Obtaining HBV core protein VLPs carrying SARS-CoV-2 nucleocapsid conserved fragments as vaccine candidates

Obtaining HBV core protein VLPs carrying SARS-CoV-2 nucleocapsid conserved fragments as vaccine candidates | 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 Research Article Obtaining HBV core protein VLPs carrying SARS-CoV-2 nucleocapsid conserved fragments as vaccine candidates Yadira Lobaina, Alexis Musacchio, Panchao Ai, Rong Chen, Edith Suzarte, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4740544/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Nov, 2024 Read the published version in Virology Journal → Version 1 posted 10 You are reading this latest preprint version Abstract The Hepatitis B core antigen (HBcAg) has been used as a carrierof several heterologous protein fragments based on its capacity to form virus-like particles (VLPs)and to activate innate and adaptive immune responses. In the present work, two chimeric proteins were designed as potential pancorona vaccine candidates, comprising the N- or C- terminal domain of SARS-CoV-2 nucleocapsid (N) protein fused to HBcAg. The recombinant proteins, obtained in E. coli , were named CN-1 and CND-1, respectively. The final protein preparations were able to form 10-25 nm particles, visualized by TEM. Both proteins were recognized by sera from COVID-19 convalescent donors; however,the antigenicity of CND-1 tends to be higher. The immunogenicity of both proteins was studied in Balb/C mice by intranasal route without adjuvant. After three doses, only CND-1 elicited a positive immune response, systemic and mucosal, against SARS-CoV-2 N protein. CND-1 was evaluated in a second experiment mixed with the CpG ODN-39M as nasal adjuvant. The induced anti-N immunity was significantly enhanced, and the antibodies generated were cross-reactive with N protein from Omicron variant, and SARS-CoV-1. Also, an anti-N broadcellular immune response was detected in spleen, by IFN-g ELISpot. The nasal formulation composed by CND-1 and ODN-39M constitutes an attractive component for a pancorona vaccine, by inducing mucosal immunity and systemic broad humoral and cellular responses against Sarbecovirus N protein. HBcAg nucleocapsid SARS-CoV-2 chimeric proteins pancorona vaccine intranasal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The Hepatitis B core (HBcAg) is one of the main structural antigens of hepatitis B virus (HBV) and constitutes a potent immunogen for humans and animals [ 1 ]. The extremely high immunogenicity of HBcAg can be explained by its particulate nature and its capacity to function as both T-cell-independent and T-cell-dependent antigen [ 2 ]. The natural HBcAg is assembled in particles of approximately 30nm with icosahedric geometry composed by 120 dimer of the viral capsid protein [ 3 ]. The repetitive motifs and the protuberances in the HBcAg particles surface confer the unique ability to bind and activate a high frequency of naive human and murine B cells [ 4 , 5 ]. HBcAg-specific B cells from unprimed mice are able to take up, process and present HBcAg to naive T-helper cells in vivo 10 5 times more efficiently than classical antigen presenting cells (APCs) [ 6 ]. The full length HBcAg, when is obtained as a recombinant protein in E. coli , retains its capacity to form virus-like particles (VLPs), able to encapsulate bacterial nucleic acid (mainly RNA) which confers potent Th1 adjuvant properties [ 7 , 8 ]. The HBcAg has been widely used in preclinical studies as carrier of heterologous epitopes forming chimeric proteins [ 9 – 15 ]. The most frequently used site for heterologous insertion has been the immunodominant c/e1 epitope, located in the center of the HBc primary sequence, which comprises a solvent-exposed loop that tolerates insertions of flexible peptide sequences [ 10 , 11 ]. Several data are available about the evaluation of such chimeric constructs in animal models [ 11 , 12 ]. It has been demonstrated that a heterologous sequence inserted into this internal loop is significantly more immunogenic than such fragment in the context of its native protein [ 13 ]. Nevertheless, due to the insertion in the loop implies structural constrains (length and particular conformation) of the heterologous motif, the N and C terminus fusions sites has been also explored with successful results [ 14 , 15 ]. In the present work two recombinant chimeric proteins including two fragments from SARS-CoV-2 nucleocapsid (N) protein fused to the C-terminus of HBcAg were designed and obtained. N protein is a conserved molecule among coronaviruses, which has emerged as an attractive antigen to be included in novel generation of pancorona vaccines [ 16 ]. The study published by Matchett et al., 2021, demonstrated that N protein, presented in the Ad5 platform, protected mice against SARS-CoV-2 challenge [ 17 ]. Dangi et al, 2021, also proved that the addition of N protein in a spike vaccine formulation, improved distal protection in mouse brain [ 18 ]. The two chimeric proteins, named CN-1 and CND-1, were obtained as recombinant proteins in E. coli , carrying fragments of SARS-CoV-2 N-terminal domain (NTD) (139 aa length) and C-terminal dimerization domain (CTD) (123 aa length), respectively. After the purification processes, preparations with more than 90% of purity were obtained and the presence of spherical particles of 10–25 nm were visualized by transmission electron microscopy (TEM). The antigenicity of both proteins was confirmed using a panel of SARS-CoV-2 convalescent’s sera and immunogenicity studies by intranasal route in Balb/C mice were done. CND-1 showed a higher antigenicity, and accordingly, it was also the most immunogenic. The immune response generated by intranasal administration of CND-1 protein was improved when the ODN-39M was added as adjuvant in the formulation. Importantly, the induced systemic humoral and cellular immune response was cross-reactive until SARS-CoV-1 level. 2. Materials and Methods 2.1 Biological and synthetic reagents Escherichia coli BL21 (DE3) : F– ompT gal dcm lonhsdSB(rB- mB-) k(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) was used for gene expression [ 19 ]. For plasmid propagation, E. coli strain XL1-blue [F’::Tn10 proAþBþ lacIq D(lacZ)M15/recA1 endA1gyrA96(NaIr) thi hsdR17(rk mþk) supE44 relA1 lac) was employed [ 20 ]. For the evaluation of animal samples, the following recombinant antigens were purchased from Sino Biological (China). N proteins from: SARS-CoV-2 Delta (40588-V07E29), and Omicron (40588-V07E34) variants; SARS-CoV-1 (40143-V08B), MERS-CoV (40068-V08B). In addition, the peptide N 351 − 365 from SARS-CoV-2 (ILLNKHIDAYKTFPP) was synthesized with ≥ 97% purity by Zhejiang Peptides Biotech (China). The ODN-39M, a 39 mer, whole phosphodiester backbone CpG ODN (5’-ATC GAC TCT CGA GCG TTC TCG GGG GAC GAT CGT CGG GGG-3’), was synthesized by Sangon Biotech (China). To evaluate the protein antigenicity the following antibody reagents were used. The anti-SARS-CoV-2 nucleocapsid polyclonal antibody (40588-T62) was purchased from Sino Biologicals (China). A monoclonal antibody anti-HBcAg (ab8637) was purchased from Abcam (USA). A panel of human sera from COVID-19 convalescent (N = 27) and negative (N = 10) donors were collected as part of a study approved by the Institutional Ethics Committee from The Eighth People’s Hospital of Dongguan (Guangdong Province, China). Informed written consent was obtained from each participant. The study description was already reported [ 21 ]. 2.2 Obtaining of the chimeric constructs CN-1 and CND-1 The following chimeric genes were chemically synthesized: CN-1: Truncated HBcAg core (1-149) + linker (GSSGGSSG) + N fragment (40–179) from SARS-CoV-2 (Delta variant) CND-1: Truncated HBcAg core (1-149) + linker (GSGGSG) + N fragment (248–371) from SARS-CoV-2 (Delta variant) Each chimeric gene was amplified by polymerase chain reaction (PCR) using the corresponding primers. The amplified band was purified and cloned into pGEM-T Easy Vector (Promega, USA). Positive clones were tested by restriction analysis, and sequencing. Each recombinant fragment was then cloned into pET28a plasmid. Positive clones were identified by restriction analysis and defined as pCN-1 and pCND-1. The E. coli strain BL21 (DE3) was transformed with each recombinant plasmid by electroporation. Each clone was later inoculated, at 0.05 of Optical Density (OD), in ZY medium supplemented with Kanamycin (50 µg/ml) and let to grow for 18 h at 28°C and 170 rpm-min (THZ-300, Blue Pard Inst, China). 2.3 Purification processes of CN-1 and CND-1 For both proteins, CN-1 and CND-1, a similar purification protocol was implemented, with some modifications. The transformed E. coli was grown during 18 h under the conditions described above, and the biomass was harvested by centrifugation at 5 000 x g for 15 min at 4 ° C. For cell disruption, 0.5 g of cells were resuspended in 50 mL of TE buffer (0.05M Tris-HCl, 5mM EDTA, pH 9.0) and treated at 4°C, with 20 cycles (30 seconds with 30 seconds rest) in a ultrasonic machine (FS-200T, Shanghai Sonxi US Inst, China), with 50% power rate and 20.3 kHz frequency. The resultant sample was centrifuged at 10, 000 x g for 15 min at 4 ° C. The supernatant was collected and filtered throughout 0.45 µm for subsequent purification steps. As a high resolution step, the ion exchange chromatography was selected based on the particular features of the HBcAg. A volume of 20 mL from the biomass disruption soluble fraction was ½ diluted with TE buffer and applied into Q Sepharose and SP Sepharose -fast flow ion exchangers (Cytiva, Sweeden) connected in tandem, and previously equilibrated with TE buffer. The loading volume of the sample was 10% and the employed flow rate was 3 cm/h. After application, columns were separated, washed independently with TE buffer and the bound proteins to SP Sepharose fast flow matrix eluted with 0.1M NaCl step gradient. The Q Sepharose fast flow matrix was employed to remove most of the protein and DNA contaminants present on the sample while CN-1 protein was eluted from the SP Sepharose fast flow matrix with high purity. The CN-1 protein fraction was collected at 0.3M NaCl. Gel filtration chromatography was used for final purification step. Sephacryl S-200 HR matrix (Cytiva, Sweden) was equilibrated with 0.05M Tris HCl, 5 mM EDTA, 0.15M NaCl, pH 9.0, at 15cm/h. The CN-1 fraction eluted at 0.3M NaCl from the SP Sepharose column was applied into the gel filtration chromatography. The CN-1 protein was collected as unique peak, which was later concentrated using Amicon system (USA), filtered by 0.22µm and stored at 4°C. In the case of CND-1, the protein was eluted from the SP Sepharose fast flow matrix at 0.1M NaCl with high purity. The CND-1 fraction collected was then applied to a gel filtration chromatography following the same conditions previously described for this step. The peak corresponding to CND-1 protein was then concentrated using Amicon system (USA), filtered by 0.22µm and stored at 4°C. The detection of the protein signal was followed by the absorbance at 280 nm in all steps 2.4 Analysis of protein samples BCA assay (Pierce, USA), was used to determine the protein concentration in all samples. The identity of each chimeric protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and dot blotting, using specific antibodies. Protein samples were subjected to 12% of acrylamide gel. SDS-PAGE gels were stained with Coomassie blue and scanned (iBright 1500, Invitrogen). Densitometry analysis, using Image J (1.41 version) software was employed to define the percentage corresponding with the protein band of interest. For Western blotting, protein samples were electro transferred from an acrylamide gel to a Immobilon-P membrane (Merck-Millipore, IRL), as described [ 22 ]. In turn, samples were applied directly to the membrane for dot blotting. The membrane from the two previous procedures was blocked with 5% skim milk in phosphate buffered saline (PBS) for 1 h at room temperature (RT), washed three times with – PBS − 0.05% Tween solution (PBS-T). The reaction with the anti-SARS-CoV-2 N polyclonal Ab generated in rabbit (SinoBiologicals, China) at 1:2500 dilution (or anti-HBcAg Mab (Abcam, USA) 1:1000 diluted), occurred during 1 h at RT. After proper washing, the incubation of the membrane with the peroxidase-conjugated goat anti-rabbit IgG (Chemicon, USA) at a 1/300 dilution, or anti-mouse IgG-peroxidase, respectively, occurred during 1 h at RT. Antibodies used during the whole procedure were diluted in 1% skim milk in PBS-T. Afterwards, upon membrane washing, the reaction was detected by incubation with Aminoethyl Carbazole (AEC) substrate solution (0.2mg/ml AEC and 0.03% H 2 O 2 in 50mM NaAc solution) at RT. 2.5 Visualization of Capsid Like Particles (CLPs) by Transmission Electron Microscopy (TEM) ETest company (Changsha, China) provided the specialized service of transmission electron microscopy analysis. CN-1 and CND-1 samples, at concentration of 0.25 mg/ml, were placed on a freshly glow-discharged, 400-mesh copper grid coated with Formvar and Carbon. After sample absorption and water washing, Uranyl Acetate stain was added. After 4 min of staining, grids were wick dried with Whatman no. 1 filter paper and later, during 20 min, were allowed to air dry. The Transmission Electron Microscope HT 7800 (Hitachi, Japan) with an acceleration voltage of 120 Kv and three magnifications: 25 000 x, 50 000 x, and 100 000 x, was the equipment used for sample visualization. For each sample, eight different fields were analyzed. The Image J software (Maryland, USA) was used to estimate the average particle size. 2.6 Characterization of CN-1 and CND-1 proteins, by ELISA, using human sera positive to SARS-CoV-2 Ninety-six-well high-binding polystyrene plates (Costar, USA) were coated with 3 µg/mL of each protein (CN-1 and CND-1), in sodium carbonate-sodium bicarbonate buffer, and incubated overnight at 4°C. Unspecific binding of the antibodies was avoided by blocking with 5% skim milk (Oxoid, UK) 1 h at 37°C. After five times washing with PBS-T, 100 µL of diluted serum sample in 2% skim milk -PBS-T were added and incubated for 2 hours at 37°C. After washing five times with PBS-T, bound antibodies were detected with a goat anti-human IgG antibody conjugated to horseradish peroxidase (Sigma-Aldrich, Germany) at 1:20000 dilution. After incubation for 1 hour at 37°C and five PBS-T washes, 100 µL of OPD substrate solution (Sigma-Aldrich, Germany) were added to each well and the mixture was incubated for 10 min in the dark at RT. The reaction was stopped by adding 0.2N Sulphuric Acid, and the optical density (O.D) at 492 nm was measured in a multiplate reader (FilterMax F3, Molecular Devices, USA). The data is represented as O.D measures. 2.7 Animal experiments in Balb/C mice Beijing Vital River Laboratory Animal Technology Co., Ltd, and Hunan Prima Drug Research Center Co., Ltd, conducted the mice experiments. Both animal facilities complied with the national standard of the People's Republic of China GB14925-2010. Each experimental protocol was subjected to analysis and approval by the Institutional Animal Care and Use Committee. Three doses of immunogens were intranasally (in) inoculated in each group of mice (N = 5 or N = 6), according to the defined design. Ten µg of each protein (CN/1 or CND-1) was administered per animal. All the immunogens were dissolved in sterile PBS in a volume of 50 µL. As negative controls, placebo groups were included in the experiments. In the first animal study, mice were distributed in three groups of six animals each. Group 1 was inoculated with CN-1 protein, Group 2 received CND-1 protein, and PBS was administered to the Group 3, as negative control. The administration schedule was 0, 15 and 30 days. Twenty-seven days after the last dose, mice were sacrificed. For the other study, mice were distributed in three groups of five animals each. Groups 1 and 2 received CND-1 protein and CND-1 + 15ug ODN-39M, respectively. Group 3 corresponded to the Placebo group, similar to the previous study. The administration schedule was 0, 7 and 21 days. Thirty days after the last dose, mice were sacrificed. Three different samples were obtained at the indicated time points: sera, spleens, and bronchoalveolar fluid (BALF). 2.8 Determination of antibody response by ELISA The Ab response in sera and BALF was measured by anti-IgG, IgG subclasses, and, -IgA ELISAs [ 23 ]. Briefly, 96 well high-binding plates (Costar, USA) were coated with N or HBcAg protein (3µg/mL) and blocked with 2% skim milk solution. Samples were evaluated in duplicates starting from 1/100 dilution of sera. BALF were tested without dilution. Specific horseradish peroxidase conjugates (Sigma, USA) and OPD (Sigma, USA)/hydrogen peroxide substrate solution were employed. The reaction was stopped using 2 N Sulphuric acid and multiplate reader (FilterMax F3, Molecular Devices, USA) was used to measure O.D at 492nm. In the graphics corresponding with sera Ab response log10 titers are represented. The arbitrary units of titers were calculated by plotting the O.D values obtained for each sample in a standard curve (hyper-immune serum of known titer). The positivity cut-off was established as 2 times the average of O.D obtained for a pre-immune sera pool. In the case of BALF, the antibody response was represented as O.D at 492 nm. 2.9 Evaluation of cell-mediated immunity by IFN-γ ELISpot The Mouse IFN-γ ELISpot antibody pair (Mabtech, Sweden) was employed to perform the ELISpot assay. Splenocytes (from five mice per group) were isolated in RPMI culture medium (Gibco, US) and processed individualized. In the case of Placebo group, splenocytes were processed as a pooled sample of five animals. Duplicates cultures (5x10 5 and 1x10 5 splenocytes per well) were incubated for 48 h at 37 ° C and 5% CO 2 , in a 96 well round-bottom culture plate (Costar, USA) with 10 µg/mL of each stimulating agent: N 351 − 365 peptide, N proteins, and Concanavalin A (ConA). Control wells of cells without stimulus (medium) were included for all samples. After the incubation period, the whole content of each culture plate was transferred to ELISpot pre-coated plates (Merck-Millipore, USA) and incubated for 16–20 h at 37 ° C and 5% CO 2 . The following steps were performed according to the manufacturer´s recommendation. For spot counting, a stereoscopic microscope (AmScope SM-1TSZ, USA) coupled to a digital camera was employed. 2.10 Statistical methods and analysis The GraphPad Prism version 5.00 statistical software (Graph-Pad Software, USA) was used for all the analyses. To reach a normal distribution, antibody titers were transformed to log10. For the non sero-converting sera, an arbitrary titer of 1:50 was assigned for statistical processing. As a parametric test, the One-way Anova test followed by a Tukey's post-test was selected for multiple group comparisons. For the non- parametric multiple comparisons, the Kruskal Wallis test and Dunns post-tests was used. P values were considered as: ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. 3. Results 3.1 Obtaining of CN-1 and CND-1 constructs Figure 1 a represents the design of the two chimeric proteins, CN-1 and CND-1. The DNA sequence corresponding to each chimeric gene was cloned into the PET-28a vector. E. coli BL21 (DE3) was transformed with each recombinant plasmid and grown in ZY auto-induction medium. An overexpressed band of molecular weight (MW) around 32.7 kDa and 31.1 kDa, matching with the theoretical size of the CN-1 and CND-1 proteins respectively, was detected by SDS-PAGE, accounting for the 5% of the total cellular proteins (Fig. 1 b and 1 c). In addition, the identity was confirmed by western blot assay. Each band was immune-identified with anti-SARS-CoV-2 nucleocapsid polyclonal Ab (Fig. 1 b and 1 c, right panel). After expression of each recombinant construct, the biomass from the bacterial culture was disrupted. Under established conditions, both chimeric proteins were mainly associated to the cell disruption soluble fraction (Fig. 1 d and 1 e). The subsequent high resolution purification steps were similar for both proteins (Fig. 1 f), with slight modifications. In general, a sample for each disruption process (for CN-1 and CND-1) was applied into two ionic exchange chromatographies coupled in tandem (Q and SP Sepharose fast flow). Contaminants were attached to the first anion exchange matrix whereas each target protein was bound to the cation one. Since the elution from the SP Sepharose was conducted by step gradient, additional contaminants were removed at low molarities of NaCl. The CN-1 and CND-1 proteins were eluted at 0.5M and 0.1M NaCl, respectively, with a high level of purity. As final polishing purification step, the gel filtration chromatography was introduced using Sephacryl S-200 HR. Both proteins were obtained with more than 90% of purity and were properly immune-identified by both, polyclonal Abs anti-SARS-CoV-2 N and the anti-HBcAg Mab (Figs. 1 g, 1 h and 1 i). The characterization by SDS-PAGE and Western blotting of each purified protein, under native conditions, is shown in Figs. 1 j and 1 k. Interestingly, aggregated forms of high MW were visualized for both proteins although the highest MW species were only visualized in the CND-1 sample. 3.2 Visualization of Capsid like Particles TEM was selected as the analytical method to define the ability of each chimeric protein to form CLPs. Three pictures, visualized with three magnification factors are represented in Fig. 2 . Particles with similar spherical morphology were detected in the samples of the two chimeric proteins. The particles visualized for CND-1 and CN-1 samples have an average size of 12.2 nm and 19.4 nm, respectively. On the other hand, the vaccine preparation composed by CND-1 mixed with ODN-39M adjuvant showed a similar pattern of particles with mean size of 15.2 nm (data not shown). No clear evidences of dimeric or aggregated structures were observed in the evaluated samples. 3.3 Recognition of CN-1 and CND-1 by SARS-CoV-2 positive sera A panel of human sera (positive and negative against SARS-CoV-2 antigens) was used to determine the recognition of the chimeric proteins CN-1 and CND-1. The Fig. 3 represents the results obtained. Both recombinant proteins were recognized by the positive human sera although the recognition of CN-1 tended to be lower. Furthermore, three sera samples belonging to the negative donors group showed a positive recognition for both chimeric proteins. These specific sera samples were additionally tested against HBcAg, by ELISA, and showed a positive recognition to hepatitis B capsid protein (data not shown). 3.4 CN-1 and CND-1 immunogenicity in Balb/C mice by intranasal route A mice experiment was conducted to explore the immunogenicity of each chimeric protein administered by intranasal route. Three doses were administered without adjuvant (Fig. 4 a). In sera, only the group receiving the CND-1 protein elicited an IgG antibody (Ab) response specific against N protein from SARS-CoV-2 and HBcAg, as shown in Fig. 4 b and 4 d respectively. Accordingly, a similar pattern was obtained when the specific IgA Abs were measured in BALF samples (Fig. 4 c and 4 d). To test the CMI, spleen cells were in vitro stimulated with the conserved peptide N 351 − 365 and the N protein from SARS-CoV-2. The frequency of IFN-γ secreting cells was measured by ELISpot assay. According to the humoral immune response, mice receiving CND-1 by intranasal route exhibited a positive response against the peptide N 351 − 365 (Fig. 4 f). Of note, no response was detected for any group upon stimulation with N protein from SARS-CoV-2 (data not shown). 3.5 Immunogenicity of the mixture CND-1 + ODN-39M Based on the nasal immunogenicity results previously described, CND-1 protein was selected to combine with the mucosal adjuvant ODN-39M as a potential nasal vaccine candidate. Three doses of each immunogen (CND-1 alone or CND-1 + ODN-39M) were intranasally administered (Fig. 5 a). As expected, the group receiving CND-1 + ODN-39M elicited high levels of IgG Abs in sera against N protein from SARS-CoV-2, with statistical differences compared to titers induced in the group inoculated with CND-1 without adjuvant (Fig. 5 b). In turn, upon IgG subclass pattern analysis, similar and high levels of IgG1 and IgG2a were elicited in animals of the group receiving the protein with adjuvant, indicating the induction of a Th1-like pattern of response against N protein from SARS-CoV-2 (Fig. 5 c). On the other hand, similar to the IgG Abs in sera, the levels of mucosal IgA against N protein from SARS-CoV-2 measured in BALF were higher in the group receiving CND-1 with adjuvant (p < 0.05) (Fig. 5 d). Furthermore, the HBcAg-specific antibody response exhibited a similar behavior. The IgG levels in sera and IgA levels in BALF, against the carrier protein, were higher in the group receiving the combination of CND-1 + ODN-39M showing statistical differences compared to the group receiving the chimeric protein without adjuvant (Fig. 5 e and 5 f). Samples were additionally tested against N proteins from SARS-CoV-2 Omicron variant, SARS-CoV-1 and MERS-CoV so as to determine the scope of the systemic humoral immune response. As shown in Fig. 6 a, high levels of IgG were detected against N proteins from SARS-CoV-2 Omicron variant, and SARS-CoV-1, for the group immunized with the adjuvated preparation, whereas no response was obtained against N protein from MERS-CoV. Finally, to test the CMI, spleen cells were in vitro stimulated with the conserved peptide N 351 − 365 and the N protein from SARS-CoV-2 Delta and Omicron variants, and the N protein from SARS-CoV-1. In line with the humoral immune response generated, mice intranasally inoculated with the mixture CND-1 + ODN-39M exhibited the higher response. The 100% of animals in this group were positive against the peptide N 351 − 365 , and the N protein from SARS-CoV-2 Delta and Omicron variants. On the other hand, the IFN-γ response specific against the N protein from SARS-CoV-1 was positive in two out 5 animals. 