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Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: passive intravenous immunotherapy using pseudomonas globulin
Serodiu,qnosis
and Immunotherapy
( 1987)
1, 153-I
67
Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: passive intravenous immunotherapy using pseudomonas globulin
Ian Alan Holder*tf
*Departments Medicine,
qf Microbiology and tshriners
and Alice N. Neelyt
and Surgery. Burns Institute,
University Cincinnati,
of Cincinnati Ohio
45219,
College U.S.A.
qf
Two immunoglobulin preparations were tested for their ability to confer passive immunity when given early after infection or post-sepsis to burned, Pseudomonas aeruginosa infected mice. Used early after infection, P. aeruginosa hyperimmune globulin (HIg) was 10 times more protective than normal human globulin (NIg) with median protective doses of 0.4 and 4.5 mg, respectively. Giving 1 mg HIg to mice 1 h post burn and infection with IO” P. aeruginosa of severaldifferent 0 serotypesallowed
70-100% of the mice to survive for IO days; 7(tlOO% of untreated controls (IO’ challenge)died by day 4. Additional HIg given 48 h later further enhancedsurvival. Used post-sepsis, HIg provided
good early protection,
but varied in IO-day survival,
dependingon the strain of P. aeruginosa used. Additional HIg did not increase survival of the septicmice. Hence, both HIg and NIg. usedearly in the infection course, prevented deaths of burned mice infected with P. aeruginosa of various 0 serotypes. HIg used therapeutically gave significant short-term and some long-term protection. Kqword.~:
hyperimmune
globulin
treatment,
pseudomonas
infection,
burns
Introduction Passiveimmunization for protection of burned patients from infections by Pseudomonus has had a long history I--J. Initially, immunoglobulin (Ig) had to be given intramuscularly because of vasomotor reactions when administered intravenously. However, pain on injection, the inability to obtain optima1 blood levels becauseof massvolume considerations, and slow absorption rates limited Ig use via the intramuscular route. The introduction of pooled. normal human Ig (NIg) or pseudomonas hyperimmune Ig (HIg) preparations, modified for intravenous use5, has renewed interest in passive immunization. These modified preparations contain opsonizing antibodies to seven immunotype specific lipopolysaccharides of P. aeruginosa plus antibodies to exotoxin A$-‘. Studies by Pollack’ and Collins and co11eagues9~‘n, plus work from our laboratory”, demonstrate that protection is afforded to burned, P. aeruginosa infected aeruginnsu
$ Correspondence Ohio 45219. U.S.A.
to: Ian Alan
Holder.
Ph.D.,
Shriners
Burns
Institute
. 202 Goodman
Street,
Cincinnati.
153 0888-0786/87/040153+
IO $03.00/O
~1’; 1987 Academic
Press Inc. (London)
Limited
IS4
1. A. Holder and A. N. Neely
mice when treated with preparations of NIg prepared for intravenous use, The protection is dose dependent and requires large amounts of Nlg. In the present study, we compared the protection afforded to burned, P. cwruginostr infected mice treated with either NIg or a preparation of native Hlg. We assessed.in addition, the efficacy of HIg given at various points during the progression of the infection. Materials and methods Microorganisms
and challenge
inoculum
aeruginosa strains SBI-N, 1071, 1998 and 2993 were clinical isolates obtained from the wounds of burn patients at the Shriners Burns Institute. Strain 6782 was donated by Wellcome Biotechnology. All strains were typed for their somatic (0) antigens according to the antigenic scheme recommended by the Serotyping Committee of the Japan Pseudomonas aeruginosa Society (JPAS)“, using antisera purchased from Denka Seiken Co., Ltd, Tokyo, Japan. The strains, their 0 antigen according to our typing system and their equivalent Habs and Fisher serotype designations are given in Table I.
Pseudomonas
1. 0 serotypesof and NIg and HIg titers against ~seudomonus aeruginosa strainsused in this study
Table
Titers*
Serotype designations Strain
JPAS
Habs
Fisher
SBI-N 1071 1998 2993 6782
G B A I E
6 2/s 3 I II
I 317 NE14 2
* ELISA titers provided Washington, PA. U.S.A. tNE = no equivalent
by
Amour
Nk
Hk
400 1600 800 800 800
1600 3200 1600 3200 3200
Pharmaceutical
Co..
