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Impact of molecular biology on Pseudomonas aeruginosa immunization
Journal of Hospital Infection (1988)
Impact
11 (Supplement
of molecular aeruginosa
A), 96102
biology on Pseudomonas immunization
J. E. Pennington Department of Medicine, University of California cisco, California 94143 and Cutter Laboratories,
San Francisco, San FranBerkeley, California 94710
Summary: Persisting high mortalities from Pseudomonas aeruginosa infection have led to new strategies for treatment. In vitro and animal studies indicate that antibodies against P. aeruainosa antigens increase host defense against this infectious agent. The most-effective immunogen is lipopolysaccharide (LPS) antigen: however. LPS vaccines are noorlv tolerated. Furthermore, the LPS molecule does not lend itself well-to production by genetic engineering. Pseudomonas aeruginosa protein antigens which might be amenable to recombinant DNA production are outer membrane proteins and exotoxin A, modified to decrease toxicity but maintain immunogenicity. P. aeruginosa antibodies is Another strategy for immunization with anti-LPS passive administration of either hyperimmune immunoglobulins (polyclonal) or monoclonal antibodies. Passive immunization offers the dual advantage of rapid protection or treatment and is well tolerated. Several monoclonal antibodies against LPS P. aeruginosa antigens have been described, including both murine and human types. Studies in animal models of infection indicate that P. aeruginosa monoclonal antibodies do protect, thus, the most feasible application of molecular biology to the problem of P. aeruginosa infection appears to be production of immunotype-specific monoclonal antibodies for immune therapy.
Introduction
The rapidly advancing discipline of molecular biology has provided immunochemists with new horizons for vaccine development. Production of viral protein antigens using recombinant DNA technology circumvents the need to use potentially dangerous native antigen vaccines (e.g., HIV). Furthermore, production of anti-idiotype antibodies (anti-id) utilizes protein mimicry to produce vaccines which may be more immunogenic than the native, non-protein antigen (e.g., Haemophilus injluenzae and pneumococcus capsular polysaccharides, as applied to infants). Finally, cloning techniques for monoclonal antibodies provide a modern technique for passive immunization. How then might such methods apply to immunization against Gram-negative bacterial infections?
01954701/88/02A096+07
(Q 1988 The Hospml
SOS.OO/O
96
Infection
Society
Molecular
biology
and pseudomonas
immunization
97
Current analysis of Gram-negative bacterial infections suggests that these pathogens persist as important sources of nosocomial infection (Stamm, Martin & Bennett, 1977; Noso&mial Infection Surveillance, 1984). Among gram-negative bacilli, Pseudo onas aeruginosa is associated with the highest mortalities (Stevens et al., 19 d ; Bryan, Reynolds & Brenner, 1983; Bryan & Reynolds, 1984; Young, 1984). Although immunological methods for cross-protection against all Gram-negative bacilli have been advocated (Ziegler et al., 1982; Baumgartner, 1985), there is evidence that serotype-specific immunization may be more protective for P. ueruginosu (Pennington & Menkes, 1981; Sadoff et al., 1982). Experience has accumulated in P. ueruginosu vaccine development (Cryz, 1984; Pennington, lt)83), but so far little information has been presented regarding molecular’methods for development of P. ueruginosu vaccines. One likely reason is that rDNA methods apply to protein antigens, while the major virulence factor of P. ueruginosu is lipopolysaccharide (LPS) (Cryz et al., 1984b). In fact, numerous comparative studies have identified LPS serotype-specific antibodies as the most protective type of antibody against P. ueruginosu (Pennington & Menkes, 1981; Cryz, Furer & Germanier, 1983u,b, 1984~; Sawada, 1984). While even less information is available regarding anti-id vaccines for P. ueruginosu, this might represent an alternative strategy. Table I provides a list of the most important P. ueruginosu virulence factors (antigens) and outlines the feasibility and priority for genetically engineered vaccine development. As an alternative to active immunization, passive transfer of LPS type-specific antibodies may be considered for treatment or prophylaxis of P. ueruginosu infections. This approach may be particularly useful for intensive care unit or immunosuppressed patients in whom active immunization may be too slow or may not promote an adequate humoral immune response. Monoclonal antibodies directed against P. ueruginosu antigens should be considered as candidates for this approach. The following is a description of feasibility studies for monoclonal versus polyclonal antibodies in the treatment of P. ueruginosu infection.
