Clinical & Translational Immunology 2021; e1269. doi: 10.1002/cti2.1269 www.wileyonlinelibrary.com/journal/cti

ORIGINAL ARTICLE Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2 Daniel Watterson1,2,3,†, Danushka K Wijesundara1,2,†, Naphak Modhiran1,2,†, Francesca L Mordant4 , Zheyi Li5, Michael S Avumegah1,2, Christopher LD McMillan1,2, Julia Lackenby1,2, Kate Guilfoyle6, Geert van Amerongen6, Koert Stittelaar6, Stacey TM Cheung1, Summa Bibby1, Mallory Daleris2, Kym Hoger2, Marianne Gillard2, Eve Radunz2 , Martina L Jones2 , Karen Hughes2, Ben Hughes2, Justin Goh2, David Edwards2,JudithScoble7,LesleyPearce7, Lukasz Kowalczyk7,TramPhan7, Mylinh La7, Louis Lu7, Tam Pham7,QiZhou7, David A Brockman8, Sherry J Morgan9,CoraLau10, Mai H Tran8, Peter Tapley8, Fernando Villalon-Letelier 4, James Barnes11,AndrewYoung1,2, Noushin Jaberolansar1,2, Connor AP Scott1, Ariel Isaacs1, Alberto A Amarilla1, Alexander A Khromykh1,3, Judith MA van den Brand12, Patrick C Reading4,11, Charani Ranasinghe5, Kanta Subbarao4,11, Trent P Munro1,2, Paul R Young1,2,3 & Keith J Chappell1,2,3 1School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia 2The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia 3Australian Infectious Disease Research Centre, Global Virus Network Centre of Excellence, The University of Queensland, Brisbane, QLD, Australia 4Department of Microbiology and Immunology, The University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia 5Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia 6Viroclinics Xplore, Schaijk, The Netherlands 7CSIRO Manufacturing, Parkville, VIC, Australia 8TetraQ, The University of Queensland, St Lucia, QLD, Australia 9StageBio, Mason, OH, USA 10University of Queensland Biological Resources, The University of Queensland, St Lucia, QLD, Australia 11WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia 12Division of Pathology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Correspondence Abstract D Watterson, PR Young and KJ Chappell, School of Chemistry and Molecular Objectives. Efforts to develop and deploy effective vaccines Biosciences, The University of Queensland, against severe acute respiratory syndrome coronavirus 2 (SARS- St Lucia, QLD 4072, Australia. CoV-2) continue at pace. Here, we describe rational antigen design E-mails: [email protected] (DW); through to manufacturability and vaccine efficacy of a prefusion- [email protected] (PRY) or [email protected] (KJC) stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 ‘MF59C.1’ (Seqirus, Parkville, Australia). † Equal contributors Methods. A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead Received 23 February 2021; vaccine candidate. The structure of this antigen was determined Revised 4 March 2021; by cryo-electron microscopy and assessed in mouse Accepted 5 March 2021 immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. Results. In mice, the Sclamp vaccine doi: 10.1002/cti2.1269 elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T

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Clinical & Translational Immunology cells in vivo. In the Syrian hamster challenge model (n = 70), 2021; 10: e1269 vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. Conclusion. The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2–8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Keywords: Molecular Clamp, neutralising antibodies, polyfunctional T cells, rapid response, SARS-CoV-2, subunit vaccine

INTRODUCTION other platforms owing primarily to the need to tailor the manufacturing process to each antigen. The scientific community, including critical However, protein vaccines paired with clinically industry and academic partnerships, has embarked validated adjuvants remain an attractive option to on an unprecedented race to develop and counter a global pandemic, as such pairings have produce effective COVID-19 vaccine(s) for global already been proven to confer a tailored immune use.1 As of February 2021, there are 74 individual response to result in efficacy with an excellent vaccine candidates in clinical trials and many more safety profile, and can therefore be utilised with in preclinical development.2 At the beginning of existing global manufacturing capacity to enable 2020, our vaccine platform, termed the Molecular rapid production scale-up to the levels required to Clamp, was at an early stage of development as a counter a pandemic threat. rapid response pipeline, supported through a To date, the most advanced subunit programme 2018 call for platform technologies to rapidly described is the Novavax candidate produced via respond to Disease X by the Coalition for insect cells as a detergent-stabilised nanoparticle in Epidemic Preparedness Innovations (CEPI). Acting combination with a saponin-based adjuvant.3 Our in response to the outbreak, and with support candidate vaccine, SARS-CoV-2 Sclamp, consists of from CEPI, we immediately refocused our efforts the recombinant viral S glycoprotein stabilised in its towards the rapid development of a molecular prefusion trimeric form, through the incorporation clamp stabilised vaccine against severe acute of the ‘Molecular Clamp’ stabilisation domain, and respiratory syndrome coronavirus 2 (SARS-CoV-2), produced in mammalian cells (Chinese hamster the causative agent of COVID-19. This manuscript ovary).4 Stabilisation of the prefusion antigen is a describes the entire preclinical development of major goal of next-generation vaccines that target our vaccine programme (timeline provided in enveloped viruses. This approach ensures that Supplementary figure 1), which is now advancing vaccines elicit the production of specific antibodies in partnership with CSL for further manufacturing that recognise a wider array of epitopes, including and pivotal stage clinical development. conformational epitopes, that are displayed on the Vaccine candidates in clinical trials in virion surface.5,6 Prefusion-stabilised spike protein development include inactivated viruses, viral from the related Middle East respiratory syndrome vectored approaches, nucleic acid-based vaccines coronavirus (MERS-CoV) has previously been shown and subunit vaccines. As each platform has to elicit a higher level of neutralising antibodies intrinsic strengths and weaknesses around aspects compared with the native MERS-CoV S.7 of manufacturability, ease of transport, safety and Severe acute respiratory syndrome coronavirus 2 efficacy, it is crucial that multiple programmes are Sclamp is one of seven candidate subunit vaccines taken forward to allow selection of the most comprising the entire ectodomain of spike that is favorable approaches to meet the global currently being evaluated in clinical trials2. Similar challenge of the COVID-19 pandemic. Adjuvanted subunit vaccines being evaluated include alternate protein-based, subunit vaccines represent an stabilisation methods including site-directed important class of vaccine and its development mutagenesis of two proline residues,3,8–10 or the has somewhat been lagged compared with the incorporation of an alternate trimerisation domain

2021 | Vol. 10 | e1269 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 2 Australian and New Zealand Society for Immunology, Inc. D Watterson et al. Molecular clamp vaccine for SARS-CoV-2 based on human collagen.11 The Molecular Clamp timeline of research and development and an trimerisation domain we have employed comprises overview of the Molecular Clamp platform, heptad repeat regions 1 and 2 (HR1/2) of including two proof-of-concept examples are glycoprotein 41 (gp41)(aminoacids540–576 and included in Supplementary figures 1 and 2). 619–656) from human immunodeficiency virus 1 Without a high-resolution structure available, the (HIV-1). These sequences encode a common fold candidate screen focussed on three main features of found in many viral families (Supplementary the molecule: (1) the signal peptide, (2) modification figure 2) and assemble into an extremely stable six- of the furin cleavage site and (3) fusion of the helix bundle that in the native context confers molecular clamp sequence. Within 34 days, we had stability to the post-fusion form of class I viral expressed and screened > 200 antigens proteins. We have repurposed this feature and have incorporating truncations and modifications at reengineered it as a modular tag capable of these key sites to identify a lead candidate that stabilising the prefusion form of a wide range of demonstrated suitable levels of trimer expression trimeric viral antigens. We have validated the while maintaining affinity to the S-specific platform against established human pathogens monoclonal antibody (mAb), CR302217 (Figure 1a–c including respiratory syncytial virus (RSV) and and Supplementary table 1). Our screen revealed influenza, where it is able to stabilise the prefusion that the most critical element for expression yield conformation without the need for additional site- was modification of the furin-like cleavage site. specific mutations common to other stabilisation Without modification of this site, no spike protein strategies (Supplementary figure 2d).12,13 could be recovered from transfected culture Furthermore, we utilise an antibody to the supernatants (Figure 1a). Given that early spike molecular clamp to enable immunoaffinity comparative sequence analysis against related SARS- purification, thereby exploiting a consistent like viruses revealed an unusual extended and poly- methodology to purify novel antigens as a rapid basic furin 1 site, we examined both site-targeted response platform to pandemic threats.14 mutations and larger deletions of the whole site. In the ongoing phase I clinical trial investigating While most of the constructs showed similar SARS-CoV-2 Sclamp, initial findings have expression to the single P1 site R685A mutation, two demonstrated favorable safety and the induction of constructs that utilised a deletion of aa680–690 and a strong neutralising immune response.15 However, either a GSG or GGSGG flexible linker were observed the HIV sequences included within the Molecular to express significantly higher yields. We examined a Clamp were also found to stimulate an immune second generation of these constructs, screening response that could cross-react with some HIV through both the entire 80 aa membrane proximal diagnostic tests. To avoid the issue of HIV diagnostic region and testing alternative signal sequences, but interference, we are in the process of re- determined that the original length and native engineering the sequences used in the Molecular signal sequence were optimal. The selected lead Clamp trimerisation; however, herein we describe construct incorporated the S protein native signal the rapid development of the Sclamp vaccine, peptide, replacement of aa680–690 with a glycine including antigen design and characterisation, linker (GSG), truncation at aa1204 and followed by production, and animal immunogenicity, safety and the Molecular Clamp coding sequence as an efficacy studies which permitted the transition to a extension of the carboxy-terminal sequence. This human Phase I trial. These results serve as an construct was originally termed M1GSG and then important proof-of-principle demonstration of the subsequently referred to as ‘Sclamp’ (Supplementary underlying platform technology. figure 3 and Supplementary table 1). We utilised several, patient-derived, SARS-CoV-2 17 18 RESULTS cross-reactive mAbs (CR3022, 309, H4 and B3819), to demonstrate high-affinity binding to the Sclamp protein as an indicator of the required Selection of the prefusion conformed conformational integrity (CR3022 kD = 0.3 nM; Sclamp lead candidate S309 kD = 0.08 nM;H4kD= 0.6 nM; B38 The virus genomic sequence first became available kD = 1.3 nM). Without stabilisation via the on 12 January 2020,16 and we immediately Molecular Clamp, or an alternate methodology commenced development of a candidate subunit such as the insertion of structure stabilising vaccine utilising the Molecular Clamp platform (a mutations,8 the spike protein dissociates into