Ft
For challenge inocula, strains were grown for 20 h at 35°C on a shaker, in brain-heart infusion broth. Cells were harvested by centrifugation, washed three times with and diluted in 0.9% sodium chloride solution to a density of 95 units (IO9 cfu ml-‘) using a Klett-Summerson Photoelectric Colorimeter. Dilutions were made from this and 0.1 ml of the appropriate dilution was used for challenge. Burned
mouse model
The burned mouse model of Stieritz & Holder” was used. Female Crl:CF-1 BR NonSwiss mice (22-25 g; Carsworth Farms, New York) received a partial thickness (IO s) alcohol flame burn covering approximately 30% of the total body surface. Immediately after burning, animals were given 0.5 ml of sterile 0.9% sodium chloride as fluid replacement therapy. The burn alone is not lethal; P. aeruginosa (see Fig. 1 and Tables for strain and amount) were injected subcutaneously directly into the burn site. For the Ig dose-response experiment, 20 mice were treated per dose. For all subsequent
Immunotherapy
experiments, the tables. I+: Pwpurotion
using
pseudomonas
globulin
155
the results from two groups of five mice each were pooled and reported in
and treatment
Two types of intravenous immune globulin preparations were used. Each was supplied by The Armour Pharmaceutical Co. (Ft. Washington, PA) as 5% gamma globulin in 2.5% albumin and 5.0% sucrose. Pooled, normal human globulin (NIg) was prepared from a commercial size lot of plasma obtained from non-immunized donors. Pseudomonas hyperimmune globulin (HIg) was fractionated from sera of 15 high titer responders who were immunized with a 16-valent Pseudomonas extract vaccine using procedures described hy Jones rf a1.‘.‘“.‘5m which O-5ml of a 16 valent pseudomonasextract vaccine was in.iected subcutaneously on days 0, 7 and 14, and blood collected on day 35. The major antigen(s) contained in this vaccine is smooth lipopolysaccharide”“. although biochemical and immunological analyses suggest the presence of outer membrane proteins and flagellar antigens’(‘. The vaccine produces high titer antibody to each of the monovalent extracts contained in the polyvalent preparation when rabbits are immunizedlh. Likewise. human burn patientsI and normal human volunteers” produced high titer antibodies when immunized with this vaccine. HIg showed from two- to greater than four-fold higher titers than NIg against the five P. ueruginosa serotypes (Table I) used for challenge in this study. Similar or greater differences (two- to eight-fold) in titers for HIg versus NIg were found for all other P. ueruginosu serotypes (unpubl. results). Since Pollack found greater than 90% kill when he tested six other Ig preparations for their opsonophagocytotic ability. we assumethat the Ig preparations used in this study have similar opsonic capacity. Likewise, since Pollack” has measured antibody to exotoxin A in over 30 other Ig preparations and found titers ranging from 1:32 to 1:256. we assumethat these Ig preparations contain a similar amount of exotoxin A antibody, For the dose-responsecurve, burned mice were infected with IO’ cfu of P. uerugino.su and treated with either 0.20, 0.35 and 0.60 mg HIg or 3.0. 4.5 and 6.6mg NIg intravenously 1 h post burn and challenge. The median protective dose (PD,,) was calculated from data obtained on the fifth day post treatment. The fifth day was chosen to make the PD,,, plot since. of the total 64 mice who died in this experiment, 58 were dead ar day 5 and the occasional additional death made little difference in the final results. In all other experiments, mice were burned, infected (with P. ueruginosu strain and cfu indicated in the Tables) and treated intravenously with HIg and NIg at different times after challenge. Albumin served as a control for the effect of non-specific protein. Results
Dose- responsecurves showing the effect of early (I h post burn and infection) NIg and HIg treatment on survival of burned. P. ueruginosu SBI-N infected mice, are shown in Fig. 1. These results show that HIg was about 10 times more protective than NTg, as the PD,,, values calculated from the dose-responsecurve were 0.4 and 4.5 mg, respectively. Survival of burned mice infected with various 0 serotype strains of P. aeruginosu and treated with 1 mg HIg, 1 h post burn and infection, is shown in Table 2. In all cases. infection of control groups using a low infecting dose of P. ueruginosu (IO’cfu) lead to a progressive and highly lethal outcome, with only 0- 30% of survival 5 days post burn and
156
I. A. Holder
and A. N. Keely
loo I-
20 -
0 0
1
2
3
4 w
5
6
7
W
Figure 1. Dose response curves for the effect of hyperimmune globulin (0) on the survival of burned mice infected with lo5 cfu Pseudomonas aeruginosu mice.
and normal human SBI-N. Each point
globulin ( r: ) represents 20
infection compared to between 70 and 100% survival at 10 days in the HIg treated groups. This was true in spite of the fact that the HIg treated groups were infected, initially, with a challenge dose ( lo5 cfu) 1OOO-fold higher than controls. The significant enhancement of survival accorded by the HIg treatment occurred regardless of the somatic antigen type of the challenge strain. When burned, infected mice were treated with 1 mg HIg at both 1 and 48 h post infection, the IO-day survival was increased to 9&100% and was uniformly 100% up until day 9 (Table 3). In this experiment, treatment using NIg at a IO-fold higher dose than HIg also provided significant enhancement of survival. Control (albumin treated) animals showed no survival advantage when given 1O-fold higher (10 mg) albumin (data not shown). Pseudomonas aeruginosa infection in burn patients starts with initial colonization/ infection of the burn wound by low numbers of organisms followed progressively by multiplication at the local site, bacteremia and sepsisand, in the absenceof treatment, death. This clinical circumstance is mimicked most closely in the burned mouse model, by our low (1O*cfu) initial infecting dose. This procedure was used for the next seriesof experiments to determine the effectiveness of HIg treatment at various times during the infection progression. When treatment with HIg was delayed for 8 h, a significant degree of protection occurred, with survival ranging betwwen 60 and 100% at 10 days depending upon the challenge strain used or the treatment dosage(Table 4). Further delay in treatment until 18 h post burn and challenge resulted in considerable loss in protection (Table 5) against some, but not all, strains of P. aeruginosa. In all cases,however, treatment provided an early (days 334) survival advantage. In one case(strain 1998) the higher treatment dose appeared to have a deleterious effect on survival compared with the lower treatment group. Table 6 presents results of an experiment in which mice received HIg at 18 h (4 mg) and 48 h (2 or 4 mg) after burn and infection. The additional 2-mg 48-h dose improved survival with every challenge strain over that observed with 4-mg treatment at 18 h alone
Immunotherapy
using
pseudomonas
globulin
147
158 Table 3. Survtval. immunoglnbulin
I. A. Holder and A. I\i. Neely after early trcatmcnt (Hlg). of burned mice
with normal infected with
hum;tn globulin (Nip) ;i varictv of 0 scrotvpcs
or hyperimmunc of P.sr,fctli~,l?,~rztr\
iww,~ino.str
“h Survivalday Challenge strain
0 serotype
1071
1998
2993
Treatment*
B
A
I
6782
* I mg i.v. of Hlg or 10 mg i.v. of NIg + 10 mice per group.
