key: cord-0988270-3ghhmhnp
authors: McMillan, Christopher L.D.; Choo, Jovin J.Y.; Idris, Adi; Supramaniam, Aroon; Modhiran, Naphak; Amarilla, Alberto A.; Isaacs, Ariel; Cheung, Stacey T.M.; Liang, Benjamin; Bielefeldt-Ohmann, Helle; Azuar, Armira; Acharya, Dhruba; Kelly, Gabrielle; Fernando, Germain J.P.; Landsberg, Michael J.; Khromykh, Alexander A.; Watterson, Daniel; Young, Paul R.; McMillan, Nigel A.J.; Muller, David A.
title: Complete protection by a single dose skin patch delivered SARS-CoV-2 spike vaccine
date: 2021-05-31
journal: bioRxiv
DOI: 10.1101/2021.05.30.446357
sha: 436306056c92a17ad4820cbad4cbab72a50bac46
doc_id: 988270
cord_uid: 3ghhmhnp

SARS-CoV-2 has infected over 160 million people and resulted in more than 3.3 million deaths, and we still face many challenges in the rollout of vaccines. Here, we use the high-density microarray patch to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show the vaccine, dry-coated on the patch is thermostable, and delivery of spike via HD-MAP induced greater cellular and antibody immune responses, with serum able to potently neutralize clinically relevant isolates including those from the B.1.1.7 and B.1.351 lineages. Finally, a single dose of HD-MAP-delivered spike provided complete protection from a lethal virus challenge, demonstrating that HD-MAP delivery of a SARS-CoV-2 vaccine is superior to traditional needle-and-syringe vaccination and has the potential to greatly impact the ongoing COVID-19 pandemic.

greater cellular and antibody immune responses, with serum able to potently neutralize clinically relevant isolates including those from the B.1.1.7 and B.1.351 lineages. Finally, a single dose of HD-MAP-delivered spike provided complete protection from a lethal virus challenge, demonstrating that HD-MAP delivery of a SARS-CoV-2 vaccine is superior to traditional needleand-syringe vaccination and has the potential to greatly impact the ongoing COVID-19 pandemic.

SARS-CoV-2 is a novel betacoronavirus that emerged in 2019 in Wuhan, China (1), before rapidly spreading around the globe causing the COVID-19 pandemic. As of the 19 th of May 2021, SARS-CoV-2 has caused over 163 million infections resulting in over 3.3 million deaths (https://covid19.who.int). The rapid spread of SARS-CoV-2 was accompanied by the rapid 5 development of multiple vaccines. Many vaccine modalities were trialed including mRNA (2) (3) (4) , DNA (5), protein subunit (6, 7) , viral vectored (8) (9) (10) (11) , nanoparticle (12) and inactivated virus (13, 14) , and several have subsequently been granted emergency authorization for use in humans.

However, the emergence of SARS-CoV-2 variants of concern which possess varying degrees of resistance to neutralization by sera from vaccinated individuals is raising concerns of vaccine 10 escape, highlighting the need for further vaccine research and development (15) (16) (17) (18) . Furthermore, some of these vaccines require ultra-low temperature storage and show only limited stability at 2-8 ˚C (19) , making transport and global distribution challenging, especially to resource limited countries. Meeting the demand of vaccine doses for global vaccination coverage in a pandemic has also proven problematic, and dose-sparing methods could prove useful. 15 One such approach to improving vaccine stability, dose-sparing and ease of distribution is the high-density microarray patch (HD-MAP). The HD-MAP is a 1 x 1 cm solid polymer This vaccine modality offers considerable advantages in thermostability and antigenicity (20, 24) with dose-sparing of a large range of vaccines previously observed (25) (26) (27) (28) (29) (30) (31) (32) . Additionally, HD-MAP application avoids the generation of sharps waste and offers advantages in terms of ease of application, potentially negating the need for highly trained healthcare workers (33, 34) .

