Associated or combined vaccination of Brazilian infants with a conjugate Haemophilus influenzae type b (Hib) vaccine, a diphtheria–tetanus–whole-cell pertussis vaccine and IPV or OPV elicits protective levels of antibodies against Hib

Vaccine 19 (2001) 367±375

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Associated or combined vaccination of Brazilian infants with a conjugate Haemophilus in¯uenzae type b (Hib) vaccine, a diphtheria±tetanus±whole-cell pertussis vaccine and IPV or OPV elicits protective levels of antibodies against Hib Orlei O. ArauÂjo a, Eduardo Forleo-Neto b, Glaucia N.R. Vespa b,*, Rosana F. Puccini a, Lilly W. Weckx a, Eduardo S. Carvalho a, Calil K. Farhat a a

Departamento de Pediatria, Universidade Federal de SaÄo Paulo/Escola Paulista de Medicina, Rua Loefgreen, 1998, SaÄo Paulo, SP, CEP 04040-003, Brazil b Pasteur MeÂrieux Connaught do Brasil, Rua do Rocio, 351, 10 andar, SaÄo Paulo, SP, CEP 04552-905, Brazil Received 24 June 1999; received in revised form 11 February 2000; accepted 8 March 2000

Abstract This study investigated the immunogenicity and safety of including a Haemophilus in¯uenzae type b vaccine (polyribosylribitol phosphate conjugated to tetanus toxoid, PRP-T) in three di€erent vaccination schemes: (1) PRP-T reconstituted with a combined diphtheria±tetanus±pertussis-inactivated poliovirus vaccine (DTP-IPV//PRP-T); (2) PRP-T reconstituted with DTP and administered concomitantly with an oral poliovirus vaccine (DTP//PRP-T+OPV); and (3) PRP-T administered concomitantly with DTP at a di€erent injection site and OPV (DTP+PRP-T+OPV). Vaccines were given at 2, 4, and 6 months of age. A total of 252 infants were enrolled, and randomly assigned to one of the three vaccination groups (84 infants in each group); 241 infants were followed until the end of the study. Antibody production against PRP, diphtheria, tetanus and pertussis antigens was satisfactory for each vaccination scheme used. A good response to Hib vaccine was elicited in each group, and 3 months after the third vaccine dose, at least 97% of children in each group had levels of PRP antibody considered to be seroprotective (>0.15 mg/ml), and over 90% of children in each group had levels over 1.0 mg/ml. The solicited local and systemic adverse events following vaccination were mild in all groups and resolved within 4 days without medical intervention. With the exception of fever, which was more common after the second dose in children who received DTP-IPV//PRP-T, local and systemic reactions did not di€er between the vaccination groups. Due to the practical advantages of combined vaccines, their use in routine immunization programs in developing countries is highly desirable. Our results show that Hib conjugate vaccine can be included in routine immunization programs that include either OPV or IPV with satisfactory immunogenicity and safety pro®les. This ¯exible approach should facilitate the inclusion of the Hib conjugate vaccine in routine immunization programs on a world-wide scale. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Haemophilus in¯uenzae type b vaccine; Poliomyelitis vaccine; Diphtheria±tetanus±pertussis vaccine

1. Introduction Widespread vaccination has successfully protected against several infectious diseases, saving many lives * Corresponding author. Tel.: +55-11-3048-0416; fax: +55-11-8204140.

every year [1]. Unfortunately, among the vaccine-preventable diseases that remain, many are still leading world-wide causes of death [1,2]. The Brazilian National Immunization Program has successfully implemented all vaccines recommended in the Expanded Programme on Immunization (EPI) of the World Health Organization (WHO). Oral poliovirus vaccine

