scholarly journals Identification of Two Neutralizing Regions on the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Produced from the Mammalian Expression System

2005 ◽  
Vol 79 (3) ◽  
pp. 1906-1910 ◽  
Author(s):  
Shixia Wang ◽  
Te-hui W. Chou ◽  
Pavlo V. Sakhatskyy ◽  
Song Huang ◽  
John M. Lawrence ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
High Titer ◽  
Expression System ◽  
Antibody Responses ◽  
Effective Vaccine ◽  
S Protein ◽  
Mammalian Expression ◽  
C Terminus ◽  
N Terminus ◽  
Specific Immunoglobulin

ABSTRACT The Spike (S) protein of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) plays important roles in viral pathogenesis and potentially in the development of an effective vaccine against this virulent infectious disease. In this study, the codon-optimized S gene of SARS-CoV was synthesized to construct DNA vaccine plasmids expressing either the full-length or segments of the S protein. High titer S-specific immunoglobulin G antibody responses were elicited in rabbits immunized with DNA against various segments of the S protein. Two neutralizing domains were identified on the S protein, one at the N terminus (Ser12-Thr535) and the other near the C terminus (Arg797-Ile1192).

scholarly journals A Review of SARS-CoV-2, Responsible for COVID-19: History, Biology, Infection Mechanism, Antigenic Vaccines, their Risks and How an Alternate RBD Vaccine is Safer?

2021 ◽  
Author(s):  
Z.M.G. Sarwar Jahangir ◽  
Arleta Helena Marnik
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Somatic Cell ◽  
Antibody Production ◽  
Spike Protein ◽  
Protein Antigen ◽  
Effective Vaccine ◽  
S Protein ◽  
Infection Mechanism ◽  
Fast Acting ◽  
Stimulate Antibody

The SARS (severe acute respiratory syndrome)-CoV (Coronavirus)-2 S(spike)-protein mRNA/cDNA currently being used as vaccines are antigenic but not antigens against SARS-CoV-2, that causes COVID (Coronavirus Disease) -19. Furthermore, the mRNA and cDNA antigenic vaccines also have potentials for homologous as well as heterologous recombination, primarily into the somatic cell DNA of the vaccine recipients. On the contrary, a SARS-CoV-2 RBD-protein antigen, a part of the S-protein, will directly stimulate antibody production against SARS-CoV-2. Hence, a vaccine composed of SARS-CoV-2 RBD-protein as a safer, fast acting, and effective vaccine against SARS-CoV-2 and thus against COVID-19. This is also useful for some immune compromised individuals.

scholarly journals Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

2021 ◽  
Vol 17 (7) ◽  
pp. e1008864
Author(s):  
Duncan N. Ndegwa ◽  
Prasun Kundu ◽  
Jessica B. Hostetler ◽  
Alejandro Marin-Menendez ◽  
Theo Sanderson ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Culture System ◽  
Vaccine Development ◽  
Expression System ◽  
Critical Role ◽  
Blood Stage ◽  
Effective Vaccine ◽  
Mammalian Expression ◽  
Close Phylogenetic Relationship

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.

scholarly journals Evaluation of Hemagglutinin Protein-Specific Immunoglobulin M for Diagnosis of Measles by an Enzyme-Linked Immunosorbent Assay Based on Recombinant Protein Produced in a High-Efficiency Mammalian Expression System

1998 ◽  
Vol 36 (12) ◽  
pp. 3509-3513 ◽  
Author(s):  
Fabienne B. Bouche ◽  
Nicolaas H. C. Brons ◽  
Sophie Houard ◽  
Francois Schneider ◽  
Claude P. Muller
Keyword(s):  
Recombinant Protein ◽  
Immunosorbent Assay ◽  
Measles Virus ◽  
High Efficiency ◽  
Expression System ◽  
Immunoglobulin M ◽  
Initial Time ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mammalian Expression ◽  
Specific Immunoglobulin

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.

scholarly journals Characterization of self-assembled virus-like particles of ferret hepatitis E virus generated by recombinant baculoviruses

2013 ◽  
Vol 94 (12) ◽  
pp. 2647-2656 ◽  
Author(s):  
Tingting Yang ◽  
Michiyo Kataoka ◽  
Yasushi Ami ◽  
Yuriko Suzaki ◽  
Noriko Kishida ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Hepatitis E ◽  
Electron Microscopic ◽  
Antigenic Analysis ◽  
Virus Like Particles ◽  
C Terminus ◽  
N Terminus ◽  
Self Assembled

