Evaluation of a reassortant rhesus rotavirus vaccine in young children Takeshi Tajima*, Juliette Thompson*, Peter F. Wright*, Yasuo Kondo* Sharon J. Toilefson*, James King* and Albert Z. Kapikiant A candidate live attenuated rotavirus vaccine representative o f serotype 1 was administered orally to 26 children, 14 o f whom were undergoing primary exposure to rotavirus. The vaccine was derived by reassortment between rhesus rotavirus strain, M M U 18006, and the human serotype 1 strain Wa. The resultant virus has the gene coding for the major surface glycoprotein VP-7 from the human strain and all other genes from the attenuated rhesus parent which is a serotype 3 strain. Prior natural exposure to rotavirus determined the infectivity and immunogenicity o f the vaccine. Only two o f 12 seropositive children had evidence o f reinfection while all 14 seronegative children were infected. Mild febrile illness was seen in vaccinees, however there was no evidence o f gastrointestinal disease. As determined by neutralization o f the human strains, the resultant serum antibody was entirely strain specific. However, heterotypic neutralization was seen when the rhesus strains were used, suggesting that neutralizing antibody can be directed to shared components o f the donor and reassortant strain presumably VP-4, the other major surface protein. Keywords: Rhesus rotavirus vaccine; rotavirus; neutralizing antibody
Introduction Infection with rotavirus is the leading cause of severe diarrhoea and resultant dehydration in both developing and industrialized countries 1'2. For this reason rotavirus has become an important target for vaccine development 3. Prevention of disease has become potentially more complex with recognition that there are at least four epidemiologically important distinct serotypes of human rotavirus 4. Serotypic specificity as determined by neutralization with hyperimmune serum is conferred by VP-7. However, additional neutralizing epitopes are contained within VP-4, another outer capsid protein formerly designated as VP-35. Rhesus rotavirus, strain MMU18006, is a promising vaccine candidate for the prevention of rotaviral disease 6'v. However, experience to date with rhesus rotavirus, which contains a VP-7 similar to human serotype 3, has suggested inconsistent protection against subsequent wild type challenge s. Only when challenge was with a human serotype 3 strain, as in a recent study in Venezuela, was substantial protection seen 9. The segmented nature of the rotavirus genome permits selection under laboratory conditions of single gene substitutions of VP-7 from human isolates of the other three major serotypes 1°. The clinical and immunological response to such reassortants is the next logical step in evaluation of rhesus rotavirus vaccines with an ultimate goal of preparing a multivalent vaccine. The present study is concerned with safety, infectivity, antigenicity and transmissibility ofa reassortant containing the VP-7 from a human serotype 1 strain, designated D, and remaining genes from the attenuated rhesus master strain. The *Department of Pediatrics, Vanderbilt University, Nashville, TN 37232, USA. tLaboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, USA. (Received 23 January 1989; accepted 28 July 1989) 0264-410X/90/080070q35 $03.00 © 1990 Butterworth & Co. (Publishers) Ltd
70
Vaccine, Vol. 8, February 1990
behaviour of this vaccine in comparison with serotype 3 master strain RRV provides important clues as to the significance and sources of serotypic differences.
Methods Clinical evaluation Children were observed for 12 consecutive days in a day care facility maintained in the Clinical Research Center at Vanderbilt University. Groups of 2-7 healthy children of progressively younger age groups were studied with collection and characterization of stool specimens, daily physical exams, and observation of behaviour over at least a 6 h period. Each child returned home to his family in the evening where a continued record of fever, illness and number of stools was kept. The definition of illness included any of the following: a fever of >38.3°C (101°F); diarrhoea as defined by > 3 stools per day or any watery or unformed stools; any vomiting; or any signs or symptoms of respiratory infection leading to a clinical diagnosis of upper respiratory illness or acute otitis media. Children were randomly assigned to receive either vaccine or an indistinguishable placebo. The code remained blinded to investigators and participants until completion of the study. Vaccine or placebo were administered on the third day of observation to children who had remained without clinical symptoms. Vaccine was administered orally in a volume of 1 ml of a 1:10 or 1:100 dilution of the virus. Virus was given, just after 300rag of sodium bicarbonate to neutralize gastric acidity. The undiluted virus, (rhesus rotavirus with the VP-7 from strain D, designated RRV x D) had a titre of 5 x 106m1-1. Written informed consent was obtained from all parents and all protocols were reviewed and approved
Evaluation of a reassortant rhesus rotavirus vaccine in young children: T. Tajima et al.
