Immunology 1991 73 388-393
ADONIS 0019280591001 70S
Oral immunization with xenogeneic antibodies stimulates the production of systemic and mucosal anti-idiotypic antibodies A. M. COLLINS,*t§ D. M. ROBERTON,*l C. S. HOSKING* & G. R. FLANNERYt *Royal Children's Hospital, Melbourne, tLa Trobe University, Bundoora, Melbourne and jAdelaide Children's Hospital, Adelaide, Australia
Acceptedfor publication 9 April 1991
SUMMARY The humoral and mucosal immune responses to oral immunization with xenogeneic antibodies were studied using an animal model in which female rabbits were fed daily doses of the MOPC-3 15 murine IgA antibody, and were mated during the course of the feeding programme. Serum and colostrum samples were assayed for the presence of anti-idiotypic antibodies by ELISA assay, before and after depletion of anti-IgA antibodies, by affinity chromatography using another murine IgA idiotype. It was shown that all animals responded to exposure to the MOPC-3 15 idiotype with the production of serum anti-murine immunoglobulin antibodies and that four of six animals produced serum anti-idiotypic antibodies. That the immune response included antibodies directed against the antigen-binding site was confirmed by competition ELISA assay. Mucosal IgG and IgA antiimmunoglobulin antibodies were present in milk from all antibody-fed rabbits tested, and IgA antiidiotypic antibodies were detectable in the colostrum of one rabbit. The results provide some support for the hypothesis that human exposure to xenogeneic antibodies, most commonly bovine milk immunoglobulins, may provoke the production of anti-idiotypic antibodies, and that such exposure may lead to disturbances of immune regulation.
INTRODUCTION Fifteen years after the possibility of idiotypic network regulation was first proposed,' controversy persists as to the functional importance of idiotypic network interactions, but the veracity of experimental manipulations of the immune response using idiotypes and anti-idiotypes is beyond question.2 It has frequently been observed that it is far easier to generate an antiidiotypic immune response by immunization with xenogeneic antibodies than it is by immunization with allogeneic or isologous idiotypes,2'3 but the fact that exposure to xenogeneic antibodies is a normal feature of human life seems to have been overlooked. This exposure is principally a consequence of bovine milk consumption, and it has recently been proposed by one of us (A. M. Collins) that this may have important implications for human health. It has been suggested that antibodies directed against dietary xenogeneic immunoglobulins should include anti-idiotypic antibodies, and that such antiidiotypic antibodies could disturb the normal regulation of the immune response in an idiotype-specific way.4 The hypothesis rests on the demonstrated survival of bovine milk antibodies during commercial pasteurisation,5 and the identification of bovine antibodies directed against common
human allergens (A. M. Collins et al., manuscript submitted for publication). It also requires the survival of bovine milk antibodies in the proteolytic environment of the gut. This survival is well established in a number of species, including humans6 and rabbits.7 Although bovine milk IgGl is not protected from proteolysis by its attachment to a secretory component, as is the case with the predominant milk antibodies of non-ruminants,8 it nevertheless seems relatively resistant to proteolytic degradation.9 In addition to the stability which might be conferred by their protein chemistry, bovine milk antibodies may additionally be protected from proteolysis by the presence of trypsin inhibitors in milk,'0 by the buffering action of other milk proteins," and in the neonate by low gastric acidity and the rapid transit of whey proteins to the more benign environment of the duodenum. 12 As part of a series of studies investigating the hypothesis, an animal model was established to investigate the nature of the immune response to oral immunization with xenogeneic antibodies. A rabbit model was chosen because both humans and rabbits sometimes produce circulating IgG antibodies in response to ingested proteins. 13''4 There is also evidence that rabbits are able to mount an immune response to dietary bovine milk gammaglobulins,'5 and it has been shown that young rabbits are able to respond to specific determinants on ingested allogeneic milk antibodies. 16 A model was developed in which adult female rabbits were exposed to murine IgA anti-DNP antibodies produced by the
§ Present address and correspondence: Dept. of Microbiology and Immunology, University of New South Wales, P.O. Box 1, Kensington, NSW 2033, Australia.
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Oral immunization with xenogeneic antibodies MOPC-3 15 mouse myeloma cell line,'7 and weekly serum samples were collected for the investigation of systemic responsiveness. Six weeks after the commencement of feeding, the rabbits were mated, and colostrum and early milk samples were collected at parturition for the investigation of the mucosal immune response to the feeding programme. Results show that rabbits are capable of mounting both systemic and mucosal immune responses upon oral immunization with xenogeneic antibodies, and that anti-idiotypic antibodies form a detectable component of the response, at least in some rabbits. MATERIALS AND METHODS Animals Rabbits were provided by the Animal Research Laboratories, Royal Children's Hospital, Melbourne, Australia. All rabbits were part of their closed breeding colony, being descended from NZW, Belgian and Australian rabbits. BALB/c mice were obtained from the Department of Genetics and Human Variation, La Trobe University, Melbourne and from the Animal Research Laboratory of the Royal Children's Hospital, Melbourne, Australia. Cell culture and ascites preparation Growth of MOPC-31517 and J558 cells'8 was initiated and maintained in DMEM (Flow Laboratories, North Ryde, Australia), containing 20% heat-inactivated foetal calf serum (FCS) (Commonwealth Serum Laboratories, Melbourne, Australia). Ascites was raised in BALB/c mice primed with 10 ml of pristane (Sigma, St Louis, MO) 10-14 days previously, and immunized i.p. with 1 0 x 107-1 5 x 107 cells.
Purification of antibodies MOPC-315 and J558 IgA antibodies were purified by ionexchange chromatography using DEAE-Sepharose CL-6B beads. The MOPC-315 antibody solution obtained was diluted to a concentration of 125 pg/ml in casein buffer (phosphatebuffered saline containing 25 mg/ml casein hydrolysate), and aliquots were frozen at -20°. MOPC-3 15 antibodies for parenteral immunization and for ELISA plate coating were further purified by affinity chromatography using human secretory component.'9 Feeding schedule Six parous female rabbits were fed purified MOPC-315 antibodies by naso-gastric feeding tubes on a 28-day cycle of 14 days of feeding and 14 days of rest. On feeding days, each rabbit received 0-5 mg of MOPC-3 15 antibody, preceded and followed by 5 ml of casein buffer. Feeding continued through pregnancy and to the end of the fourth week of lactation. Mating of rabbits Rabbits were mated at the end of the second 14-day feeding period. One rabbit was mated again shortly after the birth of its first litter and antibody-feeding continued until after the birth of the second litter.
Sample collection Blood samples (3-5 ml) were collected weekly by ear-bleed. Colostrum and early milk samples were taken from five of the six rabbits on the feeding programme. The sixth rabbit failed to
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carry a pregnancy to term. Milk flow was encouraged by the injection of 0-2 ml of oxytocin (Syntocinon; Sandoz, Basle, Switzerland). One rabbit gave birth twice while on the feeding programme, and colostrum was collected from this rabbit within 24 hr of each birth. Serum, colostrum and early milk samples were also collected from six control rabbits.
