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. Author manuscript; available in PMC: 2014 Nov 19.
Published in final edited form as: Vaccine. 2013 Oct 1;31(48):10.1016/j.vaccine.2013.09.036. doi: 10.1016/j.vaccine.2013.09.036

A T Cell Epitope-Based Vaccine Protects Against Chlamydial Infection in HLA-DR4 Transgenic Mice

Weidang Li a, Ashlesh K Murthy b, Gopala Krishna Lanka a, Senthilnath L Chetty a, Jieh-Juen Yu a, James P Chambers a, Guangming Zhong c, Thomas G Forsthuber a, M Neal Guentzel a, Bernard P Arulanandam a,*
PMCID: PMC3843363  NIHMSID: NIHMS527454  PMID: 24096029

Abstract

Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4+ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4+ T cells in host defense against Chlamydia. In this study, we identify CD4+ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4+ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4+ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4+ clones could be detected in the mouse spleen following C. muridarum and a human C. trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4+ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4+ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model.

1. Introduction

Chlamydia trachomatis causes the most common bacterial sexually transmitted disease worldwide [1, 2]. Despite the many efforts that have been made over several decades to develop a protective C. trachomatis vaccine, there currently is no efficacious vaccine available. In the early vaccine trials, use of whole chlamydial agents as Chlamydia vaccines proved to be of limited value since protection was short lived and some vaccines produced exaggerated ocular pathology over the long term. [3, 4]. This prompted the search for the formulation of a subunit vaccine, which would elicit a specific immune response to unique protein(s) and provide protection without stimulating pathological immunity. Several potential vaccine candidates (MOMP, CPAF, PMP, PorB) eliciting reactive CD4+ T cells and/or antibody responses have been evaluated in a mouse model [5–9] and all showed partial protection against genital Chlamydia infection. Thus, the identification of specific-protective epitopes that could be used either as an alternative equivalent to those already characterized proteins, or in combination with multi-epitopes from multi-antigens, becomes a sound strategy for developing an effective Chlamydia vaccine.

Chlamydia muridarum, originally isolated from the lungs of mice but sharing a very high similarity in gene order and content with the human pathogen C. trachomatis serovar D (average only ~10% difference in orthologous genes [10]), was used to establish the mouse genital chlamydial infection model [11] which mimics many aspects of acute genital infection with C. trachomatis in women [12]. Using this murine model, we have demonstrated that the recombinant chlamydial protease-like activity factor (rCPAF) is an effective Chlamydia vaccine antigen. Specifically, we showed that vaccination with rCPAF derived from the human infectious C. trachomatis L2 serovar (approximately 82% identity and 90% similarity to C. muridarum ortholog), provided protection (acceleration of bacterial clearance, reduction of upper genital tract pathology, and preservation of fertility) against intravaginal (i.vag.) C. muridarum challenge in conventional C57BL/6 mice and the HLA-DR4 transgenic mice (HLA-DR4tg), suggesting that CPAF may also induce a protective immune response in humans [7, 13–19]. We further demonstrated that rCPAF mediated protection was dependent upon CD4+ T cells and required IFN-γ [6, 20].

Here, we report the mapping of CPAF T cell eptipotes and the application of the identified epitopes in the development of a novel fusion-peptide vaccine and in the detection of CPAF-specific T cell clones, following chlamydial infection or CPAF vaccination, using MHC class II tetramers. By identifying CPAF epitopes and tracking CD4+ T cells during Chlamydia infection, we may be able to provide new insights into protective mechanisms and enhance the development of a potential effective T cell epitope vaccine against Chlamydia associated diseases.

2. Materials and methods

2.1. Bacteria and cells

Chlamydia muridarum Nigg was grown on confluent HeLa cell monolayers. Elementary bodies (EBs) were purified by discontinuous density gradient centrifugation as described previously [15]. EBV-transfected HLA-DR4 (DRB1*0401) homozygous B cells were used for HLA-DR4 molecular purification [21].

