Efficacy of a novel antigen-decorated adenoviral vaccine platform against porcine respiratory coronavirus infection in a large natural host

Efficacy of a novel antigen-decorated adenoviral vaccine platform against porcine respiratory coronavirus infection in a large natural host | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Efficacy of a novel antigen-decorated adenoviral vaccine platform against porcine respiratory coronavirus infection in a large natural host Sanis Wongborphid, Emily J Briggs, Rebecca A. Russell, Shafiyeel Chowdhury, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7978161/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Porcine respiratory coronavirus (PRCV) infection in pigs provides a physiologically and immunologically relevant large-animal model for acute respiratory coronavirus disease and vaccine evaluation. We investigated a replication-defective adenovirus (Ad) vaccine platform that enables display of antigens on the Ad capsid surface using the DogTag/DogCatcher protein superglue system. Ad vectors encoding the PRCV135 Spike (S) and Nucleocapsid (N) proteins were evaluated with or without surface decoration with the PRCV135 Spike receptor-binding domain (RBD). Both Ad(S-N) and RBD-decorated Ad(S-N)-RBD135 vaccines were protective against PRCV135 challenge. RBD135 decoration significantly enhanced neutralizing antibody titers in serum and bronchoalveolar lavage. In contrast, aerosol immunization with Ad(N) induced robust T cell responses but no protection. A multivalent cocktail of RBD-decorated Ad vectors targeting PRCV, porcine hemagglutinating encephalomyelitis virus (PHEV), and porcine deltacoronavirus (PDCoV) elicited antibodies against all three pathogens. This study demonstrates the versatility and potency of antigen-decorated Ad vectors as a platform for next-generation coronavirus vaccines in a relevant large natural host model. 158 words Biological sciences/Biotechnology Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Porcine respiratory coronavirus pig nucleocapsid aerosol DogTag/DogCatcher adeno viral vector spike nucleocapsid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Respiratory coronaviruses (CoV) are a significant global health threat and have been responsible for three major outbreaks of severe respiratory disease in humans, most notably the SARS-CoV-2 pandemic. Each of these events has been linked to zoonotic transmission from animals to humans 1 , 2 . Livestock species, including pigs, are also susceptible to CoV infections causing significant economic losses 3 , 4 . In addition to the major impact on pig health of multiple emerging CoVs, pigs may act as a conduit for future zoonotic transmission to humans as they do for influenza A viruses. Several CoVs that infect pigs have been identified 5 . These include members of the Alphacoronavirus genus: Transmissible Gastroenteritis Virus (TGEV), its mutant variant Porcine Respiratory Coronavirus (PRCV), Porcine Epidemic Diarrhoea Virus (PEDV) and Swine Acute Diarrhoea Syndrome Coronavirus (SADS-CoV). Porcine Hemagglutinating Encephalomyelitis Virus (PHEV) belongs to the Betacoronavirus genus, and Porcine Deltacoronavirus to the Deltacoronavirus genus 6 . PDCoV is believed to have originated from an avian Deltacoronavirus that acquired the ability to infect pigs, raising concerns that further mutations could enable PDCoV to infect humans 7 . Indeed, PDCoV has been detected in human plasma samples from Haitian children suffering from acute febrile illness, most likely resulting from zoonotic spill over from pigs 8 . The genetic diversity, complex evolutionary history, and high mutation rates of porcine CoVs together with the emergence of novel strains with substantial impact on both animal health and potential human health risks, highlight the urgent need for improved control strategies. There is no cross-protective immunity among the different porcine CoVs (with the exception of TGEV and PRCV), emphasising the need for either virus-specific vaccines or broadly protective platforms 5 , 7 . A similar challenge exists in humans, where ongoing evolution of SARS-CoV-2 enables partial escape from humoral immunity. Novel vaccine strategies that incorporate conserved internal proteins delivered to the respiratory tract may promote cross-reactive T cell immunity, however it remains to be determined whether this is protective 9 . Alternatively, delivery of multiple Spike antigens from different strains may offer the potential to elicit broad neutralizing antibody responses or protective antibodies against each component of the vaccine. We and others have performed a detailed analysis of PRCV pathogenesis and immune responses following infection in pigs with strains of differing pathogenicity, PRCV135 and ISU-1 10–12 . The interstitial pneumonia and lung pathology in PRCV-infected pigs closely resemble those observed in human SARS-CoV patients 10 , 11 . These findings support the use of the PRCV pig system as a robust large natural host model for testing novel vaccines and therapeutics, as well as for studying immune responses to respiratory CoV 12 . Replication-defective recombinant adenovirus (Ad) is an efficacious, highly scalable, and rapidly deployable vaccine technology. However, while conventional Ad vaccines induce potent T cell immunity (particularly CD8 + T cells), induction of humoral immunity is typically modest compared to other technologies including protein nanoparticles and messenger RNA (mRNA), particularly after homologous boosting 13 . Here we employed the PRCV pig model to assess the ability of a novel replication-defective, Ad based vaccine platform to prevent or reduce lung pathology and viral load. In this platform the Ad capsid surface can be covalently decorated with vaccine antigens using a protein superglue DogTag/DogCatcher, similar to the widely used SpyTag/SpyCatcher ligation system 14 , 15 . In mice, Ad decorated with the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) induced > 10-fold higher SARS-CoV-2 neutralization titers compared to a conventional undecorated Ad encoding S after homologous prime-boost regimens 15 . Importantly, decorated Ad achieved equivalent or superior T cell immunogenicity against encoded antigens compared to undecorated Ad. Capsid decoration has the added benefit of shielding Ad particles from anti-Ad neutralizing antibodies and other undesirable capsid interactors 15 . Using this system, the protective efficacy of Ad vectors encoding the PRCV135 Spike (S) and Nucleocapsid (N) proteins and decorated with the PRCV135 Spike receptor-binding domain (RBD), was compared to vectors encoding only the S and N proteins without decoration. This allowed us to assess whether capsid decoration enhances immunogenicity and protection. In parallel, the ability of an Ad vector encoding the PRCV135 N protein to induce T cell-mediated protection following aerosol delivery to the respiratory tract was investigated. Finally, we examined whether a multivalent “cocktail” of three Ad vectors, each decorated with the RBD of either PRCV135, PHEV, or PDCoV, could elicit cross-reactive antibody responses in this large, natural host model. Results Generation of PRCV135 vaccines and immunogenicity in mice. To assess the effect of decoration of the Ad viral vector with RBD (RBD135) on immune responses against S and N the following vaccine constructs were generated: i) Ad(S-N), an undecorated Ad encoding full-length PRCV135 S and N within the adenoviral genome and ii) Ad(S-N)-RBD135, an Ad encoding S and N and decorated with RBD135 (via covalent attachment of RBD135 to the capsid surface). SDS-PAGE analysis of decorated virions demonstrated a high degree of RBD135 coverage on the Ad capsid; ~70% of total Ad hexon protein was covalently attached to RBD135 via DogTag/DogCatcher ( Suppl. Fig. S1 ). In addition, to test whether expression of encoded N protein alone induces protective immune responses we generated Ad(N), an undecorated Ad encoding only N. Mice were immunized intramuscularly (IM) with 5 × 10⁷ infectious units (IU) of each vaccine and received a homologous boost three weeks later. Two weeks after the boost, mice were culled for immunological analysis (Fig. 1 A). Antibody responses were assessed by endpoint ELISA using recombinant RBD135 protein. Capsid decoration with RBD in Ad(S-N)-RBD135 significantly enhanced anti-RBD antibody titers compared to Ad(S-N) (Fig. 1 B). Cellular responses were measured by IFNg ELISpot using splenocytes stimulated with overlapping peptide pools spanning the S and N proteins included in the vaccines. Ad(S-N)-RBD135 immunization significantly enhanced responses to the S3 peptide pool, while responses to S1 were significantly reduced. The total summed S responses were slightly reduced after Ad(S-N)-RBD135 vaccination compared to Ad(S-N) although this trend did not reach statistical significance (Fig. 1 C). All three vaccines encoding N, Ad(S-N), Ad(S-N)-RBD135, and Ad(N), induced comparable N-specific IFNg ELISpot responses. Antibody responses to N were not measured. These data indicated that all vaccines were immunogenic and that surface presentation of RBD135 significantly increased antibody responses. Protection following prime-boost immunization in PRCV pig model. We next evaluated the contribution of RBD135, S and N antigens in the induction of protective immune responses, in the large natural host PRCV pig model. Twenty pigs were randomly assigned into four groups (n = 5 per group). Three groups were immunized intramuscularly with either Ad(S-N), Ad(S-N)-RBD135, or an undecorated Ad encoding GFP, Ad(GFP), as a control vaccine (Fig. 2 A). The fourth group was immunized by aerosol with Ad(N) using a vibrating mesh nebulizer ensuring distribution to the whole respiratory tract as previously described 16 . All animals were given a homologous booster immunization using the same route on day 25. Twenty-four days post-boost, pigs were intranasally infected with PRCV135. Nasal swabs were collected daily to monitor viral shedding. Four days after the viral challenge, all animals were humanely culled, and tissues were collected for virological, pathological, and immunological analyses. Pigs in the control Ad(GFP) group exhibited significant gross and histopathological lung lesions, with N protein immunohistochemical (IHC) staining in airway epithelial cells and evidence of inflammatory infiltrates in both the airways and lung parenchyma (Figs. 2 B–E). Pigs in the aerosol Ad(N) group showed comparable levels of gross and histopathological changes to the control group. In contrast, both the Ad(S-N) and Ad(S-N)-RBD135 intramuscularly immunized animals demonstrated markedly reduced gross pathology, with largely absent histopathological lesions (Figs. 2 D, E). Representative images of gross pathology, histopathology, and IHC staining (including brown N protein immunolabeling) in lung sections are shown in Fig. 2 F. Viral shedding was analyzed in nasal swabs by plaque assay (Fig. 3 A). There was a significant decrease in viral shedding after PRCV infection in both Ad(S-N) and Ad(S-N)-RBD135 immunized animals as assessed by the area under the curve. No virus was detected in tracheal swabs, BAL and lungs at 4 days post infection (dpi) of the Ad(S-N)-RBD135 group except for one animal in trachea and BAL (Figs. 3 B-E). Viral load in BAL and tracheal swabs was also significantly reduced in the Ad(S-N) group compared to control. Ad(S-N)-RBD135 immunization significantly reduced viral load in deep nasal swabs collected at postmortem (53 days post-prime compared to Ad(S-N)) (Fig. 3 B). No virus was detected in the lungs of 4 out of the 5 Ad(N) immunized animals as detected by plaque assays. These results indicate that immunization with either Ad(S-N) or Ad(S-N)-RBD135 is highly efficient in reducing viral shedding and viral load in tissues as well as eliminating lung pathology following PRCV135 virus challenge. In most measures of viral load Ad(S-N)-RBD135 showed the greatest reduction compared to controls, but there was no statistically significant difference between Ad(S-N) and Ad(S-N)-RBD135 immunized animals except for the viral load in deep nasal swabs at postmortem. Aerosol immunization with Ad(N) did not reduce lung pathology or viral load following PRCV challenge. Antibody responses following prime boost immunization and infection in pigs. Circulating antibody responses were measured by ELISA in serum at regular intervals throughout the study and in BAL at 53 days post prime against full-length recombinant Spike (ISU-1 strain) and N proteins (Figs. 4 A-C). Intramuscular immunization with Ad(S-N) or Ad(S-N)-RBD135 induced significantly higher serum Spike-specific IgG titers than control or Ad(N) groups. IgG levels also increased post-boost and remained elevated, at 49 and 53 days post prime. Serum IgA titers were lower than IgG; they declined after priming, but were boosted after the second immunization, followed by decline and stabilizing at 49 and 53 days post prime. The Ad(S-N)-RBD135 group exhibited significantly higher IgG and IgA responses compared to Ad(S-N) (Figs. 4 A, B ). We also assayed the responses to recombinant RBD135, which as expected were lower to those observed against full length Spike ( Suppl. Fig S2 ). In BAL, a similar pattern was observed, with Ad(S-N)-RBD135 and Ad(S-N) immunization inducing the highest S-specific IgG responses compared to control and Ad(N) groups (Fig. 4 C). Notably, no IgA responses were detected in BAL following IM immunization, suggesting that high serum S-specific IgG may transudate into the lung, while local delivery may be required to induce mucosal IgA. Serum neutralization mirrored the ELISA results, with Ad(S-N)-RBD135 showing higher 50% inhibition titers compared to Ad (S-N) as determined by the area under the curve (AUC) (Fig. 4 D). Only 2 out of the 5 Ad(S-N)-RBD135 immunized animals exhibited neutralizing activity in BAL. This may be due to the dilution of BAL fluid during the sampling process. Serum from Ad(S-N)-RBD135 and Ad(S-N) immunized animals also cross-neutralized multiple PRCV strains (PRCV137, ISU-1, 310) and TGEV, with Ad(S-N)-RBD135 consistently showing significantly higher neutralizing titers compared to Ad(S-N) (Fig. 4 F). The reduction in histopathology and viral load in nasal swabs, tracheal swabs and BAL correlated with neutralizing and Spike-specific IgG titers in serum and BAL (Fig. 4 G). N-specific IgG titers were detected in the serum of one or two animals from the Ad(S-N), Ad(S-N)-RBD135, and Ad(N) immunized groups, but at very low levels compared to S-specific responses. In BAL, responses were detected also in only one or two animals from each group (data not shown). However it is clear that no neutralizing antibodies were induced by Ad(N) immunization. In summary, IM prime boost immunization with Ad(S-N) and Ad(S-N)-RBD135 induced S-specific IgG antibodies in serum and BAL, with IgA titers detected in serum only, but not BAL. Decoration of the Ad virus vector with RBD135 increased significantly the neutralizing titers against a range of PRCV strains and TGEV compared to Ad(S-N) vaccine. All immunization regimes induced N- specific IgG antibodies in serum and BAL, although at a much lower level compared to S-specific responses Cytokine responses following immunization and PRCV challenge in pigs. IFNg ELISpot analysis was performed to quantify IFNg producing cells in PBMC, BAL and tracheobronchial lymph nodes (TBLN), following stimulation with either live PRCV135 or peptide pools covering the S and N proteins (Figs. 5 , 6 ) . Intramuscular immunization with Ad(S-N) and Ad(S-N)-RBD135 induced significantly greater S-specific IFNg ELISpot responses in PBMC compared to the control Ad(GFP) and Ad(N) groups, with a clear boosting effect following the second immunization (Fig. 5 A and Suppl Fig S3) . All three vaccines, Ad(S-N), Ad(S-N)-RBD135, and Ad(N), induced comparable N- and PRCV- specific IFNg ELISpot responses (Figs. 5 B, C). Importantly Ad(N) although given by AE induced both N- and PRCV- specific responses in blood. Responses to S, N and PRCV were significantly greater in the immunized groups compared to control Ad(GFP) as evaluated by AUC (Figs. 5 D,E,F). T cell responses were also analyzed by intracellular cytokine staining (ICS) in PBMC (Figs. 5 G-J). IFNg and TNF production by CD4 + and CD8β + T cells was measured following PRCV135 virus or S and N peptide stimulation (gating strategy shown in Suppl. Fig. S4 ). Ad(S-N) induced significantly greater PRCV specific IFNg producing CD4 + cells compared to the control (Fig. 5 G), while Ad(S-N)-RBD135 immunization induced significantly higher S- specific IFNg and TNF CD8 T cell responses compared to the control group (Figs. 5 I, J). The blood S-specific IFNg ELISpot responses were associated with reduced histopathology (Fig. 5 K). Ad(S-N) and Ad(S-N)-RBD135 groups exhibited S-specific IFNg ELISpot responses in BAL (Figs. 6 A-D). Ad(S-N), Ad(S-N)-RBD135 and Ad(N) elicited significant N- and PRCV-specific responses compared to the control (Figs. 6 E, F). ICS revealed a significantly higher proportion of N- specific IFNg and TNF cytokine producing cells in the Ad(N) group compared to the other immunization regimes. (Fig. 6 G-J). The BAL S-specific IFNg ELISpot responses correlated with reduced lung gross and histopathology and BAL viral load (Fig. 6 K ). IFNg ELISPOT cytokine responses were also detected in the TBLN, which drain the site of PRCV infection ( Suppl Figs S5 A-F ). Significantly higher responses to the S and peptide pool antigens were induced in the Ad(S-N) immunized animals compared to the Ad(GFP) control. In contrast, no statistically significant increase in responses was observed in the Ad(S-N)- RBD135- or Ad(N)-immunized animals versus controls. Greater proportions of S-specific IFNg and TNF CD8 producing cells compared to the Ad(GFP) controls were also detected in Ad(S-N) immunized animals by ICS ( Suppl Figs S5 G-J ). These differences may reflect distinct antigen presentation pathways and tissue tropism of the vaccine vectors. These data indicate that intramuscular prime-boost immunization with Ad(S-N) or Ad(S-N)-RBD135 induced S-specific cellular responses, including IFNg ELISpot and IFNg and TNF production in PBMCs, BAL and TBLN. All three vaccine regimes, Ad(S-N), Ad(S-N)-RBD135 and Ad(N), elicited N-specific responses, with Ad(N) producing the most robust CD8 + IFNg and TNF cytokine producing CD4 and CD8 T cells in BAL. Blood and BAL and TBLN S- specific IFNg ELISpot responses were associated with reduced gross and histopathology following PRCV135 challenge. Immunogenicity of multivalent “cocktail” in mice and pigs . Having shown that antibody responses to the PRCV135 S correlate with protection, we next wished to determine whether a cocktail of four Ad vaccine constructs, each decorated