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Vaccines and antisera against Gram-negative bacilli
Journal of Hospital lizfection (1981) 2, 105-111
LEADING
Vaccines
ARTICLE
and antise;;c;fginst
Gram-negative
The increasing frequency and number of deaths due to infections caused by Gramnegative bacilli (GNB) are a major problem in most large U.K. hospitals today. The bacteria causing these infections belong to the families Enterobactericeae (Eschericia cola’, Klebsiella aerogenes, Enterobacter cloacae, Proteus spp., Sewatia marcescens) and Pseudomonadaceae (Pseudomonas aeruginosa) and they attack patients who are already ill with cancer, respiratory damage, renal or hepatic diseases or patients who have been compromized by burning, neutropenia or by surgical and other invasive procedures. The limited success of control measures and of antibiotics in the treatment of infections due to highly resistant Gramnegative bacilli has led to an interest in immunoprophylaxis as a possible alternative or complementary form of treatment. At first the diversity of antigenic types found among the opportunistic GNB makes the task of preparing vaccines and antisera seem daunting. There are 76 different capsular types of K. aerogenes, 16 serological types of Ps. aeruginosa and known strains of E. coli exhibit 92 ‘K’ antigens and 148 ‘0’ antigens. It is not surprising, therefore, that research workers have looked for protective antigens common to more than one species of GNB to try and reduce the number of vaccines needed to achieve wide protective cover.
Lipopolysaccharides
(LPS) and core glycolipids
(CGL)
One of the most immunogenic components found in the cell walls of all GNB are lipopolysaccharides (LPS), but at high dosage LPS are toxic to man and animals causing intravascular coagulation, low blood pressure, renal cortical necrosis and lethal endotoxic shock (Braude, Douglas and David, 1973). In 1959 Freedman investigating the immunogenic properties of LPS found that serum from rabbits made tolerant to LPS from one species of bacteria would passively immunize rabbits against LPS from another bacterial species. This heterologous protection was explained by Luderitz et al. (1966) by the discovery of a core LPS possessing common antigens shared by endotoxins from antigenically unrelated species of GNB. Antisera against the core glycolipid (CGL) gave protection against both endotoxin (Tate, Douglas and Braude, 1966) and lethal GNB challenges (Chedid et al, 1965). Later McCabe et al. (1977) working with a CGL from an Re chemotype mutant of Salmonella minnesota composed solely of 2-keto-3-deoxyoctonate 0195~6701/81/020105
+ o,
$or.oo/o
0
105
1981 Academic
Press
Inc.
(London)
Limited
106
R. J. Jones
(KDO) and lipid A, found it afforded significant active and passive protection against challenge by K. aerogenes, PT. morganii and Salmonella typhimurium. The lipid A fraction on its own showed no protective activity and KDO has not been tested on its own for protective activity as it proved difficult to refine. Although antibody to CGL of the Re mutant was almost as effective as type-specific antibody (antibody against the whole bacterial cell) in offsetting challenges with endotoxin it was inferior to type-specific antibody in protecting against live bacterial challenges. It was found that antibody against CGL of Re mutant produced a 160-fold increase in the number of K. aerogenes required to produce an LD,, whereas immunization with type-specific antibody against K. aerogenes induced a lO,OOO-fold increase in resistance to challenge (McCabe et al., 1973). Further evidence that GNB share a common protective antigen unrelated to their ‘0’ antigenic determinants was reported by Braude et al. (1977) who found that rabbits immunized with antisera against CGL of an E. coli J5 mutant were protected against death after intravenous administration of endotoxins from Salm. abortusequi and Ser. marcescens. Although antibodies against LPS and CGL were shown to protect animals against experimental GNB infections (Sanford, Hunter and Souda, 1962; Kaijser and Olling, 1973) it proved difficult to confirm these findings in humans infected with GNB (Zinner and McCabe, 1976). High titres of ‘0’ specific antibody against the patients’ own infecting strains did not correlate with decreased frequency of shock or death in bacteraemia (McCabe, Kreger and Johns, 1972). It was found subsequently @inner and McCabe, 1976) that the haemagglutination test used for detecting antibody against LPS measured preferentially antibody in the IgM fraction of serum. Once IgM was removed from the patients’ serum samples by pre-treatment with 2-mercapto-ethanol, then antibody against LPS measured by the haemagglutination test in the IgG fraction of serum correlated with improved survival rate in patients infected with GNB, Any consideration of the mechanism of protection by antisera against common protective antigens of GNB must take into account the opsonic and antitoxic properties of the antisera. Antisera against LPS facilitates the removal of GNB in viva by enhancing the phagocytic activity of the reticula-endothelial system (RES). Benacerraf et al. (1959) f ound that the phagocytosis of E. coli in vivo by RES was directly proportional to the amount of antibody against E. co& LPS. The RES also exhibits cross-reacting phagocytic activity as shown by the accelerated clearance of E. coli 04 from the circulation of agranulocytic rabbits treated with antisera against CGL of E. cola’ J5 mutant (Ziegler et al., 1973). Winkelstein and leucocytes Drachman (1974) sh owed the importance of active polymorphonuclear to patients in the fight against bacteraemia due to GNB, an increased incidence of infection by GNB was correlated with a depressed level of polymorphs and impaired functional ability of polymorphs. Although decreased bactericidal activity in polymorphs is often found in patients with bacteraemia caused by GNB (Koch, 1972), Weinstein and Young (1976) suggest that decreased activity of polymorphs is only part of the explanation, the other part being that patients who are susceptible to GNB bacteraemia lack opsonins.