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Figure 1. Antigen design and analysis. (a) In vitro screening of S1/S2 linker modifications for yield and CR3022 affinity (KD), untransfected cell supernatant included as control. (b) Linear representation of recombinant spike antigen. (c) Sequence details for the furin site-modified constructs assayed in a. (d) Size-exclusion chromatography analysis of non-stabilised wild-type spike and Sclamp. (e) Antibody binding affinities 10 (KD) for unstabilised vs Sclamp. (f) Cryo-TEM reconstruction of antigen structure, fitted with prefusion spike structure PDB:6VXX shown in rainbow cartoon for one S monomer. Representative top and side-on 2D classes shown below, with a density for clamp indicated. (g) Production yield from CHO cell culture transient expression, stable pools and clones in flasks or bioreactors. Red—antigen concentration in cell culture supernatant estimated by BiaCore. Black—protein recovery following immunoaffinity purification as measured by absorbance at 280 nm. (h) Percentage trimer analysis of stability assessed using SE-HPLC for the development batch and clinical trial batch of Sclamp following incubation of the protein at 4, 25 or 40°C for up to 13 weeks. monomers and the binding affinity of spike smaller trimer peak was determined to 5 A conformation-specific monoclonal antibodies to resolution (Fourier shell correlation 0.143) by cryo- the monomers is reduced (Figure 1d and e). electron microscopy (cryo-EM) and found to be Sclamp was also shown to bind to soluble consistent with the previously defined closed recombinant angiotensin-converting enzyme 2 trimeric prefusion conformation of SARS-CoV-2 8 (ACE2) receptor (kD = 56 nM Æ 5). spike (Figure 1f and Supplementary figure 5). No Using size-exclusion high-performance liquid clamp density was resolved in the final structure, chromatography (SE-HPLC), we demonstrated that likely due to flexibility in the long C-terminal the expressed antigen separates into two peaks spike stalk that is disordered in published both of which have sizes corresponding to trimers structures,10,20 and has been shown to be highly with slightly different hydrodynamic radiuses and flexible in native virions.21 However, the clamp that exposure to varying pH or temperature can could be readily identified in 2D classification drive transition between the two forms images of the cryo-EM particle set (Figure 1f, (Supplementary figure 4). The structure of the inset). The structure of the larger trimer peak was

2021 | Vol. 10 | e1269 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 4 Australian and New Zealand Society for Immunology, Inc. D Watterson et al. Molecular clamp vaccine for SARS-CoV-2 unable to be resolved by cryo-EM, but it is Alhydrogel or MF59 produced a strong serum consistent with an open prefusion conformation neutralising antibody response against the matched previously defined,8 and reports that SARS-CoV-2 strain of SARS-CoV-2 as assessed by a traditional spike exists in an equilibrium between these two microneutralisation (MN) assay (Figure 2c).25 conformations.22 Further analysis of the sera and bronchoalveolar Transient gene expression in CHO cells was lavage (BAL) collected on day 42 from Alhydrogel- initially utilised to allow rapid screening of or MF59-adjuvanted Sclamp-vaccinated mice using antigen design panels with CHO stable cell lines plaque reduction neutralisation test (PRNT50) being established following lead candidate analysis showed that both 614D and the now selection. Transitioning to CHO stable pools and dominant 614G SARS-CoV-2 variants26 were using a fed-batch bioprocess resulted in increased similarly neutralised (Figure 2d–g). Serum PRNT50 expression yields in shaker flasks of 100–150 mg L–1 analysis also included a WHO-recommended and in bioreactors up to 500 mg L–1 (Figure 1g). reference serum (NIBSC code 20:130)27 to allow This level of mammalian cell-based protein benchmarking of the neutralising titre to other expression has the potential to generate many platforms. In this assay, sera from mice vaccinated millions of doses per bioreactor run using industry with MF59-adjuvanted Sclamp neutralised SARS- standard mammalian cell bioprocessing facilities. CoV-2 with a ~ 2-fold higher geometric mean titre Subsequent studies have shown that Sclamp (GMT) compared with the reference serum expressed in the CHO stable pools is decorated (Figure 2d and e). with additional sialylation, likely contributing to Clamp-specific immune response was assessed at maximising the trimer form (data not shown). day 42 for 25 mice each receiving two doses of Furthermore, a downstream refined bioprocess was Sclamp with MF59 adjuvant (Figure 2h). Based on able to achieve batch-to-batch consistency (data comparison of EC50 values in ELISA, we produced an not shown). We also confirmed that the trimeric estimate of the relative immune response directed conformation of Sclamp is unaffected by storage at against the spike ectodomain and the molecular 4°C for 3 months and is well retained even after clamp domain for each of 25 individual mice prolonged exposure to heat stress (Figure 1h and (Figure 2i). The geometric mean of these values Supplementary figure 6). suggests that 76% of IgG directed against the spike ectodomain (ranging between 38 and 92% for individual mice) and 24% of IgG to the molecular MF59-adjuvanted Sclamp vaccination clamp domain. Interestingly, while the relative elicited neutralising antibodies against percentage of the immune response to the clamp SARS-CoV-2 itself varied between individual animals, this non- Purified protein-based vaccines require adjuvant spike specific response was not associated with a co-administration to enhance immunogenicity statistically significant correlation with MN50 titres and to reduce the total amount of antigen (Figures 2j). In summary, the level of clamp-specific required to provide protective immunity.23 The response had no impact on the serum neutralisation squalene-oil-in-water adjuvant MF59 (Seqirus, response. Parkville, Australia) was selected for use with our vaccine because of its well-established MF59-adjuvanted Sclamp vaccination safety record, use in existing licensed vaccines elicited robust S-specific and its ability to stimulate a balanced T helper + 24 polyfunctional/cytotoxic CD4 and (Th1/Th2) cell response. Alhydrogel (Croda) + CD8 T-cell immunity was included as a comparator adjuvant. BALB/c mice received intramuscular (IM) injections of We evaluated SARS-CoV-2 S-specific CD4+ and CD8+ PBS (placebo) or two doses of Sclamp with or T-cell responses in vivo using a fluorescent target without Alhydrogel or MF59 (Figure 2a). array (FTA) analysis and complementary intracellular Vaccinated mice developed a robust antigen- cytokine staining (ICS) analysis to determine type 1 specific IgG response after a single dose with vs type 2 immunity.28,29 For the FTA analysis, the either adjuvant, which was boosted following targets in the FTA were prepared following serial the second dose (Figure 2b). labelling with titrated concentrations of cell Analysis of serum samples on day 42 showed that proliferation dye efluorTM 670 (CPD) and cell trace vaccination with two doses of Sclamp with either violet (CTV) that allowed the labelled cells to retain

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(a) (b) Antigen Specific Immune Response Vaccination groups p < 0.0001 p < 0.0001 PBS dosed (placebo) 10 5 SARS-CoV-2 Sclamp (5 μg/dose) p<0.0001 p< p= p<0.005 SARS-CoV-2 Sclamp (5 μg/dose) + Alhydrogel (50 μg/dose) 0.0001 0.001 10 4 p= 50 SARS-CoV-2 Sclamp (5 μg/dose) + MF59C.1 (50% vol/vol) 0.035 1st dose 2nd dose 10 3 ELISA EC Bleed Bleed Bleed 2 End 10

LoD -1 0 20 21 35 42 10 1 Days 20 35 42 Days

(c) Virus Neutralization (d) Virus Neutralization (e)Virus Neutralization (f) BAL Virus Neutralization (g) BAL Virus Neutralization SARS-CoV-2 614D SARS-CoV-2 614D SARS-CoV-2 614G SARS-CoV-2 614G SARS-CoV-2 614D p<0.0001 p<0.0001 p < 0.0001 p<0.001 10 3 10 4 10 5 p<0.001 128 128 p=0.015 64 64 10 4 ns ns 32 32 10 3 50 NIBSC 50 50 ns 50 2 16 16 10 50 20:130 10 3 NIBSC

MN 20:130 8 8 ns ns 10 2 PRNT

PRNT 2 4 4 10 BAL PRNT LoD 2 LoD BAL PRNT 2 LoD LoD 1 LoD 10 10 1 10 1 1 1

Spearman Correlation )10 5 (i) 100 (j)1000

80 800 rs =-0.29 10 4

50 (95%CI = -0.62 - 0.12) 600 p =0.16 60 50 10 3 40 MN 400 ELISA EC 10 2 20 200 Clamp Response (%) LoD 10 1 0 0 Sclamp Clamp 0 20406080 Clamp Response (%)

Figure 2. The antibody response following SARS-CoV-2 Sclamp vaccination in BALB/c mice (data from two of five repeat experiments shown).

