NIVELES DE ANTICUERPOS

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Vaccine 24 (2006) 17761785

Immunogenicity and safety of four different doses ofHaemophilusinfluenzae type b-tetanus toxoid conjugated vaccine, combined with

diphtheriatetanuspertussis vaccine (DTP-Hib), in Indonesian infants

Narain H. Punjabi a,, Emily L. Richie a, Cyrus H. Simanjuntakb, Sri Juliani Harjanto a,Ferry Wangsasaputra a, Sumarjati Arjoso b, Ainur Rofiq b, Mulyati Prijanto b,

Julitasari c, Ursula Yela d, Christian Herzog d, Stanley J. Cryz e

a U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesiab National Institute of Health Research and Development, Ministry of Health, R.I., Jakarta, Indonesia

c Communicable Disease Control, Ministry of Health, R.I., Jakarta, Indonesiad Berna Biotech Ltd., Berne, Switzerland

e Massachussetts Biologic Laboratories, University of Massachussetts Medical Center, Jamaica Plain, MA, USA.

Received 23 May 2005; received in revised form 30 September 2005; accepted 10 October 2005

Available online 27 October 2005

Abstract

Widespread use ofHaemophilus influenzae type b (Hib) conjugated vaccine in industrialized countries has resulted in a dramatic decline

in the incidence of invasive Hib diseases, but the vaccines cost has prevented its inclusion in basic immunization programs in developing

countries. To overcome this problem, combination with diphtheriatetanuspertussis (DTP) vaccine or reduction in the dose of Hib vaccine

has been proposed. To evaluate the immunogenicity and adverse reactions from lower doses of Hib-polyribosylphosphate (PRP) conjugated

with tetanus toxoid (PRP-T), a double-blind study was conducted in Jakarta, Indonesia, and its suburbs. A total of 1048 infants 6 weeks to 6

months of age received three doses of DTP vaccine combined with the usual 10 g dose or with a reduced dose of 5, 2.5 or 1.25 g of PRP-T

at two-monthly intervals. Antibodies were measured prior to the first dose and 46 weeks following the third dose. Adverse reactions were

similar among all four groups. The only significant difference was a higher rate of irritability (p < 0.02) and of temperature elevation >38 C

(p < 0.009) after doses 1 and 2 in the lowest dose group (1.25g PRP-T) compared to the other groups.

All participants tested had a 4-fold increase in antibodies against all DTP antigens. In addition, after a fourth booster dose of Hib, 99.6% of

infants produced 0.15g/ml of antibody to Hib-PRP, and 96.4% showed levels1.0g/ml after primary immunization, level that correlate

with short- and long-term immunity, respectively. Antibody titers to the PRP antigen showed no significant differences among dosage groups

with the exception of the 5.0 g group, which had a significantly higher GMC than the 1.25 g group (p < 0.012).

This study demonstrates that primary vaccination with half, one-fourth, or one-eighth of the usual dose of PRP-T, combined with DTP

vaccine, produces protective immune responses, and has side effects that are comparable to DTP vaccination alone. In these lower dosages,

PRP-T conjugate vaccine can lower vaccine costs to a level that is affordable for infant immunization programs in developing countries.

2005 Elsevier Ltd. All rights reserved.

Keywords: Haemophilus influenzae type b; Lower dose; DTP-Hib

Disclaimer: The views expressed in this paper are those of the authors

anddo notin any wayrepresentthoseof theUS Navyand theUS Department

of Defense as well the Indonesian Ministry of Health. Corresponding author.

E-mail address: [email protected] (N.H. Punjabi).

1. Introduction

It is estimated that of the 10 million annual deaths in

children less than 5 years of ageworldwide, 99% are in devel-

oping countries, and 70% are due to infectious diseases [1].

Pneumonia is the leading cause of early childhood death, and

0264-410X/$ see front matter 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.vaccine.2005.10.023


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N.H. Punjabi et al. / Vaccine 24 (2006) 17761785 1777

meningitis is among the 10 most common causes of death

from infectious diseases among children under 5 years of

age in developing countries [2,3].

Haemophilus influenzae type b (Hib) used to be an impor-

tant cause of morbidity and mortality for children under age

5 in industrialized countries [4]. The incidence of invasive

disease due to Hib declined dramatically after the inclu-sion of Hib-conjugate vaccine in routine pediatric immuniza-

tion programs in these countries [49]. For various reasons,

principally insufficient data on the incidence of Hib inva-

sive diseases and vaccine cost, this practice has not been

adopted in developing countries [4,10,11]. Many microbi-

ology laboratories in developing countries have difficulty

isolating Hib bacteria, due to lack of technological expertise,

widespread use of antibiotics prior to culture, prioritizing

isolation of other microorganisms over Hib, and the cost of

testing [10,12]. As a result, disease incidence may be under-

reported in many Asian and African countries.

