Comparison of acellular and whole-cell pertussis-component diphtheria-tetanus-pertussis vaccines in infants

Comparison of acellular and whole-cell pertussis-component diphtheria-tetanuspertussis vaccines in infants Dean A. Blumberg, MD, ChrisAnna M. Mink, MD,* James D. Cherry, MD, MSc, Candice Johnson, MD, PDD,Rachel Garber, MD, Stanley A. Plotkin, MD, Barbara Watson, MD, Gerard A. Ballanco, MD, Robert S. Daum, MD,* Bradley Sullivan, MD, Timothy R. Townsend, MD, James Brayton, MD, W. M. Gooch III, MD, David B. Nelson, MD, Blaise L. Congeni, MD, Charles G, Prober, MD, Jill G. Hackell, MD, Cornelia L, Dekker, MD,* Peter D. Christenson, PhD, and the APDT Vaccine Study Group** From the Departments of Pediatrics and Biomathematics, School of Medicine, University of California, Los Angeles; MetroHealth Medical Center and Rainbow Babies and Childrens Hospital, Cleveland, Ohio; the Division of Infectious Diseases, Children's Hospital of Phi/adelphia, Philadelphia, Pennsylvania; Rothschild Pediatric Group, Metairie, Louisiana; Pediatric Infectious Diseases, Tulane University School of Medicine, New Orleans, Louisiana; Marshfield Clinic, Marshfield, Wisconsin; Johns Hopkins Hospital, Baltimore, Maryland; Clinical Pharmacology, Primary Children's Medical Center, Salt Lake City, Utah; Department of Pediatrics, Children's Hospital of Wisconsin, Milwaukee; Children's Hospital Medical Center, Akron, Ohio; Department of Pediatrics, Stanford University Medical Center Palo Alto, California; Lederle Biologicals, Pearl River, New York

In a multicenter, double-blind, randomized, longitudinal study, 252 children received licensed Lederle diphtheria-tetanus toxolds and pertussis vaccine adsorbed (DTP) at 2, 4, and 6 months of age, and 245 children received a DTP vaccine with the Lederle/Takeda acellular pertussis component (APDT) at the same ages. Both groups of children received APDT vaccine at 18 months of age. After each of the first three immunizations, APDT vaccine recipients had fewer local and systemic reactions than did DTP vaccinees. Reactions after the 18-month APDT vaccination were minimal in severity regardless of the vaccine previously received. Antibody responses to lymphocytosis-promoting factor and agglutinogens were more pronounced in DTP recipients; however, APDT recipients had a better serologic response to filamentous hemagglutinin, and responses to the 69K protein were equivalent. This APDT vaccine produces fewer reactions than the standard whole-cell DTP vaccine. The protective significance of the serologic responses to the APDT vaccine is unknown, but the greater response to filamentous hemagglutinin and equivalent response to the 69K protein c o m p a r e d with those to DTP vaccine seem promising. (J PEDIATR1991;119:194204) Presented in part at the Fifth International Symposium on Pertussis, Copenhagen, Denmark, Sept. 22, 1988; the joint meeting of the American Pediatric Society and the Society for Pediatric Research, Washington, D.C., April 29, 1989; and the International Symposium on Pertussis: Evaluation and Research on Acellular Pertussis Vaccines, in Shizuoka, Japan, Sept. 14, 1990. (In relation to these meetings, published in part: Tokai J Exp Clin Med 1988;13:21-28 and Pediatr Res 1989;25:175A.) Submitted for publication Nov. 19, 1990; accepted Feb. 8, 1991. Reprint requests: James D. Cherry, MD, Department of Pediatrics,

194

University of California at Los Angeles School of Medicine, Los Angeles, CA 90024-1752. *Dr. Mink is now at the Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Washington, D.C.; Dr. Daum is at the University of Chicago School of Medicine, Chicago, Ill.; and Dr. Dekker is at Chiron Corp., Emeryville, Calif. **See Addendum for list of other members of the APDT Vaccine Study Group. 9/20/28583

Volumell9 Number2

APDT DTP ELISA EU FHA LPF

Compar~onofAPDT and DTP vaccines

Acellular pertussis-component DTP vaccine Diphtheria and tetanus toxoids and whole-cell pertussis-component vaccine, adsorbed Enzyme-linked immunosorbent assay ELISA unit Filamentous hemagglutinin Lymphocytosis-promoting factor

Currently licensed pertussis vaccines are associated with many reactions, 1,2 and development of a less reactogenic vaccine is desirable. In Japan, several acellular pertussiscomponent vaccines have been developed and used for routine immunization, a, 4 These vaccines produce minimal side effects, and their use is controlling epidemic pertussis in Japan. One Japanese acellular-component vaccine, manufactured by Takeda Chemical Industries, Osaka, Japan, was formulated into an APDT vaccine with diphtheria and tetanus toxoids prepared by Wyeth Laboratories, Philadelphia, Pa., and testing in the United States revealed few side effects. 510 More recently the Takeda acellular pertussis Component has been combined with Lederie Laboratories (Pearl River, N.Y.) diphtheria and tetanus toxoids. Members of our group previously compared Lederle/Takeda APDT vaccine with Lederle DTP vaccine administered as the fifth DTP immunization to 4- to 6-year-old children 11 and as the fourth DTP immunization administered to 17- to 24-month-old ehildrenl2; this APDT vaccine was immunogenic and less reactogenic than the whole-cell pertussiscomponent DTP vaccine. In this article the results of a multicenter randomized longitudinal double-blind study are reported in which Lederle/Takeda APDT vaccine was compared with Lederle DTP vaccine administered to 2-, 4-, and 6-month-old infants, with all subjects receiving APDT vaccine at 18 months of age. METHODS Subjects. Four-hundred ninety-seven healthy 2-monthold infants were enrolled at 10 study sites. In a double-blind manner, 252 children were randomly selected to receive DTP vaccine at 2, 4, and 6 months of age, and 245 children were randomly selected to receive APDT vaccine at the same ages. All children were scheduled to receive APDT vaccine at 18 months of age. Vaccine was administered in a volume of 0.5 ml into the anterior lateral thigh muscle through a 1-inch 25-gauge needle. The parents were requested to maintain a diary of local and systemic reactions for 10 days after immunization. Rectal temperature was recorded before immunization and 30 minutes and 3, 6, 24, 48, and 72 hours after immunization. The presence or absence of the following reactions was recorded at 30 minutes and at 3 and 6 hours after vaccination and daily thereafter for 10 days: local tenderness, vesicles, erythema, induration, swelling, and elevated site temperature; drowsiness, irrita-

