ISCOMS- a novel strategy for mucosal immunization? Allan McI. Mowat and Anne M. Donachie
Vaccination with individual antigens or epitopes now offers the possibility of producing customized synthetic vaccines for virtually any pathogen. It would be extremely useful if such vaccines could be given by the oral route, not only because it is a simple and acceptable way of inducing systemic immunity, but also because mucosal immunity is essential to protect against diseases of mucosal surfaces such as the intestine and respiratory tract 1,2. In addition, oral immunization may be one of the few ways of inducing vertical transmission of protection from mother to infant via the breast milk 2. Several problems have limited the development of orally-active recombinant vaccines: first, purified proteins are, in general, poorly immunogenic; second, as they are processed by the exogenous route of antigen presentation, they tend not to induce the major histocompatibility complex (MHC) class-I-restricted T cells that are important for protecting against a wide range of viral and other infectious diseases; third, they induce profound systemic tolerance when given to naive animals 3. Several strategies for overcoming oral tolerance and inducing active secretory immunity have been developed, including the use of cholera toxin (CT) as a mucosal adjuvant, the incorporation of proteins in microspheres, and the use of recombinant, auxotrophic Salmonella strains 4-6. However, the construction of such vectors is technically complicated and the efficacy variable, depending on the antigen used. Furthermore, by virtue of their physical nature or their localization within the immune system, it seems unlikely that these approaches will induce MHC class-I-restricted T-cell responses. Thus, there is a need for vectors of simple construction that
Orally-active vaccines containing purified or recombinant antigens are highly desirable, particularly in immunization against diseases of mucosal surfaces, but their development to date has been limited and fitful, hampered by a range of difficulties. Here, Allan Mowat and Anne Donachie suggest that lipophilic immune-stimulating complexes (ISCOMS) may provide an oral immunization vector for the induction of a wide range of immune responses to protein antigens.
sponses to purified protein antigens. The lipid nature of ISCOMS initially suggested that only membrane-associated or bydrophobic proteins would incorporate readily into the micelles. Hence, most early studies concentrated on mixtures of proteins obtained from viral or bacterial lysates 8,9. More recently, purified membrane or fusion proteins have been incorporated into ISCOMS (Ref. 9 and Kersten, will induce a full range of immune G.F.A., PhD thesis, 1990, Univ. of responses to orally-administered Utrecht) and strategies have been developed to allow incorporation of an protein antigens. even wider range of purified or recombinant proteins. These involve ISCOMS as adjuvants ISCOMS are negatively charged the addition of phosphatidyl choline to the mixture, together with the cage-like pentagonal dodecahedra, 30-40 nm in size 7. They form spon- exposure of hydrophobic groups on taneously on mixing cholesterol and the native protein by acidification 1° Quil A (saponin), and proteins and or palmitification of the protein before incorporation 11. Acidification other lipids, such as phosphatidyl choline, can be incorporated during has been used to form immunogenic their synthesis. First described by ISCOMS with bovine serum albuMorein and colleagues, ISCOMS min (BSA) and human immunohave been used with a number of deficiency virus (HIV) gp120, while parenterally administered antigens we have constructed immunogenic to enhance serum antibody re- ISCOMS using a large number of sponses, systemic delayed-type palmitified proteins, including ovhypersensitivity (DTH) and prolifer- albumin (OVA), cytochrome c, ative T-cell responses. In addition, the leucotoxins from Haemophilus protective immunity has been gener- haemolyticus and Pasteurella multocida, recombinant HIV p24, recomated in a variety of experimental models of infection, including sev- binant feline immunodeficiency eral viral diseases, Toxoplasmosis virus (FIV) p24, recombinant tetaand Epstein-Barr virus (EBV)- nus toxin (C fragment) and recominduced