April 18, 2023: Novel Broadly

Neutralizing SARS - C oV - 2 Monoclonal Antibodies That Bind Across The Subunits Of The Spike Protein

Anew antibody pair neutralizes all current variants of concern of SARS-CoV-2. There is an effective need for monoclonal antibody treatments for those suffering from moderate to severe Covid-19. While a number of treatments have been developed throughout the pandemic, the virus is continuously mutating, rendering many of these treatments obsolete. The key is to find antibodies that target conserved regions of the virus, allowing the antibody to neutralize a broad range of variants.

Two such broadly neutralizing antibodies were recently identified by Liu et al.Many monoclonal antibodies target the spike receptorbinding domain, the region that makes contact with the host cell's ACE2 receptor, triggering infection. The two antibodies defined by Liu et al.target different regions, the N-terminal domain, SD1, and the S2 region. Here we analyze these antibodies and discuss how their addition impacts the current monoclonal antibody treatments pool.

Isolationandcharacterizationofbroadlyneutralizingmonoclonal antibodiesagainst SARS-CoV-2variants

The researchers evaluated a panel of sera samples against a range of 11 SARS-CoV-2 variants in search of neutralizing activity. Sera from patient 12 demonstrated the highest levels of neutralization. From patient 12’s sera, they isolated 27 monoclonal antibodies, of which five neutralized Omicron BA.1. From these five, antibody candidates 12-16 and 12-19 demonstrated the strongest neutralization.

Both 12-16 and 12-19 further neutralized some of the latest and most prevalent variants of SARS-CoV-2, including BQ.1.1, XBB.1.5, and CH.1.1, all of which have highly mutated spike proteins.

In hamster models, 12-16 and 12-19 significantly reduced virus titers in subjects infected with Omicron BA.1, reducing the virus to undetectable levels within days.

Antibodies12-16and12-19target a quaternaryepitope between NTD andSD1

Liu et al. used cryo-electron microscopy to determine the binding epitope of 12-16 and 12-19 to the SARS-CoV-2 spike protein. Rather than binding the typical receptor-binding domain, the antibodies bound the N-terminal domain, subdomain 1, and a small section of the S2 region.

Most antibodies throughout the pandemic bound the receptorbinding domain from residues 333 to 527. The N-terminal domain runs from residues 14 to 305, subdomains 1 and 2 run from residues 541 to 685, and S2 is the latter half of the virus from residues 685 to 1273.

12-16 and 12-19 bind the spike at slightly different angles. 12-16 binds at a higher angle, favoring the N-terminal domain, whereas 12-19 binds at a lower angle, binding more sites on SD1. From here, we will focus on 12-19 as it binds more completely invitro than 1216, likely due to its slightly more cross-region epitope.

12-19neutralizeSARS-CoV-2bylockingRBD in thedown conformation

Akin to some antibodies seen throughout the pandemic, 12-19 blocks infection by locking the receptor-binding domain in the down conformation. Before contact with the host cell, the spike shifts from a down to an up conformation, allowing binding and enabling infection of the cell. Infection is prevented if the receptorbinding domain is locked in the down conformation.

But how can an antibody that binds the N-terminal domain, subdomain 1, and S2 impact the receptor-binding domain? Part of the 12-19 epitope binds residues that are shifted during conformation change, namely in subdomain one and a linker region between the N-terminal and receptor-binding domains. Imagine two gears in the same link. They may not necessarily be consecutive, but block the movement of one, and the whole line of gears is stalled.

By blocking the conformational shift, 12-19 impedes ACE2 binding between the receptor-binding domain and the host cell.

Another set of antibodies that uses conformation locking is the camelid nanobodies described in 2021. These bind across receptorbinding domains of a spike trimer, preventing the spring-loaded conformation switch from engaging.

Theepitope ofantibody12-19ishighlyconserved

As with any monoclonal antibody candidate, we must consider the potential escape mutations enabling a viral variant to escape neutralization from 12-19. Using deep mutational scanning libraries, Liu et al.found a few potential problem residues.

The areas most likely to produce escape mutations were the N4 loop of the N-terminal domain from residues 172-176, deletions throughout the N-terminal domain loops, namely residues 103 and 121, as well as residues 522, 561, and 577 in the subdomain 1. However, these residues are rarely mutated in currently circulating variants. The most frequent is L176F, found in only 0.2% of sequenced variants. This indicates that the epitope of 12-19 is largely conserved, at least so far in the pandemic.

Discussion

On first inspection, 12-19 would serve as a valuable antibody treatment, given its strong neutralization of the latest SARS-CoV-2 variants. An advantage it may have over other treatments is its unique binding epitope. The figure below compares the binding epitope of 12-19 to other neutralizing antibody epitopes from throughout the pandemic.

Figure 23. Schematicrepresentations ofbindingepitopes fordiffering classesofmonoclonalantibodytreatmentsthroughoutthepandemic.