195

bility, vomiting, and persistent or unusual crying. Antipyretic medication wa s not routinely administered but was recommended for rectal temperature -->39~ C. Reactogenicity and antipyretic administration data were obtained by telephone contact 1, 2, 3, and 14 days after each immunization. Medical office visits for infections and all hospitalizations were recorded between the 7-month follow-up visit and the 18-month APDT immunization. This study was approved by the institutional review boards at each institution. Informed consent was obtained from the parents or guardians after the design and purpose of the study were explained. Subject enrollment began May 1987, and the last subject completed the 19-month follow-up visit July 1989. Laboratory techniques. Serum samples were obtained by venipuncture for antibody measurements at 2 months (before the first immunization), 6 months (before the third immunization), 7 months, 18 months (before the fourth immunization), and 19 months of age. Because of insufficient serum volume, not all antibody values were determined for all children. Specific immunologic responses were assayed at Lederle Biologicals. An enzyme-linked immunosorbent assay was used to determine lymphocytosis-promoting factor and filamentous hemagglutinin antibody values, 13 and 69K protein antibodies were determined by ELISA as previously described) 2 At 2, 6, and 7 months of age, 69K protein antibody values were determined for subjects at only 2 of the 10 study sites. A microagglutination assay measured pertussis agglutinating antibodies using equal quantities of Bordetella pertussis strains 130 and 138 as the antigens. 13 Neutralizing antibodies to LPF were determined by the Chinese hamster ovary cell assay) 4 Antitoxin values for diphtheria were determined by the micro cell culture method using Vero cells, 15 and tetanus antitoxin values were determined by neutralization in mice. 16 Antibody values were excluded from analysis for subjects who were vaccinated outside of acceptable age intervals or who had blood drawn outside of acceptable postimmunization intervals. Acceptable age intervals for vaccination were defined as (number of subjects excluded in parentheses): dose 1, from 6 weeks to 3 months of age (5 subjects); dose 2, from 3.5 to 6 months and >6 weeks since first dose (32); dose 3, from 5 to 8 months and >6 weeks since second dose (48); and dose 4, from 15 to 21 months (6). The acceptable postimmunization interval was 3 to 9 weeks after the third dose for the 7-month blood draw (20 subjects), and after the fourth dose for the 19-month blood draw (27). Vaccines. The DTP vaccine was a single lot of commercially available licensed Lederle adsorbed vaccine (Tri-Immunol) containing 12.5 Lf of diphtheria toxoid and 5 Lf units of tetanus toxoid per dose, and the pertussis compo-

196

Table

Blurnberg et al.

The Journal of Pediatrics August 1991

I. F r e q u e n c y o f local and s y s t e m i c reactions within t h e first 72 hours after i m m u n i z a t i o n Dose I at 2 m o DTP (n = 252)

Dose 2 at 4 m o

APDT (n = 245)

DTP (n = 241)

APDT (n = 230)

Dose 3 al 6 m o DTP (n = 231)

Dose 4 at 18 m o

APDT (n = 223)

APDT (n = 397")

Local reactions Tenderness Erythema Any >20 mm Induration Any >20 mm Swelling >10 mm Site Temperature Warm or hot Hot Vesicles

48.1

8.6~"

27.8

4.8?

25.1

5.4?

18.3

44.1 8.3

22.9"~ 0t

44.4 7.9

28.3t 3.0:~

43.3 3,5

25,1 + 2,7

26.6 11.3

42.1 10,3 21,8

7.81" 1.2t 4.9~"

37.3 8.3 14.9

15.2t 0.9t 8,2~

37,7 4.3 15.1

16,6? 1.8 7.2~:

23,6 7.5 14.2

35.3 1.6 0.4

9.0~ 0.4 0

24,1 3.7 0.8

8,7t OCT 0

23.8 1.3 0.9

4.9~" 0 0

13,3 2.3 0,3

37.7 1.6

4.5t 1.2

34,0 3.3

9.1t 0~

39.7 6,5

14.8t" 1.4~+

21.2 2.8

38.9 7.5

19.2r 0.8t

38.6 1.2

l 7.8r 0.9

41.1 3.5

23.2"~ 0.9

15, l 0.8

31.0 7.1 5.2 5.6 4,0 0

17,61" 1.6~ 3.7 1.2:t: 0~ 0

l 9,9 1.7 4.6 3.3 0.8 0

12.6,~ 1.3 3.0 1,3 0 0

22.3 1,8 4.8 0.9 0 0.4

10,01" 0.9 0.9~ 0.9 1.4 0.4

2.8 0.3 1.5 0.3 0.3 0

Systemic reactions Rectal Temperature _>38.0 ~ C >_39.0 ~ C Irritability Any Markedw Drowsiness Any Marked Vomiting Persistent crying Unusual crying Hypotonic-hyporesponsive episode Seizures

0

0

0

0

0

0

0

Values are percentage of children vaccinated experiencing designated event. "199 subjects from the initial DTP group and I98 subjects from the initial APDT group. Sp ~0.001 DTP versus APDT. Sp <0.05 DTP versus APDT. w alteration in eating pattern.

