U.S. patent application number 14/992354 was filed with the patent office on 2016-05-05 for inactivated dengue virus vaccine with aluminium-free adjuvant.
The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS SA. Invention is credited to Benoit BARAS, Dirk GHEYSEN, Isabelle Solange Lucie KNOTT, Jean-Paul PRIEELS, Jean-Francois TOUSSAINT.
Application Number | 20160120972 14/992354 |
Document ID | / |
Family ID | 42174405 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160120972 |
Kind Code |
A1 |
BARAS; Benoit ; et
al. |
May 5, 2016 |
INACTIVATED DENGUE VIRUS VACCINE WITH ALUMINIUM-FREE ADJUVANT
Abstract
The present disclosure provides immunogenic compositions for the
prevention and/or treatment of disease caused by dengue virus.
Inventors: |
BARAS; Benoit; (Rixensart,
BE) ; GHEYSEN; Dirk; (Rixensart, BE) ; KNOTT;
Isabelle Solange Lucie; (Rixensart, BE) ; PRIEELS;
Jean-Paul; (Rixensart, BE) ; TOUSSAINT;
Jean-Francois; (Rixensart, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS SA |
Rixensart |
|
BE |
|
|
Family ID: |
42174405 |
Appl. No.: |
14/992354 |
Filed: |
January 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13148741 |
Aug 10, 2011 |
9265821 |
|
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PCT/EP2010/051882 |
Feb 16, 2010 |
|
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14992354 |
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61153060 |
Feb 17, 2009 |
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Current U.S.
Class: |
424/450 ;
424/218.1 |
Current CPC
Class: |
A61P 31/12 20180101;
A61K 39/12 20130101; A61K 2039/55572 20130101; Y02A 50/386
20180101; A61K 2039/55583 20130101; A61P 37/04 20180101; A61K
2039/5252 20130101; A61K 2039/55511 20130101; A61P 31/14 20180101;
A61K 2039/55577 20130101; A61K 2039/55 20130101; Y02A 50/30
20180101; A61K 2039/55555 20130101; C12N 2770/24134 20130101; A61K
2039/55566 20130101 |
International
Class: |
A61K 39/12 20060101
A61K039/12 |
Claims
1. An immunogenic composition comprising at least one inactivated
dengue virus antigen and an aluminum-free adjuvant, wherein the
adjuvant comprises 3D-MPL and QS21 in a liposomal formulation.
2. The immunogenic composition of claim 1, wherein the at least one
inactivated dengue virus antigen is selected from the group
consisting of a Dengue-1 virus antigen, a Dengue-2 virus antigen, a
Dengue-3 virus antigen and a Dengue-4 virus antigen.
3. The immunogenic composition of claim 1, wherein the at least one
inactivated dengue virus antigen comprises a Dengue-2 virus
antigen.
4. The immunogenic composition of claim 1, wherein the at least one
inactivated dengue virus antigen is an inactivated whole dengue
virus.
5. The immunogenic composition of claim 2, wherein the immunogenic
composition comprises a Dengue-1 virus antigen, a Dengue-2 virus
antigen, a Dengue-3 virus antigen and a Dengue-4 virus antigen.
6. The immunogenic composition of claim 1, wherein the at least one
inactivated dengue virus antigen is a purified inactivated whole
dengue virus.
7. The immunogenic composition of claim 1 wherein the immunogenic
composition comprises inactivated whole Dengue-1 virus, inactivated
whole Dengue-2 virus, inactivated whole Dengue-3 virus and
inactivated whole Dengue-4 virus.
8. The immunogenic composition of claim 1 wherein the immunogenic
composition comprises purified inactivated whole Dengue-1 virus,
purified inactivated whole Dengue-2 virus, purified inactivated
whole Dengue-3 virus and purified inactivated whole Dengue-4
virus.
9. The immunogenic composition of claim 1, wherein the adjuvant
further comprises cholesterol.
10. The immunogenic composition of claim 1 or claim 9, wherein the
adjuvant further comprises 1,
2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC).
11. The immunogenic composition of claim 1, wherein the adjuvant
comprises cholesterol, DOPC, 3D-MPL and QS21.
12. The immunogenic composition of claim 11, wherein the adjuvant
is formulated in a dose comprising: from about 0.1 to about 0.5 mg
cholesterol, from about 0.25 to about 2 mg DOPC, from about 10
.mu.g to about 100 .mu.g 3D-MPL, and from about 10 .mu.g to about
100 .mu.g QS21.
13. The immunogenic composition of claim 9, wherein the ratio of
cholesterol to QS21 is 5:1 (w/w).
14. The immunogenic composition of claim 9, wherein the ratio of
cholesterol to QS21 is 1:1 (w/w).
15. The immunogenic composition of claim 11, wherein the adjuvant
is formulated in a dose comprising: about 0.25 mg cholesterol,
about 1.0 mg DOPC, about 50 .mu.g 3D-MPL, and about 50 .mu.g
QS21.
16. A method for preventing, ameliorating or treating disease
caused by dengue virus in a subject comprising: administering the
immunogenic composition of claim 1 to the subject.
17. The method of claim 9, wherein the subject is under 5 years of
age.
18. The method of claim 9, wherein the subject is under 1 year of
age.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 13/148,741
filed 10 Aug. 2011 (allowed) which is the national stage of
international patent application no. PCT/EP2010/051882, filed 16
Feb. 2010, which claims benefit of the earlier filing date of U.S.
provisional application No. 61/153,060, filed 17 Feb. 2009, the
disclosures of which are incorporated herein by reference.
BACKGROUND
[0002] Dengue is an acute viral disease of man which is transmitted
by mosquitoes. It is endemic in the tropics and subtropics,
worldwide, where an estimated 100,000,000 cases occur annually.
Although relatively rare, dengue hemorrhagic fever (DHF) and dengue
shock syndrome (DSS) are significant causes of death in children.
At present, there is no vaccine to protect against dengue and
attempts to prevent disease by controlling the mosquito vector have
proven largely ineffective. Thus, there remains a need for a safe
and effective vaccine to protect against disease caused by dengue
virus.
BRIEF SUMMARY
[0003] The present disclosure concerns compositions that elicit an
immune response against dengue virus. More specifically, this
disclosure concerns inactivated dengue virus vaccines that include
an adjuvant. The compositions disclosed herein include an
aluminum-free adjuvant, for example, an aluminum-free adjuvant
capable of promoting a Th1 immune response. Methods for their use,
e.g., in the formulation of medicaments, for prevention of
treatment of disease caused by Dengue virus are also described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 is a bar graph illustrating Anti-Den-2 neutralizing
antibody titres in naive C57Bl/6 mice.
[0005] FIG. 2 is a bar graph illustrating Anti-Den-2 neutralizing
antibody titres in naive C57Bl/6 mice.
[0006] FIG. 3 is a bar graph illustrating Anti-Den-2 neutralizing
antibody titres in naive C57Bl/6 mice
[0007] FIG. 4 is a bar graph illustrating a characterization of the
cellular immune response by intracellular cytokine staining in
peripheral blood cells.
DETAILED DESCRIPTION
Introduction
[0008] The present invention is directed to a vaccine that
satisfies the need for a safe and effective dengue vaccine.
Purified inactivated dengue virus vaccine has a major advantage
over live-attenuated dengue virus in that inactivated viruses are
not infectious and therefore, can not revert to virulence or cause
disease. One potential drawback of a purified inactivated dengue
virus vaccine as compared to a live-attenuated is a reduced
capacity to induce high titers of dengue specific neutralizing
antibodies and the relatively short duration of the protective
immune response. These drawbacks are overcome by the formulation of
the purified inactivated dengue antigen with an appropriate
adjuvant. As disclosed herein, the adjuvant is an aluminum-free
(alum-free) adjuvant (or adjuvant system) that effectively elicits
high titers of dengue-specific neutralizing antibodies. In an
embodiment, the adjuvant elicits a predominantly Th1 response or a
balanced Th1/Th2 response characterized by production of interferon
gamma (IFN-.gamma.).
[0009] The immunogenic compositions disclosed herein include at
least one (that is, one or more than one) inactivated dengue virus
antigen, in combination with an aluminum-free adjuvant. The
inactivated dengue virus antigen can be selected from a Dengue-1
virus antigen, a Dengue-2 virus antigen, a Dengue-3 virus antigen
and a Dengue-4 virus antigen. Thus, the immunogenic composition can
be a monovalent composition including a single inactivated dengue
virus antigen from a single strain selected from Dengue-1,
Dengue-2, Dengue-3 or Dengue-4, or the composition can be a
multivalent (e.g., bivalent, trivalent, tetravalent) composition
containing inactivated antigens of more than one of these dengue
strains. In one exemplary embodiment, the immunogenic composition
includes a Dengue-2 virus antigen. For example, the immunogenic
composition can be a monovalent composition that contains an
inactivated Dengue-2 antigen. Alternatively, the immunogenic
composition can be a bivalent, trivalent or tetravalent composition
that contains an inactivated Dengue-2 virus antigen in combination
with one, two or three additional inactivated dengue virus
antigens. For example, in one embodiment, the composition is a
tetravalent composition that includes an inactivated Dengue-1 virus
antigen, an inactivated Dengue-2 virus antigen, an inactivaed
Dengue-3 virus antigen and an inactivated Dengue-4 virus
antigen.
