Note: Descriptions are shown in the official language in which they were submitted.
W O 94/19013 2 1 ~ PCT~EP94/Oo448
INFLUENZA ~ACCINE COMPOSITIONS CONTAINING 3-0-DEACYLATED MONOPHOSPHORYL
LIPID A
Cross-Reference to Other ~p~lic~t;ons
This is a continuation-in-part application of co-
pending United States patent application serial number
021,535, filed February 19, 1993.
.
Fiel~ of the Invent;on
This invention relates to vaccines useful in
preventing infection with influenza in humans.
R~ckgrol~n~ of the Invent;on
Influenza virus infection causes acute respiratory
disease in man, horses and fowl, sometimes of pandemic
proportions. Influenza viruses belong to the
orthomyxovirus family of RNA viruses and, as such, have
enveloped virions of 80 to 120 nanometers in diameter,
with two external glycoprotein spikes, hemagglutinin (HA)
and neuraminidase (NA), and five internal proteins,
nucleoprotein, matrix protein and three polymerases.
Influenza viral RNA also codes for two non-structural
proteins (NS1 and NS2) which are produced in infected
cells, but are not incorporated into infectious virions.
Three types of influenza virus, Type A, Type B and
Type C, infect humans. Type A viruses have been
responsible for the majority of human epidemics in modern
history, although there are also sporadic outbreaks of
Type B infections. Known swine, equine and fowl viruses
have mostly been Type A, although Type C viruses have
also been isolated from swine.
Within a virus, genetic variation in the surface
proteins HA and NA has resulted in three important
subtypes, designated HlN1, H2N2 and H3N2. Within Type A,
subtypes Hl ("swine flu"), H2 ("asian flu") and H3 ("Hong
Kong flu") are predominant in human infections.
Influenza viruses continually undergo genetic change
in their surface glycoproteins which affects antigenic
variation. This is most pronounced within the A virus
type, where major genetic changes in the HA or NA
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21~ G S ~
WO94/19013 PCT~4/00~ -
proteins have already occurred ("antigenic shifts"). The
emergence of these new virus subtypes have caused a
pandemic spread of infection resulting in significant
mortality and morbidity. For example, the HlN1 viruses,
prevalent before 1957, were replaced by the H2N2 virus
subtype which remained predominant until 1968, when they
were, in turn, replaced by the H3N2 subtype. Currently,
H3N2 strains are still circulating, but since 1977, HlN1
viruses have re-emerged. The ~As within a given subtype
also undergo smaller genetic changes (point mutations)
every year or two (nantigenic driftn). These are largely
restricted to antigenic determinants clustered around the
sialic acid binding site in the HA1 and result in the
emergence of new virus strains. Although this antigenic
drift does not cause serious mortality and morbidity to
the extent caused by antigenic shift, it is responsible
for yearly influenza epidemics.
Influenza vaccines are classified into three types,
whole-virion, split, and subunit. Whole-virion vaccines,
based on intact viral particles, although generally more
immunogenic, tend to be more reactogenic and are
therefore being replaced by split and subunit vaccines
which are prepared from purified viral components
obtained after disruption of the virus by treatment with
various chemical agents. The distinction between split
and subunit vaccines resides in the fact that subunit
vaccine contain almost exclusively haemagglutinin and
neuraminidase, the surface antigens of the virus, whereas
split vaccines contain in addition variable amounts of
internal components of the virus such as the
ribonucleoprotein and the matrix protein.
Currently available commercial influenza vaccines
are based on the principle that antibody to HA or NA
confers protection. They consist of non-adjuvanted,
inactivated, whole, or split virus products utilizing
virus grown in embryonated hen's eggs. All influenza
vaccines currently contain preparations from HlN1, H3N2,
and type B virus strains. Due to the annual antigenic
variation, specific virus strains are updated on a yearly
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WO94/19013 2 ~ 5 6 5 2 ~ PCT~4/oo~
basis according to WHO recommendations, which are based
on epidemiological surveillance of prelevent circulating
virus strains.
There is no "universal" influenza virus vaccine,
i.e., a non-strain specific vaccine. Recently, attempts
have been made to prepare such universal, or semi-
universal, vaccines from reassortant viruses prepared by
crossing different strains. More recently, such attempts
have involved recombinant DNA techniques focusing
primarily on the HA protein.
Influenza vaccines are under utilized for a variety
of reasons including doubts about efficacy, fear of side
effects, need for annual revaccination, and lack of
interest among providers. Present vaccines have
demonstrated efficacy ranging from approximately 60-80%
against infection with influenza viruses that are
antigenically closely related to the virus strains used
in the vaccine. This rate of protective efficacy tends
to decrease when the HA antigen of the epidemic strain HA
"drifted" away from the vaccine stràin and would fall to
zero if a "shift" in subtype occurs. In addition,
protection appears to be ~;m;n;shed in some
immunocompromised groups, such as the elderly living in
nursing homes.
Thus the major drawbacks of currently available
vaccines result from the fact that frequent antigenic
drift dictates that the component virus strains be
changed annually and individuals must undergo
revaccination.
There remains a need in the art for vaccine
formulations and compositions capable of inducing
protective responses in animals for a wide variety of
pathogens.
.,
Summ~ry of the Invent;on
In one aspect, the present invention provides a
vaccine composition capable of stimulating an enhanced
immune and protective response in a vaccinated animal
against influenza, the composition comprising a selected
-- 3 --
0 94/~ 2 a PCT~4/004~ -
influenza antigen or antigenic polypeptide and an
effective amount of 3-o-deacylated monopl~osphoryl lipid A
(3D-MPL).
In another aspect, the invention provides a vaccine
composition comprising a selected influenza antigen or
antigenic polypeptide, an effective amount of 3D-MPL and
a liposome preparation. The liposome preparation is
defined herein and, in addition to acting as a carrier,
acts as an adjuvant and offers significant manufacturing
and formulation advantages.
In a further aspect, the invention provides a method
for enhancing a vaccinee's immune response to a selected
influenza antigen. This method involves administering to
a m~mm~l, preferably a human, a vaccine composition
described above.
Other aspects and advantages of the present
invention are described further in the following detailed
description of the preferred embodiments thereof.
Rrief Descr;ption of the Dr~w;ngs
Fig. 1 is a bar graph illustrating cross-protection
for HlN1 and H2N2 subtype influenza viruses in mice
immunized with Flu D protein (SK&F 106160) in aluminum
plus 3D-MPL, as described in Example 18.
Fig. 2A is a bar graph illustrating splenic
proliferative responses pre-challenge in mice vaccinated
with flu D formulations and controls. See Example 20.
Fig. 2B is a bar graph illustrating splenic
proliferative responses post-challenge in mice vaccinated
with flu D formulations and controls. See Example 20.
Fig. 3A is a bar graph illustrating lymph node
proliferative responses obtained on day 4 in mice
vaccinated with 20 ~g flu D in aluminum (open bars) or
aluminum and 3D-MæL (cross-hatched bars). See Example
21.
Fig. 3B is a bar graph illustrating lymph node
proliferative responses obtained on day 4 in mice
vaccinated with 5 ~g flu D in aluminum (open bars) or
aluminum and 3D-MPL (cross-hatched bars). See Example
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WO94/19013 2 1~ G 5 2 ~ PCT~4/00~
21.
Fig. 3C is a bar graph illustrating lymph node
proliferative responses obtained on day 4 in mice
vaccinated with 1 ~g flu D in aluminum (open bars) or
aluminum and 3D-MPL (cross-hatched bars). See Example
21.
Fig. 4A is a bar graph illustrating proliferation on
day 2 by immune lymph nodes in mice immunized with 1 ~g D
protein vaccine formulation. See Example 21.
Fig. 4B is a bar graph illustrating proliferation on
day 3 by immune lymph nodes in mice immunized with 1 ~g D
protein vaccine formulation. See Example 21.
Fig. 4C is a bar graph illustrating proliferation on
day 4 by immune lymph nodes in mice immunized with 1 ~g D
protein vaccine formulation. See Example 21.
Fig. 4D is a bar graph illustrating IL-2 production
on day 2 by immune lymph nodes in mice immunized with 1
~g D protein vaccine formulation. See Example 21.
Fig. 4E is a bar graph illustrating IL-2 production
on day 3 by immune lymph nodes in mice immunized with 1
~g D protein vaccine formulation. See Example 21.
Fig. 4F is a bar graph illustrating IL-2 production
on day 4 by immune lymph nodes in mice immunized with 1
~g D protein vaccine formulation. See Example 21.
Fig. 5A is a graph demonstrating interferon levels
in antigen-stimulated cultures from mice immunized as
described in Example 24 below.
Fig. 5B is a graph demonstrating IL-2 levels in
antigen-stimulated cultures obtained from mice immunized
as described in Example 24 below.
Fig. 6A is graph showing virus titers determined in
the nose by MDCK microassay on days 1, 3, 5, 7 and 9
post-challenge (5 mice per group) for a control
containing alum and 3D-MPL (--square--), influenza
monovalent split vaccine containing A/PR/8 strain with no
adjuvant (--triangle--), and a strain A/PR/8 adjuvanted
with 3D-MPL (solid line and circle). See Example 28.
Fig. 6B is graph showing virus titers determined in
the nose by MDCK microassay on days 1, 3, 5, 7 and 9
2 ~ 2 ~
WO94/19013 PCT~4/004~ -
post-challenge (5 mice per group) for a control
containing alum and 3D-MæL (--square--), influenza
monovalent split vaccine~containing Singapore strain with
no adjuvant (--trian;glë--), and the Singapore strain
adjuvanted with 3D-MæL (solid line and circle). See
Example 28.
Fig. 6C is graph showing virus titers determined in
the trachea by MDCK microassay on days l, 3, 5, 7 and 9
post-challenge (5 mice per group) for the same three
vaccine formulations as in Fig. 6A.
Fig. 6D is graph showing virus titers determined in
the trachea by MDCK microassay on days l, 3, 5, 7 and 9
post-challenge (5 mice per group) for the same three
vaccine formulations as in Fig. 6B.
Fig. 6E is graph showing virus titers determined in
the lung by MDCK microassay on days l, 3, 5, 7 and 9
post-challenge (5 mice per group) for the same three
vaccine formulations as in Fig. 6A.
Fig. 6F is graph showing virus titers determined in
the lung by MDCK microassay on days l, 3, 5, 7 and 9
post-challenge (5 mice per group) for the same three
vaccine formulations as in Fig. 6B.
Det~;le~ Descr;pt;on of the Invent;on
The present invention provides vaccine compositions
capable of eliciting an enhanced immune response in
vaccinated hosts, including humans, as well as methods
for preparing and using such vaccine compositions. A
vaccine composition of this invention is characterized by
containing an effective amount of a selected influenza
antigen or antigenic polypeptide and 3-o-deacylated
monophosphoryl lipid A (3D-MPL). Optionally, a liposome
preparation may also be a component of the vaccine
compositions of this invention.
The inventors have discovered that the combination
of 3D-MPL and certain influenza antigens are effective in
achieving protective responses against influenza, which
are not achieved by the influenza antigen alone. For
example, with the antigenic polypeptide known as Flu D,
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WO94/19013 21~ 6 3 2 5 PCT~4/004~
described below, this response is such that a lower
amount of antigen is required to obtain the same results
as are achieved with purified Flu D and Complete Freunds
Adjuvant (CFA), a known strong adjuvant which is toxic to
animals.
Further, when the selected influenza antigen and 3D-
MPL are entrapped within a liposome as described herein,
a protective response which exceeds that achieved by the
antigen and any other adjuvant combination is obtained.
By the term "enhanced immune response" as used
herein, is meant that the vaccinated host produces a
stronger cellular immune response (protective T
lymphocyte production) to the vaccine composition of the
invention than is or would be produced by the host in
response to the selected antigen when not adjuvanted, or
when adjuvanted with other conventional adjuvants
suitable for internal administration. An increased
antibody (B cell) response is also anticipated by this
enhanced response.
By the term "immunologically effective amount" or
"effective amount" as used herein is meant that amount of
antigen which induces a protective immune response.
By the terms "selected antigen", "antigenic
polypeptide or protein" or "immunogen" as used herein is
meant a whole or inactivated pathogen, an immunogenic
protein, peptide or fragment from the pathogen, which is
optionally fused to another peptide or protein which is
of homologous or heterologous origin. These terms also
include a split virus, defined below. These terms may
also include non-proteinaceous biological materials from
the pathogen. The pathogens are preferably disease-
causing organisms which infect humans, although animal
pathogens may also be employed in these vaccines, where
desired for veterinary purposes. These terms refer to
the ability of the whole pathogen, split virus, peptide
or fusion protein to elicit a protective immune response
in a vaccinated host.
By the term "monovalent vaccine" is meant a vaccine
containing antigens from a single type or subtype of
-- 7
WO94119013 215 ~ ~ 2 ~ PCT~4/00~ -
influenza virus, e.g., HlNl, H2N2, H3N2 of Type A, Type B
and Type C.
By the term "multivalent vaccine" is meant a vaccine
containing antigens from;more than a single type or
subtype of influenza virus~, i.e., a trivalent vaccine may
contain antigens from any three influenza types or
subtypes, e.g., HlNl, H2N2, H3N2 of Type A, Type B and
Type C.
By the term "split virus~ is meant an influenza
virus suspension, obtained from embryonated hens' eggs
inoculated with seed lot material, which is partially
purified and concentrated. The concentrated virus
suspension is treated with a detergent, such as sodium-
desoxylcholate, to disrupt the virus particles. Removal
of viral phospholipids during this splitting process
produces an inactivated influenza antigen for which the
reactogenicity potential is greatly reduced. The split
virus suspension is completely inactivated by the
combined effect of detergent and formaldehyde.
The following disclosure of the compositions and
methods of this invention specifically describes vaccine
compositions for prophylactic use against influenza
virus.