T a b l e II. F r e q u e n c y o f a n t i p y r e t i c use within 72 hours

T a b l e Ill. S t u d y w i t h d r a w a l

of immunization Explanation

Dose No.

DTP (%)

APDT (%)

p

1 2 3 4*

12.7 13.3 I3,9 5.5

2.5 5.2 5,4 8.t

0.0001 0,003 0.002 0.32

See Table l for number of subjects in each group at each dose. *Both groups received APDT vaccine for dose 4.

nent with a p o t e n c y (as d e t e r m i n e d in t h e m o u s e protection assay 1) o f 6 units per dose. S u b j e c t s in the A P D T vaccine g r o u p received t h r e e doses o f a d s o r b e d A P D T vaccine at 2, 4, a n d 6 m o n t h s o f age f r o m one o f t h r e e lots o f A P D T vaccine. All subjects received A P D T vaccine at 18 m o n t h s of

Lost to follow-up Parent or guardian requested withdrawal Nonstudy DTP vaccine administered Poor compliance Contraindications to DTP vaccination: Development of neurological symptoms Reactions within 48 hours of vaccination Persistent or unusual crying Hypotonic-hyporesponsive episode Temperature _>40.5 ~ C Other TOTAL

DTP group

APDT group

19 10 7 4

19 1l 7 3

1

2

7 1 1 3* 53

1 1 0 35 47

*One subject each with failure to thrive, development of immunodeficiency disorder, and death. tOne subject with difficulty obtaining blood samples and two subjects with frequent illnesses.

Volume 119 Number 2

Ld (/3

Comparison o f A P D T and DTP vaccines

250

LPF(=16 T LPF(=16 /

Q----oAPDT: P r e - l m m *--* DTP: P r e - l m m

o - - - - o A P D T : P r e - l r n m LPF)16 =--= DTP: P r e - l m m LPF>16

+1

#

I--

o----oAPDT: P r e - l m m o--. DTP: P r e - l m m

CH0(=32 CH0(=32

o-----oAPDT: P r e - l m m =--= DTP: P r e - l m m

CH0)32 CH0)32

1200-

g 150

LO r

1600

+1

200

c ,< U3

Ld 03

19 7

.s +~

800-

~O

400-

100 -

L) "C_

5(3-

E

o (D C9

~ 2

6

7 Months

APDT N: DTP N:

Pre-lmm

LPF (=16 Pre-lmm LPF )16

APDT N: DTP N:

107 111 126 128

84 83 102 100

18

19

72 71 83 82

68 66 77 73

83 79 96 96

2

6

7 Months

Pre-lmm CHO (=32

APDT N: DTP N:

Pre-lmm

APDTN:

DTP N:

CHO )32

112 124 96 92

80 60 66 64

18

19

57 65 47 44

56 61 42 43

of age

77 88 61 58

B

A Ld 6O §

0

of age

125 o----oAPDT: P r e - l m m e--e DTP: P r e - l m m

FHA(=8 FHA(=8

n -oAPDT: P r e - l m m FHA>8 ,--m DTP: Pre Irnm FHA>8

o----oAPDT: P r e - t m m A g g l ( = 8 9 --* DTP: P r e - l r n m A g g l ( = 8

+1

o-----oAPDT: P r e - l m m A g g l ) 8 =--. DTP: P r e - l m m A g g l ) 8

150

100 -

C < (/3 LO

g 9

,\ ..\

,//"

25-

/"t

/ /'//~

\, ',\

c 100

o "u_

/ /" i~ /'i/--/t

\"\

.//-

/

//'

/" ~k I/I.-i/~'\

50

"oc

N E

/

//{k,

75-

E o co

50

/ /

2

o_

o 9 (.D

Qs 2

6

7 Months

Pre-lmm FHA <=8

APDT N: DTP N:

Pre-lmm FHA )8

APDT N:

DTP N:

152 178 83 61

120 130 66 53

18

19

100 109 55 44

95 97 50 42

of age

117 127 62 48

Pre-lmm Aggl(=8

APDT N:

Pre-lmm

APDI N: DTP N:

Aggl)8

DTP N:

2

6

105 111 t14 103

74 80 92 75

Month7ofs

70 73 64 75

oge

18

19

57 53 66 58

57 59 62 57

D Figure. A, Serum ELISA antibody response to LPF. B, LPF antibodies (Chinese hamster ovary [CHO] cell assay) measured by neutralization. C, Serum FHA antibodies measured by ELISA. D, Serum pertussis agglutinin response. Serologic responses in DTP and APDT recipients were based on preimmunization antibody values. Subjects with low preimmunization antibody values are indicated by circles, and those with high preimmunization antibody values are indicated by squares. Antibody values used to separate low and high preimmunization antibody value groups are noted at top of each figure. Solid symbols connected by solid lines represent DTP groups who received DTP vaccine at 2, 4, and 6 months of age and APDT vaccine at 18 months of age. Open symbols connected by dashed lines represent APDT groups who received APDT vaccine at 2, 4, 6, and 18 months of age. Number of evaluable subjects in each group at each time point is indicated below each panel.

