Towards Innovative Design and Application of Recombinant Eimeria as a Vaccine Vector

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis.

ABSTRACT

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.

INTRODUCTION

Protozoan parasites are responsible for causing a number of serious medical, veterinary, or zoonotic diseases, such as malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis, trypanosomiasis, neosporosis, babesiosis, and coccidiosis (1–8). Eimeria-caused coccidiosis produces enormous economic losses in poultry, rabbits, and other livestock industries (1, 9). Live oocyst-based anticoccidial vaccines are among the most successful vaccines against infectious diseases in animals. Chickens immunized with either a virulent or attenuated live oocyst-based coccidiosis vaccine develop durable protective immunities (10, 11). Furthermore, an Eimeria-based vector carrying antigen-coding gene(s) from another pathogenic organism that has the ability to trigger protective immune responses will greatly enhance the prevention and control of animal infectious diseases.

Vaccines represent a powerful tool for disease prevention, and the way they are delivered has a critical influence on antigen recognition and vaccine efficacy and safety. Biological delivery systems include attenuated and avirulent recombinant viral, bacterial, or protozoan vectors capable of delivering protective antigens, some of which have been commercialized. Advantages of Eimeria parasites as live vector vaccines include but are not limited to the following: (i) self-limited infection and strict host specificity of Eimeria parasites ensure high biosafety when applied as a vaccine vector (12, 13), (ii) Eimeria parasites are highly immunogenic pathogens which stimulate broad immunity, including serum antibody response, intestinal secretory antibody response, and cell-mediated immune response (CMI) (14), (iii) Eimeria parasites have relatively large genomes of between 55 and 60 Mbp, carrying 8,000 to 9,000 genes, and offer a relatively large capacity for carrying heterologous antigen genes (15, 16), (iv) Eimeria parasites are eukaryotes with the ability to maintain the native structure and immunogenicity of the heterologous antigens through posttranslation modification (17), (v) Eimeria-based vaccines can be delivered orally without losing their immunogenic potential (18), and (vi) the heterologous antigen specific immune responses could be automatically boosted and enhanced with repeated immunization of recombinant Eimeria via the fecal-oral route (1, 10, 11).

The first and essential step for developing Eimeria as a vaccine vector is to establish an effective transfection and selection system, which requires passing the following hurdles: (i) the parasite could hardly complete its life cycle in tissue culture in vitro, and (ii) the bioinformatics of Eimeria parasites are incomplete compared with Toxoplasma and Plasmodium.

Although all viral, bacterial, and protozoan vectors can be used to deliver heterologous antigens, this review focuses on the use of protozoan vectors, recombinant Eimeria as vaccine vectors in particular, which has been a topic of ongoing research in our laboratory. It is our belief that recapitulation of the key milestones in the establishment of Eimeria transfection platform and its application will help streamline the development of a parasite-based vaccine delivery vector in the future.

TRANSIENT-TRANSFECTION SYSTEM

In apicomplexan parasites, such as Toxoplasma and Plasmodium, a transfection system was established in the early 1990s (19, 20), but owing to the difficulty of completing the life cycle of Eimeria in vitro and the lack of bioinformatics, genetic manipulation of Eimeria has lagged behind (1). A preliminary study of genetic manipulation of this parasite was reported by Kelleher and Tomley in 1998, which utilized β-galactosidase (β-gal) as the reporter enzyme (21). This work demonstrated that the sporozoite stage of Eimeria parasites can allow plasmid-mediated transfection and successfully drive exogenous lacZ gene expression under the control of the Et mic-1 promoter (21). Since then, the transfection work of Eimeria was stalled for nearly 10 years due to the limited sensitivity of the 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) detection system. The introduction of the fluorescent proteins as reporters in 2007 hastened the development in this field (22).

