Eur. J. Immunol. 1990.20: 2363-2366
Polarity of imrnunogens
2363
Short paper Jose Golvanoo, Juan J. LasarteO, Pablo SarobeO, Arturo GuIlbn,O, Jesus PrietoO and Francisco Bods-Cuestao Departamento de Medicina Internan and Departamento de GeneticaO, Universidad de Navarra, Pamplona
Polarity of immunogens: implications for vaccine design* Peptide constructs have been engineered consisting of amino acid sequence determinant recognized by Tcells (TD) co-linearly linked t o haptenic peptides. It was found that high anti-hapten antibody titers were induced after immunization with those constructs which had theTD sequence in the N-terminal position with respect to the hapten. Low or zero titers were elicited when the TD was in C-terminal position. Also, a high anti-hapten antibody titer corresponded to a low or zero anti-TD antibody titer and vice versa. These results suggest that immunogens are polar and stress the relevance of searching the more adequate position of the TD within a peptide construct when designing immunogens or synthetic peptide vaccines.
1 Introduction
2 Materials and methods
Haptenic peptides can be rendered immunogenic by coupling to an amino acid sequence which binds to the MHC of type I1 (MHC-11) and which is recognized by Th cells in conjunction with this complex [l-31. Moreover, the immunogenicity of peptide haptens or of peptide immunogens can sometimes be induced or increased by linear polymerization [4]. However,we do not understand yet the details of how this sequence, recognized by T h cells, (from now on referred to as Tcell determinant or TD), confers immunogenicity to a hapten.
2.1 Synthetic peptides
It has been reported [5] that if a determinant recognized by Tcells is co-linerly linked to a determinant recognized by B cells (BD), the TD will be responsible for the induction of higher anti-BD antibody titers when theTD is in C-terminal position. This finding, in conjunction with the observation that in other co-linear peptide constructs of the type TD-BD it is the TD in N-terminal position which is responsible for the induction of anti-BD antibodies, suggests that immunogens might be polar.Thus, the position of theTD within the peptide could be of great relevance in the design of immunogenic peptides. To increase our knowledge on this subject, we synthesized eight peptide constructs of the typeTD-BD and BD-TD.We report below the immunogenicity of these peptide constructs and discuss our findings in the context of the engineering of synthetic peptide vaccines.
2.2 Immunization
[I 86211
*
This work was supported by the Gobierno de Navarra, Spain.
Correspondence: Francisco Bods-Cuesta, Departamento de Medicina Interna, Universidad de Navarra, Facultad de Medicina, Apartado 273, E-31080 Pamplona, Spain
TD: Determinant recognized by T cells BD: Determinant recognized by B cells Abbreviations:
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheirn, 1990
Peptides were synthesized by the solid-phase method of Merrifield [6] using the Fmoc alternative [7]. The synthesis was done manually and the ninhydrin test of Kaiser et al. [8] used to monitor every step. Couplings were repeated if necessary until a negative ninhydrin test was attained. All peptides were purified by HPLC on a reverse-phase C18 column.
Groups of three 4-week-old BAL,B/c mice were immunized by i.p. injection of 60 pg of peptide antigen emulsified in 200 pl CFA adjuvant and boosted at days 30 and 45 with the same dose of antigen in IFA. Mice were eye-bled on days 15, 30, 45, 52 and 60 after the first injection.
2.3 ELISA Anti-peptide antibodies were titrated by ELISA. Microtiter wells were coated with peptides by incubation with 50 pl peptide solution at 4 "C overnight (20 pg/ml peptide in 0.1 M sodium carbonate buffer). The wells were then washed three times with a solution of PBS containing 1% powdered milk and 0.1% Theen 20 (PBSMT). To block nonspecific antibody binding, the wells were incubated with the above buffer for 2 h at room temperature. After removing the PBSMT, 50 pl of different serum dilutions was added and incubated at 37 "Cfor 1h, then washed three times with PBSMT and incubated at 37 "C for 1 h with a 1/1OOOsolution of biotinylated goat anti-mouse IgG whole antibody (Amersham Int., Amersham, GB) in PBSMT. After washing three times with PBSMT, the wells were incubated with an 1/1OOO dilution of horseradish peroxidase-streptavidin (Amersham) at room temperature for 1h. After washing three times with PBS, the color reaction was started by adding 100 pl of a solution prepared by mixing: 10 ml of 0.6% acetic acid (pH 4.7); 5 pl of 33% 0014-2980/90/1010-2363$3.50+ .2510
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J. Golvano, J. J. Lasarte, F? Sarobe et al.
