A NOVEL PROBIOTIC BASED ORAL DELIVERY VACCINE FOR BOVINE TUBERCULOSIS

Project Methods
Bovine TB (bTB) is one of the most damaging diseases in agriculture worldwide. It is a zoonosis caused byMycobacterium tuberculosisvariantbovis(MBO), which primarily infects cattle and other warm-blooded animals. In the US, white-tailed deer serve as a wildlife reservoir for this disease. The rise in bTB-infected cattle herds and deer population has created a state of urgency in the last few years to Michigan agriculture, causing its status to be changed by the US Department of Agriculture from TB-accredited free zone (AFZ) to modified accredited zone (MAZ) that includes Alcona, Alpena, Montmorency, and Oscoda counties since October 13th, 2014. This change has led to restrictions imposed on domesticated animal movement from the endemic region of MI to other areas in the state or across state borders unless the cattle are being moved directly to slaughter or have a bTB-free certification. As of March 2021, 81 herds had been identified to be infected with bTB. The situation has become even more concerning since the 81stherd was identified outside the MAZ.Critically, controlling the disease in agricultural animals is a primary approach to preventing/mitigating the spread of MBOto and from wildlife and/or human populations.It is estimated to cost $1.5 million if bTB is detected in a herd of 1000 animals[13]. With >12,000 herds at risk in Michigan alone, the risk of infection is an economic threat for the farmers and the USDA, in addition to the emotional and psychological damage associated with financial and animal losses. Bovine TB is endemic in Michigan and has been maintained in white-tailed deer since first identified in 1975. Historically bTB circulating in the deer has been transmitted to those who come in close contact with the deer like hunters, venison-farmers, and consumers[14-16].BacilleCalmetteGuerin (BCG), an attenuated strain ofMBO, is the primary vaccine used for TB in humans and is effective in neonates. BCG elicits an immune response that does not always correlate with protection against progression to clinical TB in adults. Comparative genomics and deletion-complementation studies place the attenuation defect of BCG on theRD1deletion – a concept that has been garnered for differentiating infected from vaccinated animals. The RD1 deletion, however, essentially eliminates 2 major T-cell antigens that are highly conserved across theMycobacterium tuberculosisComplex (MTBC) organisms and associated with robust T-cell immunity against TB. Further,studies using BCG in animals show low protective efficacy in high-prevalence areas of the world[17]. While BCG reduces clinical disease and to some extent the severity of lesions in a proportion of vaccinated animals, it does not affect the shedding or transmission of MBO.Arecent meta-analysis reported arelatively low direct protection of BCG against infectionwith a pooled efficacy estimate of 25%, and a significant reduction in the frequency and severity of pathology of bTB in vaccinated animals but did not eliminate infection[18].An effective vaccine that blocks MBO infection and effectively interrupts the transmission of TB among infected deer (or other wildlife) and cattle is urgently needed.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 againstM. bovisin young animals, and 2) employing an in-vivo matrigel challenge system to evaluate protective immunity as the new target endpoint for defining vaccine efficacy.Aim1-Create and deliver recombinantB. subtilisspores expressing MTBC immunogens (EST-6, CFP10, Ag85B,MarP) orally. Fourhighly conserved immunogenic peptides/proteins from MTBC will be genetically engineered into the ubiquitous soil bacterium,Bacillus subtilis,to be expressed and displayed on its spores.Our workinghypothesisis that well-defined, secreted virulence antigens of MBO will generate robust and protective mucosal and systemic immune responses against BTB.The use of subunit vaccines also provides 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;Co-PI on this grant) via a recently signed MTA. His group has already created a fusion construct with Ag85B and CFP10 of MBO that isavailable for immediate use. In this collaboration, we will develop additional recombinant constructs to present other key virulence-associated peptides of MTBC (ESAT-6 and MarP) on spore coats to broaden the immune responses. No problems are expected in the completion of this aim given the combined team experience in engineering such recombinant strains.Aim2 -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.We have extensive animal and vaccine delivery and immune response evaluation expertise[19-21]that will help inform the work proposed under this aim. The matrigel-based system proposed herein is an established method and is an optimal and objective approach to evaluate local and circulating immunity and immune cell populations to identify specific correlates of protection. All in-vivo vaccination-challenge experiments and ex-vivo MBO killing work will be done at the BSL3Ag containment facilities at the University of Missouri (Laboratory of Infectious Disease Research facilities).