A Chlamydia-Specific TCR-Transgenic Mouse Demonstrates Th1 Polyfunctionality with Enhanced Effector Function.

Chlamydia is responsible for millions of new infections annually, and current efforts focus on understanding cellular immunity for targeted vaccine development. The Chlamydia-specific CD4 T cell response is characterized by the production of IFN-γ, and polyfunctional Th1 responses are associated with enhanced protection. A major limitation in studying these responses is the paucity of tools available for detection, quantification, and characterization of polyfunctional Ag-specific T cells. We addressed this problem by developing a TCR-transgenic (Tg) mouse with CD4 T cells that respond to a common Ag in Chlamydia muridarum and Chlamydia trachomatis Using an adoptive-transfer approach, we show that naive Tg CD4 T cells become activated, proliferate, migrate to the infected tissue, and acquire a polyfunctional Th1 phenotype in infected mice. Polyfunctional Tg Th1 effectors demonstrated enhanced IFN-γ production compared with polyclonal cells, protected immune-deficient mice against lethality, mediated bacterial clearance, and orchestrated an anamnestic response. Adoptive transfer of Chlamydia-specific CD4 TCR-Tg T cells with polyfunctional capacity offers a powerful approach for analysis of protective effector and memory responses against chlamydial infection and demonstrates that an effective monoclonal CD4 T cell response may successfully guide subunit vaccination strategies.

T Cell-Independent Gamma Interferon and B Cells Cooperate To Prevent Mortality Associated with Disseminated Chlamydia muridarum Genital Tract Infection.

CD4 T cells and antibody are required for optimal acquired immunity to Chlamydia muridarum genital tract infection, and T cell-mediated gamma interferon (IFN-γ) production is necessary to clear infection in the absence of humoral immunity. However, the role of T cell-independent immune responses during primary infection remains unclear. We investigated this question by inoculating wild-type and immune-deficient mice with C. muridarum CM001, a clonal isolate capable of enhanced extragenital replication. Genital inoculation of wild-type mice resulted in transient dissemination to the lungs and spleen that then was rapidly cleared from these organs. However, CM001 genital infection proved lethal for STAT1-/- and IFNG-/- mice, in which IFN-γ signaling was absent, and for Rag1-/- mice, which lacked T and B cells and in which innate IFN-γ signaling was retained. In contrast, B cell-deficient muMT mice, which can generate a Th1 response, and T cell-deficient mice with intact B cell and innate IFN-γ signaling survived. These data collectively indicate that IFN-γ prevents lethal CM001 dissemination in the absence of T cells and suggests a B cell corequirement. Adoptive transfer of convalescent-phase immune serum but not naive IgM to Rag1-/- mice infected with CM001 significantly increased the survival time, while transfer of naive B cells completely rescued Rag1-/- mice from CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important cooperation between T cell-independent B cell responses and innate IFN-γ in chlamydial host defense and suggest that interactions between T cell-independent antibody and IFN-γ are essential for limiting extragenital dissemination.

Viral-vectored boosting of OmcB or CPAF-specific T cell responses fail to enhance protection from Chlamydia muridarum in infection immune mice and elicits a non-protective CD8-dominant response in naïve mice.

A vaccine is needed to combat the Chlamydia epidemic. Replication-deficient viral vectors are safe and induce antigen-specific T cell memory. We tested the ability of intramuscular immunization with modified vaccinia virus Ankara (MVA) or Chimpanzee Adenovirus (ChAd) expressing chlamydial outer membrane protein, OmcB, or the secreted protein, CPAF, to enhance T cell immunity and protection in mice previously infected with plasmid-deficient Chlamydia muridarum CM972, and to elicit protection in naïve mice. MVA.OmcB or MVA.CPAF increased antigen-specific T cells in CM972-immune mice ∼150 and 50-fold respectively but failed to improve bacterial clearance. ChAd.OmcB/MVA.OmcB prime-boost immunization of naïve mice elicited a CD8-dominant T cell response that failed to protect. ChAd.CPAF/ChAd.CPAF prime-boost also induced a CD8-dominant response with a marginal reduction in burden. Challenge of ChAd.CPAF-immunized mice genetically deficient in CD4 or CD8 T cells showed that protection was entirely CD4-dependent. CD4-deficient mice had prolonged infection, while CD8-deficient mice had higher frequencies of CPAF-specific CD4 T cells, earlier clearance, and reduced burden compared to wild-type controls. These data reinforce the essential nature of the CD4 T cell response in protection from chlamydial genital infection in mice and the need for vaccine platforms that drive CD4-dominant responses.

Simultaneous profiling of sexually transmitted bacterial pathogens, microbiome, and concordant host response in cervical samples using whole transcriptome sequencing analysis.

Pelvic inflammatory disease (PID) is a female upper genital tract inflammatory disorder that arises after sexually transmitted bacterial infections (STI). Factors modulating risk for reproductive sequelae include co-infection, microbiota, host genetics and physiology. In a pilot study of cervical samples obtained from women at high risk for STIs, we examined the potential for unbiased characterization of host, pathogen and microbiome interactions using whole transcriptome sequencing analysis of ribosomal RNA-depleted total RNAs (Total RNA-Seq). Only samples from women with STI infection contained pathogen-specific sequences (3 to 38% transcriptome coverage). Simultaneously, we identified and quantified their active microbial communities. After integration with host-derived reads from the same data, we detected clustering of host transcriptional profiles that reflected microbiome differences and STI infection. Together, our study suggests that total RNA profiling will advance understanding of the interplay of pathogen, host and microbiota during natural infection and may reveal novel, outcome-relevant biomarkers.