Assessing Antibodies

Viola Denninger on assessing cross-reactivity of antibodies against SARS-CoV-1 and SARS-CoV-2 microfluidic antibody-affinity profiling

Covid-19 is caused by the beta coronavirus SARS-CoV-2, one of seven coronavirus species known to infect humans. Based on phylogenetic analyses, SARS-CoV-2 was shown to share remarkably high sequence similarity with SARS-CoV-1, suggesting that antibodies and compounds previously screened for use against SARS-CoV-1 could be lead candidates in the development of therapeutics against Covid-19. A rapid immune response assessment and quantification of potentially cross-reactive antibodies against SARS-CoV-2 will, therefore, be crucial in selecting the best therapeutic candidates.

SARS-CoV-2 is a single-stranded RNA-enveloped virus, and its genome encodes for several structural and functional proteins including the spike protein that covers the surface of the virus. The spike protein consists of two distinct functional subunits, S1 and S2, that mediate receptor recognition, cell attachment and fusion during infection. In the case of SARS-CoV-1 and SARS-CoV-2, homology modelling of the respective spike proteins revealed a sequence similarity of 75-80%, accounting for the fact that SARS-CoV-1 and SARS-CoV-2 share the same entry mechanisms into the host cells. Both bind to the angiotensin-converting enzyme (ACE2), a receptor that is highly expressed on the surface of human respiratory epithelial cells.

Based on the high degree of similarity between SARS-CoV-1 and SARS-CoV-2, it was therefore speculated that cross-reactive epitopes could exist, which could be exploited to rapidly repurpose existing therapeutic approaches or develop new vaccines.

Developing Antibodies

During the initial SARS outbreak in 2002, several monoclonal antibodies including CR3022 were developed against the SARS-CoV-1 spike protein with the goal to inhibit entry into the human host cell. Although the CR3022 antibody failed to neutralise SARS-CoV-2, it was shown to cross-react with a conserved epitope on SARS-CoV-2 RBD. Moreover, used in combination with another antibody, CR3014, neutralisation was achieved due to synergistic binding of different epitopes on the RBD. Such a combined antibody therapy was therefore suggested as an option in the treatment of Covid-19 patients.

To understand the underlying mechanisms that govern the functional immune response to SARS-CoV-2 scientists have used microfluidic antibody-affinity profiling (MAAP) to assess and quantify the cross-reactivity between CR3022 and the spike S1 proteins of SARS-CoV-1 and SARS-CoV-2, as well as the cross-reactivity of the two respective spike S1 proteins and a Covid-patient derived neutralising anti-SARS-CoV-2 monoclonal antibody. Their results indicate a unidirectional cross-reactivity with only CR3022 readily recognising the spike S1 subunits of both viruses. Importantly, the team behind the research could not detect any binding of the anti-SARS-CoV-2 antibody to the S1 subunit of SARS-CoV-1, emphasising the selectivity for the newly evolved virus.

By using MAAP on the Fluidity One-W Serum to determine the affinities of CR3022 and AS35 against the spike S1 subunits of SARS-CoV-1 and SARS-CoV-2, the scientists can evaluate cross-reactivity of an antibody to various antigens even in a complex background such as serum. This approach can be used to quantify the immune response of cross-reactive antibodies in patients or vaccinated individuals, as well as to rapidly evaluate therapeutic antibodies against the emerging mutant variants of SARS-CoV-2.

Viola Denninger is with Fluidic Analytics

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