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Vaccine 24 (2006) 17761785

Immunogenicity and safety of four different doses of Haemophilus inuenzae 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. Simanjuntak b , Sri Juliani Harjanto a , Ferry Wangsasaputra a , Sumarjati Arjoso b , Ainur Roq b , Mulyati Prijanto b , Julitasari c , Ursula Yela d , Christian Herzog d , Stanley J. Cryz e
U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia National Institute of Health Research and Development, Ministry of Health, R.I., Jakarta, Indonesia c Communicable Disease Control, Ministry of Health, R.I., Jakarta, Indonesia d Berna Biotech Ltd., Berne, Switzerland Massachussetts Biologic Laboratories, University of Massachussetts Medical Center, Jamaica Plain, MA, USA.
b a

Received 23 May 2005; received in revised form 30 September 2005; accepted 10 October 2005 Available online 27 October 2005

Abstract Widespread use of Haemophilus inuenzae 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 rst dose and 46 weeks following the third dose. Adverse reactions were similar among all four groups. The only signicant 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 (1.25 g 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 0.15 g/ml of antibody to Hib-PRP, and 96.4% showed levels 1.0 g/ml after primary immunization, level that correlate with short- and long-term immunity, respectively. Antibody titers to the PRP antigen showed no signicant differences among dosage groups with the exception of the 5.0 g group, which had a signicantly 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 inuenzae type b; Lower dose; DTP-Hib

1. Introduction
Disclaimer: The views expressed in this paper are those of the authors and do not in any way represent those of the US Navy and the US Department of Defense as well the Indonesian Ministry of Health. Corresponding author. E-mail address: narain@namru2.org (N.H. Punjabi). 0264-410X/$ see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.10.023

It is estimated that of the 10 million annual deaths in children less than 5 years of age worldwide, 99% are in developing countries, and 70% are due to infectious diseases [1]. Pneumonia is the leading cause of early childhood death, and

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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 inuenzae type b (Hib) used to be an important 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 inclusion of Hib-conjugate vaccine in routine pediatric immunization programs in these countries [49]. For various reasons, principally insufcient data on the incidence of Hib invasive diseases and vaccine cost, this practice has not been adopted in developing countries [4,10,11]. Many microbiology laboratories in developing countries have difculty 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 underreported in many Asian and African countries. The success of Hib conjugate vaccines in Western nations led to clinical trials and improvements in laboratory detection of Hib pathogens in developing countries; incidences of pneumonia and meningitis due to Hib were found to be similar 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 developed countries. However, few of these countries can afford to include Hib vaccine in their Expanded Program of Immunization (EPI). For example, a single dose of Hib vaccine or a bacterial culture (cerebrospinal uid 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. Administration 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 immunogenicity 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, benets, 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 limited to infants who had no history of prior immunization with any DTP or Hib vaccine (Hib vaccine is not yet available in these health centers), no history of neurological or developmental disorders including seizure or febrile convulsions, no history of systemic illness, no history of treatment with corticosteroids, immunosuppressants, blood products or investigational drugs, no evidence of immunodeciency in either the infant or the mother, no history of allergies, a normal physical examination, weight >4 kg and axillary temperature <37.5 C. Mild upper respiratory tract infection, mild skin infection, or other non-progressive conditions, such as thrush or inguinal hernia, did not exclude infants from participation. Most of the study subjects were from low to middle income families; over 90% of the participating infants were breastfed. There was strong support from and active participation by the CHCs and community health workers (CHW), who worked as volunteers and had received training on hygiene and sanitation. In coordination with the CHCs and hospitals, the CHWs assisted in recruitment of participants, follow up visits, notifying study doctors of illness among the infants. All the costs related to consultations, medication prescriptions, and hospitalization, whether or not the illness was related to vaccination were reimbursed. All children who participated and completed the primary immunization phase were offered booster immunizations with their respective Hib doses in combination with DTP vaccine at age 1518 months, after written informed consent was obtained. Children were eligible for the booster phase of this study if they met the following criteria: (1) prior participation in the primary series study; (2) written informed consent for the booster phase obtained from the parent; (3) agreement to serologic testing before the booster dose and 48 weeks after vaccination; (4) agreement to monitor body temperature and side effects at home and at a follow-up visit with research staff as performed for the primary series. Serologic testing was planned as part of the study protocol, but administrative changes at the processing laboratory resulted in postponement of the laboratory assay for uncertain time duration; therefore, evaluation of these results is deferred to a future publication. This study was approved by the Ethics Committee and the Review Board of the National Institute of Health Research and Development (LITBANGKES), Ministry of Health, Republic of Indonesia on the 25th of June 1996, and by the Committee for the Protection of Human Subjects (CPHS) of the United States Naval Medical Research Unit-Two (U.S. NAMRU-2), Jakarta, Indonesia as DoDs protocol no. 9609 on the 7th of May 1996. The study followed the GCP and WHO declaration of Helsinki for conduct of the study. 2.2. Vaccine Vaccines were manufactured and provided by the Berna Biotech Ltd. (formerly known as Swiss Serum and Vaccine Institute), Berne, Switzerland. They were packaged in

