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Use of mucosal immunization with porcine zona pellucida (PZP) in mice and rabbits
Animal Reproduction Science 93 (2006) 372–378
Short communication
Use of mucosal immunization with porcine zona pellucida (PZP) in mice and rabbits Brent J. Martin a,∗ , Mark A. Suckow b , William R. Wolter b , Thomas Berger b , John W. Turner Jr. c a
Division of Laboratory Animal Medicine, Medical University of Ohio, 3055 Arlington Avenue, Toledo, OH 43614, USA b Freiman Lifesciences Center, University of Notre Dame, Notre Dame, IN 46556, USA c Department of Physiology and Cardiovascular Genomics, Medical University of Ohio, 3055 Arlington Avenue, Toledo, OH 43614, USA Received 11 April 2005; accepted 14 September 2005 Available online 24 October 2005
Abstract Rabbits (Oryctolagus cuniculus) and two strains of mice (Mus musculus, one inbred and one outbred) were immunized against porcine zona pellucida (PZP) antigen. Alginate microspheres or cholera toxin B were used alone or in combination when mucosal immunization routes were used. Serum antibody responses and fertility were assessed. Neither rabbit or mouse groups immunized by mucosal routes generated significant antibody responses to PZP as compared to parenteral immunization (ANOVA, P > 0.05). The study shows that porcine zona pellucida is not an effective mucosal antigen in small mammals. © 2005 Elsevier B.V. All rights reserved. Keywords: Porcine zona pellucida; PZP; Mucosal immunity; Rabbit; Mouse; Alginate microsphere
1. Introduction Parenteral immunization, which primarily stimulates a systemic immune response, with porcine zona pellucida (PZP) is an effective means of reducing fertility in a variety of species (Kirkpatrick et al., 1986; Kirkpatrick and Turner, 1996; Turner et al., 1996, 2002). Although some reports have indicated success with single immunizations with long-acting pellets (Turner et al., 2001), most studies have involved multiple parenteral injections (Kirkpatrick et al., 1995). ∗
Corresponding author. Tel.: +1 419 383 4310; fax: +1 419 383 3021. E-mail address: [email protected] (B.J. Martin).
0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.09.007
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The requirement for sufficient animal access necessary to administer parenteral injections, and especially multiple injections, is a significant barrier to use of this method for animal population control. Secretive, nocturnal and abundant species, as well as those inhabiting dense cover and hostile environments, often cannot be reliably captured in sufficient numbers to allow effective population management by injection. Thus, an alternative approach focusing on development of an immunization method avoiding direct animal access could open new avenues of animal control. Previous studies using oral and intranasal vaccination to induce mucosal immunity generated efficacious immune response in many species to a variety of antigens (Bowersock et al., 1999). Many antigens are weakly immunogenic when administered to mucosal surfaces. For this reason, auxiliary materials are often incorporated to enhance the immune response. In this regard, the B-subunit of cholera toxin (CTB) is an effective adjuvant for mucosal immunity. In mice and rabbits, co-administration of cholera toxin has been shown to enhance mucosal immunity to various antigens (Liang et al., 1988; Czinn and Nedrud, 1991; Keren and Suckow, 1994; Suckow et al., 1996a). Antigens administered via the mucosa are subject to pH extremes and enzymatic action, particularly in the gastrointestinal system. Microencapsulation of antigens within a variety of polymers has been widely used to provide protection of antigens until absorption occurs. Furthermore, microencapsulation is another means of enhancing the response to vaccination at mucosal surfaces and the effect is presumed to be via augmentation of uptake by M cells of the Peyer’s patches (Florence, 1997; Singh and O’Hagan, 1998; Lemoine et al., 1998; Suckow et al., 1999). Encapsulation of antigens in alginate microspheres (MS) has been demonstrated to be an effective means of stimulating mucosal immunity following immunization in both mice (Bowersock et al., 1996) and rabbits (Suckow et al., 1996b; Jarvinen et al., 2000). Encapsulation with alginate is a mild aqueous process often selected to avoid antigen degradation (Singh and O’Hagan, 1998; Bowersock et al., 1999; Zhou et al., 2002). The objective of this study was to evaluate the efficacy of selected mucosal immunization techniques to PZP in two different species, mice and rabbits.
2. Materials and methods 2.1. Experimental animals All animal use was approved by the institutional animal care and use committees of the responsible AAALAC accredited institutions and care was consistent with the Guide for the Care and Use of Laboratory Animals. Animals were allowed to adapt to laboratory conditions for 1 week prior to use and were housed in conventional cages on a constant 12-h light/12-h dark cycle with controlled temperature and humidity and ad libitum access to food and water. Mice were adult outbred ND4 Swiss Webster and inbred C57BL/6 (Mus musculus, Harlan, Indianapolis, IN). Vendor health reports indicated that the mice were serologically negative for known murine pathogens and research facility health surveillance were confirmatory. During immunization periods, mice were housed four per cage and for breeding periods, were housed three per cage (one male per two females). Specific pathogen-free New Zealand white rabbits weighing 3.0–4.3 kg were acquired (Covance, Inc. Kalamazoo, MI) as documented proven breeders. The rabbits were housed singly, except for periods of <30 min during which the female was placed in a male’s cage for breeding.
