DTP Vaccine With Hib

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Administration of Combined Diphtheria and Tetanus Toxoids and Pertussis

Vaccine, Hepatitis B Vaccine, and Haemophilus influenzae Type b (Hib) Vaccine

to Infants and Response to a Booster Dose of Hib Conjugate Vaccine

Michael E. Pichichero and Sherry Passador From the Departments of Microbiology and Immunology, Pediatrics,and Medicine, University of Rochester Medical Center, Rochester,

New York

We compared antibody levels following separate but simultaneous administration of diphtheria

and tetanus toxoids with acellular pertussis vaccine (DTaP) containing pertussis toxoid, filamentous

hemagglutinin, and pertactin (PRN); hepatitis B vaccine; and Haemophilus influenzae type b poly-

saccharide (polyribosylribitol phosphate; PRP) vaccine conjugated to tetanus toxoid (PRP-T) with

those following administration of a combination of a DTaPhepatitis B vaccinePRP-T to infants

at 2, 4, and 6 months of age. The antibody response to a booster dose of PRP conjugate vaccine

(CRM197-OS) in infants with low (1 mg/mL) or undetectable (0.10 mg/mL) postpriming levels

of antibody to PRP was also studied. Antibody levels were quantitated before and after dose 3 by

enzyme-linked immunosorbent assay, radioimmunoassay, or neutralization assay. Seroresponse rates

were not different between the two vaccine groups except for rates of response to PRP. There was

a trend that levels of antibody to all the antigens included in the combination vaccine were lower

than those of antibody to antigens in separate vaccines; for levels of antibody to diphtheria toxoid

(P .001), PRN (P .0001), and PRP (P .0001), the differences were significant. Despite low

or undetectable postpriming levels of antibody to PRP, high-titered (geometric mean concentration,

9.02 mg/mL; range, 1.081.5 mg/mL), immunoglobulin Gpredominant antibody to PRP was pro-

duced following a booster dose of CRM197-OS, a finding consistent with a memory response.

Combination vaccines are needed to simplify the childhood sure to PRP [8 12]. This priming may occur in infants with

low or even undetectable responses of serum antibody to PRPimmunization schedule. In recent trials, diphtheria and tetanus

toxoids with acellular pertussis vaccine (DTaP) formulations after PRP conjugate vaccination. The response of infants to a

booster dose of PRP vaccine can be taken to mimic contactcombined with Haemophilus influenzae type b (Hib) conjugate

vaccines (with or without hepatitis B vaccines) administered with invading Hib organisms with a PRP capsule on their

surface and indicate the expected response to infection [13to infants at 2, 4, and 6 months of age as a primary series have

produced lower levels of antibody to the Hib polysaccharide 15]. Here, we compared antibody responses after administra-

tion of a combination of DTaPhepatitis B vaccinePRP vac-(polyribosylribitol phosphate; PRP) than simultaneous but sep-arate injections [1 5]. This appears to be a general phenome- cine conjugated to tetanus toxoid (PRP-T) with those after

separate but simultaneous administration of DTaP, hepatitis Bnon involving different DTaP formulations and different Hib

conjugate vaccines from several manufacturers. The biological vaccine, and PRP-T to infants; we also characterized the anti-

body response to PRP conjugate revaccination in those infantssignificance of the diminished levels of antibody to PRP are

uncertain [5 7]. The immune response to the components in with low (1.0 mg/mL) or undetectable (0.10 mg/mL) post-

priming levels of antibody to PRP.the DTaP formulations has been described as similar following

combination or separate injections [25].