2.2. Mice, immunizations, and infections

HLA-DRA-IEα/HLA-DRB1*0401-IEβ transgenic (HLA-DR4tg) mice [13, 22] were bred at the University of Texas at San Antonio. Animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines. Female transgenic mice (6–10 week-old) were immunized with protein antigens, including C. trachomatis L2 rCPAF [13], by subcutaneous (s.c.) or intranasal (i.n.) routes as described previously [14]. Intranasal infection with live C. muridarum EBs induces nearly-complete protective immunity against subsequent genital or respiratory challenge, and therefore was used as a positive control for vaccine efficacy in these experiments. All mice are anesthetized for i.n. administration. Chlamydia muridarum pulmonary and vaginal infection was achieved by the i.n. [23] or intravaginal (i.vag.) [7] challenge route with 5×102 and 5×104 IFU of bacteria, respectively, in 5 µl of SPG buffer. Chlamydia trachomatis infection was achieved by primary intravaginal challenge using C. trachomatis serovar D (1X106 IFU in 5 µl of SPG buffer) on day 0, followed by a secondary intravaginal CT-D challenge at 3 weeks following the primary challenge, and a tertiary challenge at 2 weeks following the secondary challenge. In order to render the mice anestrous and more receptive to the genital infection, animals were injected s.c. with 2.5 mg of Depoprovera (medroxy-progesterone acetate; Pharmacia) on day 5 before challenge. Vaginal chlamydial shedding and pulmonary Chlamydia burdens were measured as described in our previous reports [13, 23].

2.3. CPAF mapping

Overlapping CPAF peptides (20 mer) were synthesized by New England Peptide for determinant mapping according to the C. trachomatis L2 CPAF sequence. The IFN-γ ELISPOT assay was performed as described previously[24]. Briefly, 96-well MultiScreen-HTS filtration plates (Millipore) were coated overnight at 4 °C with 2 µg/ml murine IFN-γ specific mAb (eBioscience, clone AN-18). Splenocytes isolated from 3 rCPAF vaccinated, or 4 C. muridarum infected mice, in HL-1 medium (Fisher) were added separately from individual animals to the coated plates at 5×105 cells per well in the presence of individual peptides at 10 µg/ml or Chlamydia at 5×105 IFU per well. After 20 h of incubation at 37 °C with 5% CO2, the plates were washed and then incubated with biotinylated IFN-γ specific mAb (eBioscience, clone R4-6A2) at 0.5 µg/ml. This was followed by incubation with streptavidin-alkaline phosphatase (Dako) at a 1/1000 dilution. The spots were visualized with BCIP/NBT substrate (KPL) and enumerated with an ImmunoSpot Series 3 Analyzer (Cellular Technology).

2.4. rCPAFep cloning and purification

To express a novel CPAF epitopes protein (rCPAFep), we constructed an expression vector which contained 2 copies of 5 CPAF epitope genes (Cep1-5) in tandem as illustrated in Fig. 2A. Briefly, 997 nucleotides were synthesized (GenScript) to contain sequences encoding each of the 5 selected CPAF epitopes flanked by an N-terminal li-Key (LRMKLPKS) for enhancing epitope presentation and a C-terminal spacer (GPGPG) to link each epitope, as well as a His (6×) tag at the C-terminus of the rCPAFep for easy protein purification [25]. The synthetic CPAFep gene was cloned to a pET23a vector and used to transform an E. coli Rosetta strain (Novagen) for protein expression. rCPAFep expression was induced in the presence of 1 µM IPTG, with purification under denaturing conditions using an Ni-NTA Agarose column (Qiagen) according to the manufacturer’s guidelines.

Figure 2.

Figure 2

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Vaccination of HLADR4tg mice with rCPAFep induced T cell immune responses. (A) Illustration of the rCPAFep construct, which consists of 2 tandem repeats of 5 CPAF epitopes (Cep1-5; not all the duplicated peptides sequences are shown), their flanking Li-key and spacer, and a His (6×) tag. (B) rCPAFep (arrows) was over-expressed upon IPTG induction in the transformed E. coli host (left panel, commassie blue stained SDS-PAGE gel), purified to high homogeneity (middle, commassie blue stained SDS-PAGE gel), and reacted with anti-His antibody (right, Western blot). (C) Pooled purified CD4+ T cells from rCPAFep + CpG i.n. vaccinated, or CpG mock treated HLA-DR4tg, mouse (n=3 per group, day 14) were incubated with irradiated naïve monocytes (as APCs) and stimulated by rCPAF, rCPAFep, or HEL (an unrelated control antigen) for 72 h. IFN-γ in culture supernatants was measured by ELISA and presented as mean ± SD. * Significant (p < 0.05; T test) difference between the indicated groups.