with RBDs from either PRCV135, PEHV, PDCoV, or PEDV could increase immune breadth and induce robust cross-reactive antibody and T cell responses. We used the same design as before, with vaccines encoding S-N from PRCV135 in the Ad genome and decorated with RBDs from either PRCV135, PHEV, PDCoV or PEDV: Ad(S-N)-RBD135, Ad(S-N)-RBD-PHEV, Ad(S-N)-RBD-PDCoV, and Ad(S-N)-RBD-PEDV respectively. Mice were immunized intramuscularly with either Ad decorated with only PRCV135 (Ad(S-N)-RBD135) or a cocktail containing equal IU of the four different RBD decorated Ads (cocktail 4). Animals received two vaccine doses three weeks apart and were culled two weeks post boost (Fig. 7 A). Serum IgG antibody responses against each of the RBDs present in the cocktail vaccine 35 days post prime were assessed by endpoint ELISA (Fig. 7 B). Robust and comparable responses against RBD135 were observed after administration of both vaccines. IgG responses against PHEV and PDCoV were only observed after administration of the cocktail vaccine. A detectable response against PEDV was achieved in only one animal in the cocktail vaccine group. Splenic T cell responses against PRCV135 S and N antigens were measured by IFNg ELISpot (Fig. 7 C). Responses against S and N antigens were induced in both vaccine groups, with comparable magnitude between the groups. Taken together, the data confirmed an added benefit of including PHEV and PDCoV RBD decorated Ads as components of a multivalent cocktail vaccine. Due to the failure of PEDV RBD to induce detectable IgG responses in mice, this component of the cocktail was excluded for subsequent studies in pigs. Pigs were randomly assigned to two groups (n = 4 per group). One group was immunized intramuscularly with Ad(S-N)-RBD135, and the other with a cocktail containing equal IU of the three different RBD decorated Ads: Ad(S-N)-RBD135, Ad(S-N)-RBD-PHEV and Ad(S-N)-RBD-PDCoV (cocktail 3) (Fig. 8 A). All animals received a homologous booster immunization on day 28. Serum samples were collected weekly, and IgG antibody responses against each RBD in the cocktail were evaluated by endpoint ELISA. RBD-135-specific IgG were detected following administration of Ad(S-N)-RBD135 and cocktail 3 vaccines (Fig. 8 B), whereas PHEV- and PDCoV-specific responses were observed only following cocktail 3 immunization. RBD-135 responses following cocktail 3 immunization were comparable to those observed in the initial challenge study and exhibited neutralizing activity (Fig. 4 A and Fig. 8 C). Interestingly, responses to Ad(S-N)-RBD-135 were also detected in ELISA with rec PHEV or PDCoV, suggesting the induction of cross-reactive antibodies. In summary, intramuscular immunization with a cocktail of three Ad constructs displaying RBDs from PRCV135, PHEV, PDCoV induced serum IgG responses in mice and pigs. The RBD-135 responses were comparable to those elicited by immunization with the single RBD, indicating that this is a viable strategy for inducing antibodies against multiple antigens. Discussion We previously described a novel Ad-based vaccine platform utilizing the DogTag/DogCatcher protein superglue system to facilitate rapid, modular covalent decoration of Ad capsids with vaccine antigens 15 . Here, for the first time, we evaluated the immunogenicity and efficacy of an antigen decorated Ad vector vaccine in a large, natural host species. Both Ad(S-N) and the antigen decorated Ad(S-N)-RBD135 vaccines were highly efficacious, almost completely abrogating lung pathology and significantly reducing viral load following PRCV135 virus challenge. The Ad(S-N)-RBD135 immunized animals had lower viral loads in the upper respiratory tract, particularly evident from deep nasal swabs, suggesting that Ad(S-N)-RBD135 may offer improved protection against viral transmission compared to Ad(S-N), although this remains to be tested. Importantly, decoration with RBD135 significantly enhanced anti-PRCV135 neutralizing antibody responses, both in serum and BAL. Vaccine induced S-specific cytokine T cell responses were not significantly different to Ad(S-N) responses in pigs and the N-specific responses were comparable between the decorated Ad(S-N)-RBD135, undecorated Ad(S-N), and Ad(N) vaccines. Current COVID-19 vaccines, which are primarily based on the spike protein, reduce the severity of lung disease, but virus escape from humoral immunity occurs. It is therefore important to assess whether including additional antigens that induce T cell immunity could provide broader protection against antibody escape variants. Human studies suggest that T cell responses to the N protein correlate with less severe COVID-19 disease 17 , 18 . Furthermore mucosal respiratory immunization is considered to be more effective in inducing local immune responses at the site of infection 9 , 19 . Here we evaluated whether responses to the N could protect against disease. Following Ad(N) delivery to the respiratory tract by aerosol, BAL and TBLN cytokine producing cells were induced, with dominant CD8 + T cell responses. Antigen-specific cytokine producing cells were detected in blood at levels comparable to those in animals given Ad(S-N) and Ad(S-N)-RBD135 vaccines parenterally. Antibody responses against N were detected in one or two animals from the Ad(S-N) and Ad(S-N)-RBD135 and Ad(N) AE group, although at much lower titers compared to S. However, despite these local and systemic immune responses, no protection to PRCV challenge was observed - there was no reduction in viral load or lung pathology after aerosol delivery of the Ad(N) vaccine. This is in agreement with other pre-clinical studies. Immunization via the respiratory tract with recombinant parainfluenza virus expressing N did not confer protection in hamsters, while immunization with S provided complete protection against SARS-CoV challenge in the lower respiratory tract 20 . In mouse and hamster models, N-only mRNA immunization offered only modest protection, whereas combined S + N mRNA vaccination provided much stronger protection 21 . Similarly, Ad vectored N vaccine did not reduce viral load in lung or brain in K18-hACE2 mice but did so in combination with S 22 . In chickens, immunization with recombinant N protein elicited cellular immune responses but no protection, whereas S1 was protective 23 , 24 . In contrast recombinant N-protein vaccine (OVX033) induced cross-reactive T cells and protected Syrian hamsters against several variants 25 . An N-based vaccine Convacell has been licensed for human use in Russia. This squalene adjuvanted recombinant N-protein vaccine induced strong IgG and Th1/Th2 cytokine responses in mice and rabbits, T-cell responses in marmosets, and was safe/immunogenic in hamsters and non-human primates 26 . Immunized Syrian hamsters showed reduced lung histopathology, lower virus proliferation, lower relative lung weight and faster body weight recovery 27 . Several N-based vaccine prototypes are also in clinical trials but published data on their efficacy and safety remain limited 26 . Since the emergence and rapid spread of PRCV, most pigs have developed immunity to both PRCV and TGEV. In contrast, PDCoV and PEDV are rapidly expanding their geographic range 5 , 28 . PHEV, the only beta coronavirus of pigs, has a worldwide prevalence, causing severe vomiting and wasting and/or encephalomyelitis. With TGEV now rare in much of the world, fewer vaccines are available in North America and Europe, whereas parts of Asia continue to use them due to ongoing outbreaks. Vaccines for PEDV have been developed and are widely used, although continued surveillance is required due to antigenic variation 28 . No licensed PHEV or PDCoV vaccines currently exist, making the development of effective vaccines increasingly urgent. Since antibodies targeting the spike protein of PRCV135 were effective against PRCV, we evaluated the potential of a multivalent vaccine cocktail expressing the RBDs from PRCV, PDCoV, and PHEV. Multivalent display of RBD domains from diverse sarbecoviruses as a strategy to increase immune breadth has been described previously using protein nanoparticles including those based on ferritin and lumazine synthase 29 , 30 . A similar approach displaying receptor binding domains from diverse influenza A haemagglutinin proteins has also been used to design broadly neutralizing influenza vaccines 31 . In these studies, multiple different RBDs have been attached to each nanoparticle in a ‘mosaic’ design. Some studies have demonstrated that this mosaic design leads to preferential induction of antibodies that bind to conserved regions of the RBD and therefore have greater capacity to cross-react with different viral strains 29 . However, this approach presents some challenges; assessing the abundance of different RBDs on mosaic particles typically requires the availability of highly specific monoclonal antibodies for each RBD which might not be possible for RBDs from related sarbecoviruses or antigen variants. Due to subtle differences in size and structure, it is possible that some RBDs may bind preferentially and thus be overrepresented on particles compared to others. In a recent study comparing mosaic RBD nanoparticles with cocktails of homotypic RBD nanoparticles, both approaches delivered comparable immune breadth 30 . In the current study, we delivered three separate RBD Ad vaccine constructs in an equimolar cocktail to simplify quality control processes and maximise translatability. Our approach enabled particulate delivery of multiple RBDs to achieve potent humoral immunity against different porcine CoV similar to protein nanoparticle technologies. Given its established utility in evaluating vaccine candidates for influenza and SARS-CoV-2, the PRCV pig model provides a robust translational tool for assessing vaccine efficacy in a large, natural host species. The RBD-decorated Ad vaccine platform evaluated here demonstrated strong immunogenicity, inducing robust S-specific antibody and T cell responses correlating with protection as determined by reduction in viral load and lung pathology. Furthermore, delivery of a cocktail of multiple vaccine constructs, each decorated with RBDs from different CoV, induced strong serum antibody responses. These results provide valuable insights into the development of CoV vaccines in a highly relevant large animal model to inform future vaccine design for both livestock and human health. Materials and Methods Viruses, and cells. PRCV strains 86/135308 (135) and 86/137004 (137) were gifts from S. Cartwright (Animal and Plant Health Agency). 135 and 137 were both isolated from respiratory tract tissue in the UK in 1986 32 . AR310 was purchased from ATCC (ATCC VR2384). ISU-1 was purchased from Bei Resources (NR-43286). TGEV strain FS772/70 was a gift from S. Cartwright 33 . All cells and viruses were propagated as previously described 11 . Construction of Ad-DogTag vaccine vectors encoding PRCV135 S and N genes . A replication defective (E1/E3 deleted) molecular clone of Ad5 with DogTag (DIPATYEFTDGKHYITNEPIPPK) flanked by GSGGSG linkers inserted into the hexon HVR5 loop (pBAC-Ad5(GFP)DogTag) was previously described 15 . Recombinant vectors expressing PRCV135 S and N (Accession number OM830318), codon optimized for mammalian expression, were generated through cloning of gene constructs into pENTR4.CMVp and subsequent Gateway-mediated insertion into the Ad5 E1 locus of pBAC-Ad5(GFP)DogTag using Invitrogen Gateway site-specific recombination technology, replacing the green fluorescent protein (GFP) gene with the PRCV135 S and N genes. Virus was rescued and purified as described 15 . HEK293A (293A) cells (Invitrogen) were used for Ad vectors expressing GFP and 293TREX cells (Invitrogen) were used for Ad vectors expressing PRCV135 S and N genes. Ads were titered as previously described 15 . Protein production and purification . DNA sequences for expression of porcine coronavirus RBD domains with and without N terminal DogCatcher were codon optimised and cloned into mammalian protein expression plasmid pcDNA3.4. To facilitate secretion, the Igk-leader sequence METDTLLLWVLLLWVPGSTGD was introduced at the N terminus of the fusion protein, and a C-terminal C-tag (EPEA) was added to enable affinity purification. DogCatcher-porcine coronavirus RBDs were expressed in suspension ExpiCHO-S cells (Thermo Fisher); protein was harvested from culture supernatant, affinity purified using C-tag affinity resin (Thermo Fisher) using an AKTA chromatography system (GE Healthcare), and dialyzed into Tris-buffered saline (TBS) pH 7.8. RBD sequences were defined as follows: PRCV135 residues 300–449 of the S protein; PEDV (strain CV777, accession no. OR3484341) residues 504–637; PHEV1968 residues 311–608 as defined by Bahoussi et al. 34 ; and PDCoV residues 300–419 of AML40825.1, identical to OH-FD22 35 . PRCV135 N protein was transiently expressed in Expi293FTM cells according to the Gibco Expi293™ Expression System User Guide. Presence of protein was determined by detection of the C-tag by western blot ( Suppl. Fig S6 ). Following confirmation of expression, the protein was scaled up for expression at 300mL followed by purification using a C-tag affinity matrix column (ThermoFisher). Purified protein was dialysed overnight into phosphate-buffered saline (PBS) and concentrated using an Amicon Ultra Centrifugal Filter, 10 kDa MWCO to adjust the protein concentration to 1 mg/mL. The protein was quantified using Pierce™ BCA Protein Assay Kits by a BCA kit (ThermoFisher). Successful expression of N protein was confirmed by binding of DA3, a mouse monoclonal which binds the N protein of PRCV and TGEV 11 . Ad capsid decoration: conjugation reactions and vaccine preparation. Ligand-decorated vaccines were prepared by co-incubation of Ad-DogTag vectors with DogCatcher-RBD protein with 3.5µM DogCatcher-RBD for every 1 ×10 10 viral particles in sucrose storage buffer (10 mM Tris-HCl, 7.5% w/v sucrose, pH 7.8). Reactions were incubated for 16 h at 4°C during which time spontaneous conjugation of DogCatcher and DogTag occurred. To remove excess ligand, conjugated vaccines were dialyzed into sucrose storage buffer using SpectraPor dialysis cassettes with a 1000-kDa molecular weight cutoff (MWCO)(Spectrum Labs). Dialysis reduced excess ligand by at least 10-fold, as measured by densitometry on Coomassie-stained SDS-PAGE. Ligand coverage was determined by SDS-PAGE using the equation described in 15 and ranged from 55–73% hexon coverage. Ad titer after ligand-conjugation was determined as described in 15 . Mouse immunizations . All mouse procedures were performed in accordance with the terms of the UK Animals (Scientific Procedures) Act (Project License PP5949437) and approved by the Oxford University Ethical Review Body. Female BALB/c mice (aged 6–8 weeks, Envigo), housed in specific pathogen-free environments, were immunized intramuscularly (I.M.) by injection of 50 µL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 µL total). Vaccine doses used were as described in figure legends. For cocktail vaccine preparations, the constituent vaccines were mixed at an equal fractional dose prior to administration to give a total dose of 1 × 10 8 IU/animal. Endotoxin dose was < 1 EU per mouse in all studies. Experiments were performed at Biomedical Services, University of Oxford. Pig immunization and viral challenge studies . Animal studies were approved by ethical review processes at The Pirbright Institute and VetQuest and conducted under project licence PP7764821 according to regulations set out in the UK Government Animal (Scientific Procedures) Act 1986. The Pirbright Institute conforms to Animal Research: Reporting of Animal Experiments guidelines. In all experiments, animals acclimatized for at least 7 days and were randomized into different groups and pens using Excel by the animal services staff. Researchers processing the samples were only aware of the pig numbers, not the group assignments. The pathologists were blinded to the group allocation when assessing the samples for gross pathology during post-mortem examination and subsequently during the histopathological assessment. All conditions were kept the same between groups. Viral shedding and lung pathology were the main outcome variables for the studies. Immunization and PRCV viral challenge pig experiment : Twenty pigs six weeks of age, genetically composed of one-quarter Large White, one-quarter Landrace, and one-half Hampshire breeds, were obtained from a high health farm in which no PRCV antibodies could be detected. The animals were randomly assigned into four treatment groups of five pigs each. Three groups received 1x10 9 infectious units (IU) of either Ad(GFP), Ad(S-N)-RBD135, or Ad(S-N) intramuscularly (IM) with 1 ml administered in each trapezius muscle behind the ear. The fourth group received 1x10 9 IU of Ad(N) by aerosol (AE) administered over 2–5 minutes using an Aerogen Solo vibrating mesh nebulizer (Aerogen, Dangan, Galway, Ireland) following sedation with a 4 mg/kg Zoletil and 2 mg/kg Stresnil (Elanco UK AH Limited). The animals were boosted 25 days later by the same immunization route. Twenty-four days post boost, animals were inoculated intranasally with 2 x 10 7 pfu PRCV135 using a MAD300 device to deliver 2 ml into each nostril following sedation with a 3 mg/kg Zoletil and 1.5 mg/kg Stresnil. The pigs were humanely culled four days later with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. Daily nasal swabs were collected into 2ml virus transport medium (tissue culture medium 199 (Sigma-Aldrich) with 25mM Hepes, 0.035% sodium bicarbonate, 0.5% BSA, penicillin, streptomycin and nystatin) for analysis of nasal shedding. Whole blood for isolation of peripheral blood mononuclear cells (PBMCs) and serum were obtained weekly throughout the study. At postmortem blood, bronchoalveolar lavage (BAL) and tracheobronchial lymph node (TBLN), were collected for cell isolation as previously described 36 . The lung accessory lobe, BAL, tracheal and deep nasal swabs were collected for analysis of viral load in the respiratory tract. Immunization with vaccine cocktail in pigs. Eight (6–8 weeks old) pigs were randomly assigned to two groups of 4 pigs and after one week acclimatization immunized intramuscularly with: either Ad(S-N)-RBD135 or cocktail containing equal IU of the three different RBD decorated Ads: Ad(S-N)-RBD135, Ad(S-N)-RBD-PHEV and Ad(S-N)-RBD-PDCoV (cocktail 3) to give a total dose of 1 × 10 9 IU/animal.