As a separate injection, Hib conjugate vaccines are knownStudy Designto prime infants for memory responses to a subsequent expo-

Primary Immunization

Healthy infants between 6 and 12 weeks of age were re-Received 19 February 1997; revised 21 May 1997. cruited for a multicenter (Elmwood Pediatric Group in Roches-This work was presented in part at the annual meeting of the American

ter, NY; Mark Blatter, M.D., and Keith Reisinger, M.D., inSociety for Microbiology held in New Orleans in September 1996 and at the

Pittsburgh; and Larry Pickering, M.D., in Norfolk, VA), pro-annual meeting of the Society for Pediatric Research held in Washington, D.C.,in May 1996. spective, randomized trial comparing injections of DTaP

Financial support:This work was supported by SmithKline Beecham Biolog-hepatitis B vaccine PRP-T at 2, 4, and 6 months of age (group

icals (Philadelphia) and the National Institutes of Health (AI45248).1) with three separate but simultaneous injections of DTaP,Reprints or correspondence: Dr. Michael E. Pichichero, Department of Mi-

crobiology and Immunology, University of Rochester Medical Center, 601 hepatitis B vaccine, and PRP-T at 2, 4, and 6 months of ageElmwood Avenue, Box 672, Rochester, New York 14642.

(group 2). Infants were randomized three to one to groups 1Clinical Infectious Diseases 1997;25:137884 and 2, respectively, and enrollment occurred from December 1997 by The University of Chicago. All rights reserved.10584838/97/25060015$03.00 1994 to May 1995. Serum samples were obtained from infants

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1379CID 1997; 25 (December) Response to Hib Combination Vaccine

PTox, FHA, and PRN ELISAs before vaccination at 2 months of age and 1 month after the

third vaccination at 7 months of age.ELISA methods for determination of PTox, FHA, and PRN

were adopted from protocols provided by the U.S. Food and

Drug Administration (courtesy of Dr. Bruce Meade, Bethesda,PRP Conjugate Revaccination

MD) [16]. All plated volumes were 100 mL. Polystyrene 96-Infants in group 1 from Rochester (n 43), with postpriming

well microtiter plates of medium binding capacity (Dynatech

levels of antibody to PRP of1.0 mg/mL received a booster Immulon I, Chantilly, VA) were coated with the followingdose of PRP conjugate vaccine (CRM197-OS) at 9 13 months optimized concentrations of purified antigen: PTox, 0.75of age. Serum samples were obtained from these infants before

mg/mL; FHA, 1 mg/mL; and PRN, 2 mg/mL in carbonate bufferthe booster dose was administered and 1 month after the booster

(0.05 M sodium carbonate, pH 9.6). PTox- and PRN-coateddose was administered. The quantity of serum samples was

plates were incubated at 23C, and FHA-coated plates weresufficient for measurement of antibody isotype in 19 vaccinees.

incubated at 37C for 12 to 20 hours. Serum samples wereWritten informed consent was obtained from the parents or

diluted in serum incubation buffer (PBS containing 0.5% w/vguardians.

bovine serum albumin [Sigma, St. Louis], 0.05% v/v Tween

20 [Sigma], and 0.005% v/v polypropylene glycol [Aldrich

Chemical Company, Milwaukee]) and added to antigen-coatedVaccinesplates immediately after washing with wash solution (0.145 M

Primary Vaccine sodium chloride containing 0.1% v/v Tween 20); the plates

were incubated for 2 hours at 23C. For each serum sample,Group 1. Diphtheria and tetanus toxoids were manufac- eight twofold dilutions, beginning with a 1:50 dilution, weretured by the Michigan Department of Public Health (Lansing,

tested.MI) and shipped to SmithKline Beecham Biologicals in Rix-After washing, alkaline phosphataseconjugated antibody toensart, Belgium, where they were formulated with acellular

