U.S. patent number 7,932,074 [Application Number 09/743,338] was granted by the patent office on 2011-04-26 for multivalent human-bovine rotavirus vaccine.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Robert M. Chanock, Yasutaka Hoshino, Albert Z. Kapikian.
United States Patent |
7,932,074 |
Kapikian , et al. |
April 26, 2011 |
Multivalent human-bovine rotavirus vaccine
Abstract
The present invention provides vaccine compositions for
protection against human rotaviral disease without significant
reactogenicity. Human.times.bovine reassortant rotavirus comprising
each of the four clinically most important VP7 serotypes of human
rotavirus are combined in a multivalent formulation which provides
a high degree of infectivity and immunogenicity without producing a
transient febrile condition. Methods for producing an immunogenic
response without producing a transient febrile condition are also
provided.
Inventors: |
Kapikian; Albert Z. (Rockville,
MD), Chanock; Robert M. (Bethesda, MD), Hoshino;
Yasutaka (Wheaton, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
|
Family
ID: |
22245113 |
Appl.
No.: |
09/743,338 |
Filed: |
July 27, 1999 |
PCT
Filed: |
July 27, 1999 |
PCT No.: |
PCT/US99/17036 |
371(c)(1),(2),(4) Date: |
January 04, 2001 |
PCT
Pub. No.: |
WO00/06196 |
PCT
Pub. Date: |
February 10, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60094425 |
Jul 28, 1998 |
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Current U.S.
Class: |
435/235.1;
424/215.1; 424/205.1 |
Current CPC
Class: |
A61P
31/12 (20180101); A61P 1/12 (20180101); A61K
39/12 (20130101); A61K 39/15 (20130101); A61P
37/00 (20180101); A61P 31/14 (20180101); C12N
2720/12334 (20130101); A61K 2039/70 (20130101); A61K
2039/545 (20130101) |
Current International
Class: |
C12N
7/00 (20060101); A61K 39/15 (20060101) |
Field of
Search: |
;424/199.1,205.1,93.1,93.2,93.6,215.1 ;435/235.1,234 |
References Cited
[Referenced By]
U.S. Patent Documents
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4571385 |
February 1986 |
Greenberg et al. |
6113910 |
September 2000 |
Clark et al. |
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|
Primary Examiner: Chen; Stacy B
Assistant Examiner: White; Nicole Kinsey
Attorney, Agent or Firm: Kilpatrick, Townsend & Stockton
LLP
Parent Case Text
RELATED APPLICATIONS
The present application is a national stage application under 35
U.S.C. .sctn.371 of International Application No. PCT US99/17036,
filed Jul. 27, 1999, which claims the benefit of U.S. Patent
Application Ser. No. 60/094,425, filed Jul. 28, 1998.
Claims
What is claimed is:
1. An immunogenic composition comprising one or more bovine strain
reassortant rotavirus and a physiologically acceptable carrier,
wherein the one or more bovine strain reassortant rotavirus is
deposited with the American Type Culture Collection and is selected
from the group consisting of ATCC VR-2611, ATCC VR-2612, ATCC
VR-2613, ATCC VR-2614, ATCC VR-2615, ATCC VR-2616, and ATCC
VR-2617.
2. The immunogenic composition of claim 1, which comprises ATCC
VR-2611, ATCC VR-2612, ATCC VR-2616, and ATCC VR-2617.
3. The composition of claim 1, wherein the physiologically
acceptable carrier is a citrate buffer.
4. The composition of claim 1 which further comprises an adjuvant
to enhance the immune response.
5. The composition of claim 1, wherein the composition is in a
lyophilized form.
6. The composition of claim 1, wherein each bovine strain
reassortant rotavirus is formulated to provide a dosage of less
than 10.sup.6 plaque forming units.
7. A method for stimulating the immune system of an infant of less
than six months of age, the method comprising administering to the
infant the immunogenic composition of claim 1.
8. The method of claim 7, wherein the composition comprises ATCC
VR-2611, ATCC VR-2612, ATCC VR-2616, and ATCC VR-2617.
9. The method of claim 7, wherein the composition is administered
to the alimentary tract of the infant.
10. The method of claim 7, wherein the composition is administered
as a liquid suspension.
11. The method of claim 7, wherein each bovine strain reassortant
rotavirus is administered a dosage of less than 10.sup.6 plaque
forming units.
Description
BACKGROUND OF THE INVENTION
Rotaviruses are a major cause of acute dehydrating diarrhea in
infants and young children. Rotavirus disease accounts for 25% to
30% of gastroenteritis deaths in infants and young children in
developing countries and approximately 50,000-100,000
hospitalizations of children younger than five years of age in the
United States. For this reason, a safe effective vaccine is needed
to prevent severe rotavirus disease in infants and young
children.
A primary strategy for rotavirus vaccine development has been based
on a "Jennerian" approach, which takes advantage of the antigenic
relatedness of human and animal rotaviruses and the diminished
virulence of animal rotavirus strains in humans. Kapikian et al.,
in Vaccines 88, Chanock et al., eds., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., pp. 151-159 (1988). Several
candidate live oral rotavirus vaccines have been developed using
this approach, where an antigenically-related live virus derived
from a nonhuman host is used as a vaccine for immunization against
its human virus counterpart. Examples of animal rotaviruses that
have been used to vaccinate humans include bovine rotavirus strain
NCDV (RIT4237, Vesikari et al, Lancet, 2:807-811 (1983)), bovine
rotavirus strain WC3 (Clark et al., Am. J. Dis. Child., 140:350-356
(1986)) and rhesus monkey rotavirus (RRV) strain MMU 18006 (U.S.
Pat. No. 4,571,385, Kapikian et al., Vaccines 85, eds., Lerner et
al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
pp. 357-367 (1985)).
The protective efficacy among different monovalent bovine and
monovalent simian rotavirus vaccines has proved to be variable
(Vesikari in Viral Infections of the Gastrointestinal Tract
(Kapikian, Ed., Marel Dekker, Inc. pp. 419-442 (1994): Kapikian
ibid. pp. 443-470 (1994). Also, high concentrations of bovine
rotavirus have been required to produce a satisfactory immune
response in humans (10.sup.7-10.sup.8 plaque forming units (pfu))
(Vesikari et al., Ped. Inf. Dis. 4:622-625 (1985), Bernstein et al.
J. Infect. Dis. 162:1055-1062 (1990)). The variable efficacy of
these compositions can in part be attributed to the fact that the
target population of two- to five-month old infants
characteristically developed a homotypic immune response following
vaccination (Kapikian et al., Adv. Exp. Med. Biol., 327:59-69
(1992); Bernstein et al., J. Infect. Dis. 162:1055-1062 (1990);
Green et al. J. Infect. Dis. 161:667-679 (1990); and Vesikari,
Vaccine. 11:255-261 (1993)).
Clinically relevant human rotaviruses are members of the Group A
rotaviruses. These viruses share a common group antigen mediated by
VP6, a protein located on the virus intermediate shell. Also,
serotype specificity depends on the presence of the VP4 (protease
sensitive or P type) and VP7 (glycoprotein or G type) proteins
located on the virus outer shell (also often referred to as the
virus capsid), both of which independently induce neutralizing
antibodies. Kapikian et al. In Virology, Fields, ed. pps. 1353-1404
(1995).
Group A rotaviruses that infect humans have been classified into
ten distinct VP7 serotypes by neutralization assays. Amino acid
sequence analysis has indicated that within each serotype amino
acid identity within two major variable regions was high (85-100%);
however, amino acid identity between strains of different serotypes
was significantly less (Green et al., Virol. 168:429-433 (1989);
Green et al., Virol. 161:153-159 (1987); and Green et al., J.
Virol. 62:1819-1823 (1988)). Concordance between relationships
among rotaviruses as determined by virus neutralization assay or
sequence analysis of VP7 has been demonstrated. Therefore, a
reference strain can be routinely used in clinical studies as a
representative of rotavirus strains within its serotype.
To achieve protection against each of the four epidemiologically
and clinically important G serotypes (VP7) (numbered 1, 2, 3, and
4), the Jennerian approach has been modified by the production of
reassortant rotaviruses. Reassortant rotavirus strains were
constructed by coinfecting tissue culture cells with a rotavirus of
animal origin (i.e., rhesus or bovine rotavirus) and a human
rotavirus strain. Reassortant viruses produced during coinfection
that contained a single human rotavirus gene encoding VP7 from the
human strain and the 10 remaining rotavirus genes from the animal
strain were selected by exposing the progeny of the coinfection to
a set of monoclonal antibodies directed to the VP7 of the animal
strain. (See, for example. U.S. Pat. No. 4,571,385; Midthun et al.
J. Clin. Microbiol. 24:822-826 (1986); and Midthun et al. J. Virol.
53:949-954 (1985)).
Studies of human.times.rhesus rotavirus reassortants and
human.times.bovine reassortants containing the VP7 gene from a
human strain have demonstrated that the VP4 neutralization protein
of the animal rotavirus parent dominates the immune response in
infants vaccinated with these human.times.animal rotavirus
reassortants. This probably reflects the absence of animal
rotavirus VP4 antibodies among the antibodies transferred from the
mother to the infant in utero. Nevertheless, the immune response to
human rotavirus VP7 that is partially blunted by maternally derived
VP7 antibodies is sufficient to provide protection and thus VP7
antibodies form the basis of the modified Jennerian approach
(Flores et al., J. Clin. Microbiol. 27:512-518 (1989); Perez-Schael
et al., J. Clin. Microbiol. 28:553-558 (1990); Flores et al., J.
Clin. Microbiol. 31:2439-2445 (1993); Christy et al., J. Infect.
Dis. 168:1598-1599 (1993); Clark et al., Vaccine 8:327-332 (1990);
Treanor et al., Pediatr. Infect. Dis. J. 14:301-307 (1995); Madore
et al. J. Infect. Dis. 166:235-243 (1992); and Clark et al., J.
Infect. Dis. 161:1099-1104 (1990).
In studies using a single rhesus rotavirus reassortant bearing a
single human rotavirus gene, namely the gene that encodes VP7, it
was observed that the protective immunological response of such a
reassortant was characteristically homotypic in infants less than
six months of age (Green et al., J. Inf. Dis. 161:667-679 (1990)).
This observation provided further evidence for the importance of
VP7-associated immunity in immunization against rotavirus
disease.
The general experience with monovalent and quadrivalent
human.times.rhesus rotavirus reassortant vaccines has been that a
transient low-level febrile episode occurs in about one-third of
young infants 3 to 4 days after vaccination. Bernstein et al., JAMA
273:1191-1196 (1995); Flores et al., Lancet 336:330-334 (1995);
Perez-Schael et al., J. Clin. Microbiol. 28:553-558 (1990); Flores
et al., J. Clin. Microbiol. 31:2439-2445 (1990); Halsey et al., J.
Infect Dis. 158:1261-1267 (1988); Taniguichi et al., J. Clin.