4. Discussion In the present work the N protein from SARS-CoV-2 (Delta strain) was selected as the source of the heterologous fragments to be fused to HBcAg since it is a conserved coronavirus antigen and target of CMI response in humans [ 24 ]. Accordingly, several TCD4 + and TCD8 + epitopes have been mapped on it [ 25 – 27 ]. Particularly, the two regions selected for fusing to HBcAg, NTD and CTD fragments, exhibit high percentage of identity at sarbecovirus level (> 90%) and their lengths and structures are compatible to be fused at the C-terminus site of the HBcAg. Despite the most used carrier site in the HBcAg is the immunodominant c/e1 epitope, we discard it due to its lack of compatibility with the structure of the N fragments. In turn, the length of each N selected fragment (139 aa and 123 aa for CN-1 and CND-1, respectively) inserted in such site would affect the capacity of the resultant chimeric protein to form capsid-like particles, a crucial feature of the HBcAg which correlates with its high immunogenicity by intranasal route [ 28 ] As a first signal of proper conformation, both chimeric proteins (CN-1 and CND-1) were mainly associated to the soluble fraction after biomass disruption. In addition, a scale up purification process, without using chaotropic agents, could be established for each construct. After analysis by TEM, particles of 10–20 nm were visualized, indicating that the fusion of the heterologous fragments from SARS-CoV-2 N protein does not limit the particle formation ability of the HBcAg. This result is in accordance to several reports describing the formation of CLPs upon fusion of large heterologous cargoes at the C terminus of HBcAg, such as the Dom III of the envelope protein from Zika virus (100 aa), a region from Staphylococcus aureus nuclease (163aa), and the Hepatitis C core fragment (91aa) [ 15 , 29 , 30 ]. In general, the CLPs average size obtained here for both chimeric proteins is on the range, but a little lower to the previously reported for the full-length HBcAg antigen. It is widely known that the full length HBcAg (183 aas), expressed as recombinant protein in E.coli , is able to form VLPs with a size ranging between 25–30 nm [ 31 ]. However, these VLPs show an electro-dense core, corresponding with the presence of encapsulated bacterial nucleic acids, which confers the extremely high immunogenicity of HBc particles. In our work we selected the truncated HBcAg variant (149 aas) as carrier for the N fragments, considering the remaining capacity of this variant to form CLPs structures and also the big size of the cargoes. In this case the presence of associated bacterial nucleic acid, after the obtaining of the chimeric proteins in E. coli , was not observed for none of the proteins as it is shown in the TEM photographs and also after agarose gel electrophoresis analysis (data not shown). The truncated HBcAg lacks the Arginine-rich domain (naturally located the C-terminal end) which is the main responsible for the nucleic acid binding during the natural DNA encapsulating role of the HBV nucleocapsid protein [ 32 ]. In the design of the CN-1 and CND-1 chimeric proteins we hypothesized that the SARS-CoV-2 N fragments included could contribute to the nucleic acid binding effect, considering the reported RNA binding capacity of these domains [ 33 ]. However, the final outcome didn’t show evidences of bacterial nucleic acid association in the purified preparations of both chimeric proteins. This could explain in part the lower immunogenicity observed for both antigens after intranasal administration. On the other hand, it was interesting the tendency to a lower recognition of CN-1 protein compared with CND-1 protein, when they were tested against a panel of SARS-CoV-2 convalescent sera. This fact can be explained by the higher immunogenicity of the CTD region of SARS-CoV-2 nucleocapsid protein compared with the NTD region, or due to a better conformation obtained for the CND-1 chimeric protein. On the other hand, it was interesting the aggregation profile of both proteins, observed by SDS-PAGE and Western blot under native conditions. Both of them form species of high molecular weight though bands with the highest MW were only visualized for CND-1. It indicates that CTD region tends to form highly aggregated structures, in line with the report by Lo YS, et al , 2013, where authors demonstrated the oligomerization capacity of the CTD from the human coronavirus 229E nucleocapsid protein [ 33 ]. Taking the advantage of the capacity of HBcAg to induce mucosal Abs when it is administered by intranasal route [ 28 ], as well as the relevance to induce an anti-SARS-CoV-2 broad mucosal immune response for cutting the transmission upon viral entry or at least, to decrease the infection levels in lungs, the intranasal route was selected for evaluation the chimeric proteins in Balb/C mice. The results reveal the superior immunogenicity of CND-1 compared to CN-1 when they were administered by intranasal route without adjuvants. CN-1 neither induces humoral nor CMI response anti-N protein from SARS-CoV-2 under these conditions. This result is in accordance to the lower recognition of this protein by human sera and the absence of high MW aggregates by SDS-PAGE. Accordingly, none or a very low Ab response against HBcAg was obtained for CN-1. On the contrary, CND-1 was immunogenic, measured in both systemic humoral immunity and CMI against N protein from SARS-CoV-2 and the N 351 − 365 peptide, respectively. Of note, the recombinant SARS-CoV-2 N protein obtained also in E. coli , recently evaluated by our group, was not able to induce such immunity in mice without adjuvant by intranasal route [ 34 ]. Results indicate that the presentation to the immune system of the CTD fragment is favored on the context of HBcAg, probably due to the highly aggregated nature of the resultant chimeric protein, and the intrinsic adjuvant properties of the HBcAg scaffold [ 4 , 5 , 7 , 8 ]. It is known that the presence of particulate structures or protein aggregates in the nanometric size range result better immunogens after nasal administration [ 35 ]. Despite 100% of responders were obtained in the CND-1 group by anti-N IgG ELISA in sera and ELISpot assay using the conserved T CD4 + N peptide (N 351 − 365 ) as stimulating agent, in BALF, only two animals exhibited IgA anti-N positive response. In turn, also by ELISpot assay, no response was detected when the N protein of SARS-CoV-2 was used as stimulating agent. Based on these results, we decided to study the inclusion of the ODN-39M as adjuvant in the CND-1 nasal formulation. The ODN-39M is a CpG ODN which bind to and activate Toll-like receptor 9 (TLR9) for initiating the innate immune response and consequently, enhance the adaptive immune response [ 36 ]. CpG ODNs are being evaluated in more than 100 clinical trials focused on preventing or treating allergy, infectious diseases and cancer [ 37 ]. Particularly, the ODN-39M has been previously evaluated in combination with four different viral recombinant capsid proteins proving its adjuvant effect [ 34 , 38 – 40 ]. In accordance, in the present work, the addition of ODN-39M to the CND-1 protein preparation for nasal administration significantly enhanced the humoral immunity (systemic and mucosal) and systemic CMI induced against N protein from SARS-CoV-2. In turn, the systemic IgG subclasses suggest a Th1 pattern of response, similar to that described for viral infections. Actually, the anti-N immunity, comprising both arms of the immune response, has been correlated with protection against coronavirus in mice, monkeys, and humans [ 17 , 41 , 24 ]. For humoral immunity, Dangi et al, 2002 proved that anti-N Abs passively transferred to a relevant animal model decreased the viral load upon SARS-CoV-2 challenge [ 42 ]. Accordingly, in humans, anti-N Abs have been administered to treat COVID-19 patients with promising results against the severity of the disease [ 43 ]. On the other hand, N protein is as target of cross-reactive memory T cells, which were associated to protect SARS-CoV-2 naïve contacts from infection, thereby supporting the inclusion of this protein antigen in a new vaccine generation [ 24 ]. The breath of the systemic anti-N immunity, induced by intranasal administration of the CND-1 + ODN-39M preparation, revealed its potentiality to be considered as an attractive component of a pancorona vaccine. The humoral immune response reached cross-reactivity until SARS-CoV-1 level, whereas for CMI, high level of response was obtained when the conserved peptide N 351 − 365 was used as simulating agent. Importantly, this peptide spans a conserved region among sarbecovirus which is immunodominant in SARS-CoV-2 Balb/C infected mice. In addition, it was able to partially protect mice from SARS-CoV-2 infection when administered in the context of Venezuelan equine encephalitis replicon particles vector. This experiment provided direct evidence about the protective role of memory T-cells against the conserved peptide N 351 − 365 [ 44 ]. The homology level of SARS-CoV-2 nucleocapsid protein within the beta coronavirus genus supports the results obtained in the present work. The identity of N protein within sarbecoviruses subgenus is in the range of 87–99% whereas the identity between nucleocapsid of SARS-CoV-2 and MERS-CoV is only 48% [ 45 ]. Despite the response obtained in our mice experiments did not react with the N protein from MERS-CoV, we consider that cross-immunity at sarbecoviruses levels is very important. Currently, more than 50 SARS-related coronaviruses have been identified in 10 species of bat [ 46 ]. Bat-borne SARS-related coronaviruses are considered target of potential pandemics due to their diversity. Accordingly, Crook JM et al, 2021 expressed that the highest the probability for homologous recombination of sarbecoviruses through co-infection, the biggest the possibility of novel zoonotic emergence [ 47 ]. It is important to highlight that the immunogenicity obtained for the preparation of CND-1 + ODN-39M, nasally administered, is similar to that generated by the recombinant N protein combined with the same adjuvant. In a previous work, the N + ODN-39M preparation, administered by intranasal route, induced anti-N CMI response in spleen and also elicited broad humoral immunity in both, sera and lungs [ 34 ]. Nevertheless, CND-1 shows a higher nasal immunogenicity, being able to induce anti-N immune response when it is administered without any adjuvant. Such an intrinsic response is potentiated by the inclusion of the ODN-39M in the preparation. Finally, since the chimeric protein CND-1 use the HBcAg as scaffold or platform, the anti-HBcAg humoral immunity was also determined. Interestingly, positive response of IgG in sera and IgA in BALF against HBcAg were enhanced after combination with ODN-39M. It is known that the immune response against HBcAg partially contributes to the protection against HBV infection [ 48 , 49 ], therefore, since CND-1 is also able to generate such anti-HB immunity, it constitutes a further advantage of this antigenic preparation. In addition, it has been recently reported the use in humans of a nasal administered preparation based on HBcAg as non-specific innate immune stimulator to preventively train the system for fight potential respiratory infections or as early prophylaxis treatment [ 50 ]. In that work the authors found that the HBcAg, when administered by nasal route, is able to increase the expression of several IFN-stimulated genes. This effect could be another advantage of the use of the CND-1 + ODN nasal vaccine preparation. The results obtained in the present work demonstrate that the heterologous CTD fragment of N protein from SARS-CoV-2 virus can be fused to HBcAg C-terminus, without affecting its capacity of forming aggregates and CLPs. The resultant chimeric protein is immunogenic when it is administered in Balb/C mice by nasal route without adjuvants, being able to generate systemic and mucosal immune response against N and HBcAg. The nasal immunogenicity of CND-1 protein is enhanced by the addition of the mucosal adjuvant ODN-39M. The preparation CND-1 + ODN constitutes a promising vaccine candidate to face future coronavirus infections or as booster option to enhance the current immunity. In addition, the strategy of employ the HBcAg as carrier for other antigens of interest, related to threatening respiratory infections, results appealing considering its particular mucosal and systemic immunity activation capacity, and the relatively easy obtaining in E. coli . Declarations Author Contributions : Conceptualization, L.H., Y.L., G.C, E.S., Y.P.; Supervision, L.H., Y.P., G.G., R.S., W.L.; Investigation, Y.L., R.C., P.A., A.M., E.S., Y.Lan., M.Z., Z.Z.; Formal analysis, L.H., Y.L., E.S., Y.P. Funding acquisition, L.H., K.Y.; Writing - Original Draft, L.H., Y.L., Writing - Review & Editing, L.H., Y.L. Project administration, L.H., K.Y., W.L., Y.P. Funding : This work was supported by MOST "National key R&D program of China (2021YFE0192200)", "PNCT CITMA, Cuba", "Hunan Provincial Base for Scientific and Technological Innovation Cooperation (2019CB1012)", "The Science and Technology Innovation Program of Hunan Province, (2020RC5035)", "Hunan Provincial Innovative Construction Program (2020WK2031) Institutional Review Board Statement : The human sera from COVID-19 convalescent and negative individuals were collected at The Eighth- and Ninth-People’s Hospital of Dongguan city (Guangdong Province, China). The study protocol was approved by the Institutional Ethics Committee from both hospitals and was carried out in accordance with the principles of Helsinki declaration. Informed consent was obtained from all donors. Statement for Research involving animals: The studies in mice were conducted at Beijing Vital River Laboratory Animal Technology Co., Ltd, and Hunan Prima Drug Research Center Co., Ltd. Both animal facilities complied with the national standard of the People's Republic of China GB14925-2010 and the institutional guidelines. Each experimental protocol was subjected to analysis and approval by the Institutional Ethics Committee (Protocols: PANCOV04 approved on 12.04.2022, and HNSE2023(3)012 approved on 20.02.2023). In all the studies inhaled isoflurane, at regular dose or overdose, was employed as anesthesia or euthanasia method, respectively. Data Availability Statement : Not applicable. Conflicts of Interest : The authors declare no conflict of interest. Acknowledgments: We acknowledge to Dr. Jiang from Guangdong Eighth People's Hospital, Guangdong Province, China, for providing the samples from COVID-19 convalescent donors. We acknowledge to Dr. Alejandro Martín for the assistance on the Molecular biology work. References Cao T, Lazdina U, Desombere I, Vanlandschoot P, Milich DR, Sällberg M, Leroux-Roels G. 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Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Alexis","middleName":"","lastName":"Musacchio","suffix":""},{"id":338610660,"identity":"2ecccb0e-8c8c-4585-b011-ea41a9b5a9ec","order_by":2,"name":"Panchao Ai","email":"","orcid":"","institution":"China Cuba Biotechnology Joint Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Panchao","middleName":"","lastName":"Ai","suffix":""},{"id":338610661,"identity":"a01c3275-c177-4666-9138-3d994320c6f5","order_by":3,"name":"Rong Chen","email":"","orcid":"","institution":"China Cuba Biotechnology Joint Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Chen","suffix":""},{"id":338610662,"identity":"588cfff9-13c7-4572-8427-6f4c89d3d191","order_by":4,"name":"Edith Suzarte","email":"","orcid":"","institution":"Center for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Edith","middleName":"","lastName":"Suzarte","suffix":""},{"id":338610663,"identity":"998b1266-5432-4fc5-8cbf-44274ebbbce0","order_by":5,"name":"Glay Chinea","email":"","orcid":"","institution":"Center for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Glay","middleName":"","lastName":"Chinea","suffix":""},{"id":338610664,"identity":"1b68edc7-5fc4-40dd-a8f7-fd8465827438","order_by":6,"name":"Miaohong Zhang","email":"","orcid":"","institution":"Hunan PRIMA Drug Research Center Co., Ltd, National Liuyang Economic and Technological Development Zone","correspondingAuthor":false,"prefix":"","firstName":"Miaohong","middleName":"","lastName":"Zhang","suffix":""},{"id":338610665,"identity":"096103b7-2340-4a83-83ec-a2a019997305","order_by":7,"name":"Zhiqiang Zhou","email":"","orcid":"","institution":"Hunan PRIMA Drug Research Center Co., Ltd, National Liuyang Economic and Technological Development Zone","correspondingAuthor":false,"prefix":"","firstName":"Zhiqiang","middleName":"","lastName":"Zhou","suffix":""},{"id":338610669,"identity":"0bc8c3e6-d874-4eae-a752-1c40412bf5d1","order_by":8,"name":"Yaqin Lan","email":"","orcid":"","institution":"China Cuba Biotechnology Joint Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Yaqin","middleName":"","lastName":"Lan","suffix":""},{"id":338610673,"identity":"679438a4-93ef-4c0d-bd81-b06cff2606a6","order_by":9,"name":"Ricardo Silva","email":"","orcid":"","institution":"BioCubaFarma","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Silva","suffix":""},{"id":338610674,"identity":"026dbdcf-e1db-4e44-8fef-dc62b1db2f01","order_by":10,"name":"Gerardo Guillén","email":"","orcid":"","institution":"Center for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Gerardo","middleName":"","lastName":"Guillén","suffix":""},{"id":338610675,"identity":"3a04b245-7b2b-4794-a8b3-748785019c64","order_by":11,"name":"Ke Yang","email":"","orcid":"","institution":"China Cuba Biotechnology Joint Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Yang","suffix":""},{"id":338610677,"identity":"3c030dc2-ed6f-44f0-8769-516eccf2e56a","order_by":12,"name":"Wen Li","email":"","orcid":"","institution":"China Cuba Biotechnology Joint Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Li","suffix":""},{"id":338610681,"identity":"b28cd143-a717-4f77-9ddd-a18efb782069","order_by":13,"name":"Yasser Perera","email":"","orcid":"","institution":"China Cuba Biotechnology Joint Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Yasser","middleName":"","lastName":"Perera","suffix":""},{"id":338610682,"identity":"f6b2450c-bbc1-46a8-8622-81c501bfc37d","order_by":14,"name":"Lisset Hermida","email":"data:image/png;base64,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","orcid":"","institution":"China Cuba Biotechnology Joint Innovation Center","correspondingAuthor":true,"prefix":"","firstName":"Lisset","middleName":"","lastName":"Hermida","suffix":""}],"badges":[],"createdAt":"2024-07-15 03:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4740544/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4740544/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12985-024-02583-9","type":"published","date":"2024-11-29T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62247898,"identity":"01253558-c939-41b3-a1be-fa5f3146e7f3","added_by":"auto","created_at":"2024-08-12 05:20:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":246706,"visible":true,"origin":"","legend":"\u003cp\u003eExpression, purification and characterization of CN-1 and CND-1 proteins (a) Simplified diagram of CN-1 and CND-1 constructs. Expression analysis, by SDS-PAGE and Western blotting using anti-SARS-CoV-2 nucleocapsid (N) polyclonal Ab, of samples from CN-1 (b) and CND-1 (c) 1: MW molecular weight marker, 2: Cell extract of \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) transformed with each recombinant plasmid. Analysis of the disruption process by SDS-PAGE, of CN-1 (d) and CND-1 (e) samples. 1: MW marker, 2: Cell extract of \u003cem\u003eE. coli\u003c/em\u003eBL21 (DE3) transformed with each recombinant plasmid, 3: Supernatant after disruption, 4: Pellet after disruption. (f) Diagram of the purification process. (g) Dot blotting of purified samples of CN-1 and CND-1 proteins, using 1: anti-HBcAg MAb and 2: anti-N polyclonal Ab. Analysis by SDS-PAGE of purified proteins CN-1 (h) and CND-1 (i) 1: MW marker, 2: Sample of elution from Sephacryl S-200 HR. (j) Analysis by SDS-PAGE under native conditions, and Western blot using polyclonal anti-N Abs (k), of samples of each purified protein 1: MW marker, 2: CN-1, 3: CND-1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/d36cb88ec05a11e2eec555fe.png"},{"id":62247899,"identity":"b60593c2-c3d9-40ae-98ed-a60340df5b05","added_by":"auto","created_at":"2024-08-12 05:20:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":241227,"visible":true,"origin":"","legend":"\u003cp\u003eTEM using HT7800 Hitachi microscope. Three pictures corresponding with 25 000, 50 000 and 100 000 x magnifications, respectively, are shown. Left panel: CN-1, Right panel: CND-1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/645f92b1cc4b2b306cde991c.png"},{"id":62247900,"identity":"92df318c-819d-4260-a0a2-bf1a1ea55aee","added_by":"auto","created_at":"2024-08-12 05:20:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25265,"visible":true,"origin":"","legend":"\u003cp\u003eRecognition of CN-1 (left panel) and CND-1 (right panel) recombinant chimeric proteins by SARS-CoV-2 convalescent human sera. Data is presented as O.D\u003csub\u003e492nm\u003c/sub\u003e values from each individual serum. Horizontal bar represents the mean of the group in each case. (●) Convalescent and (■) negative sera.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/41cf62fe98f79b130819d304.png"},{"id":62247902,"identity":"7e5f969e-bc39-42f5-9f47-d2a1f1b90a9e","added_by":"auto","created_at":"2024-08-12 05:20:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97573,"visible":true,"origin":"","legend":"\u003cp\u003eImmunogenicity of CN-1 and CND-1 proteins, in Balb/C mice, administered by intranasal (in) route. Six- to eight-week-old mice were immunized with three doses of each formulation: Group 1: CN-1, Group 2: CND-1, Group 3: PBS. Twenty-seven days after the third immunization, mice were sacrificed. Antibody responses in the serum and bronchoalveolar lavage fluid (BALF) as well as CMI in spleen, were evaluated. (a) Diagram of immunization. Humoral immune response measured by ELISA, anti-N protein from SARS-CoV-2, (b) IgG in sera, (c) IgA in BALF; anti-HBcAg (d) IgG in sera and (e) IgA in BALFs. Data of IgG are represented as log10 of the titers. Data from IgA are expressed as O.D\u003csub\u003e492nm. \u003c/sub\u003e(f) Frequency of IFN-γ secreting cells, by ELISpot, in splenocytes after \u003cem\u003ein vitro\u003c/em\u003e stimulation with the conserved peptide N\u003csub\u003e351-365\u003c/sub\u003e. Graph showsthe response of individual mice. In all graphics the horizontal bar represents the mean value of the group. The statistical analysis was done by One-Way Anova followed of Tukey’s multiple comparison test. **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/1613aa419e63844d940143ee.png"},{"id":62248215,"identity":"c63819e2-e97b-49d2-b341-99efe151cfe1","added_by":"auto","created_at":"2024-08-12 05:28:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104860,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral immunity elicited by CND-1 alone and combined with ODN-39M, administered by intranasal route in Balb/C mice. Six- to eight-week-old mice were immunized with three doses of each formulation: Group 1: CND-1, Group 2: CND-1 + ODN-39M, Group 3: PBS. Thirty days after the third immunization, mice were sacrificed. Antibody responses in the serum and BALF were evaluated. (a) Diagram mouse immunization. Humoral immune response anti-N protein from SARS-CoV-2 measured by ELISA (b) IgG and (c) IgG1 and IgG2a in sera, and (d) IgA in BALFs. Humoral immune response anti-HBcAg measured by ELISA (e) IgG in sera, and (f) IgA in BALF. Data of IgG are represented as log10 of the titers. Data from IgA are expressed as O.D\u003csub\u003e492nm . \u003c/sub\u003eThe horizontal bar represents the mean value. The statistical analysis was done by One-Way Anova followed of Tukey’s multiple comparison test. **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/7f0f184a0371f8ff9fb193e6.png"},{"id":62247905,"identity":"081762cf-e95f-4892-a599-76d1b4533f7c","added_by":"auto","created_at":"2024-08-12 05:20:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":77330,"visible":true,"origin":"","legend":"\u003cp\u003eCross-reactivity of the systemic immune response generated in Balb/C mice by the intranasal administration of CND-1 alone and combined with ODN-39M. Thirty days after the third immunization mice were sacrificed. (a) IgG antibody response against N protein from SARS-CoV-1, MERS-CoV, and SARS-CoV-2 Omicron variant were measured by ELISA in sera at 1:100 dilution. Data are expressed as O.D values, horizontal bar represents the mean value. (b) Cell-mediated immune response. Spleens cells were isolated and \u003cem\u003ein vitro \u003c/em\u003estimulated with the conserved peptide N\u003csub\u003e351-365\u003c/sub\u003e and N proteins from SARS-CoV-2, Delta and Omicron variants, and SARS-CoV-1. The frequency IFN-γ secreting cells was measured by ELISpot. Square dots represent a pool of placebo samples. The horizontal bar represents the mean value for each group (n = 5).