Alginate microencapsulation of an attenuated O-antigen mutant of Francisella tularensis LVS as a model for a vaccine delivery vehicle.

Francisella tularensis is the etiologic agent of tularemia and a Tier I Select Agent. Subspecies tularensis (Type A) is the most virulent of the four subspecies and inhalation of as few as 10 cells can cause severe disease in humans. Due to its niche as a facultative intracellular pathogen, a successful tularemia vaccine must induce a robust cellular immune response, which is best achieved by a live, attenuated strain. F. tularensis strains lacking lipopolysaccharide (LPS) O-antigen are highly attenuated, but do not persist in the host long enough to induce protective immunity. Increasing the persistence of an O-antigen mutant may help stimulate protective immunity. Alginate encapsulation is frequently used with probiotics to increase persistence of bacteria within the gastrointestinal system, and was used to encapsulate the highly attenuated LVS O-antigen mutant WbtIG191V. Encapsulation with alginate followed by a poly-L-lysine/alginate coating increased survival of WbtIG191V in complement-active serum. In addition, BALB/c mice immunized intraperitoneally with encapsulated WbtIG191V combined with purified LPS survived longer than mock-immunized mice following intranasal challenge. Alginate encapsulation of the bacteria also increased antibody titers compared to non-encapsulated bacteria. These data suggest that alginate encapsulation provides a slow-release vehicle for bacterial deposits, as evidenced by the increased antibody titer and increased persistence in serum compared to freely suspended cells. Survival of mice against high-dose intranasal challenge with the LVS wildtype was similar between mice immunized within alginate capsules or with LVS, possibly due to the low number of animals used, but bacterial loads in the liver and spleen were the lowest in mice immunized with WbtIG191V and LPS in beads. However, an analysis of the immune response of surviving mice indicated that those vaccinated with the alginate vehicle upregulated cell-mediated immune pathways to a lesser extent than LVS-vaccinated mice. In summary, this vehicle, as formulated, may be more effective for pathogens that require predominately antibody-mediated immunity.

The Application of a Nanomaterial Optical Fiber Biosensor Assay for Identification of Brucella Nomenspecies.

Bacteria in the genus Brucella are the cause of brucellosis in humans and many domestic and wild animals. A rapid and culture-free detection assay to detect Brucella in clinical samples would be highly valuable. Nanomaterial optical fiber biosensors (NOFS) are capable of recognizing DNA hybridization events or other analyte interactions with high specificity and sensitivity. Therefore, a NOFS assay was developed to detect Brucella DNA from cultures and in tissue samples from infected mice. An ionic self-assembled multilayer (ISAM) film was coupled to a long-period grating optical fiber, and a nucleotide probe complementary to the Brucella IS711 region and modified with biotin was bound to the ISAM by covalent conjugation. When the ISAM/probe duplex was exposed to lysate containing ≥100 killed cells of Brucella, or liver or spleen tissue extracts from Brucella-infected mice, substantial attenuation of light transmission occurred, whereas exposure of the complexed fiber to non-Brucella gram-negative bacteria or control tissue samples resulted in negligible attenuation of light transmission. Oligonucleotide probes specific for B. abortus, B. melitensis, and B. suis could also be used to detect and differentiate these three nomenspecies. In summary, the NOFS biosensor assay detected three nomenspecies of Brucella without the use of polymerase chain reaction within 30 min and could specifically detect low numbers of this bacterium in clinical samples.