Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs.

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Vaccine xxx (2015) xxx–xxx

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Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs Natsumi Takeyama a,b , Yoshikazu Yuki b,∗ , Daisuke Tokuhara b,c , Kazuki Oroku a , Mio Mejima b , Shiho Kurokawa b , Masaharu Kuroda d , Toshiaki Kodama a , Shinya Nagai a,e , Susumu Ueda a , Hiroshi Kiyono b,f a

Research Department, Nippon Institute for Biological Science, Japan Division of Mucosal Immunology, The Institute of Medical Science, The University of Tokyo, Japan c Department of Pediatrics, Osaka City University Graduate School of Medicine, Japan d Rice Physiology Research Team, National Agriculture Research Center, Japan e Nisseiken Co. Ltd., Japan f International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, Japan b

a r t i c l e

i n f o

Article history: Received 16 March 2015 Received in revised form 10 July 2015 Accepted 13 July 2015 Available online xxx Keywords: Mucosal immunity Oral vaccine Pig ETEC Plant vaccine

a b s t r a c t Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in both neonatal and weaned pigs. Because the cholera toxin B subunit (CTB) has a high level of amino acid identity to the ETEC heat-labile toxin (LT) Bsubunit (LTB), we selected MucoRice-CTB as a vaccine candidate against ETEC-induced pig diarrhea. When pregnant sows were orally immunized with MucoRice-CTB, increased amounts of antigen-specific IgG and IgA were produced in their sera. CTB-specific IgG was secreted in the colostrum and transferred passively to the sera of suckling piglets. IgA antibodies in the colostrum and milk remained high with a booster dose after farrowing. Additionally, when weaned minipigs were orally immunized with MucoRice-CTB, production of CTB-specific intestinal SIgA, as well as systemic IgG and IgA, was induced. To evaluate the cross-protective effect of MucoRice-CTB against ETEC diarrhea, intestinal loop assay with ETEC was conducted. The fluid volume accumulated in the loops of minipigs immunized with MucoRice-CTB was significantly lower than that in control minipigs, indicating that MucoRice-CTB-induced cross-reactive immunity could protect weaned pigs from diarrhea caused by ETEC. MucoRice-CTB could be a candidate oral vaccine for inducing both passive and active immunity to protect both suckling and weaned piglets from ETEC diarrhea. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea in both humans and farm animals. ETEC causes economic losses in the livestock industry [1–3].

Abbreviations: CAYE, Casamino acids–yeast extract; CFU, colony forming unit; CT, cholera toxin; CTB, cholera toxin B subunit; ETEC, enterotoxigenic Escherichia coli; GM1, monosialotetrahexosylganglioside; LT, heat labile toxin; LTB, heat labile toxin B subunit; SIgA, secretory IgA; ST, heat stable toxin; PBST, Tween 20 PBS; TMB, 3,3 ,5,5 -tetramethylbenzidine; WT, wild type. ∗ Corresponding author at: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 5449 5274; fax: +81 3 5449 5411. E-mail address: [email protected] (Y. Yuki).

Although the risk of death upon single infection with ETEC in swine is not high, complex infections with ETEC, rotavirus, and coccidia are often found in piglets at both the neonatal and the post-weaning stage [4]. Therefore, hygiene measures or vaccination programs are needed to reduce the risk of enteric disease from ETEC invasion in farm animals. There is a protocol conflict in commercialized programs for the parenteral vaccination of pregnant sows against pig ETEC because of the antibody subtypes produced against ETEC antigen and the timing of the onset of ETEC diarrhea [2,5]. Young piglets are more susceptible than adults to ETEC infection, and suckling and weaning are times when particular attention needs to be paid to disease risk. As ETEC are non-invasive pathogens that multiply in the gut lumen [6], continuous SIgA supply to piglets via the milk is required for passive immunity. Parenteral vaccines do not induce adequate antigen-specific SIgA, and mucosal vaccination of young piglets is likely a better strategy against ETEC [5,7]. Additionally, piglets

http://dx.doi.org/10.1016/j.vaccine.2015.07.074 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Takeyama N, et al. Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.074

