A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice
© Zheng et al; licensee BioMed Central Ltd. 2009
Received: 30 May 2008
Accepted: 12 February 2009
Published: 12 February 2009
Developing vaccines for the prevention of human infection by H5N1 influenza viruses is an urgent task. DNA vaccines are a novel alternative to conventional vaccines and should contribute to the prophylaxis of emerging H5N1 virus. In this study, we assessed whether a single immunization with plasmid DNA expressing H5N1 hemagglutinin (HA) could provide early protection against lethal challenge in a mouse model.
Mice were immunized once with HA DNA at 3, 5, 7 days before a lethal challenge. The survival rate, virus titer in the lungs and change of body weight were assayed to evaluate the protective abilities of the vaccine. To test the humoral immune response induced by HA DNA, serum samples were collected through the eye canthus of mice on various days after immunization and examined for specific antibodies by ELISA and an HI assay. Splenocytes were isolated after the immunization to determine the antigen-specific T-cell response by the ELISPOT assay.
Challenge experiments revealed that a single immunization of H5N1 virus HA DNA is effective in early protection against lethal homologous virus. Immunological analysis showed that an antigen-specific antibody and T-cell response could be elicited in mice shortly after the immunization. The protective abilities were correlated with the amount of injected DNA and the length of time after vaccination.
A single immunization of 100 μg H5 HA DNA vaccine combined with electroporation was able to provide early protection in mice against homologous virus infection.
The outbreak of human infections of H5N1 influenza in 1997 in Hong Kong and in 2003–2004 in most Asian countries demonstrated that purely avian viruses could be transmitted to humans and cause severe disease . Prior to the Hong Kong outbreak, H5 influenza viruses had been isolated only from avian species . They exist in a non-pathogenic form in wild aquatic birds in different regions of the world and in domestic ducks in Southern China [2–4]. Highly pathogenic avian influenza (HPAI) H5N1 viruses are now enzootic in several countries and are presently undergoing unprecedented geographic expansion among wild and domestic birds [1, 5–8]. Some person-to-person transmissions in family clusters have been observed in Vietnam, Thailand and Indonesia [9–11]. Although all H5N1 viruses isolated from humans retain characteristic features of avian influenza viruses and are not currently transmissible among humans, the potential for a pandemic caused by H5N1-HPAIV is increasing [8, 12].
To prevent influenza, a protective immunity must be induced, in advance, by vaccination. Immunization with inactivated vaccines has been the main technique used to prevent avian influenza for a long time. Some studies demonstrated that inactivated H5 vaccines could protect chickens and mice against the challenge with the homologous virus [7, 13, 14]. Meanwhile, it has been reported that immunizations with avian influenza H5N1 inactivated vaccines induced protective antibodies in humans [5, 15, 16]. Immunization with DNA vaccines is also one of the strategies for preventing avian influenza. Many studies showed that DNA vaccines could provide protection for chickens and mice against avian influenza types H3, H5, H7 and H9 [7, 17–22]. Our previous studies also showed that both hemagglutinin (HA)- and neuraminidase (NA)-DNA vaccines could protect mice from the challenge with either influenza A or B viruses [23–28].
In this study, an avian influenza virus strain A/Chicken/Henan/12/2004 (H5N1) was isolated from a farmed chicken in Henan province, China. The H5 virus was found to be able to replicate in BALB/c mice without adaptation and caused mortality, which demonstrated the infectivity of influenza H5N1 virus among species. The HA gene was cloned from the virus and the abilities of an HA DNA vaccine to provide protection for BALB/c mice against homologous virus infection were explored. We showed that a single immunization of H5N1 DNA vaccine was able to provide early protection in mice against homologous virus infection.
The virus A/Chicken/Henan/12/2004(H5N1) was isolated from a farmed chicken in Henan province, China. Viral isolates were identified by the hemagglutination assay after inoculating the allantoic cavity of 10 day-old specific pathogen-free (SPF) chicken embryos. Three days after the inoculation, allantoic fluids from infected eggs were harvested, aliquoted and stored in at -80°C. The 50% embryo lethal dose (ELD50) was determined for each stock and the viruses were subsequently isolated in a Biosafety Level 3 (BSL-3) facility. The viral RNA from the isolates propagated in 10-day-embryonated eggs was extracted by the cleavage of viruses with Trizol LS Reagent (Life Technologies, Inc.). The RNA was reverse-transcribed into single-stranded cDNA with a first strand cDNA synthesis kit (AMV) (Roche Diagnostics). The viral HA gene was amplified by PCR using the Expand High Fidelity PCR System (Roche Diagnostics) with virus-specific primers (F Primer 5'-GGTCTCGAGTGTCAAAATGGAGAAAATAGTGCTT-3', XhoI site and start codon in bold; R Primer, 5'-TCTCCCGGGACAAATTTAAAT GCAAATTCTGCAT-3', Sma I site and stop codon in bold), then sequenced by the dideoxy method using an ABI PRISM 377 DNA Sequencer (Applied Biosystems).
