To stimulate increased breadth of immunogenicity and protection against Flu viruses, a 3M2e gene containing three types of M2e sequences from human, swine, and avian influenza viruses were synthesized ( Supplementary Table ) and fused to the COOH-terminus of Soc to generate Soc-3M2e. A flexible GGSSGGSS linker was introduced between each M2e segment as depicted in Figure 1B , to minimize any interference in the folding of the M2e domains. Hexa-histidine tags were also added to both termini of the Soc-3M2e protein. The fusion protein was expressed in E. coli and purified by Ni²⁺ affinity chromatography followed by size-exclusion chromatography. The major peak corresponding to a molecular weight of ~21.8 kDa (monomeric Soc-3M2e) was collected ( Figure 1B , blue profile). The purity of the recombinant Soc-3M2e proteins was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), which showed a single major band with a molecular mass of ~22 kDa, equivalent to the Soc-3M2e fusion protein ( Figure 1B ).

The 3M2e-decorated T4 nanoparticles (3M2e-T4 nanoparticles) were prepared by incubation of the purified Soc-3M2e protein with the CsCl-purified Hoc^-Soc^- T4 phages as previously described (44) ( Figure 1C ). To optimize the copy number of 3M2e, ~5×10¹⁰ T4 phages were incubated with different quantities of the Soc-3M2e protein ( Figure 1C ). The presence of bound Soc-3M2e was determined by SDS-PAGE analysis of 3M2e-T4 nanoparticles isolated by high-speed centrifugation. The data show that the Soc-3M2e protein bound efficiently to the Hoc^-Soc^- T4 phages, even at a 1:1 ratio of Soc-3M2e molecules to Soc binding sites, and reached saturation at ratio of 10:1 ( Figure 1C ). The copy number of bound Soc-3M2e per capsid (B_max) calculated from the binding curve was 427, and the apparent binding constant (K_d) was 305 nM ( Figure 1D ). Since each Soc-3M2e protein contains three tandem copies of M2e peptide, there are ~1,281 M2e molecules assembled on each T4 nanoparticle, which is remarkably higher than any VLPs reported so far. The diameter distribution and zeta-potential of 3M2e-T4 nanoparticles was determined using Zetasizer Nano ZS. The average diameter of 3M2e-T4 nanoparticles is 150.9 nm, which is larger than the diameter of Hoc^-Soc^- T4 phages (mean = 120.8 nm) ( Figure 1E ), indicating the binding of Soc-3M2e. We didn’t observe significant difference in zeta-potentials between Hoc^-Soc^- T4 (-26.7 ± 0.4 mV) and M2e-T4 nanoparticles (-24.3 ± 0.12 mV) ( Figure S1 ).

To determine the immunogenicity of 3M2e-T4 nanoparticles, mice were intramuscularly immunized with 3M2e-T4 nanoparticles displaying a total of 15μg 3M2e antigen on day 0, 14, and 28 ( Figure 2A ). Mice immunized with PBS, 15μg of Soc-3M2e soluble proteins, or a simple mixture of 15μg Soc-3M2e antigen and the same number of T4 phage nanoparticles (Soc-3M2e+T4) were used as controls. To minimize binding prior to immunization, the Soc-3M2e antigen was mixed with phage T4 at the time of immunization (most of the Soc-3M2e did not bind to capsid, see Materials and Methods for the details). Sera were collected according to the scheme shown in Figure 2A , and the titers of M2e-specific antibodies were determined by enzyme-linked immunosorbent assay (ELISA). All mice immunized with PBS were negative for M2e-specific IgG even at a low sera dilution of 50. The Soc-3M2e soluble proteins induced very low levels of M2e-specific IgG antibodies. However, 3M2e-T4 nanoparticles), without any adjuvant, induced very high levels of 3M2e-specific IgG antibodies, with end point titers of ~4×10⁵ ( Figure 2B ). Even a simple mixture of Soc-3M2e and T4 phage (Soc-3M2e+T4 mixture) generated high titers (~1×10⁵) of M2e-specific antibodies when compared to the very low titers generated by the soluble Soc-3M2e antigen ( Figure 2B , p<0.0001, ANOVA). These data point to the remarkable immune stimulatory effect of the T4 phage nanoparticles.