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pre-lled 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 toxoid, 5.8 IU of inactivated Bordetella pertussis whole cells, and one of four doses of Haemophilus inuenzae type b polyribosylphosphate conjugated to tetanus toxoid (PRP-T), namely 10, 5, 2.5, or 1.25 g. All syringes with pre-lled vaccine had similar physical appearance and the cold chain was maintained from Switzerland until administration to the infant. All vaccines were adsorbed to AlPO4 , with the average quantity of AlPO4 adjuvant per 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 DTPHib 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 maintained throughout the primary and booster immunization studies. The investigators, staff, and participants were not aware of the composition of any infants vaccine. Vaccine was administered 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 illness or absence of the infant necessitated postponing vaccination. 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 study physician. Immunization was postponed for any febrile illness within the preceding 24 h (axillary temperature >37.5 C), or for signicant inter-current illnesses such as otitis media, pneumonia, gastroenteritis, or severe malnutrition. Infants were excluded from continued participation for any of the following events after previous immunization: febrile convulsion, seizure, encephalopathy, meningitis or other neurological disorders, hypotonic/hyporesponsiveness or anaphylactic episodes within 48 h of immunization, chronic systemic illness 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 immunization. The solicited specic symptoms and signs were rated as either absent, mild, moderate or severe: irritability, anorexia, seizure, hyporesponsivehypotonic episode, inconsolable crying, vomiting and diarrhea. The diameter of erythema and induration at the injection site were measured in millimeters. Any inter-current illness was also evaluated and treated. Children who were excluded from further participation were referred to their CHC for continuation of their immunizations 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. The vaccine code 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 manner. Total anti-PRP serum antibody was measured using a Farr-type radioimmunoassay with intrinsically labeled 3 HPRP supplied by the University of Rochester, Rochester, NY, as previously described [19,20]. In summary: a constant amount of 3 H 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 precipitated with 50% cold saturated ammonium sulfate solution. The precipitated antibodyantigen complex is collected by ltration through a 0.45 m lter. 36 Chlorine (36Cl) is added to the reactant mixture as a control to determine the efciency of the ltration step. The amount of 3 H radioactivity retained by the lter corresponds to the amount of specic anti-PRP presented in the reactant. The results are expressed as micrograms of anti-PRP antibody per milliliter of serum. A reference serum (calibrated 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 13.01 g/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 Toxoid 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 afnity puried goat anti-human IgG (Art. Nr. 0741002, Kirkegaard and Perry). Briey, 100 l of a 1 g/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% tween20 (PBS-T). To each well was added 100 l of serum sample

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serially diluted in PBS-T containing 2 mg/ml casein (PBST-C). After incubation for 3 h at ambient temperature, the plates were washed three times with PBS-T. Anti-human IgG, (Kirkegaard and Perry, Gaithesberg, MD), diluted 1:2500 in PBS-T-C was added (100 l 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) was added and the color was allowed to develop for 30 min. A405 was read on a Dynatech (Embach, Switzerland) MR5000 ELISA reader. A standard antiserum containing a known amount of specic 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 of 0.1 IU/ml. Anti-pertussis toxin and anti-FHA antibody levels are reported in micrograms per milliliter. B. pertussis whole cell agglutinating antibody was determined 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 yield an A530 of 1.21.4. Pre-diluted (50 l 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 50 l of the B. pertussis 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 dened 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. The agglutinin titer is dened as the reciprocal of the highest serum dilution giving a thin sheet of cells with slight button. The human reference serum was Tosuman ZL, Berna Biotech AG. This was used as a control for the test. 2.5. Statistical analysis Differences between geometric mean concentrations (GMCs) were determined using a two-tailed t-test on logtransformed 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 concentration (GMC) were also determined using ANOVA and non-parametric test KruskalWallis (H test).

