key: cord-0839286-c9wdpoyv
authors: Halfmann, Peter J.; Castro, Ana; Loeffler, Kathryn; Frey, Steven J.; Chiba, Shiho; Kawaoka, Yoshihiro; Kane, Ravi S.
title: Potent neutralization of SARS‐CoV‐2 including variants of concern by vaccines presenting the receptor‐binding domain multivalently from nanoscaffolds
date: 2021-09-09
journal: Bioeng Transl Med
DOI: 10.1002/btm2.10253
sha: da4f3a7ee85b6e43de9adb90ddbd7caef9a75c84
doc_id: 839286
cord_uid: c9wdpoyv

The persistence of the global severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic has brought to the forefront the need for safe and effective vaccination strategies. In particular, the emergence of several variants with greater infectivity and resistance to current vaccines has motivated the development of a vaccine that elicits a broadly neutralizing immune response against all variants. In this study, we used a nanoparticle‐based vaccine platform for the multivalent display of the receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike (S) protein, the primary target of neutralizing antibodies. Multiple copies of RBD were conjugated to the SpyCatcher‐mi3 protein nanoparticle to produce a highly immunogenic nanoparticle‐based vaccine. RBD‐SpyCatcher‐mi3 vaccines elicited broadly cross‐reactive antibodies that recognized the spike proteins of not just an early isolate of SARS‐CoV‐2, but also three SARS‐CoV‐2 variants of concern as well as SARS‐CoV‐1. Moreover, immunization elicited high neutralizing antibody titers against an early isolate of SARS‐CoV‐2 as well as four variants of concern, including the delta variant. These results reveal the potential of RBD‐SpyCatcher‐mi3 as a broadly protective vaccination strategy.

already led to more than 3 million deaths worldwide. 1 Numerous vaccine candidates are therefore being developed to provide protection against SARS-CoV-2, including nucleic acid-based vaccines, viral vectorbased vaccines, subunit vaccines, and inactivated vaccines. 2 Most of the vaccines in development aim to elicit a protective immune response that targets the spike (S) protein of SARS-CoV-2. The receptor-binding domain (RBD) of the trimeric S protein, which initiates infection by binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2), 3 is the primary target of neutralizing antibodies elicited by vaccination or infection. These antibodies are able to neutralize the virus by either binding to the receptor-binding motif to directly inhibit binding to ACE2 or by binding to the RBD in a manner that locks it in an unstable state, leading to dissociation of the trimer.

Although several vaccines have been approved for clinical use and have demonstrated high effectiveness against the original strain of the virus, the recently emerged "variants of concern" are better able to escape neutralization by vaccine-induced humoral immunity, leading to a decrease in vaccine potency. The emergence of variants has motivated the design and testing of booster shots that can provide protection against these new circulating strains. While this is a reasonable near-term approach, it would be desirable to develop a vaccine that would provide broad protection against emerging SARS-CoV-2 variants.

While S-targeting vaccines in current use are based on the fulllength S protein, vaccines based on the RBD, 4-8 the primary target of neutralizing antibodies, are worth exploring. Moreover, parts of the RBD are conserved, not just between the SARS-CoV-2 variants, but also between SARS-CoV-2 and SARS-CoV-1. Antibodies binding to these conserved regions have already been shown to neutralize SARS-CoV-2 as well as SARS-CoV-1 pseudoviruses. 9 , 10 Lederer et al. 4 recently compared two RBD vaccine platforms-an mRNA vaccine and recombinant RBD formulated with AddaVax, an MF59-like adjuvant-and reported that the mRNA vaccines were superior at eliciting SARS-CoV-2 specific germinal center B-cell responses. This work, however, used monomeric recombinant RBD; in contrast, several groups have reported robust protective immune responses upon vaccination with RBDs presented multivalently from nanoparticle scaffolds 5, 7, 8 and at least one such candidate is in clinical trials. 11 Tan et al. 5 used SpyCatcher/SpyTag chemistry for the assembly of the SARS-CoV-2 RBD on SpyCatcher003-mi3 nanoparticles and showed that a prime-boost regimen elicited strong neutralizing antibody responses in mice and pigs that were superior to those in convalescent human sera. Cohen et al. 7 designed mosaic nanoparticles co-displaying SARS-CoV-2 RBD along with RBDs from other animal betacoronaviruses that elicited antibodies with cross-reactive recognition of heterologous RBDs. Kang et al. designed three different RBD-conjugated nanoparticles and reported higher neutralizing antibody titers against authentic SARS-CoV-2 virus for the resulting antisera relative to those for mice immunized with monomeric RBD. 6 Recently, Sanders et al. 8 showed that macaque immunization with a multimeric SARS-CoV-2 RBD nanoparticle elicited crossneutralizing antibody responses against SARS-CoV-2, the variants of concern (B.1.1.7, P.1, and B.1.351), SARS-CoV-1, and bat coronaviruses.

