Soluble ACE2 microbody blocks SARS-CoV-2 invasion into lung cells

As the COVID-19 pandemic continues to spread in many parts of the world, there is an urgent need for preventative vaccines and improved treatments. New research by researchers NYU Langone Medical Center And Weill Cornell Medical College Published on preprint server bioRxiv* In September 2020, we will report the potential of angiotensin converting enzyme 2 (ACE2)-a new form of antibody Fc complex that appears to provide more significant inhibition of SARS-CoV-2.

ACE2 has become one of the major therapeutic and vaccine development targets due to its essential role in viral attachment and invasion of host cells via spike glycoproteins. By blocking this first step, you can completely prevent the infection. Also, because the target is on the cell surface, intervention is easy and there is no need for cell invasion.

Soluble ACE2 receptor

In early studies, HIV-1 solubility in HIV-1 because viral binding to the CD4 receptor via the envelope glycoprotein gp120 competitively inhibits binding to the target CD4 T cell receptor and prevents viral invasion. We considered the use of the receptor. In vitro studies have confirmed this mechanism of virus suppression.

Fusing the protein to the Fc portion of the immunoglobulin molecule to form immunoadhesin resulted in the formation of the gp120 dimer, which significantly increased binding strength. This has been shown to prolong the survival of proteins in vivo.

Then another change was made to add a peptide derived from the viral co-receptor CCR5 to CD4-Ig immunoadhesin. It is a potent virus inhibitor and has been shown to prevent infection in animal models.

The current paper deals with a similar soluble receptor approach that blocks SARS-CoV-2 entry, based on recombinant human soluble ACE2 protein (hrsACE2) or hrsACE2-IgG with ACE2 bound to Ig-Fc. .. Both have previously been shown to prevent infection with SARS-CoV and SARS-CoV-2 in mouse models.

In Phase 1 and Phase 2 clinical trials, the protein showed partial blockade of viral activity, but was quickly removed from the circulation. With the addition of the Fc portion, the half-life was improved in vivo. However, this raises concerns about the potential for serious illness due to increased antibody dependence due to the presence of Fc. Fc receptors occur naturally in immune cells and are the site of attachment of anti-spike antibodies, increasing the invasion of the virus into these cells.

ACE2 microbody protein

To prevent this, current researchers have investigated using a soluble human ACE2 “microbody” in which the ACE2 molecule is fused to the Ig heavy chain Fc via the former external domain. Ectodomains retain cysteine ​​residues, form protein dimers, and increase their affinity for binding to viruses while reducing their molecular weight. Soluble ACE2 dimers and ACE2 microbody dimers are linked by non-disulfide and disulfide bonds.

Researchers have found that this microbody was unable to bind to Fc receptors on the surface of immune cells, resulting in no ADE. The antiviral activity of the microbody was 10 times that of soluble ACE2, but both were dimers. It also had a high affinity for virion binding. In tissue culture, it lasted longer than soluble ACE2.

It also blocked virus invasion in a range of other cell lines in the betacoronavirus spike protein panel, as well as the original spike variant and the dominant D614G variant.

Prevention of ACE2 catalytic activity

Researchers have found that the introduction of certain H345A mutations to prevent soluble ACE2 from exerting its catalytic enzyme activity does not affect its affinity for the S protein. This is necessary to prevent the effects on blood pressure.

Pseudoviruses incorporate increased infectivity into DS

Pseudovirus expressing a deletion mutant of Spike protein The S protein, which removes the putative ER retention sequence that prevents the transfer of the S protein to the cell surface, was at levels more than 20-fold higher than those expressing the full-length S protein. But the number of billions was similar. The increased efficiency of DS packaging may be due to the loss of inhibition of the ER cytoplasmic tail in the uptake of S into virions. This is because both are expressed almost equally on the cell surface.

Researchers also found that pseudoviruses expressing the S protein specifically bind to soluble ACE2 microbody proteins. This binding was inhibited by high convalescent human serum. Antibody titer.. Mutant ACE2 microbodies (both wild-type and soluble) bound more efficiently to virions than wild-type microbody proteins, even in the absence of mutated amino acids that interact with spike proteins.