Table
I. Candidates
for genetically
engineered pseudomonas
vaccines
Feasibility Antigen Lipopolysaccharide Outer membrane protein Exotoxin A Polysaccharide (O-specific) Proteases P&/flagella
rDNA
Anti-id
No Yes Yes No Yes Yes
Yes Yes Yes Yes Yes Yes
Protective potency of antibody High Medium/Low Low Medium Low ?
J. E. Pennington
98
Materials
and methods
Harley strain guinea pigs (400 g) were obtained from Charles Animals River Breeding Laboratories, Inc., Wilmington, Mass. Animals were housed in standard cages and fed guinea pig chow (Ralston-Purina, St. Louis, MO.). Bacteria Pseudomonas aeruginosa, strain 220 was employed for experimental pneumonias. This clinical isolate was Fisher immunotype-1 (IT-l) (Fisher, Devlin & Gnabasik, 1969). The characteristics of strain 220 have been described fully elsewhere (Pennington & Miller, 1979). Antibody preparations Hyperimmune P. globulin aeruginosa (PA-IGIV) (lot PR301 l), enriched in antibodies against LPS IT-l, -2, -4, and -6, was supplied by Cutter Laboratories, Berkeley, California. This preparation was supplied as 5% protein in 10% maltose and was rendered suitable for iv infusion by acidification to pH 4.25. Details regarding plasma donor screening and the immunological properties of this material have been provided elsewhere (Collins & Roby, 1984). Preparations of murine monoclonal antibodies (MAb) directed against P. aeruginosa IT-1 and IT-4 LPS antigens, as well as against P. aeruginosa outer membrane protein F (porin), were made and characterized using previously described methods (Pennington et al., 19863). Serological assays Concentrations of antibodies against IT-l and IT-4 P. aeruginosa antigens were determined by a radioimmune antigen binding assay previously described in detail (Pier, Markham & Eardley, 1981). Table II demonstrates that the MAb preparations contained greater concentrations of immunotype-specific antibody than did the polyclonal preparation. Experimental pneumonia Pneumonia was induced according to Pennington (1979) and Pennington & Miller (1979). Briefly, animals were anaesthetized (intraperitoneal pentobarbital), and their tracheas were isolated by a small midline neck incision. Samples (0.5 ml) of P. aeruginosa in isotonic saline were then instilled via a needle into the tracheobronchial tree. Incisions were sutured and animals awakened within 2 to 3 h. Preliminary studies established that 5 x lo6 cfus of strain 220 was the minimum inoculum which resulted in uniformly fatal pneumonia in this model. This inoculum was used for lung challenges in this study. Table
II.
Pseudomonas
LPS antibody and MAb preparations
aeruginosa
concentrations in PA-ZGIV
Antibody Preparation PA-IGIV IT-1 MAb IT-4 MAb Porin MAb
Anti-IT-l 7.6 478 Cl.0 Cl.0
cont.