Here, we explore HD-MAP delivery of a subunit vaccine candidate -a recombinant SARS-CoV- 5 2 spike glycoprotein, termed HexaPro which has been stabilized in its prefusion conformation by removal of the furin cleavage site and the inclusion of six stabilizing proline mutations(35). We found that immunization of mice with spike via HD-MAP application induced significantly higher antibody levels than intradermal needle-and-syringe delivery, with neutralization of virus seen after just one dose. Additionally, immunization of transgenic mice expressing human angiotensin- 10 converting enzyme 2 (ACE2) with spike protein via HD-MAP delivery and adjuvanted with QS21 provided complete protection from a lethal SARS-CoV-2 challenge after just one dose, with no virus replication observed in the lungs or brain of the mice.

HexaPro (35), a stabilized SARS-CoV-2 spike protein containing six proline substitutions and a mutated furin cleavage site (hereafter referred to as spike) was recombinantly expressed using the Expi293-F™ expression system and purified via immunoaffinity chromatography. After initial in vitro characterization to confirm integrity and conformational folding of the protein (Fig. S1 ), we 20 investigated formulation and coating optimization on the HD-MAP (Fig. 1A) . Initial experiments focused on the selection of generally regarded as safe (GRAS) excipients that stabilize the protein in both the dry-down process and storage over time. Excipients tested included sugars, proteins and denaturing agents, which have previously been shown to stabilize vaccines on the HD-MAP (27, 29) . Stability was assessed by ELISA, through the binding of a conformational dependent, spike-reactive antibody S309 (36) . While minimal reduction in antigenicity was seen during the dry down process (measured following immediate reconstitution), storage for 1 or 7 days at 4 ˚C resulted in considerable loss of antigenic reactivity for some excipients (Fig. S2) . However, all 5 formulations containing human serum albumin (HSA) resulted in antigenic stability at all timepoints.

Next, we investigated the stability of spike-HSA formulations at elevated temperatures and to further optimize the concentration of HSA required to confer stability. To achieve this, spike was 10 dried with or without HSA (0.25 -0.75% w/v) and stored at 25 or 40 ˚C and assessed for antigenic stability by ELISA as before. The losses upon storage at both 25 and 40 ˚C ranged between 1 and 13% after 1 week when HSA was included in the formulation, with higher losses in antigenic integrity of more than 20% at 40 ˚C observed with spike alone (Fig. 1B) . Given the minimal differences in stabilization afforded by all HSA concentrations tested, we chose to proceed with 15 the lowest effective concentration of 0.25% for future formulations to minimize the solid mass required to be dried down on the patch projections. The effects of storage over an extended time period was also assessed. After 1 month at 25 ˚C in the presence of 0.25% HSA, full antigenic reactivity was recovered, suggesting excellent stability (Fig. 1C) . While one week of storage at 40 ˚C resulted in minimal antigenic loss, 1 month storage at this elevated temperature resulted in 20 limited antigenic recovery (Fig. 1C) . As the addition of HSA greatly improved antigenic stability at all temperatures tested, HSA at 0.25% was included in all subsequent HD-MAP vaccine formulations.

We next assessed the delivery efficiency of the vaccine from the coated HD-MAP to mouse skin.

When applying spike-coated HD-MAPs to the skin of mice, removal of the vaccine coating can be 5 seen by scanning electron microscopy (SEM) of the patches pre-and post-delivery (Fig. 1D) . We also included the saponin-based adjuvant QS21, based on previous data demonstrating its potent adjuvanting effects and compatibility for coating onto the HD-MAP(29, 37). Efficient removal of vaccines containing QS21 into the skin of mice was also observed. This vaccine removal corresponded to an average delivery of 50-60% of the payload as assessed via ELISA of spike able 10 to be eluted off the patch post-application (Fig. 1E) and was accompanied by visual signs (erythema and petechiae) of HD-MAP engagement with the depilated skin of the animal (Fig. 1F ).

We next sought to assess the immunogenicity of SARS-CoV-2 spike delivered with or without 15 adjuvant, and either via HD-MAP application or intradermal (i.d.) injection using needle-andsyringe (N&S). We chose intradermal, rather than intramuscular vaccination as a more relevant comparator to HD-MAP delivery of vaccines to the immune cell-rich environment of the skin. BALB/c mice were immunized twice at 21-day intervals with 2 µg of spike alone or with the addition of QS21 ( Fig. 2A) . HD-MAPs coated with excipients only were included as controls.

