Insect Cell Culture Research Articles

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning.

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.

Chronic depletion of vertebrate lipids in Aedes aegypti cells dysregulates lipid metabolism and inhibits innate immunity without altering dengue infectivity.

Aedes aegypti is the primary vector of dengue virus (DENV) and other arboviruses. Previous literature suggests that vertebrate and invertebrate lipids and the nutritional status of mosquitoes modify virus infection. Here, we developed a vertebrate lipid-depleted Ae. aegypti cell line to investigate if chronic depletion of vertebrate lipids normally present in a blood meal and insect cell culture medium would impact cell growth and virus infection. Chronic depletion of vertebrate lipids reduced cell size and proliferation, although cells retained equivalent total intracellular lipids per cell by reducing lipolysis and modifying gene expression related to sugar and lipid metabolism. Downregulation of innate immunity genes was also observed. We hypothesized that chronic depletion of vertebrate lipids would impact virus infection; however, the same amount of DENV was produced per cell. This study reveals how Ae. aegypti cells adapt in the absence of vertebrate lipids, and how DENV can replicate equally well in cells that contain predominately vertebrate or invertebrate lipids.

Dunaliella salina as a Potential Biofactory for Antigens and Vehicle for Mucosal Application

The demand for effective, low-cost vaccines increases research in next-generation biomanufacturing platforms and the study of new vaccine delivery systems (e.g., mucosal vaccines). Applied biotechnology in antigen production guides research toward developing genetic modification techniques in different biological models to achieve the expression of heterologous proteins. These studies are based on various transformation protocols, applied in prokaryotic systems such as Escherichia coli to eukaryotic models such as yeasts, insect cell cultures, animals, and plants, including a particular type of photosynthetic organisms: microalgae, demonstrating the feasibility of recombinant protein expression in these biological models. Microalgae are one of the recombinant protein expression models with the most significant potential and studies in the last decade. Unicellular photosynthetic organisms are widely diverse with biological and growth-specific characteristics. Some examples of the species with commercial interest are Chlamydomonas, Botryococcus, Chlorella, Dunaliella, Haematococcus, and Spirulina. The production of microalgae species at an industrial level through specialized equipment for this purpose allows for proposing microalgae as a basis for producing recombinant proteins at a commercial level. A specie with a particular interest in biotechnology application due to growth characteristics, composition, and protein production capacity is D. salina, which can be cultivated under industrial standards to obtain βcarotene of high interest to humans. D saline currently has advantages over other microalgae species, such as its growth in culture media with a high salt concentration which reduces the risk of contamination, rapid growth, generally considered safe (GRAS), recombinant protein biofactory, and a possible delivery vehicle for mucosal application. This review discusses the status of microalgae D. salina as a platform of expression of recombinant production for its potential mucosal application as a vaccine delivery system, taking an advance on the technology for its production and cultivation at an industrial level.

CULTIVO in vitro DE CÉLULAS EMBRIONARIAS DE COCHINILLA (Dactylopius coccus Costa) A DIFERENTES pH

Con el objetivo de determinar el pH óptimo al que cultivos celulares de cochinilla (Dactylopius coccus Costa) registran la máxima producción de ácido carmínico in vitro, se evaluaron varios niveles de pH (4.5, 5.0, 5.5, 6.0 y 6.5) y de fracciones de centrifugado (0, 2, 5 y 10 min) de células maceradas de embriones de cochinilla establecidos en medio nutritivo de Schneider. El contenido de ácido carmínico presente en cada muestra fue determinado mediante cromatografía líquida de alta resolución. La extracción del pigmento de las muestras se realizó con una solución de HCl 2M. Los límites de detección del pigmento fueron de 1.0 mg L-1 a 120.0 mg L-1. No se observó un efecto significativo del pH en el contenido de ácido carmínico ni en el número de líneas celulares, aunque el pH intermedio (5.5) tendió a dar mejores resultados.

A new continuous cell line from the pest insect, Anomala cuprea (Coleoptera; Scarabaeidae): emergence of contractile cells.