In one preferred embodiment of this invention, a
vaccine composition capable of eliciting an enhanced
immune response protective against infection with
influenza virus contains at least a selected influenza
antigenic polypeptide, such as NSl1_p1HA26s_222 (referred to
herein as Flu D, the D protein or the Flu D protein),
adjuvanted with 3D-MPL.
Currently, flu D protein is one preferred influenza
antigenic polypeptide for use in the vaccine compositions
of this invention because it is the most easily purified
of the influenza fusion proteins which contain the entire
carboxy-terminal region of HA2 portion of the
hemagglutinin region. D protein comprises the first 8l
amino acids of NSl fused to amino acid 65 of the
truncated HA2 subunit (amino acids 65-222). Optionally,
as is the case with the other NSl-HA2 fusions proteins
-- 8
~1.56~2~
WO94/19013 PCT~4/00
disclosed herein, a linker sequence may be inserted
between the two fused sequences.
In a presently preferred embodiment, the DNA coding
sequence for flu D protein is prepared by as described in
EP 0366238 by restricting the HA2 coding sequence with
PvuII and ligating the C-terminal region of the NcoI site
between amino acids 81 and 82 in the NS1 coding sequence
via a synthetic oligonucleotide linker. This linker
sequence codes for glutamine-isoleucine-proline. D
protein, for which a 90-95% purity has been achieved,
requires application of the purification methods
described herein to substantially remove the host cell
(E. coli) proteins and other contaminants.
The flu D protein and the recombinant expression and
purification thereof are disclosed in detail in co-
pending U. S. Patent Application SN 07/751,899 and in its
corresponding European Patent Application No. 366,238,
published May 2, 1990 and U. S. Patent Application Sn
07/387,558 and in European Patent Application No.
366,239, published May 2, 1990. These applications are
incorporated by reference for the purpose of describing
this protein, its expression and purification.
Other suitable influenza antigenic polypeptides in
addition to flu D, may be used in the vaccine
compositions of this invention including those described
in European Patent Applications 366,238 and EP 366,239,
both published May 2, 1990 and in co-pending U. S. Patent
Applications SN 07/751,898, 07/751,896 and 07/837,773.
Such proteins include ~M, ~M+, A, C, C13, C13 short, and
AD. Other suitable include Cys-less D, HA266222, and
NSlH3HA2 constructs, such as those described in co-
pending U.S. Patent Applications Ser. No. 07/751,898,
07/751,896, and 07/837,773 and WO 93/15763 incorporated
by reference herein. Particularly desirable are the
H3HA2 constructs referenced above. Recent studies by the
inventors have shown that mice immunized with a
"cocktail" containing both NS1-HlHA2 and NSl-H3HA2 fusion
proteins were protected from lethal challenge with both
Hl and H3-virus subtypes.
g _
WO94/19013 2 ~ 5 6 3 2 ~ PCT~4/oo~ ~
.-. ~, i..
Coding sequences for the HA2, NS1 and other viral
proteins of influenza virus can be prepared synthetically
or can be derived from viral RNA by known techniques, or
from available cDNA-containing plasmids as described in
the above-incorporated published European applications.
For example, in addition to the above-cited references, a
DNA coding sequence for HA from the A/Japan/305/57 strain
was cloned, sequenced and reported by Gething et al,
N~tllre, ~ 301-306 (1980); an HA coding sequence for
strain A/NT/60/68 was cloned as reported by Sleigh et al,
and by Both et al, both in Developments in Cell ~;olo~y,
Elsevier Science Publishing Co., pages 69-79 and 81-89,
tl980); an HA coding sequence for strain A/WSN/33 was
cloned as reported by Davis et al, ~.~ne, lQ:205-218
(1980); and by Hiti et al, V;rology, 111:113-124 (1981).
An HA coding sequence for fowl plague virus was cloned as
reported by Porter et al and by Emtage et al, both in
Developments in C~ll R; olo~y, cited above, at pages 39-49
and 157-168.
Systems for cloning and expressing the vaccinal
polypeptide in various microorganisms and cells,
including, for example, E. coli, Bacillus, Streptomyces,
Saccharomyces, m~mm~lian and insect cells, are known and
available from private and public laboratories and
depositories and from commercial vendors.
The D protein can be purified by the process
described below in Example 13. Various conventional
procedures can be employed in connection with the
purification of the proteins of the compositions of the
present invention, although such other procedures are not
necessary to achieve a highly purified, pharmaceutical
grade product. Such procedures can be employed between,
before or after the above described process steps. One
such optional step is diafiltration, a form of continuous
dialysis which is extremely effective in achieving many
buffer exchanges. Diafiltration is preferably carried
out across a cellulosic membrane or ultrafilter.
Suitable membranes/filters are those having from about a
1000 molecular weight (MW) cut-off to those having pore
-- 10 --
~15G~25
WO 94/19013 PCT/EP94/00448
size up to 2.4 llm diameter. A number of different
systems adaptable to diafiltration are commercially
available, such as the lOK Amicon dual spiral cartridge
system. In the process of the present invention,
diafiltration using a 20 mM Tris buffer at about pH 8 can
be effectively employed in the purification and
subsequent concentration of the polypeptide to be
employed in the vaccine composition of this invention.
In another preferred embodiment, the antigen used in
a vaccine composition of the invention is a whole
inactivated pathogen, such as a split virus, described in
detail in Example 25. A monovalent split influenza
vaccine containing a split virus or a multivalent (e.g.
trivalent) split influenza vaccine containing more than
one split virus, may also be adjuvanted with 3D-MPL. In
one formulation, the vaccine contains the split virus
prepared from an HlN1 strain, such as Singapore/6/86
[Sachsisches Serumwerkl GmbH (SSW), Dresden, German and
the National Institute for Biological Standards and
Control (NIBSC, London, England)] which is currently used
in conventional flu vaccines. Alternatively, other HlN1
split viruses may be prepared from A/PR/8/34 (also called
A/PR/8) described in T. Francis, Proc. Soc. FX~?. R; ol .
~, ;~2:1172 (1935) and available from the American Type
Culture Collection, Rockville, Maryland, USA under ATCC
No. VR-95. The monovalent vaccine contains antigens from
one strain of influenza type virus. Alternatively, the
vaccine may be multivalent, containing more than one
influenza antigen, e.g., two or three split viruses, to
increase the reactivity against more than one influenza
type virus. An example of a trivalent vaccine includes,
for example, the HlN1 Singapore/6/86 strain, with an H3N2
strain Beijing/32/92 [SSW, Dresden, Germany], and a Type
B strain, Panama/45/90 [SSW, Dresden, Germany].
Influenza viruses, which can be prepared as split
viruses by known means as described in Example 25 include
any strains, subtypes and types, particularly those
recommended by WHO, many of which are available from
clinical specimens and from public depositories, such as
-- 11 --
WO94/19013 21~ ~ $ 2 5 PCT~4/004~ ~
the American Type Culture Collecti~n, 12301 Parklawn
Drive, Rockville, Maryland, 20852, USA (ATCC) and NIBSC.
For example, other suitable H3N2 virus strains include,
without limitation, A~Vlctoria/H3/75 described in Fiers
et al, Cell, 1~: 683-696 (1980); A/Udorn described in C.
J. Lai et al, Proc~ N~tl. Ac~. Sc;., USA, 77:210-214
(1980); Palese and Schulman, V;rol., 57:227-237 (1974);
and A/HK/8/68, described in WHO Weekly Epid. Record, vol.
43:401 (1968) and available from the ATCC as No. VR-544
as well as Beijing!32/92. Other suitable Type B strains
include, without limitation, those known as B/Lee/40
described by Krystal et al, Proc. N~tl. Ac~. Sc;. USA,
79:4804-4900 (1982) and B/Taiwan, available from the ATCC
as No. VR-295, Panama/45/90, and B/Yamaghta strains.
H2N2 viruses may also be useful in these vaccines.
It should be understood that in addition to other
influenza viruses or inactivated viral preparation, other
antigenic materials from other pathogens are anticipated
to be employed in addition to the exemplified antigens
following the teachings contained herein.
Another component of a vaccine composition of this
invention is the adjuvant, 3D-MPL. 3D-MPL is described
in detail in U. S. Patent No. 4,912,094, incorporated by
reference herein, and is commercially available from RIBI
Immunochem Research Inc., Hamilton, Montana. Briefly,
3D-MPL is a derivative of the endotoxin, mono-phosphoryl
Lipid A (MPL), a Lipid A analog isolated from a
heptoseless, RE mutant of a Gram-negative bacteria, such
as Salmonella minnesota. MPL lacks a phosphate group at
the C-1 position of glucosamine. Treatment of the MPL
molecule to remove the acyl chain at position 3 of
glucosamine, yields 3D-MPL. 3D-MPL is non-toxic, in
contrast to other enterobacterial lipopolysaccharides,
but retains the antigenic activity of the parent
endotoxin. This molecule is useful in preventing Gram-
negative sepsis and endotoxemia.
3D-MPL can be dissolved in water to yield solutions
of vesicular aggregates, which are presumably composed of
lipid bilayer membranes. Thus, it is not likely that 3D-
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WO94/19013 21~ 6 5 2 5 PCT~4/004~
MæL is seen as individual molecules by the immune system,but rather interacts by membrane-membrane contacts,
involving cell surfaces and vesicle surfaces.
In a preferred embodiment, the particle size of the
MæL is 'small' and in general does not exceed 120nm.
To make 3 deacylated monophosphoryl lipid A with a
small particle size, in general not exceeding 120nm the
procedure described in GB 2 220 211 may be followed (or
commercial MPL of larger particle size may be purchased
from Ribi Immunochem.) and the product may then be
sonicated until the suspension is clear. The size of the
particles may be estimated using dynamic light scattering
as described hereinbelow.
Preferably the size of the particles is in the range
60-120nm.
Most advantageously the particle size is below
lOOnm.
According to the present invention, the inventors
have determined that an influenza antigenic polypeptide,
e.g., flu D protein, when highly purified, is neither
immunogenic nor protective in the absence of adjuvant.
However, when adjuvanted with 3D-MPL, as described herein
and illustrated in Examples 14-24 below, the protein is
capable of inducing protection against influenza
infection. When the selected antigen is a split virus in
a monovalent or trivalent composition, a composition of
the present invention demonstrates an increase in
immunogenicity and cross-reactivity.
Further, the inventors have discovered that when an
influenza antigenic polypeptide, for example, flu D
protein, is adjuvanted with 3D-MPL, a lower dose of flu D
protein is required to achieve the same level of immune
response obtained when the protein is adjuvanted with an
aluminum adjuvant only. When the selected antigen is a
split influenza virus in a monovalent or trivalent
composition, it is anticipated that reactogenicity of the
composition will be decreased by the use of less antigen
in this embodiment of the invention.
Additional adjuvants may also be included in the
- 13 -
WO94/19013 215 ~ S 2 S PCT~W4/00*~ ~
vaccine compositions of the invention. One desirable
additional adjuvant is alum, or aluminum hydroxide or
aluminum phosphate.~ When flu D protein is adjuvanted
with a combination of aluminum and 3D-MPL, a level of
potency is reached equivalent to that seen with Complete
Freunds Adjuvant (CFA), the classic adjuvant for
supporting T cell responses but which is not suitable for
internal administration in humans.
To prepare a preferred vaccine composition of this
invention containing an antigenic polypeptide, e.g., flu
D protein and 3D-MPL, a desired amount of the flu D
protein in admixed with a suitable amount of the 3D-MæL,
as described in more detail below. Optionally,
lyophilized lipid A is admixed with the pre-liposome gel
described in detail below prior to the antigen. Most
preferably, a molar ratio of phosphatides to lipid A is
66:l. However, the density of the agent may be varied to
the desired level.
Other suitable agents for addition to the vaccine
composition include, for example, IL-2, QS21 [C. R.
Kensil et al, J. I~mllnol., 146(2):431-437 (l99l)], and
muramyl dipeptides (MDP). In addition, other water
soluble or insoluble chemicals or drugs and/or adjuvants
described above can be incorporated into the vaccine
compositions of this invention. For example, muramyl
dipeptides may also be used at similar ratios as
described above or as desired. Other drugs which may
form part of this vaccine may include any substance that
when taken into the vaccinee modifies one or more of its
functions, for example as recited in an official
pharmacopeia, or a substance used in the treatment or
prevention of an infection.
The vaccine compositions of this invention may
further contain suitable carriers which are well known to
those of skill in the vaccine art and can be readily
selected. Fxemplary carriers include sterile saline,
lactose, sucrose, calcium phosphate, gelatin, dextrin,
agar, pectin, peanut oil, olive oil, sesame oil, squalene
and water. Additionally, the carrier or diluent may
- 14 -
WO94/19013 2 ~ 5 G 5 2 ~ PCT~4/004~
include a time delay material, such as glyceryl
monostearate or glyceryl distearate alone or with a wax.
Optionally, suitable chemical stabilizers may be used to
improve the stability of the pharmaceutical preparation.
5 Suitable chemical stabilizers are well known to those of
skill in the art and include, for example, citric acid
and other agents to adjust pH, chelating or sequestering
agents, and antioxidants. Alternatively, when a
liposomal delivery system is part of the vaccine
composition, no carriers are necessary.
Another preferred vaccine composition of this
invention comprises a selected antigen, e.g., the flu D
protein as described above, and 3D-MPL, which components
are entrapped or intercalated in a liposome preparation.
Optionally, the liposome, flu D protein, and 3D-MPL-
containing vaccine composition may also contain one or
more additional influenza antigens or other antigens or
desirable adjuvants and agents as described above. In
one preferred formulation, the vaccine contains the
antigenic polypeptide Flu D protein and 3D-MPL placed
into a carrier liposome. In another formulation, the
vaccine contains a mono- or multi-valent influenza
antigen similarly involved with a carrier liposome.
The inventors have discovered that the liposome
preparations described herein are capable of functioning
not only as carriers, but also as adjuvants, and are
particularly advantageous because making them does not
require organic solvents or high shear fields which is a
significant advantage for protein drugs, and all
30 ingredients for the liposomes are considered safe for
internal administration. Because of the simplicity and
flexibility of the preparative method, these liposome
preparations are suitable for large scale manufacturing.