age from one of three subsequent lots of APDT vaccine. Lots of APDT vaccine were formulated by Lederle Biologicals in such a way that each dose contained 7.5 Lf of diphtheria toxoid, 5 Lf of tetanus toxoid, and 300 hemagglutinating units of Takeda acellular pertussis component. The diphtheria and tetanus toxoids were produced by Lederle Biologicals. The acellular pertussis component was manufactured by Takeda Chemical Industries from the Tohama strain of B. pertussis. 3 Components of the vaccine include toxoided LPF (3.0 #g/dose), FHA (35 #g/dose), aggluti-

nogens (0.7/~g/dose), and 69K protein (4.0 #g/dose). Antigenic components have not been quantified for the wholecell DTP vaccine; many antigens are part of the whole cell and are not accessible to measurement. Statistics. All antibody data were log-transformed before statistical analysis. Differences between APDT and DTP vaccine recipients in clinical reactions and fold change in antibody values were determined by the Fisher Exact Test. Reaction rates as a function of dose number were compared by repeated-measures weighted least-squares regression. 17

19 g

Blumberg et al.

The Journal of Pediatrics August 1991

Table IV, Serologic responses to pertussis antigens in D T P and A P D T recipients Antibody

LPF ELISA antibodies

V a c c i n e group

DTP APDT p Value

LPF neutralization titer

DTP APDT p Value

FHA ELISA antibodies

DTP APDT p Value

Agglutinin titer

DTP APDT p Value

69K protein ELISA antibodies

DTP APDT p Value

At 2 mo

At 6 mo

19.7 (15.0, 25.8) n = 239 19.9 (15.1, 26.3) n = 235 0.95

35.7 (27.7, 46.0) n = 185 23.9 (19.3, 29.5) n = 189 0.02

45.5 (39.2, 52.8) n = 216 44.3 (39.0, 50.3) n = 208 0.79

84.8 (69.0, 104.2) n = 160 78.2 (68.9, 88.8) n = 164 0.51

4.0 (3.4, 4.6) n = 239 5.3 (4.5, 6.2) n = 235 0.01

3.7 (3.3, 4.0) n = 185 19.0 (16.8, 21.5) n = 189 <0.00001

10.5 (8.7, 12.7) n = 214 11.5 (9.7, 13.7) n = 219 0.48

I7.4 (13.8, 22.0) n = 171 5.7 (4.7, 6.8) n = 176 <0.00001

4.4 (2.8, 6.9) n = 34 6.8 (4.0, 11.3) n = 32 0.21

8.7 (5.4, 14.2) n = 29 14.3 (9.6, 21.3) n = 28 0.12

Values arc geometric mean values (95% confidcnceinterval in parentheses). The DTP group received DTP vaccine at 2, 4, and 6 months of age. and APDT vaccine at 18 months of age. The APDT group received APDT vaccine at 2, 4, 6, and 18 months of age.

Antibody comparisons of geometric means between vaccine groups were m a d e by t test (all acceptable antibody values included); the paired t test was used to compare consecutive antibody values over time within vaccine groups (in which case only subjects with antibody values at both time points were included). After the 18-month A P D T immunization, the 19-month serologic values of subjects who initially received D T P vaccine were compared with those of subjects who initially received A P D T vaccine, after a d j u s t m e n t for differences in antibody values at 7 and 18 months by analysis of covariance. 18 Serologic responses to each antigen at each age in each vaccine group were compared a m o n g study sites with Gabriel's method of multiple comparisons. 19 No other formal corrections were m a d e for multiple comparisons.

tion rates were noted between children who had previously been immunized with D T P vaccine and those who had previously received A P D T vaccine. Reaction rates were equivalent when compared by race or gender. The three lots of A P D T vaccine administered at 2, 4, and 6 m o n t h s were associated with similar reaction rates, and reaction rates were similar a m o n g the three lots of A P D T vaccine at the 18-month immunization. Reaction rates were equivalent a m o n g study sites with the exception of the " a n y e r y t h e m a " category. One study site had m u c h higher rates of " a n y e r y t h e m a " in both vaccine groups at each immunization. S t a n d a r d observation techniques were utilized at this site, and other reaction rates were not unusual. T h e most likely explanation for this finding is the patient population at this site: a homogeneous group with very fair skin.

RESULTS

For the first three doses of D T P vaccine, increasing dose n u m b e r resulted in significant decreases in rates of injection site warmth, e r y t h e m a > 2 0 ram, induration > 2 0 mm, tenderness, marked irritability, any and m a r k e d drowsiness, and persistent crying (p <0.05). T h e A P D T recipients also had decreases in rates of injection site w a r m t h and any

Clinical reactions. After each of the first three immunizations, A P D T recipients had a significantly lower frequency of virtually all local and systemic reactions t h a n D T P recipients (Table I). A t 18 months, when all children received A P D T vaccine, no significant differences in reac-

