Immunogenicity of Haemophilus influenzae type b polysaccharide-outer membrane protein conjugate vaccine in patients who acquired Haemophilus disease despite previous vaccination with type b polysaccharide vaccine

ORIGINAL ARTICLES

Immunogenicity of t-laemophilus influenzae type b polysaccharide-outer membrane protein conjugate vaccine in patients who acquired Haemophilus disease despite previous vaccination with type b polysaccharide vaccine Dan M. Granoff, MD, A n i t a C h a c k o , MD, K a t h l e e n R. L o t t e n b a c h , BA, a n d K a t h e r i n e E. Sheetz, RN From the Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, and the Division of Infectious Diseases, St. Louis Children's Hospital, St, Louis To investigate the basis of the immune d e f e c t in children who a c q u i r e invasive Haemophilus disease despite previous v a c c i n a t i o n with Haemophilus influen. zae type b (Hib) p o l y s a c c h a r i d e v a c c i n e , we d e t e r m i n e d the ability of v a c c i n e failure patients with low levels of serum anticapsular a n t i b o d y ( < I #g/ml) to respond to reimmunization. Thirty-four patients, ranging in a g e from 27 to 61 months, were v a c c i n a t e d with either Hib p o l y s a c c h a r i d e (n = 20) or Hib p o l y s a c c h a r i d e - o u t e r m e m b r a n e protein c o n j u g a t e v a c c i n e (n = 14). All but three of the children had normal serum concentrations of immunoglobulins, including IgG2. The g e o m e t r i c mean serum anticapsular a n t i b o d y concentration of the group given p o l y s a c c h a r i d e v a c c i n e increased from 0.27 ~g/ml before v a c c i n a t i o n to 0.65 ~g/ml 1 month later (p <0.05), but the m a g n i t u d e of the response was nearly 10-fold less than that of 31 a g e - m a t c h e d control children given p o l y s a c c h a r i d e v a c c i n e (6.3 ~g/ml, p <0.001). In contrast, all 14 patients with v a c c i n e failure who were given c o n j u g a t e v a c c i n e showed increases of fivefold or more in serum anticapsular a n t i b o d y ( g e o m e t r i c means 0.35 and 12.8 ~g/ml, respectively; p <0.001). All patients with v a c c i n e failure who did not respond to p o l y s a c c h a r i d e v a c c i n e were subsequently given c o n j u g a t e v a c c i n e , and all had high a n t i b o d y responses. Most patients tested showed increases in c o m p l e m e n t - m e d i a t e d serum b a c t e r i c i d a l activity. These d a t a suggest that immunization with c o n j u g a t e v a c c i n e confers protection a g a i n s t H i b disease to children who, b e c a u s e of g e n e t i c or other reasons, c a n n o t respond to the u n c o n j u g a t e d form of the p o l y s a c c h a r i d e v a c c i n e . (J PEDIATR 1989;114:925-33)

Supported by U.S. Public Health Service grant No. AI 17962 and by a grant from Merck Sharp & Dohme Research Laboratories, West Point, Pa. The results of this study were presented at the annual meeting of the Society for Pediatric Research, Washington, D.C., May 5, 1988. Submitted for publication Oct. 24, 1988; accepted Dec. 6, 1988. Reprint requests: Dan M. Granoff, MD, St. Louis Children's Hospital, 400 South Kingshighway Blvd., St. Louis, MO 63110.

Invasive Haemophilus influenzae type b disease has been reported in children previously vaccinated with Hib polysaccharide vaccine. 1-3 Most of the children who acquired the disease had been healthy and had normal serum concentrations of immunoglobulins, including IgG2? '3 They also had normal serum levels of antibody to the tetanus toxoid protein, but many had evidence of impaired

925

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The Journal of Pediatrics June 1989

See related article, p. 1006.

