Recombinant Ascaris 16-Kilodalton Protein-Induced Protection against Ascaris suum Larval Migration after Intranasal Vaccination in Pigs

MAJOR ARTICLE Recombinant Ascaris 16-Kilodalton Protein–Induced Protection against Ascaris suum Larval Migration after Intranasal Vaccination in Pigs Naotoshi Tsuji,1 Takeharu Miyoshi,1 M. Khyrul Islam,1 Takashi Isobe,1 Shinobu Yoshihara,1 Takeshi Arakawa,2 Yasunobu Matsumoto,3 and Yuichi Yokomizo1 1 National Institute of Animal Health, National Agricultural Research Organization, Tsukuba, Ibaraki, 2Division of Molecular Microbiology, Center of Molecular Biosciences, University of the Ryukyus, Nishihara, Okinawa, and 3Laboratory of Global Animal Resource Science, Graduate School of Agricultural Life Science, University of Tokyo, Tokyo, Japan Geohelminth parasites of the genus Ascaris are the causative agent of ascariasis, a major medical and veterinary problem worldwide. The human ascarid, Ascaris lumbricoides, is estimated to infect 11.5 billion people, mostly in tropical and subtropical countries [1]. The pig homologue, Ascaris suum, is responsible for heavy production losses in pig farms all over the world [2]. Recent studies have revealed that A. suum of swine origin can develop in human hosts, indicating its possible emergence as an important zoonotic parasite [1, 3–5]. Although numerous studies have characterized Received 2 February 2004; accepted 27 May 2004; electronically published 30 September 2004. Financial support: Zoonosis Control Program from the Ministry of Agriculture, Forestry, and Fisheries; Program for Promotion of Basic Research Activities for Innovative Biosciences. Reprints or correspondence: Dr. Naotoshi Tsuji, Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan (). The Journal of Infectious Diseases 2004; 190:1812–20  2004 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2004/19010-0014$15.00 1812 • JID 2004:190 (15 November) • Tsuji et al. the 2 species of parasites on morphological, immunological, and biochemical bases, species discrimination between A. lumbricoides and A. suum has been controversial [6–8]. It has recently been suggested that human and swine Ascaris species reproduce in a closed population [9]. The control of pig ascariasis may thus be required for advancing measures to decrease human ascariasis. Prior studies have shown that pigs can be rendered immune to A. suum infection by immunization with radiation-attenuated infective larvae or by chemically abbreviated infection [10–13]. In mice, passive transfer of serum from immune mice is effective for larval killing and the prevention of stunting [14]. Crude larval antigens can induce protective immunity, suggesting that larval stages possess protective antigen against A. suum infection. In recent vaccine strategies, mucosal immunization is a critical goal in the development of vaccines against intestinal pathogens. Bacterial toxins such as cholera toxin (CT) are now available as mucosal adjuvants [15], and CT can induce antigen-specific sys- We recently cloned a protective antigen that is commonly expressed in Ascaris species that infect humans and pigs. We evaluated the vaccinal effects of this 16-kilodalton protein (As16) in pigs, the natural host of Ascaris suum, by intranasal immunization. Pigs that received Escherichia coli–expressed recombinant As16 (rAs16) coupled with cholera toxin (CT) had significantly elevated levels of rAs16-specific serum immunoglobulin G (IgG) and mucosal-associated IgA antibodies. rAs16 evoked a type II immune response characterized by elevated levels of interleukin-4 and -10 in the culture supernatants of peripheral blood mononuclear cells of the vaccinated pigs. An increased level of rAs16-specific serum IgG1 was also detected. Pigs vaccinated with rAs16CT were protected from migration of A. suum larvae through the lungs, as indicated by a 58% reduction in the recovery of lung-stage third-stage larvae (L3), compared with that in nonvaccinated controls. Purified immunoglobulin from rAs16-CT–vaccinated pigs inhibited survival of infective L3 and interrupted the molting of lung-stage L3. Immunofluorescence studies revealed that this immunoglobulin bound to the digestive tracts of L3, suggesting that it might inactivate functions of the gut tissues of Ascaris species. We conclude that rAs16 is a promising mucosal vaccine candidate for pig and human ascariasis. MATERIALS AND METHODS Pigs. Six-week-old crossbred piglets (Landrace ⫻ Whitelarge ⫻ Duroc) from a pathogen-free colony were used for vaccination and challenge infection studies. Animal experiments were approved by the National Institute of Animal Health Animal Care and Use Committee (approval no. 361). Parasites. Adult A. suum were obtained from infected pigs at a slaughterhouse in Shimotsuma, Japan. Unembryonated and embryonated eggs were obtained essentially as described elsewhere [22]. Infective third-stage larvae (L3) for in vitro culture were obtained as described elsewhere [23]. The lung-stage L3 used for immunohistochemical analyses and in vitro cultures were obtained from the lungs of infected pigs used in the vaccination studies, as described below. Recombinant As16 protein. Recombinant As16 protein was prepared as described elsewhere [22]. The entire coding region of As16 except the signal sequence was subcloned into a plasmid expression vector, pTrcHisB (Invitrogen). The plasmid was transformed into Escherichia coli strain TOP10F
(Invitrogen), and the purification process was monitored by SDS-PAGE with use of a T7 Taq monoclonal antibody (Novergen). The recombinant protein was purified with use of AKTA equipped with a HiTrap chlelating HP column (Amersham Pharmacia Biotech). Histidine-tagged As16 was dialyzed against PBS in an Aside-A-Lyzer Dialysis Cassette (Pierce). Vaccination and challenge infection. Recombinant As16 was coupled with CT (C-8052; Sigma) by conjugation in darkness at 4C for 16 h. Nine pigs were divided into 3 groups. The rAs16-CT–immunized group was inoculated inl with 500 mg of rAs16 coupled with 25 mg of CT in a volume of 1 mL of PBS (0.5 mL/nostril). On day 21, a booster inoculation of 300 mg of rAs16 coupled with 15 mg of CT was given. The final boost of 300 mg of rAs16 coupled with 15 mg of CT was given on day 35. The second and third groups were inoculated with the same dose of CT or rAs16 alone, respectively, on the same days as the immunized group. One week after the final immunization, pigs were inoculated orally with 104 A. suum embryonated eggs. During immunization, blood and nasal swab samples were collected at various time points to monitor antirAs16 antibodies. The swabs were mixed with 500 mL of PBS containing a protein inhibitor cocktail (Complete; Boehringer Mannheim) and vortexed, the mixture was centrifuged at 26,000 g for 60 min at 4C, and the supernatant was stored at ⫺80C. The pigs were killed on day 7 as follows: pigs were lightly anesthetized with a combination of ketamine hydrochlor (...truncated)