turnouts. ISCOMS are also binant Bordetella pertussis p69. This effective in a large number of species, technique is simple and reliable and including mice, cats, seals, cattle, should extend the applicability of primates and sheep, and an fSCOMS as protein carriers. Its suclSCOMS-based commercial vaccine cess also suggests that a similar stratagainst equine influenza is available egy, namely adding lipid tails, will in Sweden. These studies have been allow smaller epitopes or peptides reviewed extensively elsewhere s,9 to be incorporated. Very low and here we will focus on recent amounts of antigen are immunogenic novel findings that indicate that in ISCOMS, with as little as ISCOMS have unique properties as l ~ g protein being required for mucosal adjuvants and induce an optimal responses after parenteral unusually wide range of T-cell re- immunization8~9,11. © 1991,ElsevierSciencePublishersLtd, UK. 0167--4919/91/$02.00
ImmunologyToday
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Vol le No ~l 1991
Immunogenicity of parenterallyadministered ISCOMS
be destroyed in the harsh environment of the gut. In addition, an Antigens incorporated in earlier report indicated that oral imISCOMS have long been known to munization with influenza meminduce very high serum antibody re- brane proteins in ISCOMS was sponses, higher, often, than those unsuccessful 14. However, saponin itfound after natural infection with self has adjuvant effects when given the relevant infectious organism. orally ~s and it has emerged that In parallel, excellent proliferative ISCOMS are extremely resistant to T-cell responses and systemic DTH acid and bile salts (Kersten, G.F.A., have been recorded 8,9, even after PhD thesis, Univ. of Utrecht). a single immunizing dose. Indeed Furthermore, our studies over the OVA in ISCOMS induces cell- last year have shown that ISCOMS mediated immune responses that are are indeed immunogenic by the oral much larger than those found route, although larger amounts of using complete Freund's adjuvant ~2. antigen are required for optimal Clearly, ISCOMS strongly enhance stimulation than when using parentimmune responses that are depen- eral immunization. dent on MHC class-II-restricted T A single feed of OVA produces cells. systemm tolerance; a single feed of More surprisingly, ISCOMS con- OVA in ISCOMS induces primary taining OVA or HIV gp120 also in- serum immunoglobulin G (IgG) duce strong cytotoxic T lymphocyte and systemic DTH responses, with (CTL) responses in mice 12n3. These cell-mediated responses being more CTL are classical MHC class-I- effectively stimulated 11. Small restricted CD8 ÷ T cells that appear to amounts of MHC class-l-restricted recognize epitopes generated by ceils CTL activity can also be found in the synthesizing the protein endogen- spleen within a few days and this can ously. Our preliminary evidence be boosted to very high levels by suggests that OVA ISCOMS also feeding repeatedly. After repeated stimulate proliferation in CD8 ÷ T feeding, CTL are also generated in cells that recognize the same epi- the mesenteric lymph node and topes (authors' unpublished obser- strong secretory lgA antibody revations). This explains the earlier sponses can be detected in intestinal findings that immunization with in- washes (authors' unpublished obserfluenza A glycoproteins in ISCOMS vations)." OVA-specific igA is also primed a population of lymphocytes found in serum. Thus, oral immunizthat can be restimulated to gener- ation with ISCOMS provokes both ate cytolytic effector cells using live local and systemic immunity to proinfluenza virus in vitroLL Thus, tein antigens and encompasses the ISCOMS allow soluble antigens to full range of immune responses. enter the endogenous pathway of Once again, these effects are entirely antigen processing and appear to do dependent on the presence of so in a physiologically relevant man- saponin and cannot be reproduced ner. The basis of this action remains by palmitified antigen alone. Similar to be elucidated, but it should be intestinal IgA responses and serum noted that Quil A (saponin) is essen- IgG antibody responses have been tial for the induction of MHC class- detected after local immunization of I-restricted T cells by ISCOMS, as isolated loops of rat intestine with simple tiposomes do not have the