Immunogenicity and safety of rhesus-human rotavirus reassortant vaccines with serotype 1 or 2 VP7 specificity Timo Vesikari *++, Tiina Varis*, Kim Green t, Jorge Flores t and Albert Z. Kapikian t Rhesus-human rotavirus reassortants incorporating the gene expressing the VP7 surface protein o f human rotavirus serotypes 1 or 2, and the remaining ten genes from rhesus rotavirus (RRV) were evaluated as candidate oral vaccines in 2-4-month-old infants. A single dose of the serotype 1 reassortant vaccine which had a titre of 104 plaque-forming units (p.f.u.) induced a fourfold or greater antibody response in 81% o f the recipients by a combination o f ELISA and neutralization assays; 51% o f the vaccinees developed a neutralizing antibody response to the vaccine strain. A single dose o f the serotype 2 vaccine (104 p.f.u.) induced a seroresponse in all vaccinees by the combination of assays whereas 67% developed neutralizin 9 antibodies to the vaccine strain. A combination o f these two vaccines (0.5 x 104 p.f.u, o f each) induced an overall seroresponse in 95% o f the recipients but only 48% and24% response in neutralizing antibodies to serotypes I and2, respectively. A trivalent combination which included the two reassortants and R R V (0.33 x 104p.f.u. o f each strain) induced an overall response in 82% o f the vaccinees, but only 30%, 20% and 65% developed a neutralizing antibody response to serotype 1, serotype 2, and RRV, respectively. Febrile reactions on days 2-5 after vaccination were seen in 23-45% o f the infants receivin 9 the various vaccines and combinations and in 5% o f the placebo group. It is concluded that rhesus-human reassortant rotaviruses may be combined with each other and with R R V as a polyvalent vaccine, but the VP7-specific neutralizing antibody responses are likely to be lower after combined vaccination than following vaccination with a single reassortant rotavirus. Keywords:Diarrhoea; rotavirus; vaccine; genetic reassortants
INTRODUCTION Rhesus rotavirus strain MMU-18006 was recently introduced as a live oral candidate vaccine (RRV-1) against human rotavirus diarrhoea L2. The RRV-1 vaccine, titre 104 plaque-forming units (p.f.u.), was found to be 64% protective against all and 100% protective against severe rotavirus diarrhoea associated predominantly with serotype 3 rotavirus, the same serotype as the vaccine strain, in a trial in Venezuela 3. In other trials in the USA 4-5, Finland 6 and Sweden 7, vaccine protection was, however, lower or even absent; in these studies the prevalent rotavirus was either serotype 1 or strains other than the vaccine serotype. A larger dose of the vaccine (105p.f.u.) was moderately protective for serotype 1 rotavirus diarrhoea in older infants (5-12 months) in *Department of Clinical Sciences, University of Tampere, SF33520 Tampere, Finland. tLaboratory of Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, USA. tTo whom correspondence should be addressed. (Received 29 May 1990; accepted 11 October 1990) 0264--410)(/91/050334-06 © 1991 Butterworth-HeinemannLtd 334
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Sweden a but a high rate of postvaccine febrile reactions was observed; this dose was not evaluated further in this age group. Based on experience from efficacy trials of the RRV-1 vaccine and from studies on natural rotavirus infection l°-13, it is believed that VP7 serotype-specific antibodies are important for protection against rotavirus diarrhoea. In order to protect against rotavirus diarrhoea caused by rotavirus serotypes other than 3, reassortants between the rhesus rotavirus and human rotavirus serotypes 1, 2 and 4 have been developed as potential vaccine candidates s' ~~' 12. These reassortants incorporate RNA genome segment 8 or 9, encoding the VP7 surface protein of human rotavirus into the rhesus rotavirus genome. The reassortants therefore should induce a VP7-specific neutralizing antibody response against the respective human rotaviruses. In this study the immunogenicity and safety of the rhesus-human rotavirus reassortants corresponding to serotypes 1 and 2 were evaluated in 2-4-month-old Finnish infants. The candidate vaccine strains were tested individually, in combination with each other and in a
Reassortant rotavirus vaccines: T. Vesikari et al.
trivalent combination with the rhesus rotavirus. More recently, tetravalent vaccine combinations of serotypes 1, 2 and 4 reassortants and rhesus rotavirus have been developed and evaluated 14. METHODS
Subjects The vaccinees were 2-4-month-old healthy infants, whose parents had given written consent for participation in the study. The parents were given written and oral information on the vaccine and the study, after which one parent of every child signed written consent. The study protocol and consent form had been reviewed and approved by the Ethical Committee of the Tampere University Central Hospital and by the Human Research Sub-panel of the National Institute of Allergy and Infectious Disease (NIAID), of the National Institutes of Health (NIH), USA. The infants had a physical examination prior to vaccination; only those found healthy were enrolled in the study. A mild rhinitis (runny nose without fever) was not regarded as an exclusion criterion; such children had a careful ear examination to rule out otitis media. All participants received orally 40 ml soy milk buffered with 400 mg sodium bicarbonate; 30 ml was given before and 10ml immediately after oral administration of vaccine or placebo. Breast-feeding was withheld for 1 h before and I h after 'vaccination', Vaccine or placebo (lml) was placed in the back of the mouth with a tuberculin syringe without a needle. After vaccination, clinical reactions were homemonitored for 7 days. The parents were requested to take a rectal temperature once a day, or more often if the child was found to have fever. They also observed the number of bowel motions and graded the consistency of each stool passed by the child as solid, loose or watery. These and other observations (vomiting, respiratory symptoms) were recorded on a check list that was returned to the investigators at the end of the observation period. A nurse and a paediatrician were available for consultation during the entire study. Venous blood specimens were collected before and 1 month after 'vaccination'. The sera were separated and stored at - 2 0 ° C until shipment to NIH.
