c mice

Antiviral Research 111 (2014) 1–7

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Protection against influenza H7N9 virus challenge with a recombinant NP–M1–HSP60 protein vaccine construct in BALB/c mice Penghui Yang a,b,1, Wenjuan Wang b,c,1, Hongjing Gu b,1, Zhiwei Li a, Keming Zhang a, Zhouhai Wang a, Ruisheng Li a, Yueqiang Duan b, Shaogeng Zhang a,⇑, Xiliang Wang b,⇑ a b c

Beijing 302 Hospital, Beijing 100039, China Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, China Department of Clinical Laboratory, Jiujiang University Hospital, Jiujiang, Jiangxi 332000, China

a r t i c l e

i n f o

Article history: Received 27 March 2014 Revised 11 August 2014 Accepted 14 August 2014 Available online 27 August 2014 Keywords: Influenza A virus Nucleoprotein M1 protein HSP60 H7N9 vaccine

a b s t r a c t A novel influenza virus of H7N9 subtype circulated throughout China in 2013. The high fatality rate, appearance of several family clusters, and transmission in animal models observed during this outbreak accelerated efforts to identify effective strategies to prevent the spread of this influenza subtype. In this study, the recombinant protein NP–M1–HSP60, a fusion of the nucleoprotein and M1 matrix protein of the A/PR/8/34 (H1N1) influenza virus strain and HSP60, was effectively expressed in Escherichia coli and purified as a candidate component for an influenza vaccine. Intranasal immunization of female BALB/c mice with NP–M1–HSP60 in combination with an oil-in-water adjuvant twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses. Moreover, this immunization strategy completely protected mice from lethal influenza H7N9 virus challenge and significantly inhibited viral replication in the challenged mouse lung. These data suggest that this vaccine construct has great potential for the basic development of an influenza H7N9 vaccine. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction In spring 2013, the emergence of avian influenza H7N9 viruses in humans in China raised concern of a potential pandemic, spurring efforts to take measures against future outbreaks of this strain. Despite a decrease in infections during the summer of 2013, a significant increase in the number of H7N9 cases has been reported in the 2013/14 winter season. A vaccine is the most effective method of preventing influenza (Liu et al., 2012b). The H7N9 outbreaks emphasized the limitations of current vaccines and suggested that a universal influenza vaccine might be more effective in preventing a future influenza outbreak. Influenza A virus consists of eight negative-sense singlestranded viral genomic RNA segments. These genomic RNAs are incorporated into virions as ribonucleoprotein complexes, which consist of the viral RNA associated with three viral polymerase subunit proteins and nucleoprotein (NP). NP is a basic protein composed of 498 amino acids and containing several regions highly conserved among influenza A, B, and C viruses (Heiny et al., ⇑ Corresponding authors. E-mail addresses: [email protected] (S. Zhang), [email protected] (X. Wang). 1 All these authors contributed equally to this work. http://dx.doi.org/10.1016/j.antiviral.2014.08.008 0166-3542/Ó 2014 Elsevier B.V. All rights reserved.

2007; Mena et al., 1999). NP was previously considered an attractive candidate for a universal flu vaccine (Ohba et al., 2007; Price et al., 2010), but vaccines developed based on NP have failed to provide protection (Chen et al., 1998; Jamali et al., 2010; Lawson et al., 1994). Influenza A virus RNA segment 7 encodes two proteins, the matrix proteins M1 and M2. The M1 protein is a highly conserved 252-amino-acid protein (Heiny et al., 2007; Reid et al., 2002), which has also been used to develop a universal influenza vaccine (Antrobus et al., 2012; Choi et al., 2012; Quan et al., 2012). Influenza vaccines based on NP, M1, or both have been investigated in animal models. However, in clinical trials, only licensed vaccines have shown promise. Currently, several strategies have been proposed to improve the immunogenicity and protective efficacy of NPand M1-based vaccines, including the administration of high-dose formulations of the vaccine, combination of modified vectored vaccine backbones, or the use of adjuvants (Antrobus et al., 2014). Heat shock proteins (HSP), highly conserved across species, are generally considered to have protective functions in situations of cellular stress (Hartl, 1996). HSPs, such as HSP60, act directly on antigen-presenting cells (APC) to link innate and adaptive immune responses (Breloer et al., 2001; More et al., 2001). In such conditions, we speculated that HSP60 could potentially complement NP and M1-induced immunity to produce an efficient vaccine.

