Vol. 19, No. 9, Sept., 2000 THE PEDIATRIC INFECTI OUS DISEASE JOURNAL 855 as a combined injection with a Haemophilus influenzae type b vaccine, to UK

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1 Pediatr Infect Dis J, 2000;19: Vol. 19, No. 9 Copyright 2000 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. Immunogenicity and reactogenicity of a pneumococcal conjugate vaccine administered combined with a Haemophilus influenzae type b conjugate vaccine in United Kingdom infants SHARON CHOO, MBBS, LYNN SEYMOUR, RN, RHONWEN MORRIS, RN, SALLY QUATAERT, PHD, STEVE LOCKHART, MRCP, KEITH CARTWRIGHT, FRCPATH AND ADAM FINN, FRCP, FRCPCH, PHD Background. Streptococcus pneumoniae is a major disease burden in young children and the incidence of antibiotic-resistant pneumococcal strains is increasing. Multivalent pneumococcal saccharide-protein conjugate vaccines have recently been developed. Objectives. To assess the immunogenicity and reactogenicity of a 7-valent pneumococcal conjugate vaccine (7VPnC) administered as a separate injection or as a combined injection with Haemophilus influenzae type b vaccine (HbOC) at 2, 3 and 4 months of age. Methods. Randomized controlled trial of 368 healthy UK infants receiving routine vaccines only (control group), routine vaccines and 7VPnC as a separate injection (separate group), or routine vaccines and 7VPnC combined with HbOC (combined group) at 2, 3 and 4 months. The control group received 7VPnC at 5, 6 and 7 months. All groups received pneumococcal polysaccharide vaccine at 13 to 16 months. Anticapsular IgG antibodies to 7VPnC serotypes were measured at 2, 5, 13 and 14 months and safety data collected. Results. IgG antibody concentrations at 5 months were higher in the two treatment groups compared with the controls for all 7VPnC serotypes (P < 0.001) and higher in the separate group than the combined group for five 7VPnC serotypes (P < 0.05). For both treatment groups antibody concentrations were higher at 14 Accepted for publication June 7, From the Sheffield Institute for Vaccine Studies, Division of Child Health, University of Sheffield, UK (SC, LS, AF); the Public Health Laboratory, Gloucester Royal Hospital, Gloucester, UK (RM, KC); and Wyeth-Lederle Vaccines and Pediatrics, UK and US (SQ, SL). Key words: Streptococcus pneumoniae, conjugate vaccine, pneumococcal vaccine. Address for reprints: Dr. Sharon Choo, Division of Child Health, Sheffield Children's Hospital, Sheffield S10 2TH, UK. Fax ; s.choo@sheffield.ac.uk. 854 months (range, 6.6 to 25.3 µg/m1) than at 5 months (range, 0.6 to 2.5 pg/m1) for all 7VPnC serotypes (P < 0.001). Conclusion. 7VPnC was well-tolerated, safe and immunogenic when administered as a separate or as a combined 7VPnC/HbOC injection. Although antibody responses were lower in the infants who received the combination compared with those who received 7VPnC as a separate injection, marked anamnestic responses to polysaccharide challenge were observed, suggesting that both groups were immunologically primed. INTRODUCTION Streptococcus pneumoniae is a major cause of childhood morbidity and mortality. This pathogen is responsible for a wide spectrum of clinical diseases, with infants at particular risk. In England and Wales the mean annual incidences of pneumococcal bacteremia and pneumococcal meningitis in children younger than 1 year of age are estimated to be 19.4 per and 9.4 per , respectively.1 S. pneumoniae is also a common cause of community-acquired bacterial pneumonia in young children and the major bacterial cause of acute otitis media (AOM), accounting for 30 to 60% of middle ear fluid culture-positive episodes in children.2 Pneumococcal polysaccharide vaccines are generally of poor immunogenicity in children younger than 2 years of age. Concern about the increasing incidence of antibiotic-resistant pneumococci in many countries1' 3' 4 has prompted the recent development of new 7-valent, 9-valent and 11-valent polysaccharideprotein pneumococcal conjugate vaccines. Both 7-valent and 9-valent pneumococcal conjugate vaccines have been shown to be immunogenic in infants when given as a separate injection concomitantly with routine infant vaccines at 2, 4 and 6 months of ages-7 and 6, 10 and 14 weeks of age,8 respectively. This randomized controlled study assessed the immunogenicity and reactogenicity of a 7-valent pneumococcal conjugate vaccine administered concomitantly with routine vaccines, either as a separate injection or PTAB PAGE 1/9 MERCK EXHIBIT 1042

