Skip to main content
NCBI home page
Virulence logoLink to Virulence
. 2025 Sep 16;15(1):2284515. doi: 10.1080/21505594.2023.2284515

Protective efficacy of a novel recombinant influenza virus carrying partial human metapneumovirus F protein epitopes

Fang Sun a,#, Guanglin Lei a,#, Chongyu Tian a,#, Zhifang Bai a, Changqing Bai a,b, Yufeng Li a, Zhuoya Deng a, Hao Yang a, Cong Li a, Zhongpeng Zhao c, Yan Niu d, Penghui Yang a,✉
PMCID: PMC12506902  PMID: 37974334

ABSTRACT

Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infections in infants and children. Currently, no approved HMPV vaccine is available. We developed a novel recombinant influenza virus, which carried partial HMPV F protein (HMPV-F) epitopes, utilizing reverse genetics. The novel single-stranded RNA virus, termed rFLU-HMPV/F-NA, was synthesized in the neuraminidase (NA) fragment of influenza virus A/PuertoRico/8/34 (PR8). The morphological characteristics of rFLU-HMPV/F-NA were consistent with the wild-type flu virus. The virus could passage in specific pathogen-free (SPF) chicken embryos for at least five consecutive generations with haemagglutinin (HA) titres of 28–9 or 8–9LogTCID50/mL. BALB/c mice were intranasally immunized at 21-day intervals with 104 TCID50 (low-dose group) or 106 TCID50 (high-dose group) rFLU-HMPV/F-NA, and PBS or PR8 vaccine was used for the control group. rFLU-HMPV/F-NA induced robust humoral, mucosal, and cellular immune responses in vivo in a dose-dependent manner. More importantly, wt clinical HMPV isolate challenge studies showed that rFLU-HMPV/F-NA provided significant immune protection against HMPV infection compared to the PBS or PR8 vaccine control group, as shown by improved histopathological changes and reduced viral titres in the lungs of immunized mice post-challenge. These findings demonstrate that rFLU-HMPV/F-NA has potential as a promising HMPV candidate vaccine and warrants further investigation into its control of HMPV infection.

KEYWORDS: Influenza virus, reverse genetics, HMPV, HMPV vaccine

Introduction

Human metapneumovirus (HMPV) is the leading cause of death for children under five years of age and seriously affects the health of more than 86% of children under five years of age worldwide [1,2]. The respiratory symptoms of an HMPV infection comprise a typical Th17 immune response, with increased secretion of interleukin (IL)-6 and tumour necrosis factor (TNF)-α in the lung. It is a causative agent of acute respiratory tract infection (ARI), which was first isolated in 2001 [3–5]. Structurally, HMPV virions are polymorphic negative-strand particles containing eight genes and encoding nine proteins [6]. The F protein of HMPV (HMPV-F) is a class I fusion protein that exists as homo-trimers on the surface of HMPV virions and infected host cells, which has been considered an immune response target [7]. HMPV-F is highly conserved, it represents the primary antigen for neutralizing antibodies [8]. During the non-influenza season, HMPV infections are second only to hRSV infections in children; however, no vaccines or anti-HMPV strategies are available [9–11]. Hence, it is imperative to develop a safe and effective HMPV vaccine.

Influenza A virus (IAV) is a segmented negative-sense RNA virus for which reverse genetic (RG) technologies are well-established. The insertion of non-IAV sequences into the coding sequences of haemagglutinin (HA), neuraminidase (NA), PB2, M2, or NS1 can induce host antibody responses and T cell activation through exogenous antigen exposure [12,13]. Mouse and ferret models have been used to evaluate the immunoprotective effects of recombinant IAV [14–16], highlighting its ability to induce protective immunity against vector viruses and exogenous pathogens, including parainfluenza virus (PIV), tuberculosis (TB), West Nile virus (WNV), and human immunodeficiency virus (HIV). A vaccine against HMPV based of an IAV reverse genetics has not been made to date [12,13,17,18].

Previously, we successfully generated a recombinant influenza virus rFLU/HAdV/hexon, carrying the human adenovirus epitope on the backbone of the influenza virus A/PR/8/34, which elicited robust protective immunity [19]. Previous studies identified B cell epitopes in HMPV-F, which contain some known antibody binding cell epitopes, such as CTL cell epitopes 228ARAVSNMPTSAGQIK242 and predicted B cell epitopes 290PSCSGKKGNY299 and 88QLAREEQIENPRQSR102, are reportedly significant for epitope vaccines [20–24]. To obtain improved protective efficacy for an HMPV candidate vaccine, we selected a portion of the F gene coding region (nucleotides [nt] 1–909), which contained almost all known reported B cell epitopes. We inserted the first 909nt of the HMPV-F coding region into the surface-exposed NA gene fragment of IAV (H1N1) PR8 according to the structural prediction model based on gene full-length sequences(Fig S2), creating rFLU-HMPV/F-NA. The novel recombinant virus was comprehensively characterized in vitro and in vivo to evaluate its immunogenicity and immunoprotective effects against HMPV.

Materials and methods

Cells lines and viruses

COS-1, MDCK, and Vero-E6 cells were purchased from the Chinese Academy of Sciences and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum. Wild-type influenza virus A/PR/8/34 (PR8) and A/China/BJMY011/2020 (H1N1) were grown in 9–11-day-old specific pathogen-free (SPF) chicken embryos (Beijing Laboratory Animal Center, Beijing, China). The HMPV used in this study was isolated from the nasopharyngeal secretions of children with respiratory tract infections diagnosed in the Emergency Department of Chinese PLA General Hospital. The secretions were collected and transferred to the laboratory on ice. After regular treatment and centrifugation, the supernatant was inoculated within 1 h on Vero-E6 cells for HMPV separation. When the cytopathic effect (CPE) occurred, RT-PCR was performed to amplify the HMPV F gene. The amplified products were sequenced and analysed by Sangon Biotech (Shanghai, China). The sequencing result was compared with the sequence of the HMPV isolate in GenBank. If the sequence homology was > 99%, it was determined to be an HMPV, named HMPV-BJ1812. The PR8 virus vaccine used in this study was prepared in our laboratory; it was inactivated using 0.1% formalin, and its inactivated effects were confirmed as described previously [25].

