effect of schedule on reactogenicity and antibody persistence of acellular and whole-cell pertussis...

Elsevier

Vaccine, Vol. 15, No. 1, pp. 51-60, 1997 Copyright 0 1997 Elsevier Science Ltd. All rights reserved

Printed in Great Britain

ELSEVIER PII: SO264-41OX(96)00112-0 0264-410X/97 $17+0.00

Effect of schedule on reactogenicity and antibody persistence of acellular and whole-cell pertussis vaccines: value of laboratory tests as predictors of clinical performance

E. Miller*q, L.A.E. Ashworth-f-, K. Redheadf, C. Thornton?, P.A. Waight* and T. Coleman§

The performance of four acellular pertussis vaccines containing between two and five pertussis antigens combined with diphtheria and tetanus toxoids was compared with that of British whole-cell diphtherialtetanuslpertussis (DTP) vaccine both in laboratory assays for potency, toxicity and immunogenicity, and for reactogenicity and immunogenicity in infants. Clinical responses were evaluated in double blind randomized Phase II trials using 31.519 month and 21314 month schedules. The acellular DTPs had much lower toxicity than whole-cell DTP in laboratory tests and were signtjicantly less pyrogenic than whole-cell DTP under both schedules. Local reactions were not consistently lower in acellular than whole-cell vaccinees and varied with the source of the diphtheria and tetanus antigens used. DifSerences in endotoxin level and content of active pertussis toxin (PT) between acellular DTP vaccines were not clinically significant. The reactogenicity advantage of the acellular vaccines was substantially reduced under the 21314 month schedule due to the reduced reactogenicity of the whole-cell DTP vaccine when given at a younger age. There was no relationship between antigen content measured in micrograms per dose and ELISA antibody responses to filamentous haemagglutinin (FHA) and PT in infants, nor was murine immunogenicity predictive of immunogenicity in humans. Antibody response to PT was attenuated in the whole-cell group under the 21314 month schedule but was unafiected in the group receiving acellular vaccines with individually purified components; antibody response to pertactin (69 kDa antigen) was similar in recipients of the whole-cell and component acellular vaccines under the 21314 month schedule. PT antibody persistence until 4-5 years of age was signtjicantly better in recipients of the component acellular than either the whole-cell vaccine or the co-purified acellular vaccine under the 31519 month schedule. However, diphtheria antitoxin levels were reduced in acellular vaccine recipients under both schedules. Despite signtjicantly lower tetanus potencies of the acellular vaccines in laboratory tests, no differences were found in tetanus anti-toxin responses in children. Copyright 0 1997 Elsevier Science Ltd.

Keywords: Schedule for acellular and whole-cell pertussis vaccines; laboratory performance of acellular and whole-cell pertussis vaccines; antibody persistence to acellular and whole-cell pertussis vaccines

*Public Health Laboratory Service, lmmunisation Division, Communicable Disease Surveillance Centre, Colindale, London, NW9 5EQ, UK. tCentre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK. INational Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. $Hereford Public Health Laboratory, County Hospital, Hereford, HRl 2ER, UK. vo whom correspondence should be addressed. (Received 25 January 1996; revised 30 April 1996; accepted 7 May 1996)

Acellular pertussis vaccines offer the prospect of improved immunogenicity and reduced reactogenicity relative to whole-cell vaccines’. The constituents of Bordetella pertussis most widely considered for the formulation of acellular vaccines are pertussis toxin (PT), the major systemic toxin; filamentous haemagglutinin (FHA), an adhesin; Pertactin, a 69 kDa outer membrane component and the fimbrial antigens, agglutinogens 2 and 3 (AGG2+3). Acellular vaccines containing aluminium adjuvant and diphtheria and

Vaccine 1997 Volume 15 Number 1 51

Reactogenicity and antibody persistence of pertussis vaccines: E. Miller et al.

Table 1 Acellular vaccines evaluated in UK trials

Manufacturer Antigen content (ug per dose)

Toxoiding agent Adjuvant DT component

Porton

Merieux

Lederle

PT: 10 FHA: 10 Agg 2,3: 10

PT: 25 FHA: 25

PT: 3 FHA: 36 Agg 2: 0.7 69 kDa: 1.6

Formaldehyde

Gluteraldehyde

Formaldehyde

Aluminium hydroxide

Aluminium hydroxide

Aluminium phosphate

Trial 1: Wellcome Trial 2: CSL (Australia)

Trial 1: Merieux Trial 2: Merieux

Trial 1: Lederle

Connaught PT: 20 FHA: 20 Agg 2,3: 5 69 kDa: 3

Formaldehyde Aluminium phosphate Trial 2: Connaught

tetanus toxoids, in addition to various proportions of the pertussis components, have been tested for reactogenicity and immunogenicity in a number of trials, initially as boosters in children already vaccinated with whole-cell diphtheria/tetanus/pertussis (DTP) vaccine, then as primary immunizations in infants from the age of 2 months2-5.

Several Phase III efficacy trials of acellular vaccines using 21416 month or 3/5/12 month immunization schedules have recently been completed or are in progress in a number of countries. Two, conducted in Sweden and Italy, were double blind randomized placebo controlled in design and included an American whole-cell DTP vaccine as a contro16.‘. The efficacy estimates for the whole-cell and two component PT/ FHA vaccines were low, but the three component PT/ FHA/69 kDa and five component PT/FHA/69 kDa/ AGG2+3 vaccines gave good protection against laboratory confirmed clinically typical pertussi@.‘. A second Swedish Phase III trial using a 3/5/12 month schedule is now being conducted in which a British whole-cell vaccine made by Wellcome is being compared with the five and one of the three component acellular vaccines.

In order to extrapolate the results of the various Phase III trials to countries using schedules different to those evaluated in the trials, the effects on immunogenicity and reactogenicity of giving acellular vaccines across the schedule range must be evaluated. In England, a Phase II clinical trials programme to evaluate acellular DTP vaccines relative to the Wellcome whole-cell DTP vaccine was started in 1988 when the national schedule was 3/5/10 months’.“. In June 1990, an accelerated 21314 month schedule was introduced and the Phase II trials were repeated using this regimen. Serological follow up, including measurement of antibodies to diphtheria and tetanus, has been completed until 4-5 years of age for the first trial cohort and until 18 months of age for the second trial cohort. Immunogenicity and reactogenicity results from these two studies are presented here, and compared with the results obtained in laboratory assays of potency and toxicity with the same batches of vaccine.