Preparation of a polyclonal anti-MOPC-315 antiserum A polyclonal anti-idiotypic antiserum was raise by immunizing a rabbit with purified MOPC-3 15 antibodies emulsified in Freund's complete adjuvant (FCA) (Commonwealth Serum Laboratories). One milligram of antibody was injected subcuta-. neously into the hindquarter of the rabbit, and it was boosted every fortnight for 6 weeks with 0-5 mg of antibody emulsified in incomplete Freund's adjuvant (IFA) (Commonwealth Serum Laboratories). Absorption of anti-allotypic and anti-isotypic antibodies by affinity chromatography Purified J558 murine IgA antibodies were coupled to CNBractivated Sepharose 4B beads (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions, and were used to absorb anti-mouse IgA antibodies from both serum and colostrum samples.
Biotinylation Cappel goat anti-rabbit IgA antibodies (Organon Teknika, West Chester, PA) were biotinylated using biotin N-hydroxysuccinimide ester (Calbiochem, La Jolla, CA) and the method of Goding.20 ELISA assays Rabbit anti-MOPC-315 antibodies were detected in serum and colostrum samples by a direct binding ELISA assay2' in which purified MOPC-315 at a concentration of 10 ,ug/ml in coating buffer was bound to the plate overnight at 4°. Peroxidaselabelled anti-rabbit IgG (Organon Teknika) at a dilution of 1: 1000 and biotinylated anti-rabbit IgA at a dilution of 1:50 were used for the detection of plate-bound anti-MOPC-315 antibodies. Streptavidin-biotin-peroxidase complex (Amersham International, Amersham, Bucks, U.K.) was used at a dilution of 1:500. Tetramethylbenzadine (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used as the substrate and plates were read in a Titertek Multiscan MC Plate Reader (Flow Laboratories, Irvine, Ayrshire, U.K.) at 450 nm. Rabbit IgG and IgA anti-J558 ELISA assays were identical to the MOPC315 ELISA assays except that J558 antibodies were coated to the microtitre plates at a concentration of 10 Mg/ml. An unabsorbed serum sample from the parenterally immunized rabbit was chosen as the reference sample for assays of serum antibodies and was assigned 2-56 x 105 ELISA units (EU)/ml. Duplicate doubling dilutions of the reference standard were used to produce a standard curve against which sample results could be calculated. Absorbed and unabsorbed rabbit serum samples were assayed as triplicates at dilutions from I:1 to 1:2000. A milk sample collected after the birth of the first litter of Rabbit I served as the milk IgA reference standard. It was shown that the reference standard had almost four times the IgA activity against MOPC-315 as it had against J558 antibodies, and was therefore assigned 1000 EU/ml of anti-MOPC-315
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Figure 1. IgG anti-MOPC-315 and anti-J558 antibodies in J558-absorbed and unabsorbed serum samples from Rabbits 1-6 (a-f, respectively) during antibody-feeding experiments, as measured by MOPC-315 and J558 ELISA assays. Feeding began at Day 0. Results are expressed with respect to a polyclonal anti-MOPC-3 15 antiserum standard. (0) MOPC-3 15 absorbed; (-) J558 absorbed; (-) MOPC-315 unabsorbed; (0) J558 unabsorbed.
activity, and 250 EU/ml of anti-J558 IgA activity. Triplicate samples were assayed at dilutions of 1:4 (Absorbed) and 1:9 (Unabsorbed). Duplicate doubling dilutions of the reference standard were assayed from an initial dilution of 1:9. A selection of unabsorbed milk samples was assayed for IgG antibodies directed against the MOPC-315 antibody using the MOPC-315 IgG ELISA. Duplicate samples were assayed as doubling dilutions from initial dilutions of 1:1. Milk from the first litter of Rabbit 1 was again used as the reference standard, and was assigned 1000 EU/ml of IgG anti-MOPC-3 15 activity. Competition ELISA assays Anti-idiotypic binding was confirmed using a competition ELISA assay. Fifty-microlitre aliquots of doubling dilutions of serum samples were added to microtitre plates to which DNPBSA had been bound at a concentration of 25 mg/ml. Fiftymillilitre aliquots of mouse MOPC-315 idiotype were then added to the plates at a constant concentration of 100 ng/ml, and the plates were incubated for 1 hr at 37°. Plate-bound murine IgA antibodies were then detected using peroxidaselabelled goat anti-mouse IgA antibodies at a dilution of 1: 1000, as has been previously outlined. Pre- and post-feeding samples from each rabbit were tested in duplicate. Each plate also included duplicate rows to which dilutions of polyclonal anti-idiotypic antiserum was added, and serum-free MOPC-3 15 cell culture supernatant which served as a reference standard against which all other results could be determined.
RESULTS
Systemic immune response The results of the MOPC-315 and J558 IgG ELISA assays for anti-idiotypic antibodies and other antibodies in antibody-fed rabbits are presented separately for each rabbit in Fig. la-f. All rabbits produced anti-MOPC-3 15 antibodies in response to oral immunization. While unabsorbed serum from Rabbit 1 contained almost 1-7 x 105 EU/ml in the MOPC-315 ELISA at the height of its response, unabsorbed serum from Rabbit 4 contained barely 600 EU/ml of activity at the cessation of feeding. Only Rabbits 1, 2, 3 and 5 showed clear evidence of anti-idiotypic antibodies in absorbed serum samples. Activity ranged from 1 10 EU/ml (Rabbit 1) to 5650 EU/ml (Rabbit 2). Serum samples generally showed more activity in the J558 ELISA than in the MOPC-315 ELISA. This was probably a consequence of the choosing of our reference standard. On the assumption that the reference standard, an early post-immunization sample, contained a small proportion of anti-idiotypic antibodies, it was assigned 2-56 x 105 EU/ml in both assays, but the presence of a higher proportion of anti-idiotypic antibodies was subsequently shown. This would have the consequence of lowering the measured response in the MOPC-315 ELISA in comparison to the J558 ELISA. Mucosal immune response The results of the MOPC-315 and J558 IgA ELISA assays of rabbit milk samples are given in Fig. 2a, b, respectively. Figure
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Figure 3. ELISA titration curves of IgG anti-MOPC-3 15 antibodies in unabsorbed milk samples from four MOPC-315 antibody-fed rabbits and two control rabbits, as measured by MOPC-315 ELISA assays. Results express means of duplicate sample assayed as doubling dilutions from initial dilutions of 1:1.
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Figure 2. IgA anti-MOPC-315 (a) and J558 (b) antibodies in J558absorbed (-) and unabsorbed (0) milk samples from five antibody-fed rabbits and six control rabbits, as measured by MOPC-315 and J558 ELISA assays. Results express means of triplicates in EU/ml with respect to the milk standard (Rabbit 1.1).
