Dual roles of influenza B virus neuraminidase mRNA vaccine in enhancing cross-lineage protection by supplementing inactivated split vaccination

Abstract

The current influenza vaccine is based on immunity to hemagglutinin (HA) and provides poor cross-protection. Here, we generated mRNA vaccine encoding influenza B virus (IBV) neuraminidase (NA) conjugated to influenza A virus M2 ectodomain (M2e), encapsulated in lipid nanoparticles (LNP), capable of inducing cross-lineage IBV protection in a dose-dependent pattern. The combination of low-dose NA mRNA and inactivated split IBV vaccines was found to induce significantly higher levels of cross-reactive IgG responses, NA and HA inhibition titers, effector and memory cellular immune responses as well as cross-lineage protection than either NA mRNA or split vaccine alone. This study suggests that the NA mRNA vaccine not only provides cross-lineage protection with a high dose but also enhances the cross-protective efficacy of the combined low-dose NA mRNA and split vaccines. Our findings support a new strategy of using mRNA LNP-supplemented conventional vaccination to enhance cross-protection.IMPORTANCEThis study highlights a significant advancement in influenza vaccination strategies. To test a new vaccination strategy, we developed an influenza B virus (IBV) neuraminidase (NA) mRNA vaccine which could provide cross-lineage protection at a high dose. More importantly, the co-administration of NA mRNA and split IBV vaccine at low doses was found to significantly enhance the hemagglutinin and NA immunity as well as cross-lineage protection of seasonal IBV vaccines. This proof-of-concept study provides evidence for a novel strategy to enhance the immunogenicity and cross-protective efficacy of conventional vaccines by supplementing with new targets of mRNA vaccines.

Keywords: NA mRNA-LNP; cross-protection; influenza virus; split IBV vaccines.

Fig 1

Influenza B virus chimeric NA mRNA vaccine expresses M2e-NA conjugate protein. (A) Chimeric B NA mRNA construct design. Each domain amino acid (aa) sequence information is provided in Fig. S1. (B–G) HEK 293T cells were transfected with chimeric NA mRNA or eGFP mRNA to characterize protein expression. The reactivity to M2e and NA expressed by B NA mRNA vaccine construct was measured using 14C2 monoclonal antibody (mAb) (B) and NA-specific polyclonal antisera (C) by ELISA. NC: negative (no mRNA) control. (D, E) Expression of chimeric NA protein by NA mRNA transfected HEK 293T cells was assessed under a fluorescence microscope, with no mRNA transfection as negative control (F), eGFP mRNA transfection as positive control (PC) (G). All results are presented as mean ± standard error mean (SEM) with individual dots. Statistical analysis was performed using two-way ANOVA and Tukey’s post-multiple comparison tests. P-value is significant at P < 0.05; ****P < 0.0001.

Fig 2

NA mRNA LNP vaccine confers cross-lineage protection against IBV in a dose-dependent pattern. Mice were vaccinated (i.m., n = 10) with NA mRNA (1 or 3 µg) LNP or Control (SARS-CoV-2 spike) mRNA (C-mRNA 3 µg) at two doses (prime-boost, 4 weeks interval). (A, B) IgG antibody responses (P-B: prime-boost sera) to NA proteins from different IBV strains (A) and inactivated IBV antigens (B). (C–F) NAI (n = 5) activities against B/Florida/4/2006 (Y), B/Phuket/3037/2013 (Y), B/New York City/PV00094/2017 (Y), and B/Malaysia/2056/2004 (V) in boost sera. (G–I) Weight changes and survival rates in vaccinated and control mice after challenge with B/Florida/2006 (Y, 1.07 × 10⁵ EID₅₀, n = 3), B/Hong Kong/330/2001 (V, 1.4 × 10⁵ EID₅₀, n = 3), B/Malaysia/2004 (V, 1.58 × 10⁵ EID_50, n = 5). All results are presented as mean ± standard error mean (SEM) with individual dots. Statistical analysis was performed using two-way ANOVA and Tukey’s post-multiple comparison tests. P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 3

Low-dose NA mRNA + sFL vaccination enhances IgG to NA and IBV. (A) Vaccine groups, timeline for immunization and assays: Mice (n = 10) were vaccinated intramuscularly (i.m.) with sFL 0.3 µg ± 1 µg of NA mRNA LNP, NA mRNA 1 µg, or control (SARS-CoV-2 spike) mRNA 1 µg (C-mRNA) LNP. IgG antibodies to NA protein derived from different IBV strains post-prime and boost (B–D), inactivated IBVs (E–H) in boost sera. Statistical analysis was performed using two-way ANOVA and Tukey’s post-multiple comparison tests. P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 4

Low-dose NA mRNA + sFL vaccination enhances cross-lineage NAI and homo- and hetero HAI titers. The vaccine groups are the same as in Fig. 3. (A–D) NAI titers in serial serum dilutions and NAI comparisons by bar graphs at a representative dilution. (A) NAI against B/Florida/2006 (Y) at serial and 1280× dilutions. (B) NAI against B/Phuket/2013 (Y) at serial and 640× dilutions. (C) NAI against B/New York/2017 (Y) at serial and 640× dilutions. (D) NAI against B/Malaysia/2004 (V) at serial and 160× dilutions. (E) HAI titers against B/Florida/2006 (Y) in prime and boost sera. (F) HAI titers against B/New York/2017 (Y), B/Phuket/2013 (Y), and B/Malaysia (V) in boost sera. All results (n = 5 mice/group) are presented as mean ± standard error mean (SEM) with individual dots. Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple comparison tests (A–D), two-way ANOVA and Tukey’s post-multiple comparison tests (A–F). P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 5

