key: cord-0726721-bi0xelzd
authors: Li, Lei; Honda-Okubo, Yoshikazu; Baldwin, Jeremy; Bowen, Richard; Bielefeldt-Ohmann, Helle; Petrovsky, Nikolai
title: Covax-19/Spikogen® vaccine based on recombinant spike protein extracellular domain with Advax-CpG55.2 adjuvant provides single dose protection against SARS-CoV-2 infection in hamsters
date: 2022-04-18
journal: Vaccine
DOI: 10.1016/j.vaccine.2022.04.041
sha: 830c6ffcea2131b80b1be3274fe8c13f9592ac73
doc_id: 726721
cord_uid: bi0xelzd

COVID-19 presents an ongoing global health crisis. Protein-based COVID-19 vaccines that are well-tolerated, safe, highly-protective and convenient to manufacture remain of major interest. We therefore sought to compare the immunogenicity and protective efficacy of a number of recombinant SARS-CoV-2 spike protein candidates expressed in insect cells. By comparison to a full length (FL) spike protein detergent-extracted nanoparticle antigen, the soluble secreted spike protein extracellular domain (ECD) generated higher protein yields per liter of culture and when formulated with either Alum-CpG55.2 or Advax-CpG55.2 combination adjuvants elicited robust antigen-specific humoral and cellular immunity in mice. In hamsters, the spike ECD when formulated with either adjuvant induced high serum neutralizing antibody titers even after a single dose. When challenged with the homologous SARS-CoV-2 virus, hamsters immunized with the adjuvanted spike ECD exhibited reduced viral load in day 1 -3 oropharyngeal swabs and in day 3 nasal turbinate tissue and had no recoverable infectious virus in day 3 lung tissue. The reduction in lung viral load correlated with less weight loss and lower lung pathology scores. The formulations of spike ECD with Alum-CpG55.2 or Advax-CpG55.2 were protective even after just a single dose, although the 2-dose regimen performed better overall and required only half the total amount of antigen. Pre-challenge serum neutralizing antibody levels showed a strong correlation with lung protection, with a weaker correlation seen with nasal or oropharyngeal protection. This suggests that serum neutralizing antibody levels may correlate more closely with systemic, rather than mucosal, protection. The down-selected spike ECD with Advax-CpG55.2 formulation (Covax-19® vaccine) was selected for human clinical development.

protection. This suggests that serum neutralizing antibody levels may correlate more closely with systemic, rather than mucosal, protection. The down-selected spike ECD with Advax-CpG55.2 formulation (Covax-19® vaccine) was selected for human clinical development.

Almost two years from the initial outbreak, the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global public health crisis. As at 1

April 2022, SARS-CoV-2 had infected over 470 million people globally, with many countries now entering a fourth or even fifth disease wave [1] . Vaccines are a key tool to control the spread and impact of SARS-CoV-2, with various candidates having received emergency use authorization [2] .

However, these COVID-19 vaccines are not without technical limitations with new technologies, such as mRNA-based platforms, being highly temperature sensitive [3] with strict cold storage requirements [4] . Developing regions, e.g. Africa, continue to have limited access to vaccines [5, 6] and lack the required extreme cold storage infrastructure required for mRNA vaccines. In addition, the adenovirus vector and mRNA vaccines have been associated with adverse reactions including anaphylaxis [7] , central venous thrombosis [8] and myocarditis [9, 10] . Altogether this has impacted on global vaccine coverage, highlighting the need for a diversity of vaccine platforms to tackle this still-evolving global crisis.

Recombinant protein subunit vaccines have been highly successful against many viral and nonviral diseases [11] . A potential limitation of protein-based vaccines is their low immunogenicity, necessitating an adjuvant to boost their immunogenicity [12] . Traditional aluminum hydroxide (alum) and oil emulsion adjuvants remain prominent in COVID-19 vaccines in development [13] .

Advax-CpG is a proprietary adjuvant formulation which combines Advax, a non-reactogenic adjuvant based on delta inulin, with a human toll-like receptor 9 (TLR-9) agonist oligonucleotide (CpG55.2) that was developed using artificial intelligence [14, 15] . Advax-CpG adjuvant has been tested in many preclinical studies [16] [17] [18] [19] and also in human clinical trials, including against Influenza [NCT03945825; NCT03038776] and Hepatitis B [NCT01951677], where it was shown to enhance vaccine immunogenicity while maintaining a satisfactory safety profile. Advax-CpG adjuvant was previously whown to be effective in enhancing coronavirus vaccine protection, as demonstrated in models of severe acute respiratory syndrome (SARS) [20] and Middle East respiratory syndrome (MERS) [21] .

