Pseudomonas immunotherapy

Serodiugnosis

( 1988)

and Immunotherapy

2, 7-l 6

Review

Pseudomonas

article

immunotherapy

For the past 30 years infections by PseudomoNeruginosa have caused frequent, severe, often fatal sepsis in burned and other immunocompromised hospital patients’.‘. In addition the reported mortality for P. aeruginosa pneumonia is as high as 50-80°h2m5. Even with the development of newer antibiotics, resistance remains a problem necessitating combined antibiotic treatment for severe P. aeruginoscl infections6.‘. Alternate therapy for the management of severe P. aeruginosa have been evaluated for many years. Immunotherapy has been the alternative most extensively explored, and in earlier years, the major efforts in this area were directed toward its potential use in patients with burns. During the 1960s a variety of experimental vaccines were prepared and tested for the prevention of P. aeruginosu infections in burned ammaW “I_ The materials used as antigens were either the bacterial cells or substances fractionated from cell supernatant fluids. These materials provided varying degrees of protection to burned P. rreruginosa-infected animals via active immunization. Passive immunization also provided protection. but the protection varied with the time gap between burn and infection hcfore lreatmenl was given. In all cases protection was scrotype-specific. Although the vaccines and passive immunotherapy were never widely used clinically, they were the basis for later immunological studies in patients. The most promising of the vaccines tested were the heptavalent lipopolysaccharide (LPS) preparation. Pseudogen.lM from the ParkeDavis Company” I7 and the l6-valent, LPScontaining “surface extract” vaccine, subsequently to be known as PEV-01 I’. Both preparations stimulated significant antibody titers to all of the 0-serotype strains contained in the vaccine when tested in burned patients or healthy volunteers’5.‘h, and both provided reduced incidence of P. aeruginosa sepsis and nas

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decreased mortality from this organism in burn patient vaccinees’7m’0. Passive transfer of antibody (IgG) from serum of human volunteers immunized with these vaccines also was protective”,“. It was not surprising that P. aeruginosa-specific IgG was successful in reducing P. aeruginosa sepsis in burned patients, since it had been shown years before that normal human sera contained Pseudomonas-specific antibody”. Therefore pools of normal IgG were used successfully in burn patients to reduce septicemia and mortality from P. aerugi~zosc~~~.~~. Clinical studies with PseudogeniM vaccine were also carried out in cystic fibrosis:h and cancer populations”7,2”. and in intensive care patients with respiratory failure”“. In cases where it was assessed. patients responded with modest to large increases in antibody titer to all 0-serotypes of P. aeruginosa contained in the vaccine, but the results in regard to protection from infection ranged from “significant but limited” to “none noted”” z”. In experimental animal models of acute pneumonia, however, both PseudogenTM and PEV-01 vaccination significantly increased antibody titers, enhanced lung clearance of organisms, and increased survival when challenge organisms were instilled directly into the lower respiratory tract”‘.“. Furthermore, PseudogenrM-vaccinated animals appeared to have a “local protective response in respiratory tissue”. since both gross and microscopic findings exhibited less pathology in the vaccinated as compared to the control group”. Similarly, PEV-01 vaccinated rats showed less lung damage compared with controls when a chronic lung infection was established by instilling in the lung P. wrugrrzosu imbedded in agar beads’?.“. In one study. however, while protection was seen, there was no reduction in microbial load in the lungs ot the immunized animals. leading the authors to speculate that PEV-01 vaccine “may contatn components of cell surface proteins and viru8~’ 1988 Academic

