Transgenic Eimeria tenella Expressing Enhanced Yellow Fluorescent Protein Targeted to Different Cellular Compartments Stimulated Dichotomic Immune Responses in Chickens

Abstract

Eimeria tenella, one of the seven species of chicken coccidia, elicits protective immunity against challenge infection with both homologous and heterologous strains. We endeavor to use recombinant E. tenella as a vaccine vehicle for expressing and delivering pathogen Ags and investigate immune responses against these foreign Ags. In this study, two lines of transgenic E. tenella expressing a model Ag, enhanced yellow fluorescent protein (EYFP), targeted to the micronemes and to the cytoplasm of the recombinant parasites were constructed to study the impact of Ag compartmentalization on immunogenicity. The MTT assay, intracellular cytokine staining, and real-time PCR were performed to detect the EYFP-specific proliferation and effector functions of splenic lymphocytes of immunized chickens. ELISA was used to measure anti-EYFP IgG and IgA responses. The results showed that both lines of transgenic parasites stimulated EYFP-specific lymphocyte proliferation and IFN-γ expression in CD4 and CD8 T cells, whereas a higher level of Ag-specific lymphocyte proliferation was elicited by the transgenic line expressing microneme-targeted EYFP. Furthermore, this line stimulated stronger IgA response than the one expressing cytoplasm-targeted EYFP after the second immunization. Our findings are encouraging for further investigation of the effect of Ag compartmentalization in transgenic Eimeria on immunogenicity and for the development of a eukaryotic vaccine vector using genetically modified Apicomplexa parasites.

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

“…Moreover, Liu et al [11] generated parasites expressing the M2 protein of the avian influenza virus, but found no evidence of antibody or cellular responses. The only immune responses reported in chickens raised by transgenic Eimeria has been against a fluorescent reporter protein (EYFP) [23], therefore, this is the first time that antibodies against viral proteins expressed in transgenic Eimeria have been reported. The antibodies were detected by Western blotting only when sera were used at low dilution and none were positive in standard commercial ELISA kits, which use sera at higher dilutions.…”

“…Prior to conducting an in vivo challenge the consistency and magnitude of immune response stimulated by transgenic Eimeria vaccination should be improved. Options include the use of promoters capable of inducing stronger transgene expression, or addition of targeting signals for delivery to the cell surface or secretion, which could to improve the exposition of the antigen and therefore its recognition [23, 34]. Optimization of the number of parasites per vaccinating dose or transference of the technology to more fecund Eimeria species might also encourage stimulation of more significant immune response.…”

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle

“…The use of live Eimeria vaccines as vectors to express immunoprotective antigens from additional poultry pathogens could potentially add significant value by rendering vaccines multivalent and able to protect chickens against a range of different diseases. A number of studies have tested the ability of Eimeria tenella to express heterologous antigens with variable results based on the detection of specific antibodies 3, 4 or the development of immune protection against challenge 5 . Considerable work is needed to optimise antigen delivery and better characterise immune responses induced by transgenic parasites against the foreign antigens.…”

“…Secreted antigens are much more efficient in stimulating and activating both CD8 +  6 and CD4 +  7 T-cells, compared to intracellular antigens (expressed in the cytosol or in non-secreting organelles). For transgenic E. tenella expressing enhanced yellow fluorescent protein (EYFP), both compartmentalized and cytosolic EYFP induced antigen-specific cell proliferation, however, the former showed higher levels of antigen specific lymphocyte proliferation 3 , indicating that the location of the expressed antigen may be crucial for Eimeria to function as an effective vaccine vector.…”

Characterization of the Eimeria maxima sporozoite surface protein IMP1

“…Numerous reports exist in the literature on the ability of recombinant Eimeria proteins to elicit strong cellular immune responses that confer some level of protection against Eimeria challenge infection (Yin et al, 2013(Yin et al, , 2014Zhang et al, 2014). These recombinant Eimeria proteins have been generated in a variety of bacterial and eukaryotic expression vectors, and even as transgenic Eimeria, and have been delivered to the avian immune system by different routes (Huang et al, 2011). However, reproducible and complete protection against clinical parameters has not been reported to date for any recombinant Eimeria protein (McDonald and Shirley, 2009).…”