2nd and 3rd dose, and 608 children or 47% of those who received the rst 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 rst, second, third, and booster doses were 96, 161, 227, and 548 days, respectively. There were no signicant 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 vaccination 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 signicant differences in geometric mean concentrations (GMCs) to vaccine antigens among the groups, with the exception of lamentous hemagglutinin antibody (FHA) titers in the Hib 1.25 g group: these were with 0.63 g/ml, signicantly higher than the GMCs in the 2.5 and 5.0 g groups (0.44 and 0.46 g/ml, respectively, p < 0.02). Similarly, protective antibody levels pre-immunization showed signicantly higher percentages of infants with anti-tetanus toxoid levels of 0.1 IU/ml in the 1.25 g group (93%) versus the 10 g group (85%). In the 1.25 g group, however, a lower percentage of infants (32%) had pre-immunization anti-PRP-Hib levels 0.15 g/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 levels. Immune responses to Hib antigen demonstrated a signicantly lower post-immunization anti-PRP GMC in the 1.25 g 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 0.15 g/ml, and 96.498.2% reached 1.0 g/ml. There were no signicant differences among the four Hib dosage groups (Table 4). For anti-tetanus toxoid GMCs, the 2.5 g Hib group showed a signicantly lower GMC (18.15) than in the 1.25 g group (22.88). There were no signicant differences in B. pertussis agglutinating antibody GMCs. However, GMCs of anti-pertussis toxin differed: the 1.25 g group achieved a signicantly higher GMC (11.40) than the 2.5 g (6.94), 5.0 g (7.55) and 10 g groups (8.64). In addition, the

3. Results The study was conducted over 3.5 years: primary immunization from June 1996 through May 1999 and booster immunization from September 1997 until January 2000. A total of 1294 children were enrolled and received the rst dose of vaccine. Totals of 1146 (89%) and 1048 (81%) received

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547 (463774)

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Dose III (N = 1057)

230 (150480)

50:50

6.8 (5.09.9)

1.25 g group had a signicantly higher GMC of lamentous hemagglutinating (FHA) antibodies (6.35) as compared to the 2.5 g (4.83) and 5.0 g groups (4.88). Although some of the above differences in post immunization titers to DTP antigens were found to be statistically signicant, they are unlikely to be of clinical importance. With respect to the percent of infants with antibody levels at or above protective levels ( 0.1 U/ml) after immunization, serologic results for DPT antigens were similar in all vaccine groups. The only signicant difference seen was a higher percentage with antibody levels to pertussis toxin 0.1 IU/ml in the infants who received 1.25 g 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 10.0 g PRP-T (6.38) was signicantly lower than in those who received 1.25 or 2.5 g (8.68 and 8.37, respectively, p = 0.0154). Additionally, in the 1.25 g group, a higher GMC of anti-pertussis toxin was seen than in the other three groups, a higher GMC of antibodies against pertussis lamentous hemagglutinin (FHA) than in the 2.5 or 10.0 g groups, and a higher GMC of anti-tetanus toxoid than in the 2.5 g 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 convulsions; two for rash after vaccination, possibly allergic; one for brief respiratory difculty after the rst injection (the child recovered without any sequelae); one for chills 30 min after the second injection; three for fever 39.5 C after vaccination (one after the rst dose and three after the third); one for sterile abscess at the injection site; and three infants were excluded for unrelated medical problems (colostomy, abnormal 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 signicant difference between different groups of vaccine dosages or between different injection number. This generally resolved in a week and nobody experienced long term sequalae. For the systemic reactions, the incidence of inconsolable crying among groups of children that received different vaccine dosages was between 1.4 and 5.8%, with no signicant different regarding the incidence of this adverse reaction between different groups of vaccine dosages or between different injection number.

50:50

548 (454843) 547 (459837) 551 (454704)

Dose IV (N = 608)

51:49

549 (459843)

50:50

226 (154434)

48:52

227 (145501) 225 (145414) 230 (152501)

1.25 g (N = 149)

49:51

50:50

161 (93431) 160 (93308) 162 (100395)

Table 1 Demographic characteristics of vaccinated children by dose and Hib-PRP-T dosages

Dose II (N = 1146)

49:51

165 (94431)

52:48

96 (45198)

50:50

1.25 g 2.5 g (N = 190) (N = 370) Sex distribution (M:F, %)

96 (40198) 95 (40179) 96 (45178)

51:49

Dose I (N = 1294)

Mean weight range (kg)

Mean age range (days)

5.3 (3.88.2)

99 (40176)

47:53

5.5 (3.89.5) 5.6 (4.08.7) 5.6 (3.99.4)