METHODS AND MATERIALS

Trial vaccines The acellular vaccines evaluated in Trials 1 and 2 are

shown in Table 1; manufacturers’ data on composition

52 Vaccine 1997 Volume 15 Number 1

of the acellular vaccines is given. The Porton [made at the Centre for Applied Microbiology and Research (CAMR)], Merieux and Connaught products were made from individually purified antigens; the Lederle vaccine consisted of co-purified antigens. Lederle declined to enter Trial 2, and the Connaught vaccine was not available at the time Trial 1 was conducted. Different batches of whole-cell and Porton and Merieux acellular vaccines were used in Trials 1 and 2. All vaccines were dispensed in identical single dose ampoules indistinguishable by eye from each other.

Laboratory evaluation of trial vaccines The whole cell and acellular DTP vaccines were tested

for the following:

(1)

(2)

(3)

residual PT activity in vitro by CHO cell assay” and in vivo by measurements in the mouse of leukocytosis promotion”, hyperinsulinaemia induction” (Trial 1 vaccines only) and histamine sensitization13; endotoxin content by limulus amyboecyte lysate (LAL) assay I4 and pyrogenicity in the rabbit’” (Trial 1 vaccines only); toxicity by the mouse weight gain test16.

Immunogenicity of Trial 1 vaccines was tested by injecting mice with 0.2 of a single human dose (SHD) and measuring serum antibodies to PT. FHA, tetanus toxoid and diphtheria toxoid by ELISA” after 35 days; results for antibodies to PT and FHA were expressed as units with reference to Japanese National Institute of Health antiserum, those for tetanus and diphtheria were expressed relative to murine reference sera assigned arbitrary unitages. Antibodies to PT were also measured by neutralization of CHO-cell clustering”, and results expressed as the reciprocal of the highest serum dilution to show neutralization. Serum anti-fimbrial antibodies were measured by micro-agglutination’* of a B. pertussis strain of serotype 1, 2. 3, 35 days after injection of mice with 1 SHD of the vaccines; results were expressed as the reciprocal of the highest serum dilution giving agglutination. For Trial 2 vaccines, murine antibody responses were measured by a single point radio-immunoassay (SPRIA) as previously described”. Protective potency of the vaccines was assessed by a mouse intracerebral challenge test I6 with challenge at 2 weeks for the whole cell vaccine and at 3 weeks for the acellular vaccines, and


by a mouse aerosol challenge test*’ with percentage protection expressed relative to a reference whole cell vaccine (66/84) 14 days after challenge*‘. Protective potencies against diphtheria and tetanus toxins were also measured by standard procedures16 and results expressed in international units (IU)/SHD.

Study population

Subjects were infants attending clinics for primary immunization with whole-cell DTP vaccine in North Hertfordshire District Health Authority between March 1988 and January 1994. Subjects were excluded if they had had laboratory-confirmed pertussis or if they had a history of neurological disorder or serious chronic disease.

Trial protocols

Subjects were scheduled to receive the first vaccine dose at 3 months, the second at 5 months and the third at 8-10 months in Trial 1, and at 2, 3 and 4 months in Trial 2. A conjugate vaccine against Haemophilus injkenzae type b was given simultaneously at a different injection site in Trial 2. Contraindications to further doses were those specified by the Joint Committee on Vaccination and Immunisation”. The trials were approved by the North Hertfordshire District Ethics Committee and written informed consent was obtained from parents. Vaccines were randomly allocated to sequential study numbers by computer program and infants were assigned a study number in order of their attendance at clinics. Parents of all study subjects and field, laboratory and coordinating staff were ignorant of the vaccine codes until completion of data analysis.

Evaluation of reactogenicity

Clinical reactions were assessed by parents, who measured infants’ axillary temperatures and local reactions at specified times during the 7 days after vaccination, and by study nurses who made home visits at 24 h and 7 days to measure rectal temperatures and local reactions and to administer a standard symptom questionnaire. Due to supply problems, different digital thermometers were used in the two trials, those in Trial 1 reading in “F and those in Trial 2 in “C. Slight calibration differences were found between the two types of thermometer, precluding direct comparisons between temperatures measured in the two trials.

Measurement of antibody responses

Blood samples were collected by heel prick before the first dose and 6-8 weeks after the third dose in both trials; an intermediate sample was taken 6-8 weeks after the second dose in Trial 1. Further samples were taken 12-18 months after the third dose in both trials and at 4-5 years of age in Trial 1, the latter by venepuncture. Sera were assigned random code numbers and stored at 4°C until assay at CAMR.

ELISA for serum antibodies to pertussis FHA, PT and AGG2+3 were as described previously8,9. The antigens used to coat ELISA plates were prepared at CAMR, Porton and tested to ensure that comparison among acellular vaccines would be unbiased’. Serum antibodies to 69 kDa were measured in Trial 2 by a

Table 2 Laboratory tests of toxicity of the trial vaccines

PT by Serum CHO-cell Leukocytosisa Endotoxin insulins assay (mmm3xl 0e4) content (uU ml-‘)

Vaccine (ng per SHD) (S.E.M.) (IU per SHD) (S.E.M.)

We//come whole-cell: Trial 1 800 4.44 (0.38) 1250-6400 315.6 (71.4) Trial 2 256 4.33 5600 N.A.

Porton: Trial 1 32 1.33 (0.10) 125-300 82.7 (26.7) Trial 2 250 1.95 40 N.A.

Merieux: Trial 1 8 1.95 (0.28) 40 212.5 (52.3) Trial 2 48 1.39 12 N.A.

Ledei-le: Trial 1 32 1.75 (0.15) 10 42.2 (13.3)

Connaught: Trial 2 <32 1.72 6 N.A.