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should have active immunity against ETEC after weaning because passive immunity does not protect against subsequent ETEC infection [8–10]. The diarrhea associated with ETEC infection is caused by two major classes of enterotoxin produced by pig ETEC: heat-labile toxin (LT), which has a high molecular weight, and heat-stable toxins (STa and STb) [11–13]. Experimental deletion of the LT gene from F4-positive ETEC strains, which are frequent cause of pig post-weaning diarrhea, abrogated diarrheic symptoms in pigs less than 2 weeks old; this was not the case with STb gene deletion, indicating that LT is a more important contributor than STb [14]. Moreover, both epidemiologic studies and early vaccine studies have suggested that previous exposure to LT or cholera toxin (CT) can afford protection against subsequent infection with LT-producing ETEC [15]. Despite the variation in types of ETEC fimbriae, the LT produced by ETEC does not vary. Therefore, the induction of toxin-specific neutralization antibodies rather than adhesin-specific antibodies is an important strategy for protecting against ETEC diarrhea. We recently developed a rice-based cholera vaccine expressing the CT B subunit (CTB) (MucoRice-CTB) [16–18]. This vaccine has the advantages of being suited to more than 3 years’ storage without the need for a cold chain, and delivery of the vaccine antigen is needle- and syringe-free [16]. Oral administration of MucoRice-CTB to rodents and macaques induces CT-neutralizing antibodies [16,17]. Additionally, the LT of ETEC is structurally and biologically similar to CT, and several studies have demonstrated cross-protective immunity between CT and LT [19–22]. Our previous study also demonstrated that CT-specific mucosal IgA induced by oral MucoRice-CTB vaccine could provide crossprotective immunity against ETEC-induced diarrhea in mice [18]. We are newly exploiting these characteristics of MucoRice-CTB in a pig ETEC vaccine, as an oral vaccine for inducing both passive and active immunity in piglets. These new findings should enable MucoRice-CTB to be used as an oral vaccine against LT-ETEC in farm animals.

2.2. MucoRice-CTB vaccine MucoRice-CTB/N, a rice-expressed CTB [23] with an unaltered amino acid sequence, was used throughout the study. MucoRiceCTB/N was grown in a plant growth chamber (Panasonic Co. Ltd., Tokyo, Japan) with a cycle of 12 h of lights on (27 ◦ C) and 12 h of lights off (22 ◦ C). MucoRice-CTB and the seeds were ground to a fine powder in a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan). 2.3. ETEC ETEC strain S7 was a kind gift of the National Institute of Animal Health, Tsukuba, Japan. ETEC-S7 was specified as an LT+, STb+, STa−, Stx2−, F18-fimbriae-positive strain, and LT production ability was checked by culture in Casamino acids–yeast extract (CAYE) medium under aerobic conditions [24]. The ETEC-S7 strain was grown in CAYE medium with 16 h of vigorous shaking. The CFU of ETEC-S7 was calculated according to the formation of colonies on Luria-Broth agar plates, and 106 CFU/mL of ETEC-S7 solution was prepared by dilution with PBS. 2.4. Immunization Two pregnant NIBS minipig sows (#1 and #2) were each given 670 mg of MucoRice-CTB with their feed three times at 2-week intervals from 6 weeks before the calculated date of farrowing. Booster immunization with the same dose of MucoRice-CTB was given to the sows 5 days after farrowing. Serum and colostrum/milk samples were periodically collected from the vaccinated sows and their newborn piglets. Five 2-month-old female NIBS minipigs were given 670 mg of MucoRice-CTB in 10 mL of PBS intragastrically by catheter (#1 to #3) or orally mixed with the feed (#4 and #5) 4 times at 2-week intervals. In the control group, NIBS minipigs were given 670 mg of nontransgenic WT rice (Nipponbare) in PBS intragastrically (#6 to #8, n = 3) or orally mixed with the feed (#9). 2.5. ELISA