The virus was found to be able to directly replicate in BALB/c mice without adaptation and caused mortality (data not shown). To prepare adapted virus to mice, lung-to-lung passages were performed. Briefly, the BALB/c mice were anesthetized and inoculated with 20 μl of the above-mentioned H5N1 viral suspension by intranasal drip. At the 3rd day after inoculation, the mice were sacrificed, and their trachea and lungs were taken out and washed 3 times with a total of 2 ml of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). The bronchoalveolar washings were collected and used for infecting the next batch of mice after removing the cellular debris by centrifugation . The lung-to-lung passages were repeated at least three times and the adapted viruses were harvested, aliquoted, and stored in at -80°C. The 50% mouse lethal dose (MLD50) of each stock was determined using the Reed-Muench method .
The plasmid pCAGGSP7/HA (HA DNA) was constructed by cloning the PCR product of the HA gene from A/Chicken/Henan/12/2004 (H5N1) influenza virus strain into the expression vector pCAGGSP7, as described previously [23, 30]. The nucleotide sequence of the HA gene was confirmed by the dideoxy method using ABI PRISM 377 DNA Sequencer (Applied Biosystems). The plasmid was propagated in Escherichia coli XL1-blue bacteria and purified using QIAGEN Purification Kits (QIAGEN-tip 500).
Nucleotide sequence accession numbers
The HA gene sequence from A/Chicken/Henan/12/2004 (H5N1) has been deposited in GenBank. The accession number is AY950232.
Immunization by in vivo electroporation
For immunization, the BALB/c mice were anaesthetized with a mixture of ketamine and lobelanine and injected with HA DNA into the right quadriceps muscle. A pair of 5-mm-apart electrode needles (26-gauge) was then inserted into the muscle to cover the DNA injection sites and electrical pulses were delivered using an electric pulse generator (Electro Square Porator T830 M; BTX, San Diego, CA). Three pulses of 100 V each were delivered to the injection site at a rate of one pulse per second, each lasting for 50 ms . Three pulses of the opposite polarity were then applied. All procedures described above have been reviewed and approved by Animal Care Committee of Wuhan Institute of Virology, Chinese Academy of Sciences.
The mice were anesthetized and challenged with the mouse-adapted strain A/Chicken/Henan/12/2004(H5N1) (5 LD50) by intranasal administration with 20 μl of the viral suspension at 3, 5 or 7 days after immunization. This infection caused rapid and widespread viral replication in the lung and death of the control non-immunized mice within 7–10 days .
Blood samples were collected from either medial or lateral canthus of mice on various days after immunization for antibody detection. For lung virus titration, mice were sacrificed three days after the viral challenge. A ventral incision was made along the median line from the xiphoid process to the point of the chin. The trachea and lungs were taken out and washed twice by injecting 2 ml of PBS containing 0.1% BSA. The bronchoalveolar wash was used for virus titration after removing cellular debris by centrifugation .
Antibody detection by ELISA and HI assay
The serum IgM and IgG Abs against HA were measured by ELISA. ELISA was performed sequentially in a 96-well polystyrene microtiter plate with reagents consisting of: 1) HA molecules purified from the H5N1 virus ; 2) serial two-fold dilutions of sera from mice; 3) goat anti-mouse IgG or IgM Ab (Southern Biotechnology Associates, Inc. USA) conjugated with biotin; 4) streptavidin conjugated with alkaline phosphatase (Southern Biotechnology Associates, Inc. USA); and 5) p-nitrophenyl-phosphate. The amount of chromogen produced was measured based on absorbance at 414 and 405 nm in a Labsystems Multiskan Ascent Autoreader (model 354, Finland). The Ab-positive cut-off values were set as mean + 2 SD of non-immunized sera. An ELISA Ab titer was expressed as the highest serum dilution giving a positive reaction.