Since M2e-induced immune protection mainly depends on antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), the efficiencies of which are different between IgG subtypes (45–47), we determined the titers of M2e-specific IgG1 (T_H2-biased) and IgG2a (T_H1-biased). The Soc-3M2e soluble antigen mainly induced IgG1 antibodies, whereas mice immunized with 3M2e-T4 nanoparticles or Soc-3M2e+T4 mixture produced similar levels of both IgG1 and IgG2a antibodies ( Figures 2C – E ). Elicitation of balanced T_H1 and T_H2 biased immune responses is a significant feature of the T4 vaccine delivery platform and generally important for protection against infectious disease. We have also determined the 3M2e-specific IgA antibodies in sera. Although 3M2e-T4 nanoparticles induced IgA antibodies in some of the mice, the data were not statistically significant (p>0.05, ANOVA. Figure 2F ).

To determine whether 3M2e-T4 nanoparticles induced antibodies recognize M2e on influenza virions, A/Puerto Rico/8/34 (H1N1) virus was inactivated with β-propiolactone and used as the coating antigen in ELISA assays. The data revealed that these antibodies, regardless of the IgG subtype, specifically bound to the influenza virions ( Figures 3A – C ). Consistent with the end point titers ( Figure 2 ), balanced levels of virion-binding titers were observed for both the IgG1 and IgG2a subtypes. As expected, sera from PBS immunized mice showed negative results even at a low sera dilution of 10 ( Figures 3A – C ). Since M2e-induced immune protection mainly depends on ADCC and ADCP, we then examined whether M2e presented in plasma membranes of influenza virus-infected cells can be recognized by 3M2e-T4 induced antibodies. Madin-Darby canine kidney (MDCK) cells infected with A/Puerto Rico/8/34 (H1N1) influenza virus at a multiplicity of infection (MOI) of 1 were tested for the binding of M2e-specific antibodies by indirect immunofluorescence assay ( Figure 3D ). The data indicated that the sera from 3M2e-T4 VLPs immunized mice showed significant binding to influenza virus-infected cells, but not to mock-treated cells ( Figure 3D ). Together, these data demonstrated that the 3M2e-T4 nanoparticle can efficiently induce M2e-specific antibodies that recognize M2e presented both on influenza virions and on virus-infected cells, indicating their potential protection against influenza virus infection.

To investigate whether the 3M2e-T4 nanoparticles elicited M2e-specific cellular immune responses, mice were sacrificed 7 days after the second boost, and spleens were collected to isolate peripheral blood mononuclear cells (PBMCs). The number of IFN-γ and IL-4 secreting cells were analyzed by ELISPOT using 10μg/ml M2e peptide as a stimulus. Neither the mice immunized with Soc-3M2e soluble antigen nor those immunized with the Soc-3M2e+T4 mixture generated IFN-γ and IL-4 secreting cells, while the mice immunized with 3M2e-T4 nanoparticles generated significant numbers of M2e-specific IFN-γ ( Figure 4A ) and IL-4 secreting cells ( Figure 4B ). These data demonstrate that assembly of 3M2e on T4 nanoparticles is essential to stimulate the cellular arm of the host immune system. This seems to be not that critical for the humoral arm because, as shown above, substantial induction of antibodies was evident with the Soc-3M2e+T4 mixture ( Figures 2B – D ).

The mucosal surfaces of the respiratory tract are major ports of entry for influenza viruses, and previous studies indicated that both mucosal IgG and serum IgG are conducive for efficient protection (48). To determine if the 3M2e-T4 nanoparticles stimulated mucosal antibodies, bronchoalveolar lavage fluid (BALF) was collected 7 days post last immunization, and the presence of IgG and IgA antibodies were detected using ELISA. As shown in Figure 5A , soluble Soc-3M2e was able to induce low levels of M2e-specific IgG antibodies, whereas the 3M2e-T4 nanoparticles induced the highest levels. As in the case of serum IgG, the Soc-3M2e+T4 mixture could also induce mucosal IgG, much higher than the soluble antigen. As expected, the PBS control mice had no detectable levels of anti-M2e IgG antibodies ( Figure 5A ). Subtype analysis of the IgG antibodies showed that Soc-3M2e soluble antigens only elicited M2e-specific mucosal IgG1, whereas mice immunized with 3M2e-T4 nanoparticles or Soc-3M2e+T4 mixture induced balanced levels of both mucosal IgG1 and IgG2a antibodies ( Figures 5B, C ). However, all the groups failed to develop M2e-specific IgA antibodies in BALF ( Figure 5D ).