2. Materials and methods 2.1. Animals Female NIBS minipigs (Nisseiken Co. Ltd., Tokyo, Japan) (2 months old) and pregnant NIBS minipigs (12 months old) were used (Table 1). All minipigs were housed individually with free access to tap water and feed daily (NS-1; Nisseiken Co. Ltd.). All animal experiments were performed in accordance with the Nippon Institute for Biological Science’s Guidelines for Use and Care of Experimental Animals and approved by the Institute’s Animal Care and Use Committee.

CTB and LTB were respectively expressed in Bacillus brevis and Brevibacillus choshinensis and were purified by using immobilized d-galactose (Thermo Fisher Scientific Inc., Waltham, MA). Recombinant CTB or LTB (1 ␮g/mL) was coated onto 96-well plates (Nunc, Wiesbaden, Germany). Antigens were blocked with 3% skim milk (Wako, Osaka, Japan) in PBS, and samples of serum or whey were appropriately diluted in 3% skim milk–0.1% Tween 20 PBS (PBST). Serum samples were diluted to 1:200 for IgG measurement and 1:40 for IgA measurement. Whey samples were diluted 1:50, and intestinal lavage fluids were diluted 1:2 for IgA measurement. Plates were then treated with HRP-conjugated anti-pig IgG Fc (Nordic Immunology, Einhoven, The Netherlands)

Table 1 Minipigs used for MucoRice-CTB oral study. Minipigs (age)

Antigen (rice weight, route)

Sow #1 (12 mo, pregnant) Sow #2 (12 mo, pregnant) #1 (2 mo) #2 (2 mo) #3 (2 mo) #4 (2 mo) #5 (2 mo) #6 (2 mo) #7 (2 mo) #8 (2 mo) #9 (2 mo)

MucoRice-CTB (670 mg, with feed) MucoRice-CTB (670 mg, with feed) MucoRice-CTB (670 mg, intragastric) MucoRice-CTB (670 mg, intragastric) MucoRice-CTB (670 mg, intragastric) MucoRice-CTB (670 mg, with feed) MucoRice-CTB (670 mg, with feed) WT rice (670 mg, intragastric) WT rice (670 mg, intragastric) WT rice (670 mg, intragastric) WT rice (670 mg, with feed)

Samples analyzed

Intestinal loop assay

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serum

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Fig. 1. CTB-specific IgG and IgA secretion in sows’ sera, colostrum, and milk. Sow #1 (A and B) and sow #2 (C and D) were orally immunized three times with MucoRice-CTB; the third immunization was given 1 week before farrowing. A booster of the same amount of MucoRice-CTB was given to the sows on day 5 after farrowing. Sera and colostrum/milk from both sows were collected before and after farrowing and subjected to ELISA for CTB-specific antibody detection. MucoRice-CTB induced CTB-specific serum IgG (A and C) and IgA (B and D) production in the sows’ sera before farrowing, and the colostrum contained much higher amounts of both IgG and IgA than did the sera. Antibody titers in the milk gradually declined after farrowing but were increased by the booster immunization (arrowheads). In the milk, the boosting effect on IgA production was greater than that on IgG.

