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Natural variants of the sabin type 1 vaccine strain of poliovirus and correlation with a poliovirus neutralization site
VIROLOGY
143, 33’7-341 (1986)
Natural Variants of the Sabin Type 1 Vaccine Strain of Poliovirus and Correlation with a Poliovirus Neutralization Site BRADFORD
A.
JAMESON
*sl JUTTA
BONIN,*
ECKARD
WIMMER,*~
AND
OLEN
M. KEW~
*Department of Microbiology, State University of New York, School of Medicine, Stony Brook, New York 11794, and ~Division of Viral Diseases, Center for Iqfectims Diseases, Centers fur Disease Control, Atlanta, Georgia 30333 Received Septewdm 5, 1984 accepted December 17, 1984 Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VPl, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 14’7 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize >99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection. o 1985 Academic press. he.
It has been known for nearly two decades that synthetic peptides, linked to a carrier macromolecule, can induce the production of antibodies capable of neutralizing virus infectivity in experimental animals (1, 2). With the rapid expansion of information on the primary sequences of viral genes and gene products, the use of synthetic peptides as neutralizing antigens has received much renewed interest (32, 33, 37). Synthetic peptide antigens may offer safe and effective alternatives to conventional virus vaccines (al), and, moreover, may be critically important to the control of viral diseases whose etiologic agents cannot be economically propagated for large-scale vaccine manufacture. In any event, the three serotypes of polioviruses, still incompletely controlled agents of serious human disease in most parts of the world (3), represent a favorable system for study in these areas. The bases of immune protection to poi Present address: Max von Pettenkofer University of Munich, Pettenkofer Str. Munich 2, West Germany. 2 Author to whom requests for reprints addressed.
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337
liomyelitis have been extensively studied. Either of two immunologic states is sufficient to prevent paralytic disease (a) The presence of poliovirus type-specific neutralizing antibodies, or (b) the establishment of type-specific immunologic memory (27,28). Both inactivated (28) and live (27) poliovirus vaccines confer solid immunity. While numerous natural antigenic variants exist for each of the three poliovirus serotypes (5), all strains within a serotype exhibit strong antigenic cross-reactivities. Thus complete protection can be established by immunization with a set of representative antigens from each serotype. The polioviruses are well characterized at the level of genetic organization and basic virion structure. The complete genomic sequences of representatives of each serotype are known (4, 19, 24, 26, 30, 34) along with the amino acid sequences and proteolytic processing pathways of the viral proteins (7, 8, 20, 25, 31). Approximately 40% of the genome encodes the virus structural proteins, VPl, VP2, VP3, and VP4; 60 copies of each form the icosahedral capsid (14). Numerous experiments have shown that the dominant 0042-6822185 $3.00 Copyright All rights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
338
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neutralizing antigenicity of the virus is contained within the largest, most exposed capsid protein, VP1 (9, 10, 22, 35). VP2 and VP3 also contain neutralization sites (6, 11, 36), and at least one site on each protein has been identified (11; Jameson, Bonin, and Wimmer, unpublished), VP4 is apparently unexposed and in close association with the viral RNA (38, 39). At present, there exist few guidelines for the prediction of neutralizing antigenic sites from primary sequence data. Walter and Doolittle (37) and Hopp and Woods (15) suggested that the best “first approach” to this problem is to scan for contiguous sequences, five to nine residues in length, containing highly hydrophilic amino acids, charged sequences most likely to be exposed at the protein surface. Emini et aZ. (10) have further refined this approach to include regions of interserotypic sequence divergence (34), which presumably reflect some of the differences that encode serotype specificity. An alternative approach is to locate and identify residues in mutants having altered antigenicity, following the principle that many, if not most, of the mutations alter amino acid residues contained within or which directly affect the antigenie site. The most frequently used method is to select and characterize mutants resistant to neutralization by mouse neutralizing monoclonal antibodies (9, 13, 22,40). Whereas this method has identified a N-Ag in VP1 of poliovirus, type 3 (13), none of the mutants selected for by type