The inventors have also discovered that 3D-MæL can
be readily incorporated into the liposomal structures
described herein in combination with influenza antigens
to obtain results superior to that found when influenza
antigens are combined with known conventional adjuvants.
Significantly, a vaccine composition containing D protein
- 15 ~
215~25
WO94/19013 PCT~4/00~ -
in a 3D-MPL and liposome formulation has even greater
potency than D protein in CFA. See, e.g~, Examples 20
and 22 below.
The liposome preparations useful in the vaccine
compositions and methods~of this invention are described
in co-pending United Stàtes Patent Application Ser. No.
07/714,984, (US 5230899) incorporated herein by
reference. Generally, the word "liposome" has been
proposed and accepted as the term to be used in the
scientific literature to describe synthetic,
oligolamellar lipid vesicles. Such vesicles are usually
comprised of one or more concentric natural or synthetic
lipid bilayers surrounding an internal aqueous phase.
Specifically as defined herein and according to the
incorporated reference, the liposome preparations useful
in the vaccine composition of the invention are prepared
by dispersing in an aqueous medium in a manner adequate
to form liposomes, a composition comprising a liposome-
forming material containing a long chain aliphatic or
aromatic-based acid or amine; a hydrating agent of charge
opposite to that of the acid or amine, which agent is
present in a molar ratio of between 1:20 and 1:0.05
relative to the acid or amine; and water in an amount up
to 300 moles relative to the solids.
The preparation of the liposomal adjuvant carriers
useful in the invention follows. Examples of liposome-
forming materials include saponifiable and non-
saponifiable lipids, e.g., the acyl glycerols, the
phosphoglycerides, the sphingolipids, the glycolipids,
etc. The fatty acids include saturated or unsaturated
alkyl (C8-C24) carboxylic acids, mono-alkyl (C8~C27) esters
of C4~ClO dicarboxylic acids (e.g., cholesterol hemi-
succinic acid and fatty acid derivatives of amino acids
in which any N-acyl carboxylic acids also are included
(e.g., N-oleoyl threonine, N-linoleoyl serine, etc.).
Mono- or di-alkyl (C8~C24) sulfonate esters and mono- or
di-alkyl (C8~C24) phosphate esters can be substituted for
the fatty acids. Furthermore, mono- or di-acyl (C8~C24)
glycerol derivatives of phosphoric acids and mono- or di-
- 16 -
~ WO94/19013 215 ~ ~ 2 S PCT~4/00~
acyl (C8~C2~) glycerol derivatives of sulfuric acids can be
used in place of the fatty acids.
Additionally, the fatty acids also can be replaced
by amines (e.g., C8~C24 NH2), C8~C24 fatty acid derivatives
of amines (e.g., C8~C24 CONH~NH2), C8~C24 fatty alcohol
derivatives of amino acids (e.g., C8~C24 OOC~NH2), and
C8~C24 fatty acid esters of amines (e.g., C8~C24 COO~NH2).
Photopolymerizable lipids and/or fatty acids (or
amines) (e.g., diacetylenic fatty acids) also can be
included, which can provide a sealed liposome with cross-
linked membrane bilayers upon photo-initiation of
polymerization.
A long chain aliphatic and/or aromatic-based acid or
amine includes an acid or amine having an open chain
structure and consisting of paraffin, olefin and
acetylene hydrocarbons and their derivatives, i.e.,
saturated and unsaturated hydrocarbons or the backbone of
such chain contains an where aromatic substituent. Such
acids and amines may have more than one such function.
The term "long chain" means that the backbone of the
aliphatic chain of the acid or amine has ten or more
carbon atoms. If the chain contains an aromatic group,
such as phenyl, the chain will comprise at least a five
carbon backbone in conjunction with that aromatic group.
The chain of carbon atoms comprising the backbone may be
variously substituted with saturated or unsaturated
aliphatic or aromatic functions.
The terms "acid" or "amine" are conventionally
defined chemical functionalities. For example, an acid
function may be a carboxylate acid, or a phosphorous or
sulfur derived acid function such as phosphate, phosphite
or pyrophosphate or sulfate, sulfite, thiosulfate, or
similarly constituted phosphorous or sulfur-based acid.
Amines must be sufficiently basic so as to have an
ionizable hydrogen or be capable of forming quaternary
salts which have an ionization constant such that they
are capable of forming the requisite hydrate complex.
As used in the vaccine composition and relative to
the amount of water employed in the liposome composition,
- 17 -
WO94/19013 2 ~ 5 6 ~ 2 5 PCT~4/004~
the term "solids~ f~rsSto the liposome-forming
materials and the acid or amine component, hydrating
agents, the 3D-MPL, alum, and the selected antigenic
material to be encapsulated.
For the purpose of this invention, a hydrating agent
means a compound having at least two ionizable groups,
preferably of opposite charge, one of which is capable of
forming an easily dissociative ionic salt, which salt can
complex with the ionic functionality of the acid or amine
in the liposome-forming material. The hydrating agent
inherently does not form liposomes in and of itself.
Such an agent will also be physiologically acceptable,
i.e., it will not have any untoward or deleterious
physiological effect on the host to which it is
administered in the context of its use in this invention.
The preferred hydrating agents are alpha amino acids
having an ionizable omega substitution such as a
carboxylate, amino, and guanidino function and those
compounds represented by the formula:
X-(CH2)n-Y
wherein
X is H2N-C(NH)-NH-, H2N-, Z03S-~ Z2O3P-, or ZO2C-
wherein Z is H or an inorganic or organic cation;
Y is -CH(NH2)-CO2H, -NH2, -NH-C(NH)-NH2-COOH,
CH(NH2) S03z or ZH(NH2)PO3Z2 wherein Z is defined above; and
n is the integer l-10; or
a pharmaceutically acceptable salt thereof.
Also included in the list of preferred compounds are the
N,N'-dialkyl substituted arginine compounds and similar
compounds where the alkyl chain length is varied.
More preferred hydrating agents are the omega-
substituted, alpha amino acids such as arginine, its N-
acyl derivatives, homoarginine, gamma-aminobutyric acid,
asparagine, lysine, ornithine, glutamic acid, aspartic
acid or a compound represented by the following formulas:
H2NC(NH)-NH-(CH2)n-CH(NH2)COOH II
H2N-(CH2)n-CH(NH2)COOH III
H2N-(CH2)n-NH2 IV
H2NC(NH)-NH-(CH2)n-NH-CH(NH)-NH2 V
- 18 -
WO94/19013 2 ~ 5 ~ ~ 2 5 PCT~4/004~
HOOC-(CH2)n-CH(NH2)COOH VI
HOOC-(CH2)n-COOH VII
HO3S-(CH2)n-CH(NH2)COOH VIII
H2o3s-(cH2)n-cH(NH2)cooH IX
HO3S-(CH2)n-cH(NH2)sO3H X
H2O3S~(CH2)n~CH(NH2)PO3H2 XI
wherein n is 2-4.
The most preferred compounds are arginine,
homoarginine, gamma-aminobutyric acid, lysine, ornithine,
glutamic acid or aspartic acid. The hydrating agents of
this invention may be used alone or as a mixture. No
limitation is implied or intended in the use of mixtures
of these hydrating materials.
The hydrating agents of this invention are listed in
the catalogue of many chemical producers, can be custom
manufactured by such producers, or can be made by means
known in the art. Arginine, homoarginine, lysine,
glutamic acid, aspartic acid, and other naturally
occurring amino acids may be obtained by the hydrolysis
of protein and separation of the individual amino acids
or from bacterial sources.
The compounds of formula II can be made by the
method of Eisele, K. et al, Justusliehi~s. Ann. Chem., p.
2033 (1975). Further information on several
representative examples of these compounds is available
through their respective Chemical Abstracts Service
numbers as follows: nonarginine, CAS X14191-90-3;
arginine, CAS #74-79-3; and homoarginine, CAS #151-86-5.
For representative examples of formula III, see for 2,4-
diaminobutonic acid CAS #305-62-4 and for lysine CAS #56-
87-1. Methods for making representative compounds of
formula IV are available from Chemical Abstracts as
follows: ethane diamine, CAS ~305-62-4; propane diamine -
54618-94-9; and 1,4-diaminobutane, CAS ~52165-57-8. See
specifically Johnson, T.B., J. Am. Chem. Soc., 38:1854
(1916).
Of the compounds of formula VI, glutamic acid is
well known in the art and is available from many
commercial sources. Descriptions of how to make other
-- 19 --
21~ 32~
WO94/19013 PCT~4/00
representative compounds is contained in the literature,
for example: 2-aminohexandioc acid - CAS # 62787-49-9 and
2-aminoheptandioc acid - CAS # 32224-51-0. Glutamic
acid, the compound of formula VII where n is 2 is well
known in the art and can be~made by the method of Maryel
and Tuley, Org. Syn., ~:69 (1925). Other representative
compounds in this group can be made according to the art
as referenced by the following CAS numbers: hexadioic
acid, CAS # 123-04-9 and heptadioic acid, CAS # 111-16-0.
Homocysteic acid is known in the art referenced by CAS #
56892-03-6. The compound 3-sulfovaline is described in
the literature referenced by CAS ~23405-34-2.
Mixtures of the hydrating agent, liposome-forming
materials and discrete amounts of water form a gel-like
mass. When in this gel form, the hydrating agent and
acid or amine, in conjunction with all liposome
forming materials, arrange into a "hydrate complex" which
is a highly ordered liquid crystal. Hydrate complex
means the complex formed between the hydrating agent and
the acid or amine in the liposome-forming material.
Complexing in this context denotes the formation of
dissociative ionic salts where one functionality
associates with the ionic functionality of the liposome-
forming material and the other functionality has
hydrophilic properties which impart water-solubility to
the resulting complex. While the liquid crystal
structure of the hydrate complex varies with pH and
amount of hydrating agent, the liquid crystal structure
remains. NMR spectroscopy confirms that this crystal
structure consists of multilamellar lipid bilayers and
hydrophilic layers stacked together in alternating
fashion. The 3lP-NMR spectrum exhibits an anisotropic
peak, confirming the existence of multilamellar bilayers.
Although the primary components of these liposomes
will be lipids, phospholipids, other fatty acids, there
may also be added various other components to modify the
liposomes' permea~ility. There may be added, for
example, non-ionic lipid components such as polyoxy
alcohol compounds, polyglyceral compounds or esters of
- 20 -
WO94/19013 ~ 5 6 5 2 S PCT~4/004~
polyoles, polyoxyalcolinolated alcohols; the esters ofpolyoles and synthetic lipolipids, such as cerebrosides.
Other materials, such as long chain alcohols and diols,
sterols, long chain amines and their quaternary ammonium
. ~ 5 derivatives; polyoxyethylenated fatty amines, esters of
long chain amino alcohols and their salts and quaternary
ammonium derivatives; phosphoric esters of fatty
alcohols, polypeptides and proteins.
The composition of the liposome can be made of more than
one component of the various kinds of lipids, the fatty
acids, alkyl amines, or the like, and the hydrating
agents.
If the lipid component itself or antigenic material
and any other materials to be added to the vaccine
composition to be encapsulated possess the aforementioned
properties, the lipid composition may not require the
inclusion of the fatty acids (or the amines) or the
hydrating agents to form the "pre-liposome gel~ or
liposomes. For example, the mixture of
dipalmitoylphosphatidylcholine (DPPC) and distearoyl
phosphatidylethanolamine forms the "pre-liposome gel" or
liposomes with aqueous glutamic acid solution and the
mixture of DPPC and oleic acid with aqueous epinephrine
solution forms the "pre-liposome gel" and liposomes.
For use in the vaccine composition as an adjuvanting
material, the liposome preparation preferably includes
phospholipids, oleic acid (or phosphatidyl-ethanolamine)
and arginine or lysine (or glutamic acid and/or aspartic
acid).
About l:20 molar ratio of hydrating agent relative
to the liposome-forming material will provide the
salutory effects of this invention with an upper limit of
about l:0.05. The preferred concentration range for the
hydrating agent is between a l:2 to l:0.5 molar ratio of
the hydrating relative to the liposome-forming material.
As a practical matter, thus a matter of preference,
if liposomes are prepared with a long chain aliphatic or
aromatic-based acid, it is preferred to use hydrating
agents which contain at least one ionizable nitrogen,
- 21 -
2~5~ ~2~
WO94/19013 PCT~4/00~ -
,s -
such as arginine, homoarginine, lysine, ornithine, and
the like. Conversely, if the amphipatic materials used
to form the liposomes contain a long chain aliphatic or
aromatic-based amine, it is preferred to use a di-acid
such as glutamic acid, aspartic acid; any of the alkyl
di-acids such as the simple di-acids such as valeric
acid, caprylic, caproic, capric or the like; or those di-
acids having two phosphate, or sulfate functionalities;
or those di-acids having mixed -COOH/-SO3H or -COOH/-PO3H2
functions.
Certain aliphatic and aromatic-based acids and
amines are preferred in the practice of this invention.
Such compounds can have multiple functions such as having
two or more acid or amine groups or combinations thereof.
For example, one could use a di-acid, a di-amine or a
compound having an acid and an amine function. The
preferred compounds are those with one or two acid or
amine functions. More preferred are the fatty mono-acids
of 10-20 carbons, saturated and unsaturated. Most
preferred are the alkyl and alkenyl acids of 10 to 20
carbon atoms, particularly oleic acid.
Mixtures of liposome-forming materials, a long chain
aliphatic or aromatic-based acid or amine, one or more
hydrating agents with up to 300 moles of water relative
to the total solids, with or without a selected amount of
antigenic material, produces a gel which forms liposomes
directly therefrom upon addition of an aqueous solution.
