In the ongoing coronavirus disease 2019 (COVID-19) pandemic, much research has focused on the role of neutralizing antibodies in countering infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Most of these studies have been based on genome sequencing, showing matches between the Hypervariable Region (HVR) and the antigen’s receptor-binding domain (RBD). The results have been difficult to interpret, however, because of many factors, such as the rapid waning of antibody titers, as well as the shared antigenic sequences with many other viruses such as the earlier SARS (89% shared sequences) and influenza viruses.
_-_1200_width.jpg "Study: Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody. Image Credit: NIAID/ Flickr")
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Study details
A new preprint on the bioRxiv* server describes the interactions between the binding spike protein of the SARS-CoV-2 virus with the antibodies. Earlier studies have addressed the similarity in the 3D structures of the spike protein in the two SARS viruses as seen on cryo-EM. When the amino acid sequences of the spike protein of the two viruses were compared, they showed a 77% similarity. Since this does not show the differences in binding, or the effect of temperature and other factors, or the nature of bonding, these studies have not been too successful.
The current study compared the binding strength of the SARS-CoV-2 and SARS-CoV spike protein-antibody complexes. Since it is thought that viral interactions with its angiotensin-converting enzyme 2 (ACE2) receptor or its IgM/IgG antibodies are via hydrogen bonding between carboxyl and amino groups, the investigators sought to use infrared spectroscopy (FTIR) to understand the nature of bonding as well as the number of bonds. This would help to estimate the intensity of viral attack.
Unusual temperature dependence
Surprisingly, they found almost equal numbers of bonds for either virus at room temperature, or below 27°C. In other words, non-specific antibody reactivity was observed, with the accuracy being less than 95%. When the binding occurred at human body temperature, that is, at 37°C, or above 31°C, antibody specificity improved according to the number of available hydrogen bonds, at 19 and 12, respectively.
This was because of the further unfolding of the quaternary structure of the protein at higher temperatures. Thermal agitation leads to the thermal fracture of the van der Waals bonds between the protein strands, which reduces the number of such bonds. This led to the slow exposure of more hydrogen bonding sites.
FTIR showed a similar absorbance at 1550 cm-1 for both mixtures, of the SARS-CoV-2 spike protein/SARS-CoV-2 antibody and the SARS-CoV-2 112 spike protein/ SARS-CoV-1 antibody, at 0.5% and 0.4%, respectively. In other words, the bonds between the antibodies and the spike proteins did not become stronger, but rather, a greater number of bonds were formed at higher temperatures. This accounts for the higher specificity, since the higher ratio of bonds leads to an effective blockade of binding for non-specific antibodies.
These results agree with recently published research that confirms the number of hydrogen bonds in both these virus-antibody complexes by amino acid sequencing and by comparing the heavy chains of the antibody molecules targeting both viruses.
![Comparison of antibody 2 and antibody 1 amino acid sequence with respect to the binding difference to the S protein. The IgM were depicted by the blue ribbon in the middle, and the S proteins represented by the screw ribbons in the top and bottom, where some of representative epitope residues in the S protein were labelled. The sequence of the antibodies were obtained from the literature [32,33]. Both antibodies form only 2+ hydrogen bonds at room temperature due to the folding of protein structure, while such number would increase to 19 and 12, respectively, at human body temperature. As can be calculated by the Arrhenius equation, around 5% of the S protein would bind with antibody 1 at room temperature, resulting in unavoidable testing inaccuracy which could only be eliminated at body temperature.](https://d2jx2rerrg6sh3.cloudfront.net/image-handler/picture/2020/12/2020.12.21.423787v1_-_1200_width.jpg "Comparison of antibody 2 and antibody 1 amino acid sequence with respect to the binding difference to the S protein. The IgM were depicted by the blue ribbon in the middle, and the S proteins represented by the screw ribbons in the top and bottom, where some of representative epitope residues in the S protein were labelled. The sequence of the antibodies were obtained from the literature [32,33]. Both antibodies form only 2+ hydrogen bonds at room temperature due to the folding of protein structure, while such number would increase to 19 and 12, respectively, at human body temperature. As can be calculated by the Arrhenius equation, around 5% of the S protein would bind with antibody 1 at room temperature, resulting in unavoidable testing inaccuracy which could only be eliminated at body temperature.")
Comparison of antibody 2 and antibody 1 amino acid sequence with respect to the binding difference to the S protein. The IgM were depicted by the blue ribbon in the middle, and the S proteins represented by the screw ribbons in the top and bottom, where some of representative epitope residues in the S protein were labelled. The sequence of the antibodies were obtained from the literature [32,33]. Both antibodies form only 2+ hydrogen bonds at room temperature due to the folding of protein structure, while such number would increase to 19 and 12, respectively, at human body temperature. As can be calculated by the Arrhenius equation, around 5% of the S protein would bind with antibody 1 at room temperature, resulting in unavoidable testing inaccuracy which could only be eliminated at body temperature.
What are the implications?
Despite the close phylogenetic relationship and highly similar sequences found in SARS-CoV and SARS-CoV-2, infrared spectroscopy demonstrated the presence of different numbers of hydrogen bonds between the respective spike and antibody molecules. They found a temperature-dependent rise in binding strength, probably due to the unfolding of the protein quaternary structure. This was found to be due to an increase in binding sites, and hence in the number of hydrogen bonds between the carboxyl and amino ends of the different proteins.
Their findings were supported by the absorbance findings at 37°C, when the number of hydrogen bonds of the two types of antibody, to SARS-CoV-2 and SARS-CoV respectively, was found to be 19 vs 11. At 27°C, the bond ratio calculated by thermodynamic exponentials is about 20:1, accounting for the non-specificity of binding at the lower temperature. Such information will help to estimate the accuracy of the newer Covid-19 IgM/IgG rapid antibody tests, that are intended to help with faster and broader tracking of vaccine responses, and of research on the virus, in general.
Our results undoubtedly calls for the necessity of simulating human body temperature in the future antibody diagnosis, especially during the search for the possible vaccines, as the binding uniqueness, or the specificity of SARS-CoV-2 IgM/IgG, could only be fully obtainable at a warmer temperature, rather than under the usual lab/room temperature of 20°C - 25°C, where most of the vaccine research is conducted.”
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Article Revisions
- Apr 3 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
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