Short Papers Protection of guinea-pigs against foot-and-mouth disease virus by immunization with a PhoE-FMDV hybrid protein Marja Agterberg*$ Henrii~tte Adriaanse*, Simon Barteling*, Kees van Maanen t and Jan Tommassen *~ A hybrid protein was constructed containing two antigenic determinants of the structural protein VP1 of foot-and-mouth disease virus, inserted in a cell surface-exposed region of Escherichia coli outer membrane protein PhoE. Immunization of guinea-pigs with partially purified protein resulted in high levels of neutralizing antibodies and complete protection against challenge with the virus. Keywords: P h o E
p r o t e i n ; c a r r i e r s y s t e m ; f o o t - a n d - m o u t h disease virus, vaccine
Recombinant DNA techniques can be used for the high-level expression of potentially protective antigens or antigenic determinants of pathogenic organisms in a safe and fast growing organism such as Escherichia coli K-12. The expression of these antigenic determinants at the bacterial cell surface is expected to be advantageous for the development of new types of (live oral) vaccines or serodiagnostic reagents. Therefore, we are interested in developing a carrier system based on the well characterized outer membrane protein PhoE of E. coli K-12. PhoE protein is an abundant outer membrane protein of E. coli K-12. Its synthesis is induced when cells are grown under phosphate limitation ~. In its functional trimeric form the protein forms transmembrane channels through which small hydrophilic solutes can pass 2. According to a model for the folding of PhoE protein 3'~, the polypeptide traverses the outer membrane 16 times in antiparallel /~-sheet structure, thereby exposing eight regions at the cell surface. Theoretically, all these exposed regions can be used for the insertion of antigenic determinants, provided that the assembly of the hybrid proteins is not disturbed by the insertions. To explore the possibilities of PhoE protein as an exposure vector, we have chosen two antigenic determinants of the structural protein VP 1 of foot-and-mouth disease virus (FMDV), type A10. The determinants comprise amino acids 141-153 and 200 207 of VP1 protein and are recognized by the virus-neutralizing monoclonal antibodies MA 11 and MA 18, respectively 5. Previously, we described the insertion of different *Institute of Molecular Biology and Medical BJotechnology and Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584-CH Utrecht, The Netherlands. *Central Veterinary Institute, Houtribweg 39, 8221-RA Lelystad, The Netherlands. *+Present address: Department of Medical Chemistry, Free University of Amsterdam, van de Boechorststr. 7, 1007 MC Amsterdam, The Netherlands. ~To whom correspondence should be addressed (Recetved 27 November 1989; revised 20 February 1990; accepted 22 February 1990 0264-410X/90/050438-03 ~;, 1990 Butterworth-Hememann Ltd
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Vaccme, Vol. 8, October 1990
combinations of these two antigenic determinants into the fourth and the fifth exposed region of PhoE protein 6'v. The hybrid proteins were correctly incorporated into the outer membrane and the inserted epitopes were exposed at the cell surface. Immunization experiments in mice. using partially purified proteins, showed that the FMDV determinants in all constructs were immunogenic 7. One construct, which contains a tandem repeat of the MAl8-epitope plus the MAll-epitope inserted in the fourth exposed region of PhoE (Figure 1), elicited weak but significant levels of neutralizing antibodies in some animals. This hybrid protein was selected to perform protection experiments in guinea-pigs. Trimers of wild-type PhoE and P h o E - F M D V hybrid protein were isolated from derivatives of the porin-less E. coli strain CE12488 carrying the plasmids pMR059 and p M R I 4 v, respectively. Due to a phoR mutation, the plasmid-encoded PhoE and the P h o E - F M D V hybrid protein are produced constitutively in this strain. Cell envelopes were isolated from 31 cultures grown overnight at 37°C in L-broth I° by ultrasonic disintegration of the cells followed by differential centrifugation ll. Protein-peptidoglycan complexes were isolated by extraction of the cell envelopes for 30 rain at 40°C in a buffer containing 2 % SDS, followed by ultracentrifugation 12. To dissociate PhoE from the peptidoglycan, the complexes were incubated for 30rain at 60°C in the 2% SDS buffer supplemented with 0.6M NaC113. After ultracentrifugation, the trimers were isolated from the supernatant bv ethanol precipitation. For further purification the trimer preparations were applied on a Sepharose CL-4B column and the column was eluted with a buffer containing 0.1% SDS t4. Figure 2 shows the protein patterns of whole cells and of the purified proteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ~1 NR
YKQKIIAP MAt8
YKQKIIAP MA18
SRRGDLGSLAPRV MAt I
KGGD
Figure 1 Amino acid sequence of the inserted epttopes m the PhoE-FMDV hybrid protein In bold, the arnmo acid residues 157-159 of PhoE are mdtcated. The MA18- and MA11-epltopes consist el the amino acids 200-207 and 141-153 of VP1 protem, respectwely
Short Paper: M. Agterberg
et al.
