SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma

Significance This work shows that, under strong immune pressure, SARS-CoV-2 can use mutations in both the N-terminal domain and the receptor-binding domain to escape potent polyclonal neutralizing responses. Indeed, after a long period under immune selective pressure, SARS-CoV-2 evolved to evade the immunity of a potent polyclonal serum from a COVID-19 convalescent donor. Only three mutations were sufficient to generate this escape variant. The new virus was resistant to 70% of the neutralizing antibodies tested and had a decreased susceptibility to all convalescent sera. Our data predict that, as the immunity in the population increases, following infection and vaccination, new variants will emerge, and therefore vaccines and monoclonal antibodies need to be developed to address them.

Plasma samples from 20 convalescent patients with confirmed COVID-19 infection were collected for this study. All plasmas were collected between March and May 2020 where only the original Wuhan virus and D614G variants were circulating. All plasmas, tested by enzyme-linked immunosorbent assay (ELISA), were found to bind the SARS-CoV-2 S-protein trimer, and most of them also bound the S1 and S2 subunits, and the RBD. However, a broad range of reactivity profiles were noticed, ranging from weak binders with titers of 1/10 to strong binders with titers of 1/10,240 (Table 1 and SI Appendix, Fig. S1A). PT008, PT009, PT015, PT122, and PT188 showed the strongest binding toward the S trimer, and, among them, PT188 had also the highest binding to the S1-S2 subunits and among the highest binding titers against the RBD (1/1,280). All but one plasma sample (PT103) were able to bind the S-protein S1 subunit, while three plasma samples (PT103, PT200, and PT276) were negative for binding to Significance This work shows that, under strong immune pressure, SARS-CoV-2 can use mutations in both the N-terminal domain and the receptor-binding domain to escape potent polyclonal neutralizing responses. Indeed, after a long period under immune selective pressure, SARS-CoV-2 evolved to evade the immunity of a potent polyclonal serum from a COVID-19 convalescent donor. Only three mutations were sufficient to generate this escape variant. The new virus was resistant to 70% of the neutralizing antibodies tested and had a decreased susceptibility to all convalescent sera. Our data predict that, as the immunity in the population increases, following infection and vaccination, new variants will emerge, and therefore vaccines and monoclonal antibodies need to be developed to address them.
the RBD. Neutralization activity tested against the SARS-CoV-2 WT and D614G variant also showed variable titers. Most of the plasma samples neutralized the viruses with titers ranging from 1/ 20 to 1/320. Four samples had extremely low titers (1/10), whereas sample PT188 showed extremely high titers (1/10,240). Four plasma samples did not show neutralization activity against the SARS-CoV-2 WT and SARS-CoV-2 D614G variant. Plasma from subject PT188, which had the highest neutralizing titer and ELISA binding reactivity (Table 1 and SI Appendix, Fig. S1 B-D), was selected to test whether SARS-CoV-2 can evolve to escape a potent humoral immunity.
Evolution of SARS-CoV-2 Convalescent Plasma Escape Mutant. Twofold dilutions of plasma PT188 ranging from 1/10 to 1/20,480 were coincubated with 10 5 median tissue culture infectious dose (TCID 50 ) of the WT virus in a 24-well plate. This viral titer was approximately 3 logs more than what is conventionally used in microneutralization assays (16)(17)(18)(19)(20). The plasma/virus mixture was coincubated for 5 d to 8 d. Then, the first well showing cytopathic effect (CPE) was diluted 1:100 and incubated again with serial dilutions of plasma PT188 ( Fig. 1A and SI Appendix, Table  S1). For six passages and 38 d, PT188 plasma neutralized the virus with a titer of 1/640 and did not show any sign of escape. However, after seven passages and 45 d, the neutralizing titer decreased to 1/320. Sequence analyses revealed a deletion of the phenylalanine in position 140 (F140) on the S-protein NTD N3 loop in 36% of the virions (Fig. 1 B and C and SI Appendix, Table  S1). In the subsequent passage (P8), this mutation was observed in 100% of the sequenced virions, and an additional twofold decrease in neutralization activity was observed, reaching an overall neutralization titer of 1/160. Following this initial breakthrough, a second mutation occurred after 12 passages and 80 d of plasma/virus coincubation (P12). This time, the glutamic acid in position 484 of the RBD was substituted with a lysine (E484K). This mutation occurred in 100% of sequenced virions and led to a fourfold decrease in neutralization activity which reached a titer of 1/40 ( Fig. 1 B and C and SI Appendix, Table S1). The E484K substitution was rapidly followed by a third and final change comprising an 11-amino acid insertion between Y248 and L249 in the NTD N5 loop ( 248a KTRNKSTSRRE 248k ). The insertion contained an N-linked glycan sequon ( 248d NKS 248f ), and this viral variant resulted in complete abrogation of neutralization activity by the PT188 plasma sample. Initially, this insertion was observed in only 49% of the virions, but, when the virus was kept in culture for another passage (P14), the insertion was fully acquired by the virus (Fig. 1 B and C and SI Appendix, Table S1).