2 Vol. 19, No. 9, Sept., 2000 THE PEDIATRIC INFECTI OUS DISEASE JOURNAL 855 as a combined injection with a Haemophilus influenzae type b vaccine, to UK infants at 2, 3 and 4 months of age. METHODS Subjects. The protocol was approved by local research ethics committees in two UK study centers, Sheffield and Gloucester. Healthy male and female infants age 6 to 10 weeks were recruited in both study sites between June, 1997, and May, Parents of infants were approached by research nurses with information about the study, either on postnatal wards after delivery or in the community. Written informed consent was obtained from the parent or legal guardian of each child before enrollment into the study. Infants with a birth weight of <2000 g, any severe or chronic illness or a history of previous immunizations with the routine infant vaccines or study vaccine were excluded from the study. Vaccines and immunizations. A previously prepared computerized randomization list was used to allocate the infants to one of three groups (Fig. 1). The infants received their routine vaccines [diphtheria, tetanus, whole cell pertussis vaccine, 0.5 ml (Evans/ Wellcome; DTwP), H. influenzae type b-crm197 conjugate vaccine (lyophilized) (Wyeth-Lederle; HbOC) and oral polio vaccine (SmithKline Beecham; OPV)] at 2, 3 and 4 months of age. In addition the two treatment groups received 7VPnC [7-valent pneumococcal conjugate vaccine (0.5 ml) containing 2 tig of serotypes 4, 6B, 9V, 14, 19F and 23F polysaccharides, 2 lig of serotype 18C oligosaccharide, 4 aug of serotype 6B polysaccharide and 20 Ag of CRM197 (Wyeth-Lederle)] at 2, 3 and 4 months, and the controls received 7VPnC at 5, 6 and 7 months. HbOC was supplied as a lyophilized powder and reconstituted immediately before administration. The separate group received HbOC reconstituted with Control Left leg: DTwP/HbOC 7VPnC at 5, 6 and 7 months PRIMARY IMMUNIZATIONS: 2, 3 and 4 months of age Separate (7VPnC) Left leg: DTwP/HbOC Right leg: 7VPnC BOOSTER IMMUNIZATION: 13 months of age All Groups Left leg: MMR Right leg: PPS Combined (7VPnC/HbOC) Left leg: DTwP Right leg: 7VPnC/HbOC FIG. 1. Outline of study. All infants received OPV at 2, 3 and 4 months of age. DTwP (0.5 ml) as a combined injection and 7VPnC as a separate injection, and the combined group received HbOC reconstituted with 7VPnC (0.5 ml) as a combined injection and DTwP as a separate injection. All infants received a 23-valent pneumococcal polysaccharide vaccine booster containing 25 pg of each polysaccharide (Wyeth-Lederle; PPS) and measles, mumps, rubella vaccine (Pasteur Merieux MSD; MMR) at 13 to 16 months of age. All vaccines except OPV were administered intramuscularly into the anterolateral aspect of the thigh. Immunogenicity. Venous blood samples were obtained before the first immunization, 1 month after the third immunization, before the booster immunization and 1 month after the booster. Standard enzymelinked immunosorbent assay methods were used to quantitate anticapsular IgG antibodies to the seven pneumococcal vaccine serotypes (4, 6B, 9V, 14, 18C, 19F, 23F) with the use of standard reference serum, 89SF, as previously described.9 IgG antibodies to pertussis (pertussis toxoid, filamentous hemagglutinin, fimbriae 2), diphtheria toxoid, tetanus toxoid and H. influenzae type b (Hib)-polyribosylribitol phosphate (PRP) antigens were measured with standardized enzyme-linked immunosorbent assays Serology was performed at Wyeth-Lederle Laboratories (Rochester, NY). Reactogenicity. Parents were asked to record local and systemic reactions in a diary for 3 days after each vaccination and to measure their infant's axillary temperature with a digital thermometer twice a day for the 3 days. Adverse event data were collected for 1 month after each immunization visit. Statistical analysis. For the purpose of statistical analysis, concentrations below the detection limit of the assays were assigned to one-half that limit by the laboratory. IgG antibody concentrations and fold rises were transformed (logarithm to base 10), and geometric mean concentrations (GMCs) and 95% confidence intervals (95% CI) were calculated. The proportion of infants at or above defined antibody levels ( and _1.0 µg/ml) for each of the seven 7VPnC serotypes was also determined. Differences between GMCs for the three groups were tested with a two-way ANOVA model and an arbitrary significance level of 0.05, with factors for vaccine group (three levels), study site (two levels) and group by site interaction. Pairwise comparisons were made if significant differences were found (Bonferroni method). This approach was also undertaken for the other vaccine antigens. The proportions of infants achieving defined pneumococcal, Hib and diphtheria antibody concentrations were compared by Pearson's chi square test (or Fisher's exact test if >20% of cells had expected values <5). If significant differences were found, pairwise comparisons were made with PTAB PAGE 2/9 MERCK EXHIBIT 1042

