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A study of the antibody response to antigenic preparations derived from Pseudomonas aeruginosa Johnston, Linda Joan 1971

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A STUDY OF THE ANTIBODY RESPONSE TO ANTIGENIC PREPARATIONS DERIVED FROM PSEUDOMONAS AERUGINOSA by LINDA JOAN JOHNSTON A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Microbiology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this for financial gain shall not be allowed without my written permission. The University of British Columbi Vancouver 8, Canada Department ABSTRACT Several cellular and subcellular fractions were prepared from Pseudomonas aeruginosa'strain PA-^ - Those found to. be immunogenic in rabbits included.a heat-stable 1ipopolysaccharide, a protein-1ipopolysaccharide complex, a cell wall preparation arid a formalin-killed whole cell vaccine. However, a 1ipopolysaccharide preparation extracted with phenol and water was found to be a poor immunogen in rabbits. The cell wall fraction proved to be the most'effective immunogen in terms of the amount of antibody evoked, and of the duration of the serum antibody response. Hyperimmune sera produced against a l l four antigens were found to contain a mixed population of 2-mercaptoethanol sensitive and 2-mercaptdethanol resistant antibodies. Gel f i l t r a t i o n and ion exchange chromatography studies established the presence of both IgM and IgG immunoglobulins in a l l four'types of hyperimmune serum. Whole immune serum, as well as the IgM and IgG serum fractions, afforded passive protection to mice challenged with twenty or more L D ^ Q of viable organisms. There was an indication that the IgG fract of two of the four serum types provided -better protection than did: the IgM fraction, but precipitation studies indicated that this may have,been due to greater numbers of IgG immunoglobulins. In addition serum containing a high proportion of 2-mercaptoethanol resistant antibody-was found to promote faster clearance of injected bacteria than did serum taken earlier in the response. Immunodiffusion studies indicated that al1 four. antigenic prep-arations contained at least one common immunogen; moreover, a l l serum types were able to react with sheep red blood cells coated with the heat-stable 1ipopolysaccharide preparation in passive hemagglut inat ion and hemolys in tests. TABLE OF CONTENTS Page INTRODUCTION . . . . 1 LITERATURE REVIEW. . . . . 3 I. Pseudomonas aerug?nosa as a Human Pathogen. . . . 3 II. Nature of the Infection . . . . . . 5 III. Host Defences Against Pseudomonas aeruginosa. . . 7 IV. Therapeutic Measures 10 MATERIALS AND METHODS 15 I. Organisms and. Media 15 II. Preparation of Antigens 16 111. Experimental Animals. 19 IV. Immunization Procedures 19 V. Anti sera 21 1. Collection and Storage. 21 2. determination of Antibody Titer . 21 3- Fractionation of Antisera 23 a) Dissociation of Macroglobulin with 2-Mercaptoethanol . 23 b) Gel Fi ltration 23 c) Ion Exchange Chromatography. 2k VI. Passive Protection Studies 2k VI t. Clearance Studies 25 V TABLE OF CONTENTS (Continued) Page VIII. In Vitro Tests •. . 26 1. Bactericidal Assay . . . 26 2. Immunodiffusion Tests 27 3- Quantitative Precipitin Test 27 IX. Electron Microscopy. . . 28 RESULTS AND DISCUSSION 30 I. Antibody Response to the Antigenic Preparations. 30 II.' Properties of the Antigenic Preparations . . . . 37 III. Fractionation of Rabbit Antisera kj a) Sensitivity to 2-Mercaptoethanol k7 b) Fractionation of.Rabbit Serum Proteins by Gel Filtration and Ion Exchange Chromatography 49 IV. Passive Protection Tests 53 a) Whole Serum 53 b) Fractionated Serum 58 V. Clearance Studies. 62 GENERAL DISCUSSION . . 68 BIBLIOGRAPHY 72 v i LIST OF FIGURES Page Figure 1. Rabbit antibody response to'the cell wall preparation of Pseudomonas aeruginosa strain PA-7. Figure 2. Rabbit antibody response -to heat-stable lipo-polysaccharide preparation from Pseudomonas  aeruginosa strain PA-7. Figure 3- Rabbit antibody response to phenol-water extracted 1ipopolysaccharide from Pseudomonas  aeruginosa strain PA-7. Figure k. Rabbit antibody response to protein-1ipopoly-saccharide from Pseudomonas aerug i nosa strain PA-7. Figure 5- Rabbit antibody response to formalin-killed vaccine of Pseudomonas aeruginosa strain PA-7. Figure 6. Immunodiffusion analysis of 1ipopolysaccharide preparations from Pseudomonas aeruginosa.' Figure 7- Electron micrographs of cell wall preparations from Pseudomonas aerug i nosa. Figure 8. Detection of 2-mercaptoethanol resistant anti-body activity in rabbit serum. Figure 3. Elution profile of whole rabbit serum from Sephadex G-200. Figure 10. Elution profile of IgM-containing serum fract-ion from Sephadex G-200. Figure 11. Elution profile of whole rabbit serum from the DEAE-cel1ulose column. Figure 12. Precipitation analysis of purified IgM and IgG serum fractions. 31 32 33 3k 35 ko kk k8 51 52 Sk 63 LIST OF TABLES Rabbit immunization schedule for the various antigenic preparations from Pseudomonas aeruginosa strain PA-7-Protection conferred by the passive immunization of mice with rabbit anti-sera directed against various fractions from Pseudomonas aerug i nosa Passive protection in size of the challenge Passive protection the IgM-containing rabbit serum. Passive protection the IgG-containing rabbit serum. relation to the dose. of mice provided by fraction of immune of mice provided by fraction of immune Clearance of challenge organisms from mice passively protected by ' immune rabbit serum. ACKNOWLEDGEMENTS I would like to thank Dr. D. Syeklocha, my research supervisor, for her help and criticism during the course of my research, and during the preparation of this thesis, and Dr. J.J.R. Campbell for his expressed interest in my research project. I would also like to thank Mrs. Teresa Walters for the electron microscopy, arid Mr^  B i l l Page for the drawing of'the figures in this manuscript. Lastly, I should like to express my thanks to the faculty, staff and students of this department for their interest and en-couragement throughout the course of this study. INTRODUCTION The characteristics of the immune response to various bacteria has received a great deal of attention in recent years. A humoral antibody response, involving both IgM and IgG immunoglobulins, has been demonstrated for a large number of pathogenic bacteria (Pike and Schulze, 1964; Yoshida and Ekstedt, 1968; Smith, J.W., et_ aj_. 1970). Both active and passive immunization studies have shown that the immune serum is able to protect the host animal against infection by the homologous microorganism; one or the other of the two major immuno-globulin classes hasbeen demonstrated to be more effective in this regard, depending upon the bacterium involved (Yoshida and Ekstedt, 1968; Dolby and Dolby, 1969)- Much of the recent work has been directed toward the development of protective antigenic fractions from cell extracts of various bacteria (Alms and Bass, 1967; McGhee and Freeman, 1970). Studies by Bass and McCoy (1970 and by Schwarzmann and Boring (1971) have shown that a fraction isolated from the slime layer of Pseudomonas aeruginosa induces the formation of antibodies which are protective against infection by Pseudomonas aeruginosa. The object of this investigation was to examine several crude fractions prepared from a strain of Pseudomonas aeruginosa with regard to their abil i t y to stimulate the production of protective antiserum. The classes of immunoglobulin e l i c i t e d , their activities in several \n vitro tests, the duration of the Response, and the relative capacities of the different fractions to evoke antisera capable of protecting mice against challenge infections by homologous and heterologous strains of Pseudomonas aeruginosa were also investigated. LITERATURE REVIEW I. Pseudomonas aeruginosa as a Human•Pathogen In recent years, Pseudomonas aeruginosa strains have approached or.replaced those of Staphylococcus aureus in frequency of occurrence as the causative agents of human infection in the hospital environ-r ment (Huang, et a 1 . 1961; Farmer andHermah, 1969). Patients debil-itated by disease processes, burns or surgery, and individuals on antibiotic therapy a l l seem particularly prone to infection by this organ i sm (Kefa.l ides, et a l . 1964). Victims of cystic fibrosis have been found to be'particular1y susceptible to chronic infections of -the respiratory tract by mucoid variants of common serotypes of Pseudomonas aeruginosa (Doggett, 1969; Diaz et a 1. 1970). Eye infections caused by Pseudomonas aeruginosa strains are not uncommon, nor are urinary tract infections involving this organism (Ay1iffe, et_a_K. 1966; Klyhn and Gor i l l , 1967). Numerous sources of Pseudomonas aeruginosa have been found in hospital surroundings. The organism's a b i l i t y to exist in moist en-vironments is well documented; for example, Pseudomonas aeruginosa has been cultured from soap dishes, hand cream, respirators, sinks, floors and mops (Wahba, 1965: Wormald, 1970). In this context, Emmanoui1idou-Arseni and Kommentaleou (1964) showed that numerous r stra ins of Pseudomonas aerugi nosa, were able to survive in d i st i1 led water held at several different temperatures for at least three hundred days. Findings such as these point to the great d i f f i c u l t i e s s t i l l being encountered in attempting to suppress infections in hospitals. Similarly, the discovery that Pseudomonas aeruginosa is part of the intestinal flora of a small percentage of the population has disclosed another potential source of infection, at least in the hospital environment (Wahba, 1965)• The emergence during the past thirty years of Pseudomonas  aeruginosa as an increasingly frequent human pathogen is no doubt due in part to the concomitant increase in antibiotic therapy (Huang et a 1. 1961; Finland, 1970). Strains of this bacterium not only resist the inhibitory effects of most antibiotics, but also are unaffected by many antiseptics in present use (Emmanoui1idou-Arsehi and Kommen-taleou, 1964; Adair et_ a_J_. 1971). In vitro studies have shown that of the c l i n i c a l l y available antibacterial agents, only polymyxin B, coliston, gentamycin and carbenici11in are effective in inhibiting the growth of Pseudomonas aeruginosa (Hedberg and Mil l e r , 1969; Lindberg et al . 1970); although these antibiotics are used c l i n i c a l l y to combat Pseudomonas infections, reservations'have been placed upon their efficacy, due to solubility and toxicity problems, and to the development of strains which are resistant to these drugs (Smith, C.B. et a l . 1970). An excellent summary of the d i f f i c u l t i e s to be en-countered in antibiotic therapy of Pseudomonas aeruginosa infections can be found in reports by Smith, C.B. et a 1. (1970), Lindberg et a l . (1970) and others in the Symposium on Carbenici11 in (Kirby, 1970). 