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2.2. Immunizations Utilizing pig ovaries obtained from a commercial slaughterhouse, porcine zona pellucida was isolated as previously described (Liu et al., 1989). Briefly, frozen-thawed ovaries were minced, filtered and ground in the presence of phosphate buffered saline (PBS, pH 7.2). Recovered zonae were heat solubilized at 70 ◦ C for 30 min in PBS. Characteristics of the PZP isolate were determined by gel electrophoresis and the preparation was screened for viral and bacterial contamination (ZooMontana, Billings, MT). Alginate microspheres were prepared to contain PZP with or without cholera toxin B-subunit (Sigma Chemical, St. Louis, MO) as previously described (Suckow et al., 1999). Briefly, sodium alginate, medium viscosity (Keltone LV® , Monsanto, St. Louis, MO) was dissolved in distilled water at a 2% (w/v) concentration. PZP and/or CTB were slowly added. During rapid stirring, the solution was dropped into 1.5% (w/v) CaCl2 via a 30-gauge needle. The resultant MS were isolated by low-speed centrifugation and placed in 0.05% poly-l-lysine (MW 100,000; Sigma Chemical Co., St. Louis, MO) with stirring for 30 min at room temperature. Approximately, 75% of the particles were 5 m or less in diameter as measured under light microscopy. Microspheres containing PZP were prepared with either 100 or 20 g PZP/100 l MS suspension for use in mice and rabbits, respectively. Microspheres containing CTB were prepared with either 20 or 200 g CTB/100 l MS suspension for use in mice and rabbits, respectively. Mice receiving PZP were exposed to 100 g/dose as previously determined to be efficacious in parenteral immunization studies (Mahi-Brown et al., 1992; Bynum, personal communication). The mucosal immunization techniques were developed from previous successful studies (Bowersock et al., 1999; Seong et al., 1999). Groups of both mouse strains were immunized once a week for 3 weeks. Each group consisted of five female mice. Test groups were: alginate MS; alginate MS with cholera toxin (20 g CTB/dose); PZP in PBS; PZP with alginate MS; PZP in MS with 20 g CTB. Immunizations were by oral gavage (200 l total volume) in all test groups. Additionally, five mice received subcutaneous injections of PZP (in 100 l PBS) emulsified with 100 l CFA or IFA for primary and booster immunizations, respectively. Males were added to cages for breeding 3 days after the final immunization dose. All mice were euthanized with CO2 and exsanguination by cardiac puncture 17 days later. Fetuses and implantation sites were enumerated in each animal to determine litter sizes. Serum was separated and frozen at −20 ◦ C until assayed. Rabbits receiving PZP were exposed to 20 g/dose as previously determined to be efficacious in parenteral immunization studies (Dietl et al., 1982; D. Wilkening, personal communication). The mucosal immunization techniques were developed from previous studies with similar antigens (Suckow et al., 1996b, 2002; Bowersock et al., 1999; Jarvinen et al., 2000). Rabbits were immunized with PZP (20 g/dose) in volumes of 0.4 ml PBS combined with an equal volume of adjuvant. Groups of six female rabbits were immunized intranasally three times at 2-week intervals. Immunization groups included alginate MS with CTB (200 g) and PZP in alginate MS with CTB (200 g). An additional group was immunized intramuscularly with PZP (in 125 l PBS) emulsified with 125 l CFA initially and with PZP in IFA 28 days later. Immunized does were natural bred 50 days after the initial immunization by placing each in a cage with a proven breeder male rabbit. A 1 ml blood sample was collected via marginal ear vein venipuncture just prior to introduction of the male and serum frozen at −20 ◦ C until analyzed. Does were euthanized 24 days later and uteri examined for fetus number.
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2.3. ELISA assays Serum samples were analyzed by ELISA for antibody binding to PZP. The mouse ELISA was validated via mouse anti-PZP antibody from a previous study (Bynum, personal communication). Mouse sera were analyzed in duplicate at a dilution of 1:50 and compared to the PZP-CFA immunized control serum at 1:400 or 1:1600. The rabbit ELISA was performed by a reference laboratory (ZooMontana, Billings, MT). Rabbit sera were analyzed in duplicate at 1:1280 and compared to a laboratory standard PZP immunized rabbit serum at 1:1280. Microtiter plates (96-well) were coated with 12 g native PZP and blocked with 0.1% gelatin. Experimental antibody was detected with goat anti-mouse or goat anti-rabbit antibody (Kirkegaard & Perry Labs, Gaithersburg, MD 20879) conjugated with alkaline phosphatase followed by p-nitrophenyl phosphate. Absorbance was measured at 405 nm on microplate reader. 2.4. Analysis Litter sizes were reported as within group means (S.D.) and analyzed by treatment using oneway ANOVA (SPSS for Windows, 2002). Anti-PZP antibody binding was reported as the mean percentage of each group (mean (S.D.)) as compared to a PZP-CFA immunized control sera. In mice, the relative antibody binding was compared to the PZP-CFA immunized group and the remaining five groups’ means were analyzed by ANOVA. In rabbits, the relative antibody binding comparison was made to a laboratory standard PZP-CFA immunized rabbit sera (ZooMontana, Billings, MT) and the data analysis (ANOVA, SPSS for Windows, 2002) encompassed the three groups in this study. Significance was set at P < 0.05. Control group (receiving no PZP) mean percent binding + 3S.D. of the control group data was considered the threshold value to identify specific anti-PZP antibody responses in individual animals. 