by the Vanderbilt Committee for the Protection of Human Subjects
30 min at 4°C and then pelletting the supernatant on to a 40% sucrose cushion containing 10mM CaCI 2 by centrifugation at 83 000g for 2 h at 4°C. The resulting pellet was resuspended in 100mM Tris-hydrochloride (pH 7.4) containing 10mM CaC12 and adjusted to 4 haemagglutinin units per 0.025 ml. Receptor destroying enzyme, RDE, treated serum was used in the assay which employed fresh adult human type 0, Rh negative erythrocytes in a 0.5% bovine serum albumin phosphate buffered saline. The assay was performed in V-shaped 96 well microtitre polates (Dynatech Laboratories, Alexandria, Virginia) using standard HAI techniques. With a previously described ELISA assay IgA antibodies were determined in stool specimens collected before and three weeks after vaccination v.
Laboratory evaluation To determine the presence of rotavirus in the stool, daily stools were tested for rotaviral antigen using an ELISA antigen detection system (Rotazyme, Abbott Laboratories, North Chicago). To culture rotavirus from the stool a 10% suspension was made in Hank's basic salt solution with antibiotics as previously described 7. The stool suspension was subsequently clarified by low speed centrifugation, pretreated with trypsin and inoculated on to MAI04 tissue culture cells. Cells were incubated in a roller drum apparatus at 35°C and observed for cytopathic effect at 3-day intervals. Supernatant fluid from tubes exhibiting cytopathology were tested by Rotazyme. A blind passage of all negative tubes was carried out. To quantitate the amount of virus present frozen original specimens of stools containing rotavirus were serially diluted and inoculated on to MA104 monolayers with an agar overlay as previously described 7. Virus titre was expressed as plaque forming units per ml of 10% stool suspension. Because of the potential for other viruses to cause intercurrent illness and interfere with infectivity of rotavirus vaccine, stools were examined every third day for cultivatable viruses using primary human embryonic kidney, HEp-2, primary rhesus kidney, WI-38, and 293-E cells. Enteroviruses were typed by the Tennessee State Virology laboratory. Bacterial pathogens were also sought at 3 day intervals using standard laboratory techniques for Sahnonella, Shigella, Campylobacter and Yersinia. Stools were examined for ova and parasites at the beginning of each trial. Serum was drawn before, 3 and 6 weeks after, vaccination for determination of antibodies to rotavirus. Neutralizing antibodies to RRV and RRV x D were measured by a previously described plaque reduction assay with an initial serum dilution of 1:8 v. A prototype human type 1 strain, Wa, and type 3 strain, P, were also used in a plaque neutralization test with a starting serum dilution of 1:20. Antibodies were also determined using an ELISA assay with plates coated with purified RRV virus as previously described 7. Additionally a haemagglutination inhibition, HAI, assay was developed to measure antibodies to VP-4, the viral haemagglutinin, of RRV. Antigen for this assay was prepared by initially removing cellular debris by centrifugation at 60009 for Table 1
Results Clinical observations Children over 2 years of age were rarely infected with RRV x D. Only one of eight older vaccinees shed virus. As would be expected, no difference in clinical illness was seen between these older vaccinees and eight age-matched controls (data not shown). Eight of 13 children under 2 years of age studied in the playroom setting had evidence of rotavirus infection. Illness rates in the 10 days after vaccination are compared between eight infected vaccinees, five uninfected vaccinees, and five age-matched controls, Table I. The frequency of gastrointestinal and respiratory illness did not differ between the three groups; however, low-grade fever was more commonly observed in those vaccinees who were infected with the rotavirus vaccine, p=0.014. One 7month-old vaccinee had a rash resembling erythema multiforme without mucosal involvement. This child was concurrently infected with ECHO 11 and Giardia lambia. Five additional children aged 3-10 months received vaccine as outpatients at the conclusion of the day-care trials. There were no reports of gastrointestinal illness or fever in these children who were observed by their parents daily and by the study team on days 3 and 6 after vaccination. Other intercurrent infections were frequently seen, particularly with enteric viruses, in the younger children. Whereas only one of 16 children over 2 years of age had an enteric virus recovered, 13 of 18 children under 2 years shed entero- or adenoviruses during the trial. Spread of enteric viruses among participants in the trial was routinely observed. The isolation of enteroviruses
Clinical observations in children under 2 years of age given reassortant rhesus rotavirus vaccine (RRV x D) Days of symptoms
No.
