Development and characterization of influenza M2 ectodomain and/or HA stalk-based DC-targeting vaccines for different influenza infections

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein (EboGP) DC-targeting domain (EΔM) fusion protein technology. We fused influenza hemagglutinin stalk (HAcs) and extracellular matrix protein (M2e) or four copies of M2e (referred to as tetra M2e (tM2e)) with EΔM to generate EΔM-HM2e or EΔM-tM2e, respectively, and revealed that EΔM facilitates DC/macrophage targeting in vitro. In a mouse study, EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles (PVPs) induced significantly higher titers of anti-HA and/or anti-M2e antibodies. We also developed recombinant vesicular stomatitis virus (rVSV)-EΔM-HM2e and rVSV-EΔM-tM2e vaccines that resulted in rapid and potent induction of HA and/or M2 antibodies in mouse sera and mucosa. Importantly, vaccination protects mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that recombinant rVSV-EΔM-HM2e and rVSV-EΔM-tM2e are efficacious and protective universal vaccines against influenza.

Influenza is a highly contagious airborne disease that attacks the respiratory system and occurs in seasonal epidemics and pandemics. The influenza pandemic in 1918 killed approximately 50 million people globally 1,2 , and to date, influenza virus infection is still posing a substantial threat to the health sector worldwide 3 . Circulating influenza vaccines are associated with some issues, including the level of effectiveness of annual vaccines protecting against the specific influenza strain in the particular seasonal epidemic and the psychological effect on the population who must receive a flu shot every year for their lifetime. Based on these findings, the Centers for Disease Control (CDC), as part of their recent recommendations, emphasizes a need for a universal vaccine against influenza viral infection 4 .
The universal vaccine is characterized by the ability to protect individuals from different strains of the influenza virus. In addition to the four different types of influenza, each type is composed of a population of different strains. Variation in influenza strains occurs at hemagglutinin (HA) and neuraminidase (NA). For instance, influenza A, which is the most prominent family, has eighteen known HA subtypes and eleven identified NA subtypes with different host ranges, including humans, birds, bats, and swine 4,5 . The difficulty in producing a universal vaccine against the influenza virus is due to the antigenic shift or antigenic drift caused by reassortment or mutation of HA or NA. These mechanisms allow the influenza virus to continuously escape host immune defenses 6 . Additionally, the current vaccine in circulation is short-lived and narrow 7 . Therefore, as a method to develop a universal vaccine, the conserved components on the surface protein of influenza could be utilized to elicit broad immune responses specific for all the present and future strains of the influenza virus.
The influenza HA protein, which is responsible for the cell attachment and entry of the viruses, is a promising epitope in the development of influenza vaccines. Although the globular head of HA induces the production of neutralizing antibodies 8,9 , it is difficult to use when developing a universal vaccine due to the large number of HA subtype variations. However, the HA stalk and the highly conserved extracellular matrix protein (M2e) have been found to be promising in the development of a universal vaccine for influenza viral infection due to their durability and stability 10,11 These subunit proteins of influenza induce broader neutralization and participate in either antibody-dependent cellular phagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC), which subsequently eliminate influenza virus or destroy the cells already infected 12,13 . Numerous studies have used different approaches to develop universal influenza vaccines, including fusion of influenza M2e polypeptides 14 , targeting conserved broadly reactive epitopes on the HA stalk 15 , developing fusion proteins between influenza M2e and bacterial flagellin 16 , expressing recombinant HA in virus-like particles 17 and using VSV to deliver HA antigens 18 . However, although some of these approaches are being investigated in clinical trials 19 , varying limitations still exist, with the majority having relatively low immune responses, except for VSV that is used with adjuvants such as MF59 and ASO3 6 . Therefore, new and efficient universal vaccine(s) with broad protection against various strains of the influenza virus must be developed.
The dendritic cell (DC)-targeting vaccine has recently received global attention since this approach is effective because DCs function as antigen-presenting cells (APCs) that stimulate adaptive immune responses and regulate innate immune responses 20 21 . The usage of this technology is in the pipeline for the development of various vaccines against viral pathogens and cancers 20 21 22 . A study showed that targeting influenza HA and chemokine receptor Xcr1 + to DCs induces immune responses and confers protection against the influenza virus 23 . A group of scientists also targeted influenza M2e to DCs by fusing M2e with anti-Clec9 24 , while HA of influenza was infused with an artificial adjuvant vector cell targeting DCs to induce CD4 + T cells and CD4 + Tfh cells 25 .
Ebola virus glycoprotein (EboGP) is the viral protein expressed on the Ebola virus (EBOV) surface that preferentially binds to DCs, monocytes and macrophages 26,27 . As shown in our recent study, the incorporation of EboGP into HIV PVPs indeed facilitates DC and macrophage targeting and significantly enhances HIV-specific immune responses 28,29 . These observations indicated that EboGP has the potential to direct an HIV antigen toward DCs to facilitate effective anti-HIV immune responses. Notably, a highly glycosylated mucin-like domain (MLD) encompassing residues 313 to 501 is located at the apex and the sides of each EboGP monomer 30 . However, some studies have shown that removing this MLD region does not impede EboGP-mediated lentiviral vector entry 31 and was dispensable for EBOV infections in vitro 32,33 . Our laboratory has recently developed the EboGP mucin-like domain replacement system and shown the great potential of this vaccine technology to deliver heterologous polypeptides in vivo and stimulate innate and adaptive immune responses 34 .
In this study, we used this EboGP DC-targeting domain-based fusion protein technology to fuse conserved HA stalk regions (HAcs) and M2e or four copies of M2e (referred to as tetra M2e (tM2e)) within the MLD-deleted EboGP (EboGPΔM). By incorporating these fusion proteins into HIV-based pseudotyped viral particles (PVPs) or a recombinant vesicular stomatitis virus (rVSV) vector, we characterized their DC-targeting ability and investigated their potential for eliciting host immune responses and their abilities to protect against H1N1 and H3N2 influenza infections.