2.5. Antigen-specific CD4+ T cell responses

HLA-DR4tg mouse spleens (n=3) were removed at day 14 post i.n. vaccination with rCPAFep plus 10 µg CpG containing oligodeoxynucleotide (CpG) or CpG alone (mock), and single splenocyte cell suspensions were prepared. CD4+ T cells were enriched using a CD4 Enrichment Kit (STEMCELL) and the purity was determined to be at least 90% by flow cytometry. A pool of naïve monocytes treated with radiation (3000 rads) was used as a source of APCs. The purified CD4+ T cells (5×105 cells/well) were cultured with APCs (1×105 cells/well) and stimulated for 72 h in vitro with rCPAF (2 µg/ml), rCPAFep (2 µg/ml), or the unrelated antigen hen egg lysozyme (HEL, 2 µg/ml), in 96 well culture plates. Supernatants from the triplicate culture wells were analyzed for IFN-γ production using a BD OptELISA kit (BD Pharmingen).

2.6. HLA-DR4 tetramer staining and flow cytometry analysis

HLA-DR4 (DRB1-0401) tetramers containing PE fluorochrome labeled CPAF peptides (Cep 2 and Cep3; sequences described in supplementary Table 1) or control peptide (CLIP) were generated by the NIH Tetramer Core Facility. Cep2 tetramer and Cep3 tetramer were synthesized individually and were mixed together only to better stimulate/bind CD4+ T cells. Spleens were removed from HLA-DR4tg mice on day 21 post subcutaneous rCPAFep (100 µg) plus CFA (1 mg/ml emulsion) vaccination, or on day 12 after Chlamydia challenge (i.vag, 5×104 IFU). Single splenocyte cell suspensions were prepared. CD4+ T-cells were enriched and stimulated with rCPAFep protein or peptides in the presence of irradiated autologous APCs (antigen presenting cells) in complete RPMI 1640 medium containing rhIL-2 (5 ng/ml) and rhIL-7 (1 ng/ml) for 8 days for T cell expansion before tetramer staining. Antigen stimulated CD4+ T cells were incubated in complete RPMI 1640 medium for 3 h with tetramers (4 µg/ ml) at 37 °C, unless otherwise indicated, and subjected to flow cytometry analysis (ISRII, BD Biosciences) to identify the CPAF-specific CD4+ T cell population with FITC-conjugated anti-CD4 Ab (Bio-legend).

2.7. Statistical analyses

Sigma Stat (Systat Software Inc.) was used to perform all tests of significance. Student's t test was used for comparisons between two groups and analysis of variance (ANOVA) between multiple groups for vaginal chlamydial shedding. Differences between groups were considered statistically significant if p values were <0.05. All data shown are representative of 2–3 independent experiments and each experiment shown was analyzed independently.

3. Results

3.1. Identification of putative MHC class II-restricted CPAF epitopes using HLA-DR4tg mice

Chlamydia CPAF protein was expressed in mice during chlamydial genital tract infection, as seen in the columnar cells of the endocervix and endometrium (Supplementary Fig.1), and has been shown to stimulate robust host immune responses [13, 26]. To identify CPAF CD4+ T cell epitopes restricted by human HLA-DR4 molecules, HLA-DR4tg mice were immunized s.c. with the full length rCPAF protein derived from C. trachomatis plus CFA adjuvant. Twenty-one days after the injection, splenocytes were used for in vitro sensitization with synthetic overlapping peptides derived from CPAF. In order to select highly reactive T cell epitopes, we screened for IFN-γ production in ELISPOT assays. This selection was based on an initial screening of peptides that stimulated greater than 25 IFN-γ spots per 5 × 105 splenocytes (Fig. 1, upper panel). Among the 9 peptides identified, 8 were overlapping reactive fragments thereby representing only 4 non-overlapping peptides (Cep1-4) and Cep5 was a single strong reactive peptide without adjacent reactive overlapping peptides. Collectively, we selected a total of 5 peptides (aa sequences are listed in Supplementary Table 1) from initial screening for further evaluation of immunogenicity and protective immunity. Minimal IFN-γ secreting spots were detected upon stimulation with unrelated peptides, in contrast to high numbers of IFN-γ spots in response to stimulation with overlapping CPAF peptides, indicating that CPAF epitopes presented by HLA-DR4 molecule were specifically recognized by T cells. Splenocytes from mock (CFA) vaccinated HLA-DR4 mice produced a baseline of IFN-γ spots following stimulation by synthetic overlapping CPAF peptides (data not shown). Although C. muridarum Cep1-5 aa sequences are slightly different from the C. trachomatis derived rCPAF (Supplementary Table 1), these 5 CPAF peptides also can stimulate the T cells obtained from the C. muridarum infected mice to produce IFN-γ, suggesting that Cep1-5 are processed by APCs and presented to T cells during primary C. muridarum infection (Fig. 1, lower panel).