Previously, we reported on a vaccine based on the SARS-CoV-2 spike protein ECD which when formulated with Advax-CpG55.2 adjuvant provided protection in ferrets [22] . In the current study, we describe the results from our screening of a range of vaccine formulations, comparing;

1. ECD versus full length (FL) spike protein both manufactured in insect cells, 2. Advax-CpG versus alum-CpG55.2 adjuvant, and 3. a single versus double dose regimen. Effects of these vaccine formulations on humoral and cellular immunity were first evaluated in mice, with their ability to protect against a challenge with the homologous virus then being confirmed in the hamster model. The hamster model has a number of advantages as SARS-CoV-2 replicates more efficiently in the lungs of hamsters and they display clinical signs of disease, i.e. weight loss and lung pathology, that can be quantified to assess vaccine efficacy and potency [23, 24] .

The FL vaccine antigen corresponded to the full-length of the SARS-CoV-2 spike of the original Wuhan-01 strain (accession number: NC 045512), whilst the ECD construct corresponded to aa 14 -1213 of the Wuhan-01 spike protein sequence with the furin cleavage site deleted (see Figure   1A ). The constructs were produced using a Bac-to-Bac baculovirus expression system as previously described [22] . The size and purity of the recombinant FL and ECD spike proteins were confirmed by SDS-PAGE gels. Endotoxin was measured using a PyroGene Endotoxin Detection System (Cat. No. 50-658U, LONZA, Walkersville, MD, USA) and residual DNA content in final vaccine product was measured using a Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher, P7589) following the manufacturer's instructions. Advax, Advax-CpG55.2, and Alum-CpG55.2 adjuvants were from Vaxine Pty Ltd (Adelaide, Australia).

The murine studies were carried out at Flinders University, Australia as approved by the Animal Welfare Committee of Flinders University conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013). Female, BALB/c and C57BL/6 (BL6) mice (6-10 weeks old) were supplied by the central animal facility of Flinders University. Mice were immunised intramuscularly (i.m.) in the thigh muscle at weeks 0 and 2 with 0.5, 1, 2.5, 5 or 10 µg recombinant spike protein (rSp) ECD or FL formulated with either Advax-CpG55.2 (1 mg/10 µg) or Alum-CpG55.2 (50 µg/10 µg) comprising Alhydrogel (Croda, Denmark) formulated with CpG55.2. Blood samples were collected by cheek vein bleeding 2 weeks after each immunisation. Serum was separated by centrifugation and stored at -20˚C prior to use.

Animals were sacrificed at week 4, and spleens were collected and used immediately for cellbased assays (CBA and ELISPOT).

Spike-specific antibodies were determined by ELISA. Briefly, 1 µg/ml rSp [corresponding to SARS-CoV-2 (Wuhan) reference sequence Q13 to P1209] or 0.5 µg/ml receptor-binding domain (RBD)

[corresponding to SARS-CoV-2 (Wuhan) reference sequence R319 to F541] antigen in PBS were used to coat 96-well ELISA plates (100 µl/well). After blocking, 100 µl of diluted serum samples were added followed by biotinylated anti-mouse IgG (Sigma-Aldrich) with horseradish peroxidase (HRP)-conjugated Streptavidin (BD Biosciences) for 1 h. After washing, 100 µl of TMB substrate (KPL, SeraCare, Gaithersburg, MD, USA) was added and incubated for 10 min before the reaction was stopped with 100 µl 1M phosphoric acid (Sigma-Aldrich). The optical density was measured at 450 nm (OD 450 nm) using a VersaMax plate reader and analysed using SoftMax Pro Software.

Average OD 450 nm values obtained from negative control wells were subtracted.

A replication-deficient SARS-CoV-2 Spike pseudotyped lentivirus-based neutralisation assay was developed to measure neutralising activity of murine immune sera. Single-cell sorted HEK-293T stable cell line expressing human ACE2 on the plasma membrane was maintained in DMEM-10 medium [25] . Expression cassette of human-codon optimized SARS-CoV-2 spike with C-terminal 18aa truncation of the original stain and different variants was cloned into pCAGGS vector. Spikepseudotyped lentiviral particles were produced by co-transfecting HEK-293T cells with firefly luciferase encoding 3rd generation lentiviral vector pCDH-EF1-Luc-IRES-Puro, packaging plasmid psPAX2 and spike expressing plasmid pCAGGS-Spike using Lipofectamine 2000 according to the product manual and recombinant virus particles were harvested at 72h post transfection.

