T Vesikari 1, J Joensuu 1, M Baer 2,HKäyhty 4, R-M Ölander 4, H Sormunen 4, A Miettinen 3, RL Ward 5 and T Guillot 6

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1 Acta Pñdiatr 88: 513± Concurrent administration of rhesus rotavirus tetravalent (RRV-TV) vaccine with pentavalent diphtheria pertussis tetanus Haemophilus influenzae b-inactivated polio and hepatitis B vaccines T Vesikari 1, J Joensuu 1, M Baer 2,HKäyhty 4, R-M Ölander 4, H Sormunen 4, A Miettinen 3, RL Ward 5 and T Guillot 6 University of Tampere Medical School 1, Departments of Paediatrics 2 and Clinical Microbiology 3, Tampere University Hospital, Tampere, National Public Health Institute (KTL) 4, Helsinki, Finland, Children s Hospital Medical Center 5, Cincinnati, Ohio, USA, and Wyeth- Ayerst Research 6, Paris, France Vesikari T, Joensuu J, Baer M, Käyhty H, Ölander R-M, Sormunen H, Miettinen A, Ward RL, Guillot T. Concurrent administration of rhesus rotavirus tetravalent (RRV-TV) vaccine with pentavalent diphtheria pertussis tetanus Haemophilus influenzae b-inactivated polio and hepatitis B vaccines. Acta Pædiatr 1999; 88: Stockholm. ISSN To investigate the incorporation of oral rhesus human reassortant rotavirus tetravalent (RRV-TV) vaccine into a routine immunization programme, RRV-TV or oral placebo was coadministered with a pentavalent diphtheria tetanus whole-cell pertussis Haemophilus influenzae b (Hib) inactivated polio vaccine and hepatitis B vaccine following a mo schedule in a double-blind trial involving 249 infants. Seroconversion rates after 3 doses of rotavirus vaccine were 80% for rotavirus immunoglobulin A (IgA) and 93% for RRV neutralizing antibodies. Rotavirus vaccine did not interfere with the immune responses to diphtheria, tetanus, pertussis, Hib, poliovirus 1, 2 and 3, or hepatitis B. Following the first, second and third doses of vaccine, fever >38 C on the day of vaccination was seen in 31%, 24% and 24%, respectively, with no difference between RRV-TV- and placebo-vaccinated children. This fever was presumably due to the whole-cell pertussis vaccine. Those vaccinees who received concomitant RRV-TV vaccine had another peak of fever around d 4 after the first dose, when 25% of them had fever >38 C and 3% >39 C. It is concluded that RRV-TV rotavirus vaccine can be given concurrently with other childhood immunizations following a mo vaccination schedule. However, febrile reactions to RRV- TV rotavirus vaccine are common when the first dose is given at the age of 3 mo. & Combined vaccinations, fever, pertussis vaccine, rhesus rotavirus, rotavirus vaccine T Vesikari, University of Tampere, Medical School, P.O. Box 607, Tampere, Finland (Tel , fax , . lltive@uta.fi) Oral rhesus human reassortant tetravalent (RRV-TV) rotavirus vaccine has been licensed in the USA and may also be licensed in the European Union in the near future. In the USA, the Advisory Committee on Immunization Practices and the American Academy of Pediatrics have recommended universal immunization of healthy children with RRV-TV vaccine between 6 wk and 6 mo of age, concurrently with routine childhood immunizations. In Europe, it is likely that many countries will eventually introduce rotavirus vaccination, as the vaccine could prevent a major proportion of morbidity associated with rotavirus. The efficacy of RRV-TV vaccine, at a titre level of plaque-forming units (PFU), has been established in trials in the USA, Venezuela and Finland. In the USA the vaccine showed 49% overall efficacy for rotavirus gastroenteritis and 100% efficacy for dehydration associated with rotavirus gastroenteritis (1). In Venezuela, the efficacy for any rotavirus disease was 48% and in countering the need for hospitalization because of rotavirus gastroenteritis 70% (2). In Finland, the RRV-TV vaccine was 68% efficacious overall and 100% efficacious for rotavirus gastroenteritis requiring hospital admission (3). In the Finnish study, the RRV- TV vaccine induced febrile reactions on d 3 5 after vaccination in about 30% of the vaccinees (3, 4); a much lower reactogenicity was seen in the USA (1, 5) and in Venezuela (2). For routine vaccination of all healthy children, the RRV-TV vaccine should be given concurrently with other childhood immunizations. The present study was conducted with the primary objective of determining whether RRV-TV vaccine might interfere with immune responses to routine childhood vaccines. A pentavalent combination vaccine including diphtheria, tetanus, whole-cell pertussis, Haemophilus influenzae b (Hib) and inactivated polioviruses types 1, 2 and 3 (Pentacoq 1, Pasteur Mèrieux, France) was chosen for the study, as this vaccine, or an equivalent combination, is or may in the future be used in several European Scandinavian University Press ISSN

