Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
Attenuated Dengue-1 Virus Vaccine
This application claims the benefit of priority
under 35 U.S.C. ~119(e) from U.S. application serial
no. 60/126,317 filed on March 26, 1999, still pending,
and U.S. application serial no. 60/182,064 filed on
February 11, 2000, still pending.
INTRODUCTION
Dengue fever is caused by any of four serotypes
of dengue virus, dengue-1, dengue-2, dengue-3, and
dengue-4, which are transmitted to humans by
mosquitoes. In adults, dengue infections typically
cause self-limited but incapacitating acute illness
with fever, muscle pains, headache and an occasional
rash. The illness may be complicated by hemorrhagic
fever, which may be manifested by a positive
tourniquet test, spontaneous petechiae, frank
bleeding, and/or shock. Dengue hemorrhagic fever is
fatal in about 0.5% of cases. Patients who have
antibody from an earlier dengue infection who are
subsequently infected by another dengue strain have
been shown to be at higher risk for dengue hemorrhagic
fever.
The mosquito vectors of dengue viruses are found
in all tropical and sub-tropical areas of the world
and in some temperate areas of the United States,
Europe, Africa, and the Middle East. In recent years,
endemic and epidemic dengue infections have occured in
Central and South Ameria, Southeast Asia, India,
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Africa, the Caribbean and Pacific regions. Vector
control is impractical.
A concerted investigation was undertaken at the
WR.AIR to select four attenuated dengue vaccine
candidates, one for each serotype. As with other
successful human vaccines, it was planned that
passaged virus would be tested at the highest and
lowest passage levels available. One or another of
these extremes might be found suitable. If necessary,
further intermediate pasage levels could be developed
for testing. In this approach, there was no intent
to predict which, if any biological markers will
correlate with virulence of virus in human beings.
The identification of a successful human vaccine for
one DEN type might validate biological markers of
attenuation and permit improved selection of other
attenuated viruses. The empiric approach to separate
evaluation of multiple passage levels is based upon
the precedent of modern attenuated virus vaccines; for
example rubella strains that differed by only a few
duck embryo passages varied markedly in human
virulence (Halstead et al., 1970, JAMA 211, 911-916).
The early vaccine candidates were grown in cells.
Attenuated vaccines were prepared by adaptation to
growth in primary dog kidney (PDK) cells, a
nonpermissive cell for dengue virus replication
(Halstead 1978, Asian J. Infect. Dis. 978, 112-117).
Preliminary clinical studies demonstrated that dengue
virus strains could be attenuated for humans by
passage in PDK cells (Eckels, 1984 , Am J Trop Med Hyg
33, 679-683; Bhamarapravati, 1987, Bull WHO 65, 189-
195). PDK passage therefore provides an excellent
model for those who wish to study the empirical
process of selective attenuation. But, just as PDK
serial passage exerts a cumulative selection process,
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the further passage in another cell substrate provides
its own selective pressure. It is not known whether
or not FRhL passge increases or decreases the
virulence of virus for humans. The use of stable cell
lines that must be fully characterized only one time
is appealing. However, the published experience with
FRhL cells suggests that these cels may reverse or
destabilize biological properties acquired during
serial passage in PDK (Halstead et al., 1984, Am J
Trop Med Hyg 33, 654-665; Halstead et al., 1984, Am J
Trop Med Hyg 33, 666-671; Halstead et al., 1984, Am J
Trop Med Hyg 33, 672-678; Halstead et al., 1984, Am J
Trop Med Hyg 33, 679-683; Eckels et al, 1984, Am J
Trop Med Hyg 33, 679-683).
Experimental vaccines were prepared from each
candidate strain of dengue virus at multiple passage
levels in PDK cells; the passages empirically selected
for vaccine preparation were approximately 10, 20, 30,
40, and 50. The safety and immunogenicity of various
serotypes of dengue vaccine strains at one or more
passage levels was then tested in volunteers. The
purpose of these clinical investigations was to select
candidate attenuated dengue vaccines for development
as a monovalent vaccine and possible combination into
a multicomponent vaccine. In this application is
described the testing and selection of attenuated
dengue type 2, 3, and 4 vaccines. The selection of the
dengue 1 candidate vaccine has already been described
in detail elsewhere (Edelman, 1994, J Infect Dis 170,
1448-1455).
SUMMARY OF THE INVENTION
The present invention satisfies the need
discussed above. The present invention relates to
vaccine composition comprising attenuated dengue-1
virus. The attenuated virus is provided in an amount
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sufficient to induce an immune response in a human
host, in conjuction with a physiologically acceptable
carrier and may optionally include an adjuvant to
enhance the immune response of the host.
Therefore, it is one object of the present
invention to provide an attenuated dengue-1 virus
derived from serial passaging of a virulent dengue-1
isolate.
It is another object of the present invention to
provide methods for stimulating the immune system of
an individual to induce protection against dengue-1
virus. These methods comprise administering to the
individual an immunologically sufficient amount of
dengue-1 which has been attenuated by serial passage.
The attenuated dengue-1 virus of the present invention
was derived from West Pac 74;45AZ5 isolate and has
been deposited under the terms of the Budapest Treaty
with the American Type Culture Collection (ATCC) of
10801 University Boulevard, Manassas, Virginia 20110-
2209, U.S.A., and was granted the accession number of
VR-2648.
It is yet another object of the present invention
to provide pure cultures of attenuated dengue-1 virus.
The attenuated virus may be present in a cell culture
supernatant, isolated from the culture, or partially-
or completely purified. The virus may also be
lyophilized, and can be combined with a variety of
other components for storage or delivery to a host, as
desired.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Occurrence of >Grade 1 symptoms as a
result of vaccine administration.
Figure 2: Frequency of distribution of
reactogenicity index by serotype.
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Figure 3: Table showing results of dose-ranging
tetravalent dengue vaccine studies.
Figure 4: Table showing Immunogenicity of full-
dose tetravalent dengue vaccine in 10 subjects.
Figure 5: Table showing details of selected
formulations of tetravalent vaccine studies.
Figure 6, A-H: Interferon 'y production by PBMC
collected from vaccine volunteers and stimulated with
serotype specific virus. All volunteers received only
one serotype of vaccine. Graphs on the left (A-D)
show results from volunteers that were given the
second dose around day 32. Graphs on the right (E-H)
show results from volunteers that received the second
dose around day 92. A response over 1000 pg/ml was
seen just prior to the second dose in most volunteers.
Only four volunteers had a response over 1000 pg/ml
within the first 15 days of receiving the first
vaccine dose.
Figure 7, A-D: Interferon y production of PBMC
collected from vaccine volunteers receiving
tetravalent vaccine. The PBMC were stimulated
individually with each serotype of virus. Individual
lines in each graph represent responses of one
volunteer's PBMC to individual serotypes of virus. As
with the monovalent vaccine recipients, late responses
were noted.
Figure 8, A and B: Granzyme B mRNA production of
PBMC collected from monovalent and tetravalent vaccine
volunteers. Cells were collected from all individuals
whose PBMC secreted >_ 1000 pg IFNy/ml at any time.
This is a semiquantitative representation of the
amount of mRNA detected by RTPCR. The upper chart (A)
describes the intensity of bands seen for all samples.
The lower gel (B) is from selected volunteers to show
examples of positive and negative RTPCR assays.
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DETAILED DESCRIPTION
The present invention provides dengue-1 virus
suitable for vaccine use in humans. The dengue-1
virus described herein is produced by serial passaging
of an infectious dengue-1 virus isolate in a suitable
host cell line such as primary dog kidney cells so
that mutations accumulate that confer attenuation on
the isolate. Serial passaging refers to the infection
of a cell line with a virus isolate, the recovery of
the viral progeny from the host cells, and the
subsequent infection of fresh host cells with the
viral progeny to generate the next passage.
The dengue-1 West Pac 74 isolate was prepared
from a patient in Nairu in 1974. The isolate was
passaged 20 times in Fetal Rhesus monkey lung cells
with plaque selection and mutagenization with 5
azacytidine in order to recover a virus that was
attenuated and suitable for human vaccination.
Following vaccination of two human volunteers, the
vaccine was discontinued due to Dengue fever illness
in one of the volunteers. This virus was ussed as
starting seed for passage in primary dog kidney (PDK)
cell cultures. Virus from PDK passages 10, 20, and 27
was used to inoculate fetal rhesus monkey lund diploid
cell cultures (DBS-FRhL-2). The harvested virus was
tested for attenuation and vaccine potential as
described in the Examples below.
Serial passaging of a virulent (disease-causing)
strain of dengue-1 results in the isolation of
modified virus which are attenuated, i.e., infectious,
yet not capable of causing disease. These modified
viruses are tested in monkeys for reduced infectivity.
Those that have reduced infectivity are subsequently
tested in humans. Humans are the only primate that
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will exhibit signs of clinical disease. The viruses
that cause minimum to no clinical reactivity but still
infect and induce an immune response are attenuated.
In one embodiment of the invention, a virulent
dengue-1 isolate was serially passaged in primary dog
kidney (PDK) cells to derive the attenuated strain.
Serial passaging was performed by infecting PDK cells
with the virulent strain, incubating the infected
cells for several days, and collecting the supernatant
culture fluids containing virus. The harvested virus
was then applied to fresh PDK cells to generate the
next passage.
Various passages in the series were tested in
monkeys and then humans for clinical effect after
final passage in fetal Rhesus monkey lung cells
(FRhl). FRhL cells were used to optimize virus
titers. FRhL passage 1 is considered master seed,
FRhl passage 2 is considered production seed, and FRhL
passage 3 is considered vaccine lot. Attenuation of
the virus could only be determined by monkey and human
testing. The virulence of a passaged virus, i.e., the
ability to cause disease, was assessed by daily
monitoring of symptoms including temperature,
headache, rash, and such. A passage was attenuated,
as judged by the inability of this virus to elicit
clinical signs of dengue-1 disease.
Propagation of the attenuated virus of the
invention may be in a number of cell lines which allow
for dengue-1 virus growth. Dengue-1 virus grows in a
variety of human and animal cells. Preferred cell
lines for propagation of attenuated dengue-1 virus for
vaccine use include DBS-FRhL-2, Vero cells, and other
monkey cells. Highest virus yields are usually
achieved with heteroploid cell lines such as Vero
cells. Cells are typically inoculated at a
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multiplicity of infection ranging from about 0.005 to
0.01, and are cultivated under conditions permissive
for replication of the virus, e.g., at about 30-37°C
and for about 5-7 days, or as long as necessary for
virus to reach an adequate titer. Virus is removed
from cell culture and separated from cellular
components, typically by well known clarification
procedures, e.g., centrifugation, and may be further
purified as desired using procedures well known to
those skilled in the art. Preferably, care must be
taken to maintain temperature of 2-10°C during
purification to maintain viability of the virus.
The isolation of an attenuated virus may be
followed by a sequence analysis of its genome to
determine the basis for the attenuated phenotype.
This is accomplished by sequencing the viral DNA and
identifying nucleotide changes in the attenuated
isolate relative to the genomic sequence of a control
virus. Therefore, the molecular changes that confer
attenuation on a virulent strain can be characterized.
In an embodiment of the invention, the sequence
of the RNA genome isolated from the attenuated virus
is determined and compared to a control sequence of
either the prototype strain or parent strain.
Nucleotide sequence variations between the virulent
strain and the attenuated strain can be identified.
The invention provides for attenuated dengue-1
viruses which have one or more sequence alterations
relative to the sequence of the control wild-type
dengue-1.
One embodiment of the invention provided herein,
includes the introduction of sequence changes at any
of the positions listed in the table above, alone or
in combination, in order to generate attenuated virus
progeny. Viral genomes with such alterations can be
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produced by any standard recombinant DNA techniques
known to those skilled in the art (Ausubel et al.,
Current Protocols in Molecular Bioloav, Greene
Publishing Associates & Wiley Interscience, New York,
1989) for introduction of nucleotide changes into
cloned DNA. A genome may then be ligated into an
appropriate vector for transfection into host cells
for the production of viral progeny.
The ability to generate viral progeny through
plasmid-mediated introduction of a viral genome can
also be used to produce viruses with defined molecular
changes. In this embodiment of the invention, stable
virus stocks can be produced that contain altered
sequences that confer desired properties on the virus,
for example, reduced virulence. This approach can
also be used to assess the effect of molecular changes
on various properties of the virus, i.e. antigenic
type, virulence, or attenuation by introducing desired
sequence changes into the viral genome, producing
virus progeny from the genome, and recovering the
virus progeny for characterization. In addition, this
approach can be used to construct a virus with
heterologous sequences inserted into the viral genome
that are concurrently delivered by the virus to
generate an immune response against other diseases.
Construction of viral genomes with defined
molecular changes can be accomplished using standard
techniques such as oligonucleotide-directed, linker-
scanning or polymerase chain reaction-based
mutagenesis techniques known to those skilled in the
art (Zoller and Smith, 1984, DNA 3,479-488; Botstein
and Shortle, 1985, Science 229, 1193). Ligation of
the genome into a suitable vector for transfer may be
accomplished through standard techniques known to
those skilled in the art. Transfection of the vector
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into host cells for the production of viral progeny
may be done using any of the standard techniques such
as calcium-phosphate or DEAF-dextran mediated
transfection, electroporation, protoplast fusion, and
other techniques known to those skilled in the art
(Sambrook et al., Molecular Cloning: A laboratory
Manual, Cold Spring Harbor Laboratory Press, 1989).
For vaccine use, the attenuated virus of the
invention can be used directly in vaccine
formulations, or lyophilized, preferably in a
stabilizer (Hoke, 1990, Am J Trop Med Hyg 43, 219-
226), as desired, using lyophilization protocols well
known to the artisan. Lyophilized virus will
typically be maintained at about 4°C. When ready for
use, the lyophilized virus is reconstituted in water,
or alternatively a stabilizing solution, e.g., saline
or comprising Mg+' and HEPES, with or without adjuvant,
as further described below.