Press Limited

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Review

lencc exoproducts”“, Because of the endotoxic complications associated with the PseudogenTM vaccine, the unknown nature of the antigens contained in the “surface extract” PEV-01 vaccine, and because of questions raised about the experimental design of some of the clinical studies using these materials, neither vaccine gained popularity among clinicians, While the idea of passive pseudomonas immunotherapy was appealing, the detrimental vasomotor reactions that resulted from IgG being given intravenously required it to be given intramuscularly. However, because pain on injection, slow absorption and the inability to obtain optimal blood levels of antibody due to mass-volume considerations limited its use, intramuscular IgG therapy was not widely utilized. In the past decade, a resurgence of interest in pseudomonas immunotherapy has occurred. It was fueled by two events: advances in the technology of IgG preparations and studies defining the role(s) of various cell-associated factors and exoproducts in the virulence of P. aeruginosa infections. In recent years. IgG fractions from the pooled serum of normal individuals have been prepared for intravenous use. These preparations can be administered safely, in large doses and with more rapid and higher blood levels attained than were possible using intramuscular formulations’4,‘5. All of these preparations contain antibodies to all of the 0-serotypes of P. aeruginosa tested”, all demonstrate some opsonic activity against P. aeruginosa’“, and if given soon after infection, all confer dose dependent protection in P. rtc,ruXinosu-infected normal or burned mic?” I’. When IgG treatment is delayed until animals become septic, the efficacy of therapy decreases and becomes more challenge strain dependent”‘+‘. Intravenous IgG is prepared by a variety of methods. The method used for preparation can modify some of the effector functions of the particular IgG product, e.g. opsonic acbactericidal tivity’h”‘. complement-dependent with Fc receptors on activity4’, interaction monocytes and macrophage?. and animal protection’h.4’. These factors may explain contradictory reports of passive IgG therapy having little effectjh or high levels of serotypespecific protective capacity” in granulocytopenit P. ueruginosa-infected mice. Pseudomonas hyperimmune IgG is up to IOfold more protective, for burned P. aeruginostrinfected animals than is pooled normal IgG’b4UJ’. The same protection was confirmed in a guinea pig model of acute pneumonia4’.

Two major methods arc used to prepare hypcrimmune globulin: pooling high individual Oserotype titer sera obtained from large numbers of plasma donors”’ or pooling sera from human volunteers actively immunized using a polyvalent “surface-extract” vaccine4’. In the case of IgG prepared by the latter method, antibodies to antigens other than the serotype-specific lipopolysaccharides are present”’ and may explain at least part of the protection observed when this preparation is used to treat burned P. cteruginosa-infected mice4’.“. Both hyperimmune and normal pools of intravenous globulin preparations contain, in addition to antilipopolysaccharide antibodies, antibodies to exotoxin A’h.4X. Many antibiotics act synergistically with IgG treatment’” ‘“. When IgG is used in combination with B-lactam antibiotics to treat infections with B-lactamase producing organisms, the IgG may potentiate the action of the antibiotic due to the anti-(3 lactamase activity attributed to the IgG4”. However, the dual benefit of IgG plus antibiotics does not hold for all antibiotic:IgG combinations and the beneficial aspects of such treatment appear to vary with the virulence of the infecting strain of P. oeruginosa and the antibiotic, at least when used in the treatment of septic animals”‘. While there is ample experimental data showing that intravenous IgG treatment, particularly using hyperimmune globulin, may be useful in treating P. ueruginosa infections in a variety of experimental animal systems. many questions have to be answered before these preparations can be used widely in patients. Would the results obtained in animal studies be the same in humans‘? Would thcrc bc a rcasonable cost/bcncfit ratio for these products lo bc used prophylactically? Could they bc used to treat patients already septic from P. wruginosu? Even if positive results wcrc obtained from IgG trcatmcnt of burned patients. would the results be the same in cystic fibrosis patients, in cancer patients? Obviously, the answers to these and other relevant questions can bc found only through well-controlled, randomized clinical studies in large numbers of patients with various immunocompromising underlying diseases that are subject to P. wruginosa infections. For the past dozen years enormous progress has been made in the elucidation of the role(s) that various cell surface and extracellular products play in the virulence of P. mmginosu infections. Exotoxin A. alkaline protease. elastase and flagella were shown to be