I
Nk
100’
Hk
100
2
3
‘I
5
IO
100 100
90 100
90 IO0
80 I 00
x0 90
W
100
Hig
100
100 100
80 100
80 100
80 100
60 100
Nk Hk
100 100
100 100
100 100
100 100
100 100
100 100
Nk HIg
100
100
100
100
100 100
100 100
100 100
90 100
is given
I and 48 h post burn
and challenge
with
1O’cfu.
(Table 5). However, the additional 4-mg 48-h dosesprovided no additional protection over that seenwith 2 mg. In fact, as in the experiment shown in Table 5, higher dosage treatment of mice infected with strain 1998had a detrimental effect. A similar, but much smaller negative effect of the higher treatment dosage, occurred in mice challenged with strains 1071 and 6782 (Table 6).
Table 4. Survival of burned, pseudomonas infected miceafter delay of hyperimmuneglobulin
treatment for 8 h % Survival/day Challenge strain
Treatment amount* (mg)
I
2 4
loot 100
1998
2 4
2993
1071
6782
* Treatment given iv., t 10 mice per group.
3
4
5
10
100 100
90 100
90 100
90
100
60 90
100 100
100 100
100 100
100 100
100 90
80 80
2 4
100 100
100 100
100 100
100 100
100 100
100 100
2 4
100
100 100
100 100
100 100
100 100
100 100
8 h post burn
2
100 and challenge
with
IO’cfu
Immunotherapy Table
using
pseudomonas
IS9
globulin
5. Survival of burned, pseudomonas infected mice after delay of hyperimmune globulin treatment for 1Xh ‘!b Survival/day Treatment amount* (mg)
I
z
3
4
5
IO
1071
2 4
loot 100
90 80
30 40
‘0 40
10 40
IO '0
19%
2 4
100 100
80 90
50 40
50 30
50 20
30 0
3,993
2 4
100 100
60 80
40 80
30 80
30 X0
30 80
67X2
2 4
100 100
100 100
90 90
90 90
80 90
70 90
Challenge strain
* Treatment given i.v.. 18h post burn and challengewith I@ cfu t IO mm per group.
Discussion The dose-response curves established that early treatment with both NIg and HIg protected burned mice against lethal P. aeru@nosa infections with PD,, values of 0.4 and 4.5 mg for HIg and NIg, respectively. Treatment with HIg, using approximately two times the PD,,, dose established with mice burned and challenged with P. aeruginom Table
6.
Survival of burned, pseudomonas infected mice after delay of initial treatment for IX h followed by a subsequent treatment at 48 h Treatment* amount given at 48 h
I
2
3
4
5
IO
1071
2 4
loot 100
80 80
70 70
60 50
50 50
30 20
199x
2
100
60
60
50
50 30
30
100
100 100
60
4 3993
2 4
100 100
100 100
100 100
100 100
100 100
90 90
6782
2 4
100 100
100
100 100
100 90
90 90
90 90
Challenge strain
* 4 mg treatment given i.v.. listed. t IO mice per group.
93,Survival/day
18h post burn and challenge
100 with
IO’cfu;
48 h I.Y. treatment
IO
given in amounts
160
I. A. Holder and A. N. Neely
strain SBI-N, a G somatic serotype, provided significant protection to mice challenged with 1000LD,,, challenge dosesof P. ucvuginosa strains with somatic antigen other than G (Table 2). These results show that a protective dose establishedagainst one 0 serotype had relevance when other serotypes were used for challenge. This protection could be enhanced by a second treatment of HIg given 48 h post burn and infection (Table 3). Nlg given according to the same treatment schedule also provided significant protection for the burned, infected mice, but required IO-fold more globulin (Table 3). Delayed treatment provided varied protection depending upon the length of the treatment delay and the challenge strain used. Substantial survival occurred when treatment was delayed for 8 h (Table 4). At this time, the burned, infected mice are preseptic, since previous studies using this model demonstrated that the bacterial counts 8 h post challenge are below 10’ cfu per gram of skin”. Moncrief & Teplitz” showed, many years ago, that sepsisdoes not occur until bacterial counts are in excessof IO5cfu per gramme of skin. This number is exceeded by more than 1 log,,, at 18 h after challenge’3. Therefore, animals treated at this time have been septic for several hours. and are very sick. Hence, treatment at 18 h constitutes therapeutic rather than prophylactic treatment used in the previous experiments. When given post-sepsis, the protection afforded by the HIg (Tables 5 and 6) was less dramatic and more variable than when given early after infection (Tables 2 and 3). Also, when usedpost-sepsis,the higher treatment dosagestended to predispose some of the mice to an earlier death and even to provide less overall survival during the IO-day experimental period (Table 6). The decreasein protection with the higher therapeutic dosagesin the septic mice may be caused by blockade of the reticuloendothelial system following thermal injury, sepsis and the additional HIg treatmentI or may be caused by the burn and sepsismaking the mice too depressedmetabolically to handle the additional intravenous protein load. The detrimental effects of sepsisplus increased protein load is illustrated in the following experiment. Mice were burned, infected and not treated until 18h post burn and infection. All of the mice receiving 2 mg of HIg were alive on day 2, while 50% of the mice receiving 2 mg HIg plus 10 mg of albumin were dead, suggestingthat total protein load, not Ig per se, contributed to death of septic animals. In preseptic mice, on the other hand, 20 mg of NIg injected I h post-burn and infected gave increased survival over lower NIg treatments, suggesting that the preseptic mouse’s reticuloendothelial system and/or metabolism could handle large amounts of protein. Generally, the protection afforded by Ig, particularly when given post-sepsis, was dependent upon the strain of P. urruginosa used for challenge. Titers of opsonizing antibodies against the various 0 serotype strains of P. aeruginosacould not explain these differences in protection (Table 1). However, it is possiblethat the amount of exotoxin A produced by the individual strains used for challenge varied. While pools of human Ig are known to contain small amounts of antibody to exotoxin AX, there may not be enough to neutralize the amounts of toxin that are elaborated by some strains during 18 h of unrestricted growth in the skin. The animals may die of intoxication even though opsonizing antibodies given as post-sepsis treatment significantly reduces the total microbial load in the tissues. Snell et ~1.