To evaluate the protective efficacy of each formulation, immunized mice were challenged with 5LD₅₀ of A/Puerto Rico/8/34 (H1N1) virus and monitored daily for body weight and survival for 14 days. As shown in Figure 6A , infection of influenza virus resulted in substantial weight loss of PBS control mice, or mice immunized with either the Soc-3M2e soluble antigen, or Soc-3M2e+T4 mixture, three days post infection. All the mice in the PBS control group died 10 days post challenge and five of the six mice immunized with the soluble Soc-3M2e died 9 days post challenge ( Figure 6B ), whereas 67% of the mice vaccinated with Soc-3M2e+T4 mixture recovered and survived ( Figure 6B ). In contrast, all the mice immunized with 3M2e-T4 nanoparticles not only survived the lethal challenges with the H1N1 virus infections ( Figure 6B ), but also, remarkably, showed no significant body weight loss ( Figure 6A ).

The protection efficacy was further evaluated by pathological analysis of the lungs of the immunized mice 5 days post-challenge. Figure 6C shows representative results of the lung lesions (column I) and pathological changes of alveoli (column II), bronchi (column III), and pulmonary vessels (column IV). Overall, mice immunized with Soc-3M2e+T4 mixture showed obvious, though less severe, lesions in the lungs when compared to the PBS control mice or mice immunized with soluble Soc-3M2e antigen, whereas no obvious lesions were found in 3M2e-T4 nanoparticle immunized mice (column I). The alveolar walls of PBS or soluble Soc-3M2e immunized mice were severely thickened (column II), and a large number of inflammatory cells infiltrated around the bronchi (column III) and pulmonary blood vessels (column IV). Mice immunized with Soc-3M2e+T4 mixture also exhibited similar but less severe pathological changes. However, in contrast, the mice vaccinated with 3M2e-T4 nanoparticles showed relatively normal alveolar wall thickness and negligible inflammatory infiltration ( Figure 6C ).

All animal experiments were approved by the Research Ethics Committee (HZAUMO-2021-0023), Huazhong Agricultural University, Hubei, China and performed in the Laboratory Animal Center of Huazhong Agricultural University strictly in accordance with the Guidelines for the Care and Use of Laboratory Animals, Huazhong Agricultural University.

The plasmid pET-RbSoc was constructed by inserting Soc gene of RB69 phage into pET28b expression vector using NheI and XhoI restriction sites. The 3M2e gene ( Table S1 ) encoding three tandem copies of M2e from human, swine, and avian influenza viruses separated by two flexible linkers (GGSSGGSS) was synthesized and cloned into pUC19 vector using SalI and XhoI restriction sites. The 3M2e gene fragment was cut from pUC19 plasmid using SalI and XhoI and subcloned into pET-RbSoc plasmid at the XhoI site to generate the expression plasmid pRbSoc-3M2e, in which the 3M2e gene was fused to COOH-terminus of RB69 Soc.

The pRbSoc-3M2e expression plasmid was transformed into E. coli BL21 (DE3) competent cells (Novagen), and a single colony was cultured overnight in LB medium supplemented with 50 µg/ml kanamycin. Ten ml of the overnight culture was inoculated into 1 L of fresh LB medium containing the antibiotic, and the expression of Soc-3M2e was induced with 1mM isopropyl-β-D-thiogalactoside (IPTG) at 30°C for 2 hours when the OD600 of culture reached 0.8. E. coli cells were collected by centrifugation at 4,300g for 15 min and resuspended with binding buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole, and 5 μg/ml DNase I). The cells were lysed by high-pressure cell disruptor at 4°C, and cell debris was removed by high-speed centrifugation (35,000g, 20 min, 4°C). The supernatant containing the recombinant Soc-3M2e proteins was passed through a 0.22 μm filter and loaded onto HisTrap column (Yeasen, Shanghai, China). After washing with 40 ml washing buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 20 mM imidazole), the Soc-3M2e protein was eluted with elution buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 400 mM imidazole). The peak fractions were collected and further purified by size-exclusion chromatography (Hi-load 16/60 Superdex 200 column, GE Healthcare Life Sciences) using gel filtration buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl). The purity of eluted Soc-3M2e protein was analyzed by SDS-PAGE, and protein concentration was determined with BSA as a standard. The endotoxin level present in purified Soc-3M2e protein was 0.05 EU/ml tested using Limulus Amebocyte Lysate (LAL) Endotoxin Quantitation Kit (Xiamen Bioendo Technology Co., Ltd, Xiamen China).

