[show abstract][hide abstract] ABSTRACT: Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ(+)-producing CD4(+) T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4(+) T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.
The Journal of Immunology 02/2011; 186(6):3615-21. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The immune correlates of protection for most of the currently used vaccines are based on long-lived humoral immunity. Vaccines based on humoral immunity alone are unlikely to protect against infections caused by intracellular pathogens and today's most pressing infectious diseases of public health importance are caused by intracellular infections that not only include Chlamydia trachomatis but also tuberculosis, malaria, and HIV/AIDS. For these infections, vaccines that induce cellular immune responses are essential. Major impediments in developing such vaccines include difficulty in identifying relevant T cell antigens and delivering them in ways that elicit protective cellular immunity. In turn this is compounded by the complexity and plasticity of T cell developmental pathways that often correlate with specific aspects of protective immunity. Genomics and proteomics now provide tools to allow unbiased selection of candidate T cell antigens. This review mainly focuses on an immunoproteomic approach used in our laboratory to identify Chlamydia T cell antigens and how these T cell antigens can be developed into a future human Chlamydia vaccine.
Human vaccines 10/2010; 6(8):676-80. · 3.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: We investigated the phenotypic basis for genetically determined differences in susceptibility and resistance to Chlamydia muridarum pulmonary infection using BALB/c and C57BL/6 mice. Following C. muridarum intranasal inoculation, the intensity of infection was very different between BALB/c and C57BL/6 beginning as early as 3 days post-infection. Intrapulmonary cytokine patterns also differed at early time-points (days 2 and 4) between these two strains of mice. The early recruitment of neutrophils to lung tissue was greater in BALB/c than in C57BL/6 mice and correlated with a higher number of inclusion forming units (IFU) of C. muridarum. At day 12 post-infection, BALB/c mice continued to demonstrate a greater burden of infection, significantly higher lung cytokine levels for tumour necrosis factor-alpha and interleukin-17 (IL-17) and a significantly lower level for interferon-gamma than did C57BL/6 mice. In vitro, bone-marrow-derived dendritic cells (BMDCs) from BALB/c mice underwent less functional maturation in response to C. muridarum infection than did BMDCs from C57BL/6 mice. The BMDCs of BALB/c mice expressed lower levels of activation markers (CD80, CD86, CD40 and major histocompatibility complex class II) and secreted less IL-12 and more IL-23 than BMDCs from C57BL/6 mice. Overall, the data demonstrate that the differences exhibited by BALB/c and C57BL/6 mice following C. muridarum pulmonary infection are associated with differences in early innate cytokine and cellular responses that are correlated with late differences in T helper type 17 versus type 1 adaptive immune responses.
[show abstract][hide abstract] ABSTRACT: Major impediments to developing a Chlamydia vaccine lie in identifying immunologically relevant T-cell antigens and delivery in a manner to stimulate protective immunity. Using an immunoproteomic approach, we previously identified three immunodominant Chlamydia T-cell antigens (PmpG-1, PmpE/F-2, and RplF). Because RplF has high homology to a human ortholog, it may not be suitable for human vaccine development. Therefore, in this study, we evaluated protection against Chlamydia infection in the genital tract in C57BL/6 mice immunized with Chlamydia-specific membrane proteins PmpG-1, PmpE/F-2, and major outer membrane protein (MOMP; as a reference) or a combination of them formulated with one of three adjuvants, CpG oligodeoxynucleotide (CpG-ODN), AbISCO-100 (AbISCO), or DDA/TDB (dimethyldioctadecylammonium bromide/D-(+)-trehalose 6,6'-dibehenate). The results show that immunization with the CpG-ODN formulation failed to provide protection against Chlamydia infection; the AbISCO formulation conferred moderate protection, and the DDA/TDB formulation showed the highest degree of protective efficacy. The combination of PmpG-1, PmpE/F-2, and MOMP proteins formulated with DDA/TDB exhibited the greatest degree of protection among all vaccine groups studied. Moreover, this vaccine combination also engendered significant protection in BALB/c mice, which have a different major histocompatibility complex (MHC) background. We measured cell-mediated immune cytokine responses in mice immunized with PmpG-1 mixed with each of the three adjuvants. The results demonstrate that mice immunized with the DDA/TDB formulation induced the strongest gamma interferon (IFN-gamma) and interleukin-17 (IL-17) responses, characterized by the highest frequency of IFN-gamma/tumor necrosis factor alpha (TNF-alpha) and IFN-gamma/IL-17 double-positive CD4(+) T cells. In conclusion, a Chlamydia vaccine based on the recombinant proteins PmpG-1, PmpE/F-2, and MOMP delivered in a DDA/TDB adjuvant conferred protection against infection that correlated with IFN-gamma/TNF-alpha and IFN-gamma/IL-17 double-positive CD4(+) T cells.
