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1.
Curr Microbiol ; 79(6): 167, 2022 Apr 23.
Article in English | MEDLINE | ID: covidwho-1802672

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread all over the world and became a pandemic that named coronavirus disease-2019 (COVID-19). At present, several intramuscular vaccines have been successfully developed and mass vaccination has progressed in many countries. The aim of the study is to develop and examine an oral vaccine against COVID-19 with recombinant Lactococcus lactis IL1403, a strain of lactic acid bacteria, expressing SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) S1 subunit as an immunizing antigen. PBS or cell extracts from recombinant L. lactis were orally administered into mice (control VS treatment), and formation of antigen-specific antibodies and changes in the gut microbiome were analyzed. Intracellular antigen was detected, but its secretion was not successful. After immunization, antigen-specific serum IgG and fecal IgA levels were 1.5-fold (P = 0.002) and 1.4-fold (P = 0.016) higher in the immunized mice (treatment) than control, respectively. Gut microbiome profiles were clearly separated between the two groups when analyzed for beta diversity with overall similarity. At the genus level, while Coprococcus (P = 0.036) and unclassified genus of Ruminococcaceae (P = 0.037) in treatment were more abundant than control, rc4-4 (P = 0.013) and Stenotrophomonas (P = 0.021) were less abundant. Our results indicate that cell extract containing SARS-CoV-2 antigen can induce mice to produce antigen-specific antibodies without overall changes in the gut microbiome. This strategy may be useful for the development of other oral viral vaccines.


Subject(s)
COVID-19 Vaccines , COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , Cell Extracts , Humans , Immunization , Lactococcus lactis/genetics , Mice , Mice, Inbred BALB C , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
2.
Int J Biol Macromol ; 209(Pt A): 984-990, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1796725

ABSTRACT

MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.


Subject(s)
Coronavirus 3C Proteases , Middle East Respiratory Syndrome Coronavirus , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Middle East Respiratory Syndrome Coronavirus/enzymology , Middle East Respiratory Syndrome Coronavirus/genetics , Protein Conformation , Recombinant Proteins
3.
Biochemistry (Mosc) ; 87(4): 319-330, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1784775

ABSTRACT

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , Disulfides , Epitopes , Humans , Immunization , Mice , Recombinant Proteins/genetics , SARS-CoV-2
4.
J Clin Lab Anal ; 36(5): e24328, 2022 May.
Article in English | MEDLINE | ID: covidwho-1763246

ABSTRACT

OBJECTIVES: The spike protein has been reported as one of the most critical targets for vaccine design strategies against the SARS-CoV-2 infection. Hence, we have designed, produced, and evaluated the potential use of three truncated recombinant proteins derived from spike protein as vaccine candidates capable of neutralizing SARS-CoV-2 virus. METHODS: In silico tools were used to design spike-based subunit recombinant proteins (RBD (P1 ), fusion peptide (P2 ), and S1/S2 cleavage site (P3 )). These proteins were checked for their ability to be identified by the anti-SARS-CoV-2 antibodies by exposing them to COVID-19 serum samples. The proteins were also injected into mice and rabbit, and the antibody titers were measured for 390 days to assess their neutralization efficiency. RESULTS: The antibodies that existed in the serum of COVID-19 patients were identified by designed proteins. The anti-spike antibody titer was increased in the animals injected with recombinant proteins. The VNT results revealed that the produced antibodies could neutralize the cultured live virus. CONCLUSION: Truncated subunit vaccines could also be considered as robust tools for effective vaccination against COVID-19. Using a combination of in silico, in vitro, and in vivo experiments, it was shown that the injection of spike-based truncated recombinant proteins could stimulate long-lasting and neutralizing antibody responses.


