The recently published article describing the discovery of a MAP mutation that may lead to the development of a MAP vaccine for both animals and humans was received by our community with great interest. One of the researchers, Dr. William C. Davis, has provided Human Para with a summary of this highly technical article. A huge thanks to Dr. Davis and his colleagues for this piece, and for their contribution to MAP science.

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The long road to the development of a peptide-based vaccine for Mycobacterium avium paratuberculosis.

by Dr. William C. Davis

 

Definitions

Antigenic Component: Part of an Antigen, which is a molecular structure on the surface of pathogens that are recognized by the immune system and can trigger an immune response.

Attenuated Vaccine: A live vaccine which uses a weakened, harmless form of the germ that causes the disease.

CD8 cytotoxic T cell: A type of white blood cell that kills defective cells in the body such as cancerous cells, cells that have become infected with viruses or bacteria, or cells that are damaged in some way.

Effector T cell: The group of white blood cells that respond to a stimulus (like a bacteria). A cytotoxic T cell is one type of effector T cell.

Intracellular Bacteria: Bacteria that live inside the host’s cells in an attempt to survive and avoid destruction. Map is known to do this.

Macrophage: A type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the type of proteins specific to healthy body.

Map: Mycobacterium avium subspecies paratuberculosis.

Peptide: A small protein, composed of two or more amino acids. Examples include hormones, antibiotics, insulin and enzymes.

Prophylactic: a medicine used to prevent disease.

relA: A protein coding gene.

 

The recent publication of the article A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria is a summary report of our efforts to develop a peptide-based vaccine for Map. It has been a long road and we are close to achieving this goal.

As described in the report, there were many incremental steps to reach this stage in developing a peptide-based vaccine. Methods had to be developed to study the immune response to Map in the natural host. This was followed by development of methods to search for genes used by Map to establish a persistent infection and cause clinical disease. The search revealed that one gene, relA, is an Achilles heel for Map. Deletion of relA disrupts the ability of Map to establish a persistent infection. Analysis of the reason why Map couldn’t establish a persistent infection showed that deletion of relA allowed for the development of an immune response that cleared the infection with the mutant.

The next step required some innovation to develop methods to study the immune response to the mutant in tissue culture where it would be possible to examine the functional activity of the effector T cells responsible for clearance of the mutant. These studies revealed CD8 cytotoxic T cells develop with the ability to kill intracellular bacteria, an essential criterion for the development of a vaccine to intracellular pathogens.

The studies up to this point showed we had identified a Map mutant for potential use as an attenuated vaccine for Map. Although this was a major advance, the findings with the mutant indicated that the cytotoxic T cell response had to be directed towards a specific antigenic component of Map that allowed the cytotoxic T cells to recognize cells infected with Map and deliver a lethal hit. The ability to study the immune response to Map in tissue culture allowed us to dissect the immune response and identify the target of the cytotoxic T cells. A vaccinated steer provided a continuous source of cells to conduct the studies.

Culture of T cells from the vaccinated steer with the relA mutant stimulated multiplication of the memory cytotoxic T cells specific for relA. The cytotoxic T cells killed wild type Map present in infected macrophages without killing the macrophage.

With this tissue culture system in place we were able to determine what the cytotoxic T cells were recognizing. We were very lucky. Previous studies by one of the co-authors, John Bannantine, suggested the target of the immune response to the relA mutant might be directed towards a membrane protein, MMP. Analysis of the memory cytotoxic T cell response revealed vaccination with relA stimulated development of cytotoxic T cells specific for MMP. Culture of cytotoxic memory T cells from the vaccinated steer, with either relA or MMP, stimulated multiplication of the cytotoxic memory T cells. This exciting finding led to the most recent results. With a minor modification of the in vitro culture system, we found that it was possible to generate the entire immune response to the Map relA mutant. We then found the same cytotoxic T cells response could be elicited by MMP alone.

Although it is clear that an effective vaccination program in livestock would remove Map from the food stream and human exposure to Map, there is also a potential use of the vaccine in humans, but with a different strategy for use: immunotherapy.

Infection with Map is like infection with M. tuberculosis in that infection doesn’t always lead to clinical disease. Infection leads to a latent persistent infection under the control of the immune system. In humans, Mtb immune protection may last a lifetime with only about 10% of infected individuals developing clinical disease. This is associated with a breakdown in protective immunity. This also occurs with Map in humans also but is less well documented because of the prevalent belief that Map is not a zoonotic pathogen.

Extensive surveys have only been conducted in India where Map is endemic in livestock and present in the environment. Studies with livestock in India have shown the immune system can be boosted in animals showing clinical signs of disease by vaccination with a killed vaccine, resulting in transient reversal of disease. These findings suggest a better vaccine could provide a permanent reversal of disease by restoring the immune response to Map. An effective vaccine could also restore protective immunity in humans and reverse the progression of disease associated with Map infection. However, efficacy would have to be established before this immunotherapy would be available to the public.

The studies needed to validate the prophylactic and immunotherapeutic use of a peptide-based vaccine can be performed with cattle. For use as a vaccine, studies need to be conducted with newborn calves to demonstrate clear immunity is induced before environmental exposure to Map. For use in immunotherapy, studies need to be conducted with cattle at an advanced stage of infection where infection has disrupted protective immunity.

The results at this stage of our studies are very promising. The finding that deletion of a single gene cripples Map’s ability to establish a persistent infection led to the discovery of a peptide with potential for development of a peptide-based vaccine. Studies with the peptide in tissue culture show vaccination leads to development of immune cells that can kill Map inside macrophages, an essential requirement for a vaccine against intracellular pathogens. While this is being investigated in cattle, it could have implications for human health as well.