Protection against influenza A virus by vaccination with a recombinant fusion protein linking influenza M2e to human serum albumin (HSA)

Accepted Manuscript Title: Protection against influenza A virus by vaccination with a recombinant fusion protein linking influenza M2e to human serum albumin (HSA) Author: Xupeng Mu Kebang Hu Mohan Shen Ning Kong Changhao Fu Weiqun Yan Anhui Wei PII: DOI: Reference:

S0166-0934(15)00377-8 http://dx.doi.org/doi:10.1016/j.jviromet.2015.11.014 VIRMET 12915

To appear in:

Journal of Virological Methods

Received date: Revised date: Accepted date:

25-11-2014 18-11-2015 19-11-2015

Please cite this article as: Mu, X., Hu, K., Shen, M., Kong, N., Yan, C.F., ,Weiqun, Wei, A.,Protection against influenza A virus by vaccination with a recombinant fusion protein linking influenza M2e to human serum albumin (HSA), Journal of Virological Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights

1. A novel vaccine that link M2e of influenza A viruses with HSA was constructed. 2. HSA/M2e could induce strong anti-M2e specific humoral immune responses. 3. HSA/M2e was able to reduce viral load in the mice lungs and protect mice against

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challenge with influenza A virus.

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Protection against influenza A virus by vaccination with a recombinant fusion protein linking influenza M2e to human serum albumin (HSA)

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Xupeng Mua, Kebang Hub, Mohan Shenc, Ning Kongd, Changhao Fud,Weiqun Yand, Anhui Weid,*[email protected] a

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Department of Central Laboratory, China-Japan Union Hospital, Jilin University, Changchun, China b Department of Urology, The first Hospital, Jilin University, Changchun, China c Department of Infectious Disease, The Second Affiliated Hospital, China Medical University, Shenyang, China d Department of Biological Engineering, College of Pharmacy, Jilin University, Changchun, China *Corresponding author. Fax: +86 431 85650627

Abstract

The highly conserved extracellular domain of M2 protein (M2e) of influenza A viruses has limited immunogenicity on its own. Hence, aiming to enhance immunogenicity of M2e protein, optimal approaches remain to be established. In this study, we created recombinant fusion protein vaccines by linking M2e consensus sequence of influenza A viruses with C-terminal domain of human serum albumin (HSA). Then HSA/M2e recombinant fusion protein was studied. Our results showed that HSA/M2e could induce strong anti-M2e specific humoral immune responses in the established mice model. Administration of HSA/M2e with Freund’s adjuvant resulted in a higher number of IFN-γ-producing cells compared to HSA/M2e or M2e peptide emulsified in Freund’s adjuvant. Furthermore, HSA/M2e was able to reduce viral load in the mice lungs and provide significant protection against lethal challenge with an H1N1 or an H3N2 virus compared to controls. In conclusion, this study has demonstrated a potential vaccine that could provide protection in preventing the threat of influenza outbreak because of rapid variation of the influenza virus.

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Keywords: Influenza A virus; M2e; HSA; Immune protection; Vaccine