[0010] The one or more inactivated dengue virus antigens are
formulated with an adjuvant that is free of aluminum or aluminum
salts, that is, an aluminum-free adjuvant or adjuvant system. In
one embodiment, the adjuvant includes an oil-in-water emulsion. For
example, the oil-in-water emulsion can include an oil phase that
incorporates a metabolisable oil, and optionally includes an
additional oil-phase component, such as a tocol. The oil-in-water
emulsion also contains an aqueous component, such as a buffered
saline solution (e.g., phosphate buffered saline). In addition, the
oil-in-water emulsion typically contains an emulsifier. In one
embodiment, the metabolizable oil is squalene. In one embodiment,
the tocol is alpha-tocopherol. In one embodiment, the emulsifier is
a nonionic surfactant emulsifier (such as polyoxyethethylene
sorbitan monooleate, TWEEN80.TM.). In exemplary embodiments, the
oil-in-water emulsion contains squalene and alpha tocopherol in a
ratio which is equal or less than 1 (w/w).
[0011] In one specific example, the immunogenic composition
includes an oil-in-water emulsion adjuvant system formulated in a
dose comprising: from about 2% to about 10% squalene; from about 2%
to about 10% alpha-tocopherol; and from about 0.3% to about 3%
polyoxyethethylene sorbitan monooleate For example, the immunogenic
composition can include an adjuvant formulated in a dose
comprising: from about 10 mg to about 12 mg squalene; from about 10
mg to about 12 mg alpha-tocopherol; and from about 4 mg to about 6
mg polyoxyethethylene sorbitan monooleate. In one specific example,
the adjuvant includes in a single (whole) dose: 10.68 mg squalene;
11.86 mg tocopherol; 4.85 mg polyoxyethethylene sorbitan
monooleate. In other embodiments, the immunogenic composition is
formulated with a fractional dose (that is a dose, which is a
fraction of the preceding single dose formulations, such as one
half of the preceding quantity of components, 1/4 of the preceding
quantity of components, or another fractional dose, e.g., 1/3, 1/6,
etc.) of the preceding quantity of components.
[0012] In certain embodiments, the inactivated dengue virus antigen
is formulated with an alum-free adjuvant system that includes
3-Deacylated monophosphoryl lipid A (3D-MPL) and/or QS21. In one
embodiment, the adjuvant system includes 3D-MPL and QS21. For
example, in one embodiment, the adjuvant contains 3D-MPL and QS21
in a liposomal formulation. Optionally, the adjuvant system also
contains cholesterol. In one specific embodiment, the adjuvant
includes QS21 and cholesterol. Optionally, the adjuvant system
contains 1, 2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC). For
example, in one specific adjuvant system to be formulated with the
inactivated dengue virus antigen contains cholesterol, DOPC, 3D-MPL
and QS21.
[0013] In one specific example, the immunogenic composition
includes an adjuvant formulated in a dose that includes: from about
0.1 to about 0.5 mg cholesterol; from about 0.25 to about 2 mg
DOPC; from about 10 .mu.g to about 100 .mu.g 3D-MPL; and from about
10 .mu.g to about 100 .mu.g QS21. In one embodiment, the ratio of
cholesterol to QS21 in the adjuvant is 5:1 (w/w). For example, the
ratio of cholesterol to QS21 in the adjuvant can be approximately
or exactly 1:1 (w/w). In one specific formulation, the adjuvant is
formulated in a single dose that contains: about 0.25 mg
cholesterol; about 1.0 mg DOPC; about 50 .mu.g 3D-MPL; and about 50
.mu.g QS21. In other embodiments, the immunogenic composition is
formulated with a fractional dose (that is a dose, which is a
fraction of the preceding single dose formulations, such as one
half of the preceding quantity of components (cholesterol, DOPC,
3D-MPL and QS21), 1/4 of the preceding quantity of components, or
another fractional dose, e.g., 1/3, 1/6, etc.) of the preceding
quantity of components.
[0014] In another aspect, this disclosure concerns a method for
producing a dengue vaccine comprising the following steps:
providing at least one purified inactivated dengue virus antigen;
and, formulating the at least one purified inactivated dengue virus
antigen with an aluminum-free adjuvant. For example, the
immunogenic composition can be formulated with a whole virus
antigen produced from either a virulent or attenuated strain, which
has been inactivated or killed. For example, the live (virulent or
attenuated) virus can be killed or inactivated, rendering it
incapable of replication, using chemical agents, such as
formaldehyde, betapropiolactone (BPL), or hydrogen peroxide, or
using ultraviolet irradiation, or by using a combination of two or
more inactivation steps (which can be the same or different, e.g.,
formaldehyde and BPL, formaldehyde and UV irradiation, BPL and UV
irradiation, hydrogen peroxide and BPL, hydrogen peroxide and UV
irradiation, etc., in any combination). Optionally, the inactivated
dengue virus is subjected to additional processing, such as
splitting, or further purification of antigenic subunits.
[0015] In another aspect, this disclosure concerns a method for
preventing, ameliorating or treating disease caused by dengue virus
in a subject comprising: administering an immunogenic composition
(e.g., vaccine) containing at least one inactivated dengue virus
antigen in combination with an alum-free adjuvant (as described
herein). In one embodiment, the method involves administering the
immunogenic composition to a child, such as a child under 5 years
or age, or under 1 year of age. In one embodiment, the immunogenic
composition is administered to a naive subject under 1 year of age.
In other embodiments, the immunogenic composition is administered
to an adult subject, such as an adult subject, such as an elderly
subject over about 60 or 65 years of age. Such subjects can be
previously exposed to dengue virus. Typically, the vaccine is
administered parentally, e.g., intramuscularly. In another aspect,
this disclosure concerns an immunogenic composition containing at
least one purified inactivated dengue virus antigen in combination
with an alum-free adjuvant for use in medicine, e.g., for the
prevention, ameleioration or treatment of Dengue virus infection
and/or Dengue virus induced disease, such as hemorrhagic fever
(DHF) and dengue shock syndrome (DSS).
TERMS
[0016] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of terms are provided.
Additional terms and explanations can be provided in the context of
this disclosure.
[0017] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
Definitions of common terms in molecular biology can be found in
Benjamin Lewin, Genes V, published by Oxford University Press, 1994
(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of
Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN
0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8).
[0018] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. The term "plurality" refers to two or
more. It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description. Additionally, numerical limitations given
with respect to concentrations or levels of a substance, such as an
antigen, are intended to be approximate. Thus, where a
concentration is indicated to be at least (for example) 200 pg, it
is intended that the concentration be understood to be at least
approximately (or "about" or ".about.") 200 pg.
[0019] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
this disclosure, suitable methods and materials are described
below. The term "comprises" means "includes." Thus, unless the
context requires otherwise, the word "comprises," and variations
such as "comprise" and "comprising" will be understood to imply the
inclusion of a stated compound or composition (e.g., nucleic acid,
polypeptide, antigen) or step, or group of compounds or steps, but
not to the exclusion of any other compounds, composition, steps, or
groups thereof. The abbreviation, "e.g." is derived from the Latin
exempli gratia, and is used herein to indicate a non-limiting
example. Thus, the abbreviation "e.g." is synonymous with the term
"for example."
[0020] An "immunogenic composition" is a composition of matter
suitable for administration to a human or animal subject (e.g., in
an experimental setting) that is capable of eliciting a specific
immune response, e.g., against a pathogen, such as dengue virus. As
such, an immunogenic composition includes one or more antigens (for
example, whole purified virus or antigenic subunits, e.g.,
polypeptides, thereof) or antigenic epitopes. An immunogenic
composition can also include one or more additional components
capable of eliciting or enhancing an immune response, such as an
excipient, carrier, and/or adjuvant. In certain instances,
immunogenic compositions are administered to elicit an immune
response that protects the subject against symptoms or conditions
induced by a pathogen. In some cases, symptoms or disease caused by
a pathogen is prevented (or treated, e.g., reduced or ameliorated)
by inhibiting replication of the pathogen (e.g., dengue virus)
following exposure of the subject to the pathogen. In the context
of this disclosure, the term immunogenic composition will be
understood to encompass compositions that are intended for
administration to a subject or population of subjects for the
purpose of eliciting a protective or palliative immune response
against dengue (that is, vaccine compositions or vaccines).
[0021] The term "purification" (e.g., with respect to a pathogen or
a composition containing a pathogen) refers to the process of
removing components from a composition, the presence of which is
not desired. Purification is a relative term, and does not require
that all traces of the undesirable component be removed from the
composition. In the context of vaccine production, purification
includes such processes as centrifugation, dialization,
ion-exchange chromatography, and size-exclusion chromatography,
affinity-purification or precipitation. Thus, the term "purified"
does not require absolute purity; rather, it is intended as a
relative term. Thus, for example, a purified virus preparation is
one in which the virus is more enriched than it is in its
generative environment, for instance within a cell or population of
cells in which it is replicated naturally or in an artificial
environment. A preparation of substantially pure viruses can be
purified such that the desired virus or viral component represents
at least 50% of the total protein content of the preparation. In
certain embodiments, a substantially pure virus will represent at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%,
or at least 95% or more of the total protein content of the
preparation.
[0022] An "isolated" biological component (such as a virus, nucleic
acid molecule, protein or organelle) has been substantially
separated or purified away from other biological components in the
cell and/or organism in which the component occurs or is produced.
Viruses and viral components, e.g., proteins, which have been
"isolated" include viruses, and proteins, purified by standard
purification methods. The term also embraces viruses and viral
components (such as viral proteins) prepared by recombinant
expression in a host cell.
