J. ALLERGY
Paris et al.
21. Wide L, Bennich H, JohanssonSGO. Diagnosis of allergy by an in vitro test for allergen antibodies. Lancet 1967;2:1105-7. 22. Vijay HM, Young NM, Bernstein IL. Studies on Alternarin allergens. VI. Stability of the allergen componentsof Alternaria tenuis extracts under a variety of storageconditions. Int Arch Allergy Appl Immunol 1987;83:325-8. 23. Vijay HM, Young NM, JacksonGED, White GP, Bernstein IL. Studieson Alternaria allergens. V. Comparative biochemical and immunological studies of three isolates of Alternaria tenuis cultured on synthetic media. Int Arch Allergy Appl Immunol 1985;78:37-42. 24. BengtssonA, Karlsson A, Rolfsen W, EinarssonR. Detection of allergens in mould and mite preparationsby a nitrocellulose electroblotting technique. Int Arch Allergy Appl Immunol 1986:80:383-90.
CLIN. IMMUNOL. MAY 1990
25. Tee RD, Gordon DJ, Newman Taylor AJ. Cross-reactivity betweenantigensof fungal extracts studiedby RAST inhibition and immunoblot technique. J ALLERGY CL~ IMMUNOL 1987; 79~627-33. 26. Kroutil LA, Bush RK. Detection of Alternariu allergens by Westernblotting. J ALLERGY CLIN IMMUNOL 1987;80:170-6.
27. Vijay HM, Huang H, Young NM, Bernstein IL. Studies on Alternuria allergens. I. Isolation of allergens from Alternaria teds and Altermria solani. Int Arch Allergy Appl Immunol 1979;60:229-39. 28. Bush RK, Yunginger JW. Standardizationof fungal allergens. Clin Rev Allergy 1987;5:3-21.
Response to a Haemophilus influenzae type b diphtheria CRM,,, conjugate vaccine in children with a defect of antibody production to Haemophilus influenzae type b polysaccharide Lynda C. Schneider, MD,* Richard A. Insel, MD, Gail Howie, RN, Date V. Madore, PhD, and Raif S. Geha, MD Boston, Mass., and Rochester, N.Y. A defect in antibody response to immunization with Haemophilus influenzae type b (Hib) capsular polysaccharide vaccine has been reported in children with recurrent infections and normal immunoglobin levels. We identified 15 children, aged 2 to 6 years, with this defect, and we evaluated their response to immunization with an Hib capsular oligosaccharide diphtheria CRM,,, protein-conjugate vaccine (HbOC). The children received a series of three vaccines: HbOC at 0 and 8 weeks, ana’ the Hib polysaccharide vaccine at 16 weeks. Levels of antibody to the Hib capsular polysaccharide (polyribosyl ribitol phosphate, PRP) and to diphtheria toxoid were obtained before and 4 weeks after each vaccination. The geometric mean serum anti-PRP concentration was 0.17 pgiml before immunization and 29.3 pglml after the second HbOC immunization (week 12). All 15 children had postvaccination anti-PRP antibody levels >I .O pglml after receiving the second HbOC (week 12). In addition, booster responses were observed after the second Hib conjugate in I3 of the patients and after the Hib polysaccharide in four of the patients. All patients with low preimmunization diphtheria titers had a response to the diphtheria toxoid. These results suggest that conjugation of Hib polysaccharide with diphtheria CRM,,, overcomes the defective antibody response to Hib oligosaccharide in children who are initially observed with recurrent infections and inability to respond to the Hib polysaccharide vaccine. (J ALLERGY CLANIMMUNOL 1990;85:948-53.)
From the Division of Immunology, The Children’s Hospital, and the Depattmem of Pediatrics, Harvard Medical School, Boston, Mass.; the Department of Pediatrics and Microbiology, University of RochesterSchool of Medicine and Dentistry, Rochester, N.Y.; and Praxis Biologics, Rochester,N.Y. Supportedby National Institutes of Health Grant AIl7217. Received for publication Aug. 15, 1989. Revised Dec. 22, 1989.