human IgG (Kirkegaard and Perry Laboratories, Gaithersburg,pertussis tricomponent vaccine (DTaP) consisting of purifiedMD) diluted in conjugate incubation buffer (serum incubationpertussis toxin (PTox), filamentous hemagglutinin (FHA), per- buffer containing 2% v/v fetal bovine serum) was added totactin (PRN), and hepatitis B vaccine (SmithKline Beechameach well. Conjugated antibody to human IgG was used at anBiologicals). PRP-T was manufactured by Pasteur Merieuxoptimal dilution for PTox, FHA, and PRN ELISAs. Following(Lyon, France). The liquid DTaPhepatitis B vaccine was useda 12- to 20-hour incubation at 23C, the plates were washed,to reconstitute lyophilized PRP-T.and substrate solution (1 mg of p-nitrophenyl phosphate/mLGroup 2. DTaP used for this vaccine cohort consisted of[Sigma] in 1 M Tris, pH 9.8, containing 1 mM MgCl2) wasdiphtheria and tetanus toxoids manufactured by Chiron Behringadded to each well. After a 60-minute incubation at room tem-GmbH (Markburg, Germany) and was formulated at Smith-

perature, 50 mL of 3 N NaOH was added, and the plates wereKline Beecham Biologicals with the same lot of acellular read at dual wavelengths (absorbance, 405 and 630 nm).pertussis tricomponent vaccine used for group 1. Hepatitis BFor each ELISA, a minimum level of detection was defined.vaccine and PRP-T were from the same lots and vaccine manu-

This value was defined as the minimum amount of antibody,facturers that were used for group 1.in units, that had to be present for the serum to have at leastWhen combined with the same acellular pertussis vaccineone point within the linear range of the reference serum dose-product, different diphtheria and tetanus toxoids were includedresponse curve. This value was estimated to be 2 ELISA unitsin the vaccines given to group 1 infants vs. group 2 infants to(EU)/mL for PTox, 3 EU/mL for FHA, and 4 EU/mL for PRN.bridge safety and serology results for the two manufacturingThe coefficients of variation for the assays were 21% for PToxsources. Oral poliovirus vaccine was administered to all vaccin-(180 determinations), 13% for FHA (165 determinations), andees in both groups concurrently at 2, 4, and 6 months of age.14% for PRN (175 determinations).All vaccines were from a single lot.

Booster Dose of Vaccine ELISAs for Diphtheria and Tetanus Toxoids

The vaccine used for booster doses was CRM197-OS, which ELISAs for diphtheria and tetanus toxoids were performedconsisted of a mutant variant of diphtheria toxin conjugated by methods similar to those described by Melville-Smith andto PRP oligosaccharide (Lederle-Praxis Biologicals, Wyeth- Balfour [17] and Melville-Smith et al. [18]. Ninety-six-wellLederle, Rochester, NY). microtiter plates of medium binding capacity (Dynatech Immu-

lon III, Chantilly, VA) were coated with 100 mL of diphtheria

and tetanus toxoids (concentrations, 10 and 1 Lf unit/mL, re-Assay Methods

spectively) in sterile PBS (0.01 M sodium phosphate/0.14 M

sodium chloride, pH 7.4) at 37C for 1620 hours. (Antigen-All assays were performed at the University of Rochester

(Rochester, NY) in the laboratory of the investigators. coated plates can be stored at 4C for up to 1 month.) Coated

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1380 Pichichero and Passador CID 1997; 25 (December)

plates were washed with wash solution (PBS/0.1% Tween 20) IU/mL; hepatitis B surface antigens, mIU/mL; PTox, FHA, and

PRN, EU/mL; and PRP, mg/mL.and incubated for 1 hour at room temperature; twofold serial

dilutions (initial dilution, 1:50) of serum samples in PBS/0.05%

Tween 20 were tested. After washing, the plates were incubatedStatistical Analysisfor 1 hour at room temperature with 100 mL of alkaline phos-

phataselabeled goat antibody to human IgG in PBS/0.05%For statistical calculations, serum samples in which the anti-

Tween 20 (Tago, Camarillo, CA). The optimal dilution of the body concentration was less than the minimum detectable levelantibody to human IgG to be used was determined empirically

were assigned a value equal to one-half of that level. Geometricfor each assay.