Blood was collected 20 days after each immunization and the serum assessed for spike-specific IgG by ELISA. After one immunization, both unadjuvanted and QS21-adjuvanted spike delivered by HD-MAP induced significantly higher (p < 0.0001) IgG levels compared to their i.d.-delivered controls (Fig. 2B ). This trend was mirrored in the response after 2 doses, with levels approximately 1.6-fold higher after a second immunization in both HD-MAP groups. Promisingly, serum from the unadjuvanted HD-MAP spike group had significantly higher (p < 0.0001) IgG titers compared to QS21-adjuvanted i.d.-delivered spike, highlighting the immune-enhancing attributes of the HD-MAP alone. Serum from day 41 also contained high levels of IgG able to bind to the receptor- 5 binding domain (RBD) and N-terminal domain (NTD) of the spike protein (Fig. S3 ), indicating broad coverage of epitopes known to be bound by neutralizing antibodies (36, (38) (39) (40) . Again, IgG levels were higher in HD-MAP immunized animals compared to their i.d. counterparts. This was especially apparent for NTD-specific IgG levels, where complete seroconversion in both unadjuvanted and adjuvanted groups was seen in HD-MAP groups compared to only partial 10 seroconversion for i.d.-delivered spike (0% for unadjuvanted and 62.5% for adjuvanted).

To investigate the functionality of the induced immune response, we assessed the serum for its ability to neutralize SARS-CoV-2 virus via plaque-reduction neutralization test (PRNT) against an early SARS-CoV-2 isolate, QLD02, matching closely to the original Wuhan-Hu-1 strain. 15 Following one dose, HD-MAP-spike immunized mice had detectable levels of neutralizing antibodies (nAbs) in the serum, which were boosted ~1.7-fold after a second immunization ( An important site of immunity for respiratory infections such as SARS-CoV-2 is at mucosal surfaces (41) of the respiratory tract, and an ideal vaccine would induce immunity at these surfaces. splenocytes, which was significantly higher compared to i.d. delivery of the same formulation (p < 0.0001, Fig. 2F ). With no adjuvant, an average of 65 SFU per 10 6 splenocytes was seen when delivered via HD-MAP, compared to only 6 for i.d. delivery. This is a good initial indication that cellular immunity is induced by HD-MAP immunization, which complements the high IgG titers in the serum and at mucosal sites.

Serum was further analyzed for neutralization of SARS-CoV-2 variants of concern that have recently emerged (15) (16) (17) (18) . These variants, which included an isolate containing the D614G 15 With the observation that HD-MAP immunization induced significantly higher IgG and cellular immune responses relative to N&S i.d. delivery, we next assessed HD-MAP vaccine potency in a SARS-CoV-2 challenge model. Transgenic mice expressing human ACE2 driven by the cytokeratin-18 (K18) promoter (K18-hACE2 mice), have been shown to be an effective model for SARS-CoV-2 infection (47) and were used in this study. Mice (n=8) were immunized via HD-20 MAP with either a single dose or a prime/boost regime at 21-day intervals, with or without QS21 as an adjuvant, or left unimmunized as naïve controls (Fig. 4A) . Blood was taken for analysis of serum IgG levels via ELISA and PRNT prior to challenge, which occurred 21 days after the final immunization. At this time, all immunized mice had seroconverted with high levels of spikespecific IgG, and all had high levels of nAbs against 614D, 614G and B.1.1.7 virus isolates, though nAb levels against the B.1.351 isolate were lower (Fig. 4B, Fig S5) . Mice were then challenged with 10 4 PFU of an early isolate of SARS-CoV-2 (hCoV-19/Australia/VIC01/2020, GISAID Accession ID EPI_ISL_406844, matching to the Wuhan-1 reference virus (48) (47, 49) . The mice receiving a single dose of unadjuvanted spike had detectable virus levels in the brain (log-transformed titer of 6.8 PFU/g), albeit at significantly lower levels (p = 0.0005) than naïve mice, but no detectable virus in the lungs ( Fig. 4F and G) . These mice had however been sacrificed due to signs of infection. Mice in this group that did not show symptoms were not analyzed at this timepoint. Conversely, all other 20 immunization regimes (a single dose of QS21-adjuvanted spike, two doses of adjuvanted spike and two doses of unadjuvanted spike) completely inhibited infection as evidenced by no detectable virus in the lungs at day 6 post-infection ( Fig. 4F and G) . Histopathological analysis of lung and brain tissue revealed mild to moderate perivascular and interstitial leukocyte infiltration in the lungs of naïve and single-dose immunized mice (Fig. S6A) . This was reduced in mice receiving two doses of vaccine. Similar trends were seen in brain tissue, with perivascular cuffing, neutrophil infiltration, neural degeneration and gliosis seen in naïve and single-dose mice, with mice from two dose groups appearing normal (Fig. S6B ).