1. Introduction

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Influenza virus is the pathogen of influenza, which is a highly contagious virus and has a high lethality rate among all of the infectious diseases. Vaccination is the best way to prevent influenza infection. All current commercial human influenza vaccines contain hemagglutinin (HA) and neuraminidase (NA) as their main viral antigens (Fiers et al., 2004). Although conventional influenza vaccines can offer protection, they must be formulated every year to match the prevailing seasonal viral strains. The antibodies induced by vaccination or exposure to previously circulating virus do not efficiently prevent infection by newly emerged influenza virus strains. To overcome the threat of influenza virus a universal vaccine which could protect against all influenza virus strains and provide long-lasting immunity will be an attractive solution. In order to achieve the broad-spectrum immunity, an HA DNA vaccine together with other influenza antigens NA, NP, and M2 were co-administered. The study found that co-administering certain antigens could offer better or comparable protection to HA alone, however, combining additional antigens may lead to unfavourable immune responses (Patel et al., 2012). Next to HA and NA, the M2 protein is also a transmembrane protein of the influenza A virus that is expressed at the plasma membrane of influenza virus-infected cells. The extracellular domain of the influenza M2 protein (M2e) is conserved in all known human influenza strains, except A/PR/8/34, A/Brevig Mission/1/18 and A/Fort Monmouth/1/47 (Ito et al., 1991; Reid et al., 2002). Studies showed that anti-M2e mAb could restrict virus growth in vitro and reduce the spread of different influenza virus strains (Zebedee et al., 1988). In particular, the N-terminus of M2e can induce inhibitory antibodies against influenza virus replication in vitro (Liu et al., 2003). However, M2 protein alone can only induce lower protective immunity against virus infection compared with other viral antigens, including HA, NA (Patel et al., 2009). Reports have also confirmed that anti-M2e-specific antibody responses following influenza A virus infection were weak and indicated that humans currently lack optimal M2e-specific protection (Zhong et al., 2014; Feng et al., 2006; Black et al., 1993). Carrier molecules, such as hepatitis B virus core (Neirynck et al., 1999; Filette et al., 2006), T7 bacteriophage nanoparticles (Hashemi et al., 2012), rotavirus fragment NSP4 protein (Andersson et al., 2012), Brucella abortus lumazine synthase protein (BLS) (Alvarez et al., 2013), tuftsin (Liu et al., 2012), have been used to increase the immunogenicity of the M2e. Meanwhile, potent adjuvants have also been used to enhance the protective immune response, except from those that used Freund’s adjuvant, AS04 or cholera toxin (CT) (Fu et al., 2009; Lee et al., 2014; Mozdzanowska et al., 2007). Importantly, several studies have demonstrated that influenza vaccines based on M2e showed effective and broad spectrum protective immunity-potentially making this one of the important target genes for influenza vaccines (Neirynck et al., 1999; Filette et al., 2006). Human serum albumin (HSA) is the major component of serum, which is involved in 2

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maintenance of osmolarity and plasma volume. It is a natural carrier involved in the endogenous transport and delivery of various natural as well as therapeutic molecules, and has a long half-life in human beings. After fusing with HSA, the fusion protein can reduce its bioavailability and prolong its half-time in vivo to boost therapeutic effect. HSA may be used as a suitable carrier for M2e as well as to boost the immunogenicity of M2e peptide (Chuang et al., 2002). In previous reports, rabbit antibodies induced by M2e-BSA conjugates could inhibit influenza virus replication in vitro (Liu et al., 2003). Meanwhile, HSA fusion protein could achive high-level expression in Pichia pastoris and hybrid protein was less in culture supernatant so it was easy to be purified. Reports have shown that M2e could only induce low immunologic reaction due likely to its small molecular weight (Lamb et al., 1985). In this study, we used HSA as a carrier protein to present the M2e peptide to the immune system of mice. Our results confirmed that the M2e vaccine could exhibit excellent immunogenicity and protect mice effectively against challenge with influenza A virus.

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2. Materials and Methods 2.1. Viruses, mice and peptide

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Influenza viruses used in this study included a mouse-adapted influenza A/New Caledonia/20/1999 (H1N1) virus and A/Wisconsin/67/2005 (H3N2) virus. The influenza viruses were propagated in 10 day-old embryonated chicken eggs and adapted in mice as described previously (Quan et al., 2010). These viruses were frozen at -80 ℃ until use. Specific-pathogen-free female BALB/c mice, aged 6-8 weeks, were purchased from the Animal Resource Center of Norman Bethune College of Medicine, Jilin University, China. The peptide corresponding to influenza A virus M2e (SLLTEVETPIRNEWGCRCNGSSD) was synthesized at Sangon Biotech (China). All animal experiments were approved by the Animal Welfare and Research Ethics Committee of Jilin University. Mice were housed in a temperature-controlled room with proper darkness-light cycles and fed with a regular diet. All efforts were made to minimize suffering. 2.2. Expression and purification of recombinant HSA/M2e fusion protein

Construction of plasmid pPICZα-HSA/M2e as well as expression and coupling procedure were performed as previously described (Mu et al., 2010). Briefly, the plasmid pPICZα-HSA/M2e was transformed into Pichia pastoris. The pH of the fermentation supernatant was adjusted to 3.0 with 5 M hydrochloric acid and was diluted with an equal volume of 200 mmol/L NaAc-HAc (pH 3.0). This was then loaded onto the SP Sepharose FF column which was equilibrated with 20 mmol/L NaAc-HAc (pH 3.0) buffer. The bound protein was eluted with a linear salt gradient (0.5-1 M NaCl) and was monitored by measuring the absorbance at 280 nm. The column effluent containing HSA/M2e was ultrafiltrated to remove the salt and concentrated, the concentrate was then diluted with an equal volume of 200 mmol/L Tri-HCl (pH 8.0). This was then loaded onto Q Sepharose FF column equilibrated with 20 mmol/L Tri-HCl (pH 8.0) buffer for further purification. The column was eluted with a linear salt gradient (0-0.5 M NaCl). The elution fractions containing 3