Microbiol. 29:483-487 (1991); Simasathien et al., Pediatr. Infect.
Dis. J. 13:590-596 (1994); Madore et al., J. Infect. Dis.
166:235-243 (1992); and Joensuu et al., Lancet 350:1205-1209
(1997).
Results of studies in humans with bovine rotavirus strains NCDV and
WC3 (VP7 serotype 6) indicate that these particular bovine
rotavirus strains do not appear to cause fever or other reactions.
It should be noted that serotype 6 VP7 is not known to be present
on human rotaviruses that are important in human rotavirus disease.
Also, a bovine rotavirus was not found to be as immunogenic as the
rhesus rotavirus when administered to humans. The bovine rotavirus
strain NCDV (RIT4237 vaccine) has been evaluated in more than five
efficacy trials in infants and young children. In these trials, the
bovine RIT4237 vaccine was administered at a dose range of
10.sup.7.8 to 10.sup.8.3 tissue culture infectious doses .sub.50
(TCID.sub.50), with the usual dosage exceeding 10.sup.8.0
TCID.sub.50. Also, in a dose-response study, Vesikari et al., Ped.
Infect. Dis., 4:622-625 (1985)) observed that 15% (2/13) of four-
to six-month old infants developed a homotypic antibody response
when the vaccine was administered at a dose of 10.sup.6.3
TCID.sub.50; 71% (10/14) when administered at a dose of 10.sup.7.2
TCID.sub.50, and 100% when administered at a dose of 10.sup.8.3
TCID.sub.50. Thus, the dose for this bovine rotavirus strain that
produced an optimal immunogenicity was determined to be in the
range of 10.sup.8.0 TCID.sub.50.
In a direct comparison of the infectivity and immunogenicity of
rhesus rotavirus and bovine rotavirus in humans, 10.sup.5 plaque
forming units (pfu) of rhesus rotavirus (RRV vaccine) or 10.sup.8.3
pfu of RIT4237 was administered to children six to eight months of
age. (Vesikari et al., J. Infect. Dis. 153:832-839 (1986)). The RRV
vaccine induced a homotypic neutralizing antibody response in 81%
of vaccinees, whereas the two thousand fold greater dose of the
bovine RIT4237 vaccine induced homotypic neutralizing antibodies in
only 45% of vaccinees, which was a statistically significant
difference.
Efficacy trials were also conducted with the WC3 bovine rotavirus
strain. In these trials, the WC3 strain was administered to infants
and young children at a dose range of 10.sup.7.0 to 10.sup.7.3 pfu.
(Clark et al., Am. J. Dis. Child. 140:350-356 (1986)). Although
data regarding the dose required for significant immunogenicity was
not provided, Clark et al. noted that the WC3 strain appears to
possess safety characteristics similar to those of RIT4237, yet was
immunogenic at a dose at least five fold less than that used with
bovine RIT4237, though this immunogenicity still required a dose
that was considerably greater than that of rhesus rotavirus
vaccine.
The WC3 rotavirus strain has been used as one of the parent strains
for generating reassortants with various human rotavirus strains.
(Clark et al., J. Infect. Dis. (suppl.) 174:73-80 (1996)). In one
efficacy trial, 10.sup.73 pfu of a monovalent reassortant of WC3
and a human rotavirus VP7 serotype 1 was administered on a three
dose schedule to infants and young children. (Treanor et al., Ped.
Inf. Dis. J. 14:301-307 (1995)). Immunogenicity data was not
reported for this trial. In another efficacy study, a quadrivalent
formulation was used which contained three human VP7 reassortants
of bovine rotavirus WC3 with a human rotavirus VP7 serotype of 1,
2, or 3 and as a fourth component, a human x bovine reassortant
bearing a human rotavirus VP4 protein with the remaining genes
derived from the bovine rotavirus WC3. Each of the three VP7
reassortants was used at a dose of 10.sup.7.0 pfu, while the VP4
reassortant was administered at a dosage of 5.times.10.sup.6.0 pfu.
(Clark et al., Arch. Virol. (suppl.) 12:187-198 (1996); Clark et
al. J. Infect. Dis. (suppl.) 174:73-80 (1996); Vesikari et al.
Arch. Virol. (suppl.) 12:177-186 (1996)). Immunogenicity data for
this trial also was not reported, but these studies indicate that
to characteristically produce a protective response similar to that
obtained with the rhesus rotavirus or human.times.rhesus
reassortant vaccines a dosage of 10.sup.7 to 10.sup.8.3 pfu was
required. (Clark et al. Arch. Virol. (suppl.) 12:187-198 (1996);
Vesikari et al. Arch. Virol. (suppl.) 12:177-186 (1996)). This
dosage is 10 to 100 times higher than that for the rhesus rotavirus
and human.times.rhesus rotavirus reassortant vaccine
compositions.
Multivalent rotavirus vaccine compositions have been developed. In
particular, three human.times.rhesus rotavirus reassortants
representing human serotypes 1, 2 and 4 have been combined with a
rhesus rotavirus strain (RRV) (the latter sharing neutralization
specificity with human serotype 3) to form a quadrivalent vaccine
composition (Perez-Schael et al., J. Clin. Microbiol. 28:553-558
(1990), Flores et al., J. Clin. Microbiol. 31:2439-2445 (1993)). As
with the monovalent rhesus rotavirus, the human.times.rhesus
reassortant rotavirus vaccine compositions were found to produce a
transient low level febrile condition in approximately 15% to 33%
of the infants vaccinated (Perez-Schael et al. supra). This
transient febrile episode or condition, although generally
considered acceptable by the parents and health care providers of
the clinical trial, could possibly be a deterrent in certain
situations, such as, in premature infants who may have low levels
of passively acquired maternal antibodies to rotavirus and the
like.
Although the animal rotavirus-based rotavirus vaccine composition
presently licensed by the United States Food and Drug
Administration provides an important level of protection in humans
against rotavirus infection, a multivalent vaccine composition with
both high infectivity and which produce little or no febrile
response is desirable, especially for certain clinical situations.
Surprisingly, the present invention fulfills these and other
related needs.
SUMMARY OF THE INVENTION
The present invention provides an immunogenic composition of
human.times.bovine reassortant rotaviruses. The human.times.bovine
reassortants are provided in multivalent compositions in an amount
sufficient to induce an immune response to each serotype of human
rotavirus of current and future clinical importance in a dose of
sufficient infectivity to overcome previous practical limitations
of the art without causing a transient low level fever in a human
host. Further components of the immunogenic composition can include
a physiologically acceptable carrier and optionally an adjuvant to
enhance the immune response of the host. In certain embodiments,
the human.times.bovine reassortant rotavirus VP7 antigen is derived
from a human parent rotavirus strain. e.g., from a human rotavirus
of serotype 1, serotype 2, serotype 3, serotype 4, serotype 5,
serotype 9, or from a bovine parent rotavirus strain of serotype
10. The remaining genes which encode the other rotavirus proteins
are derived from a bovine rotavirus strain. In a preferred
embodiment the bovine rotavirus UK-Compton strain is used. A
particularly preferred immunogenic composition which provides
coverage for VP7 serotypes 1, 2, 3 and 4 comprise a quadrivalent
composition which includes human.times.bovine rotavirus
reassortants D.times.UK, DS-1.times.UK, P.times.UK and
ST3.times.UK, respectively.
In further embodiments, additional human.times.bovine rotavirus
reassortants corresponding to human rotavirus VP7 serotypes 5,
and/or 9, or a bovine.times.bovine reassortant crossreactive with
human rotavirus VP7 serotype 10, or a human.times.bovine
reassortant rotavirus containing a human rotavirus VP4 serotype 1A
can be included to provide an immunogenic composition with a
broader range of use. Of particular interest are a pentavalent
composition comprising rotavirus reassortants D.times.UK,
DS-1.times.UK, P.times.UK, ST-3.times.UK and Wa(VP4).times.UK
stains; a hexavalent composition comprising the pentavalent
composition noted above, plus a VP7, serotype 9.times.UK strain,
and a septavalent composition comprising the hexavalent composition
noted above plus a VP7 serotype 5.times.UK strain or the
septavalent composition noted above plus a VP7 serotype 10.times.UK
strain. Additional strains of rotavirus as they are recognized to
produce significant disease in humans can also be made into bovine
rotavirus reassortants and added to an immunogenic composition of
the present invention. The immunogenic composition of the present
invention will typically be formulated in a dose of less than about
10.sup.6.0 plaque forming units of each rotavirus VP7 or VP4
serotype reassortant. It is particularly preferred that the dosage
is between about 10.sup.5 to less than about 10.sup.6 plaque
forming units.
In other embodiments, the invention provides methods for
stimulating the immune system to produce an immunogenic response to
human rotavirus antigens with little or no accompanying transient
low level fever. These methods comprise administering to an
individual an immunogenically sufficient amount of a multivalent
human.times.bovine reassortant rotavirus composition comprising at
least four VP7 serotypes of human rotavirus. In a preferred
embodiment the human.times.bovine reassortant rotavirus which
comprise the composition include a human rotavirus serotype
1.times. bovine rotavirus strain UK, a human rotavirus serotype
2.times. bovine rotavirus strain UK, a human rotavirus serotype
3.times. bovine rotavirus strain UK, and a human rotavirus serotype
4.times. bovine rotavirus strain UK. The multivalent composition
can also include, but is not limited to, i.e., a human.times.bovine
reassortant rotavirus of serotype 5, and/or serotype 9, or a
bovine.times.bovine reassortant rotavirus with human rotavirus VP7
serotype 10 specificity, or a human rotavirus serotype VP4
1A.times.bovine rotavirus UK reassortant and the like. Further, as
additional rotavirus serotypes are recognized as important in human
disease, they too can be added to an immunogenic composition of the
present invention and used in methods for stimulating the immune
system to produce an immunogenic response to currently recognized
and newly recognized rotaviruses of clinical significance.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention provides immunogenic rotavirus compositions
for use in humans. The compositions described herein are produced
by combining monovalent reassortant human.times.animal rotaviruses
so as to provide one of each of the most clinically relevant
serotypes of group A human rotavirus in a formulation which induces
a rotavirus-specific antibody response without an attendant
transient low level febrile response.
Thus, the immunogenic compositions of the invention specifically
comprise a combination of reassortant human.times.bovine
rotaviruses and a physiologically acceptable carrier to form a
multivalent composition. In a particular embodiment, the
multivalent immunogenic composition comprises a combination of four
reassortant human.times.bovine rotaviruses of the clinically
relevant serotypes of human rotavirus that are most prevalent
world-wide, to form a quadrivalent composition. The immunogenic
composition is administered in an immunogenically sufficient amount
to an individual in need of immunological protection against
rotavirus, such as, e.g. an infant, child or adult. The composition
elicits the production of an immune response that is at least
partially protective against symptoms of serious rotaviral disease,
such as severe diarrhea and dehydration, when the individual is
subsequently infected with a wild-type human rotavirus strain. As
the reassorted viruses of the immunogenic composition infect the
host alimentary tract, some mild disease may occur as a result of
the vaccination, but typically the immunogenic composition of the
present invention will not cause clinically relevant fever or
reaction in the vaccinee. Following vaccination, there are
detectable levels of host engendered serum antibodies which are
capable of neutralizing the serotypes of rotavirus that make up the
immunogenic composition. In particular, the multivalent immunogenic
composition of the present invention will produce an immunological
response to most, if not all, of the clinically relevant group A
human rotaviruses prevalent in different settings. The teachings of
the present invention are not limited to those human rotavirus
serotypes currently recognized of clinically relevant, but also
include those serotypes of human rotavirus that emerge as
clinically relevant in the future.