Faithful expression of a heterologous gene requires adequate 5′ and 3′ flanking sequences that are derived from Eimeria or its closely related Toxoplasma gondii genes (23). The monocistronic nature of transcription in Eimeria and Toxoplasma facilitates the identification of promoter elements that are usually in close proximity to the transcription start site. Numerous promoters suitable for driving reporter or exogenous gene expression from both Eimeria and Toxoplasma are currently available (23–26). They can be chosen appropriately according to the purpose of the application, as they exhibit a diverse range of strength and stage specificities (25, 27, 28). It is thought that a strong promoter, e.g., SAG13 (surface antigen 13) or His4 (histone protein 4), will be suitable when using Eimeria as a vaccine vector carrying heterologous antigens for eliciting an efficient immune response (29, 30). The 3′ untranslated region (3′ UTR) is very important in regulating exogenous gene expression in genetically modified organisms, although the mechanism is still unclear. It has been confirmed that an Actin, His4, Mic2 (microneme protein 2), or SAG13 promoter efficiently triggers reporter gene expression with the 3′ UTR derived from the Actin gene. The final expression pattern of the exogenous gene in the transgenic parasite is a result of multiple factors, including the promoter, signal sequence, and property of the gene product (Fig. 1). For example, the reporter enhanced yellow fluorescent protein (EYFP) fused with the signal sequence of dense granule antigen 8 (GRA8) of T. gondii was mainly secreted to the parasitophorous vacuole (PV) in Eimeria tenella controlled by the Actin promoter (25, 31).

FIG 1.

Tagging subcellular compartments with fluorescent protein markers in Eimeria. Schematic drawing of an intracellular parasite with the subcellular structures and organelles which are successfully tagged with fluorescent protein markers (black) or not (gray). Micronemes: EYFP fused with E. tenella microneme protein 1 (EtMic1) signal sequence (Mic1ss-EYFP) (18), mCherry fused with EtMic2 (Mic2-mCherry) (58), or mCherry fused with EtMic2 signal peptide (SP2-mCherry) (59); dense granules: YFP fused with T. gondii dense granule protein 7 (GRA7-YFP) (26). *, the YFP signal is concentrated in small particles of the sporozoites when they are extracellular. However, whether the small particles are dense granules still needs to be further confirmed. PV: RFP fused with T. gondii dense granule protein 8 signal sequence (GRA8ss-RFP) (25) or YFP fused with P. falciparum repetitive interspersed family protein (RIFIN) signal sequence (RIFINss-YFP) (31); nucleus: YFP fused with E. tenella histone protein 4 nuclear localization sequence (nls-YFP) (31, 60); outer membrane: mCherry fused with E. tenella microneme protein 2 signal peptide and surface antigen 1 GPI-anchor sequence (SP2-mChe-GPI) (59).

Electroporation is one of the most efficient methods for introducing exogenous DNA or RNA into the cell. For Eimeria, transfection of sporozoites or merozoites has been achieved by electroporation with circular or linear plasmids or PCR products (21, 24, 32, 33). Earlier research on the transient transfection of Eimeria was conducted by using complete cytomix buffered with a closed circular plasmid electroporation system, but its efficiency was low (21, 24). The efficiency is improved about 200 times after transfection with a linear plasmid in the presence of a restriction enzyme, termed restriction enzyme-mediated integration (REMI) (33). A very high transfection efficiency was achieved in chicken, rabbit, or rodent Eimeria sporozoites or merozoites by using the AMAXA nucleofection system with REMI (32–35). The expression of reporter fluorescent protein in transfected sporozoites and the transfection efficiency can be easily monitored and counted using a fluorescence microscope after culturing in primary chicken kidney cells or Madin-Darby bovine kidney (MDBK) cells in vitro. Thus, the transient-transfection system of Eimeria provides an efficient platform for selecting and optimizing the promoter, signal sequence, or other regulators and for the subsequent establishment of the stable transgenic Eimeria constructs (22–24, 36, 37).

