- Open Access
Prokaryote-expressed M2e protein improves H9N2 influenza vaccine efficacy and protection against lethal influenza a virus in mice
© Kim et al.; licensee BioMed Central Ltd. 2013
Received: 25 August 2012
Accepted: 22 March 2013
Published: 3 April 2013
Influenza vaccines are prepared annually based on global epidemiological surveillance data. However, since there is no method by which to predict the influenza strain that will cause the next pandemic, the demand to develop new vaccination strategies with broad cross-reactivity against influenza viruses are clearly important. The ectodomain of the influenza M2 protein (M2e) is an attractive target for developing a vaccine with broad cross-reactivity. For these reasons, we investigated the efficacy of an inactivated H9N2 virus vaccine (a-H9N2) mixed with M2e (1xM2e or 4xM2e) proteins expressed in Escherichia coli, which contains the consensus of sequence the extracellular domain of matrix 2 (M2e) of A/chicken/Vietnam/27262/09 (H5N1) avian influenza virus, and investigated its humoral immune response and cross-protection against influenza A viruses.
Mice were intramuscularly immunized with a-H9N2, 1xM2e alone, 4xM2e alone, a-H9N2/1xM2e, or a-H9N2/4xM2e. Three weeks post-vaccination, mice were challenged with lethal homologous (A/ chicken /Korea/ma163/04, H9N2) or heterosubtypic virus (A/Philippines/2/82, H3N2 and A/aquatic bird/Korea/maW81/05, H5N2). Our studies demonstrate that the survival of mice immunized with a-H9N2/1xM2e or with a-H9N2/4xM2e (100% survival) was significantly higher than that of mouse-adapted H9N2 virus-infected mice vaccinated with 1xM2e alone or with 4xM2e alone (0% survival). We also evaluated the protective efficacy of the M2e + vaccine against infection with mouse-adapted H5N2 influenza virus. Protection from death in the control group (0% survival) was similar to that of the 1×M2e alone and 4xM2e alone-vaccinated groups (0% survival). Only 40% of mice vaccinated with vaccine alone survived challenge with H5N2, while the a-H9N2/1×M2e and a-H9N2/4×M2e groups showed 80% and 100% survival following mouse-adapted H5N2 challenge, respectively. We also examined cross-protection against human H3N2 virus and found that the a-H9N2/1×M2e group displayed partial cross-protection against H3N2 (40% survival), whereas vaccine alone, 1×M2e alone, 4×M2e alone, or H9N2/1×M2e groups showed incomplete protection (0% survival) in response to challenge with a lethal dose of human H3N2 virus.
Taken together, these results suggest that prokaryote-expressed M2e protein improved inactivated H9N2 virus vaccine efficacy and achieved cross-protection against lethal influenza A virus infection in mice.
Comparison of M2e sequence among vaccine and challenge strains
M2e sequence homology
A/ chicken /Vietnam/27262/2009 (H5N1)
A/ chicken /Korea/04163/2004 (H9N2)
MSLLTEVETPTRNG WECK CSDSSD
MSLLTEVETPTRNG WECK CSDSSD
A/aquatic bird/Korea/maW81/05 (H5N2)
MSLLTEVETPTRNG WECK CSDSSD
MSLLTEVETPI RNEWG CRCN DSSD
In this study, we investigated the efficacy of inactivated H9N2 virus vaccine (a-H9N2) mixed with 1×M2e or 4×M2e proteins expressed in Escherichia coli without adjuvant and were administered via the intramuscular route. Mice immunization and challenge experiments demonstrated that prokaryote-expressed M2e (1×M2e and 4×M2e) protein itself improved the efficacy of inactivated H9N2 virus vaccine and achieved cross-protection against lethal influenza A virus in mice.
