WO2002079244A2 - Chlamydial protease-like activity factor responsible for degradation of host transcription factor - Google Patents

Abstract

Microbial pathogens have been selected for the capacity to evade or manipulate host responses in order to survive after infection. Chlamydia, an obligate intracellular pathogen and the causative agent for many human diseases, can escape T lymphocyte immune recognition by degrading host transcription factors required for major histocompatibility complex (MHC) antigen expression. We have now identified a chlamydial protease-like activity factor (CPAF) that is secreted into the host cell cytosol and that is both necessary and sufficient for the degradation of host transcription factors RFX5 and upstream stimulation factor 1 (USF-1). The CPAF gene is highly conserved among chlamydial strains, but has no significant overall homology with other known genes. Thus, CPAF represents a unique secreted protein produced by an obligate intracellular bacterial pathogen to interfere with effective host adaptive immunity.

Description

CHLAMYDIAL PROTEASE-LIKE ACTIVITY FACTOR RESPONSIBLE FOR DEGRADATION OF HOST TRANSCRIPTION FACTOR

PRIOR APPLICATION

This application claims priority under 35 USC § 119(e) to USSN 60/279,744, filed March 30, 2001.

This invention was made with government support under National Institutes of Health funding. The United States government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of bacterial diseases.

BACKGROUND OF THE INVENTION

Chlamydia has to replicate within a cytoplasmic vacuole of eukaryotic cells and has adapted so well that it can persist in its host for a long period of time. Although chlamydial infection is known to be responsible for many severe human diseases ranging from trachoma after ocular infections to life-threatening complications after urogenital infections, the mechanism of chlamydial pathogenesis is still not clear. It is thought that the persistent infection is a major cause of chlamydia-induced diseases in humans (Beatty et al., 1994, Microbiol Rev 58: 686-699; Askienazy-Elbhar and Suchet, 1999, Infect Dis Obstet Gynecol 7: 31-34). Chlamydia-infected cells are able to continuously release inflammatory cytokines (Rasmussen et al., 1997, J Clin Invest 99: 77-87). It is thought that the continuous release of inflammatory cytokines by persistently infected cells may play a major role in chlamydial pathogenesis (Beatty et al., 1994, Microbiol Rev 58: 686-699; Rasmussen et al., 1997, J Clin Investig 99: 77-87). One of the hallmarks of C. pneumoniae infection is persistence (Airenne et al., 1999, Infect Immun 67: 1445-1449; Gabriel et al., 1998, Eur Heart J 19: 1321-1327; Gieffers et al., 2001 , Circulation 103: 351- 356). Unfortunately, the tissue-damaging inflammatory responses induced by chlamydial infection often fail to efficiently eliminate the chlamydial organisms that are hidden inside the persistently infected host cells. We have hypothesized that chlamydia may have evolved strategies for evading host defense mechanisms in order to establish and to maintain a persistent infection.

There are two major chlamydial species that cause human diseases (Grayston and Wang, 1975, J Infect Dis 132: 87-105; Kuo et al., 1995, Clin Microbiol Rev 8: 451-461). The Chlamydia trachomatis species is a leading cause of trachoma and sexually transmitted diseases (Grayston and Wang, 1975), while the C. pneumoniae species causes various respiratory infections (Grayston et al., 1993, J Infect Dis 168: 1231-1235). Although the C. pneumoniae-induced respiratory infections are often asymptomatic, its association with atherosclerosis has attracted the attention of many investigators (Grayston, 2000, J Infect Dis 181(supp 3): S402-S410). C. pneumoniae organisms have been detected in a large proportion of atherotic plaques but not in nonatherotic cardiovascular tissues (Ouchi et al., 2000, J Infect Dis 181: (Suppl 3): S441-S443; Taylor- Robinson and Thomas, 2000, J Infect Dis 181 (Suppl 3): S437-S440). In cell culture, C. pneumoniae infection was able to transform macrophages into foam cells (Kalayoglu et al., 2000, J Infect Dis 181 (Suppl 3): S483-S489), a hallmark of atherosclerosis. Several groups including ourselves have demonstrated that respiratory infection with C. pneumoniae organisms can greatly enhance atherosclerotic lesion development in animal models (Campbell et al., 2000, J Infect Dis 181 (Suppl 3): S508-S513; Fong et al., J Infect Dis 181 (Suppl 3): S519-S520; Hu et al., 1999, J Clin Investig 103: 747-753; Liu et al., 2000, Mol Cell Biochem2\5: 123-128). More importantly, antibiotic treatment of the infected animals can prevent the C. pneumoniae exacerbation of atherosclerosis (Fong, 2000, J Infect Dis 181 (Suppl 3): S514-S518; Muhlestein, 2000, J Infect Dis 181 (Suppl 3): S505-S507). Despite the important role of C. pneumoniae infection in atherosclerosis, the mechanism of the C. pneumoniae atherogenicity is still not clear.

Multicellular organisms have evolved various responses for controlling microbial infections, which, in turn, select for microbial pathogens with the ability to evade these same host defenses. Our previous studies have shown that chlamydia possesses varied strategies for interfering with or preventing both immune recognition and immune effector activity (Zhong et al., 2000, J Exp Med 191: 1525-1534; Zhong et al., 1999, J Exp Med 189: 1931-1938; Fan et al., 1998, J Exp Med 187: 487-496). Chlamydial infection suppressed both MHC class I and class II antigen expression in the infected cells. The inhibition of MHC antigen expression was correlated with the degradation of host transcription factors RFX5 and upstream stimulation factor 1 (USF-1). Furthermore, degradation of both RFX5 and USF-1 was caused by a chlamydia-dependent proteasome- like activity (CPA) in the cytosol of chlamydia-infected cells and the CPA was inhibited only by an irreversible proteasomal inhibitor lactacystin among all the inhibitors tested (Zhong et al., 2000; Zhong et al., 1999). A logical extension of these previous studies is to identify the factor(s) responsible for the chlamydial proteasome-like activity. SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a composition comprising purified CPAF.

According to a second aspect of the invention, there is provided a purified polypeptide having CPAF activity.

According to a third aspect of the invention, there is provided a method of identifying a compound that reduces degradation of transcription factors by CPAF comprising: contacting purified CPAF and at least one CPAF substrate with a test compound; and determining whether CPAF activity is reduced in the presence of the test compound by detecting reduction in CPAF substrate levels, said reduction being an indication that the compound inhibits CPAF activity.

According to a fourth aspect of the invention, there is provided a process for making a compound that inhibits CPAF activity comprising: carrying out the method described above to identify a compound that inhibits CPAF; and manufacturing the compound.

According to a fifth aspect of the invention, there is provided a method of eliciting an immune response in an animal comprising: introducing into the animal a composition comprising CPAF and CPAF fragments in purified, recombinant or plasmid forms. Especially, adjuvants such as interleukin 12 will be used to enhance IgA production against CPAF.