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In our previous studies, a universal influenza virus vaccine construct consisting of a NP–M1–HSP60 fusion protein was expressed in Escherichia coli. We aimed to evaluate the efficacy of the NP– M1–HSP60 fusion protein in combination with oil-in-water SP01 adjuvant in terms of conferring protection against novel H7N9 virus challenge in mice. The protective value of NP–M1–HSP60 was evaluated by measuring serum and mucosal antibody titers, residual lung virus titers, and survival rates. 2. Materials and methods 2.1. Viruses and cells The influenza A (H7N9) virus used in this study was A/Anhui/ 01/2013 (abbreviation for Ah01/H7N9) (Li et al., 2012; Yang et al., 2010). Live-virus experiments were performed in Biosafety Level 3 facilities under governmental and institutional guidelines. Viruses were propagated by inoculation into 10-day-old SPF chicken eggs via the allantoic route and titered in MDCK cells. MDCK (CCL-34) cells were purchased from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin G, and 100 lL/mL streptomycin sulfate. 2.2. NP–M1–HSP60 expression, purification, and verification The complete sequences of NP and M1 from A/PR/8/34 (H1N1) joined with HSP60 coding region by a seven-amino-acid linker, GGGPGGG, were synthesized and inserted into the bacterial expression vector pET28a (Novagen) for expression as a fusion protein with His tags at both the N and C termini. To ensure that C-terminal His tags were non-functional, the terminal code TAA was introduced before the restriction endonuclease EcoRI site. The target protein was purified using AKTA Purifier (GE) with a

Ni-chromatography column (GE) and detected by Western blotting. 2.3. Immunization and virus challenge Female BALB/c mice aged 4–6 weeks were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (AMMS), Beijing, China. Groups of mice (n = 26) were vaccinated intranasally (i.n.) with 10-lg NP–M1–HSP60 vaccine antigen, NP–M1, or HSP60 with oil-in-water SP01 adjuvant twice, 2 weeks apart. The SP01 oil-in-water emulsion adjuvant containing squalene, polyether, and castor oil (Sigma) was combined with the antigen by mixing 1:1 v/v, as described previously (Yang et al., 2012). Blood, nasal wash, and bronchoalveolar lavage fluid (BALF) samples were collected 10 days after boost, and the spleens of vaccinated mice were harvested for assessment of cellular immune responses. Two weeks after the second immunization, mice (n = 18) were i.n. challenged with a lethal dose of influenza virus Ah01/H7N9 (50LD50). Procedures, including intranasal inoculation of live virus and bronchoalveolar lavage (BAL), were performed under sodium pentobarbital anesthesia (60–80 mg/kg). Challenged mice (n = 10) were monitored daily for 14 days for body weight and survival. Mice showing 30% body weight loss were considered to have reached the experimental endpoint approved by the Institutional Animal Care and Use Committee of AMMS and were humanely euthanized. Lung tissues were collected 4 days post-infection for analysis of lung viral titers and histopathological changes. 2.4. ELISA Antigen-specific IgG and isotype (IgG1, IgG2a) levels in sera and sIgA in nasal wash and BALF were determined by indirect ELISA as described previously (Liu et al., 2012a). 96-well plates were coated overnight with 10 lg/mL purified inactivated Ah01/H7N9 antigen

Fig. 1. Construction of plasmids and identification of recombinant proteins. (A) Synthetic genes (NP–M1–HSP60, NP–M1 or HSP60) were cloned into the pET28a vector. (B) Recombinant NP–M1–HSP60 or HSP60 proteins expressed in E. coli were purified by His-tag affinity chromatography and detected by Western blotting using an anti-HSP60 monoclonal antibody.

