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Immunogenicity of an acellular pertussis vaccine composed of genetically inactivated pertussis toxin combined with filamentous hemagglutinin and pertactin in infants and children
CLINICAL AND LABORATORY OBSERVATIONS
Immunogenicity of an acellular pertussis vaccine composed of genetically inactivated pertussis toxin combined with filamentous hemagglutinin and pertactin in infants and children A u d i n o P o d d a , MD, Erminia C a r a p e l l a De Luca, MD, Lucina Titone, MD, A n n a Maria C a s a d e i , MD, A n t o n i o C a s c i o , MD, M a r c e l l a Bartalini, G i a n f r a n c o Volpini, S a m u e l e P e p p o l o n i , PhD, Ilio Marsili, PhD, L u c i a n o Nencioni, PhD, a n d Rino Rappuoli, PhD From Biocine-Sclavo R&D Vaccines, Siena, Italy, the Istituto di Puericultura, UniversitO "La Sapienza," Rome, Italy, and the Clinica di Malattie Infettive, Universita di Palermo, Palermo, Italy We studied the immunogenicity of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin, and a 69-kilodalton protein, pertactin, in 30 children a g e d 12 to 24 months and in 80 infants a g e d 2 to 4 months. A significant increase of the neutralizing titer and of the titers against pertussis toxin, filamentous hemagglutinin, and pertactin, as determined by enzyme-linked immunosorbent assay, was achieved after three doses of vaccine in all the children; a significant increase of these antibody titers was obtained in 100%, 96.1%, 93.5%, and 98.7% of the infants, respectively. (J PEDIATR1993;123:81-4) We recently reported the safety and immunogenicity, in infants and children, of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin. 1 This and other studies have shown that PT-9K/129G toxin is safe and more immunogenic than chemically detoxified PT molecules. 1-6 Following the suggestion that acellular vaccines should induce immunity against PT and against one or more of the molecules involved in bacterial adhesion, such as filamentous hemagglutinin, pertactin (a 69-kilodalton protein), and the agglutinogens, we constructed a vaccine containing PT-9K/129G toxin, FHA, and the 69 kd protein] We report here the immunogenicityof this vaccine in infants and children.
PT-9K/129G toxin, 10 #g FHA, 10 #g pertactin, 0.05 mg thimerosal, and 0.5 mg aluminum hydroxide. Study design. The vaccine was tested in 30 children aged 12 to 24 months (group 1) and 80 infants aged 2 to 4 months (group 2). The children, enrolled at the infectious disease department of the University of Palermo, Palermo, Italy, had no history of pertussis or pertussis immunization and received intramuscularly two doses of vaccine 8 weeks apart; 8 of the 30 children received a third dose 8 weeks afELISA FHA kd PT
Enzyme linked immunosorbent assay Filamentous hemagglutinin Kilodalton Pertussistoxin
METHODS Vaccine. The acellular pertussis vaccine used in this study, Acelluvax, was prepared at Biocine Sclavo Laboratories, Siena, Italy. Each dose (0.5 ml) contained 7.5 ~zg Submitted for publication Dec. 8, 1992; accepted March 4, 1993. Reprint requests: Rino Rappuoli, PhD, Biocine-SclavoR&D Vaccines, Via Fiorentina 1, 53100 Siena, Italy. Copyright 9 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/22/46916
ter the second one. The 80 infants were enrolled both in Palermo and at the child health department of the University "La Sapienza," of Rome, Italy. All infants, born at term after an uneventful pregnancy, received intramuscularly three doses of vaccine 8 weeks apart; eight of the infants enrolled in Rome received a booster dose about 1 year after the third one. Venous blood samples were taken before each dose of vaccine and 1 month after the last dose. Informed consent was obtained from the parents of each child.