3 856 THE PEDIATRIC INFECTI OUS DISEASE JOURNAL Vol. 19, No. 9, Sept., 2000 Fisher's exact test (two sided). A paired sample t test (two-sided) was used to compare pneumococcal antibody concentrations at 14 months with those at 5 months for each of the treatment groups. The frequency of local postimmunization reactions at the 7VPnC injection site (separate group) was compared with that of the 7VPnC/HbOC site (combined group) with Fisher's exact test (two sided), and McNemar's test used to compare local reactions within the separate group. The incidences of systemic reactions in the three groups were compared by Pearson's chi square test (or Fisher's exact test if >20% of cells had expected values <5); and if significant differences were found, pairwise comparisons were made with Fisher's exact test (two sided). A11 statistical analyses were performed using SPSS for Windows Version 9.0 (SPSS, Inc., Chicago, IL). Power calculations performed before study commencement showed that a sample size of 120 subjects per group would provide 80% power to detect a fold difference in pneumococcal IgG antibody concentrations between the treatment groups and the control group at 5 months for any of the seven 7VPnC serotypes. The primary objective of this study was to compare the pneumococcal anticapsular IgG antibody responses to the seven 7VPnC serotypes 1 month after the primary immunizations in the three groups. The secondary objectives were (1) to compare the pneumococcal IgG antibody concentrations after booster immunization with concentrations after primary immunization for the 7VPnC serotypes and (2) to compare IgG antibody responses to the concomitant vaccine antigens after the primary vaccinations in the three groups. RESULTS Subjects. Three hundred sixty-eight infants (267 from Sheffield and 101 from Gloucester) entered the study; 120 were randomized to the control group, 124 to the separate group and 124 to the combined group. There were no significant differences among the three groups with respect to gender and median age at each immunization. At the end of the primary phase 360 infants remained in the study, 115 in the control group, 121 in the separate group and 124 in the combined group. The main reasons for withdrawal from the study were withdrawal of consent (6 infants) and loss to follow-up (1 infant). These study withdrawals were not caused by adverse events. Three hundred twenty-six infants entered in the booster phase of the study and received the pneumococcal plain polysaccharide vaccine. A11 available blood samples were assayed. Immunogenicity data from infants receiving three doses of 7VPnC at the correct time points were included in the statistical analysis. The numbers of infants included in the immunogenicity analysis were 355 at age 2 months (age range, 9 to 10 weeks), 348 at age 5 months (range, 3 to 9 weeks after third immunization), 317 at age 13 months (age range, 13 to 16 months) and 308 at age 14 months (range, 3 to 9 weeks after booster immunization). Safety data were analyzed on an intention to treat basis. Immunogenicity. Because the conclusions following per protocol and intention to treat analyses were the same, only per protocol results will be presented. Table 1 shows pneumococcal IgG anticapsular antibody GMCs for the primary phase of the study. Before the first vaccination at 2 months of age, there were no significant differences in GMCs among the three groups. At 5 months of age there were significantly higher GMCs in the treatment groups compared with controls for the seven 7VPnC serotypes (all P < 0.001). GMCs at 5 months ranged from 1.1 to 2.5 µg/ml in the separate group and from 0.6 µg/m1 to 2.1 µg/ml in the combined group, with 6B the least immunogenic vaccine serotype and 19F the most immunogenic serotype. GMCs were significantly higher in the separate group than the combined group at 5 months for serotypes 6B (P < 0.01), 9V (P < 0.01), 14 (P < 0.05), 19F (P < 0.01) and 23F (P < 0.001). The mean fold rises in pneumococcal