(Fig. 8 A). All pigs received a homologous booster immunization on day 28. Serum samples were collected weekly for evaluation of antibody titers. Lung gross pathology, histopathology, and immunohistochemistry. The percentage of pulmonary consolidation, including both dorsal and ventral aspects was assessed blindly by a veterinary pathologist at necropsy 11 . Formalin fixed tissues were processed by a routine histology method. Haematoxylin and eosin staining and immunohistochemistry (IHC) against PRCV nucleoprotein (N) using a monoclonal antibody were performed on serially sectioned formalin-fixed paraffin embedded tissues 11 . Determination of infectious virus by plaque assays. Tissues were homogenised in PBS containing 0.1% BSA using C-tubes (Miltenyi) and an Octo-MACS tissue dissociator. Homogenised tissue was clarified by low-speed centrifugation, and the supernatant titrated by plaque assay in ST cells. 2g each of lung, trachea and eyelid were processed. Swine testis (ST) cells were seeded in sterile 12-well tissue culture plates and allowed to grow until confluent. Ten-fold serial dilutions of sample were prepared in PRCV growth medium (EMEM supplemented with 0.02% yeast extract, 10% tryptose-phosphate broth, 2mM L-glutamine, 10,000 U/ml penicillin, 10,000 µg/ml streptomycin, and 1% nystatin solution). Cells were washed with PBS, then 250µl of each dilution applied to the cells in duplicate. After 1 hour incubation at 37°C, the inoculum was removed and 1ml N,N-Bis(2-hydryoxyethyl)-2aminoethanesulphonic acid (BES) medium containing 1% agar added. Cells were fixed with 4% formaldehyde in PBS 48 hours post-infection and plaques visualised using 0.1% crystal violet. Virus neutralization PRCV135. Serum harvested weekly throughout the animal studies and BAL collected at post-mortem were diluted 1:2 in triplicate in PRCV growth media, then serially diluted 1:2 across a 96-well plate. 100µl of PRCV growth media containing 1x10 3 pfu of TGEV or PRCV 135, 137, 310 or ISU-1 was added to 100µl of each serum dilution and incubated at room temperature for 30 minutes. 200µl of virus/serum mix was added to ST cells and incubated at 37°C for 48 hours. Cells were fixed with 4% formaldehyde and visualised with 0.1% crystal violet, before wells were marked as cytopathic effect (no neutralization) or no cytopathic effect (neutralization). Neutralization titer resulting in 50% neutralization per ml (VNT 50 ) was calculated using the Reed-Muench calculation 37 . ELISA. Mice : ELISA analysis of mouse serum samples was performed as described previously (Biswas et al 2011). Briefly, 96-well ELISA plates were coated with porcine coronavirus RBDs at 2 µg/mL. Plates were blocked with 10% Milk powder in 0.05% PBS-Tween20. Serum was diluted 3-fold in 0.05% PBS-Tween20 with a starting dilution of 1 in 100. Secondary antibody (STAR117A, BioRad) was diluted 1 in 5000 and the signal was developed in p -nitrophenylphosphate (pNPP) solution. The signal was allowed to develop for 30 minutes and plates were read at 405 nm on an Infinite M Plex (Tecan). Data were analyzed in Prism v10. Pigs : Maxisorp 96-well plates (ThermoScientific) were coated with 100µl/well of 0.5µg/ml recombinant full-length spike protein form ISU-1 as previously described diluted in PBS overnight at 4°C 11,12 . The full-length S protein and RBD only of PRCV 135 and PRCV ISU-1 were aligned using blastp (NCBI), indicating 95% sequence identity within the RBD and 97% sequence identity across the whole spike protein ( Suppl Fig. S7 ). Recombinant RBDs from PRCV135, PEHV and PDCoV or N were produced as described above. Plates were washed with PBS containing 0.05% Tween20 and blocked for one hour at room temperature with block buffer (PBS containing 0.05% Tween20, 4% w/v Marvel milk powder). Serum was serially diluted 2-fold in block buffer and 100µl/well applied to the plate for 1 hour at room temperature. Serum was washed off the plate, then 100µl/well of secondary antibody (anti-porcine IgG H&L-HRP or anti-porcine IgA-HRP, Biorad) diluted 1:20,000 in block buffer added for 1 hour at room temperature. The assay was developed using 50µl/well of TMB high-sensitivity substrate solution (Biolegend), and the reaction terminated using equal volume of 1M sulphuric acid. Optical density at 450nm and 630nm was measured, and endpoint titer determined. IFN g ELISpot assay. Mice : For mouse spleen IFNg ELISpot analysis, ELISPOT plates with hydrophobic PVDF membranes (MERCK) were coated overnight at 4°C with 5 mg/mL monoclonal rat anti-mouse IFNg antibody (AN18 Mabtech) in carbonate-bicarbonate coating buffer (MERCK). The next day the anti-mouse IFNg antibody was removed and the plates blocked in 100 mL aMEM (MERCK) supplemented with 10% foetal bovine serum, 100 U/ml penicillin and streptomycin, 4 mM L-Glutamine and 50 mM 2-Mercaptoethanol). Splenocytes were seeded in duplicate at 250,000 cells/well, 125,000 cells/well and 62,500 cells/well and stimulated with the PRCV S and N peptide at a final concentration of 5 µg/ml (Mimotopes, Melbourne, Australia − 16-mer peptides overlapping by 12 residues with three pools for the S protein (residues 1-100, 101–200, and 201–305) and one pool of 94 peptides for N) pools. As a negative control duplicate wells containing 250,000 cells for each mouse remained unstimulated. After 18 hours incubation at 37 o C with 5% CO 2 , the plates were washed and incubated with 1 µg/ml Biotin Rat anti-Mouse IFNg (Mabtech) for 2 hours at room temperature. The plates were washed before incubating with 1 µg/ml of Streptavidin alkaline phosphatase (Mabtech) for 1 hour at room temperature. The assay was developed using AP conjugate substrate kit (Biorad). Spots were counted using an AID ELISpot reader (AID). Results were expressed as the number of IFNg-producing cells per 10 6 stimulated cells after subtracting the average number of spots from the unstimulated wells. Pigs : ELISpot plates with mixed cellulose esters (MCE) membrane (MERCK) were coated overnight at 4°C with 0.5 µg/ml purified Mouse Anti-Pig IFNg (559961, BD Pharmingen™). The plates were washed with PBS, and blocked for 2 hours at 37°C with RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% foetal calf serum and 100 U/ml penicillin-streptomycin (Thermofisher)). Cryopreserved cells were thawed, washed, seeded in triplicate at 250,000 cells/well and stimulated with either PRCV135 virus (MOI = 1) or PRCV S or N peptide pools (Mimotopes, Melbourne, Australia) as previously described 11 , 12 . Sixteen-mer peptides overlapping by 12 residues with three pools for the S protein (residues 1-100, 101–200, and 201–305) and one pool of 94 peptides for N at a final concentration of 5 µg/ml were used. Triplicate wells of cells were also stimulated with 6 µg/ml ConA (Sigma-Aldrich) as a positive control and with medium as a negative control. After 48 hours the plates were washed followed by an incubation with 0.5 µg/ml Biotin Mouse Anti-Pig IFNg (559958, BD Pharmingen™) for 2 hours at room temperature. The plates were washed before incubating with 2 µg/ml of Streptavidin alkaline phosphatase (S921, Invitrogen) for 1 hour at room temperature. The assay was developed using AP conjugate substrate kit (Biorad). Spots were counted using an AID ELISpot reader (AID) and Immunospot ELISpot plate reader (Cellular Technology Limited). Results were expressed as the number of IFNg -producing cells per 10 6 stimulated cells after subtracting the average number of spots in medium-stimulated control wells. Intracellular cytokine staining (ICS). Cell suspensions obtained from PBMC, BAL and TBLN were thawed and seeded in duplicate wells of a 96-well plate (1 x 10 6 cells per well). Cells were stimulated overnight with either PRCV 135 (MOI 0.5), or they were treated with RPMI-1640 medium containing stable glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FCS) as a negative control. Alternatively, some cells were stimulated for 5 hours with 2 µg/ml of overlapping peptides spanning the S and N proteins as described above. Positive control wells were stimulated with a cocktail containing phorbol 12-myristate 13-acetate (PMA) and ionomycin (BioLegend). Brefeldin A (GolgiPlug™, BD Biosciences) was added to all wells and incubated for 4 hours. Subsequently, cells were centrifuged for 5 minutes at 500g and washed twice with PBS. All cells were stained with Near-Infrared Fixable LIVE/DEAD stain (Invitrogen), CD4-PerCP-Cy5.5 (74-12-4, BD Biosciences) and CD8β-FITC (PPT23, Bio-Rad Laboratories) for 20 mins at 4°C. Cells were washed and were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) for 20 mins at 4°C. Cells were washed with BD Perm/Wash™ buffer (BD Biosciences) and incubated with anti-IFNg (P2G10, BD Biosciences) and anti-TNF (MAb11, BioLegend) antibodies. Following two more washes and resuspension in PBS, cytokine producing CD4 + and CD8 + T cells were analyzed using a MACSquant Analyzer 16 (Miltenyi). Wells containing only cells and medium for each animal were considered as a negative control (unstimulated), and the frequency of cytokine-producing cells was determined by subtracting the values from unstimulated cells. Data analysis was performed using FlowJo software version 10.10.0. Statistical Analysis. GraphPad Prism 10.2.0 was used to perform statistical analyses. First, Normality tests (D’Agostino-Pearson omnibus, Anderson-Darling, Shapiro-Wilk and Kolmogorov-Smirnov test) were done to test whether the data were normally distributed. The normally distributed data were analyzed using either One-way ANOVA or Two-way ANOVA and Bonferroni’s multiple comparisons test while the data that were not normally distributed were analyzed using Mann-Whitney test. Mouse data were analyzed using non-parametric Kruskal-Wallis test or Mann-Whitney test, depending on the number of comparisons being performed. Significant differences are presented on each graph (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). To assess correlations between immune parameters and virological/pathological measures, a non-parametric Spearman correlation coefficient ( ρ ) was computed for each pair of measures (viral load, antibody response, T cell responses and pathology) using Graphpad Prism v 10.0.3. All Ad(S-N), Ad(S-N)-RBD135, Ad(N) and Ad(GFP) samples were included in the analysis. Declarations Conflict of interest. R.A.R., B.G.C, L.M.R, R.D, L.A.H.B, and M.D.J.D are employees of SpyBiotech Ltd. S.B. is CSO and co-founder of SpyBiotech Ltd. The other authors declare no conflict of interests. Acknowledgements. We are grateful to the animal staff at VetQuest and the Pirbright Institute for providing excellent animal care. We thank the Immunological Toolbox and Flow Cytometry Scientific Technology Platforms at the Pirbright Institute. Data availability. The datasets generated and/or analysed during the current study are provided as a source data file. Any further data may be provided by contacting the corresponding author upon reasonable request. Funding. This work was supported by the UKRI Biotechnology and Biological Sciences Research Council (BBSRC) BB/X014266/1 and the Pirbright Institute’s Strategic Programme Grants (ISPGs) BBS/E/PI/230001C, BBSRC National Bioscience Research Infrastructure: High Containment and Low Containment Services and Science Platforms grants BBS/E/PI/23NB0003; BBS/E/PI/23NB0004. Clinical trial number: not applicable. Author Contributions S.K., S.B., M.D.J.D. and E.T. designed the study. S.W., E.J.B., R.A.R., S.C., M.D.J.D. and E.T. curated the data. Formal data analysis was performed by S.W., E.J.B., R.A.R., S.C., F.J.S., B.P., S.K., S.B., M.D.J.D., and E.T. E.T., S.K., E.B., G.F., S.B., and M.D.J.D. obtained funding. Investigation was performed by S.W., E.J.B., R.A.R., S.C., A.V., B.G.C., L.M.R., R.D., L.A.H.B., C.R., C.F.H., D.M., J.R.S., M.I., E.B., E.M., T.C., F.J.S., B.P., S.K., S.B., M.D.J.D., and E.T. Methodology was developed by S.W., E.J.B., R.A.R., F.J.S., B.P., S.K., S.B., M.D.J.D., and E.T. Resources were acquired by E.T., S.B., M.D.J.D., and S.K., and supervision provided by E.T., S.B. and M.D.J.D. Data was validated by S.W., E.J.B., R.A.R., S.C., B.P., S.K., M.D.J.D., and E.T., and visualized by S.W., E.J.B., R.A.R., F.J.S., S.K., M.D.J.D., and E.T. S.W., E.J.B., R.A.R., S.K., M.D.J.D., and E.T. wrote the original draft of the manuscript, with additional review and editing provided by C.R., D.M., J.R.S., M.I., E.B., F.J.S., B.P. and S.B. References Piret, J. & Boivin, G. Pandemics Throughout History. Frontiers in Microbiology Volume 11–2020 (2021). https://doi.org:10.3389/fmicb.2020.631736 Saif, L. J. Animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? Rev Sci Tech 23, 643–660 (2004). https://doi.org:10.20506/rst.23.2.1513 Saif, L. J. & Jung, K. Comparative Pathogenesis of Bovine and Porcine Respiratory Coronaviruses in the Animal Host Species and SARS-CoV-2 in Humans. J Clin Microbiol 58 (2020). https://doi.org:10.1128/jcm.01355-20 Turlewicz-Podbielska, H. & Pomorska-Mól, M. Porcine Coronaviruses: Overview of the State of the Art. Virol Sin 36, 833–851 (2021). https://doi.org:10.1007/s12250-021-00364-0 Vlasova, A. N. et al. Porcine coronaviruses.. Emerging and Transboundary Animal Viruses , 79–110 (2020). Woo Patrick, C. Y. et al. Discovery of Seven Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and Avian Coronaviruses as the Gene Source of Gammacoronavirus and Deltacoronavirus. Journal of Virology 86, 3995–4008 (2012). https://doi.org:10.1128/jvi.06540-11 Wang, Q., Vlasova, A. N., Kenney, S. P. & Saif, L. J. Emerging and re-emerging coronaviruses in pigs. Curr Opin Virol 34, 39–49 (2019). https://doi.org:10.1016/j.coviro.2018.12.001 Lednicky, J. A. et al. Independent infections of porcine deltacoronavirus among Haitian children. Nature 600, 133–137 (2021). https://doi.org:10.1038/s41586-021-04111-z Tscherne, A. & Krammer, F. A review of currently licensed mucosal COVID-19 vaccines. Vaccine 61, 127356 (2025). https://doi.org:10.1016/j.vaccine.2025.127356 Jung, K. et al. Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus. J Virol 81, 13681–13693 (2007). https://doi.org:10.1128/jvi.01702-07 Keep, S. et al. Porcine respiratory coronavirus as a model for acute respiratory coronavirus disease. Front Immunol 13:867707 (2022). Sedaghat-Rostami, E. et al. Porcine respiratory coronavirus as a model for acute respiratory disease: mechanisms of different infection outcomes. The Journal of Immunology 214, 1643–1660 (2025). https://doi.org:10.1093/jimmun/vkaf066 McDonald, I., Murray, S. M., Reynolds, C. J., Altmann, D. M. & Boyton, R. J. Comparative systematic review and meta-analysis of reactogenicity, immunogenicity and efficacy of vaccines against SARS-CoV-2. NPJ Vaccines 6, 74 (2021). https://doi.org:10.1038/s41541-021-00336-1 Keeble, A. H. et al. DogCatcher allows loop-friendly protein-protein ligation. Cell Chem Biol 29, 339–350.e310 (2022). https://doi.org:10.1016/j.chembiol.2021.07.005 Dicks, M. D. J. et al. Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2. Mol Ther 30, 3639–3657 (2022). https://doi.org:10.1016/j.ymthe.2022.08.002 Martini, V. et al. Distribution of Droplets and Immune Responses After Aerosol and Intra-Nasal Delivery of Influenza Virus to the Respiratory Tract of Pigs. Front Immunol 11, 594470 (2020). https://doi.org:10.3389/fimmu.2020.594470 Peng, Y. et al. An immunodominant NP(105–113)-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease. Nat Immunol 23, 50–61 (2022). https://doi.org:10.1038/s41590-021-01084-z Kundu, R. et al. Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts. Nat Commun 13, 80 (2022). https://doi.org:10.1038/s41467-021-27674-x Chew, C. K. et al. Safety, efficacy and immunogenicity of aerosolized Ad5-nCoV COVID-19 vaccine in a non-inferiority randomized controlled trial. NPJ Vaccines 9, 209 (2024). https://doi.org:10.1038/s41541-024-01003-x Buchholz, U. J. et al. Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc Natl Acad Sci U S A 101, 9804–9809 (2004). https://doi.org:10.1073/pnas.0403492101 Hajnik, R. L. et al. Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Sci Transl Med 14, eabq1945 (2022). https://doi.org:10.1126/scitranslmed.abq1945 Dangi, T., Class, J., Palacio, N., Richner, J. M. & Penaloza MacMaster, P. Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2. Cell Rep 36, 109664 (2021). https://doi.org:10.1016/j.celrep.2021.109664 Ignjatovic, J. & Galli, L. The S1 glycoprotein but not the N or M proteins of avian infectious bronchitis virus induces protection in vaccinated chickens. Archives of Virology 138, 117–134 (1994). https://doi.org:10.1007/BF01310043 Meir, R. et al. Immune responses to mucosal vaccination by the recombinant A1 and N proteins of infectious bronchitis virus. Viral Immunol 25, 55–62 (2012). https://doi.org:10.1089/vim.2011.0050 Primard, C. et al. OVX033, a nucleocapsid-based vaccine candidate, provides broad-spectrum protection against SARS-CoV-2 variants in a hamster challenge model. Front Immunol 14, 1188605 (2023). https://doi.org:10.3389/fimmu.2023.1188605 Rak, A., Isakova-Sivak, I. & Rudenko, L. Overview of Nucleocapsid-Targeting Vaccines against COVID-19. Vaccines 11, 1810 (2023). Rabdano, S. O. et al. Immunogenicity and In Vivo Protective Effects of Recombinant Nucleocapsid-Based SARS-CoV-2 Vaccine Convacell(®). Vaccines (Basel) 11 (2023). https://doi.org:10.3390/vaccines11040874 Gerdts, V. & Zakhartchouk, A. Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. Vet Microbiol 206, 45–51 (2017). https://doi.org:10.1016/j.vetmic.2016.11.029 Cohen, A. A. et al. Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models. Science 377, eabq0839 (2022). https://doi.org:10.1126/science.abq0839 Liu, C. et al. Mosaic RBD nanoparticle elicits immunodominant antibody responses across sarbecoviruses. Cell Rep 43, 114235 (2024). https://doi.org:10.1016/j.celrep.2024.114235 Kanekiyo, M. et al. Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses. Nat Immunol 20, 362–372 (2019). https://doi.org:10.1038/s41590-018-0305-x Brown, I. & Cartwright, S. New porcine coronavirus? Vet Rec 119, 282–283 (1986). https://doi.org:10.1136/vr.119.11.282 Garwes, D. J. & Pocock, D. H. The polypeptide structure of transmissible gastroenteritis virus. J Gen Virol 29, 25–34 (1975). https://doi.org:10.1099/0022-1317-29-1-25 Bahoussi, A. N. et al. Genetic Characteristics of Porcine Hemagglutinating Encephalomyelitis Coronavirus: Identification of Naturally Occurring Mutations Between 1970 and 2015. Front Microbiol 13, 860851 (2022). https://doi.org:10.3389/fmicb.2022.860851 Ji, W. et al. Structures of a deltacoronavirus spike protein bound to porcine and human receptors. Nature Communications 13, 1467 (2022). https://doi.org:10.1038/s41467-022-29062-5 Morgan, S. B. et al. Aerosol Delivery of a Candidate Universal Influenza Vaccine Reduces Viral Load in Pigs Challenged with Pandemic H1N1 Virus. J Immunol 196, 5014–5023 (2016). https://doi.org:10.4049/jimmunol.1502632 REED, L. J. & MUENCH, H. A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS12. American Journal of Epidemiology 27, 493–497 (1938). https://doi.org:10.1093/oxfordjournals.aje.a118408 Additional Declarations Competing interest reported. R.A.R., B.G.C, L.M.R, R.D, L.A.H.B, and M.D.J.D are employees of SpyBiotech Ltd. S.B. is CSO and co-founder of SpyBiotech Ltd. The other authors declare no conflict of interests. Supplementary Files SupplementaryFiguresamended.