The vaccines The serotype 1 reassortant was prepared from the D strain (serotype 1 VP7) of human rotavirus and the MMU-18006 strain of rhesus rotavirus (RRV) whereas the serotype 2 reassortant was prepared from the DS-1 strain (serotype 2 VP7) of human rotavirus and the same strain of RRV. Both reassortants were made at the Laboratory of Infectious Diseases, NIAID, NIH, as previously described 12. The experimental vaccine lots for human use were made by Flow Laboratories, Inc. (McLean, VA, USA), which also performed the necessary safety tests. The bulk RRV vaccine and the D × RRV reassortant vaccine had a titre of 106p.f.u. ml-1; the DS-1 x RRV reassortant vaccine had a titre of 104p.f.u. m1-1. The bulk viruses were shipped on dry ice to Tampere and stored at - 7 0 ° C . In the first phase of the study, individual reassortants D x RRV or DS-1 x RRV were evaluated with a placebo. The coded vaccine preparations for the study were made
in Tampere by a person not otherwise involved in the study. The bulk vaccines were thawed and the D x RRV vaccine was diluted 1:100 in minimum essential medium (MEM) to yield 104 p.f.u, m l - 1, whereas the DS-I × RRV vaccine was not diluted since its titre was only 104 ml- 1. both vaccines were aliquoted into 1 ml capped vials. M E M was used for placebo, and 1 ml aliquots were likewise prepared in vials. The vaccine and placebo vials were number coded according to a randomization list, and stored at - 7 0 ° C until use. Shortly before vaccinations on each day the number of vials required were thawed and kept on crushed ice until administered. The combined vaccine evaluations were carried out without a placebo control. For the bivalent combination, 0.5ml 1:100 dilution of D x RRV bulk (prepared as above) and 0.5 ml undiluted DS-1 × RRV were administered successively. For the trivalent vaccination, 0.33 ml each of the 1:100 dilution of D x RRV and RRV, and 0.33 ml undiluted DS-1 x RRV, were administered successively. Thus, for each evaluation (monovalent, bivalent or trivalent) the total amount of vaccine given was 104p.f.u. in 1 ml volume.
Laboratory procedures Rotavirus ELISA IgA antibody determinations 15 and plaque reduction neutralization (PRN) tests 16'17 were carried out as described previously. RRV was used as antigen in the ELISA test. The PRN tests of paired sera obtained from the monovalent vaccine study were performed against the reassortant vaccine strains D x RRV and DS-1 x RRV. A subset of these sera and all available paired sera from the bivalent and trivalent vaccine study were tested against the human rotavirus strain Wa (serotype 1 VP7) and P (serotype 3 VP7) and against the RRV (serotype 3 VP7). A fourfold or greater rise was regarded as significant in both ELISA and P R N tests.
RESULTS In the initial phase of the study, which compared monovalent serotype 1 and 2 reassortant vaccines and placebo, a total of 89 infants were enrolled. Of these, 85 completed the study, 37 infants received the serotype 1 reassortant vaccine, 24 the serotype 2 reassortant vaccine, and 24 the placebo. One child in the placebo group developed diarrhoea and mild fever 2 days after vaccination and rotavirus was demonstrated in the stools with an ELISA antigen detection kit (Dakopatts A.S., Glostrup, Denmark). The same child also had a PRN antibody response to serotype 1 reassortant rotavirus. A child in the serotype 1 reassortant vaccine group developed a fever of 39.1 °C and diarrhoea on day 3 after vaccination, and rotavirus was demonstrated in the stools by ELISA. This was regarded as a natural rotavirus infection since it has been shown previously that the level of RRV excretion after vaccination is too low to be detected by this test ~s. These two children are not included in the calculations of serological responses or side reactions. No other clinically apparent cases of natural rotavirus diarrhoea were seen in the study group during the follow-up period. In addition, one child who received the DS-1 × RRV vaccine developed an RS-virus infection in the week after vaccination and follow-up was discontinued; this child is not included in the results.
