Recombinant Infectious Bronchitis Coronavirus Beaudette with the Spike Protein Gene of the Pathogenic M41 Strain Remains Attenuated but Induces Protective Immunity

ABSTRACT We have replaced the ectodomain of the spike (S) protein of the Beaudette strain (Beau-R; apathogenic for Gallus domesticus chickens) of avian infectious bronchitis coronavirus (IBV) with that from the pathogenic M41 strain to produce recombinant IBV BeauR-M41(S). We have previously shown that this changed the tropism of the virus in vitro (R. Casais, B. Dove, D. Cavanagh, and P. Britton, J. Virol. 77:9084-9089, 2003). Herein we have assessed the pathogenicity and immunogenicity of BeauR-M41(S). There were no consistent differences in pathogenicity between the recombinant BeauR-M41(S) and its apathogenic parent Beau-R (based on snicking, nasal discharge, wheezing, watery eyes, rales, and ciliostasis in trachea), and both replicated poorly in trachea and nose compared to M41; the S protein from the pathogenic M41 had not altered the apathogenic nature of Beau-R. Both Beau-R and BeauR-M41(S) induced protection against challenge with M41 as assessed by absence of recovery of challenge virus and nasal exudate. With regard to snicking and ciliostasis, BeauR-M41(S) induced greater protection (seven out of nine chicks [77%]; assessed by ciliostasis) than Beau-R (one out of nine; 11%) but less than M41 (100%). The greater protection induced by BeauR-M41(S) against M41 may be related to the ectodomain of the spike protein of Beau-R differing from that of M41 by 4.1%; a small number of epitopes on the S protein may play a disproportionate role in the induction of immunity. The results are promising for the prospects of S-gene exchange for IBV vaccine development.

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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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Vaccine Design against Coronavirus Spike (S) Glycoprotein in Chicken: Immunoinformatic and Computational Approaches

10.21203/rs.3.rs-25375/v2 ◽

2020 ◽

Author(s):

Eman Ali Awadelkareem ◽

Sumaia Awad Elkariem Ali

Keyword(s):

T Cell ◽

Rna Virus ◽

Protein S ◽

Economic Loss ◽

Protein Characterization ◽

Spike Protein ◽

T Cell Epitopes ◽

Resistance Response ◽

S Protein ◽

Infectious Bronchitis

Abstract Background: Infectious bronchitis (IB) is a highly contagious respiratory disease in chickens and produces economic loss within the poultry industry. This disease is caused by a single stranded RNA virus belonging to Cronaviridae family. This study aimed to design a potential multi-epitopes vaccine against Infectious bronchitis virus spike protein (S). Protein characterization was also performed for IBV spike protein.Methods: The present study used various tools in Immune Epitope Database (IEDB) to predict conserved B and T cell epitopes against IBV spike (S) protein that may perform a significant role in provoking the resistance response to IBV infection. Results: In B cell prediction methods, three epitopes (1139KKSSYY1144, 1140KSSYYT1145, 1141SSYYT1145) were selected as surface, linear and antigenic epitopes. Many MHCI and MHCII epitopes were predicted for IBV S protein. Among them 982YYITARDMY990 and 983YITARDMYM991 epitopes displayed high antigenicity, no allergenicity and no toxicity as well as great linkage with MHCI and MHCII alleles. Moreover, docking analysis of MHCI epitope produced strong binding affinity with BF2 alleles. Conclusion: Five conserved epitopes were expected from spike glycoprotein of IBV as the best B and T cell epitopes due to high antigenicity, no allergenicity and no toxicity. In addition, MHC epitopes showed great linkage with MHC alleles as well as strong interaction with BF2 alleles. These epitopes should be designed and incorporated and then tested as multi-epitope vaccine against IBV.

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Impacts of Coronavirus on Farm and Pet Animals

10.20944/preprints202008.0473.v2 ◽

2020 ◽

Author(s):

Sachin Subedi ◽

Sulove Koirala ◽

Lilong Chai

Keyword(s):

Infectious Bronchitis Virus ◽

Vaccine Development ◽

Rna Virus ◽

Farm Animals ◽

Effective Vaccine ◽

Nasal Discharge ◽

Infectious Bronchitis ◽

Gastrointestinal Lesions ◽

On Farm ◽

The Impact

Coronaviruses are positive sense RNA virus belonging to the Coronaviridae family, which are further subdivided into four genera: Alpha, Beta, Gamma, and Delta Coronaviruses. Infectious bronchitis virus and SARS-CoV belong to Beta Coronaviridae family. Infectious bronchitis virus causes respiratory and nephritic signs that includes tracheal rales, urate crystals, lethargy and nasal discharge. In livestock and pets, the Coronavirus infection causes mostly gastrointestinal lesions, which may be prevented through vaccination and biosecurity. Recent infections of SARS-CoV-2 (also known as COVID-19) on farm and pet animals were summarized in this study. Besides, zoo animals were reported with infections in some countries/regions. Although the damage of COVID-19 has not been reported as serious as highly pathogenic avian influenza (HPAI) and African Swine Fever (ASF) on farm animals so far, the transmission mechanism of COVID-19 among group animals/farms and its long-term impacts are still not clear. The impact of Coronavirus on animals and potential prevention strategies, such as vaccine development and farm biosecurity measures, were discussed. Prior to the development of the effective vaccine, the biosecurity measures (e.g., conventional disinfection strategies and innovated technologies) may play roles in preventing potential spread of diseases/viruses.