Volume 119 Number 2

Comparison of APDT and D TP vaccines

At 7 mo

A t 18 m o

A t 19 m o

140.0 (109.3, 179.4) n = 177 37.7 (30.5, 46.6) n=182

29,0 (23.7, 35.4) n=156 12.3 (9.6, 15.8) n = 158

176.4 (152.9, 203.4) n = 142 45.0 (37.7, 53,6) n=146

<0.0000l

<0.00001

<0.00001

406.7 (321.5,514.3) n = 164 122.7 (104.2, 144.4) n = 155 <0.0000 l

123.3 (99.7, 152.5) n = 120 63.3 (52.8, 75.8) n = 121 <0.0000 t

886.1 (694.8, 1130.0) n = 115 270.4 (223.8, 326.7) n = 114 <0.00001

11.7 (10.1, 13.5) n = 177 60.5 (53.6, 68.2) n = 182 <0.0000 l

3.7 (3.I, 4.5) n = 156 7.0 (5.7, 8.6) n = 158 <0.00001

37.4 (32.0, 43.7) n = 142 81.7 (70.7, 94.5) n = 146 <0.00001

87.2 (69.0, 110.2) n = 163 9.7 (7.8, 11.9) n = 165 <0.0000l

12.8 (10.l, 16.3) n = 122 5.4 (4.2, 6.9) n = 131 <0.00001

115.5 (87.4, 152.7) n = 129 19.4 (15.2, 24.7) n = 125 <0.00001

29.7 (20.3, 43.5) n=27 43.9 (30.2, 63.6) n=28 0.14

9.2 (7.6, 11.2) n = 156 12.5 (10.3, 15.1) n = 158 0.03

126.8 (103.2, 155.8) n = 142 130.7 (111.7, 152.8) n = 146 0.82

drowsiness, but had increasing rates of local erythema >20 ram, any induration, and temperature >_38 ~ C with subsequent doses (p <0.05). More DTP recipients than A P D T vaccinees received antipyretic agents after immunization (Table II). There were similar rates of antipyretic administration after the A P D T immunization at 18 months. Two children had hypotonic-hyporesponsive episodes. A 6-month-old girl was noted to be lethargic and limp 2 hours after her third DTP immunization. She was occasionally "glassy eyed" but responsive, and the episode had resolved by the next morning, 14 hours later, Another 6-month-old girl received her third A P D T immunization and 25 hours later was noted to be pale and difficult to arouse. Within 30 minutes, she appeared to be behaving normally, and her vital signs and physical examination findings were normal when she was examined by investigators 90 minutes after the episode began. Accidental death occurred in one child who had received two DTP immunizations; he was strangled by a pacifier cord at 51/2 months of age. The numbers of medical office visits for infections and hospitalizations between the 7-month and 18-month visits were similar in the two vaccine groups. The D T P group had

19 9

3.5 office visits for infections per child-year; the A P D T group had 3.6 office visits for infections per child-year. One child required hospitalization for a serious bacterial disease: a 10-month-old girl who had received three DTP immunizations was hospitalized for preseptal eellulitis caused by Streptococcus pneumoniae. Withdrawal from the study occurred with equal frequency in the A P D T and DTP vaccine groups (Table III). Fourteen children were thought to have contraindications to further pertussis immunization: 10 subjects in the DTP group and four in the A P D T group, Three of these subjects experienced neurologic disorders unrelated to immunization and were withdrawn from the study. These children are discussed below. In one subject in the DTP vaccine group, a partial complex seizure disorder was diagnosed at 7 months of age; no medication was prescribed, and results of follow-up electroencephalography and examination were normal. One subject in the A P D T vaccine group had a simple febrile seizure at 17 months of age. In one subject in the A P D T vaccine group, spasmus nutans and macroencephaly were diagnosed at 16 months of age; at 21 months of age the neuroIogic examination findings were normal.

20 0

Blumberg et aL

Serologic responses. Pertussis antibody values in vaccinees are shown in Table IV, and proportions of vaccinees achieving preset antibody values and fourfold antibody rises are shown in Table V. The DTP recipients had significantly higher antibody values against LPF (as measured by ELISA or the neutralization assay) than APDT recipients had (Table IV). Similarly, more members of the DTP vaccine group had LPF antibody values >--30 ELISA units at 7, 18, and 19 months of age (Table V). Because the preimmunization (2-month) LPF ELISA antibody values were higher than expected, some serum samples with high antibody values were reexamined. Some hemolysis was noted, and some samples contained debris (presumably blood cell stroma). Because of this observation, LPF antibody values were determined in a subset of sera before and after recentrifugation (Table VI). The LPF ELISA antibody values decreased after further centrifugation, and the 2-month serum samples showed a greater proportional decrease than the 6- or 7-month samples. These results were not affected by vaccine group (analysis of variance for differences between the DTP and APDT groups of before- and after-recentrifugation values over time: p = 0.59). The relative comparisons of the APDT and DTP groups were similar regardless of which values were used: in this subset of subjects the groups were equivalent at 2 months, but the DTP group had LPF ELISA values about two times higher than the APDT group at 6 months and about four times higher at 7 months. The 2-, 6-, and 7-month LPF ELISA antibody values correlated better with LPF neutralization titers after recentrifugation (r = 0.87,p <0.0001; n = 184) than before recentrifugation (r = 0.42, p <0.0001; n = 184). When the subjects were 18 and 19 months of age, some of the serum samples were recentrifuged before laboratory analysis. The LPF ELISA antibody values of the reeentrifuged samples did not differ from the values of the samples that were not recentrifuged; this equivalency was present within each vaccine group at 18 and 19 months of age. The APDT recipients had much higher F H A antibody values in response to immunization (Table IV). The F H A antibody values >-30 EU were attained by more APDT recipients than DTP vaccinees at 7 months of age, and this difference persisted after the 18-month APDT vaccination (Table V). The DTP vaccinees had higher agglutinin titcrs in response to immunization (Tables IV and V). The 69K protein antibody values were similar in response to DTP or APDT vaccine (Tables IV and V). The APDT and DTP vaccine groups both had significant increases (p <0.00001) in all pertussis serologic values assayed at 7 compared with 6 months, and at 19 compared with 18 months. At the 18-month APDT immunization, the