Eur. J. Immunol. 1990.20: 2363-2366
(w/v) hydrogen peroxide and 100 pl of 40 mM water solution of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). After 30 min the plates were read at 405 nm in aTitertek Multiscan MKII (Flow Laboratories, Puteaux, France).
3 Results 3.1 Anti-peptide antisera titration The amino acid sequences of peptides used in the present study are shown in Table 1. Peptides FERFEIFPKE and FISEAIIHVLHSR are proven T D in BALB/c mice [9]. They correspond to sequences 111-120 and 106-118 from influenza hemagglutinin and sperm whale myoglobin, respectively. Sequences LKCNNKTFNGTGPCTNV and LGIWGCSGKLICITAV correspond to amino acids 238-254 and 605-620 from the gp160 envelope protein of HIV-1 virus (amino acid positions according to [lo]). The first eight peptides of this table were used to immunize BALBk mice and the antisera thus obtained were titrated by ELISA against peptides coating the microtiter plates. To measure the proportion of antibodies that were specific for the BD and TD regions of each construct, every anti-peptide antiserum was titrated against several peptides sharing with the construct only the region to be titrated. Thus, to measure the antibodies induced by LGI-FER against region LGI, the anti-LGI-FER antiserum was titrated against peptides LGI, LGI-FIS and FIS-LGI. Similarly, peptides LKC-FER and FER-LKC were used to measure those elicited by LGI-FER against region FER. In the same manner, to measure anti-LKC antibodies, the anti-FIS-LKC antiserum was titrated against peptides LKC, LKC-FER and FER-LKC, whereas peptides FIS-LGI and LGI-FIS were used to titrate the anti-FIS antibodies; and so on for the other peptides representing different combinations of these two BD and TD. Each antiserum was titrated with several peptides not only to map the immune response directed against different regions of the immunogen, but also to measure changes in antibody recognition of a region due to structural changes of this region induced by different peptide environments.
FIS-u(c RS-LGl FER-UC FER-LGI LKC-FIS LGI-FIS LKCFER LGI-FER
Figure 1. Antibody titers against ED (above) and TD (below) contained in peptide constructs FIS-LKC, FIS-LGI, FER-LKC, FER-LGI,LKC-FIS, LGI-FIS, LKC-FER and LGI-FER. For the sake of clarity only one set of titers is shown: the one at day 60 in which the antibodies against a given region are titrated using a peptide containing this region in the same relative position as that of the peptide used for immunization. Each bar represents the titer against a given region of the immunogen. It is plotted facing the three-letter code corresponding to this region. TD are marked in
bold.
Fig. 1shows the antibody titers against BD: LGI and LKC (upper part of the figure) and against TD: FIS and FER (lower part). Higher anti-BD antibody titers are obtained when the T D of the peptide used for immunization is situated in N-terminal position. The polarity of our peptide constructs is, thus, opposite to that reported by Cox et al. [5]. Indeed, if the T D of our peptides is situated in a C-terminal position, no anti-BD antibodies are obtained after immunization with LKC-FER and low with LGIFER, whereas low titers are obtained for FER-LGI and high for FER-LKC. Immunization with LGI-FER and with LKC-FIS affords antibody titers that are much lower than the ones elicited with FIS-LGI and FIS-LKC, respectively. In all cases the anti-BD antibodies recognized the BD better in those peptides having the BD in the same relative position (N terminal or C terminal, as the case might be) as the peptide used for immunization (data not shown).