Construction and characterization of EΔM-tM2e and EΔM-HM2e chimeric fusion proteins
The HAcs and M2e proteins of the influenza virus are highly conserved among the strains of the influenza virus, and previous studies have attempted to develop HAcs-or M2e-based universal vaccines for influenza virus 8,9,10,11,35 . We used the EboGPΔM Replacement System 34 to insert 4 copies of M2e (tM2e) (aa 92) into EΔM and form the EΔM-tM2e fusion protein as a method to improve the DC-targeting ability of HAcs-or M2e-based vaccines (Fig. 1A, C). The four copies of influenza M2e consisted of M2e from human (2 copies), swine (1 copy) and bird (1 copy) strains and were linked with a GGGS linker (Fig. 1A). Additionally, we combined a copy of M2e from the human influenza strain with the HAcs from influenza H5N1 using a GSA linker (HM2e) (aa 179) and inserted it into EΔM to form EΔM-HM2e as a fusion protein (Fig.   1B, C).
We first produced PVPs that incorporated the EΔM-tM2e or EΔM-HM2e fusion protein and investigated their abilities into enter DCs/macrophages. Briefly, EΔM-tM2e-or EΔM-HM2e-expressing plasmids were cotransfected with an HIV Gag-Pol packaging plasmid Δ8.2 and a multigene (reverse transcriptase/integrase/envelope)-deleted HIV vector (ΔRI/ΔE/Gluc) in HEK-293T cells as previously described 28,36 . In ΔRI/ΔE/Gluc, the nef gene was replaced with a Gaussia luciferase (Gluc) gene that was expressed and released when the virus entered cells. and PVPs was also detected using a rabbit anti-HIV p24 antibody ( Fig. 1D and E). Overall, these results indicated that EΔM-HM2e and EΔM-tM2e fusion proteins were efficiently expressed and incorporated into PVPs.

The ability of EΔM-HM2e-or EΔM-tM2e-PVPs to enter macrophages and dendritic cells (DCs)
EboGP has been shown to play a critical role during the infection of DCs and macrophages by facilitating viral attachment, fusion, and entry 28,38,39 . Therefore, we investigated whether the fusion of influenza-conserved HM2e or tM2e with EΔM would impede or affect the ability of EΔM to enter DCs and macrophages. Briefly, we infected human monocyte-derived macrophages (MDMs) or monocyte-derived DCs (MDDCs) with equal amounts (adjusted with HIV p24 levels) of EΔM-tM2e-or EΔM-HM2e-pseudotyped Gluc + PVPs. In parallel, the EΔM-pseudotyped Gluc + PVPs were used as controls. On different days after infection, the ability of the PVPs to enter cells was monitored by detecting the Gluc activity in the supernatant of infected cells. As expected, EΔM-tM2e-and EΔM-HM-PVPs displayed more efficient entry than EΔM-PVPs (Fig. 1 F and G), suggesting that the fusion of influenza HAcs and/or M2e with EΔM did not affect the cell entry efficiency. Overall, the fusion of HM2e or tM2e to EΔM still maintains the DC-and macrophage-targeting ability of PVPs.

