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. 2014 Dec;88(23):13769-80.
doi: 10.1128/JVI.02232-14. Epub 2014 Sep 17.

Effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness

Jianhua Sui  1 , Meagan Deming  2 , Barry Rockx  2 , Robert C Liddington  3 , Quan Karen Zhu  1 , Ralph S Baric  4 , Wayne A Marasco  5
Affiliations

Effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness

Jianhua Sui et al. J Virol. 2014 Dec.

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.

Importance: The 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.

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Figures

FIG 1
FIG 1
Neutralization of human and animal SARS-CoVs and neutralization escape mutants by a panel of nAbs. (A) Amino-acid differences in the RBDs of human and animal SARS-CoVs. (B) Neutralization of viruses listed in panel A by 80R, its derivative antibodies, and 11A (GD03 specific and not a derivative of 80R) in a microneutralization assay. The 50% neutralization titer was defined as the Ab dilution at which at least 50% of wells showed no CPE. The Ab concentration in the stock solution used for dilution was 20 μg/ml. (C) Neutralization efficacy against wild-type (WT) and neutralization escape mutants generated in a previously reported study (34) by the same panel of nAbs tested in panel B. The neutralization assay was performed similarly to that in panel B.
FIG 2
FIG 2
Prophylactic treatment of SARS-CoV infections in 12-month-old BALB/c mice by 80R and fm6 nAbs. Body weights of mice infected with icGD03 or icUrbani were measured daily after passive administration of 12.5 mg/kg (∼250 μg/mouse) nAbs. Lung tissues of mice infected with icGD03 or icUrbani were harvested on day 4 postinfection and assayed for infectious virus by a plaque assay using Vero E6 cells. Error bars indicate standard deviations (n = 5). *, P < 0.01, **, P < 0.001, and ***, P < 0.0001, compared with the PBS group.
FIG 3
FIG 3
Neutralization escape mutants of 80R derivatives and generation of new antibodies to neutralize escape mutants. (A) Sequence comparison of 80R derivative nAbs in their light-chain complementarity-determining regions (LCDR1 to -3) (38) and their escape mutations in the RBD. (B) Cross-neutralization of escape mutant viruses listed in panel A by fm6. The neutralization assay was performed similarly to that in Fig. 1. 80R was included as a control. (C) Sequence comparison of human monoclonal Abs against the fm6 escape mutant Urbani-Y436H in their LCDRs. A dot indicates the same residue as that of 80R. These Abs were isolated from selection of nonimmune phage display antibody library (Abs 12 and 22) and 80R-cs Ab-phage display libraries (all other Abs) (38) using Tor2-Y436H-S1 protein coupled onto magnetic beads or the immunotube as the panning targets. (D) Neutralization of S protein of SARS-CoV-pseudotyped lentiviruses with antibodies listed in panel C. Ab Y12 and Y112A (highlighted in blue) have broadly neutralization activity against all five viruses tested.
FIG 4
FIG 4
Broadly neutralizating activity of nAbs fm6 and Y112A. Neutralization titers against five different SARS-CoVs or escape mutants were determined in a plaque reduction neutralization assay. Abs were serially diluted 2-fold as indicated, and 100 PFU of the different icSARS-CoV strains was used (see Materials and Methods). At the end of the assay, plaques were stained and counted for calculation of plaque reduction efficiency. Each value shown represents the average of duplicate samples. GD03-MA is a mouse-adapted strain encoding the 2004 human GD03 S protein with Y436H mutation in the RBD (39).
FIG 5
FIG 5
Effects of escape mutations within the Urbani background on the binding to ACE2 and viral growth in vitro and in vivo. (A) List of all critical amino acid changes associated with escape mutation. (B) The locations of the amino acids listed in panel A are shown in the cocrystal structure of the SARS-CoV RBD (blue) and its receptor, human ACE2 (yellow) (PDB code 2AJF). (C) Binding affinity and kinetics measurement of the RBD of the escape mutations listed in panel A to human ACE2. Binding kinetics were evaluated using a 1:1 Langmuir binding model. Each Ka, Kd, and KD value represents the mean and standard error of two independent experiments run on Biacore. (D) In vitro growth characteristics of neutralization escape mutant SARS-CoV. Cultures of Vero E6 cells were infected in duplicate with icUrbani WT and neutralization escape mutants as indicated at a multiplicity of infection (MOI) of 1, as described in Materials and Methods. Virus titers at different time points were determined by a plaque assay using Vero E6 cells. A dotted line indicates the lowest detectable virus titer. (E) Escape mutants of cs5, cs39, and fm6 in aged mice (12 months old). (Left) Weight loss. All mice were inoculated with 105 PFU of viruses as indicated. Body weights of infected mice were measured on a daily basis (5 mice per group). Weight changes are expressed as the mean percent changes for infected animals relative to the initial weights at day 0. *, P < 0.05, and **, P < 0.01, compared with the icUrbani WT, by two-way analysis of variance. (Right) Lung titers. Lung tissues were harvested from infected mice on day 4 after infection and were assayed for virus titer by a plaque assay using Vero E6 cells. A dotted line indicates the lowest detectable virus titer.
FIG 6
FIG 6
Effect of escape mutations within the GD03 background on the binding to ACE2 and in vivo virus growth in mouse model. (A) A table lists escape mutants generated with icGD03 or icGD03-MA (Y436H) viruses for nAb Y12 and Y112A and the binding kinetics of the RBD of these escape mutants with human ACE2 and mouse ACE2 (mACE2). (B) Escape mutants of Y12 and Y112A in young mice (10 weeks old). The experiment was performed similarly to that in Fig. 5E. (Left) Virus titer in lungs. Lung tissues were harvested from infected mice on day 4 after infection for virus titer analysis. Error bars denote standard deviations. *, P < 0.05, and ***, P < 0.0001, compared with the GD03-MA strain. A dotted line indicates the lowest detectable virus titer. (Right) Weight loss. Body weights of infected mice were monitored at different time points (5 mice per group). Weight changes are expressed as the mean percent changes for infected animals relative to the initial weights at day 0.
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