In a recent study posted to bioRxiv*, researchers evaluated how angiotensin-converting enzyme 2 (ACE2) expression in target cells impacts neutralization.
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 (pseudo or live virus). It has been reported that neutralizing activity of antibodies varies markedly based on the type of viral system.
The study and findings
In the present study, researchers systematically assessed how ACE2 expression on target cells impacts the neutralizing activity of antibodies in polyclonal sera. They engineered human embryonic kidney (HEK) 293T cells to express variable ACE2 levels (very low, low, medium, or high). Approximately a 30-fold difference in ACE2 expression was observed across the 293T cell clones.
The clones with very low ACE2 expression (30-fold less ACE2) had about eight-fold less infectivity. First, antibodies targeting SARS-CoV-2 spike protein’s receptor-binding domain (RBD) were depleted from polyclonal sera collected from individuals infected in 2020 and later vaccinated with Pfizer’s or Moderna’s vaccine in early 2021.
SARS-CoV-2 spike pseudo-typed lentiviral neutralization assays were performed on the four 293T clones using RBD antibody-depleted and non-depleted sera. The authors found that RBD-directed antibodies accounted for most of the neutralization when cells expressed high ACE2 levels. Anti-RBD antibodies were responsible for almost all the neutralization with high-ACE2-expressing cells.
In contrast, anti-RBD antibodies contributed only 90% of neutralization using low or very-low ACE2-expressing cells. These results implied that anti-RBD antibodies provide a majority of neutralizing activity of polyclonal sera regardless of ACE2 expression level. However, the relative contribution of these antibodies to neutralizing activity is higher in high-ACE2 expressing cells than low ACE2 cells.
The authors also noted a modest trend of the neutralization titers to be greater in cells expressing less ACE2. This was evident for both RBD antibody-depleted and non-depleted sera, albeit the effect was more prominent for the antibody-depleted sera. RBD antibody-depleted sera always had detectable neutralizing activity in very low, low, and medium ACE2-expressing cells, while some serum samples lost detectable neutralization in high-ACE2 cells.
Next, the research team analyzed neutralization by monoclonal antibodies (mAbs) directed to distinct spike epitopes. mAbs included S309, Ly-CoV555, and 4A8. 4A8 binds to the spike’s N-terminal domain (NTD), Ly-CoV555 binds to the RBD’s receptor-binding motif (RBM) and competes with ACE2 binding, and S309 binds to RBD outside RBM without competing with ACE2 binding.
mAbs binding to spike outside of RBM was less potent on cells expressing high ACE2. Specifically, Ly-CoV555 was the only mAb slightly affected by the ACE2 expression levels of cells. Ly-CoV555 exhibited neutralizing activity on all target cells, albeit a five-fold reduction in half-maximal inhibitory concentration (IC50) was noted with high-ACE2 cells relative to very-low ACE2 cells. In contrast, S309 and 4A8 could not exhibit complete neutralization on high-ACE2 cells, even at a very high antibody concentration. S309 and 4A8 had improved neutralization in lower ACE2 cells.
In summary, the findings showed that anti-RBD antibodies contribute to most of the neutralization of target cells expressing high levels of ACE2. The experiments with mAbs revealed that antibodies directed to epitopes outside the RBM were less potent on high ACE2 cells. Neutralization by Ly-CoV555 was only marginally affected by ACE2 levels on target cells. However, S309 and 4A8 had poor neutralizing activity on cells with high ACE2 expression.
Thus, the contribution of antibodies, directed at different spike epitopes, to neutralizing activity differs with ACE2 expression on target cells. Nevertheless, it remains unclear why the 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 on target cells is a critical variable for SARS-CoV-2 neutralization assays.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.