INFECTION AND IMMUNITY, Apr. 1991, p. 1251-1254
Vol. 59, No. 4
0019-9567/91/041251-04$02.00/0 Copyright © 1991, American Society for Microbiology
Protection of Immunosuppressed Mice against Infection with Pseudomonas aeruginosa by Recombinant P. aeruginosa Lipoprotein I and Lipoprotein I-Specific Monoclonal Antibodies MATHIAS FINKE,1 GUNTHER MUTH,2 THOMAS REICHHELM,1 MARTIN THOMA,' MICHAEL DUCHENE,3t KLAUS-DIETER HUNGERER,2 HORST DOMDEY,3 AND BERND-ULRICH VON SPECHTl* Chirurgische Universitatsklinik, Chirurgische Forschung, Hugstetterstrasse 55, 7800 Freiburg im Breisgau,' Behringwerke
AG, Marburg, and Laboratorium fur Molekulare Biologie, Genzentrum der Ludwig Maximilians-Universitat Munchen, 8033 Martinsried,3 Federal Republic of Germany Received 17 October 1990/Accepted 4 January 1991
Outer membrane protein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. The protective effect of OprI vaccination and that of three OprI-specific monoclonal antibodies (MAbs) against infection with P. aeruginosa were tested in immunosuppressed mice. The combination of OprI and MAb 2A1 protected the mice against a challenge with a 96-fold 50% lethal dose. The binding site of MAb 2A1 was mapped, resulting in the identification of a protective epitope (amino acids 7 to 20).
Pseudomonas aeruginosa bacteremia is a frequent cause of gram-negative sepsis in the neutropenic subject (22). The two major antigenic cell envelope components of P. aeruginosa are the lipopolysaccharides and the outer membrane proteins (OPRs) (8, 15, 16). We have been interested in the potential of P. aeruginosa OPRs as an immunoprophylactic tool because OPRs are antigenically cross-reactive among all 17 known serogroups of the International Antigenic Typing Scheme (14, 16, 21). We have cloned the genes coding for OprF and OprI of P. aeruginosa (4, 5) and have used OprI expressed in Escherichia coli for a successful vaccination of immunocompetent mice (6). The disadvantage of the immunocompetent mouse model is, however, that the state of neutropenia and the reduction of the antibody synthesizing capacity, which are both critical parameters for the multiplication of P. aeruginosa in immunosuppressed hosts, are not simulated. As an alternative, Cryz et al. (3) have described a leukopenic mouse model in which the 50% lethal dose (LD50) for cyclophosphamide-treated mice is as low as 20 living P. aeruginosa cells. We have used this model previously to prove that our OprI-specific monoclonal antibody (MAb) 2A1 provides a significant yet rather low degree of protection against P. aeruginosa infection (17). With this model, we are now able to show that preimmunization with recombinant OprI in combination with the application of an OprI-specific MAb can be very effective to prevent potentially lethal P. aeruginosa infections in immunosuppressed individuals. In addition, we provide evidence that our MAbs bind to different epitopes of the OprI molecule and that the epitope which is recognized by MAb 2A1 (amino acids [aa] 7 to 20) seems to be the most important for protection.
DNA, preparation of DNA fragments, DNA ligations, and transformation into E. coli were carried out as described by Maniatis et al. (13). For expression of OprI fusion proteins, vectors of the pSEM series (10) or vectors of the pEX series (19) were used. Restriction sites were engineered by using the polymerase chain reaction (18). Plasmid pPIX1 encodes aa 2 to 57 fused to the MS2 polymerase, while plasmids pPI12 (aa 21 to 64) and pPI13 (aa 7 to 62) carry the NH2-terminal part of the 3-galactosidase gene as a fusion partner.