948
Accepted for publication Dec. 27, 1989. Reprint requests:L. Schneider,MD, Division of Immunology, Children’s Hospital, 300 Longwood Ave., Boston, MA 02115. *Lynda C. Schneider, MD, received National Institutes of Health Training Grant HD-07321 and SandozFellowship of the Immune Deficiency Foundation. 1111191345
VOLUME NUMRER
Responseto HbOC by HbPs nonrestmnders 949
85 5
Abbreviations used Hib: Haemophilus injluenzae type b
PRP: Polyribosyl ribitol phosphate HbOC: Hib capsular oligosaccharide diphtheria CRM,,, protein conjugate vaccine HbPs: Hib polysaccharide vaccine GM: Geometric mean A0 : Antibody
A defect in AB responseto immunization with capsular HbPs has been identified in children with recurrent infections and normal immunoglobulin levels. This poor responseto the HbPs has been proposedas a marker for poor AB responseto a variety of viral and bacterial antigens’ and has been associatedwith IgGZsubclass deficiency.’ This appears to be a selective polysaccharide defect because children with normal subclasslevels and many children with IgG2subclass deficiency respond normally to the pneumococcalvaccine.’ However, a recent article has suggested that impaired responseto pneumococcal vaccine may accompany a defective responseto H~Ps.~ Impaired AB responseto Hib vaccine has also been reported in children who developedinvasive Hib diseasein spite of vaccination with HbPs.4Recently Hib protein-conjugate vaccines have been developed and have resulted in improved AB response in normal infants and children,5-*as well asin children who were vaccine failures.’ In this study we wished to determine whether a unique group of children who sufferedfrom recurrent sinopulmonary infections and had not respondedto the Hib polysaccharide vaccine could develop significant AB levels to Hib by immunization with HbGC. The results demonstrate that immunization with the diphtheria CRM,Wconjugate Hib vaccine overcomesthe inability to respond to Hib polysaccharidein this group of children.
mm Patient
population
We determined the responseto the Hib polysaccharide vaccine of children referred for recurrent infections to the Immunology Practice Group at the Children’s Hospital in Boston. We identified 15 children (eight boys and seven giris), aged 2 to 6 years, who had documented recurrent infections including otitis (more than four episodes),sinusitis, and/or pneumonia and who had a 3- to 5-week postvaccination anti-PRP AB level of CO.5 pg/ml after immunization with the Hib polysaccharide vaccine as part of their evaluation in our office. The children were enrolled in the study 1 to 6 months after receiving the Hib polysac-
charide vaccine. Most of the children had also received at least one previous dose of the Hib polysaccharide vaccine at their pediatricians before the vaccination performed as part of their immunology evaluation. We comparedthen AB responsesto the responsesof healthy control subjects, age matchedto within 2 to 3 months, who were mmunized one time with the Hib polysaccharidevaccine. Vaccination
schedule
After informed consent was obtained from the parents, the eligible children were enrolled in a 24-week study. The study design is presentedin Table I. Vaccineswere obtained from Praxis Biologics, Rochester, N.Y. The Hib diphtheria CRM,,, conjugate vaccine (HbGC, HIBTITER) consists of 10 pg of PRP oligosaccharidesdirectly coupled to 25 p,g of a nontoxic cross-reactivemutant diphtheria toxin (CRM,,,). The patients were immunized via the intramuscular route with a series of three vaccines: I-lib diphtheria CRM,,, conjugate vaccine (HbOC) at 0 and 8 weeks and the Hib polysaccharide vaccine (HbPs, commercial name B Capsa1) at 16 weeks. Before and4 weeksafter, diphtheria and PRP AB levels were measuredfor each vaccine. AntiPRP AB levels were also measured8 weeks after the final vaccination. Vaccination of one of the children was delayed a month becauseof a febrile illness. Three sera were lost in laboratory handling. Antibody
quantitation
Serum immunoglobulins and IgG subclasseswere obtained aspart of the routine evaluationof the patients. Serum levels of IgG, IgA, and IgM were quantitated by radial immunodiffusion. IgG subclasseswere measuredby radial immunodiffusion in the Children’s Hospital of Bostan Clinical Immunology Laboratory with a commercially available kit from the Binding Site, Inc. (San Diego, Calif.). Levels of AB to the Hib capsular polysaccharide (PRP) were measuredby radioantigen-binding assay,aspreviously described,‘Oand calibrated with the standardantiserumfrom the Office of Biologics (United StatesFood and Drug Administration). This assayhad a lower limit of detection of 0.040 pg AB per milliliter. IgGI- and IgG2-subclassHib polysaccharide AB levels and diphtheria toxoid AB levels were determinedby enzyme immunoassayas described.” Subclassdeterminations were done with blinded samplesin the laboratory of Praxis Biologics. The lower limit of detection for the IgGl assaywas 0.075 kg/ml, and for the IgG2 assay,0.15 kg/ml. RESULTS Patients’ characteristics
The mean patients’ age was 42 months (range, 26 to 77 months). Ail patients had normal serum levels of IgG, IgA, and IgM. All patients had IgG 1- and
IgG3-subclasslevels within 2 SD of the meanfor their age. Two of these 15patientshad IgGZsubclass levels 2 SD or more below the mean for their age (patient No. 9, lgG2, 44 mg/dl; patient No. 13, lgG2, 25
Schneider et al.
