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Controlled Study of aNew Five-Component Acellular Pertussis Vaccine in Adults and Young Children J. A. Englund, W. P. Glezen, and L. Barreto
Departments ofMicrobiology and Immunology and ofPediatrics. Baylor College ofMedicine. Houston. Texas; Connaught Laboratories. Ltd.. Wi//owdale, Canada
A new five-component acellular pertussis (AP) vaccine containing 10 jLg of pertussis toxoid,S jLg of filamentous hemagglutinin,S jLg of combined agglutinogens 2 and 3, and 3 jLg of pertactin was evaluated in adults and young children. AP vaccine was compared with saline placebo in 31 adults, and AP vaccine combined with diphtheria and tetanus toxoids (ADTP) was compared with whole cell DTP in 41 children, ages 16-20 months, who had received whole cell DTP during infancy. AP was mildly to moderately reactogenic in adults, with pain noted within 72 hand 5-8 days after immunization. ADTP was less reactogenic than DTP in children, with significantly decreased pain, redness, irritability, and fever and less use of acetaminophen reported. No late reactions were observed in any child. The multicomponent ADTP was immunogenic, with fourfold or greater antibody rises to at least four pertussis antibody assays in allIS immunized adults. Pertussis-specific antibody responses in children who received ADTP and DTP were similar. The multicomponent ADTP vaccine is currently being studied in a National Institute of Allergy and Infectious Diseases-sponsored efficacy study in Sweden. Advances in the characterization of the antigenic and biologically active factors of Bordetella pertussis have resulted in the production of new acellular pertussis vaccines prepared from more purified pertussis antigens. Early acellular pertussis vaccines have included varying concentrations of purified pertussis toxoid (PT, also called lymphocytosis promoting factor [LPFD with or without purified filamentous hemagglutinin (FHA). PT has been shown to play an important role in the development of disease and immunity to pertussis [I]; FHA is known as the dominant adhesin for adherence of B, pertussis to ciliated respiratory cells [2], Antigens other than PT and FHA may also induce protective immunity against B. pertussis and are therefore candidates for inclusion in acellular pertussis vaccines. Agglutinogens (Aggs) 1,2, and 3 are surface antigens associated with the bacterial fimbriae and are promising candidates for acellular vaccines, since agglutinating antibody has long been correlated with immunity to pertussis, Studies in mice have demonstrated protection after immunization with purified Aggs [3]. It has been postulated that the low protective effi-
Received 23 March 1992; revised 20 July 1992. Presented in part: 31st Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, October 1991. Informed consent was obtained from all patients or from their parents or guardians. Guidelines for human experimentation of the US Department of Health and Human Services and the Institutional Review Board for Human Research, Baylor College of Medicine, were followed. Financial support: Connaught Laboratories, Willowdale, Canada. Reprints or correspondence: Dr. Janet A. Englund. Department ofMicrobiology and Immunology, Baylor College of Medicine, One Baylor Plaza. Houston, TX 77030. The Journal of Infectious Diseases 1992;166:1436-41 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0038$01.00
cacy of whole cell vaccine used in the 1960s in the United Kingdom was due to a lack of Agg 3in the vaccine [4]; an increased incidence of infection with B, pertussis containing that serotype occurred during that period. Currently, the World Health Organization recommends that pertussis vaccines include 'the three major Aggs, although the need for Agg I in vaccines has not yet been demonstrated. Antibody to a 69-kDa adenylate cyclase-associated protein, a protein originally postulated to be important because of the role of adenylate cyclase in disease pathogenesis, has been found in human sera after natural infection with pertussis or immunization with whole cell vaccine [5]. Immunization with this purified protein or passive immunization with specific monoclonal antibodies directed against the protein can protect infant mice from a lethal respiratory challenge with B, pertussis [6]. A combination ofthis protein and FHA is also protective in the mouse intracerebral challenge potency test in the absence of PT [7]. This newly identified protein, now called pertactin (Per) because it is involved in the attachment or invasion of eukaryotic cells, appears to play an important role in protective immunity. We report the first controlled study of a multicomponent vaccine prepared by the independent purification of PT, FHA, Aggs 2 and 3, and Per. Study results enabled the evaluation of this five-component acellular vaccine in a National Institute of Allergy and Infectious Diseases (NIAID) Multicenter Acellular Infant Pertussis Trial [8], and this vaccine is now being studied in the NIAID-sponsored Swedish Efficacy Trial, which began in March 1992.
