Single‐Dose Intranasal Chimeric Influenza A–B Nucleoprotein Vaccine Confers Dual Protection in a Mouse Model

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NP sequences mutate slowly and share > 90% amino‑acid homology within a virus type; NP is recognized by cytotoxic T lymphocytes (CTLs), which clear infected cells and contribute to broad heterosubtypic immunity. Intranasal NP‑based vaccines have previously protected mice against diverse influenza A subtypes (H1N1, H3N2, H5N2, H7N9 and H9N2) and two lineages of influenza B (B/Yamagata and B/Victoria lineages), yet NP is type‑specific, and vaccines often target only one type. To achieve cross‑type protection, we engineered recombinant adenoviruses expressing NP from influenza A (A‑NP), influenza B (B‑NP) or chimeric constructs linking the two sequences (AB‑NP and BA‑NP). Four replication‑deficient adenoviruses encoding A‑NP, B‑NP, AB‑NP, or BA‑NP were produced. BALB/c mice were anaesthetized and immunized intranasally once with either a mixture of A‑NP plus B‑NP (A + B), or a single chimeric vaccine (AB‑NP or BA‑NP). All regimens induced robust systemic specific IgG, mucosal IgA responses, and CTLs, reactive to both A and B NP. Each group showed complete protection from both influenza A and B challenges compared with controls. Of note, the AB‑NP fusion vaccine conferred the highest survival rate and the least morbidity. Our data demonstrate that fusing influenza A and B NP sequences into a single recombinant antigen induces cross‑type immunity after a single intranasal dose. This extends previous findings that NP‑based vaccines protect against multiple influenza A subtypes and both B lineages and suggests that chimeric NP vaccines could form the basis of a real universal influenza vaccine strategy. Biological sciences/Immunology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 I. Introduction Influenza is a contagious respiratory disease caused by influenza viruses. According to the World Health Organization (WHO), each year influenza causes 3–5 million cases of severe illness and between 290,000 and 650,000 deaths worldwide, posing a persistent threat to human health. The virus primarily infects the respiratory mucosa, with typical symptoms including fever, chills, cough, sore throat, myalgia, headache and fatigue [https://www.cdc.gov/flu/signs-symptoms/index.html]. Influenza viruses are classified into four types—A, B, C and D—of which influenza A (IAV) and influenza B (IBV) cause seasonal outbreaks in humans 1 . These viruses frequently undergo antigenic drift and reassortment, generating novel strains and raising the risk of pandemics 2 , which can impose substantial economic and social burdens beyond individual morbidity. For example, during the 2009 H1N1 pandemic, global demand for antiviral drugs and vaccines surged, leading to a dramatic rise in healthcare costs 3 . It is therefore essential to develop measures that can effectively prevent pandemic influenza resulting from antigenic variation. Vaccination remains the most effective and cost‑efficient strategy for preventing influenza infection, reducing disease burden, and establishing herd immunity 4 . Seasonal influenza vaccines are reformulated annually to match predicted circulating strains, primarily targeting the surface antigens haemagglutinin (HA) and neuraminidase (NA). These proteins are highly immunogenic and abundant on the viral surface, eliciting robust humoral and T‑cell responses upon infection. However, continual antigenic changes in HA and NA result in mismatches between vaccine strains and circulating viruses, diminishing vaccine effectiveness 2,5,6 . Consequently, there is a pressing need to develop universal influenza vaccines that target conserved antigens and provide broader and more durable protection against seasonal and pandemic strains 7-9 . To achieve this goal, attention has turned from the mutationally labile HA and NA to the internal nucleoprotein (NP), a highly conserved viral protein essential for genome transcription and replication 10 . NP is conserved not only among different strains within a subtype but also across IAV subtypes and between IAV and IBV lineages 11,12 . NP‑derived peptides are presented by MHC class I and II molecules on infected cells, eliciting cytotoxic CD4+ and CD8+ T‑cell responses that clear infected cells. These properties make NP an attractive target for T‑cell‑based universal vaccines capable of inducing broad cross‑protection. In the present study, we developed an adenoviral‑vector vaccine encoding the influenza NP antigen. Adenoviral vectors offer several advantages for vaccine development: their genome is well characterized and easily manipulated for foreign gene insertion, they have broad host tropism facilitating efficient gene delivery, and they are immunogenic across species including humans 13 . We aimed to create a vaccine that simultaneously protects against both IAV and IBV by incorporating NP into an adenoviral platform. Intranasal administration was selected to enhance mucosal immunogenicity and promote sustained respiratory mucosal immunity against diverse respiratory pathogens. Previous work has demonstrated that intranasal delivery of recombinant adenovirus expressing NP (rAd/NP) establishes tissue‑resident memory CD8+ T cells in the airways—a population not observed after intramuscular injection—and confers complete protection against influenza virus challenge 14-16 . This suggests that the mucosal route leverages the natural tropism of adenovirus for the nasal mucosa, yielding high antigen delivery efficiency 13 . Here, we evaluate the immunogenicity and protective efficacy of our NP‑based recombinant adenoviral vaccine administered intranasally. Specifically, we assess NP‑specific CD8+ T‑cell responses and protection against IAV and IBV challenge in mice. Our findings demonstrate that the NP‑fused recombinant adenovirus confers dual protection against IAV and IBV, indicating improved efficacy compared with conventional rAd/NP vaccines and offering valuable insights for advancing universal influenza vaccine development. II. Materials and Methods 1. Construction of Recombinant Adenoviral Vaccines Expressing Influenza Nucleoproteins To generate cDNA linking the NP of A/PR8 and the NP of B/Yamagata, PCR was performed using rAd/ANP and rAd/BNP as templates (2X PCR Master Mix Solution, iNtRON Biotechnology, Korea). Primer information is provided on the supplementary table 1. The cDNA was used to produce recombinant adeno virus using the AdEasy vector system (Qbiogene, Inc., USA). The pShuttle vector was linearized with Pme1 and co-electroporated