Thus, dengue-1 virus vaccines of the invention
contain as an active ingredient an immunogenically
effective amount of an attenuated dengue-1 virus as
described herein. The attenuated virus may be
introduced into a subject, particularly humans, with a
physiologically acceptable vehicle and/or adjuvant.
Useful vehicles are well known in the art, and
include, e.g., water, buffered water, 0.4% saline,
0.3o glycine, hyaluronic acid and the like. The
resulting aqueous solutions may be packaged for use as
is, or lyophilized, the lyophilized preparation
rehydrated prior to administration, as mentioned
above. The compositions may contain pharmaceutically
accepatable auxilliary substances as required to
approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting
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agents, wetting agents and the like, for example,
sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, and the like.
Administration of the live attenuated viruses
disclosed herein may be carried out by any suitable
means, including both parenteral injection (such as
intraperitoneal, subcutaneous, or intramuscular
injection), by in ovo injection in birds, orally and
by topical application of the virus (typically carried
in the pharmaceutical formulation) to an airway
surface. Topical application of the virus to an
airway surface can be carried out by intranasal
administration (e.g. by use of dropper, swab, or
inhaler which deposits a pharmaceutical formulation
intranasally). Topical application of the virus to an
airway surface can also be carried out by inhalation
administration, such as by creating respirable
particles of a pharmaceutical formulation (including
both solid particles and liquid particles) containing
the virus as an aerosol suspension, and then causing
the subject to inhale the respirable particles.
Methods and apparatus for administering respirable
particles of pharmaceutical formulations are well
known, and any conventional technique can be employed.
As a result of the vaccination the host becomes at
least partially or completely immune to dengue-1 virus
infection, or resistant to developing moderate or
severe dengue-1 viral infection.
The vaccine composition containing the attenuated
dengue-1 virus of the invention are administered to a
person susceptible to or otherwise at risk of dengue-1
virus infection to enhance the individual's own immune
response capabilities. Such an amount is defined to
be a "immunogenically effective dose". In this use,
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the precise amount again depends on the subject's
state of health and weight, the mode of
administration, the nature of the formulation, etc.,
but generally range from about 104 to 105 pfu virus per
subject. In any event, the vaccine formulations
should provide a quantity of attenuated dengue-1 virus
of the invention sufficient to effectively protect the
subject against serious or life-threatening dengue-1
virus infection.
The attenuated dengue-1 virus of the invention of
one particular serotype can be combined with
attenuated viruses of other serotypes of dengue virus
to achieve protection against multiple dengue viruses.
Typically the different modified viruses will be in
admixture and administered simultaneously, but may
also be administered separately.
In some instances it may be desirable to combine
the attenuated dengue-1 virus vaccines of the
invention with vaccines which induce protective
responses to other agents.
Single or multiple administration of the vaccine
compositions of the invention can be carried out.
Multiple administration may be required to elicit
sufficient levels of immunity. Levels of induced
immunity can be monitored by measuring amount of
neutralizing serum antibodies, and dosages adjusted or
vaccinations repeated as necessary to maintain desired
levels of protection. For example, the vaccine can be
administered at 0 and 6 months.
In yet another embodiment, the invention
relates to a method for detecting the presence of
dengue-1 infection in a sample. Using standard
methodology well known in the art, a diagnostic assay
can be constructed by coating on a surface (i.e. a
solid support) for example, a microtitration plate or
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a membrane (e.g. nitrocellulose membrane), all or a
unique portion of dengue 1. A sample from a subject
suspected of having an dengue-1 infection is brought
in contact with the plate or membrane. The presence
of a resulting complex formed between the virus and
its antigen specific therefor in the sample can be
detected by any of the known methods common in the
art, such as fluorescent antibody spectroscopy or
colorimetry. This method of detection can be used,
for example, for the diagnosis of dengue-1 infection.
The following examples are provided by way of
illustration, not limitation.
The following MATERIALS AND METHODS were used in
the examples that follow
Materials and Methods for vaccine production.
Virus strains. DEN viruses were passaged in
primary dog kidney (PDK) cell cultures following
isolation from human and mosquito sources. Table 1
lists the strains that were adapted and passaged in
PDK cells. After passage in PDK cells, virus strains
were further adapted to FRhL cells for seed and
vaccine production. This consisted of an additional
3-4 passages for final vaccine lot preparation.
Parental virus strains, also listed in Table 1, were
derived from low, cell culture passages in cells that
were permissive for DEN virus replication.
Vaccine production. DEN vaccines for all four
serotypes were prepared in FRhL cell culture using a
similar procedure. FRhl cells, banked and pre-tested
(see Table 2 for testing results) were removed from
liquid nitrogen storage and plated in 150 cm2 flasks
in Eagle's minimum essential media (EMEM)
(Biowhittaker, Waldersville, MD) cell medium
supplemented with non-essential amino acids, fetal
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bovine serum, FBS (20) (Biowhittaker, Waldersville,
MD), and antibiotics. After the flasks reached
confluency, medium was removed and flasks inoculated
with DEN production seed diluted for an input of 0.01
MOI, and allowed to adsorb at 32°C for 1 hr.
Following adsorption and feeding with fresh EMEM
medium, flasks were returned to 32°C for 4 days. On
day 4 post-inoculation, medium from all flasks was
discarded and cell monolayers were washed 3 times with
100 ml of Hanks BSS (Biowhittaker, Waldersville, MD).
After washing, flasks were fed with EMEM medium
containing 0.25% human serum albumin (HSA, Alpha
Therapeutic Corp, Los Angeles, CA) replacing FBS.
After an additional two days of incubation at 32°C,
supernatant culture fluids were removed from all
flasks and pooled. After sampling for safety tests,
the remaining culture fluids were pooled and clarified
by filtration through a 0.45 micron, non-protein
binding membrane filter. The filtered fluids were
pooled and mixed with an equal volume of stabilizer
containing 150 lactose and 5% HSA. The bulk,
stabilized fluids were stored at -70°C until freeze-
dried. For final vialing, bulk, stabilized fluids
were thawed rapidly at 41°C and aliquoted in 3 ml
volumes in serum vials. Trays of vials were frozen to
a temperature of -40°C in a Hull freeze-dryer,
followed by drying for 1 day. Following capping,
vials were stored at -20°C in a monitored freezer.
Vaccine testing. All cell banks used for virus
preparations as well as seed and vaccine lots were
tested for the presence of contaminating agents. The
test articles and results are listed in Table 2. No
detectable contaminants were found in any of the
products.
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Rhesus monkev inoculation. Adult, male and female
rhesus monkeys (6-15 kg) were immunized with the DEN
vaccine lots or parent viruses by subcutaneous
inoculation of 0.5 ml in the upper arm. Blood for
virus isolation and antibody tests was drawn from the
femoral vein prior to inoculation and every day for 14
days following inoculation. Blood was also drawn at
30 and 60 days following immunization. Virus
challenges were performed similarly.
Virus isolation by amplification in C6/36 cells.
Virus isolation by C6/36 cell culture amplification
has been described in Putnak et al, 1996 (J. Infect
Dis 174, 1176-1184). Briefly, following inoculation
of monkeys, daily blood specimens were obtained from
days 1 to 14. Serum was separated and frozen at -
80°C. For recovery of virus from sera, thawed sera
were diluted 1:3 in cell culture medium and used to
inoculate 25 cm2 flasks containing monolayers of C6/36
mosquito cells. Following adsorption of virus, flasks
were maintained at 28°C in EMEM maintenance medium.
After 7 days, medium was changed and flasks incubated
an additional 7 days. On day 14 post inoculation,
supernatant culture fluids were decanted and frozen at
-80°C after mixing with an equal volume of heat-
inactivated fetal bovine serum (FBS). Frozen
specimens were later assayed for infectious virus by
plaque assay.
All documents cited herein supra or infra are
hereby incorporated in their entirety by reference
thereto.
CA 02365728 2001-09-21
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CA 02365728 2001-09-21
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CA 02365728 2001-09-21
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CA 02365728 2001-09-21
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CA 02365728 2001-09-21
WO 00/57908 21 PCT/US00/08201
Plague assays. Infectious virus was titrated
from amplified viremia isolates or directly from
monkey sera by plaque assay in Rhesus monkey kidney
(LLC-Mk2, ATCC CCL7) cells following the procedure of
Sukhavachana et al. 1966 (Bull WHO 35, 65-66).
Assays in C6/36 cells was performed as described in
Putnak et al, 1996, supra.
Neutralization tests. DEN neutralizing
antibodies were measured from monkey sera using a
plaque reduction neutralization test similar to that
used by Russell et al, 1967 (J Immunol 99, 285-290).
Parent viruses listed in Table 1 were used to measure
the plaque reduction 50o endpoint (PRNT50) in serum
specimens.
Example 1
DEN virus modification in PDK cells and vaccine
lot production. DEN virus strains selected for
vaccine development had a variety of passage histories
prior to PDK passage. In the case of DEN-4 341750
there was just one mosquito passage before inoculation
of PDK cell culture, while DEN-1 West Pac 74 strain
had a history of twenty FRhL cell passages prior to
PDK passage (Table 1). With the exception of DEN-3,
all strains adapted after a small number of PDK
passages. For DEN-3, additional efforts were required
to increase viral input in early passages in order to
adapt this strain to PDK cells. As a general case
after adaptation to PDK cells, DEN virus titers were
found to be in the 104-105 PFU/ml range. Attempts to
increase titers were not successfull and alternative
cell substrates were sought for vaccine production.
DBS-FRhL-2 (FRhL) cells were selected for this purpose
for several reasons: 1) DEN viruses replicate to
titers of ca 106 PFU/ml allowing manufacture of DEN
CA 02365728 2001-09-21
WO 00/57908 22 PCT/US00/08201
vaccines in these cells; 2) the cells have been used
for the preparation of several DEN vaccines that have
been tested in Phase I clinical trials without adverse
reactions that may be related to the vaccine cell
substrate; 3) FRhL cells are normal, rhesus monkey
lung diploid cells that have no tumorigenic potential
and are free of reverse transcriptase activity and
contaminating agents; 4) since the cells are "normal"
diploid cells there is no regulatory or other
requirement to purify the vaccines; 5) FRhL cell banks
can be established at cell generations usable for
vaccine manufacture starting with available, low
passage cells. PDK passage therefore provides an
excellent model for those who wish to study the
empirical process of selective attenuation. But, just
as PDK serial passage exerts a cumulative selection
process, the further passage in another cell substrate
provides its own selective pressure. It is not known
whether or not FRhL passge increases or decreases the
virulence of virus for humans. The use of stable cell
lines that must be fully characterized only one time
is appealing. However, the published experience with
FRhL cells suggests that these cells may reverse or
destabilize biological properties acquired during
serial passage in PDK (Halstead et al., 1984, Am J
Trop Med Hyg 33, 654-665; Halstead et al., 1984, Am J
Trop Med Hyg 33, 666-671; Halstead et al., 1984, Am J
Trop Med Hyg 33, 672-678; Halstead et al., 1984, Am J
Trop Med Hyg 33, 679-683; Eckels et al, 1984, Am J
Trop Med Hyg 33, 679-683).
Adaptation of PDK-passaged viruses to FRhL was
uniformly successfull for all strains of DEN virus and
was not dependent on PDK passage. Viral titers from
harvests of FRhL passages 1-4 ranged from 105-106
PFU/ml. By the third-fourth FRhL passage, vaccine
CA 02365728 2001-09-21
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lots of all of the DEN strain set viruses were
prepared and tested as listed in Table 2. Data is
also provided in Table 2 for the FRhL cell bank
testing as well as the master and production seed
testing. Results of these tests, required to ensure
the safety and the freedom from contamination, were
negative, or fell within allowable specifications.
For the DEN-4 341750 PDK-20 production seed, monkey
neurovirulence tests were performed. Results of this
study can be found in Hoke, 1990 (Am J Trop Med Hyg
43, 219-226). The DEN-4 production seed as well as
the DEN-4 parent virus that was used for comparison
were not neuropathogenic. Whether the remaining
candidate DEN vaccines need to be evaluated for
neurovirulence remains questionable based on data from
this experience as well as other tests of DEN monkey
neurovirulence (personal comunication).
Example 2
Rhesus monkevs inoculated with PDK-passaaed DEN
viruses. The infectivity of DEN viruses passaged in
PDK cells and designated as "strain sets" was compared
to parental, unmodified viruses for each serotype.
Table 3 lists the results of these studies where the
degree of infectivity for monkeys was measured by the
number of days of viremia that could be found in
sequentially drawn serum two weeks following
inoculation. Parental virus inoculation of monkeys
resulted in 6.8, 5, 3, and 4.7 mean days of viremia in
groups of 3-4 monkeys inoculated with DEN-1, DEN-2,
DEN-3, and DEN-4, respectively. For DEN-2 parent,
additional data (not shown) has substantiated that
infection with measurable viremia is very reproducible
over time using similar monkeys and isolation
techniques. Unfortunately, only partial data exists
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on viral titers in monkey sera. Most of the data that
exists comes from experience with the DEN-2 parent
virus where monkey viremic blood was titrated in
mosquito cell culture. Peak viral titers at 4-8 days
post inoculation resulted in titers reaching 105
PFU/ml of serum (Putnak et al, 1996, supra).
For each strain set, PDK passage results in
modification of DEN virus as shown by reduced capacity
of the virus to infect monkeys. For several of the
strain sets this was clearly evidenced by the complete
lack of viremia at the highest PDK passage.
Inoculation of monkeys with DEN-1 at PDK passage 27
resulted in 0 days of viremia in 4 monkeys. This
translates to 0 isolations out of a total of 56
bleedings tested. A similar result was found for DEN-
3 PDK-20 and PDK-30. At PDK-30 for this virus, all
evidence of monkey infectivity was lost, i.e., no
viremia and no evidence of seroconversion in the
monkeys inoculated with 106 PFU of virus. The DEN-2
strain required the greatest number of PDK passages to
attain modification of monkey infectivity. With this
virus, at least 40 passages in PDK cell culture were
required for reduced viremia. To contrast this
experience, the DEN-4 strain 341750 only required 6
passages in PDK cells for a modified monkey infection.