Neutralisation activity of immune sera was then measured with a single round transduction of 293T-hACE2 cells with Spike-pseudotyped lentiviral particles. Briefly, prior to infecting cells, immune sera was serially diluted and incubated with pseudotyped virus particle for 1 h at 37°C.

Then 100 µL of virus-serum was added into 50 µL of 293T-hACE2 cells freshly plated at 125,000 cells per well in a 96-well white tissue culture plate. The cells were then cultured at 37°C for 72h, followed by removing of the culture medium and replacing of 30 µL of Phenol red-free DMEM medium. Then 30 µL of ONE-Glo EX (Promega) reagent was added into each well and incubated at RT with shaking at 400rpm on a thermoblock before luciferase activity reading on BMG FluoStar plate reader. Neutralization was calculated by reduction in % Luciferase units relative to pseudotyped virus alone group without any serum treatment. Neutralization antibody titers were then calculated using Sigmoidal 4PL robust fit regression method in GraphPad Prism Ver. 9.

BALB/c mice were sacrificed 2 weeks after the last immunization, and individual spleens were collected aseptically. Single-cell suspension in sterile 3% FCS in PBS was prepared using a 70 µm easy strainer (Greiner Bio-One) with a 5 ml syringe plunger. Isolated spleen cells were pelleted and incubated in red blood cell (RBC) lysis buffer for 10 min. For Cytometric Bead Array (CBA) assay, splenocytes were cultured at 5 x 10 5 cells/well in 96-well plates with 3 µg/ml of rSp antigen [corresponding to SARS-CoV-2 (Wuhan) reference sequence Q13 to P1209] at 37°C and 5% CO 2 .

Two days later, the supernatants were harvested and cytokine concentrations determined by mouse Th1/Th2/Th17 CBA kit (BD) and analysed by FCAP array Software (BD). In addition to CBA assay, enzyme-linked immune absorbent spot (ELISPOT) assay was performed using mouse 

All hamsters were transferred to a Biosafety Level-3 animal facility at the Regional Biocontainment Lab at Colorado State University prior to live virus challenge. At day 35 following prime immunization, animals were challenged with 1e4 PFU of SARS-CoV-2 (isolate USAWA1/ 2020) acquired originally through BEI Resources (product NR-52281) and passaged twice in Vero E6 cells as previously described [26] . In brief, the hamsters were first lightly anesthetized with a 10:1 mixture of ketamine hydrochloride and xylazine hydrochloride. Each hamster was administered virus via pipette into the nares (50uL/nare) for a total volume of 100 uL per hamster. Virus back-titration was performed on Vero cells immediately following inoculation.

Hamsters were observed until fully recovered from anesthesia. All hamsters were maintained for three days then humanely euthanized and necropsied. Oropharyngeal swabs were also taken on days 1-3 after challenge to evaluate viral shedding. Swabs were placed in BA-1 medium (Trisbuffered MEM containing 1% BSA) supplemented with antibiotics then stored at -80 °C until further analysis. Tissues were collected for virus quantification and histopathology. For virus quantitation, approximately 100mg of the right cranial lung lobe and nasal turbinates from each hamster were homogenized in 9 volumes of BA-1 media with antibiotics then frozen to -80 °C for later analysis. The tissue homogenates were briefly centrifuged and virus titers in the clarified fluid was determined by plaque assay. Viral titers of tissue homogenates are expressed as pfu/100 mg (log10). For histopathology, portions of the left and right medial lung lobes were fixed in 10% neutral buffered formalin for seven days then paraffin embedded, sectioned and stained with hematoxylin and eosin using routine methods for histological examination.

Plaque assays were used to quantify infectious virus in oropharyngeal swabs and tissue homogenates. Briefly, 10-fold serial dilutions were prepared in BA-1 media supplemented with antibiotics. Confluent Vero E6 cell monolayers were grown in 6-well tissue culture plates. The growth media was removed from the cell monolayers and each well was inoculated with 0.1 mL of the appropriate diluted sample. The plates were rocked every 10-15 min for 45 min and then overlaid with 0.5% agarose in in MEM without phenol red and incubated for 1 day at 37 °C, 5% CO2. A second overlay with neutral red dye was added at 24-30 h and plaques were counted at 48-72 h post-plating. Viral titers are reported as the pfu per swab or per 100mg of tissue. Samples were considered negative for infectious virus if viral titers were below the limit of detection (LOD). For oropharyngeal swabs the LOD was 10 pfu/swab. For tissues the LOD was 10 pfu/100 mg.

