Development and clinical application of new polyvalent combined paediatric vaccines

Vaccine 17 (1999) 1620±1627

Development and clinical application of new polyvalent combined paediatric vaccines Francis E. Andre * SmithKline Beecham Biologicals, rue de l'Institut 89, 1330 Rixensart, Belgium

Abstract The availability of combined vaccines containing protective antigens against the majority of (ideally all) diseases for which universal immunization is recommended in infancy would simplify the implementation, increase the acceptance, reduce the global cost of immunization programmes and improve disease control, while o€ering the possibility of disease elimination or even pathogen eradication. The desirability of combined vaccines is further enhanced, and made more urgent, because of the increasing number of diseases that can be prevented by vaccination. The complicated logistics of administering di€erent vaccines that each require several inoculations is a signi®cant barrier to successful immunization of a population. Furthermore, interest in immunization is continuously gaining momentum since it is now generally recognised that vaccines are among the safest and most cost-e€ective medical interventions for infectious diseases that continue, in spite of the widespread use of ecacious antimicrobial drugs, to be an important cause of morbidity and mortality. This burden is likely to increase due to the development of antimicrobial resistance. Basic research on new vaccines or improvement of existing ones such as the use of new technologies may be carried out in academic or other non-industrial laboratories but development work, including the necessary extensive clinical testing, that lead to products that can be approved for routine use is usually co-ordinated and ®nanced by commercial companies. The decision to develop any particular combined vaccine will therefore be in¯uenced not only by its medical desirability and technical feasibility but also the potential ®nancial returns that the required investments in time and resources may bring to the company. All major vaccine manufacturers are currently working, either alone or through strategic alliances, towards developing more polyvalent vaccines by adding antigens such as inactivated polio virus, conjugated Haemophilus in¯uenzae type b polysaccharide and hepatitis B surface antigen to the diphtheria±tetanus±pertussis vaccine either in its `classical' (whole-cell) or more puri®ed (acellular) formulations. Experience is showing that the development of combined vaccines involves much more than the simple mixing of existing antigens. Possible incompatibilities or mutual interferences between the antigens themselves, or between excipients, preservatives, adjuvants, residual contaminants, stabilisers and suspending ¯uids make it mandatory that each formulation be thoroughly tested for quality, stability, ecacy and safety. Furthermore the ability to produce and control it consistently must be established before it can be licensed for commercial use. The progress being made in this ®eld is reviewed. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Combined paediatric vaccines

1. Introduction Vaccination is recognised as being the most coste€ective medical intervention when used in properly implemented rational population-wide programmes [1, 2]. The ability of vaccines to control the considerable disease burden in¯icted by infectious diseases is well documented [3]. The most acclaimed * Tel.: +32-2-656-8335; fax: +32-2-656-9134; e-mail: [email protected]. 0264-410X/99/$19.00 # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 8 ) 0 0 4 2 6 - 5

victory of medical science, the eradication of smallpox, was achieved through the use of the ®rst vaccine developed over 200 years ago [4, 5]. However, it is also clear that, currently, the potential for disease control, elimination and eradication o€ered by vaccines is not being fully exploited in the world. The reasons for this failure are obviously very complex. They include political and socio-economic traditions that tend to favour, in healthcare as in other areas, short-term therapeutic approaches over the more fundamental but less glamorous long-term approach of prevention. Other barriers to optimal implementation of vaccination