Infection and immunity 03/2010; 78(5):2272-82. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Using a combination of affinity chromatography and tandem mass spectrometry, we recently identified 8 MHC class II (I-A(b)) -bound Chlamydia peptides eluted from dendritic cells (DCs) infected with Chlamydia muridarum. In this study we cloned and purified the source proteins that contained each of these peptides and determined that three of the eight peptide/protein Ags were immunodominant (PmpG-1, RplF, and PmpE/F-2) as identified by IFN-gamma ELISPOT assay using splenocytes from C57BL/6 mice recovered from C. muridarum infection. To evaluate whether the three immunodominant Chlamydia protein Ags were also able to protect mice against Chlamydia infection in vivo, we adoptively transferred LPS-matured DCs transfected ex vivo with the cationic liposome DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate) and individual PmpG-1(25-500aa), RplF, or PmpE/F-2 (25-575 aa) proteins. The results showed that the transfected Chlamydia proteins were efficiently delivered intracellularly into DCs. Mice vaccinated with DCs transfected with individual Chlamydia protein PmpG-1(25-500), RplF, or PmpE/F-2(25-575) exhibited significant resistance to challenge infection as indicated by reduction in the median Chlamydia inclusion forming units in both the lung and genital tract models. The major outer membrane protein was used as a reference Ag but conferred significant protection only in the genital tract model. Overall, vaccination with DCs transfected with PmpG-1(25-500) exhibited the greatest degree of protective immunity among the four Chlamydia Ags tested. This study demonstrates that T cell peptide Ags identified by immunoproteomics can be successfully exploited as T cell protein-based subunit vaccines and that PmpG-1(25-500) protein may be a suitable vaccine candidate for further evaluation.
The Journal of Immunology 03/2009; 182(3):1602-8. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DCs) appear to orchestrate much of the immunobiology of Chlamydia infection, but most studies of Chlamydia-DC interaction have been limited by the availability and heterogeneity of primary bone marrow-derived DCs (BMDCs). We therefore evaluated the immunobiology of Chlamydia muridarum infection in an immortal DC line termed JAWS II derived from BMDCs of a C57BL/6 p53-knockout mouse. JAWS II cells were permissive to the developmental cycle of Chlamydia. Infection-induced cell death was 50 to 80% less in JAWS II cells than in BMDCs. Chlamydia infected JAWS II cells and yielded infectious progeny 10-fold greater than that with primary BMDCs. JAWS II cells showed an expression pattern of cell activation markers and cytokine secretion following Chlamydia infection similar to that of primary BMDCs by up-regulating the expression of CD86, CD40, and major histocompatibility complex class II and secreting significant amounts of interleukin-12 (IL-12) but not IL-10. JAWS II cells pulsed with Chlamydia stimulated immune CD4(+) T cells to secrete gamma interferon. Adoptive transfer of ex vivo Chlamydia-pulsed JAWS II cells conferred levels of immunity on C57BL/6 mice similar to those conferred by primary BMDCs. Taken together, the data show that JAWS II cells exhibit immunobiological characteristics and functions similar to those of primary BMDCs in terms of Chlamydia antigen presentation in vitro and antigen delivery in vivo. We conclude that the JAWS II cell line can substitute for primary BMDCs in Chlamydia immunobiological studies.
Infection and immunity 07/2008; 76(6):2392-401. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.
The Journal of Immunology 03/2008; 180(4):2459-65. · 5.52 Impact Factor