- Email: [email protected]
Immunization against nosocomial infection
Immunization
Against Nosocomial
ABRAHAM I. BRAUDE. M.D. ELIZABETH J. ZIEGLER, M.D. J. ALLEN McCUTCHAN, M.D. HERNDON DOUGLAS, B.A.
San Diego, California
From the Department of Medicine and Pathology, University of California, San Diego, California. This study was supported by Army Contract No, MED/DADA 17-69-C-9161 and USPHS Grant AI-10108. It was presented at the 2nd International Conference on Nosocomial Infections, held August 5-8.1980, in Atlanta, Georgia. Requests for reprints should be addressed to Dr. Abraham I. Braude, University Hospital, 225 W. Dickinson Street, San Diego, CA 92103.
Infection
Overwhelming infection with gram-negative bacteremia has become the most serious nosocomial infection in compromised patients. Because gram-negative bacteria share a common core lipopolysaccharide, we tried to develop a single vaccine or antiserum that might control these infections regardless of species. We used a mutant of Escherichia coli 0111 (J5) deficient in u&line diphosphate-galactose (UDP-GAL) epimerase and thus unable to attach “0” side chains, so that core lipopolysaccharide was exposed. A vaccine composed of this mutant produced antibody that gave broad protection against lethal infections by different gram-negative bacteria in immunosuppressed animals. The J5 vaccine protected against 98 percent lethal doses of Pseudomonas aeruginosa, and J5 antiserum improved survival tenfold in animals dying of Esch. coli, Klebsiella and Pseudomonas bacteremia. The protection with vaccine or prophylactic antiserum was undiminished in animals challenged six weeks after immunization. Encouraged by these results, we conducted a double-blind trial in patients with gram-negative bacteremia. In those given J5 antiserum, the mortality rate was cut in half and survival from deep shock increased from 28 percent to 82 percent. Because of these preliminary results in 136 patients, the study has been extended to 300 patients and the double blind code will be examined again to see if the early favorable results are confirmed and extended. In the last 20 years gram-negative bacteria have become the leading agents of fatal bacterial infections in hospitals and their preeminence as a cause of death has been explained by two properties. One is their toxic lipopolysaccharides or endotoxins; another is a versatility in achieving antibiotic resistance, which is not encountered with the pathogenic gram-positive organisms. Because the clinical picture of shock from gram-negative bacterial septicemia is identical to that induced experimentally with endotoxin, many students of this problem refer to shock in gram-negative bacteremia as “endotoxin shock.” In order to test the idea that endotoxin contributes to the disease and mortality in infections by gram-negative bacteria, we prepared antitoxins against endotoxin and examined them for their ability to neutralize the effects of endotoxin and to lower mortality from bacteremia. Our basic premise was that the endotoxin carried on the surface of the outer membrane of gram-negative bacteria is in a position to react with body fluids and cause the same disturbances as those seen after injection of the extracted lipopolysaccharide. There are at least three major antigenic components in endotoxins and each is a theoretic target for a protective antibody or anti-endotoxin. These antigenic components are lipid A, core polysaccharide and “0” polysaccharide. Although the lipid A and core polysaccharide of most gram-negative
February 1981
The American Journal of Medicine
Volume 70
463
IMMUNIZATION
AGAINST NOSOCOMIAL
INFECTION-BRAUDE
bacteria share a similar, if not identical, structure, the “0” antigens vary with each species and serologic type of bacteria, and would require a separate antiserum for each serotype. In rough bacteria the “0” antigens are lost through a mutation that deprives the bacteria of the enzymes required to synthesize the “0” antigen or attach them to the core. We exploited this genetic change in order to uncover the core region for purposes of developing antiserums against that antigen on the assumption that antibody to the core of lipopolysaccharide would react uniformly with the endotoxins of all gram-negative bacteria because their core antigens are more or less the same. This is especially true of the “backbone” which contains only heptose, phosphate, ketodeoxyoctonate (KDO) and hexosamine. This uniform structure of the core lipopolysaccharide of virtually all pathogenic gram-negative bacteria provides a plausible basis for developing one antiserum that might be active against all gram-negative bacteria responsible for fatal nosocomial infection. We postulated that vaccination of healthy men with a rough mutant of Esch. coli 0111 (J5) would produce such a core antiserum for neutralizing and protecting patients from the endotoxins in all bacteria causing human septicemia. The human antiserum against core glycolipid would then be of value not only for prophylaxis of lethal gram-negative bacteremia but also for treatment after the bacteremia developed. In order to produce antibody to core glycolipid we prepared a vaccine from a rough mutant of Esch. coli 0111, known as