An interesting result is obtained when the humoral response against the T D is titrated. The general trend is that higher anti-TD titers are induced by those peptides eliciting lower anti-BD titers. Comparison of the upper part with the lower part of Fig. 1gives the impression of a mirror image. In particular, peptide FIS-LKC is capable of eliciting very Table 1. Amino acid sequences of peptide constructs,correspond- high anti-LKC titers (> 1OOOOO) without inducing anti-FIS ing to different co-linear combinations of BD and TD antibodies. This last result is of practical relevance because the absence of antibodies anti-FIS would probably insure Peptide sequences Abbreviation that FIS could be used in multiple vaccinations in the same animal, without fear of suppression of this T D by anti-FIS LGI-FER antibodies. Obviously this would be valid if FIS-BD and not LGIWGCSGKLCITAV-FERFEFElFPKE LKC-FER LKCNNKTFWGTGPCI’NV-FERFEIFPKE BD-FIS immunogens were injected. LGIWGCSGKLICITAV--FAIIHVL€SR LGI-FIS LKCNNKTFNGTGPCTNV-FAIIHVLHSR LKC-FIS As mentioned above, for all eight peptides tested, higher FER-LKC FERFEIFPKE-LGIWGCSGKLICITAV anti-BD antibody titers were obtained when the TD was in FER-LKC FERFEIFPKE-LKCNNKTFNGTGPCTNV an N-terminal position. Also, a low antibody titer against FISEAIIHVLHSR-LGIWGCSGKLICITAV FIS-LGI theTD was induced to a high antibody titer induced against FISEAIIHVLHSR-LKCNNKTFNGTGPCTNV FIS-LKC the BD and vice versa.These result in conjunction with that LGIWGCSGKLICTTAV LGI of Cox et a]. [5] suggest that certain immunogens have LKCNNKTFWGTGPCI’NV LKC TD-BD polarity and others BD-TD. However, as we discuss
Eur. J. Irnmunol. 1990. 20: 2363-2366
Polarity of irnmunogens
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external TD or carrier). Also, they both induced Tcell proliferation. For these reasons, BD1 and BD2 should be considered as TD. Thus, the anti-BD1 and anti-BD2 titers induced by constructs TD1-BD1 and TD1-BD2 might at At the time of writing this manuscript two reports describ- least be partly due toTcell help provided by B Dl and BD2, ing results similar to ours have come to our attention* [ll]. respectively. However, the much higher anti-BD1 and In a peptide construct consisting of a T D from the pre-S(2) anti-BD2 induced by the constructs, with respect to those region of hepatitis B virus surface protein linked to another induced by BD1 and BD2 alone, suggests that most of theT amino acid sequence , the construct induced antibodies cell help might be due toTD1. Since, in the construct that against the adjacent sequence to the T D only when the T D had theTD1 in C-terminal position, the BD1 was linked to was in N-terminal position*. Similarly, Levely et al. [ l l ] TD1 with a bifunctional reagent, it can not be concluded safely that this TD1 would had provided less T cell help to synthesized two linear constructs: TD1-BD1, TDl:BD2, where TD1 is a determinant recognized by Tcells corres- BD1 if linearly linked to BD1 in a construct of the type ponding to amino acids 45-60 from protein 1 A of respira- BD1-TD1. We do not know if the putative BD used in the tory syncytial virus (RS), and BD1 and BD2 correspond to (pre-S2)-BD construct* might have had potential TD amino acids 151-164 from glycoprotein G and amino acids character. 221-236 from glycoprotein F of RS virus, respectively. In both constructs, high antibody titers were induced against the region adjacent to TD1 but relatively low titers against 4 Discussion TD1. Moreover, Levelyet al. [ l l ] cross-linked BD1 to T Dl with a bifunctional reagent to obtain a construct in which We believe that our results, in conjunction with those of TD1 is in C-terminal position with respect to BD1. This others* [5,11], clearly prove the importance of choosing construct induced high antibody titers against TD1 and low the right relative position of the T D for an adequate induction of antibodies against the BD in linear constructs against BD1. of the type: TD-BD and BD-TD. These results suggest that immunogens are probably polar. Although we do not clearly understand the origin of this polarity, we suspect it 3.2 T cell proliferation assays might be due to enzymatic processing of the antigens.Thus, We knew that FIS and FER were proven T D in BALBlc [91 theTD that we have used in our own work (FISand FER), but we did not know if the two putative BD that had been like those used* in [ll],contain Arg or Lys near their C chosen (LKC and LGI sequences of the eight constructs of terminus. Similarly,it has been reported [ 121that in peptide Table 1) might also behave as TD. Thus, the induction of 120-145 of the pre-S(2) hepatitis B surface antigen there is anti-LKC or anti-LGI antibodies by these constructs might a T D at the N terminus (residues 120-132) and a BD at the have been due to: (a) T D help provided by FIS and FER, or C terminus (residues 133-145). Also in this case there is an Arg in position 135 near the