Outer membrane proteins preferentially load MHC class II peptides: Implications for as a Chlamydia trachomatis T cell vaccine

Abstract

CD4 T cell immune responses such as interferon-γ and tumor necrosis factor-α secretion are necessary for Chlamydia immunity. We used an immunoproteomic approach in which Chlamydia trachomatis and Chlamydia muridarum-derived peptides presented by MHC class II molecules on the surface of infected dendritic cells (DCs) were identified by tandem mass spectrometry using bone marrow derived DCs (BMDCs) from mice of different MHC background. We first compared the C. muridarum immunoproteome in C3H mice to that previously identified in C57BL/6 mice. Fourteen MHC class II binding peptides from 11 Chlamydia proteins were identified from C3H infected BMDCs. Two C. muridarum proteins overlapped between C3H and C57B/6 mice and both were polymorphic membrane proteins (Pmps) which presented distinct class II binding peptides. Next we studied DCs from C57BL/6 mice infected with the human strain, C. trachomatis serovar D. Sixty MHC class II binding peptides derived from 27 C. trachomatis proteins were identified. Nine proteins were orthologous T cell antigens between C. trachomatis and C. muridarum and 2 of the nine were Pmps which generated MHC class II binding epitopes at distinct sequences within the proteins. As determined by antigen specific splenocyte responses outer membrane proteins PmpF, -G and -H and the major outer membrane protein (MOMP) were antigenic in mice previously infected with C. muridarum or C. trachomatis. Furthermore a recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with MOMP formulated with a Th1 polarizing adjuvant significantly accelerated (p < 0.001) clearance in the C57BL/6 mice C. trachomatis transcervical infection model. We conclude that Chlamydia outer membrane proteins are important T cell antigens useful in the development of a C. trachomatis subunit vaccine.

1. Indrouction

CD4 T cell-mediated immunity is a major component of host defense against Chlamydia infection [1] and the identification of epitopes presented by MHC class II molecules should enable the development of a Chlamydia T cell vaccine [2]. Dendritic cells (DCs) are at the centre of initiation of T cell mediated immune responses [3]. DCs capture antigen in the periphery and migrate to regional lymph nodes where they present processed antigen on MHC molecules to naïve T cells to induce T cell mediated immune responses. Since T cells mainly recognize protein antigens, protective vaccine candidates are likely to be found within the proteome of an organism. An approach called immunoproteomics [4], in which peptides presented by immunoaffinity purified MHC molecules from infected DCs are identified by tandem mass spectrometry (MS/MS) allow genomic information to guide the delineation of the T cell immunoproteome of an organism.

We previously used immunoproteomics to identify epitopes presented by MHC class II molecules from C57BL/6 bone marrow derived DCs (BMDCs) infected with Chlamydia muridarum [2,5]. Chlamydia-specific CD4 T cells harvested from mice recovered from C. muridarum infection recognized these MHC class II-bound peptides in vitro [6] and the source proteins of these MHC class II-bound peptides accelerated clearance of C. muridarum genital tract infection when formulated as vaccine with a Th1 polarizing adjuvant consisting of cationic liposome and modified mycobacterial cord factor [7].

We are interested in identifying Chlamydia trachomatis proteins presented by MHC class II molecules. In this study we investigated the C. trachomatis immunoproteome using infected C57BL/6 murine DCs and compared the findings to the C. muridarum immunoproteome identified in two different inbred strains of mice (C57BL/6 and C3H). We found that outer membrane proteins were commonly identified as source proteins encoding MHC class II binding peptides in all three experimental conditions. When used as vaccine with a Th1 polarizing adjuvant recombinant outer membrane proteins accelerated clearance of C. trachomatis from transcervically infected C57BL/6 mice. We conclude that outer membrane proteins are important T cell antigens in both C. trachomatis and C. muridarum capable of presentation by multiple MHC class II molecules and which elicit protective immunity. They are therefore useful for vaccine development.

2. Methods

2.1. Chlamydia strains

C. muridarum strain Nigg and C. trachomatis serovar D were grown in HeLa 229 cells in Eagle's essential medium supplemented with 10% fetal calf serum (FCS). Elementary bodies (EBs) were purified from HeLa 229 cells on discontinuous density gradients of Renografin-76 (Squib Canada) as described previously [8].