Neisseria gonorrhoeae pore protein same effect, nor does free palmitified 1B (Kersten, G.F.A., PhD thesis, antigen (authors' unpublished obser- 1990, Univ. of Utrecht). vations). Interestingly, as with other mucosal immunogens 2, combined ISCOMS as vectors for mucosal parenteral and oral immunization immunization with ISCOMS is extremely effective The second novel feature of at inducing immunity in the gut and ISCOMS to emerge from recent elsewhere, offering the potential for work is that they confer immuno- combining vaccination routes. It genicity on proteins delivered by the remains to be determined whether oral route. This was initially surpris- oral immunization with ISCOMS ing, as it might have been assumed also stimulates immunity at other that the lipophilic ISCOMS would mucosal sites, such as the respiratory Immunology Today
384
tract or breast, or if orallyadministered ISCOMS generate protective immunity. However, ISCOMS do induce protective immunity against influenza in mice when given intranasally, indicating that the adjuvant effect of ISCOMS is likely to be operative in a variety of mucosal tissues 13,14. These effects of oral ISCOMS are much greater and of wider scope than those found using other mucosal adjuvants such as CT, and underline the potential usefulness of this strategy. In addition, the oral route has two practical advantages for immunization with ISCOMS. First, the occasional toxic effects of ISCOMS are virtually never found after feeding. Second, mice primed orally with OVA ISCOMS do not appear to be susceptible to local T-cell-mediated hypersensitivity reactions after refeeding the antigen (authors' unpublished observations). Such responses have been shown to occur under circumstances in which the sensitization of mucosal T cells occurs due to a breakdown in oral tolerance 4. The reason for the failure of ISCOMS to induce this potentially hazardous response is unknown, but may be related to their ability to provoke efficient secretory lgA antibody responses. Future directions
Several issues need to be addressed before ISCOMS-based, oral vaccines could be considered for human use. First, it needs to be shown that oral immunization with ISCOMS confers protective immunity against mucosal and systemic pathogens. Second, a practical concern is the occasional toxicity observed in mice given large doses of ISCOMS parenterally. Although larger animals such as cats, horses and primates appear less susceptible to these effects and our own experience is that toxicity is reduced considerably when using the oral route, this problem potentially limits the amount of ISCOMS (and hence antigen) which can be given. The toxicity can be largely attributed to the saponins present in ISCOMS, which, as noted above, are essential for the adjuvant properties. However, recent work has shown that the saponins in Quil A can be separated into discrete fractions, some of which retain full
Vot 12 No. lJ 1991
iiiiiiiiii adjuvanticity, but display no toxicity, in vivo 16. Thus, it may be possible to use purified saponin fractions to construct functional ISCOMS that have no side effects, particularly if used orally. It will also be necessary to define dosage schedules that are optimal for inducing a full range of immune responses after oral immunization with 1SCOMS. A single feed of OVA in ISCOMS generates poor serum and intestinal antibody responses and relatively low CTL responses. Although these can be raised to very high levels by repeated feedings, multiple dose vaccines could present practical difficulties in use. A related problem is that the primary responses induced by oral or intestinal immunization seem to wane after three-to-four weeks. Although it is possible that memory responses might be observed for longer than this, it seems likely that new strategies will be needed to obtain more intense and more prolonged immunity in animals fed ISCOMS. These could include techniques for incorporating larger amounts of antigen into ISCOMS or the use of a variety of different antigens from a single pathogen. Alternatively, it may be feasible to construct 'hybrid' antigenbearing ISCOMS that include agents that might assist the adjuvanticity of
iiiiii!!iiiii