Review important for Pseudomonas virulence in burned animals” “; exotoxin A, exoenzyme S and proteases in both acute and chronic lung infectionsTx hi, and protease and exotoxin A in cornea] infections”‘,6’. In all cases studied, Pseudomonas virulence appeared to be multifactorial; no single virulence product or characteristic is responsible for all aspects of virulence. Indeed, antibodies to several virulence factors. alkaline protease, elastase and exotoxin A. are found in the serum of Pseudomonasinfected cystic fibrosis patients”3.N, and antibodies to exotoxin A as well as antibodies to Pscwkmonas lipopolysaccharide antigens arc found in the serum of bacteremic patientshi.“‘. In fact, high anti-toxin and anti-lipopolysaccharide titers correlate with enhanced survival in Psrudonmzas septic patients”‘. Antibodies to cxotoxin A and both proteases were found in the serum of cancer patients and passive transfer of patient sera with high exotoxin A titers protected mice against challenge with purified exotoxin Ah’. Antibody to outer membrane proteins, and perhaps flagella, were prcscnt in the serum of a burn patient infected by I’. afwc‘~irlosa6~. Taking advantage of the knowledge of the roles that virulence factors play in pseudomonas infections, that these substances are good immunogens. and that antibodies to them are found in the serum of infected patients, investigators explored virulence factors alone or in combination, for active immunization or passive transfer of antibody. One of the earliest multicomponent vaccines used combinations of toxoided alkaline protease (proteoid). elastase (elastoid) and “original endotoxin protein” (OEP) from P. aerugin0.w. “Original cndotoxin protein” was reported to be a common protective antigen that could be used to prevent infections due to P. acw,qinosa regardless of the 0-serotype of the infecting strain@‘.‘“. In experimental cornea] infections. three component vaccination, as well as passive three-component IgG therapy, was more protective than single component therapy. especially when combined with antibiotic treatment”. On the other hand, mink immunized with the multicomponent vaccine, while better protected from experimental hemorrhagic pneumonia than animals immunized with OEP alone, were no more protected than mink immunized with proteoid or elastoid. alone or in combination”. Burned mice were protected significantly more following elastoid vaccination than by vaccination with OEP or proteoid alone or immunization using

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the three component vaccine”. Active and passive immunization of burned mice using exotoxin A toxoid or antitoxin has given mixed results which range from extension of survival timer2.‘4’5 to no effect with passive therapy’h. While antitoxin or antibiotic treatment alone each extended the mean time to death of burned P. aeruginosa-infected mice in one study, only when they were given together did long term survival occurs’. This suggests that. at least in burned animals, immunological neutralization of a virulence exoproduct alone is not the best means to protect hosts from pseudomonas infection; simultaneous reduction of microbial load plus neutralization appears to be more effective, since unrestricted growth of the infecting strain might produce more of the exoproduct than the anti-exoproduct therapy alone has the capacity to neutralize. Flagella have been used successfully as vaccines to prevent P. aeruginosu infections in burned anima1s7’.‘k. Protection appears to be flagella antigen-specific and O-lipopolysaccharidc non-specific. This type of vaccine holds appeal because P. aeruginosa contains only two major antigenic types of flagella compared to between seven and 16 lipopolysaccharide serotypes. Furthermore, the protein nature of the flagella make them more preferable for active immunization than lipopolysaccharide antigens. Both Pseudomona.t lipopolysaccharidc and purified elastase have been used to immunize rabbits who subsequently received cornea] challenge with viable P. urruginosa’“. While animals immunized with the LPS wcrc better protected than those immunized with elastasc only. animals given elastase only immunization showed significantly less cornea1 damage than controls. This also was true. when antiserum prepared against purified elastasc was administered. In burned mice, hohcver, passive immunization using anti-protcase serum gibe disparate results. In one study it provided substantial protection”. while in another it was withouI effect“‘. Because of the well-known side-effects 01 LPS vaccines, alternative non-toxic derivatives have been sought. High and low molecular weight polysaccharides, which share the immunotype specific antigenic determinants of the LPS 0 side chain from which they are prepared, appear to be such materiaWO “‘. Both materials produced high titers of antibodies in immunized animals, and active or passive immunization provided 0 serotype-specific protection in