Rescue of recombinant IAV engineered to express the HMPV-F protein

Recombinant viruses were produced using reverse genetics. The coding sequences of HMPV-F were downloaded from GenBank, and full-length sequences of NA (Fig. S1) were optimized and synthesized by Sangon Biotech. The first 909 nt of HMPV-F were inserted into the NA of A/PR/8/34 (PR8) (Figure 1). We used the bidirectional expression vector pHW2000 to clone HMPV/F-NA. Sequence-verified recombinant plasmid pFLU-HMPV/F-NA were then co-transfected with the other seven plasmids of the PR8 skeleton (pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW-197 M, and pHW198-NS) in MDCK cells and COS-I cells (ratio 1:2) using Effectene (Qiagen Hilden, Germany). rFLU-HMPV/F-NA was harvested following confirmation of HA positivity. Titers of rFLU-HMPV/F-NA were determined from the 50% tissue culture infectious dose (TCID50) in MDCK cells.

Figure 1.

Figure 1.

Open in a new tab

Construction and characterization of recombinant rFLU-HMPV/F-NA. (a) schematic representation of the IAV reverse genetics system used to produce pFLU-HMPV/F-NA. MDCK and COS-I cells were co-transfected with pFLU-HMPV/F-NA, pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW-197 M, and pHW198-NS (ratio 1:2) using Effectene. rFLU-HMPV/F-NA was harvested from cell supernatants upon confirmation of HA positivity. Titers of rFLU-HMPV/F-NA were determined by the TCID50. (b) construction of the pFLU-HMPV/F-NA plasmid. The first 909nt of HMPV-F were inserted into NA of A/PR/8/34 (PR8). The bidirectional expression vector pHW2000 was used to clone HMPV/F-NA. (c) pFLU-HMPV/F-NA was verified by gel electrophoresis. M: Trans15K DNA marker. (d) gel-based detection of the eight transgenic segments of IAV, which were used for transfection. Representative gel images are shown.

Western blotting and electron microscopy

The recombinant rFLU-HMPV/F-NA virus was characterized by Western blotting and electron microscopy. rFLU-HMPV/F-NA was concentrated by ultracentrifugation and purified on a 30–60% sucrose density gradient. Purified rFLU-HMPV/F-NA was obtained following deglycosylation. The white membrane layer between the 30 and 60% sucrose density gradient in the Overspeed centrifuge tube was rFLU-HMPV/F-NA and was suspended in 2 mL of phosphate-buffered saline (PBS). rFLU-HMPV/F-NA was added to the hyperisolated tube, sterilized with PBS, and centrifuged at 4°C and 12,400 g for 2 h. The supernatant was discarded, and the virus precipitate was resuspended with 1 mL of sterile PBS, packaged, and stored at −80°C until use. The virions were negatively stained to visualize their morphology and size on a transmission electron microscope (TEM) (HT7700; Hitachi Ltd., Tokyo, Japan) and were compared with naive IAV (PR8).

Immunofluorescence assays

MDCK cells were grown to monolayers in 24-well plates and digested. The cells were infected for 24 h with purified third generation rFLU-HMPV/F-NA at a multiplicity of infection (MOI) of 1 (200 µL/well). Mock-infected and PR8-only virus controls were included in each experiment. At 24 hours post-infection (hpi), the cells were fixed in 4% formaldehyde for 30 min and permeabilized with 1% Triton X-100 for 15 min. The cells were then blocked in 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature and labelled with primary anti-IAV NP (ab128193) and anti-HMPV F (ab94800) antibodies (1:50 dilution) in 1% BSA in PBS overnight at 4°C. The cells were washed and stained with goat anti-rabbit IgG (H+L) (Alexa Fluor®488, ab150077) and goat anti-mouse IgG (DyLight594, ab150113) for 1 h in the dark. Finally, the cells were stained with DAPI (HXC0065) for 10 min and loaded onto slides using an anti-fluorescence quencher (HXJ3109). Each sample was repeated in triplicate. The cells were imaged on an Olympus confocal microscope (Olympus Corp., Tokyo, Japan).

Stability of recombinant rFLU-HMPV/F-NA

rFLU-HMPV/F-NA was collected from allantoic fluid, inoculated onto 9-day-old SPF chicken embryos, and subjected to HA and TCID50 assays for five generations. HA tests were performed in 96-well plates with 2-fold serial dilutions of the virus. Chicken red blood cells (1%) were added at room temperature, and HA positivity was recorded after 30 min.

The TCID50 titres of rFLU-HMPV/F-NA were obtained from MDCK cells seeded into 96-well plates (2 × 105/mL). The cells were infected with 10-fold serial dilutions of rFLU-HMPV/F-NA in serum-free DMEM for 5 days. The supernatants were then collected, added to HA plates, and assayed as described above. The Reed-Muench method was used to calculate the TCID50 of each virus generation. These experiments were repeated in triplicate.

Animal experiments

All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee and Ethics Committee of Chinese PLA General Hospital. All facilities were approved by the Animal Care and Ethics Committee of Chinese PLA General Hospital.

Groups of six-week-old female BALB/c mice were randomly divided into four groups: (1) rFLU-HMPV/F-NA virus 104TCID50 (low-dose group, 15 mice), (2) rFLU-HMPV/F-NA virus 106TCID50 (high-dose group, 15 mice), (3) PBS control group (15 mice), and (4) PR8 vaccine control group (106 TCID50, 15 mice). The mice were anesthetized with isoflurane and immunized using a nasal drip (20 µL per mouse). Booster immunizations were performed 28 days after priming. Blood was collected from the fundus vein of each mouse as a negative serum before immunization or from five mice from each group post-immunization. On day 14 post-boost, three mice from each group were subject to ocular dissection for blood collection. Lung lavage fluid, nasal lavage fluid, and mouse spleen tissue were also collected and analysed.

