Heterosubtypic protective immunity against widely divergent influenza subtypes induced by fusion protein 4sM2 in BALB/c mice

For the expression of target proteins with his-tag fusion at the N-term, monomer or multimer consensus sM2 plasmids (sM2 and 4sM2, respectively) were constructed into pRSET A vector. The recombinant proteins were expressed mainly as inclusion bodies in E. coli. Refolded inclusion bodies containing the recombinant proteins were purified by His-tag affinity chromatography and dialyzed using permeable cellulose membrane and confirmed by SDS-PAGE and immunoblotting. As shown in Figure 1B, proteins were electrophoresed on the SDS-polyacrylamide gel and stained with Coomassie brilliant blue. After destaining, proteins were observed at the expected molecular weight of 60 kDa (4sM2, Figure 1B) and 15 kDa (sM2, data not shown). Additionally, reactions of 4sM2 protein with mouse anti-Histidin (C-term, Figure 1C) and rabbit anti-M2 antibodies (Figure 1D) were confirmed by immunoblotting. With Coomassie staining and immunoblotting, no other proteins were observed around the expected band which indicate, there were no conformational changes of proteins after refolding from inclusion bodies.

Upon confirmation of protein expression and subsequent purification, groups of mice were immunized intramuscularly (i.m.) with 30 μg of 4sM2 protein at day’s 0 and 14, and sera were collected at day’s -1, 7, and 21 to assess the antibody titer. Serum antibodies were measured by ELISA using the sM2 protein (Figure 2A), M2 peptide (Figure 2B) or inactivated purified virions as a coating antigen. The levels of serum IgG absorbance increased around 10 fold after the second application of 4sM2 protein compared with those observed before immunization (Figure 2A and B). As shown in Figure 2D immune sera collected from mice boosted with 4sM2 showed significant level of antibodies cross reactivities to influenza subtypes H5N1, H5N2 and H9N2 viruses. Importantly 4sM2 immune sera also showed significant levels of cross reactivities to H1N1 and H7N3 subtypes which contains sM2 sequences of 8 and 2 amino acid mismatches respectively (Table 1). Control group of mice that were immunized with 0.85% saline (NC) did not show any responses against protein, peptide or inactivated whole viruses. Therefore this result suggests that 4sM2 is capable to induce antibody which is cross reactive to different subtypes of influenza virus.

M2 specific IgG1 and IgG2a were also tested and found that both IgG1 and IgG2a increased significantly compare to negative control (NC) after the booster immunization (Figure 2C), and IgG2a was predominant than IgG1. These results suggest that the 4sM2 recombinant protein is strongly immunogenic and is also capable of producing both Th1 and Th2 inducing M2 specific IgG1 and IgG2a antibodies.

To investigate the broad protective immune mechanism, 4sM2 induced IFN-γ and IL-4 secreting cells in the spleen were determined by ELISPOT. Cells were collected 10 days after the boost immunization and stimulated with sM2 protein or M2 peptide. Significant numbers of IFN-γ secreting cells were observed in the spleen following stimulation with both the sM2 protein (Figure 2E, left) and M2 peptide (Figure 2E, right). We also observed a detectable level of IL-4 secreting splenocytes following stimulation with both the sM2 protein (Figure 2F, left) and the M2 peptide (Figure 2F, right). Mock (un-immunized) mice showed a background level of spot for both the sM2 protein and M2 peptide stimulation. These findings indicate that four tandem copies of consensus sM2 can induce M2-specific IFN-γ and IL-4 secreting T cell responses, which may contribute to the protective immunity.

The efficacies of the 4sM2 vaccine against the divergent influenza virus subtypes were investigated. Mice were immunized i.m. twice at 2 weeks interval. Two weeks after the final immunization, mice were challenged intranasally (i.n.) with the 10MLD₅₀ of A/EM/Korea/W149/06(H5N1) influenza subtype that contains 2 mismatched amino acids against the sM2 consensus sequence (Table 1). Protective efficacy and morbidity (measured by survival rates and weight losses, respectively) were monitored every other day for 13 days post-infection (dpi); mice were euthanized and considered dead if the original body weight is reduced by >25%. As shown in Figure 3A, immunized mice lost 5–10% of their body weight but conferred 100% survival by 13 dpi after lethal challenge of H5N1 virus (Figure 3A). In contrast, remarkable losses of body weight were observed in unimmunized mice and none of them survived due to lethal infection of H5N1 virus (Figure 3A, bottom).