F.E. Andre / Vaccine 17 (1999) 1620±1627

programmes are the complicated and expensive logistics, including the maintenance of a cold chain, required to administer to all children many thermolabile vaccines according to multi-injection schedules. The number of injections required to fully immunise a child against all the diseases for which vaccines exist has already reached a level which is becoming unacceptable to parents and healthcare personnel. Unjusti®ed fear of side e€ects is also a€ecting acceptance [6]. The current boom in vaccine research, fuelled by the spectacular breakthroughs in immunology, molecular biology and genomics, will lead to many new vaccines [7] that it will be hard to add to the already overcrowded immunisation calendar for young children. The obvious solution to this growing problem is combination, in a multivalent vaccine, of antigens that induce immunity against several diseases. This will reduce the number of inoculations and medical visits required to achieve full immunisation. Combined vaccines are not new and combinations like DTP (triple vaccine against diphtheria, tetanus and pertussis), trivalent oral (OPV) and injectable (IPV) polio, measles/mumps/rubella (MMR), trivalent in¯uenza, polyvalent pneumococcal and meningococcal vaccines have been extensively used for many decades. The advantages of combined vaccines include increased convenience for all users, higher compliance by recipients, wider coverage of the population, better disease control and, because of the simpli®ed logistics of vaccine delivery, reduced administrative costs [8]. Over the last ten years, SmithKline Beecham Biologicals (SB BIO), the vaccine manufacturer that employs the author, has been engaged in developing new paediatric vaccines using DTP as the cornerstone on which to build more polyvalent-vaccines. The diculties encountered, progress made, results obtained and lessons learned will be surveyed, in chronological fashion.

1621

2. Historical retrospective In 1988, the decision was taken to develop a combined DTP hepatitis B (DTPw±HB) vaccine based on the classic (whole-cell) DTP vaccine. Hopes for a rapid successful outcome were high as simultaneous administration of these antigens had already been demonstrated to be possible [9]. International support for this combination came from the Children's Vaccine Initiative that recommended development of this vaccine as a priority in order to facilitate incorporation of hepatitis B immunisation in the WHO's Expanded Programme on Immunisation [10]. However, results in the initial clinical studies were poor for the HB component [11] and considerable pharmaceutical work had to be undertaken to develop a formulation that gave good results [12] (Table 1). The vaccine was ®nally licensed in the European Union in July 1996 after extensive clinical testing, involving more than 2500 healthy infants, showed that it could be used according to di€erent schedules without reduction of the immunogenicity of any of the components (Table 2) [13]. Reactogenicity was slightly increased but judged clinically acceptable. This vaccine can also be used to reconstitute lyophilised conjugated Haemophilus in¯uenzae type b (Hib) SB BIO vaccine just before injection [14].

3. Development of acellular pertussis vaccine In the early 1990's emphasis switched towards the development of puri®ed antigens for the pertussis component in the DTP vaccine. After the good tolerability, safety and protective ecacy of acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTPa) had been demonstrated in large scale clinical

Table 1 Anti-HBs response in the ®rst four studies with di€erent types of formulation and lots of DTPw±HB vaccine (adapted from Ref. [11]) Study

1 2 3 4

Several

Vaccine

Schedule (month)

type

lot

I I I I I I II III IV

A B A A A B C D E

DTPw + Engerix±B

3, 4, 5 3, 4, 5 2, 4, 6 2, 4, 6

2, 4, 6 (opposite arms)

N

44 13 30 29 18 11 21 18 25 130

Anti-HBs one month after third dose SC (%)

SP (%)

GMT (IU/l)

93.2 92.3 93.3 93.1 100 100 90.5 94.4 92.0

79.5 69.2 93.3 89.7 100 100 90.5 94.4 92.0

45 34 63 67 157 229 271 193 1794

100

100

1782

SC = seroconversion (anti-HBs>1 IU/l). SP = seroprotection (anti-HBs>10 IU/l). GMT = geometric mean titre in seroconverters.