mean concentrations (GMCs) of antibody were compared byAfter incubation, the plates were washed, and 100 mL of

means of the Students t test. Differences in attaining definedsubstrate solution (1 mg of p-nitrophenyl phosphate/mL in

antibody levels between vaccine groups were compared by1 M diethanolamine, pH 9.8, containing 1 mM MgCl2) was

using the x2 test.added to each well. Plates were incubated for 1 hour at room

temperature, and then 50 mL of 3 NNaOH solution was added

to each well; the plates were assayed for absorbance at wave-Results

lengths of 405 nm and 630 nm. The minimum level of detection

for diphtheria and tetanus toxins was estimated to be 0.05 Two hundred fifty-one children received the combination of

DTaPhepatitis B vaccinePRP-T (group 1), and 80 childrenIU/mL. The coefficients of variation for the assays were 15%

for diphtheria toxoid (370 determinations) and 11% for tetanus received separate injections of DTaP, hepatitis B vaccine, and

PRP-T (group 2). Preimmunization antibody levels were simi-toxoid (480 determinations).lar in the two cohorts for the primary series (data not shown).

Levels of antibody to tetanus toxoid, hepatitis B surface anti-PRP ELISA

gen, PTox, and FHA were not significantly different in group

1 than in group 2 following primary vaccination (table 1).Antibody to PRP was quantitated by ELISA, as described

by Phipps et al. [19], with use of Hib oligosaccharidehuman Levels of antibody to diphtheria toxoid (P .001), PRN

(P .0001), and PRP (P .0001) were lower in group 1 thanserum albumin conjugate (lower limit of detection, 0.10 mg

antibody/mL). The coefficient of variation for the assay was in group 2 (table 1). The percentage of infants with a presumed

protective level of0.1 IU/mL for diphtheria toxoid [22] was12% (420 determinations). The secondary antibody in the

ELISA was alkaline phosphatase labeled goat antibody to hu- 100% in both vaccine groups, and the rate of seroresponse to

PRN was comparable (92.8% vs. 95.9%, respectively) betweenman Ig (IgG, IgA, and IgM), goat antibody to human IgG, or

goat antibody to human IgM (Tago). group 1 and group 2. However, the percentage of infants

achieving a level of antibody to PRP of 0.15 mg/mL and

1.0 mg/mL was significantly lower in group 1 than in groupHepatitis B Surface Antigen RIA2 (P .007 and P .0001, respectively).

The postpriming, prebooster dose, and 1-month postboosterAntibody to hepatitis B surface antigen was measured by

RIA that was performed as described in a commercial kit dose levels of antibody to PRP in the 43 group 1 children from

Rochester who had low or undetectable levels of antibody to(AUSUB kit, Abbott Laboratories, North Chicago, IL). The

lower limit of detection was 10 mIU/mL, and the coefficient PRP after administration of DTaP hepatitis B vaccine

PRP-T are shown in table 2. From postpriming to the preboosterof variation was 18% (60 determinations).

dose, all but two of the 43 children had similar or diminished

levels of antibody to PRP (table 2), a finding suggesting thatReference Sera and Antibody Quantitation

exposure to wild-type Hib or cross-reacting bacterial antigens

did not occur. All of the vaccinees had a rise in the level ofReference sera for diphtheria and tetanus toxoids were cali-

brated against an international (World Health Organization) antibody to PRP following the booster dose of CRM197-OS

(table 2). Following the booster dose, the GMC rose to 9.02standard. Reference sera for PTox, FHA, and PRN were inter-

nal references calibrated against the U.S. Food and Drug Ad- mg/mL, and the levels of antibody to PRP were 0.15 mg/mL

in 100% of the children and1.0 mg/mL in 100% of theministration reference lot number 3 (PTox and FHA) and lot

number 4 (PRN). Reference sera for PRP were the CBER children who did not have antibodies previously (table 3).

We also evaluated the isotype of antibody to PRP in a subsetstandard lot 1983 which contained 70 mg/mL.

