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1.
Viruses ; 15(1)2022 Dec 28.
Article in English | MEDLINE | ID: covidwho-2234402

ABSTRACT

Baculoviruses are enveloped, insect-specific viruses with large double-stranded DNA genomes. Among all the baculovirus species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied. Due to its characteristics regarding biosafety, narrow host range and the availability of different platforms for modifying its genome, AcMNPV has become a powerful biotechnological tool. In this review, we will address the most widespread technological applications of baculoviruses. We will begin by summarizing their natural cycle both in larvae and in cell culture and how it can be exploited. Secondly, we will explore the different baculovirus-based protein expression systems (BEVS) and their multiple applications in the pharmaceutical and biotechnological industry. We will focus particularly on the production of vaccines, many of which are either currently commercialized or in advanced stages of development (e.g., Novavax, COVID-19 vaccine). In addition, recombinant baculoviruses can be used as efficient gene transduction and protein expression vectors in vertebrate cells (e.g., BacMam). Finally, we will extensively describe various gene therapy strategies based on baculoviruses applied to the treatment of different diseases. The main objective of this work is to provide an extensive up-to-date summary of the different biotechnological applications of baculoviruses, emphasizing the genetic modification strategies used in each field.


Subject(s)
COVID-19 , Nucleopolyhedroviruses , Animals , Humans , Baculoviridae/genetics , COVID-19 Vaccines/metabolism , Nucleopolyhedroviruses/genetics , Spodoptera
2.
J Biosci Bioeng ; 134(5): 432-440, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2041897

ABSTRACT

The production of recombinant proteins using insect cells has been widely used for over 30 years, which contributing to life science research and biotechnology. Insect cells exhibiting enhanced N-glycosylation and recombinant protein productivity enhance the productivity of the baculovirus-insect cell system (BICS). A new highly proliferative insect cell strain, 2g2, was established from the Mamestra brassicae pupa ovary cell strain NIAS-MB-32 (RCB0413) to address the problem of Sf-rhabdovirus and to explore the newly available possibilities in BICS as well as Sf9, such as increased protein production and recombinant baculovirus amplification. The high-growth cell strain 2g2 was examined for its recombinant protein production ability and baculovirus productivity; moreover, the activity of the produced recombinant proteins was examined using Sf9 as a benchmark. Recombinant protein productivity and virus production by BICS in 2g2 was confirmed as equivalent to that of Sf9. Furthermore, we produced the severe acute respiratory syndrome coronavirus 2 spike protein in a baculovirus-free system and compared its productivity, binding activity with human angiotensin-converting enzyme 2, and N-glycosylation. The productivity and bioactivity were found to be equal to or better than that of Sf9. Moreover, N-glycosylation analysis revealed that the glycans derived from the 2g2-produced glycoproteins were mostly of the high mannose type as Sf9. Therefore, 2g2 may have the same N-glycosylation ability as Sf9. Finally, the Sf-rhabdovirus was confirmed to be negative in 2g2. Our results demonstrated that the novel insect cell strain 2g2 can serve as a protein production tool in scientific research and industrial biotechnology.


Subject(s)
Baculoviridae , COVID-19 , Animals , Humans , Baculoviridae/genetics , Baculoviridae/metabolism , Recombinant Proteins/metabolism , Insecta , Spodoptera/metabolism
3.
Front Immunol ; 12: 771011, 2021.
Article in English | MEDLINE | ID: covidwho-1639016

ABSTRACT

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is an ongoing pandemic. Detection and vaccination are essential for disease control, but they are distinct and complex operations that require significant improvements. Here, we developed an integrated detection and vaccination system to greatly simplify these efforts. We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based system to accurately detect patient serum. Notably, although well-recognized by our newly developed detection system in which S-displaying insect cells acted as antigen, anti-S antibodies from many patients were barely detectable by Western blot, evidencing that COVID-19 patients primarily produce conformation-dependent anti-S antibodies. Furthermore, the same baculovirus constructs can display N (N-Bac) or S (S-Bac) on the baculovirus envelope and serve as vector vaccines. Animal experiments show that S-Bac or N-Bac immunization in mice elicited a strong and specific antibody response, and S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination.