2 514 T Vesikari et al. ACTA PÆDIATR 88 (1999) countries. Hepatitis B vaccine was given separately in the opposite buttock; at the time the study was initiated no hexavalent combination including hepatitis B vaccine was available. The study schedule was mo, which is the programme currently followed in Finland and some other European countries, including Austria, Belgium and The Netherlands (6). In addition, the study provided an opportunity to evaluate the reactogenicity of RRV-TV vaccine given concurrently with the pentavalent vaccine and, conversely, the reactogenicity of the pentavalent vaccine with or without RRV-TV vaccine. Methods The study protocol was approved by the ethics committees of the National Public Health Institute (KTL), Finland, and Tampere University Hospital, Tampere, Finland. The study was conducted in 30 well-baby clinics around or near the city of Tampere. Before enrolment of the child, one of the parents signed an informed consent form. Vaccines RRV-TV vaccine, PFU per dose consisting of PFU of each monovalent component (rhesus rotavirus and rhesus human reassortant rotaviruses corresponding to rotavirus G-types 1, 2 and 4) was supplied by Wyeth-Ayerst Research (W-AR, Philadelphia, PA, USA). The vaccine was a lyophilized product, stored at 2 8 C and resuspended in 2.5 ml of diluent shortly before use. The diluent contained 9.6 mg ml 1 citric acid and 25.6 mg ml 1 sodium bicarbonate as buffer. The placebo, also supplied by W-AR, was an identical preparation without the active vaccine viruses. The pentavalent diphtheria tetanus whole-cell pertussis Hib inactivated poliovirus vaccine (Pentacoq, batch no. R ) was received by W-AR from the manufacturer Pasteur-Mèrieux, and supplied to the study site. The vaccine was a combination of lyophilized H. influenzae type b conjugate vaccine (10 mg dose 1 ) which before use was reconstituted with Tetracoq 1 suspension containing diphtheria (30 IU dose 1 ) and tetanus (60 IU dose 1 ) toxoids, inactivated Bordetella pertussis whole cells (4 IU ml 1 ) and inactivated polio viruses types 1, 2 and 3 (20, 4 and 16 D-antigen units dose 1, respectively). The hepatitis B vaccine (Engerix-B 1 20 mg dose 1, batch no. ENG 1553A4), manufactured by SmithKline Beecham, Rixensart, Belgium, was supplied likewise through W-AR. Study design The study design was double-blind and randomized for RRV-TV vaccine and placebo. A randomization schedule was generated by the Biostatics section of W-AR. Each enrolled infant was assigned a code-labelled package of three doses of RRV-TV vaccine or placebo. The study vaccine (RRV-TV or placebo) was always administered with the other vaccines at the same visit, in the following order: oral study vaccine, Pentacoq in the left buttock, and Engerix in the right. The vaccines were to be administered at 3, 4 and 5 mo of age. The permitted age for the first dose was wk, and the time interval for the next 2 doses d. The maximum permissible age for the last dose was 26 wk. Infants in good general health with a birthweight greater than 2200 g could be enrolled; it was also required that the family had a telephone. Vaccination was delayed if the infant or a household member had diarrhoea or vomiting within 3 d of the scheduled vaccine administration, or if the infant was being treated with an antibiotic. Postvaccination safety surveillance was conducted on d 1 (day of vaccination) to d 5 after each vaccination. The parents recorded daily on diary cards rectal temperature, vomiting, diarrhoea, respiratory symptoms, decreased appetite, abdominal cramping, irritability and the infant s activity level. Parents received a digital thermometer and were taught its proper use. Rectal temperatures were taken in the afternoon or evening. If the evening temperature was higher than 37.5 C, the temperature was also taken the next