For another DEN-1 strain, 1009, even after 50 PDK
passages there was no evidence of modified monkey
infection when compared to parental virus (data not
shown). In conclusion, PDK cell passage appears to be
an effective empirical method for modification and
attenuation of various DEN isolates. This is an
unnatural host for DEN that probably places selection
pressure for virus populations that are suited for PDK
replication but not necessarily for replication in
target cells in monkeys and humans.
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Materials and Methods for Candidate Vaccine Studies in
Humans
Volunteers. Healthy male and female volunteers
ages 18-45 were examined and screened by a panel of
tests, including blood chemistries, hematology,
prothrombin time, partial thromboplastin time,
urinalysis, rapid plasma reagin antibody, and serology
for hepatitis B surface antigen and antibody to HIV.
Volunteers were excluded on the basis of persistent
significant abnormality or positive test. Female
volunteers were eligible to participate if they had a
negative pregnancy test within 48 hours of vaccination
and were willing to sign a consent form stating that
they avoid conception using conventional contraception
for the 3 months following vaccination. In addition,
volunteers were excluded if they had previous
flavivirus immunity, which may affect responses to
dengue vaccines (Scott, 1983, J Infect Dis 148, 1055-
1060) or a history of allergy to neomycin,
streptomycin, or gentamycin. Prior flavivirus immunity
was defined as having no detectable hemagglutination
inhibition antibodies (at a 1:10 serum dilution)
against dengue types 1-4, Japanese encephalitis, or
yellow fever and no history of yellow fever vaccine or
flavivirus infection.
Volunteers scored >- 700 on a written exam
designed to test knowledge of all aspects of the
clinical trial. Informed consent was subsequently
obtained from each volunteer in compliance with US 21
CFR Part 50-Protection of Human Subjects. The
clinical protocol conformed to all relevant regulatory
requirements, including the Declaration of Helsinki
(Protocol), and Army Regulations 70-25-Use of
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Volunteers as Subjects of Research, and 40-7-Use of
Investigational Drugs in Humans and the Use of
Schedule I Controlled Substances. The studies were
approved by the Human Subject Research Review Board,
Office of the Surgeon General, U.S. Army, the WRAIR
Human Use Research Committee, and the Institutional
Review Board, University of Maryland at Baltimore.
Study Vaccines. The study vaccines are listed in
table 4. Vaccine viruses were passaged repeatedly in
primary dog kidney cells and then in fetal rhesus
monkey lung (FRhL) continuous diploid cell culture as
three terminal passages to prepare seed and vaccine.
Each candidate, before trial in volunteers, was
confirmed to elicit substantially reduced viremia
compared to its wild-type parent virus in vaccinated
rhesus monkeys. Adequate attenuation measured by
infection of rhesus monkeys indicated that the dengue
vaccine strains were appropriate vaccines for human
testing.
Immediately before immunization, a vial of
lyophilized vaccine was reconstituted with sterile
water for injection (USP). After immunization, unused
portions of rehydrated vaccine were maintained on ice
and titrated within 4 hours in LLC-MK2 cell monolayers
(Sukhavachana et al. 1966, Bull WHO 35, 65-66). Each
volunteer received between 1.0 x 105 and 4.5 x 106 pfu
of virus, depending on the candidate vaccine injected
(Table 4). The passage history of the individual
study vaccines is summarized below.
35
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Table 4. WRAIR LIVE ATTENUATED DENGUE VACCINES
Vaccine PDK Year Study Number Dose
Passage Site of (x 105
* Voluntee pfu)
rs
Dengue 27 1991 CVD 10 4.4 - 45
1
(45AZ5)
20 1991 CVD # 10 7.7 - 38
1992
10 1991 CVD 9 2.8 - 3.5
1992
0 1984 USAMRIID 2 ?
##
Dengue 50 1991 CVD 3 6.8
2
(S16803)
40 1996 USAMRIID 3 5
30 1991 CVD 10 5.6 - 10
1992
Dengue 20 1992 CVD 6 1.0 - 1.4
3
(CH53489
10 1992 CVD 3 3.8
0 1986 USAMRIID 2 ?
Dengue 20 1989 USAMRIID 8 1.0
4
(341750)
15 1991 CVD 3 4.8
TOTAL 10 - - 69 -
* Primary dog kidney passage level
# Center for Vaccine Development, University of
Maryland, Baltimore
## United States Army Medical Research Institute of
Infectious Diseases, Frederick, MD
Denaue 1 45AZ5 Vaccine: DEN-1 strain West Pac 74
was isolated from a human case of DEN fever on Nairu
Island (Western Pacific) in 1974. The isolate was
passaged 20 times in FrhL cell culture and a vaccine
lot was prepared. Passages included mutagenization
and plaque selection to recover a virus that was
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attenuated and suitable for human vaccination.
Following vaccination of two human volunteers, the
decision was made to discontinue use of the vaccine
due to DEN illness in one of the volunteers. The
vaccine was further attenuated by passage in PDK and
FrhL cell cultures. The current, candidate vaccine is
DEN-1 45AZ5 PDK-20.
Dengue 2 S16803 Vaccine: The dengue 2 strain
S16803 virus was derived from a Thai virus isolate
from a patient with dengue fever. The virus was
subjected to a total of 50 PDK passages, with terminal
passage in fetal rhesus monkey lung diploid cells
(DBS-FRhL-2) for seed and vaccine production. Two
vaccine candidates were initially prepared at the 30th
and 50th PDK passage levels and selected for testing.
Another vaccine candidate was developed at the WRAIR
from the same dengue 2 parent strain 516803 virus and
produced at the 40th passage level by the Salk
Institute (Swiftwater, PA).
Denaue Tvt~e-3 CH53489 Vaccine: Dengue type-3
strain CH53489 virus was derived from a Thai strain,
passaged 30 times in primary dog kidney (PDK) cells
after initial passage in primary green monkey kidney
(PGMK) and C6/36 insect cells. Virus from PDK
passages 10, 20, and 30 was used to inoculate fetal
rhesus monkey lung diploid cell cultures.
Dengue 4 341750 Carib Vaccine: The dengue 4
vaccine candidate was derived from a Caribbean strain
of Dengue 4 (Columbia, 1982), passaged at the
University of Hawaii, and manufactured at the WR.AIR
(Marchette, 1990, Am J Trop Med Hyg 43, 212-218).
Antibody to the parent virus neutralizes other dengue
4 virus strains including H-241, the prototype strain.
Attenuation of the human isolate was achieved by
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passage 20 times in primary canine kidney (PDK) cell
cultures.
Study Desian. A standard randomized, single
blind inpatient clinical protocol was used for all
pilot studies. The majority of the studies were
conducted at the Center for Vaccine Development,
University of Maryland, Baltimore MD. The pilot
studies of dengue 2 S16803 PDK 40 vaccine and dengue 4
CH341750 PDK 20 vaccine were performed at the Medical
Division, United States Army Medical Research
Institute of Infectious Diseases (USAMRIID), Ft
Detrick, MD.
In the initial clinical studies of a vaccine, the
highest available passage for a particular strain was
tested first in three volunteers. Symptoms were
monitored closely for three weeks, and if the
volunteers remained well, the next lower passage was
tested. If one or more of the volunteers became ill,
testing of lower passages of the vaccine strain was
not performed, as it was presumed lower passages were
likely to be less attenuated. After testing of all
acceptable passage levels in three volunteers, the
lowest level that did not cause illness was selected
for further testing in up to seven additional
volunteers.
To allow careful observation, prevent exposure to
extraneous infectious diseases, and to prevent the
possible infection of vector mosquitoes, volunteers
were confined to the research ward from three days
prior to inoculation until 20 days after immunization.
All adverse experiences occurring within this period
following administration of each vaccine were
recorded, irrespective of severity or whether or not
they are considered vaccination-related. Acceptable
safety of a vaccine was defined in advance as the
CA 02365728 2001-09-21
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absence of the following serious adverse events: any
severe clinical illness not explained by a diagnosis
unrelated to the vaccination; persistent fever (oral
temperature of >_38.5°C for 4 determinations over 24
hours, a maximum daily oral temperature of >_38.5°C on
three successive days, or temperature exceeds 40°C on
any individual determination); thrombocytopenia (fewer
than 100,000 platelets/mm3) or leukopenia (absolute
neutrophil count < 1000) on 2 consecutive deter-
minations; or serum amino alanine transferase (ALT)
level of more than 4 times normal on 3 or more
successive days which is otherwise unexplained. In
addition, any experience which would suggest any
significant side effect that may be associated with
the use of the vaccine were documented as a serious
event.
Volunteers were inoculated subcutaneously with
0.5 ml of undiluted vaccine on day 0. After
immunization, vital signs were recorded every 6 hours.
The injection site was examined and the maximum
diameter of erythema and induration measured and
recorded daily. Clinical signs (fever [>37.8°C],
rash, vomiting, petechiae, and liver and splenic
enlargement) and symptoms (malaise, headache, myalgia,
arthralgia, nausea, and eye pain or photophobia) were
assessed daily for the first 20 days after
immunization. Symptoms were graded as mild (noticed
symptom but continued ward activity) or severe (forced
to bed by symptom). If requested by the volunteer,
painful symptoms were treated with propoxyphene
hydrochloride; antipyretics were not used.
Observations were recorded on a standard checklist of
symptoms and physical findings. Volunteers were
discharged from the study ward on day 21, and
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requested to return for serologic studies 1, 6, 12,
and 24 months after inoculation.
Two healthy flavivirus-immune volunteers were
immunized at USAMRIID with the parent strain of the
dengue 1 45AZ5 vaccine and two years later with the
parent strain of the dengue 3 CH53489 vaccine.
Medical records from the study were reviewed for
presence or absence of the following signs and
symptoms: fever, rash, malaise, headache, myalgia,
arthralgia, and eye pain or photophobia. Viremia was
measured daily. In contrast to the present trials,
symptoms were not systematically recorded, and the
intensity of symptoms was not graded. In addition,
clinical experience with the dengue 4 341750 Carib PDK
20, given to 8 volunteers at USAMRIID during a later
study, was extracted and summarized to compare with
those of the current vaccinees (Hoke, 1990, supra).
Laboratory Evaluation. Blood was collected from
volunteers every other day and on day 31 for routinely
available medical tests for hemoglobin and hematocrit,
white blood cell count with differential count,
platelet count, and aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) levels. In
addition, blood was collected every other day through
day 20 for virus isolation and antibody studies.
Blood (20 ml) was allowed to clot at 4°C for <_ 2
hours, sera was decanted into 1-ml aliquots, frozen
and stored at -70°C until study.
Virus Isolation. For determination of dengue
viremia, serum was thawed and inoculated onto C6/36
mosquito cell monolayers and incubated at 28°C for 14
days. Supernatant culture fluid harvests were assayed
for virus by plaque assay on LLC-MKZ cells
(Sukhavachana et al. 1966, Bull WHO 35, 65-66). To
WO 00/57908 32 PCT/US00/08201
quantitate the amount of virus in serum, a plaque
assay was performed on the C6/36 clone of Aedes
albopictus mosquito cells (Hoke, 1990, supra). Cell
culture flasks were inoculated with dilutions of
plasma and adsorbed at 35°C for 1-2 hours. An overlay
medium consisting of Hank's Balanced Salt Solution and
0.750 agarose, 5~ lactalbumin hydrolysate, 0.12 M
NaHC03, and antibiotics was added and all flasks were
incubated at 35°C. After 7 days, the flasks were
stained with 50 liquid neutral red for 3-5 hours.
Excess stain was removed and the plaques read after 18
hours.
Seroloav. Antibody tests included ELISA, HAI,
and plaque reduction neutralization tests (PRNT)
performed using a dengue virus of the same serotype as
the strain in the vaccine being tested. Detection of
anti-dengue IgM antibodies was performed by
modification of an ELISA, where values >0.10 OD units
were considered positive (Innis, 1989, supra). The HAI
test was performed by the standard technique modified
to microvolumes using 4-8 units of individual
antigens, using serum extracted with acetone to remove
inhibitors 9Clarke and Casals, 1958, Am J Trop Med Hyg
7, 561-573). PRNT assays were performed by the method
described by Russell et al. (Russell, 1967, supra).
Statistical Analvsis. The relationship between
passage level and the frequency and severity of
reactogenicity was analyzed, for dengue 2 vaccine
S16803 (PDK 30, 40 and 50) and for dengue 3 vaccine
CH53489 (PDK 10 and 20), using the Cochran-Armitage
test for trend and Spearman's correlations,
respectively. The symptoms and signs independently
analyzed included the presence or absence, and the
number of days experiencing eye symptoms, headache,
CA 02365728 2001-09-21
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WO 00/57908 33 PCT/US00/08201
malaise, myalgia, arthralgia, rash and fever
(temperature > 37.8°C). The null hypothesis, that
higher PDK level was not associated with lower
reactogenicity, was evaluated at a probability of five
percent. By inspection of the data, the optimal
passage level for each virus was determined based on
the clinical and immunological responses of each
volunteer. The passage level which caused no
unacceptable side effects but which immunized about
800 of volunteers was selected for further development
by the U.S. Army Medical Research and Development
Command's Flavivirus Vaccine Steering Committee.
Definition of Infection by the Vaccine.
Infection by vaccine is defined as replication of
dengue virus in the volunteer, detected by presence of
serum type-specific neutralizing antibody or IgM anti-
dengue antibody after immunization. Viremia was not
included as necessary for diagnosis of infection as it
was never detected in the absence of an antibody
response. A vaccine failure is defined as an
unacceptable adverse clinical response or failure to
develop convalescent IgM or PRNT antibodies.
Example 3
Clinical Responses to Attenuated Denaue Vaccines
Dengue 2 516803 Vaccine
The dengue 2 strain 516803 virus produced from
the 50th passage in PDK cells was tested in three
volunteers. The volunteers did well, with no oral
temperatures > 38.0°C. Two of 3 volunteers had
transient mild symptoms of malaise, headache, and eye
symptoms (eye pain or photophobia). Laboratory
findings included mild ALT elevations (<2x normal) in
2 of 3, and mild leukopenia in 1 of 3 volunteers.