HA inhibition (HI) assays

Blood was taken and placed at 37°C for 1 h before the first and second immunizations, centrifuged at 4°C for 1 h at 8000 rpm for 10 min, and then at 12,000 rpm for 2 min. Serum samples were mixed with RDE (cholera bacteriocin) at a ratio of 1:4 and incubated at 37°C for 16–18 h. Samples were heated at 56°C for 50 min and stored at 4°C. Virus samples were serially diluted to four HA units and assayed on HI reaction plates for 1 h at room temperature to determine the HA titres of A/China/BJMY011/2020 (H1N1). We analysed the HI titres in the immunized mice using GraphPad Prism 6.0 statistical software (GraphPad Software, San Diego, CA, USA).

Neutralization assays

Mouse sera from each treatment group were diluted in antibody-free and serum-free MEM cell culture media (1:10, 1:40, 1:160, and 1:640) and added to HMPV (MOI 100) supernatants at a 1:1 ratio for 1 h at 37°C. Next, 2 × 105 cells/mL of virus supernatant/serum sample were added to the VERO-E6 cell suspensions in 24-well plates at 37°C for 2 h. The cells were cultured in complete media for 7 days at 37°C in a 5% CO2 incubator. On day 7, 100 µL of supernatant was collected and analysed via qRT-PCR. Ct values were recorded, and the potency of anti-HMPV neutralizing antibodies in the immunized mice was calculated using the Reed-Muench method.

Serum anti-HMPV IgG titers

Serum samples were serially diluted in 1% BSA in PBS (1:200, 1:400, 1:800, and 1:1600 up to 1:102400) and added to HMPV virus-coated enzyme-linked immunosorbent assay (ELISA) plates (coated with 5 µg/mL of HMPV, 100 µL/well) overnight at 4°C. Horseradish peroxidase (HRP)-labelled sheep anti-mouse IgG in 1% BSA was added to each well (1:4000 dilution) for 1 h at 37°C. H2SO4 (2 M) was added to terminate the reaction. Absorbance values were read at 450 nm on a Synergy H4 ELISA plate reader. The mean values of the experimental wells were ≥ 2.1-fold higher than those of the negative control groups.

Mucosal anti-HMPV IgA titers

Lung and nasal lavage samples were diluted in 1% BSA and assayed on HMPV ELISA plates as described. The virus was detected using HRP-conjugated sheep anti-mouse secretory IgA (sIgA) diluted 1:4000 in BSA.

Flow cytometry

The spleens of rFLU-HMPV/F-NA-immunized mice were collected from each group on day 14 after the boost. The spleens were ground in 6 mL of saline and diluted to 2 × 105/mL in a splenic lymphocyte suspension. The samples were treated with 5 mL of mouse splenic lymphocyte stimulating solution (200 µL saline +0.5 µL stimulating agent [BioLegend, San Diego, CA, USA] + 0.08 µg HMPV) at 37°C for 6 h. The cells were labelled with FITC-CD4 antibodies in the dark for 30 min at 37°C and fixed for 20 min at room temperature. Next, the cells were labelled with primary antibodies against TNF-α (MAB11–502913), interferon (IFN)-γ (W19227A–383304), IL-2 (BV421), and IL-4 (PE/CY-740540) for 40 min in the dark at room temperature, washed in PBS, and centrifuged at 8000 rpm for 1 min. The supernatants were discarded, and the cell pellets were resuspended in PBS before analysis on a BD Flow Cytometer (Beckman Coulter Inc., Brea, CA, USA).

Wild-type HMPV challenge

rFLU-HMPV/F-NA 104TCID50, 106TCID50, PBS, and PR8 inactivated vaccine control mice (12 mice per group) were infected with 106 PFU/mL of HMPV-BJ1812 via a nasal drip (20 µL per mouse) under isoflurane anaesthesia on day 14 after the boost. The mice were observed for changes in body weight and/or respiratory symptoms. Previously, we have established an animal model of Wild-type HMPV infection, respectively, the results showed that the virus load was highest on the 6th day post-infection. Therefore, five mice from each group were euthanized on day 6, and tissue samples were collected from the left lung to determine the HMPV loads. Tissue was collected from the right lung for pathological examination via haematoxylin and eosin (H&E) staining, and the pathological scores were analysed from each group according to the histopathology scoring system [26]. The remaining mice were assessed for survival rates and weight changes over 14 days.

Wild-type A/China/BJMY011/2020 (H1N1) challenge

Groups of mice (12 per group) immunized with rFLU-HMPV/F-NA 104TCID50, 106TCID50, PBS, and PR8 inactivated vaccine. They were then infected with 15LD50/mL of A/China/BJMY011/2020 (H1N1) via a nasal drip (20 µL/mouse) under isoflurane anaesthesia on day 14 after the boost. The mice were observed for changes in body weight, survival, and/or respiratory symptoms until 14 days post-challenge. Previously, we have established an animal model of Wild-type A/China/BJMY011/2020 (H1N1) infection, respectively, the results showed that the virus load was highest on the 4th day post-infection. Therefore, on day 4 post-infection, five mice from each group were euthanized and tissue samples were collected from the left lung to determine the viral loads. Tissue was collected from the right lung for pathological examination with H&E staining. The pathological scores were summarized from each group according to the histopathology scoring system. The survival rates and weight changes of the mice were recorded and analysed over 14 days post-challenge.