morning. Study nurses called the families on d 2 to remind them of the surveillance. If required, the infant was seen by the study physician. Venous blood specimens were collected before the first vaccination and approximately 1 mo after the third. The serum obtained was divided into 6 aliquots and stored at 20 C until shipped on dry ice to the respective laboratories. Laboratory methods Rotavirus antibody determinations were carried out at the Children s Hospital Medical Center, Cincinnati, OH, USA. Serum rotavirus immunoglobulin A (IgA) was determined by an enzyme-linked immunosorbent assay (ELISA) using a standard curve procedure essentially as described previously (7). For this, the standard was pooled adult human sera which was assigned a rotavirus IgA titre of units ml 1. The limit of detection by this assay was 30 units ml 1. Neutralizing antibody titres to RRV were determined by an ELISA-based assay (NELISA) described previously (8, 9). In brief, a specified concentration of RRV grown in MA104 cells (approximately 2000 infectious viruses) was neutralized with a series of twofold dilutions of the antisera starting at dilutions of 1:10. Neutralizing antibody titres were defined as the reciprocal of the dilution that reduced the final optical densities by 60% relative to cells infected with non-neutralized virus. Seroconversions were defined as at least a fourfold increase in the titre of antibody from before until after

3 ACTA PÆDIATR 88 (1999) Rotavirus vaccination 515 Table 1. Seroconversion by anti-rrv neutralizing antibody test and levels of antibodies before and after three doses of RRV-TV vaccine. Seroconversion for RRV-3 Yes: 114/122 (93%) No: 8/122 (7%) GMT (10 90%) Before vaccination 8.67 ( ) ( ) After third dose ( ) ( ) GMT: geometric mean titre. Table 2. Distribution of (postdose 3) antibody levels to diphtheria, tetanus and pertussis when these vaccines were coadministered with RRV- TV or placebo. Seroconverted for RRV-3 RRV-TV Placebo Antigen Yes (n = 114) No (n = 8) No (n = 117) Diphtheria GMT (IU ml 1 ) th 90th centile Tetanus GMT (IU ml 1 ) th 90th centile Pertussis GMT (titre) th 90th centile Hib a GMT (mg ml 1 ) th 90th centile Polio 1 GMT (titre) th 90th centile Polio 2 GMT (titre) th 90th centile Polio 3 GMT (titre) th 90th centile Hepatitis B b GMT (miu ml 1 ) th 90th centile Number of sera studied: a vaccine seroconverters 110, placebo 116; b vaccine seroconverters 109, placebo 115. GMT: geometric mean titre. immunization. This value was selected because it is the conservative standard traditionally used for such studies. Clearly, serum neutralizing antibody of maternal origin present at the time of vaccination will have delayed considerably between blood collections. Therefore, the fourfold increases will have been detected on a decreasing background, thus effectively requiring potentially much greater than fourfold increases to be classified as a seroconversion based on neutralizing antibody titres. The diphtheria, tetanus, pertussis, Hib and poliovirus antibody determinations were conducted at the National Public Health Institute (KTL), Helsinki, Finland. IgG antibodies to diphtheria and tetanus toxoids were measured using a double-antigen ELISA method as described previously (10). Tetanus and diphtheria antitoxin concentrations were expressed in International Units (IU ml 1 ). The quantitation limit was IU ml 1 for both antigens; a concentration of 0.01 IU ml 1 was considered protective. A standard assay for the microagglutination of B. pertussis was used to detect agglutinating antibodies (11). Activity was expressed as the reciprocal of the highest dilution giving complete agglutination. An enzyme immunoassay described previously (12) with a few modifications (13) was used to measure total specific antibodies to Hib capsular polysaccharide. The lower detection level was 0.1 mg ml 1. The results are given as geometric mean concentration and as percentages of children with antibody concentration 1.0 mg ml m1. This concentration is associated with long-term protection after vaccination with plain Hib polysaccharide vaccine (14) and has been used widely as a cut-off when assessing the immunogenicity of new Hib conjugate vaccines and vaccine combinations. The microneutralization assay for poliovirus antibodies was carried out using a standard procedure (15): duplicate twofold dilutions of sera, 100 CCID 50 each of poliovirus types 1, 2 and 3, and overnight neutralization at 36 C with vero cells wk 1. The titre was scored according to the reciprocal of last serum dilution with 50% inhibition of viral cytopathic effect on d 6. A titre 1:8 was defined as protective. Hepatitis B antibodies (HbsAb) were determined at the Department of Clinical Microbiology, Tampere University Hospital, using a commercial semiquantitative enzyme immunoassay (Cobas Core Anti-HBsEIA,