According to a sixth aspect of the invention, there is provided a purified antibody that binds specifically to CPAF.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Purification and sequence identification of CPAF. (A) A cytosolic protein preparation from chlamydia-infected HeLa cells (HeLa L2 S100) was subjected to three consecutive column separations as listed in the figure. Fractions collected from each column were monitored for both degradation activity in a cell-free assay and total protein profiles on SDS-polyacrylamide gels. Fractions with degradation activity were pooled and loaded to the next column for further fractionation. Two dominant protein bands that correlated with the degradation activity from the final Mono Q column were excised for protein sequence identification. "+" indicates partial digestion of the substrate and "++" indicates complete loss of substrate. "-" indicates that the substrate band is intact compared with the substrate alone sample. (B) List of cryptic peptides that match the chlamydial ORF CT858-encoded protein under GENBANK accession no. A71461. Amino acid residues are represented using the single letter codes and numbered according to their positions in CPAFct sequence. Tryptic fragments derived from the top band match the COOH-terminal portion whereas those from the bottom band match the NH₂-terminal portion of CPAFct. The top band was designated as CPAFc and the bottom band as CPAFn.

Figure 2. CPAF is both necessary and sufficient for degradation of host transcription factors. (A) HeLa L2 S100 was subjected to Superdex 200 size exclusion column analysis as described previously (Zhong et al., 2000). The fractions were assayed for both RFX5 degradation activity and the presence of either host 20S proteasome subunits or CPAFn fragment. The presence of CPAFn but not host 20S proteasomes correlated with RFX5 degradation activity. (B) The effect of antibody depletion of CPAF on RFX5 degradation activity in chlamydia-infected HeLa cell lysates. Both the anti-CPAFn (54b) and anti-MOMP (MC22) antibodies were used to precipitate proteins from chlamydia-infected HeLa cell lysate. The intact (total) lysate, the supernatant after antibody precipitation, and the proteins precipitated by the antibodies (pellet) were all examined for their ability to degrade RFX5 in a cell-free assay. The anti-CPAFn antibody precipitated RFX5 degradation activity from supernatants to pellets. (C) Radioimmunoprecipitation analysis of proteins precipitated by anti-CPAFn or anti-MOMP antibodies. HeLa cells infected with chlamydia were metabolically labeled and the proteins in the labeled cell lysate were precipitated with antibodies. The supernatant after the first precipitation (I) was subjected to a second immunoprecipitation (II) with the same antibodies. Both the anti-CPAFn and anti-MOMP antibodies completely removed the corresponding molecules from the lysates during I precipitation. The ratio of cell lysate vs. antibody was the same as in Fig. 2 B, (D) Degradation of transcription factors RFX5 and USF-1 by chlamydia-synthesized and bacterium-expressed CPAF in a cell-free assay with or without the inhibitor lactacystin. HeLa L2 S100 (containing chlamydia-synthesized CPAF) was used as positive control, and HeLa S100 was used as a negative control. Bacterium-expressed GST-CPAF was used as a final concentration of either 0.2 (low; sufficient for RFX5 degradation) or 0.6 uM (high; sufficient for both RFX5 and USF-I degradation). The degradation was inhibitable by the proteasome inhibitor lactacystin (100 uM). A fusion protein containing GST and the COOH-terminal portion of CPAF (GST- CPAFc) failed to degrade any of the nuclear factors even at 4 uM. The anti-USF-2 antibody can detect both USF-1 and USF-2. Ns, nonspecific binding. (E). Purification of the recombinant human RFX5 from a bacterial expression system. The GST-RFX5 fusion protein was purified with glutathione-agarose beads. Different amounts of the purified protein were loaded to a 12% polyacrylamide SDS gel. After electrophoresis, the gel was stained with Coomassie blue dye. The GST-RFX5 fusion protein has a MW of 100 kDa. (F). Degradation of the purified human recombinant RFX5 by CPAF in a cell-free assay. The cell-free assay was carried out in the exact same way as described in D, except that 0.5 ug of the purified GST-RFX5 instead of the NEs was used as substrate in some reactions as indicated in the figure. RFX5 in NEs is defined as endogenous and the bacterium-expressed GST-RFX5 fusion protein defined as recombinant.

Figure 3. Intracellular distribution of CPAF during chlamydial infection. (A) Infected or uninfected HeLa cells were fractionated into cytosolic (SI00) and nuclear (NE) fractions. The fractionated cellular samples and purified chlamydial organisms were evaluated for RFX5 degradation activity in a cell-free assay (top row) or analyzed for the presence of CPAFn (second row), MOMP (third row), or host heat shock protein 70 (HSP70; last row) using the corresponding antibodies. HeLa L2 SI00 contained the highest RFX5 degradation activity, with a small amount of activity detected in the NE prepared from the same infected cells (lane HeLa L2 NE). The purified chlamydial organisms in the form of either the infectious elementary body (lane L2 EB) or the metabolically active but noninfectious reticulate body (lane L2 RB) contained no CPAF or CPAF activity. (B) Immunofluorescence detection of CPAF in chlamydia-infected cells. HeLa cell monolayer was infected with chlamydia organisms for 30 h. The monolayer was stained with Hoechst 32258 for DNA (blue), anti-MOMP antibody MC22 (probed with an FITC-conjugated, mouse lgG3-specific secondary antibody; green), and anti-CPAFn antibody 54b (probed with a Cy3-conjugated, mouse lgG1 -specific secondary antibody; red). Images were acquired individually for each stain in gray (top row) using a Cooker digital camera connected to an AX70 Olympus microscope and the single-color images were merged in frame into the triple-color image (bottom) using the software Image Pro. Note that the anti- CPAFn antibody only stained the cytosol (red) of the cells with chlamydial inclusions (green).

Figure 4. Degradation of RFX5 by recombinant CPAFcp. CPAFcp was expressed as a fusion protein with GST as the fusion partner and the purified GST-CPAFcp was tested for its ability to degrade either the endogenous RFX5 in HeLa cell NE (A) or a recombinant human RFX5 (GST-RFX5) purified from a bacterial expression system (B). The degradation was carried out in a cell free assay. CPAFcp was used at final concentrations of 0.05 (low), 0.2 (med), or 0.6 (high) μM, while the proteasome inhibitor lactacystin was used at 200μM. A fusion protein containing GST and just the COOH- terminal portion of CPAFcp (GST-CPAFcp C terminus) failed to degrade either the endogenous or the recombinant RFX5 even at a final concentration of 2 μM. DMSO, dimethyl sulfoxide (a solvent used for dissolving lactacystin); ns, nonspecific binding.

Figure 5. Correlation of CPAFcp secretion by C. pneumoniae with host transcription factor degradation in C. pneumoniae-infected cells. (A) An immunofluorescence staining approach was used to identify CPAFcp in C. pneumoniae- infected HeLa cells. HeLa monolayer cells were infected with C. pneumoniae at a low MOI so that only a small portion of cells was infected. The infection was allowed for 72 h in the presence of 2μg of cycloheximide/ml. The processed monolayer cells were costained with Hoechst 32258 for DNA (blue), anti-AR39 organism . rabbit antiserum (green), and anti- CPAFcp mouse antiserum (red). Images were acquired individually for each stain in gray (top row), and the single-color images were merged in frame into the triple-color image (bottom). Note that the anti-CPAFcp antibody only stained the cytosol (red) of the cells harboring C. pneumoniae inclusions (green). (B) A Western blot assay was used to compare the levels of CPAFcp and RFX5 in HeLa cells alone or HeLa cells infected with C. pneumoniae at various MOIs as indicated in the figure. Cell lysates from 2 x 10⁵ cells were loaded into each lane. After transfer onto nitrocellular membrane, CPAFcp and RFX5 were detected with the corresponding antibodies. Note the inverse correlation between the levels of CPAFcp and RFX5.