2.2. Mice

Female C57BL/6 (H2^b) and C3H/HeNCrl (C3H) (H2^k) mice (8 to 10 weeks old) were purchased from Charles River Canada (Saint Constant, Canada). The mice were maintained and used in strict accordance with University of British Columbia guidelines for animal care.

2.3. Generation of BMDCs

Bone marrow derived dendritic cells (BMDCs) were generated as previously described [9]. Briefly, bone marrow cells flushed from the femurs of female C57BL/6 or C3H mice were cultured in Falcon petri dishes at 4 × 10⁷ cells in 50ml DC medium. DC medium was IMDM supplemented with 10% FCS, 0.5 mM 2-ME, 4mM l-glutamine, 50μg/ml gentamicin, and 5% of culture supernatant of murine GM-CSF-transfected plasmacytoma X63-Ag8 and 5% of culture supernatant of murine IL-4 transfected plasmacytoma X63-Ag8 which contained 10ng/ml GM-CSF and 10ng/ml IL-4, respectively. On day 3, half of culture supernatants were removed and fresh DC medium was added. On day 5, nonadherent cells (purity of >50% CD11c⁺) were harvested and cultured in fresh DC medium for Chlamydia infection.

2.4. Purification of MHC class II-bound peptides

MHC class II-bound peptides were purified as described previously [2]. Briefly, 5 × 10⁹ immature BMDCs were infected at a 1:1 multiplicity of infection with C. muridarum or C. trachomatis serovar D for 12 or 24 h. BMDCs were then solubilized in lysis buffer (1% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 150mM NaCl, 20 mM Tris–HCl, pH 8, 0.04% sodium azide, protease inhibitors). MHC class II molecules were isolated using allele-specific anti-MHC monoclonal antibody affinity columns containing the monoclonal antibodies Y-3P (specific to I-A MHC class II allele of both C57BL/6 and C3H) and 14-4-4S (specific to I-E MHC class II allele of C3H). Purified MHC class II molecules were separated from the peptides using 0.2 N acetic acid and subjected to ultrafiltration through a 10-kDa cut-off membrane to remove high molecular weight material [10].

2.5. Identification of MHC class II-bound peptides

The MHC class II-bound peptides were further purified, concentrated, filtered and desalted using STop And Go Extraction tips [11]. Peptides were then analyzed by LC/MS/MS using an LTQ-Orbitrap Velos (Thermo Electron) on-line coupled to Agilent 1200 Series nanoflow HPLCs using nanospray ionization sources (Proxeon Biosystems, Odense, Denmark). Analytical columns were packed into 15 cm long, 50 μm inner diameter fused silica emitters using 3 μm diameter ReproSil Pur C₁₈ AQ beads (Dr. Maisch, www.Dr-Maisch.com), joint with 2-cm-long, 100-μm-inner diameter fused silica trap column packed with 5 μm-diameter Aqua C-18 beads (Phenomenex, www.phenomenex.com) and a 20-μm-inner diameter fused silica gold coated spray tip with 6-μm-diameter opening. LC buffer A consisted of 0.5% acetic acid and buffer B consisted of 0.5% acetic acid and 80% acetonitrile. Gradients were run from 10% B to 32% B over 51 min, then from 32% B to 40% B in the next 5 min, then increased to 100% B over 2 min period, held at 100% B for 2.5 min, and then dropped to 0% B for another 20 min to recondition the column. The Velos was set to acquire a full range scan at 60,000 resolution in the Orbitrap, from which the ten most intense multiply-charged ions per cycle were isolated for fragmentation in the LTQ. Centroided fragment peak lists were processed with Proteome Discoverer v. 1.2 (ThermoFisher Scientific). The search was performed with Mascot algorithm v. 2.4 against a database comprised of the protein sequences from the mouse and Chlamydia proteome. The estimated false discovery rate was below 2%.