Source:ACCESSHealthInternational

Compounding the unique binding epitope of 12-19 is it's reaching across spike domains to lock conformation. We believe conformation locking is among the most potent mechanisms by which an antibody can neutralize a virus.

The 12-19 antibody is not our first encounter with conformationlocking antibodies. In fact, some of our most antibody candidates to other dangerous pathogens employ a similar method. Lassa virus antibody 8.9F binds across the three faces of the Lassa trimer, locking the glycoproteins into place. Ebola antibodies 1C3 and 1C11 use a similar mechanism, locking the whole of the Ebola structure in place. Even parasites such as Malaria can be overcome with conformation-locking antibodies, such as CIS43, which prevents a cleavage function required for malaria infection.

The need for effective monoclonal antibody treatments is urgent. We should constantly look for antibodies that employ this conformation-locking tactic against SARS-CoV-2 and other major pathogens. These treatments consistently provide strong neutralization and protection. Their addition to the antibody arsenal would be well worth the effort to find them.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Novel Broadly Neutralizing SARS-CoV-2 Monoclonal AntibodiesThatBindAcrossTheSubunitsOfTheSpikeProtein

April 25, 2023: Are We Leaving Useful Antibodies Behind? The Value Of NonNeutralizing Protective Monoclonal Antibodies

While common sense may suggest that antibodies that do not neutralize the SARS-CoV-2 virus are of little value, recent studies show they can still confer protection against infection. Monoclonal antibodies have been a very effective method for preventing and treating Covid-19. Unfortunately, the current generation of virus variants has mutated to a degree that currently approved monoclonal antibodies that were previously successful are no longer effective.

There is a clear need for new approaches to antibody therapies. One method we have described is the use of combination antibodies that target highly conserved sites. Here we outline a different approach: using antibodies that bind, but are non-neutralizing, yet still protective in high doses. Part one will describe how this method was uncovered, and part two will describe more recent progress in this approach.

A recent study from Swedish scientists Bahnan et al. was the first to show the potential of non-neutralizing antibodies. What is a protective non-neutralizing antibody? These antibodies bind the virus spike, but do not prevent infection in vitro . However, they do protect from infection in live animal models.

How can that be? The virus still enters the cells as the antibody does not block infection, but the infected cells are killed before the virus spreads throughout the body. Bahnan et al.found that high levels of non-neutralizing monoclonal antibodies mediate the interactions between infected cells and immune system monocytes. The following study describes this discovery.

IsolationofSpike-ReactiveHuman MonoclonalAntibodies

They isolated the antibodies found in the sera of 20 Covid-19 patients. Ten of the 96 they isolated were reactive when exposed to the in vitro spike protein. Those most notably reactive were Ab11, 57, 59, 66, 77, 81, 94, and 95.

They identified the binding epitopes using cryo-electron microscopy to narrow down those that may be neutralizing antibodies. Of those most strongly reactive, they identified six antibodies that bound the spike receptor-binding domain (Ab11, 57, 59, 66, 77, 81) and one that binds both the receptor-binding and Nterminal domains (Ab94). However, they note that only Ab59’s epitope allows for virus neutralization. The rest were nonneutralizing.

The most important factor in our typical search for monoclonal antibody treatments is their neutralizing capacity. As suggested by their binding epitopes, only Ab59 showed a reduction of virus titers.

HighLevelsofNon-NeutralizingAntibodiesCanProtectAgainst SARS-CoV-2Infection

Given the varying levels of neutralization among the 96 isolated antibodies, Bahnan et al. introduced each antibody individually to spike-monocyte interactions to determine the role of antibodies as mediators of immune interactions.

They found that modulated interactions were correlated with the dosage of monoclonal antibodies involved in the interaction. The highest dosage yielded a reduction in spike-monocyte interaction. Ab59, the only neutralizing antibody among the set of 96, showed the same trend of modulating monocyte interaction as other nonneutralizing antibodies, suggesting that this phenomenon is independent of neutralizing capacity.

Using humanized ACE2 mice subjects, they found that subjects treated with a large dose (250 µg) of non-neutralizing Ab94 were better protected from Covid-19 using weight loss as the measure as compared to the calculated protective dose (100 µg) of neutralizing Ab59.

Discussion

While neutralizing monoclonal antibodies will continue to be the focus of Covid-19 treatment, this discovery unlocks a new world of potential for antibody treatment. There have been dozens, if not hundreds, of antibody candidates in the past three years that either were not completely neutralizing initially, or lost neutralization as emerging variants developed. At the right dosage, some of these antibodies may prove to be valuable protective treatments.

In part two, we will describe another study that explores the use of monoclonal antibodies outside of their traditional role as neutralizing agents against the SARS-CoV-2 spike. With any luck, this new avenue may yield new treatments for those continuing to suffer from Covid-19 sooner rather than later.