FIG. 6. CPAFcp is necessary for degradation of the host transcription factor RFX5. (A) CEs of HeLa cells alone (HeLa-CE) or HeLa cells infected with C. pneumoniae AR39 strain (AR39-CE) or infected with C. irachomatis LGV2 strain (L2-CE) were used as the sources of enzymes for degrading RFX5 in HeLa cell NE in a cell-free degradation assay as described above. Equivalent amounts of CEs were used for each reaction. RFX5 was detected with a rabbit anti-RFX5 antiserum. Note that both AR39 and L2 CEs degraded RFX5, but HeLa-CE failed to do so. (B) AR39-CE was absorbed with an anti-CPAFcp or control antibody, and both the intact AR39-CE and the final remaining supernatants after antibody absorption were measured for their ability to degrade RFX5 in a HeLa cell NE by using the cell-free degradation assay. Note that AR39-CE depleted with the anti-CPAFcp antibody lost most of its RFX5 degradation activity, whereas the AR39-CE depleted with the control antibody maintained the same level of degradation activity as the intact AR39- CE. Table 1 - To monitor the purification efficiency, the pooled fractions from each column purification were titrated for determining both protein concentration and degradation activity. The activity was measured by serial dilution of the pooled fractions and expressed as the highest dilution factors per mg of protein, under which the degradation activity was still detectable. These measurements were further used for calculating the recovery and enrichment of CPAF activity. ""' -Final volumes of pooled fractions as indicated in Fig. 1 A (prior to dilution). "$" CPAF activity was estimated based on the highest dilution of the pooled fractions when the CPAF degradation activity was still detectable and expressed as the highest dilution factors per mg of protein, hdf, highest dilution factors. "§"The recovery of CPAF activity was calculated based on the total activity recovered after each purification step using the total activity in L2 S100 as 100%. The enrichment was calculated based on CPAF activity per mg of protein (as described for Estimated CPAF activity above) using the activity in L2 S100 as the base.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

DEFINITIONS

As used herein, "CPAF" refers to a polypeptide or nucleic acid encoding Chlamydial protease-iike activity. It is of note that this definition includes native CPAF, recombinant CPAF and bioactive fragments thereof.

As used herein, "CPAF activity" refers to a transcription factor degrading activity isolated from Chlamydia which is inhibited by lactacystin.

As used herein, "CPAFct" refers to CPAF isolated from C. trachomatis, the DNA sequence of which is shown in SEQ ID No. 1. CPAFct polypeptide is encoded by nucleotides 43-1872 of SEQ ID No. 1.

As used herein, "CPAFcp" refers to CPAF isolated from C. pneumoniae. the DNA sequence of which is shown in SEQ ID No. 2. CPAFcp polypeptide is encoded by nucleotides 16-1875 of SEQ ID No. 2. As used herein, "CPAFci" refers to CPAF isolated from C. psittaci, the DNA sequence of which is shown in SEQ ID No. 3. CPAFcp polypeptide is encoded by nucleotides 1-1782 of SEQ ID No. 3.

As used herein, "purified" does not require absolute purity but is instead intended as a relative definition. For example, purification of starting material or natural material to at least one order of magnitude, preferably two or three orders of magnitude is expressly contemplated as falling within the definition of "purified".

As used herein, the term "treating" in its various grammatical forms refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causitive agent other abnormal condition.

As used herein, "immunogenic fragment" refers to a polypeptide capable of eliciting a specific immune response.

As used herein, "bioactive fragment" refers to a fragment of a biomolecule which retains measurable activity compared to the full-length biomolecule.

Described herein is the identification of a family of Chlamydial protease-like activity factors (CPAF) responsible for the degradation of host transcription factors. Specifically, genes encoding CPAF activity in C. psittachi (CPAFci), C. irachomatis (CPAFct) and C. pneumoniae (CPAFcp) have been identified. Furthermore, CPAF has been purified in both native and recombinant forms, as discussed below. As will be appreciated by one of skill in the art, purified CPAF has many uses. Specifically, as described below, purified CPAF may be used as a vaccine or may be used as a pharmaceutical for treating diseases requiring expression of host transcription factors RFX5 and USF-1. Also described are antibodies directed against CPAF which may be used as pharmaceutical compositions as described below or may be used for screening samples for Chlamydial infections.

It is of note that CPAF may be purified as described herein, for example, using antibody-based purification methods wherein the antibodies are directed against either native CPAF or against a fusion partner when purifying recombinant CPAF, as discussed below.

The CPAFct open reading frame (ORF) was previously designated as CT858 which describes the position of the gene relative to other chlamydial genes, but has no functional meaning. Previously, CT858 was listed as a hypothetical ORF containing a DHR domain found in a group of C-terminus proteases. The function of the DHR domain is not known and therefore this did not suggest that CT858 had protease activity. More importantly, there are many proteases in chlamydia. These proteases may be required for chlamydial biosynthesis but not for chlamydial interactions with host cells. Finally, it was not possible to know what substrates CT858 cleaved based on the sequence information. Thus, the discovery that CT858 is responsible for CPAF-mediated degradation of host transcriptional factors required for immune molecule expression is novel as is the discovery that CPAF is secreted, as described below.

As discussed above, CPAF degrades the host transcription factors that are required for host immune molecules (defense molecules) expression. It is also of note that a defect in these transcriptional factors is known to cause immune deficiency diseases. Our finding is that chlamydia has evolved CPAF to degrade these host transcription factors for the purpose of evading host defense mechanisms.

It is of note that CPAF is a better vaccine candidate than other chlamydial proteins. As will be apparent to one of skill in the art, proteins that are exposed on the surface of the pathogens are considered as the prime candidates based on the belief that antibodies raised to these surface-exposed proteins can neutralize the infectivity of the pathogens. This may be true for many extracellular pathogens. However, antibodies to chlamydia surface-exposed proteins may not work since chlamydia is an obligate intracellular pathogen, e.g. chlamydia replicates itself inside cells. One may ask why antibodies can effectively control viral infection since viruses have to replicate inside host cells and thus are also intracellular pathogens. The key is that chlamydia replicates in a closed cytoplasmic vacuole while viruses replicate freely in the cytoplasm of host cells. It is now known that some form of antibodies such as IgA can get into the cytoplasm space of host cells, which allow these antibodies to catch the viral components that are synthesized in the cytosol. However, although IgA can efficiently get into host cytosol, IgA may not be able to get into vacuoles in which chlamydia resides. Therefore, antibodies to chlamydia organism surface exposed proteins, such as the well studied major outer membrane protein (MOMP), may not be effective in controlling chlamydial infection. This is because these antibodies can not even reach their targets even after these antibodies get inside cells, which may explain why there has been no effective chlamydial vaccines despite the tremendous amount of efforts made in the field. CPAF is a much better vaccine candidate since CPAF is secreted from chlamydial vacuoles into host cytosol. For example, IgA antibodies raised with CPAF vaccines can get inside cells and bind to CPAF released in the cytosol of the chlamydia-infected cells. After CPAF function is blocked by CPAF- specific IgAs, the infected cells may be able to gain the ability to present chlamydial peptides either from CPAF or other chlamydial proteins via MHC expression (we have shown that a primary function of CPAF is to suppress MHC expression by degrading the key transcription factors required for MHC gene activation). Once the chlamydial peptide: MHC complexes are displayed on the surface of the infected cells, T lymphocytes will be able to detect the infected cells and activate various immune effector mechanisms such as enhancing phagocytosis of the intracellular chlamydia (mainly by CD4+ T cells) or lysis of the infected cells (by CD8+ T cells), which will lead to elimination of chlamydial organisms. In short, CPAF-based vaccines can elicit all three major arms of immune responses (antibodies, CD4+ and CD8+ T cells) to control chlamydial infections even when chlamydia hides inside a cytoplasmic vacuole.

