Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.

Novel bridge multi-species ELISA for detection of SARS-CoV-2 antibodies.

Considering the course of the current SARS-CoV-2 pandemic, it is important to have serological tests for monitoring humoral immune response against SARS-CoV-2 infection and vaccination. Herein we describe a novel bridge enzyme-linked immunosorbent assay (b-ELISA) for SARS-CoV-2 antibodies detection in human and other species, employing recombinant Spike protein as a unique antigen, which is produced at high scale in insect larvae. METHODS: Eighty two human control sera/plasmas and 169 COVID-19 patients' sera/plasmas, confirmed by rRT-PCR, were analyzed by the b-ELISA assay. In addition, a total of 27 animal sera (5 horses, 13 rats, 2 cats and 7 dogs) were employed in order to evaluate the b-ELISA in other animal species. RESULTS: Out of the 169 patient samples, 129 were positive for IgG anti-SARS-CoV-2 and 40 were negative when they were tested by ELISA COVIDAR® IgG. When a cut-off value of 5.0 SDs was established, 124 out of the 129 COVID-19 positive samples were also positive by our developed b-ELISA (sensitivity: 96.12%). Moreover, the test was able to evaluate the humoral immune response in animal models and also detected as positive a naturally infected cat and two dogs with symptoms, whose owners had suffered the COVID-19 disease. CONCLUSION: The obtained results demonstrate that the method developed herein is versatile, as it is able to detect antibodies against SARS-CoV-2 in different animal species without the need to perform and optimize a new assay for each species.

Rapid and cost-effective process based on insect larvae for scale-up production of SARS-COV-2 spike protein for serological COVID-19 testing.

Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this study, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.

Supramolecular effect of acetate on chitin gelling medium: Structural properties and protein interaction.

In this work, the influence of Sodium Acetate Trihydrate (SAT) on the gelling stage of a chitin hydrogel was studied. Characterization techniques, such as FTIR, Raman, solid-state NMR, Dielectric Spectroscopy, Small-angle X-ray scattering (SAXS), Wide-angle X-ray scattering (WAXS), and X-ray diffraction (XRD) were used to study the effect of SAT on the micro and nanostructure of the material in the wet, dry and freeze-dried states. It was demonstrated that the amount of SAT in the gelling solution can induce a variation in the supramolecular interaction among the polysaccharide chains, which leads to a change in the structural characteristics. In addition, it was observed that the polymer-water interactions are also altered by this structural ordering. Also, the affinity interaction with lysozyme was evaluated and an influence on the adsorption capacity was evidenced with the use of SAT. This could be an advance for biotechnological, biomedical, and food applications.

Single-step purification of equine chorionic gonadotrophin directly from plasma using affinity chromatography.

Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficient affinity chromatographic matrix for eCG purification directly from plasma was developed. The matrix consisted of chitosan mini-spheres with immobilized wheat germ agglutinin (WGA). The matrix allowed 98% adsorption of eCG directly from plasma without any pre-treatment with an overall yield of around 60%. The matrix chosen was able to maintain the efficient performance of the purification process for three consecutive cycles. Also, the process was scaled-up 500 times in volume and tested over seven consecutive cycles maintaining its chromatographic performance. The results presented here suggest the potential application of this matrix to one-step purification of eCG from plasma.

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus.

Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNß) was expressed in insect larvae that constitute important agronomic plagues. rFeIFNß accumulated in the hemolymph of Spodoptera frugiperda larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNß was recovered after purification with a specific activity of 1 × 106 IU mg-1. By this method, we obtained 8.9 × 104 IU of purified rFeIFNß per larva. The product was biologically active in vitro, with an antiviral activity of 9.5 × 104 IU mL-1, as well as a potent antitumor activity comparable to that of the commercial FeIFNω. The glycosylation of rFeIFNß was confirmed by peptide-N-glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFNß production in laboratory research or veterinary medicine applications.

Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligand.

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase.

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.