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Evaluation of booster doses of Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in 18-month-old children
~UTTERWORTH I-~E I N E M A N N
Vaccine, Vol. 13, No. 1, pp. 104-108, 1995 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0264.-410X/95 $10.00+ 0.00
Evaluation of booster doses of Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in 18-month-old children David W. Scheifele *~, William Meekison*, Roland Guasparini*, Ann Roberts t, Luis Barreto $, John Thipphawong J; and Sharon Wiltsey $ A booster dose o f H a e m ophi l us influenzae type b conjugate vaccine in the second year of life is the final step in the recommended series of doses to protect infants from invasive infection. This study assessed the safety and immunogenicity o f P R P - T conjugate vaccine booster doses (Act-HIB ®, Connaught Laboratories Ltd). The participants were 367 healthy children who had taken part in a study of primary immunization with PRP-T. At 18-19 months old, subjects were randomly assigned to receive diphtheria-pertussis-tetanus (DPT) and PRP- T vaccines either mixed in one syringe (n = 183) or separately in opposite limbs (n = 184). Adverse events were monitored for 48 h after immunization. Blood was obtained prior to vaccination in half of the subjects (combined injections group) and following vaccination in all subjects to test for antibodies to each o f the antigens administered. Local adverse reactions were infrequent with P R P - T alone and equally frequent at sites of D P T or D P T / P R P - T injection, except for redness >>,25 mm in diameter which was more.frequent after the combined vaccines (25.1 versus 14.1%, p
During 1992, all provinces in Canada implemented programmes for vaccination of young infants against Haemophilus influenzae type b (Hib) invasive infections. These programmes have been remarkably effective in reducing the frequency of Hib disease 1. The vaccines selected by the provinces are HibTITER ~m (HbOC, Lederle Cyanamid) or Act-HIB ® (PRP-T, Connaught Laboratories Ltd). The recommended series z for each product consists of three primary doses given at 2, 4 and *Vaccine Evaluation Center, BC's Children's Hospital, Vancouver, Canada. tCalgary Health Services, Calgary, Alberta, Canada and tConnaught Laboratories, Willowdale, Ontario, Canada. ~To whom correspondence should be addressed at Vaccine Evaluation Center, 950 West 28th Avenue (Trailer), Vancouver, British Columbia V5Z 4H4, Canada. (Received 30 March 1994; revised 24 May 1994; accepted 24 May 1994)
104 Vaccine 1995 Volume 13 Number 1
6 months of age, with a booster dose at 18 months. Both products are intended to be given simultaneously with diphtheria-pertussis-tetanus (DPT) vaccine: Act-HIB ® (PRP-T) can be mixed with DPT vaccine produced by the same manufacturer and given as a single injection. Act-HIB ® was evaluated in Canadian infants aged 2-6 months 3 prior to licensure, but attributes of the booster dose were inferred from studies done elsewhere4'5. We report here one of the first studies to assess the performance of the booster dose of PRP-T vaccine given mixed in one syringe with DPT, in comparison with simultaneous separate injections of these vaccines.
METHODS Children who had participated in a study of primary immunization with PRP-T 3 were invited to participate
Hib booster vaccination: D.W. Scheifele et al.
in this randomized controlled trial of booster doses. All children had received PRP-T at 2, 4 and 6 months of age. All had developed anti-PRP (polyribosylribitol phosphate) levels t>0.15 #gml-1 by 7 months of age, at which time no difference in levels was evident between those who received PRP-T mixed in one syringe with DPT vaccine and those who received separate concurrent injections. Children were eligible for enrolment in t h e current study if they were healthy, had completed the three-dose primary series and had no contraindications to receiving additional doses of DPT or PRP-T vaccine, according to published criteria 6. Written informed consent was obtained from their parents prior to enrolment. The study was approved by the ethics committees of the University of British Columbia and the Calgary Board of Health. Participants were vaccinated at 18-19 months of age with DPT, PRP-T and OPV vaccines. They were randomly (1:1) assigned to receive either (i) a combined injection of DPT and PRP-T mixed in one syringe or (ii) separate injections of these products in opposite limbs. The new randomization was stratified to produce two equivalent groups balanced in terms of previous interventions (mixed or separate modes of vaccination, PRP-T lot given). In the combined vaccinations group, liquid DPT vaccine was added to lyophilized PRP-T vaccine to create the mixture prior to use. In the separate vaccinations group, a saline diluent supplied by the manufacturer was used to rehydrate PRP-T vaccine. One lot of PRP-T vaccine (S-2440) was used, along with one lot of DPT vaccine (3951-11). OPV lot derivation was not controlled. The DPT vaccine was prepared by Connaught Laboratories Limited (Willowdale, Ontario), as was the OPV vaccine. DPT contained 5 Lf tetanus toxoid, 25 Lf diphtheria toxoid, 4-12 protective units of pertussis vaccine and 1.5 mg aluminium phosphate per 0.5 ml dose. The PRP-T vaccine was prepared by Pasteur Merieux Srrums et Vaccins and contained 10 #g of polyriboseribosylphosphate (PRP) covalently linked to 20#g of tetanus toxoid in each dose. The vaccination technique was standardized between the three field teams that conducted this study. PRP-T and DPT vaccines were administered intramuscularly into the deltoid area in all but six children, in whom the thigh was preferred. Parents were asked to observe and record any changes in their child's health during the 48 h after vaccination. They were given celluloid templates to measure areas of redness or swelling at injection sites. Fever prophylaxis with acetaminophen was advocated routinely. Temperatures were to be taken at intervals for 48 h. Parents' observations were systematically reviewed during telephone interviews with study staff 24 and 48 h after immunization, as previously described 3. Compliance with the protocol was monitored at intervals by the sponsor. Preimmunization antibody trough levels were measured in a representative sample of subjects only because of the large number available. Either of the newly randomized groups could have been selected as they were carefully balanced: we chose to bleed those assigned to receive combined vaccines, thereby minimizing painful procedures. Blood was obtained from all participants 1 month after immunization. Sera were tested for (i) diphtheria antitoxin by the microneutralization test; (ii)