end of both epitopes. This (b) T D help provided by LKC or LGI. suggests the possibility of an proteolytic-cleavage at the C To test which of the above two alternatives was responsible terminus of Arg or Lys in contructs of the type TD-BD but for the induction of antibodies against LKC and LGI, we not in constructs BD-TD. In the first case (but not in the inoculated mice with the above constructs. We isolated the second) an activeTD would be released as a consequence of LN of these mice and carried out cell proliferation assays antigen processing which would provide Tcell help to the using synthetic peptides containing different combinations clone that internalized the immunogen TD-BD. O n the of BD and TD.These assays (data not shown) proved that: contrary, in constructs where the T D in the C-terminal (a) FIS is a strong T D and FER a much weaker one, (b) position is better suited for the induction of anti-BD LKC is in no case responsible for cell proliferation, thus had antibodies [5], the Lys residue is near the N terminus (and no T D character, (c) LGI is probably the main TD in not near the C terminus like in the constructs discussed above). Thus, the active TD might be more easily released construct LGI-FER. when situated at the C terminus of the immunogen, in Since LGI seemed to be the mainTD of peptide LGI-FER agreement with the polarity reported by Cox et al. [ S ] . it is likely that LGI was responsible for the T D help Similarly, glucagon, a natural peptide hormone of 29 amino necessary to induce antibodies against FER following acids has two Arg residues in position 17 and 18which might immunization with LGI-FER. In this case also, the immune provide a cleavage site just between the well-characterized system would prefer the position of the T D in the N- BD (residues 1-17) and T D (residues 19-29) regions of this terminal region of the immunogen. Since LKC did not hormone [13]. If the processing hypothesis was right, stimulate T cells, anti-FER antibodies induced by LKC- inserting Arg or Lys residues (or other amino acids for other FER or FER-LKC immunization are most likely due toTD specificities) between the T D and the BD would provide a help provided by FER. That is, FER behaves both as T D cleavage site to insure the release of the TD. In this way and BD. In an analogous manner, FIS behaves as T D and immunogens could be engineered which have the TD in either N-terminal or C-temrinal position. We are carrying BD in construct LKC-FIS. out experiments to test this hypothesis. The putative BD used by Levely et al. [ l l ] (here referred to as BD1 and BD2) were immunogenic by themselves Another possibility for bypassing the effect of polarity (induced antibodies without T cell help provided by an might be to prepare immunogens by linear polymerization of peptides TD-BD or BD-TD as described [4]. This would afford compounds of the type TD-BD-TD-BD-TD . . . * D. Millich, personal communication. where theTD is both N-terminal and C-terminal respect to
below, to prove the validity of this hypothesis it was necessary to show that the putative BD of our constructs had no T D character by themselves.
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J. Golvano, J. J. Lasarte, F? Sarobe et al.
the BD. Thus, the TD within the polymer might be able to use either of the two alternatives to provideTcel1help to the BD. While waiting for a clearer understanding of the molecular mechanism to explain the immunogen polarity, the most adequate position of the TD to induce high anti-BD antibody titers in peptide constructs or subunit vaccines of the type TD-BD or BD-TD should be assessed experimentally. Received June 6, 1990.
5 References 1 Good, M. F., Malloy, W. L., Lunde, M. N., Margalit, H., Cornette, J. L., Smith, G. L., Moss, B., Miller, L. H. and Berzofsky, J. A., Science 1987. 235: 1059.
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2 Leclerck, C., Przewlocki, G., Chutze, M. P. and Chedid, L., Eur. J. Immunol. 1987. 17: 269. 3 Borras-Cuesta, F., Petit-Camurdan, A. and Fedon,Y., Eur. J. Immunol. 1987. 17: 1213. 4 Borras-Cuesta, F., Fedon,Y. and Petit-Camurdan, A. Eur. J. Immunol. 1988. 18: 199. 5 Cox,J. H., Ivanyi, J. ,Young, D. B., Lamb, J. R., Syred, A. D. and Francis, M . J., Eur. 1. Immunol. 1988. 18: 2015. 6 Merrifield, R. B., J. Am. Chem. SOC. 1963. 85: 2149. 7 Atherton, E., Logan, J. C. andSheppard, C. R., J. Chem. SOC. Perkin. Trans. 1981. 1: 538. 8 Kaiser, E., Colescott, R. L., Bossinger, C. D. and Cook, F? I., Anal. Biochem. 1970. 34: 595. 9 Rothbard, J. B. and Taylor, W. R., EMBO J. 1988. 7: 93. 10 Wain-Hobson, S., Sonigo, F?, Danos, O., Cole, S. and Muon, I? F?, Cell 1985. 40: 9. 11 Levely, M. E., Mitchell, M. A. and Nicholas, J. A., Cell. Immunol. 1990. 125: 65. 12 Milich, D.R., McLachlan, A., Chisari, F.V. and Thornton, G. B., J. Exp. Med. 1986. 164: 532. 13 Senyk, G.,Williams, E. B., Nitecki, D. E. and Goodman, J.W., J. Exp. Med. 1971. 133: 1294.