SDS-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of OprI fusion proteins. Sodium dodecyl sulfate (SDS)-polyacrylamide (15%) slab gel electrophoresis and Western blotting were performed as described previously (11, 12, 20). OprI fragments were incubated with the OprI-specific MAbs 2A1, 6A4, and 5B4. Binding was visualized as described recently (21). MAbs. The induction preparation, and purification of the mouse MAb designated 2A1 (immunoglobulin [Ig]G2b), 6A4 (IgG2a), and 5B4 (IgG2a) have been described (14, 17). Enzyme-linked immunosorbent assay. Polyclonal antibodies and MAbs against P. aeruginosa were measured by an enzyme-linked immunosorbent assay as described in detail in a previous paper (6). Bacterial strains and growth conditions. P. aeruginosa International Antigenic Typing Scheme serogroup 6 (ATCC 33353) was obtained from A. Bauernfeind, Max von Pettenkofer-Institut, University of Munich, Munich, Federal Republic of Germany. Bacteria were grown and adjusted to the required concentration as described previously (6). For the expression of recombinant proteins, E. coli K-12 W3110 lacIQ L8 (1) was used. Immunosuppression. For immunosuppression, the leukopenic mouse model described by Cryz et al. (3) was used. The animals received three injections of 150 ,ug of cyclophosphamide (Serva, Heidelberg, Federal Republic of Germany) per g of body weight in 0.25 ml of phosphate-buffered saline (PBS) on days 0, 2, and 4. Protection experiments. (i) Effect of immunization with OprI alone compared with immunization with OprI plus MAb. Three hundred BALB/c mice (female, 10 to 12 weeks old) were divided into five groups (A through E). The
MATERIALS AND METHODS
Preparation of recombinant OprI. OprI was purified from transformed E. coli K-12 JE5513 (4) by the method of Inouye et al. (9) with slight modifications as described recently (6). Expression of OprI fusion proteins. Isolation of plasmid Corresponding author. t Present address: Institut fur Allgemeine und Experimentelle Pathologie der Universitat Wien, Vienna, Austria. *
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animals of groups B to E were immunized intraperitoneally on days 0, 7, and 21 with 50 ,ug of recombinant OprI (1 mg/ml) suspended in an equal volume of 1.5% AI(OH)3. The animals were bled from the tail vein on day 26 for serum collection to determine antibody titers in individual mice. One day later, all animals (A through E) received immunosuppression as described above (days 27, 29, and 31). On day 30, the animals of groups B, C, and D received intraperitoneally 2 mg of MAbs 2A1, 6A4, and 5B4, respectively; in groups A and E, the animals were injected with PBS. On days 31, 32, 33, and 34, all mice received 2 mg of azlocillin intraperitoneally (Securopen; Bayer, Leverkusen, Federal Republic of Germany) twice a day. On day 31, each group (A through E) was randomized into three subgroups (I, II, and III) containing 20 animals per subgroup. The mice of groups B to E received subcutaneously either 1 x 103 (subgroup I), 5 x 103 (subgroup II), or 5 x 104 (subgroup III) CFU of P. aeruginosa serogroup 6. The controls (group A) received 100 (subgroup I) or 103 (subgroup II) CFU. Ten noninfected mice of the subgroup AIII were used to control the leukocyte counts on days 33 and 34 to confirm a state of leukopenia. The other 10 animals of this subgroup served as controls to exclude nonspecific infection. The survival of all animals was monitored for 10 days after infection. (ii) Protective effect of immunization with MAb alone compared with immunization with MAb plus OprI. In this experiment, groups of BALB/c mice were immunized with either OprI (group G) or injected with Al(OH)3 only (groups F and H) and immunosuppressed as described above. On day 30, mice of groups G and H received 2 mg of MAb 2A1 intraperitoneally. Subgroups I, II, and III of the groups F and G were challenged as described above on day 31 with either 1.6 x 102 (I), 8 x 103 (II), or 8 x 104 (III) CFU of P. aeruginosa serogroup 6. Statistics. Survival curves were compared by the logit regression model (7). The LD50 values were calculated by probit analysis (7). RESULTS AND DISCUSSION In both experiments described above, immunization of mice with OprI induced comparable antibody titers against P. aeruginosa serogroup 6 in the sera of individual mice as measured by an enzyme-linked immunosorbent assay. In experiments (i) and (ii) described above, median antibody titers of 18% (Q1IQ3: 12/29.5) (n = 240) and 18% (Q1IQ3: 13/24) (n = 60), respectively, compared with the polyclonal standard serum against OPRs, were measured (Ql and Q3 represent the upper and lower quartiles). Compared with nonimmunized controls, OprI immunization before immunosuppression provided significant protection (P c 0.001) and increased considerably the resistance of the animals against infection with P. aeruginosa serogroup 6, i.e., towards a 36-fold LD50 (Table 1, group E). Previous control experiments exclude the possibility that the contaminating lipopolysaccharide from E. coli contributes significantly to the observed protection (data not shown). Additional treatment with MAb 2A1, but not with MAb 6A4 or 5B4, induced a statistically significant increase of the survival rate (P < 0.05) in comparison with the group immunized only with OprI. The protection provided by MAb 2A1 alone was found to be significant concerning survival time as analyzed by a proportional hazard model (2) (P = 0.019) but not significant as analyzed by use of a logit regression model (P = 0.0566). The results (Table 1) show that, in spite of the fact that the polyclonal antibodies induced by OprI as well as MAb 2A1
TABLE 1. Calculated LD50 values after protection by OprI immunization and/or MAb and challenge with P. aeruginosaa Group
Treatment
A B C D E F G H
Control OprI + MAb 2A1 OprI + MAb 6A4 OprI + MAb 5B4 OprI Control OprI + MAb 2A1 MAb 2A1
LD50 x 101 x 103 x 103 x 103 x 103 x 101 x 103
7.8 7.5 5.2 3.4 2.9 4.5 3.7 1.6
x 102
a For experimental details, see Materials and Methods.
