The high variability of influenza virus causes difficulties in the control and prevention of influenza, thus seeking a promising approach for dealing with these problems is a hot topic. Haemagglutinin (HA) and neuraminidase (NA) are major surface antigens of the influenza virus, and provide effective protection against lethal challenges with this virus. We constructed a DNA vaccine (pHA-IRES2-NA) that co-expressed both HA and NA, and compared its protective efficacy and immunogenic ability with that of singly expressed HA or NA, or a mixture of the two singly expressed proteins. Our findings showed that both HA and NA proteins expressed by pHA-IRES2-NA could be detected in vivo and in vitro. The protection of DNA vaccines was evaluated by serum antibody titres, residual lung virus titres and survival rates of the mice. In the murine model, immunization of pHA-IRES2-NA generated significant anti-HA and anti-NA antibody, increased the percentage of CD8+ cells and gamma interferon-producing CD8+ cells and the ratio of Th1/Th2 (T helper) cells, which was comparable to the effects of immunization with HA or NA DNA alone or with a mixture of HA and NA DNA. All the mice inoculated by pHA-IRES2-NA resisted the lethal challenge by homologous influenza virus and survived with low lung virus titre. In addition, previous studies reported that co-expression allowed higher-frequency transduction compared to co-transduction of separated vector systems encoding different genes. The novel HA and NA co-expression DNA vaccine is a successful alternative to using a mixture of purified HA and NA proteins or HA and NA DNA.
AlheimM.,
LazdinaU.,
MilichD. R.,
SallbergM.2001; Flow cytometric determination of cytokine production and proliferation in hepatitis B core antigen specific murine CD4 cells: lack of correlation between number of cytokine producing cells and cytokine levels in supernatant. J Immunol Methods 258:157–167[CrossRef]
Aymard-HenryM.,
ColemanM. T.,
DowdleW. R.,
LaverW. G.,
SchildG. C.,
WebsterR. G.1973; Influenzavirus neuraminidase and neuraminidase-inhibition test procedures. Bull World Health Organ 48:199–202
BonaC.,
RaduD.,
KoderaT.2004; Molecular studies on the diversification of haemagglutinin-specific human neonatal repertoire subsequent to immunization with naked DNA. Vaccine 22:1624–1630[CrossRef]
BotA.,
AntohiS.,
BotS.,
Garcia-SastreA.,
BonaC.1997; Induction of humoral and cellular immunity against influenza virus by immunization of newborn mice with a plasmid bearing a hemagglutinin gene. Int Immunol 9:1641–1650[CrossRef]
ChenJ.,
LeeK. H.,
SteinhauerD. A.,
StevensD. J.,
SkehelJ. J.,
WileyD. C.1998; Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation. Cell 95:409–417[CrossRef]
ChenJ.,
FangF.,
LiX.,
ChangH.,
ChenZ.2005; Protection against influenza virus infection in BALB/c mice immunized with a single dose of neuraminidase-expressing DNAs by electroporation. Vaccine 23:4322–4328[CrossRef]
De MareA.,
BungenerL. B.,
RegtsJ.,
de Vries-IdemaJ.,
Van der ZeeA. G.,
WilschutJ.,
DaemenT.2008; The effect of pre-existing immunity on the capacity of influenza virosomes to induce cytotoxic T lymphocyte activity. Vaccine 26:2314–2321[CrossRef]
FeltquateD. M.,
HeaneyS.,
WebsterR. G.,
RobinsonH. L.1997; Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization. J Immunol 158:2278–2284
HeX. S.,
MahmoodK.,
MaeckerH. T.,
HolmesT. H.,
KembleG. W.,
ArvinA. M.,
GreenbergH. B.2003; Analysis of the frequencies and of the memory T cell phenotypes of human CD8+ T cells specific for influenza A viruses. J Infect Dis 187:1075–1084[CrossRef]
JohanssonB. E.,
GrajowerB.,
KilbourneE. D.1993; Infection-permissive immunization with influenza virus neuraminidase prevents weight loss in infected mice. Vaccine 11:1037–1039[CrossRef]
JohnsonP. A.,
ConwayM. A.,
DalyJ.,
NicolsonC.,
RobertsonJ.,
MillsK. H.2000; 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 81:1737–1745
LambkinR.,
NovelliP.,
OxfordJ.,
GelderC.2004; Human genetics and responses to influenza vaccination: clinical implication. Am J Pharmacogenomics 4:293–298[CrossRef]
LiX.,
FangF.,
SongY.,
YanH.,
ChangH.,
SunS.,
ChenZ.2006; Essential sequence of influenza neuraminidase DNA to provide protection against lethal viral infection. DNA Cell Biol 25:197–205[CrossRef]
McDonaldN. J.,
SmithC. B.,
CoxN. J.2007; Antigenic drift in the evolution of H1N1 influenza A viruses resulting from deletion of a single amino acid in the haemagglutinin gene. J Gen Virol 88:3209–3213[CrossRef]
OranA. E.,
RobinsonH. L.2003; DNA vaccines, combining form of antigen and method of delivery to raise a spectrum of IFN- γ and IL-4-producing CD4+ and CD8+ T cells. J Immunol 171:1999–2005[CrossRef]
PertmerT. M.,
RobertsT. R.,
HaynesJ. R.1996; 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 70:6119–6125
PlotkinJ. B.,
DushoffJ.,
LevinS. A.2002; Hemagglutinin sequence clusters and the antigenic evolution of influenza A virus. Proc Natl Acad Sci U S A 99:6263–6268[CrossRef]
RobinsonH. L.,
HuntL. A.,
WebsterR. G.1992; Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 11:957–960
SadS.,
MarcotteR.,
MosmannT. R.1995; Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity 2:271–279[CrossRef]
SasakiS.,
JaimesM. C.,
HolmesT. H.,
DekkerC. L.,
MahmoodK.,
KembleG. W.,
ArvinA. M.,
GreenbergH. B.2007; Comparison of the influenza virus-specific effector and memory B-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines. J Virol 81:215–228[CrossRef]
UlmerJ. B.,
DonnellyJ. J.,
PakerS. E.,
RhodesG. H.,
FelgnerP. L.,
DwarkiV. J.,
GromkowskiS. H.,
DeckR. R.,
DeWittC. M.other authors1993; Heterologous protection against influenza by infection of DNA encoding a viral protein. Science 259:1745–1749[CrossRef]
WilsonI. A.,
SkehelJ. J.,
WileyD. C.1981; Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289:366–373[CrossRef]
ZhangF.,
ChenJ.,
FangF.,
ZhouY.,
WuJ.,
ChangH.,
ZhangR.,
WangF.,
LiX.other authors2005; Maternal immunization with both hemagglutinin and neuraminidase expressing DNAs provides an enhanced protection against a lethal influenza virus challenge in infant and adult mice. DNA Cell Biol 24:758–765[CrossRef]
ZhangW.,
LiM.,
CaoK.,
YangJ.,
ShiQ.,
WangB.,
JiangZ.,
LiH.2006; Construction of eukaryotic expressing plasmids encoding HA and HA 1 of influenza A virus and their transient expression in HEK293 cells. J Huazhong Univ Sci Technolog Med Sci 26:225–230[CrossRef]