1622

F.E. Andre / Vaccine 17 (1999) 1620±1627

Table 2 Geometric mean titres (GMT) and seroprotection rates (SP) or response rate one month after DTPw±HB vaccine using three immunisation schedules (adapted from Ref. [13]) Schedule (months)

N

Antibodies one month after third dose against HB

3, 4, 5 3, 4.5, 6 2, 4, 6 a

389 91 167

D

T

PT

GMT (IU/l)

SPa (%)

GMT (IU/ml)

SPb (%)

GMT (IU/ml)

SPb (%)

GMT (mIU/ml)

response rate (%)

549 1398 1526

98.7 100.0 100.0

1.78 1.98 2.10

99.7 98.9 100.0

3.38 4.48 4.98

100.0 100.0 100.0

92 134 149

99.5 100.0 92.2

Anti-HBs>10 IU/l. bSpeci®c antibodies>0.1 IU/ml.

studies [15] attention was focused on developing combined vaccines based on DTPa. The antigens considered for inclusion in the combinations were HB, IPV and Hib. At this point some advised to concentrate on working exclusively on the most polyvalent combination (i.e. DTPa±HB±IPV/Hib) while others, possibly anticipating the technical diculties ahead, preferred, in view of the many adverse interferences that could occur between antigens, adjuvants, excipients, stabilisers, preservatives, residual contaminants and suspending solutions, to add antigens one by one. Deleterious interactions may lead to one or more of the following: reduced immunogenicity, increased reactogenicity, lower stability. In the end the cautious view prevailed particularly when it became obvious, at an international conference, that there was little consensus on the combinations most needed in di€erent European countries [16]. The company therefore embarked upon developing 7 DTPa-based combined vaccines (viz. DTPa±HB, DTPa/Hib, DTPa±IPV, DTPa±HB/Hib, DTPa±HB±IPV, DTPa±IPV/Hib and DTPa±HB±IPV/Hib) in order to satisfy all potential customers. This has proven a very arduous undertaking over the last few years. Results published and on ®le on the so far licensed combinations (DTPa±HB, DTPa/Hib, DTPa±IPV, DTPa±IPV/Hib) are summar-

ised below. The other combinations are approaching completion of their clinical testing. Results available on these are encouraging [17±19] but will not be reported here.

4. Licensure of DTPa-based combined vaccines Four DTPa-based combined vaccines (DTPa±HB, DTPa±IPV, DTPa/Hib, DTPa±IPV/Hib) developed by SB BIO have so far been licensed. When and where the ®rst license was obtained for these are shown in Table 3. Since the ®rst licenses were obtained more countries have approved these vaccines and they are being introduced more widely. 4.1. Clinical results on DTPa-based combined vaccines The clinical studies summarised in this review were conducted by experienced investigators in many countries worldwide under conditions in accordance with Current Good Clinical Practices. 4.1.1. Reactogenicity Local and general signs and symptoms were recorded in the usual manner in randomised groups of

Table 3 Countries and dates of ®rst registration of DTPa-based SBBIO combined vaccines Vaccine

DTPa±IPV DTPa/Hib DTPa±HB DTPa±IPV/Hib

Country of 1st registration

France Germany EU (n = 15) France Germany

Approval date for indication boosters

primary course

August 1996 October 1996 ± July 1997 April 1998

not developed October 1996 July 1997 submitted April 1998

F.E. Andre / Vaccine 17 (1999) 1620±1627

1623

Table 4 Incidence of local and general signs and symptoms after primary vaccination with DTPa and HB administered separately and DTPa±HB (adapted from Ref. [20])

Local symptoms (%) Any pain Severe pain Redness>2 cm Swelling>2 cm General symptoms (%) Fever (q388C) Fever (q39.58C)

DTPa±HB (n = 51)

DTPa

26.7 0.0 17.2 18.3

13.3 0.0 14.4 10.0

2.8 0.6

+

HB (n = 58) 3.3 0.0 2.8 0.6

5.0 0.0

subjects receiving the combined vaccine intramuscularly in one arm (or leg) or DTPa (or DTPa-based combined vaccine) and the corresponding monovalent (additional) vaccine in the opposite limb. Although the frequency of reported adverse events (solicited or unsolicited) varied widely between studies, di€erences between groups in the same study were not clinically relevant. A typical result is shown in Table 4. The combined DTPa±HB vaccine did not cause more general reactions than the DTPa and HB vaccines given simultaneously but separately and the slight increase in local reactions after the combined vaccine in comparison with DTPa is acceptable in view of the fact that the HB vaccine caused some local reactions in the other limb. In any case, all DTPa-based combined vaccines were signi®cantly less reactogenic than the corresponding DTPw-based combinations (Tables 5 and 6) [20, 21]. Whereas DTPw-based combined vaccines cause more reactions when used as a booster than when used for primary immunisation DTPa±IPV/Hib did not Table 5 Incidence of local and general signs and symptoms after primary vaccination with DTP±HB and DTPw±HB (adapted from Ref. [20]) DTPa±HB (n = 7,613)