Next the protection efficiency of 4sM2 vaccine against A/PR/8/34(H1N1) subtype of influenza virus that contains 8 mismatched with sM2 consensus sequence was evaluated. Set of immunized mice were challenged with 10MLD₅₀ of the H1N1 virus and protection efficacy were measured as before. Unimmunized mice lost >25% of body weight and all died by 9 dpi whereas mice immunized with 4sM2 showed negligible body weight loss and 80% were survived (Figure 3B). Another set of vaccinated mice were infected with A/Aquatic bird/Korea/W81/2005(H5N2) influenza virus to better understand the degree of cross protection by 4sM2 vaccine. The sM2 sequence of H5N2 contains 1 mismatched against sM2 consensus sequence. All mice in the control group became severely ill (lost weight >25%) and eventually died by 9 dpi. In contrast, the 4sM2 immunized group experienced 19% loss in body weight within 3 to 9 dpi, but started to recover thereafter; 100% of the vaccinated mice were survived the H5N2 virus infection (Figure 3C). The breadths of cross protection of the 4sM2 vaccine against divergent influenza subtypes were further examined. For this, 2 sets of immunized mice were challenged with A/Aquaticbird/Korea/W44/2005(H7N3) or A/Chicken/Korea/116/2004(H9N2) that contain 2 and 3 amino acid mismatched with sM2 consensus sequence respectively. In both cases unimmunized mice were severely ill (lost weight >25%) and died by 9 to 11 dpi. Although, mice immunized with 4sM2 showed little body weight loss, but recovered gradually and finally 60% and 80% survived the H7N3 and H9N2 virus challenges respectively (Figure 3D and E). Taken together, the results showed that immune responses induced by highly conserved 4sM2 vaccine conferred the protection against divergent subtypes of influenza virus lethal infection either it is complete or partial.

Virus titers in the lungs of challenged mice were measured to estimate the virus clearance at 3 and 5 dpi. The 4sM2 immunized mice had significantly reduced lung virus titers at day 3 and had completely cleared the infection by day 5 in case of H5N1 virus challenge (Figure 4A). Similarly, the immunized mice elicited significant reduction of lung virus titers in compare to unimmunized mice by 5 dpi in case of H1N1 and H5N2 influenza virus (Figure 4B and4C). The clearances of viruses from the lung after challenge with H7N3 (Figure 4D) and H9N2 (Figure 4E) subtypes were also assessed. Both immunized and unimmunized mice showed high lung virus titers at 3 dpi. In contrast, virus titers decreased significantly in immunized group at 5 dpi which correlate the survival result of both H7N3 and H9N2 lethal infection. A histopathological examination was also performed to correlate the virus clearance in the lungs. Representative lungs samples were collected after challenge with H5N2 virus and process to examine under light microscope. As shown in Figure 4F, clear signs of profound pulmonary inflammations were observed in unimmunized mice lung, whereas the mice immunized with 4sM2 showed no significant pulmonary inflammation (Figure 4F). These results demonstrate that consensus 4sM2 protein vaccine induced immune responses strong enough to completely clear the H5N1 and H5N2 subtypes and significantly reduce the virus titers of H1N1, H7N3 and H9N2 influenza subtypes in vivo.

Duration of protection ability is an important criterion for the potential vaccine. For this, the longevity of cross protection by the 4sM2 vaccine was investigated. Mice were immunized according to the schedule mentioned previously, and sera were collected at -1 (pre), 21 (2^nd), and 180 days after vaccination. Consistent levels of serum IgG specific to sM2 were determined even at 180 days after the final vaccination (Figure 5A). Mice were then challenged with A/Aquatic bird/Korea/W81/2005(H5N2) influenza subtype; morbidity and mortality were checked until 13 dpi. The unimmunized mice showed >25% body weight loss (Figure 5B) and all mice died at 9 dpi whereas the immunized mice survived 80% with negligible body weight loss which was recovered by 13 dpi (Figure 5C). This result demonstrates that 4sM2 vaccine conferred protection even after 6 months of final vaccination against heterosubtype lethal infection.

