Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

Abstract

Antibodies targeting the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present a promising approach to combat the coronavirus disease 2019 (COVID-19) pandemic; however, concerns remain that mutations can yield antibody resistance. We investigated the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared after in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Escape mutants were not generated after treatment with a noncompeting antibody cocktail.

Fig. 1. Escape mutant screening protocol.

(A) A schematic is displayed of the VSV-SARS-CoV-2-S virus genome encoding residues 1-1255 of the spike protein in place of the VSV glycoprotein. N, nucleoprotein, P, phosphoprotein, M, matrix, and L, large polymerase. (B) A total of 1.5 × 10⁶ pfu of the parental VSV-SARS-CoV-2-S virus was passed in the presence of antibody dilutions for 4 days on Vero E6 cells. Cells were screened for virus replication by monitoring for virally induced cytopathic effect (CPE). Supernatants and cellular RNAs were collected from wells under the greatest antibody selection with detectable viral replication (circled wells; ≥20% CPE). For a second round of selection, 100uL of the P1 supernatant was expanded for 4 days under increasing antibody selection in fresh Vero E6 cells. RNA was collected from the well with the highest antibody concentration with detectable viral replication. The RNA was deep sequenced from both passages to determine the selection of mutations resulting in antibody escape. (C) The passaging results of the escape study are presented with the qualitative percentage of CPE observed in each dilution (red ≥ 20%CPE and blue < 20% CPE). Black boxes indicate dilutions that were passaged and sequenced in P1 or sequenced in P2. A no antibody control was sequenced from each passage to monitor for tissue culture adaptations.

Fig. 2. Deep sequencing of passaged virus identifies escape mutations.

VSV-SARS-CoV-2-S virus was mixed with either individual or combinations of anti-spike mAbs. Viral RNA from wells with the highest mAb concentration and detectable cytopathic effect (CPE) on passage 1 or 2 (collected 4 days post-infection) was isolated and RNAseq analysis was performed to identify changes in spike protein sequence relative to input virus. For passage 2, viral RNA was isolated and sequenced from wells with high mAb concentrations (>10ug/ml) with subsequently validated escape; if no validated escape was seen at these high mAb concentrations and no virus was grown, ND is shown as no virus RNA was isolated. All mutated amino acid residues within the spike protein are shown. Specific condition (concentration in ug/ml) of the well that was selected for sequencing is shown in the left-hand column (refer to Fig. 1 for outline of the experiment). Red boxes highlight residues that were mutated relative to input virus under each condition specified in the left-hand column. Percentage in each box identifies % of sequencing reads that contained the respective mutant sequence. Residues mapping to the RBD domain are highlighted in blue.