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/53cf14ab43fc39512bb568e2.png"},{"id":70382837,"identity":"27310ff0-1c50-4de0-ae2a-a0e474cf12fc","added_by":"auto","created_at":"2024-12-02 16:32:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1480503,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/2163a2f2-b3b2-4541-9b3b-8b771691b349.pdf"},{"id":62247903,"identity":"44ea5c8b-5983-452b-9b0a-141d43606681","added_by":"auto","created_at":"2024-08-12 05:20:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":712317,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileFigure1uncroppedversion.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4740544/v1/2ad6d71bbcfc49627e85c05f.pdf"}],"financialInterests":"Competing interest reported. Y.Lobaina, A.M., P.A., R.C., E.S., Y.Lan, G.C., R.S., G.G, K.Y, Y.P., L.H., are authors of a patent (recently presented in China) related to the content of the manuscript.","formattedTitle":"Obtaining HBV core protein VLPs carrying SARS-CoV-2 nucleocapsid conserved fragments as vaccine candidates","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe Hepatitis B core (HBcAg) is one of the main structural antigens of hepatitis B virus (HBV) and constitutes a potent immunogen for humans and animals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The extremely high immunogenicity of HBcAg can be explained by its particulate nature and its capacity to function as both T-cell-independent and T-cell-dependent antigen [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The natural HBcAg is assembled in particles of approximately 30nm with icosahedric geometry composed by 120 dimer of the viral capsid protein [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The repetitive motifs and the protuberances in the HBcAg particles surface confer the unique ability to bind and activate a high frequency of naive human and murine B cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. HBcAg-specific B cells from unprimed mice are able to take up, process and present HBcAg to naive T-helper cells in vivo 10\u003csup\u003e5\u003c/sup\u003e times more efficiently than classical antigen presenting cells (APCs) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The full length HBcAg, when is obtained as a recombinant protein in \u003cem\u003eE. coli\u003c/em\u003e, retains its capacity to form virus-like particles (VLPs), able to encapsulate bacterial nucleic acid (mainly RNA) which confers potent Th1 adjuvant properties [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe HBcAg has been widely used in preclinical studies as carrier of heterologous epitopes forming chimeric proteins [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The most frequently used site for heterologous insertion has been the immunodominant c/e1 epitope, located in the center of the HBc primary sequence, which comprises a solvent-exposed loop that tolerates insertions of flexible peptide sequences [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several data are available about the evaluation of such chimeric constructs in animal models [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been demonstrated that a heterologous sequence inserted into this internal loop is significantly more immunogenic than such fragment in the context of its native protein [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nevertheless, due to the insertion in the loop implies structural constrains (length and particular conformation) of the heterologous motif, the N and C terminus fusions sites has been also explored with successful results [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present work two recombinant chimeric proteins including two fragments from SARS-CoV-2 nucleocapsid (N) protein fused to the C-terminus of HBcAg were designed and obtained. N protein is a conserved molecule among coronaviruses, which has emerged as an attractive antigen to be included in novel generation of pancorona vaccines [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The study published by Matchett et al., 2021, demonstrated that N protein, presented in the Ad5 platform, protected mice against SARS-CoV-2 challenge [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Dangi et al, 2021, also proved that the addition of N protein in a spike vaccine formulation, improved distal protection in mouse brain [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe two chimeric proteins, named CN-1 and CND-1, were obtained as recombinant proteins in \u003cem\u003eE. coli\u003c/em\u003e, carrying fragments of SARS-CoV-2 N-terminal domain (NTD) (139 aa length) and C-terminal dimerization domain (CTD) (123 aa length), respectively. After the purification processes, preparations with more than 90% of purity were obtained and the presence of spherical particles of 10\u0026ndash;25 nm were visualized by transmission electron microscopy (TEM). The antigenicity of both proteins was confirmed using a panel of SARS-CoV-2 convalescent\u0026rsquo;s sera and immunogenicity studies by intranasal route in Balb/C mice were done. CND-1 showed a higher antigenicity, and accordingly, it was also the most immunogenic. The immune response generated by intranasal administration of CND-1 protein was improved when the ODN-39M was added as adjuvant in the formulation. Importantly, the induced systemic humoral and cellular immune response was cross-reactive until SARS-CoV-1 level.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Biological and synthetic reagents\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli BL21 (DE3)\u003c/em\u003e: F\u0026ndash; ompT gal dcm lonhsdSB(rB- mB-) k(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) was used for gene expression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For plasmid propagation, \u003cem\u003eE. coli\u003c/em\u003e strain XL1-blue [F\u0026rsquo;::Tn10 proA\u0026thorn;B\u0026thorn; lacIq D(lacZ)M15/recA1 endA1gyrA96(NaIr) thi hsdR17(rk m\u0026thorn;k) supE44 relA1 lac) was employed [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the evaluation of animal samples, the following recombinant antigens were purchased from Sino Biological (China). N proteins from: SARS-CoV-2 Delta (40588-V07E29), and Omicron (40588-V07E34) variants; SARS-CoV-1 (40143-V08B), MERS-CoV (40068-V08B). In addition, the peptide N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e from SARS-CoV-2 (ILLNKHIDAYKTFPP) was synthesized with \u0026ge;\u0026thinsp;97% purity by Zhejiang Peptides Biotech (China).\u003c/p\u003e \u003cp\u003eThe ODN-39M, a 39 mer, whole phosphodiester backbone CpG ODN (5\u0026rsquo;-ATC GAC TCT CGA GCG TTC TCG GGG GAC GAT CGT CGG GGG-3\u0026rsquo;), was synthesized by Sangon Biotech (China).\u003c/p\u003e \u003cp\u003eTo evaluate the protein antigenicity the following antibody reagents were used. The anti-SARS-CoV-2 nucleocapsid polyclonal antibody (40588-T62) was purchased from Sino Biologicals (China). A monoclonal antibody anti-HBcAg (ab8637) was purchased from Abcam (USA). A panel of human sera from COVID-19 convalescent (N\u0026thinsp;=\u0026thinsp;27) and negative (N\u0026thinsp;=\u0026thinsp;10) donors were collected as part of a study approved by the Institutional Ethics Committee from The Eighth People\u0026rsquo;s Hospital of Dongguan (Guangdong Province, China). Informed written consent was obtained from each participant. The study description was already reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Obtaining of the chimeric constructs CN-1 and CND-1\u003c/h2\u003e \u003cp\u003eThe following chimeric genes were chemically synthesized:\u003c/p\u003e \u003cp\u003eCN-1: Truncated HBcAg core (1-149)\u0026thinsp;+\u0026thinsp;linker (GSSGGSSG)\u0026thinsp;+\u0026thinsp;N fragment (40\u0026ndash;179) from SARS-CoV-2 (Delta variant)\u003c/p\u003e \u003cp\u003eCND-1: Truncated HBcAg core (1-149)\u0026thinsp;+\u0026thinsp;linker (GSGGSG)\u0026thinsp;+\u0026thinsp;N fragment (248\u0026ndash;371) from SARS-CoV-2 (Delta variant)\u003c/p\u003e \u003cp\u003eEach chimeric gene was amplified by polymerase chain reaction (PCR) using the corresponding primers. The amplified band was purified and cloned into pGEM-T Easy Vector (Promega, USA). Positive clones were tested by restriction analysis, and sequencing. Each recombinant fragment was then cloned into pET28a plasmid. Positive clones were identified by restriction analysis and defined as pCN-1 and pCND-1.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e strain BL21 (DE3) was transformed with each recombinant plasmid by electroporation. Each clone was later inoculated, at 0.05 of Optical Density (OD), in ZY medium supplemented with Kanamycin (50 \u0026micro;g/ml) and let to grow for 18 h at 28\u0026deg;C and 170 rpm-min (THZ-300, Blue Pard Inst, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Purification processes of CN-1 and CND-1\u003c/h2\u003e \u003cp\u003eFor both proteins, CN-1 and CND-1, a similar purification protocol was implemented, with some modifications.\u003c/p\u003e \u003cp\u003eThe transformed \u003cem\u003eE. coli\u003c/em\u003e was grown during 18 h under the conditions described above, and the biomass was harvested by centrifugation at 5 000 x g for 15 min at 4\u003csup\u003e\u0026deg;\u003c/sup\u003eC. For cell disruption, 0.5 g of cells were resuspended in 50 mL of TE buffer (0.05M Tris-HCl, 5mM EDTA, pH 9.0) and treated at 4\u0026deg;C, with 20 cycles (30 seconds with 30 seconds rest) in a ultrasonic machine (FS-200T, Shanghai Sonxi US Inst, China), with 50% power rate and 20.3 kHz frequency. The resultant sample was centrifuged at 10, 000 x g for 15 min at 4\u003csup\u003e\u0026deg;\u003c/sup\u003eC. The supernatant was collected and filtered throughout 0.45 \u0026micro;m for subsequent purification steps.\u003c/p\u003e \u003cp\u003eAs a high resolution step, the ion exchange chromatography was selected based on the particular features of the HBcAg. A volume of 20 mL from the biomass disruption soluble fraction was \u0026frac12; diluted with TE buffer and applied into Q Sepharose and SP Sepharose -fast flow ion exchangers (Cytiva, Sweeden) connected in tandem, and previously equilibrated with TE buffer. The loading volume of the sample was 10% and the employed flow rate was 3 cm/h. After application, columns were separated, washed independently with TE buffer and the bound proteins to SP Sepharose fast flow matrix eluted with 0.1M NaCl step gradient. The Q Sepharose fast flow matrix was employed to remove most of the protein and DNA contaminants present on the sample while CN-1 protein was eluted from the SP Sepharose fast flow matrix with high purity. The CN-1 protein fraction was collected at 0.3M NaCl.\u003c/p\u003e \u003cp\u003eGel filtration chromatography was used for final purification step. Sephacryl S-200 HR matrix (Cytiva, Sweden) was equilibrated with 0.05M Tris HCl, 5 mM EDTA, 0.15M NaCl, pH 9.0, at 15cm/h. The CN-1 fraction eluted at 0.3M NaCl from the SP Sepharose column was applied into the gel filtration chromatography. The CN-1 protein was collected as unique peak, which was later concentrated using Amicon system (USA), filtered by 0.22\u0026micro;m and stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eIn the case of CND-1, the protein was eluted from the SP Sepharose fast flow matrix at 0.1M NaCl with high purity. The CND-1 fraction collected was then applied to a gel filtration chromatography following the same conditions previously described for this step. The peak corresponding to CND-1 protein was then concentrated using Amicon system (USA), filtered by 0.22\u0026micro;m and stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe detection of the protein signal was followed by the absorbance at 280 nm in all steps\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analysis of protein samples\u003c/h2\u003e \u003cp\u003eBCA assay (Pierce, USA), was used to determine the protein concentration in all samples. The identity of each chimeric protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and dot blotting, using specific antibodies. Protein samples were subjected to 12% of acrylamide gel. SDS-PAGE gels were stained with Coomassie blue and scanned (iBright 1500, Invitrogen). Densitometry analysis, using Image J (1.41 version) software was employed to define the percentage corresponding with the protein band of interest. For Western blotting, protein samples were electro transferred from an acrylamide gel to a Immobilon-P membrane (Merck-Millipore, IRL), as described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In turn, samples were applied directly to the membrane for dot blotting. The membrane from the two previous procedures was blocked with 5% skim milk in phosphate buffered saline (PBS) for 1 h at room temperature (RT), washed three times with \u0026ndash; PBS \u0026minus;\u0026thinsp;0.05% Tween solution (PBS-T). The reaction with the anti-SARS-CoV-2 N polyclonal Ab generated in rabbit (SinoBiologicals, China) at 1:2500 dilution (or anti-HBcAg Mab (Abcam, USA) 1:1000 diluted), occurred during 1 h at RT. After proper washing, the incubation of the membrane with the peroxidase-conjugated goat anti-rabbit IgG (Chemicon, USA) at a 1/300 dilution, or anti-mouse IgG-peroxidase, respectively, occurred during 1 h at RT. Antibodies used during the whole procedure were diluted in 1% skim milk in PBS-T. Afterwards, upon membrane washing, the reaction was detected by incubation with Aminoethyl Carbazole (AEC) substrate solution (0.2mg/ml AEC and 0.03% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 50mM NaAc solution) at RT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Visualization of Capsid Like Particles (CLPs) by Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eETest company (Changsha, China) provided the specialized service of transmission electron microscopy analysis. CN-1 and CND-1 samples, at concentration of 0.25 mg/ml, were placed on a freshly glow-discharged, 400-mesh copper grid coated with Formvar and Carbon. After sample absorption and water washing, Uranyl Acetate stain was added. After 4 min of staining, grids were wick dried with Whatman no. 1 filter paper and later, during 20 min, were allowed to air dry.