A gene encoding the consensus sM2 containing residues of extracellular and cytoplasmic domain without the transmembrane domain from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database was chemically synthesized (Figure 1A). Plasmid sM2 and 4sM2 were constructed by cloning as described previously[36]. The sM2 gene was modified by adding a Nhe I site at the 5′ terminal and Bam H I and Hin d III sites as well as the termination codons TAA and TGA at the 3′ terminal for cloning into the pRSET A vector. The polymerase chain reaction (PCR) was employed to amplify the gene using the primer pair 5′-CTA GCT AGC ATG TCA TTA TTA ACA-3′ (sense 1), 5′-GAA GAT CTA TGT CAT TAT TAA CA- 3′ (sense 2) and 5′-AAG CTT TTA TCA GGA TCC ACC TGA ACC ACC TGA ACC ACC TGA ACC ACC TTC AAG TTC-3′ (anti sense). Two different primer senses were simultaneously used during this multi-cloning process. The sM2 (sense 1) was ligated with pRSET A using a CoreBio 96 plus thermocycler (CoreBio L&B, Seoul, Korea), whereas sM2 (sense 2) was ligated into the T Easy Vector (Invitrogen, Seoul, Korea). Each plasmid was linearized by RE digestion using Bam H I, Hin d III for the pRSET A vector and Bgl II, Hin d III for the T Easy Vector at 37°C for 2 h and purified by phenol/chloroform/ isoamylalcohol treatment. The linearized plasmids were electrophoresed on a 0.9% agarose gel and recovered using a QIAquick Gel Extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The pRSET A vector and sM2 insert (sense 2) were ligated with T4 ligase (TaKaRa Bio, Seoul, Korea) at 16°C for 4 h. Sense 1 for sM2 was fused to sense 2 to produce 2sM2. Consequently, 4sM2 was produced by combining 2sM2 (sense 1) to 2sM2 (sense 2). The ligated products were transformed into E. coli JM83 competent cells using an electroporation method described previously. The recombinant plasmids were recovered by plasmid DNA extraction following the manufacturer’s instructions using Accuprep Plasmid Mini-prep (Bioneer, Daejeon, Korea). The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing (Solgent, Seoul, Korea).

Proteins sM2 and 4sM2 were generated using an E. coli expression system as described previously[37, 38]. Briefly, recombinant plasmids were introduced into the E. coli BL21 (DE3) strain using the heat shock method of transformation. A colony was seeded in 5 ml LB broth supplemented with 100 μg/ml ampicillin and 35 μg/ml chloramphenicol and grown at 37°C with shaking. The overnight culture was transferred to 800 ml fresh LB medium and cultured at 37°C with 200 rpm shaking. When the culture reached an optical density (OD) of 600 nm (OD₆₀₀) at 0.6, expression of the target proteins were induced by adding 2 mM isopropyl-β-D-thiogalactopyranoside (99% purity; Bio Basic, Ontario, Canada) and incubating for another 12 h at 30°C. Cultures were then harvested by centrifugation at 6,000 × g for 20 min at 4°C. The cell pellets were stored at -20°C overnight.

For the isolation of inclusion bodies (IBs), cell pellets were thawed in ice and re-suspended in 20 ml cold buffer containing 20 mM Tris-HCL, 0.5 M NaCl, 10% glycerol and protease inhibitor (1 mM phenyl methyl sulfonyl fluoride, Sigma-Aldrich, Seoul, Korea). Bacterial lyses was performed by sonication for 3 min with an interval of 2 s pulses and 1 s resting at 25% amplitude and centrifuged (12,000 × g, 20 min) at 4°C. Supernatant was removed and remaining pellet retained as IBs. The collected pellet was resuspended with denaturing buffer (20 mM Tris-HCl, 0.5 M NaCl, 10% glycerol and 6 M urea) followed by sonication for 1 min and centrifuged as before. Debris supernatant was discarded; remaining pellet was resuspended with denaturing buffer II (20 mM Tris-HCl, 0.5 M NaCl, 8 M urea, pH 8.0) and kept in 4°C with shaking overnight. The pellet was sonicated and centrifuged as before. Supernatant was separated as rescued protein from IBs, which is ready for purification[39].