I

Hib

Haemophilus influenzae type b

I

serum antibody responses to the type b capsular polysaccharide after recovery from Hib disease? Several new Haemophilus conjugate vaccines, consisting of type b capsular polysaccharide coupled to different carrier proteins, are under investigation (reviewed in reference 4). In December 1987 the first of these conjugates, Hib conjugate vaccine (diphtheria toxoid conjugate), was licensed for use in the United States? Considerable data indicate that conjugate Hib vaccines are more immunogenic in infants and children than Hib polysaccharide vaccine? 9 Further, after priming with conjugate, booster antibody responses are observed after reinjection with either conjugate or conventional Hib polysaccharide vaccinesJ ~ In contrast, vaccination with unconjugated Hib polysaccharide vaccine does not prime the immune system for a booster antibody response. 1~,~3 These data suggest that Hib conjugate vaccines can activate and prime the immune system to produce an antibody response in infants whose immature B cells are otherwise unresponsive to vaccination with unconjugated Hib polysaccharide vaccine. Most children in whom Hib disease develops after vaccination with Hib polysaccharide vaccine are 24 months of age or older at the time of immunization and are in otherwise good health. 13 Despite intensive study, ]4 the immunologic defect underlying the impaired antibody responses of these patients to Hib polysaccharide remains unknown. We wanted to determine whether serum anticapsular antibody develops in these children when they are revaccinated with Hib polysaccharide vaccine or are vaccinated with a prototype Hib conjugate vaccine. METHODS Vaccines. The conjugate vaccine used in this study was supplied by Merck Sharp and Dohme Research Laboratories (West Point, Pa.) and consisted of Hib polysaccharide covalently coupled to an outer membrane protein complex of Neisseria meningitidis, group B. 14aIn our study two 10ts of conjugate vaccine were used: Lot No. 1069/C-P241 was given to 10 children (20 injections), and each 0.75 ml dose contained 15 #g of polysaccharide and 148 #g of protein. Lot No. 1072/C-P298 was given to 18 children (35 injections), and each 0.5 ml dose contained 15 ~zg of polysaccharide and 131 #g of protein. There were no significant differences in the incidence of side reactions to the two lots of vaccine or in the magnitude of the antibody

responses. Therefore the data from the two lots were combined. The lyophilized conjugate vaccine was reconstituted with a diluent containing aluminum hydroxides and was used within 4 hours. Commercially available unconjugated Hib polysaccharide vaccine (Lederle, Praxis, or Connaught) was administered to 20 children. Each 0.5 ml dose of this vaccine contained 25 tzg of capsular polysaccharide. Both the polysaccharide vaccine and the conjugate vaccine were administered intramuscularly. Patient selection. The protocol was approved by the human studies committee of Washington University School of Medicine. The population eligible for the study included 11 l children, 24 to 61 months of age, in whom invasive Hib disease developed >--21 days after they were vaccinated with the Hib polysaccharide vaccine and who had a serum sample obtained 2 to 24 months after the onset of their disease. These children resided in 30 states, the District of Columbia, and two provinces in Canada. Forty-two (38%) of the children were excluded from the study because they had convalescent serum anticapsular antibody levels to type b polysaccharide >2 lzg/ml as measured by a radioantigen binding assay. Of the remaining 69 patients, 34, from 19 states, were enrolled with their families' permission at a mean interval _+ SD of 12 _+ 6 months after the onset of Hib disease (range 2 to 24 months). All 34 patients who were enrolled had serum anticapsular antibody concentrations <1 #g/ml, whereas (37%) of the 35 eligible children who were not enrolled had antibody concentrations between 1 and 2 #g/ml. Thus the children enrolled in the trial were selected from the group of patients with the lowest concentrations of serum antibody after recovery from disease. Vaccination. Enrollment in the study was deferred if the child had received any vaccination in the previous week or had a history of fever in the preceding 72 hours. After vaccination, parents were contacted by telephone within 48 hours to ascertain the incidence of adverse reactions. In addition, parents were provided a thermometer and clinical follow-up forms anal were instructed to record axillary temperature and possible adverse reactions for 5 consecutive days after vaccination. No significant local or systemic adverse reactions were noted with either vaccine. Of the 34 children, 30 were assigned randomly to receive either one dose of polysaccharide vaccine (16 children) or two doses of conjugate vaccine separated by 2 months (14 children). The remaining four children were enrolled before the availability of the conjugate vaccine and received one dose of the polysaccharide vaccine. The clinical characteristics of these four children and their antibody responses were indistinguishable from those of the other 16 children assigned randomly to receive the

