Immune Response in Cattle to Brucella Abortus RB51 Over-Expressing Protective Antigens

NON-TECHNICAL SUMMARY: Current vaccines against brucellosis do not afford specific protection against other cattle diseases. the development of a multivalent vaccine to protect against brucellosis and other cattle diseases would make vaccination programs more cost effective. This project assesses the ability of a second generation vaccine derived from vaccine strain RB51 to induce antibodies or cellular immune responses consistent with protection against mycobacterium infections.

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APPROACH: Our ability to raise healthy cattle, free of zoonotic diseases such as Brucella spp., has had a positive impact on both the health and expenditures of producers and consumers of animal foods in Virginia and the USA. Since the implementation of the Brucellosis eradication program in 1950s, it is estimated that the steady reduction over the past 50 years and virtual elimination of brucellosis in cattle herds in 2000 has saved the US millions of dollars annually in terms of cattle and human costs (1). There are still concerns about other zoonotic agents including Mycobacterium bovis, M. paratuberculosis for which no effective vaccines are available for cattle and threaten the health of both cattle and humans. Thus new vaccines that can take advantage of proven vaccines to deliver protective antigens represent an efficient means to immunize susceptible populations. In the proposed work, the USDA approved cattle vaccine, Brucella abortus RB51, will be assessed for its ability to act as a platform to deliver specific antigens to induce immune responses in cattle that correlate with protection against specific pathogens i.e. in addition to Brucella spp. If such protective immune responses (e.g. humoral and cellular) are observed, then additional funding will be sought at the federal level to set up challenge studies in cattle. The development of such a platform-based vaccine will allow for the prevention of several diseases with one one dose of an attenuated vaccine. This type of technology will be cost effective and efficient and minimize side effects seen with multiple vaccination schemes. In addition, a platform based vaccine that can be rapidly formulated and prevent several types of diseases will allow us to respond quickly to biological threats whether they be initiated by natural or terrorist means.
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PROGRESS: 2005/07 TO 2006/06 <BR>
Heifers (4-5 female Holsteins, 8-10 months of age per group) were immunized subcutaneously with either saline, vaccine strains B. abortus RB51, strain RB51/WboA or RB51/SOD/Esat6. 30ml of blood per calf was collected via jugular venipuncture every other week for six weeks. Plasma was harvested from 10mL of whole blood and stored for evaluation of antibody production using antigen specific ELISA. From the remaining 20ml of blood collected, we separated lymphocytes and measured proliferation in response to the vaccine strains as assessed by Alamar blue colorimetric assay as well as the amount of interferon gamma produced as measured by ELISA. At 2, 4 and 6 weeks post immunization relative to the saline treatment, the serum IgG immune responses exhibited a progressive titer increase in all heifers receiving the vaccines: RB51 - 5, 15, 21 mg/dl; RB51/wboA - 6, 8, 14 mg/dl; RB51/SOD/Esat6 - 6, 5, 14 mg/dl respectively. The corresponding interferon-gamma levels relative to saline treatment (measured in supernatants of stimulated lymphocytes) showed a maximum at 2 weeks post-immunization in all groups regardless of the Brucella extract (RB51, RB51/wboA, RB51/SOD/Esat6) used to stimulate the proliferation; the ability to stimulate interferon gamma levels from lymphocytes decreased at 4 and 6 weeks. The extract derived from strain RB51/wboA stimulated the highest level of interferon-gamma (7-9 ng/ml at 2 weeks) production in lymphocytes independent of the Brucella vaccine used to immunize. In contrast, the extract derived from strain RB51/SOD/Esat6 stimulated the smallest level of interferon-gamma and was significantly less than that stimulated by extract from strain RB51; this suggests an immunosuppressive effect. There were no statistically significant differences in leukocyte differential counts (remained normal) or in serum cortisol levels as a function of the Brucella vaccines at 2, 4 and 6 weeks post-immunization. We conclude that these novel platform based B. abortus RB51 based vaccines (RB51/wboA and RB51/SOD/Esat6) are not detrimental to the calves. It appears that strain RB51/wboA is able to better stimulate interferon gamma production in lymphocytes than either strain RB51 or RB51/SOD/Esat6. It will be necessary to perform challenge studies to determine if strain RB51/wboA is able to more effectively protect cattle that strain RB51 against a virulent Brucella challenge. In addition, because of the low levels of gamma-interferon stimulated relative to strain RB51, it appears that strain RB51/SOD/Esat6 would not be a good candidate to protect cattle against a Mycobacterium bovis infection.
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IMPACT: 2005/07 TO 2006/06<BR>
These results show that it is possible to modifiy a USDA/APHIS approved vaccine to prevent brucellosis in cattle and improve its ability to protect against brucellosis but not tuberculosis. It will be necessary to perform challenge studies of cattle immunized with the novel RB51 vaccine to show efficacy in the field. If proven to be effective, it may be possible to lower the effective dose of the vaccine and pass this savings on to the livestock producers.