EΔM-HM2e-or EΔM-tM2e-PVP immunization induced significantly higher titers of specific anti-influenza HA and M2 antibodies in mice
Since EΔM-HM2e or EΔM-tM2e PVPs significantly target DCs and macrophages, we next investigated whether EΔM-HM2e or EΔM-tM2e PVPs strongly stimulated influenza HA and M2e immune responses in vivo. For this experiment, we subcutaneously immunized Balb/c mice with equal amounts (100 ng HIV p24) of EΔM-HM2e-, EΔM-tM2e-PVPs, or native HA/NA/M2-PVPs. The body weight of all immunized mice was monitored at 0, 28 and 56 days, and no statistically significant differences were observed ( Fig. 2A and B). On Day 63 postimmunization, sera from mice were collected as described in the Materials and Methods, and the anti-M2e and anti-HA specific humoral responses were determined using ELISAs in plates coated with M2e peptides or recombinant HA from H5N1. Influenza M2e-specific humoral immune responses were detected in mice injected with EΔM-tM2e, EΔM-HM2e, and native HA/NA/M2-PVPs (Fig.2C). Interestingly, our results revealed that EΔM-tM2e PVP immunization elicited robust production of influenza M2e-specific antibodies. Additionally, antibody titers induced in the mice immunized with EΔM-HM2e-PVPs were significantly higher than those induced by native HA/NA/M2-PVPs on Day 63 (Fig. 2C). Meanwhile, immunization with EΔM-HM2e-PVPs induced significantly higher anti-HA humoral responses than immunization with HA/NA/M2-PVPs (Fig. 2D). Collectively, these results indicate that EΔM-tM2e-PVP and EΔM-HM2e-PVP immunization resulted in significantly stronger specific humoral antibodies against influenza M2e and/or HA.
Since both EΔM-tM2e-PVPs and EΔM-HM2e-PVPs contain the HIV-1 Gag protein and EbolaGP, we therefore tested the anti-EbolaGP-and anti-HIV P24-specific humoral immune responses in immunized mice. As expected, we only observed a significantly higher anti-EbolaGP antibody titer in the sera of mice immunized with EΔM-tM2e-and EΔM-HM2e-PVPs We therefore further investigated whether the expression of EΔM-tM2e or EΔM-HM2e in the rVSV vector would induce strong immune responses against the influenza virus.

Construction and characterization of rVSV expressing the EΔM-tM2e or EΔM-HM2e chimeric protein.
The rVSV vector represents a safe and potent vaccine development platform for stimulating both innate and adaptive immune responses 41,42,43 . In this study, we further investigated whether the expression of EΔM-tM2e or EΔM-HM2e in the rVSV vectors also induced the production of anti-M2e and anti-HA antibodies, respectively, in vivo. For this purpose, we inserted the genes encoded by EΔM-tM2e and EΔM-HM2e into an rVSV vector to the position where the VSV-G gene sequence was located (Fig. 3A). Then, the attenuated replicating rVSV expressing either EΔM-tM2e or EΔM-HM2e, named rVSV/EΔM-tM2e or rVSV/EΔM-HM2e, was rescued in VeroE6 cells via reverse genetics technology 44   The anti-HA (including H1, H3 and H5) antibody titers in rVSV-EΔM-HM2e-immunized mice were also evaluated. The results revealed that rVSV-EΔM-HM2e immunization induced robust anti-HA-specific IgG against recombinant HA derived from H1N1, H3N2 and H5N1 (Fig.   4E). Meanwhile, analyses of IgG subsets revealed that rVSV-EΔM-HM2e induced similar levels of anti-HA IgG1 against H1, H3 and H5 but lower levels of Ig2a against H3 and H5 than those against H1 (Fig. 4F). Taken together, rVSV-EΔM-M2e or rVSV-EΔM-HM2e immunization induced robust production of specific anti-M2e and/or anti-HA IgG antibodies in mice.
As shown in previous studies, higher serological and mucosal IgA responses are associated with a better flu disease prognosis and decreased influenza transmission 46,47,48,49 .
These results indicated the presence of M2e and HA IgA antibodies in the respiratory tracts of the immunized mice.
Finally, we evaluated the cell-mediated immune responses in immunized mice. For this experiment, splenocytes isolated from rVSV-EΔM-tM2e-or rVSV-EΔM-HM2e-immunized mice were cultured and stimulated with M2e peptides or HA peptides, and the released cytokines and chemokines were quantified using an MSD V-plex mouse cytokine kit, as described in the Materials and Methods. Our results (Fig. 5. E, F) revealed that both rVSV-EΔM-tM2e-and rVSV-EΔM-HM2e-immunized mouse splenocytes expressed significantly higher levels of interferon (IFN)-γ and interleukin (IL)-2, which are involved predominantly in the cellular immune response. Meanwhile, a moderate increase in IL-4 levels and abundant production of IL-5 that may be linked to tissue protection were detected. Immunization using the two vaccine candidates also resulted in the production of macrophage inflammatory protein-1 (MIP-1α).
We then increased the challenge dose of H1N1 to 1.4X10 3 PFU. The mice vaccinated with PBS experienced morbidity after challenge, as evidenced by a 15 to 30% loss of body mass (Fig. 6B).