2.6. Molecular cloning, expression and purification of C. trachomatis recombinant proteins

The recombinant proteins CT143, CT144, CT375, CT424, RplF (L6), CT619, PmpG, PmpF, PmpE, PmpH, major outer membrane protein (MOMP) and GroEL1 (Hsp60) were cloned, expressed and purified as follows: CT143, CT144, CT375, CT424, rplF, CT619, pmpG, pmpF, pmpE, pmpH, MOMP and groEL1 DNA fragments were generated by PCR using genomic DNA isolated from C. trachomatis. PCR reactions were carried out using Herculase Enhanced DNA polymerase (Agilent Technologies). The PCR product was purified with the QIAquick PCR purification kit (Qiagen) and the purified DNA fragments were cloned into pET32a expression vector (Novagen) after restriction enzyme digestion with BamHI/NotI using standard molecular biology techniques. For pmpE, pmpF, pmpG and pmpH, only the first half of the gene (representing amino acids 18–520, 26–585, 25–512, and 24–520, respectively) were cloned into the vector for expression because of MHC binding peptides were all found within this domain. The accuracy of the sub cloned genes were confirmed by sequencing. Plasmids containing the CT143, CT144, CT375, CT424, rplF, CT619, pmpG, pmpF, pmpE, pmpH, MOMP and groEL1 genes were transformed into the E. coli strain BL21(DE3) (Strategene) where protein expression was carried out by inducing the lac promoter for expression of T7 RNA polymerase using isopropyl-β-d-thiogalactoside pyranoside. The expressed CT143, CT144, CT375, CT424, RplF, CT619, PmpG, PmpF, PmpE, PmpH, MOMP and GroEL1 proteins with N-terminal His-tag were purified by nickel column using the His bind purification system (Qiagen). LPS removal of these proteins was carried out by adding 0.1% Triton-114 in one of the wash buffers during purification.

2.7. Transcervical infection with C. trachomatis serovar D in mice and determination of Chlamydia titer

Mice were infected with C. trachomatis serovar D transcervically after two s.c. injections with 2.5 mg of medroxyprogesterone acetate (Depo-Provera; Pharmacia and Upjohn) at day 3 and day 10 prior to infection. Transcervical inoculation of C. trachomatis was performed using NSET (Non-Surgical Embryo Transfer Device for Mice, ParaTechs Product no. 60010) as described by Gondek et al. [12] Briefly, a speculum was inserted into the mouse vaginal tract and the NSET tip directly inserted into the upper genital tract. The C. trachomatis inoculum (2 × 10⁷ IFU in 10 μl) was pipetted into the upper genital tract and the NSET device and speculum were removed. At day 6 post infection, vaginal swabs and whole genital tracts were collected and stored at −80°C for titration. Chlamydia titers in homogenates of genital tract tissue and in vaginal swabs were measured by inclusion counts on Hela cells as described previously [13].

2.8. ELISPOT assay

The IFN-γ ELISPOT assay was performed as described previously [14]. Briefly, 96-well MultiScreen-HA filtration plates (Millipore) were coated overnight at 4 °C with 2 μg/ml of murine IFN-γ specific monoclonal antibody (BD PharMingen, Clone R4-6A2). Mice were transcervically infected into the uterine cavity with 2 × 10⁷ IFU live C. trachomatis serovar D. Fourteen or 21 days after infection, the splenocytes were harvested and stimulated in vitro with 1 μg/ml individual Chlamydia proteins or 5 × 10⁵ IFU/ml heat killed-EB as a positive control. After 20 h incubation at 37×C and 5% CO2, the plates were washed and then incubated with biotinylated murine IFN-γ specific monoclonal antibodies (BD PharMingen, Clone XMG1.2) at 2 μg/ml. This was followed by incubation with streptavidin-alkaline phosphatase (BD PharMingen) at a 1:1000 dilution. The spots were visualized with a substrate consisting of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Sigma-Aldrich).

2.9. Statistical analysis

The data were analyzed with the aid of GraphPad Prism software program. Data are presented as means ± standard errors of the means (SEM) (ELISPOTs assay). Wallis test was performed to analyze data on IFU (Chlamydia titer) from multiple groups and Mann-Whitney U test to compare median between pairs. p Values of <0.05 were considered significant.

3. Results

3.1. Identification of C. muridarum derived MHC class II-bound peptides from C3H mice

Proteins which contain epitopes that can be presented by multiple MHC class II alleles are ideal vaccine candidates. Such proteins may present sequence specific or “promiscuous” epitopes and appear to have properties favorable to entering the antigen processing and presentation system [15,16]. Using the immunoproteomic approach we previously identified 17 Chlamydia T cell antigens when C57BL/6 BMDCs were infected with the murine strain C. muridarum [2,5]. Thirteen of these antigens were tested in a murine genital tract model and eleven elicited protective immunity against C. muridarum genital tract infection [17]. We now use the immunoproteomic approach to identify C. muridarum derived MHC class II-bound peptides presented on BMDCs from C3H mice (H2 I-A^k and I-E^k MHC alleles). A total of 14 peptides derived from 11 C. muridarum source proteins were identified (Table 1). Two proteins, PmpG and PmpF, overlapped with the source protein antigens identified using BMDC derived from C57BL/6 mice. The peptide epitopes presented by the Pmps were different in sequence between C57BL/6 and C3H mice.

