Enhanced murine antigen-specific gamma interferon and immunoglobulin G2a responses by using mycobacterial ESAT-6 sequences in DNA vaccines.

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.

Cloning and analysis of the gene for a major surface antigen of Mycoplasma gallisepticum.

Myplasma gallisepticum infects a wide variety of gallineaceous birds including chickens, turkeys, and pheasants. Infection occurs both horizontally and vertically. Thus, control of the spread of M. gallisepticum to noninfected flocks is difficult. Continual monitoring is necessary to identify infected flocks even under the most stringent infectious control practices. Monitoring, however, is usually performed by measuring hemagglutination activity (HA) in serum, an insensitive and variable test. Variability in the HA test arises differences in agglutination antigen, changes in antigenic profiles of the M. gallisepticum strain, and variability in reading the agglutination reaction. Enzyme-linked immunosorbent assays (ELISAs) are the preferred method of testing because of the ease in obtaining sera and the sensitivity and reproducibility of the assays, but the ELISA suffers from a lack of standardization in the test antigen. The ELISA test will be more easily accepted once the test antigen has been standardized. To this end, we have identified, cloned, and characterized the gene for an antigen that has potential as a species-specific antigen for M. gallisepticum The gene codes for a 75-kD protein, P75, that is recognized during natural infections. Recombinant P75 is not recognized in immunoblots by convalescent sera produced in chickens infected with Mycoplasma synoviae, Mycoplasma gallinarum, and Mycoplasma gallinaceum or in turkeys infected with Mycoplasma meleagridis.

Mycobacterial ESAT-6 protein enhances mouse IFN-gamma responses to Mycoplasma hyopneumoniae P71 protein.

Type 1 immune responses play an important role in the resolution of diseases with infectious or oncogenic etiologies. Vaccines for production animals frequently target humoral immune responses and are often ineffective in protecting against disease. In order to shift the immune response more toward cellular immunity (i.e., type 1 response), we tested the ability of a mycobacterial protein, early secretory antigenic target (ESAT-6), to enhance interferon-gamma (IFN-gamma) secretion during the recall response with a second antigen. The Mycoplasma hyopneumoniae membrane protein P71 was used as a test antigen in murine vaccination studies. The ESAT-6 open reading frame (ORF) was fused to DNA encoding P71 to produce a recombinant protein that was used to immunize BALB/c mice. Control mice immunized with P71 alone demonstrated a splenic response characterized by release of interleukin-10 (IL-10) and a balanced antigen-specific IgG1/IgG2a antibody response. The presence of ESAT-6 as a fusion partner with P71 during immunization, however, resulted in an enhanced P71-specific IFN-gamma response, decreased release of IL-10, and significantly greater (p < 0.05) IgG2a antibody levels in comparison to immunizing with P71 alone. These results demonstrate that ESAT-6 can modify the profile of an immunologic response to an accompanying immunogen.