Aerosol immunization with influenza matrix, nucleoprotein, or both prevents lung disease in pig

Seasonal influenza vaccines primarily induce strain-specific neutralizing antibodies to hemagglutinin (HA), but rapid antigenic drift leads to loss of protection and frequent vaccine updates. Broadly protective influenza vaccines (BPIVs) may be achievable by targeting conserved internal antigens that elicit CD4+ and CD8+ T-cell responses, notably polymerase basic protein 1 (PB1), nucleoprotein (NP), and matrix 1 (M1), which correlate with reduced viral shedding and disease severity in humans. Induction of lung-localized immunity via respiratory tract vaccination can be particularly effective against respiratory pathogens, because severe influenza disease is driven by infection of the lower respiratory tract. Viral-vectored platforms such as replication-deficient chimpanzee adenovirus (ChAdOx) and modified vaccinia Ankara (MVA) can boost M1- and NP-specific T cells in humans. Neuraminidase (NA), while variable, evolves more slowly than HA and can contribute to broader protection, especially when delivered mucosally. Pigs are a relevant natural large-animal model with close immunological and physiological similarity to humans and are infected by similar influenza subtypes. Prior work in pH1N1 pre-exposed pigs showed that aerosol prime-boost immunization with ChAdOx2-NPM1-NA2 followed by MVA-NPM1-NA2 abolished viral shedding and lung pathology after heterologous H3N2 challenge, but the matched NA precluded dissecting the relative roles of T cells versus antibodies and of individual antigens. The present study evaluates: (1) whether a single aerosol dose of ChAdOx2-NPM1-NA2 is protective in pre-exposed pigs, and (2) whether both NP and M1 are necessary, by directly comparing single-antigen (NP or M1) versus fusion (NPM1) aerosol prime-boost regimens.

Evidence from human challenge and community cohort studies demonstrates that preexisting T-cell responses to conserved internal influenza proteins (e.g., NP, M1, PB1) are associated with reduced illness severity and viral shedding. Heterologous prime-boost vaccination with adenovirus and poxvirus vectors can boost NP/M1-specific T cells in adults. Strategies targeting NA are promising, as NA drifts more slowly than HA and mucosal NA immunization induces protective immunity across animal models. Universal vaccine efforts often focus on conserved HA stem regions or mosaic/multivalent HA approaches, but internal antigen-focused T-cell vaccines offer a complementary route to heterosubtypic protection. Prior pig studies showed strong protection with aerosolized ChAdOx2-NPM1-NA2/MVA-NPM1-NA2 in pH1N1 pre-exposed animals, but NA matching complicated attribution of protective mechanisms. Differences between mice and pigs in vaccine-induced protection have been noted, underscoring the need for large animal models for translational relevance.

Vaccine constructs: ChAdOx2 and MVA vectors expressing NP, M1, their fusion (NPM1), and a combined construct with NA2 (from A/swine/Ohio/A01354299/2017 H3N2) were produced as described previously. Single-antigen ChAdOx2 vectors (NP or M1) were generated by removing the alternate ORF from NPM1 shuttle plasmids via inverse PCR and recombining into ChAdOx2 destination plasmids. MVA single-antigen vectors were generated similarly under the F11 promoter. NP and M1 ORFs derived from A/swine/England/1353/2009.

Mouse immunogenicity: Female 6-week-old Balb/c mice (n=5/group) received ChAdOx2-NP, -M1, or -NPM1 at 1×10^5 IU by either intramuscular (IM; 50 µL) or intranasal (IN; 30 µL drop-wise) routes under isoflurane anesthesia, followed 4 weeks later by homologous MVA boost at 1×10^6 PFU (same route). Four weeks post-boost, splenocytes and lung cells were harvested for IFN-γ ELISpot and intracellular cytokine staining (ICS) following stimulation with overlapping NP or M1 peptides. Lung tissue-resident memory (TRM) phenotyping (CD103, CD69) was performed by spectral flow cytometry.

Pig study 1 (single-dose ChAdOx2-NPM1-NA2): Twelve 6-week-old female Landrace×Large White pigs, seronegative for pH1N1 and H3N2, were intranasally pre-exposed with 5×10^6 PFU A/swine/England/1353/2009 (pH1N1) (2 mL/nostril). After 24 days, six pigs were immunized by aerosol with 5×10^7 IU ChAdOx2-NPM1-NA2 (1 mL in PBS over 2–5 min) using an Aerogen Solo vibrating mesh nebulizer; six remained unimmunized controls. Four weeks later all pigs were intranasally challenged with 1.2×10^7 PFU A/swine/Ohio/A01354299/2017 (H3N2) (2 mL/nostril). Daily nasal swabs (D1–D4) were collected for plaque assay. On D4 post-challenge, pigs were euthanized; bronchoalveolar lavage (BAL), lungs, and PBMCs were collected for virology (plaque assays), pathology (gross, histopathology, NP immunohistochemistry with Iowa scoring), and immunology (ELISAs for serum and BAL IgG/IgA against pH1N1, H3N2, recNA2; IFN-γ ELISpot in BAL) analyses.