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normal”’ or burned mice8’~“‘. However, the low molecular weight polysaccharide had to be conjugated to protein, in this case serum albumin, for antibodies to be generateda’. In one studyHL, IOOO-fold higher molecular weight polysaccharide had to be given, compared to LPS, to provide an equivalent degree of protection to immunized mice. Some caveats about polysaccharide vaccines include the fact that. as with LPS vaccines, for complete protection against P. ueruginosa infections regardless of the 0-serotype of the infecting strain, a multivalent vaccine would have to be used. Furthermore, different mouse strains respond differently to immunizations with high molecular weight polysaccharides; one mouse strain required 50 times more antigen to generate antibody and less protection was observed than when another mouse strain was usedn4. Additionally, antibodies generated to high molecular weight polysaccharide immunization, using a preparation made from a Fisher immunotype 1 P. aeruginosa strain and collected from one mouse strain, reacted with only type 1 polysaccharide, whereas antibodies collected from another mouse strain immunized with the same immunogen cross-reacted with both type I and type 2 polysaccharides. If human genetic counterparts of these different mouse strains exist, then the implications of this study could be considerable. As was mentioned previously, procedures which reduce microbial load while simultaneously neutralizing virulence exoproducts might provide better protection against P. aeruginosa than either factor alone. In an immunological approach which addresses this point, a low molecular weight polysaccharide conjugated to exotoxin A, as the protein moiety, was prepared”‘. The conjugate was non-toxic for mice, non-pyrogenic for rabbits, and generated both serotype-specific antipolysaccharide and anti-toxin IgG. Anti-conjugate IgG neutralized the cytotoxic effects of exotoxin A in vitro and immunization of mice significantly raised both the LD,, from challenge with the homologous 0-serotype strain as well as the mean lethal dose of exotoxin A. As was pointed out earlier, antibodies to outer membrane proteins were found in the serum of a burned patient infected by P. ueruginos#. Antibodies to these proteins also are found in the serum of cystic fibrosis patients infected by P. ueruginosa*6,87. Since some of these proteins are antigenically related in all Oserotype strains of P. aeruginosaR8~R9 they have become candidates for vaccines. Active immu-

nization with purified outer membrane proteins, as we]] as local infection with P. ueruginosa, increased antibody titers to outer membrane proteins, especially Porin F, in the vaccinated or infected animals,KH.“’ ‘):, and active immunization using purified Porin F protein provided protection from heterologus strain challenge in both normal and burned miceHE.“. Passive immunization using polyclonal antiserum was protective for normal mice9’, and passive immunization using monoclonal antibody was protective for both normal and burned mice9:. Monoclonal antibody increased non-complement dependent phagocytosis when tested in an in vitro system92. However, studies comparing passive immunization using monoclonal antibodies to outer membrane proteins versus monoclonal antibodies to LPS demonstrated that LPS monoclonal antibody treatment provided significantly better protection, on a weight basis, than outer membrane monoclonalsy3. This was true for both infected normal and burned mice. While data using outer membrane proteins for immunotherapy of P. ueruginosu infections appears promising, interpretation of data from outer-membrane immunization-protection experiments must be done cautiously due to the difficulty in purifying these proteins free of LPSv’J’“. Monoclonal antibodies can be prepared against specific LPS antigens of P. ueruginosu9’ and a monoclonal antibody has been prepared which reacts with a “new common polysaccharide antigen of P. aeruginosa”9”. This antibody bound to about 80% of the various Oserotype strains of P. aeruginosa testedgh. The value of these materials in protection from P. ueruginosa infections awaits the appropriate experimental data. In addition, monoclonal antibodies have been prepared against exotoxin A“‘. Monoclonal antibodies can be prepared that react with “two discrete structural domains of P. aeruginosu toxin A”. One antibody neutralized the cytotoxic and lethal properties without affecting ADP-ribosyl transferase activity, while the other neutralized the ADPribosyl transferase activity of toxin but did not interfere with the binding of toxin to membrane receptors. Anti-binding monoclonals were protective in toxin A challenged L cells and toxin injected mice, whereas anti-ADP ribolylating activity monoclonals were not. Results of this study have significant relevance to any study which proposes to prepare as treatment substances for P. ueruginosa infections, monoclonal antibodies to a variety of Pseudomonas exoproducts, such as proteases or exoenzyme S,