DTPw±HB (n = 21,161)

Local symptoms (%) Any pain Severe pain Redness Redness = 2 cm Swelling Swelling>2 cm

8.1 0.5 21.3 1.2 16.4 1.7

45.9 3.2 44.4 5.0 37.6 9.2

General symptoms (%) Fever (q388C) Fever (q39.58C)

10.7 0.2

40.0 0.6

(Table 6). It can be concluded that DTPa-based combined vaccines are clinically well tolerated.

5. Immunogenicity 5.1. DTPa±HB vaccine Two feasibility studies conducted in Turkey [22] and Lithuania [23] established that there was no reduction in the immune response of infants to any antigen in the DTPa±HB vaccine when compared to separate administration of the two vaccines in opposite limbs. Further studies in Italy [24, 25] and elsewhere [20] showed that ``Infanrix2 Hep B provides at least the same level of immunogenicity compared to diphtheria, tetanus, acellular pertussis (DTPa) and hepatitis B vaccines administered separately'' (Ref. [20], p. 28). The peak antibody levels post-vaccination depend upon the administration schedule (Table 7) but in all tested schedules (2, 4 and 6; 3, 4 and 5; 3, 5 and 7; 3, 5 and 11±12 months) the responses were satisfactory (Ref. [20], p. 29). The combination can also be used for preterm infants starting at the same chronological age as term infants [25]. As mentioned earlier, this combination has been approved by the European Medicines Evaluation Agency. 5.2. DTPa/Hib vaccine DTPa and Hib vaccines injected simultaneously into opposite limbs elicit comparable immune responses to those obtained when they are administered separately [26]. However, in the same study, it was shown that after two doses at 4 and 6 months there was about a 10fold drop in geometric mean concentration of Hib antibodies when it was administered mixed with DTPa or both DTPa and IPV as compared with simul-

1624

F.E. Andre / Vaccine 17 (1999) 1620±1627

Table 6 Type and frequency of adverse events in infants receiving 4 doses of DTPa±IPV/Hib or DTPw±IPV/Hib (Pentacoq1, PMC) at 2, 4, 6 and 12 months (adapted from Ref. [21]) After ®rst 3 doses

After booster dose

DTPa±IPV/Hib (n = 286)

DTPw±IPV/Hib (n = 276)

DTPa±IPV/Hib (n = 92)

DTPw±IPV/Hib (n = 87)

Local symptoms (%) Any pain Severe pain Redness Redness>2 cm Swelling Swelling>2 cm

15 0 9 1.7 9 2.4

35a 0.7 25a 5.1a 30a 9.4a

9 1.1 15 5.4 15 1.1

59a 17.2a 43a 17.2a 47a 17.2a

General symptoms (%) Fever (q388C) Fever (q39.58C) Any restlessness Severe restlessness Any anorexia Severe anorexia

22 1.4 28 0.3 15 0.7

48a 5.4a 41a 0.7 24a 0.4

16 2.2 25 3.3 15 4.3

62a 4.6 62a 18.4a 44a 9.2

a

Statistically signi®cant increase compared to DTPa±IPV/Hib group.