Volume 114 Number 6

polysacchride vaccine. Therefore the results from the 20 children were combined. Serum was obtained before vaccination and again 1 to 2 months after each injection of vaccine for measurement of antibody to type b polysaccharide. Of the 20 children given polysaccharide vaccine, 14 had <2 #g/ml of serum anticapsular antibody after vaccination and were crossed over to the conjugate arm of the study. (All but one of these 14 subjects had antibody concentrations <1 #g/ml.) Control subjects. For a control group we initially considered enrolling patients who had Hib disease after 24 months of age but who had never received Hib vaccine. However, our previous studies indicated that, in nearly all unvaccinated patients of this age, serum anticapsular antibody concentrations >2 #g/ml develop after recovery from disease, ~,14and therefore these children would not be eligible for enrollment. Thus the serum antibody responses of the 20 vaccine failure patients reimmunized with Hib polysaccharide vaccine were compared with those of 31 healthy children from Missouri and Illinois who had been vaccinated with Hib polysaccharide vaccine as part of previous studies. 15,16 The control subjects ranged in age from 24 to 61 months (mean _+ SD = 41 _+ 10) and were selected to correspond to the ages of the study patients. In addition, all the control children selected had <2/zg/ml of anticapsular antibody in prevaccination serum samples to correspond with the prevaccination serum antibody levels of the patients whose vaccination failed. (Nine control subjects with higher levels of antibody in preimmunization sera were excluded.) Additional control sera were obtained from 13 healthy children, 24 to 48 months of age (mean _+ SD = 32 _+ 8), who were given one injection of conjugate vaccine as part of immunogenicity studies sponsored by Merck Sharp and Dohme Research Laboratories and conducted at several other study sites in the United States. These control sera were provided by Dr. Philip P. Vella and were selected only on the basis of the age of the subject (>2 years), availability of sufficient sample volumes, and presence of antibody concentrations in prevaccination sera of <2 /zg/ml as determined in our laboratory. Serologic studies. All sera were assayed at Washington University. Serum concentrations of IgG, IgA, and IgM were measured by nephelometry (ICS, Beckman Instruments Inc., Brea, Calif.). Serum concentrations of IgG2 were measured by a particle concentration fluorescence immunoassay17 with the murine monoclonal antibody GOM1 (see later discussion). Total serum antieapsular antibody concentrations to Hib polysaccharide were measured by a radioantigen binding assay. ~,18 Prevaccination and postvaccination serum samples from each subject were

Immunogenicity o f conjugate vaccine

927

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AGE (MONTHS) Fig. I. Serum antibody response to Hib polysaccharide vaccine in relation to age of vaccination. Each circle represents antibody concentration of an individual patient. Lines represent best fit for mean § 95% confidence interval for antibody concentrations of immunized control children of different ages. Patients with vaccine failure had lower antibody responses to reimmunization than healthy control children immunized for first time (geometric means of 0.65 vs 6.3/zg/ml, p <0.001).