J. ALLERGY
4
0
12
8 Thne (Weeks)
A HbOC
CLIN. IMMUNOL. MAY 1990
A HbOC
FIG. 1. GM AB titer to diphtheria toxoid in 14 children immunized with HbOC.
TABLE I. Study design Visit (wk) 0 Hboc
X
Hib polysaccharide Anti-PM levels Antidiphtheriatiter
X X
4
12
8
18
28
24
X X
X
X
X
X X
X X
X X
Patients received a series of three vaccines. Anti-PRP levels were measuredevery 4 weeks during the study.
mg/dl). Six patients had IgG4 levels 1.0 pg / ml after receiving the second HbOC (week 12). Booster responseswere observed in 13 children after the secondHbOC (week 12); the two children (patients Nos. 3 and 9) who failed to obtain a booster responsehad high preimmunization AB levels (40 and 65 p,g/rnl). Eight of the 15 patients (Nos. 2, 4, 6, 8, 9, 11, 13, and 15) had high AB levels (~20 @g/ml) before receiving the Hib polysaccharide vaccine (week 16) and had negligible change in their postvaccine levels. Of the remaining seven children, four (Nos. 3, 5, 12, and 14) had a boosterresponseto the Hib polysaccharide. The other three children (Nos. 1,7, and 10) had either a decrease or no changein their anti-PRP AB levels. The lowest postimmunization AB concentration occurred in patient No. 7 who had a history of transient hypogammaglobulinemia. He had a maximum level of 1.2 p,g/ml at week 12, but this fell to 0.44 p,g/ml at week 24 of the study and to 0.18 pg/ ml 13 months later. IgGl- and IgGZsubclass ABs to PRP were measuredin 14 of the children. Immunization with HbOC
VOLUME NUMBER
85 5
Response
to HbOC by HbPs nonresponders
951
TABLE Il. Response to Hib polysaccharide vaccine in children with recurrent infections compared to healthy age-matched control children ---.--__ Anti-PRP
level after immunization
(pglml) -..-I_____
Patients
No. 1
2 3 4 5 6 7 8 9 IO 11 12 13 14 15 GM
Control
Subjeats
Age ho)
Before HbPs
After HbPs
Arae (mo)
Before HbPs
After HbPs
47 37 36 36 26 35 53 60 77 35 48 32 41 35 31
0.04 0.04 0.05 0.08 0.22 0.15 0.06 0.06 0.10 0.04 0.12 0.06 0.15 0.04 0.04 0.07
0.10 0.04 0.18 0.22 0.27 0.42 0.22 0.35 0.11 0.06 0.47 0.43 0.27 0.04 0.06 0.16
47 37 36 32 26 35 53 59 77 33 44 32 40 34 31
0.80 1.30 0.76 0.54 0.14 0.08 0.36 0.10 1.10 0.42 0.34 0.11 0.45 0.75 0.36 0.37
3.30 32.00 4.60 3.20 16.82 1.33 13.70 7.60 950 2.80 8.02 -~40.00 :s40.00 16.40 0.11 9.14 --
AB levels were obtained 3 to 5 weeks after each immunization.