This gel can be labelled a pre-liposome gel because its
structural characteristics are essentially those of
liposomes and, the gel has the facility for being
converted into liposomes upon dilution with an aqueous
solution. Aqueous solution in excess of about 300 molar
equivalents cause the beginning of liposome formation.
The structure of this gel is a highly ordered liquid
crystal which forms an optically clear solution. The X,
Y, and Z dimensions of the liquid crystal vary with the
concentrations of hydrating agent at constant pH as well
as with the pH of the solution. By varying the hydrating
agent concentration at constant pH or changing the pH
- 22 -
~ WO94/19013 215 ~ ~ 2 ~ PCT~4/00~
while maintaining the percentage of hydrating agent, the
size and number of lamellae structures of the lipid
bilayers of the subsequent liposome vesicles can be
controlled.
The gel structure itself can accommodate up to
approximately 300 moles of water per mole of solid
without disturbing the stability of the gel structure.
The structure of the gel as determined by proton nuclear
magnetic resonance (NMR) spectroscopy is comprised of
multilamellae lipid bilayers and hydrophilic layersstacked together in an alternating fashion. The 3lP-NMR
spectrum of the same gel exhibits an anisotropic peak
further confirming that the gel consists of a
multilamellar bilayer. This gel can be autoclaved, a
convenient means of sterilization. Furthermore, the gel
shows no discoloration and remains clear at room
temperature for at least one year after being autoclaved.
If desirable and feasible, the gel can optionally further
be sterilized by filtration through an appropriate
sterilization filter. Upon dispersion of the gel into an
aqueous solution, liposomes are efficiently and
spontaneously produced.
The pre-liposome gel, with or without the 3D-MPL and
antigenic material to be encapsulated, also can be
dehydrated (e.g. lyophilized) and the powder rehydrated
to form liposomes spontaneously, even after a long period
of storage. This capability provides the vaccine
compositions particularly useful for administering water-
sensitive antigenic materials where long term pre-use
storage is needed.
The pre-liposomal gel is prepared as follows. A
semisolid liquid crystalline gel referred to as the pre-
liposomal gel is prepared by combining three basic
ingredients: a phospholipid, a fatty acid, and a
hydrating agent in water. Depending upon the lipid
composition desired, a variety of phospholipids or
mixtures thereof ranging in gel-liquid transition
temperatures (Tm) may be employed. The fatty acid can be
chosen based on degree of saturation or chain length and
- 23 -
2156525
WO94/19013 PCT~ ~4/00~ -
is usually mixed with the phospholipid into a thick
paste. Cholesterol may be added to the lipid mix to
control the bilayer character. (Cholesterol in some
instances increases the Tm of the membrane thereby
influencing its permeability to entrapped agents).
The hydrating agent, preferably an alpha amino acid,
such as arginine, is added to the lipid mix as a solution
in water at a slow rate until a homogeneous paste is
achieved. One desirable formulation employs egg
phosphatidyl choline:oleic acid at a 1:1~2 molar ratio.
The arginine is added in water at approximately 1:1.2
phospholipid to arginine molar ratio.
The concentration of L-arginine in the aqueous phase
component of the gel governs the diameter of the stable
liposome particle which eventually forms. The size of
the particle depends on the route of administration and
whether or not it is desired to target macrophage cells.
For example, for oral administration, a liposome particle
size of between about 1 to about 5 micrometers is
desired. To target to lymphocytes, a particle size of
between about 200 to 500 nm is preferred. For a depot
effect, a liposome particle size between about 5 to about
10 micrometers is desired. Various size particles may be
easily tested and selected by one of skill in the art.
The final pre-liposomal gel can contain up to about
65 to about 70% water by weight, has the consistency of
an ointment, and has the appearance of a typical liquid
crystal when observed under a polarizing light
microscope. The pre-liposomal gel can be stored under an
inert atmosphere, or lyophilized to a dry powder for long
term storage.
In the formation of the vaccine compositions of this
invention, the same steps of liposome preparation are
followed with the addition of an immunologically
effective amount of the selected antigen, e.g., an
antigenic polypeptide, particularly the flu D protein,
and 3D-MPL, and optionally, one or more additional
immunogenic proteins, peptides or fragments from a
selected pathogen mixed with the liposome preparation.
- 24 -
~ WO94/19013 2 ~ 5 ~ ~ 2 5 PCT~4/004~
To prepare such vaccine compositions, the selected
antigen is encapsulated or intercalated within the
liposome preparation by mixing therewith.
There are two methods by which the 3D-MPL and the
. - 5 selected antigen can be incorporated into the liposome
composition to prepare a vaccine composition according to
this invention. One method involves the incorporation of
the antigen with liposomes by hydration of the pre-
liposomal gel, or the hydrated lyophilized powder, with a
solution of the antigen in an appropriate buffer. The
other method involves the physical mixing of the pre-
liposomal gel with a lyophilized preparation of the
antigen. This mixture is subsequently hydrated which
causes the spontaneous formation of liposomes.
The method selected is the one which results in the
largest level of antigen-liposome association with the
smallest loss of antigen as unassociated fraction and is
dependent upon the physico-chemical properties of the
antigen in the presence of the lipid. Generally,
however, the ratios of the components in such a vaccine
mixture are 20 mg antigenic protein: 2 mg 3D-MPL: 300 mg
liposomal gel. For instance, when the antigen is flu D,
the second method is employed because the antigen has
significant hydrophobic character and a low aqueous
solubility and, as below described, to form a flu-D
composition according to this invention, 300 mg of pre-
liposomal gel was mixed with 20 to 30 mg flu-D protein
and 0.5 to 2 mg 3D-MPL.
It is considered that one of skill in the art given
the teachings herein can readily determine the selected
amount of the antigenic material to use in the practice
of this invention.
Optionally, other agents may be added co-entrapped
with the selected antigen, e.g., flu D protein, and 3D-
MPL in a liposome composition. Suitable other agents aredescribed above.
Once the liposomes are formed in the presence of the
3D-MPL, selected antigen, and any optionally additional
components, any unassociated antigen may be removed by
- 25 -
215~5~
WO94/19013 PCT~ ~4/00
various means. Typically, the liposomes are centrifugedserially at approximateIy ~00,000 x g and the
supernatants are discarded. The final liposome pellet is
reconstituted in an acceptable liquid, for example, 5%
dextrose, normal saline, or buffered solution, for
injection at a desired lipid or antigen concentration.
Other methods known to those of skill in the art, such as
gel filtration or dialysis, may be employed for removal
of unassociated antigen.
The description of the selected antigen, 3D-MPL and
liposome formulation also encompasses the use of the
split viruses described herein in place of, or in
addition to, Flu D.
The present invention also provides a method of
enhancing an immune response, particularly a T-cell
mediated response, in a human or other m~mm~ l, to the
selected antigen in the vaccine composition. This method
involves administering to the human or other mammal a
vaccine composition of the invention containing 3D-MPL
and the selected antigen. Optionally a liposome
preparation may be part of the composition as described
above. This method is not limited to any particular
antigen exemplified herein. In a preferred embodiment,
the method is useful in eliciting an enhanced protection
effective against influenza infection. In such an
embodiment, the vaccine composition comprises an
effective amount of flu D and 3D-MPL, as described above.
In another embodiment the vaccine composition comprises
an effective amount of one or more split influenza
viruses and 3D-MPL, as described above. Other vaccines
may be prepared and administered in effective amounts in
a similar manner.
As indicated by the examples, particularly Examples
25-30, the vaccine composition employing split viruses
adjuvanted with 3D-MPL results in superior virus
clearance particularly in the lung, the stimulation of
higher neutralizing antibody titers; the occurrence of
heterosubtypic (H1N1) cross-reactivity in the absence of
neutralizing activity; and altered progression of disease
- 26 -
~ WO94/19013 21 S 6 5 2 ~ PCT~4/00~
from upper to lower respiratory track (via trachea).
The dosage and administration protocols can be
optimized in accordance with standard vaccination
practices for these vaccine compositions. Typically, the
vaccines will be administered intramuscularly, although
other routes of administration may be used, such as the
subcutaneous, colonic, oral, pulmonary, intradermal,
intraperitoneal, or intravenous routes. The route chosen
may be dictated by the type of immune response desired.
For example, a subcutaneous route may provide the
classical "depot effect" or more prolonged stimulation
than others. It is also possible that a given route of
administration may generate a stronger antibody response
than a cellular response, or vice versa. For example,
the oral route of administration may permit generation of
an enhanced IgA response that is useful for local
immunity.
Based on what is known about other polypeptide
vaccines, it is expected that a useful single dosage for
average adult humans of an influenza antigenic
polypeptide vaccine, such as the flu D protein containing
vaccine of this invention is in the range of between
about 1 to about 1000 micrograms of D protein, preferably
about 50 to about 500 micrograms protein, and most
preferably about 100 ~g, in admixture with suitable
amounts of 3D-MPL adjuvant. When these doses of flu D
protein are used, the amounts of 3D-MPL in the vaccine
composition are between about 1 to about 500 micrograms
3D-MæL/~g viral protein, more preferably about 10 to
about 50 ~g 3D-MPL/~g viral protein, and most preferably
about 50 ~g 3D-MPL.
When flu D and 3D-MPL are administered via a
liposome carrier as described herein, the preferred
amount of D protein in the vaccine composition is between
about 50 to about 500 ~g and the amount of 3D-MPL is
between about 10 to about 50 ~g. Such a 3D-MPL
adjuvanted flu D vaccine composition will contain about 1
to about 10 mg, and preferably about 3 mg, liposome
material per about 0.2 mg antigenic protein.
- 27 -
2 1 ~
WO94/19013 PCT~4/00
When the flu D and 3D-MPL vaccine formulation
optionally contains another adjuvant, such as aluminum or
aluminum hydroxide, the preferred dosage of D protein is
between about 10 to about 500 ~g protein; the preferred
amount of aluminum or aluminum hydroxide is between about
10 to about 500 ~g.
Similarly, based on wha~ is known about other split
virus vaccines, it is expected that a useful single
dosage for average adult humans of an influenza
monovalent or multivalent split virus vaccine, such as
those described herein, is about 15 micrograms of
hemagglutinin (HA) per strain, with total protein ranging
from about 80-300 ~g/ml, in admixture with suitable
amounts of 3D-MPL adjuvant. When these doses of split
virus are used, the amount of 3D-MPL in the vaccine
composition is preferably about 50 micrograms 3D-MPL per
dose. Of course, these amounts of virus protein and 3D-
MæL may be altered by one of skill in the art.
When one or more split virus and 3D-MPL are
administered via a liposome carrier as described herein,
the preferred amount of each split virus in the vaccine
composition is likely to be less than about 15 ~g
HA/strain and the amount of 3D-MPL is between about 10 to
about 50 ~g. Such a 3D-MPL adjuvanted influenza split
virus vaccine composition will contain about 1 to about
10 mg, and preferably about 3 mg, liposome material per
about 0.2 mg viral protein.
When the split virus and 3D-MPL vaccine formulation
optionally contains another adjuvant, such as aluminum or
aluminum hydroxide, the preferred dosage of each split
virus in the vaccine composition may be adjusted
downward. A preferred amount of aluminum or aluminum
hydroxide is similar to that described above for the
antigenic polypeptide.
Any of the vaccines described by this invention can
be administered (preferably in 0.5 mls dosage units)
initially in late summer or early fall and can be
readministered two to six weeks later, if desirable, or
periodically as immunity wanes, for example, every two to
- 28 -
~ WO94/19013 215 6 ~ 2 ~ PCT~4/004~
five years.
The following examples illustrate the preferred
methods for preparing vaccine compositions of the
invention. These examples are illustrative only and do
not limit the scope of the invention.
F.X~Dl e 1 - Gel Prep~r~t;on
Dipalmitoylphosphatidylcholine, 3.0 grams, was
weighed into a 50 ml beaker. Oleic acid 1.2 grams was
added and mixed together to form a uniform paste.
Arginine 0.72 grams in 30 ml of distilled deionized
water was added to the dipalmitoylphosphatidyl-
cholineoleic acid paste and heated to 45C. With mixing
by hand, the mixture formed a clear stable gel. The gel
was stored and liposomes later formed by diluting the get
with phosphate buffered saline.
F.X~ e ~ - Prep~r~t;on of T.; po~o~es
Dipalmitoylphosphatidylcholine, 120 mg, and 24 mg of
oleic acid were added together and mixed thoroughly until
a white homogeneous paste was observed.
Then 20 mg of arginine was dissolved into 60 ml of
phosphate buffered saline (ionic strength = 0.15, pH =
7.4). The arginine-saline solution was added to the
paste and heated to 40C for ~ hour, or until a slightly
turbid solution was observed.
F~ le 3 - T~rge Sc~le Gel ~n~ T;poso~e Prep~r~t;on
i) Gel M~nuf~ctllre: To 50 grams of egg phosphatide
powder type 20 (Asahi Chemicals) was added 20 grams of
oleic acid N.F. Mixing gave a white paste which was
cooled to 4C and ground into a fine powder. This powder
was added to an aqueous solution containing 20 grams of
arginine and 500 grams of distilled deionized water. The
mixture was mixed with a spatula as the solution was
heated to about 35C to help hydrate phospholipids. A
homogeneous, slightly yellow get was formed. This gel
can be autoclaved and stored at 4C or can be frozen and
later reconstituted.
- 29 -
WO94/19013 2 1 ~ 6 ~ 2 3 PCT~4/00~ ~
ii) M~nllf~ctl]re of T~iPosomes: The gel prepared in the
preceding paragraph was taken from cold storage and
returned to room temperature. It was then mixed with 2
liters of phosphate buffered saline, pH 7.4. A white
opaque liposome solution was formed.
F.X~pl e 4 - I;poso~e For~tion from the Gel
A homogenous paste of 1.0 gram of
dipalmitoylphosphatidycholine (DPPC) and 400 mg of oleic
10 acid was formed. Then 300 mg arginine was mixed in 10 mL
of phosphate buffered saline, heated to 45C and added to
the DPPC/oleic acid paste to form liposomes.