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2a shows that anti-MOPC-3 15 antibodies were detected only in the milk of Rabbits I and 3, and anti-idiotypic antibodies only in the milk of Rabbit 1. The results of the J558 ELISA, however, are more difficult to interpret. In particular, some anti-J558 activity was detected in unabsorbed samples from Controls 1 and 6 (Cl, C6), while unabsorbed milk from Rabbit 5 showed high activity in the J558 ELISA but not in the MOPC-315 ELISA. J558 antibodies carry many determinants, and it may be that some rabbits produce mucosal antibodies which cross-react with some of these determinants regardless of their exposure to murine MOPC-315 antibodies. The rabbits (1, 3 and 5) whose milk showed evidence of a mucosal response to MOPC-315 antibodies also showed the highest systemic immune responses to oral immunization with idiotype. A limited number of unabsorbed samples were assayed for anti-MOPC-3 15 milk IgG antibodies using the MOPC-3 15 IgG ELISA assays. Titration curves for these samples are presented as Figure 3. The two control samples showed less than 1 EU/ml of activity compared to the 1000 EU/ml of activity in the reference standard (Rabbit 1.1). Anti-MOPC-315 activity in Rabbit 5 milk was measured as 258 EU/ml. Anti-MOPC-315 activity was calculated to be 15 and 21 EU/ml, respectively, for samples from Rabbits 4 and 6. Characterization of anti-idiotypes by competition assay The results of the idiotype/anti-idiotype competition assays are presented as Fig. 4a, b. The presence of antibodies competing with antigen for the antigen-binding site of the MOPC-315 antibody was confirmed in sera from Rabbits 1, 3 and 5, and also in the polyclonal anti-idiotypic antiserum (P-aId). Inhibition of idiotype binding was seen with serum from Rabbit at a dilution of 1:320, from Rabbits 3 and 5 at dilutions of 1: 15, while serum from the parenterally immunized rabbit was inhibitory at a dilution of 1:2560.
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Figure 4. Competition ELISA titration curves of murine IgA MOPC315 antibody binding to plate-bound DNP-BSA in the presence of doubling dilutions of pre- and post-feeding serum samples from MOPC-315 antibody-fed rabbits (1-6) and serum (P-aId) before and after parenteral immunization with MOPC-3 15. Results express means of duplicate wells containing samples at doubling dilutions from initial dilutions of 1:9 (Standard and Rabbit I) and 1:1 (Rabbits 2-6).
DISCUSSION The results reported here demonstrate that oral immunization with xenogeneic antibodies results in both systemic and mucosal anti-immunoglobulin responses. The responses of four of six antibody-fed rabbits included detectable serum IgG antiidiotypic antibodies, and a component of these anti-idiotypic antibodies were binding-site directed. Both IgA and IgG antiimmunoglobulin antibodies were also detected in the milk of antibody-fed rabbits, but not in the milk of six control rabbits, and IgA anti-idiotypic antibodies were detected in the milk of one rabbit. There were insufficient sample volumes to more fully investigate the mucosal IgG response. The demonstration of
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both IgG and IgA anti-mouse immunoglobulin antibodies reflects the fact that both isotypes are important components of rabbit milk,8 and that IgG, as well as IgA, milk antibodies can be produced by rabbits in response to dietary proteins.2223 We believe these results represent the first demonstration of such responsiveness, for a search of the literature failed to locate a single study which attempted to elicit an anti-idiotypic response from the mucosal immune system, or to generate such a response from either the systemic or mucosal immune systems by oral immunization. There is, however, indirect evidence for the involvement of anti-idiotypic antibodies in the development of mucosal responsiveness. In a study investigating a neonatal immune response in the apparent absence of antigen exposure, it was suggested that the presence of secretory IgA and IgM antiEscherichia coli 0 and anti-poliovirus antibodies in secretions of the neonate could be a result of exposure to maternal antiidiotypic antibodies.24 The anti-idiotypic responses observed in antibody-fed rabbits were generally very low, and the biological significance of these responses could be questioned. While a more precise quantification in absolute rather than in relative units might be informative, modulation of the immune response has been achieved with remarkably low concentrations of anti-idiotypic antibodies-particularly when administered to neonates.25 We have therefore chosen to investigate the biological implications of oral immunization with xenogeneic antibodies by examining the antigen-specific (anti-DNP) responsiveness of the offspring of female rabbits exposed to MOPC-315 antibodies throughout pregnancy and lactation. Preliminary results suggest an antigenspecific modulation of both the IgG and IgE responses in these rabbits. In addition to the support the results reported here lend to the hypothesis that exposure to xenogeneic antibodies may lead to disturbances of immune function, the finding that oral immunization with xenogeneic antibodies can lead to the production of systemic and mucosal anti-idiotypic antibodies is an interesting phenomenon in its own right, and one which may have some practical implications. The results point to the need for greater caution in the application of some passive immunization therapy. Recently passive oral immunization has been demonstrated to be effective in the treatment of a variety of enteric bacterial,26 viral27 and protozoan28 infections. The results reported here suggest that such a therapeutic strategy may lead to the generation of anti-idiotypic antibodies, as has previously been noted after xenogeneic monoclonal29 and polyclonal30 antibody therapy. In fact, after multiple infusions of therapeutic xenogeneic antibodies, the anti-idiotypic component of the response may come to predominate.3' Such a response, after either parenteral or oral passive immunization, could have implications for later immunological responsiveness to the pathogens in question. The results also raise the possibility that the oral route may by an effective one for the administration of anti-idiotypic vaccines. Such a protocol might be adopted, for example, by those attempting to provoke a protective response in the neonate. Anti-idiotypic vaccines have been identified as having special potential in the neonate, for they avoid exposure of the neonate to pathogen, and they may induce responsiveness to carbohydrate antigens-to which neonates are poor responders.32 In addition, oral immunization with anti-idiotypic vaccines directly challenges the mucosal immune system, which
develops earlier in life than the systemic immune system.33 Such a protocol may therefore allow earlier immunization of the infant. It has been said that the hypothesis that human consumption of xenogeneic antibodies leads to idiotype-specific pertubations of immune responsiveness will be 'hard to prove or disprove'.34 Certainly the work reported here fails to 'prove' the hypothesis, but it has considerably strengthened arguments in favour of its continued investigation.
ACKNOWLEDGMENTS We thank Magdy Sourial, Shane Osterfield and Marcia Riordan for their assistance with the experimental work, and Dr H. Ward and Dr Michael Shelton for their suggestions and discussion.