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dissolved in pre-coating buffer. After washing and blocking, serial dilutions of anti-sera were added in triplicate and incubated. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or its isotypes (IgG1, IgG2a) or sIgA (Bethyl) were used as secondary antibodies. Next, 3,30 ,5,50 -tetramethybenzidine was added, the reaction was stopped with 2 M H2SO4, and absorbance at 450 nm was measured.

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Fig. 2. Immunization schedule and specific IgG antibody titers in serum. (A) Groups of mice (n = 26) were primed with 10 lg of vaccine (NP–M1–HSP60, NP–M1, HSP60, or SP01) or PBS intranasally (i.n.) at the start of the vaccination protocol. Two weeks later, a boost i.n. immunization with the same antigen was given. Ten days postboost, mice (n = 8) were sacrificed for analysis of the immune response. Two weeks post-boost, the remaining mice (n = 18) were challenged with influenza Ah01/H7N9 virus. Four days post infection, mice (n = 8) were sacrificed for virus titer and lung pathology assays. In the remaining mice, body weight and survival (n = 10) were monitored daily until 14 days post infection. Serum samples of each group were collected at days 10 and 24 and diluted 1:100 for ELISA in triplicate. (B) IgG levels. (C) IgG1 and IgG2a levels. Dashed line indicates the limit of detection. Results are expressed as means ± SD. ⁄⁄p < 0.001.

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Fig. 3. Mucosal antibody response in BALB/c mice. Secretory IgA levels were assessed by ELISA in nasal (A) and lung (B) lavage fluid collected 10 days post boost from mice immunized i.n. with NP–M1–HSP60 vaccine construct. Dashed line indicates the limit of detection. Results are expressed as means ± SD; ⁄⁄p < 0.001; n = 8.

solution. Spots were counted using an automated ELISPOT reader system and the ImmunoSpot 3 Software (Cellular Technology Ltd.). 2.6. Virus titers Nasal turbinate, lung, and brain were harvested 4 days postinfection and homogenized in DMEM containing antibiotics at 10% w/v tissue. Tissue homogenates, first clarified by low-speed centrifugation, were titrated in 96-well culture plates containing MDCK cells, and titers were expressed as Log10TCID50/gram tissue.

2.5. ELISPOT assay

2.7. Histopathology

To measure cytokine levels, spleens from immunized mice were removed, homogenized, and re-suspended at 1  105 cells/100 ll in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. The cells were cultured in triplicate and plated in enzymelinked immunosorbent spot (ELISPOT) plates (BD Pharmingen) that were previously coated with IL-4 or IFN-c capture antibody (BD Pharmingen) overnight at 4 °C, stimulated with purified inactivated Ah01/H7N9 antigen at 5 lg/mL, and incubated for 48 h at 37 °C under 5% CO2. Spot-forming cells were detected by addition of biotinylated anti-IFN-c or anti-IL-4 antibody followed by the addition of streptavidin–HRP and development with AEC substrate

Four days post-challenge, lungs of infected mice were removed, immediately fixed in 10% neutral-buffered formalin, and embedded in paraffin. Sections (4–6 lm) were mounted on slides, stained with H&E, and observed by microscopy. 2.8. Statistical analysis Survival data were analyzed by Kaplan–Meier survival analysis. Measurements at single time points were subjected to ANOVA. A multiple comparison test was performed after the ANOVA procedure using the GraphPad Prism ver. 5.0 software.

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3. Results 3.1. Expression of soluble recombinant proteins The NP–M1–HSP60, NP–M1, and HSP60 genes were chemically synthesized and inserted into a pET28a vector to His-tag the target proteins at both the N and C termini (Fig. 1A). Soluble proteins expressed in the E. coli BL21 (DE3) strain were purified by HisTag affinity chromatography and confirmed by Western blotting using an anti-HSP60 monoclonal antibody (Fig. 1B). This approach yielded greater than 90% pure recombinant proteins with the expected molecular masses.

