Production of equine sera as a potential immunotherapy against COVID-19

Emerging viruses such as the COVID-19-inducing virus, SARSCoV- 2, represent a threat to human health, unless effective vaccines, drugs or alternative treatments, such as passive immunization, become accessible. Animal-derived immunoglobulins, such as equine immunoglobulins might be useful as immunoprophylaxis or immunotherapy against this viral disease. Therapeutic antibodies (Abs) for SARS-CoV-2 were obtained from hyperimmune equine plasma using the Spike protein receptor binding domain (RBD) as an immunogen. The presence of anti-RBD antibodies was evaluated by ELISA and the titres of neutralizing antibodies were determined in viral cell culture. Immunized horses generated high-titre of anti-RBD antibodies with antiviral neutralizing activity on Vero-E6 cells of 1/1,000. To minimize potential adverse effects, the immunoglobulins were digested with pepsin, and purified to obtain the F(ab’)2 fragments with the protocol standardized by Biotecfar C.A for the production of snake antivenom. Pre-immune serum displayed an unexpected anti-RBD reactivity by ELISA (titre up to 1/900) and Western Blot, but no angioneutralizing activity. Modelling of the RBD of equine coronavirus showed that some of the known epitopes of SARS-CoV-2 RBD were structurally conserved in the equine coronavirus protein. This might suggest that some of the reactivity observed in the pre-immune serum to the SARS-CoV-2 RBD might be due to a previous exposure to equine coronavirus.

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Protein Footprinting, Conformational Dynamics and Interface-Adjacent Neutralization ‘Hotspots’ in the SARS-CoV-2 Spike Protein Receptor Binding Domain / Human ACE2 Interaction

10.26434/chemrxiv.13385387 ◽

2020 ◽

Author(s):

Dominic Narang ◽

Matthew Balmer ◽

D. Andrew James ◽

Derek Wilson

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Conformational Dynamics ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Additional Information ◽

Protein Receptor ◽

Hdx Ms

This study provides an HDX-MS based analysis of the interaction between the SARS-CoV-2 spike protein and the human Angiotensin Converting Enzyme 2. <div><br></div><div>- The data agree exactly with the X-ray co-crystal structure of this complex, but provide additional information based on shifts in dynamics that are observed just outside the interface. </div><div><br></div><div>- These dynamic changes occur specifically in regions that are the primary targets of neutralizing antibodies that target spike protein, suggesting that the neutralization mechanism may result from suppression of dynamic shifts in the spike Receptor Binding Domain (RBD) that are necessary for favorable binding thermodynamics in the spike / ACE2 interaction.</div>

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Passive Immunization of Piglets Using Equine Plasma Containing PRRS Virus-Neutralizing Antibodies

10.31274/ans_air-180814-171 ◽

2008 ◽

Author(s):

Mark Mogler ◽

D.L. Hank Harris

Keyword(s):

Neutralizing Antibodies ◽

Passive Immunization ◽

Prrs Virus ◽

Equine Plasma

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Versatile, Multivalent Nanobody Cocktails Efficiently Neutralize SARS-CoV-2

10.1101/2020.08.24.264333 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yufei Xiang ◽

Sham Nambulli ◽

Zhengyun Xiao ◽

Heng Liu ◽

Zhe Sang ◽

...

Keyword(s):

Crystal Structure ◽

Global Health ◽

Neutralizing Antibodies ◽

Cost Effective ◽

Structural Proteomics ◽

Receptor Binding Domain ◽

Health Crisis ◽

Protein Receptor ◽

Large Repertoire ◽

Century Health

AbstractThe outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly efficacious therapeutics are urgently needed. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered multiple elite Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration, more potent than some of the best human neutralizing antibodies. We determined a crystal structure of such an elite neutralizing Nb in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. Structural characterization facilitated the bioengineering of novel multivalent Nb constructs into multi-epitope cocktails that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization. These promising agents are readily translated into efficient, cost-effective, and convenient therapeutics to help end this once-in-a-century health crisis.