The Journal of Pediatrics August 1991 DTP group had better responses to LPF (measured by ELISA and neutralization) and agglutinogens, an equivalent response to the 69K protein, and a poorer response to F H A than those subjects who had received APDT vaccine for the first three immunizations. These differences persisted even after allowances were made for the serologic values at 7 and 18 months of age. Seventy-three percent of the subjects were white and 18% were black; the racial distribution was similar in both vaccine groups. When subjects were grouped by race, black children in both vaccine groups had better serologic responses than white children to all pertussis antigens assayed, except for similar agglutinin values in the APDT vaccine group. (The 69K protein antibody responses were not compared by race at 6 and 7 months because of the low number of sera analyzed at 2, 6, and 7 months.) These differences were not consistently statistically significant. No consistent differences were noted in the serologic responses to the different lots of APDT vaccine. Similarly, no consistent significant differences were noted when subjects were grouped by gender or study site. The APDT and DTP recipients were grouped by low versus high prevaccination antibody values at 2 months of age, and their responses to vaccination were compared (Figure). For LPF ELISA antibodies, infants with high preimmunization values in the DTP group had higher values at 7 months than those in the DTP group with low preimmunization values (Figure, panel A); 18- and 19-month values were equivalent. The LPF ELISA antibodies in APDT recipients with low and high preimmunization values followed a pattern similar to that in DTP vaccinees. In contrast, DTP vaccinees with lower preimmunization LPF neutralizing antibody had higher LPF neutralizing antibody values after three immunizations than subjects with high preimmunization antibody values (Figure, panel B). The differences between the groups persisted at 18 and 19 months of age. The APDT recipients with high and low preimmunization LPF neutralizing antibodies did not have differing responses. When F H A responses were evaluated, the APDT recipients with lower preimmunization antibody values had a better response to immunization than those subjects with high initial values, whereas the DTP groups had equivalent responses (Figure, panel C). For both vaccine groups, agglutinin responses did not appear to correlate with preimmunization antibody values (Figure, panel D). These relationships were consistent at a variety of preimmunization antibody values utilized to separate infants into high and low antibody groups. (The 69K protein antibody responses were not analyzed in the above manner because of the low number of preimmunization sera assayed.) Subjects in the DTP vaccine group had higher diphtheria and tetanus antibody values in response to immunization

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Comparison o f APD T and D TP vaccines

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T a b l e V. Antibody fold changes and attainment of threshold Antibody

Criterion

APDT group

DTP group

p

LPF

->30 EU at 7 mo >_30 EU at 18 mo ->30 EU at 19 mo Neutralization ->4-fold rise 6 to 7 mo Neutralization ->4-fold rise 18 to 19 mo

54.9 31.0 59.6 17.7 73.3

81.4 51.3 98.6 58.9 82.7

<0.0001 <0.0001 <0.0001 <0.0001 0.10

FHA

->30 EU at 7 mo ->30 EU at 18 mo _>30 EU at 19 mo

82.4 12.0 90.4

15.3 7.7 59.9

<0.0001 0.26 <0.0001

Agglutinins

->1:32 at 7 mo ->4-fold rise 6 to 7 mo ->1:32 at 18 mo ->1:32 at 19 mo >_4-fold rise 18 to 19 mo

29.7 32.2 24.4 51.2 51.9

83.4 59.5 36.9 85.3 73.8

<0.0001 <0.0001 0.04 <0.0001 0.001

69K protein

-->30 EU at 7 mo ->30 EU at 18 mo ->30 EU at 19 mo

67.9 22.8 93.8

51.9 15.4 87.3

0.28 0.11 0.07

Values arc percentages of subjects in each group. Number of subjects in each group at each time point for each antibody test is shown in Table IV.

than A P D T recipients had. At 7 months, 97.0% of DTP recipients and 95.9% of A P D T recipients had diphtheria antibody values >_0.01 I U / m l , and all subjects had tetanus antibody values _>0.01 I U / m l . At 19 months, all subjects in both vaccine groups had tetanus and diphtheria antibody values _>0.01 I U / m l . DISCUSSION This A P D T vaccine was well tolerated and was significantly less reactogenic than the standard D T P vaccine. The frequency of reactions after DTP vaccination in this study was similar to that in previous studies, 2, 2o except that fever and irritability occurred less often. The A P D T vaccine was also well tolerated at the immunization given at 18 months, regardless of previously administered vaccine. This is in contrast to findings by Blennow and Granstr6m 21 with a different A P D T vaccine; they found an increased rate of local reactions in children previously immunized with that A P D T vaccine compared with that in children previously immunized with a DTP vaccine. The cause of reactions associated with pertussis vaccination is unclear. A causative role for L P F has been postulated, but no evidence has been found linking biologically active L P F to severe reactions associated with DTP vaccine. 22 Increasing D T P vaccine endotoxin content correlates with an increasing rate of febrile reactions, z3 Acellular pertussis vaccines have less endotoxin, 3 thus accounting for the decreased frequency of fever observed after immunization. Persistent crying is initiated by painful local reactions in a subset of children22; the reduced rate of local