Table I Protectton of guinea-pigs against challenge wuth FMDV, type A10 Holland Neutralization tttres % PhoEFMDV ~ PhoE
Antigen
Animal
day 21
WT-PhoE PhoE-FMDV
1-5 1 2 3 4 5
p r o t e i n ; c a r r i e r s y s t e m ; f o o t - a n d - m o u t h disease virus, vaccine
Recombinant DNA techniques can be used for the high-level expression of potentially protective antigens or antigenic determinants of pathogenic organisms in a safe and fast growing organism such as Escherichia coli K-12. The expression of these antigenic determinants at the bacterial cell surface is expected to be advantageous for the development of new types of (live oral) vaccines or serodiagnostic reagents. Therefore, we are interested in developing a carrier system based on the well characterized outer membrane protein PhoE of E. coli K-12. PhoE protein is an abundant outer membrane protein of E. coli K-12. Its synthesis is induced when cells are grown under phosphate limitation ~. In its functional trimeric form the protein forms transmembrane channels through which small hydrophilic solutes can pass 2. According to a model for the folding of PhoE protein 3'~, the polypeptide traverses the outer membrane 16 times in antiparallel /~-sheet structure, thereby exposing eight regions at the cell surface. Theoretically, all these exposed regions can be used for the insertion of antigenic determinants, provided that the assembly of the hybrid proteins is not disturbed by the insertions. To explore the possibilities of PhoE protein as an exposure vector, we have chosen two antigenic determinants of the structural protein VP 1 of foot-and-mouth disease virus (FMDV), type A10. The determinants comprise amino acids 141-153 and 200 207 of VP1 protein and are recognized by the virus-neutralizing monoclonal antibodies MA 11 and MA 18, respectively 5. Previously, we described the insertion of different *Institute of Molecular Biology and Medical BJotechnology and Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584-CH Utrecht, The Netherlands. *Central Veterinary Institute, Houtribweg 39, 8221-RA Lelystad, The Netherlands. *+Present address: Department of Medical Chemistry, Free University of Amsterdam, van de Boechorststr. 7, 1007 MC Amsterdam, The Netherlands. ~To whom correspondence should be addressed (Recetved 27 November 1989; revised 20 February 1990; accepted 22 February 1990 0264-410X/90/050438-03 ~;, 1990 Butterworth-Hememann Ltd
438
Vaccme, Vol. 8, October 1990
combinations of these two antigenic determinants into the fourth and the fifth exposed region of PhoE protein 6'v. The hybrid proteins were correctly incorporated into the outer membrane and the inserted epitopes were exposed at the cell surface. Immunization experiments in mice. using partially purified proteins, showed that the FMDV determinants in all constructs were immunogenic 7. One construct, which contains a tandem repeat of the MAl8-epitope plus the MAll-epitope inserted in the fourth exposed region of PhoE (Figure 1), elicited weak but significant levels of neutralizing antibodies in some animals. This hybrid protein was selected to perform protection experiments in guinea-pigs. Trimers of wild-type PhoE and P h o E - F M D V hybrid protein were isolated from derivatives of the porin-less E. coli strain CE12488 carrying the plasmids pMR059 and p M R I 4 v, respectively. Due to a phoR mutation, the plasmid-encoded PhoE and the P h o E - F M D V hybrid protein are produced constitutively in this strain. Cell envelopes were isolated from 31 cultures grown overnight at 37°C in L-broth I° by ultrasonic disintegration of the cells followed by differential centrifugation ll. Protein-peptidoglycan complexes were isolated by extraction of the cell envelopes for 30 rain at 40°C in a buffer containing 2 % SDS, followed by ultracentrifugation 12. To dissociate PhoE from the peptidoglycan, the complexes were incubated for 30rain at 60°C in the 2% SDS buffer supplemented with 0.6M NaC113. After ultracentrifugation, the trimers were isolated from the supernatant bv ethanol precipitation. For further purification the trimer preparations were applied on a Sepharose CL-4B column and the column was eluted with a buffer containing 0.1% SDS t4. Figure 2 shows the protein patterns of whole cells and of the purified proteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ~1 NR
YKQKIIAP MAt8
YKQKIIAP MA18
SRRGDLGSLAPRV MAt I
KGGD
Figure 1 Amino acid sequence of the inserted epttopes m the PhoE-FMDV hybrid protein In bold, the arnmo acid residues 157-159 of PhoE are mdtcated. The MA18- and MA11-epltopes consist el the amino acids 200-207 and 141-153 of VP1 protem, respectwely
Short Paper: M. Agterberg
et al.
Table I Protectton of guinea-pigs against challenge wuth FMDV, type A10 Holland Neutralization tttres % PhoEFMDV ~ PhoE
Antigen
Animal
day 21
WT-PhoE PhoE-FMDV
1-5 1 2 3 4 5