5 I I. Nature of the Infection The exact means by which Pseudomonas aeruginosa exerts its pathogenicity is not completely understood at present. Under certain conditions, the organism is known to be invasive; moreover, pulmonary involvement and urinary tract infections encountered subsequent to primary invasions in other parts of the body, indicate that Pseudomonas infections often become systemic (Jones, 1970; Jordan et a l . 1970; Smith, C.B. et a 1. 1970). Support for these findings comes from animal experiments which demonstrated that the deaths of mice which had received an intravenous injection of live cells was due to i renal failure caused by lodgement and multiplication of the bacteria in the kidneys (Klyhn and G o r i l l , 1967). However, as yet, insuffi-cient evidence has been presented to attribute the pathological effects of Pseudomonas aeruginosa solely to invasiveness, without any con-sideration of the possible roles of the various cellular and extra-cellular factors produced by this organism. Several extracellular enzymes are known to be produced by strains of Pseudomonas aeruginosa which may aid the organism in its establishment and subsequent growth in the infected s i t e . These include a hemolysin, a lecithinase, one or more proteases, and several others (Berk, 1964; Carney and Jones, 1968). Several of these proteins have been partially purified and shown to be lethal for experimental animals (Berk, 1964; Johnson et a l . 1967). Meinke and coworkers (1970) described localized hemmorhage and necrosis upon 6 autopsy of mice injected with varying concentrations of a partially purified protease with elastolytic a c t i v i t y . Relatively large doses of an enzyme containing fractions obtained from culture supernatant produced an eye exudate, hemmorhage and inflammation of the intestine when injected into mice (Carney and Jones, 1968). Although not a l l of the enzymes are necessarily produced by any one strain, qualitative and quantitative differences in enzyme production by strains of this organism may influence their pathogenicity (Liu, 1964; Carney and Jones, 1968). A further extracellular toxin has also been implicated which is believed to be separate from these enzymes, and which is also lethal for mice (Liu and Hsieh, 1969)- The role of this fraction in the pathological process has yet to be evaluated. The extracellular siime or capsular material has been shown to be toxic for mice (Liu et a 1. 1961; Cetin et a 1. 1965), especially after mild acid hydrolysis, or deoxyribonuc1 ease degradation (Callahan et a l . 1964). However, recent evidence has shown that slime exerted no effect on the v i a b i l i t y of leukocytes in vitro at least (Schwarzmann and Boring, 1971) -The endotoxin moiety of Pseudomonas aeruginosa has been shown to be somewhat similar in chemical composition to those of the Enterobacteriaceae (Michaels and Eagon, 1969; Fensom and Gray, 1969). However, conflicting reports have been published concerning its importance in the overall infectious process (Liu et a l . 1961; Klyhn and G o r i l l , 1967). The protein portion of the molecule is 7 capable of Inducing a Schwartzman reaction, and a pyrogenic response; pri the other hand, the intact complex appears to be relatively non-toxic, since large numbers of whole cells have been injected into mice with relatively l i t t l e effect on the host (Braun and Elrod, 1941 ; Liu et_ aj_. 1961; Laborde and de Fajardo, 1 9 6 5 ) . An important consideration in the evaluation of the role of the endotoxin and of the other extracellular products in the disease'process is the number of bacterial cells actually achieved . in the infected animal. As noted by Roantree (1967) in the case of Salmonella infections, only if the host's defences are overcome sufficiently to allow the presence of a large number of bacteria within the body can one expect to achieve the concentrations of endotoxin, slime, or enzymes which have been shown necessary to produce biological effects. III. Host Defences Against Pseudomonas aeruginosa Bacterial infections are prevented in various ways by the human body. External and internal surfaces are protected by the fatty acid content of the skin, the lysozyme in tears, saliva, and acidic' secretions, a l l of which are mi Idly bactericidal. The resident flora of the body also aids in suppressing the multiplication of potential pathogens. If organisms do penetrate these defences, and begin to multiply within the tissues, the body's inflammatory response may prevent further spread of the invading bacteria. Normal human serum is bactericidal to some bacterial species, including Pseudomonas aeruginosa; in the latter case, this antibacterial activity has been shown to be enhanced at temperatures siightly above 37 C (Muschel et 1969). In addition, complement factors of the serum aid in the phago cytosis of many bacterial species. Pseudomonas aeruginosa is known to stimulate antibody formation in.humans and experimental animals. Normal human serum may contain agglutinins to the organism (Gaines and Landy, 1955); individuals who have survived an acute Pseudomonas infection, as well as those chronically infected by the organism, have been shown to possess elevated antibody titers (Gaines and Landy, 1955; Diaz et a l . , 1970; Young et a l . 1970). This response may persist in humans for some time after recovery from infection (Young et_ a_l_. 1970), although the antibody response to Pseudomonas aeruginosa antigens injected into experimental animals appears to be quite transitory (Laborde and de Fajardo, 1965; Bass and McCoy, 1971). The importance of phagocytosis in determining the outcome of bacterial infections has been the object of a considerable study in recent years (Cohn and Hirsch, 196*5; Rowley et_ aj_. 1968) . Re-sistance to ingestion and destruction by phagocytic cells of the reticuloendothelial system seems to be related to the virulence of the bacterial strain for the animal host (Wells and Hsu, 1970; Nakamura et a l . 1970; Yee and Buffenmeyer, 1970). Surface anti-genic composition, as well as lyt i c substances which may be pro-duced by the microorganisms, can enable bacterial cells to resist 9 phagocytosis and/or post-phagocytic destruction (Cohn and Hirsch, 1965; Roantree, 1967; Medearis et_ a|_. 1968) . In vitro stud ies have shown that rabbit peritoneal macrophages phagocytbse and k i l l Pseudomonas aeruginosa c e l l s , although the slime produced by mucoid variants of Pseudomonas strains was found to inhibit ingestion of the i bacterial cells by the leukocytes (Schwarzmann and Boring, 1971). Phagocytic efficiency may be increased in the presence of serum antibodies specific for the microorganism. Salmonella typhimurium cells have been found to be more susceptible to phagocytosis in vitro in the presence of immune seruirT (Wells and Hsu, 1970); increased phagocytosis of Escherichia colj cells in previously immunized animals has also been reported , (Benecerraf et a l . 1959; Whitby and Rowley, 1959)-Immune serum was found to counteract the antiphagocytic effect of the slime of mucoid Pseudomonas aerug i nosa . var iants (Schwarzmann and Boring, 1971). Thus the correlation between detectable serum antibodies and protection from infection which has been observed for several bacterial species, including Pseudomonas aeruginosa (Jones and Lowbury, .1965; Roantree, 1967; Rowley et a 1. 1968), is in al l likelihood a reflection of the increased efficiency of phagocytosis and intracellular destruction of the organism in the presence of specific antiserum. The liver and spleen appear to be the principal organs of sequest-ration and elimination of bacterial cells injected intraperitoneally or intravenously as well as in natural infections which have become systemic (Benecerraf et a l . 1959). Which of the two is most active In phagocytosing the invading microorganisms seems to be determined by the amount and class of specific antibody present (Benecerraf, et a 1. 1959; Schulkind and Rabins, 1971). The liver appears to sequester the majority of the injected organisms in immune animals; in contrast, enhanced uptake of the organisms by the spleen is seen in non-immune animals; or those in the early stages of the immune response (Schulkind and Rabins, 1971). IV. Therapeutic Measures Because of the obstacles which have been encountered in attempts to combatVPseudomonas aeruginosa infections with antibiotics, much emphasis has been placed upon the development of some form of immuno-therapy to supplement or replace the use of antibacterial drugs. Both active and passive immunization procedures have been considered as possible approaches. The preparation of a polyvalent vaccine or of an immunogen with a protective antigenic determinant common to a l l of the frequently encountered serotypes has thus far met with l i t t l e success, a fact which tends to argue against the development of an active immunization program (de Fajardb and Laborde, 1968; Fisher, et a 1. 1969). Oh the other hand, antiserum specifically prepared against the infecting serotype or serotypes (Lindberg et a 1. 1970) could be passively administered to help counteract already established infections. A potential hazard in this type of treatment, of course, is the possibility of e l i c i t i n g allergic reactions to the foreign serum or serum proteins admin i stered. Whole c e l l s , viable or killed by various methods, cell extracts, culture f i l t r a t e s and the slime layer of Pseudomonas aeruginosa have al l been shown to be immunogenic in experimental animals. Liu and his coworkers (1961) found that the slime layer and whole cells evoked antibodies capable of protecting mice against lethal numbers of live c e l l s . Alexander and coworkers (1966) and Alms and Bass (1967) have shown that antiserum specific for an ethanol-pre-cipitable material in the slime layer is capable of protecting mice against an experimental Pseudomonas aeruginosa infection. Bass and McCoy (1971) recently showed that killed cells or the aIcohol-pre-, cipitated fraction from the slime layer e l i c i t cross-protection against infection by various strains which correlates well with the heat-stable "0" serotypes as defined by Verder and Evans ( 1 9 6 1 ) . This finding is in agreement with an earlier proposal that protection obtained by using whole cells as the immunogen is directed against a part of the slime layer which cannot be removed from the cells (Liu et a l . 1 9 6 1 ) , but is in contrast to the results obtained by Fisher and coworkers ( 1 9 6 9 ) , who found l i t t l e correlation between protective activity of an antiserum and its "0" agglutinin content. Jones (1968) has also found that the antiserum directed against an enzyme containing fraction from culture f i l t r a t e s protects burned mice against colonization by lethal numbers of Pseudomonas aeruginosa c e l l s ; in this case, there was also some cross-protection when strains of different serotypes were used to infect the serum-protected mice. Whole cells of some Gram-negative organisms, as well as their somatic antigens, have long been known to stimulate humoral antibody production. The response to a single injection of organisms or of their "0" antigen has been found to consist mainly of antibodies of the IgM class (Pike and Schulze, 1964; Landy et_ al_. 