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7978161","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":543447659,"identity":"886b8e59-c713-43f8-b495-b7ab6df67981","order_by":0,"name":"Sanis Wongborphid","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Sanis","middleName":"","lastName":"Wongborphid","suffix":""},{"id":543447660,"identity":"fe099c0d-25a7-4ad8-b2b0-2ab07b423679","order_by":1,"name":"Emily J Briggs","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"J","lastName":"Briggs","suffix":""},{"id":543447661,"identity":"cfa48fd7-75c8-4549-8e98-96d89f771ebe","order_by":2,"name":"Rebecca A. Russell","email":"","orcid":"","institution":"SpyBiotech Limited","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"A.","lastName":"Russell","suffix":""},{"id":543447662,"identity":"b0ae4b44-bf68-4c28-96d9-40dcbed75950","order_by":3,"name":"Shafiyeel Chowdhury","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Shafiyeel","middleName":"","lastName":"Chowdhury","suffix":""},{"id":543447663,"identity":"78cfd6f6-7be3-4690-96b0-873ff6e62ec1","order_by":4,"name":"Ashutosh Vats","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Ashutosh","middleName":"","lastName":"Vats","suffix":""},{"id":543447664,"identity":"738392cb-2ada-4237-a463-c68f3dfac1a0","order_by":5,"name":"Bethany G. Charlton","email":"","orcid":"","institution":"SpyBiotech Limited","correspondingAuthor":false,"prefix":"","firstName":"Bethany","middleName":"G.","lastName":"Charlton","suffix":""},{"id":543447665,"identity":"fc64709b-43ba-48e2-8b24-b3b135896575","order_by":6,"name":"Louisa M. Rose","email":"","orcid":"","institution":"SpyBiotech Limited","correspondingAuthor":false,"prefix":"","firstName":"Louisa","middleName":"M.","lastName":"Rose","suffix":""},{"id":543447666,"identity":"139f83df-c618-4129-b4f3-3f000218385e","order_by":7,"name":"Rosa D'Agostino","email":"","orcid":"","institution":"SpyBiotech Limited","correspondingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"","lastName":"D'Agostino","suffix":""},{"id":543447667,"identity":"042807ff-8851-40d1-9d77-b39bbf5702ba","order_by":8,"name":"Lesley A. H. Bowman","email":"","orcid":"","institution":"SpyBiotech Limited","correspondingAuthor":false,"prefix":"","firstName":"Lesley","middleName":"A. H.","lastName":"Bowman","suffix":""},{"id":543447668,"identity":"372b9e36-94cc-4979-b959-90f057c7d0ca","order_by":9,"name":"Christine Rollier","email":"","orcid":"","institution":"University of Surrey","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"","lastName":"Rollier","suffix":""},{"id":543447669,"identity":"0a15c8e9-54b3-41a2-b493-7ba6e96a36a8","order_by":10,"name":"Catherine F. Hatton","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"F.","lastName":"Hatton","suffix":""},{"id":543447670,"identity":"ac4dbe73-5e98-4352-af22-871535bb016e","order_by":11,"name":"Jean-Remy Sadeyen","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Jean-Remy","middleName":"","lastName":"Sadeyen","suffix":""},{"id":543447671,"identity":"b332471e-f2c3-4d51-84dc-6a781cc35bc1","order_by":12,"name":"Munir Iqbal","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Munir","middleName":"","lastName":"Iqbal","suffix":""},{"id":543447672,"identity":"cc29ef0e-06d6-4fd7-b58f-3a3aa799fc7a","order_by":13,"name":"Elliot Moorhouse","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Elliot","middleName":"","lastName":"Moorhouse","suffix":""},{"id":543447673,"identity":"ba28a93b-46f8-45ea-816e-bfabcb3eb4b4","order_by":14,"name":"Tiphaine Cayol","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Tiphaine","middleName":"","lastName":"Cayol","suffix":""},{"id":543447674,"identity":"53d40dfd-e5e8-44da-9c52-c3c81f704c41","order_by":15,"name":"Erica Bickerton","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Erica","middleName":"","lastName":"Bickerton","suffix":""},{"id":543447675,"identity":"a01a970d-25d9-494b-8d27-22aee3911f81","order_by":16,"name":"Danish Munir","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Danish","middleName":"","lastName":"Munir","suffix":""},{"id":543447676,"identity":"d0073d8f-67c9-49fd-991a-dd5b2cb808be","order_by":17,"name":"Francisco J. Salguero","email":"","orcid":"","institution":"UK Health Security Agency","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"J.","lastName":"Salguero","suffix":""},{"id":543447677,"identity":"baa90779-b96a-4aca-8747-73fcd76dabaa","order_by":18,"name":"Basudev Paudyal","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Basudev","middleName":"","lastName":"Paudyal","suffix":""},{"id":543447678,"identity":"325ed441-6064-4137-a9d1-a5313e1cd9cc","order_by":19,"name":"Sarah Keep","email":"","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Keep","suffix":""},{"id":543447679,"identity":"4bbc491b-b3f3-4f3d-a996-e4bc605af632","order_by":20,"name":"Sumi Biswas","email":"","orcid":"","institution":"SpyBiotech Limited","correspondingAuthor":false,"prefix":"","firstName":"Sumi","middleName":"","lastName":"Biswas","suffix":""},{"id":543447680,"identity":"5fd143a2-5ae6-4af5-bbea-e2fe82c1c7e7","order_by":21,"name":"Matthew D. J. Dicks","email":"","orcid":"","institution":"SpyBiotech Limited","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"D. J.","lastName":"Dicks","suffix":""},{"id":543447681,"identity":"e06271c9-8c54-4139-8e6d-b34e15a86be2","order_by":22,"name":"Elma Tchilian","email":"data:image/png;base64,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","orcid":"","institution":"The Pirbright Institute","correspondingAuthor":true,"prefix":"","firstName":"Elma","middleName":"","lastName":"Tchilian","suffix":""}],"badges":[],"createdAt":"2025-10-29 09:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7978161/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7978161/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96245166,"identity":"5403cb18-396a-45af-aba9-7c903ba5e774","added_by":"auto","created_at":"2025-11-19 07:19:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3467338,"visible":true,"origin":"","legend":"","description":"","filename":"PRCVimmunisationmanuscriptrevised31Oct.docx","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/c3129c6fd755f5a414dcff8c.docx"},{"id":95954500,"identity":"0b38afe3-3a62-44c6-ae4e-29d5f46e0011","added_by":"auto","created_at":"2025-11-14 20:56:41","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22316,"visible":true,"origin":"","legend":"","description":"","filename":"9d025a9cf26a4bd18e922a870863d1d3.json","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/a4cb4a87877b81d020638988.json"},{"id":96243592,"identity":"0cd1fa35-b536-4857-a1e6-ce4a119369b7","added_by":"auto","created_at":"2025-11-19 07:16:41","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":797023,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresamended.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/4e6a6337b82249621d8fc5da.pdf"},{"id":95954502,"identity":"22c479cd-2dff-4532-8151-d53ac5da6b68","added_by":"auto","created_at":"2025-11-14 20:56:41","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136370,"visible":true,"origin":"","legend":"","description":"","filename":"9d025a9cf26a4bd18e922a870863d1d31enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/4c8512f3b481eb878a32144b.xml"},{"id":95954506,"identity":"0fbf34e8-88b0-42a6-83d5-8149d4d80077","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124870,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/5f0634ff6ae85b2c1fdf4222.png"},{"id":96245300,"identity":"2d34af18-e764-4656-a09c-e3ad761dd84b","added_by":"auto","created_at":"2025-11-19 07:20:17","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1074736,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/c5027250cb6bc9967a7d1b7f.jpeg"},{"id":95954505,"identity":"d42a4848-4114-435a-a899-7112d9049cbf","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":191290,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/f96d1d63c15de670f38e2743.jpeg"},{"id":96243739,"identity":"fa3aa009-8fae-4583-b315-5a51620f992f","added_by":"auto","created_at":"2025-11-19 07:16:56","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":431580,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/dbdbc2b38e82d24c785d233e.jpeg"},{"id":95954511,"identity":"00efb9d3-3c9c-40f8-8152-8655b1ccbb3a","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":480378,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/e6360479089fc4dc0deb6248.jpeg"},{"id":96244575,"identity":"58e54980-4852-4f32-9776-2564ffe21fce","added_by":"auto","created_at":"2025-11-19 07:18:53","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":424130,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/d42c1678a3ad9292263334d8.jpeg"},{"id":95954514,"identity":"7f8dfc74-7884-48d7-af2d-2bfc412369e8","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":296822,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/2ae71b283c54be1f3d688e47.jpeg"},{"id":95954528,"identity":"95f87f78-c845-4123-993a-1bcda4c7437e","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":292276,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/e918773f053459b95a9fd6e6.jpeg"},{"id":95954522,"identity":"e4e364db-a545-475d-a157-937161e03b98","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31730,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/56a7299cca01fc40adc1d6a6.png"},{"id":96244223,"identity":"965d7cb0-547b-4198-8bec-d8a9e0f0388e","added_by":"auto","created_at":"2025-11-19 07:17:57","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127105,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/cef1799c2158f89dddefe751.png"},{"id":95954509,"identity":"4d6a2bf1-77c6-4a8b-91ab-96a77ab2a8fe","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17473,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/9c2413c3e1438c91e7d51813.png"},{"id":95954520,"identity":"9f1baa96-eec5-404d-8803-4c25e789f17e","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44283,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/ff51e73006d751bd97ce7bf7.png"},{"id":96245287,"identity":"d0fc4867-2564-479d-bc20-b5765ab49ef8","added_by":"auto","created_at":"2025-11-19 07:20:16","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45278,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/8fdfc7a975e01c1592ffa28b.png"},{"id":95954523,"identity":"67a5a459-a298-45f8-955e-acd477370425","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39117,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/b1f9e425bda60accb5aba1d5.png"},{"id":95954516,"identity":"85c6cd05-514d-4132-9adc-b3a6960b6f75","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29558,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/8928e085c7d63633806998b5.png"},{"id":95954518,"identity":"2081fae3-174f-4813-a1ce-ae70896ef62e","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":28220,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/896ce9ed29f64b2503615731.png"},{"id":95954524,"identity":"3dd421db-dec5-4dd2-8ef6-7c66473d8fd5","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134271,"visible":true,"origin":"","legend":"","description":"","filename":"9d025a9cf26a4bd18e922a870863d1d31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/0b2bb6658412f7e2c45d272a.xml"},{"id":96244274,"identity":"3964994d-08a7-4e7e-a1b9-3acb4bd45b9d","added_by":"auto","created_at":"2025-11-19 07:18:03","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150561,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/dababa6f40c1d3018ba8946e.html"},{"id":95954497,"identity":"dca39e2e-6a40-4c8b-b252-7e5b9b056377","added_by":"auto","created_at":"2025-11-14 20:56:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-RBD antibody production and S and N-specific T cell responses following mouse immunization with decorated and undecorated Ad5 vectors encoding PRCV135 S and N proteins. \u003c/strong\u003e(A) Schematic immunization schedule. Balb/C mice (six per group) were immunized intramuscularly (IM) in homologous prime boosts regimens with 5 x 107 infectious units/mouse of undecorated Ad5 encoding PRCV135 S and N proteins, (Ad5(S-N)), Ad5(S-N) decorated with PRCV135 RBD (Ad5(S-N)-RBD135) and undecorated Ad5 encoding PRCV135 N protein only (Ad5(N)). (B) Serum IgG antibody responses to PRCV135 RBD at Day 20 and Day 35 measured by endpoint ELISA. (C) IFNg-ELISpot responses in spleens at Day 35 against the PRCV135 S and N proteins antigen using 4 different peptide pools (pp, 3 against S and 1 against N). Dotted line represents limit of detection (LOD). Median responses shown by a horizontal line. Responses expressed as spot-forming cells (SFC) per 106 cells. Statistical analyses performed by post-hoc Mann-Whitney test where only two comparisons are performed and Kruskal-Wallis with Dunn’s test for multiple comparisons where more than two comparisons are performed, *p \u0026lt; 0.05; **p \u0026lt; 0.01; ns, not significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/6e3cb6962c0ae9b2f3a55686.png"},{"id":96246090,"identity":"89a65feb-c3b4-449e-9871-d5873e75d528","added_by":"auto","created_at":"2025-11-19 07:24:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":316218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design and lung pathology. \u003c/strong\u003ePigs were immunized intramuscularly (IM) with 1x109 infectious units of either Ad(GFP), Ad(S-N)-RBD, or 1x109 infectious units by aerosol (AE) with Ad(N). Twenty five days later they received homologous boost and after further 24 days were challenged with PRCV. Pigs were culled four days later (A). Lungs were examined for percent area of consolidation (B), gross pathology (C), histopathology Morgan (D) and histopathology including immunohistochemistry Iowa (E). Representative images of the macro and histopathology (H\u0026amp;E) and IHC detection of N protein. Areas of consolidation (arrows) are observed mostly in apical and medial lung lobes of Ad(GFP) and Ad(N) AE groups, described as broncho-interstitial pneumonia by histopathology (H\u0026amp;E) showing abundant N protein positively immunostained cells (IHC brown stain, DAB) within the airway epithelia and inflammatory cell infiltrates. Bar = 100 µm (F). Data in B-E are presented as mean ± SEM, with symbols indicating individual animals. Normally distributed data (C) were analysed by one-way ANOVA followed by Dunnet’s post-hoc test, while data not normally distributed (B, D, E) were analysed by Kruskal Wallis and Dunn’s multiple comparison test. Significant difference (p \u0026lt; 0.05) to Ad-(GFP) control is indicated by * (* p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/67b1bc05d93eeb535682ca7f.png"},{"id":95954498,"identity":"fe71b38b-6d0f-43b2-95b6-df6af32d31f3","added_by":"auto","created_at":"2025-11-14 20:56:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVirus load following challenge with PRCV 135. \u003c/strong\u003eViral load in daily nasal swabs (A), deep nasal swabs (B), tracheal swabs (C), homogenized lung (D) and bronchoalveolar lavage (E). Data are presented as mean ± SEM, with symbols indicating individual animals. Normally distributed data (A, B) were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test, while data not normally distributed (C - E) were analyzed by Kruskal Wallis and Dunn’s multiple comparison test. Significant differences to Ad-(GFP) control are indicated by * (p \u0026lt; 0.05), ** (p \u0026lt; 0.01), **** (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/10394f9c8b7bfed3cfd967dc.png"},{"id":95954515,"identity":"419c2a8b-40e2-409f-a77e-b661a32a49e6","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":159986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody responses to immunization and challenge. \u003c/strong\u003eSpike-specific IgG and IgA responses in the serum (A, B). Spike-specific IgG responses in the bronchoalveolar lavage (C). Neutralizing antibody titres against PRCV 135 in serum (D) and bronchoalveolar lavage (E). Neutralizing antibody titres at 36 days post-prime against PRCV 135, ISU-1, PRCV 137, 310, and TGEV (F). Spearman rank correlation between respiratory pathology/viral load and antibody responses from immunized infected animal (G). Data are presented as mean ± SEM, with symbols indicating individual animals or the mean of all animals in a group. Normally distributed data (A,B,D,F) were analyzed by two-way ANOVA followed by Tukey’s HSD post-hoc test or t test, while data not normally distributed (A-E) were analyzed by Kruskal Wallis and Dunn’s multiple comparison test. Asterisks represent significant correlation (*p\u0026lt;0.05, **p\u0026lt;0.01). Significant differences to Ad-(GFP) control (C) or between groups (A, B, D, F) are indicated by * (p \u0026lt; 0.05), ** (p \u0026lt; 0.01), *** (p \u0026lt; 0.001), **** (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/f6f0076415f335b9fda33dd9.png"},{"id":96245949,"identity":"c34791b5-6118-4ff5-baf9-d9597fe432ae","added_by":"auto","created_at":"2025-11-19 07:23:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":181938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT cell responses in PBMC. \u003c/strong\u003eCells producing IFNg in response to peptide pools (pp) spanning S (A,D) or N (B,E) proteins, or live PRCV135 virus (C,F) were measured by ELISpot. Symbols in A-F represent mean spot-forming units (SFU) per group, with error bars indicating SEM. IFNg- and TNF-producing CD4+ and CD8+ T cells were measured by intracellular cytokine staining following stimulation with whole virus or S or N peptides (G-J). Data are presented as mean ± SEM, with symbols indicating individual animals (D-J) or the mean of all animals in a group (A-C). Normally distributed data (D-F) were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test, while ICS data were analyzed and compared using two-way ANOVA and Dunnett’s test (G-J). Significant differences to Ad-(GFP) control are indicated by * (p \u0026lt; 0.05), ** (p \u0026lt; 0.01), *** (p \u0026lt; 0.001), **** (p \u0026lt; 0.0001). Spearman rank correlation between respiratory pathology/viral load and blood T cell responses from immunized infected animals (K). Asterisks represent significant correlation (*p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/f54e8d49897b5c0e268b77b2.png"},{"id":96246078,"identity":"2d7d6414-2bd6-496f-9eb9-759e0597bda0","added_by":"auto","created_at":"2025-11-19 07:24:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":156524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT cell responses in BAL.