pertussis agglutinins by the microagglutination method; (iii) tetanus antitoxin by enzyme-linked immunoassay (ELISA); (iv) PRP antibody by radioimmunossay; and (v) subcellular pertussis antigens, including pertussis toxin, filamentous haemagglutinin, 69 kDa protein and fimbrial agglutinogens, by ELISA. The methods used have been described previously3. The tests were performed at Connaught Laboratories Ltd on codelabelled samples. Data were assembled and analysed at the Vaccine Evaluation Center in Vancouver. Case report forms were checked for accuracy and completeness upon receipt and corrections effected if necessary. Accuracy of data entry was verified through programming checks and manual verification of key data for all files. The incidence rate of injection site redness was designated as a primary outcome measure because it is most likely to be ascertained reliably by parents. Analyses of intergroup difference in antibody responses were based on absolute titres achieved as well as the proportions exceeding predetermined levels of particular significance. Geometric mean titres (GMTs) were calculated from log-transformed values, negative values being assigned an arbitrary value just below the lower detection limit. Means of normally distributed antibody data were compared using Student's t test. The •2 test with continuity correction was preferred for comparison of proportional data; Fisher's exact test was also used. Calculations were performed using SAS/STAT software (SAS Institute, Cary, NC, USA). RESULTS Of the 423 children who completed primary immunization with PRP-T vaccine and were eligible to receive booster doses, 367 (86.8%) participated in this study. The combined injection group contained 183 subjects, the separate injection group contained 184. A total of 180 infants were enrolled at Vancouver site A, 77 at Vancouver site B and 110 in Calgary. Most infants (90.7%) were 18 months old at the time of the booster dose, the remainder being 19 months old. Fifty-one per cent were female. A telephone follow-up was obtained for all but one child at 24 h and for all but two at 48 h following immunization. Groups were closely matched in terms of age at immunization and sex distribution both by centre and by treatment assignment. No immediate adverse reactions were encountered. Local adverse reactions to immunization are summarized in Table 1. Morbidity was infrequently observed after PRP-T vaccine. Twenty-four hours following vaccination, children who received combined injections of DPT/PRPT more often had redness 1>25 mm in diameter (25.1%) than did those who received DPT separately (14.1%, p = 0.008, Fisher's exact test). Apart from this, other local changes occurred at a similar frequency between DPT and DPT/PRP-T sites. Acetaminophen prophylaxis utilization rates exceeded 95% in both groups. Additional treatment doses were given to 54% and 61% of subjects (difference not significant). Antipyretic use could have influenced assessment of adverse reactions such as fever. Moreover, intercurrent illnesses (principally cough and cold syndromes) were reported within 48 h after 36 immunizations (9.8%), with similar distribution between groups. Affected infants were not removed from the analysis.
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Hib booster vaccination: D.W, Scheifele et al.
Table 1
Local adverse reactions to the booster immunizations
Observation
Combined injections of DPT/PRP-T (%)
Separate injection of DPT (%)
Separate injection of PRP-T (%)
Redness at 24 h Any />25 mm
80 (43.7) 46 (25.1)*
83 (45.1) 26 (14.1)*
12 (6.5) 3 (1.6)
Redness at 48 h Any >/25 mm
57 (31.1) 39 (21.3)
60 (32.6) 31 (16.8)
4 (2.2) 3 (1.6)
Swelling at 24 h Any ~>25 mm
43 (23.5) 27 (14.8)
49 (26.6) 23 (12.5)
8 (4.3) 1 (0.5)
Swelling at 48 h Any
25 (13.7)
29 (15.8)
1 (0.5)
Tenderness at 24 h Mild Moderate Severe
64 (35.0) 40 (21.9) 17 (9.3)
60 (32.6) 37 (20.1) 18 (9.8)
37 (20.1) 15 (8.2) 5 (2.7)
183 (100)
184 (100)
Total number
184 (100)
*p=0.008, Fisher's exact test
Table 2
Anti-PRP levels in study participants
Anti-PRP levels a (/~g m1-1)
Combined injection group n (%)
Separate injections group n (%)
Pre-immunization Total assayed <0.06 0.06-0.149 0.15--0.999 >~1.00 > 10.00 Geometric mean titre t~ 95% confidence interval
180 (100) 32 (17.8) 16 (8.9) 80 (44.4) 52 (28.9) 3 (1.7) 0.41 0.33-0.51
Not performed
Post-immunization Total assayed 179 (100) <0.149 0 0.15-0.99 0 7>1.00 179 (100) > 10.00 157 (87.7) Geometric mean titre b 38.1 95% confidence interval 32.3-44.9
Table 3 Responses to diphtheria, tetanus and pertussis antigens in study participants
179 (100) 0 3 (1.7) 176 (98.3) 154 (86.0) 33.9 28.5-40.2
aMeasured by radioimmunoassay t)For anti-PRP levels <0.06 iLg ml 1, the value was assumed to be 0.05 #g ml 1
Parents complied well with temperature measurements, obtained in 88.8% of infants. Most (87%) preferred axillary measurements. During the first 24h after immunization, a temperature/> 38.0°C occurred in tested children (n=326) at a frequency of 30.6%, with 7.6% exceeding 39.0°C. Fever was detected between 24 and 48 h in 6.8% of children tested (n=252). No significant difference in fever patterns existed between groups. Similarly, no intergroup differences occurred in rates of irritability (mean 54.8 %), crying more than usual (40.9 %), lethargy (16.4%) or vomiting (5.4%) during the first 24 h after immunization. One child developed seizures after separately administered vaccines. This child had several brief convulsive episodes 2 h after immunization, in
106
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association with fever of 40°C. The event was attributed to vaccination. No hypotonic-hyporesponsive episodes were noted. Eight children were seen by physicians for adverse events within 48 h of vaccination, including some with intercurrent illnesses. Serum samples were obtained as intended prior to immunization from 180 of 183 children in the combined injections group. Serum samples were obtained from both groups 4 weeks following immunization: sufficient serum for anti-PRP tests was obtained from 358 children (97.5%). Prior to booster immunization, the geometric mean anti-PRP level in those tested (Table 2) was 0.41 #g m l - 1 (95% confidence interval (CI95): 0.33-0.51). No measurable anti-PRP (<0.06/~gm1-1) was detected in 32 samples (17.8%) while an additional 16 (8.9%) had levels between 0.06 and 0.149/~g m l - 1. Levels />0.15/~g nil- 1 were present in 132 subjects (73.3%). For the group whose pre- and postimmunization levels can be compared, G M T s increased from 0.41 to 38.1/~g ml-1, a rise of over 90-fold. Comparable levels were seen following immunization in both groups. All children in both groups had postimmunization anti-PRP levels 1>0.15/~g m l - 1 and over 98% had levels > 1.00/~g m1-1 In the group tested before and after booster immunization, those who had less than 0.15/~g ml-1 of anti-PRP prior to immunization responded less well than those who had greater levels, with postbooster G M T s of 13.4 and 55.2 #g ml-1, respectively (p < 0.0001). Nevertheless, all children with low preimmunization levels had postimmunization anti-PRP levels ~> 1.00/~g m l - 1 and 66% had levels ~>10.0/~g ml- 1. Responses to other vaccine antigens are summarized
Serological test
Combined injection group n (%)
Separate injections group n (%)
Diphtheria antitoxin (IU ml-1) a Prebooster (n) />0.01 />0.10 GMT (CI95) Postbooster (n) />0.10 /> 1.00 GMT (CI95)
180 (100) 176 (97.8) 79 (43.9) 0.10 (0.08-0.13) 181 (100) 181 (100) 180 (99.4) 10.8 (9.3-12.5)
Not done
Tetanus antitoxin (IU ml-1) a Prebooster (n) ~>0.01 />0.10 GMT (CI95) Postbooster (n) >~0.10 >~1.00 GMT (CI9~)
179 (100) 170 (95.0) 98 (54.7) 0.10 (0.08-0.12) 181 (100) 181 (100) 153 (84.5) 2.2 (1.9-2.4)
Pertussis agglutinins a Prebooster (n) GMT (CI95) Postbooster (n) GMT (CI95)
180 (100) 33.1 (27.4-40.1) 181 (100) 544.4 (467.6-633.7)
aSee text for assay method used
182 (100) 181 (99.5) 179 (98.4) 9.3 (7.8-11.0)
Not done
181 (100) 180 (99.5) 160 (88.4) 2.3 (2.1-2.7)
Not done 183 (100) 554.4 (470.9-652.7)
Hib booster vaccination: D.W. Scheifele et al.