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Table I. Geometric mean titers of toxin-neutralizing antibodies, IgG anti-PT, IgG anti-FHA, and IgG anti-pertactin (69 kd protein), before and after each injection of Acelluvax in children of two age groups G e o m e t r i c m e a n titers in serum A g e group (too)
Neutralizing titer (CHO assay)
12-24 2-4
IgG anti-PT (ELISA)
12-24 2-4
IgG anti-FHA (EL1SA)
12-24 2-4
lgG anti-pertactin'~ (ELISA)
12-24 2-4
Before dose
1.1 (0.9-1.4) 1.2 (I.1-1.4) 2.1 (1.4-3.2) 5.2 (4.3-6.3) 7.6 (4.9-11.9) 17.4 (14.1-21.4) 1.8 (1.0-3.4) 3.5 (2.6-4.6)
After dose I
2.6 (1.7-3.8) 4.5 (3.4-6.1) 27.6 (17.2-44.6) 27.5 (21.6-35.2) 82.4 (49.5-137.4) 50.3 (39.1-64.7) 9.6 (6.1-15.1) 11.8 (9.3-14.8)
After dose 2
106.6 (68.1-166.9) 93.4 (76.8-113.6) 133.8 (79.4-225.5) 90.8 (73.6-112.1) 691.3 439.2-1088.0) 258.4 203.9-327.4) 189.4 121.0-296.6) 88.5 (66.6-117.5)
After dose 3
269.1" (150.0-482.9) 240.7 * (209.8-276.1) 106.5 (3%8-300.4) 132.1 (108.9-160.2) 759.7 (285.1-2024.0) 474.3 (387.1-581.1) 276.4 (185.4-412.0) 188.8 (154.2-231.2)
Values in parentheses are the 95% confidence intervals. CHO, Chinese hamster ovary cells. *p <0.05 versus post-second-dose value. "~Pertactin, a 69 kd protein.
Immunogenicity assays. IgG antibodies to PT, FHA, and pertaetin were measured by enzyme-linked immunosorbent assay as previously described. 2, 7.8 Toxin-neutralizingtiters were expressed as the reciprocal of the highest serum dilution causing complete inhibition of the clustering activity induced by the native toxin on Chinese hamster ovary cells. 9 For the neutralization assay an arbitrary value of I was attributed to negative samples to obtain the mean values. The U.S. Reference Human Pertussis Antiserum, kindly provided by the Center for Biologics Evaluation and Research, Bethesda, Md., and containing IgG antibodies to PT, 200 ELISA units/ml, igG antibodies to FHA, 200 ELISA units/mll and 640 toxin-neutralizing units, was used as a standard for ELISA and Chinese hamster ovary cell assays. In the case of the 69 kd protein, an "internal standard" immune serum, which was assigned a value of 200 ELISA units of IgG antibodies to 69 kd per milliliter, was Used. At the end of the study a new lot of standard antiserum, also containing anti-69-kd antibodies, was made available by the U.S. Food and Drug Administration (lot No. 4). One unit of this antiserum contained approximately 2.2 units of the internal standard used in this study. Statistical analysis. Differences in the mean antibody titers were evaluated by analysis of variance, with log-transformed data.
RESULTS All children and infants enrolled completed the trial. No major adverse event was reported during the trial; reactogenicity was substantially similar to that reported in our previous trial, 1 in which a vaccine containing the PT-9K/ 129G toxin alone was used (data not shown). lmmunogenieity. In group 1 we could not obtain blood from two children after the first dose and from one child after the second dose. In group 2 we did not obtain sera from four infants before the first vaccine injection, from six after the first one, from four after the second, from seven after the third, and from one before the booster dose. As reported in Table I, after two doses of vaccine a fourfold increase of the neutralizing antibody titer and of the anti-FHA ELISA titer was achieved by all the children in group 1, whereas a fourfold increase of the anti-PT and anti-69-kd ELISA titers was observed in 97% of them. A third dose in eight children of this group induced a further significant increase of the mean neutralizing titer (p <0.05). Most of the infants in group 2 had an increase of the antibody titers against all the vaccine components after the first dose. The titer further increased after the second and third doses. After the third dose a fourfold increase was observed in 100% of the infants for the neutralizing titer, in 93.5% for the FHA titer, in 96.1% for the PT titer, and in