Subject(s)
COVID-19 Vaccines , COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , Humans , Mice , Rabbits , Recombinant Proteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology
5.
Sci Rep ; 12(1): 2505, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1747189

ABSTRACT

Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Binding Sites , COVID-19/pathology , COVID-19/virology , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Docking Simulation , Nitriles/chemistry , Nitriles/metabolism , Nitriles/pharmacology , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/metabolism , Quinazolines/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Virus Replication/drug effects
6.
Viral Immunol ; 35(3): 254-258, 2022 04.
Article in English | MEDLINE | ID: covidwho-1740746

ABSTRACT

Data on the human immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins have been applied to vaccine development and diagnosing coronavirus disease 2019 (COVID-19), but little research has been done on the relationship between the human immune response and COVID-19 severity. We herein sought to determine whether there is a correlation between the immunoglobulin level and COVID-19 severity. Clinical samples were collected from 102 patients with COVID-19. Of these, 65 and 37 patients had mild and severe symptoms, respectively. An enzyme-linked immunosorbent assay using the recombinant SARS-CoV-2 nucleocapsid (N) protein, spike (S) protein, and synthetic peptides covering N and S as antigens was performed to measure the IgM and IgG levels. The correlation between the immunoglobulin level and COVID-19 severity was then analyzed. A significant difference in the level of IgG antibodies against N and of IgM antibodies against the receptor binding domain of the S protein was observed between patients with nonsevere and severe COVID-19 symptoms, and the level of IgG antibodies against N was found to be higher in patients with severe symptoms whereas the level of IgM antibodies against the S peptides was higher in patients with nonsevere symptoms. The level of specific antibodies against SARS-CoV-2 structural proteins might correlate with COVID-19 severity. If so, this fact may be useful for predicting the prognosis of the disease and in determining the appropriate treatment with greater precision.


Subject(s)
COVID-19 , Nucleocapsid Proteins , Antibodies, Viral , COVID-19/diagnosis , Humans , Immunoglobulin G , Immunoglobulin M , Peptides , Recombinant Proteins , SARS-CoV-2
7.
Sci Adv ; 8(7): eabl6242, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1714333

ABSTRACT

Large segmental osseous defects heal poorly. Recombinant, human bone morphogenetic protein-2 (rhBMP-2) is used clinically to promote bone healing, but it is applied at very high doses that cause adverse side effects and raise costs while providing only incremental benefit. We describe a previously unexplored, alternative approach to bone regeneration using chemically modified messenger RNA (cmRNA). An optimized cmRNA encoding BMP-2 was delivered to critical-sized femoral osteotomies in rats. The cmRNA remained orthotopically localized and generated BMP locally for several days. Defects healed at doses ≥25 µg of BMP-2 cmRNA. By 4 weeks, all animals treated with 50 µg of BMP-2 cmRNA had bridged bone defects without forming the massive callus seen with rhBMP-2. Moreover, such defects recovered normal mechanical strength quicker and initiated bone remodeling faster. cmRNA regenerated bone via endochondral ossification, whereas rhBMP-2 drove intramembranous osteogenesis; cmRNA provides an innovative, safe, and highly translatable technology for bone healing.


Subject(s)
Bone Morphogenetic Protein 2 , Bone Regeneration , Animals , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/pharmacology , Femur , Osteogenesis , RNA, Messenger/genetics , Rats , Recombinant Proteins/pharmacology , Wound Healing
8.
Hematology ; 27(1): 318-321, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1713441