with the pAd-Easy vector into BJ5183 cells (Omics Biotech, Republic of Korea) to produce recombinant pAd AB-NP and BA-NP vectors. The produced vectors were amplified in DH5a cells to yield sufficient amounts for subsequent experiments. Each vector, linearized with Pac1, was transfected into HEK293 cell lines and plaque formation was awaited. When all cells in the dish detached, showing CPE, the cells were harvested and used to obtain recombinant adeno virus particles from lysate by freezing-thawing. The lysates were used for further infection to scale up harvesting the recombinant adeno virus particles. The physical titer of the adenoviruses was determined by OD 260 nm absorbance, and the infectious titer was calculated by TCID50 to obtain PFU/mL. 2. Western Blot Analysis To confirm the expression of AB-NP and BA-NP, HEK293 cells were transduced with each recombinant adenovirus at a multiplicity of infection (MOI) of 10. After 48 hours, the cells were harvested and lysed in RIPA buffer supplemented with PMSF, pepstatin, and leupeptin. Protein samples were mixed with sampling dye containing DTT and boiled prior to SDS-PAGE. Separated proteins were transferred onto a PVDF membrane and blocked with 5% skim milk in TBST. For detection, a polyclonal anti-ANP antibody (1:1000, #PA5-32242, Invitrogen, Carlsbad, CA, USA) and a laboratory-generated polyclonal anti-BNP antibody were used as primary antibodies. HRP-conjugated goat anti-rabbit IgG (1:5000, AbC-5003, AbClone, Republic of Korea) and HRP-conjugated rabbit anti-mouse IgG (1:5000, ab6728, Abcam, Cambridge, UK) were used as secondary antibodies. Protein bands were visualized using the Amersham™ ECL™ Prime Western Blotting Detection Reagent (Cytiva, Little Chalfont, UK) and imaged with the ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA). 3. Mice and Ethical Statement 5 weeks old C57BL/6 mice (Orient Bio Inc., Seongnam, Republic of Korea) were housed a week under specific pathogen-free (SPF) conditions in the Laboratory Animal Facility of the College of Pharmacy, Ewha Womans University. All animal experiments were conducted in accordance with the guidelines for animal care and use, as approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University (approval number: 22-058). 4 . Vaccination and Virus Challenge Six-week-old mice were lightly anesthetized with isoflurane (Hana Pharm. Co. Ltd., Seoul, Republic of Korea) and intranasally immunized with 3×10 7 PFU of recombinant adenoviral vaccines expressing influenza A (rAd/ANP) or B (rAd/BNP) NP genes, diluted in 50 µL PBS. The vaccine constructs rAd/ANP and rAd/BNP were prepared as previously described 17 . The novel constructs AB-NP and BA-NP were administered in the same dose and volume via the intranasal route. Three weeks post-immunization, mice were challenged intranasally with 10 LD 50 of influenza A/Puerto Rico/8/34 (H1N1) or B/Yamagata/16/1988 virus to evaluate vaccine-induced protection. 6. Tissue and Blood Collection and lung cell isolation Two weeks post-immunization, blood was collected from the facial vein. 1X cold PBS with heparin were then added right after blood collection and inverted several times to prevent blood clotting. For CD marker staining, blood cells were blocked with streptavidin and anti-mouse CD16/CD32. Red Blood Cell lysing buffer were not used for blood cell staining. For intravital labeling, 10 μg of APC-conjugated anti-CD45 antibody in 200 μL PBS was administered via the tail vein. After 15 minutes, mice were euthanized, and lungs were perfused with 1X cold PBS containing heparin before harvested. Lungs were excised and dissociated into single-cell suspensions using a 93070 strainer (SPL) in RPMI 1640 medium (Welgene, Republic of Korea) supplemented with 10% FBS and 1% penicillin/streptomycin. To facilitate single-cell preparation, 1 mg/mL collagenase type II (Worthington) and 100 µg/mL DNase I (Sigma) were added and incubated for a half hour in 37’C incubator. Red blood cells were lysed using Red Blood Cell Lysing Buffer (Sigma) and 10% FBS supplemented RPMI 1640 was used as stop solution. Cell number and viability of the single-cell suspension were assessed using Muse count & Viability kit (Luminex), and based on these results, staining was performed using 1×10 6 cells. 7. Flow Cytometry Lung single-cell suspensions were centrifuged at 1600 rpm for 5 minutes at 4°C. Every washing step was conducted with FACS buffer (1X PBS with 0.5% FBS and 0.1% NaN₃). Cells were incubated with purified anti-mouse CD16/CD32 (BD Pharmingen, San Diego, CA, USA) to block Fc receptors, and 50 µg/mL streptavidin (Invitrogen) for 20 minutes at 4°C. After blocking, the cells were washed with 1× cold PBS, followed by staining with Zombie dye. The cells were then washed with FACS buffer and subjected to CD marker staining. The following fluorochrome-conjugated antibodies were used: Zombie Violet™ Fixable Viability Dye (#423114), APC/Cy7-anti-CD3 (clone 17A2), FITC-anti-CD8 (clone 53-6.7), APC-anti-CD45 (clone 30-F11) and all antibodies were purchased from biolegend (San Diego, CA, USA). Cells were stained for 30 minutes at 4°C with fluorescent antibodies and MHC tetramers (Db/BNP352–360 and Db/ANP366–374 tetramers Db/NP352–360 and Db/NP366–374 tetramers were prepared as previously described 18 . After incubation, the cells were washed twice and fixed with BD FACS Lysing Solution (BD Pharmingen) at room temperature for 20 minutes. Samples were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter) and FlowJo software (TreeStar Inc.). 8. Statistical Analysis Statistical analyses were performed using GraphPad Prism versions 5.0 and 9.0 (GraphPad Software, Inc.). Comparisons between groups were made using unpaired two-tailed Student’s t-tests or one-way ANOVA as appropriate. Statistical significance was defined as p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***). III. Results A. Construction of Adenoviral Vectors Co-expressing Conserved NP Antigens from Influenza A and B Viruses To develop a candidate universal influenza vaccine, we generated recombinant adenoviral vectors expressing the nucleoprotein (NP) antigens of both influenza A and B viruses, which are known to elicit T-cell responses. First, the NP gene sequences from influenza A and B viruses were obtained and amplified by PCR as linear fragments containing restriction enzyme sites. These amplified NP fragments were then ligated into plasmid backbones. To investigate whether the gene order of antigens influences protein expression, two different recombinant plasmids were constructed: one with the influenza A NP sequence positioned upstream of the