Viral titers

Lung tissue samples from infected mice were ground, centrifuged, and assayed using qRT-PCR primers targeting HMPV-F and GAPDH (Shanghai Biotech). The primer sequences were as follows: HMPV-F Forward Primer: 5ˈ-CTTTGGACTTAATGACAGATG-3ˈ, HMPV-F Reverse Primer: 5ˈ-GTCTCCCTGTGCTAACTTTG-3ˈ, GAPDH Forward Primer: 5ˈ-AGGTCGGTGTGAACGGATTTG-3ˈ, and GAPDH Reverse Primer: 5ˈ-TGTAGACCATGTAGTTGAGGTCA-3ˈ. qRT-PCR assays were performed using a PowerUp™ SYBR™ Green Master Mix Kit (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s, and 72°C for 30 s.

Pathology

On day 6 post-HMPV challenge, the individual lungs of the mice were removed and fixed in 4% formaldehyde. The lungs of the immunized mice were isolated, fixed, dehydrated, and embedded in paraffin. H&E staining was used to observe peribronchiolar, perivascular, and alveolar inflammation.

Statistical analysis

Statistical analyses were performed using GraphPad Prism v9.0.0. A Student’s t-test was used to analyse differences between groups. A one-way analysis of variance was performed for multiple group comparisons, and a two-sided p < 0.05 was defined as statistically significant.

Results

Generation of rFLU-HMPV/F-NA using RG technology

We aimed to establish a recombinant influenza virus carrying a partial HMPV-F, the primary target of neutralizing antibodies. Following bioinformatics analyses, sequence optimization, and literature reviews, we deemed the major antigenic epitope of HMPV-F to be positioned between nt 1–909 in the coding region. This sequence was inserted into the NA gene of IAV-PR8 to form a recombinant 1152-base pair fragment (Figure 1b). This recombinant sequence was inserted into pHW2000 by Shanghai Biotech to generate pFLU-HMPV/F-NA, which was then co-transfected with the other seven plasmids of the PR8 skeleton (pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW-197 M, and pHW198-NS) into COS-1 and MDCK cells (Figure 1a,d). The rescued recombinant viruses were named rFLU-HMPV/F-NA.

Characterization of rFLU-HMPV/F-NA

We assessed the expression of HMPV-F and PR8-NP in IAV and rFLU-HMPV/F-NA-infected cells by immunofluorescence to fully characterize rFLU-HMPV/F-NA. Robust IAV-NP staining was detected in PR8 and rFLU-HMPV/F-NA cells. No fluorescence was observed in the uninfected cell controls (NC; Figure 2a). Importantly, rFLU-HMPV/F-NA infection led to high levels of HPMV expression that was absent in PR8 and uninfected cells, confirming the successful production of the virus (HMPV-F, red staining; Figure 2a). The expression of target protein HMPV/F was confirmed by Western blotting, which showed that the main 35 kDa protein interacts with positive serum from HPMV patients (Figure 2b). Observation using a TEM showed the rFLU-HMPV/F-NA virions as being spherical and intact (Figure 2c) and with an identical size distribution as native PR8-IAV (Figure 2d). rFLU-HMPV/F-NA could be passaged through SPF chicken embryos for five consecutive generations with a stable HA potency of 28–9 for P1–P5 (Figure 2e) and a TCID50 titre of 108–9 TCID50/mL (Figure 2f). These results validated the use of rFLU-HMPV/F-NA for subsequent studies.

Figure 2.

Figure 2.

Open in a new tab

Rescue and identification of rFLU-HMPV/F-NA. (a) immunofluorescence analysis of IAV NP and HMPV-F expression (60×). MDCK cells were infected for 24 h with purified third-generation rFLU-HMPV/F-NA (MOI: 1) and labelled 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 with an Olympus confocal microscope. (b) size of PR8 and rFLU-HMPV/F-NA as found by Western blotting. (c) morphology and (d) size distribution of PR8 and rFLU-HMPV/F-NA as found using a TEM. (e) passaged rFLU-HMPV/F-NA (P1–P5) was assessed for HA positivity. (f) P1–P5 viral titres of rFLU-HMPV/F-NA were assessed based on the TCID50.

The humoral anti-HMPV immune response following immunization with rFLU-HMPV/F-NA

We hypothesized that rFLU-HMPV/F-NA would induce potent immunogenicity in vivo. For confirmation, BALB/c mice were intranasally immunized with rFLU-HMPV/F-NA (the time course of animal dosing is shown in Figure 3a). We measured the HI antibody titres against the circulating influenza virus isolate A/China/BJMY011/2020 (H1N1) in the sera of mice immunized at 28 days after priming and at 14 days after boosting. The low- and high-dose rFLU-HMPV/F-NA groups could induce significant HI titres compared to the PBS group after priming and boosting in a dose-dependent manner (Figure 3b; p < 0.01). Additionally, 14 days after the second vaccination, the mean HI antibody titres increased sharply to 101.6 and 104.0 in the 104 TCID50 and 106 TCID50 groups, respectively. Meanwhile, the HI titres were higher in the high-dose group than in the low-dose group, though the differences were not statistically significant (p > 0.05). Thus, rFLU-HMPV/F-NA can effectively induce HI antibodies against A/China/BJMY011/2020 (H1N1), a clinical IAV isolate.

Figure 3.

Figure 3.

Open in a new tab

Immunogenicity of rFLU-HMPV/F-NA. (a) schematic outlining the animal immunization schedule. (b) HI titres against IAV in mice immunized with rFLU-HMPV/F-NA. Values are the means ± standard error (SE) of three independent experiments (*** p < 0.001). (c) neutralizing titres against HMPV in mice immunized with rFLU-HMPV/F-NA. (d) IgG titres against HMPV in mice immunized with rFLU-HMPV/F-NA.

