Progress 01/01/05 to 12/31/05
Outputs
Since attenuated Brucella unmarked deletion mutants exhibiting different rates of clearance protect mice and goats against subsequent infection to varied degrees, we hypothesized that vaccine efficacy is due to the persistence of the vaccine as well as varied immune responses elicited by the strain upon initial administration or as part of the memory response. To determine the host immune profile developed from vaccination with Brucella mutants, two attenuated strains were selected: virB2 deleted, a highly attenuated mutant, as well as asp24 deleted, a more persistent though attenuated mutant. Mice were vaccinated i.p. with the mutants and sacrificed at selected timepoints early in infection. Ascites and splenocytes were collected for FACS analysis, in vitro cytokine production, and bacterial colonization to determine differences elicited by the vaccines. Mice were also vaccinated with the strains and allowed to rest for >12 weeks then euthanized, or challenged with
wild type Brucella for 1 hour or 48 hours. Naive mice, as well as naive then challenged mice, were used as controls. Ascites and splenocytes were collected for FACS analysis, in vitro cytokine production, and protection against infection. FACS analysis revealed significant differences between groups of mice for CD4, CD8, MHCII, CD14, CD11c, and CD19. In particular, populations of macrophages (MHCII and CD14 positive) and dendritic cells (CD11c positive) decrease over the course of infection, suggesting depletion or trafficking of these cell types to lymphoid tissue. Characterization of cytokines as a correlate of host memory response is in progress. Neither vaccine strain demonstrated marked differences in host cell elicitation after extended periods of vaccination. Protection is believed to be due to the initial host responses immediately after vaccination and the duration of persistence of the vaccine strain. Future plans include evaluating these initial responses (in progress) as
well as varying the administration route with special emphasis on aerosol inoculation to determine differences in protection due to the initial bacterial presentation to the host.
Impacts
The protection provided by these vaccine strains in the mouse model and in pregnant goats suggests differences in immunity, as a result of prolonged survival of the vaccine strain. Since prolonged survival of vaccine strains could result in unforeseen side-effects experiments are in progress are designed to identify the differences in immune response of the host that provide superior immune protection. Alternative approaches will be employed to stimulate these responses using short-lived and safer vaccine candidates. One such approach is microencapsulation.
Publications
- Kahl, M., Tsolis, R. M., and Ficht, T.A. 2006. Evaluation of Protection Afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice. Submitted to Infect. Immun.
- Kahl, M, Davis, D.S., Elzer, P.H., Hagius, S., Adams, L.G., and Ficht, T.A. 2006. A Novel Unmarked Deletion Mutant of B. melitensis does not Colonize Fetal Tissues and Affords Significant Protection Against Infection in the Pregnant Goat Model. Submitted to Vaccine.
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Progress 04/01/99 to 03/31/05
Outputs
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 S19ΔluxR 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-γ) 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 S19ΔluxR 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.
Impacts
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.
Publications
- Kahl, M., Tsolis, R. M., and Ficht, T.A. 2006. Evaluation of Protection Afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice. Infect. Immun. 74:4048-57.
- Kahl, M, Davis, D.S., Elzer, P.H., Hagius, S., Adams, L.G., and Ficht, T.A. 2006. A Novel Unmarked Deletion Mutant of B. melitensis does not Colonize Fetal Tissues and Affords Significant Protection Against Infection in the Pregnant Goat Model. Vaccine. 24:5169-77.