1 specific neutralizing monoclonal antibodies map to the site at which the neutralizing monoclonal antibodies bind (D. C. Diamond et aZ., submitted for publication). In the case of poliovirus type 1 the alteration of the epitopes that render them unable to function in neutralization occurs apparently by conformational changes elicited by mutations distant to the antibody binding site. Genetic variation of poliovirus occurs rapidly during replication in the virus’ natural host (17). Nakano et al. (23) have reported that natural antigenic variants of type 1 poliovirus can be obtained as late isolates (10 days postvaccination) from healthy infants who had received monovalent type 1 oral poliovaccine. The changes in antigenicity of the mutants were characterized by analysis with polyclonal (23) and monoclonal (5) sera and by sequencing the regions of the virion encoding VP1 (18). Mutations encoding substitutions in amino acid residues 99 to 106 were detected (la), a region in VP1 shown previously to be an important neutralization antigenic site (10). Here we report that substitution mutations can occur also in VP1 in amino acids 142 and 14’7 in natural variants of poliovirus isolated from vaccinated infants (Fig. 1). This region is not considered to be highly hydrophilic when analyzed according to Hopp and Woods (EJ), an observation that made it unlikely that this region is immunologically active. To examine whether this region is in-
140
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FIG. 1. Sequences of VP1 codons 140 to 149 of Mahoney (parent to Sabin l), Sabin 1, and two independent antigenic variants of Sabin 1, isolated 10 or 28 days postvaccination from healthy infants fed monovalent type 1 oral vaccine. The sequences of Mahoney and Sabin 1 are identical over this interval. Base substitutions and deduced amino acid substitutions in.the.-antigenkv&+gd& Id;kii&@&p~:&n~s&;i;& &@. v. lrion RNAs were sequen~e&~bgr~IW%&&~ ~WiQR%XW&~~~~~&~~di~ tkimh&Obrjl~tigd3
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147
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FIG. 2. The amino acid sequence of peptide No. 14. The underlined sequence is colinear with the VP1 amino acid Nos. 141-14’7 of poliovirus type 1. The peptide was synthesized, conjugated to bovine serum albumin (BSA), and injected into female New Zealand white rabbits exactly as outlined by Emini et al. (10).
volved in neutralization we synthesized a peptide (designated peptide No. 14) corresponding to amino acid sequence 141 to 14’7 of VP1 (Fig. 2). We conjugated the peptide to a bovine serum albumin carrier, and tested for its ability to stimulate production of poliovirus type 1 specific neutralizing antibodies in rabbits. Antibodies produced in response to the carrierconjugated peptide No. 14 were capable of neutralizing 90% of a poliovirus type 1 (Mahoney) population (Table 1). This ob-
servation indicates that the peptide can assume a conformation resembling that of a virion epitope albeit at low frequency. Additionally, the neutralizing response was probably of the IgM class as the antisera were not, or were only weakly, capable of immunoprecipitating virions in the presence of Staphylococcus aweus A cells. It was found, however, that peptide No. 14 did not react with antibodies contained in acute convalescent patient sera using an ELISA (data not shown), an observation suggesting that this region in VP1 may normally not be recognized in an anti-poliovirus response in humans. Although the neutralizing antibody response to the peptide No. 14 conjugate alone was weak, we examined whether immunological memory to the poliovirusspecific peptide and to virion-specific epitopes had been established. Emini et aL (10) have used poliovirus-specific synthetic
TABLE CHARACTERIZATION
OF ANTISERA
Plaque reduction titers prior viral boost (log,, PW* Rabbit A B
PVl
PV2
PV3
1.0 1.0
<0.5 <0.5
<0.5 1.0
Plaque reduction titers past viral boost (log,, PFW
A B
1
GENERATED
Antibody boost PVl 1:2 1:2
Antibody boost
339
IN RESPONSE TO PEPTIDE
titers prior viral (100 TCIDw)C
No. 14”
Immunoprecipitation by antiserumd
PV2
PV3
PVl
PV2
PV3
<1:2 1:2
<1:2 1:2
(+I (+)
-
-
titers past viral (100 TCID&
PVl
PV2
PV3
PVl
PV2
PV3
5.0 5.6
1.9 1.6
<0.5 2.5
1:64 1:64
1:4 1:4
<1:2 1:16
Immunoprecipitation by antiserum PVl
PV2
PV3
+ +
(+) (+I
-
“Fiveto six-week-old female New Zealand white rabbits were inoculated with 1 mg bovine serum albumin-linked peptide (1:l in complete Freund’s adjuvants). The inoculation routes were intradermal (id), subcutaneous (SC) 3 weeks later, and intramuscular (im) after another week. Seven and fourteen days after the last peptide injection the rabbits were bled and the neutralization titers of the obtained sera were determined. bThe standard plaque reduction neutralization assays were done as described by Emini et al. (IO). The sera samples used for these assays were obtained 14 days after the final peptide inoculation. ‘The titers of neutralizing antibodies were determined by a standard neutralization assay of HeLa cells using 100 TCID,, per well. d The immunoprecipitations using S. aureus protein A were done according Emini et al. (10). ’ Sera samples were obtained 36 days following the single subimmunogenic viral boost.
