Performing Department
VET MED DEAN
Non Technical Summary
ovine tuberculosis (bTB), caused by Mycobacterium tuberculosis variant bovis (MBO), is one of the most damaging diseases in agriculture worldwide. The rise in BTB infected deer and cattle herds in Michigan, has led to a state of urgency due to associated animal trade and movement restrictions. Controlling the disease in animals is a primary approach to preventing/mitigating spread of MBO into and from wildlife or human populations. This study proposes a novel vaccine design around a central paradigm of microbial pathogenesis that posits that immune responses against virulence factors of M. bovis are sufficient and effective to protect animals against MBO infection. These highly conserved immunogenic peptides/proteins from M. tuberculosis Complex will be genetically engineered into the ubiquitous soil bacterium (and a probiotic), Bacillus subtilis, to be expressed and displayed on its spores. Recombinant spores are heat- stable, easily stored, and easily administered mucosal (oral or intranasal) vaccine. Recombinant spores will be tested, in this study, for their ability to elicit anti-MBO cellular and humoral immune responses and in-vivo tested for bacterial killing using in a oral vaccination-challenge study design.
Animal Health Component
100%
Research Effort Categories
Basic
60%
Applied
35%
Developmental
5%
Goals / Objectives
Bovine tuberculosis (bTB), caused by Mycobacterium tuberculosis variant bovis (MBO), is one of the most damaging diseases in agriculture worldwide. The rise in BTB infected deer and cattle herds in Michigan, has led to a state of urgency due to associated animal trade and movement restrictions. Controlling the disease in animals is a primary approach to preventing/mitigating spread of MBO into and from wildlife or human populations. Attenuated Mycobacterium bovis BCG has long been used as a vaccine against TB and BTB, and a recent meta-analysis of ~1,300 studies reported a relatively low direct protection of BCG against infection, with a pooled efficacy estimate of 25%, and significant reduction in the frequency and severity of pathology of BTB in vaccinated animals but did not eliminate infection. Thus, we propose a novel subunit vaccine design around a central paradigm of microbial pathogenesis that posits that immune responses against virulence factors, such as ESAT-6, CFP-10, MarP, and Ag85b, are sufficient and effective to protect animals against MBO infection. These highly conserved immunogenic peptides/proteins from M. tuberculosis Complex will be genetically engineered into the ubiquitous soil bacterium, Bacillus subtilis, to be expressed and displayed on its spores. Recombinant spores are heat- stable, easily stored, and easily administered mucosal (oral or intranasal) vaccine. Recombinant spores will be tested, in this study, for their ability to elicit anti-MBO cellular and humoral immune responses and in-vivo tested for bacterial killing using a Matrigel system, after intranasal or oral immunization of calves.Aims: This proposal addresses a novel mucosal delivery vaccine concept through 1) testing our next generation oral subunit vaccines packaged in a probiotic bacterium to target mucosal immune responses against M. bovis in young animals, and 2) employing an in-vivo matrigel challenge system to evaluate protective immunity as the new target endpoint for defining vaccine efficacy.
Project Methods
Create and deliver recombinant B. subtilis spores expressing MTBC immunogens (EST-6, CFP10, Ag85B, MarP) orally. Four highly conserved immunogenic peptides/proteins from Mycobacterium tuberculosis Complex (MTBC) will be genetically engineered into the ubiquitous soil bacterium, Bacillus subtilis, to be expressed and displayed on its spores. Our working hypothesis is that well defined, secreted and virulence antigens of MBO are sufficient to generate robust and protective mucosal and systemic immune responses against bTB. Use of subunit vaccines also provide the option of differentiating vaccinated from infected animals (DIVA), a critical need in controlling chronic infections such as bTB. The vector engineering proposed for this aim is available through co-PI on this grant, Dr. Dhandayuthapani (Texas Tech University, El Paso). A fusion of Ag85B and CFP10 of MBO has already been constructed and is available for immediate use. In this collaboration we will develop additional recombinant constructs to present other key virulence associated peptides of MTC (ESAT-6 and MarP) on spore coats to broaden the immune responses. At completion of this aim, we expect to have developed a universal platform for oral vaccine delivery targeting bovine TB (this study) and amenable for use for TB in multiple species as well as to other infectious diseases of animals and humans.Packaging recombinant proteins on Spores of Bacillus subtilis: Bacillus spores do not require special storage and can be administered orally or intranasally. Using plasmid vectors 6 open reading frames were cloned