The success of Hib conjugate vaccines in Western nations

led to clinical trials and improvements in laboratory detec-tion of Hib pathogens in developing countries; incidences of

pneumonia and meningitis due to Hib were found to be simi-

lar to or higher than in industrialized countries [4,10,1315].

These studies show that disease occurs at earlier ages and is

associated with higher morbidity and mortality in less devel-

oped countries. However, few of these countries can afford

to include Hib vaccine in their Expanded Program of Immu-

nization (EPI). For example, a single dose of Hib vaccine or

a bacterial culture (cerebrospinal fluid or blood) would each

cost over US$ 10. This remains unaffordable for developing

world families whose monthly incomes are about US$ 30

[1618].Vaccine costs can be reduced through several strategies,

such as combinations with other vaccine(s), multi-dose vials,

and lowering the amounts of immunizing agents. Adminis-

tration of reduced doses of Hib in combination with DPT

produces protective antibody levels because of the adjuvant

effect of the whole cell pertussis component [4,1720].

The objective of this study was to evaluate the immuno-

genicity and safety of primary immunization with three lower

doses of PRP-T (5, 2.5, or 1.25 g) combined with DTP

(whole cell killed pertussis), compared to DTP combined

with the standard Hib dose (10 g), in Indonesian infants.

2. Materials and methods

2.1. Study participants

The mothers of infants aged 6 weeks to 6 months at seven

community health centers (CHCs, also called PUSKESMAS)

in Jakarta and surrounding areas were offered to have their

infant participate in the study. The study purpose, risks, ben-

efits, and procedures were explained prior to enrollment, and

written informed consent was obtained from a parent prior to

enrollment.

Only healthy infants were enrolled. Eligibility was lim-

ited to infants who had no history of prior immunization

with any DTP or Hib vaccine (Hib vaccine is not yet avail-

able in these health centers), no history of neurological or

developmental disorders including seizure or febrile convul-

sions, no history of systemic illness, no history of treatment

with corticosteroids, immunosuppressants, blood products orinvestigational drugs, no evidence of immunodeficiency in

eitherthe infantor themother,no history of allergies, a normal

physical examination, weight >4 kg and axillary temperature


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1778 N.H. Punjabi et al. / Vaccine 24 (2006) 17761785

pre-filled syringes that were labeled with a unique number

corresponding to the participants study number. Each 0.5 ml

dose contained 25 Lf of diphtheria toxoid, 5 Lf of tetanus tox-

oid, 5.8IU of inactivatedBordetellapertussis wholecells, and

one of four doses ofHaemophilus influenzae type b polyribo-

sylphosphate conjugated to tetanus toxoid (PRP-T), namely

10, 5,2.5, or1.25g. All syringes with pre-filled vaccine hadsimilar physical appearance and the cold chain was main-

tained from Switzerland until administration to the infant.

All vaccines were adsorbed to AlPO4, with the average

quantity of AlPO4 adjuvantper dose of being 2 mg/0.5 ml.For

primary immunization, only one batch of DTP with varying

dosages of PRP-T was used. Two different batches of DTP-

Hib vaccine were used for booster immunization.

2.3. Trial design

The study was conducted in a double-blind manner. All

infants who met all inclusion criteria were sequentially

assigned a unique study number, which was maintainedthroughout the primary and booster immunization studies.

The investigators, staff, and participants were not aware of

the composition of any infants vaccine. Vaccine was admin-

istered intramuscularly in the anterolateral thigh, alternating

right and left for each dose. Primary immunization consisted

of three doses of vaccine given at 79-week intervals; longer

intervals between vaccine doses occurred if inter-current ill-

ness or absence of the infant necessitated postponing vacci-

nation. Data analysis for immune responses excluded results

for infants whose vaccine doses were more than 12 weeks

apart. Infants were observed for at least 30 min after each

vaccination, and were released after being checked by a studyphysician.

Immunization was postponed for any febrile illness within

the preceding 24 h (axillary temperature >37.5 C), or for

significant inter-current illnesses such as otitis media, pneu-

monia, gastroenteritis, or severe malnutrition. Infants were

excluded from continued participation for any of the fol-

lowing events after previous immunization: febrile convul-

sion, seizure, encephalopathy, meningitis or other neurologi-

cal disorders, hypotonic/hyporesponsiveness or anaphylactic

episodes within 48 h of immunization, chronic systemic ill-

ness requiring regular medication, and persistent crying (>3 h

within 48 h of immunization). Venous blood samples were

obtained before immunization and 46 weeks after the third

vaccination. The serum was separated into tubes labeled with

the date and study number and then frozen at 70 C.