Ferret hepatitis E virus (HEV), a novel hepatitis E-like virus, has been identified in ferrets in The Netherlands. Due to the lack of a cell-culture system for ferret HEV, the antigenicity, pathogenicity and epidemiology of this virus have remained unclear. In the present study, we used a recombinant baculovirus expression system to express the 112-N-terminus and 47-C-terminus-amino-acid-truncated ferret HEV ORF2 protein in insect Tn5 cells, and found that a large amount of a 53 kDa protein (F-p53) was expressed and efficiently released into the supernatant. Electron microscopic analysis revealed that F-p53 was self-assembled into virus-like particles (ferret HEV-LPs). These ferret HEV-LPs were estimated to be 24 nm in diameter, which is similar to the size of G1, G3, G4 and rat HEV-LPs derived from both the N-terminus- and C-terminus-truncated constructs. Antigenic analysis demonstrated that ferret HEV-LPs were cross-reactive with G1, G3, G4 and rat HEVs, and rat HEV and ferret HEV showed a stronger cross-reactivity to each other than either did to human HEV genotypes. However, the antibody against ferret HEV-LPs does not neutralize G3 HEV, suggesting that the serotypes of these two HEVs are different. An ELISA for detection of anti-ferret HEV IgG and IgM antibodies was established using ferret HEV-LPs as antigen, and this assay system will be useful for monitoring ferret HEV infection in ferrets as well as other animals. In addition, analysis of ferret HEV RNA detected in ferret sera collected from a breeding colony in the USA revealed the genetic diversity of ferret HEV.

scholarly journals Immune Responses in Mice Vaccinated with Virus-Like Particles of Western Equine Encephalitis Virus from an Insect Cell-Based Expression System

2021 ◽  
Author(s):  
Jinzhu Ma ◽  
Hualei Wang ◽  
Xuexing Zheng ◽  
Hongxia Wu Wu ◽  
Songtao Yang ◽  
...  
Keyword(s):  
Insect Cells ◽  
High Titer ◽  
Expression System ◽  
Encephalitis Virus ◽  
Effective Vaccine ◽  
Virus Like Particles ◽  
Equine Encephalitis Virus ◽  
Important Challenge ◽  
Western Equine Encephalitis Virus ◽  
Western Equine Encephalitis

Western equine encephalitis virus (WEEV) can cause lethal encephalitis in humans and equines and represents a serious public health threat in many countries. Therefore, development of efficient vaccines against WEEV remains an important challenge in the field of disease control. This study described for the first time successful production of WEEV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is very suitable for production of WEEV VLPs. The immune experiment herein in mice showed that the VLPs formulated with 206-adjuvant were responsible for the stronger-VLP-specific cellular immune response, and were able to induce the secretion of IL-2, IL-4, IFN-γ and production of high titer antibodies that can effectively neutralize the WEEV pseudoviruses. The WEEV VLPs from insect cells could provide a new, safe, non-replicating and effective vaccine candidate against WEEV infections.

scholarly journals A Review of SARS-CoV-2, Responsible for COVID-19: History, Biology, Infection Mechanism, Antigenic Vaccines, Their Risks and how an Alternate RBD Vaccine is Safer?

2021 ◽  
Author(s):  
Z.M.G. Sarwar Jahangir ◽  
Arleta Helena Marnik
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Somatic Cell ◽  
Antibody Production ◽  
Spike Protein ◽  
Protein Antigen ◽  
Effective Vaccine ◽  
S Protein ◽  
Infection Mechanism ◽  
Fast Acting ◽  
Stimulate Antibody

The SARS (severe acute respiratory syndrome)-CoV (Coronavirus)-2 S(spike)-protein mRNA/cDNA currently being used as vaccines are antigenic but not antigens against SARS-CoV-2, that causes COVID (Coronavirus Disease) -19. Furthermore, the mRNA and cDNA antigenic vaccines also have potentials for homologous as well as heterologous recombination, primarily into the somatic cell DNA of the vaccine recipients. On the contrary, a SARS-CoV-2 RBD-protein antigen, a part of the S-protein, will directly stimulate antibody production against SARS-CoV-2. Hence, a vaccine composed of SARS-CoV-2 RBD-protein as a safer, fast acting, and effective vaccine against SARS-CoV-2 and thus against COVID-19. This is also useful for some immune compromised individuals.

scholarly journals Amino Acids 270 to 510 of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Are Required for Interaction with Receptor

2004 ◽  
Vol 78 (9) ◽  
pp. 4552-4560 ◽  
Author(s):  
Gregory J. Babcock ◽  
Diana J. Esshaki ◽  
William D. Thomas ◽  
Donna M. Ambrosino
Keyword(s):  
Amino Acids ◽  
Severe Acute Respiratory Syndrome ◽  
Receptor Binding ◽  
Expression System ◽  
Viral Envelope ◽  
Receptor Binding Site ◽  
Host Cell Membrane ◽  
Viral Attachment ◽  
Amino Terminal ◽  
Mammalian Expression