Because of the acceptable safety profile of the PDK 50
CA 02365728 2001-09-21
WO 00/57908 3 4 PCT/US00/08201
vaccine, the next lower available passage, PDK 30, was
selected for clinical evaluation.
The PDK 30 vaccine, tested in 10 subjects, was
underattenuated and produced symptoms compatible with
mild to moderate dengue. Four volunteers (400)
developed low grade fever, to Tmax 38.5°C, over days 9
-14 post vaccination (median day 12). Eighty percent
developed rash. The majority of volunteers experienced
eye symptoms (10/10), headaches (9/10), and malaise
(9/10), while 70 percent had >_ 1 severe symptom of
headache, eye pain and photophobia, malaise, or
myalgia. Three volunteers had mild elevation of their
alanine aminotransferase (ALT), a measure of liver
pathology.
Because the PDK 30 vaccine was considered too
reactogenic to test further in volunteers, the PDK 40
vaccine was produced from the master seed. Two of
three volunteers inoculated with PDK 40 developed a
mild dengue-like syndrome 9-10 days after vaccination,
with low-grade temperatures (<38.1°C), rash, myalgias,
and headache. Symptoms resolved spontaneously over
several days without disability or requirement for
medication. Accompanying symptoms was an unanticipated
rise in serum liver enzymes, to a maximum ALT level of
199 IU/ml in one (4 times normal) and 77 IU/ml maximum
ALT for the other (1.5 fold elevation from normal).
The third volunteer remained asymptomatic but also
developed two-fold elevations in ALT (to max 102). All
laboratory abnormalities resolved within days without
intervention, and all volunteers were discharged in
good health 21 days after receipt of the vaccine.
Because of the unusual frequency of hepatitis events
associated with PDK 40 vaccine, no further development
is planned for the product.
CA 02365728 2001-09-21
WO 00/57908 3 5 PCT/US00/08201
Table 5 summarizes the initial clinical
experience with the WR.AIR dengue 2 vaccine.
Decreased frequency of signs of fever and rash are
apparent between passage level 30 and 50 vaccines.
Furthermore, there is a decline in oral temperature
from Tmax 38.5°C towards normal with increasing
passage, but no change in duration of fever beyond one
day. For the dengue 2 vaccine, the frequency and
duration of eye symptoms, rash, headache, malaise and
myalgia were significantly associated with passage
level.
CA 02365728 2001-09-21
WO 00/57908 PCT/US00/08201
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CA 02365728 2001-09-21
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CA 02365728 2001-09-21
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CA 02365728 2001-09-21
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CA 02365728 2001-09-21
WO 00/57908 40 PCT/US00/08201
Dengue 3 CH53489 Vaccine. A dengue 3 vaccine
(CH53489, PDK 0) developed at WR.AIR was administered
to two healthy yellow fever-immune male volunteers as
a 0.5 ml subcutaneous inoculation of 2 x 104 pfu of
virus. The immediate post immunization course was
uneventful. By day 6, both volunteers were ill with
moderately severe dengue fever characterized by high
fever, chills, myalgias, headache, malaise, and a
diffuse erythematous rash. Both volunteers developed
thrombocytopenia and leukopenia but there were no
signs of hemorrhagic fever. After a febrile period
lasting five days, both men rapidly recovered and were
well by day 21. Because of the severe illnesses
experienced by both subjects, no further testing of
this passage level was undertaken. Subsequently, PDK
10 and PDK 20 passage levels were prepared as vaccine
candidates.
The PDK 20 vaccine was given to 6 volunteers and
resulted in mild reactogenicity. One subject
experienced an early febrile illness on day 3 with
transient fever (Tmax 38.2°C), pharyngitis, and
cervical lymphadenopathy. No dengue virus was isolated
from the volunteer's serum. This subject was felt to
have had an intercurrent illness with fever, which was
not directly related to vaccination. Four out of 6
volunteers developed short-lived mild dengue symptoms
without rash; arthralgia, eye pain, and headache were
the most frequent complaints. However, one volunteer
had more severe symptoms of headache, malaise, and eye
pain for three days. He also developed leukopenia and
sustained elevation in ALT levels; these laboratory
abnormalities had resolved on follow-up at day 31.
Another volunteer had mild and reversible elevation of
ALT alone, to less than 2x normal. Because the PDK 20
vaccine was safe with marginally acceptable
CA 02365728 2001-09-21
WO 00/57908 41 PCT/US00/08201
reactogenicity, the next lowest available passage
vaccine virus (PDK 10) was tested.
The PDK 10 virus proved too reactogenic in
recipients. One of three volunteers developed low-
s grade fever on days 10 and 11 (Tmax 38.3°C), and a
florid rash for 13 days. Another volunteer developed
persistent pruritus associated with waxing and waning
hives on days 6 to 9 post vaccination, and tender
cervical and axillary lymph nodes. He subsequently
developed a maculopapular rash with malaise, headache,
and myalgia on days 10-12. This volunteer may have
had an idiosyncratic allergic reaction to the vaccine,
followed by a typical dengue-like illness. These two
volunteers also had laboratory abnormalities of
leukopenia and elevation of ALT levels to <2x normal,
which resolved on followup on day 31.
Table 6 summarizes the response to dengue 3
CH53489 vaccines. Although there was a trend for less
frequent and shorter duration signs and symptoms with
passage, no passage reached statistical significance
in either analysis.
Dengue 4 342750 Vaccine. Eight volunteers
received 105 PFU of PDK 20 vaccine (Hoke, 1990 supra).
Five volunteers developed a scarcely noticeable
macular, blanching rash and minimal temperature
elevation (max 38.1°C). Viremia and antibody response
also developed in these five volunteers (630).
A new DEN-4 341750 candidate vaccine was
prepared from PDK passage 15, anticipating that the
lower passage might be more infective. Three
volunteers received this vaccine and two experienced
minimal symptoms. The third volunteer became ill
abruptly on day 8 with fever, edematous swelling of
the face and extremities, severe lassitude, rash, eye
pain, photophobia, and arthralgias. Over the next
CA 02365728 2001-09-21
WO 00/57908 42 PCT/US00/08201
three days, fever persisted with Tmax of 39.6°C, but
signs and symptoms resolved spontaneously. Because of
this serious adverse reaction to vaccination, further
use of PDK-15 vaccine was terminated and PDK-20 was
chosen for further evaluation.
Example 4
Viremia and Immune Responses to Attenuated Dengue
Vaccines
Table 7 describes viremia and immune responses
with the WRAIR dengue vaccines. The infectivity of
the individual vaccines is summarized below.
Dengue 2 516803 Vaccine. No recipients of the
PDK 50 vaccine developed viremia, yet two of 3
developed low-titer neutralizing antibody by day 60.
These findings suggested that the vaccine virus was
diminished in infectivity for humans. By contrast,
two of 3 dengue 2 PDK 40 vaccinees had demonstrable
viremia, and all developed high titer antibody after
vaccination. As expected, infectivity of the dengue 2
PDK 30 vaccine was highest: viremia was detected in
all 10 volunteers and all subjects seroconverted with
neutralizing antibody titers of >1:60 by day 60.
Dengue 3 CH53489 Vaccine. Dengue-3 virus
retaining temperature sensitivity and small plaque
phenotype of the vaccine virus was recovered for 6 and
7 days in the 2 yellow fever immune recipients of the
dengue 3 PDK 0 vaccine. Subsequently, high titered
PRNT50 and hemagglutination inhibition (HAI)
antibodies with a secondary-infection-like cross
reactivity was measured in serum collected on days 30
and 60 from both volunteers. Infectivity was similar
in subjects who received the dengue 3 PDK 10
attenuated vaccine: 2 of 3 developed viremia and
vaccination induced neutralizing antibodies in all.
CA 02365728 2001-09-21
WO 00/57908 43 PCT/US00/08201
In contrast, 2 of 6 dengue 3 PDK 20 vaccinees had
detectable viremia and three volunteers subsequently
seroconverted, reflecting diminished infectivity.
Dengue 4 341750 T7accine. Eight volunteers received
105 PFU of the PDK 20 vaccine, and viremia and
antibody response developed in five (63~). The vaccine
prepared from a lower passage of this candidate, PDK
15, was more infective. Virus was isolated from a
single volunteer, on days 8 and 10 following
vaccination, with maximum titer of 15 pfu/ml. This
volunteer subsequently developed a neutralizing
antibody titer of 450 with a secondary HAI response,
and was found to have been previously exposed to St.
Louis encephalitis virus (PRNT titer 1:20 before
vaccination). The two volunteers without detectable
viremia developed neutralizing titers of 1:10 and 1:40
by day 30 after vaccination.
Example 5
Selection of Candidate Vaccines
The extended program of safety testing of the
WR.AIR PDK-attenuated vaccines is shown in Table 8,
which lists the salient features of the vaccines for
each serotype. Increasing PDK passage resulted in
decreasing mean illness score, which assesses duration
and number of symptoms per volunteer. In addition,
rising PDK passage was also associated with decreased
mean days of viremia, with the exception of dengue 4
vaccines. Of the tested dengue 2, 3, and 4 vaccines,
only one passage level was judged safe and acceptably
reactogenic, and suitable for expanded clinical study:
dengue 2 PDK 50, dengue 3 PDK 20, and dengue 4 PDK 20.
However, the percentage of recipients infected
declined with increasing PDK passage level.
Seroconversion, defined as percentage with
CA 02365728 2001-09-21
WO 00/57908 44 PCT/US00/08201
neutralizing antibody titer _> 1:10 similarly declined
within broad confidence intervals.
Discussion
The WRAIR has longstanding involvement in the
development of live-attenuated dengue vaccines. Both
the WRAIR and Mahidol dengue vaccine programs have
developed several live vaccines by attenuation through
several passages (repeated growth in tissue culture)
in dog kidney (PDK) cells. The results of the pilot
testing in small numbers of volunteers established the
safety of WRAIR candidate vaccines. No volunteers
among 65 recipients required emergent treatment of
sustained serious injury. Three volunteers suffered
transient idiosyncratic reactions associated with
dengue vaccination, resulting in withdrawal of the
vaccines they received from further clinical
development. Experimental infection with
underattenuated vaccines, while uncomfortable, was
tolerable.
The clinical experience showed that increasing
PDK passage of vaccine viruses increased attenuation
for volunteers. This effect is best seen with dengue
1 and dengue 3 viruses, where parental unpassaged
viruses resulted in unmodified dengue fever and
subsequent 20 PDK passages acceptable reactogenicity.
However, increasing PDK passage decreased infectivity
of vaccine viruses, resulting in diminished
immunogenicity. Furthermore, diminished viremia with
vaccine viruses in humans appear to correlate with
those in rhesus monkeys (with the exception of dengue
4 PDK 15). These findings suggest that infectiousness
of an attenuated dengue virus vaccine in volunteers
proved equivalent to immunogenicity.
The relationship between passage level and
reactogenicity should be interpreted with caution,
CA 02365728 2001-09-21
WO 00/57908 45 PCT/US00/08201
because subjects who experienced one symptom were
likely to experience several symptoms. As our
analytic methods assume independence of these
symptoms, interpretations based on independent p-
values can be tenuous. Still, we believe rash showed
a strong association with passage level (independent p
- 0.009 for presence, p = 0.01 for duration). This is
bolstered by a lack of significant correlation between
rash and other symptoms, for either Dengue 2 or 3
vaccine (Spearman's tests).
Only vaccines with acceptable safety profiles
were selected for expanded clinical testing: dengue 1
45AZ5 PDK 20, dengue 2 S16803 PDK 50, dengue 3 CH53489
PDK 20, and dengue 4 341750 PDK 20. Because of the
broad confidence intervals in seroconversion due to
small numbers of volunteers, subsequent studies sought
to increase the number of recipients of each of the
four selected vaccines. In addition, further tests
will seek to determine whether immunogenicity of these
attenuated vaccines can be boosted through
administration of two doses instead of the single dose
used for these studies.
Example 6
Expanded studv of monovalent vaccines;
monovalent vaccines given as two doses; and monovalent
vaccines mixed as a tetravalent formulation given as
one and two doses
Study Design: The objectives of these were to
evaluate the safety and immunogenicity of the four
monovalent vaccines given as a single dose and then by
two-dose vaccination schedules. Subsequently safety
and immunogenicity of the combination tetravalent
vaccine were evaluated. Subjects were separately
recruited from two sites, University of Maryland at
CA 02365728 2001-09-21
WO 00/57908 46 PCT/US00/08201
Baltimore and the WRAIR, Washington DC. The first
group of 22 subjects were divided into 4 groups of 4
or 5 persons who each received either a single dose of
monovalent dengue or yellow fever 17D virus
(Connaught). The 17D yellow fever vaccinees served as
control and benchmark for reactogenicity. Another 31
subjects were divided into 4 groups of 7-8 persons who
were given two doses of one monovalent vaccine, half
at 1 month and the other half at 3 months. Finally 10
volunteers were given 2 or 3 doses of the tetravalent
vaccine. The first 4 tetravalent recipients received
vaccination at 0 and 1 month. The latter 6
tetravalent recipients were vaccinated at 0,1 and 4
months. All subjects except the 10 tetravalent
vaccine recipients were given a vaccine serotype at
random and in double-blinded fashion.
Subjects: Subjects were normal healthy adults
age 18-50. All subjects were seronegative for
hepatitis B, C and HIV. All subjects were
seronegative for dengue 1-4, JE, SLE, and YF by
hemagglutination inhibition assay before entry into
the study.
Vaccines: The four serotype vaccine candidates
were originally isolated from humans with clinical
disease. Each were then modified by serially passage
in primary dog kidney (PDK) and then fetal rhesus lung
cells as described above. These candidates were
selected based on previous small pilot studies in
human volunteers. Each lyophilized monovalent
vaccines were reconstituted with sterile water and
given in a volume of 0.5 cc. The doses of serotypes
1-4 were 106, 106, 105 and 105 pfu of Dengue 1, 2, 3,
and 4 respectively. The tetravalent vaccine dose was
prepared by mixing 0.25 cc of each reconstituted
monovalent and given in a final volume of lcc. The
CA 02365728 2001-09-21
WO 00/57908 4~ PCT/US00/08201
dose of the tetravalent vaccine was 1.1-2.8 x 106 pfu.