[0023] An "antigen" is a compound, composition, or substance that
can stimulate the production of antibodies and/or a T cell response
in an animal, including compositions that are injected, absorbed or
otherwise introduced into an animal. The term "antigen" includes
all related antigenic epitopes. The term "epitope" or "antigenic
determinant" refers to a site on an antigen to which B and/or T
cells respond. The "dominant antigenic epitopes" or "dominant
epitope" are those epitopes to which a functionally significant
host immune response, e.g., an antibody response or a T-cell
response, is made. Thus, with respect to a protective immune
response against a pathogen, the dominant antigenic epitopes are
those antigenic moieties that when recognized by the host immune
system result in protection from disease caused by the pathogen.
The term "T-cell epitope" refers to an epitope that when bound to
an appropriate WIC molecule is specifically bound by a T cell (via
a T cell receptor). A "B-cell epitope" is an epitope that is
specifically bound by an antibody (or B cell receptor
molecule).
[0024] In the context of the present disclosure, a dengue virus
antigen is typically a whole killed or inactivated virus. The term
"inactivated" in the context of a dengue virus vaccine means that
the antigenic component (e.g., virus) is incapable of replication
in vivo or in vitro. For example, the term inactivated encompasses
a virus that has been replicated, e.g., in vitro, and then killed
using chemical or physical means such that it is no longer capable
of replicating. The term can also include antigens produced by
further processing (e.g., splitting, fractionation, and the like),
and components produced by recombinant means, e.g., in cell
culture.
[0025] An "adjuvant" is an agent that enhances the production of an
antigen-specific immune response as compared to administration of
the antigen in the absence of the agent. Common adjuvants include
aluminum containing adjuvants that include a suspensions of
minerals (or mineral salts, such as aluminum hydroxide, aluminum
phosphate, aluminum hydroxyphosphate) onto which antigen is
adsorbed. In the context of the present disclosure the adjuvants
are aluminum-(alum-)free adjuvants, which are formulated in the
absence of any such aluminum salts. Alum-free adjuvants include oil
and water emulsions, such as water-in-oil, and oil-in-water (and
variants thereof, including double emulsions and reversible
emulsions), liposaccharides, lipopolysaccharides, immunostimulatory
nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like
Receptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9
agonists), and various combinations of such components.
[0026] An "immune response" is a response of a cell of the immune
system, such as a B cell, T cell, or monocyte, to a stimulus. An
immune response can be a B cell response, which results in the
production of specific antibodies, such as antigen specific
neutralizing antibodies. An immune response can also be a T cell
response, such as a CD4+ response or a CD8+ response. In some
cases, the response is specific for a particular antigen (that is,
an "antigen-specific response"). If the antigen is derived from a
pathogen, the antigen-specific response is a "pathogen-specific
response." A "protective immune response" is an immune response
that inhibits a detrimental function or activity of a pathogen,
reduces infection by a pathogen, or decreases symptoms (including
death) that result from infection by the pathogen. A protective
immune response can be measured, for example, by the inhibition of
viral replication or plaque formation in a plaque reduction assay
or ELISA-neutralization assay, or by measuring resistance to
pathogen challenge in vivo.
[0027] A "Th1" biased immune response is characterized by the
presence of CD4+ T helper cells that produce IL-2 and IFN-.gamma.,
and thus, by the secretion or presence of IL-2 and IFN-.gamma.. In
contrast, a "Th2" biased immune response is characterized by a
preponderance of CD4+ helper cells that produce IL-4, IL-5, and
IL-13.
[0028] A "subject" is a living multi-cellular vertebrate organism.
In the context of this disclosure, the subject can be an
experimental subject, such as a non-human animal, e.g., a mouse, a
cotton rat, or a non-human primate. Alternatively, the subject can
be a human subject.
[0029] The immunogenic compositions disclosed herein are suitable
for preventing, ameliorating and/or treating disease caused by
infection with dengue virus.
[0030] The immunogenic compositions disclosed herein include one or
more purified inactivated dengue virus antigen. For example, the
immunogenic compositions can include a single strain of dengue
virus (i.e., a monovalent composition), or they can contain more
than one strain of dengue virus (i.e., a multivalent composition).
Typically, a multivalent composition contains strains selected from
different serotypes. Because there are four serotypes of dengue
virus which can cause disease, that is, dengue type one (DEN-1),
dengue type two (DEN-2), dengue type three (DEN-3) and dengue type
four (DEN-4), and because cross-reactive non-neutralizing
antibodies are predisposing to more severe forms of dengue disease,
one representative of each serotype can be selected for inclusion
into the final vaccine in order to guarantee protection against
disease from any of the four serotypes. Thus, in one embodiment,
the immunogenic composition is a tetravalent composition that
includes strains selected from each of the four serotypes of dengue
virus.
[0031] The viruses used as antigens can be selected from
essentially any strain (or strains) of dengue virus. For example, a
virus strain can be selected for each serotype, which is chosen
based on its conformity to a defined (e.g., consensus) sequence for
the serotype, such as a DEN-1 consensus sequence, a DEN-2 consensus
sequence, a DEN-3 consensus sequence, or a DEN-4 consensus
sequence. Such a virus can be naturally occurring or synthetic.
Alternatively, a virus strain can be selected to correlate with a
strain prevalent in the area or population in which the vaccine is
intended to be administered. Another option is to select strains
for each serotype as a matter of convenience based on availability
or prior experience. For example, exemplary strains are described
in U.S. Pat. No. 6,254,873, which is incorporated by reference
herein. Additional suitable strains are disclosed, e.g., in U.S.
Pat. No. 7,226,602. Additional strains can be found, for example,
in the VBRC viral genome database
(http://athena.bioc.uvic.ca/organisms/Flaviviridae/Dengue/Curated
genes), and the Dengue Virus Database
(http://www.broad.mit.edu/annotation/viral/Dengue/ProjectInfo.html).
[0032] In the context of a purified inactivated dengue virus
vaccine, either virulent or attenuated strains can be used.
Typically virulent strains propagate to higher titer in host cells,
facilitating production at commercial scale. However, virulent
strains require special care in handling to prevent infection of
personnel involved in manufacturing. Attenuated strains, e.g.,
developed by adaptation to production in cultured cells and
selection for reduced virulence and/or reduced replication in the
mosquito vectors of dengue, require fewer handling precautions but
can be difficult to produce. Exemplary attenuated strains suitable
for use in the context of an immunogenic composition containing an
inactivated dengue virus and an aluminum-free adjuvant are
described in WO 00/57907 and U.S. Pat. No. 6,638,514, and WO
00/58444 and U.S. Pat. No. 6,613,556, which are incorporated herein
by reference. Thus, the strain(s) selected are typically chosen
from among the numerous strains available to replicate in cells
that are suitable for production of materials intended for human
use (e.g., cells that are certified free of pathogens). For
example, strains can be screened to identify those viruses that
grow to the highest titers, for example from a titer of at least
about 5.times.10.sup.6 pfu/ml, preferably at least 1.times.10.sup.7
pfu/ml or more in the cell line(s) of choice; (ii) selecting those
strains of dengue virus which grow to the highest titers in the
cell line(s) of choice; and (iii) further adapting those selected
strains for enhanced growth by additional passage from one to
several times in the cell line(s) of choice. The selected viruses
(for example, chosen from the four serotypes of dengue viruses) can
be further adapted to grow to high titers by additional cell
culture passages or by genetic manipulation to make high-titered
master and production seed lots.
[0033] Suitable cell lines for propagating dengue virus include
mammalian cells, such as Vero cells, AGMK cells, BHK-21 cells,
COS-1 or COS-7 cells, MDCK cells, CV-1 cells, LLC-MK2 cells,
primary cell lines such as fetal Rhesus lung (FRhL-2) cells, BSC-1
cells, and MRC-5 cells, or human diploid fibroblasts, as well as
avian cells, chicken or duck embryo derived cell lines, e.g., AGE1
cells, and primary, chicken embryo fibroblasts, and mosquito cell
lines, such as C6/36. Preferably, the chosen cell(s) are adapted to
grow in the absence of serum or serum-derived proteins, and can
maintain dengue virus replication at high titers under serum-free
(and/or protein-free) growth conditions.
[0034] To propagate virus in cell culture, the selected dengue
virus strain is used to infect the host cell (for example, selected
from among the suitable cell types listed above). After virus
adsorption, the cultures are fed with medium capable of supporting
growth of the cells. Preferably, the medium does not contain serum
or serum-derived proteins or other animal-derived proteins, or
serum-free media can be used to replace serum-containing media
during production. Numerous formulations of serum-free medium are
available commercially. A detailed description of methods for
producing virus in cells maintained under serum-free conditions can
be found, for example, in published US Patent Application No.
20060183224, which is incorporated herein by reference.
[0035] The host cells are maintained in culture for several days
until the desired virus titer is achieved. Optionally, the cells
are maintained in a continuous perfusion system from which virus
can be intermittently or continuously obtained over the course of
several days or more. Under non-continuous culture conditions, a
virus titer of at least about 10.sup.6 to 10.sup.7 PFU/ml by 3-7
days post infection, is desirable. In some host cells, the titer
remains high for several days, and virus can be recovered at
multiple timepoints to maximize yield. For example, virus can be
harvested from these cultures daily, from about 3 to about 13 days
post infection by collecting the supernates and refeeding the
cells. Optionally, the supernates can be pooled prior to additional
processing. In other host cells, virus can be grown to a higher
titer, but over a shorter period of time. In such a case, the virus
can be harvested at peak titer as determined empirically.