ABSTRACT A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S1190). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S1190 binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S1190 maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S1190 glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.

scholarly journals Vaccinomic approach for novel multi epitopes vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yassir A. Almofti ◽  
Khoubieb Ali Abd-elrahman ◽  
Elsideeq E. M. Eltilib
Keyword(s):  
Binding Energy ◽  
T Lymphocytes ◽  
Severe Acute Respiratory Syndrome ◽  
Tertiary Structure ◽  
Effective Vaccine ◽  
S Protein ◽  
Grand Average ◽  
Aliphatic Index ◽  
Novel Coronavirus ◽  
B And T Lymphocytes

Abstract Background The spread of a novel coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in China and other countries is of great concern worldwide with no effective vaccine. This study aimed to design a novel vaccine construct against SARS-CoV-2 from the spike S protein and orf1ab polyprotein using immunoinformatics tools. The vaccine was designed from conserved epitopes interacted against B and T lymphocytes by the combination of highly immunogenic epitopes with suitable adjuvant and linkers. Results The proposed vaccine composed of 526 amino acids and was shown to be antigenic in Vaxigen server (0.6194) and nonallergenic in Allertop server. The physiochemical properties of the vaccine showed isoelectric point of 10.19. The instability index (II) was 31.25 classifying the vaccine as stable. Aliphatic index was 84.39 and the grand average of hydropathicity (GRAVY) was − 0.049 classifying the vaccine as hydrophilic. Vaccine tertiary structure was predicted, refined and validated to assess the stability of the vaccine via Ramachandran plot and ProSA-web servers. Moreover, solubility of the vaccine construct was greater than the average solubility provided by protein sol and SOLpro servers indicating the solubility of the vaccine construct. Disulfide engineering was performed to reduce the high mobile regions in the vaccine to enhance stability. Docking of the vaccine construct with TLR4 demonstrated efficient binding energy with attractive binding energy of − 338.68 kcal/mol and − 346.89 kcal/mol for TLR4 chain A and chain B respectively. Immune simulation significantly provided high levels of immunoglobulins, T-helper cells, T-cytotoxic cells and INF-γ. Upon cloning, the vaccine protein was reverse transcribed into DNA sequence and cloned into pET28a(+) vector to ensure translational potency and microbial expression. Conclusion A unique vaccine construct from spike S protein and orf1ab polyprotein against B and T lymphocytes was generated with potential protection against the pandemic. The present study might assist in developing a suitable therapeutics protocol to combat SARSCoV-2 infection.

scholarly journals Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

2000 ◽  
Vol 348 (3) ◽  
pp. 597-606 ◽  
Author(s):  
Jian-Ting ZHANG
Keyword(s):  
Multidrug Resistance ◽  
Expression System ◽  
Critical Role ◽  
Signal Recognition Particle ◽  
Dependent Manner ◽  
Membrane Translocation ◽  
C Terminus ◽  
N Terminus ◽  
Polytopic Membrane Protein

Multidrug-resistance-associated protein (MRP) is a member of the ATP-binding cassette (ABC) membrane-transport superfamily and is responsible for multidrug resistance in cancer cells. Distinct from other members of the ABC superfamily, MRP has three membrane-spanning domains (MSDs) and the N-terminus is located extracellularly. It has been shown that the first MSD (MSD1) with an extracellular N-terminus is important for MRP function. To address what ensures the generation of this structural organization of MRP and to understand in general the molecular mechanism of membrane folding of polytopic proteins with extracellular N-termini, the biogenesis of MSD1 in human MRP1 was examined using an in vitro expression system. Surprisingly, the second transmembrane segment (TM2) in MSD1 was found to play a critical role in the correct membrane translocation and folding of MSD1 in human MRP1. TM2 not only plays an essential role to ensure the N-terminus-outside/C-terminus-inside orientation of TM1 with an extracellular N-terminus, it can also translocate into membranes post-translationally in a signal-recognition particle and ribosome-dependent manner to provide an additional insurance for correct folding of MSD1 in MRP. These findings suggest that TM2 in a polytopic membrane protein with an extracellular N-terminus may play a critical role in controlling correct membrane translocation and folding of the protein in general.

scholarly journals Structural Basis and Function of the N Terminus of SARS-CoV-2 Nonstructural Protein 1

2021 ◽  
Author(s):  
Kaitao Zhao ◽  
Zunhui Ke ◽  
Hongbing Hu ◽  
Yahui Liu ◽  
Aixin Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nonstructural Protein ◽  
Protein Translation ◽  
Host Protein ◽  
Structural Basis ◽  
Pathogenic Factor ◽  
Nonstructural Protein 1 ◽  
C Terminus ◽  
N Terminus ◽  
And Function

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive.

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