Pig study 2 (antigen contribution; prime-boost): Twenty-four similar pigs were pre-exposed intranasally with 3×10^6 PFU pH1N1. After 4 weeks they were randomized (n=6/group) to receive aerosol ChAdOx2-NP, ChAdOx2-M1, or ChAdOx2-NPM1 at 5×10^10 IU in 1 mL; controls were unimmunized. Four weeks later, homologous aerosol MVA boost at 1.5×10^6 PFU (MVA-NP, -M1, or -NPM1). Four weeks post-boost, all were challenged intranasally with 5.7×10^6 PFU H3N2 (4 mL total). Nasal swabs (D1–D4) were collected for viral load (plaque assay). On D4, euthanasia and collection of BAL and PBMCs were performed for IFN-γ ELISpot (NP, M1 peptides; live pH1N1/H3N2 stimulation), ICS of BAL T cells (CD4/CD8 IFN-γ and TNF after pH1N1, H3N2, NP, M1 stimulation), and ELISAs for serum and BAL antibodies (pH1N1, H3N2, NP). Gross pathology (Halbur), percentage lung involvement by image analysis, histopathology (Morgan), and NP-IHC (Iowa) were scored.

Assays: ELISAs included serum IgG to pH1N1, H3N2, and recNA2; BAL IgG/IgA to pH1N1/H3N2; and commercial NP IgG kit with endpoint titer calculation. ELISpot measured IFN-γ spot-forming cells per 10^6 cells. ICS quantified cytokine-producing CD4+ and CD8+ T cells in BAL following antigen stimulation. Statistics used normality testing, t tests or Mann–Whitney for two-group comparisons, and two-way ANOVA with Bonferroni or Kruskal–Wallis with Dunn’s tests for multiple groups/timepoints. Spearman correlations between 16 immune parameters and 5 virological/pathological measures were computed across all groups.

Single-dose ChAdOx2-NPM1-NA2 aerosol in pre-exposed pigs:

• Trend to reduced H3N2 nasal shedding on D2 and D3 post-challenge (p=0.058 and p=0.099), with no detectable virus in BAL or lung in either group.
• Significantly reduced percentage lung involvement by image analysis versus controls; histopathology scores significantly lower; NP-IHC (Iowa) significantly reduced in immunized animals.
• Serum IgG to pH1N1, H3N2, and recNA2 increased post-vaccination (not statistically significant). BAL antibody trends higher (IgG H1N1/H3N2; IgA H3N2) without significance. BAL IFN-γ ELISpot trends higher after M1 (p=0.06) and pH1N1 (p=0.07) stimulation.

Antigen contribution (prime-boost ChAdOx2→MVA):

• Viral shedding: Significant reduction in nasal shedding AUC for M1 and NPM1 versus controls. No H3N2 detected in BAL or lungs (likely dilution by lavage).
• Pathology: Gross pathology scores reduced significantly for NP and M1; histopathology and NP-IHC (Iowa) significantly reduced for all immunized groups, with M1 showing the lowest scores across measures. Percentage lung involvement reduced in all immunized groups; M1 alone gave the greatest reduction versus controls, with no significant differences among NP, M1, and NPM1.
• Antibodies: NP group had significantly higher serum IgG titers to pH1N1, H3N2, and NP after MVA boost than control and M1; NPM1 also boosted serum IgG versus control and M1 (Table 1). In BAL, NP induced significantly higher IgG-pH1N1, IgG-H3N2, and IgA-H3N2 than M1 and control; NPM1 trended higher without significance. No differences in pH1N1-specific BAL IgA among groups. M1 immunization did not boost virus- or M1-specific antibody responses.
• T cells: PBMC IFN-γ ELISpot showed M1-specific responses increased after ChAdOx2-M1 prime and MVA-M1 boost; NP-specific responses highest in NP group after boost and at study end (D87) for pH1N1, H3N2, and NP stimulations. In BAL, NP-specific IFN-γ was highest in NP group; M1-specific IFN-γ highest in M1 group. All immunized groups had significantly higher pH1N1-specific BAL IFN-γ versus controls; H3N2-specific BAL IFN-γ significantly higher in NP and NPM1 groups. ICS revealed higher frequencies of cytokine-producing CD8+ than CD4+ T cells in BAL; NP and NPM1 induced strong CD8 IFN-γ to pH1N1/NP (and H3N2 for NP). M1 induced M1-specific CD4 and CD8 IFN-γ/TNF, though some did not differ significantly from pre-exposed controls.
• Correlations: % M1-specific TNF+ CD8+ T cells negatively correlated with all five virological/pathological measures (−0.74<ρ<−0.45). % M1-specific IFN-γ+ CD8+ T cells and M1 ELISpot in BAL negatively correlated with 4 of 5 measures (−0.68<ρ<−0.52 and −0.56<ρ<−0.43). NP ELISpot in PBMC (D63) and BAL, % M1-specific IFN-γ+ CD4+ and % NP-specific TNF+ CD4+ also showed negative correlations with at least one outcome. No correlation with NP-specific serum IgG titers, though Fc-mediated functions may still contribute.