0264-410X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 0 9 3 - 1

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(OPV) has been highly e€ective in e€orts to eradicate this disease, with the last Brazilian case of poliomyelitis being reported in 1989 [3]. On the other hand, vaccination coverage against diphtheria, tetanus and pertussis (DTP) is close to 75% in Brazil, and although these diseases are under control, they have not been eliminated [4]. Over the last few years, several countries have started routinely using Haemophilus in¯uenzae type b (Hib) conjugated vaccines, in which the capsular polysaccharide (polyribosylribitol) is conjugated to a carrier protein. This initiative has enabled participating countries to virtually eliminate invasive disease due to Hib [5±9]. Due to the remarkable performance of the Hib vaccines in protecting infants against serious invasive Hib infections such as meningitis and pneumonia [9,10], anti-Hib vaccination is about to be implemented on a national level in Brazil. While the administration of combined vaccines usually simpli®es immunization logistics and enhances vaccine acceptance within a community, environmental and genetic factors that modulate the immune system might lead to di€erences in the speci®c response to a vaccine among populations from distinct geographic regions. Studies conducted in Venezuela, for example, reported antibody titers after Hib vaccination that were superior to those observed outside of Latin America [11,12]. Since it is not known if this phenomenon would occur in another Latin American nation, the present study was undertaken in Brazilian infants to investigate the immune response elicited by PRP-T, whether administered with DTP separately or in combination, and with either IPV or OPV, at 2, 4, and 6 months of age. In Brazil, the preferred injection site for infant vaccination is the buttocks (except for hepatitis B vaccine), so, in addition, this study evaluated the immunogenicity and safety of the vaccines injected at this site.

2. Methods 2.1. Subjects From December 1995 to March 1997, healthy infants of either sex, aged between 8 and 12 weeks who were being brought for routine primary immunization to a Primary Health Care Center in Embu, a city within the metropolitan region of SaÄo Paulo, Brazil, were recruited for the study. Written informed consent was obtained from each parent or guardian before enrolment. The following were considered non-inclusion criteria: 1. previous vaccination with one of the study vaccines; 2. weight <2500 g at time of birth;

3. history of chronic illness or of an immuno-depressive disorder; 4. therapy with immuno-suppressive agents or immunoglobulins; or 5. any contraindication to pertussis vaccination, such as acute febrile illness, hypersensitivity to any vaccine component, or an evolving central nervous system disorder.

2.2. Vaccines 2.2.1. PRP-T The vaccine (Act-HIB2; Pasteur MeÂrieux Connaught (PMC), France; Batches L0171 and L0318) contains 10 mg of PRP covalently linked to 20 mg of tetanus toxoid. The PRP-T vaccines were supplied in lyophilized form in single-dose vials. Reconstitution was carried out at the time of injection with 0.5 ml of diluent (saline 0.4% w/v). 2.2.2. DTP The vaccine (D.T.COQ2/D.T.P.2; PMC, France; Batch K0231) contains puri®ed diphtheria toxoid (r30 international units, IU), puri®ed tetanus toxoid (r60 IU) and inactivated Bordetella pertussis whole organism (r4 IU). The antigens were adsorbed onto aluminum hydroxide (R1.25 mg of aluminum) and presented in a 0.5-ml pre®lled syringe. 2.2.3. DTP//PRP-T (TETRAct-HIB2; PMC, France; Batch L0745) PRP-T vaccine in lyophilized form was reconstituted at the time of injection with 0.5 ml of DTP in a pre®lled syringe. Quantities of antigen in DTP and PRPT vaccines are as described above. 2.2.4. DTP-IPV//PRP-T (PENTAct-HIB2; PMC, France; Batch 0866) PRP-T vaccine in lyophilized form was reconstituted at the time of injection with 0.5 ml of TETRACOQ2 (DTP-IPV) provided in a pre®lled syringe. The IPV vaccine in PENTAct-HIB2 contains inactivated poliovirus type 1 (40 D-Ag U), inactivated poliovirus type 2 (8 D-Ag U), and inactivated poliovirus type 3 (32 DAg U). Quantities of antigen in DTP and PRP-T vaccines are as described above. 2.2.5. OPV The trivalent oral poliovirus vaccine (Pasteur MeÂrieux Connaught, France; Batch L515P) contains 3  105 TCID 50, 1  105 TCID 50, and 3  105 TCID 50 of poliovirus types 1, 2, and 3, respectively. The OPV vaccines are supplied in single-dose ampoules of a volume of 0.5 ml.