Saline: Trial 1 0 1.77 (0.14) 0 31.5 (17.8)

aAfter vaccination with 1 single human dose (SHD)

similar ELISA using antigen given by Connaught Laboratories Ltd (Willowdale, Ontario, Canada). Pre- vaccination and post-vaccination sera from each subject were assigned randomly to the same assay plate. Plates were read and data stored for each antigen indepen- dently to avoid any possibility of operator bias when titres were calculated using interactive software. Results for the coded sera were expressed as titres corrected against a reference preparation [National Institute of Biological Standards and Control (NIBSC) 89/530] used also as a working standard. Samples without detectable antibody were assigned a titre of 10. Antibodies to diphtheria and tetanus were measured by ELISA as previously and the results expressed as IU ml- ’ “; sera from children in Trial 1 were assayed at Hereford Public Health Laboratory and at CAMR for children in Trial 2.

Statistical analysis

The trials were designed to compare geometric mean antibody levels and adverse event rates in recipients of acellular and whole cell vaccines. To achieve 90% power it was decided to recruit a minimum of 80 participants per group. Geometric mean titres (GMTs) were compared using t-tests applied to the logged titres. The proportions without antibody were compared using the x2 test with continuity correction, or where appropriate, Fisher’s exact test. The relationship between pre- and post-vaccination antibody responses was investigated by Normal errors regression of the log post-vaccination titres on the log pre-vaccination titres, adjusting for schedule effect.

RESULTS

Laboratory evaluation of the vaccines

Toxicity. The whole cell vaccine batches gave significantly higher values in all the toxicity tests than each of the acellular vaccines (Table 2). None of the acellular vaccines used in either trial had significant levels of



Table 3 Pertussis potencies and murine immunogenici~ of vaccines used in Trial 1

Vaccine

Pertussis potency i.ca JU or U per SHD (fiducial limits)

Anti-PT CHO-cell titre

Anti-PT ELISA U (S.E.M.)

Anti-FHA ELlSA U (S.E.M.)

Agglutinin titre vs serotype 1, 2, 3

Whole-cell 7.0 (3.8-l 3.5) 0 c2 49 (14) 256 Porton 1.6 (0.8-3.2) 64 148 (59) 122 (15) 32 Merieux 0.4 (0.0-0.9) 64 100 (30) 93 (6) 0 Lederle 1.7 (0.9-3.4) 64 161 (63) 1351 (320) 0

?U per SHD for whole cell vaccine for which challenge was at 2 weeks; U per SHD for acellular vaccines for which challenge was at 3 weeks

Table 4 Potencies and murine immunogenicity of the diphtheria and tetanus components of the vaccines used in Trial 1

Vaccine Diphtheria potency IU per SHD (fiducial limits}

Anti-diphtheria ELISA U (S.E.M.)

Tetanus potency IU per SHD (fiducial limits)

Anti-tetanus ELISA U (S&M.)

Whole-cell 237 (170-331) 0.59 (0.06) 314 (236-420) 9.15 (2.0) Porton 108 (78-155) 0.04 (0.02) 94 (72-l 23) 0.83 (0.17) Merieux 89 (63-l 24) 0.08 (0.01) 111 (84-145) 1.87 (0.23) Lederle 128 (93-l 80) 0.09 (0.05) 140 (105-186) 1.85 (0.36)

active PT as measured by histamine sensitization (data not shown). Of the acellular vaccines, the batch of Porton vaccine used in Trial 2 was estimated to have the highest active PT content as measured by CHO cell assay. This batch had been stored for 2 years before use and had shown no detectable active PT in the initial CHO cell assay 2 years earlier, indicating some reversion to toxicity on prolonged storage. The Merieux batch in Trial 2 had also been stored for 2 years and showed a smaller increase in active PT, from ~1 to 48 ng per SHD. The endotoxin content of the Porton batch decreased on storage from 250 to 40 IU per SE-ID; the respective figures for Merieux batch were 5 and 12 IU per SHD. The leucocytosis results and serum insulin tests on these batches were not repeated. The batch of Merieux vaccine used in Trial 1 led to a marked increase in serum insulin level. The pyrogenic responses of rab- bits to the four Trial 1 vaccines, given in doses related to their endotoxin content, reflected the LAL results (data not shown). All vaccines passed the mouse weight gain test.

Pertussis potency and immunogenicity. The results of murine pertussis potency and immunogenicity tests of the vaccines used in Trial 1 are shown in Table 3. Despite the use of the modified intracerebraf challenge test for the acellular vaccines, none of the batches used in Trial 1 showed significant protection in this assay. For Trial 2 vaccines, the Merieux and Connaught products gave no protection but the Porton vaccine achieved a value of 6.3 U per SHD (fiducial limits 3.2-13.0); the Porton vaccine was tested before the prolonged period of storage and the test was not repeated. The potency of the whole-cell batch used in Trial 2 was similar to the batch used in Trial 1, 7.80 IU per SHD (fiducial limits 4.9-12.5). All the acellular vaccines showed some potency in clearing infection from the lungs of mice challenged with an aerosol of B. pertussis. The percentage protection values for the various batches were as follows: Porton 33% (Trial 1 batch), 48% (Trial 2 batch); Merieux 24% (Trial 1 batch), 33% (Trial 2 batch); Lederle 38% (Trial 1 batch); whole cell 100% both batches; (Connaught vaccine not tested). When expressed as percentage clearance at day 5 relative to the


organisms recovered on day 1, there was little discrimi- nation between vaccines all giving a value of 94% or above. Only the acellular vaccines gave rise to a detectable anti-P?: response by CHO cell assay. Anti-FHA responses by ELBA were also better with the acellular than whole-cell vaccines (Table 3). Similar results were obtained for Trial 2 vaccines using the radio- immunoassay method. Of the Trial 1 acellular vaccines, only Porton’s gave an agglutinin response. though this was four to eightfold lower than that to whole-cell vaccine.

Diphtheria and tetanus potenc_y and immunogenicity Murine immune responses to diphtheria and tetanus components of the Trial 1 vaccines measured by ELISA, were similar for each of the acellular vaccines and much lower than those given by whole-cell vaccine (Table 4). In tests of protective potency against diphtheria and tetanus toxins, the acellular vaccines in Trial 1 gave similar results to each other, all of which were two- to threefold lower than those given by Wellcome vaccine. Similar results were obtained for potency and murine immunogenicity of the tetanus component with vaccines for Trial 2 but, for the diphtheria component, differences between whole-cell and acellular vaccines were less pronounced, the whole-cell vaccine having a potency of 93 (fiducial limits 59-151) compared with 57 (34-92). 64 (39-103) and 103 (68-103) for the Merieux, Porton and Connaught vaccines. respectively.