in Table3. Pre-immunization diphtheria and tetanus antitoxin levels were above the minimum protective levels (>10.01 IUm1-1) in over 95% of children tested. After the booster dose, antitoxin levels were substantially higher: no difference in GMTs was noted between recipients of separate or combined vaccines. Postimmunization GMTs of pertussis agglutinins were virtually identical in the two groups. Following the booster dose, no intergroup differences reaching statistical significance (p<0.05) were observed in concentrations of antibodies to subcellular pertussis antigens including filamentous haemagglutinins (GMTs for combined and separate groups were 43.8 and 47.2 enzyme units (EU)m1-1, respectively), 69kDa protein (22.5 and 21.9 EU ml- 1), fimbrial agglutinogens (710.1 and 787.1 EUm1-1) or pertussis toxin (54.8 and 56.4 EU ml- 1). DISCUSSION This study demonstrates that DPT and PRP-T vaccines can be combined in one syringe for convenient administration without evidence of interference with the immune response to individual antigens. With 367 participants, the study had about 90% power to detect differences between groups amounting to 15% or more in selected postimmunization antibody concentrations (~ = 0.05, two-sided): no differences were detcted for any of the antigens tested, including PRP, tetanus and diphtheria antitoxins, pertussis agglutinins and 4 subcellular pertussis antigens. In both study groups, high levels of anti-PRP antibody were present in sera obtained 4 weeks after immunization. A 90-fold increase was evident in those tested serially. The geometric mean anti-PRP levels were 33.9 and 38.1/~g ml-1 in the two groups following immunization (difference not significant), much higher than observed at completion of the primary series a when the GMT was 4.24 ~tg ml- 1. The sequential data indicate a true booster effect. In contrast, children first vaccinated with PRP-T at 18 months of age reportedly develop GMTs in the range of 3.6-6.4/~g ml- 1 (Ref. 7). Given separately, PRP-T booster doses caused only occasional, minor, local adverse effects. No increase in the frequency of local reactions was evident when compared with reactions observed after the primary series of doses 2'8. As expected 9'1°, local reactions to DTP vaccine were encountered more frequently after booster doses than after primary doses 3. The addition of PRP-T to DPT was associated with a greater rate of redness ~>25 mm in diameter 24 h after immunization (25.1% compared with 14.1% after DPT alone, p--0.008). This was not associated with an increased rate of local tenderness so the phenomenon may be of no practical importance to vaccinees. The basis for increased local erythema after the combined vaccines is uncertain but it might result from the additional quantity of tetanus protein administered. Systemic adverse effects were mild and infrequent in both groups, although our observations could have been influenced by near-universal use of acetaminophen prophylaxis in our subjects. A booster dose is recommended for all of the Hib conjugate vaccines given to infants because anti-PRP levels decline with time following the primary series s'l 1,12. The recommended time of its administration varies
among products and with national immunization schedules. In Canada, PRP-OMP (PedvaxHIB ®) is recommended for use at 12 months of age whereas HbOC (HibTITER ®) and PRP-T are recommended at 18 months. The recommendation for PRP-OMP is based on the pattern of declining anti-PRP levels 13 and possible reduction in protective efficacy 14. The recommendations for HbOC and PRP-T reflect the desire to conveniently link their administration to DPT vaccination at 18 months of age and are consistent with observed rates of anti-PRP decay. A recent prospective study 12 in the USA showed that after a primary series with HbOC or PRP-T vaccines, anti-PRP levels declined from 7 to 15 months of age by 75-81%. Another study4 that utilized an early pre-production lot of PRP-T measured a decline in anti-PRP levels between 8 and 18 months of age amounting to 85.4%, to a GMT of 1.6 pg ml-1. Levels had fallen below 0.15 ~tg ml- 1 in 99% of subjects. In our 18-month-old subjects, anti-PRP levels had declined by 90% from values observed at 7 months 3 and in 27% they had fallen below 0.15/~g m1-1. The explanation for the lower trough levels in our subjects is most probably the additional 3-month interval beyond 15 months. Subjects tested represented a balanced sampling of vaccine lots and regimens (mixed versus separate vaccinations) used for primary doses. No prebooster vaccination failures have been reported to the manufacturer in Canada at the present time. Anti-PRP levels ~>0.15/~gm1-1 were previously demonstrated 15 to correlate with short-term protection against Hib diseases following use of a vaccine that did not induce good immunological memory. Similar observations were made in agammaglobulinaemic subjects passively protected using immune serum globulin. A more dynamic situation exists following immunization with Hib conjugate vaccines which typically induce immunological memory. In our study, children with low preimmunization anti-PRP levels ( < 0.15/tg ml- 1) responded less vigorously to the booster dose of PRP-T than did children with higher preimmunization levels (13.4 versus 55.2/.tg ml- 1, p < 0.0001). However, their responses were greater than those seen in children first immunized with PRP-T at 18 months of age 7, whose geometric mean responses in recent US studies ranged from 3.6 to 6.4 ~tg ml-1, suggesting that the responses were of the anamnestic type. All had produced antibody after the primary immunization series. The anti-PRP levels achieved in the lower responder group were consistent with long-term protection. We conclude that booster doses of PRP-T are well tolerated and highly immunogenic, even when mixed with DPT vaccine for single injection convenience. Our observations refer to a specific vaccination schedule and to a DPT product made in Canada. This product results in lower anti-pertussis toxin antibody levels but higher anti-fimbriae and anti-69kDa protein antibody levels than widely used American vaccines 16. Other DPT preparations demonstrate greater 17,18 or lesser 19 degrees of interference with responses to constituents of the DPT/PRP-T mixture upon completion of the primary series. Although these effects are not sufficient to impair short-term protection, the immunobiology of booster doses may vary in detail when other schedules or DPT products are used.