Vaccines
and antisera Capsular
against
Gram-negative
bacilli
107
polysaccharides
Sometimes the possession of a capsular antigen is sufficient to enable GNB to resist opsonization (Dijk et al., 1980) showing that inherent characteristics of bacteria also play a part in their virulence to patients. Polysaccharide capsular antigens are shared by many species of bacteria, E. coli, K. aerogenes and Streptococcus pneumoniae and antibodies against these polysaccharides act as opsonins (Young and Stevens, 1977). Of the 15 pneumonococcal antigens present in contemporary vaccines at least 12 are similar to antigens found in capsular types of K. aerogenes and some are known to be similar to E. coli antigens, as determined by precipitin reactions (Heidelberger and Nimmich, 1976). Since E. coli and K. aerogenes are the two main species of bacteria responsible for bacteraemia caused by GNB (Stamm, Martin and Bennett, 1977) i t may be worthwhile considering vaccination with pneumococcal vaccine as a possible method of overcoming GNB bacteraemia even if the cross-protection following pneumococcal immunisation extends to only some of the strains of E. coli or K. aerogenes. Ribosomal
vaccines
Vaccines based on LPS are highly immunogenic but can be toxic. To avoid the potential toxicity of LPS vaccines, studies have been made with the non-toxic ribosomal vaccines. Youmans and Youmans (1966) were the first to discover that ribosome rich fractions of Mycobacterium tuberculosis were immunogenic and since then these findings have been extended to a number of other bacterial species, Ps. 1978). aeruginosa, Neissel-ia meningitidis, Salm. typhimurium, etc. (Lieberman, There are conflicting reports concerning the role of active substances in ribosomal vaccines. Immunogenicity has been attributed to highly purified RNA (Venneman, 1972), protein attached to RNA (J 0h nson, 1973), glycoprotein or mucopolysaccharide (Houchens and Wright, 1973) or to LPS contaminant (Hoops et al., 1975). Treatment of ribosomal vaccine with ribonuclease or trypsin had no effect on immunogenicity of the vaccine suggesting that neither RNA nor protein were essential for immunogenicity of the vaccine. Current opinion has opted for LPS contaminant as the main immunogenic ingredient of ribosomal vaccines (Lieberman, 1978) as pure ribosomal fractions make weak immunogens and ribosome fractions contaminated with LPS are strongly immunogenic. From a practical viewpoint ribosomal vaccines suffer from a major disadvantage in that they only induce protection against the strain of bacteria from which they were made. Pseudomonas
vaccines
and immunoglobulins
Pseudomonas aeruginosp, the most invasive of the opportunistic GNB is infamous for its ability to become resistant to many topical and systemic treatments. The uncertainty of controlling pseudomonas infections with some contemporary treatments instigated the production of several vaccines and antisera against Ps. aeruginosa infections which have been tested in patients with burns, cystic fibrosis, acute myeloid leukaemia and cancer. Early pseudomonas vaccines were monovalent (Feller et al., 1964) but as bacteria from any of the 16 different serological types of
108
R. J. Jones
Ps. muginosa can cause infection, then multivalent vaccines were produced. A protective antigen (OEP) common to all 16 serotypes of Ps. aeruginosa is being investigated in animals by Abe, Tanamoto and Homma (1977) and looks promising but vaccines made from exotoxin A (Walker et al., 1979) and from ribosomes (Lieberman, 1978) have restricted protectivity, the former fails to prevent bacterial proliferation while the ribosomal vaccine induces low-grade strain-specific protection. In patients the most widely tested pseudomonas vaccines are Pseudogen (ParkeDavis, heptavalent vaccine) and PEV-01 (Wellcome Research Laboratories, 16valent vaccine). Both vaccines were extracted from the cell walls of Ps. aeruginosa, Pseudogen was extracted with phenol (Hanessian et al., 1971), PEV-01 with an EDT.A/glycine mixture (Miler et al., 1977) from a representative strain of each of the 16 serological types of Ps. aeruginosa. Although both vaccines are highly immunogenic, they vary in their toxicity for man. Pseudogen at protective doses produced minor local and systemic reactions (Alexander and Fisher, 1974) while PEV-01 produced no adverse reactions (Jones et al., 1976). In a trial of Pseudogen in 322 patients with burns greater than 20 per cent of their body surfaces (Alexander and Fisher, 1974), the degree of protection induced by Pseudogen was directly related to the dose of vaccine given. None of the patients given 25 ug of Pseudogen/kg died from pseudomonas septicaemia while 12.5 per cent of lower dosed vaccinees died and 14.7 per cent of unvaccinated control patients died with pseudomonas septicaemia. Young, Meyer and Armstrong (1973) vaccinated patients with leukaemia, lymphoma and with solid tumours using varying doses of Pseudogen. They noted a statistically significant improvement in the survival of vaccinated patients, a result confirmed in other trials by Haghbin, Armstrong and Murphy (1973) and Pennington et al. (1975). Patients with cystic fibrosis showed decreased coughing for several months after treatment with Pseudogen but Ps. aeruginosa was not eradicated from the sputum (Pennington, 1979). Controlled trials of