Thus, native or recombinant CPAF or an immunogenic fragment thereof may be used to elicit an immune response in an individual. In these embodiments, purified CPAF may be combined with adjuvants known in the art as well as molecules intended to modulate the immune response, for example, interleukin 12. In other embodiments, a DNA vaccine comprising a suitable expression vector operably linked to DNA encoding CPAFct (SEQ ID No. 1), CPAFcp (SEQ ID No. 2) or CPAFci (SEQ ID No. 3) or an immunogenic fragment thereof is used to elicit an immune response in an individual.

In other embodiments, purified CPAF, either recombinant or native, or a bioactive fragment thereof is used in a screening assay for isolating compounds that reduces degradation of transcription factors by CPAF. In these embodiments, CPAF and at least one CPAF substrate, for example, RFX5 or USF1 are mixed with a test compound. The mixture is then kept under conditions suitable for CPAF degradation of the host transcription factors. The effect of the test compound on CPAF activity is then determined by detecting reduction in transcription factor levels, said reduction being an indication that the compound inhibits CPAF activity. It is of note that other CPAF substrates may be used within this screening method. It is also of note that compounds isolated by this screening method may be combined with pharmaceutical carriers as discussed below. As will be apparent to one of skill in the art, suitable CPAF inhibitors may be used in a similar manner as antibodies directed against CPAF. That is, the inhibitors and the antibodies will reduce CPAF activity, thereby counteracting Chlamydia's mechanism for evading the host immune system.

As will be apparent to one knowledgeable in the art, a therapeutically effective amount of the purified CPAF, an immunogenic fragment thereof or a bioactive fragment thereof or antibodies directed against CPAF is the amount sufficient to achieve the desired result. The amount administered will vary according to the concentration of the active agent and the body weight of the patient. Other factors include the degree of infection, the body weight and the age of the patient.

The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the condition of the patient, as well as body weight or surface area of the patient to be treated. Administration may be accomplished by a single dose or divided doses.

In some embodiments, the purified CPAF or fragments thereof and/or antibodies directed against CPAF discussed above may be combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non- biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, mathacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethycellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example, Remington: The Science and Practice of Pharmacy, 2000, Gennaro, AR ed., Eaton, PA: Mack Publishing Co.

As will be apparent to one knowledgeable in the art, specific carriers and carrier combinations known in the art may be selected based on their properties and release characteristics in view of the intended use. Specifically, the carrier may be pH-sensitive, thermo-sensitive, thermo-gelling, arranged for sustained release or a quick burst. In some embodiments, carriers of different classes may be used in combination for multiple effects, for example, a quick burst followed by sustained release.

In other embodiments, the purified CPAF, fragments thereof and/or antibodies directed against CPAF at concentrations or dosages described above may be encapsulated for delivery. Specifically, the purified CPAF, fragments thereof and/or antibodies directed against CPAF may be encapsulated in biodegradable microspheres, microcapsules, microparticles, or nanospheres. The delivery vehicles may be composed of, for example, hyaluronic acid, polyethylene glycol, poly(lactic acid), gelatin, poly(E- caprolactone), or a poly(lactic-glycolic) acid polymer. Combinations may also be used, as, for example, gelatin nanospheres may be coated with a polymer of poly(lactic-glycolic) acid. As will be apparent to one knowledgeable in the art, these and other suitable delivery vehicles may be prepared according to protocols known in the art and utilized for delivery of the purified CPAF and/or antibodies directed against CPAF. Alternatively, the delivery vehicle may be suspended in saline and used as a nanospray for aerosol dispersion onto an area of interest.

In some embodiments, the purified CPAF, fragments thereof and/or antibodies directed against CPAF in any suitable form as described above, may be combined with biological or synthetic targetting molecules, for example, site-specific binding proteins, antibodies, lectins or ligands, for targetting the purified CPAF, fragments thereof and/or antibodies directed against CPAF to a specific region or location. Specifically, in some embodiments, antibodies against CPAF may be linked to toxic compounds for targeting cells infected with Chlamydia.

As discussed herein, CPAF inhibitors isolated as discussed above and the antibodies directed against CPAF may be used to treat, prevent or reduce the severity of Chlamydia infections. Specifically, these compounds will interfere with CPAF activity, thereby preventing degradation of host transcription factors and. allowing MHC antigen expression.

As discussed above, purified CPAF, either native or recombinant, or a bioactive fragment thereof may be used to treat or reduce the severity of diseases associated with over-expression of transcription factors USF1 and RFX5. As will be apparent to one of skill in the art, CPAF would reduce the levels of these transcription factors, thereby reducing severity of the disease.

The invention provides kits for carrying out the methods of the invention. Accordingly, a variety of kits are provided. The kits may be used for any one or more of the following (and, accordingly, may contain instructions for any one or more of the following uses): treating: C. trachomatis, C. pneumoniae, C. psittaci and/or C. pecorum infection in an individual; reducing severity of one or more symptoms associated with C. trachomatls, C. pneumoniae, C. psittaci and/or C. pecorum infection in an individual; or reducing recurrence of one or more symptoms associated with C. trachomatls, C. pneumoniae, C. psittaci and/or C. pecorum infection.

The kits of the invention comprise one or more containers comprising the purified CPAF or antibodies directed against CPAF., a suitable excipient as described herein and a set of instructions, generally written instructions although electronic storage media (e.g., magnetic diskette or optical disk) containing instructions are also acceptable, relating to the use and dosage of the purified CPAF or antibodies directed against CPAF for treating, preventing or ameliorating Chlamydial infections and/or diseases associated therewith. The instructions included with the kit generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers of the the purified CPAF or antibodies directed against CPAF may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.

The purified CPAF and/or antibodies directed against CPAF of the kit may be packaged in any convenient, appropriate packaging. For example, if the composition is a freeze-dried formulation, an ampoule with a resilient stopper is normally used, so that the drug may be easily reconstituted by injecting fluid through the resilient stopper. Ampoules with non-resilient, removable closures (e.g., sealed glass) or resilient stoppers are most conveniently used for injectable forms of the purified CPAF or antibodies directed against CPAF.

The invention will now be described further by way of examples. However, it is important to note that the invention is not in any way restricted by these examples.