[0036] For example, Vero cells can be amplified in VPSFM medium
with Ficin up to passage 141. One additional passage can be
performed in the presence of porcine trypsin. Cells can be seeded
at approximately 0.7.times.10.sup.6 cells/ml in a Bioreactor (4 L)
and cultivated on micro-carriers (cytodex-1) for 5 days in
VPSFM+Pluronic 0.1% under perfusion conditions (e.g., D0.fwdarw.: 0
Volume/day; D1.fwdarw.D2: 1 v/d; then 1.5 v/d for the 3 next days).
Cells at approximately 3.times.10.sup.6 cells/ml are then infected
(at 35.degree. C.) with dengue virus, e.g., at a MOI 0.01 in
DMEM+Glutamine 4 mM++FBS 2% for 2 h with a reduced medium volume (2
L) at 37.degree. C. The medium volume in the Bioreactor is then
adjusted to 4 L. After 3 days, a large part of the medium (around 3
L) is removed and replaced by DMEM medium+Glutamin 4 mM. At 7 days
post-infection, virus can be harvested.
[0037] To recover virus, the virus is harvested by common methods
known in the art including slow-speed centrifugation (for example
at 1500.times.g for 10 min), or by filtration through a filter of
pore size of 0.45 .mu.m. Methods for concentrating said virus(es)
are within the scope of a person with ordinary skill in the art and
include, for example, ultrafiltration (e.g., with a membrane of no
greater than 300 kDa pore size), or precipitation with polyethelene
glycol (PEG) 8000. Methods for purifying viruses are known to a
person with ordinary skill in the art and include continuous or
multi-step sucrose gradients, purification by column chromatography
using size exclusion, ion exchange, adsorption, or affinity
columns, or purification by partitioning in polymer two-phase or
multi-phase systems, and any combination thereof. Methods for
assaying for virus positive fractions include plaque assay,
hemagglutination (HA) assay, and/or antigen assays such as
immunoassays.
[0038] For example, dengue virus can be concentrated from culture
supernate by polyethylene glycol (PEG) precipitation. Supernate
fluids from infected cells are clarified by centrifugation
(1,500.times.g) for 10 min. The clarified supernate is adjusted to
6% PEG 8000 (Sigma) and 0.5 M NaCl and held at 4.degree. C. with
gentle stirring for 45 min. The precipitate is collected by
centrifugation at 5000.times.g for 50 min and resuspended in STE
buffer (0.1 M NaCl, 10 mM tris, 1 mM EDTA, pH 7.6). The virus
suspension can then be clarified by centrifugation (12,000.times.g
for 10 min at 4.degree. C.). The pellet is discarded, and the
supernate is then centrifuged (170,000.times.g, 80 min, 4.degree.
C.) to pellet virus. This virus pellet is resuspended in STE buffer
at the desired concentration.
[0039] Alternatively, or additionally, dengue virus can be
concentrated from cell supernates by tangential flow
ultrafiltration. Supernate fluids from infected cells is clarified
by centrifugation at low speed as described above, then filtered
through a 0.45 .mu.m CN filter (Nalgene). The filtered supernate is
concentrated by tangential flow ultrafiltration using a
low-protein-binding 100 kDa-cutoff membrane (e.g., omega 100K
screen channel, Filtron, Inc.). Concentration is carried out at
4.degree. C. using a flow rate of 400 ml per min, a filtration rate
of approximately 100 ml per min and a pressure of 20-30 psi.
[0040] Further purification can be achieved using sucrose gradient
ultracentrifugation. Dengue virus can be purified on sucrose
gradients essentially as described previously (Srivastava et al.
Arch. Virol. 96: 97-107, 1987) with minor modifications. Fifteen ml
sucrose gradients can be made in 1''.times.3.5'' (40 ml)
ultracentifuge tubes (ULTRA-CLEAR.TM., Beckman, Inc.) by stepwise
addition of the following w/w sucrose solutions in phosphate
buffered saline, pH 7.4 (PBS, without Ca and Mg, Whittaker MA
Bioproducts): 2 ml 60%, 2 ml 55%, 2 ml 50%, 2 ml 45%, 2 ml 40%, 2
ml 35%, 2 ml 30% and 1 ml 15%. A smooth gradient is formed by
allowing the tubes to stand for 2-4 hrs at room temperature. Up to
25 ml of concentrated virus is applied to each tube.
Ultracentrifugation is then carried out in (e.g., in a SW 28 rotor
(Beckman) at 17,000 rpm for 18 hrs at 4.degree. C.). Following
centrifugation, 1 to 2 ml fractions can be collected from the
bottom of the tubes. Fractions can be assayed for total protein,
virus HA, and virus antigen as desired. Positive gradient fractions
are typically pooled, and diluted to 10% or less sucrose with
Medium 199 (M199, Gibco-BRL) or PBS. Optionally, prior to
inactivation, virus pools can be filtered through a 0.22 .mu.m
low-protein-binding filter (GV type, Millipore).
[0041] Following purification, the recovered viruses are
inactivated by a means selected to preserve their antigenicity and
immunogenicity while destroying their infectivity. In one example,
an effective quantity of an agent, such as formalin or
beta.-propriolactone is added to the virus, and the mixture is
incubated with the inactivating agent until inactivated. For
example, formalin (37% formaldehyde) is diluted 1:40 in PBS, and
the pH is adjusted to approximately 7.4 with 1 N NaOH. The solution
is then sterilized by passage through a 0.22 .mu.m CN filter
(Nalgene). This formalin solution is added to purified virus (1:50)
for a final formalin concentration of 0.05%. Inactivation is
carried out at between 15 and 25.degree. C., for 48 hrs up to 14
days (typically, 10 days or less). Optionally, virus is filtered
through a 0.22 um GV type filter and transferred to a fresh
container. At the completion of inactivation, free formalin in the
bulk culture is neutralized, e.g., by addition of an equimolar
amount of sterile 10% w/v sodium bisulfite.
[0042] Alternatively, inactivation can be achieved by irradiating
the virus with a radioactive source until the virus is inactivated.
One favorable example of a radioactive source is cobalt-60, at
doses sufficient to inactivate the infectivity of the viruses while
preserving the antigenicity essentially intact. Examples of useful
doses are those which fall within the range from 5.5 to 7.0 Mrads.
For example, virus aliquots of 50 .mu.l in 1.5 ml sterile
polypropylene tubes are frozen and placed on dry ice in the gamma
cell of a .sup.60Co source, for a period sufficient to deliver the
desired irradiation.
[0043] Alternatively, ultraviolet irradiation can be employed to
inactivate dengue virus. Suitable devices for ultraviolet
irradiation of virus in a commercial setting are well known in the
art, and devices are commercially available, e.g., from Bayer,
which expose the supernate (or other fluid containing the virus) to
a UV-C light source at approximately 254 nm, for a time sufficient
to inactivate virus while preserving immunogenicity.
[0044] Alternatively, the dengue virus can be inactivated using
hydrogen peroxide as described, e.g., in published US Patent
Application No. 20070031451, which is incorporated herein by
reference.
[0045] If desired, two or more activation steps can be employed.
When two or more inactivation steps are employed the steps can be
the same or different. For example, a combination of any suitable
inactivation process, such as any of the preceding inactivation
processes, can be employed during the purification and inactivation
of dengue virus for formulation into an immunogenic composition for
administration to a human subject.
[0046] Quality of the virus preparation can be monitored by a
variety of methods known in the art. For example, virus can be
monitored prior to inactivation in a plaque titration assay. Virus
is amplified on the same or a different host cell as used for
production, and used to infect a suitable cell line, such as
LLC-MK2 cells or Vero cell monolayers, and the number of viral
plaques can be assessed to determine infectivity of the recovered
virus (see, e.g., Sukhavachana et al. WHO Bull. 35: 65-6, 1966).
Another method for assessing quantity and quality of recovered
antigen is by virus hemagglutination (HA) and
hemagglutination-inhibition (HI) assays. Virus HA and HI assays can
be performed as previously described (Clarke & Casals, Am. J.
Trop. Med. Hyg. 7: 561-73, 1958). Total protein is determined
essentially as described by Bradford (Anal. Biochem. 72: 248,
1976), using a commercially-available kit (BioRad, Hercules,
Calif.) and bovine serum albumin (BSA) or gamma globulin as a
standard.
[0047] Alternatively, antigen can be detected and/or quantitated
after inactivation, e.g., in an antigen spot blot assay. To detect
and quantitate antigen in inactivated virus preparations virus
samples are diluted out serially, typically by two-fold dilution,
and spotted onto nitrocellulose paper. The papers are air-dried,
blocked with 5% casein in PBS, and incubated with a specific
antibody or antiserum followed by enzyme-linked secondary antibody.
Virus can also be detected by western blotting. In brief, antigen
preparations are solubilized in SDS-PAGE sample buffer containing
1% SDS, 66 mM Tris-HCl, pH 6.8, 1% glycerol and 0.7% bromphenol
blue at 22 C. for 10 min and electrophoresed on 12.5%
polyacrylamide gels (e.g., as described by Feighny et al. Am. J.
Trop. Med. Hyg. 50(3): 322-8, 1994). Resolved proteins are
transferred electrophoretically to nylon or nitrocellulose
membrane. Proteins can then be detected by staining with colloidal
gold and viral antigens can be identified immunologically using a
non-isotopic modification of the Western blot procedure.
Immunogenic Compositions and Methods
[0048] The inactivated dengue virus(es) is mixed with a suitable
aluminum-free adjuvant to produce an immunogenic composition
suitable for immunizing human subjects in order to elicit high
titers of virus neutralizing antibodies and protect the immunized
human from disease caused by dengue virus. Typically, the
inactivated dengue virus(es) are formulated in a pharmaceutically
acceptable carrier or excipient.