Clinical evaluation

Follou~ up of trial cohorts. A total of 432 infants were recruited into Trial 1, of whom 179 received whole-cell, 94 Porton, 74 Merieux and 85 Lederle vaccine. Some ampoules of each of the manufacturers’ vaccines were lost due to a problem with clinic refrigerators and, because of limited supplies, resulted in unequal numbers in the different acellular vaccine groups. A total of 405 infants were recruited into Trial 2, of whom 139 received whole cell, 88 Porton, 89 Merieux and 89 ~onnaught vaccine. The larger size of the whole-cell group in both


Tabie 5 Percentage of infants with an adverse event reported at the 24 h follow-up fall doses ~om4jned~

Adverse event within 24 h

Wellcome whole-cell Portan

N,=523 A!,=278 N,=412 A$=262

Merieux

N,=276 A$=263

Lederfe

PI,=244

Connaught

N,=263

Rectal temperaturti 3, 5, 9 months: ~100.4”F 2, 3, 4 months: 238°C

23 systemic symptomsb 3, 5, Q months 2, 3, 4 months

local erythema 22.5 cm 3, 5, 9 months 2, 3, 4 months

tocai seething 42.5 cm 3, 5, 9 months 2, 3, 4 months

12.1 11.2

22.9 9.0 16.2 8.6 18.4 14.5 12.5 N.A.

21.6 3.9

22.3 18.7 7.4 7.4 3.6 2.3 0.6 N.A.

3.0 3.5 5.3 3.1

20.9 11.1 11.9 5.0 2.3 N.A.

0.9 N.A. N.A. 2.3

N,=Number of doses given in Trial 1 (3, 5, 9 months); N,=number of doses given in Trial 2 (2, 3, 4 months). aDenominator=number of rectal temperatures taken; bfeeding less, disturbed night, irritable, sleepy, crying more than usual

r’~ *~,****_._ E S?

98-i ,:

~ 3~~ Ne;;;L $? 37

_I ..-I .\ at

f._*- *\ *

$ z? 5

m

l% z

I 97.5- - Whole-ceil vaccine :

36,5- - Whole-cell vaccine

S! - - - All acellular vaccines 9 - - - All acellular vaccines

97 /, 36; i , I , , , , , , , , 3hrs pm ,, am “DO” pm ,, am pm , pm pm pm pm Bhrs pm , am mm pm am pm J pm pm pm pm

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 1 day 2 day 3 day 4 day 5 day 6 day 7

Time after vaccine Time after vaccine

Figure I Mean axilfary temperatures measured by Darents after the third dose of vaccine for whole-cell and acellular vaccine groups under the 31519 (old) and 21314 {new) schedules

trials resulted from the late introduction of some of the acelluiar vaccines and the necessity to ensure continua- tion of the whole-cell arm through the trial.

Of the 837 infants recruited into the two trials, 35 (4.2%) failed to receive aif three doses of vaccine, 15 because of contraindications and 20 who moved or who were given non-trial vaccine by mistake. There were no significant differences between acellular and whole-cell groups in the incidence of adverse events contraindicat- ing further doses of vaccine; 81253 (3.2%) vs 31179 (1.70/), respectively in Trial 1 (P=O.54), and 21266 ~0.7%) vs 2f139 (1.4%), respectively in Trial 2 (P=O.14). The mean age at first, second and third doses in Trial 1 was 14, 22 and 38 weeks, respectively compared with 8, 13 and 18 weeks, respectively in Trial 2. Within trials there was no differences in age at vaccination between acellular and whole cell groups.

Reactogwzicif_v. Post-vaccination temperatures were lower after acellular than whole-cell vaccine under both schedules (R&e 5) although the differences were less marked at 21314 months due to reduced pyrogenicity of the whole-cell vaccine (Figure I). Under both schedules

there was an increase in pyrogenicity with dose; for the whole oetl vaccine given at 213f4 months, the proportion with rectal temperatures 38°C at 24 h increased from 31137 (2.2%) at the first dose to 251132 (18.9%) at the third; the increase with acellular vaccines was less marked, from 31266 (1 .I%) to 131261 (5.0%) and was not significantly different from the background rate of pyrexia in the study population, as represented by the proportion of all infants with temperatures 38°C at the 7 day follow-up after each dose, No signi~cant differences in post-vaccination teI~~ratures were found between different acellular vaccines under either schedule, any mean temperature changes being consistent with normal diurnal variation. The difference between acellular and whole-cell vaccines in the incidence of systemic symptoms was less marked than the difference in pyrogenicity (Tub/e 5). For whole-cell and acellular vaccines, the incidence of large local reactions was considerably reduced under the accelerated schedule (F&we 2 and Table 5). The incidence of local reactions to the two vaccines which contained the same DT components (the whole-cell vaccine and the Porton vaccine used in Trial 1) was similar under the same schedule.



Old schedule New schedule

m 1st DTP m 1st DTP

aa 2nd DTP

m 3rd DTP

40 40

5 30 30

E

$

E 2 2 a” 20 a” 20

10 10

0 0 Whole-cell Porton Merieux Lederie Whole-cell Porton Merieux Connaught

Figure 2 Proportion of infants with local erythema 2.5 cm in diameter 24 after vaccination acccording to dose and vaccine group under the 3/4/9 (old) and 2/3/4 (new) schedules

Table 6 Antibody responses to pertussis antigens. Geometric mean antibody titres (GMTs)s and numbers without detectable antibody

Vaccine

PT FHA AGG2+3 69 kDa

Sera GMT GMT GMT GMT tested Neg. (95% C.l.)= Neg. (95% Cl.)” Neg. (95% C.l.)a Neg. (95% C.l.)a

