A live-attenuated chlamydial vaccine protects against trachoma in nonhuman primates

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

Effective, Broad Spectrum Control of Virulent Bacterial Infections Using Cationic DNA Liposome Complexes Combined with Bacterial Antigens

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

Antibody signature of spontaneous clearance of Chlamydia trachomatis ocular infection and partial resistance against re-challenge in a nonhuman primate trachoma model.

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.Genomic 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.Our 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

Frameshift Mutations in a Single Novel Virulence Factor Alter the In Vivo Pathogenicity of Chlamydia trachomatis for the Female Murine Genital Tract▿ ¶ ‖

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

Antibody signature of <i>C. trachomatis</i> infected macaques following primary infection and re-challenge.

<p>Three monkeys were infected ocularly with <i>C. trachomatis</i> strain A2497 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002248#pntd.0002248-Kari2" target="_blank">[7]</a>. Monkeys cleared the infection 9–14 wpi (RML134: 13 wpi; RML124: 8 wpi; RML126: 14 wpi). Clinical disease remained severe to moderately severe during the periods of culture positivity and continued until about 30 wpi. Three months after resolution of disease monkeys were re-challenged and all monkeys exhibited partial protection characterized by a more rapid clearance of organism (3–7 wpi) with an accompanying reduction in the intensity and duration of ocular inflammation (11 wpi). Sera collected from monkeys during primary infection and following re-challenge were analyzed by RIP using intrinsically labeled chlamydial proteins prepared from non-denaturing detergent lysed infected McCoy cells. Sera from all three macaques produced a similar yet simple antigen recognition profile that differed in complexity over the period of primary infection and re-challenge. A total of 10 proteins were immunoprecipitated with molecular masses of 166, 90, 72, 60, 40, 29, 25, 23, 16 and 15 kDa. There was a noticeable temporal and recall recognition pattern of the antigens in all three monkeys. The MOMP (40 kDa) recognition was strong very early post infection (2–4 weeks) and consistently sustained throughout the entire period of infection and disease. In contrast immunoprecipitation of the other chlamydial proteins occurred later following primary infection, with the strongest immunoprecipitation reactions occurring at the time of infection eradication and disease resolution. There was a recognizable increase in the immunoprecipitation of all of these proteins by all three animals following re-challenge.</p