Allison C. Rice-Ficht

Brucellosis: The Case for Live, Attenuated Vaccines

The successful control of animal brucellosis and associated reduction in human exposure has limited the development of human brucellosis vaccines. However, the potential use of Brucella in bioterrorism or biowarfare suggests that direct intervention strategies are warranted. Although the dominant approach has explored the use of live attenuated vaccines, side effects associated with their use has prevented widespread use in humans. Development of live, attenuated Brucella vaccines that are safe for use in humans has focused on the deletion of important genes required for survival. However, the enhanced safety of deletion mutants is most often associated with reduced efficacy. For this reason recent efforts have sought to combine the optimal features of a attenuated live vaccine that is safe, free of side effects and efficacious in humans with enhanced immune stimulation through microencapsulation. The competitive advantages and innovations of this approach are: (1) use of highly attenuated, safe, gene knockout, live Brucella mutants; (2) manufacturing with unique disposable closed system technologies, and (3) oral/intranasal delivery in a novel microencapsulation-mediated controlled release formula to optimally provide the long term mucosal immunostimulation required for protective immunity. Based upon preliminary data, it is postulated that such vaccine delivery systems can be storage stable, administered orally or intranasally, and generally applicable to a number of agents.

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1 *

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.

Early Phase Morphological Lesions and Transcriptional Responses of Bovine Ileum Infected with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johnes disease) and a possible etiopathologic agent in human Crohns disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5–12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric β-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohns disease.

Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge.

ABSTRACT The development of safe and efficacious immunization systems to prevent brucellosis is needed to overcome the disadvantages of the currently licensed vaccine strains that restrict their use in humans. Alginate microspheres coated with a protein of the parasite Fasciola hepatica (vitelline protein B [VpB]) and containing live Brucella melitensis attenuated mutant vjbR::Tn5 (BMEII1116) were evaluated for vaccine efficacy and immunogenicity in mice. A single immunization dose in BALB/c mice with the encapsulated vjbR mutant improved protection against wild-type B. melitensis 16M challenge compared to the nonencapsulated vaccine strain (P < 0.05). The encapsulated mutant was also shown to induce a sustained elevation of Immunoglobulin G levels. Cytokine secretion from spleen cells of mice vaccinated with the encapsulated vjbR::Tn5 revealed elevated secretion of gamma interferon and interleukin-12, but no interleukin-4, suggesting an induction of a T helper 1 response reflecting the enhanced immunity associated with microencapsulation. Together, these results suggest that microencapsulation of live attenuated organisms offers the ability to increase the efficacy of vaccine candidates.

Use of helper T cells to identify potential vaccine antigens of Babesia bovis

Babesia bovis is an economically important hemoprotozoon parasite o f cattle that is prevalent in many, tropical and subtropical regions o f the world. Effective vaccines against this tick-transmitted parasite consist o f live organisms attenuated by passage through splenectomized calves. However, the nature o f acquired resistance to challenge infection with heterologous isolates of this parasite has not been clearly defined. Unsuccessful attempts to select protective antigens have relied upon the use of antibodies to identify immunodominant proteins. In this review, Wendy Brown and Allison Rice-Ficht discuss the limitations of this approach and the rationale behind using helper T cells to select potential vaccine antigens.

Characterization of a cDNA encoding bovine interleukin 10: kinetics of expression in bovine lymphocytes

A cDNA encoding bovine interleukin 10 (IL10) was cloned and sequenced using total cellular RNA derived from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). A cDNA was produced with reverse transcriptase using oligo(dT) primers, and was amplified using primers chosen from consensus regions of the mouse and human IL10 genes. The nucleotide sequence derived from this cDNA shares 84, 79 and 78% homology with the human, mouse and rat cDNAs, respectively. The deduced amino-acid sequence shares an overall 77, 71 and 74% homology with the human, mouse and rat IL10 proteins, respectively. Northern blot analysis of the bovine IL10 mRNA reveals expression of a single 1.8-kb transcript, reaching maximal levels between 8 and 24 h, in ConA-stimulated peripheral T-cells, and weak expression in lipopolysaccharide-activated macrophages.

The Brucella abortus S19 ΔvjbR Live Vaccine Candidate Is Safer than S19 and Confers Protection against Wild-Type Challenge in BALB/c Mice When Delivered in a Sustained-Release Vehicle

ABSTRACT Brucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. A vjbR knockout was generated in the S19 vaccine and investigated for its potential use as an improved vaccine candidate. Vaccination with a sustained-release vehicle to enhance vaccination efficacy was evaluated utilizing the live S19 ΔvjbR::Kan in encapsulated alginate microspheres containing a nonimmunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B). BALB/c mice were immunized intraperitoneally with either encapsulated or nonencapsulated S19 ΔvjbR::Kan at a dose of 1 × 105 CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses postvaccination indicate that the vaccine candidate was able to elicit an anti-Brucella-specific immunoglobulin G response even when the vaccine was administered in an encapsulated format. The safety was revealed by the absence of splenomegaly in mice that were inoculated with the mutant. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the nonencapsulated vaccine. These results suggest that S19 ΔvjbR::Kan is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained-release manner.

Babesia bovis: gene isolation and characterization using a mung bean nuclease-derived expression library

Genomic DNA prepared from erythrocyte cultures of Babesia bovis merozoites was digested with mung bean nuclease and used to construct a lambda gt11 expression library of B. bovis recombinants. Immunoscreening with two polyclonal antibody probes detected multiple recombinants from which two, designated Bb-1 and Bb-3, were chosen for further analysis. Monospecific immunoglobulins isolated from the screening sera using nitrocellulose-bound fusion proteins were employed to determine the native molecular weight and the intracellular location of the babesial proteins encoded by the recombinants. Clone Bb-1 encodes an antigen of 77,000 Da located at the apical end of the intraerythrocytic parasite. A protein of 75,000 Da encoded by clone Bb-3 is associated with the infected red blood cell cytoplasm and/or membrane but not with the merozoite.

A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4+ T lymphocyte responses in B. bovis-immune cattle ☆

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.

On the diameter and size distributions of Bovine Serum Albumin (BSA)-based microspheres

A fluid mechanics-based correlation for the average size of Bovine Serum Albumin (BSA) microspheres, prepared using a water-in-oil emulsion technique, is presented. The correlation is formulated based on the theory of turbulent dispersion and a non-dimensional Weber number. Measured average diameters of the BSA microspheres prepared in olive oil at different stirring speeds are used to construct the correlation formula. The correlation gives good agreement with experimentally measured average diameters for a wide range of Weber numbers. This correlation is particularly useful to the pharmaceutical industry for predicting the size of encapsulated microspheres used in drug delivery prior to microsphere preparation. The effect of additives on microsphere size was also explored. The average diameter of the BSA microspheres was doubled by the addition of a bioadherent vitelline protein B solution. In addition, a Rosin-Rammler statistical distribution function is shown to accurately represent the microsphere size distribution obtained at different stirring speeds.