Cells

The human embryonic kidney (HEK) 293 T cells, Spodoptera frugiperda (Sf9) insect cells, and Madin Darby Canine Kidney (MDCK) cells were purchased from the American Type Culture Collection (ATCC). The HEK293T and MDCK cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, U.S.) supplemented with 10% fetal bovine serum (FBS, Gibco, U.S.) and 1% penicillin-streptomycin solution (Gibco, U.S.) at 37 °C with 5% CO2. The Sf9 cells were cultured in SF-SFM medium (Suzhou world-medium Biotechnology Co., Ltd., Suzhou, China) supplemented with 1% penicillin-streptomycin solution (Gibco, U.S.), at 27 °C in the dark, using a cell shaker setting at 110 rpm.

Virus and recombinant NA protein

All influenza viruses were grown in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. Influenza A virus was cultured at 37 °C for 48 h, while influenza B virus was cultured at 33 °C for 72 h. Then the eggs were chilled at 4 °C overnight. The next day, the allantoic fluid was harvested, filtered, and centrifuged at the speed of 10,000 × g at 4 °C for 10 min. Finally, the virus was stored at −80 °C. Virus titer was determined by TCID50 assay. To prepare the virus for the challenge experiment, the collected allantoic fluid was ultracentrifuged on an Optima XPN-100 ultracentrifuge (Beckman, U.S.) at the speed of 130,000 × g at 4 °C for 2 h to precipitate the virus. The virus pellet was resuspended in 1 × phosphate-buffered saline (PBS) and further purified by passing through a 30% sucrose cushion. The purified virus was aliquoted and stored at −80 °C.

All recombinant NAs were expressed in the Sf9 cells, as described in the previous study47. Recombinant NAs were purified by Ni+ Sepharose high-performance chromatography.

CircRNA design and synthesis

CircRNA was generated using the PIE splicing strategy23. Linear precursor RNAs were transcribed from linearized plasmid templates using T7 RNA polymerase (Thermo, U.S.). After treatment with DNase I (New England Biolabs, USA), RNAs were purified using an RNA purification kit (Magen Biotechnology Co., Ltd., Guangzhou, China). For the RNA cyclization reaction, purified RNA was first heated to 70 °C for 5 min and immediately placed on ice. The reaction solution was added, containing a final concentration of 2 mM Guanosine triphosphate (GTP), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT. The RNA cyclization reaction was performed at 55 °C for 8 min, then RNase R (New England Biolabs, USA) was added to the reaction and incubated at 37 °C for 15 min to remove uncirculated RNA. CircRNA was finally column purified using the RNA purification kit (Magen Biotechnology Co., Ltd., Guangzhou, China) and stored at −80 °C. According to the previous study23, the quality and integrity of circRNAs were assessed by 1.5% agarose gel electrophoresis at 160 V for 30 min.

LNP encapsulated circRNA

LNPs were generated by rapidly mixing the organic and aqueous phases (1:3, v/v) through the iNanoE microfluidic system (Micro & Nano (Shanghai) Biologics Co. Ltd., Shanghai, China) at a total flow rate of 12 mL/min. The organic phase was formed by mixing SM102, 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC), cholesterol, and DMG-PEG2000 (AVT (Shanghai) Pharmaceutical Tech Co., Ltd) in ethanol with a molar ratio of 50:10:38.5:1.5. The circRNA was dissolved in 50 mM citric acid buffer (pH = 4). The N/P ratio of ionizable lipids and circRNA was 4:1. The formed LNPs were diluted 40-fold in PBS and concentrated using a 100 kDa ultrafiltration tube (Millipore, U.S.). The encapsulation efficiency of LNP was determined following the method described previously48. To characterize particle size and PDI, LNPs were added to the cuvette after 40-fold dilution by PBS, and dynamic light scattering (DLS) was performed using Zetasizer Pro (Malvern Panalytical, England), with three replicates of measurements for each sample, and PBS as a negative sample control.

CircRNA-LNP transfection and flow cytometric analyses

Before transfection, HEK293T cells were seeded in 24-well plates at a density of 6 × 105. The following day, 500 ng of each LNP encapsulated circRNA was diluted in DMEM and added to cells. After 48 h, the cells were harvested and washed with 1% Bovine Serum Albumin (BSA) Fraction V in 1 × PBS. Then cells were incubated with 10 μg/mL anti-NA antibodies: 1G01, 229-2C06, or CC61 for 20 min on ice. After that, cells were washed twice with 1% BSA PBS and incubated with 1: 2000 dilution of rabbit anti-human IgG(H + L) FITC (Southern Biotech, U.S.) for 20 min on ice in the dark. Cells were washed twice and resuspended. Flow cytometric data were acquired on CytoFLEX S flow cytometer (Beckman, U.S.). Approximately 50,000 events were collected per sample.

