In a recent study published in bioRxiv*, researchers assessed how the expression of angiotensin-converting enzyme 2 (ACE2) in target cells affects neutralization.
Study: Receptor-binding domain (RBD) antibodies contribute more to SARS-CoV-2 neutralization when target cells express high levels of ACE2. Image credit: Kateryna Kon/Shutterstock
background
Neutralization assays are commonly used to determine immunity elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination. These assays use different target cells (cells expressing endogenous ACE2 or those engineered to overexpress ACE2) and viral systems (pseudoviruses or live viruses). The neutralizing activity of antibodies has been reported to vary markedly depending on the type of viral system.
The study and conclusions
In the present study, the researchers systematically evaluated how ACE2 expression in target cells affects the neutralizing activity of antibodies in polyclonal sera. They engineered human embryonic kidney (HEK) 293T cells to express varying levels of ACE2 (very low, low, medium, or high). An approximately 30-fold difference in ACE2 expression was observed between the 293T cell clones.
Clones with very low ACE2 expression (30-fold less ACE2) had approximately eightfold less infectivity. First, antibodies directed at the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were depleted from polyclonal sera collected from individuals infected in 2020 and subsequently vaccinated with the Pfizer or Moderna vaccine in early 2021.
SARS-CoV-2 spike pseudotyped lentiviral neutralization assays were performed on the four 293T clones using RBD antibody-depleted and non-depleted sera. The authors found that RBD-targeted antibodies accounted for most of the neutralization when cells expressed high levels of ACE2. Anti-RBD antibodies were responsible for almost all neutralization with cells expressing a high level of ACE2.
In contrast, anti-RBD antibodies only contributed to 90% of the neutralization by cells expressing low or very low ACE2. These results implied that anti-RBD antibodies provide most of the neutralizing activity of the polyclonal sera, regardless of the level of ACE2 expression. However, the relative contribution of these antibodies to the neutralizing activity is higher in high ACE2 expressing cells than in low ACE2 cells.
The authors also observed a modest tendency for neutralization titers to be greater in cells expressing less ACE2. This was evident for both RBD antibody-depleted and nondepleted sera, although the effect was more prominent for antibody-depleted sera. RBD antibody-depleted sera always had detectable neutralizing activity in very low, low, and medium ACE2 expressing cells, whereas some serum samples lost detectable neutralization in high ACE2 cells.
The research team then analyzed neutralization using monoclonal antibodies (mAb) directed at different spike epitopes. mAbs included S309, Ly-CoV555, and 4A8. 4A8 binds to the N-terminal domain (NTD) of the spike, Ly-CoV555 binds to the receptor-binding motif (RBM) of RBD and competes with ACE2 binding, and S309 binds to RBD outside of RBM without competing with ACE2 binding.
mAbs binding to the non-RBM spike were less potent in cells expressing high ACE2. Specifically, Ly-CoV555 was the only mAb that slightly affected the ACE2 expression levels of the cells. Ly-CoV555 showed neutralizing activity on all target cells, although a five-fold reduction in mean maximal inhibitory concentration (IC50) was observed with high ACE2 cells relative to very low ACE2 cells . In contrast, S309 and 4A8 failed to show complete neutralization in high ACE2 cells, even at a very high antibody concentration. S309 and 4A8 had enhanced neutralization in lower ACE2 cells.
Conclusions
In summary, the findings showed that anti-RBD antibodies contribute to the majority of neutralization of target cells expressing high levels of ACE2. Experiments with mAbs revealed that antibodies directed at epitopes outside the RBM were less potent in high ACE2 cells. Neutralization by Ly-CoV555 was only marginally affected by ACE2 levels in target cells. However, S309 and 4A8 had little neutralizing activity in cells with high ACE2 expression.
Thus, the contribution of antibodies, directed at different spike epitopes, to neutralizing activity differs with ACE2 expression in target cells. However, it remains unclear why neutralizing potency varies with ACE2 expression. The findings cannot answer the fundamental question it raises, what level of ACE2 expression provides a viable measure of SARS-CoV-2 neutralization? Overall, the present work suggests that ACE2 expression in target cells is a critical variable for SARS-CoV-2 neutralization assays.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.