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HSA/M2e were dialyzed and filtrated through 0.22 µm filters. The final purified HSA/M2e solution was stored under sterile conditions at -80 ℃. 2.3. Analysis of recombinant HSA/M2e fusion protein

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SDS-PAGE analysis was performed using a 10% gel according to the method of Laemmli (Laemmli, 1970). For Western blotting, the proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked overnight with 5% skim milk in PBS containing 0.1% Tween-20 (PBST) at 4 ℃, followed by incubation with mouse anti-M2e monoclonal antibody [14C2] (Abcam) for 3 h at room temperature. After washing five times, the membrane was incubated with the goat anti-mouse IgG conjugated to HRP (Dingguo, China). The bound antibody was detected using 3, 3’-diaminobenzidine (DAB).

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2.4. Animal vaccination and virus challenge

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For animal experiments, 6 to 8-week-old female BALB/c mice were intraperitoneally (i.p.) immunized with the recombinant fusion protein HSA/M2e or M2e peptide formulated with or without Freund’s adjuvant (CFA/IFA). Mice immunized with 10 ug of M2e peptide plus Freund’s adjuvant were used as positive controls. Mice immunized with HSA or Freund’s adjuvant were used as negative controls. Three immunizations were administered at an interval of 2 weeks. In the case of adjuvanted HSA/M2e or M2e peptide, the antigen was administered in CFA for priming only and in IFA for both the second and third boost. The spleens of vaccinated mice were harvested 2 weeks after the last boost for detection of T cell responses. At one week after the third immunization, 15 mice in each group were lightly anesthetized with sodium pentobarbital and inoculated simultaneously by the intranasal route with five lethal doses (5 LD50) of influenza H1N1 virus or H3N2 virus. Among them, 5 mice in each group were sacrificed by cervical dislocation after 5 days of virus challenge, lung tissues were used for titration of virus. We observed the details of the mice regarding the weight and t mortality for 2 weeks after virus challenge. 2.5. Serum antibody determination

Blood samples were obtained 1 day before the first injection and 1 week after each of the three subsequent injections to determine M2e-specific IgG antibody responses. The blood was incubated for 1 hour at 37 ℃ followed by placing in ice overnight. The sera were collected by centrifugation for 15 min at 3000 rpm and stored at -20 ℃ until use. 96-well ELISA microplates (Nunc) were precoated overnight at 4 ℃ with 100 µl/well M2e peptide in PBS (5 µg/ml). After blocking with 5% skim milk in PBS and washed with PBST, 1/10 serial diluted sera were added to the plates and allowed to react for 2 h at 37 ℃, starting with 1/100 dilution. After washing with PBST, HRP-labeled rabbit anti-mouse IgG (Sigma), rat anti-mouse IgG1 or IgG2a (BD Biosciences) was added. Tetramethylbenzidine (TMB) substrate was added after 1 hour and the reaction was stopped by adding 50 ul of 1 M H2SO4. Sera obtained from naive mice were used as negative controls. All serum samples from individual mice were assessed in triplicate. Titers were expressed as the highest dilution that yielded an optical OD450 greater than twice that of similarly diluted control sera. OD450 was measured on a microplate spectrophotometer. 4

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2.6. IFN-γ ELISPOT assay

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Two weeks after the last immunization, five mice from each group were sacrificed. Splenocytes were harvested after homogenizeation and red blood cells were lysed using a Tris-ammonium chloride buffer (Tris-NH4Cl, pH 7.2). Finally, splenocytes were resuspended in RPMI-1640 medium containing 10% FBS (Invitrogen). 96-well ELISPOT plates (BD Biosciences) were coated with 100 µl/well of diluent rat anti-mouse IFN-γ mAb and stored at 4 ℃ overnight. The plates were washed and then blocked with RPMI-1640 medium containing 10% FBS for 2 h at room temperature. Splenocytes were added to wells (2×105 cells /well in 100 ul) in triplicate, stimulated with synthetic M2e peptide, and incubated for 24 h at 37 C with 5% CO2, which had earlier been found to be optimal. The concanavalin A (conA) was added to positive control wells at final concentration of 10 ug/ml and PBS was added as negative control. After incubation for 24 h, plates were washed and treated sequentially with a biotinylated monoclonal antibody, streptavidin-HRP and AEC (Sigma) substrate solution. Finally, the plates were washed with deionized water and dried at room temperature for 2 h in the dark. Spots were counted under an automated ELISPOT reader system. 2.7. Determination of viral loads in lungs