Discussion
Despite the progress achieved in developing a universal vaccine for influenza viral infection, an FDA-approved universal vaccine for influenza viral infection is still unavailable. In this study, we generated PVP and rVSV-based vaccine candidates expressing the ectodomain of influenza matrix protein (M2e) and/or hemagglutinin stalk regions (HAcs) that were fused with the DC-targeting domain of EboGP (EΔM) and revealed their abilities to efficiently elicit antiinfluenza immune responses and protect against different strains of influenza virus.
Recent advances in influenza virus vaccine research have shown that targeting the highly conserved epitopes in the HA stalk domain or M2e of influenza virus is a promising approach for developing a universal vaccine 13 50, 51, 52 . However, methods to enhance the antigenicity of these large polypeptides are still an important issue to be addressed. According to our recent study, the replacement of the mucin-like domain (MLD) of EboGP with heterologous large polypeptides still maintains the ability of EboGP to target human macrophages/dendritic cells (DCs) and induce robust immune responses against the inserted polypeptides 53 . Therefore, in this study, we fused four copies of M2e from human (2 copies), avian (one copy) and swine (one copy) influenza strains with EΔM or M2e (from human influenza virus) and influenza HAcs derived from H5N1 (aa 156) 54 with EΔM to generate EΔM-tM2e or EΔM-HM2e fusion proteins, respectively ( Fig. 1A-C), with increased immunogenicity. Because the mucin-like domain is located at the apex of the sides of the EboGP monomer 34, 55 , the inserted heterologous polypeptides, such as HM2e and tM2e, are also expected to be exposed to the apex of the sides of the EΔM-HM2e and EΔM-M2e fusion proteins and may not interfere with the cell targeting and entry of EΔM. Indeed, our study showed that EΔM-HM2e-and EΔM-tM2e-PVPs exhibited a strong ability to target and enter DCs and macrophages compared to native HA/NA/M2-PVPs ( Fig. 1F-G). This finding also correlated with our recently published data revealing that the incorporation of the EΔM-V3 fusion protein or EboGP into HIV Env pseudotyped VLPs facilitated DC targeting compared to HIV-Env PVPs, indicating that EΔM accommodates large polypeptides without losing its DC and macrophage targeting ability 28,53 .
As the presence of EboGP or EΔM-V3 fusing protein in either PVP-or rVSV-based vaccine candidates substantially enhances their immunogenicity 28, 34 , we also tested whether EΔM-M2e or EΔM-HM2e-PVPs displayed strong immunogenicity. For this experiment, the mice were immunized intramuscularly with EΔM-M2e-or EΔM-HM2e-PVPs, and the results clearly showed that the anti-M2e and/or anti-HA antibody titers in EΔM-tM2e-or EΔM-HM2e-PVP-immunized mice were significantly higher than those in HA/NA/M2-PVP-immunized mice ( Fig. 2C-E). The anti-p24 antibody titer in EΔM-tM2e-PVP-or EΔM-HM2e-PVP-immunized mice was also higher than that in HA/NA/M2-PVP-immunized mice (Fig. 2F). Moreover, our results clearly showed that EΔM-M2e-PVPs resulted in remarkably higher anti-M2 antibody levels that exceeded the anti-M2 antibody titer produced following EΔM-HM2e-PVP immunization ( Fig. 2C and D). Overall, the aforementioned observations provide evidence to support our hypothesis that the presence of EΔM-tM2e or EΔM-HM2e on the surface of PVPs induces efficient antibody responses to M2 and HA. The rVSV vector system as a vaccine platform has attracted global attention as a vaccine delivery system for multiple viral proteins that induces strong humoral and cell-mediated immune responses against viral proteins 53, 56, 57, 58 59 . Interestingly, a recent study showed that rVSV expressing HA stalk as an influenza vaccine confers rapid protection against different H5 influenza strains 18 . In this study, we generated rVSVs encoding EΔM-tM2e or EΔM-HM2e in place of VSV-G and found that the replication of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e was significantly attenuated compared with VSVwt (Fig. D). This observation is important for the safety of rVSV-based vaccines. In the animal study, a single dose of rVSV immunization induced relatively high levels of anti-M2e and anti-HA antibody responses on Day 20. Following the booster immunization, both rVSV-EΔM-tM2e and rVSV-EΔM-HM2e induced robust antiinfluenza HA and M2e IgG production, including IgG2a and IgG1, and significantly higher levels of anti-influenza IgA antibodies in both the sera and nasal mucosa of mice (Figs. 4 and 5).