3. Results Neither the alginate MS nor MS with CTB when administered in combination with PZP elicited significant anti-PZP antibody binding compared to PZP-CFA (ANOVA, P > 0.05) in either mouse strain (Table 1). All PZP-CFA immunized mice had strong, specific anti-PZP antibody responses. Four mice immunized via mucosal routes demonstrated specific anti-PZP antibody responses. An ND4 mouse immunized with PZP in PBS demonstrated a very weakly positive antibody binding (6.6% relative binding as compared to PZP-CFA, litter size = 12). Another ND4 mouse immunized using both MS and CTB demonstrated 53.6% relative binding but had a litter size of 14. Two C57BL/6 mice demonstrated specific anti-PZP antibody responses. One immunized with PZP-MS had 16.6% antibody binding and a litter size of 8 and the other immunized with PZP-MS-CTB had 35% antibody binding and a litter of 9. Immunization had no significant effect on litter size for any group (ANOVA, P > 0.05; Table 1) including the PZP-CFA group with mean (S.D.) of 9.5 (1.9) and 7.0 (1.2) for the ND4 and C57BL/6 strains, respectively. Three ND4 mice (one received MS alone, another PZP in PBS and the last PZP-CFA) had no litters; the former two also lacked specific anti-PZP antibody. As copulatory plugs were absent in some mice, the litter size results from these mice were not used in Table 1. All six rabbits immunized with PZP-CFA developed a strong specific anti-PZP antibody response, which was significantly different from the rabbits immunized with PZP-MS-CTB (ANOVA, P < 0.01; Table 2). Three PZP-CFA immunized rabbits demonstrated relative antibody binding >60% of the laboratory standard serum; two bred but had no litters and the third bred
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Table 1 Mouse relative antibody binding and litter sizes Mouse strain groupa
ND4
C57BL/6
Percent binding (S.D.)b,d 1. Alginate MS (PO) 2. MS-CTB (PO) 3. PZP (PO) 4. PZP in MS with CTB (PO) 5. PZP and alginate MS (PO) 6. PZP and CFA (SC)
Litter size (S.D.)c,e
2.5 (1.4) 2.8 (1.4) 3.4 (2.1) 11.9 (23.3) 1.6 (0.6) 100 (–)
9.0 (0.8) 9.4 (3.0) 9.0 (2.4) 10.0 (2.4) 9.8 (2.5) 9.5 (1.9)
Percent binding (S.D.)b,f
Litter size (S.D.)g
3.2 (1.1) 4.0 (1.3) 5.3 (1.6) 10.1 (14.0) 4.9 (6.7) 100 (–)
8.0 (1.2) 8.2 (1.3) 8.2 (0.4) 8.6 (0.9) 7.2 (2.6) 7.0 (1.2)
a MS, alginate microspheres; CTB, cholera toxin B; PZP, porcine zona pellucida; CFA, Freund’s complete and incomplete adjuvant; PO, gavage; SC, subcutaneous. b Titer values are a percentage of PZP-CFA immunized mice. c Excludes mice that did not breed. d Values for Groups 1–5 are not significantly different (ANOVA, P = 0.539). e Values for Groups 1–5 are not significantly different (ANOVA, P = 0.982). f Values for Groups 1–5 are not significantly different (ANOVA, P = 0.582). g Values for Groups 1–5 are not significantly different (ANOVA, P = 0.465).
Table 2 Rabbit relative antibody binding and litter sizes Groupa
Percent binding (S.D.)b
Litter size (S.D.)c
1. Alginate MS and CTB (IN) 2. PZP, MS and CTB (IN) 3. PZP and CFA (IM)
0.8 (0.9)d 3.2 (2.9)d 53.7 (20.4)e
8.8 (1.8) 6.4 (2.5) 5.3 (5.6)
Laboratory standard
100 (–)
ND
a MS, alginate microspheres; CTB, cholera toxin B; PZP, porcine zona pellucida; CFA, Freund’s complete and incomplete adjuvant; IN, intranasal; IM, intramuscular; laboratory standard is from rabbits immunized IM with PZP in CFA. b Titer values are a percentage of a laboratory standard serum from a PZP/Freunds immunized rabbit. c Excludes rabbits that did not accept the buck; ND, not done; values are not significantly different (ANOVA, P = 0.359). d Values are not significantly different (P = 0.273). e Group 3 percent binding is significantly different from Groups 1 and 2 (P < 0.01).
and had one fetus. The mucosal immunization utilizing alginate MS and CTB elicited specific anti-PZP antibody in two of the six rabbits. The antibody responses were weak at 4.3 and 8.4% of the standard and did not impact fertility (litter sizes of 7 and 8, respectively). 4. Discussion The results of this study indicate that porcine zona pellucida was not an effective antigen for mucosal immunization of small mammals compared to PZP in CFA. Using an effective immunization technique (Bynum, personal communication), the mice in this study generated adequate antibody titers when CFA was used as an adjuvant. A similar dosage of PZP augmented with mucosal immunostimulants failed to generate significant antibody responses. When cholera toxin was used as the mucosal adjuvant, some individual mice of both mouse strains appeared to generate a detectable antibody response. Freunds adjuvant induced a much stronger antibody response but failed to impact fertility. Fertility reduction has not been