ABSTRACT

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse drug reaction associated with thrombosis. Clinical scoring systems and the presence of anti-platelet factor 4 (anti-PF4)/heparin antibodies determine the diagnosis. CASE PRESENTATION: A 57-year-old man who was treated with acenocoumarol due to a chronic left ventricular thrombus was admitted to the hospital for severe SARS-CoV-2 pneumonia and pulmonary embolism. The patient was started on bemiparin and discharged. Left lower limb acute arterial ischemia and thrombocytopenia were diagnosed 18 days later. Computed tomography angiography revealed a large left ventricular thrombus and multiple arterial thrombi. Left femoral-popliteal thromboembolectomy was performed. Anti-PF4/heparin antibodies confirmed an HIT diagnosis. Fondaparinux (7.5 mg/24 h) was initiated, but cardiac surgery was necessary. Bivalirudin was used during surgery, with an initial load (1.25 mg/kg) and maintenance infusion (2.5 mg/kg/h). The cardiac thrombus was extracted, but the patient experienced a postsurgical myocardial infarction. Percutaneous cardiovascular intervention (PCI) required a bivalirudin load (0.75 mg/kg) and maintenance infusion (1.75 mg/kg/h). No coronary lesions were detected, and argatroban was started afterwards (0.5 µg/kg/min). When the platelet count exceeded 100 × 109/L, acenocoumarol was initiated. Thereupon, acetylsalicylic acid (100 mg/24 h) was added. No other complications have been reported to date. CONCLUSION: The clinical presentation of intraventricular and multiple arterial thrombi is remarkable. SARS-CoV-2 infection likely contributed to a hypercoagulable state. The management of patients with HIT undergoing cardiac surgery is challenging. If surgery cannot be delayed, then treatment with bivalirudin is recommended. Additionally, this drug is recommended for PCI. Bivalirudin is safe and well-tolerated in both procedures.


Subject(s)
Acenocoumarol/administration & dosage , Anticoagulants/administration & dosage , Arginine/analogs & derivatives , COVID-19/drug therapy , Heparin , Hirudins/administration & dosage , Peptide Fragments/administration & dosage , Percutaneous Coronary Intervention , Pipecolic Acids/administration & dosage , SARS-CoV-2 , Sulfonamides/administration & dosage , Thrombocytopenia , Thrombosis , Arginine/administration & dosage , COVID-19/complications , Heparin/administration & dosage , Heparin/adverse effects , Humans , Male , Middle Aged , Recombinant Proteins/administration & dosage , Thrombocytopenia/chemically induced , Thrombocytopenia/therapy , Thrombosis/chemically induced , Thrombosis/therapy
9.
Int J Mol Sci ; 23(4)2022 Feb 16.
Article in English | MEDLINE | ID: covidwho-1708485

ABSTRACT

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.


Subject(s)
COVID-19 Vaccines/chemistry , COVID-19 Vaccines/pharmacology , Immunity, Humoral/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Animals , Binding Sites , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Dextrans/chemistry , Female , HEK293 Cells , Humans , Mice, Inbred BALB C , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spermidine/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Vaccines, DNA/pharmacology , Vero Cells
10.
Protein Expr Purif ; 194: 106075, 2022 06.
Article in English | MEDLINE | ID: covidwho-1703723

ABSTRACT

Brevibacillus choshinensis is a gram-positive bacterium that is known to efficiently secrete recombinant proteins. However, the expression of these proteins is often difficult depending upon the expressed protein. In this study, we demonstrated that the addition of arginine hydrochloride and proline to the culture medium dramatically increased protein expression. By culturing bacterial cells in 96-well plates, we were able to rapidly examine the expression conditions and easily scale up to 96 mL of culture for production. Although functional expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein without any solubility-enhancing tag in bacterial strains (including Escherichia coli) has not been reported to date, we succeeded in efficiently producing RBD which showed a similar CD spectrum to that of RBD produced by eukaryotic cell expression systems. Furthermore, RBD from the omicron variant (B.1.1.529) was also produced. Physicochemical analyses indicated that omicron RBD exhibited markedly increased instability compared to the wild-type. We also revealed that the Fab format of the anti-SARS-CoV-2 antibody C121 can be produced in large quantities using the same expression system. The obtained C121 Fab bound to wild-type RBD but not to omicron RBD. These results strongly suggest that the Brevibacillus expression system is useful for facilitating the efficient expression of proteins that are difficult to fold and will thus contribute to the rapid physicochemical evaluation of functional proteins.


