Virus neutralizing assays (VNAs) are the gold standard for screening serum for neutralizing antibodies in vitro. These are being used, in many cases, to treat early coronavirus disease 2019 (COVID-19) and prevent disease progression and death from COVID-19.
Regeneron and Eli Lilly have produced neutralizing monoclonal antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and many others are also under development.
In contrast to VNAs that require Biosafety Level 3 (BSL-3) settings, pseudovirus neutralization assays (pVNAs) can be done at BSL-2 levels. Both require several days for completion. Sandwiched enzyme-linked immunosorbent assays (ELISAs) or those based on surface plasmon resonance are high-throughput assays, but lump neutralizing and binding antibodies together.
These concerns make it imperative to develop a more rapid and accessible alternative. A new paper from scientists at the University of Michigan presents a technique called rapid in vitro inhibition assay (RIVIA), whereby potential antibody and nanobody candidates can be rapidly screened. The basis for screening is the ability of the molecule to produce competitive inhibition of the interaction between the SARS-CoV-2 spike and the host cell angiotensin-converting enzyme 2 (ACE2) receptor.
The RIVIA assay
The study, published in the Annals of the Chemical Society: Analytical Chemistry, reported on the results of the RIVIA assay in screening for neutralizing antibodies. The assay aimed to achieve the same results as a VNA but with a shorter turnaround time, and with the same inhibition efficacy. The researchers used four SARS-CoV-2 spike component antigens, namely, the receptor-binding domain (RBD), the S1 spike subunit, the S-extracellular domain (S-ECD) and the S-ECD homotrimer. The aim was to find the optimal spike component that would be closest to the pVNA results.
They also used seven anti-RBD monoclonal antibodies (mAbs) with different affinities and binding epitopes to produce a result that could be generalized to a range of antibodies directed against the virus. These included rabbit, mouse and human mAbs with strong neutralizing activity against the virus, as well as one with high binding but low neutralizing efficacy, CR3022.
These were first incubated with the various spike components, and then allowed to react with ACE2 in a microfluidics chamber. By balancing the total incubation time with the turnaround time, they found that the assay could be completed within 90 minutes even with one hour of incubation. Simultaneously, they used the same reagents in a SARS-CoV-2 pVNA set-up, to achieve a direct comparison.
The results showed that RIVIA produces comparable results to the pVNAs only with the S-ECD, as expected since it has the right configuration when compared to the actual viral spike protein. R001 had the greatest neutralizing capacity. The control antibody D006 is non-neutralizing, while CR3022 neutralizes the pseudovirus only at very high concentrations (~20,000 ng/mL).
In fact, inhibition of neutralization in 50% of the cells (IC50) with CR3022 is achieved only >1,000 ng/mL. With RIVIA and the S-ECD, therefore, non-neutralizing and weakly neutralizing antibodies can be screened out from the strongly neutralizing antibodies, all of which show a similar IC50 compared to pVNA results.
The RBD, S1 and even the S-ECD monomer could not duplicate this effect due to their smaller size, which allowed for overestimation of the neutralizing efficacy of the weakly neutralizing CR3022. In fact, with the RBD, even the control D006 antibody inhibited RBD binding by over 50% at high concentrations, either because of the small size or due to binding of a secondary epitope.
In addition, they found that the slope of the inhibition of binding correlated with binding affinity for the spike protein.
Nanobody screening with RIVIA
Coming to nanobodies, that is, proteins with a single domain able to bind a single specific antigen, these small, light proteins are easy to produce by standard approaches, and can be modified according to need. They have already been used in imaging, biosensor and therapeutic research over the last few years.
The ability of nanobodies fused to the crystallizable fraction (Fc) of a specific antibody to inhibit viral infection has been tested. This approach was evaluated with respect to SARS-CoV-2 as well using RIVIA.
The results showed a successful screening of strongly neutralizing nanobodies by RIVIA, with somewhat comparable results to those of the pVNAs. The strongest neutralizing antibody (R001) was outranked for potency by the strongest nanobody (Nb21), which had a lower IC50.
When tested against the three major variants of the virus, namely, Alpha, Beta and Gamma, the RIVIA assay showed that ACE2 binding affinity in the absence of antibodies or nanobodies was lowest with the wildtype S-ECD homotrimer, and highest for the Alpha variant, corroborating earlier findings. The IC50 of all antibodies and nanobodies was higher for the Alpha variant compared to the wildtype, but it did not show the ability to escape immune neutralization.
With the other two variants, immune escape was significant, as shown by the failure of many of the antibodies and nanobodies to neutralize them effectively. This includes Nb21 failure, while R001 could only produce neutralization at above 50,000 ng/mL. Almost none of the others did not show neutralizing efficacy.
Since different nanobodies have different binding epitopes on the RBD, the scientists also tested a panel of nanobodies with known escape mutations, to screen for their effects on antibody binding to the S-ECD homotrimer.
When tested against the Beta variant, which had the greatest immune escape capability, they found that Nb21 failed to neutralize it. In comparison, Nb34, with a lower neutralizing efficacy against the wildtype virus, was able to neutralize the Beta variant with the IC50 being 10 ng/mL. Nb93 and Nb95 also showed neutralizing efficacy at nanogram concentrations.
Since all four tested Nbs bind to different epitopes, we infer that the mutations in the epitope of Nb21 may be related to the immune evasion ability of the β variant.”
The study shows that RIVIA compares favorably with pVNAs in the time taken to screen for neutralizing antibodies and nanobodies. It requires only 90 minutes to complete, versus up to 48 hours for the latter. It requires only 8 μL of reagent because of its microfluidic set-up. This reduces the cost considerably, providing quantitative efficacy results at a very high speed, in comparison to the more unwieldy pVNAs, and the non-quantitative lateral flow assays.
The unsuitability of the RBD for quantitative in vitro VNAs is shown, probably due to its small size which leads to false-positive results. Finally, they also showed that,
Some of these agents maintain nearly the same inhibition activities, suggesting that their binding epitopes may be less affected by prevalent RBD mutations and thus can potentially be used as broader neutralizers against SARS-CoV-2 variants.”
Future research will show how RIVIA performs when used to screen neutralizing antibodies for other variants of this virus. The speed at which S-ECD homotrimers can be adapted to screen neutralizing antibodies is higher than pseudovirus variants. This could make RIVIA a very useful tool in the development of new neutralizing antibodies to viral variants.