RECOMBINANT EIMERIA SELECTION SYSTEM

Transfected sporozoites (E. tenella and Eimeria mitis) or merozoites (Eimeria necatrix) were inoculated into chicks via the cloaca (24, 28, 32) or intravenous injection route (Eimeria acervulina) (unpublished data) depending on the location where the sporozoites or merozoites invade the intestine, ceca for E. tenella and E. necatrix versus duodenum for E. acervulina. Inoculation into the intestine of the transfected sporozoites of rabbit coccidia via operation is also an efficient way to obtain the transgenic parasites (34, 38). Recombinant Eimeria oocysts expressing reporter fluorescent protein can be obtained after several generation selections in vivo using fluorescence activated cell sorting (FACS) (24, 37, 39). Many factors are known to limit efficient production of recombinant Eimeria oocysts with FACS only. A mutant T. gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3, TgDHFR)-based pyrimethamine resistance selection system allows the highly efficient selection of recombinant Plasmodium and Toxoplasma (39, 40). In combination with FACS, this drug selection system greatly enhances recombinant Eimeria selection (24, 37). Two strategies are used for transgenic parasite selection. One strategy is transfecting the sporozoites with two plasmids each containing TgDHFR or fluorescent reporter expression cassette (37); the low efficacy of cotransfection of Eimeria sporozoites with two plasmids partly limits its routine application. To overcome this limitation, the other strategy utilizes fused expression of TgDHFR and fluorescent reporter in one expression cassette (41). More than 90% of a transgenic Eimeria population can be obtained within 5 generations using the one plasmid system (41, 42). Sporocyst selection by the one plasmid system further improves the selection efficiency (24, 43). An uncertain proportion of transgenic negative sporocysts often appears among the 4 sporocysts in the transgenic-positive oocysts (24), which may help explain the reduced effects of negative sporocysts using a sporocyst-based selection system (24, 43).

After several generations of selection, the recombinant Eimeria parasites usually stably express exogenous gene(s). The exogenous plasmid is successfully integrated into the genome of the parasites, although the integration sites are variable in one transgenic population, as revealed by genome walking or plasmid rescue method (28, 37). Only one integration site was analyzed in the transgenic population obtained with double pressures, drug and FACS, and the results indicated that the transgenic population tends to stabilize after continuous selections (42). One integration site was also examined in the single oocyst progeny of transgenic Eimeria, and the reporter gene was stably expressed without the selection pressures (27). Moreover, only one copy of exogenous DNA was identified by Southern blotting in the genome of the single sporocyst clone progeny (44). The various integration sites of exogenous plasmids in the genome of different recombinant Eimeria lines possibly resulted in the difference in fecundity, i.e., transgenic E. tenella TE1 showed reduced fecundity (37), whereas EtM2e, another transgenic Eimeria line, showed 2-fold increase in fecundity compared with its parental strain (44). The capacity of heterologous pathogen antigen expression in the recombinant Eimeria is the first and critical criterion for developing Eimeria as a novel vaccine delivery vehicle (Fig. 2). The expression of a reporter gene or gene of interest (GOI) in recombinant Eimeria can be detected by fluorescence microscopic observation or immunofluorescence assay and Western blotting. The reporter gene is constitutively expressed throughout the life cycle of the recombinant Eimeria driven by the His4 or Actin promoter but not in the unsporulated oocyst stage when driven by the Mic2 or SAG13 promoter (25, 27). Similar expression patterns of GOI, e.g., immune mapped protein 1 of Eimeria maxima (EmIMP1), with reporter regulated by the same elements, were detected by Western blotting and indirect immunofluorescence assay (29). In addition, the overexpression of heterologous IMP1 did not interfere with the expression and distribution of its parental homologous IMP1 (29).

FIG 2.

The scheme of recombinant Eimeria production and the use of Eimeria vectors to vaccinate against heterologous pathogens. (I) Construction of recombinant Eimeria stably expressing heterologous antigen(s). Immunodominant or immunoprotective antigen genes (GOIs) are amplified from cDNA, which is synthesized according to the pathogens’ mRNA. In some cases, the GOIs are optimized according to the Eimeria codon preference. The genes are subsequently cloned into a shuttle plasmid which contains the elements for replication and selection in E. coli, whereas the flanking sequences that regulated GOI and selection gene (drug resistance and/or fluorescent genes) expression in Eimeria, e.g., promoter, 3′ UTR and signal sequence, are derived from apicomplexan parasites’ gene(s). The wild-type Eimeria sporozoites or merozoites are transfected with linearized shuttle plasmid using REMI. Heterologous antigen expressing recombinant Eimeria can be obtained after continuous drug and/or FACS selection in vivo and can be identified and analyzed by PCR, genome walking, Western blotting (WB), and indirect immunofluorescence assay (IFA). (II) Immunities elicited by the recombinant Eimeria. Heterologous antigen-specific humoral, cellular, or mucosal immune responses can be detected and analyzed by enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT) assay, real-time PCR, and intracellular cytokine staining. (III) Protection provided by the recombinant Eimeria against target pathogens. Recombinant Eimeria parasite-immunized hosts are challenged with the target pathogen, and the protection can be analyzed by clinical symptoms, pathogen loads, and survival rates.

