Lipids are an important source of energy metabolism, including fatty acids, triglycerides, and cholesterol. The tricarboxylic acid cycle and β-oxidation process are the most important ways for most organisms to obtain energy. Fatty acid β-oxidation metabolism mainly involves acyl CoA and acetyl CoA. In the process of β-oxidation of fatty acids, 3-hydroxyacyl-CoA dehydrogenase (EGR-03347) is involved in the process of removing two carbon atoms from long-chain fatty acids, and is one of the key enzymes in fatty acid metabolism. This study used EGR-03347 to lay the foundation for the successful development of a canine anti-E. granulosus vaccine.
The development of vaccines against multicellular parasites, such as E. granulosus, is challenging compared with that for single celled organisms. However, the development of bioinformatics in recent decades has contributed to the improvement of the development of parasite vaccines, and immunoinformatic methods have promoted research into multi epitope vaccines. Multi epitope vaccines could induce a wide range of immune responses because of the presence of B cell and T cell epitopes. Therefore, the study of epitopes has played an important role in the construction of E. granulosus vaccines [16, 17]. The results of the present study indicated that the amino acid sequence of EGR-03347 included 11 potential B cell linear epitopes, which suggested that EGR-03347 has great antigenic potential. The homology alignment of the EGR-03347 protein sequence showed that it was in the same branch with E. multilocularis, and its homology with other species was much lower. We observed that the EGR-03347 gene was expressed in the E. granulosus protoscolex and, especially, in adults. Moreover, immunofluorescence localization of EGR-03347 in the larvae and adults of E. granulosus showed that EGR-03347 was mainly distributed in the epidermis and sucker of PSCs, and in the scolex, mature, and gravid proglottis of the tapeworm, suggesting that EGR-03347 might play an important role in controlling and maintaining the specificity of the parasite life cycle . Tapeworms like E. granulosus lack an intestinal tract, only possessing microvilli or microfibrils, which are the main sites for nutrient absorption and waste excretion . Furthermore, the location of EGR-03347 in the suction cups and hooks of protocercus might be related to the energy demand attached to the host intestine, thus EGR-03347 might be involved in the interaction between the parasite and its host [20, 21]. This suggested that EGR-03347 plays a key role in the growth and physiological activities of E. granulosus . Tapeworms contain the complete metabolic pathways of fat, which could be used for the formation and development of the ascus and PSC [23, 24].
The western blotting results showed that the serum from mouse injected with rEGR03347 could recognize EGR-03347 among the natural proteins of PSCs, and the positive serum from dogs infected with E. granulosus could also recognize rEGR-03347, which indicated that rEGR-03347 had good immunoreactivity. In particular, the recognition of rEGR-03347 by the positive serum from dogs indicated that EGR03347 might be a component of excretory/secretory (ES) products of the E. granulosus. However, the predicted amino acid sequence of EGR-03347 did not contain signal peptide; therefore, we speculated that EGR-03347 was an exocrine secretory protein, being released into extracellular secretion through exosomes .
In the present study, we used the mixed vaccine of EGR-03347 and Quil A to vaccinate the dogs. The Quil A adjuvant has been proven to be a key component in today's vaccines. Adding adjuvants to vaccine candidates could enhance the efficacy of the antigen, reduce the required dosage of the vaccine, make the host immune response more rapid, make T helper cells bind more effectively to optimize the quality and persistence of the antibody response, or induce effector CD4 + or CD8 + T cells to kill pathogens in cells . Therefore, this experiment used rEGR-03347 mixed with the Quil A adjuvant to induce a more effective immune response. After the third immunization, 100 000 protoscoleces were taken orally in the PBS group as the control. The IgG titer of each immunization group was monitored. We observed that the antibody levels of the vaccine group increased gradually with prolonged immunization time, indicating that the specific antibodies were produced by stimulation with rEGR-03347.
We noted that rEGR-03347 induced a Th1/Th2 cytokine response, and both Th1 and Th2 cytokines play an important role in the occurrence and development of echinococcosis. The Th2 response plays an important role in the emergence and growth of cysts, and plays a key role in the escape from 6immune surveillance , which is an important step in the development of a prophylactic E. granulosus vaccine. In the development of hydatid cysts, the Th1 immune response is dominant in the early stage of hydatid infection, and is related to protective immunity [28, 29]. Th2 mainly produces IL-4, IL-5, and IL-6 cytokines, while Th1 mainly produces IL1 and IFN-γ. In echinococcosis, a large number of studies have shown that Th1 type and Th2 type cytokines co-exist. The Th2 response is conducive to the growth of the parasite, and the Th1 response inhibits the growth of the parasite and plays an immunoprotective role in the host [30, 31].
Li  found that by immunizing mice with rEg14-3-3, the secretion of IFN-γ and IL-2 increased significantly, suggesting that Th1 might play a major role in the resistance to E. granulosus infection. Mohammad  immunized sheep and dogs with the rEGVac vaccine, and the IL-4 level in sheep and dogs increased significantly two weeks after immunization; however, the IL-4 level in serum decreased significantly after booster immunization, indicating that rEGVac has a significant inhibitory effect on the growth and development of adult PSCs.
In the present study, compared with the PBS control group, the secretion of IFNγ and IL-4 in the serum of the enhanced immunized dogs increased significantly increased, which showed a downward trend after 28 days of protoscolex challenge. One of the characteristics of parasite infection is change the cytokine spectrum develop towards the Th2 type, such as producing IL-4, IL-5, and IL-6 . In fact, the results may help to prevent host resistance to parasite resistance . The increase in the total IgG level in dogs infected with protocercariae indicated that there may be a good humoral response. Eosinophils had shown the ability to clear protocercariae, and a large number of host reaction sites exist in some degenerated cysts, indicating that a Th2 type immune response might also play an important role in the anti-parasite response . IFN-γ can activate macrophages to release NO, enhance the expression of MHC-II molecules and the antigen presentation ability, thus inhibiting the protoscolex of E. granulosus. This supports the view that the rEGR-03347 vaccine plays an important role in anti E. granulosus infection.
According to a previous study , recombinant EgM9 and EgM123 were mixed with Quil A to vaccinate dogs. At 45 days after infection, fewer worms (17.1%) developed 4 segments in the EgM9 vaccinated dogs compared with that in the control dogs, and EgM123 induced a significant protective efficacy in terms of worm burden reduction and suppression of worm growth and egg production at day 45 postinfection. No worms developed to the 4 segment stage and no worms produced eggs. In the present study, all dogs were sacrificed 28 days post challenge, before the appearance of eggs, for safety reasons. Compared with the PBS control group, the worm reduction rate was 87.19 %, It was better than the Salmonella vaccine EgA31-EgTrp(70%-80%)[38, but it was slightly lower than the recombinant vaccine EgM123(89.2%). and the worm development to the mature stage was significantly decreased in rEGR-03347 group, with an inhibition rate of 67.7 %. These results indicated that the rEGR-03347 vaccine could inhibit the growth and development of the tapeworms in canine intestines, which supported rEGR-03347 as a potential candidate vaccine for canine against E. granulosus infection.