\u003c/strong\u003e Cells producing IFNg in response to peptide pools (pp) spanning S (A-D) or N (E) proteins, or live PRCV 135 virus (F) were measured by ELISpot. Symbols in A-F represent mean spot-forming units (SFU) per group, with error bars representing SEM. IFNg- and TNF-producing CD4+ and CD8+ T cells were measured following stimulation with whole virus or S or N peptides by intracellular cytokine staining (G-J). Data is presented as mean ± SEM, with symbols indicating individual animals (A-J). Data not normally distributed was analyzed by Kruskal Wallis and Dunn’s multiple comparison test (A-F). ICS data were analyzed and compared using Two-way ANOVA and Dunnett’s test (G-J). Significant difference to Ad-(GFP) control is indicated * (p \u0026lt; 0.05), ** (p \u0026lt; 0.01), *** (p \u0026lt; 0.001), **** (p \u0026lt; 0.0001). Spearman rank correlation between respiratory pathology/viral load and BAL T cell responses from immunized infected animals (K). Asterisks represent significant correlation (*p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/0eda8123e678271db0c15b0c.png"},{"id":95954517,"identity":"cae31d03-7853-4428-840f-5fda986338cf","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":81429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-RBD antibody production and S and N-specific T cell responses following mouse immunization with cocktails of decorated Ad5 vectors encoding PRV135 S and N proteins.\u003c/strong\u003e (A) Schematic immunization schedule. Balb/C mice (six per group) were immunized intramuscularly (IM) in homologous prime boosts regimens with a total of 1x 108 IU /mouse. Mice received either 1x 108 IU of Ad5 encoding PRCV135 S and N proteins decorated with PRCV135 RBD, Ad5(S-N)-PRCV RBD or a cocktail of 2.5 x 107 IU each of Ad5 encoding PRCV135 S and N proteins decorated with either PRCV135 RBD, PEDV RBD, PDCoV RBD or PHEV RBD (cocktail 4). (B) Serum IgG antibody responses to the 4 different RBD proteins at D35 measured by endpoint ELISA. (C) IFNg-ELISpot responses in spleens at D35 against the PRCV135 S and N proteins using 4 different peptide pools (3 against S and 1 against N). Dotted line represents limit of detection (LOD). Median responses shown by a horizontal line. Statistical analyses performed by post-hoc Mann-Whitney test, *p \u0026lt; 0.05; ns, not significant.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/cd2b38c4b39288926cd8a13b.png"},{"id":95954521,"identity":"c357ef1f-5e73-4d6a-8a24-379dcbc28557","added_by":"auto","created_at":"2025-11-14 20:56:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":78813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody responses following cocktail immunization in pigs.\u003c/strong\u003ePigs were immunized twice 28 days apart with either Ad5(S-N)-RBD135 or a cocktail 3 of each of Ad5 decorated with either PRCV135 RBD, PDCoV RBD or PHEV RBD (A). The animals were euthanized at day 49. Serum IgG antibody responses to different RBD proteins over the time course measured by endpoint ELISA (B). Neutralizing antibody titres against PRCV 135 in serum at days 37 and 49 (C).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/8e52a67c3e1698b8e46722a0.png"},{"id":98007945,"identity":"ef8eb309-e5c8-40f8-a6ff-6f6f2e923ee6","added_by":"auto","created_at":"2025-12-11 17:24:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2374861,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/1fc5aefd-5768-4834-a2ad-d261abfd2122.pdf"},{"id":95954504,"identity":"667d52cc-0a08-4a50-8e63-7bc196a9d2bc","added_by":"auto","created_at":"2025-11-14 20:56:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":797023,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresamended.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7978161/v1/0c150f33e7627828eea7b0fa.pdf"}],"financialInterests":"Competing interest reported. R.A.R., B.G.C, L.M.R, R.D, L.A.H.B, and M.D.J.D are employees of SpyBiotech Ltd. S.B. is CSO and co-founder of SpyBiotech Ltd. The other authors declare no conflict of interests.","formattedTitle":"Efficacy of a novel antigen-decorated adenoviral vaccine platform against porcine respiratory coronavirus infection in a large natural host","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRespiratory coronaviruses (CoV) are a significant global health threat and have been responsible for three major outbreaks of severe respiratory disease in humans, most notably the SARS-CoV-2 pandemic. Each of these events has been linked to zoonotic transmission from animals to humans \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Livestock species, including pigs, are also susceptible to CoV infections causing significant economic losses \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In addition to the major impact on pig health of multiple emerging CoVs, pigs may act as a conduit for future zoonotic transmission to humans as they do for influenza A viruses.\u003c/p\u003e\u003cp\u003eSeveral CoVs that infect pigs have been identified \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These include members of the \u003cem\u003eAlphacoronavirus\u003c/em\u003e genus: Transmissible Gastroenteritis Virus (TGEV), its mutant variant Porcine Respiratory Coronavirus (PRCV), Porcine Epidemic Diarrhoea Virus (PEDV) and Swine Acute Diarrhoea Syndrome Coronavirus (SADS-CoV). Porcine Hemagglutinating Encephalomyelitis Virus (PHEV) belongs to the \u003cem\u003eBetacoronavirus\u003c/em\u003e genus, and Porcine Deltacoronavirus to the \u003cem\u003eDeltacoronavirus\u003c/em\u003e genus \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. PDCoV is believed to have originated from an avian Deltacoronavirus that acquired the ability to infect pigs, raising concerns that further mutations could enable PDCoV to infect humans \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Indeed, PDCoV has been detected in human plasma samples from Haitian children suffering from acute febrile illness, most likely resulting from zoonotic spill over from pigs \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe genetic diversity, complex evolutionary history, and high mutation rates of porcine CoVs together with the emergence of novel strains with substantial impact on both animal health and potential human health risks, highlight the urgent need for improved control strategies. There is no cross-protective immunity among the different porcine CoVs (with the exception of TGEV and PRCV), emphasising the need for either virus-specific vaccines or broadly protective platforms \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. A similar challenge exists in humans, where ongoing evolution of SARS-CoV-2 enables partial escape from humoral immunity. Novel vaccine strategies that incorporate conserved internal proteins delivered to the respiratory tract may promote cross-reactive T cell immunity, however it remains to be determined whether this is protective \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Alternatively, delivery of multiple Spike antigens from different strains may offer the potential to elicit broad neutralizing antibody responses or protective antibodies against each component of the vaccine.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe and others have performed a detailed analysis of PRCV pathogenesis and immune responses following infection in pigs with strains of differing pathogenicity, PRCV135 and ISU-1 \u003csup\u003e10\u0026ndash;12\u003c/sup\u003e. The interstitial pneumonia and lung pathology in PRCV-infected pigs closely resemble those observed in human SARS-CoV patients \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These findings support the use of the PRCV pig system as a robust large natural host model for testing novel vaccines and therapeutics, as well as for studying immune responses to respiratory CoV \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eReplication-defective recombinant adenovirus (Ad) is an efficacious, highly scalable, and rapidly deployable vaccine technology. However, while conventional Ad vaccines induce potent T cell immunity (particularly CD8\u0026thinsp;+\u0026thinsp;T cells), induction of humoral immunity is typically modest compared to other technologies including protein nanoparticles and messenger RNA (mRNA), particularly after homologous boosting \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Here we employed the PRCV pig model to assess the ability of a novel replication-defective, Ad based vaccine platform to prevent or reduce lung pathology and viral load. In this platform the Ad capsid surface can be covalently decorated with vaccine antigens using a protein superglue DogTag/DogCatcher, similar to the widely used SpyTag/SpyCatcher ligation system \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In mice, Ad decorated with the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) induced\u0026thinsp;\u0026gt;\u0026thinsp;10-fold higher SARS-CoV-2 neutralization titers compared to a conventional undecorated Ad encoding S after homologous prime-boost regimens \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Importantly, decorated Ad achieved equivalent or superior T cell immunogenicity against encoded antigens compared to undecorated Ad. Capsid decoration has the added benefit of shielding Ad particles from anti-Ad neutralizing antibodies and other undesirable capsid interactors \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Using this system, the protective efficacy of Ad vectors encoding the PRCV135 Spike (S) and Nucleocapsid (N) proteins and decorated with the PRCV135 Spike receptor-binding domain (RBD), was compared to vectors encoding only the S and N proteins without decoration. This allowed us to assess whether capsid decoration enhances immunogenicity and protection. In parallel, the ability of an Ad vector encoding the PRCV135 N protein to induce T cell-mediated protection following aerosol delivery to the respiratory tract was investigated. Finally, we examined whether a multivalent \u0026ldquo;cocktail\u0026rdquo; of three Ad vectors, each decorated with the RBD of either PRCV135, PHEV, or PDCoV, could elicit cross-reactive antibody responses in this large, natural host model.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGeneration of PRCV135 vaccines and immunogenicity in mice.\u003c/b\u003e To assess the effect of decoration of the Ad viral vector with RBD (RBD135) on immune responses against S and N the following vaccine constructs were generated: i) Ad(S-N), an undecorated Ad encoding full-length PRCV135 S and N within the adenoviral genome and ii) Ad(S-N)-RBD135, an Ad encoding S and N and decorated with RBD135 (via covalent attachment of RBD135 to the capsid surface). SDS-PAGE analysis of decorated virions demonstrated a high degree of RBD135 coverage on the Ad capsid; ~70% of total Ad hexon protein was covalently attached to RBD135 via DogTag/DogCatcher (\u003cb\u003eSuppl. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). In addition, to test whether expression of encoded N protein alone induces protective immune responses we generated Ad(N), an undecorated Ad encoding only N. Mice were immunized intramuscularly (IM) with 5 \u0026times; 10⁷ infectious units (IU) of each vaccine and received a homologous boost three weeks later. Two weeks after the boost, mice were culled for immunological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Antibody responses were assessed by endpoint ELISA using recombinant RBD135 protein. Capsid decoration with RBD in Ad(S-N)-RBD135 significantly enhanced anti-RBD antibody titers compared to Ad(S-N) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Cellular responses were measured by IFNg ELISpot using splenocytes stimulated with overlapping peptide pools spanning the S and N proteins included in the vaccines. Ad(S-N)-RBD135 immunization significantly enhanced responses to the S3 peptide pool, while responses to S1 were significantly reduced. The total summed S responses were slightly reduced after Ad(S-N)-RBD135 vaccination compared to Ad(S-N) although this trend did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). All three vaccines encoding N, Ad(S-N), Ad(S-N)-RBD135, and Ad(N), induced comparable N-specific IFNg ELISpot responses. Antibody responses to N were not measured.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese data indicated that all vaccines were immunogenic and that surface presentation of RBD135 significantly increased antibody responses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtection following prime-boost immunization in PRCV pig model.\u003c/b\u003e We next evaluated the contribution of RBD135, S and N antigens in the induction of protective immune responses, in the large natural host PRCV pig model. Twenty pigs were randomly assigned into four groups (n\u0026thinsp;=\u0026thinsp;5 per group). Three groups were immunized intramuscularly with either Ad(S-N), Ad(S-N)-RBD135, or an undecorated Ad encoding GFP, Ad(GFP), as a control vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The fourth group was immunized by aerosol with Ad(N) using a vibrating mesh nebulizer ensuring distribution to the whole respiratory tract as previously described \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. All animals were given a homologous booster immunization using the same route on day 25. Twenty-four days post-boost, pigs were intranasally infected with PRCV135. Nasal swabs were collected daily to monitor viral shedding. Four days after the viral challenge, all animals were humanely culled, and tissues were collected for virological, pathological, and immunological analyses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePigs in the control Ad(GFP) group exhibited significant gross and histopathological lung lesions, with N protein immunohistochemical (IHC) staining in airway epithelial cells and evidence of inflammatory infiltrates in both the airways and lung parenchyma (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;E). Pigs in the aerosol Ad(N) group showed comparable levels of gross and histopathological changes to the control group. In contrast, both the Ad(S-N) and Ad(S-N)-RBD135 intramuscularly immunized animals demonstrated markedly reduced gross pathology, with largely absent histopathological lesions (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Representative images of gross pathology, histopathology, and IHC staining (including brown N protein immunolabeling) in lung sections are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF.\u003c/p\u003e\u003cp\u003eViral shedding was analyzed in nasal swabs by plaque assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was a significant decrease in viral shedding after PRCV infection in both Ad(S-N) and Ad(S-N)-RBD135 immunized animals as assessed by the area under the curve. No virus was detected in tracheal swabs, BAL and lungs at 4 days post infection (dpi) of the Ad(S-N)-RBD135 group except for one animal in trachea and BAL (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). Viral load in BAL and tracheal swabs was also significantly reduced in the Ad(S-N) group compared to control. Ad(S-N)-RBD135 immunization significantly reduced viral load in deep nasal swabs collected at postmortem (53 days post-prime compared to Ad(S-N)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). No virus was detected in the lungs of 4 out of the 5 Ad(N) immunized animals as detected by plaque assays.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results indicate that immunization with either Ad(S-N) or Ad(S-N)-RBD135 is highly efficient in reducing viral shedding and viral load in tissues as well as eliminating lung pathology following PRCV135 virus challenge. In most measures of viral load Ad(S-N)-RBD135 showed the greatest reduction compared to controls, but there was no statistically significant difference between Ad(S-N) and Ad(S-N)-RBD135 immunized animals except for the viral load in deep nasal swabs at postmortem. Aerosol immunization with Ad(N) did not reduce lung pathology or viral load following PRCV challenge.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibody responses following prime boost immunization and infection in pigs.\u003c/b\u003e Circulating antibody responses were measured by ELISA in serum at regular intervals throughout the study and in BAL at 53 days post prime against full-length recombinant Spike (ISU-1 strain) and N proteins (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Intramuscular immunization with Ad(S-N) or Ad(S-N)-RBD135 induced significantly higher serum Spike-specific IgG titers than control or Ad(N) groups. IgG levels also increased post-boost and remained elevated, at 49 and 53 days post prime. Serum IgA titers were lower than IgG; they declined after priming, but were boosted after the second immunization, followed by decline and stabilizing at 49 and 53 days post prime. The Ad(S-N)-RBD135 group exhibited significantly higher IgG and IgA responses compared to Ad(S-N) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B\u003cb\u003e).\u003c/b\u003e We also assayed the responses to recombinant RBD135, which as expected were lower to those observed against full length Spike (\u003cb\u003eSuppl. Fig S2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn BAL, a similar pattern was observed, with Ad(S-N)-RBD135 and Ad(S-N) immunization inducing the highest S-specific IgG responses compared to control and Ad(N) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Notably, no IgA responses were detected in BAL following IM immunization, suggesting that high serum S-specific IgG may transudate into the lung, while local delivery may be required to induce mucosal IgA.\u003c/p\u003e\u003cp\u003eSerum neutralization mirrored the ELISA results, with Ad(S-N)-RBD135 showing higher 50% inhibition titers compared to Ad (S-N) as determined by the area under the curve (AUC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Only 2 out of the 5 Ad(S-N)-RBD135 immunized animals exhibited neutralizing activity in BAL. This may be due to the dilution of BAL fluid during the sampling process. Serum from Ad(S-N)-RBD135 and Ad(S-N) immunized animals also cross-neutralized multiple PRCV strains (PRCV137, ISU-1, 310) and TGEV, with Ad(S-N)-RBD135 consistently showing significantly higher neutralizing titers compared to Ad(S-N) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The reduction in histopathology and viral load in nasal swabs, tracheal swabs and BAL correlated with neutralizing and Spike-specific IgG titers in serum and BAL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). N-specific IgG titers were detected in the serum of one or two animals from the Ad(S-N), Ad(S-N)-RBD135, and Ad(N) immunized groups, but at very low levels compared to S-specific responses. In BAL, responses were detected also in only one or two animals from each group (data not shown). However it is clear that no neutralizing antibodies were induced by Ad(N) immunization.