are directed against epitopes which are located on the same antigen, a 96-fold increase of the LD50 can be obtained by the combined treatment protocol, compared with the application of MAb 2A1 alone (3-fold LD50 increase) or immunization with OprI alone (36-fold LD50 increase). These results show that the effect of OprI immunization can be significantly increased by MAbs but that this effect is more or less restricted to MAb 2A1 because application of even larger amounts of MAb 6A4 (4 mg) did not induce a higher rate of protection (data not shown). To investigate whether these different observed rates of additional protection can be explained by the fact that the MAbs are directed against different epitopes or whether the protective ability of the MAbs is a distinct feature of the MAb itself like the IgG subclass, epitope mapping was performed. Recombinant fusion proteins were prepared, representing overlapping parts of the OprI sequence. These fusion proteins were analyzed in Western blot (20) experiments with MAbs 6A4, 5B4, and 2A1 (Fig. 1). Because three MAbs reacted with the fusion proteins derived from pPIX1 and pPI13, it was evident that the MAbs did not react with the N-terminal part of OprI including the lipid moiety nor with the C-terminal five amino acids. MAbs 6A4 and 5B4 reacted with fusion proteins encoded by pPIX1, pPI12, and pPI13, indicating that both of them were directed against an epitope located in the central part of the OprI protein (aa 21 to 57). MAb 2A1, however, showed a reaction only with the fusion proteins from pPIX1 and pPI13, not with the fusion protein encoded by pPI12. Obviously, MAb 2A1 recognizes an OprI epitope that is not present in fusion protein from pPI12. Therefore, we conclude that MAb 2A1 is directed against an epitope between aa 7 and 20 of the OprI protein. If a polyclonal response induced in mice by OprI immunization covers all of the epitopes which are recognized by the different MAbs, one could speculate that the epitope that is recognized by MAb 5B4 is less effective for protection, since MAb 5B4 did not decrease the OprI-induced polyclonal protection by blocking a protective epitope. With the binding site of MAb 2A1, a highly protective epitope (aa 7 to 20) could be defined on the OprI protein. This epitope has been demonstrated to be present in all P. aeruginosa serotypes according to the International Antigenic Typing Scheme by use of immunofluorescence and Western blotting techniques with MAb 2A1 (14). Work is in progress to investigate if this relatively short sequence of OprI alone might be suitable as a protective vaccine. ACKNOWLEDGMENTS This work was supported by grants to B. U. v. S. from the Deutsche Forschungsgemeinschaft (Sp 170/2-2), the Bundesminister fur Verteidigung (InSan I-1-0987-V-5490), and from the Bundesmi-
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FIG. 1. Identification of epitopes of OprI by Western blotting with different OprI-specific MAbs. (A to D) The fusion proteins containing different parts of OprI were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and either stained directly with Coomassie blue (A) or incubated with MAbs as indicated (B, C, and D). Binding of the MAbs was visualized by using alkaline phosphatase-linked goat anti-mouse antibodies. Lanes: a, pPI12; b, pPI13, c, pSEM3; d, pPIX1; e, pEX32b; m, marker proteins (lowmolecular-weight marker kit) (Sigma, Taufkirchen, Federal Republic of Germany). (E) A schematic recognition pattern of MAbs 2A1, 6A4, and 5B4 to the different OprI fragments and to the corresponding amino acid sequence of OprI is shown. 1253
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FINKE ET AL.