resulted in a rise in anti-PRP AB levels of both subclass levels. The prestudy AB levels (week 0) and after the secondHbOC immunization (week 12) titers are presentedin Table IV. Preimmunization IgGl and IgG2 AB levels were close to the lower limit of the sensitivity of the assays. This precluded analysis of the ratio (IgGl :IgG2) of these values. After immunization, the GM IgGl AB level was approximately five times greater than the GM IgG2 AB level, which is approximately in proportion to the normal serum distribution of these two subclasses.‘2~ I3 All patients except for patients No. 7 (with the history of transient h~~~li~rnia) and No. 12 (an IgG2 subclass-delicient patient) had postvaccination IgG2 anti-PIW levels >1.5 pg/ml. As illustrated in Fig. 1, immunization with HbOC also resulted in a rise in diphtheria antibody titer. The GM titers were 0.07 ELISA units before vaccination, 0.77 ELISA units after the first HbOC immunization, and 0.56 ELISA units after the second HbOC immunization. All children had a rise in their diphtheria titers. However, patient No. 7 had a lower diphtheria titer than most other patients (0.17 ELISA units after first HbOC immunization versus GM, 0.77 ELISA units). There were two IgG2 subclass-deficient children (patients Nos. 9 and 12) included in our study. One
IgG2deficient patient (No. 9) had levels similar to the other stndy patients. The seco& I&&deficient patient (No. 12) had slightly lower anti-PRP levels than other children in the study. His maximum antiPRP titer was 8.0 p,g/ml after the secondHbOC immunization (compared to GM, 29.3 pg/ml), and his final anti-PRP concentration (week 24) was 1.2 pg/ml (GM, 12.1 kg/ml), which s&&d be high enoughto confer protection. I4His IgG2 anti-PRPlevel was0.34 pg / ml after the secondHb0C immunization in comparisonto a GM of 4.72 p,g/ ml. His diphtheria titer was 0.24 ELISA units after the first HbGC immunization in comparison to a GM of 0.77 ELISA units. An impaired responseto Hib ~~~o~~de vaccine has been demonstratedin ~liildmn ysuiq~erthan 2 yesrs of age,“+*l5 and in &i&en with recurre5t tions and normaI i~u~l~~ 1eveIs. Adults and children have also been described who s&Wed from twxrrent infections and who lacked an I@3 repe to all polysacchasidesmeasuti. I67” In addition, previously healthy children have dev&oped ittva.siveHib diseasein spite of immunization with poiysac~haride Hib vaccines.4.I4 Thus, these patients with impaired
J. ALLERGY
952 Schneider et al.
TABLE III. Response to HbOC in children
with defect in AB production Anti-PRP
AB concentration
wk0 Patient No.
wk4
CLIN. IMMUNOL. MAY 1990
to Hib polysaccharide
after immunization
(pglml)
wk 12
wk 16
wk 20
Age
Before HbGC No. 1
After HbOC No. 1
After HbOC No. 2
Before HbPs
After HbPs
47 37 36 36 26 35 53 60 77 35 48 32 41 35 31
0.04 0.06 0.03 0.48
6.0 1.2 40.0 3.0 5.0 28.0 0.5 8.0 65.0 5.8
22.0 75.0 34.0 44.0 128.0 175.0 1.2 30.0 60.0 12.0 44.0 8.0 28.0 9.2 78.0 29.3
12.0 50.0 10.0 29.0 16.0 61.0 0.9 31.0 60.0 2.1 22.0 1.8 48.0 7.2 25.0 14.3
7.8 11.0 22.0 44.0 85.0 74.0 0.8 33.0 68.0 2.4 19.0 3.6 58.0 23.0 20.0 17.4
(mol
1 2 3 4 5 6 7* 8 9t 10 11 127 13 14 15 GM
0.10 0.55
0.20 0.52 0.11 0.50 0.16 0.15 0.06 0.23 0.06 0.17
10.0
5.0 20.0 1.3 1.4 5.9
AB levels were obtained 4 weeks after each immunization. *Patient 7 had a history of transient hypogammaglobulinemia. TPatients 9 and 12 had IgGZ-subclass deficiency.
TABLE IV. Subclass
responses
to HbOC GMIAB to PRP hglml)
Before study (week 0) After HbOC No. 2 (week 12) p Value*
IgGl
lgG2
0.10 23.20 1.O pg/mI (GM antibody titer, 12.1 pg/rnl). Thus, the conjugation of Hib oligosaccharide with diphtheria
CRM,,, results in a more immunogenic vaccine for this group of patients. Booster responseswere observed after the second HbOC immunization in all our patients except for two patients who had very high anti-PRP levels after the primary HbOC (65 and 40 u.g of ABs per milliliter). These results are similar to studies of HbOC’* I9 and other conjugates in infants”23 and support the use of a series of Hib vaccines. The third vaccine of our series, the Hib polysaccharide, was administeredto determine if, after priming with conjugate, the patientscould respondto polysaccharide. The patients’ responsesto this vaccine were heterogeneous.Most of the children had high AB titers (>20 p,g/ml) before the Hib polysaccharide vaccine and consequently did not have a booster response. Of seven children with levels
Paris et al.
