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Immunologic responses to Hemophilus influenzae meningitis
February, 1972 T h e J o u r n a l of P E D I A T R I C S
209
Immunologic responses to Hemopbilus influenzae meningitis Twenty-nine children with meningitis caused by H. influenzae, type b were studied for their hemagglutinating and bactericidal antibody responses. No antibody rises were observed in 22 (76 per cent), of whom 21 were under 2 years of age. Among 7 patients with fourfold or greater antibody titer increases, 6 were over 2 years of age. The mean age for antibody responders was 54 months while for nonresponders it was 11 months (p ~ 0.001). Among 7 children with no antibody responses during hospitalization, none had either hemagglutinating or bactericidal antibodies 6 to 23 months later.
Carl W. Norden, M.D.,* Marion Melish, M.D., James C. Overall, Jr., M.D.,
and John Baum, M.D.,
R o c h e s t e r , N . Y.
HEMOPHILUS INFLUENZAE, type b is the most frequent cause of bacterial meningitis in children under the age of 4 years in the United States? A recent increase in the absolute as well as the proportional frequency of meningitis due to this organism has been reported. 2 In spite of antibiotic therapy, the mortality rate remains at about 10 per cent and neurologic sequelae persist in a substantial proportion of survivors? In 1933, Fothergill and WrighP described the occurrence of H . influenzae, type b menFrom the Departments of Medicine and Pediatrics, University of Rochester School of Medicine and Dentistry. Supported by P H S / G R S G Research Grant Number F R 05403, and by Research Grant CC 00613 from the Center for Disease Control, Atlanta, Ga. ~Address: Montefiore Hospital, 3459 Filth Ave. Pittsburgh, Pa. 15213.
ingitis to be inversely related to the presence of bactericidal activity of blood for this organism. They pointed out that while the incidence of influenzal meningitis was highest in children 2 to 36 months of age, unusual in neonates, and rare in older children and adults, blood bactericidal activity was usually present at birth, declined at about 2 months, and remained absent until about 3 See related article, p. 204. years. An increasing proportion of children over 3 years of age acquired such activity so that by age 10 or above the blood of all tested individuals demonstrated bactericidal antibodies. Only a few reports of antibody levels in individuals infected with H . influenzae, type b have been published. 5-s I n this paper, we will describe studies of the antibody reVol. 80, No. 2, pp. 209-214
2 10
Norden et al.
sponses of 29 children to H. influenzae, type b meningitis. T h e persistence of such antibodies was also determined in some repeat sera obtained as long as 23 months after the acute illnesses. METHODS
Study population. Twenty-nine children with meningitis due to H. influenzae, type b were studied during a 2 year period (1969 and 1970). There were 21 boys and 8 girls, ranging in age from 3 months to 11 years (median t3 months). One male, age 5 years, died on the fourth hospital day, but all other patients survived. One child was left with hemiparesis and motor retardation; another is totally deaf. "Acute sera" were obtained during hospital days I to 5 and "convalescent sera" on day 11 or later. I n a few cases, the initial sera were obtained during hospital days 6 to 9. Sera were stored at -20 ~ C. Serologic tests. Bactericidal. Sera were diluted serially in Dulbecco's phosphate-buffered saline (PBS) 9 in U-shaped microtiter plates (Microbiological Associates). T o t h e 0.05 ml. of diluted serum e" in each well was added 0.025 ml. of human complement (undiluted serum) from a donor with hypogammaglobulinemia. Complement was stored in 0.5 ml, aliquots at -70 ~ C. until used; it contained 120 units of complement activity (normal 80 to 160) and had no antibacterial activity against H. influenzae, type b. The strain of H. influenzae, type b used had been isotated from the spinal fluid of a child with meningitis. Multiple aliquots of a 4 hour broth culture grown from a single colony of this organism had been frozen at -70 ~ C. For each test, a frozen culture was grown for 18 hours in Levinthal's broth and subcultured into fresh broth which was incubated for 4 hours in a 37 ~ C. water bath. Colony counts of the 4 hour culture were ~'Penicillinase (Difeo), 0.05 ml., was added to 0.95 ml. ~f serum obtained from children receiving ampicillin. T h e addition of penieillinase eliminated bactericidal activity due to resldual antibiotic, but did not affect bac~e:ieidal antibodies.
