Weidang Li

Tumor necrosis factor alpha production from CD8+ T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum Infection

ABSTRACT The immunopathogenesis of Chlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin−/− mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α−/− mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8+ T cells, we evaluated the role of CD8+ T cells during genital Chlamydia muridarum infection and oviduct sequelae. Following vaginal chlamydial challenge, (i) mice deficient in TAP I (and therefore the major histocompatibility complex [MHC] I pathway and CD8+ T cells), (ii) wild-type mice depleted of CD8+ T cells, and (iii) mice genetically deficient in CD8 (CD8−/− mice) all displayed similar levels of vaginal chlamydial clearance but significantly reduced hydrosalpinx, compared to those of wild-type C57BL/6 mice, suggesting a role for CD8+ T cells in chlamydial pathogenesis. Repletion of CD8−/− mice with wild-type or perforin−/−, but not TNF-α−/−, CD8+ T cells at the time of challenge restored hydrosalpinx to levels observed in wild-type C57BL/6 mice, suggesting that TNF-α production from CD8+ T cells is important for pathogenesis. Additionally, repletion of TNF-α−/− mice with TNF-α+/+ CD8+ T cells significantly enhanced the incidence of hydrosalpinx and oviduct dilatation compared to those of TNF-α−/− mice but not to the levels found in wild-type mice, suggesting that TNF-α production from CD8+ T cells and non-CD8+ cells cooperates to induce optimal oviduct pathology following genital chlamydial infection. These results provide compelling new evidence supporting the contribution of CD8+ T cells and TNF-α production to Chlamydia-induced reproductive tract sequelae.

Antigen-Specific CD4+ T Cells Produce Sufficient IFN-γ to Mediate Robust Protective Immunity against Genital Chlamydia muridarum Infection

Chlamydia has been shown to evade host-specific IFN-γ-mediated bacterial killing; however, IFN-γ-deficient mice exhibit suboptimal late phase vaginal Chlamydia muridarum clearance, greater dissemination, and oviduct pathology. These findings introduce constraints in understanding results from murine chlamydial vaccination studies in context of potential implications to humans. In this study, we used mice deficient in either IFN-γ or the IFN-γ receptor for intranasal vaccination with a defined secreted chlamydial Ag, chlamydial protease-like activity factor (CPAF), plus CpG and examined the role of IFN-γ derived from adoptively transferred Ag-specific CD4+ T cells in protective immunity against genital C. muridarum infection. We found that early Ag-specific IFN-γ induction and CD4+ T cell infiltration correlates with the onset of genital chlamydial clearance. Adoptively transferred IFN-γ competent CPAF-specific CD4+ T cells failed to enhance the resolution of genital chlamydial infection within recipient IFN-γ receptor-deficient mice. Conversely, IFN-γ production from adoptively transferred CPAF-specific CD4+ T cells was sufficient in IFN-γ-deficient mice to induce early resolution of infection and reduction of subsequent pathology. These results provide the first direct evidence that enhanced anti-C. muridarum protective immunity induced by Ag-specific CD4+ T cells is dependent upon IFN-γ signaling and that such cells produce sufficient IFN-γ to mediate the protective effects. Additionally, MHC class II pathway was sufficient for induction of robust protective anti-C. muridarum immunity. Thus, targeting soluble candidate Ags via MHC class II to CD4+ T cells may be a viable vaccine strategy to induce optimal IFN-γ production for effective protective immunity against human genital chlamydial infection.