The target proteins were purified by His-tag affinity chromatography (Qiagen, USA), dialyzed using a permeable cellulose membrane (molecular cut-off: 12–14 kDa, Spectrum Laboratories, East Tamaki, Auckland, USA) for 24 h at 4°C. The dialyzed target proteins were quantified using the Bradford assays (Bio-Rad, Hercules, CA, USA) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For immune detection of protein, the membranes were probed with mouse anti-histidine antibodies (1:500, Invitrogen, Carlsbad, CA, USA) and rabbit anti-M2 antibodies (1:1000). Rabbit anti-M2 antibody used in this experiment was generated by i.m. inoculation of KLH conjugated M2 peptide to the rabbit, two times of 2 weeks interval. Membranes were reacted with a 1:1000 dilution of anti-mouse or anti-rabbit IgG HRP. Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System (iNtRON Biotechnology, Gyeonggi-do, South Korea) and visualized by enhanced chemiluminescence (ECL). The purified proteins were used as a vaccine[23].

Preliminary experiment was conducted to determine the doses and efficacy of sM2 and 4sM2 protein on influenza virus infection. Mice vaccinated with sM2 (10 μg and 30 μg) showed 40% and 60% survival respectively while the mice vaccinated with 4sM2 (10 μg) registered 60% survival and the 4sM2 (30 μg) showed 100% survival against lethal infection of H5N1 (data not shown). In this study, monomeric sM2 protein was used as coating antigen for ELISA and stimulator in ELISPOT, multimeric 4sM2 protein used for vaccine study. For this, a total of 138 female BALB/c mice (5 weeks old) were purchased from Samtako (Seoul, Korea) and acclimated for 7 days at room temperature prior to use. Mice were divided into six experimental sets (Table 2). Four sets contained two groups of 11 mice each. One set had two groups of 17 (6 mice for lung histopathology at 3 and 5 dpi) mice each. The remaining sets had two groups containing eight mice each (for the long lasting and CTL response experiment). Mice were vaccinated i.m. with 30 μg of 4sM2 protein or 0.85% saline at 2 weeks interval as illustrated in Figure 1D. Mice inoculated with 0.85% saline considered as a negative control (NC). The first injection included Freund’s complete adjuvant, and 2 weeks later mice were given a boost with 4sM2 or 0.85% saline in Freund’s incomplete adjuvant.

The highly pathogenic (HPAI) A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird /Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3) and A/Chicken/Korea/116/2004(H9N2) influenza subtypes used in this study, were obtained from the virus collection at the College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea. All viruses were propagated in allantoic fluid from 10-day-old chicken embryos.

Blood were collected to analyze serum antibody levels at 0, 7, 21, and 180 days after vaccination. Blood were collected from the retro-orbital plexus, incubated at room temperature for 30 min; sera were separated by centrifugation (12,000 × g, 5 min) and stored at -20°C until analysis. Ether narcosis-anesthetized mice were bled from the heart with a syringe, dissected to expose the thoracic cavity, and the lungs were collected aseptically to determine lung virus titer and lung histopathology. Samples for the lung virus titer were stored at -70°C, and the histopathology samples were fixed in 10% formalin until analysis[36, 40].

The antibody level specific to sM2 was evaluated by ELISA as described previously[23, 41]. Briefly, 96-well immunosorbent plates (Nunc-Immuno Plate MaxiSorp; Nunc Life Technologies, Basel, Switzerland) were sensitized with 3 μg/ml of sM2 protein, M2 peptide or inactivated purified virions for 12 h at 4°C then washed three times with PBS (pH 7.4) containing 0.05% Tween 20 (PBS-T). The wells were blocked with 300 μl of 10% skim milk in PBS for 2 h at room temperature followed by washing again with PBS-T. Sera were diluted 1: 500 for protein and peptide and 1:100 for virus coated plates, added in triplicate, and incubated for 2 h at 37°C. Following another round of washing rabbit anti-mouse IgG HRP antibody (Sigma, Seoul, Korea) was added to each well (1:1000), and incubated for an additional 2 h at 37°C. Substrate solutions containing tetramethylbenzidine and H₂O₂ were added after final washing. The reaction performed at room temperature and terminated immediately with stop solution (2 N H₂SO₄). Optical density was measured at 450_nm using an ELISA auto reader (Molecular Devices, Sunnyvale, CA, USA).

