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Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the world’s population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.


INTRODUCTION 38
On 11 March 2020 the World Health Organisation declared the COVID-19 outbreak, caused by 39 the SARS-CoV-2 virus, a pandemic (Cucinotta and Vanelli, 2020). Since then, COVID-19 and the 40 efforts to contain it have changed the lives of unprecedented numbers of people. For example, 41 in April 2020 3.9 billion people were affected by lockdown measures aimed to cut or at least 42 reduce the chain of transmission with widespread negative impacts on employment, education 43 and other health issues. According to the Johns Hopkins University there have so far been 44 151M confirmed COVID-19 cases globally (May 2021) and virtually every country has been 45 affected. Officially, 3.2M people have died from SARS-CoV-2 infection (2021a, 2021b). 46 SARS-CoV-2 belongs to the family of Coronaviridae, which contain a positive-stranded RNA 47 genome (Pal et al., 2020). The RNA is enveloped by a membrane that harbours four coat 48 proteins (Fig. 1A). On the inside of the virus, the nucleocapsid protein (NP) is crucial for RNA 49 packaging and viral release from host cells (Zeng et al., 2020). The Spike protein, which is 50 embedded in the virus' membranous envelope, is essential for the interaction with human 51 angiotensin-converting enzyme 2 (hACE2) (Ke et al., 2020). It is the interaction with hACE2 that 52 is thought to initiate the process that leads to cell entry of viral RNA and infection (Shang et 53 al., 2020). The Spike protein is translated as a single polypeptide that is proteolytically 54 processed into its two subunits, S1 and S2. The Spike of SARS-CoV-2 is a trimer consisting of 55 three S1-S2 heterodimers . For membrane fusion between the cell and the 56 virus to occur, two cleavage events within the Spike complex are required (Ke et al., 2020). A 57 protease cleavage site located between S1 and S2 is cleaved by the producer cell's proprotein 58 convertase furin during virus assembly (Papa et al., 2021) (Fig. 1A). The second cleavage site is 59 located in the S2 domain at position R797, and its hydrolysis by the target cell's surface 60 protease TMPRSS2 triggers membrane fusion and cell entry (Papa et al., 2021). 61 The SARS-CoV-2 receptor-binding domain (RBD) is located within the S1 subunit of the Spike. 62 It is the RBD that interacts directly with the host cell via the hACE2 receptor (Ke et al., 2020). 63 It is therefore not surprising that antibodies directed against the RBD or overlap with the ACE2 64 binding region are strongly neutralising, making the RBD a promising subunit vaccine candidate 65 (Ke et al., 2020;Seydoux et al., 2020). The RBD is glycosylated and contains four disulphide 66 bridges that contribute to its stability, necessitating its expression in mammalian cells, as is 67 also the case for the Spike. 68 To end the pandemic, vaccines are by far the most promising approach and vaccine 69 developments, clinical trials, approvals and mass roll-outs are in progress. So far, until May 70 2021, 89 COVID-19 vaccines have been tested in clinical trials. Of those, 36 are undergoing 71 safety trials, 27 are in the phase of large-scale testing, 6 vaccines are authorised for limited 72 use, and 8 vaccines are fully approved (2021a). All approved vaccines show good-to-excellent 73 protection against severe illness and preliminary data have shown that virus transmission is 74 significantly reduced in vaccinated individuals (Mahase, 2020; Thompson et al., 2021). Most of 75 the approved vaccines and those in late-stage trials are mRNA-based, vector-based, 76 inactivated viruses or DNA vaccines (Mahase, 2020). Vector-and RNA-based vaccines can often 77 be rapidly developed because they deliver the immunogen coding sequence rather than the 78 immunogen itself. Currently, only one vaccine candidate in late phase trials is a protein-based 79 subunit vaccine, Novavax (Mahase, 2021). Some subunit vaccines are amenable to processes 80 such as lyophilisation that remove the need for a complex storage and distribution cold-chain. 