Induction of Cross-Serovar Protection against Genital Chlamydial Infection by a Targeted Multisubunit Vaccination Approach

ABSTRACT An important consideration for antichlamydial vaccine development is the induction of cross-serovar protection, since multiple serovars (D to L) of Chlamydia trachomatis cause genital infections. We have shown previously that vaccination with C. trachomatis-derived recombinant chlamydial protease-like activity factor (rCPAF) induced significant earlier resolution of Chlamydia muridarum infection and reduced oviduct pathology. However, the vaccinated mice continued to shed chlamydiae for up to 2 weeks after challenge. In this study, C. trachomatis serovar D recombinant proteins, such as recombinant major outer membrane protein (rMOMP), recombinant inclusion membrane protein A (rIncA), and rCPAF were administered intranasally, individually or in combinations, with murine interleukin-12 (IL-12) as an adjuvant, and cross-species immunity against intravaginal C. muridarum infection was examined. Immunization with rCPAF plus IL-12 (rCPAF+IL-12), compared to immunization with rIncA+IL-12 or rMOMP+IL-12, induced the greatest antigen-specific gamma interferon production from purified CD4+ T cells and concurrently enhanced serum antibody production. All (100%) the animals vaccinated with rCPAF+IL-12 alone or in any combination completely resolved the infection by day 18 after challenge compared to animals vaccinated with rIncA+IL-12 (50%), rMOMP+IL-12 (33%), or phosphate-buffered saline (mock vaccinated; 0%). Moreover, oviduct pathology in mice vaccinated by any regimen that included rCPAF, but not rMOMP+IL-12 or rIncA+IL-12 alone, was markedly reduced compared to mock-immunized animals. The addition of rMOMP and/or rIncA did not significantly enhance the rCPAF+IL-12-induced effect on bacterial clearance or oviduct pathology. These results suggest a greater conservation of protective linear antigenic epitopes within CPAF than MOMP or IncA across the examined serovars and the need to identify other highly conserved antigens for use with rCPAF in a multisubunit recombinant vaccine.

A limited role for antibody in protective immunity induced by rCPAF and CpG vaccination against primary genital Chlamydia muridarum challenge

Mice deficient in B cells (micromT mice) were used to evaluate the role of antibody in enhanced chlamydial clearance and reduction of pathology afforded by vaccination with recombinant chlamydial protease-like activity factor (rCPAF). Enhanced, but comparable, chlamydial clearance was observed in micromT and wild-type (WT) mice after rCPAF+CpG vaccination. Chlamydia-induced pathology was present in mock-immunized animals, but at significantly greater levels in micromT than WT mice, whereas vaccinated micromT and WT mice exhibited similar reductions in pathology. Thus, antibodies may play a role in protection against chlamydial pathology after primary infection, but were largely dispensable in rCPAF+CpG-induced chlamydial clearance and reduction in pathology.

Vaccination with the Defined Chlamydial Secreted Protein CPAF Induces Robust Protection Against Female Infertility Following Repeated Genital Chlamydial Challenge

We previously have shown the efficacy of recombinant (r) chlamydial protease-like activity factor (CPAF) vaccination against hydrosalpinx development following primary genital chlamydial challenge. In this study, we evaluated further the protection induced by rCPAF vaccination against infertility. Following primary challenge, fertility levels were not significantly different between the mock- and CPAF-vaccinated and Chlamydia alone challenged mice. However, following secondary genital chlamydial challenge, mock (PBS) immunized mice displayed a significant reduction of fertility compared to age-matched naïve mice, while mice vaccinated intranasally with rCPAF+CpG displayed significant prevention of infertility. These results suggest that hydrosalpinx may be a reliable indicator of impending infertility, and that rCPAF is a promising candidate to prevent infertility resulting from repeated genital chlamydial infections.

Immunization with a Combination of Integral Chlamydial Antigens and a Defined Secreted Protein Induces Robust Immunity against Genital Chlamydial Challenge