We wanted to test if this exciting result showing broad protection could be generalized to other scaffolds displaying RBD multivalently.

We therefore designed and tested vaccine constructs based on SpyCatcher-mi3 nanoscaffolds, which have been used to display a variety of different antigens through SpyTag-SpyCatcher conjugation, 12 including the SARS-CoV-2 RBD. 5, 6 By addition of a SpyTag to the C-terminus of RBD, we were able to irreversibly attach multiple copies of the RBD to each SpyCatcher-mi3 particle. The efficacy of the vaccine construct was then tested against a panel of variants. Immunization studies demonstrated the production of a strong and broadly cross-reactive humoral response against SARS-CoV-2 and SARS-CoV-1. Furthermore, the immunization elicited high neutralizing antibody titers, not just against an early isolate of SARS-CoV-2, but also against four important "variants of concern" including the delta variant (B.1.617.2).

To begin, we produced protein nanoparticles that use SpyTag-SpyCatcher technology for the multivalent display of RBD ( Figure 1 ).

The SpyCatcher-mi3 scaffold is based on a mutated aldolase protein from a thermophilic bacterium fused to the SpyCatcher protein that self-assembles into a dodecahedral 60-mer virus-like particle. 12 The SpyCatcher allows for the conjugation of SpyTagged proteins through the formation of an isopeptide bond, making it a versatile and efficient platform. The SpyCatcher-mi3 was expressed in BL21 (DE3) competent Escherichia coli cells. After cell lysis, Spycatcher-mi3 was purified with a CaptureSelect C-tag affinity column and size exclusion chromatography (SEC). Purity was assessed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 2A ).

Next, we generated an RBD construct to be conjugated to SpyCatcher-mi3. The SpyTag sequence (AHIVMVDAYKPTK) was inserted at the C-terminus of the RBD (amino acids 319-541 of SARS-CoV-2 S protein) followed by a 6xHis-tag. The SpyTag enables conjugation to SpyCatcher-mi3, while the 6xHis-Tag enables purification by with AddaVax, a vaccine adjuvant consisting of an oil-in-water nanoemulsion, was administered to mice (n = 3), followed by a boost 25 days later. Mice were bled before the boost (25 days after the initial immunization) then terminally bled (47 days after the initial immunization) to collect sera and characterize the breadth of the antibody response. High titers against an early isolate of SARS-CoV-2 (S-614D) and three variants of concern-P.1, B.1.1.7, and B.1.351-were seen after a single immunization with RBD-SpyCatcher-mi3 ( Figure S2 ). A second immunization boosted antibody titers against these SARS-CoV-2 variants and also elicited high antibody titers against SARS-CoV-1 ( Figure 3A and Table 1 ). Importantly, we also observed high neutralizing antibody titers against the early isolate of SARS-CoV-2 as well as 4 SARS-CoV-2 variants of concern-P.1, B.1.1.7, B.1.351, and B.1.617.2 ( Figure 3B and Table 1 ). RBD-SpyCatcher-mi3 demonstrated significantly higher neutralization titers against an early isolate of SARS-CoV-2 compared to those previously reported for an

AddaVax-adjuvanted monomeric RBD. 4 There was no significant difference in the endpoint antibody titers and neutralizing antibody titers against the different strains ( Figure 3A ,B).