Neutralizing antibody levels were determined by plaque reduction neutralization test (PRNT).

Briefly, sera were first heat-inactivated for 30 min at 56 °C in a waterbath, then a series of twofold dilutions in BA-1 media prepared in a 96-well plate starting at a 1:5 dilution. An equal volume of SARS-CoV-2 virus (isolate USA-WA1/2020) was added to the serum dilutions and the samplevirus mixture was gently mixed. The plates were incubated for 1 h at 37 °C. Following incubation, serum-virus mixtures were plated onto Vero E6 plates as described for virus plaque assays.

Antibody titers were recorded as the reciprocal of the highest dilution in which >90% of virus was neutralized. All hamsters were tested for the presence of antibodies against SARS-CoV-2 prior to vaccination.

Histopathology was blindly interpreted by a veterinary pathologist (HBO). H&E-stained lung tissue sections were examined and microphotographed using a Nikon Eclipse 50i microscope equipped with a Nikon DS-Fi1 microscope and NIS-Elements F4.60.00 software. The H&E-stained slides were assessed for morphological evidence of inflammatory-mediated pathology in lung and trachea and reduction or absence of pathological features used as an indicator of vaccineassociated protection. Each hamster was assigned a score of 0-5 based on absent, mild, moderate, or severe manifestation, respectively, for each manifestation of pulmonary pathology including overall lesion extent, bronchitis, alveolitis, pneumocyte hyperplasia and vasculitis as previously described 37 and then the sum of all scores for each hamster calculated.

GraphPad Prism 8.3.1 for Windows was used for drawing graphs and statistical analysis (GraphPad Software, San Diego, CA, USA). Murine neutralization titers, antibodies, T cell based assays and tissue severity scores were evaluated using Kruskal-Wallis test with Dunn correction for multiple comparisons between groups. The limit of detection for viral plaque titres was 10 PFU, so titers lower than this were given a value of 5 PFU (half the limit of detection) for the purposes of statistical analysis. PRNT90 are presented as geometric mean titres, the minimum dilution tested for neutralizing antibody was 1:10, and a titer of 5 was used for a negative result.

T-test was used for analysis of daily weight change. Statistical analyses of the correlation of PRNT90, total weight loss, viral load titers and tissue severity scores was performed by linear regression, PRNT90 and viral titers were log transformed for the purposes of the analysis. For all comparisons, p<0.05 was considered to represent a significant difference. In figures * = p < 0.05; ** = p < 0.01; *** = p < 0.001 and ****, p < 0.0001.

Adjuvanted spike protein vaccine provides robust cellular and humoral immunity

In the current study we evaluated two vaccine antigens constructs, a full length (FL) spike protein versus the extracellular domain (ECD), both with the furin cleavage site deleted (see Figure 1A ).

The final proteins had a purity of >90% as confirmed by SDS-PAGE ( Figure 1B ) and were sterile with negligible endotoxin and residual host-cell DNA content. The secreted ECD antigen gave significantly higher protein yields of 15-20 mg per liter of insect cell culture compared to the FL protein which had yields of only <4 mg per litre (data not shown).

To initially cmpare the cellular and humoral immunogenicity of the FL versus ECD protein, mice were immunized twice 2 weeks apart with either ECD or FL antigen (0.5-10 g) alone or with control adjuvant, Alum-CpG55.2. At week 2, the ECD (10µg) construct showed a trend towards increased total IgG (anti-spike) compared to equivalent dose of FL protein (10µg) ( Figure 1C) . ECD (10µg) or FL alone (10µg) induced similar levels of anti-RBD total IgG at both week 2 and 4. The addition of the Alum-CpG55.2 adjuvant had an antigen-dose sparing effect but didn't change the overall pattern of response.

Cytokine production was measured using a CBA assay in culture supernatants of rSp-stimulated splenocytes obtained from immunised mice ( Figure 1D ). IFN-γ release was significantly higher in the adjuvanted ECD group compared to the adjuvanted FL group. TNFα and IL-6 trended higher in the adjuvanted ECD compared to the FL group, but did not reach statistical significance. Other cytokines, such as IL-2, IL-4, IIL-10, IL-17, were comparable between both the ECD and FL protein immunized mice. Cytokine ELISPOT results on rSp-stimulated splenocytes showed a similar trend to a higher frequency of IFN-γ secreting T cells in the splenocytes from the adjuvanted ECD group compared to the adjuvanted FL group, although this was not statistically significant ( Figure 1E ).

