A chimeric influenza virus vaccine expressing fusion protein epitopes induces protection from human metapneumovirus challenge in mice

Abstract

Human metapneumovirus (HMPV) is a common virus associated with acute respiratory distress syndrome in pediatric patients. There are no HMPV vaccines or therapeutics that have been approved for prevention or treatment. In this study, we constructed a novel recombinant influenza virus carrying partial HMPV fusion protein (HMPV-F), termed rFLU-HMPV/F-NS, utilizing reverse genetics, which contained (HMPV-F) in the background of NS segments of influenza virus A/PuertoRico/8/34(PR8). The morphological characteristics of rFLU-HMPV/F-NS were consistent with the wild-type flu virus. Additionally, immunofluorescence results showed that fusion proteins in the chimeric rFLU-HMPV/F-NS could work well, and the virus could be stably passaged in SPF chicken embryos. Furthermore, intranasal immunization with rFLU-HMPV/F-NS in BALB/c mice induced robust humoral, mucosal and Th1-type dominant cellular immune responses in vivo. More importantly, we discovered that rFLU-HMPV/F-NS afforded significant protective efficacy against the wild-type HMPV and influenza virus challenge, with significantly attenuated pathological changes and reduced viral titers in the lung tissues of immunized mice. Collectively, these findings demonstrated that chimeric recombinant rFLU-HMPV/F-NS as a promising HMPV candidate vaccine has potentials for the development of HMPV vaccine.

Keywords: HMPV; HMPV vaccine; RG technology; fusion protein; influenza virus.

Figure 1

Construction and characterization of pFLU-HMPV/F-NS. (A) Construction strategy of plasmid pFLU-HMPV/F-NS; (B) Detection of transgenic segments in pFLU-HMPV/F-NS. M: Trans15K DNA Marker; (C) Schematic representation of the IAV reverse genetics system used for the production of rFLU-HMPV/F-NS. MDCK and COS I cells were co-transfected with pHW-PB2, pHW-PB1, pHW-PA, pHW-HA, pHW-NP, pHW-NA, and pHW-M (ratio 1:2), using Effectene. rFLU-HMPV/F-NS was harvested from cell supernatants upon confirmation of HA positivity. Titers of rFLU-HMPV/F-NS were determined by TCID₅₀.

Figure 2

Identification of recombinant virus rFLU-HMPV/F-NS. (A) Immunofluorescent analysis of IVA and HMPV-F protein expression (60×). MDCK cells were infected for 24 h with purified 3rd generation rFLU-HMPV/F-NS (MOI: 1) and labeled with primary anti-IAV NP and anti-HMPV F antibodies. Cells were washed and stained with goat anti-rabbit IgG H&L (Alexa Fluor®488) and Goat Anti-Mouse IgG (DyLight594) for 1 h. Cells were imaged on an OLYMPUS confocal microscope. (B) Morphology and (C) size distribution of rFLU-HMPV/F-NS analyzed via TEM. (D) Growth curve of the recombinant virus. The HA titer of the recombinant virus increased to 2^8–9 at 72 h and decreased to 2^6–7 at 96 h. (E) Passaged rFLU-HMPV/F-NS (P1~P5) was assessed for HA positivity. (F) P1 ~ P5 viral titers of rFLU-HMPV/F-NS were assessed via TCID₅₀.

Figure 3

The immunogenicity of recombinant virus rFLU-HMPV/F-NS. (A) Schematic outlining the animal immunization schedule. (B) HI antibody titers against A/China/BJMY011/2020 (H1N1) in mice immunized with rFLU-HMPV/F-NS. Values are the mean ± SE of three independent experiments (^**p < 0.01, ^***p < 0.001). (C) Neutralizing antibody titers against HMPV in mice immunized with rFLU-HMPV/F-NS. (D) IgG titers against HMPV in mice immunized with rFLU-HMPV/F-NS.

Figure 4

sIgA antibody titers against HMPV in mice immunized with rFLU-HMPV/F-NS. (A) Line bars and (B) graphical representation of HMPV-induced sIgA values calculated from the serum samples of immunized mice by ELISA plates. Values are the mean ± SE of three independent experiments (^***p < 0.001).

Figure 5

The level of cytokines in mice immunized with rFLU-HMPV/F-NS. Cytokine expression of HMPV-specific Th1/Th2 cytokines (TNF-α, IFN-γ, IL-2, and IL-4) in mouse spleen lymphocytes were assessed by flow cytometry. (A) Representative flow cytometric gating. (B) Graphical representation of the number of cells per 10⁵ solenocytes Values are the mean ± SE of three independent experiments; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 6

Immunoprotective effects of rFLU-HMPV/F-NS following HMPV challenge. (A) Weight changes; (B) Survival curves; (C) HMPV viral load (^***p < 0.001) and (D) Histopathology (×200) in the lung of immunization mice against HMPV.

Figure 7

Immunoprotective effects of rFLU-HMPV/F-NS following A/China/BJMY011/2020 (H1N1) challenge. (A) Weight changes; (B) Survival curves; (C) Viral titers (^***p < 0.001) and (D) Histopathology (×200) in the lung of immunization mice against A/China/BJMY011/2020 (H1N1).