REFERENCES 1. JERNE N.K. (1974) Towards a network theory of the immune system. Ann. Immunol. (Inst. Pasteur), 125, 373. 2. COHN M. (1986) The concept of functional idiotype network for immune regulation mocks all and comforts none. Ann. Immunol. (Inst. Pasteur), 137, 64. 3. LING N.R., ELLIOTT D. & LOWE J. (1987) Modulation of the murine immune response to human IgG by complexing with monoclonal antibodies. II. Antibody responses to idiotopes of the human IgG paraprotein and of the mouse monoclonal antibodies. Immunology, 62, 7. 4. COLLINS A.M. (1988) Xenogeneic antibodies and atopic disease. Lancet, i, 734. 5. YOLKEN R.H., LOSONSKY G.A., VONDERFECHT S., LEISTER F. & WEE S. (1985) Antibody to human rotavirus in cow's milk. N. Engl. J. Med. 312, 605. 6. HILPERT H., BRUssow H., MIETENS C., SIDOTI J., LERNER L. & WERCHAU H. (1987) Use of bovine milk concentrate containing antibody to rotavirus to treat rotavirus gastroenteritis in infants. J. Infect. Dis. 156, 158. 7. MCCLEAD R.E. & GREGORY S.A. (1984) Resistance of bovine colostral anti-cholera toxin antibody to in vitro and in vivo proteolysis. Infec. Immun. 44, 474. 8. WATSON D.L. (1980) Immunological functions of the mammary gland and its secretion-a comparative review. Aust. J. biol. Sci. 33, 403. 9. BROCK J.H., ARZABE F.R., PINEIRO A. & OLIVITO A. (1977) The effect of trypsin and chymotrypsin on the bactericidal activity and specific antibody activity of bovine colostrum. Immunology, 32,207. 10. MCLEAN B.S. & HOLMES I.H. (1981) Effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection. J. clin. Microbiol. 13, 22. 11. MORGAN K.L., BOURNE F.J., NEWBY T.J. & BRADLEY P.A. (1981) Humoral factors in the secretory immune system of ruminants. Adv. exp. Med. Biol. 137, 391. 12. MYLREA P.J. (1966) Digestion of milk in young calves. I. Flow and acidity of the contents of the small intestine. Res. Vet. Sci. 7, 733. 13. SILVERMAN G.A., PERI B.A. & ROTHBERG R.M. (1982) Systemic antibody responses of different species following ingestion of soluble protein antigens. Dev. Comp. Immunol. 6, 737. 14. PERI B.A. & ROTHBERG R.M. (1986) Transmission of maternal antibody prenatally and from milk into serum of neonatal rabbits. Immunology, 57, 49. 15. HANGLOW A.C., WELSH C.J.R., CONN P. & COOMBS R.R.A. (1985) Early rheumatoid-like synovial lesions in rabbits drinking cow's milk. Int. Archs Allergy appl. Immun. 78, 152. 16. ADLER F.L. & ADLER L.T. (1982) Consequences of prenatal exposure to maternal alloantigens. Ann. NY Acad. Sci. 392, 266.
Oral immunization with xenogeneic antibodies 17. EISEN H.N., SIMMs E.S. & POTTER M. (1968) Mouse myeloma proteins with anti-hapten antibody activity. The protein produced by plasma cell tumour MOPC 315. Biochemistry, 11, 4126. 18. CARSON D. & WEIGERT M. (1973) Immunochemical analysis of the cross-reacting idiotypes of mouse myeloma proteins with antidextran activity and normal anti-dextran antibody. Proc. natl. Acad. Sci. U.S.A. 70, 235. 19. JONES C.L., GEORGIOU G.M., FOWLER K.J., WAJNGARTEN P.I. & ROBERTON D.M. (1987) Purification of polymeric immunoglobulin from cell culture supernatants by affinity chromatography using secretory component. J. immunol. Meth. 104, 237. 20. GODING J.W. (1986) Monoclonal Antibodies: Principles and Practice, 2nd edn. Academic Press, London. 21. ENGVALL E. & PERLMANN P. (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzymelabelled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109, 129. 22. MONTGOMERY P.C., COHN J. & LALLY E.T. (1973) The induction and characterization of secreting IgA antibodies. In: The Immunoglobulin A system. (eds J. Mestecky and A. R. Lawton), pp. 453, Plenum Press, New York. 23. PERI B.A. & ROTHBERG R.M. (1986) Transmission of maternal antibody prenatally and from milk into serum of neonatal rabbits. Immunology, 57, 49. 24. MELLANDER L., CARLSSON B. & HANSON L.A. (1986) Secretory IgA and IgM antibodies to E. coli 0 and poliovirus type I antigens occur in amniotic fluid, meconium and saliva from newborns. A neonatal immune response without antigenic exposure: a result of antiidiotypic induction? Clin. exp. Immunol. 63, 555. 25. RUBINSTEIN L.J., YEH M. & BONA C.A. (1982) Idiotype-antiidiotype network. II. Activation of silent clones by treatment at birth with idiotypes is associated with the expansion of idiotypespecific helper T cells. J. exp. Med. 156, 506. 26. TACKET C.O., LOSONSKY G., LINK H., HOANG Y., GUESRY P.,
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HILPERT H. & LEVINE M.M. (1988) Protection by milk immunoglobulin concentrate against oral challenge with enterotoxigenic Escherichia coli. N. Engl. J. Med. 318, 1240. 27. DAVIDSON G.P., DANIELS E., NUNAN H., MOORE A.G., WHYTE P.B.D., FRANKLIN K., MCCLOUD P.I. & MOORE D.J. (1989) Passive immunisation of children with bovine colostrum containing antibodies to human rotavirus. Lancet, ii, 709. 28. TZiPORI S., ROBERTON D. & CHAPMAN C. (1986) Remission of diarrhoea due to cryptosporidiosis in an immunodeficient child treated with hyperimmune bovine colostrum. Br. Med. J. 293, 1276. 29. AUSTIN E.B., ROBINS R.A., DURRANT L.G., PRICE M.R. & BALDWIN R.W. (1989) Human monoclonal anti-idiotypic antibody to the
tumour-associated antibody 791T/36. Immunology, 67, 525. 30. HERLYN D., WETrENDORF M., SCHMOLL E., ILIOPOULOS D., SCHEDEL I., DREIKHAUSEN V., RAAB R., Ross A.H., JAKSCHE H., SCRIBA
M. & KoPROWSKI H. (1987) Anti-idiotypic immunization of cancer patients. Modulation of the immune response. Proc. nati. Acad. Sci. U.S.A. 84, 8055. 31. SHAWLER D.L., BARTHOLOMEW R.M., SMITH L.M. & DILLMAN R.O. (1985) Human immune response to multiple injections of murine monoclonal IgG. J. Immunol. 135, 1530. 32. CAPRA J.D. & BONA C. (1988) Idiotypes: practical advances from fundamental concepts. Immunol. Today, 9, 98. 33. HANSON L.A., ANDERSSON B., MELLANDER L., SODERSTROM T. &
EDEN C.S. (1985) Protective factors in milk and the development of the immune system. Pediatrics, 75 (suppl), 172. 34. HUSBY S. (1988) Xenogeneic antibodies, idiotypes, and atopic disease. Lancet, i, 1233. Note added in proof A paper has recently come to our attention (S. Jackson et al., Infect. Immun. 58, 1932; 1990) of a protective secretory immune response after oral immunization with an anti-idiotypic vaccine.