The .[ournal o[ Pediatrics February 1972
consistently 3 to 4 x 10 ~ ml. The bacterial suspension was diluted I:1000 in phosphatebuffered saline, and 0.025 ml. (about 7 x 10 '~ colony-forming units per milliliter) was added to each well. No significant differences in bactericidal activities were observed over an inoculum range of 7 x i02 to 7 x 10 '~ organisms. Immediately after mixing, and after one hour of incubation at 37 ~ C., the contents of each well were streaked onto chocolate agar plates with a standard milk loop calibrated to deliver 0.001 ml. T h e plates were incubated for 24 hours at 37 ~ C. in 5 per cent carbon dioxide. A colony count reduction of 50 per cent or greater was required for bactericidal activity to be recorded as positive; almost all plates showed either no reduction or 100 per cent killing. The coefficient of variation for colony counts from a single well was 15 per cent. All sera were tested in duplicate; titers were reproducible within a single 2-fold serum dilution. Each set of tests included control plates of complement and bacteria alone, and bacteria and phosphate-buffered saline only, to identify killing not due to specific antibodies. Hemagglutination. H u m a n type O, Rhnegative erythrocytes were fixed with glutaraldehyde 1~ and stored as a 30 per cent suspension in phosphate-buffered saline with 0.1 per cent Merthiolate at 4 ~ C. for as long as 30 days. The titers of standard sera were not altered when tested with cells stored for this long a period. A solution of polyribose phosphate (kindly provided by Drs. David Smith and Porter Anderson, Children's Hospital, Boston, Mass.) prepared from the capsular material of H. influenzae, type b was the antigen used to sensitize the red ceils. To accomplish this, 1 ml. of a 2.5 per cent suspension of red cells was added to 1 ml. of polyribose phosphate (0.5 /~g per milliliter) and shaken for 1 hour at 37 ~ C. Sensitized cells were washed 5 times in phosphate-buffered saline and then added in 0.025 ml. volumes to equal amounts of serum serially diluted in phosphate-buffered saline containing 0.5 per cent bovine serum albumin in the micro-
Volume 80 Number 2
Immunologic responses to H. influenzae meningitis
,5'
211
~AC'~E~tCtDAt TITER HA
TI'rER
.4-
O (3O CN
/t , 4 / ',
9:3"
.2"
O /~\3
~1 / ---4
2'0
'/
3'0
/ "~" 4'0 TUBE
/
",
/
/
, . ~ 512 i on
! I |
to HUMBER
,
6'o
Fig. 1. Convalescent serum separated by Sephadex G-200 gel filtration into pools represented by numbers 1 to 4. Pool 1 contained all recovered IgM and Pool 3 the major portion of IgG; these pools were tested for bactericidal and hemagglutinating activity. Pool 1 was devoid of bacter:cidal activity; hemagglutinatlng activity was present in both pools.
titer U-shaped plates. After mixing by tapping, the plates were incubated for one hour at 37 ~ C. They were read 4 hours later for agglutination. The titer of the sera was taken to be the highest dilution in which agglutination was visible. Controls for each set of tests included commercial antiserum against H. influenzae, type b (Hyland Laboratories--titer 1.:2048 to 1:4096), unsensitized cells plus serum, and sensitized cells with diluent only. Sera were tested in duplicate and their titers were reproducible within one 2-fold serum dilution. When sera exhibiting hem.agglutination (HA) titers were absorbed with polyribose phosphate-sensitized red cells, their titers decreased to 1:2 or less. Consequently, titers greater than 1:2 were considered to be indicative of antibody activity. Fractionation of serum. Serum ( 1 ml.) was placed on a 2.7 x 105 cm. Sephadex G-200 column equilibrated with phosphatebuffered saline, p H 7.4 (kindly performed by Dr. George Abraham, Department of Medicine, University of Rochester School of Medicine). The fractions obtained were divided into 4 pools corresponding to the elution profile (Fig. 1), concentrated by pervaporation to the original volume and then tested for the presence of IgG and IglV[ by immunodiffusion in agar with class-specific
antisera. Pool 1 contained all tile recovered IgM and Pool 3 the major portion of the IgG; both were tested for hemagglutinating and bactericidal activity. RESULTS Bactericidal and hemagglutinating antibody. Table I summarizes the data from 22 patients who showed no rises in either bactericidal (BA) or hemagglutinating (HA) antibody titers and the 7 patients who had 4-fold or greater antibody rises. Among the latter, 5 had increases in both HA and BA, 1 H A only, and 1 BA only. Bactericidal titers rose from 0 in acute sera to a range of 4 to 64 in convalescent sera from 5 patients; one patient (11 years old) with a BA titer of 32 in the acute serum had an increase in titer to 128 in convalescent serum. H A titers rose from less than 2 in acute sera to a range of 32 to 512 in convalescent sera from 5 patients. The l 1-year-old girl with bactericidal antibodies in acute serum had an H A titer of 64 in acute serum which subsequently rose to 512. No differences were observed between the two groups (those with antibody responses and those without) in the frequency of bacteremia, peak leukocyte counts, spinal fluid glucose levels, duration of symptoms before institutio:: of antibiotic therapy, and dura-
2 12
Norden et al.