The propagation and purification of Hoc^-Soc^- phage T4 were carried out as previously described (44, 59, 60). Briefly, an overnight culture of E. coli P301 was inoculated into LB/M9CA medium and incubated at 37°C until the cell density reaches 1.5-2.0×10⁸ cells/ml. E. coli cells were then infected with Hoc^-Soc^- phage T4 at a multiplicity of infection (MOI) of 0.2-0.4, and cultured at 37°C for another 2-3 h. The cultures were harvested by centrifugation at 30,000g for 30 min, and the pellet containing phages was suspended in Pi-Mg buffer (26 mM Na₂HPO₄, 22 mM KH₂PO₄, 79 mM NaCl, 1 mM MgSO₄) containing chloroform and DNase I. After 20 min incubation at 37°C, the suspension was centrifuged at 4,300g for 20 min to remove cell debris, and phages in the supernatant were collected by high-speed centrifugation (30,000g, 30 min). The phage pellet was resuspended in 1 ml Pi-Mg buffer and purified by CsCl step density gradient centrifugation. Finally, the phages were dialyzed against dialysis solution I (10 mM Tris pH 8.0, 200 mM NaCl, and 5 mM MgCl₂) for 5 h followed by dialysis solution II (10 mM Tris pH 8.0, 50 mM NaCl, and 5 mM MgCl₂) for overnight at 4°C. The endotoxin level present in purified Hoc^-Soc^- phages was 1.53 EU/ml, which is well under the maximum recommended endotoxin levels, 20 EU/ml, in subunit vaccines (61).

In vitro assembly of proteins on Hoc^-Soc^- T4 phages was performed as described previously (26, 44). To optimize the binding of Soc-3M2e, about 5 × 10¹⁰ phage particles were incubated at 4°C for 45 min with Soc-3M2e proteins at different ratios of antigen molecules to Soc binding sites (1:1 to 30:1, Figure 1C ). The unbound Soc-3M2e proteins were removed by centrifugation at 21,130 g for 30 min, and the pellet of phage particles containing bound proteins were washed twice with PBS. The Soc-3M2e decorated phage particles were finally resuspended in PBS, transferred to a new tube, and analyzed by SDS-PAGE. The gp23* and Soc-3M2e proteins in each lane were quantified by Image-Pro Plus software using BSA as a standard. The copy number of bound Soc-3M2e proteins was determined using gp23* (49 kDa, 930 copies per capsid) as internal control. The saturation binding curve was generated using Prism GraphPad software as previously described (26, 27, 43). The y-axis showed the copy number at the different ratios, while x-axis represented the concentration of unbound Soc-3M2e. The apparent binding affinity (K_d) and the maximal number of bound molecules (B_max) were determined by nonlinear regression analysis with the methods of one site specific binding with a Hill slope. The diameter distribution and zeta-potential of the nanoparticles were determined using Zetasizer Nano ZS (Malvern Panalytical, UK).

Six- to eight-week-old female BALB/c mice were purchased from Laboratory Animal Center of Huazhong Agricultural University, Hubei, China. The 3M2e-T4 nanoparticles were prepared as described above and injected intramuscularly into mice (15μg 3M2e per dose) at week 0, 2, and 4. Each batch of sample was analyzed by SDS-PAGE for consistency in the copy number of displayed antigens before injection ( Figure S4 ). Mice immunized with the mixture of Soc-3M2e proteins (15μg 3M2e per dose) and T4 phages (9×10¹¹ particles) were used as controls (Soc-3M2e+ T4). To minimize the binding of Soc-3M2e, the protein was mixed with T4 phages right before each immunization. Other control groups included PBS, Soc-3M2e soluble proteins (15μg 3M2e per dose), and T4 phages (9×10¹¹ particles). Two weeks post the third immunization, mice (n=6) were anaesthetized with ether and intranasally infected with 5 LD₅₀ of influenza A/Puerto Rico/8/1934 virus. All mice were monitored daily for morbidity and mortality for 14 days. Animals with 30% or greater body weight loss were euthanized immediately and considered as death.

