Live Attenuated Human Rotavirus Vaccine, Rotarix™

David I. Bernstein, MD, MA

Rotavirus infections are the leading cause of severe gastroenteritis in young children
worldwide. Recently two new rotavirus vaccines have entered the world market. This review
provides a summary of the rationale, development, and evaluation of one of these vaccines,
Rotarix*. Rotarix* is a live oral rotavirus vaccine developed from a single protective human
strain following multiple passages in tissue culture to attenuate the strain. The vaccine is
administered as two oral doses at approximately 2 and 4 months of age. Large safety and
efficacy trials have shown the vaccine is safe, not associated with intussusception, and
effective against the most common circulating human serotypes. Efficacy against severe
rotavirus gastroenteritis and hospitalization have ranged from 85 to 100 percent.
Semin Pediatr Infect Dis 17:188-194 © 2006 Elsevier Inc. All rights reserved.

S ince its discovery in the 1980s and the recognition that

rotavirus infections are the leading cause of severe gastro-
enteritis in infants and young children in developed and de-
veloping countries, rotavirus has been a leading target for
vaccine development. Simply put, almost every child will
acquire a rotavirus infection before reaching the age of 5
years. This prevalence leads to at least 50,000 hospitaliza-
tions in the United States,1-3 a number that may represent a
substantial underestimation,4 and 352,000 to 592,000 deaths
around the world, mostly in underdeveloped countries.5,6
This review provides a summary of the development of
Rotarix™ (GlaxoSmithKline Immunotherapeutics, Need-
ham, MA), a live human attenuated rotavirus vaccine that is
currently available in the European Union and many other
countries, including most of Latin America. To better under-
stand the rationale for this vaccine, it is important to under-
stand the immunologic basis that suggested such an ap-
proach would provide broad protection against the common
serotypes, a hypothesis that has been confirmed in recent
clinical trials.
Rotavirus Structure
The rotavirus particle is a triple-layered particle with icosa-
hedral symmetry (Fig. 1).7 The outer shell is composed of the
VP4 and VP7 proteins. Antibodies to these two proteins and
Division of Infectious Diseases, Department of Pediatrics Cincinnati Chil-
dren’s Hospital Medical Center, University of Cincinnati College of Med-
icine, Cincinnati, OH.
Address reprint requests to David I. Bernstein, MD, MA, Cincinnati Children’s
Hospital Medical Center, University of Cincinnati College of Medicine, Di-
vision of Infectious Diseases, Department of Pediatrics, 3333 Burnet Ave.
ML 6014, Cincinnati, OH 45229. E-mail: David.Bernstein@cchmc.org
only these two are capable of neutralizing the virus. Group A
rotaviruses are further classified by type. Genotypic classifi-
cation is based on nucleic acid sequence analysis, whereas
serotypes are determined by similarities based on neutraliza-
tion. At least 15 types of the VP7 glycoprotein (G) exist, with
five common in human disease (G1-4 and G9) and three
others (G8, G12, and G5) recently detected. G1 viruses are
detected most often. The genotype and serotypes generally
are equivalent for VP7, but this is not true for the VP4 or P
(for protease-sensitive) protein. Thus, the nomenclature for
VP4 includes both genotypes, based on gene sequencing and
serotypes. The P serotype usually is indicated by an open
number, whereas the genotype is noted with a bracketed
number: for example, the most common type is P1A[8]. At
least 25 P types exist, with three seen in humans: P1A[8],
P1B[4], and P2[6]. P1A and P1B share some cross-reactive
epitopes, which may account for some cross-protection.8 The
most common combinations are G1P[8], G3P[8], G4P[8],
G9P[8], G9P[6], and G2P[4]. This terminology is similar
to that of the influenza virus nomenclature, with the in-
fluenza H and N types corresponding to the G and P types
of rotavirus. However, cross-protection appears to be
more common after rotavirus infection than after influenza
virus infection.
The inner layer of rotavirus is composed of virus protein
VP2, which is surrounded by the middle layer composed of
the most abundant structural virus protein, designated VP6
(Fig. 1). VP6 is a highly conserved protein among the group
A rotaviruses being discussed. The inner shell surrounds 11
segments of double-stranded RNA, which code for 11 pro-
teins, of which six are structural proteins (VP1, 2, 3, 4, 6, and
7) and five are nonstructural (NSP 1-5). Of interest is the
possible role of NSP4 as an enterotoxin.9