the complexes. Candidates for this might include adjuvant peptides and cytokines, or molecules that could target ISCOMS more efficiently to the intestinal immune system, such as CT or antibodies directed at specific antigen-presenting cells (APCs). Ultimately, however, the successful development of ISCOMSbased oral vaccines will require a better fundamental understanding of the unique adjuvant effects. At present, nothing is known of how ISCOMS traverse the intestine, how they enter the immune system or how they are processed by APCs. Elucidation of these issues will provide information of direct interest not only for vaccine development, but also for the understanding of the mucosal immune system generally and of the functions of APCs. Allan Mowat and Anne Donachie are at the Dept of Immunology, University of Glasgow, Western Infirmary, Glasgow G l l 6NT, UK. References
1 Bloom, B.R. (1989) Nature 342, 15-20 2 Challacombe, S.J. (1987) in Food Allergy and Intolerance (Brostoff,J. and Cballacombe, S.J., eds), pp. 269-285, W.B. Saunders
3 Mowat, A.McI. (1987) Immunol. Today 8, 93-98 4 Lycke,N. and Holmgren, J. (1986) Immunology 59, 301-308 5 Poirier, T.P., Kehoe, M.A. and Beacbey, E.H. (1988)J. Exp. Med. 168, 25-32 6 Eldridge, J.H., Hammond, C.J., Meulbroek, J.A. et al. (1990) J. Control. Release 11,205 70zel, M., Hoglund, S., Gelderbrom, H.R. and Morein, B. (1989) J. Ultrastruct. Mol. Struct. Res. 102, 240-248 8 Morein, B., L6vgren, K., H6glund, S. and Sundquist, B. (1987) Immunol. Today 8,333-338 9 Morein, B., Fossum, C., Lovgren, K. and Hoglund, S. (1990) Semin. Virol. 1, 49-55 10 Morein, B., Ekstrom, J. and L6vgren, K. (1990)J. lmmunol. Meth. 128,177-181 11 Mowat, A.McI., Donachie, A.M., Reid, G. and Jarrett, O. (1991) immunology 72, 317-322 12 Takahashi, H., Takeshita, T., Morein, B. et al. (1990) Nature 344, 873-875 13 Jones, P.D., Tha Hla, R., Morein, B., L6vgren, K. and Ada, G.L. (1988) Scan& J. Immunol. 27, 645-652 14 L6vgren, K. (/988) Scan&.l: lmmunol. 27, 241-245 15 Chavali, S.R. and Campbell, J.B. (1987) Immunobiology 174, 347-359 16 Kensil, C.R., Patel, U., Lennick, M. and Marciani, D. (1991)J. Immunol. 146,431-437
': Naturally-processed peptides
Immunology Today
385
rot. 12 No. 11 1991
Vaccination with individual antigens or epitopes now offers the possibility of producing customized synthetic vaccines for virtually any pathogen. It would be extremely useful if such vaccines could be given by the oral route, not only because it is a simple and acceptable way of inducing systemic immunity, but also because mucosal immunity is essential to protect against diseases of mucosal surfaces such as the intestine and respiratory tract 1,2. In addition, oral immunization may be one of the few ways of inducing vertical transmission of protection from mother to infant via the breast milk 2. Several problems have limited the development of orally-active recombinant vaccines: first, purified proteins are, in general, poorly immunogenic; second, as they are processed by the exogenous route of antigen presentation, they tend not to induce the major histocompatibility complex (MHC) class-I-restricted T cells that are important for protecting against a wide range of viral and other infectious diseases; third, they induce profound systemic tolerance when given to naive animals 3. Several strategies for overcoming oral tolerance and inducing active secretory immunity have been developed, including the use of cholera toxin (CT) as a mucosal adjuvant, the incorporation of proteins in microspheres, and the use of recombinant, auxotrophic Salmonella strains 4-6. However, the construction of such vectors is technically complicated and the efficacy variable, depending on the antigen used. Furthermore, by virtue of their physical nature or their localization within the immune system, it seems unlikely that these approaches will induce MHC class-I-restricted T-cell responses. Thus, there is a need for vectors of simple construction that
Orally-active vaccines containing purified or recombinant antigens are highly desirable, particularly in immunization against diseases of mucosal surfaces, but their development to date has been limited and fitful, hampered by a range of difficulties. Here, Allan Mowat and Anne Donachie suggest that lipophilic immune-stimulating complexes (ISCOMS) may provide an oral immunization vector for the induction of a wide range of immune responses to protein antigens.
