Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on Severe Acute Respiratory Syndrome Coronavirus Neutralization Escape and Fitness

Abstract

The receptor binding domain (RBD) of the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major target of protective immunity in vivo. Although a large number of neutralizing antibodies (nAbs) have been developed, it remains unclear if a single RBD-targeting nAb or two in combination can prevent neutralization escape and, if not, attenuate viral virulence in vivo. In this study, we used a large panel of human nAbs against an epitope that overlaps the interface between the RBD and its receptor, angiotensin-converting enzyme 2 (ACE2), to assess their cross-neutralization activities against a panel of human and zoonotic SARS-CoVs and neutralization escape mutants. We also investigated the neutralization escape profiles of these nAbs and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral strains, including neutralization escape viruses derived from other nAbs; however, no single nAb or combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani background) or increased virulence (GD03 background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single nAbs or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint. IMPORTANCEThe emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has resulted in severe human respiratory disease with high death rates. Their zoonotic origins highlight the likelihood of reemergence or further evolution into novel human coronavirus pathogens. Broadly neutralizing antibodies (nAbs) that prevent infection of related viruses represent an important immunostrategy for combating coronavirus infections; however, for this strategy to succeed, it is essential to uncover nAb-mediated escape pathways and to pioneer strategies that prevent escape. Here, we used SARS-CoV as a research model and examined the escape pathways of broad nAbs that target the receptor binding domain (RBD) of the virus. We found that neither single nAbs nor two nAbs in combination blocked escape. Our results suggest that targeting conserved regions with less plasticity and more structural constraint rather than the SARS-CoV RBD-like region(s) should have broader utility for antibody-based immunotherapy.

Molecular Basis of Coronavirus Virulence and Vaccine Development

“…A combination of two neutralizing mAbs could prevent the emergence of neutralization escape mutants or at least attenuate viral virulence in vivo. However, although neutralizing mAbs directed against epitopes located at the interface between the viral S protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2), proved to have great potency and breadth in neutralizing multiple viral strains (Sui et al, 2014), both the single and combined use of one or two mAbs, respectively, failed to prevent the emergence of antibody escape variants. Therefore, the use of one or two neutralizing mAbs that target a structurally flexible SARS-CoV epitope may be of limited value for in vivo immunotherapies and should be combined with neutralizing mAbs that bind a second conserved epitope with low structural plasticity.…”

Analysis of preferred codon usage in the coronavirus N genes and their implications for genome evolution and vaccine design

“…For instance, its large parts are cleaved during infection by cellular proteases and expose the receptors to activate viral attachment to the host (Fiscus, 1987;Wu et al, 2004aWu et al, , 2004bMaache et al, 2006;Gao et al, 2013). Additionally, the S protein is prone to mutations, especially in the amino acids associated with the spike protein-cell receptor interface, in order to overcome host immunity (Wu and Yan, 2005;Sui et al, 2014). In an interesting study, the N gene of the CoV was found to be more effective for evaluating the codon usage bias than the S gene (Ahn et al, 2009).…”

Middle East respiratory syndrome coronavirus vaccines: current status and novel approaches

“…Thus far, the variability in the amino acid sequence of the spike protein observed among MERS-CoV strains is low [71], and circulating MERS-CoV strains did not show any significant variation in the serological reactivity [25,59], implying that the development of a vaccine that is effective against one strain is likely to be protective against all circulating strains. Another risk posed from the development of antibody escape variants is still present [72,73], although this is not likely to happen as mutations in two epitopes may be required, and mutants that develop may have reduced viral fitness [73,74].…”