1965; Fukazawa et_ aj_. 1970); IgG molecules appear in appreciable quantities only late in the immune response, if at a l l . "0" antigens from different strains of Proteus mi rab i 1 i s, however, have been found to evoke either an IgM or an IgG response after a single dose (Smith, J.W. et a 1. 1 9 7 0 ) . Hyperimmunization with whole cells or somatic antigen preparations, on the other hand, e l i c i t s an IgM response, but also leads to earlier formation of, and, larger quantities of IgG immuno-globulin (Pike and Schulze, 1964; Landy et a l . 1965; Bjornson and Michael , 1 9 7 0 ) . The relative a b i l i t i e s of the two major immunoglobulin fractions to prevent infection by bacteria have been compared by a number of investigators. Yoshida and Ekstedt (1968) found that the protective activity of hyperimmune serum against infection by Staphy1ococcus  aureus was associated with the IgM fraction. Dolby and Dolby ( 1 9 6 9 ) , however, demonstrated that IgG was more effective in protecting mice against a lethal intracerebral challenge of Bordetella pertussis than was the IgM fraction of the serum. Antibodies isolated early in the immune response to^Sal'mone Ha typhi murium increased sequestration of the injected organisms by the spleen; "late" antibodies seemed to aid the liver in taking up the organisms (Schulkind and Rabins, 1 9 7 1 ) . However, the protective a b i l i t i e s of these sera against an experi-mental Salmonella infection was not reported. Hyperimmunization of rabbits with the protective antigen isolated from the slime of Pseudomonas aeruginosa evoked a mixed IgM and IgG response; IgG antibodies were found to be more effective, on a weight basis, than IgM molecules in the protection of mice against a lethal intra-peritoneal challenge of 1ive organisms (Bjornson and Michael, 1 9 7 0 ) . No reports concerning the types of immunoglobulins evoked by immuniza-tion with Pseudomonas aeruginosa cells or somatic antigen are known. In conclusion, then, there are a-mumber of factors to consider in attempting to develop an antigen which could be used immunothera-peutically to combat Pseudomonas aeruginosa infections. The amount of antigen given in a single injection has been shown, in studies with Serratia marcescens, to be important in determining the duration of the antibody response (Field et a 1. 1 9 7 0 ) . The type of antigenic preparation administered is known to affect not only the length of the response, but also the class of immunoglobulin evoked (Laborde and de Fajardo, 1965; Smith, J.W. et_ aj_. 1 9 7 0 ) . Bass and McCoy (1971) have found that booster injections of Pseudomonas aeruginosa antigens f a i l to produce antisera of higher titer than that achieved in the original response, but do maintain the antibody level for a longer period of time. Sublethal doses of ful l y virulent live Pseudomonas aeruginosa cells have been shown to evoke a prolonged antibody response (Laborde and de Fajardo, 1965; Young et^ a_l_. 1970) j-but the potential toxicity of this type of vaccine renders it unsuitable for c l i n i c a l use. Perhaps immunization with viable cells of relatively avirulent mutant strains such as the rough Salmonella typhimurium strains used in studies by Germanier ( 1 9 7 0 ) , might also induce a relatively long period of antibody production. The type of immunoglobulin produced in response to a given anti-gen, as well as the amount of cross-reactivity of the protection induced, are also important considerations in the development of a suitable vaccine. Ideally, one would hope to find an immunogenic fraction which is relatively noh-toxic, stimulates the production of high levels of protective antibody, and which exhibits at least some cross-specificity in the protection conferred. MATERIALS AND METHODS 1 . Organisms and Media The organism used in this study was a strain of Pseudomonas  aeruginosa designated PA-7. This strain was obtained from Dr. P.V. Liu, of the Department of Microbiology, University of Louisville, School of Medicine, Louisville 2, Kentucky, in 1968. The original stock culture was maintained in the lyophilized state; once a year, a fresh vial was reconstituted to be used as the working stock. This culture was kept at h C on trypticase soy (BBL) or brain heart infusion (Difco) agar slants and was transferred to a fresh slant every six to eight weeks. At intervals of approximately three months, this working stock was serially subcultured onto human blood agar plates to maintain its virulence for mice, and its slime pro-ducing capacity (personal observation). Pseudomonas aeruginosa strains PA-1 and PA-479 were also used in some experiments. PA-1 was obtained from Dr. P.V. Liu and PA-479 was obtained as a cli n i c a l isolate at the Department of Microbiology, University of British Columbia. For the production of the antigenic preparations, PA-7 was grown in trypticase soy broth (TSB) on a shaking water bath (Metabolyte Water Bath Shaker, New Brunswick Scientific Co., Inc., New Brunswick, N.J.) at 37 C for 12-14 hours. I I. Preparation of Antigens 1. Formalin-killed whole cell vaccine. Cells were washed three times in sterile saline, and resuspended in saline. The optical density at 660 nm was measured with a Beckman Spectronic 20 (Beckman Instruments, Inc., Fullerton, C a l i f . ) . The concentration of cells was then adjusted to 10 O.D. and formalin was added to a concentration of 0.3 percent. The vaccine was left at room tempera-ture for at least 2k hours, after which time s t e r i l i t y tests were carried out. The cells were then sedimented by centrifugation, resuspended in sterile saline, and stored at VC until needed. This preparation will be referred to hereafter as Form Vacc. 2. Heat-stable 1ipopolysaccharide. The method used for the extraction of this fraction was one modified from that of Suzuki and his coworkers (1964). Broth-grown cells were washed several times in sterile saline, then resuspended ' in d i s t i l l e d water to a final concentration of 0.5 gm wet weight of cells per ml. This suspension was heated at 100 C for 90 minutes. After cooling, the cellular debris was removed by.centrifugation, at 10,000 x g_ for 20 minutes. The volume of the supernatant was measured, and solid NaCl was added to obtain a concentration of 0.1 M. Five volumes of 95 percent ethanol were added slowly with s t i r r i n g , and the re-sulting suspension was left at room temperature for 30 minutes. The precipitate was collected by centr ifugation at 15,000 x g_ for 20 minutes, redissolved in 0.1 m NaCl, and reprecipitated twice more with ethanol. The final precipitate obtained was dissolved in 0.1 M phosphate buffer, pH 7-0, containing 20 yg each of deoxyribo-nuclease (Worthington Biochemica1 Corp., Freehold, N.J.) and ribo-nuclease (Calbiochem, Los Angeles, C a l i f . ) ; and 2 yg of MgCl^ per nil. This mixture was incubated at 37 C for one hour, then centri-fuged at 25,000 x g_ for 90 minutes. The pellet was resuspended in d i s t i l l e d water, washed once with d i s t i l l e d water, and stored in the lyophilized state in a desiccator at room temperature. This material was designated later in the text as H-S LPS. 3. Extraction of 1ipopolysaccharide with the phenol-water. The method for the extraction of this fraction was taken from Nowotny, 1969- TSB grown cells were washed several times in saline, then resuspended in d i s t i l l e d water. The cell suspension was heated to 70 C, and an equal volume of warm phenol (70 C) was added. This mixture was maintained at 70 C with stirring for 10 minutes, then cooled and centrifuged at 3,000 x g_ for 30 minutes. The two phases of the supernatant were separated, and the phenol phase was extracted with disti1 led water twice more, as described above. The three water phases were combined, and the volume was reduced by flash evaporation. Exhaustive dialysis of the remaining material against d i s t i l l e d water removed the remaining traces of phenol. Three volumes of cold methanol containing 0.2 percent MgC^ were added to the non-d ia 1 ysabl e material. The resulting precipitate was collected by centrifugation at 3,000 x g for 30 minutes, redissolved in d i s t i l l e d water, and reprecipitated twice with cold methanol without MgCl^- Repeated flash evaporations re-moved the methanol. The material was lyophilized, reconstituted r in 0.1 M phosphate buffer, and incubated with the nucleases as described above for the heat-stable 1ipopolysaccharide fraction. The 1ipopolysaccharide material was collected by centrifugation at 25,000 x g_ for 90 minutes; the pellet was resuspended and washed once in d i s t i l l e d water, and lyophilized for storage.' This pre-paration is designated later as P-WLPS. k. Cell walls. The method followed was essentially that of Bobo and Eagon, 1968. Broth-grown cells were washed several times in saline, resuspended in 0.1 M phosphate buffer, pH 5 . 5 , and passed twice through a precooled French pressure cell (Aminco, Silver Springs, Md.). The resulting suspension was centrifuged at 3,000 x g_ for 10 minutes, and the pellet was discarded. The supernatant was centri-fuged at 15,000 x g_ for 20 minutes. This pellet was suspended in phosphate buffer, pH 7 -0 , with.20 ug of deoxyribonuc1 ease and ribo-nuclease, and 2 yg of MgC^ per ml. After 1-2 hours incubation at 37 C, the cell walls were pelleted by centr ifugation at 15,000 x g_ for 20 minutes, washed twice in pH 7-0 phosphate buffer, twice in d i s t i l l e d water, and lyophilized for storage. 5. Protein-1ipopolysaccharide complex. The method de-scribed by Rogers et a 1. 1969, was followed for the isolation of this fraction. Cell walls isolated as previously described were suspended to a concentration of 1.5 mg per ml In 0.033 M Tris-HCl. buffer, pH 8.0, containing 1 ymole of ethylenediaminetetraacetate (EDTA) per ml. The suspension was incubated at 25 C with stirring for 30 minutes, then centrifuged at 37,000 x £ for 60 minutes at 0 C;, The supernatant was passed through a 0.45 f i l t e r (Millipore Corp., Bed-ford, Mass.), concentrated by lyophi1ization, dialyzed for 96 hours at k C against several changes of 0.033 M Tris HC1 buffer, pH 8.0, and relyophi1ized for storage. This preparation is later called Pr-LPS. III. Experimental Animals All animals used were obtained from the Animal Unit, Faculty of Medicine, University of British Columbia, Vancouver, B.C. White, female, Swiss-bred mice weighing between 20 and 25 gm were used in a l l experiments. White, female rabbits used for immunization with the Pseudomonas fractions weighed 2-3 kg at the start of the immunization schedule. IV. Immunization Procedures Rabbit immunization schedules are shown in Table I. Two routes of injection were used. For intravenous (i.v.) injections, the antigens were diluted to the appropriate concentration in pyrogen-free saline (Baxter Laboratories of Canada, Ltd., A l l i s t o n , Ont.) and injected via the marginal ear vein. For subcutaneous (s.c.) injections, 20 Tab1e I. Rabbit Immunization Schedule for the Various Antigenic Preparations from Pseudomonas aeruginosa Strain PA-7-lay of iect ion H-S LPS Dosea P-W LPS Pr-LPS Cel 1 Wal Is Form. Vacc. 0 10 10 10 10 5 x 10 8 k .10 10 - - 1 x 10 9 5 - - 10 10 -8 20 20 - - 2 x 10 9 10 - - 20 20 -12 20 20 - - 2 x 109 15 - - 20 20 -16 50 50 - - -20 50 50 50 50 -2k 100 100. - - -25 - - 50 50 -30 - - 100 100 -kk - 2 x 1 03t> - - -57 50- - - -63 - - - - 2 x 10 9 79 - - - 50 -96 - - 2 x 103b - -a- Antigen dose, in ug, except in the case of the formalin-killed vaccine, where the dose refers to the number of cells injected. 