IgG antibody levels ranged from 2.0 to 19.3 in the separate group and from 1.1 to 18.4 in the combined group. Table 2 shows the pneumococcal anticapsular IgG Pneumococcal Serotype 4 6B 9V 14 18C 19F 23F TABLE 1. GMCs of pneumococcal anticapsular IgG antibodies (µ,g/m1) at 2 and 5 months Control (n = ) 0.10 ( )* 0.50 ( ) 0.21 ( ) 0.41 ( ) 0.21 ( ) 0.55 ( ) 0.33 ( ) GMC: geometric mean concentration. Numbers in parentheses, 95% CI. 2 mo 5 mo Mean Fold Rise Separate (7VPnC) (n = ) 0.12 ( ) 0.53 ( ) 0.29 ( ) 0.31 ( ) 0.25 ( ) 0.58 ( ) 0.33 ( ) Combined (7VPnC/HbOC) (n = ) 0.11 ( ) 0.57 (0, ) 0.28 ( ) 0.40 ( ) 0.24 ( ) 0.62 ( ) 0.36 ( ) Control (n = ) 0.03 ( ) 0.11 ( ) 0.07 ( ) 0.12 ( ) 0.06 ( ) 0.14 ( ) 0.09 ( ) Separate (7VPnC) (n = ) 2.40 ( ) 1.11 ( ) 1.50 ( ) 2.23 ( ) 1.42 ( ) 2.45 ( ) 1.52 ( ) Combined (7VPnC/HbOC) (n = ) 2.06 ( ) 0.64 ( ) 1.06 ( ) 1.39 ( ) 1.18 ( ) 1.58 ( ) 0.82 ( ) Separate (7VPnC) (n = ) 19.3 ( ) 2.0 ( ) 5.0 ( ) 7.5 ( ) 5.6 ( ) 4.1 ( ) 4.5 ( ) Combined (7VPnC/HbOC) (n = ) 18.4 ( ) 1.1 ( ) 3.8 ( ) 3.4 ( ) 4.8 ( ) 2.5 ( ) 2.2 ( ) PTAB PAGE 3/9 MERCK EXHIBIT 1042

4 Vol. 19, No. 9, Sept., 2000 THE PEDIATRIC INFECTIOUS DISEASE JOURNAL 857 TABLE 2. GMCs of pneumococcal anticapsular IgG ant ibo dies (µg/m1) at 13 and 14 months Mean Fold Rise ct, =1 * o 75 I 2A 28 II C.) a1 Cr) 79 tl 4 ' II C.) O I II Co r- oo ^ r- cc '-' cc; ca CO co 6 CV CO I I I ai A 'elb r4 r4 0 M CV,, C9 O 0 Cr) 0 CO CD CO 07 ud 4 ui 06 4 c.ci c i CV CV t- ud -c -4 CV CO 6 l4 6,-i )CV CV CD CV C- CV CV c t- 0 CO 0- CD 07 CV 00 CO CO CV CD CV ;ccccv CO CV CO CO CO OD CO oo CO CV CV t- CV 0- CV CV c) 0 o- oo oo cy OD OD [-- CV OD OD 0 CV QD CV r4 O O 6 CV N U7 CO co C.- r, ry m C; nt CI; 0t t0)m CV CV ud CV 47 t C o Mm 00 QD o,1" CD QD QD rn ud 6,4 c4 c5 6 6 PP TH cr>,n m Cr) r, CV antibody GMCs for the booster phase. Control infants had received 7VPnC at 5, 6 and 7 months. Pneumococcal antibody GMCs decreased between 5 and 13 months. GMCs at 13 months were 0.3 to 1.0 and 0.2 to 0.9 µg/ml in the separate and combined groups, respectively, and GMCs were generally higher (0.5 to 2.7 µg/ml) in the control group. At 14 months the GMCs rose to 6.4 to 18.8 µg/ml (control group), 7.0 to 22.5 µ,g/m1 (separate group) and 6.2 to 22.9 µg/ml (combined group). 18C and 23F were the least immunogenic serotypes, and 6B and 19F were the most immunogenic serotypes after booster immunization. The mean fold rise in pneumococcal IgG levels after the booster ranged from 14.3 to 51.7 in the separate group and from 12.3 to 46.4 in the combined group. For both of these groups GMCs were significantly higher at 14 months than at 5 months for all 7VPnC serotypes (all P < 0.001). Reverse cumulative distribution curves for the seven 7VPnC serotypes after primary immunizations and after booster immunization are shown in Figure 2. At 5 months the proportion of infants achieving pneumococcal IgG anticapsular antibody concentrations of µg/ml and 1.0 µg/ml was significantly higher in the treatment groups than the control group for all 7VPnC serotypes (all P < 0.001). The percentage of infants with pneumococcal IgG antibody concentrations of µg/ml at 5 months was greater in the separate group than the combined group for serotype 14 (P < 0.05) and not significantly different in the two treatment groups for the remaining serotypes. However, the proportion of infants achieving pneumococcal IgG concentrations of >1.0 µg/ml at 5 months was significantly higher in the separate group than the combined group for serotypes 6B, 9V, 14, 19F (all P < 0.01) and 23F (P < 0.001). We also used analyses advocated by the Food and Drug Administration for assessment of the noninferiority of combination vaccines. For all the 7VPnC serotypes the ratio of the GMCs in the combined group to the separate group was >0.5. In addition, when the lower 95% CIs of the proportions of subjects achieving 0.15 µg/ml specific antibody in the two treatment groups were compared, there was <10% difference for each of the seven serotypes. However, when 1.0 µg/ml was used as the correlate of protection, there were.1.0% differences for serotypes 6B, 9V, 14, 19F and 23F. Table 3 shows IgG antibody responses to the Hib and diphtheria antigens administered at 2, 3 and 4 months of age. PRP IgG antibody GMCs at 5 months were significantly higher in the treatment groups than in controls (P < 0.01 and P < 0.001, respectively). The proportion of infants achieving PRP IgG antibody concentrations of µg/ml were similar for all groups whereas the percentage of infants achieving levels of PTAB PAGE 4/9 MERCK EXHIBIT 1042