PEV-01 in burned patients in New Delhi have shown the value of immunoprophylaxis against Ps. aeruginosa in the saving of lives of patients, with burns greater than 15 per cent of their body surfaces (Jones, Roe and Gupta, 1978, 1979 and 1980). In New Delhi where the risk of death from pseudomonas septicaemia is high, 40.6 per cent (13/32) unvaccinated adults died while only 6.6 per cent (2/30) of adult vaccinees died. In a trial at Birmingham burns unit where pseudomonas septicaemia occurs less frequently than at New Delhi, vaccinated patients showed no Ps. aeruginosa in their blood cultures, had increased levels of antipseudomonas antibody and showed enhanced phagocytic activity against pseudomonas compared with unvaccinated controls. In 1980, Jones, Roe and Gupta found that an immunoglobulin, made from plasma of human volunteers vaccinated with PEV-01 given prophylactically to burned patients on arrival at the hospital prevented death of all 18 passively immunized children compared with a mortality of 21 per cent (9/42) in the unimmunized control children. Pseudomonas immunoglobulin is a potent weapon in the fight against Ps. aerugitiosa where this organism poses a major threat to patients’ lives. The protec-
Vaccines
and antisera
against
Gram-negative
bacilli
109
tion given by immunoglobulin seems to be effective against only strains of Ps. aerugilzosa. Fisher (1977) found in experiments with animals that immunoglobulin raised in human volunteers against Pseudogen gave no protection against challenge by E. c&i, Ent. cloacae, PT. mirabilis nor K. aerogenes, but there was some protection against Ser. maxescens. Comment
Much of the work withvaccines and antisera against GNB is at an experimental stage but the pseudomonas vaccines and immunoglobulin show how successful immunological treatments against GNB could be. The way forward should be to produce vaccines against GNB that raise opsonins and restrict the spread of the bacteria. Where this approach is impracticable immunoglobulins, raised in healthy volunteers which are rich in opsonins and antitoxins, should be used prophylactically or therapeutically. Certain epidemiological studies are required. The immunologist needs to know which patients are at greatest risk of developing each type of GNB infection. Do patients become infected with more than one type of GNB? Can high risk groups of patients who are potential candidates for immunotherapy be recognized? When during hospitalization do infections with GNB occur? Answers to these questions together with a continuing search for protective antigens against GNB should obtain maximum benefit from immunological treatments in the future. R. J. Jones Medical Research Council Unit, Birmingham Accident Hospital References Abe, C., Tanamoto, antigen (OEP)
K. & Homma, J. Y. (1977). Infection protective of Pseudomonas aeruginosa and its chemical
Journal of Experimental
property of the common composition. Japanese
Medicine 47, 393-402.
Alexander, J. W. & Fisher, M. W. (1974). I mmunisation against pseudomonas infection after thermal injury. Journal of Infectious Diseases 130, 152-l 58. Benacerraf, B., Kivy-Rosenberg, E., Sebestyen, M. M. & Zweifach, B. W. (1959). The effect of high doses of X-irradiation on the phagocytic, proliferative and metabolic properties of the RES. Journal of Experimental Medicine 110, 49-64. Braude, A. I., Douglas, H. & David, C. E. (1973). Treatment and prevention of intravascular coagulation with antiserum to endotoxin. Journal of Infectious Diseases 128, (Supplement), 5157-5164. Braude, A. I., Ziegler, E. J., Douglas, H. & McCutchan, J. A. (1977). Antibody to cell wall glycolipid of Gram-negative bacteria: induction of immunity to bacteraemia and endotoxaemia. Journal of Infectious Diseases 136, (Supplement), 5167-5173. Chedid, L., Parant, M., Parant, F. & Boyer, F. (1968). A proposed mechanism for natural immunity to enterobacterial pathogens. Journal of Immunology 100, 292-301. Dijk, W. C., Verburgh, E. & Tol, R. (1980). Interaction of phagocytic and bacterial cells in patients with bacteraemia caused by Gram-negative bacteria. j’ournal of
hfectious Feller,
Diseases 141, 441-449.
I., Burgess, V., Callahan, W. & Waldyke, J. (1964). Use of vaccine and hyperimmune serum for protection against pseudomonas septicaemia. Journal of Trauma 4,451-459. Fisher M. V. (1977). A polyvalent human 7 globulin immune to Pseudomonas ueruginosa: passive protection of mice against lethal infection. Journal of Infectious Diseases 136,
S181-186.
R. J. Jones
110
Freedman, H. H. (1959). Passive transfer of protection against lethality of homologous and heterologous endotoxins. Proceedings of the Society of Experimental Biology and Medicine
102, 504-506. Haghbin,
M.,
Armstrong,
D. & Murphy, M. L. (1973). Controlled in children with acute leukaemia.
Pseudomonas aeruginosa vaccine 32, 761-769. -
prospective trial of Cancer (Philadelphia)
Hanessian, S., Regan, W. D. & Haskell, T. H. (1971). Isolation and characterisation of antigenic components of a new heptavalent pseudomonas vaccine. Nature 229,209-210. Heidelberger, M. H. & Nimmich, W. (1976). Immunochemical relationship between bacteria belonging to two separate families pneumococci and klebsiella. Immunochemistry 13, h7-80 -, --.
Hoops, P., Prather, L. J., Berry, J. & Ravel, een in effective ribosomal vaccines
immunity
13, 1184-l 192.