-

Trial 7: 3, 5, 9 months 6 weeks after second dose

Trial 1: 3, 5, 9 months 6 weeks after third dose

Trial 2: 2, 3, 4 months 6 weeks after third dose

Whole-cell 164

Porton a7

Merieux 69

Lederle 74

Whole-cell 154

Porton 83

Merieux 66

Lederle 74

Whole-cell 133

Porton 83

Merieux 87

Connaught 85

17

2

0

2

1

1

2

3

13

0

0

0

451 4 (344-591) 2070 0 (1551-2763) 4592 0 (3939-5354) 1371 2 (1046-l 797)

1439 1 5164 (1169-l 770) (425336270) 4345 0 4688 (3390-5569) (3844-5718) 4385 1 24547 (3375-5697) (17817-33819) 1324 1 8954 (1001-1752) (6768-l 1845)

407 0 $09534)

0 (2695-3797) 6486 0 (5489-7665) 5188 0 (4592-5860)

4008 (3500-4589) 2897 (2376-3533) i9187 (16458-22369) 7798 (6872-8848)

1047 (851-1289) 1245 (945-l 639) 7638 (6435-9067) 4488 (3332-6044)

4

0

19

2

11482 N.A (8334-15818) 35563 (26522-47687) 166 (100-275) 3990 (2541-6266)

27925 N.A (22284-34995) 53333 (44726-63597) 908 (570-l 445) 5248 (3691-7462)

23388 1 4560 I1 9263-28397) (3739-5562) 53456 ’ 15 (45032-63457) $3-99) ?:3-70) 22 59

(46-76) 21428 0 3319 (17023-26974) (2637-4178)

a95% confidence interval

Serum pertussis antibod_y responses. Within each due to impurities in the vaccine. Merieux vaccine gave trial, there were no sigmficant differences between no response to fimbrial antigens in Trial 2, post-third vaccine groups in the pre-immunization GMTs to any dose titres being consistent with persisting maternal antigen. Significant differences between trials in GMTs antibodies. Other responses in the two schedules were were only found for antibodies to FHA; GMT 367 in broadly similar except for PT in the whole-cell group. Trial 1 vs 561 in Trial 2 (WO.001). Antibody responses Thus, under the accelerated schedule the GMT to PT 6 weeks after the second and third doses of vaccine in was significantly lower (P<O.OOOl), and the proportion Trial 1, and 6 weeks after the third dose in Trial 2, are of infants without antibodies to PT significantly higher given in Table 6. The GMTs to each antigen increased (P<O.OOl) than in Trial 1. The GMT to PT, and the with the third dose in Trial 1, with the exception of the proportions negative, after the third dose of whole cell anti-PT responses in Merieux and Lederle groups. After vaccine under the accelerated schedule were similar to the third dose in Trial 1, all three acellular vaccines had those after the second dose under the extended schedule induced responses to FHA and PT as great as, or (Table 6 and Figure 3). The PT antibody response to greater, than those in the whole-cell group (Table 6). The the component acellular vaccines was not attenuated origin of the anti-fimbrial (AGG2+3) response to the by accelerated immunization (Figure 3). The antibody Merieux vaccine in Trial 1 is uncertain but was possibly response to 69 kDa was slightly greater in the whole-cell



PT titre in base 10 log scale

Figure 3 Anti-PT response expressed as a reversed cumulative distribution curve: group 1, post-third dose titres in recipients of component acellular vaccines groups under the old and new schedules; group 2, post-third dose titres in recipients of whole-cell and Lederle co-purified acellular vaccines under the old schedule; group 3, post-second dose titres under the old schedule and post-third dose titres under the new schedule in recipients of whole-cell vaccine

Table 7 Persistence of antibody to pertussis antigens after vaccination. GMTs and numbers without detectable antibody

Vaccine

PT FHA AGG2+3 69 kDa

Sera GMT GMT GMT GMT tested Neg. (95% C.l.)= Neg. (95% C.l.)a Neg. (95% C.l.)a Neg. (95% C.l.)=

Trial 1: 3, 5, 9 months 12-18 months after third dose Whole-cell

Porton

Merieux

Lederle

Trial 7: 3, 5, 9 months 4.5 years of age Whole-cell

Porton

Merieux

Lederle

Trial 2: 2, 3, 4 months 12-18 months after third dose Whole-cell

Porton

Merieux

Connaught

70 17

30 1

34 7

38 14

77 18

52 9

37 6

43 14

64 5

48 0

48 0

39 1

179 2 1042 4 4055 N.A. (114-281) (750-l 449) (2606-6310) 920 0 1648 0 7396 (601-1406) (1026-2647) (5875-9310) 299 0 3388 19 108 (155-579) (2372-4830) (140-289) 147 1 2027 12 321 (72-302) (1285-3192) (135-764)

100 4 (72-l 38) 160 2 (106240) 248 0 (141-436) 97 0 (53-l 80)

185 0 (142-241) 352 0 (277-440) 837 0 (61 O-l 148) 370 0 (281-487)

787 0 (539-l 150) 653 2 (403-l 059) 3715 8 (2324-5940) 1482 7 (936-2349)

1224 0 ?9ll7$634)

0 (754-l 368) 3854 4 (2662-5581) 711 0 (578-875)

2070 N.A. (1565-2739) 2000 (1332-3002) 189 (101-352) 231 (131-407)

2636 0 774 (2075-3348) (564-l 062) 2471 10 66 (1843-3314) (46-96) 148 9 77 (106-205) (55-l 09) 1786 0 675 (1273-2506) (481-946)

a95% confidence interval

than the Connaught acellular vaccine under the accelerated schedule (P=O.O37), a difference which was still seen at 12-18 months.

In both trials there was a negative correlation (WO.001) between pre-immunization antibody levels to PT and levels after the third dose in the whole-cell but not the acellular groups.