188 1045-1870/06/$-see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1053/j.spid.2006.08.006
Live attenuated human rotavirus vaccine, Rotarix™
Figure 1 Rotavirus structure illustrating 11 genome segments and the proteins they encode on the left. Schematic
diagram of structure in the middle and computer-generated image on the right.
Rationale for
Vaccine Development
That induction of immune responses occurs at the initial site
of rotavirus exposure, the mucosal epithelium, is increasingly
being recognized; therefore, almost all of the vaccines devel-
oped against rotavirus disease have been live attenuated oral
vaccines. Oral vaccines are used to best induce protection at
the site of infection and the major site of pathology, the
intestinal mucosa. Replication in intestinal epithelial cells
leads to the primary symptoms of rotavirus infection: fever,
diarrhea, and vomiting.10 Although viral antigen and live
virus have been found in the blood,11,12 no evidence indicates
that systemic spread plays a major role in the pathogenesis of
the infection. Viremia, however, probably accounts for the
rare complications of extra-intestinal disease.
Rotavirus vaccines are designed to induce a protective re-
sponse that mimics that induced by natural infection. Thus,
in an early study, Bishop and coworkers13 reported that neo-
natal infection with rotavirus provided significant protection
against subsequent disease but not reinfection. In perhaps
the best study of protection after natural infections reported
to date, investigators showed that children can be reinfected
with rotavirus but that each infection lowers the risk for the
development of both a subsequent infection and a subse-
quent infection with diarrhea.14 In this trial, no child had
moderate to severe diarrhea due to rotavirus after having two
previous rotavirus infections, whether or not the previous rota-
virus infections were symptomatic. This protection was seen
despite the fact that four of the major rotavirus serotypes (G1-
G4) circulated during the period of this study. These findings
contributed to the rationale for developing a two-dose vaccine.
189
In trials conducted in Cincinnati, a previous rotavirus in-
fection provided significant protection from both asymptom-
atic and symptomatic reinfections. However, serotype G1
rotaviruses predominated in all years.15 In a large multicenter
U.S. vaccine study, the protective efficacy of a previous rota-
virus infection against a symptomatic reinfection was 93 per-
cent (95% confidence interval 50-99%).16 In this trial, sero-
type G1 infections predominated in year 1 (93% of isolates),
but in year 2, 34 percent of isolates were nonserotype G1,
suggesting that heterotypic protection was seen, at least
based on the VP7 serotype (G type) of the infecting strains. In
this trial, symptomatic nonserotype G1 reinfection was not
detected in subjects with natural rotavirus infections in the
first year.
Further, in a study in Bangladesh, children who presented
with a clinically significant rotavirus illness had substantially
fewer rotavirus-specific neutralizing antibodies in general
than did age-matched well children, but only neutralizing
antibodies to G heterologous rotavirus strains correlated to
protection in multivariate logistic regression analysis.17 Sim-
ilarly, other studies also have failed to detect a correlation
between G serotype-specific serum neutralizing antibodies
and protection after vaccination.18-22 In some of these studies,
a correlation with the presence of rotavirus IgA antibodies
was detected.14,24 However, one should note that second ep-
isodes of severe rotavirus diseases are associated more often
with reinfection with virus of a different serotype than rein-
fection with the same serotype.14,24 Further, early vaccine
trials with RIT 4237, WC3, and RRV vaccine virus strains
showed inconsistent results, with some convincing evidence
for protection against homologous but not heterologous in-
fections (reviewed in Ward and Franco et al23,25,26).
190
Immunology
This discussion of immunologic mechanisms requires that
one understand that most of the studies evaluating correlates
of protection use serum antibody levels and that the local
intestinal response could differ (reviewed in Ward and
Franco et al23,25-27). Also important to understand is that if
protection is mediated by neutralizing antibodies, these an-
tibodies may be induced by either the VP7 or VP4 protein, as
discussed previously.7 Indeed, after a protective natural in-
fection or immunization has been established, most of the
antibodies produced are directed against the VP4 and not the
VP7 protein.28,29 True heterologous protection would be
against a virus that differs in both of these proteins. For
example, protection induced by the most common serotype
G1P[8] and the one found in Rotarix™ against a G2P[6]
inoculation would be an example of heterologous protection,
whereas protection of G1P[8] against other common types,
that is, G3P[8], G4P[8], and G9 P[8], would not.
The reassortant vaccines RotaTeq™ (see companion arti-
cle in this issue) and Rotashield (previously licensed but now
