Performing Department
(N/A)
Non Technical Summary
Anaplasma marginaleis an intraerythrocytic rickettsial pathogen and causes the most prevalent tick-borne infection of cattle both worldwide and within the U.S. In the U.S., the major impact is episodic outbreaks in which morbidity is due to severe anemia and the mean case fatality rate has been reported at 36%. Economic losses due to anaplasmosis are estimated at ~$300 million/year. There are no vaccines currently licensed in the U.S. Those used elsewhere carry the risk of transmitting emerging pathogens and rely on the cold chain. Outer Membrane Protein (OMP) preparations and OMP complexes have been shown to induce robust protection. However, these vaccination regimes require technical expertise, a cold chain, and are not practical for large scale production. We propose to develop a vaccine against anaplasmosis based on the OMP preparations/complexes. We will test the hypothesis that the protective capacity of OMP preparations/complexes can be recapitulated with a defined set of vaccine candidates known to be present in the OMP preparations. We will deliver a cocktail of vaccine candidates as a genetic (or DNA) vaccine using a gene gun. This platform can deliver as many candidates as needed and does not rely on a cold chain. We will employ tick challenges to ensure rigorous testing of our vaccine. The project will incorporate homologous, heterologous and field challenge scenarios. We will test whether we can reduce the number of immunogens in the vaccine without losing efficacy, and whether a vaccine based on US variants will provide protection in a global context.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
Bovine anaplasmosis continues to be an unresolved problem the world over, placing a heavy burden on the cattle industry. Prevention of tick-borne diseases, such as anaplasmosis, relies on acaricides, which pose environmental hazards, are costly and ultimately will lead to the development of acaricide resistant ticks.It is well established that outer membrane proteins from Anaplasma marginale provide protective immunity, however, moving from this costly, time-consuming preparation to a vaccine has been an elusive goal due to the perceived need to reduce the number of required antigens to allow for large-scale vaccine production.We have been at the forefront of vaccine development for anaplasmosis for the past 20 years and have brought cutting edge methodologies to bear on anaplasmosis research - providing the first genome sequence for this pathogen, which brought research for this organism into the molecular age. We have performed innovative bioinformatics analyses and comparative genomic studies as well as intricate proteomic analyses that have helped to define the surface proteome. We now have a short list of vaccine candidates that are ripe for testing. To date, candidate antigens tested individually have failed to elicit protective immunity, therefore we propose to test a "cocktail" of vaccine candidates composed of 10 antigens. This moves away from the plodding, slow progress of testing individual candidates, and should also provide a robust and broadly based immune response that cannot be easily circumvented by single gene mutation after deployment. Our approach will test the hypothesis that the protective capacity of OMP preparations can be recapitulated with a defined set of vaccine candidates.We propose to use a genetic (DNA) vaccine approach as these have been shown to induce the full spectrum of immune responses, are inexpensive to produce, heat stable and therefore easy to deploy in rural settings.Additionally, we will employ tick challenge throughout the testing process. Tick-borne transmission is fundamentally different from needle challenge, and this is one of the reasons why apparently successful vaccine candidates have failed when tested in the field. Simulating natural challenge conditions is the best way to test protection, therefore this is an important aspect of the plan.Our strong collaboration with the University of Pretoria will allow us to assess our vaccine formulation in 1) field trials, 2) in heterologous challenge situations and 3) in geographically distant locales to determine how universally relevant our vaccine will be.Our goals will be achieved with the following specific aims:Aim 1: Vaccine trial with homologous challenge.In this aim, we will use a gene gun to immunize cattle with our vaccine candidates and do tick challenges with the same strain of A. marginale that the vaccine candidate came from.Aim 2: Vaccine RefinementIf we achieve robust protection, we will determine if we can use fewer antigens and achieve the same result. If robust protection is not achieved, we can augment the vaccine cocktail with additional antigens and/or adjuvants and test for protection as in Aim 1.Aim 3: Vaccine trial with heterologous challengeOnce we have arrived at the optimal vaccine formulation, we will repeat the immunization and challenge with a genetically different strain of A. marginale.Aim 4: Field trials to test geographic relevancyOur South African partners will clone local variants of our vaccine candidates. One set of cattle will be immunized with the SA variants and another with the US variants and we will determine if substantial differences in protection are seen when the cattle are put in the field.This comprehensive approach addresses the major constraints of developing a vaccine to prevent bovine anaplasmosis, and will result in a defined vaccine with a robust delivery mechanism that induces a potent, protective immune response that can be readily deployed in a multitude of settings - across the US and in rural smallholder farming situations.
Project Methods
The project expects to test formulations of a genetic vaccine for A. marginale.Outer Membrane Protein preparations of A. marginale are known to be protective, however, these are not a good vaccine as they are difficult to prepare, expensive, and require the cold chain.Aim1: A cocktail of endotoxin free DNA for 10 genes mixed in equimolar amounts will be delivered by gene gun once every three weeks three times. Sera (10 ml blood draw) will be collected after each immunization, and western blots will be used to verify that the cattle have an antibody response to each of the genes/proteins in the immunogen. Three weeks after the last immunization, the cattle will be challenged. A group of sham immunized animals will be used as a negative control, and OMP immunized animals will used as a positive control. No adjuvant will be used. At peak parasitemia after challenge, blood will be collected for RNASeq analysis for immune correlates of immune protection.Aim 2: This aim allows for vaccine refinement - if we have good results in aim1 - can we achieve the same with fewer candidates?If we have poor results in aim 1, can we adjust the vaccine delivery. We can use information from Western analysis performed in Aim 1 - which immunogens stimulated good responses, which did not. We can add additional candidates to the cocktail, and or remove candidates. We can add an adjuvant guided by the RNASeq data. Once the cocktail has been adjusted, immunization schedule will be the same, perhaps with an adjuvant added. Adjuvants that may be used in this aim include DNA encoding proteins that will be delivered with the vaccine candidates or monophosphoryl lipid A (MPL; L8695, Sigma) to be applied topically to the site of gene gun inoculation.Aim 3:This aim tests performance in heterologous challenge. Immunization and challenge strategy will be the same as in aim 2 except with a different strain of A. marginale.Aim 4: In this aim cattle will be immunized as inaim 2, however, 1 set will be immunized with South African versions of the vaccine candidates and another set will be immunized with the US version of the candidates. The cattle will be released on an experimental farm on the University of Pretoria campus and followed to see if and when they become infected with A. marginale. The two sets of vaccine candidates (local vs US) will be compared for efficiacy.