Potential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice

Veterinary Research Potential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice Yue Xie 0 3 Sijie Chen 2 Yubo Yan 2 Zhihe Zhang 1 Desheng Li 5 Hua Yu 2 Chengdong Wang 5 Xiang Nong 0 3 Xuan Zhou 0 3 Xiaobin Gu 0 3 Shuxian Wang 0 3 Xuerong Peng 4 Guangyou Yang 0 3 0 Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University , Ya'an 625014 , China 1 The Sichuan Key Laboratory for Conservation Biology on Endangered Wildlife-Developing toward a State Key Laboratory for China, Chengdu Research Base of Giant Panda Breeding , Chengdu, Sichuan 610081 , China 2 Sichuan Entry-Exit Inspection and Quarantine Bureau , Chengdu 610041 , China 3 Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University , Ya'an 625014 , China 4 Department of Chemistry, College of Life and Basic Science, Sichuan Agricultural University , Ya'an 625014 , China 5 China Conservation and Research Center for Giant Panda , Wolong 623006 , China The intestinal nematode Baylisascaris schroederi is an important cause of death for wild and captive giant pandas. Inorganic pyrophosphatases (PPases) are critical for development and molting in nematode parasites and represent potential targets for vaccination. Here, a new PPase homologue, Bsc-PYP-1, from B. schroederi was identified and characterized, and its potential as a vaccine candidate was evaluated in a mouse challenge model. Sequence alignment of PPases from nematode parasites and other organisms show that Bsc-PYP-1 is a nematode-specific member of the family I soluble PPases. Immunohistochemistry revealed strong localization of native Bsc-PYP-1 to the body wall, gut epithelium, ovary and uterus of adult female worms. Additionally, Bsc-PYP-1 homologues were found in roundworms infecting humans (Ascaris lumbricoides), swine (Ascaris suum) and dogs (Toxocara canis). In two vaccine trials, recombinant Bsc-PYP-1 (rBsc-PYP-1) formulated with Freund complete adjuvant induced significantly high antigen-specific immunoglobulin (Ig)G but no IgE or IgM responses. Analysis of IgG-subclass profiles revealed a greater increase of IgG1 than IgG2a. Splenocytes from rBsc-PYP-1/FCA-immunized mice secreted low levels of T helper (Th)1-type cytokines, interferon- and interleukin (IL)-2, while producing significantly high levels of IL-10 and significantly elevated levels of IL-4 (Th2 cytokines) after stimulation with rBsc-PYP-1 in vitro. Finally, vaccinated mice had 69.02-71.15% reductions (in 2 experiments) in larval recovery 7 days post-challenge (dpc) and 80% survival at 80 dpc. These results suggest that Th2-mediated immunity elicited by rBsc-PYP-1 provides protection against B. schroederi, and the findings should contribute to further development of Bsc-PYP-1 as a candidate vaccine against baylisascariasis. - Introduction Baylisascariasis is a neglected zoonotic helminthic disease caused by parasitic nematodes of the genus Baylisascaris (Nematoda: Ascaridida) with great medical and veterinary significance worldwide [1-3]. The causative pathogens, Baylisascaris spp., are widely distributed in the giant panda (Ailuropoda melanoleuca), red panda (Ailurus fulgens), raccoon (Procyon lotor), Ursid species (Ursus maritimus, Ursus arctos pruinosus, Selenartos thibetanus mupinensis and Ursus arctos lasiotus) and other mammals including humans and can lead to severe clinical visceral (VLM), ocular (OLM) and neural larva (NLM) migrans in these definitive or intermediate hosts [4-7]. Among them, Baylisascaris schroederi is the only endoparasite that appears to be consistently found in the giant panda, a flagship species for wildlife conservation in China, and represents a significant threat to both wild and captive populations [8,9]. In nature, B. schroederi infection rates among wild pandas may reach between 50100%, making it one of the leading causes of death from primary and secondary infection in wild populations [4,10,11]. Zhang et al. demonstrated that the probability of death of wild pandas caused by this pathogen increased significantly between 1971 and 2005, and the associated VLM was the most important cause of death during the recent period 20012005 [8]. As with all ascarid species, B. schroederi infection follows a trophic pathway by ingestion with life cycle completion without intermediate hosts. This parasite (at the adult stage) usually inhabits the intestines of the giant panda, while its migrating larvae may disseminate into various body tissues. B. schroederi can induce extensive inflammation and scarring of the intestinal wall and parenchyma of the liver and lung (mainly caused by larvae), as well as intestinal obstruction, inflammation and even death (caused by adults) in giant pandas [9,12-14]. Until now the control of B. schroederi infection in pandas is relied chiefly on chemotherapy, and treatment with antiparasitic drugs requires multiple doses until the animal ceases to expel worms or shed eggs in feces [9]. However, alternative preventative and treatment strategies are needed due to the rapid emergence of multidrug-resistant ascarids and pollution of the food chain and the environment from chemotherapy, as well as the persistent exposure of host animals to parasites of different stages in their surroundings. Vaccines, particularly target antigens that play crucial roles in the survival, development and reproduction of parasitic nematodes would be an ideal control strategy. Inorganic pyrophosphatases (PPases, EC 3.6.1.1), a class of cytosolic enzymes catalyzing the hydrolysis of inorganic pyrophosphate (PPi) to ortho-phosphate (Pi), are widely distributed among living cells and function in energy metabolism, lipid metabolism and some biosynthetic reactions [15]. PPases are essential for the growth and development of prokaryotes, fungus, nematodes and plants (e.g., tobacco and potato) [16-22]. For parasites, such as Ascaris roundworms, PPases are believed to be further involved in molting, as indicated in Ascaris suum by RNAmediated interference and enzyme activity inhibition assays [23,24]. Considering the important roles of PPases in life processes of various organisms including parasitic nematodes, some of these enzymes have been selectively targeted for pharmaceutical and vaccine purposes [25]. For example, an adjuvanted recombinant PPase antigen from ascarids was recently demonstrated to induce a high level of protection (>70%) against A. suum challenge in mice, and its potential for use as a candidate vaccine against ascariasis is further suggested in pigs [23]. However, no information on PPases of B. schroederi is available to date. More importantly, B. schroederi-specific protein antigens as potential targets for vaccines and/or chemotherapeutic agents are still scarce with only three antigen molecules (Bs-Ag1, Bs-Ag2 and Bs-Ag3) available [12,13,26], and the precise mechanism of protective immunity against B. schr (...truncated)