J. M. (1976). Evidence for an extrinsic immunofrom Salmonella typhimurium. Infection and __
Houchens, D. P. & Wright, G. L. (1973). Immunity to Salmonella typhimurium infection: characteristics of antigens in active protection. Infection and Immunity 7, 507-511. Johnson, W. (1973). Ribosomal vaccines. Infection and Immunity 8, 395400. Jones, R. J., Roe, E. A. & Gupta, J. L. (1978). Low mortality in burned patients in a pseudomonas vaccine trial. Lancet 2, 401-403. Jones, R. J., Roe, E. A. & Gupta, J. L. (1979). Controlled trials of a polyvalent pseudomonas vaccine in burns. Lancet 2, 977-983. Jones, R. J., Roe, E. A. & Gupta, J. L. (1980). Controlled trial of pseudomonas immunoglobulin and vaccine in burned patients. Lancet 2, 1263-1265. Jones, R. J., Roe, E. A., Lowbury, E. J. L., Miler, J. M. & Spilsbury, J. F. (1976). A new pseudomonas vaccine: preliminary trial in human volunteers. Journal of Hygiene 76,
429-439. Kaijser,
B.
&
Olling,
S. (1973).
Experimental haematogenous pyelonephritis response and its protective capacity.
Escherichia coli in rabbits : the antibody Infectious Diseases 128, 41-49.
due
to
Journal of
Koch,
C. (1974). Acquired defect in bactericidal function of neutrophil granulocytes during bacterial infection. Acta Pathologica Microbiologica Scandinavia (B) 82, 439447. Lieberman, M. M. (1978). Pseudomonas ribosomal vaccines: preparation, properties and immunogenicity. Infection and Immunity 21, 76-86. McCabe, W. R., Bruins, S. C., Craven, D. E. &Johns, M. (1977). Cross-reactive antigens: their potential for immunisation induced immunity to Gram-negative bacteria. Journal of Infectious Diseases 136, (Supplement), Sl61-S166. McCabe, W. R., Greely, A., Di Genio, J. & Johns, M. A. (1973). Humoral immunity to type-specific and cross-reactive antigens of Gram-negative bacilli. Journal of Infectious Diseases 128, (Supplement), S284-S289. McCabe W. R., Kreger, B. E. & Johns, M. (1972). Type specific and cross-reactive antibodies in Gram-negative bacteraemia. New England Journal of Medicine 287, 261-267. Miler, J. M., Spilsbury, J. F., Jones, R. J., Roe, E. A. & Lowbury, E. J. L. (1977). A new polyvalent pseudomonas vaccine. Journal of Medical Microbiology 10, 19-27. Pennington, J. E. (1979). Immunotherapy of Pseudomonas aeruginosa. In Pseudomonas aeruginosa: Clinical Manifestations of Infection and Current Therapy (Doggett, R. G., Ed.), pp. 192-215. Academic Press, London. Pennington, J. E., Reynolds, H. Y., Wood, R. E., Robinson, R. A. & Levin, A. S. (1975). Use of a Pseudomonas aeruginosa vaccine in patients with acute leukaemia and cystic fibrosis. American Journal of Medicine 58, 629-638. Sanford, J. P., Hunter, B. W. & Souda, L. L. (1976). The role of immunity in the pathogenesis of experimental haematogenous pyelonephritis. Journal of Experimental
Medicine 115, 383-410. Stamm, W. E., Martin, S. M. & Bennett, J. V. (1977). Epidemiology of nosocomial infections due to Gram-negative bacilli: aspects relevant to the developmental use of vaccines. Jburnal of Infectious Diseases 136, (Supplement), S151-160. Tate, W. J., Douglas, H. & Braude, A. I. (1966). Protection against lethality of Escherichia coli endotoxin with ‘0’ antiserum. Annals of the New York Academy of Sciences 133,
746-762. Venneman,
M.
R. (1972).
Salmonella typhimurium.
Purification
of immunogenically
Infection and Immunity
active
5, 269-282.
RNA
preparations
o
Vaccines
and
antisera
against
Gram-negative
bacilli
111
Walker, H. L., McLoed, C. G., Leppla, S. H. & Mason, A. D. (1979). Evaluation of Pseudomonas aeruginosa Toxin A in experimental rat burn wound sepsis. Infection and Immunity 25, 828-830. Weinstein, R. J. & Young, L. S. (1976). Neutrophil function in Gram-negative rod bacteraemia. Yournal of Clinical Investieation 58. 190-199. Winkelstein, J. A: & Drackman, R. H. (1674). Phagocytosis. The normal process and its clinically significant abnormalities. Paediatrics Clinic North America 21, 551-559. Youmans, A. S. & Youmans, G. P. (1966). Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulphate. Journal of Bacteriology 91, 2139-2145. Young, L. S., Meyer, R. D. & Armstrong, D. (1973). Pseudomonas vaccine in cancer patients. Annals of Internal Medicine 79, 518-528. Young, L. S. & Stevens, P. (1977). Cross-protective immunity to Gram-negative bacilli. Journal of Infectious Disease 136, (Supplement), S174-S180. Ziegler, E. J., Douglas, H., Sherman, J. E., Davis, C. E. & Braude, A. I. (1973). Treatment of EscPlerichia coli and klebsiella bacteraemia in agranulocytic animals with antiserum to a UDP-Gal epimerase-deficient mutant. Journal of Immunology 111,433438. Zinner, S. H. & McCabe, W. R. (1976). Effects of IgM and IgG antibody in patients with bacteraemia due to Gram-negative bacilli. Journal of Infectious Diseases 133, 3745.
