- Email: [email protected]
[6] Immunization with phage-displayed mimotopes
[6]
IMMUNIZATION
WITH PHAGE-DISPLAYED
MIMOTOPES
109
2. Detach cells from the flask by a 20-min incubation in PBS-2 mM EDTA followed by scraping. 3. Centrifuge (10 rain, 130 g) and resuspend the cells in TBSC at a final concentration of 10 6 cells/ml. 4. Add 5/zl fluoresceinated TAG3 peptide ligand (10 -5 M)41 to 50/zl calmodulin-tagged scFv antibody (e.g., 2 × 10 -6 M ) and incubate for 5 rain. 5. Add cells (500/zl) and incubate on ice for 20 min. 6. Centrifuge cells (5 min, 300 g) and resuspend in 1 ml TBSC. 7. Analyze on a Becton-Dickinson FAC scan.
[6] I m m u n i z a t i o n
with Phage-Displayed
Mimotopes
B y GIOVANNI GALFRI~, PAOLO MONACI, ALFREDO NICOSIA, ALESSANDRA LUZZAGO, FRANCO FELICI, a n d RICCARDO CORTESE
Phage-displayed mimotopes (phagotopes) are selected from random peptide libraries because they bind to specific antibodies, thus behaving as antigenic mimics that resemble to some extent the original antigen. If the positive image of the antigen (Ag) these mimotopes provide is accurate enough to induce the production of specific antibodies (Ab) in animals, the mimotopes prove to be suitable substitute immunogens, totally distinct from the original antigen (that raised the antibodies used to identify the phagotopes). At present, the only feasible way of assessing this capability is the active immunization of experimental animals. Filamentous phages are known to be excellent immunogens; de la Cruz and collaborators 1 first proposed using filamentous phage as immunological carriers by cloning short peptides derived from the malaria circumsporozoite at the N terminus of plII. Greenwood et aL 2 cloned malaria peptides at the N terminus of the major coat protein VIII, and Minenkova et al. 3 cloned an antigenic determinant of human immunodeficiency virus (HIV) gag protein fused to pVIII and used the recombinant phage to successfully immunize rabbits. Similarly, when Willis et al. 4 immunized mice with malaria peptides fused to pVIII, they not only obtained an antibody response, but also demonstrated that the response in mice is T-cell dependent and undergoes class switching from immunoglobulin M (IgM) to IgG. t V. F. de la Cruz, A. A. Lal, and T. F. McCutchan, J. Biol. Chem. 263, 4318 (1988). 2 j. Greenwood, A. E. Willis, and R. N. Perham, J. Mot. Biol. 220, 821 (1991). 3 O. O. Minenkova, A. A. Ilyichev, G. P. Kishchenko, and V. A. Petrenko, Gene 128, 85 (1993). 4 A. E. Willis, R. N. Perham, and D. Wraith, Gene 128, 79 (1993).
METHODS IN ENZYMOLOGY, VOL. 267
Copyright © 1996 by Academic Press. Inc. All rights of reproduction in any form reserved.
110
PHAGEDISPLAYLIBRARIES
[6]
Mimotopes selected by using either hepatitis B virus surface antigen (HBsAg)-specific monoclonal antibodies or human sera have been used to induce HBsAg-specific antibodies in both mice and rabbitsJ -7 Immunization Protocols In preparing immunization protocols, numerous variables have to be considered, particularly the site and route of administration, the number of boosts and the interval between them, and the dose and quality of antigen to be administered at each boost. The optimal conditions will vary considerably according to the species of animal to be immunized. The immunization protocols reported in this chapter essentially derive from our experience using phagotopes as immunogens rather than as a systematic analysis of the just-described parameters.