(a) Prime/boost vaccination and bleed schedule for the study. (b) SARS-CoV-2 Sclamp-specific IgG EC50 titre (reciprocal EC50) in vaccinated mice (n = 10) 20, 35 and 42 days following IM injection of the first dose. (c) Day 42 serum MN titre against infectious SARS-CoV-2 614D as evaluated by MN50 assay (n = 10). (d) Day 42 serum MN titre against SARS-CoV-2 614D as evaluated by PRNT50 assay (n = 10). (e) Day 42 serum MN titre against SARS-CoV-2 614G as evaluated by PRNT50 assay (n = 10). (f) Day 42 BAL MN titre against SARS-CoV-2 614D as evaluated by PRNT50 assay (n = 10). (g) Day 42 BAL MN titre against SARS-CoV-2 614G as evaluated by PRNT50 assay. (h) Day 42 serum total Sclamp-specific IgG

EC50 titre and clamp-specific IgG EC50 titre (n = 25). (i) Relative percentage of total IgG response directed to the clamp domain, and spike epitopes, calculated based on EC50 titres in h. (j) Spearman correlation between relative percentage clamp-specific IgG and MN50 titre. P-values were calculated following transformation of the neutralisation titres to log10 using (1) one-way ANOVA with Tukey’s multiple comparison post hoc test for normally distributed and homoscedastic data, (2) Welch’s ANOVA with Games–Howell post hoc analysis for all heteroscedastic data and (3) the Kruskal–Wallis H-test for non-normally distributed and homoscedastic data sets.

a unique, easily distinguishable fluorescent bar-code S492–519,S683–707,S741–770,S785–802 and S885–1273; following flow cytometry analysis (Figure 3a and b). Figure 3a and b). Following intravenous challenge This allowed us to accurately pulse each fluorescent of mice with the FTA, up-regulation of CD69 on bar-coded cell population with 7 peptide pools that peptide-pulsed B220+ FTA cells recovered from collectively span the SARS-CoV-2 S1–1226 sequence, challenged mice is dependent on antigen-specific + S16–554 (S1), S673–1218 (S2), S1–1226 pool (Total) and the CD4 Th cell responses and the killing of cognate Peptivator array that comprise in silico mapped peptide-pulsed targets is a result of cytotoxic T 30,31 human immunodominant peptides (S304–338,S421–475, lymphocyte (CTL) responses in vivo. Our findings

2021 | Vol. 10 | e1269 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 6 Australian and New Zealand Society for Immunology, Inc. D Watterson et al. Molecular clamp vaccine for SARS-CoV-2 in the current study revealed that Alhydrogel- and challenged with intranasal (IN) inoculation of MF59-adjuvanted regimens were more effective in SARS-CoV-2 following either a single-dose eliciting S-specific Th cell and CTL responses (Figure 4a) or a prime/boost vaccination regimen compared with Sclamp (Ag) only and placebo (Figure 5a). The challenge dose selected was control groups with the MF59-adjuvanted regimen based on the ability to induce disease and to be being superior in eliciting the highest magnitude of relevance to natural exposure, rather than an and the broadest S-specific Th cell and CTL responses artificially high dose as has been used in a range – in vivo (Figure 3c and Supplementary tables 2 and of non-human primate (NHP) studies.33 36 Both 3). The superior immunogenicity of the MF59- studies included a formalin-inactivated virus adjuvanted regimen was evident for Th cell combined with Alhydrogel (Croda) as a responses to S185–362 (P2), S353–530 (P3), S521–698 (P4), comparator based on the initial concern that S689–886 (P5), S857–1070 (P6), S1041–1226 (P7), S1, S2, total vaccines that drive a Th2 response and/or a poorly pool and Peptivator, but not for S1–194 (P1). CTL neutralising response may result in immunity followed a similar trend, but not with immunopathology as has previously been responses to P6 and P7. observed for MERS-CoV and SARS-CoV.37,38 Of The ICS analysis showed that the mice note is that more recent studies have now shown vaccinated with MF59-adjuvanted Sclamp that there is no compelling evidence for this developed the highest number of S-specific CD4+ undesired effect or antibody-dependent or CD8+ T cells that expressed mono- or enhancement of infection for inactivated SARS- polyfunctional interferon (IFN)-c, tumor necrosis CoV-2 vaccines adjuvanted with aluminium-based factor (TNF)-a and/or interleukin (IL)-2 (Figure 3d, adjuvants.39 To further validate protection data Supplementary figure 7 and Supplementary following vaccination, we also included an tables 4 and 5). MF59- and Alhydrogel-adjuvanted additional group in the prime/boost study Sclamp-vaccinated mice developed higher whereby hamsters were infected (intranasally) + numbers of S-specific CD4 T cells that produced with SARS-CoV-2 and then allowed to recover IL-13 and a more subtle elevation in the numbers over a 7-week period prior to a second challenge. of S-specific CD8+ T cells that produced IL-4 or IL- A single vaccination in hamsters with MF59- 13 was also observed in the MF59-adjuvanted adjuvanted Sclamp resulted in modest but Sclamp regimen compared with the placebo and/ statistically significant induction of neutralising or Sclamp only (Figure 3d, and Supplementary antibodies (MN100 GMT = 9.6) while the figure 7 and Supplementary tables 4 and 5). inactivated virus did not (Figure 4b). Two doses of Overall, using 2 independent T-cell assays, we either vaccine elicited a more potent neutralising confirmed that MF59-adjuvanted Sclamp antibody response (MN100 GMT = 40.6), as did vaccination elicited S-specific CD4+ and CD8+ T-cell prior infection and recovery from SARS-CoV-2 responses with the highest magnitude and infection (MN100 GMT = 33.1; Figure 5b). Note breadth of recognition encompassing type 1 (IFN- that the NIBSC convalescent human serum control c, TNF-a and IL-2) and type 2 (IL-4 and IL-13) (20:130) in this MN100 assay format has a responses, albeit with the type 2 responses being GMT = 30 (results not shown). less prominent compared with type 1. The highly permissive nature of the hamster model was initially observed in a study that evaluated viral replication in daily throat swabs Sclamp vaccination induced protection collected following IN challenge up to 4 days against pathogenic SARS-CoV-2 challenge following a single dose and 8 days following a Given that vaccination with Sclamp adjuvanted prime-boost regimen (Figures 4c and 5c). These with MF59 elicited the most robust antibody and studies showed that infectious virus titre, as

T-cell immunity, we further evaluated this measured by TCID50, peaked at 1–2 days post- regimen relative to parameters deemed important challenge and then gradually decreased to for safety evaluation (i.e. vaccine-mediated undetectable levels between 5 and 8 days post- enhancement of disease) and protection in the challenge (Figures 4c and 5c). Vaccination with established hamster model of SARS-CoV-2 either one or two doses of MF59-adjuvanted infection.32 To investigate the possibility of Sclamp or inactivated virus with Alhydrogel vaccine-enhanced respiratory disease, we resulted in a decrease in both the peak virus titre completed two studies in which hamsters were detected in throat swabs and total virus shedding

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(a) P1 (b) P2 P4 P3 P5 Nil CPD CPD S2 P7 S1 P6 CPD Total FSC-H

B220 CTV Peptivator CTV

Nil P1 P2 P3 P4 P5 Ag + MF59C.1 Ag + Alhydrogel

Ag only P6 P7 S1 S2 Total Peptivator Placebo CTV FMO

(c) Placebo Ag only Ag + Alhydrogel Ag + MF59C.1 Th cell response CTL response 2000 ** 100 ** ** ** 1500 75 ** ** ** 1000 ** ** **** ** **** ns ns ** ** ** 50 * ** ns * * 600 ** ** ns ns * ns ns ns * ns ns ** ** ** ** ** ns ns ns ns ns 400 ns %killed 25 ns ns ns ns ns * ** ** ** ** * ns ns ns 200 ns ns * ns * ns ns ** GMFI of CD69 0 0 1 2 1 3 4 5 1 2 P P P3 P4 P5 P6 P7 S1 S2 tal P P2 P P P P6 P7 S S tal To To tivator tivator p e Pep P (d) Placebo Ag only Ag + Alhydrogel Ag + MF59C.1

4000 Type 14000 Type 2 + + 3000 3000

CD4 ** CD4 ** + + ** ns **** 2000 ns ** 2000 ns ns ** ** ** ns * ns ns **** ns ns * 1000 ns 1000 ** ns ** ns ns ns #ofCD3 #ofCD3 None 0 0 IFN-γ + - - + + - + IFN-γ - - - TNF-α - + - + - + + IL-4 + - + IL-2 - - + - + + + IL-13 - + +

+ 4000 * + 4000

CD8 3000 CD8 3000 + ** + ns 2000 2000 * ns * ns ns **** ** ns ns * ns ns ns ns ns *** 1000 ns ns * 1000 ns ns # of CD3 ns ns ns #ofCD3 None 0 0 IFN-γ + - - + + - + IFN-γ - - - TNF-α - + - + - + + IL-4 + - + IL-2 - - + - + + + IL-13 - + +