downstream of the cotC, which encodes spore coat protein C, and the rrno of B. subtilis by our collaborator at TTU. These constructs are integrated into the chromosome of B. subtilis through homologous recombination. Strains carrying this fusion construct (first generation) have been created (and named MTB antigen 1 or MTAG1 carrying a fusion of Ag85B and CFP-10 that are expressed on spore coats, MTAG2 was constructed to express these proteins in the cytosol of vegetative B. subtilis), tested in mice. Recombinant proteins, such as ESAT-6 and MarP, can be passively adsorbed on the spores for delivery as described.Aim 2 - Determine the protective efficacy of the polyvalent mucosal delivery vaccine formulation in a subcutaneous matrigel-based challenge model. Our working hypothesis is that MBO virulence proteins delivered, as an intranasal aerosol or orally will provide maximum protective efficacy in calves. WeAnimal Vaccination-Challenge Design - Vaccination challenge experiments will be performed in Holstein bull calves (n = 10/group) randomly allocated to - spore vaccine group (oral, intranasal, and intranasal+oral groups), BCG (current vaccine control), and unvaccinated control. Oral and/or intranasal delivery of vaccine (108 recombinant spores carrying each combination of antigens/dose) or sham (vector control) or saline will be performed at weaning (Day 0). A booster dose of the vaccine or sham is performed at day 14. For the spore vaccine group, we will combine 108 spores carrying AG85B-CFP10 fusion with spores expressing ESAT-6 and MarP at the same dose and administered mucosally. Matrigel alone or an aliquot instilled with M. bovis will be administered subcutaneously behind each ear of animals, respectively, on day 28, to evaluate immune correlates of protection as described below. Blood samples are collected at baseline (day 0), day 14, 28, 31, and 40 to define circulating M. bovis-specific T-cells as well as antibody responses elicited by mucosally delivered antigens. Five animals per group will be euthanized in a staggered manner between days 31 and 40 to evaluate mycobacterial decay and immune cell populations within matrigels.A sample size of 10 animals per vaccine group is proposed to first establish correlates of protection using a novel mucosal delivery vaccine against MTBC infections. Since these studies need to be performed in a BSL3Ag facility, larger sample sizes would exceed the budgets offered by AFRI. Thus, the currently proposed study would provide sufficient biological plausibility data and aid in defining larger sample sizes based on protection conferred in the natural host, for future studies.Blood will be collected at day 0 and subsequently at weekly intervals to evaluate antigen specific T-cell responses. Matrigel with live M. bovis (Strain 10-7428 - a clinical isolate from an outbreak whose genome we have sequenced recently) or no antigen will be used as test and control in the same animal. Matrigel (with MBO and another aliquot without MBO) will be placed subcutaneously behind each ear of vaccinated and control animals to delineate T-cell trafficking, and local immunity conferred among vaccinates. Each animal, therefore, serves as its own control in terms of evaluating non-specific matrigel effects versus MBO specific immune responses elicited by mucosal vaccines. The gel is left in-situ for 72-hrs before the first set of 10 animals per group are sampled. Subsequently, matrigel is harvested for immune response evaluations.Evaluation of Immune Responses and Protection conferred: To identify correlates of protection, we will analyze functional differences in T cell subsets within matrigels, between vaccinated and control animals, and correlate these with differences in mycobacterial burden and matrigel histopathology scores of matrigels.We will evaluate ex-vivo cell mediated immunity using the standard Interferon Gamma Release Assay (IGRA) [Quantiferon Gold, Qiagen], ELISpot, cell mediated bacterial killing (ex-vivo) , and a Luminex based antigen-specific cytokine release profiles for vaccinated animals at baseline, post vaccination, post a booster dose, and finally post challenge. All cytokine assays will be targeted at antigen specific responses of the buffy coat (white blood) cells that are purified and incubated with subunit antigens (CFP-10, ESAT6, Ag85B, and MarP).Data Analysis - Antigen presentation on the bacterial spores will be tested with antigen specific antibodies applied both in-situ and in Western blots.Each animal will serve as its own control in terms of matrigel based responses. Vaccinated and control animal bacterial loads will be compared on a log scale to evaluate efficacy of bacterial killing in matrigels. ELISPOT, Quantiferon GOLD (Qiagen), and cytokine analyses will be analyzed by group using an ANOVA and repeated measures analyses. We will work in consultation with Center for Statistics (CSTAT) at Michigan State University - CVM has a an embedded statistician in for consultation and statistical analysis help.