4 516 T Vesikari et al. ACTA PÆDIATR 88 (1999) Fig. 1. (a d) Recorded symptoms on d 1 5 after concurrent vaccination with oral RRV-TV rotavirus vaccine or oral placebo and pentavalent diphtheria tetanus pertussis Hib inactivated polio virus vaccine and hepatitis B vaccine. F. Hoffmann-La Roche). A protective level of HBsAb was held at 10 miu ml 1. Statistical analysis Interference of RRV-TV with the immune responses to other vaccines was assessed by comparing the number of subjects with a protective level of antibodies for each of the antibody assays, using Fisher s Exact test. A Wilcoxon Rank-Sum test was carried out for the results of each antibody assay to determine whether the distributions of antibody levels were similar across treatment groups. Antibody levels for each assay were summarized by treatment group using geometric means and the 10th and 90th centiles. The safety data were analysed using Fisher s exact test. All statistical analyses were performed at the Biostatistics Section of W-AR. Results A total of 249 infants was enrolled and 243 completed the study, i.e. received 3 doses of study vaccine (RRV- TV or placebo) plus 3 doses of Pentacoq and Engerix-B. Of the 126 children who received the study vaccine, 4

5 ACTA PÆDIATR 88 (1999) Rotavirus vaccination 517 Fig. 1. Continued. were withdrawn for reasons discussed below and 122 completed the study. Of the 123 children who received the placebo, 2 were withdrawn and 121 completed the study. Immunogenicity of RRV-TV vaccine After the third dose of vaccine 97 out of the 122 (80%) RRV-TV vaccine recipients and none of the 118 placebo recipients tested had seroconverted by rotavirus IgA ELISA antibody. All subjects, including the 25 who did not seroconvert, were rotavirus IgA ELISA negative before the first dose of vaccine. By measuring neutralizing antibodies to RRV, 114 out of the 122 (93%) RRV-TV vaccine recipients and 1 of the 118 (1%) placebo recipients showed seroconversion. Because sera were not collected between vaccinations, it was not possible to determine after which dose the children had seroconverted. However, the eight subjects who failed to seroconvert by neutralizing antibody had significantly higher prevaccination anti- RRV antibody levels than those who seroconverted (Table 1). Serological responses to other vaccines Table 2 summarizes the antibody responses to diphtheria, tetanus, pertussis, Hib, polioviruses 1, 2 and 3, and

6 518 T Vesikari et al. ACTA PÆDIATR 88 (1999) hepatitis B in the vaccinees who received RRV-TV vaccine concomitantly and seroconverted by RRV antibody (n = 114) or did not seroconvert (n = 8), or who received placebo and did not seroconvert (n = 117). There was no difference in postvaccination geometric mean antibody levels to each of these antigens between the three groups and, of particular note, the levels were not lower in the infants who had received concomitant RRV-TV and seroconverted than in those who had received placebo. After the third dose of Pentacoq all participants had achieved minimum protective antitoxin levels (0.01 IU ml 1 ) for diphtheria and tetanus. For Hib antibodies 112 out 118 (94.9%) RRV-TV vaccine recipients and 108 out of 117 (92.3%) placebo recipients reached a level of 1.0 mg ml 1 or greater (ns). All subjects had poliovirus types 1, 2 and 3 neutralizing antibodies at a titre of 1:8 or higher after 3 doses of vaccine. One subject in the RRV-TV vaccine group failed to seroconvert to hepatitis B; accordingly, 99% of the RRV-TV vaccine and 100% of placebo recipients had protective levels of hepatitis B antibody after 3 doses of Engerix B vaccine. The geometric mean levels of postvaccination antibodies to diphtheria, tetanus, pertussis, Hib, polioviruses 1, 2 and 3, and hepatitis Bs