assayed in parallel. The U.S. Office of Biologics postimmunization adult serum pool was the reference standard. The mean variation in values for replicate samples tested on different days over an 18-month period was 10.3%. The IgG, IgG1, and IgG2 antibody responses were measured by enzyme-linked immunosorbent assay, 15.19 with Hib polysaccharide coupled to poly-L-lysine as the antigen on the plate. Biotinylated goat-antihuman IgG was used to detect IgG. The reagent used to measure IgG1 antibody was a murine monoclonal antibody, HG11, prepared at Washington University School of Medicine.2~For measurement of IgG2, a mixture of two murine monoclonal antibodies+ HP6014 at a 1:10,000 dilution and GOM1 at a 1:500 dilution, was used (ICN Biomedicals, Lisle, I11.)?t The antibody concentrations in the test sera were quantirated in micrograms of antibody per milliliter of serum as described elsewhereY 9,22 Complement-mediated bactericidal activity was measured as previously described23.24 against log-phase cells of H. influenzae type b strain Eagan. All test sera were heat inactivated at 56 ~ C for 1 hour before use. The complement source was serum from an adult with agammaglobulinemia and normal complement levels as described previ-

9 28

Granoff et al.

The Journal of Pediatrics June t989

T a b l e I. Characteristics of study groups

Polysaccharide vaccine group (n = 20)

White race Male sex Disease Meningitis Epiglottitis Other Serum IgM, IgA, IgG, or igG2 > 2 SD helow mean for age Primary immunization with po!ysaccharide vaccine Onset of Haemophilus disease Reimmunization (entry into study)

No.

%

15 11 13 4 3 2

Age'

Conjugate vaccine group (n = 14) No.

%

75 55

12 6

86 43

65 20 15 10

11

78

27 + 5 (2t-41) 32 + 5 (22-43) 43 _+ 7 (27-54)

1

7

2

14

1

7

Age*

26 _+7 (18-44) 32 + 7 (21-45) 46 _+ 9 (31-61)

*Mean numberof months_+SD (range).

ously23; the complement was used at a final concentration of 20%. The precentage of survival of bacteria was calculated by comparing colony counts initially and after 60 minutes' incubation in the assay mixture. Bactericidal titers were defined as ,~he dilution of test serum yielding >90% killing. Each assay included the following controls: samples with complement alone and samples with test sera and heat-inactivated complement: During the incubation period the colony counts increased in these control vials (i.e., > 100% survival). Statistical analysis. Analysis of antibody response was performed on logarithmically transformed data. Previous analysis of the antibody responses of children given Hib polysaccharide vaccine indicated a significant regression of the log postimmunization antibody concentration on age in months? 5 Therefore we used covariance analysis to compare the geometric mean concentrations of serum antibody of the vaccine failure and control children given Hib polysaccharide, with age as a covariate. However, in analyzing the antibody responses to the conjugate vaccine, we did not find age to be a significant covariate in children vaccinated at >24 months of age. Therefore the Student t test was used to compare the geometric mean antibody levels between groups given conjugate vaccine. A paired t test was used to compare antibody concentrations in prevaccination and postvaccination samples within a group. RESULTS Clinical characteristics. Table I summarizes the clinical characteristics and age distribution of the vaccine failure

subjects assigned to receive polysaccharide or conjugate vaccine. Both groups were predominantly white, and meningitis was the most common diagnosis. There were no significant differences in the two groups with respect to the mean ages of primary immunization With Hib polysaccharide vaccine, mean ages of onset of Hib disease, or mean ages of reimmunization at the time of entry into our study. With the exception of their episodes of Hib disease, nearly all the children in both vaccine groups were healthy and none had a history of other episodes of bacteremia. One child assigned to the polysaccharide vaccine group had sickle cell disease, and one child in the conjugate group had a ventriculoperitoneal shunt in place. Two patients in the p01ysaccharide vaccine group (10%), and one in the conjugate group (7%) had low serum concentrations of IgG (259, 351, and 387 mg/dl, respectively; lower limit of 95% confidence interval for children 24 to 47 months, 424 mg/dl); the child in the conjugate also had low serum IgM and IgA concentrations (46 mg/dl and 17 mg/dl, respectively). None of the 34 study patients had low levels of serum igG2 (<40 mg/dl). Antibody response to Hib polysaccharide vaccine. Before vaccination the geometric mean antibody concentration in the 20 patients with vaccine failure who were assigned to receive polysaccharide vaccine was 0.27 ~g/ml (Table II). One month after vaccination the geometric mean serum antibody concentration had increased significantly to 0.65 ~g/ml (p <0.05). Fig. 1 shows the ages of the 20 patients at the time of vaccination and the coresponding anticapsular antibody concentrations in sera obtained 1 to 2 months after vaccination. The patients with vaccine failure had