The procedure for quantitating antibodies was the straight- of serum samples obtained before and after the booster dose

of CRM197-OS. When antibody to PRP was detected in pre-line method for the ELISAs [20] and the Hollinger formula for

the hepatitis B surface antigen RIA [21]. Assays were per- booster-dose serum samples, it was always IgG, and no child

had measurable levels of IgM (table 4). Following the boosterformed at the University of Rochester on coded sera to maintain

blinding during performance of assays. Antibody concentra- dose of CRM197-OS, the rises were primarily in IgG levels,

with very low or no rise in IgM levels.tions were expressed as follows: diphtheria and tetanus toxoids,

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Table 1. GMCs of antibody and seroresponses in infants 1 month after three doses of vaccine.

Antibody, variables Group 1 (n 251) Group 2 (n 80)

Antibody to diphtheria toxoid

GMC (95% CI) 0.87* (0.789 0.947) 1.18* (0.998 1.397)

0.1 IU/mL (95% CI) 100 (98.1 100) 100 (94.3 100)

Antibody to tetanus toxoid

GMC (95% CI) 2.31 (2.12 2.53) 2.55 (2.163.01)

0.1 IU/mL (95% CI) 100 (98.1 100) 100 (94.3 100)

Antibody to PTox

GMC (95% CI) 61.8

(56.10 68.02) 71.9

(59.5286.93)

5 EU/mL (95% CI) 99.3 (96.8 99.9) 100 (94.3 100)

Seroresponsex 232/237 (97.9) 72/73 (98.6)

Antibody to FHA

GMC (95% CI) 112.0# (102.5 122.4) 127.7# (109.23149.25)

5 EU/mL (95% CI) 99.6 (97.5 100) 100 (94.3 100)

Seroresponse 228/237 (96.2) 72/73 (98.6)

Antibody to PRN

GMC (95% CI) 77.0** (70.03 84.69) 126.6** (104.02 153.90)

5 EU/mL (95% CI) 100 (98.1 100) 100 (94.3 100)

Seroresponse 220/237 (92.8) 70/73 (95.9)

Antibody to HBsAg

GMC (95% CI) 473.3 (383.6 583.8) 699.7 (528.8925.7)10 mIU/mL (95% CI) 94.6 (93.1 98.2) 98.8 (92.3 99.9)

Antibody to PRP

GMC (95% CI) 1.16** (0.937 1.433) 5.45** (3.787 7.832)

0.15 mg/mL (95% CI) 81.3 (75.8 85.8) 93.8 (85.497.7)

1.0 mg/mL (95% CI) 58.2** (51.9 64.5) 87.5** (77.8 93.5)

Antibody to poliovirus type 1

GMC (95% CI) 21.9 (18.36 26.03) 27.85 (19.8239.13)

1:8 IU/mL (95% CI) 99.6 (97.4 100) 98.7 (92.2 99.9)


GMC (95% CI) 64.1xx (57.69 71.41) 72.89xx (58.9990.06)

1:8 IU/mL (95% CI) 100 (98.1 100) 100 (94.2 100)


GMC (95% CI) 5.33## (4.67 6.07) 4.58## (3.585.86)

1:8 IU/mL (95% CI) 99.6 (97.4 100) 100 (94.2 100)

NOTE. Group 1 infants who received combination of DTaPhepatitis B vaccinePRP-T at 2, 4, and 6 monthsof age; group 2 infants who received separate injections of DTaP, hepatitis B vaccine, and PRP-T at 2, 4, and 6months of age. Abbreviations: DTaP diphtheria and tetanus toxoids with acellular pertussis vaccine; EU ELISAunit; FHA filamentous hemagglutinin; GMC geometric mean concentration; HBsAg hepatitis B surfaceantigen; n no. of assayed serum samples that differ for different antigens because of serum quantities;PRN pertactin; PRP polyribosylribitolphosphate; PRP-T PRP vaccine conjugated to tetanus toxoid;PTox pertussis toxin.