Figure 1.

Figure 1

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Mapping of HLA-DR4 (DRB1*0401)-restricted CPAF epitopes. Six- to 10-wk old HLA-DR4tg mice were immunized s.c. with rCPAF plus CFA (n=3) or challenged i.vag. with C. muridarum (n=4). Single cell suspensions prepared from individual spleens (21 days after treatment) were stimulated with CPAF overlapping peptides (20-mer), unrelated peptides or positive control stimuli (αCD3 and rCPAF) and subjected to ELISPOT assay to measure the frequencies of epitope-specific IFN-γ producing T cells. * indicates 5 selected CPAF T cell reactive epitope peptides for the construction of rCPAFep, a novel CPAF epitope fusion protein. # indicates 2 peptides chosen for the generation of HLA-DR4 tetramers.

3.2. Resolution of genital C. muridarum challenge in HLA-DR4 tg mice after vaccination with rCPAFep

We expressed a novel CPAF epitope fusion protein (rCPAFep) consisting of 2 copies of each Cep1-5 and a C-terminus His (6×)-tag as illustrated in Figure 2A. Then a 34 kDa protein was over-expressed in transformed E coli upon IPTG induction, purified to high homogeneity by Nickel affinity chromatography and shown to have positive reactivity with anti-His-tag antibody (Figure 2B).

We then assessed the immunogenicity of rCPAFep and its protective efficacy against genital chlamydial challenge. First, HLA-DR4tg mice were vaccinated i.n. with rCPAFep plus CpG, or CpG alone, and CD4+ T-cells were enriched from splenocytes at day 14 for cellular cytokine recall assay. As shown in Figure 2C, rCPAFep primed splenocytes secreted high levels of IFN-γ when stimulated with either the full length rCPAF (518±148 pg/ml) or the combined eptitope protein rCPAFep (1225.3 ± 394.2 pg/ml), in contrast to a negligible reaction with HEL suggesting that vaccination with rCPAFep may induce robust antigen-specific Th1 type mediated immune responses required for Chlamydia clearance. Next, we compared the protective efficacy mediated by rCPAF and rCPAFep vaccination. As shown in Figure 3, CpG (mock)-vaccinated mice displayed high levels of vaginal chlamydial shedding initially and progressively cleared the infection with complete clearance by day 30 after challenge. Consistent with our previous observations [15], live EB vaccinated mice started to clear the infection as early as day 3 with marked reduction of chlamydial shedding at day 6, and complete clearance in all mice by day 9. rCPAF+CpG vaccinated mice displayed significant reductions in chlamydial shedding, with clearance of the infection in 83% of the mice as early as day 18, and in all mice by day 21 after challenge. Similarly, 50% of rCPAFep+CpG vaccinated mice cleared the infection at day 18 with all mice free of vaginal bacterial shedding by day 24.

Figure 3.

Figure 3

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Vaccination of HLADR4tg mice with rCPAFep protects against genital chlamydial infection comparable to full-length CPAF immunization. Groups (6 per group) of HLA-DR4tg mice were immunized i.n. with rCPAF/CpG, rCPAFep/CpG, or CpG (mock), and boosted twice at a two week intervals. Another group (n = 6) of mice received one live EB i.n. vaccination. One month after the final immunization, mice were challenged i.vag. with 5 × 104 IFU of C. muridarum. Chlamydial shedding was monitored every third day after challenge for 1 month, and presented as mean ±SD for each group at each time point (A). * Significant reductions (P < 0.05; one-way ANOVA) in bacterial shedding between the indicated group and CpG-immunized (mock) mice are indicated. (B). Percentage of mice shedding Chlamydia after genital challenge for each vaccination group is summarized.