Table 7 Seropositivity rates (SR) and geometric mean titres (GMTs) of antibodies in infants vaccinated with DTPa±HB vaccine according to two schedules (adapted from Ref. [24]) Schedule (months)

N

Antibodies one month after third dose against D

2, 4, 6 3, 5, 11 a

172 196

T

PT

FHA

PRN

HB

GMT IU/ml

SRa %

GMT IU/ml

SRa %

GMT EIU/ml

SRb %

GMT EIU/ml

SRb %

GMT EIU/ml

SRb %

GMT IU/l

SRc %

0.19 1.71

100 100

>0.2 >0.2

100 100

56.1 65.3

100 100

153 232

100 100

240 372

100 100

949 5554

99.4 100

Speci®c antibodies>0.01 IU/ml. bSpeci®c antibodies>5 EIU/ml. cAnti-HBs>10 IU/l.

taneous but separate injections of Hib vaccine with DTPa (or IPV) vaccines or mixed DTPa and IPV vaccines. The subjects in that study have been given a third (booster) dose of vaccine at 24 months of age. Very high concentrations of Hib antibodies (GMC's 23±73 mcg/ml) were elicited in all 4 groups of the study indicating that all subjects had been fully primed Table 8 Geometric mean anti-PRP concentrations (GMC) and percentage of vaccinees with antibody concentrations above 1.0 mcg/ml one month after administration at 3, 4 and 5 months (adapted from Ref. [28]) Vaccine

N

GMC (mcg/ml)

>1.0 mcg/ml (%)

DTPa + Hib DTPa/Hib

185 387

7.20 2.02

91.4 71.8

and that immunological memory for Hib had persisted for 2 years [27]. These ®ndings and conclusions were con®rmed in a large randomised, multi-centre study in healthy infants [28] as shown in Table 8. Although mixing Hib vaccine with DTPa before administration clearly signi®cantly reduces the antibody response to PRP (but not to D, T, PT, FHA and PRN [28]) the DTPa/Hib vaccine was found acceptable because: . Anti-PRP levels of >0.15 mcg/ml (the presumed protective level) were elicited in over 95% of vaccinees after primary vaccination. . These levels were higher than those of a vaccine that gave 90% protection [29] and were in the same range as those obtained with 4 commercial monovalent vaccines [30].

F.E. Andre / Vaccine 17 (1999) 1620±1627

1625

Table 9 Booster response to DTPa/Hib vaccine administered 1 year after 3-dose primary course (adapted from Ref. [28]) Primary vaccine

DTPa + Hib DTPa + Hib DTPa/Hib

Booster vaccine

N

DTPa + Hib DTPa/Hib DTPa/Hib

GMC (mcg/ml)

36 10 83

>0.15 mcg/ml (%)

pre

post

pre

post

1.1 1.4 0.3

84.8 34.6 40.2

94.4 100 63.9

100 100 100

Table 10 Immune responses to a booster dose of DTPa±IPV/Hib, DTPa±IPV + Hib and DTPw±IPV/Hib (Pentacoq1, PMC) vaccines in primed children 15 to 24 months old (adapted from Ref. [32]) Vaccine

Antibody levels 34210 days after booster dose Hib

DTPa±IPV/Hib (n = 56±57) DTPa±IPV + Hib (n = 37) DTPw±IPV/Hib (n = 42±46)

60.4 60.0 39.3

D

T

5.7 4.8 3.2

PT

13.3 15.9 6.9

FHA

84.5 73.5 21.0

505 456 156

PRN

812 790 257

polio type 1

2

3

4693 5261 2763

2859 3853 2117

5405 5161 4031

Table 11 Antibody levels one month after primary vaccination (2, 4, 6 months) and booster vaccination (month 12) with DTPa±IPV/Hib and DTPw±IPV/ Hib vaccines (adapted from Ref. [21]) Vaccine

DTPa±IPV/Hib (n = 80±92) DTPw±IPV/Hib (Pentacoq1, PMC) (n = 80±92)

Antibody levels one month after primary series/booster dose against D (IU/ml)

T (IU/ml)

PT (EIU/ml)

FHA (EIU/ml)

PRN (EIU/ml)

Hib (mcg/ml)

1.2/4.3 1.2/3.8

2.1/6.1 3.0/6.6

57/112 16/21

149/374 38/107

157/525 87/222

5.1/23 6.7/14

The geometric mean titres for polio types 1, 2 and 3 were signi®cantly higher at all time points (months 6, 7, 12 and 13) in the DTPa±IPV/ Hib group.