81 As such, they provide substantial advantages over nucleic-acid based vaccines in the quest for 82 but that use the same type of vaccine, could be problematic. This is because immunity is 87 generated against the vaccine vector itself, neutralising it before it can deliver its immunogen 88 cargo (Bottermann et al., 2018). It is anticipated that in future, several different types of 89 vaccines will be required to cope with emerging variants of SARS-CoV-2. 90 Previous work has shown that protein-based subunit vaccines directed against SARS-CoV-2 91 deliver high antibody responses in animal models (Tan et (Gauss et al., 2006). Immunisation using SARS-CoV-2 RBD linked to Dps (RBD-S-Dps) 99 proved to be highly effective in eliciting an immune response and to produce neutralising 100 antibodies that inhibit cell entry in vitro. Furthermore, transgenic K18-hACE2 mice infected 101 with SARS-CoV-2 were completely protected from serious illness following a single 102 immunisation with RBD-S-Dps. 103 the structural integrity of Ag-S-Dps complexes after the coupling reactions. We note that no 165 aggregation was observed for Spike-S-Dps, indicating that the co-transfection approach 166 produced mostly trimeric Spike proteins with only one SpyTag2 present. Taken together, we 167 showed that the scaffold and the three antigens could be produced easily and at high yields 168 and resulted in biochemically pure and defined Ag-S-Dps proteins that display 12 antigens on 169 each Dps scaffold. 170 To determine whether the coupled Ag-S-Dps complexes were stable in blood plasma for 171 immunisations, we mixed the RBD-S-Dps complex with human serum (clotted, not heat 172 inactivated, at a 1:3 volume ratio). The RBD-S-Dps complex was remarkably stable, with 50% 173 remaining intact after 37 h at 37 °C (Suppl. Fig. 1A & B). Given the stability of the Dps scaffold 174 both in serum and when exposed to denaturing conditions (SDS-PAGE, "RT" lane) (Fig. 2C), we 175 next investigated whether the coupled RBD-S-Dps sample would survive lyophilisation and 176 subsequent re-solubilisation. A lyophilised, dry sample would facilitate prolonged storage even 177 in the absence of refrigeration. We therefore freeze-dried RBD-S-Dps and after rehydration 178 found no evidence of precipitation or significantly reduced protein concentration by SDS-PAGE 179 (Suppl. Fig. 1C). There was also no disappearance of the SDS-stable high-molecular weight 180 band, indicating Dps sphere integrity was maintained after re-hydration. Finally, electron 181 microscopy analysis showed the rehydrated sample to be indistinguishable from the starting 182 material with no evidence of disintegration or aggregation (Suppl. Fig. 1D). 183

Multimerisation by Dps greatly enhances immunogenicity, especially for RBD 184
Having obtained the three multimerised antigen-Dps (Ag-S-Dps), we tested whether they 185 induce a stronger immune response than their monomeric equivalents. We immunised mice 186 with the following protocol: five male C57BL/6J mice per group were given 50 µg protein 187 subcutaneously on days zero and 23, and 25 µg on day 64 (using CpG 1668 as an adjuvant) (Fig.  188   3A). Blood samples were taken on days 13 (1 st bleed), 34 (2 nd bleed) and 74 (3 rd bleed). After 189 the 1 st boost on day 34, antigen-specific antibodies were detected in the sera from the mice 190 by ELISA (Fig. 3B). Substantially higher antibody titres were detected with multimerised Dps-191 fused RBD and NP. Multimerisation only improved Spike titres modestly, which may be 192 expected given that Spike is already a trimer without Dps. After 74 days, and the second boost, 193 the specific antibody titres were further increased. Spike induced the weakest response and 194 multimerisation had the smallest effect. In contrast, RBD-S-Dps and NP-S-Dps induced 195 substantial increases in antibody titres compared to the non-multimerised versions. We also 196 analysed sera for antibodies against the scaffold protein itself (SpyC-Dps). Sera from mice 197 immunised with coupled Ag-S-Dps complexes showed measurable but low antibody titres 198 against SpyC-Dps. Anti-SpyC-Dps responses remained low even after the second boost, 199 suggesting that the scaffold itself is poorly immunogenic and that in the context of the fusions 200 the antibody response is largely directed against the viral antigens displayed on the surface. 