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Hib b o o s t e r vaccination: D.W. Scheifele et al.
ACKNOWLEDGEMENTS This study was funded by Connaught Laboratories Ltd. The investigators are grateful to members of the three field teams (led by T. Arcand, K. Jacobsen and E. Grapentin) and the data management team (led by G. Bjornson) for their excellent support and performance. Participants from Connaught included J. Bevilacqua and L. Young of the serology department. Dr Scheifele is a recipient of an Investigatorship Award from BC's Children's Hospital Foundation.
9 10 11
12
13
REFERENCES 1 Scheifele, D., Gold, R., Law, B., D~ry, P., Halperin, S., Marchessault, V. and Duclos, P. Decline in Haemophilus influenzae type b invasive infections at five Canadian pediatric centres. Can. Commun. Dis. Rep. 1983, 19, 88-91 2 National Advisory Committee on Immunization. Statement on Haemophilus influenzae type b conjugate vaccines for use in infants and children. Can. Med. Assoc. J. 1993, 148, 199-204 3 Scheifele, D., Barreto, L., Meekison, W., Guasparini, R. and Friesen, B. Can Haemophilus influenzae type b-tetanus toxoid conjugate vaccine be combined with diphtheria toxoid-pertussis vaccine-tetanus toxoid? Can. Med. Assoc. J. 1993, 149, 1105-1112 4 Parke, J., Schneerson, R., Reimer, C., Black, C., Welfare, S., Bryla, D. et al. Clinical and immunologic responses to Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in infants injected at 3, 5, 7 and 18 months of age. J. Pediatr. 1991,118,184-190 5 Claesson, B., Schneerson, R., Lagergard, T., Trollfors, B., Taranger, J., Johansson, J. et al. Persistence of serum antibodies elicited by Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in infants vaccinated at 3, 5 and 12 months of age. Pediatr. Infect. Dis. J. 1991, 10, 560-564 6 National Advisory Committee on Immunization. Canadian Immunization Guide, 3rd edn, Health and Welfare Canada, Ottawa, 1989 7 Data on file with Connaught Laboratories Inc, Swiftwater, Pennsylvania, USA, from multicentre US trials involving children who were 17-24 months of age when given PRP-T vaccine 8 Fritzell, B. and Plotkin, S. Efficacy and safety of a Haemophilus influenzae type b capsular polysaccharide-tetanus protein conjugate vaccine. J. Pediatr. 1992, 121,355-362
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14
15 16
17
18
19
Cody, C., Baraff, L., Cherry, J., Marcy, S. and Manclark, C. Nature and rates of adverse reactions associated with DPT and DT immunizations in infants and children. Pediatrics 1981,98, 6,50-.660 Meekison, W., Arcand, T., Scheifele, D. and Humphreys, G. Local adverse reactions to adsorbed DPT vaccine - Surrey, British Columbia. Can. Dis. Wkly Rep. 1989, 15, 73-74 Bulkow, L., Wainwright, R., Letson, G., Chang, S. and Ward, J. Comparative immunogenicity of four Haemophilus influenzae type b conjugate vaccines in Alaska Native infants. Pediatr. Infect. Dis. J. 1993, 12, 484-492 Decker, M., Edwards, K., Bradley, R. and Palmer, P. Responses of children to booster immunization with their primary conjugate Haemophilus influenzae type b vaccine or with polyribosylribitol phosphate conjugated with diphtheria toxoid. J. Pediatr. 1993, l~'J, 410-413 Santosham, M., Hill, J., Wolff, M., Reid, R., Lukaos, L. and Ahonkhai, V. Safety and immungenicity of a Haemophilus influenzae type b conjugate vaccine in a high risk American Indian population. Pediatr. Infect. Dis. J. 1991, 10, 113-117 Santosham, M., Wolff, M., Reid, R., Hoehnboken, M., Bateman, M., Goepp, J. et al. The efficacy in Navajo infants of a conjugate vaccine consisting of Haemophilus influenzae type b polysaccharide and Neisseria meningitidis outer membrane protein complex. N. Engl. J. Med. 1991, 324, 1762-1767 Kayhty, H., Peltola, H., Karanko, V. and Makela, P. The protective level of serum antibodies to the capsular polysaccharide of Haemophilus influenzae type b. J. Infect. Dis. 1983, 147, 1100 Baker, J.D., Halperin, S.A., Edwards, K., Miller, B., Decker, M. and Stephens, D. Antibody response to Bordetella pertussis antigens after immunization with American and Canadian whole-cell vaccines. J. Pediatr. 1991, 121,523-527 Clemens, J.D., Ferreccio, C., Levine, M.M., Horwitz, I., Rao, M.R., Edwards, K.M. and Fritzell, B. Impact of Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine on responses to concurrently administered diphtheria-tetanuspertussis vaccine. JAMA 1992, 267, 673-678 Ferreccio, C., Clemens, J., Avendano, A., Horwitz, I., Flores, C., Avila, L e t al. The clinical and immunological responses of Chilean infants to Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine co-administered in the same syringe with diphtheria-tetanus toxoids-pertussis vaccine at two, four and six months of age. Pediatr. Infect. Dis. J. 1991, 10, 764-771 Avendano, A., Ferreccio, C., Lagos, R., Horwitz, I., Cayazzo, M., Fritzell, B. et al. Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine does not depress serologic responses to diphtheria, tetanus or pertussis antigens when co-administered in the same syringe with diphtheria-tetanuspertussis vaccine at two, four and six months of age. Pediatr. Infect. Dis. J. 1993, 12, 638-643
Vaccine, Vol. 13, No. 1, pp. 104-108, 1995 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0264.-410X/95 $10.00+ 0.00
Evaluation of booster doses of Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in 18-month-old children David W. Scheifele *~, William Meekison*, Roland Guasparini*, Ann Roberts t, Luis Barreto $, John Thipphawong J; and Sharon Wiltsey $ A booster dose o f H a e m ophi l us influenzae type b conjugate vaccine in the second year of life is the final step in the recommended series of doses to protect infants from invasive infection. This study assessed the safety and immunogenicity o f P R P - T conjugate vaccine booster doses (Act-HIB ®, Connaught Laboratories Ltd). The participants were 367 healthy children who had taken part in a study of primary immunization with PRP-T. At 18-19 months old, subjects were randomly assigned to receive diphtheria-pertussis-tetanus (DPT) and PRP- T vaccines either mixed in one syringe (n = 183) or separately in opposite limbs (n = 184). Adverse events were monitored for 48 h after immunization. Blood was obtained prior to vaccination in half of the subjects (combined injections group) and following vaccination in all subjects to test for antibodies to each o f the antigens administered. Local adverse reactions were infrequent with P R P - T alone and equally frequent at sites of D P T or D P T / P R P - T injection, except for redness >>,25 mm in diameter which was more.frequent after the combined vaccines (25.1 versus 14.1%, p