J5. The antiserum obtained after immunization with this vaccine, is designated J5 antiserum. During the past eight years we have shown that J5 antiserum can prevent the toxic actions of endotoxins from various gram-negative bacteria and protect against lethal bacteremia in immunosuppressed animals [l-7]. We have also examined human J5 antiserum for its value in the therapy and prophylaxis of gram-negative bacteremia and shock in patients [8]. The following results were obtained. Antitoxic Properties of J5 Antiserum. Protection against the toxic action of lipopolysaccharide by antiserum: J5 antiserum can protect against all of the toxic properties of endotoxin,. It is a powerful antitoxin that can prevent each of the following forms of toxicity from lipopolysaccharide: (1) Death fr’omintravenous lipopolysaccharide. The core antiserum sharply lowers the mortality rate from endotoxin in experimental animals [1,9]. (2) The local Shwartzman reaction. The incidence of local (dermal) Shwartzman reactions dropped from 100 percent in rabbits given nonimmune serum to only 8 percent in those protected with J5 antiserum [2]. (3) The generalized Shwartzman reaction (disseminated intravascular coagulation (DIC). Antiserum injected intravenously 2.2hours after the preparative dose of endotoxin (treatment of DIG], or 24 hours before (prevention), decreased the incidence of renal cortical necrosis from 90 percent to 18 percent. At the same time
464
February 1961
The American Journal of Medicine
ET AL.
antiserum to lipopolysaccharide prevented the sharp drop in fibrinogen and platelets that accompanies the deposition of fibrin in the glomeruli and other vessels J31. The J5 antiserum prevents all toxic manifestations of gram-negative bacteremia that are attributed to endotoxin. Moreover, the ability of J5 antiserum to protect against smooth endotoxins proves that “0” antibody is not necessary for such antitoxic activity [2,3]. The protective antiserums have high titers against core glycolipid as measured by passive hemagglutination and enzyme-linked immunoabsorbent assays (ELISA) [lo]. The antitoxic protective factor is found both in the IgG and IgM fractions of antiserum and provides protection for at least one month after administration intravenously or subcutaneously. Protection against bacteremia in animals: The uniform effectiveness of antiserum to the core region of lipopolysaccharide in neutralizing toxic manifestations of all endotoxins provided a reasonable basis for developing one antiserum that might be active against all gram-negative bacteria involved in serious human infections. With this goal in mind we set out to test the J5 antiserum against the bacteremia in experimental animals. Core antiserums and vaccines have given even more impressive protection against bacteremia than against endotoxin. Studies with endotoxin have been limited to passive protection before challenge. Bacteremia studies, on the other hand, have dealt not only with protection before challenge, but also with treatment after bacteremia is underway. The rabbit has been the best experimental animal for these studies because it can be used in models of natural infection and bacteremia that closely mimic the clinical infection that overwhelms immunosuppressed patients [13,i4]. Mice have been less satisfactory because it is necessary to challenge them with huge inoculums of bacteria that produce unacceptably steep dose-response curves, often with less than one log difference between sublethal and 100 percent lethal doses. In addition, most studies have been carried out with rabbit serums which are toxic in mice and could nullify some protective properties. Thus it is not surprising that conflicting results have been obtained in mouse studies. The standard model for bacteremia in this work is the neutropenic rabbit challenged by feeding various species of gram negative bacteria. The bacteria penetrate the gut and produce high fever with overwhelming bacteremia and fatal shock [ll]. Pseudomonas will do the same when dropped into the conjunctival sac of neutropenic rabbits and produces the classic picture of Pseudomonas vasculitis, ecthyma gangrenosum and death from bacteremia [12]. The neutropenic rabbit model thus mirrors closely the endogenous source of the bacteremia and the clinical manifestations in immunosuppressed patients, and avoids the unrealistic injection of huge inoculums of gram-negative bacteria that
’Volume 70
IMMUNIZATION
are needed to kill healthy mice. It also avoids the problems of heterologous antiserum because potent rabbit antiserum can be produced conveniently in large volumes. Immunization with core vaccines or passive immunization with core antiserums have consistently protected rabbits against the wide range of heterologous gram-negative bacteria encountered in lethal human bacteremia and shock. Core antiserum prepared in rabbits immunized with either boiled bacteria or purified J5 lipopolysaccharide prevents death from bacteremia in neutropenic rabbits when given therapeutically after the onset of bacteremia due to Ps.,aeruginosa, Esch. coli and K. pneumoniae [4,5]. In experiments with survival rates under 10 percent in animals given nonimmune rabbit serum, survival rose to 40 to 70 percent