F.X~pl e 5 - Pre-T;poso~e Gel
One gram of dipalmitoylphosphatidylcholine (DPPL)
was mixed with 400 mg of oleic acid to form a homogeneous
paste. 300 mg of arginine was mixed with 2 ml of water
at 45C until dissolved. The arginine solution was mixed
with the DPPC/oleic acid paste at about 45C to give a
thick gel. Liposomes formed when this gel was diluted
with phosphate buffered saline.
F.~mpl e 6 - V~r;ous Tiposo~e Form~ tions
A. Cholesterol Cont~;n;ng Tiposo~es
Cholesterol, 15 mg, was mixed with 100 mg
dipalmitoylphosphatidylcholine (DPPC) to form a
homogeneous powder. Then 23 mg of oleic acid was added
to the powder and thoroughly mixed to form a homogeneous
paste. To make liposomes, 30 mg of arginine was added to
10 ml of phosphate buffered saline, heated to 40C and
added to the DPPC/cholesterol/oleic acid paste. The
combination was mixed at 40C to obtain liposomes.
B. p~lmitlc Ac;~-Cont~;n;ng T.iposo~es
Dipalmitoylphosphatidylcholine (DPPC) 250 mg was
mixed with 25 mg of palmitic acid to form a uniform
powder. Then 80 mg of oleic acid was mixed with this
powder and heated to 45C with constant stirring until a
uniform paste was formed. Arginine 100 mg was dissolved
in 25 ml of distilled deionized water and heated to 45C.
- 30 -
WO94/19013 215 6 ~ 2 ~ PCT~4/004~
This arginine solution was added to the paste at 45C and
mixed until a uniform homogeneous gel was formed. The
gel was diluted ten fold with phosphate buffered saline
to form liposomes.
C. Isoste~r;c Ac;~-Cont~;n;n~ T;poso~es
Dipalmitoylphosphatidylcholine lO0 mg was mixed with
50 mg of isostearic acid to form a uniform homogeneous
paste. An arginine solution of 50 mg of arginine in 2.0
ml of distilled deionized water was made and added to the
isostearic acid paste and heated to 45C. This mixture
was mixed until a clear gel was formed. Liposomes are
formed upon dilution with phosphate-buffered saline.
D. O1eoyl Threonine Cont~;n;ng Tiposomes
Dipalmitoylphosphatidylcholine 125 mg and 75 mg of
oleoyl threonine were added together and heated to 40C to
form a paste. Then 2 ml of distilled deionized water was
added with constant mixing at 40C.
A clear gel was formed which can be diluted with
phosphate buffer saline at pH 5 to form liposomes.
E. ~yr;styl ~m; ne cont~;n;ng T.; posomes
Dipalmitoylphosphatidylcholine 192 mg was added to
72 mg of myristyl amine and heated with constant mixing
until a uniform paste was formed. Glutamic acid 65 mg in
5 ml of distilled deionized water was added to the paste
and heated until a gel was formed. Phosphate buffered
saline was added to the get to form liposomes.
F. DTPC Cont~;n;ng T.; posomes
Dilaurylphosphatidylcholine (DLPC) 50 mg was mixed
with 20 mg oleic acid to form a homogeneous paste.
Arginine 20 mg was added to lO ml of phosphate buffered
saline, added to the paste and hand mixed until a turbid
liposome solution formed.
G. Phosph~ti~y1eth~nol~m;ne-Glut~m;c Aci~ Tiposomes
L-glutamic acid 32 mg was dissolved in 2.0 ml of
distilled deionized water and the pH adjusted to 5.2 with
l.0 N sodium hydroxide. This solution was heated to 60C,
and lO0 mg of phosphatidylethanolamine added. The
solution was kept to 60C with constant mixing until a
uniform viscous gel was observed.
- 31 -
2~6~2~
WO94119013 PCT~4/00
The phosphatidylethanolamine-glutamic acid gel was
diluted l/l0 by phosphate ~uffered saline. Vesicular-
like structures are observed under phase contrast light
microscopy.
Fx~Dle 7 - ~ff~ct of Arg;n;ne Concentr~t;on on T;poso~e
To 502 mg of dipalmitoylphosphatidylcholine (DPPC)
was added l0 microliters of (2-palmitoyl-l-C14) (0.l
mCi/ml) dipalmitoylphosphatidylcholine. Chloroform was
added to effect complete mixing of the radioactivity
and then evaporated. Oleic acid (OA), 195 mg, was then
mixed into the lipid to form a paste. Five ml of
distilled water containing ll9 mg of arginine was added
and mixed at 45C to form a clear gel.
One gram of the gel was weighed into four different
vials and arginine was added as indicated in the
following Table l.
T~hle l
Sample Composition
Sample ID DPPC:OA:Arg
Vial l + l ml water (l:l:l)
25 Vial 2 + l ml of
50 mg/ml Arg in H2O ~l:l:3)
Vial 3 + l ml of
84 mg/ml Arg in H2O (l:l:5)
Vial 4 + l ml of
192 mg/ml Arg in H2O (l:l:l0)
One-half gram of each solu~ion was diluted in 50 ml
of phosphate buffered saline of pH 7.8.
The estimated average diameter was obtained from a
Sephracyl S-l000 column chromatographic analysis
employing 14C-isotope labelled DPPC (i.e. diameters were
determined based on intensity of scattering using photon
correlation spectroscopy (PCS)). The effects are given
- 32 -
WO94/19013 21~ ~ 5 2 5 PCT~4/00~
in the following Table 2.
T~hle ~
Fffects of ~rg;n;ne Concentr~t;on on Ves;cle S;ze
Estimated
System Ratio pH Diameter (nm)
DPPC:OA:Arg 1:1:1 7.8 -220
DPPC:OA:Arg 1:1:3 7.8 -140
DPPC:OA:Arg 1:1:5 7.8 -90
DPPC:OA:Arg 1:1:10 7.8 ~20
Fx~ple 8 - pH Fffect on Ves;cle S;7e
Additionally, the vesicle size can be varied by
varying the pH of the aqueous buffer solution.
To 100 mg of dipalmitoylphosphatidylcholine (DPPC)
was added 25 microliters of (2-palmitoyl-1-Cl4) (0.1
mCitml) dipalmitoylphosphatidylcholine. Chloroform was
added to effect complete mixing of the radioactivity and
then evaporated. Oleic acid (OA), 40.1 mg, was then
mixed into the lipid to form a paste. One ml of a
solution containing 24 mg/ml arginine in water was added
to the lipid mixture and mixed at 45C to form a clear
gel.
Two 100 mg aliquots of this gel were diluted in 10
ml of phosphate buffer at pH 9.0 and 7.4 respectively.
Again, the estimated average diameter was obtained
from the Sephracyl S-1000 column chromatographic analysis
employing l4C-isotope labelled dipalmitoyl-
phosphatidylcholine. Results are given in the following
Table 3.
WO94/19013 2 ~ ~ 6 ~ ~ ~ PCT~4/00~ ~
T~hle 3
pH ~ffects OA Ves;cle S;7e
~ - Estimated
~ystem R~t;o ~ pH D;~meter (nm)
DPPC:OA:Arg 1:1:1 7.4 ~<300
DPPC:OA:Arg 1:1:1 7.8 -220
DPPC:OA:Arg 1:1:1 9.0 ~25.4
Thus, a desired size of the liposomal vesicles can
be prepared by varying the arginine concentration or the
pH of the aqueous buffer solution.
Fx~ple 9 - Tiposo~e St~h;l;ty
Sterile liposomes may be prepared from the heat
sterilized pre-liposome gel. Alternatively, the liposome
gel or the liposomes may be sterile filtered through an
appropriate sterilizing filter.
Liposomes prepared from DPPC:OA:Arg ~1:1:2) at pH
8.0 were heat sterilized and stored at room temperature
for approximately one year without adding antimicrobial
agents and anti-oxidants. No bacterial growth,
discoloration and precipitation were observed. Negative
stain electron microscopic examination of the one year
old liposomes revealed that the liposomal vesicles are
stable.
F.x~m~l e 10 - Sucrose T~tency
Encapsulated sucrose latency was measured using Clq~
sucrose encapsulated with the DPPC:OA:Arg (1:1:1)
liposome system in aqueous phosphate buffer solution at
pH 7.8. The result was presented in the following Table
4.
34 -
~ WO94/19013 21~ G 5 2 ~ PCT~4100~
T~hle 4
Days % Sucrose Latency
-5 0 100
1 97.4
3 93.4
7 91.4
Thus, the present liposome system has an excellent
latency for drug delivery.
F.x~m~le 11 - T~oph;l;zeA T.;posomes
Oleic acid, 30.0 gm, and 7.5 gm of cholesterol
U.S.P. were confected. Then 75.0 gm of phosphatide type
20 powder (Asahi Chemical Co.) was mixed with the oleic
acid/cholesterol mixture until an homogeneous paste was
formed.
Then 15.0 gm of arginine (free base) was dissolved
in 183 gm of distilled, deionized water. This arginine
solution was mixed slowly with the lipid paste to form a
homogeneous gel. The gel pH was adjusted to 7.4 using
5.0 N HC1.
A 10.0 gm, aliquot of this pre-liposome gel was
transferred to a 10 ml vial and lyophilized. The
resulting powder formed liposomes when diluted with 5 ml
of phosphate buffered saline.
F.x~m~le 1~ - Constrllctlon of Flll D F.xpress;on Pl~sm;~.~
Plasmid pAPR701 is a pBR322-derived cloning vector
which carries coding regions for the Ml and M2 influenza
virus proteins (A/PR/8/34). It is described by Young et
al, in The Or;g;n of P~nAem;c Influenz~ V;rl~ses, 1983,
edited by W. G. Laver, Elsevier Science Publishing Co.
Plasmid pAPR801 is a pBR322-derived cloning vector
which carries the NS1 coding region (A/PR/8/34). It is
described by Young et al, cited above.
Plasmid pAS1 is a pBR322-derived expression vector
- 35 -
WO94/19013 215 6 a 2 a PCT~4/00~ ~
which contains the PL promoter, an N utilization site (torelieve transcriptional polarity effects in the presence
of N protein) and the cII ribosome binding site including
the cII translation initiation codon followed immediately
by a BamHI site. It is described by Rosenberg et al,
Metho~ F.n7~ol ., 101:123-138 (1983).
Plasmid pASldeltaEH was prépared by deleting a non-
essential EcoRI-HindIII region of pBR322 origin from
pAS1. A 1236 base pair BamHI fragment of pAPR801,
containing the NS1 coding region in 861 base pairs of
viral origin and 375 base pairs of pBR322 origin, was
inserted into the BamHI site of pASldeltaEH. The
resulting plasmid, pASldeltaEH/801 expresses authentic
NSl (230 amino acids). This plasmid has an NcoI site
between the codons for amino acids 81 and 82 and an NruI
site 3' to the NS sequences. The BamHI site between
amino acids 1 and 2 is retained.
Plasmid pASl~EH/801 was cut with BglII, end-filled
with DNA polymerase I (DNApolI; Klenow) and ligated
closed, thus eliminating the BglII site. The resulting
plasmid pBgl~ was digested with NcoI, end-filled with
DNApolI (Klenow) and ligated to a BglII linker. The
resulting plasmid, pB4, contains a BglII site within the
NS1 coding region. Plasmid pB4 was digested with BglII
and ligated to a synthetic DNA linker as described in
Example 4 of EP 0366238.
The resulting plasmid, pB4+, permits insertion of
DNA fragments within the linker following the coding
region for the first 81 amino acids of NSl followed by
termination codons in all three reading frames. Plasmid
pB4+ was digested with XmaI (cuts within linker), end-
filled (Klenow) and ligated to a 520 base pair
PvuII/HindIII, end-filled fragment derived from the HA2
coding region. The resulting plasmid, pD, codes for a
protein comprised of the first 81 amino acids of NSl,
three amino acids derived from the synthetic DNA linker
(Gln-Ile-Pro), followed by amino acids 65-222 of the HA2,
as shown in Fig. 2 of published European Patent
Application No. 366,238.
- 36 -
21~6a25
WO94119013 PCT~W4/004
To facilitate plasmid selection in production
fermentations, a BamHI fragment derived from the pD
expression plasmid encoding the recombinant flu D
protein, was ligated into the BamHI site of a pAS1
plasmid derivative containing a kanamycin resistance gene
from Tn903 for selection [Berg et al, ~;croh;ology, ed.
D. Schlessinger, pp. 13-15, American Society for
Microbiology (Washington, DC 1978); Nomura et al, The
S;ngle-Str~n~e~ DN~ Ph~es, ed. D. Denhardt et al,
pp.467-472, Cold Spring Harbor Laboratory (New York
1978); Castellazzi et al, Moleclll. Gen. G~net., 117:211-
218 (1982)]. This results in the vector, pCl3(H6s-222)Kn
As described in Shatzman and Rosenberg, Meth.
Fnzy~ol., 152:661-673 (1987), pOTS207 is a pAS derived
cloning vector which carries the kanamycin resistance
gene from Tn903 [Berg, cited above; Nomura, cited above;
Castellazzi, cited above]. It was constructed by
digesting plasmid pUC8 ~Yanisch-Perron et al, Ç~n~,
33:103-119 (1985)], with BamHI and ligated to a BclI
fragment containing the kanamycin gene from Tn903. The
resulting plasmid, pUC8-Kan, was digested with EcoRI and
PstI, and the fragment containing the kanamycin gene was
inserted between the EcoRI and PstI sites of pOTSV
[Shatzman and Rosenberg, cited above]. The resulting
plasmid is pOTS207.
A 520 bp fragment encoding the HA2 coding sequence
was isolated from pJZ102 [a pBR322-derived cloning vector
which carries a coding region for the entire HA protein
(A/PR/8/34) (described by Young et al, The Or;g;n of
30 P~n~ml c Influenz~ V;rllses, ed. W.G. Laver, Elsevier
Science Publishing Co. (1983) ] and inserted into pB4+
which had been cut with XmaI and end filled. The
resulting plasmid, pCl3H65_222 was digested with BamH1 and
the fragment encoding the flu D protein isolated from
35 this fragment was then ligated into the BamH1 site of
pOTS207 to produce the plasmid pCl3(H6s222)Kn [SmithKline
Beecham].