antigen are shown in Table 2. There were no significant differences between the RRV-TV vaccine and placebo recipients in the mean antibody levels to any of these antigens. Reactogenicity Febrile reactions were common after vaccinations, and typically occurred in two phases (Fig. 1). The first peak, presumably due to the Pentacoq vaccine and, more specifically, to the whole-cell pertussis component, was seen on the day of vaccination and the next day. The second peak, characteristic of rhesus rotavirus vaccine, was seen on d 3 5 after the first vaccination in the recipients of RRV-TV vaccine. After the first vaccination, rectal temperature > 38 C was recorded on d 1 in 31% and on d 2 in 11% of the vaccinees; there was no significant difference between the RRV-TV and the placebo recipients. After each of the second and third doses fever >38 C was recorded in 24% of the vaccinees on the day of vaccination. About 2% of the vaccinees had fever >39 C on d 1 after each dose of vaccine. On d 4 after the first vaccination, rectal temperatures >38 C were recorded in 25% and >39 C in 3% of the RRV-TV vs none of the placebo recipients (p < ). After the second vaccination there was no difference in fever between the RRV-TV and placebo recipients on d 4, but after the third vaccination 6% of the RRV-TV vaccine and 1% of the placebo recipients had fever >38 C (p = 0.03). On the day of vaccination (d 1), irritability of the infant was recorded in as many as 75%, 61% and 58% of the vaccinees after the first, second and third dose, respectively; there was no difference between the RRV-TV and placebo recipients. Decreased activity on d 1 was recorded in 46%, 32% and 24% after the first, second and third dose of vaccine, with no difference between RRV-TV and placebo groups. On d 4, at the time of the presumed febrile response due to rhesus rotavirus vaccine, there was no significant difference in reported irritability between RRV-TV and placebo recipients (23% and 17%, respectively), but decreased activity was more common in the RRV- TV vaccinees (17%) than in the placebo recipients (2%; p = ). Decreased activity was seldom reported on d 4 after the second and third vaccinations and there was no difference between the RRV-TV and placebo recipients. Decreased appetite on d 4 was reported in 11% and 4% of the RRV-TV and placebo recipients, respectively (p = 0.036), after the first vaccination (Fig. 1). Abdominal cramping was reported after the first vaccination on d 4 and 5, respectively, in 18% and 13% of the RRV-TV vaccinees and 13% and 8% of the placebo recipients; these differences were marginally significant. Diarrhoea and vomiting were not increased around d 4 after vaccination, and there was no difference between the RRV-TV and placebo vaccine recipients in respect of the occurrence of these symptoms. Respiratory symptoms were equally common in the RRV-TV and placebo groups, were not clustered on any particular day, and were not different after the first, second or third vaccination (data not shown). Medical intervention was required in five cases after the first, in none after the second, and in six cases after the third vaccination. Of the 5 cases after the first vaccination, 4 (3%) were in the RRV-TV group and occurred between d 3 and 5 after vaccination; the 1 case in the placebo group was on d 2. Of the six cases after the third vaccination, five were in the RRV-TV group. One of these was on the day of vaccination and 4 were on d 3 and 4. The only case in the placebo group occurred on d 2. In addition to these physician visits by children who continued the study, there were four withdrawals in the RRV-TV group and two in the placebo group, all totally or in part for medical reasons. Two children in the RRV- TV group were not given the third Pentacoq vaccine because the first two doses had resulted in a marked febrile reaction. Two further children were withdrawn by the parents; one for high fever on d 1 and the other for decreased appetite and abdominal cramping starting from d 1 after the first vaccination. In the placebo group, one child was withdrawn because of diarrhoea starting on d 3 after the second dose and lasting for 12 d. Another child in the placebo group was withdrawn 3 wk after dose 2 at the parents request; the child had a prolonged cough.