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Polarity of imrnunogens
2363
Short paper Jose Golvanoo, Juan J. LasarteO, Pablo SarobeO, Arturo GuIlbn,O, Jesus PrietoO and Francisco Bods-Cuestao Departamento de Medicina Internan and Departamento de GeneticaO, Universidad de Navarra, Pamplona
Polarity of immunogens: implications for vaccine design* Peptide constructs have been engineered consisting of amino acid sequence determinant recognized by Tcells (TD) co-linearly linked t o haptenic peptides. It was found that high anti-hapten antibody titers were induced after immunization with those constructs which had theTD sequence in the N-terminal position with respect to the hapten. Low or zero titers were elicited when the TD was in C-terminal position. Also, a high anti-hapten antibody titer corresponded to a low or zero anti-TD antibody titer and vice versa. These results suggest that immunogens are polar and stress the relevance of searching the more adequate position of the TD within a peptide construct when designing immunogens or synthetic peptide vaccines.
1 Introduction
2 Materials and methods
Haptenic peptides can be rendered immunogenic by coupling to an amino acid sequence which binds to the MHC of type I1 (MHC-11) and which is recognized by Th cells in conjunction with this complex [l-31. Moreover, the immunogenicity of peptide haptens or of peptide immunogens can sometimes be induced or increased by linear polymerization [4]. However,we do not understand yet the details of how this sequence, recognized by T h cells, (from now on referred to as Tcell determinant or TD), confers immunogenicity to a hapten.
2.1 Synthetic peptides
It has been reported [5] that if a determinant recognized by Tcells is co-linerly linked to a determinant recognized by B cells (BD), the TD will be responsible for the induction of higher anti-BD antibody titers when theTD is in C-terminal position. This finding, in conjunction with the observation that in other co-linear peptide constructs of the type TD-BD it is the TD in N-terminal position which is responsible for the induction of anti-BD antibodies, suggests that immunogens might be polar.Thus, the position of theTD within the peptide could be of great relevance in the design of immunogenic peptides. To increase our knowledge on this subject, we synthesized eight peptide constructs of the typeTD-BD and BD-TD.We report below the immunogenicity of these peptide constructs and discuss our findings in the context of the engineering of synthetic peptide vaccines.
2.2 Immunization
[I 86211
*
This work was supported by the Gobierno de Navarra, Spain.
Correspondence: Francisco Bods-Cuesta, Departamento de Medicina Interna, Universidad de Navarra, Facultad de Medicina, Apartado 273, E-31080 Pamplona, Spain
TD: Determinant recognized by T cells BD: Determinant recognized by B cells Abbreviations:
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheirn, 1990
Peptides were synthesized by the solid-phase method of Merrifield [6] using the Fmoc alternative [7]. The synthesis was done manually and the ninhydrin test of Kaiser et al. [8] used to monitor every step. Couplings were repeated if necessary until a negative ninhydrin test was attained. All peptides were purified by HPLC on a reverse-phase C18 column.
Groups of three 4-week-old BAL,B/c mice were immunized by i.p. injection of 60 pg of peptide antigen emulsified in 200 pl CFA adjuvant and boosted at days 30 and 45 with the same dose of antigen in IFA. Mice were eye-bled on days 15, 30, 45, 52 and 60 after the first injection.
2.3 ELISA Anti-peptide antibodies were titrated by ELISA. Microtiter wells were coated with peptides by incubation with 50 pl peptide solution at 4 "C overnight (20 pg/ml peptide in 0.1 M sodium carbonate buffer). The wells were then washed three times with a solution of PBS containing 1% powdered milk and 0.1% Theen 20 (PBSMT). To block nonspecific antibody binding, the wells were incubated with the above buffer for 2 h at room temperature. After removing the PBSMT, 50 pl of different serum dilutions was added and incubated at 37 "Cfor 1h, then washed three times with PBSMT and incubated at 37 "C for 1 h with a 1/1OOOsolution of biotinylated goat anti-mouse IgG whole antibody (Amersham Int., Amersham, GB) in PBSMT. After washing three times with PBSMT, the wells were incubated with an 1/1OOO dilution of horseradish peroxidase-streptavidin (Amersham) at room temperature for 1h. After washing three times with PBS, the color reaction was started by adding 100 pl of a solution prepared by mixing: 10 ml of 0.6% acetic acid (pH 4.7); 5 pl of 33% 0014-2980/90/1010-2363$3.50+ .2510
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J. Golvano, J. J. Lasarte, F? Sarobe et al.
