Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained weight and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design
Using a high-throughput rapid system for antibody discovery, we isolated more than 1000 mAbs from three convalescent donors by memory B cell selection using SARS-CoV-2 S or RBD recombinant proteins. About half of the mAbs isolated could be expressed, and they also bound effectively to S and/or RBD proteins. Only a small fraction of these Abs were neutralizing, which highlights the value of deep mining of responses to access the most potent Abs.
A range of nAbs were isolated to different sites on the S protein. The most potent Abs, reaching single-digit nanogram per milliliter IC50 values in PSV assays, are targeted to a site that, judged by competition studies, overlaps the ACE2 binding site. Only one of the Abs, directed to RBD-B, neutralized SARS-CoV-1 PSV, as may be anticipated given the differences in ACE2 contact residues between the two viruses (fig. S13) and given that the selections were performed with SARS-CoV-2 target proteins. Abs that are directed to the RBD but not competitive with soluble ACE2 (although they may be competitive in terms of an array of membrane-bound ACE2 molecules interacting with an array of S proteins on a virion) are generally less potent neutralizers and tend to show incomplete neutralization, plateauing well below 100% neutralization. The one exception is the cross-reactive RBD-B antibody, mentioned above. Similarly low potency and incomplete neutralization are observed for Abs to the S protein that are not reactive with recombinant RBD. The cause(s) of these incomplete neutralization phenomena is unclear but presumably originates in some S protein heterogeneity that is either glycan, cleavage, or conformationally based. Regardless, the RBD-A nAbs that directly compete with ACE2 are clearly the most preferred for prophylactic and therapeutic applications and as reagents to define nAb epitopes for vaccine design. Note that, even for a small sampling of naturally occurring viral variants, two were identified that showed notable resistance to individual potent nAbs to the WA1 strain, which suggests that neutralization resistance will need to be considered in planning for clinical applications of nAbs. Cocktails of nAbs may be required.
In terms of nAbs as passive reagents, the efficacy of a potent anti-RBD nAb in vivo in Syrian hamsters is promising in view of the positive attributes of this animal model and suggests that human studies are merited. Nevertheless, as for any animal model, there are many limitations, including, in the context of antibody protection, differences in effector cells and Fc receptors between humans and hamsters. The failure of the non-RBD S-protein nAb to protect in the animal model is consistent with its lower potency and, likely most importantly, its inability to fully neutralize challenge virus. In the context of human studies, the following antibody engineering goals could be considered: improving the potency of protective nAbs by enhancing binding affinity to the identified RBD epitope, improving half-life, and reducing Fc receptor binding to minimize potential antibody-dependent enhancement (ADE) effects if they are identified. As observed for heterologous B cell responses against different serotypes of flavivirus infection, there is a possibility, but no current experimental evidence, that subtherapeutic vaccine serum responses or subtherapeutic nAb titers could potentially exacerbate future coronavirus infection disease burden by expanding the viral replication and/or cell tropism of the virus. If ADE is found for SARS-CoV-2 and operates at subneutralizing concentrations of neutralizing antibodies, as it can for dengue virus (13), then it would be important, from a vaccine standpoint, to carefully define the full range of nAb epitopes on the S protein, as we have begun to do here. From a passive antibody standpoint, it would be important to maintain high nAb concentrations or appropriately engineer nAbs.
The nAbs described here have very low somatic hypermutation (SHM), typically only one or two mutations in the VH gene and one or two in the VL gene. Such low SHM may be associated with the isolation of the nAbs relatively soon after infection, perhaps before affinity-maturation has progressed. Low SHM has also been described for potent nAbs to Ebola virus, RSV, Middle East respiratory syndrome coronavirus, and yellow fever virus (14–17) and may indicate that the human naïve repertoire is often sufficiently diverse to respond effectively to many pathogens with little mutation. Of course, nAb efficacy and titer may increase over time, as described for other viruses, and it will be interesting to see if even more potent nAbs to SARS-CoV-2 evolve in our donors in the future.
What do our results suggest for SARS-CoV-2 vaccine design? First, they suggest a focus on the RBD—strong nAb responses have indeed been demonstrated by immunizing mice with a multivalent presentation of RBD (18). The strong preponderance of non-neutralizing antibodies and the very few nAbs to S protein that we isolated could arise for a number of reasons, including the following: (i) The recombinant S protein that we used to select B cells is a poor representation of the native spike on virions. In other words, there may be many nAbs to the S protein, but we failed to isolate them because of the selecting antigen. (ii) The recombinant S protein that we used is close to native, but non-neutralizing antibodies bind to sites on the S protein that do not interfere with viral entry. (iii) The S protein in natural infection disassembles readily, generating a strong Ab response to viral debris that is non-neutralizing, because the antibodies recognize protein surfaces that are not exposed on the native spike. The availability of both neutralizing and non-neutralizing antibodies generated in this study will facilitate evaluation of S protein immunogens for presentation of neutralizing and non-neutralizing epitopes and will promote effective vaccine design. The design of an immunogen that improves on the quality of nAbs elicited by natural infection may well emerge as an important goal of vaccine efforts.
In summary, we describe the very rapid generation of neutralizing antibodies to a newly emerged pathogen. The antibodies can find clinical application and will aid in vaccine design.
Reference & Source Information: https://science.sciencemag.org/
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