64 (60±84) 64 (60±85) 64 (60±72) 63 (60±83) 64 (60±87) 65 (60±94) 84 80 77 84 83 81 84 84 84 5.3 (20.6) 5.3 (20.7) 5.4 (20.7) 41 44 45 Mean (+SD). Number of children that received the vaccine. c Mean of the interval between dose 1 and dose 2. d Mean of the interval between dose 2 and dose 3.

a

b

84 84 84 DTP-IPV//PRP-T (Group 1) DTP//PRP-T+OPV (Group 2) DTP+PRP-T+OPV (Group 3)

64.5 (24.8) 64.5 (24.8) 63.8 (23.8)

Interval 2d [Days (range)] Interval 1d [Days (range)] 3rd dosec 2nd dosec 1st dosec Weight at enrollmentb (kg) Male

The measurement of anti-PRP antibodies was performed according to a Farr-type radio-immunoassay [13]. The results were expressed as mg/ml (0.15 and 1.0 mg/ml were considered as seroprotective thresholds for data analysis). Anti-diphtheria (anti-D) and anti-tetanus (anti-T) antibody assays were measured by enzyme-linked immunosorbent assay (ELISA) as previously described [14,15]. The results were expressed as IU/ml with reference to international standards. The lower limit of quanti®cation (LLOQ) in serum was 0.006 IU/ml, and the seroprotective threshold was set at 0.01 IU/ml for both anti-D and anti-T antibodies. Pertussis antibody levels were measured by serum agglutination of a suspension of B. pertussis (strain Ra€a) as detailed previously [16]. The titer of the serum was expressed as the reciprocal of the highest dilution of serum showing de®nite agglutination. In the absence of a serological correlate of protection against pertussis, the seropositivity threshold was considered to be r10 IU/ml. The antibodies against poliovirus type 1, 2 and 3 were measured by a neutralization assay, based on the ability of speci®c antibodies to neutralize virus infectiv-

Age at enrollmenta (days)

2.4. Laboratory methods

Infants enrolled

This was an open, randomized, comparative study. The study protocol was approved by the local Ethics Committee of the Federal University of SaÄo Paulo before initiation, and procedures complied with the latest version of the Declaration of Helsinki. Parenteral vaccines were injected intramuscularly into the buttock, and OPV was administered by the oral route. Children were randomly assigned to one of three groups as follows: single injection of DTP-IPV// PRP-T vaccine; single injection of DTP//PRP-T vaccine and one dose of OPV; and two injections, one with DTP vaccine and the other in the opposite buttock with PRP-T vaccine, and one dose of OPV. The vaccines were given at 2, 4, and 6 months of age. All vaccinees were closely observed at the center for 30 min after each vaccination. Other adverse events were monitored by means of a home visit 3±5 days after vaccination, during which time parents or guardians were asked to report all signs and symptoms with the help of a questionnaire. Two blood samples were collected, one just before the ®rst immunization and the second 3 months after the last injection, when infants were brought back to the center for their routine measles vaccination. Blood samples were kept for 30 min at room temperature then centrifuged, and the serum stored at ÿ208C until further analysis.

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Group

2.3. Vaccination groups

Table 1 Characteristics of 252 Brazilian infants enrolled in a clinical study to investigate vaccination with a Hib conjugate vaccine, given according to three di€erent schemas at 2, 4 and 6 months of age

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ity for a susceptible host cell, as described elsewhere [17]. The titer of neutralizing antibodies was expressed as the inverse of the highest dilution of serum that inhibited the cytopathic e€ect of the virus. A titer of poliovirus type 1, 2, or 3 neutralizing antibodies r5 was considered as seroprotective. All assays were performed at the Clinical Sero-Immunology Laboratory, Pasteur MeÂrieux Connaught (Val de Reuil, France). A unique number was used for each sample, and the code was unknown to the technician performing the laboratory tests. 2.5. Statistical analysis The mean 2 SD for the age, weight and height of the infants at enrolment, and the interval between each vaccine dose were calculated. Geometric mean titer (GMT) values and the 95% con®dence intervals (CI95%) were calculated at each blood sampling time. For GMT calculations for the pertussis and polio vaccine components, an antibody concentration below the LLOQ was given an arbitrary value of one-half this value. The comparison between the GMTs of antibodies before and after vaccination was analyzed by Wilcoxon's rank-sum test. GMTs between groups were compared by the Kruskal±Wallis test followed by multiple pairwise comparisons (Dunn's procedure). Seroprotection and seropositivity rates (i.e. percentage of subjects with antibody levels greater than or equal to the prede®ned threshold values), and the reported local and systemic adverse events after vaccination, were compared between groups by the chi-square test. Statistical comparisons were considered signi®cant when p < 0.05. 3. Results A total of 252 infants were enrolled, with 84 included in each of the three groups. Among these, 241 were followed until the end of the study. The

