scholarly journals SARS-CoV-2 Spike Protein Extrapolation for COVID Diagnosis and Vaccine Development

2021 ◽  
Vol 8 ◽  
Author(s):  
Yashpal S. Malik ◽  
Prashant Kumar ◽  
Mohd Ikram Ansari ◽  
Maged G. Hemida ◽  
Mohamed E. El Zowalaty ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Sequence Similarity ◽  
Molecular Diagnostic ◽  
Post Exposure Prophylaxis ◽  
Molecular Diagnostic Techniques ◽  
Virus Envelope ◽  
High Sequence Similarity ◽  
S Protein

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to coronavirus disease 2019 (COVID-19) pandemic affecting nearly 71.2 million humans in more than 191 countries, with more than 1.6 million mortalities as of 12 December, 2020. The spike glycoprotein (S-protein), anchored onto the virus envelope, is the trimer of S-protein comprised of S1 and S2 domains which interacts with host cell receptors and facilitates virus-cell membrane fusion. The S1 domain comprises of a receptor binding domain (RBD) possessing an N-terminal domain and two subdomains (SD1 and SD2). Certain regions of S-protein of SARS-CoV-2 such as S2 domain and fragment of the RBD remain conserved despite the high selection pressure. These conserved regions of the S-protein are extrapolated as the potential target for developing molecular diagnostic techniques. Further, the S-protein acts as an antigenic target for different serological assay platforms for the diagnosis of COVID-19. Virus-specific IgM and IgG antibodies can be used to detect viral proteins in ELISA and lateral flow immunoassays. The S-protein of SARS-CoV-2 has very high sequence similarity to SARS-CoV-1, and the monoclonal antibodies (mAbs) against SARS-CoV-1 cross-react with S-protein of SARS-CoV-2 and neutralize its activity. Furthermore, in vitro studies have demonstrated that polyclonal antibodies targeted against the RBD of S-protein of SARS-CoV-1 can neutralize SARS-CoV-2 thus inhibiting its infectivity in permissive cell lines. Research on coronaviral S-proteins paves the way for the development of vaccines that may prevent SARS-CoV-2 infection and alleviate the current global coronavirus pandemic. However, specific neutralizing mAbs against SARS-CoV-2 are in clinical development. Therefore, neutralizing antibodies targeting SARS-CoV-2 S-protein are promising specific antiviral therapeutics for pre-and post-exposure prophylaxis and treatment of SARS-CoV-2 infection. We hereby review the approaches taken by researchers across the world to use spike gene and S-glycoprotein for the development of effective diagnostics, vaccines and therapeutics against SARA-CoV-2 infection the COVID-19 pandemic.

scholarly journals Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1597
Author(s):  
Thuong Thi Ho ◽  
Van Thi Pham ◽  
Tra Thi Nguyen ◽  
Vy Thai Trinh ◽  
Tram Vi ◽  
...  
Keyword(s):  
High Pressure ◽  
Neutralizing Antibodies ◽  
H5n1 Virus ◽  
Vaccine Development ◽  
Particle Characterization ◽  
Innovative Strategy ◽  
Specific Igg ◽  
Positively Charged

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40–70 nm (ND40) and ~100–250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use.

Chimeric virus-like particles of universal antigen epitopes of coronavirus and phage Qβ coat protein trigger the production of neutralizing antibodies

2021 ◽  
Vol 21 ◽  
Author(s):  
Yukun Guo ◽  
Ruizhen Guo ◽  
Yingxian Ma ◽  
Wenru Chang ◽  
Shengli Ming ◽  
...  
Keyword(s):  
Coat Protein ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Genetic Recombination ◽  
Fluorescent Label ◽  
Salting Out ◽  
Virus Like Particles ◽  
Chimeric Vlps ◽  
Universal Epitope