among rabbits treated with one injection of core antiserum after the onset of bacteremia. These animals were given no antibiotics. Gamma globulin from core antiserum was also effective therapeutically. Active or passive protection remains undiminished for at least one month and is extremely effective also against P.s. aeruginosa. The survival rate from Pseudomonas bacteremia is increased from 13 to 92 percent (P = <0.0005] after vaccination with the J5 core antigen, even though no significant protection against Pseudomonas bacteremia occurs after immunization with a vaccine composed of Esch. coli 0111, the parent of J5 [5]. This failure of Esch. coli 0111 vaccine fits with the concept that the core lipopolysaccharide is concealed by 0:lll oligosaccharide antigen so that core antibodies are not generated upon vaccination. J5 Antiserum Therapy of Gram-Negative Bacteremia and Shock in Patients. Our preliminary results in a double-blind study on the use of J5 human antiserums for the treatment of gram-negative bacteremia also suggest that core antibody lowers the mortality rate [8]. In these studies the J5 antiserum was prepared by vaccinating human subjects. Healthy young men aged 18 to 30 years have been immunized with a J5 vaccine composed of log or 5 X log killed cells. Subjects receiving single or multiple subcutaneous injections of the vaccine were carefully observed for untoward effects for over three years, and nothing more than temporary local reactions occurred at the site of injection. The human antiserum was as good as rabbit antiserum in treating bacteremic rabbits. One intravenous injection of human J5 antiserum into rabbits dying of severe Pseudomonas bacteremia improved survival from 3 percent to 55 percent. These results extend the therapeutic range of human J5 antiserum to encompass essentially all gram-negative bacteria that cause septicemia or shock in patients. We also found that when J5 human antiserum was administered to gravely ill bacteremic patients, the death rate from bacteremia was cut virtually in half, as compared to controls. The death rate was lowered from 26 percent in controls to 14 percent in patients treated
February
AGAINST
NOSOCOMIAL
INFECTION-BRAUDE
ET AL.
with J5 antiserum [lo]. These differences were not significant at the 5 percent level, indicating that more patients were needed for the study. Among patients in profound gram-negative shock, the rate of recovery rose from 29 percent in controls to 82 percent in those treated with J5 antiserum (P = 0.02 before and 0.074 after Yates correction). These favorable results encouraged us to expand the therapeutic trial and we have now given J5 antiserum or control serum to 320 patients with a diagnosis of gram-negative bacteremia. We are now ready to break the code a second time and make a final analysis of the results. Prophylaxis with J5 Antiserum in Patients. The purpose of these studies was to prevent bacteremia in patients predisposed to infection by giving them antiserum before the infection occurred. These prophylactic studies are different from the therapeutic trials (described in the previous section) in which antiserum was given after bacteremia occurred. The prophylactic trial was set up in neutropenic patients with leukemia and lymphoma in two phases. The first phase of the study has been completed with very favorable results [l3]. The study was conducted by assigning patients at random to receive either J5 antiserum or pre-immune control serum on entering the trial. Each patient received 1 U (3 ml/kg) of serum intravenously every 21 days while neutropenic [ <500 polymorphonuclear leukocytes/ mm3]. Observations were made on the frequency of febrile attacks and bacteremia. The J5 antiserum sharply reduced the occurrence of fever so that the incidence of febrile days (over 38.0%) fell from 70/180 (44 percent) in controls to 35/194 (18 percent) in those given antiserum (P <0.005) [lo]. These studies indicate that prophylactic J5 antiserum reduces the fever, morbidity and bacteremia in neutropenic patients with gram-negative infections. COMMENTS From the theoretic and experimental considerations presented in this report, we think a good case can be made to consider a shift in emphasis from antimicrobial drugs to immunologic methods in the control of gramnegative bacteremia in hospitalized patients. Whereas our preliminary observations on the use of J5 antiserum in the treatment of bacteremia have been encouraging, our ongoing and projected studies for preventing bacteremia in predisposed patients may be more useful. Patients whose immune responses are not yet suppressed by either their disease or treatment might be actively immunized with J5 vaccine. Experimental animals can be protected against the occurrence of lethal bacteremia during periods of immunosuppression and neutropenia by vaccinating them with core vaccines when their immunity is normal [8]. For this reason, patients given vaccines against gram-negative bacteremias before receiving immunosuppressive drugs might also mount an immune response that would carry over and prevent bacteremia when they become immunosup-
1981
The American Journal of Medicine
Volume 70
465
IMMUNIZATION AGAINST NOSOCOMIAL INFECTION-BRAUDE
ET AL.