5 858 THE PEDIATRIC INFECTIOUS DISEASE JOURNAL Vol. 19, No. 9, Sept., Proportion of su bje cts ( %) Pneumococcal anticapsular IgG concentration (ug/ml) FIG. 2. Reverse cumulative distribution curves of pneumococcal anticapsular IgG antibody concentrations at 5 and 14 months in the separate group (7VPnC) (A) and the combined group (7VPnC/HbOC) (B). 1.0 Ag/m1 was significantly higher in the treatment groups than the controls (both P < 0.001). Diphtheria IgG antibody GMCs were also significantly higher in the treatment groups compared with the controls at 5 months (both P < 0.001). The proportion of infants achieving diphtheria IgG antibody concentrations of -_0.01IU/m1 and 0.1U/m1 was 100% in all groups. There were no significant differences between the treatment groups in terms of PRP and diphtheria responses. IgG antibody GMCs to tetanus and pertus- PTAB PAGE 5/9 MERCK EXHIBIT 1042

6 Vol. 19, No. 9, Sept., 2000 THE PEDIATRIC INFECTIOUS DISEASE JOURNAL 859 TABLE 3. IgG antibodies to Haemophilus influenzae type b and diphtheria toxoid antigens at 5 months Hib PRP Diphtheria N GMC (mg/ml) No. of subjects No. of subjects mg/ml mg/ml N GMC (IU/ml) No. of subjects No. of subjects IU/ml IU/ml Control ( )*t 101 (94)$ 63 (58)$* ( )$1I 106 (100)$ 106 (100)$ Separate (7VPnC) ( )* 112 (97) 92 (80) ( )1 112 (100) 112 (100) Combined (7VPnC/HbOC) ( )* 121 (98) 101 (82) ( (1000) 119 (100) Combined us. control, P < 0.001; separate vs. control, P < t Numbers in parentheses, 95% CI. Numbers in parentheses, percent. Combined us. control, P < 0.001; separate us. control, P < II Combined us. control, P < 0.001; separate us. control, P < sis antigens after the primary immunizations were not significantly different among the three groups. Reactogenicity. The most frequently reported local reaction at the 7VPnC injection site in the Separate group was redness (22 to 25%), followed by pain (19 to 24%) and swelling (17 to 21%). The incidence of redness and swelling was significantly less at the 7VPnC site than at the DTwP/HbOC site for all three doses, and pain was significantly less common at the 7VPnC site for Doses 2 and 3. The incidence of local reactions at the 7VPnC and 7VPnC/HbOC injection sites was not significantly different. Table 4 shows the frequency of reported systemic reactions in the three groups after each primary immunization. Irritability, drowsiness and crying more than usual were the most frequently reported systemic reactions in the treatment groups, accounting for 59 to 71%, 44 to 58% and 52 to 70% of infants in the control, separate and combined groups, respectively. Crying more than usual was significantly more common in the separate group than controls for Dose 1 and significantly more frequent in the combined group than controls for Doses 1 and 3 (all P < 0.01). There were no significant differences among the three groups for other systemic reactions, and fever was uncommon. Most of the local and systemic reactions were reported to be of mild severity. Twelve serious adverse events occurred during the primary phase of the study within 1 month of immunization, 4 per vaccine group. These events consisted of hospitalizations for bronchiolitis (3), upper respiratory infection (2), croup (1), urinary tract infection (1), suspected meningococcal disease (1), blood in stool (1), irritability (1) and elective surgery (2). All these events were considered unrelated to 7VPnC and the infants made a complete recovery. DISCUSSION The seven serotypes included in this pneumococcal conjugate vaccine account for 80% of pediatric pneumococcal invasive disease in the US.13' 14 The efficacy of 7VPnC for vaccine-type pneumococcal invasive disease and for clinically diagnosed, radiologically confirmed pneumonia has recently been shown to be 97 and 73%, respectively, in US infant' 15 and 57% for TABLE 4. Number of infants experiencing systemic reactions in the 3 days following each immunization Control (n = ) Separate (7VPnC) (n = ) Combined (7VPnC/HbOC) (n = 124) Dose 1 Irritability 70 (60)* 82 (68) 88 (71) Drowsiness 61 (52) 66 (56) 69 (58) Crying more than usual 59 (51)t 82 (69)t 87 (70)t Feeding less than usual 30 (26) 39 (34) 36 (31) Fever.38 C 2 (2) 5 (4) 2 (2) Analgesic/antipyretic use 45 (39) 45 (37) 60 (48) Dose 2 Irritability 65 (57) 80 (68) 84 (68) Drowsiness 42 (37) 55 (46) 62 (50) Crying more than usual 58 (50) 72 (62) 76 (62) Feeding less than usual 30 (26) 41 (35) 37 (30) Fever.38 C 3 (3) 2 (2) 2 (2) Analgesic/antipyretic use 38 (33) 56 (47) 49 (40) Dose 3 Irritability 55 (50) 68 (59) 75 (63) Drowsiness 41 (38) 50 (44) 58 (50) Crying more than usual 44 ( (52) 69 (58)$ Feeding less than usual 28 (26) 24 (21) 34 (29) Fever n38 C 5 (5) 1 (1) 7 (6) Analgesic/antipyretic use 31 (27Y 46 (38) 55 (46)$ Numbers in parentheses, percent. I- Dose 1: P < 0.01 for crying more than usual (separate us. control, combined us. control). Dose 3: P < 0.01 for crying more than uaual and analgesic/antipyretic use (combined us. control). PTAB PAGE 6/9 MERCK EXHIBIT 1042