All vaccinations were given subcutaneously in the
upper arm.
Clinical safety: Reactions to vaccinations
were assessed by combination of daily symptom diaries
and periodic physician evaluations during the 3 weeks
after each vaccination. Subjects were housed in study
quarters for close observation for 5-7 days past the
incubation period of 1 week after vaccination, a time
period during which reactions and viremia were most
likely . Subjects were examined and queried
specifically for symptoms of feverishness, chills,
headache, retroorbital pain, myalgia, arthralgia, rash
and others. Each symptom was graded on a scale of 0
(none), 1 (did not affect normal activity; did not
require medications), 2 (required medication or change
in activity), or 3 (required bedrest or unrelieved by
medication). The most common symptoms were grouped
into four categories. These categories were:
1)subjective fever and chills, 2)headache and
retroorbital pain, 3)myalgia and arthralgia and
4)gastrointestinal complaints which included nausea,
vomiting and abdominal pain. A symptom index of each
category was calculated by the product of the highest
symptom grade for each day and the duration of the
symptom expressed in days. If symptom occurred at all
during 24 hours it is assigned duration of 1 day. The
Reactogenicity Index (RI) is simply the sum of the
symptom indices for each category. The RI summarized
the vaccine reactions of each subject. The symptom
category indices and RI allow for semi-quantitative
comparison of vaccine reactions among subjects and
vaccine serotypes.
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Subjects were monitored for hematologic and
liver toxicities by serial CBC, platelet counts, AST
and ALT during the study.
Serious adverse events were defined as severe
illness lacking other likely causes, fever >38.5°C
continuously for over 24 hours or Tmax >38.5°C for 3
consecutive days or a single oral temperature >104°C,
neutropenia of <1,000/ml or thrombocytopenia of
<90,000/ml on 2 consecutive determinations, or serum
ALT or AST > 5 times normal.
Immunogenicity: Method of hemagglutination
inhibition assay was done by method of Clarke and
Cassals, 1958 (Am J Trop Hyg 7, 561-573) Dengue IgM
and IgG were measured by capture ELISA in all but the
last 6 tetravalent subjects. Dengue and yellow fever
neutralizing antibodies were measured on Day 0 and 30
after each vaccination by plaque reduction
neutralization test. The study endpoint determination
was measurement of any neutralizing antibody 30 days
after last vaccination. Neutralizing antibody
seroconversion is defined as 50o reduction in plaques
at minimum of 1:5 serum dilution. Viremia was
determined on sera from days 7-14 after initial and
second vaccination. Method used for virus isolation
was a delayed plaque method adapted from Yuill, 1968
(Am J Trop Med Hyg 17, 441-448) using LLC-MK2 or C6/36
cells for amplification and Vero for plaque formation.
Data from the single-dose and two-dose studies
were combined for this report. The subject
characteristics are shown in Table 9. Total of fifty
nine normal subjects were given dengue virus vaccines;
forty nine received monovalent test articles and ten
received tetravalent vaccine. Four received licensed
17D yellow fever vaccination (Connaught).
CA 02365728 2001-09-21
WO 00/57908 49 PCT/US00/08201
Table 9. Subject Characteristics
Vaccine No . Sax Race Mean
Subjects Age
(No.
received 2
doses)
Den-1 12 (8) 7M/5F 6W/6B 32
Den-2 12 (8) 7M/5F 7W/5B 36
Den-3 13 (8) 9M/4F 8W/5B 36
Den-4 12 (7) 6M/6F 4W/6B/1H/lAmI 33
Tetravalent4 (4) 3M/1F 4W 26
YF 17D 4 (0) 3M/1F 3W/1B 30
Example 7
REACTOGENICITY
Local reaction. Nineteen of 59 (32%) dengue
vaccine recipients reported mild arm pain at injection
site. Of these 7 received DEN-1, 4 DEN-2, 1 DEN-3, 1
DEN-4 and 5 received tetravalent. Only 5 reported any
injection site pain after 24 hours. None affected use
of the arm.
Systemic reactions. 200 of 59 dengue recipients
reported no symptoms at all with their first
vaccination while 700 of subjects were asymtomatic
with the second vaccination. The four subjects who
received a third dose reported no symptoms associated
with it. The most commonly reported reactions from
dengue vaccination were headache and myalgias. They
occurred in varying severity. Figure 1 shows
occurrence of > Grade 1 symptoms from the first
vaccination causing change in daily activities or
taking of medications for relief. After the first
dose of vaccine, five (80) subjects, one serotype 1,
one serotype 4, and three tetravalent, reported one
severe grade 3 symptom of either chills, myalgia,
headache or nausea for less than 1 day duration. No
subjects reported any grade 3 symptoms with
revaccination.
CA 02365728 2001-09-21
WO 00/57908 5 0 PCT/US00/08201
The RI ranged from 0 to 35. Table 10 compares
the reported reactogencity of each vaccine. The DEN-1
monovalent and tetravalent vaccines were associated
with more reactogenicity. The second or third dose of
all dengue vaccines uniformly caused few reactions,
even in those subjects with moderate to severe
symptoms from the initial vaccination.
Table 2. Mean Reactogenicity
Index
Vaccine Total Dose Dose 2 RI Dose 3
1
Subject RI (n) (n) RI (n)
s
Den-1 12 7.4 (12)0.5 (8) -
Den-2 12 3.8 (12)0.3 (8) -
Den-3 13 2.9 (13)0.8 (8) -
Den-4 12 3.7 (12)0.5 (6) -
Tetravalent 10 9.3 ( 1.9 ( 10) 0.0 (4)
10)
YF 17D 4 3.8 (4) - -
- = not done
Figure 2 shows the frequency distribution of RI
by serotype. Eight subjects (140) developed fever
(>100.4° F). Of the eight 4 received DEN-1, 1 DEN-2,
1 DEN-3 and 2 tetravalent. Highest and longest fever
ocurred in a DEN-1 recipient with T~ of 103.3° F and
fever of 3 days. Only one other subject, who also
received DEN-1, had more than one day of fever. Seven
of the eight episodes of fever occurred following the
first vaccination.
Sixteen subjects(27o) developed a generalized
rash, involving the trunk and extremities from their
first vaccination. Rash was usually erythematous,
macular papular and mildly pruritic. Only 7 of the 16
with generalized rash had fever. Of the 16 subjects
with rash five received DEN-1, two DEN-2, one DEN-3,
three DEN-4 and five tetravalent. Rash typically
became noticeable by day 8-10 after vaccination and
resolved in 3 ?4 days. No subjects developed any
CA 02365728 2001-09-21
WO 00/57908 51 PCT/US00/08201
petechiae, purpura or scarring. No subjects developed
rash from revaccination.
Gastrointestinal symptoms were relatively common,
occurring in a third of subjects, but they were mild
and brief, lasting less than 24 hours. One DEN-4
recipient developed severe nausea associated with
crampy abdominal pain for one day.
Six subjects(10~), 5 dengue and 1 yellow fever
17D recipient, developed transient neutropenia with
absolute neutrophil count less than 1000/ml. The
lowest was 288 in a DEN-1 subject. Neutropenia
typically resolves in 2-3 days. No subject developed
thrombocytopenia. There were no clinically
significant elevations in AST or ALT.
As expected of this group of non-immune adult
receiving their first dengue virus exposure none
developed any clinical evidence of dengue hemorrhagic
fever.
Example 8
IMMUNOGENICITY
Viremia was detected in 10 subjects (170), one
received DEN-2, four DEN-3, one DEN-4 and four
tetravalent. No DEN-1 viremia was detected. The
serotype(s) of the virus isolated from the tetravalent
subjects have yet to be identified. All detected
viremias occurred after the first dose of virus.
Curiously fever ocurred with viremia only in 3
tetravalent recipients. All viremic subjects
developed neutralizing antibody. One did not develop
IgM or IgG response even with viremia.
Table 11 summarizes the antibody responses to
monovalent vaccination. Neutralizing antibodies were
detected more frequently than the IgM and IgG. No
seroconversion was detected by IgM or IgG that was not
also found by the PRNTso of 1:10 serum dilution. then
CA 02365728 2001-09-21
WO 00/57908 52 PCT/US00/08201
present, IgM were positive in 41o by 14 days after
vaccination, in 17% by 21 days and 42o by 30 days.
IgM typically peaked by day 30 after first
vaccination. A single exception was in a tetravalent
recipient whose IgM peaked three days after his second
vaccination. IgM can persist for more than 3 months.
The seroconversion rates by neutralizing antibody were
1000, 920, 54o and 58o for monovalent serotypes 1,2,3
and 4 respectively. When present, neutralizing
antibody was typically detectable by day 30 after
first vaccination. No time points between day 0 and
30 were assessed for neutralizing antibody. The
second dose of vaccine boosted DEN-2 GMT by over four-
fold, which was not seen with the other serotypes.
Two DEN-3 subjects seroconverted after a second dose
of vaccine, one at 1 month and the other at 3 months.
They had not developed neutralizing antibody after one
dose. Interestingly the IgM/IgG patterns of these two
subjects suggest a secondary response after their
second dose suggesting they were immunologically
sensitized by the first dose.
Despite pre-entry negative hemagglutination
inhibition assay for dengue, SLE, JE and YF 5 of 53
(90) subjects tested developed a secondary antibody
response pattern with IgM to IgG ratio of <1.8. All 5
were negative for homologous dengue neutralizing
antibody prior to vaccination. This suggests a
previous occult exposure to flavivirus. We found no
significant difference between the mean RIs of
secondary and primary antibody responders (9.6 vs 5.8,
p=0.19).
There were 12 monovalent subjects who did not
develop IgM/IgG or neutralizing antibody. One
received DEN-2, six DEN-3 and five DEN-4. The mean
reactogenicity index for this group of antibody non-
CA 02365728 2001-09-21
WO 00/57908 53 PCT/US00/08201
responders was less than 1 which was significantly
different from the mean RI of type 2,3 and 4
neutralizing antibody responders. (0.9 vs 4.9,
p<0.003).
Our studies included 25 blacks and 31 Caucasian
subjects. There was no significant difference between
the mean RIs of these two racial groups. This is of
interest because there is some epidemiologic evidence
suggesting milder dengue disease severity among
blacks.
CA 02365728 2001-09-21
WO 00/57908 54 PCT/US00/08201
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CA 02365728 2001-09-21
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CA 02365728 2001-09-21
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Age, Sex
Table 12 shows PRNT seroconversion results
from the ten tetravalent vaccine subjects. The
first 4 subjects received two vaccinations at 0 and
1 month. One subject missed his second vaccination
on day 30 and was vaccinated on day 60. Six more
subjects were to be vaccinated at 0 and 1 month and
if response was incomplete a third vaccination at 4
month was administered. Two subjects developed
neutralizing antibody to all 4 serotypes after a
single dose. Another two tetravalent recipients
seroconverted to all 4 serotypes after vaccination
at 4 months. Two others developed trivalent
responses. A second dose of the tetravalent given
at 1 or 2 months did not significantly increase
seroconversions. The overall seroconversion rates
in these 10 tetravalent subjects were 100, 800,
80% and 40o for DEN-1,2,3 and 4 respectively.
Example 9
A study was designed to evaluate interaction
of each serotype component in tetravalent vaccine
by a 2-level 24 factorial design.
Fifty-four subjects were given 15 permutations
of 2 dose levels of each serotype. Results are
shown in Figure 3. H, high dose, indicates
undiluted vaccine, ranging between 105-106 pfu/ml;
L, low dose, indicates a 1:30 dilution of undiluted
vaccine resulting in about 103'5 -104'5 pfu/ml.
Six subjects were given full-dose tetravalent
vaccine at 0 and 1 month. If subject did not make
tetravalent neutralizing antibody response, a third
dose at 4 months was given. Results are shown in
Figure 4.
Four human subjects were given syringe-mixed
full-dose tetravalent vaccine at time 0 and 1
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month. Endpoints were clinical safety and
neutralizing antibody at 1 month after second
vaccination. T-cell responses were measured in the
first 4 subjects. Results are shown in Figure 5.
Results indicate that tetravalent vaccine (16
formulations) were found to be safe in 64 non-
immune American volunteers. Reactogenicity varied.
Four formulations elicited trivalent or tetravalent
neutralizing antibody responses in all volunteers.
In concordance with monovalent experience, a second
dose of tetravalent vaccine at 1 month did not
induce significant reactogenicity but also did not
augment neutralizing antibody responses. End
titration of neutralizing antibody responses is in
progress. Memory interferon-gamma responses in T-
cells can be measured in the absence of
neutralizing antibody. Dosing intervals >_ 4 months
may result in improved tetravalent seroconversion.
Discussion
These vaccines appear attenuated in humans
when compared with historical descriptions of
experimental infections with wild-type dengue.
(Simmons et al., 1931, Manila Bureau of Printing)
We used a numeric scale based on self-reported
symptom duration and severity to quantify
reactogenicity. Such method tends to over estimate
vaccine-related reactions. Ideally it should be
validated with cases of natural dengue infection.
However, imprecise the RI allowed us to reasonably
compare symptoms between individuals and groups.
Results from testing the monovalent vaccines showed
the degree of attenuation to be variable among the
four dengue vaccine candidates. 45AZ5 PDK20 is the
least attenauted, highest titer and resulted in
uniform seroconversion. The DEN-2 candidate,
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S16803 PDK50, similarly resulted in nearly 100%
seroconversion with a benign reactogenicity
profile. The Den-3 and Den-4 had low
reactogenicity profiles but seroconversion rates
were only 50-60%. It should be noted that the
doses of type 3 and 4, the less immunogenic
strains, are ten-fold less than that of types 1 and
2.