pressed. Many leukemic and other compromised patients reach a state of immunosuppression, however, when active immunization would no longer be effective. For them, the only alternative form of prophylaxis will be passive immunization. The striking effectiveness of passive immunotherapy with J5 antiserum in rabbits, and the promising results with it in patients, suggest that antiserum prophylaxis may also be useful.
ment-humoral and cellular-be combined. J5 antiserum is remarkably effective in neutropenic animals,
The promising results with leukocyte transfusions [14] in lowering the death rate from bacteremia in neutropenic patients suggest that the two forms of replace-
probably because mononuclear phagocytes may continue to function after neutrophils are gone. Since there is room for improvement in the results obtained with either granulocyte or antibody transfusion, there is reason to suspect they would work better together. Immunization of prospective leukocyte donors with core glycolipid vaccines might provide full replacement for the immunologic deficiencies that pave the way for lethal nosocomial bacteremias.
REFERENCES 1.
2. 3. 4.
5.
Tate WJ III. Douglas H. Braude AI: Protection against lethality of E. cob endotoxin with “0” antiserum. Ann NY Acad Sci 1966: 133: 746-762. Braude AI. Douglas HJ: Passive immunization against the local Shwartzman reaction. 1 Immunol 1971; 108: 505512. Braude AI, Douglas H. Davis CE: Treatment and prevention of intravascular coagulation with antiserum to endotoxin. J Infect Dis 1973; 128: S157-164. Ziegler El. Douglas H. Sherman iE. Davis CE. Braude AI: Tieatment ofE. coli and Klebsiella bacteremia in agranulocvtic animals with antiserum to a UDP-gal epimerasedeficient mutant. J Immunoll973; 111: 433-438: Ziegler EJ. Douglas H. Braude AI: Prevention of lethal Pseudomonas bacteremia with e imerase-deficient E. coli antiserum. Trans Assoc Am P!lysicians 1975; 88: lOl-
9.
10.
11. 12.
108.
6.
7. 6.
466
Braude AI, Ziegler EJ, Douglas H, McCutchan JA: Antibody to cell wall glycolipid of gram negative bacteria: induction of immunity to bacteremia and endotoxemia. J Infect Dis 1977; 136: S167-S173. Braude AI. Ziegler El. Do&as H. McCutchan IA: Protective properties orantisera to”R core. Microbiology 1977; 1977: 253-256. Ziegler EJ, McCutchan JA, Braude AI: Clinical trial of core
February 1961 The American Journal of Medicine
13.
14.
glycolipid antibody in gram negative bacteremia. Trans Assoc Am Physicians 1978: 91:‘253-258. Ito JI. Wunderlich AC, Davis CE. Guiney DG, Braude AI: The importance of magnesium in the enzyme-linked immunosorbent assay for Iipopolysaccharides of rough E. cob 1151and aonococci. In 1Infect Dis fin mess\. Davis CE,Brown KR. Douglas H. Tate’ WJ III. Braude AI: Prevention of death from endotoxin with antisera. I. The risk of fatal anaphylaxis to endotoxin. J Immunoll969; 192: 563-572. Braude AI. Douglas H. Jones J: Experimental production of lethal E. coli bacteremia of pelvic origin. J Bacterial 1969: 90: 979-991. Ziegler EJ, Douglas H: Pseudomonas aeruginosa vasculitis and bacteremia following conjunctivitis: a simple model of fatal Pseudomonas infection in neutropenia. J Infect Dis 1979; 139: 288-296. Wolf JL, McCutchan JA, Ziegler EJ, Braude AI: Prophylactic antibody to core lipopolysaccharide in neutropenia. In: Nelson JD, Grassi C, eds., Current chemotherapy and infectious disease Washington, D.C.: American Society for Microbiology, 1980; 1439-1441. Alavi IB. Root RK. Dierassi I. et al.: A randomized trial of gran’uiocyte transfusions for infection in acute leukemia. N Engl J Med 1977; 296: 766-711.