3.3. Acceleration of Chlamydia clearance in the lungs by rCPAFep vaccination

A second chlamydial infection model was used to evaluate the protective efficacy of rCPAFep against C. muridarum. We observed that Chlamydia replicated in the lungs during the first week of pulmonary infection for all vaccinated (s.c. rCPAF, s.c. rCPAFep, i.n. Live EB) groups and PBS mock (s.c.) treated mice (Fig. 4). While the bacterial numbers in mock treated mice continued to increase at day 10, a significant decrease of bacterial burdens was evident in both rCPAF and rCPAFep vaccinated mice and almost no viable Chlamydia could be detected in EB vaccinated mice at day 10. By day 14, all vaccinated mice cleared the infection while mock treated mice still sustained a high bacterial load in the lungs. The comparable protective efficacy of the peptide-fusion rCPAFep and full length rCPAF against pulmonary chlamydial infection was consistent with their comparable protection against genital infection.

Figure 4.

Figure 4

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Vaccination with rCPAFep protects against pulmonary chlamydial infection comparable to full-length CPAF immunization. Groups (15 per group) of mice were immunized s.c. with rCPAF, rCPAFep, or PBS (mock), and boosted twice at a two week intervals. Another group (n = 15) of mice received one live EB i.n. vaccination. One month after the final immunization, mice were challenged i.n. with 5 × 102 IFU of C. muridarum. Chlamydial burdens in the lungs were measured at the indicated times and presented as mean ±SD for each group at each time point. * Significant reductions (P < 0.05; one-way ANOVA) in bacterial shedding between the indicated group and mock-immunized (mock) mice are indicated.

3.4. Direct detection of specific CD4+ T cells after rCPAFep vaccination or C. muridarum infection

One of the applications of identified T cell epitopes is to generate MHC tetramers for identifying/tracking antigen-specific T cells. To serve this purpose, we chose Cep2 and 3 (Cep2/3) in this study for further characterization since they are part of the mature protein (Cep1 was within the predicted signal peptide), and stimulated T cell activation better than Cep4 and 5 (Fig. 1, lower panel) with chlamydial infection. Cep2 and Cep3 were shown to tightly bind in the peptide-binding groove of HLA-DR4 with a Kd of 0.30 µM and 0.85 µM, respectively (Supplementary Fig. 2). Cep2, Cep3/HLA-DR4 tetramers were generated to detect CPAF-specific CD4+ T cells. Since antigen-specific T cell frequency is very low, we combined Cep2 and 3 tetramers (both PE-labeled and mixed at a 1:1 ratio) in this study to maximize the detection capability. As shown in Figure 5A, direct staining of purified CD4+ T cells, following rCPAFep+CFA s.c. vaccination or C. muridarum vaginal infection, with Cep2/3 tetramers showed 3.6% of CD4+ T cells bound with Cep2 and Cep3 HLA-DR4 tetramers from a representative rCPAFep vaccinated mouse and 2.3% of CD4+ T cells from a C. muridarum challenged mouse. The frequency of CD4+ T cells binding in either the mock (CFA) vaccinated or mock (PBS) challenged mice was significantly lower (0.5% and 0.3%, respectively). The calculated frequency of Cep2/3 positive CD4+ T-cells from all analyzed mice (3–4 per group) are summarized in the Figure 5A bar graph, indicating the presence of 3.5± 0.1% and 1.9±0.6% CPAF-specific T cells in the rCPAFep vaccinated and C. muridarum infected mice, respectively. These results demonstrated that the novel Cep tetramers are useful reagents to identify and enumerate the CPAF-specific CD4+ T cells induced by the rCPAFep vaccine or by Chlamydia infection.

Figure 5.