The sequence of the kanamycin resistant C13(H65-
222)Kn plasmid was derived as follows: nucleotide
- 37 -
W094/19013 21~ ~ ~ 2 a PCT~4/004~ ~
positions 1-31, 3136-3964, 4021-4343, 4533-7166 were
derived from pBR322 [Young et al, cited above];
nucleotide positions 32-1879, 4344-4532 were derived from
~ phage, nucleotide positions 1880-2122, 2682-3135 were
derived from the NS1 gene, nucleotide positions 2123-
2132, 2660-2681 were derived from a synthetic linker,
nucleotide positions 2133-2659 were derived from the HA2
gene, nucleotide positions 3965-4020 were derived from
the pCV1 polylinker, and nucleotide positions 7167-8601
were derived from the pUCKanl2 (Tn903:Knr). The DNA
sequence of the coding region for the flu D protein
derivative was confirmed by the dideoxy chain termination
method of sequencing DNA [Sanger et al, Proc. N~tl. Ac~.
Sci. USA, 74:5463-5467 (1977)].
This Cl3(H6s222)Kn plasmid is essentially the same as
the plasmids which are described in co-pending U. S.
Patent Application Serial No. 07/387,200, its
corresponding published EPA No. 366,238, and co-pending
U. S. Patent Application Serial No. 07/387,558, its
corresponding published EPA No. 366,239, with the
exception that the ~-lactam marker has been removed and
replaced with a Kanamycin marker. The above applications
are incorporated for reference for their description of
other appropriate vectors.
The pC13(H6s222)Kn plasmid was transformed into E. coli
expression strain AR58 [SmithKline Beecham]; and
production of the flu D protein was confirmed by
immunoblot analysis [Towbin et al, Proc. N~tl. Ac~. Sci.
r~sA~ 76:4350 (1979)] which revealed a major
immunoreactive species at the predicted molecular weight
of 27.7 kD.
F.~le 13 - Pllrific~t;on of D Prote;n
The NSll_8l-HA26s_222 or D protein (MW 27.7 kD), was
purified as described in detail in published European
Patent Application N0. 366,239, corresponding to U.S.
Patent Application Ser. No. 07/387,558.
Following the induction of synthesis of D protein by
any E. coli host strain AR58 [SmithKline Beecham] using
- 38 -
WO94/19013 21~ 6 ~ 2 ~ PCT~4/00~
the pC13~H65z22)Kn system described in Example 12, the
bacterial cells were collected by centrifugation and
frozen at -70C until used. This pellet was thawed and
resuspended in 50 mM Tris, 2 mM EDTA, 0.1 mM
5 dithiothreitol (DTT), 5~ glycerol, at pH 8. The
resulting suspension was treated with lysozyme (final
< concentration of at least about 0.2 mg/ml) for about 1
hour under ambient conditions.
This suspension was then lysed on a Manton Gaulin
10 homogenizer [APV Gaulin, Inc., Everett, Massachusetts] at
8,000 psi in two passes. The resulting suspension was
treated with Triton X-100 (1% final concentration) and
deoxycholate (DOC; 0.1% final concentration). The pellet
containing D protein was suspended in 50 mM Gly-NaOH
buffer containing 2 mM EDTA and 5% glycerol at pH 10.5
using a Turrax homogenizer. The resulting suspension
after addition of Triton X-100 (1% final concentration)
was stirred for about 1 hour in a 4C cold room, then
centrifuged. The pellet containing D protein was
dissolved in 8 M urea, 50 mM Tris at pH 8.0, overnight at
4C. The supernatant contained the D protein.
This supernatant, after the addition of DTT (to 50
mM final concentration), was stirred under ambient
conditions for about 1 hour then loaded onto a DEAE Fast
Flow Sepharose [Pharmacia] column equilibrated with 8M
urea and 50 mM Tris buffer at pH 8. D protein was eluted
with a 0 to 0.3 M NaCl gradient (over five column volumes
or greater) in equilibration buffer.
Fractions containing D protein were concentrated on
a Minisette tangential flow apparatus [Pharmacia],
treated with 10 mg SDS per mg of protein, DTT [Sigma] (to
a final concentration of 50 mM) and stirred at ambient
temperature for about 1.5 hour.
The resulting solution was loaded onto a Superose-12
column [Pharmacia], equilibrated with 25 mM Tris-Glycine
buffer at pH 8,6, containing 1% SDS. The D protein was
eluted at its predicted monomer MW in an isocratic
gradient of equilibration buffer. Fractions containing D
protein of adequate purity were pooled, concentrated,
- 39 -
WO94/19013 215 6 5 2 ~ PCT~4/004~ ~
treated with 10 mg SDS per mg of protein and DTT (to 50
mM final concentration) and then chromatographed on a
Superose-12 column under the same conditions described
immediately above.
Fractions containing D protein of the highest purity
were pooled, concentrated and loaded onto a G25 Sephadex
fine column [Pharmacia], equilibrated with 50 mM Tris
buffer at pH 8.0 containing 8 M urea. D protein was
eluted isocratically with equilibration buffer and
fractions containing D protein free of SDS were pooled
and concentrated. This highly purified sample of D
protein was then concentrated, dialyzed against 20 mM
Tris, 1 mM EDTA, pH 8.0 and sterile filtered.
Fx~m~le 14 - Fv~lu~t;on of Flu D V~cclne Co~os;tions _
Detailed descriptions of the D protein, and methods
used for ln v;tro T cell assays and protection studies
are described in S. B. Dillon et al, V~cc; ne, lQ:309
(1992), incorporated by reference herein.
CTL and proliferation assays were performed as
described previously [S. B. Dillon et al, cited above].
IL-2 was measured on an IL-2 dependent CTL line (CTLL)
and IFN~ was measured by a commercial ELISA kit.
Vaccine compositions according to this invention
containing superose purified D protein (Example 13) in
aluminum hydroxide adjuvant, with or without 3D-MPL, were
prepared as follows: 3D-MPL (RIBI Immunochemical,
Hamilton, MT) was reconstituted in sterile water for
injection to a working concentration of 1 mg/ml. This
stock solution was sonicated for 30 minutes, and
subsequent dilutions, made in 5% dextrose, were sonicated
for an additional 10 minutes prior to addition to
mixtures of the D protein in aluminum hydroxide, prepared
as described previously [Dillon et al, V~cc;ne,
10(5):309-318 (1992)].
The antigen doses were titrated from 0.01, 0.1, 1.0,
10, 20, to 100 ~g/dose of injectioni and the 3D-MæL doses
were titrated from 0.025, 0.25, 2.5, 25, to 50 ~g/dose of
injection. The ratio of 3D-MPL:antigen was maintained at
- 40 -
WO94/19013 2 ~ 2 ~ PCT~4/00448
2.5:1 (w/w) for all antigen doses except the 100 ~g dose,
as shown in Table 5 below. Aluminum hydroxide was 100 ~g
per dose in all cases. A control vaccine composition
contained 20 ~g antigen mixed with CFA, as described in
Dillon et al, V~cc;ne, 1~(5):309-318 (1992). The final
injection volumes were 0.2 ml per mouse.
For these protection studies, mice (CB6Fl; H-2~)
were injected subcutaneously with a selected vaccine
dose, at weeks 0 and 3, and challenged intranasally
(under metofane anesthesia) at week 7 with 2 to 5 LDso
doses of A/Puerto Rico/8/34 [A/PR/8/34 (influenza type A,
HlNl)] virus.
Detailed descriptions of the methods used for
protection studies are described in S. Dillon ~ ~l,
V~cc;ne, 10(5): 309-318 (1992), incorporated by reference
herein. Percent survival at day 21 post-challenge was
compared between groups by the Fischers exact probability
test.
- 41 -
WO94/19013 21~ ~ ~ 2 ~ PCT~4/00~ -
T~hle 5
~t;~en Dose (U~) 3D-~PT. Dose (U~ %Sl~rviv~l (D~y ~1)
100 50 27
100 0 53+
86+~++
0 40
67+
0 40
1.0 2.5 80+~++
1.0 0 7
0.1 0.25 20
0.1 0 20
0.01 0.025 20
0.01 0 13
0.0 50 13
0.0 0 0
+ p < 0.003 vs adjuvant control.
p < 0.01 vs adjuvant control.
++ p < 0.01 vs aluminum formulation at same antigen dose.
The results of Table 5 show that the minimum dose of
flu D antigen required for significant protection from
lethal challenge was reduced from 10 ~g (with aluminum
only) to 1 ~g when 3D-MPL was included in the vaccine
composition. Also, the percentage of mice surviving
challenge was increased when the 20 ~g (p < 0.01), 10 ~g
(P 2 0.05; NS) and 1 ~g (p < 0.01) antigen doses were
administered with 3D-MPL as well as aluminum vs. aluminum
only in the vaccine composition. Survival in a CFA
control group at 20 ~g antigen was 73% in this
- 42 -
WO94/19013 21~ ~ ~ 2 5 PCT~4/00~
experiment.
F.x~Dle 15 - F.v~ t;on of Fl-l D V~ccine Co~os;t;ons
This experiment was performed essentially as
described in Example 14 above except that the dose of 3D-
MæL was titrated and administered either alone, with the
minimal effective dose of flu D antigen (l ~g) and the
aluminum adjuvant (lO0 ~g), or with flu D antigen (lO~g)
and the aluminum adjuvant (lO0 ~g). The results reported
in Table 6 identify 2 ~g 3D-MPL as the minimal effective
dose.
T~hle 6
~ntigen Dose (~g) 3D-MPT. Dose (~g) % Sl~rv;v~l (D~y 71)
80+
2.0 73+
0.4 53+
0.0 53~
l lO 80+~++
l 2.0 87+~++
l 0.4 33
l 0.0 33
0 10 0
0 2.0 0
0 0.4 0
0 0.0 7
+ p < O.OOl vs adjuvant control.
p < 0.02 vs adjuvant control.
++ p < O.Ol vs aluminum formulation at same antigen dose.
F.x~ple 16 - F~.v~ t;on of Flu D V~ccine Co~pos;t;ons
In this experiment, the potency of liposomes plus
3D-MPL was compared to liposomes (Lipo) without 3D-M2L
- 43 -
WO94/19013 215 8 ~ 2 5 PCT~4/oo~ ~
[Ribi Immunochem], and to Al(OH), or CFA, by determining
the level of protection seen after challenge with 2, 10
or 50 LDso doses of virus. The vaccine used was the same
as in Table 9 below.
Mice were injected at weeks 0 and 3 and challenged
with A/PR/8/34 at week 7. Survival;is shown in Table 7
below through day 21.
.-
T~hle 7
% Survival
Dose Ch~llenge Dose (TnsoL
~ntigen (~g) A~ v~nt ~ 10 ~Q
D protein 50 Lipo 53 53b 7
0 Lipo 27 0 0
D protein 50 Lipo/3D-MPLa loob 73b,c 47b,c
O Lipo/3D-MPL 13 0 0
20 D protein 50 Al3+ 8 6b 20 13
0 Al3+ 7 13 0
D protein 50 CFA 86b 30b,c 13
- 0 CFA 33 0 0
a The amount of 3D-MPL per liposome was not determined.
b p < 0.003 vs. adjuvant control.
c p < 0.003 vs. aluminum hydroxide formulation at same
antigen dose.
The results showed that significant protection
against a 50 LDso challenge was achieved with the
liposome/3D-MPL formulation but not with any of the other
ad~uvant formulations tested (Table 7). ~ignificant
protection was achieved against the 2 LDso challenge with
all formulations, and all formulations except aluminum
were protective against the 10 LDso challenge dose.
Therefore, the D protein in the 3D-MPL/liposome
formulation constitutes a vaccine with even greater
- 44 -
WO94/19013 ~15 6 ~ 2 ~ PCT~4/00~
potency than the D protein in CFA, which is the classic
adjuvant for supporting T cell response, but is not
suitable for use in humans.
Fx~ple 17 - Fv~ t;on of Flll D V~cc;ne Com~os;t;ons
In this experiment, a vaccine composition of the
invention containing flu D protein, 3D-MPL, and liposomes
was evaluated in comparison to a vaccine composition
containing flu D, 3D-MPL and aluminum adjuvant. The
actual amount of 3D-MPL incorporated into liposomes was
measured.
The flu D protein (superose purified) in 25 mM
Tris/1 mM EDTA, pH 8, at a total protein concentration of
2.49 mg/mL was dialyzed against 20 mM ammonium
bicarbonate pH 8 and lyophilized to a powder in 20 mg
aliquots.
Egg phosphatides (Asahi Type 5) and oleic acid
(Sigma) were admixed at approximately a 1:1.2 molar
ratio. L-arginine (free base), 0.38 M in water was added
(1:1 molar ratio phosphatide:arginine) to the
phosphatide/fatty acid mixture and mixed until a smooth
and homogeneous gel is formed. This gel was the pre-
liposomal gel.
The lyophilized protein was combined with the pre-
liposomal gel (0.06-0.08 protein/gel (w/w) ratio) and
mixed at room temperature until a homogeneous paste is
formed. Monophosphoryl lipid A (Ribi/Immunochem) was
added at a ratio of 1 ~mole of lipid A to 66 ~mole of
phosphatides. This was a transparent colorless system
composed of nanoparticulate (submicron in diameter)
structures of aggregated 3D-MPL monomers. The resulting
liposome suspension was further diluted with 25 mM Tris/1
mM EDTA, pH 8.0 buffer and homogenized. The liposome
preparation was then serially centrifuged (3x) to remove
unincorporated protein and 3D-MæL. Each time the pellet
was resuspended with the Tris/EDTA buffer. The final
liposome suspension was analyzed and kept stored under N2
at 5C while not in use.