1^  Subcutaneous injection of the antigen in Freund's complete ad-juvant. All other injections were intravenous: Abbreviations: H-SLPS, heat-stable 1ipopolysaccharide; PTWLPS, phenol' water 1ipopolysaccharide; Pr-LPS, protein-1ipopolysaccharide; Form. Vacc, formal in-ki 1 led vaccine. the saline solution was mixed with an equal volume of complete Freund's adjuvant (Difco) and 0.2 ml of the emulsion were injected into each of five sites on the shaved area of the animal's back. V. Ant ? sera 1. Col 1ection.and Storage Rabbits were bled from the marginal ear vein or by cardiac puncture. Blood samples were stored overnight at k C to allow for clot retraction. The sera were separated, cleared of any residual red blood cells by low speed centrifugation, then heated to 56 C for 30 minutes to inactivate the complement. Sera were stored in small volumes at -20 C. 2. Determination of Antibody Titer All sera were tested routinely for antibody activity by. the passive hemagglutination and hemolysin tests; many were also tested for their bacterial agglutinin content. Passive hemagglutination and hemolysin tests were carried out using the Microtiter dilution technique (Cooke Engineering Co., Alexandria, Va.) . 25 yl samples of serum were serially .diluted with 25 yl of saline in the dilution plates. 25 yl of a 2.5 percent suspension of 1ipopolysaccharide-coated sheep red blood cells were then added to each well in the passive hemagglutination assay; for passive hemolysis, 25 yl of a 1/30 dilution of guinea pig complement were added to each antiserum dilution, and the plate was incubated at 37 C for 30 minutes before the coated sheep red blood cells were added. Plates were incubated at 37 C for 2 hours, and at k C for 18 hours before the results were read. The highest antiserum dilution showing at least 50 percent hemagglutination or hemolysis of the added red blood cells upon visual inspection was taken as the end point of the ti t r a t i o n . . Appropriate saline and normal red blood cel 1 .tcontrol s were included in al l assays; normal rabbit serum controls were occasionally included. Washed sheep red blood cells were coated with alkali-modified heat-stable 1ipopolysaccharide according to the method of Neter and his coworkers (1956). Lipopolysaccharide was dissolved in saline to a concentration of 500-1,000 yg per ml.. The pH was adjusted to 8.5 -9.0 with 0.2 N NaOH. After heating the solution at 56 C for 30 minutes, the pH was readjusted to 7-2 with 0.2 N HC1. An equal volume of 5 percent sheep red blood celIs was added, and the mixture incubated at 37 C for 30 minutes with occasional shaking. The red cells were then collected by low speed centrifugation, washed twice in saline, and resuspended to a concentration of 2.5 percent, in saline. Formalin-killed vaccine was used as the antigen in bacterial agglutinin titrations. Serial two-fold dilutions in saline were made of the serum under test. The vaccine was diluted with saline to equal in opacity the third tube of the Brown opacity tube series (Burroughs, Wellcome and Co., London, Eng.); plate counts demonstrated q that this opacity is equal to 2-3 x 10 PA-7 cells per ml. A volume of this antigen equal to that of the antiserum di1utions,was added to each tube, and to a saline control tube. The highest antiserum dilution in which visible agglutination was detectable was taken as the bacterial agglutinin t i t e r . 3. Fractionation of Antisera a) Dissociation of Macroglobulin with 2-Mercaptoethanol The procedure for this step was taken from Nowotny (1969). Equal volumes of the serum and of 0.2 M 2-mercaptoethanol (in saline) were mixed and allowed to stand at room temperature for one hour. This mixture was then used in the place of serum in the passive hemagglutination and hemolysin and bacterial agglutination tests as described above. b) Gel Filtration Whole rabbit serum was applied in 2-3 ml samples at 4 C to a 2.5 x kS cm Sephadex G-200 column (Pharmacia, Uppsala, Sweden). Flow was in the upward direction, at a rate of 0.15 ml per minute. The buffer was 0.05 M phosphate buffer,.pH 7-3i containing 2.2% NaCl and 0.2% sodium azide. Fractions of 1.5 ml were collected and assayed for protein content by measuring the optical density at 280 nm with a Beckman DBG (Beckman Instruments Inc., Fullerton, C a l i f . ) . In order to achieve a better separation of IgG and IgM immuno-globulins, further fractionation of the antibody-containing protein peaks were attempted. The leading front of the f i r s t peak eluted (see Figure 8); was concentrated to a volume of 2-3 ml by ultra-f i l t r a t i o n (Amicon Ultrafiltration C e l l , Amicaon Corp., Lexington ,Mass.) , and refractionated on Sephadex G-200 under the conditions described above. The leading front of the protein peak thus obtained (Figure 9) was again concentrated by u l t r a f i l t r a t i o n to 2-3 ml. Ring tests and gel diffusion tests were performed on this fraction with goat anti-rabbit-y-gjobulin (Meloy Laboratories, Springfield, Va.) to check for the presence of any contaminating IgG antibodies, c) Ion exchange Chromatography The method used was. taken from the procedure of Nowotny (19&9). Whole rabbit serum which had been dialyzed against 0.005 M sodium phosphate buffer for 48 hours at 4 C, was applied in 5 ml samples to a 2.5 x 45 diethylaminoethyl (DEAE)-cel1ulose column, also at 4 C. The serum was eluted with an 800 ml continuous gradient, in which the starting buffer was 0.005 M sodium phosphate buffer, pH 7.0 and the final buffer was 0.05 M Nah^PO^ in 0.05 M NaCl. The flow rate was one rri per minute. Fractions of 5 ml were col 1 ected and assayed for protein content by measuring the optical density at'280 nm with a Beckman DBG spectrophotometer. Protein peaks were pooled, each was concentrated to the original serum volume by.ultra-f i l t r a t i o n , and was tested for antibody content by the passive hemagglutination test. V1 . Passive Protection Studies The method adopted for these studies was modified from those used by Bass and McCoy (1971) and Jones and his coworkers (1971). Mice that had received an intraperitoneal injection of 0.2 ml of whole or fractionated rabbit serum, or of saline, were challenged by the same route k hours later with various numbers of viable bacteria suspended in 10 percent TSB in saline. The serum fractions were dialysed against saline before use, and all serum dilutions were made in pyrogen-free sa1ine,. Groups of 10 mice were used for each dilution tested. The challenge organisms were grown in TSB on a shaking water bath at 37 C for 6 hours, sedimented by centrifugation, and resus-pended to the original volume in 10 percent TSB-saline. All further dilutions of the organisms were also made in 10 percent TSB-saline. A plate count was performed on the washed bacterial suspension in all experiments to determine the number of organisms injected. Mortality was recorded in most experiments only for the 72 hour period following injection of the cha11enge bacteria; in some cases, however, deaths were recorded for periods of up to 2 weeks post-challenge., VII. Clearance Studies To determine the effect of the rabbit antiserum on the persist-ence of the injected organisms in passively protected mice, those animals which had survived the 72 hour test period were sacrificed at various intervals thereafter, and samples of their heart blood and peritoneal washings were plated on trypticase soy agar to check for the presence of viable organisms. The 1iver and spleen of each animal were also removed aseptically, cut into small pieces, and spread on trypticase soy agar plates. The plates were incubated at 37 C for -24 hours , after which time they were inspected for typical colonial morphology. Representative colonies were tested by slide agglutination with rabbit anti-Pseudomonas serum to ensure that the organisms isolated were PA-7. To confirm the validity of the assay method used, in some ex-periments samples taken from the mice were divided; one half was treated as described above, and the other half was added to TSB in tubes, and incubated at 37 C for at least 48 hours. Growth in these tubes was plated, and the resulting colonies were tested as above. VIII. In Vitro tests 1. Bactericidal Assay The method for this assay was developed from one described by Bjornson and Michael (1970). Cells were grown in TSB on a shaking water bath at 37 C for 6 hours, sedimente.d by centrifugation, washed once in sterile saline, and resuspended in saline to the original volume of the culture. The concentration of cells per ml of this suspension was determined by a surface plate count. The cell suspension was diluted by a factor of 10 ^, and various volumes of the diluted material were added to mixtures of whole or fractionated antiserum and guinea pig complement (Hyland).. Normal rabbit serum and saline controls were also included. The mixtures' were incubated at 37 C for one hour, then 0.1 ml volumes were plated in duplicate for counting. 2. Immunodiffusion Tests Double diffusion plates were prepared according to the method of Campbell et a 1. ( 1 9 6 4 ) . The plates contained 0.85 percent lonager No. 2 (Oxoid) in borate-sa1ine, pH 8; 0.1 percent Merthiolate' (Eli L i l l y and Co., Indianapolis) was added to the borate-saline solution, but the trypan blue stain was omitted. Antigen preparations and antisera were diluted in borate-saline, and 0.1 ml volumes were added, to each wel 1 . The plates were incubated in a moist chamber at 37 C for at least one week, and were observed daily for the development of precipi-tation lines. When the development of the lines was complete, the plates were rinsed with several changes of saline to remove unreacted protein and antigen, sealed and stored at I C until photographed. 