Mutation profile of SARS-CoV-2 spike protein and identification of potential multiple epitopes within spike protein for vaccine development against SARS-CoV-2

AbstractDevelopment of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

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SARS-CoV-2 full length spike protein for COVID-19 vaccine development and diagnostic testing

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2020.12.546 ◽

2021 ◽

Vol 147 (2) ◽

pp. AB152

Author(s):

Crystal Richardson ◽

Mayuresh Abhyankar ◽

Jillian Bracaglia ◽

Sayeh Agah ◽

Zachary Schuhmacher ◽

...

Keyword(s):

Vaccine Development ◽

Diagnostic Testing ◽

Full Length ◽

Spike Protein

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SARS-CoV-2 RBD in vitro evolution parrots and predicts contagious mutation spread

10.21203/rs.3.rs-183310/v1 ◽

2021 ◽

Author(s):

Gideon Schreiber ◽

Jiri Zahradník ◽

Shir Marciano ◽

Maya Shemesh ◽

Eyal Zoler ◽

...

Keyword(s):

South African ◽

Convergent Evolution ◽

Vaccine Development ◽

Spike Protein ◽

Structural Basis ◽

Receptor Binding Domain ◽

In Vitro Evolution ◽

High Affinity ◽

Future Drug

Abstract SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the “European” (20E-EU1), “British” (501.V1),”South African” (501.V2), and Brazilian variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9Å Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

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Identification of a novel neutralizing epitope on the N-terminal domain of the HCoV-229E spike protein

Journal of Virology ◽

10.1128/jvi.01955-21 ◽

2021 ◽

Author(s):

Jiale Shi ◽

Yuejun Shi ◽

Ruixue Xiu ◽

Gang Wang ◽

Rui Liang ◽

...

Keyword(s):

Epitope Mapping ◽

Vaccine Development ◽

Selective Pressure ◽

Spike Protein ◽

Neutralizing Epitope ◽

Change Over Time ◽

S Protein ◽

Terminal Domain ◽

Neutralizing Epitopes ◽

Over Time

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. Importance Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

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Engineering a Live Attenuated Porcine Epidemic Diarrhea Virus Vaccine Candidate via Inactivation of the Viral 2'-O-Methyltransferase and the Endocytosis Signal of the Spike Protein

Journal of Virology ◽

10.1128/jvi.00406-19 ◽

2019 ◽

Vol 93 (15) ◽

Cited By ~ 10

Author(s):

Yixuan Hou ◽

Hanzhong Ke ◽

Jineui Kim ◽

Dongwan Yoo ◽

Yunfang Su ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Vaccine Development ◽

Vaccine Candidate ◽

Spike Protein ◽

Economic Losses ◽

High Dose ◽

Type I ◽

Viral Pathogen ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2′-O-methyltransferase (2′-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2′-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A. Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2′-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2′-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

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Generating stable chinese hamster ovary cell clones to produce a truncated SARS-CoV spike protein for vaccine development

Biotechnology Progress ◽

10.1002/btpr.480 ◽

2010 ◽

Vol 26 (6) ◽

pp. 1733-1740 ◽

Cited By ~ 4

Author(s):

Shih-Chang Lin ◽

Chih-Hsiang Leng ◽

Suh-Chin Wu

Keyword(s):

Chinese Hamster Ovary Cell ◽

Chinese Hamster Ovary ◽

Vaccine Development ◽

Chinese Hamster ◽

Spike Protein ◽

Ovary Cell ◽

Cell Clones

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Clinical Profile of COVID-19 in Children and Research Progress on Angiotensin-converting Enzyme 2: A Mini-review

Journal of Nepal Medical Association ◽

10.31729/jnma.5436 ◽

2020 ◽

Vol 58 (230) ◽

Author(s):

Qian Gao ◽

Jing Liu ◽

Zhilong Mu ◽

Xianpeng Yan ◽

Jay Narayan Shah ◽

...

Keyword(s):

Clinical Characteristics ◽

Vaccine Development ◽

Spike Protein ◽

Acute Injury ◽

Research Progress ◽

Converting Enzyme ◽

Future Prospects ◽

Strategic Information ◽

Angiotensin Converting Enzyme 2 ◽

Short Course

The cases of coronavirusdisease 2019 in children have been increasing with the ongoing pandemic.The finding suggests children have mild symptoms and a short course of the disease. Angiotensinconverting enzyme-2 mediates entry of the virus into the cell, the combination of virus and ACE2 leads to an increase in activity of angiotensin II, resulting in acute injury to lungs, myocardium and other organs. The infection causes down-regulation of ACE2 expression. The ACE2 plays an important role in the infection progression and clinical characteristics of COVID-19. Works on ACE2 and virus spike protein have future prospects of strategic information on prevention, management as well as vaccine development.

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Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model

10.1101/2020.09.16.300970 ◽

2020 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Raveen Rathnasinghe ◽

Michael Schotsaert ◽

Lynda Coughlan ◽

...

Keyword(s):

Mouse Model ◽

Cleavage Site ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Middle Eastern ◽

Host Cells ◽

Spike Protein ◽

Optimized Design ◽

Angiotensin Converting Enzyme 2 ◽

Viral Target

AbstractThe spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the PP mutations completely protected from challenge in this mouse model.ImportanceA vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validates the choice of antigens that contain the PP mutation and suggests that deletion of the polybasic cleavage site could lead to a further optimized design.

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Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption

10.1101/2021.08.24.457448 ◽

2021 ◽

Author(s):

Wenlin Ren ◽

Jun Lan ◽

Xiaohui Ju ◽

Mingli Gong ◽

Quanxin Long ◽

...

Keyword(s):

The Netherlands ◽

Active Surveillance ◽

Functional Properties ◽

Genetic Variant ◽

Vaccine Development ◽

Binding Mode ◽

Spike Protein ◽

Adaptive Mutation ◽

Steric Clash ◽

Initial Transmission

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020.Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans.Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry.Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

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A Mycobacteriophage-Based Vaccine Platform: SARS-CoV-2 Antigen Expression and Display

Microorganisms ◽

10.3390/microorganisms9122414 ◽

2021 ◽

Vol 9 (12) ◽

pp. 2414

Author(s):

Krista G. Freeman ◽

Katherine S. Wetzel ◽

Yu Zhang ◽

Kira M. Zack ◽

Deborah Jacobs-Sera ◽

...

Keyword(s):

Single Particle ◽

High Purity ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Antigen Expression ◽

Spike Protein ◽

Infectious Agents ◽

Protein Antigens ◽

Vaccine Platform ◽

Excellent Safety Profile

The explosion of SARS-CoV-2 infections in 2020 prompted a flurry of activity in vaccine development and exploration of various vaccine platforms, some well-established and some new. Phage-based vaccines were described previously, and we explored the possibility of using mycobacteriophages as a platform for displaying antigens of SARS-CoV-2 or other infectious agents. The potential advantages of using mycobacteriophages are that a large and diverse variety of them have been described and genomically characterized, engineering tools are available, and there is the capacity to display up to 700 antigen copies on a single particle approximately 100 nm in size. The phage body may itself be a good adjuvant, and the phages can be propagated easily, cheaply, and to high purity. Furthermore, the recent use of these phages therapeutically, including by intravenous administration, suggests an excellent safety profile, although efficacy can be restricted by neutralizing antibodies. We describe here the potent immunogenicity of mycobacteriophage Bxb1, and Bxb1 recombinants displaying SARS-CoV-2 Spike protein antigens.

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