ABSTRACT We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF; a secreted chlamydial protein) in inducing antigen-specific CD4+ T cell/gamma interferon (IFN-γ)-mediated but not antibody-mediated chlamydial clearance and reduction of upper genital tract (UGT) pathological sequelae. Since chlamydial integral antigens may induce neutralizing antibody protection, we further evaluated induction of protective immunity using a combination of rCPAF and UV-inactivated chlamydial elementary bodies (UV-EB) against vaginal chlamydial challenge in comparison to immunization with the individual components or live EB. The rCPAF-UV-EB immunization induced a significantly enhanced anti-UV-EB cellular and antibody response and a reduced anti-CPAF cellular and antibody response, compared to immunization with the respective individual components. Moreover, vaccination with UV-EB and rCPAF-UV-EB induced serum antibodies that neutralized chlamydial infectivity. The rCPAF-UV-EB immunization resulted in a significant reduction of vaginal chlamydial shedding and induced earlier bacterial clearance than vaccination of mice with the individual components. Importantly, the UGT sequelae were significantly reduced in mice immunized with rCPAF or rCPAF-UV-EB, but not in those immunized with UV-EB alone, and approached the levels of protection induced by live EB. These results collectively suggest that a combination of neutralizing antibodies induced by integral chlamydial antigens and cell-mediated responses induced by secreted proteins such as CPAF induces optimal protective immunity against genital chlamydial infections.

Heat denatured enzymatically inactive recombinant chlamydial protease-like activity factor induces robust protective immunity against genital chlamydial challenge.

We have shown previously that vaccination with recombinant chlamydial protease-like activity factor (rCPAF) plus interleukin-12 as an adjuvant induces robust protective immunity against primary genital Chlamydia muridarum challenge in mice. Since CPAF is a protease, we compared the effects of enzymatically active and inactive (heat denatured) rCPAF to determine whether proteolytic activity is expendable for the induction of protective immunity against chlamydial challenge. Active, but not inactive, rCPAF immunization induced high levels of anti-active CPAF antibody, whereas both induced robust splenic CPAF-specific IFN-gamma production. Vaccination with active or inactive rCPAF induced enhanced vaginal chlamydial clearance as early as day 6 with complete resolution of infection by day 18, compared to day 30 in mock-vaccinated and challenged animals. Importantly, significant and comparable reductions in oviduct pathology were observed in active and inactive rCPAF-vaccinated mice compared to mock-vaccinated animals. Thus, rCPAF induced anti-chlamydial immunity is largely independent of enzymatic activity and secondary or higher order protein conformation.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection

BACKGROUND We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. METHODS In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. RESULTS TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. CONCLUSIONS Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

Perforin- and Granzyme-Mediated Cytotoxic Effector Functions Are Essential for Protection against Francisella tularensis following Vaccination by the Defined F. tularensis subsp. novicida ΔfopC Vaccine Strain

ABSTRACT A licensed vaccine against Francisella tularensis is currently not available. Two Francisella tularensis subsp. novicida (herein referred to by its earlier name, Francisella novicida) attenuated strains, the ΔiglB and ΔfopC strains, have previously been evaluated as potential vaccine candidates against pneumonic tularemia in experimental animals. F. novicida ΔiglB, a Francisella pathogenicity island (FPI) mutant, is deficient in phagosomal escape and intracellular growth, whereas F. novicida ΔfopC, lacking the outer membrane lipoprotein FopC, which is required for evasion of gamma interferon (IFN-γ)-mediated signaling, is able to escape and replicate in the cytosol. To dissect the difference in protective immune mechanisms conferred by these two vaccine strains, we examined the efficacy of the F. novicida ΔiglB and ΔfopC mutants against pulmonary live-vaccine-strain (LVS) challenge and found that both strains provided comparable protection in wild-type, major histocompatibility complex class I (MHC I) knockout, and MHC II knockout mice. However, F. novicida ΔfopC-vaccinated but not F. novicida ΔiglB-vaccinated perforin-deficient mice were more susceptible and exhibited greater bacterial burdens than similarly vaccinated wild-type mice. Moreover, perforin produced by natural killer (NK) cells and release of granzyme contributed to inhibition of LVS replication within macrophages. This NK cell-mediated LVS inhibition was enhanced with anti-F. novicida ΔfopC immune serum, suggesting antibody-dependent cell-mediated cytotoxicity (ADCC) in F. novicida ΔfopC-mediated protection. Overall, this study provides additional immunological insight into the basis for protection conferred by live attenuated F. novicida strains with different phenotypes and supports further investigation of this organism as a vaccine platform for tularemia.

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

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.