‘~ have shown that treatment of burned P. aeruginosa infected mice with either antibiotic, to reduce the microbial load, or antitoxin to neutralize exotoxin A, extended the mean time to death but did not result in increased long-term survival. Only when both treatment modalities were used simultaneously was long-term survival achieved. Data presented in this report demonstrate that both NIg and HIg were highly
lmmunotherapy
using
pseudomonas
161
globulin
protective, when given prophylactically, to burned P. ueruginos~ infected mice. The HIg was many times more effective on a weight basis. Protection occurred regardless of the 0 serotype of the challenge strain. Post-sepsis treatment using HIg also provided a significant survival advantage to burned, P. cleruginnsa infected mice, but the protection appeared more challenge strain dependent. and there might have been a problem of protein overload or reticuloendothelial blockade when septic mice were given large amounts of Ig. Antibiotic treatment in conjunction with Ig therapy has been shown to act synergistically in P. aerugz’nosa infections in both burned and neutropenic mice’, and the addition of antibiotics to our post-sepsis Hlg treatment may enhance survival further. Similarly, survival may be extended if we supplement the HIg therapy with antitoxin treatment. Experiments to test these hypotheses are in progress. Acknowledgement
This study was supported,
in part, by Grant
No. 34028
from
the National
Institute
of
General Medical Sciences. References I. 2. 3. 4.
.’
6.
-,I.
8. 9.
IO.
II.
I?. 13.
Alexander JW. Fisher MW. Immunization against Pseudomonas infection after thermal injury. J Infect Dis 1984; 130: 515228. Feller I. Pierson C. Pseudomonas vaccine and hyperimmune plasma for burned patients. Arch Surg 1968; 97: 225-9. Jones FJ, Roe EA. Controlled trial of Pseudomonas immunoglobulin and vaccine in burn patients. Lancet 1980; Dee 13: 126335. Kefalides NA, Arana JA, Barzen A cl? al. Role of infection in mortality from severe burns: Evaluation of plasma gammaglobulin, albumin and saline-solution therapy in a group of Peruvian children. N Engl J Med 1962; 267: 317-23. Finlayson, JS. History of immunoglobulin use. In Alving BS. Finlayson JS. eds. Immunoglobulins: characteristics and use of intravenous preparations. U.S. Department of Health and Human Services publication no. FDA-8@ 9005. Washington, D.C.: Government Printing Office. 1980: ix-x. Davis E. Efficacy of modified human immune serum globulin in the treatment of experimental murine infections with seven immunotypes of Pseudomonas aeruginosa. J Infect Dis 1975; 340: 717 21. Maclntyre S, Lucken R. Owen P. Smooth lipopolysaccharide is the major protective antigen for mice in the surface extract from IATS serotype 6 contributing to the polyvalent Pseudomonas aeruginosa vaccine PEV. Infect Immun 1986; 52: 7684. Pollack M. Antibody activity against Pseudomonas aeruginosa in immune globulins prepared for intravenous use in humans. J Infect Dis 1983; 141: 109&8. Collins MS, Dorsey JH. Comparative anti-pseudomonas aeruginosa activity of chemically modified and native immunoglobulin G (human), and potentiation of antibiotic protection against P.yeudomonas aeruginosa and Group B Streptococcus in vivo. Am J Med 1984: 76(3A): 15.5-60. Collins MS, Roby RE. Protective activity of an intravenous immune globulin (human) enriched in antibody against lipopolysaccharidc antigens of Pseudomonas aeruginosa. Am J Med 1984; 76(3A): 168-74. Holder IA, Naglich JG. Experimental studies of the pathogenesis of infections due to Pswdomonas aeruginosa: treatment with intravenous immune globulin. Am J Med 1984; 763A): 161-7.
HommaJY. A newantigenicand live-cellslide-agglutinationprocedurefor the infrasubspecifit. serologic classification of Pseudomonas aerzginosa. Jpn J Exp Med 1976; 46: 329-36. Stieritz DD, Holder IA. Experimental studies of the pathogenesis of infections due to P.wudomonas aeruginosa: description of a burned mouse model. J Infect Dis 1978: 131: 688 91.
162
I. A. Holder and A. N. Keely
14. Jones FJ. Roe EA, Gupta JL. Controlled trial of Pseudomonas immunoglobulin and vaccine in burn patients. Lancct 1980; Dee I?: 1263-j. 1. Jones RJ, Roe EA. Lowbury EJL. Miller JJ. Spilsbury JF. A new Pseudomonas vaccine Preliminary trial on human volunteers. J Hyg Camb 1976; 76: 429-39. 16. MacIntyre S, McVeigh T, Owen P. Immunochcmical and biochemical analysis of the polyvalent Pseudutmturs nerugino,srr vaccine PEV. Infect lmmun 1986; 51: 675-~86. 17. Moncrief JA, Teplitz C. Changing concepts in burn sepsis. J Trauma 1964; 4: 233 4.5. 18. Rittenbury MS, Hanback LD. Phagocytic depression in thermal injuries. J Trauma 1967; 7: 523 40. 19. Snell K. Holder IA, Leppla SA, Saelinger CB. Role of exotoxin A and protease as possible virulence factors in experimental infections with Pseuhtutnu.s aerugitmw. Infect Immun 1978; I’): 83945.