Parents and CHWs were instructed about possible side

effects, how to reach study doctors, and when to go to the

CHC or hospital if their infant was ill. Parents were asked

to complete an adverse reactions report form for 7 days

after each immunization, including measurement of daily

temperature. For this they were provided a thermometer

and instructed how to use it. A study physician reviewed

adverse events reported on the form and examined each

infant, recorded body temperature and examination results,

on days 1, 2 and either 5, 6, or 7 following each immu-

nization. The solicited specific symptoms and signs were

rated as either absent, mild, moderate or severe: irritability,

anorexia, seizure, hyporesponsivehypotonic episode, incon-

solable crying, vomiting and diarrhea. The diameter of ery-

thema and induration at the injection site were measured in

millimeters. Any inter-current illness was also evaluated andtreated.

Children who were excluded from further participation

were referred to their CHC for continuation of their immu-

nizations according to Indonesian Ministry of Health policy.

Data on demographics, adverse reactions, and immune

responses were double entered using EpiInfo in a blinded

manner. The randomization list was prepared at Berna

Biotech Ltd. Thevaccinecode was broken only after analyses

of both the safety and serological data were performed.

2.4. Serological assays

All serological assays were performed in a blinded man-ner. Total anti-PRP serum antibody was measured using a

Farr-type radioimmunoassay with intrinsically labeled 3H-

PRP supplied by the University of Rochester, Rochester,

NY, as previously described [19,20]. In summary: a con-

stant amount of 3H labeled PRP is reacted with dilutions

of a reference anti-PRP antibody solution or test sera to form

a soluble antibodyantigen complex. This complex is precip-

itated with 50% cold saturated ammonium sulfate solution.

The precipitated antibodyantigen complex is collected by

filtrationthrough a 0.45m filter. 36 Chlorine (36Cl)is added

to the reactant mixture as a control to determine the efficiency

of the filtration step. The amount of3

H radioactivity retainedby the filter corresponds to the amount of specific anti-PRP

presented in the reactant.

The results are expressed as micrograms of anti-PRP

antibody per milliliter of serum. A reference serum (cali-

brated against a standard supplied by the Center for Biologies

Evaluation and Research, Food and Drug Administration,

Bethesda, MD), anti-PRP solution, Nosocuman Lot 1, SSIV,

with an assigned value of 13.01g/ml. A protective antibody

concentration was assigned a value of >0.15 g IU/ml.

Anti-diphtheria toxin, anti-tetanus toxin, anti-pertussis

toxin and anti-FHA antibodies were examined by ELISA

method. Antigens used for coating ELISA were Tetanus Tox-

oid Lot 153, Berna Biotech AG, FHA Lot 702293, Berna

Biotech AG, Diphtheria Toxin, Lot 300-1, Berna Biotech

AG, Pertussis Toxin, Lot 0623P, Berna Biotech AG. The

secondary antibody which has been used is a peroxidase

labeled affinity purified goat anti-human IgG (Art. Nr. 074-

1002, Kirkegaard and Perry). Briefly, 100l of a 1g/ml

antigen solution in phosphate buffered saline (PBS) was

used to coat each microliter well (72 h, 4 C). The coating

solution was removed and the wells blocked with a casein

solution (2 mg/ml in PBS) for 1 h at 37 C. The plates were

then washed repeatedly with PBS containing 0.05% tween-

20 (PBS-T). To each well was added 100l of serum sample


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serially diluted in PBS-T containing 2 mg/ml casein (PBS-

T-C). After incubation for 3 h at ambient temperature, the

plateswere washed three times with PBS-T. Anti-human IgG,

(Kirkegaard and Perry, Gaithesberg, MD), diluted 1:2500

in PBS-T-C was added (100l per well). After incubation

for 2 h at 22 C, the wells were washed with PBS-T, 100 l

of ABTS substrate (Boehringer, Mannheim, Germany) wasadded and the color was allowed to develop for 30 min. A405was read on a Dynatech (Embach, Switzerland) MR5000

ELISA reader. A standard antiserum containing a known

amount of specific antibody was run in parallel and the values

of the test samples were determined. Antibodies to tetanus

and diphtheria toxins are expressed in international units

(IU) per milliliters. A protective titer was assigned a value

of0.1 IU/ml. Anti-pertussis toxin and anti-FHA antibody

levels are reported in micrograms per milliliter.