B NP sequence (AB-NP), and the other with the reverse arrangement (BA-NP) (Figure 1A). Each recombinant plasmid was introduced into the adenoviral vector backbone through co-transformation to generate recombinant adenoviruses. HEK293 cells were transduced with the resulting adenoviral constructs, and NP protein expression was evaluated by western blot analysis. As shown in Figure 1B, both AB-NP and BA-NP constructs successfully expressed the NP proteins, confirming efficient in vitro expression of the antigenic components. B. Intranasal Administration of Adenoviral NP Fusion Vaccines Induces Antigen-Specific CD8+ T-Cell Responses in Mice A previous study demonstrated that intranasal administration of recombinant adenoviral vaccines can elicit CD8+ T cell responses that contribute to protection against influenza virus infection 16 . To evaluate whether the newly constructed NP-fusion adenoviral vaccines can induce cytotoxic T-cell responses in the lungs or circulation, female C57BL/6 mice (5–6 weeks old) were randomly divided into three groups (n = 4 per group). Mice were intranasally immunized once with an equal dose of AB-NP or BA-NP, and two weeks post-vaccination, mice were euthanized to analyze antigen-specific CD8+ T cells in blood and lung tissues by flow cytometry. For comparison, control groups were similarly immunized with A+B (A+B) using the same intranasal route and PFU dose. Antigen-specific CD8+ T cell responses were analyzed two weeks post-immunization using the same gating strategy described in Supplementary Figure S1 and S2 (Figure 2A). On day 14 post-immunization, we first assessed the frequency of circulating CD8+ T cells in the peripheral blood. NP-specific CD8+ T cells targeting influenza B virus were detected at low frequencies across all groups. In contrast, influenza A NP-specific CD8+ T cells were relatively more abundant in the AB-NP group compared to the other groups; however, the increase did not reach statistical significance (Figures 2B and 2C). These findings suggest that intranasal immunization may preferentially induce T-cell activation and proliferation, resulting in lower levels of detectable cytotoxic T cells in the bloodstream 19 . As expected, lung-resident NP-specific CD8+ T cells were robustly induced in all vaccinated groups. Notably, the highest frequencies of influenza A NP-specific CD8+ T cells were observed in the AB-NP group. In the case of influenza B NP-specific CD8+ T cells, the strongest responses were observed in the A+B group, followed by AB-NP and BA-NP in descending order (Figures 2D and 2E). Collectively, these results indicate that intranasal administration of NP fusion adenoviral vaccines (AB-NP and BA-NP) can induce pulmonary antigen-specific CD8+ T cell responses comparable to those elicited by single-NP adenoviral vaccines (A+B). C. Protective Efficacy of NP Fusion Adenoviral Vaccines Against Influenza A and B Virus Challenge To evaluate the protective efficacy of the NP fusion adenoviral vaccines against lethal influenza virus infection, mice were divided into four groups: a non-vaccinated naïve group, groups immunized intranasally with A+B, and groups immunized with AB-NP or BA-NP (Figures 3A and 3B). Three weeks post-immunization, all mice were challenged intranasally with a lethal dose of either influenza A virus (A/PR8) or B virus (B/Yamagata), and body weight and survival were monitored daily for 14 days. Following A/PR8 challenge, all mice in the naïve control group succumbed to infection within 8 days. The BA-NP group showed 60% survival, accompanied by substantial weight loss up to day 8, followed by gradual recovery. In contrast, both the A+B and AB-NP groups exhibited 100% survival. Notably, mice vaccinated with the NP fusion vaccine AB-NP experienced less body weight loss compared to the rAd/ANP group, suggesting superior protection. These results indicate that among the tested constructs, AB-NP provided the most effective protection against influenza A virus infection (Figure 3C). In the B/Yamagata challenge model, the control group showed 20% survival, while all three vaccinated groups demonstrated complete (100%) protection. Body weight loss was minimal in all vaccinated groups. These findings suggest that both AB-NP and BA-NP conferred protective efficacy comparable to that of A+B. Taken together, the results demonstrate that the AB-NP fusion vaccine provided protective immunity against both influenza A and B virus strains, highlighting its potential as a broadly protective adenoviral influenza vaccine candidate. IV. Discussion In the present study, we developed and evaluated recombinant adenoviral vaccines encoding nucleoprotein (NP) antigens from both influenza A and B viruses, aiming to induce cross-reactive T cell responses that contribute to broad-spectrum protection. Our findings demonstrate that the co-expression of conserved NP antigens from both virus types in a single adenoviral vector is feasible and immunogenic when delivered intranasally. NP is a highly conserved internal protein that has been extensively studied as a target for T cell–based influenza vaccines due to its ability to induce cross-protective CD8+ T cell responses. Both AB-NP and BA-NP vectors successfully expressed the respective NP fusion proteins in vitro and elicited antigen-specific CD8+ T cells in vivo. Notably, AB-NP induced the highest frequency of NP-specific CD8+ T cells in the lungs, which are crucial for local immune protection in the respiratory tract. Although systemic T-cell responses in peripheral blood were relatively low, this likely reflects the recruitment of effector cells to the mucosal tissues following intranasal vaccination. Our challenge experiments confirmed that AB-NP conferred robust protection against both A/PR8 (H1N1) and B/Yamagata strains. In the case of A/PR8, AB-NP achieved complete protection with minimal body weight loss, outperforming BA-NP and showing comparable or improved efficacy relative to A+B. Against B/Yamagata, AB-NP and BA-NP provided equivalent protection to A+B, indicating that the NP fusion constructs retained full immunogenic potential for both antigens. Interestingly, the order of NP antigen expression in the construct appeared to influence the immune outcome. AB-NP, in which the influenza A NP precedes the B NP, consistently elicited stronger T cell responses and better protection, especially against influenza A virus. This may reflect a hierarchical immunodominance effect, in which the first expressed antigen may be preferentially processed or presented. These findings suggest that rational design of antigen order in multivalent