IgG isotyping was performed under the same conditions described for the IgG ELISA except for the secondary antibody. After the primary antibody reaction, the ELISA plates were incubated with 1000-fold diluted rabbit anti-mouse IgG1 HRP and IgG2a HRP (Zymed, San Francisco, CA, USA) for 2 h at 37°C. Subsequent steps were performed as described for the IgG ELISA.

Cytokine ELISPOTs were developed and counted as described previously to detect and compare the T-cell response to the 4sM2 protein[42, 43]. Briefly, BD ELISPOT 96-well plates were coated with anti-mouse interferon IFN-γ or interleukin IL-4 capture antibodies in 100 μl PBS/well and incubated at 4°C overnight. The plates were blocked with complete RPMI 1640 medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), and incubated in RT for 2 h. Freshly isolated splenocytes were added at 5 × 10⁴ cells/well in media containing the sM2 protein (1 μg/well) or M2 peptide (1 μg/well) or only medium (negative control), or 5 μg/ml phytohemagglutinin (positive control, Invitrogen, Carlsbad, CA, USA). Plates were incubated for 48 h at 37°C in 5% CO₂. After discarding the cells, the plates were treated sequentially with biotinylated anti-mouse IFN-γ and IL-4 antibodies, streptavidin-HRP, and substrate solution. Finally, the plates were washed with deionized water and dried for at least 2 h in the dark. Spots were counted automatically using an Immuno Scan Entry analyzer (Cellular Technology Ltd., Shaker Heights, OH, USA).

Mice were anesthetized by ether narcosis and infected intranasally with 10MLD₅₀ of challenge viruses in 20 μl PBS. The MLD₅₀ of A/EM/Korea/W149/06(H5N1), A/PuertoRico/8/34(H1N1), A/Aquatic bird /Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses were determined in 8 week old naive BALB/c mice. Lungs were collected at 3 and 5 dpi to measure lung virus titers and assess lung histopathology. Remaining mice were monitored for body weight changes and survival. Three mice from one set were selected at random 10 days after the final vaccination to analyze the T-cell immune responses. The remaining mice from the same set were challenged at 180 days (after final vaccination) for the long lasting protection assay.

Lung virus titers were determined as the 50% tissue culture infectious dose (TCID₅₀) as described previously[44, 45]. Briefly, lung tissues were homogenized in 500 μl PBS containing antibiotic and antimycotic compounds (Gibco, Grand Island, NY, USA). The supernatants were collected after centrifugation (12,000 × g, 15 min) of mechanically homogenized lung samples. MDCK cells were inoculated with a 10-fold serial dilution of sample and incubated at 37°C in a humid atmosphere of 5% CO₂ for 1 h. After 1 h of absorption, media was removed and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) trypsin (Thermo Fisher Scientific, Rockford, USA) was added to the infected cells and incubated for 3 days. Viral cytopathic effects were observed daily, and titers were determined by the HA test. For HA, chicken red blood cells (0.5%) were added to 50 μl of cell supernatant and incubated for 30 min. The virus titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method.

Lungs were collected aseptically at 3 and 5 days post infection. Tissues were fixed in 10% formalin solution, embedded in paraffin, sectioned, and stained with eosin. Tissue sections were examined under a light microscope to assess the pathological changes[46].

The research protocols for the use of mice in this study were conducted following approval from the Institutional Animal Care and Use Committee of Bioleaders Corporation (Reference number BLS-ABSL-10-011). Animal experiments were conducted in bio-safety level BSL-2 and BSL-3⁺ laboratory facilities.