Pertussis antibody persistence. A total of 175 children from the Trial 1 cohort and 196 from the Trial 2

cohort were bled 12-18 months after the third dose of vaccine (mean interval 66 weeks after third dose in Trial 1 and 78 weeks in Trial 2). The reduction in GMT compared with the 6 week post-third dose sample varied from ca 3-l%fold, so that differences between groups were greatly diminished (Table 7). However, under both schedules, antibody persistence to PT was better in the component acellular than in the whole-cell or co-purified acellular vaccine groups. The boderline positivity for the antibody to 69 kDa in recipients of the Merieux and



Table 8 Persistence of diptheria and tetanus antibodies in two trials

Vaccine

Diphtheria antibody (IU ml-‘)

No. <O.la tested No. (%)

GMT (95% Cl)

Tetanus antibody (IU ml-‘)

No. <O.Ol a GMT tested No. i%) (95% Cl)

(a) Persistence of diphtheria and tetanus antibodies at school age: Trial 1 children

Whole-cell 69 17 (25) 0.25

Porton 44 20 ;O.$-0.35) 0.12 (0.08-o. 18)

Merieux 29 12 (41) 0.12

Lederle 39 16 fq8-0.17) 0.12 (0.09-0.18)

(b) Persistence of diphtheria and tetanus antibodies at 12-18 months: Trial 2 ch~idre~

Whole-cell 54 26 (46) 0.16 (0.11-0.22)

Porton 40 25 (63) 0.08 (0.06-O. 11)

Merieux 37 18 (49) 0.20 (0.14-0.28)

Connaught 39 29 (74) 0.06 (0.05-0.08)

75

48

36

42

54

40

37

39

(-_)

(-_)

(-_)

i-_)

(11)

(8)

(19)

(3)

0.43 bqO;-0.56)

kyj228-0.50)

(0.21-0.49) 0.32 (0.24-0.43)

0.28 (0.20-0.39) 6.33 , LO.&0.43)

(6.29-0.52) 0.25 (0.1 Q-0.33)

=Protective antibody level by ELISA taken as 0.1 IU ml-’ for diphtheria and 0.01 for tetanus

Porton vaccines at 6 weeks was also seen at 12-l 8 months.

A total of 209 children in Trial 1 were bled before school entry at a mean age of 4.25 years (Table 7). Persistence of antibody to PT was significantly better in the component acellular vaccine groups than in the whole-cell group; Porton vs whole-cell groups P=O.Ol: Merieux vs whole-cell P<O.OOOl. There was no significant difference in persistence of antibody to PT between the Lederle and whole-cell groups, nor between the Porton and whole-cell groups in persistence of antibody to AGG2+3. Two of the Lederle vaccinees bled at school age had a history of clinical pertussis. In these subjects, si~i~cant increases were found in IgG antibodies to the pertussis test antigens compared with levels seen in the 6 week or 18 month blood samples; IgA antibodies were also detected. Significant increases in titre and/or the development of IgA antibodies to two or three pertussis antigens were seen in a further 14 children none of whom had a clinical history of pertussis; these children comprised four whole-cell, four Porton, four Merieux and two Lederle recipients). Thus, serological evidence of pertussis infection acquired in the pre-school years was found in 161209 (8%) of children. Omission of antibody data from these subjects had little effect on the GMTs for any antigen.

~ipbtberi~ and tetanus antibo& persistence. In children in Trial 1, diphtheria and tetanus antitoxin titres were only measured before school entry; in Trial 2 children, antitoxin titres were measured at the 12-18 month follow-up. Within trials, there were no significant differences between acellular and whole-cell groups in tetanus antibody levels (Table 8). However, diphtheria antibody levels in Trial 1 children were significantly lower at school age in the acellular than whole-cell group and higher proportions of children had levels ~0.1 IU ml-- (P=O.O2 acellular vs whole-cell group) (Table 8a). For Trial 2 children, diplltheria antitoxin levels in two of the three acellular groups at 12-18


months were significantly lower than in the whole-cell group (Porton vs whole-cell, P40.01; Connaught vs whole-cell, P _< 0.001). In the whole-cell group the GMTs to diphtheria and tetanus were lower in Trial 2 children at 12-18 months than in Trial l’children at 4-5 years.

DISCUSSION

In this study the laboratory and clinical performance of four acellular DTP vaccines was compared directly with whole-cell DTP vaccine thereby allowing the potential value of control tests as predictors of clinical performance to be evaluated. Laboratory tests showed all four acellular vaccines to be considerably less toxic than the currently used UK whole-cell DTP vaccine, particularly with regard to endotoxin content. This was paralleled by differences in the proportion of infants with fevers, which was significantly lower in all acellular vaccine groups under both schedules. These results are consistent with those of Baraff et al.” who found a direct relationship between endotoxin content and pyro- genie response to whole-cell vaccines. The amount of active PT. as measured by the CHO-cell assay did not appear to be correlated with clinical reactions, as shown by the low reactogenicity of the batch of Porton vaccine used in Trial 2, for which the level of active PT was not far below that of whole-cell vaccine. The relatively small differences measured between acellular vaccines in levels of endotoxin and active PT were not clinically significant. nor was the high result for the Merieux vaccine in the hyperinsulinaemia test. The frequency of systemic symptoms such as excessive crying or irritability was generally lower in acellular than whole-cell groups, although differences were not marked, suggesting that the substantially greater toxicity of the UK whole-cell vaccine as measured in laboratory tests may have few behavioural consequences in infants. Local reactions were not consistently lower in the acellular than whole- cell group and appeared to vary with the source of


the DT toxoids used. Overall, the relative lack of reactogenicity of acellular vaccines was less apparent under the accelerated schedule as a result of the sub- stantial reduction in fevers and local reactions in the whole-cell group that occurred when immunization was completed at a younger age.

The absolute amount of each pertussis antigen in the vaccines was not predictive of the antibody responses in either laboratory tests or human trials. For example, in mice given a single dose of vaccine, a far higher anti-FHA titre was obtained with Lederle (36 pug dose-‘) than with Merieux (25 pug dose-‘) vaccine, whereas in children Merieux vaccine induced higher FHA antibody levels than Lederle at all stages of Trial 1. For PT the non-predictive nature of murine immunogenicity results was just as pronounced. It is clear therefore that the immunogenicity of each manufacturer’s purified acellular components must be established in clinical trials and cannot be inferred from laboratory assays of antigen content or animal immunogenicity studies. Once established, however, these laboratory tests can form part of the batch control procedures for each manufacturer’s product. With the exception of the Porton vaccine used in Trial 2, none of the acellular vaccines showed significant protection in the modified intracerebral challenge test. Its value as a control test for acellular vaccines is therefore doubtful. The aerosol challenge test needs standardization of the method used to define protection before its value as a predictor of efficacy can be established.