During 1992, all provinces in Canada implemented programmes for vaccination of young infants against Haemophilus influenzae type b (Hib) invasive infections. These programmes have been remarkably effective in reducing the frequency of Hib disease 1. The vaccines selected by the provinces are HibTITER ~m (HbOC, Lederle Cyanamid) or Act-HIB ® (PRP-T, Connaught Laboratories Ltd). The recommended series z for each product consists of three primary doses given at 2, 4 and *Vaccine Evaluation Center, BC's Children's Hospital, Vancouver, Canada. tCalgary Health Services, Calgary, Alberta, Canada and tConnaught Laboratories, Willowdale, Ontario, Canada. ~To whom correspondence should be addressed at Vaccine Evaluation Center, 950 West 28th Avenue (Trailer), Vancouver, British Columbia V5Z 4H4, Canada. (Received 30 March 1994; revised 24 May 1994; accepted 24 May 1994)
104 Vaccine 1995 Volume 13 Number 1
6 months of age, with a booster dose at 18 months. Both products are intended to be given simultaneously with diphtheria-pertussis-tetanus (DPT) vaccine: Act-HIB ® (PRP-T) can be mixed with DPT vaccine produced by the same manufacturer and given as a single injection. Act-HIB ® was evaluated in Canadian infants aged 2-6 months 3 prior to licensure, but attributes of the booster dose were inferred from studies done elsewhere4'5. We report here one of the first studies to assess the performance of the booster dose of PRP-T vaccine given mixed in one syringe with DPT, in comparison with simultaneous separate injections of these vaccines.
METHODS Children who had participated in a study of primary immunization with PRP-T 3 were invited to participate
Hib booster vaccination: D.W. Scheifele et al.
in this randomized controlled trial of booster doses. All children had received PRP-T at 2, 4 and 6 months of age. All had developed anti-PRP (polyribosylribitol phosphate) levels t>0.15 #gml-1 by 7 months of age, at which time no difference in levels was evident between those who received PRP-T mixed in one syringe with DPT vaccine and those who received separate concurrent injections. Children were eligible for enrolment in t h e current study if they were healthy, had completed the three-dose primary series and had no contraindications to receiving additional doses of DPT or PRP-T vaccine, according to published criteria 6. Written informed consent was obtained from their parents prior to enrolment. The study was approved by the ethics committees of the University of British Columbia and the Calgary Board of Health. Participants were vaccinated at 18-19 months of age with DPT, PRP-T and OPV vaccines. They were randomly (1:1) assigned to receive either (i) a combined injection of DPT and PRP-T mixed in one syringe or (ii) separate injections of these products in opposite limbs. The new randomization was stratified to produce two equivalent groups balanced in terms of previous interventions (mixed or separate modes of vaccination, PRP-T lot given). In the combined vaccinations group, liquid DPT vaccine was added to lyophilized PRP-T vaccine to create the mixture prior to use. In the separate vaccinations group, a saline diluent supplied by the manufacturer was used to rehydrate PRP-T vaccine. One lot of PRP-T vaccine (S-2440) was used, along with one lot of DPT vaccine (3951-11). OPV lot derivation was not controlled. The DPT vaccine was prepared by Connaught Laboratories Limited (Willowdale, Ontario), as was the OPV vaccine. DPT contained 5 Lf tetanus toxoid, 25 Lf diphtheria toxoid, 4-12 protective units of pertussis vaccine and 1.5 mg aluminium phosphate per 0.5 ml dose. The PRP-T vaccine was prepared by Pasteur Merieux Srrums et Vaccins and contained 10 #g of polyriboseribosylphosphate (PRP) covalently linked to 20#g of tetanus toxoid in each dose. The vaccination technique was standardized between the three field teams that conducted this study. PRP-T and DPT vaccines were administered intramuscularly into the deltoid area in all but six children, in whom the thigh was preferred. Parents were asked to observe and record any changes in their child's health during the 48 h after vaccination. They were given celluloid templates to measure areas of redness or swelling at injection sites. Fever prophylaxis with acetaminophen was advocated routinely. Temperatures were to be taken at intervals for 48 h. Parents' observations were systematically reviewed during telephone interviews with study staff 24 and 48 h after immunization, as previously described 3. Compliance with the protocol was monitored at intervals by the sponsor. Preimmunization antibody trough levels were measured in a representative sample of subjects only because of the large number available. Either of the newly randomized groups could have been selected as they were carefully balanced: we chose to bleed those assigned to receive combined vaccines, thereby minimizing painful procedures. Blood was obtained from all participants 1 month after immunization. Sera were tested for (i) diphtheria antitoxin by the microneutralization test; (ii)
pertussis agglutinins by the microagglutination method; (iii) tetanus antitoxin by enzyme-linked immunoassay (ELISA); (iv) PRP antibody by radioimmunossay; and (v) subcellular pertussis antigens, including pertussis toxin, filamentous haemagglutinin, 69 kDa protein and fimbrial agglutinogens, by ELISA. The methods used have been described previously3. The tests were performed at Connaught Laboratories Ltd on codelabelled samples. Data were assembled and analysed at the Vaccine Evaluation Center in Vancouver. Case report forms were checked for accuracy and completeness upon receipt and corrections effected if necessary. Accuracy of data entry was verified through programming checks and manual verification of key data for all files. The incidence rate of injection site redness was designated as a primary outcome measure because it is most likely to be ascertained reliably by parents. Analyses of intergroup difference in antibody responses were based on absolute titres achieved as well as the proportions exceeding predetermined levels of particular significance. Geometric mean titres (GMTs) were calculated from log-transformed values, negative values being assigned an arbitrary value just below the lower detection limit. Means of normally distributed antibody data were compared using Student's t test. The •2 test with continuity correction was preferred for comparison of proportional data; Fisher's exact test was also used. Calculations were performed using