Insect contractile cells frequently appear at an early phase of cell culture, but in most cases, they disappear before a continuous cell line is established, so the cell line ceases to contract. Continuous contractile insect cell lines are currently available from only one species each of Hymenoptera and Diptera. Here, we obtained a new cell line that contracted long after being established as a continuous cell line. The cell line contracted for a short period at an early phase of insect cell culture before a continuous cell line was established, but then did not contract again for several years. After this cell line entered the continuous growth phase, it produced spontaneously contractile tissues for about 4 mo but stopped contracting again. This is the first instance of a cell line that contracted after its establishment as a non-contractile continuous cell line. It is unclear whether the contractile cells survive or die after contraction ceases at an early phase of cell culture, and our results indicate that potential contractile cells survive for years after they stop to contract. The cells of this line sometimes produced complex contractilestructures, such as sheet-like tissues. Only a few continuous cell lines have been derived from scarabaeid beetles. The new continuous cell line was derived from the culture of the fat bodies of the scarab beetle Anomala cuprea, which is a pest in the agriculture and forestry of Japan. The population doubling time of the new cell line was 2.5 d and thus it grows very rapidly among coleopteran continuous cell lines. Our new cell line will facilitate research on the physiology and pathology of Coleoptera, including scarab beetles, and may also contribute to research on invertebrate muscles.

Functional characterization of piggyBac-like elements from Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

PiggyBac is a transposable DNA element originally discovered in the cabbage looper moth (Trichoplusia ni). The T. ni piggyBac transposon can introduce exogenous fragments into a genome, constructing a transgenic organism. Nevertheless, the comprehensive analysis of endogenous piggyBac-like elements (PLEs) is important before using piggyBac, because they may influence the genetic stability of transgenic lines. Herein, we conducted a genome-wide analysis of PLEs in the brown planthopper (BPH) Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), and identified a total of 28 PLE sequences. All N. lugens piggyBac-like elements (NlPLEs) were present as multiple copies in the genome of BPH. Among the identified NlPLEs, NlPLE25 had the highest copy number and it was distributed on five chromosomes. The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals, as well as a single open reading frame transposase encoding 546 amino acids. Furthermore, NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision. A cross-recognition between the NlPLE25 transposon and the piggyBac transposon was also revealed in this study. These findings provide useful information for the construction of transgenic insect lines.

Effect of sericin, a silk derived protein, on the amplification of Zika virus in insect and mammalian cell cultures

Sericin, a silk-derived non-immunogenic protein, has been used to improve cell culture performance by increasing viability, cell concentration, and promoting adherence of several cell lines. Here, we hypothesized that the properties of sericin can enhance the amplification of flaviviruses in cell cultures. The propagation of flavivirus is inefficient and limits scientific research. Zika virus (ZIKV) is an important human pathogen that has been widely studied because of its high impact on public health. There is a need to amplify Zika virus both for research and vaccine development. In this work, we show that sericin improves ZIKV amplification in insect (C6/36) and mammalian (Vero) cell cultures, and that it has a cryoprotectant capacity. Supplementation of cell culture media with sericin at 80 µg/mL resulted in a significant increase of 1 log in the concentration of ZIKV infectious particles produced from both cell lines. Furthermore, final virus yields increased between 5 and 10-fold in Vero cells and between 7 and 23-fold in C6/36 cells when sericin was supplemented, compared to control conditions. These results show that sericin is an effective supplement to increase ZIKV production by Vero and C6/36 cells. Additionally, sericin was a suitable cryoprotective agent, and hence an alternative to FBS and DMSO, for the cryopreservation of C6/36 cells but not for Vero cells.

Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

The effect of different insect cell culture media on the efficiency of protein production by Spodoptera frugiperda cells

BackgroundSpodoptera frugiperda (Sf-9) cells are often used to produce recombinant proteins by transient expression. The culture medium contributes to the efficiency of protein production, but the effect of modern insect cell culture media on Sf-9 cells has not been evaluated for different transient expression systems. Accordingly, we compared the baculovirus expression vector system (BEVS) and lipopolyfection (LP) for the production of schistosomal ALDH and the antimicrobial peptide BR033, respectively. We used Sf-9 cells adapted to four commercially available media: TriEx ICM, ExpiSf CDM, Sf-900 II SFM and IS Sf Insect ACF. ResultsDuring cell maintenance, the highest viable cell density was achieved using IS Sf Insect ACF, but the shortest doubling time was observed in TriExICM. All four media supported transient expression with standard protocols for LP and BEVS. We confirmed the typical switch in metabolic rates (glucose, lactate and amino acids) after transfection or infection, comparable to previous reports. The maximum protein production rates were 0.26 ± 0.04 amol/(cellh) for LP and 4.63 ± 1.85 amol/(cell h) for BEVS, both achieved using Sf-900 II SFM. ConclusionsThe experiments demonstrate that the most suitable medium for transient production in Sf-9 cells depends on the experimental objectives or production framework. We therefore recommend implementing the screening of different media during process development.How to cite: Käßer L, Harnischfeger J, Salzig D, et al. The effect of different insect cell culture media on the efficiency of protein production by Spodoptera frugiperda cells. Electron J Biotechnol 2022;56. https://doi.org/10.1016/j.ejbt.2022.01.004.

Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells.

With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.

Insect Cells-Baculovirus System for the Production of Difficult to Express Proteins: From Expression Screening for Soluble Constructs to Protein Quality Control.

Rapid preparation of proteins for functional and structural analysis is a major challenge both in academia and industry. The number potential targets continuously increases and many are difficult to express proteins which, when produced in bacteria, result in insoluble and/or misfolded recombinant proteins, protein aggregates, or unusable low protein yield. We focus here on the baculovirus expression vector system which is now commonly used for heterologous production of human targets. This chapter describes simple and cost-effective protocols that enable iterative cycles of construct design, expression screening and optimization of protein production. We detail time- and cost-effective methods for generation of baculoviruses by homologous recombination and titer evaluation. Handling of insect cell cultures and preparation of bacmid for cotransfection are also presented.

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.

Chemical Treatments for Insect Cell Differentiation: The Effects of 20-Hydroxyecdysone and Veratridine on Cultured Spodoptera frugiperda (Sf21) Insect Cell Ultrastructure

Simple SummaryCultured insect Sf21 cells treated with the hormone 20-hydroxyecdysone grow long processes and resemble neurons. They also make physical contact with one another and appear to have the potential to form synapses, areas in which nerve cells are in close contact and communicate with one another electrically or by the release of chemical transmitters. This study uses electron microscopy to look for structural evidence of synapses in 20-hydroxyexdysone treated Sf21 cell cultures. Unfortunately, no evidence of synaptic structures were observed, suggesting that other factors are required for the formation of functional synapses in these cultures.Previous studies have shown that insect cell cultures stop dividing, form clumps, and can be induced to grow processes reminiscent of axons, when the culture medium is supplemented with 20-hydroxyecdysone, insulin, or an agent that mimics their action, such as the ecdysone agonist, methoxyfenozide. Those cell growing processes resemble nerve cells, and the present study evaluates the ultrastructure of these cultures by transmission electron microscopy. Sf21 cells treated with 20-hydroxyecdysone (with or without veratridine amendment) and subjected to ultrastructural analysis had a similar somatic appearance to control cells, with slight changes in organelles and organization, such as a greater number of cytoplasmic vacuoles and mitochondrial granules. Finger-like projections were observed between control and treated cells. However, no structural markers of synaptic contacts (e.g., vesicles or synaptic thickenings) were observed in controls, 20-hydroxyecdysone, or 20-hydroxyecdysone + veratridine treated cells. It is concluded that additional agents would be required to induce functional synaptogenesis in Sf21 cells.

Fluid dynamics of a pilot‐scale OrbShake bioreactor under different operating conditions

AbstractBACKGROUNDFor several years, orbitally shaken cylindrical bioreactors (OSRs) have become a new approach for propagation of mammalian cells cultivated under suspension. With new commercial cell culture media available today, allowing exceptionally high cell densities, the identification of suitable operating conditions presents a special challenge. Balancing hydrodynamic stress and other factors in reactors, this is particularly true for gas transfer rates providing sufficient oxygen (O2) to cultures that can attain ten‐fold greater biomasses than just a decade ago. This challenge is more profound the larger the working volume of the reactor becomes.RESULTFor 10‐L working volume cylindrical OSRs, we established a computational fluid dynamics model to evaluate the impacts of different operational conditions. Both fluid velocity distributions and O2 transfer coefficients (kLa) were calculated for different filling volumes and shaking speeds. Also, hydrodynamic stress parameters were investigated considering the most critical working conditions.CONCLUSIONThe fluid velocity decreased with larger working volumes and, as expected, was found to increase with higher shaking frequency. Gas transfer rates between 10 and 54 h−1 were found in working volumes from 2.5 to 10 L and under shaking frequencies from 100–160 rpm. Critically assessing the obtained data, with no liquid‐submerged gas supply, we conclude that an air flow into the head‐space of a disposable bag appears to be satisfactory under certain operating conditions to support the culture of the most sensitive mammalian or insect cell cultures towards high cell density, presently in fed‐batch cultures achieving ≤30 × 106 cells mL–1. Shear stress parameters were found to be exceptionally low. © 2021 Society of Chemical Industry (SCI).

