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<OL> <LI> To characterize at the genetic level the molecular mechanisms responsible for survival and virulence of the intracellular pathogen Brucella. <LI> To develop and evaluate attenuated Brucella mutants incapable of persistent survival in macrophages and in the mouse model. <LI> To develop and evaluate Brucella mutants possessing selected deletions of virulence genes as vaccine candidates in domestic animals (sheep, goats and cattle).
NON-TECHNICAL SUMMARY: Brucellosis in cattle and wildlife causes economic and public health concerns. The purpose of this project is to construct attennated vaccine strains based on our knowledge of the genetic factors necessary for survival in the host. Such vaccine strains will be tested using the mouse model to establish their ability to stimulate immune protection despite their reduced survival.
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APPROACH: Brucellosis in a zoonotic disease affecting small ruminants, cattle and humans in Israel. Infection of pregnant animals leads to abortion and massive numbers of infectious organisms are released into the environment. Humans contract brucellosis through contaminated milk and diary products. The simplest means to control brucellosis is pasteurization and the slaughter of infected animals. Attenuated live strains offer a means of protecting valuable animals and are superior to killed organisms or subunit vaccines. B. melitensis strain Rev 1 works best as a vaccine in sheep and goats, while B. abortus strain 19 is optimal in cattle. Widespread use of Rev 1 has led to several unforeseen problems, including abortion, attenuated immunity, persistent survival of the vaccine strain, persistent humoral response, horizontal transmission, reversion to virulence and recently the appearance of a typical virulent strains. These findings indicated the need to develop alternative vaccines for cattle, sheep and goats in the Middle East. In this proposal the characterization of virulence genes will be used to create improved vaccine strains via recombinant DNA techniques. Fortification of immunity in adult animals may also be performed via revaccination with recombinant subunit/DNA vaccines based on Brucella immunodominant/virulence antigens.
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PROGRESS: 1999/04 TO 2005/03<BR>
A luxR knockout was created 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∆luxR in encapsulated alginate microspheres containing a non-immunogenic eggshell precursor protein of the parasite Fasciola hepatica (Vitelline protein B, VpB). BALB/c mice were immunized intraperitoneally with either encapsulated or unencapsulated S19ÄluxR at a dose of 1x105 CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses post-vaccination indicate that the vaccine candidate was able to elicit an anti-Brucella specific IgG response even when the vaccine was administered in an encapsulated format. In addition, circulating Interferon Gamma (IFN-ã) and Interleukin 12 (IL-12) were elicited, suggesting and induction of a T helper 1 response (Th1). Enhanced safety was revealed by the absence of splenomegaly in mice that were vaccinated with the mutant and following challenge. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the unencapsulated vaccine. These results suggest that S19ÄluxR is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained release manner. Development of improved vaccine strains against Brucella species for use in animals as well as in humans must consider the possibility of infection via aerosol to evaluate protective efficacy. Deep lung tissue of BALB/c mice was infected with Brucella melitensis utilizing a Madison aerosol chamber to elicit systemic infections. Mice were shown to inhale an average of 1.3x104 CFU/lungs and develop a chronic infection of the lungs and peripheral organs. Experiments have demonstrated that unmarked deletion mutants of the asp24 gene consistently confer superior protection against homologous and heterologous aerosol challenge, particularly in mouse spleens when delivered parenterally. However, persistence of the organism was prolonged in the lung regardless of vaccine strain, indicating that the lung may serve as a source of chronic infection to exacerbate the disease. To enhance mucosal immunity for improved protection in lung tissue, intranasal vaccination of mice was performed. Colonization of lungs, livers, and spleens was evaluated at various doses to determine optimal vaccine concentration and to monitor clearance from the host of mutants deficient in the asp24 gene, as well as ∆manBA and ∆mucR. Among these the ∆mucR mutants clears from mouse tissues at a rapid rate when administered parenterally. Intranasal vaccination of mice against aerosol exposure is under investigation to identify viable vaccine candidates against aerosol brucellosis.
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IMPACT: 1999/04 TO 2005/03<BR>
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.