Volume 70
Against Nosocomial
ABRAHAM I. BRAUDE. M.D. ELIZABETH J. ZIEGLER, M.D. J. ALLEN McCUTCHAN, M.D. HERNDON DOUGLAS, B.A.
San Diego, California
From the Department of Medicine and Pathology, University of California, San Diego, California. This study was supported by Army Contract No, MED/DADA 17-69-C-9161 and USPHS Grant AI-10108. It was presented at the 2nd International Conference on Nosocomial Infections, held August 5-8.1980, in Atlanta, Georgia. Requests for reprints should be addressed to Dr. Abraham I. Braude, University Hospital, 225 W. Dickinson Street, San Diego, CA 92103.
Infection
Overwhelming infection with gram-negative bacteremia has become the most serious nosocomial infection in compromised patients. Because gram-negative bacteria share a common core lipopolysaccharide, we tried to develop a single vaccine or antiserum that might control these infections regardless of species. We used a mutant of Escherichia coli 0111 (J5) deficient in u&line diphosphate-galactose (UDP-GAL) epimerase and thus unable to attach “0” side chains, so that core lipopolysaccharide was exposed. A vaccine composed of this mutant produced antibody that gave broad protection against lethal infections by different gram-negative bacteria in immunosuppressed animals. The J5 vaccine protected against 98 percent lethal doses of Pseudomonas aeruginosa, and J5 antiserum improved survival tenfold in animals dying of Esch. coli, Klebsiella and Pseudomonas bacteremia. The protection with vaccine or prophylactic antiserum was undiminished in animals challenged six weeks after immunization. Encouraged by these results, we conducted a double-blind trial in patients with gram-negative bacteremia. In those given J5 antiserum, the mortality rate was cut in half and survival from deep shock increased from 28 percent to 82 percent. Because of these preliminary results in 136 patients, the study has been extended to 300 patients and the double blind code will be examined again to see if the early favorable results are confirmed and extended. In the last 20 years gram-negative bacteria have become the leading agents of fatal bacterial infections in hospitals and their preeminence as a cause of death has been explained by two properties. One is their toxic lipopolysaccharides or endotoxins; another is a versatility in achieving antibiotic resistance, which is not encountered with the pathogenic gram-positive organisms. Because the clinical picture of shock from gram-negative bacterial septicemia is identical to that induced experimentally with endotoxin, many students of this problem refer to shock in gram-negative bacteremia as “endotoxin shock.” In order to test the idea that endotoxin contributes to the disease and mortality in infections by gram-negative bacteria, we prepared antitoxins against endotoxin and examined them for their ability to neutralize the effects of endotoxin and to lower mortality from bacteremia. Our basic premise was that the endotoxin carried on the surface of the outer membrane of gram-negative bacteria is in a position to react with body fluids and cause the same disturbances as those seen after injection of the extracted lipopolysaccharide. There are at least three major antigenic components in endotoxins and each is a theoretic target for a protective antibody or anti-endotoxin. These antigenic components are lipid A, core polysaccharide and “0” polysaccharide. Although the lipid A and core polysaccharide of most gram-negative
February 1981
The American Journal of Medicine
Volume 70
463
IMMUNIZATION
AGAINST NOSOCOMIAL
INFECTION-BRAUDE
bacteria share a similar, if not identical, structure, the “0” antigens vary with each species and serologic type of bacteria, and would require a separate antiserum for each serotype. In rough bacteria the “0” antigens are lost through a mutation that deprives the bacteria of the enzymes required to synthesize the “0” antigen or attach them to the core. We exploited this genetic change in order to uncover the core region for purposes of developing antiserums against that antigen on the assumption that antibody to the core of lipopolysaccharide would react uniformly with the endotoxins of all gram-negative bacteria because their core antigens are more or less the same. This is especially true of the “backbone” which contains only heptose, phosphate, ketodeoxyoctonate (KDO) and hexosamine. This uniform structure of the core lipopolysaccharide of virtually all pathogenic gram-negative bacteria provides a plausible basis for developing one antiserum that might be active against all gram-negative bacteria responsible for fatal nosocomial infection. We postulated that vaccination of healthy men with a rough mutant of Esch. coli 0111 (J5) would produce such a core antiserum for neutralizing and protecting patients from the endotoxins in all bacteria causing human septicemia. The human antiserum against core glycolipid would then be of value not only for prophylaxis of lethal gram-negative bacteremia but also for treatment after the bacteremia developed. In order to produce antibody to core glycolipid we prepared a vaccine from a rough mutant of Esch. coli 0111, known as J5. The antiserum obtained after immunization with