. Immunological priming to PRP was induced with persistent immunological memory. Primed subjects will respond anamnestically to exposure to Haemophilus in¯uenzae type b thus providing protection [31]. The DTPa/Hib vaccine can boost to high levels antiHib in subjects primed with the same vaccine or DTPa + Hib (Table 9). 5.3. DTPa IPV This combination has been licensed only in France for boosting children who were immunised against diphtheria, tetanus, pertussis and poliomyelitis in

infancy. In a study into which 145 children aged 15 to 24 months previously immunised in infancy with DTPw±IPV (Tetracoq1, (Pasteur MeÂrieux Connaught) (PMC) were enrolled one dose of either DTPa±IPV (+Hib in the other arm) or DTPa±IPV/Hib or DTPw±IPV/Hib (Pentacoq1, PMC) was given. A serum sample was obtained 34 2 10 days (range 24±92 days) later [32]. In comparison to the reference vaccine (Pentacoq1) which is used routinely in France for this indication both DTPa±IPV + Hib and DTPa±IPV/ Hib elicited higher antibody levels against all the 9 antigens in the vaccines (Table 10). Comparable levels were obtained in the groups receiving DTPa±IPV/Hib and DTPa±IPV + Hib showing that no deleterious interference occurred when

1626

F.E. Andre / Vaccine 17 (1999) 1620±1627

Hib was reconstituted with DTPa±IPV for use as a booster. This vaccine can also be used to boost preadolescents aged 10±13 years [33]. 5.4. DTPa-IPV/Hib The study by BeÂgue et al. [32] had shown that this combined vaccine produced better antibody responses than a reference DTPw±IPV/Hib vaccine when both were used as a booster. The results of a study [21] comparing the antibody responses when these two combined vaccines were administered at 2, 4, 6 and 12 months of age are shown in Table 11. Clearly DTPa±IPV/Hib is as immunogenic as the corresponding reference whole-cell pertussis combined vaccine both for primary and booster vaccination [34]. It was licensed in Germany in April 1998 for both indications. 6. Comment The advantages of combined vaccines in immunisation programmes are so obvious that all major manufacturers of vaccines have in the past 10 years devoted considerable energy and resources to developing more polyvalent vaccines based on either the classic (wholecell) or acellular DTP vaccines. Experience has shown that the development of a new combined vaccine is time-consuming and expensive [11]. There are many diculties of a technical, regulatory, commercial and political nature to be overcome [8]. However in the past 2 years SB BIO has introduced six new combined vaccines of a conventional type. The clinical results are described in this review. In the near future three additional combinations (DTPa±HB/Hib, DTPa±HB± IPV 2 Hib) will be made available. More long term, new technologies will no doubt make the development of other combined vaccines possible [35]. Such vaccines will considerably facilitate the use of immunisation to reduce disease burden. References [1] The World Bank. World development report 1993. Investing in health. World development indicators. New York, USA: Oxford University Press, 1993. [2] Tengs TO, Adams ME, Pliskin JS et al. Five hundred life-saving interventions and their cost-e€ectiveness. Risk Anal. 1995;15:369±90. [3] WHO, UNICEF. State of the World's vaccines and immunization, 1996. Geneva: World Health Organisation, 1996. WHO/ GPV/9604. [4] Jenner E. An inquiry into the causes and e€ects of the variolae vaccinae, a disease discovered in some of the western countries of England, particularly Gloustershire, known by the name of the cow pox. London, 1798.