EXOGENOUS ANTIGEN-SPECIFIC IMMUNE RESPONSES

The heterologous pathogen antigen expressed by the recombinant Eimeria delivered to and recognized by the host immune system represents the second criterion for developing Eimeria as the vaccine vector (Fig. 2). Using the EYFP as a model antigen, EYFP-specific lymphocyte proliferation and interferon gamma (IFN-γ) expression in CD4 and CD8 T cells were detected in the transgenic Eimeria-immunized chickens (18). A higher level of EYFP-specific lymphocyte proliferation and a stronger IgA production were detected in the transgenic line expressing microneme-targeted EYFP immunized birds than those expressing cytoplasm-targeted EYFP (18). These findings indicated that the compartmentalization of heterologous antigen in recombinant Eimeria affects the immune responses and should be a considering factor for constructing recombinant Eimeria vaccines. Various designs that target the heterologous protein to different cell organelles or the sporocyst wall (41) in the recombinant Eimeria have been described (Fig. 1). However, due to the lack of antibodies against proteins from specific organelles (e.g., rhoptries and dense granules), the acute localization of foreign proteins is difficult to determine by colocalization experiments. For example, the YFP fused with T. gondii dense granule protein 7 (GRA7-YFP) is expressed in PV when the parasite is intracellular and in “small particles” when extracellular, which is predicted to express in the dense granules (26). Whether the small particles are dense granules still needs to be further confirmed because the antibodies and structural evidence of dense granules of Eimeria sporozoite are not available.

Immunodominant or immunoprotective antigens from viruses, bacteria, or protozoa expressed in the recombinant Eimeria are recognizable by the host immune system and trigger immune responses. For example, immunization with recombinant Eimeria expressing the VP2 protein from infectious bursal disease virus or glycoprotein I from infectious laryngotracheitis virus induced low titer, transgene-specific antibodies in chickens (45). Recombinant Eimeria expressing surface antigen 1 of T. gondii (Et-TgSAG1) elicited TgSAG1-specific IgY antibody production and IFN-γ-secreting peripheral blood mononuclear cell proliferation after a single oral immunization in chickens (30). Interestingly, intraperitoneal immunization of Et-TgSAG1 sporozoites efficiently triggered the humoral immune response with predominant IgG2a production in mice, and the antisera recognized the native SAG1 in T. gondii tachyzoites (30). Parallel experiments showed that recombinant E. tenella expressing EmIMP1 elicited EmIMP1-specific antibody production, the titer of the antibodies was increased after a booster immunization, and these antibodies showed reactivity with native EmIMP1 antigens. E. maxima solubilized protein extracts that enhance specific IFN-γ-secreting peripheral blood mononuclear cell proliferation were also detected in recombinant Eimeria immunized birds (29). In contrast, immunization with recombinant Eimeria expressing another immunoprotective antigen, apical membrane antigen of E. maxima (EmAMA1), showed variable antibody reactivity against recombinant EmAMA1 protein but did not show reactivity against native EmAMA1 in E. maxima solubilized protein extracts (46). However, immunization with this recombinant Eimeria induced low levels of serum interleukin 10 (IL-10) production which showed positive correlation with reduced oocyst output (46).

