An M2e-based synthetic peptide vaccine for influenza A virus confers heterosubtypic protection from lethal virus challenge
- Ji-Hong Ma†1,
- Fu-Ru Yang†1,
- Hai Yu1,
- Yan-Jun Zhou1,
- Guo-Xin Li1,
- Meng Huang1,
- Feng Wen1 and
- Guangzhi Tong1Email author
© Ma et al.; licensee BioMed Central Ltd. 2013
Received: 10 November 2012
Accepted: 4 July 2013
Published: 9 July 2013
Vaccination is considered as the most effective preventive method to control influenza. The hallmark of influenza virus is the remarkable variability of its major surface glycoproteins, HA and NA, which allows the virus to evade existing anti-influenza immunity in the target population. So it is necessary to develop a novel vaccine to control animal influenza virus. Also we know that the ectodomain of influenza matrix protein 2 (M2e) is highly conserved in animal influenza A viruses, so a vaccine based on the M2e could avoid several drawbacks of the traditional vaccines. In this study we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope, and then investigated its immune responses.
Our results show that the M2e-MAP induced strong M2e-specific IgG antibody,which responses following 2 doses immunization in the presence of Freunds’ adjuvant. M2e-MAP vaccination limited viral replication substantially. Also it could attenuate histopathological damage in the lungs of challenged mice and counteracted weight loss. M2e-MAP-based vaccine protected immunized mice against the lethal challenge with PR8 virus.
Based on these findings, M2e-MAP-based vaccine seemed to provide useful information for the research of M2e-based influenza vaccine. Also it show huge potential to study vaccines for other similarly viruses.
Influenza virus is a globally important respiratory pathogen which causes a high degree of morbidity and mortality in humans and animals annually . Influenza virus typically infects 10~20% of the total worldwide population during seasonal epidemics, resulting in three to five million cases of severe illness and 250,000 to 500,000 deaths per year . Moreover, novel influenza strains appear occasionally in the human population, causing pandemics. In 2009, the world confronted the first influenza pandemic of the 21st century, which iscaused by a novel influenza A H1N1 virus . Antigenic and genetic analysis has suggested that this pandemic H1N1 virus is a product of reassortment between genes of the human, avian and swine influenza strains .
To date, vaccination is considered as the most effective preventive measure to control influenza. However, conventional vaccines have many drawbacks, the most important is the uncertainty of virus selection strains to be included in each year’s vaccine formulation . The hallmark of influenza virus is the remarkable variability of its major surface glycoproteins, HA and NA, which allows the virus to evade existing anti-influenza immunity in the target population . The potential shortage of pandemic influenza vaccines and the absence of specific-immunity in the human population make the development of a cross-protective influenza vaccine, which is based on conserved antigens, a promising prophylactic strategy. M2e, the ectodomain of the M2 protein, is highly conserved across influenza a subtypes and has become an attractive antigen target for producing a cross-protective influenza vaccine conferring broad spectrum prevention [7–9]. In contrast to BALB/c mice, Wolf AI, et al. show that immunization of other inbred and outbred mouse strains did not induce protective Abs. So it suggested that it correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs .
In this study we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope. The vaccine can provide heterosubtypic protection against lethal virus challenge.
M2e-MAP immunization could induce high titers of M2e-specific IgG antibodies
M2e-MAP vaccination limited replication of virus and attenuated virus-induced lung pathology
Virus isolation and titrations in lungs on day 3 post challenge
Protection against challenge and virus titerb
M2e-MAP vaccination provided effective protection from lethal challenge with PR8 virus
The considerable antigenic variation in influenza A virus and other virus such as FMDV and HIV presents problems in their control by vaccination . In previous researches anti-M2e Abs induced by M2e-MAPs shows highly cross-reactive and can mediate protection to various viruses . In the case of influenza A virus, an attractive alternative approach would be to develop a cross-protective influenza vaccine based on conserved antigens such as M2e. Several studies have shown that immunization with M2e can protect against influenza A virus infection . Different results and conclusions from previous studies may be due to the difference of carrier proteins, adjuvant or routes of administration. The vaccine described in the present study has several advantages. The researchers showed that branched MAP based vaccine is better than single peptide based vaccine, and results also demonstrated that MAP-based vaccine could give high-titer antibody responses . And vaccines with T-helper epitope can induce an antibody response . So we have designed the vaccine by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope.
In this study, virus titers were determined by two methods, the first one is the traditional way by SPF eggs, and the second method was real time PCR which was showed previously . Bothexperiments showed that the M2e-MAP could induce strong M2e-specific IgG antibody, which responses following 2 doses immunization in the presence of Freund and limited viral replication. Also it could attenuate histopathological damage in the challenged mice lungs and was able to counteract weight loss and protect mice from lethal challenge with PR8 virus. Therefore, the induction of M2e-specific antibody responses was necessary for M2e-based vaccines in the prevention of these three virus infection.