or HRP-conjugated anti-pig IgA (ACRIS Antibodies GmbH, Herford, Germany) at a dilution of 1:10,000 in 3% skim milk–PBST. The color was developed by adding 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate, and the absorbance was measured at a wavelength of 450 nm. Measured OD was normalized by using the equation: normalized OD = measured OD/positive control OD (1:200 dilution of CTB-immunized/ETEC-challenged pig serum). 2.6. ETEC intestinal loop challenge As an in vivo bacterial challenge we performed ligatedintestinal-loop tests in 2-month-old minipigs. Immunized and control minipigs were starved for 24 h but had free access to water. Minipigs were anesthetized by combined intramuscular injection of medetomidine (0.05 mg/kg) (Dormitor: Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) and midazolam (0.3 mg/kg) (Dormicam: Astellas Pharma Inc., Tokyo, Japan) and continuous supply of 0.1% isoflurane (Escain: Mylan Pharmaceuticals Inc., Pittsburgh, PA) via an anesthetic gas scavenging system. The first loop in the intestine started at a distance of about 10 cm from the cecum. The length of one loop was 6 cm, with 2 cm between the loops. ETEC at 106 CFU or physiological saline was administered in triplicate into different loops of the same individual. The minipigs were kept awake after surgery and periodically administered carprofen (Rimadyl; Zoetis, Tokyo, Japan). Eighteen hours after surgery, the challenged

minipigs were euthanized and the volume of accumulated fluids in each intestinal loop recorded. 2.7. GM1-binding assay To analyze the neutralizing effect of the whey from immunized sows, samples were subjected to monosialotetrahexosylganglioside (GM1)-binding assay, with some modifications [25]. Samples were serially diluted and were treated for 1 h with 50 ng/mL of CT. They were then added to 96-well plates coated with 5 ␮g/mL of GM1 (Sigma–Aldrich, St. Louis, MO). The color was developed by adding TMB substrate, and the absorbance was measured at a wavelength of 450 nm. 2.8. Data analysis Analyses for statistically significant differences were performed with Student’s t-test. 3. Results 3.1. MucoRice-CTB induces pig maternal cross-reactive immunity Because pigs have no mechanism for transplacental transfer of immunoglobulins, the first choice of a MucoRice-CTB


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C

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1

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Substitute milk Milk from MucoRice-CTB -immunized sows

0.4 0.1 0.2 0 day1

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Fig. 2. Neutralization activity and transfer of maternal CTB-specific antibodies. (A) Colostrum and milk from two sows were subjected to GM1-CT binding inhibition assay. The neutralizing index was calculated from the OD450 obtained by GM1-ELISA. A strong neutralizing effect of colostrum on day 0 after farrowing and of milk after booster immunization was found. Arrowhead shows the time at which the MucoRice-CTB booster was given to the sows (on day 5 after farrowing). (B and C) IgG (B) and IgA (C) antibody transfer from sow to offspring via colostrum. Newborn piglets from sow #2 were allocated to two groups after birth. One group was fed artificial milk (n = 2) and the other group was fed milk from the MucoRice-CTB-immunized sow (n = 3). On days 1, 3, 10, and 15 after birth, piglet blood samples were collected to check for the retention of CTB-specific IgG and IgA antibodies in the sera. All piglets in the sow-milk-fed group were positive for CTB-specific antibodies (solid lines, open plots), whereas in the sera of piglets from the same litter fed the artificial milk (solid lines, closed plots) we detected no CTB-specific antibodies. Thus CTB-specific IgG and IgA were transferred from sow to offspring via the milk. Serum titers gradually decreased with time, regardless of CTB-specific antibody secretion in the sows’ milk.

vaccination program to protect newborn piglets from ETECassociated diarrhea is maternal immunization. Two sows 6 weeks before farrowing were immunized orally three times with a 670mg dose of MucoRice-CTB containing nearly 1.9 mg of CTB protein. Both sows produced CTB-specific IgG and IgA antibodies in their sera 3 weeks after primary immunization (Fig. 1). On farrowing day, the colostrum from both sows had greater concentrations of CTBspecific IgG and IgA antibodies than did the sera (Fig. 1). Serum IgG levels remained stable after farrowing, whereas CTB-specific IgG in the milk declined dramatically to reach its lowest level by day 5 (sow #1) or day 10 (sow #2). CTB-specific IgA levels in the sows’ sera showed a different trend and decreased moderately after farrowing. When the sows were orally boosted with an additional dose of MucoRice-CTB on day 5 after farrowing, the levels of CTB-specific IgG and IgA antibodies in both the serum and the milk increased. The elevation in IgG titer in the milk was milder than that of IgA. In sow #1, the milk IgA level had recovered to approximately the level in the colostrum by day 7, and in sow #2 by day 10 (Fig. 1B and D). In addition, CTB-specific antibodies in the milk of immunized sows reacted with LTB (Supplemental Fig. 1), suggesting that MucoRiceCTB could induce cross-protective immunity against ETEC-LT. To confirm the neutralizing activity of colostrum and milk, we performed a CTB binding inhibition assay against GM1. Colostrum obtained from both the immunized sows showed almost more than 80% binding inhibition (Fig. 2A). In line with the decline in CTBspecific antibody titers in the milk, the neutralizing effect of the milk declined to a minimum by day 5 (sow #1) or day 7 (sow #2) (Figs 1 and 2A). However, a short time after the MucoRice-CTB booster immunization on day 5 the levels recovered to as high as those in the colostrum.