nisterium fur Forschung und Technologie (OIK I 8910/4), and to H.D. from the Bundesministerium fur Forschung und Technologie (BCT0372-8). REFERENCES 1. Brent, R., and M. Ptashne. 1981. Mechanism of action of the lexA gene product. Proc. Natl. Acad. Sci. USA 78:4204-4208. 2. Cox, D. R. 1972. Regression models and life tables (with discussion). J. R. Statist. Soc. B 34:187-220. 3. Cryz, J. R., S. C. E. Furer, and R. Germanier. 1983. Passive protection against Pseudomonas aeruginosa infection in an experimental leucopenic mouse model. Infect. Immun. 40:659-664. 4. Duchene, M., C. Barron, A. Schweizer, B. U. von Specht, and H. Domdey. 1989. Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli. J. Bacteriol. 171:4130-4137. 5. Duchene, M., A. Schweizer, F. Lottspeich, G. Krauss, M. Marget, K. Vogel, B. U. von Specht, and H. Domdey. 1988. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J. Bacteriol. 170:155-162. 6. Finke, M., M. Duchene, A. Eckhardt, H. Domdey, and B. U. von Specht. 1990. Protection against experimental Pseudomonas aeruginosa infection by recombinant P. aeruginosa lipoprotein I expressed in Escherichia coli. Infect. Immun. 58:2241-2244. 7. Finney, D. J. 1971. Probit analysis. Cambridge University Press, Cambridge. 8. Hancock, R. E. W., and A. M. Carey. 1979. Outer membrane of Pseudomonas aeruginosa: heat- and 2-mercapto-ethanol-modifiable proteins. J. Bacteriol. 140:902-910. 9. Inouye, S., K. Takeishi, N. Lee, M. De Martini, A. Hirashima, and M. Inouye. 1976. Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form. J. Bacteriol. 127:555-563. 10. Knapp, S., M. Broker, and E. Amann. 1990. pSEM vectors: high level of expression of antigenic determinants and protein domains. Biotechniques 8:280-281. 11. Lugtenberg, B., J. Meijers, R. Peters, P. van der Hoek, and L. van Alphen. 1975. Electrophoretic resolution of the major outer membrane proteins of E. coli K 12 into 4 bands. FEBS Lett. 58:245-258.
INFECT. IMMUN. 12. Mancini, G., A. Carbonara, and J. F. Heremans. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. J. Immunochem. 2:235-254. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Marget, M., A. Eckhardt, W. Ehret, B. U. von Specht, M. Duchene, and H. Domdey. 1989. Cloning and characterization of heavy and light chain cDNAs from Pseudomonas aeruginosa outer membrane protein I specific monoclonal antibody. Gene 74:335-345. 15. Mutharia, L. M., and R. E. W. Hancock. 1985. Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can. J. Microbiol. 31:381-386. 16. Mutharia, L. M., T. I. Nicas, and R. E. W. Hancock. 1982. Outer membrane proteins of Pseudomonas aeruginosa serotyp strains. J. Infect. Dis. 146:770-779. 17. Rahner, Ch., A. Eckhardt, M. Duchene, H. Domdey, and B. U. von Specht. 1990. Protection of immunosuppressed mice against infection with Pseudomonas aeruginosa by monoclonal antibodies to outer membrane protein OprI. Infection 18:242-245. 18. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Ehrlich, and N. Arnheim. 1985. Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354. 19. Strebel, K., E. Beck, K. Strohmaier, and H. Schaller. 1986. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J. Virol. 57:983-991. 20. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 21. von Specht, B. U., G. Strigl, W. Ehret, and W. Brendel. 1987. Protective effect of an outer membrane vaccine against Pseudomonas aeruginosa infection. Infection 15:408-412. 22. Wade, J. C., and S. C. Schimpff. 1988. Epidemiology and prevention of infection in the compromised host, p. 467-501. In R. H. Rubin and L. S. Young (ed.), Plenum Medical Book Co., New York.