[0049] Pharmaceutically acceptable carriers and excipients are well
known and can be selected by those of skill in the art. For
example, the carrier or excipient can favorably include a buffer.
Optionally, the carrier or excipient also contains at least one
component that stabilizes solubility and/or stability. Examples of
solubilizing/stabilizing agents include detergents, for example,
laurel sarcosine and/or polyoxyethethylene sorbitan monooleate.
Alternative solubilizing/stabilizing agents include arginine, and
glass forming polyols (such as sucrose, trehalose and the like).
Numerous pharmaceutically acceptable carriers and/or
pharmaceutically acceptable excipients are known in the art and are
described, e.g., in Remington's Pharmaceutical Sciences, by E. W.
Martin, Mack Publishing Co., Easton, Pa., 5th Edition (1975).
[0050] Accordingly, suitable excipients and carriers can be
selected by those of skill in the art to produce a formulation
suitable for delivery to a subject by a selected route of
administration.
[0051] Suitable excipients include, without limitation: glycerol,
Polyethylene glycol (PEG), Sorbitol, Trehalose, N-lauroylsarcosine
sodium salt, L-proline, Non detergent sulfobetaine, Guanidine
hydrochloride, Urea, Trimethylamine oxide, KCl, Ca.sup.2+,
Mg.sup.2+, Mn.sup.2+, Zn.sup.2+ and other divalent cation related
salts, Dithiothreitol, Dithioerytrol, and B-mercaptoethanol. Other
excipients can be detergents (including: polyoxyethethylene
sorbitan monooleate, Triton X-00, NP-40, Empigen BB,
Octylglucoside, Lauroyl maltoside, Zwittergent 3-08, Zwittergent
3-0, Zwittergent 3-2, Zwittergent 3-4, Zwittergent 3-6, CHAPS,
Sodium deoxycholate, Sodium dodecyl sulphate,
Cetyltrimethylammonium bromide).
[0052] The immunogenic compositions disclosed herein also include
an adjuvant. In the context of an immunogenic composition suitable
for administration to a subject for the purpose of eliciting a
protective immune response against dengue, the adjuvant is an
aluminum-free adjuvant selected to elicit a balanced Th1/Th2
response or a Th1 biased immune response. Such an immune response
is characterized by the production of .gamma.-IFN.
[0053] The adjuvant is typically selected to enhance a balanced
Th1/Th2 response or a Th1 biased immune response in the subject, or
population of subjects, to whom the composition is administered.
For example, when the immunogenic composition is to be administered
to a subject of a particular age group susceptible to (or at
increased risk of) dengue infection, the adjuvant is selected to be
safe and effective in the subject or population of subjects. Thus,
when formulating an immunogenic composition containing an
inactivated dengue virus antigen for administration in neonatal, or
infant subjects (such as subjects between birth and the age of one
year), or in a child (e.g., such as subjects between birth and five
years of age), the adjuvant is selected to be safe and effective in
neonates and infants and/or children.
[0054] Additionally, the adjuvant is typically selected to enhance
a Th1 immune response when administered via a route by which the
immunogenic composition is administered. For example, when the
immunogenic composition is formulated for intramuscular
administration, adjuvants including one or more of 3D-MPL, squalene
(e.g., QS21), liposomes, and/or oil and water emulsions are
favorably selected.
[0055] One suitable adjuvant for use in combination with
inactivated dengue antigens is a non-toxic bacterial
lipopolysaccharide derivative. An example of a suitable non-toxic
derivative of lipid A, is monophosphoryl lipid A or more
particularly 3-Deacylated monophoshoryl lipid A (3D-MPL). 3D-MPL is
sold under the name MPL by GlaxoSmithKline Biologicals N.A., and is
referred throughout the document as MPL or 3D-MPL. See, for
example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and
4,912,094. 3D-MPL primarily promotes CD4+ T cell responses with an
IFN-.gamma. (Th1) phenotype. 3D-MPL can be produced according to
the methods disclosed in GB2220211 A. Chemically it is a mixture of
3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated
chains. In the compositions of the present invention small particle
3D-MPL can be used. Small particle 3D-MPL has a particle size such
that it can be sterile-filtered through a 0.22 .mu.m filter. Such
preparations are described in WO94/21292.
[0056] A lipopolysaccharide, such as 3D-MPL, can be used at amounts
between 1 and 100 .mu.g per human dose of the immunogenic
composition. Such 3D-MPL can be used at a level of about 50 .mu.g,
for example between 40-60 .mu.g, suitably between 45-55 .mu.g or
between 46 and 54 .mu.g or between 47 and 53 .mu.g or between 48
and 52 .mu.g, or between 49 and 51 .mu.g, or at 50 .mu.g. In
another embodiment, for example for administration to an infant or
child, the dose of the immunogenic composition comprises a
fractional dose of 3D-MPL at a level of about 25 .mu.g per human
dose of the immunogenic composition. Such 3D-MPL can be used at a
level of about 25 .mu.g, for example between 20-30 .mu.g, suitably
between 21-29 .mu.g or between 22 and 28 .mu.g or between 23 and 27
.mu.g or between 24 and 26 .mu.g, or 25 .mu.g. In other embodiment
(e.g., suitable for administration to very young infants), the
human dose of the immunogenic composition comprises an a fractional
dose containing an even lower amount of 3D-MPL at a level of about
10 .mu.g, for example between 5 and 15 .mu.g, suitably between 6
and 14 .mu.g, for example between 7 and 13 .mu.g or between 8 and
12 .mu.g or between 9 and llpg, or 10 .mu.g. In a further
embodiment, the human dose of the immunogenic composition comprises
3D-MPL at a level of about 5 .mu.g, for example between 1 and 9
.mu.g, or between 2 and 8 .mu.g or suitably between 3 and 7 .mu.g
or 4 and .mu.g, or 5 .mu.g.
[0057] In other embodiments, the lipopolysaccharide can be a
.beta.(1-6) glucosamine disaccharide, as described in U.S. Pat. No.
6,005,099 and EP Patent No. 0 729 473 B1. One of skill in the art
would be readily able to produce various lipopolysaccharides, such
as 3D-MPL, based on the teachings of these references. Nonetheless,
each of these references is incorporated herein by reference. In
addition to the aforementioned immunostimulants (that are similar
in structure to that of LPS or MPL or 3D-MPL), acylated
monosaccharide and disaccharide derivatives that are a sub-portion
to the above structure of MPL are also suitable adjuvants. In other
embodiments, the adjuvant is a synthetic derivative of lipid A,
some of which are described as TLR-4 agonists, and include, but are
not limited to: OM174
(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phos-
phono-.beta.-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-.quadra-
ture.-D-glucopyranosyldihydrogenphosphate), (WO 95/14026); OM 294
DP
(3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-h-
ydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)
(WO 99/64301 and WO 00/0462); and OM 197 MP-Ac DP
(3S-,9R)-3-.quadrature.(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-
-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate
10-(6-aminohexanoate) (WO 01/46127).
[0058] Other TLR4 ligands which can be used are alkyl Glucosaminide
phosphates (AGPs) such as those disclosed in WO 98/50399 or U.S.
Pat. No. 6,303,347 (processes for preparation of AGPs are also
disclosed), suitably RC527 or RC529 or pharmaceutically acceptable
salts of AGPs as disclosed in U.S. Pat. No. 6,764,840. Some AGPs
are TLR4 agonists, and some are TLR4 antagonists. Both are thought
to be useful as adjuvants.
[0059] Other suitable TLR-4 ligands, capable of causing a signaling
response through TLR-4 (Sabroe et al, JI 2003 p 1630-5) are, for
example, lipopolysaccharide from gram-negative bacteria and its
derivatives, or fragments thereof, in particular a non-toxic
derivative of LPS (such as 3D-MPL). Other suitable TLR agonists
are: heat shock protein (HSP) 10, 60, 65, 70, 75 or 90; surfactant
Protein A, hyaluronan oligosaccharides, heparan sulphate fragments,
fibronectin fragments, fibrinogen peptides and b-defensin-2, and
muramyl dipeptide (MDP). In one embodiment the TLR agonist is HSP
60, 70 or 90. Other suitable TLR-4 ligands are as described in WO
2003/011223 and in WO 2003/099195, such as compound I, compound II
and compound III disclosed on pages 4-5 of WO2003/011223 or on
pages 3-4 of WO2003/099195 and in particular those compounds
disclosed in WO2003/011223 as ER803022, ER803058, ER803732,
ER804053, ER804057, ER804058, ER804059, ER804442, ER804680, and
ER804764. For example, one suitable TLR-4 ligand is ER804057.
[0060] Other adjuvants that can be used in immunogenic compositions
with an inactivated dengue virus antigens, e.g., alternatively to,
or in combination with, 3D-MPL, or another adjuvant described
herein, are saponins, such as QS21.
[0061] Saponins are taught in: Lacaille-Dubois, M and Wagner H.
(1996). A review of the biological and pharmacological activities
of saponins. Phytomedicine vol 2 pp 363-386). Saponins are steroid
or triterpene glycosides widely distributed in the plant and marine
animal kingdoms. Saponins are noted for forming colloidal solutions
in water which foam on shaking, and for precipitating cholesterol.
When saponins are near cell membranes they create pore-like
structures in the membrane which cause the membrane to burst.
Haemolysis of erythrocytes is an example of this phenomenon, which
is a property of certain, but not all, saponins.