Reduced Susceptibility to Convalescent Plasma and Monoclonal
Antibodies. To evaluate the ability of the SARS-CoV-2 PT188 escape mutant (PT188-EM) to evade the polyclonal antibody response, all 20 plasma samples from COVID-19 convalescent patients were tested in a traditional CPE-based neutralization assay against this viral variant using the virus at 100 TCID 50 . All samples showed at least a twofold decrease in neutralization activity against SARS-CoV-2 PT188-EM ( Fig. 2A, Table 1, and SI Appendix, Fig. S1 B-D). As expected, the plasma used to select the escape mutant showed the biggest neutralization decrease against this escape mutant with a 256-fold decrease compared to WT SARS-CoV-2. Plasma PT042, PT006, PT005, PT012, and PT041 also showed a substantial drop in neutralization efficacy (Table 1). In addition, we observed that a higher response toward the S-protein S1 subunit correlates with loss of neutralization activity against SARS-CoV-2 PT188-EM (see SI Appendix, Fig. S2A), whereas a high response toward the S-protein S2 subunit did not show correlation (see SI Appendix, Fig. S2B).
We also tested a previously identified panel of 13 neutralizing mAbs (nAbs) by CPE-based neutralization assay to assess their neutralization efficacy against SARS-CoV-2 PT188-EM. These antibodies were classified into three groups based on their binding profiles to the S protein. Group I nAbs were able to bind the S1-RBD, group II targeted the S1 subunit but not the RBD, and group III nAbs were specific for the S-protein trimer ( Table 2). These antibodies also showed a variable neutralization potency against the SARS-CoV-2 WT and D614G viruses ranging from 3.9 ng/mL to 500.0 ng/mL (Fig. 2B, Table 2, and SI Appendix, Fig.  S1 E-G). The three mutations selected by SARS-CoV-2 PT188-EM to escape the highly neutralizing plasma completely abrogated the neutralization activity of two of the six tested RBD-directed antibodies (F05 and G12) (Fig. 2B, Table 2, and SI Appendix, Fig. S1 E-G), suggesting that their epitopes include E484. In contrast, the extremely potent neutralizing antibody J08 was the most potently neutralizing antibody against this escape mutant, with an IC 100 of 22.1 ng/mL. Interestingly, the S1-RBD−directed antibody C14 showed a twofold increase in neutralization activity compared to the SARS-CoV-2 WT virus, whereas I14 and B07 showed a 16-fold and twofold decrease, respectively. All tested antibodies derived from group II (S1-specific not RBD) and group III (S-protein trimer specific) completely lost their neutralization ability against SARS-CoV-2 PT188-EM (Fig. 2B, Table 2, and SI Appendix, Fig. S1 E-G). To better understand the abrogation of activity of some of the tested antibodies, J13, I21, and H20 were cocomplexed with SARS-CoV-2 WT S protein and structurally evaluated by negativestain EM. Two-dimensional (2D) class averages of the three tested antibodies showed that they all bind to the NTD of the S protein (Fig. 2C). A 3D reconstruction for the J13 Fab complex provided further evidence that this antibody binds to the NTD (Fig. 2D).