Methods Vaccines. The five-component acellular pertussis vaccine used in the adult trial contained 10 JLg ofPT, 5 JLg of FHA, 5 JLg
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Concise Communications
of combined Aggs 2 and 3, and 3 ILg of Per, adsorbed with 1.5 mg of aluminum phosphate and 0.6% phenoxyethanol. In the pediatric study, the acellular pertussis vaccine was combined with adsorbed diphtheria toxoid (15 limit of flocculation [Lf) units) and tetanus toxoid (5 Lf). Pertussis antigens were produced from B. pertussis 10536 grown in submerged culture. After completion of growth, the culture broth was separated by centrifugation and PT and FHA were purified by sequential chromatography over perlite and hydroxylapatite. The Agg antigens were prepared from bacteria that had been disrupted with urea, heat-treated, and purified by precipitation and chromatography. Per was isolated from the run-through of the perlite chromatography step by ammonium sulfate precipitation and subsequent purification. A single lot of commercially available whole cell diphtheriatetanus-pertussis (DTP) vaccine (Connaught Laboratories, Swiftwater, PA) containing 6.7 Lfunits of diphtheria toxoid, 5 Lfunits of tetanus toxoid, 0.25 mg of aluminum potassium sulfate, thimerosal (1: 10,000) as preservative, and sufficient pertussis antigens to meet required potency standards (15 opacity units/dose) was used as a centrol vaccine in the pediatric study. The diphtheria toxoid content of the whole cell DTP vaccine (6.7 Lf) was substantially lower than that in the acellular DTP (ADTP) vaccine (15 Lf). Production methods used to prepare tetanus and diphtheria toxoids were similar but not identical. Subjects. Thirty-one healthy adults, ages 21-60 years, were enrolled in May 1990 in the placebo-controlled adult trial. Follow-up clinical and laboratory studies were completed for all subjects. Children eligible for whole cell DTP, based on guidelines of the American Academy of Pediatrics, were recruited for this study from private pediatric offices in Houston. Forty-one healthy children, ages 16-20 months, who had received primary immunizations with whole cell DTP during infancy, were enrolled between June and October 1990. Reaction rates were recorded in all participants and sufficient sera for serologic evaluation were available in 37 children (90%). Study design. Adult subjects were randomized at entry to receive either placebo or acellular pertussis (AP) vaccine and immunized with a 25-gauge, 2.54-cm needle in the deltoid muscle. Subjects were evaluated by blinded study personnel at 6 h and at 1,2, 3 or 4, 7, 10, and 28 days after immunization. A reaction diary was kept by study participants for 14 days to record pain, graded as mild (tenderness on touching), moderate (pain on touching but not increased with motion), and severe (pain with decreased mobility of extremity); redness and swelling (size in centimeters); and systemic symptoms. Oral temperature was recorded daily using a digital thermometer supplied by the investigators. Blood samples were obtained at each clinic visit for determination of glucose level, insulin level, and white blood cell count and differential. Antibody concentrations to vaccine antigens were determined before immunization and 10 and 28 days after immunization. Children were randomized to receive either licensed whole cell DTP or five-component ADTP in a ratio of 1:2 after a preimmunization blood draw. Vaccine was administered intramuscularly in the thigh with a 25-gauge, 2.54-cm needle and blood was collected 4 weeks after immunization. Parents were interviewed by telephone by a blinded investigator on days 1, 3, and 10 after immunization. Parents were requested to fill out a
1437
reaction diary daily for 14 days after immunization and to record the rectal temperature, obtained with a digital thermometer supplied by the investigators. Specific parameters queried included redness and swelling (area measured by a study gauge) and pain as assessed by the caretaker: mild, pain only when injection site was touched; moderate, withdrawal from touch but good mobility ofextremity; and severe, pain at rest or mobility decreased. Other systemic symptoms were also recorded by parents and investigators (decreased appetite, vomiting, drowsiness, use ofacetaminophen, fussiness, crying, and upper respiratory symptoms). Laboratory methods. Laboratory studies in adults (hemoglobin level, white blood cell count and differential, platelet count, and serum glucose level) were done at a reference laboratory on days 0, 1, 2, 3 or 4, 7, and 28. Insulin levels