SAS/STAT software (SAS Institute, Cary, NC, USA). RESULTS Of the 423 children who completed primary immunization with PRP-T vaccine and were eligible to receive booster doses, 367 (86.8%) participated in this study. The combined injection group contained 183 subjects, the separate injection group contained 184. A total of 180 infants were enrolled at Vancouver site A, 77 at Vancouver site B and 110 in Calgary. Most infants (90.7%) were 18 months old at the time of the booster dose, the remainder being 19 months old. Fifty-one per cent were female. A telephone follow-up was obtained for all but one child at 24 h and for all but two at 48 h following immunization. Groups were closely matched in terms of age at immunization and sex distribution both by centre and by treatment assignment. No immediate adverse reactions were encountered. Local adverse reactions to immunization are summarized in Table 1. Morbidity was infrequently observed after PRP-T vaccine. Twenty-four hours following vaccination, children who received combined injections of DPT/PRPT more often had redness 1>25 mm in diameter (25.1%) than did those who received DPT separately (14.1%, p = 0.008, Fisher's exact test). Apart from this, other local changes occurred at a similar frequency between DPT and DPT/PRP-T sites. Acetaminophen prophylaxis utilization rates exceeded 95% in both groups. Additional treatment doses were given to 54% and 61% of subjects (difference not significant). Antipyretic use could have influenced assessment of adverse reactions such as fever. Moreover, intercurrent illnesses (principally cough and cold syndromes) were reported within 48 h after 36 immunizations (9.8%), with similar distribution between groups. Affected infants were not removed from the analysis.
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Table 1
Local adverse reactions to the booster immunizations
Observation
Combined injections of DPT/PRP-T (%)
Separate injection of DPT (%)
Separate injection of PRP-T (%)
Redness at 24 h Any />25 mm
80 (43.7) 46 (25.1)*
83 (45.1) 26 (14.1)*
12 (6.5) 3 (1.6)
Redness at 48 h Any >/25 mm
57 (31.1) 39 (21.3)
60 (32.6) 31 (16.8)
4 (2.2) 3 (1.6)
Swelling at 24 h Any ~>25 mm
43 (23.5) 27 (14.8)
49 (26.6) 23 (12.5)
8 (4.3) 1 (0.5)
Swelling at 48 h Any
25 (13.7)
29 (15.8)
1 (0.5)
Tenderness at 24 h Mild Moderate Severe
64 (35.0) 40 (21.9) 17 (9.3)
60 (32.6) 37 (20.1) 18 (9.8)
37 (20.1) 15 (8.2) 5 (2.7)
183 (100)
184 (100)
Total number
184 (100)
*p=0.008, Fisher's exact test
Table 2
Anti-PRP levels in study participants
Anti-PRP levels a (/~g m1-1)
Combined injection group n (%)
Separate injections group n (%)
Pre-immunization Total assayed <0.06 0.06-0.149 0.15--0.999 >~1.00 > 10.00 Geometric mean titre t~ 95% confidence interval
180 (100) 32 (17.8) 16 (8.9) 80 (44.4) 52 (28.9) 3 (1.7) 0.41 0.33-0.51
Not performed
Post-immunization Total assayed 179 (100) <0.149 0 0.15-0.99 0 7>1.00 179 (100) > 10.00 157 (87.7) Geometric mean titre b 38.1 95% confidence interval 32.3-44.9
Table 3 Responses to diphtheria, tetanus and pertussis antigens in study participants
179 (100) 0 3 (1.7) 176 (98.3) 154 (86.0) 33.9 28.5-40.2
aMeasured by radioimmunoassay t)For anti-PRP levels <0.06 iLg ml 1, the value was assumed to be 0.05 #g ml 1
Parents complied well with temperature measurements, obtained in 88.8% of infants. Most (87%) preferred axillary measurements. During the first 24h after immunization, a temperature/> 38.0°C occurred in tested children (n=326) at a frequency of 30.6%, with 7.6% exceeding 39.0°C. Fever was detected between 24 and 48 h in 6.8% of children tested (n=252). No significant difference in fever patterns existed between groups. Similarly, no intergroup differences occurred in rates of irritability (mean 54.8 %), crying more than usual (40.9 %), lethargy (16.4%) or vomiting (5.4%) during the first 24 h after immunization. One child developed seizures after separately administered vaccines. This child had several brief convulsive episodes 2 h after immunization, in
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association with fever of 40°C. The event was attributed to vaccination. No hypotonic-hyporesponsive episodes were noted. Eight children were seen by physicians for adverse events within 48 h of vaccination, including some with intercurrent illnesses. Serum samples were obtained as intended prior to immunization from 180 of 183 children in the combined injections group. Serum samples were obtained from both groups 4 weeks following immunization: sufficient serum for anti-PRP tests was obtained from 358 children (97.5%). Prior to booster immunization, the geometric mean anti-PRP level in those tested (Table 2) was 0.41 #g m l - 1 (95% confidence interval (CI95): 0.33-0.51). No measurable anti-PRP (<0.06/~gm1-1) was detected in 32 samples (17.8%) while an additional 16 (8.9%) had levels between 0.06 and 0.149/~g m l - 1. Levels />0.15/~g nil- 1 were present in 132 subjects (73.3%). For the group whose pre- and postimmunization levels can be compared, G M T s increased from 0.41 to 38.1/~g ml-1, a rise of over 90-fold. Comparable levels were seen following immunization in both groups. All children in both groups had postimmunization anti-PRP levels 1>0.15/~g m l - 1 and over 98% had levels > 1.00/~g m1-1 In the group tested before and after booster immunization, those who had less than 0.15/~g ml-1 of anti-PRP prior to immunization responded less well than those who had greater levels, with postbooster G M T s of 13.4 and 55.2 #g ml-1, respectively (p < 0.0001). Nevertheless, all children with low preimmunization levels had postimmunization anti-PRP levels ~> 1.00/~g m l - 1 and 66% had levels ~>10.0/~g ml- 1. Responses to other vaccine antigens are summarized
Serological test
Combined injection group n (%)
Separate injections group n (%)
Diphtheria antitoxin (IU ml-1) a Prebooster (n) />0.01 />0.10 GMT (CI95) Postbooster (n) />0.10 /> 1.00 GMT (CI95)
180 (100) 176 (97.8) 79 (43.9) 0.10 (0.08-0.13) 181 (100) 181 (100) 180 (99.4) 10.8 (9.3-12.5)
Not done
Tetanus antitoxin (IU ml-1) a Prebooster (n) ~>0.01 />0.10 GMT (CI95) Postbooster (n) >~0.10 >~1.00 GMT (CI9~)
179 (100) 170 (95.0) 98 (54.7) 0.10 (0.08-0.12) 181 (100) 181 (100) 153 (84.5) 2.2 (1.9-2.4)
Pertussis agglutinins a Prebooster (n) GMT (CI95) Postbooster (n) GMT (CI95)
180 (100) 33.1 (27.4-40.1) 181 (100) 544.4 (467.6-633.7)
aSee text for assay method used
182 (100) 181 (99.5) 179 (98.4) 9.3 (7.8-11.0)
Not done
181 (100) 180 (99.5) 160 (88.4) 2.3 (2.1-2.7)
Not done 183 (100) 554.4 (470.9-652.7)
Hib booster vaccination: D.W. Scheifele et al.