The lower tetanus potencies for the acellular vaccines as measured in the laboratory assays were not reflected in lower antitoxin titres in children. It has already been shown that whole-cell’“, but not acellular vaccinesZ4, have an adjuvanting effect on the response to tetanus toxoid in the mouse; our study shows that the potenti- ating effect of whole-cell pertussis vaccine on tetanus responses does not occur in children. In contrast, the generally lower diphtheria potencies measured in mice for acellular than whole-cell DTP vaccines appeared to reflect lower immunogenicity in children, with the exception of the batch of Merieux vaccine used in Trial 2. Lower diphtheria antitoxin levels in acellular than whole-cell vaccinees have also been found in trials in the USZ5. Although diphtheria potencies were not reported for the vaccines used in the US trials, the diphtheria antigen content, as measured by flocculation, was at least as high in the acellular as in the whole-cell vaccines studied”‘. The clinical relevance of the lower diphtheria antitoxin titres in acellular vaccinees is unclear. Our assumption of a protective level of 0.1 IU ml-’ by ELBA is conservative, and measurement of antitoxin levels by the Vero cell neutralization assay for which the protective level is considered to be 0.01 EU ml-‘, is likely to be more accurate in identifying potentially susceptible children. The ability of a vaccine to prime for a subsequent booster response to diphtheria toxoid may also be a more relevant marker of protection than ELISA antibody levels persisting after vaccination. Further studies which allow systematic comparison of antitoxin titres in children, preferably measured by Vero cell neutralization, with potency in laboratory control tests would be helpful in deciding the minimum diphtheria potency requirements for acellular DTP vaccines. The lower diphtheria and tetanus antitoxin titres in children in Trial 2 compared with Trial 1 are

consistent with the attenuation of the response to these components of Wellcome DTP vaccine already reported under the accelerated schedule”.

Our study shows a marked reduction in the response to PT in the whole-cell vaccine group under the accelerated schedule, with around 10% of infants failing to develop detectable PT antibody after the third dose. Pre-immunization (maternal) PT antibody titres were negatively correlated with post-vaccination titres in both Trials 1 and 2 for whole-cell but not acellular vaccinees. However, the lower antibody level to PT found in whole-cell vaccinees under the accelerated schedule was not the result of an inhibitory effect of higher pre- immunization levels of maternal antibody as there were no differences between schedules in infants’ PT antibody levels before i~unization. The clinical significance of the lower PT antibody response under the accelerated schedule is uncertain, as no differences between the schedules were found in antibody persistence at 1 year and a good antibody response was maintained to 69 kDa, an antigen which has been shown to increase protection when added to a two component PT/FHA vaccine’. Four children who were PT antibody negative after the third dose of whole-cell DTP vaccine at 4 months of age received a single booster dose of Lederle monovalent acellular pertussis vaccine and all developed high PT antibody levels within 4 weeks (titre range 567-1525). The ability of the different schedules to prime for a subsequent booster response, or the induction of a cell-mediated immune response. may be more relevant measures of successful primary immunization than ELISA antibody levels immediately after the third dose.

Insufficient amounts of purified AGG2 and AGG3 were available to allow separate measurement of antibodies to these fimbrial antigens in all the trial sera. Assays of a limited number of post-vaccination sera using purified AGG2 and AGG3 showed that whole-cell and Porton acellular vaccines induced antibodies to both fimbrial types’. This is important in view of the epi- demiologi~al evidence for a role for serotype-specific anti-~mbrial antibodies (agglutinins) in protection against pertussis infectiot?‘. Lederle vaccine contained only a small amount of co-purified fimbrial antigen and gave rise to much lower titres of antibody than the Wellcome, Porton and Connaught vaccines. Moreover, only antibody to AGG2 would be expected in recipients of Lederle vaccine. The finding of some AGG2+3 antibody in the majority of Merieux vaccine recipients in Trial 1 was surprising in view of the supposed lack of fimbrial antigen in the vaccine but it has previously been reported as giving rise to agglutinins’.

Our study showed that boosting of antibody levels by sub-clinical infection can occur in children who have responded to primary immunization with either whole- eel1 or acellular DTP vaccines. Such boosting may be an important component of the long-term efficacy of pertussis immunization but becomes less likely as the chance of contact with pertussis disease diminishes. Unlike in North America and many European countries, no booster dose of pertussis vaccine is given in the UK after completion of the primary course. With the dra- matic decline in pertussis incidence in the UK in recent years26 it may become necessary to ensure protection through the early school years by giving pertussis vaccine at school enty. The use of acellular DTP vaccines in this age group is currentIy being investigated as


Reactoge~ic~~ and antibody persistence of pertussis vaccines: E. Mh?r et al.

whole-cell vaccine has been shown to be unacceptably reactogenic when introduced at pre-school ageZ7. This study provides good evidence that component acellular vaccines retain their high immunogeni~ity and excellent safety over a wide schedule range. In this respect they have an advantage over the Wellcome whole-cell vaccine which shows an increasing reactoge~i~~ty with age and attenuation of the anti-PT response it induces under an accelerated schedule. The encouraging findings with acellular vaccines imply that the results of the various Phase III trials can be extrapo- lated to countries using different primary immunization schedules. The suitability of acellular DTP vaccines for administration as combined vaccines with other paedi- atric vaccines such as H. ~~~ue~~~e b, inactivated polio- virus, m~ningococcal, pnenmococ~al and hepatitis vaccines will now need to be assessed over a similar schedule range.

ACKNOWLEDGEMENTS

We thank the general practitioners and families in North Hertfordsl~ire for participating in the trials, the study nurses for their excellent work, Joan Vurdien for admin- istrative support and Dr Farrington for his help with the statistical analyses. We also thank Drs Robinson and Irons for the suppfy of PT, FHA and agglutinogen antigens and Connaught Laboratories Ltd for the gift of the 69 kDa antigen. The trials were conducted with financial support from the Medical Research Council and the Department of Health.