Figure 5

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Detection of CPAF specific CD4+ T cell populations induced by rCPAFep vaccination or C. muridarum infection using tetramer staining. (A) Representative flow cytometry quadrant plots showing frequency of Cep2/3 HLA-DR4 tetramer stained CD4+ T cells in the right top quadrant for a mock (CFA adjuvant alone) and rCPAFep vaccinated mouse (upper panels), or a mock (PBS) and C. muridarum infected mouse (lower panels). The frequency (mean± SD) of Cep2/3-specific CD4+ cells was calculated from 3–4 individually tested mice of each indicated group (bar graph). (B) Representative flow cytometry quadrant plots showing frequency of Cep2/3 HLA-DR4 tetramer stained CD4+ T cells upon in vitro expansion by rCPAFep stimulation or peptide. The frequency (mean± SD) of Cep2/3-specific CD4+ cells in expanded cell cultures was calculated from 3–4 individually tested mice of each indicated group. * Significant (p < 0.05; T test) difference between the indicated groups.

To further confirm that the CD4+ T cells stained by the Cep2/3-HLA-DR4 tetramers represented CPAF epitope-specific CD4+ T cell populations, we expanded the CD4+ T cells in vitro by specific-epitope stimulation. As shown in Figure 5B, Cep2/3-HLA-DR4 tetramers bound to 25% and 17% of CD4+ T cells from a representative rCPAFep vaccinated and a C. muridarum challenged mouse, respectively. Overall, the frequency of detected tetramer positive CD4+ T cells (Fig 5B, 3–4 mice per group) after in vitro stimulation of total splenic CD4+ T cells with rCPAFep demonstrated a dramatic increase in mice previously vaccinated with rCPAFep or infected with C. muridarum, but increased minimally in CFA (adjuvant only) or PBS (mock) immunized mice. This suggests that despite a low frequency of Cep2/3 positive CD4+ T cells in vivo following vaccination or infection, they can be expanded significantly by in vitro peptide stimulation demonstrating the antigen-specificity of the response.

3.5. C. trachomatis intra-vaginal infection leads to CPAF CD4+ T cell activation and proliferation

Since we successfully demonstrated that the Cep tetramers can be used to detect CPAF specific CD4+ T cells, we examined whether chlamydial CPAF was expressed in the HLA-DR4tg mice, processed and presented by APCs, and stimulated CPAF specific CD4+ T cell activation and proliferation during genital infection with the human trachomatis serovar D. The splenocytes were isolated, stimulated and stained with Cep2/3 tetramers on day 12 after the third C. trachomatis challenge. As shown in Figure 6A, following direct staining of T cells from C. trachomatis infected mice with Cep2/3 tetramers, only 0.9% CD4+ T cells bound the Cep2 and Cep3-HLA-DR4 tetramers from one of the C. trachomatis infected mice, but this increased to 6.5% after expansion in vitro. Almost no CD4+ T cells bound to the unrelated control tetramers. Overall, significantly higher numbers of CPAF-specific CD4+ T cells were detected in C. trachomatis infected mice, compared to PBS mock challenged mice (Fig. 6B). Collectively, we have provided evidence that CPAF specific CD4+ T cells were activated and may be expanded during human Chlamydia strain infection in the HLA-DR4tg mice, which strongly supports the feasibility of developing a T cell epitope-based vaccine against Chlamydia infection.

Figure 6.

Figure 6

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Detection of CPAF specific CD4+ T cell populations induced by C. trachomatis infection. (A) Representative flow cytometry quadrant plots showing frequency of Cep2/3 HLA-DR4 tetramer stained CD4+ T cells in the right top quadrant for a mock (PBS) and C. trachomatis infected mouse (n=3 per group; tested individually) without (upper panels) or with (lower panels) in vitro expansion by rCPAFep stimulation or peptides. (B) The frequency (mean± SD) of Cep2/3-specific CD4+ cells was calculated from 3 individually tested mice of each indicated group. * Significant (p < 0.05; T test) difference between the indicated groups.

4. Discussion

In previous studies, our laboratory has demonstrated that vaccination of HLA-DR4tg mice with rCPAF can significantly accelerate the resolution of genital C muridarum infection, whereas similarly vaccinated MHC class II deficient mice failed to resolve the infection [13]. These results indicated the presence of protective HLA-DR4 epitopes within the CPAF protein, and provided a viable platform for mapping those CPAF CD4+ T cell epitopes. We now report the identification of 5 CPAF derived T cell reactive epitopes (Cep1-5) which, when expressed as a fusion protein (rCPAFep), provide a comparable protective efficacy against pulmonary and genital chlamydial infection to full length CPAF, suggesting that these 5 peptides are the dominant protective epitopes of the CPAF protein.