constructs can modulate the magnitude and quality of the immune response. Intranasal delivery of adenoviral vaccines offers several advantages, including direct stimulation of mucosal immunity, localized T cell activation, and the potential for needle-free administration. Our study reinforces previous reports showing that mucosal vaccination is particularly effective in inducing resident memory T cells in the lungs, which are instrumental in rapid response against respiratory pathogens. Moreover, T cell–oriented vaccine design provides a complementary approach to traditional antibody-based vaccines, especially in light of antigenic drift and the limited breadth of current seasonal influenza vaccines. Taken together, our data suggest that AB-NP is a promising universal influenza vaccine candidate capable of eliciting potent T cell responses and conferring protection against both influenza A and B viruses. The use of conserved internal antigens such as NP circumvents the issue of frequent antigenic variation seen in surface glycoproteins like HA and NA. The single-vector design encoding dual NP antigens simplifies manufacturing and enables broad coverage with a single dose. This study has some limitations. First, while protection was clearly observed in the murine model, further evaluation in additional models and human-relevant systems is necessary. Second, neutralizing antibody responses were not assessed, although NP is not expected to induce such responses. Future work should examine long-term memory, tissue-resident T cell phenotypes, and combinatorial strategies integrating NP with other conserved antigens, such as M2 or HA stem regions. In conclusion, this study provides preclinical evidence that a dual-NP-expressing adenoviral vaccine, particularly AB-NP, is an effective platform for inducing cross-protective T cell immunity. These findings support its further development as a component of a broadly protective, next-generation influenza vaccine. Declarations V. Data Availiability The datasets analyzed in this study are available from the corresponding author upon reasonable request. VI. Acknowledgments This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant number: HV23C0053). VII. Author Contributions YC, JYH, and JC conceived and designed the experiments. YC and JYH performed the experiments. YC, JYH, and JC analyzed the data. JYH and JC wrote the main manuscript text, and YC and JC reviewed the manuscript. VIII. Competing Interests The authors declare no competing interests. References Nuwarda, R. F., Alharbi, A. A. & Kayser, V. An Overview of Influenza Viruses and Vaccines. Vaccines (Basel) 9 (2021). https://doi.org/10.3390/vaccines9091032 Trombetta, C. M., Kistner, O., Montomoli, E., Viviani, S. & Marchi, S. Influenza Viruses and Vaccines: The Role of Vaccine Effectiveness Studies for Evaluation of the Benefits of Influenza Vaccines. Vaccines (Basel) 10 (2022). https://doi.org/10.3390/vaccines10050714 Peasah, S. K., Azziz-Baumgartner, E., Breese, J., Meltzer, M. I. & Widdowson, M. 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PLoS One 5, e13162 (2010). https://doi.org/10.1371/journal.pone.0013162 Altman, J. D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996). https://doi.org/10.1126/science.274.5284.94 Uddback, I. et al. Long-term maintenance of lung resident memory T cells is mediated by persistent antigen. Mucosal Immunol 14, 92–99 (2021). https://doi.org/10.1038/s41385-020-0309-3 Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":113041,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction and expression of adenoviral vectors encoding conserved NP antigens from influenza A and B viruses.\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of DNA constructs encoding the nucleoprotein (NP) sequences from influenza A virus (IAV) and influenza B virus (IBV). (B) HEK293 cells were transfected with AB-NP or BA-NP constructs, and cell lysates were analyzed by western blot to confirm NP protein expression. Control lysates were prepared from HEK293 cells infected with rAd/ANP or rAd/BNP at an MOI of 10. NP expression was detected using anti-A_NP primary antibody and HRP-conjugated secondary antibody.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7764215/v1/8495bf406fdc87869f0f3412.png"},{"id":96171633,"identity":"44c9599f-4507-4377-bbb6-8bb755b2d9ec","added_by":"auto","created_at":"2025-11-18 10:44:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143856,"visible":true,"origin":"","legend":"\u003cp\u003eAntigen-specific CD8+ T cell responses following intranasal immunization with NP fusion adenoviral vaccines.\u003c/p\u003e\n\u003cp\u003e(A) Experimental timeline. C57BL/6 mice were divided into three groups and intranasally immunized with 3×10⁷PFU of BA-NP (G1), AB-NP (G2), or rAd/ANP and rAd/BNP (G3). (B) Representative dot plots of antigen-specific CD8+ T cells in peripheral blood at day 14 post-vaccination, stained with IAV NP–specific tetramer (left) and IBV NP–specific tetramer (right). (C) Frequency of epitope-specific CD8+ T cells among total blood leukocytes. (D) Dot plots of lung parenchymal CD8+ T cells gated on CD3+CD8+CD45- and stained with either ANP- or BNP-specific tetramers. (E) Total number of NP-specific CD8+ T cells in the lung parenchyma. Data represent mean ± SD from four mice per group. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7764215/v1/52ffcde42cc8acca6d44a67d.png"},{"id":96171639,"identity":"36e060d5-e534-4bdf-b595-03018aba71b0","added_by":"auto","created_at":"2025-11-18 10:44:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":173970,"visible":true,"origin":"","legend":"\u003cp\u003eProtective efficacy of NP fusion adenoviral vaccines against lethal influenza A and B virus challenge.\u003c/p\u003e\n\u003cp\u003e(A, B) Timeline of challenge experiments. C57BL/6 mice were divided into four groups and intranasally immunized with 3×10⁷ PFU of AB-NP, BA-NP, rAd/ANP, or rAd/BNP. Naïve mice served as negative controls. (C) Body weight changes and survival following challenge with 10 LD\u003csub\u003e50\u003c/sub\u003e of influenza A virus (A/PR8). (D) Body weight and survival following challenge with 10 LD\u003csub\u003e50\u003c/sub\u003e of influenza B virus (B/Yamagata). Data represent mean ± SEM from 4–5 mice per group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7764215/v1/040e07aceaa8f8b10e9496a1.png"},{"id":100858883,"identity":"82d55f30-e8d6-4b83-a31a-cbda9c60afc8","added_by":"auto","created_at":"2026-01-22 07:24:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":751478,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7764215/v1/14bf2447-24c1-4d94-89b8-9fca2e5ee064.pdf"},{"id":96171638,"identity":"cbff19df-f5fa-4fe4-841d-304c6d8b113e","added_by":"auto","created_at":"2025-11-18 10:44:20","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":230026,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7764215/v1/844f6d35a0fe5e981b1f1f54.