(pg ml-‘) Anti-IT-4 44.9 Cl.0 74.0 Cl.0
Molecular
biology
and pseudomonas
immunization
99
Experiments to compare the t,herapeutic efficacy of the PA-IGIV preparation versus the MAb preparations involved cohorts of guinea pigs infected with equal inocula o P. aeruginosa and then treated 2 h after infection with a single iv infu 9fon of antibody preparation. Control groups received infusion of 5% bovine serum albumin (BSA) in 10% maltose. Infusion volumes (5 ml) were kept constant for all study groups. The methodology for iv infusions via the external jugular vein has been described before (Pennington, Pier & Small, 1986~). For survival studies, animals were observed for 4 days and were considered long-term survivors if they lived beyond this period (Pennington et al., l(b86a). I n selected studies, animals were sacrificed at timed intervals after &fection by intraperitoneal pentobarbital. Lungs were removed surgically, homogenized, and cultured quantitatively, as described before (Pennington, 1979, and Pennington & Miller, 1979). In addition, heart blood was obtained after thoractomy, and blood was cultured in trypticase soy broth (BBL Microbiology Systems, Cockeysville, Maryland) (Pennington et al., 1986~). Statistical analysis Differences in survival from pneumonia were compared by the Chi-Square analysis with Yates’s correction. The paired Student’s t-test was employed for analyses of quantitative bacteriology. Results Polyclonal and monoclonal antibody preparations were successful in reducing mortality from infection, and this effect was clearly dose-dependent (Table III). Importantly, the monoclonal preparation promoted higher, thus more protective, antibody titers using considerably lower protein dosages. It was also of interest that the monoclonal antibody preparations provided LPS serotype-specific protection (Table IV). Furthermore, protection was greater when a MAb directed against LPS antigen was used as compared to a MAb against outer membrane protein (Table IV). Bacteriological clearance studies supported the findings in the survival studies (Figure 1). Table
III.
Dose-dependent antibody-mediated
Hyperimmune Dose (mg kg-‘) 100 250 500 -
protection from Pseudomonas Monoclonal
globulin
Serum concentration antibody (pg ml-‘)?
Survival* W)
Cl.3 1.38 2.69 -
* 12 per group. t One hour after infusions (anti-IT-l). $6 per group.
33 -
Dose (mg kg-‘) 1.0 2.5 5.0 20.0
antibody
Serum concentration antibody (pg ml-‘)t 1.77 4.65 6.15 14.60
pneumonia (IT-l) Survivalj (%)
75 100
100
J. E. Pennington Table
IV.
LPS type-specific monoclonal antibody protection from type 1 Pseudomonas
Monoclonal Type
No. survived/ No. infected
antibody
1
Porin Type 4(outer membrane Albumin control
protein
F)
after
infection
% Survival
9/12
75
2112
:::
o/12
Time
Figure
pneumonia
0
(h)
1. Intrapulmonary
bacterial killing in guinea pigs infected with IT-l strain treated 2 h later with intravenous infusion of albumin, 500 mg kg-’ (0), MAb to outer membrane protein F (porin), 5 mg kg-’ (Cl), PA-IGIV, 500 mg kg-’ ( l ), or MAb to IT-l LPS antigen, 5 mg kg-’ (A). Four animals per time point per group. Positive blood cultures 9 h after infection; 4/4, controls; l/4; porin MAb; O/4 PA-IGIV; O/4 IT-l MAb.
Pseudomonas aeruginosa, then
Discussion
These studies using monoclonal antibodies for passive immune therapy in an experimental model of P. aeruginosa lung infection are encouraging. Furthermore, they suggest that passive immunotherapy against P. aeruginosa should employ antibodies directed against O-type-specific LPS
Molecular
biology
and pseudomonas
immunization
101