Route of Administration Mice immunization with phagotopes through intraperitoneal (ip) injection is the most practical route of administration and gave good results in our tests. In rabbits, the protocol used is based on general knowledge, and is a combination of intradermal (id) priming followed by a first subscapular boost (ss) and several intramuscular (im) boosts to maintain antibody production over a long period when necessary. Immunization in monkeys (Macaca fascicularis) has been attempted through a series of subcutaneous (sc) injections. These were sufficient to raise good titers of antibodies to wild-type phage protein, but nonetheless were unable to generate specific antimimotope response, s This result is probably attributable to other factors (see below) and not to the route of administration used. However, an antimimotope response was successfully raised in chimpanzees through im administration. 8
Number and Spacing of Boosts In mice, 5 days after ip priming, IgG antibodies specific for phage coat proteins were already present at a good concentration (titers of 1/3005 C. Motti, M. Nuzzo, A. Meola, G. Galfrr, F. Felici, R. Cortese, A. Nicosia, and P. Monaci, Gene 146, 191 (1994). 6 Ao Folgori, R. Taft, A. Meola, F. Felici, G. Galfrb, R. Cortese, P. Monaci, and A. Nicosia, EMBO J. 13, 2236 (1994). 7 A. Meola, P. Delmastro, P. Monaci, A. Luzzago, A. Nicosia, F. Felici, R. Cortese, and G. Galfr~, J. Immunol. 154, 3162 (1995). 8 G. Galfr~, unpublished observations (1994).
[6]
IMMUNIZATION WITH PHAGE-DISPLAYED MIMOTOPES
111
1/900), and no consistent production of IgM was detected. A short interval was therefore sufficient for the second injection to induce a very strong antiphage response. The induction of mimotope-specific antibodies is, however, a much more difficult process and varies considerably from one mimotope to another. For most immunogenic mimotopes, some degree of response is detectable after the first boost, but in most cases a longer period of immunization is required, and some animals start to respond only after three or four boosts; in the latter case, longer intervals between boosts seem to be of some benefit. Our experience of immunization in rabbits is more limited, but essentially reflects what was observed in mice. Positive responses can be sustained through occasional boosts for more than 1 year. In one chimpanzee a remarkably quick response was obtained with a good titer of antigen cross-reactive antibodies only 15 days after the initial priming. The titer improved considerably after the first and second boosts, and was maintained over a 1-year period by occasional boosts.
Dose Response Doses ranging from l09 t o 1012 phage particles per injection were used in mice. Even with the smallest dose, all the animals produce antibodies to wild-type phage, and about half of them show a specific response to the mimotopes. Increasing the dose results in higher titers in a shorter time (fewer boosts are necessary) and also increases the percentage of animals responding to the specific mimotope. The dose-response curve is very fiat, while the different mimotopes often present considerable fluctuation in immunogenicity. Approximately 1011 phage particles (corresponding in pC89 phagemid-derived libraries 9'1° to about 1.3 tzg of protein) per injection are sufficient for most of the mice. In rabbits of about 1.5 kg of weight, 1013 phage (about 130 ~g) per injection were used with good results. With monkeys weighing about 3 kg, 6 × 1013 phage (about 800/.~g) were used and produced the results described earlier. In a chimpanzee of about 30 kg, good immunization was obtained using 2 × l014 phage particles (about 2.7 mg).
Quality of Antigen The purity of the antigen is relevant only if impurities are immunodominant or if antibodies to the contaminant interfere with the assays used to 9 F. Felici, L. Castagnoli, A. Musacchio, R. Jappelli, and G. Cesareni, J. MoL Biol. 222, 301 (1991). 10A. Luzzago, F. Felici, A. Tramontano, A. Pessi, and R. Cortese, Gene 128, 51 (1993).
112
PHAGEDISPLAYLIBRARIES
[6]
demonstrate the specific response. Immunization with phagotopes inevitably induces a strong response to the phage wild-type antigens and, in the majority of cases, to the specific mimotope. Phage that had been purified either by polyethylene glycol (PEG) precipitation or through CsC1 gradient were used to immunize mice, and both preparations elicited equally good antibody responses to the phage protein and to the specific mimotope displayed (i.e., antibodies cross-reactive with the original antigen). As expected, sera from mice immunized with (PEG)purified phage showed higher background when tested by enzyme-linked immunosorbent assay (ELISA) on recombinant proteins purified from bacterial extract; generally speaking, this does not present a problem if proper controls are included in the experiments. CsC1 purification of phage reduces, but does not completely eliminate, the induction of antibodies to contaminant bacterial antigens.
Use of Adjuvants It has been reported 4 that mimotopes displayed on phage behave as Tcell-dependent antigens and that adjuvants are not required for immunization. Although we have also obtained a strong antibody response in mice when injecting phage in physiological solution, we did find that using Freund's adjuvant (FA) considerably amplifies this response. In general, the use of adjuvant results in higher titers in a shorter time in most of the animals, and this effect is particularly marked for poorly immunogenic mimotopes. In monkey, immunization without adjuvant led to a response against wild-type phage coat proteins, but not against the mimotope. In chimpanzee, one animal injected with phage formulated in Freund's incomplete adjuvant (IFA) showed a prompt and strong mimotope-specific response, while immunizing another two animals utilizing a weaker adjuvant failed.
