We used HLA-DR4 tg mice to examine whether the presentation of CPAF epitopes on human MHC-II molecules can induce protective immunity against genital chlamydial challenge. Intranasal vaccination of HLA-DR4 tg mice with CPAF plus IL-12 elicited a Th1 cellular immune response and robust antibody production and significantly accelerated the resolution of genital C. muridarum infection, similar to what was observed for vaccinated conventional C57BL/6 animals. This protection was not evident in CPAF-plus-IL-12-immunized MHC-IIΔ/Δ animals. Results from this study suggest the presence of protective HLA-DR determinants on CPAF and support the translational value of this candidate vaccine antigen for humans.
To our knowledge, this is the first study using HLA-DR4 tg mice to examine immunity against chlamydial infections. HLA-DR4 tg mice were originally generated with HLA-DRA-IEa and HLA-DRB1*0401-IEβ chimeric genes and then backcrossed to MHC-IIΔ/Δ
mice to eliminate any effect of endogenous MHC class II proteins (10
). Therefore, all MHC class II-restricted responses in HLA-DR4 tg animals are induced via the human HLA-DR4 molecules. Chlamydia
-infected HLA-DR4 tg mice developed a C. muridarum
-specific Th1-type cellular response, as indicated by high IFN-γ production, and resolved the infection by approximately 30 days after challenge. The immune response and resolution kinetics in these mice were qualitatively and quantitatively comparable to those in similarly treated conventional C57BL/6 mice, in which immunity against primary genital C. muridarum
infection has been extensively characterized (14
). Chlamydial infections typically induce a Th1-type immune response in challenged C57BL/6 animals, and such responses, specifically IFN-γ, have been demonstrated to be important in the resolution of the infection (2
). Challenged MHC-IIΔ/Δ
animals in the present study developed only minimal cellular and humoral Chlamydia
-specific responses and failed to resolve the infection within the 30-day period monitored, indicating that protective immunity was induced primarily via the HLA-DR4 molecules in challenged HLA-DR4 tg animals. Collectively, these results suggest that HLA-DR4 tg mice are a good model for studying immune responses against genital chlamydial infections.
CPAF expression has been detected as early as 8 to 12 h after chlamydial challenge by Western blot analyses (4
) and at approximately 30 h by immunofluorescence staining (28
). We recently identified in situ CPAF expression in the genital tracts of C. muridarum
-infected HLA-DR4 tg mice (Murthy and Arulanandam, unpublished). CPAF was detected in the columnar cells of the endocervix and endometrium of the genital tract of infected HLA DR4 tg mice at 4 days after challenge. These results are supported by our observations from in vitro experiments in which low levels of CPAF have been shown to degrade USF-1 and RFX-5, host transcription factors for MHC antigen expression (28
), which may allow infected epithelial cells to evade immune detection. More recently, CPAF was also shown to degrade keratin-8, thus potentially allowing expansion of the chlamydial vacuole inside the host cell and also possibly prolonging survival of the infected cells (4
). Therefore, neutralization of such CPAF activity in vivo could result in immune-mediated elimination of infected cells and/or prevention of chlamydial vacuolar expansion, thus leading to an abortive infection in such cells. In fact, we recently demonstrated that intranasal vaccination with CPAF and IL-12 induces a robust Th1-type CPAF-specific immune response and leads to significantly accelerated resolution of a genital chlamydial infection in conventional BALB/c animals (Murthy and Arulanandam, unpublished).
HLA-DR4 tg mice are a useful model for examining the immune responses against bacterial antigens in the context of human as opposed to murine MHC-II molecules. Intravaginal C. muridarum
infection in HLA-DR4 tg mice induced a strong CPAF-specific Th1 cellular response and antibody production. These results are consistent with the findings of Sharma et al. (21
) that Chlamydia
-seropositive individuals and C. trachomatis
-infected conventional BALB/c mice exhibit antibody responses against CPAF. Additionally, in our studies, similarly challenged MHC-IIΔ/Δ
animals displayed only a minimal anti-CPAF immune response. Collectively, these results indicate that CPAF molecules are processed and presented to T cells during an active genital chlamydial infection and that such HLA-DR4 determinants are present within CPAF. An important implication of this finding is the possibility of inducing protective immunity in humans by using CPAF as a vaccine candidate.
Characterization of the immune responses to chlamydial vaccine candidate antigens has been widely pursued, both as a means to better understand the underlying T-cell-mediated immunity and to develop better strategies for vaccine development. However, such studies of humans are limited by difficulties in sampling and by technical difficulties in analyzing the ensuing T-cell response directly ex vivo without introducing culture artifacts. As a consequence, chlamydial vaccine candidates are routinely tested in murine models. The T-cell responses against these antigens occur in the context of murine MHC molecules, whose determinant binding characteristics differ from those of the corresponding human MHC alleles (6
). Specifically, MHC molecules are highly polymorphic, and each allelic MHC product has a unique peptide binding motif that makes it specific for a unique peptide fragment of the antigen (7
). Since individuals within a population that express different MHC alleles exhibit unique peptide binding properties, rules of determinant recognition established for one allele do not apply to another allele and determinant presentation data from one species cannot necessarily be extrapolated to another (7
These constraints can be overcome by using mice that lack endogenous murine MHC class II molecules but express a human HLA allele (HLA-DR4 tg mice). The chimeric MHC molecules show the same peptide binding specificity as the HLA-DR4 molecules and are capable of presenting antigens to human T cells (10
). In addition, since the cytoplasmic tail of the HLA-DR-IEα construct of the transgenic mice was derived from the murine MHC class II molecule, signaling and sorting of the MHC class II molecules via the cytoplasmic tail of the HLA-DR constructs can be assumed to be normal, without affecting the functioning of the antigen-presenting cells. The production of significant amounts of IFN-γ, but not IL-4 recall responses, by splenocytes of CPAF-immunized mice in this study indicates the induction of a Th1-biased anti-CPAF cellular immune response. Furthermore, CPAF-vaccinated HLA-DR4 tg mice, but not MHC-IIΔ/Δ
animals, resolved the infection significantly earlier than the corresponding challenged mock-immunized animals. This clearly demonstrates that (i) CPAF-specific immune responses after vaccination induce protection against genital chlamydial challenge and, more importantly, (ii) CPAF contains protective determinants that are processed and presented by human HLA-DR4 molecules. This observation implies great importance for CPAF as a potential vaccine candidate for humans and warrants further mapping of protective CPAF epitopes that are restricted by human MHC class II alleles. In this regard, HLA-DR4 tg mice have been utilized to identify T-cell-reactive peptides against the outer surface protein (OspA) of Borrelia burgdorferi
, the agent of Lyme disease (8
). The immunodominant peptides identified using the HLA-DR4 tg mice were shown to generate immune responses similar to those generated in treatment-resistant Lyme-induced arthritis patients. These results reinforce the relevance of using HLA-DR4 tg mice to identify MHC-related epitopes that have relevance to human disease.
Our study indicates that HLA-DR4 tg mice that express the human HLA allele provide an appropriate model for studying immune responses against genital C. muridarum infection and constitute a viable platform for mapping candidate CD4+ T-cell antigens, such as CPAF, that may have translational value in humans.