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Titers of infectious virus were determined by titration of tissue homogenates in MDCK cell cultures (Liang et al., 1994). Five mice from each group were sacrificed five days after the last challenge and the amount of residual lung viruses were estimated. Lungs were aseptically rinsed and homogenized in 2 ml ice-cold PBS. The supernatants from lung homogenates were stored at -20 ℃ until analysis. MDCK cells in 96-well plates were inoculated with suspensions of lung homogenates in DMEM containing TPCK-treated trypsin (Invitrogen) and serially diluted tenfold starting from 1:10. After incubation for 72 h at 37 ℃, the culture supernatants were tested by mixing 50 ul of supernatant with 50 ul of a 0.5% suspension of pig RBCs. The virus titer of each specimen, expressed as the 50% tissue culture infection dose (TCID50), was calculated by the Reed-Muench method (Chen et al., 2000). 2.8. Data statistical analysis

All values were expressed as standard error of the mean and controls were performed using the two-tailed student’s-test. Values of p<0.05, were considered statistically significant. 3. Results 3.1. Purification and characterization of recombinant HSA/M2e fusion protein Cloning of HSA/M2e sequence into the multiple cloning site of pPICZα plasmid resulted in the expression of HSA/M2e fusion protein, under the control of P. pastoris AOX1 promoter (Fig. 1). The total soluble protein in the supernatant was 1.08 g/L culture. The supernatants collected were purified by SP Sepharose FF exchange chromatography, ultrafiltration and Q Sepharose FF exchange chromatography. Following these procedures, the purity of HSA/M2e was approximately 95% based on SDS-PAGE (Fig. 2A), the final recovery of the recombinant protein was 67%. As shown in Fig. 2A, the protein samples of HSA/M2e displayed only one band with molecular weight corresponding to about 68 kDa. In Western blotting, 14C2 recognizing M2e epitopes were incorporated into HSA (Fig. 2B). These results suggested that recombinant

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HSA/M2e fusion proteins maintain its antigenicity after purification. 3.2. HSA/M2e fusion proteins elicited strong humoral immune responses with high titers of M2e-specific antibodies

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The humoral immune responses induced by HSA/M2e fusion proteins were evaluated by measuring the M2e-specific IgG antibodies with the sera of the vaccinated mice by ELISA. As shown in Fig. 3A, the titers in the sera continued to increase from the first boost and reached the highest titers after the last immunization. As shown in Fig.3B, immunization with HSA/M2e fusion proteins without adjuvant elicited high titers of M2e-specific IgG antibodies following three immunizations, which means that the carrier HSA did not suppress the induction of an M2e-specific IgG response in mice. Meanwhile, HSA/M2e with adjuvant significantly increased M2e-specific IgG antibodies compared to HSA/M2e without adjuvant or M2e peptide plus adjuvant (P<0.01). Moreover, mice immuned by HSA/M2e with adjuvant induced higher titers of anti-M2e IgG2a antibodies than adjuvant-free immunization (P<0.01), indicating the induction of a Th1-dominant immune response, whereas no significant difference was observed in IgG1 titers. Vaccination with HSA alone induced only background level of M2e-specific antibodies at the lower limit of detection in mice. These results suggest that HSA/M2e antigen could induce humoral immune responses in mice. 3.3. Detection of M2e-specific IFN-γ-secreting lymphocytes

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To detect the ability of M2e-based HSA/M2e fusion proteins to elicit specific cellular immune responses, IFN-γ-producing cells were determined by ELISPOT. Splenocytes were stimulated in vitro with synthetic M2e peptide. As shown in Fig. 4, mice immunized with HSA/M2e (with or without adjuvant) or M2e peptide plus adjuvant induced a significantly higher number of IFN-γ-producing cells compared to control groups. Moreover, HSA/M2e plus adjuvant induced a significant increase in IFN-γ production compared to HSA/M2e without adjuvant or M2e peptide plus adjuvant (P<0.01). The number of IFN-γ-producing cells in control mice was low. These results demonstrate that HSA/M2e antigen could activate Th1 cytokine production. 3.4. Virus clearance from the lungs