The reassorted rotavirus which is a component of the multivalent
immunogenic composition of the present invention is in an isolated
and typically purified form. By isolated is meant to refer to
reassorted rotavirus that has been separated from other cellular
and viral products of its manufacture, such as wild type virus and
other heterologous components of a cell culture or other
systems.
Generally, rotavirus reassortants are produced by coinfection of
mammalian cells in culture with a tissue culture-adapted animal
rotavirus. i.e., bovine, rhesus, and the like, and a tissue
culture-adapted human rotavirus. Typically. African green monkey
kidney (AGMK) cells are used as the host cells for co-infection.
Following co-infection with the animal and human rotavirus strains,
selection of the desired reassortant is typically achieved by
exposing the growth yield of co-infected cultures to neutralizing
antibodies specific for the protein product of the animal rotavirus
gene that is to be replaced by the human rotavirus gene (See, U.S.
Pat. No. 4,571,385, incorporated herein by reference). In
particular, polyclonal serum or monoclonal antibody specific for
bovine rotavirus VP7 and/or VP4 proteins can be used. After several
rounds of plaque purification and subculture, selected reassortants
are characterized for serotype and genotype. Serotype is typically
determined by plaque reduction neutralization (PRN) assay or enzyme
immunoassay. Genotype is typically determined by gel
electrophoresis and RNA-RNA hybridization of the viral genome.
Rotavirus reassortants having only the human VP7 or VP4 gene are
typically selected for the present multivalent immunogenic
compositions. Reassortants comprising multiple human rotavirus
genes can also be used. In this regard, reassortant rotaviruses of
interest are particularly those encoding the human rotavirus VP7
and/or the human rotavirus VP4 gene products.
In the present invention, particularly preferred rotavirus
reassortants are human rotavirus and bovine rotavirus reassortants
comprising the human rotavirus gene encoding VP7 and the remaining
ten rotavirus genes of bovine rotavirus origin. The bovine
rotavirus strain UK (Woode et al., Res. Vet. Sci. 16:102-105
(1974); Bridger and Woode, Br. Vet. J., 131:528-535 (1975)) is
particularly preferred because of its pedigree and as demonstrated
by the present invention its higher level of infectivity in humans.
The high infectivity level of the UK bovine rotavirus reassortants
demonstrated herein means a lower dose is needed, and consequently
the manufacturing cost per dose should be significantly less than
other presently known bovine rotavirus reassortant vaccines. Other
animal rotavirus strains can also be used to make reassortant
rotavirus as long as the compositions are capable of inducing a
serologic response in a vaccinee when administered at a dosage of
less than 10.sup.6.0 plaque forming units for each rotavirus
serotype and do not produce a transient low level febrile response.
For example, in certain embodiments the reassortant rotavirus
comprises an animal VP7 antigen which is immunologically
cross-reactive with human VP7 serotype 10. This reassortant
rotavirus can be a bovine.times.bovine reassortant.
In an alternative embodiment, reassortant rotavirus of a specific
serotype can be produced using a previously obtained reassortant.
For example, to produce additional bovine UK reassortants the human
rotavirus VP7 serotype 1 D strain.times.bovine UK reassortant
HD/BRV-1 (ATCC VR-2069) can be used to produce human
rotavirus.times.bovine UK reassortants having human VP7 serotypes
of 2, 3, 4, 9, and/or bovine rotavirus VP7 serotype 10. The methods
used are similar to those described above except polyclonal or
monoclonal neutralizing antibody specific for the VP7 serotype of
the parental human rotavirus reassortant is used to select for new
reassortants of the desired human (and/or bovine) rotavirus VP7
serotype. It is also contemplated as part of the present invention
that as other clinically relevant human VP4 or VP7 serotypes are
isolated and identified reassortant rotavirus of the newly
discovered serotype can be produced by the described methods.
Propagation of the reassorted rotavirus can be in a number of cell
cultures which support rotavirus growth. Preferred cell cultures
for propagation of rotavirus reassortants for vaccine use include
primary or secondary simian African green monkey kidney cells
(AGMK), qualified diploid simian FRhL-2 cells and qualified simian
heteroploid Vero cells. Cells are typically inoculated with
rotavirus reassortants at a multiplicity of infection ranging from
about 0.1 to 1.0 per cell, or more, and are cultivated under
conditions appropriate for viral replication, for about 3-5 days,
or as long as necessary for virus to reach an adequate titer.
Rotavirus reassortants are harvested from infected cell culture and
separated from cellular components, typically by well known
clarification procedures, e.g., centrifugation, and may be purified
as desired using procedures well known to those skilled in the
art.
In a preferred embodiment for use as an immunogenic composition, a
human x bovine reassortant rotavirus of serotype 1, serotype 2,
serotype 3 and serotype 4 are used as a quadrivalent vaccine.
Typically, the human.times.bovine reassortant rotavirus of each of
the four serotypes will be admixed to form a combined composition
for simultaneous administration. The final ratio of each rotavirus
serotype is determined by the immunogenicity of the individual
rotavirus reassortants. Although not preferred, each
human.times.bovine reassortant, or a combination thereof, can also
be administered in a sequential manner to provide an effective
vaccine formulation.
In other preferred embodiments the human.times.bovine reassortant
rotavirus of serotype 1, serotype 2, serotype 3 and serotype 4 are
combined with a human.times.bovine reassortant rotavirus of VP7
serotype 5 and/or 9, a bovine.times.bovine reassortant rotavirus of
VP7 serotype 10, and/or a human.times.bovine reassortant rotavirus
of VP4 serotype 1A to yield a multivalent immunogenic composition.
The additional reassortant rotaviruses just described can be used
in any combination for use as a hexavalent, septavalent, or
octavalent immunogenic composition.
Human.times.bovine reassortant rotavirus multivalent immunogenic
compositions of the present invention contain as an active
ingredient an immunogenically effective amount of each of at least
the four clinically most important VP7 serotypes of human rotavirus
as described herein. In particular, each antigenically distinct
human rotavirus reassortant is administered at a dosage of less
than 10.sup.6.0 plaque forming units. The immunogenic composition
may be introduced into a host, particularly humans, with a
physiologically acceptable carrier and/or adjuvant. Useful carriers
include.e.g., citrate-bicarbonate buffer, buffered water, normal
saline, and the like. The resulting aqueous solutions may be
packaged for use as is, or lyophilized, as desired, using
lyophilization protocols well known to the artisan. Lyophilized
virus will typically be maintained at about 4.degree. C. When ready
for use the lyophilized preparation is combined with a sterile
solution prior to administration, as mentioned above.
The compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, tri-ethanolamine
oleate, citrate-bicarbonate, or the like. When the composition is
administered orally it may also be necessary to provide the
individual a buffer solution to partially neutralize stomach acid
and protect the reassortant rotavirus while passing to the
intestine. Buffer solutions appropriate for this use include sodium
bicarbonate, citrate bicarbonate, or the like. Upon immunization
with a human.times.bovine reassortant rotavirus composition of the
present invention, particularly via the oral route, the immune
system of the host responds to the composition by producing both
local secretory and serum antibodies specific for the rotavirus
proteins. As a result of the administration of the composition, the
host becomes at least partially or completely immune to human
rotavirus disease caused by a wild-type strain that corresponds to
the immunizing serotype(s). If wild-type virus infection does
occur, the host is resistant to developing moderate or severe
rotaviral disease, particularly of the gastrointestinal tract.
The multivalent immunogenic compositions of the present invention
containing the human.times.bovine reassortant rotaviruses are
administered to a person, particularly an infant, susceptible to or
otherwise at risk of rotavirus disease to induce the individual's
own immune response capabilities. Such an amount is defined to be
an "immunogenically effective dose." Immunogenicity or
"immunogenically effective dose" as used in the present invention
means the development in a vaccinee of a cellular and/or antibody
mediated immune response to the vaccine composition. Usually such a
response consists of the vaccinee producing serum antibodies, B
cells, helper T cells, suppressor T cells, and/or cytotoxic T cells
directed specifically to an antigen or antigens included in the
vaccine composition of the present invention. A four-fold or
greater rise above a preinoculation antibody titer following
immunization measured by a rotavirus group-specific, or rotavirus
serotype-specific assay is considered a significant response.
In this use, the precise amount of each human.times.bovine
reassortant rotaviral serotype in a particular immunogenic
composition depends on the patient's age, state of health and
weight, the mode of administration, the nature of the formulation,
etc., but generally the range was from about 10.sup.4 to about
10.sup.6 plaque forming units, preferably from about 10.sup.5 to
less than 10.sup.6 plaque forming units (pfu) of each serotype per
patient.
In any event, the formulations for the immunogenic composition
should provide a quantity of each human.times.bovine reassortant
rotavirus of the invention sufficient to induce an individual's
immune response against rotavirus disease. Preferably, this immune
response will effectively protect the individual against serious or
life-threatening rotavirus disease without being "reactogenic." As
used herein, "reactogenic" or reactogenicity denote a mild
transient fever occurring during the week following administration
of the immunogenic composition. A fever is defined in the context
of the present invention as the development of an oral temperature
of greater than or equal to 38.degree. C. in an adult, or a rectal
temperature of greater than or equal to 38.1.degree. C. in a
pediatric vaccinee.
In some instances it may be advantageous to combine the preferred
quadrivalent human.times.bovine reassortant rotaviral compositions
of the present invention with other serotypes of human rotavirus or
other infectious agents, particularly, other gastrointestinal
viruses. For example, the quadrivalent human.times.bovine
reassortant rotaviral compositions of the present invention can
further include, for example, human.times.bovine reassortant
rotavirus of serotype 5 (Timenetsky et al., J. General Virol.
78:1373-1378 (1997)), and/or serotype 9 (Nakagomi et al. Microbiol.
Immunol. 34:77-82 (1990)), and/or bovine.times.bovine reassortant
rotavirus which is cross reactive with human rotavirus serotype 10
and/or human.times.bovine reassortant rotavirus of VP4 serotype 1A.
Administration can be simultaneous (but typically separately) or
sequentially with another possible gastrointestinal virus vaccine,
such as a human calicivirus (e.g., Norwalk virus) or related
vaccine.