Subject(s)
Brevibacillus , COVID-19 , Antibodies, Viral , Arginine/metabolism , Brevibacillus/genetics , Brevibacillus/metabolism , Humans , Proline/metabolism , Recombinant Proteins/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
11.
Viruses ; 14(2)2022 02 21.
Article in English | MEDLINE | ID: covidwho-1702211

ABSTRACT

In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that will allow a comprehensive analysis of the viral proteome, which will allow further understanding of SARS-CoV-2 pathogenesis and the design of therapeutic strategies.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Viral/pharmacology , SARS-CoV-2/immunology , Viral Proteins/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Antibodies, Monoclonal/classification , Antibodies, Viral/immunology , COVID-19/therapy , COVID-19/virology , HEK293 Cells , Humans , Recombinant Proteins/immunology , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/immunology
12.
Viruses ; 13(12)2021 12 17.
Article in English | MEDLINE | ID: covidwho-1702075

ABSTRACT

BACKGROUND: The SARS-CoV-2 spike protein mediates attachment of the virus to the host cell receptor and fusion between the virus and the cell membrane. The S1 subunit of the spike glycoprotein (S1 protein) contains the angiotensin converting enzyme 2 (ACE2) receptor binding domain. The SARS-CoV-2 variants of concern contain mutations in the S1 subunit. The spike protein is the primary target of neutralizing antibodies generated following infection, and constitutes the viral component of mRNA-based COVID-19 vaccines. METHODS: Therefore, in this work we assessed the effect of exposure (24 h) to 10 nM SARS-CoV-2 recombinant S1 protein on physiologically relevant human bronchial (bro) and alveolar (alv) lung mucosa models cultured at air-liquid interface (ALI) (n = 6 per exposure condition). Corresponding sham exposed samples served as a control. The bro-ALI model was developed using primary bronchial epithelial cells and the alv-ALI model using representative type II pneumocytes (NCI-H441). RESULTS: Exposure to S1 protein induced the surface expression of ACE2, toll like receptor (TLR) 2, and TLR4 in both bro-ALI and alv-ALI models. Transcript expression analysis identified 117 (bro-ALI) and 97 (alv-ALI) differentially regulated genes (p ≤ 0.01). Pathway analysis revealed enrichment of canonical pathways such as interferon (IFN) signaling, influenza, coronavirus, and anti-viral response in the bro-ALI. Secreted levels of interleukin (IL) 4 and IL12 were significantly (p < 0.05) increased, whereas IL6 decreased in the bro-ALI. In the case of alv-ALI, enriched terms involving p53, APRIL (a proliferation-inducing ligand) tight junction, integrin kinase, and IL1 signaling were identified. These terms are associated with lung fibrosis. Further, significantly (p < 0.05) increased levels of secreted pro-inflammatory cytokines IFNγ, IL1ꞵ, IL2, IL4, IL6, IL8, IL10, IL13, and tumor necrosis factor alpha were detected in alv-ALI, whereas IL12 was decreased. Altered levels of these cytokines are also associated with lung fibrotic response. CONCLUSIONS: In conclusion, we observed a typical anti-viral response in the bronchial model and a pro-fibrotic response in the alveolar model. The bro-ALI and alv-ALI models may serve as an easy and robust platform for assessing the pathogenicity of SARS-CoV-2 variants of concern at different lung regions.


Subject(s)
Lung/metabolism , Respiratory Mucosa/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/metabolism , Cytokines/metabolism , Gene Expression Profiling , Humans , Models, Biological , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism
13.
Biochem Biophys Res Commun ; 601: 129-136, 2022 04 23.
Article in English | MEDLINE | ID: covidwho-1699331

ABSTRACT

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.