Background: Virus-like particles (VLPs) are non-genetic multimeric nanoparticles synthesized through in vitro or in vivo self-assembly of one or more viral structural proteins. Immunogenicity and safety of VLPs make them ideal candidates for vaccine development and efficient nanocarriers for foreign antigens or adjuvants to activate the immune system. Aims: The present study aimed to design and synthesize a chimeric VLP vaccine of the phage Qbeta (Qβ) coat protein presenting the universal epitope of the coronavirus. Methods: The RNA phage Qβ coat protein was designed and synthesized, denoted as Qbeta. The CoV epitope, a universal epitope of coronavirus, was inserted into the C-terminal of Qbeta using genetic recombination, which was designated as Qbeta-CoV. The N-terminal of Qbeta-CoV was successively inserted into the TEV restriction site using mCherry red fluorescent label and modified affinity-purified histidine label 6xHE, which was denoted as HE-Qbeta-CoV. Isopropyl β-D-1-thiogalactopyranoside (IPTG) assessment revealed the expression of Qbeta, Qbeta-CoV, and HE-Qbeta-CoV in the BL21 (DE3) cells. The fusion protein was purified by salting out using ammonium sulfate and affinity chromatography. The morphology of particles was observed using electron microscopy. The female BALB/C mice were immunized intraperitoneally with the Qbeta-CoV and HE-Qbeta-CoV chimeric VLPs vaccines. Their sera were collected for the detection of antibody level and antibody titer using ELISA. The serum is used for the neutralization test of the three viruses of MHV, PEDV, and PDCoV. Results: The results revealed that the fusion proteins Qbeta, Qbeta-CoV, and HE-Qbeta-CoV could all obtain successful expression. Particles with high purity were obtained after purification; the chimeric particles of Qbeta-CoV and HE-Qbeta-CoV were found to be similar to Qbeta particles in morphology and formed chimeric VLPs. In addition, two chimeric VLP vaccines induced specific antibody responses in mice, and the antibodies showed certain neutralizing activity. Conclusion: The successful construction of the chimeric VLPs of the phage Qβ coat protein presenting the universal epitope of coronavirus provides a vaccine form with potential clinical applications for the treatment of coronavirus disease.

scholarly journals Enhanced Prokaryotic Expression of Dengue Virus Envelope Protein

2013 ◽  
Vol 16 (4) ◽  
pp. 609 ◽  
Author(s):  
Advaita Ganguly ◽  
Ravindra B. Malabadi ◽  
Dipankar Das ◽  
Mavanur R. Suresh ◽  
Hoon H Sunwoo
Keyword(s):  
Dengue Virus ◽  
Envelope Protein ◽  
Vaccine Development ◽  
Virus Envelope ◽  
E Coli ◽  
C Terminus ◽  
Virus Envelope Protein ◽  
Biological Functionality ◽  
Dengue Diagnostics

Purpose. To highlight the expression and purification of the recombinant dengue virus type-1 antigen exploiting the codon optimized full length envelope for increased yield in E. coli. Methods. A 6x His tag was inserted at the C terminus to facilitate purification. The purified protein was recognized in Western blot by Monoclonal antibody specific for the tag. The in vitro refolded recombinant protein was used to immunize mice for the development of hybridomas and also analyzed for its biological functionality with heparan sulfate binding assay. Results. The polyclonal anti-sera from the immunized mice were found to recognize the envelope protein thereby establishing the immunogenicity of the protein. Conclusion. The purified envelope protein could potentially be used towards dengue diagnostics and vaccine development efforts. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

scholarly journals Incorporation of apolipoprotein E into HBV–HCV subviral envelope particles to improve the hepatitis vaccine strategy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elsa Gomez-Escobar ◽  
Julien Burlaud-Gaillard ◽  
Clara Visdeloup ◽  
Adeline Ribeiro E. Silva ◽  
Pauline Coutant ◽  
...  
Keyword(s):  
Apolipoprotein E ◽  
Neutralizing Antibodies ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Vaccine Strategy ◽  
S Protein ◽  
Hepatitis Vaccine ◽  
Protein Immunization ◽  
Neutralizing Potential