Single or multiple administrations of the immunogenic compositions
of the invention can be carried out. In neonates and infants,
multiple administrations may be required to elicit a sufficient
level of immunity, particularly where there are high levels of
maternally derived antibodies specific for rotavirus.
Administration should begin within the first 2-4 months of life,
and continue at intervals such as one to two months or more after
the initial immunization, or as necessary to induce and maintain
sufficient levels of immunity against human rotavirus infection.
Similarly, adults who are particularly susceptible to repeated or
serious rotavirus disease, such as, for example, health care
workers, day care workers, family members of young children, the
elderly, etc. may require multiple immunizations to establish
and/or maintain an effective immune response. Levels of induced
immunity can be monitored by measuring amounts of rotavirus
group-specific antibodies or serotype-specific neutralizing
antibodies in serum and secretions, and dosages adjusted or
vaccinations repeated with one or more serotypes of a multivalent
reassortant rotavirus composition of the present invention when
necessary to maintain desired levels of immunity.
The following examples are offered by way of illustration, not by
way of limitation.
Example I
This Example describes the production of rotavirus reassortants
derived from human rotavirus strains D (VP7:1), DS-1 (VP7:2), P
(VP7:3) and ST3 (VP7:4), and bovine UK Compton (UK) rotavirus and
the evaluation of the safety, immunogenicity and reactogenicity of
each reassortant individually in adults, children, and infants.
Human.times.bovine reassortant rotavirus strains representing VP7
serotypes 1, 2, 3 and 4 were derived from the bovine UK Compton
(UK) strain and from human rotavirus strains D (VP7 serotype 1,
ATCC VR-970), DS-1 (VP7 serotype 2; Wyatt et al., Perspect. Virol.
10:121-145 (1978)) and P (VP7 serotype 3; Wyatt et al., Science
207:189-171 (1980)), and ST3 (VP7 serotype 4; Banatvala et al., J.
Am. Vet. Med. Assoc. 173:527-530 (1978)). Human rotavirus strains
D, DS-1, and P were recovered from stools of children hospitalized
with diarrhea; Strains D and DS-1 were propagated and passaged in
gnotobiotic calves (Wyatt et al., 1978, supra; and Midthun et al.,
1985, J. Virol. 53:949-954) and later grown only in tissue culture,
while strain P was grown only in AGMK tissue culture. Human
rotavirus strain ST3 was isolated from a stool of an asymptomatic
neonate and passaged in AGMK cells. The bovine UK Compton rotavirus
strain was isolated in primary calf kidney cells from the stool of
a colostrum-deprived calf with diarrhea. (Woode et al., Res. Vet.
Sci. 16:102-105 (1974)). The further passage of this virus in
primary calf kidney cells was carried out by Flewett et al., at the
Regional Virus Laboratory, East Birmingham Hospital, Birmingham,
England and sent to the National Institutes of Health, Bethesda,
Md. At the NIH the virus was serially passaged in primary bovine
embryonic kidney cells, primary AGMK cells, and in diploid simian
DBS-FRhL cells. The seed pool contained virus that was plaque
purified in AGMK cells and passaged in primary calf kidney cell
culture.
The individual human.times.bovine rotavirus reassortants with a
single VP7 encoding gene derived from human rotavirus D, DS-1, P or
ST3 strain and the remaining 10 genes derived from the bovine UK
strain (lot BR-3, clone 22) have been described (Midthun et al. J.
Clin. Microbiol. 24:822-826 (1986) and Midthun et al., J. Virol.
53:949-954 (1985), U.S. Pat. No. 4,571,385 all of which are
incorporated herein by reference). The D.times.UK, DS-1.times.UK.
P.times.UK and ST3.times.UK vaccine suspensions used in these
clinical trials. i.e., lot HD BRV-1, clone 47-1-1 (ATCC VR-2069 and
ATCC VR-2617), 10.sup.5.8 pfu/ml; lot HDS1 BRV-1, clone 66-1-1
(ATCC VR-2616), 10.sup.5.3 pfu/ml; lot HP BRV-2, clone 22-1-1 (ATCC
VR-2611), 10.sup.5.3; and lot ST3 BRV-2, clone 52-1-1 (ATCC
VR-2612), 10.sup.5.8 pfu/ml respectively, were prepared and
successfully safety tested to confirm freedom from adventitious
agents in accordance with the guidelines of the U.S. Food and Drug
Administration as well known to the skilled artisan.
All pediatric studies and one study in adults with the D.times.UK
human.times.bovine reassortant rotavirus were conducted in a
randomized, placebo-controlled manner to assess the safety and
immunogenicity of each candidate rotavirus vaccine strain. The
safety of each human.times.bovine reassortant rotavirus was
evaluated sequentially in adults 18 to 45 years of age, in children
6 to 60 months of age, and finally in infants 1.5 to 5.9 months of
age. The various studies were carried out at either the Johns
Hopkins University Center for Immunization Research, Baltimore, Md.
or the Vaccine Clinic, Vanderbilt University, Nashville, Tenn.
The criteria for selection of adult and pediatric subjects for
rotavirus vaccine trials have been described in Halsey et al., J.
Infect. Dis. 158:1261-1267 (1988). An undiluted dose of each
rotavirus reassortant was evaluated in adults initially.
Subsequently, a 1:10 dilution of each reassortant and later an
undiluted dose (10.sup.5.3 pfu) of P.times.UK were evaluated in
children 6 to 60 months of age. After the safety of each
reassortant had been demonstrated in these children, a 1:10 dose
and an undiluted dose of D.times.UK and DS-1.times.UK were also
evaluated sequentially in infants <6 months old. Since it
appeared that an undiluted dose of these reassortants was required
to infect the majority of the young infants, the P.times.UK or
ST3.times.UK reassortant was administered undiluted to infants
<6 months old.
Initially, the safety of 10.sup.5.8 pfu of the D.times.UK
reassortant rotavirus strain was evaluated in five healthy adult
volunteers who had a low level of VP7 serotype 1 specific
neutralizing antibodies in their serum. The clinical procedures for
the studies with adults were those previously described in Halsey
et al., supra, with a few exceptions. Briefly, all subjects fasted
for at least 1 hour before and after each feeding of rotavirus.
Each adult volunteer drank 120 ml of distilled water with 2 g of
NaHCO.sub.3, followed 1 min. later by 1 ml of undiluted candidate
vaccine suspended in 30 ml of buffered solution or 31 ml of placebo
(buffered solution without the vaccine). Oral temperature was
recorded twice daily and any elevated temperature was rechecked
within 20 minutes. Stool samples were collected for 7 days
following the administration of rotavirus and the consistency and
number of stools recorded and any symptoms were also recorded daily
for 7 days after vaccination.
Most of the clinical procedures for the pediatric studies were also
identical to those described by Halsey (supra.), with a few
exceptions. Briefly, routine childhood immunizations appropriate
for the child's age were given on schedule, and at least two weeks
before or after administration of rotavirus or placebo. After
fasting one hour, each pediatric subject was randomized to receive
rotavirus or placebo in a 2:1 ratio. Each child drank 30 ml of
infant formula (Similac; Ross Laboratories, Columbus, Ohio) mixed
with 0.4 g of NaHCO.sub.3, and then drank 1 ml of rotavirus
reassortant or placebo (buffered formula or Eagle's Minimal
Essential Medium). Infants <6 months of age who received the
10.sup.5.8 pfu of D.times.UK rotavirus reassortant were offered a
second dose of this virus 4 to 12 weeks after the first dose in an
attempt to increase immunogenicity.
In studies of the D.times.UK and DS-1.times.UK reassortants, rectal
temperatures were taken once or twice a day, and symptoms, if any,
were recorded daily. Parents were instructed to collect a stool
sample daily and record the number and consistency of stools passed
by their child daily. Procedures for pediatric studies of
P.times.UK and ST-3.times.UK were similar with slight
modifications.
Study subjects were considered to have "rotavirus-like illness."
(i.e., an illness that could possibly be caused by a rotavirus, if
they had diarrhea, or any episode of frank vomiting or fever during
the 7-day period after oral administration of rotavirus. Diarrhea
was defined as three or more unformed stools within 48 hours. Fever
was defined as an oral temperature .gtoreq.37.8.degree. C. in
adults or a rectal temperature .gtoreq.38.1.degree. C. in pediatric
subjects, confirmed within 10-20 minutes.
Blood was collected from each study participant before and 4-6
weeks after administration of rotavirus for measurement of
rotavirus-specific antibodies and serum alanine aminotransferase
(ALT) level: the latter was used to ascertain whether the vaccine
adversely affected liver functions. In adults, an additional blood
specimen was also collected one week after administration of
rotavirus and used for measurement of ALT level.
Prevaccination and postvaccination sera were tested for
rotavirus-specific IgA and IgG antibodies by ELISA, using rhesus
rotavirus as a group-specific antigen as described in Midthun et
al., J. Clin. Microbiol. 27:2799-2804 (1989) and Hoshino et al., J.
Clin. Microbiol. 21:425-430 (1985); each incorporated by reference
herein. Paired sera were also tested by plaque reduction
neutralization (PRN) antibody assay as described in Midthun et al.,
J. Clin. Microbiol. 27:2799-2804 (1989). Rotaviruses used in the
PRN assay included: Wa (serotype 1), DS-1 (serotype 2), P (serotype
3) and ST3 or VA70 (serotype 4) human rotavirus strains plus:
D.times.UK, DS-1.times.UK, P.times.UK, and ST3.times.UK reassortant
strains and the UK (Compton) bovine rotavirus strain. A fourfold or
greater rise in antibody titer in the postvaccination serum
compared to the prevaccination serum measured by ELISA IgA or ELISA
IgG, or PRN antibody assay was considered a significant
response.
Frozen stool samples were thawed and made into 10% stool
suspensions in veal infusion broth. The stool suspensions were
inoculated onto simian MA104 cell culture tubes and incubated in a
roller drum at 37.degree. C. for 7 days. The supernatant from the
cell culture was blind passaged onto fresh simian MA104 cell
culture tubes and incubated at 37.degree. C. for 7 days. The 10%
stool suspension and the supernatants from each set of cultures
were stored at -20.degree. C., until later when they were thawed
and tested for rotavirus by ELISA. Selected rotavirus positive
stool specimens collected following vaccination were serotyped by
polymerase chain reaction to determine the serotype of rotavirus
shed (Gouvea et al., J. Clin. Microbiol. 28:276-282 (1990) and
Gouvea et al. J. Clin. Microbiol. 32:1333-1337 (1994), each
incorporated by reference herein).
Diarrheal stools of study subjects were examined for ova and
parasites, and they were tested for salmonella, shigella,
campylobacter, aeromonas, yersinia, enterovirus, adenovirus, and
rotavirus. Diarrheal stools were also examined by electron
microscopy for rotavirus and other viral particles. To detect
adventitious agents associated with intercurrent illness, nasal
swabs or nasal wash specimens were collected from study subjects
who had fever and respiratory symptoms during the 7-day observation
period in studies of P.times.UK and ST3.times.UK reassortants, and
these specimens were tested in cell culture for respiratory
viruses.