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Reassortant rotavirus vaccines: T. Vesikari et al. Table 1 Serological responses to vaccination with monovalent rhesus-human rotavirus reassortants as determined by ELISA and plaque reduction neutralization (PRN) assay No. (%) with positive response"/no, tested by indicated assay PRN Vaccine
ELISA IgA
D x RRV
DS-1 x RRV
Any test
D x RRV (serotype 1) DS-1 x RRV (serotype 2) Placebo
25/37 (70%) 21/24 (88%) 1/23 (4%)
19/37 (51%) ~ 9/23 (39%) c 3/23 (13%)
14/36 (39%) 16/24 (67%) 0/23 (0%)
30/37 (81%) 24/24 (100%) 4/23 (17%)
aFourfold or greater antibody rise; ~p=0.003, Fisher's exact test; °p=0.09, Fisher's exact test; all other comparisons between vaccines and placebo p38°C, ~1, and ~>39°C, PPJ)in the recipients of combined vaccination with (a), serotype 1 and 2 rhesus-human reassortant rotavirus vaccines and (b), serotype 1 and 2 reassortant plus rhesus rotavirus vaccine
20
unusual irritability and/or abdominal cramps on day 3 after vaccination. 10
0
DISCUSSION
1
2
3
4
5
6
7
Time a f t e r vaccination ( d a y s l Flgure I Febrile reactions (rectal temperature ~>38°C, m, and ~>39°C, 1721) in the recipients of single serotype rhesus-human reassortant rotavirus vaccines (a), D x RRV (serotype I ) ( b ) , DS-I x RRV (serotype 2) and (c), placebo
The present study indicated that both the immunogenicity and reactogenicity of rhesus-human reassortant rotaviruses with VP7 specificity of serotype 1 or 2 were remarkably similar to the corresponding properties of the parent virus, rhesus rotavirus strain MMU-18006. In previous studies, 105p.f.u. of rhesus rotavirus vaccine induced a seroresponse in 88-100% of the recipients 15'17'19 and .104p.f.u. induced a response in 76 100°,/o20-22 . These figures are comparable to the
Vaccine, Vol. 9, May 1991 337
R e a s s o r t a n t r o t a v i r u s v a c c i n e s : T. V e s i k a r i et al.
seroresponse rates of 81% and 100% for serotype 1 and 2 reassortants, respectively, observed in this study. The rate of transient febrile reactions to rhesus rotavirus, typically seen on days 3 and 4 after vaccination 23, has varied greatly in different studies depending on the age and pre-existing rotavirus antibody status of vaccinees. However, in a comparable population of children in Finland, the rhesus rotavirus vaccine was associated with a 26% rate of febrile reactions 6, which is similar to that found in the present study with serotype 1 and 2 reassortant rotavirus vaccines. Altogether, it may be concluded that the reactogenicity of the rhesus-human reassortant rotavirus vaccines does not appear to be diminished or increased when compared with their parent rhesus rotavirus strain. While the overall take rate of the rhesus-human reassortant candidate rotavirus vaccines was satisfactory, the VPV-specific neutralizing antibody responses were low. About one half of the vaccinees who developed an antibody rise failed to demonstrate a neutralizing antibody response to the homologous VP7 antigen. This finding cannot be compared with experience of rhesus rotavirus vaccine, since studies of the latter have not measured VPV-specific antibody responses separately but rather the total neutralizing antibody, which includes responses directed to VP4, VP7 or both. In this study, measurement of PRN antibody responses to both RRV and the P strain suggested that the response to the VP4 component of RRV was more efficient than that to its VPV. Recent studies using an epitope-blocking assay have also suggested that the RRV VP4 is a more efficient immunogen 24. The vaccine combinations used in the present study were made by mixing one half-dose of each component (D x RRV and DS-I x RRV) for a bivalent serotype 1 and 2 combination, and a one-third dose of each component (D × RRV, DS-1 x RRV and RRV) to make a trivalent serotype 1 + 2 + 3 combination. Thus, the total amount of virus was the same as in the single serotype vaccines, i.e. 104p.f.u. Not surprisingly, the overall seroconversion rates and febrile reaction rates were comparable to vaccination with single serotype reassortant vaccines. The VPV-specific neutralizing antibody responses were generally lower after vaccination with combination vaccines than with single serotype vaccines. The seroconversion rates were lower after the trivalent vaccine, although the difference between the bivalent and trivalent vaccine was not significant (Table 3). The small reduction in dose (one half or one third compared to monovalent vaccine) is unlikely to provide a full explanation. Rather, it is probable that the vaccine components interfere with and compromise the take of one another. This has also been shown in recent studies of a quadrivalent combination vaccine (rhesus-human reassortants for serotypes 1, 2 and 4 plus RRV) in Venezuela ~4. In these studies the VPV-specific neutralization antibody responses were dependent on the dose, being substantially lower for a dose of 0.25 x 104p.f.u. of each component than a dose of 1.0 × 104 p.f.u, of each component. However, even with the latter 'full' dose, the VPV-specific responses to all four serotypes were lower than those observed in a previous study in which a dose of 104p.f.u. of serotype 1 or 2 reassortants was evaluated xa. Therefore, some interference between the vaccine components was apparent. These immunogenicity and safety studies may be
338
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regarded as part of the effort to develop an optimal composition for a live oral rotavirus vaccine capable of inducing VPV-specific neutralizing antibodies for the most important human rotavirus serotypes. The experience accumulated suggests that combining several viruses for a multivalent vaccine leads to decreased serological responses to individual antigens. This problem can be partly overcome by increasing the dose of each component in the combination, but this may result in increased reactogenicity. The present level of febrile reactions (20-30%) is probably the upper limit for acceptance in this study population. However, much lower reaction rates have been observed in studies of the rhesus rotavirus or human-RRV reassortants in Venezuela ~4'~s'22, Peru (C. Lanata, personal communication), and Israel (R. Dagan, personal communication). A probable explanation for this difference is that infants in those countries usually have higher levels of maternallyacquired rotavirus antibody than infants in Finland. In such populations the titre of a rhesus-human reassortant rotavirus vaccine could perhaps be substantially increased from the present level to reach higher seroconversion rates without a concomitant increase in reactogenicity. Alternatively, multiple doses of a vaccine with a titre of 104 p.f.u., as used in the present study, may induce a higher rate of seroresponse. Studies are currently under way to investigate the immunogenicity, safety and efficacy of three doses of a tetravalent rhesus-human rotavirus reassortant vaccine composed of 1.0 × 104 p.f.u, of each component.