Review which may have both binding and enzymatitally active domains. Recently a novel approach to anti-pseudomonas immunological therapy was established by the development of temperature-sensitive mutants as vaccine strains. These mutants grow at the permissive temperature of 27”C, but their growth is limited to two to five replications (“coasting”) at the non-permissive (36°C) temperature which would be found in the human or animal host99. Active immunization with both two- and five-replication temperature-sensitive “coasting” mutants protected mice, immunized intraperitoneally, from challenge with the wild type parent98.99. The more extensive “coaster” strain (five-replication) provided equal protection with a significantly reduced immunizing dose compared with the limited (two-replication) “coaster” and generated significantly higher levels of serotype-specific IgG as we1Y. Furthermore, intranasal immunization provided enhanced lung clearance when mice were challenged with the homologous wild type strain by aerosolization9Y.‘00. While temparature-sensitive mutants generate serotype-specific anti-LPS IgG, therefore requiring a polyvalent preparation for broad spectrum anti-Pseudomonas immunization, they have the appeal that during their limited replication in the host they also would generate non-toxic but perhaps still immunogenic amounts of antigenit exoproducts. Concern about reversion to wild type could be removed by combining several temperature sensitive mutations in one strain: “Because the reversion rate of a strain containing three temperature sensitive mutations, each of identical phenotype, is the product of the rates of the individual strains, mutations with reversion frequencies as high as IO-’ can be combined to make a strain with a reversion frequency of IO-“. This would place the vaccine strain within the same safety limits as those set down for recombinant DNA strdins”vq. Two additional approaches to P. aeruginosa immunotherapy which have engendered some controversy are the use of ribosomal vaccine and the Gram negative common core glycolipid. A P. aeruginosu ribosomal vaccine has been described and shown to be immunogenic”“. Active immunization using this preparation resulted in serotypic specific protection in normal mice given intraperitoneal challenge”” and in burned rats challenged subcutaneously

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in the burn site”“. Passive ribosomal vaccine induced antiserum or immunoglobulin fractions were protective to normal mice and burned rats”‘?.““. However. LPS components have been found in the ribosomal vaccine preparations”“, and protection is not diminished when the preparations arc treated with trypsin or ribonuclease’“‘. These facts cast doubt that ribosomes are, in fact, the immunizing material in thcsc preparations. Appropriate judgements about the value of Ps~udon~mas ribosomal vaccine cannot be made until this question ih resolved. Another unusual approach to immunotherapy of P. aeruginosa infections is the use of the E. coli J5 mutant. This mutant lacks O-specific side chains because it is deficient in uridine-5’diphosphate-galactose-4-epimerase activity which reduces incorporation of exogenous galactose into LPS. Thus antiserum prepared against its LPS contains antibody directed against the exposed core region that is shared among numerous Gram-negative bacteria. Antiserum prepared against J5 provided enhanced survival when used to treat neutropenit rabbits infected with unrelated E. cd. Kkhsiella pneumoniue and P. uw,qirwsa’O” lilh, Active immunization was protective as well’““. in fact it was more protective than passive immunization’“‘. A clinical study which used passive J5 antiserum therapy to treat patients with Gram-negative bacteremia showed that there were significantly fewer deaths in the treated groups compared to controIs”‘X. While P. wrw ~:innsa was the second leading cause of bactercmia, after E. cd. in both the non-immunized and immunized patients in this study, the survival rates in relationship to ~u~~MFZO~~U.Sinfected patients were not discussed. Therefore. JS immunization as it relates specifically to survival of P. rrcvu@zoso-infected patients could not be assessed. While antibody levels to anti-core IgG. as well as anti-toxin A and total IgG levels correlated individually with survival in patients with pseudomonas septicemia, “none augmented the prognostic power of type-specific antibodies in combination with anti-core IgM, which together predicted outcome accurately 73.5% at the time”‘09, suggesting that “cross protective activity against P. ueruginosa of naturally occurring antibodies to the endotoxin core of E. coli anti-core antibodies, particularly of the IgM isotype. appear to augment the more specific protective immunity engendered bv antibodies to the 0 specific side chains
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In contrast to studies in which J5 immunotherapy was effective in treating P. aevu@,zo.su infections. others report that active J5 immunization was not successfuI in enhancing survival in burned rats”” nor did this immunization provide anything better than weak cross protection against experimental hemorrhagic pseudomonas pneumonia in guinea-pigs”‘. Even more discouraging. JS immunization provided no protection for the guinea-pigs when the pneumonia was induced using either E. coli or K. pneumoniae. Thus, disparity of results among various research studies using J5 immunization make any definitive statement about the potential usefulness of this approach difIicult, at best. A recently published report may have relevance in this regard”‘. In this study it was found that in preparing core reactive monoclonal antibody to LPS. some preparations were contaminated with low levels of endotoxin. The authors demonstrated that low doses (I .O ng per mouse) of endotoxin could protect mice from infection from various species of bacteria, that “spiking” non-immune rabbit immunoglobulin with LPS rendered the previously ineffective IgG efficacious against infection, and that the same was true when monoclonal antibody to LPS core that was contaminated with endotoxin was used in the study. Therefore, the extent of LPS contamination of J5 vaccine preparations or the antiserum prepared from the vaccines that were used in the various studies described above might explain, at least partially. the different results obtained. Obviously, before any of the immunological approaches to P. aeruginosa infections just described can be put into general use, the same questions raised for the use of intravenous IgG therapy must be answered. However. because of the renewed interest in Pseudomonas immunotherapy in the past decade. the recent intense research activity in this area, and the diversity of approaches being tried currently, clinically useful immunotherapy for P. aeruginosa infections appears to be closer than ever. IAN ALAN HOLDER of Surgery, Microbiology and Molecular Genetics, University of‘ Cincinnati College of’ Medicine and Shriners Burns Institute, Cincinnuti. Ohio 45219. U.S.A Departments