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consistently observed in mice immunized against zona pellucida antigen (Sacco et al., 1981; Lou et al., 1995; Sun et al., 1999). The PZP antigen dosage (Dietl et al., 1982) and immunization technique (D. Wilkening personal communication) have been successfully used in rabbits. Immunization with PZP-CFA also generated good antibody responses in this study. However, the combined mucosal immunostimulants of CTB and alginate MS did not reliably induce an antibody response in these rabbits. Antigens administered with the immunostimulant cholera toxin typically stimulate a powerful antibody response at mucosal surfaces and in serum (Liang et al., 1988; Czinn and Nedrud, 1991; Suckow et al., 1996a). An alternative approach to the use of adjuvants is to encapsulate antigens into microspheres. Alginate MS and CTB, either alone or in combination, were not effective in mice and rabbits when used to augment immune response to PZP. These results are in contrast to earlier studies that demonstrated enhancement of mucosal immunity to antigens when microencapsulated in alginate microspheres and/or when co-administered with cholera toxin (Suckow et al., 1996b; Jarvinen et al., 2000). The mechanism for the unresponsiveness observed in the present study is undefined. In conclusion, while mucosal immunization against PZP could open new avenues of population control, this study shows that neither rabbits nor two strains of mice demonstrate reliable antibody responses and fertility effects to mucosal immunization with PZP in alginate MS using CTB as the immunostimulant. Other species or immunization techniques may offer different results. A recent study reported the use of chitosan, another polysaccharide mucosal adjuvant, with a PZP DNA vaccine (Sun et al., 2004). The authors reported that immunized mice synthesized intestinal PZP protein but no immunological responses were reported. References Bowersock, T.L., HogenEsch, H., Suckow, M., Turek, J., Davis-Snyder, E., Borie, D., Jackson, R., Park, H., Park, K., 1996. Administration of ovalbumin encapsulated in alginate microspheres to mice. In: Ottenbrite, R.M., Huang, S.J., Park, K. (Eds.), Hydrogels and Biodegradable Polymers for Bioapplications. American Chemical Society, Washington, DC, pp. 58–67. Bowersock, T.L., HogenEsch, H., Suckow, M., Guimond, P., Martin, S., Borie, D., Torregrosa, S., Park, H., Park, K., 1999. Oral vaccination of animals with antigens encapsulated in alginate microspheres. Vaccine 17, 1804–1811. Czinn, S.J., Nedrud, J.G., 1991. Oral immunization against Helicobacter pylori. Infect. Immun. 59, 2359–2363. Dietl, J., Freye, J., Mettler, L., 1982. Fertility inhibition using low-dose immunization with porcine zonae pellucidae. Am. J. Reprod. Immunol. 2, 153–156. Florence, A.T., 1997. The oral absorption of micr- and nanoparticulates: niether exceptional nor unusual. Pharm. Res. 14, 259–266. Jarvinen, L.Z., HogenEsch, H., Suckow, M.A., Bowersock, T.L., 2000. Intranasal vaccination of New Zealand white rabbits against Pasteurellosis using alginate-encapsulated Pasteurella multocida toxin and potassium thiocyanate extract. Comp. Med. 50, 263–269. Keren, D.F., Suckow, M.A., 1994. Animal models for immunoglobulin A secretion. Methods Enzymol. 235, 140–155. Kirkpatrick, J.F., Calle, P.P., Kalk, P., Liu, I.K.M., Bernoco, M., Turner Jr., J.W., 1986. Immunocontraception of captive exotic species. II Formosan sika deer (Cervus nippon taiouanus), axis deer (Cervus axis), Himalayan tahr (Heritragus jemlahicus), Roosevelt elk (Cervus elaphus roosevelti), Reeves’ muntjac (Muntiacus reevesi), and sambar deer (Cervus unicolor). J. Zoo. Wildl. Med. 27, 482–495. Kirkpatrick, J.F., Naugle, R., Liu, I.K.M., Bernoco, M., Turner Jr., J.W., 1995. Effects of seven consecutive years of porcine zona pellucida contraception on ovarian function in feral mares. Biol. Reprod. Monogr. Ser. 1: Equine Reprod. VI, 411–418. Kirkpatrick, J.F., Turner Jr., J.W., 1996. Fertility control in wildlife management: a review. In: Cohn, P.N., Plotka, E.D., Seal, U.S. (Eds.), Contraception in Wildlife. Book 1. Edwin Meelon Press, Lewiston, NY, pp. 133–155. Lemoine, D., Wauters, S., Bouchend’homme, S., Preat, V., 1998. Preparation and characterization of alginate microspheres containing a model antigen. Int. J. Pharm. 176, 9–19.
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Liang, X.P., Lamm, M.E., Nedrud, J.G., 1988. Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus. J. Immunol. 141, 1495–1501. Liu, I.K.M., Bernoco, M., Feldman, M., 1989. Contraception in mares heteroimmunized with porcine zona pellucida. J. Reprod. Fertil. 85, 19–29. Lou, Y., Ang, J., Thai, H., McElveen, F., Tung, K.S.K., 1995. A zona pellucida 3 peptide vaccine induces antibodies and reversible infertility without ovarian pathology. J. Immunol. 155, 2715–2720. Mahi-Brown, C.A., McGuinness, R.P., Moran, F., 1992. The cellular immune response to immunization with zona pellucida antigens. J. Reprod. Immunol. 21, 29–46. Sacco, A.G., Subramanian, M.G., Yurewicz, E.C., 1981. Active immunization of mice with porcine zonae pellucidae: immune response and effect on fertility. J. Exp. Zool. 218, 405–418. Singh, M., O’Hagan, D., 1998. The preparation and characterization of polymeric antigen delivery systems for oral administration. Adv. Drug Deliv. Rev. 34, 285–304. Seong, S.Y., Cho, N.H., Kwon, I.C., Jeong, S.Y., 1999. Protective immunity of microsphere-based mucosal vaccines against lethal intranasal challenge with Streptococcus pneumoniae. Infect. Immun. 67, 3587–3592. Suckow, M.A., Bowersock, T.L., Nielsen, K., Grigdesby, C.F., 1996a. Enhancement of respiratory immunity to Pasteurella multocida by cholera toxin in rabbits. Lab. Anim. 30, 120–126. Suckow, M.A., Bowersock, T.L., Park, H., Park, K., 1996b. Oral immunization of rabbits against Pasteurella multocida with an alginate microsphere delivery system. J. Biomater. Sci. Polym. Ed. 8, 131–139. Suckow, M.A., Siger, L., Bowersock, T.L., Turek, J.J., Van Horne, D., Borie, D., Taylor, A., Park, H., Park, K., 1999. Alginate microparticles for vaccine delivery. In: El-Nokaly, M.A., Soini, H.A. (Eds.), Polysaccharide Applications: Cosmetics and Pharmaceuticals. American Chemical Society, Washington, DC, pp. 1–14. Suckow, M.A., Jarvinen, L.Z., HogenEsch, H., Park, K., Bowersock, T.L., 2002. Immunization of rabbits against a bacterial pathogen with an alginate microparticle vaccine. J. Control Release 85, 227–235. Sun, C., Pan, S., Xie, Q., Xiao, L., 2004. Preparation of chitosan-plasmid DNA nanoparticles encoding zona pellucida glycoprotein-3␣ and its expression in mouse. Mol. Reprod. Dev. 68, 182–188. Sun, W., Lou, Y.H., Dean, J., Tung, K.S.K., 1999. A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida. Biol. Reprod. 