EXAMPLE I - Column Chromatography

For purification and sequence identification of CPAF, cytosolic protein preparation from 200 large flasks (150 cm²) of chlamydia-infected HeLa cells (HeLa L2 S100) (Fan et al., 1998) was subjected to the following three consecutive column separations: DEAF, heparin, and Mono Q (all columns were from Amersham Pharmacia Biotech). An AKTA purifier 10 (Amersham Pharmacia Biotech) was used to run the columns. Fractions collected from each column were monitored for both degradation activity in a cell-free assay as we have established previously (Zhong et al., 2000), and total protein profiles on SDS-polyacrylamide gels. Fractions with degradation activity were pooled and both the protein concentration and degradation activity of the pooled fractions were carefully titrated. These measurements were used to calculate the purification efficiency. The pooled fractions were loaded to the next column for further fractionation. However, the pooled fractions collected from DEAE column were diluted 1 :2 with H₂0 to reduce the salt concentration before the heparin column purification. Two dominant protein bands that correlated with the degradation activity from the final Mono Q column were excised for protein sequence identification using both mass spectrometry analysis (Biorealis Biosciences Inc.) and NH₂-terminal sequence determination (University of Victoria, British Columbia, Canada). For chromatographic analysis of CPA, a size exclusion column was loaded with HeLa L2 S100 and eluted with PBS (Zhong et al., 2000). The eluted fractions were subjected to both the RFX5 degradation activity measurement and Western blot detection of host 20S proteasome and CPAF components.

EXAMPLE II - Western Blot.

The Western blot assay was carried out as we described previously (Zhong et al., 1997, PNAS USA 94: 13856-13861 ). Mouse antibodies were used to detect the NH₂- terminal portion of CPAF (CPAFn) fragment (CPAFn546; mlgG1 ), chlamydial major outer membrane protein (MOMP) (MC22; mlgG3), CPAFcp C terminus (antiserum was generated by immunizing mice with a GST fusion protein containing the C-terminal half of the CPAFcp), eukaryotic HSP70 (lgG2a; Santa Cruz Biotechnology, Inc.), and 20S proteasome subunits (AFFINITI Research Products Limited). Rabbit antibodies were used to detect RFX5 (Rockland immunochemicals), USF-1 , and USF-2 (Santa Cruz Biotechnology, Inc.).

Samples from the various cell-free degradation assays were directly subjected to sodium dodecyl sulfate-polyacrylamide gel separation and Western blot analysis. Samples from infection dose titration experiments were obtained as follows: HeLa cells were infected with C. pneumoniae AR39 strain at various multiplicities of infection (MOIs) as indicated in the legend to Fig. 2 in the presence of 2 p.g of cycloheximide/ml. At 48 h after infection, the cell culture was washed and replenished with fresh growth medium without cycloheximide in order to allow host cells to recover their ability to synthesize new proteins. After an additional 24 h of culture, the cell samples were harvested for sodium dodecyl sulfate-polyacrylamide gel separation and Western blot analysis. Mouse antibodies were used to detect CPAFcp C terminus (antiserum was generated by immunizing mice with a GST fusion protein containing the C-terminal half of the CPAFcp [data not shown]). Rabbit antibodies were used to detect RFX5 (Rockland Immunochemicals, Gilbertsville, Pa.). EXAMPLE 111 - Cell free Degradation Assay.

The cell-free degradation assay was performed as described previously (Zhong et al., 2000). To generate fusion proteins for the cell-free assay, chlamydial DNA sequences coding for CPAF or CPAF fragments were cloned into a pGEX vector (Amersham Pharmacia Biotech) and expressed as fusion proteins with the glutathione S-transferase (GST) as fusion partner. The fusion proteins were purified with glutathione-conjugated agarose beads as described in the manufacturer's manual (Amersham Pharmacia Biotech). The following procedure was used to prepare nuclear extracts (NEs) as substrate (containing RFX5 and USF-1 and USF-2) for the cell-free assay. Normal HeLa cells were dounced to break cytoplasmic membranes and the pellets were repeatedly washed with a buffer consisting of 1 % NP-40 and 150 mM NaCl in 50 mM Tris, pH 8.0, plus a protease inhibitor cocktail to remove cytosol/membrane proteins as much as possible. The final washed nuclear pellets were extracted with a buffer consisting of 0.5 M NaCl and 1 % Triton X-100 in 20 mM Tris, pH 8.0. The NEs thus prepared are essentially free of 20S proteasome components as detected on Western blot. The human RFX5 gene from pREP-4/RFX5 plasmid was cloned into the pGEX vector and RFX5 was expressed in the JM109 Escherichia coli strain as a GST fusion protein. The GST-RFX5 fusion protein was purified to homogeneity using glutathione-conjugated agarose beads as described above. The purified GST-RFX5 was used as substrate in the cell-free degradation assays.

A cytosolic extract (CE) of either chlamydia-infected or normal HeLa cells was made with a buffer consisting of 1% NP-40 and 150 mM NaCl in 50 mM Tris (pH 8,0) plus a protease inhibitor cocktail. The CE thus prepared were used as the source of enzymes. To generate fusion proteins for the cell-free assay, C. pneumoniae AR39 DNA sequences coding for CPAFcp or CPAFcp fragments were cloned into a pGEX vector (Pharmacia) and expressed as fusion proteins with glutathione S-transferase (GST) as the fusion partner. The fusion proteins were purified with glutathione-conjugated agarose beads as described in the manufacturer's manual (Pharmacia). The degradation activity of the purified protein was measured in the cell-free assay. The following procedure was used to prepare nuclear extracts (NEs) as substrate (containing RFX5) for the cell-free assay. Normal HeLa cells were homogenized to break cytoplasmic membranes and the residual pellets were repeatedly washed with the NP-40 buffer as described above to remove cytosol or membrane proteins as much as possible. The final washed nuclear pellets were extracted with a buffer consisting of 0.5 M NaCl and 1 % Triton X-100 in 20 mM Tris (pH 8.0). To prepare the purified RFX5 as substrate, the human RFX5 gene from pREP- 4/RFX5 plasmid was cloned into the pGEX vector, and the fusion protein GST-RFX5 was expressed and purified to homogeneity by using glutathione-conjugated agrose beads. The purified GST-RFX5 was used as a substrate in the cell-free degradation assays.

EXAMPLE IV - Immunofuorescence Staining Assay

Immunofluorescence detection of CPAF in chlamydia-infected cells was carried out as described previously (Fan et al., 1998; Zhong et al., 1997). In brief, HeLa cell monolayer was infected with Chlamydia trachomatis L2 for 30 h. The monolayer, after it was fixed with paraformaldehyde (Sigma-Aldrich) and permeabilized with Saponin (Sigma- Aldrich), was costained with Hoechst 32258 (blue), anti-MOMP antibody MC22 (probed with an FITC-conjugated, mouse lgG3-specific secondary antibody), and anti-CPAFn antibody 54b (probed with a Cy3-conjugated, mouse IgGI-specific secondary antibody). Images were acquired individually for each stain in gray using a Cooker digital camera connected to an AX70 Olympus microscope, and the single-color images were merged in frame into the triple-color image using the software Image Pro.

Immunoflorescence detection of CPAFcp in C. pneumoniae-infected cells was carried out as follows: HeLa cell monolayer was infected with C. pneumoniae AR39 for 72 h. The monolayer, after fixation with paraformaldehyde (Sigma, St. Louis, Mo.) and permeabilization with Saponin (Sigma), was costained with Hoechst 32258 (blue), a rabbit anti-AR39 antiserum (raised with purified AR39 elementary bodies [data not shown]; probed with a Cy2-conjugated goat anti-rabbit immunoglobulin G [IgG]). and a mouse anti- CPAFcp antiserum (probed with a CY3conjugated goat anti-mouse lg(i). Images were acquired individually for each stain by using a Hamamatsu digital camera connected to an AX70 Olympus microscope, and the single-color images were merged in frame into the triple-color image by using the software SimplePCI.