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single‐Dose Intranasal Chimeric Influenza A–B Nucleoprotein Vaccine Confers Dual Protection in a Mouse Model","fulltext":[{"header":"I. Introduction","content":"\u003cp\u003eInfluenza is a contagious respiratory disease caused by influenza viruses. According to the World Health Organization (WHO), each year influenza causes 3–5 million cases of severe illness and between 290,000 and 650,000 deaths worldwide, posing a persistent threat to human health. The virus primarily infects the respiratory mucosa, with typical symptoms including fever, chills, cough, sore throat, myalgia, headache and fatigue [https://www.cdc.gov/flu/signs-symptoms/index.html]. Influenza viruses are classified into four types—A, B, C and D—of which influenza A (IAV) and influenza B (IBV) cause seasonal outbreaks in humans \u003csup\u003e1\u003c/sup\u003e. These viruses frequently undergo antigenic drift and reassortment, generating novel strains and raising the risk of pandemics \u003csup\u003e2\u003c/sup\u003e, which can impose substantial economic and social burdens beyond individual morbidity. For example, during the 2009 H1N1 pandemic, global demand for antiviral drugs and vaccines surged, leading to a dramatic rise in healthcare costs \u003csup\u003e3\u003c/sup\u003e. It is therefore essential to develop measures that can effectively prevent pandemic influenza resulting from antigenic variation.\u003c/p\u003e\n\u003cp\u003eVaccination remains the most effective and cost‑efficient strategy for preventing influenza infection, reducing disease burden, and establishing herd immunity \u003csup\u003e4\u003c/sup\u003e. Seasonal influenza vaccines are reformulated annually to match predicted circulating strains, primarily targeting the surface antigens haemagglutinin (HA) and neuraminidase (NA). These proteins are highly immunogenic and abundant on the viral surface, eliciting robust humoral and T‑cell responses upon infection. However, continual antigenic changes in HA and NA result in mismatches between vaccine strains and circulating viruses, diminishing vaccine effectiveness \u003csup\u003e2,5,6\u003c/sup\u003e. Consequently, there is a pressing need to develop universal influenza vaccines that target conserved antigens and provide broader and more durable protection against seasonal and pandemic strains \u003csup\u003e7-9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo achieve this goal, attention has turned from the mutationally labile HA and NA to the internal nucleoprotein (NP), a highly conserved viral protein essential for genome transcription and replication \u003csup\u003e10\u003c/sup\u003e. NP is conserved not only among different strains within a subtype but also across IAV subtypes and between IAV and IBV lineages \u003csup\u003e11,12\u003c/sup\u003e. NP‑derived peptides are presented by MHC\u0026nbsp;class\u0026nbsp;I and II molecules on infected cells, eliciting cytotoxic CD4+ and CD8+ T‑cell responses that clear infected cells. These properties make NP an attractive target for T‑cell‑based universal vaccines capable of inducing broad cross‑protection.\u003c/p\u003e\n\u003cp\u003eIn the present study, we developed an adenoviral‑vector vaccine encoding the influenza NP antigen. Adenoviral vectors offer several advantages for vaccine development: their genome is well characterized and easily manipulated for foreign gene insertion, they have broad host tropism facilitating efficient gene delivery, and they are immunogenic across species including humans \u003csup\u003e13\u003c/sup\u003e. We aimed to create a vaccine that simultaneously protects against both IAV and IBV by incorporating NP into an adenoviral platform. Intranasal administration was selected to enhance mucosal immunogenicity and promote sustained respiratory mucosal immunity against diverse respiratory pathogens. Previous work has demonstrated that intranasal delivery of recombinant adenovirus expressing NP (rAd/NP) establishes tissue‑resident memory CD8+ T cells in the airways—a population not observed after intramuscular injection—and confers complete protection against influenza virus challenge \u003csup\u003e14-16\u003c/sup\u003e. This suggests that the mucosal route leverages the natural tropism of adenovirus for the nasal mucosa, yielding high antigen delivery efficiency \u003csup\u003e13\u003c/sup\u003e .\u003c/p\u003e\n\u003cp\u003eHere, we evaluate the immunogenicity and protective efficacy of our NP‑based recombinant adenoviral vaccine administered intranasally. Specifically, we assess NP‑specific CD8+ T‑cell responses and protection against IAV and IBV challenge in mice. Our findings demonstrate that the NP‑fused recombinant adenovirus confers dual protection against IAV and IBV, indicating improved efficacy compared with conventional rAd/NP vaccines and offering valuable insights for advancing universal influenza vaccine development.\u003c/p\u003e"},{"header":"II. Materials and Methods","content":"\u003cp\u003e1. Construction of Recombinant Adenoviral Vaccines Expressing Influenza Nucleoproteins\u003c/p\u003e\n\u003cp\u003eTo generate cDNA linking the NP of A/PR8 and the NP of B/Yamagata, PCR was performed using rAd/ANP and rAd/BNP as templates (2X PCR Master Mix Solution, iNtRON Biotechnology, Korea). Primer information is provided on the supplementary table 1. The cDNA was used to produce recombinant adeno virus using the AdEasy vector system (Qbiogene, Inc., USA). The pShuttle vector was linearized with Pme1 and co-electroporated with the pAd-Easy vector into BJ5183 cells (Omics Biotech, Republic of Korea) to produce recombinant pAd AB-NP and BA-NP vectors. The produced vectors were amplified in DH5a cells to yield sufficient amounts for subsequent experiments. Each vector, linearized with Pac1, was transfected into HEK293 cell lines and plaque formation was awaited. When all cells in the dish detached, showing CPE, the cells were harvested and used to obtain recombinant adeno virus particles from lysate by freezing-thawing. The lysates were used for further infection to scale up harvesting the recombinant adeno virus particles.\u003c/p\u003e\n\u003cp\u003eThe physical titer of the adenoviruses was determined by OD 260 nm absorbance, and the infectious titer was calculated by TCID50 to obtain PFU/mL.