MUNANA assay

LNP transfected cells were diluted at the density 1 × 106 in MUNANA buffer (33.3 mM MES, 4 mM CaCl2, pH 6.5). The cells were two-fold serially diluted using MUNANA buffer in a black 96-well plate to make a 50 μL final volume. Then, 50 μL of 300 μΜ of MUNANA substrate was added. After incubating at 37 °C for 1 h, 100 μL of stop solution (138.6 mM NaOH in absolute ethanol) was added. The plate was read using a SYNERGY H1 (BioTek, US) multimode microplate reader with 355 nm excitation and 460 nm emission.

Vaccination and virus challenge

6–8-week-old female BALB/c mice (n = 5 per group) were intramuscularly injected with 100 μL circRNA vaccine diluted in PBS. Trivalent circRNA vaccines were formulated with equal amounts of three NA circRNA vaccines. Boost vaccination was performed 28 days after prime vaccination. Twenty-eight days after boost vaccination, mice were anesthetized with avertin (250 mg/kg) and intranasally infected with 5 × LD50 influenza virus in 30 μL PBS. The body weight was monitored for 14 days after infections, and mice that lost over 25% of their initial weight were humanely euthanized. The weighing order was randomly assigned each day, and each group of mice was weighed in a different order each day. The animals were grouped and manipulated by different individuals. The experimental operators were unaware of the specific grouping. All mice were kept in a SPF environment, and all virus challenge experiments were performed in an animal biosafety level 2 laboratory.

For the euthanasia of mice, animals were initially anesthetized with Avertin (250 mg/kg) via intraperitoneal injection. Subsequently, euthanasia was carried out by cervical dislocation after confirming their lack of responsiveness to pain and stimuli (deep anesthesia). Euthanasia was carried out following the American Veterinary Medical Association (AVMA) Guidelines.

ELISA

The 96-well ELISA plates were coated with recombinant NA proteins (50 μL, 200 ng/well) or trivalent inactivated vaccine (TIV, Southern hemisphere, 2022-2023) in PBS at 4 °C overnight. The following day, the plates were blocked with 150 μL 3% BSA at 37 °C for 1 h. The sera were serially diluted 3-fold, starting with a 1:300 dilution (or 1:100 in the prime group for Fig. 6), and then added to the plates. The plates were subsequently incubated at 37 °C for 1 h. Following washing with PBST (PBS added with 0.05% TWEEN 20), 75 μL of 1: 4000 dilution of HRP-conjugated anti-mouse IgG/IgG1/IgG2a secondary antibody (Southern Biotech, U.S.) was added to plates and incubated at 37 °C for 1 h. The readout was developed using 2,2’-Azino-bis (3-Ethylbenzthiazoline-6-Sulfonic Acid, ABTS) ELISA substrate (sigma, U.S.). After 20 min of incubation, absorbance was measured at 405 nm on a Spectramax ABS Plus (Molecular Devices, U.S.) microplate reader. Endpoint titers were defined as the dilution fold with OD value exceeding 2 × background (without sera, but the secondary antibody was added). Monoclonal antibody 1G0110 was used as positive control.

Enzyme-linked lectin assay (ELLA)

The 96-well ELISA plates were pre-coated with 100 μL of fetuin (Sigma, U.S.) at 25 μg/mL in PBS and incubated at 4 °C for 24 h, then the plates were washed three times with PBST. Heat-inactivated sera were 2-fold serially diluted starting with a 1:50 dilution (in some groups were 1:100) in DPBS-T-BSA buffer (Dulbecco’s phosphate-buffered saline containing 0.133 g/L CaCl2 and 0.1 g/L MgCl2 with 0.05% Tween-20 and 1% BSA) and mixed with an equal volume of virus or NA recombinant proteins. The mixtures were incubated at 37 °C for 2 h and then transferred to the fetuin-coated plates. The plates were incubated at 37 °C for 18 h and washed six times with PBST subsequently. Then 100 μL/well of HRP-conjugated peanut agglutinin lectin (Sigma, U.S.) in PBS was added to the plates and left in the dark for 2 h at room temperature. After adding the ABTS ELISA substrate and 15 min of incubation, the absorbance of the samples was read at 405 nm on a Spectramax ABS Plus (Molecular Devices, U.S.) microplate reader. The samples that did not reach 50% inhibition at the initial sera dilution were considered negative.

Microneutralization assay

For the microneutralization assay, sera were treated with the receptor-destroying enzyme (RDE, Denka Seiken, Japan) and then inactivated at 56 °C for 30 min. Sera were then diluted 2-fold serially starting at 1:10 dilutions in infection media (DMEM supplemented with 1 μg/mL TPCK-treated trypsin, 0.1 mM MEM non-essential amino acid, 1% penicillin-streptomycin solution). Next, 60 μL of diluted sera were mixed with 60 μL of 100 × TCID50 virus and incubated at 37 °C for 1 h. Then the virus-sera mixtures were transferred to the PBS-washed MDCK cells and incubated at 37 °C with 5% CO2 for 1 h. After washing with PBS twice, 100 μL of infection medium with the same sera dilution was added to the cells. Following incubation at 37 °C for 48 h (for influenza A virus) or 33 °C for 72 h (for influenza B virus). Virus replication was detected using a hemagglutination assay. Briefly, 50 μL of cell supernatant was transferred into a 96-well V-bottom plate, followed by the addition of 25 μL of 1% chicken red blood cells (RBCs). After incubating at room temperature for 15 mins, the presence of virus was determined by observing hemagglutination of the RBCs. The neutralization titer of sera was determined as the highest dilution at which no virus was detected.