EXAMPLE V - Immunoprecipitation Assay

The immunoprecipitation assays were carried out as described previously (Zhong et al., 2000; Zhong et al, 1996, J Exp Med 184: 2061-2066). For antibody depletion of CPAF and the MOMP, both the anti-CPAFn and anti-MOMP antibodies were used to precipitate proteins in lysates of chlamydia-infected HeLa cells without radiolabeling (Zhong et al, 2000). The intact lysate, the supernatant after antibody precipitation, and the proteins precipitated by the antibodies were all examined for their ability to degrade RFX5 in a cell-free assay. To visualize the proteins precipitated by the above antibodies, a radioimmunoprecipitation assay was performed (Zhong et al, 1996). In brief, HeLa cells infected with chlamydia were metabolically labeled with [S³⁵]methionine/cysteine (ICN Biomedicals) and the proteins in the labeled cell lysate were precipitated with the corresponding antibodies. The supernatant after the first precipitation (I) was subjected to a second immunoprecipitation (II) with the same antibodies. Both the anti-CPAFn and anti- MOMP antibodies completely removed the corresponding molecules from the lysates during I precipitation.

A mouse antiserum raised with the C-terminal fragment of CPAFcp was used for depleting CPAFcp and a control antiserum from mouse similarly immunized with . an unrelated GST fusion protein for mock depletion. Then, 5 μl of each antiserum was conjugated to protein G-agarose beads, and the antibody-bead complexes were used to absorb 20 μl of cytosol extracts of AR39-infected HeLa cells (AR39-CE) in a total volume of 60 ill for 1 h at room temperature. After a second absorption, 15 μl of the final remaining supernatant was compared with 5 μl of control AR39-CE (without absorption) for their ability to degrade RFX5 in a cell-free assay.

EXAMPLE VI - Purification and Sequence Identification of CPAF.

A chlamydial protease-like activity (designated as CPA) has been detected in the cytosol of chlamydia-infected cells and its presence correlated with degradation of host transcription factors (Zhong et al, 2000). To better understand the molecular mechanisms underlying chlamydial evasion of host immune recognition, we used a column chromatography approach to identify the factor(s) responsible for CPA (Fig. 1 A). Although it was not possible to correlate any obvious protein bands with degradation activity in fractions eluted from either DEAE or heparin column, these two purification steps allowed us to remove most of the unwanted proteins and to dramatically enrich the degradation activity (Table I). The final Mono Q column purification resulted in 1 , 000-fold increase in specific activity (Table I). Indeed, two predominant protein bands in fractions eluted from a final step Mono Q column correlated with the nuclear protein degradation activity (Fig. 1 A). Both protein bands were excised for sequence identification using mass spectrometry (Fig. 1 B). The sequences of a total of eight tryptic fragments derived from the bottom band matched the sequence of the NH₂-terminal portion of a chlamydial protein encoded by open reading frame (ORF) CT858 (sequence data are available from GenBank EMBL/DDBJ under accession no. AE001359; Stephens et al, 1998, Science 282: 754-759), whereas the sequences of 17 tryptic fragments from the top band matched the sequence of the COOH-terminal portion of the same chlamydial protein (Fig. 1 B). These observations indicate that the two purified protein bands are encoded by a single ORF in the chlamydial genome. We have thus designated the bottom band protein as CPAFn and the top as CPAFc (Fig. 1 ). The sequence identity of CPAF was further confirmed using conventional NH₂-temninal sequencing. The CPAF gene has no significant overall homology to any other known genes despite its high conservation among chlamydial strains (Stephens et al, 1998; Kalman et al, 1999, Nat Genet 21: 385-389; Read et al, 2000, Nucleic Acids Res 28: 1397-1406). Although a short CPAF sequence ₍ ⁴⁸⁸ picVLINEQDFSCADFFVVLKDNDRALIVGTRT AGAG⁵²⁵) was found to share a significant homology with the COOH-terminal sequences of tail-specific proteinases from various species, the function of the conserved sequence domain is not yet known. Because of the limited homology, the CPAF gene was previously designated as either hypothetical ORF or "predicted protease containing interphotoreceptor retinoid-binding protein (IRBP) and 13,14-dihydroxy-retinol (DHR) domains" (Stephens et al, 1998; Kalman et al, 1999; Read et al, 2000), as discussed above.

EXAMPLE VII - CPAF Is Both Necessary and Sufficient for Degradation of Host Transcription Factors

We have previously demonstrated that the chlamydial proteasome-like activity does not reflect the function of host proteasome components (Zhong et al, 2000). When fractions obtained from a size exclusion chromatography column were probed with an antibody recognizing CPAFn, the chlamydial proteasome-like activity measured with RFX5 degradation was found to correlate very well with the presence of the CPAFn fragment but not host proteasome components (Fig. 2 A). To test whether CPAF is necessary for the chlamydial proteasome-like activity, we used the anti-CPAFn antibody to precipitate CPAFn from chlamydia-infected HeLa cell lysate and measured the RFX5 degradation activity of the precipitated pellets as well as the activity of the supernatants after the antibody depletion (Fig. 2 B). The total lysate of chlamydia-infected HeLa cells efficiently degraded RFX5. However, the supernatants remaining after antibody-mediated depletion of CPAFn completely lost the ability to degrade RFX5, whereas RFX5 degradation activity was readily detected in the precipitated material. Depletion of RFX5 degradation activity by anti-CPAFn was specific, because a control antibody recognizing the MOMP failed to precipitate this activity from the lysates. To monitor the efficiency of the antibody O 02/079244

19 precipitation, radiolabeled lysate from chlamydia-infected HeLa cells was similarly precipitated with anti-CPAFn (I precipitation) and the supernatant after the first precipitation was reprecipitated (II precipitation) with the same reagent (Fig. 2 C). Autoradiography after SDS-PAGE of the material in both the I and II immunoprecipitates revealed that the anti-CPAFn antibody effectively removed both the CPAFn and CPAFc fragments from the lysates during the I precipitation, because the II immunoprecipitate showed minimal amounts of CPAF fragments. The control anti-MOMP antibody efficiently precipitated MOMP but not CPAF molecules from the lysates (Fig. 2 C). Together, these experiments demonstrate that CPAF or CPAF-associated material is necessary for RFX5 degradation activity in chlamydia-infected cell lysates.

To evaluate whether CPAF is sufficient for degrading host transcription factors, the protein encoded by the full-length CPAF gene was expressed as a fusion with GST, using a bacterial system. The purified GST-CPAF was evaluated for its ability to degrade RFX5 in a cell-free assay in comparison to material extracted from infected cells (HeLa L2 S100; Fig. 2 D). The HeLa L2 S100 degraded both the RFX5 and USF-1 transcription factors in NEs, whereas the material from uninfected HeLa cells (HeLa S 00) failed to do so. Furthermore, even though the chlamydia proteolytic activity could be fractionated away from host cell 20S proteasomes (Fig. 2 A), the degradation activity of the HeLa L2 S100 was completely inhibited by lactacystin, an irreversible inhibitor of proteasoma^ proteases. These observations suggest that a chlamydial proteasome-like activity was being measured in this assay, as reported previously (Zhong et al, 2000). More significantly, the purified GST-CPAF was able to degrade both RFX5 and USF-1 and the degradation was inhibitabie by lactacystin. A GST fusion protein containing only the COOH-terminal portion of CPAF (GST-CPAFc) failed to degrade either of the host nuclear proteins even at a concentration 20 times higher than that of GST-CPAF (Fig. 2 D). These observations demonstrated that the purified CPAF is sufficient to mediate the degradation of host transcription factors in HeLa cell NEs. However, this set of experiments failed to address whether other components in the NEs also contributed to the degradation activity.