Furthermore, the splenocytes of rVSV-EΔM-tM2e-and rVSV-EΔM-HM2e-immunized mice released large amounts of MIP-1α, IL-2, IL-5, IL-6, IL-10 and IFN-γ upon stimulation with M2e peptide or HA peptide, indicating that immunization with these rVSV vaccine candidates also induced significantly higher levels of cell-mediated immunity (Fig. 5E, F). Unfortunately, we still do not know whether these vaccine candidates might induce stronger cell-mediated immunity than the wild-type influenza HA or M2 protein due to a lack of rVSV expressing the wild-type influenza HA or M2 protein in our experimental system.
Another interesting observation was that rVSV-EΔM-tM2e elucidated a remarkably higher level of the anti-M2e antibody than rVSV-EΔM-HM2e (Fig. 4). This result was consistent with the data from EΔM-tM2e-PVPs (Fig. 2C). M2e has low immunogenicity due to its small size (aa 24) and small number of copies in the virus. We increased its immunogenicity by creating PVPs or rVSV containing four M2e tandem repeats that increase the M2e copy number on the surface of viral particles and/or the cells. This strategy resulted in significant increases in the production of M2e-specific antibodies and cell-mediated immune response. The intranasal and intramuscular immunizations of rVSV-EΔM-tM2e provided 100% protection against both H1N1 and H3N2 challenges (Fig. 6). Thus, using EboGP DC-targeting domain (EΔM) fusion protein technology 34 , we can insert multiple copies of large polypeptides in EΔM to induce significantly stronger immune responses against targeted polypeptides. Additionally, this study provides evidence for the efficacy of the rVSV-EΔM-tM2e vaccine candidate and for M2e-based vaccines as a universal vaccine approach.
Our study also showed that rVSV-EΔM-HM2e induced high levels of antibody responses to HA from different influenza strains, including H1N1, H3N2 and H5N1 (Fig. 4E and Fig. 5B), indicating its broad anti-HA responses. The rVSV-HAcs-M2e vaccine immunization also provided 100% protection against challenge with a lethal dose of H3N1 and partial (25%) protection against lethal H1N1 challenge (Fig. 6). Based on these results, the rVSV-tM2e vaccine candidate appears to provide broader or more efficient protection than rVSV-HM2e. A universal influenza vaccine should be effective against all influenza viruses, regardless of any antigenic mutation or HA/NA subtypes. Further studies are required to investigate the protective effects of these candidate vaccines on other strains of influenza virus and on nonhuman primates.
However, the results presented in this study have provided strong evidence for a universal influenza vaccine platform.
Overall, this study is the first to show that infusion of the influenza HA stalk region and/or conserved M2e with EΔM elicited robust influenza immune responses and protected against different strains of influenza virus. The novelty of this study is the use of EboGP-based DC-targeting domain (EΔM) fusion protein technology to present the conserved antigens of influenza virus (HA stalk regions and conserved M2e region) and induce a stronger immune response against influenza virus infections. This study also provides convincing evidence for the EboGP-based DC-targeting domain (EΔM) fusion technology that can be broadly used to develop vaccine strategies that protect against other emergent and re-emergent infectious diseases.