Mouse immunogenicity: IM generally induced higher splenic responses than IN for M1 and NPM1. IN induced lung CD8 IFN-γ/TNF to NP and M1, including TRM (CD103+CD69+). NPM1 dampened M1-specific responses compared to M1 alone, while NP responses were similar between NP and NPM1.

Targeting conserved internal antigens via aerosol-delivered viral vectors elicited robust local T-cell immunity and reduced disease following heterologous challenge in a large natural host model. A single aerosol dose of ChAdOx2-NPM1-NA2 significantly reduced lung pathology with a trend toward reduced shedding, though less dramatically than prior heterologous prime-boost regimens; optimizing dose or employing two aerosol doses may enhance protection. Because the single-dose construct’s NA matched the challenge strain, the relative contributions of NA antibodies versus T-cell responses could not be definitively resolved, but the internal antigens plus NA approach merits further study.

Critically, direct comparison of single-antigen versus fusion constructs in pre-exposed pigs showed that either NP or M1 alone sufficed to reduce viral shedding and lung pathology, with M1 alone achieving the greatest reduction in multiple lung pathology metrics. M1 vaccination generated no detectable antibody responses, yet induced strong M1-specific CD4 and CD8 T-cell responses that correlated with protection, indicating T cell–mediated mechanisms. NP (and NPM1) boosted non-neutralizing antibodies and T-cell responses but did not confer superior protection over M1. Negative correlations between M1-specific CD8 cytokine responses and virological/pathological readouts further support a protective role for these T cells. The data suggest that, in pre-exposed hosts with existing immunity, boosting mucosal T-cell responses—particularly against M1—can mitigate disease following heterologous challenge.

These findings inform BPIV design: inclusion of both NP and M1 may not be necessary for protection in pre-exposed populations, which could free vector capacity for NA and/or HA antigens to add strain-specific antibody protection. Aerosol delivery effectively targets the respiratory tract and may be operationally feasible, with precedence from aerosolized adenoviral and measles vaccines. Differences between murine and porcine responses underscore the importance of testing in relevant large animal models and fine-tuning the induction of protective lung-resident memory T cells while limiting immunopathology.

This study provides the first direct evidence in pre-exposed pigs that aerosol vaccination inducing T-cell responses against conserved internal influenza proteins protects against heterologous challenge-induced lung disease. A single aerosol dose of ChAdOx2-NPM1-NA2 reduced lung pathology, and prime-boost immunization with either NP or M1 alone reduced viral shedding and pathology, with M1 alone conferring the greatest reduction and correlating with M1-specific T-cell responses. There was no clear advantage to including both NP and M1, suggesting simplified antigen compositions are feasible and leaving space to incorporate NA and/or HA for added antibody-mediated protection. Future work should optimize dosing and schedules (including two-dose aerosol regimens), incorporate appropriate vector controls, increase group sizes for power, evaluate durability of mucosal immunity over longer intervals, and dissect contributions of NA and Fc-mediated antibody functions to protection.

• No empty vector or irrelevant antigen control groups to exclude non-specific vector effects; only unimmunized pre-exposed controls were used due to logistical constraints.
• In the single-dose study, NA in the vaccine matched the challenge H3N2 strain, confounding attribution of protection mechanisms between NA antibodies and T-cell responses.
• Group sizes (n=6) limit statistical power; several immune and virological trends did not reach significance.
• Virus was not detected in BAL or lungs, likely due to dilution from lavage, limiting assessment of lower-airway viral replication.
• Only about 30% of the aerosolized dose deposits in pig lungs, potentially underestimating vaccine efficacy of single-dose regimens.
• Pre-challenge assessment of respiratory immune boosting after ChAdOx2 was not performed (would require additional animals); durability beyond the study timeline was not assessed.
• Short interval to necropsy (4 days post-challenge) limits evaluation of longer-term protection and resolution.