Irradiation of UVC LED at 277 nm inactivates coronaviruses by photodegradation of spike protein.

To interrupt SARS-CoV-2 transmission chains, Ultraviolet-C (UVC) irradiation has emerged as a potential disinfection tool to aid in blocking the spread of coronaviruses. While conventional 254-nm UVC mercury lamps have been used for disinfection purposes, other UVC wavelengths have emerged as attractive alternatives but a direct comparison of these tools is lacking with the inherent mechanistic properties unclear. Our results using human coronaviruses, hCoV-229E and hCoV-OC43, have indicated that 277-nm UVC LED is most effective in viral inactivation, followed by 222-nm far UVC and 254-nm UVC mercury lamp. While UVC mercury lamp is more effective in degrading viral genomic content compared to 277-nm UVC LED, the latter results in a pronounced photo-degradation of spike proteins which potentially contributed to the higher efficacy of coronavirus inactivation. Hence, inactivation of coronaviruses by 277-nm UVC LED irradiation constitutes a more promising method for disinfection.

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Antigen-based multiplex strategies to discriminate SARS-CoV-2 natural and vaccine induced immunity from seasonal human coronavirus humoral responses

10.1101/2021.02.10.21251518 ◽

2021 ◽

Author(s):

Eric D. Laing ◽

Spencer L. Sterling ◽

Stephanie A. Richard ◽

Nusrat J. Epsi ◽

Si’Ana Coggins ◽

...

Keyword(s):

De Novo ◽

Natural Infection ◽

Strong Relationship ◽

Spike Protein ◽

Antibody Detection ◽

Natural Immunity ◽

Serum Samples ◽

Multiplex Immunoassay ◽

Human Coronaviruses ◽

Spike Proteins

ABSTRACTSensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 sero-surveillance. With the rollout of SARS-CoV-2 vaccines, such assays must be able to distinguish vaccine from natural immunity to SARS-CoV-2 and related human coronaviruses. Here, we developed and implemented multiplex microsphere-based immunoassay strategies for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2 and the endemic seasonal human coronaviruses (HCoV), enabling high throughout measurement of pre-existing cross-reactive antibodies. We varied SARS-CoV-2 antigen compositions within the multiplex assay, allowing direct comparisons of the effects of spike protein, receptor-binding domain protein (RBD) and nucleocapsid protein (NP) based SARS-CoV-2 antibody detection. Multiplex immunoassay performance characteristics are antigen-dependent, and sensitivities and specificities range 92-99% and 94-100%, respectively, for human subject samples collected as early as 7-10 days from symptom onset. SARS-CoV-2 spike and RBD had a strong correlative relationship for the detection of IgG. Correlation between detectable IgG reactive with spike and NP also had strong relationship, however, several PCR-positive and spike IgG-positive serum samples were NP IgG-negative. This spike and NP multiplex immunoassay has the potential to be useful for differentiation between vaccination and natural infection induced antibody responses. We also assessed the induction of de novo SARS-CoV-2 IgG cross reactions with SARS-CoV and MERS-CoV spike proteins. Furthermore, multiplex immunoassays that incorporate spike proteins of SARS-CoV-2 and HCoVs will permit investigations into the influence of HCoV antibodies on COVID-19 clinical outcomes and SARS-CoV-2 antibody durability.

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Immunological imprinting of the antibody response in COVID-19 patients

Nature Communications ◽

10.1038/s41467-021-23977-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Teresa Aydillo ◽

Alexander Rombauts ◽

Daniel Stadlbauer ◽

Sadaf Aslam ◽

Gabriela Abelenda-Alonso ◽

...

