Identification of a neutrophil chemotactic factor from Tritrichomonas foetus as superoxide dismutase.

Antibodies to a neutrophil chemotactic factor from Tritrichomonas foetus were used to screen a T. foetus cDNA expression library in lambda gt11. All positive clones were identified as homologs of iron-containing superoxide dismutase (SOD). Native gel electrophoresis showed that the antibodies indeed recognized T. foetus antigens with SOD activity. Two SOD genes were found in T. foetus, and cloned and sequenced as parts of larger genomic segments of 3844 and 4089 base pairs. Transcription initiated between the first and second methionine codons of each genomic open reading frame, generating mRNAs with 5' untranslated regions of 11-15 bases, and encoding proteins of 195 amino acids. The two SOD coding sequences lacked obvious introns. They were 79% identical at both the nucleotide and amino acid levels. Both SOD genes were inserted into a eukaryotic expression vector and stably expressed in mammalian cells; both proteins were recognized by the antibodies, and both assumed a cytosolic, extranuclear distribution in these cells. Histidine-tagged forms of both T. foetus SODs were expressed in E. coli and after purification, found to have neutrophil chemotactic activity similar to the non-recombinant factor purified from T. foetus. Identification of this neutrophil chemotactic factor as SOD provides additional insight into the host-parasite interaction.

Rapid internalization and degradation of surface-bound antibodies by Tritrichomonas foetus.

Tritrichomonas foetus is a protozoan parasite of cattle that can be cultured axenically. Three monoclonal antibodies specific for surface antigens of T. foetus were found to be rapidly internalized and degraded by these cells after binding. Degradation was not due to secreted or artificially liberated proteases but depended on targeting to internal degradative compartments. Radiolabeled catabolites of the antibodies were subsequently incorporated into the parasite's own proteins. Antibody degradation could be inhibited by certain protease inhibitors or lowered temperatures; a sharp reduction in degradation between 20 C and 15 C was similar to a well documented block in endocytic transport to degradative compartments of mammalian cells. Growth and proliferation of T.foetus in the continuous presence of the antibodies appeared unhindered, but there was a general shift toward expression of both more and less of each epitope among cells within each population. Subclones of these populations always exhibited striking variability in epitope expression levels, with patterns similar to the parent cultures. These findings may lead to a better understanding of how T. foetus resists host immune responses.

A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge.

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150microg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150microg rPA, 150microg rPA+150microg of a conjugated 10-mer peptide representing the Bacillus anthracis capsule (conj), or 150microg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA+conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p=0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA+conj immunized groups (p=0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later.

An intranasal vaccine targeting both the Bacillus anthracis toxin and bacterium provides protection against aerosol spore challenge in rabbits.

An intranasal vaccine targeting the Bacillus anthracis toxin and vegetative bacterium was tested for the ability to protect immunized rabbits against aerosol B. anthracis spore exposure. Rabbits were vaccinated intranasally with PA-based vaccines formulated as dry powders with or without chitosan (ChiSys, Archimedes Development Limited), a compound that exhibits muco-adhesive properties, or as a liquid. Formulations also contained MPL adjuvant and PA. Some vaccines contained PA conjugated to a 10-mer peptide of the poly-d-glutamic acid capsule of B. anthracis. Rabbits were immunized on days 0 and 28 and aerosol challenged with an average 250LD50 Ames spores on day 85. Serum antibody was measured before and after challenge. Significant anti-PA serum IgG levels were obtained, particularly with use of ChiSys based formulations. PA-Conj induced significant anti-capsule responses, although a formulation containing free capsule peptide did not. All immunized rabbits survived the challenge, but differences in morbidity, as evidenced by anorexia, between vaccine groups were observed. Only rabbits immunized with PA+PA-Conj appeared normal throughout the post-challenge observation period (14 days), while all that received PA with the free capsule peptide appeared ill at times as evidenced by a failure to eat normally. One negative control rabbit received a lower inhaled spore dose (183LD50) and survived the challenge, although it was anorexic post-challenge. It also had a high level of anti-LF antibodies in its convalescent serum (5400 U/ml), indicating an extensive infection. In contrast, 75% of the immunized rabbits had no LF-specific antibody in their post-challenge sera, and the rest had low levels (< or = 138 U/ml), indicating that infections resulting in toxin production were avoided or greatly reduced. Thus, intranasal immunization with a chitosan-based powder vaccine combining PA and capsule epitopes provided superior protection against B. anthracis infection compared to a single antigen (PA) vaccine, as evidenced by a reduction in morbidity and prevention of death.

Transient invagination of flagella by Tritrichomonas foetus.

We describe an experimental system for the study of rapid and reversible formation of pseudocysts, which are spherical forms that lack a true cyst wall, by Tritrichomonas foetus, a trichomonad parasite of the bovine genitourinary tract. It highlights the dynamics of the plasma membrane and cytoskeleton of this parasite, which is perpetually devoid of any sort of protective cell wall, and can reflect a responsive survival mechanism. We have found that cooling of axenic cultures of T. foetus from their normal 37 degrees C to below about 16 degrees C can trigger pseudocyst formation. The three anterior flagella and the single recurrent flagellum can be fully internalized within 1 3 min at 37 degrees C, with the axonemes and flagellar membranes remaining intact within the cell body. Electron microscopy confirms that the internalized flagella are surrounded by a separate membrane. At 37 degrees C the internalized flagella can resume beating movements and become externalized as quickly as within 10 min. We have begun to elucidate the mechanisms of this unusual phenomenon, characterizing its temperature dependence and exploring the effects of agents that interfere with various aspects of the cytoskeleton, phagocytosis, endocytosis, and exocytosis.