7 860 THE PEDIATRIC INFECTI OUS DISEASE JOURNAL Vol. 19, No. 9, Sept., 2000 serotype-specific, culture-confirmed AOM in Finnish priming induced by 7VPnC. Previous studies have infants.16 To date published studies have assessed the demonstrated poor specific anticapsular antibody reimmunogenicity of this vaccine in a 2, 4 and 6 month sponses to pneumococcal polysaccharide vaccines in US immunization schedule, administered concomi- children years of age particularly with respect to tantly with routine infant vaccines as a separate injec- serotypes 6B, 14, 19F and 23F In our study tion.5' 6 In our study 7VPnC was given at 2, 3 and 4 >10-fold increases in anticapsular antibody concentramonths of age, either as a separate injection or as a tions were demonstrated in both treatment groups combined injection with HbOC. Infants who received after 23-valent pneumococcal polysaccharide vaccine 7VPnC as a separate injection had antibody responses (0.5 ml) containing 25 µg of each polysaccharide comparable to those of US infants immunized at 2, 4 booster immunization for all the 7VPnC serotypes, and 6 months of age.5-7 consistent with a memory response. Postbooster GMCs Serologic correlates of protection are not yet defined (range, 6.2 to 25.3 µ,g/m1) were generally higher than in for pneumococcal disease. However, specific IgG anti- US infants primed and boosted with 7VPnC (range, 1.8 capsular antibody concentrations of 0.15 and to 10.5,u,g/m1),5-7 suggesting that the higher dose of µ,g/ml, thought to be consistent with short term and polysaccharides contained in the plain polysaccharide long term protection from invasive Hib disease,17' 18 vaccine may have induced larger memory responses. are widely used in the evaluation of pneumococcal This finding has implications for future primary immuvaccines. In the US efficacy study >97% of 7VPnC nization schedules, given that primary schedules conrecipients achieved anticapsular antibody levels of sisting of three priming doses of pneumococcal conju µ,g/m1 for all 7VPnC serotypes after the primary gate vaccine and a booster dose of pneumococcal series, which correlated with an observed efficacy of polysaccharide vaccine would be a cheaper option than 97.3%. In our study 92 to 99% and 88 to 99% of infants four doses of pneumococcal conjugate vaccine and in the separate and combined groups, respectively, might induce greater and broader protection. achieved anticapsular antibody concentrations of In the UK booster doses of Hib conjugate vaccine are 0.15 µ,g/m1 after the primary immunizations. In the not routinely given, and the incidence of Hib invasive US and Finnish efficacy trials, 7VPnC efficacy was disease remains extremely low.27 Heath et al.28 dempoor for serotype 19F with respect to A0M7' 19 but was onstrated antibody persistence at the age of 3 to 4 years high for invasive disease in the US study, suggesting in UK children primed with Hib vaccine at 2, 3 and 4 that serum anticapsular antibody titers may not be months of age, with Hib antibody titers of 0.15 p,g/m1 good correlates of protection for AOM. Mucosal im- in 92% of the children studied, and marked responses mune responses to the vaccine may be more important to a plain polysaccharide booster in the remaining for the prevention of otitis and were also measured in children, suggesting the presence of immunologic this study.2 memory. Likewise pneumococcal vaccine boosters may Significantly higher anticapsular antibody responses not be required at all in future schedules. However, if were observed in the separate group compared with the less immunogenic combined regimens are adopted, this combined group after the primary series with respect could influence the need for boosters. Further studies to five of the seven 7VPnC serotypes. However, there are therefore required to assess the need for, and was a marked response to booster immunization in optimal timing