REFERENCES Kapikian, A.Z., Midthun, K., Hoshino, Y. et al. Rhesus rotavirus: a candidate vaccine for prevention of human rotavirus disease. In: Vaccines 85. Molecular and Chemical Basis of Resistance to Parasitic, Bacterial and Viral Diseases (Eds Lerner, R.A., Chanock,
R.M. and Brown, F.) Cold Spring Harbor Laboratory, New York, 1985, pp 357-367 Kapikian, A.Z., Flores, J., Hoshino, Y. et al. Rotavirus: the major etiologic agent of severe infantile diarrhoea may be controllable by a 'Jennerian' approach to vaccination. J. Infect. Dis. 1986,153, 815 Flores, J., Perez-Schael, I., Gonzalez, M. et al. Protection against severe rotavirus diarrhoea by rhesus rotavirus vaccine in Venezuelan infants. Lancet 1987, i, 982 Christy, C., Madore, H.P., Pichichero, M.E. et al. Field trial of rhesus rotavirus: rotavirus vaccine in infants. Pediatr. Infect. Dis. J. 1988, 7, 645 Rennels, M., Losonsky, G.A., Young, A.E. et al. An efficacy trial of the rhesus rotavirus vaccine in Maryland. Am. J. Dis. Child. 1990, 144, 601 Vesikari, T., Rautanen, T., Varis, T. et al. Clinical trial of rhesus rotavirus candidate vaccine (strain MMU 18006) in children vaccinated between 2 and 5 months of age. Am. J. Dis. Child. 1990, 144, 285 Gothefors, L., Wadell, G., Juto, P., Taniguchi, K., Kapikian, A.Z. and Glass, R.I. Prolonged efficacy of rhesus rotavirus vaccine in Swedish children. J. Infect. Dis. 1989, 159, 753 Edelman, R. Perspective on the development and deployment of rotavirus vaccines. Pediatr. Infect. Dis. J. 1987, 6, 704 Kapikian, A.Z., Wyatt, R.G., Levine, M.M et al. Oral administration of human rotavirus to volunteers: induction of illness and correlates of resistance. J. Infect. Dis. 1983, 147, 95 10 Chiba, S., Yokoyama, T., Nakata, S. et al. Protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies. Lancet 1986, ii, 417 Midthun, K., Greenberg, H.B., Hoshino, Y., Kapikian, A.Z., Wyatt, R.G. and Chanock, R.M. Reassortant rotaviruses as potential vaccine candidates. J. Virol. 1985, 53, 949 12 Kapikian, A.Z., FIores, J., Midthun, K. et al. Development of a rotavirus vaccine by a 'Jennerian' and modified 'Jennerian' approach. In: Vaccines 88 (Eds Ginsburg, H., Brown, F., Lerner,
Reassortant
13
14
15
16
17
18
R.A. and Chanock, R.M.) Cold Spring Harbor Laboratory, New York, 1988, pp 151-158 Hoshino, Y., Saif, L.J., Soreno, M.M. et al. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 1988, 62, 744 Perez-Schael, I., Blanco, M., Vilar, M. et al. Clinical studies of a quadrivalent rotavirus vaccine in Venezuelan infants. J. Clin. Microbiol. 1990, 28, 553 Losonsky, G.A., Rennels, M.B., Lim, Y. et al. Systemic and mucosal immune responses to rhesus rotavirus vaccine MMU 18006. Pediatr. Infect. Dis. J. 1988, 7, 388 Wyatt, R.G., Greenberg, H.B., James, W.D. et al. Definition of human rotavirus serotypes by plaque reduction assay. Infect. Immun. 1982, 37, 110 Hoshino, Y., Wyatt, R.G., Greenberg, H.B. eta/. Serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization. J. Infect. Dis. 1984, 149, 694 Flores, T., Perez-Schael, I., Blanco, M. eta/. Reactions to antigenicity of two human-rhesus rotavirus reassortant vaccine candidates of
r o t a v i r u s v a c c i n e s : T~ V e s i k a r i et al.