The levels of antigen-specific antibodies in sera and bronchoalveolar lavage fluid (BALF) were quantified by enzyme-linked immunosorbent assay (ELISA). Blood samples (n=6) were collected on days 0, 14, 28, and 35. BALF samples (n=5) were collected 7 days after the third immunization. Briefly, the lungs from sacrificed mice were flushed three times with 1ml PBS, which was then centrifugated at 3,500 g for 10 min. The supernatant was harvested for analysis of M2e-specific antibody titers. The ELISA plates were coated overnight at 4°C with 200 ng/well of M2e peptide pool consisting of equal amounts of peptides from human, swine, and avian influenza viruses or β-propiolactone-inactivated influenza A virus (1×10⁶ PFU/well) or Hoc^-Soc^- T4 phages (1×10⁹ PFU/well). The plates were blocked with 3% BSA in PBS-T (PBS containing 0.05% Tween-20) for 1 hour at 37°C. Serially diluted sera and BALF (1% BSA in PBS-T) were then added to each well of plates, which were incubated at 37°C for 1 hour. After washing 5 times with PBS-T, the plates were incubated with secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a, and IgA) at 37°C for 1 hour. Following five washes, 100 μl of TMB (3,3′,5,5′-tetramethylbenzidine) substrate was added to each well, and the reaction was stopped with 2 M H₂SO₄. The absorbance atOD450 was determined by a microplate reader.

About 2×10⁵ MDCK cells in 500 μl of growth medium (DMEM supplemented with 10% fetal bovine serum) were seeded into an each well of 24-well plate and allowed to adhere overnight. The growth medium was removed, and cells were washed twice with PBS and mock infected or infected with A/Puerto Rico/8/1934 virus in serum-free DMEM at a MOI of 1 for 1 h. The cells were then washed three times with PBS and cultured in serum-free DMEM containing 1 μg/ml TPCK-trypsin for 20h. After washing with PBS, cells were fixed with 10% formalin for 10 min, permeabilized with 0.1% Triton X-100 for 20 min, and blocked with 5% BSA in PBS-T (PBS containing 0.05% Tween-20) for 1 hour at 37 °C. The cells were then incubated with the sera from immunized mice at a dilution of 1:100 for 1h. After 5 times wash with PBS-T, Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific) were used as secondary antibodies at a dilution of 1:1000. Following five washes, nuclei were counterstained with 1 μg/ml DAPI (BD Biosciences) for 5 min in the dark. Photography was performed on an inverted fluorescence microscope (Thermo Fisher Scientific).

ELISPOT assay was performed to determine the number of M2e-specific IFN-γ and IL-4 secreting cells in spleen according to the manufacturer’s protocol (DAKAWE, China). Briefly, 7 days post the last immunization, mice were sacrificed and the spleens were harvested to prepare single-cell suspensions. Around 3×10⁵ splenocytes were seeded to each well of plates and stimulated with M2e peptides (equal amounts of M2e peptide from human, swine, and avian influenza viruses) at a final concentration of 10 μg/ml. After 32-34 hours of culture at 37°C, 5% CO₂, the splenocytes were removed by cell-cracking buffer of ice-cold deionized water. The plates were then incubated with biotinylated antibodies followed the addition of HRP-conjugated streptavidin. After washing 5 times, the reaction was developed with AEC (3-amino-9-ethylcarbazole) substrate and stopped with flowing water. Plates were dried at room temperature and spot-counted (DAKAWE, Wuhan, China).

Mice (n=3) were immunized and challenged as described above. Five days after challenge with 5 LD₅₀ of influenza A/PR/8/1934 virus, mice were sacrificed, and lungs were isolated. Lung tissues were fixed in 10% formalin, dehydrated through a graded series of ethanol, embedded in paraffin wax, and cut into 4 µm-thick sections. After deparaffinization, sections were stained with hematoxylin-eosin and observed under an optical microscope (Nikon, Japan).

All the statistical analyses were performed using GraphPad Prism software. Comparisons among different groups were evaluated by one-way ANOVA. In all cases, p< 0.05 was considered as statistically significant difference.