Mice Immunization Protocol On the basis of the findings and considerations described earlier, the following protocol was designed. Day 0: obtain a sample of preimmune serum and priming ip injection of 1011 phage in CFA Day 21: boost ip with 10ll phage in IFA Day 31: first test bleed Day 42: second boost with 1011 phage in IFA Day 52: second test bleed In most cases, high titers of antibodies to wild-type phage are already present in the first bleed. For strongly immunogenic mimotopes, half of
[6]
IMMUNIZATION WITH PHAGE-DISPLAYED MIMOTOPES
113
the animals present titers ranging from 1/100 to 1/1000 against mimicked antigens in the second bleed. For the less immunogenic mimotopes, the cycle of 3-week boosts followed by test bleeds 10 days after immunization is repeated. BALB/c, C57BL, and MF1 mice strains were used. Although MF1 mice in general respond marginally better, the variations observed were not statistically significant.
Rabbit Immunization Protocol Day 0: obtain a sample of preimmune serum and priming id injection of 1013 phage in CFA Day 21: boost ss with 1013 phage in IFA Day 36: boost im with 1013 phage in physiological solution Day 37: boost im with 1013 phage in physiological solution Day 47: first test bleed Day 65: boost im with 1013 phage in physiological solution Day 75: second bleed As for the mice, the cycle of 4-week interval boosts followed by test bleeds 10 days after immunization is repeated for the less immunogenic mimotopes. If necessary, the positive response can be maintained for several months with occasional im boosts once it has been achieved.
Analysis of Immune Response
Response to Wild-Type Phage Coat Proteins It is often convenient to assess the response to immunization in terms of quantifying the production of antibodies to wild-type phage. As a rule, high titers are easily achieved and often correlate to the feasibility of obtaining a mimotope-specific response. The most practical test to assess titers against wild-type phage is by ELISA, following one of the numerous protocols widely described in the literature. H Briefly, a 96-well ELISA plate is coated with the phage by incubating 100 /zl/well of a solution of 10 /zg/ml of wild-type phage in phosphatebuffered saline (PBS) for at least 1 hr at room temperature. The coating solution is discarded and the plate is washed once with PBS. Add 100/zl/ well of sera dilutions to be tested and incubate on ice for 1 to 4 hr. Discard the solution and wash once with PBS and three times with distilled water. 11 E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, "Current Protocols in Immunology." Wiley, New York, 1991.
114
PHAGEDISPLAYLIBRARIES
[6]
Add 100 /zl/well of peroxidase-labeled anti-mouse Ig (or other species specificity, as required) at a proper dilution and incubate on ice for 1 to 4 hr. Discard the solution and wash twice with PBS and five times with distilled water. Add 100 ~l/well of peroxidase substrate. After 15-30 min, or when sufficient color develops, add 50/zl/well of 2 M H 2 8 0 4 . Mix as thoroughly as possible by carefully shaking the plate and read the optical density at 450 nm. The use of 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) as a substrate is suggested as it is not a carcinogen. The TMB substrate is prepared by first dissolving 10 mg/ml TMB (Sigma, St. Louis, MO) in dimethyl sulfoxide. One hundred microliters of this stock solution is diluted prior to use in 10 ml of 0.1 M acetate-citrate buffer at pH 6, with the addition of 20/zl of 3% (w/v) H202. The other most commonly used enzyme for ELISA is alkaline phosphatase. The protocol used is essentially the same, but p-nitrophenyl phosphate disodium is used as the substrate; ready to use tablets of this substrate are commercially available (Sigma).
Response to Mimotopes The antibody response to a phagotope is complex, largely recognizing the wild-type coat proteins. It is hence difficult to assess if part of this response is directed at the mimotope displayed. Utilizing a synthetic peptide containing the mimotope sequence can be a useful strategy, but may lead to problems because some peptide sequences are not recognized by specific antibodies when they are no longer phage displayed. 7J2 A phagotope's capability of immunogenic mimicry is thus generally assessed by testing the relevant antisera for the presence of antibodies that are cross-reactive with the antigen it is assumed to mimic. This is often done by ELISA whenever the antigen is available in sufficiently purified form or by protein immunoblotting if the antigen is not easy to purify. Protein immunoblotting (Western blotting) protocols are extensively discussed in the literature u and only need to be adapted to the specific antigen under study.