Five mice from each group were used to determine viral clearance five days after influenza A virus challenge. Lung homogenates were used to titrate the virus in MDCK cell cultures. As shown in Fig.5, decreased levels of viral titers were found in mice vaccinated with HSA/M2e in contrast to control groups. Moreover, HSA/M2e with Freund’s adjuvant significantly enhanced the antiviral effects which was shown by significantly lower lung virus titer compared to HSA/M2e without adjuvant or M2e peptide plus adjuvant (P<0.05). Mice inoculated with HSA, PBS and Freund’s adjuvant did not have significant reduction in virus titers. These suggested that HSA/M2e vaccines have induced protective immunity against viral replication and enhanced virus removal in the local lung tissues. 3.5. HSA/M2e fusion proteins provided cross-protection from lethal challenge with influenza A viruses

To examine the potential cross-protective immunity induced by HSA/M2e fusion proteins against lethal challenge of H1N1 or H3N2 viruses, mice were separately challenged with 5 LD50 of influenza H1N1 virus or H3N2 virus one week after the last 6

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immunization. The cross-protective ability of HSA/M2e was further evaluated by daily observation of clinical signs, including weight loss and survival rates of the vaccinated mice over the course of 2 weeks. As shown in Fig. 6A and Fig. 6C, all mice began to loss weight 2-3 days after injection of virus. The body weight in the control groups showed continuous decrease till to die, while the weight in HSA/M2e (with or without adjuvant) group and M2e peptide with Freund’s adjuvant group began to increase after 6 days which is a significant improvement as compared to the control groups. In addition, almost no mice in the control groups survived for more than 14 days, the mortality of mice were 100% (Fig. 6B and Fig. 6D ), whereas 80% of the vaccinated mice survived for more than 14 days. The protection rate reached 80% (Fig. 6B and Fig. 6D). The survival rate of HSA/M2e or M2e peptide with Freund’s adjuvant-vaccinated mice were significantly higher than that of mice vaccinated with Freund’s adjuvant or PBS (P<0.01). These results further demonstrated that HSA/M2e could confer cross protection against two divergent lethal influenza virus strains. 4. Discussion

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Influenza pandemics are both unpredictable and unpreventable. Passive administration of anti-M2e mAb (14C2) could reduce the level of replication of influenza A virus in the lungs of mice (Treanor et al., 1990). Several studies have demonstrated that influenza vaccines based on M2e may provide effective cross-protective immunity and offer an attractive solution to overcome the threat of influenza A virus (Neirynck et al., 1999; Filette et al., 2006). Recently, TCN-032, a human anti-M2e mAb, has been found that could provide immediate immunity and therapeutic benefit in experimental human influenza A infection, with no apparent emergence of resistant virus (Ramos et al., 2015). In this study, we fused M2e with HSA and analyzed in detail the potential of the influenza A vaccine based on M2e. The exact mechanisms by which the universal influenza vaccines based on M2e induced protection remain unclear. In some reports, M2e-specific antibodies were suggested to play an important role. Studies found that the M2e-hepatitis core (M2e-HBc) particles protected mice against influenza virus challenge by inducing M2e-specific antibodies, and passive administration of antibodies could provide sufficient protection (Neirynck et al., 1999; Jegerlehner et al., 2004). Another study further indicated that M2e-specific antibodies could restrict virus replication in trachea and lung, and M2e-specific T cells contributed to protection in the upper respiratory tract (Mozdzanowska et al., 2007). A recent report showed that IgG1 and IgG2 antibodies induced by M2e-HBc might mediate protection of mice from influenza virus (Bakkouri et al., 2011). Other studies have found that antibodies of IgG2a isotypes could restrict viral infections which were better than IgG1 antibodies in mice (Markine-Goriaynoff et al., 2002). In conclusion, cross-protective immunity induced by influenza vaccines based on M2e could be influenced by carrier proteins, adjuvant or routes of administration (Filette et al., 2005). In this study, we investigated whether HSA/M2e fusion protein could induce protective immunity against a lethal challenge with influenza viruses in mice. M2e peptide was stably expressed and purified HSA/M2e fusion protein was able to induce 7