PROTECTION AGAINST HETEROLOGOUS PATHOGEN

The third and final criterion for developing Eimeria as a vaccine vector is that the recombinant Eimeria can elicit high-level protection against heterologous pathogen infections (Fig. 2). The protection provided by different recombinant Eimeria lines against heterologous pathogens was evaluated. In the study of using E. tenella as the vaccine vector expressing Campylobacter jejuni vaccine candidate CjaA, single or serial oral vaccination of specific-pathogen-free (SPF) chickens with the E. tenella-CjaA induces 91% and 86% immune protection against subsequent C. jejuni challenge infection compared with unvaccinated and wild-type E. tenella vaccinated birds (43). Immunization with recombinant Eimeria expressing TgSAG1 efficiently protects chickens against T. gondii infection, significantly reducing inflammation in the peripheral immune organ compared with naive and wild-type parasite-immunized birds. Moreover, Et-TgSAG1-immunized mice show a prolonged survival time (24 to 96 hours) compared with naive and wild-type parasite-immunized mice after challenge with virulent T. gondii tachyzoites (30). Immunization with recombinant Eimeria expressing either EmIMP1 or EmAMA1 can reduce oocyst production after the heterologous E. maxima challenge (29, 46). The protection against heterologous pathogen infection was enhanced when coimmunized with double-recombinant Eimeria lines (47). Use of Eimeria as vaccine vector expressing immunoprotective antigens from heterologous Eimeria species represents the first instance of cross-protection against different Eimeria species by immunization with one species of Eimeria and opens a new avenue for developing coccidiosis vaccines.

MODIFICATION OF EIMERIA FOR USE AS A VACCINE VECTOR AND BEYOND

Members of the Eimeria genus are major causative agents of coccidiosis in a wide range of livestock and birds. While the vaccines consisting of live oocysts have proven a great success against coccidiosis in chickens and turkeys, there are never-ending attempts to decrease their pathogenicity but retain their immunogenicity and to explore their potential as vaccine vectors.

One traditional method for vaccine production involves controlled passage of the first oocysts produced during infection in chickens and repeated selection (48). This has been the most efficient way for producing live-attenuated vaccine strains of Eimeria (so called “precocious” lines) (48). This method has broad utility with Eimeria parasites in avian and rabbits. The precocious parasite shows a marked attenuation of virulence compared with the parent parasite, but each precocious line has a significantly lower reproductive capacity (49).

Another traditional method for producing attenuated vaccine strains is through serial passage of Eimeria parasites by inoculation of in vitro-released sporozoites into the allantoic cavity of embryonated chicken eggs. This strategy commonly results in a line that is significantly less pathogenic for chickens than wild-type strains (50). However, to date, only E. tenella has proven efficacious in producing the attenuated egg-adapted line (14, 49). The two traditional methods efficiently reduce the pathogenicity of the Eimeria parasites and maintain their immunogenicity compared with their parental parasites; however, the fecundity of the attenuated lines is significantly reduced. This undoubtedly increases the cost for developing coccidiosis vaccines as well for developing Eimeria as vaccine vectors based on the attenuated strains.

Enhancing the immunogenicity of the low or intermediate immunogenic Eimeria species (e.g., E. necatrix and E. tenella) and reducing their pathogenicity via genetic modification offer a promising approach for developing the next-generation coccidiosis vaccines. The modified Eimeria strains are also useful as vaccine vectors because of their increased efficiency and safety. Molecular adjuvants, such as cytokines and immune ligands, expressed by vaccine vectors or coimmunized with recombinant or DNA subunit vaccines efficiently improve the protective immunity compared with those without modification by the adjuvants (51, 52). Several studies have shown that molecular adjuvants expressed by the Eimeria vector significantly enhance the immunogenicity of recombinant Eimeria. The type of molecular adjuvants may sometimes influence the nature of immune responses. For example, secreted chicken IL-2 continuously expressed by recombinant Eimeria throughout the life cycle enhances parasite soluble antigen-specific cell-mediated immunity, whereas IL-2 elicits no or only slight effects on humoral immunity (42). Consequently, immunization with the IL-2-expressing Eimeria improves the protection of chickens against subsequent wild-type parasite infection (42). Recombinant Eimeria-expressing immunoglobulin Fc fragment with surface display that is fused with the glycosylphosphatidylinositol (GPI) anchor at its C terminus also enhances the protection of chickens against wild-type Eimeria infection (53). It was found that antigen derived from immunogenic species of Eimeria can serve as an adjuvant enhancing the immunogenicity of other Eimeria species, i.e., recombinant E. tenella expressing E. maxima profilin increases the immunogenicity of E. tenella, which elicits stronger cell-mediated immunity and provides better protection against E. tenella infection (54). All these findings indicate that it is feasible to genetically modify Eimeria for use as a novel vaccine vector and a safe and efficient coccidiosis vaccine.