B. pertussis whole cell agglutinating antibody was deter-

mined as follows: Lyophilized B. pertussis strain 460 (200

US opacity units per milliliter) was reconstituted and diluted

1:10 in saline containing 0.01% (w/w) thimerosal to yieldan A530 of 1.21.4. Pre-diluted (50l at a 1:20 dilution) test

sera were serially diluted (2-fold) using saline in U-bottomed

micro titer plates. To each well was added 50loftheB. per-

tussis cell suspension and the plates were vigorously shaken

for 1 min using an MA69 Micro shaker (Cooke Microtiter

System, Dynatech, Embach, Switzerland). The plates were

then sheathed with plastic foil and incubated at 35 C for

approximately 18 h. The agglutin titer was defined as the

reciprocal of the highest serum dilution resulting in a thin

sheet of cells with a slight button. A human reference serum

was run in parallel. Theagglutinin titer is defined as the recip-

rocal of the highest serum dilution giving a thin sheet of cellswith slight button. The humanreference serumwas Tosuman

ZL, Berna Biotech AG. This was used as a control forthe test.

2.5. Statistical analysis

Differences between geometric mean concentrations

(GMCs) were determined using a two-tailed t-test on log-

transformed values. The differences between adverse events

associated with immunization among different groups of

Hib-PRP-T dosages were determined using ANOVA and

the non-parametric test KruskalWallis (H test). The values

of GMC were log-transformed and the differences between

groups attaining a given antibody level/geometric mean con-

centration (GMC) were also determined using ANOVA and

non-parametric test KruskalWallis (H test).

3. Results

The study was conducted over 3.5 years: primary immu-

nization from June 1996 through May 1999 and booster

immunization from September 1997 until January 2000. A

total of 1294 children were enrolled andreceivedthe first dose

of vaccine. Totals of 1146 (89%) and 1048 (81%) received

2nd and 3rd dose, and 608 children or 47% of those who

received the first dose of vaccine, received the 4th (booster)

doses, respectively. All of them had complete monitoring of

the side effects. Of the 1048 infants who completed primary

immunization, 1006 (96%) had blood drawn for serological

studies. The mean ages at the time of the first, second, third,

and booster doses were 96, 161, 227, and 548 days, respec-tively. There were no significant differences among vaccine

groups in mean ages or gender distribution at the time of any

of the three immunizations (Table 1). A total of 215 children

had serologic testing before booster immunization, and 184

had repeat testing after vaccination.

3.1. Immunogenicity

Immunogenicity data are presented for the primary vacci-

nation series; sera drawn before and after the booster doses

are pending upon serological testing; these results are to be

published subsequently.

3.1.1. Pre-immunization serology

Prior to vaccination, there were no significant differences

in geometric mean concentrations (GMCs) to vaccine anti-

gens among the groups, with the exception of filamentous

hemagglutinin antibody (FHA) titers in the Hib 1.25g

group: these were with 0.63g/ml, significantly higher than

the GMCsin the 2.5 and 5.0g groups (0.44 and 0.46g/ml,

respectively, p < 0.02). Similarly, protective antibody levels

pre-immunization showed significantly higher percentages of

infants with anti-tetanus toxoid levels of0.1 IU/ml in the

1.25g group (93%) versus the 10 g group (85%).

In the 1.25g group, however, a lower percentage ofinfants (32%) had pre-immunization anti-PRP-Hib levels

0.15g/ml than either the 5.0 or 10.0 g groups (45 and

43%; p < 0.05).

3.1.2. Post-immunization serology

Tables 2 and 4 present pre- and post-immunization GMC

data and the rate of infants achieving protective antibody lev-

els.

Immune responses to Hib antigen demonstrated a sig-

nificantly lower post-immunization anti-PRP GMC in the

1.25g group (12.48) than in the higher dose groups (15.62,

17.28, and 15.10) respectively;however,all infants developed

protective antibody levels. Similarly, primary immunization

generated high levels of anti-PRP antibody seroprotection

rates: 99.6100% reached 0.15g/ml, and 96.498.2%

reached 1.0g/ml. There were no significant differences

among the four Hib dosage groups (Table 4).