Figure 3. MF59-adjuvanted SARS-CoV-2 Sclamp vaccination elicits potent T-cell responses in mice (data from two separate experiments shown). (a) Representative flow cytometry plots show the gating strategy for analysis of S-specific Th cell responses based on the up-regulation of CD69 on peptide-pulsed B220+ single cells in the FTA. BALB/c mice vaccinated as in Figure 2a were challenged 19 days after boost with the FTA comprising S peptide-pulsed, CPD and CTV-labelled targets. FMO—fluorescent minus one control. (b) Dot plots showing all targets recovered from a representative FTA-challenged mouse which were used to analyse the S-specific CTL responses as described in the Methods. (c) Bar graphs show the mean (n = 5) and SEM of the geometric mean fluorescent intensity (GMFI) of CD69 on gated B220+ cells and the percentage of peptide-pulsed targets that were killed in the FTA. (d) Mean (n = 5) and SEM of the number of the indicated cytokine-producing S1–1226-specific CD4+ or CD8+ T cells. For the ICS analysis, splenocytes recovered 24 days following boost from mice prime/boost vaccinated at 2-weekly intervals were stimulated for 16 h with the total pool. P-values for all the comparisons are shown in Supplementary tables 2–5 and the P-values shown indicate significance relative to placebo (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

2021 | Vol. 10 | e1269 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 8 Australian and New Zealand Society for Immunology, Inc. D Watterson et al. Molecular clamp vaccine for SARS-CoV-2

(a) Single Dose Study Challenge Vaccination groups sts 2 1 dose 1x10 TCID50 PBS dosed (placebo) Inactivated virus (50 μg/dose) + Alhydrogel (85 μg/dose) End Sclamp (5 μg/dose) + MF59C.1 (50% vol/vol) 0 28 32 (*n=10)

(b) Hamster Single dose (c) Virus Shedding - Time course Virus Shedding - Peak Virus Shedding - Total p < 0.001 p = 0.004 0.007 ns ns 0.044 p = 0.003 128 <0.001 ns ns ns 1011 ns 104 104 1010 64 109 p<0.001 103 103 108 /ml) /ml) /ml)

titre 32 ns 50 50 50 107 100 102 102 106

MN 16 (TCID (TCID (TCID Throat swab Throat 105 8 101 101 104

LoD 1-4 day swabs throat Peak LoD Sum of throat swabs day 1-4 day swabs throat of Sum 103 LoD 01234 Days post infection

(d) Nasal Turbinate Day 4 Lung Day 4 (e) Histopathology Analysis at 4 days Post-Infection ns p=<0.001 109 109 ns ns p=0.021 p=0.011 p=0.023 p=<0.001 ns ns ns ns ns 108 108 3 107 107 106 106 /g /g 2 50 50 105 105

4 4 TCID TCID 10 10 1

3 3

10 10 Score Severity 0 102 102 Rhinitis Trachetis Bronchitis Bronchiolitis Alveolitis 101 LoD 101 LoD

Figure 4. Protection of hamsters following a single dose of MF59-adjuvanted Sclamp. (data from a single experiment shown). (a) Single-dose study schedule. Syrian hamsters (n = 10/group) were vaccinated with a single dose of either a placebo, 50 lg inactivated virus with Alhydrogel or

5 lg SARS-CoV-2 Sclamp with MF59 prior to IN challenge with 100 TCID50 of SARS-CoV-2. (b) Average SARS-CoV-2 MN100 titre measured using hamster serum collected at day 21 and 28. (c) Kinetics of infectious viral load in daily throat swabs on days 1–4 post-challenge (left panel), the peak virus titre from the kinetics analysis (middle panel), and the sum of virus titre for swabs collected on days 1–4 post-challenge (right panel). (d) Viral loads in nasal turbinates and lung tissue at day 4 post-challenge. (e) Severity score of inflammatory response based on histology at day 4 post-challenge: 0 = no inflammatory cells, 1 = few inflammatory cells, 2 = moderate number of inflammatory cells and 3 = many inflammatory cells. P-values were calculated using a Kruskal–Wallis ANOVA with Dunn’s multiple comparison tests or a two-way ANOVA with Dunnett’s multiple comparison tests. Error bars show SD. as measured by summation of virus detected in this purpose, all animals were sacrificed at 4 days daily throat swabs collected between days 1 and 4 post-challenge (n = 10) in the single-dose study, (Figures 4c and 5c). Prior exposure to SARS-CoV-2 and in the prime/boost study, half were sacrificed infection reduced peak and total virus shedding at day 4 (n = 5) and half at day 8 (n = 5) to better by a greater level than either vaccination regimen evaluate resolution of infection. Placebo- (Figure 5c). This was expected given that vaccinated animals sacrificed at 4 days post- immunity in the upper respiratory tract is more challenge had high levels of virus present in both efficiently elicited during natural, IN exposure of lung and nasal turbinate tissue (Figures 4d and the virus as opposed to IM vaccination. These 5d). This was completely cleared from the lungs of findings further validate the hamster model in control animals by day 8 and 3/5 hamsters had recapitulating infection outcomes in humans. low levels of detectable virus that remained in the Next, we investigated virus replication and nasal turbinate tissue (Figure 5g). associated pathology in the nasal turbinates and A single dose of either the inactivated virus at more distal sites (i.e. the lungs) as these with Alhydrogel or MF59-adjuvanted Sclamp parameters can be indicative of a productive resulted in a significant reduction in virus load in infection that can impact disease progression. For the lungs at day 4 post-challenge (P < 0.001) but

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(a) Prime/Boost Study Challenge Vaccination groups sts nd 2 1 dose or infection 2 dose 1x10 TCID50 PBS dosed (placebo)

End Inactivated virus (50 μg/dose) + Alhydrogel (85 μg/dose) Sclamp (5 μg/dose) + MF59C.1 (50% vol/vol) 0 21 49 53 57 SARS-CoV-2 Infection and Recovery (*n=5)(*n=5)

(b) Virus Neutralization (c) Virus Shedding - Time course Virus Shedding - Peak Virus Shedding - Total p<0.0001 ns ns <0.001 0.006 0.003 ns ns ns p<0.001 p<0.001 4 p<0.0001 10 ns ns 0.004 ns ns ns ns ns p=0.046 ns 128 1011 ns ns 0.002 <0.001 0.006 0.005 ns ns ns 104 p=0.045 p=0.049 1010 64 3 10 109 103 /ml) 8

titre 32 10 50 /ml) /ml) 50

100 2 7 10 50 10 2

MN 16 (TCID 10 6 Throat swab Throat 10 (TCID (TCID LoD 5 8 101 10 LoD 1 10 104 LoD Peak throat swabs day 1-4 day swabs throat Peak 01234568 Sum of throat swabs day 1-4 103 LoD Days post infection

(d)Nasal Turbinate Day 4 Lung Day 4 (e) Gross Pathology Day 4 (f) Histopathology at Day 4 p=0.001 ns 109 ns 109 p=0.007 100% p=0.002 p=0.025 p=0.009 ns ns p=0.036 ns p=0.001 ns ns 8 8 ns 10 10 ns p=0.037 p=0.009 ns ns 80% 107 107 3 6 106 10 /g /g 60% 50 50 105 105 2

4 40%

4 TCID TCID 10 10 1 103 103 20%

LoD Score Severity 102 102 Affected Lung Tissue 0 LoD 101 101 0% Rhinitis Trachetis Bronchitis Bronchiolitis Alveolitis

(g) Nasal Turbinate Day 8 Lung Day 8 (h) Gross Pathology Day 8 (i) Histopathology at Day 8 p=0.004 ns ns ns p=0.036 p=0.002 9 ns 9 ns 100% 10 10 p=0.021 ns ns ns p=0.036 p=0.002 108 108 ns ns ns ns p=0.048 p=0.010 80% 107 107 3 106 106 60% /g /g 2 50 105 50 105 40% 4 4 TCID 10 TCID 10 1 103 103

20% Severity Score

LoD Affected Lung Tissue 102 102 0 LoD 101 101 0% Rhinitis Trachetis Bronchitis Bronchiolitis Alveolitis

Figure 5. Protective outcomes following prime/boost vaccination (data from a single experiment shown). (a) Prime/boost study schedule. Syrian hamsters (n = 10/group) were vaccinated with two doses of either a placebo, 50 lg inactivated virus with Alhydrogel or 5 lg of SARS-CoV-2 2 Sclamp with MF59, or were infected with 10 TCID50 SARS-CoV-2 via IN administration and allowed to recover prior to challenge. (b) Average

SARS-CoV-2 MN100 titre measured using serum collected at day 42 and 49. (c) Kinetics of infectious viral load in daily throat swabs on days 1–8 post-challenge (left panel), the peak virus titre from the kinetics analysis (middle panel) and the sum of virus titre for swabs collected on days 1–4 post-challenge (right panel). (d, g) Viral loads in nasal turbinate and lung tissue at day 4 and day 8 post-challenge. (e, h) Extent of affected lung tissue damage as assessed by gross pathology at day 4 and day 8 post-challenge. (f, i) Severity score of inflammatory response based on histology at day 4 and day 8 post-challenge: 0 = no inflammatory cells, 1 = few inflammatory cells, 2 = moderate number of inflammatory cells and 3 = many inflammatory cells. P-values were calculated using a Kruskal–Wallis ANOVA with Dunn’s multiple comparison tests or a two-way ANOVA with Dunnett’s multiple comparison tests. Error bars show SD. no reduction in viral titre within the nasal although this was not statistically significant turbinate tissue (Figure 4d). In the prime/boost (Figure 5d). Two doses of inactivated virus with study, administration of two doses of MF59- Alhydrogel reduced viral titre in the lungs but not adjuvanted Sclamp resulted in complete absence within the nasal turbinate (Figure 5d). Prior of detectable virus in the lungs for all but one infection and recovery from SARS-CoV-2 infection animal at 4 days post-challenge (Figure 5d). The resulted in the lowest viral titres in both lung and two-dose MF59-adjuvanted Sclamp regimen nasal turbinate tissue although the effect was not reduced viral geometric mean TCID50 titres in the statistically significant in the nasal turbinate nasal turbinates from 2.9 9 107 to 3.8 9 106, (P = 0.053 relative to placebo) (Figure 5c).