Eur. J. Immunol. 1990.20: 2363-2366
(w/v) hydrogen peroxide and 100 pl of 40 mM water solution of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). After 30 min the plates were read at 405 nm in aTitertek Multiscan MKII (Flow Laboratories, Puteaux, France).
3 Results 3.1 Anti-peptide antisera titration The amino acid sequences of peptides used in the present study are shown in Table 1. Peptides FERFEIFPKE and FISEAIIHVLHSR are proven T D in BALB/c mice [9]. They correspond to sequences 111-120 and 106-118 from influenza hemagglutinin and sperm whale myoglobin, respectively. Sequences LKCNNKTFNGTGPCTNV and LGIWGCSGKLICITAV correspond to amino acids 238-254 and 605-620 from the gp160 envelope protein of HIV-1 virus (amino acid positions according to [lo]). The first eight peptides of this table were used to immunize BALBk mice and the antisera thus obtained were titrated by ELISA against peptides coating the microtiter plates. To measure the proportion of antibodies that were specific for the BD and TD regions of each construct, every anti-peptide antiserum was titrated against several peptides sharing with the construct only the region to be titrated. Thus, to measure the antibodies induced by LGI-FER against region LGI, the anti-LGI-FER antiserum was titrated against peptides LGI, LGI-FIS and FIS-LGI. Similarly, peptides LKC-FER and FER-LKC were used to measure those elicited by LGI-FER against region FER. In the same manner, to measure anti-LKC antibodies, the anti-FIS-LKC antiserum was titrated against peptides LKC, LKC-FER and FER-LKC, whereas peptides FIS-LGI and LGI-FIS were used to titrate the anti-FIS antibodies; and so on for the other peptides representing different combinations of these two BD and TD. Each antiserum was titrated with several peptides not only to map the immune response directed against different regions of the immunogen, but also to measure changes in antibody recognition of a region due to structural changes of this region induced by different peptide environments.
FIS-u(c RS-LGl FER-UC FER-LGI LKC-FIS LGI-FIS LKCFER LGI-FER
Figure 1. Antibody titers against ED (above) and TD (below) contained in peptide constructs FIS-LKC, FIS-LGI, FER-LKC, FER-LGI,LKC-FIS, LGI-FIS, LKC-FER and LGI-FER. For the sake of clarity only one set of titers is shown: the one at day 60 in which the antibodies against a given region are titrated using a peptide containing this region in the same relative position as that of the peptide used for immunization. Each bar represents the titer against a given region of the immunogen. It is plotted facing the three-letter code corresponding to this region. TD are marked in
bold.
Fig. 1shows the antibody titers against BD: LGI and LKC (upper part of the figure) and against TD: FIS and FER (lower part). Higher anti-BD antibody titers are obtained when the T D of the peptide used for immunization is situated in N-terminal position. The polarity of our peptide constructs is, thus, opposite to that reported by Cox et al. [5]. Indeed, if the T D of our peptides is situated in a C-terminal position, no anti-BD antibodies are obtained after immunization with LKC-FER and low with LGIFER, whereas low titers are obtained for FER-LGI and high for FER-LKC. Immunization with LGI-FER and with LKC-FIS affords antibody titers that are much lower than the ones elicited with FIS-LGI and FIS-LKC, respectively. In all cases the anti-BD antibodies recognized the BD better in those peptides having the BD in the same relative position (N terminal or C terminal, as the case might be) as the peptide used for immunization (data not shown).
