A novel DNA vaccine expressing the Ag85A-HA2 fusion protein provides protection against influenza A virus and Staphylococcus aureus

Abstract

Secondary pneumonia due to Staphylococcus aureus (S. aureus) causes significant morbidity and mortality. The aim of the research was designed a novel DNA vaccine encoding the Mycobacterium tuberculosis secreted antigen Ag85A fused with the influenza A virus (IAV) HA2 protein to provide protection against both influenza and secondary infection with S. aureus. The DNA vaccine vector efficiently expressed the encoded antigen in mammalian cells, as determined by RT-PCR, Western blotting and immunofluorescence analysis. Mice were immunized with the vaccine by intramuscular injection before challenge with IAV and S. aureus. The pulmonary and the splenocyte culture IFN-γ levels were significant higher in immunized mice than their respective controls. Although the antibody titer in the HI test was low, the sera of mice immunized with the novel vaccine vector were effective in neutralisation assay in vitro. The vaccine could reduce the loss of body weight in mice during IAV challenge. Both Western blotting and RT-PCR showed that the vaccine markedly enhanced toll like receptor 2 (TLR2) expression in splenocytes after the secondary infection with S. aureus. The survival rate of mice with high TLR2 expression (pEGFP/Ag85A-HA2 or iPR) was significantly increased compared with mice immunized with pEGFP/HA2 after challenge with S. aureus. However, the pulmonary IL-10 concentration and S. aureus titer were significantly decreased in immunized mice, and expression of TLR2 was increased after challenge with S. aureus. These results demonstrated that Ag85A could strengthen the immune response to IAV and S. aureus, and TLR2 was involved in the host response to S. aureus.

Figure 1

Expression of HA2 or Ag85A in HEK293 cells. (A) Expression of HA2 or Ag85A in HEK293 cells. HEK293 cells were transfected with plasmids encoding individual HA2 or Ag85A proteins or the Ag85A-HA2 fusion protein, and expression was evaluated by GFP fluorescence. Shown are cells transfected with pEGFP/HA2 (a, b), pEGFP/Ag85A (c, d), pEGFP/C2 (e, f), pEGFP/Ag85A-HA2 (g, h) or untransfected cells (i, j). Fluorescent images (a, c, e, g, i) and phase contrast images (b, d, f, h, j) are shown. (B) Expression was evaluated by RT-PCR. (7) cells transfected with pEGFP/ HA2; (3,6,9) Trans2K Plus DNA Marker; (2,5,8) cells transfected with pEGFP-C2; (4) cells transfected with pEGFP/Ag85A; (1) cells transfected with pEGFP/ Ag85A-HA2. Fragments of approximately 1500 bp (1), 750 bp (4, 7) and 500 bp (1, 2, 4, 5, 7 and 8) were separated by 2% agarose gel electrophoresis following RT-PCR. GADPH: Fragments of approximately 500 bp; pEGFP/Ag85A: Fragments of approximately 750 bp; pEGFP/ HA2: Fragments of approximately 750 bp; pEGFP/ Ag85A-HA2: Fragments of approximately 1500 bp. (C) Expression was evaluated by Western blotting. (a) cells transfected with pEGFP/ Ag85A-HA2; (b) cells transfected with pEGFP-C2; (c) cells transfected with PBS; (d) cells transfected with pEGFP/ HA2; (e) cells transfected with pEGFP/Ag85A.

Figure 2

Comparison of serum antibody titers (log 2 dilution) giving 50% CPE inhibition assay in MDCK cells. (*P < 0.05). The high NAb titers were detected in mice immunized with iPR, pEGFP/Ag85A-HA2 and pEGFP/HA2. The NAb titer from the pEGFP/Ag85A-HA2 vaccinated group was higher than those of the pEGFP-C2, pEGFP/HA2 and pEGFP/Ag85A vaccinated groups (P < 0.05). The NAb titer of the pEGFP/HA2 vaccinated group was higher than those of the pEGFP-C2, PBS and pEGFP/Ag85A vaccinated groups (P < 0.05).

Figure 3

IFN-γproduction in splenocyte culture 72 h after induced by HA in vitro. *P < 0.05. HA induced higher IFN-γ titers in the pEGFP/Ag85A-HA2 group (P < 0.05). Splenocytes from mice vaccinated with pEGFP/Ag85A-HA2 produced 1.5-fold higher IFN-γ in response to HA than those from mice vaccinated with pEGFP/HA2 (P < 0.05).

Figure 4

Cytokine levels in mouse lung homogenates. IFN-γ (*P < 0.05) (A) and IL-10 (*P < 0.05) (B) of lung homogenates day 8 after infection with A/PR/8/34 and on day 2 after infection with S. aureus. The pulmonary IFN-γ levels of mice vaccinated with pEGFP/Ag85A-HA2 were significantly higher than those vaccinated with pEGFP/HA2 on day 8 after infection with A/PR/8/34 (P < 0.05). The pulmonary IFN-γ levels of mice vaccinated with pEGFP/HA2 were significantly higher than those vaccinated with pEGFP-C2 or PBS (P < 0.05). The pulmonary levels of the anti-inflammatory cytokine IL-10 were significantly higher in the PBS group and the pEGFP-C2 vaccinated group than those in the other groups at day 2 after infection with S. aureus (P < 0.05).

Figure 5

Expression of TLR2 in splenocytes of different vaccinated groups on day 2 after infection with S. aureus. (A) Western blot detection of TLR2 in splenocytes. Lane A: PBS group; lane B pEGFP-C2 group; lane C pEGFP /HA2 group; lane D pEGFP/Ag85A group; lane E pEGFP/Ag85A-HA2 group; lane F iPR group. (B) RT-PCR detection of TLR2 in splenocytes. Lanes 1, 8: pEGFP/Ag85A-HA2 group; lanes 2, 9: pEGFP-C2 group; lanes 3, 10: pEGFP /HA2 group; lanes 4, 11: pEGFP/Ag85A group; lanes 5, 12: PBS group; lanes 7, 13: iPR group. Lanes 1–5, 7: TLR2 (410 bp); lanes 8–13: GADPH (531 bp); lanes 6, 14: marker.

Figure 6

A Body weight of mice. Weight was calculated relative to day 0. Data are mean ± SEM of four mice per group. B Survival rate of mice after infection with IAV. Survival rate was calculated from infection with IAV to infection with S. aureus.

Figure 7

Survival rate of mice from day 3 after infection with S. aureus. The survival rate of mice vaccinated with pEGFP/Ag85A-HA2 or iPR was significantly higher than their respective controls (P < 0.05).

Figure 8

Bacterial outgrowth during postinfluenza pneumonia. (*P < 0.05). S. aureus titration was performed on day 2 after infection with S. aureus. The S. aureus titer in mice vaccinated with pEGFP/Ag85A-HA2 was lower than that in mice vaccinated with pEGFP/HA2.