This study is an extension of a previous study in which we were unable to confirm the protective effect of C. pneumoniae
vaccine candidate genes identified by expression library immunization. This result was presumably due to a Th2 immune shift caused by biolistic genetic immunization of individual instead of complex mixtures of genes. [14
]. In this project, we first evaluated the promotion of Th1-biased immunity by use of a detoxified version of the E. coli
heat-labile enterotoxin as genetic immunization adjuvant. The results showed a strong Th1-promoting effect of the LT genetic adjuvant, and we subsequently proceeded to re-test the best C. pneumoniae
vaccine candidate genes. We had assayed all 1,263 putative ORFs of the C. pneumoniae
genome by ELI for vaccine candidates before [14
], and have now confirmed several ORFs that effectively protect mice against C. pneumoniae
lung infection. Most significantly, vaccination with C. pneumoniae
and Cpn0420 significantly protected mice against C. pneumoniae
mediated death, reduced lung disease, and increased elimination of the C. pneumoniae
organisms in the mouse lungs. Four other genes, ide
, and Cpn0095, also mediated considerable protection against one or more readouts of disease but not all three. While not top candidates, these genes may be more protective in other host backgrounds, or be useful in combination with other antigens.
Of the six candidate genes selected from the literature that have been reported to confer protection against chlamydial infections, only copN, Momp and gatA protected mice from C. pneumoniae-induced death; none ameliorated lung disease, or facilitated clearance of the pathogens.
Reactivity to some genes tested in this study, such as fabD
, glgX, atoC
, and hsp60
, resulted in deaths of several mice between 4 to 9 days after the challenge infection. This is typically the result of shock precipitated by an uncontrolled release of cytokines (“cytokine storm”) during a strongly polarized Th1 immune response [23
]. This immune response elicited by these genes in surviving mice, however, did not result in later protection from disease or efficient elimination of C. pneumoniae
. Thus, use of these genes in a vaccine is not advisable.
Conversely, vaccination with genes Cpn0020, npt1, copN, Momp, and gatA provided complete protection from death; however, they did not reduce subsequent disease or C. pneumoniae lung loads. This suggests that these genes did elicit a limited Th2 response that was protective early in infection, but this response was not sufficient to clear the chlamydiae or the disease that resulted from the continuous chlamydial presence. Thus, these genes are also not preferable as vaccine candidates.
The protective antigens, used individually or preferably in combination, must be further evaluated in vaccine formulations that are appropriate for administration to humans. Two main issues apply: i) do the C. pneumoniae
vaccine candidates identified in the mouse have functionally the same role during human infection, and thus can they serve similarly in humans as highly visible target to an effective immune response, and ii) are the vaccine candidates similarly presented by different MHC-II molecules? While no data are available for humans, we have demonstrated that protective C. abortus
genes identified by ELI in a mouse model are also protective in cows [17
]. This suggests a high probability that the C. pneumoniae
vaccine candidates identified here will function similarly in humans. Presentation of the vaccine candidate proteins by different MHC-II molecules can be tested in inbred mouse lines. Our unpublished data show that the C. abortus
vaccine candidates identified in BALB/c mice (haplotype d) are equally protective in A/J (haplotype a) and C57BL/6J (haplotype b) mice. Furthermore, the protection mediated in outbred cattle also suggests that they function equally on different MHC-II backgrounds. Nevertheless, it may be a good strategy to include several full- length vaccine candidates in a human vaccine to maximize the probability that any of their peptides will be presented on any possible MHC-II molecule of an outbred vaccinee population, as suggested by Igietseme et al. [24
]. For example, Ifere et al. [25
] have shown that a vaccine composed of MOMP and PorB (porin B) induced a higher Th1 response than single subunit vaccines. It is likely that a combination of the candidate genes identified in this study may provide pronounced protection that is close or equal to the level of protection mediated by prior natural infection.
It is very important to identify suitable adjuvants, since they can selectively induce appropriate immune responses and improve protective efficacy by facilitating specific presentation of the antigens to macrophages or dendritic cells, or facilitate consistent release of the antigens [26
]. Aluminum salts have been shown to be effective, but are not preferred for this vacccine because they induce a Th2-biased humoral immune response. Liposomes and MF59, a squalene-based sub-micron emulsion, have also been tested [27
]. Arrington et al. [15
] used cholera toxin (CT) and the E. coli
heat-labile enterotoxin (LT) as genetic adjuvants, and both elicited a strong Th1-biased immune response, typically effective against intracellular pathogens. Our previous tests also have confirmed that the LT adjuvant boosts Th1 immunity as evidenced by elevated mouse IgG2a/IgG1 ratios, but also increases overall levels of antibodies (data not shown).
In design of efficacious vaccines against intracellular Chlamydia
pathogens, an efficient delivery system is critical for mediation of a long term protective immunity [28
]. Some researchers have used bacteria or bacterial antigens as delivery vehicles and achieved considerable success. As an example, He et al. [29
] used a live attenuated recombinant influenza A/PR8/34 virus as a vaccine vector for intranasal delivery of a subunit vaccine (a chlamydial epitope) against C. trachomatis
infection, and a strong Th1 response against chlamydial EBs was detected. Additionally, C. trachomatis
shedding was decreased and long-term protective immunity correlated with the preservation of specific Th1 cells and elevated IgG2a in genital secretions.
Another potential avenue for delivery of a C. pneumoniae
vaccine is protein and gene vaccine formulations. These subunit vaccines are safer alternatives to killed or live attenuated whole organism vaccines, and genetic formulations are more stable and amenable to multi-component inocula. Unfortunately, they have been less effective in humans and livestock than in mouse models [26
]. However, in the genetic vaccines, more recent use of electroporation and gene gun delivery protocols have yielded much better results than needle injections. Nevertheless, work will continue to identify optimal strategies for protein and gene vaccine delivery, adjuvantation, and antigen presentation.
In conclusion, this study has identified the best suited subunit vaccine candidate antigens among all annotated C. pneumoniae
proteins. These antigens will be the basis for further formulation of an experimental vaccine in a commercially viable format. This vaccine will then be tested in animal models of lung disease as well as C. pneumoniae
-enhanced insulin resistance [5
], and ultimately may enter human trials.