were obtained by standardized RIA. All sera were stored at -20°C until shipped on dry ice to Connaught Laboratories (Willowdale ) for antibody determination. Tetanus toxoid and diphtheria toxoid antibody were determined by an ELISA based on the method of Voller et al. [9]. Antibody assays to pertussis antigens were done on blinded sera based on the ELISA of Manclark et al. [10]. ELiSAs to the pertussis antigens included in the vaccine (PT, FHA, Aggs2 and 3, and Per) were done as was the CHO cell neutralization assay. In the ELISA, the antigens used for coating were prepared as described for the component pertussis vaccine. Peroxidase-conjugated affinity-purified heavy chain goat anti-human IgG, IgM, and IgA (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for determination of immunoglobulin subclass. US reference pertussis human antiserum (lot 3; Center for Drugs and Biologics, Food and Drug Administration, Bethesda, MD) was used as a control, with the following reference values: IgG anti-FHA, 200 units/ml.; IgG anti-PT, 200 units/ml.; IgM antiFHA, 15 units/rnl.; IgM anti-PT, 15 units/ml.; IgA anti-FHA, 100 units/ml.; IgA anti-PI', 15 units/ml.; and CHO cell neutralization assay demonstrating complete neutralization of clustering induced by 0.5 ng/mL LPI; at a dilution of 1:640. Functional neutralizing antibody to PT or CHO cell antibody was determined in a microplate tissue culture assay of CHO cells [11]. The titers were expressed as the reciprocal of the highest dilution giving 80% neutralization of the clustering effect of PT. Seropositivity was defined as a titer of ;;.4 and a significant change as fourfold or greater. Statistics. Reaction rates of patients were compared using the two-tailed Fisher's exact test. Serologic calculations were done on logarithmically transformed data, reported as the antilogarithm. The mean geometric titers of each group were calculated from the mean ofthe log-transformed data. Between-group comparisons were made using the t test for independent samples and within group comparisons were made using either the t test or paired t test.
Results Reactions. Of the 31 adults in the study, 16 received placebo (mean age, 29.7 years) and 15 received AP vaccine (mean age, 31.1 years). The AP vaccine was generally well tolerated by study participants with no serious systemic reactions (e.g., fever, muscle aches, or malaise) observed in any
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subject (table 1). By contrast, local reactions consisting of pain and swelling in the injection area unaccompanied by fever were noted in 11 of 15 AP recipients compared with 2 of 16 placebo recipients (P < .005). Local reactions in AP recipients occurred at two distinct time points: early, days 0-4 (10/15 recipients), and late, days 5-8 (6/15 recipients). The majority of reactions consisted of minimal pain (11/15), although 5 recipients reported moderate pain, such as pain associated with movement. The late reaction was also char-
Table 1. Adverse reactions in adults and children (16-20 months old) after placebo, acellular pertussis (AP), whole cell diphtheriatetanus-pertussis (DTP), or acellular DTP (ADTP) vaccine during l4-day follow-up after vaccination. Adults Placebo Subjects completing study Fever ;;.38.6°C 1st72 h After72 h Local redness, 0.25-2.0 em' >2 em' Local swelling ,,;2 crrr' in first 72 h >2 em' after 72 h Painduring first 72 h Anylocalized Mild (hurt when touched) Moderate (withdrawal with touch) Severe, decreased mobility Limp or refusal to walk Duration >48 h Painafter 72 h Any localized Mild (hurt when touched) Moderate (withdrawal with touch) Severe, decreased mobility Axillary swelling/tenderness, first 72 h After72 h Drowsiness, first 72 h After72 h Vomiting, first 72 h After 72 h Decreased appetite, first 72 h After72 h Increased irritability, first 72 h After72 h Increased crying, first 72 h After72 h Acetaminophen use, first 72 h After 72 h
Children AP
DTP
ADTP
16(100) 15(100) 13(100) 28 (100) 0 0 0 I (6)
0 0 2 (13) 0
4 (31) 5 (38) 6 (40) 0
2 (7) 4 (14) 3 (11)* 2 (7)
0 0
0 4 (27)
0 0
0 0
O'
2 (12)*
II (73) 8 (5)
12(92) 4 (31)
4 (14)' 3 (II)
0
2 (13)
2 (15)
I (4)
0 0 0
0 0 0
6 (46) 3 (23) 8 (62)
0*
0' 0
6 (40) 3 (20)
4 (31) 3 (23)
I (4) 0
0
3 (20)
I (7)
I (4)
0
0
0
0
0 0 2 (12) 0 0 0 0 0 0 0
I 3 (20) I (7) 0 0 0 0 0 0 0
2 (12) 5 (31)
2 (13) 3 (20)
7 (54) 7 (54) 0 I (8) 4 (31) I (8) 10(77) II (85) 5 (38) 0 7 (54) I (8)
O'
I (4)*
5 (18) 7 (25) 0 4 (14) 9 (32) 5 (18) 7 (25)' 13(46) 0* I (4) 4 (14)* 5 (18)
NOTE. Dataare no. (%). , Significantly decreased reaction compared with othergroup, P < .05.