\u003c/p\u003e \u003cp\u003eThe Transmission Electron Microscope HT 7800 (Hitachi, Japan) with an acceleration voltage of 120 Kv and three magnifications: 25 000 x, 50 000 x, and 100 000 x, was the equipment used for sample visualization. For each sample, eight different fields were analyzed. The Image J software (Maryland, USA) was used to estimate the average particle size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Characterization of CN-1 and CND-1 proteins, by ELISA, using human sera positive to SARS-CoV-2\u003c/h2\u003e \u003cp\u003eNinety-six-well high-binding polystyrene plates (Costar, USA) were coated with 3 \u0026micro;g/mL of each protein (CN-1 and CND-1), in sodium carbonate-sodium bicarbonate buffer, and incubated overnight at 4\u0026deg;C. Unspecific binding of the antibodies was avoided by blocking with 5% skim milk (Oxoid, UK) 1 h at 37\u0026deg;C. After five times washing with PBS-T, 100 \u0026micro;L of diluted serum sample in 2% skim milk -PBS-T were added and incubated for 2 hours at 37\u0026deg;C. After washing five times with PBS-T, bound antibodies were detected with a goat anti-human IgG antibody conjugated to horseradish peroxidase (Sigma-Aldrich, Germany) at 1:20000 dilution. After incubation for 1 hour at 37\u0026deg;C and five PBS-T washes, 100 \u0026micro;L of OPD substrate solution (Sigma-Aldrich, Germany) were added to each well and the mixture was incubated for 10 min in the dark at RT. The reaction was stopped by adding 0.2N Sulphuric Acid, and the optical density (O.D) at 492 nm was measured in a multiplate reader (FilterMax F3, Molecular Devices, USA). The data is represented as O.D measures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Animal experiments in Balb/C mice\u003c/h2\u003e \u003cp\u003eBeijing Vital River Laboratory Animal Technology Co., Ltd, and Hunan Prima Drug Research Center Co., Ltd, conducted the mice experiments. Both animal facilities complied with the national standard of the People's Republic of China GB14925-2010. Each experimental protocol was subjected to analysis and approval by the Institutional Animal Care and Use Committee.\u003c/p\u003e \u003cp\u003eThree doses of immunogens were intranasally (in) inoculated in each group of mice (N\u0026thinsp;=\u0026thinsp;5 or N\u0026thinsp;=\u0026thinsp;6), according to the defined design. Ten \u0026micro;g of each protein (CN/1 or CND-1) was administered per animal. All the immunogens were dissolved in sterile PBS in a volume of 50 \u0026micro;L. As negative controls, placebo groups were included in the experiments.\u003c/p\u003e \u003cp\u003eIn the first animal study, mice were distributed in three groups of six animals each. Group 1 was inoculated with CN-1 protein, Group 2 received CND-1 protein, and PBS was administered to the Group 3, as negative control. The administration schedule was 0, 15 and 30 days. Twenty-seven days after the last dose, mice were sacrificed.\u003c/p\u003e \u003cp\u003eFor the other study, mice were distributed in three groups of five animals each. Groups 1 and 2 received CND-1 protein and CND-1\u0026thinsp;+\u0026thinsp;15ug ODN-39M, respectively. Group 3 corresponded to the Placebo group, similar to the previous study. The administration schedule was 0, 7 and 21 days. Thirty days after the last dose, mice were sacrificed.\u003c/p\u003e \u003cp\u003eThree different samples were obtained at the indicated time points: sera, spleens, and bronchoalveolar fluid (BALF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of antibody response by ELISA\u003c/h2\u003e \u003cp\u003eThe Ab response in sera and BALF was measured by anti-IgG, IgG subclasses, and, -IgA ELISAs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, 96 well high-binding plates (Costar, USA) were coated with N or HBcAg protein (3\u0026micro;g/mL) and blocked with 2% skim milk solution. Samples were evaluated in duplicates starting from 1/100 dilution of sera. BALF were tested without dilution. Specific horseradish peroxidase conjugates (Sigma, USA) and OPD (Sigma, USA)/hydrogen peroxide substrate solution were employed. The reaction was stopped using 2 N Sulphuric acid and multiplate reader (FilterMax F3, Molecular Devices, USA) was used to measure O.D at 492nm. In the graphics corresponding with sera Ab response log10 titers are represented. The arbitrary units of titers were calculated by plotting the O.D values obtained for each sample in a standard curve (hyper-immune serum of known titer). The positivity cut-off was established as 2 times the average of O.D obtained for a pre-immune sera pool. In the case of BALF, the antibody response was represented as O.D at 492 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Evaluation of cell-mediated immunity by IFN-γ ELISpot\u003c/h2\u003e \u003cp\u003eThe Mouse IFN-γ ELISpot antibody pair (Mabtech, Sweden) was employed to perform the ELISpot assay. Splenocytes (from five mice per group) were isolated in RPMI culture medium (Gibco, US) and processed individualized. In the case of Placebo group, splenocytes were processed as a pooled sample of five animals. Duplicates cultures (5x10\u003csup\u003e5\u003c/sup\u003e and 1x10\u003csup\u003e5\u003c/sup\u003e splenocytes per well) were incubated for 48 h at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e, in a 96 well round-bottom culture plate (Costar, USA) with 10 \u0026micro;g/mL of each stimulating agent: N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e peptide, N proteins, and Concanavalin A (ConA). Control wells of cells without stimulus (medium) were included for all samples. After the incubation period, the whole content of each culture plate was transferred to ELISpot pre-coated plates (Merck-Millipore, USA) and incubated for 16\u0026ndash;20 h at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e. The following steps were performed according to the manufacturer\u0026acute;s recommendation. For spot counting, a stereoscopic microscope (AmScope SM-1TSZ, USA) coupled to a digital camera was employed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical methods and analysis\u003c/h2\u003e \u003cp\u003eThe GraphPad Prism version 5.00 statistical software (Graph-Pad Software, USA) was used for all the analyses. To reach a normal distribution, antibody titers were transformed to log10. For the non sero-converting sera, an arbitrary titer of 1:50 was assigned for statistical processing. As a parametric test, the One-way Anova test followed by a Tukey's post-test was selected for multiple group comparisons. For the non- parametric multiple comparisons, the Kruskal Wallis test and Dunns post-tests was used. P values were considered as: ns, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Obtaining of CN-1 and CND-1 constructs\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea represents the design of the two chimeric proteins, CN-1 and CND-1. The DNA sequence corresponding to each chimeric gene was cloned into the PET-28a vector. \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) was transformed with each recombinant plasmid and grown in ZY auto-induction medium. An overexpressed band of molecular weight (MW) around 32.7 kDa and 31.1 kDa, matching with the theoretical size of the CN-1 and CND-1 proteins respectively, was detected by SDS-PAGE, accounting for the 5% of the total cellular proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In addition, the identity was confirmed by western blot assay. Each band was immune-identified with anti-SARS-CoV-2 nucleocapsid polyclonal Ab (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, right panel).\u003c/p\u003e \u003cp\u003eAfter expression of each recombinant construct, the biomass from the bacterial culture was disrupted. Under established conditions, both chimeric proteins were mainly associated to the cell disruption soluble fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The subsequent high resolution purification steps were similar for both proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), with slight modifications. In general, a sample for each disruption process (for CN-1 and CND-1) was applied into two ionic exchange chromatographies coupled in tandem (Q and SP Sepharose fast flow). Contaminants were attached to the first anion exchange matrix whereas each target protein was bound to the cation one. Since the elution from the SP Sepharose was conducted by step gradient, additional contaminants were removed at low molarities of NaCl. The CN-1 and CND-1 proteins were eluted at 0.5M and 0.1M NaCl, respectively, with a high level of purity. As final polishing purification step, the gel filtration chromatography was introduced using Sephacryl S-200 HR. Both proteins were obtained with more than 90% of purity and were properly immune-identified by both, polyclonal Abs anti-SARS-CoV-2 N and the anti-HBcAg Mab (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eThe characterization by SDS-PAGE and Western blotting of each purified protein, under native conditions, is shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek. Interestingly, aggregated forms of high MW were visualized for both proteins although the highest MW species were only visualized in the CND-1 sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Visualization of Capsid like Particles\u003c/h2\u003e \u003cp\u003eTEM was selected as the analytical method to define the ability of each chimeric protein to form CLPs. Three pictures, visualized with three magnification factors are represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Particles with similar spherical morphology were detected in the samples of the two chimeric proteins. The particles visualized for CND-1 and CN-1 samples have an average size of 12.2 nm and 19.4 nm, respectively. On the other hand, the vaccine preparation composed by CND-1 mixed with ODN-39M adjuvant showed a similar pattern of particles with mean size of 15.2 nm (data not shown). No clear evidences of dimeric or aggregated structures were observed in the evaluated samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Recognition of CN-1 and CND-1 by SARS-CoV-2 positive sera\u003c/h2\u003e \u003cp\u003eA panel of human sera (positive and negative against SARS-CoV-2 antigens) was used to determine the recognition of the chimeric proteins CN-1 and CND-1. The Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e represents the results obtained. Both recombinant proteins were recognized by the positive human sera although the recognition of CN-1 tended to be lower. Furthermore, three sera samples belonging to the negative donors group showed a positive recognition for both chimeric proteins. These specific sera samples were additionally tested against HBcAg, by ELISA, and showed a positive recognition to hepatitis B capsid protein (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 CN-1 and CND-1 immunogenicity in Balb/C mice by intranasal route\u003c/h2\u003e \u003cp\u003eA mice experiment was conducted to explore the immunogenicity of each chimeric protein administered by intranasal route. Three doses were administered without adjuvant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In sera, only the group receiving the CND-1 protein elicited an IgG antibody (Ab) response specific against N protein from SARS-CoV-2 and HBcAg, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed respectively. Accordingly, a similar pattern was obtained when the specific