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Figure 6. Representative histopathology images from the lungs of placebo and Sclamp + MF59-vaccinated hamsters in the prime/boost study (109 objective; data from a single experiment are shown). (a, b) Representative images used for bronchitis scoring on day 4 and day 8 post- challenge. (c, d) Representative images used for alveolitis scoring on day 4 and day 8 post-challenge.

Histopathological assessment of placebo- there was also evidence of alveolar oedema and vaccinated hamsters at day 4 and at day 8 haemorrhage (Supplementary table 6). Of note, provided an opportunity to follow disease these findings of increased inflammation and progression. At day 4 post-challenge, associated pathologies were evident despite the inflammation was primarily confined to the upper apparent clearance of virus from the lung by day respiratory tract and larger airways (trachea and 8 (Figure 5g). bronchus); however, by day 8 severe inflammation A single dose of MF59-adjuvanted Sclamp was was present in the smaller airways (bronchioles shown to provide a significant level of protection and alveoli) (Figure 5f and i, and representative against bronchitis, bronchiolitis and alveolitis histopathology images in Figure 6). At day 8 post- (Figure 4e). While a single dose of inactivated challenge, the overall percentage of lung tissue virus with Alhydrogel did not provide significant affected was increased (Figure 5e and h) and protection against disease pathology, neither did

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(a) Virus Shedding vs Neutralizing titre 1011

10 10 Placebo 109 rs = NA 108 Inactivated virus + Alum rs = -0.40 (95%CI = -0.72 to 0.07); p = 0.08 /ml) 107 50 SARS-CoV-2 Sclamp + MF59 106 rs = -0.20 (95%CI = -0.60 to 0.28); p = 0.40

(TCID 105 Infection + Recovery rs = -0.69 (95%CI undetermined); p = 0.03 104 Combined dataset 3 10 rs = -0.64 (95%CI = -0.76 to -0.46); p <0.0001 Sum of throat swabs day 1-4 day swabs throat of Sum 102 101 102

MN100 Titre

(b) Lung titre vs Neutralizing titre 108

107 Placebo rs = NA 6 10 Inactivated virus + Alum rs = -0.06 (95%CI = -0.57 to 0.48); p = 0.84 /g) 105 50 SARS-CoV-2 Sclamp + MF59 r p 104 s = -0.59 (95%CI = -0.85 to -0.10); = 0.02

(TCID Infection + Recovery 3 r

Lung Titre day 4 day Titre Lung 10 s = NA Combined dataset 102 rs = -0.71 (95%CI = -0.83 to -0.53); p <0.0001 101 101 102

MN100 Titre

Figure 7. Correlation between neutralising antibodies and protection in hamster (data from two experiments shown). (a) Log-transformed À1 MN100 titre was compared against the log-transformed measure of total virus shedding (sum of throat swabs day 1–4 TCID50 mL ) for all 70 animals included in the single-dose and prime/boost challenge studies. (b) Log-transformed MN100 titre was compared against the log- transformed measure of virus in lung tissue at day 4 post-challenge for 50 animals sacrificed at day 4 included in the single-dose and prime/boost challenge studies. Spearman non-parametric correlation and associated values were calculated for each treatment and the combined dataset using GraphPad Prism 8.4.3. it result in increased inflammation despite the Consistent with preclinical trials of all other failure to elicit any detectable level of vaccine candidates, sterilising protection in the neutralising antibody (Figure 4b and e). upper respiratory tract was not observed; Two doses of MF59-adjuvanted Sclamp or however, reduction in viral shedding was seen. In inactivated virus with Alhydrogel, or prior order to assess correlates of protection in the infection and recovery, were each able to provide hamster model, we compared viral MN100 titre a high level of protection against rhinitis, prechallenge with total virus shedding post- tracheitis and bronchitis at day 4, as well as challenge (sum of throat swabs collected on days bronchiolitis and alveolitis at day 8 (Figure 5f and 1–4) on a combined dataset including all hamsters i, and representative histopathology images in in the single and prime/boost studies (n = 70) Figure 6). Pre-existing immunity from vaccination (Figure 7a) and with virus lung titre at day 4 post- either with MF59-adjuvanted Sclamp, inactivated challenge on a combined dataset including virus with Alhydrogel, or from prior infection, hamsters in the single and prime/boost studies reduced the overall percentage of affected lung (n = 50; Figure 7b). There was a clear correlation at day 8 (Figure 5h) and reduced the findings of between neutralising antibody level and a peribronchial and perivascular cuffing, alveolar reduction in virus shedding (rs = À0.64; 95% oedema and alveolar haemorrhage CI = À0.76 to À0.46; P < 0.0001) and lower virus

(Supplementary table 6). lung titre at day 4 (rs = À0.71; 95% CI = À0.83 to

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À0.53; P < 0.0001). Interestingly, all hamsters In mice, vaccination with MF59-adjuvanted within the group that were previously infected Sclamp was found to elicit a robust humoral and recovered from SARS-CoV-2 infection showed immune response that efficiently neutralised both substantially lower levels of virus shedding than the original SARS-CoV-2 strain and the D614G that expected based on linear regression of the variant which has since become the dominant entire data set. global circulating strain. A major issue in comparing results between vaccine platforms is DISCUSSION that there is no standardised method for assessing virus neutralisation. To address this issue, WHO Vaccine development in response to the COVID-19 has recommended the use of a convalescent pandemic has been unprecedented in terms of patient reference serum (NIBSC code 20:130) until both speed and the diversity of the underlying an international standard can be made available technology platforms. These approaches have at the end of 2020.27 Serum from mice that included inactivated viruses, nucleic acid-based received two doses of MF59-adjuvanted Sclamp vaccines, viral vectors and protein subunits.2 While was found to neutralise SARS-CoV-2 at twice the each of these platforms comes with its own level of this reference serum. To our knowledge, strengths and limitations, this diversity should also no other programmes have reported neutralising increase the likelihood of success and allow for titres relative to this reference serum; however, as the selection of the optimal vaccine(s) to progress these data provide a useful cross-platform into widespread global use based on key comparator for vaccine developers and regulatory parameters, including safety and efficacy.40 The authorities, we would encourage others to utilise utility of the SARS-CoV-2 spike protein as the this resource and openly present the data. target for an effective vaccine has also been To assess whether an anticipated immune strengthened through the release of efficacy data response to the clamp itself would detract from for multiple vaccines in phase 3 studies; however, the protective immune response to the spike data around longevity and type of protection will ectodomain, we quantified the relative response take longer to determine. to each domain in mice. The data showed that on Driven by the rationale that any improvement average roughly 76% of antigen-specific IgG were in protein yield directly translates into a future specific to spike. We also assessed whether serum increase in dose availability, we dedicated from mice with higher antibody responses to the considerable effort to the selection of an optimal clamp domain was less capable of neutralising candidate and development of a high-yielding SARS-CoV-2 virus but found no significant and scalable bioprocess. The ability to correlation. The preclinical studies showed that manufacture at large scale, ensuring consistent the molecular clamp in the Sclamp antigen is product quality, together with supply chain immunogenic and while cross-reactivity of the stability, will be a critical differentiator in clamp response to HIV-1 GP41 present in some achieving global vaccine supply.40 Furthermore, diagnostic reagents was recognised as a we exploit an antibody to the clamp domain for possibility, this was considered to likely have immunoaffinity purification that allows high-level minimum impact due to the exclusion of the purification using a predefined and standard immunodominant region (aa577–618).34 For this protocol and reagents that are independent of reason and the urgency to develop a vaccine for antigen, therefore speeding vaccine development. the COVID-19 pandemic, progression to the phase Selective engineering of an antigen for high I clinical trial was deemed warranted and expression can increase production, while approved by an independent ethics committee.15 selection of a homogeneous and stable antigen In addition to the humoral immune response, reduces product loss during the purification type 1 biased S-specific T-cell immunity is likely to process. Our streamlined process for screening of contribute to protection in convalescent COVID-19 clamp-stabilised candidate antigens,14 facilitated patients.41 Broadly reactive and/or type 1 the development of a manufacturing process that cytokine-producing T-cell responses are also can yield 100s of milligrams per litre of culture, characteristic of DNA, adenovirus serotype 26 and which potentially translates to 10s of millions of chimpanzee adenovirus vaccines that protected doses per production run using industry standard, non-human primates against SARS-CoV-2 mammalian cell bioproduction platforms. challenge and are in clinical development.33,35,36,42