The liposomes were subsequently assayed to determine
- 45 -
WO94/19013 2 ~ 2 ~ PCT~4/004~ ~
protein content by the modified Lowery colorimetric
assay, the phospholipid concentration by the Barlett
assay tBartlett, G. R.j J. ~iol. Chem., 2~:466-468
(1959)] and liposome~-size by photon correlation
spectroscopy tMalvern Model 4700]. All assays were
performed by standard techniques. Liposomes were not
analyzed for lipid A content, however, it is likely that
all of the lipid A remains in the final liposome
fraction.
In the following Tab]e 8, D protein LA and control
LA-I was prepared by incorporating solid lipid A into the
lipid phase. Control LA-II was prepared by hydrating
with a solution of lipid A for incorporation.
Table 8
Liposome Phospholipid Protein 3D-MPL Diameter Z-
Sample (~mole/mL) (mg/mL) (~g/mL) Avg Mean
(nm)
Control 4.37 not not 200.8
detected determined
25 Control+ not
3D-MPL 7.87 0.02 determined 201.3
not
Flu D 21.8 4.89 detected 487.0
FluD+
3D-MPL 14.8 4.06 270 502.4
-
Mice were immunized s.c. with 0.2 ml of the
antigen/carrier/3D-MPL formulation at weeks 0 and 3, and
challenged intranasally on week 7 with 5 LDso of A/PR/8/34
virus. The "carrier" was either aluminum hydroxide (Al)
or liposomes (Lipo). Survival was monitored for 21 da~s.
These results are illustrated in Table 9 below. Unless
- 46 -
21~6~25
WO94/19013 PCT~4/00~
otherwise indicated, the p Value is measure vs. a 3D-MPL
control.
T~l e 9
5 Antigen Dose %
nose C~rr;er 3D-~nT ~urv;v~l p V~lue
1.0 ~g Al - 33 0.084
1.0 ~g 2.5 ~g 80 0.000/0.001
0.1 ~g A1 - 33 0.084
0.1 ~g 2.5 ~g 93 0.000/0.001
0 ~g Al 2.5 ~g 7
1.0 ~g Lipo - 73 0.001
1.0 ~g 0.05 ~g80 0.000
0.1 ~g Lipo - 46 0.054
20 0.1 ~g 0.005 ~g 87 0.000/0.020
0 ~g Lipo 0.05 ~g13
a p value vs. homologous dose without 3D-MPL.
This data indicates that by using liposomes as a
carrier, the required dose of 3D-MPL (0.005 - O.05 ~g)
can be dramatically reduced vs. the amount required with
aluminum (about 2 ~g). (See also Table 6 above).
Fx~ple 18 - Fv~ tion of V~cc;ne Co~po~;t;on
CB6Fl mice (12 per group) were injected sc at weeks 0
and 3 with a vaccine composition comprising 100 ~g flu D
in aluminum (100 ~g) plus 3D-MæL (10 ~g). At week 7, the
mice were challenged with 3-5 LDso doses of the viruses
shown in Fig. 1. Survival was monitored for 21 days
post-challenge.
The results of Fig. 1 demonstrate that the antigenic
specificity of the vaccine was equivalent to that
- 47 -
WO94/19013 215 ~ S ~ S PCT~4/00~ -
demonstrated earlier with the A1~3 adjuvant [S. B. Dillon
et al, cited above] (i.e., cross-protective for both H1
and H2 subtypes, but lack of efficacy for H3 or Type B).
Survival was also greater in the vaccinated group
challenged with the heterologous HlNl virus, A/Tai/86,
although the results were not significant, p < 0.09. (See
Fig. 1).
Fx~ple 19 - ~v~ t;on of Flll D V~c;ne Co~os;t;on
CB6Fl mice were injected subcutaneously (sc) with a
vaccine composition comprising 1 ~g D protein in aluminum
(100 ~g) plus 3D-MPL (2.5 ~g) or with aluminum only at 0
and 3 weeks. Eighteen mice were challenged at week 7
with 5 LDso doses of A/PR/8/34 virus. Five mice were
sacrificed at day 7 post-challenge for lung titers.
(Death in control groups at day 7 was 60~ for Al/3D-MPL
and 40% for CFA). Spleens were removed from 2 mice per
group pre-challenge and 3 mice per group at day 6 post-
challenge for proliferation and cytokine assays (Table 15
below). Table 10 reports survival (n=10/group) and lung
virus clearance (n=5/group) after 5 LDso challenge. Lung
virus titer was recorded on day 7 in the fourth column of
Table 10.
The results in Table 10 show that the reduction in
lung virus titers in mice given a 5 LDso challenge was of
greater magnitude with 3D-MPL vs. CFA (2.4 log10 vs. 0.9
log10), although survival was equivalent in these groups.
- 48 -
~ WO94/19013 21~ 6 ~ 2 5 PCT~4/004~
T~hle 10
________________________________________________________
Ant;~en (~g) A~juv~nt Sl~rv;v~l logl0 TcID5r/lllng
1.0 Al/3D-MPL86% 4.41 + 0.83+
0 Al/3D-MPL 0$ 6.80 + 0.45
1.0 Al 33% 6.39 + 0.41+
0 Al 0% 7.39 + 0.41
1.0 CFA 86% 6.00 + 0.50++
0 CFA 26% 6.90 + 0.72
p < 0.001 vs. adjuvant control group.
~ p < 0.01 vs. adjuvant control group.
+ p < 0.003 vs. adjuvant control group.
++ p < 0.03 vs. adjuvant control group.
Fx~ple ~0 - Fv~ tion of Flu D V~ccine Co~os;t;on
To determine if splenic T cell proliferation, ~IFN
production were correlated with protection, these
responses were monitored in the spleens of mice immunized
with 1 ~g of D protein in Al vs Al/3D-MPL (2.5 ng 3D-MPL)
adjuvants (from the study shown in Table 10) pre- and
post-challenge. At week 7, two mice were sacrificed 6
days after virus challenge. Spleens were co-cultured
with D protein and pulsed with 3H-thymidine on day 3.
Culture supernatants were harvested 8 hours later.
Overall, splenic proliferative responses were
similar pre- vs. post-challenge for groups vaccinated
with either D protein in the Al or Al/3D-MPL formulation
(maximal cpm in unstimulated cultures equalled 2750 cpm)
(Fig. 3). The magnitude of the proliferative response
was also similar for either adjuvant group with the
maximum stimulation index equally 3.0 or lower (Fig. 2).
Pre-challenge gamma-interferon levels in antigen-
stimulated culture supernatants were only modestly (about
2 fold) elevated and the two adjuvant groups were
- 49 -
21 ~2~
WO94/19013 PCT~P94/004
equivalent as shown in Table 11.
However, in contrast, gamma-interferon production
was increased greater than 7 fold in antigen-stimulated
cultures from the Al/3D-MPL:group post-challenge; whereas
post-challenge levels did not rise in the group
vaccinated with Alt3 only (Table 11). These results thus
provide evidence that interferon-gamma production is
correlated with reduced lung titers and survival post-
challenge, and further show that T cell proliferation per
se does not reflect the di.fference between the two
adjuvant groups.
T~hle 11
ng/ml IFN-gamma
Injection ~g/ml Pre- Post-
Anti~en Dose A~juv~nt in vitro chl]nge chlln~e
D 1 ~g Al+3/3D-MPL 0 <0.9 <0.9
1 1.0 >7.0
1.8 >7.0
- - Al+3/3D-MPL 0 <0.9 <0.9
1 <0.9 1.0
0.94 <0.9
D 1 ~g Al 0 <0.9 <0.9
1.9 1.0
1.9 1.0
- - Al 0 <0.9 1.2
1 <0.9 1.7
1.1 1.0
- 50 -
WO94/19013 21~ 6 ~ 2 S PCT~4/004~
Fx~Dle ~l - Fv~ t;on of Flu D V~ccine Co~posit;on
To further investigate a role for CD4 cells,
antigen-specific proliferation and cytokine production
were compared in mice vaccinated with the vaccine
compositions containing flu D and aluminum adjuvant (Al)
vs. vaccine compositions containing flu D and Al/3D-MPL
formulations.
Lymph nodes from mice given a single injection of l,
5 or 20 ~g D protein in Al/3D-MPL (ratio of 3D-
MPL:antigen at 2.5:1 w/w) or Al+ adjuvant (lO0 ~g) 7 daysearlier were cultured with 0-30 ~g/ml purified D protein.
The results are illustrated in Figs. 3 and 4.
Proliferation was clearly increased in the groups that
received Al/3D-MPL adjuvant, and the difference was
greatest at the lowest in vivo antigen dose (l ~g). In
Fig. 3 maximal cpm in unstimulated cultures equalled 1368
cpm. In Fig. 4 m~X; m~l cpm in unstimulated cultures
equalled 1600 cpm; and maximal cpm of CTLL (an IL-2
dependent CTL line) cells cultured with supernatants from
unstimulated cultures equalled 195 cpm). Peak (48 hours)
I7L-2 activity was greater in the groups vaccinated with
the Al/3D-MPL formulations, although the levels were
generally low in all cultures (Fig. 4).
Table 12 provides the results of the analysis of
interferon-gamma in supernatants from the same antigen-
stimulated cultures. Interferon levels i~ supernatants
from adjuvant control cultures were all <l ng/ml.
Interferon-gamma was measured with Gibco-BRL ELISA kit.
This cytokine, which was highest at day 4 of culture, was
up to 5-fold greater in the Al/3D-MPL group.
WO94/19013 21~ 6 ~ 2 5 PCT~4/00~ ~
T~hle 12
~g antigen antigen IFN-~mm~ (ng/~l)
(in vivo) Adjuvant in vitro day 2 day 3 day 4
(~g/mL)
1.0 Al~ 0 <1 <1 <1
'1 <1 <1 <1
<1 <1 1 . 1
1.0A1/3D-MPL 0 <1 <1 <1
1 <1 <1 1.9
1.1 2.9 4.9
The results from Table 11 and 12 collectively
support a role for interferon gamma in the mechanism of
action of MPL adjuvant.
Fx~ple 22 - Fv~lu~tion of Flu D V~ccine Co~pos;t;on
Since the above examples indicate that 3D-MPL
improves potency of the vaccine, a study was done to
determine if the booster injection was necessary for
protection. Mice were vaccinated with a single sc
injection of 50 ~g D and challenged 7 weeks later with 2
LD50 A/PR/8/34. Alternatively, a second group was given a
booster injection with the same antigen dose at 3 weeks.
The 3D-MPL dose was 125 ~g. The flu D was adjuvanted
with either aluminum hydroxide, aluminum plus 3D-MPL, or
CFA. Controls for each adjuvant were performed.
The results in Table 13 below (shown in Example 23)
show that the incorporation of 3D-MPL (125 ~g) into a
vaccine formulation with 50 ~g of antigen significantly
increased survival in mice given either one or two
injections when compared to the same dose of the vaccine
protein adsorbed to aluminum. Again, survival in the
Al/MæL group was comparable to that seen in the CFA.
- 52 -
WO 94/19013 21~ 6 ~ 2 a PCT~4/004~
F.X~pl e 23 - Cytotox;c T T~ hocyte A~s~y.s
Detailed descriptions of the methods used for in
vitro T cell assays are described in S. Dillon ~
cited above. Assays to detect memory CTL were performed
after secondary in vitro stimulation with virus as
described previously [See, F. Ennis et al, T~ncet,
II:887-891 (1981); A. Yamada et al, J. FXp, Me~.,
152:663-674 (1985); and K. Kuwono et al, J. F~xD. Me~.,
169:1361-1371 (1989)]. Briefly, spleen or lymph node
cells from immune or control mice were cultured at a
ratio of 6:1 with influenza virus-infected, syngeneic
spleen cells for 5 days. Culture medium was RPMI 1640
supplemented with 10% fetal calf serum [Hyclone
Laboratories, Logan, UT], 2 mM glutamine, 5 x 10-5 M 2-ME,
10 mM HEPES, penicillin and streptomycin.
The spleens from mice given one or two sc
injections of 50 ~g D protein (0 and 3 weeks), were
removed at week seven and restimulated in vitro as
described above. One lytic unit (LU3s) is defined as the
number of effector cells in the chromium release assay
required for 35% lysis (determined by regression
analysis). Table 13 reports the results of
HlNl-specific CTL activity in these spleens of mice
immunized with SK&F 106160 (D Protein) in aluminum
hydroxide and 3D-MæL.
WO 94/19013 2 ~ 2 5 PCT/EPg4/00448
+ +
o o
.~
-- ,
V ,,,
,~ ~
a) - + +. .,
~u. ;+~aaa
0~. . .
~U~ZZZ
V
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,, r a)
~ -, h
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U~ ~ ) V V V O ~
~ h `I ~,
1~ h Q~
~ E~ ~ ~ ~ " ~, ~, a O ~
U~ ~ C~ V V V
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a~ ~ ~ ~ C~ ~ _, s
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+ ~ u ~+
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c 3
a a a ~ ~ + +~
- 54 -
WO94/19013 21~ PCT~4/00
In the experiments of this example, 3D-MPL did not
potentiate class I restricted memory CD8+ CTL relative to
the response generated with aluminum hydroxide (indicated
as Al~3) as reported in Table 13. In addition, the
results in Table 13 show that splenic CTL were comparable
in mice given 1 vs. 2 sc injections, whereas protection
t was clearly improved in mice given a second injection.
Therefore it appears that a mechanism other than
CD8+ CTL must be responsible for the improved immunity
seen when 3D-MPL is included in the vaccine composition
containing the antigenic peptide and aluminum adjuvant,
or when mice are given a booster injection.