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T a b l e II. Geometric mean titers of toxin-neutralizing antibodies, IgG anti-PT, IgG anti-FHA, and IgG anti-pertactin (69 kd protein) before and after each injection of Acelluvax in eight infants 2 to 4 months old who received a primary series of three doses of vaccine 8 weeks apart and a booster dose 1 year after the third one G e o m e t r i c m e a n titers in serum Before dose
Neutralizing titer (CHO assay) IgG anti-PT (ELISA) IgG anti-FHA (ELISA) IgG anti-pertactin'~ (ELISA)
2.1 (0.5-8.1) 10.4 (4.3-25.0) 29.0 (11.1-75.7) 6.0 (2.5-14.2)
After dose I
7.9 (3.0-20.6) 40.12 (11.6-138.5) 71.2 (23.1-218.9) 19.50 (7.1-53.5)
After dose 2
After dose 3
Before dose 4
After dose 4
80.0 (46.7-136.8) 150.5 (55.0-411.6) 266.6 (106.2-668.7) 180.9 (83.6-391.1)
269.1 (147.6-490.2) 257.4 (70.3-942.5) 476.1 (232.4-975.1) 280.3 (176.2-445.8)
44.2 (16.1-121.1) 24.6 (15.0-40.0) 27.6 (9.7-78.4) 37.0 (11.7-117.1)
830.0* (488.1-1411) 226.9 (136.6-376.7) 355.l (176.9-712.7) 1104.9" (523.4-2332.3)
Values in parentheses are the 95% confidenceintervals. CHO, Chinese hamster ovary cells. *p <0.0i versuspost-third-dosevalue. ~'Pertactin, a 69 kd protein.
98.7% for 69 kd titers. Eight infants tested 1 year after the third dose of vaccine had a significant decrease of the antibody titers against all vaccine components (Table II). The booster dose induced an anamnestic antibody response: the toxin-neutralizing antibody and the anti-69-kd E L I S A titers were much higher than those observed after the third dose (p <0.001). The levels of the ELISA titers against F H A and PT were comparable to those achieved after the third dose of vaccine. DISCUSSION As already reported in the phase-1 study, we observed that the P T - 9 K / 1 2 9 G toxin can be combined with F H A and pertactin without altering its immunogenicity. We also found that the toxin-neutralizing titer was comparable to that obtained with the acellular pertussis vaccine containing only the P T - 9 K / 1 2 9 G t o x i n ] despite the reduced PT content (7.5 vs 15 ~g) in Acelluvax. Eight infants examined 1 year after the third vaccine injection showed a decreased antibody titer against all vaccine components. A booster dose increased the antibody titers to a level similar to or higher than that obtained after the third dose; the toxin-neutralizing antibody and the anti-69-kd titers reached values significantly higher than those previously observed. After the booster dose, a peculiar behavior of the anti-PT antibodies was observed. The anti-PT ELISA titer was comparable to that induced by the third vaccine dose, whereas the toxin-neutralizing titer was significantly higher. This finding may be due to an affinity maturation of the toxin-neutralizing antibodies or to an increased proportion
of toxin-specific-memory B cells producing neutralizing antibodies. We conclude that a pertussis vaccine containing the genetically detoxified PT, combined with F H A and the 69 kd protein, is safe and immunogenic. We expect that this vaccine, combined with diphtheria and tetanus toxoids, may be licensed for mass vaccination whenever the efficacy studies are completed. Moreover, at present in countries such as Italy, where whole-cell diphtheria-tetanus-pertussis vaccine is poorly accepted because of its reactogenicity, a monovalent acellular pertussis vaccine, similar to that evaluated in this trial, could be used, at the same time or subsequent to diphtheria-tetanus toxoid vaccination, to increase pertussis vaccine coverage. REFERENCES 1. Podda A, Carapella De Luca E, Titone L, et al. Acellular pertussis vaccine composed of genetically inactivated pertussis toxin: safety and immunogenicity in 12- to 24- and 2- to 4-month-old children. J PEDIATR1992;120:680-5. 2. Podda A, Nencioni L, De Magistris MT, et al. Metabolic, humoral and cellular responses in adult volunteers immunized with the genetically inactivated pertussis toxin mutant PT9K/129G. J Exp Med 1990;172:861-8. 3. Krantz I, Sekura R, Trollfors B, et al. Immunogenicity and safety of a pertussis vaccine composed of pertussis toxin inactivated by hydrogen peroxide, in 18- to 23-month-old children. J PEDIATR1990;116:539-43. 4. Nencioni L, Volpini G, Peppoloni S, "De Magistris MT, Marsili I, Rappuoli R. Properties of the pertussis toxin mutant PT9K/129G after formaldehyde treatment. Infect Immun 1991;59:625-30. 5. Englund JA, Edwards KM, Decker M, Anderson E, Pichichero M, Steinhoff M. Multicenter study of acellular (AC) and