Putative Structural Effects Enabling Viral Escape. Computational modeling and simulation of the WT and PT188-EM spikes provides a putative structural basis for understanding antibody escape. The highly antigenic NTD is more extensively mutated, containing the F140 deletion as well as the 11-amino acid insertion in loop N5 that introduces a novel N-glycan sequon at position N248d ( Fig. 3 A-C). In contrast, the single mutation in the RBD (E484K) swaps the charge of the sidechain, which would significantly alter the electrostatic complementarity of antibody binding to this region (Fig. 3D). Upon inspection of molecular dynamics (MD) simulations of the NTD escape mutant model, we hypothesize that the F140 deletion alters the packing of the N1, N3, and N5 loops (see SI Appendix, Fig. S3), where the loss of the bulky aromatic sidechain would overall reduce the stability of this region (Table 1). Subsequently, the extensive insertion within the N5 loop appears to remodel this critical antigenic region, predicting substantial steric occlusion with antibodies targeting this epitope, such as antibody 4A8 (Fig. 3B) (21). Furthermore, introduction of a new N-glycan at position N248d (mutant numbering scheme) would effectively eliminate neutralization by such antibodies (Fig. 3B and SI Appendix, Fig. S4).   at day 0 and day 1 (see SI Appendix, Fig. S5C). Finally, strong correlations between viral titers and RdRp/N-gene levels were observed for both SARS-CoV-2 WT and PT188-EM (see SI Appendix, Fig. S5 D and E).

Discussion
We have shown that the authentic SARS-CoV-2, if constantly pressured, has the ability to escape even a potent polyclonal serum targeting multiple neutralizing epitopes. These results are remarkable because SARS-CoV-2 shows a very low estimated evolutionary rate of mutation, as this virus encodes a proofreading exoribonuclease machinery, and, therefore, while escape mutants can be easily isolated when viruses are incubated with single mAbs, it is usually believed that a combination of two mAbs is sufficient to eliminate the evolution of escape variants (22)(23)(24)(25). The recent isolation of SARS-CoV-2 variants in the United Kingdom, South Africa, Brazil, and Japan with deletions in or near the NTD loops shows that what we describe here can occur in the real world. The ability of the virus to adapt to the host immune system was also observed in clinical settings where an immunocompromised COVID-19 patient, after 154 d of infection, presented different variants of the virus, including the E484K substitution (26). Therefore, we should be prepared to deal with virus variants that may be selected by the immunity acquired from infection or vaccination. This can be achieved by developing secondgeneration vaccines and mAbs, possibly targeting universal epitopes and able to neutralize emerging variants of the virus.
A limitation of this study is that viral evolution of SARS-CoV-2 was evaluated only for one plasma sample, limiting the observation of possible spike protein mutations only to a specific polyclonal response. In fact, PT188-EM impacted our plasma samples differently, where PT188, used to pressure the virus in vitro, was the most impacted sample (256-fold decrease), while the remaining 15 neutralizing plasmas showed a median neutralization titer reduction of ∼sevenfold.
Our data also confirm that the SARS-CoV-2 neutralizing antibodies acquired during infection target almost entirely the NTD and the RBD. In the RBD, the possibility to escape is limited, and the mutation E484K that we found is one of the most frequent mutations to escape mAbs (22) and among the most common RBD mutations described in experimental settings (27). Remarkably, the evolution of the E484K substitution observed in our experimental setting was replicated a few months later in the real world by the emergence of E484K variants in South Africa, Brazil, and Japan (14). This is likely due to residue E484 being targeted by antibodies derived from IGHV3-53 and closely related IGHV3-66 genes, which are the most common germlines for antibodies directed against the RBD (28). Recently, this mutation has also been shown to reduce considerably the neutralizing potency of vaccine-induced immunity and to escape mAbs already approved for emergency use by the Food and Drug Administration (29)(30)(31).
On the other hand, the NTD loops can accommodate many different changes, such as insertions, deletions, and amino acid alterations. Interestingly, in our case, the final mutation contained an insertion carrying an N-glycosylation site which has the potential to hide or obstruct the binding to neutralizing epitopes. The introduction of a glycan is a well-known immunogenic escape strategy described in influenza (32), HIV-1, and other viruses (33)(34)(35), although this finding presents a patient-derived escape mutant utilizing this mechanism for SARS-CoV-2. Surprisingly, only three mutations, which led to complete rearrangement of NTD N3 and N5 loops and substitution to a key residue on the RBD, were sufficient to eliminate the neutralization ability of a potent polyclonal serum. Fortunately, not all plasma and mAbs tested were equally affected by the three mutations, suggesting that natural immunity to infection can target additional epitopes that can still neutralize the PT188-EM variant. Vaccine-induced immunity, which is more robust than natural immunity, is likely to be less susceptible to emerging variants. Indeed, so far, the virus has not mutated sufficiently to completely avoid the antibody response raised by current vaccines (36,37). Going forward, it will be important to continue to closely monitor which epitopes on the S protein are targeted by the vaccines against SARS-CoV-2 that are being deployed in hundreds of millions of people around the world. SARS-CoV-2 Authentic Virus Neutralization Assay. The mAbs and plasma neutralization activity was evaluated using a CPE-based assay as previously described (17,20). Further details are available in SI Appendix, Materials and Methods.