Production and characterization of PVPs or rVSV containing EΔM-tM2e and EΔM-HM2e
To produce EΔM-tM2e or EΔM-HM2e pseudotyped viral particles (PVPs) for in vitro study of viral entry, 293T cells were co-transfected pCAGGS-EΔM-tM2e or pCAGGS-EΔM- The Gaussia luciferase (GLuc) assay was done at various times of infection by collecting supernatants from the cell cultures. A 10µl of sample was mixed with a 50µl portion of GAR-1 reagent (targeting Systems) and then measured in the luminometer (Promega, USA) 36 .

Immunofluorescence assay
As previously described 45

Mice immunization experiment
Female BALB/c mice aged 4-6 weeks used in this study were obtained from the Central Animal Care Facility, the University of Manitoba (with the animal study protocol approval No. For influenza viral challenge in mice, the mouse-adapted strain of A/Puerto Rico/8/34 (H1N1) or H3N2 virus strains were used. Each group of mice (5 for each group) were intramuscularly immunized with 1x10 7 TCID 50 of rVSV-EΔM-tM2e, rVSV-EΔM-HM2e or PBS and boosted with 5x10 6 TCID 50 of VSV or PBS on day 21. Meanwhile, One group of mice were intranasal immunization with 1x10 6 TCID 50 of EΔM-tM2e and was boosted with 1x10 3 TCID 50 on day 21. After two weeks of the final immunization, the challenge was done by infecting the mice intranasally with 2.1X10 3 PFU or 7X10 2 PFU H1N1, or 1.4X10 4 PFU of H3N2, while mice injected with PBS were challenged as a negative control. Weight loss or gain of the mice were monitored daily for 2 weeks after the challenge.

Assay (ELISA)
To determine influenza HA and M2 specific antibodies in mice sera, ELISA plates (NUNC Maxisorp, Thermo Scientific) were coated with 100 µl of M2e peptide or rHA (H1, H3 or H5) proteins (1µg or 0.5 µg/ml respectively) in a coupling buffer (0.05M carbonate-bicarbonate of pH 9.6) overnight at 4˚C. To measure the HIV Gag-specific or EboGP-specific antibody in sera, the plate was coated with HIV-1 IIIB p24 recombinant protein (0.5µg/ml) or recombinant glycoprotein (0.5µg/ml) as described previously 53 . The plates were washed twice after the incubation with 1X PBST and blocked with blocking buffer (1% BSA in PBS) at 37˚C for 1 hr.
The serum samples were diluted with primary antibody diluent (1:100-1:10 9 ), then 100µl of the diluted mouse serum samples were added into each well of the plates and incubated for 2 hrs at 37˚C. Dishes were immediately washed five times, and 100 µl peroxidase-conjugated goat antimouse immunoglobulin G (IgG) (GE Healthcare), IgG1, IgG2a or IgA secondary antibodies were added and incubated for 1 h at 37˚C. The plates were washed five times, and 3',3',5',5' Tetramethylbenzidine (TMB) (Mandel Scientific) was added and incubated for 15 min at room temperature in a dark room. To stop the reaction, 100 µl of 1N HCl was added to each well, and the absorbance was measured at 450nm optical density 18 .

Cytokine detection
Splenocytes from immunized mice have collected aseptically and placed into the cell strainer and were mashed through the cell strainer inside a sterile 50 ml tube using the plunger end of the syringe to make single-cell suspensions. Red blood cells were removed using Ammonium-Chloride-Potassium (ACK) buffer, and the suspended cell was cultured in 48-well plates at a density of 2x10 6 /125µl with DMEM containing either M2 or HA peptide (1 or 2 µg/peptide/ml) respectively. After 3 days of culturing, supernatants were collected and stored at -70˚C for a cytokine detection assay. Cytokine (IFN-g, IL-2, IL-4, IL-5, and MIP-1a) levels were measured in supernatants using the 8-plex mouse MSD V-plex kit (Mesoscale Discovery, USA) following the manufacturer procedure.

Statistics
Statistical analysis of cytokine levels, including the results of GLuc assay, influenza M2, HA and ELISA, and various cytokine/chemokines, were performed using the unpaired t-test (considered significant at P≥0.05) by GraphPad Prism 5.01 software.    Statistical significance between the two groups was determined using an unpaired t-test, and significant p values are represented with asterisks, *≤0.05, **≤0.01. No significance (ns) was not shown.