The serum neutralization activity was measured by hemagglutination inhibition assays. Receptor destroying enzyme (RDE)-treated sera were serially diluted (2 fold) in V-bottom 96-well plates. Approximately 4 HA units of viral antigen was incubated with the serum for 30 min at room temperature, followed by the addition of 0.5% cRBCs and incubation at room temperature for 40 min. The inhibition of hemagglutination at the highest serum dilution was considered as the HI titer of the serum.
Specific cellular immune responses were assessed by IFN-γ and IL-4 ELISPOT assays using mouse splenocytes. Assays were performed according to the instruction manual (U-CyTech, Netherlands). Briefly, ELISPOT 96-well plates Multiscreen Assay System (Millipore) were coated with anti-mouse IFN-γ and IL-4 capture Abs and incubated for 24 h at 4°C. On the following day, the plates were washed and blocked for 2 h with 1% BSA. 2 × 105 splenocytes from the immunized mice were added to each well and stimulated overnight at 37°C in 5% CO2 in the presence of RPMI 1640 (negative control), Con A (positive control), or 2 μg/ml purified HA-protein . After 18 h of stimulation, the cells were washed and incubated for 1 h at 37°C with biotinylated detector antibodies (U-CyTech). The plates were washed, and streptavidin-HRP Conjugate (U-CyTech) was added to each well and incubated for 1 h at 37°C. The plate was washed, and AEC coloring system (U-CyTech) was added to each well. The plates were then rinsed with distilled water and dried at room temperature. Spots were counted by an automated ELISPOT Bioreader 4000 (Bio-Sys Limited Germany) . The results were expressed as the number of spot forming cells (SFC) per 106 splenocytes cells in the ELISPOT experiment
The bronchoalveolar wash was diluted 10-fold serially starting from a dilution of 1:10, inoculated on Madin Darby canine kidney (MDCK) cells, incubated at 37°C and examined for cytopathic effect 2 days later. The virus titer of each specimen, expressed as the 50% tissue culture infection dose (TCID50), was calculated by the Reed-Muench method . The virus titer in each experimental group is represented by the mean ± SD of the virus titer per ml of specimens from all mice in each group.
Statistical parameters (average values and standard deviations) were calculated using spss10.0 software. For comparisons of experimental groups, statistical analysis was performed using one-way ANOVA.spss for windows 10.0. p < 0.05 was considered significant. For survival, the probability was calculated by using Fisher's exact test, comparing the rate of survival in mice immunized with HA DNA to that of the control groups.
Immunization with one HA DNA dose provided early protection in BALB/c mice against lethal dosage of H5N1 virus challenge
Protection of mice immunized with HA DNA vaccine against lethal H5N1 virus challengea
Lung virus titerb
(No. of survivors/no. tested)
6.25 ± 0.35
4.80 ± 0.28c
4.25 ± 0.35c
5.75 ± 0.35
4.55 ± 0.07c
4.00 ± 0.70c
5.75 ± 0.35
4.55 ± 0.07c
3.25 ± 0.35c
6.25 ± 0.35
Antibody response induced in mice at early stage when immunized once with various dosages of HA DNA
Cell-mediated immune response induced in mice at an early stage when immunized once with various dosages of HA DNA
To prepare for a pandemic influenza outbreak, any human isolate of a novel subtype should be considered as a potential hazard against humans and the feasibility of a vaccine production should be evaluated [36, 37]. To control avian influenza, plasmid DNA vaccines have been considered as alternatives to inactivated vaccine. Many studies have shown that H1-, H3-, H5-, H7- and H9-DNA vaccines could protect mice and chickens against challenge with the homologous influenza virus [20–22, 28, 35].
In this study, BALB/c mice were immunized once by electroporation with HA DNA at different dosages. Three, 5 or 7 days after the immunization, the mice were challenged with a lethal dose (5 LD50) of H5N1 virus. The results showed that immunization once with 50 μg or 100 μg of HA DNA provided partial protection for mice against homologous H5N1 virus 5 days after immunization and that immunization once with 100 μg provided good protection against the H5N1 virus 7 days after immunization. We demonstrated that the immunity could be established shortly after the vaccination with a single dose of H5N1 DNA vaccine and that it afforded early protection against H5N1 virus in mice.