Next, we examined the transfer of CTB-specific IgG and IgA antibodies from colostrum to the piglets’ sera. The offspring of sow #2 were allocated randomly to an artificial milk-fed group (n = 2) and a sow-suckled group (n = 3) immediately after birth. The piglets that received artificial milk had no CTB-specific antibodies in their sera throughout the experimental period (Fig. 2B and C). Conversely, all three piglets in the sow-fed group had CTB-specific IgG and IgA antibodies in their sera on day 1 after birth (Fig. 2B and C), indicating that there had been successful transition of maternal antibody from the colostrum. Serum IgG antibody levels in these piglets gradually declined within 2 weeks to almost half of the original titers. Serum IgA antibody levels declined more rapidly over the 2-week period (Fig. 2B and C). This suggested that there were differences in the half-lives of the two classes of antibody in the piglets’ blood. 3.2. Oral MucoRice-CTB induces active mucosal immunity in minipigs We next evaluated whether weaned minipigs developed CTB-specific antibodies after being immunized orally with MucoRice-CTB. Two-month-old minipigs were immunized 4 times at 2-week-intervals either intragastrically (n = 3; #1 to #3) or orally (n = 2; #4 and #5) with MucoRice-CTB (Table 1). CTB-specific IgG and IgA antibodies were induced in the sera by both immunization methods, whereas WT rice produced no IgG or IgA response (Fig. 3A and B). Most minipigs produced CTB-specific antibody after the second immunization, although minipig #2 responded after the third immunization. In either case, once the CTB-specific IgG and IgA antibody levels rose they remained high for the rest of the experimental period. Comparison of the immunization


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Fig. 3. Immune response to CTB in minipigs orally immunized with MucoRice-CTB. Two-month-old minipigs were immunized four times either intragastrically (n = 3: #1 to #3) or orally (n = 2: #4 and #5) with a 670-mg dose of MucoRice-CTB. Sera or intestinal lavage fluids were used to compare, by ELISA, the CTB-specific immune responses of immunized minipigs with those of minipigs given WT rice (n = 3: #6 to #8). (A and B) Oral immunization was done at 0 weeks and at 2, 4, and 6 weeks after primary immunization. CTB-specific IgG (A) or IgA antibody (B) levels in the sera were elevated in both the intragastrically vaccinated group (#1 to #3) and the orally vaccinated group (#4 and #5) after two or three sequential immunizations. No CTB-specific antibody production was observed in the minipigs given WT rice (#6 to #8). (C) Jejunal and ileal lavage fluids were collected from minipigs #2, #3, #4, and #5 (MucoRice-CTB-immunized) and #7 and #8 (WT-rice-immunized) 1 week after final immunization. CTB-specific IgA was secreted into the lumens of both the jejunum (open columns) and the ileum (closed columns) in the MucoRice-immunized minipigs.