Vol. 59, No. 4
0019-9567/91/041251-04$02.00/0 Copyright © 1991, American Society for Microbiology
Protection of Immunosuppressed Mice against Infection with Pseudomonas aeruginosa by Recombinant P. aeruginosa Lipoprotein I and Lipoprotein I-Specific Monoclonal Antibodies MATHIAS FINKE,1 GUNTHER MUTH,2 THOMAS REICHHELM,1 MARTIN THOMA,' MICHAEL DUCHENE,3t KLAUS-DIETER HUNGERER,2 HORST DOMDEY,3 AND BERND-ULRICH VON SPECHTl* Chirurgische Universitatsklinik, Chirurgische Forschung, Hugstetterstrasse 55, 7800 Freiburg im Breisgau,' Behringwerke
AG, Marburg, and Laboratorium fur Molekulare Biologie, Genzentrum der Ludwig Maximilians-Universitat Munchen, 8033 Martinsried,3 Federal Republic of Germany Received 17 October 1990/Accepted 4 January 1991
Outer membrane protein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. The protective effect of OprI vaccination and that of three OprI-specific monoclonal antibodies (MAbs) against infection with P. aeruginosa were tested in immunosuppressed mice. The combination of OprI and MAb 2A1 protected the mice against a challenge with a 96-fold 50% lethal dose. The binding site of MAb 2A1 was mapped, resulting in the identification of a protective epitope (amino acids 7 to 20).
Pseudomonas aeruginosa bacteremia is a frequent cause of gram-negative sepsis in the neutropenic subject (22). The two major antigenic cell envelope components of P. aeruginosa are the lipopolysaccharides and the outer membrane proteins (OPRs) (8, 15, 16). We have been interested in the potential of P. aeruginosa OPRs as an immunoprophylactic tool because OPRs are antigenically cross-reactive among all 17 known serogroups of the International Antigenic Typing Scheme (14, 16, 21). We have cloned the genes coding for OprF and OprI of P. aeruginosa (4, 5) and have used OprI expressed in Escherichia coli for a successful vaccination of immunocompetent mice (6). The disadvantage of the immunocompetent mouse model is, however, that the state of neutropenia and the reduction of the antibody synthesizing capacity, which are both critical parameters for the multiplication of P. aeruginosa in immunosuppressed hosts, are not simulated. As an alternative, Cryz et al. (3) have described a leukopenic mouse model in which the 50% lethal dose (LD50) for cyclophosphamide-treated mice is as low as 20 living P. aeruginosa cells. We have used this model previously to prove that our OprI-specific monoclonal antibody (MAb) 2A1 provides a significant yet rather low degree of protection against P. aeruginosa infection (17). With this model, we are now able to show that preimmunization with recombinant OprI in combination with the application of an OprI-specific MAb can be very effective to prevent potentially lethal P. aeruginosa infections in immunosuppressed individuals. In addition, we provide evidence that our MAbs bind to different epitopes of the OprI molecule and that the epitope which is recognized by MAb 2A1 (amino acids [aa] 7 to 20) seems to be the most important for protection.
DNA, preparation of DNA fragments, DNA ligations, and transformation into E. coli were carried out as described by Maniatis et al. (13). For expression of OprI fusion proteins, vectors of the pSEM series (10) or vectors of the pEX series (19) were used. Restriction sites were engineered by using the polymerase chain reaction (18). Plasmid pPIX1 encodes aa 2 to 57 fused to the MS2 polymerase, while plasmids pPI12 (aa 21 to 64) and pPI13 (aa 7 to 62) carry the NH2-terminal part of the 3-galactosidase gene as a fusion partner.
