CpG-modified plasmid DNA encoding flagellin improves immunogenicity and provides protection against Burkholderia pseudomallei infection in BALB/c mice

Abstract

The plasmid DNA encoding the fliC gene of Burkholderia pseudomallei combined with CpG oligodeoxynucleotide (ODN) was injected intramuscularly into BALB/c mice, resulting in the increased production of certain humoral antibodies and flagellin-specific spleen cell clonal expansion. CpG ODN, as an immunoadjuvant, was added to the plasmid containing the fliC gene in order to obtain ongoing expression in muscle for a long period. Functional expression of flagellin from the constructed CpG-modified plasmid in transfected peritoneal exudate cells of BALB/c mice was shown by reverse transcription-PCR and Western blotting. Furthermore, BALB/c mice immunized with the modified plasmid had relatively higher resistance to B. pseudomallei infection in vivo than did mice immunized with unmodified plasmid DNA. The time course of restricted bacterial growth in spleen and liver and changes in the cytokine profiles of immunized mice suggested that the stimulated phagocytic cells would be able to kill the bacteria eventually, possibly as a consequence of the induction of Th-1-type immune polarization in vivo. Th-1-type immune polarization was detected in response to flagellin induction in mice immunized with CpG-modified plasmid DNA by the appearance of increased levels of immunoglobulin G2a antibodies and gamma interferon-secreting cells specific to flagellin. The exogenous CpG motifs added to the fliC gene would contribute to an adjuvant-like response that enhances the flagellin-specific immunogenicity and provides protection against B. pseudomallei infection. This CpG-modified plasmid DNA vaccination is an important potential strategy that should be developed to protect against melioidosis.

FIG. 1.

An illustration of plasmid pcDNA3/CpG-fliC construction. The ODN (5′-TCT CCC AGC GTG CGC CAT-3′) was added onto the plasmid pcDNA3/fliC (driven by the cytomegalovirus promoter, pCMV) using BamHI linkers.

FIG. 2.

Improvement of mouse immune response by plasmid DNA in the presence of CpG ODN. The BALB/c mice were immunized with pcDNA3/fliC (DNA only) or in a combination with CpG ODN (doses of CpG ODN ranging from 1 μg to 100 μg). As controls, the mice were immunized with pcDNA3. After an 8-week immunization, the humoral antibodies in the sera (a) and the level of cellular proliferation in spleen cells (b), both specific for flagellin, were determined. Each set of data is based on measurements deriving from six mice.

FIG. 3.

Expression of flagellin in PECs. Plasmid pcDNA3/CpG-fliC was transfected into PECs using Lipofectin reagent. (a) After a 48-h transfection, the flagellin-specific mRNA was measured from total RNA extracted in transfectants. The molecular size markers used herein (lane M) included 1.5 kb, 1.2 kb, and ladders from 1 to 0.1 kb. As examples of PECs transfected with pcDNA3/fliC, the RT-PCR products for the β-actin gene and for the fliC gene are shown in lanes 1 and 2, respectively. As examples of PECs transfected with pcDNA3/CpG-fliC, the RT-PCR products for the β-actin gene and for CpG-fliC gene are shown in lanes 3 and 4, respectively. As controls (nontransfected PECs), the RT-PCR products for the β-actin gene, the fliC gene, and the CpG-fliC gene are shown in lanes 5, 6, and 7, respectively. (b) These transfectants were lysed and immunoreacted with antiflagellin antibody. Lane 1, nontransfected PECs; lane 2, PECs transfected with pcDNA3/CpG-fliC; lane 3, PECs transfected with pcDNA3/fliC.

FIG. 4.

Infection by B. pseudomallei of immunized BALB/c mice. The six immunized mice, which were seropositive for flagellin, were infected with 10⁵ CFU of B. pseudomallei by intravenous injection in the tail. After the indicated time, the bacterial survival in the spleen and liver from the pcDNA3/fliC (▴)- or pcDNA3/CpG-fliC (•)-immunized mouse groups was determined. The mice were immunized with pcDNA3 as a control (○). Means and standard deviations for bacterial survival in these two organs were calculated by averaging the measurement of (duplicate) samples deriving from three mice (three mice were used in each experiment).

FIG. 5.

Changes in cytokine profiles. The BALB/c mice were immunized with pcDNA3/fliC, pcDNA3/CpG-fliC, or pcDNA3. The immunized mice, which were seropositive for flagellin, were infected with 10⁵ CFU of B. pseudomallei by intravenous injection in the tail. Subsequent to the indicated time delay (in days), extracted spleen cells were used to examine the intracellular level of cytokine-specific mRNA by RT-PCR.

FIG. 6.

Production of IgG subclasses and IFN-γ-secreting cells. The experimental BALB/c mice were immunized with pcDNA3/CpG-fliC (CpG-fliC), pcDNA3/fliC (fliC), pcDNA3 (vector), or free CpG ODN (CpG ODN). After 8 weeks of immunization, the antibody titers for IgG1 and IgG2a in the sera (a) and the IFN-γ-secreting cells from the spleen (b) specific to flagellin were determined. Each set of data is based upon the results derived from a sample of six mice.