major reasons for dropout from the study were infants whose families moved away from the municipality and infants for whom parents or guardians refused to allow further blood sampling. No signi®cant di€erence was observed among the three groups of vaccinees with regard to demographic characteristics or time interval between vaccination visits (Table 1). 3.1. Serologic response to PRP-T The anti-PRP antibody responses are shown for each group in Table 2. Although some baseline di€erences in anti-PRP GMT values were noted between the groups, these di€erences did not achieve statistical signi®cance and are not considered of clinical signi®cance. Anti-PRP antibody titers in each vaccine group increased substantially after vaccination. Three months after the third dose of vaccine, at least 97% of children in each group achieved seroprotective levels of PRP antibody (r0.15 mg/ml), and over 90% of infants had antibody levels considered to confer long-term protection (r1.0 mg/ml). The anti-PRP antibodies levels among children in the DTP+PRP-T+OPV group, who received all the vaccines separately, appeared to be higher than in the other two groups. Nevertheless, the observed di€erence in GMTs only achieved statistical signi®cance between DTP+PRP-T+OPV (6.25 mg/ ml) and DTP//PRP-T+OPV (3.89 mg/ml) ( p < 0.05). 3.2. Serologic response to diphtheria, tetanus and pertussis The antibody responses induced by DTP vaccine administered through di€erent immunization protocols are summarized in Table 3. Prior to vaccination, more than 80% of the infants had antibody levels against the three vaccine components that were above the thresholds of seroprotection or seropositivity. Pre-vaccination antibody levels did not di€er signi®cantly between groups. Three months after the third dose of vaccine,

Table 2 Serum anti-PRP antibody concentrations before the ®rst injection (Pre-) and 3 months after the third injection (Post-) in Brazilian infants given a Hib conjugate vaccine according to three di€erent schemas at 2, 4 and 6 months of age

N r0.15 mg/mla r1.0 mg/mla GMT (mg/ml)b (Cl95%) a b

DTP-IPV//PRP-T Group 1

DTP//PRP-T+OPV Group 2

DTP+PRP-T+OPV Group 3

Pre-

Post-

Pre-

Post-

Pre-

Post-

84 39.0% 7.0% 0.11 (0.08±0.14)

84 100% 95.0% 4.94 (3.56±6.16)

84 39% 7.5% 0.49 (0.37±0.65)

80 100% 91.0% 3.89 (3.06±4.94)

84 40.0% 3.0% 0.11 (0.08±0.14)

77 97.0% 93.5% 6.25 (4.54±8.61)

There is no signi®cant di€erence between the groups regarding the number of infants with anti-PRP >0.15 mg/ml or >1.0 mg/ml ( p > 0.05). Pre-vaccination: Group 1=Group 2=Group 3; post-vaccination: Group 3 > Group 2, Group 1=Group 2, Group 1=Group 3.

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100% of infants in each group achieved or maintained antibody levels superior to those considered protective or indicative of seropositivity. Comparing between the groups, the GMT values for speci®c antibody to each of the three components of DTP tended to be greater when DTP had not been combined with other vaccines (DTP+PRP-T+OPV). Between-group di€erences in GMT values achieved statistical signi®cance for diphtheria ( p < 0.001), tetanus ( p < 0.001) and pertussis ( p < 0.01).

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3.4. Safety 3.4.1. Immediate adverse reactions Immediate reactions to vaccination observed during the 30 min immediately following vaccination occurred infrequently, with just one child in the DTP+PRPT+OPV group presenting with redness and tenderness at each injection site after the ®rst dose. 3.4.2. Local adverse reactions Almost all adverse reactions at the injection site appeared between 12 and 48 h after vaccination. In general, local reactions were more frequent after the ®rst dose than after the second or third doses. The percentages of children presenting at least one local reaction after the ®rst, second and third doses, respectively, were 29%, 18%, and 19% in the DTPIPV//PRP-T group; 36%, 18%, and 18% in the DTP// PRP-T+OPV group; and 36%, 22%, and 21% in the DTP+PRP-T+OPV. Globally, the most common reactions were pain and induration. However, the frequency of these two reactions never exceeded 26% in any group, irrespective of the vaccine dose. No signi®cant di€erence was observed between the three groups regarding the occurrence of pain, tenderness, induration, warmth, redness, swelling, or ecchymosis. All local symptoms resolved within 4 days without medical intervention (data not shown).

