HSI to influenza virus can be successfully induced by live or inactivated whole influenza viruses or DNA vaccines in different kinds of animals
[9–13]. In our research, we primed mice with NDV-based recombinant virus vaccine expressing HA gene of influenza A/H5N1 virus(rLH5), and induced cross-protection to influenza A/H1N1 virus infection from severe disease, death and viral replication in the lungs. Ge et al.
 demonstrated that the NDV portion of rLH5 vaccine protected mice from NDV infection and could not provide protection from influenza A/H5N1 virus infection. We also found that the NDV portion of vaccine does not elicit cross-protection against influenza A/H1N1 virus infection(data not shown). Therefore, the protective efficacy of HSI in our experiments was induced by gene of influenza virus in rLH5 vaccine.
Current vaccines generate neutralizing antibodies directed against viral HA and NA surface glycoproteins of homologous influenza viruses
. According to Genbank (ADG59526.1 and AAC53844.1), the amino acid homology of HA of A/Font Monmouth/1/1947 (H1N1) and A/Bar-headed goose/Qinghai/3/2005 (H5N1) was 62%. In this study, antibodies against influenza A/H1N1 virus in sera were determined by HI assay after infection. The results showed a strong antibody response to influenza A/H1N1 virus in group 3 (H1N1-H1N1) that played a critical role in immune responses to homologous influenza virus infection. We observed low or undetectable levels of antibodies to influenza A/H1N1 virus in the other groups. It indicated a negative correlation between HSI and antibody levels.
We found that the IFN-γ and IL-10 levels in the lung samples from group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1) were significantly high after infection in comparison to the other groups . IFN-γ, a Th1 cytokine, can enhance the phagocytic function of macrophages and the cytotoxic effect of NK cells. CTL, which can lyse target cells and prevent spread of influenza virus in vivo, can be activated by IFN-γ
[8, 16–18]. IL-10 is an anti-inflammatory cytokine. Although IL-10 is a Th2 cytokine, it can inhibit the generation of inflammatory mediators and prevent imbalances of the inflammatory reaction
. High levels of IFN-γ and IL-10 can help clear influenza virus, combat excessive inflammation and promote recovery.
T-cell-mediated immune responses are an important factor in HSI to influenza virus. IFN-γ+CD4+ T cells are Th1 cells and IFN-γ+CD8+ T cells are effector CTL that are critical features of an adaptive immune system
[20–22]. In this study, high frequencies of activated IFN-γ+CD4+ and IFN-γ+CD8+ T cells in mice of group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1) were elicited by influenza A/H1N1 virus challenge. It indicated that T-cell-mediated immunity might play an important role in cross-protection between different subtypes of influenza virus.
Vaccines that broadly induce cross-reactive T cell responses are usually directed against conserved viral epitopes of internal proteins, such as NP proteins
[23, 24]. De Groot et al.
 reported that the HA also possesses cross-conserved T-cell epitopes
[26–28]. We had found that there was no correlation between the levels of antibody and HSI against influenza virus. And our results showed that the levels of IFN-γ and the percentages of both IFN-γ+CD4+ and IFN-γ+CD8+ T cells in group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1) increased significantly after infection. We speculate that the cross-protection against heterosubtypic influenza A/H1N1 viruses in mice may be due to cross-conserved T-cell epitope sequences in HA.
Some researchers have used various adjuvants such as IL-2, IL-12, GM-CSF or the heat-labile enterotoxin from enterotoxigenic Escherichia coli to improve immune responses
[29, 30]. In this study, we selected rIL-2 as an adjuvant to enhance HSI. We found that the mice in the group that co-administered with IL-2 had higher survival rates, lower viral loads and greater amount of IFN-γ production. Henke et al.
 reported that IL-2 increased the efficacy of DNA immunization to prevent influenza virus infections, IL-2 as an adjuvant for vaccination can stimulate the proliferation and activation of T cells, NK cells and macrophage. It can also enhance cellular immunity
. Further studies will be done to clarify the mechanism that IL-2, as an adjuvant, enhance HSI.
In summary, rLH5 vaccine induced cross-protective immune response against heterosubtypic influenza A/H1N1 virus infection in mice, and this cross-protection was improved with IL-2 as an adjuvant. T cell immunity may play an important role in HSI against influenza virus. Further studies will be necessary to determine the most effective cross-conserved T-cell epitope to HSI and the precise mechanism of HSI.