Volume 114 Number 6

lrnmunogenicity o f conjugate vaccine

929

Table IL Summary of antibody responses to vaccination

Vaccine Vaccine failure patients Polysaccharide Conjugate Randomly assigned group Crossover group~: Healthy subjects Polysaccharide Conjugate

Age at immunization (mo)

Anticapsular antibody (ug/ml)*

No. tested

Mean _+ SD

Range

Prevaccination (total)

20

44 _+ 9

27-54

0.27

14 I4

45 _+ 8 43 _+ 8w

31-61 29-54

0.35 0.28

31 13

41 _+ 10 32 _+ 8

24-61 24-48

0.36 0.66

After dose

After 2 doses

Total

IgG

Total

igG

0.65


ND

ND

12.8 5.2

5.8 2.2

16.2 i2.7

9.1 7.6

6.3 19.1

3.5 9.5

ND ND

ND ND

ND, Not done. *Numbers representgeometricmeans.The vaccinefailurepatientsgivenpolysaccharidevaccinehad significantlylowerantibodyresponsesthan the Controls givenpolysaccharidevaccine(p < 0.001) or the vaccinefailurepatientsgivenconjugatevaccine(p < 0.001). After a secondinjectionof conjugate,there was a significantincreasein antibody(p < 0.05 by paired t test). For statistical analysisof other comparisons,see text. "~Belowlimitoi"IgG assay. :~Frompolysaccharidegroup;selectedbasedon failureto respondwith serumantibodyconcentration>1.9 tzg/ml. w at time of first conjugatedose.

significantly lower serum antibody responses to the polysaccharide vaccine than the healthy children (geometric mean antibody concentrations of 0.65 vs 6.3 #g/ml, p <0.001 by analysis of covariance with age as a covariate). Antibody levels >1 #g/ml developed in none of eight patients with vaccine failure who were revaccinated between 27 and 40 months of age, compared with 12 of 15 control subjects (p <0.001). Among the 12 vaccine failure patients revaccinated between 40 and 61 months of age, antibody levels >1 #g/ml developed in 6, in comparison with 15 of 16 control subjects of similar age immunized for the first time (p <0.03). The 6 patients with vaccine failure whose serum antibody responses were >1 ~g/ml did not differ significantly (p >0.05) from the 14 patients with vaccine failure whose serum antibody responses were <1 ~zg/ml with respect to the distribution of diagnoses of meningitis, epiglottitis, or other Hib disease (data not shown), their respective mean ages of primary immunization with Hib polysaccharide vaccine (27 vs 27 months), or mean ages at onset of Hib disease (31 vs 33 months). Of the 14 patients, 2 (14%) with <1 #g/ml of antibody in postvaccination sera had low serum concentrations of total IgG, whereas none of 6 patients with higher antibody responses did (p >0.5). Antibody response to conjugate vaccine. Before vaccination the geometric mean serum antibody concentration of the 14 vaccine failure patients assigned to receive conjugate vaccine was 0.35/~g/ml. Fig. 2 shows preimmunization and postimmunization serum antibody concentrations of the individual vaccine failure patients given conjugate. For comparison the corresponding antibody concentrations of the vaccine failure patients given Hib polysaccharide

vaccine are shown. After one injection of conjugate vaccine all 14 patients had at least fivefold increases in serum antibody levels, and the geometric mean increased to 12.8 ~g/ml (p <0.00i, compared with the postimmunization geometric mean antibody concentration of the vaccine failure patients given polysaccharide vaccine), o f the 14 patients in the conjugate vaccine group, 13 received a second injection of conjugate Vaccine, and there was a small further increase in the geometric mean antibody to 16.2 #g/ml (p <0.05 by paired t test). As summarized in Table II, the geometric mean antibody responses of the 14 vaccine failure patients given the first injection of conjugate vaccine did not differ significantly from those of 13 healthy control children given one injection of conjugate vaccine. The antibody responses of the vaccine failure patients to a second injection of conjugate vaccine could not be compared With those of the control children because the latter received only one injection.