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peptides to show that some conjugated peptides, while by themselves unable to elicit circulating neutralizing antibodies, can prime the immune system for a highlevel, IgG-type neutralizing antibody response. Apparently there is some, as yet unknown, mechanism whereby the immune system can fix a memory of the “neutralizing conformation,” and a subsequent subimmunogenic exposure to intact virus can then elicit an enhanced level response. When the peptide-primed rabbits were inoculated with a subimmunogenic dose of poliovirus type 1 (5 log,, PFU at Day 17 past the last peptide inoculation), they produced increased levels (>5 log,, PFU/ml plaque titer reduction) of IgG-type neutralizing antibodies (Table 1). An increased antipeptide titer, as deduced by endpoint dilutions in an ELISA, was also observed concomitantly with the increase in neutralizing antibody titer. Although the neutralizing activity was predominantly type specific, the antisera also contained antibodies capable of binding to and neutralizing type 2 and type 3 polioviruses. We have also observed a cross-reacting immune response to typespecific peptides with peptides of regions of VP1 other than the region 141 to 147 (16). This phenomenon has also been reported to occur with poliovirus-specific peptides and neutralizing activity against human hepatitis virus A (12). The data do not permit us to determine whether the region of residues 141 to 147 of poliovirus type 1 is involved in recognition processes between cells of the immune system or is indeed a weak neutralization antigenic site. We favor the latter, which suggests that the mutations observed in the variants isolated from vaccinated infants are not simply neutral changes that can be structurally accommodated, but arise in response to selective antibody. Because antigenic drift frequency occurs during replication of the oral poliovaccines in humans (H), sequence analysis of the variants isolated from vaccine recipients may be useful for the recognition of polypeptide sequences active in immune re. _the neutralizing sponse in humans.
ACKNOWLEDGMENTS We thank David Diamond for helpful discussions, and Marlies Schmidt for technical assistance. This work was supported by Public Health Service Grants AI-15122 and CA-28146 from the National Institutes of Health. REFERENCES 1. ANDERER, F. A., and SCXLUMBERGER, H. D., Biochim. Biophys Actu 97, 503-509 (1965). 2. ANDERER, F. A., and SCHLUMBERGER, H. D., Biochim. Biophys. Acta 115,222-224 (1966). 3. ASSAAD, F., and LJUNGARS-ESTEVES, K., Reu. Iqf Dis. 6,302-307 (1934). 4 CANN, A. J., STANWAY, G., HAUPTMANN, R., MINOR, P. D., SCHILD, G. C., and ALMOND, J. W., Nucl. Acids Res. 11, 126’7-1281 (1983). 5. CRAINIC, R., COUILLIN, P., BLONDEL, B., CABAU, N., BOUE, A., and HORODNICEANU, F., Infect. Immun. 41, 121’7-1225 (1983). 