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whole cell (WC) pertussis vaccines in young children. Program and Abstracts of the 3 l st Interscience Conference on Antimicrobial Agents and Chemotherapy, McCornick Place Convention Center, Chicago Illinois, 1991. 6. Edwards KM, Meade BD, Decker MD, et al. Comparison of thirteen acellular pertussis vaccines: serologic response. Proceedings of the American Pediatric Society/Society for Pediatric Research Congress, 1992 [Abstract 16687]. 7. Podda A, Nencioni L, Marsili I, et al. Phase I clinical trial of an acellular pertussis vaccine composed of genetically detox-
The Journal of Pediatrics July 1993
ified pertussis toxin combined with FHA and 69 kDa. Vaccine 1991;9:741-5. Manclark CR, Meade BD, Burstyn DG. Serological response to Bordetella pertussis. In: Rose NR, Friedman H, Fahey JL, eds. Manual of clinical laboratory immunology. Washington, D.C.: American Society for Microbiology, 1986:388-94. Hewlett EL, Sauer KT, Myers GA, Cowell JL, Guerrant RL. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect Immun 1983;40:1198203.
Diagnostic use of B-cell alloantigen D8/J17 in rheumatic chorea Brian M. F e l d m a n , MD, John B. Zabriskie, MD, Earl D. S i l v e r m a n , MD, a n d R o n a l d M. Laxer, MD From the Department of Paediatrics, Division of Rheumatology, Hospital for Sick Children, Toronto, Ontario, Canada, and Rockefeller University Hospital, New York, New York
Two unrelated patients with a family history of rheumatic fever had isolated, acquired chorea. Both index cases, as well as affected family members, had increased expression of the rheumatic B-cell alloantigen D8/17. This test may help differentiate Sydenham chorea from lupus chorea. (J PEDIATR1993;123:84-6)
Systemic lupus erythematosus and r h e u m a t i c ( S y d e n h a m ) chorea are the most likely causes of isolated acquired chorea. Differentiation can be difficult, and each diagnosis requires different t r e a t m e n t . A recently identified B-cell alloantigen, D 8 / 1 7 , is expressed more frequently in patients with r h e u m a t i c fever t h a n in control subjects1; its expression in family m e m b e r s is at an i n t e r m e d i a t e level. W e postulated t h a t the presence of this antigen in elevated levels might be used to differentiate patients with r h e u m a t i c chorea from those with SLE. W e describe two patients with isolated chorea in w h o m a clinical diagnosis of r h e u m a t i c
Drs. Laxer and Silverman are associates of the Arthritis Society (Canada), and Dr. Feldman is an Ontario Ministry of Health Research Fellow. Submitted for publication Oct. 6, 1992; accepted Feb. 12, 1993. Reprint requests: Brian M. Feldman, MD, Department of Paediatrics, Division of Rheumatology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Copyright | 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/22/46465
chorea was associated with personal a n d family studies showing an increased expression of D 8 / 1 7 . CASE REPORTS Patient 1. A 10-year-old boy had a 2-week history of chorea manifested by facial jerking, slurred speech, lack of coordination, and a progressive increase in involuntary movements that disappeared during sleep. He had been well except for a mild sore throat about 6 weeks before being seen. He had no history of intoxication, medication use, or travel to an area endemic for Lyme disease. Although there was no family history of chorea, rheumatic fever with carditis had been diagnosed in a maternal uncle during childhood. Physical examination revealed a restless, emotionally labile boy. There was no sign of pharyngitis, and the remainder of the systemic examination findings were normal. Neurologic examination showed diffuse hypotonia, bilateral choreiform movements, and characteristic deep-tendon reflexes. Findings of the remainder of the neurologic examination were normal. The results of the following tests were normal or negative: complete blood cell count, differential cell count, determination of the erythrocyte sedimentation rate, copper studies, complement, liver function tests, immunoglobulins, thyroid function tests, electrocardiography, radiography of the chest, slit-lamp examination for