and Immunotherapy
( 1987)
1, 153-I
67
Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: passive intravenous immunotherapy using pseudomonas globulin
Ian Alan Holder*tf
*Departments Medicine,
qf Microbiology and tshriners
and Alice N. Neelyt
and Surgery. Burns Institute,
University Cincinnati,
of Cincinnati Ohio
45219,
College U.S.A.
qf
Two immunoglobulin preparations were tested for their ability to confer passive immunity when given early after infection or post-sepsis to burned, Pseudomonas aeruginosa infected mice. Used early after infection, P. aeruginosa hyperimmune globulin (HIg) was 10 times more protective than normal human globulin (NIg) with median protective doses of 0.4 and 4.5 mg, respectively. Giving 1 mg HIg to mice 1 h post burn and infection with IO” P. aeruginosa of severaldifferent 0 serotypesallowed
70-100% of the mice to survive for IO days; 7(tlOO% of untreated controls (IO’ challenge)died by day 4. Additional HIg given 48 h later further enhancedsurvival. Used post-sepsis, HIg provided
good early protection,
but varied in IO-day survival,
dependingon the strain of P. aeruginosa used. Additional HIg did not increase survival of the septicmice. Hence, both HIg and NIg. usedearly in the infection course, prevented deaths of burned mice infected with P. aeruginosa of various 0 serotypes. HIg used therapeutically gave significant short-term and some long-term protection. Kqword.~:
hyperimmune
globulin
treatment,
pseudomonas
infection,
burns
Introduction Passiveimmunization for protection of burned patients from infections by Pseudomonus has had a long history I--J. Initially, immunoglobulin (Ig) had to be given intramuscularly because of vasomotor reactions when administered intravenously. However, pain on injection, the inability to obtain optima1 blood levels becauseof massvolume considerations, and slow absorption rates limited Ig use via the intramuscular route. The introduction of pooled. normal human Ig (NIg) or pseudomonas hyperimmune Ig (HIg) preparations, modified for intravenous use5, has renewed interest in passive immunization. These modified preparations contain opsonizing antibodies to seven immunotype specific lipopolysaccharides of P. aeruginosa plus antibodies to exotoxin A$-‘. Studies by Pollack’ and Collins and co11eagues9~‘n, plus work from our laboratory”, demonstrate that protection is afforded to burned, P. aeruginosa infected aeruginnsu
$ Correspondence Ohio 45219. U.S.A.
to: Ian Alan
Holder.
Ph.D.,
Shriners
Burns
Institute
. 202 Goodman
Street,
Cincinnati.
153 0888-0786/87/040153+
IO $03.00/O
~1’; 1987 Academic
Press Inc. (London)
Limited
IS4
1. A. Holder and A. N. Neely
mice when treated with preparations of NIg prepared for intravenous use, The protection is dose dependent and requires large amounts of Nlg. In the present study, we compared the protection afforded to burned, P. cwruginostr infected mice treated with either NIg or a preparation of native Hlg. We assessed.in addition, the efficacy of HIg given at various points during the progression of the infection. Materials and methods Microorganisms
and challenge
inoculum
aeruginosa strains SBI-N, 1071, 1998 and 2993 were clinical isolates obtained from the wounds of burn patients at the Shriners Burns Institute. Strain 6782 was donated by Wellcome Biotechnology. All strains were typed for their somatic (0) antigens according to the antigenic scheme recommended by the Serotyping Committee of the Japan Pseudomonas aeruginosa Society (JPAS)“, using antisera purchased from Denka Seiken Co., Ltd, Tokyo, Japan. The strains, their 0 antigen according to our typing system and their equivalent Habs and Fisher serotype designations are given in Table I.
Pseudomonas
1. 0 serotypesof and NIg and HIg titers against ~seudomonus aeruginosa strainsused in this study
Table
Titers*
Serotype designations Strain
JPAS
Habs
Fisher
SBI-N 1071 1998 2993 6782
G B A I E
6 2/s 3 I II
I 317 NE14 2
* ELISA titers provided Washington, PA. U.S.A. tNE = no equivalent
by
Amour
Nk
Hk
400 1600 800 800 800
1600 3200 1600 3200 3200
Pharmaceutical
Co..
