Launching the first vaccination programs for a beneficial, pollinating insect.

BACKGROUND OF THE PROJECT: We discovered how queen bees can achieve a vaccine effect in their larvae with the protein Vitellogenin: the queens stick bits of bacteria to Vitellogenin and transfer the protein to the eggs. The bacterial bits activate the innate immune system, and the larvae have disease protection when they hatch. The project studies this process and puts it to use. DELIVERABLES RELATED TO THE PROJECT DESCRIPTION: We have completed Objective 1 (Aim 1), and conclude that the worker bees of colonies are important for the queen's ability to vaccinate her larvae. Sub-objective 2 (Aim 2) asks about the significance of structural variation in the protein Vitellogenin for binding to bacteria. Our work centers on two issues: A) What genetic variation exists for Vitellogenin, and how does this variation affect the protein's structure and properties? And, B) How can Vitellogenin find bacteria and attack/attach to these? The sub-goal was completed through a PhD thesis, with the defense held February 18th 2022. Sub-goal 3 (Aim 3) focuses on vaccine development. Here, our work has provided the basis for the creation of a company named Dalan Animal Health, see https://www.dalan.com, i.e. through our former postdoc Dalial Freitak, now professor in Graz, Austria. At NMBU, we have also developed a prototype for registration modules with sensors and cameras that log data from processes in the hive, so that vaccine effects can be observed at the colony level. Master students from NMBU EIK-lab have contributed to the work (https://www.nmbu.no/eik-lab). The programming and software created for the purpose is open source, available here: https://github.com/Eik-Lab /Bee-CTRL. We have received incentive funds from NMBU to continue this innovation project in 2023. The last sub-goal (Aim 4) looks at the cost-effectiveness of vaccine and its distribution in Europe and USA. Here we have documented physiological costs of vaccination for the queen's offspring (worker bees), see article by Leponiem, Amdam & Freitak 2021, reported earlier. And, we have now completed a manuscript that reveals a social cost through weakening of queen pheromone profiles in US colonies (Orlova, Harwood, Freitak & Amdam, submitted 2022). These findings provide a more complex basis for the calculations than we envisioned. We originally planned to model benefits (bee health) and costs (production and distribution of the vaccine in various countries) with a focus on optimizing vaccine pricing. However, our findings indicate that the focus must shift to assessments of health, where benefits of disease protection is weighed against physiological and social costs, which may not be the same in Europe and the USA. This issue is now being taken on by Freitak's company, Dalan Animal Health. DELIVERIABLES RELATED TO PROJECT EXTENSION: Under Objective 2, we achieved a complete analysis of bees from 16 countries with over 540 informative individuals in which both Vitellogenin alleles could be sequenced. This is a unique dataset in structural biology and genetic biogeography that documents 121 protein variants of Vitellogenin in bees. Through the extension of the project, the former PhD student was employed as a postdoc from February to September 2022. Through this initiative, we published the article entitled "Identification of 121 variants of honey bee Vitellogenin protein sequences with structural differences at functional sites", in the journal Protein Science. From there, the postdoc worked on the data set together with Dr. Reed Cartwright at Arizona State University. They study the biogeographical distribution of alleles in light of selection on and commercial movement of bees. They have generated a database that codes Vitellogenin's sequence variants sorted by breed, geographical location, colony and individual. This database is the basis for an article that we will submit to PLoS Genetics in January 2023, called "Genetic relationship in European honey bee populations based on the informative marker Vitellogenin". As a bonus, the analysis shows a unique genetic mutation in Nordic bees. The mutation can be of interest for beekeeping in Norway and nearby areas. With this and other work as a foundation, the postdoc applied for a Mobility Grant from NFR in 2022 (not awarded, but it received the high grade 6 (excellent) for all criteria), and later also for the European Marie Sklodowska-Curie Actions Postdoctoral Fellowship with a deadline of 14 September 2022. In these applications, Vitellogenin from salmon is included to increase relevance and impact for industry and business development. This has given the postdoc unique contacts towards Scale Aquaculture through Dr. Torstein Kristensen at Nord University. We are very pleased with this development of our research, and are excited about what we can contribute! Overall, we have been very successful with this project. We are proud of our deliverables and grateful for the award!