Immunofluorescence A particularly useful methodology in studying the mimicry of posttranscriptionally modified antigenic proteins is that of their expression in suitable eukaryotic cells lines followed by detection by indirect immunofluorescence. a2 F. Felici, A. Luzzago, A. Folgori, and R. Cortese, Gene 128, 21 (1993).
[6]
IMMUNIZATION WITH PHAGE-DISPLAYED M1MOTOPES
] 15
Standard protocols can be adopted for this purpose; we, for example, have successfully used a vaccinia virus transient expression system. Cell monolayers of transfected mammalian cells on sterile coverslips were prepared; 36 hr after infection and transfection the cells were fixed with 3.7% formaldehyde and permeabilized by immersion for 10 min in 0.1% Triton X-100. Cells were incubated in proper dilutions of antisera to test, rinsed in l x PBS, and incubated with a secondary fluorescein or rhodaminelabeled anti-Ig antibody. After rinsing, coverslips were mounted in Moviol and examined under a UV microscope.
Study of Cross-Reactions As previously mentioned, reactivity with the original antigen in ELISA of the antisera from animals immunized with phagotopes is not always conclusive proof of antigenic mimicry. To further verify this hypothesis, a convenient method is the study of specific binding cross-inhibition. This can be accomplished through a standard ELISA assay as described earlier, but in the first incubation fixed amounts of antisera are mixed with increasing amounts of inhibitors. If the original antigen is coated on the plate, complete inhibition should be achieved with the same antigen in solution or with the specific phagotope, but not with irrelevant antigens, wild-type phage, or unrelated phagotope. In addition to studying the sera specificity, this technique is generally useful in studying cross-reactions among different mimotopes mimicking the same or overlapping epitopes in inhibiting the binding to the original antigen of serum antibodies elicited by them. 7 Acknowledgments We thank the members of our laboratories for contributing to various aspects of this work and Ms. Janet Clench for reviewing the manuscript.
IMMUNIZATION
WITH PHAGE-DISPLAYED
MIMOTOPES
109
2. Detach cells from the flask by a 20-min incubation in PBS-2 mM EDTA followed by scraping. 3. Centrifuge (10 rain, 130 g) and resuspend the cells in TBSC at a final concentration of 10 6 cells/ml. 4. Add 5/zl fluoresceinated TAG3 peptide ligand (10 -5 M)41 to 50/zl calmodulin-tagged scFv antibody (e.g., 2 × 10 -6 M ) and incubate for 5 rain. 5. Add cells (500/zl) and incubate on ice for 20 min. 6. Centrifuge cells (5 min, 300 g) and resuspend in 1 ml TBSC. 7. Analyze on a Becton-Dickinson FAC scan.
[6] I m m u n i z a t i o n
with Phage-Displayed
Mimotopes
B y GIOVANNI GALFRI~, PAOLO MONACI, ALFREDO NICOSIA, ALESSANDRA LUZZAGO, FRANCO FELICI, a n d RICCARDO CORTESE
Phage-displayed mimotopes (phagotopes) are selected from random peptide libraries because they bind to specific antibodies, thus behaving as antigenic mimics that resemble to some extent the original antigen. If the positive image of the antigen (Ag) these mimotopes provide is accurate enough to induce the production of specific antibodies (Ab) in animals, the mimotopes prove to be suitable substitute immunogens, totally distinct from the original antigen (that raised the antibodies used to identify the phagotopes). At present, the only feasible way of assessing this capability is the active immunization of experimental animals. Filamentous phages are known to be excellent immunogens; de la Cruz and collaborators 1 first proposed using filamentous phage as immunological carriers by cloning short peptides derived from the malaria circumsporozoite at the N terminus of plII. Greenwood et aL 2 cloned malaria peptides at the N terminus of the major coat protein VIII, and Minenkova et al. 3 cloned an antigenic determinant of human immunodeficiency virus (HIV) gag protein fused to pVIII and used the recombinant phage to successfully immunize rabbits. Similarly, when Willis et al. 4 immunized mice with malaria peptides fused to pVIII, they not only obtained an antibody response, but also demonstrated that the response in mice is T-cell dependent and undergoes class switching from immunoglobulin M (IgM) to IgG. t V. F. de la Cruz, A. A. Lal, and T. F. McCutchan, J. Biol. Chem. 263, 4318 (1988). 2 j. Greenwood, A. E. Willis, and R. N. Perham, J. Mot. Biol. 220, 821 (1991). 3 O. O. Minenkova, A. A. Ilyichev, G. P. Kishchenko, and V. A. Petrenko, Gene 128, 85 (1993). 4 A. E. Willis, R. N. Perham, and D. Wraith, Gene 128, 79 (1993).