The Journal of Pediatrics February 1972
Table I. Antibody responses of children with H. influenzae, type b meningitis
Antibody Number 22 7
response
No Yesi"
Number with presence of antibodies
Age range~mean (months) 3- 25/11 9-132/54
Acute 0 1
BA I ]Convalescent 0 6
Acute 1 1
HA I Convalescent 1" 6
'~tIA titer 1:16 in both acute and convalescent sera. t F o u r f o l d or greater
increase in convalescent sera
of either H A or BA titers.
Table II. Serologic data 6 to 23 months after meningitis
lnitial antibody response No Yes~"
Number 7 3
Number with antibodies in repeat sera BA t HA 0 0 2 1
"~Ag in Table I.
tion of antibiotic therapy. All children were treated with ampicillin 300 mg. per kilogram, intravenously, for approximately 14 days during hospitalization. The most striking difference between the two groups was in their ages. A mean of 11 + 7 months was noted for the 22 nonresponders and 54 + 39 months for the responders (t = 4.8 p < 0.001). Among 22 children less than 24 months of age, only 1 had an antibody response, whereas 6 of 7 over 24 months responded (Fisher's exact test, p = 0.0001). Twelve children, 4 to 20 months of age, who had no antibody response to H. influenzae, type b were found to have significant HA titers to tetanus and diphtheria and normal isohemagglutinin titers. (These tests were kindly performed by Dr. Robert H. Schwartz, Department of Pediatrics, University of Rochester School of Medicine. ) Bactericidal antibodies with patients' own organisms. Acute and convalescent sera from 5 children who showed no bactericidal antibodies with our standard H. influenzae, type b strain also were tested with organisms isolated from their own cerebrospinal fluids. These tests were performed as were all the others, and none demonstrated antibody. Follow-up studies. Sera were obtained
from 10 children 6 to 23 months after their acute illnesses. None of the 7 children who had had no antibody responses during their hospitalizations had either H A or BA titers in subsequent bleedings (Table I I ) . Two of 3 children who had had antibody titer increases during hospitalization had persistent BA; one also had a significant HA titer. The third child had rises in both H A and BA during hospitalization, hut neither test was positive 18 months later (Table I I ) . Serum fractionation. Convalescent sera obtained from 2 patients were fractionated and Pools 1 ("IgM rich") and 3 ("IgG rich") were tested for BA and H A antibodies. The results for one serum are shown in Fig. 1; the results with the other were similar. The BA titer of the " I g G " fraction was equal to that of the whole serum (64) while the " I g M " fraction was negative. H A titers of 1:512 (equal to the whole serum) and 1:4 were obtained with the " I g M " and " I g G " fractions, respectively. DISCUSSION That 22 of 29 children with H. influenzae, type b meningitis failed to produce either BA or HA antibodies was surprising; even more so was the finding that all but one of the 22 were less than 2 years of age. No antibodies were found in 7 of these children who were retested 6 to 23 months after their acute illnesses. There are several possihle expianations for the failure of 76 per cent of the study population to develop antibodies to H. in/tuenzae, type b and for the striking differences between the antibody responses of those less than and those more than 2 years of age. First, the techniques for measuring
Volume 80 Number 2
Immunologic responses to H. influenzae meningitis
these antibodies may be too insensitive to detect small quantities. This possibility cannot be excluded, but seems unlikely since antibodies were consistently detected in older children. A second explanation is that the strain selected for bactericidal antibody assays was unusually resistant to serum activity. However, the paired sera from 5 children were tested for bactericidal activity against both their own as well as the standard culture and in no case were the results discordant. A third possibility is that the final sera were obtained too early to permit the detection of rising antibody titers. In 11 of the 22 children without antibody responses, the second bleedings were performed 14 days or more after hospitalization; additionally, 7 of the 22 still had no detectable antibodies 6 to 23 months later. T h a t maternal antibodies to H. influenzae, type b persisted in some children and inhibited their own antibody responses is a fourth possibility. This hypothesis seems untenable in the 10 children over one year of age, a time when maternal antibodies ordinarily are no longer present. The possibility that these children are immunologically immature or incompetent can be discounted since they had adequate antibody responses to diphtheria and tetanus antigens and normal isohemagglutinin titers. Early treatment with antimicrobial agents has been shown to suppress the formation of bacteriostatic antibodies to beta hemolytic streptococci? 1 This possibility could not explain the age-related failure to form BA or HA antibodies to H. influenzae, type b since all patients, regardless of age, were treated at approximately the same time in the course of their illnesses. H. influenzae, type b could be a poor immunizing agent, but even if true, that would not explain the age-related difference in antibody response to infection. Preliminary studies 8, ~ with subcutaneous injection of purified polysaccharides from the capsules of this organism showed that they are potent antibody inducers in adults, supporting the conclusion that the failure of most children
2 13
with influenzal meningitis to acquire H A or BA antibodies is not related to the antigenie properties of this organism. Finally, the phenomenon of "immune paralysis" as an explanation for the failure of younger children to show an antibody response to influenzal meningitis should be considered. Felton and associates is reported that the administration of pneumococcal polysaccharides in quantities 500 times greater than immunizing doses produced longlasting and total inhibition of the ability of adult mice to produce neutralizing antibodies to them. There is no evidence that this phenomenon has been induced in man during natural infections. Both meningitis and bacteremia caused by H. inlTuenzae, type b result in the dissemination of a large number of organisms and their antigenically potent potyribose phosphate capsular material. Younger and smaller children might be overwhelmed by this antigenic load with "immune paralysis" while a comparable antigenic load could be handled with appropriate antibody responses by older children. The initial failure to produce antibodies appeared to be persistent 6 to 23 months after the acute illness; this could be in keeping with Felton and associates' experimental observations. Some clarification might be obtained by measuring the effect of injecting such nonresponding children with purified H. influenzae, type b polysaccharide. If the hypothesis of "immune paralysis" is correct, such children would be expected to again fail to show antibody responses to immunization with the polysaccharide. We were unable to obtain purified polysaccharide material with which to conduct such a trial. " I m m u n e paralysis," as originally described, persisted for the life-span of the adult mouse. It was postulated 13 that in order for "immune paralysis" to persist, antigen must remain in the tissues. It is conceivable that in younger children with H. influenzae meningitis, the failure to respond with formation of BA and HA antibodies could be temporary and related to the large antigenic load, and that if chal-
2 14
Norden et al.