The breadth of the immune responses induced by the DNA vaccine included both cell-mediated and humoral immunities. Although most studies with HA DNA have reported that effective antibody responses were induced only after a booster immunization [38–40], a single dose of a DNA vaccine had also been proved effective . Tsang et al recently demonstrated that a single intramuscular DNA vaccination, when combined with electroporation, significantly enhanced both the onset and duration of primary antibody response and possibly the duration of immune memory . We obtained similar results in our previous experiments with H9 virus HA DNAs [27, 28]. The present study demonstrated that a single dose of H5 virus HA DNA could induce specific IgM and IgG antibodies in mice (Figures 2 and 3). The IgM antibody, which appears prior to other kinds of antibodies during the immune response, was detectable in mice on the 3rd day after immunization, reached its highest value on the 10th day and then gradually disappeared. The IgG antibody appeared a little later. It was detectable on the 5th day and maintained a high level over a 4-week measurement. It indicated that a single dose of H5 virus HA DNA could induce a primary antibody response and that the level of the induced IgG antibody was long-lasting.
The cellular immune response is mediated by antigen specific CD4+ and CD8+ T cells. T cells cannot recognize free pathogens, but instead identify infected cells and exert effector functions, including direct cytotoxic effects and cytokine release [43, 44]. It has been reported that muscle inoculation of DNA preferentially elicits a Th1-type response with relatively high CTL activity [45, 46]. Our results demonstrated, besides circulating anti-influenza virus antibodies, a potent systemic cellular immune response following a single immunization of H5N1 HA DNA. The mice immunized once with HA DNA in dosages ranging from 10 μg to 100 μg raised significant amounts of IFN-γ-producing cells, but low levels of IL-4-producing cells, in response to the stimulation with H5-HA protein (Figure 5). These findings suggested that H5N1 DNA vaccine might be a potent inducer of systemic type 1 T cells, which were capable of mediating protection in the respiratory tract. The H5 HA DNA vaccine might, to some degree, induce a cytotoxic response in a naive population, although the cytotoxic functionality of the CD8+ T cells was not measured.
A series of human clinical trials with inactivated H5N1 vaccines demonstrated that inactivated H5N1 vaccines, with or without adjuvant, could elicit neutralization antibodies and cross-reactive neutralization antibodies to currently circulating variant H5N1 strains [47–50]. These clinical trials also showed us some unexpected findings. Nolan et al. recently demonstrated the better immune response for children compared to adults when they received two doses of inactivated, split H5N1 vaccine and the lower seroresponse to the H5N1 vaccines in those who had been previously vaccinated against seasonal influenza [49, 50]. These findings suggested that pre-existing antibodies to human influenza viruses might influence the immunogenicity of H5N1 vaccines. It is worthy to know if these findings are only limited to inactivated vaccines. Furthermore, although the H5 HA DNA vaccine was shown in the present study to provide early protection against lethal challenge in mice model, how it would behave in a human body remains unknown.
We have demonstrated that a single immunization of 100 μg of an H5 HA DNA vaccine, combined with electroporation, elicited both humoral and cellular immune responses, which provide mice the early protection against avian influenza H5N1 virus. It might be helpful for the ideal of providing rapid onset of protection against AIV infection with a "single shot" vaccine.
This study was supported by National 973 Project (2005CB523007, 2006CB933102), European commission 6th framework programme (FP6-2005-SSP-5-B-INFLUENZA), Chinese Academy of Sciences (KSCX1-YW-R-14), Hunan Provincial Science and Technology Department (2006NK2003) and National Key Technology R&D Program of China (2006BAD06A03).