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Progress 01/01/04 to 12/31/04
Outputs
Of the available vaccines that provide protection against animal brucellosis the common feature is prolonged survival in the host, and it has been assumed that this is a key feature in providing protective immunity. In contrast, although safe and efficacious in their primary target species, B. abortus S19 (cattle) and B. melitensis Rev-1 (sheep/goats) have proved ineffective and in some cases fully virulent in other hosts. Prolonged survival may be associated with unnecessary side effects including fetal/placental colonization. In ongoing studies, attenuated vaccine candidates were identified using the mouse model and macrophage culture to screen signature-tagged mutant banks and identified two groups of Brucella genes important for the infection of mice: those required for establishing infection and those required for maintaining chronic infection. Mutants carrying transposon insertions in the genes required for establishing infection are reduced in number as early
as two weeks post infection in mice. Mutants attenuated for chronic persistence, endure in the spleen at reduced levels for at least eight weeks post infection. In order to determine whether effective protection was a property of prolonged survival four mutants representing both classes were evaluated for their protective immune response. Two (virB and manBA) are required for establishing infection and are cleared early (less than 10 weeks) from the spleens of mice. Two others (gcvH and asp24) are required for maintaining a chronic infection and are persist beyond 10 weeks post inoculation. In order to demonstrate the level of protection provided by virB10 and gcvH mutant strains, mice were vaccinated with either one of the mutant strains or the bovine vaccine strain S19 at 8, 16, & 24 weeks prior to the challenge infection with wild-type B. abortus S2308. Overall, protection coincided with persistence of the vaccine candidates when compared to the vaccine strain S19 (where asp24>gcvH
> S19 > virB10). The cytokine responses observed were consistent with the levels of protective immunity observed, i.e., the longer a strain persisted the greater the level of pro-inflammatory cytokines (IFNg, IL2, etc) when measured early after vaccination. Attempts to measure cytokine expression at later times revealed greatly reduced levels of proinflammatory cytokines in response to Brucella antigen that may reflect a decline in memory T-cells capable of responding. Elevated levels of the anti-inflammatory cytokine IL10 were detected, yet these vaccines still maintained significant protection against challenge. Experiments in pregnant goats confirmed these results with longer lasting vaccines providing better protection against colonization and abortion than short-lived vaccines.
Impacts
The significance of these findings lies in the apparent change in cytokine responses observed. Although a proinflammatory response is thought to be desirable at late times these results demonstrate little evidence of such response. In fact there is a shift toward an anti-inflammatory response that belies the protection observed. Future experiments will explore the potential expansion of memory T cell populations following challenge (or boosting)as well as the use of microencapsulation techniques to enhance the protective immune response elicited by short-lived, highly attenuated Brucella through controlled release strategies.
Publications
- Banai, M., L. G. Adams, L.J. Dangott, M. Frey and T. A. Ficht. 2002. Brucella attenuation and relevance to vaccine properties. Small Ruminant Research. 45: 129-137.
- Hong, P.C.1, Pei, J.W.1, Kahl, M.M.1, Tsolis, R.M.2, and Ficht, T.A. 2005. Brucella abortus mutants defective for maintaining chronic infection confer protection in BALB/c mice. Manuscript in preparation.
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Progress 01/01/03 to 12/31/03
Outputs
Work is in progress 1. to characterize at the genetic level the molecular mechanisms responsible for survival and virulence of the intracellular pathogen Brucella. 2. To develop and evaluate attenuated Brucella mutants incapable of persistent survival in macrophages and in the mouse model. 3. To develop and evaluate Brucella mutants possessing selected deletions of virulence genes as vaccine candidates in domestic animals (sheep, goats and cattle). In this research the characterization of virulence genes is being 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
Impacts
Development of vaccines against brucellosis effective in wildlife and agricultural species.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
To be effective, live vaccines against brucellosis must persist long enough to elicit protective immunity, but should be cleared as quickly as possible to avoid unnecessary side effects. In previous studies, we have used the mouse model to screen a signature-tagged mutant bank and identified two groups of Brucella genes important for the infection of mice: those required for establishing infection and those required for maintaining chronic infection. Mutants carrying transposon insertions in the genes required for establishing infection are reduced in number as early as two weeks post infection in mice. Mutants attenuated for chronic persistence, endure in the spleen at reduced levels for at least eight weeks post infection. Two mutants (virB10 & gcvH) defective in the ability to persist chronically were evaluated for their persistence in a mouse model. Mutants virB10 & gcvH were cleared from the spleen at 16 weeks and at least 24 weeks post infection, respectively.