ADONIS 0019280591001 70S
Oral immunization with xenogeneic antibodies stimulates the production of systemic and mucosal anti-idiotypic antibodies A. M. COLLINS,*t§ D. M. ROBERTON,*l C. S. HOSKING* & G. R. FLANNERYt *Royal Children's Hospital, Melbourne, tLa Trobe University, Bundoora, Melbourne and jAdelaide Children's Hospital, Adelaide, Australia
Acceptedfor publication 9 April 1991
SUMMARY The humoral and mucosal immune responses to oral immunization with xenogeneic antibodies were studied using an animal model in which female rabbits were fed daily doses of the MOPC-3 15 murine IgA antibody, and were mated during the course of the feeding programme. Serum and colostrum samples were assayed for the presence of anti-idiotypic antibodies by ELISA assay, before and after depletion of anti-IgA antibodies, by affinity chromatography using another murine IgA idiotype. It was shown that all animals responded to exposure to the MOPC-3 15 idiotype with the production of serum anti-murine immunoglobulin antibodies and that four of six animals produced serum anti-idiotypic antibodies. That the immune response included antibodies directed against the antigen-binding site was confirmed by competition ELISA assay. Mucosal IgG and IgA antiimmunoglobulin antibodies were present in milk from all antibody-fed rabbits tested, and IgA antiidiotypic antibodies were detectable in the colostrum of one rabbit. The results provide some support for the hypothesis that human exposure to xenogeneic antibodies, most commonly bovine milk immunoglobulins, may provoke the production of anti-idiotypic antibodies, and that such exposure may lead to disturbances of immune regulation.
INTRODUCTION Fifteen years after the possibility of idiotypic network regulation was first proposed,' controversy persists as to the functional importance of idiotypic network interactions, but the veracity of experimental manipulations of the immune response using idiotypes and anti-idiotypes is beyond question.2 It has frequently been observed that it is far easier to generate an antiidiotypic immune response by immunization with xenogeneic antibodies than it is by immunization with allogeneic or isologous idiotypes,2'3 but the fact that exposure to xenogeneic antibodies is a normal feature of human life seems to have been overlooked. This exposure is principally a consequence of bovine milk consumption, and it has recently been proposed by one of us (A. M. Collins) that this may have important implications for human health. It has been suggested that antibodies directed against dietary xenogeneic immunoglobulins should include anti-idiotypic antibodies, and that such antiidiotypic antibodies could disturb the normal regulation of the immune response in an idiotype-specific way.4 The hypothesis rests on the demonstrated survival of bovine milk antibodies during commercial pasteurisation,5 and the identification of bovine antibodies directed against common
human allergens (A. M. Collins et al., manuscript submitted for publication). It also requires the survival of bovine milk antibodies in the proteolytic environment of the gut. This survival is well established in a number of species, including humans6 and rabbits.7 Although bovine milk IgGl is not protected from proteolysis by its attachment to a secretory component, as is the case with the predominant milk antibodies of non-ruminants,8 it nevertheless seems relatively resistant to proteolytic degradation.9 In addition to the stability which might be conferred by their protein chemistry, bovine milk antibodies may additionally be protected from proteolysis by the presence of trypsin inhibitors in milk,'0 by the buffering action of other milk proteins," and in the neonate by low gastric acidity and the rapid transit of whey proteins to the more benign environment of the duodenum. 12 As part of a series of studies investigating the hypothesis, an animal model was established to investigate the nature of the immune response to oral immunization with xenogeneic antibodies. A rabbit model was chosen because both humans and rabbits sometimes produce circulating IgG antibodies in response to ingested proteins. 13''4 There is also evidence that rabbits are able to mount an immune response to dietary bovine milk gammaglobulins,'5 and it has been shown that young rabbits are able to respond to specific determinants on ingested allogeneic milk antibodies. 16 A model was developed in which adult female rabbits were exposed to murine IgA anti-DNP antibodies produced by the
§ Present address and correspondence: Dept. of Microbiology and Immunology, University of New South Wales, P.O. Box 1, Kensington, NSW 2033, Australia.
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Oral immunization with xenogeneic antibodies MOPC-3 15 mouse myeloma cell line,'7 and weekly serum samples were collected for the investigation of systemic responsiveness. Six weeks after the commencement of feeding, the rabbits were mated, and colostrum and early milk samples were collected at parturition for the investigation of the mucosal immune response to the feeding programme. Results show that rabbits are capable of mounting both systemic and mucosal immune responses upon oral immunization with xenogeneic antibodies, and that anti-idiotypic antibodies form a detectable component of the response, at least in some rabbits. MATERIALS AND METHODS Animals Rabbits were provided by the Animal Research Laboratories, Royal Children's Hospital, Melbourne, Australia. All rabbits were part of their closed breeding colony, being descended from NZW, Belgian and Australian rabbits. BALB/c mice were obtained from the Department of Genetics and Human Variation, La Trobe University, Melbourne and from the Animal Research Laboratory of the Royal Children's Hospital, Melbourne, Australia. Cell culture and ascites preparation Growth of MOPC-31517 and J558 cells'8 was initiated and maintained in DMEM (Flow Laboratories, North Ryde, Australia), containing 20% heat-inactivated foetal calf serum (FCS) (Commonwealth Serum Laboratories, Melbourne, Australia). Ascites was raised in BALB/c mice primed with 10 ml of pristane (Sigma, St Louis, MO) 10-14 days previously, and immunized i.p. with 1 0 x 107-1 5 x 107 cells.
Purification of antibodies MOPC-315 and J558 IgA antibodies were purified by ionexchange chromatography using DEAE-Sepharose CL-6B beads. The MOPC-315 antibody solution obtained was diluted to a concentration of 125 pg/ml in casein buffer (phosphatebuffered saline containing 25 mg/ml casein hydrolysate), and aliquots were frozen at -20°. MOPC-3 15 antibodies for parenteral immunization and for ELISA plate coating were further purified by affinity chromatography using human secretory component.'9 Feeding schedule Six parous female rabbits were fed purified MOPC-315 antibodies by naso-gastric feeding tubes on a 28-day cycle of 14 days of feeding and 14 days of rest. On feeding days, each rabbit received 0-5 mg of MOPC-3 15 antibody, preceded and followed by 5 ml of casein buffer. Feeding continued through pregnancy and to the end of the fourth week of lactation. Mating of rabbits Rabbits were mated at the end of the second 14-day feeding period. One rabbit was mated again shortly after the birth of its first litter and antibody-feeding continued until after the birth of the second litter.