3. Quantitative Precipitation Test The amount of antibody in each type of purified fraction was quantitatively determined by the precipitation test. Equal volumes of undiluted serum fractions from serum prepared against the cell wall fraction, and of saline dilutions of heat-stable 1ipopolysaccharide were mixed, and incubated at 37 C for 1-2 hours. The tubes were then refrigerated for 48-72 hours; after this time, the tubes were centrifuged in the cold at 2500 rpm, and the supernatant carefully removed and tested for antigen or antibody excess by ring tests. The precipitated material was washed twice in cold saline to remove any unreacted protein, and dissolved in 0.1 N NaOH to the original volume of the mixture. The protein content of these solutions was determined by the method of Lowry and coworkers (1951). iX. Electron Microscopy Cell wall suspensions which had been treated with deoxyribo-nuclease and ribonuclease as described above were suspended in dis-t i l l e d water and frozen until needed for electron microscopic studies. In addition, samples of the cell wall preparation were subjected to further enzymatic treatments for comparative purposes. One set of cell walls was incubated at 37 C for 3 hours in 0.05 M sodium phosphate buffer, pH 8.0, with 500 yg/ml of trypsin (Calbiochem, Los Angeles, C a l i f . ) , sedimented by centrifugation, washed several times in d i s t i l l e d water, and stored as an aqueous suspension at -20 C. The other sample was suspended in 0.1 M sodium acetate buffer to which 500 yg/ml of lipase (Miles Laboratories, Inc., Elkhart, Ind.) had been added, and was incubated at 37 C for 3 hours. These cell walls were then washed as described above, and frozen. Cell wall preparations were fixed and stained by several dif-ferent methods. One set was fixed in 2.3 percent glutaraldehyde in phosphate buffer for 30 minutes, then in 1 percent 0s0, in phosphate buffer for 15 minutes, and lastly in uranyl acetate in sucrose-acetate buffer for 30 minutes (Pease, 1964; Silva et a l . 1968). Another sample was fixed in OsO^, and post-fixed in uranyl acetate (Silva et_ aj_. 1968) . A third set was fixed in glutaraldehyde, and post-fixed in OsO^ (Pease, 1964). Samples were dehydrated in ethanol and propylene oxide, and embedded in Epon. Sections were stained with a saturated alcohol solution of uranyl acetate for one minute, rinsed with d i s t i l l e d water, and stained for 2 minutes with lead citrate (Fahmey, 1967)- After drying, the preparations were examined in a Phillips 300 electron microscope at 60 KV. RESULTS AND DISCUSSION The purpose o f t h i s s t u d y was t o p r e p a r e d an a n t i g e n i c f r a c t i o n from PA-7 w h i c h would be c a p a b l e o f s t i m u l a t i n g t h e p r o d u c t i o n o f p r o t e c t i v e a n t i s e r u m i n e x p e r i m e n t a l a n i m a l s . A l t h o u g h o t h e r w o r k e r s have been s u c c e s s f u l , by t h e i n j e c t i o n o f whole c e l l v a c c i n e s , i n s t i m u l a t i n g an a g g l u t i n i n r e s p o n s e (Laborde and de F a j a r d o , 1965) and p r o t e c t i v e serum (Jones et_'aj_. 1971) i n m i c e , we f a i l e d t o evoke m a r k e d l y e l e v a t e d p a s s i v e h e m a g g l u t i n i n l e v e l s i n t h e s e a n i m a l s . The r a b b i t , however, was found t o be more s u i t a b l e f o r t h e p r o d u c t i o n o f a n t i s e r u m c o n t a i n i n g h i g h l e v e l s o f p a s s i v e h e m a g g 1 u t i n a t i n g a n t i b o d i e s . I. A n t i b o d y Response t o t h e A n t i g e n i c P r e p a r a t i o n s The p a s s i v e h e m a g g l u t i n i n and h e m o l y s i n r e s p o n s e s t o e a c h o f t h e p r e p a r a t i o n s i n j e c t e d i n t o r a b b i t s a r e shown i n F i g u r e s 1 - 5-A l l o f t h e p r e p a r a t i o n s t e s t e d were found t o be immunogenic w i t h t h e t y p e o f i n j e c t i o n s c h e d u l e used (see T a b l e I o f the M a t e r i a l s ' and Methods) and no t o x i c e f f e c t s were noted i n t h e r a b b i t s a t any t i m e . Humoral a n t i b o d y l e v e l s e l i c i t e d by a l l o f t h e p r e p a r a t i o n s , w i t h t h e e x c e p t i o n of t h e p h e n o l - w a t e r e x t r a c t , were q u i t e h i g h (peak p a s s i v e h e m a g g l u t i n i n t i t e r s o f 1/2048 or g r e a t e r ) . The most 1 0 2 0 3 0 T I M E . ( D A Y S ) 4 0 5 0 Figure 1. Rabbit antibody response to cell preparations from Pseudomonas aeruginosa strain PA-7. Rabbits were injected with the cell wall preparation according to the schedule shown in Table I (Materials and Methods). Titers shown refer to A, passive hemolysin and B, passive hemagglutinin levels. Symbols: Q Q, rabbit 1; 0 — — 0 , rabbit 2; a - f i r s t injection of series. 1 1 i r . 1 2 - ® 1 0 -8 -Figure 2. Rabbit antibody response to heat-stable 1ipopolysaccharide preparation from Pseudomonas aeruginosa strain PA-7-Rabbits were injected with the heat-stable 1ipopolysaccharide preparation according to the schedule shown in Table I (Materials and Methods). Titers shown refer to A, passive hemolysin, and B, passive hemagglutinin levels. Symbols: £ > — • , rabbit 1; 0 — 0 , rabbit 2; a - f i r s t injection of series. 2 0 3 0 T I M E ( D A Y S ) 4 0 F i g u r e 3 Rabbft antibody response to phenol-water extracted lipo-polysaccharide from Pseudomonas aeruginosa strain PA-7-Rabbits were injected with the phenol-water lipopoly-saccharide preparation according to the schedule shown in Table t (Materials and Methods). Titers shown refer to A, passive hemolysin and B, passive hemagglutinin levels, Symbols: • • , rabbft 1; 0 — 0 , rabbit 2; a - f i r s t injection of series. Figure k. Rabbit antibody response to protein -1ipopolysaccharide from Pseudomonas aeruginosa s t r a i n PA'-7.. Rabbits were injected with the protein -1ipopolysaccharide preparation according to the schedule shown in Table 1 (Materials and Methods). T i t e r s shown refer to A, passive hemolysin and B, passive hemagglutinin l e v e l s . Symbols: D-—• , rabbit 1; 0 — 0 , rabbit 2; a - f i r s t i n j e c t i o n in s e r i e s . T T I M E ( D A Y S ) Figure 5. Rabbit antibody response to f o r m a l i n - k i l l e d vaccine of Pseudomonas aeruginosa s t r a i n PA-7. Rabbits were injected with f o r m a l i n - k i l l e d c e l l s according to the schedule shown in Table I (Materials and Methods). Titers shown refer to A, passive hemolysin, and B, passive hemagglutination l e v e l s . Symbols: D — • , rabbit 1; 0 — 0 , rabbit 2, a - f i r s t i n j e c t i o n in s e r i e s . prolonged response of high titer antibodies was stimulated by the cell wall preparation (Figure 1). The reason for the poorer re-sponse to the phenol-water extract (Figure 3) is not known, although the relatively low protein content of this preparation in comparison with that of the other 1ipopolysaccharide preparations may be a factor.' Other workers '(Whang et a 1. 1971) have also noted that phenol-water extracted 1ipopolysaccharides are .poor immunogens, and have mentioned the lack of aggregation of the molecules as a contributing factor. Although the antibody.responses to the recall injections (Table I) are not shown in Figures 1 "5, the intravenous boosters of the cell wall and heat-stable 1ipopolysaccharide preparations, and of the formalin-killed vaccine resulted in rapid rises in the titers of both the passive hemagglutinatfng and hemolysing anti-bodies. Peak titers equalled or exceeded those achieved during the earlier hyperimmunization regime; only the cell wall preparation, however, stimulated a response which persisted for longerthan two weeks after the booster injection. The recall injections of the phenol-water extracted 1ipo-polysacchar ide and the protein-1ipopolysaccharide preparations were administered subcutaneously in Freund1s complete adjuvant in order:to determine the effect of this type of injection upon the response to a relatively good and ,a relatively poor immunogen. Neither preparation stimulated the production of detectable antibody f o r approx imate ly s i x weeks a f t e r t h e i n j e c t i o n . A f t e r t h i s t ime , the l e v e l of serum a n t i b o d i e s rose s l ow ly f o r about a month in the r a b b i t s which had been i n j ec ted w i t h p ro te i n-1 i popo l ysaccha r i de ; the l eve l obta ined was c l o s e to the peak t i t e r o f the e a r l i e r response, arid remained constant fo r over two months. No a p p r e c i a b l e r i s e in a n t i b o d y - t i t e r was.noted o v e r . a 2 month p e r i o d , , in the serum of r a b b i t s i n j ec ted w i th a booster dose of the pheno l -Water e x t r a c t in F reund 1 s ad juvan t . Because of the low l eve l of an t ibody syn thes i s s t imu la ted by the phenol-water e x t r a c t , no f u r t h e r work was' done on t h i s serum. The ant iserum prepared aga ins t "the c e l l wa l l f r a c t i o n was used in a l l subsequent t e s t s , s i nce i t was fel.t.. t ha t . t h i s f r a c t i o n would con ta i n an t igens most r e p r e s e n t a t i v e of those that would be en -countered in the na tu ra l s i t u a t i o n . A n t i s e r a s p e c i f i c fo r the heat -s t a b l e 1 i.popol ysacchar ide and pro te i n-1 i popol ysacchar ide p r e p a r a t i o n s , and f o r the f o r m a l i n - k i l l e d vacc ine were a l s o used fo r comparat ive purposes.. I I . . P r o p e r t i e s of the Ant igen P r e p a r a t i o n s . The average y i e l d of the a n t i g e n i c f r a c t i o n s c a l c u l a t e d as a percentage of the dry weight of the c e l l s used in t h e i r p repa ra t i on was 2-3 percent fo r the h e a t - s t a b l e and phenol-water 1 ipopol.y-sacchar ide p r e p a r a t i o n s , 0.5 percent fo r the p ro te in-1 i popb l ysac -cha r i de f r a c t i o n , and 8 percent f o r t h e c e l l w a l l s . Thus, not on ly does the cell wall preparation appear to be the most effective immunogen, but also i t . is the most efficient "fraction to produce in terms of yield per unit weight of c e l l s . In addition, toxicity studies have indicated that the.lethal doses for the heat-stable' 1 i pbpol ysacchar ide, protei n-1.i popo.l ysacchar ide, and cell wall pre-parations are all greater than 8 mg per kg of mouse body weight; however, this dose level was found to be toxic in a l l cases, with diarrhea, eye "exudates, a hunched position, and immobility of the affected animals being noted. Therefore, it was concluded from these studies that the amounts of each preparation used for the immunization did not approach the lethal dose; furthermore, no obvious adverse affects on the rabbits were noticed, even when they received the recall injections. The reason for the low toxicity of Pseudomonas aeruginosa c e l l s , cell walls and 1 ? popol ysacchar ide preparat ions is" not known. Fensom and Gray (l969) showed that the chemical composition of 1 the 1ipopolysaccharide molecule of Pseudomonas aeruginosa is basically similar to that of the Enterobacteriaceae. The cell walls were also found to'resemble, qualitatively and quantitatively,the cell walls of other Gram-negative bacteria (Clarke et a 1. 1 9 6 7 ) . However, for the Enterobacteriaceae, LD50 levels of 0.1 - 0.2 mg of endotoxin (lipopolysaccharide) per kg of body weight have been reported for mice (Davis et a 1 . 