To further confirm that CPAF alone is sufficient for the degradation activity, we expressed the human transcription factor RFX5 in a prokaryotic system as a GST fusion protein. The GST-RFX5 was purified to homogeneity (Fig. 2 E) and the purified protein was used as the substrate for measuring the degradation activity of the cloned CPAF in a cell-free assay (Fig. 2 F), The cloned CPAF completely degraded the endogenous RFX5 in HeLa cell NEs and the degradation activity was inhibited by lactacystin, which is consistent with the observations described above in Fig. 2 D. More importantly, the cloned CPAF also effectively degraded the recombinant human RFX5 purified from a bacterial expression system (Fig. 2 F). Degradation of the recombinant RFX5 was inhibited by lactacystin, suggesting that the same enzymatic activity was responsible for the degradation of both endogenous and recombinant RFX5. A similarly purified control GST- CPAFc fusion protein failed to degrade the recombinant RFX5, suggesting that degradation of RFX5 measured in the assay was not contributed by bacterial contaminants (if there was any) or the GST fusion partner. These observations clearly demonstrate that the chlamydial CPAF alone is sufficient for degrading host RFX5.

EXAMPLE Vlil - Intracellular Distribution of CPAF during Chlamydial Infection.

Although CPAF is encoded by the chlamydial genome, the host transcription factor degradation activity was detected in the cytosol of the infected cells (Zhong et al, 2000), suggesting that CPAF is secreted into host cell cytosol. We next used cell fractionation followed by immunoblotting to further evaluate the subcellular distribution of CPAF during chlamydial infection (Fig. 3 A). The majority of RFX5 degradation activity was associated with the cytosolic fraction of HeLa cells infected with chlamydia (Fig. 3 A, top row; lane HeLa L2 S100), with a small amount of activity in the NE fraction (lane HeLa L2 NE). RFX5 degradation activity correlated very well with the presence of CPAFn fragment. The cytosolic fraction of uninfected HeLa cells had no protease activity (lane HeLa S100). Surprisingly, the purified chlamydial organisms themselves also had no capacity to degrade RFX5. These findings suggest that CPAF is mainly localized in the cytosolic fraction of the infected HeLa cells and does not accumulate to any appreciable extent in chlamydia organisms themselves.

An immunofluorescence assay was used to further confirm the intracellular distribution of CPAF (Fig. 3 B). CPAF was only detected in the cells containing chlamydial inclusion bodies but not the adjacent normal HeLa cells growing on the same coverslip, indicating that CPAF is restricted to chlamydia-infected cells. As expected from the cell fractionation studies, CPAF was mainly detected in the cytoplasmic portion of the infected cells, even though CPAF is encoded by the chlamydial genome. These observations suggest that the chlamydia-rsynthesized CPAF is secreted into the host cell cytoplasm, allowing it to access host proteins.

EXAMPLE IX - A recombinant chlamydial protease-like activity factor cloned from the C. pneumoniae genome is sufficient for degrading host transcription factor RFX5. Sequence homology searching (http: //www.nebi. nlm.nih.gov/blast/Blast.cgi) was used to identify a conserved hypothetical open reading frame (cpn1016; http: //violet.berkeley.edu:4231/cpn/p1016.htm1) in the C. Pneumoniae genome (Kalman et al, 1999, Nat Genet 21 : 385-389; Read et al, 2000, Nucleic Acids Res 28: 1397-1406) that encodes a homologue of CPAFct. The C. pneumoniae homologue is designated CPAFcp. There is 48% amino acid sequence identity between CPAFct and CPAFcp (http:/Avww.ncbi.nlm.nih.gov/blast/Blast.cgi#7388442). We expressed CPAFcp as a fusion protein with GST as the fusion partner and tested the purfied GST-CPAFcp fusion protein for its ability to degrade host transcription factor RFX5 in a cell-free degradation assay (Fig. 4). The GST-CPAFcp readily degraded RFX5 in a HeLa cell NE, and the degradation activity was inhibited by lactacystin but not the solvent dimethyl sulfoxide alone (Fig. 4A). A GST fusion protein containing just the C terminus of CPAFcp did not degrade RFX5. These observations demonstrated that CPAFcp possesses a proteolytic activity similar to that of CPAFct. To exclude the possible contribution of other components in the HeLa NEs to the CPAFcp degradation activity, we used a recombinant RFX5 purified from a bacterial expression system as the substrate to further evaluate the CPAFcp degradation activity in a ceil-free assay (Fig. 4B). CPAFcp effectively degraded the recombinant RFX5, and the degradation was also inhibited by lactacystin, suggesting that the same enzymatic activity is responsible for degrading both the endogenous and recombinant RFX5. Again, a fusion protein containing the C terminus of CPAFcp did not degrade the recombinant RFX5, suggesting that bacterial contaminants did not contribute to the degradation activity. Together, these observations demonstrate that CPAFcp alone is sufficient for degrading RFX5. Correlation of CPAFcp secretion by C. pneumoniae organisms with host transcription factor degradation in C. pneumoniae-infected cells. Although we have demonstrated that the recombinant CPAFcp purified from a bacterial expression system is sufficient for degrading RFX5, it is not known whether CPAFcp is actually produced by C. pneumoniae organisms and whether the C. pneumoniae-synthesized CPAFcp is functional. We first used a CPAFcp-specfic antibody to detect endogenous CPAFcp in C. pneumoniae-infected HeLa cells (Fig. 5A). CPAFcp was found predominantly in the cytosol of the infected cells, suggesting that C. pneumoniae organisms not only produce CPAFcp but also secrete the CPAFcp into host cell cytoplasm to allow CPAFcp to access host proteins. The fact that CPAFcp is only detected in the infected cells but not in the adjacent uninfected cells suggests that CPAFcp is restricted to the infected cells only. To correlate CPAFcp production with the host transcription factor degradation activity in the \

22

infected cells, we used a Western blot assay to compare the levels of CPAFcp and transcription factor RFX5 in HeLa cells alone or HeLa cells infected with C. pneumoniae at various MOIs (Fig. 5B). The level of CPAFcp produced by C. pneumoniae increased, while the level of the host transcription factor RFX5 decreased in an infection dose-dependent manner. The inverse relationship between CPAFcp and RFX5 suggests that CPAFcp may be responsible for the disappearance of RFX5.

EXAMPLE IX - CPAFcp is required for the degradation of RFX5 in C. pneumoniae- infected cells.