ACE2 microbody blocks SARS-CoV-2 pseudoviral infection

Researchers used fluorescent dye markers to investigate the ability of soluble ACE2 and ACE2 microbodies to prevent SARS-CoV-2 DS pseudoviral replication. They showed that soluble ACE2 showed moderate antiviral activity, while the microbody showed much stronger inhibitory activity, while the mutated microbody showed very strong inhibitory activity. Effective antiviral concentrations EC50 were 1.24 μg / ml, 0.36 μg / ml, and 0.15 μg / ml for soluble ACE2, ACE2 microbody, and mutated ACE2 microbody, respectively.

The activity of soluble ACE2 is similar to that of convalescent serum and explicitly inhibits the SARS-CoV-2 spike protein.

They infected cell lines with live engineered SARS-CoV-2 containing the fluorochrome marker gene in ORF7, allowing their replication to be monitored. They found that at serial dilution, the ACE2 microbody retained 1-0.125 μg of antiviral activity. Soluble ACE2 with an EC50 of 1 μg lost activity at 0.5 μg. Here both wild-type and mutant ACE2 microbodies show similar EC50s and are superior to soluble ACE2.

They subsequently determined that the microbody protein was also active when exposed to the virus at a later time, with the former being added simultaneously or by 6 hours after infection. This resulted in 80% inhibition when both were added simultaneously, about the same 30 minutes after infection, and 55% suppression in 2 hours when about 10% of the infectious virus bound to the cells. .. After that, the activity declined.

Therefore, the ACE2 microbody can block infection if it is present before the virus binds to target cells, or at the time of exposure, and even up to 2 hours after exposure. This time frame shows the period during which the virus has not yet significantly bound to the cell.

In addition, incubating with the ACE2 microbody for 30 minutes, even shortly after virus binding, can reduce infection rates. The virus’s binding rate to the ACE2 receptor was found to be time-dependent, probably initially with little spike-ACE2 interaction, followed by additional S protein in the binding over time. .. When the latter occurs, the ACE2 microbody is unable to block the invasion of the virus.

ACE2 Microbody Block D614G Variant

A variant of SARS-CoV-2 containing a D614G point mutation in the S protein is known to be the major variant whenever it appears rapidly. It is associated with increased infectivity. DS pseudoviruses containing this mutation are also moderately blocked by soluble ACE2, but both wild-type and mutant ACE2 microbodies are more potent due to the inhibition of viral replication by this variant. Comparing the two pseudoviruses for binding, we found that soluble ACE2 binds the D614G spike-carrying mutant more efficiently.

ACE2 microbody binds to spike proteins of other betacoronaviruses

The ACE2 and H345A microbody proteins prevented the invasion of all pseudoviruses carrying various beta coronavirus spike proteins, and although soluble ACE2 had low antiviral activity, it showed a broad spectrum of antibeta coronaviruses.

Implications

The ACE2 microbody is 10 times more active against SARS-CoV-2 than soluble ACE2 and 4 times more compatible with viral particles. Its human origin means its low immunogenicity. It also appears to have a wide range of effects on emerging spike variants, including the D614G mutation.

Researchers introduced a specific mutation into the ACE2 microbody to eliminate the catalytic activity of the enzyme while preserving its antiviral activity. This mutation appeared to increase the activity of the microbody, but did not increase its inhibitory capacity in live viral assays.

The researchers also suggest that the microbody may have more significant antiviral activity than was observed here, as most of the experiments were performed on cells expressing high levels of ACE2. The antiviral activity of the ACE2 microbody was high when assayed on cell lines with very low ACE2 levels.

The investigator summarizes, “The disulfide-bonded ACE2 microbody protein inhibits the invasion of the lentivirus SARS-CoV-2 spike protein pseudovirus, activates SARS-CoV-2 10 times more potent than the unmodified soluble ACE2, and is the first to enter the cell. Showed activity after virus binding.. “

The study also showed that the D614GS protein provides high infectivity and high affinity for ACE2. However, the ACE2 microbody was able to neutralize the spike proteins of other betacoronaviruses as well as the pseudoviruses containing this spike variant. This may indicate that this technology can be used to neutralize new emergences. Coronavirus It also binds to the ACE2 receptor and “Ready-made reagents that can be deployed quickly“

*Important Notices

bioRxiv As it publishes unpeer-reviewed preliminary scientific reports, it should not be considered definitive and should not guide clinical / health-related behavior or treat it as established information.