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In order to assess the T-cell response in detail, we develop severe pneumonia and lung injury similar evaluated the magnitude and breadth of the T- to COVID-19 patients,45 and (4) protection can be cell response in vivo to complement the in vitro T- reproducibly evaluated based on infectious viral cell stimulation and cytokine production analysis. load rather than viral genomic copy levels.32,44,45 Although such analyses are expected to be In the light of the extensive safety record for beneficial when targeting mutable RNA viruses, MF59C.1 and subunit vaccines and the ethics of SARS-CoV-2 vaccines tested in preclinical models NHP experiments that do not provide more previously have relied on in vitro analysis with informative data, we considered the limited indications about the breadth of the T-cell immunogenicity data from mice, the protection response. Using the in vivo FTA and ICS analysis, data from Syrian hamsters and toxicology data our study for the first time demonstrates that an from Sprague Dawley rats sufficient to support MF59-adjuvanted subunit vaccine, Sclamp, can moving this product to the clinic without further elicit a robust, broad and predominantly type 1 testing in NHPs. cytokine-producing polyfunctional T-cell response. A single dose of MF59-adjuvanted Sclamp or We also identified several novel immunodominant inactivated virus with Alhydrogel afforded a regions following in vivo functional analysis of Th level of protection to hamsters from SARS-CoV-2 cell and CTL responses to benefit the future infection, with a significant decrease in peak development of SARS-CoV-2 vaccines. viral shedding detected in throat swabs and in MF59-adjuvanted Sclamp vaccination elicited virus detected within lung tissue upon sacrifice some type 2 cytokine (i.e. IL-4 and mostly IL-13)- at 4 days post-infection. A single dose of MF59- producing T cells as has also been shown in mice adjuvanted Sclamp showed significant vaccinated with a SARS-CoV-2-stabilised spike protection against disease severity as measured mRNA construct.35 The type 2 T-cell immunity by the level of inflammation within the elicited following MF59-adjuvanted Sclamp bronchus, bronchioles and alveoli. While a vaccination is likely to be beneficial for promoting single dose of the inactivated virus with a neutralising antibody response, but not for Alhydrogel adjuvant did not provide a triggering immune-mediated pathology for the significant level of protection against disease following reasons: (1) There was no evidence of severity, there was a trend towards reduced vaccine-enhanced disease in the hamster model pathology. The absence of an increased and the hamsters were protected following inflammatory response as well as decreased vaccination; (2) pathological/excessive production virus titres following administration of of type 2 cytokines is characteristic of poorly inactivated virus with Alhydrogel, despite the cytolytic and low-quality T-cell responses,28,43 absence of detectable virus neutralising which was not the case in the current study, and antibodies, adds further weight to the (3) there is no evidence to suggest that exposure mounting evidence against antibody-dependent to type 2 cytokines during SARS-CoV-2 infection is enhancement (ADE) or vaccine-associated detrimental. enhanced respiratory disease (VAERD) being of As a result of their genetic similarity to humans, significant concern for SARS-CoV-2.46,47 NHPs, particularly Rhesus macaque, are thought to Administration of two doses of either MF59- be a relevant model for SARS-CoV-2 vaccine adjuvanted Sclamp or inactivated virus with development. However, infection kinetics are Alhydrogel in the prime/boost study further poorly correlated and NHPs experience only mild reduced viral titres within the lung at day 4 post- disease.33–36 Multiple studies,32,44,45 including our infection compared with the single dose. In the own, suggest that the Syrian hamster provides a case of MF59-adjuvanted Sclamp, there was also more effective animal model to evaluate SARS- an apparent reduction in viral load within nasal CoV-2 vaccine efficacy for the following reasons: turbinate tissue, suggesting that protection may 2 3 (1) low-dose (i.e. 10 –10 TCID50) intranasal extend to the upper respiratory tract; however, administration of SARS-CoV-2 more accurately this trend did not reach statistical significance. reflects likely natural transmission dynamics and Both vaccines decreased virus shedding collected results in replication of infectious virus to high in throat swabs; however, neither was to the titres in the respiratory tract (lungs and nasal same level as the group exposed to prior SARS- turbinates), (2) hamsters are permissive to CoV-2 infection. This was expected as prior communicable transmission,32 (3) hamsters can intranasal infection can elicit robust, localised

2021 | Vol. 10 | e1269 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 14 Australian and New Zealand Society for Immunology, Inc. D Watterson et al. Molecular clamp vaccine for SARS-CoV-2 immunity (e.g. IgA) to rapidly prevent re-infection this work, we have shown that the immune resulting from IN exposure of SARS-CoV-2.48 response to the HIV-1 sequences presents within the Histopathological assessment of lungs from clamp trimerisation domain. This finding has na€ıve (placebo-vaccinated) animals at day 8 post- prevented progression into later-stage clinical trials infection revealed severe inflammation affecting a until re-engineering of the molecular clamp high percentage of total lung tissue and evidence trimerisation domain is completed to alleviate HIV of alveolar oedema and haemorrhage. Both diagnostic interference. Despite this issue, this vaccines, as well as prior SARS-CoV-2 infection, candidate vaccine has significant advantages that were able to significantly reduce disease severity demonstrate the suitability of the underlying assessed at both 4 and 8 days post-infection. In platform technology to meet the global response to particular, the observation of severe lung the ongoing COVID-19 pandemic as well as novel inflammation at day 8 has important parallels to viruses that may emerge in the future. Most notably, the pathogenic proinflammatory cytokine storm these advantages include accelerated progression observed in patients suffering the most severe from sequence to product in the absence of consequences of infection.49 We propose that the structural information and compatibility of the protection against severe lung pathology at this manufacturing process with industry standard time point in the hamster model could be highly mammalian cell-based bioprocess facilities and relevant to support vaccine-mediated protection favorable liquid stability for future global against the severe consequences of SARS-CoV-2 distribution. infection and also vaccine safety against the potential of VAERD. METHODS Total virus shedding as measured by summation of the TCID SARS-CoV-2 titres from daily throat 50 Constructs and plasmids swabs collected between 1 and 4 days post- infection provided a quantitative measure of To express the prefusion S ectodomain, codon-optimised productive viral replication. There was a high SARS-CoV-2 S (GenBank accession number: MN908947) gene degree of correlation between higher virus with variations including (1) substitution at the furin cleavage site, (2) substitution at signal peptide, (3) truncation at C- neutralisation titre prior to challenge and terminal domain was generated with primers containing reduction in total virus shedding. Neutralising overlapping sequence by PCR mutagenesis using Phusion antibodies are the primary goal of most SARS- polymerase (New England Biolabs, Ipswich, MA, USA). These CoV-2 vaccination strategies; however, to our amplicons were introduced upstream of the clamp knowledge this is the first reported evidence that trimerisation motif. Variable domains of heavy and light chain of CR3022,17 S309,18 B38,18 H4,50 CB6,18 G4 (anti-MERS S)51 or neutralising antibody level is correlated with anti-clamp HIV128152 were cloned into the mammalian reduced virus shedding in a relevant animal expression vector, pNBF-Hv or pNBF-Lv in-frame with IgK model. Importantly, our findings also suggest that signal peptide. neutralising antibodies are not the only correlate of protection as animals previously exposed to Recombinant protein expression SARS-CoV-2 infection and reinfected showed substantially lower virus shedding than predicted The ExpiCHO-S expression system (ThermoFisher Scientific, by neutralising antibody levels alone. It will be Waltham, MA USA) was used for transient spike protein and antibody expression. CHO cells were cultured in useful to understand additional mediators of ExpiCHO-S Expression Medium (GibcoTM, ThermoFisher protection, which could potentially target other Scientific) and transfection was conducted following the SAR-CoV-2 proteins, to inform second-generation manufacturer’s protocols for 5 or 7 days prior to harvest of vaccine approaches. This will be particularly the culture supernatant and protein purification. Stable cell relevant should the leading vaccine candidates lines were generated using the Lonza GS Xceed System (Lonza, Basel, Switzerland). CHOK1SV GS-KO cells were already in clinical stages fail to elicit sufficient transfected via electroporation with linearised GS immunity to break the transmission cycle and end expression vector encoding SARS-CoV-2 Sclamp as per the COVID-19 pandemic. manufacturer’s instructions (GS Xceed manual, version 06 Overall, this work demonstrates the ability of 2019). Approximately 24 h later, enriched pools were µ MF59-adjuvanted Sclamp to provide protection selected using 50 M L-methionine sulphoximine (MSX) over a period of 3–4 weeks. Stable pool shaker flask expression against SARS-CoV-2 infection and supports was assessed over 12 days via Lonza’s abridged fed-batch continued development and progression through shake flask screen (v8.10), and clone selection was human clinical trials. Subsequently to completion of performed using the Beacon Optofluidic platform (Berkeley