of, pneumococcal vaccine boosters in both groups, suggesting that 7VPnC had successfully future immunization schedules. primed for an anamnestic response. Postbooster anti- The increasing number of vaccine antigens available capsular antibody concentrations of 1.0 µg/ml were for infants has resulted in a need for more combination achieved in 93 to 100% and 92 to 99% of infants in the vaccines. Dose-dependent carrier-induced suppression separate and combined groups, respectively. The lower of Hib and tetanus antibody responses has been obanticapsular antibody concentrations observed in the served with a pneumococcal vaccine conjugated to combined group at 5 months may have little biologic tetanus toxoid, given concomitantly with DTwP and a significance in the context of such priming. However, Hib-tetanus toxoid conjugate vaccine at 2, 4 and 6 theoretical concerns remain that a humoral anamnes- months of age.29 On the other hand studies of pneumotic immune response may not occur rapidly enough to coccal CRM197 conjugate vaccines administered conprotect children with poorer responses from invasive comitantly with DTwP and HbOC have shown endisease.21 hancement of Hib and diphtheria responses after Studies to date have used a fourth dose of 7VPnC as primary immunizations at 2, 4 and 6 months3 or 6, 10 a booster vaccine.5-7 In the present study 23-valent and 14 weeks.8 However, when 7VPnC was adminispneumococcal polysaccharide vaccine (0.5 ml) contain- tered to US infants concomitantly with diphtheriaing 25 11,g of each polysaccharide (Wyeth-Lederle) was tetanus-acellular pertussis vaccine and HbOC at 12 to used as the booster challenge to assess immunologic 15 months,6 lower PRP and diphtheria responses were PTAB PAGE 7/9 MERCK EXHIBIT 1042

8 Vol. 19, No. 9, Sept., 2000 THE PEDIATRIC INFE CTIOUS DISEASE JOURNAL 861 observed than when it was given on a different occasion from HbOC and diphtheria-tetanus-acellular pertussis vaccine, although this was as a booster and in this sense not strictly comparable with primary responses. In our study significantly higher Hib and diphtheria IgG antibody GMCs were noted in the treatment groups compared with controls at 5 months. This increased immunogenicity of HbOC may represent a CRM197 carrier-induced enhancement of specific humoral immune responses to PRP antigen. 7VPnC was well-tolerated, causing local reactions less frequently than DTwP/HbOC. The incidence of systemic reactions in the treatment groups was generally comparable with that in controls. The proportion of infants experiencing local and systemic postimmunization reactions was similar to that seen in other studies.5-7 This study has demonstrated the safety and immunogenicity of a 7-valent pneumococcal conjugate vaccine administered to UK infants at 2, 3 and 4 months of age. Specific anticapsular antibody responses were similar to those of US infants immunized at 2, 4 and 6 months. Marked rises in anti-pneumococcal IgG antibody concentrations were observed in 7VPnC-primed infants after plain pneumococcal polysaccharide challenge, suggesting the induction of immunologic memory. Pneumococcal anticapsular antibody responses were reduced for five of the seven 7VPnC serotypes in 7VPnC/HbOC recipients, but this may be clinically unimportant given that their response to polysaccharide boosting suggested good priming. New combination vaccines containing pneumococcal conjugate vaccines will require further study. ACKNOWLEDGMENTS We thank the children and their parents who took part in this study. We also thank Peter Richmond and Frank Bell (research fellows); Ann Duffes (research coordinator); Lorna Ward, Anne Marie Wright, Karen McMurtrie, Jan Everard, Gail Breeze, Anne Maher, Wendy Nedoma and Diane Webb (research nurses); Alan S. Rigby (statistician); Barbara Watson (Gloucester PHL); and Anne McDonough, Samantha Gault and Diane Piper (Wyeth- Lederle). This study was supported by a grant from Wyeth- Lederle Vaccines and Pediatrics. REFERENCES 1. Laurichesse H, Grimaud O, Waight P, Johnson AP, George RC, Miller E. Pneumococcal bacteraemia and meningitis in England and Wales, 1993 to Commun Dis Public Health 1998;1: Fedson DS, Musher DM, Eskola J. Pneumococcal vaccine. In: Plotkin SA, Orenstein WA, eds. Vaccines. Philadelphia: Saunders, 1999: Butler JC, Hofmann J, Cetron MS, Elliott JA, Facklam RR, Breiman RF. 