methods revealed that slightly higher IgA and lower IgG responses occurred in the case of oral immunization than with intragastric immunization (Fig. 3B). In contrast, piglets intramuscularly vaccinated with recombinant CTB together with Freund’s adjuvant exhibited high levels of CTB-specific IgG production in their sera, but no specific IgA production at all (Supplemental Fig. 2). To examine whether CTB-specific IgA antibody was produced at mucosal sites, we collected intestinal lavage fluids from 4 minipigs in the MucoRice-CTB immunized group and 2 control minipigs in week 7 of the MucoRice-CTB intragastric/oral immunization experiment, at the time of the intestinal loop assay. Lavage fluids from all four minipigs immunized with MucoRice-CTB contained CTB-specific IgA (#2 to #5), whereas fluids from WT-rice immunized minipigs (#7 and #8) contained no antibody (Fig. 3C). These data suggested that oral immunization with MucoRice-CTB could induce both systemic and mucosal immunity in minipigs. 3.3. Oral MucoRice-CTB-induced LT cross-protective immunity provides protection against pig ETEC diarrhea Pigs are natural hosts for ETEC, and many farm pigs are negatively affected by this pathogen. Cross-protection against LT induced by MucoRice-CTB is an absolute necessity if MucoRiceCTB is to be a suitable ETEC vaccine. Equivalent concentrations of

recombinant CTB and LTB were used as ELISA antigens. Although the OD values in ELISA were significantly lower (P < 0.05) in the LTB reactions than in the CTB ones; both IgG and IgA antibodies of MucoRice-CTB-immunized minipigs showed cross-reactivity with recombinant LTB (Fig. 4A and B). The best cross-reactivity to LTB for IgG antibody was 50.5% (OD450 of LTB/OD450 of CTB) in piglet #3; for IgA antibody it was 68.6% in piglet #4. We next investigated the protective effect of MucoRice-CTB vaccination. As an alternative to ETEC oral challenge, to examine whether orally induced IgA secretion into the intestinal lumen would suppress signs of diarrhea, we administered ETEC directly into the intestinal loops of MucoRice-CTB-immunized minipigs 1 week after final immunization. ETEC-S7 (an LT-producing STb-gene-positive strain with F18 fimbriae) was injected at 106 CFU/loop into the ligated intestinal loops. Data on triplicate loops in each minipig (MucoRice-CTB group: #2 to #5; control group: #7 to #9) were obtained. Significant differences in fluid accumulation per loop were observed between control and immunized pigs (Fig. 4C). The average volumes in the control group were 8.8 mL, 23.9 mL, and 27.3 mL, whereas the immunized group accumulated average volumes of only 5.4 mL, 4.7 mL, 2.7 mL, and 2.7 mL—significantly less than in the controls (P < 0.01). The fluid volume in the loops of all minipigs that received physiological saline without ETEC was 0 mL. These data suggested that




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Fig. 4. Cross-reactivity of anti-CTB antibodies against LTB. (A and B) An equivalent concentration of solubilized recombinant CTB or LTB (1 ␮g/mL) was coated onto ELISA plates, and MucoRice-CTB-immunized minipig (#2, #3, #4, and #5) sera collected 1 week after final immunization were analyzed. Both IgG (A) and IgA (B) of MucoRiceCTB-immunized minipigs cross-reacted with recombinant LTB. (C) Enterotoxigenic Escherichia coli strain S7 (106 CFU/dose) was injected into each intestinal loop of minipigs under anesthesia. Eighteen hours after surgery, the fluids accumulated in the loops were quantified. Data were obtained from triplicate loops per individual (open circles), and bars show averages. Fluid volumes in the loops of the MucoRice-CTB-immunized minipigs (#2 to #5) were suppressed at 106 CFU/dose of bacteria compared with those in the loops of minipigs given WT rice (#7 to #9). Statistically significant between groups; *, P < 0.01; **, P < 0.05.