We have compared these titers with some previously published reports for RBD-based vaccines. We stress, however, that the assays mice with two doses of RBD mRNA and also reported neutralizing antibody titers two-logs higher than those for mice immunized with monomeric RBD protein. 4 Kang et al. reported a similar enhancement in immunogenicity for RBD-nanoparticle constructs compared to monomeric RBD. 6 The reciprocal of the dilution required for 50% neutralization for sera from mice immunized with the RBD-conjugated nanoparticles adjuvanted with AddaVax after the second boost was 10 3 and these neutralizing titers were 10 to 120-fold greater than those for sera from animals immunized with the monomeric RBD.

Thus, our neutralizing antibody titers after a prime and boost ( (Table 1) .

We have also compared our neutralizing antibody titers against the variants of concern with those obtained from sera of immunized as well as three variants of concern. These studies strongly support the further testing of RBD-based vaccines for clinical use as a vaccine that elicits broadly neutralizing antibodies.

Stamatatos et al. 13 recently reported that the immunization of those previously infected with SARS-CoV-2 can significantly boost neutralizing antibody titers against all variants, with the neutralization attributed to antibodies targeting the RBD. In light of the robust and broadly protective responses seen in naïve mice by immunization with RBD-SpyCatcher-mi3, it will be interesting to characterize how the response is influenced by pre-existing immunity due to infection or immunization.

With an increasing percentage of the population already infected or vaccinated, characterization of the role of pre-existing immunity will be an increasingly important consideration in vaccine design.

Construct 2019-nCoV RBD-SpyTag, encoding amino acids 319 to 541 from the SARS-CoV-2 S protein sequence (UniProt P0DTC2) followed by a GGSGG spacer, a SpyTag, and a 6xHis-Tag, was opti- 

Cells transformed with the DNA coding for SpyCatcher-mi3 were used for a 5 ml starter culture which was further scaled up (after growing for 12-16 h) to 1 L of 2xYT media containing kanamycin. Cells were grown at 37 C until the OD 600 reached 0.8. The temperature was reduced to 22 C and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. Cells were allowed to grow overnight before harvest and were then centrifuged at 7000g for 7 min.

Cell lysis and protein purification were performed according to the protocol described by Bruun et al. 12 In brief, the cell pellet was 

Small scale reactions between RBD constructs and SpyCatcher-mi3

were initially set up to determine optimal stoichiometric ratios. Mixtures were allowed to react overnight and the extent of RBD conjuga- 

ELISAs were performed using recombinant spike antigens produced from codon-optimized cDNA expressed in Expi293F cells (Thermo Fisher Scientific). Recombinant proteins with a C-terminal HIS-tag were purified by using TALON metal affinity resin. 16 

The following viruses were used in the neutralization assays: Virus neutralization titers were determined as the reciprocal of the highest serum dilution that completely prevented cytopathic effects. 

Re-emerging Infectious Disease grant (JP19fk0108113) from the

COVID-19) Dashboard. World Health Organization

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Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

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A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

Rapid development of SARS-CoV-2 spike protein receptor-binding domain self-assembled nanoparticle vaccine candidates

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice

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Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

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Engineering a rugged nanoscaffold to enhance plug-and-display vaccination

Human neutralizing antibodies elicited by SARS-CoV-2 infection

Comparison of neutralizing antibody titers elicited by mRNA and adenoviral vector vaccine against SARS-CoV-2 variants

A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV

A serological assay to detect SARS-CoV-2 seroconversion in humans

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Potent neutralization of SARS-CoV-2 including variants of concern by vaccines presenting the receptor-binding domain multivalently from nanoscaffolds

The authors declare that there is no conflict of interest. 

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

https://orcid.org/0000-0003-3084-4098