The importance of cross-neutralizing antibodies for recently developed variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is obvious in light of the fact that these variants are highly transmissible, as well as the possibility of avoiding an immune response to earlier versions of this virus.
New research paper on preprint published on bioRxiv* The server reports the ability of a series of neutralizing muscle antibodies to protect mice against multiple SARS-CoV-2 lineages.
Antibodies and virus neutralization
Twenty vaccines have been approved for use worldwide, in addition to several monoclonal antibodies, in combination, for the prevention and treatment of SARS-CoV-2 infection.
This virus enters the host cells to establish a successful infection by binding via the spike protein to the receptor of the host cell, the enzyme converter angiotensin 2 (ACE2). The part of the spike that binds to the receptor is called the receptor-binding domain (RBD) and makes an attractive target for antibodies, both therapeutic and preventive.
Neutralizing antibody titers are highly correlated with reinfection protection, but since vaccination will take time to cover the global population, there is an urgent need for therapeutic antibodies and drugs to treat and prevent serious diseases.
Currently available monoclonal antibodies (mAbs) have been raised to the top of the original Wuhan virus variant. However, the occurrence of escape mutations, especially in the RBD spike region, threatens the utility of these mAbs.
Because most neutralizing antibodies act by blocking virus entry, targeting RBD, mutations in RBD can reduce their mAb efficacy.
The N-terminal domain (NTD) is another region that is often targeted in newer variants because it is neutralized by many antibodies. Therefore, these variants can avoid vaccine-induced antibodies.
Objectives and details of the study
Researchers from Krammer’s laboratory, Icahn School of Medicine in Mount Sinai, aimed to evaluate the ability to bind and neutralize 14 mAb mice collected against viral RBD on both RBD and protein spike. They also assessed the potential of non-neutralizing mAbs to reduce viral load.
Third, the study examined the binding of these mAbs to RBD mutants containing point and clustered mutations that define the three main variants of concern (VOC) – UK (B1.1.7), South African (SA) variant (B.1.351) and Brazilian variant ( P.1).
Finally, mAbs were tested for neutralization relative to the UK and SA variants.
What were the results?
Antibodies to mouse immunoglobulin G (IgG) were raised in hybridoma cells. All 14 were found to bind to RBD with high affinity, indicating very low minimum binding concentrations (MBC).
When tested against full spike protein, most bound well, but three had higher MBCs compared to RBD. The researchers hypothesize that this could be due to the overlap of RBD-binding epitopes on the solid spike compared to RBD expressed in isolation.
Ten of the 14 mAbs were specific for SARS-CoV-2 RBD, but four also showed cross-reactivity for SARS-CoV RBD.
When tested with live virus in a microneutralization test, where the ability of antibodies to block viral infection of culture cells is determined as a reciprocal dilution value at which cells remain completely uninfected, six showed a high neutralizing ability.
In all six cases, the concentration at which 50% of the cells remained uninfected (IC50) with live virus was very low, 0.1-1 ug / ml. This suggests that they can prevent virus entry and replication at high dilutions, especially the two that had the lowest IC50.
Reduction of virus titer in mice
The researchers tested mAbs on their ability to block virus entry and reduce viral loads in engineered mice expressing the human ACE2 gene. MAbs were administered two hours before challenge in mice with SARS-CoV-2.
Plaque reduction neutralization tests showed that only six mAbs with neutralizing capacity could protect the animal from virus entry and replication. Three of the six reduced viral load by two logs on day 3, while the other two caused a significantly more significant reduction in viral titer.
On day 5, no animal receiving neutralizing mAb showed signs of significant viral presence in the lungs, although two had residual virus
Reduced lung pathology
Lung tissue was taken on day 4 for histopathology and immunohistochemistry (IHC). This showed some evidence of interstitial pneumonia, perhaps due to the high dose of the virus.
Some mild inflammation was found to be the result of injection of the adenoviral vector hACE2, but the scores of inflammation in mice that received the vector and virus were higher. There were no signs of an increase in antibody-dependent disease (ADE).
Nucleoproteins were almost undetected, showing that all neutralizing mAbs prevented the virus from entering the cell and thus lowered the virus titer in the lungs.
Binding to RBD variants
The study also shows that some RBD variants such as K487R and N487R caused one mAb to lose complete binding. Mutations E484K, F486A and F490K, also reduced binding to the third mAb. Grouped mutations on the SA variant of RBD also caused a loss of binding to the same mAb.
However, five mAbs retained 50% binding affinity for at least all mutated RBDs, and some had higher binding than for wild-type RBDs. Researchers comment, “The ability to bind all RBDs could be a function of antibody affinity that, when high, can allow an antibody to maintain its trace. ”
Overall, neutralizing mAbs bound wild and mutated RBDs to somewhat equivalent levels.
Of particular interest, four of the six neutralizing mAbs bound both wild-type and SA RBD variant, while all six neutralized the UK RBD variant. The latter has only one RBD mutation, N501Y.
Of the four that maintained the binding and neutralization of the SA variant, the IC50 increased from 2.5-fold to 5-fold compared to the wild-type virus or the UK version.
The reasons for the loss of neutralization of the SA variant could be lower binding to RBD containing E484, for one mAb, and altered epitope display on the spike relative to RBD for the other.
Inhibition of ACE2-RBD binding
The researchers also structural analysis of six neutralizing mAbs found that two of them failed to form stable complexes with RBD and could not be visualized. Three of them showed overlap with the ACE2 binding site which could explain how they prevented ACE2-RBD binding.
They bind to RBD at different angles, indicating that they bind to different epitopes.
What are the implications?
The study shows that helical RBD can experience multiple mutations while retaining the ability to bind to the ACE2 receptor and cause infection of the host cell.
“The plasticity of RBD is alarming because extensive changes in RBD could reduce the efficacy of current vaccines, and additional enhanced vaccinations with updated vaccines may be needed in the future.. ”
The lack of protection with non-neutralizing mAbs is attributed to their IgG1 isotype, which in mice excludes effector-mediated Fc receptor (FcR) interactions. Thus, antibodies that do not activate FcR and do not have the ability to neutralize lack protective efficacy.
The two antibodies that best protected against virus entry were IgG2a, a subtype associated with Fc-Fc interactions in mice, supporting the hypothesis that antibodies associated with effector FcR activation are key to the protective effects of antibodies.
In humans, IgG2 antibodies interact strongly with FcRs, and most antibodies to this virus belong to this isotype.
The study suggests the potential that humanized forms of these neutralizing mAbs can be developed as therapeutics with strong activity against wild-type viruses, as well as UK and SA variants.
It’s all in a long but great title:
“Mouse monoclonal antibodies against RBD SARS-CoV-2 neutralize the authentic wild-type SARS-CoV-2 as well as viruses B.1.1.7 and B.1.351 and protect in vivo in a mouse model in a way that depends on neutralization” https: // t.co/Lbw4seLU4D
– Dr Kevin Purcell (@kevinpurcell) April 6, 2021
* Important notice
bioRxiv publishes preliminary scientific reports that have not been reviewed and should therefore not be considered final, direct clinical practice / health-related behavior, or treated as established information.