\u003c/p\u003e\n\u003cp\u003e2. Western Blot Analysis\u003c/p\u003e\n\u003cp\u003eTo confirm the expression of AB-NP and BA-NP, HEK293 cells were transduced with each recombinant adenovirus at a multiplicity of infection (MOI) of 10. After 48 hours, the cells were harvested and lysed in RIPA buffer supplemented with PMSF, pepstatin, and leupeptin. Protein samples were mixed with sampling dye containing DTT and boiled prior to SDS-PAGE. Separated proteins were transferred onto a PVDF membrane and blocked with 5% skim milk in TBST. For detection, a polyclonal anti-ANP antibody (1:1000, #PA5-32242, Invitrogen, Carlsbad, CA, USA) and a laboratory-generated polyclonal anti-BNP antibody were used as primary antibodies. HRP-conjugated goat anti-rabbit IgG (1:5000, AbC-5003, AbClone, Republic of Korea) and HRP-conjugated rabbit anti-mouse IgG (1:5000, ab6728, Abcam, Cambridge, UK) were used as secondary antibodies. Protein bands were visualized using the Amersham™ ECL™ Prime Western Blotting Detection Reagent (Cytiva, Little Chalfont, UK) and imaged with the ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e\n\u003cp\u003e3. Mice and Ethical Statement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5 weeks old C57BL/6 mice (Orient Bio Inc., Seongnam, Republic of Korea) were housed a week under specific pathogen-free (SPF) conditions in the Laboratory Animal Facility of the College of Pharmacy, Ewha Womans University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the guidelines for animal care and use, as approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University (approval number: 22-058).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cs\u003e4\u003c/s\u003e. Vaccination and Virus Challenge\u003c/p\u003e\n\u003cp\u003eSix-week-old mice were lightly anesthetized with isoflurane\u0026nbsp;(Hana Pharm. Co. Ltd., Seoul, Republic of Korea) and intranasally immunized with 3×10\u003csup\u003e7\u003c/sup\u003e PFU of recombinant adenoviral vaccines expressing influenza A (rAd/ANP) or B (rAd/BNP) NP genes, diluted in 50 µL PBS. The vaccine constructs rAd/ANP and rAd/BNP were prepared as previously described \u003csup\u003e17\u003c/sup\u003e. The novel constructs AB-NP and BA-NP were administered in the same dose and volume via the intranasal route. Three weeks post-immunization, mice were challenged intranasally with 10 LD\u003csub\u003e50\u003c/sub\u003e of influenza A/Puerto Rico/8/34 (H1N1) or B/Yamagata/16/1988 virus to evaluate vaccine-induced protection.\u003c/p\u003e\n\u003cp\u003e6. Tissue and Blood Collection and lung cell isolation\u003c/p\u003e\n\u003cp\u003eTwo weeks post-immunization, blood was collected from the facial vein. 1X cold PBS with heparin were then added right after blood collection and inverted several times to prevent blood clotting. For CD marker staining, blood cells were blocked with streptavidin and anti-mouse CD16/CD32. Red Blood Cell lysing buffer were not used for blood cell staining.\u003c/p\u003e\n\u003cp\u003eFor intravital labeling, 10 μg of APC-conjugated anti-CD45 antibody in 200 μL PBS was administered via the tail vein. After 15 minutes, mice were euthanized, and lungs were perfused with 1X cold PBS containing heparin before harvested. Lungs were excised and dissociated into single-cell suspensions using a 93070 strainer (SPL) in RPMI 1640 medium (Welgene, Republic of Korea) supplemented with 10% FBS and 1% penicillin/streptomycin. To facilitate single-cell preparation, 1 mg/mL collagenase type II (Worthington) and 100 µg/mL DNase I (Sigma) were added and incubated for a half hour in 37’C incubator. Red blood cells were lysed using Red Blood Cell Lysing Buffer (Sigma) and 10% FBS supplemented RPMI 1640 was used as stop solution. Cell number and viability of the single-cell suspension were assessed using Muse count \u0026amp; Viability kit (Luminex), and based on these results, staining was performed using 1×10\u003csup\u003e6\u003c/sup\u003e cells.\u003c/p\u003e\n\u003cp\u003e7. Flow Cytometry\u003c/p\u003e\n\u003cp\u003eLung single-cell suspensions were centrifuged at 1600 rpm for 5 minutes at 4°C. Every washing step was conducted with FACS buffer (1X PBS with 0.5% FBS and 0.1% NaN₃). Cells were incubated with purified anti-mouse CD16/CD32 (BD Pharmingen, San Diego, CA, USA) to block Fc receptors, and 50 µg/mL streptavidin (Invitrogen) for 20 minutes at 4°C. After blocking, the cells were washed with 1× cold PBS, followed by staining with Zombie dye. The cells were then washed with FACS buffer and subjected to CD marker staining. The following fluorochrome-conjugated antibodies were used: Zombie Violet™ Fixable Viability Dye (#423114), APC/Cy7-anti-CD3 (clone 17A2), FITC-anti-CD8 (clone 53-6.7), APC-anti-CD45 (clone 30-F11) and all antibodies were purchased from biolegend (San Diego, CA, USA). Cells were stained for 30 minutes at 4°C with fluorescent antibodies and MHC tetramers (Db/BNP352–360 and Db/ANP366–374 tetramers Db/NP352–360 and Db/NP366–374 tetramers were prepared as previously described \u003csup\u003e18\u003c/sup\u003e. After incubation, the cells were washed twice and fixed with BD FACS Lysing Solution (BD Pharmingen) at room temperature for 20 minutes. Samples were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter) and FlowJo software (TreeStar Inc.).\u003c/p\u003e\n\u003cp\u003e8. Statistical Analysis\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism versions 5.0 and 9.0 (GraphPad Software, Inc.). Comparisons between groups were made using unpaired two-tailed Student’s t-tests or one-way ANOVA as appropriate. Statistical significance was defined as p \u0026lt; 0.05 (*), p \u0026lt; 0.01 (**), or p \u0026lt; 0.001 (***).\u003c/p\u003e"},{"header":"III. Results","content":"\u003cp\u003eA. Construction of Adenoviral Vectors Co-expressing Conserved NP Antigens from Influenza A and B Viruses\u003c/p\u003e\n\u003cp\u003eTo develop a candidate universal influenza vaccine, we generated recombinant adenoviral vectors expressing the nucleoprotein (NP) antigens of both influenza A and B viruses, which are known to elicit T-cell responses. First, the NP gene sequences from influenza A and B viruses were obtained and amplified by PCR as linear fragments containing restriction enzyme sites. These amplified NP fragments were then ligated into plasmid backbones. To investigate whether the gene order of antigens influences protein expression, two different recombinant plasmids were constructed: one with the influenza A NP sequence positioned upstream of the B NP sequence (AB-NP), and the other with the reverse arrangement (BA-NP) (Figure 1A).