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.Brucella, an obligate intracellular bacterium, is the causative agent of brucellosis, a zoonosis of nearly worldwide distribution (8). Among the six different Brucella species, Brucella melitensis, B. suis, and B. abortus are pathogenic and virulent not only for sheep, goats, swine, and cattle, respectively, but also for humans. Despite the availability of live vaccine strains for cattle (S19 and RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including interference with diagnosis, resistance to antibiotics, and residual virulence, that prevent the use of these vaccines in humans (4, 6, 28). Numerous attempts to develop safe and more effective vaccines, including the use of killed organisms, cell extracts, or recombinant proteins, has had limited success (18,22,27,28,33).In the absence of defined protective immunogens, the use of attenuated vaccine strains offers the best approach. As an alternative, we have investigated the ability to combine an attenuated live vaccine delivered in a controlled release vehicle. For this purpose, alginate, a naturally occurring biopolymer, offers advantages, including biocompatibility and relatively mild conditions required to produce an alginate matrix or particle (7). Extensive investigation has shown the efficacy of this release system when used to encapsulate protein agents such as insulin, erythropoietins, and chemokines (20,25,32). To further increase the efficacy of the capsular delivery, a novel recombinant form of the vitelline protein B (VpB) derived from the eggshell precursor of the parasite Fasciola hepatica was incorporated into the capsules (26). VpB possesses an unusual resistance to enzymatic and chemical breakdown that is expected to extend the time frame of erosion and release of the capsule content (34).Ongoing research in our laboratory has identified B. mel...

Quorum-Sensing and BvrR/BvrS Regulation, the Type IV Secretion System, Cyclic Glucans, and BacA in the Virulence of Brucella ovis: Similarities to and Differences from Smooth Brucellae

“…4 and 5), the ⌬vjbR mutant of B. melitensis 16M has been reported to be severely impaired with regard to survival in macrophages and was almost completely cleared from the spleens of infected mice at 4 weeks p.i. (3,14). In contrast, survival of the ⌬vjbR mutant of the B. abortus S19 vaccine was only slightly attenuated in J774.A1 macrophages (2) and it showed levels of spleen infection and persistence in the mouse model similar to those of the parental strain and remarkably higher than those reported for the B. melitensis 16M mutant (2,3,14).…”

“…(3,14). In contrast, survival of the ⌬vjbR mutant of the B. abortus S19 vaccine was only slightly attenuated in J774.A1 macrophages (2) and it showed levels of spleen infection and persistence in the mouse model similar to those of the parental strain and remarkably higher than those reported for the B. melitensis 16M mutant (2,3,14). These results suggest variability among the brucellae regarding the impact of VjbR on virulence, but other vjbR mutants should be obtained in the genus Brucella to clarify this point.…”

Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice

“…Mice were bled from the orbital sinus during the immunization schedule and sera were stored at 270 uC until tested. The IgG and IgG subclass antibodies against whole-cell antigens (WCAs) of RB51 and B. canis S26 were measured by indirect ELISA as described previously (Arenas-Gamboa et al, 2008). Briefly, ELISA plates were coated with 25 mg per well WCAs of each strain in carbonate buffer overnight at 4 uC.…”

Extended Safety and Efficacy Studies of the Attenuated Brucella Vaccine Candidates 16MΔvjbRand S19ΔvjbRin the Immunocompromised IRF-1−/−Mouse Model

“…VjbR is required for adequate VirB expression, which is essential for survival in phagocytes and virulence in mice (32). The vjbR mutants of B. melitensis and B. abortus are highly attenuated in mice and macrophages while conferring high levels of protective immunity, making such mutants ideal vaccine candidates (2,3).…”