One of the advantages of administrating a rCPAFep epitope-based vaccine over full length rCPAF is that the former, with reduction of non-necessary protein sequences, may induce less undesirable effects while retaining the capability to induce comparable protective immunity against Chlamydia infection. In addition, this vaccine also could be combined with other potent chlamydial antigens for formulating a better composite vaccine candidate. For example, the combination of rCPAFep with B-cell epitopes from MOMP, which has been shown to induce neutralizing antibody [27], may provide a higher degree of protective efficacy against a primary chlamydial infection and limit immunopathological consequences.

The chlamydial HLA-DR4tg mouse infection model is an excellent surrogate experimental animal system to develop a vaccine against human Chlamydia diseases. In this model, all MHC class II-presented responses are induced via human HLA-DR4 molecules. rCPAFep vaccinated HLA-DR4tg mice exhibited significantly accelerated resolution of the genital infection and CD4+ T cells also exhibited the highest antigen–specific IFN-γ production upon stimulation with rCPAFep, compared to mock immunized mice, indicating that protective immunity was induced primarily via the HLA-DR4 molecules. Thus, this model overcomes the often doubted human relevance of testing chlamydial vaccine candidates for protection via endogenous mouse MHC class II molecules.

HLA-DR4/epitope tetramers are useful reagents for direct visualization, characterization and isolation of Chlamydia-specific CD4+ T cells, which may serve as an immune correlate for effective vaccine induced protection. Our present data provided the first direct evidence that CPAF epitope-specific CD4+ T cells were induced in the spleen after either vaccination or Chlamydia infection. Although the percentage of tetramer stained positive splenic CD4+ T cells was low, rCPAFep vaccination still provided protection against C. muridarum infection. In this regard, a recent report showed that only 1000 Chlamydia-specific T cells constitute the lower limit needed for significant protection in mice; although, those mice were infected by a newly developed transcervical (intrauterine) inoculation method [28]. Thus, the small population of Chlamydia-specific CD4+ T cells induced by rCPAFep vaccination may be sufficient to confer protective immunity following Chlamydia challenge.

Several studies have shown that i.vag. challenge with C. trachomatis in mice induced less immune cell infiltration and did not produce strong protective immunity [29–31]. However, adaptive immune responses could be mounted after repeated C. trachomatis challenge in mice. Thus, we were able to detect 0.9% of CD4+ T cells bound to Cep2 and Cep3/HLA-DR4 tetramers in HLA-DR4tg mice after 3 C. trachomatis serovar D challenges. This is the first evidence that a conserved CPAF epitope is presented by the host following both C. muridarum and C. trachomatis infection.

Supplementary Material

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    HLA-DR4 restricted T cell epitopes of CPAF were mapped

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    CPAF epitope fusion protein protects against genital and pulmonary chlamydial challenge

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    CPAF-specific CD4+ T cells were identified using HLA-DR tetramers

Acknowledgments

We thank the NIH Tetramer Core Facility for providing tetramers Cep2 and Cep3 for this study. This work was supported by National Institutes of Health Grant 1RO1AI074860 and the Army Research Office of the Department of Defense under Contract No. W911NF-11-1-0136. This project also was supported by a grant from the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health.

Abbreviations used in this paper

CPAF

chlamydial protease-like activity factor

rCPAFep

recombinant CPAF-epitope fusion protein

IFU

inclusion-forming unit

HLA-DR4tg mice

HLA-DR4 transgenic mice

Footnotes

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Conflict of interest: The authors have no potential conflicts of interest to report.

Authors’ contribution

WL, AKM, JPC, GZ, TGF, MNG and BPA conceived and designed the study; WL, GKL, SLC and JPC performed the study; WL, JY, MNG, and BPA wrote the paper. All authors have read and approved the final manuscript.

References

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Supplementary Materials

01
NIHMS527454-supplement-01.pdf (38.4KB, pdf)
02
NIHMS527454-supplement-02.pdf (12.2KB, pdf)
03
NIHMS527454-supplement-03.pdf (59.1KB, pdf)
04
NIHMS527454-supplement-04.pdf (71.5KB, pdf)

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