Neutralising antibodies in Spike mediated SARS-CoV-2 adaptation

Abstract

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, particularly in immunocompromised individuals where prolonged viral replication occurs. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences by both short and long read technologies over 23 time points spanning 101 days. Although little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days, N501Y in Spike was transiently detected at day 55 and V157L in RdRp emerged. However, following convalescent plasma we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility, but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type and appeared to compensate for the reduced infectivity of D796H. Consistent with the observed mutations being outside the RBD, monoclonal antibodies targeting the RBD were not impacted by either or both mutations, but a non RBD binding monoclonal antibody was less potent against ΔH69/ΔV70 and the double mutant. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with reduced susceptibility to neutralising antibodies.

Keywords: COVID-19; Convalescent plasma; SARS-CoV-2; antibody escape; evasion; immune suppression; mutation; neutralising antibodies; resistance.

Figure 1:

Clinical time line of events with longitudinal respiratory sample CT values. CT- cycle threshold

Figure 2.. Analysis of 23 Patient derived whole SARS-CoV-2 genome sequences in context of national sequences and other cases of chronic SARS-CoV-2 shedding.

A. Circularised maximum-likelihood phylogenetic tree rooted on the Wuhan-Hu-1 reference sequence, showing a subset of 250 local SARS-CoV-2 genomes from GISAID. This diagram highlights significant diversity of the case patient (green) compared to three other local patients with prolonged shedding (blue, red and purple sequences). All “United Kingdom / English” SARS-CoV-2 genomes were downloaded from the GISAID database and a random subset of 250 selected as background. B. Close-view maximum-likelihood phylogenetic tree indicating the diversity of the case patient and three other long-term shedders from the local area (red, blue and purple), compared to recently published sequences from Choi et al (orange) and Avanzato et al (gold). Control patients generally showed limited diversity temporally, though the Choi et al sequences were found to be even more divergent than the case patient. Environmental samples (patient’s call bell, and patient’s mobile phone) are indicated.

Figure 3.. Serum SARS-CoV-2 antibody levels and virus population changes in chronic SARS-CoV-2 infection.

A. Anti SARS-CoV-2 IgG antibodies in patient and pre/post convalescent plasma compared to RNA+ Covid19 patients and prepandemic healthy controls: Red, grey and gold: IgG antibodies to SARS-CoV2 nucleocapsid protein (N), trimeric S protein (S) and the receptor binding domain (RBD) were measured by multiplexed particle based flow cytometry (Luminex) in RNA+ Covid 19 patients (N=20, red dots), Pre-pandemic healthy controls (N=20, grey dots) and in the convalescent donor plasma (orange dots); Results are shown as mean fluorescent intensity (MFI) +/− SD. Patient sera over time in blue: Anti SARS-CoV2 IgG to N (blue squares), S (blue circles) and RBD (blue triangles). Timing of CP units is also shown B. Highlighter plot indicating nucleotide changes at consensus level in sequential respiratory samples compared to the consensus sequence at first diagnosis of COVID-19. Each row indicates the timepoint the sample was collected (number of days from first positive SARSCoV-2 RT-PCR). Black dashed lines indicate the RNA-dependent RNA polymerase (RdRp) and Spike regions of the genome. There were few nucleotide substitutions between days 1–54, despite the patient receiving two courses of remdesivir. The first major changes in the spike genome occurred on day 82, following convalescent plasma given on days 63 and 65. The amino acid deletion in S1, ΔH69/V70 is indicated by the black lines. Sites: Endotracheal aspirate (ETA) or Nose/throat swabs (N+T).

Figure 4.. Whole genome variant trajectories showing amino acids and relationship to treatments.

Data based on Illumina short read ultra deep sequencing at 1000x coverage. Variants shown reached a frequency of at least 10% in at least 2 samples. Treatments indicated are convalescent plasma (CP) and Remdesivir (RDV). Variants described in the text are designated by labels using the same colouring as the position in the genome. A. Variants detected in the patient from days 1–81. B. Variants detected in the patient from day 81–101. C. Multiple sequence alignment of 10 randomly selected SARS-CoV-2 Genomes containing the Δ69/70 deletion. In all cases, six nucleotides are deleted that are out of frame.

Figure 5.. Longitudinal variant frequencies and phylogenetic relationships for virus populations bearing six Spike (S) mutations

A. At baseline, all four S variants (Illumina sequencing) were absent (<1% and <20 reads). Approximately two weeks after receiving two units of convalescent plasma (CP), viral populations carrying ΔH69/V70 and D796H mutants rose to frequencies >90% but decreased significantly four days later. This population was replaced by a population bearing Y200H and T240I, detected in two samples over a period of 6 days. These viral populations were then replaced by virus carrying W64G and P330S mutations in Spike, which both dominated at day 93. Following a 3^rd course of remdesivir and an additional unit of convalescent plasma, the ΔH69/V70 and D796H virus population re-emerged to become the dominant viral strain reaching variant frequencies of >90%. Pairs of mutations arose and disappeared simultaneously indicating linkage on the same viral haplotype. CT values from respiratory samples are indicated on the right y-axis (black triangles) B. Maximum likelihood phylogenetic tree of the case patient with day of sampling indicated. Spike mutations defining each of the clades are shown ancestrally on the branches on which they arose. On dates where multiple samples were collect, these are indicated as endotracheal aspirate (ETA) and Nose + throat swabs (N+T).

Figure6:. Spike mutant D796H + ΔH69/V70 has infectivity comparable to wild type but is less sensitive to multiple units of convalescent plasma (CP).

A. western blot of virus pellets after centrifugation of supernatants from cells transfected with lentiviral pseudotyping plasmids including Spike protein B. Single round Infectivity of luciferase expressing lentivirus pseudotyped with SARS-CoV-2 Spike protein (WT versus mutant) on 293T cells co-transfected with ACE2 and TMPRSS2 plasmids. Infectivity is corrected for reverse transcriptase activity in virus supernatant as measured by real time PCR C–E. convalescent plasma (CP units 1–3) neutralization potency. against pseudovirus virus bearing Spike mutants D796H, ΔH69/V70 and D796H + ΔH69/V70 F, G patient serum neutralisation potency against pseudovirus virus bearing Spike mutants D796H, ΔH69/V70 and D796H + ΔH69/V70. Patient serum was taken at indicated Day (D). Indicated is serum dilution required to inhibit 50% of virus infection (ID50). Data points represent means of technical replicates and each data point is an independent exp.

Figure 7:. Neutralization potency of a panel of monoclonal antibodies targeting the RBD is not impacted by Spike mutations.

Lentivirus pseudotyped with SARS-CoV-2 Spike protein: WT (D614G background), D796H, ΔH69/V70, D796H+ΔH69/V70 were produced in 293T cells and used to infect target Hela cells stably expressing ACE2 in the presence of serial dilutions of indicated monoclonal antibodies. Data are representative of at least two independent experiments. RBD: receptor binding domain. Classes of RBD binding antibodies are indicated based Bouwer et al.

Figure 8.. Location of Spike mutations ΔH69/Y70 in S1 and D796H in S2.

Amino acid residues H69 and Y70 deleted in the N-terminal domain (red) and D796H in subunit 2 (orange) are highlighted on a SARSCoV-2 spike trimer (PDB: 6ZGE Wrobek et al., 2020). Each of the three protomers making up the Spike homotrimer are coloured separately in shades of grey (centre). Close-ups of ΔH69/Y70 (above) and D796H (below) are shown in cartoon, stick representation. Both mutations are in exposed loops.