Ft
For challenge inocula, strains were grown for 20 h at 35°C on a shaker, in brain-heart infusion broth. Cells were harvested by centrifugation, washed three times with and diluted in 0.9% sodium chloride solution to a density of 95 units (IO9 cfu ml-‘) using a Klett-Summerson Photoelectric Colorimeter. Dilutions were made from this and 0.1 ml of the appropriate dilution was used for challenge. Burned
mouse model
The burned mouse model of Stieritz & Holder” was used. Female Crl:CF-1 BR NonSwiss mice (22-25 g; Carsworth Farms, New York) received a partial thickness (IO s) alcohol flame burn covering approximately 30% of the total body surface. Immediately after burning, animals were given 0.5 ml of sterile 0.9% sodium chloride as fluid replacement therapy. The burn alone is not lethal; P. aeruginosa (see Fig. 1 and Tables for strain and amount) were injected subcutaneously directly into the burn site. For the Ig dose-response experiment, 20 mice were treated per dose. For all subsequent
Immunotherapy
experiments, the tables. I+: Pwpurotion
using
pseudomonas
globulin
155
the results from two groups of five mice each were pooled and reported in
and treatment
Two types of intravenous immune globulin preparations were used. Each was supplied by The Armour Pharmaceutical Co. (Ft. Washington, PA) as 5% gamma globulin in 2.5% albumin and 5.0% sucrose. Pooled, normal human globulin (NIg) was prepared from a commercial size lot of plasma obtained from non-immunized donors. Pseudomonas hyperimmune globulin (HIg) was fractionated from sera of 15 high titer responders who were immunized with a 16-valent Pseudomonas extract vaccine using procedures described hy Jones rf a1.‘.‘“.‘5m which O-5ml of a 16 valent pseudomonasextract vaccine was in.iected subcutaneously on days 0, 7 and 14, and blood collected on day 35. The major antigen(s) contained in this vaccine is smooth lipopolysaccharide”“. although biochemical and immunological analyses suggest the presence of outer membrane proteins and flagellar antigens’(‘. The vaccine produces high titer antibody to each of the monovalent extracts contained in the polyvalent preparation when rabbits are immunizedlh. Likewise. human burn patientsI and normal human volunteers” produced high titer antibodies when immunized with this vaccine. HIg showed from two- to greater than four-fold higher titers than NIg against the five P. ueruginosa serotypes (Table I) used for challenge in this study. Similar or greater differences (two- to eight-fold) in titers for HIg versus NIg were found for all other P. ueruginosu serotypes (unpubl. results). Since Pollack found greater than 90% kill when he tested six other Ig preparations for their opsonophagocytotic ability. we assumethat the Ig preparations used in this study have similar opsonic capacity. Likewise, since Pollack” has measured antibody to exotoxin A in over 30 other Ig preparations and found titers ranging from 1:32 to 1:256. we assumethat these Ig preparations contain a similar amount of exotoxin A antibody, For the dose-responsecurve, burned mice were infected with IO’ cfu of P. uerugino.su and treated with either 0.20, 0.35 and 0.60 mg HIg or 3.0. 4.5 and 6.6mg NIg intravenously 1 h post burn and challenge. The median protective dose (PD,,) was calculated from data obtained on the fifth day post treatment. The fifth day was chosen to make the PD,,, plot since. of the total 64 mice who died in this experiment, 58 were dead ar day 5 and the occasional additional death made little difference in the final results. In all other experiments, mice were burned, infected (with P. ueruginosu strain and cfu indicated in the Tables) and treated intravenously with HIg and NIg at different times after challenge. Albumin served as a control for the effect of non-specific protein. Results
Dose- responsecurves showing the effect of early (I h post burn and infection) NIg and HIg treatment on survival of burned. P. ueruginosu SBI-N infected mice, are shown in Fig. 1. These results show that HIg was about 10 times more protective than NTg, as the PD,,, values calculated from the dose-responsecurve were 0.4 and 4.5 mg, respectively. Survival of burned mice infected with various 0 serotype strains of P. aeruginosu and treated with 1 mg HIg, 1 h post burn and infection, is shown in Table 2. In all cases. infection of control groups using a low infecting dose of P. ueruginosu (IO’cfu) lead to a progressive and highly lethal outcome, with only 0- 30% of survival 5 days post burn and
156
I. A. Holder
and A. N. Keely
loo I-
20 -
0 0
1
2
3
4 w
5
6
7
W
Figure 1. Dose response curves for the effect of hyperimmune globulin (0) on the survival of burned mice infected with lo5 cfu Pseudomonas aeruginosu mice.
and normal human SBI-N. Each point
globulin ( r: ) represents 20
infection compared to between 70 and 100% survival at 10 days in the HIg treated groups. This was true in spite of the fact that the HIg treated groups were infected, initially, with a challenge dose ( lo5 cfu) 1OOO-fold higher than controls. The significant enhancement of survival accorded by the HIg treatment occurred regardless of the somatic antigen type of the challenge strain. When burned, infected mice were treated with 1 mg HIg at both 1 and 48 h post infection, the IO-day survival was increased to 9&100% and was uniformly 100% up until day 9 (Table 3). In this experiment, treatment using NIg at a IO-fold higher dose than HIg also provided significant enhancement of survival. Control (albumin treated) animals showed no survival advantage when given 1O-fold higher (10 mg) albumin (data not shown). Pseudomonas aeruginosa infection in burn patients starts with initial colonization/ infection of the burn wound by low numbers of organisms followed progressively by multiplication at the local site, bacteremia and sepsisand, in the absenceof treatment, death. This clinical circumstance is mimicked most closely in the burned mouse model, by our low (1O*cfu) initial infecting dose. This procedure was used for the next seriesof experiments to determine the effectiveness of HIg treatment at various times during the infection progression. When treatment with HIg was delayed for 8 h, a significant degree of protection occurred, with survival ranging betwwen 60 and 100% at 10 days depending upon the challenge strain used or the treatment dosage(Table 4). Further delay in treatment until 18 h post burn and challenge resulted in considerable loss in protection (Table 5) against some, but not all, strains of P. aeruginosa. In all cases,however, treatment provided an early (days 334) survival advantage. In one case(strain 1998) the higher treatment dose appeared to have a deleterious effect on survival compared with the lower treatment group. Table 6 presents results of an experiment in which mice received HIg at 18 h (4 mg) and 48 h (2 or 4 mg) after burn and infection. The additional 2-mg 48-h dose improved survival with every challenge strain over that observed with 4-mg treatment at 18 h alone
Immunotherapy
using
pseudomonas
globulin
147
158 Table 3. Survtval. immunoglnbulin
I. A. Holder and A. I\i. Neely after early trcatmcnt (Hlg). of burned mice
with normal infected with
hum;tn globulin (Nip) ;i varictv of 0 scrotvpcs
or hyperimmunc of P.sr,fctli~,l?,~rztr\
iww,~ino.str
“h Survivalday Challenge strain
0 serotype
1071
1998
2993
Treatment*
B
A
I
6782
* I mg i.v. of Hlg or 10 mg i.v. of NIg + 10 mice per group.