LEADING
Vaccines
ARTICLE
and antise;;c;fginst
Gram-negative
The increasing frequency and number of deaths due to infections caused by Gramnegative bacilli (GNB) are a major problem in most large U.K. hospitals today. The bacteria causing these infections belong to the families Enterobactericeae (Eschericia cola’, Klebsiella aerogenes, Enterobacter cloacae, Proteus spp., Sewatia marcescens) and Pseudomonadaceae (Pseudomonas aeruginosa) and they attack patients who are already ill with cancer, respiratory damage, renal or hepatic diseases or patients who have been compromized by burning, neutropenia or by surgical and other invasive procedures. The limited success of control measures and of antibiotics in the treatment of infections due to highly resistant Gramnegative bacilli has led to an interest in immunoprophylaxis as a possible alternative or complementary form of treatment. At first the diversity of antigenic types found among the opportunistic GNB makes the task of preparing vaccines and antisera seem daunting. There are 76 different capsular types of K. aerogenes, 16 serological types of Ps. aeruginosa and known strains of E. coli exhibit 92 ‘K’ antigens and 148 ‘0’ antigens. It is not surprising, therefore, that research workers have looked for protective antigens common to more than one species of GNB to try and reduce the number of vaccines needed to achieve wide protective cover.
Lipopolysaccharides
(LPS) and core glycolipids
(CGL)
One of the most immunogenic components found in the cell walls of all GNB are lipopolysaccharides (LPS), but at high dosage LPS are toxic to man and animals causing intravascular coagulation, low blood pressure, renal cortical necrosis and lethal endotoxic shock (Braude, Douglas and David, 1973). In 1959 Freedman investigating the immunogenic properties of LPS found that serum from rabbits made tolerant to LPS from one species of bacteria would passively immunize rabbits against LPS from another bacterial species. This heterologous protection was explained by Luderitz et al. (1966) by the discovery of a core LPS possessing common antigens shared by endotoxins from antigenically unrelated species of GNB. Antisera against the core glycolipid (CGL) gave protection against both endotoxin (Tate, Douglas and Braude, 1966) and lethal GNB challenges (Chedid et al, 1965). Later McCabe et al. (1977) working with a CGL from an Re chemotype mutant of Salmonella minnesota composed solely of 2-keto-3-deoxyoctonate 0195~6701/81/020105
+ o,
$or.oo/o
0
105
1981 Academic
Press
Inc.
(London)
Limited
106
R. J. Jones
(KDO) and lipid A, found it afforded significant active and passive protection against challenge by K. aerogenes, PT. morganii and Salmonella typhimurium. The lipid A fraction on its own showed no protective activity and KDO has not been tested on its own for protective activity as it proved difficult to refine. Although antibody to CGL of the Re mutant was almost as effective as type-specific antibody (antibody against the whole bacterial cell) in offsetting challenges with endotoxin it was inferior to type-specific antibody in protecting against live bacterial challenges. It was found that antibody against CGL of Re mutant produced a 160-fold increase in the number of K. aerogenes required to produce an LD,, whereas immunization with type-specific antibody against K. aerogenes induced a lO,OOO-fold increase in resistance to challenge (McCabe et al., 1973). Further evidence that GNB share a common protective antigen unrelated to their ‘0’ antigenic determinants was reported by Braude et al. (1977) who found that rabbits immunized with antisera against CGL of an E. coli J5 mutant were protected against death after intravenous administration of endotoxins from Salm. abortusequi and Ser. marcescens. Although antibodies against LPS and CGL were shown to protect animals against experimental GNB infections (Sanford, Hunter and Souda, 1962; Kaijser and Olling, 1973) it proved difficult to confirm these findings in humans infected with GNB (Zinner and McCabe, 1976). High titres of ‘0’ specific antibody against the patients’ own infecting strains did not correlate with decreased frequency of shock or death in bacteraemia (McCabe, Kreger and Johns, 1972). It was found subsequently @inner and McCabe, 1976) that the haemagglutination test used for detecting antibody against LPS measured preferentially antibody in the IgM fraction of serum. Once IgM was removed from the patients’ serum samples by pre-treatment with 2-mercapto-ethanol, then antibody against LPS measured by the haemagglutination test in the IgG fraction of serum correlated with improved survival rate in patients infected with GNB, Any consideration of the mechanism of protection by antisera against common protective antigens of GNB must take into account the opsonic and antitoxic properties of the antisera. Antisera against LPS facilitates the removal of GNB in viva by enhancing the phagocytic activity of the reticula-endothelial system (RES). Benacerraf et al. (1959) f ound that the phagocytosis of E. coli in vivo by RES was directly proportional to the amount of antibody against E. co& LPS. The RES also exhibits cross-reacting phagocytic activity as shown by the accelerated clearance of E. coli 04 from the circulation of agranulocytic rabbits treated with antisera against CGL of E. cola’ J5 mutant (Ziegler et al., 1973). Winkelstein and leucocytes Drachman (1974) sh owed the importance of active polymorphonuclear to patients in the fight against bacteraemia due to GNB, an increased incidence of infection by GNB was correlated with a depressed level of polymorphs and impaired functional ability of polymorphs. Although decreased bactericidal activity in polymorphs is often found in patients with bacteraemia caused by GNB (Koch, 1972), Weinstein and Young (1976) suggest that decreased activity of polymorphs is only part of the explanation, the other part being that patients who are susceptible to GNB bacteraemia lack opsonins.
