Background
Human infection of highly pathogenic avian H5N1 influenza virus was first reported in Hong Kong in 1997, causing six deaths [
1]. Since then, human cases of H5N1 virus infection have been continually laboratory-confirmed in many countries, with approximately 60% death rate [
2]. Probable limited human-to-human spread of H5N1 subtype virus is believed to have occurred as a result of prolonged and very close contact [
3]. Owing to the universal lack of pre-existing immunity to H5N1 virus in the population, pandemic caused by the virus may outbreak. Vaccination is the preferred approach for the prevention of influenza infection. Inactivated H5N1 influenza vaccines have been proved to be effective in eliciting neutralizing antibodies against the virus in clinic trials, but proved to have poor immunogenicity [
4]. Novel strategies, including DNA vaccines, should be developed to cope with the H5N1 influenza virus that may cause potential pandemics.
Seasonal influenza A subtypes H1N1 and H3N2 have globally circulated in humans for a few decades. There are rare people that have no history of exposure to these viruses [
5,
6]. Although it is necessary to annually update vaccine strains to ensure effective protection against seasonal influenza infection in humans due to the frequent antigenic drift of the virus strains, seasonal human influenza-specific CTLs, mostly targeting conserved internal proteins, e.g., NP and M1, have been demonstrated to offer T cell cross-reactivity more or less against avian influenza H5N1 virus [
6‐
8]. The memory T cells established by seasonal human influenza A infection could not provide adequate protection, but could alleviate symptoms of influenza H5N1 virus infection [
7].
DNA vaccines based on various genes of H5N1 virus have already been explored previously, demonstrating that, when DNA vaccines encoding NP or M1 were used to immunize mice, multi-dose injection would be needed to provide effective protection [
9]. In this study, a single dose of vaccination with NP, M1 or NP + M1 DNAs from A/chicken/Henan/12/2004(H5N1) virus strain was evaluated in mice pre-exposed to A/PR8(H1N1) virus, which showed that DNA vaccination might be a quick and effective strategy against H5N1 infection in individuals innaive to influenza A virus.
Discussion
Seasonal influenza A subtypes H1N1 and H3N2, as well as type B virus, have globally co-circulated in the human population for a few decades. Infection of influenza virus induces specific immune responses in the human body, including both humoral and cell-mediated immune responses. Due to antigenic drift that is a continuous ongoing process in type A influenza virus, the immunity induced by a certain strain is usually limited. However, more and more recent researches have demonstrated that specific CTLs, established by influenza exposure and mostly targeting the virus internal proteins, provide some level of cross-protection against not only antigenically distinct viruses of the same subtype (drift variants) but also different subtypes [
10‐
13]. In vitro testing with T cells isolated from healthy volunteers has been proved that the T cells could cause host cells infected with swine or avian influenza virus to undergo lysis [
7,
8]. In vivo experiments using a mouse model have also testified the cross-protection offered by influenza T cell responses against lethal challenge with heterologous virus [
14,
15]. Similar results were obtained in our present study. After exposed to A/PR8(H1N1) virus, mice gained partial protection against lethal A/chicken/Henan/12/2004(H5N1) virus challenge. Four of the total 12 mice survived (Table
2). Transfer of anti-H1N1 antiserum to naive mice could fully protect mice against H1N1 virus challenge, but had no use in defending them against H5N1 virus challenge (Table
1). These indicate that the partial intersubtypic cross-protection mainly relies on the cell-mediated immune responses induced by infection.
Though the cross-protection provided by infection could play a role in alleviating symptoms of H5N1 infection and reducing death, it is after all very limited. Vaccination is an indispensable way to fight against human infection with avian H5H1 virus. Various kinds of vaccines to H5N1 influenza virus have been tried preclinically or clinically, including inactivated whole-virion vaccines [
16,
17], split vaccines [
18,
19] and subunit vaccines [
20]. However, these vaccines induce only humoral responses and are mainly based on the virus surface protein HA, which are time-consuming on preparation and have been proved to be low immunogenic. Adjuvant addition and increased dose of antigen have to be adopted to increase the immune effect [
21,
22]. Compared with these conventional vaccines, DNA vaccine has lots of advantages. It induces balanced immune responses and can be prepared in a short time and on a large scale, with high purity and stability [
23]. It seems that DNA vaccine is a suitable candidate for pandemic vaccines. According to our previous studies, influenza DNA vaccines based on the surface protein, hemagglutinin or neuraminidase, could provide good protection against lethal challenge with homologous virus, including H5 and other subtypes, whereas those based on the internal protein, either NP or M1, failed to offer satisfactory protection even with multi-dose injection [
24‐
27]. In our present study, after mice had been infected beforehand with A/PR8 (H1N1) to mimic the seasonal influenza virus infection, they were immunized once with H5N1 virus NP DNA, M1 DNA or NP + M1 DNAs, and were then challenged with a lethal dose of the homologous H5N1 virus. The results are somehow unexpected (Table
2). The survival rates offered by a single dose of H5N1 NP DNA or NP + M1 DNA vaccination in pre-exposed mice reached 83% (10/12) and 100% (12/12), respectively. The protective ability (as expressed by survival rate, bodyweight loss and lung virus titer) in these two subgroups of mice had significant difference as compared with that in pre-exposed but unimmunized control group.