lenged subsequently with the same organism, they would be capable of responding. Although the explanation for the age-related failure to produce antibodies to H. influenzae meningitis is still unclear, " i m m u n e paralysis" is an attractive speculation which might relate patient age, size of the infecting inoculum, a n d antibody response. ADDENDUM Following the preparation of this paper, Peter and associates (Abstract, Society for Pediatric Research, 1971, p. 76) reported that only 5 of 17 children hospitalized with H. influenzae, type b meningitis, epiglottitis, or arthritis acquired antibodies to their infections. Their ages in relation to antibody responses were not provided in the abstract, but during the presentation, it was stated that the children with antibody responses were all over 2 years of age, which is in agreement with our findings. We are indebted to Mrs. Phyllis Meyer and Mrs. Linda Witinski for their excellent technical assistance and to Dr. Harry A. Feldman for his advice and critical review of the manuscript. We would like to express our appreciation to Drs. David Smith and Porter Anderson for helpful discussions concerning methodology. REFERENCES
1. Sell, S.: The clinical importance of Hemophilus infiuenzae infections in children, Pediatr. Clin. North Am. 17: 415, 1970. 2. Turk, D., and May, J.: Hemophilus influenzae: Its clinical importance, London, 1967, The English Universities Press, Ltd.
The Journal of Pediatrics February 1972
3. Sproles, F., Azerrad, J., Williamson, C., et al.: Meningitis due to Hemophilus influenzae: Long term sequelae, J, PEma:rm 75: 782, 1969. 4. Fothergill, L. D., and Wright, J.: Influenzal meningitis: The relation of age incidence to the bactericidal power of blood against the causal organism, J. Immunol. 24: 273, 1933. 5. Collier, A. M., Connor, J. D., and Nyhan, W. L.: Systemic infection with Hemophilus influenzae in very young infants, J. PEDIATR. 70: 539, 1967. 6. Mathies, A. W., Hodgman, J., and Ivler, D.: tiemophilus influenzae meningitis in a premature infant, Pediatrics 35: 791, 1965. 7. Norden, C., Callerame, M., and Baum, J.: Hemophilus influenzae meningitis in an adult: A study of bactericidal antibodies and immunoglubulins, N. Engl. J. Med. 282: 190, 1970 8. Robbins, J., Schneerson, R., Rodrigues, L., et al.: Characterization of "natural" infection and immunization-induced anticapsular antibodies to H. influenzae, type b, Pediatr. Res. 40: 376, 1970. (Abst.) 9. Dulbecco, R., and Vogt, M.: Plaque formation and isolation of pure lines with poliomyelitis viruses, J. Exp. Med. 99: 167, 1954. 10. Bing, D., Weyland, J., and Stavltsky, A.: Hemagglutination with aldehyde-fixed erythrocytes for assay of antigens and antibodies, Proc. Soc. Exp. Biol. Med. 121: 1166, 1967. If. Daikos, G., and Weinstein, L.: Streptococcal bacteriostatic antibody in patients treated with penicillin, Proc. Soc. Exp. Biol. Med. 78: 160, 1951. 12. Smith, D., Anderson, P., Johnston, R., et al.: The role of polyribophosphate in immunity to Hemophilus influenzae b, J. Clin. Invest. 49: 90a, 1970. (Abst.) 13. Felton, L., Kaufman, G., Prescott, B., et al.: Studies on the mechanism of the. immunological paralysis induced in mice by pneumoeoccal polysaccharides, J. Immunol. 74: 17, 1955.