The rates of illness of vaccinees and placebo recipients and the
rates of serologic response for these groups within each age group
and in each study were compared using a two-tailed Fisher's exact
test.
The percentages of adults, children and infants who had rotavirus
detected in their stools or developed a fourfold or greater rise in
serum antibody titer(s) after a single oral administration of each
of the VP7-serotype-specific human.times.UK bovine rotavirus
reassortants are shown in Table 1. Rotavirus was not recovered in
cell culture from the stools of any of the adult vaccinees, infants
<6 months old fed undiluted P.times.UK reassortant, or placebo
recipients. Only a small proportion of (i) the children 6-60 months
old given a 1:10 dilution of the D.times.UK, DS-1.times.UK or
P.times.UK reassortant or (ii) the infants under 6 months of age
administered an undiluted dose of the D.times.UK or DS-1.times.UK
strain shed rotavirus. In most cases rotavirus was detected after
the second cell culture passage of only one or two stool samples.
In contrast, the ST3.times.UK virus was recovered from stools of
the infants and young children more frequently and for a longer
period (usually stools collected over a period of 3 or more days,
especially during days 5-7 postvaccination). The ST3.times.UK virus
was isolated from the stool of one 23-month-old child on day 30
postvaccination and confirmed by PCR. Quantitation of the virus
recovered from stools of nine ST3.times.UK rotavirus reassortant
recipients indicated the maximum amount of virus shed was
4.7.times.10.sup.2 plaque forming units (pfu) per ml of the 10%
stool suspension.
Tests by PCR of 10% stool suspensions (9 of 9) or tissue culture
passages of stools (1 of 1) from ten vaccine recipients confirmed
the shedding of the ST3.times.UK rotavirus reassortant in all ten
vaccinees. However, three of these vaccinees also shed a wild-type
rotavirus: a VP7 serotype 1 human rotavirus strain (one infant) or
a VP7 serotype 3 strain (one child and one infant). Data from these
three pediatric patients, who did not have rotavirus-like illness,
were excluded from the serologic analysis.
Serologic responses to rotavirus were detected less often in adults
and older children (who probably had been infected with wild-type
rotavirus previously) than in young infants (Table 1). Serologic
responses were detected in 23%, 18% and 15% of the adults who were
fed the D.times.UK, DS-1.times.UK or ST3.times.UK reassortant,
respectively, but in none of the adults inoculated with the
P.times.UK reassortant. Among the children 6-60 months-old who
received a 1:10 dilution, serologic responses to rotavirus were
detected in 33% of D.times.UK recipients. 40% of DS-1.times.UK
recipients, and 57% of ST3.times.UK recipients. The 10.sup.4' pfu
dose of P.times.UK reassortant failed to elicit any antibody
responses in children between 6 and 60 months of age, but a
ten-fold higher dose of this composition was moderately
immunogenic, with antibody responses detected in 5 of 11 (45%)
children in this age group who were given this reassortant
orally.
TABLE-US-00001 TABLE 1 Virologic and serologic responses of
infants, children and adults following a single dose of human VP7
serotype-specific-bovine UK rotavirus reassortant vaccines % With a
Fourfold or Greater Antibody Rise by Indicated Assay Plaque
Reduction Neutralization % Who Human ELISA Age Group Dose Given No.
of % Shed Vaccine- Rotavirus Any Vaccine Strain (log.sub.10pfu)
subjects Infected Rotavirus Strain Parent IgA IgG Assay Infants,
1.5-5.9 months D x UK 4.8 8 63 38 50 50 63 38 63 D x UK 5.8 20 50
15 30 30 40 35 50 DS-1 x UK 4.3 8 63 0 38 NT 38 50 63 DS-1 x UK 5.3
11 82 18 82 0 18 18 82 P x UK 5.3 10 80 10 70 10 10 10 80 ST3 x UK
5.8 14 93** 64 92 9.dagger. 25 44 92 Children, 6-60 months D x UK
4.8 9 33 11 22 33 11 11 33 DS-1 x UK 4.3 10 40 10 40 NT 40 40 40 P
x UK 4.3 10 10 10 0 NT 0 0 0 P x UK 5.3 11 45 0 36 NT 9 27 45 ST3 x
UK 4.8 8 63** 63 43 17.dagger. 43 57 57 Adults, 18-45 years D x UK
5.8 13 23 0 8 8 15 8 23 DS- I x UK 5.3 11 18 0 9 NT 9 18 18 P x UK
5.3 12 0 NT NT NT 0 0 18 ST3 x UK 5.8 20 15 0 0 0 10 10 15 Note:
pfu -- plaque forming units; NT = not tested. Infection was defined
as evidence of virus shedding of a fourfold or greater rise in
titer of serum rotavirus-specific antibody measured by ELISA IgA or
IgG assay or plaque reduction neutralization assay. *The rotavirus
vaccine and human rotavirus strains used in plaque reduction
neutralization assays were D x UK and Wa; DS-1 x UK and DS-1; P x
UK and P; and ST3 x UK and ST3 strains. .dagger.Serum specimens
from one infant and two children were not tested. ** One child and
two infants who shed rotavirus reassortant who had evidence of
wild-type rotavirus in their stool by PCR analysis. The serologic
results of these three vaccinees were excluded from the analysis of
antibody responses.
Among infants <6 months of age who were fed rotavirus, serologic
responses, (a measure of immunogenicity), were detected more often
(93%) in recipients of an undiluted dose of human.times.bovine UK
reassortant derived from the ST3 human rotavirus strain than in
those who received a 1:10 or undiluted dose of the
human.times.bovine UK reassortants derived from the D (50-63%),
DS-1 (82%), or P (63-80%) human rotavirus strain. These differences
with regard to dose were not statistically significant. Overall,
antibody responses were detected more often by plaque reduction
neutralization (PRN) assay (using the homologous reassortant virus
as antigen) than by the ELISA IgA or IgG assay (using rhesus
rotavirus as a group-specific antigen) in all vaccine groups except
for the D.times.UK vaccine group in which antibody responses were
detected most often by the IgA ELISA. For three of the
reassortants, the neutralizing antibodies in the postimmunization
sera of the vaccinated infants were directed more often against the
reassortant rotavirus than the human rotavirus parent strain,
suggesting that the VP4 neutralization antigen of the bovine
rotavirus was immunodominant. This was particularly evident in the
results of PRN tests on postimmunization sera of 11 infants who
were fed 10.sup.5.3 pfu of DS-1.times.UK and who developed a
significant increase in neutralizing antibodies against the UK
bovine rotavirus parent strain (data not shown) or the
DS-1.times.UK reassortant at a rate of 55% and 82%, respectively,
whereas none of them developed a significant increase in
neutralizing antibodies to the DS-1 human rotavirus parent
strain.
This absolute dissociation of VP7 and VP4 responses was not the
rule, however, because infant vaccinees who received a single dose
of the type 1(D) human.times.bovine rotavirus reassortant developed
a VP7-specific neutralizing antibody response to the human
rotavirus serotype 1 parent in 50% of instances. In addition, a
homotypic VP7 neutralizing antibody response was also observed,
albeit at a lower frequency, for human rotavirus type 3 (10%) and
type 4 (8%). It should be noted that, as in Example II for the
quadrivalent formulation infra, immunogenicity of the
human.times.bovine rotavirus reassortants was considerably greater
when three sequential doses of the reassortants were administered
at two month intervals. Thus, this expanded immunization schedule
induced a VP7-specific neutralizing antibody response to each of
the human rotavirus parents of the reassortants that in the case of
serotypes 2, 3, and 4 significantly exceeded the response observed
following a single dose of reassortant. Specifically. 32% of infant
vaccinees responded to serotype 2. 33% to serotype 3 and 42% to
serotype 4. Improvement was not noted for serotype 1 where
immunogenicity was already high after a single dose of this
reassortant.
There was no evidence that rotavirus was shed in stools of placebo
recipients, nor was there evidence for a rotavirus serologic
response in this group with the exception of one 6-month-old
placebo recipient who had a four fold rise in titer of ELISA IgG
antibody, but no other serologic response in the other assays.
Each of the rotavirus reassortants appeared to be safe and well
tolerated, as evidenced by the absence of gastrointestinal illness
in adults, and the lack of a statistically significant increase in
the rate of "rotavirus-like illnesses" between pediatric vaccinees
and placebo recipients in each vaccine group and age group (Table
2). Only three adult volunteers (recipients of P.times.UK or
ST3.times.UK vaccine) had any symptoms (fever) that met the
criteria for "rotavirus-like illness;" however, none of these ill
vaccinees had evidence of rotavirus infection. All three adults who
developed a fever following administration of rotavirus had a
concomitant respiratory illness or shed a respiratory virus that
was detected by tissue culture assay of a nasopharyngeal swab. One
P.times.UK recipient with sinusitis, cough and rhinorrhea had a
positive culture for influenza A virus; another P.times.UK
recipient had a positive culture for respiratory syncytial virus
(RSV); and one ST3.times.UK recipient had cough, rhinorrhea and
hoarseness. The concomitant respiratory illnesses, recovery of
respiratory pathogens and lack of evidence of rotavirus infection
in these volunteers suggest that these fevers probably were due to
an intercurrent respiratory tract infection.
Intercurrent illness also occurred during most of the studies of
the four rotavirus vaccine candidates in pediatric subjects.
Vomiting in association with coughing, and fever associated with
otitis media or respiratory symptoms were common. Despite the high
background of intercurrent illnesses, the rate of "rotavirus-like
illnesses" (fever, diarrhea or vomiting) in 6-60 month-old children
and infants <6 months of age within each vaccine group was not
statistically significantly different from that of the placebo
recipients (Table 2). Overall, 8 of 48 children 6-60 months old who
were given a reassortant rotavirus, and 3 of 27 recipients of the
placebo experienced "rotavirus-like illness" within 7 days after
inoculation. Only two of these rotavirus-like illnesses were
associated with rotavirus infection. Both illnesses, which occurred
after oral administration of the DS-1.times.UK reassortant, were
mild and self-limited: one child had fever (maximum, 38.5.degree.
C.) on day 2; the other child vomited three times on days 2 and
3.
TABLE-US-00002 TABLE 2 Frequency of illness in adults and infants
after oral administration of human VP7 serotype-specific bovine UK
rotavirus reassortant vaccines, or placebo. Dose of Admin- % of
Subjects with Indicated Findings Vaccine Vaccine istered Any
Respiratory Evaluated, (log.sub.10 pfu) or Vaccine/ % Rotavirus-
Illness or Otitis ALT Age Group Placebo Placebo Infected Fever
Diarrhea Vomiting like Illness Media Elevation* D x UK 18-40 yrs.