For anti-tetanus toxoid GMCs, the 2.5g Hib group

showeda significantlylower GMC(18.15) than in the1.25g

group (22.88). There were no significant differences in B.

pertussis agglutinating antibody GMCs. However, GMCs of

anti-pertussis toxin differed: the 1.25g group achieved a

significantly higher GMC (11.40) than the 2.5g (6.94),

5.0g (7.55) and 10g groups (8.64). In addition, the


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Table1

DemographiccharacteristicsofvaccinatedchildrenbydoseandHib-PRP-Tdosages

DoseI(N=1294)

DoseII(N=1146)

DoseIII(N=1057)

DoseIV(N=608)

1.2

5g

(N=190)

2.5g

(N=370)

5.0g

(N=367)

10.0

g

(N=367)

1.2

5g

(N=164)

2.5g

(N=328)

5.0g

(N=331)

10.0g

(N=323)

1.2

5g

(N=149)

2.5g

(N=303)

5.0g

(N=312)

10.0g

(N=293)

1.2

5g

(N=76)

2.5g

(N=169)

5.0g

(N=176)

10.0g

(N=187)

Sexdistribution(M:F,

%)

669:625

590:556

539:518

322:2

86

47:53

51:49

57:43

50:50

52:48

49:51

56:44

50:50

50:50

49:51

55:45

48:52

50:50

51:49

60:40

50:50

Meanagerange(days)

96(40198)

161(93431)

227(145

501)

548(454

843)

99(40176)

95(40179)

96(45178)

96(45

198)

165(94431)

160(93308)

162(100395)

160(98405)

230(150480)

225(145414)

230(152

501)

226(154434)

549(459843)

547(459837)

551(454704)

547(463774)

Meanweightrange(kg)

5.5

(3.89.5

)

6.5

(4.111.5

)

7.1

(5.011.5

)

9.3

(6.2

14.0

)

5.3

(3.88.2

)

5.6

(4.08.7

)

5.6

(3.99.4

)

5.5

(3.99.5

)

6.2

(4.18.2

)

6.5

(4.510.3

)

6.6

(4.711.5

)

6.5

(4.810.0

)

6.8

(5.09.9

)

7.1

(511.0

)

7.2

(5.2

11.5

)

7.3

(5.411.0

)

9.4

(7.413.5

)

9.1

(6.213.0

)

9.3

(6.912.7

)

9.2

(6.514.0

) 1.25g group had a significantly higher GMC of filamen-

tous hemagglutinating (FHA) antibodies (6.35) as compared

to the 2.5g (4.83) and 5.0g groups (4.88). Although some

of the above differences in post immunization titers to DTP

antigens were found to be statistically significant, they are

unlikely to be of clinical importance.

With respect to the percent of infants with antibody levelsat or above protective levels ( 0.1 U/ml)after immunization,

serologic results for DPT antigens were similar in all vaccine

groups. The only significant difference seen was a higher

percentage with antibody levels to pertussis toxin0.1 IU/ml

in the infants who received 1.25g Hib than in the other Hib

dosage groups. Protective levels (0.1 IU/ml) were seen in

almost all infants after primary immunization: 98.9100%

for anti-diphtheria toxin; 99.6100% for anti-tetanus toxoid;

7486% for anti-pertussis toxin; 7274% for anti-FHA; and

9798% for B. pertussis agglutinating antibody.

The GMC of antibody to diphtheria toxin in the group

that received 10.0g PRP-T (6.38) was significantly lower

than in those who received 1.25 or 2.5 g (8.68 and 8.37,respectively, p = 0.0154). Additionally, in the 1.25g group,

a higher GMC of anti-pertussis toxin was seen than in the

other three groups, a higher GMC of antibodies against per-

tussis filamentous hemagglutinin (FHA) than in the 2.5 or

10.0g groups, and a higher GMC of anti-tetanus toxoid

than in the 2.5g group.

3.2. Adverse events

During the primary immunization phase, a total of 38

infants were excluded from further participation. Of these

exclusions, 25 were for medical reasons: 14 for febrile con-vulsions; two for rash after vaccination, possibly allergic;

one for brief respiratory difficulty after the first injection (the

child recovered without any sequelae); one for chills 30 min

after the second injection; three for fever 39.5 C after vac-

cination (one after the first dose and three after the third); one

for sterile abscess at the injection site; and three infants were

excluded for unrelated medical problems (colostomy, abnor-

mal head circumference, and tuberculosis). Nine infants were

excluded because they received non-study vaccine, and four

infants were excluded because they erroneously received a

dose not corresponding to their randomization number.

The adverse event data are presented in Table 3. The local

reaction to the injection were mild, with only between 0.6 and

4.6% of parents reported induration of 10 mm or greater at the

injection site, with no significant difference between differ-

ent groups of vaccine dosages or between different injection

number. This generally resolved in a week and nobody expe-

rienced long term sequalae.

For the systemic reactions, the incidence of inconsolable

crying among groups of children that received different vac-

cine dosages was between 1.4 and 5.8%, with no significant

different regarding the incidence of this adverse reaction

between different groups of vaccine dosages or between dif-

ferent injection number.