The frequencies of IL-2, IL-4 and IL-17 secreting T cells were comparable between ECD and FL proteins.

Next we compared the effect of two different combination adjuvants on immune responses to ECD or FL spike protein. Advax-CpG and alum-CpG adjuvants induced comparable levels of spikebinding and RBD-binding IgG that were several fold (1.5-3) higher than with either antigen injected alone without adjuvant ( Figure 1F) . The neutralizing activity of antibodies induced by the different adjuvanted formulations was assessed using a pseudoneutralization assays ( Figure   1G ). Formulations of the ECD protein showed higher 50% inhibitory concentration (IC 50 ) when adjuvanted with Advax-CpG as compared to alum-CpG, whereas the FL protein did better with the alum-CpG adjuvant.

Golden Syrian hamsters were immunized intramuscularly either once with a single vaccine dose, or with two doses 3 weeks apart. Sera was obtained 2 weeks after the final vaccination for measurement of SARS-CoV-2 neutralizing antibodies against the ancestral strain using a plaque reduction neutralization test with a 90% cutoff (PRNT90) ( Figure 2B ). As expected, none of the saline nor adjuvant-alone control injected hamsters developed neutralizing antibodies. For the spike ECD 2-dose groups, only those animals which received spike protein formulated with Advax-CpG or alum-CpG adjuvants achieved neutralizing titers >1:640 in all animals in the group.

In the single-dose Advax-CpG adjuvanted vaccine groups, the spike ECD protein induced higher neutralization titers (GMT = 640) than the spike FL protein (GMT = 113). A similar pattern was seen in the 2-dose groups, with spike ECD + Advax-CpG inducing greater than 5-fold higher neutralization titers (GMT = 761) than spike FL + Advax-CpG (GMT = 135).

At day 35, all hamsters were challenged intranasally with 1e 4 PFU of SARS-CoV-2 (isolate USAWA1/ 2020). The control groups (adjuvant alone and saline) lost an average of 6.4 -6.8% body weight during the first two days post-challenge and with further weight loss at termination on day 3, while the spike immunized animals only lost 4.1 -6.4% weight up to Day 2 then had begun to recover weight by day 3 (Figure 2C ). Cumulative weight loss throughout the 3 days was lower for all spike vaccine immunized groups compared to either control group, but with no significant differences between the various spike vaccine groups ( Figure 2D ). There was a strong negative correlation (R=-0.8063 p<0.0001) between the serum PRNT90 titer pre-challenge (day 35) and cumulative weight loss after challenge (Supplementary Figure 1i ). This suggests that the level of serum neutralizing antibodies prior to virus exposure may play an important role in determining systemic disease severity.

To assess vaccine effects on viral replication and shedding over time, oropharyngeal swabs were taken daily from hamsters on days 1-3 after challenge and viral loads measured using a viral plaque assay (Figure 2Ei-iv) . Viral loads in swabs from control groups reached a peak on day 2 before beginning to decrease on day 3. By contrast, the viral loads in all the spike immunized groups fell progressively from day 1 to day 3. Only the Spike ECD+Alum-CpG group had significantly lower throat swab viral load titers on day 1 (GMT = 7; p<0.05) and day 2 (GMT = 8.4; p<0.01) compared to the saline control group (GMT = 383) (Figure 2Eii) . There was also a large reduction in the throat swab viral load titers in the Spike ECD+Advax-CpG group on days 2 (GMT= 31; p=0.1) compared to saline group control (GMT = 383), however was not statistically significantly (Figure 2Eiii-iv) . By day 3, several immunized animals had no detectable throat swab virus, including 100% in FL+Advax-CpG group, 75% in 1-dose ECD+Advax-CpG and 2-dose ECD+Alum-CpG and 2-dose ECD+Advax and 50% in ECD+Advax-CpG, ECD alone and 1-dose FL+Advax-CpG groups.

For cumulative throat swab virus load over Days 1-3, the 2-dose ECD+Alum-CpG had the lowest overall viral load (viral titer=28) compared to saline control group (viral titer = 7310) followed by 2-dose ECD + Advax-CpG (viral titer =159, p<0.05). A reduction in viral load was also observed in the 2-dose ECD + Advax (viral titer = 377) and the 2-dose ECD alone (viral titer = 40) groups compared to saline control group, however, these differences were not statistically significant ( Figure 3A ). There was a negative correlation between PRNT90 antibodies pre-challenge and throat swab viral load, with the highest negative correlation between PRNT90 and throat swab viral load on day 3 (R=-0.6528, p<0.0001) and day 2 (R=-0.6526, p<0.0001) followed by day 1 (R= -0.4777, p=0.0018) (Supplementary Figure 1ii-v) .