METHODS IN ENZYMOLOGY, VOL. 267
Copyright © 1996 by Academic Press. Inc. All rights of reproduction in any form reserved.
110
PHAGEDISPLAYLIBRARIES
[6]
Mimotopes selected by using either hepatitis B virus surface antigen (HBsAg)-specific monoclonal antibodies or human sera have been used to induce HBsAg-specific antibodies in both mice and rabbitsJ -7 Immunization Protocols In preparing immunization protocols, numerous variables have to be considered, particularly the site and route of administration, the number of boosts and the interval between them, and the dose and quality of antigen to be administered at each boost. The optimal conditions will vary considerably according to the species of animal to be immunized. The immunization protocols reported in this chapter essentially derive from our experience using phagotopes as immunogens rather than as a systematic analysis of the just-described parameters.
Route of Administration Mice immunization with phagotopes through intraperitoneal (ip) injection is the most practical route of administration and gave good results in our tests. In rabbits, the protocol used is based on general knowledge, and is a combination of intradermal (id) priming followed by a first subscapular boost (ss) and several intramuscular (im) boosts to maintain antibody production over a long period when necessary. Immunization in monkeys (Macaca fascicularis) has been attempted through a series of subcutaneous (sc) injections. These were sufficient to raise good titers of antibodies to wild-type phage protein, but nonetheless were unable to generate specific antimimotope response, s This result is probably attributable to other factors (see below) and not to the route of administration used. However, an antimimotope response was successfully raised in chimpanzees through im administration. 8
Number and Spacing of Boosts In mice, 5 days after ip priming, IgG antibodies specific for phage coat proteins were already present at a good concentration (titers of 1/3005 C. Motti, M. Nuzzo, A. Meola, G. Galfrr, F. Felici, R. Cortese, A. Nicosia, and P. Monaci, Gene 146, 191 (1994). 6 Ao Folgori, R. Taft, A. Meola, F. Felici, G. Galfrb, R. Cortese, P. Monaci, and A. Nicosia, EMBO J. 13, 2236 (1994). 7 A. Meola, P. Delmastro, P. Monaci, A. Luzzago, A. Nicosia, F. Felici, R. Cortese, and G. Galfr~, J. Immunol. 154, 3162 (1995). 8 G. Galfr~, unpublished observations (1994).
[6]
IMMUNIZATION WITH PHAGE-DISPLAYED MIMOTOPES
111
1/900), and no consistent production of IgM was detected. A short interval was therefore sufficient for the second injection to induce a very strong antiphage response. The induction of mimotope-specific antibodies is, however, a much more difficult process and varies considerably from one mimotope to another. For most immunogenic mimotopes, some degree of response is detectable after the first boost, but in most cases a longer period of immunization is required, and some animals start to respond only after three or four boosts; in the latter case, longer intervals between boosts seem to be of some benefit. Our experience of immunization in rabbits is more limited, but essentially reflects what was observed in mice. Positive responses can be sustained through occasional boosts for more than 1 year. In one chimpanzee a remarkably quick response was obtained with a good titer of antigen cross-reactive antibodies only 15 days after the initial priming. The titer improved considerably after the first and second boosts, and was maintained over a 1-year period by occasional boosts.
Dose Response Doses ranging from l09 t o 1012 phage particles per injection were used in mice. Even with the smallest dose, all the animals produce antibodies to wild-type phage, and about half of them show a specific response to the mimotopes. Increasing the dose results in higher titers in a shorter time (fewer boosts are necessary) and also increases the percentage of animals responding to the specific mimotope. The dose-response curve is very fiat, while the different mimotopes often present considerable fluctuation in immunogenicity. Approximately 1011 phage particles (corresponding in pC89 phagemid-derived libraries 9'1° to about 1.3 tzg of protein) per injection are sufficient for most of the mice. In rabbits of about 1.5 kg of weight, 1013 phage (about 130 ~g) per injection were used with good results. With monkeys weighing about 3 kg, 6 × 1013 phage (about 800/.~g) were used and produced the results described earlier. In a chimpanzee of about 30 kg, good immunization was obtained using 2 × l014 phage particles (about 2.7 mg).