Vaccines containing M2e protein induced cross-reactive humoral immune response in mice
Hemagglutination inhibition antibody titer of sera collected 2 week after boost immunization
HI titers (GMT)a
Inactivated vaccine (H9N2)
1xM2e + vaccine
4xM2e + vaccine
1×M2e or 4×M2e mixed with inactivated a-H9N2 vaccine induced protection against a mouse-adapted H9N2 avian influenza virus
1×M2e and 4×M2e proteins induced viral clearance in mice challenged with lethal dose of influenza viruses
During the last decade, H9N2 avian influenza viruses circulated worldwide in poultry populations causing mild respiratory disease and reductions in egg production [23–26]. However, H9N2 viruses do not appear to replicate efficiently or cause severe disease until in April 1999 when two World Health Organization (WHO) reference laboratories independently confirmed the isolation of avian H9N2 influenza A (A/HK/1073/99) viruses for the first time in humans . Following that year, another strain of H9N2 virus has been isolated repeatedly from the human population in mainland China [27, 28]. Other reports also indicated continuous interspecies transmission of H9N2 avian influenza virus from avian to mammalian hosts [27, 29]. Therefore, WHO declared H9N2 influenza virus as a potential candidate for the next influenza pandemic . Currently available influenza virus vaccines only induce humoral immunity by boosting anti-influenza antibodies whose targets are limited to the surface glycoproteins, HA and NA . Accordingly, contemporary universal influenza vaccines were developed mainly based on conserved sequences in M2, HA1, HA2, and NP proteins of the influenza virus . Because it is highly conserved in all types of influenza A viruses, M2e has been studied as a universal influenza vaccine target. A number of studies with M2e vaccines have already been conducted [17, 20, 32–34] and recently, phase I clinical studies have been carried out with chemically or genetically produced M2e fusion proteins . Tompkins et al. proposed that various M2e sequences of M2 expression constructs could be used as vaccines. Despite substantial sequence divergence, H5-derived vaccines might also protect against circulating H1N1 and H3N2 subtypes. Here, we investigated the potential of vaccines containing prokaryotic expressed monomer or polymer of M2e proteins (1×M2e and 4×M2e, respectively) without adjuvant, to contribute to cross-protective immunity against several influenza virus subtypes. 1×M2e and 4×M2e clones were generated by using consensus M2e gene from an H5N1 avian virus without its trans-membrane domain (Table 1). In contrast to adjuvanted M2e vaccine studies, our serologic assays revealed that receipt of the prokaryotic cell-expressed M2e protein alone did not exhibit neutralizing activity against homologous or heterologous viruses indicating that our M2e formulation might not be sufficient to prevent morbidity. Similar results were also observed in a report that utilized baculovirus-expressed M2 VLPs . Surprisingly, apart from providing homologous protection, an inactivated H9N2 (a-H9N2) vaccine in combination with the 4×M2e protein elicited enhanced cross-protection against a mouse-adapted H5N2 avian virus A/aquatic bird/Korea/maW81/05 and appeared to extend against a human H3N2 (A/Philippines/2/82) virus. Although sterile immunity was not achieved in any of our vaccination strategies, our data demonstrated potentially interesting enhancement in cross-protection.
Neutralization of influenza viruses has been primarily attributed as a function of antibodies directed against the HA surface glycoprotein antigen. However, anti-NA antibodies could also produce apparent neutralization by steric inhibition of virus adsorption and by interfering with viral release [37, 38]. Comparison of the deduced N2 amino acid sequences of the three viruses showed 91.9% and 83.8% homology between H9N2 and the H5N2 and H3N2 viruses, respectively. Therefore, we could not completely rule out the role of N2-derived antibodies in the cross-neutralization and protection observed in Figures 2 and 4. Apparently though, mixture of the a-H9N2 vaccine with monomer and polymer M2e exhibited improved serologic and survival values particularly those with the a-H9N2/4×M2e vaccine group. M2e-specific antibodies have been shown to induce humoral immunity and mediate protection against influenza infection in vivo[39, 40]. Furthermore, M2e-specific antibodies could promote antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-mediated cytotoxicity (CDC) [41, 42]. Therefore, we speculate that the difference in cross-protectivity afforded by the 1×M2e and 4×M2e, albeit administration of similar antigen concentrations, was mediated by the multiple copies of the M2e proteins that induced more robust cross-reactive antibodies.