To evaluate viral load in lungs and nasal turbinate tissues, animals were euthanized on day 3 and portions of right cranial lung lobe and nasal turbinate were collected for virus quantification using a plaque assay. Both control groups had extremely high day 3 viral loads in the range of Altogether, these results suggest that lung viral load determines COVID-19 disease severity in hamsters with serum neutralizing antibodies playing the most important role in controlling lung viral replication and disease severity.

To evaluate whether spike protein vaccines could protect against lung pathology, portions of the lung were collected from all animals at Day 3 for H&E staining. Figure 3 shows representative lung section images from a normal uninfected control lung (Figure 4Ai) , an infected saline control animal (total histopathology score 25) (Figure 4Aii) , and protected animals from the ECD+Advax-CpG and ECD+Alum-CpG groups (total histopathology score <10) (Figure 4Aiii -iv) at 20x and 100x magnification. Tissue scoring showed lower total lung scores between the immunised and control groups ( Figure 4B) , with the 2-dose ECD+Alum-CpG group having the lowest overall lung pathology score (1.75, p<0.01) followed by 2-dose ECD+Advax (6.75, p = 0.09), ECD+Advax-CpG (10.25) and ECD alone (10.5). Some immunized animals had a total lung score of 0, indicating their lungs were free of any signs of disease, including 75% of animals in ECD+Alum-CpG group and 50% of animals in the ECD+Advax-CpG group ( Figure 4B) . The 1-dose ECD+Advax-CpG and 2dose ECD+Advax-CpG groups had a similar mean total lung severity score of 9 and 10.25, respectively, consistent with absence of detectable day 3 lung virus in both these groups.

Conversely, the 2-dose FL+Advax-CpG group, which had the highest day 3 lung virus load of the immunized groups, also had the highest mean lung severity scores of the immunized groups with a mean lung severity score of 17.25.

Total lung pathology scores were highly correlated with cumulative day 1-3 weight loss 

The COVID-19 pandemic remains highly active with 0.5-3 million new cases per day, globally [1] .

Control measures such as quarantine protocols and lockdowns have had variable success [27] . Antigen yield is a critical parameter in pandemic vaccine selection and the ECD protein produced much higher protein yields than the FL construct per liter of insect cell culture. In murine immunogenicity studies, when both FL and ECD proteins were formulated with a control alum-CpG adjuvant, the ECD protein induced slightly higher rSp-binding IgG against after the first dose and also induced higher T cell IFNγ recall responses than the FL protein. Spike ECD when formulated with Advax-CpG adjuvant induced levels of rSp-and RBD-binding IgG and neutralizing antibody not significantly different to those induced by alum-CpG adjuvant. Interestingly, the alum-CpG adjuvant did yield slightly higher neutralizing antibody levels for the FL antigen suggesting some adjuvant effects might be antigen specific. Overall, based on all the data and considering the higher protein yields obtained for the ECD construct, we selected the spike ECD protein formulated with Advax-CpG adjuvant as our lead Covid-19 vaccine candidate going forward.

We next sought to assess the efficacy of the down-selected vaccine candidate, which was now named Covax-19®/Spikogen®, against viral replication/shedding and prevention of disease pathology in the well-established hamster model [26, 28] . The candidate ECD vaccine was compared against the FL protein and the Advax-CpG adjuvant was compared to the alum-CpG adjuvant formulation. Both single and 2-dose regimens of adjuvanted spike protein provided robust protection of hamsters against lung infection and pathology, with a high correlation seen between serum neutralizing antibody levels pre-challenge and lung protection post-challenge.

Other Covid-19 studies in animals and humans have similarly shown a good correlation between serum spike antibody levels and protection against systemic disease [29] [30] [31] .