SDS-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of OprI fusion proteins. Sodium dodecyl sulfate (SDS)-polyacrylamide (15%) slab gel electrophoresis and Western blotting were performed as described previously (11, 12, 20). OprI fragments were incubated with the OprI-specific MAbs 2A1, 6A4, and 5B4. Binding was visualized as described recently (21). MAbs. The induction preparation, and purification of the mouse MAb designated 2A1 (immunoglobulin [Ig]G2b), 6A4 (IgG2a), and 5B4 (IgG2a) have been described (14, 17). Enzyme-linked immunosorbent assay. Polyclonal antibodies and MAbs against P. aeruginosa were measured by an enzyme-linked immunosorbent assay as described in detail in a previous paper (6). Bacterial strains and growth conditions. P. aeruginosa International Antigenic Typing Scheme serogroup 6 (ATCC 33353) was obtained from A. Bauernfeind, Max von Pettenkofer-Institut, University of Munich, Munich, Federal Republic of Germany. Bacteria were grown and adjusted to the required concentration as described previously (6). For the expression of recombinant proteins, E. coli K-12 W3110 lacIQ L8 (1) was used. Immunosuppression. For immunosuppression, the leukopenic mouse model described by Cryz et al. (3) was used. The animals received three injections of 150 ,ug of cyclophosphamide (Serva, Heidelberg, Federal Republic of Germany) per g of body weight in 0.25 ml of phosphate-buffered saline (PBS) on days 0, 2, and 4. Protection experiments. (i) Effect of immunization with OprI alone compared with immunization with OprI plus MAb. Three hundred BALB/c mice (female, 10 to 12 weeks old) were divided into five groups (A through E). The
MATERIALS AND METHODS
Preparation of recombinant OprI. OprI was purified from transformed E. coli K-12 JE5513 (4) by the method of Inouye et al. (9) with slight modifications as described recently (6). Expression of OprI fusion proteins. Isolation of plasmid Corresponding author. t Present address: Institut fur Allgemeine und Experimentelle Pathologie der Universitat Wien, Vienna, Austria. *
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animals of groups B to E were immunized intraperitoneally on days 0, 7, and 21 with 50 ,ug of recombinant OprI (1 mg/ml) suspended in an equal volume of 1.5% AI(OH)3. The animals were bled from the tail vein on day 26 for serum collection to determine antibody titers in individual mice. One day later, all animals (A through E) received immunosuppression as described above (days 27, 29, and 31). On day 30, the animals of groups B, C, and D received intraperitoneally 2 mg of MAbs 2A1, 6A4, and 5B4, respectively; in groups A and E, the animals were injected with PBS. On days 31, 32, 33, and 34, all mice received 2 mg of azlocillin intraperitoneally (Securopen; Bayer, Leverkusen, Federal Republic of Germany) twice a day. On day 31, each group (A through E) was randomized into three subgroups (I, II, and III) containing 20 animals per subgroup. The mice of groups B to E received subcutaneously either 1 x 103 (subgroup I), 5 x 103 (subgroup II), or 5 x 104 (subgroup III) CFU of P. aeruginosa serogroup 6. The controls (group A) received 100 (subgroup I) or 103 (subgroup II) CFU. Ten noninfected mice of the subgroup AIII were used to control the leukocyte counts on days 33 and 34 to confirm a state of leukopenia. The other 10 animals of this subgroup served as controls to exclude nonspecific infection. The survival of all animals was monitored for 10 days after infection. (ii) Protective effect of immunization with MAb alone compared with immunization with MAb plus OprI. In this experiment, groups of BALB/c mice were immunized with either OprI (group G) or injected with Al(OH)3 only (groups F and H) and immunosuppressed as described above. On day 30, mice of groups G and H received 2 mg of MAb 2A1 intraperitoneally. Subgroups I, II, and III of the groups F and G were challenged as described above on day 31 with either 1.6 x 102 (I), 8 x 103 (II), or 8 x 104 (III) CFU of P. aeruginosa serogroup 6. Statistics. Survival curves were compared by the logit regression model (7). The LD50 values were calculated by probit analysis (7). RESULTS AND DISCUSSION In both experiments described above, immunization of mice with OprI induced comparable antibody titers against P. aeruginosa serogroup 6 in the sera of individual mice as measured by an enzyme-linked immunosorbent assay. In experiments (i) and (ii) described above, median antibody titers of 18% (Q1IQ3: 12/29.5) (n = 240) and 18% (Q1IQ3: 13/24) (n = 60), respectively, compared with the polyclonal standard serum against OPRs, were measured (Ql and Q3 represent the upper and lower quartiles). Compared with nonimmunized controls, OprI immunization before immunosuppression provided significant protection (P c 0.001) and increased considerably the resistance of the animals against infection with P. aeruginosa serogroup 6, i.e., towards a 36-fold LD50 (Table 1, group E). Previous control experiments exclude the possibility that the contaminating lipopolysaccharide from E. coli contributes significantly to the observed protection (data not shown). Additional treatment with MAb 2A1, but not with MAb 6A4 or 5B4, induced a statistically significant increase of the survival rate (P < 0.05) in comparison with the group immunized only with OprI. The protection provided by MAb 2A1 alone was found to be significant concerning survival time as analyzed by a proportional hazard model (2) (P = 0.019) but not significant as analyzed by use of a logit regression model (P = 0.0566). The results (Table 1) show that, in spite of the fact that the polyclonal antibodies induced by OprI as well as MAb 2A1
TABLE 1. Calculated LD50 values after protection by OprI immunization and/or MAb and challenge with P. aeruginosaa Group
Treatment
A B C D E F G H
Control OprI + MAb 2A1 OprI + MAb 6A4 OprI + MAb 5B4 OprI Control OprI + MAb 2A1 MAb 2A1
LD50 x 101 x 103 x 103 x 103 x 103 x 101 x 103
7.8 7.5 5.2 3.4 2.9 4.5 3.7 1.6
x 102
a For experimental details, see Materials and Methods.