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Morrison AJ. Jr. Wenzel RP. Epidemiology ol infections due to Pseudomonas ueruginosa. Rev Infect Dis 1984; 6 (suppi.): 627 -42. Linares HA. A report of I I5 consecutive autopsies in burned children. Burns 1981: 8: 263- 70. Pennington JE, Reynolds HY. Carbonc PP. Pseudomonas pneumonia: a retrospective study of 36 cases. Am J Med 1973; 55: 155 60. Gatmaitan BG. Garruthers MM, Lcrner, AM. Gentamicin in treatment of primary gram-negative pneumonias, Am J Med Sci 1970: 260: 90- 4. Stevens RM, Teres D. Skillman JJ, Feingold DS. Pneumonia in an intensive care unit. A 30 month experience. Arch Intern Med 1974: 134: 106 II. Bodey GP. Bolivar R, Fainstein V. Jadeja L. Infections caused by Pseudomonus ueruginosa. Rev Infect Dis 1983; 5: 279 313. Macmillan BG, Holder, IA, Alexander JW. Infections of burn wounds; in Bennett JV. Brachman PS, eds. Hospital Infections. Boston: Little, Brown, Co., 1986: 143 64. Alms TH, Bass JA. Induction of protection by an alcohol-precipitated fraction from the slime layer. J Infect Dis 1967: 117: 249 56. Feller I, Bial AB. Callahan W. Waldvke J. Use of vaccine and hyperimmunc serum for protection against Pseudomona septicemia. J Trauma 1964: 4: 451 6. Markley K, Smallman E. Protection by vaccination against Pseudomonas infection after thermal injury. J Bact 1968; 96: 867--74. Alexander JW, Fisher MW, MacMillan BG, Altemeier WA. Prevention of invasive pseudomonas infection in burns with a new vaccine. Arch Surg 1969: 99: 249 56. Alexander JW, Brown W. Walter HL. et al. Studies on the isolation of an infection protective antigen from Pseudomonus ueruginosa. Surg Gyn Obst 1966: 123: 956 77. Fisher MW, Devlin HB. Gnabasik FA. New immunotype schema for Pseudomonas aerugirn~sls~~based on protective antigens. J Bact 1969: 98: 835-6.