in Table3. Pre-immunization diphtheria and tetanus antitoxin levels were above the minimum protective levels (>10.01 IUm1-1) in over 95% of children tested. After the booster dose, antitoxin levels were substantially higher: no difference in GMTs was noted between recipients of separate or combined vaccines. Postimmunization GMTs of pertussis agglutinins were virtually identical in the two groups. Following the booster dose, no intergroup differences reaching statistical significance (p<0.05) were observed in concentrations of antibodies to subcellular pertussis antigens including filamentous haemagglutinins (GMTs for combined and separate groups were 43.8 and 47.2 enzyme units (EU)m1-1, respectively), 69kDa protein (22.5 and 21.9 EU ml- 1), fimbrial agglutinogens (710.1 and 787.1 EUm1-1) or pertussis toxin (54.8 and 56.4 EU ml- 1). DISCUSSION This study demonstrates that DPT and PRP-T vaccines can be combined in one syringe for convenient administration without evidence of interference with the immune response to individual antigens. With 367 participants, the study had about 90% power to detect differences between groups amounting to 15% or more in selected postimmunization antibody concentrations (~ = 0.05, two-sided): no differences were detcted for any of the antigens tested, including PRP, tetanus and diphtheria antitoxins, pertussis agglutinins and 4 subcellular pertussis antigens. In both study groups, high levels of anti-PRP antibody were present in sera obtained 4 weeks after immunization. A 90-fold increase was evident in those tested serially. The geometric mean anti-PRP levels were 33.9 and 38.1/~g ml-1 in the two groups following immunization (difference not significant), much higher than observed at completion of the primary series a when the GMT was 4.24 ~tg ml- 1. The sequential data indicate a true booster effect. In contrast, children first vaccinated with PRP-T at 18 months of age reportedly develop GMTs in the range of 3.6-6.4/~g ml- 1 (Ref. 7). Given separately, PRP-T booster doses caused only occasional, minor, local adverse effects. No increase in the frequency of local reactions was evident when compared with reactions observed after the primary series of doses 2'8. As expected 9'1°, local reactions to DTP vaccine were encountered more frequently after booster doses than after primary doses 3. The addition of PRP-T to DPT was associated with a greater rate of redness ~>25 mm in diameter 24 h after immunization (25.1% compared with 14.1% after DPT alone, p--0.008). This was not associated with an increased rate of local tenderness so the phenomenon may be of no practical importance to vaccinees. The basis for increased local erythema after the combined vaccines is uncertain but it might result from the additional quantity of tetanus protein administered. Systemic adverse effects were mild and infrequent in both groups, although our observations could have been influenced by near-universal use of acetaminophen prophylaxis in our subjects. A booster dose is recommended for all of the Hib conjugate vaccines given to infants because anti-PRP levels decline with time following the primary series s'l 1,12. The recommended time of its administration varies
among products and with national immunization schedules. In Canada, PRP-OMP (PedvaxHIB ®) is recommended for use at 12 months of age whereas HbOC (HibTITER ®) and PRP-T are recommended at 18 months. The recommendation for PRP-OMP is based on the pattern of declining anti-PRP levels 13 and possible reduction in protective efficacy 14. The recommendations for HbOC and PRP-T reflect the desire to conveniently link their administration to DPT vaccination at 18 months of age and are consistent with observed rates of anti-PRP decay. A recent prospective study 12 in the USA showed that after a primary series with HbOC or PRP-T vaccines, anti-PRP levels declined from 7 to 15 months of age by 75-81%. Another study4 that utilized an early pre-production lot of PRP-T measured a decline in anti-PRP levels between 8 and 18 months of age amounting to 85.4%, to a GMT of 1.6 pg ml-1. Levels had fallen below 0.15 ~tg ml- 1 in 99% of subjects. In our 18-month-old subjects, anti-PRP levels had declined by 90% from values observed at 7 months 3 and in 27% they had fallen below 0.15/~g m1-1. The explanation for the lower trough levels in our subjects is most probably the additional 3-month interval beyond 15 months. Subjects tested represented a balanced sampling of vaccine lots and regimens (mixed versus separate vaccinations) used for primary doses. No prebooster vaccination failures have been reported to the manufacturer in Canada at the present time. Anti-PRP levels ~>0.15/~gm1-1 were previously demonstrated 15 to correlate with short-term protection against Hib diseases following use of a vaccine that did not induce good immunological memory. Similar observations were made in agammaglobulinaemic subjects passively protected using immune serum globulin. A more dynamic situation exists following immunization with Hib conjugate vaccines which typically induce immunological memory. In our study, children with low preimmunization anti-PRP levels ( < 0.15/tg ml- 1) responded less vigorously to the booster dose of PRP-T than did children with higher preimmunization levels (13.4 versus 55.2/.tg ml- 1, p < 0.0001). However, their responses were greater than those seen in children first immunized with PRP-T at 18 months of age 7, whose geometric mean responses in recent US studies ranged from 3.6 to 6.4 ~tg ml-1, suggesting that the responses were of the anamnestic type. All had produced antibody after the primary immunization series. The anti-PRP levels achieved in the lower responder group were consistent with long-term protection. We conclude that booster doses of PRP-T are well tolerated and highly immunogenic, even when mixed with DPT vaccine for single injection convenience. Our observations refer to a specific vaccination schedule and to a DPT product made in Canada. This product results in lower anti-pertussis toxin antibody levels but higher anti-fimbriae and anti-69kDa protein antibody levels than widely used American vaccines 16. Other DPT preparations demonstrate greater 17,18 or lesser 19 degrees of interference with responses to constituents of the DPT/PRP-T mixture upon completion of the primary series. Although these effects are not sufficient to impair short-term protection, the immunobiology of booster doses may vary in detail when other schedules or DPT products are used.