- Lipatov AS, Govorkova EA, Webby RJ, Ozaki H, Peiris M, Guan Y, Poon L, Webster RG: Influenza: emergence and control. J Virol. 2004, 78 (17): 8951-8959. 10.1128/JVI.78.17.8951-8959.2004.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen H, Deng G, Li Z, Tian G, Li Y, Jiao P, Zhang L, Liu Z, Webster RG, Yu K: The evolution of H5N1 influenza viruses in ducks in southern China. Proc Natl Acad Sci USA. 2004, 101 (28): 10452-10457. 10.1073/pnas.0403212101.View ArticlePubMedPubMed CentralGoogle Scholar
- Hinshaw VS, Webster RG, Turner B: The perpetuation of orthomyxoviruses and paramyxoviruses in Canadian waterfowl. Can J Microbiol. 1980, 26 (5): 622-629.View ArticlePubMedGoogle Scholar
- Sharp NJ, Sullivan M, Harvey CE, Webb T: Treatment of canine nasal aspergillosis with enilconazole. J Vet Intern Med. 1993, 7 (1): 40-43.View ArticlePubMedGoogle Scholar
- Horimoto T, Kawaoka Y: Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol. 2005, 3 (8): 591-600. 10.1038/nrmicro1208.View ArticlePubMedGoogle Scholar
- Xu X, Subbarao , Cox NJ, Guo Y: Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology. 1999, 261 (1): 15-19. 10.1006/viro.1999.9820.View ArticlePubMedGoogle Scholar
- Kodihalli S, Haynes JR, Robinson HL, Webster RG: Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin. J Virol. 1997, 71 (5): 3391-3396.PubMedPubMed CentralGoogle Scholar
- Shortridge KF: Pandemic influenza: a zoonosis?. Semin Respir Infect. 1992, 7 (1): 11-25.PubMedGoogle Scholar
- Tran TH, Nguyen TL, Nguyen TD, Luong TS, Pham PM, Nguyen VC, Pham TS, Vo CD, Le TQ, Ngo TT, et al: Avian influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med. 2004, 350 (12): 1179-1188. 10.1056/NEJMoa040419.View ArticlePubMedGoogle Scholar
- Ungchusak K, Auewarakul P, Dowell SF, Kitphati R, Auwanit W, Puthavathana P, Uiprasertkul M, Boonnak K, Pittayawonganon C, Cox NJ, et al: Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med. 2005, 352 (4): 333-340. 10.1056/NEJMoa044021.View ArticlePubMedGoogle Scholar
- Normile D: Avian influenza. Human transmission but no pandemic in Indonesia. Science. 2006, 312 (5782): 1855-10.1126/science.312.5782.1864.View ArticleGoogle Scholar
- Lin YP, Shaw M, Gregory V, Cameron K, Lim W, Klimov A, Subbarao K, Guan Y, Krauss S, Shortridge K, et al: Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc Natl Acad Sci USA. 2000, 97 (17): 9654-9658. 10.1073/pnas.160270697.View ArticlePubMedPubMed CentralGoogle Scholar
- Guan Y, Shortridge KF, Krauss S, Webster RG: Molecular characterization of H9N2 influenza viruses: were they the donors of the "internal" genes of H5N1 viruses in Hong Kong?. Proc Natl Acad Sci USA. 1999, 96 (16): 9363-9367. 10.1073/pnas.96.16.9363.View ArticlePubMedPubMed CentralGoogle Scholar
- Lu X, Tumpey TM, Morken T, Zaki SR, Cox NJ, Katz JM: A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans. J Virol. 1999, 73 (7): 5903-5911.PubMedPubMed CentralGoogle Scholar
- Middleton D, Bingham J, Selleck P, Lowther S, Gleeson L, Lehrbach P, Robinson S, Rodenberg J, Kumar M, Andrew M: Efficacy of inactivated vaccines against H5N1 avian influenza infection in ducks. Virology. 2007, 359 (1): 66-71. 10.1016/j.virol.2006.08.046.View ArticlePubMedGoogle Scholar
- Chen H, Smith GJ, Li KS, Wang J, Fan XH, Rayner JM, Vijaykrishna D, Zhang JX, Zhang LJ, Guo CT, et al: Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control. Proc Natl Acad Sci USA. 2006, 103 (8): 2845-2850. 10.1073/pnas.0511120103.View ArticlePubMedPubMed CentralGoogle Scholar
- Kodihalli S, Kobasa DL, Webster RG: Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines. Vaccine. 2000, 18 (23): 2592-2599. 10.1016/S0264-410X(99)00485-5.View ArticlePubMedGoogle Scholar
- Kodihalli S, Goto H, Kobasa DL, Krauss S, Kawaoka Y, Webster RG: DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice. J Virol. 1999, 73 (3): 2094-2098.PubMedPubMed CentralGoogle Scholar