Quality of Antigen The purity of the antigen is relevant only if impurities are immunodominant or if antibodies to the contaminant interfere with the assays used to 9 F. Felici, L. Castagnoli, A. Musacchio, R. Jappelli, and G. Cesareni, J. MoL Biol. 222, 301 (1991). 10A. Luzzago, F. Felici, A. Tramontano, A. Pessi, and R. Cortese, Gene 128, 51 (1993).
112
PHAGEDISPLAYLIBRARIES
[6]
demonstrate the specific response. Immunization with phagotopes inevitably induces a strong response to the phage wild-type antigens and, in the majority of cases, to the specific mimotope. Phage that had been purified either by polyethylene glycol (PEG) precipitation or through CsC1 gradient were used to immunize mice, and both preparations elicited equally good antibody responses to the phage protein and to the specific mimotope displayed (i.e., antibodies cross-reactive with the original antigen). As expected, sera from mice immunized with (PEG)purified phage showed higher background when tested by enzyme-linked immunosorbent assay (ELISA) on recombinant proteins purified from bacterial extract; generally speaking, this does not present a problem if proper controls are included in the experiments. CsC1 purification of phage reduces, but does not completely eliminate, the induction of antibodies to contaminant bacterial antigens.
Use of Adjuvants It has been reported 4 that mimotopes displayed on phage behave as Tcell-dependent antigens and that adjuvants are not required for immunization. Although we have also obtained a strong antibody response in mice when injecting phage in physiological solution, we did find that using Freund's adjuvant (FA) considerably amplifies this response. In general, the use of adjuvant results in higher titers in a shorter time in most of the animals, and this effect is particularly marked for poorly immunogenic mimotopes. In monkey, immunization without adjuvant led to a response against wild-type phage coat proteins, but not against the mimotope. In chimpanzee, one animal injected with phage formulated in Freund's incomplete adjuvant (IFA) showed a prompt and strong mimotope-specific response, while immunizing another two animals utilizing a weaker adjuvant failed.
Mice Immunization Protocol On the basis of the findings and considerations described earlier, the following protocol was designed. Day 0: obtain a sample of preimmune serum and priming ip injection of 1011 phage in CFA Day 21: boost ip with 10ll phage in IFA Day 31: first test bleed Day 42: second boost with 1011 phage in IFA Day 52: second test bleed In most cases, high titers of antibodies to wild-type phage are already present in the first bleed. For strongly immunogenic mimotopes, half of
[6]
IMMUNIZATION WITH PHAGE-DISPLAYED MIMOTOPES
113
the animals present titers ranging from 1/100 to 1/1000 against mimicked antigens in the second bleed. For the less immunogenic mimotopes, the cycle of 3-week boosts followed by test bleeds 10 days after immunization is repeated. BALB/c, C57BL, and MF1 mice strains were used. Although MF1 mice in general respond marginally better, the variations observed were not statistically significant.
Rabbit Immunization Protocol Day 0: obtain a sample of preimmune serum and priming id injection of 1013 phage in CFA Day 21: boost ss with 1013 phage in IFA Day 36: boost im with 1013 phage in physiological solution Day 37: boost im with 1013 phage in physiological solution Day 47: first test bleed Day 65: boost im with 1013 phage in physiological solution Day 75: second bleed As for the mice, the cycle of 4-week interval boosts followed by test bleeds 10 days after immunization is repeated for the less immunogenic mimotopes. If necessary, the positive response can be maintained for several months with occasional im boosts once it has been achieved.