withdrawn) were designed to induce neutralizing antibodies
to multiple human serotypes by inclusion of reassortant
strains that include the VP7 and, in the case of Rotateq, also
the G[8] VP4. However, a discrepancy appears to exist in the
high level of protection but modest induction of detectable
serum neutralizing antibodies to these human strains, espe-
cially for Rotashield.30-32 This finding indicates that perhaps
it is the magnitude of the immune response, rather than
specific virus-neutralizing antibody titers, that best correlates
with level of protection, or that factors other than serum
neutralizing antibodies, such as mucosal IgA, T cells, or cy-
tokines, can provide protection (reviewed in Ward et al and
Franco et al22,23,25,26,33). Support for protection by each of
these three immune mechanisms can be found in mouse
experiments and gnotobiotic pig experiments, which have
been reviewed by other authors.23,25,26,33,34
Studies of experimental rotavirus infection in mice, pigs, and
calves have revealed conflicting results regarding the absolute
role of neutralizing antibodies in protection.24-26,35-39 Some
studies have favored a role for transcytosed IgA antibodies
and intracellular neutralization. For example, protection can
be provided by passive administration of a monoclonal anti-
body to the most abundant rotavirus protein VP6, but this
antibody is one that does not possess neutralizing activity in
classical in vitro virus neutralization assays. In one study, an
IgA isotype, but not an IgG isotype monoclonal antibody to
VP6, provided protection40,41; in another study, an IgA
monoclonal antibody to VP6 provided protection via intra-
cellular neutralization but not immune exclusion.42 In other
murine studies, mucosal VP6 immunization with adjuvant or
virus-like particles (VLP) composed of VP2 and VP6 pro-
vided protection.43-45 The mechanism of this protection is not
entirely clear but appears to be mediated by CD4⫹
T lymphocytes46 and can occur in the absence of IgA anti-
bodies.47 Other evidence suggests that protection can be pro-
vided by CD4⫹ T lymphocytes,48,49 CD8⫹ T cells,48,50,51 and
cytokines.52,53 In a recent study, passively-transferred IgG
D.I. Bernstein
antibodies also provided protection in a monkey model of
rotavirus, contradicting other studies.54
In summary, vaccine-induced protection could be pro-
vided by antibodies induced by either shared VP7 or VP4
neutralizing epitopes, by non-neutralizing IgA or IgG anti-
bodies, or by T-cell-mediated mechanisms. Thus, vaccine
strategies that induce high levels of IgA antibodies or that
induce cross-reactive T cells probably could provide homol-
ogous and heterologous protection. Indeed other researchers
have stated that IgA antibody appears to be the best correlate
of protection.23
Vaccine Development
Because of the initial success achieved using naturally atten-
uated animal rotaviruses, such as rhesus rotavirus, as human
vaccine candidates, the classical Jennerian technique, the
dominant approach has been to use bovine or monkey rota-
viruses that were modified later to include human rotavirus
gene segments (discussed in companion article). More re-
cently, attenuation has been accomplished by repeated pas-
sage of natural human isolates in tissue culture.
Development of
Rotavirus Vaccine 89-12
The first indication that the 89-12 strain could provide pro-
tection from subsequent rotavirus infections, especially rota-
virus disease, came from a study conducted in Cincinnati in
1988. Although the WC3 vaccine that was evaluated proved
to be ineffective, the study provided an opportunity to follow
the subjects prospectively through a subsequent rotavirus
season to evaluate protection from the natural infections that
occurred in the first year. Fortunately, the 1988/89 rotavirus
season in Cincinnati was unusual in that only one strain of a
G1[P8] rotavirus, as identified by electropherotyping, circu-
lated that year.55 This occurrence allowed the opportunity to
evaluate the protection afforded by this single strain of rotavi-
rus.15
Protection in the infants infected in the first year was 70
percent against symptomatic rotavirus infection and 81 per-
cent against any rotavirus reinfection. Curiously, both infants
who developed symptoms after reinfection had not devel-
oped a detectable rotavirus antibody response after their pri-
mary infection. It appeared, therefore, that the strain circu-
lating in Cincinnati in 1989 could provide protection from
rotavirus even if the initial infection were asymptomatic. Fur-
ther infection with 89-12 induced more neutralizing anti-
bodies to the VP4 protein than the VP7 protein,28 with evi-
dence of neutralizing antibodies to at least the four major
rotavirus serotypes G1-4.56 Recognizing that developing a
vaccine from a human rather than an animal rotavirus strain
might have advantages, researchers decided that this strain
would form the basis for a vaccine candidate.
After several options to attenuate this strain were dis-
cussed, the decision was made to use multiple cell culture
passages as an attenuation strategy. One isolate from an in-
Live attenuated human rotavirus vaccine, Rotarix™ 191