Subject(s)
COVID-19/prevention & control , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Binding Sites/genetics , COVID-19/virology , Enzyme Stability , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Mutation , Protein Conformation , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA Helicases/chemistry , RNA Helicases/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Temperature , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
14.
Clin Infect Dis ; 74(4): 567-574, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1699244

ABSTRACT

BACKGROUND: Neutropenia is commonly encountered in cancer patients. Recombinant human granulocyte colony-stimulating factor (G-CSF, filgrastim), a cytokine that initiates proliferation and differentiation of mature granulocytes, is widely given to oncology patients to counteract neutropenia, reducing susceptibility to infection. However, the clinical impact of neutropenia and G-CSF use in cancer patients with coronavirus disease 2019 (COVID-19) remains unknown. METHODS: An observational cohort of 379 actively treated cancer patients with COVID-19 was assembled to investigate links between concurrent neutropenia and G-CSF administration on COVID-19-associated respiratory failure and death. These factors were encoded as time-dependent predictors in an extended Cox model, controlling for age and underlying cancer diagnosis. To determine whether the degree of granulocyte response to G-CSF affected outcomes, the degree of response to G-CSF, based on rise in absolute neutrophil count (ANC) 24 hours after growth factor administration, was also incorporated into a similar Cox model. RESULTS: In the setting of active COVID-19 infection, outpatient receipt of G-CSF led to an increased number of hospitalizations (hazard ratio [HR]: 3.54, 95% confidence interval [CI]: 1.25-10.0, P value: .017). Furthermore, among inpatients, G-CSF administration was associated with increased need for high levels of oxygen supplementation and death (HR: 3.56, 95% CI: 1.19-10.2, P value: .024). This effect was predominantly seen in patients that exhibited a high response to G-CSF based on their ANC increase post-G-CSF administration (HR: 7.78, 95% CI: 2.05-27.9, P value: .004). CONCLUSIONS: The potential risks versus benefits of G-CSF administration should be considered in neutropenic cancer patients with COVID-19, because G-CSF administration may lead to worsening clinical and respiratory status.


Subject(s)
COVID-19 , Neoplasms , Neutropenia , COVID-19/complications , COVID-19/drug therapy , Filgrastim/therapeutic use , Granulocyte Colony-Stimulating Factor/therapeutic use , Humans , Neoplasms/complications , Neoplasms/drug therapy , Neutropenia/complications , Neutropenia/drug therapy , Recombinant Proteins/therapeutic use , SARS-CoV-2
15.
Front Immunol ; 12: 838082, 2021.
Article in English | MEDLINE | ID: covidwho-1674340

ABSTRACT

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative to conventional monoclonal antibodies also for clinical applications. Furthermore, they do not solely represent a substitute for monoclonal antibodies but their unique features allow expanding the applications of biotherapeutics and changes the pattern of disease treatment. Nanobodies possess the double advantage of being small and simple to engineer. This combination has promoted extremely diversified approaches to design nanobody-based constructs suitable for particular applications. Both the format geometry possibilities and the functionalization strategies have been widely explored to provide macromolecules with better efficacy with respect to single nanobodies or their combination. Nanobody multimers and nanobody-derived reagents were developed to image and contrast several cancer diseases and have shown their effectiveness in animal models. Their capacity to block more independent signaling pathways simultaneously is considered a critical advantage to avoid tumor resistance, whereas the mass of these multimeric compounds still remains significantly smaller than that of an IgG, enabling deeper penetration in solid tumors. When applied to CAR-T cell therapy, nanobodies can effectively improve the specificity by targeting multiple epitopes and consequently reduce the side effects. This represents a great potential in treating malignant lymphomas, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma and solid tumors. Apart from cancer treatment, multispecific drugs and imaging reagents built with nanobody blocks have demonstrated their value also for detecting and tackling neurodegenerative, autoimmune, metabolic, and infectious diseases and as antidotes for toxins. In particular, multi-paratopic nanobody-based constructs have been developed recently as drugs for passive immunization against SARS-CoV-2 with the goal of impairing variant survival due to resistance to antibodies targeting single epitopes. Given the enormous research activity in the field, it can be expected that more and more multimeric nanobody molecules will undergo late clinical trials in the next future. Systematic Review Registration.


