The ΔC2 wild-type construct behaved similar to porcine cardiac α-actin purified from native Sus scrofa heart tissue and all ΔC2 constructs showed improved fluorescent labeling. Further analysis of untruncated and ΔC2 constructs showed that while neither the A21V nor the D26N mutation affects nucleotide binding, they cause a similar slowing of the rate of filament formation as well as a reduction in the thermal stability of monomeric and filamentous cardiac α-actin. In vitro motility assays and transient-kinetic studies probing the interaction of the actin variants with cardiac β-myosin revealed perturbed actomyosin interactions and a reduced motile activity for the p.D26N variant. Addition of the small molecule effector EMD 57033, which targets cardiac β-myosin, rescued the approximately 40% drop in velocity observed with the p.D26N constructs and activated the motile activity of wild-type and p.D26N to the same level of 1100 nm s-1 .

Mutation in PgABCC2 confers low-level resistance to Cry1Ac in pink bollworm.

With the increasing incidence of pest resistance to transgenic crops producing Bacillus thuringiensis (Bt) proteins in the field, elucidating the molecular basis of resistance is important for monitoring, delaying and countering pest resistance. Previous work revealed that mutation or down-regulated expression of the cadherin gene (PgCad1) is associated with pink bollworm (Pectinophora gossypiella) resistance to Cry1Ac, and 20 mutant PgCad1 alleles (r1-r20) were characterized. Here, we tested the hypothesis that the ABC transporter PgABCC2 is a functional receptor for the Bt toxin Cry1Ac and that a mutation is associated with resistance. We identified and characterized the first resistance allele (rC2 ) of PgABCC2 in the laboratory-selected Cry1Ac-resistant strain AQ-C2 of pink bollworm. The rC2 allele had a one-base deletion in exon20, resulting in a frameshift and the introduction of a premature stop codon. This resulting PgABCC2 protein had a truncated C-terminus, including the loss of the NBD2 domain. AQ-C2 exhibited 20.2-fold greater resistance to Cry1Ac than the susceptible strain, and its inheritance of Cry1Ac resistance was recessive and genetically linked to PgABCC2. When produced in cultured insect cells, recombinant wild-type and rC2 mutant PgABCC2 proteins localized within the cell plasma membrane, although substantial cytoplasmic retention was also observed for the mutant protein, while the mutant PgABCC2 caused a 13.9-fold decrease in Cry1Ac toxicity versus the wild-type PgABCC2. PgABCC2 is a functional receptor of Cry1Ac and the loss of its carboxyl terminus (including its NBD2 domain) confers low-level resistance to Cry1Ac in both larvae and in cultured cells. © 2024 Society of Chemical Industry.

Broad antibody and T cell responses to Ebola, Sudan, and Bundibugyo ebolaviruses using mono- and multi-valent adjuvanted glycoprotein vaccines

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion (“IVAX-1”) was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.

Pi-seq—A customizable multichannel syringe pump for microfluidics

The advent of single cell technologies resulted in growing demand for microfluidics in the biological sciences. Commercial platforms have remained expensive, inflexible, and non-customizable black boxes. We developed an open source, multichannel, zero-backflow microfluidics device based on syringe pumps controlled by a Raspberry Pi computer. It uses both readily available and 3D-printed parts as well as a custom PCB and is easily serviceable. Moreover, it is fully customizable for various applications. Total cost is under €600. We equipped one channel with a custom Peltier-based temperature controller for precise heating or cooling and a mixer mechanism to prevent sedimentation of the cells within the syringe. Depending on the cells in the sample, heating and cooling can be useful to maintain a beneficial environment or to slow down cellular processes and cell death, respectively. Combined with microfluidics consumables and a microscope, the device is capable of integration into a high quality droplet-based single cell RNA sequencing workflow as shown here. Analysis of a mixture of human and insect cells resulted in a dataset of 17,769 single cells and demonstrates reliable operation and separation.

A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency

A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency

Optimized Recombinant Expression and Purification of the SARS-CoV-2 Polymerase Complex.

An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.

Serological characterisation of Lagovirus virus-like particles originating from native and mutated VP60 of rabbit haemorrhagic disease virus 2 and European brown hare syndrome virus

Abstract Introduction Since lagoviruses cannot be cultivated in vitro, using expression systems is an alternative and promising way of producing diagnostic viral antigens. It opens up their use as active immunogens for vaccine production. Material and Methods Virus-like particles (VLPs) were produced in a baculovirus expression system in Spodoptera frugiperda 9 (Sf9) insect cells based on wild-type and mutated variants of the virus capsid VP60 protein from a Polish strain of European brown hare syndrome virus (EBHSV) and wild-type and mutated versions of this protein from a Polish strain of rabbit haemorrhagic disease virus 2 (RHDV2). The mutations were the substitution of an arginylglycylaspartic acid (Arg-Gly-Asp/RGD) motif in the P2 subdomain and, in the S or P2 domain, the substitution of three lysines. The VLPs were purified with sucrose gradient ultracentrifugation. Results Protein production was confirmed by Western blot analysis using rabbit or hare sera and ELISA tests with different types of monoclonal antibody. The haemagglutination properties of some VLPs were also evaluated. Electron microscopy of wild-type EBHSV, wild-type RHDV2 and the four VP60 variants produced in this experiment revealed the formation of characteristic VLP structures. Conclusion For the first time, mutated VLPs of RHDV2 with an RGD motif in the VP60 sequence were obtained, which could potentially be used to deliver cargo to eukaryotic cells. Virus-like particles based on the VP60 proteins of EBHSV and RHDV with a three-lysine substitution in the S or P2 domains were also obtained. Potential exists for VLPs of EBHSV and RHDV2 as vaccine candidates.