Table 1.

MHC class II (I-A^k or I-E^k)-bound C. muridarum-derived peptides and their source proteins identified when C3H bone marrow derived dendritic cells were infected with live C. muridarum.

Peptide	Presenting MHC class II allele	Chlamydia muridarum locus#	Source protein	Protein abbreviation
DIVLSDMSISRQHAK	I-Ek	TC0035	Hypothetical protein	TC0035
LPVGNPAEPSLM	I-Ak	TC0052	Major outer membrane protein	MOMP
LPVGNPAEPSLMIDG
KKFISYAL	I-Ek	TC0243	ABC transporter, permease protein	TC0243
NISSDLQAHMIASKTHNQ NISSDLQAHMIASKTHNQIK ISSDLQAHMIASKTHNQI	I-Ek	TC0262	Polymorphic membrane protein F	PmpF
KDEGVVFFSKNIAAGKG	I-Ek	TC0263	Polymorphic membrane protein G	PmpG
EDFYKQMRAFRR	I-Ek	TC0294	Signal recognition particle protein	Ffh
IAAAGTGLL	I-Ak	TC0392	Hypothetical protein	TC0392
ITGIVPISPGVRA	I-Ak	TC0408	Hypothetical protein	TC0408
KLGKVLGPRNL	I-Ek	TC0592	50S Ribosomal protein L1	RplA
VTNKVTLAMQGQKLEG	I-Ek	TC0741	Translocated actin recruiting phosphoprotein33	Tarp
GSFAPVGLN	I-Ak	TC0910	Hypothetical protein	TC0910

3.2. Identification of C. trachomatis derived MHC class II-bound peptides from C57BL/6 mice

In order to formulate a vaccine against human C. trachomatis infection, we need to identify its T cell antigens and therefore extended the immunoproteomic approach to C. trachomatis. Sixty peptides derived from 27 different C. trachomatis proteins were identified when BMDCs derived from C57BL/6 mice were infected with C. trachomatis serovar D for 12 h (Table 2). Similar to the peptide repertoire identified in C. muridarum, the chromosomal location of genes encoding source proteins of the C. trachomatis derived epitopes were spread uniformly throughout the C. trachomatis genome. Interestingly, none of the source proteins for T cell antigens were encoded by genes located in the plasticity zone, the highly variable chromosomal region between C. muridarum and C. trachomatis genomes that encodes several of the species and biovar specific virulence factors [18].

Table 2.

MHC class II (I-Ab)-bound C. trachomatis serovar D-derived peptides and their source proteins identified when C57BL/6 bone marrow derived dendritic cells were infected with live C. trachomatis for 12 h.