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vaccine. J. Med Microbial 1977; IO: 19 27. Schemmer KE, Alexander JW. Fisher MW. Immunological response of severely burned patients to P.seudomonas vaccination. Surg Forum 1969; 20: 69 ~7 I. JJ. Spilsbury JF. A new Pseudomonas 16. Miler vaccine: preliminary trial on human volunteers. J Hyg Camb 1976; 76: 429 39. 17. Alexander JW, Fisher MW, MacMillan BG. Immunological control of Pseudomonas infection in burn patients: A clinical evaluation. Arch Surg 1971; 102: 31 5. JW. Fisher MW. Immunisation 18. Alexander against P.wudomontr.s infection after thermal injury. J Infect. Dis. 1974; 130 (suppl.): 5152 X. I5

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Review 19. Jones RJ. Roe EA. Gupta JL. Low mortality in a P.veudomonas vaccine trial. Lancet 1978; ii: 401m 3. 20. Jones RJ. Roe EA. Gupta JL. Controlled trials of polyvalent Pseudomonas vaccine in burns. Lancet 1979; ii: 977-83. 21. Jones RJ. Alexander JW. Fisher MW. Clinical evaluation of Pseudomonas hyperimmune glohulin and plasma. Surg Forum-1970: XXI 22. Jones RJ. Roe EA. Controlled trial of Pwudomonas immunoglobulin and vaccine in burn patients. Lancet 1980; 1263-5. 23. Gaines S. Landy M. Prevalence of antibody to Pseudomonas in normal human sera. J Bact 1955; 69: 628-33. 14. Kefalides NA, Arana JA, Bazan A, Velarde N, Rosenthal SM. Evaluation of antibiotic prophylaxis and gamma-globulin, plasma, albumin and saline-solution therapy in severe burns. Ann Surg 1964: 159: 496506. 15. Kefalides NA, Arana JA, Bazan A et al. Evaluation of plasma. gamma-globulin, albumin and saline-solution therapy in a group of Peruvian children. N Engl J Med 1962; 267: 317 23. 26. Pennington JE. Immunotherapy or Psrudomonu.~ aeruginosa infection. In: Doggert RE, ed. P.reudomonas aeruginosa: clinical manifestations of infection and current therapy. New York: Academic Press, 1976: 192~ 215. 27. Young LS. Meyer RD. Armstrong D. Pseudomonas ueruginosa vaccine in cancer patients. Ann Intern Med 1973; 79: 518-27. 38. Haghbin M, Armstrong D, Murphy ML. Controlled prospective trial of Pseudomonas aerugino.~a vaccine in children with acute leukemia. Cancer 1973; 32: 761-6. 19. Polk HC, Jr., Borden S. Aldrete JA. Prevention of P,seudomonas respiratory infection in a surgical intensive care unit. Ann Surg 1973; 177: 607 15. 30. Pennington JE. Pier GB. Efficacy of cell wall P.wudomonas aeruginosa vaccines for protection against experimental pneumonia. Rev Infect Dis 1983: 5: S852Z7. 31. Pennington JE. Lipopolysaccharide Pseudomonay vaccine: efficacy against pulmonary infection with Pseudomonas aeruginosa. J Infect Dis iY79; 140: 73 80. 32. Klinger JD. Cash HA, Wood RE. Miler JJ. Protective immunization against chronic Pseudomonus aeruginosa pulmonary infection in rats. Infect Immun 1983; 39: 1377 84. 33. Pennington JE, Hickey WF, Blackwood LL, Amaut MA. Active immunization with lipopolysaccharide Pseudomonas antigen for chronic Pseudomonas bronchopneumonia in guinea pigs. J Clin Invest 1981; 68: II40 8. 34. Finlayson JS. History of immunoglobulin use. In: Alving BM, Finlayson JS eds. Immunoglohulin: characteristics and uses of intravenous preparations. U.S. Department of Health and Human Services publication no. FDA-80-9005. Washington D.C.: Government Printing Office. DC. 1980; ix Y