To further detect the neutralizing titres of rFLU-HMPV/F-NA against HMPV, immunized mice were assayed via micro-neutralization tests, and viral loads were assessed via qRT-PCR analysis (Figure 3c). Antibody neutralization in the PBS group was lower than in the high- and low-dose primary and booster immunization groups, and the following booster immunization was significantly higher in the high- and low-dose groups compared to the primary immunization group (p < 0.01). The mean neutralizing titres increased sharply to 106.9 and 102.6 in the 104 TCID50 and 106 TCID50 groups following the second vaccination. Meanwhile, the neutralizing titres in the high-dose group exceeded those of the low-dose group following primary and booster immunization, but the differences were not significant (p > 0.05). These data suggest that rFLU-HMPV/F-NA can effectively induce the production of anti-HMPV neutralizing antibodies, which may protect mice from HMPV infection.

To evaluate the humoral immune responses induced following rFLU-HMPV/F-NA immunization in mice, we measured the HMPV-specific IgG antibody levels by ELISAs. As shown in Figure 3d, compared with the PBS group, the primary and booster immunization in the low- and high-dose groups produced highly effective HMPV-specific IgG antibodies (p < 0.01), the titres of which were significantly higher following the booster compared to the primary immunization (p < 0.01). The mean titres of IgG antibodies increased sharply to 103.6 and 104.0 in the 104 TCID50 and 106 TCID50 groups following the second vaccination. Meanwhile, the titres of IgG antibodies in the high-dose group exceeded those of the low-dose group following the primary immunization, but the differences were not significant (p > 0.05). However, the titres of IgG antibodies in the high-dose group were significantly higher than those in the low-dose group following the booster immunization (p < 0.05).

Mucosal anti-HMPV immune responses following immunization with rFLU-HMPV/F-NA

To evaluate the mucosal immune responses induced by rFLU-HMPV/F-NA immunization, we measured mucosal sIgA antibodies against HMPV in the nasal and lung lavage fluids of immunized mice 14 days after the boost. The levels of HMPV sIgA antibodies in the nasal lavage fluid of immunized mice in the low- and high-dose groups were significantly higher than those in the PBS control group (p < 0.01). The mean titres of HMPV-specific sIgA antibodies increased sharply to 101.5 and 102.1 in the 104 TCID50 and 106 TCID50 groups at 14 days after the boost (Figure 4a). Meanwhile, the titres of HMPV-specific sIgA antibodies in the high-dose group were significantly higher than those in the low-dose group (p < 0.05). The levels of HMPV sIgA antibodies in the lung lavage fluid of immunized mice in the low- and high-dose groups were significantly higher than those in the PBS control group (p < 0.01) (Figure 4b). The mean titres of HMPV-specific sIgA antibodies increased sharply to 102.2 and 102.3 in the 104 TCID50 and 106 TCID50 groups at 14 days after the boost. Meanwhile, the titres of HMPV-specific sIgA antibodies in the high-dose group were significantly higher than those in the low-dose group (p < 0.05). These data highlight the ability of rFLU-HMPV/F-NA to induce mucosal immune responses.

Figure 4.

Figure 4.

Open in a new tab

sIga antibody titres in mice immunized with rFLU-HMPV/F-NA. (a) HMPV-induced IgA values calculated from the nasal lavage fluid of immunized mice by ELISA, and (b) HMPV-induced IgA values calculated from the lung lavage fluid of immunized mice by ELISA. Values are the means ± SE of three independent experiments (*** p < 0.001).

rFLU-HMPV/F-NA induces robust cellular immune responses

We examined the levels of HMPV-specific Th1/Th2 cytokines (TNF-α, IFN-γ, IL-2, and IL-4) in mouse spleen lymphocytes at 14 days after the boost using flow cytometry to verify whether the immune protection observed was due to a bias towards Th1-type immune responses (Figure 5a). Compared to the PBS group, the low-dose group produced significantly higher levels of the HMPV-specific cytokines TNF-α, IFN-γ (Th1-type), and IL-4 (Th2-type) (p < 0.01) and significantly increased IL-2 (Th1-type) levels (p < 0.05). The high-dose group had elevated levels of TNF-α, IFN-γ, IL-2 (Th1-type), and IL-4 (Th2-type) (Figure 5b; p < 0.01).These data suggest that rFLU-HMPV/F-NA induces a Th1/Th2 immune response with a tendency towards Th1 and provide a novel explanation of the balance between Th1/Th2 immune responses in response to recombinant HMPV vaccines.

Figure 5.

Figure 5.

Open in a new tab

Cytokine levels in mice immunized with rFLU-HMPV/F-NA. 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) raw FACs traces. (b) graphical representation of the number of cells per 105. Solenocyte values are the means ± SE of three independent experiments (** p < 0.01, *** p < 0.001).

rFLU-HMPV/F-NA afforded significant immune protection against a wild-type HMPV challenge

We evaluated whether rFLU-HMPV/F-NA could protect mice against an HMPV challenge by recording body weight changes and survival for 14 days. The body weights of mice in the high-dose group showed no significant changes (Figure 6a). Therefore, rFLU-HMPV/F-NA effectively protected mice following HMPV infection. We measured the expression of HMPV-F by qRT-PCR to detect the viral loads in mouse lung tissue following the HMPV challenge. The lung tissues of mice were excised at day 6 post-HMPV challenge, and the viral loads in the lung homogenates were measured (Figure 6b). The rFLU-HMPV/F-NA-immunized groups could effectively inhibit HMPV replication in lung tissue compared to the PR8 and PBS groups. More significant inhibitory effects were observed in the high-dose group (p < 0.01).

Figure 6.

Figure 6.

Open in a new tab

Immunoprotective effects of rFLU-HMPV/F-NA following an HMPV challenge. (a) weight changes (means ± SE; n = 12). (b) HMPV titres (*** p < 0.001). (c) histopathology (×200) was assessed in immunized mice (n = 5) following HMPV infection. (d) summary of the mouse group pathological scores. (*p < 0.05,**p < 0.01, ***p < 0.001).