7 ACTA PÆDIATR 88 (1999) Rotavirus vaccination 519 Discussion The first objective of this study was to investigate whether oral administration of three doses of RRV-TV might interfere if given concomitantly with routine immunizations. The study design followed the standard Finnish diphtheria tetanus pertussis (DTP) immunization schedule of 3, 4 and 5 mo, also applied in some other European countries, but used a pentavalent combination vaccine, licensed and used in France, plus a hepatitis B vaccine which is not part of the routine immunization programme in Finland. This was done to obtain results more representative than those including only the routine vaccines in the Finnish immunization programme; at the same time the study provided an opportunity to compare the immunogenicity and reactogenicity of the test vaccines with those currently used in Finland. With regard to a possible interaction of oral RRV-TV vaccine with injectable vaccines given concurrently, there was no indication that RRV-TV might reduce immune responses to any of the other six vaccines or vaccine components. A corresponding study was published from the USA after the submission of this paper. In this study no indication of interference of RRV-TV vaccine with antibody responses to diphtheria, pertussis, tetanus, Hib or oral poliovirus vaccine was observed (16). Concomitant administration of the RRV-TV likewise appeared to have no effect on the reactogenicity profile of the pentavalent DTP-Hib-inactivated polio virus combination vaccine. Most of the reactions attributable to this vaccine and, more specifically, most probably to its whole-cell pertussis component, were seen on the day of vaccination and the following day. These febrile reactions were equally common in the RRV-TV and placebo recipients. This is in accordance with our earlier experience of giving RRV-TV vaccine together with DTPw (whole-cell pertussis) manufactured by the National Public health Institute (KTL) of Finland. However, in this retrospective comparison, the Pentacoq vaccine was clearly more reactogenic than the DTPw vaccine made by KTL. Fever >38 C on the day of vaccination was seen in 31%, 28% and 24% after the first, second and third dose, respectively, as compared with 10%, 9% and 9%, respectively, after the corresponding doses of the Finnish DTPw vaccine, given at 2, 3 and 5 mo of age (4). It is probable that the difference is due to the greater reactogenicity of the pertussis component in the Pentacoq vaccine and explained by variability in the potency standardization of the whole-cell pertussis vaccines (17). The reactogenicity of RRV-TV in this study was similar to that in our previous experience. Febrile reactions >38 C and >39 C on d 4 were seen in this study in 25% and 3% of the vaccinees, respectively, after the first dose, compared with 30% and 2% in our earlier study (3, 4). After the second vaccination there was no increased febrile response rate on d 4 in the RRV-TV vaccine recipients, but after the third dose some children again showed a febrile response on that day. These could have been children who did not respond to the first but did respond to the third dose of RRV-TV. The reactogenicity of RRV-TV rotavirus vaccine in terms of febrile responses was similar to that of wholecell pertussis vaccine after the first, but not the second and third doses of vaccine. However, the reactogenicity of whole-cell pertussis vaccine was greater in that decreased activity or general irritability of the child was commonly associated with the febrile response induced by the whole-cell pertussis but not rotavirus vaccine. In judging the clinical significance of these febrile reactions one cannot ignore the observation that the mother of one child decided to discontinue participation because of apparently Pentacoq vaccine-associated reactions, and two more were withdrawn by the investigator. No children were withdrawn because of RRV-TV vaccine-associated reactions, but as many as eight were seen by a physician for fever probably or possibly related to RRV-TV vaccine. A key to the lower reactogenicity of the RRV-TV vaccine