[0062] Saponins are known as adjuvants in vaccines for systemic
administration. The adjuvant and haemolytic activity of individual
saponins has been extensively studied in the art (Lacaille-Dubois
and Wagner, supra). For example, Quil A (derived from the bark of
the South American tree Quillaja Saponaria Molina), and fractions
thereof, are described in U.S. Pat. No. 5,057,540 and "Saponins as
vaccine adjuvants", Kensil, C. R., Crit Rev Ther Drug Carrier Syst,
1996, 12 (1-2):1-55; and EP 0 362 279 B1. Particulate structures,
termed Immune Stimulating Complexes (ISCOMS), comprising fractions
of Quil A are haemolytic and have been used in the manufacture of
vaccines (Morein, B., EP 0 109 942 B1; WO 96/11711; WO 96/33739,
and U.S. Pat. No. 6,846,489). The haemolytic saponins QS21 and QS17
(HPLC purified fractions of Quil A) have been described as potent
systemic adjuvants, and the method of their production is disclosed
in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1, which are
incorporated herein by reference. Such saponins, or fractions
thereof, can be used alone or in combination, and optionally can be
formulated as ISCOMS. Other saponins which have been used in
systemic vaccination studies include those derived from other plant
species such as Gypsophila and Saponaria (Bomford et al., Vaccine,
10(9):572-577, 1992).
[0063] QS21 is an Hplc purified non-toxic fraction derived from the
bark of Quillaja Saponaria Molina. A method for producing QS21 is
disclosed in U.S. Pat. No. 5,057,540. Non-reactogenic adjuvant
formulations containing QS21 are described in WO 96/33739. The
aforementioned references are incorporated by reference herein.
Said immunologically active saponin, such as QS21, can be used in
amounts of between 1 and 50 .mu.g, per human dose of the
immunogenic composition. Advantageously QS21 is used at a level of
about 1 and 100 .mu.g per human dose of the immunogenic
composition. For example, QS21 can be used at a level of about 50
.mu.g, for example between 40-60 .mu.g, suitably between 45-55
.mu.g or between 46 and 54 .mu.g or between 47 and 53 .mu.g or
between 48 and 52 .mu.g, or between 49 and 51 .mu.g, or at 50
.mu.g. In another embodiment, for example for administration to an
infant or child, the dose of the immunogenic composition comprises
a fractional dose 25 .mu.g, for example between 20-30 .mu.g,
suitably between 21-29 .mu.g or between 22-28 .mu.g or between
23-27 .mu.g or between 24-26 .mu.g, or 25 .mu.g. In another
embodiment, the human dose of the immunogenic composition comprises
QS21 at a level of about 10 .mu.g, for example between 5 and 15
.mu.g, suitably between 6-14 .mu.g, for example between 7-13 .mu.g
or between 8-12 .mu.g or between 9-11 .mu.g, or 10 .mu.g. In a
further embodiment, the human dose of the immunogenic composition
comprises QS21 at a level of about 5 .mu.g, for example between 1-9
.mu.g, or between 2-8 .mu.g or suitably between 3-7 .mu.g or 4-6
.mu.g, or 5 .mu.g. Such formulations comprising QS21 and further
comprising cholesterol (suitably in a liposomal formulation) have
been shown to be successful Th1 stimulating adjuvants when
formulated together with an antigen. Thus, for example, an
inactivated dengue virus antigen can favorably be employed in
immunogenic compositions with an adjuvant comprising a combination
of QS21 and cholesterol. Optionally such an adjuvant also contains
1, 2-Dioleoyl-sn-Glycero-3-phophocholine (DOPC). Saponins, such as
QS21, can also be formulated with one or more additional
immunostimulating agents (such as 3D-MPL as discussed herein, or
e.g., CpG oligonucleotides, or other TLR agonists).
[0064] Combinations of different adjuvants, such as those mentioned
hereinabove, can also be used in compositions with inactivated
dengue virus antigens. For example, as already noted, QS21 can be
formulated together with 3D-MPL. The ratio of QS21:3D-MPL will
typically be in the order of 1:10 to 10:1; such as 1:5 to 5:1, and
often substantially 1:1. Typically, the ratio is in the range of
2.5:1 to 1:1 3D-MPL:QS21. Optionally, the combination of QS21 and
3D-MPL also contains one or both of cholesterol and DOPC.
Additional details regarding formulations and dosages of such
combinations, especially doses (fractional doses) suitable for
administration to infants and children, can be found in
WO2007068907 and US 20080279926, which are incorporated herein by
reference. When formulated in combination, this combination can
enhance an antigen-specific Th1 immune response.
[0065] In some instances, the adjuvant formulation includes an
oil-in-water emulsion.
[0066] One example of an oil-in-water emulsion comprises a
metabolisable oil, such as squalene, a tocol such as a tocopherol,
e.g., alpha-tocopherol, and a surfactant, such as polysorbate 80 or
TWEEN.TM. 80, in an aqueous carrier, and does not contain any
additional immunostimulants(s). The aqueous carrier can be, for
example, phosphate buffered saline. One favorable example of such
an oil-in-water emulsion is designated herein AS03. Additionally
the oil-in-water emulsion can contain sorbitan trioleate (SPAN.TM.
85) and/or lecithin and/or tricaprylin. Another example of an
oil-in-water emulsion is MF59, which is a squalene based emulsion
system sold by Novartis Vaccine and Diagnostics.
[0067] In another embodiment of the invention there is provided a
vaccine composition comprising an antigen or antigen composition
and an adjuvant composition comprising an oil-in-water emulsion,
wherein said oil-in-water emulsion comprises 0.25-10 mg
metabolisable oil (suitably squalene), 0.25-11 mg tocol (suitably a
tocopherol, such as alpha-tocopherol) and 0.125-4 mg emulsifying
agent. In some instances, e.g., for administration to children, the
oil-in-water emulsion is present in a fractional dose of 1/2, 1/4,
or 1/8 of the standard adult dose. Additional details regarding
fractional doses of oil-in-water emulsions suitable for use in
combination with an inactivated dengue virus vaccine can be found
in WO 2008043774, U.S. Ser. No. 12/445,090, which is incorporated
herein by reference.
[0068] In particular formulations using an oil-in-water emulsion,
such an emulsion can include additional components, for example,
such as cholesterol, squalene, alpha tocopherol, and/or a
detergent, such as polyoxyethethylene sorbitan monooleate
(TWEEN.TM. 80) or sorbitan trioleate. In exemplary formulations,
such components can be present in the following amounts: from about
1-50 mg cholesterol, from 2 to 10% squalene, from 2 to 10% alpha
tocopherol and from 0.3 to 3% polyoxyethethylene sorbitan
monooleate (all volume/volume). Typically, the ratio of
squalene:alpha tocopherol is equal to or less than 1 as this
provides a more stable emulsion. In some cases, the formulation can
also contain a stabilizer.
[0069] Optionally, the oil and water emulsion adjuvant includes one
or more further immunostimulant. In one specific embodiment, the
adjuvant formulation includes 3D-MPL prepared in the form of an
emulsion, such as an oil-in-water emulsion. In some cases, the
emulsion has a small particle size of less than 0.2 .mu.m in
diameter, as disclosed in WO 94/21292. For example, the particles
of 3D-MPL can be small enough to be sterile filtered through a 0.22
micron membrane (as described in European Patent number 0 689 454).
Alternatively, the 3D-MPL can be prepared in a liposomal
formulation. Optionally, the adjuvant containing 3D-MPL (or a
derivative thereof) also includes an additional immunostimulatory
component.
[0070] For example, when an immunogenic composition with an
inactivated dengue virus antigen is formulated for administration
to an infant, the dosage of adjuvant is determined to be effective
and relatively non-reactogenic in an infant subject. Generally, the
dosage of adjuvant in an infant formulation is lower than that used
in formulations designed for administration to adult (e.g., adults
aged 65 or older). For example, the amount of 3D-MPL is typically
in the range of 1 .mu.g-200 .mu.g, such as 10-100 .mu.g, or 10
.mu.g-50 .mu.g per dose. An infant dose is typically at the lower
end of this range, e.g., from about 1 .mu.g to about 50 .mu.g, such
as from about 2 .mu.g, or about 5 .mu.g, or about 10 .mu.g, to
about 25 .mu.g, or to about 50 .mu.g. Typically, where QS21 is used
in the formulation, the ranges are comparable (and according to the
ratios indicated above). For adult and elderly populations, the
formulations typically include more of an adjuvant component than
is typically found in an infant formulation.
[0071] An immunogenic composition typically contains an
immunoprotective quantity (or a fractional dose thereof) of the
antigen and can be prepared by conventional techniques. Preparation
of immunogenic compositions, including those for administration to
human subjects, is generally described in Pharmaceutical
Biotechnology, Vol. 61 Vaccine Design--the subunit and adjuvant
approach, edited by Powell and Newman, Plenum Press, 1995. New
Trends and Developments in Vaccines, edited by Voller et al.,
University Park Press, Baltimore, Md., U.S.A. 1978. Encapsulation
within liposomes is described, for example, by Fullerton, U.S. Pat.
No. 4,235,877. Conjugation of proteins to macromolecules is
disclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and by
Armor et al., U.S. Pat. No. 4,474,757.