An interesting result is obtained when the humoral response against the T D is titrated. The general trend is that higher anti-TD titers are induced by those peptides eliciting lower anti-BD titers. Comparison of the upper part with the lower part of Fig. 1gives the impression of a mirror image. In particular, peptide FIS-LKC is capable of eliciting very Table 1. Amino acid sequences of peptide constructs,correspond- high anti-LKC titers (> 1OOOOO) without inducing anti-FIS ing to different co-linear combinations of BD and TD antibodies. This last result is of practical relevance because the absence of antibodies anti-FIS would probably insure Peptide sequences Abbreviation that FIS could be used in multiple vaccinations in the same animal, without fear of suppression of this T D by anti-FIS LGI-FER antibodies. Obviously this would be valid if FIS-BD and not LGIWGCSGKLCITAV-FERFEFElFPKE LKC-FER LKCNNKTFWGTGPCI’NV-FERFEIFPKE BD-FIS immunogens were injected. LGIWGCSGKLICITAV--FAIIHVL€SR LGI-FIS LKCNNKTFNGTGPCTNV-FAIIHVLHSR LKC-FIS As mentioned above, for all eight peptides tested, higher FER-LKC FERFEIFPKE-LGIWGCSGKLICITAV anti-BD antibody titers were obtained when the TD was in FER-LKC FERFEIFPKE-LKCNNKTFNGTGPCTNV an N-terminal position. Also, a low antibody titer against FISEAIIHVLHSR-LGIWGCSGKLICITAV FIS-LGI theTD was induced to a high antibody titer induced against FISEAIIHVLHSR-LKCNNKTFNGTGPCTNV FIS-LKC the BD and vice versa.These result in conjunction with that LGIWGCSGKLICTTAV LGI of Cox et a]. [5] suggest that certain immunogens have LKCNNKTFWGTGPCI’NV LKC TD-BD polarity and others BD-TD. However, as we discuss
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Polarity of irnmunogens
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external TD or carrier). Also, they both induced Tcell proliferation. For these reasons, BD1 and BD2 should be considered as TD. Thus, the anti-BD1 and anti-BD2 titers induced by constructs TD1-BD1 and TD1-BD2 might at At the time of writing this manuscript two reports describ- least be partly due toTcell help provided by B Dl and BD2, ing results similar to ours have come to our attention* [ll]. respectively. However, the much higher anti-BD1 and In a peptide construct consisting of a T D from the pre-S(2) anti-BD2 induced by the constructs, with respect to those region of hepatitis B virus surface protein linked to another induced by BD1 and BD2 alone, suggests that most of theT amino acid sequence , the construct induced antibodies cell help might be due toTD1. Since, in the construct that against the adjacent sequence to the T D only when the T D had theTD1 in C-terminal position, the BD1 was linked to was in N-terminal position*. Similarly, Levely et al. [ l l ] TD1 with a bifunctional reagent, it can not be concluded safely that this TD1 would had provided less T cell help to synthesized two linear constructs: TD1-BD1, TDl:BD2, where TD1 is a determinant recognized by Tcells corres- BD1 if linearly linked to BD1 in a construct of the type ponding to amino acids 45-60 from protein 1 A of respira- BD1-TD1. We do not know if the putative BD used in the tory syncytial virus (RS), and BD1 and BD2 correspond to (pre-S2)-BD construct* might have had potential TD amino acids 151-164 from glycoprotein G and amino acids character. 221-236 from glycoprotein F of RS virus, respectively. In both constructs, high antibody titers were induced against the region adjacent to TD1 but relatively low titers against 4 Discussion TD1. Moreover, Levelyet al. [ l l ] cross-linked BD1 to T Dl with a bifunctional reagent to obtain a construct in which We believe that our results, in conjunction with those of TD1 is in C-terminal position with respect to BD1. This others* [5,11], clearly prove the importance of choosing construct induced high antibody titers against TD1 and low the right relative position of the T D for an adequate induction of antibodies against the BD in linear constructs against BD1. of the type: TD-BD and BD-TD. These results suggest that immunogens are probably polar. Although we do not clearly understand the origin of this polarity, we suspect it 3.2 T cell proliferation assays might be due to enzymatic processing of the antigens.Thus, We knew that FIS and FER were proven T D in BALBlc [91 theTD that we have used in our own work (FISand FER), but we did not know if the two putative BD that had been like those used* in [ll],contain Arg or Lys near their C chosen (LKC and LGI sequences of the eight constructs of terminus. Similarly,it has been reported [ 121that in peptide Table 1) might also behave as TD. Thus, the induction of 120-145 of the pre-S(2) hepatitis B surface antigen there is anti-LKC or anti-LGI antibodies by these constructs might a T D at the N terminus (residues 120-132) and a BD at the have been due to: (a) T D help provided by FIS and FER, or C terminus (residues 133-145). Also in this case there is an Arg in position 135 near the end of both