Influenza vaccines based on internal proteins induce specific CTL responses that can kill infected cells and help the host recovery from the infection. The antibodies induced by NP or M1 contribute little to providing protective ability, as shown in our and many other researches [
23,
24,
28,
29]. In our present study, the level of cellular immune responses, as reflected by the number of the IFN-γ secreting splenocytes in mice, were correlated with degree of protection (Figure
2 and Table
2). A single dose of H5N1 NP DNA or NP + M1 DNAs significantly enhanced the specific cellular response in mice pre-exposed to H1N1 virus, compared with that in the corresponding unexposed mice. In spite of this, we noticed that, though the residue lung virus titers were significantly reduced in pre-exposed mice immunized with NP DNA or NP + M1 DNAs (Table
2), they were not as low as those in mice immunized with HA or NA DNA, as shown in our previous experiments [
9]. This may be due to the lack of effective specific Abs to prevent virus from attaching to and releasing among host cells.
The concern about safety of DNA vaccines always exists, including potential integration of plasmid into host genome, induction of autoimmune responses or immunologic tolerance, and so on, but DNA vaccines have been approved to use in animals such as horses [
30] and dogs [
31]. DNA vaccines have also entered the clinic for initial safety and immunogenicity testing in humans for various infectious diseases, like HIV infections [
32], influenza virus infections [
33], malaria [
34] and hepatitis B infections [
35]. All DNA vaccines tested so far were well tolerated with no local or systemic serious adverse effects [
36].
Methods
Viruses and mice
Influenza virus strains used in this study included a mouse-adapted A/PR/8/34(H1N1) virus and an H5N1 virus A/chicken/Henan/12/2004(H5N1), which had been through repeated lung-to-lung passages and adapted in mice as described in our previous studies [
25,
38]. They were frozen at -70°C until use. All the experiments with live H5N1 virus were performed in a biosafety level 3 containment facilities. SPF female BALB/c mice, aged 6-8 weeks old, were purchased from the Center for Disease Control and Prevention in Hubei Province, China. They were bred and maintained in SPF conditions all along. All the performances on mice in this study followed the Chinese Regulations for the Administration of Laboratory Animals.
DNA vaccines and peptides
Plasmids pCAGGSP7/NP, pCAGGSP7/M1 were constructed by cloning the PCR products of NP and M1 genes from the A/chicken/Henan/12/2004(H5N1) influenza virus strain into the plasmid expression vector pCAGGSP7, respectively, as described previously [
9,
24]. The plasmids were propagated in E. coli XL1-blue bacteria and purified using QIAGEN purification kits (QIAGEN-tip 500). The peptide RAVKLYKKLKRE for M1 protein [
39] and the peptide TYQRTRALV for NP protein [
40], which were used for IFN-γ ELISPOT assay, were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, China.
Virus infection and challenge
The virus pre-exposure mouse model was achieved by intranasal infection with 5 μl of the viral suspension containing 5LD
50 influenza virus A/PR/8/34 six weeks before immunization. For challenge experiments, the mice were anesthetized and challenged with 20 μl of the viral suspension containing 20LD
50 influenza virus A/chicken/Henan/12/2004(H5N1) or A/PR8(H1N1) by intranasal route. The small volume of the virus suspension induced local infection, which was not lethal. On the other hand, the large volume induced total respiratory infection that caused virus shedding from the lung and led to death from viral pneumonia 5 - 10 days later [
41].
Immunization
Mice were immunized with NP DNA, M1 DNA or a mixture of the two DNAs dissolved in 50 μl of Tris-EDTA buffer at a dosage of 50 μg (25 μg each in the mixture of two DNAs) by injection into the quadriceps muscles. After injection, a pair of electrode needles with 5 mm apart was inserted into the muscle to cover the DNA injection site and electric pulses were delivered using an electric pulse generator (Electro Square Porator T830 M; BTX, San Diego, CA). Three pulses of 100 V each, followed by three pulses of the opposite polarity, were delivered to each injection site at a rate of one pulse per second. Each pulse lasted for 50 ms.