201 Taken together the data show that multimerised Ag-S-Dps complexes produce substantial 202 improvements in antibody titres over the uncoupled antigens. Overall, the strongest response 203 was observed for RBD-S-Dps. 204 Next, we tested the neutralisation activity of antibodies produced by the mice immunised with 205 RBD-S-Dps, RBD-SpyT2, Spike-S-Dps, and Spike-SpyT2. The mouse sera within each group were 206 pooled at day 34 (2 nd bleed) or 74 (3 rd bleed) and analysed using a pseudovirus infection assay 207 (note that NP-directed sera will not have an effect in this assay because pseudotyped viruses 208 do not contain NP). In this assay, a lentiviral vector expressing GFP is pseudotyped with Spike 209 protein from SARS-CoV-2 to obtain virions that display Spike in their envelope and infect cells 210 in an ACE2-dependent manner. As seen in Figure 3C, the day 34 sera pool of the multimerised 211 RBD-S-Dps group protected against pseudovirus infection up to a dilution of 1:400, whereas 212 the monomeric RBD-SpyT2 only showed a protective effect at a 1:100 dilution, and even then 213 only reduced infection by ~50%. Sera from mice immunised with multimeric Spike-S-Dps also 214 protected against infection, whilst Spike-SpyT2 sera were unable to neutralise at any of the 215 dilutions tested. The sera taken after 74 days had substantially increased neutralisation activity 216 ( Fig. 3D). The sera from RBD-S-Dps-immunised mice gave the strongest protection: even at a 217 1:6400 dilution only ~10% infection could be detected. At this 1:6400 dilution, the monomeric 218 RBD-SpyT2 and Spike-S-Dps sera gave very little neutralisation. While pseudoviruses are widely 219 used to test the neutralisation activity of SARS-CoV-2 antisera, they are based on a lentiviral 220 rather than coronavirus particle and do not recapitulate live virus replication. We therefore 221 tested whether antibodies raised against multimeric RBD-S-Dps were capable of blocking a 222 spreading infection of a primary clinical isolate of SARS-CoV-2. Viral replication was measured 223 by RT-qPCR using probes against NP (gRNA) or E (sgRNA). RBD-S-Dps antisera from five 224 different animals all potently inhibited SARS-CoV-2 (Suppl. Fig. 2A & B). In contrast, the potency 225 of antisera raised against RBD-SpyT2 varied considerably between mice. We conclude that 226 immunisation with RBD-S-Dps not only produces the highest titre antibodies (Fig. 3B), but also 227 the most neutralising ( Fig. 3C & D) and with reliable potency against live virus (Suppl. Fig. 2A  Suppl. Fig. 3A & B). We attempted to correlate this with differences in antibody titres, but while 265 there was a trend towards lower titres in male mice, particularly just before and just after the 266 challenge, this was not significant (Suppl. Taken together, these data indicate that immunisation with RBD-S-Dps is highly protective 287 against SARS-CoV-2 in hACE2-expressing mice, even after a single dose, whilst monomeric RBD-288 SpyT2 is not. 289

DISCUSSION 290
Here we have shown that the ferritin-like protein Dps from the hyperthermophile S. islandicus 291 possesses exceptional qualities as a SARS-CoV-2 subunit vaccine scaffold. We combined Dps 292 with the SpyCatcher/SpyTag system in order to create a "plug-and-play" system that allows 293 the rapid and facile synthesis of highly stable multimeric subunit vaccines. Mixing the 294 SpyCatcher-Dps protein with any compatible SpyTag antigen leads to the assembly of highly 295 monodisperse nanoparticles displaying exactly 12 antigens. Using this approach, we have 296 produced subunit vaccines based on Spike, RBD or NP from SARS-CoV-2 and tested them in 297 immunisation and viral challenge experiments. In each case, the Dps-displayed antigens out-298 performed their non-multimerised equivalents and induced a more rapid and potent antibody 299 response. 300 Subunit vaccines offer distinct advantages in cost, simplicity, production capacity, storage, 301 transport and administration over nucleic-acid based vaccines (Pollet et al., 2021). Principle 302 amongst these considerations is stability, with currently used vaccines such as those produced 303 by Pfizer-BioNTech, Moderna and Oxford-AstraZeneca requiring a -80°C or -20°C cold-chain. 