60, 900–907. Turner Jr., J.W., Kirkpatrick, J.F., Liu, I.K.M., 1996. Effectiveness, reversibility and serum antibody titers associated with immunocontraception in white-tailed deer. J. Wildl. Manag. 60, 45–51. Turner Jr., J.W., Liu, I.K.M., Flanagan, D.R., Rutberg, A.T., Kirkpatrick, J.F., 2001. Immunocontraception in feral horses: one inoculation provides one year of infertility. J. Wildl. Manag. 65, 235–241. Turner Jr., J.W., Liu, I.K.M., Flanagan, D.R., Bynum, K.S., Rutberg, A.T., 2002. Porcine zona pellucida (PZP) immunocontraception of wild horses (Equus caballus) in Nevada: a 10-year study. Reprod. Suppl. 60, 177–186. Zhou, S., Deng, X., Yuan, M., Li, X., 2002. Investigation on preparation and protein release of biodegradable polymer microspheres as drug-delivery system. J. Appl. Polym. Sci. 84, 778–784.
Short communication
Use of mucosal immunization with porcine zona pellucida (PZP) in mice and rabbits Brent J. Martin a,∗ , Mark A. Suckow b , William R. Wolter b , Thomas Berger b , John W. Turner Jr. c a
Division of Laboratory Animal Medicine, Medical University of Ohio, 3055 Arlington Avenue, Toledo, OH 43614, USA b Freiman Lifesciences Center, University of Notre Dame, Notre Dame, IN 46556, USA c Department of Physiology and Cardiovascular Genomics, Medical University of Ohio, 3055 Arlington Avenue, Toledo, OH 43614, USA Received 11 April 2005; accepted 14 September 2005 Available online 24 October 2005
Abstract Rabbits (Oryctolagus cuniculus) and two strains of mice (Mus musculus, one inbred and one outbred) were immunized against porcine zona pellucida (PZP) antigen. Alginate microspheres or cholera toxin B were used alone or in combination when mucosal immunization routes were used. Serum antibody responses and fertility were assessed. Neither rabbit or mouse groups immunized by mucosal routes generated significant antibody responses to PZP as compared to parenteral immunization (ANOVA, P > 0.05). The study shows that porcine zona pellucida is not an effective mucosal antigen in small mammals. © 2005 Elsevier B.V. All rights reserved. Keywords: Porcine zona pellucida; PZP; Mucosal immunity; Rabbit; Mouse; Alginate microsphere
1. Introduction Parenteral immunization, which primarily stimulates a systemic immune response, with porcine zona pellucida (PZP) is an effective means of reducing fertility in a variety of species (Kirkpatrick et al., 1986; Kirkpatrick and Turner, 1996; Turner et al., 1996, 2002). Although some reports have indicated success with single immunizations with long-acting pellets (Turner et al., 2001), most studies have involved multiple parenteral injections (Kirkpatrick et al., 1995). ∗
Corresponding author. Tel.: +1 419 383 4310; fax: +1 419 383 3021. E-mail address: [email protected] (B.J. Martin).
0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.09.007
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373
The requirement for sufficient animal access necessary to administer parenteral injections, and especially multiple injections, is a significant barrier to use of this method for animal population control. Secretive, nocturnal and abundant species, as well as those inhabiting dense cover and hostile environments, often cannot be reliably captured in sufficient numbers to allow effective population management by injection. Thus, an alternative approach focusing on development of an immunization method avoiding direct animal access could open new avenues of animal control. Previous studies using oral and intranasal vaccination to induce mucosal immunity generated efficacious immune response in many species to a variety of antigens (Bowersock et al., 1999). Many antigens are weakly immunogenic when administered to mucosal surfaces. For this reason, auxiliary materials are often incorporated to enhance the immune response. In this regard, the B-subunit of cholera toxin (CTB) is an effective adjuvant for mucosal immunity. In mice and rabbits, co-administration of cholera toxin has been shown to enhance mucosal immunity to various antigens (Liang et al., 1988; Czinn and Nedrud, 1991; Keren and Suckow, 1994; Suckow et al., 1996a). Antigens administered via the mucosa are subject to pH extremes and enzymatic action, particularly in the gastrointestinal system. Microencapsulation of antigens within a variety of polymers has been widely used to provide protection of antigens until absorption occurs. Furthermore, microencapsulation is another means of enhancing the response to vaccination at mucosal surfaces and the effect is presumed to be via augmentation of uptake by M cells of the Peyer’s patches (Florence, 1997; Singh and O’Hagan, 1998; Lemoine et al., 1998; Suckow et al., 1999). Encapsulation of antigens in alginate microspheres (MS) has been demonstrated to be an effective means of stimulating mucosal immunity following immunization in both mice (Bowersock et al., 1996) and rabbits (Suckow et al., 1996b; Jarvinen et al., 2000). Encapsulation with alginate is a mild aqueous process often selected to avoid antigen degradation (Singh and O’Hagan, 1998; Bowersock et al., 1999; Zhou et al., 2002). The objective of this study was to evaluate the efficacy of selected mucosal immunization techniques to PZP in two different species, mice and rabbits.