IgA Abs were measured in BALF samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eTo test the CMI, spleen cells were \u003cem\u003ein vitro\u003c/em\u003e stimulated with the conserved peptide N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e and the N protein from SARS-CoV-2. The frequency of IFN-γ secreting cells was measured by ELISpot assay. According to the humoral immune response, mice receiving CND-1 by intranasal route exhibited a positive response against the peptide N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Of note, no response was detected for any group upon stimulation with N protein from SARS-CoV-2 (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Immunogenicity of the mixture CND-1\u0026thinsp;+\u0026thinsp;ODN-39M\u003c/h2\u003e \u003cp\u003eBased on the nasal immunogenicity results previously described, CND-1 protein was selected to combine with the mucosal adjuvant ODN-39M as a potential nasal vaccine candidate. Three doses of each immunogen (CND-1 alone or CND-1\u0026thinsp;+\u0026thinsp;ODN-39M) were intranasally administered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAs expected, the group receiving CND-1\u0026thinsp;+\u0026thinsp;ODN-39M elicited high levels of IgG Abs in sera against N protein from SARS-CoV-2, with statistical differences compared to titers induced in the group inoculated with CND-1 without adjuvant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In turn, upon IgG subclass pattern analysis, similar and high levels of IgG1 and IgG2a were elicited in animals of the group receiving the protein with adjuvant, indicating the induction of a Th1-like pattern of response against N protein from SARS-CoV-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). On the other hand, similar to the IgG Abs in sera, the levels of mucosal IgA against N protein from SARS-CoV-2 measured in BALF were higher in the group receiving CND-1 with adjuvant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eFurthermore, the HBcAg-specific antibody response exhibited a similar behavior. The IgG levels in sera and IgA levels in BALF, against the carrier protein, were higher in the group receiving the combination of CND-1\u0026thinsp;+\u0026thinsp;ODN-39M showing statistical differences compared to the group receiving the chimeric protein without adjuvant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eSamples were additionally tested against N proteins from SARS-CoV-2 Omicron variant, SARS-CoV-1 and MERS-CoV so as to determine the scope of the systemic humoral immune response. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, high levels of IgG were detected against N proteins from SARS-CoV-2 Omicron variant, and SARS-CoV-1, for the group immunized with the adjuvated preparation, whereas no response was obtained against N protein from MERS-CoV.\u003c/p\u003e \u003cp\u003eFinally, to test the CMI, spleen cells were \u003cem\u003ein vitro\u003c/em\u003e stimulated with the conserved peptide N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e and the N protein from SARS-CoV-2 Delta and Omicron variants, and the N protein from SARS-CoV-1. In line with the humoral immune response generated, mice intranasally inoculated with the mixture CND-1\u0026thinsp;+\u0026thinsp;ODN-39M exhibited the higher response. The 100% of animals in this group were positive against the peptide N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e, and the N protein from SARS-CoV-2 Delta and Omicron variants. On the other hand, the IFN-γ response specific against the N protein from SARS-CoV-1 was positive in two out 5 animals.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the present work the N protein from SARS-CoV-2 (Delta strain) was selected as the source of the heterologous fragments to be fused to HBcAg since it is a conserved coronavirus antigen and target of CMI response in humans [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Accordingly, several TCD4\u003csup\u003e+\u003c/sup\u003e and TCD8\u003csup\u003e+\u003c/sup\u003e epitopes have been mapped on it [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Particularly, the two regions selected for fusing to HBcAg, NTD and CTD fragments, exhibit high percentage of identity at sarbecovirus level (\u0026gt;\u0026thinsp;90%) and their lengths and structures are compatible to be fused at the C-terminus site of the HBcAg. Despite the most used carrier site in the HBcAg is the immunodominant c/e1 epitope, we discard it due to its lack of compatibility with the structure of the N fragments. In turn, the length of each N selected fragment (139 aa and 123 aa for CN-1 and CND-1, respectively) inserted in such site would affect the capacity of the resultant chimeric protein to form capsid-like particles, a crucial feature of the HBcAg which correlates with its high immunogenicity by intranasal route [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAs a first signal of proper conformation, both chimeric proteins (CN-1 and CND-1) were mainly associated to the soluble fraction after biomass disruption. In addition, a scale up purification process, without using chaotropic agents, could be established for each construct. After analysis by TEM, particles of 10\u0026ndash;20 nm were visualized, indicating that the fusion of the heterologous fragments from SARS-CoV-2 N protein does not limit the particle formation ability of the HBcAg. This result is in accordance to several reports describing the formation of CLPs upon fusion of large heterologous cargoes at the C terminus of HBcAg, such as the Dom III of the envelope protein from Zika virus (100 aa), a region from Staphylococcus aureus nuclease (163aa), and the Hepatitis C core fragment (91aa) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In general, the CLPs average size obtained here for both chimeric proteins is on the range, but a little lower to the previously reported for the full-length HBcAg antigen. It is widely known that the full length HBcAg (183 aas), expressed as recombinant protein in \u003cem\u003eE.coli\u003c/em\u003e, is able to form VLPs with a size ranging between 25\u0026ndash;30 nm [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, these VLPs show an electro-dense core, corresponding with the presence of encapsulated bacterial nucleic acids, which confers the extremely high immunogenicity of HBc particles. In our work we selected the truncated HBcAg variant (149 aas) as carrier for the N fragments, considering the remaining capacity of this variant to form CLPs structures and also the big size of the cargoes. In this case the presence of associated bacterial nucleic acid, after the obtaining of the chimeric proteins in \u003cem\u003eE. coli\u003c/em\u003e, was not observed for none of the proteins as it is shown in the TEM photographs and also after agarose gel electrophoresis analysis (data not shown). The truncated HBcAg lacks the Arginine-rich domain (naturally located the C-terminal end) which is the main responsible for the nucleic acid binding during the natural DNA encapsulating role of the HBV nucleocapsid protein [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the design of the CN-1 and CND-1 chimeric proteins we hypothesized that the SARS-CoV-2 N fragments included could contribute to the nucleic acid binding effect, considering the reported RNA binding capacity of these domains [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, the final outcome didn\u0026rsquo;t show evidences of bacterial nucleic acid association in the purified preparations of both chimeric proteins. This could explain in part the lower immunogenicity observed for both antigens after intranasal administration.\u003c/p\u003e \u003cp\u003eOn the other hand, it was interesting the tendency to a lower recognition of CN-1 protein compared with CND-1 protein, when they were tested against a panel of SARS-CoV-2 convalescent sera. This fact can be explained by the higher immunogenicity of the CTD region of SARS-CoV-2 nucleocapsid protein compared with the NTD region, or due to a better conformation obtained for the CND-1 chimeric protein. On the other hand, it was interesting the aggregation profile of both proteins, observed by SDS-PAGE and Western blot under native conditions. Both of them form species of high molecular weight though bands with the highest MW were only visualized for CND-1. It indicates that CTD region tends to form highly aggregated structures, in line with the report by Lo YS, \u003cem\u003eet al\u003c/em\u003e, 2013, where authors demonstrated the oligomerization capacity of the CTD from the human coronavirus 229E nucleocapsid protein [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaking the advantage of the capacity of HBcAg to induce mucosal Abs when it is administered by intranasal route [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], as well as the relevance to induce an anti-SARS-CoV-2 broad mucosal immune response for cutting the transmission upon viral entry or at least, to decrease the infection levels in lungs, the intranasal route was selected for evaluation the chimeric proteins in Balb/C mice.\u003c/p\u003e \u003cp\u003eThe results reveal the superior immunogenicity of CND-1 compared to CN-1 when they were administered by intranasal route without adjuvants. CN-1 neither induces humoral nor CMI response anti-N protein from SARS-CoV-2 under these conditions. This result is in accordance to the lower recognition of this protein by human sera and the absence of high MW aggregates by SDS-PAGE. Accordingly, none or a very low Ab response against HBcAg was obtained for CN-1. On the contrary, CND-1 was immunogenic, measured in both systemic humoral immunity and CMI against N protein from SARS-CoV-2 and the N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e peptide, respectively. Of note, the recombinant SARS-CoV-2 N protein obtained also in \u003cem\u003eE. coli\u003c/em\u003e, recently evaluated by our group, was not able to induce such immunity in mice without adjuvant by intranasal route [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Results indicate that the presentation to the immune system of the CTD fragment is favored on the context of HBcAg, probably due to the highly aggregated nature of the resultant chimeric protein, and the intrinsic adjuvant properties of the HBcAg scaffold [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is known that the presence of particulate structures or protein aggregates in the nanometric size range result better immunogens after nasal administration [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite 100% of responders were obtained in the CND-1 group by anti-N IgG ELISA in sera and ELISpot assay using the conserved T CD4\u0026thinsp;+\u0026thinsp;N peptide (N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e) as stimulating agent, in BALF, only two animals exhibited IgA anti-N positive response. In turn, also by ELISpot assay, no response was detected when the N protein of SARS-CoV-2 was used as stimulating agent. Based on these results, we decided to study the inclusion of the ODN-39M as adjuvant in the CND-1 nasal formulation. The ODN-39M is a CpG ODN which bind to and activate Toll-like receptor 9 (TLR9) for initiating the innate immune response and consequently, enhance the adaptive immune response [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. CpG ODNs are being evaluated in more than 100 clinical trials focused on preventing or treating allergy, infectious diseases and cancer [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Particularly, the ODN-39M has been previously evaluated in combination with four different viral recombinant capsid proteins proving its adjuvant effect [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In accordance, in the present work, the addition of ODN-39M to the CND-1 protein preparation for nasal administration significantly enhanced the humoral immunity (systemic and mucosal) and systemic CMI induced against N protein from SARS-CoV-2. In turn, the systemic IgG subclasses suggest a Th1 pattern of response, similar to that described for viral infections. Actually, the anti-N immunity, comprising both arms of the immune response, has been correlated with protection against coronavirus in mice, monkeys, and humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For humoral immunity, Dangi et al, 2002 proved that anti-N Abs passively transferred to a relevant animal model decreased the viral load upon SARS-CoV-2 challenge [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Accordingly, in humans, anti-N Abs have been administered to treat COVID-19 patients with promising results against the severity of the disease [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. On the other hand, N protein is as target of cross-reactive memory T