5.8 13 23 0 0 0 0 0 15 18-40 yrs. 0 8 0 0 0 0 0 0 0 6-60 mos. 4.8 9
33 11 0 11 22 22 0 6-60 mos. 0 5 0 0 20 0 20 0 20 1.5-5.9 mos. 4.8
8 63 13 0 0 13 13 0 1.5-5.9 mos 0 6 0 0 17 0 17 17 0 1.5-5.9 mos.
5.8 20 50 30 15 30 50 15 0 1.5-5.9 mos. 0 10 0 10 20 20 30 0 0 DS-1
x UK 18-40 yrs. 5.3 11 18 0 0 0 0 0 0 6-60 mos. 4.3 10 40 20 0 20
30 20 0 6-60 mos. 0 6 13 33 0 0 33 17 0 1.5-5.9 mos. 4.3 8 63 13 0
13 25 25 0 1.5-5.9mos. 0 4 0 0 0 0 0 0 0 1.5-5.9 mos. 5.3 11 82 9
18 27 45 9 0 1.5-5.9 mos. 0 4 0 25 0 25 50 25 0 P x UK 18-40 yrs.
5.3 12 0 17 0 0 17 8 0 6-60 mos. 4.3 10 10 20 0 10 20 10 0 6-60
mos. 0 5 0 0 0 0 0 40 0 6-60 mos. 5.3 11 45 0 0 0 0 0 0 6-60 mos. 0
6 0 0 0 0 0 0 0 1.5-5.9 mos. 5.3 10 80 0 0 0 0 0 0 1.5-5.9 mos. 0 5
0 20 0 20 20 0 0 ST3 x UK 18-45 yrs. 5.8 20 15 5 0 0 5 25 0 6-60
mos. 4.8 8 63 13 0 0 13 25 0 6-60 mos. 0 5 0 0 0 0 0 80 0 1.5-5.9
mos. 5.8 14 93 21 7 14 36 57 7 1.5-5.9 mos. 0 7 0 0 0 14 14 14 14
Note: Rotavirus-like illness was defined as the presence of fever,
diarrhea or vomiting, as defined in the methods section. *Results
were based on ALT levels measured in blood collected 4-6 after
inoculation.
Coxsackie B5 or an echovirus and cytomegalovirus were isolated from
two children 6-60 months old who were fed ST3.times.UK. Also, an
adenovirus or parainfluenza type 3 virus was isolated from three
placebo recipients in the same study.
Among study subjects <6 months of age, 23 of 71 vaccine
recipients and 8 of 36 placebo recipients experienced
"rotavirus-like illness" within 7 days after the oral
administration of the first dose of rotavirus reassortant. Fever in
infants without respiratory symptoms or otitis media was lower
(range, 38.2-38.3.degree. C.) than in those with respiratory
symptoms or otitis media (range, 38.4-40.degree. C.). The majority
(8 of 12) of infants who vomited had only one or two episodes; none
had vomiting that interfered with feeding or resulted in
dehydration. The rates of "rotavirus-like illness" and respiratory
tract illness or otitis media in infants (classified as infected
vaccinees, uninfected vaccinees or placebo recipients) are shown in
Table 3. Infected vaccinees were vaccine recipients who had
evidence of rotavirus infection after vaccination; uninfected
vaccinees were vaccine recipients in whom there was no evidence of
rotavirus infection after vaccination. There was no consistent
pattern of symptoms among infected vaccinees, nor were there
significant differences between the rates of illnesses in infected
vaccinees and uninfected vaccinees or placebo recipients for each
vaccine group. Suggesting that the observed symptoms were
manifestations of intercurrent illness as elaborated below.
Among the <6 months-old vaccinees with rotavirus-like illness.
14 were vaccine responders. All but three of the vaccine responders
had mild rotavirus-like illnesses with one or two symptoms; two had
fever alone; three had fever with respiratory illness or otitis
media; nine vomited one or more times (maximum 6 times) and three
of them vomited after coughing. Three vaccine responders had
moderate-to-severe "rotavirus-like illness" following feeding of
10.sup.5.8 pfu of ST3.times.UK, but they also had an intercurrent
respiratory virus infection. Adenovirus and cytomegalovirus were
isolated from nasal wash specimens. In addition, the appropriate
rotavirus reassortant was recovered from the stools of one child
who developed high fever (maximum, 40.degree. C.) for 3 days, but
this child also had rhinorrhea, cough, and otitis media for 5 days.
This child was hospitalized and treated empirically with vancomycin
and cephalosporin until sepsis was ruled out; she recovered
uneventfully. Parainfluenza virus and adenovirus were cultured from
nasal specimens of one infant with fever (maximum, 39.3.degree. C.)
and otitis media for two days: this child also shed the appropriate
rotavirus reassortant in stools. Influenza A virus (but not
rotavirus) was isolated from another infant with fever (maximum,
38.6.degree. C.), diarrhea (9 watery stools) over 3 days, and
wheezing, cough and rhinorrhea for 3-4 days.
TABLE-US-00003 TABLE 3 Illness and adventitious agents identified
in infants <6 months-old who were infected or not infected with
a human-bovine UK reassortant rotavirus vaccine or who received
placebo. Percentage of Subjects with Respiratory Reassortant Virus
Fever "Rotavirus-like Illness or Adventitious Agents Evaluated,
Subjects (no.) >38.1* Vomiting Diarrhea Illness" Otitis Media
Identified (no. of subjects) D x UK Infected vaccinees (14) 14 36 7
36 7 -- Uninfected vaccinees (14) 36 7 14 43 21 Aeromonas
hydrophilia (1) Placebo (16) 6 13 19 25 6 -- DS-1 x UK Infected
vaccinees (14) 0 21 7 29 14 -- Uninfected vaccinees (5) 40 40 20 60
20 Campylohacter jejuni (1) Placebo (8) 13 13 0 25 13 -- P x UK
Infected vaccinees (8) 0 0 0 0 0 Uninfected vaccinees (2) 0 0 0 0 0
-- Placebo (5) 20 20 0 20 0 ST3 x UK Infected vaccinees (13) 23 15
8 38 62 RSV (2), parainfluenza (1); adenovirus (2), CMV (1);
influenza (1) Uninfected vaccinees (1) 0 0 0 0 0 -- Placebo (7) 0
14 0 14 14 RSV (1) Note: Infants were considered infected with the
rotavirus reassortant virus administered if they shed rotavirus
and/or had a fourfold or greater increase in serum
rotavirus-specific antibody titer. Rotavirus-like illness was
defined as fever, vomiting or diarrhea. RSV = respiratory syncytial
virus; CMV = cytomegalovirus.
As shown in Table 3, nine of 22<6 months-old vaccinees who had
no evidence of rotavirus infection experienced "rotavirus-like
illness." Campylobacter jejuni was isolated from one infant who had
fever and 34 dysenteric stools after receiving 10.sup.5.3 pfu of
DS-1.times.UK. Aeromonas hvdrophila was isolated from diarrheal
stools of another infant who also had fever and otitis media after
receiving 10.sup.5.8 pfu of D.times.UK. Respiratory syncytial virus
was also isolated from two infants who received the D.times.UK
reassortant as well as from one placebo recipient, each of whom had
rhinorrhea with or without wheezing.
There was no evidence of liver damage resulting from infection with
the rotavirus reassortants. The proportion of pediatric
participants with an ALT elevation 4-6 weeks postinoculation was no
greater in vaccinees than in placebo recipients. Only two pediatric
vaccinees (one infant vaccinated once with ST3.times.UK and another
who received a second dose of D.times.UK) had an elevated ALT
value. This value was less than twice normal and was normal when
repeated within a week. Two placebo recipients also had mildly
elevated ALT values 4-6 weeks postinoculation. Transient, mild
elevations in ALT values were occasionally detected in adult
volunteers (some of whom reported alcohol consumption) after
feeding of the D.times.UK or ST3.times.UK reassortant. None of
these volunteers had evidence of rotavirus infection. Four adults
had ALT elevations one week after administration of D.times.UK;
their ALT levels were normal or less than twice the normal value
(two volunteers) when repeated three weeks later. Two other adults
had an elevated ALT level one week after receiving the ST3.times.UK
reassortant; their ALT values were normal 4 weeks after
administration of rotavirus.
Although only 10 of 20 (50%) infants who received 10.sup.5.8 pfu of
D.times.UK reassortant rotavirus developed rotavirus-specific
antibodies after one dose, a booster immunization of 14 infants
with this reassortant 4-12 weeks later elicited a fourfold or
greater increase in antibody titer in 12 of the 14 (86%) infants,
including 7 infants who had mounted an antibody response after the
first dose. Among the 14 infants who received both doses, the net
effect of the second dose was to elicit antibodies in all 14
vaccinees and to boost the level of neutralizing antibodies against
the rotavirus reassortant, from a geometric mean titer of 1:66
after the first dose, to 1:336 after the second dose.
After booster immunization with D.times.UK, only one rotavirus
reassortant recipient shed rotavirus and this was for only one day
after which virus could not be recovered from the child. Only two
of the 14 infants (five of whom had not been infected after the
first dose) experienced rotavirus-like illness after the second
dose. One child, who had not been infected with the rotavirus
reassortant after the first dose, had mild fever (maximum
38.1.degree. C.) along with rhinorrhea after the second dose.
Another child, who had been infected by the reassortant virus after
the first dose, vomited 4 times after receiving the second dose.
One of the vaccinees in this group had an elevated ALT value after
the booster dose, which was normal when repeated.
Example II
This example describes a quadrivalent human.times.bovine
reassortant rotavirus immunogenic composition that was evaluated
for its clinical safety and immunogenicity in adults, young
children, and infants.
The four human.times.bovine reassortant rotaviruses described in
Example I were combined in equal volumes to form a single
quadrivalent vaccine composition. All studies were conducted in a
placebo-controlled manner to assess the safety and immunogenicity
of the combined composition. All serologic and microbiological
testing were carried out as described in Example I.
A single dose of undiluted quadrivalent human (VP7 serotypes 1, 2,
3, and 4)-bovine UK rotavirus vaccine containing 10.sup.5.3 to
10.sup.5.8 PFU per reassortant was evaluated in 17 adults (11
vaccine recipients and 6 placebo recipients) at the Johns Hopkins
University Center for Immunization Research. Study subjects fasted
for at least one hour before and after administration of vaccine or
placebo. They were fed 120 ml of a buffer solution (sodium
bicarbonate) to neutralize gastric acidity followed one minute
later by the quadrivalent immunogenic composition mixed with the
buffer, or the placebo. One of the 11 adult vaccinees reported a
single episode of vomiting and three diarrheal stools during days 2
and 3 and pharyngitis and rhinorrhea between days 4 and 5
postvaccination. This volunteer had no evidence of rotavirus
infection, his stools were negative for rotavirus by culture and
electron microscopic examination, and a serologic response to
rotavirus was not detected. Bacterial cultures of the diarrheal
stools were also negative. The other 10 vaccinees and 6 placebo
recipients were asymptomatic postvaccination. None of the vaccine
or placebo recipients had an elevated ALT postvaccination, nor was
rotavirus detected in their stools. Rotavirus antibody responses
were detected in sera of 6 (55%) of eleven vaccinees (5 by ELISA
IgA assay and 4 by ELISA IgG assay). Thus, the quadrivalent vaccine
appeared to be safe and immunogenic enabling further evaluation in
children 6 to 60 months of age.