\u003c/p\u003e\n\u003cp\u003eEach recombinant plasmid was introduced into the adenoviral vector backbone through co-transformation to generate recombinant adenoviruses. HEK293 cells were transduced with the resulting adenoviral constructs, and NP protein expression was evaluated by western blot analysis. As shown in Figure 1B, both AB-NP and BA-NP constructs successfully expressed the NP proteins, confirming efficient in vitro expression of the antigenic components.\u003c/p\u003e\n\u003cp\u003eB. Intranasal Administration of Adenoviral NP Fusion Vaccines Induces Antigen-Specific CD8+ T-Cell Responses in Mice\u003c/p\u003e\n\u003cp\u003eA previous study demonstrated that intranasal administration of recombinant adenoviral vaccines can elicit CD8+ T cell responses that contribute to protection against influenza virus infection \u003csup\u003e16\u003c/sup\u003e. To evaluate whether the newly constructed NP-fusion adenoviral vaccines can induce cytotoxic T-cell responses in the lungs or circulation, female C57BL/6 mice (5–6 weeks old) were randomly divided into three groups (n = 4 per group). Mice were intranasally immunized once with an equal dose of AB-NP or BA-NP, and two weeks post-vaccination, mice were euthanized to analyze antigen-specific CD8+ T cells in blood and lung tissues by flow cytometry. For comparison, control groups were similarly immunized with A+B (A+B) using the same intranasal route and PFU dose. Antigen-specific CD8+ T cell responses were analyzed two weeks post-immunization using the same gating strategy described in Supplementary Figure S1 and S2 (Figure 2A).\u003c/p\u003e\n\u003cp\u003eOn day 14 post-immunization, we first assessed the frequency of circulating CD8+ T cells in the peripheral blood. NP-specific CD8+ T cells targeting influenza B virus were detected at low frequencies across all groups. In contrast, influenza A NP-specific CD8+ T cells were relatively more abundant in the AB-NP group compared to the other groups; however, the increase did not reach statistical significance (Figures 2B and 2C). These findings suggest that intranasal immunization may preferentially induce T-cell activation and proliferation, resulting in lower levels of detectable cytotoxic T cells in the bloodstream \u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs expected, lung-resident NP-specific CD8+ T cells were robustly induced in all vaccinated groups. Notably, the highest frequencies of influenza A NP-specific CD8+ T cells were observed in the AB-NP group. In the case of influenza B NP-specific CD8+ T cells, the strongest responses were observed in the A+B group, followed by AB-NP and BA-NP in descending order (Figures 2D and 2E). Collectively, these results indicate that intranasal administration of NP fusion adenoviral vaccines (AB-NP and BA-NP) can induce pulmonary antigen-specific CD8+ T cell responses comparable to those elicited by single-NP adenoviral vaccines (A+B).\u003c/p\u003e\n\u003cp\u003eC. Protective Efficacy of NP Fusion Adenoviral Vaccines Against Influenza A and B Virus Challenge\u003c/p\u003e\n\u003cp\u003eTo evaluate the protective efficacy of the NP fusion adenoviral vaccines against lethal influenza virus infection, mice were divided into four groups: a non-vaccinated naïve group, groups immunized intranasally with A+B, and groups immunized with AB-NP or BA-NP (Figures 3A and 3B). Three weeks post-immunization, all mice were challenged intranasally with a lethal dose of either influenza A virus (A/PR8) or B virus (B/Yamagata), and body weight and survival were monitored daily for 14 days.\u003c/p\u003e\n\u003cp\u003eFollowing A/PR8 challenge, all mice in the naïve control group succumbed to infection within 8 days. The BA-NP group showed 60% survival, accompanied by substantial weight loss up to day 8, followed by gradual recovery. In contrast, both the A+B and AB-NP groups exhibited 100% survival. Notably, mice vaccinated with the NP fusion vaccine AB-NP experienced less body weight loss compared to the rAd/ANP group, suggesting superior protection. These results indicate that among the tested constructs, AB-NP provided the most effective protection against influenza A virus infection (Figure 3C).\u003c/p\u003e\n\u003cp\u003eIn the B/Yamagata challenge model, the control group showed 20% survival, while all three vaccinated groups demonstrated complete (100%) protection. Body weight loss was minimal in all vaccinated groups. These findings suggest that both AB-NP and BA-NP conferred protective efficacy comparable to that of A+B.\u003c/p\u003e\n\u003cp\u003eTaken together, the results demonstrate that the AB-NP fusion vaccine provided protective immunity against both influenza A and B virus strains, highlighting its potential as a broadly protective adenoviral influenza vaccine candidate.\u003c/p\u003e"},{"header":"IV. Discussion","content":"\u003cp\u003eIn the present study, we developed and evaluated recombinant adenoviral vaccines encoding nucleoprotein (NP) antigens from both influenza A and B viruses, aiming to induce cross-reactive T cell responses that contribute to broad-spectrum protection. Our findings demonstrate that the co-expression of conserved NP antigens from both virus types in a single adenoviral vector is feasible and immunogenic when delivered intranasally.\u003c/p\u003e\n\u003cp\u003eNP is a highly conserved internal protein that has been extensively studied as a target for T cell\u0026ndash;based influenza vaccines due to its ability to induce cross-protective CD8+ T cell responses. Both AB-NP and BA-NP vectors successfully expressed the respective NP fusion proteins in vitro and elicited antigen-specific CD8+ T cells in vivo. Notably, AB-NP induced the highest frequency of NP-specific CD8+ T cells in the lungs, which are crucial for local immune protection in the respiratory tract. Although systemic T-cell responses in peripheral blood were relatively low, this likely reflects the recruitment of effector cells to the mucosal tissues following intranasal vaccination.\u003c/p\u003e\n\u003cp\u003eOur challenge experiments confirmed that AB-NP conferred robust protection against both A/PR8 (H1N1) and B/Yamagata strains. In the case of A/PR8, AB-NP achieved complete protection with minimal body weight loss, outperforming BA-NP and showing comparable or improved efficacy relative to A+B. Against B/Yamagata, AB-NP and BA-NP provided equivalent protection to A+B, indicating that the NP fusion constructs retained full immunogenic potential for both antigens.