sponses to purified protein antigens. The lipid nature of ISCOMS initially suggested that only membrane-associated or bydrophobic proteins would incorporate readily into the micelles. Hence, most early studies concentrated on mixtures of proteins obtained from viral or bacterial lysates 8,9. More recently, purified membrane or fusion proteins have been incorporated into ISCOMS (Ref. 9 and Kersten, will induce a full range of immune G.F.A., PhD thesis, 1990, Univ. of responses to orally-administered Utrecht) and strategies have been developed to allow incorporation of an protein antigens. even wider range of purified or recombinant proteins. These involve ISCOMS as adjuvants ISCOMS are negatively charged the addition of phosphatidyl choline to the mixture, together with the cage-like pentagonal dodecahedra, 30-40 nm in size 7. They form spon- exposure of hydrophobic groups on taneously on mixing cholesterol and the native protein by acidification 1° Quil A (saponin), and proteins and or palmitification of the protein before incorporation 11. Acidification other lipids, such as phosphatidyl choline, can be incorporated during has been used to form immunogenic their synthesis. First described by ISCOMS with bovine serum albuMorein and colleagues, ISCOMS min (BSA) and human immunohave been used with a number of deficiency virus (HIV) gp120, while parenterally administered antigens we have constructed immunogenic to enhance serum antibody re- ISCOMS using a large number of sponses, systemic delayed-type palmitified proteins, including ovhypersensitivity (DTH) and prolifer- albumin (OVA), cytochrome c, ative T-cell responses. In addition, the leucotoxins from Haemophilus protective immunity has been gener- haemolyticus and Pasteurella multocida, recombinant HIV p24, recomated in a variety of experimental models of infection, including sev- binant feline immunodeficiency eral viral diseases, Toxoplasmosis virus (FIV) p24, recombinant tetaand Epstein-Barr virus (EBV)- nus toxin (C fragment) and recominduced turnouts. ISCOMS are also binant Bordetella pertussis p69. This effective in a large number of species, technique is simple and reliable and including mice, cats, seals, cattle, should extend the applicability of primates and sheep, and an fSCOMS as protein carriers. Its suclSCOMS-based commercial vaccine cess also suggests that a similar stratagainst equine influenza is available egy, namely adding lipid tails, will in Sweden. These studies have been allow smaller epitopes or peptides reviewed extensively elsewhere s,9 to be incorporated. Very low and here we will focus on recent amounts of antigen are immunogenic novel findings that indicate that in ISCOMS, with as little as ISCOMS have unique properties as l ~ g protein being required for mucosal adjuvants and induce an optimal responses after parenteral unusually wide range of T-cell re- immunization8~9,11. © 1991,ElsevierSciencePublishersLtd, UK. 0167--4919/91/$02.00
ImmunologyToday
383
Vol le No ~l 1991
Immunogenicity of parenterallyadministered ISCOMS
be destroyed in the harsh environment of the gut. In addition, an Antigens incorporated in earlier report indicated that oral imISCOMS have long been known to munization with influenza meminduce very high serum antibody re- brane proteins in ISCOMS was sponses, higher, often, than those unsuccessful 14. However, saponin itfound after natural infection with self has adjuvant effects when given the relevant infectious organism. orally ~s and it has emerged that In parallel, excellent proliferative ISCOMS are extremely resistant to T-cell responses and systemic DTH acid and bile salts (Kersten, G.F.A., have been recorded 8,9, even after PhD thesis, Univ. of Utrecht). a single immunizing dose. Indeed Furthermore, our studies over the OVA in ISCOMS induces cell- last year have shown that ISCOMS mediated immune responses that are are indeed immunogenic by the oral much larger than those found route, although larger amounts of using complete Freund's adjuvant ~2. antigen are required for optimal Clearly, ISCOMS strongly enhance stimulation than when using parentimmune responses that are depen- eral immunization. dent on MHC class-II-restricted T A single feed of OVA produces cells. systemm