Novavax's Nuvaxovid recombinant Covid-19 vaccine is based on FL spike protein nanoparticles [32] , and thereby bears the greatest similarity to the FL protein used in our study. Covax-19/Spikogen vaccine is distinct to Nuvaxoid as whereas Nuvaxoid is a FL protein expressed in SF9 insect cells that forms nanoparticles with lipid and detergent components, Covax-19/Spikogen vaccine is based on a secreted soluble spike ECD expressed in Tni insect cells. Our spike ECD antigen has the furin cleavage site removed to inhibit transition from pre-fusion to post-fusion conformation [33] as maintaining the pre-fusion state is hypothesized to lead to better neutralizing antibody production [34, 35] Interestingly, in addition to the large protein yield differences, we saw some immunogenicity differences between the spike ECD and FL proteins. In both mice and hamsters, the spike ECD protein induced slightly higher spike antibody titers than the FL protein, and in the murine immunogenicity studies it induced higher T cell IFN- recall responses. All animals that received either single or two-dose spike ECD had neutralization titers of 1:160 or greater, whilst in the FL groups, 75% of animals had titers equal to or below 1:160. This titer of 1:160 may be significant as it is a key selection threshold for convalescent plasma to be considered therapeutic for COVID-19 patients [36] . Serum neutralization titers were highest in the 2-dose ECD with Advax-CpG or alum-CpG groups which achieved titers of >1:640 in all animals. Reassuringly, no adverse effects of the Advax-CpG adjuvanted rSp protein formulations were observed in either the mice or hamsters, suggesting its suitability for further development.

In the hamster study there was a strong correlation between high serum neutralizing antibody titers prior to challenge and reduced weight loss and lung severity scores post-challenge. The 2dose ECD groups with Advax-CpG or Alum-CpG had the highest percentage of animals with no lung pathology, being 50% and 75%, respectively. Eosinophilic lung immunopathology which was a feature of alum-adjuvanted SARS CoV vaccines [20] was not observed in any group in this SARS-CoV-2 study. This suggests that either SARS-CoV-2 does not cause eosinophilic lung immunopathology or that any Th2 bias in our study imparted by the alum adjuvant may have been countered by its co-formulation with CpG55.2, which drives a strong Th1 signal.

One aim of this study was to evaluate whether any of the vaccine formulations could confer single-dose protection. A vaccine candidate that can provide robust protection after a single dose would be highly desirable in a pandemic as it could speed the global vaccine rollout. One dose of ECD with Advax-CpG conferred protection, comparable to protection provided the 2-dose regimen, with similar attenuation of weight loss (5.3% vs. 5.1%), absent lung virus on day 3 and lung severity scores (9 vs 10). However, the single-dose vaccine did use twice the total amount of antigen. Nevertheless, we would caution against overinterpreting this result, as 2 vaccine doses were required for maximum protection in our earlier ferret challenge study [22] , so high levels of single-dose protection may just be a feature of easily protected small rodent models and make not translate to larger animals including monkeys and humans.

The viral load in lungs and nasal turbinate strongly correlated with weight-loss and lung pathology scores. Serum neutralizing antibody levels prior to challenge strongly correlated with reduction in viral load in lung and nasal turbinate, consistent with other studies suggesting that serum neutralizing antibody plays an important role in systemic virus control of coronaviruses [37] .

Interestingly, throat swab viral loads were a poor predictor of disease severity and showed only a low correlation with weight loss or lung scores. Serum neutralizing antibody levels prechallenge similarly only showed a modest negative correlation with throat swab viral loads. This suggests that serum antibody levels may be less effective against mucosal infection.

Nevertheless, it would appear that serum neutralizing antibodies may help restrict the virus to the upper respiratory tract, thereby preventing severe lower respiratory tract infection.

Interfering with viral replication in the nasal mucosa might not only protect the lower respiratory tract against infection, but might also assist in blocking spread of disease and community transmission. All ECD and FL protein immunized groups in the hamster study had a trend towards reduced nasal turbinate viral loads and sum of throat swab viral loads compared to the saline control group, although only the 2-dose ECD with Advax-CpG or alum-CpG adjuvant groups were significantly lower than the control group. Future transmission studies are planned to examine whether the vaccine can prevent virus transmission from a vaccinated animal to a naïve recipient.

How a parenteral vaccine might control mucosal virus replication is not known and may involve trafficking of memory T or B cells to the mucosa or some other mechanism. Notably, in a previous mouse immunogenicity study, the addition of Advax-CpG adjuvant to SARS-CoV-2 spike protein produced a balanced Th1/Th2 response, increased the breadth of serum neutralizing antibody to cover the alpha variant of concern, and induced a strong cellular response characterized by polyfunctional T cells and robust in vivo cytotoxic T lymphocyte activity against spike proteinlabelled target cells [22] . Furthermore, in the ferret challenge model, susceptibility to lung infection was dependent on the number of vaccine doses, with only a 2-dose vaccine schedule, but not the 1-dose schedule, able to prevent lung infection with SARS-CoV-2. Hence, multiple features are likely to contribute to mucosal protection. Human studies all suggest the importance of two or more doses of Covid-19 vaccine for maximal protection, with only modest and shorterlasting protection seen with single dose vaccines [38, 39] .