serotypes 1 to 2 in Venezuelan infants. J. Clin. Microbiol. 1989, 21, 512 19 Losonsky, G.A., Rennels, M.B., Kapikian, A.Z. et al. Safety, infectivity, transmissability and immunogenicity of rhesus rotavirus (MMU 18006) in infants. Pediatr. Infect. Dis. 1986, 5, 25 20 Anderson, E.L., Belshe, R.B., Bartram, J. et al. Evaluation of rhesus rotavirus vaccine (MMU 18006) in infants and young children. J. Infect. Dis. 1986, 153, 823 21 Wright, P.F., Tajima, T., Thompson, J. eta/. Candidate rotavirus vaccine (rhesus rotavirus strain) in children: an evaluation. Pediatrics 1987, 80, 473 22 Perez-Schael, I., Gonzalez, M., Daoud, N. eta/. Reactogenicity and antigenicity of the rhesus rotavirus vaccine in Venezuelan children. J. InfeCt. Dis. 1987, 155, 334 23 Vesikari, T., Kapikian, A.Z., Delem, A. et a/. A comparative trial of rhesus monkey (RRV-1) and bovine (RIT 4237) rotavirus vaccines in young children. J. Infect. Dis. 1986, 153, 832 24 Green, K.Y., Taniguchi, K., Mackow, E.R. and Kapikian, A.Z. Homotypic and heterotypic epitope-specific antibody responses in adult and infant rotavirus vaccinees: Implications for vaccine development. J. Infect. Dis. (in press)
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INTRODUCTION Rhesus rotavirus strain MMU-18006 was recently introduced as a live oral candidate vaccine (RRV-1) against human rotavirus diarrhoea L2. The RRV-1 vaccine, titre 104 plaque-forming units (p.f.u.), was found to be 64% protective against all and 100% protective against severe rotavirus diarrhoea associated predominantly with serotype 3 rotavirus, the same serotype as the vaccine strain, in a trial in Venezuela 3. In other trials in the USA 4-5, Finland 6 and Sweden 7, vaccine protection was, however, lower or even absent; in these studies the prevalent rotavirus was either serotype 1 or strains other than the vaccine serotype. A larger dose of the vaccine (105p.f.u.) was moderately protective for serotype 1 rotavirus diarrhoea in older infants (5-12 months) in *Department of Clinical Sciences, University of Tampere, SF33520 Tampere, Finland. tLaboratory of Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, USA. tTo whom correspondence should be addressed. (Received 29 May 1990; accepted 11 October 1990) 0264--410)(/91/050334-06 © 1991 Butterworth-HeinemannLtd 334
Vaccine, Vol. 9, May 1991
Sweden a but a high rate of postvaccine febrile reactions was observed; this dose was not evaluated further in this age group. Based on experience from efficacy trials of the RRV-1 vaccine and from studies on natural rotavirus infection l°-13, it is believed that VP7 serotype-specific antibodies are important for protection against rotavirus diarrhoea. In order to protect against rotavirus diarrhoea caused by rotavirus serotypes other than 3, reassortants between the rhesus rotavirus and human rotavirus serotypes 1, 2 and 4 have been developed as potential vaccine candidates s' ~~' 12. These reassortants incorporate RNA genome segment 8 or 9, encoding the VP7 surface protein of human rotavirus into the rhesus rotavirus genome. The reassortants therefore should induce a VP7-specific neutralizing antibody response against the respective human rotaviruses. In this study the immunogenicity and safety of the rhesus-human rotavirus reassortants corresponding to serotypes 1 and 2 were evaluated in 2-4-month-old Finnish infants. The candidate vaccine strains were tested individually, in combination with each other and in a
Reassortant rotavirus vaccines: T. Vesikari et al.
trivalent combination with the rhesus rotavirus. More recently, tetravalent vaccine combinations of serotypes 1, 2 and 4 reassortants and rhesus rotavirus have been developed and evaluated 14. METHODS
Subjects The vaccinees were 2-4-month-old healthy infants, whose parents had given written consent for participation in the study. The parents were given written and oral information on the vaccine and the study, after which one parent of every child signed written consent. The study protocol and consent form had been reviewed and approved by the Ethical Committee of the Tampere University Central Hospital and by the Human Research Sub-panel of the National Institute of Allergy and Infectious Disease (NIAID), of the National Institutes of Health (NIH), USA. The infants had a physical examination prior to vaccination; only those found healthy were enrolled in the study. A mild rhinitis (runny nose without fever) was not regarded as an exclusion criterion; such children had a careful ear examination to rule out otitis media. All participants received orally 40 ml soy milk buffered with 400 mg sodium bicarbonate; 30 ml was given before and 10ml immediately after oral administration of vaccine or placebo. Breast-feeding was withheld for 1 h before and I h after 'vaccination', Vaccine or placebo (lml) was placed in the back of the mouth with a tuberculin syringe without a needle. After vaccination, clinical reactions were homemonitored for 7 days. The parents were requested to take a rectal temperature once a day, or more often if the child was found to have fever. They also observed the number of bowel motions and graded the consistency of each stool passed by the child as solid, loose or watery. These and other observations (vomiting, respiratory symptoms) were recorded on a check list that was returned to the investigators at the end of the observation period. A nurse and a paediatrician were available for consultation during the entire study. Venous blood specimens were collected before and 1 month after 'vaccination'. The sera were separated and stored at - 2 0 ° C until shipment to NIH.