reactions associated with this A P D T vaccine would be expected to result in less persistent crying. The occurrence of some reactions, such as seizures or hypotonic-hyporesponsive episodes, is sufficiently rare (1 in 1750 vaccinations 2) that meaningful frequencies or comparisons cannot be made in a study of this size. Seizures associated with DTP vaccine probably represent febrile convulsions.22, 24 The decreasing rate and severity of febrile reactions associated with the A P D T vaccine may result in fewer pertussis vaccine-associated convulsions. Unfortunately, a hypotonic-hyporesponsive episode was noted after both A P D T and DTP vaccination. The pathogenesis of hypotonic-hyporesponsive episodes is unknown, and A P D T use will apparently not eliminate the occurrence of these reactions. Many different pertussis serologic assays have been studied, but no absolute antibody level, type, or combination has been determined to be protective. Sato and Sato 2s suggested that E L I S A values greater than 20 or 30 E U in their laboratory against L P F or F H A were protective. Granstr6m et al. z6 followed subjects with pertussis and postulated that L P F neutralizing antibodies mediated disease protection. However, the recent Swedish vaccine efficacy trial in which an L P F toxoid vaccine and an L P F toxoidF H A vaccine were evaluated found no correlation between L P F antibody and protectionY In that study the mean postimmunization antibody value against L P F was as high in children who subsequently had pertussis as in those who did not. Further analysis of data from the Swedish trial found that children who received the L P F toxoid vaccine

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The Journal of Pediatrics August 1991

Table VI. Comparison of LPF ELISA antibody values before and after recentrifugation G e o m e t r i c m e a n value" (EU)

AI 2 m o

At 6 m o

At 7 m o

DTP vaccinees (n = 34) Before recentrifugation After recentrifugation Ratio (before/after recentrifugation)~" p (before vs after recentrifugation)

29.3 (13.1, 65.6) 3.0 (1.6, 5.6) 9.7 (4.6, 20.7)
43.4 (25.2, 75.0) 12.2 (6.5, 23.0) 3.6 (2.1, 6.0)
140.2 (91.7, 214.6) 39.2 (21.2, 72.4) 3.6 (2.0, 6.3)
APDT vaccinees (n = 31) Before recentrifugation After recentrifugation Ratio (before/after recentrifugation):) p (before vs after recentrifugation)

36.1 (15.1, 86.5) 2.5 (1,6, 3.8) 14.4 (5.6, 37.3) <0.0001

18.8 (11.8, 29.9) 5.6 (4.1, 7.8) 3.3 (2.2, 5.0) <0.0001

38.5 (23.7, 62.6) 8.4 (5.6, 12.7) 4.6 (2.7, 7.6) <0.0001

Ratio of DTP to APDT vaccinees Before recentrifugation p (DTP vs APDT) After recentrifugation p (DTP vs APDT) Ratio (before/after recentrifugation) p (before vs after recentrifugation)

0.8 (0.2, 2.6) 0.72 1.2 (0.6, 2.5) 0.62 0.7 (0.2, 2.2) 0.51

2.3 (1.1, 4.7) 0.02 2.2 (1.1, 4.4) 0.03 1.1 (0.6, 2.0) 0.83

3.6 (1.9, 6.8) 0.0001 4.6 (2.2, 9.6) <0.0001 0.8 (0.4, 1.7) 0.52

*95% confidenceintervalin parentheses. tComparison of ratio differencesover time: 2 versus 6 months,p = 0.003; 2 versus 7 months,p = 0.03; 6 versus 7 months,p = 0.98. ~Comparisonof ratio differencesover time: 2 versus 6 months,p = 0.0005; 2 versus 7 months,p = 0.02; 6 versus 7 months,p = 0.18.

had no protection against infection or colonization. 28 However, children who received the vaccine with both LPF toxold and F H A had some protection against infection. Studies done about 40 years ago found that serum agglutinin titers correlated with postimmunization protection.29, 30 The decreased response to aggtutinogens in A P D T recipients in this study is cause for concern. However, in the early studies it was also found that some children who did not develop agglutinin antibodies were also protected, suggesting that the measured agglutinin titers acted as markers of protection rather than as protective antibodies. The 69K protein of B. pertussis is a recently described outer membrane protein that elicits protection when administered as a vaccine in a murine model of pertussis. 3j Antibody to a similar outer membrane protein is protective against Bordetella bronchiseptica in a piglet model. 32 Infants were noted to have relatively different serologic responses to this APDT vaccine than older children. In previous studies of 17- to 24-month-old and 4- to 6-year-old children comparing this A P D T vaccine with DTP vaccine, we found that the ELISA LPF and agglutinin responses were approximately equivalent or better in DTP recipients, whereas the F H A and 69K protein responses were higher in A P D T recipients. TM 12,33 In this study of infants, A P D T vaccinees had reduced responses to LPF and aggtutinogens, a better response to FHA, and an equivalent response to the 69K protein compared with DTP recipients. An unexpected finding was the decrease in LPF ELISA values after recentrifugation of the 2-, 6-, and 7-month se-

rum samples. The cause of the difference in values is unknown. Hemolysis and blood cell debris may have falsely elevated the LPF ELISA values. Supporting this interpretation is the improved correlation noted between the LPF neutralization assay results and the recentrifuged L P F ELISA values. The greater difference observed at 2 months may be due to the increased difficulty of drawing blood at younger ages, resulting in more hemolysis, or to a passively transferred maternal factor. Alternatively, antibodies may have been removed by recentrifugation (perhaps by removal of debris with adsorbed antibodies), thus falsely decreasing the values. Nevertheless, the assays were performed in a blinded manner, the relationships between the A P D T and DTP vaccine groups were constant before and after recentrifugation, and therefore we believe that the relative comparisons of the A P D T and DTP vaccine groups are valid. Host factors influence serologic responses to pertussis vaccines. Investigators at Vanderbilt University noted that higher preimmunization LPF ELISA antibody values were correlated with lower values after three DTP vaccinations than in subjects with lower preimmunization values. 34 Responses to A P D T were found to be independent of preimmunization antibody values, In contrast, the DTP recipients in this study with higher preimmunization LPF ELISA antibody values also had higher values at 7 months, after three immunizations, than those children with lower preimmunization values; 18- and 19-month values were equivalent. The A P D T vaccinees with high and low preimmunization values followed a pattern similar to that in the DTP recip-