Potential limitations of this study were the small sample size and the short duration of the virus challenge period, i.e., 3 days prior to termination. However, there was very high consistency of responses within each group and significant differences were apparent in all major parameters including virus load and clinical signs between the vaccine and the control unimmunized groups.

The hamster immunogenicity results were also consistent with the murine immunogenicity results. The termination at day 3 post-challenge was based on findings in previous studies that even unimmunized hamsters show peak SARS-CoV-2 viral titers by day 2 with subsequent rapid clearance by day 4, making later time points not useful for assessing vaccine effects [40] . Another limitation was that similar to other COVID-19 hamster studies [41] [42] [43] [44] , our hamsters were challenged just 2 weeks after the final immunization [45] . Emerging data suggests that immunity induced by adenoviral and mRNA vaccines might rapidly wane over months, leading to loss of protection [46] . We recognize it will be important to undertake future studies to evaluate the long-term durability of Covax-19® protection. While only a homologous SARS-CoV-2 virus was used in this study to perform the infection challenges, future studies will examine the ability to provide durable protection against variant strains.

In summary, we show in mice and hamsters that spike ECD when formulated with Advax-CpG55.2 adjuvant gives high serum neutralization titers, enhances T cell IFNγ responses, and provides robust protection against SARS-CoV-2 infection. The candidate vaccine provided single-dose protection against the homologous virus and was well-tolerated. This data supports the use of spike protein ECD with Advax-CpG adjuvant as a promising Covid-19 vaccine candidate.

We thank Sakshi Piplani for assisting with generating the SARS-CoV-2 spike protein FL and ECD structures shown in Figure 1 . pathology. Correlation between total lung severity score versus viral load of (i) cranial lung, (ii) nasal turbinate and (iii) accumulative and (iv-vi) individual daily throat swabs day 1-3 (n=40, groups indicated in legend). Viral titers are presented as log 10 .

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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FIGURE LEGENDS: Figure 1: Spike protein vaccine immunogenicity in mice and effects of adjuvants. (A) 3D models were produced of SARS-CoV-2 extracellular domain (ECD) and full length (FL) spike protein

BALB/c mice were immunised i.m. twice at 2-week intervals with 0.5-10 µg rSp ECD or FL with alum-CpG or Advax-CpG adjuvant. (C) ELISA results for rSp-and RBD-binding IgG (mean + SD) in mice immunized with 2.5-10µg rSp ECD or FL alone or with alum-CpG adjuvant 2 weeks after first and second immunization. (D) CBA cytokine levels and (E) ELISPOT results for rSp-stimulated splenocytes from mice immunized with 10µg ECD or FL rSp alone or with alum-CpG adjuvant. (F) ELISA results for serum rSp-and RBD-binding IgG (O.D. mean + SD) in BL6 mice immunized with 0

COV-2 spike pseudotyped lentivirus neutralisation titers for sera from BL6 mice immunized twice with 1µg ECD or FL vaccine antigen with Advax-CpG or alum-CpG adjuvant. Statistical analysis was performed using Kruskal-Wallis test

Adjuvanted SARS-CoV-2 ECD protein induces strong neutralizing antibodies in hamsters and reduces clinical disease. (A) Hamsters (n = 4) were vaccinated at day 0 and boosted at day 21 with SARS-CoV-2 extracellular domain (ECD) or Full length (FL) spike protein at either 2.5 or 10 µg alone or formulated with Advax-CpG (2-0.2 mg), Advax (2 mg)

A 1-dose group was administered 10µg of ECD or FL spike protein formulated with Advax

Blood was collected on day 35 and then the animals were challenged nasally with 1e 4 PFU of SARS-CoV

Oropharyngeal swabs were collected each day and on day 3 post-infection animals were sacrificed and nasal turbinates and cranial lobe of lungs were collected to determine SARS-CoV-2 viral loads. (B) Neutralization activity of sera at day 35 was determined by a PRNT90 assay. Results are presented as GMT. (C) Daily weight change normalized as percentage of starting weight. (D) Cumulative weight loss and (E) daily viral titers in throat swabs for 3 days

Multiple T-test and PRNT90, cumulative weight loss and viral titers was performed using Kruskal-Wallis test with Dunn correction for multiple comparisons (*; p < 0.05 and **