In order to demonstrate the level of protection provided by virB10 and gcvH mutant strains, mice were vaccinated with either one of the mutant strains or the bovine vaccine strain S19 at 8, 16, & 24 weeks prior to the challenge infection with wild-type B. abortus S2308. Overall, protection coincided with persistence of the vaccine candidates when compared to the vaccine strain S19 (where gcvH > S19 > virB10). The cytokine responses observed for these two strains were consistent with the levels of protective immunity observed, i.e., the longer a strain persisted the greater the level of inflammatory cytokines (IFN?, IL2, etc). However, interpretation of protective immunity as a function of the duration of survival among these mutants may be misleading, since alternate trafficking within the cells may alter the immune response and vaccine potential due to altered antigen processing and presentation. For this reason the vaccine potential of a single strain BA102, has been evaluated at
different times post inoculation after artificially clearing the organism with deoxycyclin treatment. In this case, it was shown that that the protective efficacy of BA102 was optimal at 16 weeks post inoculation and that protection declined, but remained significant as long as 24 weeks post-inoculation.
Impacts
Development of vaccines against brucellosis effective in wildlife and agricultural species.
Publications
- 1. Hong, P.C.1, Pei, J.W.1, Kahl, M.M.1, Tsolis, R.M.2, and Ficht, T.A. 2003. Brucella abortus mutants defective for maintaining chronic infection confer protection in BALB/c mice. Manuscript in preparation.
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Progress 01/01/01 to 12/31/01
Outputs
The objective of this research is to construct deletion mutants in Brucella strains lacking essential genes necessary for virulence. One method employs the use of antibiotic selection in the first step to identify recombinants and sucrose back selection to identify the organisms in which an excision event has eliminated the virulence gene and any recombinant DNA. This method has not proven successful in our hands. Although integrants were obtained (single-crossovers), their resolution (double crossover) always yield the wild-type strain rather than the knockout. This has been tried most extensively with asp24 and we cannot eliminate the essential nature of this gene. We will transform with a second plasmid containing an intact copy of asp24 to control for this possibility. In an alternative approach we plan to use the flp/frt system to excise a kan cassette built into appropriate knockouts. The system requires a temperature sensitive plasmid for delivery of the
recombinase and is subsequently eliminated by growth at the restrictive temperature. The vaccine strains lacking foreign DNAs may then be used in field trials without concern for uncontrolled dispersal of recombinant DNA in environmental organisms. Target genes of interest (cyd, asp24, manB, virB) have been identified in work supported by other funding. Knockout mutants are being constructed in several genetic backgrounds (Rev-1, S19 and virulent field strains, etc.). Rough variants are a prime area of interest due to their potential value as vaccines that do not interfere with diagnostic testing. The contribution of Brucella O-antigen (LPS) to virulence is well established and appears to contribute to the extracellular survival of the organism. The contribution of O-antigen to intracellular survival is far more contentious. Field trials of the manB mutant are ongoing in Mexico and South Africa in cattle and sheep. The manB mutants (BruVacT) are highly attenuated and neither cause
abortion nor persist in pregnant animals. Naturally occurring rough variants in B.melitensis exhibited polymorphisms at the manBA locus that may be promoted by the Brucella repeat elements located at the 3' end of manB and downstream from manA. The overall GC content of this region is 55% and is consistent with a highly conserved portion of the genome. The changes occur in this locus in approximately 50% of the rough variants. The remaining rough organisms have not been characterized, but may differ in other loci important in O-antigen biosynthesis. The changes in the manBA locus do not appear to correspond to IS711 insertion. Sequences sharing homology with a jump start sequence is found just upstream from the manBA locus and may contribute to regulated expression from this locus or two the DNA rearrangements.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
The objective of this research is to construct deletion mutants in Brucella strains lacking essential genes necessary for virulence. The method employs the use of antibiotic selection in the first step to identify recombinants and sucrose back selection to identify the organisms in which an excision event has eliminated the virulence gene and any recombinant DNA. The vaccine strains lacking foreign DNAs may then be used in field trials without concern for uncontrolled dispersal of recombinant DNA in environmental organisms. Target genes of interest (cyd, asp24, manB, virB) have been identified in work supported by another funding agency. Knockout mutants are being constructed in several genetic backgrounds (Rev-1, S19 and virulent field strains, etc.). Rough variants are another area of interest due to their potential value as vaccines that do not interfere with diagnostic testing. The contribution of Brucella O-antigen (LPS) to virulence is well established and
appears to contribute to the extracellular survival of the organism. The contribution of O-antigen to intracellular survival is far more contentious. Field trials of the manB mutant are ongoing in Mexico and South Africa in cattle and sheep. The manB mutants (BruVac trademark) are highly attenuated and neither cause abortion nor persist in pregnant animals. Naturally occurring rough variants in B. melitensis exhibited polymorphisms at the manBA locus that may be promoted by the Brucella repeat elements located at the 3' end of manB and downstream from manA. The overall GC content of this region is 55 percent and is consistent with a highly conserved portion of the genome. The changes occur in this locus in approximately 50 percent of the rough variants. The remaining rough organisms have not been characterized, but may differ in other loci important in O-antigen biosynthesis. The changes in the manBA losu do not appear to correspond to IS711 insertion. Sequences sharing homology with
a jump start sequence is found just upstream from the manBA locus and may contribute to regulated expression from this locus or two the DNA rearrangements.