are directed against epitopes which are located on the same antigen, a 96-fold increase of the LD50 can be obtained by the combined treatment protocol, compared with the application of MAb 2A1 alone (3-fold LD50 increase) or immunization with OprI alone (36-fold LD50 increase). These results show that the effect of OprI immunization can be significantly increased by MAbs but that this effect is more or less restricted to MAb 2A1 because application of even larger amounts of MAb 6A4 (4 mg) did not induce a higher rate of protection (data not shown). To investigate whether these different observed rates of additional protection can be explained by the fact that the MAbs are directed against different epitopes or whether the protective ability of the MAbs is a distinct feature of the MAb itself like the IgG subclass, epitope mapping was performed. Recombinant fusion proteins were prepared, representing overlapping parts of the OprI sequence. These fusion proteins were analyzed in Western blot (20) experiments with MAbs 6A4, 5B4, and 2A1 (Fig. 1). Because three MAbs reacted with the fusion proteins derived from pPIX1 and pPI13, it was evident that the MAbs did not react with the N-terminal part of OprI including the lipid moiety nor with the C-terminal five amino acids. MAbs 6A4 and 5B4 reacted with fusion proteins encoded by pPIX1, pPI12, and pPI13, indicating that both of them were directed against an epitope located in the central part of the OprI protein (aa 21 to 57). MAb 2A1, however, showed a reaction only with the fusion proteins from pPIX1 and pPI13, not with the fusion protein encoded by pPI12. Obviously, MAb 2A1 recognizes an OprI epitope that is not present in fusion protein from pPI12. Therefore, we conclude that MAb 2A1 is directed against an epitope between aa 7 and 20 of the OprI protein. If a polyclonal response induced in mice by OprI immunization covers all of the epitopes which are recognized by the different MAbs, one could speculate that the epitope that is recognized by MAb 5B4 is less effective for protection, since MAb 5B4 did not decrease the OprI-induced polyclonal protection by blocking a protective epitope. With the binding site of MAb 2A1, a highly protective epitope (aa 7 to 20) could be defined on the OprI protein. This epitope has been demonstrated to be present in all P. aeruginosa serotypes according to the International Antigenic Typing Scheme by use of immunofluorescence and Western blotting techniques with MAb 2A1 (14). Work is in progress to investigate if this relatively short sequence of OprI alone might be suitable as a protective vaccine. ACKNOWLEDGMENTS This work was supported by grants to B. U. v. S. from the Deutsche Forschungsgemeinschaft (Sp 170/2-2), the Bundesminister fur Verteidigung (InSan I-1-0987-V-5490), and from the Bundesmi-
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a
a
fusioni proteins: pPIXI pPIP
epitope:
...................
mAb:
2A I
Oprl 0 !
L~~~~.
.
.
.
.
6A4//5B4
20
10 .
.
.
.
.
.
.
.
.
30 .
.
.
.
.
50
40 .
.
.
.
60 aa
.