Subject(s)
Single-Domain Antibodies/chemistry , Single-Domain Antibodies/therapeutic use , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/therapy , Communicable Diseases/immunology , Communicable Diseases/therapy , Humans , Immunomodulation , Molecular Imaging , Molecular Targeted Therapy , Neoplasms/diagnostic imaging , Neoplasms/immunology , Neoplasms/therapy , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Single-Domain Antibodies/immunology
16.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Article in English | MEDLINE | ID: covidwho-1671749

ABSTRACT

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.


Subject(s)
COVID-19/drug therapy , Interferon-alpha/pharmacology , SARS-CoV-2/drug effects , Transcriptome , Virus Replication/drug effects , Animals , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Cloning, Molecular , Disease Models, Animal , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Profiling , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Interferon-alpha/genetics , Interferon-alpha/immunology , Mice , Protein Isoforms/classification , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/pharmacology , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Signal Transduction , Vero Cells
17.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1666759

ABSTRACT

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Immunoglobulin G/blood , Inclusion Bodies/chemistry , Protein Refolding , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Hydrostatic Pressure , Immunoglobulin G/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Solubility
18.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1650946

ABSTRACT

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.


Subject(s)
Amino Alcohols/pharmacology , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/pharmacology , Phenyl Ethers/pharmacology , Receptors, Virus/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Sulfhydryl Compounds/pharmacology , Allosteric Regulation , Amino Alcohols/chemistry , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Cell Line , Disulfides/antagonists & inhibitors , Disulfides/chemistry , Disulfides/metabolism , Dose-Response Relationship, Drug , Humans , Molecular Docking Simulation , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Oxidation-Reduction , Phenyl Ethers/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Sulfhydryl Compounds/chemistry
19.
Lancet Infect Dis ; 22(5): 636-648, 2022 05.
Article in English | MEDLINE | ID: covidwho-1650654