1967); since our preparations are only partially iPurified, however, caution must be used in attempting to compare, these values. Immunodiffusion analyses of antigenic preparations are pre-sented in Figure 6. Schematic representations are presented in addition to photographs, since some of the lines disappeared very quickly, and others are very faint. Figure 6 A shows the reaction of an,t iserum prepared against the heat-stable 1ipopolysaccharide preparation with different concentrations of heat-stable 1ipopolysaccharide, phenol-water 1ipopolysaccharide, and protein-1ipopolysaccharfde; 1ines of identity between the heat-stable 1ipopolysaccharide and the phenol-water 1ipopolysaccharide (Wells, 1 S 2, k S 5); and between the heat-stable 1ipopolysaccharide and the protein-1ipopolysac-charide (Wells, 3 & 4 ) , indicate that there are common antigenic determinants "in these preparations. Figure 6 B-D show that each of the three 1ipopolysaccharide preparations is able to react with antisera prepared against the heat-stable 1ipopolysaccharide, the protein 1ipopolysaccharide, and the cell walls; although antiserum prepared against the formalin-killed vaccine reacts only with the prbtein-1ipopolysaccharide here, other studies have demonstrated that it is also able to react with the heat-stable and phenol-water 1ipopolysaccharide preparations (Figure 6 E ). The cross-reactivity seen in these figures is to be expected because of the nature of the antigen fractions; however, the lack of correlation between antigenicity and immunogenic!ty in the case of "the phenol-water extract is interesting. It may be that the antigen shared by the F i g u r e 6 A . Figure 6. Immunodiffusion a n a l y s i s of IIpopolysaccharide preparations from Pseudomonas aeruginosa. Plates contain 0.35 percent lona<jcr No.2 In borate-sal Ine b u f f e r , pH 8.0. Antigens and an t i s e r a were added to the wells In 0.1 M volumes. Plates were Incubated at 37 C for 7 - 1 n days. A. Antiserum (AS) was prepared against heat-stable 1Ipo-polysacchar Ide. Well 1 contains 1 mg/nl of heat-stable 11popolysaccharIde; well 2, 1 mg/ml of phenol-water 1Ipo-polysacchar Jde; well 3, 1 mg/ml of proteln-lIpopoly-sacchar Ide; wells 4-6, 0.5 mg/ml of heat-stable l l p o -polysaccharMe, phenol-water I Ipopolysaccharlde, and proteln -1Ipopolysaccharlde r e s p e c t i v e l y . B-jD. V e i l 1 contains antl-heat-stable I Ipopolysaccharlde antiserum; well 2, a n t i - c e l l wall serum; well 3» a n t l -protcln-lIpopolysaccharlde serum; well 4, antl-formalIn-k l l l e d vaccine antiserum; well 5, normal rabbit serum; well 6, s a l i n e . Antigens (Ag) are heat-stable 1Ipopoly-saccharlde (B), proteln -1IpopolysaccharIde (C), and nhenol-water 1IpopolysaccharIde (D), at 1 mg/ml E. Antiserum (AS) v/as prepared against the formalIn-klI led vaccine. I. A n t l i c n ( A g ) Is heat-stable 1Ipopolysacchar-lde at a concentration of 1 mg/ml. l l . Antigen (Ag) Is phenol-water IIpopolysaccharIde at a concentration of 1 mg/ml. Symbols: /wv\ , D l l f u s e l i n e ; , sharp l i n e . © © F i g u r e 6 B . © © © F i g u r e 6 C . protein -1ipopolysaccharide and the heat-stable 1Ipopolysaccharide, but missing from the phenol-water preparation (Figure 6 A , Wells, 3 - 5 ) is the immunogenic moiety. It is of course, also possible that the two lines at well k of Figure 6 A merely represent d i f f e r e n t degrees of aggregation of the molecule, and that only the more aggregated species of the heat-stable 1ipopolysaccharide . molecule (outer 1ine) is capable of acting as an immunogen (see . Whang .et/al_. 1971). Electron micrographs of the c e l l wall preparations are shown in Figure 7. Hubert and his coworkers (1971) have published electron micrographs showing the appearance of the c e l l wall and c e l l membrane in cross-sections of whole c e l l s of Pseudomonas  aeruginosa. They showed the external t r i p l e layered c e l l wall and the t r i p l e layered plasma membrane which are typical of Gram-negative organisms. Kellogg and coworkers (1971), in a study of the p a r t i c u l a t e f r a c t i o n s of Neisseria gonorrheae, show cel1 wall fragments which resemble c l o s e l y those shown in Figure 7 B of our preparations.. . * A t r i p l e layered structure with two electron dense layers (as described by Hubert et a l . 1971) is seen only in those pre-parations in which the c e l l walls were treated with the nucleases, . followed by 1ipase digestion (Figures 7 E & F ) . Nuclease treat-ment alone resulted in the i s o l a t i o n of a structure containing three electron dense layers (Figures 7 A S B) while trypsin appeared Figure 7- Electron micrographs of cell wall preparations from Pseudomonas aeruginosa. Isolated cell wall preparations were fixed and stained as outlined in the Materials and Methods. A. Fixed in osmium tetraoxide, post-fixed in uranyl acetate. The various sizes of cell wall fragments can be noted, x 15,250. B. Fixed in osmium tetraoxide, post-fixed in uranyl acetate. Note the three electron dense layers, x 58,000. C7D. Fixed in 2.3 percent glutaraldehyde, then post-fixed in one percent osmium tetraoxide. Note the loss of organized structural layers. C- x 73,200; D- xi51,000. E-F. Fixed in 2.3 percent glutaraldehyde, post-fixed in one percent osmium tetraoxide. Note the loss of one electron dense layer. E- x 73,200; F- x 151,000. 46 to have simply caused a disorganization of the structural layers (Figures 7 C £ D). It was decided, on the basis of these photographs, to employ in further studies the cell wall preparation, which had undergone only nuclease digestion. This was felt to be the most represent-ative antigenic sample, since trypsin appeared to. d i sorgan i ze the natural structure of the cel1 envelope, while it was not possible, without further chemical analysis, to determine whether the electron-dense layer removed by the lipase digestion, was part of the cell membrane or of the cell wall. III. Fractionation of Rabbit Antisera a) Sensitivity to 2-Mercaptoethanol The sequence of appearance of 2-mercaptoethanol sensitive (2-MES) and 2-mercaptoethanol resistant (2-MER) passive hemag-glutinins in the serum of rabbits immunized with the cell wall preparation (Table l) is shown in Figure 8. Aliquots of the serum samples taken during the course of the response (Figure 1) were treated with 2-mercaptoethanol as described in the Materials and Methods in order to determine the proportion of 2-mercapto-ethanol resistant antibody at various stages during the response. The 2-mercaptoethanol resistant activity was found in appreciable quantities only late in the response to the hyperimmunizing series of injections; in this regard, the results are very similar to data 20 4 0 6 0 TIME (DAYS) 8 0 \ b 100 Figure 8. Detection of 2-mercaptoethanol resistant antibody activity in rabbit serum. Aliquots of antiserum from rabbits injected with the cell wall preparation according to the schedule shown in Table I (Materials and Methods) were mixed with equal volumes of 0.2 M 2-mercaptoethanol, incubated for one hour at room temperature, and used as the serum source in the passive hemagglutination test. Original titers are shown in Figure 1. obtained by Landy and his coworkers (1965) and by Pike and Schulze (I36h) in analogous studies with other Gram-negative organisms. The recall injection, on the other hand, was found to stimulate not only an increase in the rate of overa11 antibody synthesis, but also in the rate of increase of the 2-mercaptoethanol resis-tant fraction of the antibody a c t i v i t y . In addition to the above studies, antisera directed against the other "three antigenic fractions were also treated .with 2-mercaptoethanol to determine the" percentage of 2-mercaptoethanol-sensitive and 2-mercaptoethanol resistant antibody a c t i v i t y . A large percentage of the passive hemagglutinatirig activity in sera obtained late in the response to al l of these antigenic fractions was also found to be resistant to 2-mercaptoethanol treatment. Although sensitivity and resistance to 2-mercaptoethanol may not be a completely -valid measure of the IgM or IgG content of an immune serum, the results obtained in these studies were helpful in estimating the periods during the antibody responses which would yield the most IgM or IgG antibody upon fractionation of the sera. b) Fractionation of Rabbit Serum Proteins by Gel Filtration and Ion Exchange Chromatography -Attempts were made to separate the IgM and IgG . immunoglobu1in fractions of whole rabbit serum by passage of serum samples through Sephadex G-200. A typical elution p r o f i l e , expressed as the optical densities of the fractions measured at 2 8 0 nm is shown in Figure 9 -Each of the three protein peaks, when pooled arid concentrated to" the original serum volume, was found to contain some passive hema-gglutinating a c t i v i t y . A large proportion of this activity was located in the f i r s t protein peak, and was" mainly 2-mercaptoethanol sensitive antibody. Interfacial tests performed with goat-anti-rabbit yglobulin and the concentrated material from the shaded area of the f i r s t protein peak (Figure 9 ) demonstrated that there was some IgG globulin coritaminating this fraction. Accordingly, this material was refractionated on Sephadex G - 2 0 0 , and the elution profile illustrated in Figure 10 was obtained. The leading front of the protein peak (shaded area) was again concentrated, and ring tests with goat ant i-rabbi tvy-jg lobul i n did not detect any remaining IgG in the fraction. Passive hemagglutination tests performed on samples of this fraction, with and without prior treatment with 2-mercaptoethanol, indicated that not al l of the antibody activity was sensitive to the action of 2-mercaptoethanol (Table h); however, this material was used as the IgM fraction in subsequent tests. Since the IgG immunoglobulins appeared to be eluted from the Sephadex G-200 column in a broad band, whole rabbit serum which had been shown to be rich iri 2-mercaptoethanol resistant antibody activity was chromatographed on DEAE-cellulose, and the IgG was eluted by means of a continuous gradient. A typical elution pattern 1 2 3 COLUMN VOID VOLUMES Figure 9. Elution profile of whole rabbit serum from Sephadex G-200. A 2 - 3 ml sample of whole rabbit serum, taken early in the response, was applied to a 2.5 x 45 cm Sephadex G-200 1column at 4 C. The flow rate was 0.1 ml per minute and the column void volume was 70 ml. 3 ml fractions were collected, and the optical density at 280 nm measured with a Beckman DBG-spectrophotometer. Fractions within the cross-hatched area were pooled and concentrated for refractionation. 52 COLUMN VOID VOLUMES Figure 10. Elut ion profile of Sephadex G-200. IgM-containing serum fraction from The f i r s t protein peak eluted when whole serum was passed through Sephadex G-200, was concentrated by u l t r a f i l t r a t i o n and repassaged through Sephadex G-200 at k C. The column void volume was 70 ml. Fractions within the shaded area of the protein peaks were pooled, concentrated to a volume of 2 - 3 ml, and used as the IgM fraction. is shown in Figure 1 1 . Material under, the f i r s t protein peak was pooled and concentrated to the original serum volume; interfacial tests with goat anti-rabbit y~91obu1in demonstrated the presence of IgG molecules in the fraction, and passive hemagglutination tests showed that al1 of the antibody activity was resistant to 2-mercaptoethanol treatment. This material was used as the IgG fraction in the subsequent tests. IV. Passive Protection Tests a) Whole Serum Passive protection tests were performed with mice, using rabbit antisera prepared against the four antigenic preparations (heat-stable 1ipopolysaccharide, protein-1ipopolysaccharide, cell walls and formalin-killed vaccine) to determine their capacity to e l i c i t antisera protective against a lethal challenge of PA-7. 