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Table 2

Antibody response to diphtheria, tetanus, and pertussis vaccine components

Antigen g PRP per vaccine dose Baseline 7 Months (after third dose)

GMC %0.1 IU/ml GMC % 1 IU ml %4-fold rise in titer

Diphteria (IU/ml) 1.25 0.13 60 8.68 100 NA

2.5 0.14 56 8.37 100 NA

5 0.14 58 7.69 98.9 NA10 0.13 52 6.38a 99.2 NA

Tetanus (IU/ml) 1.25 1.05 93 22.88 100 NA

2.5 0.91 88 18.15b 100 NA

5 0.87 87 19.55 99.6 NA

10 0.76 85c 20.17 100 NA

Pertussis toxin (g IgG/ml) 1.25 0.58 NA 11.4d NA 86e

2.5 0.60 NA 6.94 NA 74

5 0.59 NA 7.55 NA 74

10 0.64 NA 8.64 NA 76

FHA (g IgG/ml) 1.25 0.63f NA 6.35g NA 73

2.5 0.44 NA 4.83 NA 74

5 0.46 NA 5.34 NA 74

10 0.51 NA 4.88 NA 72

Pertussis 1.25 10.79 NA 481.09 NA 97

Agglutinating 2.5 11.05 NA 505.81 NA 97

Titer 5 10.95 NA 515.28 NA 97

10 10.81 NA 543.60 NA 98

a p < 0.05 vs. dose 2.5g, p = 0.05 vs. dose 1.25g.b p < 0.05 vs. dose 1.25g.c p < 0.05 vs. dose 1.25g.d p < 0.001 vs. dose 2.5g, p < 0.002 vs. dose 5g, p < 0.05 vs. dose 10g.e p < 0.007 vs. dose 2.5g, p < 0.007 vs. dose 5g, p < 0.02 vs. dose 10g.f p < 0.005 vs. dose 2.5g, p < 0.02 vs. dose 5g.g p < 0.05 vs. dose 2.5g, p = 0.05 vs. dose 10g.

Table 3

Adverse events associated with immunization

Adverse event Dose Percent reporting event

1.25g 2.5g 5.0g 10.0g

Fever (38 C) 1 51.6* 38.1 43.1 42.5

2 56.7* 37.2 45.0 35.0

3 38.9** 41.3 35.3 33.4

4 26.3*** 11.8 23.3 17.1

Irritability 1 60.0 56.5 64.3 62.7

2 72.0# 57.0 59.8 64.1

3 75.8$ 62.7 64.1 64.2

4 75.0 65.1 63.6 65.8

Inconsolable crying 1 4.7 4.3 4.9 4.6

2 4.3 3.7 5.1 5.0

3 4.7 5.6 5.8 4.4

4 1.4 2.4 2.3 1.7

Induration (10 mm) 1 4.2 2.7 2.7 3.3

2 3.7 2.7 2.1 4.6

3 1.3 3.0 3.2 3.4

4 1.3 0.6 2.8 2.1

* p < 0.008 vs. 2.5, 5.0, and 10.0g doses.** p < 0.009 vs. 10.0g dose.

*** p < 0.031 vs. all doses.# p < 0.02 vs. 2.5g dose, and p < 0.007 vs. 5.0g.$ p < 0.001 and 0.005 vs. 2.5, 5.0, and 10.0g, respectively.

For irritability after injection, the overall incidence washigh as reported by theparents,between 56.5 and75.8%, with

thegroupthat received theDPT with 1.25gHibaftersecond

and third injection having significantly higher incidence in

comparison to the other groups of DPT with higher dosages

of Hib.

Fever of 38 C or higher was reported among 11.8 and

56.7% of children. Infants who received DTP containing

the 1.25g dose of Hib had the highest proportion of fever

38 C, a difference which was significant for the 1st, 2nd,

and4th immunizationcomparedto allother doses,and signif-

icantly higherthan forthe 5.0and 10.0g doses after thethird

vaccination. Irritability was seen more often among infants

who received the 1.25g dose of Hib as compared to those

in the 2.5, 5.0, and 10.0g groups; this was significant for

the 2nd and 3rd immunization.

There were a total of 40 incidences of febrile seizures:

eight occurred after the first, six after the second, and 26

after the fourth (booster) dose of vaccine; these differences

were statistically not significant, and no seizures resulted in

any permanent sequelae. A total of 15 infants developed a

rash after vaccination, but only two of these were excluded

from further participation.