2. Materials and methods 2.1. Experimental animals All animal use was approved by the institutional animal care and use committees of the responsible AAALAC accredited institutions and care was consistent with the Guide for the Care and Use of Laboratory Animals. Animals were allowed to adapt to laboratory conditions for 1 week prior to use and were housed in conventional cages on a constant 12-h light/12-h dark cycle with controlled temperature and humidity and ad libitum access to food and water. Mice were adult outbred ND4 Swiss Webster and inbred C57BL/6 (Mus musculus, Harlan, Indianapolis, IN). Vendor health reports indicated that the mice were serologically negative for known murine pathogens and research facility health surveillance were confirmatory. During immunization periods, mice were housed four per cage and for breeding periods, were housed three per cage (one male per two females). Specific pathogen-free New Zealand white rabbits weighing 3.0–4.3 kg were acquired (Covance, Inc. Kalamazoo, MI) as documented proven breeders. The rabbits were housed singly, except for periods of <30 min during which the female was placed in a male’s cage for breeding.
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2.2. Immunizations Utilizing pig ovaries obtained from a commercial slaughterhouse, porcine zona pellucida was isolated as previously described (Liu et al., 1989). Briefly, frozen-thawed ovaries were minced, filtered and ground in the presence of phosphate buffered saline (PBS, pH 7.2). Recovered zonae were heat solubilized at 70 ◦ C for 30 min in PBS. Characteristics of the PZP isolate were determined by gel electrophoresis and the preparation was screened for viral and bacterial contamination (ZooMontana, Billings, MT). Alginate microspheres were prepared to contain PZP with or without cholera toxin B-subunit (Sigma Chemical, St. Louis, MO) as previously described (Suckow et al., 1999). Briefly, sodium alginate, medium viscosity (Keltone LV® , Monsanto, St. Louis, MO) was dissolved in distilled water at a 2% (w/v) concentration. PZP and/or CTB were slowly added. During rapid stirring, the solution was dropped into 1.5% (w/v) CaCl2 via a 30-gauge needle. The resultant MS were isolated by low-speed centrifugation and placed in 0.05% poly-l-lysine (MW 100,000; Sigma Chemical Co., St. Louis, MO) with stirring for 30 min at room temperature. Approximately, 75% of the particles were 5 m or less in diameter as measured under light microscopy. Microspheres containing PZP were prepared with either 100 or 20 g PZP/100 l MS suspension for use in mice and rabbits, respectively. Microspheres containing CTB were prepared with either 20 or 200 g CTB/100 l MS suspension for use in mice and rabbits, respectively. Mice receiving PZP were exposed to 100 g/dose as previously determined to be efficacious in parenteral immunization studies (Mahi-Brown et al., 1992; Bynum, personal communication). The mucosal immunization techniques were developed from previous successful studies (Bowersock et al., 1999; Seong et al., 1999). Groups of both mouse strains were immunized once a week for 3 weeks. Each group consisted of five female mice. Test groups were: alginate MS; alginate MS with cholera toxin (20 g CTB/dose); PZP in PBS; PZP with alginate MS; PZP in MS with 20 g CTB. Immunizations were by oral gavage (200 l total volume) in all test groups. Additionally, five mice received subcutaneous injections of PZP (in 100 l PBS) emulsified with 100 l CFA or IFA for primary and booster immunizations, respectively. Males were added to cages for breeding 3 days after the final immunization dose. All mice were euthanized with CO2 and exsanguination by cardiac puncture 17 days later. Fetuses and implantation sites were enumerated in each animal to determine litter sizes. Serum was separated and frozen at −20 ◦ C until assayed. Rabbits receiving PZP were exposed to 20 g/dose as previously determined to be efficacious in parenteral immunization studies (Dietl et al., 1982; D. Wilkening, personal communication). The mucosal immunization techniques were developed from previous studies with similar antigens (Suckow et al., 1996b, 2002; Bowersock et al., 1999; Jarvinen et al., 2000). Rabbits were immunized with PZP (20 g/dose) in volumes of 0.4 ml PBS combined with an equal volume of adjuvant. Groups of six female rabbits were immunized intranasally three times at 2-week intervals. Immunization groups included alginate MS with CTB (200 g) and PZP in alginate MS with CTB (200 g). An additional group was immunized intramuscularly with PZP (in 125 l PBS) emulsified with 125 l CFA initially and with PZP in IFA 28 days later. Immunized does were natural bred 50 days after the initial immunization by placing each in a cage with a proven breeder male rabbit. A 1 ml blood sample was collected via marginal ear vein venipuncture just prior to introduction of the male and serum frozen at −20 ◦ C until analyzed. Does were euthanized 24 days later and uteri examined for fetus number.