During a subsequent study, twenty infants and children 6 to 60
months of age were fed a buffered formula followed by a single dose
of undiluted quadrivalent human VP7 serotype 1.times.UK, human VP7
serotype 2.times.UK, human VP7 serotype 3.times.UK, and human VP7
serotype 4.times.UK rotavirus reassortant vaccine (12 children) or
placebo (8 children) at least two weeks before or after receiving
routine childhood immunizations appropriate for the individual's
age. "Rotavirus-like illness" was observed in one vaccinee (fever
during the first 24 hours after receiving virus and one episode of
vomiting on day 4) and in one placebo recipient (fever on day 2 as
well as rhinorrhea and cough on days 2-7). Another vaccinee had
rhinorrhea on day 7. None of the ill children demonstrated evidence
of rotavirus infection. Rotavirus was only detected in two stool
specimens collected from two asymptomatic vaccinees on a single
day. Rotavirus was not detected by electron microscopy or by
culture of stools of the other vaccinees or placebo recipients,
including four children who were siblings of vaccine recipients.
None of the vaccinees or placebo recipients had an ALT elevation
after administration of the quadrivalent rotavirus preparation.
Rotavirus antibody responses were detected in 6 of 12 vaccinees (4
by ELISA IgA assay and 6 by ELISA IgG assays). Altogether 7 of 12
vaccinees had evidence of rotavirus infection. Thus, the candidate
vaccine appeared to be safe and immunogenic, enabling progression
to the target population of infants of less than six months of age
who received three doses of vaccine.
The safety and immunogenicity of three doses of the undiluted
quadrivalent human.times.bovine rotavirus reassortant vaccine or
placebo was next evaluated in 30 young infants who received their
routine pediatric immunizations concurrently at approximately 2, 4,
and 6 months of age. Twenty infants were randomized to receive the
candidate rotavirus vaccine and ten infants to receive placebo. One
vaccinee and one placebo recipient were withdrawn from the study
prior to the second vaccination for medical reasons. After the
first, second or third dose of vaccine or placebo along with
routine childhood immunizations (including whole cell pertussis
vaccination), fever was reported in 1 of 20, 6 of 19, and 6 of 19
vaccinees, and in 2 of 10, 0 of 9, and 3 of 9 placebo recipients,
respectively. All episodes of fever occurred within the first 48
hours after oral administration of the quadrivalent rotavirus
formulation that coincided with multiple routine pediatric
immunizations, except for three febrile episodes, two of which were
accompanied by respiratory illness in vaccinees while the third
episode occurred in a placebo recipient. Diarrhea was reported in
one placebo recipient, but not in any vaccinee. One vaccinee had
two episodes of vomiting, as well as respiratory symptoms and
conjunctivitis, on day 7 after the second dose. Another child had a
single episode of vomiting precipitated by coughing during the
first 24 hours after vaccination. Only one vaccinee with mild fever
(38.2.degree. C.) had rotavirus detectable in a stool sample,
suggesting that the fever was associated with rotavirus infection.
Respiratory symptoms or rashes were seen in 4 of 19 vaccinees after
the second and after the third doses, and in 1 of 10 placebo
recipients after the first dose and in 1 of 9 placebo recipients
after the second and third doses. All illnesses reported were mild.
ALT was slightly elevated before and after the first vaccination in
5 of 20 and 2 of 20 vaccinees, respectively, and in 1 of 10 and 2
of 10 placebo recipients. Rotavirus was detected in stools by tests
in cell culture for 2 of 20 vaccinees after the first dose, in 0 of
19 vaccinees after the second dose, as well as 2 of 19 vaccinees
after the third dose.
Based on evidence of rotavirus shedding and/or a virus-specific
serum antibody response, 12 of 19 (63%) vaccinees were infected
with rotavirus after receiving the first dose and 19 of 19 (100%)
after receiving three doses. (Sera were not collected after the
second vaccination). Only a few infants had rotavirus detected in
their stools that were collected on days 3, 5 and 7 after the first
dose and on day 4 after the second and third doses. Specifically, 3
of 20 (15%) shed rotavirus after the first dose of vaccine and 1 of
19 (11%) after the third dose. Neither of the two infants (one
vaccinee and one placebo recipient) who were withdrawn from the
study because of an elevated ALT before and after the first dose
had any evidence of rotavirus infection. Among the 19 vaccinees who
received 3 doses of vaccine, antibody responses were detected by
the following assays following the first and/or third dose of
vaccine: ELISA IgA (50%). ELISA IgG (63%), and plaque reduction
neutralization assay against the UK bovine strain (100%) or human
rotavirus type 1 (Wa, 32%), 2 (DS-1.32%), 3 (P. 32%), or 4 (VA70,
32%). (Table 4)
TABLE-US-00004 TABLE 4 Serologic Responses in Individuals who
Received Three Doses of Approximately 4 .times. 10.sup.5 PFU of
Quadrivalent Bovine (UK) Rotavirus-based Vaccine or Placebo in
Infants Vaccinated at Approximately 2.4 and 6 Months of Age No.
with 4-fold or greater serum antibody response.sup.a by by
neutralization versus indicated virus/no. tested ELISA Wa DS-1 P
VA-70 UK Group IgA* / IgG* (1)** (2) (3) (4) (6) Vaccine 9/18/12/29
8/19 6/19 6/19 6/19 19/19 (50%)/(63%) (42%) (32%) (32%) (32%)
(100%) Placebo 0/8/1/9 0/9 0/9 0/9 0/9 0/9 .sup.aFollowing first
and/or third dose *Rotavirus group specific response **VP7
Serotype
At the conclusion of the study, 7 placebo recipients aged 8.25 to
9.25 months were given a single dose of the quadrivalent rotavirus
vaccine. This vaccine appeared to be well tolerated. Rotavirus-like
illness was observed in only one infant who had a fever on days 3,
4 and 7 as well as rhinorrhea on days 3 through 7. Respiratory
illness (without fever or gastroenteritis) was observed in 5 other
vaccinees. The absence of significant development of fever for the
bovine UK rotavirus-based vaccine in this older age group is of
considerable importance because a monovalent rhesus rotavirus
vaccine had been shown to be considerably more prone to induce a
febrile response in infants 6 to 8 months of age, a time when most,
if not all, passively acquired maternal antibodies to rotavirus
have been lost.
When these serologic responses to the bovine UK-based quadrivalent
composition were compared to the neutralizing antibody responses
induced in infants vaccinated at 2, 4, and 6 months of age with the
rhesus rotavirus-based quadrivalent vaccine administered at
10.sup.5.0 pfu of each component (Rennels et al., Pediatrics
97:7-13 (1996)), several important features were noted. The
neutralizing antibody responses induced by the rhesus
rotavirus-based vaccine included a response to rhesus rotavirus,
one of the parent strains of the reassortants, in 90% of the
infants vaccinated. Also, a neutralizing antibody response was
induced to human rotavirus serotype 1 in 14% of the children, to
human serotype 2 in 31%, to human serotype 3 in 29%, and to human
serotype 4 in 14%. Therefore, the bovine UK rotavirus-based human
reassortant immunogenic composition induced a significantly greater
frequency of neutralizing antibody responses to serotype 1
(P<0.005 Fisher exact test) and to serotype 4 (P<0.05 Fisher
exact test) than the rhesus rotavirus tetravalent composition.
Responses to human rotavirus serotype 2 and 3 strains and the
homotypic animal rotavirus parental strain were not significantly
different.
The equivalence and possible superiority of immunogenicity of the
tetravalent human.times.bovine UK rotavirus reassortant vaccine
when compared to the human.times.rhesus rotavirus reassortant
vaccine assumes significance when viewed in the context of the high
level of protective efficacy conferred by the rhesus
rotavirus-based vaccine.
In a multicenter efficacy trial in the United States, the rhesus
rotavirus vaccine was demonstrated to have a protective efficacy of
80% against very severe rotavirus diarrhea and 100% efficacy
against dehydration caused by rotavirus (Rennels et al. Pediatrics
97:7-13 (1996)). This vaccine was licensed by the Food and Drug
Administration in August 1998 after its clinical profile had been
approved by the FDA Advisory Committee in December 1997. This
vaccine was recommended for routine immunization of infants at 2,
4, and 6 months of age by the U.S. Advisory Committee for
Immunization Practices in June 1997, pending licensure. On May,
1999, it was licensed in the fifteen countries of the European
community.
The bovine UK rotavirus-based multivalent immunogenic composition
does not appear to induce a transient low level fever in humans. A
bovine UK rotavirus-based multivalent composition might be
preferred in some clinical situations. Thus, the bovine UK
rotavirus-based multivalent immunogenic compositions of the present
invention provide a unique constellation of properties including
(1) infectivity and immunogenicity similar to the licensed
quadrivalent rhesus rotavirus vaccine; (2) reduced ability to
induce a transient low level fever; (3) attenuation similar to that
previously described for bovine rotavirus-based vaccine
compositions, but with significantly greater infectivity and
immunogenicity as judged from the lower dosage required; and (4)
antigenic coverage for all of the human rotavirus serotypes of
major clinical importance in severe rotaviral disease.
Example III
In this example a summary is provided of a preliminary interim
analysis of data from an ongoing clinical trial that allowed a
comparison of a preferred tetravalent human-bovine reassortant
rotavirus composition of the present invention with the licensed
tetravalent rhesus-human rotavirus reassortant vaccine. ROTASHIELD.
The analysis examined the rate of low level fever response and the
protective efficacy against rotaviral diarrhea of the two
compositions.
The interim two year clinical study is currently in progress to
compare a tetravalent human-bovine rotavirus composition of the
present invention with the tetravalent rhesus-human rotavirus
reassortant vaccine (ROTASHIELD, recently licensed for use in the
United States and the 15 countries of the European Community) for
safety and protective efficacy against rotaviral diarrhea. The
study is being performed in Finland and includes 172 tetravalent
human-bovine rotavirus reassortant recipients, 86 corresponding
placebo controls, 161 ROTASHIELD vaccinees and 79 corresponding
placebo recipients. For logistical reasons the study was performed
in two adjacent small Finnish cities, Tampere and Lahti and
therefore it was necessary to employ two placebo groups. Two
placebo groups were also required when individuals in both study
groups were in the same location, because the containers for the
compositions differed in appearance.
The study was established to run concurrently in both cities in
order to recruit sufficient study subjects for each composition
that could detect with 80% power a decreased incidence (from 30% to
15%) of a febrile response to a composition (assuming an
insignificant difference between placebo groups, a two-tailed test
at 0.05 significance level and 10% dropouts). The tetravalent
human-bovine rotavirus reassortant composition and its
corresponding placebo were randomized only in Tampere, whereas, the
ROTASHIELD vaccine and its corresponding placebo were randomized in
both Lahti and Tampere.