Vaccines
and antisera Capsular
against
Gram-negative
bacilli
107
polysaccharides
Sometimes the possession of a capsular antigen is sufficient to enable GNB to resist opsonization (Dijk et al., 1980) showing that inherent characteristics of bacteria also play a part in their virulence to patients. Polysaccharide capsular antigens are shared by many species of bacteria, E. coli, K. aerogenes and Streptococcus pneumoniae and antibodies against these polysaccharides act as opsonins (Young and Stevens, 1977). Of the 15 pneumonococcal antigens present in contemporary vaccines at least 12 are similar to antigens found in capsular types of K. aerogenes and some are known to be similar to E. coli antigens, as determined by precipitin reactions (Heidelberger and Nimmich, 1976). Since E. coli and K. aerogenes are the two main species of bacteria responsible for bacteraemia caused by GNB (Stamm, Martin and Bennett, 1977) i t may be worthwhile considering vaccination with pneumococcal vaccine as a possible method of overcoming GNB bacteraemia even if the cross-protection following pneumococcal immunisation extends to only some of the strains of E. coli or K. aerogenes. Ribosomal
vaccines
Vaccines based on LPS are highly immunogenic but can be toxic. To avoid the potential toxicity of LPS vaccines, studies have been made with the non-toxic ribosomal vaccines. Youmans and Youmans (1966) were the first to discover that ribosome rich fractions of Mycobacterium tuberculosis were immunogenic and since then these findings have been extended to a number of other bacterial species, Ps. 1978). aeruginosa, Neissel-ia meningitidis, Salm. typhimurium, etc. (Lieberman, There are conflicting reports concerning the role of active substances in ribosomal vaccines. Immunogenicity has been attributed to highly purified RNA (Venneman, 1972), protein attached to RNA (J 0h nson, 1973), glycoprotein or mucopolysaccharide (Houchens and Wright, 1973) or to LPS contaminant (Hoops et al., 1975). Treatment of ribosomal vaccine with ribonuclease or trypsin had no effect on immunogenicity of the vaccine suggesting that neither RNA nor protein were essential for immunogenicity of the vaccine. Current opinion has opted for LPS contaminant as the main immunogenic ingredient of ribosomal vaccines (Lieberman, 1978) as pure ribosomal fractions make weak immunogens and ribosome fractions contaminated with LPS are strongly immunogenic. From a practical viewpoint ribosomal vaccines suffer from a major disadvantage in that they only induce protection against the strain of bacteria from which they were made. Pseudomonas
vaccines
and immunoglobulins
Pseudomonas aeruginosp, the most invasive of the opportunistic GNB is infamous for its ability to become resistant to many topical and systemic treatments. The uncertainty of controlling pseudomonas infections with some contemporary treatments instigated the production of several vaccines and antisera against Ps. aeruginosa infections which have been tested in patients with burns, cystic fibrosis, acute myeloid leukaemia and cancer. Early pseudomonas vaccines were monovalent (Feller et al., 1964) but as bacteria from any of the 16 different serological types of
108
R. J. Jones
Ps. muginosa can cause infection, then multivalent vaccines were produced. A protective antigen (OEP) common to all 16 serotypes of Ps. aeruginosa is being investigated in animals by Abe, Tanamoto and Homma (1977) and looks promising but vaccines made from exotoxin A (Walker et al., 1979) and from ribosomes (Lieberman, 1978) have restricted protectivity, the former fails to prevent bacterial proliferation while the ribosomal vaccine induces low-grade strain-specific protection. In patients the most widely tested pseudomonas vaccines are Pseudogen (ParkeDavis, heptavalent vaccine) and PEV-01 (Wellcome Research Laboratories, 16valent vaccine). Both vaccines were extracted from the cell walls of Ps. aeruginosa, Pseudogen was extracted with phenol (Hanessian et al., 1971), PEV-01 with an EDT.A/glycine mixture (Miler et al., 1977) from a representative strain of each of the 16 serological types of Ps. aeruginosa. Although both vaccines are highly immunogenic, they vary in their toxicity for man. Pseudogen at protective doses produced minor local and systemic reactions (Alexander and Fisher, 1974) while PEV-01 produced no adverse reactions (Jones et al., 1976). In a trial of Pseudogen in 322 patients with burns greater than 20 per cent of their body surfaces (Alexander and Fisher, 1974), the degree of protection induced by Pseudogen was directly related to the dose of vaccine given. None of the patients given 25 ug of Pseudogen/kg died from pseudomonas septicaemia while 12.5 per cent of lower dosed vaccinees died and 14.7 per cent of unvaccinated control patients died with pseudomonas septicaemia. Young, Meyer and Armstrong (1973) vaccinated patients with leukaemia, lymphoma and with solid tumours using varying doses of Pseudogen. They noted a statistically significant improvement in the survival of vaccinated patients, a result confirmed in other trials by Haghbin, Armstrong and Murphy (1973) and Pennington et al. (1975). Patients with cystic fibrosis showed decreased coughing for several months after treatment with Pseudogen but Ps. aeruginosa was not eradicated from the sputum (Pennington, 1979). Controlled trials of