CircRNA based multivalent neuraminidase vaccine induces broad protection against influenza viruses in mice

Abstract

Developing broad-spectrum influenza vaccines is crucial for influenza control and potential pandemic preparedness. Here, we reported a novel vaccine design utilizing circular RNA (circRNA) as a delivery platform for multi-subtype neuraminidases (NA) (influenza A N1, N2, and influenza B Victoria lineage NA) immunogens. Individual NA circRNA lipid nanoparticles (LNP) elicited robust NA-specific antibody responses with neuraminidase inhibition activity (NAI), preventing the virus from egressing and infecting neighboring cells. Additionally, the administration of circRNA LNP induced cellular immunity in mice. To achieve a universal influenza vaccine, we combined all three subtypes of NA circRNA-LNPs to generate a trivalent circRNA vaccine. The trivalent vaccine elicited a balanced antibody response against all three NA subtypes and a Th1-biased immune response in mice. Moreover, it protected mice against the lethal challenge of matched and mismatched H1N1, H3N2, and influenza B viruses, encompassing circulating and ancestral influenza virus strains. This study highlights the potential of delivering multiple NA antigens through circRNA-LNPs as a promising strategy for effectively developing a universal influenza vaccine against diverse influenza viruses.

Fig. 1. Design and characterization of circRNA-LNP encoding NA.

a Schematic diagram of circRNA vaccine production. Linear RNA precursors encoding N1, N2, and IBV NA were self-spliced by group I catalytic intron to form circRNA. CircRNA was then encapsulated into lipid nanoparticles (LNP). The figure was created by the author using Adobe Illustrator 2020. b Verify the self-splicing junction site of circRNA using Sanger sequencing of the junction site after reverse transcription and PCR amplification. c CircRNA and linear precursor were separated on 1.5% agarose gel. Lane L: Linear precursor. Lane L + R: Linear precursor digested with RNase R. Lane C: circRNA not digested with RNase R. Lane C + R: circRNA digested with RNase R. d MUNANA assay for measuring NA enzyme activity in LNP-transfected cells. The curve shows the fluorescence intensity versus the density of cells in 50 μL. Data was shown as means of four repeated experiments ± SD. e Dynamic light scattering (DLS) to characterize the particle size and dispersion of circRNA-LNP. Representative images of each LNP were shown. f Frequency of NA expression in 293 T cells transfected with NA LNP, Blank LNP as a negative control. IRES: internal ribosome entry site. E1: exon fragment 1 upstream of 5’intron. E2: exon fragment 2 downstream of 3’intron.

Fig. 2. Monovalent circRNA vaccine induces antibody response in mice.

a Five mice per group were immunized with two doses of monovalent circRNA vaccine (N1, N2, IBV) at a four-week interval. Sera were collected 3 weeks after both the prime and boost immunization. A blank LNP group was used as a control. The figure was created by the author using Adobe Illustrator 2020. Binding antibodies against Mich15 (b), SL32 (c), and BCOL17 (d) NA recombinant proteins were detected by ELISA. ELISA was performed to assess the binding ability of N1 monovalent (e), N2 monovalent (f), IBV monovalent (g) circRNA immune sera to heterologous N1, N2, IBV NA recombinant protein. The radar diagram shows the geometric mean titer (GMT) of each group (n = 5). The abbreviation of the virus strain was the same as in Fig. S1. NAI antibodies against Mich15 (h), SL32 (i), and BCOL17 (j) were detected by ELLA. Using recombinant protein as antigen. Neutralization titers of boost immune sera against CA09 (k), SL32 (l), and BCOL17 (m) virus were detected by microneutralization assay. Data are presented as the mean ± SD (n = 5), and each symbol represents one animal. The dashed lines correspond to the lowest initial dilution. Statistical significance was performed by One-way ANOVA; Unpaired Student’s t tests were used to compare antibody titers between prime and boost sera within the same dosage group of mice; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Fig. 3. CircRNA vaccination induced NA-specific cellular responses.

a–c Mice were vaccinated with 10 μg of circRNA-LNP encoding N1, N2, or IBV NA, following a prime-boost vaccination scheme. Ten days post boost vaccination, the spleen was harvested and spot-counting for IFN-γ/IL-4 -secreting cells by ELISpot assay after stimulating with N1 (a), N2 (b) or IBV NA (c) recombinant protein. Representative walls of IFN-γ (d) or IL-4 (e) ELISpot. The proportion of IFN-γ (f), TNF-α (g), or IL-2 (h) secreting CD4⁺ and CD8⁺ T cells was assessed by FASC. Splenocytes from the blank LNP group were used as a control. Data are presented as the mean ± SD (n = 5), and each symbol represents one animal. Statistical significance was performed by unpaired t test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Fig. 4. Trivalent circRNA vaccine induced a balanced immune response against N1, N2, and influenza B NA.

a Trivalent circRNA vaccine immunization and challenge regimen. Mice received a prime-boost immunization program with 4 weeks intervals, 4 weeks after boost vaccination mice were challenged with 5 × LD₅₀ or 1 × LD₅₀ influenza virus. Body weight and survival rate were monitored for 14 days. Some mice were euthanized, and lungs were collected at 4 dpi. A blank LNP group was used as a control. The figure was created by the author using Adobe Illustrator 2020. ELISA was used to measure the binding antibody titers against Mich15 (b), SL32 (c), and BCOL17 (d) in the serum samples from the trivalent vaccine group. e The binding breadth of trivalent vaccine immune sera after boost vaccination. The radar diagram shows each group’s geometric mean titer (GMT) (n = 5). The abbreviation of the virus strain was the same as in Fig. S1. NAI titer of trivalent immune sera. NA inhibition antibody titers against Mich15 (f), SL32 (g), and BCOL17 (h) were detected by ELLA. Neutralization titer of boost immune sera for trivalent vaccine. Microneutralization titers against CA09 (i), SL32 (j), and BCOL17 (k) virus were detected by microneutralization assay. Data are presented as the mean ± SD (n = 5), and each symbol represents one animal. The dashed lines correspond to the lowest initial dilution. Statistical significance was performed by One-way ANOVA; Unpaired Student’s t tests were used to compare antibody titers between prime and boost sera within the same dosage group of mice; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Fig. 5. Trivalent circRNA vaccine protects mice from heterologous virus challenge.

a–e Mice (n = 5) were immunized with 6 μg or 30 μg trivalent CircRNA vaccine in two doses at 28-day intervals. Blank LNP was used as a control. Twenty-eight days post boost vaccination, mice were challenged 5 × LD₅₀ of A/California/04/2009(H1N1) (a), A/Puerto Rico/8/1934 (H1N1) (b), A/Gui Zhou/54/89 (H3N2) (c), A/Hong Kong/8/1968 (H3N2) (d), B/Colorado/06/2017(Victoria) (e). Body weight and survival rate were monitored for 14 days. Body weight was shown as the mean of five mice ± SD.

Fig. 6. Protective efficacy of a single dose of circRNA vaccine.

a Immunization schedule for the challenge experiment in the prime-only group. The figure was created by the author using Adobe Illustrator 2020. b–d Antibody titers against the NA recombinant proteins from Mich15 (c), SL32 (d), and BCOL17 (e) were measured using ELISA. The data present results from five mice per group. Dashed lines indicate the lowest initial dilution. Statistical significance was determined using One-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns denotes not significant. e Mice (n = 5) in the prime-only group were challenged with 5 × LD₅₀ of B/Colorado/06/2017 (Victoria) virus. Body weight and survival rate were monitored for 14 days. Body weight was shown as the mean of five mice ± SD.