Peptide	Chlamydia trachomatis locus#	Source proteins	Protein abbrev.
YKLVYQNALSNFSGKK	CT045	Leucyl aminopeptidase	PepA
GPKGRHVVIDKSFGSPQVTKDGVT	CT110	Chaperonin GroEL1 41	Hsp60
EERVVGQPFAIAAVSDS	CT113	Clp Protease ATPase	ClpB
DLKVTGPTIHTDLD	CT143	Hypothetical protein 33	CT143
GKLIVTNPKSDISFGG	CT144	Hypothetical protein 60	CT144
GSPGQTNYAAAKAGIIGFS	CT237	3-Ketoacyl-(acyl-carrier-protein) reductase	FabG
GTKTPIGTPIAVFSTEQ	CT247	Dihydrolipoamide acetyltransferase 51	PdhC
SPKEAAIAAARASLSPEEKR	CT289	Hypothetical protein	CT289
YDHIIVTPGANADILPE	CT375	Predicted d-amino acid dehydrogenase	CT375
FDGEKASVGAPTVGNAVVKG	CT420	50S ribosomal protein L21 33	Rl21
KLDGVSSPAVQESISESL	CT424	Sigma regulatory factor	RsbV
TPSAVNPLPNPEIDS	CT472	Hypothetical protein	CT472
DSTHGSFAPQATFSDG	CT505	Glyceraldehyde-3-phosphate dehydrogenase	GapA
VKGNEVFVTPAAHVVDRPG	CT514	50S ribosomal protein L6	RplF
ETPGAAEGAEAQTASEQPSKENAEKQEENNED	CT559	Yop proteins translocation lipoprotein	CdsJ
ADVLLLSPKASVSPGG	CT561	Type III secretion translocase 46	CdsL
IPFAKPDANLSAED	CT619	Hypothetical protein	CT619
KAPQFGYPAVQNSADS	CT622	CHLPN 76 kDa homolog	CT622
KEGEEDTAESAANEEPKAEASQEEE	CT664	FHA domain; homology to adenylate cyclase	CT664
IFDTTTLNPTIAGAGDVK	CT681	Major outer membrane protein 45	MOMP
TPVESTTPVAPEISVVNAK	CT759	Muramidase (invasin repeat family)	NlpD
QVFQLITQVTGRSG	CT778	Primosome assembly protein	PriA
ISYDYSSGNAEASSHN	CT837	Hypothetical protein	CT837
DAGVPIKAPVAGIAMG	CT842	Polyribonucleotide Nucleotidyltransferase	Pnp
GSVVFSGATVNSADFH	CT869	Polymorphic membrane protein E	PmpE
AMANEAPIAFIANVAG	CT871	Polymorphic membrane protein G	PmpG
AEKGGGAIYAPTIDISTNGGS	CT872	Polymorphic membrane protein H	PmpH

There are 924 and 894 proteins encoded by the genomes of C. muridarum and C trachomatis, respectively, and in principle all of these could be processed and presented by antigen presenting cells. Strikingly only 3% (27 of 894) of the C. trachomatis proteome is actually detected via MHC class II molecules following infection of DCs by our method, a percentage similar to what we previously observed with C. muridarum [2]. The reasons for the limited presentation of Chlamydia peptides by antigen presenting cells are unknown and may be confounded by the sequestration of antigens within the Chlamydia inclusion of infected DCs [19].

Nine source proteins identified from C57BL/6 MHC class II–bound peptides overlap between the C. muridarum and C. trachomatis proteome: Comparison of source proteins among the MHC class II-bound Chlamydia peptides revealed 9 overlapping source proteins from the C. muridarum and C. trachomatis immunoproteome in C57BL/6 mice (Table 3 and Fig. 1). For T cell proteins (defined as containing MHC binding peptides) which overlapped between the C. trachomatis and C. muridarum proteome we expected that similar or identical epitopes would be derived from shared proteins which were highly conserved in sequence while different epitopes would be presented from proteins less conserved in sequence. Five of the six highly conserved proteins (>75% sequence identity) presented the same or very similar epitopes (identical: FabG, RsbV, CT375; similar: PdhC, RplF) although one source protein (GapA) from the highly sequence conserved group presented a completely different epitope despite its 95% amino acid sequence identity. Two of the three less conserved (<71% sequence identity) proteins (PmpE and PmpG) presented distinct epitopes located at different sequence positions within the surface exposed passenger domain of these outer membrane proteins (Fig. 2).

Table 3.

Nine overlapping source proteins between C. trachomatis and C. muridarum. Identical amino acid residues of the epitopes between the two strains are underlined and in bold. Percentage protein identity is for the whole length proteins of each comparison.

C. trachomatis Locus#/protein abbreviation	C. trachomatis-derived peptides	C. muridarum locus#	C. muridarum-derived peptides	C. trachomatis/C. muridarum protein identity
CT143	DLKVTGPTIHTDLD	TC0420	DLNVTGPKIQTDVD	75%
CT237 (FabG)	GSPGQTNYAAAKAGIIGFS	TC0508	SPGQTNYAAAKAGIIGFS	90%
CT247(PdhC)	GTKTPIGTPIAVFSTEQ	TC0518	EGTKIPIGTPIAVFSTEQN	87%
CT424 (RsbV)	KLDGVSSPAVQESISESL	TC0707	KLDGVSSPAVQESISE	96%
CT375	YDHIIVTPGANADILPE	TC0654	YDHIIVTPGANADIL	85%
CT505 (GapA)	DSTHGSFAPQATFSDG	TC0792	MTTVHAATATQSVVD	95%
CT514 (RplF)	VKGNEVFVTPAAHVVDRPG	TC0801	VKGNEVFVSPAAHIIDRPG	96%
CT869 (PmpE)	GSVVFSGATVNSADFH	TC0261	SRALYAQPMLAISEA	69%
CT871 (PmpG)	AMANEAPIAFIANVAG	TC0263	NAKTVFLSNVASPIYVDPA ASPIYVDPAAAGGQPPA	71%

Fig. 1.