Sample collection Blood samples (3-5 ml) were collected weekly by ear-bleed. Colostrum and early milk samples were taken from five of the six rabbits on the feeding programme. The sixth rabbit failed to
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carry a pregnancy to term. Milk flow was encouraged by the injection of 0-2 ml of oxytocin (Syntocinon; Sandoz, Basle, Switzerland). One rabbit gave birth twice while on the feeding programme, and colostrum was collected from this rabbit within 24 hr of each birth. Serum, colostrum and early milk samples were also collected from six control rabbits.
Preparation of a polyclonal anti-MOPC-315 antiserum A polyclonal anti-idiotypic antiserum was raise by immunizing a rabbit with purified MOPC-3 15 antibodies emulsified in Freund's complete adjuvant (FCA) (Commonwealth Serum Laboratories). One milligram of antibody was injected subcuta-. neously into the hindquarter of the rabbit, and it was boosted every fortnight for 6 weeks with 0-5 mg of antibody emulsified in incomplete Freund's adjuvant (IFA) (Commonwealth Serum Laboratories). Absorption of anti-allotypic and anti-isotypic antibodies by affinity chromatography Purified J558 murine IgA antibodies were coupled to CNBractivated Sepharose 4B beads (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions, and were used to absorb anti-mouse IgA antibodies from both serum and colostrum samples.
Biotinylation Cappel goat anti-rabbit IgA antibodies (Organon Teknika, West Chester, PA) were biotinylated using biotin N-hydroxysuccinimide ester (Calbiochem, La Jolla, CA) and the method of Goding.20 ELISA assays Rabbit anti-MOPC-315 antibodies were detected in serum and colostrum samples by a direct binding ELISA assay2' in which purified MOPC-315 at a concentration of 10 ,ug/ml in coating buffer was bound to the plate overnight at 4°. Peroxidaselabelled anti-rabbit IgG (Organon Teknika) at a dilution of 1: 1000 and biotinylated anti-rabbit IgA at a dilution of 1:50 were used for the detection of plate-bound anti-MOPC-315 antibodies. Streptavidin-biotin-peroxidase complex (Amersham International, Amersham, Bucks, U.K.) was used at a dilution of 1:500. Tetramethylbenzadine (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used as the substrate and plates were read in a Titertek Multiscan MC Plate Reader (Flow Laboratories, Irvine, Ayrshire, U.K.) at 450 nm. Rabbit IgG and IgA anti-J558 ELISA assays were identical to the MOPC315 ELISA assays except that J558 antibodies were coated to the microtitre plates at a concentration of 10 Mg/ml. An unabsorbed serum sample from the parenterally immunized rabbit was chosen as the reference sample for assays of serum antibodies and was assigned 2-56 x 105 ELISA units (EU)/ml. Duplicate doubling dilutions of the reference standard were used to produce a standard curve against which sample results could be calculated. Absorbed and unabsorbed rabbit serum samples were assayed as triplicates at dilutions from I:1 to 1:2000. A milk sample collected after the birth of the first litter of Rabbit I served as the milk IgA reference standard. It was shown that the reference standard had almost four times the IgA activity against MOPC-315 as it had against J558 antibodies, and was therefore assigned 1000 EU/ml of anti-MOPC-315
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Figure 1. IgG anti-MOPC-315 and anti-J558 antibodies in J558-absorbed and unabsorbed serum samples from Rabbits 1-6 (a-f, respectively) during antibody-feeding experiments, as measured by MOPC-315 and J558 ELISA assays. Feeding began at Day 0. Results are expressed with respect to a polyclonal anti-MOPC-3 15 antiserum standard. (0) MOPC-3 15 absorbed; (-) J558 absorbed; (-) MOPC-315 unabsorbed; (0) J558 unabsorbed.
activity, and 250 EU/ml of anti-J558 IgA activity. Triplicate samples were assayed at dilutions of 1:4 (Absorbed) and 1:9 (Unabsorbed). Duplicate doubling dilutions of the reference standard were assayed from an initial dilution of 1:9. A selection of unabsorbed milk samples was assayed for IgG antibodies directed against the MOPC-315 antibody using the MOPC-315 IgG ELISA. Duplicate samples were assayed as doubling dilutions from initial dilutions of 1:1. Milk from the first litter of Rabbit 1 was again used as the reference standard, and was assigned 1000 EU/ml of IgG anti-MOPC-3 15 activity. Competition ELISA assays Anti-idiotypic binding was confirmed using a competition ELISA assay. Fifty-microlitre aliquots of doubling dilutions of serum samples were added to microtitre plates to which DNPBSA had been bound at a concentration of 25 mg/ml. Fiftymillilitre aliquots of mouse MOPC-315 idiotype were then added to the plates at a constant concentration of 100 ng/ml, and the plates were incubated for 1 hr at 37°. Plate-bound murine IgA antibodies were then detected using peroxidaselabelled goat anti-mouse IgA antibodies at a dilution of 1: 1000, as has been previously outlined. Pre- and post-feeding samples from each rabbit were tested in duplicate. Each plate also included duplicate rows to which dilutions of polyclonal anti-idiotypic antiserum was added, and serum-free MOPC-3 15 cell culture supernatant which served as a reference standard against which all other results could be determined.
RESULTS
Systemic immune response The results of the MOPC-315 and J558 IgG ELISA assays for anti-idiotypic antibodies and other antibodies in antibody-fed rabbits are presented separately for each rabbit in Fig. la-f. All rabbits produced anti-MOPC-3 15 antibodies in response to oral immunization. While unabsorbed serum from Rabbit 1 contained almost 1-7 x 105 EU/ml in the MOPC-315 ELISA at the height of its response, unabsorbed serum from Rabbit 4 contained barely 600 EU/ml of activity at the cessation of feeding. Only Rabbits 1, 2, 3 and 5 showed clear evidence of anti-idiotypic antibodies in absorbed serum samples. Activity ranged from 1 10 EU/ml (Rabbit 1) to 5650 EU/ml (Rabbit 2). Serum samples generally showed more activity in the J558 ELISA than in the MOPC-315 ELISA. This was probably a consequence of the choosing of our reference standard. On the assumption that the reference standard, an early post-immunization sample, contained a small proportion of anti-idiotypic antibodies, it was assigned 2-56 x 105 EU/ml in both assays, but the presence of a higher proportion of anti-idiotypic antibodies was subsequently shown. This would have the consequence of lowering the measured response in the MOPC-315 ELISA in comparison to the J558 ELISA. Mucosal immune response The results of the MOPC-315 and J558 IgA ELISA assays of rabbit milk samples are given in Fig. 2a, b, respectively. Figure
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Figure 2. IgA anti-MOPC-315 (a) and J558 (b) antibodies in J558absorbed (-) and unabsorbed (0) milk samples from five antibody-fed rabbits and six control rabbits, as measured by MOPC-315 and J558 ELISA assays. Results express means of triplicates in EU/ml with respect to the milk standard (Rabbit 1.1).