The second dose of virus was associated with
remarkably little reactions. However, the benefit
of a second dose of monovalent vaccine at 1 or 3
month is small. Den-1 and 2 were already near
uniformly immunogenic such that an additional dose
may be superfluous. Nevertheless the GMT of Den-2
was boosted over four-fold. This may be evidence
of low level viral replication after the second
dose or the dose contains sufficient antigenic mass
to elicit a booster response. This pattern of
neutralizing antibody response has also been seen
with second vaccination with 17D YF. (Wisseman,
1962, Am J Trop Med Hyg 11, 570-575) The first
dose of Den-3 may have sensitized the two
monovalent subjects who seroconverted after the
second dose with secondary antibody response
pattern. This suggests that our neutralizing
antibody assay may not be sensitive enough to
detect the appropriate immune response to type 3
vaccine candidates. The second dose did not add
any new seroconverters to type 4. There was no
obvious additional benefit in giving a second dose
of monovalent DEN-1 or DEN-4 with the dose and
schedule tested.
Twelve monovalent subjects who did not make
neutralizing antibody response to monovalent
vaccines also did not respond with measurable
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dengue IgM or IgG. All these non-responders
received viable virus from the same vial that
clearly replicated in other subjects. They
developed no reactions to the vaccinations. Thus,
by all indications there was no evidence of virus
replication in these subjects. The mechanism for
this nonresponsiveness is unknown. It may be the
result of lack of host substrate necessary for
infection or an effective innate immunity.
The value of multiple dosing may be more
apparent in combination live-attenuated vaccines as
a strategy to circumvent viral interference. Here
dose of each component as well as the dosing
interval may be important. Interference and
enhancement can potentially occur when dengue
viruses are given in combination. Four subjects
developed neutralizing antibody to all 4 serotypes,
two after the first dose, and two after a third
dose at 4 months. Four of five volunteers who
received revaccination at 4 months seroconverted to
3 or more serotypes. The explanation of this
difference may be that at one month after
vaccination there is sufficient cross-reactive
neutralizing antibodies to suppress replication of
heterotypic viruses in the vaccine. Sabin found
that there was such transient cross protection
lasting up to 3 months when human subjects were
given one serotype virus. (Sabin, 1959, Viral and
Rickettsial Infections of Man. Philadelphia: JB
Lippincott Company). Our future tetravalent
studies will use a 0,6 month vaccination schedule.
The poor immunogenicity of of DEN-3 and 4 may
be that at 105 pfu/ml Den-3 and Den-4 doses are at
replicative disadvantage compared to DEN-1 and 2,
both of which are at 106 pfu in the tetravalent
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formulation. We are exploring alternative
production strategies to increase titers of DEN-3
and DEN-4.
Without detecting viremia of all 4 viruses in
the tetravalent responders one cannot be certain
that the presence of neutralizing antibody
necessarily imply replication of all 4 serotypes.
Measured neutralizing antibodies may be cross
reactive and of low avidity. This problem should be
addressed by looking at long-term persistence of
antibody against each serotype. A sensitive and
serotype-specific RT-PCR assay would be useful to
determine polyvalent viremia as evidence of viral
replication.
Only two of the tetravalent vaccinees
developed neutralizing antibody to all 4 serotypes
after one vaccination. Such incomplete response to
tetravalent vaccine raises questions about risk of
dengue hemorrhagic fever in the setting of exposure
to virulent heterologous serotypes. If antibody-
dependent enhancement is the pathophysiologic
mechanism for DHF risk may be present even when all
four serotype antibodies are elicited by
vaccination but one or more serotype antibody wanes
differentially below neutralizing threshold. We
report below that TH1 T-cell response can be
measured in these tetravalent vaccinees even in the
absence of neutralizing antibody. Would that be
sufficient to protect? These questions may only be
answered by careful long-term field testing of
tetravalent vaccines in endemic areas.
In conclusion, our results indicate that the
four serotypes are variably reactogenic as
monovalent vaccines with type 1 more so than
serotypes 2,3 and 4. Serotypes 1 and 2 elicited
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neutralizing antibody in >90~ while serotypes 3 and
4 are less immunogenic. The tetravalent
combination is safe, reasonably well-tolerated and
induced neutralizing antibody to all 4 serotypes in
four of ten subjects. Two doses of tetravalent
vaccine did not improve seroconversion rates at the
one or two-month dosing intervals tested. A longer
dosing interval of over 4 months may improve
seroconversion rate.
Example 10
Material and Methods for T-cell response to denaue
vaccines
Subjects. Thirty-five healthy adult volunteers
ages 18-50 (21 males, 14 females) participated in a
phase I clinical trial, conducted by the Walter Reed
Army Institute of Research, involving candidate dengue
virus vaccines. The participants were selected from a
group of volunteers based upon the absence of
circulating anti-flavivirus antibody. Additional
selection criteria was HIV negative status and good
health based upon a physical exam and responses to a
questionnaire.
Vaccine aroups. Thirty individuals randomly
received two doses of a live attenuated monovalent
vaccine; four received two doses of a live attenuated
tetravalent vaccine. One monovalent recipient
(volunteer ID 1) quit the study after only receiving
the first dose. Prior to vaccination, there was no
detectable hemagglutination-inhibiting serum antibody
to dengue virus types 1-4, Japanese encephalitis
virus, St. Louis encephalitis virus, or yellow fever
virus in any of the volunteers. Each dose was given as
a 0.5 ml subcutaneous injection of undiluted
virus(es).
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PBMC collection. Peripheral blood (8 ml) was
collected from each volunteer by venipuncture into
Vacutainer Cell Preparation Tubes (CPT) (Becton-
Dickinson, Franklin Lakes, NJ) on day 0 and at five
time points after the first dose but prior to the
second dose(days 3, 7, 9, 14, 28/ 30/ 31/ 60 or 91).
Blood was also collected on the day of the second dose
and at four time points afterwards (days 3, 7, 9 and
14 post second dose). The time of administration of
the second dose, depending on the volunteer, thus was
approximately 1-3 months after the first dose.
Variation in collection times around 1 month occurred
due to variation in volunteer scheduling. Cells were
separated from whole blood by centrifugation at 1000
xg for 30 minutes. PBMC were collected (the cell layer
above the gel in the CPT tube) and washed twice in
Hank's balanced salt solution (Life Technologies,
Rockville, MD) with centrifugations at 500 xg.
Isolated PBMC were resuspended in 4 ml (per CPT tube)
of Cell Freezing Media/ DMSO (Sigma, St. Louis, MO)
and frozen in 1 ml aliquots overnight at -70°C. The
PBMC were then transferred to vapor phase liquid
nitrogen for long term storage.
Vaccine viruses. The following live attenuated
dengue virus strains described above were used in the
monovalent vaccines: 45AZ5PDK20 (DEN 1), S16803PDK50
(DEN 2), CH53489 (DEN 3), 341750PDK20 (DEN 4). The
tetravalent vaccine was an equal mixture of all four
of these strains.
Cell culture viruses. The following dengue
viruses, propagated in Vero cells, were used for PBMC
stimulation in culture: Westpac 74 (DEN 1), S16803
(DEN 2), CH53489 (DEN 3), and TVP360 (DEN 4). All four
serotypes were provided by Dr. Robert Putnak in 1 ml
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aliquots and stored at -70°C until use. The virus
titers ranged from .30- 2.4 x 10 6 pfu/ml.
Bulk culture of PBMC and stimulation with live
virus. Frozen vials of PBMC were removed from liquid
nitrogen storage and gently thawed at 37°C. PBMC were
washed twice with RPMI medium 1640 (Life Technologies,
Rockville, MD) and suspended in complete media
containing 10o human male AB serum (Sigma) plus
supplements [penicillin (100 U/ml)-streptomycin (0.1
mg/ml)-fungisone (0.25 mg/ml) [Sigma], 2 mM L-
glutamine (Life Technologies), and 0.5 mM 2-
mercaptoethanol (Sigma)]. The cells were suspended at
a concentration of 2.5 million cells/ ml. Some assays
required 3.25 million cells/ml. The PBMC (100 ml)
were added to individual wells of a 96-well V-bottom
plate (Costar, Acton, MA). An equal volume of dengue
virus 1, 2, 3, or 4 diluted in 10% complete media at a
concentration of 3000 to 24000 pfu/100 ml was added to
each well. Control wells received an equal volume of
medium without virus. The cells were then cultured at
37°C in 5% C02 for four days .
Immunoas sav .
A chemiluminescent immunoassay was done to
determine the quantity of lymphokine secreted in
tissue culture supernatant at the end of 4 days of
culture. A 96 well immunoassay plate, Microlite 2
(Dynatech Laboratories, Inc., Chantilly, Virginia) was
coated overnight with 50 ul/well of 10 mg/ml unlabeled
anti-lymphokine (IL-4, IL-10, or Interferon y)
antibody (Pharmingin San Diego, CA) in a 0.1 M
potassium bicarbonate buffer. The plates were washed
and 100 ul I-block buffer (Tropix, Bedford, MA ) was
added for one hour. Standards (Recombinant IL-4, IL-10
and Interferon 'y, Pharmingen, San Diego, CA) were pre
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diluted in I-block beginning with a concentration of
ng/ml. Eight-three fold dilutions of the standard
were made. Samples, controls and standards were
diluted in an equal volume of I-block buffer. Aliquots
5 of 50 ul were added to each assay plate. The samples
incubated for 1 hour at room temperature. The plates
were washed. Secondary biotinlyated antibody was
diluted 1:1000 in I-block and 50 ul/well was added to
the assay plates. The plates were washed and 50
10 ul/well of avidin-alkaline phosphatase (Avidix AP,
Tropix, Bedford, MA) was added to the assay plates.
The plates were incubated for one hour at room
temperature. The washed plates were incubated twice
for one minute with assay buffer (Tropix). The CDP-
Star substrate (Tropix) was added to each well (100
ul/well). After 10 minutes the plates were read on a
MD2250 luminometer (Dyatech, Chantilly, VA). The
first specimens were assayed using a modified
protocol. Instead of a detector step using avidin-
alkaline phosphatase, avidin-aequorin (Sealite
Sciences, Atlanta, GA) was used. This material became
unavailable during the study so the protocol was
modified. Results using standard and control
specimens were identical for the two assay formats.
Serotvt~e cross-reactivity. To examine serotype
specificity, PBMC collected on days 42, 45, or 105
from selected recipients of the monovalent attenuated
vaccines (see results) were stimulated for four days @
250,000 cells/ well with each serotype of virus in
independent cultures. Culture supernatants were then
analyzed using the chemiluminescent lymphokine ELISA.
T cell subset depletions. To examine the specific
cellular source of lymphokine production, PBMC were
depleted of CD3+ or CD8+ T lymphocytes prior to
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stimulation. Selected PBMC were washed twice with
RPMI medium 1640 and suspended at 3.25 million
cells/ml in 5o complete media (30~ more PBMC were used
as input to compensate for cell loss during the
depletion procedure). For the negative depletion,
cells (650,000 PBMC) were incubated with washed
antibody coated magnetic beads. Two types of beads
were used, M-450 anti CD3 and anti CD8 beads (Dynal,
Oslo, Norway). The anti CD3 beads were used at a
concentration of 5.2 million particles/tube giving an
approximate 20:1 bead to target cell ratio. The anti-
CD8 beads were used at a concentration of 4.0 million
particles per tube giving an approximate 31:1 bead to
target cell ratio.) DYNABEADSTM (Dynal) in 1.5 ml
microcentrifuge tubes. The cells were incubated at
4°C for 30 minutes with moderate agitation. Non-
depleted PBMC were used as controls. Using an MPC-2
magnetic particle concentrator (Dynal) labeled cells
were removed from the cell mixture. CD3+ and CD8+
negatively selected PBMC were transferred to fresh
microcentrifuge tubes. To remove any residual unbound
cells, the concentrated Dynabeads were washed once
with 200 ul complete medium. After transfer, the final
volume (400 ul) was divided equally into two wells of
a 96-well V-bottom culture plate. Depleted and control
PBMC culture supernatants were analyzed after four
days using the chemiluminescent lymphokine ELISA. In
addition, the cultured PBMC were assayed for
intracellular granzyme B mRNA (see below). CD4+
depletion was performed similarly but the separation
was done after stimulation using M-450 CD4+ (28.6 ml/
4.004 million particles, an approximate 31:1 bead to
target cell ratio) dynabeads. CD4+ negatively selected
PBMC were assayed only for intracellular granzyme B
mRNA.
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Flow cvtometrv. Depletion efficiency (measured as
o depletion) was determined using FACS analysis after
dual staining of a randomly selected, unstimulated
PBMC population (both non-depleted control and CD3+ or
CD8+ depleted sets). The cells were incubated with PE
labeled anti-CD4+ or anti-CD8+ and FITC labeled anti-
CD3+ antibodies (Becton-Dickinson) for 30 minutes at
4°C. Labeled PBMC were then washed three times with
fluorescence buffer [PBS (Sigma), 0.050 Na Azide, 1~
Fetal Bovine Serum (Summit Biotechnology, Boulder, CO)
and preserved in fluorescence fixative [PBS, 10
Formalin, 0.050 Na Azide] prior to analysis. Depletion
efficiency, using the CD4+ Dynabeads, was not
measured.
Granzyme B assay. Non-depleted control PBMC and T
cell subset depleted PBMC were assayed for
intracellular granzyme B mRNA, after four days of
stimulation with wild-type virus. A Reverse
Transcriptase Polymerase Chain Reaction (RT-PCR) assay
in a 96-well plate format was used.
The mRNA purification was done using the
"Straight A's" mRNA Isolation System (Novagen,
Madison, WI). After centrifugation and removal of PBMC
culture supernatants for lymphokine ELISA analysis,
pelleted PBMC were lysed using 200 ul/ well of lysis
buffer containing 10 mM dithiothreitol and then
incubated with 200 mg/ well of washed oligo dT
magnetic beads for 30 minutes at room temperature.
After thoroughly washing the beads with eight volumes
of wash buffer using a MPC-96 (Dynal) magnetic
particle concentrator to remove DNA, proteins, and
cellular debris, mRNA was eluted at 70°C for 20
minutes with 200 ul/ well of HZO. The eluate was
transferred to a 1.5 ml microcentrifuge tube and a
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second round elution performed with an additional 200
ml/ well of H20. The 400 ul of eluate was next
precipitated using 50 ul of 3M sodium acetate (pH
5.2), 20 mg of glycogen (Novagen), and 300 ul of
isopropanol. After a final wash with 70°s cold
ethanol, the mRNA pellet was suspended in 30 ul of
Hz 0 .