fected child, designated 89-12, was selected and serially pas- Dose 1
saged 26 times in primary African Green Monkey kidney cells 100
(AGMK), then seven times in a serially passaged AGMK cell 90 RIX4414
line.57 To determine whether the passages in cell culture had 80 Placebo

70
attenuated the virus, researchers conducted studies in adults
60
followed by studies in children with previous rotavirus infec-
50

%
tions.57 After these trials provided evidence of attenuation 40
based on a lack of signs or symptoms of rotavirus disease, the 30

trial proceeded to infants. After the initial safety trial in a 20

10
small number of infants and dose-ranging studies were com-
0
pleted, the evaluation proceeded to a multicenter efficacy Fever ( 38°C) Diarrhea Vomiting Irritability Loss of
Appetite
trial.
In the initial randomized placebo-controlled, double-blind Dose 2

efficacy trial, 213 healthy infants, 10 to 16 weeks of age, were

100
given two oral doses of either 89-12 (1 ⫻ 105 PFU/mL) or 90 RIX4414
placebo after administration of an antacid.58 The vaccine was 80 Placebo
safe, although a mild fever of short duration was detected in 19 70
percent of vaccine recipients compared with 5 percent of pla- 60

cebo recipients after the first dose (P ⬍ 0.001). Rotavirus-spe- 50

%
cific antibody responses developed in 94.4 percent of vaccine 40

recipients, and vaccine virus was detected in 75 percent of stools 30

20
obtained on day 7 after immunization. In the first year after
10
vaccination was given, vaccine efficacy was 89 percent against
0
any rotavirus disease, the highest reported for any multicenter Fever ( 38°C) Diarrhea Vomiting Irritability Loss of appetite