3.3. Serologic response to polio vaccines The polio vaccines were highly immunogenic for all poliovirus types (Table 4). The proportion of vaccinated children presenting protective levels of poliovirus-neutralizing antibodies (reciprocal dilution r5) ranged between 91 and 100% depending on the vaccine group and the poliovirus type. Some betweengroup di€erences were observed in terms of GMT values, with the antibody responses to poliovirus types 1 and 3 elicited by DTP-IPV being higher than those in the two groups who received OPV ( p < 0.01 and p < 0.001, respectively). On the other hand, OPV appeared to induce higher levels of speci®c antibodies against poliovirus type 2. The only statistically signi®cant between-group di€erence in GMT values observed for poliovirus type 2 was between DTP//PRP-T+OPV (GMT=545) and DTP-IPV//PRP-T (GMT=311) ( p < 0.01).

3.4.3. Systemic adverse reactions All systemic adverse events reported up to 5 days after vaccination were mild and resolved within 4 days. There were no reports of any immediate anaphy-

Table 3 Serum anti-diphtheria, tetanus and pertussis antibody concentrations before the ®rst injection (Pre-) and 3 months after the third injection (Post) in Brazilian infants given a Hib conjugate vaccine either combined with an IPV-containing combination vaccine (DTP-IPV) or combined or associated with DTP, in association with OPV, at 2, 4 and 6 months of age

N Anti-De Anti-T Anti-PT

r0.01 IU/mla GMTb (CI95%) r0.01 IU/mla GMTc (CI95%) r10 IU/mla GMTd (CI95%)

DTP-IPV//PRP-T Group 1

DTP//PRP-T+OPV Group 2

DTP+PRP-T+OPV Group 3

Pre-

Post-

Pre-

Post-

Pre-

Post-

84 89% 0.11 (0.07±0.18) 82% 0.11 (0.07±0.18) 80% 33 (25±43)

84 100% 0.18 (0.14±0.23) 100% 1.17 (0.97±1.42) 100% 609 (536±758)

84 94% 0.14 (0.09±0.21) 90% 0.15 (0.10±1.69) 83% 37 (29±48)

80 100% 0.26 (0.21±0.33) 100% 1.69 (1.43±1.99) 100% 668 (589±758)

84 94% 0.14 (0.08±0.23) 86% 0.10 (0.06±0.15) 88% 33 (26±41)

77 100 % 0.54 (0.44±0.67) 100% 10.91 (1.56±2.32) 100% 809 (716±913)

a There is no signi®cant di€erence between the groups regarding the number of infants that reached seroprotective antibody levels (r0.01 IU/ ml) ( p > 0.05). b Anti-D post-vaccination: Group 3 > Group 1, Group 3 > Group 2, Group 1=Group 2. c Anti-T post-vaccination: Group 3 > Group 1, Group 2 > Group 1, Group 3=Group 2. d Anti-PT post-vaccination: Group 3 > Group 1, Group 3=Group 2, Group 1=Group 2. e Anti-D, anti-diphtheria antibodies; anti-T, anti-tetanus antibodies, anti-PT, anti-pertussis toxoid antibodies.

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lactic or anaphylactoid reaction, any episode of prolonged, inconsolable crying or hypotonic±hyporesponsive episode, or any seizure. The most common reaction was fever (i.e. 29±44% after the ®rst dose, 18±37% after the second dose, and 19±30% after the third dose), which had its onset within 2 days of vaccination, and never persisted for longer than 48 h. The only di€erence between groups concerned fever after the second dose, which was more common among children who had received DTP-IPV//PRP-T (37.3%) compared with the other two groups (DTP//PRPT+OPV=18.0% and DTP+PRP-T+OPV=31.0%; p = 0.02). The frequency of other systemic reactions (i.e. irritability, drowsiness, nausea, vomiting and diarrhea) did not di€er between the three groups after any vaccine dose (data not shown). 3.4.4. Serious adverse event One death due to pneumonia occurred 52 days after the third dose of DTP-IPV//PRP-T. In view of the temporal relationship with vaccine administration, there appeared to be no causal relation between this event and vaccination. However, as the etiological agent was not identi®ed, the available data were not considered to be sucient by the investigator and sponsor to attribute a causality relationship to this event (relationship unknown). 4. Discussion Our study aimed to evaluate the immune response elicited by a conjugated Hib vaccine, PRP-T, when administered at 2, 4 and 6 months of age, either as-