209
Immunologic responses to Hemopbilus influenzae meningitis Twenty-nine children with meningitis caused by H. influenzae, type b were studied for their hemagglutinating and bactericidal antibody responses. No antibody rises were observed in 22 (76 per cent), of whom 21 were under 2 years of age. Among 7 patients with fourfold or greater antibody titer increases, 6 were over 2 years of age. The mean age for antibody responders was 54 months while for nonresponders it was 11 months (p ~ 0.001). Among 7 children with no antibody responses during hospitalization, none had either hemagglutinating or bactericidal antibodies 6 to 23 months later.
Carl W. Norden, M.D.,* Marion Melish, M.D., James C. Overall, Jr., M.D.,
and John Baum, M.D.,
R o c h e s t e r , N . Y.
HEMOPHILUS INFLUENZAE, type b is the most frequent cause of bacterial meningitis in children under the age of 4 years in the United States? A recent increase in the absolute as well as the proportional frequency of meningitis due to this organism has been reported. 2 In spite of antibiotic therapy, the mortality rate remains at about 10 per cent and neurologic sequelae persist in a substantial proportion of survivors? In 1933, Fothergill and WrighP described the occurrence of H . influenzae, type b menFrom the Departments of Medicine and Pediatrics, University of Rochester School of Medicine and Dentistry. Supported by P H S / G R S G Research Grant Number F R 05403, and by Research Grant CC 00613 from the Center for Disease Control, Atlanta, Ga. ~Address: Montefiore Hospital, 3459 Filth Ave. Pittsburgh, Pa. 15213.
ingitis to be inversely related to the presence of bactericidal activity of blood for this organism. They pointed out that while the incidence of influenzal meningitis was highest in children 2 to 36 months of age, unusual in neonates, and rare in older children and adults, blood bactericidal activity was usually present at birth, declined at about 2 months, and remained absent until about 3 See related article, p. 204. years. An increasing proportion of children over 3 years of age acquired such activity so that by age 10 or above the blood of all tested individuals demonstrated bactericidal antibodies. Only a few reports of antibody levels in individuals infected with H . influenzae, type b have been published. 5-s I n this paper, we will describe studies of the antibody reVol. 80, No. 2, pp. 209-214
2 10
Norden et al.
sponses of 29 children to H. influenzae, type b meningitis. T h e persistence of such antibodies was also determined in some repeat sera obtained as long as 23 months after the acute illnesses. METHODS
Study population. Twenty-nine children with meningitis due to H. influenzae, type b were studied during a 2 year period (1969 and 1970). There were 21 boys and 8 girls, ranging in age from 3 months to 11 years (median t3 months). One male, age 5 years, died on the fourth hospital day, but all other patients survived. One child was left with hemiparesis and motor retardation; another is totally deaf. "Acute sera" were obtained during hospital days I to 5 and "convalescent sera" on day 11 or later. I n a few cases, the initial sera were obtained during hospital days 6 to 9. Sera were stored at -20 ~ C. Serologic tests. Bactericidal. Sera were diluted serially in Dulbecco's phosphate-buffered saline (PBS) 9 in U-shaped microtiter plates (Microbiological Associates). T o t h e 0.05 ml. of diluted serum e" in each well was added 0.025 ml. of human complement (undiluted serum) from a donor with hypogammaglobulinemia. Complement was stored in 0.5 ml, aliquots at -70 ~ C. until used; it contained 120 units of complement activity (normal 80 to 160) and had no antibacterial activity against H. influenzae, type b. The strain of H. influenzae, type b used had been isotated from the spinal fluid of a child with meningitis. Multiple aliquots of a 4 hour broth culture grown from a single colony of this organism had been frozen at -70 ~ C. For each test, a frozen culture was grown for 18 hours in Levinthal's broth and subcultured into fresh broth which was incubated for 4 hours in a 37 ~ C. water bath. Colony counts of the 4 hour culture were ~'Penicillinase (Difeo), 0.05 ml., was added to 0.95 ml. ~f serum obtained from children receiving ampicillin. T h e addition of penieillinase eliminated bactericidal activity due to resldual antibiotic, but did not affect bac~e:ieidal antibodies.