21. Wide L, Bennich H, JohanssonSGO. Diagnosis of allergy by an in vitro test for allergen antibodies. Lancet 1967;2:1105-7. 22. Vijay HM, Young NM, Bernstein IL. Studies on Alternarin allergens. VI. Stability of the allergen componentsof Alternaria tenuis extracts under a variety of storageconditions. Int Arch Allergy Appl Immunol 1987;83:325-8. 23. Vijay HM, Young NM, JacksonGED, White GP, Bernstein IL. Studieson Alternaria allergens. V. Comparative biochemical and immunological studies of three isolates of Alternaria tenuis cultured on synthetic media. Int Arch Allergy Appl Immunol 1985;78:37-42. 24. BengtssonA, Karlsson A, Rolfsen W, EinarssonR. Detection of allergens in mould and mite preparationsby a nitrocellulose electroblotting technique. Int Arch Allergy Appl Immunol 1986:80:383-90.
CLIN. IMMUNOL. MAY 1990
25. Tee RD, Gordon DJ, Newman Taylor AJ. Cross-reactivity betweenantigensof fungal extracts studiedby RAST inhibition and immunoblot technique. J ALLERGY CL~ IMMUNOL 1987; 79~627-33. 26. Kroutil LA, Bush RK. Detection of Alternariu allergens by Westernblotting. J ALLERGY CLIN IMMUNOL 1987;80:170-6.
27. Vijay HM, Huang H, Young NM, Bernstein IL. Studies on Alternuria allergens. I. Isolation of allergens from Alternaria teds and Altermria solani. Int Arch Allergy Appl Immunol 1979;60:229-39. 28. Bush RK, Yunginger JW. Standardizationof fungal allergens. Clin Rev Allergy 1987;5:3-21.
Response to a Haemophilus influenzae type b diphtheria CRM,,, conjugate vaccine in children with a defect of antibody production to Haemophilus influenzae type b polysaccharide Lynda C. Schneider, MD,* Richard A. Insel, MD, Gail Howie, RN, Date V. Madore, PhD, and Raif S. Geha, MD Boston, Mass., and Rochester, N.Y. A defect in antibody response to immunization with Haemophilus influenzae type b (Hib) capsular polysaccharide vaccine has been reported in children with recurrent infections and normal immunoglobin levels. We identified 15 children, aged 2 to 6 years, with this defect, and we evaluated their response to immunization with an Hib capsular oligosaccharide diphtheria CRM,,, protein-conjugate vaccine (HbOC). The children received a series of three vaccines: HbOC at 0 and 8 weeks, ana’ the Hib polysaccharide vaccine at 16 weeks. Levels of antibody to the Hib capsular polysaccharide (polyribosyl ribitol phosphate, PRP) and to diphtheria toxoid were obtained before and 4 weeks after each vaccination. The geometric mean serum anti-PRP concentration was 0.17 pgiml before immunization and 29.3 pglml after the second HbOC immunization (week 12). All 15 children had postvaccination anti-PRP antibody levels >I .O pglml after receiving the second HbOC (week 12). In addition, booster responses were observed after the second Hib conjugate in I3 of the patients and after the Hib polysaccharide in four of the patients. All patients with low preimmunization diphtheria titers had a response to the diphtheria toxoid. These results suggest that conjugation of Hib polysaccharide with diphtheria CRM,,, overcomes the defective antibody response to Hib oligosaccharide in children who are initially observed with recurrent infections and inability to respond to the Hib polysaccharide vaccine. (J ALLERGY CLANIMMUNOL 1990;85:948-53.)
From the Division of Immunology, The Children’s Hospital, and the Depattmem of Pediatrics, Harvard Medical School, Boston, Mass.; the Department of Pediatrics and Microbiology, University of RochesterSchool of Medicine and Dentistry, Rochester, N.Y.; and Praxis Biologics, Rochester,N.Y. Supportedby National Institutes of Health Grant AIl7217. Received for publication Aug. 15, 1989. Revised Dec. 22, 1989.