Plant-derived VLP: a worthy platform to produce vaccine against SARS-CoV-2.

After its emergence in late 2019 SARS-CoV-2 was declared a pandemic by the World Health Organization on 11 March 2020 and has claimed more than 2.8 million lives. There has been a massive global effort to develop vaccines against SARS-CoV-2 and the rapid and low cost production of large quantities of vaccine is urgently needed to ensure adequate supply to both developed and developing countries. Virus-like particles (VLPs) are composed of viral antigens that self-assemble into structures that mimic the structure of native viruses but lack the viral genome. Thus they are not only a safer alternative to attenuated or inactivated vaccines but are also able to induce potent cellular and humoral immune responses and can be manufactured recombinantly in expression systems that do not require viral replication. VLPs have successfully been produced in bacteria, yeast, insect and mammalian cell cultures, each production platform with its own advantages and limitations. Plants offer a number of advantages in one production platform, including proper eukaryotic protein modification and assembly, increased safety, low cost, high scalability as well as rapid production speed, a critical factor needed to control outbreaks of potential pandemics. Plant-based VLP-based viral vaccines currently in clinical trials include, amongst others, Hepatitis B virus, Influenza virus and SARS-CoV-2 vaccines. Here we discuss the importance of plants as a next generation expression system for the fast, scalable and low cost production of VLP-based vaccines.

Ambient Temperature Stable, Scalable COVID-19 Polymer Particle Vaccines Induce Protective Immunity.

There is an unmet need for safe and effective severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccines that are stable and can be cost‐effectively produced at large scale. Here, a biopolymer particle (BP) vaccine technology that can be quickly adapted to new and emerging variants of SARS‐CoV‐2 is used. Coronavirus antigen‐coated BPs are described as vaccines against SARS‐CoV‐2. The spike protein subunit S1 or epitopes from S and M proteins (SM) plus/minus the nucleocapsid protein (N) are selected as antigens to either coat BPs during assembly inside engineered Escherichia coli or BPs are engineered to specifically ligate glycosylated spike protein (S1‐ICC) produced by using baculovirus expression in insect cell culture (ICC). BP vaccines are safe and immunogenic in mice. BP vaccines, SM‐BP‐N and S1‐ICC‐BP induced protective immunity in the hamster SARS‐CoV‐2 infection model as shown by reduction of virus titers up to viral clearance in lungs post infection. The BP platform offers the possibility for rapid design and cost‐effective large‐scale manufacture of ambient temperature stable and globally available vaccines to combat the coronavirus disease 2019 (COVID‐19) pandemic.

Novel constructs and 1-step chromatography protocols for the production of Porcine Circovirus 2d (PCV2d) and Circovirus 3 (PCV3) subunit vaccine candidates

Porcine circovirus type 2 (PCV2) has been a major problem for the pig production industry worldwide for decades. While the majority of commercially available vaccines are based on the original PCV2a genotype, the current dominant genotype is PCV2d. The notable differences between genotypes could lead to incomplete cross-protection. Moreover, most current subunit PCV2 vaccines are generated from expensive insect cell culture technology. In this work, we present a new workflow for production of an updated and relatively inexpensive PCV2d vaccine candidate. After expression in fed-batch Escherichia coli fermentation systems with a simple one-step ion-exchange chromatography purification protocol, the yield of purified PCV2d-based antigen reached over 1 g per litre bacterial culture. Using similar procedures, we also demonstrated even higher PCV2d-based antigen yields from a chimeric PCV2d-PCV3 capsid construct, which is cleaved during fermentation to release PCV2d- and PCV3-related polypeptides. Although the PCV2d-based recombinant protein from this protocol did not form viral-like particles as analysed by size-exclusion chromatography, it could effectively induce capsid-specific and PCV2d-neutralising antibodies in immunised animals, indicating significant potential as a new vaccine candidate that can be easily manufactured at commercial scale.

Molecular and Functional Characterization of Pyrokinin-Like Peptides in the Western Tarnished Plant Bug Lygus hesperus (Hemiptera: Miridae).