PERSPECTIVES

The genetic manipulation of an apicomplexan parasite, namely, members of the Eimeria genus, and its application as vaccine vectors or in biological studies have advanced by leaps and bounds in the last decade. To date, a transient-transfection and stable-selection system has been successfully developed in chicken, rabbit, and rodent Eimeria. Although laboratory studies have yielded promising results and confirmed feasibility, there is still some way to go before coccidia can be used as an efficient vaccine vector or commercial vaccine. The challenges for developing Eimeria as vaccine vectors include, but are not limited to, the following: (i) the in vitro culture system of Eimeria remains inefficient; (ii) the transfection efficiency of Eimeria is still much lower than that of other apicomplexan parasites, such as Toxoplasma or Plasmodium spp., let alone bacteria and mammalian cell lines; (iii) the complex mechanism of host immunity against Eimeria infection and reinfection is not fully understood; and (iv) the immunoprotective antigen(s) of Eimeria that elicits high-level protective immunities remains to be identified.

While a transient and stable transfection platform has been successfully developed in four Eimeria species of chicken (E. tenella, E. mitis, E. necatrix, and E. acervulina), current work centers on using E. tenella as vaccine vector expressing a heterologous antigen. Partial protection against a heterologous pathogen can be provided by recombinant E. tenella expressing the target antigen. As various Eimeria species parasitize on different sites of the intestine, specific antigens expressed by the four or more Eimeria species and coimmunization with the mixed recombinant Eimeria species offer a promising way for improving the protection against target pathogens (47). Thus, establishment of the transfection platform on other species, especially E. maxima, the most immunogenic species among chicken coccidia, is extremely urgent.

Genetic editing in Eimeria represents another bottleneck. Our recent studies confirmed the CRISPR/Cas9 system efficiently mediated target gene deletion and insertion (unpublished data). Using this system, virulent genes could be deleted to create a safer vaccine vector. We could also accurately link a heterologous antigen to the immunodominant antigen of Eimeria in conjugated vaccines which help improve recognition of the heterologous antigen by the host immune system.

Recombinant Eimeria as vaccine vector has many advantages and offers a promising and feasible approach for developing novel veterinary biological products. Some vectors (e.g., herpesvirus, adenovirus, and Salmonella spp.) are already in commercial use (55–57), and we can learn from these examples to better optimize the Eimeria vector for commercial application. Notable challenges are related to target antigen selection, heterologous antigen expression level, the strength and type of immune responses, and protection against heterologous pathogens for commercialization of Eimeria vector vaccines. Nonetheless, rapid development and revolution of biotechnology will help overcome these challenges, realize the potential of recombinant Eimeria as vaccine vector, and open the way for developing other vaccine vectors in animals and humans.

Biographies

Xinming Tang completed his Ph.D. at China Agricultural University in Prof. Xun Suo’s lab, studying Eimeria immunology and its application as vaccine vectors. During his graduate work, he improved the reverse genetic manipulation platform for Eimeria. He is now a postdoctoral fellow at Institute of Animal Science, Chinese Academy of Agricultural Sciences. He is currently investigating the mechanisms of sporogony of Eimeria and its potential application for developing novel vaccine strains.

Xianyong Liu received his Ph.D. degree (2009) in Prof. Xun Suo’s lab at the College of Veterinary Medicine, China Agricultural University, working on developing genetic manipulation tool for Eimeria and the application of transgenic eimerian parasites as vaccine vector. Then, he was enrolled as a lecturer of veterinary parasitology and continues research on Eimeria and Toxoplasma there. He is working on deciphering the developmental regulation mechanisms of Eimeria and Toxoplasma.

Xun Suo graduated in Beijing Agricultural University majoring in Veterinary Medicine, where he also earned his master’s degree working on Eimeria parasites of rabbits. He serves 30 years for this university (now China Agricultural University). His laboratory focuses on basic and applied research on Eimeria, Trypanosoma, Toxoplasma, and Cryptosporidium. He is the recipient of the National Distinguished Teachers. He is the President (2019 to 2023) of Chinese Society for Parasitology under China Zoological Society. His current orientation is to develop safer vaccines against coccidiosis in poultry and livestock and an oral vaccine against Toxoplasma in cats and to construct transgenic Eimeria live oocyst-based vaccine-producing and delivering vaccines against coccidiosis and other major viral and bacterial infectious diseases.