8 The challenge dose of 2 0 L D 5 0 (3 x 1 0 cells) was chosen after preliminary experiments showed that this dose was lethal to a l l mice injected with normal rabbit serum, whereas at least 50 percent of groups of. mice protected by low dilutions of immune rabbit serum survived the challenge. The data presented in Table II shows that each of the four types of whole antiserum was able to protect mice against this challenge dose. In most cases, experiments were terminated after 72 hours, since simultaneous virulence tests dem-onstrated' that a l l of the unprotected animals died within 48 hours 54 C O L U M N V O I D V O L U M E S Figure 11. E l u t i o n p r o f i l e of whole rabbit serum from the DEAE-cellulose column. A 5 ml sample of whole rabbit serum was applied to a 2.5 x 45 DEAE-cellulose column at 4 C. The serum was eluted with an 800 ml continuous gradient from 0.005 M sodium phosphate b u f f e r , pH 7-0, to 0.05 M Nah^PO/j in 0.05 M NaCl. The column void volume was 120 ml; b ml f r a c t i o n s were c o l l e c t e d and monitored at 280 nm for protein content. Table II. Protection conferred by the passive immunization of mice with rabbit antisera directed against various fractions from Pseudomonas aeruginosa.3 Immun iz fng Ant i serum Ant i serum Cha11enge Percent Agent Titer D f1ut ion Dose Surviva1 (LD 50) PHA BA Heat-stable 1/1024 1 /640 1/2 20 67 L i popolysac- 1/10 20 58 char ide Protei n -1i po- 1/4096 1/640 1/2 20 92 pol ysacchar i de 1/10 20 80 (serum from day 30) Protein-1ipo- 1/512 1/5120 1/2 20 85 polysacchar ide 1/10 20 69 (serum from day' 1/50 20 23 160) 1/100 20 8 Cell Walls 1/1024 1/640 1/2 20 93 (serum from day 1/10 20 73 11) 1/20 20 66 Cell Walls 1/4096 1/640 1/2 20 77 (serum from day 1/50 20 64 3 D Forma 1 i T i - 1/2048 1/1280 1/1 20 100 ki lied 1/10 20 43 vacc'i ne 1/1024 1/256 1/10 20 30 _b - - 1/1 20 0 Mice were injected intraperitonea11y with 0.2 ml of rabbit serum d i l -uted in s t e r i l e , pyrogen-free saline. Four hours later, viablePA-7 cells suspended in 10 percent TSB-saline were injected intraperiton-e a l ^ . Deaths were recorded for a 72 hours period post-challenge. Normal rabbit serum was injected in place of the immune serum as described above. of challenge;' moreover, very few deaths in the groups of serum-prOtected animals occurred after the i n i t i a l three-day period post-challenge. The results show that the degree to which an antiserum sample can be dfluted before a significant loss in the protection afforded is incurred, can be estimated on the basis of the passive hema-gglutinating antibody content-of the serum (with the exception of the antiserum prepared against the formalin-killed vaccine). In addition, the timeduring the response from which the antiserum was obtained did not seem to affect the relationship between passive hemagglutinin content and passive protection a b i l i t y . This is evident from the data for serum prepared against cell walls and protein-1ipopolysaccharide. The lower protective capacity of serum directed against the formalin-killed vaccine is puzzling; it might be suggested that formalin adversely affects the imrriuno-genicity of the protective antigen alone, without alteririg other immunogenic structures In the cell responsible for the stimulation of agglutinating and passive hemagglutinating a c t i v i t i e s . This seems unlikely, however, in view of the relationship between passive hemagglutination and protection noted above. Some experiments also involved the injection of different sizes of challenge doses -into mice passively protected as previously de-scribed. Table III shows results obtained when mice were given various numbers of cells after injection of antisera prepared against Table 1(1. Passive protection in relation to the size of the challenge dose.a Immunizing, Titer of Cha 11 e'nge Percent Agent Ant i serum Dose (LD 50) Surv iva1 PHAb BA Cell Wall 1/204 1/32 20 77 50 6b 100 0 Heat-stable 1/102 1/64 10 90 . 1i popoly- 20 58 saccharide 1/52 1/32 20 80 100 8 Mice were passively immunized with 0.2 ml immune rabbit serum diluted in saline, then challenged with viable PA-7 suspended in 10 percent TSB-salirie. Deaths were recorded for a 72 hour period after challenge. • Original serum titer was divided by the serum dilution performed. Cell wal1 antiserum was diluted 1/20, heat-stable lipopoly-saccharide sera diluted 1/10. cell walls or heat-stable 1ipopolysaccharide. As expected, the degree of protection afforded by a given antiserum was inversely related to the size of the cha11enge•dose." These data demonstrate that all of ,the four types of antisera can afford protection against moderately large challenges (20 - 50 LD 50) of PA-7, but are of l i t t l e use against very severe challenges (100 LD 5 0 ) . It was also of,some interest to determine whether the immune rabbit serum prepared against PA-7 antigens could provide pro-tection for mice against challenge by two other strains of Pseudomonas  aerugi.nosa. Bacterial agglutination tests with the formalin-killed vaccines of strains PA-7, PA-1 and PA-479 as antigens,. showed that none of the four major antigenic preparations used stimulated the production of antibodies which agglutinated PA-1 or PA-479 vaccines. Moreover, passive protection studies in which antiserum directed against the cel1 wall preparation was used to protect the mice, showed that antiserum dilutions which protected 60 - 75 percent of the mice chal1enged with PA-7 protected less than 20 percent of the mice challenged with PA-1' or PA-479- Fisher and his coworkers (1969) have shown a lack of correlation between agglutination and protection among different strains of Pseudomonas aeruginosa; how-ever, our results seem to agree more closely with the findings of Bass and McCoy ( 1 9 7 1 ) , who showed that cross-agglutination and cross-protection were analogous. b) Fractionated Serum IgM and IgG serum fractions'pur ified as described in the Materials 59 and Methods were also used in passive protection studies with mice.. The results presented in Tables IV and V (IgM and IgG .fractions respectively) demonstrate that both immunpglobulin types provide passive protection against infection by PA-7. When the passive hemagglutination titers of the fractions were considered, neither of the two fractions from cell wall or protein-1ipopolysaccharide appeared to be more efficient than the other; the data obtained from sera prepared against the heat-stable 1ipopolysaccharide and formalin-killed vaccine, however, indicate that IgG globulins in these sera provide better protection than do the IgM globulins, Bjornson and Michael (1970), working with immunoglobulin fractions obtained from the serum of rabbits immunized with purified muco-polysaccharide from the slime layer, also showed that the IgG fraction offered superior protection to mice against infection by Pseudomonas. aerug inosa. Because of the well-known superiority of IgM globulin in participating in agglutination-type reactions, the increased efficiency of protection afforded by the IgG fraction of immune serum may simply reflect a much larger number of IgG molecules than there are IgM molecules in a given.volume of their'respective fractions. The precipitin' results obtained with IgM and IgG serum fractions from a sample of anti-cell wall serum (day kk) support this hypo-thesis, since there was found to be substantially greater amount of IgG than IgM antibody, on a molar ratio, in the two fractions, which Table IV. Passive protection fraction of '. immune of. mice provided rabbit serum.3 by the IgM-cOntaining Immunizing Agent Titer of IgM Fract ion Di 1 ution of Fract ion Challenge Dose (LD 50) Percent Surv ival PHA 2-MePHAb Cel 1 1/64 1/1 20 60 Wa 1 1 s 1/10 20 60 Protein- 1/16 1/2 20 60 1i popoly- 1/10 20 20 sacchar ide Heat-stable 1/512 1/32 1/2 20 10 1ipopoly- 1/10 20 10 sacchar ide 1/50 20 o Forma 1 in- , 1/256 1/64 1/2 10 60 kilied 1/10 10 60 vaccine Mice were injected intraperitoneally with 0.2 ml of the IgM-con-taining fraction o f immune rabbit serum diluted in s t e r i l e , pyrogen-free saline. Four hours later, viable PA-7 cells suspended in 0.2 ml of 10 percent TSB-saline were injected intraperitoneally, Deaths were recorded for a 72 hour period post-challenge. Aliquots'of the serum fractions were incubated with equal volumes of 0.2 M 2-mercaptoethanol immediately before use in the passive hemagglutination test (PHA) as described in the Materials and Methods, Table V. Passive protection of mice provided by the' I gG-containing fraction of immune rabbit serum.3 Immun iz ing Titer of IgG Dilution of C ha 11enge Percent Agent Fract ion Fract ion Dose (LD 50) Surv iva1 PHA 2-MePHAb Cel1 wal1s 1/256 1/256 1/2 20 83 1/10 20 70 1/30 20 50 1/1024 1/1024 1/1.0 20 100 1/25 20 67 1/50 20 25 Protein 1/512 1/512 1/10 20 67 1i popoly-1/50 20 0 saccharide Heat-stable 1/32 1/32 1/2 20 44 1ipopoly-1/10 20 56 sacchar ide Forma 1i n- 1/32 1/32 1/2 20 78 killed 1/10 20 78 vacc i ne Mice were injected intraperitoneal1y with 0.2 ml of the IgG- contain-ing fractions diluted in s t e r i l e , pyrogen-free saline. Four hours later, the animals received the challenge organisms suspended in 0.2 ml of 10 percent TSB-saline. Deaths were recorded for a 72-hour period after the challenge injection. Aliquots of the serum fractions were incubated with equal volumes of 0.2 M 2-mercaptoethanol immediately before use in the passive hemagglutination test (PHA) as described in the Materials and Methods afforded similar degrees of protection to mice against a Pseudomonas  aerug i nosa'infect ion. Figure 12 shows the precipitin curves ob-tained when heat-stable 1ipopolysaccharide was precipitated by these serum fractions. At the zone of equivalence, 66 yg of IgG antibody was precipitated while only 16 yg' of IgM antibody was precipitated at the equivalence point; since the molecular weight of the IgM globulins is approximately 6 times that of IgG molecules, the above values show that the IgG serum fraction contains 15 " 20 times as many immunoglobulin molecules per unit volume, as does the IgM fraction. Thus it seems reasonable to suppose that a larger number of immunoglobulins in the IgG fraction may be" responsible for the enhanced protective effect noted generally for the IgG fractions. V. Clearance Stud ies Preliminary clearance tests were performed on mice which had survived (for 72 hours) an intraperitoneal challenge of viable cells numbering less.than, or equal to, the LD50. The spleen, liver peritoneal f l u i d , and heart blood were assayed for the presence of viable PA-.7. Previous studies have shown that the majority of bacterial cells injected intraperitoneally into mice are sequestered and cleared by.the liver and spleen (Benecerraf et_ a]_. 