nn 1992; 166 (December)
acterized by axillary tenderness in 4 of 6 participants and by palpable axillary lymphadenopathy in I. Five of the 6 AP recipients who developed late local reactions also reported early mild (n = 4) or moderately painful (n = 1) reactions. Forty-one children who had previously received three doses of whole cell DTP vaccine were randomized to receive either ADTP (n = 28) or whole cell DTP (n = 13). No significant adverse reactions, such as prolonged or high fever, highpitched crying, or seizures, were noted in either vaccine group. Reactions associated with the receipt of vaccine included fever, pain, limping, redness, swelling, irritability, prolonged crying, and decreased appetite (table 1). No significant difference was seen in the number of children with a fever ;;.38.6°C. Severe pain, characterized by decreased mobility or limping, was more common in DTP recipients (6/13 vs. 0/28, P < .001). Duration of pain in the DTP group ranged from 48 h in 3 children; all pain resolved within 48 h in ADTP recipients. Other reactions significantly more frequent in the DTP group included localized redness, irritability, drowsiness within the first 72 h, acetaminophen use in the first 72 h, and prolonged crying (not high pitched) during the first 72 h. Laboratory studies. Among adult subjects, laboratory studies revealed no significant differences in fasting serum glucose levels, insulin levels, lymphocyte counts, and platelet counts before and 6 h through 7 days after receipt of vaccine or placebo. A significantly higher total white blood cell count was noted in vaccine than in placebo recipients before and during the first 2 study days, but there was no difference in pre- versus postimmunization white blood cell counts within the placebo or AP group and no difference in total lymphocyte counts at any time between the two groups. Serology. All 15 adult AP recipients had a fourfold or greater increase in IgG antibody to at least four of the five laboratory assays for pertussis antibody; no controls seroconverted to any antigen. Two adults (l each) did not show a fourfold increase in FHA or PT antibodies; these 2 subjects had high antibody levels to these antigens before immunization and a subsequent antibody increase that was slightly less than a fourfold rise. Thirteen of 15 subjects had a significant antibody rise to pertussis antigens by day 10, indicating a brisk antibody response. Geometric mean IgG titers of antibody to PT, FHA, Agg, and Per and results of the CHO cell neutralization assay were not different in the placebo and AP groups before immunization but were significantly higher in the AP group at both days 10 and 28 (table 2). No differences in geometric mean titer or frequency of fourfold antibody rise was noted in IgM anti-P'T or IgM anti-FHA, but significant increases in IgA antibody directed against PT, FHA, Agg, and Per were seen at both days 10 and 28 in AP recipients (data not shown). No change in antibody to tetanus or diphtheria toxoids was detected in either group. No significant differences were noted in preimmunization concentrations ofIgG or IgA antibody to PT, FHA, Agg, or Per
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lID 1992;166 (December)
Table 2. IgG antibody responses to pertussis antigens and diphtheria and tetanus toxoids in adults and young children after inoculation with placebo or acellular pertussis (AP), diphtheria-tetanus-pertussis (DTP), or multicomponent acellular DTP (ADTP) toxoids. Adults Beforeimmunization
Pertussis toxoid
Filamentous hemagglutinin Agglutinogens Pertactin
Children
Postimmunization day 28
Before immunization
After immunization
Placebo
AP
Placebo
Ap*
DTP
ADTP
DTP*
ADTP*
16.45 (9.46-28.62)
22.78 (12.11-42.86)
16.56 (9.08-30.22)
415.87 (243.91-709.09)
43.71 ( 14.29-133.88)
15.45 (8.50-28.10)
221.32 (99.83-490.67)
306.55 ( 155.84-603.03)
15.24 (10.28-22.60) 21.26 ( 12.14-37.23) 7.89 (4.00-15.56)
23.59 ( 15.59-35.69) 28.64 (12.20-67.21) 11.47 (6.41-20.55)
13.36 (7.71-2316) 27.0 (15.37-47.78) 7.46 (3.51-15.87)
317.37 (243.05-414.41) 2048.00 ( 1025.62-4089.55) 855.13 (396.41-1844.67)
2.93 (1.81-4.73) 26.72 (16.94-42.15) 6.54 (2.79-15.33)
3.86 (3.03-4.93) 29.24 (13.63-62.75) 9.45 (5.50-16.23)
30.06 (11.82-76.46) 315.2 (127.4-779.9) 60.13 (24.59-147.04)
29.86 (16.51-53.99) 1243.3 (594.8-2603.5) 116.16 (57.87-233.19)
12.30 (6.97-21.68)
Controlled Study of aNew Five-Component Acellular Pertussis Vaccine in Adults and Young Children J. A. Englund, W. P. Glezen, and L. Barreto
Departments ofMicrobiology and Immunology and ofPediatrics. Baylor College ofMedicine. Houston. Texas; Connaught Laboratories. Ltd.. Wi//owdale, Canada