sociated or combined with other pediatric vaccines used routinely in many countries, namely DTP and IPV or OPV. Three months after the third vaccine dose, we observed some between-group di€erences concerning the GMT values of PRP-speci®c antibodies, with a tendency for a stronger response in the group who had received all vaccines separately. It is widely accepted that a child with a serum anti-PRP antibody concentration r0.15 mg/ml is likely to be protected against Hib infection, and concentrations r1.0 mg/ml are reported to correlate with long term protection [18±21]. Despite the observed di€erences in GMT values, 3 months after immunization, adequate rates of seroprotection were obtained in each group, with over 97% of infants who received Hib conjugate vaccine, either associated or combined with DTP and IPV or OPV, being seroprotected against Hib, irrespective of the vaccine scheme used, and over 90% with PRP antibody levels indicative of long-term protection. There were no di€erences between groups in terms of the proportions of children who were seroprotected. There is good evidence to indicate that children given primary vaccination with PRP-T, in association or combination with other routine childhood vaccines, are primed to elicit a booster response upon subsequent encounter with the pathogen. A recent study demonstrated that infants given primary vaccination with PRP-T produced a vigorous anamnestic response to a dose of unconjugated PRP polysaccharide when given at 12±14 months of age, a time at which this vaccine is only immunogenic in children who have been immunologically primed [22]. Moreover, this anamnestic response was observed even in children with anti-PRP titres <0.15 mg/ml, suggesting that these

Table 4 Serum anti-Poliovirus antibody concentrations before the ®rst immunization (Pre-) and 3 months after the third injection (Post-) in Brazilian infants given a Hib conjugate vaccine either combined with an IPV-containing combination vaccine or in association with OPV at 2, 4 and 6 months of age

N Type 1 Type 2 Type 3

r5a GMTb (CI95%) r5a GMTc (CI95%) r5a GMTb (CI95%)

DTP-IPV//PRP-T Group 1

DTP//PRP-T+OPV Group 2

DTP+PRP-T+OPV Group 3

Pre-

Post-

Pre-

Post-

Pre-

Post-

84 64% 10 (7±13) 69% 12 (9±16) 45% 5 (4±7)

84 96% 240 (173±335) 96% 311 (211±458) 99% 672 (480±942)

84 59% 8 (6±10) 57% 9 (6±11) 35% 5 (4±6)

80 99% 125 (88±178) 100% 545 (427±696) 95% 135 (102±179)

84 64% 11 (8±15) 67% 12 (9±16) 54% 7 (5±9)

77 91% 115 (79±166) 100% 465 (351±616) 95% 99 (70±138)

a Reciprocal dilution. There is no signi®cant di€erence between groups regarding the number of infants that reached the seroprotective threshold of poliovirus-neutralizing antibodies ( p > 0.05). b Polio types 1 and 3 post-vaccination: Group 1 > Group 3, Group 1 > Group 2, Group 2=Group 3. c Polio type 2 post-vaccination: Group 2 > Group 1, Group 2=Group 3, Group 1=Group 3.