* P .001. Percentage of samples with indicated antibody level. P .30.

P .14.x No. of samples with seroresponse/total no. tested (%). Seroresponse definition: if initially seronegative, a postvacci-

nation titer of5 EU/mL; if initially seropositive, there was at least maintenance of the prevaccination titer. Therewere only 237 and 73 samples for groups 1 and 2, respectively, because paired serum samples were not alwaysavailable.

# P .15.

** P

.0001. P .06. P .007.

P .19.xx P .26.## P .27.

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Table 2. Individual levels of antibody to PRP in infants following Table 3. GMCs of antibody to PRP for 43 children with low levelsof antibody to PRP who received a booster dose of CRM197-OS.a booster dose of CRM197-OS.

Antibody level (mg/mL) Percentage of children with

Time of indicated antibody level

antibody levelSubject Prebooster Postbooster

no. Postpriming dose dose determination GMC* (95% CI) 0.15 mg/mL 1.0 mg/mL

Postpriming 0.54 (0.47 0.61) 65.1 01 0.44 0.12 5.2

2 0.22 0.13 4.2 Prebooster dose 0.36 (0.30 0.42) 56 0

Postbooster dose 9.02 (3.52 14.52) 100 1003 0.60 0.10 18.8

4 0.33 0.10 3.2 NOTE. CRM197-OS PRP conjugate vaccine; GMC geometric mean5 0.28 0.10 4.1

concentration; PRPHaemophilus influenzae type b polysaccharide (polyribo-6 0.27 0.10 3.8sylribitolphosphate).7 0.10 0.10 66.9

* Data are mg/mL.8 0.10 0.10 0.5

9 0.10 0.11 4.9

10 0.10 0.10 2.0 because a decrease in the response of antibody to PRP has11 0.66 0.10 9.3 been observed when an Hib vaccine is mixed in the same12 0.10 0.10 14.7

13 0.10 0.10 13.9 syringe with DTwP (diphtheria and tetanus toxoids with whole14 0.83 0.10 3.2 cell pertussis) [24] and DTaP [25]. We studied the combina-15 0.10 0.10 7.2

tion of DTaPhepatitis B vaccinePRP-T (which infants in16 0.97 0.10 8.0 group 1 received) and found generally lower antibody levels17 0.73 0.15 7.3when comparing the combination vaccine with separate but18 0.10 0.10 13.3

19 0.22 0.10 5.7 simultaneous injections of DTaP, hepatitis B vaccine, and20 0.10 0.17 31.5 PRP-T (which infants in group 2 received). The clinical rele-21 0.58 0.18 9.8

vance of these differences is unknown, since seroresponses22 0.58 0.19 49.3

were observed in nearly 100% of all vaccinees in both groups23 0.10 0.22 2.41 and 2 and known levels presumed to correlate with protection24 0.42 0.23 7.3

25 0.68 0.28 3.3 were generally exceeded (except for antibody to PRP).26 0.65 0.28 10.3

27 0.91 0.29 5.9

28 0.69 0.31 8.0 Table 4. Levels of total Ig, IgG, and IgM antibodies to PRP in29 0.41 0.32 7.0 infants before and after a booster dose of CRM 197-OS.30 0.94 0.33 81.5

31 0.52 0.34 6.0 Antibody level (mg/mL)32 0.27 0.35 42.7

33 0.10 0.35 49.9 Prebooster dose Postbooster dose34 0.70 0.40 8.7 Subject

35 0.36 0.42 2.0 no. Total Ig IgG IgM Total Ig IgG IgM36 0.51 0.43 20.7

37 0.90 0.44 18.5 2 0.10 0.10 0.10 4.2 3.2 0.2438 0.10 0.44 9.2 3 0.10 0.10 0.10 18.8 10.7 1.139 0.10 0.52 41.3 4 0.10 0.10 0.10 3.2 3.2 0.1040 0.87 0.69 34.2 5 0.10 0.10 0.10 4.1 3.1 0.1741 0.76 0.70 26.0 6 0.10 0.10 0.10 3.8 3.0 0.1042 0.84 0.78 1.0 21 0.18 0.19 0.10 9.8 8.8 1.7443 0.10 0.95 21.6 23 0.22 0.18 0.10 2.4 1.8 0.10