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2.3. ELISA assays Serum samples were analyzed by ELISA for antibody binding to PZP. The mouse ELISA was validated via mouse anti-PZP antibody from a previous study (Bynum, personal communication). Mouse sera were analyzed in duplicate at a dilution of 1:50 and compared to the PZP-CFA immunized control serum at 1:400 or 1:1600. The rabbit ELISA was performed by a reference laboratory (ZooMontana, Billings, MT). Rabbit sera were analyzed in duplicate at 1:1280 and compared to a laboratory standard PZP immunized rabbit serum at 1:1280. Microtiter plates (96-well) were coated with 12 g native PZP and blocked with 0.1% gelatin. Experimental antibody was detected with goat anti-mouse or goat anti-rabbit antibody (Kirkegaard & Perry Labs, Gaithersburg, MD 20879) conjugated with alkaline phosphatase followed by p-nitrophenyl phosphate. Absorbance was measured at 405 nm on microplate reader. 2.4. Analysis Litter sizes were reported as within group means (S.D.) and analyzed by treatment using oneway ANOVA (SPSS for Windows, 2002). Anti-PZP antibody binding was reported as the mean percentage of each group (mean (S.D.)) as compared to a PZP-CFA immunized control sera. In mice, the relative antibody binding was compared to the PZP-CFA immunized group and the remaining five groups’ means were analyzed by ANOVA. In rabbits, the relative antibody binding comparison was made to a laboratory standard PZP-CFA immunized rabbit sera (ZooMontana, Billings, MT) and the data analysis (ANOVA, SPSS for Windows, 2002) encompassed the three groups in this study. Significance was set at P < 0.05. Control group (receiving no PZP) mean percent binding + 3S.D. of the control group data was considered the threshold value to identify specific anti-PZP antibody responses in individual animals. 3. Results Neither the alginate MS nor MS with CTB when administered in combination with PZP elicited significant anti-PZP antibody binding compared to PZP-CFA (ANOVA, P > 0.05) in either mouse strain (Table 1). All PZP-CFA immunized mice had strong, specific anti-PZP antibody responses. Four mice immunized via mucosal routes demonstrated specific anti-PZP antibody responses. An ND4 mouse immunized with PZP in PBS demonstrated a very weakly positive antibody binding (6.6% relative binding as compared to PZP-CFA, litter size = 12). Another ND4 mouse immunized using both MS and CTB demonstrated 53.6% relative binding but had a litter size of 14. Two C57BL/6 mice demonstrated specific anti-PZP antibody responses. One immunized with PZP-MS had 16.6% antibody binding and a litter size of 8 and the other immunized with PZP-MS-CTB had 35% antibody binding and a litter of 9. Immunization had no significant effect on litter size for any group (ANOVA, P > 0.05; Table 1) including the PZP-CFA group with mean (S.D.) of 9.5 (1.9) and 7.0 (1.2) for the ND4 and C57BL/6 strains, respectively. Three ND4 mice (one received MS alone, another PZP in PBS and the last PZP-CFA) had no litters; the former two also lacked specific anti-PZP antibody. As copulatory plugs were absent in some mice, the litter size results from these mice were not used in Table 1. All six rabbits immunized with PZP-CFA developed a strong specific anti-PZP antibody response, which was significantly different from the rabbits immunized with PZP-MS-CTB (ANOVA, P < 0.01; Table 2). Three PZP-CFA immunized rabbits demonstrated relative antibody binding >60% of the laboratory standard serum; two bred but had no litters and the third bred
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Table 1 Mouse relative antibody binding and litter sizes Mouse strain groupa
ND4
C57BL/6
Percent binding (S.D.)b,d 1. Alginate MS (PO) 2. MS-CTB (PO) 3. PZP (PO) 4. PZP in MS with CTB (PO) 5. PZP and alginate MS (PO) 6. PZP and CFA (SC)
Litter size (S.D.)c,e
2.5 (1.4) 2.8 (1.4) 3.4 (2.1) 11.9 (23.3) 1.6 (0.6) 100 (–)
9.0 (0.8) 9.4 (3.0) 9.0 (2.4) 10.0 (2.4) 9.8 (2.5) 9.5 (1.9)
Percent binding (S.D.)b,f
Litter size (S.D.)g
3.2 (1.1) 4.0 (1.3) 5.3 (1.6) 10.1 (14.0) 4.9 (6.7) 100 (–)
8.0 (1.2) 8.2 (1.3) 8.2 (0.4) 8.6 (0.9) 7.2 (2.6) 7.0 (1.2)
a MS, alginate microspheres; CTB, cholera toxin B; PZP, porcine zona pellucida; CFA, Freund’s complete and incomplete adjuvant; PO, gavage; SC, subcutaneous. b Titer values are a percentage of PZP-CFA immunized mice. c Excludes mice that did not breed. d Values for Groups 1–5 are not significantly different (ANOVA, P = 0.539). e Values for Groups 1–5 are not significantly different (ANOVA, P = 0.982). f Values for Groups 1–5 are not significantly different (ANOVA, P = 0.582). g Values for Groups 1–5 are not significantly different (ANOVA, P = 0.465).
Table 2 Rabbit relative antibody binding and litter sizes Groupa
Percent binding (S.D.)b
Litter size (S.D.)c
1. Alginate MS and CTB (IN) 2. PZP, MS and CTB (IN) 3. PZP and CFA (IM)
0.8 (0.9)d 3.2 (2.9)d 53.7 (20.4)e
8.8 (1.8) 6.4 (2.5) 5.3 (5.6)
Laboratory standard
100 (–)
ND
a MS, alginate microspheres; CTB, cholera toxin B; PZP, porcine zona pellucida; CFA, Freund’s complete and incomplete adjuvant; IN, intranasal; IM, intramuscular; laboratory standard is from rabbits immunized IM with PZP in CFA. b Titer values are a percentage of a laboratory standard serum from a PZP/Freunds immunized rabbit. c Excludes rabbits that did not accept the buck; ND, not done; values are not significantly different (ANOVA, P = 0.359). d Values are not significantly different (P = 0.273). e Group 3 percent binding is significantly different from Groups 1 and 2 (P < 0.01).