6. DERNICK, R., HEUKESHOVEN, J., and HILBRIG, M., Virw 130, 243-246 (1983). 7. DORNER, A. J., DORNER, L. F., LARSEN, G. R., WIMMER, E., and ANDERSON, C. W., J. viral 42.1017-1028 (1982). 8. EMINI, E. A., ELZINGA, M., and WIMMER, E., J. Viral 42, 194-199 (1982a). 9. EMINI, E. A,, JAMESON, B. A., LEWIS, A. J., LARSEN, G. R., and WIMMER, E., J. Viral 43, 997-1005 (1982b). 10. EMINI, E. A., JAMESON, B. A., and WIMMER, E., Nature (London) 304, 666-703 (1983). 11. EMINI, E. A., JAMESON, B. A., and WIMMER, E., J. Vim! 52, 719-721 (1984). 12. EMINI, E. A., BOGER, J., HUGHS, J. V., MITRA, S. W., and LINEMAYER, D. L., In “Modern Approaches to Vaccines” (R. Chanock and R. Lerner, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., in press. 1.6. EVANS, D. M. A., MINOR, P. D., SCHILD, G. C., and ALMOND, J. W., Nature (London) 304,459462 (1933). f.& HOGLE, J. M., In “Modern Approaches to Vaccines” (R. Chanock and R. Lerner, eds.), pp. 7-11. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1984. 15. HOPP, T. P., and WOODS, K. R., Proc Natl. Acd Sci. USA 78, 3824-3828 (1982). 16. JAMESON, B. A., BONIN, J., MURRAY, M., KEW, 0. M., and WIMMER, E., In “Modern Approaches to Vaccines” (R. Chanock and R. Lerner, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., in press. 27. KEW, 0. M., NOI-TAY, B. K., HATCH, M. H., NAKANO, J. H., and OBIJESKI, J. F., J. Gen viral. 56.307-317 (1981). 28. KEW, 0. M., and NOTTAY, B. K., In “Modern Approaches to Vaccines” (R. Chanock and R.
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Lerner, eds.), pp. 357-367. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1984. 19. KITAMURA, N., SEMLER, B. L., ROTHBERG, P. G., LARSEN, G. R., ADLER, J., DORNER, A. J., EMINI, E. A., HANECAK, R., LEE, J. J., VAN DER WERF, S., ANDERSON, C. W., and WIMMER, E. Nature (London) 291, 547-553 (1981). 20. LARSEN, G. R., ANDERSON, C. W., DOWER, A. J., SEMLER, B. L., and WIMMER, E., J. Viral 41, 340-344 (1982). 21. LERNER, R. A., Nature (London) 299, 592-596 (1982). 22. MINOR, P. D., SCHILD, G. C., BOOTMAN, J., EVANS, D. M. A., FERGUSON, M., REEVE, P., SPITZ, M., STANWAY, G., CANN, A. J., HAUPTMANN, R., CLARKE, D. L., MO~NTFORD, R. C., and ALMOND, J. W., Nature (Mm) 301, 674-679 (1983). 23. NAKANO, J. H., GELFAND, H. M., and COLE, J. T., Amer. .J. Hyg. 78, 215-221 (1963). 24. NOMOTO, A., OMATA, T., TOYODA, H., KUGE, S., HORIE, H., KATAOKA, Y., GENBA, Y., NAKANO, J. H., and IMURA, N., Proc Natl. Acad Sci USA 79, 5793-5797 (1982). 25. PALLANSCH, M., KEW, 0. M., SEMLER, B. L., OMILIANOWSKI, D. R., ANDERSON, C. W., WIMMER, E., and RUECKERT, R., J. Vird 49, 873880 (1984). 26. RACANIELLO, V. R., and BALTIMORE, D., Proc. Nat1 Ad Sci. USA 78, 4887-4891 (1981). 27. SABIN, A. B., Rev. InJ Die. 6, 391-396 (1984). 28. SALK, J., Rev. IrLf Dk. 6,444-450 (1984). 29. SMITH, A. J. H., In “Methods in Enzymology” (K. Moldave and L. Grossman, eds.), Vol. 65, pp. 560-580. Academic Press, New York, 1980.