A total of sixinfants required hospitalization: onefor chills

(described above), two for a respiratory infection which was


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notsymptomatic prior to vaccination, andthreefor high fever,

diarrhea, and cough, with onset 28 days after immunization.

There was no significant difference in the rates of hospi-

talization between the vaccine groups.

There were a total of four deaths, but all were consid-

ered unrelated to vaccine administration. Three infants died

at home, having not sought medical care prior to death. One3-month-old died 1 month after the first DTP-Hib 5g vacci-

nation; another infantdied at age6 months, 2 weeks following

the second DTP-Hib 5g vaccination; both were well before

immunization andduringthe 7-day follow-up period; the par-

ents account of the illness did not clarify the probable cause

of death. A third infant died from pneumonia at age 9 months,

4 monthsafter receiving histhird DTP-Hib 2.5g. Thefourth

infant died at age 3 months, about 18 h after his first vaccina-

tion with DTP-Hib 2.5g. He had been healthy and afebrile

prior to vaccination, but developed fever and irritability sev-

eral hours later, refusing to breast-feed. The parents did not

contact the CHW, and he died at home in the early morn-

ing. A post mortem examination was offered but the parentsdeclined.

A total of 162 infants were not brought for the complete

series of primary immunizations, 102 (7.8%) for the second

dose and60 (5%) for the third dose; reasons included families

having left the area (in part due to the economic crisis that

affected Indonesia in the middle of the study).

Adverse events after booster immunization did not dif-

fer among dosage groups, with the exception of DTP with

the 1.25g Hib dose group, in which fever was seen sig-

nificantly more often (26.3%, p < 0.005) than in the other

three groups (12, 23, and 17%). Irritability, induration, and

inconsolable crying were not significantly different amongthe dosage groups.

4. Discussion

The history of infectious diseases and public health have

established the role of vaccination in the prevention of infec-

tious diseases, resulting in dramatic declines or even, as in the

case of small-pox, eradication of disease [13,2125]. This

success story has been repeated with the virtual elimination

of Hib invasive diseases in North American and European

countries following the inclusion of Hib-conjugate vaccines

in national immunization programs [49].

In developing countries, a reluctance to adopt similar

strategies was based on low reported incidences of invasive

Hib diseases, high vaccine costs, and the much higher burden

of other infectious diseases that were more easily diagnosed

[1014]. The incidence has been underestimated because ofdiagnostic difficulties such as the lack of pathognomonic

signs and symptoms, incorrect laboratory procedures, and

avoidance of cultures because of cost or cultural fear of

lumbar puncture [4,5]. Recent studies and improvements in

laboratory diagnosis have shown incidences of Hib inva-

sive diseases that are higher in many developing compared

to industrialized countries, with the exception of epiglottitis

[4].

Despite these data, national immunization programs in

developing countries have not yet adopted Hib vaccine in

their EPI, due to its high cost. Strategies to make the vac-

cine affordable include the use of combined vaccines, multi

dose presentation, or incorporation of lower antigen doses[4,1618].

This study demonstrates that one-eighth of the standard

10g dose of PRP-T elicits protective antibody levels, with

99.6% of infants developing antibody levels of0.15g/ml

and 96.4% of1.0g/ml, levels which correlate with short-

and long-term protection, respectively. These results are com-

parable to standard dosage Hib conjugate vaccines and are

comparable to the results obtained in Chile by Lagos et al.

[26]. In addition, these lower dose Hib-DTP vaccines elicited

antibody levels against the DTP antigens equivalent to stan-

dard dose vaccine (Table 4).

The hospitalizations and adverse events observed wereevenly distributed among all the groups. The four deaths

observed during the 3.5-year study duration can be extrap-

olated to an annual infant mortality rate (IMR) of slightly

more than one per 1000. In comparison, the annual IMR is

43/1000 for Indonesia, and 27/1000 for the Jakarta province,

indicating a favorable effect of participation in the vaccine

study, and of regular medical care, in comparison to the

general infant population [27]. It would be safe to assume

that if the infants who died had been taken to the hospital in

a timely fashion, some or all of the deaths could have been

prevented. Three of the four infants were doing well on day 7

Table 4

Anti-PRP antibody response following immunization with DPT-PRP-T vaccines

g PRP/dose Geometric mean anti-PRP (g/ml) Total (%)

Baseline Post-immunization 0.15g/ml 1g/ml

Baseline Post-immunization Baseline Post-immunization

1.25g 0.12* 12.48 32** 100 8 96.4

2.5g 0.16 15.62 41 99.6 11 97.4

5.0g 0.15 17.28 45 99.6 7 98.2

10.0g 0.14 15.10 43 99.6 7 96.9

* p < 0.02 for comparison with dose 5.0g.** p < 0.05 for comparison with doses 5.0g and 10.0g.