RT-PCR steps were performed in 96-well plates.
Oligonucleotide primers (22 bp), which correspond to
exons of the human granzyme B (CTLA-1) and amplify a
120 by region, were synthesized by Dr. Stuart Cohen at
the Walter Reed Army Institute of Research. The
primers had the following sequences: grb2a (sense)
5'AGC CGA CCC AGC AGT TTA TCC C (SEQ ID N0:1), grb2b
(anti-sense) 5'C TCT GGT CCG CTT GGC CTT TCT (SEQ ID
N0:2).
For each reverse transcriptase reaction, the
total reaction volume was 40 ul and included the
following: MgCl2 (5 mM), 10X buffer II (10 mM Tris-
HCL, 50 mM KCL, pH 8.3), dNTPs (1 mM each), and RNase
inhibitor (40 Units) [Perkin Elmer, Norwalk, CT] AMV
reverse transcriptase (10 Units) [Siekagaku], grb2b
primer (3 pmoles), sH20, and 4 ml of mRNA template.
RT incubation steps were done in a 9600 thermocycler
(Perkin Elmer) with parameters set at 42°C (90
minutes), 99°C (5 minutes), 4°C (indefinitely). For
each PCR, the total reaction volume was 50 ul and
included the following: MgCl2 (2 mM), 10X buffer II
(same as above), dNTPs (.4 mM each), amplitaq gold
(1.25 Units), grb2a and grb2b primers (1 pmole each),
sH20, and 5 ul of cDNA template. PCR incubation steps
were also done in a 9600 thermocycler with parameters
set at 95°C initial denaturation/ enzyme activation
(10 minutes), 30 cycles: [95°C denaturing (30 seconds
with a 10 second ramp)/ 60°C annealing (30 seconds
CA 02365728 2001-09-21
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with a 30 second ramp)/ 72°C extension (30 seconds
with a 30 second ramp)], 72 °C final extension (7
minutes), 4°C (indefinitely).
Using electrophoresis, final amplified PCR
products (10 ul) were separated on ethidium bromide
stained 2% agarose (SeaKem)/ 1X TAE (Tris-Acetate-
EDTA) gels and analyzed using a digital camera
(Scientific Imaging Systems, New Haven, CT).
It was reasoned that if a booster response to a
booster dose of live vaccine could be demonstrated, a
more attenuated live virus vaccine could be used. The
booster response sought was both an antibody and a T
cell response.
In~hile T cell responses to dengue vaccines have
been measured, fewer measurements of T cell responses
have been made than antibody responses. Therefore,
the T cell response to administration of live dengue
vaccine is less well characterized. One goal of this
study was to determine the nature of the T cell
response to the vaccines in terms of T helper
response, serotype specificity and cytotoxic
potential.
The predominating T cell response to these
vaccines was a Th1 response. This was determined by
the secretion of interferon 'y by peripheral blood
mononuclear cells (PBMC) stimulated by live dengue
virus in a four day culture. The interferon y was
secreted by CD3+CD8- T cells. The T cell response was
dengue virus serotype specific with some cross-
reactive response. An anamnestic response was noted
in some of the individuals and not others.
Lvmphokine secretion by dengue stimulated cells.
Live dengue virus was used to stimulate PBMC
cultures. The serotype of stimulating virus used in
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culture was the same as the serotype of the vaccine
virus. After four days, the tissue culture
supernatants were assayed for the presence of
interferon 'y, IL-4 and IL-10. In all cultures, IL-4
and IL-10 were consistently negative. Two assay
controls were used to insure that the assay was
working properly. First, the standard curve used
recombinant lymphokine and second, a control sample
was used to insure that the lymphokines could be
detected in the presence of tissue culture
supernatant.
In contrast to the negative expression of IL-4
and IL-10, high levels of interferon Y were detected
in several of the culture supernatants. Figure 6
shows the kinetics of interferon ~y expression of cells
collected from volunteers receiving monovalent
vaccines. Overall, the highest interferon ~y responses
were by PBMC collected from recipients of dengue 1 and
dengue 2 candidate vaccines, though there were a few
high responses in dengue 3 and 4 recipients. The
interferon 'y was occasionally detected by the 14th day
after the first inoculation but often the expression
was not detected until just prior to or just after
administration of the second dose. The kinetics of
secretion was therefore much slower than expected. In
regard to booster responses for the monovalent
recipients in this study, there were no consistent
patterns. Depending on the individual, interferon 'y
levels either increased or decreased after
administration of the second dose.
Unstimulated PBMC from all volunteers at all
collection points showed undetectable levels of
interferon 'y. The mean expression from stimulated day
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zero cells was 127 pg/ml with a standard deviation of
230 pg/ml.
For the monovalent vaccine recipients, there were
16 positive and 14 negative interferon y responders
(mean ~ 3 standard deviations). Sixteen of thirty
monovalent vaccine recipients had PBMC cultures with
interferon g results >1000 pg/ml for at least one time
point. Twelve had sustained interferon g secretion at
>1000 pg/ml for two or more consecutive time points.
Also, twelve of thirty had secretion >1000 pg/ml on
the last time point assayed.
Four volunteers received tetravalent vaccines (an
equal mixture of all four monovalent strains). Figure
7 shows the interferon y production by PBMC collected
from these tetravalent recipients. The PBMC were
stimulated in separate cultures using one of each of
the four serotypes of dengue virus. The PBMC from
volunteers #33 and #36 secreted significant amounts of
interferon Y, >1000 pg/ml, for at least one time point
after stimulation with each of the four of the
serotypes. The PBMC from volunteer #35 secreted
significant amounts of interferon y in response to
three of the four serotypes (not dengue 3). The PBMC
from volunteer #34 secreted significant interferon-
gamma only in response to dengue 2 virus. Highest
responses were predominantly to DEN 1 and 2. The
kinetics of interferon y production was delayed in the
tetravalent vaccine volunteers as it was in the
monovalent volunteers. High levels of interferon y
were detected just prior to and after inoculation of
the second dose. In regard to booster responses, as
with the monovalent recipients, there were no
consistent interferon secretion ~y patterns after
administration of the second dose.
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In aggregate, these results indicate that the
predominant T lymphocyte response in both monovalent
and tetravalent vaccine recipients was an antigen
specific Th1 response.
Example 11
SerotSroe cross-reactivity. PBMC from twelve of
the monovalent vaccine recipients were examined for
the presence of dengue serotype-specific and cross-
reactive responses. Based on kinetics, those
individuals who secreted >1000 pg/ml of interferon y
in PBMC culture supernatants on the last time point
(second to last collection day) were chosen. PBMC from
the last collection day were stimulated in independent
cultures for four days with each dengue serotype
followed by analysis of secreted interferon y in
culture supernatants. Although there was some serotype
cross-reactivity, the highest response was always seen
in PBMC stimulated with the same serotype virus as the
original vaccination (Table 14). Thus the interferon
y responses seen in PBMC from these selected
monovalent vaccine recipients were dengue serotype-
specific.
Cross reactive responses were half (or less) of
the serotype specific response. For Dengue 2 vaccine
recipients, the highest cross-reactive response was
with dengue 4 virus. For dengue 4 vaccine recipients,
the highest cross-reactive response was with dengue 2
virus. For dengue 1 vaccine recipients, the cross-
reactive responses varied. There was only one dengue
3 vaccine recipient in this group and that response
was serotype specific.
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Table 14. Serotype specific and cross-reactive interferon 'y
expression by PBMC collected from monovalent vaccine
recipients. The PBMC collected from individuals receiving
monovalent attenuated dengue vaccines were separately
stimulated in culture with each of the four serotypes of dengue
virus. A subgroup of cells was selected based upon an
interferon 'y production of at least 1000 pg/ml in other assays.
Serotype specific responses were always the highest, however
cross-reactive responses also were noted. Results are shown as
supernatant interferon y in picograms/ml.
Voluntee SerotypeDengue Dengue Dengue Dengue
1 2 3 4
4 1 1030 202 419 129
10 1 6 4 8 58 42 73
1 1 63 0 15 0
1 1 1731 51 25 506
22 1 5 4 6 200 159 4
29 1 1 6 8 0 26 0
31 1 375 0 0 0
11 2 690 5175 96 261
2 2 797 6101 85 962
3 3 0 0 71 0
12 4 1239 1987 1067 4 410
13 4 445 1391 11 4 81 8
Example 12
T cell subset depletions. To verify that this
was a Th1 response, the identity of the cells
secreting the interferon 'y was determined. This was
done by depleting T cells or T cell subsets prior to
culture . The cells used in this study were mixed
PBMC separated from whole blood using density gradient
centrifugation. The predominant cells in PBMC
populations include T cells, B cells, monocytes and NK
cells. For this assessment, we chose the time point
of the highest interferon y response based on kinetics
in 13 monovalent and 3 tetravalent volunteers.
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Cells were removed from PBMC using immunomagnetic
cell separation. The depletion efficiency was
assessed using flow cytometzy in test depletions.
Analysis of the cultured PBMC was not done because of
the small number available. In the test depletions,
removal of CD3+ cells using CD3 monoclonal antibody
resulted in a 92o reduction of CD3+ cells relative to
non-depleted PBMC controls. The CD3 depletion was
monitored using dual labels for CD3 and CD4, dual
labels for CD3 and CD8, and single label for CD3. The
CD3 depletion was more thorough for CD4+ cells than
CD8+ cells with 980 of the CD3/CD4 T cells being
depleted and 900 of the CD3/CD8 cells being depleted
in the CD3 depleted groups. Removal of CD8+ cells
using CD8 monoclonal antibody resulted in a 99.90
reduction of CD8+ cells.
Selected PBMC were depleted of CD3+ or CD8+ T
lymphocytes, stimulated in culture with dengue virus
for four days, and then examined for secreted
interferon ~y. Results were compared to those obtained
from non-depleted PBMC controls cultured at the same
time. CD4+ T lymphocytes were not depleted prior to
stimulation because other cell populations need CD4+ T
help for production of interferon y.
Removal of CD3+ cells prior to culture
substantially reduced the production of interferon ~y
as shown in Table 15. The range for reduction in
interferon y after CD3+ depletion was 59-100
Reduced but significant interferon y production was
seen in some CD3+ depleted cultures. This residual
production indicates that either the small amount of
residual CD3+ cells remaining after immunomagnetic
cell separation are secreting interferon y and/or
another population of cells is also secreting
interferon 'y.
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Table 15. Lymphocytes secreting (or inducing the
secretion of) interferon y are CD3+ CD8-T cells.
Selected lymphocyte subsets were negatively depleted
using immunomagnetic cell separation techniques. The
remaining cells were stimulated with live dengue virus
for four days and the culture supernatant was assayed
for interferon 'y. Depletion of CD3+ lymphocytes prior
to culture negatively influenced the production of
interferon y.
Control CD3 de CD8 de leted
leted
InterferonInterferon%Change Interferon %Change
Y y y
Volunteer(pg/ml)
3 883 0 100 565 36
4 3084 1038 6 5559 [80]
10 4295 1781 59 4271 0.6
1 1 525 88 83 633 [21 ]
12 10000 101 90 1000 0
13 5977 385 9 8392 [40]
1365 255 81 191 [40]
16 1861 84 95 2113 [14]
17 576 42 93 1265 [120]
4614 1329 71 4235 8
22 1303 39 89 1349 [3.5]
29 2478 5 99 5681 [129]
31 995 37 63 305 [207]
33T 2393 9 99 211 12
35T 10000 202 98 963 4
36T 3542 469 87 325 8
Except in one individual, removal of CD8+ cells
prior to culture did not reduce the production of
interferon 'y. In 9 of the 16 cultures, removal of
15 CD8+ cells actually increased its production, possibly
due to removal of suppression by these cells or by
reducing the killing of infected antigen presenting
cells by CD8+ cytotoxic lymphocytes.
Together, these results indicate that the
20 interferon ~y seen in these PBMC cultures is either
secreted by CD4+ T lymphocytes and/or by cells
influenced by CD4+ T lymphocytes. This supports the
finding of a Th1 response.
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Example 13
Granzyme B. A Th1 response is associated with,
among other things, a cytotoxic lymphocyte response.
In an effort to see if cells capable of cell mediated
killing were present in these vaccine volunteers,
granzyme B mRNA was measured in the PBMC cultured for
the depletion experiments. After removal of culture
supernatants for lymphokine analysis, the cells were
pelleted and lysed for extraction of mRNA. Granzyme B
specific primers were used for RT-PCR. The PCR
product was analyzed by agarose gel electrophoresis.
Gel band intensity was converted into a +, - scale
using a reference photograph (Fig 8) for comparison.
Extra cells from seven of the volunteers were cultured
without virus. The unstimulated PBMC, from these
seven volunteers, had little (- or +) granzyme B mRNA
expression. With antigen-specific stimulation,
expression was substantially upregulated in all 16 of
the selected vaccine recipients (Figure 8). T cell
subset depletion using CD8 monoclonal antibody did not
significantly reduce granzyme B expression relative to
control PBMC. There were 3 individuals (ID 16, 22,
and 33) whose granzyme B expression was reduced in the
CD8 depleted group. In one (ID 33), the decrease was
substantial. In contrast, T cell subset depletion
using CD3 monoclonal antibody reduced expression in 14
of the volunteers. In 8 of the monovalent volunteers
and in all 3 tetravalent volunteers, the decrease was
substantial. Four of the interferon 'y non-responders
were also examined for granzyme B mRNA. All showed low
levels of expression (data not shown).
In cells from seven of the volunteers, T cell
subset depletion using CD4 monoclonal antibody was
done after the four days of culture. The depletion
was done after culture in order to provide T helper
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activity to all cells needing help during culture.