341
30. STANWAY, G., HUGHES, P. J., MOUNTFORD, R. C., REEVE, P., MINOR, P. D., SCHILD, G. C., and ALE~OND, J. W., Proc Nat1 Acad Sci. USA 81, 1539-1543 (1984). 31. SEMLER, B. L., ANDERSON, C. W., KITAMURA, N., ROTHBERG, P. G., WISHART, W. L., and WIMMER, E., Proc. Natl. Ad Sci USA 78, 34643468 (1981). 32. SUTCLIFFE, J. G., SHINNICK, T. M., GREEN, N., and LERNER, R. A., Science (Washington, D. C.) 219, 660-666 (1983). 33. TODD, P. E. E., EAST, I. J., and LEACH, J., Trends Biochem Sci 7, 212-216 (1982). $4 TOYODA, H., KOHARA, M., KATAOKA, Y., SUGAMURA, T., OMATA, T., IMURA, N., and NOMOTO, A., J. MoL Biol. 174, 561-585 (1984). 35. VAN DER WERF, S., WYCHOWSKI, C., BRUNEAU, P., BLONDEL, B., CRAINIC, R., HORODNICEANU, F., and GIRARD, M., Proc. Nat1 Acad Sci USA 80, 5080-5084 (1983). 36. VAN DER MAREL, P., HAZENMINK, T. G., HENNEKE, M. A. C., and VAN WEZEL, A. L., Vaccine 1, 17-22 (1984). 37. WALTER, G., and DOOLI~E, R. F., In “Genetic Engineering” (J. K. Setlow and A. Hollaender, eds.), Genetic Engineering, Vol. 5, pp. 61-91. Plenum, New York/London, 1983. 38. WETZ, K., and HABERMEHL, K.-O., J. Gen I%ol. 44, 525-534 (1979). 39. WETZ, K., and HABERMEHL, K.-O., J. Gen ViTol 59, 397-401 (1983). 40. YEWDELL, J. W., and GERHARD, W., Ann. Rev. Micmbiol 35, 185-206 (1981). 41. ZIMMERN, D., and KAESBERG, P., Proc. Nat1 Ad Sci. USA 75, 4257-4261 (1978).
143, 33’7-341 (1986)
Natural Variants of the Sabin Type 1 Vaccine Strain of Poliovirus and Correlation with a Poliovirus Neutralization Site BRADFORD
A.
JAMESON
*sl JUTTA
BONIN,*
ECKARD
WIMMER,*~
AND
OLEN
M. KEW~
*Department of Microbiology, State University of New York, School of Medicine, Stony Brook, New York 11794, and ~Division of Viral Diseases, Center for Iqfectims Diseases, Centers fur Disease Control, Atlanta, Georgia 30333 Received Septewdm 5, 1984 accepted December 17, 1984 Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VPl, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 14’7 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize >99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection. o 1985 Academic press. he.
It has been known for nearly two decades that synthetic peptides, linked to a carrier macromolecule, can induce the production of antibodies capable of neutralizing virus infectivity in experimental animals (1, 2). With the rapid expansion of information on the primary sequences of viral genes and gene products, the use of synthetic peptides as neutralizing antigens has received much renewed interest (32, 33, 37). Synthetic peptide antigens may offer safe and effective alternatives to conventional virus vaccines (al), and, moreover, may be critically important to the control of viral diseases whose etiologic agents cannot be economically propagated for large-scale vaccine manufacture. In any event, the three serotypes of polioviruses, still incompletely controlled agents of serious human disease in most parts of the world (3), represent a favorable system for study in these areas. The bases of immune protection to poi Present address: Max von Pettenkofer University of Munich, Pettenkofer Str. Munich 2, West Germany. 2 Author to whom requests for reprints addressed.