trial. Importantly, vaccine efficacy was 100 percent against very

severe infections and those requiring medical attention. Fol-
low-up of the children continued for a second year and revealed
an efficacy of 76 percent (95% CI 45-85) against any rotavirus
disease and continued to be 100 percent for very severe disease
and those requiring medical intervention.59
Rotarix™ (RIX4414)
Further development of the vaccine, initially named
RIX4414, was performed by GlaxoSmithKline after they ac-
quired the rights to the vaccine. The vaccine was developed
further by limiting dilution cloning of 89-12 in Vero cell
culture and continued passage of the strain. The resulting
vaccine then was formulated as a lyophilized preparation to
be given after reconstitution with a liquid calcium carbonate
buffer produced by GlaxoSmithKline.60
The initial trials of RIX4414 established its safety in adults
and previously infected toddlers before a dose-escalation
study was performed in infants.61 In this trial, increases were
not detected in any solicited symptom, including fever, diar-
rhea, or vomiting, at any vaccine dose. Thus, the increased
passage in tissue culture and/or the clonal selection process
appeared to produce a vaccine strain that was not associated
with the increase in mild fever seen after inoculation with
89-12. The immune response was influenced by the dose,
with seroconversion detected in 73 to 96 percent of infants
after the second dose. Vaccine virus shedding was detected in
38 to 60 percent of recipients after the first dose.
Evaluation of RIX4414 began in Asia shortly thereafter, mark-
ing the initiation of what would become a global effort to eval-
uate this vaccine. Thus, Rotarix™ studies have been part of an
intense effort, mainly centered in Latin America, to evaluate the
Figure 2 Percentage of RIX4414 and placebo recipients with solicited
symptoms during the 15 days after vaccination. (A) After dose one. (B)
After dose two. Reprinted with permission from De Vos et al.60
epidemiology and disease burden of rotavirus and intussuscep-
tion, as well as the economic impact of rotavirus disease.
In a dose-ranging trial conducted in Singapore in 2464
infants, the vaccine was administered concurrently with rou-
tine childhood immunizations.62 Again, the vaccine was
found to be safe and did not produce an increase in the
incidence of side effects, even at the highest dose of 106.1
fluorescent focus units (ffu). Similar to the results of the
Finish study, the vaccine was highly immunogenic, with
“take” rates (defined as seroconversion or vaccine virus shed-
ding) of 97.8 to 100 percent depending on the dose. Inter-
ference with the other routine childhood vaccines was not
detected. More safety and immunogenicity trials have been
conducted in South Africa, but results are not yet available.
In a multicenter study performed in the United States,
infants 5 to 15 weeks of age received either placebo or 105.2 or
106.4 ffu of RIX4414 concomitantly with their other child-
hood vaccines.63 The vaccine again did not increase fever,
vomiting, or diarrhea or any unsolicited adverse event (Fig. 2).
Vaccine take was detected in 81.5 or 88 percent of the low- or
high-dose vaccine recipients, respectively, after receiving two
doses. Further, the rotavirus vaccine did not interfere with
the immune response to the other routine immunizations.
In the largest trial conducted to date, safety was evaluated in
more than 60,000 infants in 11 Latin American countries and
Finland, while efficacy was evaluated in a subgroup of more
than 20,000 infants.64 Infants received two doses of either vac-
cine at a dose of 106.5 or placebo at approximately 2 and 4
192
Table 1 Efficacy of RotarixTM Against Rotavirus Gastroenteritis
in Latin America and Finland
Rotarix™ Placebo
(n ⴝ 9009) (n ⴝ 8858) Efficacy
Severe disease 12 77 85
Hospitalization 9 59 85
Vesikari scale >11 11 71 85
Vesikari scale >19 0 16 100
G1 P[8] 3 36 92
G3, G4 or G9 and 4 31 87
P[8]
G2 P[4] 6 10 41*
*In a large meta-analysis, efficacy was 67%.
months of age. Infants received routine immunizations accord-
ing to local regulations, but oral polio was provided at least 2
weeks before or after administration of the vaccine. In the safety
cohort, significantly fewer serious adverse events and hospital-
izations were reported in the vaccine group than in the placebo
group.
Efficacy
The first efficacy study with RIX4414 was conducted in Fin-
land during two rotavirus epidemic seasons, 2001 to 2002,
with a relatively low, suboptimal, dose of vaccine virus (104.7
ffu). In this randomized, double-blind, placebo-controlled
trial of 405 infants, the vaccine was well tolerated and immu-
nogenic.65 In this trial, efficacy against any episode of rotavi-
rus gastroenteritis was 73 percent, whereas efficacy against
any severe episode of rotavirus gastroenteritis was 90 percent
in the first season and 72 or 85 percent, respectively, over the
entire follow-up period. Thus, no decrease in efficacy was
seen in the second year compared with the first. Thirty-five of
the 38 cases of rotavirus gastroenteritis were caused by virus
of the same G1 serotype as the vaccine.