I
Nk
100’
Hk
100
2
3
‘I
5
IO
100 100
90 100
90 IO0
80 I 00
x0 90
W
100
Hig
100
100 100
80 100
80 100
80 100
60 100
Nk Hk
100 100
100 100
100 100
100 100
100 100
100 100
Nk HIg
100
100
100
100
100 100
100 100
100 100
90 100
is given
I and 48 h post burn
and challenge
with
1O’cfu.
(Table 5). However, the additional 4-mg 48-h dosesprovided no additional protection over that seenwith 2 mg. In fact, as in the experiment shown in Table 5, higher dosage treatment of mice infected with strain 1998had a detrimental effect. A similar, but much smaller negative effect of the higher treatment dosage, occurred in mice challenged with strains 1071 and 6782 (Table 6).
Table 4. Survival of burned, pseudomonas infected miceafter delay of hyperimmuneglobulin
treatment for 8 h % Survival/day Challenge strain
Treatment amount* (mg)
I
2 4
loot 100
1998
2 4
2993
1071
6782
* Treatment given iv., t 10 mice per group.
3
4
5
10
100 100
90 100
90 100
90
100
60 90
100 100
100 100
100 100
100 100
100 90
80 80
2 4
100 100
100 100
100 100
100 100
100 100
100 100
2 4
100
100 100
100 100
100 100
100 100
100 100
8 h post burn
2
100 and challenge
with
IO’cfu
Immunotherapy Table
using
pseudomonas
IS9
globulin
5. Survival of burned, pseudomonas infected mice after delay of hyperimmune globulin treatment for 1Xh ‘!b Survival/day Treatment amount* (mg)
I
z
3
4
5
IO
1071
2 4
loot 100
90 80
30 40
‘0 40
10 40
IO '0
19%
2 4
100 100
80 90
50 40
50 30
50 20
30 0
3,993
2 4
100 100
60 80
40 80
30 80
30 X0
30 80
67X2
2 4
100 100
100 100
90 90
90 90
80 90
70 90
Challenge strain
* Treatment given i.v.. 18h post burn and challengewith I@ cfu t IO mm per group.
Discussion The dose-response curves established that early treatment with both NIg and HIg protected burned mice against lethal P. aeru@nosa infections with PD,, values of 0.4 and 4.5 mg for HIg and NIg, respectively. Treatment with HIg, using approximately two times the PD,,, dose established with mice burned and challenged with P. aeruginom Table
6.
Survival of burned, pseudomonas infected mice after delay of initial treatment for IX h followed by a subsequent treatment at 48 h Treatment* amount given at 48 h
I
2
3
4
5
IO
1071
2 4
loot 100
80 80
70 70
60 50
50 50
30 20
199x
2
100
60
60
50
50 30
30
100
100 100
60
4 3993
2 4
100 100
100 100
100 100
100 100
100 100
90 90
6782
2 4
100 100
100
100 100
100 90
90 90
90 90
Challenge strain
* 4 mg treatment given i.v.. listed. t IO mice per group.
93,Survival/day
18h post burn and challenge
100 with
IO’cfu;
48 h I.Y. treatment
IO
given in amounts
160
I. A. Holder and A. N. Neely
strain SBI-N, a G somatic serotype, provided significant protection to mice challenged with 1000LD,,, challenge dosesof P. ucvuginosa strains with somatic antigen other than G (Table 2). These results show that a protective dose establishedagainst one 0 serotype had relevance when other serotypes were used for challenge. This protection could be enhanced by a second treatment of HIg given 48 h post burn and infection (Table 3). Nlg given according to the same treatment schedule also provided significant protection for the burned, infected mice, but required IO-fold more globulin (Table 3). Delayed treatment provided varied protection depending upon the length of the treatment delay and the challenge strain used. Substantial survival occurred when treatment was delayed for 8 h (Table 4). At this time, the burned, infected mice are preseptic, since previous studies using this model demonstrated that the bacterial counts 8 h post challenge are below 10’ cfu per gram of skin”. Moncrief & Teplitz” showed, many years ago, that sepsisdoes not occur until bacterial counts are in excessof IO5cfu per gramme of skin. This number is exceeded by more than 1 log,,, at 18 h after challenge’3. Therefore, animals treated at this time have been septic for several hours. and are very sick. Hence, treatment at 18 h constitutes therapeutic rather than prophylactic treatment used in the previous experiments. When given post-sepsis, the protection afforded by the HIg (Tables 5 and 6) was less dramatic and more variable than when given early after infection (Tables 2 and 3). Also, when usedpost-sepsis,the higher treatment dosagestended to predispose some of the mice to an earlier death and even to provide less overall survival during the IO-day experimental period (Table 6). The decreasein protection with the higher therapeutic dosagesin the septic mice may be caused by blockade of the reticuloendothelial system following thermal injury, sepsis and the additional HIg treatmentI or may be caused by the burn and sepsismaking the mice too depressedmetabolically to handle the additional intravenous protein load. The detrimental effects of sepsisplus increased protein load is illustrated in the following experiment. Mice were burned, infected and not treated until 18h post burn and infection. All of the mice receiving 2 mg of HIg were alive on day 2, while 50% of the mice receiving 2 mg HIg plus 10 mg of albumin were dead, suggestingthat total protein load, not Ig per se, contributed to death of septic animals. In preseptic mice, on the other hand, 20 mg of NIg injected I h post-burn and infected gave increased survival over lower NIg treatments, suggesting that the preseptic mouse’s reticuloendothelial system and/or metabolism could handle large amounts of protein. Generally, the protection afforded by Ig, particularly when given post-sepsis, was dependent upon the strain of P. urruginosa used for challenge. Titers of opsonizing antibodies against the various 0 serotype strains of P. aeruginosacould not explain these differences in protection (Table 1). However, it is possiblethat the amount of exotoxin A produced by the individual strains used for challenge varied. While pools of human Ig are known to contain small amounts of antibody to exotoxin AX, there may not be enough to neutralize the amounts of toxin that are elaborated by some strains during 18 h of unrestricted growth in the skin. The animals may die of intoxication even though opsonizing antibodies given as post-sepsis treatment significantly reduces the total microbial load in the tissues. Snell et ~1.