The .[ournal o[ Pediatrics February 1972
consistently 3 to 4 x 10 ~ ml. The bacterial suspension was diluted I:1000 in phosphatebuffered saline, and 0.025 ml. (about 7 x 10 '~ colony-forming units per milliliter) was added to each well. No significant differences in bactericidal activities were observed over an inoculum range of 7 x i02 to 7 x 10 '~ organisms. Immediately after mixing, and after one hour of incubation at 37 ~ C., the contents of each well were streaked onto chocolate agar plates with a standard milk loop calibrated to deliver 0.001 ml. T h e plates were incubated for 24 hours at 37 ~ C. in 5 per cent carbon dioxide. A colony count reduction of 50 per cent or greater was required for bactericidal activity to be recorded as positive; almost all plates showed either no reduction or 100 per cent killing. The coefficient of variation for colony counts from a single well was 15 per cent. All sera were tested in duplicate; titers were reproducible within a single 2-fold serum dilution. Each set of tests included control plates of complement and bacteria alone, and bacteria and phosphate-buffered saline only, to identify killing not due to specific antibodies. Hemagglutination. H u m a n type O, Rhnegative erythrocytes were fixed with glutaraldehyde 1~ and stored as a 30 per cent suspension in phosphate-buffered saline with 0.1 per cent Merthiolate at 4 ~ C. for as long as 30 days. The titers of standard sera were not altered when tested with cells stored for this long a period. A solution of polyribose phosphate (kindly provided by Drs. David Smith and Porter Anderson, Children's Hospital, Boston, Mass.) prepared from the capsular material of H. influenzae, type b was the antigen used to sensitize the red ceils. To accomplish this, 1 ml. of a 2.5 per cent suspension of red cells was added to 1 ml. of polyribose phosphate (0.5 /~g per milliliter) and shaken for 1 hour at 37 ~ C. Sensitized cells were washed 5 times in phosphate-buffered saline and then added in 0.025 ml. volumes to equal amounts of serum serially diluted in phosphate-buffered saline containing 0.5 per cent bovine serum albumin in the micro-
Volume 80 Number 2
Immunologic responses to H. influenzae meningitis
,5'
211
~AC'~E~tCtDAt TITER HA
TI'rER
.4-
O (3O CN
/t , 4 / ',
9:3"
.2"
O /~\3
~1 / ---4
2'0
'/
3'0
/ "~" 4'0 TUBE
/
",
/
/
, . ~ 512 i on
! I |
to HUMBER
,
6'o
Fig. 1. Convalescent serum separated by Sephadex G-200 gel filtration into pools represented by numbers 1 to 4. Pool 1 contained all recovered IgM and Pool 3 the major portion of IgG; these pools were tested for bactericidal and hemagglutinating activity. Pool 1 was devoid of bacter:cidal activity; hemagglutinatlng activity was present in both pools.
titer U-shaped plates. After mixing by tapping, the plates were incubated for one hour at 37 ~ C. They were read 4 hours later for agglutination. The titer of the sera was taken to be the highest dilution in which agglutination was visible. Controls for each set of tests included commercial antiserum against H. influenzae, type b (Hyland Laboratories--titer 1.:2048 to 1:4096), unsensitized cells plus serum, and sensitized cells with diluent only. Sera were tested in duplicate and their titers were reproducible within one 2-fold serum dilution. When sera exhibiting hem.agglutination (HA) titers were absorbed with polyribose phosphate-sensitized red cells, their titers decreased to 1:2 or less. Consequently, titers greater than 1:2 were considered to be indicative of antibody activity. Fractionation of serum. Serum ( 1 ml.) was placed on a 2.7 x 105 cm. Sephadex G-200 column equilibrated with phosphatebuffered saline, p H 7.4 (kindly performed by Dr. George Abraham, Department of Medicine, University of Rochester School of Medicine). The fractions obtained were divided into 4 pools corresponding to the elution profile (Fig. 1), concentrated by pervaporation to the original volume and then tested for the presence of IgG and IglV[ by immunodiffusion in agar with class-specific
antisera. Pool 1 contained all tile recovered IgM and Pool 3 the major portion of the IgG; both were tested for hemagglutinating and bactericidal activity. RESULTS Bactericidal and hemagglutinating antibody. Table I summarizes the data from 22 patients who showed no rises in either bactericidal (BA) or hemagglutinating (HA) antibody titers and the 7 patients who had 4-fold or greater antibody rises. Among the latter, 5 had increases in both HA and BA, 1 H A only, and 1 BA only. Bactericidal titers rose from 0 in acute sera to a range of 4 to 64 in convalescent sera from 5 patients; one patient (11 years old) with a BA titer of 32 in the acute serum had an increase in titer to 128 in convalescent serum. H A titers rose from less than 2 in acute sera to a range of 32 to 512 in convalescent sera from 5 patients. The l 1-year-old girl with bactericidal antibodies in acute serum had an H A titer of 64 in acute serum which subsequently rose to 512. No differences were observed between the two groups (those with antibody responses and those without) in the frequency of bacteremia, peak leukocyte counts, spinal fluid glucose levels, duration of symptoms before institutio:: of antibiotic therapy, and dura-
2 12
Norden et al.
