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The Mycobacterium tuberculosis protein ESAT-6 has unusual immune stimulating activities, has been implicated in the recall of long-lived immunity, and induces protection against tuberculosis in mice. For many diseases caused by bacterial or viral pathogens, a strong cell-mediated immune (i.e., type 1) response is often required for recovery or protection. Therefore, it is important to design immunization regimens that induce agent-specific type 1 immunity. We have shown in previous studies that ESAT-6 could enhance antigen-specific type 1 immune responses in BALB/c mice against a second antigen when presented as a purified fusion protein. It was also of interest to determine if ESAT-6 could enhance the type 1 response against a second antigen beyond that afforded by DNA vaccination through CpG motifs. This was tested by using gene fusions of ESAT-6 and the Mycoplasma hyopneumoniae surface antigen P71. Modified P71 gene sequences were cloned with or without ESAT-6 sequences into a DNA vaccine vector and were used to immunize mice. Splenic lymphocytes from vaccinated mice were tested for gamma interferon (IFN-gamma) and interleukin-10 (IL-10) secretion. Serum antibodies were examined for P71 antigen-specific isotype responses. When stimulated in vitro with purified P71 antigen, splenocytes from the ESAT-6:P71 vaccinates secreted higher levels of IFN-gamma and lower levels of IL-10 compared to those of vaccinates receiving the P71 construct alone. Furthermore, the immunoglobulin G2a serum antibody levels were significantly higher in the ESAT-6:P71 vaccinates compared to those of the vaccinates receiving P71 alone. In conclusion, ESAT-6 was shown to enhance antigen-specific type 1 immune responses in BALB/c mice when used in DNA vaccines.

Enhanced Murine Antigen-Specific Gamma Interferon and Immunoglobulin G2a Responses by Using Mycobacterial ESAT-6 Sequences in DNA Vaccines