Venn diagram depicting the overlap of T cell antigens identified when BMDCs generated from mice of two different MHC background are infected with the human strain C. trachomatis and the mouse strain C. muridarum.

Fig. 2.

Location of MHC class II-bound peptides derived from C. muridarum (filled star) and C. trachomatis (open star) within the predicted passenger domains of the Pmp proteins identified using BMDCs from C57 (black star) or C3H (red star). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Determination of antigenicity of the Chlamydia proteins based on IFN-γ responses in C57BL/6 mice following C. trachomatis transcervical infection: Twelve proteins containing MHC class II-bound peptides identified by the immunoproteomic approach were evaluated for antigenicity in the C. trachomatis transcervical infection model. These included six overlapping source proteins between C. muridarum and C. trachomatis (CT143, CT375, CT424, RplF, PmpG, PmpE), two novel hypothetical source proteins (CT144, CT619), and PmpF, PmpH, MOMP and GroEL1 only identified in C. trachomatis (Table 2). To determine antigenicity in the context of in vivo infection, we performed IFN-γ ELISPOT assay using splenocytes from C57BL/6 mice following transcervical C. trachomatis infection since immunity in this model is due to IFN-γ secreting CD4 T cells [12]. Two and three weeks after transcervical inoculation of C trachomatis, mice were sacrificed and splenocytes were harvested and stimulated in vitro with the indicated recombinant protein. Media was used as a negative control and heat killed C. trachomatis serovar D elementary bodies (HK-EB) as a positive control. As shown in Fig. 3A, immune cells exposed to HK-EB developed the largest numbers of IFN-γ secreting cells where more than 1000 IFN-γ secreting cells were detected per 10⁶ spleen cells. Cells stimulated with media as negative control showed nearly blank background levels indicating that IFN-γ secreting cells detected in the experimental system are Chlamydia antigen-specific. Relative to the negative control immune cells stimulated with individual Chlamydia protein exhibited positive IFN-γ responses (Fig. 3A). The IFN-γ responses in immune cells following stimulation with CT144, RplF (L6), CT169, PmpF, PmpH, MOMP and GroEL1 protein were strong (>50 spot forming cells [SFC] per 10⁶ splenocytes); other antigens (PmpG, PmpE, CT143, CT375 and CT424) stimulated weak IFN-γ responses (<50 SFCs per 10⁶ splenocytes). We conclude that outer membrane proteins such as MOMP, PmpF and PmpH are among the dominant C. trachomatis T cell antigens detected in this model system.

Fig. 3.

(A). T cell antigenicity of C. trachomatis (Ct) membrane protein antigens (left panel) and other identified T cell antigens (right panel) in immune C57BL/6 mice identified by IFN-γ ELISPOT assay. Mice were transcervically infected with 2 × 10⁷ IFU live C. trachomatis serovar D. Fourteen or 21 days after infection, the splenocytes were harvested and stimulated in vitro for 20 h with 1 μg/ml individual Chlamydia proteins or 5 × 10⁵ IFU/ml heat killed-EB as a positive control. The results represent the average of duplicate wells and are expressed as the means ± SEM of Chlamydia Ag-induced IFN-γ -secreting cells per 10⁶ splenocytes for groups of five mice. (B). T cell antigenicity of C. muridarum (Cm) Pmps and MOMP in immune C57BL/6 (left panel) and C3H (right panel) mice identified by IFN-γ ELISPOT assay. Mice were intravaginally infected with 1500 IFU of C. muridarum. Three weeks later, splenocytes were harvested and stimulated in vitro for 20 h with 1 μg/ml individual Chlamydia proteins or heat killed-EB as a positive control.

