Like other intracellular pathogens, chlamydial biological complexity underscores the likely need of multiple target antigens in a vaccine. Use of whole inactivated chlamydial agents appears to be because of the presence of im-munopathogenic components, plus the fact that early trials indicated that trachoma was exacerbated following episodes of C. trachomatis infection. On the other hand, a live attenuated strain of C. psittaci is used in veterinary medicine as a safe way to prevent abortion in ewes, suggesting that there might be hope for an attenuated vaccine. A live vaccine replicates like the target pathogen and promotes processing and presentation of antigens most similar to a natural infection. Furthermore, while replicating, a live vaccine presumably expresses all or most of its important target immunogens. This may be important for chlamy-diae, which exist in (at least) two developmental forms. Live attenuated vaccines could also stimulate mucosal immune responses and are capable of inducing systemic humoral and cell-mediated responses. Major obstacles in generating and isolating attenuated C. trachomatis strains are the inability to genetically manipulate the organism and to isolate and propagate clonal lineages. Plaque cloning techniques have been recently developed and will facilitate the isolation of clonal lineages of chlamydiae.(35) However, efficient genetic transformation systems for chlamydiae still need development.

Dendritic cells (DC) pulsed with chlamydial antigens or infected with C. trachomatis can induce protection when transferred to naive animals.(36) This method is based on the use of DCs that will be activated and present chlamydial epitopes on their MHC class I and II. Such cells are then used in adoptive cell transfers to autologous recipients. DC vaccines could be used as tools to unravel protective antigens, co-stimulatory molecules, and homing requirements. The ability of dendritic cells to produce IL-12 upon chlamydial-organism stimulation is required for the induction of DC vaccine protection against chlamydial infection. Murine DCs undergo phenotypic maturation to become activated upon exposure to type I interferons (type I IFNs) in vivo or in vitro and can also be matured by different adjuvants and used as such to stimulate adaptive responses.(37) Although unlikely to constitute an alternative to other types of immunotherapy because of methodological constraints, protection conferred by DC vaccines constitute a way to obtain the proof of principle for possibilities to develop a chlamydial vaccine. Moreover, induction of protective immunity occurred following chlamydial vaccination when coinoculated with a transgene-based GM-CSF adjuvant, probably allowing DCs to migrate to the site of immunization.(38)

To date there has been little progress in the identification of promising candidate chlamydia vaccine antigens. The most studied antigen here is MOMP. C. trachomatis MOMP is a predominant disulfide cross-linked surface protein and an immunodominant B cell antigen. MOMP is also the primary serotyping antigen. Antibodies specific to MOMP neutralize infectivity by blocking chlamydial attachment to host cells, suggesting a role of MOMP as a chlamydial ad-hesin (reviewed in ref. 39). MOMP has thus been the focus of many vaccination studies because of these important immunological properties and implication in chlamydial pathogenesis. Recombinant MOMP, MOMP synthetic peptides, DNA vaccines encoding MOMP, and the passive transfer of MOMP-specific monoclonal antibodies have all been evaluated for protective efficacy.(40) However, they have all yielded disappointing results with no or at best partial protective immunity. This is also true with chlamydial vaccines using other antigens such as OMP-2, Hsp60, Cap-1, ADP/ATP translocase.(8,15,30,41,42) The reason for the ineffectiveness of MOMP as a vaccine is not known, but it may result from the use of MOMP immunogens that do not mimic the native structure of the protein. Studies of the C. pneumoniae MOMP antigenic structure have produced a portrayal of exposure and immunogenicity different from the MOMP of C. trachomatis. Whereas C. trachomatis MOMP varies antigenically among many strains, the C. pneumoniae MOMP is antigenically constant, suggesting that the selective pressure responsible for antigenic variation seen in C. trachomatis is not active for C. pneumoniae. C. pneumoniae probably behave like a more timepersistent intracellular organism being less exposed to anti-MOMP-neutralizing antibodies. Like the C. trachomatis homologue, C. pneumoniae MOMP is exposed on the surface of the bacteria and conformational epitopes can be recognized by species-specific neutralizing monoclonal antibodies as well as human sera.(26)