Institute, 9a, D-8000 should
be
337
liomyelitis have been extensively studied. Either of two immunologic states is sufficient to prevent paralytic disease (a) The presence of poliovirus type-specific neutralizing antibodies, or (b) the establishment of type-specific immunologic memory (27,28). Both inactivated (28) and live (27) poliovirus vaccines confer solid immunity. While numerous natural antigenic variants exist for each of the three poliovirus serotypes (5), all strains within a serotype exhibit strong antigenic cross-reactivities. Thus complete protection can be established by immunization with a set of representative antigens from each serotype. The polioviruses are well characterized at the level of genetic organization and basic virion structure. The complete genomic sequences of representatives of each serotype are known (4, 19, 24, 26, 30, 34) along with the amino acid sequences and proteolytic processing pathways of the viral proteins (7, 8, 20, 25, 31). Approximately 40% of the genome encodes the virus structural proteins, VPl, VP2, VP3, and VP4; 60 copies of each form the icosahedral capsid (14). Numerous experiments have shown that the dominant 0042-6822185 $3.00 Copyright All rights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
338
SHORT
COMMUNICATIONS
neutralizing antigenicity of the virus is contained within the largest, most exposed capsid protein, VP1 (9, 10, 22, 35). VP2 and VP3 also contain neutralization sites (6, 11, 36), and at least one site on each protein has been identified (11; Jameson, Bonin, and Wimmer, unpublished), VP4 is apparently unexposed and in close association with the viral RNA (38, 39). At present, there exist few guidelines for the prediction of neutralizing antigenic sites from primary sequence data. Walter and Doolittle (37) and Hopp and Woods (15) suggested that the best “first approach” to this problem is to scan for contiguous sequences, five to nine residues in length, containing highly hydrophilic amino acids, charged sequences most likely to be exposed at the protein surface. Emini et aZ. (10) have further refined this approach to include regions of interserotypic sequence divergence (34), which presumably reflect some of the differences that encode serotype specificity. An alternative approach is to locate and identify residues in mutants having altered antigenicity, following the principle that many, if not most, of the mutations alter amino acid residues contained within or which directly affect the antigenie site. The most frequently used method is to select and characterize mutants resistant to neutralization by mouse neutralizing monoclonal antibodies (9, 13, 22,40). Whereas this method has identified a N-Ag in VP1 of poliovirus, type 3 (13), none of the mutants selected for by type
1 specific neutralizing monoclonal antibodies map to the site at which the neutralizing monoclonal antibodies bind (D. C. Diamond et aZ., submitted for publication). In the case of poliovirus type 1 the alteration of the epitopes that render them unable to function in neutralization occurs apparently by conformational changes elicited by mutations distant to the antibody binding site. Genetic variation of poliovirus occurs rapidly during replication in the virus’ natural host (17). Nakano et al. (23) have reported that natural antigenic variants of type 1 poliovirus can be obtained as late isolates (10 days postvaccination) from healthy infants who had received monovalent type 1 oral poliovaccine. The changes in antigenicity of the mutants were characterized by analysis with polyclonal (23) and monoclonal (5) sera and by sequencing the regions of the virion encoding VP1 (18). Mutations encoding substitutions in amino acid residues 99 to 106 were detected (la), a region in VP1 shown previously to be an important neutralization antigenic site (10). Here we report that substitution mutations can occur also in VP1 in amino acids 142 and 14’7 in natural variants of poliovirus isolated from vaccinated infants (Fig. 1). This region is not considered to be highly hydrophilic when analyzed according to Hopp and Woods (EJ), an observation that made it unlikely that this region is immunologically active. To examine whether this region is in-
140
MohoneylSobinl
A GCA
A!”
“LC
A:”
Infant
1: Day
10
A GCA
At”
&C
A&J
Infant
3: .Day 28
A GCA
A;”
“‘UC
A:”
FIG. 1. Sequences of VP1 codons 140 to 149 of Mahoney (parent to Sabin l), Sabin 1, and two independent antigenic variants of Sabin 1, isolated 10 or 28 days postvaccination from healthy infants fed monovalent type 1 oral vaccine. The sequences of Mahoney and Sabin 1 are identical over this interval. Base substitutions and deduced amino acid substitutions in.the.-antigenkv&+gd& Id;kii&@&p~:&n~s&;i;& &@. v. lrion RNAs were sequen~e&~bgr~IW%&&~ ~WiQR%XW&~~~~~&~~di~ tkimh&Obrjl~tigd3
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3vsd
zhm-ni
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SHORT I41
COMMUNICATIONS
147
Y-G-G-N-F-T-E-T-N-t&,H
FIG. 2. The amino acid sequence of peptide No. 14. The underlined sequence is colinear with the VP1 amino acid Nos. 141-14’7 of poliovirus type 1. The peptide was synthesized, conjugated to bovine serum albumin (BSA), and injected into female New Zealand white rabbits exactly as outlined by Emini et al. (10).