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Development of equine antisera with high neutralizing activity against SARS-CoV-2

10.21203/rs.3.rs-83582/v1 ◽

2020 ◽

Author(s):

Gajanan Sapkal ◽

Anil Yadav ◽

Gururaj Rao Deshpande ◽

Pragya D. Yadav ◽

Ketki Deshpande ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Passive Immunization ◽

Cost Effective ◽

Viral Disease ◽

Effective Vaccine ◽

Effective Treatment Option ◽

Bacterial Diseases ◽

Igg Binding ◽

Effective Product

Abstract The pandemic of COVID -19 caused by SARS-CoV-2 is leading to a humongous impact on the mankind with over a million people succumbing to it worldwide. Although there are few drugs approved for the treatment, there is not yet a safe and effective vaccine available for COVID-19. Also, the passive immunization therapy with convalescent plasma, though potentially an effective treatment option for other viral disease has limitation of availability. The prior use of immunoglobulins generated in animals has proven to be effective in several viral and bacterial diseases. Here, we report the development and evaluation of equine hyper immune globulin raised against inactivated SARS-CoV-2 virus. Post immunization neutralization titres of the equines demonstrated high neutralizing antibodies. To minimize the adverse effects, the immunoglobulins were digested with pepsin, and purified to obtain the F(ab’)2 fragments. The average nAb titre of the purified bulk was 22,927 and correlated with high IgG binding efficiency in ELISA. The quality control assessments of the different batches proved to have consistent nAb titres. The study provides evidence of the potential of generating highly purified F(ab’)2 from equines against SARS-CoV-2 that can demonstrate consistent and high neutralization activity. Further, in-vivo testing for efficacy of this indigenously developed, cost effective product will pave the way to clinical evaluation.

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Titers, Prevalence, and Duration of SARS-CoV-2 Antibodies in a Local COVID-19 Outbreak and Following Vaccination

Vaccines ◽

10.3390/vaccines9060587 ◽

2021 ◽

Vol 9 (6) ◽

pp. 587

Author(s):

Jodi F. Hedges ◽

Macy A. Thompson ◽

Deann T. Snyder ◽

Amanda Robison ◽

Matthew P. Taylor ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Natural Infection ◽

Herd Immunity ◽

Antibody Responses ◽

Receptor Binding Domain ◽

Specific Antibodies ◽

Neutralization Capacity ◽

Protein Receptor ◽

Asymptomatic Infections ◽

Antibody Levels

Information concerning the development of neutralizing antibodies and their duration will be critical to establishing herd immunity for COVID-19. We sought to evaluate SARS-CoV-2 spike protein receptor-binding domain (RBD)-specific antibodies, their duration, and capacity for SARS-CoV-2 neutralization in volunteers while the pandemic spread within our community starting in March 2020. Those participants with the highest starting titers had the longest-lasting response, up to 12 months post-diagnosis. SARS-CoV-2 neutralization capacity was correlated with anti-RBD antibody levels. The majority of our participants with confirmed COVID-19 diagnosis had very mild or asymptomatic infections. We also detected low and largely non-neutralizing anti-RBD IgG titers in a few participants with no known COVID-19 diagnosis. Finally, we found that antibody responses induced by vaccination were significantly higher than those induced by natural infection. Thus, our study suggests that vaccination is still critical even for those naturally infected or diagnosed with COVID-19.

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Recombinant Receptor-Binding Domains of Multiple Middle East Respiratory Syndrome Coronaviruses (MERS-CoVs) Induce Cross-Neutralizing Antibodies against Divergent Human and Camel MERS-CoVs and Antibody Escape Mutants

Journal of Virology ◽

10.1128/jvi.01651-16 ◽

2016 ◽

Vol 91 (1) ◽

Cited By ~ 34

Author(s):

Wanbo Tai ◽

Yufei Wang ◽

Craig A. Fett ◽

Guangyu Zhao ◽

Fang Li ◽

...