antigens. It is also evident that monoclonal antibodies may provide protective concentrations of antibodies using protein dosages which are considerably lower than tho required for plasma-derived polyclonal antibody preparations. Addit’ r nal advantages of monoclonal antibodies as compared to plasma-derived globulins may include better availability and avoidance of potential risks attendant with plasma-based products. Nevertheless, problems also exist for monoclonal antibody therapy. Obviously, the foreign protein associated with murine monoclonals might sensitize recipients. The recent development of human monoclonal antibodies against P. aeruginosa LPS antigens may reduce this risk (Hector et al., 1987). Anothe clinical dilemma is the need for rapid identification of the infecting pathog %n prior to use of a serotype-specific passive immune reagent. Recent studies of monoclonal antibodies for rapid diagnosis of P. aeruginosa infection (Rubin et al., 1985) are encouraging. References Baumgartner, J. D., Glauser, M. P., McCutchan, J. A., Ziegler, E. J., Van Melle, G., Klauber, M. R., Vogt, M., Muehlen, E., Luethy, R., Chiolero, R. & Geroulanos, S. (1985). Prevention of gram-negative shock and death in surgical patients by antibody to endotoxin core glycolipid. Lancet ii, S-3. Bryan, C. S. & Reynolds, K. L. (1984). Bacteremic nosocomial pneumonia. American Review
of Respiratory Diseases 129, 668-671. E. R. (1983). Analysis of 1,186 episodes of Bryan, C. S., Reynolds, K. L. & B renner, gram-negative bacteremias in non-university hospitals: the effects of antimicrobial therapy. Reviews of Infectious Diseases 5, 629-638. Collins, M. S. & Roby, R. E. (1984). Protective activity of an intravenous immune globulin (human) enriched in antibody against lipopolysaccharide antigens of Pseudomonas aeruginosa. American Journal of Medicine 76, 168-l 74. Cryz, S. J. (1984). Pseudomonas aeruginosa infections. In Bacterial Vaccine, pp. 317-351. Academic Press, Inc., London. Cryz, S. J., Furer, E. & Germanier, R. (1983a). Protection against Pseudomonas oerugin#osa infection in a murine burn wound sepsis model by passive transfer of antitoxin A, Infection and Immunity 39, 1072-1079. antielastase, and antilipopolysaccharide. Crvz. S. 1.. Furer. E. & Germanier. R. (19836). Passive protection against Pseudomonas aeruginosa infection in an experimental leukopenic mouse model. Infection and Immunity 40,659664. R. (1984~). Protection against fatal Pseudomonas Cryz, S. J., Furer, E. & Germanier, aeruginosa burn wound sepsis by immunization with lipopolysaccharide and high-molecular-weight polysaccharide. Infection and Immunity 43, 795-799. in virulence of Cryz, S. J., Furer, E. & Germanier, R. (19843). R o 1e of lipopolysaccharide _,
“,
I
.
Pseudomonas aeruginosa. Infection and Immunity Fisher,
M. W.,
44, 5088513.
N. H. & Gnabasik, F. J. (1969). New immunotype scheme Pseudomonas aeruainosa based on nrotective antigens. I I7oburnal of Bacteriologv-_
835-836.
Devlin,
-
for
98.,
Hector,
R. F., Trawinski, J., Besemer, D. J., Collins, M. S. & Pennington, J. E. (1987). In vitro and in vivo evaluation of a human IeM monoclonal antibodv to Pseudomonas aeruginosa immunotype 3. 27th Interscience Conference on Antimiciobial Agents and
Chemotherapy, New York, NY,
1987.
Infection Surveillance. 1984 (1986). CDC Morbiditv and Mortalitv Weeklv Report 35, 17ss-29s~. Pennington, J. E. (1979). Lipopolysaccharide pseudomonas vaccine: efficacy against pulmonary infection with Pseudomonas aeruginosa. Journal of Infectious Diseases 140, Nosocomial
73-80.
102
J. E. Pennington
Pennington, J. E. (1983). Pseudomonas aeruginosa pneumonia: the potential for immune intervention. In Seminars in Znfectious Diseases (Weinstein, L. & Fields, B. N., Eds), pp. 71-80. Thieme-Stratton, New York. Pennington, J. E. & Menkes, E. (1981). Type-specific versus cross-protective vaccination for gram-negative pneumonia. Journal of Znfectious Diseases 144, 599-603. Pennington, J. E. & Miller, J. J. (1979). Evaluation of a new polyvalent pseudomonas vaccine in respiratory infections. Infection and Immunity 25, 1029-1034. Pennington, J. E., Pier, G. B. & Small, G. J. (1986a). Efficacy of intravenous immune globulin for treatment of experimental Pseudomonas aeruginosa pneumonia. Journal of Critical Care 1, 4-10. Pennington, J. E., Small, G. J., Lostrom, M. E. & Pier, G. B. (19866). Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia. Infection and Immunity 54, 239-244. Pier, G. B., Markham, B. & Eardley, D. D. (1981). Correlation of the biological responses of C3H/HeJ mice to endotoxin with the chemical and structural properties of the lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli. Journal of Immunology 127. 184-I 97. Rubin, R. H.,Strauss, H. W., Nedelman, M., Barlai-Kovach, M., Callahan, R., Wilkinson, R., Nellis, M. & Young, L. S. (1985). Imaging of localized type I Pseudomonas aeruginosa infection with specific radiolabelled monoclonal antibody. Clinical Research 33, 565A. Sadoff, J. C., Futrovsky, S. L., Sidberry, H. F., Iglewski, B. H. & Seid, R. C., Jr. (1982). Seminars in Infectious Diseases 4. 346-354. Sawada, S., Suzuki, M., Kawamura; T., Fujinaga, S., Masuho, Y. & Tomibe, K. (1984). Protection against infection with Pseudomonas aeruginosa by passive transfer of monoclonal antibodies to lipopolysaccharides and outer membrane proteins. Journal of Infectious Diseases 150, 570-576. Stamm, W. E., Martin, S. M. & Bennett, J. V. (1977). Epidemiology of nosocomial infections due to gram-negative bacilli: aspects relevant to development and use of vaccines. Journal of Infectious Diseases 136 (Suppl.), 151-160. Stevens, R. M., Teres, D., Skillman, J. J. & Feingold, D. S. (1974). Pneumonia in an intensive care unit. Archives of Znternal Medicine 134, 106-I 11. Young, L. S. (1984). The clinical challenge of infections due to Pseudomonas aeruginosa. Reviews of Znfectious Diseases 6 (Suppl.), 603607. Zeigler, E. J., McCutchan, J. A., Fierer, J., Glauser, M. P., Sadoff, J. C., Douglas, H. & Braude, A. I. (1982). Treatment of gram-negative bacteremia and shock with human antiserum to a mutant Escherichia coli. New England ‘Journal of Medicine 307, 12251230.
Impact
11 (Supplement
of molecular aeruginosa
A), 96102
biology on Pseudomonas immunization
J. E. Pennington Department of Medicine, University of California cisco, California 94143 and Cutter Laboratories,
San Francisco, San FranBerkeley, California 94710
Summary: Persisting high mortalities from Pseudomonas aeruginosa infection have led to new strategies for treatment. In vitro and animal studies indicate that antibodies against P. aeruainosa antigens increase host defense against this infectious agent. The most-effective immunogen is lipopolysaccharide (LPS) antigen: however. LPS vaccines are noorlv tolerated. Furthermore, the LPS molecule does not lend itself well-to production by genetic engineering. Pseudomonas aeruginosa protein antigens which might be amenable to recombinant DNA production are outer membrane proteins and exotoxin A, modified to decrease toxicity but maintain immunogenicity. P. aeruginosa antibodies is Another strategy for immunization with anti-LPS passive administration of either hyperimmune immunoglobulins (polyclonal) or monoclonal antibodies. Passive immunization offers the dual advantage of rapid protection or treatment and is well tolerated. Several monoclonal antibodies against LPS P. aeruginosa antigens have been described, including both murine and human types. Studies in animal models of infection indicate that P. aeruginosa monoclonal antibodies do protect, thus, the most feasible application of molecular biology to the problem of P. aeruginosa infection appears to be production of immunotype-specific monoclonal antibodies for immune therapy.
Introduction
The rapidly advancing discipline of molecular biology has provided immunochemists with new horizons for vaccine development. Production of viral protein antigens using recombinant DNA technology circumvents the need to use potentially dangerous native antigen vaccines (e.g., HIV). Furthermore, production of anti-idiotype antibodies (anti-id) utilizes protein mimicry to produce vaccines which may be more immunogenic than the native, non-protein antigen (e.g., Haemophilus injluenzae and pneumococcus capsular polysaccharides, as applied to infants). Finally, cloning techniques for monoclonal antibodies provide a modern technique for passive immunization. How then might such methods apply to immunization against Gram-negative bacterial infections?