ABSTRACT

BACKGROUND: We evaluated our SARS-CoV-2 prefusion spike recombinant protein vaccine (CoV2 preS dTM) with different adjuvants, unadjuvanted, and in a one-injection and two-injection dosing schedule in a previous phase 1-2 study. Based on interim results from that study, we selected a two-injection schedule and the AS03 adjuvant for further clinical development. However, lower than expected antibody responses, particularly in older adults, and higher than expected reactogenicity after the second vaccination were observed. In the current study, we evaluated the safety and immunogenicity of an optimised formulation of CoV2 preS dTM adjuvanted with AS03 to inform progression to phase 3 clinical trial. METHODS: This phase 2, randomised, parallel-group, dose-ranging study was done in adults (≥18 years old), including those with pre-existing medical conditions, those who were immunocompromised (except those with recent organ transplant or chemotherapy) and those with a potentially increased risk for severe COVID-19, at 20 clinical research centres in the USA and Honduras. Women who were pregnant or lactating or, for those of childbearing potential, not using an effective method of contraception or abstinence, and those who had received a COVID-19 vaccine, were excluded. Participants were randomly assigned (1:1:1) using an interactive response technology system, with stratification by age (18-59 years and ≥60 years), rapid serodiagnostic test result (positive or negative), and high-risk medical conditions (yes or no), to receive two injections (day 1 and day 22) of 5 7mu;g (low dose), 10 7mu;g (medium dose), or 15 7mu;g (high dose) CoV2 preS dTM antigen with fixed AS03 content. All participants and outcome assessors were masked to group assignment; unmasked study staff involved in vaccine preparation were not involved in safety outcome assessments. All laboratory staff performing the assays were masked to treatment. The primary safety objective was to describe the safety profile in all participants, for each candidate vaccine formulation. Safety endpoints were evaluated for all randomised participants who received at least one dose of the study vaccine (safety analysis set), and are presented here for the interim study period (up to day 43). The primary immunogenicity objective was to describe the neutralising antibody titres to the D614G variant 14 days after the second vaccination (day 36) in participants who were SARS-CoV-2 naive who received both injections, provided samples at day 1 and day 36, did not have protocol deviations, and did not receive an authorised COVID-19 vaccine before day 36. Neutralising antibodies were measured using a pseudovirus neutralisation assay and are presented here up to 14 days after the second dose. As a secondary immunogenicity objective, we assessed neutralising antibodies in non-naive participants. This trial is registered with ClinicalTrials.gov (NCT04762680) and is closed to new participants for the cohort reported here. FINDINGS: Of 722 participants enrolled and randomly assigned between Feb 24, 2021, and March 8, 2021, 721 received at least one injection (low dose=240, medium dose=239, and high dose=242). The proportion of participants reporting at least one solicited adverse reaction (injection site or systemic) in the first 7 days after any vaccination was similar between treatment groups (217 [91%] of 238 in the low-dose group, 213 [90%] of 237 in the medium-dose group, and 218 [91%] of 239 in the high-dose group); these adverse reactions were transient, were mostly mild to moderate in intensity, and occurred at a higher frequency and intensity after the second vaccination. Four participants reported immediate unsolicited adverse events; two (one each in the low-dose group and medium-dose group) were considered by the investigators to be vaccine related and two (one each in the low-dose and high-dose groups) were considered unrelated. Five participants reported seven vaccine-related medically attended adverse events (two in the low-dose group, one in the medium-dose group, and four in the high-dose group). No vaccine-related serious adverse events and no adverse events of special interest were reported. Among participants naive to SARS-CoV-2 at day 36, 158 (98%) of 162 in the low-dose group, 166 (99%) of 168 in the medium-dose group, and 163 (98%) of 166 in the high-dose group had at least a two-fold increase in neutralising antibody titres to the D614G variant from baseline. Neutralising antibody geometric mean titres (GMTs) at day 36 for participants who were naive were 2189 (95% CI 1744-2746) for the low-dose group, 2269 (1792-2873) for the medium-dose group, and 2895 (2294-3654) for the high-dose group. GMT ratios (day 36: day 1) were 107 (95% CI 85-135) in the low-dose group, 110 (87-140) in the medium-dose group, and 141 (111-179) in the high-dose group. Neutralising antibody titres in non-naive adults 21 days after one injection tended to be higher than titres after two injections in adults who were naive, with GMTs 21 days after one injection for participants who were non-naive being 3143 (95% CI 836-11 815) in the low-dose group, 2338 (593-9226) in the medium-dose group, and 7069 (1361-36 725) in the high-dose group. INTERPRETATION: Two injections of CoV2 preS dTM-AS03 showed acceptable safety and reactogenicity, and robust immunogenicity in adults who were SARS-CoV-2 naive and non-naive. These results supported progression to phase 3 evaluation of the 10 7mu;g antigen dose for primary vaccination and a 5 7mu;g antigen dose for booster vaccination. FUNDING: Sanofi Pasteur and Biomedical Advanced Research and Development Authority.


Subject(s)
COVID-19 Vaccines , COVID-19 , Adjuvants, Immunologic , Adolescent , Adult , Aged , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines/adverse effects , Double-Blind Method , Female , Humans , Immunogenicity, Vaccine , Lactation , Middle Aged , Recombinant Proteins , SARS-CoV-2 , Vaccines, Synthetic , Young Adult
20.
PLoS One ; 17(1): e0262868, 2022.
Article in English | MEDLINE | ID: covidwho-1643287

ABSTRACT

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.


Subject(s)
Antibodies, Viral/chemistry , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/epidemiology , COVID-19 Serological Testing/standards , Coronavirus Nucleocapsid Proteins/chemistry , Epidemiological Monitoring , Female , Humans , Immunoglobulin A/chemistry , Immunoglobulin A/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin M/chemistry , Immunoglobulin M/immunology , Male , Middle Aged , New York/epidemiology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , SARS-CoV-2/classification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/chemistry
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