Here we report the combination of a SARS-CoV-2 spike subunit protein vaccine candidate with the HD-MAP vaccine delivery platform, resulting in a stable and effective vaccine candidate against SARS-CoV-2. With the HD-MAP platform, dry-coated vaccine is delivered directly to the immune-rich epidermis and upper dermal layers of the skin, while simultaneously causing 10 localized cell death and inflammation, both of which serve to enhance vaccine-induced immunity (22, 50) . This platform delivery system offers a number of other benefits, including ease of administration, and potentially self-administration or administration by individuals who are not trained health-care professionals, reduced cold chain dependence, no requirement for reconstitution and improved thermostability. We showed that the dry-coated spike protein was 15 thermostable (as measured by conformational epitope integrity), with stability observed for up to a month at 25 ˚C and a week at 40 ˚C (Fig 1B and C) . Should this measure of stability translate to biophysical stability, this represents a marked improvement over other SARS-CoV-2 vaccine candidates, where limited stability (2-24 hours) at room temperature is seen (19) . This lends itself well to improvements in vaccine transport and delivery to patients, especially in a "last-mile" 20 context where appropriate infrastructure may be limited. This is particularly relevant to low-to-middle income countries (LMICs), where there is an urgent need to facilitate vaccination of populations against SARS-CoV-2 (51, 52).

HD-MAP-delivered spike resulted in a robust neutralizing IgG and cellular immune response in mice, which was superior to delivery of the same vaccine via intradermal needle-and-syringe 5 immunization. This is consistent with previous studies utilizing the HD-MAP where significant improvements in immunogenicity were observed (27, 29, 53, 54) . The inclusion of QS21, which is known to broaden the immune response and enhance cell-mediated immunity (37) (15-18, 45, 46) . These collective findings are promising given the importance of cellular immunity in SARS-CoV-2 disease resolution and protection from emerging 15 viral variants (42, 44) . Additionally, the HD-MAP appears to provide improved immunogenicity and cellular responses to SARS-CoV-2 vaccine candidates when compared to immunization with other lower density microarray patch delivery systems (55, 56) . Taken together, this data suggests HD-MAP vaccination could provide improved cellular immune responses and increased breadth of IgG, both of which would limit the potential for escape from protection by emerging viral variants.

Immunization of K18-hACE2 mice with spike via HD-MAP resulted in complete protection from disease after just a single QS21-adjuvanted dose. The protective immune response resulted in 5 abrogation of virus replication in both the lungs and brain tissue assessed on day 6 post-infection, in contrast to naïve mice which had high levels of replicating virus in these organs ( Fig. 4F and   G ). This is notable as SARS-CoV-2 infection has been shown to infect the brain of humans and mice, causing meningitis and encephalitis (49, 57, 58) . Protection with a single dose would provide significant benefits for rollout of mass vaccination campaigns during a pandemic. Only requiring 10 a single dose to achieve protection would mean fewer doses to be manufactured and more rapid vaccine coverage of populations, in contrast to most current SARS-CoV-2 vaccines which require 2 doses 3-12 weeks apart to achieve protection (59-62). Although some adenovirus-vectored vaccines have been shown to provide single-dose protection from SARS-CoV-2 infection in mice and humans (63, 64) , issues surrounding safety and undesired adverse events have impacted their 15 clinical rollout (65) . Subunit vaccines could offer an improved and more desirable safety profile.