Development of influenza M2e vaccines based on prokaryotic expression system without adjuvant is significant since E. coli-expressed M2e can be easily produced, safe and practical for animal and public health use. One concern about M2-based vaccines is the possibility that escape mutants may arise. However, a study of forced escape mutants found limited diversity  indicating that structural constraints, perhaps due to the requirements of the M1 structure encoded by the same segment, may limit drift . It is noteworthy that our vaccination strategy showed that H9N2/4×M2e could protect the immunized host against a range of the viruses containing mismatched amino acid sequence (ranging from 0 to 3 out of 24 amino acids) of the M2e protein from A/chicken/Vietnam/27262/09 (H5N1) strain (Table 2). A previous report has shown that the monoclonal anti-M2e 14C2 (IgG1) antibody inhibits plaque growth of some influenza strains in vitro. In addition, another study showed that M2 VLPs (eukaryotic expression system) provides complete cross protection against influenza A virus . However, producing the VLP-based M2 proteins is relatively tedious and expensive compared to prokaryote-expressed ones. In addition, most of the studies conducted so far used M2e proteins in combination with various adjuvants. Therefore such reports may not have appreciated the additive effect of the M2e proteins (alone) which we observed when combined with an inactivated whole-virus vaccine.
Recently, there have been some concerns regarding the possible emergence of a new influenza pandemic by avian H5N1, H9N2, and H3N2 variants. Furthermore, the number of reported cases of human infections with a novel triple reassortant A (H3N2)v (isolated from North American swine)  has been increasing since July 2012 [45, 46] indicating a potential public health risk. Therefore, the development of universal influenza vaccines against various subtypes is urgently needed. In this study, we have demonstrated the efficacy of E. coli-expressed M2e proteins in providing cross-protection against lethal influenza virus infection. We provide evidence that an inactivated a-H9N2 vaccine containing M2e proteins could be potential candidate for inducing cross-protection, as shown against avian A/ chicken /Korea/ma163/04(H9N2) and A/Aquatic bird/Korea/maW81/05(H5N2) and human A/Philippines/2/82(H3N2) influenza viruses. The cross-reactivity and protective efficacy of the M2e protein suggests that polymer M2e protein, which in our case 4×M2e, could potentially promote protection against other influenza viruses.
Overall, our results demonstrate that prokaryote-expressed 1×M2e and 4×M2e protein immunization with an inactivated vaccine are efficacious against influenza A virus in mice. Although sterile immunity was not achieved in any of our vaccination strategies, our data demonstrated potentially interesting enhancement in cross-protection. These findings may offer an approach to control epidemic and pandemic influenza viruses.
Materials and methods
Construction of plasmids expressing 1×M2e or 4×M2e protein
The list and sequence of primers used for PCR analysis
5′-AAGCTT TAATGA GGATCC ACCTGAACCACCTGAACCACCTGAACCACCTTCAAGTTC-3
Mice and viruses
Five-week-old female BALB/c (H-2d) mice were purchased from SAMTAKO (Pyungteack, Korea). The A/chicken/Korea/ma163/04 (ma163/H9N2), A/aquatic bird/Korea/maW81/05 (maW81/H5N2), and A/Philippines/2/82 (Phil82/H3N2) were grown for two days at 37°C in the allantoic cavities of 10-day-old fertile chicken eggs. Clarified allantoic fluids were aliquoted and then stored at -70°C.
Madin-Darby Canine Kidney (MDCK) cells obtained from ATCC were maintained in EMEM (LONZA, Inc., Allendale, NJ) supplemented with 5% fetal bovine serum (LONZA, Inc.), 1% penicillin/streptomycin (Gibco-Invitrogen, Inc., Carlsbad, CA), and 1% non-essential amino acids (Gibco-Invitrogen, Inc.).