Total days of observation
Mean no. of stools/day
5
42
2.0
5
39
1.8
nfecte0 874vaccnees Uninfected vaccinees Controls
Fever />38 =
9
(i "
>--39~
0
Diarrhoea
Vomiting
Respiratory illness
26
3
19
0
15
2
17
0
12
1
26
*Does not include 5 children given vaccine as outpatients "p=0.16 Two-tailed Fisher's ) Pooling of days of fever in uninfected vaccinees and controls yields a p value of 0.014 when compared with "p=0.049 Two-tailed Fisher's ) infected vaccinees
Vaccine, Vol. 8, February 1990
71
Evaluation of a reassortant rhesus rotavirus vaccine in young children: T. Tajima et al. Table 2
Viral and serological responses in children receiving reassortant D x rhesus rotavirus (RRV x D) No. woth ~>4 fold rise Virus shedding
Serooeoa*,ve,,4,
Vaccinees (26)"
:°}
ELISA
"}
c
Seropositive (12) Controls (13)
1 0
0
~No. in group given in parentheses bOnly the 9 children studied in the day Cp = 0.0004 Two-tailed Fisher exact ~p=0.000001 Two-tailed Fisher exact ep=0.0002 Two-tailed Fisher exact 'p=0.00001 Two-tailed Fisher exact
~
, 2 0
Hemagglutination inhibition
,4} 1 0
~
Total
'i}
,
0
care centre had indicated procedure test test test test
D
of pre-existing serum antibody was the major determinate of virus shedding, Table 2. Only one of 12 seropositive vaccinees shed virus while eight of nine seronegatives studied daily shed virus. The seropositive child was given the higher, 1 :I0, dilution of vaccine and shed virus on a single day. The striking contrast in virus shedding between seropositive and seronegative children is shown in Figure I. The presence of intercurrent viral agents had no effect on the shedding of RRV x D. As in a previous study of non-reassortant rhesus rotavirus, replication of RRV x D in the respiratory tract was sought on two occasions, days 4 and 7, in each child after vaccination. No rotavirus was recovered from respiratory secretions. No evidence of transmission of rotavirus to 13 controls, five of whom were seronegative, was seen in spite of close daily contact with infected vaccinees. Only four of 39 stools from which virus was recovered were positive for rotaviral antigen by Rotazyme which is indicative of lower titres of vaccine virus replication in comparison with natural infection.
"I"
i!
l o
~ooo &
u~ -i
u~ l
Plaque neutralization
•
._c 1O0
T 5
10
Vaccine immunogenicity
' 0
v
I
~
C
C
""
^
^
""
0
2
3
q
5
6
7
8
9-I0
Time a f t e r v a c c i n e a d m i n i s t r a t i o n
,,~tD-
21
(RRV-D)(days)
Figure 1 Virus shedding in children receiving RRV × D. Significant differences in virus shedding between the groups are indicated by an * (p < 0.05). Preserum antibody (ELISA): Q, < 200, n = 9; O, > 200, n = 12
was significantly associated with diarrhoea in study participants. As diarrhoea occurred in both vaccinees and controls, it was not a confounding factor in analysis of vaccine associated illness. Non-viral pathogens identified included Giardia lambia in three children and Campylobacter in one patient.