Low-dose NA mRNA + sFL co-vaccination induces enhanced cross-lineage protection. Immunization and experimental design were the same as in Fig. 3. (A) Body weight changes and survival rates in two-dose (boost)-vaccinated mice after intranasal (i.n.) challenge with homologous B/Florida/4/2006 virus (Y, 1.43 × 10⁶ EID₅₀). (B) Weight changes and survival rates in boost-vaccinated mice following i.n. challenge with cross-lineage B/Malaysia/2506/2004 (V, 1.58 × 10⁵ EID₅₀). (C) The percentages of BW loss in vaccinated and control mice reached a peak at 5 dpi with B/Malaysia/2004. (D) Lung viral titers in embryonated egg substrates at 5 dpi with B/Malaysia/2004. The data are presented as mean ± standard error mean (SEM) with individual dots. Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple comparison tests (C, D), two-way ANOVA and Tukey’s post-multiple comparison tests (A, B). P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 6

Low-dose NA mRNA + sFL vaccination protects from cross-lineage B/Malaysia virus-induced inflammatory responses. Vaccinated mice (i.m., n = 5) with NA mRNA 1 µg + sFL combination vaccines or sFL alone (Prime-Boost, 4 weeks interval) were challenged with B/Malaysia/2506/2004 (V) virus as described in Fig. 5. The lung samples were harvested on 5 dpi to determine inflammatory responses. (A–F) Inflammatory cytokines and chemokines in lung extracts at 5 dpi. (G–L) Inflammatory cellular infiltrates into the lungs. (G) Neutrophils: CD45⁺F4/80ˉCD11b⁺Ly6c^low; (H) Plasmacytoid DCs (pDCs): CD45⁺F4/80ˉCD11c⁺MHC II^highB220⁺; (I) CD11b⁺mDCs: CD45⁺F4/80ˉCD11c⁺MHC II^highCD103⁻CD11b⁺; (J) CD103⁺mDCs: CD45⁺F4/80ˉCD11c⁺MHC II^highCD103⁺CD11b⁻; (K) CD11b + resident DCs (rDCs): CD45⁺F4/80ˉCD11c⁺MHC II^low CD8a⁻CD11b⁺; (L) Alveolar macrophages (AMs): CD45⁺F4/80⁺CD11bˉCD11c⁺. All results are presented as mean ± standard error mean (SEM) with individual dots. Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple comparison tests. P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 7

Low-dose NA mRNA + sFL vaccination significantly enhances cellular immune responses. Cellular immune responses were characterized by flow cytometry at 5 dpi with B/Malaysia/2004 virus in the spleens (A–F), mLNs (G–J), and lungs (K–R) from the same vaccine groups as in Fig. 5 (n = 5 mice). (A) The percentages of germinal center B (GC B) cells: CD45⁺CD3⁻B220⁺CD19⁺CD4⁻GL7⁺. (B) memory B (MBCs) cells: CD45⁺CD3⁻IgGD⁻B220⁻CD138⁺. (C) plasma cells (PCs): CD45⁺CD3⁻IgGD⁻CD19⁺B220⁺ CD138⁻GL7⁻. (D) T follicular helper (Tfh) cells: CD45⁺CD3⁺CD4⁺PD1⁺CXCR5⁺CD25⁻Foxp3⁻, (E) Effector memory T (T_EM) cells in total CD45⁺ splenocytes: CD4 T_EM: CD3⁺CD8⁻CD4⁺CD62L⁻CD44⁺. (F) CD8 T_EM: CD3⁺CD4⁻CD8⁺CD62L⁻CD44⁺. Effector IFN-γ⁺ CD8 and CD4, and IL-4⁺ CD8 and CD4 T cells upon in vitro stimulation with B/Florida/2004 inactivated virus (G–N) or NA protein (O–R) were analyzed by flow cytometry. All results are presented as mean ± standard error mean (SEM) with individual dots. Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple comparison tests. P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 8

NA mRNA priming and dose effects on enhancing IgG responses to combined sML (V) + NA mRNA vaccination. (A) Split sML ± mRNA vaccine groups with differential boost doses to determine NA mRNA priming and boost dose effects (n = 5 mice). (B, C) IgG antibody responses to NA proteins (B) and different IBV antigens (C) in prime sera. (D) IgG antibodies for NA proteins and (E) different IBV antigens in boost sera. All results are presented as mean ± standard error mean (SEM) with individual dots. Statistical analysis was performed using two-way ANOVA and Tukey’s post-multiple comparison tests. P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 9

NA mRNA prime and boost dose effects on enhancing cross-reactive humoral immunity and cross-lineage protection after sML (V) + NA mRNA vaccination. (A) HAI Titers against B/Malaysia/2004 (V) prime and boost sera. (B) HAI titers against B/Texas (V) and B/Hong Kong/2003 (V) in boost sera. (C, D) NAI titers in serial dilutions of boost sera and NAI comparisons by bar graphs at a representative dilution. (C) NAI against homologous B/Malaysia/2004 (V) virus at serial and 1,250× dilutions. (D) NAI activities against B/New York/2017 (Y) virus at serial and 250× dilutions. (E) NAI activities against B/Florida/2004 (Y) virus at serial and 1,250× dilutions. (F) BW changes and survival rates, and (G) comparison of peak weight loss in the differential boost vaccination groups after challenge with cross-lineage B/New York/2017 (Y, 7.9 × 10³ EID50) virus. All results are presented as mean ± standard error mean (SEM) with individual mice (n = 5). Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple comparison tests (A), two-way ANOVA and Tukey’s post-multiple comparison tests (B–G). P-value is significant at P < 0.05; *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.