- Ljungberg K, Wahren B, Almqvist J, Hinkula J, Linde A, Winberg G: Effective construction of DNA vaccines against variable influenza genes by homologous recombination. Virology. 2000, 268 (2): 244-250. 10.1006/viro.2000.0199.View ArticlePubMedGoogle Scholar
- Ljungberg K, Kolmskog C, Wahren B, van Amerongen G, Baars M, Osterhaus A, Linde A, Rimmelzwaan G: DNA vaccination of ferrets with chimeric influenza A virus hemagglutinin (H3) genes. Vaccine. 2002, 20 (16): 2045-2052. 10.1016/S0264-410X(02)00049-X.View ArticlePubMedGoogle Scholar
- Lee CW, Senne DA, Suarez DL: Development of hemagglutinin subtype-specific reference antisera by DNA vaccination of chickens. Avian Dis. 2003, 47 (3 Suppl): 1051-1056. 10.1637/0005-2086-47.s3.1051.View ArticlePubMedGoogle Scholar
- Cherbonnel M, Rousset J, Jestin V: Strategies to improve protection against low-pathogenicity H7 avian influenza virus infection using DNA vaccines. Avian Dis. 2003, 47 (3 Suppl): 1181-1186. 10.1637/0005-2086-47.s3.1181.View ArticlePubMedGoogle Scholar
- Chen Z, Sahashi Y, Matsuo K, Asanuma H, Takahashi H, Iwasaki T, Suzuki Y, Aizawa C, Kurata T, Tamura S: Comparison of the ability of viral protein-expressing plasmid DNAs to protect against influenza. Vaccine. 1998, 16 (16): 1544-1549. 10.1016/S0264-410X(98)00043-7.View ArticlePubMedGoogle Scholar
- Chen Z, Kadowaki S, Hagiwara Y, Yoshikawa T, Matsuo K, Kurata T, Tamura S: Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase. Vaccine. 2000, 18 (28): 3214-3222. 10.1016/S0264-410X(00)00149-3.View ArticlePubMedGoogle Scholar
- Kadowaki S, Chen Z, Asanuma H, Aizawa C, Kurata T, Tamura S: Protection against influenza virus infection in mice immunized by administration of hemagglutinin-expressing DNAs with electroporation. Vaccine. 2000, 18 (25): 2779-2788. 10.1016/S0264-410X(00)00087-6.View ArticlePubMedGoogle Scholar
- Chen Z, Kadowaki S, Hagiwara Y, Yoshikawa T, Sata T, Kurata T, Tamura S: Protection against influenza B virus infection by immunization with DNA vaccines. Vaccine. 2001, 19 (11–12): 1446-1455. 10.1016/S0264-410X(00)00351-0.View ArticlePubMedGoogle Scholar
- Chen J, Fang F, Li X, Chang H, Chen Z: Protection against influenza virus infection in BALB/c mice immunized with a single dose of neuraminidase-expressing DNAs by electroporation. Vaccine. 2005, 23 (34): 4322-4328. 10.1016/j.vaccine.2005.03.035.View ArticlePubMedGoogle Scholar
- Qiu M, Fang F, Chen Y, Wang H, Chen Q, Chang H, Wang F, Wang H, Zhang R, Chen Z: Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin- or neuraminidase-expressing DNA in BALB/c mice. Biochem Biophys Res Commun. 2006, 343 (4): 1124-1131. 10.1016/j.bbrc.2006.03.088.View ArticlePubMedGoogle Scholar
- Reed LJ, Muench H: A simple method of estimating fifty per cent endpoints. AmJHyg. 1938, 27: 493-497.Google Scholar
- Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991, 108 (2): 193-199. 10.1016/0378-1119(91)90434-D.View ArticlePubMedGoogle Scholar
- Aihara H, Miyazaki J: Gene transfer into muscle by electroporation in vivo. Nat Biotechnol. 1998, 16 (9): 867-870. 10.1038/nbt0998-867.View ArticlePubMedGoogle Scholar
- Tamura SI, Asanuma H, Ito Y, Hirabayashi Y, Suzuki Y, Nagamine T, Aizawa C, Kurata T, Oya A: Superior cross-protective effect of nasal vaccination to subcutaneous inoculation with influenza hemagglutinin vaccine. Eur J Immunol. 1992, 22 (2): 477-481. 10.1002/eji.1830220228.View ArticlePubMedGoogle Scholar
- Phelan MA, Mayner RE, Bucher DJ, Ennis FA: Purification of influenza virus glycoproteins for the preparation and standardization of immunological potency testing reagents. J Biol Stand. 1980, 8 (3): 233-242. 10.1016/S0092-1157(80)80039-4.View ArticlePubMedGoogle Scholar