Subject(s)
Baculoviridae/immunology , COVID-19 Vaccines/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Aged, 80 and over , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Baculoviridae/genetics , COVID-19/immunology , COVID-19/prevention & control , Cell Line , Cell Surface Display Techniques , Coronavirus Nucleocapsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Phosphoproteins/genetics , Phosphoproteins/immunology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spodoptera , Vaccination , Young Adult
4.
PLoS One ; 16(11): e0257736, 2021.
Article in English | MEDLINE | ID: covidwho-1511815

ABSTRACT

Since 2016, fall armyworm (FAW) has threatened sub-Saharan 'Africa's fragile food systems and economic performance. Yet, there is limited evidence on this transboundary pest's economic and food security impacts in the region. Additionally, the health and environmental consequences of the insecticides being used to control FAW have not been studied. This paper presents evidence on the impacts of FAW on maize production, food security, and human and environmental health. We use a combination of an agroecology-based community survey and nationally representative data from an agricultural household survey to achieve our objectives. The results indicate that the pest causes an average annual loss of 36% in maize production, reducing 0.67 million tonnes of maize (0.225 million tonnes per year) between 2017 and 2019. The total economic loss is US$ 200 million, or 0.08% of the gross domestic product. The lost production could have met the per capita maize consumption of 4 million people. We also find that insecticides to control FAW have more significant toxic effects on the environment than on humans. This paper highlights governments and development partners need to invest in sustainable FAW control strategies to reduce maize production loss, improve food security, and protect human and environmental health.


Subject(s)
Agriculture/economics , Insecticide Resistance/genetics , Insecticides/pharmacology , Spodoptera/pathogenicity , Africa, Northern , Animals , Ethiopia , Humans , Insecticides/economics , Larva/genetics , Larva/parasitology , Socioeconomic Factors , Spodoptera/drug effects , Zea mays/growth & development , Zea mays/parasitology
5.
Nat Commun ; 12(1): 6103, 2021 10 20.
Article in English | MEDLINE | ID: covidwho-1475296

ABSTRACT

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/isolation & purification , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Cell Line, Tumor , Crystallography, X-Ray , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/ultrastructure , Spodoptera , Surface Plasmon Resonance , Virus Attachment , Virus Internalization
6.
Sci Rep ; 10(1): 21393, 2020 12 07.
Article in English | MEDLINE | ID: covidwho-1387456

ABSTRACT

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/biosynthesis , Antigens, Viral/biosynthesis , Cloning, Molecular , Recombinant Proteins/biosynthesis , SARS-CoV-2/immunology , Animals , HEK293 Cells , Humans , Protein Stability , Spodoptera
7.
Biotechnol Bioeng ; 118(10): 4129-4137, 2021 10.
Article in English | MEDLINE | ID: covidwho-1310445

ABSTRACT

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.


Subject(s)
COVID-19 Serological Testing , COVID-19/diagnosis , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/biosynthesis , Animals , Larva/metabolism , Larva/virology , Nucleopolyhedroviruses , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , SARS-CoV-2/metabolism , Sf9 Cells , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spodoptera
8.
J Insect Physiol ; 132: 104248, 2021 07.
Article in English | MEDLINE | ID: covidwho-1303601

ABSTRACT

Since 2016, the fall armyworm (FAW, Spodoptera frugiperda) has invaded large parts of Africa and Asia, impacting millions of hectares of maize crops and thereby posing a major threat to food security. The rapid geographic spread and outbreak dynamics of S. frugiperda are tied to its unique dispersal ability and long-distance migration capability. Yet, up till present, limited research has been conducted on the physiological determinants of S. frugiperda flight and migration. In this study, we used laboratory experiments to assess whether mating and oviposition affect S. frugiperda flight ability and wingbeat frequency. During 2019-2020, migratory FAW females were trapped in Yunnan (China) and dissected to assess ovarian development. Tethered flight assays showed that gravid S. frugiperda females exhibited strong flight ability at 1-3 days following the onset of oviposition. Flight distance and duration negatively correlated with the number of deposited eggs. Ovarian dissections further showed that over 50% of migrant females were mated and 46-54% had initiated oviposition. Our study shows the complex, yet nuanced effects of reproductive status on flight capacity, with possibly a facultative trade-off between flight and reproduction. These novel insights into S. frugiperda physiology and migration behavior can guide future monitoring and integrated pest management (IPM) programs against this newly-invasive pest in China and abroad.