Immunogenicity of an acellular pertussis vaccine composed of genetically inactivated pertussis toxin combined with filamentous hemagglutinin and pertactin in infants and children A u d i n o P o d d a , MD, Erminia C a r a p e l l a De Luca, MD, Lucina Titone, MD, A n n a Maria C a s a d e i , MD, A n t o n i o C a s c i o , MD, M a r c e l l a Bartalini, G i a n f r a n c o Volpini, S a m u e l e P e p p o l o n i , PhD, Ilio Marsili, PhD, L u c i a n o Nencioni, PhD, a n d Rino Rappuoli, PhD From Biocine-Sclavo R&D Vaccines, Siena, Italy, the Istituto di Puericultura, UniversitO "La Sapienza," Rome, Italy, and the Clinica di Malattie Infettive, Universita di Palermo, Palermo, Italy We studied the immunogenicity of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin, and a 69-kilodalton protein, pertactin, in 30 children a g e d 12 to 24 months and in 80 infants a g e d 2 to 4 months. A significant increase of the neutralizing titer and of the titers against pertussis toxin, filamentous hemagglutinin, and pertactin, as determined by enzyme-linked immunosorbent assay, was achieved after three doses of vaccine in all the children; a significant increase of these antibody titers was obtained in 100%, 96.1%, 93.5%, and 98.7% of the infants, respectively. (J PEDIATR1993;123:81-4) We recently reported the safety and immunogenicity, in infants and children, of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin. 1 This and other studies have shown that PT-9K/129G toxin is safe and more immunogenic than chemically detoxified PT molecules. 1-6 Following the suggestion that acellular vaccines should induce immunity against PT and against one or more of the molecules involved in bacterial adhesion, such as filamentous hemagglutinin, pertactin (a 69-kilodalton protein), and the agglutinogens, we constructed a vaccine containing PT-9K/129G toxin, FHA, and the 69 kd protein] We report here the immunogenicityof this vaccine in infants and children.
PT-9K/129G toxin, 10 #g FHA, 10 #g pertactin, 0.05 mg thimerosal, and 0.5 mg aluminum hydroxide. Study design. The vaccine was tested in 30 children aged 12 to 24 months (group 1) and 80 infants aged 2 to 4 months (group 2). The children, enrolled at the infectious disease department of the University of Palermo, Palermo, Italy, had no history of pertussis or pertussis immunization and received intramuscularly two doses of vaccine 8 weeks apart; 8 of the 30 children received a third dose 8 weeks afELISA FHA kd PT
Enzyme linked immunosorbent assay Filamentous hemagglutinin Kilodalton Pertussistoxin
METHODS Vaccine. The acellular pertussis vaccine used in this study, Acelluvax, was prepared at Biocine Sclavo Laboratories, Siena, Italy. Each dose (0.5 ml) contained 7.5 ~zg Submitted for publication Dec. 8, 1992; accepted March 4, 1993. Reprint requests: Rino Rappuoli, PhD, Biocine-SclavoR&D Vaccines, Via Fiorentina 1, 53100 Siena, Italy. Copyright 9 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/22/46916
ter the second one. The 80 infants were enrolled both in Palermo and at the child health department of the University "La Sapienza," of Rome, Italy. All infants, born at term after an uneventful pregnancy, received intramuscularly three doses of vaccine 8 weeks apart; eight of the infants enrolled in Rome received a booster dose about 1 year after the third one. Venous blood samples were taken before each dose of vaccine and 1 month after the last dose. Informed consent was obtained from the parents of each child.