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high titres of M2e-specific IgG antibodies when vaccinated with or without adjuvant. HSA/M2e successfully induced both IgG1/IgG2a and T cell responses in mice after three immunizations. Moreover, 80% survival was achieved when mice were immunized with HSA/M2e and subsequently challenged with influenza A virus. When HSA/M2e was emulsified in adjuvant, significantly higher M2e-specific IgG titers (titers ＞ 100,000) were obtained and mice were completely protected against challenge with influenza A virus. Therefore, HSA/M2e vaccines could induce M2e-specific antibody in mice. Meanwhile, mice from this group had significantly higher numbers of IFN-γ secreting splenocytes compared to control groups (300±18 versus 20±4, respectively) (P<0.05). This finding suggests that M2e-specific T cells may contribute to cellular immunity in the mice model, in addition to M2e-specific humoral immunity. Furthermore, 80% of the mice immunized with HSA/M2e survived upon lethal challenge with two different mouse-adapted influenza viruses (H1N1 and H3N2) indicating a desirable cross-protection. The results obtained in this work suggested that both kinds of immune responses may be eventually involved. Broad-spectrum, protective immunity has been described in mice after vaccination with M2e-HBc fusion protein, produced in E. coli, together with adjuvant (Filette et al., 2006; Schotsaert et al., 2009). The adjuvants such as AS04, Onchocerca volvulus activation associated protein-1 (ASP-1), Toll-like receptor (TLR) ligand flagellin, papaya mosaic virus or CTA1-DD, have been demonstrated to improve the M2e-based vaccines as they were able to enhance the anti-M2e immunity (Lee et al., 2014; Zhao et al., 2010; Huleatt et al., 2008; Eliasson et al., 2008). The HSA/M2e vaccine described here has several advantages. The well-defined HSA/M2e could be produced and purified efficiently with the Pichia pastoris expression system and the process could be scaled up readily. Moreover, compared with other carriers, HSA has little or no immunogenicity in human body, which is more likely to become approved for use in humans (Kratz et al., 2008). In summary, fusing with HSA, recombinant M2e-based vaccine formulations effectively induced immune responses in the vaccinated mice and provided potent protection from virus challenge. Such a vaccine might have the advantage of providing protection against most known human influenza A strains.

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Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Lamb, R. A., Zebedee, S. L., Richardson, C. D., 1985. Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell 40, 627-633. Lee, Y. N., Kim, M. C., Lee, Y. T., Hwang, H. S., Cho, M. K., Lee, J.S., Ko, E.J., Kwon, Y. M., Kanq, S. M., 2014. AS04-adjuvanted virus-like particles containing multiple M2 extracellular domains of influenza virus confer improved protection. Vaccine 21, 485-492. Liang. S., Mozdzanowska. K., Palladino. G., Gerhard. W., 1994. Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity. J. Immunol. 152, 1653-1661. Liu, W., Li, H., Chen, Y. H., 2003. N-terminus of M2 protein could induce antibodies with inhibitory activity against influenza virus replication. FEMS. Immunol. Med. Microbiol. 35, 141-146. Liu, X., Guo, J., Han, S., Yao, L., Chen, A., Yang, Q., Bo, H., Xu, P., Yin, J., Zhang, Z., 2012. Enhanced immune response induced by a potential influenza A vaccine based on branched M2e polypeptides linked to tuftsin. Vaccine 30, 6527-6533. Markine-Goriaynoff, D., Coutelier, J. P., 2002. Increased efficacy of the immunoglo-bulin G2a subclass in antibody-mediated protection against lactate dehydrogenase-elevating virus-induced polioencephalomyelitis revealed with switch mutants. J. Virol. 76, 432-435. Mozdzanowska. K., Zharikova. D., Cudic. M., Otvos. L., Gerhard. W., 2007. Roles of adjuvant and route of vaccination in antibody response and protection engendered by a synthetic matrix protein 2-based influenza A virus vaccine in the mouse. Virol. J. 4, 118. Mu, X. P., Wei, A. H., Shen, M. H., Yan, W. Q., 2010. Secretory expression of HSA/M2e fusion protein in pichia pastoris. Chinese. Journal. of Gerontology. 30, 617-619. Neirynck, S., Deroo, T., Saelens, X., Vanlandschoot, P., Jou, W. M., Fiers, W., 1999. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat. Med. 5, 1157-1163. Patel, A., Tran, K., Gray, M., Li, Y., Ao, Z., Yao, X., Kobasa, D., Kobinger, G.P., 2009. Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses. Vaccine 27, 3083-3089. Patel, A., Graya, M., Li, Y., Kobasa, D., Yao, X., Kobinger, G.P., 2012. Co-administration of certain DNA vaccine combinations expressing different H5N1 influenza virus antigens can be beneficial or detrimental to immune protection.Vaccine 30,626-636. Quan, F.S., Vunnava, A., Compans, R.W., Kang, S.M., 2010.Virus-like particle vaccine protects against 2009 H1N1 pandemic influenza virus in mice. PLoS ONE 5, e9161.