1959). Organisms isolated from the peritoneal fluid in our studies were considered to indicate persistence and probably multiplication of the injected bacteria, while Pseudomona s aerug i nosa 2 5 5 0 75 j j g A N T I G E N A D D E D 100 Figure 12. P r e c i p i t a t i o n a n a l y s i s of p u r i f i e d IgG and IgM serum f r a c t ions. Serum prepared against the c e l1 wall preparation was fr a c t i o n a t e d on Sephadex G-200 and DEAE-cel1ulose as described in the Materials and Methods to obtain p u r i f i e d immunoglobulin f r a c t i o n s . These f r a c t i o n s were used in the p r e c i p i t a t i o n tests as described in the Materials and Methods. Symbols: O—-O, IgG; • Q, IgM. in the heart blood was thought to indicate inadequate clearance of the injected organisms;, allowing for seeding of the bacteria from the peritoneal f l u i d , and possibly from the.spleen and l i v e r , with genera 1ized "spread throughout the body. Most of these control mice were found to be cleared of injected cells" by 3 ~ 5 days after the cha11enge. The passive protection data presented earlier had shown that most deaths in the immune serum-protected groups occurred within the f i r s t three day post-challenge period; it was thus considered of interest to determine whether or not the challenge organisms were cleared from the survivors of this 72 hour period. Table Vf presents the results obtained when mice passively protected by immune rabbit sera were sacrificed at different time intervals after the challenge injection, and their organs assayed for PA-7. Al-though there" is a certain amount of variation in the clearance patterns among, different'animals within the same group, definite trends were seen. Experiments involving cell wall antisera were interesting because of the faster rate of clearance of organisms from animals protected by serum taken later in the response (day hk) , as opposed to that observed,in mice protected by antiserum taken at an earlier date (day 11). Figure 8 showed that no 2-mercaptoethanol resistaht'antibody was detectable in serum obtained on day 11, whereas approximately 50 percent of the passive hemag-glutinating antibody was resistant to the action of 2-mercaptoethanol Table VI. Clearance of challenge organisms from mice passive protected by immune rabbit serum.3 Immunizing Anti serum Cha11enge k Agent D i1ut ion Dose C1earance U (LD 50) Days Organ Post Cha1lenge Spleen L i ver PF HB Cell 1/2 20 3 0/5 0/5 3/5 5/5 Wal 1 6 k/k k/k \lk 3/k (serum from 1/20 20 3 0/k 0/k Z/k k/k day 11) 6 k/k k/k .3/k k/k Cal 1 1/2 20 3 k/k k/k k/k k/k Wal 1 (serum from 1/10 20 3 k/k k/k k/k k/k day kk) Protein-1ipo- 1/2 20 5 3/5 2/5 k/5 5/5 polysacchar ide 8 2/5 2/5 3/5 k/5 Formalin-ki1 led 1/1 20 7 k/k k/k k/k k/k vacc i ne 1/2 20 7 0/k 0/k NDC ND 1/2 10 7 k/k k/k ND ND Organs and fluids were removed from survivors of the 72-hour test period iii the passive protection tests, and qualitatively assayed for the presence of PA-7 c e l l s . Reported as the number of the particular organ free of viable PA-7, over the number of mice assayed. N.D. - not determined Abbreviations: PF - peritoneal f l u i d ; HB - heart blood in the serum obtained on day kh. It is unci ear ,why the later anti-serum promoted an increased rate of clearance, particularly since Bjornson and Michael (1970) have reported that IgM molecules appear to be at least as efficient as IgG globulins in an opsonophagocytos assay. This data does correlate well, however, with passive pro-tection studies involving the purified immunoglobulin' fractions (Tables IV and V) in which the IgG fractions were found generally to offer better protection. Data from experiments in which antiserum prepared against the formalin-killed vaccine was used as the source of protecting antibodies (Table Vl) would appear to indicate that both the size of the challenge dose and the antiserum dilution affect the rate of "clearance of the injected bacterial c e l l s , suggesting that there is an optimal ratio of organisms to antibody molecules within the body of the mouse for maximum clearance. The reason for the poor clearance of PA-7 from mice protected by serum prepared against the protein-1ipopolysaccharide fraction, in spite of the good protective abi1fty of this serum, is not known It is possible that this serum is lacking in specific opsonins, and simply exerts a delaying action on the lethal activity of the in-jected bacteria; an effect of this sort was noted by Laborde and de Fajardo (1969) when some of a group of mice which had been pro-tected by immune rabbit "serum prepared against whole cell vaccines died 10 - 20 days after challenge with Pseudomonas aeruginosa. The poorer clearing potential of this serum may also have been due to a lack of bactericidal a c t i v i t y . Bjornson and Michael (1970) and Muschel et_ aj_. (1969) have reported that some strains of Pseudomonas aeruginosa are sensitive to a complement dependent bactericidal activity in normal and immune serum. However, using the methods outlined by these workers, we were unable to demonstrate significant bactericidal a c t i v i t y , even with undiluted whole serum (prepared against the cell wall fraction). Thus, we were not able to test the hypothesis proposed above, and thus unable to offer an explanation for the lack of clearance of injected organisms in the mice protected by serum prepared against the protein-1ipopolysacchari GENERAL DISCUSSION Four antigenic fractions, namely the heat-stable 1 ipopoly-sacchar ide, protein-1ipopolysaccharide, and cell wall preparations, and the formalin-killed vaccine, were prepared from a strain of Pseudomonas aeruginosa and compared, not only in terms of their a b i l i t y to stimulate serum antibodies, but also in terms of their capacity to e l i c i t the production of protective antiserum. All four antigens were found to be immunogenic in rabbits, although differences in the level and duration of the response elicited were noted. In addition,.antisera prepared in rabbits against a l l four fractions were found to be protective for mice challenged with the homologous strain. Studies by Jones and his coworkers (1971) have demonstrated that antiserum taken from mice three days after immunization with a fraction from the culture f i l t r a t e of Pseudomonas aerug i nosa were able to protect mice against challenge by the homologous strain. No agglutinating antibody activity was demonstrable in this serum, although the protective activity could be removed from the serum by absorption with bacterial cells of the homologous strain. It seems likely then, that the protection afforded by the early mouse serum was due to antibody, but that the titer was too low to be detected by the bacterial agglutination test. In fact, because the protective factor was associated with the macro-globulin serum fraction, Jones and coworkers proposed that early IgM production was responsible for the protective effect. Bass and McCoy (1971) also demonstrated that the protective a b i l i t y of a serum was directly related to its' ant i-Pseudomonas ant ibody content. The results presented in this study show a direct relationship between the antibody content of a serum, and its protective a b i l i t y ; moreover, this antibody activity can be most easily and sensitively measured by means of the passive hemagglutination test. There is some question as to the mechanism by which immune serum exerts its protective effect and also as to which of the two major immunoglobulin classes is the more important in this regard. IgM globulin molecules have been reported to be more e f f i c i e n t , on a molar basis, in in vitro tests such as opsonophagocytosis, complement-dependent bactericidal reactions, and agglutination-type reactions (Pike and Chandler, 1969; Bjornson and Michael, 1970), and thus might be expected to be more" efficient in promoting the elimination of injected organisms by the reticuloendothelial system. However, at the same time, the IgG antibodies appear to be better able to protect'mice against experimental bacterial in-fections (Dolby and Dolby, 1969; Bjornson and Michael, 1970). One explanation which has been offered for this anomaly is that the smaller IgG molecules are able to diffuse more rapidly.than IgM globulins, and thus enter the circulation much more quickly to help counteract the spread of organisms from the infection s i t e , (Bjornson and Michael, 1970). Our results lend support to this hypothesis, since they indicated that serum containing a high pro-portion of 2-mercaptoethanol resistant antibody activity was more effective in eliminating injected organisms than was serum taken earlier in the response and containing mainly IgM antibody. Landy and his coworkers (1965), working with SaImonella  ent e r i t i d i s a n t igens in rabbits, have reported that, whereas a sing1e - injection of antigen stimulated serum antibody production, a hyperimmunizing series of injections was necessary to stimulate an IgG response. The data presented in this report also shows that IgG antibodies make up a significant portion of the antibody re-sponse to the cell wall antigenic fraction (and seemingly to the other antigenic preparations as well) only late in the response to the series of injections; recall injections, however, stimulated a much more rapid rise in the IgG t i t e r . On the basis of the results obtained by Bjornson and Michael (1970), and upon consideration of our data which suggests that, for two of the four types of antisera at least, IgG globulins may be more protective, it would appear that antigens capable of stimulating the production of IgG antibody may be desirable for further studies. All four of our antigenic fractions could thus be considered to be useful antigens, since a l l were found to stimu-late the production of both IgM and IgG antibodies in rabbit serum; in addition, a l l four preparations are relatively non-toxic for experimental animals: The cell wall fraction might be considered to be the most comp1ete antigen, rendering it useful for comparative purposes with the soluble antigenic preparations. In fact, immunodiffusion analysis has indicated that there is at least one common antigen in a l l of these preparations. That this antigen may be identifiable with the heat-stable 1ipopolysaccharide fraction which coats onto sheep red blood cells is suggested by the ab i l i t y of a 11 four1 anti-serum types to react with this antigen. It is as yet unclear, though, whether or not this common antigen is protective, and also, what, if any, is its relationship to the protective antigen of Pseudomonas aeruginosa which can be isolated from the slime layers (Alms and Bass, 1967)• BIBLIOGRAPHY Adair, F.W., S.G. Geftic and.J. Gelzer. 1971. Resistance of Pseudomonas to quaternary ammonium compounds. Appl. Microbiol. 21_: 1058-1063-Alexander, J.W., W. Brown, H. Walker, A.D. Mason and J.A. Moncrief. 1966. Protection studies done with various fractions of Pseudomonas aeruginosa. Surg. Gynec. Obstet. 123.: 965"977. Alms, T.H. and J.A. Bass. 1967- Immunization against Pseudomonas  aeruginosa. I. Induction of protection by an alcohol-precipitated fraction from the slime layer. J. infect. Dis. 177: 249-256. 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