\u003c/p\u003e\u003cp\u003eIn summary, IM prime boost immunization with Ad(S-N) and Ad(S-N)-RBD135 induced S-specific IgG antibodies in serum and BAL, with IgA titers detected in serum only, but not BAL. Decoration of the Ad virus vector with RBD135 increased significantly the neutralizing titers against a range of PRCV strains and TGEV compared to Ad(S-N) vaccine. All immunization regimes induced N- specific IgG antibodies in serum and BAL, although at a much lower level compared to S-specific responses\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytokine responses following immunization and PRCV challenge in pigs.\u003c/b\u003e IFNg ELISpot analysis was performed to quantify IFNg producing cells in PBMC, BAL and tracheobronchial lymph nodes (TBLN), following stimulation with either live PRCV135 or peptide pools covering the S and N proteins (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Intramuscular immunization with Ad(S-N) and Ad(S-N)-RBD135 induced significantly greater S-specific IFNg ELISpot responses in PBMC compared to the control Ad(GFP) and Ad(N) groups, with a clear boosting effect following the second immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand Suppl Fig S3)\u003c/b\u003e. All three vaccines, Ad(S-N), Ad(S-N)-RBD135, and Ad(N), induced comparable N- and PRCV- specific IFNg ELISpot responses (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Importantly Ad(N) although given by AE induced both N- and PRCV- specific responses in blood. Responses to S, N and PRCV were significantly greater in the immunized groups compared to control Ad(GFP) as evaluated by AUC (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD,E,F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eT cell responses were also analyzed by intracellular cytokine staining (ICS) in PBMC (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). IFNg and TNF production by CD4\u0026thinsp;+\u0026thinsp;and CD8β\u0026thinsp;+\u0026thinsp;T cells was measured following PRCV135 virus or S and N peptide stimulation (gating strategy shown in \u003cb\u003eSuppl. Fig. S4\u003c/b\u003e). Ad(S-N) induced significantly greater PRCV specific IFNg producing CD4\u0026thinsp;+\u0026thinsp;cells compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), while Ad(S-N)-RBD135 immunization induced significantly higher S- specific IFNg and TNF CD8 T cell responses compared to the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J). The blood S-specific IFNg ELISpot responses were associated with reduced histopathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK).\u003c/p\u003e\u003cp\u003eAd(S-N) and Ad(S-N)-RBD135 groups exhibited S-specific IFNg ELISpot responses in BAL (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). Ad(S-N), Ad(S-N)-RBD135 and Ad(N) elicited significant N- and PRCV-specific responses compared to the control (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). ICS revealed a significantly higher proportion of N- specific IFNg and TNF cytokine producing cells in the Ad(N) group compared to the other immunization regimes. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-J). The BAL S-specific IFNg ELISpot responses correlated with reduced lung gross and histopathology and BAL viral load (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIFNg ELISPOT cytokine responses were also detected in the TBLN, which drain the site of PRCV infection (\u003cb\u003eSuppl Figs S5 A-F\u003c/b\u003e). Significantly higher responses to the S and peptide pool antigens were induced in the Ad(S-N) immunized animals compared to the Ad(GFP) control. In contrast, no statistically significant increase in responses was observed in the Ad(S-N)- RBD135- or Ad(N)-immunized animals versus controls. Greater proportions of S-specific IFNg and TNF CD8 producing cells compared to the Ad(GFP) controls were also detected in Ad(S-N) immunized animals by ICS (\u003cb\u003eSuppl Figs S5 G-J\u003c/b\u003e). These differences may reflect distinct antigen presentation pathways and tissue tropism of the vaccine vectors.\u003c/p\u003e\u003cp\u003eThese data indicate that intramuscular prime-boost immunization with Ad(S-N) or Ad(S-N)-RBD135 induced S-specific cellular responses, including IFNg ELISpot and IFNg and TNF production in PBMCs, BAL and TBLN. All three vaccine regimes, Ad(S-N), Ad(S-N)-RBD135 and Ad(N), elicited N-specific responses, with Ad(N) producing the most robust CD8\u0026thinsp;+\u0026thinsp;IFNg and TNF cytokine producing CD4 and CD8 T cells in BAL. Blood and BAL and TBLN S- specific IFNg ELISpot responses were associated with reduced gross and histopathology following PRCV135 challenge.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunogenicity of multivalent \u0026ldquo;cocktail\u0026rdquo; in mice and pigs\u003c/b\u003e. Having shown that antibody responses to the PRCV135 S correlate with protection, we next wished to determine whether a cocktail of four Ad vaccine constructs, each decorated with RBDs from either PRCV135, PEHV, PDCoV, or PEDV could increase immune breadth and induce robust cross-reactive antibody and T cell responses. We used the same design as before, with vaccines encoding S-N from PRCV135 in the Ad genome and decorated with RBDs from either PRCV135, PHEV, PDCoV or PEDV: Ad(S-N)-RBD135, Ad(S-N)-RBD-PHEV, Ad(S-N)-RBD-PDCoV, and Ad(S-N)-RBD-PEDV respectively. Mice were immunized intramuscularly with either Ad decorated with only PRCV135 (Ad(S-N)-RBD135) or a cocktail containing equal IU of the four different RBD decorated Ads (cocktail 4). Animals received two vaccine doses three weeks apart and were culled two weeks post boost (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Serum IgG antibody responses against each of the RBDs present in the cocktail vaccine 35 days post prime were assessed by endpoint ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Robust and comparable responses against RBD135 were observed after administration of both vaccines. IgG responses against PHEV and PDCoV were only observed after administration of the cocktail vaccine. A detectable response against PEDV was achieved in only one animal in the cocktail vaccine group. Splenic T cell responses against PRCV135 S and N antigens were measured by IFNg ELISpot (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Responses against S and N antigens were induced in both vaccine groups, with comparable magnitude between the groups. Taken together, the data confirmed an added benefit of including PHEV and PDCoV RBD decorated Ads as components of a multivalent cocktail vaccine. Due to the failure of PEDV RBD to induce detectable IgG responses in mice, this component of the cocktail was excluded for subsequent studies in pigs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePigs were randomly assigned to two groups (n\u0026thinsp;=\u0026thinsp;4 per group). One group was immunized intramuscularly with Ad(S-N)-RBD135, and the other with a cocktail containing equal IU of the three different RBD decorated Ads: Ad(S-N)-RBD135, Ad(S-N)-RBD-PHEV and Ad(S-N)-RBD-PDCoV (cocktail 3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). All animals received a homologous booster immunization on day 28. Serum samples were collected weekly, and IgG antibody responses against each RBD in the cocktail were evaluated by endpoint ELISA. RBD-135-specific IgG were detected following administration of Ad(S-N)-RBD135 and cocktail 3 vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), whereas PHEV- and PDCoV-specific responses were observed only following cocktail 3 immunization. RBD-135 responses following cocktail 3 immunization were comparable to those observed in the initial challenge study and exhibited neutralizing activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Interestingly, responses to Ad(S-N)-RBD-135 were also detected in ELISA with rec PHEV or PDCoV, suggesting the induction of cross-reactive antibodies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn summary, intramuscular immunization with a cocktail of three Ad constructs displaying RBDs from PRCV135, PHEV, PDCoV induced serum IgG responses in mice and pigs. The RBD-135 responses were comparable to those elicited by immunization with the single RBD, indicating that this is a viable strategy for inducing antibodies against multiple antigens.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe previously described a novel Ad-based vaccine platform utilizing the DogTag/DogCatcher protein superglue system to facilitate rapid, modular covalent decoration of Ad capsids with vaccine antigens \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Here, for the first time, we evaluated the immunogenicity and efficacy of an antigen decorated Ad vector vaccine in a large, natural host species. Both Ad(S-N) and the antigen decorated Ad(S-N)-RBD135 vaccines were highly efficacious, almost completely abrogating lung pathology and significantly reducing viral load following PRCV135 virus challenge. The Ad(S-N)-RBD135 immunized animals had lower viral loads in the upper respiratory tract, particularly evident from deep nasal swabs, suggesting that Ad(S-N)-RBD135 may offer improved protection against viral transmission compared to Ad(S-N), although this remains to be tested. Importantly, decoration with RBD135 significantly enhanced anti-PRCV135 neutralizing antibody responses, both in serum and BAL. Vaccine induced S-specific cytokine T cell responses were not significantly different to Ad(S-N) responses in pigs and the N-specific responses were comparable between the decorated Ad(S-N)-RBD135, undecorated Ad(S-N), and Ad(N) vaccines.\u003c/p\u003e\u003cp\u003eCurrent COVID-19 vaccines, which are primarily based on the spike protein, reduce the severity of lung disease, but virus escape from humoral immunity occurs. It is therefore important to assess whether including additional antigens that induce T cell immunity could provide broader protection against antibody escape variants. Human studies suggest that T cell responses to the N protein correlate with less severe COVID-19 disease \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Furthermore mucosal respiratory immunization is considered to be more effective in inducing local immune responses at the site of infection \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Here we evaluated whether responses to the N could protect against disease. Following Ad(N) delivery to the respiratory tract by aerosol, BAL and TBLN cytokine producing cells were induced, with dominant CD8\u0026thinsp;+\u0026thinsp;T cell responses. Antigen-specific cytokine producing cells were detected in blood at levels comparable to those in animals given Ad(S-N) and Ad(S-N)-RBD135 vaccines parenterally. Antibody responses against N were detected in one or two animals from the Ad(S-N) and Ad(S-N)-RBD135 and Ad(N) AE group, although at much lower titers compared to S.\u003c/p\u003e\u003cp\u003eHowever, despite these local and systemic immune responses, no protection to PRCV challenge was observed - there was no reduction in viral load or lung pathology after aerosol delivery of the Ad(N) vaccine. This is in agreement with other pre-clinical studies. Immunization via the respiratory tract with recombinant parainfluenza virus expressing N did not confer protection in hamsters, while immunization with S provided complete protection against SARS-CoV challenge in the lower respiratory tract \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In mouse and hamster models, N-only mRNA immunization offered only modest protection, whereas combined S\u0026thinsp;+\u0026thinsp;N mRNA vaccination provided much stronger protection \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Similarly, Ad vectored N vaccine did not reduce viral load in lung or brain in K18-hACE2 mice but did so in combination with S \u003csup\u003e22\u003c/sup\u003e. In chickens, immunization with recombinant N protein elicited cellular immune responses but no protection, whereas S1 was protective \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn contrast recombinant N-protein vaccine (OVX033) induced cross-reactive T cells and protected Syrian hamsters against several variants \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. An N-based vaccine Convacell has been licensed for human use in Russia. This squalene adjuvanted recombinant N-protein vaccine induced strong IgG and Th1/Th2 cytokine responses in mice and rabbits, T-cell responses in marmosets, and was safe/immunogenic in hamsters and non-human primates \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Immunized Syrian hamsters showed reduced lung histopathology, lower virus proliferation, lower relative lung weight and faster body weight recovery \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Several N-based vaccine prototypes are also in clinical trials but published data on their efficacy and safety remain limited \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSince the emergence and rapid spread of PRCV, most pigs have developed immunity to both PRCV and TGEV. In contrast, PDCoV and PEDV are rapidly expanding their geographic range \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. PHEV, the only beta coronavirus of pigs, has a worldwide prevalence, causing severe vomiting and wasting and/or encephalomyelitis. With TGEV now rare in much of the world, fewer vaccines are available in North America and Europe, whereas parts of Asia continue to use them due to ongoing outbreaks. Vaccines for PEDV have been developed and are widely used, although continued surveillance is required due to antigenic variation \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. No licensed PHEV or PDCoV vaccines currently exist, making the development of effective vaccines increasingly urgent. Since antibodies targeting the spike protein of PRCV135 were effective against PRCV, we evaluated the potential of a multivalent vaccine cocktail expressing the RBDs from PRCV, PDCoV, and PHEV.\u003c/p\u003e\u003cp\u003eMultivalent display of RBD domains from diverse sarbecoviruses as a strategy to increase immune breadth has been described previously using protein nanoparticles including those based on ferritin and lumazine synthase \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. A similar approach displaying receptor binding domains from diverse influenza A haemagglutinin proteins has also been used to design broadly neutralizing influenza vaccines \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In these studies, multiple different RBDs have been attached to each nanoparticle in a \u0026lsquo;mosaic\u0026rsquo; design. Some studies have demonstrated that this mosaic design leads to preferential induction of antibodies that bind to conserved regions of the RBD and therefore have greater capacity to cross-react with different viral strains \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, this approach presents some challenges; assessing the abundance of different RBDs on mosaic particles typically requires the availability of highly specific monoclonal antibodies for each RBD which might not be possible for RBDs from related sarbecoviruses or antigen variants. Due to subtle differences in size and structure, it is possible that some RBDs may bind preferentially and thus be overrepresented on particles compared to others. In a recent study comparing mosaic RBD nanoparticles with cocktails of homotypic RBD nanoparticles, both approaches delivered comparable immune breadth \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In the current study, we delivered three separate RBD Ad vaccine constructs in an equimolar cocktail to simplify quality control processes and maximise translatability. Our approach enabled particulate delivery of multiple RBDs to achieve potent humoral immunity against different porcine CoV similar to protein nanoparticle technologies.\u003c/p\u003e\u003cp\u003eGiven its established utility in evaluating vaccine candidates for influenza and SARS-CoV-2, the PRCV pig model provides a robust translational tool for assessing vaccine efficacy in a large, natural host species. The RBD-decorated Ad vaccine platform evaluated here demonstrated strong immunogenicity, inducing robust S-specific antibody and T cell responses correlating with protection as determined by reduction in viral load and lung pathology. Furthermore, delivery of a cocktail of multiple vaccine constructs, each decorated with RBDs from different CoV, induced strong serum antibody responses. These results provide valuable insights into the development of CoV vaccines in a highly relevant large animal model to inform future vaccine design for both livestock and human health.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eViruses, and cells.\u003c/b\u003e PRCV strains 86/135308 (135) and 86/137004 (137) were gifts from S. Cartwright (Animal and Plant Health Agency). 135 and 137 were both isolated from respiratory tract tissue in the UK in 1986\u003csup\u003e32\u003c/sup\u003e. AR310 was purchased from ATCC (ATCC VR2384). ISU-1 was purchased from Bei Resources (NR-43286). TGEV strain FS772/70 was a gift from S. Cartwright \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. All cells and viruses were propagated as previously described \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of Ad-DogTag vaccine vectors encoding PRCV135 S and N genes\u003c/b\u003e. A replication defective (E1/E3 deleted) molecular clone of Ad5 with DogTag (DIPATYEFTDGKHYITNEPIPPK) flanked by GSGGSG linkers inserted into the hexon HVR5 loop (pBAC-Ad5(GFP)DogTag) was previously described \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Recombinant vectors expressing PRCV135 S and N (Accession number OM830318), codon optimized for mammalian expression, were generated through cloning of gene constructs into pENTR4.CMVp and subsequent Gateway-mediated insertion into the Ad5 E1 locus of pBAC-Ad5(GFP)DogTag using Invitrogen Gateway site-specific recombination technology, replacing the green fluorescent protein (GFP) gene with the PRCV135 S and N genes. Virus was rescued and purified as described \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. HEK293A (293A) cells (Invitrogen) were used for Ad vectors expressing GFP and 293TREX cells (Invitrogen) were used for Ad vectors expressing PRCV135 S and N genes. Ads were titered as previously described \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein production and purification\u003c/b\u003e. DNA sequences for expression of porcine coronavirus RBD domains with and without N terminal DogCatcher were codon optimised and cloned into mammalian protein expression plasmid pcDNA3.4. To facilitate secretion, the Igk-leader sequence METDTLLLWVLLLWVPGSTGD was introduced at the N terminus of the fusion protein, and a C-terminal C-tag (EPEA) was added to enable affinity purification. DogCatcher-porcine coronavirus RBDs were expressed in suspension ExpiCHO-S cells (Thermo Fisher); protein was harvested from culture supernatant, affinity purified using C-tag affinity resin (Thermo Fisher) using an AKTA chromatography system (GE Healthcare), and dialyzed into Tris-buffered saline (TBS) pH 7.8. RBD sequences were defined as follows: PRCV135 residues 300\u0026ndash;449 of the S protein; PEDV (strain CV777, accession no. OR3484341) residues 504\u0026ndash;637; PHEV1968 residues 311\u0026ndash;608 as defined by Bahoussi \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e34\u003c/sup\u003e; and PDCoV residues 300\u0026ndash;419 of AML40825.1, identical to OH-FD22 \u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePRCV135 N protein was transiently expressed in Expi293FTM cells according to the Gibco Expi293\u0026trade; Expression System User Guide. Presence of protein was determined by detection of the C-tag by western blot (\u003cb\u003eSuppl. Fig S6\u003c/b\u003e). Following confirmation of expression, the protein was scaled up for expression at 300mL followed by purification using a C-tag affinity matrix column (ThermoFisher). Purified protein was dialysed overnight into phosphate-buffered saline (PBS) and concentrated using an Amicon Ultra Centrifugal Filter, 10 kDa MWCO to adjust the protein concentration to 1 mg/mL. The protein was quantified using Pierce\u0026trade; BCA Protein Assay Kits by a BCA kit (ThermoFisher). Successful expression of N protein was confirmed by binding of DA3, a mouse monoclonal which binds the N protein of PRCV and TGEV \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAd capsid decoration: conjugation reactions and vaccine preparation.