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2a shows that anti-MOPC-3 15 antibodies were detected only in the milk of Rabbits I and 3, and anti-idiotypic antibodies only in the milk of Rabbit 1. The results of the J558 ELISA, however, are more difficult to interpret. In particular, some anti-J558 activity was detected in unabsorbed samples from Controls 1 and 6 (Cl, C6), while unabsorbed milk from Rabbit 5 showed high activity in the J558 ELISA but not in the MOPC-315 ELISA. J558 antibodies carry many determinants, and it may be that some rabbits produce mucosal antibodies which cross-react with some of these determinants regardless of their exposure to murine MOPC-315 antibodies. The rabbits (1, 3 and 5) whose milk showed evidence of a mucosal response to MOPC-315 antibodies also showed the highest systemic immune responses to oral immunization with idiotype. A limited number of unabsorbed samples were assayed for anti-MOPC-3 15 milk IgG antibodies using the MOPC-3 15 IgG ELISA assays. Titration curves for these samples are presented as Figure 3. The two control samples showed less than 1 EU/ml of activity compared to the 1000 EU/ml of activity in the reference standard (Rabbit 1.1). Anti-MOPC-315 activity in Rabbit 5 milk was measured as 258 EU/ml. Anti-MOPC-315 activity was calculated to be 15 and 21 EU/ml, respectively, for samples from Rabbits 4 and 6. Characterization of anti-idiotypes by competition assay The results of the idiotype/anti-idiotype competition assays are presented as Fig. 4a, b. The presence of antibodies competing with antigen for the antigen-binding site of the MOPC-315 antibody was confirmed in sera from Rabbits 1, 3 and 5, and also in the polyclonal anti-idiotypic antiserum (P-aId). Inhibition of idiotype binding was seen with serum from Rabbit at a dilution of 1:320, from Rabbits 3 and 5 at dilutions of 1: 15, while serum from the parenterally immunized rabbit was inhibitory at a dilution of 1:2560.
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Figure 4. Competition ELISA titration curves of murine IgA MOPC315 antibody binding to plate-bound DNP-BSA in the presence of doubling dilutions of pre- and post-feeding serum samples from MOPC-315 antibody-fed rabbits (1-6) and serum (P-aId) before and after parenteral immunization with MOPC-3 15. Results express means of duplicate wells containing samples at doubling dilutions from initial dilutions of 1:9 (Standard and Rabbit I) and 1:1 (Rabbits 2-6).
DISCUSSION The results reported here demonstrate that oral immunization with xenogeneic antibodies results in both systemic and mucosal anti-immunoglobulin responses. The responses of four of six antibody-fed rabbits included detectable serum IgG antiidiotypic antibodies, and a component of these anti-idiotypic antibodies were binding-site directed. Both IgA and IgG antiimmunoglobulin antibodies were also detected in the milk of antibody-fed rabbits, but not in the milk of six control rabbits, and IgA anti-idiotypic antibodies were detected in the milk of one rabbit. There were insufficient sample volumes to more fully investigate the mucosal IgG response. The demonstration of
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both IgG and IgA anti-mouse immunoglobulin antibodies reflects the fact that both isotypes are important components of rabbit milk,8 and that IgG, as well as IgA, milk antibodies can be produced by rabbits in response to dietary proteins.2223 We believe these results represent the first demonstration of such responsiveness, for a search of the literature failed to locate a single study which attempted to elicit an anti-idiotypic response from the mucosal immune system, or to generate such a response from either the systemic or mucosal immune systems by oral immunization. There is, however, indirect evidence for the involvement of anti-idiotypic antibodies in the development of mucosal responsiveness. In a study investigating a neonatal immune response in the apparent absence of antigen exposure, it was suggested that the presence of secretory IgA and IgM antiEscherichia coli 0 and anti-poliovirus antibodies in secretions of the neonate could be a result of exposure to maternal antiidiotypic antibodies.24 The anti-idiotypic responses observed in antibody-fed rabbits were generally very low, and the biological significance of these responses could be questioned. While a more precise quantification in absolute rather than in relative units might be informative, modulation of the immune response has been achieved with remarkably low concentrations of anti-idiotypic antibodies-particularly when administered to neonates.25 We have therefore chosen to investigate the biological implications of oral immunization with xenogeneic antibodies by examining the antigen-specific (anti-DNP) responsiveness of the offspring of female rabbits exposed to MOPC-315 antibodies throughout pregnancy and lactation. Preliminary results suggest an antigenspecific modulation of both the IgG and IgE responses in these rabbits. In addition to the support the results reported here lend to the hypothesis that exposure to xenogeneic antibodies may lead to disturbances of immune function, the finding that oral immunization with xenogeneic antibodies can lead to the production of systemic and mucosal anti-idiotypic antibodies is an interesting phenomenon in its own right, and one which may have some practical implications. The results point to the need for greater caution in the application of some passive immunization therapy. Recently passive oral immunization has been demonstrated to be effective in the treatment of a variety of enteric bacterial,26 viral27 and protozoan28 infections. The results reported here suggest that such a therapeutic strategy may lead to the generation of anti-idiotypic antibodies, as has previously been noted after xenogeneic monoclonal29 and polyclonal30 antibody therapy. In fact, after multiple infusions of therapeutic xenogeneic antibodies, the anti-idiotypic component of the response may come to predominate.3' Such a response, after either parenteral or oral passive immunization, could have implications for later immunological responsiveness to the pathogens in question. The results also raise the possibility that the oral route may by an effective one for the administration of anti-idiotypic vaccines. Such a protocol might be adopted, for example, by those attempting to provoke a protective response in the neonate. Anti-idiotypic vaccines have been identified as having special potential in the neonate, for they avoid exposure of the neonate to pathogen, and they may induce responsiveness to carbohydrate antigens-to which neonates are poor responders.32 In addition, oral immunization with anti-idiotypic vaccines directly challenges the mucosal immune system, which
develops earlier in life than the systemic immune system.33 Such a protocol may therefore allow earlier immunization of the infant. It has been said that the hypothesis that human consumption of xenogeneic antibodies leads to idiotype-specific pertubations of immune responsiveness will be 'hard to prove or disprove'.34 Certainly the work reported here fails to 'prove' the hypothesis, but it has considerably strengthened arguments in favour of its continued investigation.
ACKNOWLEDGMENTS We thank Magdy Sourial, Shane Osterfield and Marcia Riordan for their assistance with the experimental work, and Dr H. Ward and Dr Michael Shelton for their suggestions and discussion.