[0072] Typically, the amount of antigen in each dose of the
immunogenic composition is selected as an amount which induces an
immunoprotective response without significant, adverse side effects
in the typical subject. Immunoprotective in this context does not
necessarily mean completely protective against infection; it means
protection against symptoms or disease, especially severe disease
associated with the virus. The amount of antigen can vary depending
upon which specific immunogen is employed. Generally, it is
expected that each human dose will comprise 0.05-100 .mu.g of
inactivated virus, such as from about 0.1 .mu.g (e.g., 0.1, 0.2,
0.3, 0.4, or 0.5 .mu.g) to about 50 .mu.g, for example, from about
0.5 .mu.g to about 30 .mu.g, such as about 2 .mu.g, about 3 .mu.g,
about 4 .mu.g, about 5 .mu.g, about 10 .mu.g, about 15 .mu.g, about
20 .mu.g, or about 25 .mu.g, of each strain of inactivated dengue
virus. The amount utilized in an immunogenic composition is
selected based on the subject population (e.g., infant). An optimal
amount for a particular composition can be ascertained by standard
studies involving observation of antibody titres and other
responses in subjects. Following an initial vaccination, subjects
can receive a boost after a suitable interval (e.g., in about 4
weeks).
[0073] It should be noted that regardless of the adjuvant selected,
the concentration in the final formulation is calculated to be safe
and effective in the target population. For example, immunogenic
compositions for eliciting an immune response against dengue virus
in humans are favorably administered to infants (e.g., infants
between birth and 1 year, such as between 0 and 6 months, at the
age of initial dose). Immunogenic compositions for eliciting an
immune response against dengue are also favorably administered to
adult humans (e.g., alone or in a combination with antigens of
other pathogens for example in the context of a "traveler's"
vaccine). It will be appreciated that the choice of adjuvant can be
different in these different applications, and the optimal adjuvant
and concentration for each situation can be determined empirically
by those of skill in the art.
[0074] Methods for eliciting an immune response against dengue in a
subject are also a feature of this disclosure. Such methods include
administering an immunologically effective amount of a composition
comprising an inactivated dengue virus antigen and an aluminum-free
adjuvant to a subject, such as a human subject. For example, the
composition includes an adjuvant that elicits a Th1 biased immune
response. The composition is formulated to elicit an immune
response specific for dengue, that is, the composition is
formulated to, and results in, a Th1 biased immune response that
reduces or prevents infection with dengue virus and/or reduces or
prevents a pathological response following infection with a dengue
virus.
[0075] Typically, vaccines are prepared as injectables, either as
liquid solutions or suspensions; solid form suitable for solution
in, or suspension in, liquid prior to injection may also be
prepared. Although the composition can be administered by a variety
of different routes, most commonly, the immunogenic compositions
are delivered by an intramuscular, subcutaneous or intradermal
route of administration. Generally, the vaccine may be administered
subcutaneously, intradermally, or intramuscularly in a dose
effective for the production of neutralizing antibody and
protection. The vaccines are administered in a manner compatible
with the dosage formulation, and in such amount as will be
prophylactically and/or therapeutically effective. The quantity to
be administered, which is generally in the range of 0.05-100 .mu.g
of each strain of inactivated virus per dose, depends on the
subject to be treated, capacity of the subject's immune system to
synthesize antibodies, and the degree of protection desired.
Precise amounts of the vaccine to be administered may depend on the
judgment of the practitioner and may be peculiar to each
subject.
[0076] The vaccine may be given in a single dose schedule, or
preferably a multiple dose schedule in which a primary course of
vaccination may be with 1-10 separate doses, followed by other
doses given at subsequent time intervals required to maintain and
or reinforce the immune response, for example, at 1-4 months for a
second dose, and if needed, a subsequent dose(s) after several
months or years. The dosage regimen will also, at least in part, be
determined by the need of the individual and be dependent upon the
judgment of the practitioner. Examples of suitable immunization
schedules include: a first dose, followed by a second dose between
7 days and 6 months, and an optional third dose between 1 month and
two years post initial immunization, or other schedules sufficient
to elicit titers of virus-neutralizing antibodies expected to
confer protective immunity, for example selected to correspond to
an established pediatric vaccine schedule. The generation of
protective immunity against dengue with an inactivated virus
vaccine may reasonably be expected after a primary course of
immunization consisting of 1 to 3 inoculations. These could be
supplemented by boosters at intervals (e.g., every two years)
designed to maintain a satisfactory level of protective
immunity.
EXAMPLES
Example I
Immunogenicity of Exemplary Immunogenic Compositions that Contain a
Dengue Purified Inactivated Virus (1 .mu.g and 10 .mu.g) and an
Aluminum-Free Adjuvant
[0077] Groups of 15 naive adult female C57Bl/6 mice were vaccinated
intramuscularly with two doses of an exemplary vaccine candidate
containing purified inactivated virus (PIV) of a Dengue-2 (Den-2)
strain. The vaccine was administered in a total volume of 50 Mice
were immunized with formulations containing Dengue PIV alone or
formulations containing Dengue PIV vaccine adjuvanted with Alum
(alum hydroxide), 3-Deacylated monophoshoryl lipid A (3D-MPL)
adsorbed onto aluminum hydroxide (AS04D), an oil-in-water emulsion
(AS03A), and 3D-MPL and QS21 in a liposomal formulation (AS01B)
(see groups in Table 1 below). AS03A and AS01B are two non-limiting
examples of aluminum (alum)-free adjuvants. The Dengue PIV antigen
used for formulation was a purified bulk obtained essentially as
described herein from a culture of a wild-type Den-2 virus,
inactivated with 0.185% of formalin for 7 days at 22.degree. C.
[0078] Throughout the examples described herein, statistical
analyses were performed on post vaccination CD4+ frequencies and
neutralizing titres by UNISTAT. The protocol applied for analysis
of variance can be briefly described as follow: .quadrature.Log
transformation of data; Shapiro-Wilk test on each population
(group) in order to verify the normality of groups distribution;
Cochran test in order to verify the homogenicity of variance
between the different populations (groups); Analysis of variance on
selected data; Test for interaction of one-way ANOVA; Tukey-HSD
Test for multiple comparisons.
TABLE-US-00001 TABLE 1 PIV Den-2 (Conc Total Gr Antigen/Formulation
prot/dose) Other treatment 1 PIV Den-2 Plain (non-adjuvanted) 10
.mu.g Days 0 and 21 2 PIV Den-2 + AS22A (Al(OH)3) 10 .mu.g Days 0
and 21 3 PIV Den-2 + AS04D 10 .mu.g Days 0 and 21 4 PIV Den-2 +
AS03A 10 .mu.g Days 0 and 21 5 PIV Den-2 + AS01B 10 .mu.g Days 0
and 21 6 PIV Den-2 + Plain (non-adjuvanted) 1 .mu.g Days 0 and 21 7
PIV Den-2 + AS22A (Al(OH)3) 1 .mu.g Days 0 and 21 8 PIV Den-2 +
AS04D 1 .mu.g Days 0 and 21 9 PIV Den-2 + AS03A 1 .mu.g Days 0 and
21 10 PIV Den-2 + AS01B 1 .mu.g Days 0 and 21 11 PBS Days 0 and
21
[0079] The humoral immune response was measured 21 days after the
first immunization (set as Day 0) and the second immunization (Day
21), that is, on days 21 and 42, post immunization. Serum samples
were tested using an anti-Dengue neutralization assay. In brief, to
measure mouse serum neutralizing titers against Dengue virus,
serial dilutions of filtered and heat inactivated serum were
incubated with a fixed amount of monospecific Dengue virus
(Dengue-2). The mixture of serum and virus was then added to a
monolayer of Vero cells (from a WHO cell bank) and incubated for 4
days. Viral infection inhibition by serum samples was measured
using an ELISA that detects cell-associated viral antigens on Vero
cells adhered to a 96-well microplate. The resulting optical
density readings were automatically processed into an Excel
spreadsheet that uses a log mid-point linear regression program
model to derive a virus percent reduction of infection (referred to
as microneutralization 50 percent reduction, or MN50). The virus
neutralization titer (MN50) is defined as the reciprocal of the
serum dilution giving 50% reduction in the absorbance readout of
the assay when compared to the virus dose control without serum
(TV).
[0080] Neutralizing antibody titers were determined on pooled sera
for each group at day 21, and on individual sera at day 42. Results
are illustrated in FIG. 1. The same immunological profile was
observed for both doses tested (1 and 10 .mu.g total protein). The
neutralizing antibody titers were enhanced after two
administrations of 1 .mu.g or 10 .mu.g of Dengue PIV vaccine as
compared to the response after a single dose of the composition.
For both doses of Dengue PIV vaccine tested, significantly lower
neutralizing antibody responses were observed with the
non-adjuvanted vaccine compared to the adjuvanted Dengue PIV
vaccine (p<0.00001). At the 10 .mu.g dose of total protein,
Dengue PIV vaccine adjuvanted with AS04D induced significantly
higher neutralizing antibody response compared to the response
induced by the Dengue PIV vaccine adjuvanted with alum alone
(AS22A) (p=0.0027). No difference was observed between these two
adjuvants at the dose of 1 .mu.g total protein (p>0.05). As
shown in FIG. 1, compositions adjuvanted with either alum-free
adjuvant induced significantly higher neutralizing antibody
responses (AS03A (p<0.0173) or AS01B (p<0.0001)) than did the
compositions with AS04D or AS22A. Dengue PIV vaccine adjuvanted
with AS03A and AS01B induced similar levels of neutralizing
antibody titers (p>0.05).
[0081] These results demonstrate that at the dose of 1 or 10 .mu.g
total protein of Dengue PIV, higher neutralizing antibody titres
were observed in mice immunized with compositions containing an
adjuvant system that does not contain alum (as exemplified by AS03A
or AS01B shown here) compared to the response induced by the same
antigen in combination with an alum-containing adjuvant (e.g.,
AS22A or AS04D).
