ANAVAC - A combined tissue culture and reverse vaccinology approach to develop a vaccine against Anaplasma phagocytophilum in sheep.

Anaplasma phagocytophilum causes tick-borne fever (TBF) in small ruminants and the bacterium has been detected in several mammalian species, including humans. In Norway, approximately 850.000 sheep are exposed to ticks on pastures annually, of which 500.000 are lambs. Estimates show that approximately 300.000-400.000 lambs are infected with the bacterium each year. Lambs and sheep that have not been exposed to the bacterium are especially vulnerable to become severely sick. The main disease problems associated with TBF in ruminants are large economic- and welfare implications. The bacterium suppresses the immune system of the animal so that secondary infections may follow, and result in losses from deaths and crippling. Vaccines against A. phagocytophilum are currently not available. Long-acting and broad-spectrum antibiotics have traditionally been used in treatment and prevention of TBF. However, there is a growing concern for the environmental safety and human health associated with antibiotic use and resistance in veterinary medicine and agriculture. Thus, new approaches for preventing tick-borne infections are needed. The current project have worked to vaccine candidates against TBF in sheep. The project aimed to identify vaccine candidates by studying the immunizing effect of bacterial surface proteins produced in recombinant E. coli, live whole cell bacteria and genetically manipulated organisms expressing protective proteins. The efficacy of different vaccine candidates were evaluated based on their ability to produce a measurable immunological response upon vaccination in sheep and their safety in vaccinated animals. In addition, the project studied pathogen-host interactions by analyzing the immunological interplay between the sheep and the bacterium. This was explored using global RNA sequencing technology and bio-informatics analyses. The project conducted two animal experiments. The first animal trial was conducted in the fall of 2016 in which surface proteins from Anaplasma phagocytophilum were enriched in E. coli cultures before injected as a vaccine to lambs. After two vaccinations, the lambs were inoculated with live wild type Anaplasma. Blood samples were taken regularly during the trial and the clinical response to vaccination and infection was closely monitored. The results indicate a serological response to the proteins. However, there are no convincing indications that the proteins are protective against infection with wild bacteria. This is interesting and important since the specific role of the selected proteins are less likely to have a function in the host immunity than earlier expected. Other studies have shown that proteins creating a strong serological immune response may not be important for protection against infection. This is especially so for surface proteins that undergo antigenic variation in the duration of a persistent infection. The second animal trial was carried out in the fall of 2017. Genetically modified Anaplasma, expressing fluorescent proteins, were used as live vaccines in lambs. The genome of the transformed organisms have been sequenced to localize the inserted genes. When a new gene is inserted somewhere in the bacterial genome, it will interfere with other genes and may ultimately weaken the bacterium. This process of attenuation may make it suitable in a vaccine trial because the bacterium expresses most of the important surface proteins as the wild type strain does, but may be less virulent. The results of the live bacterial vaccine trial indicate that the bacterium was not particularly attenuated by the mutation process as it gave a fairly typical disease episode to the vaccinated lambs. Thereafter the lambs responded by a new disease sequel after challenge, indicating that the vaccination did not result in a protective immunity. One group was vaccinated with the wild type bacterium and then challenged 28 days later with the same bacterium. This group showed clinical immunity, meaning that they did not develop clinical symptoms when they were re-infected. This was in spite of a bacteremia being present. The reason why the mutants did not provide the same clinical immunity even though they originated from the same bacterial wild type bacterium, is not known, but they were grown in cell cultures deprived of any stimulation by immune systems. The wild type bacterium was propagated in a sheep. Further, the project studied the host-pathogen interactions of Anaplasma in lambs. RNA (ribonucleic acids) are codes to how the organism responds to a stimulus. Thus in the case of an infection with Anaplasma, the lamb will respond by activating genes that are important in the cascades of immune responses. By studying RNA and gene expression, we identifed important molecular interactions between the bacterium and the host, which will guide us in new vaccine developing strategy.

The project had international and interdisciplinary collaboration between Norway, Sweden and the United States within the field of Rickettsial vaccinology. Exchange of people and knowledge has been one primary goal of this project, that has been achieved with great success. One PhD student visited two of the most experienced research groups in the field across the world in both Florida and Minnesota. One leading researcher from Florida visited Norway to assist in inoculation of lambs with mutated bacteria. In addition, several physical visits have been made between Norway and Sweden and the results of the project has been presented at international conferences in Europe and the US. The consortium has earned international attention through the project and interesting potential collaboration has been established parallel to the project. Skills and knowledge related to vaccinology of Anaplasma has been gained that will be basis for future projects in collaboration between nations.

The tick-borne rickettsia Anaplasma phagocytophilum has been detected in several mammalian species, including sheep and humans. In Norway approximately 850.000 sheep are exposed to ticks on pastures of which 500.000 are lambs. Estimates show that approximately 300.000-400.000 lambs are infected annually The main disease problems associated with TBF in ruminants are seen in young animals and result in large economic and welfare implications due to immunosuppressive effect of the bacterium which is accompanied by secondary bacterial infections. Estimates have shown that the disease leads to total indirect losses of more than 30 mill. NOK annually. However, the total cost due to TBF in Norway may be much higher due to direct losses from deaths and crippling staphylococcal infection. Vaccines against A. phagocytophilum are currently not available. Prophylaxis is mostly done by a variety of pour-on applications and long-acting tetracycline has also been used as a prophylactic measure. However, there is a growing concern about the environmental safety and human health, rising antibiotic resistance in target- and non-target bacteria, increasing costs of chemical control and the increasing resistance of ticks to pesticides. The main knowledge need is to find bacterial components that can be used to develop an effective vaccine against the higly virulent and prevalent strains of A. phagocytophilum in Norwegian sheep production. The project will identify vaccine candidates by studying the immunizing effect of live whole cell bacteria, as well as recombinant organisms expressing protective protein subunits, using a reverse vaccinology approach. The efficacy of different vaccine candidates will be evaluated based on their ability to produce a measurable immunological response upon vaccination, their safety in vaccinated animals, and their ability to prevent colonization and persistence of the organism upon challenge. The project will apply global RNA sequencing as a method to study the host-pathogen interface using mutated organisms and wild type bacteria as exposure factors. The goal is to detect changes in the gene expression related to the animal immune system when meeting a bacterium that is authentic or mutated. By studying the expression profiles, the goal is to find genes that are either up-regulated or down-regulated as a response to the genome structure in the bacterium. If a genetic change in the bacterium is able to shift a part of the immune system of the host in either direction, we may be able to manipulate or construct an antigen that can be used as a vaccine candidate with a known function.