24 0.23 0.15 0.10 7.3 3.2 2 NOTE. Postpriming levels of antibody to PRP were determined when 25 0.28 0.18 0.10 3.3 1.5 0.67

vaccinees were 7 months old; prebooster dose levels were determined at 9 27 0.29 0.24 0.10 5.9 4.9 0.2813 months of age, and postbooster dose levels were determined 1 month later. 28 0.31 0.46 0.10 8.0 6.2 0.11CRM197-OS PRP conjugate vaccine; PRP Haemophilus influenzae type

29 0.32 0.17 0.10 7.0 5.9 0.76b polysaccharide (polyribosylribitolphosphate).

31 0.34 0.34 0.10 6.0 4.1 0.10

32 0.35 0.38 0.10 42.7 39.4 0.74

34 0.40 0.50 0.10 8.7 6.0 0.1

38 0.44 0.44 0.10 18.5 14.4 0.19Discussion41 0.69 0.78 0.10 34.2 19.9 0.8

43 0.78 0.37 0.10 1.0 0.7 0.10Concerns have been raised about possible cross-interference44 0.95 1.11 0.10 21.6 20.2 0.12between several different antigens when administered simulta-

neously in a single injection of vaccine [1, 57, 23, 24]. This NOTE. CRM197-OS PRP conjugate vaccine; PRP Haemophilus in-fluenzae type b polysaccharide (polyribosylribitolphosphate).concern is particularly important with regard to Hib vaccines

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GMCs of antibody to diphtheria toxoid and the PRN compo- taining DTaP and Hib vaccine (with or without hepatitis B

vaccine) to infants is needed. We selected an Hib conjugatenent in the acellular pertussis vaccine were significantly lower

after dose 3 in group 1 than in group 2. However, all infants vaccine with a different protein carrier (CRM197) than the car-

rier used in the primary series of vaccinations (tetanus toxoid)in both vaccine groups had levels of antibody to diphtheria

toxoid after dose 3 of0.1 IU/mL, and 92.8% achieved serore- to minimize the effect of carrier-induced T helper cells. In

addition, CRM197-OS does not give rise to high levels of anti-sponses to PRN after dose 3. Therefore, the differences in the

observed levels of antibody to diphtheria toxoid are of uncertain body to PRP after a single injection [27 29]. Comparisons ofthe levels of antibody to PRP in CRM197-OS-vaccinated chil-clinical importance, since all patients achieved a level of anti-

dren from our study with those in historical controls would not body associated with protection against disease [22]. Because

be appropriate. The only historical antibody data available ona protective level of antibody to PRN following administration

administration of a single dose of CRM197-OS to 9- to 13-of DTaP has not been established, the clinical importance of

month-old children involved prelicensure CRM197-OS, and thethe observed differences in GMCs between the study groups

prelicensure product induces antibody to PRP at different levelsmay be of importance but cannot be assessed at this time.

and with different characteristics than does the licensedGMCs of antibody to PRP were lower in group 1 than in

CRM197-OS that we used [30]; infants receiving postlicensuregroup 2 (P .0001), and the presumed protection level ofCRM197-OS at 5 to 6 months of age achieve a GMC of antibody0.15 mg/mL was achieved in 81.3% of infants in group 1 vs.to PRP of 1.30 mg/mL (95% CI 0.295.71) [27].93.8% in group 2 (P .007).