Simple SummaryNeuropeptides regulate most insect biological functions. One such group of peptides, the pyrokinins (PKs), are distinguished by a C-terminal FXPRLamide. While widely distributed in most insects, they are poorly characterized in plant bugs. To address this limitation, we identified the PK transcript in the western tarnished plant bug (Lygus hesperus) and examined its expression. The Lygus PK transcript is predicted to yield three PK-like peptides but only two (LyghePKa and LyghePKb) have the characteristic C-terminal amide. The transcript is expressed throughout development and is most abundant in heads. A custom FXPRLamide antibody revealed immunoreactive cells throughout the Lygus central nervous system consistent with typical neuropeptide expression. To assess potential functional roles of the peptides, a fluorescence-based Ca2+ influx assay using cultured insect cells stably expressing a moth PK receptor was performed. LyghePKa was unable to stimulate receptor activation, whereas LyghePKb triggered a robust response. The in vivo pheromonotropic activity of the two peptides was likewise assessed using three different moth species. Like the cell culture system, only the LyghePKb peptide was active. The study suggests evolutionary divergence of the PK gene in plant bugs and provides critical insights into likely biological functions in the western tarnished plant bug.The pyrokinin (PK) family of insect neuropeptides, characterized by C termini consisting of either WFGPRLamide (i.e., PK1) or FXPRLamide (i.e., PK2), are encoded on the capa and pk genes. Although implicated in diverse biological functions, characterization of PKs in hemipteran pests has been largely limited to genomic, transcriptomic, and/or peptidomic datasets. The Lygus hesperus (western tarnished plant bug) PK transcript encodes a prepropeptide predicted to yield three PK2 FXPRLamide-like peptides with C-terminal sequences characterized by FQPRSamide (LyghePKa), FAPRLamide (LyghePKb), and a non-amidated YSPRF. The transcript is expressed throughout L. hesperus development with greatest abundance in adult heads. PRXamide-like immunoreactivity, which recognizes both pk- and capa-derived peptides, is localized to cells in the cerebral ganglia, gnathal ganglia/suboesophageal ganglion, thoracic ganglia, and abdominal ganglia. Immunoreactivity in the abdominal ganglia is largely consistent with capa-derived peptide expression, whereas the atypical fourth pair of immunoreactive cells may reflect pk-based expression. In vitro activation of a PK receptor heterologously expressed in cultured insect cells was only observed in response to LyghePKb, while no effects were observed with LyghePKa. Similarly, in vivo pheromonotropic effects were only observed following LyghePKb injections. Comparison of PK2 prepropeptides from multiple hemipterans suggests mirid-specific diversification of the pk gene.

OdTx12/GNA, a chimeric variant of a β excitatory toxin from Odontobuthus doriae, reveals oral toxicity towards Locusta migratoria and Tenebrio molitor

OdTx12, a beta excitatory toxin from Odontobothus doriae had previously been identified and characterized. It had been shown that OdTx12 causes significant lethal effects on insects by injection but does not show any toxicity on mice. Due to the natural ineffectiveness of scorpion toxins to act as oral toxins, OdTx12 was fused to Galanthus nivalis agglutinin (GNA), a protein with the potential to cross the insect gut. The sequence of OdTx12/GNA gene was chemically synthesized, cloned in Escherichia coli, and expressed. The effect of the purified fusion protein (OdTx12/GNA) was assessed on the insect and mammalian cell lines, insect larvae and mice. Toxicity assay on insect cell culture (SF9 cell line) showed comparable toxicity between OdTx12 and OdTx12/GNA (LD50 of 0.0030 and 0.0048 μM, respectively). Also very similar mortality rates were observed by injecting OdTx12 and OdTx12/GNA to Locusta migratoria and Tenebrio molitor. Oral administration of OdTx12/GNA, after five days of feeding, resulted in 96.6% and 98.3% mortality of L. migratoria and T. molitor larvae with an LC50 of 0.69 and 0.43 nmol/g of insect food, respectively, while OdTx12 alone did not cause any toxic effects on the larvae orally, suggesting the role of GNA in delivering the toxin to the insect's haemolymph. No toxicity or mortality was observed after toxicity testing of OdTx12/GNA on a mammalian cell line (HEP-2) or any mortality in vivo, by testing the protein in the laboratory mouse.Herein, we demonstrated that the fusion protein OdTx12/GNA could be considered an effective toxin for the biological control of insects.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.