For each composition dose, study subjects were monitored for 7 days
post immunization for fever, defined as a rectal temperature equal
to or greater than, 38.degree. C. Monitoring was also maintained
for episodes of vomiting, loose stools, irritability and other
systemic events during the 7 days following immunization.
Monitoring for gastroenteritis, serious adverse events, or
hospitalization was to be maintained for the entire term of the two
year study. The study was planned to include two seasons of
gastroenteritis.
Preliminary data has been obtained on the rate of a febrile
response following the first dose of each tetravalent reassortant
composition or placebo without unblinding members of the study
team. Further, only data for each group, as a whole, has been
analyzed to compare the two compositions, i.e., the tetravalent
human-bovine rotavirus reassortant composition and ROTASHIELD, for
occurrence of fever and for protective efficacy. Comparability
between the study groups was maintained with respect to the cell
culture substrate by formulating both compositions to contain
rotavirus grown in qualified diploid simian fetal rhesus lung
(FRhL) cells.
Following randomization, the tetravalent human-bovine rotavirus
reassortant composition (10.sup.5.3 to 10.sup.5.8 pfu per
component), ROTASHIELD (10.sup.5 pfu per component), or placebo was
administered orally to healthy 2 month old infants and again 2
months later. Parents of the infants recorded rectal temperatures
once, or twice a day, and symptoms were recorded daily. Stool
samples were collected and a record of the number and consistency
of the stools passed was maintained. Available data summarizing
cumulative rates of fever occurring during the seven day period
following the first dose of rotavirus reassortant composition and
preliminary data summarizing protective efficacy during the first
season of rotaviral gastroenteritis are provided in Table 5 and
Table 6 and summarized below.
Among the ROTASHIELD recipients, 46.2% developed a low level
transient febrile response (.gtoreq.38.degree. C. (100.4.degree.
F.)) during the week following administration of the first dose of
reassortant composition (Table 5). This frequency was significantly
greater than that of its placebo group whose rate of fever was
11.4% (p<0.0001). In contrast, the rate of fever
(.gtoreq.38.degree. C.) observed for the tetravalent human-bovine
rotavirus reassortant composition recipients (15.2%) did not differ
significantly from that of its placebo group (11.0%). The frequency
of a fever of >38.4.degree. C. (101.1.degree. F.) for the
ROTASHIELD vaccinees was still appreciable (20.3%) and was
significantly greater (p<0.0001) than that of its placebo group
(1.3%). In contrast, the frequency of fever of >38.4.degree. C.
experienced by recipients of the tetravalent human-bovine rotavirus
reassortant composition was 1.8%, which was not significantly
different from that of its placebo group (0%). Thus, in these two
comparisons, ROTASHIELD induced fever with an appreciable frequency
that was significantly higher than that of its placebo group,
whereas febrile response to the tetravalent human-bovine rotavirus
reassortant composition did not differ from that of its placebo and
was significantly less than observed for ROTASHIELD. Overall,
ROTASHIELD was associated with significantly more fever during the
seven days following immunization than was the tetravalent
human-bovine rotavirus reassortant composition, (p<0.0001),
whether fever was defined as .gtoreq.38.degree. C. or
>38.4.degree. C. The essentially total lack of a febrile
response to the human.times.bovine reassortant composition in the
Finnish trial is reflective of the absence of background illnesses
in the study group. This is in sharp contrast to Examples I and II
where background illnesses were quite frequent. In the Finnish
study, the relative absence of background illness allowed the
evaluation of the development of fever, or a febrile response with
greater precision than was possible in the earlier clinical
trials.
Preliminary data was also obtained relating to the comparative
protective efficacy against rotavirus diarrhea of the tetravalent
human-bovine rotavirus reassortant composition and ROTASHIELD. This
data was particularly important because with the lack of observed
febrile response of the former composition and the use of a 10 to
100-fold lower virus dose than had been used for the previous
bovine rotavirus and human-bovine reassortant compositions, it was
conceivable that the current composition would be too attenuated to
induce protection against naturally-occurring rotavirus illness. On
the other hand, the human-rhesus rotavirus reassortant vaccine,
ROTASHIELD, has characteristically been found to exhibit a
protective efficacy of 80% to 100% against severe rotaviral
disease.
An estimate for protective efficacy was obtained from the present
study by analyzing the distribution of a total of 48 episodes of
rotaviral gastroenteritis of any severity that were documented
among the study subjects during the first season of surveillance.
Diagnosis of rotaviral gastroenteritis was established by
identification of rotavirus in the feces of a study subject with
gastroenteritis. Identification was made by: (1) a standard
immunological method, the ELISA technique, employing rotavirus
group-specific and serotype-specific antisera (Joensuu et al.
Lancet 350:1205-1209 (1997); and Hoshino et al. J. Clin. Microbiol.
21:425-430 (1985)), and (2) PCR employing primers that recognize
rotavirus group-specific conserved sequences or rotavirus
serotype-specific conserved sequences (Gouvea et al J. Clin.
Microbiol. 28:276-282 (1990)).
Analysis of protective efficacy during the first season, was
conducted in such a manner that the site study team, as well as the
clinical and study monitoring staff, remained blinded to the
assignment of individual subjects to a study group (i.e.,
tetravalent human-bovine rotavirus reassortant composition,
ROTASHIELD, or corresponding placebo group) and they will remain
blinded for the remaining term of the study. This restriction was
applied to this preliminary analysis to permit continued
surveillance and collection of data until the termination of the
study at the finish of the second gastroenteritis season. As a
consequence, the analysis of vaccine efficacy by severity of
disease remains to be performed after the termination of the two
year surveillance. Nevertheless, the rate of rotavirus
gastroenteritis of any severity for each study group during the
first season could be determined and compared to that of the other
groups without unblinding the trial.
The rate of rotavirus gastroenteritis episodes of any severity for
the two placebo groups during the first season was remarkably
similar, 17.7% and 17.4%, indicating the comparability of the two
study sites and of the epidemiology of rotavirus infection at these
sites. When compared to its placebo group, ROTASHIELD exhibited a
protective efficacy of 65% for rotavirus gastroenteritis of any
severity. The comparable estimate for the tetravalent human-bovine
rotavirus reassortant composition was 70%, a protective efficacy
similar to the licensed vaccine. This level of protective efficacy
was more than satisfactory considering the fact that efficacy could
not be analyzed according to severity of disease. It is very likely
that the protective effect of the human-bovine rotavirus
reassortant composition against severe disease will be
significantly greater than 70% based on the experience gained
during previous ROTASHIELD clinical trials wherein rotavirus
vaccine efficacy increased with increasing severity of disease.
Characteristically with ROTASHIELD, a protective efficacy of 80% to
100% was observed for the most severe disease, while the protective
efficacy calculated for any rotavirus illness of any severity
reached only 48 to 68%.
TABLE-US-00005 TABLE 5 Cumulative Rates of Fever (Rectal) Occurring
During the 7 Day Period Following First Dose of ROTASHIELD or
Tetravalent Human-Bovine Reassortant Composition (TBRC) (Mean and
95% Confidence Interval (CI)). Rectal P Value for Temperature No in
Subjects with Fever 95% CI Indicated (.degree. C.) Group Group No.
Rate (%) (% 2 sided) Comparison .gtoreq.38 ROTASHIELD.sup.1 158 73
46.2 38.2-54.3 {close oversize bracket} <0.0001 Placebo 79 9
11.4 5.3- 20.5 {close oversize bracket} <0.0001.sup.a TBRC.sup.2
165 25 15.2 10-21.6 {close oversize bracket} NS Placebo 82 9 11
5.1-19.8 >38.4 ROTASHIELD 158 32 20.3 14.3-27.4 {close oversize
bracket} <0.0001 Placebo 79 1 1.3 0-6.9 {close oversize bracket}
<0.0001.sup.a TBRC 165 3 1.8 0.4-5.2 {close oversize bracket} NS
Placebo 82 0 0 0-4.4 >39.1 ROTASHIELD 158 3 1.9 0.4-5.3 {close
oversize bracket} NS Placebo 79 0 0 0-4.6 {close oversize bracket}
NS TBRC 165 0 0 0-2.2 {close oversize bracket} NS Placebo 82 0 0
0-4.4 .sup.110.sup.5 pfu per component of the tetravalent vaccine
.sup.210.sup.5.3-10.sup.5.8 pfu per component of the tetravalent
composition NS = Not significant .sup.aComparison of occurrence of
fever in ROTASHIELD vs TBRC groups
TABLE-US-00006 TABLE 6 Preliminary Report: A phase II Double Blind
trial of the safety and immunogenicity of tetravalent human-bovine
rotavirus reassortant composition and tetravalent rhesus rotavirus
vaccine (ROTASHIELD). Distribution of first season rotaviral
gastroenteritis illness of any severity by study group. No. who
developed RV gastroenteritis of any severity Protective Location
Study Group No. of Subjects (%) Efficacy Lahti ROTASHIELD 161 10
(6.2) 65% and Placebo 79 14 (17.7) (P < 0.02) Tampere Tampere
TBRC 172 9 (5.2) 70% Placebo 86 15 (17.4) (P < 0.003)
The vaccine of this invention exhibits all of the advantageous
properties of the quadrivalent rhesus rotavirus formulation with
regard to immunogenicity and protective efficacy. Similarly, the
multivalent immunogenic compositions of the invention shares the
advantageous property of lack of significant febrile response
exhibited by the previously described bovine rotavirus
formulations. However, it does not exhibit the disadvantageous
features of the quadrivalent rhesus rotavirus vaccine regarding the
development of transient low level febrile response or of the
previously described bovine rotavirus formulations regarding their
low infectivity.
Microorganism Deposit Information
The human rotavirus strains were deposited with the American Type
Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, Jun. 4, 1998, under the conditions of the Budapest
Treaty and designated as follows.
TABLE-US-00007 Reassortant Designation ATCC Accession Number HD
.times. BRV, clone 47-1-1 (VP7:l [D]) ATCC VR-2617 HDS1 .times.
BRV-1, clone 66-1-1 (VP7:2 [DS-1] ATCC VR-2616 HP .times. BRV,
clone 22-1-1 (VP7:3 [P]) ATCC VR 2611 HST3 .times. BRV-2, clone
52-1-1 (VP7:4 [ST3]) ATCC VR-2612 IAL28 .times. UK, clone 33-1-1
(VP7:5 [IAL28]) ATCC VR-2613 AU32 .times. UK, clone 27-1-1 (VP7:9
[AU32]) ATCC VR-2614 KC-1 .times. UK, clone 32-1-1 (VP7:10 [KC-1])
ATCC VR-2615
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims. The scope of the invention should, therefore, be determined
not with reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
All publications and patent documents cited in this application are
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication or patent document
were so individually denoted.
* * * * *