Comparative antigenicity of the Chlamydia outer membrane proteins based on IFN-γ responses in C57BL/6 and C3H mice following C. muridarum intravaginal infection: After identifying source proteins from Chlamydia that generate MHC class II binding epitopes in two different genetic background, we determined antigenicity in the context of in vivo infection via IFN-γ ELISPOT assay using splenocytes from C57Bl/6 and C3H mice following intravaginal C. muridarum infection. As shown in Fig. 3B, immune cells exposed to HK-EB developed the largest number of IFN-γ secreting cells where more than 1000 IFN-γ secreting cells were detected per 10⁶ spleen cells. The results demonstrate that IFN-γ responses in immune cells following stimulation with C. muridarum PmpG, PmpF and MOMP protein were immunodominant in both C57/BL6 and C3H.

3.3. Recombinant outer membrane protein vaccine accelerates clearance of C. trachomatis transcervical infection

We previously evaluated the protective efficacy of a C. muridarum outer membrane protein vaccine composed of Pmps and MOMP using the C. muridarum genital tract infection model in three different strains of mice and the multisubunit vaccine elicited protection as measured by accelerated clearance [20]. C. trachomatis does not infect the lower murine genital tract as efficiently as does C. muridarum and its clearance is mainly the result of innate immune response [21]. We therefore utilized the transcervical infection model established by Gondek et al. using C. trachomatis serovar D where CD4 T cells have been demonstrated to be essential to clearance [12]. We found that transcervical inoculation of C. trachomatis serovar D in C57BL/6 mice exhibited much higher Chlamydia loads detected by assaying homogenates of genital tract tissue in comparison to vaginal swabs (Fig. 4a). Transcervical infection by C. trachomatis serovar D was able to equally infect C57BL/6, Balb/c and C3H mice (Fig. 4b). Thus we used the transcervical infection mouse model to evaluate T cell vaccine efficiency against C. trachomatis serovar D challenge.

Fig. 4.

Transcervical infection of the upper genital tract with C. trachomatis serovar D. (a) Comparison of C. trachomatis serovar D (Ct.D) titer in homogenate of genital tract tissue with that of vaginal surface swab at day 6 post infection in C57BL/6 mice. Mice were infected with 2 × 10⁷ IFU of C. trachomatis serovar D transcervically after two treatments with Depo-Provera at day 3 and day 10. Vaginal swabs and whole genital tracts were collected at day 6 post infection. Chlamydia titers in homogenates of genital tracts and in vaginal swab were measured by inclusion counts on Hela 229 cells. (b) Transcervical infection with C. trachomatis serovar D in Balb/c, C57BL/6 and C3H mice. Mice were infected with 2 × 10⁷ IFU of C. trachomatis serovar D transcervically after two treatments with Depo-Provera at day 3 and day 10. Whole genital tracts were isolated and homogenized at day 6 post infection. Chlamydia titers in homogenates of genital tracts were measured by inclusion counts on Hela 229 cells.

C. trachomatis serovars that cause human diseases can be clinically divided into three pathovars depending on their site of infection; trachoma (serovars A, B, Ba and C), sexually transmitted genital tract infection (serovars D through K) and lymphogranuloma venereum (serovars L1, L2 and L3). Pmps are an important component for chlamydial adhesion to host cells and sequences for six of the 9 Pmps correlate with the pathovar clustering [22]. MOMP is the serovar typing antigen, the major porin protein and the dominant antigen containing both B and T cell sites. Immunity directed to MOMP is determined by both serovar specific and serogroup specific antigenic determinants within the protein sequence [23]. We used four Pmps from C. trachomatis serovar D and MOMP from serovars D, J and F to represent serogroups B-complex, C-complex and GF-complex, respectively in a recombinant vaccine. We tested the efficacy of the multisubunit vaccine with the Th1 polarizing adjuvant DDA/MPL [24]. We tested both the Pmp/MOMP combination vaccine and the four Pmps and the three MOMPs as individual antigen components. Four weeks after the final immunization, C57BL/6 mice were challenged transcervically with C. trachomatis serovar D. Genital tracts were isolated and homogenized at day 6 post infection and C. trachomatis shedding was measured by inclusion counts on Hela 229 cells. Mice immunized with PBS were used as a negative control, and mice previously transcervically infected with C. trachomatis serovar D were used as a positive control. Strikingly mice previously infected with C. trachomatis serovar D were resistant to challenge infection, confirming that this model could be used to evaluate C. trachomatis vaccine candidates. All three vaccine groups demonstrated a significant reduction in C. trachomatis shedding compared to the PBS group (p< 0.001) (Fig. 5).