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children were also primed and therefore protected against exposure to the pathogen. After PRP-T immunization, the anti-PRP antibody concentration declines with an estimated half-life of 15.8 weeks [23]. On extrapolation from the results obtained in this study, it can be estimated that the PRP antibody GMT attained 3 months after the last dose of vaccine would take at least 6 years to decline to 0.15 mg/ml. Therefore in each group, the anti-PRP antibody levels elicited by the administration of Hib conjugate vaccine either associated or combined with other routine vaccines appeared satisfactory, being suf®cient to guarantee suitably protective levels of Hibspeci®c antibodies throughout early childhood, in particular during the ®rst year of childhood when most Hib invasive disease occurs [24±26], and up to the time when a booster dose of conjugated Hib vaccine may be given during the second year of life. Finally, previous studies have already examined the immunogenicity of simultaneous administration of vaccines against Hib, DTP, and polio in the same syringe or by separate injections [27±33], and interference among the antigens has also been observed in several of these investigations [31±33]. Nevertheless, as in our study, no di€erence was observed in the percentage of infants that reached antibodies levels considered protective [31±33]. Furthermore, there is good serological data to con®rm that DTP//PRP-T or DTP-IPV//PRPT is immunogenic and e€ective in countries that have adopted these combined vaccines in their national immunization programs [10,32±36]. Use of a Hib conjugate vaccine in infant immunization programs, whether combined or associated with routine vaccines, would also provide herd immunity, which has been found to be an added bene®t obtained with the implementation of routine immunization programs in other countries, inhibiting the carriage state among vaccinated infants and reducing exposure of the susceptible population (i.e. non-vaccinated individuals or those that respond less e€ectively to immunization) [37±40]. In the present study, blood samples were drawn 3 months after the third dose of the primary series, instead of the usual blood draw one month after completion of the primary series. This approach was adopted to reduce the chance of drop-outs by pro®ting from the fact that children would be returning to the health care center for measles immunization, currently recommended in Brazil at 9 months of age. Due to the decline in antibody levels observed after primary immunization, this late sampling time makes it dicult to compare directly the absolute antibody levels obtained in our study with published results using the standard one month post-immunization sampling time. Nevertheless, the high humoral responses to Hib observed in Venezuelan infants [11] were not seen in

373

our study, even taking into account the longer time between immunization and blood sampling. The antiPRP antibody titers observed in our population of Brazilian infants seem more compatible with those observed in studies performed in the United States and Europe [41±43]. Concerning antibody responses to diphtheria, tetanus and pertussis, despite some between-group di€erences in GMT values, three months after immunization, all children had either achieved or maintained seroprotective or seropositive antibody levels. A variety of procedures are employed to determine the serological response following pertussis disease or immunization with pertussis vaccine, including agglutination assays, bactericidal assays, complement ®xation and ELISA [44]. Although no speci®c serological procedure is available that has been proved to be a direct measure of the host's immunity to infection or disease, early studies found that protection from pertussis correlated with high titers of agglutinating antibody [45±47]. We thus used the agglutination technique to obtain a rapid con®rmation of the presence of adequate titers of agglutinating antibodies after vaccination and to be able to compare between the di€erent study groups. This technique is often used when the immunogenicity of whole-cell pertussis vaccines is under study (see [48,49]). Both IPV and OPV induced seroprotective levels of poliovirus-neutralizing antibodies in the great majority of infants, if not all (91±100%). Injection into the buttock has been associated with decreased immunogenicity of certain antigens in children and adults [50]. Due to the potential risk of sciatic nerve injury [51], a recommended site for intramuscular injection in infants is the antero-lateral aspect of the upper thigh [52,53]. Nevertheless, in Brazil, arguments and e€orts to change this habit are not readily accepted either by parents or by health professionals, and the buttock is still the preferred injection site. Since vaccinators in Brazil are experienced with injections in this anatomical area Ð making it the ®rst choice for local health care providers Ð we decided to evaluate this route of administration in our study. We con®rmed that a satisfactory antibody production was obtained, with no clinically important di€erence in frequency of adverse events compared with a typical intramuscular injection into the upper aspect of the thigh as in other published reports [16,33,34]. With the exception of fever, which after the second dose was most common among children in the group that received the pentavalent combination (DTP-IPV//PRP-T), systemic reactions did not di€er between vaccination schemes. This study among Brazilian infants reinforces the concept that combined vaccines are immunogenic and safe. Reducing the number of injections has many

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practical advantages, especially for developing countries, as it simpli®es immunization logistics and enables a wider acceptance and compliance with vaccination schedules, with a consequential increase in coverage. As demonstrated by our results, the Hib conjugate vaccine can be included in routine immunization programs, irrespective whether OPV or IPV is used. The Hib conjugate vaccine can be combined safely and e€ectively with either an IPV-containing tetravalent combination or a DTP vaccine with OPV given in association. This ¯exibility should facilitate the inclusion of the Hib conjugate vaccine in routine immunization programs on a world-wide scale.

[11]

[12]

[13]

[14]

Acknowledgements The authors are grateful to Mark Fletcher and Susan Wood for assistance in the manuscript preparation. We also acknowledge Christine Blondeau and Olivier Chappey for serological analyses. The study was supported by a grant from Pasteur MeÂrieux Connaught.

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