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Lights, Emeryville, CA, USA). Stable pools were loaded onto ARM 300 (JEOL, Tokyo, Japan) equipped with a K3 detector the OptoSelectTM 1750b Chip (Berkeley Lights) as single cells. (Gatan, Pleasanton, CA, USA). 50 frame, 5-s movies were Cells were cultured on a chip for 3–5 days before pens were collected using JADAS software (JEOL) at a magnification of analysed for the secretion of SARS-CoV-2 Sclamp using 50 0009, corresponding to a pixel size of 0.48 A per pixel À À fluorescently tagged anti-clamp IgG. Selected pens were and a dose rate of 9e pix 1 s 1. Movies were binned 29 then exported into 96-well plates and scaled up into shaker during motion correction using MotionCor2 (v1.1.0),53 flasks. Clones were further assessed via Lonza’s abridged giving a final pixel size of 0.96 A. The contrast transfer fed-batch shake flask screen (v8.10). function (CTF) parameters of each image were determined using CTFFIND (v. 4.1).54 Initial 2D references were generated using particles manually picked in RELION 3.1.55 Recombinant protein purification A60A filtered map from EMD-21452 was used as an initial model for 3D classification using C1 symmetry, and the Severe acute respiratory syndrome coronavirus 2-stabilised most ordered class was further refined using the RELION 3D spike protein was purified using immunoaffinity € auto-refine procedure with C3 symmetry. CTF refinement chromatography on an AKTA pure protein purification and particle polishing were performed using RELION. The system (Cytiva, Marlborough, MA, USA). This was achieved final half-maps were masked with a soft, extended mask using an in-house made immunoaffinity chromatography and Fourier shell correlation calculated using the gold — column the anti-clamp mAb HIV1281 coupled to 1- or 5- standard Fourier shell correlation cut-off of 0.143. mL HiTrap-NHS-activated HP columns (Cytiva). CHO expression cultures were centrifuged at 4000 g for 10 min at 4°C and resultant supernatant was filtered through a Thermal stability testing filter unit (0.22 µm pore size). Supernatant was added to an anti-clamp protein affinity column to purify clamp-tagged Purified antigen was sterile-filtered and diluted in PBS to a proteins or protein A HP column (Cytvia) to purify final concentration of 0.18 mg mLÀ1. 250-µL aliquots were antibodies. Eluates following purification from culture added to 1.5-mL sterile microcentrifuge tubes which were supernatant were neutralised immediately and buffer- then stored at either 4, 25 or 40°C for 1, 2, 4 or 8 weeks. At exchanged into PBS using Merck Amicon Ultra-4 or Ultra-15 each designated time point, samples were removed from centrifugal filter units. Protein concentration was incubation and assessed by ELISA and SE-HPLC. determined using the NanoDrop One (ThermoFisher Scientific) or via the Pierce BCA protein assay kit (ThermoFisher Scientific). Homogeneity of purified proteins Mouse vaccinations and immune analysis was analysed using SDS/PAGE and visualised following staining with Coomassie blue. Female, 5- to 7-week-old BALB/c mice were purchased from the Australian Resource Centre, Perth, and housed in individually ventilated HEPA-filtered cages at the University SE-HPLC analysis of Sclamp of Queensland Biological Resources facility, The Australian Institute for Bioengineering and Nanotechnology. Mice High-resolution assessment of the oligomeric state of were acclimatised for at least 5 days prior to vaccination via Sclamp was conducted using an Agilent 1200 HPLC. the IM route into the hind leg muscle with 50 µL of PBS Duplicate 95-µL samples were injected onto a Waters X- (placebo) or 5 µg per mouse of SARS-CoV-2 Sclamp with or Bridge 300-mm column precalibrated with PBS mobile without Alhydrogel (50 µg per mouse; Croda, Snaith, UK) or – phase and using a flow rate of 0.8 mL min 1. MF59 (50% vol:vol of SARS-CoV-2 Sclamp:MF59 as recommended by the manufacturer) under anaesthesia. Blood was collected via the tail vein prior to each Negative staining electron microscopy of vaccination and by cardiac puncture at the study end point. prefusion Sclamp protein After overnight incubation of blood samples at 4°C, serum was collected via centrifugation for 10 min at 10 000 g, Severe acute respiratory syndrome coronavirus 2 proteins heat-inactivated at 56°C for 30 min and stored at À80°C À1 were diluted at ~ 10 µgmL in PBS. Diluted proteins (4 lL) prior to analysis. were adsorbed onto carbon-coated grids (ProSciTech, Bronchoalveolar lavage collection was performed by Thuringowa, QLD, Australia) for 2 min and glow-discharged inserting a catheter into the trachea and flushing the lungs for 5 s in 25 mA. The grids were blotted and washed three with 400 µL of PBS. After centrifugation at 300 g for 7 min times in water and stained twice with 1% uranyl acetate with at 4°C, clarified BAL supernatants were frozen at À80°C blotting in between. The grids were air-dried and imaged prior to analysis. using a Hitachi HT7700 microscope operated at 120 Kv. To assess T-cell responses in mice at the study end point, splenocyte suspensions depleted of red blood cells (RBC) were analysed using ICS or the FTA as described below. Cryo-EM analysis of Sclamp

Severe acute respiratory syndrome coronavirus 2 proteins ELISA À were diluted to 0.5 mg mL 1 in PBS. Diluted proteins (4 lL) were adsorbed onto glow-discharged quantifoil grids (Q2/1) A capture ELISA was used to screen ExpiCHO-S supernatants and plunge frozen using an EMGP2 system (Leica, Mount and purified proteins for expression of Sclamp vaccine Waverley, VIC, Australia). Grids were imaged on a CRYO candidates. Nunc MaxiSorp ELISA plates were coated with

2021 | Vol. 10 | e1269 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 16 Australian and New Zealand Society for Immunology, Inc. D Watterson et al. Molecular clamp vaccine for SARS-CoV-2

– 2 µgmL1 of the anti-clamp mAb HIV1281 in PBS overnight described.57 Briefly, serial dilutions of mouse sera and BAL at 4°C. Plates were then blocked with 150 µL per well of were incubated with ~ 6000 focus-forming units mLÀ1 of 5% KPL milk diluent/blocking solution concentrate SARS-CoV-2 for 1 h at 37°C. Serum virus mixtures were (SeraCare, Milford, MA, USA) in PBS with 0.05% Tween-20 added to Vero E6 cell monolayers (preseeded at 40 000 cells for 1 h at room temperature (RT). Blocking buffer was per well in 96-well plates and incubated overnight) and removed and plates were incubated with serial dilutions of incubated at 37°C for 30 min. Subsequently, cells were harvested ExpiCHO-S supernatant for 1 h at 37°C. Plates overlaid with 1% (w/v) medium viscosity carboxymethyl were washed three times with water before incubation cellulose in M199 (ThermoFisher Scientific) supplemented with 5 µgmLÀ1 of an in-house produced recombinant with 2% heat-inactivated foetal bovine serum CR3022 mAb17 with a mouse IgG1 backbone for 1 h at supplemented with 1% penicillin–streptomycin (Sigma- 37°C. Following another wash step, plates were incubated Aldrich, St. Louis, MO, USA) and incubated at 37°Cin5% with a 1:2000 dilution of a horseradish peroxidase (HRP)- CO2 for 14 h. After incubation, overlay was removed and conjugated goat anti-mouse secondary antibody cells fixed with 80% cold-acetone in PBS for 30 min at (ThermoFisher Scientific) for 1 h at 37°C. After a final wash, À20°C. Plates were then dried, blocked with blocking plates were developed for 5 min using TMB single solution buffer (1xKPL in 0.1% PBS–Tween-20) for 1 h and then À chromogen/substrate (ThermoFisher Scientific) before the incubated with 1 µgmL 1 of CR3022 anti-S mAb and À1 reaction was stopped by addition of 2 N H2SO4. Absorbance 0.2 µgmL IR-Dye800-conjugated goat anti-human IgG (LI- at 450 nm was then read on a Spectramax 190 Microplate COR Biosciences, Lincoln, NE, USA) in blocking buffer. Plates reader (Molecular Devices, San Jose, CA, USA). were washed 3 times after antibody incubations by For ELISA analysis of mouse serum from placebo- or Sclamp- submerging in PBS with 0.1% Tween-20. Plates were then immunised mice, Nunc Maxisorp ELISA plates were coated dried prior to visualising using Odyssey (LI-COR Biosciences). with 2 µgmLÀ1 of Sclamp antigen or an alternate clamp- Immunoplaques were manually counted in blinded fashion. stabilised viral glycoprotein (influenza virus haemagglutinin (HA)clamp) and blocked as above. The blocked plates were FTA analysis incubated with serial dilutions of each mouse serum at 37°C for 1 h. The plates were then washed, developed and read as Fluorescent target array analysis was performed using a well- described above. EC50 values were calculated by three- established method as previously described.30,31 In brief, na€ıve parameter curve fitting using nonlinear regression in autologous BALB/c splenocytes were evenly split and serially GraphPad Prism (version 8.3.1) (GraphPad, San Diego, CA, labelled with 0.12, 0.46, 1.7, 6.2, 23 or 85 µM of CTV USA). The limit of detection (LoD) was defined as the (Invitrogen) and 10 or 39 µM of CPD (Invitrogen) for 5 min at reciprocal of the highest concentration of sera tested and any RT. Each of the dye-labelled populations was pulsed with values falling below the LoD were reported as ½ LoD. DMSO (nil) or 10 µgmLÀ1 peptide of the indicated peptide pools composed of 15–18 aa peptides (10–11 aa overlap ° MN assay between adjacent peptides) for 4 h at 37 Cwith5%CO2. Overlapping peptides spanning the SARS-CoV-2 S1–1226 and Neutralising activity against infectious SARS-CoV-2 was the Peptivator array used for peptide pulsing were purchased assessed by a traditional MN assay as described for SARS- from Shanghai RoyoBiotech (Shanghai, China) and Miltenyi CoV.25 Briefly, SARS-CoV-2 isolate CoV/Australia/VIC01/ Biotec (Bergisch Gladbach, Germany), respectively. The FTA 202056 was passaged in Vero cells and stored at À80°C. was then injected intravenously into placebo or vaccinated 6 6 Serum samples were heat-inactivated at 56°C for 30 min mice such that each mouse received 24 9 10 cells (2 9 10 and serially diluted 1:20 to 1:10 240 before addition of 100 cells from each fluorescent bar-coded target cell population) µ TCID50 of SARS-CoV-2 in MEM/0.5% BSA and incubation at in 200 LofPBS. RT for 1 h. Residual virus infectivity in the plasma/virus Fifteen hours after the injection, RBC-depleted mixtures was assessed in quadruplicate wells of Vero cells splenocytes from FTA-challenged mice were stained with À incubated in serum-free media containing 1 lgmL 1 of PE-Cy7-conjugated anti-mouse CD69 (clone H1.2F3; BD