EDUCATION: The project has built the expertise and experience of PhD students and Postdocs, thereby contributing to their successful career development: Dr. Dalial Freitak is our previous postdoc -- now associate professor at the University of Graz Austria in addition to the Founder & Chief Scientific Officer at Dalan Animal Health, Inc, Dr. Jane Ludvigsen is also a previous postdoc on the project -- now Team leader at Fürst Medical Laboratory, Oslo. Dr. Vilde Leipart is our former PhD student and final postdoc on the project. She continues her outstanding work and development in our new FRIPRO project awarded in 2022. In addition, the closing project has contributed to career development for Dr. Gyan Harwood who was PhD student at our partnering institution Arizona State University. He is now a lead Ecotoxicologist at Corteva Agriscience, USA. Likewise, the project has inspired groundbreaking work by PhD student Mateu M. Canals at our partnering institution University of Oslo. NEW FUNDING: The project has led to a new FRIPRO 2022 award, thereby facilitating the continuation of our world-leading research on Vitellogenin. Specifically, the finding of 121 protein variants of Vitellogenin during the project period was leveraged as a unique resource when building the new project application. INNOVATION: The project has facilitated the incorporation of a new company by Dr. Freitak, Dalan Animal Heath. This company is planning to launch honey bee vaccines commercially in 2023. We have also developed a prototype for registration modules with sensors and cameras that log data from processes in the hive, so that vaccine effects can be observed at the colony level. We have received incentive funds from NMBU to continue this innovation project. DATA RESOURCES: The project produced the first full-length Vitellogenin structural model. To do this, we used the novel Artificial Intelligence platform AlphaFold; leading the way for new insights into the 3D shapes of Vitellogenin molecules. Moreover, we have established the most comprehensive allele collection for any Vitellogenin-encoding gene, with 121 uniquely coding variants. Finally, our prototype monitoring system for honey bee hives generates massive data of interest to the educational mission of NMBU. Here, courses that are in need of BIG datasets for training students in complex analyses. UNIQUE NORDIC MUTATION: We have uncovered a distinctive Vitellogenin deletion mutation in the native Nordic bee (the Brown bee) with possible implications for apiculture and bee breeding in the Nordic countries. .

Vaccination programs for honey bees (Apis mellifera) is a completely novel, unprecedented effort, as there are no current or former vaccines available for this pollinating insect. Honey bee health is of enormous international concern after nearly a decade of considerable decline. Bacterial pests are a constant threat to young bee larvae that has been met with antibiotics in some countries. Several bacterial strains are now antibiotic resistant, and this treatment strategy is not sustainable. Historically, vaccination of beneficial insects like bees was deemed unfeasible since these animals lack antibody-based, acquired immune systems. Yet, our work shows how the basic, innate immune system of bees enables transfer of immune elicitors (bacterial pieces) from queens to eggs. Our preliminary data (this proposal) demonstrate that vaccination of honey bee queens with bacterial pieces triggers protective immunity in their hatching larvae. With our partners, we will make ground-breaking progress for pollinator health: We will map out mechanisms of larval immune priming in honey bees including social facilitation and specificity (Aims 1-2). We use these insights to develop effective vaccines against the most devastating larval pests of bees: American foulbrood (Paenibacillus larvae) and European foulbrood (Melissococcus plutonius) (Aim 3). And, we conduct detailed analyses of the costs and benefits of building vaccination programs for honey bees in Europe and the USA (Aim 4). This proposal, thereby, provides the launch-pad for the world's first vaccination programs for a beneficial, pollinating insect. Our work is motivated by the US Presidential Memorandum on creating a federal strategy to promote the health of honey bees and other pollinators, and by the Communication from the European Commission on bee health. It provides positive environmental impacts by improving pollinator health and by developing an alternative to the use of antibiotics in the beekeeping industry.