5.0 g (N = 367)

669:625 57:43

5.5 (3.99.5)

10.0 g (N = 367)

6.2 (4.18.2)

1.25 g (N = 164)

6.5 (4.111.5) 6.5 (4.510.3) 6.6 (4.711.5)

5.0 g (N = 331)

2.5 g (N = 328)

590:556 56:44

6.5 (4.810.0)

160 (98405)

10.0 g (N = 323)

7.1 (5.011.5) 7.1 (511.0) 7.2 (5.211.5)

5.0 g (N = 312)

2.5 g (N = 303)

539:518 55:45

7.3 (5.411.0)

10.0 g (N = 293)

9.4 (7.413.5)

1.25 g (N = 76)

9.3 (6.214.0) 9.1 (6.213.0) 9.3 (6.912.7)

5.0 g (N = 176)

2.5 g (N = 169)

322:286 60:40

9.2 (6.514.0)

10.0 g (N = 187)

N.H. Punjabi et al. / Vaccine 24 (2006) 17761785 Table 2 Antibody response to diphtheria, tetanus, and pertussis vaccine components Antigen g PRP per vaccine dose Baseline GMC Diphteria (IU/ml) 1.25 2.5 5 10 1.25 2.5 5 10 1.25 2.5 5 10 1.25 2.5 5 10 1.25 2.5 5 10 0.13 0.14 0.14 0.13 1.05 0.91 0.87 0.76 0.58 0.60 0.59 0.64 0.63f 0.44 0.46 0.51 10.79 11.05 10.95 10.81 % 0.1 IU/ml 60 56 58 52 93 88 87 85c NA NA NA NA NA NA NA NA NA NA NA NA 7 Months (after third dose) GMC 8.68 8.37 7.69 6.38a 22.88 18.15b 19.55 20.17 11.4d 6.94 7.55 8.64 6.35g 4.83 5.34 4.88 481.09 505.81 515.28 543.60 % 1 IU ml 100 100 98.9 99.2 100 100 99.6 100 NA NA NA NA NA NA NA NA NA NA NA NA

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% 4-fold rise in titer NA NA NA NA NA NA NA NA 86e 74 74 76 73 74 74 72 97 97 97 98

Tetanus (IU/ml)

Pertussis toxin ( g IgG/ml)

FHA ( g IgG/ml)

Pertussis Agglutinating Titer


a b c d e f g

p < 0.05 vs. dose 2.5 p < 0.05 vs. dose 1.25 p < 0.05 vs. dose 1.25 p < 0.001 vs. dose 2.5 p < 0.007 vs. dose 2.5 p < 0.005 vs. dose 2.5 p < 0.05 vs. dose 2.5

g, p = 0.05 vs. dose 1.25 g. g. g. g, p < 0.002 vs. dose 5 g, p < 0.05 vs. dose 10 g. g, p < 0.007 vs. dose 5 g, p < 0.02 vs. dose 10 g. g, p < 0.02 vs. dose 5 g. g, p = 0.05 vs. dose 10 g.

Table 3 Adverse events associated with immunization Adverse event Fever (38 C) Dose Percent reporting event 1.25 g 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 51.6* 56.7* 38.9** 26.3*** 60.0 72.0# 75.8$ 75.0 4.7 4.3 4.7 1.4 4.2 3.7 1.3 1.3 2.5 g 38.1 37.2 41.3 11.8 56.5 57.0 62.7 65.1 4.3 3.7 5.6 2.4 2.7 2.7 3.0 0.6 5.0 g 43.1 45.0 35.3 23.3 64.3 59.8 64.1 63.6 4.9 5.1 5.8 2.3 2.7 2.1 3.2 2.8 10.0 g 42.5 35.0 33.4 17.1 62.7 64.1 64.2 65.8 4.6 5.0 4.4 1.7 3.3 4.6 3.4 2.1

Irritability

Inconsolable crying

Induration (10 mm)

* ** *** # $

p < 0.008 vs. 2.5, 5.0, and 10.0 g doses. p < 0.009 vs. 10.0 g dose. p < 0.031 vs. all doses. p < 0.02 vs. 2.5 g dose, and p < 0.007 vs. 5.0 g. p < 0.001 and 0.005 vs. 2.5, 5.0, and 10.0 g, respectively.