A new five-component acellular pertussis (AP) vaccine containing 10 jLg of pertussis toxoid,S jLg of filamentous hemagglutinin,S jLg of combined agglutinogens 2 and 3, and 3 jLg of pertactin was evaluated in adults and young children. AP vaccine was compared with saline placebo in 31 adults, and AP vaccine combined with diphtheria and tetanus toxoids (ADTP) was compared with whole cell DTP in 41 children, ages 16-20 months, who had received whole cell DTP during infancy. AP was mildly to moderately reactogenic in adults, with pain noted within 72 hand 5-8 days after immunization. ADTP was less reactogenic than DTP in children, with significantly decreased pain, redness, irritability, and fever and less use of acetaminophen reported. No late reactions were observed in any child. The multicomponent ADTP was immunogenic, with fourfold or greater antibody rises to at least four pertussis antibody assays in allIS immunized adults. Pertussis-specific antibody responses in children who received ADTP and DTP were similar. The multicomponent ADTP vaccine is currently being studied in a National Institute of Allergy and Infectious Diseases-sponsored efficacy study in Sweden. Advances in the characterization of the antigenic and biologically active factors of Bordetella pertussis have resulted in the production of new acellular pertussis vaccines prepared from more purified pertussis antigens. Early acellular pertussis vaccines have included varying concentrations of purified pertussis toxoid (PT, also called lymphocytosis promoting factor [LPFD with or without purified filamentous hemagglutinin (FHA). PT has been shown to play an important role in the development of disease and immunity to pertussis [I]; FHA is known as the dominant adhesin for adherence of B, pertussis to ciliated respiratory cells [2], Antigens other than PT and FHA may also induce protective immunity against B. pertussis and are therefore candidates for inclusion in acellular pertussis vaccines. Agglutinogens (Aggs) 1,2, and 3 are surface antigens associated with the bacterial fimbriae and are promising candidates for acellular vaccines, since agglutinating antibody has long been correlated with immunity to pertussis, Studies in mice have demonstrated protection after immunization with purified Aggs [3]. It has been postulated that the low protective effi-
Received 23 March 1992; revised 20 July 1992. Presented in part: 31st Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, October 1991. Informed consent was obtained from all patients or from their parents or guardians. Guidelines for human experimentation of the US Department of Health and Human Services and the Institutional Review Board for Human Research, Baylor College of Medicine, were followed. Financial support: Connaught Laboratories, Willowdale, Canada. Reprints or correspondence: Dr. Janet A. Englund. Department ofMicrobiology and Immunology, Baylor College of Medicine, One Baylor Plaza. Houston, TX 77030. The Journal of Infectious Diseases 1992;166:1436-41 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0038$01.00
cacy of whole cell vaccine used in the 1960s in the United Kingdom was due to a lack of Agg 3in the vaccine [4]; an increased incidence of infection with B, pertussis containing that serotype occurred during that period. Currently, the World Health Organization recommends that pertussis vaccines include 'the three major Aggs, although the need for Agg I in vaccines has not yet been demonstrated. Antibody to a 69-kDa adenylate cyclase-associated protein, a protein originally postulated to be important because of the role of adenylate cyclase in disease pathogenesis, has been found in human sera after natural infection with pertussis or immunization with whole cell vaccine [5]. Immunization with this purified protein or passive immunization with specific monoclonal antibodies directed against the protein can protect infant mice from a lethal respiratory challenge with B, pertussis [6]. A combination ofthis protein and FHA is also protective in the mouse intracerebral challenge potency test in the absence of PT [7]. This newly identified protein, now called pertactin (Per) because it is involved in the attachment or invasion of eukaryotic cells, appears to play an important role in protective immunity. We report the first controlled study of a multicomponent vaccine prepared by the independent purification of PT, FHA, Aggs 2 and 3, and Per. Study results enabled the evaluation of this five-component acellular vaccine in a National Institute of Allergy and Infectious Diseases (NIAID) Multicenter Acellular Infant Pertussis Trial [8], and this vaccine is now being studied in the NIAID-sponsored Swedish Efficacy Trial, which began in March 1992.