this vaccine, is designated J5 antiserum. During the past eight years we have shown that J5 antiserum can prevent the toxic actions of endotoxins from various gram-negative bacteria and protect against lethal bacteremia in immunosuppressed animals [l-7]. We have also examined human J5 antiserum for its value in the therapy and prophylaxis of gram-negative bacteremia and shock in patients [8]. The following results were obtained. Antitoxic Properties of J5 Antiserum. Protection against the toxic action of lipopolysaccharide by antiserum: J5 antiserum can protect against all of the toxic properties of endotoxin,. It is a powerful antitoxin that can prevent each of the following forms of toxicity from lipopolysaccharide: (1) Death fr’omintravenous lipopolysaccharide. The core antiserum sharply lowers the mortality rate from endotoxin in experimental animals [1,9]. (2) The local Shwartzman reaction. The incidence of local (dermal) Shwartzman reactions dropped from 100 percent in rabbits given nonimmune serum to only 8 percent in those protected with J5 antiserum [2]. (3) The generalized Shwartzman reaction (disseminated intravascular coagulation (DIC). Antiserum injected intravenously 2.2hours after the preparative dose of endotoxin (treatment of DIG], or 24 hours before (prevention), decreased the incidence of renal cortical necrosis from 90 percent to 18 percent. At the same time
464
February 1961
The American Journal of Medicine
ET AL.
antiserum to lipopolysaccharide prevented the sharp drop in fibrinogen and platelets that accompanies the deposition of fibrin in the glomeruli and other vessels J31. The J5 antiserum prevents all toxic manifestations of gram-negative bacteremia that are attributed to endotoxin. Moreover, the ability of J5 antiserum to protect against smooth endotoxins proves that “0” antibody is not necessary for such antitoxic activity [2,3]. The protective antiserums have high titers against core glycolipid as measured by passive hemagglutination and enzyme-linked immunoabsorbent assays (ELISA) [lo]. The antitoxic protective factor is found both in the IgG and IgM fractions of antiserum and provides protection for at least one month after administration intravenously or subcutaneously. Protection against bacteremia in animals: The uniform effectiveness of antiserum to the core region of lipopolysaccharide in neutralizing toxic manifestations of all endotoxins provided a reasonable basis for developing one antiserum that might be active against all gram-negative bacteria involved in serious human infections. With this goal in mind we set out to test the J5 antiserum against the bacteremia in experimental animals. Core antiserums and vaccines have given even more impressive protection against bacteremia than against endotoxin. Studies with endotoxin have been limited to passive protection before challenge. Bacteremia studies, on the other hand, have dealt not only with protection before challenge, but also with treatment after bacteremia is underway. The rabbit has been the best experimental animal for these studies because it can be used in models of natural infection and bacteremia that closely mimic the clinical infection that overwhelms immunosuppressed patients [13,i4]. Mice have been less satisfactory because it is necessary to challenge them with huge inoculums of bacteria that produce unacceptably steep dose-response curves, often with less than one log difference between sublethal and 100 percent lethal doses. In addition, most studies have been carried out with rabbit serums which are toxic in mice and could nullify some protective properties. Thus it is not surprising that conflicting results have been obtained in mouse studies. The standard model for bacteremia in this work is the neutropenic rabbit challenged by feeding various species of gram negative bacteria. The bacteria penetrate the gut and produce high fever with overwhelming bacteremia and fatal shock [ll]. Pseudomonas will do the same when dropped into the conjunctival sac of neutropenic rabbits and produces the classic picture of Pseudomonas vasculitis, ecthyma gangrenosum and death from bacteremia [12]. The neutropenic rabbit model thus mirrors closely the endogenous source of the bacteremia and the clinical manifestations in immunosuppressed patients, and avoids the unrealistic injection of huge inoculums of gram-negative bacteria that
’Volume 70
IMMUNIZATION
are needed to kill healthy mice. It also avoids the problems of heterologous antiserum because potent rabbit antiserum can be produced conveniently in large volumes. Immunization with core vaccines or passive immunization with core antiserums have consistently protected rabbits against the wide range of heterologous gram-negative bacteria encountered in lethal human bacteremia and shock. Core antiserum prepared in rabbits immunized with either boiled bacteria or purified J5 lipopolysaccharide prevents death from bacteremia in neutropenic rabbits when given therapeutically after the onset of bacteremia due to Ps.,aeruginosa, Esch. coli and K. pneumoniae [4,5]. In experiments with survival rates under 10 percent in animals given nonimmune rabbit serum, survival rose to 40 to 70 percent among rabbits treated