epitopes. This (b) T D help provided by LKC or LGI. suggests the possibility of an proteolytic-cleavage at the C To test which of the above two alternatives was responsible terminus of Arg or Lys in contructs of the type TD-BD but for the induction of antibodies against LKC and LGI, we not in constructs BD-TD. In the first case (but not in the inoculated mice with the above constructs. We isolated the second) an activeTD would be released as a consequence of LN of these mice and carried out cell proliferation assays antigen processing which would provide Tcell help to the using synthetic peptides containing different combinations clone that internalized the immunogen TD-BD. O n the of BD and TD.These assays (data not shown) proved that: contrary, in constructs where the T D in the C-terminal (a) FIS is a strong T D and FER a much weaker one, (b) position is better suited for the induction of anti-BD LKC is in no case responsible for cell proliferation, thus had antibodies [5], the Lys residue is near the N terminus (and no T D character, (c) LGI is probably the main TD in not near the C terminus like in the constructs discussed above). Thus, the active TD might be more easily released construct LGI-FER. when situated at the C terminus of the immunogen, in Since LGI seemed to be the mainTD of peptide LGI-FER agreement with the polarity reported by Cox et al. [ S ] . it is likely that LGI was responsible for the T D help Similarly, glucagon, a natural peptide hormone of 29 amino necessary to induce antibodies against FER following acids has two Arg residues in position 17 and 18which might immunization with LGI-FER. In this case also, the immune provide a cleavage site just between the well-characterized system would prefer the position of the T D in the N- BD (residues 1-17) and T D (residues 19-29) regions of this terminal region of the immunogen. Since LKC did not hormone [13]. If the processing hypothesis was right, stimulate T cells, anti-FER antibodies induced by LKC- inserting Arg or Lys residues (or other amino acids for other FER or FER-LKC immunization are most likely due toTD specificities) between the T D and the BD would provide a help provided by FER. That is, FER behaves both as T D cleavage site to insure the release of the TD. In this way and BD. In an analogous manner, FIS behaves as T D and immunogens could be engineered which have the TD in either N-terminal or C-temrinal position. We are carrying BD in construct LKC-FIS. out experiments to test this hypothesis. The putative BD used by Levely et al. [ l l ] (here referred to as BD1 and BD2) were immunogenic by themselves Another possibility for bypassing the effect of polarity (induced antibodies without T cell help provided by an might be to prepare immunogens by linear polymerization of peptides TD-BD or BD-TD as described [4]. This would afford compounds of the type TD-BD-TD-BD-TD . . . * D. Millich, personal communication. where theTD is both N-terminal and C-terminal respect to
below, to prove the validity of this hypothesis it was necessary to show that the putative BD of our constructs had no T D character by themselves.
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J. Golvano, J. J. Lasarte, F? Sarobe et al.
the BD. Thus, the TD within the polymer might be able to use either of the two alternatives to provideTcel1help to the BD. While waiting for a clearer understanding of the molecular mechanism to explain the immunogen polarity, the most adequate position of the TD to induce high anti-BD antibody titers in peptide constructs or subunit vaccines of the type TD-BD or BD-TD should be assessed experimentally. Received June 6, 1990.
5 References 1 Good, M. F., Malloy, W. L., Lunde, M. N., Margalit, H., Cornette, J. L., Smith, G. L., Moss, B., Miller, L. H. and Berzofsky, J. A., Science 1987. 235: 1059.
Eur. J. Immunol. 1990. 20: 2363-2366
2 Leclerck, C., Przewlocki, G., Chutze, M. P. and Chedid, L., Eur. J. Immunol. 1987. 17: 269. 3 Borras-Cuesta, F., Petit-Camurdan, A. and Fedon,Y., Eur. J. Immunol. 1987. 17: 1213. 4 Borras-Cuesta, F., Fedon,Y. and Petit-Camurdan, A. Eur. J. Immunol. 1988. 18: 199. 5 Cox,J. H., Ivanyi, J. ,Young, D. B., Lamb, J. R., Syred, A. D. and Francis, M . J., Eur. 1. Immunol. 1988. 18: 2015. 6 Merrifield, R. B., J. Am. Chem. SOC. 1963. 85: 2149. 7 Atherton, E., Logan, J. C. andSheppard, C. R., J. Chem. SOC. Perkin. Trans. 1981. 1: 538. 8 Kaiser, E., Colescott, R. L., Bossinger, C. D. and Cook, F? I., Anal. Biochem. 1970. 34: 595. 9 Rothbard, J. B. and Taylor, W. R., EMBO J. 1988. 7: 93. 10 Wain-Hobson, S., Sonigo, F?, Danos, O., Cole, S. and Muon, I? F?, Cell 1985. 40: 9. 11 Levely, M. E., Mitchell, M. A. and Nicholas, J. A., Cell. Immunol. 1990. 125: 65. 12 Milich, D.R., McLachlan, A., Chisari, F.V. and Thornton, G. B., J. Exp. Med. 1986. 164: 532. 13 Senyk, G.,Williams, E. B., Nitecki, D. E. and Goodman, J.W., J. Exp. Med. 1971. 133: 1294.
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