Methods Vaccines. The five-component acellular pertussis vaccine used in the adult trial contained 10 JLg ofPT, 5 JLg of FHA, 5 JLg
JID 1992;166 (December)
Concise Communications
of combined Aggs 2 and 3, and 3 ILg of Per, adsorbed with 1.5 mg of aluminum phosphate and 0.6% phenoxyethanol. In the pediatric study, the acellular pertussis vaccine was combined with adsorbed diphtheria toxoid (15 limit of flocculation [Lf) units) and tetanus toxoid (5 Lf). Pertussis antigens were produced from B. pertussis 10536 grown in submerged culture. After completion of growth, the culture broth was separated by centrifugation and PT and FHA were purified by sequential chromatography over perlite and hydroxylapatite. The Agg antigens were prepared from bacteria that had been disrupted with urea, heat-treated, and purified by precipitation and chromatography. Per was isolated from the run-through of the perlite chromatography step by ammonium sulfate precipitation and subsequent purification. A single lot of commercially available whole cell diphtheriatetanus-pertussis (DTP) vaccine (Connaught Laboratories, Swiftwater, PA) containing 6.7 Lfunits of diphtheria toxoid, 5 Lfunits of tetanus toxoid, 0.25 mg of aluminum potassium sulfate, thimerosal (1: 10,000) as preservative, and sufficient pertussis antigens to meet required potency standards (15 opacity units/dose) was used as a centrol vaccine in the pediatric study. The diphtheria toxoid content of the whole cell DTP vaccine (6.7 Lf) was substantially lower than that in the acellular DTP (ADTP) vaccine (15 Lf). Production methods used to prepare tetanus and diphtheria toxoids were similar but not identical. Subjects. Thirty-one healthy adults, ages 21-60 years, were enrolled in May 1990 in the placebo-controlled adult trial. Follow-up clinical and laboratory studies were completed for all subjects. Children eligible for whole cell DTP, based on guidelines of the American Academy of Pediatrics, were recruited for this study from private pediatric offices in Houston. Forty-one healthy children, ages 16-20 months, who had received primary immunizations with whole cell DTP during infancy, were enrolled between June and October 1990. Reaction rates were recorded in all participants and sufficient sera for serologic evaluation were available in 37 children (90%). Study design. Adult subjects were randomized at entry to receive either placebo or acellular pertussis (AP) vaccine and immunized with a 25-gauge, 2.54-cm needle in the deltoid muscle. Subjects were evaluated by blinded study personnel at 6 h and at 1,2, 3 or 4, 7, 10, and 28 days after immunization. A reaction diary was kept by study participants for 14 days to record pain, graded as mild (tenderness on touching), moderate (pain on touching but not increased with motion), and severe (pain with decreased mobility of extremity); redness and swelling (size in centimeters); and systemic symptoms. Oral temperature was recorded daily using a digital thermometer supplied by the investigators. Blood samples were obtained at each clinic visit for determination of glucose level, insulin level, and white blood cell count and differential. Antibody concentrations to vaccine antigens were determined before immunization and 10 and 28 days after immunization. Children were randomized to receive either licensed whole cell DTP or five-component ADTP in a ratio of 1:2 after a preimmunization blood draw. Vaccine was administered intramuscularly in the thigh with a 25-gauge, 2.54-cm needle and blood was collected 4 weeks after immunization. Parents were interviewed by telephone by a blinded investigator on days 1, 3, and 10 after immunization. Parents were requested to fill out a
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reaction diary daily for 14 days after immunization and to record the rectal temperature, obtained with a digital thermometer supplied by the investigators. Specific parameters queried included redness and swelling (area measured by a study gauge) and pain as assessed by the caretaker: mild, pain only when injection site was touched; moderate, withdrawal from touch but good mobility ofextremity; and severe, pain at rest or mobility decreased. Other systemic symptoms were also recorded by parents and investigators (decreased appetite, vomiting, drowsiness, use ofacetaminophen, fussiness, crying, and upper respiratory symptoms). Laboratory methods. Laboratory studies in adults (hemoglobin level, white blood cell count and differential, platelet count, and serum glucose level) were done at a reference laboratory on days 0, 1, 2, 3 or 4, 7, and 28. Insulin levels