Impacts
(N/A)
Publications
- Mock, J., Allen, C.A., L.G. Adams, and T.A. Ficht. 2000. DNA sequence analysis of the Brucella abortus rfb locus: An explanation for the frequent appearance of rough variants. Infect. Immun. In preparation.
- Endley, S., McMurray, D., and T.A. Ficht. 2000. Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection. J. Bacteriol. Submitted . Ficht, T.A. 2000. Adaptation and survival of Brucella abortus in acidic environments. Infect. Immun. Submitted.
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Progress 01/01/99 to 12/31/99
Outputs
Acidification of Brucella containing vacuoles is necessary for survival and replication. Exposure and persistence in low pH environments has been confirmed in recent reports that document trafficking of Brucella within autophagic vacuoles in Vero cells and phagosome acidification observed within infected macrophages. The work reported here describes acid resistance mechanisms in B.abortus S2308 that potentially enhance intracellular survival. Exponentially growing broth cultures of B. abortus were resistant to low pHs with greater than 90% survival in media at pH greater than or equal to 3.6 after 2 hours. A rapid decline in acid resistance presumably due to a loss of proton homeostasis occurred in media at lower pHs (greater than 0.0001% survival at pH 3.4). Acid tolerance defined as increased resistance to pH 3.4 occurred during normal growth as B.abortus entered late-log growth phase (greater than 5 x 109 cfu/ml), but was subsequently lost during stationary phase.
An adaptive acid tolerance response was observed during early-log growth phase (greater than4 x 109 cfu/ml) when exposed to medium of intermediate pHs 4.4 to 4.8. Both mechanisms increased survival of B. abortus down to a pH greater than or equal to 3.4. Since exposure of B. abortus to pH conditions lower then pH 4.0 appears unlikely, both responses must be designed to protect B. abortus against physiologically significant pHs (4.0-4.5) for extended times, i.e., greater than 2 hours. Protection against other environmental stresses has not been characterized and cannot be ruled out. Although, cytochrome d oxidase mutants exhibit no acid tolerance and are rapidly cleared from the spleens of mice, it was not possible to draw a direct correlation between acid sensitivity and attenuated survival. Another mutant defective in the expression of the 24 kDa acid shock protein (cbp24) exhibits no late log phase acid tolerance, but maintains a prolonged adaptive response. The attenuated (1-2
logs) phenotype of this strain in the mouse model suggests that the early- and late-log phase responses do not complement each other completely. Acid tolerance responses may contribute directly or indirectly to attenuated intracellular survival.
Impacts
(N/A)
Publications
- Allen, C.A., L.G. Adams, B.A. Sowa, and T.A. Ficht. 1998. Characterization of the role of O-antigen in survival and virulence of Brucella abortus. Infect. Immun. 66: 1008-1016.
- Miller, W.G., L.G. Adams, T. Ficht, N. Cheville, J. Payeur, D. Harley, S. Ridgway. 1999. First case report of Brucella abortion in a bottlenose dolphin Tursiops truncatus. J. Zoo Wildl. Med. 30: 100-110.
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