cells, which were associated to protect SARS-CoV-2 na\u0026iuml;ve contacts from infection, thereby supporting the inclusion of this protein antigen in a new vaccine generation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe breath of the systemic anti-N immunity, induced by intranasal administration of the CND-1\u0026thinsp;+\u0026thinsp;ODN-39M preparation, revealed its potentiality to be considered as an attractive component of a pancorona vaccine. The humoral immune response reached cross-reactivity until SARS-CoV-1 level, whereas for CMI, high level of response was obtained when the conserved peptide N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e was used as simulating agent. Importantly, this peptide spans a conserved region among sarbecovirus which is immunodominant in SARS-CoV-2 Balb/C infected mice. In addition, it was able to partially protect mice from SARS-CoV-2 infection when administered in the context of Venezuelan equine encephalitis replicon particles vector. This experiment provided direct evidence about the protective role of memory T-cells against the conserved peptide N\u003csub\u003e351\u0026thinsp;\u0026minus;\u0026thinsp;365\u003c/sub\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe homology level of SARS-CoV-2 nucleocapsid protein within the beta coronavirus genus supports the results obtained in the present work. The identity of N protein within sarbecoviruses subgenus is in the range of 87\u0026ndash;99% whereas the identity between nucleocapsid of SARS-CoV-2 and MERS-CoV is only 48% [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Despite the response obtained in our mice experiments did not react with the N protein from MERS-CoV, we consider that cross-immunity at sarbecoviruses levels is very important. Currently, more than 50 SARS-related coronaviruses have been identified in 10 species of bat [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Bat-borne SARS-related coronaviruses are considered target of potential pandemics due to their diversity. Accordingly, Crook JM et al, 2021 expressed that the highest the probability for homologous recombination of sarbecoviruses through co-infection, the biggest the possibility of novel zoonotic emergence [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is important to highlight that the immunogenicity obtained for the preparation of CND-1\u0026thinsp;+\u0026thinsp;ODN-39M, nasally administered, is similar to that generated by the recombinant N protein combined with the same adjuvant. In a previous work, the N\u0026thinsp;+\u0026thinsp;ODN-39M preparation, administered by intranasal route, induced anti-N CMI response in spleen and also elicited broad humoral immunity in both, sera and lungs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Nevertheless, CND-1 shows a higher nasal immunogenicity, being able to induce anti-N immune response when it is administered without any adjuvant. Such an intrinsic response is potentiated by the inclusion of the ODN-39M in the preparation.\u003c/p\u003e \u003cp\u003eFinally, since the chimeric protein CND-1 use the HBcAg as scaffold or platform, the anti-HBcAg humoral immunity was also determined. Interestingly, positive response of IgG in sera and IgA in BALF against HBcAg were enhanced after combination with ODN-39M. It is known that the immune response against HBcAg partially contributes to the protection against HBV infection [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], therefore, since CND-1 is also able to generate such anti-HB immunity, it constitutes a further advantage of this antigenic preparation. In addition, it has been recently reported the use in humans of a nasal administered preparation based on HBcAg as non-specific innate immune stimulator to preventively train the system for fight potential respiratory infections or as early prophylaxis treatment [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In that work the authors found that the HBcAg, when administered by nasal route, is able to increase the expression of several IFN-stimulated genes. This effect could be another advantage of the use of the CND-1\u0026thinsp;+\u0026thinsp;ODN nasal vaccine preparation.\u003c/p\u003e \u003cp\u003eThe results obtained in the present work demonstrate that the heterologous CTD fragment of N protein from SARS-CoV-2 virus can be fused to HBcAg C-terminus, without affecting its capacity of forming aggregates and CLPs. The resultant chimeric protein is immunogenic when it is administered in Balb/C mice by nasal route without adjuvants, being able to generate systemic and mucosal immune response against N and HBcAg. The nasal immunogenicity of CND-1 protein is enhanced by the addition of the mucosal adjuvant ODN-39M. The preparation CND-1\u0026thinsp;+\u0026thinsp;ODN constitutes a promising vaccine candidate to face future coronavirus infections or as booster option to enhance the current immunity. In addition, the strategy of employ the HBcAg as carrier for other antigens of interest, related to threatening respiratory infections, results appealing considering its particular mucosal and systemic immunity activation capacity, and the relatively easy obtaining in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Conceptualization, L.H., Y.L., G.C, E.S., Y.P.; Supervision, L.H., Y.P., G.G., R.S., W.L.; Investigation, Y.L., R.C., P.A., A.M., E.S., Y.Lan., M.Z., Z.Z.; Formal analysis, L.H., Y.L., E.S., Y.P. Funding acquisition, L.H., K.Y.; Writing - Original Draft, L.H., Y.L., Writing - Review \u0026amp; Editing, L.H., Y.L. Project administration, L.H., K.Y., W.L., Y.P. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by MOST \"National key R\u0026amp;D program of China (2021YFE0192200)\", \"PNCT CITMA, Cuba\", \"Hunan Provincial Base for Scientific and Technological Innovation Cooperation (2019CB1012)\", \"The Science and Technology Innovation Program of Hunan Province, (2020RC5035)\", \"Hunan Provincial Innovative Construction Program (2020WK2031)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe human sera from COVID-19 convalescent and negative individuals were collected at The Eighth- and Ninth-People’s Hospital of Dongguan city (Guangdong Province, China). The study protocol was approved by the Institutional Ethics Committee from both hospitals and was carried out in accordance with the principles of Helsinki declaration. Informed consent was obtained from all donors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement for Research involving animals:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies in mice were conducted at Beijing Vital River Laboratory Animal Technology Co., Ltd, and Hunan Prima Drug Research Center Co., Ltd. Both animal facilities complied with the national standard of the People's Republic of China GB14925-2010 and the institutional guidelines. Each experimental protocol was subjected to analysis and approval by the Institutional Ethics Committee (Protocols: PANCOV04 approved on 12.04.2022, and HNSE2023(3)012 approved on 20.02.2023). In all the studies inhaled isoflurane, at regular dose or overdose, was employed as anesthesia or euthanasia method, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: The authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We acknowledge to Dr. Jiang from Guangdong Eighth People's Hospital, Guangdong Province, China, for providing the samples from COVID-19 convalescent donors. We acknowledge to Dr. Alejandro Martín for the assistance on the Molecular biology work.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCao T, Lazdina U, Desombere I, Vanlandschoot P, Milich DR, S\u0026auml;llberg M, Leroux-Roels G. 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Microbes Infect 2020, 22, 188\u0026ndash;194, doi:10.1016/j.micinf.2020.04.002.\u003c/li\u003e\n\u003cli\u003eRavelomanantsoa, N.A.F.; Guth, S.; Andrianiaina, A.; Andry, S.; Gentles, A.; Ranaivoson, H.C.; Brook, C.E. The Zoonotic Potential of Bat-Borne Coronaviruses. Emerg Top Life Sci 2020, 4, 353\u0026ndash;369, doi:10.1042/ETLS20200097.\u003c/li\u003e\n\u003cli\u003eCrook, J.M.; Murphy, I.; Carter, D.P.; Pullan, S.T.; Carroll, M.; Vipond, R.; Cunningham, A.A.; Bell, D. Metagenomic Identification of a New Sarbecovirus from Horseshoe Bats in Europe. Sci Rep 2021, 11, 14723, doi:10.1038/s41598-021-94011-z.\u003c/li\u003e\n\u003cli\u003eMaini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, King AS, Herberg J, Gilson R, Alisa A, Williams R, Vergani D, Naoumov NV, Ferrari C, Bertoletti A. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med. 2000 Apr 17;191(8):1269-80. doi: 10.1084/jem.191.8.1269.\u003c/li\u003e\n\u003cli\u003eWebster GJ, Reignat S, Brown D, Ogg GS, Jones L, Seneviratne SL, Williams R, Dusheiko G, Bertoletti A. Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol. 2004 Jun;78(11):5707-19. doi: 10.1128/JVI.78.11.5707-5719.2004. PMID: 15140968; PMCID: PMC415806.\u003c/li\u003e\n\u003cli\u003eAguiar JA, Marrero MA, Figueroa DA, Aguilar A, Idavoy A, Martinez S, Moran I, et al. Preparing for the Next Pandemic: Increased Expression of Interferon-Stimulated Genes After Local Administration of Nasalferon or HeberNasvac. DNA AND CELL BIOLOGY 2024, Volume 43, Number 2, DOI: 10.1089/dna.2023.0283\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HBcAg, nucleocapsid, SARS-CoV-2, chimeric proteins, pancorona vaccine, intranasal","lastPublishedDoi":"10.21203/rs.3.rs-4740544/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4740544/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Hepatitis B core antigen (HBcAg) has been used as a carrierof several heterologous protein fragments based on its capacity to form virus-like particles (VLPs)and to activate innate and adaptive immune responses. In the present work, two chimeric proteins were designed as potential pancorona vaccine candidates, comprising the N- or C- terminal domain of SARS-CoV-2 nucleocapsid (N) protein fused to HBcAg. The recombinant proteins, obtained in \u003cem\u003eE. coli\u003c/em\u003e, were named CN-1 and CND-1, respectively. The final protein preparations were able to form 10-25 nm particles, visualized by TEM. Both proteins were recognized by sera from COVID-19 convalescent donors; however,the antigenicity of CND-1 tends to be higher. The immunogenicity of both proteins was studied in Balb/C mice by intranasal route without adjuvant. After three doses, only CND-1 elicited a positive immune response, systemic and mucosal, against SARS-CoV-2 N protein. CND-1 was evaluated in a second experiment mixed with the CpG ODN-39M as nasal adjuvant. The induced anti-N immunity was significantly enhanced, and the antibodies generated were cross-reactive with N protein from Omicron variant, and SARS-CoV-1. Also, an anti-N broadcellular immune response was detected in spleen, by IFN-g ELISpot. The nasal formulation composed by CND-1 and ODN-39M constitutes an attractive component for a pancorona vaccine, by inducing mucosal immunity and systemic broad humoral and cellular responses against Sarbecovirus N protein.\u003c/p\u003e","manuscriptTitle":"Obtaining HBV core protein VLPs carrying SARS-CoV-2 nucleocapsid conserved fragments as vaccine candidates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-12 05:20:48","doi":"10.21203/rs.3.rs-4740544/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-03T22:37:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-03T22:32:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317145820134777110101658587120516870475","date":"2024-09-23T11:38:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-16T21:56:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300206780422835775166549262738937924759","date":"2024-08-28T15:27:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207443645187829709884450913445467349888","date":"2024-08-10T03:18:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-28T03:44:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-17T23:16:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-17T23:03:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2024-07-15T03:36:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48cb9b2b-02f9-4d6e-b028-fcbdb6fc5398","owner":[],"postedDate":"August 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T16:03:52+00:00","versionOfRecord":{"articleIdentity":"rs-4740544","link":"https://doi.org/10.1186/s12985-024-02583-9","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2024-11-29 15:57:57","publishedOnDateReadable":"November 29th, 2024"},"versionCreatedAt":"2024-08-12 05:20:48","video":"","vorDoi":"10.1186/s12985-024-02583-9","vorDoiUrl":"https://doi.org/10.1186/s12985-024-02583-9","workflowStages":[]},"version":"v1","identity":"rs-4740544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4740544","identity":"rs-4740544","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}