PEV-01 in burned patients in New Delhi have shown the value of immunoprophylaxis against Ps. aeruginosa in the saving of lives of patients, with burns greater than 15 per cent of their body surfaces (Jones, Roe and Gupta, 1978, 1979 and 1980). In New Delhi where the risk of death from pseudomonas septicaemia is high, 40.6 per cent (13/32) unvaccinated adults died while only 6.6 per cent (2/30) of adult vaccinees died. In a trial at Birmingham burns unit where pseudomonas septicaemia occurs less frequently than at New Delhi, vaccinated patients showed no Ps. aeruginosa in their blood cultures, had increased levels of antipseudomonas antibody and showed enhanced phagocytic activity against pseudomonas compared with unvaccinated controls. In 1980, Jones, Roe and Gupta found that an immunoglobulin, made from plasma of human volunteers vaccinated with PEV-01 given prophylactically to burned patients on arrival at the hospital prevented death of all 18 passively immunized children compared with a mortality of 21 per cent (9/42) in the unimmunized control children. Pseudomonas immunoglobulin is a potent weapon in the fight against Ps. aerugitiosa where this organism poses a major threat to patients’ lives. The protec-
Vaccines
and antisera
against
Gram-negative
bacilli
109
tion given by immunoglobulin seems to be effective against only strains of Ps. aerugilzosa. Fisher (1977) found in experiments with animals that immunoglobulin raised in human volunteers against Pseudogen gave no protection against challenge by E. c&i, Ent. cloacae, PT. mirabilis nor K. aerogenes, but there was some protection against Ser. maxescens. Comment
Much of the work withvaccines and antisera against GNB is at an experimental stage but the pseudomonas vaccines and immunoglobulin show how successful immunological treatments against GNB could be. The way forward should be to produce vaccines against GNB that raise opsonins and restrict the spread of the bacteria. Where this approach is impracticable immunoglobulins, raised in healthy volunteers which are rich in opsonins and antitoxins, should be used prophylactically or therapeutically. Certain epidemiological studies are required. The immunologist needs to know which patients are at greatest risk of developing each type of GNB infection. Do patients become infected with more than one type of GNB? Can high risk groups of patients who are potential candidates for immunotherapy be recognized? When during hospitalization do infections with GNB occur? Answers to these questions together with a continuing search for protective antigens against GNB should obtain maximum benefit from immunological treatments in the future. R. J. Jones Medical Research Council Unit, Birmingham Accident Hospital References Abe, C., Tanamoto, antigen (OEP)
K. & Homma, J. Y. (1977). Infection protective of Pseudomonas aeruginosa and its chemical
Journal of Experimental
property of the common composition. Japanese
Medicine 47, 393-402.
Alexander, J. W. & Fisher, M. W. (1974). I mmunisation against pseudomonas infection after thermal injury. Journal of Infectious Diseases 130, 152-l 58. Benacerraf, B., Kivy-Rosenberg, E., Sebestyen, M. M. & Zweifach, B. W. (1959). The effect of high doses of X-irradiation on the phagocytic, proliferative and metabolic properties of the RES. Journal of Experimental Medicine 110, 49-64. Braude, A. I., Douglas, H. & David, C. E. (1973). Treatment and prevention of intravascular coagulation with antiserum to endotoxin. Journal of Infectious Diseases 128, (Supplement), 5157-5164. Braude, A. I., Ziegler, E. J., Douglas, H. & McCutchan, J. A. (1977). Antibody to cell wall glycolipid of Gram-negative bacteria: induction of immunity to bacteraemia and endotoxaemia. Journal of Infectious Diseases 136, (Supplement), 5167-5173. Chedid, L., Parant, M., Parant, F. & Boyer, F. (1968). A proposed mechanism for natural immunity to enterobacterial pathogens. Journal of Immunology 100, 292-301. Dijk, W. C., Verburgh, E. & Tol, R. (1980). Interaction of phagocytic and bacterial cells in patients with bacteraemia caused by Gram-negative bacteria. j’ournal of
hfectious Feller,
Diseases 141, 441-449.
I., Burgess, V., Callahan, W. & Waldyke, J. (1964). Use of vaccine and hyperimmune serum for protection against pseudomonas septicaemia. Journal of Trauma 4,451-459. Fisher M. V. (1977). A polyvalent human 7 globulin immune to Pseudomonas ueruginosa: passive protection of mice against lethal infection. Journal of Infectious Diseases 136,
S181-186.
R. J. Jones
110
Freedman, H. H. (1959). Passive transfer of protection against lethality of homologous and heterologous endotoxins. Proceedings of the Society of Experimental Biology and Medicine
102, 504-506. Haghbin,
M.,
Armstrong,
D. & Murphy, M. L. (1973). Controlled in children with acute leukaemia.
Pseudomonas aeruginosa vaccine 32, 761-769. -
prospective trial of Cancer (Philadelphia)
Hanessian, S., Regan, W. D. & Haskell, T. H. (1971). Isolation and characterisation of antigenic components of a new heptavalent pseudomonas vaccine. Nature 229,209-210. Heidelberger, M. H. & Nimmich, W. (1976). Immunochemical relationship between bacteria belonging to two separate families pneumococci and klebsiella. Immunochemistry 13, h7-80 -, --.
Hoops, P., Prather, L. J., Berry, J. & Ravel, een in effective ribosomal vaccines
immunity
13, 1184-l 192.