REFERENCES

Robinson, A. and Ashworth, L.A.E. Acellular and defined- component vaccines against pertussis. In: Pafhogenesis and /mmwity in Pen’ussis -(Eds Wardtaw, A.C. and Patton, R.). Wiley and Sons, Chichester, 7988, pp. 399-427 Edwards, K.M., Bradley, R.B., Decker, M.D. et af. E~al~a~~Q~ of a new highly purified pertussis vaccine in infants and children. J. tnfect. Dis. 1989, 160. 832-836 Cherry, J.D., Mo~imer, EA., Hackeil, J.G., Scott, J.V. and the Multi~enter APDT Vaccine Study Groups. Clinical trials in the United States and Japan with the Lederle-Takeda and Takeda acellular ~~ussis-d~phthe~~tetanus JAPDT) vaccines. fnter- national Symposium on Pertussisr Evaluation and Research on Acellular Pertussis Vaccines. Qev. Biol. Standard 1991, 73, 51-58 Pichichero, M.E., Francis, A.B., Blatter, M.M. et al. Acellular pertussis vaccination of 2-month-old infants in the United States. Pediatrics 1992, 89, 882-887 Vanura, H., Just, M., Ambrosch, F. et al. Study of pertussis vaccines in infants: comparison of response to acellular Pertussis vaccines containing 25 ,ug of FHA and either 25 or 8 /Lg of PT with response to whole-cell pertussis OTP vaccine. Vaccine 1994, 12,2%_-214 Gustafsson, L., Hallander, HO., Olin, P, Reizenstein, E. and Storsaeter, &A. A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N. Engl. J. Med. 1996, 334, 349-355 Greco, D., Salamaso, S., Mastrantonio, P. et a/. Clinical efficacy, immunogenicity and safety of two acellular and one whole-celf pertusiss vaccines: results from the Italian Trial. N. Engl. J. Med. 1996,334,341-348 Miller, E., Miller, D.L., Ashworth, L.A.E., Waight, P.A. and Harbert, K.A. Preliminary comparison of antibody responses

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10

11

12

13

14

75

16

$7

18

19

20

21

22

23

24

25

26

27

and symptoms following primary immunisation with British whole cell and three acellular DTP vaccines. Proceedings of Stvth int~rnat~o~af Symposium on Pertussis (Ed. Man~ta~, CR.). Department of Health and Human Services, US. Public Heafth Service, Bethesda, USA, 1990, pp. 303-309 Miller, E., Ashworth, L.A.E., Robinson, A., Waight, P.A. and Iran% L.I. Double blind Phase 2 trial in three month old infants of whole cell pertussis vaccine and an acellular vaccine containing aggiutinogens. Lancet 1991, 337, 70-73 Glllenius, P., J@tmaa, E., Askelof, P., Granstr~m, M. and Tim, M. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. J. Biol. Standard 1985, 13, 61-66 Redhead, K. and Seagroatt, V. The effects of purified components of Bordetefla per&s& in the weight gain test for the toxicity testing of pertussis vaccines. J. Biot. Standard 1986,14, 57-65 Hewlett, EL., Roberts, CO., Wolf, J. and Manclark, CR. Biphasic effect of pertussis vaccine on serum insulin in mice. lffPect lmmun. 1983, 41, 137-144 Ishida, S., Kurokawa, M. and Asakawa, S. A new biological assay method for histamine-sensitizing factor using survival time as a resoonse. JaD. J. Med. Sci. Biol. 1976, 29, 139-150

Ray, A., Redhead, K., Selkirk, S. and Poole, S. Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis. FEMS Micrabiol. Lett, 1991, 79, 211- 218 British fharmacopoeja, Vol Ii. Test for pyrogens, Appendix XIV, A172-A173. HMSO, London, 1993

W.H.0. Expert Committee on Biological Standardization. Technical Report Series, No. 800. W.H.O., Geneva, 1990

Melville-Smith, M.E., Seagroatt, V.A. and Watkins, J.T. A comparison of enzyme-linked ~mm~noso~ent assay (EFISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera. J. Biof. Standard 1983,11,137-144 Zhang, J.M., Cowell, J.L., Steven, A.C. and Manclark, CR. Purification of serotype 2 fimbriae of Bordetella pertussis and their identification as a mouse protective antigen. Dev. Biof. Standard 1985, 61, 173-l 85 Ramsay, M.E.B., Rao, M., Begg, N.T., Redhead, K. and Attwell, A.M. Antibody response to accelerated immunisation with diphtheria, tetanus, pertussis vaccine. Lancet 1993, 342, 203-206 Redhead, K., Watkins, J., Barnard, A. and Mills, K.H.G. Effective immunization against Bordefe//a pertussis respiratory infection in mice is dependent on induction of celf-mediated immunity. Infect. fmmun. 1983,61, 31903798 Depa~ment of Health, Welsh Office and Scottish Home and Heafth Depa~ment. lmmun~satio~ agajnsi Infectious Disease. HMSO, London, 1988 Baraft, L.J., Manclark, CR., Cherry, J.D., Christenson. P. and Marcy, S.M. Analysis of adverse reactions to diphtheria and tetanus toxoids and pertussis vaccine by vaccine lot, endotoxin content, pertussis vaccine potency and percentage of mouse weight gain. Pediafr. Infect. Dis. J. 1989, 8, 502-507 Mussett, M.V. and Sheffield, F.A. Collaborative investigation of methods proposed for the potency assay of adsorbed diphtheria and tetanus toxoids in European Pharmacopoeja. J. &of. Standard ‘I 973.1, 259-283 Redhead, K., Hill, T.A. and Watkins, J.T. Potentiation of the potency of tetanus toxoid by pertussis vaccine. J. Med. ~icrobio~. 1991, 35, $87-188 Edwards, K.M., Meade, B.D., Decker, M.D. et&. Comparison of 13 acellular pertussis vaccines: Overview and serotoggic response. Paediatfics 1995,96,548-557 Miller, E., Vurdien, J.E. and White, J.M. The epidemiology of pertussis in England and Wales. Communicable Dis. F?ep. Rev. 1992,2, R152-R154 Miller, E., Rush, M., Ashwo~h, L.A.E. et a/. Antibody responses and reactions to the whole cell pertussis component of a combined diptheriaftetanuslpertussis vaccine given at school entry. Vaccine 1995, 13, 1183-l 186

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