3.2. NP–M1–HSP60 protein vaccine construct induced high reactive antibody titers in mice Humoral immune responses induced by i.n. immunization of NP–M1–HSP60 proteins were assessed by measuring IgG antibodies in the serum of vaccinated mice by indirect ELISA. Each group of

IFN-γ producing cells per 1× 105 splenocytes

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3.3. Improved mucosal immune responses from immunization with NP–M1–HSP60 protein vaccine construct Following i.n. immunization with NP–M1–HSP60 protein vaccine construct, sIgA antibody levels in nasal and lung lavage fluids were determined by ELISA. As shown in Fig. 3, sIgA was detected in nasal and lung lavage fluids in NP–M1–HSP60- and NP–M1-vaccinated mice, whereas no antibodies were detected in those vaccinated with HSP60, SP01, or PBS. sIgA titers of NP–M1–HSP60vaccinated groups in nasal lavage and BALF were higher than those of the control groups. Together, these results imply that intranasal immunization of mice with NP–M1–HSP60 not only induces systemic immunity but also a strong mucosal antibody response.

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3.4. NP–M1–HSP60 protein vaccine construct effectively induce cellular immune responses in mice

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To further estimate the ability of the NP–M1–HSP60 protein to elicit a cellular immune response, the number of IFN-c- and IL-4producing cells was determined by ELISPOT assays. Splenocytes were prepared on day 24 and stimulated with NP–M1–HSP60 pro-

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BALB/c mice was immunized i.n. on days 0 and 14, respectively (Fig. 2A). As shown in Fig. 2B, NP–M1–HSP60 and NP–M1 induced significantly stronger antibody responses than HSP60, SP01, or PBS, particularly on day 10 after boost. There was no significant difference in IgG titers between the NP–M1–HSP60 and NP–M1 vaccinated groups. Additionally, both antigens induced higher IgG1 and IgG2a titers than those in the HSP60, SP01, or PBS groups. The IgG1 and IgG2a titers of NP–M1–HSP60-vaccinated mice were 3.4  104 ± 0.01 and 1.8  104 ± 0.36, respectively. These data indicate that vaccination with NP–M1–HSP60 induced a relatively balanced IgG1 and IgG2a response, suggesting stimulation of both Th2- (IgG1) and Th1- (IgG2a) associated immune responses.

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Days post challenge Fig. 4. Analysis of IFN-c and IL-4 by ELISPOT assays. On day 10 after the second immunization, mice were sacrificed and single-cell suspensions were prepared from the spleen, cultured for 48 h, and stimulated with lg/mL purified inactivated Ah01/H7N9 antigen. IFN-c (A) and IL-4 (B) secretion by splenic lymphocytes was detected by ELISPOT in triplicate wells. Representative images are presented. Values and bars represent means ± SD. ⁄⁄p < 0.001 compared to HSP60, SP01 or PBS.

Fig. 5. Protection efficacy of NP–M1–HSP60 vaccine construct against a lethal challenge of live influenza Ah01/H7N9 virus. Two weeks after the second immunization, vaccinated mice were i.n. infected with Ah01/H7N9 (50LD50) and monitored daily for 2 weeks post challenge. (A) Body weight change (%); (B) survival rate (%). Each point represents mean ± SD. ⁄⁄p < 0.001.

P. Yang et al. / Antiviral Research 111 (2014) 1–7

tein in vitro. As shown in Fig. 4A, NP–M1–HSP60 vaccination led to significantly higher levels of IFN-c-producing T cells than HSP60, SP01, or PBS (p < 0.001). Notably, there was no significant difference in cytokine-producing T cells between NP–M1–HSP60- and NP–M1-vaccinated mice. Similarly, we found that NP–M1–HSP60 induced significantly more IL-4–producing T cells than did control vaccinations (p < 0.001) (Fig. 4B). The number of IFN-c-secreting cells and IL-4-secreting cells were essentially equal in the NP– M1–HSP60 vaccinated group, indicating that both types of immune responses were induced by NP–M1–HSP60 vaccination.