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after vaccination, so it is very unlikely that these deaths were

related to vaccination. The fourth death occurred within 24 h

after vaccination; the relationship between this death and

vaccination remained unclear.

Many publications have confirmed Hib vaccine to be

one of the safest vaccines ever used, whether alone or in

combination, in a standard or in a reduced dose [2835].Similar observations have been made regarding diphtheria

and tetanus vaccines. The side effects of DTP combined

with conjugated Hib vaccines are similar to those seen

after DTP alone, and were most likely attributable to the

killed whole cell pertussis component [27]. In the compar-

ison of placebo versus Hib added to whole cell DTP vac-

cine, no significant increase in adverse reactions was found

[36].

Killed whole cell pertussis vaccine is known to cause

significant adverse reactions, including neurological side

effects such as seizures. The latter events have been consid-

ered acceptable because of the effectiveness and low cost

of whole-cell pertussis vaccine, as described by Edwardsand Decker in their editorial concerning two acellular vac-

cine trials [3740]. The decline in the use of whole cell

killed pertussis vaccine in the 1970s in Japan, Sweden, and

United Kingdom was followed by a resurgence of pertus-

sis cases and then the development of the acellular vaccine

[39,41,42]. Many countries continue to use whole cell killed

pertussis vaccine in their immunization programs, a policy

that is supported by the World Health Organization (Global

Programme for Vaccine and Immunization, Expanded Pro-

gramme on Immunization (EPI), 1996) [43]. This policy is

also maintained by the Indonesian National Immunization

Program and by Immunization Authorities in other countries[4447].

The killed whole cell pertussis vaccine was incorporated

into the vaccine used for this trial because of the adjuvant

effect that it provides for the PRP-Hib antigen. Less satis-

factory immune responses to Hib antigens are seen when

it is combined with the acellular pertussis vaccine [48]. In

developing countries with high infant mortality rates, pneu-

monia is the leading cause of death. In developed countries,

Streptococcus pneumoniae and Haemophilus influenzae are

the principal pathogens in bacterial pneumonia [1]. Another

study comparing DTP-Hib with DTP, conductedon the island

of Lombok in Indonesia, to evaluate the incidence of vaccine-

preventable Haemophilus influenzae type b pneumonia and

meningitis, observed 1449 deaths or 2.6% among 55,073

children enrolled, with 77% of all deaths occurring else-

where than in hospital [49]. The difference between this and

our study was that the Lombok study areas were more rural

regarding the population and the availability of health care

facilities.

In conclusion, reduced doses of Hib PRP-T combined

with killed whole cell pertussis DTP vaccine, when used for

primary immunization, produce immune responses and side

effects that are comparable to those observed after admin-

istration of the 10g PRP-Hib-DTP combination. DTP-Hib

vaccines containing 5, 2.5, or 1.25 g of PRP-Hib could be

affordable for developing countries, and would be expected

to reduce infant and childhood morbidity and mortality if

included in their EPI programs.

Berna Biotech Ltd., Berne, Switzerland, does not manu-

facture DTP or Hib vaccines any more.

Acknowledgements

This study was supported by grant from the Berna Biotech

Ltd. (formerly known as Swiss Serum and Vaccine Institute),

Berne, Switzerland, by the Indonesian Ministry of Health,

and by the U.S. Naval Medical Research and Development

Command, Navy Department.

For their support and encouragement for the study, the

authors would like to thank the Director and staff of National

Institute of Health Research and Development (NIHRD or

Badan Penelitian dan Pengembangan Kesehatan Departe-

men Kesehatan) R.I., Ministry of Health of Indonesia, thestaff of the Bacteriology Laboratory, Center for Infectious

Diseases Research NIHRD RI, the Commanding Officer and

personnel of U.S. Naval Medical Research Unit (NAMRU)

No. 2, Jakarta, Indonesia, the Directors, medical and nursing

staffs of the PUSKESMAS Cibinong, Cirimekar, Citeureup

and Gunung Putri in Kab. Bogor and PUSKESMAS Kapuk,

Penjaringan, Koja and Sunter Agung in North Jakarata, the

Directors and staff of the Infectious Diseases Hospital Prof.

Dr. Sulianti Saroso of Jakarta, Atmajaya Hospital, Koja and

Bhakti Husada Hospitals, DR Yulitasari and Drs. Maya, Mar-

taria Dhiana, Shinta Laurencia, Nurmiati Nasution, Cynthia,

Robert Polowidjaja and Mr. Maman Supriatman for theirassistance in the study implementation.

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