\u003c/b\u003e Ligand-decorated vaccines were prepared by co-incubation of Ad-DogTag vectors with DogCatcher-RBD protein with 3.5\u0026micro;M DogCatcher-RBD for every 1 \u0026times;10\u003csup\u003e10\u003c/sup\u003e viral particles in sucrose storage buffer (10 mM Tris-HCl, 7.5% w/v sucrose, pH 7.8). Reactions were incubated for 16 h at 4\u0026deg;C during which time spontaneous conjugation of DogCatcher and DogTag occurred. To remove excess ligand, conjugated vaccines were dialyzed into sucrose storage buffer using SpectraPor dialysis cassettes with a 1000-kDa molecular weight cutoff (MWCO)(Spectrum Labs). Dialysis reduced excess ligand by at least 10-fold, as measured by densitometry on Coomassie-stained SDS-PAGE. Ligand coverage was determined by SDS-PAGE using the equation described in \u003csup\u003e15\u003c/sup\u003e and ranged from 55\u0026ndash;73% hexon coverage. Ad titer after ligand-conjugation was determined as described in \u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMouse immunizations\u003c/b\u003e. All mouse procedures were performed in accordance with the terms of the UK Animals (Scientific Procedures) Act (Project License PP5949437) and approved by the Oxford University Ethical Review Body. Female BALB/c mice (aged 6\u0026ndash;8 weeks, Envigo), housed in specific pathogen-free environments, were immunized intramuscularly (I.M.) by injection of 50 \u0026micro;L of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 \u0026micro;L total). Vaccine doses used were as described in figure legends. For cocktail vaccine preparations, the constituent vaccines were mixed at an equal fractional dose prior to administration to give a total dose of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e IU/animal. Endotoxin dose was \u0026lt;\u0026thinsp;1 EU per mouse in all studies. Experiments were performed at Biomedical Services, University of Oxford.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePig immunization and viral challenge studies\u003c/b\u003e. Animal studies were approved by ethical review processes at The Pirbright Institute and VetQuest and conducted under project licence PP7764821 according to regulations set out in the UK Government Animal (Scientific Procedures) Act 1986. The Pirbright Institute conforms to Animal Research: Reporting of Animal Experiments guidelines. In all experiments, animals acclimatized for at least 7 days and were randomized into different groups and pens using Excel by the animal services staff. Researchers processing the samples were only aware of the pig numbers, not the group assignments. The pathologists were blinded to the group allocation when assessing the samples for gross pathology during post-mortem examination and subsequently during the histopathological assessment. All conditions were kept the same between groups. Viral shedding and lung pathology were the main outcome variables for the studies.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImmunization and PRCV viral challenge pig experiment\u003c/span\u003e: Twenty pigs six weeks of age, genetically composed of one-quarter Large White, one-quarter Landrace, and one-half Hampshire breeds, were obtained from a high health farm in which no PRCV antibodies could be detected. The animals were randomly assigned into four treatment groups of five pigs each. Three groups received 1x10\u003csup\u003e9\u003c/sup\u003e infectious units (IU) of either Ad(GFP), Ad(S-N)-RBD135, or Ad(S-N) intramuscularly (IM) with 1 ml administered in each trapezius muscle behind the ear. The fourth group received 1x10\u003csup\u003e9\u003c/sup\u003e IU of Ad(N) by aerosol (AE) administered over 2\u0026ndash;5 minutes using an Aerogen Solo vibrating mesh nebulizer (Aerogen, Dangan, Galway, Ireland) following sedation with a 4 mg/kg Zoletil and 2 mg/kg Stresnil (Elanco UK AH Limited). The animals were boosted 25 days later by the same immunization route.\u003c/p\u003e\u003cp\u003eTwenty-four days post boost, animals were inoculated intranasally with 2 x 10\u003csup\u003e7\u003c/sup\u003e pfu PRCV135 using a MAD300 device to deliver 2 ml into each nostril following sedation with a 3 mg/kg Zoletil and 1.5 mg/kg Stresnil. The pigs were humanely culled four days later with an overdose of pentobarbital sodium anaesthetic, confirmed by the permanent cessation of circulation. Daily nasal swabs were collected into 2ml virus transport medium (tissue culture medium 199 (Sigma-Aldrich) with 25mM Hepes, 0.035% sodium bicarbonate, 0.5% BSA, penicillin, streptomycin and nystatin) for analysis of nasal shedding. Whole blood for isolation of peripheral blood mononuclear cells (PBMCs) and serum were obtained weekly throughout the study. At postmortem blood, bronchoalveolar lavage (BAL) and tracheobronchial lymph node (TBLN), were collected for cell isolation as previously described \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The lung accessory lobe, BAL, tracheal and deep nasal swabs were collected for analysis of viral load in the respiratory tract.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImmunization with vaccine cocktail in pigs.\u003c/span\u003e Eight (6\u0026ndash;8 weeks old) pigs were randomly assigned to two groups of 4 pigs and after one week acclimatization immunized intramuscularly with: either Ad(S-N)-RBD135 or cocktail containing equal IU of the three different RBD decorated Ads: Ad(S-N)-RBD135, Ad(S-N)-RBD-PHEV and Ad(S-N)-RBD-PDCoV (cocktail 3) to give a total dose of 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e IU/animal.(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). All pigs received a homologous booster immunization on day 28. Serum samples were collected weekly for evaluation of antibody titers.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLung gross pathology, histopathology, and immunohistochemistry.\u003c/b\u003e The percentage of pulmonary consolidation, including both dorsal and ventral aspects was assessed blindly by a veterinary pathologist at necropsy \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Formalin fixed tissues were processed by a routine histology method. Haematoxylin and eosin staining and immunohistochemistry (IHC) against PRCV nucleoprotein (N) using a monoclonal antibody were performed on serially sectioned formalin-fixed paraffin embedded tissues \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of infectious virus by plaque assays.\u003c/b\u003e Tissues were homogenised in PBS containing 0.1% BSA using C-tubes (Miltenyi) and an Octo-MACS tissue dissociator. Homogenised tissue was clarified by low-speed centrifugation, and the supernatant titrated by plaque assay in ST cells. 2g each of lung, trachea and eyelid were processed. Swine testis (ST) cells were seeded in sterile 12-well tissue culture plates and allowed to grow until confluent. Ten-fold serial dilutions of sample were prepared in PRCV growth medium (EMEM supplemented with 0.02% yeast extract, 10% tryptose-phosphate broth, 2mM L-glutamine, 10,000 U/ml penicillin, 10,000 \u0026micro;g/ml streptomycin, and 1% nystatin solution). Cells were washed with PBS, then 250\u0026micro;l of each dilution applied to the cells in duplicate. After 1 hour incubation at 37\u0026deg;C, the inoculum was removed and 1ml N,N-Bis(2-hydryoxyethyl)-2aminoethanesulphonic acid (BES) medium containing 1% agar added. Cells were fixed with 4% formaldehyde in PBS 48 hours post-infection and plaques visualised using 0.1% crystal violet.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVirus neutralization PRCV135.\u003c/b\u003e Serum harvested weekly throughout the animal studies and BAL collected at post-mortem were diluted 1:2 in triplicate in PRCV growth media, then serially diluted 1:2 across a 96-well plate. 100\u0026micro;l of PRCV growth media containing 1x10\u003csup\u003e3\u003c/sup\u003e pfu of TGEV or PRCV 135, 137, 310 or ISU-1 was added to 100\u0026micro;l of each serum dilution and incubated at room temperature for 30 minutes. 200\u0026micro;l of virus/serum mix was added to ST cells and incubated at 37\u0026deg;C for 48 hours. Cells were fixed with 4% formaldehyde and visualised with 0.1% crystal violet, before wells were marked as cytopathic effect (no neutralization) or no cytopathic effect (neutralization). Neutralization titer resulting in 50% neutralization per ml (VNT\u003csub\u003e50\u003c/sub\u003e) was calculated using the Reed-Muench calculation \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eELISA.\u003c/b\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMice\u003c/span\u003e: ELISA analysis of mouse serum samples was performed as described previously (Biswas et al 2011). Briefly, 96-well ELISA plates were coated with porcine coronavirus RBDs at 2 \u0026micro;g/mL. Plates were blocked with 10% Milk powder in 0.05% PBS-Tween20. Serum was diluted 3-fold in 0.05% PBS-Tween20 with a starting dilution of 1 in 100. Secondary antibody (STAR117A, BioRad) was diluted 1 in 5000 and the signal was developed in \u003cem\u003ep\u003c/em\u003e-nitrophenylphosphate (pNPP) solution. The signal was allowed to develop for 30 minutes and plates were read at 405 nm on an Infinite M Plex (Tecan). Data were analyzed in Prism v10.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePigs\u003c/span\u003e: Maxisorp 96-well plates (ThermoScientific) were coated with 100\u0026micro;l/well of 0.5\u0026micro;g/ml recombinant full-length spike protein form ISU-1 as previously described diluted in PBS overnight at 4\u0026deg;C \u003csup\u003e11,12\u003c/sup\u003e. The full-length S protein and RBD only of PRCV 135 and PRCV ISU-1 were aligned using blastp (NCBI), indicating 95% sequence identity within the RBD and 97% sequence identity across the whole spike protein (\u003cb\u003eSuppl Fig. S7\u003c/b\u003e). Recombinant RBDs from PRCV135, PEHV and PDCoV or N were produced as described above. Plates were washed with PBS containing 0.05% Tween20 and blocked for one hour at room temperature with block buffer (PBS containing 0.05% Tween20, 4% w/v Marvel milk powder). Serum was serially diluted 2-fold in block buffer and 100\u0026micro;l/well applied to the plate for 1 hour at room temperature. Serum was washed off the plate, then 100\u0026micro;l/well of secondary antibody (anti-porcine IgG H\u0026amp;L-HRP or anti-porcine IgA-HRP, Biorad) diluted 1:20,000 in block buffer added for 1 hour at room temperature. The assay was developed using 50\u0026micro;l/well of TMB high-sensitivity substrate solution (Biolegend), and the reaction terminated using equal volume of 1M sulphuric acid. Optical density at 450nm and 630nm was measured, and endpoint titer determined.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIFN\u003c/b\u003eg \u003cb\u003eELISpot assay.\u003c/b\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMice\u003c/span\u003e: For mouse spleen IFNg ELISpot analysis, ELISPOT plates with hydrophobic PVDF membranes (MERCK) were coated overnight at 4\u0026deg;C with 5 mg/mL monoclonal rat anti-mouse IFNg antibody (AN18 Mabtech) in carbonate-bicarbonate coating buffer (MERCK). The next day the anti-mouse IFNg antibody was removed and the plates blocked in 100 mL aMEM (MERCK) supplemented with 10% foetal bovine serum, 100 U/ml penicillin and streptomycin, 4 mM L-Glutamine and 50 mM 2-Mercaptoethanol). Splenocytes were seeded in duplicate at 250,000 cells/well, 125,000 cells/well and 62,500 cells/well and stimulated with the PRCV S and N peptide at a final concentration of 5 \u0026micro;g/ml (Mimotopes, Melbourne, Australia \u0026minus;\u0026thinsp;16-mer peptides overlapping by 12 residues with three pools for the S protein (residues 1-100, 101\u0026ndash;200, and 201\u0026ndash;305) and one pool of 94 peptides for N) pools. As a negative control duplicate wells containing 250,000 cells for each mouse remained unstimulated. After 18 hours incubation at 37\u003csup\u003eo\u003c/sup\u003eC with 5% CO\u003csub\u003e2\u003c/sub\u003e, the plates were washed and incubated with 1 \u0026micro;g/ml Biotin Rat anti-Mouse IFNg (Mabtech) for 2 hours at room temperature. The plates were washed before incubating with 1 \u0026micro;g/ml of Streptavidin alkaline phosphatase (Mabtech) for 1 hour at room temperature. The assay was developed using AP conjugate substrate kit (Biorad). Spots were counted using an AID ELISpot reader (AID). Results were expressed as the number of IFNg-producing cells per 10\u003csup\u003e6\u003c/sup\u003e stimulated cells after subtracting the average number of spots from the unstimulated wells.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePigs\u003c/span\u003e: ELISpot plates with mixed cellulose esters (MCE) membrane (MERCK) were coated overnight at 4\u0026deg;C with 0.5 \u0026micro;g/ml purified Mouse Anti-Pig IFNg (559961, BD Pharmingen\u0026trade;). The plates were washed with PBS, and blocked for 2 hours at 37\u0026deg;C with RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% foetal calf serum and 100 U/ml penicillin-streptomycin (Thermofisher)). Cryopreserved cells were thawed, washed, seeded in triplicate at 250,000 cells/well and stimulated with either PRCV135 virus (MOI\u0026thinsp;=\u0026thinsp;1) or PRCV S or N peptide pools (Mimotopes, Melbourne, Australia) as previously described \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Sixteen-mer peptides overlapping by 12 residues with three pools for the S protein (residues 1-100, 101\u0026ndash;200, and 201\u0026ndash;305) and one pool of 94 peptides for N at a final concentration of 5 \u0026micro;g/ml were used. Triplicate wells of cells were also stimulated with 6 \u0026micro;g/ml ConA (Sigma-Aldrich) as a positive control and with medium as a negative control. After 48 hours the plates were washed followed by an incubation with 0.5 \u0026micro;g/ml Biotin Mouse Anti-Pig IFNg (559958, BD Pharmingen\u0026trade;) for 2 hours at room temperature. The plates were washed before incubating with 2 \u0026micro;g/ml of Streptavidin alkaline phosphatase (S921, Invitrogen) for 1 hour at room temperature. The assay was developed using AP conjugate substrate kit (Biorad). Spots were counted using an AID ELISpot reader (AID) and Immunospot ELISpot plate reader (Cellular Technology Limited). Results were expressed as the number of IFNg -producing cells per 10\u003csup\u003e6\u003c/sup\u003e stimulated cells after subtracting the average number of spots in medium-stimulated control wells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntracellular cytokine staining (ICS).\u003c/b\u003e Cell suspensions obtained from PBMC, BAL and TBLN were thawed and seeded in duplicate wells of a 96-well plate (1 x 10\u003csup\u003e6\u003c/sup\u003e cells per well). Cells were stimulated overnight with either PRCV 135 (MOI 0.5), or they were treated with RPMI-1640 medium containing stable glutamine, 100 IU/ml penicillin, 100 \u0026micro;g/ml streptomycin, and 10% FCS) as a negative control. Alternatively, some cells were stimulated for 5 hours with 2 \u0026micro;g/ml of overlapping peptides spanning the S and N proteins as described above. Positive control wells were stimulated with a cocktail containing phorbol 12-myristate 13-acetate (PMA) and ionomycin (BioLegend). Brefeldin A (GolgiPlug\u0026trade;, BD Biosciences) was added to all wells and incubated for 4 hours. Subsequently, cells were centrifuged for 5 minutes at 500g and washed twice with PBS.\u003c/p\u003e\u003cp\u003eAll cells were stained with Near-Infrared Fixable LIVE/DEAD stain (Invitrogen), CD4-PerCP-Cy5.5 (74-12-4, BD Biosciences) and CD8β-FITC (PPT23, Bio-Rad Laboratories) for 20 mins at 4\u0026deg;C. Cells were washed and were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) for 20 mins at 4\u0026deg;C. Cells were washed with BD Perm/Wash\u0026trade; buffer (BD Biosciences) and incubated with anti-IFNg (P2G10, BD Biosciences) and anti-TNF (MAb11, BioLegend) antibodies. Following two more washes and resuspension in PBS, cytokine producing CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells were analyzed using a MACSquant Analyzer 16 (Miltenyi). Wells containing only cells and medium for each animal were considered as a negative control (unstimulated), and the frequency of cytokine-producing cells was determined by subtracting the values from unstimulated cells. Data analysis was performed using FlowJo software version 10.10.0.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analysis.\u003c/b\u003e GraphPad Prism 10.2.0 was used to perform statistical analyses. First, Normality tests (D\u0026rsquo;Agostino-Pearson omnibus, Anderson-Darling, Shapiro-Wilk and Kolmogorov-Smirnov test) were done to test whether the data were normally distributed. The normally distributed data were analyzed using either One-way ANOVA or Two-way ANOVA and Bonferroni\u0026rsquo;s multiple comparisons test while the data that were not normally distributed were analyzed using Mann-Whitney test. Mouse data were analyzed using non-parametric Kruskal-Wallis test or Mann-Whitney test, depending on the number of comparisons being performed. Significant differences are presented on each graph (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). To assess correlations between immune parameters and virological/pathological measures, a non-parametric Spearman correlation coefficient (\u003cem\u003eρ\u003c/em\u003e) was computed for each pair of measures (viral load, antibody response, T cell responses and pathology) using Graphpad Prism v 10.0.3. All Ad(S-N), Ad(S-N)-RBD135, Ad(N) and Ad(GFP) samples were included in the analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;R.A.R., B.G.C, L.M.R, R.D, L.A.H.B, and M.D.J.D are employees of SpyBiotech Ltd. S.B. is CSO and co-founder of SpyBiotech Ltd. The other authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u003c/strong\u003e We are grateful to the animal staff at VetQuest and the Pirbright Institute for providing excellent animal care. We thank the Immunological Toolbox and Flow Cytometry Scientific Technology Platforms at the Pirbright Institute.