Specimens
Three days after the challenge, six mice from each group were randomly taken out for sample collection. The mice were anaesthetized with chloroform and then bled from the heart with a syringe. The sera were collected from the blood and used for specific IgG Ab assay. After bleeding, the mice were incised ventrally along the median line from the xiphoid process to the point of the chin. The trachea and lungs were taken out and washed 3 times by injecting with a total of 2 ml of PBS containing 0.1% BSA. The bronchoalveolar washes were used for virus titration after removing cellular debris by centrifugation.
Ab assay by ELISA
The concentrations of IgG Abs against H1N1 virus, NP or M1 protein were measured by ELISA. ELISA was performed sequentially from the solid phase using a series of reagents consisting of first, inactivated H1N1 vaccine, NP or M1 protein prepared by Shanghai Institute of Biological Products; second, serial 2-fold dilutions of sera from each group of immunized or preimmunized mice; third, goat anti-mouse IgG Ab (γ-chain specific) (Southern Biotechnology Associates) conjugated with biotin; fourth, streptavidin conjugated with alkaline phosphatase (Southern Biotechnology Associates); and finally, p-nitrophenyl-phosphate. The amount of chromogen produced was measured based on absorbance at 405 - 450 nm in an ELISA reader (Labsystems Multiskan Ascent). Ab-positive cut-off values were set as means + 2 × SD of preimmunized sera. An ELISA Ab titer was expressed as the highest serum dilution giving a positive reaction.
HI assay
The anti-HA Ab titers were measured by HI assay. Receptor destroying enzyme-treated sera were serially diluted (twofold) in V-shaped 96-well plates. Four hemagglutination units of virus were added to the test and incubated at room temperature for 15 min, followed by addition of 0.5% red blood cells and incubation at room temperature for 30 min. The HI titer is the reciprocal of the highest serum dilution that completely inhibits hemagglutination.
Passive serum transfer
Naive mice were passively immunized by tail vein injection with 300 μl of pooled serum from mice infected with A/PR8 (H1N1) influenza virus six weeks before or from mice uninfected. One day after the serum transfer, mice were challenged, as described above, with 20LD50 of H5N1 or H1N1 influenza virus.
IFN-γ ELISPOT assay
Spleen cells were isolated from mice for ELISPOT assays at 6 weeks after the vaccination. According to the instruction manual (U-CyTech, Netherlands), 96-well PVDF plates (Millipore, Bedford, MA) were coated with 100 μl of 10 μg/ml rat anti-mouse IFN-γ Ab in PBS and incubated at 4°C overnight. The plates were washed 3 times with sterile PBS and then blocked with 200 μl of blocking solution R and incubated at 37°C for 1 h. Next, 1 × 105 lymphocytes isolated from the spleen cells were added to the wells in triplicate, stimulated with 2 μg/ml of a synthesized influenza virus peptide, and incubated at 37°C for 18 h. The lymphocytes were then removed, and 100 μl of biotinylated anti-mouse IFN-γ Ab was added to each well and incubated at 37°C for 1 h. Subsequently, 100 μl of properly diluted Streptavidin-HRP conjugate solution was added and incubated at room temperature for 2 h after washing 5 times with PBS. Finally, the plates were treated with 100 μl of AEC substrate solution and incubated at room temperature for 20 min in the dark. The reaction was stopped by washing with dematerialized water. The plates were air-dried at room temperature and read using an ELISPOT reader (Bioreader 4000; Bio-sys, Germany).
Virus titrations
To examine cytopathic effect, the bronchoalveolar washes, diluted 10-fold serially starting from a dilution of 1:10, were inoculated onto the MDCK cells at 37°C for 2 days. The virus titer of each specimen, expressed as TCID50, was calculated by the Reed-Muench method. The virus titer in each experimental group was represented by the mean ± SD of the virus titer per ml of specimens from six mice in each group.
Statistics
The data from test groups were evaluated by Student's t-test; if P-value was less than 0.05, the difference was considered significant. The survival rates of mice in test and control groups were compared by using Fisher's exact test.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HYC carried out most of the experiments and wrote the manuscript. JW, WJZ and FF did part of the experiment and participated in manuscript preparation. CYH participated in antibody detection and lung virus titration. FYW participated in its design and coordination. ZC was the main designer of the experiment and revised the manuscript. All authors read and approved the final manuscript.