Keyword(s):

Immune Response ◽

Severe Acute Respiratory Syndrome ◽

Antibody Response ◽

Humoral Immune Response ◽

Nucleocapsid Protein ◽

Spike Protein ◽

Ongoing Research ◽

Variable Regions ◽

Humoral Immune ◽

Human Coronaviruses

AbstractIn addition to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), humans are also susceptible to six other coronaviruses, for which consecutive exposures to antigenically related and divergent seasonal coronaviruses are frequent. Despite the prevalence of COVID-19 pandemic and ongoing research, the nature of the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here we longitudinally profile the early humoral immune response against SARS-CoV-2 in hospitalized coronavirus disease 2019 (COVID-19) patients and quantify levels of pre-existing immunity to OC43, HKU1 and 229E seasonal coronaviruses, and find a strong back-boosting effect to conserved but not variable regions of OC43 and HKU1 betacoronaviruses spike protein. However, such antibody memory boost to human coronaviruses negatively correlates with the induction of IgG and IgM against SARS-CoV-2 spike and nucleocapsid protein. Our findings thus provide evidence of immunological imprinting by previous seasonal coronavirus infections that can potentially modulate the antibody profile to SARS-CoV-2 infection.

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Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain

10.21203/rs.3.rs-59058/v1 ◽

2020 ◽

Author(s):

Mohamed Raef Smaoui ◽

Hamdi Yahyaoui

Keyword(s):

Receptor Binding ◽

Single Point ◽

Protein Structures ◽

Point Mutations ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Point Mutant ◽

The Stability ◽

Spike Proteins

Abstract The interaction between the receptor-binding domain of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explore attempts at affecting the binding interaction between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the receptor-binding domain and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on potential mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3,800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing an effective vaccine.

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Mapping of UV-C dose and SARS-CoV-2 viral inactivation across N95 respirators during decontamination

Scientific Reports ◽

10.1038/s41598-021-98121-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alisha Geldert ◽

Alison Su ◽

Allison W. Roberts ◽

Guillaume Golovkine ◽

Samantha M. Grist ◽

...

Keyword(s):

Optical Simulation ◽

Pathogen Inactivation ◽

Viral Inactivation ◽

Flexible Form ◽

Measurement Tools ◽

Ultraviolet C ◽

N95 Respirators ◽

Uv C ◽

Dose Difference

AbstractDuring public health crises like the COVID-19 pandemic, ultraviolet-C (UV-C) decontamination of N95 respirators for emergency reuse has been implemented to mitigate shortages. Pathogen photoinactivation efficacy depends critically on UV-C dose, which is distance- and angle-dependent and thus varies substantially across N95 surfaces within a decontamination system. Due to nonuniform and system-dependent UV-C dose distributions, characterizing UV-C dose and resulting pathogen inactivation with sufficient spatial resolution on-N95 is key to designing and validating UV-C decontamination protocols. However, robust quantification of UV-C dose across N95 facepieces presents challenges, as few UV-C measurement tools have sufficient (1) small, flexible form factor, and (2) angular response. To address this gap, we combine optical modeling and quantitative photochromic indicator (PCI) dosimetry with viral inactivation assays to generate high-resolution maps of “on-N95” UV-C dose and concomitant SARS-CoV-2 viral inactivation across N95 facepieces within a commercial decontamination chamber. Using modeling to rapidly identify on-N95 locations of interest, in-situ measurements report a 17.4 ± 5.0-fold dose difference across N95 facepieces in the chamber, yielding 2.9 ± 0.2-log variation in SARS-CoV-2 inactivation. UV-C dose at several on-N95 locations was lower than the lowest-dose locations on the chamber floor, highlighting the importance of on-N95 dose validation. Overall, we integrate optical simulation with in-situ PCI dosimetry to relate UV-C dose and viral inactivation at specific on-N95 locations, establishing a versatile approach to characterize UV-C photoinactivation of pathogens contaminating complex substrates such as N95s.

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Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens

Processes ◽

10.3390/pr8121539 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1539

Author(s):

Paco Pino ◽

Joeri Kint ◽

Divor Kiseljak ◽

Valentina Agnolon ◽

Giampietro Corradin ◽

...