were obtained by standardized RIA. All sera were stored at -20°C until shipped on dry ice to Connaught Laboratories (Willowdale ) for antibody determination. Tetanus toxoid and diphtheria toxoid antibody were determined by an ELISA based on the method of Voller et al. [9]. Antibody assays to pertussis antigens were done on blinded sera based on the ELISA of Manclark et al. [10]. ELiSAs to the pertussis antigens included in the vaccine (PT, FHA, Aggs2 and 3, and Per) were done as was the CHO cell neutralization assay. In the ELISA, the antigens used for coating were prepared as described for the component pertussis vaccine. Peroxidase-conjugated affinity-purified heavy chain goat anti-human IgG, IgM, and IgA (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for determination of immunoglobulin subclass. US reference pertussis human antiserum (lot 3; Center for Drugs and Biologics, Food and Drug Administration, Bethesda, MD) was used as a control, with the following reference values: IgG anti-FHA, 200 units/ml.; IgG anti-PT, 200 units/ml.; IgM antiFHA, 15 units/rnl.; IgM anti-PT, 15 units/ml.; IgA anti-FHA, 100 units/ml.; IgA anti-PI', 15 units/ml.; and CHO cell neutralization assay demonstrating complete neutralization of clustering induced by 0.5 ng/mL LPI; at a dilution of 1:640. Functional neutralizing antibody to PT or CHO cell antibody was determined in a microplate tissue culture assay of CHO cells [11]. The titers were expressed as the reciprocal of the highest dilution giving 80% neutralization of the clustering effect of PT. Seropositivity was defined as a titer of ;;.4 and a significant change as fourfold or greater. Statistics. Reaction rates of patients were compared using the two-tailed Fisher's exact test. Serologic calculations were done on logarithmically transformed data, reported as the antilogarithm. The mean geometric titers of each group were calculated from the mean ofthe log-transformed data. Between-group comparisons were made using the t test for independent samples and within group comparisons were made using either the t test or paired t test.
Results Reactions. Of the 31 adults in the study, 16 received placebo (mean age, 29.7 years) and 15 received AP vaccine (mean age, 31.1 years). The AP vaccine was generally well tolerated by study participants with no serious systemic reactions (e.g., fever, muscle aches, or malaise) observed in any
ConciseCommunications
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subject (table 1). By contrast, local reactions consisting of pain and swelling in the injection area unaccompanied by fever were noted in 11 of 15 AP recipients compared with 2 of 16 placebo recipients (P < .005). Local reactions in AP recipients occurred at two distinct time points: early, days 0-4 (10/15 recipients), and late, days 5-8 (6/15 recipients). The majority of reactions consisted of minimal pain (11/15), although 5 recipients reported moderate pain, such as pain associated with movement. The late reaction was also char-
Table 1. Adverse reactions in adults and children (16-20 months old) after placebo, acellular pertussis (AP), whole cell diphtheriatetanus-pertussis (DTP), or acellular DTP (ADTP) vaccine during l4-day follow-up after vaccination. Adults Placebo Subjects completing study Fever ;;.38.6°C 1st72 h After72 h Local redness, 0.25-2.0 em' >2 em' Local swelling ,,;2 crrr' in first 72 h >2 em' after 72 h Painduring first 72 h Anylocalized Mild (hurt when touched) Moderate (withdrawal with touch) Severe, decreased mobility Limp or refusal to walk Duration >48 h Painafter 72 h Any localized Mild (hurt when touched) Moderate (withdrawal with touch) Severe, decreased mobility Axillary swelling/tenderness, first 72 h After72 h Drowsiness, first 72 h After72 h Vomiting, first 72 h After 72 h Decreased appetite, first 72 h After72 h Increased irritability, first 72 h After72 h Increased crying, first 72 h After72 h Acetaminophen use, first 72 h After 72 h
Children AP
DTP
ADTP
16(100) 15(100) 13(100) 28 (100) 0 0 0 I (6)
0 0 2 (13) 0
4 (31) 5 (38) 6 (40) 0
2 (7) 4 (14) 3 (11)* 2 (7)
0 0
0 4 (27)
0 0
0 0
O'
2 (12)*
II (73) 8 (5)
12(92) 4 (31)
4 (14)' 3 (II)
0
2 (13)
2 (15)
I (4)
0 0 0
0 0 0
6 (46) 3 (23) 8 (62)
0*
0' 0
6 (40) 3 (20)
4 (31) 3 (23)
I (4) 0
0
3 (20)
I (7)
I (4)
0
0
0
0
0 0 2 (12) 0 0 0 0 0 0 0
I 3 (20) I (7) 0 0 0 0 0 0 0
2 (12) 5 (31)
2 (13) 3 (20)
7 (54) 7 (54) 0 I (8) 4 (31) I (8) 10(77) II (85) 5 (38) 0 7 (54) I (8)
O'
I (4)*
5 (18) 7 (25) 0 4 (14) 9 (32) 5 (18) 7 (25)' 13(46) 0* I (4) 4 (14)* 5 (18)
NOTE. Dataare no. (%). , Significantly decreased reaction compared with othergroup, P < .05.