and had one fetus. The mucosal immunization utilizing alginate MS and CTB elicited specific anti-PZP antibody in two of the six rabbits. The antibody responses were weak at 4.3 and 8.4% of the standard and did not impact fertility (litter sizes of 7 and 8, respectively). 4. Discussion The results of this study indicate that porcine zona pellucida was not an effective antigen for mucosal immunization of small mammals compared to PZP in CFA. Using an effective immunization technique (Bynum, personal communication), the mice in this study generated adequate antibody titers when CFA was used as an adjuvant. A similar dosage of PZP augmented with mucosal immunostimulants failed to generate significant antibody responses. When cholera toxin was used as the mucosal adjuvant, some individual mice of both mouse strains appeared to generate a detectable antibody response. Freunds adjuvant induced a much stronger antibody response but failed to impact fertility. Fertility reduction has not been
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consistently observed in mice immunized against zona pellucida antigen (Sacco et al., 1981; Lou et al., 1995; Sun et al., 1999). The PZP antigen dosage (Dietl et al., 1982) and immunization technique (D. Wilkening personal communication) have been successfully used in rabbits. Immunization with PZP-CFA also generated good antibody responses in this study. However, the combined mucosal immunostimulants of CTB and alginate MS did not reliably induce an antibody response in these rabbits. Antigens administered with the immunostimulant cholera toxin typically stimulate a powerful antibody response at mucosal surfaces and in serum (Liang et al., 1988; Czinn and Nedrud, 1991; Suckow et al., 1996a). An alternative approach to the use of adjuvants is to encapsulate antigens into microspheres. Alginate MS and CTB, either alone or in combination, were not effective in mice and rabbits when used to augment immune response to PZP. These results are in contrast to earlier studies that demonstrated enhancement of mucosal immunity to antigens when microencapsulated in alginate microspheres and/or when co-administered with cholera toxin (Suckow et al., 1996b; Jarvinen et al., 2000). The mechanism for the unresponsiveness observed in the present study is undefined. In conclusion, while mucosal immunization against PZP could open new avenues of population control, this study shows that neither rabbits nor two strains of mice demonstrate reliable antibody responses and fertility effects to mucosal immunization with PZP in alginate MS using CTB as the immunostimulant. Other species or immunization techniques may offer different results. A recent study reported the use of chitosan, another polysaccharide mucosal adjuvant, with a PZP DNA vaccine (Sun et al., 2004). The authors reported that immunized mice synthesized intestinal PZP protein but no immunological responses were reported. References Bowersock, T.L., HogenEsch, H., Suckow, M., Turek, J., Davis-Snyder, E., Borie, D., Jackson, R., Park, H., Park, K., 1996. Administration of ovalbumin encapsulated in alginate microspheres to mice. In: Ottenbrite, R.M., Huang, S.J., Park, K. (Eds.), Hydrogels and Biodegradable Polymers for Bioapplications. American Chemical Society, Washington, DC, pp. 58–67. Bowersock, T.L., HogenEsch, H., Suckow, M., Guimond, P., Martin, S., Borie, D., Torregrosa, S., Park, H., Park, K., 1999. Oral vaccination of animals with antigens encapsulated in alginate microspheres. Vaccine 17, 1804–1811. Czinn, S.J., Nedrud, J.G., 1991. Oral immunization against Helicobacter pylori. Infect. Immun. 59, 2359–2363. Dietl, J., Freye, J., Mettler, L., 1982. Fertility inhibition using low-dose immunization with porcine zonae pellucidae. Am. J. Reprod. Immunol. 2, 153–156. Florence, A.T., 1997. The oral absorption of micr- and nanoparticulates: niether exceptional nor unusual. Pharm. Res. 14, 259–266. Jarvinen, L.Z., HogenEsch, H., Suckow, M.A., Bowersock, T.L., 2000. Intranasal vaccination of New Zealand white rabbits against Pasteurellosis using alginate-encapsulated Pasteurella multocida toxin and potassium thiocyanate extract. Comp. Med. 50, 263–269. Keren, D.F., Suckow, M.A., 1994. Animal models for immunoglobulin A secretion. Methods Enzymol. 235, 140–155. Kirkpatrick, J.F., Calle, P.P., Kalk, P., Liu, I.K.M., Bernoco, M., Turner Jr., J.W., 1986. Immunocontraception of captive exotic species. II Formosan sika deer (Cervus nippon taiouanus), axis deer (Cervus axis), Himalayan tahr (Heritragus jemlahicus), Roosevelt elk (Cervus elaphus roosevelti), Reeves’ muntjac (Muntiacus reevesi), and sambar deer (Cervus unicolor). J. Zoo. Wildl. Med. 27, 482–495. Kirkpatrick, J.F., Naugle, R., Liu, I.K.M., Bernoco, M., Turner Jr., J.W., 1995. Effects of seven consecutive years of porcine zona pellucida contraception on ovarian function in feral mares. Biol. Reprod. Monogr. Ser. 1: Equine Reprod. VI, 411–418. Kirkpatrick, J.F., Turner Jr., J.W., 1996. Fertility control in wildlife management: a review. In: Cohn, P.N., Plotka, E.D., Seal, U.S. (Eds.), Contraception in Wildlife. Book 1. Edwin Meelon Press, Lewiston, NY, pp. 133–155. Lemoine, D., Wauters, S., Bouchend’homme, S., Preat, V., 1998. Preparation and characterization of alginate microspheres containing a model antigen. Int. J. Pharm. 176, 9–19.
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