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M1–HSP60 protein vaccine completely protects mice against live influenza H7N9 virus. Next, we examined whether NP–M1– HSP60 could improve viral clearance after challenge with live influenza H7N9 virus. 3.6. NP–M1–HSP60 protein vaccine restricts viral replication and attenuates H7N9-induced lung pathology For histopathological examination, lung tissues were collected on day 4 post-challenge with live Ah01/H7N9 virus (Fig. 6A). Severe histopathological damage, including slight pulmonary vascular dilatation and congestion, fragmentation of alveolar walls and infiltration of lymphocytes, were observed in mice injected with HSP60 or SP01 alone or PBS, whereas only mild inflammatory changes were seen in mice immunized with NP–M1–HSP60 or NP– M1 (Fig. 6A), indicating that these vaccines prevented lung pathology caused by the influenza H7N9 virus. To quantify viral replication, nasal turbinates, lungs, and brains were collected and used to inoculate cultured cells (Fig. 6B). Virus titers in nasal turbinate and lungs were significantly (p < 0.01) lower in the group vaccinated with NP–M1–HSP60 and NP–M1 than in those from mice injected with HSP60, SP01 or PBS. No virus was observed in brain tissues from any group. These data indicate that NP–M1–HSP60 vaccine construct efficiently induce protective immunity against live influenza H7N9 virus infection of the respiratory tract.

3.5. NP–M1–HSP60 protein provides effective protection from lethal challenge with live influenza H7N9 virus To further evaluate the protective efficacy of the NP–M1–HSP60 protein vaccine against live influenza H7N9 virus, mice were challenged with wild-type Ah01/H7N9 (50-fold the LD50) at 2 weeks after the last immunization. All mice immunized with NP–M1– HSP60 survived for at least 14 days after influenza Ah01/H7N9 virus challenge, and body weight recovered quickly 7 days post infection (Fig. 5). In contrast, in NP–M1-vaccinated mice, body weight decreased acutely on the fifth day after challenge, and 90% survived. Of note, there were statistically significant differences between the two groups in weight loss at 7 days post challenge. As a comparison, all mice vaccinated with HSP60, SP01, or PBS died within 9–11 days. These results indicate that the NP–

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Fig. 6. Histopathological changes in lung and virus titers in nasal turbinates, lung, and brain of vaccinated mice following challenge with live influenza Ah01/H7N9 virus. Lung tissues were collected 4 days after challenge. (A) Representative images of histopathological damage from H&E-stained lungs in three mice per group. (B) Virus titers in nasal turbinates, lung, and brain of infected mice. TCID50 assays in MDCK cells were performed in triplicate 4 days post infection and expressed as Log10 TCID50/g tissue. N.D., none detected. Data are expressed as means ± SD. ⁄⁄p < 0.001.

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4. Discussion In this study, we investigated the potential of recombinant NP– M1–HSP60 protein as an influenza H7N9 vaccine. This construct induced protective immunity against influenza H7N9 virus in a mouse model. The vaccine construct, consisting of full-length NP and M1 proteins fused to HSP60, was expressed in E. coli and purified by chromatography, yielding ample highly purified protein. Vaccination with NP–M1–HSP60 elicited high titers of specific IgG antibodies and Th1/Th2-associated immune responses. NP– M1–HSP60 protein vaccine construct provide complete protection against lethal challenge by live influenza H7N9 virus, as evidenced by increased survival rates, a significant decrease in viral replication, and marked alleviation of lung pathology in challenged mice. Previous studies have shown that NP- and M1-based immunization protect mice against lethal challenge with influenza virus (Boyd et al., 2012; Donnelly et al., 1997; Zheng et al., 2013). However, most reported NP- and M1-based vaccines are DNA(Antrobus et al., 2014; Donnelly et al., 1997; Ohba et al., 2007; Zhirnov et al., 2007) or viral-skeleton-based (Antrobus et al., 2012; Choi et al., 2012; Lillie et al., 2012; Price et al., 2010; Quan et al., 2012), and other papers indicated that NP-based vaccines fail to provide complete protection against influenza virus (Chen et al., 1998; Jamali et al., 2010; Krammer and Palese, 2014; Lawson et al., 1994). The matrix protein (M1, M2) and nucleoprotein (NP) of influenza virus have highly conserved sequences, and are the major target antigens of current universal influenza vaccine studies. To date, the mechanism of conserved influenza protein-induced protection has not been elucidated. Several reports have indicated that antibody responses induced by NP or M1 fail to provide protection against influenza challenge (Chen et al., 2009; Huang et al., 2012; Sui et al., 2010; Wang et al., 2012). T-cell responses play a more important role in protection against various strains of influenza virus (Sun et al., 2011). The number of IFN-c-secreting cells has been reported to be correlated with protection and survival (Berthoud et al., 2011; Chen et al., 2009; Ohba et al., 2007; Sipo et al., 2011; Sui et al., 2010). Here, immunization with NP– M1–HSP60 likely stimulated IFN-c-secreting T cells specific for both NP and M1, enhancing protective efficacy. This suggests that T-cell responses to conserved internal antigens of influenza have the potential to modify or prevent disease and virus shedding (Berthoud et al., 2011). NP- and M1-specific IgA was detected in the nasal and lung washes, which could result from the adjuvant; SP01 is a potent Th1 adjuvant used in influenza and other vaccines because it induces effective mucosal and systemic immune responses (Yu et al., 2012). Whether mucosal IgA contributes to protection remains unclear; protection by this vaccine might be a synergistic effect of mucosal, humoral, and cell-mediated immunity. In any case, an effective and safe influenza H7N9 vaccine should produce strong systemic as well as mucosal immune responses. Importantly, we also evaluated the protective efficacy of an NP–M1 protein construct and found that it provided only partial protection against Ah01/H7N9 virus challenge (Fig. 5). Furthermore, in this study, with the HSP60 derived from Helicobacter pylori (Hp), very low anti-HSP60 titers in the serum of vaccinated mice were detected after vaccination (data not shown). As the protein is highly conserved, whether human vaccination could trigger production of auto-antibodies that induce autoimmune disease should be further investigated. In addition, further research is also required to determine the potential of NP–M1–HSP60 as an influenza H7N9 candidate vaccine, including vaccination strategy, duration of immunity, examination of its protective efficacy against other H3, H5, H7, and H9 subtypes, and confirmation of these protective effects in ferrets and monkeys.