01954701/88/02A096+07
(Q 1988 The Hospml
SOS.OO/O
96
Infection
Society
Molecular
biology
and pseudomonas
immunization
97
Current analysis of Gram-negative bacterial infections suggests that these pathogens persist as important sources of nosocomial infection (Stamm, Martin & Bennett, 1977; Noso&mial Infection Surveillance, 1984). Among gram-negative bacilli, Pseudo onas aeruginosa is associated with the highest mortalities (Stevens et al., 19 d ; Bryan, Reynolds & Brenner, 1983; Bryan & Reynolds, 1984; Young, 1984). Although immunological methods for cross-protection against all Gram-negative bacilli have been advocated (Ziegler et al., 1982; Baumgartner, 1985), there is evidence that serotype-specific immunization may be more protective for P. ueruginosu (Pennington & Menkes, 1981; Sadoff et al., 1982). Experience has accumulated in P. ueruginosu vaccine development (Cryz, 1984; Pennington, lt)83), but so far little information has been presented regarding molecular’methods for development of P. ueruginosu vaccines. One likely reason is that rDNA methods apply to protein antigens, while the major virulence factor of P. ueruginosu is lipopolysaccharide (LPS) (Cryz et al., 1984b). In fact, numerous comparative studies have identified LPS serotype-specific antibodies as the most protective type of antibody against P. ueruginosu (Pennington & Menkes, 1981; Cryz, Furer & Germanier, 1983u,b, 1984~; Sawada, 1984). While even less information is available regarding anti-id vaccines for P. ueruginosu, this might represent an alternative strategy. Table I provides a list of the most important P. ueruginosu virulence factors (antigens) and outlines the feasibility and priority for genetically engineered vaccine development. As an alternative to active immunization, passive transfer of LPS type-specific antibodies may be considered for treatment or prophylaxis of P. ueruginosu infections. This approach may be particularly useful for intensive care unit or immunosuppressed patients in whom active immunization may be too slow or may not promote an adequate humoral immune response. Monoclonal antibodies directed against P. ueruginosu antigens should be considered as candidates for this approach. The following is a description of feasibility studies for monoclonal versus polyclonal antibodies in the treatment of P. ueruginosu infection.
Table
I. Candidates
for genetically
engineered pseudomonas
vaccines
Feasibility Antigen Lipopolysaccharide Outer membrane protein Exotoxin A Polysaccharide (O-specific) Proteases P&/flagella
rDNA
Anti-id
No Yes Yes No Yes Yes
Yes Yes Yes Yes Yes Yes
Protective potency of antibody High Medium/Low Low Medium Low ?
J. E. Pennington
98
Materials
and methods
Harley strain guinea pigs (400 g) were obtained from Charles Animals River Breeding Laboratories, Inc., Wilmington, Mass. Animals were housed in standard cages and fed guinea pig chow (Ralston-Purina, St. Louis, MO.). Bacteria Pseudomonas aeruginosa, strain 220 was employed for experimental pneumonias. This clinical isolate was Fisher immunotype-1 (IT-l) (Fisher, Devlin & Gnabasik, 1969). The characteristics of strain 220 have been described fully elsewhere (Pennington & Miller, 1979). Antibody preparations Hyperimmune P. globulin aeruginosa (PA-IGIV) (lot PR301 l), enriched in antibodies against LPS IT-l, -2, -4, and -6, was supplied by Cutter Laboratories, Berkeley, California. This preparation was supplied as 5% protein in 10% maltose and was rendered suitable for iv infusion by acidification to pH 4.25. Details regarding plasma donor screening and the immunological properties of this material have been provided elsewhere (Collins & Roby, 1984). Preparations of murine monoclonal antibodies (MAb) directed against P. aeruginosa IT-1 and IT-4 LPS antigens, as well as against P. aeruginosa outer membrane protein F (porin), were made and characterized using previously described methods (Pennington et al., 19863). Serological assays Concentrations of antibodies against IT-l and IT-4 P. aeruginosa antigens were determined by a radioimmune antigen binding assay previously described in detail (Pier, Markham & Eardley, 1981). Table II demonstrates that the MAb preparations contained greater concentrations of immunotype-specific antibody than did the polyclonal preparation. Experimental pneumonia Pneumonia was induced according to Pennington (1979) and Pennington & Miller (1979). Briefly, animals were anaesthetized (intraperitoneal pentobarbital), and their tracheas were isolated by a small midline neck incision. Samples (0.5 ml) of P. aeruginosa in isotonic saline were then instilled via a needle into the tracheobronchial tree. Incisions were sutured and animals awakened within 2 to 3 h. Preliminary studies established that 5 x lo6 cfus of strain 220 was the minimum inoculum which resulted in uniformly fatal pneumonia in this model. This inoculum was used for lung challenges in this study. Table
II.
