Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

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The receptor binding domain of SARS-CoV-2 spike is the key target of neutralizing antibody in human polyclonal sera

10.1101/2020.08.21.261727 ◽

2020 ◽

Cited By ~ 2

Author(s):

Tara L. Steffen ◽

E. Taylor Stone ◽

Mariah Hassert ◽

Elizabeth Geerling ◽

Brian T. Grimberg ◽

...

Keyword(s):

Antibody Response ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Natural Infection ◽

Neutralizing Antibody ◽

Binding Domain ◽

Antigenic Determinants ◽

Receptor Binding Domain ◽

Neutralization Capacity

AbstractNatural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.

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Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development

Journal of Virology ◽

10.1128/jvi.02357-13 ◽

2013 ◽

Vol 87 (21) ◽

pp. 11963-11963 ◽

Cited By ~ 4

Author(s):

L. Du ◽

G. Zhao ◽

Z. Kou ◽

C. Ma ◽

S. Sun ◽

...

Keyword(s):

Middle East ◽

Receptor Binding ◽

Vaccine Development ◽

Binding Domain ◽

Middle East Respiratory Syndrome ◽

The Novel ◽

Receptor Binding Domain ◽

Human Coronavirus ◽

S Protein

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Biochemical, Biophysical, and Functional Characterization of Bacterially Expressed and Refolded Receptor Binding Domain ofPlasmodium vivaxDuffy-binding Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m101531200 ◽

2001 ◽

Vol 276 (20) ◽

pp. 17111-17116 ◽

Cited By ~ 69

Author(s):

Sanjay Singh ◽

Kailash Pandey ◽

Rana Chattopadhayay ◽

Syed Shams Yazdani ◽

Andrew Lynn ◽

...

Keyword(s):

Receptor Binding ◽

Binding Protein ◽

Functional Characterization ◽

Binding Domain ◽

Receptor Binding Domain

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Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

10.1101/2021.03.15.435528 ◽

2021 ◽

Author(s):

Alexandra C Walls ◽

Marcos C Miranda ◽

Minh N Pham ◽

Alexandra Schaefer ◽

Allison Greaney ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Polyclonal Antibodies ◽

Clinical Stage ◽

Binding Domain ◽

Single Shot ◽

Receptor Binding Domain ◽

Negative Consequences ◽

Neutralizing Activity

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

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Comparative Immunomodulatory Evaluation of the Receptor Binding Domain of the SARS-CoV-2 Spike Protein; a Potential Vaccine Candidate Which Imparts Potent Humoral and Th1 Type Immune Response in a Mouse Model

Frontiers in Immunology ◽

10.3389/fimmu.2021.641447 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tripti Shrivastava ◽

Balwant Singh ◽

Zaigham Abbas Rizvi ◽

Rohit Verma ◽

Sandeep Goswami ◽

...

Keyword(s):

Immune Response ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Vaccine Candidate ◽

Unmet Need ◽

Binding Domain ◽

Spike Protein ◽

Cell Immune Response ◽

Receptor Binding Domain

The newly emerged novel coronavirus, SARS-CoV-2, the causative agent of COVID-19 has proven to be a threat to the human race globally, thus, vaccine development against SARS-CoV-2 is an unmet need driving mass vaccination efforts. The receptor binding domain of the spike protein of this coronavirus has multiple neutralizing epitopes and is associated with viral entry. Here we have designed and characterized the SARS-CoV-2 spike protein fragment 330-526 as receptor binding domain 330-526 (RBD330-526) with two native glycosylation sites (N331 and N343); as a potential subunit vaccine candidate. We initially characterized RBD330-526 biochemically and investigated its thermal stability, humoral and T cell immune response of various RBD protein formulations (with or without adjuvant) to evaluate the inherent immunogenicity and immunomodulatory effect. Our result showed that the purified RBD immunogen is stable up to 72 h, without any apparent loss in affinity or specificity of interaction with the ACE2 receptor. Upon immunization in mice, RBD generates a high titer humoral response, elevated IFN-γ producing CD4+ cells, cytotoxic T cells, and robust neutralizing antibodies against live SARS-CoV-2 virus. Our results collectively support the potential of RBD330-526 as a promising vaccine candidate against SARS-CoV-2.

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