The Journal of Pediatrics February 1972
Table I. Antibody responses of children with H. influenzae, type b meningitis
Antibody Number 22 7
response
No Yesi"
Number with presence of antibodies
Age range~mean (months) 3- 25/11 9-132/54
Acute 0 1
BA I ]Convalescent 0 6
Acute 1 1
HA I Convalescent 1" 6
'~tIA titer 1:16 in both acute and convalescent sera. t F o u r f o l d or greater
increase in convalescent sera
of either H A or BA titers.
Table II. Serologic data 6 to 23 months after meningitis
lnitial antibody response No Yes~"
Number 7 3
Number with antibodies in repeat sera BA t HA 0 0 2 1
"~Ag in Table I.
tion of antibiotic therapy. All children were treated with ampicillin 300 mg. per kilogram, intravenously, for approximately 14 days during hospitalization. The most striking difference between the two groups was in their ages. A mean of 11 + 7 months was noted for the 22 nonresponders and 54 + 39 months for the responders (t = 4.8 p < 0.001). Among 22 children less than 24 months of age, only 1 had an antibody response, whereas 6 of 7 over 24 months responded (Fisher's exact test, p = 0.0001). Twelve children, 4 to 20 months of age, who had no antibody response to H. influenzae, type b were found to have significant HA titers to tetanus and diphtheria and normal isohemagglutinin titers. (These tests were kindly performed by Dr. Robert H. Schwartz, Department of Pediatrics, University of Rochester School of Medicine. ) Bactericidal antibodies with patients' own organisms. Acute and convalescent sera from 5 children who showed no bactericidal antibodies with our standard H. influenzae, type b strain also were tested with organisms isolated from their own cerebrospinal fluids. These tests were performed as were all the others, and none demonstrated antibody. Follow-up studies. Sera were obtained
from 10 children 6 to 23 months after their acute illnesses. None of the 7 children who had had no antibody responses during their hospitalizations had either H A or BA titers in subsequent bleedings (Table I I ) . Two of 3 children who had had antibody titer increases during hospitalization had persistent BA; one also had a significant HA titer. The third child had rises in both H A and BA during hospitalization, hut neither test was positive 18 months later (Table I I ) . Serum fractionation. Convalescent sera obtained from 2 patients were fractionated and Pools 1 ("IgM rich") and 3 ("IgG rich") were tested for BA and H A antibodies. The results for one serum are shown in Fig. 1; the results with the other were similar. The BA titer of the " I g G " fraction was equal to that of the whole serum (64) while the " I g M " fraction was negative. H A titers of 1:512 (equal to the whole serum) and 1:4 were obtained with the " I g M " and " I g G " fractions, respectively. DISCUSSION That 22 of 29 children with H. influenzae, type b meningitis failed to produce either BA or HA antibodies was surprising; even more so was the finding that all but one of the 22 were less than 2 years of age. No antibodies were found in 7 of these children who were retested 6 to 23 months after their acute illnesses. There are several possihle expianations for the failure of 76 per cent of the study population to develop antibodies to H. in/tuenzae, type b and for the striking differences between the antibody responses of those less than and those more than 2 years of age. First, the techniques for measuring
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Immunologic responses to H. influenzae meningitis
these antibodies may be too insensitive to detect small quantities. This possibility cannot be excluded, but seems unlikely since antibodies were consistently detected in older children. A second explanation is that the strain selected for bactericidal antibody assays was unusually resistant to serum activity. However, the paired sera from 5 children were tested for bactericidal activity against both their own as well as the standard culture and in no case were the results discordant. A third possibility is that the final sera were obtained too early to permit the detection of rising antibody titers. In 11 of the 22 children without antibody responses, the second bleedings were performed 14 days or more after hospitalization; additionally, 7 of the 22 still had no detectable antibodies 6 to 23 months later. T h a t maternal antibodies to H. influenzae, type b persisted in some children and inhibited their own antibody responses is a fourth possibility. This hypothesis seems untenable in the 10 children over one year of age, a time when maternal antibodies ordinarily are no longer present. The possibility that these children are immunologically immature or incompetent can be discounted since they had adequate antibody responses to diphtheria and tetanus antigens and normal isohemagglutinin titers. Early treatment with antimicrobial agents has been shown to suppress the formation of bacteriostatic antibodies to beta hemolytic streptococci? 1 This possibility could not explain the age-related failure to form BA or HA antibodies to H. influenzae, type b since all patients, regardless of age, were treated at approximately the same time in the course of their illnesses. H. influenzae, type b could be a poor immunizing agent, but even if true, that would not explain the age-related difference in antibody response to infection. Preliminary studies 8, ~ with subcutaneous injection of purified polysaccharides from the capsules of this organism showed that they are potent antibody inducers in adults, supporting the conclusion that the failure of most children
2 13
with influenzal meningitis to acquire H A or BA antibodies is not related to the antigenie properties of this organism. Finally, the phenomenon of "immune paralysis" as an explanation for the failure of younger children to show an antibody response to influenzal meningitis should be considered. Felton and associates is reported that the administration of pneumococcal polysaccharides in quantities 500 times greater than immunizing doses produced longlasting and total inhibition of the ability of adult mice to produce neutralizing antibodies to them. There is no evidence that this phenomenon has been induced in man during natural infections. Both meningitis and bacteremia caused by H. inlTuenzae, type b result in the dissemination of a large number of organisms and their antigenically potent potyribose phosphate capsular material. Younger and smaller children might be overwhelmed by this antigenic load with "immune paralysis" while a comparable antigenic load could be handled with appropriate antibody responses by older children. The initial failure to produce antibodies appeared to be persistent 6 to 23 months after the acute illness; this could be in keeping with Felton and associates' experimental observations. Some clarification might be obtained by measuring the effect of injecting such nonresponding children with purified H. influenzae, type b polysaccharide. If the hypothesis of "immune paralysis" is correct, such children would be expected to again fail to show antibody responses to immunization with the polysaccharide. We were unable to obtain purified polysaccharide material with which to conduct such a trial. " I m m u n e paralysis," as originally described, persisted for the life-span of the adult mouse. It was postulated 13 that in order for "immune paralysis" to persist, antigen must remain in the tissues. It is conceivable that in younger children with H. influenzae meningitis, the failure to respond with formation of BA and HA antibodies could be temporary and related to the large antigenic load, and that if chal-
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Norden et al.