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Hib b o o s t e r vaccination: D.W. Scheifele et al.
ACKNOWLEDGEMENTS This study was funded by Connaught Laboratories Ltd. The investigators are grateful to members of the three field teams (led by T. Arcand, K. Jacobsen and E. Grapentin) and the data management team (led by G. Bjornson) for their excellent support and performance. Participants from Connaught included J. Bevilacqua and L. Young of the serology department. Dr Scheifele is a recipient of an Investigatorship Award from BC's Children's Hospital Foundation.
9 10 11
12
13
REFERENCES 1 Scheifele, D., Gold, R., Law, B., D~ry, P., Halperin, S., Marchessault, V. and Duclos, P. Decline in Haemophilus influenzae type b invasive infections at five Canadian pediatric centres. Can. Commun. Dis. Rep. 1983, 19, 88-91 2 National Advisory Committee on Immunization. Statement on Haemophilus influenzae type b conjugate vaccines for use in infants and children. Can. Med. Assoc. J. 1993, 148, 199-204 3 Scheifele, D., Barreto, L., Meekison, W., Guasparini, R. and Friesen, B. Can Haemophilus influenzae type b-tetanus toxoid conjugate vaccine be combined with diphtheria toxoid-pertussis vaccine-tetanus toxoid? Can. Med. Assoc. J. 1993, 149, 1105-1112 4 Parke, J., Schneerson, R., Reimer, C., Black, C., Welfare, S., Bryla, D. et al. Clinical and immunologic responses to Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in infants injected at 3, 5, 7 and 18 months of age. J. Pediatr. 1991,118,184-190 5 Claesson, B., Schneerson, R., Lagergard, T., Trollfors, B., Taranger, J., Johansson, J. et al. Persistence of serum antibodies elicited by Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in infants vaccinated at 3, 5 and 12 months of age. Pediatr. Infect. Dis. J. 1991, 10, 560-564 6 National Advisory Committee on Immunization. Canadian Immunization Guide, 3rd edn, Health and Welfare Canada, Ottawa, 1989 7 Data on file with Connaught Laboratories Inc, Swiftwater, Pennsylvania, USA, from multicentre US trials involving children who were 17-24 months of age when given PRP-T vaccine 8 Fritzell, B. and Plotkin, S. Efficacy and safety of a Haemophilus influenzae type b capsular polysaccharide-tetanus protein conjugate vaccine. J. Pediatr. 1992, 121,355-362
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14
15 16
17
18
19
Cody, C., Baraff, L., Cherry, J., Marcy, S. and Manclark, C. Nature and rates of adverse reactions associated with DPT and DT immunizations in infants and children. Pediatrics 1981,98, 6,50-.660 Meekison, W., Arcand, T., Scheifele, D. and Humphreys, G. Local adverse reactions to adsorbed DPT vaccine - Surrey, British Columbia. Can. Dis. Wkly Rep. 1989, 15, 73-74 Bulkow, L., Wainwright, R., Letson, G., Chang, S. and Ward, J. Comparative immunogenicity of four Haemophilus influenzae type b conjugate vaccines in Alaska Native infants. Pediatr. Infect. Dis. J. 1993, 12, 484-492 Decker, M., Edwards, K., Bradley, R. and Palmer, P. Responses of children to booster immunization with their primary conjugate Haemophilus influenzae type b vaccine or with polyribosylribitol phosphate conjugated with diphtheria toxoid. J. Pediatr. 1993, l~'J, 410-413 Santosham, M., Hill, J., Wolff, M., Reid, R., Lukaos, L. and Ahonkhai, V. Safety and immungenicity of a Haemophilus influenzae type b conjugate vaccine in a high risk American Indian population. Pediatr. Infect. Dis. J. 1991, 10, 113-117 Santosham, M., Wolff, M., Reid, R., Hoehnboken, M., Bateman, M., Goepp, J. et al. The efficacy in Navajo infants of a conjugate vaccine consisting of Haemophilus influenzae type b polysaccharide and Neisseria meningitidis outer membrane protein complex. N. Engl. J. Med. 1991, 324, 1762-1767 Kayhty, H., Peltola, H., Karanko, V. and Makela, P. The protective level of serum antibodies to the capsular polysaccharide of Haemophilus influenzae type b. J. Infect. Dis. 1983, 147, 1100 Baker, J.D., Halperin, S.A., Edwards, K., Miller, B., Decker, M. and Stephens, D. Antibody response to Bordetella pertussis antigens after immunization with American and Canadian whole-cell vaccines. J. Pediatr. 1991, 121,523-527 Clemens, J.D., Ferreccio, C., Levine, M.M., Horwitz, I., Rao, M.R., Edwards, K.M. and Fritzell, B. Impact of Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine on responses to concurrently administered diphtheria-tetanuspertussis vaccine. JAMA 1992, 267, 673-678 Ferreccio, C., Clemens, J., Avendano, A., Horwitz, I., Flores, C., Avila, L e t al. The clinical and immunological responses of Chilean infants to Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine co-administered in the same syringe with diphtheria-tetanus toxoids-pertussis vaccine at two, four and six months of age. Pediatr. Infect. Dis. J. 1991, 10, 764-771 Avendano, A., Ferreccio, C., Lagos, R., Horwitz, I., Cayazzo, M., Fritzell, B. et al. Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine does not depress serologic responses to diphtheria, tetanus or pertussis antigens when co-administered in the same syringe with diphtheria-tetanuspertussis vaccine at two, four and six months of age. Pediatr. Infect. Dis. J. 1993, 12, 638-643