Mechanisms of Protection Associated with Rough Brucella Strains in a Respiratory Model

NON-TECHNICAL SUMMARY: The protective immune response to respiratory challenge of Brucella abortus 2308 is not known, nor is it known whether current Brucella vaccines can protect against respiratory challenge. We will determine the protective immune response to respiratory challenge of Brucella abortus 2308, and we will determine the differential ability for current Brucella vaccines to protect against challenge. Information from these studies may allow Brucella spp to be used as vectors for multivalent vaccines against bioterrorist agents.

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APPROACH: We will generate Brucella expressing GFP (green fluorescent protein), RFP (red) and hemagglutinin (HA) strains for the intracellular detection of Brucella, as well as the ability to assess epitope specific responses. We will optimize intranasal (IN) doses of strains 2308, RB51 and RB51SOD. We will then determine the differential ability of Brucella vaccines to stimulate innate and adaptive immunity and to correlate these with enhanced protection via challenge experiments. Mice will be vaccinated IN and challenged as described, using optimized doses. Mice vaccinated with strain 2308 will be used to compare smooth vs. rough induced vaccination responses. There will be 21 mice per group. Four mice per group will be euthanized at days, 5, 14, 28, and 42 post-vaccination. There will be 5 mice in each group remaining. At day 49 following initial inoculation, these five mice per group will be challenged IN with 2308 (2 x 103 CFU). Experiment will be repeated for statistical strength. To determine the differential ability of Brucella vaccines to stimulate innate response. Analysis will consist of histopathology, cytokine and chemokine analysis, as well as DC recruitment and activation. Innate response will be assessed based on cytokine and chemokine production on bronchoalveolar lavage (BAL) samples as measured by ELISAs. Dendritic cell activation between Brucella strains will be assessed based on upregulation of activation markers. Cells will be isolated from spleen, MLN and BAL. Four color staining, using antibodies from Pharmingen, will be used to enumerate infected cells (CD11c, CD11b, CD4, CD8) as detected by GFP or RFP expression. Histopathology of spleen and lung sections will be performed to determine the presence of and severity of histopathological changes associated with vaccination and challenge, particularly neutrophil infiltrate in the lung. <P>
Objective 2: To determine the differential ability of Brucella vaccines to stimulate adaptive immunity. Analysis will consist of serology, antigen specific proliferation and cytokine production. Humoral response (IgG2a, IgG1, IgA) will be measured on serum using indirect ELISAs. Antigen specific responses will be measured by IFN-gamma and IL-4 production in response to specific Brucella (killed 2308, RB51 or RB51SOD) and HA challenge. Cytokines (IFN-gamma) will be measured by ELISAs as well as intracellular cytokine staining. HA specific tetramers will be used to measure the antigen specific CD4 and CD8 response. Naive, memory and effector populations of CD4 and CD8 cells in spleen, blood and MLN will be assessed by flow cytometry. Clearance of vaccine strains (CFU) will be determined on lung, (MLN) and spleen. <P>Objective 3: To determine the differential ability of Brucella spp. vaccines to protect against respiratory B. abortus 2308 challenge. To determine how vaccine stimulated innate and adaptive immune response correlate with protection, strain RB51,RB51SOD and 2308 immunized mice will be challenged IN with strain 2308 as described. Analysis will consist of histopathology, and the above described endpoints for innate and adaptive immunity.
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PROGRESS: 2004/10 TO 2007/09<BR>
OUTPUTS: From our studies, we have had some very interesting but unanticipated results. We have focused on developing the respiratory model of vaccination and protection, because this is mandatory to perform additional studies. We have determined after several challenges studies, that solely intranasal vaccination (with RB51 or RB51SOD) does not protect against respiratory challenge. For all experiments in this summary, mice vaccinated with saline were used for controls. Mice were vaccinated intranasally with rough vaccine strain and clearance was confirmed 6-7 wks later prior to respiratory challenge with strain 2308. Mice were euthanized 14 days later. There was a trend towards protection in the spleen (1 log difference in clearance between control and vaccinated group), but mice were not protected in the mediastinal lymph nodes or lung. The experiment was repeated to confirm clearance as well as to assess the effects of boosting the mice and also to assess whether clearance/protection was delayed. Mice were vaccinated with strain RB51SOD. Half the mice were boosted. All mice were challenged with strain 2308. Half of the mice were killed at 14 days PI; the other half of the mice were killed at 42 days PI. Protection was not significantly improved at 42 days PI. There was a trend (p=0.10) for boosting in clearance in the spleens, but protection was not significant in the lungs. We hypothesized that systemic priming was needed prior to boosting. We then assessed the effect of route. Mice were vaccinated IP, ID (dermal), IM (muscular), subcutaneously (SQ), or IN with strain RB51SOD. Mice were challenged IN with strain 2308, and mice were euthanized 14 days PI. Protection in the spleen was significant in IM, ID, IP and SQ, with IM and ID providing the best protection in lung and MLN as well. We also reassessed the optimal IN dose for boosting mice (following systemic priming). Subsequently, we performed a challenge study to assess the effects of systemic priming as well as IN boosting on protection. Mice were vaccinated with RB51SOD either IM, ID or IN. Half the mice were boosted IM, ID or IN. All the mice were boosted IN, and then all the mice were challenged with strain 2308. There was a trend towards protection with IM and ID, but the back calculation of the challenge strain demonstrated that the challenge strain was too low. These experiments will be repeated. In these studies, we have also determined that vaccine strain RB51 induces a greater innate response than strains RB51SOD and strain 2308. Rough vaccine strain RB51 induced significantly higher percentages of dendritic cells (DC) expressing MHC class II high in BAL compared to PBS at all time points and compared to RB51SOD groups at days 5 and 7 PI. Strain RB51 induced significantly greater percentages of DC expressing MHC class II high compared to PBS in MLN at days 3, 7, and 14 PI. Strain RB51 stimulated significant upregulation of CD40 expression on DC in BAL at day 7 PI compared to PBS control group. DC functional analysis and histopathological analysis is pending. <BR> TARGET AUDIENCES: Please see citations. We have or will be presenting data from this project at 3 conferences. We will also be using the data from these experiments as preliminary data for larger grants. <BR> PROJECT MODIFICATIONS: Because we did not see the protection that we anticipated with intranasal vaccination, we had to focus on the development of the respiratory model of vaccination and challenge.
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IMPACT: 2004/10 TO 2007/09 <BR>
From our studies we have determined that IN vaccination solely will not provide protection. We are determining the effects of systemic priming on protection. We are also very interested in determining the mechanisms of "poor immunity" induced by intranasal vaccination. Recent studies demonstrate that some IN vaccination induces Th2 immune response, whereas a Th1 response is needed for overall protection. We will investigate additional studies to determine the whether IN vaccination induces a Th2 response. We have submitted a grant to Wake Forest to continue studies extending from those described here. We also plan on submitting other proposals both to assess mechanisms of protection, inability of intranasal vaccination to protect, as well as mechanisms of subversion to NIH for funding consideration.