Vaccination and challenge study
Five-week-old female inbred BALB/c mice were used for all experiments. Groups of 19 mice were intramuscularly (i.m.) immunized with 2 μg of inactivated H9N2 vaccine (a-H9N2), only 1×M2e (15 μg), only 4×M2e (15 μg), inactivated H9N2 + 1×M2e (a-H9N2/1×M2e) and inactivated H9N2 + 4×M2e (a-H9N2 vaccine/4×M2e) with two doses at three week intervals. Two weeks after the final immunization, mice were lightly anaesthetized and challenged intranasally (i.n.) with 2LD50 of A/chicken/Korea/ma163/04 (ma163/H9N2), A/aquatic bird/Korea/maW81/05 (maW81H5N2), or A/Philippines/2/82 (Phil82/H3N2) in a volume of 30 μl. Following infection, three mice were sacrificed 3, 5, and 7 dpi for lung viral titrations whereas the remaining ten mice were monitored daily for morbidity assessed by measuring body weight loss and survival for up to 14 dpi. Individual body weights were recorded for each mouse on various days post-infection.
Hemagglutination inhibition (HI) test
Total lung homogenate samples were treated with receptor-destroying enzyme (RDE, Denka Seiken, Japan) at 37°C overnight, followed by heat-inactivation at 56°C for 30 min. RDE-treated lung samples were serially diluted two-fold and incubated with 25 μl of ma163/H9N2, maW81/H5N2, or Phil82/H3N2 virus in U-bottom microtiter plates (Nunc, Corning, NY) for 30 min, followed by incubation with 50 μl of 0.5% turkey red blood cells (tRBCs) for 30 min.
Twenty-five microliters of Phosphate buffer saline (PBS) was dispensed in a 96-well microplate. Heat-inactivated serum samples (at 25 ul volume) were added in the first wells and serially diluted two-fold. An equal volume (25 ul) of live influenza virus at a concentration of 102 TCID50/ml was added to all samples. The mixture of sera and virus was incubated at 37°C for 1 h, loaded onto near confluent MDCK cells in a 96-well tissue culture plate, and incubated for two days at 37°C in 5% CO2. The plates were incubated for 2 days and the cytopathic effect was visually assessed using an inverted microscope. 50 μl of either cell supernant in U-bottom microtiter plate (Nunc, NY, USA), followed by incubation with 50 μl of 0.5% tRBCs for 30 min.
Virus titers in lung tissues
To determine titers of infectious virus in lungs of infected mice, lung samples from three mice per group were collected 3, 5, or 7 dpi. Lung tissues from euthanized mice were aseptically extracted and homogenized in minimal essential medium (MEM). Antibiotics were added to achieve 10% (w/v) suspensions of lungs. Ten-fold serial dilutions of samples were added in quadruplicate to a monolayer of MDCK cells seeded in 96-well cell culture plates 18 h before infection, and allowed to absorb for 2 h at 37°C. Fresh medium was then added to the cells, which are incubated back at 37°C for 48 h. Virus cytopathic effect (CPE) was observed daily and the viral titer was determined by the hemagglutinin (HA) test as follows. Fifty μl of 0.5% tRBCs were added to 50 μl of cell culture supernatant and incubated at room temperature for 30 min. Wells showing HA activity were scored as positive. The virus titer was calculated by the Reed and Muench method  and expressed as log10TCID50/ml of lung tissue.
The data were analyzed using GraphPad Prism version 5.00 for Windows (GraphPad Software, La Jolla, CA). p values of less than 0.05 (p < 0.05) were considered to be statistically significant.
The research protocol for the use of mice in this study were conducted in strict accordance and adherence to relevant policies regarding animal handling as mandated under the Guidelines for Animal Use and Care of the Korea Center for Disease Control (K-CDC) and was approved by the Medical Research Institute (approval number CBNU-IRB-2012-GM01). Animal care and use in an enhanced biosafety level 3 containment laboratory was approved by the Animal Experiment Committee of Bioleaders Corp. (permit number BLS-ABSL-12-010).
This study was supported in part by a Top Brand Project grant from the Korea Research Council of Fundamental Science and Technology, Korea Research Institute of Bioscience and Biotechnology (KRIBB) Initiative Program (KGM3111013) and a 2010–0024405 from National Research Foundation of Korea.
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