After correlating C. pneumoniae secretion of CPAFcp with the degradation of RFX5 in C. pneumoniae-infected cells, we next measured the RFX5 degradation activity in the cytosol of C. pneumoniae-infected cells by using a cell-free degradation assay (Fig. 6A). A CE from C. trachomatis LGV2 strain-infected cells (L2-CE) was used as positive control since L2-CE has been previously shown to contain the RFX5 degradation activity, as discussed above. A CE from uninfected HeLa cells (HeLa-CE) was used as negative control. The CE from C. pneumoniae AR39 strain-infected cells (AR39-CE) completely degraded RFX5, whereas the negative control HeLa-CE failed to do so. The RFX5 degradation activity in AR39-CE was inhibited by lactacystin, suggesting that AR39-CE possesses a proteolytic activity similar to that of the recombinant CPAFcp (see Fig. 4).

To directly assess whether CPAFcp is responsible for the RFX5 degradation activity in the cytosol of infected cells, we used a CPAFcp-specific antibody to perform a depletion experiment (Fig. 6B). AR39-CE but not HeLa-CE degraded RFX5. More importantly, the AR39-CE supernatant after being absorbed with a CPAFcp-specific antibody conjugated to agarose beads could no longer degrade RFX5, while the AR39-CE supernatant similarly absorbed with a control antibody still maintained the RFX5 degradation activity. This result demonstrates that CPAFcp is necessary for the RFX5 degradation activity in the C. pneumoniae-infected cell cytosol.

Chlamydial growth occurs strictly within a modified cytoplasmic vacuole of eukaryotic cells (Hackstadt et al, 1997, Trends Microbiol 5: 288-293), and the possibility of communication between chlamydial vacuoles and host cellular compartments has frequently been discussed (Hackstadt et al, 1995, PNAS USA 92: 4877-4881 ; Stephens,- 1994, Trends Microbiol 2: 99-101 ; Hatch, 1998, Science 282: 638-639; Wylie et al, 1997, J Bacteήol 179: 7233-7242). At least one purpose of such transfer of chlamydia-derived proteins to the invaded cell appears to be protection from host immune recognition (Zhong et al, 2000; Zhong et al, 1999) and attack (Fan et al, 1998). Our previous studies suggested that chlamydia may secrete a CPAF into the host cell cytosol to degrade transcription factors required for MHC gene activation, thus limiting expression of these key proteins involved in T cell antigen recognition (Zhong et al, 2000; Zhong et al, 1999). We have now identified the gene encoding CPAF that is secreted into host cell cytosol and demonstrated that CPAF is both necessary and sufficient for the degradation of the transcription factors RFX5 and USF-1. Interestingly, CPAF was identified as two separate polypeptides encoded by a single ORF in the chlamydial genome (Fig. 1 A). Both the CPAFn and CPAFc fragments are coprecipitated from chlamydia-infected cell lysates by an antibody that only recognizes the CPAFn fragment (Fig. 2 C). These observations suggest that CPAF may function in the form of intramolecular dimers. Although the details of the activation and secretion of CPAF remain to be fully determined, it is clear that by secreting a single factor into the cytosol, chlamydia can suppress both MHC class I and class II antigen expression. Many viruses are known to cause suppression or degradation of cellular proteins required for mounting host defense responses (Scheffner et al, 1990, Cell 63: 1129-1136; Shamu et al, 1999, J Cell Biol 147: 45-58; Tortorella et al, 2000, Annu Rev Immunol 18: 861-926). However, these viral strategies are often dependent on the function of host proteasomes (Hughes et al, 1997, PNAS USA 94: 1896-1901 ; Tortorella et al, 1998, J Cell Biol 142: 365-376). We have demonstrated that CPAF is both necessary and sufficient for degrading host transcription factors required for MHC antigen expression. Thus, this report identifies a novel molecular mechanism by which a nonviral intracellular pathogen interacts with its host and manipulates immune responses for its benefit.

Evasion of host defense is likely advantageous for chlamydia to survive for long periods of time in its host. Our results demonstrate that CPAFcp by itself is sufficient for degrading the host transcription factor RFX5 in a cell-free degradation assay with a purified human RFX5 as substrate. We have also shown that CPAFcp is required for the RFX5 degradation activity in the C. pneumoniae-infected cells by using an antibody depletion experiment. There is no system for genetic transformation of chlamydia, so definitive gene knockout experiments are not possible. However, antibody depletion of CPAFcp and blocking CPAFcp function with specific protease inhibitors clearly indicate the biological function of CPAFcp. These experiments together have provided the first line of evidence demonstrating that C, pneumoniae has indeed evolved specific strategies for evading host adaptive immunity. Although there is only 48% amino acid sequence identity between CPAFct and CPAFcp (http://www.ncbi.nlm.nih.gov/ blast£last.cgi#7388442), the proteolytic activity of both CPAFs is conserved, suggesting that degradation of host transcription factors required for MHC antigen expression may be essential for chlamydial survival in its hosts. Our studies on CPAFct and CPAFcp have shown that CPAF is mainly secreted into host cell cytosol without an obvious presence in the organisms themselves, suggesting that the main purpose for chlamydial synthesis of CPAF is to use CPAF to manipulate host cells. However, secretion of chlamydial proteins into host cell cytosol is itself a danger for the chlamydial organisms since microbial products present in the host cell cytosol can be processed and presented to host T lymphocytes. These lymphocytes can potentially detect and attack the infected cells (Germain, 1994, Cell 76: 287-299).

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

Table I. Summary of CPAF Purfi ication

, Estimated Estimated total Estimated Estimated

Purification Total CPAF CPAF activity activity step Volume* protein activity* activity recovery- enrichment"

ml mg hdf/mg hdf % fold

L2 S100 100 5,310 0.094 499 100 1

DEAE 70 214 0.97 208 42 10

Heparin 150 13 11.49 149 30 122

Mono Q 5 0.5 90 45 9 957 ,

Claims

1. A composition comprising purified CPAF.

2. The composition according to claim 1 wherein the CPAF is CPAFct.

3. The composition according to claim 1 wherein the CPAF is CPAFcp.

4. The composition according to claim 1 wherein the CPAF is CPAFci.

5. The composition according to claim 1 including an adjuvant.

6. The composition according to claim 1 including a pharmaceutically acceptable excipient.

7. A purified polypeptide having CPAF activity.

8. The polypeptide according to claim 7 wherein the polypeptide is from C. pneumoniae.

9. The polypeptide according to claim 7 wherein the polypeptide is from C. trachomatis.

10. The polypeptide according to claim 7 wherein the polypeptide is from C. psittaci.

11. A method of identifying a compound that reduces degradation of transcription factors by CPAF comprising: contacting purified CPAF and at least one CPAF substrate with a test compound; and determining whether CPAF activity is reduced in the presence of the test compound by detecting reduction in CPAF substrate levels, said reduction being an indication that the compound inhibits CPAF activity.

12. A process for making a compound that inhibits CPAF activity comprising: carrying out the method of claim 1 to identify a compound that inhibits CPAF; and manufacturing the compound.

13. A method of eliciting an immune response in an animal comprising: introducing into the animal a composition comprising purified CPAF.

14. A purified antibody that binds specifically to CPAF.

15. The antibody according to claim 14 wherein the CPAF is CPAFct.

16. The antibody according to claim 14 wherein the CPAF is CPAFcp.

17. The antibody according to claim 14 wherein the CPAF is CPAFci.

18. The antibody, according to any one of claims 14-17 wherein the antibody inhibits CPAF activity.