Ramos, E. L., Mitcham, J. L., Koller, T. D., Bonavia, A., Usner, D. W., Balaratnam, G., Fredlund, P., Swiderek, K. M., 2015. Efficacy and safety of treatment with an anti-M2e monoclonal antibody in experimental human influenza. J Infect Dis 211, 1038-1044.

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Reid, A. H., Fanning, T. G., Janczewski, T. A., McCall, S., Taubenberger, J. K., 2002. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J. Virol. 76, 10717-10723. Schotsaert, M., Filette, M. D., Fiers, W., Saelens, X., 2009. Universal M2 ectodomain -based influenza A vaccines: preclinical and clinical developments. Expert. Rev. Vaccines. 8, 499-508. Treanor, J.J., Tierney, E.L., Zebedee, S.L., Lamb, R.A., Murphy, B.R., 1990. Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice. J. Virol. 64, 1375-1377. Zebedee, S.L., Lamb, R.A., 1988. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J. Virol. 62, 2762-2772. Zhao, G., Du, L., Xiao, W., Sun, S., Lin, Y., Chen, M., Kou, Z., He,Y., Lustiqman, S., Jiang, S., Zheng, B.J., Zhou,Y., 2010. Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant. Vaccine 28, 7233-7240. Zhong, W., Reed, C., Blair, P. J., Katz, J. M., Hancock, K., 2014. Serum antibody response to matrix protein 2 following natural infection with 2009 pandemic influenza A (H1N1) virus in humans. J Infect Dis 209, 986-994.

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Fig. 1. Schematic representation of the recombinant expression vector. pPICZα-HSA/M2e plasmid encoded HSA/M2e fusion as a secretary protein under the control of P. pastoris AOX1 promoter. TAA stop codon (#) was inserted downstream of the M2e sequence to avoid any C-terminus extension of the M2e peptide.

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Fig. 2. Purification and characterization of recombinant HSA/M2e fusion protein. (A) SDS-PAGE analysis of purified recombinant protein. Lane 1, protein molecular weight marker (broad); Lane 2, purified HSA/M2e; Lane 3, purified HSA; (B) Western blotting analysis of purified HSA/M2e protein in P. pastoris identified by an anti-M2e antibody[14C2]. Lane 1, purified HSA/M2e; Lane 2, purified HSA. No bands were detected on the purified HSA lane indicating no reactivity of 14C2 with HSA.

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Fig. 3. Total IgG, IgG1 and IgG2a antibody titers against the influenza virus M2e peptide. (A) M2e-specific IgG antibody responses in mouse sera collected at one week after each vaccination were determined. (B) Total IgG, IgG1 and IgG2a antibody titers were detected after the last immunization. Results are expressed as an antibody endpoint titer, where the O.D. value is 3-fold higher than the background value obtained with a 1:100 dilution of pre-immune serum from the mice. Data represent the mean ±SD of represented by *p

0.05, **p

five mice. Statistically significant differences are 0.01.

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Fig. 4. M2e-specific T cell responses were measured using IFN-γ ELISPOT. Spleen lymphocytes were separated from mice 2 weeks after the last vaccination and stimulated in vitro to detect IFN-γ-producing T cells. Data for each individual mouse are represented as number of IFN-positive spot forming cells ( SFCs ) per 106 spleen lymphocytes. Statistically significant differences are represented by *p

0.05, **p

0.01.

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Fig. 5 Virus clearance from the lungs of immunized mice following 5 LD50 of influenza virus challenge. 5 days after challenge, the 50% tissue culture infection dose (TCID50) was determined for homogenized lung tissue titrated in MDCK cell cultures. Statistically significant differences are represented by *p

0.05, **p

0.01.

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Fig. 6. Body weight changes and survival rates of virus-challenged mice. Intranasal challenge was performed in immunized mice using 5 LD50 of H1N1 or H3N2 virus. Mice were monitored daily for 2 weeks post-challenge. Statistically significant 0.05, **p

0.01.

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differences are represented by *p

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