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Exosome-Mediated mRNA Delivery For SARS-CoV-2 Vaccination

10.1101/2020.11.06.371419 ◽

2020 ◽

Author(s):

Shang-Jui Tsai ◽

Chenxu Guo ◽

Nadia A. Atai ◽

Stephen J. Gould

Keyword(s):

Viral Vectors ◽

Vaccine Development ◽

Spike Protein ◽

Interleukin 4 ◽

Target Cells ◽

Durable Response ◽

Elevated Expression ◽

Cellular Immune

AbstractBackgroundIn less than a year from its zoonotic entry into the human population, SARS-CoV-2 has infected more than 45 million people, caused 1.2 million deaths, and induced widespread societal disruption. Leading SARS-CoV-2 vaccine candidates immunize with the viral spike protein delivered on viral vectors, encoded by injected mRNAs, or as purified protein. Here we describe a different approach to SARS-CoV-2 vaccine development that uses exosomes to deliver mRNAs that encode antigens from multiple SARS-CoV-2 structural proteins.ApproachExosomes were purified and loaded with mRNAs designed to express (i) an artificial fusion protein, LSNME, that contains portions of the viral spike, nucleocapsid, membrane, and envelope proteins, and (ii) a functional form of spike. The resulting combinatorial vaccine, LSNME/SW1, was injected into thirteen weeks-old, male C57BL/6J mice, followed by interrogation of humoral and cellular immune responses to the SARS-CoV-2 nucleocapsid and spike proteins, as well as hematological and histological analysis to interrogate animals for possible adverse effects.ResultsImmunized mice developed CD4+, and CD8+ T-cell reactivities that respond to both the SARS-CoV-2 nucelocapsid protein and the SARS-CoV-2 spike protein. These responses were apparent nearly two months after the conclusion of vaccination, as expected for a durable response to vaccination. In addition, the spike-reactive CD4+ T-cells response was associated with elevated expression of interferon gamma, indicative of a Th1 response, and a lesser induction of interleukin 4, a Th2-associated cytokine. Vaccinated mice showed no sign of altered growth, injection-site hypersensitivity, change in white blood cell profiles, or alterations in organ morphology. Consistent with these results, we also detected moderate but sustained anti-nucleocapsid and anti-spike antibodies in the plasma of vaccinated animals.ConclusionTaken together, these results validate the use of exosomes for delivering functional mRNAs into target cells in vitro and in vivo, and more specifically, establish that the LSNME/SW1 vaccine induced broad immunity to multiple SARS-CoV-2 proteins.

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Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

10.1101/2020.07.27.224089 ◽

2020 ◽

Author(s):

Matthew D. Beasley ◽

Sanja Aracic ◽

Fiona M. Gracey ◽

Ruban Kannan ◽

Avisa Masarati ◽

...

Keyword(s):

Vaccine Development ◽

Spike Protein ◽

Receptor Binding Domain ◽

Single Chain ◽

Single Chain Antibody ◽

High Affinity ◽

Scfv Library ◽

Antibody Libraries ◽

Prophylactic Use

AbstractAntibodies with high affinity against the receptor binding domain (RBD) of the SARS-CoV-2 S1 ectodomain were identified from screens using the Retained Display™ (ReD) platform employing a 1 × 1011 clone single-chain antibody (scFv) library. Numerous unique scFv clones capable of inhibiting binding of the viral S1 ectodomain to the ACE2 receptor in vitro were characterized. To maximize avidity, selected clones were reformatted as bivalent diabodies and monoclonal antibodies (mAb). The highest affinity mAb completely neutralized live SARS-CoV-2 virus in cell culture for four days at a concentration of 6.7 nM, suggesting potential therapeutic and/or prophylactic use. Furthermore, scFvs were identified that greatly increased the interaction of the viral S1 trimer with the ACE2 receptor, with potential implications for vaccine development.