948
Accepted for publication Dec. 27, 1989. Reprint requests:L. Schneider,MD, Division of Immunology, Children’s Hospital, 300 Longwood Ave., Boston, MA 02115. *Lynda C. Schneider, MD, received National Institutes of Health Training Grant HD-07321 and SandozFellowship of the Immune Deficiency Foundation. 1111191345
VOLUME NUMRER
Responseto HbOC by HbPs nonrestmnders 949
85 5
Abbreviations used Hib: Haemophilus injluenzae type b
PRP: Polyribosyl ribitol phosphate HbOC: Hib capsular oligosaccharide diphtheria CRM,,, protein conjugate vaccine HbPs: Hib polysaccharide vaccine GM: Geometric mean A0 : Antibody
A defect in AB responseto immunization with capsular HbPs has been identified in children with recurrent infections and normal immunoglobulin levels. This poor responseto the HbPs has been proposedas a marker for poor AB responseto a variety of viral and bacterial antigens’ and has been associatedwith IgGZsubclass deficiency.’ This appears to be a selective polysaccharide defect because children with normal subclasslevels and many children with IgG2subclass deficiency respond normally to the pneumococcalvaccine.’ However, a recent article has suggested that impaired responseto pneumococcal vaccine may accompany a defective responseto H~Ps.~ Impaired AB responseto Hib vaccine has also been reported in children who developedinvasive Hib diseasein spite of vaccination with HbPs.4Recently Hib protein-conjugate vaccines have been developed and have resulted in improved AB response in normal infants and children,5-*as well asin children who were vaccine failures.’ In this study we wished to determine whether a unique group of children who sufferedfrom recurrent sinopulmonary infections and had not respondedto the Hib polysaccharide vaccine could develop significant AB levels to Hib by immunization with HbGC. The results demonstrate that immunization with the diphtheria CRM,Wconjugate Hib vaccine overcomesthe inability to respond to Hib polysaccharidein this group of children.
mm Patient
population
We determined the responseto the Hib polysaccharide vaccine of children referred for recurrent infections to the Immunology Practice Group at the Children’s Hospital in Boston. We identified 15 children (eight boys and seven giris), aged 2 to 6 years, who had documented recurrent infections including otitis (more than four episodes),sinusitis, and/or pneumonia and who had a 3- to 5-week postvaccination anti-PRP AB level of CO.5 pg/ml after immunization with the Hib polysaccharide vaccine as part of their evaluation in our office. The children were enrolled in the study 1 to 6 months after receiving the Hib polysac-
charide vaccine. Most of the children had also received at least one previous dose of the Hib polysaccharide vaccine at their pediatricians before the vaccination performed as part of their immunology evaluation. We comparedthen AB responsesto the responsesof healthy control subjects, age matchedto within 2 to 3 months, who were mmunized one time with the Hib polysaccharidevaccine. Vaccination
schedule
After informed consent was obtained from the parents, the eligible children were enrolled in a 24-week study. The study design is presentedin Table I. Vaccineswere obtained from Praxis Biologics, Rochester, N.Y. The Hib diphtheria CRM,,, conjugate vaccine (HbGC, HIBTITER) consists of 10 pg of PRP oligosaccharidesdirectly coupled to 25 p,g of a nontoxic cross-reactivemutant diphtheria toxin (CRM,,,). The patients were immunized via the intramuscular route with a series of three vaccines: I-lib diphtheria CRM,,, conjugate vaccine (HbOC) at 0 and 8 weeks and the Hib polysaccharide vaccine (HbPs, commercial name B Capsa1) at 16 weeks. Before and4 weeksafter, diphtheria and PRP AB levels were measuredfor each vaccine. AntiPRP AB levels were also measured8 weeks after the final vaccination. Vaccination of one of the children was delayed a month becauseof a febrile illness. Three sera were lost in laboratory handling. Antibody
quantitation
Serum immunoglobulins and IgG subclasseswere obtained aspart of the routine evaluationof the patients. Serum levels of IgG, IgA, and IgM were quantitated by radial immunodiffusion. IgG subclasseswere measuredby radial immunodiffusion in the Children’s Hospital of Bostan Clinical Immunology Laboratory with a commercially available kit from the Binding Site, Inc. (San Diego, Calif.). Levels of AB to the Hib capsular polysaccharide (PRP) were measuredby radioantigen-binding assay,aspreviously described,‘Oand calibrated with the standardantiserumfrom the Office of Biologics (United StatesFood and Drug Administration). This assayhad a lower limit of detection of 0.040 pg AB per milliliter. IgGI- and IgG2-subclassHib polysaccharide AB levels and diphtheria toxoid AB levels were determinedby enzyme immunoassayas described.” Subclassdeterminations were done with blinded samplesin the laboratory of Praxis Biologics. The lower limit of detection for the IgGl assaywas 0.075 kg/ml, and for the IgG2 assay,0.15 kg/ml. RESULTS Patients’ characteristics
The mean patients’ age was 42 months (range, 26 to 77 months). Ail patients had normal serum levels of IgG, IgA, and IgM. All patients had IgG 1- and
IgG3-subclasslevels within 2 SD of the meanfor their age. Two of these 15patientshad IgGZsubclass levels 2 SD or more below the mean for their age (patient No. 9, lgG2, 44 mg/dl; patient No. 13, lgG2, 25
Schneider et al.