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Table I. Geometric mean titers of toxin-neutralizing antibodies, IgG anti-PT, IgG anti-FHA, and IgG anti-pertactin (69 kd protein), before and after each injection of Acelluvax in children of two age groups G e o m e t r i c m e a n titers in serum A g e group (too)
Neutralizing titer (CHO assay)
12-24 2-4
IgG anti-PT (ELISA)
12-24 2-4
IgG anti-FHA (EL1SA)
12-24 2-4
lgG anti-pertactin'~ (ELISA)
12-24 2-4
Before dose
1.1 (0.9-1.4) 1.2 (I.1-1.4) 2.1 (1.4-3.2) 5.2 (4.3-6.3) 7.6 (4.9-11.9) 17.4 (14.1-21.4) 1.8 (1.0-3.4) 3.5 (2.6-4.6)
After dose I
2.6 (1.7-3.8) 4.5 (3.4-6.1) 27.6 (17.2-44.6) 27.5 (21.6-35.2) 82.4 (49.5-137.4) 50.3 (39.1-64.7) 9.6 (6.1-15.1) 11.8 (9.3-14.8)
After dose 2
106.6 (68.1-166.9) 93.4 (76.8-113.6) 133.8 (79.4-225.5) 90.8 (73.6-112.1) 691.3 439.2-1088.0) 258.4 203.9-327.4) 189.4 121.0-296.6) 88.5 (66.6-117.5)
After dose 3
269.1" (150.0-482.9) 240.7 * (209.8-276.1) 106.5 (3%8-300.4) 132.1 (108.9-160.2) 759.7 (285.1-2024.0) 474.3 (387.1-581.1) 276.4 (185.4-412.0) 188.8 (154.2-231.2)
Values in parentheses are the 95% confidence intervals. CHO, Chinese hamster ovary cells. *p <0.05 versus post-second-dose value. "~Pertactin, a 69 kd protein.
Immunogenicity assays. IgG antibodies to PT, FHA, and pertaetin were measured by enzyme-linked immunosorbent assay as previously described. 2, 7.8 Toxin-neutralizingtiters were expressed as the reciprocal of the highest serum dilution causing complete inhibition of the clustering activity induced by the native toxin on Chinese hamster ovary cells. 9 For the neutralization assay an arbitrary value of I was attributed to negative samples to obtain the mean values. The U.S. Reference Human Pertussis Antiserum, kindly provided by the Center for Biologics Evaluation and Research, Bethesda, Md., and containing IgG antibodies to PT, 200 ELISA units/ml, igG antibodies to FHA, 200 ELISA units/mll and 640 toxin-neutralizing units, was used as a standard for ELISA and Chinese hamster ovary cell assays. In the case of the 69 kd protein, an "internal standard" immune serum, which was assigned a value of 200 ELISA units of IgG antibodies to 69 kd per milliliter, was Used. At the end of the study a new lot of standard antiserum, also containing anti-69-kd antibodies, was made available by the U.S. Food and Drug Administration (lot No. 4). One unit of this antiserum contained approximately 2.2 units of the internal standard used in this study. Statistical analysis. Differences in the mean antibody titers were evaluated by analysis of variance, with log-transformed data.
RESULTS All children and infants enrolled completed the trial. No major adverse event was reported during the trial; reactogenicity was substantially similar to that reported in our previous trial, 1 in which a vaccine containing the PT-9K/ 129G toxin alone was used (data not shown). lmmunogenieity. In group 1 we could not obtain blood from two children after the first dose and from one child after the second dose. In group 2 we did not obtain sera from four infants before the first vaccine injection, from six after the first one, from four after the second, from seven after the third, and from one before the booster dose. As reported in Table I, after two doses of vaccine a fourfold increase of the neutralizing antibody titer and of the anti-FHA ELISA titer was achieved by all the children in group 1, whereas a fourfold increase of the anti-PT and anti-69-kd ELISA titers was observed in 97% of them. A third dose in eight children of this group induced a further significant increase of the mean neutralizing titer (p <0.05). Most of the infants in group 2 had an increase of the antibody titers against all the vaccine components after the first dose. The titer further increased after the second and third doses. After the third dose a fourfold increase was observed in 100% of the infants for the neutralizing titer, in 93.5% for the FHA titer, in 96.1% for the PT titer, and in
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T a b l e II. Geometric mean titers of toxin-neutralizing antibodies, IgG anti-PT, IgG anti-FHA, and IgG anti-pertactin (69 kd protein) before and after each injection of Acelluvax in eight infants 2 to 4 months old who received a primary series of three doses of vaccine 8 weeks apart and a booster dose 1 year after the third one G e o m e t r i c m e a n titers in serum Before dose
Neutralizing titer (CHO assay) IgG anti-PT (ELISA) IgG anti-FHA (ELISA) IgG anti-pertactin'~ (ELISA)
2.1 (0.5-8.1) 10.4 (4.3-25.0) 29.0 (11.1-75.7) 6.0 (2.5-14.2)
After dose I
7.9 (3.0-20.6) 40.12 (11.6-138.5) 71.2 (23.1-218.9) 19.50 (7.1-53.5)
After dose 2
After dose 3
Before dose 4
After dose 4
80.0 (46.7-136.8) 150.5 (55.0-411.6) 266.6 (106.2-668.7) 180.9 (83.6-391.1)
269.1 (147.6-490.2) 257.4 (70.3-942.5) 476.1 (232.4-975.1) 280.3 (176.2-445.8)
44.2 (16.1-121.1) 24.6 (15.0-40.0) 27.6 (9.7-78.4) 37.0 (11.7-117.1)
830.0* (488.1-1411) 226.9 (136.6-376.7) 355.l (176.9-712.7) 1104.9" (523.4-2332.3)
Values in parentheses are the 95% confidenceintervals. CHO, Chinese hamster ovary cells. *p <0.0i versuspost-third-dosevalue. ~'Pertactin, a 69 kd protein.