Several distinct M2-based vaccine constructs have been developed and found that they could induce significant resistance to influenza type A virus replication in mice [15–19]. We routinely used a fairly severe viral challenge to evaluate the efficacy of the M2e-based vaccination. It may be noted that the challenge conditions used by various viruses to score protection after vaccination with an M2e-based vaccine vary widely [20–22]. In this study high titers M2e-specific antibody responses could be induced following immunization of M2e-MAP plus Freunds’ adjuvant. Using three virus strains to challenge the immuned mice and got good results. After challenged by PR8 virus, a highly lethal virus, it demonstrated a clear-cut protection against mortality. The rest two mild virulence virus strains did not allow to score for survival, but the protection was assayed by reduction of virus titer in lungs homogenates. So we can know that the M2e-MAP play important role in the development of the vaccine.
In conclusion, we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope. This study provided useful information for the further development of M2e-based influenza vaccine.
Materials and methods
Virus and peptide
Immunization and virus challenge
Six to eight weeks old female SPF BALB/c mice were purchased from the Sino-British SIPPR/BK Animal Co., Ltd (Shanghai, China) and were immunized subcutaneously (s. c.) with M2e-MAP (10μg per mouse) plus Freund’s complete adjuvant (FCA, sigma) at a final volume of 100 μL and boosted with the same amount of immuogen in Freund’s incomplete adjuvant (FIA, Sigma) at a 2-week intervals. Mice injected with Freund’s adjuvant alone were used as negative control. Mice sera were collected at 0,1,2,3 and 4 weeks post first immunization to detect specific antibody responses.
Two weeks after boost, mice were anesthetized and challenged intranasally (i.n.) with a lethal dose (10LD50) of PR8 and 106EID50 of SwGD96 and SwHLJ1. Challenged mice were observed for illness or death and weighed daily for 2 weeks. Lung tissues were collected from euthanized mice on day 3 post-challenge for further virological tests and histopathological analysis.
The M2e-specific antibody titers were determined by endpoint ELISA. Briefly, 96-well microtitre plates (Costar) were coated with peptide (0.5 μg per well) in 0.1 M carbonate buffer (pH 9.6) overnight at 4°C. After blocking with 5% skim milk in PBS for 2 h, serial diluted mice sera samples were added and incubated for 1 h at 37°C. Washing the ELISA plate, andhorseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:10,000 dilution, Beijing Zhongshan Biotechnology) was added and incubated for 1 h at 37°C. Assay was developed using 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (Amresco), and the reaction was stopped by adding 2 M H2SO4. The absorbance at 450 nm was measured by microplate autoreader (BioTek Instruments). The reciprocal of the highest dilution of the serum that is greater than twice average absorbance value of pre-vaccination serum was designated as the antibody titer.
Virus titers in lung
Lung tissues from euthanized mice were aseptically removed and homogenized in 1 ml of cold PBS. The homogenates were frozen at −80°C and later thawed for ease of handing. Solid debris was pelleted by centrifugation. Serial diluted from initial dilutions of 1:10 and then titrated for virus infectivity in 10-day-old SPF ECE. Virus titers were calculated by the Reed-Muench method and expressed as mean log10EID50 per milliliter ± standard deviation (SD).
In addition, lung tissues were also collected and tested by real-time RT-PCR assay recommended by WHO with some modifications as described . Lung tissues were homogenized in 300 μL of cold PBS and viral RNA were extracted by RNeasy Plus Mini kit (QIAGEN). Then reverse transcription were performed and viral amounts in lungs were determined by real-time PCR using the InfA primers and TaqMan probe. The specific primers and labeled fluorogenic-probe were as follows: InfA-F:5-GACCGATCCTGTCACCTCTGAC-3; InfA-R:5-AGGGCATTCTGGACAAAGCGTCTA-3; TaqMan probe:5-FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ-3.
The lung tissues of challenged mice were removed and immediately fixed in 10% neutral buffered formalin, and then embedded in paraffin wax. Sections were made at 4–6 μm thickness and mounted on slides. Histopathological changes were examined by H & E staining and observed under light microscopy.
The survival curves were calculated by Kaplan-Meier method and the significance were analyzed with log-rank test. Other data were analyzed by two-tailed Student’s t test. P < 0.0001 was considered significantly different. All analysis were performed by Graphpad Prism software.
In this study, all of the slaughter experiments were conducted in accordance with the guidelines of Shanghai Veterinary Research Institute, Shanghai, china (permit number SYXK 2011–0116). All animal procedures were approved by the Animal committee of Shanghai Veterinary Research Institute.
The study was supported by grants from NSFC-Guangdong Joint Foundation (U0931003), the Ministry of Agriculture of China (No. 2009ZX08010-022B), International Sci & Tech Cooperation Program (2010DFB33920) and the Excellent Scientist Program of Shanghai (09XD1405400).
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