To date, only limited studies investigating subunit-based SARS-CoV-2 vaccines have been performed, and while single-dose protection has been seen, this was achieved with much higher doses than the 2 µg used here. Tian et al used 10 µg of a full-length spike protein vaccine with 20 Matrix-M adjuvant, and saw protection after a single dose, though signs of infection were still observed in mice (6) . Another study utilized a spike nanoparticle vaccine in which multiple spike proteins are assembled on a nanoparticle (66) . Immunization provided protection in hamsters, though this immunity was not complete and required a dose of 100 µg, 50 times higher than required by HD-MAP immunization (66) . Other work using a prefusion-stabilized spike protein (5 µg/dose, adjuvanted with MF59) showed reduced viral load in the lungs and throat swabs of hamsters on day 4 post-infection after one or two immunizations (7), with relatively high levels of replicating virus still recovered from these samples. Additionally, neither regime decreased the 5 viral load in the nasal turbinates compared to naïve hamsters on day 4 or day 8 post-infection (7). This suggests the antibody and/or cellular immunity induced by these vaccination regimes were not high enough to inhibit viral replication in these organs.

In conclusion, this body of work demonstrates the first investigation of SARS-CoV-2 spike 10 vaccination via a microarray patch. HD-MAP spike vaccines are stable, immunogenic, and protective against virus challenge in mice after a single dose. These finding represent a substantial improvement in many areas of SARS-CoV-2 vaccination and offers a promising alternative to currently available vaccines that warrants further investigation in the context of human SARS-CoV-2 infection.

Animal ethics and animals: Female BALB/c and K18-hACE2 transgenic mice were purchased from the Animal Resources Centre (Perth, Australia) and housed in pathogen-free conditions at the Australian Institute for Bioengineering and Nanotechnology or Griffith University. All animal procedures were approved by the University of Queensland and Griffith . This construct has been modified to include a cleavage site substitution as well as six prolines for stability (35). Plasmids encoding heavy and light chains of SARS-CoV-2 S-specific antibodies, as well as spike receptor binding domain and N-terminal domain constructs were constructed inhouse as previously described (7, 67, 68) . 35 Recombinant antibody expression and purification: Plasmids encoding the heavy and light chains of monoclonal antibodies were prepared using the PureYield™ Plasmid Midi-or Maxiprep system and filter sterilized using a 0.22 µm filter. Plasmids were then transfected into ExpiCHO-S™ cells (ThermoFisher Scientific) as per the manufacturer's instructions. Culture 40 supernatant was harvest via centrifugation at 7 days post-transfection, and antibodies purified using Protein A affinity chromatography on an AKTA pure FPLC system (Cytiva) as per the manufacturer's instructions. Purified antibodies were concentrated and buffer exchanged into phosphate-buffered saline (PBS) using a 30 kDa MWCO Amicon Ultra Centrifugal Filter (Merk) and stored at -20 ˚C until use.

Fc-fusion protein expression and purification: Plasmids containing the receptor binding domain (RBD) and N-terminal domain (NTD) of SARS-CoV-2 spike fused to human Fc were prepared using the PureYield™ Plasmid Midi-or Maxi-prep system and filter sterilized using a 0.22 µm filter. Plasmids were then transfected into ExpiCHO-S™ cells (ThermoFisher Scientific) as per the manufacturer's instructions. Culture supernatant was harvested and protein purified via 5 Protein A affinity chromatography as before.

SARS-CoV-2 Spike HexaPro expression and purification: Plasmid DNA for transfection was prepared using Promega PureYield™ Plasmid Maxiprep System and filter-sterilised using a 0.22 µm filter prior to transfection into Expi293-F™ cells (ThermoFisher Scientific), as per the 10 manufacturer's instructions. Culture supernatant was clarified by centrifugation at 5,000 x g for 30 minutes at 4 ˚C before filtration through a 0.22 µm filter. Protein was purified from supernatants as previously described (7), using an in-house made immunoaffinity purification column containing the S-specific mAb 2M-10B11. After purification, proteins were concentrated and buffer exchanged into PBS as before, and stored at -20 ˚C. Protein was assessed for purity SDS-15 PAGE using a 4-12% NuPage SDS gel and size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column. Purified spike protein was also analyzed via ELISA. Briefly, SARS-CoV-2 S protein was coated onto ELISA plates at 2 µg/mL in PBS and incubated at 4 ˚C overnight. The next day, plates were blocked with 5% milk diluent blocking concentrate (Seracare) for 30 minutes at room temperature. Serial dilutions of antibodies were then added to the plates before 20 incubation at 37 ˚C for 1 hour. After washing with PBS with 0.05% tween 20, relevant HRPconjugated secondary antibodies were added (goat anti-mouse or anti-human, diluted 1:2000). After washing, TMB substrate was then added to develop the ELISA prior to stopping the reaction with 1M phosphoric acid. Absorbance was then read at 450 nm using a Varioskan LUX Microplate reader (ThermoFisher).