tolerance; a single feed of More surprisingly, ISCOMS con- OVA in ISCOMS induces primary taining OVA or HIV gp120 also in- serum immunoglobulin G (IgG) duce strong cytotoxic T lymphocyte and systemic DTH responses, with (CTL) responses in mice 12n3. These cell-mediated responses being more CTL are classical MHC class-I- effectively stimulated 11. Small restricted CD8 ÷ T cells that appear to amounts of MHC class-l-restricted recognize epitopes generated by ceils CTL activity can also be found in the synthesizing the protein endogen- spleen within a few days and this can ously. Our preliminary evidence be boosted to very high levels by suggests that OVA ISCOMS also feeding repeatedly. After repeated stimulate proliferation in CD8 ÷ T feeding, CTL are also generated in cells that recognize the same epi- the mesenteric lymph node and topes (authors' unpublished obser- strong secretory lgA antibody revations). This explains the earlier sponses can be detected in intestinal findings that immunization with in- washes (authors' unpublished obserfluenza A glycoproteins in ISCOMS vations)." OVA-specific igA is also primed a population of lymphocytes found in serum. Thus, oral immunizthat can be restimulated to gener- ation with ISCOMS provokes both ate cytolytic effector cells using live local and systemic immunity to proinfluenza virus in vitroLL Thus, tein antigens and encompasses the ISCOMS allow soluble antigens to full range of immune responses. enter the endogenous pathway of Once again, these effects are entirely antigen processing and appear to do dependent on the presence of so in a physiologically relevant man- saponin and cannot be reproduced ner. The basis of this action remains by palmitified antigen alone. Similar to be elucidated, but it should be intestinal IgA responses and serum noted that Quil A (saponin) is essen- IgG antibody responses have been tial for the induction of MHC class- detected after local immunization of I-restricted T cells by ISCOMS, as isolated loops of rat intestine with simple tiposomes do not have the Neisseria gonorrhoeae pore protein same effect, nor does free palmitified 1B (Kersten, G.F.A., PhD thesis, antigen (authors' unpublished obser- 1990, Univ. of Utrecht). vations). Interestingly, as with other mucosal immunogens 2, combined ISCOMS as vectors for mucosal parenteral and oral immunization immunization with ISCOMS is extremely effective The second novel feature of at inducing immunity in the gut and ISCOMS to emerge from recent elsewhere, offering the potential for work is that they confer immuno- combining vaccination routes. It genicity on proteins delivered by the remains to be determined whether oral route. This was initially surpris- oral immunization with ISCOMS ing, as it might have been assumed also stimulates immunity at other that the lipophilic ISCOMS would mucosal sites, such as the respiratory Immunology Today
384
tract or breast, or if orallyadministered ISCOMS generate protective immunity. However, ISCOMS do induce protective immunity against influenza in mice when given intranasally, indicating that the adjuvant effect of ISCOMS is likely to be operative in a variety of mucosal tissues 13,14. These effects of oral ISCOMS are much greater and of wider scope than those found using other mucosal adjuvants such as CT, and underline the potential usefulness of this strategy. In addition, the oral route has two practical advantages for immunization with ISCOMS. First, the occasional toxic effects of ISCOMS are virtually never found after feeding. Second, mice primed orally with OVA ISCOMS do not appear to be susceptible to local T-cell-mediated hypersensitivity reactions after refeeding the antigen (authors' unpublished observations). Such responses have been shown to occur under circumstances in which the sensitization of mucosal T cells occurs due to a breakdown in oral tolerance 4. The reason for the failure of ISCOMS to induce this potentially hazardous response is unknown, but may be related to their ability to provoke efficient secretory lgA antibody responses. Future directions