Vaccine infectivity Rotaviruses, all identified as vaccine strain by growth characteristics, were recovered from a total of nine vaccinees. The higher dose of virus (1:10 dilution of virus stock with 10 5.3 plaque forming units) was given to only five vaccinees, four in the > 2 year age group, and no substantive information on the effect of dose on infectivity or clinical reactogenicity could be found. The presence
72
Vaccine, Vol. 8, February 1990
As with infectivity, serum antibody responses to vaccination were highly correlated with the absence of pre-existing antibody. All 14 initially seronegative children had antibody rises by 6 weeks after vaccination, Table 2. Only two seropositive children had rises in antibody determinations; one of these children had shed virus. No antibody rises were seen in controls further verifying lack of transmission of vaccine virus in the study setting. The HAI assay measured antibody responses to VP-4, the viral haemagglutinin. This protein is shared by all the rotavirus reassortants and is essential to its high level of replication in tissue culture. It is a highly antigenic protein in that all 14 seronegative children undergoing primary ekposure to rotavirus developed HAI antibody. Four of seven vaccinees tested who were shedding virus had a fourfold rise in IgA antibody in stools obtained 3 weeks after vaccination. Plaque neutralization antibody responses were measured using RRV, representative of serotype 3 via VP-7, and RRV × D, representative of serotype 1 via VP-7. Responses of RRV × D vaccine recipients were virtually identical for each virus, Figure 2. To further explore type specific neutralizing antibody response to RRV× D, pre- and postimmunization serum plaque neutralizing antibody titres were determined against prototype human strains, Wa for type 1 and P for type 3. Nine children
Evaluation of a reassortant rhesus rotavirus vaccine in young children: T. Tajirna et al.
almost completely against infection by RRV x D but allowed sufficient virus replication to cause virus recovery and seroresponses in 55% of RRV recipients 7. The poor infectivity of RRV × D in seropositive children was not explained by a greater attenuation of reassortant virus. In seronegative children RRV x D had a significantly higher take rate than the original RRV.
o
b
a
O
oo 1000 oo o
o
O
ol c
OO
o
ooo o o
D C
Discussion
o
100
E D
C~
00000 0000
10
I
I
I
I
R RVx D
Wa
R RV
P
Virus
neutralized
Figure 2 Antibody response 3 weeks after vaccination with RRV x D to rhesus and human strains. (a) Serotype 1; (b) serotype 3
were initially seronegative by all antibody assays and were thus presumed to be undergoing primary infection with vaccine exposure. Eight shed virus and all had evidence of antibody responses by ELISA and HAI assays, assays which do not differentiate between serotypes. In contrast to the cross-reactive response to rhesus strains presumably generated to the shared VP-4, the response to the human strains was entirely type specific and limited to the type 1 immunizing strain,
Figure 2. Prevaccination neutralizing antibody titres of seropositives, should reflect prior natural infection with serotype 1, serotype 3 or both. With rhesus strains five of nine exhibited at least a fourfold difference in prevaccination neutralizing titre between serotypes 1 and 3. In spite of limited serotyping of human isolates from Nashville in the past 3 years having shown all ten isolates recovered to be serotype 1, four of five had higher titres to serotype 3. By neutralization of the human strains in prevaccination serum the majority of children again appeared to have been exposed to both strains. One child was identified with antibodies only to type 1 and another seropositive child by ELISA and HAI had no neutralizing antibody to 1 or 3. Presumably the latter child's infection had been with types 2 or 4. The amount of neutralizing antibody induced by recent vaccination was similar to that in seropositive children with past wild-type infection.
Comparison with R R V, serotype 3, vaccine Infectivity of RRV x D differed significantly from that previously demonstrated under the same conditions with RRV, Table 3. In spite of similar levels of pre-existing antibody to RRV x D and RRV as measured by ELISA and neutralizing antibody as measured against the homologous rhesus strain, the antibody present protected
The four epidemiologically important human serotypes of rotavirus appear to vary in frequency in different geographic locations 1. Recent isolates from the United States have been predominantly type 1 indicating the importance of including this strain in any potential vaccine s. However, it appears that a fully effective rotavirus vaccine will have to offer protection against all four strains. The initial focus in serotyping was on VP-7 as the major protein which elicits neutralizing antibody. The present four serotypic classes are based on VP-7 antigenic characteristics. Recently it has become evident that VP-4 may also contribute significantly to neutralization x2. Serotyping is further complicated by the ability of rotavirus strains to reassort and for cross-neutralization to be one way ~3. In this context, the experience with reassortant rotavirus vaccine, RRV x D, both answers and raises questions. The type 1 reassortant virus appeared to have a very similar clinical reactivity to the parental rhesus strain, RRV. Thus the addition of VP-7 of human origin did not alter the level of attenuation of the rhesus strain. As in other evaluations of the family of rhesus rotavirus vaccines, brief and largely