volved in neutralization we synthesized a peptide (designated peptide No. 14) corresponding to amino acid sequence 141 to 14’7 of VP1 (Fig. 2). We conjugated the peptide to a bovine serum albumin carrier, and tested for its ability to stimulate production of poliovirus type 1 specific neutralizing antibodies in rabbits. Antibodies produced in response to the carrierconjugated peptide No. 14 were capable of neutralizing 90% of a poliovirus type 1 (Mahoney) population (Table 1). This ob-
servation indicates that the peptide can assume a conformation resembling that of a virion epitope albeit at low frequency. Additionally, the neutralizing response was probably of the IgM class as the antisera were not, or were only weakly, capable of immunoprecipitating virions in the presence of Staphylococcus aweus A cells. It was found, however, that peptide No. 14 did not react with antibodies contained in acute convalescent patient sera using an ELISA (data not shown), an observation suggesting that this region in VP1 may normally not be recognized in an anti-poliovirus response in humans. Although the neutralizing antibody response to the peptide No. 14 conjugate alone was weak, we examined whether immunological memory to the poliovirusspecific peptide and to virion-specific epitopes had been established. Emini et aL (10) have used poliovirus-specific synthetic
TABLE CHARACTERIZATION
OF ANTISERA
Plaque reduction titers prior viral boost (log,, PW* Rabbit A B
PVl
PV2
PV3
1.0 1.0
<0.5 <0.5
<0.5 1.0
Plaque reduction titers past viral boost (log,, PFW
A B
1
GENERATED
Antibody boost PVl 1:2 1:2
Antibody boost
339
IN RESPONSE TO PEPTIDE
titers prior viral (100 TCIDw)C
No. 14”
Immunoprecipitation by antiserumd
PV2
PV3
PVl
PV2
PV3
<1:2 1:2
<1:2 1:2
(+I (+)
-
-
titers past viral (100 TCID&
PVl
PV2
PV3
PVl
PV2
PV3
5.0 5.6
1.9 1.6
<0.5 2.5
1:64 1:64
1:4 1:4
<1:2 1:16
Immunoprecipitation by antiserum PVl
PV2
PV3
+ +
(+) (+I
-
“Fiveto six-week-old female New Zealand white rabbits were inoculated with 1 mg bovine serum albumin-linked peptide (1:l in complete Freund’s adjuvants). The inoculation routes were intradermal (id), subcutaneous (SC) 3 weeks later, and intramuscular (im) after another week. Seven and fourteen days after the last peptide injection the rabbits were bled and the neutralization titers of the obtained sera were determined. bThe standard plaque reduction neutralization assays were done as described by Emini et al. (IO). The sera samples used for these assays were obtained 14 days after the final peptide inoculation. ‘The titers of neutralizing antibodies were determined by a standard neutralization assay of HeLa cells using 100 TCID,, per well. d The immunoprecipitations using S. aureus protein A were done according Emini et al. (10). ’ Sera samples were obtained 36 days following the single subimmunogenic viral boost.
340
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peptides to show that some conjugated peptides, while by themselves unable to elicit circulating neutralizing antibodies, can prime the immune system for a highlevel, IgG-type neutralizing antibody response. Apparently there is some, as yet unknown, mechanism whereby the immune system can fix a memory of the “neutralizing conformation,” and a subsequent subimmunogenic exposure to intact virus can then elicit an enhanced level response. When the peptide-primed rabbits were inoculated with a subimmunogenic dose of poliovirus type 1 (5 log,, PFU at Day 17 past the last peptide inoculation), they produced increased levels (>5 log,, PFU/ml plaque titer reduction) of IgG-type neutralizing antibodies (Table 1). An increased antipeptide titer, as deduced by endpoint dilutions in an ELISA, was also observed concomitantly with the increase in neutralizing antibody titer. Although the neutralizing activity was predominantly type specific, the antisera also contained antibodies capable of binding to and neutralizing type 2 and type 3 polioviruses. We have also observed a cross-reacting immune response to typespecific peptides with peptides of regions of VP1 other than the region 141 to 147 (16). This phenomenon has also been reported to occur with poliovirus-specific peptides and neutralizing activity against human hepatitis virus A (12). The data do not permit us to determine whether the region of residues 141 to 147 of poliovirus type 1 is involved in recognition processes between cells of the immune system or is indeed a weak neutralization antigenic site. We favor the latter, which suggests that the mutations observed in the variants isolated from vaccinated infants are not simply neutral changes that can be structurally accommodated, but arise in response to selective antibody. Because antigenic drift frequency occurs during replication of the oral poliovaccines in humans (H), sequence analysis of the variants isolated from vaccine recipients may be useful for the recognition of polypeptide sequences active in immune re. _the neutralizing sponse in humans.
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