The aim of this study was to determine whether components of ABC systems may be exploited as vaccine antigens against melioidosis. ABC system proteins were selected as potential vaccine candidates because members of this family of proteins have been shown generally to be immunogenic and to play roles in bacterial virulence (16
). In addition, some components of ABC systems have been shown to be vaccine antigens (6
). Homologues of the LolC, PotF, and OppA proteins identified in B. pseudomallei
and soluble domains were produced in E. coli
, with membrane-spanning regions removed in order to aid expression and purification for the study. Additionally, it was considered likely that the protein regions predicted to be present in the periplasm would be more likely to be involved in immunogenicity than the regions present within the inner membrane of the bacteria are.
Hamsters, diabetic rats, and outbred and inbred strains of mice have previously been used for small-animal models of melioidosis (37
). We have previously used BALB/c mice inoculated by the i.p. route to evaluate live attenuated mutants of B. pseudomallei
as vaccines (2
). In addition we have shown that it is possible to demonstrate protection of immunized mice against an intranasal challenge with B. pseudomallei
). Therefore, in the longer term we could use this model to test the ability of ABC transporter proteins to protect against pneumonic melioidosis. The initial study was designed to determine whether immunization of BALB/c mice with truncated LolC, PotF, or OppA afforded any protection against B. pseudomallei
K96243 delivered by the i.p. route. In previous work evaluating subunit vaccines against B. pseudomallei
, the MPL+TDM adjuvant was found to be effective (27
) and therefore was considered appropriate for the initial evaluation of the ABC system proteins. MPL+TDM comprises nontoxic, highly refined, monophosphoryl lipid A from Salmonella enterica
serovar Minnesota and synthetic trehalose dicorynomycolate, an analogue of trehalose dimycolate, from the cord factor of Mycobacterium tuberculosis
. This adjuvant, an oil-in-water emulsion, applies a depot effect to the antigen, resulting in retention at the immunization site for an extended period of time, as well as stimulating the immune system due to lipid A stimulation of proinflammatory cytokines (23
). In a previous study using MPL+TDM as an adjuvant for proteins, an IgG1 response was found to predominate in immunized mice (15
). However, in this study the predominant IgG isotype response to the three different proteins tested was IgG2a, reflecting a TH1-type immune response. The importance of this type of immune response has been confirmed by the requirement for IFN-γ in controlling B. pseudomallei
). Such TH1-type immunity results in the activation of macrophages, the secretion of IFN-γ and other cytokines, and the subsequent stimulation of the cell-mediated immune response likely required for protection against B. pseudomallei
In this study, both the LolC and PotF proteins offered significant protection to immunized mice against approximately 40 minimal lethal doses (MLDs) of B. pseudomallei. However, immunization with LolC was more effective at both stimulating systemic cell-mediated immunity and affording protection against infection. When a range of adjuvants were tested for improving the protection afforded by LolC, ISCOMs complexed with CpG ODN 10103 were found to offer a level of protection against a larger challenge (approximately 70 MLDs) of B. pseudomallei greater than the level of protection observed with LolC with the MPL+TDM adjuvant. Although no significant protection against 70 MLDs of B. pseudomallei was observed in mice given LolC in complex with the MPL+TDM adjuvant in this experiment, other unpublished data obtained in our laboratories have confirmed this protection afforded by this combination of protein and adjuvant against B. pseudomallei. Furthermore, we have demonstrated the equivalence in protective efficacy of the combinations compared to the live attenuated mutant of B. pseudomallei, 2D2.
ISCOMs are cage-like structures assembled from cholesterol, phospholipids, and saponins that can selectively target antigen to phagocytic cells and enhance the induction of a cell-mediated immune response (32
). Synthetically produced CpG ODNs act as immune response modifiers through activation of the innate immune response, since CpG DNA is able to up-regulate costimulatory molecules and increase cytokine secretion, antigen processing, and peptide-major histocompatibility complex stability through interaction with Toll-like receptor 9 (12
). In this study, the use of ISCOMs together with CpG ODN as an adjuvant for LolC resulted in an LolC-specific IgG2a bias similar to that observed when MPL+TDM was used, indicating that a TH1-type immune response was stimulated.
The ability to protect against heterologous strains is an important feature of vaccines designed to prevent bacterial infection. Therefore, the LolC protein with either the MPL+TDM adjuvant or the adjuvant ISCOM-CpG complex was also evaluated for protection against B. pseudomallei 576, a strain of B. pseudomallei with an LPS O-antigen type different than that of K96243. This experiment showed that LolC delivered with either adjuvant was able to protect immunized mice. The lolC ORF encoding LolC was shown by PCR screening to be present in a range of B. pseudomallei strains, indicating the potential ability of the protein to protect against melioidosis caused by different strains of B. pseudomallei. Since lolC was also present in B. mallei, it is possible that the LolC protein may protect against glanders, the disease caused by B. mallei. The protective efficacy of immunization with LolC against glanders should be tested.
The mechanism by which LolC protects mice against melioidosis is not yet understood. The components of ABC transporters that may be expected to interact with the host immune system in gram-negative bacteria are the associated outer membrane proteins. However, LolC is an inner membrane protein in B. pseudomallei
, and the truncated form used to immunize in this study is predicted to be located in the periplasm. Nevertheless, it is possible that LolC may be able to interact with the immune system since there are many examples of antibodies in convalescent-phase sera which are directed to cytoplasmic proteins of bacteria (16
). This may reflect the surface location of at least part of the protein population or that bacteria with disrupted cell walls or bacteria which are taken up by antigen-presenting cells display cytoplasmic or periplasmic protein to the immune system. Since LolC stimulates strong humoral immune responses in immunized mice, it is possible that LolC-specific antibody could directly block the Lol lipoprotein transport system that is known to be essential in E. coli
). Alternatively, LolC-specific antibody may have an opsonizing effect, or cell-mediated immunity to LolC may play a role in protection. Further work is required to determine the role of LolC in the virulence of B. pseudomallei
and to elucidate how immunization with LolC provides protection against melioidosis.
The work described here identified a novel protein antigen that offers protection against infection with heterologous strains of B. pseudomallei
. To our knowledge, this is the first example of a soluble recombinant B. pseudomallei
protein domain, the periplasmic domain of LolC, which is protective against this organism. The LolC protein is able to stimulate strong antibody and cell-mediated immune responses when it is combined with either the MPL+TDM adjuvant or an ISCOM-CpG ODN complex adjuvant. Since it is considered possible that B. pseudomallei
could be used as a biological weapon (29
), future work to assess this antigen will include an evaluation of the protection afforded against an aerosol challenge with B. pseudomallei
, as well as the cross-protective efficacy provided against B. mallei