Several issues need to be addressed before ISCOMS-based, oral vaccines could be considered for human use. First, it needs to be shown that oral immunization with ISCOMS confers protective immunity against mucosal and systemic pathogens. Second, a practical concern is the occasional toxicity observed in mice given large doses of ISCOMS parenterally. Although larger animals such as cats, horses and primates appear less susceptible to these effects and our own experience is that toxicity is reduced considerably when using the oral route, this problem potentially limits the amount of ISCOMS (and hence antigen) which can be given. The toxicity can be largely attributed to the saponins present in ISCOMS, which, as noted above, are essential for the adjuvant properties. However, recent work has shown that the saponins in Quil A can be separated into discrete fractions, some of which retain full
Vot 12 No. lJ 1991
iiiiiiiiii adjuvanticity, but display no toxicity, in vivo 16. Thus, it may be possible to use purified saponin fractions to construct functional ISCOMS that have no side effects, particularly if used orally. It will also be necessary to define dosage schedules that are optimal for inducing a full range of immune responses after oral immunization with 1SCOMS. A single feed of OVA in ISCOMS generates poor serum and intestinal antibody responses and relatively low CTL responses. Although these can be raised to very high levels by repeated feedings, multiple dose vaccines could present practical difficulties in use. A related problem is that the primary responses induced by oral or intestinal immunization seem to wane after three-to-four weeks. Although it is possible that memory responses might be observed for longer than this, it seems likely that new strategies will be needed to obtain more intense and more prolonged immunity in animals fed ISCOMS. These could include techniques for incorporating larger amounts of antigen into ISCOMS or the use of a variety of different antigens from a single pathogen. Alternatively, it may be feasible to construct 'hybrid' antigenbearing ISCOMS that include agents that might assist the adjuvanticity of
iiiiii!!iiiii
the complexes. Candidates for this might include adjuvant peptides and cytokines, or molecules that could target ISCOMS more efficiently to the intestinal immune system, such as CT or antibodies directed at specific antigen-presenting cells (APCs). Ultimately, however, the successful development of ISCOMSbased oral vaccines will require a better fundamental understanding of the unique adjuvant effects. At present, nothing is known of how ISCOMS traverse the intestine, how they enter the immune system or how they are processed by APCs. Elucidation of these issues will provide information of direct interest not only for vaccine development, but also for the understanding of the mucosal immune system generally and of the functions of APCs. Allan Mowat and Anne Donachie are at the Dept of Immunology, University of Glasgow, Western Infirmary, Glasgow G l l 6NT, UK. References
1 Bloom, B.R. (1989) Nature 342, 15-20 2 Challacombe, S.J. (1987) in Food Allergy and Intolerance (Brostoff,J. and Cballacombe, S.J., eds), pp. 269-285, W.B. Saunders
3 Mowat, A.McI. (1987) Immunol. Today 8, 93-98 4 Lycke,N. and Holmgren, J. (1986) Immunology 59, 301-308 5 Poirier, T.P., Kehoe, M.A. and Beacbey, E.H. (1988)J. Exp. Med. 168, 25-32 6 Eldridge, J.H., Hammond, C.J., Meulbroek, J.A. et al. (1990) J. Control. Release 11,205 70zel, M., Hoglund, S., Gelderbrom, H.R. and Morein, B. (1989) J. Ultrastruct. Mol. Struct. Res. 102, 240-248 8 Morein, B., L6vgren, K., H6glund, S. and Sundquist, B. (1987) Immunol. Today 8,333-338 9 Morein, B., Fossum, C., Lovgren, K. and Hoglund, S. (1990) Semin. Virol. 1, 49-55 10 Morein, B., Ekstrom, J. and L6vgren, K. (1990)J. lmmunol. Meth. 128,177-181 11 Mowat, A.McI., Donachie, A.M., Reid, G. and Jarrett, O. (1991) immunology 72, 317-322 12 Takahashi, H., Takeshita, T., Morein, B. et al. (1990) Nature 344, 873-875 13 Jones, P.D., Tha Hla, R., Morein, B., L6vgren, K. and Ada, G.L. (1988) Scan& J. Immunol. 27, 645-652 14 L6vgren, K. (/988) Scan&.l: lmmunol. 27, 241-245 15 Chavali, S.R. and Campbell, J.B. (1987) Immunobiology 174, 347-359 16 Kensil, C.R., Patel, U., Lennick, M. and Marciani, D. (1991)J. Immunol. 146,431-437
': Naturally-processed peptides
Immunology Today
385
rot. 12 No. 11 1991