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Analysis of the Role of TpUB05 Antigen from Theileria parva in Immune Responses to Malaria in Humans Compared to Its Homologue in Plasmodium falciparum the UB05 Antigen

Pathogens ◽

10.3390/pathogens9040271 ◽

2020 ◽

Vol 9 (4) ◽

pp. 271

Author(s):

Jerome Nyhalah Dinga ◽

Stephanie Numenyi Perimbie ◽

Stanley Dobgima Gamua ◽

Francis N. G. Chuma ◽

Dieudonné Lemuh Njimoh ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Immune Responses ◽

Protective Immunity ◽

Vaccine Development ◽

Theileria Parva ◽

Peripheral Blood Mononuclear ◽

Significant Difference ◽

First Time

Despite the amount of resources deployed and the technological advancements in molecular biology, vaccinology, immunology, genetics, and biotechnology, there are still no effective vaccines against malaria. Immunity to malaria is usually seen to be species- and/or strain-specific. However, there is a growing body of evidence suggesting the possibility of the existence of cross-strain, cross-species, and cross-genus immune responses in apicomplexans. The principle of gene conservation indicates that homologues play a similar role in closely related organisms. The homologue of UB05 in Theileria parva is TpUB05 (XP_763711.1), which has been tested and shown to be associated with protective immunity in East Coast fever. In a bid to identify potent markers of protective immunity to aid malaria vaccine development, TpUB05 was tested in malaria caused by Plasmodium falciparum. It was observed that TpUB05 was better at detecting antigen-specific antibodies in plasma compared to UB05 when tested by ELISA. The total IgG raised against TpUB05 was able to block parasitic growth in vitro more effectively than that raised against UB05. However, there was no significant difference between the two study antigens in recalling peripheral blood mononuclear cell (PBMC) memory through IFN-γ production. This study suggests, for the first time, that TpUB05 from T. parva cross-reacts with UB05 from P. falciparum and is a marker of protective immunity in malaria. Hence, TpUB05 should be considered for possible development as a potential subunit vaccine candidate against malaria.

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Identification of a Potent Inhibitor Targeting the Spike Protein of Pandemic Human Coronavirus, SARS-CoV-2 by Computational Methods

10.26434/chemrxiv.12197934.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

SRUTHI UNNI ◽

Snehal Aouti ◽

Padmanabhan Balasundaram

Keyword(s):

Vaccine Development ◽

Protein S ◽

Dynamics Simulation ◽

Spike Protein ◽

Simulation Studies ◽

Viral Pathogen ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Chemical Libraries ◽

Pi Interactions

<p>Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is an emerging new viral pathogen that causes severe respiratory disease. SARS-CoV-2 is responsible for an outbreak of COVID-19 pandemic worldwide. As there are no confirmed antiviral drugs or vaccines currently available for the treatment of COVID-19, discovering potent inhibitors or vaccines are urgently required for the benefit of humanity. The glycosylated Spike protein (S-protein) directly interacts with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. As the S-protein is exposed to the surface and is essential for entry into the host, the S-protein can be considered as a first-line therapeutic target for antiviral therapy and vaccine development. In-silico screening, docking and molecular dynamics simulation studies were performed to identify repurposing drugs using DrugBank and PubChem library against the RBD of S-protein. The study identified a laxative drug, Bisoxatin (DB09219), which is used for the treatment of constipation and preparation of the colon for surgical procedures. It binds nicely at the S-protein – ACE2 interface by making substantial pi-pi interactions with Tyr505 in the ‘Site 1’ hook region of RBD and hydrophilic interactions with Glu406, Ser494 and Thr500. Bisoxatin consistently binds to the protein throughout the 100 ns simulation. Taken together, we propose that the discovered molecule, Bisoxatin may be a potent repurpose drug to develop new chemical libraries for inhibiting SARS-CoV-2 entry into the host.</p>

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Identification of a Potent Inhibitor Targeting the Spike Protein of Pandemic Human Coronavirus, SARS-CoV-2 by Computational Methods

10.26434/chemrxiv.12197934 ◽

2020 ◽

Author(s):

SRUTHI UNNI ◽

Snehal Aouti ◽

Padmanabhan Balasundaram

Keyword(s):

Vaccine Development ◽

Protein S ◽

Dynamics Simulation ◽

Spike Protein ◽

Simulation Studies ◽

Viral Pathogen ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Chemical Libraries ◽

Pi Interactions

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Identification of a common deletion in the spike protein of SARS-CoV-2

10.1101/2020.03.31.015941 ◽

2020 ◽

Cited By ~ 9

Author(s):

Zhe Liu ◽

Huanying Zheng ◽

Runyu Yuan ◽

Mingyue Li ◽

Huifang Lin ◽

...

Keyword(s):

Protein Function ◽

Cleavage Site ◽

Vaccine Development ◽

Mechanistic Explanation ◽

Spike Protein ◽

Clinical Samples ◽

Genomic Changes ◽

And Function

AbstractTwo notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.

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