The vaccines The serotype 1 reassortant was prepared from the D strain (serotype 1 VP7) of human rotavirus and the MMU-18006 strain of rhesus rotavirus (RRV) whereas the serotype 2 reassortant was prepared from the DS-1 strain (serotype 2 VP7) of human rotavirus and the same strain of RRV. Both reassortants were made at the Laboratory of Infectious Diseases, NIAID, NIH, as previously described 12. The experimental vaccine lots for human use were made by Flow Laboratories, Inc. (McLean, VA, USA), which also performed the necessary safety tests. The bulk RRV vaccine and the D × RRV reassortant vaccine had a titre of 106p.f.u. ml-1; the DS-1 x RRV reassortant vaccine had a titre of 104p.f.u. m1-1. The bulk viruses were shipped on dry ice to Tampere and stored at - 7 0 ° C . In the first phase of the study, individual reassortants D x RRV or DS-1 x RRV were evaluated with a placebo. The coded vaccine preparations for the study were made
in Tampere by a person not otherwise involved in the study. The bulk vaccines were thawed and the D x RRV vaccine was diluted 1:100 in minimum essential medium (MEM) to yield 104 p.f.u, m l - 1, whereas the DS-I × RRV vaccine was not diluted since its titre was only 104 ml- 1. both vaccines were aliquoted into 1 ml capped vials. M E M was used for placebo, and 1 ml aliquots were likewise prepared in vials. The vaccine and placebo vials were number coded according to a randomization list, and stored at - 7 0 ° C until use. Shortly before vaccinations on each day the number of vials required were thawed and kept on crushed ice until administered. The combined vaccine evaluations were carried out without a placebo control. For the bivalent combination, 0.5ml 1:100 dilution of D x RRV bulk (prepared as above) and 0.5 ml undiluted DS-1 × RRV were administered successively. For the trivalent vaccination, 0.33 ml each of the 1:100 dilution of D x RRV and RRV, and 0.33 ml undiluted DS-1 x RRV, were administered successively. Thus, for each evaluation (monovalent, bivalent or trivalent) the total amount of vaccine given was 104p.f.u. in 1 ml volume.
Laboratory procedures Rotavirus ELISA IgA antibody determinations 15 and plaque reduction neutralization (PRN) tests 16'17 were carried out as described previously. RRV was used as antigen in the ELISA test. The PRN tests of paired sera obtained from the monovalent vaccine study were performed against the reassortant vaccine strains D x RRV and DS-1 x RRV. A subset of these sera and all available paired sera from the bivalent and trivalent vaccine study were tested against the human rotavirus strain Wa (serotype 1 VP7) and P (serotype 3 VP7) and against the RRV (serotype 3 VP7). A fourfold or greater rise was regarded as significant in both ELISA and P R N tests.
RESULTS In the initial phase of the study, which compared monovalent serotype 1 and 2 reassortant vaccines and placebo, a total of 89 infants were enrolled. Of these, 85 completed the study, 37 infants received the serotype 1 reassortant vaccine, 24 the serotype 2 reassortant vaccine, and 24 the placebo. One child in the placebo group developed diarrhoea and mild fever 2 days after vaccination and rotavirus was demonstrated in the stools with an ELISA antigen detection kit (Dakopatts A.S., Glostrup, Denmark). The same child also had a PRN antibody response to serotype 1 reassortant rotavirus. A child in the serotype 1 reassortant vaccine group developed a fever of 39.1 °C and diarrhoea on day 3 after vaccination, and rotavirus was demonstrated in the stools by ELISA. This was regarded as a natural rotavirus infection since it has been shown previously that the level of RRV excretion after vaccination is too low to be detected by this test ~s. These two children are not included in the calculations of serological responses or side reactions. No other clinically apparent cases of natural rotavirus diarrhoea were seen in the study group during the follow-up period. In addition, one child who received the DS-1 × RRV vaccine developed an RS-virus infection in the week after vaccination and follow-up was discontinued; this child is not included in the results.