ELISPOT assay

The day before assay, 1:100 anti-mouse IFN-γ or IL-4 antibody (U-Cytech, Netherland) was added to ELISpot plate (Millipore, U.S.) and incubated at 4 °C overnight. The Next day, plates were blocked with 10% FBS in RPMI 1640 medium at 37 °C for 1 h. Next 4 × 105 Splenocytes were added to the ELISPOT plate and stimulated by 10 μg/mL recombinant NA protein at 37 °C in 5% CO2 for 40 h. The plates were washed with PBST 6 times and incubated with 1:100 biotinylated antibody for 2 h at RT. After washing, the plates were incubated with 1:500 Streptavidin-HRP for 1 h at RT. Finally, the plates were incubated with 5-bromo-4chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT, Beyotime, China) substrate solution for 5 min, and scanned using Mabtech IRIS FluoroSpot/ELISpot reader (Mabtech, Sweden).

Intracellular cytokine staining assay

The splenocytes were isolated and plated at a density of 4 × 106 per well in a round-bottom 96-well plate and stimulated with 10 μg/mL recombinant NA protein at 37 °C, 5% CO2 for 6 h. Brefeldin A was then added to each sample and cells were incubated for an additional 4 h. After incubation, cells were washed with PBS and stained with LIVE/DEAD cell staining solution (Biolegend, U.S.) for 30 min in the dark. After washing with FASC buffer (PBS added 2% FBS), cells were then incubated with Fc Blocker (Biolegend, #101319, U.S.) for 5 min in dark and then surface-stained with the following antibodies: anti-CD3-Pacific Blue™ (Biolegend, #100214, U.S.); anti-CD4-FITC (Biolegend, #100509, U.S.); anti-CD8α-Brilliant Violet 605 (Biolegend, #100744, U.S.). After fixation with a fixation/permeabilization solution (Beyotime, China), cells were intracellularly stained with the following antibodies: anti-IFN-γ-APC (Biolegend, #505810, U.S.); anti-TNF-α-Brilliant Violet 785™ (Biolegend, #506341, U.S.); anti-IL-2-PE-Cyanine7 (Biolegend, #503832, U.S.) for 30 min in dark. Finally, cells were washed twice using permeabilization buffer and suspended in FASC buffer. Flow cytometric analysis and cell sorting were performed on CytoFLEX S Flow Cytometer (Beckman Coulter, U.S.). Analysis was performed using FlowJo software V_10.

Virus plaque assay and TCID50 assay

Mice were sacrificed and lungs were homogenized in 10% (w/v) DMEM at 4 dpi. The lung homogenate was diluted in a 1:10 series and inoculated into a 6-well plate containing a single layer of MDCK cells. After 1 h of incubation, the plates were washed twice with PBS and 2 mL overlay (2 × DMEM, 1 μg/mL TPCK-treated trypsin, 0.1 mM MEM non-essential amino acid, 1% penicillin-streptomycin solution, 0.8% low melting agar) was added. After 72 h of culture, the agar overlays were removed, and the cells were fixed and stained with a crystal violet solution.

To determine the TCID50, the lung homogenate was 3-fold serially diluted in DMEM starting at 1:10. MDCK monolayers in 96-well plates were washed twice with PBS before adding 100 μL of the diluted lung homogenate. After incubating at 37 °C with 5% CO2 for 1 h, cells were washed with PBS, followed by adding 100 μL of infection media. Influenza A virus-infected cells were further cultured at 37 °C with 5% CO2 for 48 h, while influenza B virus-infected cells were incubated at 33 °C with 5% CO2 for 72 h. Hemagglutination assay was performed as described in 10. TCID50 was calculated using the Reed-Muench method.

Phylogenetic tree analysis

All NA sequences involved in this study were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/). NA phylogenetic tree was generated by MAGAX using the Maximum Likelihood method.

Statistical analysis

Statistical analyses were performed using the Prism 9.0 software (GraphPad, U.S.). All errors are expressed as means with standard derivation (±SD). In Figs. 2 and 4, one-way ANOVA with multiple comparison tests was employed to compare antibody titers among different groups within the prime or boost immunization phases. Unpaired Student’s t tests were used to compare antibody titers between prime and boost sera within the same dosage group of mice. Unpaired Student’s t test analysis was performed to determine p values in T cell responses. Group data were considered statistically significant when p < 0.05, and*, **, ***, ****, and ns in results represent p < 0.05, p < 0.01, p < 0.001, p < 0.0001, and not significant, respectively.