with one injection of core antiserum after the onset of bacteremia. These animals were given no antibiotics. Gamma globulin from core antiserum was also effective therapeutically. Active or passive protection remains undiminished for at least one month and is extremely effective also against P.s. aeruginosa. The survival rate from Pseudomonas bacteremia is increased from 13 to 92 percent (P = <0.0005] after vaccination with the J5 core antigen, even though no significant protection against Pseudomonas bacteremia occurs after immunization with a vaccine composed of Esch. coli 0111, the parent of J5 [5]. This failure of Esch. coli 0111 vaccine fits with the concept that the core lipopolysaccharide is concealed by 0:lll oligosaccharide antigen so that core antibodies are not generated upon vaccination. J5 Antiserum Therapy of Gram-Negative Bacteremia and Shock in Patients. Our preliminary results in a double-blind study on the use of J5 human antiserums for the treatment of gram-negative bacteremia also suggest that core antibody lowers the mortality rate [8]. In these studies the J5 antiserum was prepared by vaccinating human subjects. Healthy young men aged 18 to 30 years have been immunized with a J5 vaccine composed of log or 5 X log killed cells. Subjects receiving single or multiple subcutaneous injections of the vaccine were carefully observed for untoward effects for over three years, and nothing more than temporary local reactions occurred at the site of injection. The human antiserum was as good as rabbit antiserum in treating bacteremic rabbits. One intravenous injection of human J5 antiserum into rabbits dying of severe Pseudomonas bacteremia improved survival from 3 percent to 55 percent. These results extend the therapeutic range of human J5 antiserum to encompass essentially all gram-negative bacteria that cause septicemia or shock in patients. We also found that when J5 human antiserum was administered to gravely ill bacteremic patients, the death rate from bacteremia was cut virtually in half, as compared to controls. The death rate was lowered from 26 percent in controls to 14 percent in patients treated
February
AGAINST
NOSOCOMIAL
INFECTION-BRAUDE
ET AL.
with J5 antiserum [lo]. These differences were not significant at the 5 percent level, indicating that more patients were needed for the study. Among patients in profound gram-negative shock, the rate of recovery rose from 29 percent in controls to 82 percent in those treated with J5 antiserum (P = 0.02 before and 0.074 after Yates correction). These favorable results encouraged us to expand the therapeutic trial and we have now given J5 antiserum or control serum to 320 patients with a diagnosis of gram-negative bacteremia. We are now ready to break the code a second time and make a final analysis of the results. Prophylaxis with J5 Antiserum in Patients. The purpose of these studies was to prevent bacteremia in patients predisposed to infection by giving them antiserum before the infection occurred. These prophylactic studies are different from the therapeutic trials (described in the previous section) in which antiserum was given after bacteremia occurred. The prophylactic trial was set up in neutropenic patients with leukemia and lymphoma in two phases. The first phase of the study has been completed with very favorable results [l3]. The study was conducted by assigning patients at random to receive either J5 antiserum or pre-immune control serum on entering the trial. Each patient received 1 U (3 ml/kg) of serum intravenously every 21 days while neutropenic [ <500 polymorphonuclear leukocytes/ mm3]. Observations were made on the frequency of febrile attacks and bacteremia. The J5 antiserum sharply reduced the occurrence of fever so that the incidence of febrile days (over 38.0%) fell from 70/180 (44 percent) in controls to 35/194 (18 percent) in those given antiserum (P <0.005) [lo]. These studies indicate that prophylactic J5 antiserum reduces the fever, morbidity and bacteremia in neutropenic patients with gram-negative infections. COMMENTS From the theoretic and experimental considerations presented in this report, we think a good case can be made to consider a shift in emphasis from antimicrobial drugs to immunologic methods in the control of gramnegative bacteremia in hospitalized patients. Whereas our preliminary observations on the use of J5 antiserum in the treatment of bacteremia have been encouraging, our ongoing and projected studies for preventing bacteremia in predisposed patients may be more useful. Patients whose immune responses are not yet suppressed by either their disease or treatment might be actively immunized with J5 vaccine. Experimental animals can be protected against the occurrence of lethal bacteremia during periods of immunosuppression and neutropenia by vaccinating them with core vaccines when their immunity is normal [8]. For this reason, patients given vaccines against gram-negative bacteremias before receiving immunosuppressive drugs might also mount an immune response that would carry over and prevent bacteremia when they become immunosup-
1981
The American Journal of Medicine
Volume 70
465
IMMUNIZATION AGAINST NOSOCOMIAL INFECTION-BRAUDE
ET AL.