[5] Fenner F, Henderson DA, Arita L, Jezek Z, Ladnji ID. Smallpox and its eradication. Geneva: World Health Organisation, 1988. [6] Je€erson T. Vaccination and its adverse e€ects: real or perceived?. Br. Med. J. 1998;317:159±60. [7] National Institutes of Health. The Jordan report. Accelerated development of vaccines. Bethesda, USA, 1998. [8] Rappuoli R, Locht C, Poolman J, Andre F, Dougan G. New vaccines, especially new combined vaccines. Vaccine 1996;14:691±700. [9] Coursaget P, Yvonnet B, Relyveld EH et al. Simultaneous administration of diphtheria/tetanus/pertussis/polio and hepatitis B vaccines in a simpli®ed immunization programme: immune response to diphtheria toxoid, tetanus toxoid, pertussis and hepatitis B surface antigen. Infect. Immun. 1986;51:784±7. [10] Children's Vaccine Initiative. Report of the 2nd Meeting of the Consultative Group Geneva, 16±17 November 1992. CVI/CG-2/ 92. p. 2±3. [11] Andre FE. Development of combined vaccines: manufacturers' viewpoint. Biologicals 1994;22:317±21. [12] Papaevangelou G, Karvelis E, Alexiou D et al. Evaluation of a combined tetravalent diphtheria, tetanus, whole-cell pertussis and hepatitis B candidate vaccine administered to healthy infants according to a three-dose vaccination schedule. Vaccine 1995;13:175±8. [13] Andre FE. The way forward: combined vaccines in Hepatitis B vaccination, moving forward to the next decade. Chester, England: Adis International, 1996. p. 9±11. [14] Wim KM, Aye M, Htay-Htay H, Safary A, Bock H. Comparison of separate and mixed administration of DTPwHBV and HIB vaccines: immunogenicity and reactogenicity pro®les. Int. J. Infect. Dis. 1997;2(2):79±84. [15] Bogaerts H, Capiau C, Hauser P et al. Overview of the clinical development of a diphtheria±tetanus±acellular pertussis vaccine. J. Infect. Dis. 1996;174 (Suppl 3):S276±S280. [16] Andre F, Cholat J, Furminger I, editors. Proceedings 2nd European Conference on Vaccinology: combined vaccines for Europe, pharmaceutical, regulatory and policy-making aspects. Biologicals 1994;22(4):297±436. [17] Zepp F, Schmitt H-J, Kaufhold A et al. Evidence for induction of polysaccharide speci®c B-cell-memory in the 1st year of life: plain Haemophilus in¯uenzae type b±PRP (Hib) boosters children primed with a tetanus-conjugate Hib±DTPa±HBV combined vaccine. Eur. J. Pediatr. 1997;157:18±24. [18] Yeh SH, Partridge S, Marcy SM et al. A randomised study of the safety and immunogenicity of DTPa±HB±IPV vaccine administered as three doses or in a sequential IPV/OPV schedule at 2, 4 and 6 months of age. Pediatr. Res. 1998;43(4 (Part 2 of 2)):161A. [19] Blatter MM, Reisinger KS, Terwelp DR, DelBuono FJ, Howe BJ. Immunogenicity of a combined diphtheria±tetanus±acellular pertussis (DT-tricomponent Pa)±hepatitis B (HB)±inactivated poliovirus (IPV) admixed with Haemophilus in¯uenzae type b (HIB) vaccine in infants. Pediatr. Res. 1998;43(4 (Part 2 of 2)):141A. [20] Infanrix2 Hep B Product Monograph. Belgium: SmithKline Beecham Biologicals, 1997. p 32±3. [21] Dagan R, Igbania K, Piglansky L et al. Safety and immonogenicity of a combined pentavalent diphtheria, tetanus, acellular pertussis, inactivated poliovirus and Haemophilus in¯uenzae type b, tetanus conjugate vaccine in infants, compared with a whole cell pertussis pentavalent vaccine. Pediatr. Infect. Dis. J. 1997;16:1113±21. [22] Kanra G, Ceylan M, Ecevit Z et al. Primary vaccination of infants with a combined diphtheria±tetanus±acellular pertussis± hepatitis B vaccine. Pediatr. Infect. Dis. J. 1995;14(11):998± 1000.