REFERENCES 1. JERNE N.K. (1974) Towards a network theory of the immune system. Ann. Immunol. (Inst. Pasteur), 125, 373. 2. COHN M. (1986) The concept of functional idiotype network for immune regulation mocks all and comforts none. Ann. Immunol. (Inst. Pasteur), 137, 64. 3. LING N.R., ELLIOTT D. & LOWE J. (1987) Modulation of the murine immune response to human IgG by complexing with monoclonal antibodies. II. Antibody responses to idiotopes of the human IgG paraprotein and of the mouse monoclonal antibodies. Immunology, 62, 7. 4. COLLINS A.M. (1988) Xenogeneic antibodies and atopic disease. Lancet, i, 734. 5. YOLKEN R.H., LOSONSKY G.A., VONDERFECHT S., LEISTER F. & WEE S. (1985) Antibody to human rotavirus in cow's milk. N. Engl. J. Med. 312, 605. 6. HILPERT H., BRUssow H., MIETENS C., SIDOTI J., LERNER L. & WERCHAU H. (1987) Use of bovine milk concentrate containing antibody to rotavirus to treat rotavirus gastroenteritis in infants. J. Infect. Dis. 156, 158. 7. MCCLEAD R.E. & GREGORY S.A. (1984) Resistance of bovine colostral anti-cholera toxin antibody to in vitro and in vivo proteolysis. Infec. Immun. 44, 474. 8. WATSON D.L. (1980) Immunological functions of the mammary gland and its secretion-a comparative review. Aust. J. biol. Sci. 33, 403. 9. BROCK J.H., ARZABE F.R., PINEIRO A. & OLIVITO A. (1977) The effect of trypsin and chymotrypsin on the bactericidal activity and specific antibody activity of bovine colostrum. Immunology, 32,207. 10. MCLEAN B.S. & HOLMES I.H. (1981) Effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection. J. clin. Microbiol. 13, 22. 11. MORGAN K.L., BOURNE F.J., NEWBY T.J. & BRADLEY P.A. (1981) Humoral factors in the secretory immune system of ruminants. Adv. exp. Med. Biol. 137, 391. 12. MYLREA P.J. (1966) Digestion of milk in young calves. I. Flow and acidity of the contents of the small intestine. Res. Vet. Sci. 7, 733. 13. SILVERMAN G.A., PERI B.A. & ROTHBERG R.M. (1982) Systemic antibody responses of different species following ingestion of soluble protein antigens. Dev. Comp. Immunol. 6, 737. 14. PERI B.A. & ROTHBERG R.M. (1986) Transmission of maternal antibody prenatally and from milk into serum of neonatal rabbits. Immunology, 57, 49. 15. HANGLOW A.C., WELSH C.J.R., CONN P. & COOMBS R.R.A. (1985) Early rheumatoid-like synovial lesions in rabbits drinking cow's milk. Int. Archs Allergy appl. Immun. 78, 152. 16. ADLER F.L. & ADLER L.T. (1982) Consequences of prenatal exposure to maternal alloantigens. Ann. NY Acad. Sci. 392, 266.
Oral immunization with xenogeneic antibodies 17. EISEN H.N., SIMMs E.S. & POTTER M. (1968) Mouse myeloma proteins with anti-hapten antibody activity. The protein produced by plasma cell tumour MOPC 315. Biochemistry, 11, 4126. 18. CARSON D. & WEIGERT M. (1973) Immunochemical analysis of the cross-reacting idiotypes of mouse myeloma proteins with antidextran activity and normal anti-dextran antibody. Proc. natl. Acad. Sci. U.S.A. 70, 235. 19. JONES C.L., GEORGIOU G.M., FOWLER K.J., WAJNGARTEN P.I. & ROBERTON D.M. (1987) Purification of polymeric immunoglobulin from cell culture supernatants by affinity chromatography using secretory component. J. immunol. Meth. 104, 237. 20. GODING J.W. (1986) Monoclonal Antibodies: Principles and Practice, 2nd edn. Academic Press, London. 21. ENGVALL E. & PERLMANN P. (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzymelabelled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109, 129. 22. MONTGOMERY P.C., COHN J. & LALLY E.T. (1973) The induction and characterization of secreting IgA antibodies. In: The Immunoglobulin A system. (eds J. Mestecky and A. R. Lawton), pp. 453, Plenum Press, New York. 23. PERI B.A. & ROTHBERG R.M. (1986) Transmission of maternal antibody prenatally and from milk into serum of neonatal rabbits. Immunology, 57, 49. 24. MELLANDER L., CARLSSON B. & HANSON L.A. (1986) Secretory IgA and IgM antibodies to E. coli 0 and poliovirus type I antigens occur in amniotic fluid, meconium and saliva from newborns. A neonatal immune response without antigenic exposure: a result of antiidiotypic induction? Clin. exp. Immunol. 63, 555. 25. RUBINSTEIN L.J., YEH M. & BONA C.A. (1982) Idiotype-antiidiotype network. II. Activation of silent clones by treatment at birth with idiotypes is associated with the expansion of idiotypespecific helper T cells. J. exp. Med. 156, 506. 26. TACKET C.O., LOSONSKY G., LINK H., HOANG Y., GUESRY P.,
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HILPERT H. & LEVINE M.M. (1988) Protection by milk immunoglobulin concentrate against oral challenge with enterotoxigenic Escherichia coli. N. Engl. J. Med. 318, 1240. 27. DAVIDSON G.P., DANIELS E., NUNAN H., MOORE A.G., WHYTE P.B.D., FRANKLIN K., MCCLOUD P.I. & MOORE D.J. (1989) Passive immunisation of children with bovine colostrum containing antibodies to human rotavirus. Lancet, ii, 709. 28. TZiPORI S., ROBERTON D. & CHAPMAN C. (1986) Remission of diarrhoea due to cryptosporidiosis in an immunodeficient child treated with hyperimmune bovine colostrum. Br. Med. J. 293, 1276. 29. AUSTIN E.B., ROBINS R.A., DURRANT L.G., PRICE M.R. & BALDWIN R.W. (1989) Human monoclonal anti-idiotypic antibody to the
tumour-associated antibody 791T/36. Immunology, 67, 525. 30. HERLYN D., WETrENDORF M., SCHMOLL E., ILIOPOULOS D., SCHEDEL I., DREIKHAUSEN V., RAAB R., Ross A.H., JAKSCHE H., SCRIBA
M. & KoPROWSKI H. (1987) Anti-idiotypic immunization of cancer patients. Modulation of the immune response. Proc. nati. Acad. Sci. U.S.A. 84, 8055. 31. SHAWLER D.L., BARTHOLOMEW R.M., SMITH L.M. & DILLMAN R.O. (1985) Human immune response to multiple injections of murine monoclonal IgG. J. Immunol. 135, 1530. 32. CAPRA J.D. & BONA C. (1988) Idiotypes: practical advances from fundamental concepts. Immunol. Today, 9, 98. 33. HANSON L.A., ANDERSSON B., MELLANDER L., SODERSTROM T. &
EDEN C.S. (1985) Protective factors in milk and the development of the immune system. Pediatrics, 75 (suppl), 172. 34. HUSBY S. (1988) Xenogeneic antibodies, idiotypes, and atopic disease. Lancet, i, 1233. Note added in proof A paper has recently come to our attention (S. Jackson et al., Infect. Immun. 58, 1932; 1990) of a protective secretory immune response after oral immunization with an anti-idiotypic vaccine.