might be the administration of the first dose at an earlier age, in the presence of a higher level of transplacentally acquired anti-rotavirus antibodies. Studies in Israel have shown that neonatal immunization with RRV-TV vaccine is safe (18). Our own experience in Finland likewise suggests that in neonates such reactions may not occur (Vesikari et al., unpublished). In the future, less reactogenic reassortant rotavirus vaccines based on bovine rotaviruses may be introduced. Bovine rotavirus vaccines have consistently been less reactogenic than rhesus vaccines in Finnish infants (19). References 1. Rennels MB, Glass RI, Dennehy PH, Bernstein DI, Pichichero ME, Zito ET, et al. Safety and efficacy of high-dose rhesus human reassortant rotavirus vaccines report of the National Multicenter Trial. Pediatrics 1996; 97: Pérez-Schael I, Guntinas MJ, Pérez M, Pagone V, Rojas AM, González R, et al. Efficacy of the rhesus rotavirus-based quadrivalent vaccine in infants and young children in Venezuela. N Engl J Med 1997; 337: Joensuu J, Koskenniemi E, Pang X-L, Vesikari T. Randomised placebo-controlled trial of rhesus human reassortant rotavirus vaccine for prevention of severe rotavirus gastroenteritis. Lancet 1997; 350: Joensuu J, Koskenniemi E, Vesikari T. Symptoms associated with rhesus human reassortant rotavirus vaccine in infants. Pediatr Infect Dis J 1998; 17: Dennehy PH, Rodgers GC Jr, Ward RL, Markwick AJ, Mack M, Zito ET, For the US Rhesus Rotavirus vaccine study group. Comparative evaluation of reactogenicity and immunogenicity of two dosages of oral tetravalent rhesus rotavirus vaccine. Pediatr Infect Dis J 1996; 15: Helwig H, Mertsola J, Harvey D, Nicolopoulos D, Schaack J-C,

8 520 T Vesikari et al. ACTA PÆDIATR 88 (1999) Sedlak W, on behalf of the Confederation of European Specialists in Paediatrics (CESP). Childhood immunisation in the European Union. Eur J Pediatr 1998; 157: Ward RL, Bernstein DI, Shukla R, Young EC, Sherwood JR, McNeal MM, et al. Effects of antirotavirus antibody on protection of adults administered a human rotavirus. J Infect Dis 1989; 159: Knowlton DR, Spector DM, Ward RL. Development of an improved method for measuring neutralizing antibody to rotavirus. J Virol Methods 1991; 33: Ward RL, Kapikian AZ, Goldberg KM, Knowlton DR, Watson MW, Rappaport R. Serum rotavirus neutralizing antibody titres compared by plaque reduction and enzyme-linked immunosorbent assay-based neutralization assays. J Clin Microbiol 1996; 34: Kristiansen M, Aggerbeck H, Heron I. Improved ELISA for determination of anti-diphtheria and/or anti-tetanus antitoxin antibodies in sera. APMIS 1997; 105: Manclark CR, Meade BD, Burstyn D. Serological response to Bordetella pertussis. In Rose NR, Friman H, editors. Manual of clinical laboratory immunology, 3rd ed. Washington DC: ASM, Phipps DC, West J, Eby R, Koster M, Madore DV, Quataert SA. An ELISA employing a Haemophilus influenzae type b oligosaccharide human serum albumin conjugate correlates with the radioantigen binding assay. J Immunol Methods 1990; 135: Kurikka S, Käyhty H, Saarinen L, Rönnberg P-R, Eskola J, Mäkelä PH. Immunologic priming by one dose of Haemophilus influenzae type b conjugate vaccine in infancy. J Infect Dis 1995; 172: Käyhty H, Mäkelä PH. The protective level of serum antibodies to the capsular polysaccharide of Haemophilus influenzae type b. (J Infect Dis 1984; 147: 1100). Reply to the letter of Dr Porter Anderson. J Infect Dis 1984; 149: Albrecht P, van Steenis G, van Wezel A. Standardization of poliovirus neutralizing antibody test. Rev Infect Dis 1984; 6: Markwick AJ, Rennels MB, Zito ET, Wade MS, Mack ME, for the US Rhesus Rotavirus Vaccine Study GroupRev Infect Dis J 1998; 17: Robinson A, Funnell SGP. Potency testing of acellular pertussis vaccines. Vaccine 1992; 10: Dagan R, Kassis I, Sarov B. Safety and immunogenicity of oral tetravalent human rhesus reassortant rotavirus vaccine in neonates. Pediatr Infect Dis J 1992; 11: Vesikari T, Joensuu J. Review of rotavirus vaccine trials in Finland. J Infect Dis 1996; 174 Suppl 1: S81 7 Received Oct. 5, Accepted in revised form Jan. 19, 1999