Use of unconjugated PRP vaccine as a booster dose wouldThe Hib capsular polysaccharide PRP is a T lymphocytehave been of interest because this vaccine might have betterindependent antigen [812]. Conjugation to a protein carrier

reflected natural exposure to Hib [1315]. Examination of thesupplies T cell epitopes [11]. Presentation of such epitopes tokinetics of the response of antibody to PRP with a booster doseCD4/ T helper cells by PRP-specific B cells leads to activationalso might have been helpful, since primed infants might showand clonal expansion of both carrier-specific T cells and poly-a more rapid increase in antibody levels than unprimed infants.saccharide-specific B cells [11]. This occurrence explains theUsing the same combination of DTaPhepatitis B vaccineability of PRP conjugate vaccines to induce higher antibodyPRP-T that we used but in a different schedule for the primaryresponses than unconjugated PRP vaccines as well as the abilityseries, Zepp et al. [31] described specific B cell memory-typeof conjugates to prime for booster responses.responses to a booster dose of unconjugated PRP vaccine givenA serum level of antibody to PRP of 0.15 mg/mL hasto toddlers aged 1522 months.been associated with protection against invasive Hib infection

Potential mechanisms for reduced immunogenicity that is[25]. After unconjugated PRP vaccination, a level of antibodyinduced by combining vaccine components have been dis-to PRP of1.0 mg/mL is associated with protection againstcussed by Insel [32]. Immunogenicity differences betweenHib infection [26]. The biological relevance of these antibodycombined vaccines and separate injections may be due to physi-levels in assessing the anticipated efficacy of conjugated PRP

cal or chemical interactions among the components. The addi-vaccines is questionable [57]. Pure PRP vaccine does nottion of adjuvants to combination vaccines may result in someprime for immunologic memory or a booster response, whichnormally adjuvanted components of the vaccine being adsorbedis in contrast to PRP conjugate products. For these reasons, weto the adjuvant or other normally adsorbed components beinginvestigated whether reconstituting PRP-T with liquid DTaPdisplaced from the adjuvant. An effect of the protein carrierhepatitis B vaccine for administration as one injection inter-could be hypothesized as a mechanism for reduced responses offeres with the induction of immunologic memory; to do thisantibody to PRP through carrier-induced epitope suppression.we observed the immune response to a booster dose of CRM 197-Further, with combination vaccines, antigen competition mightOS 3 to 7 months later.occur at the germinal centers of regional lymph nodes. A studyThe expected response in primed subjects should be a rapidwhere simultaneous injections of multiple vaccines in the sameanamnestic response with high GMCs of antibody dominatedextremity are compared with injections in different extremitiesby IgG [11]. We found evidence that immunologic memory isof infants could assess this possibility.induced and that a booster IgG response occurs in infants

Serological assays that correlate with efficacy will need to primed with PRP-T administered in combination with DTaP

be used as surrogates for efficacy trials. Combined vaccineshepatitis B vaccine as a single injection. Even in infants withwith some variations in immunogenicity may need to be ac-low (1.0 mg/mL) and undetectable (0.10 mg/mL) postprim-cepted, as long as protective efficacy is preserved, to meet theing levels of antibody to PRP, high-titered IgG-predominantgoals of the Childrens Vaccine Initiative: (1) to reduce the booster responses were observed. Thus, the combination ofnumber of contacts required to fully immunize the child, andDTaPhepatitis B vaccinePRP-T appears to prime the infant(2) to reduce the number of immunizations administered at

immune system for secondary-type responses of antibody toeach visit.

PRP, which is typical for separate injections of Hib conjugate

vaccine. ReferencesFurther study of the significance of lowered levels of anti- 1. Eskola J, Olander RM, Hovi T, Litmanen L, Peltola S, Kayhty H. Random-

ised trial of the effect of co-administation with acellular pertussis DTP body to PRP following administration of combinations con-

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27983.tion of a new combination vaccine that incorporates diphtheria-tetanus-

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DTP Vaccine With Hib