Pseudomonas
LPS antibody and MAb preparations
aeruginosa
concentrations in PA-ZGIV
Antibody Preparation PA-IGIV IT-1 MAb IT-4 MAb Porin MAb
Anti-IT-l 7.6 478 Cl.0 Cl.0
cont.
(pg ml-‘) Anti-IT-4 44.9 Cl.0 74.0 Cl.0
Molecular
biology
and pseudomonas
immunization
99
Experiments to compare the t,herapeutic efficacy of the PA-IGIV preparation versus the MAb preparations involved cohorts of guinea pigs infected with equal inocula o P. aeruginosa and then treated 2 h after infection with a single iv infu 9fon of antibody preparation. Control groups received infusion of 5% bovine serum albumin (BSA) in 10% maltose. Infusion volumes (5 ml) were kept constant for all study groups. The methodology for iv infusions via the external jugular vein has been described before (Pennington, Pier & Small, 1986~). For survival studies, animals were observed for 4 days and were considered long-term survivors if they lived beyond this period (Pennington et al., l(b86a). I n selected studies, animals were sacrificed at timed intervals after &fection by intraperitoneal pentobarbital. Lungs were removed surgically, homogenized, and cultured quantitatively, as described before (Pennington, 1979, and Pennington & Miller, 1979). In addition, heart blood was obtained after thoractomy, and blood was cultured in trypticase soy broth (BBL Microbiology Systems, Cockeysville, Maryland) (Pennington et al., 1986~). Statistical analysis Differences in survival from pneumonia were compared by the Chi-Square analysis with Yates’s correction. The paired Student’s t-test was employed for analyses of quantitative bacteriology. Results Polyclonal and monoclonal antibody preparations were successful in reducing mortality from infection, and this effect was clearly dose-dependent (Table III). Importantly, the monoclonal preparation promoted higher, thus more protective, antibody titers using considerably lower protein dosages. It was also of interest that the monoclonal antibody preparations provided LPS serotype-specific protection (Table IV). Furthermore, protection was greater when a MAb directed against LPS antigen was used as compared to a MAb against outer membrane protein (Table IV). Bacteriological clearance studies supported the findings in the survival studies (Figure 1). Table
III.
Dose-dependent antibody-mediated
Hyperimmune Dose (mg kg-‘) 100 250 500 -
protection from Pseudomonas Monoclonal
globulin
Serum concentration antibody (pg ml-‘)?
Survival* W)
Cl.3 1.38 2.69 -
* 12 per group. t One hour after infusions (anti-IT-l). $6 per group.
33 -
Dose (mg kg-‘) 1.0 2.5 5.0 20.0
antibody
Serum concentration antibody (pg ml-‘)t 1.77 4.65 6.15 14.60
pneumonia (IT-l) Survivalj (%)
75 100
100
J. E. Pennington Table
IV.
LPS type-specific monoclonal antibody protection from type 1 Pseudomonas
Monoclonal Type
No. survived/ No. infected
antibody
1
Porin Type 4(outer membrane Albumin control
protein
F)
after
infection
% Survival
9/12
75
2112
:::
o/12
Time
Figure
pneumonia
0
(h)
1. Intrapulmonary
bacterial killing in guinea pigs infected with IT-l strain treated 2 h later with intravenous infusion of albumin, 500 mg kg-’ (0), MAb to outer membrane protein F (porin), 5 mg kg-’ (Cl), PA-IGIV, 500 mg kg-’ ( l ), or MAb to IT-l LPS antigen, 5 mg kg-’ (A). Four animals per time point per group. Positive blood cultures 9 h after infection; 4/4, controls; l/4; porin MAb; O/4 PA-IGIV; O/4 IT-l MAb.