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability.\u0026nbsp;\u003c/strong\u003eThe datasets generated and/or analysed during the current study are provided as a source data file. Any further data may be provided by contacting the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThis work was supported by the UKRI Biotechnology and Biological Sciences Research Council (BBSRC) BB/X014266/1 and \u0026nbsp;the Pirbright Institute\u0026rsquo;s Strategic Programme Grants (ISPGs) BBS/E/PI/230001C, BBSRC National Bioscience Research Infrastructure: High Containment and Low Containment Services and Science Platforms grants BBS/E/PI/23NB0003; BBS/E/PI/23NB0004.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.K., S.B., M.D.J.D. and E.T. designed the study. S.W., E.J.B., R.A.R., S.C., M.D.J.D. and E.T. curated the data. Formal data analysis was performed by S.W., E.J.B., R.A.R., S.C., F.J.S., B.P., S.K., S.B., M.D.J.D., and E.T. E.T., S.K., E.B., G.F., S.B., and M.D.J.D. obtained funding. Investigation was performed by S.W., E.J.B., R.A.R., S.C., A.V., B.G.C., L.M.R., R.D., L.A.H.B., C.R., C.F.H., D.M., J.R.S., M.I., E.B., E.M., T.C., F.J.S., B.P., S.K., S.B., M.D.J.D., and E.T. Methodology was developed by S.W., E.J.B., R.A.R., F.J.S., B.P., S.K., S.B., M.D.J.D., and E.T. Resources were acquired by E.T., S.B., M.D.J.D., and S.K., and supervision provided by E.T., S.B. and M.D.J.D. Data was validated by S.W., E.J.B., R.A.R., S.C., B.P., S.K., M.D.J.D., and E.T., and visualized by S.W., E.J.B., R.A.R., F.J.S., S.K., M.D.J.D., and E.T. S.W., E.J.B., R.A.R., S.K., M.D.J.D., and E.T. wrote the original draft of the manuscript, with additional review and editing provided by C.R., D.M., J.R.S., M.I., E.B., F.J.S., B.P. and S.B.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePiret, J. \u0026amp; Boivin, G. Pandemics Throughout History. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e Volume 11\u0026ndash;2020 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fmicb.2020.631736\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fmicb.2020.631736\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaif, L. J. Animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? \u003cem\u003eRev Sci Tech\u003c/em\u003e 23, 643\u0026ndash;660 (2004). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.20506/rst.23.2.1513\u003c/span\u003e\u003cspan address=\"https://doi.org:10.20506/rst.23.2.1513\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaif, L. J. \u0026amp; Jung, K. Comparative Pathogenesis of Bovine and Porcine Respiratory Coronaviruses in the Animal Host Species and SARS-CoV-2 in Humans. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e 58 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1128/jcm.01355-20\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1128/jcm.01355-20\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurlewicz-Podbielska, H. \u0026amp; Pomorska-M\u0026oacute;l, M. Porcine Coronaviruses: Overview of the State of the Art. \u003cem\u003eVirol Sin\u003c/em\u003e 36, 833\u0026ndash;851 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s12250-021-00364-0\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s12250-021-00364-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVlasova, A. N. \u003cem\u003eet al.\u003c/em\u003e Porcine coronaviruses.. \u003cem\u003eEmerging and Transboundary Animal Viruses\u003c/em\u003e, 79\u0026ndash;110 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWoo Patrick, C. Y. \u003cem\u003eet al.\u003c/em\u003e Discovery of Seven Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and Avian Coronaviruses as the Gene Source of Gammacoronavirus and Deltacoronavirus. \u003cem\u003eJournal of Virology\u003c/em\u003e 86, 3995\u0026ndash;4008 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1128/jvi.06540-11\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1128/jvi.06540-11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, Q., Vlasova, A. N., Kenney, S. P. \u0026amp; Saif, L. J. Emerging and re-emerging coronaviruses in pigs. \u003cem\u003eCurr Opin Virol\u003c/em\u003e 34, 39\u0026ndash;49 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.coviro.2018.12.001\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.coviro.2018.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLednicky, J. A. \u003cem\u003eet al.\u003c/em\u003e Independent infections of porcine deltacoronavirus among Haitian children. \u003cem\u003eNature\u003c/em\u003e 600, 133\u0026ndash;137 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41586-021-04111-z\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41586-021-04111-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTscherne, A. \u0026amp; Krammer, F. A review of currently licensed mucosal COVID-19 vaccines. \u003cem\u003eVaccine\u003c/em\u003e 61, 127356 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.vaccine.2025.127356\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.vaccine.2025.127356\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJung, K. \u003cem\u003eet al.\u003c/em\u003e Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus. \u003cem\u003eJ Virol\u003c/em\u003e 81, 13681\u0026ndash;13693 (2007). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1128/jvi.01702-07\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1128/jvi.01702-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKeep, S. \u003cem\u003eet al.\u003c/em\u003e Porcine respiratory coronavirus as a model for acute respiratory coronavirus disease. \u003cem\u003eFront Immunol\u003c/em\u003e 13:867707 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSedaghat-Rostami, E. \u003cem\u003eet al.\u003c/em\u003e Porcine respiratory coronavirus as a model for acute respiratory disease: mechanisms of different infection outcomes. \u003cem\u003eThe Journal of Immunology\u003c/em\u003e 214, 1643\u0026ndash;1660 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/jimmun/vkaf066\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/jimmun/vkaf066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcDonald, I., Murray, S. M., Reynolds, C. J., Altmann, D. M. \u0026amp; Boyton, R. J. Comparative systematic review and meta-analysis of reactogenicity, immunogenicity and efficacy of vaccines against SARS-CoV-2. \u003cem\u003eNPJ Vaccines\u003c/em\u003e 6, 74 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41541-021-00336-1\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41541-021-00336-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKeeble, A. H. \u003cem\u003eet al.\u003c/em\u003e DogCatcher allows loop-friendly protein-protein ligation. \u003cem\u003eCell Chem Biol\u003c/em\u003e 29, 339\u0026ndash;350.e310 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.chembiol.2021.07.005\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.chembiol.2021.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDicks, M. D. J. \u003cem\u003eet al.\u003c/em\u003e Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2. \u003cem\u003eMol Ther\u003c/em\u003e 30, 3639\u0026ndash;3657 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.ymthe.2022.08.002\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.ymthe.2022.08.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartini, V. \u003cem\u003eet al.\u003c/em\u003e Distribution of Droplets and Immune Responses After Aerosol and Intra-Nasal Delivery of Influenza Virus to the Respiratory Tract of Pigs. \u003cem\u003eFront Immunol\u003c/em\u003e 11, 594470 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fimmu.2020.594470\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fimmu.2020.594470\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeng, Y. \u003cem\u003eet al.\u003c/em\u003e An immunodominant NP(105\u0026ndash;113)-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease. \u003cem\u003eNat Immunol\u003c/em\u003e 23, 50\u0026ndash;61 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41590-021-01084-z\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41590-021-01084-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKundu, R. \u003cem\u003eet al.\u003c/em\u003e Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts. \u003cem\u003eNat Commun\u003c/em\u003e 13, 80 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41467-021-27674-x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41467-021-27674-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChew, C. K. \u003cem\u003eet al.\u003c/em\u003e Safety, efficacy and immunogenicity of aerosolized Ad5-nCoV COVID-19 vaccine in a non-inferiority randomized controlled trial. \u003cem\u003eNPJ Vaccines\u003c/em\u003e 9, 209 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41541-024-01003-x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41541-024-01003-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuchholz, U. J. \u003cem\u003eet al.\u003c/em\u003e Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 101, 9804\u0026ndash;9809 (2004). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1073/pnas.0403492101\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1073/pnas.0403492101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHajnik, R. L. \u003cem\u003eet al.\u003c/em\u003e Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. \u003cem\u003eSci Transl Med\u003c/em\u003e 14, eabq1945 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1126/scitranslmed.abq1945\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1126/scitranslmed.abq1945\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDangi, T., Class, J., Palacio, N., Richner, J. M. \u0026amp; Penaloza MacMaster, P. Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2. \u003cem\u003eCell Rep\u003c/em\u003e 36, 109664 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.celrep.2021.109664\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.celrep.2021.109664\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIgnjatovic, J. \u0026amp; Galli, L. The S1 glycoprotein but not the N or M proteins of avian infectious bronchitis virus induces protection in vaccinated chickens. \u003cem\u003eArchives of Virology\u003c/em\u003e 138, 117\u0026ndash;134 (1994). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/BF01310043\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/BF01310043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeir, R. \u003cem\u003eet al.\u003c/em\u003e Immune responses to mucosal vaccination by the recombinant A1 and N proteins of infectious bronchitis virus. \u003cem\u003eViral Immunol\u003c/em\u003e 25, 55\u0026ndash;62 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1089/vim.2011.0050\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1089/vim.2011.0050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrimard, C. \u003cem\u003eet al.\u003c/em\u003e OVX033, a nucleocapsid-based vaccine candidate, provides broad-spectrum protection against SARS-CoV-2 variants in a hamster challenge model. \u003cem\u003eFront Immunol\u003c/em\u003e 14, 1188605 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fimmu.2023.1188605\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fimmu.2023.1188605\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRak, A., Isakova-Sivak, I. \u0026amp; Rudenko, L. Overview of Nucleocapsid-Targeting Vaccines against COVID-19. \u003cem\u003eVaccines\u003c/em\u003e 11, 1810 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRabdano, S. O. \u003cem\u003eet al.\u003c/em\u003e Immunogenicity and In Vivo Protective Effects of Recombinant Nucleocapsid-Based SARS-CoV-2 Vaccine Convacell(\u0026reg;). \u003cem\u003eVaccines (Basel)\u003c/em\u003e 11 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3390/vaccines11040874\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3390/vaccines11040874\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGerdts, V. \u0026amp; Zakhartchouk, A. Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. \u003cem\u003eVet Microbiol\u003c/em\u003e 206, 45\u0026ndash;51 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.vetmic.2016.11.029\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.vetmic.2016.11.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCohen, A. A. \u003cem\u003eet al.\u003c/em\u003e Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models. \u003cem\u003eScience\u003c/em\u003e 377, eabq0839 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1126/science.abq0839\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1126/science.abq0839\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, C. \u003cem\u003eet al.\u003c/em\u003e Mosaic RBD nanoparticle elicits immunodominant antibody responses across sarbecoviruses. \u003cem\u003eCell Rep\u003c/em\u003e 43, 114235 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.celrep.2024.114235\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.celrep.2024.114235\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanekiyo, M. \u003cem\u003eet al.\u003c/em\u003e Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses. \u003cem\u003eNat Immunol\u003c/em\u003e 20, 362\u0026ndash;372 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41590-018-0305-x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41590-018-0305-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown, I. \u0026amp; Cartwright, S. New porcine coronavirus? \u003cem\u003eVet Rec\u003c/em\u003e 119, 282\u0026ndash;283 (1986). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1136/vr.119.11.282\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1136/vr.119.11.282\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarwes, D. J. \u0026amp; Pocock, D. H. The polypeptide structure of transmissible gastroenteritis virus. \u003cem\u003eJ Gen Virol\u003c/em\u003e 29, 25\u0026ndash;34 (1975). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1099/0022-1317-29-1-25\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1099/0022-1317-29-1-25\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBahoussi, A. N. \u003cem\u003eet al.\u003c/em\u003e Genetic Characteristics of Porcine Hemagglutinating Encephalomyelitis Coronavirus: Identification of Naturally Occurring Mutations Between 1970 and 2015. \u003cem\u003eFront Microbiol\u003c/em\u003e 13, 860851 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fmicb.2022.860851\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fmicb.2022.860851\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJi, W. \u003cem\u003eet al.\u003c/em\u003e Structures of a deltacoronavirus spike protein bound to porcine and human receptors. \u003cem\u003eNature Communications\u003c/em\u003e 13, 1467 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41467-022-29062-5\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41467-022-29062-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorgan, S. B. \u003cem\u003eet al.\u003c/em\u003e Aerosol Delivery of a Candidate Universal Influenza Vaccine Reduces Viral Load in Pigs Challenged with Pandemic H1N1 Virus. \u003cem\u003eJ Immunol\u003c/em\u003e 196, 5014\u0026ndash;5023 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.4049/jimmunol.1502632\u003c/span\u003e\u003cspan address=\"https://doi.org:10.4049/jimmunol.1502632\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eREED, L. J. \u0026amp; MUENCH, H. A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS12. \u003cem\u003eAmerican Journal of Epidemiology\u003c/em\u003e 27, 493\u0026ndash;497 (1938). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/oxfordjournals.aje.a118408\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/oxfordjournals.aje.a118408\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Porcine respiratory coronavirus, pig, nucleocapsid, aerosol, DogTag/DogCatcher, adeno viral vector, spike, nucleocapsid","lastPublishedDoi":"10.21203/rs.3.rs-7978161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7978161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePorcine respiratory coronavirus (PRCV) infection in pigs provides a physiologically and immunologically relevant large-animal model for acute respiratory coronavirus disease and vaccine evaluation. We investigated a replication-defective adenovirus (Ad) vaccine platform that enables display of antigens on the Ad capsid surface using the DogTag/DogCatcher protein superglue system. Ad vectors encoding the PRCV135 Spike (S) and Nucleocapsid (N) proteins were evaluated with or without surface decoration with the PRCV135 Spike receptor-binding domain (RBD). Both Ad(S-N) and RBD-decorated Ad(S-N)-RBD135 vaccines were protective against PRCV135 challenge. RBD135 decoration significantly enhanced neutralizing antibody titers in serum and bronchoalveolar lavage. In contrast, aerosol immunization with Ad(N) induced robust T cell responses but no protection. A multivalent cocktail of RBD-decorated Ad vectors targeting PRCV, porcine hemagglutinating encephalomyelitis virus (PHEV), and porcine deltacoronavirus (PDCoV) elicited antibodies against all three pathogens. This study demonstrates the versatility and potency of antigen-decorated Ad vectors as a platform for next-generation coronavirus vaccines in a relevant large natural host model.\u003c/p\u003e\u003cp\u003e158 words\u003c/p\u003e","manuscriptTitle":"Efficacy of a novel antigen-decorated adenoviral vaccine platform against porcine respiratory coronavirus infection in a large natural host","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-14 20:56:36","doi":"10.21203/rs.3.rs-7978161/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"21e159c5-06db-43c6-a082-d6eb9041b5a6","owner":[],"postedDate":"November 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57814557,"name":"Biological sciences/Biotechnology"},{"id":57814558,"name":"Health sciences/Diseases"},{"id":57814559,"name":"Biological sciences/Immunology"},{"id":57814560,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-12-11T17:23:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-14 20:56:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7978161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7978161","identity":"rs-7978161","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}