Analysis of Immune Response
Response to Wild-Type Phage Coat Proteins It is often convenient to assess the response to immunization in terms of quantifying the production of antibodies to wild-type phage. As a rule, high titers are easily achieved and often correlate to the feasibility of obtaining a mimotope-specific response. The most practical test to assess titers against wild-type phage is by ELISA, following one of the numerous protocols widely described in the literature. H Briefly, a 96-well ELISA plate is coated with the phage by incubating 100 /zl/well of a solution of 10 /zg/ml of wild-type phage in phosphatebuffered saline (PBS) for at least 1 hr at room temperature. The coating solution is discarded and the plate is washed once with PBS. Add 100/zl/ well of sera dilutions to be tested and incubate on ice for 1 to 4 hr. Discard the solution and wash once with PBS and three times with distilled water. 11 E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, "Current Protocols in Immunology." Wiley, New York, 1991.
114
PHAGEDISPLAYLIBRARIES
[6]
Add 100 /zl/well of peroxidase-labeled anti-mouse Ig (or other species specificity, as required) at a proper dilution and incubate on ice for 1 to 4 hr. Discard the solution and wash twice with PBS and five times with distilled water. Add 100 ~l/well of peroxidase substrate. After 15-30 min, or when sufficient color develops, add 50/zl/well of 2 M H 2 8 0 4 . Mix as thoroughly as possible by carefully shaking the plate and read the optical density at 450 nm. The use of 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) as a substrate is suggested as it is not a carcinogen. The TMB substrate is prepared by first dissolving 10 mg/ml TMB (Sigma, St. Louis, MO) in dimethyl sulfoxide. One hundred microliters of this stock solution is diluted prior to use in 10 ml of 0.1 M acetate-citrate buffer at pH 6, with the addition of 20/zl of 3% (w/v) H202. The other most commonly used enzyme for ELISA is alkaline phosphatase. The protocol used is essentially the same, but p-nitrophenyl phosphate disodium is used as the substrate; ready to use tablets of this substrate are commercially available (Sigma).
Response to Mimotopes The antibody response to a phagotope is complex, largely recognizing the wild-type coat proteins. It is hence difficult to assess if part of this response is directed at the mimotope displayed. Utilizing a synthetic peptide containing the mimotope sequence can be a useful strategy, but may lead to problems because some peptide sequences are not recognized by specific antibodies when they are no longer phage displayed. 7J2 A phagotope's capability of immunogenic mimicry is thus generally assessed by testing the relevant antisera for the presence of antibodies that are cross-reactive with the antigen it is assumed to mimic. This is often done by ELISA whenever the antigen is available in sufficiently purified form or by protein immunoblotting if the antigen is not easy to purify. Protein immunoblotting (Western blotting) protocols are extensively discussed in the literature u and only need to be adapted to the specific antigen under study.
Immunofluorescence A particularly useful methodology in studying the mimicry of posttranscriptionally modified antigenic proteins is that of their expression in suitable eukaryotic cells lines followed by detection by indirect immunofluorescence. a2 F. Felici, A. Luzzago, A. Folgori, and R. Cortese, Gene 128, 21 (1993).
[6]
IMMUNIZATION WITH PHAGE-DISPLAYED M1MOTOPES
] 15
Standard protocols can be adopted for this purpose; we, for example, have successfully used a vaccinia virus transient expression system. Cell monolayers of transfected mammalian cells on sterile coverslips were prepared; 36 hr after infection and transfection the cells were fixed with 3.7% formaldehyde and permeabilized by immersion for 10 min in 0.1% Triton X-100. Cells were incubated in proper dilutions of antisera to test, rinsed in l x PBS, and incubated with a secondary fluorescein or rhodaminelabeled anti-Ig antibody. After rinsing, coverslips were mounted in Moviol and examined under a UV microscope.
Study of Cross-Reactions As previously mentioned, reactivity with the original antigen in ELISA of the antisera from animals immunized with phagotopes is not always conclusive proof of antigenic mimicry. To further verify this hypothesis, a convenient method is the study of specific binding cross-inhibition. This can be accomplished through a standard ELISA assay as described earlier, but in the first incubation fixed amounts of antisera are mixed with increasing amounts of inhibitors. If the original antigen is coated on the plate, complete inhibition should be achieved with the same antigen in solution or with the specific phagotope, but not with irrelevant antigens, wild-type phage, or unrelated phagotope. In addition to studying the sera specificity, this technique is generally useful in studying cross-reactions among different mimotopes mimicking the same or overlapping epitopes in inhibiting the binding to the original antigen of serum antibodies elicited by them. 7 Acknowledgments We thank the members of our laboratories for contributing to various aspects of this work and Ms. Janet Clench for reviewing the manuscript.