304 In countries without a highly developed logistical and medical infrastructure this represents a 305 scaffold has been shown to induce a neutralising antibody response. Our scaffold differs from 315 those previously used to deliver SARS-CoV-2 immunogens in several important aspects. First, 316 because we have used a thermostable protein it is intrinsically more stable, providing potential 317 benefits to vaccine transport and storage and also to immunogen stability in vivo. Second, it is 318 smaller than other scaffolds (< 10 nm vs > 10 nm for ferritin or 25 nm for the I3-01 319 nanoparticle), making it an easier cargo for cellular uptake. Third, it displays fewer copies than 320 ferritin or I3-01 (12 vs 24 or 60, respectively), allowing the selection of higher-affinity B cells 321 and avoiding the activation of off-target (and possibly cross-reactive) B cell competitors (Kato 322 et al., 2020). Fourth, in contrast to bona fide ferritin scaffolds, the N-and the C-termini of Dps 323 are both accessible on the outside of the sphere. This allows, at east in principle, for the 324 conjugation of two discrete antigens onto a single scaffold, for example both SARS-CoV-2 325 Spike/RBD and NP to be displayed simultaneously. 326 Importantly, we have provided here data demonstrating the benefit of antigen 327 multimerisation in inducing not just neutralising antibodies but an immune response capable 328 of providing in vivo protection. In our SARS-CoV-2 challenge experiments, we found that RBD 329 alone failed to protect mice, which displayed continued weight-loss and high viral loads in the 330 lungs. In contrast, our Dps-based vaccine displaying RBD completely protected mice from 331 SARS-CoV-2-associated pneumonia and disease after only a single immunisation. We noted 332 however a difference in Dps-RBD induced protection between male and female mice, with the 333 latter having lower viral loads and hardly any pulmonary changes. Trial data for both mRNA 334 and vector-based vaccines has not been disaggregated by sex but data on SARS-CoV-2 infection 335 show that men are more at risk of severe adverse conditions, hospitalisation and death (Klein 336

RNA extraction and DNase treatment 524
The upper lobes of the right lung were dissected and homogenised in 1 mL of TRIzol reagent 525 (TFS) using a Bead Ruptor 24 (Omni International) at 2 meters per second for 30 s. The 526 homogenates were clarified by centrifugation at 12,000 x g for 5 min before full RNA extraction 527 was carried out according to manufacturer's instructions. RNA was quantified and quality 528 assessed using a Nanodrop (TFS) before a total of 1 μg was DNase treated using the TURBO 529 DNA-free kit (TFS) as per manufacturer's instructions. GCATGCCAGAGTCTCGTTCG. Similarly, the E sgRNA standard was generated by PCR using the 545 qPCR primers. cDNA was generated using the SuperScript IV reverse transcriptase kit (TFS) and 546 PCR carried out using Q5 High-Fidelity 2X Master Mix (New England Biolabs) as per 547 manufacturer's instructions. Both PCR products were purified using the QIAquick PCR 548 Purification Kit (Qiagen) and serially diluted 10-fold from 10 10 to 10 4 copies/reaction to form 549 the standard curve. 550

Histology and immunohistology 551
The left lung lobes were fixed in formal saline for 24 h and routinely paraffin wax embedded. showing that all samples form defined and monodisperse spheres that display the antigens on 782 their surface, leading to particles of different sizes for the three differently-sized antigens. 783  Fig. 4F). In 853 male animals, multifocal inflammatory infiltrates with viral antigen expression and mild 854 vasculitis similar to the other two groups, but substantially less extensive were seen (Suppl. 855 Fig. 4G, H). Also here, macrophages were the dominant cells in the focal infiltrates (Suppl. Fig.  856 5B), followed by the T cells (Suppl. Fig. 5D). The T cell population showed a mild shift in 857 composition, as now CD8-positive cells were as numerous or more frequent than CD4-positive 858 cells (Suppl. Fig. 5F-H). CD8-positive cells were also seen within alveolar lumina (Suppl. Fig. 5B  859 inset). B cells were found in moderate numbers in the infiltrates, in one animal they also 860 formed peribronchiolar aggregates (Suppl. Fig. 5J).