Antibody response of study patients crossed over to the conjugate arm. The 14 vaccine failure patients with <2 izg/ml of serum anticapsular antibody after revaccination with polysaccharide vaccine were crossed over to the conjugate arm of the study. After one injection of conjugate vaccine, all 14 children had fourfold or greater increases in serum antibody concentrations (Table II). However, their geometric mean titer was significantly lower than that of the 14 vaccine failure children randomly assigned to receive the conjugate vaccine (p <0.01). The lower antibody responses of the patients transferred from the polysaccharide vaccine group were expected because in selecting these patients we excluded six children who had had high antibody responses to the polysaccharide vaccine.

930

Granoff et al.

The Journal of Pediatrics June 1989

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1:i9. 9. Serum anticapsular antibody concentrations before and 1 to 2 months after vaccination with Hib polysaccharide vaccine, or Hib polysaccharide-outer membrane protein conjugate vaccine. Each circle or triangle represents serum antibody concentrations of an individual patient. Dashed lines represent geometric means of antibody concentrations in each group. Patients given conjugate vaccine had higher geometric mean antibody responses than those given polysaccharide vaccine (p <.001).

Thus the children transferred to the conjugate arm were not equivalent to those assigned directly to receive the conjugate. Of 14 children crossed over to the conjugate vaccine group, 13 were given a second injection of conjugate. Twofold or greater further increases in serum antibody developed in seven (Table II). The fourteenth child in this group inadvertently was given polysaccharide vaccine for his booster dose, and therefore his response was excluded from the analysis. Although he had responded poorly to the initial reimmunization with polysacchar]de vaccine (0.38 /~g/ml of antibody), after priming with Conjugate and boosting with the polysaccharide, he had an increase in antibody from 3.2 to 12 ~g/ml. IgG responses to immunization. IgG anticapsular antibody concentrations were not measured in serum obtained before vaccination because the concentrations of total antibody in these sera were all <1/~g/ml. As summarized in Table II, the 14 vaccine failure study patients randomly assigned to receive cgnjugate vaccine showed high serum concentrations of lgG antibody after the first dose and a significant increase in IgG antibody after the second injection (p <0:05). In contrast, among the 20 vaccine failure patients given polysaccharide vaccine, the geometric mean IgG antibody concentration was <1 ~zg/ml in

serum obtained 1 month after vaccination (below the lower limit of the assay). The 14 patients crossed over from the polysaccharide group to the conjugate group also showed h i g h IgG antibody concentrations, especially after the second dose (Table II). The ratios Of IgG1 to IgG2 antibody concentrations to Hib polysaccharide were determined in postvaccination sera from patients with >1 /~g/ml of IgG antibody ("IgG responders"). Subjects with lower concentrations of IgG antibody wei'e excluded because of the high error in determining subclass ratios in sera with low concentrations of IgG antibody. Although only 4 of the 20 vaccine failure study patients iff the polysaccharide group had IgG responses of >1 #g/ml, IgG1 responses predominated in these four subjects (geometric mean ratio of IgGi to IgG2 = 6.2). Among the healthy control children given polysaccharide vaccine, a similar subclass distribution was observed in sera from the 20 who showed IgG reSponses (geometric mean IgG1 to IgG2 r a t i o = 3 . 9 ) . IgG1 responses also predominated in the vaccine failure patients assigned randomly to receive conjugate vaccine (12 IgG responders; geometric mean IgG1 to IgG2 ratio = 4.5). Among the healthy control children given conjugate Vaccine, the eight IgG responders showed predominantly IgG1 respOnses (geometric mean ratio = 10,4).