AbstractHepatitis C is a major threat to public health for which an effective treatment is available, but a prophylactic vaccine is still needed to control this disease. We designed a vaccine based on chimeric HBV–HCV envelope proteins forming subviral particles (SVPs) that induce neutralizing antibodies against HCV in vitro. Here, we aimed to increase the neutralizing potential of those antibodies, by using HBV–HCV SVPs bearing apolipoprotein E (apoE). These particles were produced by cultured stable mammalian cell clones, purified and characterized. We found that apoE was able to interact with both chimeric HBV–HCV (E1-S and E2-S) proteins, and with the wild-type HBV S protein. ApoE was also detected on the surface of purified SVPs and improved the folding of HCV envelope proteins, but its presence lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a good strategy for improving HCV vaccines based on viral envelope proteins.

scholarly journals Functional activity of antisera against recombinant Zika virus envelope protein subunits expressed in Escherichia coli

2019 ◽  
Author(s):  
Hong-Yun Tham ◽  
Man Kwan Ooi ◽  
Vinod RMT Balasubramaniam ◽  
Sharifah Syed Hassan ◽  
Hong-Wai Tham
Keyword(s):  
Zika Virus ◽  
Envelope Protein ◽  
Mammalian Cells ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Cross Reactivity ◽  
Virus Envelope ◽  
Humoral And Cellular Immunity ◽  
Progressive Development

AbstractThe global Zika virus (ZIKV) outbreak across continents has been drawing research attentions to researchers and healthcare professionals. It highlights the urgent development of ZIKV vaccines that offer rapid, precise and specific protection to those living in the high-risk regions - the tropical and subtropical regions. As a public health priority, there is a progressive development in the discovery of vaccine candidates and design in recent years. Many efforts have been placed in the in vitro development of ZIKV subunits as the vaccine candidate in various protein expression systems, including bacteria, yeast, plant cells, insect cells and mammalian cells. However, due to the lack of knowledge on humoral and cellular immune responses against virus vaccines, a commercialised vaccine against Dengue virus (DENV) has been suspended due to a health scare in Philippines. Moreover, the closely-related DENV and ZIKV has indicated serological cross-reactivity between both viruses. This has led to greater attentions to precautions needed during the design of ZIKV and DENV vaccines. In this study, we pre-selected, synthesised and expressed the domain III of ZIKV envelope protein (namely rEDIII) based on a previously-established report (GenBank: AMC13911.1). The characteristics of purified ZIKV rEDIII was tested using SDS-PAGE, Western blotting and LC-MS/MS. Since the ZIKV rEDIII has been well reported as a potential protein candidate in ZIKV vaccine development, we assessed the possible outcome of preexisting immunity against the rEDIII proteins by conducting dot-blotting assays using mice antisera pre-immunised with ZIKV particles (ZIKV strain: MRS_OPY_Martinique_PaRi_2015, GenBank: KU647676) . Surprisingly, the antisera was able to recognise the rEDIII of a different ZIKV strain (GenBank: AMC13911.1). Despite its great antigenicity in eliciting humoral and cellular immunity against ZIKV infection, our finding calls for greater attention to evaluate the details of ZIKV rEDIII as a stand-alone vaccine candidate.

scholarly journals A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in rhesus macaques

2021 ◽  
Author(s):  
Ronald R. Cobb ◽  
Joseph Nkolola ◽  
Pavlo Gilchuk ◽  
Abishek Chandrashekar ◽  
Robert V. House ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Human Monoclonal Antibody ◽  
Neutralizing Antibody ◽  
Post Exposure Prophylaxis ◽  
Human Mabs ◽  
Viral Neutralization ◽  
Exposure Prophylaxis ◽  
Human Primate

Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention, post-exposure prophylaxis, or therapy. However, the titer of neutralizing antibodies required for protection against SARS-CoV-2 infection remains poorly characterized. We previously described two potently neutralizing mAbs COV2-2130 and COV2-2381 targeting non-overlapping epitopes on the receptor-binding domain of SARS-CoV-2 spike protein. Here, we engineered the Fc-region of these mAbs with mutations to extend their persistence in humans and reduce interactions with Fc gamma receptors. Passive transfer of individual or combinations of the two antibodies (designated ADM03820) given prophylactically by intravenous or intramuscular route conferred virological protection in a non-human primate (NHP) model of SARS-CoV-2 infection, and ADM03820 potently neutralized SARS-CoV-2 variants of concern in vitro. We defined 6,000 as a protective serum neutralizing antibody titer in NHPs against infection for passively transferred human mAbs that acted by direct viral neutralization, which corresponded to a concentration of 20 microgram/mL of circulating mAb.