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9 862 THE PEDIATRIC INFECTIOUS DISEASE JOURNAL Vol. 19, No. 9, Sept., Pichichero ME, Voloshen T, Passador S. Kinetics of booster responses to Haemophilus influenzae type b conjugate after combined diphtheria-tetanus-acellular pertussis-haemophilus influenzae type b vaccination in infants. Pediatr Infect Dis J 1999;18: Pomat WS, Lehmann D, Sanders RC, et al. Immunoglobulin G antibody responses to polyvalent pneumococcal vaccine in children in the highlands of Papua New Guinea. Infect Immun 1994;62: Temple K, Greenwood B, Inskip H, Hall A, Koskela M, Leinonen M. Antibody response to pneumococcal capsular polysaccharide vaccine in African children. Pediatr Infect Dis J 1991;10: Dagan R, Melamed R, Zamir O, Leroy O. Safety and immunogenicity of tetravalent pneumococcal vaccines containing 6B, 14, 19F and 23F polysaccharides conjugated to either tetanus toxoid or diphtheria toxoid in young infants and their boosterability by native polysaccharide antigens. Pediatr Infect Dis J 1997;16: Anderson EL, Kennedy DJ, Geldmacher KM, Donnelly J, Mendelman PM. Immunogenicity of heptavalent pneumo- coccal conjugate vaccine in infants. J Pediatr 1996;128: Obaro SK, Huo Z, Banya WAS, et al. A glycoprotein pneumococcal conjugate vaccine primes for antibody responses to a pneumococcal polysaccharide vaccine in Gambian children. Pediatr Infect Dis J 1997;16: Public Health Laboratory Service. Infectious diseases in England and Wales: April to June Commun Dis Rep 1999;9(Suppl 5):S Heath PT, Bowen-Morris J, Griffiths D, Griffiths H, Crook DW, Moxon ER. Antibody persistence and Haemophilus influenzae type b carriage after infant immunisation with PRP-T. Arch Dis Child 1998;77: Dagan R, Eskola J, Leclerc C, Leroy O. Reduced response to multiple vaccines sharing common protein epitopes that are administered simultaneously to infants. Infect Immun 1998; 66: Daum RS, Hogerman D, Rennels MB, et al. Infant immunization with pneumococcal CRM197 vaccines: effect of saccharide size on immunogenicity and interactions with simultaneously administered vaccines. J Infect Dis 1997;176: Pediatr Infect Dis J, 2000;19: Vol. 19, No. 9 Copyright by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. Timing of perinatal human immunodeficiency virus type 1 infection and rate of neurodevelopment RENEE SMITH, MS, KATHLEEN MALEE, PHD, MANHATTAN CHARURAT, MHS, LARRY MAGDER, PHD, CLAUDE MELLINS, PHD, CAROL MACMILLAN, MD, JOAN HITTLEMAN, PHD, TAMAR LASKY, PHD, ANTOLIN LLORENTE, PHD AND JACK MOYE, MD FOR THE WOMEN AND INFANT TRANSMISSION STUDY GROUP* Background. Identifying HIV-1-infected children who are at greatest risk for disease-related morbidities is critical for optimal therapeutic as well as preventive care. Several factors have been implicated in HIV-1 disease onset and severity, including maternal and infant host characteristics, viral phenotype and timing of HIV-1 Accepted for publication June 13, From the University of Illinois at Chicago (RS, CM) and Northwestern University/Children's Memorial Hospital (KM), Chicago, IL; the Institute of Human Virology, Baltimore, MD (MC, LM); Columbia College of Physicians and Surgeons, New York, NY (CM); the State University of New York Health Science Center at Brooklyn, Brooklyn, NY (JH); Institute of Human Virology, Baltimore, MD (TL); Baylor College of Medicine, Houston, TX (AL); and National Institutes of Health/National Institute of Child Health and Human Development (JM). Key words: Pediatric human immunodeficiency virus type 1 infection, developmental outcome, early vs. late infant infection. Address for reprints: Renee Smith, M.S., University of Illinois- Chicago, 840 S. Wood St. M/C 856, Chicago, IL Fax ; resmith@uic.edu. infection. Early HIV-1 culture positivity, i.e. intrauterine infection, has been associated with poor immunologic, virologic and clinical outcomes in children of HIV-infected women. However, a direct effect of timing of infection on neurodevelopmental outcome in infancy has not yet been identified. Methods. Serial neurodevelopmental assessments were performed with 114 infants vertically infected with HIV-1 in a multicenter natural history, longitudinal study. Median mental and motor scores were compared at three time points. Longitudinal regression analyses were used to evaluate the neurodevelopmental functioning of children with early positive cultures and those with late positive cultures. Results. Early infected infants scored significantly lower than late infected infants by 24 months of age and beyond on both mental (P = 0.05) and motor (P = 0.03) measures. Early HIV-1 PTAB PAGE 9/9 MERCK EXHIBIT 1042