Example II
Immunogenicity of Exemplary Immunogenic Compositions that Contain a
Dengue Purified Inactivated Virus (1 .mu.g and 10 .mu.g) and an
Aluminum-Free Adjuvant at Different Dilutions of Adjuvant
[0082] Groups of 15 adult female C57Bl/6 mice were administered two
doses of the exemplary Dengue PIV vaccine intramuscularly in a
total volume of 50 Mice were immunized with formulations containing
either Dengue PIV alone or formulations containing Dengue PIV
antigen adjuvanted with Alum (AS22A, alum hydroxide), or dilution
of different adjuvant systems, e.g., such as 1/2 the standard dose
of AS04D (AS04D/2), AS03B (oil-in-water emulsion-based Adjuvant
System containing 5.93 mg tocopherol), and AS01E (half dose of
AS01B) (detailed in Table 2). The PIV used for formulation was a
purified bulk obtained from a culture of a wild-type Den-2 virus,
inactivated with 0.185% of Formalin for 7 days at 22.degree. C.
TABLE-US-00002 TABLE 2 PIV Den-2 Conc Total Gr Antigen/Formulation
prot/mouse Other treatment 1 PIV Den-2 Plain (non-adjuvanted) 1
.mu.g Days 0 and 21 2 PIV Den-2 + AS22A (Al(OH)3) 1 .mu.g Days 0
and 21 5 PIV Den-2 + AS04D/2 10 .mu.g Days 0 and 21 6 PIV Den-2 +
AS03B 10 .mu.g Days 0 and 21 7 PIV Den-2 + AS01E 10 .mu.g Days 0
and 21 8 PIV Den-2 + AS04D/2 1 .mu.g Days 0 and 21 9 PIV Den-2 +
AS03B 1 .mu.g Days 0 and 21 10 PIV Den-2 + AS01E 1 .mu.g Days 0 and
21 11 PBS Days 0 and 21
[0083] The humoral immune response to immunization was measured 21
days after the first and second immunizations (Days 21 and 42,
respectively) on 15 mice/group. Serum samples were tested by the
neutralization assay described above.
[0084] Neutralizing antibody titers on pool sera per group at day
21, and on individual sera per group at day 42, are presented in
FIG. 2. The same immunological profile was observed for both doses
tested (1 and 10 .mu.g total protein). Higher neutralizing antibody
titers were observed after two administrations of the immunogenic
composition as compared to the response induced after only a single
administration of either 1 .mu.g or 10 .mu.g of Dengue PIV antigen.
For both doses of Dengue PIV vaccine tested, significantly lower
neutralizing antibody responses were observed with the
non-adjuvanted vaccine compared to the adjuvanted Dengue PIV
formulations (p<0.00001). At the dose of 1 .mu.g protein total,
Dengue PIV vaccine adjuvanted with AS04D/2 induced similar
neutralizing antibody response compared to the response induced by
the Dengue PIV vaccine adjuvanted with alum alone (p>0.05). For
the same vaccine dose tested, Dengue PIV vaccine adjuvanted with
AS03B or AS01E induced significantly higher neutralizing antibody
titers compared to vaccine adjuvanted with AS04D (p.ltoreq.0.029
for AS03B and p.ltoreq.0.00001 for AS01E) or alum (p.ltoreq.0.0035
for AS03B and p.ltoreq.0.00001 for AS01E).
[0085] In conclusion, at a dose of either 1 or 10 .mu.g total
protein of Dengue PIV antigen, higher neutralizing antibody titres
were observed in mice immunized with compositions adjuvanted with a
dilution of adjuvant system that does not contain alum (AS03B or
AS01E) compared to the response induced by the same antigen
adjuvanted with an alum-containing adjuvant (AS22A or AS04D/2).
Example III
Immunogenicity of Exemplary Immunogenic Compositions that Contain a
Dengue Purified Inactivated Virus ((2 .mu.G and 200 ng) and an
Aluminum-Free Adjuvant at Different Dilutions of Adjuvant
[0086] Groups of 25 adult female C57Bl/6 mice were immunized
intramuscularly with two doses of an exemplary Dengue PIV vaccine
in a total volume of 50 Mice were immunized with formulations
containing Dengue PIV antigen alone or formulations containing
Dengue PIV antigen adjuvanted with Alum (AS22A, alum hydroxide) or
dilution of different adjuvant, e.g., AS04D (AS04D/2), AS03B
(oil-in-water emulsion-based Adjuvant System containing 5.93 mg
tocopherol), and AS01E (half dose of AS01B) (the groups are
detailed in Table 3). The PIV antigen used for formulation was a
purified bulk obtained from a culture of a wild-type Den-2 virus,
inactivated with 0.185% of formalin for 7 days at 22.degree. C.
TABLE-US-00003 TABLE 3 PIV Den-2 Conc Total Gr Antigen/Formulation
prot/mouse Other treatment 1 PIV Den-2 Plain (non-adjuvanted) 200
ng Days 0 and 14 2 PIV Den-2 + AS22A (Al(OH)3) 200 ng Days 0 and 14
3 PIV Den-2 + AS04D/2 2 .mu.g Days 0 and 14 4 PIV Den-2 + AS03B 2
.mu.g Days 0 and 14 5 PIV Den-2 + AS01E 2 .mu.g Days 0 and 14 6 PIV
Den-2 + AS04D/2 200 ng Days 0 and 14 7 PIV Den-2 + AS03B 200 ng
Days 0 and 14 8 PIV Den-2 + AS01E 200 ng Days 0 and 14 9 PBS Days 0
and 14
[0087] The humoral immune response to administration of the
immunogenic composition was measured 14 days after the first and
second immunizations (Days 14 and 28, respectively) on 15
mice/group. Serum samples were evaluated by the neutralization
assay described above.
[0088] Ten mice from each group were sacrificed at 7 days post
immunization in order to evaluate the cellular immune response by
ICS using 5 pools of 2 spleens each. CMI was only evaluated for the
groups of mice immunized with 2 .mu.g of Dengue PIV vaccine
adjuvanted with AS04D/2, AS03B, AS01E and in mice immunized with
200 ng of the non-adjuvanted vaccine or the vaccine adjuvanted with
AS22A (groups 1 to 5) and mice receiving PBS (group 9). Sera of 15
other mice were collected 14 days after the first immunization (day
14) and the second immunization (day 28).
[0089] Neutralizing antibody titers were determined on pooled sera
per group at day 14, and on individual sera per group at day 28.
Results are provided in FIG. 3. Higher neutralizing antibody titers
were observed after two administrations of either 2 .mu.g or 200 ng
of Dengue PIV vaccine as compared to the titers after a single
administration. Significantly higher neutralizing antibody
responses were observed with the adjuvanted Dengue PIV formulations
as compared to the non-adjuvanted formulation (p<0.0089). At the
dose of 200 ng of Dengue PIV, the formulations adjuvanted with
AS03B and AS01E elicited a substantially higher neutralizing
antibody response as compared to the response elicited by the same
antigen formulated with AS22A or AS04D/2. At the lower dose of
antigen, Dengue PIV adjuvanted with AS22A and AS04D/2 induced
comparable neutralizing antibody response at the dose of 200 ng
total protein (p>0.05). At the higher antigen dose of 2 .mu.g
total protein, the formulation with AS04D/2 induced similar
neutralizing antibody response compared to the vaccine adjuvanted
with AS03B (p>0.05). However, at lower doses of antigen (200
ng), the formulation with AS03B elicited significantly higher
neutralizing antibody titers that AS04D/2 (p=0.0012).
Interestingly, Dengue PIV adjuvanted with AS01E induced
significantly higher neutralizing antibody titers compared to all
other groups (p.ltoreq.0.0028).
[0090] The cellular immune response was also evaluated following
two administrations of Dengue PIV in combination with the
aforementioned dilutions of adjuvant. CD4+ T cell cytokine
responses were evaluated 7 days after the second immunization (Day
21) by ICS (FIG. 4).
[0091] Lower CD4+ T cell response was induced by the non-adjuvanted
Dengue PIV vaccine compared to all other groups (p.ltoreq.0.001).
Mice immunized with Dengue PIV vaccine adjuvanted with AS01E
induced the highest CD4+ T cell responses compared to mice in all
other groups (p.ltoreq.0.00001). CD4+ T cells response obtained
with Dengue PIV vaccine adjuvanted with AS22A, AS04D/2 and AS03B
were not statistically different (p>0.05).
[0092] In conclusion, higher neutralizing antibody titres were
observed in mice immunized with Dengue PIV vaccine adjuvanted with
an adjuvant system that does not contain alum (AS03B or AS01E)
compared to the response induced by this vaccine adjuvanted with
Alum hydroxide (AS22A). Of note, Dengue PIV vaccine adjuvanted with
AS01E induced higher neutralizing antibody response and higher CD4
T cell response compared to the vaccine adjuvanted with the
alum-containing adjuvant, AS04D/2.
[0093] In summary, as compared to mice immunized with Dengue PIV
formulated with Alum-containing adjuvants (AS22A, AS04D or
AS04D/2), mice immunized with a vaccine formulated with alum-free
adjuvant systems (e.g., AS01B or AS01E, which do not contain alum),
elicited substantially higher neutralizing antibody response and
CD4+ T cell responses. These results demonstrate the favorable
immunogenic attributes of Dengue PIV vaccines formulated with
alum-free adjuvants.
* * * * *
References