In contrast to the present study, DNA vaccines for M. tuberculosis
have largely been based on one or more specific genes encoding proteins that induce gamma interferon secretion (31
). It has been demonstrated that a single antigen, mycobacterial heat shock protein Hsp65, was effective in protecting mice against challenge with M. tuberculosis
, reducing infection in the lung by 2 log10
). Other studies have shown that there is reduced tissue infection after challenge in mice or guinea pigs immunized either with Hsp65, Apa, or a combination of ESAT-6 and Ag85A coding sequences (31
). The advantage of expression library immunization is that all antigens encoded in a pathogen's genome can be screened simultaneously to identify protective sequences.
Using expression library immunization (10
), a high-throughput DNA immunization technique, thousands of bacterial genes can be screened simultaneously to identify protective sequences. This immunization technique, as described above, proceeds through successive rounds of immunization and library segregation to ultimately isolate a single or select group of protective sequences. Sequencing a protective clone pool provided information about potential protective sequences in the pool. Because high-throughput sequencing is performed in a 96-well format, every sequence can be traced back to its original clone for further manipulation. The in silico screening accomplished by nucleotide sequencing resulted in two important results. First, a 64% reduction in clone number was achieved in less than 2 weeks. This level of clone reduction would have otherwise required an 8-month immunization and challenge experiment with a group of 150 mice. Second, because we obtained the nucleotide sequence of every clone in a protective clone pool, subsequent experiments could be directed with specific knowledge of which genes offered protection. Additionally, the 10 new clone arrays were generated so that any two clone arrays shared 17 clones. This was essential to account for the possible additive effects of multiple clones in providing protection. Ideally, expression library immunization aims to identify one protective sequence; however, it is more likely that multiple coding regions provide protection, and their combined use may provide even greater levels of protection.
Our preliminary studies (data not shown) demonstrated that we could deliver and express the β-galactosidase gene in mice. Expression was observed in all tissues examined, including those peripheral to the transfection site (i.e., spleen, inguinal lymph node, and mesenteric lymph node). This suggests that antigen-presenting cells were directly transfected with plasmid and migrated throughout the body, an essential component for generating a systemic immune response. In addition, detection of β-galactosidase up to 140 days postimmunization demonstrated that antigens encoded by DNA vaccines are not immediately cleared but are available for immune stimulation for prolonged periods of time. These data are supported by a previous study, which reported that plasmid-encoded luciferase was expressed for at least 19 months after intramuscular injection in mice (57
). Taken together, the systemic and prolonged antigen expression demonstrated that the pVAX-Koz vector and gene gun delivery method was effective for expressing a cloned gene in mice.
Using the protocol for DNA immunization and bacterial challenge described above, we were able to demonstrate that mice were protected against challenge with M. avium subsp. paratuberculosis when they were immunized with four different clone pools of naked DNA. Further partitioning of a protective clone pool into clone arrays resulted in effective protection as well. Bacterial infection was dramatically reduced in both the spleens and mesenteric lymph nodes of mice immunized with DNA from these clone pools compared to the infected control groups and other mouse clone pool groups. Surprisingly, the parent protective clone pool did not reduce bacterial infection in mice compared to the no-DNA, infected group in the second vaccine trial. After resampling of the parent clone pool, it is likely that a different mixture of clones was obtained from the heterogeneous population of clones present. Regardless, results from this study demonstrated that there was significant protection when mice were vaccinated with four different clone arrays.
The coding sequences that were shared by the four protective clone arrays were determined, and a detailed list of the 26 proteins that could potentially protect mice from M. avium
challenge was prepared. Among the shared proteins, the mycobacterial cell membrane-associated protease, FtsH, has been shown to induce antibody production in M. tuberculosis
). The mycobacterial major membrane protein (MMP) has been reported to induce both cellular and humoral immune responses in M. leprae
-infected individuals (42
), and a DNA vaccine study showed that MMP protects mice from leprosy challenge (36
). In addition, the MMP has recently been shown to play a role in invasion of macrophages by M. avium
). Proline-rich antigens which induce both T- and B-cell responses in M. tuberculosis
and M. leprae
have been described (28
). Furthermore, a DNA vaccine encoding the M. tuberculosis
36-kDa proline-rich antigen protected mice from challenge with live bacteria (49
). The PE family of antigens is named after their proline- and glutamic acid-rich N-terminal ends. These antigens are highly antigenic, are located on the cell wall and cell membrane, and are involved in macrophage infection and cell-to-cell interactions (9
). The macrophage cell entry protein, Mce, is a virulence factor involved in macrophage entry and survival (2
). In addition, the polyketide synthase family of genes produces lipid-like molecules with a wide variety of functions, including iron acquisition and virulence (4
). The genes mentioned above were well represented in the protective clone array pools and likely contributed to the reduced tissue colonization observed in mice challenged with M. avium
Immunoblot analysis confirmed that mice generated humoral immune responses to vaccination and challenge. Reactivity to 15-kDa and 65-kDa M. avium
proteins was consistently observed in all groups of challenged mice. While the size and identity of the 15-kDa protein is speculative, it is quite possible that the 65-kDa protein is a homologue of the M. tuberculosis
heat shock protein, Hsp65 (35
). More importantly, sera from mice vaccinated with DNA from nonprotective clone pools recognized 35-kDa, 50-kDa, and 90-kDa proteins, while sera from protective clone groups did not recognize, or weakly recognized, the same proteins. The identities of these proteins are not known, and whether the differences in immune responses were a direct result of protection from challenge remains to be determined.
In summary, the present study demonstrated that expression library immunization of mice is an effective method of vaccination. This is the first study in which DNA vaccination as a method of protection against challenge with M. avium subsp. paratuberculosis was evaluated. In the present study we were able to demonstrate protection by reduced colonization in tissues of mice after vaccination with gene pools that contained protective sequences.