We performed H&E staining on pathological lung sections from mice within each group on day 6 of infection to evaluate histopathological changes in the lung tissues in each of the mouse groups post-challenge (Figure 6c). Compared to normal mice, the lung tissues of the PBS and PR8 vaccine groups showed alveolar fusion and many inflammatory cells. Both the low- and high-dose groups showed reduced pathological damage and lower levels of inflammatory cell infiltration. The high-dose group showed the most beneficial immunoprotective effects; these were confirmed by the pathological score results, which displayed a similar tendency for each group (Figure 6d). These data highlight the protective efficacy of rFLU-HMPV/F-NA against HMPV.

rFLU-HMPV/F-NA afforded significant immune protection against a wild-type A/China/BJMY011/2020 (H1N1) challenge

Body weight changes and survival were recorded for 14 days post-A/China/BJMY011/2020 (H1N1) challenge to ascertain whether rFLU-HMPV/F-NA could protect mice against a wild type influenza virus infection. The body weights of mice in the high-dose group showed no significant changes (Figure 7a). However, mice in the PBS group began to die at 8 days following A/China/BJMY011/2020 challenge (Figure 7b). Therefore, rFLU-HMPV/F-NA effectively protected mice from clinical influenza virus A/China/BJMY011/2020 infection.

Figure 7.

Figure 7.

Open in a new tab

Immunoprotective effects of rFLU-HMPV/F-NA following a Wild-type A/China/BJMY011/2020(H1N1) challenge. (a) weight changes (mean ± SE; n = 12), (b) survival curves, (c) viral titres (*** p < 0.001), (d) histopathology (×200) was assessed in immunized mice (n = 5) following Wild-type A/China/BJMY011/2020(H1N1) infection, (e) summary of the mouse group pathological scores. (*p < 0.05, **p < 0.01, ***p < 0.001).

The lung tissues of mice were collected on day 4 post-A/China/BJMY011/2020 challenge, and the viral loads in lung homogenates were measured (Figure 7c). Both the low- and high-dose rFLU-HMPV/F-NA groups and the PR8 vaccine group effectively decreased the virus titres in the lung tissues of immunized mice compared to the PBS group (p < 0.01).

We performed H&E staining on pathological sections from mice within each group at 4 days post-infection to evaluate the histopathological changes in the lung tissues of each group of mice post-challenge (Figure 7d). The lung tissues of the PBS group showed alveolar fusion and many inflammatory cells compared to the normal mice. However, the PR8 vaccine group and low- and high-dose groups displayed reduced pathological damage and lower levels of inflammatory cell infiltration. The high-dose group showed the best immunoprotective effects. Each group had similar pathological scores (Figure 7e). These data highlight the protective efficacy of rFLU-HMPV/F-NA against influenza and HMPV infections.

Discussion

HMPV’s impact on worldwide health, the economy, and society, which is significant and must be addressed. Decades of attempts have been made to develop effective HMPV vaccines, but there is still no licenced vaccine at present [7,27]. Research has shown that genes of interest can be cloned into one of the membrane glycoproteins of IAV (NA) by reverse genetics, which can result in strong antibody responses [12]. In this study, we used RG technology to insert the HMPV-F gene into the NA recombinant plasmid, named rFLU-HMPV/F-NA. More importantly, the genetic stability of the rFLU-HMPV/F-NA vaccine candidate was also examined and inserted epitopes genes, the virus could passage stably for at least five consecutive generations. Furthermore, the safety and immunogenicity were evaluated by using the recombinant vaccine strain rFLU-HMPV/F-NA as a positive control in mice with intranasal immunization, the results showed that rFLU-HMPV/F-NA can induce robust humoral, mucosal, and cellular immune responses and provide significant immune protection against HMPV and IAV infection. These studies provide a new strategy for the development of HMPV vaccines and lay a theoretical foundation for the realization of “A single vaccine for dual-purpose” against HMPV and IAV infection.

Research has shown that Nasal vaccination represents a favourable route of vaccine delivery as it can stimulate mucosal immunity [28,29]. The mucosal system depends on sIgA production by B lymphocytes, preventing pathogens from attaching to and invading the mucosa [30]. IAV infects the upper respiratory tract, and local secretory antibodies are important in the induction of cross-protection with serum IgG antibodies. The primary function of sIgA is to prevent antigens from adhering to the mucosa, promoting the formation of immune complexes excreted by epithelial cells. Since sIgA protects against upper respiratory tract infections and serum IgG affords protection in the lower respiratory tract, immunization methods that stimulate sIgA and IgG can improve protection [31]. The neutralizing titres against IAV and HMPV in the sera of mice immunized with rFLU-HMPV/F-NA revealed effective protection, supporting the notion of a single vaccine for dual usage. Moreover, HMPV-specific sIgA titres in the nasal and lung lavage fluids of mice after a boost showed that rFLU-HMPV/F-NA could induce an effective mucosal immune response in mice.

Previous studies showed that inducing a balanced Th1/Th2 immune response during vaccine development is essential to prevent and treat infectious and autoimmune diseases [32]. Research [33,34] has shown that Th1/Th2 cytokines (TNF- α, IFN- γ, IL-2, and IL-4) are hMPV-specific. In line with these reports, the secretions of spleen lymphocytes (TNF- α, IFN- γ, IL-2, and IL-4) of the rFLU-HMPV/F-NA immune group were significantly increased and mainly biased towards the Th1-type cellular immune response, indicating that rFLU-HMPV/F-NA induced a robust Th1-type immune response and Th1/Th2 balance. When mice were challenged with HMPV 14 days after the boost, we observed no significant fluctuations in body weight. rFLU-HMPV/F-NA-immunized mice also exhibited significantly fewer lung tissue lesions and lower levels of lymphocyte infiltration, highlighting its protective effects against HMPV in lung tissues. Similarly, the viral loads in rFLU-HMPV/F-NA-vaccinated mice were significantly lower than in the control group in a dose-dependent manner. There was no significant difference in the viral loads in the PR8 vaccine and PBS groups. These data suggest that recombinant rFLU-HMPV/F-NA led to effective immune protection against HMPV infection in mice. Furthermore, previous studies showed that stimulating memory B cells with PreF antigen may produce specific antibodies to PreF [35,36], and we will explore whether HMPV Pre F can induce memory immunity in the subsequent experiments.

