Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens

Abstract

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.

Evaluation of vaccine delivery systems for inducing long-lived antibody responses to Dermanyssus gallinae antigen in laying hens

“…In the current study, we used a prototype vaccine antigen D. gallinae cathepsin D1 (Dg-CatD-1) (Bartley et al, 2012), and compared antigen delivery to laying hens by: (i) immunization with purified recombinant Dg-CatD-1 in adjuvant; (ii) recombinant DNA vaccination for expression of Dg-CatD-1 and (iii) oral challenge with populations of the live transgenic coccidial parasite Eimeria tenella expressing Dg-CatD-1. These delivery platforms were chosen to compare antigenspecific antibody titres in laying hens using tried and tested protein-in-adjuvant vaccination (Bartley et al, 2017), with new vaccine technologies which include DNA vaccination (Gupta et al, 2016;Meunier et al, 2016) and a novel experimental E. tenella antigen delivery system (Clark et al, 2012;Marugan-Hernandez et al, 2016;Pastor-Fernandez et al, 2018), in order to identify the optimal system. We assessed each delivery platform for the ability to induce specific anti-Dg-CatD-1 IgY in laying hens and monitor longevity of the response over a commercially relevant period.…”

Protective effects of a food-grade recombinant Lactobacillus plantarum with surface displayed AMA1 and EtMIC2 proteins of Eimeria tenella in broiler chickens

“…Apical membrane antigen 1 (AMA1) is a member of the highly conserved parasite surface proteins among apicomplexan protozoan, such as Toxoplasma gondii [23], Plasmodium and Coccidia [24]. The AMA1 is found on the sporozoite surface of E. tenella.…”

Laboratory Growth and Genetic Manipulation of Eimeria tenella

“…The first report describing the transient transfection of E. tenella employed the beta‐galactosidase gene as a reporter (Kelleher & Tomley, ), but due to ease of use all subsequent studies have opted to use fluorescent proteins. The expression of these reporters permits not only assessment of the efficiency of transfection by simple microscopic visualization, but also allows the selective isolation of transgenic (fluorescent) oocysts by fluorescence‐activated cell sorting (FACS) from progeny individuals (i.e., those obtained after infecting chickens with transfected sporozoites) and verification of the expression and localization of a protein of interest when it is fused to a reporter protein (Clark et al., ; Marugán‐Hernandez et al., , ; Pastor‐Fernandez et al., ; Yan et al., ). Notably, transfection efficiency does not seem to be affected by the choice of different fluorescent reporters (e.g., mCherry, tdTomato, mCitrine, YFPmYFP, or AmCyan) in E. tenella (Clark et al., ).…”

“…Earlier experiments carried out in T. gondii resulted in the generation of the DHFR‐TSm2m3 gene, a mutated form which codes for a version of the enzyme that is not inhibited by pyrimethamine treatment (Donald & Roos, ). Transfection of the DHFR‐TSm2m3 gene coupled with subsequent dietary pyrimethamine supplementation has been proven to be an efficient way to obtain stable transgenic populations of Eimeria parasites (Clark et al., ; Pastor‐Fernandez et al., ; Qin et al., ; Tang et al., ; Yan et al., ). Furthermore, dual selection approaches by transfection with genes coding for a fluorescent reporter and pyrimethamine resistance (either in a single or in two different plasmids) has been found to speed up the generation of stable populations (Clark et al., ; Hanig et al., ).…”

“…This approach could streamline commercial vaccine formulations from up to eight Eimeria species/strains, due to the lack of cross‐protective immunity, to just a few lines expressing relevant antigens from all other species (Blake & Tomley, ). The toolbox would also allow expression of additional antigens directed against other pathogens which affect poultry, even modifying their trafficking within the parasite to target antigen exposure to the host immune system (Clark et al., ; Marugán‐Hernandez et al., , ; Pastor‐Fernandez et al., ; Tang et al., ).…”