Driving potent neutralization of a SARS-CoV-2 Variant of Concern with a heterotypic boost

The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet known whether heterotypic vaccine boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein. Passive immunization with plasma sampled following this boost protected K18-hACE2 mice from lethal challenge with a 501Y.V2 clinical isolate, whereas only partial protection was afforded by plasma sampled after two Wu-Hu-1 spike immunizations.


Introduction
At least 20 candidate SARS-CoV-2 vaccines have already entered phase 3 clinical trials. A number of these demonstrated high efficacy 1-5 , significantly reducing morbidity and mortality, and are being rolled-out globally. This first generation of vaccines all encode or deliver a spike glycoprotein derived from the pandemic founder strain, Wu-Hu-1 6 . Driven by multiple evolutionary forces 8 , SARS-CoV-2 is evading immune responses and undermining our prevention and mitigation strategies. Globally, a number of VOCs are rising in frequency (see Fig 1), each harbouring spike mutations that confer resistance to prior immunity. Of particular concern is the surge of variant 501Y.V2 9 , with multiple mutations in dominant neutralizing antibody epitopes making it several fold more resistant to antibodies elicited by current vaccines [10][11][12] . This underpins the substantially reduced vaccine efficacies in South Africa, where this variant is circulating at high frequency 13,14 . Updated vaccines are likely required to protect against current and future mutated variants. Importantly, by the time these are rolled out, a significant pro- portion of the global population are likely to be seropositive as a result of either infection, or immunization with Wu-Hu-1 based vaccines. A relevant question now is whether a single dose will be sufficient to induce robust neutralising antibody responses to VOCs in seropositive individuals, and whether these boosts are sufficient to confer protection. Importantly, the first exposure to a pathogen can shape future responses to mutated variants. This immunological imprinting or original antigenic sin 15 is well-described for influenza A virus, where protection is highest against the first strain encountered and diminished against those encountered later in life 16,17 . It is crucial for the design of updated vaccines and regimens to determine if existing immunity dampens antibody responses to new VOCs, or if a heterotypic boost can efficiently recruit cross-protective memory responses.
To determine whether restoration of neutralizing antibody titers to 501Y.V2 afforded a biologically relevant improvement in protective immunity, mice transgenic for human ACE2 (K18-hACE2 21 ) were passively immunized intraperi-toneally (i.p.) with plasma samples taken either 2 weeks following the second spike immunization (N=8) ("post S"), or 1-2 weeks following the RBD booster immunization (N=8) ("post vRBD"). Passive immunization conferred titers approximately 10-fold lower than donor plasma (Supp. Fig 2), and macaque polyclonal antibodies were not rapidly cleared following xenotransfusion with an unchallenged mouse still maintaining titers >1400 after 5 days (data not shown). Mice were then challenged intranasally with 2.4x106 RNA copies of either 501Y.V2 or 'wild-type' (encoding a spike matching Wu-Hu-1) virus (corresponding to 100 PFU of 501Y.V2 or 86 PFU of wild-type), and weight -a reliable proxy for disease severity 22 -was monitored daily. Across all groups, protection was strongly correlated with the neutralizing antibody titers to the challenge virus on the day of challenge (Spearman's ρ = 0.822, p<1x10-8, Fig. 3a). All control mice that did not receive plasma (PBS only) succumbed to disease when challenged with either variant, showing precipitous weight loss starting around three days post-challenge ( Fig. 3b-d). Passive transfer of post S serum conferred protection from WT virus ( Fig. 3c) but not from 501Y.V2 (Fig. 3d), clearly demonstrating that evasion of the antibody response by this VOC was sufficient to cause disease. Notably, passive transfer of post vRBD plasma protected against both WT and 501Y.V2 (Fig. 3c,d).