In addition, previous studies showed that HMPV-F is highly conserved, which is considered an effective neutralizing antibody target [8], therefore we speculate the F protein could have a slow rate of antigenic drift and the fusion NA-F wouldn’t trimerize upon forming the chimeric virus, Of course, these results need to be validated in our subsequent work.

Conclusions

In conclusion, chimeric rFLU-HMPV/F-NA carrying partial HMPV-F epitopes could induce effective humoral, cellular, and mucosal immunity in mice. These results provide experimental data for future investigations of bivalent human influenza viral vector HMPV vaccines for the much-needed control of HMPV infections. Therefore, rFLU-HMPV/F-NA is a promising vaccine candidate which warrants further investigation.

Supplementary Material

Fig S1.tif
KVIR_A_2284515_SM4264.tif (2.5MB, tif)
Fig S2.tif
KVIR_A_2284515_SM4263.tif (4.3MB, tif)

Funding Statement

The work was supported by the National Natural Science Foundation of China [81971564].

Disclosure statement

All authors: No reported conflicts of interest.

Author contributions

S.F., G.L.L. and C.Y.T. contributed equally to the study. S.F., G.L.L., C.Y.T., Z.F.B., C.Q.B., Y.F.L., Z.Y.D., H.Y. and C.L. search the literature. S.F., G.L.L. and C.Y.T. did the main experiment. S.F., G.L.L. and C.Y.T. wrote the original manuscript. Z.Z.P., Y.N. and P.H.Y. supervised the work and contributed resources. All authors contributed and edited the manuscript.

Institutional Review Board statement

All animals used in this study were treated according to the standards of animal welfare. Procedures were reviewed by the Animal Care and Welfare Committee of the Animal Ethics Committee of the Fifth Medical Center of PLA General Hospital.

Data availability statement

The datasets generated for this study are available on request to the corresponding author.

Financial support

This study was supported by the Natural Science Foundation of China [81971564]