Stability dry down assay: Spike protein was formulated as desired and added to wells of a 96well tissue culture plate before being placed under a filtered nitrogen gas jet stream at 15 liters per minute for 15 minutes using a MICROVAP microplate evaporator (PM Separations). Plates were then sealed in foil bags containing desiccant and stored for the desired time. To reconstitute, 150 30 µL of 5% Milk diluent blocking concentrate (Seracare) diluted in PBS containing 0.05% tween 20 was added to each well. Reconstitution was achieved by pipetting and incubating for 15 minutes at 37 ˚C with shaking at 125 rpm. Proteins were then analyzed via capture ELISA as above, with samples captured on streptavidin-coated plates and probed with S-specific antibodies.

Negative stain transmission electron microscopy: Purified spike protein was diluted to ~10 µg/mL in PBS and applied to glow-discharged carbon films supported by formvar on 400-mesh copper grids (ProSciTech). Samples were applied for 2 minutes before washing three times with water. The grids were then stained with 1% uranyl acetate and air dried before imaging on a Hitachi HT7700 transmission electron microscope at 120 kV and 30k magnification. 40 High density microarray patch coating and application: High density microarray patches (HD-MAPs) were kindly provided by Vaxxas Pty Ltd (Brisbane, Australia). Prior to vaccine coating, HD-MAPs were cleaned via oxygen plasma treatment for 5 minutes at 30W power at the Australian national Fabrication Facility, Queensland Node. A vaccine coating solution consisting of various amounts of SARS-CoV-2 HexaPro spike protein, 0.25% human serum albumin (HSA), 0.75% methylcellulose, and varying amounts of the adjuvant QS21 (Desert King) was then applied to the patch. The solution was then dried using a sterile-filtered nitrogen gas stream as previously described (54) . Vaccine-coated HD-MAPs were then applied to the flank of mice using who had previously had the hair at the vaccination site removed via shaving and depilatory cream. The HD-MAPs were applied at a velocity of 18-20 m/s using a custom applicator.

Scanning electron microscopy of HD-MAPs: HD-MAPs were coated with 15 nm of platinum 5 and imaged by scanning electron microscopy (SEM) using a Hitachi SU3500 at the Centre for Microscopy and Microanalysis at the University of Queensland. Samples were at a 45 ˚C angle.

Immunization of BALB/c mice: Naïve 6-8 week old female BALB/c mice were randomly divided into 6 groups of 8 mice and vaccinated either via HD-MAP application or intradermal 10 injection with 2 µg of SARS-CoV-2 S HexaPro, 2 µg of SARS-CoV-2 S HexaPro with the adjuvant QS21 (Desert King), or vehicle only control. All mice were immunized twice at 21-day intervals. Blood was taken prior to each immunization and 21 days after the final immunization via tail bleed or cardiac puncture. A bronchoalveolar lavage (BAL) was also performed at the time of cardiac puncture. The blood was allowed to clot overnight at 4 ˚C before the serum harvested 15 by centrifugation at 10,000 x g for 10 minutes at 4 ˚C. Samples were stored at -20 ˚C until further analysis. The BAL fluid was centrifuged at 1000 x g for 5 minutes to remove debris and the supernatant harvested and stored at -80 ˚C until further analysis.

ELISpot: Spleens from mice were collected and processed into single-cell suspensions in 20 RPMI1640 media supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin (R10 media). Red blood cells were lysed using eBioscience™ RBC lysis buffer (ThermoFisher) and resuspended in R10 media to stop the lysis. Splenocytes were counted and 100,000 cells/well added to the plates of the Mouse IFN-g ELISpot PLUS (HRP) kit (MABTECH). Cells were then stimulated for 18 hours at 37 ˚C with SARS-CoV-2 S PepTivator 25 peptides (Miltenyi Biotec) or media alone as a negative control. Spots were developed as per the manufacturer's instructions. Spots were quantified by eye and plotted as spot-forming units (SFU) per million cells.