35. Schroeder DD. Tankersley DL, Lundblad JL. A new preparation of modified immune serum globulin (human) suitable for intravenous administration. VOX Sang 1981; 40: 373-82. 36. Pollack M. Antibody activity against Pseudomonas aeruginosa in immune globulins prepared for intravenous use in humans. J Infect Dis 1983; 147; 109&8. 37. Davis SD. Efficacy of modified human immune serum globulin in the treatment of experimental murine-infections with seven immunotypes of Pseudomonas aeruginosa. J Infect Dis 1975; 134: 717 21. studies of 38. Holder IA. Naglich JG. Experimental the pathogenesis of infections due to P.veudomr~ml., aeruginosu: treatment with intravenous mmune glohulin. (IgG-IV). Am J Med 1984: 161 39. Collins

MS. Roby RE. Anti-Pseudomonas ueruactivity of an intravenous human IgG oreuaration in burned mice. J Trauma 1983: 23: ‘53&t. Holder IA. Neely AN. Experimental studies of the pathogenesis of infections due to Pseudomor2a.c aeruginosa: passive intravenous immunotherapy using pseudomonas globulin. Serodiag Immunother Infect Dis 1987; I: 153-~62. Yasuda H. Yajima T. Tanii T, Ashiba T, iwaata M. Opsonic and complement dependent bactericidal activities of various immunoglobulin preparations for intravenous use. Vox Sang 19X6; 51: 27(f7. Jungi TW. Eiholzer J. Lerch PG. Barandun S. The capacity of various types of immunoglobulin for intravenous use to interact with Fc receptors of human monocytes and macro. phages. Blut 1986: 53: 32l- 32. Neely AN. Holder IA. Use of passive immunotherapy in the treatment of experimental P.wr~doI~II,~I.S uwugino.wr mfcctions in burns. In Dormg G, Botzenart K. Holder IA. eds. Basic research and clinical aspects of Pseudomonas ucrrqin0.w Basel: A. G. Karger Co., 1987: 26 40. Cryz SJ, Furer E. Germanier R. Passive protection against Pseudomonas aeruginosa infection in an experimental leukopenic mouse model. Infect lmmun 1983; 40: 659~64. Collins MS. Roby RE. Protective activity 01‘ an intravenous immune globulin (human) enriched in antibody against lipopolysaccharide antigens of Pseudomonas ueruginosu. Am J Med 1984; 14X4: 168 74. Pennington JE. Pier GB. Sadoff JC. Small GJ. Active and passive immunization strategies for Pscwdomonas aertqinosa pneumonia. Rev Infect Di\ 1986; 8: S426 33. Maclntyrc S. McVeigh T. Owen P. Immune)chemical and biochemical analysis of the pal\valcnt P.ccwd0montr.t trerugin0.w vaccine PFV. Infect Immun 1986: 51: 675- 86. Collins MS. Tsay GC. Hector RF. Roby RE. Dorsey JH. lmmunoglobulin G: potentiation ot Tobrdmycin and Azlocillin in the treatment of Phwdom0na.s sepsis in neutropenic mice and

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IS

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human antiserum to a mutant Eve /zc,r&iil~ NEJM 1982: 20: 1225 30. Pollack M, Huang. AI, Prescott RK e/ tl/ Enhanced survival in Psrudomoncr.r rrertrginow septicemia associated with high levels of circulating antibody to E,sciwkhiu cwli endotoxin core. J Clin Invest 1983: 72: IX74 XI. Sadoff JC. Futrovsky SL. Sidberry III’. Iglewski BH. Seid RC. Detoxifed lipopolysaccharide protein conjugates. Semin Infect Dis 1982; 4: 34&54. Pennington JE, Menkes E. Type-specific vs. cross-protective vaccination for gram negative bacterial pneumonia. J Infect Dis 1981: 144: 599 603. Chong KT. Huston M. Implications of endotoxin contamination in the evaluation of antibodies to lipopolysaccharides in a murine model of gram-negative sepsis, J Infect Dis 1987; 156: 713 9. coli.

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