Keyword(s):

Middle East ◽

Receptor Binding ◽

Broad Spectrum ◽

Neutralizing Antibodies ◽

Middle East Respiratory Syndrome ◽

Receptor Binding Domain ◽

Vaccine Candidates ◽

Vaccine Target ◽

Protein Receptor ◽

Escape Mutants

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via the spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014, and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing monoclonal antibodies (MAbs) and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 MAb escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains. IMPORTANCE MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and Camel-RBD, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity, and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.

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Antibodies Elicited in Response to a Single Cycle Glycoprotein D Deletion Viral Vaccine Candidate Bind C1q and Activate Complement Mediated Neutralization and Cytolysis

Viruses ◽

10.3390/v13071284 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1284

Author(s):

Maria Luisa Visciano ◽

Aakash Mahant Mahant ◽

Carl Pierce ◽

Richard Hunte ◽

Betsy C. Herold

Keyword(s):

Neutralizing Antibodies ◽

High Titer ◽

Passive Immunization ◽

Immune Serum ◽

Effective Vaccine ◽

Glycoprotein D ◽

Single Cycle ◽

Viral Vaccine ◽

Trial Outcomes ◽

Simplex Virus

Herpes simplex virus (HSV) prevention is a global health priority but, despite decades of research, there is no effective vaccine. Prior efforts focused on generating glycoprotein D (gD) neutralizing antibodies, but clinical trial outcomes were disappointing. The deletion of gD yields a single-cycle candidate vaccine (∆gD-2) that elicits high titer polyantigenic non-gD antibodies that exhibit little complement-independent neutralization but mediate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Active or passive immunization with ΔgD-2 completely protects mice from lethal disease and latency following challenge with clinical isolates of either serotype. The current studies evaluated the role of complement in vaccine-elicited protection. The immune serum from the ΔgD-2 vaccinated mice exhibited significantly greater C1q binding compared to the serum from the gD protein vaccinated mice with infected cell lysates from either serotype as capture antigens. The C1q-binding antibodies recognized glycoprotein B. This resulted in significantly greater antibody-mediated complement-dependent cytolysis and neutralization. Notably, complete protection was preserved when the ΔgD-2 immune serum was passively transferred into C1q knockout mice, suggesting that ADCC and ADCP are sufficient in mice. We speculate that the polyfunctional responses elicited by ΔgD-2 may prove more effective in preventing HSV, compared to the more restrictive responses elicited by adjuvanted gD protein vaccines.

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Non-replicating adenovirus based Mayaro virus vaccine elicits protective immune responses and cross protects against other alphaviruses

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009308 ◽

2021 ◽

Vol 15 (4) ◽

pp. e0009308

Author(s):

John M. Powers ◽

Nicole N. Haese ◽

Michael Denton ◽

Takeshi Ando ◽

Craig Kreklywich ◽

...

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Protective Efficacy ◽

Human Adenovirus ◽

Viral Disease ◽

Cross Reactivity ◽

Immune Serum ◽

Viral Dissemination ◽

Lethal Infection ◽

Mayaro Virus

Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFN⍺R1-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFN⍺R1-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses–chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFN⍺R1-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas.

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Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants

10.1101/2021.11.29.470362 ◽

2021 ◽

Author(s):

Jonathan M Labriola ◽

Shane Miersch ◽

Gang Chen ◽

Chao Chen ◽

Alevtina Pavlenco ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Protein S ◽

Receptor Binding Domain ◽

Resistant Variant ◽

S Protein ◽

High Affinity ◽

Protein Receptor ◽

Peptide Antibody ◽

N Terminus ◽

Host Infection

The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used naive phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD <1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

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Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

Nature Communications ◽

10.1038/s41467-020-19204-y ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Tânia F. Custódio ◽

Hrishikesh Das ◽

Daniel J. Sheward ◽

Leo Hanke ◽

Samuel Pazicky ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Spike Protein ◽

Receptor Binding Domain ◽

Combined Approach ◽

Emerging Viruses ◽

Medium Term ◽

Neutralization Activity ◽

High Affinity ◽

Isolation And Characterization

Abstract The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

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