The high conservation and known effect in protective immunity against influenza of NP make it a high potential target antigen for universal influenza vaccine. Extensive studies have been carried out on influenza vaccine targeting NP for a long time, among which DNA vaccine is being the most investigated. Results from our previous study indicated that NP DNA vaccine alone could achieve better protection only in the case of repeated immunization
[11, 12, 20]. Currently, heterologous prime-boost strategies have become the primary way to enhance the immunogenicity of the DNA vaccine. Immunization strategy based on DNA prime followed by a viral vector or recombinant protein boost has been widely applied in mouse model, nonhuman primate, and human clinical trials
[21, 22]. Epstein et al. have induced cross-reactive CTL by using DNA prime-adenovirus vector vaccine boost strategy based on NP and protected mice from lethal challenge of H5N1 virus
. So far, we developed NP DNA prime-intranasal protein boost strategy, and evaluated the induced immune response and protective effect in mouse model.
Our results demonstrated that NP DNA immunization followed by intranasal boosting with rNP could effectively enhance the humoral immune response by producing higher titer of NP specific antibody in serum, as compared to vaccination with either NP DNA vaccine or intranasal rNP alone. Classification and bias of antibody isotypes can somehow reflect the Th bias of induced immune response. It is generally believed in mouse model that it is a Th-1-biased immune response when the ratio IgG2a/IgG1 of serum antibody isotype titers is larger than 1, whereas IgG2a/IgG1<1 indicates a Th-2-biased immune response
. In our experiments, the Th-1-biased immune response induced by NP DNA vaccine was maintained following an intranasal boosting with rNP and the response level was raised as compared to vaccination with DNA or rNP alone. These results are consistent with those of other research groups
Several studies have demonstrated that NP-specific antibody is not directly related to protective immunity against influenza virus. Our previous serum passive immunization experiment also proved that NP-specific serum alone could not provide protection
[5, 27]. However, there were studies which revealed that non-neutralizing antibodies against NP might play a role in fighting against a sublethal dose challenge of viral infection
[28, 29]. Recently, two studies conducted by Lamere et al. suggested that NP-specific serum antibody could play a certain role in heterosubtypic immunity against influenza virus through mechanisms with both FcRs and CD8+ T cells involved
[30, 31]. Although NP-specific serum antibody is not the key factor in protective immunity, it should be taken into account in vaccine design concerning its potential protective mechanism. In this study we showed that the heterologous prime-boost strategy could induce good antibody responses.
Specific cellular immune responses targeting influenza internal conservative antigen (i.e. NP) are widely regarded as the main factor mediating cross-protection against influenza protection
[32, 33]. NP-specific CTLs can rapidly proliferate, differentiate, and be recruited to infection sites (lung tissue and nasal mucosa) after viral infection. These CTLs kill viral infected cells through direct killing, FasL dependent or TRAIL dependent pathway and mediate clearance of virus
[34, 35]. On the other hand, Th-1-type CD4+ T cells can mediate protective immunity through IFN-γ dependent or nondependent mechanism
[36, 37]. Vaccine that targeting conversed gene and immunizing through mucosal route with a valid immune strategy is one of the effective methods to induce heterosubtypic immunity against influenza. Recently, our group successfully induced heterosubtypic immunity against influenza by intranasal administration of NP, M1 and M2 protein with mucosal adjuvant
[5, 18, 19].
In our experiments, we induced high levels of NP-specific IFN-γ–producing CD8+ T cells and Th-1-biased CD4+ T cells, through a DNA prime-intranasal protein boost strategy, which was significantly different from vaccination with DNA or protein alone. In addition, the strength of such immune was actually found to be associated with the survival rate of mice after the homo- or heterologous viral challenge. Moreover, from the lung virus titer between day 3 and day 7 post-infection, it was clear that virus in lung was cleared from mice in intranasal protein boost group while there were no big changes in group vaccinated with DNA or protein alone. These results suggest that the NP DNA prime-intranasal protein boost strategy can induce higher level of NP-specific T cellular immune response, particularly CD8+ T cell response, significantly augmenting the protection efficiency of NP based DNA vaccine.
The intranasal protein boost strategy also induced high level of NP specific mucosal IgA antibody during the experiments. Meanwhile, well protection was obtained in the two mucosal boost groups (Group D1P1 and D2P1), indicating somehow that the NP-specific mucosal IgA was related to protection against influenza infection. This finding was consistent with our previous results
. Although the role of NP-specific IgA in protection against influenza infection remained unclear, an in vivo experiment conducted by Mukhtar et al. may provide some possible explanations. It was proved in their work that single-chain intracellular antibody against NP could specifically bind to newly synthesized NP in cell after influenza infection and block the interaction between NP and influenza RNA polymerase complexes. By this way, the transcription and translation of influenza viral gene were depressed. Additionally, several studies indicated that the binding of IgA to its poly-receptor on epithelial cells enabled IgA to bind the newly synthesized viral protein during its penetration through epithelial cells and thus, influenced the viral replication
[39–41]. Therefore we speculate that NP specific IgA may interfere with the viral replication to somewhat extent during its secretion in respiratory epithelium.
NP is the target to induce influenza heterosubtypic immunity. We observed in our studies that NP DNA vaccination once or twice followed by an intranasal boost with rNP was able to protect mice from lethal challenge of heterologous H9N2 virus. This type of infection-permissive heterosubtypic immunity accelerated the clearance of lung virus after infection obviously and the protection rate reached 100%. It was reported previously that in the cross-protective immunity mediated by NP specific CD8+ T cells, ideal effect was achieved only when vaccine antigen and the NP of the challenge strain shared the identical sequences of the immunodominant protective CTL epitopes
. Here we found the homology of the amino acid sequence was 93.6% between NP of PR8 (H1N1) strain and the Chicken/Jiangsu/11/2002 (H9N2) strain. They had the same immunodominant CTL epitope-NP147-155(TYQRTRALV) and differences only occurred in 1-2 amino acids of the other 3 immunodominant Th epitopes. For this reason, CTL specific to NP of PR8 virus is also able to kill H9N2 viral infected cells and mediate cross-protection. Since less is known about the B cell epitope on NP and its conservative property
, the role of NP-specific antibodies (IgG and sIgA) in cross-protection is not clear yet. The more broad-spectrum cross-protection induced by DNA prime-intranasal protein boost strategy based on NP, such as cross-protection against highly pathogenic avian influenza H5N1 virus and pandemic H1N1 2009 influenza A virus, will be further evaluated in our future study.
In conclusion, our study proved the DNA prime-intranasal protein boost strategy based on NP could effectively enhance the immune response induced by NP DNA vaccine. Meanwhile, this vaccine delivery via the mucosal route was able to induce better mucosal immune response. Immune response induced by this strategy was not only resistant to lethal challenge of homologous influenza virus, but also provided complete cross-protection. Due to its success in inducing heterosubtypic immunity against influenza virus, the DNA prime-protein intranasal boost strategy based on NP will probably be a good choice for universal flu vaccine research