Volume 114 Number 6

Immunogenicity o f conjugate vaccine

Bactericidal activity. Complement-mediated bactericidal activity against Hib was measured in serum samples obtained from 12 representative patients given conjugate vaccine. Five of the patients were assigned directly to the conjugate group, and seven patients were transferred from the polysaecharide group. Selection of these 12 children was based on availability of sufficient quantities of sera for testing. Before vaccination with conjugate only 1 of the 12 children had detectable serum bactericidal activity (titers >_ 1:2). In contrast, after receiving two doses of conjugate vaccine, 9 of the 12 children had serum bactericidal activity detected (geometric mean titer of 1:1 2). As shown in Fig. 3, after immunization with conjugate vaccine, there was no significance difference in serum bactericidal titers between the children assigned directly to the conjugate vaccine group and those crossed over from the polysaccharide group. Furthermore, in samples from children in both groups, the magnitude of the bactericidal titers correlated directly with the respective anticapsular antibody concentrations. DISCUSSION We examined the serum antibody responses to Hib capsular polysaccharide in children in whom Hib disease developed despite previous vaccination with Hib polysaccharide vaccine and who had low levels of serum antibody after recovery from disease. Most of the children had impaired antibody responses to reimmunization with the Hib polysaccharide vaccine but had normal antibody responses when immunized with an Hib polysaccharideprotein conjugate vaccine. The immunologic basis for the impaired antibody responses of these patients to the unconjugated polysaccharide vaccine is unknown. All but 2 of the 20 patients in this group had normal concentrations of total serum immunoglobulins, including IgG2. Thus the presence of hypogammaglobulinemia or IgG2 deficiency does not explain their poor antibody responses. The type b polysaccharide is a heteropolymer of ribose and ribitol phosphate? ~ The unconjugated polysaccharide vaccine is thought to be a thymic-independent antigen, 26 capable Of eliciting an antibody response largely in the absence of T cell help. 27 Most healthy infants < 18 months of age fail to produce antibody after immunization with the unconjugated Hib polysaccharide vaccinC s but do respond to conjugate vaccines. 68' 20,30 Healthy children >2 years of age usually respond to both vaccines. However, many of the vaccine failure patients in our study who were immunized between 3 and 5 years of age had an antibody response pattern more typical of infants <18 months of age. Possibly this poor response reflects a developmental delay in ability to respond to the Hib polysaccharide. Alternatively, the vaccine failure patients' inability to

93 1

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64

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1'0

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ANTICAPSULAR ANTTBOOY ~ G / N L

Fig. 3. Relation between bactericidal titer and anticapsular antibody concentration in sera from 12 representative children given two injections of conjugate vaccine. Sera were obtained 1 to 2 months after second injection of conjugate. Each point represents value for an individual patient.

produce antibody after immunization with the unconjugated Hib polysaccharide vaccine may reflect excessive T cell suppressor activity or a constitutive maturation arrest of B cells that at this age can usually be activated and clonally expanded bY the thymic-independent form of this antigen. Whether this functional inability to respond to the type b polysaccharide antigen was induced by the initial immunization with the Hib polysaecharide vaccine given at approximately 2 years of age (i.e., tolerance) or reflects an underlying constituent B cell defect cannot be determined on the basis of the existing data. Coupling the type b polysaccharide to a protein carrier confers thymic-dependent properties to the polysaccharide 3~ and greatly enhances the immunogenicity of the polysaccharide.69 The conjugate vaccine used in this study induces serum antibody in infants as young as 2 months of age,8, z9 and repeated injections boost this response? ~ 29 As previously reported concerning healthy infants immunized with this conjugate, t~ the serum antibody evoked by the conjugate vaccine in the vaccine failure patients had functional activity in vitro against the bacteria. However, three of the vaccine failure patients immunized with conjugate who developed serum anticapsular antibody after vaccination had no detectable serum bactericidal activity when the sera were tested at diIutions of 1:2 or greater. In previous studies there have been immunized healthy subjects whose serum antibody was less active in

