Scalable approach for coronavirus-like particles making based on the spike protein using Sf9 cells.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

Background: Hepatitis E virus (HEV) is able to induce fulminant hepatitis E infection in immunosuppressed individuals. Previous studies showed that the Baculovirus expression system (BES) could be used for developing hepatitis E virus-like particles (VLPs). Objectives: In the present study, the formation of VLPs of the recombinant proteins of HEV truncated open reading frame 2 (ORF2; 112 - 607) and Rotavirus non-structural protein 4 (NSP4) (OSU-a) were investigated in BES and Escherichia. coli. Methods: To construct VLPs, the truncated ORF2-NSP4 protein was expressed in BES. The expression of protein was confirmed by western blot. This protein was expressed in E. coli. The truncated ORF2-NSP4 gene was subcloned in pET28a, and then transformed into E. coli DH5α. The confirmed colonies were transformed into E. coli BL21. The solubility of protein were checked by SDS-PAGE and western blot analysis. In the final step, to verify the VLP formation in BES and E. coli, the recombinant proteins were stained with 2% uranyl acetate and checked by transmission electron microscopy (TEM). Results: The 75 KDa truncated ORF2-NSP4 protein was successfully expressed in Sf9 cells, assembled, and formed VLP according to TEM results. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully subcloned and expressed in BL21 but VLP was not detected in TEM analysis. Conclusions: The truncated ORF2-NSP4 VLP were efficiently expressed in the SF9 cells, as a potential mucosal vaccine against HEV and Rotavirus. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully expressed.

BackgroundHuman metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined.MethodsThe HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis.ResultsAnalysis of cells co-expressing the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction.ConclusionCo-expression of the F, G and M proteins leads to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

In the light of the COVID-19 pandemic, an elaborative computational analysis was conducted regarding coronaviruses, their phylogeny, and the different strains of the pathogen responsible for the disease, SARS-CoV-2. In the front, this disease looks like the common cold. However, it can lead to acute respiratory failure, septic shock, and organ failure. Like the Spanish flu pandemic in 1918, the COVID-19 pandemic also became the cause of millions of lives lost across the globe. Coronaviruses consist of four basic proteins - spike, nucleocapsid, membrane, and envelope proteins. Spike proteins (S) are structures protruding from the surface of the virus and facilitate its entry into the the host cells via interaction with the ACE-2 receptors who are also found on the surface of the host cells. For this research work, the entire sequence of the spike protein was considered, and a table was created carrying out the comparison between the spike protein sequences of the WT and 13 different variants (Alpha, Beta, Gamma, Kappa, Delta, Mu, Epsilon, Lambda, Omicron, Mu, Eta, Zeta, and Theta). This table helped detect the conserved parts of spike protein sequence across all variants, conserved mutations as well as mutations unique to certain variants. Findings from the table are then used to study phylogenetic trees which explain the emergence of new coronavirus variants and their genetic distances from the WT.

COVID-19 is an infectious disease resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged as a global pandemic in 2020. Virus-like particles (VLP) are artificially generated nanoparticles composed of a subset of viral components that closely mimic the native virus in size, structure, and surface composition. These features contribute to their robust immunogenic potential, stimulating both antibody-mediated and cell-mediated immune responses. This study aimed to evaluate the expression of SARS-CoV-2 structural proteins and the VLP production using a baculovirus–insect cell system. Two monocistronic recombinant baculoviruses, each carrying either the spike (S) or nucleocapsid (N) gene of SARS-CoV-2, were employed in two different culture systems: Schott flasks and a stirred-tank bioreactor. The Sf9 cells showed substantial differences between the Schott flask and stirred tank bioreactor in terms of metabolism, virus titer, and cell death after infection. Both proteins’ expressions were confirmed in the two culture systems assessed. However, VLP formation (69.1–78.2 nm) was only confirmed in the co-infection setup with both monocistronic recombinant baculoviruses. In contrast, infection with only the S-protein baculovirus failed to produce VLP in our findings.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped, single-stranded RNA virus, is causative of coronavirus disease 2019 (COVID-19). The viral entry process is facilitated by the highly antigenic spike protein of SARS-CoV-2, which binds to the angiotensin-converting enzyme 2 (ACE2) receptor. However, research involving live SARS-CoV-2 is limited to biosafety level (BSL)-3 facilities, hindering the progress in vaccine and therapeutic development. Pseudotyped viruses have emerged as valuable tools for studying entry inhibitors, fusion inhibitors, and neutralizing assays in BSL-2 facilities. Several modifications to the spike protein have been identified to enhance the yield of pseudotyped viruses. Notably, the removal of the last 19 amino acids from the spike protein and the D614G modification have been shown to increase pseudovirus titers. In this study, we aimed to identify an effective expression vector for pseudotyping by inserting SARS-CoV-2 S Δ19 into the intron-containing pcDNA3.1 vector. The inclusion of the β-globin intron sequence led to a significant augmentation in spike protein expression and a threefold improvement in pseudotyped virus production. Furthermore, we examined whether the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) could enhance spike protein expression by subcloning the spike protein gene into a lentiviral vector containing WPRE. Our results demonstrated that vectors containing the WPRE exhibited increased spike protein expression compared to vectors lacking the WPRE in HEK293-hACE2 cells. This study demonstrates that the inclusion of a β-globin intron and WPRE enhances the production of SARS-CoV-2 spike protein pseudotyped lentiviruses, a valuable tool for COVID-19 research.