J. ALLERGY
4
0
12
8 Thne (Weeks)
A HbOC
CLIN. IMMUNOL. MAY 1990
A HbOC
FIG. 1. GM AB titer to diphtheria toxoid in 14 children immunized with HbOC.
TABLE I. Study design Visit (wk) 0 Hboc
X
Hib polysaccharide Anti-PM levels Antidiphtheriatiter
X X
4
12
8
18
28
24
X X
X
X
X
X X
X X
X X
Patients received a series of three vaccines. Anti-PRP levels were measuredevery 4 weeks during the study.
mg/dl). Six patients had IgG4 levels 1.0 pg / ml after receiving the second HbOC (week 12). Booster responseswere observed in 13 children after the secondHbOC (week 12); the two children (patients Nos. 3 and 9) who failed to obtain a booster responsehad high preimmunization AB levels (40 and 65 p,g/rnl). Eight of the 15 patients (Nos. 2, 4, 6, 8, 9, 11, 13, and 15) had high AB levels (~20 @g/ml) before receiving the Hib polysaccharide vaccine (week 16) and had negligible change in their postvaccine levels. Of the remaining seven children, four (Nos. 3, 5, 12, and 14) had a boosterresponseto the Hib polysaccharide. The other three children (Nos. 1,7, and 10) had either a decrease or no changein their anti-PRP AB levels. The lowest postimmunization AB concentration occurred in patient No. 7 who had a history of transient hypogammaglobulinemia. He had a maximum level of 1.2 p,g/ml at week 12, but this fell to 0.44 p,g/ml at week 24 of the study and to 0.18 pg/ ml 13 months later. IgGl- and IgGZsubclass ABs to PRP were measuredin 14 of the children. Immunization with HbOC
VOLUME NUMBER
85 5
Response
to HbOC by HbPs nonresponders
951
TABLE Il. Response to Hib polysaccharide vaccine in children with recurrent infections compared to healthy age-matched control children ---.--__ Anti-PRP
level after immunization
(pglml) -..-I_____
Patients
No. 1
2 3 4 5 6 7 8 9 IO 11 12 13 14 15 GM
Control
Subjeats
Age ho)
Before HbPs
After HbPs
Arae (mo)
Before HbPs
After HbPs
47 37 36 36 26 35 53 60 77 35 48 32 41 35 31
0.04 0.04 0.05 0.08 0.22 0.15 0.06 0.06 0.10 0.04 0.12 0.06 0.15 0.04 0.04 0.07
0.10 0.04 0.18 0.22 0.27 0.42 0.22 0.35 0.11 0.06 0.47 0.43 0.27 0.04 0.06 0.16
47 37 36 32 26 35 53 59 77 33 44 32 40 34 31
0.80 1.30 0.76 0.54 0.14 0.08 0.36 0.10 1.10 0.42 0.34 0.11 0.45 0.75 0.36 0.37
3.30 32.00 4.60 3.20 16.82 1.33 13.70 7.60 950 2.80 8.02 -~40.00 :s40.00 16.40 0.11 9.14 --
AB levels were obtained 3 to 5 weeks after each immunization.