Fig. 5.

Vaccine elicited protection against C. trachomatis serovar D infection in C57BL/6 mice after immunization with different vaccine formulations with DDA/MPL adjuvant. Four weeks after the final immunization, mice were transcervically challenged with 2 × 10⁷ IFU of C. trachomatis serovar D. Genital tracts were isolated and homogenized at day 6 post infection. Chlamydia titers in homogenates were measured by inclusion counts on Hela 229 cells. Mice immunized with PBS were used as a negative control, and mice previously infected with 2 × 10⁷ IFU of C. trachomatis serovar D transcervically were used as a positive control. Horizontal bar represents median value for each group. All vaccine groups significantly reduced C. trachomatis shedding compared to the PBS group; p value <0.001.

4. Discussion

Identifying T cell antigens is of major importance to vaccinology for intracellular pathogens. The primary sequence of a protein is one determinant of T cell immunogenicity since the amino acid sequence influences antigen processing (protease susceptibility of the residues flanking the epitope) and determines anchoring of the peptide into MHC binding pockets. MHC class I and II differ in the importance of anchoring residues. In class I anchoring residues are critical to high affinity binding but for class II are less so [15]. Rather DM editing of the peptide complex is vital to high affinity class II binding [25]. Our data demonstrate that MHC allelic selection is clearly important for Chlamydia CD4 T cell antigens as seen when the C. muridarum immunoproteome is compared between C57BL/6 and C3H mice. Although 17 proteins yielded MHC class II binding peptides in C57BL/6 and 11 proteins generated binding peptides in C3H, only two C. muridarum protein (PmpG and PmpF) generated MHC class II binding epitopes of differing sequence in both strains of mice (Fig. 1). Furthermore 6 of the 9 antigenic orthologous proteins shared between the C. muridarum and C. trachomatis immunoproteome presented nearly identical peptide epitopes on the I-A^b allele of C57BL/6 mice (Table 3).

T cell immunogenicity also appears to be determined by properties intrinsic to the pathogen proteins such as cellular location, abundance, and kinetics of expression. Our results demonstrate that surface proteins are enriched among the Chlamydia T cell immunoproteome (Fig. 1). Of particular interest, MOMP and the polymorphic membrane family of proteins (PmpE, PmpF and PmpG) generated MHC class II binding epitopes at multiple sites within the sequence (Fig. 2) suggesting that these proteins are capable of presenting to the host immune system via multiple MHC binding epitopes. Pmps belong to a group of proteins known as type V autotransporters comprising an N-terminal signal sequence, a passenger domain and a translocation unit (Fig. 2) [26]. C. trachomatis and C. muridarum genomes encode nine different Pmps (PmpA to PmpI) [18,27]. A recent study reported by Becker and Hagemann showed that all nine C. trachomatis Pmps mediate adhesion to human epithelial cells [28].

Identification of four Pmps and MOMP as T cell antigens via an immunoproteomic analysis suggests that outer membrane proteins may have advantages over other groups of proteins in presenting to the immune system. This is supported by a recent quantitative proteomic analysis of C. trachomatis elementary bodies which revealed that the three Pmps we identified (PmpE, PmpG and PmpH) constituted 61% of the total Pmp protein abundance [29] and MOMP is already known to constitute over 60% of the total outer membrane protein abundance [8]. Thus high abundance proteins as well as outer membrane localization may play a role in favoring presentation.

PmpG is the most protective C. muridarum T cell antigen we identified via our immunoproteomic approach and two recent studies further validated the immunodominance of PmpG in the murine model [30,31]. These studies demonstrated that the PmpG epitope can be detected on splenic antigen presenting cells for at least 6 months after clearance of primary genital tract infection and a large fraction of the T cells activated during Chlamydia infection are PmpG-specific suggesting that PmpG in particular is intrinsically immunogenic.

In conclusion our results demonstrate both MHC class II allelic selection and intrinsic antigenic protein features contribute to T cell antigenicity of Chlamydia outer membrane proteins. MHC allele specificity appears to be more important than intrinsic features of antigenic proteins in Chlamydia perhaps because so few proteins enter the class II processing and presentation pathway from the Chlamydia vacuole. The shared T cell antigens we identified, in particular the Pmps and MOMP are likely relevant to Chlamydia immunobiology both in murine and human models and constitute potential C. trachomatis vaccine candidates.