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-Erns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 106 TCID50 virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.

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GIP005 Pharmacological study of the effect of Metronidazol

Coronavirus Disease 19 (COVID-19) emergence reveals globally a great health issue and due to the limited information and knowledge on the origin of this novel coronavirus 2019 (2019-nCoV). Therefore, this study aims to investigate the evolution and analysis of molecular epidemiology for both Spike and Envelope proteins of 20 available complete genome sequences of different bat coronaviruses including 2019-nCoV in order to find out which type of bat coronaviruses is more likely to be the origin of this new 2019-nCoV and also multiple amino acid sequences of Envelope protein for all bat coronaviruses were aligned for the purpose of finding the greater probability of novel 2019-nCoV original host among bat coronaviruses. Phylogenetic tree analysis for Spike protein revealed that all 2019-nCoV related coronaviruses isolated from these species of species are discovered in China and Hong Kong and the Middle East bat are less likely to contribute in spreading or to become the origin of 2019-nCoV and all coronaviruses that from Hong Kong and China are located into one clade next to the clade that contains 2019-nCoV coronaviruses which indicates that this group of coronaviruses are genetically different for 2019-nCoV; moreover, Hong Kong and USA bat coronaviruses does not contain the bat coronavirus from China and are located into one clade far from the clade that contains 2019-nCoV indicates that all coronaviruses are genetically very different from 2019-nCoV, and USA bat coronavirus may has no role in generating of 2019-nCoV. The phylogenetic trees analysis of Envelope protein showed that Envelope protein of different coronaviruses are more similar in comparison to Spike protein, USA bat coronavirus has a relatively closeness relationship to 2019-nCoV. Furthermore, Envelope protein alignment showed the closely related amino acid sequence which confirms that the outcomes of phylogenetic tree analysis in which that these bat coronaviruses have genetically close relationship together and more interestingly amino acid sequence (MG772934.1) shows 100% identity with the amino acid sequence of 2019-nCoV (NC 045512.2) and the same virus has a close relationship in both Spike and Envelope due to that in both phylogenetic tree analysis are neighbored with 2019-nCoV in the same clade.

Secretion of noninfectious dengue virus-like particles and identification of amino acids in the stem region involved in intracellular retention of envelope protein

Hepatitis C virus: The role of N-glycosylation sites of viral genotype 1b proteins for formation of viral particles in insect and mammalian cells

Neuraminidase (NA) is a second major surface protein of the influenza virus and has recently been suggested as a supplemental antigen to the major immunodominant hemagglutinin (HA) antigen in the influenza vaccine. NA is less affected by antigenic drift compared to the HA, induces strong anti-neuraminidase immune responses, and provides broader protection against many influenza strains. However, the NA amount in currently licensed influenza virus vaccines is much lower than that of HA, and not standardized. A platform to produce NA antigen, in the form of virus-like particles (VLPs), was thus developed, to facilitate supplementation of NA antigen in the influenza vaccine formula. Stably transformed Sf9 insect cells had been engineered to express the influenza A virus (H5N1) NA gene under a baculovirus OpMNPV IE2 promoter. Recombinant NA protein was synthesized and assembled into VLPs, in the intact cellular environment provided by insect cells. Approximately 150 µg/ml of NA-VLPs was obtained in the culture medium. Purification of the NA-VLPs was achieved by a sucrose density gradient ultracentrifugation. The purified NA-VLPs effectively induced anti-NA antibodies with neuraminidase inhibition activities in mice. This work demonstrates a simple process to produce an immunocompetent NA-VLPs antigen, exclusively made of only neuraminidase, by insect cells.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12033-022-00519-8.