The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells

doi:10.1016/j.cellimm.2006.10.002

Cellular Immunology

Volume 242, Issue 2, August 2006, Pages 110-117

https://doi.org/10.1016/j.cellimm.2006.10.002 Get rights and content

Abstract

We have previously determined the protective efficacy of intranasal vaccination with chlamydial protease-like activity factor (CPAF) against genital chlamydial infection. Since T-helper 1 (Th1) responses are important for anti-chlamydial immunity, we examined the contribution of CD4⁺ T cells in CPAF mediated immunity against intravaginal (i.vag.) Chlamydia muridarum infection in C57BL/6 mice. CPAF+IL-12 vaccination induced antigen-specific CD4⁺ T cells that secreted elevated levels of IFN-γ, and generated strong humoral responses. The protective effects of CPAF vaccination against genital chlamydial challenge were abrogated by anti-CD4 neutralizing antibody treatment. Moreover, anti-chlamydial immunity could be adoptively transferred to naïve recipients using CPAF-specific CD4⁺ T cells. Therefore, CPAF mediated anti-chlamydial immunity is highly dependent upon antigen-specific CD4⁺ T cells.

Introduction

Chlamydia trachomatis is the leading worldwide cause of sexually transmitted bacterial disease [1]. Antimicrobial therapy is available for treatment of genital chlamydial infections; however, the majority of these infections are initially asymptomatic (75% in women and 50% in men) and therefore not recognized [1], [2]. Untreated infections may ascend into the upper genital tract and cause chronic inflammatory pathology, leading to sequelae such as pelvic inflammatory disease, ectopic pregnancy and infertility [3]. The rising incidence rates of these infections and sequelae over the last decade [1] underscore the timely importance of development of an effective anti-chlamydial vaccine.

Anti-chlamydial vaccine candidates have been evaluated for protection in the murine model of genital chlamydial infection [1], [2]. Among the few candidates examined, the chlamydial major outer membrane protein (MOMP) has been extensively characterized, but found to be only partially protective [2]. Refolding of MOMP to achieve native conformation prior to immunization has been recently reported to reduce vaginal bacterial shedding and infertility rates in mice after genital Chlamydia muridarum challenge [4]. Immunization with an anti-idiotypic antibody to the chlamydial exolipid antigen also has been shown to induce partial protection against genital C. trachomatis challenge [5]. We recently have demonstrated that intranasal vaccination with the chlamydial protease-like activity factor (CPAF) and the mucosal adjuvant interleukin-12 (IL-12) [6], [7] induces enhanced bacterial clearance and robust protection against oviduct pathology following genital C. muridarum challenge [27]. The protection induced by CPAF vaccination was shown to be highly dependent on endogenous IFN-γ production. These promising results indicate the importance of additional characterization of CPAF in the induction of protective immunity against genital chlamydial infection.

There is evidence from several laboratories [1], [2], [9], [21], [22] to suggest that T helper 1 (Th1) cells acting via IFN-γ-dependent and independent pathways are important for the resolution of genital chlamydial infection. Specifically, depletion of CD4⁺ T cells, but not CD8⁺ T cells or B cells, results in marked inability of mice to resolve primary C. muridarum infection in the genital tract [8]. Furthermore, adoptive transfer of Chlamydia-specific CD4⁺ T cells, not CD8⁺ T cells or passive transfer of antibodies, confer protective immunity against chlamydial infection [9], [10], [11], [12]. Upon bacterial rechallenge, B cell deficient (μMT) mice depleted of CD4⁺ T cells, but not CD8⁺ T cells, displayed a severe inability to resolve the infection [12], suggesting the importance of CD4⁺ T cells in effective anti-chlamydial immunity. Collectively, these results underscore the importance of antigen-specific CD4⁺ T cells in anti-chlamydial immunity and suggest that vaccination strategies that elicit such responses may be highly beneficial.

In this study, we examined the role of antigen-specific CD4⁺ T cells in CPAF+IL-12 mediated immunity against genital C. muridarum infection. Depletion of CD4⁺ T cells significantly abrogated the protective effects of CPAF+IL-12 vaccination. Moreover, protective immunity was shown to be adoptively transferred by CPAF-specific CD4⁺ T cells. These results together suggest that CPAF mediated immunity is highly dependent on the induction of antigen-specific CD4⁺ T cells.

Section snippets

Mice

Female 4- to 8-week-old C57BL/6 mice were purchased from Simonsen Laboratories (Gilroy, CA) and maintained at the University of Texas at San Antonio Animal Facility. Mice were given food and water ad libitum and all animal procedures were performed in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

Bacteria

Chlamydia trachomatis mouse pneumonitis (MoPn; recently designated as the separate species C. muridarum) was grown on confluent HeLa cell monolayers. The cells were

Intranasal CPAF+IL-12 immunization induces Th1 type immune response

Splenocytes were removed at day 14 after i.n. immunization and purified CD4⁺ T cells (5 × 10⁵ cells/well) were cultured with mitomycin treated splenocytes as antigen presenting cells (5 × 10⁵ cells/well) and stimulated with CPAF. As shown in Fig. 1A, purified CD4⁺ T cells from CPAF+IL-12 vaccinated animals exhibited elevated levels of IFN-γ production, in a dose-dependent fashion (1.2 ng/ml and 1.5 ng/ml of IFN-γ, respectively), upon stimulation with 0.5 μg or 1 μg of CPAF, as compared to those from

Discussion

We previously have shown that CPAF+IL-12 vaccinated animals display faster resolution, but not resistance, to genital chlamydial infection suggesting that cellular immunity, not neutralizing antibodies, may be involved in mediating this protective effect. In the current study, we examined the role of CD4⁺ T cells in CPAF+IL-12 mediated immunity against genital chlamydial infection. CPAF+IL-12 vaccination induced the generation of IFN-γ producing antigen-specific CD4⁺ T cells and resulted in

Acknowledgments

This work was supported by National Institutes of Health Grants AR048973 and SO6GM008194-24. We also thank Wyeth (Cambridge, MA) for providing murine recombinant interleukin-12 (IL-12).

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Citation Excerpt :
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.
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 Chlamydia muridarum and a human Chlamydia 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.
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The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells

Abstract

Introduction

Section snippets

Mice

Bacteria

Intranasal CPAF+IL-12 immunization induces Th1 type immune response

Discussion

Acknowledgments

Vaccine

Cell Immunol.

Int. Immunopharmacol.

Methods Enzymol.

Nat. Rev. Immunol.

Infect. Immun.

Int. Rev. Immunol.

Infect. Immun.

Nat. Med.

J. Infect. Dis.

Infect. Immun.

Infect. Immun.

Regul. Immunol.

The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4⁺ T cells