Pseudomonas aeruginosa, then
Discussion
These studies using monoclonal antibodies for passive immune therapy in an experimental model of P. aeruginosa lung infection are encouraging. Furthermore, they suggest that passive immunotherapy against P. aeruginosa should employ antibodies directed against O-type-specific LPS
Molecular
biology
and pseudomonas
immunization
101
antigens. It is also evident that monoclonal antibodies may provide protective concentrations of antibodies using protein dosages which are considerably lower than tho required for plasma-derived polyclonal antibody preparations. Addit’ r nal advantages of monoclonal antibodies as compared to plasma-derived globulins may include better availability and avoidance of potential risks attendant with plasma-based products. Nevertheless, problems also exist for monoclonal antibody therapy. Obviously, the foreign protein associated with murine monoclonals might sensitize recipients. The recent development of human monoclonal antibodies against P. aeruginosa LPS antigens may reduce this risk (Hector et al., 1987). Anothe clinical dilemma is the need for rapid identification of the infecting pathog %n prior to use of a serotype-specific passive immune reagent. Recent studies of monoclonal antibodies for rapid diagnosis of P. aeruginosa infection (Rubin et al., 1985) are encouraging. References Baumgartner, J. D., Glauser, M. P., McCutchan, J. A., Ziegler, E. J., Van Melle, G., Klauber, M. R., Vogt, M., Muehlen, E., Luethy, R., Chiolero, R. & Geroulanos, S. (1985). Prevention of gram-negative shock and death in surgical patients by antibody to endotoxin core glycolipid. Lancet ii, S-3. Bryan, C. S. & Reynolds, K. L. (1984). Bacteremic nosocomial pneumonia. American Review
of Respiratory Diseases 129, 668-671. E. R. (1983). Analysis of 1,186 episodes of Bryan, C. S., Reynolds, K. L. & B renner, gram-negative bacteremias in non-university hospitals: the effects of antimicrobial therapy. Reviews of Infectious Diseases 5, 629-638. Collins, M. S. & Roby, R. E. (1984). Protective activity of an intravenous immune globulin (human) enriched in antibody against lipopolysaccharide antigens of Pseudomonas aeruginosa. American Journal of Medicine 76, 168-l 74. Cryz, S. J. (1984). Pseudomonas aeruginosa infections. In Bacterial Vaccine, pp. 317-351. Academic Press, Inc., London. Cryz, S. J., Furer, E. & Germanier, R. (1983a). Protection against Pseudomonas oerugin#osa infection in a murine burn wound sepsis model by passive transfer of antitoxin A, Infection and Immunity 39, 1072-1079. antielastase, and antilipopolysaccharide. Crvz. S. 1.. Furer. E. & Germanier. R. (19836). Passive protection against Pseudomonas aeruginosa infection in an experimental leukopenic mouse model. Infection and Immunity 40,659664. R. (1984~). Protection against fatal Pseudomonas Cryz, S. J., Furer, E. & Germanier, aeruginosa burn wound sepsis by immunization with lipopolysaccharide and high-molecular-weight polysaccharide. Infection and Immunity 43, 795-799. in virulence of Cryz, S. J., Furer, E. & Germanier, R. (19843). R o 1e of lipopolysaccharide _,
“,
I
.
Pseudomonas aeruginosa. Infection and Immunity Fisher,
M. W.,
44, 5088513.
N. H. & Gnabasik, F. J. (1969). New immunotype scheme Pseudomonas aeruainosa based on nrotective antigens. I I7oburnal of Bacteriologv-_
835-836.
Devlin,
-
for
98.,
Hector,
R. F., Trawinski, J., Besemer, D. J., Collins, M. S. & Pennington, J. E. (1987). In vitro and in vivo evaluation of a human IeM monoclonal antibodv to Pseudomonas aeruginosa immunotype 3. 27th Interscience Conference on Antimiciobial Agents and
Chemotherapy, New York, NY,
1987.
Infection Surveillance. 1984 (1986). CDC Morbiditv and Mortalitv Weeklv Report 35, 17ss-29s~. Pennington, J. E. (1979). Lipopolysaccharide pseudomonas vaccine: efficacy against pulmonary infection with Pseudomonas aeruginosa. Journal of Infectious Diseases 140, Nosocomial
73-80.
102
J. E. Pennington
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