asymptomatic fever was seen. The duration and amount of virus shed by seronegatives was similar with the two vaccines. In each instance vaccine virus shedding was considerably lower than that seen with natural infection where as much as 10 logs of virus per ml of 10% stool suspension is frequently detected. The lack of transmission of rhesus strains continued to be a consistent observation in this study. Thus the two vaccine virus strains appear to be indistinguishable in clinical reactivity and infectivity in seronegative children within the limitations of the small number of children studied. However, host susceptibility of children with prior rotavirus infection differed between the two strains. Pre-existing antibody did not prevent infection with serotype 3, although it substantially decreased the amount of virus shed 7. In contrast, pre-existing antibody
Table 3 Comparative infectivity of rhesus rotaviruses RRV and RRV x D in seropositive and seronegative children
Serostatus
Seropositive (ELISA >200) Seronegative (ELISA ~
Introduction Infection with rotavirus is the leading cause of severe diarrhoea and resultant dehydration in both developing and industrialized countries 1'2. For this reason rotavirus has become an important target for vaccine development 3. Prevention of disease has become potentially more complex with recognition that there are at least four epidemiologically important distinct serotypes of human rotavirus 4. Serotypic specificity as determined by neutralization with hyperimmune serum is conferred by VP-7. However, additional neutralizing epitopes are contained within VP-4, another outer capsid protein formerly designated as VP-35. Rhesus rotavirus, strain MMU18006, is a promising vaccine candidate for the prevention of rotaviral disease 6'v. However, experience to date with rhesus rotavirus, which contains a VP-7 similar to human serotype 3, has suggested inconsistent protection against subsequent wild type challenge s. Only when challenge was with a human serotype 3 strain, as in a recent study in Venezuela, was substantial protection seen 9. The segmented nature of the rotavirus genome permits selection under laboratory conditions of single gene substitutions of VP-7 from human isolates of the other three major serotypes 1°. The clinical and immunological response to such reassortants is the next logical step in evaluation of rhesus rotavirus vaccines with an ultimate goal of preparing a multivalent vaccine. The present study is concerned with safety, infectivity, antigenicity and transmissibility ofa reassortant containing the VP-7 from a human serotype 1 strain, designated D, and remaining genes from the attenuated rhesus master strain. The *Department of Pediatrics, Vanderbilt University, Nashville, TN 37232, USA. tLaboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, USA. (Received 23 January 1989; accepted 28 July 1989) 0264-410X/90/080070q35 $03.00 © 1990 Butterworth & Co. (Publishers) Ltd
70
Vaccine, Vol. 8, February 1990
behaviour of this vaccine in comparison with serotype 3 master strain RRV provides important clues as to the significance and sources of serotypic differences.
Methods Clinical evaluation Children were observed for 12 consecutive days in a day care facility maintained in the Clinical Research Center at Vanderbilt University. Groups of 2-7 healthy children of progressively younger age groups were studied with collection and characterization of stool specimens, daily physical exams, and observation of behaviour over at least a 6 h period. Each child returned home to his family in the evening where a continued record of fever, illness and number of stools was kept. The definition of illness included any of the following: a fever of >38.3°C (101°F); diarrhoea as defined by > 3 stools per day or any watery or unformed stools; any vomiting; or any signs or symptoms of respiratory infection leading to a clinical diagnosis of upper respiratory illness or acute otitis media. Children were randomly assigned to receive either vaccine or an indistinguishable placebo. The code remained blinded to investigators and participants until completion of the study. Vaccine or placebo were administered on the third day of observation to children who had remained without clinical symptoms. Vaccine was administered orally in a volume of 1 ml of a 1:10 or 1:100 dilution of the virus. Virus was given, just after 300rag of sodium bicarbonate to neutralize gastric acidity. The undiluted virus, (rhesus rotavirus with the VP-7 from strain D, designated RRV x D) had a titre of 5 x 106m1-1. Written informed consent was obtained from all parents and all protocols were reviewed and approved
Evaluation of a reassortant rhesus rotavirus vaccine in young children: T. Tajima et al.
by the Vanderbilt Committee for the Protection of Human Subjects
30 min at 4°C and then pelletting the supernatant on to a 40% sucrose cushion containing 10mM CaCI 2 by centrifugation at 83 000g for 2 h at 4°C. The resulting pellet was resuspended in 100mM Tris-hydrochloride (pH 7.4) containing 10mM CaC12 and adjusted to 4 haemagglutinin units per 0.025 ml. Receptor destroying enzyme, RDE, treated serum was used in the assay which employed fresh adult human type 0, Rh negative erythrocytes in a 0.5% bovine serum albumin phosphate buffered saline. The assay was performed in V-shaped 96 well microtitre polates (Dynatech Laboratories, Alexandria, Virginia) using standard HAI techniques. With a previously described ELISA assay IgA antibodies were determined in stool specimens collected before and three weeks after vaccination v.
