In cynomolgus macaques, ocular infection with a live trachoma strain lacking the conserved 7.5-kb plasmid induced no ocular pathology but facilitated solid or partial protection from subsequent infection with a virulent strain of trachoma.

Chlamydia trachomatis is a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model of C. trachomatis infection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivated in vitro prior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiple in vitro passages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrasting in vivo virulence characteristics, the strains exhibited no differences in the results of in vitro infectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.

Although numerous reports document a role for CD43 in T cell signaling, the direct participation of this molecule in cell activation has been questioned. In this study we show that CD43 ligation on human normal periph-

Here we report on the safety, immunogenicity, and vaccine efficacy of the naturally occurring plasmid-free attenuated Chlamydia trachomatis L2-5667R strain in a murine infection model. Intravaginal Immunization induced both chlamydial specific serum antibody and systemic CD4+ Th1 biased immune responses but failed to induce local IgA antibodies. Immunization induced no pathological changes in the urogenital tract. Protective immunity was evaluated by vaginal challenge with a natural occurring non-attenuated plasmid positive C. trachomatis urogenital strain (serovar D). Vaccinated mice were not protected from colonization/infection but exhibited a reduction in infectious burden at early time periods (1-2 weeks) post-challenge. Partial protective immunity did not protect against inflammatory disease. Thus, intravaginal vaccination with the live-attenuated L2R stain is safe, induces a systemic antibody and CD4+Th1 biased immune response, but its protective efficacy is limited to reducing chlamydial burden at early time periods post-infection.

We have performed a computer analysis to study the prevalence of DNA static curvature in the regulatory regions of Escherichia coli, detecting a large number of operons with curved DNA fragments in their 5' upstream regions. A statistical analysis reveals that all the global transcription factors identified so far in E. coli have a tendency to regulate operons with curved DNA sequences in their upstream regions. In addition to these global regulators, we also found that the PurR, ArgR, FruR, TyrR, and CytR specific regulators present a similar propensity. Interestingly, for these cases we found no previous reference describing a possible relationship with curved DNA regions. To validate our theoretical results, we performed site-directed mutagenesis to reduce the degree of DNA curvature in the regulatory sequences of the aroG, pyrC, and argCBH operons. The effects of these changes were measured by polyacrylamide gel electrophoresis assays and further evaluated in vivo by transcriptional fusions to a reporter gene. All our results point toward a more widespread role of curved DNA in gene transcription, a fact that has previously been underestimated.

Trachoma, caused by the obligate intracellular organism Chlamydia trachomatis, is the world’s leading cause of preventable blindness for which a vaccine is needed. We have previously shown that a plasmid-deficient live-attenuated trachoma vaccine delivered ocularly to macaques elicited either solid or partial protective immunity against a virulent ocular challenge. Solidly protected macaques shared the same MHC class II alleles implicating CD4+ T cells in superior protective immunity. Understandably, we sought to define T cell immune correlates in these animals to potentially improve vaccine efficacy. Here, following a two year resting period, these macaques were boosted intramuscularly with the live-attenuated trachoma vaccine and their peripheral T cell anamnestic responses studied. Both solidly and partially protected macaques exhibited a CD4+ and CD8+ T cell anamnestic response following booster immunization. CD8+ but not CD4+ T cells from solidly protected macaques proliferated against soluble chlamydial antigen. We observed a more rapid T cell inflammatory cytokine response in tears of solidly protected animals following ocular re-challenge. Most notably, depletion of CD8+ T cells in solidly protected macaques completely abrogated protective immunity. Collectively, our findings support the conclusion that CD8+ T cells play an important but unexpected role in live-attenuated trachoma vaccine mediated protective immunity.

Protection against virulent pathogens that cause acute, fatal disease is often hampered by development of microbial resistance to traditional chemotherapeutics. Further, most successful pathogens possess an array of immune evasion strategies to avoid detection and elimination by the host. Development of novel, immunomodulatory prophylaxes that target the host immune system, rather than the invading microbe, could serve as effective alternatives to traditional chemotherapies. Here we describe the development and mechanism of a novel pan-anti-bacterial prophylaxis. Using cationic liposome non-coding DNA complexes (CLDC) mixed with crude F. tularensis membrane protein fractions (MPF), we demonstrate control of virulent F. tularensis infection in vitro and in vivo. CLDC+MPF inhibited bacterial replication in primary human and murine macrophages in vitro. Control of infection in macrophages was mediated by both reactive nitrogen species (RNS) and reactive oxygen species (ROS) in mouse cells, and ROS in human cells. Importantly, mice treated with CLDC+MPF 3 days prior to challenge survived lethal intranasal infection with virulent F. tularensis. Similarly to in vitro observations, in vivo protection was dependent on the presence of RNS and ROS. Lastly, CLDC+MPF was also effective at controlling infections with Yersinia pestis, Burkholderia pseudomallei and Brucella abortus. Thus, CLDC+MPF represents a novel prophylaxis to protect against multiple, highly virulent pathogens.

Background Chlamydia trachomatis is the etiological agent of trachoma the world's leading cause of infectious blindness. Here, we investigate whether protracted clearance of a primary infection in nonhuman primates is attributable to antigenic variation or related to the maturation of the anti-chlamydial humoral immune response specific to chlamydial antigens.Methodology/Principal FindingsGenomic sequencing of organisms isolated throughout the protracted primary infection revealed that antigenic variation was not related to the inability of monkeys to efficiently resolve their infection. To explore the maturation of the humoral immune response as a possible reason for delayed clearance, sera were analyzed by radioimmunoprecipitation using intrinsically radio-labeled antigens prepared under non-denaturing conditions. Antibody recognition was restricted to the antigenically variable major outer membrane protein (MOMP) and a few antigenically conserved antigens. Recognition of MOMP occurred early post-infection and correlated with reduction in infectious ocular burdens but not with infection eradication. In contrast, antibody recognition of conserved antigens, identified as PmpD, Hsp60, CPAF and Pgp3, appeared late and correlated with infection eradication. Partial immunity to re-challenge was associated with a discernible antibody recall response against all antigens. Antibody recognition of PmpD and CPAF was destroyed by heat treatment while MOMP and Pgp3 were partially affected, indicating that antibody specific to conformational epitopes on these proteins may be important to protective immunity.Conclusions/SignificanceOur findings suggest that delayed clearance of chlamydial infection in NHP is not the result of antigenic variation but rather a consequence of the gradual maturation of the C. trachomatis antigen-specific humoral immune response. However, we cannot conclude that antibodies specific for these proteins play the primary role in host protective immunity as they could be surrogate markers of T cell immunity. Collectively, our results argue that an efficacious subunit trachoma vaccine might require a combination of these antigens delivered in their native conformation.