\u003c/p\u003e\n\u003cp\u003eInterestingly, the order of NP antigen expression in the construct appeared to influence the immune outcome. AB-NP, in which the influenza A NP precedes the B NP, consistently elicited stronger T cell responses and better protection, especially against influenza A virus. This may reflect a hierarchical immunodominance effect, in which the first expressed antigen may be preferentially processed or presented. These findings suggest that rational design of antigen order in multivalent constructs can modulate the magnitude and quality of the immune response.\u003c/p\u003e\n\u003cp\u003eIntranasal delivery of adenoviral vaccines offers several advantages, including direct stimulation of mucosal immunity, localized T cell activation, and the potential for needle-free administration. Our study reinforces previous reports showing that mucosal vaccination is particularly effective in inducing resident memory T cells in the lungs, which are instrumental in rapid response against respiratory pathogens. Moreover, T cell\u0026ndash;oriented vaccine design provides a complementary approach to traditional antibody-based vaccines, especially in light of antigenic drift and the limited breadth of current seasonal influenza vaccines.\u003c/p\u003e\n\u003cp\u003eTaken together, our data suggest that AB-NP is a promising universal influenza vaccine candidate capable of eliciting potent T cell responses and conferring protection against both influenza A and B viruses. The use of conserved internal antigens such as NP circumvents the issue of frequent antigenic variation seen in surface glycoproteins like HA and NA. The single-vector design encoding dual NP antigens simplifies manufacturing and enables broad coverage with a single dose.\u003c/p\u003e\n\u003cp\u003eThis study has some limitations. First, while protection was clearly observed in the murine model, further evaluation in additional models and human-relevant systems is necessary. Second, neutralizing antibody responses were not assessed, although NP is not expected to induce such responses. Future work should examine long-term memory, tissue-resident T cell phenotypes, and combinatorial strategies integrating NP with other conserved antigens, such as M2 or HA stem regions.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study provides preclinical evidence that a dual-NP-expressing adenoviral vaccine, particularly AB-NP, is an effective platform for inducing cross-protective T cell immunity. These findings support its further development as a component of a broadly protective, next-generation influenza vaccine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eV. Data Availiability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVI. Acknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the Korean Health Technology R\u0026amp;D Project, Ministry of Health \u0026amp; Welfare, Republic of Korea (Grant number: HV23C0053).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVII. Author Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYC, JYH, and JC conceived and designed the experiments. YC and JYH performed the experiments. YC, JYH, and JC analyzed the data. JYH and JC wrote the main manuscript text, and YC and JC reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVIII. Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNuwarda, R. F., Alharbi, A. A. \u0026amp; Kayser, V. 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D. \u003cem\u003eet al.\u003c/em\u003e Phenotypic analysis of antigen-specific T lymphocytes. \u003cem\u003eScience\u003c/em\u003e 274, 94\u0026ndash;96 (1996). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.274.5284.94\u003c/span\u003e\u003cspan address=\"10.1126/science.274.5284.94\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUddback, I. \u003cem\u003eet al.\u003c/em\u003e Long-term maintenance of lung resident memory T cells is mediated by persistent antigen. \u003cem\u003eMucosal Immunol\u003c/em\u003e 14, 92\u0026ndash;99 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41385-020-0309-3\u003c/span\u003e\u003cspan address=\"10.1038/s41385-020-0309-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7764215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7764215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe influenza nucleoprotein (NP) is conserved and type‑specific, making it a key target for universal vaccine development. NP sequences mutate slowly and share \u0026gt; 90% amino‑acid homology within a virus type; NP is recognized by cytotoxic T lymphocytes (CTLs), which clear infected cells and contribute to broad heterosubtypic immunity. Intranasal NP‑based vaccines have previously protected mice against diverse influenza A subtypes (H1N1, H3N2, H5N2, H7N9 and H9N2) and two lineages of influenza B (B/Yamagata and B/Victoria lineages), yet NP is type‑specific, and vaccines often target only one type. To achieve cross‑type protection, we engineered recombinant adenoviruses expressing NP from influenza A (A‑NP), influenza B (B‑NP) or chimeric constructs linking the two sequences (AB‑NP and BA‑NP). Four replication‑deficient adenoviruses encoding A‑NP, B‑NP, AB‑NP, or BA‑NP were produced. BALB/c mice were anaesthetized and immunized intranasally once with either a mixture of A‑NP plus B‑NP (A + B), or a single chimeric vaccine (AB‑NP or BA‑NP). All regimens induced robust systemic specific IgG, mucosal IgA responses, and CTLs, reactive to both A and B NP. Each group showed complete protection from both influenza A and B challenges compared with controls. Of note, the AB‑NP fusion vaccine conferred the highest survival rate and the least morbidity. Our data demonstrate that fusing influenza A and B NP sequences into a single recombinant antigen induces cross‑type immunity after a single intranasal dose. This extends previous findings that NP‑based vaccines protect against multiple influenza A subtypes and both B lineages and suggests that chimeric NP vaccines could form the basis of a real universal influenza vaccine strategy.\u003c/p\u003e","manuscriptTitle":"Single‐Dose Intranasal Chimeric Influenza A–B Nucleoprotein Vaccine Confers Dual Protection in a Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 10:44:15","doi":"10.21203/rs.3.rs-7764215/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b88fe0fe-a61f-4fbe-8870-05e291149f33","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58083628,"name":"Biological sciences/Immunology"},{"id":58083629,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-01-21T22:09:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 10:44:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7764215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7764215","identity":"rs-7764215","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}