Laboratory evaluation To determine the presence of rotavirus in the stool, daily stools were tested for rotaviral antigen using an ELISA antigen detection system (Rotazyme, Abbott Laboratories, North Chicago). To culture rotavirus from the stool a 10% suspension was made in Hank's basic salt solution with antibiotics as previously described 7. The stool suspension was subsequently clarified by low speed centrifugation, pretreated with trypsin and inoculated on to MAI04 tissue culture cells. Cells were incubated in a roller drum apparatus at 35°C and observed for cytopathic effect at 3-day intervals. Supernatant fluid from tubes exhibiting cytopathology were tested by Rotazyme. A blind passage of all negative tubes was carried out. To quantitate the amount of virus present frozen original specimens of stools containing rotavirus were serially diluted and inoculated on to MA104 monolayers with an agar overlay as previously described 7. Virus titre was expressed as plaque forming units per ml of 10% stool suspension. Because of the potential for other viruses to cause intercurrent illness and interfere with infectivity of rotavirus vaccine, stools were examined every third day for cultivatable viruses using primary human embryonic kidney, HEp-2, primary rhesus kidney, WI-38, and 293-E cells. Enteroviruses were typed by the Tennessee State Virology laboratory. Bacterial pathogens were also sought at 3 day intervals using standard laboratory techniques for Sahnonella, Shigella, Campylobacter and Yersinia. Stools were examined for ova and parasites at the beginning of each trial. Serum was drawn before, 3 and 6 weeks after, vaccination for determination of antibodies to rotavirus. Neutralizing antibodies to RRV and RRV x D were measured by a previously described plaque reduction assay with an initial serum dilution of 1:8 v. A prototype human type 1 strain, Wa, and type 3 strain, P, were also used in a plaque neutralization test with a starting serum dilution of 1:20. Antibodies were also determined using an ELISA assay with plates coated with purified RRV virus as previously described 7. Additionally a haemagglutination inhibition, HAI, assay was developed to measure antibodies to VP-4, the viral haemagglutinin, of RRV. Antigen for this assay was prepared by initially removing cellular debris by centrifugation at 60009 for Table 1
Results Clinical observations Children over 2 years of age were rarely infected with RRV x D. Only one of eight older vaccinees shed virus. As would be expected, no difference in clinical illness was seen between these older vaccinees and eight age-matched controls (data not shown). Eight of 13 children under 2 years of age studied in the playroom setting had evidence of rotavirus infection. Illness rates in the 10 days after vaccination are compared between eight infected vaccinees, five uninfected vaccinees, and five age-matched controls, Table I. The frequency of gastrointestinal and respiratory illness did not differ between the three groups; however, low-grade fever was more commonly observed in those vaccinees who were infected with the rotavirus vaccine, p=0.014. One 7month-old vaccinee had a rash resembling erythema multiforme without mucosal involvement. This child was concurrently infected with ECHO 11 and Giardia lambia. Five additional children aged 3-10 months received vaccine as outpatients at the conclusion of the day-care trials. There were no reports of gastrointestinal illness or fever in these children who were observed by their parents daily and by the study team on days 3 and 6 after vaccination. Other intercurrent infections were frequently seen, particularly with enteric viruses, in the younger children. Whereas only one of 16 children over 2 years of age had an enteric virus recovered, 13 of 18 children under 2 years shed entero- or adenoviruses during the trial. Spread of enteric viruses among participants in the trial was routinely observed. The isolation of enteroviruses
Clinical observations in children under 2 years of age given reassortant rhesus rotavirus vaccine (RRV x D) Days of symptoms
No.