The complete genome sequence of C. pneumoniae provides an inventory of all proteins potentially produced by this bacteria. Thus, no potential vaccine candidates need slip through the net through ignorance of its existence. However, the genome revolution has brought to the forefront immense difficulties in evaluating the large number of potential vaccine candidates. The challenge is now shifted to which on the many candidates should be taken forward. The information from the bacterial genome sequences allows those skilled in bioinformatics to classify genes and their products and identify those most potentially interesting for vaccine development. Software packages will assign function to genes, predict their key functions such as their cellular locations, MW, pI, etc. However experimental biology should always supplement this information. Stephens has classified the genome-encoded proteins into four categories(43): (1) those present in chlamydiae and other organisms, (2) those unique to chlamydiae, (3) those unique to one chlamydia species but related to those in other species, and (4) those unique to C. pneumoniae lacking identifiable relatives in other organisms.

Proteins present in chlamydiae and other organisms (type 1) would typically have metabolic functions common to most organisms, including proteins important in translation, transcription, replication, and metabolic pathways. Although generally not good candidates for vaccine—there are exceptions such as Hsp60—type III secretion structural proteins constitute attractive candidates. Those shared by different species of chlamydia but not by other species (type 2) are interesting candidates for vaccination, since, in general, they would participate in chlamydial structure or biochemistry. This category includes many outer membrane proteins, inclusion membrane proteins, and type III secretion effectors. Antigens unique to one chlamydial species but present in other bacterial species (type 3) are few and probably involved in the metabolism or virulence of the particular species. This set is not promising for vaccination. Those unique to

C. pneumoniae without relatives in other species (type 4) characterize the species and reflect exclusive biology and virulence traits. Their function is not known and thereby cannot be ruled out as vaccine candidates.

Using immune responses to indicate genes expressed in vivo which could also be used for vaccine design is logical, powerful, and specific. A direct and innovative approach is based on the finding that amplified PCR fragments can be rendered transcriptionally active when inoculated into animals (i.e., DNA immunization). Because all candidate antigens are encoded in the genome, a systematic search could be realized through construction of expression libraries. Animals will be immunized with a representative library of pathogen DNA prepared in such a way that the encoded genes are expressed and secreted, the animal will be subsequently challenged and assayed for protection.(44) Expression libraries capable of inducing protection could be subfractionated in order to identify the relevant protective DNA immunogens. Availability of the genome should facilitate selection since only few nucleotides of the insert DNA responsible for inducing protection need to be sequenced, and then matched to the whole genome sequence. The ability to identify epitopes by expression cloning is particularly useful when applied to organisms such as C. pneumoniae, where large numbers of organisms are difficult to obtain, and where techniques for genetic manipulation of the organism are unavailable. Expression libraries have been used to characterize antigens reactive with antibodies directed against chlamydia. Recently prokaryotic and eukaryotic bacterial expression libraries have been used to identify C. trachomatis antigens recognized by MHC class I and II restricted T cells.(30,45) In brief, chlamydial proteins expressed by E. coli were processed by antigen-presenting cells (APC), or macrophage cell lines infected with retroviral expression libraries and the resulting peptide/MHC complexes screened by antigen-specific T cells. Using these approaches the antigens recognized by CD4+ and CD8+ T cell lines were identified. (30,45) Selection of unknown antigens screened with immune T cells or a panel of T cell hybridomas from infected mice selected on the basis of the ability of these cells to secrete high levels of IFN-y if in contact with proper immunogens can be useful in vaccine design. This tactic could complement the approaches in which cDNA-encoding chlamydia open reading frames are used to vaccinate experimental animals to determine antigens associated with protection.

Alternative approaches involving purification and sequencing of peptides eluted from MHC molecules have been used to identify pathogen-derived antigens. This technique is more applicable for identifying antigens recognized by CD8+ T cells, where peptides of relatively restricted defined length (8-10 amino acids) are recognized, although this could also be used for identification of antigens eluted from MHC class II.

Although this technique has still not been used in identification of chlamy-dial epitopes, search for chlamydial sequences encoding potential target epi-topes ofmurine anti-C.pneumoniae CD8+T cells, among C. pneumoniae proteins likely to reach the cytosol of infected cells, has been performed.(20,21) Sequences were tested for their ability to bind MHC molecules and, when coincubated with APC, these were recognized and lysed by C. pneumoniae-specific CTLs.

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