Materials and Methods
Viral Escape Assay Using Authentic SARS-CoV-2. All SARS-CoV-2 authentic virus procedures were performed in the biosafety level 3 (BSL3) laboratories at Toscana Life Sciences in Siena (Italy) and Vismederi S.r.l., Siena (Italy). BSL3 laboratories are approved by a certified biosafety professional and are inspected every year by local authorities. To detect neutralization-resistant SARS-CoV-2 escape variants, a standard concentration of the virus was sequentially passaged in cell cultures in the presence of serially diluted samples containing SARS-CoV-2-specific antibodies. Briefly, 12 serial twofold dilutions of PT188 plasma prepared in complete Dulbecco's modified Eagle's medium 2% fetal bovine serum (starting dilution 1:10) were added to the wells of one 24-well plate. Virus solution containing 10 5 TCID 50 of authentic SARS-CoV-2 was dispensed in each antibody-containing well, and the plates were incubated for 1 h at 37°C, 5% CO 2 . The mixture was then added to the wells of a 24-well plate containing a subconfluent Vero E6 cell monolayer. Plates were incubated for 5 d to 7 d at 37°C, 5% CO 2 and examined for the presence of CPE using an inverted optical microscope. A virus-only control and a cell-only control were included in each plate to assist in distinguishing absence or presence of CPE. At each virus passage, the content of the well corresponding to the lowest sample dilution that showed complete CPE was diluted 1:100 and transferred to the antibody-containing wells of the predilution 24-well plate prepared for the subsequent virus passage. At each passage, both the virus pressured with PT188 and the virus-only control were harvested, propagated in 25-cm 2 flasks, and aliquoted at −80°C to be used for RNA extraction, RT-PCR, and sequencing.
Negative Stain Electron Microscopy. SARS-CoV-2 S protein was expressed and purified as previously described (38). Purified spike was combined with individual Fabs at final concentrations of 0.04 mg/mL and 0.16 mg/mL, respectively. Following a 30-min incubation on ice, each complex was deposited on plasma cleaned CF-400 grids (EMS) and stained using methylamine tungstate (Nanoprobes). Grids were imaged at 92,000× magnification in a Talos F200C transmission electron microscope (TEM) equipped with a Ceta 16M detector (Thermo Fisher Scientific). Contrast transfer function estimation and particle picking were performed using cisTEM (39), and particle stacks were exported to cryoSPARC v2 (40) for 2D classification, ab initio 3D reconstruction, and heterogeneous refinement.
Computational Methods. The PT188-EM spike escape mutant was modeled using in silico approaches. As the mutations are localized in two different domains of the spike, namely the NTD and the RBD, separate models were generated for each domain. In detail, two models of the PT188-EM spike NTD (residues 13 to 308) were built starting from two different cryoelectron microscopy (cryo-EM) structures of the WT S protein as templates: 1) one bearing a completely resolved NTD [Protein Data Bank (PDB) ID code 7JJI (41)], which includes all the loops from N1 to N5, and 2) one bound to the antibody 4A8 [PDB ID code 7C2L (21)], which presents only one small gap within the N5 loop. The model of the PT188-EM spike RBD was based on the cryo-EM structure of the spike's RBD in complex with ACE2 [PDB ID code 6M17 (42)]. The generated models were subsequently refined using explicitly solvated all-atom MD simulations. The systems and the simulations were visually inspected with visual molecular dynamics, which was also used for image rendering (43). Further details on the computational method analyses are reported in SI Appendix, Materials and Methods.
Data Availability. All study data are included in the article and SI Appendix.