Total days of observation
Mean no. of stools/day
5
42
2.0
5
39
1.8
nfecte0 874vaccnees Uninfected vaccinees Controls
Fever />38 =
9
(i "
>--39~
0
Diarrhoea
Vomiting
Respiratory illness
26
3
19
0
15
2
17
0
12
1
26
*Does not include 5 children given vaccine as outpatients "p=0.16 Two-tailed Fisher's ) Pooling of days of fever in uninfected vaccinees and controls yields a p value of 0.014 when compared with "p=0.049 Two-tailed Fisher's ) infected vaccinees
Vaccine, Vol. 8, February 1990
71
Evaluation of a reassortant rhesus rotavirus vaccine in young children: T. Tajima et al. Table 2
Viral and serological responses in children receiving reassortant D x rhesus rotavirus (RRV x D) No. woth ~>4 fold rise Virus shedding
Serooeoa*,ve,,4,
Vaccinees (26)"
:°}
ELISA
"}
c
Seropositive (12) Controls (13)
1 0
0
~No. in group given in parentheses bOnly the 9 children studied in the day Cp = 0.0004 Two-tailed Fisher exact ~p=0.000001 Two-tailed Fisher exact ep=0.0002 Two-tailed Fisher exact 'p=0.00001 Two-tailed Fisher exact
~
, 2 0
Hemagglutination inhibition
,4} 1 0
~
Total
'i}
,
0
care centre had indicated procedure test test test test
D
of pre-existing serum antibody was the major determinate of virus shedding, Table 2. Only one of 12 seropositive vaccinees shed virus while eight of nine seronegatives studied daily shed virus. The seropositive child was given the higher, 1 :I0, dilution of vaccine and shed virus on a single day. The striking contrast in virus shedding between seropositive and seronegative children is shown in Figure I. The presence of intercurrent viral agents had no effect on the shedding of RRV x D. As in a previous study of non-reassortant rhesus rotavirus, replication of RRV x D in the respiratory tract was sought on two occasions, days 4 and 7, in each child after vaccination. No rotavirus was recovered from respiratory secretions. No evidence of transmission of rotavirus to 13 controls, five of whom were seronegative, was seen in spite of close daily contact with infected vaccinees. Only four of 39 stools from which virus was recovered were positive for rotaviral antigen by Rotazyme which is indicative of lower titres of vaccine virus replication in comparison with natural infection.
"I"
i!
l o
~ooo &
u~ -i
u~ l
Plaque neutralization
•
._c 1O0
T 5
10
Vaccine immunogenicity
' 0
v
I
~
C
C
""
^
^
""
0
2
3
q
5
6
7
8
9-I0
Time a f t e r v a c c i n e a d m i n i s t r a t i o n
,,~tD-
21
(RRV-D)(days)
Figure 1 Virus shedding in children receiving RRV × D. Significant differences in virus shedding between the groups are indicated by an * (p < 0.05). Preserum antibody (ELISA): Q, < 200, n = 9; O, > 200, n = 12
was significantly associated with diarrhoea in study participants. As diarrhoea occurred in both vaccinees and controls, it was not a confounding factor in analysis of vaccine associated illness. Non-viral pathogens identified included Giardia lambia in three children and Campylobacter in one patient.
Vaccine infectivity Rotaviruses, all identified as vaccine strain by growth characteristics, were recovered from a total of nine vaccinees. The higher dose of virus (1:10 dilution of virus stock with 10 5.3 plaque forming units) was given to only five vaccinees, four in the > 2 year age group, and no substantive information on the effect of dose on infectivity or clinical reactogenicity could be found. The presence
72
Vaccine, Vol. 8, February 1990
As with infectivity, serum antibody responses to vaccination were highly correlated with the absence of pre-existing antibody. All 14 initially seronegative children had antibody rises by 6 weeks after vaccination, Table 2. Only two seropositive children had rises in antibody determinations; one of these children had shed virus. No antibody rises were seen in controls further verifying lack of transmission of vaccine virus in the study setting. The HAI assay measured antibody responses to VP-4, the viral haemagglutinin. This protein is shared by all the rotavirus reassortants and is essential to its high level of replication in tissue culture. It is a highly antigenic protein in that all 14 seronegative children undergoing primary ekposure to rotavirus developed HAI antibody. Four of seven vaccinees tested who were shedding virus had a fourfold rise in IgA antibody in stools obtained 3 weeks after vaccination. Plaque neutralization antibody responses were measured using RRV, representative of serotype 3 via VP-7, and RRV × D, representative of serotype 1 via VP-7. Responses of RRV × D vaccine recipients were virtually identical for each virus, Figure 2. To further explore type specific neutralizing antibody response to RRV× D, pre- and postimmunization serum plaque neutralizing antibody titres were determined against prototype human strains, Wa for type 1 and P for type 3. Nine children
Evaluation of a reassortant rhesus rotavirus vaccine in young children: T. Tajirna et al.