In conclusion, we have developed a recombinant NP–M1– HSP60 protein vaccine construct for protection against influenza H7N9 virus infection. Our results demonstrate that NP–M1– HSP60 protein, in combination with oil-in-water SP01 adjuvant, induces potent humoral, mucosal, and cell-mediated responses, alleviates lung pathology, and protects mice from lethal challenge with influenza H7N9 virus. We therefore conclude that NP–M1– HSP60 is a candidate influenza H7N9 vaccine and may represent a novel approach to the development of epidemic and pandemic influenza vaccines. Acknowledgments This work was carried out in part with funding from the National Natural Scientific Foundation (81370518), the Ministry of Science and Technology of China (2012CB518905, 2013ZX10004003 and SS2012AA020905). P.H.Y. was supported by Beijing Nova Program (No. Z141107001814054). No other potential conflict of interest relevant to this article was reported. References Antrobus, R.D., Berthoud, T.K., Mullarkey, C.E., Hoschler, K., Coughlan, L., Zambon, M., Hill, A.V., Gilbert, S.C., 2014. Coadministration of seasonal influenza vaccine and MVANP+M1 simultaneously achieves potent humoral and cell-mediated responses. Mol. Ther. 22, 233–238. Antrobus, R.D., Lillie, P.J., Berthoud, T.K., Spencer, A.J., McLaren, J.E., Ladell, K., Lambe, T., Milicic, A., Price, D.A., Hill, A.V., Gilbert, S.C., 2012. A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVANP+M1 in adults aged over 50 years. PLoS One 7, e48322. Berthoud, T.K., Hamill, M., Lillie, P.J., Hwenda, L., Collins, K.A., Ewer, K.J., Milicic, A., Poyntz, H.C., Lambe, T., Fletcher, H.A., Hill, A.V., Gilbert, S.C., 2011. Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVANP+M1. Clin. Infect. Dis. 52, 1–7. Boyd, A.C., Ruiz-Hernandez, R., Peroval, M.Y., Carson, C., Balkissoon, D., Staines, K., Turner, A.V., Hill, A.V., Gilbert, S.C., Butter, C., 2012. Towards a universal vaccine for avian influenza: protective efficacy of modified vaccinia virus Ankara and adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus. Vaccine. Breloer, M., Dorner, B., More, S.H., Roderian, T., Fleischer, B., von Bonin, A., 2001. Heat shock proteins as ‘‘danger signals’’: eukaryotic Hsp60 enhances and accelerates antigen-specific IFN-gamma production in T cells. Eur. J. Immunol. 31, 2051–2059. Chen, Q., Kuang, H., Wang, H., Fang, F., Yang, Z., Zhang, Z., Zhang, X., Chen, Z., 2009. Comparing the ability of a series of viral protein-expressing plasmid DNAs to protect against H5N1 influenza virus. Virus Genes 38, 30–38. Chen, Z., Sahashi, Y., Matsuo, K., Asanuma, H., Takahashi, H., Iwasaki, T., Suzuki, Y., Aizawa, C., Kurata, T., Tamura, S., 1998. Comparison of the ability of viral protein-expressing plasmid DNAs to protect against influenza. Vaccine 16, 1544–1549. Choi, J.G., Kim, M.C., Kang, H.M., Kim, K.I., Lee, K.J., Park, C.K., Kwon, J.H., Kim, J.H., Lee, Y.J., 2012. Protective efficacy of baculovirus-derived influenza virus-like particles bearing H5 HA alone or in combination with M1 in chickens. Vet. Microbiol.. Donnelly, J.J., Friedman, A., Ulmer, J.B., Liu, M.A., 1997. Further protection against antigenic drift of influenza virus in a ferret model by DNA vaccination. Vaccine 15, 865–868. Hartl, F.U., 1996. Molecular chaperones in cellular protein folding. Nature 381, 571– 579. Heiny, A.T., Miotto, O., Srinivasan, K.N., Khan, A.M., Zhang, G.L., Brusic, V., Tan, T.W., August, J.T., 2007. Evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets. PLoS One 2, e1190. Huang, B., Wang, W., Li, R., Wang, X., Jiang, T., Qi, X., Gao, Y., Tan, W., Ruan, L., 2012. Influenza A virus nucleoprotein derived from Escherichia coli or recombinant vaccinia (Tiantan) virus elicits robust cross-protection in mice. Virol. J. 9, 322. Jamali, A., Sabahi, F., Bamdad, T., Hashemi, H., Mahboudi, F., Kheiri, M.T., 2010. A DNA vaccine-encoded nucleoprotein of influenza virus fails to induce cellular immune responses in a diabetic mouse model. Clin. Vaccine Immunol. 17, 683– 687. Krammer, F., Palese, P., 2014. Universal influenza virus vaccines: need for clinical trials. Nat. Immunol. 15, 3–5. Lawson, C.M., Bennink, J.R., Restifo, N.P., Yewdell, J.W., Murphy, B.R., 1994. Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge. J. Virol. 68, 3505–3511. Li, C., Yang, P., Sun, Y., Li, T., Wang, C., Wang, Z., Zou, Z., Yan, Y., Wang, W., Wang, C., Chen, Z., Xing, L., Tang, C., Ju, X., Guo, F., Deng, J., Zhao, Y., Yang, P., Tang, J.,

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