932

Granoff et al.

functional assays of complement-mediated bacterial killing or opsonization than would be expected based on antibody concentrations measured in radioantigen binding assays or by enzyme-linked immunosorbent assay. I~,32,33 This phenomenon is incompletely understood, but it cannot be presumed that every vaccinated child who has a serum antibody response as measured by an antigen binding assay is necessarily protected against disease. Conversely, some infants who fail to show a serum antibody response to immunization with conjugate vaccine may be protected from disease, 34 possibly as a result of immunologic priming and the ability to develop a rapid increase in serum anticapsular antibody if the bacteria are encountered subsequently.4, 34 Vaccine failure patients undoubtedly represent a heterogeneous group. The patients in our study represent a select subgroup of low responders to Hib polysaccharide after recovery from Hib disease, and we would anticipate that this subgroup represents the most difficult one to immunize. Therefore the excellent immunogenicity of the Hib polysaccharide-outer membrane protein conjugate observed in these patients is encouraging. Preliminary data also indicate that other Hib conjugate vaccines are immunogenic in children with IgG2 deficiency35 or sickle cell disease, 36 conditions associated with poor antibody responses to unconjugated Hib polysaccharide vaccine. Recently the Haemophilus type b polysaccharide diphtheria toxoid conjugate has been reported to be immunogenic in children 18 to 23 months of age who have recovered from Hib disease? 7 Thus, although the immunologic defects responsible for the impaired antibody responses remain to be clarified, high levels of serum antibody against the type b capsule can be elicited in these patients with the use of conjugate vaccines. These results have important implications for predicting the efficacy of conjugate vaccines in preventing Hib disease, both in healthy children and in children with underlying diseases.

We are grateful to the following physicians for immunizing their patients and for obtaining the serum samples used in this study: Jan Alford, Lilburn, Ga.; Ramzy Attala, Belleville, Ilk; William Barson, Ohio State University, Columbus; Ralph Brown, Baltimore; Robert Bulten, Grand Rapids, Mich.; Lydia Caros, Minneapolis; Subhash Chaudhary, Southern Illinois University, Springfield; James Cooley, Wellesley, Mass.; Robert Daum, Tulane School of Medicine, New Orleans; Thomas Foy, Crystal City, Mo.; Vernon Goller and Donald Guenther, Pendleton, Ore.; Arthur Gower, Manassas, Va.; S. Hwong, Crawfordsville, Ind.; Steven Johnson, Mount Vernon, Wash.; Ray Kundel, Duluth, Minn.; Ashir Kurnar, Michigan State University, East Lansing; Joe McHugh, Wayzata, Minn.; Wayne McNet, Bridgewater, Va.; Carlos Menendez, Greenville, Ohio; David Moore, Charlotte, N.C.; Kevin Murphy, Omaha; Trudy Murphy, University of

The Journal of Pediatrics June 1989 Texas Health Science Center, Dallas; James Nordin, St. Paul; George Penn, Monterey, Calif.; Henry Rikkers, Watertown, Mass.; Stanley Perlman, University of Iowa, Iowa City; Eugene Shapiro, Yale University School of Medicine, New Haven, Conn.; Gary Sides, Festus, Mo.; Charles Snavely, Las Vegas; Thomas Stealy, Edina, Minn.; Andrew Thomas, St. Paul; Richard Van Schoick, Jackson, Mich.; Victor Wong and Kwang Sik Kim, Children's Hospital of Los Angeles; and Gary Wheeler, Bowman Gray School of Medicine, Winston-Salem, N.C. Moon Nahm, Washington University School of Medicine, measured the serum IgG2 concentrations of the patients. Sharon Bader and Venita Boelloeni provided expert technical assistance. Michael T. Osterholm, Minnesota Department of Health, referred a number of the patients and offered helpful suggestions. REFERENCES

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