MucoRice-CTB oral immunization could induce cross-protective immunity against LT-ETEC-associated diarrhea in piglets. 4. Discussion Pig ETEC-induced diarrhea is still a cause of mortality and morbidity in neonatal and post-weaning piglets worldwide [1–3]. Two major bottlenecks in the development of current pig ETEC vaccines are the facts that (1) parenteral immunization of pigs induces systemic but not mucosal immunity, and (2) with current vaccination programs it is hard to cover all ETECs at susceptible periods of the pig’s life. We therefore need a new strategy for developing pig ETEC vaccines that will overcome these problems. The current commercialized pig ETEC vaccines mostly offer passive immunity to piglets upon intramuscular administration to pregnant sows. The ETEC vaccine is made up of inactivated bacterial components, the majority being F4-fimbriae positive [21,24]. Vaccination of pregnant sows induces lactogenic antibody, which can be transferred to the newborn piglets via the colostrum and milk. IgGs are incorporated into the piglets’ blood via the gut Fc transporter within 48 h after birth [26]. However, continuous supply of toxin-reactive SIgA in the milk to the piglet’s gut lumen during lactation is the most promising way to defend against toxin-induced severe diarrhea. Live attenuated strains of ETEC as oral vaccines successfully induce antigen-specific IgA antibody secretion into the gut lumen because they are capable of colonizing the pig intestine for continuous stimulation of the gut mucosal immune system [27]. In contrast, in the case of non-replicating inactivated antigens of pig enteric diseases such as rotavirus, or other bacterial and viral antigens, it is hard to induce antigen-specific immunity by oral immunization [28,29]. In our study, sows orally immunized with MucoRice-CTB successfully produced CTB-specific SIgA antibody in their sera and colostrum, as well as CTB-specific IgG antibody. Additionally, SIgA was continuously secreted into the milk for 2 weeks after a booster dose 5 days after farrowing; high CT-neutralizing activity was observed. Our study shows that MucoRice-CTB is tolerated in the harsh environment of the intestine and effectively stimulates the gut immune system to generate a toxin-specific SIgA antibody response [16–18]. From this perspective, orally administered

MucoRice-CTB is appropriate for inducing mucosal immunity in pigs. We further investigated whether MucoRice-CTB could raise active immunity in newly weaned piglets. To protect newly weaned piglets, an active intestinal mucosal immune response is required. Like the pregnant sows, weaned minipigs produced CTB- and LTBcross-reactive IgG and IgA in their sera and SIgA in their intestines, strongly supporting our finding that vaccinated minipigs were protected from signs of diarrhea when ETEC was introduced into the intestinal loop. These data are consistent with those in a previously reported rodent model [16,18]. In fact, to obtain the best results from ETEC vaccination at the time of weaning, vaccination during suckling is needed [6]. In a previous study, an attenuated F18-positive ETEC strain was given orally to piglets 10 days before weaning and successfully induced the production of fimbriae-specific antibodies [30]. Altering the timing of MucoRiceCTB vaccination from weaned period to the suckling period is a matter of importance to consider in the near future. However, our study showed that MucoRice-CTB could be a key factor in inducing mucosal IgA production in sows and young piglets, thus having a greater impact than current ETEC vaccines. The use of MucoRiceCTB as an ETEC vaccine can be extended from passive mucosal immunization to active mucosal immunization. The majority of ETEC on farms worldwide are F4 or F18 positive; of these, about 57% carry the LT gene [3,31]. A couple of studies have shown that oral vaccination with purified F4 fimbriae is highly antigenic and can induce mucosal immunity in pigs [32,33]. In contrast, F18 antigen is poorly immunogenic [34]. Because CTB-based vaccines can target not only F4-type but also F18-type ETECs, in this study, we chose to focus on LT toxin rather than the adhesins. In addition, LT and ST are widespread among ETEC with different types of fimbrial antigens, including F4, or F18 [3,35,36], and ETEC possess these toxins in various combinations. However, virulence and signs of diarrhea in ETEC infection are associated more with LT than with ST [14]. Moreover, because there have been several reports of the presence of genes encoding the virulence factors EAST1 and AIDA1 in ETEC [3,37], the use of multivalent MucoRice, which expresses not only CTB but also other ETEC virulence factors including these two, may lead to more effective oral ETEC vaccination of piglets.