F.E. Andre / Vaccine 17 (1999) 1620±1627 [23] Usonis V, Bakasenas V, Willems P, Clemens R. Feasibility study of a combined diphtheria±tetanus±acellular pertusis±hepatitis B (DTPa-HBV) vaccine, and comparison of clinical reactions and immune response with diphtheria±tetanus±acellular pertussis (DTPa) and hepatitis B vaccines applied as mixed or injected into separate limbs. Vaccine 1997;15(15):1680±6. [24] Giammanco G, Moiraghi A, Zotti C et al. Safety and immunogenicity of a combined diphtheria±tetanus±acellular pertusis±hepatitis B vaccine administered according to two di€erent primary vaccination schedules. Vaccine 1998;16(7):722±6. [25] Faldella G, Alessandroni R, Magini GM et al. The preterm infant's antibody response to a combined diphtheria, tetanus, acellular pertussis and hepatitis B vaccine. Vaccine 1998; 16(17):1646±1649. [26] Eskola J, OÈlander RM, Hovi T et al. Randomised trial of the e€ect of co-administration with acellular pertussis DTP vaccine on immunogenicity of Haemophilus in¯uenzae type b conjugate vaccine. Lancet 1996;348:1688±92. [27] Eskola J, KaÈyhty H, Willems P, Bogaerts H, Kaufhold A. 16th Annual Meeting of the European Society for Paediatric Infectious Diseases (ESPID), 27±29 May 1998. Bled, Slovenia. Abstract 07. [28] Schmitt HJ, Zepp F, MuÈsschenborn S et al. Immunogenicity and reactogenicity of a Haemophilus in¯uenzae type b tetanus conjugate vaccine when administered separately or mixed with concomitant diphtheria±tetanus±toxoid and acellular pertussis vaccine from primary and for booster immunisations. Eur. J. Pediatr. 1998;157:208±14. [29] Eskola J, KaÈyhty H, Takala AK, Peltola H et al. A randomised, prospective ®eld trial of a conjugate vaccine in the protection of

[30]

[31]

[32]

[33]

[34]

[35]

1627

infants and young children against invasive Haemophilus in¯uenzae type b disease. N. Engl. J. Med. 1990; 323:1381±1387. Decker M, Edwards K, Bradley R, Palmer P. Comparative trial in infants of four conjugate Haemophilus in¯uenzae type b vaccines. J. Pediatr. 1992; 2:184±189. Grano€ DM, Holmes SJ, Osterholm MT et al. Induction of immunologic memory in infants primed with Haemophilus in¯uenzae type b conjugate vaccines. J. Infect. Dis. 1993; 168:663± 671. BeÂgue P, Stagnara J, Vie-le-Sage F et al. Immunogenicity and reactogenicity of a booster dose of diphtheria, tetanus, acellular pertussis and inactivated poliomyelitis vaccines given concurrently with Haemophilus in¯uenzae type b conjugate vaccine or as pentavalent vaccine. Pediatr. Infect. Dis. J. 1997;16(8):787± 94. BeÂgue P, Grimprel E, Giovannangeli MD, Abitbol V. Immunogenicity and reactogenicity of a booster dose of DTPa± IPV compared to one dose of DT±IPV administered to preadolescents aged 10±13 years. 16th Annual Meeting of the European Society for Peadiatric Infectious Diseases (ESPID), 27±29 May 1998. Bled, Slovenia. p. 52. Willems P, Kaufhold A. Clinical experience with a pentavalent combination vaccine: DTPA±IPV/Hib overview. 16th Annual Meeting of the European Society for Peadiatric Infectious Diseases (ESPID), 27±29 May 1998. Bled, Slovenia. p. 49. Andre FE, Stanbury WJ, Teuwen DE. Conventional and new generation combined vaccines. In: Kurstak E, editor. Modern vaccinology. New York, London: Plenum Medical Book Company, 1994. p. 41±54.