Volume 119 Number 2

ients. The different results in this study and the Vanderbilt study may be due to our discrepant initial LPF ELISA values, which may have partially misdefined the groups. In addition, the vaccines in these studies are not strictly comparable; the acellular vaccine used in this study has approximately four times less LPF per dose than the acellular vaccine used in the Vanderbilt study, and different DTP vaccines were used as well. Among DTP vaccine recipients, the LPF neutralizing antibody response in this study was better among those subjects with lower concentrations of preimmunization LPF neutralizing antibodies; a similar pattern of response to F H A was noted in the APDT vaccine group. Higher preimmunization antibody concentrations may partially neutralize the serologic response to particular antigens. Compared with the DTP vaccine used in this study, this APDT vaccine possesses increased F H A antigenicity and decreased LPF antigenicity, perhaps permitting exaggeration of differences in the immune responses in the different groups. It is unclear why preimmunization antibody correlated differently with LPF ELISA antibody and LPF neutralizing antibody responses. The delineation of more precise antibody responses, perhaps by defining epitope specificity, may shed some light on this issue. The APDT immunization administered at 18 months resulted in good responses to all antigens in both vaccine groups. However, previous DTP recipients had better responses to LPF and agglutinogens, and previous APDT recipients had better responses to FHA. These differences were not fully explained by the serologic values before the 18-month immunization (at 7 and 18 months of age), thus offering evidence that pertussis vaccines are T cell dependent. Because of high vaccination levels and the relatively low incidence of pertussis in the United States, APDT vaccine efficacy data have not been obtained in this country. In a recent efficacy study in Japan, this APDT vaccine had an efficacy Of 98% for classic pertussis and 81% for all respiratory illnesses that may have been pertussis. 35 This degree of efficacy is similar tO that of the DTP vaccines presently used in the United States. 1 Lower diphtheria and tetanus antibody values were found in APDT recipients than in DTP vaccinees. However, 100% of all vaccinees achieved protective antibody concentrations (>__0.01 IU/ml) at the conclusion of the vaccine series. The APDT vaccine contained less diphtheria toxoid per dose than the DTP vaccine used in this study. The increased immunogenicity of tetanus toxoid in the DTP vaccine may be due to an adjuvant effect secondary to endotoxin or other components in the DTP vaccine. We conclude that this APDT vaccine is immunogenic and less reactogenic than the presently used DTP vaccine. The

Comparison of APDT and DTP vaccines

203

APDT recipients had reduced responses to LPF and agglutinogens, a better response to FHA, and an equivalent response to the 69K protein than the DTP recipients, but the protective significance of different serologic responses is currently unknown. The serologic data in combination with efficacy data from Japan indicate that this APDT vaccine is a promising candidate for routine immunization in the United States. ADDENDUM

The following are additional members of the APDT Vaccine Study Group: Karen Lewis, MD, Esther F. Seeley, PNP, and Patricia Chatfield, PNP, University of California at Los Angeles Medical Center; S. Michael Marcy, MD, and Lisa Guravitz, PNP, Kaiser-Permanente, Panorama City, Calif.; Ricki G. Robinson, MD, Kathleen Smith, MD, Laura Mabie, MD, H. James Holroyd, MD, Leonard R. Baker, MD, Frank E. Dudenhoeffer, MD, and Elaina Camp, RN, Descanso Pediatrics, La Canada, Calif.; Jeffrey Blumer, MD, Leonard Rome, MD, Jennifer O'Horo, RN, and Judy Whitwell, RN, Case Western Reserve University, Cleveland, Ohio; Carol Rothenberger, RN, and Carol Pankok, RN, Children's Hospital of Philadelphia, Philadelphia, Pa.; Melanie Bergens, RN, Rothschild Pediatrics Group, Metairie, La.; Judy Zirnhelt, Marshfield Clinic, Marshfield, Wis.; Elaine Skleres, RN, John Hopkins Hospital, Baltimore, Md.; John Christianson, MD, David Johnson, MD, Elise Swenson, RPh, Darla Longhurst, MT, Gina Agy, RN, and Lisa Lenoch, RN, Primary Children's Medical Center, Salt Lake City, Utah; Gloria McGrath, RN, Pediatric Primary Care Clinic, Milwaukee, Wis.; Gayle Ma!izia, RN, Children's Hospital Medical Center of Akron, Akron, Ohio; Deborah Au, RN, Stanford University Medical Center, Stanford, Calif.; Jane V. Scott, PhD, Mason G. Stout, PhD, Joseph R. Mezzatesta, PhD, Jane F. Graveline, MS, and Gail Howard, RN, MS, Lederle Biologicals, Pearl River, N.Y. We thank Dr. M. B. J. Steinfeld for critical manuscript review and Dr. A. Sperduto for helpful discussions. REFERENCES

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