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2023.2284515

References

  • [1].Van Den Bergh A, Bailly B, Guillon P, et al. Antiviral strategies against human metapneumovirus: Targeting the fusion protein. Antiviral Res. 2022. Nov;207:105405. [DOI] [PubMed] [Google Scholar]
  • [2].Gálvez NMS, Andrade CA, Pacheco GA, et al. Alexis M Kalergis.Host components that modulate the disease caused by hMpv.Viruses. Viruses. 2021 Mar 22;13(3):519. doi: 10.3390/v13030519 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Collaborators GL. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study 2015. Lancet Infectious Diseases. 2017;17(11):1133–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].van den Hoogen BG, de Jong JC, Groen J, et al. Osterhaus AD: a newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med. 2001;7(6):719–724. doi: 10.1038/89098 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Nao N, Saikusa M, Sato K, et al. Recent molecular evolution of human metapneumovirus (HMPV): subdivision of HMPV A2b strains. Microorganisms. 2020;8(9):1280. doi: 10.3390/microorganisms8091280 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Gálvez NMS, Andrade CA, Pacheco GA, et al. Host components that modulate the disease caused by hMPV. Viruses. 2021;13(3):519. doi: 10.3390/v13030519 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Wen X, Pickens J, Mousa JJ, et al. Jardetzky TS: a chimeric pneumovirus fusion protein carrying neutralizing epitopes of Both MPV and RSV. PLoS One. 2016;11(5):e0155917. doi: 10.1371/journal.pone.0155917 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Rappazzo CG, Hsieh CL, Rush SA, et al. Potently neutralizing and protective anti-human metapneumovirus antibodies target diverse sites on the fusion glycoprotein. Immunity. 2022. 13;55(9):1710–1724. doi: 10.1016/j.immuni.2022.07.003 [DOI] [PubMed] [Google Scholar]
  • [9].Ji L, Chen L, Xu D, et al. Molecular typing and epidemiologic profiles of human metapneumovirus infection among children with severe acute respiratory infection in Huzhou, China. Mol Biol Rep. 2021;48(12):7697–7702. doi: 10.1007/s11033-021-06776-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Chupin C, Pizzorno A, Traversier A, et al. Avian cell line DuckCelt®-T17 is an efficient production System for live-attenuated human metapneumovirus vaccine candidate Metavac®. Vaccines. 2021;9(10):1190. doi: 10.3390/vaccines9101190 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Stewart-Jones GBE, Gorman J, Ou L, et al. Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses. Proc Natl Acad Sci USA. 2021;118(39): doi: 10.1073/pnas.2106196118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].de Goede AL, Boers PH, Dekker LJ, et al. Characterization of recombinant influenza a virus as a vector for HIV-1 p17Gag. Vaccine. 2009;27(42):5735–5739. doi: 10.1016/j.vaccine.2009.07.032 [DOI] [PubMed] [Google Scholar]
  • [13].Yamauchi Y. Influenza a virus uncoating. Adv Virus Res. 2020;106:1–38. [DOI] [PubMed] [Google Scholar]
  • [14].Xu L, Bao L, Deng W, et al. The mouse and ferret models for studying the novel avian-origin human influenza a (H7N9) virus. Virol J. 2013;10(1):253. doi: 10.1186/1743-422X-10-253 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Kimble JB, Wymore Brand M, Kaplan BS, et al. Vaccine-Associated Enhanced respiratory disease following influenza virus infection in ferrets recapitulates the model in pigs. J Virol. 2022;96(5):e0172521. doi: 10.1128/jvi.01725-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Roubidoux EK, Schultz-Cherry S. Animal models utilized for the development of influenza virus vaccines. Vaccines. 2021;9(7):787. doi: 10.3390/vaccines9070787 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Li Z, Gabbard JD, Mooney A, et al. Single-dose vaccination of a recombinant parainfluenza virus 5 expressing NP from H5N1 virus provides broad immunity against influenza a viruses. J Virol. 2013;87(10):5985–5993. doi: 10.1128/JVI.00120-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Stukova MA, Sereinig S, Zabolotnyh NV, et al. Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT-6 protein. Tuberculosis (Edinburgh, Scotland). 2006;86(3–4):236–246. doi: 10.1016/j.tube.2006.01.010 [DOI] [PubMed] [Google Scholar]
  • [19].Yang P, Li T, Liu N, et al. Recombinant influenza virus carrying human adenovirus epitopes elicits protective immunity in mice. Antiviral Res. 2015;121:145–151. doi: 10.1016/j.antiviral.2015.06.015 [DOI] [PubMed] [Google Scholar]
  • [20].Huang J, Diaz D, Mousa JJ, et al. Antibody recognition of the pneumovirus fusion protein trimer interface. PLOS Pathogens. 2020;16(10):e1008942. doi: 10.1371/journal.ppat.1008942 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Korsun NS, Angelova SG, Trifonova IT, et al. The prevalence and genetic Characterization of human metapneumovirus in Bulgaria, 2016-2019. Intervirology. 2021;64(4):194–202. doi: 10.1159/000516821 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Li X, Guo L, Kong M, et al. Design and Evaluation of a Multi-Epitope Peptide of Human Metapneumovirus. Intervirology. 2015;58(6):403–412. doi: 10.1159/000445059 [DOI] [PubMed] [Google Scholar]
  • [23].Bayrakdar F, Başak Altaş A, Korukluoğlu G. Phylogenetic variability of human metapneumovirus strains circulating in Turkey during two consecutive epidemic seasons. Mikrobiyol Bul. 2016. Jan;50(1):63–72. doi: 10.5578/mb.10060 [DOI] [PubMed] [Google Scholar]
  • [24].Kandeil A, Moatasim Y, Gomaa MR, et al. Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses. Vaccine. 2016;34(2):218–224. doi: 10.1016/j.vaccine.2015.11.037 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Tanabe N, McDonough JE, Vasilescu DM, et al. Pathology of idiopathic pulmonary fibrosis assessed by a combination of microcomputed tomography, histology, and immunohistochemistry. Am J Pathol. 2020;190(12):2427–2435. doi: 10.1016/j.ajpath.2020.09.001 [DOI] [PubMed] [Google Scholar]
  • [26].Cox RG, Livesay SB, Johnson M, et al. The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins. J Virol. 2012;86(22):12148–12160. doi: 10.1128/JVI.01133-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Cattoir L, Vankeerberghen A, Boel A, et al. Epidemiology of RSV and hMPV in Belgium: a 10-year follow-up. Acta Clin Belg. 2019;74(4):229–235. doi: 10.1080/17843286.2018.1492509 [DOI] [PubMed] [Google Scholar]
  • [28].Park HJ, Ferko B, Byun YH, et al. Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice. PLoS One. 2012;7(6):e39921. doi: 10.1371/journal.pone.0039921 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Lambert LC, Fauci AS. Influenza vaccines for the future. N Engl J Med. 2010;363(21):2036–2044. doi: 10.1056/NEJMra1002842 [DOI] [PubMed] [Google Scholar]
  • [30].van Riet E, Ainai A, Suzuki T, et al. Mucosal IgA responses in influenza virus infections; thoughts for vaccine design. Vaccine. 2012;30(40):5893–5900. doi: 10.1016/j.vaccine.2012.04.109 [DOI] [PubMed] [Google Scholar]
  • [31].Gianchecchi E, Manenti A, Kistner O, et al. How to assess the effectiveness of nasal influenza vaccines? Role and measurement of sIga in mucosal secretions. Influenza Other Respir Viruses. 2019;13(5):429–437. doi: 10.1111/irv.12664 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Lamichhane PP, Boonnak K, Changsom D, et al. H5N1 NS genomic segment distinctly governs the influenza virus infectivity and cytokine induction in monocytic cells. Asian Pac J Allergy Immunol. 2018;36(1):58–68. doi: 10.12932/AP0870 [DOI] [PubMed] [Google Scholar]
  • [33].Stepanova E, Matyushenko V, Rudenko L, et al. Prospects of and barriers to the development of epitope-based vaccines against human metapneumovirus. Pathogens. 2020;9(6). doi: 10.3390/pathogens9060481 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Dubois J, Pizzorno A, Cavanagh MH, et al. Strain-dependent impact of G and SH deletions provide New insights for live-attenuated HMPV vaccine development. Vaccines. 2019;7(4):164. doi: 10.3390/vaccines7040164 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Xiao X, Fridman A, Zhang L, et al. Profiling of hMPV F-specific antibodies isolated from human memory B cells. Nat Commun. 2022. 10;13(1):2546. doi: 10.1038/s41467-022-30205-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Phung E, Chang LA, Mukhamedova M, et al. Elicitation of pneumovirus-specific B cell responses by a prefusion-stabilized respiratory syncytial virus F subunit vaccine. Sci Transl Med. 2022. 22;14(650). doi: 10.1126/scitranslmed.abo5032 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig S1.tif
KVIR_A_2284515_SM4264.tif (2.5MB, tif)
Fig S2.tif
KVIR_A_2284515_SM4263.tif (4.3MB, tif)

Data Availability Statement

The datasets generated for this study are available on request to the corresponding author.

Articles from Virulence are provided here courtesy of Taylor & Francis

RESOURCES