Discussion
For many licensed vaccines, reduced efficacy has been observed against the 501Y.V2 variant 13,14,23 . Moreover, the decay of vaccine-elicited antibody titers 19,24 suggests that over time, protection will wane further. Consistent with reduced vaccine efficacy against 501Y.V2, we show that breakthrough infection and disease occurs in K18-hACE2 mice passively immunized with plasma from rhesus macaques immunized with two doses of adjuvanted wild-type spike protein.
Cross-species passive immunization and challenge experiments have been used to show antibody-mediated protection conferred by influenza vaccination (human to mouse 25 ), and more recently, to characterize protection from wild-type SARS-CoV-2 (macaque to hamster 26 ). Our data further demonstrate the utility of this approach for investigating protection against SARS-CoV-2 variants.
The ability of vaccines to broaden existing responses to new variants is still largely unclear. Despite weak immunogenicity of soluble, monomeric RBD as a priming antigen 20 , heterotypic RBD administered as a boost elicited a potent recall response in non-human primates. This was robust to the boosting dose, and effective as low as 2 µg, possibly aided by a dose-sparing effect of Matrix-M 27 . While reduced neutralization of 501Y.V2 was evident following 2 doses of Wu-Hu-1 spike, both Wu-Hu-1 and 501Y.V2 were potently neutralized following heterotypic (501Y.V2) RBD boost. In animal challenge models, neutralizing antibodies following passive immunization represented a robust correlate of protection such that the restoration of neutralizing antibody titers to 501Y.V2 also translated into protective immunity.
The potent, cross-neutralizing antibody response that arises following a heterotypic boost indicates that original antigenic sin does not represent a significant barrier to the acquisition of protective immunity against current SARS-CoV-2 VOCs. In the immunized animal sampled only 5 days post vRBD boost, neutralizing titers (against both Wu-Hu-1 and 501Y.V2) were already elevated suggesting these titers are the product of a rapidly activated population of antibody secreting cells. Further, this time course indicates that successive rounds of affinity maturation likely were not required for neutralization of 501Y.V2, but rather that vRBD-specific antibody responses could be boosted from the pool of existing memory B cells primed by Wu-Hu-1. These responses are largely consistent with recently reported results from 501Y.V2 spike mRNA (mRNA1273.351, Moderna) booster vaccinations 28,29 . The observation that immunization with RBD (and not whole spike) was capable of inducing robust neutralising antibody responses is particularly promising as RBD is a small, stable protein that can be rapidly synthesized and efficiently expressed. Taken together, these data indicate that potent, crossneutralizing and cross-protective antibody responses can be recruited with heterotypic SARS-CoV-2 immunogens following a primary exposure, and identify soluble RBD booster immunizations as an attractive strategy to broaden vaccine protection from new SARS-CoV-2 variants.    30 ) and RBD were produced by the transient transfection of Freestyle 293-F cells using FreeStyle MAX reagent (Thermo Fisher) or polyethylenimine (PEI), respectively. The HIS-tagged Spike ectodomain and RBD were purified from filtered supernatant using nickel IMAC resin (HisPur Ni-NTA, Thermo Fisher Scientific) followed by size-exclusion chromatography on a Superdex 200 (Cytiva) in PBS. On the day of immunization, indicated doses were mixed with 50 µg Matrix-M™ adjuvant (Novavax AB, Uppsala, Sweden) in a final inoculation volume of 800 µl. Macaques were immunized intramuscularly (i.m.) with half of each dose administered in each quadricep. All immunizations and blood samplings were performed under sedation with 10-15 mg/kg ketamine (Ketaminol, Intervet, Sweden) administered i.m.. Blood plasma was isolated by centrifugation, and heat inactivated at 56°C for 60 minutes. Pseudotyped virus neutralization assays. All plasma and serum samples were heat inactivated at 56°C for 60 minutes. Pseudotyped lentiviruses displaying either the SARS-CoV-2 pandemic founder variant (Wu-Hu-1) or 501Y.V2 variant spike 31 and packaging a firefly luciferase reporter gene were generated by the co-transfection of HEK293T cells using Lipofectamine 3000 (Invitrogen) per the manufacturer's protocols. Media was changed 12-16 hours after transfection, and pseudotyped viruses were harvested at 48-and 72-hours post-transfection, clarified by centrifugation, and stored at -80°C until use. Pseudotyped viruses sufficient to generate 50,000 relative light units (RLUs) were incubated with serial dilutions of plasma for 60 min at 37°C in a 96-well plate, and then 15,000 HEK293T-hACE2 cells were added to each well. Plates were incubated at 37°C for 48 hours, and luminescence was then measured using Bright-Glo (Promega) per the manufacturer's protocol, on a GM-2000 luminometer (Promega). Virus production and quantification. Viral isolates expanded from clinical samples of 'wild-type' and 501Y.V2 32 were kind gifts from Jonas Klingstrom (Karolinska Institutet) and Alex Sigal (The African Health Research Institute) respectively. Isolates were propagated in Calu-3 cells for 72 hours and harvested from the supernatant to generate replication competent SARS-CoV-2 stocks for animal challenge. All virus challenge stocks harboured no cell-culture adaptation mutations detectable by Sanger sequencing of the full-length spike gene (see Supp. Fig. 3). Viral titers were quantified by a plaque forming assay in Vero E6 cells, as previously described 33 . Viral RNA was quantified from clarified viral supernatant in TRIzol (ThermoFisher Scientific) and extracted using the manufacturer's protocol but with the following modifications: Total RNA was precipitated with isopropanol in the presence of Glycoblue (Thermo Fisher Scientific) coprecipitant for 45 min at -20C, and RNA pellets were resuspended in warm RNase-free water. RT-qPCR reactions were performed using the Superscript III one step RT-qPCR system with Platinum Taq Polymerase (Invitrogen) with 400 nM of each primer and 200 nM of probe. Primers and probes for the CoV-E gene target were as previously described 34 . Thermal cycling consisted of RT at 55C for 10 min, denaturation at 95C for 3 min, and 45 cycles of 95C for 15 seconds and 58C for 30 seconds. Reactions were carried out using a CFX96 Connect Real-Time PCR Detection System (Bio-Rad) following manufacturer instructions. To generate standard curves, a synthetic DNA template gBlock (Integrated DNA Technologies) was transcribed using the mMessage mMachine T7 Transcription Kit (Invitrogen) and serially diluted. For each viral stock, RT-qPCR was run on a 10-fold dilution series ranging from 1:10 to 1:10,000 to ensure linearity of the assay and avoid saturation at high copy numbers. K18-hACE2 mouse challenge. One day prior to challenge, K18-hACE2 mice were passively immunized with 200µl of macaque plasma, administered intraperitoneally (i.p.) under isoflurane sedation. Each plasma sample was administered to four mice (two to be challenged with wild-type virus, and two to be challenged with 501Y.V2).