scholarly journals Glycans of SARS-CoV-2 Spike Protein in Virus Infection and Antibody Production

2021 ◽  
Vol 8 ◽  
Author(s):  
Xiaohui Zhao ◽  
Huan Chen ◽  
Hongliang Wang
Keyword(s):  
Immune Response ◽  
Antibody Production ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Protein Glycosylation ◽  
The Other ◽  
Defense System ◽  
S Protein ◽  
Receptor Interactions

Viral protein glycosylation represents a successful strategy employed by the parasite to take advantage of host–cell machinery for modification of its own proteins. The resulting glycans have unneglectable roles in viral infection and immune response. The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which presents on the surface of matured virion and mediates viral entry into the host, also undergoes extensive glycosylation to shield it from the human defense system. It is believed that the ongoing COVID-19 pandemic with more than 90,000,000 infections and 1,900,000 deaths is partly due to its successful glycosylation strategy. On the other hand, while glycan patches on S protein have been reported to shield the host immune response by masking “nonself” viral peptides with “self-glycans,” the epitopes are also important in eliciting neutralizing antibodies. In this review, we will summarize the roles of S protein glycans in mediating virus–receptor interactions, and in antibody production, as well as indications for vaccine development.

scholarly journals Cellular and Humoral Immunogenicity of a Candidate DNA Vaccine Expressing SARS-CoV-2 Spike Subunit 1

Vaccines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 852
Author(s):  
Khalid A. Alluhaybi ◽  
Rahaf H. Alharbi ◽  
Rowa Y. Alhabbab ◽  
Najwa D. Aljehani ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Immune Responses ◽  
Dna Vaccine ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Cell Responses ◽  
Cellular Immune Responses ◽  
Specific Igg ◽  
Cellular Immune ◽  
Different Doses

The urgent need for effective, safe and equitably accessible vaccines to tackle the ongoing spread of COVID-19 led researchers to generate vaccine candidates targeting varieties of immunogens of SARS-CoV-2. Because of its crucial role in mediating binding and entry to host cell and its proven safety profile, the subunit 1 (S1) of the spike protein represents an attractive immunogen for vaccine development. Here, we developed and assessed the immunogenicity of a DNA vaccine encoding the SARS-CoV-2 S1. Following in vitro confirmation and characterization, the humoral and cellular immune responses of our vaccine candidate (pVAX-S1) was evaluated in BALB/c mice using two different doses, 25 µg and 50 µg. Our data showed high levels of SARS-CoV-2 specific IgG and neutralizing antibodies in mice immunized with three doses of pVAX-S1. Analysis of the induced IgG subclasses showed a Th1-polarized immune response, as demonstrated by the significant elevation of spike-specific IgG2a and IgG2b, compared to IgG1. Furthermore, we found that the immunization of mice with three doses of 50 µg of pVAX-S1 could elicit significant memory CD4+ and CD8+ T cell responses. Taken together, our data indicate that pVAX-S1 is immunogenic and safe in mice and is worthy of further preclinical and clinical evaluation.

scholarly journals A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 603
Author(s):  
Beatrice Sarah Berneck ◽  
Alexandra Rockstroh ◽  
Jasmin Fertey ◽  
Thomas Grunwald ◽  
Sebastian Ulbert
Keyword(s):  
Zika Virus ◽  
Insect Cell ◽  
Neutralizing Antibodies ◽  
Structural Similarity ◽  
Cross Reactivity ◽  
Virus Envelope ◽  
E Protein ◽  
Neutralizing Capacity ◽  
Fusion Loop

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles.

scholarly journals Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe

2020 ◽  
Vol 21 (23) ◽  
pp. 9062
Author(s):  
Barbara Kędzierska ◽  
Katarzyna Potrykus ◽  
Agnieszka Szalewska-Pałasz ◽  
Beata Wodzikowska
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Sequence Similarity ◽  
In Vitro Transcription ◽  
High Sequence Similarity ◽  
Sequence Composition ◽  
Repressor Complex ◽  
Transcriptional Fusions

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

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