Keyword(s):

Diagnostic Tests ◽

Neutralizing Antibodies ◽

Good Manufacturing Practice ◽

Spike Protein ◽

High Quality ◽

Virus Surface ◽

Corona Virus ◽

Global Demand ◽

Diagnostic Sensitivity And Specificity ◽

Spike Proteins

The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell populations and on protein purification via a two-step scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2-specific-immunoglobulin molecules was found to exceed that of spike fragments (Spike subunit-1, S1 and Receptor Binding Domain, RBD). The process described here will enable production of sufficient high-quality trimeric spike protein to meet the global demand for SARS-CoV-2 diagnostic tests and potentially vaccines.

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High-Potency Polypeptide-based Interference for Coronavirus Spike Glycoproteins

10.21203/rs.3.rs-388285/v1 ◽

2021 ◽

Author(s):

Qinghua Wang ◽

Jianpeng Ma ◽

Adam Acevedo

Keyword(s):

Amino Acid Sequence Identity ◽

Natural Infection ◽

Viral Proteins ◽

Spike Protein ◽

High Potency ◽

Viral Pathogens ◽

Native Sequence ◽

Virion Envelope ◽

Novel Concept ◽

Spike Proteins

Abstract The world is experiencing an unprecedented coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 spike protein-based vaccines are currently the main preventive agent to fight against the virus. However, several variants with extensive mutations in SARS-CoV-2 spike proteins have emerged. Some of these variants exhibited increased replication, higher transmission and virulence, and were partially resistant to antibody neutralization from natural infection or vaccination. With over 130 million confirmed cases and widespread vaccination around the globe, the emergence of new escape SARS-CoV-2 variants could be accelerated. New therapeutics insensitive to mutations are thus urgently needed. Here we have developed an inhibitor based on SARS-CoV-2 spike protein that potently reduced pseudovirus infectivity by limiting the level of SARS-CoV-2 spike proteins on virion envelope. Most importantly, the inhibitor was equally effective against other coronavirus spike proteins that shared as low as 35% amino-acid sequence identity, underscoring its extreme tolerance to mutations. The small-sized inhibitor would also allow simple delivery by, for instance, nasal spray. We expect the inhibitor reported here to be an invaluable aid to help end COVID-19 pandemic. Furthermore, the use of a partial native sequence or its homologues to interfere with the functions of the native protein represents a novel concept for targeting other viral proteins in combating against important viral pathogens.

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Harnessing the Sars-cov-2 Spike Protein Binding Affinity to Ace2 Receptor Through Narcotinic Compounds Combined With Adjuvants: an in Silico Insight

10.21203/rs.3.rs-254891/v1 ◽

2021 ◽

Author(s):

Saeedeh Mohammadi ◽

Esmail Doustkhah ◽

Nader Sakhaee ◽

Ayoub Esmailpour ◽

Mohammad Esmailpour

Keyword(s):

Binding Affinity ◽

In Silico ◽

Muramyl Dipeptide ◽

Docking Studies ◽

Spike Protein ◽

Binding Affinities ◽

Narcotic Analgesics ◽

S Protein ◽

Suitable Combination ◽

Spike Proteins

Abstract Protein products of SARS-CoV-2 spike (S) coding gene sequence, were all analyzed and compared to other SARS-CoV S proteins to elucidate structural similarities of spike proteins. A homology modeling of SARS-CoV-2 S protein was obtained and used in molecular docking studies to find binding affinities of spike protein for angiotensin-converting enzyme 2 (ACE2). The two most important binding sites of S protein, namely, RBD and CTD, critically responsible for binding interactions, were identified. Finally, binding affinity of RBD and CTD domains of S protein with narcotic analgesics are studied. Moreover, interactions of ACE2 receptor- S protein with narcotic compounds when mixed with small molecule adjuvants to improve the immune response and increase the efficacy of potential vaccines, were taken into consideration. In-silico results suggest that the combination of narcotine hemiacetal with mannide monooleate shows a stronger binding affinity with CTD, while carprofen-muramyl dipeptide and squalene have stronger binding affinities for the RBD portion of S protein. Thus, a suitable combination of these narcotic is proposed to yield potent site-blocking efficacy for ACE2 receptor against SARS-CoV-2 spike proteins.