Subject(s)
Flight, Animal/physiology , Reproduction/physiology , Spodoptera/physiology , Animal Distribution/physiology , Animals , Female , Oviposition , Pest Control
9.
Sci Adv ; 7(22)2021 05.
Article in English | MEDLINE | ID: covidwho-1247308

ABSTRACT

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/immunology , Recombinant Fusion Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Tetanus Toxoid/immunology , Animals , COVID-19/genetics , COVID-19/immunology , COVID-19 Vaccines/genetics , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/genetics , Female , Macaca fascicularis , Mice , Mice, Inbred BALB C , Mice, Transgenic , Phosphoproteins/genetics , Phosphoproteins/immunology , Protein Domains , Rats , Recombinant Fusion Proteins/genetics , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spodoptera , Tetanus Toxoid/genetics , Vero Cells
10.
Viruses ; 12(9)2020 09 22.
Article in English | MEDLINE | ID: covidwho-973229

ABSTRACT

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.


Subject(s)
Murine hepatitis virus/chemistry , Peptides/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Animals , Cell Line , Coronavirus Infections , Humans , Intracellular Membranes/metabolism , Mice , Murine hepatitis virus/genetics , Murine hepatitis virus/metabolism , Mutation , Peptides/chemical synthesis , Peptides/metabolism , Sf9 Cells , Spodoptera , Unilamellar Liposomes/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism
11.
Molecules ; 25(22)2020 Nov 18.
Article in English | MEDLINE | ID: covidwho-934509

ABSTRACT

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.


Subject(s)
Angiotensin-Converting Enzyme 2/isolation & purification , Dipeptidyl Peptidase 4/isolation & purification , Spike Glycoprotein, Coronavirus/isolation & purification , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Cloning, Molecular , Dipeptidyl Peptidase 4/biosynthesis , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Humans , Kinetics , Models, Molecular , Plasmids/chemistry , Plasmids/metabolism , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sf9 Cells , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spodoptera , Surface Plasmon Resonance
12.
Emerg Microbes Infect ; 9(1): 2076-2090, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-913103

ABSTRACT

The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 µg) with the inclusion of an aluminium adjuvant. Higher doses (20 µg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.


Subject(s)
Antigens, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Immunogenicity, Vaccine/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/immunology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19 , COVID-19 Vaccines , Cell Line , Coronavirus Infections/immunology , Cryoelectron Microscopy , Enzyme-Linked Immunosorbent Assay , Humans , Macaca fascicularis , Mice , Mice, Inbred BALB C , Neutralization Tests , Peptidyl-Dipeptidase A/metabolism , Protein Domains/genetics , Protein Domains/immunology , SARS-CoV-2 , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spodoptera , Vaccination , Viral Envelope Proteins/immunology
13.
Anal Chem ; 92(21): 14730-14739, 2020 11 03.
Article in English | MEDLINE | ID: covidwho-872627

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.


Subject(s)
Betacoronavirus/chemistry , Polysaccharides/analysis , Spike Glycoprotein, Coronavirus/chemistry , Animals , Glycosylation , HEK293 Cells , Humans , Mass Spectrometry/methods , Polysaccharides/chemistry , SARS-CoV-2 , Spodoptera/chemistry
14.
Cell ; 182(3): 722-733.e11, 2020 08 06.
Article in English | MEDLINE | ID: covidwho-628738

ABSTRACT

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Severe acute respiratory syndrome-related coronavirus/immunology , Universal Design , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/chemistry , COVID-19 , COVID-19 Vaccines , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Infections/virology , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Middle East Respiratory Syndrome Coronavirus/chemistry , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Binding , Protein Interaction Domains and Motifs/immunology , Receptors, Virus/metabolism , Severe acute respiratory syndrome-related coronavirus/chemistry , SARS-CoV-2 , Sf9 Cells , Specific Pathogen-Free Organisms , Spodoptera , Transfection , Vaccination/methods , Vero Cells , Viral Vaccines
15.
J Biol Chem ; 295(20): 6785-6797, 2020 05 15.
Article in English | MEDLINE | ID: covidwho-52576

ABSTRACT

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2'-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Betacoronavirus/enzymology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Virus Replication/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Betacoronavirus/physiology , Models, Molecular , SARS-CoV-2 , Sf9 Cells , Spodoptera
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