98.7% for 69 kd titers. Eight infants tested 1 year after the third dose of vaccine had a significant decrease of the antibody titers against all vaccine components (Table II). The booster dose induced an anamnestic antibody response: the toxin-neutralizing antibody and the anti-69-kd E L I S A titers were much higher than those observed after the third dose (p <0.001). The levels of the ELISA titers against F H A and PT were comparable to those achieved after the third dose of vaccine. DISCUSSION As already reported in the phase-1 study, we observed that the P T - 9 K / 1 2 9 G toxin can be combined with F H A and pertactin without altering its immunogenicity. We also found that the toxin-neutralizing titer was comparable to that obtained with the acellular pertussis vaccine containing only the P T - 9 K / 1 2 9 G t o x i n ] despite the reduced PT content (7.5 vs 15 ~g) in Acelluvax. Eight infants examined 1 year after the third vaccine injection showed a decreased antibody titer against all vaccine components. A booster dose increased the antibody titers to a level similar to or higher than that obtained after the third dose; the toxin-neutralizing antibody and the anti-69-kd titers reached values significantly higher than those previously observed. After the booster dose, a peculiar behavior of the anti-PT antibodies was observed. The anti-PT ELISA titer was comparable to that induced by the third vaccine dose, whereas the toxin-neutralizing titer was significantly higher. This finding may be due to an affinity maturation of the toxin-neutralizing antibodies or to an increased proportion
of toxin-specific-memory B cells producing neutralizing antibodies. We conclude that a pertussis vaccine containing the genetically detoxified PT, combined with F H A and the 69 kd protein, is safe and immunogenic. We expect that this vaccine, combined with diphtheria and tetanus toxoids, may be licensed for mass vaccination whenever the efficacy studies are completed. Moreover, at present in countries such as Italy, where whole-cell diphtheria-tetanus-pertussis vaccine is poorly accepted because of its reactogenicity, a monovalent acellular pertussis vaccine, similar to that evaluated in this trial, could be used, at the same time or subsequent to diphtheria-tetanus toxoid vaccination, to increase pertussis vaccine coverage. REFERENCES 1. Podda A, Carapella De Luca E, Titone L, et al. Acellular pertussis vaccine composed of genetically inactivated pertussis toxin: safety and immunogenicity in 12- to 24- and 2- to 4-month-old children. J PEDIATR1992;120:680-5. 2. Podda A, Nencioni L, De Magistris MT, et al. Metabolic, humoral and cellular responses in adult volunteers immunized with the genetically inactivated pertussis toxin mutant PT9K/129G. J Exp Med 1990;172:861-8. 3. Krantz I, Sekura R, Trollfors B, et al. Immunogenicity and safety of a pertussis vaccine composed of pertussis toxin inactivated by hydrogen peroxide, in 18- to 23-month-old children. J PEDIATR1990;116:539-43. 4. Nencioni L, Volpini G, Peppoloni S, "De Magistris MT, Marsili I, Rappuoli R. Properties of the pertussis toxin mutant PT9K/129G after formaldehyde treatment. Infect Immun 1991;59:625-30. 5. Englund JA, Edwards KM, Decker M, Anderson E, Pichichero M, Steinhoff M. Multicenter study of acellular (AC) and
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whole cell (WC) pertussis vaccines in young children. Program and Abstracts of the 3 l st Interscience Conference on Antimicrobial Agents and Chemotherapy, McCornick Place Convention Center, Chicago Illinois, 1991. 6. Edwards KM, Meade BD, Decker MD, et al. Comparison of thirteen acellular pertussis vaccines: serologic response. Proceedings of the American Pediatric Society/Society for Pediatric Research Congress, 1992 [Abstract 16687]. 7. Podda A, Nencioni L, Marsili I, et al. Phase I clinical trial of an acellular pertussis vaccine composed of genetically detox-