- Webster RG, Kawaoka Y, Taylor J, Weinberg R, Paoletti E: Efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens. Vaccine. 1991, 9 (5): 303-308. 10.1016/0264-410X(91)90055-B.View ArticlePubMedGoogle Scholar
- Laddy DJ, Yan J, Corbitt N, Kobasa D, Kobinger GP, Weiner DB: Immunogenicity of novel consensus-based DNA vaccines against avian influenza. Vaccine. 2007, 25 (16): 2984-2989. 10.1016/j.vaccine.2007.01.063.View ArticlePubMedPubMed CentralGoogle Scholar
- Subbarao K, Chen H, Swayne D, Mingay L, Fodor E, Brownlee G, Xu X, Lu X, Katz J, Cox N, et al: Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology. 2003, 305 (1): 192-200. 10.1006/viro.2002.1742.View ArticlePubMedGoogle Scholar
- Katz JM: The impact of avian influenza viruses on public health. Avian Dis. 2003, 47 (3 Suppl): 914-920. 10.1637/0005-2086-47.s3.914.View ArticlePubMedGoogle Scholar
- Fynan EF, Webster RG, Fuller DH, Haynes JR, Santoro JC, Robinson HL: DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci USA. 1993, 90 (24): 11478-11482. 10.1073/pnas.90.24.11478.View ArticlePubMedPubMed CentralGoogle Scholar
- Ulmer JB, Deck RR, DeWitt CM, Friedman A, Donnelly JJ, Liu MA: Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines. Vaccine. 1994, 12 (16): 1541-1544. 10.1016/0264-410X(94)90081-7.View ArticlePubMedGoogle Scholar
- Johnson PA, Conway MA, Daly J, Nicolson C, Robertson J, Mills KH: Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses. J Gen Virol. 2000, 81: 1737-1745.View ArticlePubMedGoogle Scholar
- Deck RR, DeWitt CM, Donnelly JJ, Liu MA, Ulmer JB: Characterization of humoral immune responses induced by an influenza hemagglutinin DNA vaccine. Vaccine. 1997, 15 (1): 71-78. 10.1016/S0264-410X(96)00101-6.View ArticlePubMedGoogle Scholar
- Tsang C, Babiuk S, van Drunen Littel-van den Hurk S, Babiuk LA, Griebel P: A single DNA immunization in combination with electroporation prolongs the primary immune response and maintains immune memory for six months. Vaccine. 2007, 25 (30): 5485-5494. 10.1016/j.vaccine.2007.03.009.View ArticlePubMedGoogle Scholar
- Kuklin N, Daheshia M, Karem K, Manickan E, Rouse BT: Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization. J Virol. 1997, 71 (4): 3138-3145.PubMedPubMed CentralGoogle Scholar
- Riberdy JM, Flynn KJ, Stech J, Webster RG, Altman JD, Doherty PC: Protection against a lethal avian influenza A virus in a mammalian system. J Virol. 1999, 73 (2): 1453-1459.PubMedPubMed CentralGoogle Scholar
- Pertmer TM, Roberts TR, Haynes JR: Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery. J Virol. 1996, 70 (9): 6119-6125.PubMedPubMed CentralGoogle Scholar
- Feltquate DM, Heaney S, Webster RG, Robinson HL: Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization. J Immunol. 1997, 158 (5): 2278-2284.PubMedGoogle Scholar
- Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M: Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med. 2006, 354 (13): 1343-1351. 10.1056/NEJMoa055778.View ArticlePubMedGoogle Scholar
- Leroux-Roels I, Borkowski A, Vanwolleghem T, Drame M, Clement F, Hons E, Devaster JM, Leroux-Roels G: Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial. Lancet. 2007, 370 (9587): 580-589. 10.1016/S0140-6736(07)61297-5.View ArticlePubMedGoogle Scholar
- Nolan TM, Richmond PC, Skeljo MV, Pearce G, Hartel G, Formica NT, Hoschler K, Bennet J, Ryan D, Papanaoum K, et al: Phase I and II randomised trials of the safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in healthy adults. Vaccine. 2008, 26 (33): 4160-4167. 10.1016/j.vaccine.2008.05.077.View ArticlePubMedGoogle Scholar
- Nolan T, Richmond PC, Formica NT, Hoschler K, Skeljo MV, Stoney T, McVernon J, Hartel G, Sawlwin DC, Bennet J, et al: Safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in infants and children. Vaccine. 2008, 26 (50): 6383-6391. 10.1016/j.vaccine.2008.08.046.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/17/prepub