FIG. 1. Identification of epitopes of OprI by Western blotting with different OprI-specific MAbs. (A to D) The fusion proteins containing different parts of OprI were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and either stained directly with Coomassie blue (A) or incubated with MAbs as indicated (B, C, and D). Binding of the MAbs was visualized by using alkaline phosphatase-linked goat anti-mouse antibodies. Lanes: a, pPI12; b, pPI13, c, pSEM3; d, pPIX1; e, pEX32b; m, marker proteins (lowmolecular-weight marker kit) (Sigma, Taufkirchen, Federal Republic of Germany). (E) A schematic recognition pattern of MAbs 2A1, 6A4, and 5B4 to the different OprI fragments and to the corresponding amino acid sequence of OprI is shown. 1253
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nisterium fur Forschung und Technologie (OIK I 8910/4), and to H.D. from the Bundesministerium fur Forschung und Technologie (BCT0372-8). REFERENCES 1. Brent, R., and M. Ptashne. 1981. Mechanism of action of the lexA gene product. Proc. Natl. Acad. Sci. USA 78:4204-4208. 2. Cox, D. R. 1972. Regression models and life tables (with discussion). J. R. Statist. Soc. B 34:187-220. 3. Cryz, J. R., S. C. E. Furer, and R. Germanier. 1983. Passive protection against Pseudomonas aeruginosa infection in an experimental leucopenic mouse model. Infect. Immun. 40:659-664. 4. Duchene, M., C. Barron, A. Schweizer, B. U. von Specht, and H. Domdey. 1989. Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli. J. Bacteriol. 171:4130-4137. 5. Duchene, M., A. Schweizer, F. Lottspeich, G. Krauss, M. Marget, K. Vogel, B. U. von Specht, and H. Domdey. 1988. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J. Bacteriol. 170:155-162. 6. Finke, M., M. Duchene, A. Eckhardt, H. Domdey, and B. U. von Specht. 1990. Protection against experimental Pseudomonas aeruginosa infection by recombinant P. aeruginosa lipoprotein I expressed in Escherichia coli. Infect. Immun. 58:2241-2244. 7. Finney, D. J. 1971. Probit analysis. Cambridge University Press, Cambridge. 8. Hancock, R. E. W., and A. M. Carey. 1979. Outer membrane of Pseudomonas aeruginosa: heat- and 2-mercapto-ethanol-modifiable proteins. J. Bacteriol. 140:902-910. 9. Inouye, S., K. Takeishi, N. Lee, M. De Martini, A. Hirashima, and M. Inouye. 1976. Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form. J. Bacteriol. 127:555-563. 10. Knapp, S., M. Broker, and E. Amann. 1990. pSEM vectors: high level of expression of antigenic determinants and protein domains. Biotechniques 8:280-281. 11. Lugtenberg, B., J. Meijers, R. Peters, P. van der Hoek, and L. van Alphen. 1975. Electrophoretic resolution of the major outer membrane proteins of E. coli K 12 into 4 bands. FEBS Lett. 58:245-258.
INFECT. IMMUN. 12. Mancini, G., A. Carbonara, and J. F. Heremans. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. J. Immunochem. 2:235-254. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Marget, M., A. Eckhardt, W. Ehret, B. U. von Specht, M. Duchene, and H. Domdey. 1989. Cloning and characterization of heavy and light chain cDNAs from Pseudomonas aeruginosa outer membrane protein I specific monoclonal antibody. Gene 74:335-345. 15. Mutharia, L. M., and R. E. W. Hancock. 1985. Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can. J. Microbiol. 31:381-386. 16. Mutharia, L. M., T. I. Nicas, and R. E. W. Hancock. 1982. Outer membrane proteins of Pseudomonas aeruginosa serotyp strains. J. Infect. Dis. 146:770-779. 17. Rahner, Ch., A. Eckhardt, M. Duchene, H. Domdey, and B. U. von Specht. 1990. Protection of immunosuppressed mice against infection with Pseudomonas aeruginosa by monoclonal antibodies to outer membrane protein OprI. Infection 18:242-245. 18. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Ehrlich, and N. Arnheim. 1985. Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354. 19. Strebel, K., E. Beck, K. Strohmaier, and H. Schaller. 1986. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J. Virol. 57:983-991. 20. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 21. von Specht, B. U., G. Strigl, W. Ehret, and W. Brendel. 1987. Protective effect of an outer membrane vaccine against Pseudomonas aeruginosa infection. Infection 15:408-412. 22. Wade, J. C., and S. C. Schimpff. 1988. Epidemiology and prevention of infection in the compromised host, p. 467-501. In R. H. Rubin and L. S. Young (ed.), Plenum Medical Book Co., New York.