For irritability after injection, the overall incidence was high as reported by the parents, between 56.5 and 75.8%, with the group that received the DPT with 1.25 g Hib after second and third injection having signicantly 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 1.25 g dose of Hib had the highest proportion of fever 38 C, a difference which was signicant for the 1st, 2nd, and 4th immunization compared to all other doses, and significantly higher than for the 5.0 and 10.0 g doses after the third vaccination. Irritability was seen more often among infants who received the 1.25 g dose of Hib as compared to those in the 2.5, 5.0, and 10.0 g groups; this was signicant for the 2nd and 3rd immunization. There were a total of 40 incidences of febrile seizures: eight occurred after the rst, six after the second, and 26 after the fourth (booster) dose of vaccine; these differences were statistically not signicant, 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 six infants required hospitalization: one for chills (described above), two for a respiratory infection which was

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not symptomatic prior to vaccination, and three for high fever, diarrhea, and cough, with onset 28 days after immunization. There was no signicant difference in the rates of hospitalization between the vaccine groups. There were a total of four deaths, but all were considered unrelated to vaccine administration. Three infants died at home, having not sought medical care prior to death. One 3-month-old died 1 month after the rst DTP-Hib 5 g vaccination; another infant died at age 6 months, 2 weeks following the second DTP-Hib 5 g vaccination; both were well before immunization and during the 7-day follow-up period; the parents account of the illness did not clarify the probable cause of death. A third infant died from pneumonia at age 9 months, 4 months after receiving his third DTP-Hib 2.5 g. The fourth infant died at age 3 months, about 18 h after his rst vaccination with DTP-Hib 2.5 g. He had been healthy and afebrile prior to vaccination, but developed fever and irritability several hours later, refusing to breast-feed. The parents did not contact the CHW, and he died at home in the early morning. A post mortem examination was offered but the parents declined. A total of 162 infants were not brought for the complete series of primary immunizations, 102 (7.8%) for the second dose and 60 (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 differ among dosage groups, with the exception of DTP with the 1.25 g Hib dose group, in which fever was seen signicantly more often (26.3%, p < 0.005) than in the other three groups (12, 23, and 17%). Irritability, induration, and inconsolable crying were not signicantly different among the dosage groups.

4. Discussion The history of infectious diseases and public health have established the role of vaccination in the prevention of infectious 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
Table 4 Anti-PRP antibody response following immunization with DPT-PRP-T vaccines g PRP/dose Geometric mean anti-PRP ( g/ml) Baseline Post-immunization Total (%)

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 of diagnostic difculties 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 invasive 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 vaccine 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 10 g dose of PRP-T elicits protective antibody levels, with 99.6% of infants developing antibody levels of 0.15 g/ml and 96.4% of 1.0 g/ml, levels which correlate with shortand long-term protection, respectively. These results are comparable 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 standard dose vaccine (Table 4). The hospitalizations and adverse events observed were evenly distributed among all the groups. The four deaths observed during the 3.5-year study duration can be extrapolated 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

0.15 g/ml Baseline Post-immunization 100 99.6 99.6 99.6

1 g/ml Baseline 8 11 7 7 Post-immunization 96.4 97.4 98.2 96.9

1.25 2.5 5.0 10.0


* **

g g g g

0.12* 0.16 0.15 0.14

12.48 15.62 17.28 15.10

32** 41 45 43

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

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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 conrmed 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 comparison of placebo versus Hib added to whole cell DTP vaccine, no signicant increase in adverse reactions was found [36]. Killed whole cell pertussis vaccine is known to cause signicant adverse reactions, including neurological side effects such as seizures. The latter events have been considered acceptable because of the effectiveness and low cost of whole-cell pertussis vaccine, as described by Edwards and Decker in their editorial concerning two acellular vaccine 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 pertussis 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 Programme 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 satisfactory 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, pneumonia is the leading cause of death. In developed countries, Streptococcus pneumoniae and Haemophilus inuenzae are the principal pathogens in bacterial pneumonia [1]. Another study comparing DTP-Hib with DTP, conducted on the island of Lombok in Indonesia, to evaluate the incidence of vaccinepreventable Haemophilus inuenzae type b pneumonia and meningitis, observed 1449 deaths or 2.6% among 55,073 children enrolled, with 77% of all deaths occurring elsewhere 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 administration of the 10 g 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 manufacture 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 Departemen Kesehatan) R.I., Ministry of Health of Indonesia, the staff of the Bacteriology Laboratory, Center for Infectious Diseases Research NIHRD RI, the Commanding Ofcer 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, Martaria Dhiana, Shinta Laurencia, Nurmiati Nasution, Cynthia, Robert Polowidjaja and Mr. Maman Supriatman for their assistance in the study implementation.

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