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Reassortant rotavirus vaccines: T. Vesikari et al. Table 1 Serological responses to vaccination with monovalent rhesus-human rotavirus reassortants as determined by ELISA and plaque reduction neutralization (PRN) assay No. (%) with positive response"/no, tested by indicated assay PRN Vaccine
ELISA IgA
D x RRV
DS-1 x RRV
Any test
D x RRV (serotype 1) DS-1 x RRV (serotype 2) Placebo
25/37 (70%) 21/24 (88%) 1/23 (4%)
19/37 (51%) ~ 9/23 (39%) c 3/23 (13%)
14/36 (39%) 16/24 (67%) 0/23 (0%)
30/37 (81%) 24/24 (100%) 4/23 (17%)
aFourfold or greater antibody rise; ~p=0.003, Fisher's exact test; °p=0.09, Fisher's exact test; all other comparisons between vaccines and placebo p38°C, ~1, and ~>39°C, PPJ)in the recipients of combined vaccination with (a), serotype 1 and 2 rhesus-human reassortant rotavirus vaccines and (b), serotype 1 and 2 reassortant plus rhesus rotavirus vaccine
20
unusual irritability and/or abdominal cramps on day 3 after vaccination. 10
0
DISCUSSION
1
2
3
4
5
6
7
Time a f t e r vaccination ( d a y s l Flgure I Febrile reactions (rectal temperature ~>38°C, m, and ~>39°C, 1721) in the recipients of single serotype rhesus-human reassortant rotavirus vaccines (a), D x RRV (serotype I ) ( b ) , DS-I x RRV (serotype 2) and (c), placebo
The present study indicated that both the immunogenicity and reactogenicity of rhesus-human reassortant rotaviruses with VP7 specificity of serotype 1 or 2 were remarkably similar to the corresponding properties of the parent virus, rhesus rotavirus strain MMU-18006. In previous studies, 105p.f.u. of rhesus rotavirus vaccine induced a seroresponse in 88-100% of the recipients 15'17'19 and .104p.f.u. induced a response in 76 100°,/o20-22 . These figures are comparable to the
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seroresponse rates of 81% and 100% for serotype 1 and 2 reassortants, respectively, observed in this study. The rate of transient febrile reactions to rhesus rotavirus, typically seen on days 3 and 4 after vaccination 23, has varied greatly in different studies depending on the age and pre-existing rotavirus antibody status of vaccinees. However, in a comparable population of children in Finland, the rhesus rotavirus vaccine was associated with a 26% rate of febrile reactions 6, which is similar to that found in the present study with serotype 1 and 2 reassortant rotavirus vaccines. Altogether, it may be concluded that the reactogenicity of the rhesus-human reassortant rotavirus vaccines does not appear to be diminished or increased when compared with their parent rhesus rotavirus strain. While the overall take rate of the rhesus-human reassortant candidate rotavirus vaccines was satisfactory, the VPV-specific neutralizing antibody responses were low. About one half of the vaccinees who developed an antibody rise failed to demonstrate a neutralizing antibody response to the homologous VP7 antigen. This finding cannot be compared with experience of rhesus rotavirus vaccine, since studies of the latter have not measured VPV-specific antibody responses separately but rather the total neutralizing antibody, which includes responses directed to VP4, VP7 or both. In this study, measurement of PRN antibody responses to both RRV and the P strain suggested that the response to the VP4 component of RRV was more efficient than that to its VPV. Recent studies using an epitope-blocking assay have also suggested that the RRV VP4 is a more efficient immunogen 24. The vaccine combinations used in the present study were made by mixing one half-dose of each component (D x RRV and DS-I x RRV) for a bivalent serotype 1 and 2 combination, and a one-third dose of each component (D × RRV, DS-1 x RRV and RRV) to make a trivalent serotype 1 + 2 + 3 combination. Thus, the total amount of virus was the same as in the single serotype vaccines, i.e. 104p.f.u. Not surprisingly, the overall seroconversion rates and febrile reaction rates were comparable to vaccination with single serotype reassortant vaccines. The VPV-specific neutralizing antibody responses were generally lower after vaccination with combination vaccines than with single serotype vaccines. The seroconversion rates were lower after the trivalent vaccine, although the difference between the bivalent and trivalent vaccine was not significant (Table 3). The small reduction in dose (one half or one third compared to monovalent vaccine) is unlikely to provide a full explanation. Rather, it is probable that the vaccine components interfere with and compromise the take of one another. This has also been shown in recent studies of a quadrivalent combination vaccine (rhesus-human reassortants for serotypes 1, 2 and 4 plus RRV) in Venezuela ~4. In these studies the VPV-specific neutralization antibody responses were dependent on the dose, being substantially lower for a dose of 0.25 x 104p.f.u. of each component than a dose of 1.0 × 104 p.f.u, of each component. However, even with the latter 'full' dose, the VPV-specific responses to all four serotypes were lower than those observed in a previous study in which a dose of 104p.f.u. of serotype 1 or 2 reassortants was evaluated xa. Therefore, some interference between the vaccine components was apparent. These immunogenicity and safety studies may be
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regarded as part of the effort to develop an optimal composition for a live oral rotavirus vaccine capable of inducing VPV-specific neutralizing antibodies for the most important human rotavirus serotypes. The experience accumulated suggests that combining several viruses for a multivalent vaccine leads to decreased serological responses to individual antigens. This problem can be partly overcome by increasing the dose of each component in the combination, but this may result in increased reactogenicity. The present level of febrile reactions (20-30%) is probably the upper limit for acceptance in this study population. However, much lower reaction rates have been observed in studies of the rhesus rotavirus or human-RRV reassortants in Venezuela ~4'~s'22, Peru (C. Lanata, personal communication), and Israel (R. Dagan, personal communication). A probable explanation for this difference is that infants in those countries usually have higher levels of maternallyacquired rotavirus antibody than infants in Finland. In such populations the titre of a rhesus-human reassortant rotavirus vaccine could perhaps be substantially increased from the present level to reach higher seroconversion rates without a concomitant increase in reactogenicity. Alternatively, multiple doses of a vaccine with a titre of 104 p.f.u., as used in the present study, may induce a higher rate of seroresponse. Studies are currently under way to investigate the immunogenicity, safety and efficacy of three doses of a tetravalent rhesus-human rotavirus reassortant vaccine composed of 1.0 × 104 p.f.u, of each component.