The Journal of Pediatrics July 1993
ified pertussis toxin combined with FHA and 69 kDa. Vaccine 1991;9:741-5. Manclark CR, Meade BD, Burstyn DG. Serological response to Bordetella pertussis. In: Rose NR, Friedman H, Fahey JL, eds. Manual of clinical laboratory immunology. Washington, D.C.: American Society for Microbiology, 1986:388-94. Hewlett EL, Sauer KT, Myers GA, Cowell JL, Guerrant RL. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect Immun 1983;40:1198203.
Diagnostic use of B-cell alloantigen D8/J17 in rheumatic chorea Brian M. F e l d m a n , MD, John B. Zabriskie, MD, Earl D. S i l v e r m a n , MD, a n d R o n a l d M. Laxer, MD From the Department of Paediatrics, Division of Rheumatology, Hospital for Sick Children, Toronto, Ontario, Canada, and Rockefeller University Hospital, New York, New York
Two unrelated patients with a family history of rheumatic fever had isolated, acquired chorea. Both index cases, as well as affected family members, had increased expression of the rheumatic B-cell alloantigen D8/17. This test may help differentiate Sydenham chorea from lupus chorea. (J PEDIATR1993;123:84-6)
Systemic lupus erythematosus and r h e u m a t i c ( S y d e n h a m ) chorea are the most likely causes of isolated acquired chorea. Differentiation can be difficult, and each diagnosis requires different t r e a t m e n t . A recently identified B-cell alloantigen, D 8 / 1 7 , is expressed more frequently in patients with r h e u m a t i c fever t h a n in control subjects1; its expression in family m e m b e r s is at an i n t e r m e d i a t e level. W e postulated t h a t the presence of this antigen in elevated levels might be used to differentiate patients with r h e u m a t i c chorea from those with SLE. W e describe two patients with isolated chorea in w h o m a clinical diagnosis of r h e u m a t i c
Drs. Laxer and Silverman are associates of the Arthritis Society (Canada), and Dr. Feldman is an Ontario Ministry of Health Research Fellow. Submitted for publication Oct. 6, 1992; accepted Feb. 12, 1993. Reprint requests: Brian M. Feldman, MD, Department of Paediatrics, Division of Rheumatology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Copyright | 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/22/46465
chorea was associated with personal a n d family studies showing an increased expression of D 8 / 1 7 . CASE REPORTS Patient 1. A 10-year-old boy had a 2-week history of chorea manifested by facial jerking, slurred speech, lack of coordination, and a progressive increase in involuntary movements that disappeared during sleep. He had been well except for a mild sore throat about 6 weeks before being seen. He had no history of intoxication, medication use, or travel to an area endemic for Lyme disease. Although there was no family history of chorea, rheumatic fever with carditis had been diagnosed in a maternal uncle during childhood. Physical examination revealed a restless, emotionally labile boy. There was no sign of pharyngitis, and the remainder of the systemic examination findings were normal. Neurologic examination showed diffuse hypotonia, bilateral choreiform movements, and characteristic deep-tendon reflexes. Findings of the remainder of the neurologic examination were normal. The results of the following tests were normal or negative: complete blood cell count, differential cell count, determination of the erythrocyte sedimentation rate, copper studies, complement, liver function tests, immunoglobulins, thyroid function tests, electrocardiography, radiography of the chest, slit-lamp examination for