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. 2022 Jan;77(1):230-242.
doi: 10.1111/all.15066. Epub 2021 Sep 22.

Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes

Pia Gattinger  1 , Katarzyna Niespodziana  1 , Karin Stiasny  2 , Sabina Sahanic  3 , Inna Tulaeva  1   4 , Kristina Borochova  1 , Yulia Dorofeeva  1 , Thomas Schlederer  1 , Thomas Sonnweber  3 , Gerhard Hofer  5 , Renata Kiss  6 , Bernhard Kratzer  7 , Doris Trapin  7 , Peter A Tauber  7 , Arno Rottal  7 , Ulrike Körmöczi  7 , Melanie Feichter  7 , Milena Weber  1 , Margarete Focke-Tejkl  1   8 , Judith Löffler-Ragg  3 , Bernhard Mühl  9 , Anna Kropfmüller  10 , Walter Keller  11 , Frank Stolz  6 , Rainer Henning  6 , Ivan Tancevski  3 , Elisabeth Puchhammer-Stöckl  2 , Winfried F Pickl  7   8 , Rudolf Valenta  1   4   8   12
Affiliations

Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes

Pia Gattinger et al. Allergy. 2022 Jan.

Abstract

Background: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important.

Methods: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus.

Results: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1 , and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants.

Conclusion: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2-neutralizing antibodies conferring sterilizing immunity.

Keywords: COVID-19; SARS-CoV-2; conformational epitopes; vaccine; virus neutralization.

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Conflict of interest statement

Rudolf Valenta has received research grants from HVD Life‐Sciences, Vienna, Austria, WORG Pharmaceuticals, Hangzhou, China and from Viravaxx AG, Vienna, Austria. He serves as consultant for Viravaxx AG. Renata Kiss, Frank Stolz and Rainer Henning are employees of Viravaxx AG, Vienna, Austria. The other authors have no conflict of interest to declare.

Figures

FIGURE 1
FIGURE 1
Study population analyzed for antibody reactivity to the SARS‐CoV‐2 proteome. (A) Flow chart of analyzed study subjects enrolled from April to July 2020 and historic control sera. (B) Synthetic peptides (underlined) numbered according to Table S4 (Supporting information Methods S1) derived from the amino acid sequence of the SARS‐CoV‐2 spike protein. RBD is printed in green and N‐glycosylation sites in bold. (C) Side (left) and top (right) view of the S protein trimer (surface representation) with RBD‐derived peptides (top, rainbow color code) and carbohydrate moieties (light gray) indicated. (D) Layout of SARS‐CoV‐2 microarray. Positions of SARS‐CoV‐2 protein and peptide triplicates in boxes according to Tables S3 and S4 (Supporting information Methods S1). RBD‐derived peptides are numbered in green within the green dashed box. Triplicates of control proteins according to Table S5 (Supporting information Methods S1) are within dashed lines
FIGURE 2
FIGURE 2
IgG responses of convalescent COVID‐19 patients and historic controls to microarrayed SARS‐CoV‐2 proteins and peptides. (A) Protein‐ and (B) peptide‐specific IgG levels (x‐axes; proteins, peptides, RBD‐derived peptides green; y‐axes, ISU in log10 scale) in COVID‐19–convalescent patients according to their virus neutralization titers (VNT) and in historic controls. P values <.0001 for differences to historic controls are indicated as ***
FIGURE 3
FIGURE 3
Virus neutralization titers correlate with IgG levels to folded RBD and inhibition of RBD binding to ACE2. Correlation of virus neutralization titers (VNTs) in sera of COVID‐19 convalescent subjects (x‐axes, log2 scale) with (A) levels of IgG antibodies (y‐axis: ISU values) to folded RBD, unfolded RBD and RBD‐derived peptides or with (B) percentages of inhibition of RBD binding to ACE2 (y‐axis: % inhibition). (C) Percentages of inhibition of RBD binding to ACE2 determined for COVID‐19 convalescent patients. The horizontal bar denotes the median
FIGURE 4
FIGURE 4
Patients´ IgG antibodies recognize mainly conformational epitopes on folded RBD. Patients´ IgG binding to (A) folded or unfolded proteins and (B) RBD‐derived peptides (top of Figures) without or with pre‐adsorption with folded RBD, unfolded S1 or RBD peptide mix (x‐axes). y‐axes: ISU values, log10 scale, significant differences compared to no inhibition are indicated. P values: * <.05, ** <.001, *** <.0001
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