In the large study conducted in Latin America and Finland,
efficacy was 85 percent against severe rotavirus diarrhea and
hospitalizations.64 Of note, efficacy was high (more than
86%) against severe rotavirus diarrhea caused not only by
G1P[8] strains but also by the VP4-related G3[8], G4[P8],
and G9[P8] strains (Table 1). Efficacy against the few G2P[4]
strains was less, 41.0 percent. However, in several meta-
analysis studies, the efficacy was 67 to 71 percent, with 95
percent CIs of 15 to 87 percent, indicating that the vaccine
will be efficacious against strains that do share VP4 or VP7
proteins (personal communications, GSK).66
In the most recent trial conducted in six European countries,
4274 infants were randomized in a 2:1 ratio to receive Rotarix™
or placebo concomitantly with routine vaccinations.66 Protec-
tion was 87 percent against any rotavirus gastroenteritis, 96
percent against severe disease, and 100 percent against hospi-
talization attributable to rotavirus (Table 2). Furthermore, in
agreement with the study discussed previously, efficacy against
G3, G4, and G9 was similar to that against G1 and exceeded 95
percent, whereas efficacy against G2 strains was 75 percent. Of
D.I. Bernstein
importance, efficacy against hospitalization because of gastroen-
teritis of any cause was 75 percent.
In conclusion, efficacy of this vaccine is excellent against se-
vere rotavirus disease regardless of the serotype, although effi-
cacy is reduced against strains that do not share either the VP7 or
VP4 proteins. Efficacy is somewhat increased in the most devel-
oped countries compared with lesser developed countries, as
evidenced by comparison of the efficacy in the large trial in Latin
America with the one in Europe. Thus, comparison of efficacy in
Europe with any previous vaccine trial shows that Rotarix™
meets or exceeds previous rates of protection.
Intussusception and Rotarix™
Because of the association of the previously licensed
Rotashield™ vaccine with intussusception, large safety trials
have been conducted with both Rotateq™ and Rotarix™. As
stated previously, Rotarix™ was evaluated in a randomized
placebo controlled trial of more than 63,000 infants in Latin
America.64 In that trial, 31,373 infants ages 6 to 17 weeks
were randomized to receive two doses of vaccine and 31,552
to receive placebo. The mean age at the administration of the
first dose was 8.2 weeks and at the second dose was 15.8
weeks of age. The primary endpoint of the study was the
occurrence of intussusception as defined by the Brighton
collaboration group within 30 days of receiving either dose of
vaccine. A secondary evaluation involved cases occurring
during the duration of the study. Cases were captured by
active hospital surveillance and scheduled for visits after each
dose. An independent surveillance system also was in place
to ensure that all cases were identified. Cases were reviewed
by a blinded expert and monitored by an independent data
monitoring board. Seven cases, two after the first dose was
administered, were identified in the placebo group compared
with six, one following the first dose administered, in the
vaccine group. No clustering in the initial 1 to 2 weeks was
identified after the receipt of either dose (Fig. 3). During the
duration of the study, 16 cases occurred in the placebo group
compared with nine in the vaccine group. Thus, no evidence
was found that Rotarix™ was associated with intussusception.
Current Status
Rotarix™ currently is available in the European Union, 15
Latin American countries, and 29 other countries, including
Table 2 Efficacy of RotarixTM Against Rotavirus Gastroenteritis
in Europe
Efficacy (95% CI)
Any disease 87% (80-92%)
Severe disease 96% (90-99%)
Hospitalization 100% (82-100%)
Severe disease
G1 96 (86-100%)
G3 100% (45-100%)
G4 100% (65-100%)
G9 95% (78-99%)
G2 P[4] 75% (ⴚ386 to 89%)
Live attenuated human rotavirus vaccine, Rotarix™
First Dose
Infants with Intussusception
5 10 15 20 25
Second Dose
5 10 15 20 25
Figure 3 Number of cases of intussusception after the administration of Rotarix™ or placebo.
several in Asia. Ongoing discussions with the Food and Drug
Administration will determine when approval is sought in
the United States.
Summary
The evidence to date indicates that two oral doses of Rotarix™ is
safe and immunization is not associated with either fever or
gastrointestinal side effects. Most importantly, a large safety trial
showed no association with intussusception. To date, there has
been no impact on the immunogenicity of concurrently admin-
istered vaccines, allowing the vaccine to be administered within
existing immunizations schedules. Efficacy studies conducted
in Europe and Latin America also have indicated excellent pro-
tection especially against severe disease and hospitalizations due
to rotavirus, including those caused by nonserotype I infections.
Results of future trials currently underway will be needed to
determine whether the efficacy detected in the completed stud-
ies also will be seen in the least developed nations of the world,
where rotavirus infections take their highest toll.
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