TPCK trypsin at 37°C/5% CO2; viral cytopathic effect was Biosciences, San Jose, CA, USA) and BUV395-conjugated read on day 5. The neutralising antibody titre was anti-mouse B220 (clone RA3-6B2; BD Biosciences) and fixed calculated using the Reed/Muench method as previously using 0.5% paraformaldehyde. Subsequently, stained described.25 LoD was defined as the reciprocal of the samples were acquired using the BD LSRII and analysed highest concentration of sera tested and any values falling using the FlowJo software (version 8.8.7) (BD, Franklin below the LoD were reported as ½ LoD. Lakes, NJ, USA). GMFI of CD69 plotted was calculated using the formula: B220+ peptide-pulsed target value (GMFI of + CD69) - B220 nil target value (GMFI of CD69). The PRNT50 analysis following formula was used to calculate the % killed data: [(nil target value % À peptide-pulsed target value %)/nil Two SARS-CoV-2 isolates generated from infected patients target value %] 9 100. in Queensland, Australia, were used for the PRNT assay, QLD02/2020—30/1/2020 (GISAID accession EPI_ISL_407896) and QLDID935/2020—25/03/2020 (GISAID accession ICS analysis EPI_ISL_436097) and were referred here as 614D and 614G, respectively. Both isolates were passaged three times in 2x106 RBC-depleted splenocytes from vaccinated or placebo Vero E6 cells. Viral titration and PRNT were performed on control mice were seeded into wells of a 96-well round- À1 Vero E6 cells by an immuno-plaque assay (iPA) as recently bottom plate and stimulated with 5 µgmL of S1–1226 at 37°C

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with 5% CO2 for 12 h. DMSO and PMA/ionomycin (PMA at by gross pathology as well as histopathological assessment 25 ng mL–1 and ionomycin at 1 µgmL–1) stimulations were for evidence of congestion, emphysema, presence of included as negative and positive controls, respectively. foreign body, alveolar haemorrhage, bronchioloalveolar – Following the 12-h incubation, 1 µgmL1 of brefeldin A hyperplasia and inflammation and oedema. (BioLegend, San Jose, CA, USA) was added to each well and incubated for further 4 h prior to staining the cells with fluorochrome-conjugated monoclonal antibodies. The Statistical analysis stimulated cells were stained for cell-surface markers, fixed and permeabilised using IC Fix/Perm buffer (BioLegend) prior Statistical analysis of the data was performed using the IBM to the intracellular stain to analyse cytokine expression. The SPSS (Armonk, NY, USA) Statistics Software (version 25) following fluorochrome-conjugated monoclonal antibodies (Armonk, NY, USA) or GraphPad Prism (version 8.3.1). P-values were used to stain the cells: CD3 (clone: 17A2; BioLegend), are reported for statistically significant comparisons with < > CD4 (clone: GK1.5; BioLegend), CD8 (clone: 53-6.7; P 0.05. Non-significant data with P 0.05 are denoted ‘ns’. BioLegend), IFN-c (clone: XMG1.2; BioLegend), TNF-a (clone: MP6-XT22; BioLegend), IL-2 (clone: JES6-5H4; BioLegend), IL-4 (clone: 11B11; BioLegend) and IL-13 (clone: eBio13A; ACKNOWLEDGMENTS eBioscience, San Jose, CA, USA). Stained cells were acquired This work was funded by CEPI. We thank Mike Whelan, using the BD LSRII flow cytometer and analysed using the Raafat Fahim and the rest of the project management team FlowJo software (version 10.8). from CEPI for all their expert advice and guidance. MF59 adjuvant was provided by Seqirus, and we thank CSL and Preparation of formalin-inactivated virus Seqirus for the assistance with manufacturing process development. We thank Cytiva for their development of To prepare formalin-inactivated virus, SARS-CoV-2 was the custom immunoaffinity resin, Berkeley lights for cultured in Vero E6 cells for 3 days before harvesting assistance optimising clonal cell line selection and Lonza supernatant and clarification via centrifugation. Formalin and Thermofisher for the provision of CHO cell lines. We was added to the virus stock at 1:4000 and incubated at thank Christina Henderson for administrative coordination 37°C for 3 days. The formalin-treated virus stock was and The University of Queensland Legal, Finance and centrifuged using Millipore Amicon filters (Merk, Communications teams. We thank Virginia Nink and Nadia Darmstadt, Germany) to concentrate the virus prior to De Jager at the Queensland Brain Institute for their help removal of formalin and buffer-exchange using PBS. Virus with the flow cytometry. Antigen production was was passaged on Vero E6 cells to confirm loss of infectivity. supported by the National Biologics Facility and Therapeutic Total protein concentration was quantified by BCA analysis. Innovation Australia. We thank the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Centre Hamster challenge study for Microscopy and Microanalysis and the Research Compute Centre, the University of Queensland, in particular – = Male, 10 12-week-old Syrian hamsters (n 10 per group) the assistance of Drs Roger Wepf, Lou Brillault and Matthias were vaccinated with either a single dose or two doses as Floetenmeyer. We thank Nvidia for the supply of a DGX shown in Figures 4a and 5b, respectively. Vaccination with workstation to aid computational analysis of cryo-EM data PBS alone was used as a placebo and vaccination with and Michael Landsberg for assistance. We thank NIBSC for µ 50 g of formalin-inactivated SARS-CoV-2 virus formulated the provision of the reference serum. We also thank Jake with Alhydrogel was included as a comparator vaccination. Carrol and the Research Computing Center at UQ for All vaccines were delivered by IM immunisation. For technical assistance. KS is supported by NHMRC investigator + formalin-inactivated SARS-CoV-2 Alhydrogel vaccination, grant 1177174 and generous support of the Jack Ma µ 100 L total volume was delivered per dose, while all other Foundation and the a2 Milk Company. The Melbourne µ vaccines comprised a total volume of 50 L per dose. For an WHO Collaborating Centre for Reference and Research on infection and recovery comparator group, hamsters were Influenza is supported by the Australian Government housed separately in a BSL-3 containment isolator and Department of Health. SARS-CoV-2 isolates QLD02 and 2 infected with 10 TCID50 of SARS-CoV-2 (BetaCoV/Munich/ QLD935 were provided by Queensland Health Forensic & µ BavPat1/2020) diluted in PBS to a dose volume of 100 L Scientific Services, Queensland Department of Health. delivered via IN administration. Patricia Pilling, Laura Castelli, Luisa Pontes-Braz assisted At 4 weeks after the single-dose and the boost with assay development. We also thank Jonneke de Rijck, vaccination in the two dose, or 7 weeks after infection in Guido van der Net, Stephane Nooijen and the rest of the the infection and recovery group, hamsters were Viroclinics Xplore team for their work on the hamster 2 challenged with 10 TCID50 of SARS-CoV-2 (BetaCoV/ challenge studies as well as Judith van den Brand of Munich/BavPat1/2020) diluted in PBS to a dose volume of Utrecht University who performed the gross and 100 µL delivered via IN administration. Following challenge, histopathological examinations and analysis. animal weight loss was recorded daily, and throat swabs were taken for virus quantification by TCID50 and qPCR. At day 4 or day 8 of infection, animals were sacrificed, and CONFLICT OF INTEREST tissue samples collected from the lung lobes and nasal turbinate to measure virus titre by TCID50. Samples were KJC, DW and PRY are inventors of the ‘Molecular Clamp’ also fixed and sectioned to enable the assessment of lesions patent, US 2020/0040042.

2021 | Vol. 10 | e1269 ª 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 18 Australian and New Zealand Society for Immunology, Inc. D Watterson et al. Molecular clamp vaccine for SARS-CoV-2

Young: Conceptualization; Funding acquisition; Writing- AUTHOR CONTRIBUTIONS review & editing. Keith Chappell: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Dan Watterson: Conceptualization; Data curation; Formal Methodology; Project administration; Validation; Writing- analysis; Investigation; Methodology; Project administration; original draft; Writing-review & editing. Validation; Visualization; Writing-original draft; Writing- review & editing. Danushka Wijesundara: Data curation; Formal analysis; Investigation; Methodology; Validation; Writing-original draft; Writing-review & editing. Naphak REFERENCES Modhiran: Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Writing-original 1. Amanat F, Krammer F. SARS-CoV-2 vaccines: status draft; Writing-review & editing. Francesca Mordant: Data report. Immunity 2020; 52: 583–589. curation; Formal analysis; Investigation; Methodology; 2. WHO(WHO). 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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Watterson, D; Wijesundara, DK; Modhiran, N; Mordant, FL; Li, Z; Avumegah, MS; McMillan, CL; Lackenby, J; Guilfoyle, K; van Amerongen, G; Stittelaar, K; Cheung, STM; Bibby, S; Daleris, M; Hoger, K; Gillard, M; Radunz, E; Jones, ML; Hughes, K; Hughes, B; Goh, J; Edwards, D; Scoble, J; Pearce, L; Kowalczyk, L; Phan, T; La, M; Lu, L; Pham, T; Zhou, Q; Brockman, DA; Morgan, SJ; Lau, C; Tran, MH; Tapley, P; Villalon-Letelier, F; Barnes, J; Young, A; Jaberolansar, N; Scott, CAP; Isaacs, A; Amarilla, AA; Khromykh, AA; van den Brand, JMA; Reading, PC; Ranasinghe, C; Subbarao, K; Munro, TP; Young, PR; Chappell, KJ

Title: Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2

Date: 2021-01-01

Citation: Watterson, D., Wijesundara, D. K., Modhiran, N., Mordant, F. L., Li, Z., Avumegah, M. S., McMillan, C. L., Lackenby, J., Guilfoyle, K., van Amerongen, G., Stittelaar, K., Cheung, S. T. M., Bibby, S., Daleris, M., Hoger, K., Gillard, M., Radunz, E., Jones, M. L., Hughes, K. ,... Chappell, K. J. (2021). Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2. CLINICAL & TRANSLATIONAL IMMUNOLOGY, 10 (4), https://doi.org/10.1002/cti2.1269.

Persistent Link: http://hdl.handle.net/11343/277993

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