pressed. Many leukemic and other compromised patients reach a state of immunosuppression, however, when active immunization would no longer be effective. For them, the only alternative form of prophylaxis will be passive immunization. The striking effectiveness of passive immunotherapy with J5 antiserum in rabbits, and the promising results with it in patients, suggest that antiserum prophylaxis may also be useful.
ment-humoral and cellular-be combined. J5 antiserum is remarkably effective in neutropenic animals,
The promising results with leukocyte transfusions [14] in lowering the death rate from bacteremia in neutropenic patients suggest that the two forms of replace-
probably because mononuclear phagocytes may continue to function after neutrophils are gone. Since there is room for improvement in the results obtained with either granulocyte or antibody transfusion, there is reason to suspect they would work better together. Immunization of prospective leukocyte donors with core glycolipid vaccines might provide full replacement for the immunologic deficiencies that pave the way for lethal nosocomial bacteremias.
REFERENCES 1.
2. 3. 4.
5.
Tate WJ III. Douglas H. Braude AI: Protection against lethality of E. cob endotoxin with “0” antiserum. Ann NY Acad Sci 1966: 133: 746-762. Braude AI. Douglas HJ: Passive immunization against the local Shwartzman reaction. 1 Immunol 1971; 108: 505512. Braude AI, Douglas H. Davis CE: Treatment and prevention of intravascular coagulation with antiserum to endotoxin. J Infect Dis 1973; 128: S157-164. Ziegler El. Douglas H. Sherman iE. Davis CE. Braude AI: Tieatment ofE. coli and Klebsiella bacteremia in agranulocvtic animals with antiserum to a UDP-gal epimerasedeficient mutant. J Immunoll973; 111: 433-438: Ziegler EJ. Douglas H. Braude AI: Prevention of lethal Pseudomonas bacteremia with e imerase-deficient E. coli antiserum. Trans Assoc Am P!lysicians 1975; 88: lOl-
9.
10.
11. 12.
108.
6.
7. 6.
466
Braude AI, Ziegler EJ, Douglas H, McCutchan JA: Antibody to cell wall glycolipid of gram negative bacteria: induction of immunity to bacteremia and endotoxemia. J Infect Dis 1977; 136: S167-S173. Braude AI. Ziegler El. Do&as H. McCutchan IA: Protective properties orantisera to”R core. Microbiology 1977; 1977: 253-256. Ziegler EJ, McCutchan JA, Braude AI: Clinical trial of core
February 1961 The American Journal of Medicine
13.
14.
glycolipid antibody in gram negative bacteremia. Trans Assoc Am Physicians 1978: 91:‘253-258. Ito JI. Wunderlich AC, Davis CE. Guiney DG, Braude AI: The importance of magnesium in the enzyme-linked immunosorbent assay for Iipopolysaccharides of rough E. cob 1151and aonococci. In 1Infect Dis fin mess\. Davis CE,Brown KR. Douglas H. Tate’ WJ III. Braude AI: Prevention of death from endotoxin with antisera. I. The risk of fatal anaphylaxis to endotoxin. J Immunoll969; 192: 563-572. Braude AI. Douglas H. Jones J: Experimental production of lethal E. coli bacteremia of pelvic origin. J Bacterial 1969: 90: 979-991. Ziegler EJ, Douglas H: Pseudomonas aeruginosa vasculitis and bacteremia following conjunctivitis: a simple model of fatal Pseudomonas infection in neutropenia. J Infect Dis 1979; 139: 288-296. Wolf JL, McCutchan JA, Ziegler EJ, Braude AI: Prophylactic antibody to core lipopolysaccharide in neutropenia. In: Nelson JD, Grassi C, eds., Current chemotherapy and infectious disease Washington, D.C.: American Society for Microbiology, 1980; 1439-1441. Alavi IB. Root RK. Dierassi I. et al.: A randomized trial of gran’uiocyte transfusions for infection in acute leukemia. N Engl J Med 1977; 296: 766-711.
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