lenged subsequently with the same organism, they would be capable of responding. Although the explanation for the age-related failure to produce antibodies to H. influenzae meningitis is still unclear, " i m m u n e paralysis" is an attractive speculation which might relate patient age, size of the infecting inoculum, a n d antibody response. ADDENDUM Following the preparation of this paper, Peter and associates (Abstract, Society for Pediatric Research, 1971, p. 76) reported that only 5 of 17 children hospitalized with H. influenzae, type b meningitis, epiglottitis, or arthritis acquired antibodies to their infections. Their ages in relation to antibody responses were not provided in the abstract, but during the presentation, it was stated that the children with antibody responses were all over 2 years of age, which is in agreement with our findings. We are indebted to Mrs. Phyllis Meyer and Mrs. Linda Witinski for their excellent technical assistance and to Dr. Harry A. Feldman for his advice and critical review of the manuscript. We would like to express our appreciation to Drs. David Smith and Porter Anderson for helpful discussions concerning methodology. REFERENCES
1. Sell, S.: The clinical importance of Hemophilus infiuenzae infections in children, Pediatr. Clin. North Am. 17: 415, 1970. 2. Turk, D., and May, J.: Hemophilus influenzae: Its clinical importance, London, 1967, The English Universities Press, Ltd.
The Journal of Pediatrics February 1972
3. Sproles, F., Azerrad, J., Williamson, C., et al.: Meningitis due to Hemophilus influenzae: Long term sequelae, J, PEma:rm 75: 782, 1969. 4. Fothergill, L. D., and Wright, J.: Influenzal meningitis: The relation of age incidence to the bactericidal power of blood against the causal organism, J. Immunol. 24: 273, 1933. 5. Collier, A. M., Connor, J. D., and Nyhan, W. L.: Systemic infection with Hemophilus influenzae in very young infants, J. PEDIATR. 70: 539, 1967. 6. Mathies, A. W., Hodgman, J., and Ivler, D.: tiemophilus influenzae meningitis in a premature infant, Pediatrics 35: 791, 1965. 7. Norden, C., Callerame, M., and Baum, J.: Hemophilus influenzae meningitis in an adult: A study of bactericidal antibodies and immunoglubulins, N. Engl. J. Med. 282: 190, 1970 8. Robbins, J., Schneerson, R., Rodrigues, L., et al.: Characterization of "natural" infection and immunization-induced anticapsular antibodies to H. influenzae, type b, Pediatr. Res. 40: 376, 1970. (Abst.) 9. Dulbecco, R., and Vogt, M.: Plaque formation and isolation of pure lines with poliomyelitis viruses, J. Exp. Med. 99: 167, 1954. 10. Bing, D., Weyland, J., and Stavltsky, A.: Hemagglutination with aldehyde-fixed erythrocytes for assay of antigens and antibodies, Proc. Soc. Exp. Biol. Med. 121: 1166, 1967. If. Daikos, G., and Weinstein, L.: Streptococcal bacteriostatic antibody in patients treated with penicillin, Proc. Soc. Exp. Biol. Med. 78: 160, 1951. 12. Smith, D., Anderson, P., Johnston, R., et al.: The role of polyribophosphate in immunity to Hemophilus influenzae b, J. Clin. Invest. 49: 90a, 1970. (Abst.) 13. Felton, L., Kaufman, G., Prescott, B., et al.: Studies on the mechanism of the. immunological paralysis induced in mice by pneumoeoccal polysaccharides, J. Immunol. 74: 17, 1955.