Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan, China, the virus has led to a global pandemic.
There has been much concern due to the emergence of multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that have reduced vaccine efficacy and evaded neutralizing antibody therapeutics. Thus, it is imperative to develop strategies to inhibit all known and future variants of SARS-CoV-2.
According to new research by an international team of scientists, all SARS-CoV-2 variants, including variants of concern (VOC) Alpha, Beta, Gamma, and Delta, exhibit enhanced affinity toward recombinant human soluble ACE2.
Further, a significant finding was that soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by multiple VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates.
Two independent laboratories confirmed the inhibitor's effectiveness in inhibiting SARS-CoV-2 infections. Data show that SARS-CoV-2 variants that have emerged worldwide, including the current VOC and several variants of interest, are inhibited by soluble ACE2. This provides proof of principle of a pan-SARS-CoV-2 therapeutic.
A preprint version of this in vitro study, which is yet to undergo peer review, is available on the bioRxiv* server.
Angiotensin-converting enzyme 2 (ACE2)
When SARS-CoV-2 infects host cells, the viral Spike protein binds to the main human cell-entry receptor, angiotensin-converting enzyme 2 (ACE2). This leads to the subsequent infection of host cells.
Current COVID-19 vaccines induce the production of neutralizing antibodies that inhibit the Spike/ACE2 interaction. One therapeutic approach for COVID-19 treatment is the use of approved monoclonal antibodies that block the Spike/ACE2 interaction. Due to the importance of this interaction, there is a substantial focus on the research of its molecular details.
Consequently, it is also one of the best-validated drug targets for COVID-19 treatment. In this study, the scientists used a clinical-grade recombinant human soluble ACE2 (APN01) which is already in phase 2 clinical trials.
Throughout the pandemic, several variants of SARS-CoV-2 have emerged. The World Health Organization has labeled some of the variants as VOCs because they are highly infectious and transmissible.
Many of these variants harbor mutations in the viral Spike protein that do not appear to affect the infectivity and transmissibility of the virus, but rather reduce the potency of vaccines and monoclonal antibodies therapies.
They are also responsible for breakthrough infections. For example, the Delta variant can cause infections even in doubly vaccinated individuals.
This situation presents a need for universal strategies to prevent and treat current and future VOCs. With this aim, the scientists explored the therapeutic potential of APN01.
Clinical grade recombinant human ACE2 was produced and used for this study. Spike protein receptor-binding domain (RBD) and ACE2 binding studies were performed using ELISA and surface plasmon resonance analysis.
To test the neutralization of VOCs by APN01, neutralization assays were performed in VeroE6 cells and human lung epithelial cells. These neutralization assays were conducted at NIAID Integrated Research Facility at Fort Detrick, Frederick, MD, USA. To ensure reproducibility of the data, the neutralization assays were repeated at the Karolinska Institutet, Stockholm, Sweden.
Increased affinity of APN01 interactions with SARS-CoV-2-RBD variants. (a) Schematic depicts structure of the SARS-CoV-2 spike protein S1 domain. Indicated is the amino-terminal domain (NTD), the receptor-binding domain (RBD) in blue and within the RBD the receptor binding motif (RBM) in purple. Numbers above depict domain boundaries. Mutations within the RBD/RBM are indicated below with observed amino acid exchanges. Shown in red are mutations observed in Variants of Concern (VOC). (b) PyMOL rendered visualization of the SARS-CoV-2 RBD. Rendering depicts the SARS-CoV-2 RBD with mutation sites shown in green. (c) ELISA analysis showing the binding strength of SARS-CoV-2 RBD carrying the indicated mutations to APN01. Axis labels indicate the SARS-CoV-2 RBD variant substitutions tested. (d) Surface Plasmon Resonance analysis to derive kinetic constants (ka, kd) and affinity values (KD) of SARS-CoV-2 RBD/APN01 interaction. The table lists both the tested variants and the introduced amino acid substitution as well as the designation of the respective Variants of Concern mutations tested in this study. Reference strain RBD sequence corresponds to the Wuhan SARS-CoV-2 isolate (e) Representative SPR sensorgram images for the SARS-CoV-2 RBD/APN01 interaction.
Blocks SARS-CoV-2 variants
SARS-CoV-2 evolution is focused on the Spike protein, especially the RBD. RBD mutations affect ACE2 binding. Clinical grade ACE2 showed higher affinity to the Spike RBDs of SARS-CoV-2 variants when compared to the Spike RBD of the original Wuhan SARS-CoV-2 isolate.
This increased binding affinity may be the reason for the enhanced infectivity of the VOCs. Moreover, this increased affinity was also observed with full-length Spike proteins.
Neutralization assays in VeroE6 cells with APN01 showed that APN01 neutralized all the SARS-CoV-2 isolates tested. More importantly, this inhibition was enhanced against all VOCs.
Equivalent results were obtained from a physiologically relevant cell system, the lung epithelial cells. Interestingly, the scientists observed that the neutralization potency correlated with the Spike protein/APN01 binding affinity.
In conclusion, ACE2/APN01 showed strong binding to VOC RBDs or VOC full-length Spike protein and potently inhibited viral infection by the SARS-CoV-2 isolates.
The neutralization assays were independently validated at the Karolinska Institutet. These confirmatory experiments also showed that APN01 inhibited viral infection. Significantly, APN01 inhibited infection by the VOCs with increased potency.
Limitations of this study
The scientists agree that this study has two limitations:
- This study used two different cells types. Other cell types also should be studied to observe the same effect.
- If ACE2 is to be considered a universal agent, additional variants should be tested.
The current VOCs include Alpha, Beta, Gamma, and Delta variants and the variants of interest include Iota, Kappa, Eta, Mu, and Lambda variants.
Novel variants are to be expected due to large-scale vaccination programs. Some of these variants may lead to breakthrough infections and rapid spread of the virus. Therefore, the design of universal therapeutic strategies is of paramount importance.
APN01 has undergone a phase 2 trial in severe COVID-19 patients using intravenous infusions. This testing is now being extended to a larger patient population.
The scientists propose that the data from this study in conjunction with clinical data will pave the way for the development of a universal and pan-SARS-CoV-2 therapy.
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.