Several genetically modified plants, including tobacco and Arabidopsis, have recently been developed for use in veterinary vaccines or for producing therapeutic antibodies [38,39]. Veterinary medicines—even more than human ones—need to be economical. Simplifying the manufacturing process is one way of reducing production costs. MucoRice-CTB is based on edible rice seeds and therefore expresses few allergenic antigens [40]. There is therefore potential for MucoRice-CTB to be used as a pig vaccine without antigen purification. Additionally, we have succeeded in inducing an immune response against CTB in pigs by simply mixing the vaccine into the feed; the result is comparable to that with intragastric administration. This method facilitates pig vaccination on farms and saves both veterinarians and animals from the unwanted stresses of vaccination. In summary, MucoRice-CTB could be used to protect pigs from LT-ETEC-mediated diarrhea by inducing passive and active antibodies to CTB. These results reinforce the advantages of MucoRice-CTB in both human and veterinary clinical application and show that it can protect pigs at all life stages from all types of LT-ETEC-induced diarrhea. In our next step we intend to consolidate manufacturing facilities for genetically modified rice plants. We also intend to perform an additional animal experiment aimed at the ultimate approval of MucoRice-CTB as a pig veterinary medicine. The MucoRice oral vaccination system could be applied to various animal species and various veterinary diseases. Acknowledgments This work was supported by a grant from a project of the National Agriculture and Food Research Organization of Japan (Y.Y.); the Adaptable and Seamless Technology transfer Program through target driven R&D (A-step) (AS2311903E) (T.K.); and a grant for the promotion of veterinary vaccine development from the Ministry of Agriculture, Forestry, and Fisheries of Japan (N.T.). We are grateful to Drs. Kunisuke Tanaka, Takehiro Masumura, Ai Saso, Tomonori Nochi, Yoshiko Fukuyama, Koji Kashima, and Tatsuhiko Azegami; Mr. Yuji Suzuki; and Ms. Satomi Minakawa for their useful discussions and technical support. We also thank Drs. Akira Iwata and Nobuyuki Tsutsumi. Conflicts of interest: None. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2015.07. 074 References [1] Van den Broeck W, Cox E, Goddeeris BM. Seroprevalence of F4+ enterotoxigenic Escherichia coli in regions with different pig farm densities. Vet Microbiol 1999;69:207–16. [2] Moxley RA, Duhamel GE. Comparative pathology of bacterial enteric diseases of swine. Adv Exp Med Biol 1999;473:83–101. [3] Zhang W, Zhao M, Ruesch L, Omot A, Francis D. Prevalence of virulence genes in Escherichia coli strains recently isolated from young pigs with diarrhea in the US. Vet Microbiol 2007;123:145–52. [4] Katsuda K, Kohmoto M, Kawashima K, Tsunemitsu H. Frequency of enteropathogen detection in suckling and weaned pigs with diarrhea in Japan. J Vet Diagn Invest 2006;18:350–4. [5] Melkebeek V, Goddeeris BM, Cox E. ETEC vaccination in pigs. Vet Immunol Immunopathol 2013;152:37–42. [6] Gaastra W, de Graaf FK. Host-specific fimbrial adhesins of noninvasive enterotoxigenic Escherichia coli strains. Microbiol Rev 1982;46:129–61. [7] Lin J, Mateo KS, Zhao M, Erickson AK, Garcia N, He D, et al. Protection of piglets against enteric colibacillosis by intranasal immunization with K88ac (F4ac) fimbriae and heat labile enterotoxin of Escherichia coli. Vet Microbiol 2013;162:731–9. [8] Bourne FJ, Curtis J. The transfer of immunoglobins IgG, IgA and IgM from serum to colostrum and milk in the sow. Immunology 1973;24:57–62.

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