Protection against Mycobacterium avium by DNA vaccines expressing mycobacterial antigens as fusion proteins with green fluorescent protein

Abstract

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.

FIG. 1

Expression and localization of EGFP and mycobacterial antigen-EGFP fusion proteins in vitro. Murine 3T3 fibroblasts were transiently transfected with pEGFP, p65K-EGFP, p85A-EGFP, or p85B-EGFP and visualized 3 days later by fluorescence microscopy. Two photographs are shown for each plasmid. EGFP produced a homogenous pattern in the nucleus and cytoplasm. The 65K-EGFP fusion protein produced a homogenous cytoplasmic pattern with little or no protein in the nucleus. The 85A-EGFP (not shown) and 85B-EGFP fusion proteins produced speckled cytoplasmic patterns.

FIG. 2

Expression and localization of EGFP and 85B-EGFP fusion protein in vivo. Tibialis anterior muscles of C57BL/6 mice were injected with pEGFP, p65K-EGFP, or p85B-EGFP. Four days later, the mice were euthanized, and frozen sections of the muscle were visualized by fluorescence microscopy. EGFP and 65K-EGFP (not shown) produced homogenous fluorescence, but 85B-EGFP produced a speckled pattern. The clear areas inside the muscle cells are frozen-section artifacts.

FIG. 3

Spleen weights (A) and spleen CFU (B) after M. avium infection in vaccination experiment 1. C57BL/6 mice were vaccinated with three intramuscular injections of saline, p65K-EGFP, or pCMV4.65 or with one intradermal injection of BCG vaccine and then challenged with intraperitoneal M. avium, as described in Materials and Methods. All groups contained five mice, and all data represent means ± standard errors. Each experimental group was compared to the saline group by using a two-tailed t test. NS, not significant.

FIG. 4

Spleen weights (A) and spleen CFU (B) after M. avium infection in vaccination experiment 2. C57BL/6 mice were vaccinated with three intramuscular injections of saline, p85A-EGFP, p85B-EGFP, pSodA-EGFP, or pCMV7.36 or with one intradermal injection of BCG vaccine and then challenged with intraperitoneal M. avium, as described in Materials and Methods. All groups contained five mice, and all data represent means ± standard errors. Each experimental group was compared to the saline group by using a two-tailed t test. NS, not significant.

FIG. 5

Spleen IFN-γ mRNA after M. avium infection as determined by RT-PCR. Total RNA was extracted from spleen tissue at 4 and 8 weeks after infection. Murine IFN-γ mRNA was amplified by RT-PCR. PCR products were subjected to electrophoresis on a 3% agarose gel and stained with ethidium bromide. Each lane shows the 243-bp IFN-γ PCR product from a single mouse. PCR products from mice vaccinated with pCMV4.65 (lanes 1 to 3), p65K-EGFP (lanes 4 to 6), BCG (lanes 7 to 9), and saline (lanes 10-12) are shown. IFN-γ mRNA was increased at 4 weeks in all groups and declined toward baseline levels at 8 weeks. There were no significant differences among the groups.