‘~ have shown that treatment of burned P. aeruginosa infected mice with either antibiotic, to reduce the microbial load, or antitoxin to neutralize exotoxin A, extended the mean time to death but did not result in increased long-term survival. Only when both treatment modalities were used simultaneously was long-term survival achieved. Data presented in this report demonstrate that both NIg and HIg were highly
lmmunotherapy
using
pseudomonas
161
globulin
protective, when given prophylactically, to burned P. ueruginos~ infected mice. The HIg was many times more effective on a weight basis. Protection occurred regardless of the 0 serotype of the challenge strain. Post-sepsis treatment using HIg also provided a significant survival advantage to burned, P. cleruginnsa infected mice, but the protection appeared more challenge strain dependent. and there might have been a problem of protein overload or reticuloendothelial blockade when septic mice were given large amounts of Ig. Antibiotic treatment in conjunction with Ig therapy has been shown to act synergistically in P. aerugz’nosa infections in both burned and neutropenic mice’, and the addition of antibiotics to our post-sepsis Hlg treatment may enhance survival further. Similarly, survival may be extended if we supplement the HIg therapy with antitoxin treatment. Experiments to test these hypotheses are in progress. Acknowledgement
This study was supported,
in part, by Grant
No. 34028
from
the National
Institute
of
General Medical Sciences. References I. 2. 3. 4.
.’
6.
-,I.
8. 9.
IO.
II.
I?. 13.
Alexander JW. Fisher MW. Immunization against Pseudomonas infection after thermal injury. J Infect Dis 1984; 130: 515228. Feller I. Pierson C. Pseudomonas vaccine and hyperimmune plasma for burned patients. Arch Surg 1968; 97: 225-9. Jones FJ, Roe EA. Controlled trial of Pseudomonas immunoglobulin and vaccine in burn patients. Lancet 1980; Dee 13: 126335. Kefalides NA, Arana JA, Barzen A cl? al. Role of infection in mortality from severe burns: Evaluation of plasma gammaglobulin, albumin and saline-solution therapy in a group of Peruvian children. N Engl J Med 1962; 267: 317-23. Finlayson, JS. History of immunoglobulin use. In Alving BS. Finlayson JS. eds. Immunoglobulins: characteristics and use of intravenous preparations. U.S. Department of Health and Human Services publication no. FDA-8@ 9005. Washington, D.C.: Government Printing Office. 1980: ix-x. Davis E. Efficacy of modified human immune serum globulin in the treatment of experimental murine infections with seven immunotypes of Pseudomonas aeruginosa. J Infect Dis 1975; 340: 717 21. Maclntyre S, Lucken R. Owen P. Smooth lipopolysaccharide is the major protective antigen for mice in the surface extract from IATS serotype 6 contributing to the polyvalent Pseudomonas aeruginosa vaccine PEV. Infect Immun 1986; 52: 7684. Pollack M. Antibody activity against Pseudomonas aeruginosa in immune globulins prepared for intravenous use in humans. J Infect Dis 1983; 141: 109&8. Collins MS, Dorsey JH. Comparative anti-pseudomonas aeruginosa activity of chemically modified and native immunoglobulin G (human), and potentiation of antibiotic protection against P.yeudomonas aeruginosa and Group B Streptococcus in vivo. Am J Med 1984: 76(3A): 15.5-60. Collins MS, Roby RE. Protective activity of an intravenous immune globulin (human) enriched in antibody against lipopolysaccharidc antigens of Pseudomonas aeruginosa. Am J Med 1984; 76(3A): 168-74. Holder IA, Naglich JG. Experimental studies of the pathogenesis of infections due to Pswdomonas aeruginosa: treatment with intravenous immune globulin. Am J Med 1984; 763A): 161-7.
HommaJY. A newantigenicand live-cellslide-agglutinationprocedurefor the infrasubspecifit. serologic classification of Pseudomonas aerzginosa. Jpn J Exp Med 1976; 46: 329-36. Stieritz DD, Holder IA. Experimental studies of the pathogenesis of infections due to P.wudomonas aeruginosa: description of a burned mouse model. J Infect Dis 1978: 131: 688 91.
162
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14. Jones FJ. Roe EA, Gupta JL. Controlled trial of Pseudomonas immunoglobulin and vaccine in burn patients. Lancct 1980; Dee I?: 1263-j. 1. Jones RJ, Roe EA. Lowbury EJL. Miller JJ. Spilsbury JF. A new Pseudomonas vaccine Preliminary trial on human volunteers. J Hyg Camb 1976; 76: 429-39. 16. MacIntyre S, McVeigh T, Owen P. Immunochcmical and biochemical analysis of the polyvalent Pseudutmturs nerugino,srr vaccine PEV. Infect lmmun 1986; 51: 675-~86. 17. Moncrief JA, Teplitz C. Changing concepts in burn sepsis. J Trauma 1964; 4: 233 4.5. 18. Rittenbury MS, Hanback LD. Phagocytic depression in thermal injuries. J Trauma 1967; 7: 523 40. 19. Snell K. Holder IA, Leppla SA, Saelinger CB. Role of exotoxin A and protease as possible virulence factors in experimental infections with Pseuhtutnu.s aerugitmw. Infect Immun 1978; I’): 83945.