AUTHOR CONTRIBUTIONS
The following day, mice were bled from the tail vein (to obtain serum for the characterization of neutralizing antibody titers), moved to a BSL3 facility and challenged intranasally with a standardized dose (2.4x106 RNA copies) of either 'wild-type' or 501Y.V2 virus stock in a total challenge volume of 40µl in PBS. All challenges were performed under light isoflurane sedation. Weight and general body condition was monitored daily until weight loss was evident, after which mice were monitored twice daily. Throughout the experiment, weight loss, changes in general health, breathing, body movement and posture, piloerection and eye health were monitored. Mice were euthanized when they experienced weight loss of at least 20% of their starting body weight, or when movement was greatly impaired and/or they experienced difficulty breathing that was considered to reach a severity level of 0.5 on Karolinska Institutet's veterinary plan for monitoring animal health.

20A Calu-3 72 hpi T T G G T G C A G G T A T A T G C G C T A G T T A T C A G A C T C A G A C T A A T T C T C C T C G G C G G G C A C G T A G T G T A G C
20H/501Y.V2 isolate

T T G G T G C A G G T A T A T G C G C T A G T T A T C A G A C T C A K A C T A A T T C T C C T Y G G C G G G C A C G T A G T G T A G C
20A Vero E6  Fig. SI3. Expansion of SARS-CoV-2 isolates in Vero E6 but not Calu-3 cells rapidly selected for mutations and deletions proximal to the furin cleavage site. Challenge stocks used in this study were produced in Calu-3 cells, and confirmed by Sanger sequencing to harbour no high frequency cell culture adaptation mutations in spike. Electropherograms spanning the furin cleavage site from sanger sequencing of amplified viral RNA are shown for virus cultured in Vero E6 or Calu-3 cells demonstrating the rapid loss of the furin recognition sequence upon culture in Vero E6 cells but not Calu3 cells. Received stock of 501Y.V2 ("20H/501Y.V2 isolate") had a mixture of intact/knocked-out furin site.