K18-hACE2 mouse challenge model: Female K18-hACE2 mice (6-8 weeks old) were 30 immunized via HD-MAP application as before or left unimmunized as naïve controls. Mice received 1 or 2 immunizations, 21 days apart, and were challenged with 1 x 10 4 PFU of SARS-CoV-2 (VIC01 isolate) 21 days after the final immunization via intranasal inoculation (20 µL total volume) while under isoflurane anesthesia. Blood was taken prior to each immunization and prior to challenge. Mice were monitored daily for weight loss and disease severity and culled when 35 weight loss surpassed 15% of pre-challenge body weight. After culling, lung and brain tissue were harvested for viral titer assessment immunoplaque assay. A lung lobe and brain hemisphere from each animal was fixed in 10% neutral buffered formalin and processed for paraffin embedding and hematoxylin-eosin stained sections were examined by a veterinary pathologist blinded to the treatments. 40 Serum ELISAs: Serum from mouse immunizations was assessed for SARS-CoV-2 S-specific IgG levels via ELISA as previously described (7) . Briefly, SARS-CoV-2 spike protein was coated onto ELISA plates at 2 µg/mL in PBS overnight at 4 ˚C. Plates were blocked and serial dilutions of serum were added to the plates for 1 hour at 37 ˚C. Serum binding was detected using an HRP-45 linked goat anti-mouse secondary antibody and TMB substrate as discussed previously.

Plaque reduction neutralization tests: Serum and BAL fluid was assessed for SARS-CoV-2 neutralization activity via plaque reduction neutralization test (PRNT) as previously described (67). Briefly, heat-inactivated serum or BAL fluid were serially diluted in DMEM supplemented with 2% FBS and penicillin/streptomycin (Gibco) before virus was added. Samples were incubated for 30 minutes at 37 ˚C before being added to confluent VeroE6 monolayers in 96-well plates. 5 Infection was allowed to proceed for 1 hour at 37 ˚C before virus inoculum removed and overlay medium added. Cells were fixed with 80% acetone 14-16 hours post-infection and allowed to dry prior to plaque visualization with SARS-CoV-2 spike-specific mAbs CR3022 or S309 and IRDye 800CW-conjugated goat anti-human secondary antibodies. Plates were scanned using an Odyssey CLx imaging system (LI-COR). 10 Assessment of viral titer in mouse organs: Viral titer in the lungs of mice was determined via immunoplaque assay as previously described (67). Briefly, lung and brain tissues were homogenized on a Bead Ruptor 24 Elite (Omni International, Kennesaw, GA) and centrifuged at 12,000 x g for 7.5 minutes at 4 ˚C before performing serial dilutions, which were added to VeroE6 15 cells in a 96-well tissue culture plates. Plates were fixed and plaques visualized via immunostaining.

Statistical analysis: GraphPad Prism 9 software was used for statistical analysis and generation of graphs and figures. Data are presented as mean with standard deviation or standard 20 error in the mean, as described in the figure legends. Statistical significance was determined via one-way ANOVA with Tukey's multiple comparison test, with p < 0.05 considered statistically significant.

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Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222)

SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Interim Results of a Phase 1-2a Trial of Ad26.COV2.S Covid-19 Vaccine

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower 10 Respiratory Tracts against SARS-CoV-2

Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination

One dose of COVID-19 nanoparticle vaccine REVC-128 provides protection against SARS-CoV-2 challenge at two weeks post immunization. bioRxiv

We would like to thank the Queensland Health Forensic and Scientific Services, Queensland Department of Health, as well as the Peter Doherty Institute for Infection and Immunity and 20 Melbourne Health, Victoria, Australia, for providing SARS-CoV-2 isolates. We thank A/Prof. Keith Chappell, Dr Noushin Jaberolansar and Dr Andrew Young from the University of Queensland for providing technical advice and reagents for antigen purification. We thank the staff at the University of Queensland Biological Resources facility for providing technical assistance. We acknowledge the facilities, and the scientific and technical assistance, of the Australian