nn 1992; 166 (December)
acterized by axillary tenderness in 4 of 6 participants and by palpable axillary lymphadenopathy in I. Five of the 6 AP recipients who developed late local reactions also reported early mild (n = 4) or moderately painful (n = 1) reactions. Forty-one children who had previously received three doses of whole cell DTP vaccine were randomized to receive either ADTP (n = 28) or whole cell DTP (n = 13). No significant adverse reactions, such as prolonged or high fever, highpitched crying, or seizures, were noted in either vaccine group. Reactions associated with the receipt of vaccine included fever, pain, limping, redness, swelling, irritability, prolonged crying, and decreased appetite (table 1). No significant difference was seen in the number of children with a fever ;;.38.6°C. Severe pain, characterized by decreased mobility or limping, was more common in DTP recipients (6/13 vs. 0/28, P < .001). Duration of pain in the DTP group ranged from 48 h in 3 children; all pain resolved within 48 h in ADTP recipients. Other reactions significantly more frequent in the DTP group included localized redness, irritability, drowsiness within the first 72 h, acetaminophen use in the first 72 h, and prolonged crying (not high pitched) during the first 72 h. Laboratory studies. Among adult subjects, laboratory studies revealed no significant differences in fasting serum glucose levels, insulin levels, lymphocyte counts, and platelet counts before and 6 h through 7 days after receipt of vaccine or placebo. A significantly higher total white blood cell count was noted in vaccine than in placebo recipients before and during the first 2 study days, but there was no difference in pre- versus postimmunization white blood cell counts within the placebo or AP group and no difference in total lymphocyte counts at any time between the two groups. Serology. All 15 adult AP recipients had a fourfold or greater increase in IgG antibody to at least four of the five laboratory assays for pertussis antibody; no controls seroconverted to any antigen. Two adults (l each) did not show a fourfold increase in FHA or PT antibodies; these 2 subjects had high antibody levels to these antigens before immunization and a subsequent antibody increase that was slightly less than a fourfold rise. Thirteen of 15 subjects had a significant antibody rise to pertussis antigens by day 10, indicating a brisk antibody response. Geometric mean IgG titers of antibody to PT, FHA, Agg, and Per and results of the CHO cell neutralization assay were not different in the placebo and AP groups before immunization but were significantly higher in the AP group at both days 10 and 28 (table 2). No differences in geometric mean titer or frequency of fourfold antibody rise was noted in IgM anti-P'T or IgM anti-FHA, but significant increases in IgA antibody directed against PT, FHA, Agg, and Per were seen at both days 10 and 28 in AP recipients (data not shown). No change in antibody to tetanus or diphtheria toxoids was detected in either group. No significant differences were noted in preimmunization concentrations ofIgG or IgA antibody to PT, FHA, Agg, or Per
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Concise Communications
lID 1992;166 (December)
Table 2. IgG antibody responses to pertussis antigens and diphtheria and tetanus toxoids in adults and young children after inoculation with placebo or acellular pertussis (AP), diphtheria-tetanus-pertussis (DTP), or multicomponent acellular DTP (ADTP) toxoids. Adults Beforeimmunization
Pertussis toxoid
Filamentous hemagglutinin Agglutinogens Pertactin
Children
Postimmunization day 28
Before immunization
After immunization
Placebo
AP
Placebo
Ap*
DTP
ADTP
DTP*
ADTP*
16.45 (9.46-28.62)
22.78 (12.11-42.86)
16.56 (9.08-30.22)
415.87 (243.91-709.09)
43.71 ( 14.29-133.88)
15.45 (8.50-28.10)
221.32 (99.83-490.67)
306.55 ( 155.84-603.03)
15.24 (10.28-22.60) 21.26 ( 12.14-37.23) 7.89 (4.00-15.56)
23.59 ( 15.59-35.69) 28.64 (12.20-67.21) 11.47 (6.41-20.55)
13.36 (7.71-2316) 27.0 (15.37-47.78) 7.46 (3.51-15.87)
317.37 (243.05-414.41) 2048.00 ( 1025.62-4089.55) 855.13 (396.41-1844.67)
2.93 (1.81-4.73) 26.72 (16.94-42.15) 6.54 (2.79-15.33)
3.86 (3.03-4.93) 29.24 (13.63-62.75) 9.45 (5.50-16.23)
30.06 (11.82-76.46) 315.2 (127.4-779.9) 60.13 (24.59-147.04)
29.86 (16.51-53.99) 1243.3 (594.8-2603.5) 116.16 (57.87-233.19)
12.30 (6.97-21.68)