J. M. (1976). Evidence for an extrinsic immunofrom Salmonella typhimurium. Infection and __
Houchens, D. P. & Wright, G. L. (1973). Immunity to Salmonella typhimurium infection: characteristics of antigens in active protection. Infection and Immunity 7, 507-511. Johnson, W. (1973). Ribosomal vaccines. Infection and Immunity 8, 395400. Jones, R. J., Roe, E. A. & Gupta, J. L. (1978). Low mortality in burned patients in a pseudomonas vaccine trial. Lancet 2, 401-403. Jones, R. J., Roe, E. A. & Gupta, J. L. (1979). Controlled trials of a polyvalent pseudomonas vaccine in burns. Lancet 2, 977-983. Jones, R. J., Roe, E. A. & Gupta, J. L. (1980). Controlled trial of pseudomonas immunoglobulin and vaccine in burned patients. Lancet 2, 1263-1265. Jones, R. J., Roe, E. A., Lowbury, E. J. L., Miler, J. M. & Spilsbury, J. F. (1976). A new pseudomonas vaccine: preliminary trial in human volunteers. Journal of Hygiene 76,
429-439. Kaijser,
B.
&
Olling,
S. (1973).
Experimental haematogenous pyelonephritis response and its protective capacity.
Escherichia coli in rabbits : the antibody Infectious Diseases 128, 41-49.
due
to
Journal of
Koch,
C. (1974). Acquired defect in bactericidal function of neutrophil granulocytes during bacterial infection. Acta Pathologica Microbiologica Scandinavia (B) 82, 439447. Lieberman, M. M. (1978). Pseudomonas ribosomal vaccines: preparation, properties and immunogenicity. Infection and Immunity 21, 76-86. McCabe, W. R., Bruins, S. C., Craven, D. E. &Johns, M. (1977). Cross-reactive antigens: their potential for immunisation induced immunity to Gram-negative bacteria. Journal of Infectious Diseases 136, (Supplement), Sl61-S166. McCabe, W. R., Greely, A., Di Genio, J. & Johns, M. A. (1973). Humoral immunity to type-specific and cross-reactive antigens of Gram-negative bacilli. Journal of Infectious Diseases 128, (Supplement), S284-S289. McCabe W. R., Kreger, B. E. & Johns, M. (1972). Type specific and cross-reactive antibodies in Gram-negative bacteraemia. New England Journal of Medicine 287, 261-267. Miler, J. M., Spilsbury, J. F., Jones, R. J., Roe, E. A. & Lowbury, E. J. L. (1977). A new polyvalent pseudomonas vaccine. Journal of Medical Microbiology 10, 19-27. Pennington, J. E. (1979). Immunotherapy of Pseudomonas aeruginosa. In Pseudomonas aeruginosa: Clinical Manifestations of Infection and Current Therapy (Doggett, R. G., Ed.), pp. 192-215. Academic Press, London. Pennington, J. E., Reynolds, H. Y., Wood, R. E., Robinson, R. A. & Levin, A. S. (1975). Use of a Pseudomonas aeruginosa vaccine in patients with acute leukaemia and cystic fibrosis. American Journal of Medicine 58, 629-638. Sanford, J. P., Hunter, B. W. & Souda, L. L. (1976). The role of immunity in the pathogenesis of experimental haematogenous pyelonephritis. Journal of Experimental
Medicine 115, 383-410. Stamm, W. E., Martin, S. M. & Bennett, J. V. (1977). Epidemiology of nosocomial infections due to Gram-negative bacilli: aspects relevant to the developmental use of vaccines. Jburnal of Infectious Diseases 136, (Supplement), S151-160. Tate, W. J., Douglas, H. & Braude, A. I. (1966). Protection against lethality of Escherichia coli endotoxin with ‘0’ antiserum. Annals of the New York Academy of Sciences 133,
746-762. Venneman,
M.
R. (1972).
Salmonella typhimurium.
Purification
of immunogenically
Infection and Immunity
active
5, 269-282.
RNA
preparations
o
Vaccines
and
antisera
against
Gram-negative
bacilli
111
Walker, H. L., McLoed, C. G., Leppla, S. H. & Mason, A. D. (1979). Evaluation of Pseudomonas aeruginosa Toxin A in experimental rat burn wound sepsis. Infection and Immunity 25, 828-830. Weinstein, R. J. & Young, L. S. (1976). Neutrophil function in Gram-negative rod bacteraemia. Yournal of Clinical Investieation 58. 190-199. Winkelstein, J. A: & Drackman, R. H. (1674). Phagocytosis. The normal process and its clinically significant abnormalities. Paediatrics Clinic North America 21, 551-559. Youmans, A. S. & Youmans, G. P. (1966). Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulphate. Journal of Bacteriology 91, 2139-2145. Young, L. S., Meyer, R. D. & Armstrong, D. (1973). Pseudomonas vaccine in cancer patients. Annals of Internal Medicine 79, 518-528. Young, L. S. & Stevens, P. (1977). Cross-protective immunity to Gram-negative bacilli. Journal of Infectious Disease 136, (Supplement), S174-S180. Ziegler, E. J., Douglas, H., Sherman, J. E., Davis, C. E. & Braude, A. I. (1973). Treatment of EscPlerichia coli and klebsiella bacteraemia in agranulocytic animals with antiserum to a UDP-Gal epimerase-deficient mutant. Journal of Immunology 111,433438. Zinner, S. H. & McCabe, W. R. (1976). Effects of IgM and IgG antibody in patients with bacteraemia due to Gram-negative bacilli. Journal of Infectious Diseases 133, 3745.