almost completely against infection by RRV x D but allowed sufficient virus replication to cause virus recovery and seroresponses in 55% of RRV recipients 7. The poor infectivity of RRV × D in seropositive children was not explained by a greater attenuation of reassortant virus. In seronegative children RRV x D had a significantly higher take rate than the original RRV.
o
b
a
O
oo 1000 oo o
o
O
ol c
OO
o
ooo o o
D C
Discussion
o
100
E D
C~
00000 0000
10
I
I
I
I
R RVx D
Wa
R RV
P
Virus
neutralized
Figure 2 Antibody response 3 weeks after vaccination with RRV x D to rhesus and human strains. (a) Serotype 1; (b) serotype 3
were initially seronegative by all antibody assays and were thus presumed to be undergoing primary infection with vaccine exposure. Eight shed virus and all had evidence of antibody responses by ELISA and HAI assays, assays which do not differentiate between serotypes. In contrast to the cross-reactive response to rhesus strains presumably generated to the shared VP-4, the response to the human strains was entirely type specific and limited to the type 1 immunizing strain,
Figure 2. Prevaccination neutralizing antibody titres of seropositives, should reflect prior natural infection with serotype 1, serotype 3 or both. With rhesus strains five of nine exhibited at least a fourfold difference in prevaccination neutralizing titre between serotypes 1 and 3. In spite of limited serotyping of human isolates from Nashville in the past 3 years having shown all ten isolates recovered to be serotype 1, four of five had higher titres to serotype 3. By neutralization of the human strains in prevaccination serum the majority of children again appeared to have been exposed to both strains. One child was identified with antibodies only to type 1 and another seropositive child by ELISA and HAI had no neutralizing antibody to 1 or 3. Presumably the latter child's infection had been with types 2 or 4. The amount of neutralizing antibody induced by recent vaccination was similar to that in seropositive children with past wild-type infection.
Comparison with R R V, serotype 3, vaccine Infectivity of RRV x D differed significantly from that previously demonstrated under the same conditions with RRV, Table 3. In spite of similar levels of pre-existing antibody to RRV x D and RRV as measured by ELISA and neutralizing antibody as measured against the homologous rhesus strain, the antibody present protected
The four epidemiologically important human serotypes of rotavirus appear to vary in frequency in different geographic locations 1. Recent isolates from the United States have been predominantly type 1 indicating the importance of including this strain in any potential vaccine s. However, it appears that a fully effective rotavirus vaccine will have to offer protection against all four strains. The initial focus in serotyping was on VP-7 as the major protein which elicits neutralizing antibody. The present four serotypic classes are based on VP-7 antigenic characteristics. Recently it has become evident that VP-4 may also contribute significantly to neutralization x2. Serotyping is further complicated by the ability of rotavirus strains to reassort and for cross-neutralization to be one way ~3. In this context, the experience with reassortant rotavirus vaccine, RRV x D, both answers and raises questions. The type 1 reassortant virus appeared to have a very similar clinical reactivity to the parental rhesus strain, RRV. Thus the addition of VP-7 of human origin did not alter the level of attenuation of the rhesus strain. As in other evaluations of the family of rhesus rotavirus vaccines, brief and largely asymptomatic fever was seen. The duration and amount of virus shed by seronegatives was similar with the two vaccines. In each instance vaccine virus shedding was considerably lower than that seen with natural infection where as much as 10 logs of virus per ml of 10% stool suspension is frequently detected. The lack of transmission of rhesus strains continued to be a consistent observation in this study. Thus the two vaccine virus strains appear to be indistinguishable in clinical reactivity and infectivity in seronegative children within the limitations of the small number of children studied. However, host susceptibility of children with prior rotavirus infection differed between the two strains. Pre-existing antibody did not prevent infection with serotype 3, although it substantially decreased the amount of virus shed 7. In contrast, pre-existing antibody
Table 3 Comparative infectivity of rhesus rotaviruses RRV and RRV x D in seropositive and seronegative children
Serostatus
Seropositive (ELISA >200) Seronegative (ELISA ~