resulted in a rise in anti-PRP AB levels of both subclass levels. The prestudy AB levels (week 0) and after the secondHbOC immunization (week 12) titers are presentedin Table IV. Preimmunization IgGl and IgG2 AB levels were close to the lower limit of the sensitivity of the assays. This precluded analysis of the ratio (IgGl :IgG2) of these values. After immunization, the GM IgGl AB level was approximately five times greater than the GM IgG2 AB level, which is approximately in proportion to the normal serum distribution of these two subclasses.‘2~ I3 All patients except for patients No. 7 (with the history of transient h~~~li~rnia) and No. 12 (an IgG2 subclass-delicient patient) had postvaccination IgG2 anti-PIW levels >1.5 pg/ml. As illustrated in Fig. 1, immunization with HbOC also resulted in a rise in diphtheria antibody titer. The GM titers were 0.07 ELISA units before vaccination, 0.77 ELISA units after the first HbOC immunization, and 0.56 ELISA units after the second HbOC immunization. All children had a rise in their diphtheria titers. However, patient No. 7 had a lower diphtheria titer than most other patients (0.17 ELISA units after first HbOC immunization versus GM, 0.77 ELISA units). There were two IgG2 subclass-deficient children (patients Nos. 9 and 12) included in our study. One
IgG2deficient patient (No. 9) had levels similar to the other stndy patients. The seco& I&&deficient patient (No. 12) had slightly lower anti-PRP levels than other children in the study. His maximum antiPRP titer was 8.0 p,g/ml after the secondHbOC immunization (compared to GM, 29.3 pg/ml), and his final anti-PRP concentration (week 24) was 1.2 pg/ml (GM, 12.1 kg/ml), which s&&d be high enoughto confer protection. I4His IgG2 anti-PRPlevel was0.34 pg / ml after the secondHb0C immunization in comparisonto a GM of 4.72 p,g/ ml. His diphtheria titer was 0.24 ELISA units after the first HbGC immunization in comparison to a GM of 0.77 ELISA units. An impaired responseto Hib ~~~o~~de vaccine has been demonstratedin ~liildmn ysuiq~erthan 2 yesrs of age,“+*l5 and in &i&en with recurre5t tions and normaI i~u~l~~ 1eveIs. Adults and children have also been described who s&Wed from twxrrent infections and who lacked an I@3 repe to all polysacchasidesmeasuti. I67” In addition, previously healthy children have dev&oped ittva.siveHib diseasein spite of immunization with poiysac~haride Hib vaccines.4.I4 Thus, these patients with impaired
J. ALLERGY
952 Schneider et al.
TABLE III. Response to HbOC in children
with defect in AB production Anti-PRP
AB concentration
wk0 Patient No.
wk4
CLIN. IMMUNOL. MAY 1990
to Hib polysaccharide
after immunization
(pglml)
wk 12
wk 16
wk 20
Age
Before HbGC No. 1
After HbOC No. 1
After HbOC No. 2
Before HbPs
After HbPs
47 37 36 36 26 35 53 60 77 35 48 32 41 35 31
0.04 0.06 0.03 0.48
6.0 1.2 40.0 3.0 5.0 28.0 0.5 8.0 65.0 5.8
22.0 75.0 34.0 44.0 128.0 175.0 1.2 30.0 60.0 12.0 44.0 8.0 28.0 9.2 78.0 29.3
12.0 50.0 10.0 29.0 16.0 61.0 0.9 31.0 60.0 2.1 22.0 1.8 48.0 7.2 25.0 14.3
7.8 11.0 22.0 44.0 85.0 74.0 0.8 33.0 68.0 2.4 19.0 3.6 58.0 23.0 20.0 17.4
(mol
1 2 3 4 5 6 7* 8 9t 10 11 127 13 14 15 GM
0.10 0.55
0.20 0.52 0.11 0.50 0.16 0.15 0.06 0.23 0.06 0.17
10.0
5.0 20.0 1.3 1.4 5.9
AB levels were obtained 4 weeks after each immunization. *Patient 7 had a history of transient hypogammaglobulinemia. TPatients 9 and 12 had IgGZ-subclass deficiency.
TABLE IV. Subclass
responses
to HbOC GMIAB to PRP hglml)
Before study (week 0) After HbOC No. 2 (week 12) p Value*
IgGl
lgG2
0.10 23.20 1.O pg/mI (GM antibody titer, 12.1 pg/rnl). Thus, the conjugation of Hib oligosaccharide with diphtheria
CRM,,, results in a more immunogenic vaccine for this group of patients. Booster responseswere observed after the second HbOC immunization in all our patients except for two patients who had very high anti-PRP levels after the primary HbOC (65 and 40 u.g of ABs per milliliter). These results are similar to studies of HbOC’* I9 and other conjugates in infants”23 and support the use of a series of Hib vaccines. The third vaccine of our series, the Hib polysaccharide, was administeredto determine if, after priming with conjugate, the patientscould respondto polysaccharide. The patients’ responsesto this vaccine were heterogeneous.Most of the children had high AB titers (>20 p,g/ml) before the Hib polysaccharide vaccine and consequently did not have a booster response. Of seven children with levels