REFERENCES Kapikian, A.Z., Midthun, K., Hoshino, Y. et al. Rhesus rotavirus: a candidate vaccine for prevention of human rotavirus disease. In: Vaccines 85. Molecular and Chemical Basis of Resistance to Parasitic, Bacterial and Viral Diseases (Eds Lerner, R.A., Chanock,
R.M. and Brown, F.) Cold Spring Harbor Laboratory, New York, 1985, pp 357-367 Kapikian, A.Z., Flores, J., Hoshino, Y. et al. Rotavirus: the major etiologic agent of severe infantile diarrhoea may be controllable by a 'Jennerian' approach to vaccination. J. Infect. Dis. 1986,153, 815 Flores, J., Perez-Schael, I., Gonzalez, M. et al. Protection against severe rotavirus diarrhoea by rhesus rotavirus vaccine in Venezuelan infants. Lancet 1987, i, 982 Christy, C., Madore, H.P., Pichichero, M.E. et al. Field trial of rhesus rotavirus: rotavirus vaccine in infants. Pediatr. Infect. Dis. J. 1988, 7, 645 Rennels, M., Losonsky, G.A., Young, A.E. et al. An efficacy trial of the rhesus rotavirus vaccine in Maryland. Am. J. Dis. Child. 1990, 144, 601 Vesikari, T., Rautanen, T., Varis, T. et al. Clinical trial of rhesus rotavirus candidate vaccine (strain MMU 18006) in children vaccinated between 2 and 5 months of age. Am. J. Dis. Child. 1990, 144, 285 Gothefors, L., Wadell, G., Juto, P., Taniguchi, K., Kapikian, A.Z. and Glass, R.I. Prolonged efficacy of rhesus rotavirus vaccine in Swedish children. J. Infect. Dis. 1989, 159, 753 Edelman, R. Perspective on the development and deployment of rotavirus vaccines. Pediatr. Infect. Dis. J. 1987, 6, 704 Kapikian, A.Z., Wyatt, R.G., Levine, M.M et al. Oral administration of human rotavirus to volunteers: induction of illness and correlates of resistance. J. Infect. Dis. 1983, 147, 95 10 Chiba, S., Yokoyama, T., Nakata, S. et al. Protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies. Lancet 1986, ii, 417 Midthun, K., Greenberg, H.B., Hoshino, Y., Kapikian, A.Z., Wyatt, R.G. and Chanock, R.M. Reassortant rotaviruses as potential vaccine candidates. J. Virol. 1985, 53, 949 12 Kapikian, A.Z., FIores, J., Midthun, K. et al. Development of a rotavirus vaccine by a 'Jennerian' and modified 'Jennerian' approach. In: Vaccines 88 (Eds Ginsburg, H., Brown, F., Lerner,
Reassortant
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14
15
16
17
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R.A. and Chanock, R.M.) Cold Spring Harbor Laboratory, New York, 1988, pp 151-158 Hoshino, Y., Saif, L.J., Soreno, M.M. et al. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 1988, 62, 744 Perez-Schael, I., Blanco, M., Vilar, M. et al. Clinical studies of a quadrivalent rotavirus vaccine in Venezuelan infants. J. Clin. Microbiol. 1990, 28, 553 Losonsky, G.A., Rennels, M.B., Lim, Y. et al. Systemic and mucosal immune responses to rhesus rotavirus vaccine MMU 18006. Pediatr. Infect. Dis. J. 1988, 7, 388 Wyatt, R.G., Greenberg, H.B., James, W.D. et al. Definition of human rotavirus serotypes by plaque reduction assay. Infect. Immun. 1982, 37, 110 Hoshino, Y., Wyatt, R.G., Greenberg, H.B. eta/. Serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization. J. Infect. Dis. 1984, 149, 694 Flores, T., Perez-Schael, I., Blanco, M. eta/. Reactions to antigenicity of two human-rhesus rotavirus reassortant vaccine candidates of
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serotypes 1 to 2 in Venezuelan infants. J. Clin. Microbiol. 1989, 21, 512 19 Losonsky, G.A., Rennels, M.B., Kapikian, A.Z. et al. Safety, infectivity, transmissability and immunogenicity of rhesus rotavirus (MMU 18006) in infants. Pediatr. Infect. Dis. 1986, 5, 25 20 Anderson, E.L., Belshe, R.B., Bartram, J. et al. Evaluation of rhesus rotavirus vaccine (MMU 18006) in infants and young children. J. Infect. Dis. 1986, 153, 823 21 Wright, P.F., Tajima, T., Thompson, J. eta/. Candidate rotavirus vaccine (rhesus rotavirus strain) in children: an evaluation. Pediatrics 1987, 80, 473 22 Perez-Schael, I., Gonzalez, M., Daoud, N. eta/. Reactogenicity and antigenicity of the rhesus rotavirus vaccine in Venezuelan children. J. InfeCt. Dis. 1987, 155, 334 23 Vesikari, T., Kapikian, A.Z., Delem, A. et a/. A comparative trial of rhesus monkey (RRV-1) and bovine (RIT 4237) rotavirus vaccines in young children. J. Infect. Dis. 1986, 153, 832 24 Green, K.Y., Taniguchi, K., Mackow, E.R. and Kapikian, A.Z. Homotypic and heterotypic epitope-specific antibody responses in adult and infant rotavirus vaccinees: Implications for vaccine development. J. Infect. Dis. (in press)
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