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Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations

Nature Immunology ◽

10.1038/s41590-021-01068-z ◽

2021 ◽

Author(s):

Vincent Dussupt ◽

Rajeshwer S. Sankhala ◽

Letzibeth Mendez-Rivera ◽

Samantha M. Townsley ◽

Fabian Schmidt ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Low Dose ◽

Viral Escape ◽

Spike Protein ◽

Receptor Binding Domain ◽

Viral Inactivation ◽

Angiotensin Converting Enzyme 2 ◽

Therapeutic Monoclonal Antibodies ◽

Potent Protection

AbstractPrevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

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Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies

10.1101/2021.02.18.431897 ◽

2021 ◽

Author(s):

Takuya Tada ◽

Belinda M. Dcosta ◽

Hao Zhou ◽

Ada Vaill ◽

Wes Kazmierski ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Receptor Binding ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Neutralization Activity ◽

Protein Mutations ◽

Spike Proteins

AbstractMonoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide protection against severe COVID-19. The emergence of SARS-CoV-2 variants with heavily mutated spike proteins raises the concern that the therapy could become less effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study, we tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising concerns of reduced efficacy in the treatment of patients infected with variant viruses. The results suggest that there is a need to develop additional monoclonal antibodies that are not affected by the current spike protein mutations.

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Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform

10.1101/2021.03.23.21253460 ◽

2021 ◽

Author(s):

Sidhartha Chaudhury ◽

Jack Hutter ◽

Jessica S Bolton ◽

Shilpa Hakre ◽

Evelyn Mose ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Cross Reactivity ◽

Multiplex Assay ◽

Spike Protein ◽

Contact Tracing ◽

Epitope Specificity ◽

Army Hospital ◽

Protein Receptor ◽

Healthcare Administrators ◽

Human Coronaviruses

AbstractSerological assessment of SARS-CoV-2 specific responses are an essential tool for determining the prevalence of past SARS-CoV-2 infections in the population especially when testing occurs after symptoms have developed and limited contact tracing is in place. The goal of our study was to test a new 10-plex electro-chemiluminescence-based assay to measure IgM and IgG responses to the spike proteins from multiple human coronaviruses including SARS-CoV-2, assess the epitope specificity of the SARS-CoV-2 antibody response against full-length spike protein, receptor-binding domain and N-terminal domain of the spike protein, and the nucleocapsid protein. We carried out the assay on samples collected from three sample groups: subjects diagnosed with COVID-19 from the U.S. Army hospital at Camp Humphreys in Pyeongtaek, South Korea; healthcare administrators from the same hospital but with no reported diagnosis of COVID-19; and pre-pandemic samples. We found that the new CoV-specific multiplex assay was highly sensitive allowing plasma samples to be diluted 1:30,000 with a robust signal. The reactivity of IgG responses to SARS-CoV-2 nucleocapsid protein and IgM responses to SARS-CoV-2 spike protein could distinguish COVID-19 samples from non-COVID-19 and pre-pandemic samples. The data from the three sample groups also revealed a unique pattern of cross-reactivity between SARS-CoV-2 and SARS-CoV-1, MERS-CoV, and seasonal coronaviruses HKU1 and OC43. Our findings show that the CoV-2 IgM response is highly specific while the CoV-2 IgG response is more cross-reactive across a range of human CoVs and also showed that IgM and IgG responses show distinct patterns of epitope specificity. In summary, this multiplex assay was able to distinguish samples by COVID-19 status and characterize distinct trends in terms of cross-reactivity and fine-specificity in antibody responses, underscoring its potential value in diagnostic or serosurveillance efforts.

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