Removal of CD4+ cells after stimulation did not affect
granzyme B expression relative to non-depleted
controls in the seven volunteers analyzed. Thus,
although there is an antigen dependent production of
granzyme B mediated by CD4+ Th1 cells, the actual
cells that produce the granzyme B appear to be cells
other than T cells. Whether this is production by NK
cells or macrophages is unknown.
Discussion
Two objectives of this study were to determine if
there was a measurable T cell response in the vaccine
recipients and if a cell mediated response to the
second dose of vaccine could be seen. For those
objectives, T cell response kinetics were measured by
re-stimulating cells collected at intervals around the
two doses. The re-stimulation was done with live
virus in bulk cultures of PBMC collected during the
study.
A third objective of this study was to determine
the nature of the T cell response in terms of 1. cell
type defined by lymphokine repertoire, 2. dengue
serotype specific and cross-reactive responses, and
3. a measure of cytotoxic potential, granzyme B
production. These responses were measured in PBMC
from both monovalent and tetravalent vaccine
recipients. In regard to the tetravalent vaccine
recipients, it was important to determine if a
response could be detected to all four serotypes of
dengue virus.
Human and mouse T helper responses can be divided
into two groups based upon their pattern of lymphokine
expression 5. T helper 1 (Th1) cells are
characterized by the secretion of IL-2 and interferon
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y. Of those two lymphokines, interferon y is the most
important in terms of identifying Th1 cells. T helper
2 (Th2) cells are characterized by the secretion of
IL-4, IL-5, IL-6 and IL-10. In mixed populations of
cells or PBMC bulk culture, one of the two secretion
patterns usually predominates.
One factor influencing the Th1 vs Th2 response is
the nature of the assaulting infection. Viral
infections, and some bacterial infections such as
Listeria and Mycobacterium (Peters, 1996, Hepatology
23, 909-916) often induce a Th1 response while some
parasitic infections will induce a Th2 response
(Conrad et al., 1990, J Exp Med 171, 1497-1508). The
proportion of the two responses may vary during the
course of the infection. For instance, even though
viral infections usually begin with a Th1 response, a
Th2 response can be produced later in the infection.
The initial Th1 response may augment CTL responses and
direct immunoglobulin isotype switching while the
following Th2 response may augment antibody production
by B cells.
In natural dengue infection, one study showed a
Th1 response in most individuals. The Th1 response
was associated with an effective immune response
without associated severe pathogenesis. In contrast,
some individuals developed a Th2 response that was
associated with greater pathogenesis.
In spite of the association of a Th1 response
with an effective anti-dengue immune response, the key
lymphokine of a Th1 response, interferon y, has both
positive and negative influences on the immune
response. In Thailand, Kurane found high levels of
interferon y in the serum of DHF patients in
comparison to lower levels in the serum of DF patients
(Kurane et al., 1991, J Clin Invest 88, 1473-1480).
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The increased interferon 'y may be a measure of immune
activation. Interferon y is needed to activate and
maintain activation of cytotoxic cells (CD4+ T cells,
CD8+ T cells and NK cells). While this mechanism may
contribute to pathogenesis in severe infections, the
same response may be beneficial in milder infections
by reducing the number of virally infected cells
through antigen specific cytolysis. The positive
role of interferon y in controlling dengue virus
infection is demonstrated in a recent mouse knockout
model deficient in interferons a, ~i and 'y. The knock-
out mice were susceptible to lethal infection by
dengue viruses in contrast to normal adult controls
that were resistant to infection (Johnson and Roehrig,
1999, J Virol 73, 783-786).
Alternatively, interferon ~y may contribute to the
pathogenesis of dengue virus infection. One mechanism
for the pathogenesis may be by immune enhancement due
to increasing the infection of one major target cell,
the macrophage. In culture, interferon 'y increased
the antibody-mediated infection of a macrophage cell
line U937 by increasing the number of Fc receptors on
the surface of the cells (Kontny et al., 1988, J Virol
62, 3928-3933). Although another study using normal
cultured macrophages showed the opposite effect of
decreasing the infection (Sittisombut et al., 1995, J
Med Viro1 45, 43-49). Given these conflicting
results, it is unclear whether interferon y
contributes to increased infection of macrophages.
In this study, a Th1 response was the predominant
response. Assays for IL-4 and IL-10 were consistently
negative indicating a lack of TH2 response. High
levels of interferon Y were detected in the
supernatants of many of the cultures, indicating the
presence of a Th1 response in those cultures.
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Since the stimulated cells were whole PBMC, the
cells responsible for secretion of the interferon 'y
needed to be determined. This was done by depleting T
cell subsets using an immunomagnetic procedure.
Negative depletion was done prior to culture with
antibodies recognizing either CD3 or CD8. Since CD3
depletion resulted in abrogation of interferon ~y
secretion and CD8 depletion did not, it was concluded
that CD3+ CD8- lymphocytes were the cell population
secreting the interferon y or at least controlling the
secretion of interferon 'y. This confirms that the
interferon y was the result of a Th1 response.
Residual interferon y in some cultures after depletion
may have been due to some remaining CD4+ T lymphocytes
after depletion or other cells in the culture,
possibly Natural Killer cells or macrophages.
The peak interferon y response was serotype
specific. V~lhen cells from monovalent vaccine
recipients were stimulated separately by each of the
four serotypes of dengue viruses, the peak interferon
y production was in response to stimulation by dengue
virus homologous to the vaccine virus. Lesser, cross-
reactive responses to other dengue viruses were noted
in several of the cultures. This is similar to the
results obtained by others using a different
measurement, lymphocyte proliferation. In one study,
cells from individuals receiving a dengue 2 vaccine
exhibited the greatest response to dengue 2 virus but
cross-reactive responses were noted (Dharakul, J
Infect Dis 170, 27-33). This was confirmed at the
clonal level where the majority of clones obtained
from a dengue 3 vaccine recipient responded best to
dengue 3 antigen but had cross reactive responses to
the other three dengue antigens (Kurane et al., 1989,
J Exp Med 170, 763-775). The conclusion of the latter
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study was that primary dengue virus infection produces
predominantly cross-reactive CD4+ lymphocyte responses
(proliferation and interferon y production).
In this study, cross-reactive responses of
monovalent vaccine recipients' PBMC were usually half
or less of the serotype specific response. In the
tetravalent vaccine recipients, interferon 'y secretion
in response to individual serotypes of dengue virus
was significant in three out of four tetravalent
vaccine recipients. The responses varied within
individual vaccine recipients enough that it was not
possible to determine if the lower responses were
serotype specific responses or cross-reactive
responses.
The kinetics of T cell activation as indicated by
interferon y secretion was slower than expected. In a
few instances, responses could be detected by day 14.
However in most cases, responses were not detected
until just prior to administration of the second
vaccine dose. It is unclear what the reason is for
the delayed kinetics. One explanation could be that
antigen production by vaccine virus infected cells is
slow and persistent. However, it is equally possible
that the methods preferentially detected memory
responses rather than acute responses. For instance,
if active CD8+ cells were inhibiting a CD4+ response
in PBMC collected during early infection, a measurable
response may be attenuated. In cultures where the
CD8+ lymphocytes were depleted, interferon y secretion
by the remaining lymphocytes was increased in more
than half of the cultures. This inhibition may have
been greater during early infection.
Others have observed more acute lymphokine
production kinetics. Serum lymphokines, including
serum interferon y were measured for 17 days after
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inoculation with an attenuated dengue vaccine. An
acute response was noted in that study that peaked
during the time of viremia (Kurane et al., 1995, J
Clin Lab Immunol 46, 35-40).
The response to the second dose was mixed. Some
individuals showed an increase in interferon y
production while others showed a decrease. The
interferon y production by cells collected from
vaccine recipients just prior to the second dose was
high enough that it may have masked any anamnestic
response to the second dose. In addition, the late
interferon y response may have made the measurement of
an anamnestic T cell response more difficult. It is
clear that some individuals responded to the second
dose. This may indicate that there is some localized
virus growth in the presence of an active immune
response.
In summary, the predominant T cell response to
administration of these live attenuated dengue viruses
was a Th1 response. This was demonstrated by the
secretion of interferon 'y by re-stimulated PBMC
collected from vaccine recipients. None of the PBMC
cultures from vaccine recipient's cells had
significant IL-4 or IL-10 secretion into the culture
supernatant after re-stimulation. The Th1 response
was verified by showing that CD3+ CD8- lymphocytes
were secreting the interferon y. The Th1 response was
predominantly dengue serotype specific but smaller
cross-reactive responses were noted.
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Example 14
Clinical and immunoloaical evaluations of four
dengue viruses as challenge strains in immune and
susceptible volunteers.
The primary objective of this study is to
characterize clinical responses to each of 4 candidate
dengue challenge viruses in susceptible and immune
volunteers to judge their suitability as challenge
strains for human vaccine efficacy studies. The
secondary objective of the study is to generate
hypotheses regarding the immune correlates of
protection for dengue fever.
Dose, Schedule and Route: All volunteers will
receive either 1 of 4 dengue challenge viruses or
placebo in a single dose of 0.5m1 subcutaneously in
the deltoid region on study day 0.
Study Groups:
Volunteer Set #1 (susceptible): to receive
either DEN-1, DEN-2, DEN-3, DEN-4 or placebo
Volunteer Set #2 (immune): to receive either DEN
virus (serotype corresponding to previously received
vaccine) or placebo
General Eligibility Criteria: Age 18-35,
excellent health without any chronic medical
conditions, score of >75% on written study-
comprehension examination, informed consent,
availability for the study period, letter of approval
for participation from chain of command (military
only), serologic conversion in response to previous
dengue vaccination (volunteer set #2 only)
Statistics: Data analysis will be primarily
descriptive in this pilot study given the small number
of volunteers in each test article group. The primary
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concern will be to document the frequency of clinical
events (pre-specified and unexpected) within the four
study groups as compared to placebo.
Pre-challenge immune measures and post-challenge
immune responses of all challenged volunteers who
develop dengue fever will be compared to those of all
challenged volunteers who remain well, to develop
hypotheses about immune correlates of protection.
Application of the Human Dengue Challenge Model:
In contrast to most historical human dengue challenge
experiments which were designed either to characterize
dengue illness or to evaluate the attenuation of live
vaccine candidates, this challenge study will aim to
1) validate 4 dengue viruses as challenge strains in
flavivirus-naive volunteers (volunteer set #1), and 2)
identify correlates of immunity in recipients of
monotypic dengue vaccine when subsequently challenged
with homotypic dengue virus (volunteer set #2).
If the clinical response in volunteer set #1
suggests these strains are suitable for challenge,
then in future controlled experiments, these challenge
strains will be administered to recipients of dengue
vaccine candidates or placebo to select the most
promising vaccine candidates for further development.
If the immunological response in volunteer set #2
suggests that some aspect of pre-challenge immunity
(antibodies and/or T cell memory) correlate with
protection, such correlates of protection could
simplify dengue vaccine development.
Defining Criteria for Dengue Viruses to be Tested
as Challenge Strains: A suitable dengue challenge
virus will 1) reproducibly cause uncomplicated dengue
fever lasting 3-7 days in volunteer set #1, 2) be
produced in compliance with Good Manufacturing
Practices (GMP) and be free of adventitious agents or
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reactogenic non-viral components, and 3) be available
as lyophilized virus in sufficient quantity (>100
doses). Challenge Viruses include DEN-1 45AZ5 (PDK-
0) inactivated, DEN-2 516803 (PDK-10), DEN-3 cl 24/28
(PDK-0), DEN-4 341750 (PDK-6) (PDK = primary dog
kidney cells).
Dose of each challenge virus in plaque forming
units (pfu.) will be 0.5 x titer.
The study challenge viruses meet the latter two
criteria. This study aims to demonstrate that the
study candidate challenge strains meet the first
criterion. We have some evidence that the 4
challenge viruses to be tested in this study are
appropriately pathogenic. The DEN-1 and DEN-3
challenge viruses have already been shown to cause
uncomplicated febrile illness in volunteers. Though
the DEN-2 and DEN-4 challenge viruses to be
administered in this study are untested in volunteers,
they are believed to be pathogenic, as they are the
precursors of dengue virus vaccine candidates that
were rejected because they caused febrile illnesses in
volunteers. The only reason for rejecting any of the
4 study candidate dengue challenge viruses is if they
cause either no illness in flavivirus-naive volunteers
(volunteer set #1) or excessive illness in any
volunteer (volunteer sets #1 and #2).
Volunteer Set #1: Ten healthy flavivirus-naive
volunteers will be randomized to receive dengue virus
challenge with 1 of 4 serotypes (2 volunteers per
serotype) or placebo. The volunteers and
investigators will remain blinded to the inoculum. We
expect that the 8 volunteers who receive dengue
challenge viruses will become moderately ill with 3-7
days of fever, severe headache and myalgias. Full
recovery may take as long as 14 days from onset of
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illness. Each challenge virus will be deemed
suitable based on the clinical responses of the 2
recipients and must satisfy the study definition of
dengue fever. Dengue fever is defined as . an illness
with: 2 or more of the following: headache, myalgia,
erythematous rash, retro-orbital pain, arthralgias,
and sustained fever for 48 hours or more allowing for
periods of decreased temperature due to acetaminophen
use, and tissue response during period of fever and
days thereafter manifest by neutropenia or
thrombocytopenia or liver injury, and evidence of
dengue viremia during the period of fever..
Volunteer Set #2: Up to twelve young, healthy
immune volunteers will receive homotypic dengue
challenge virus (N=10, regardless of serotype) or
placebo (N=2). Immune volunteers are previous
recipients of monovalent, live-attenuated dengue
vaccines who had a primary neutralizing antibody
response. Section 23.3 summarizes the clinical and
immunologic data regarding these monovalent dengue
vaccine recipients. Immune volunteers are expected to
remain well. Measures of their pre-challenge immune
status or immune activation intra-challenge may
identify correlates of protection.
30
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SEQUENCE LISTING
5'AGC CGA CCC AGC AGT TTA TCC C (SEQ ID N0:1),
grb2b (anti-sense) 5'C TCT GGT CCG CTT GGC CTT TCT
(SEQ ID N0:2).