F.X~pl e 24 - Depletion Stu~ies
To further determine which T cell subset is best
correlated with the activity of the 3D-MPL adjuvant,
depletion studies were done with anti-CD-4 or anti-CD-8
monoclonal antibodies (Mabs). An initial study was
performed using a post-vaccination protocol for T cell
subset depletion as follows. Mice (15/group) were
immunized with two subcutaneous injections of 50 ug flu D
(SK&F 106160) in Al/3D-MPL at 0 and 21 days, and
challenged with 5 LD50 doses of A/PR/8/34 on day 42.
Antibodies (300 ~g/mouse) were administered ip post-
vaccination on days 32, 33, 34, 39, 40 and 41 pre-
challenge and on days 43, 48 and 53 post-challenge. In
another study, mice were treated with Mabs to deplete T
cell subsets prior to the first injection of vaccine
(pre-vaccination protocol) on days -3, -2, -1. After the
vaccination on day 0, mice were further treated with Mabs
on days, 7, 14, 18, 19 and 20, and then boosted with
vaccine on day 21. Mice were further treated with Mabs
on days 28, 35, 39, 40 and 41 prior to virus challenge on
day 42. Mice were further treated with Mabs post-
challenge on days 43, 49 and 54. The results of these Tcell subset depletion experiments are shown in Table 14
below. The results show that the effectiveness of
vaccination was reduced when mice were depleted of either
the CD4+ or CD8+ T cell subsets, the effect was more
- 55 -
WO94/19013 2 15 6 3 2 5 PCT~4/00~ ~
pronounced when Mab treatment was initiated prior to
vaccination. Therefore, these results definitively show
that the mechanism of action of the flu D vaccine in
Al/MPL formulation is T cell mediated, and both T cell
subsets are required for activity.
Since production of gamma interferon correlated with
protection (Table 11), a study was performed to determine
the phenotype of T cell responsible for producing this
cytokine in vaccinated mice. Mice werë depleted of CD4
or CD8+ T cell subsets by injectin~ anti-CD4 or anti-CD8
Mabs ip daily for 3 days (300 ~g/injection). Four days
after the last Mab injection, mice were immunized with 20
~g flu D protein in the aluminum hydroxide (100 ~g) and
3D-MPL (20 ~g) formulation, and lymph nodes were removed
7 days later. The lymph node cells were restimulated in
vitro with 0, 1 or 10 ~g/ml D protein, and supernatants
collected on days 1-4 for interferon and IL-2 assays.
The results in Fig. 5 show that IL-2 production was
completely eliminated by anti-CD4 treatment, but was also
partially reduced in anti-CD-8 treated mice (Fig. 5B).
Peak IFN~ production (day 4 supernatants) was reduced by
approximately 50% in anti-CD4 or anti-CD8 treated mice
(Fig. 5C). Therefore, both CD4+ and CD8+ T cell subsets
produced IL-2 and IFN~.
- 56 -
WO94/19013 215 ~ ~ 2 S PCT~4100448
T~hle 14
% Survival % Survival
Antigen 3D-MPL Mab (Mabs Post- (Mabs Pre-
5(~g) (~g) Treatment Vaccination) Vaccination)
none 70 80
lO50 lO anti-CD4 30 20+
anti-CD8 50 0
anti-CD4+
anti-CD8 30 lO+
0 lO none 0 0
20p < 0.002 vs. adjuvant control (group 5)
p < 0.02 vs. adjuvant control (group 5)
+ p < O.Ol vs. untreated control (group l)
The examples presented above demonstrate that a
recombinant influenza HlNl vaccine formulated with
aluminum plus 3D-MPL facilitated virus clearance and
survival, and reduced the antigen dose required for
significant protection against lethal challenge, reaching
a level of potency equivalent to that seen with CFA. The
data collected to date provides evidence that the
mechanism by which 3D-MPL acts to potentiate the activity
of this recombinant influenza vaccine appears to be via
the selective potentiation of CD4+ T cell responses, and
may be restricted to THl-type cells which produce IL-2
and IFN~.
Fx~le ~5 - Prep~ration of S~lit Virus
Split viruses, such as those produced by Sachsisches
Serumwerkl GmbH (SSW) (Dresden, Germany), may be prepared
~ 57 -
WO94/19013 ~ ~ 5 ~ i" PCT~4/004~ -
by well known methods, such as those documented in, for
example, European Pharmacopoeia PA/PH/Exp 3 (1992); DAB
10 "Vaccines Influenzae ex virorum fragmentis
praeparatum" and the World Health Organization draft
revised requirements for Influenza vaccines (Inactivated)
(1990). Such split viruses are prepared using one or
more influenza virus strains recommended by WHO and the
EEC, such as A/Singapore/6/86, A/Beijing/32/92 and
B/Panama/45/90. These strains can change depending on
the strains popular in a particular year.
As described in more detail elsewherer influenza
viruses are obtained from embryonated hens' eggs
inoculated with seed lot material. These virus
suspensions are partially purified and concentrated. The
concentrated virus suspension is treated with a
detergent, sodium desoxylcholate, to disrupt (or "split")
the virus particles. Following the removal of viral
phospholipids during the splitting process, the
reactogenicity potential is greatly reduced. The split
virus suspension is completely inactivated by the
combined effect of the detergent and formaldehyde.
More specifically, the process for producing a split
virus of this invention is as follows.
A. Prep~r~tion of monov~lent whole viru.~ inoculllm
On the day of inoculation of embryonated eggs a
fresh inoculum is prepared by mixing the influenza
working seed lot with a phosphate buffer containing
gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25
~g/ml. The virus inoculum is kept at 2-8C.
Nine to eleven day old embryonated eggs are used for
virus replication. The eggs are incubated at the farms
before arrival at the manufacturing plant and enter into
the production rooms after decontamination of the shells.
The eggs are inoculated with 0.2 ml of the virus inoculum
on an automatic egg inoculation apparatus. The inoculum
is injected at a pressure of + O.03 MPa. The inoculated
eggs are incubated at the appropriate temperature (virus
strain-dependent) for 50 to 96 hours. At the end of the
incubation period, the embryos are killed by cooling the
- 58 -
~ WO94/19013 215 6 ~ 2 S PCT~4/00~
eggs and storage for 12-60 hours at 2-8C.
The allantoic fluid from the chilled embryonated
eggs is harvested by appropriate egg harvesting machines.
Usually, 8 to 10 ml of crude allantoic fluid can be
collected per egg. To the crude monovalent virus bulk is
add 0.100 mg/ml thiomersal.
The harvested allantoic fluid is clarified by flow
through centrifugation at a volume of 100-200 L/hour and
a centrifugal force of 10-17,000 g. This preclarified
liquid can be further clarified on a 6-8 ~m membrane
filter.
To obtain a CaHPO4 gel in the clarified virus pool,
0.5 M Na2~PO4 and 0.5 M CaCl2 solutions are added to reach
a final concentration of CaHPO4 of 1.5 g to 3.5 g
CaHPO4/liter depending on the virus strain. After
sedimentation for at least 10 hours, the supernatant is
removed and the sediment containing the Influenza virus
is resolubilized by addition of a 0.26 M EDTA solution.
The concentration of EDTA varies between 4.5 and 10
g/liter of the original harvest volume.
The resuspended sediment is filtered on a 6 to 8 ~m
filter membrane.
The Influenza virus is concentrated by isopycnic
centrifugation in a linear sucrose gradient (0-55%) at
25 90,000 g. The flow through volume is from 8-12
liters/hour. At the end of the centrifugation, the
content of the rotor is recovered by four different
fractions (the sucrose is measured in a refractometer):
- fraction 1 55-52% sucrose
- fraction 2 52-38% sucrose
- fraction 3 38-20% sucrose
- fraction 4 20-0% sucrose
For further vaccine preparation, only fractions 2
and 3 are used. Fraction 3 is diluted in order to reduce
the sucrose content to approximately 6%. The Influenza
virus present in this diluted fraction is pelleted at
53,000 g to remove soluble contaminants. The pellet is
resuspended and thoroughly mixed to obtain a homogenous
suspension. Fraction 2 and the resuspended pellet of
- 59 -
WO94/19013 21~ 6 ~ 2 ~ PCT~4/00~ ~
fraction 3 are pooled and phosphate buffer is added to
obtain a volume of 30 liters. At this stage, the product
is called ~monovalent whole virus concentrate".
B. Spl;t v;rlls ~onov~lent hlllk
The selected Influenza virus, preferably the
monovalent whole virus concentrate of Pàrt A above, is
disrupted by centrifugation at 70,000~g through a Nadoc
linear sucrose gradient of 0-55% containing a linear
distribution of sodiumdesoxycholate from 1,2-1,5%. Tween
80 is present at 0.1~ in the gradient. The virus
concentrate is pumped at the rate of 5 liters/hour. At
the end of the centrifugation, the content of the rotor
is collected in 3 different fractions:
Fraction 1: 55-40% sucrose
Fraction 2: 40-13% sucrose
Fraction 3: 13-0% sucrose
The haemagglutinin is concentrated in fraction 2.
Phosphate buffer containing thiomersal at 0.01% and Tween
80 at 0.01% is added to dilute the fraction four times (+
5 liters).
The diluted fraction 2 is filtered on filter
membranes ending with a 0.2 ~m membrane. At the end of
the filtration, the filters are washed with phosphate
buffer containing 0.01% thiomersal and 0.01% Tween 80.
As a result, the final volume of the filtered fraction 2
is 5 times the original fraction volume. Depending on
the virus strain, a brief sonication of the split virus
material can be introduced to facilitate the sterile
filtration.
The filtered monovalent split material is incubated
at 22 + 2C for at least 84 hours to allow inactivation of
viruses and mycoplasma by the effect of sodium
desoxycholate. After this incubation time, phosphate
buffer containing 0.01% thiomersal and 0.01% Tween 80 is
added in order to bring the total protein content down to
maximum of 250 ~g/ml. Formaldehyde is added at the rate
of 50 ~g/ml and the inactivation takes place at 4C + 2C
for at least 72 hours.
The inactivated split virus material is
- 60 -
~ WO94/19013 215 6 ~ ~ ~ PCT~4/00~
ultrafiltered on membranes having a mean pore size of20,000 daltons. During ultrafiltration, the content of
formaldehyde, NaDOC and saccharose is greatly reduced.
The volume remains constant during ultrafiltration
(diafiltration) by adding phosphate buffer containing
0.01% thiomersal and 0.01% Tween 80.
The ultrafiltered split material is filtered on
membranes ending with a 0.2 ~m, depending on the virus
strain the last filtration membrane can be 0.8 ~m. At
this stage, the product is called: "monovalent final
bulk". The monovalent final bulk is stored at 2-8C for a
maximum of 18 months.
Fx~ple 26 - S~l;t V;rlls V~cc;ne Co~pos~t~on
15 (a) Preparation of MPL with a particle size of 60 - 120
nm
Water for injection is injected in vials containing
lyophilised 3 deacylated monophosphoryl lipid A (MPL)
from Ribi Immunochem, Montana using a syringe to reach a
concentration of 1 to 2 mg per ml. A preliminary
suspension is obtained by mixing using a vortex. The
content of the vials is then transferred into 25 ml Corex
tubes with round bottoms (10 ml suspension per tube) and
the suspension is sonicated using a water bath sonicator.
When the suspension has become clear, the size of the
particles is estimated using dynamic light scattering
(Malvern Zetasizer 3). The treatment is continued until
the size of the MPL particles is in the range 60 - 120
nm, and preferably below 100nm.
Suspensions can in some cases be stored at 4 degrees
C without significant aggregation up to 5 months.
Isotonic NaCl (0.15M) or isotonic NaCl plus 10mM
phosphate induces a rapid aggregation (size >3-5~m).
(b) The split virus vaccine composition of this
invention is prepared as follows.
A final bulk-buffer is prepared by adding to water
for injection the concentrated salt solutions: NaCl 4 mg;
Na2HPO4 0.52 mg; KH2PO4 0.19 mg; KCl 0.1 mg; and MgCl2 0.05
- 61 -
WO94/19013 21~ ~ ~ 2 a PCT~4/004~ ~
mg, and Thiomersal 50 ~g. The resulting solution is
stirred 15 minutes before further use.
Monovalent split virus bulk (15 ~g HA) is then mixed
with 3D-MPL 50 ~g and the resulting mixture is stirred
for about l hour at room (or ambient) temperature.
The buffer mixture and;thé virus bulk mixture are
then mixed together. After 30 minutes stirring at room
temperature, the pH is brought to 7.15 + 0.1. The
resulting final vaccine is stored at +2 - +8C.
To prepare a multivalent split virus vaccine, such
as a trivalent vaccine, monovalent split virus pools of
the selected strains prepared as described above are
mixed to constitute the final multivalent vaccine. The
resulting pooled material is stirred for 30 minutes at
room temperature (pH 7.15 + 0.1). The resulting final
vaccine may be stored at temperatures between about 2C to
about 8C. The vaccine is a colourless, light opalescent
aqueous suspension of purified split infiuenza virus.
For purposes of comparison in the tests of the
following examples, a vaccine preparation was prepared
without the adjuvant. For this control vaccine, a final
monovalent bulk-buffer is prepared by adding to water for
injection the concentrated salt solutions: NaCl 4 mg;
Na2HPO4 0.52 mg; KH2PO~ 0.19 mg; KCl 0.1 mg; MgCl2 0.05 mg;
and Thiomersal 50 ~g. The resulting solution is stirred
15 minutes before further use. The mixture is stirred
for 15 minutes at room temperature. Monovalent bulk (15
~g HA) is added, followed by 30 minutes stirring at room
temperature (pH 7.15 + 0.1). The resulting final vaccine
is stored at temperatures between +2C - ~8C.
Two monovalent vaccine compositions of this
invention and monovalent control vaccines were prepared
for the following examples using the HlN' strains, A/PR/8
and Singapore mixed with 3D-MPL 50~g (pa-ticle size <
lOOnm as obtained from Example 26 (a)).
Fx~ple 27 - Teth~l Ch~llenge ;n M;ce
The immunogenicity of the monovalent vaccine
formulation of this invention has been evaluated and
- 62 -
~ WO94fl9013 215 6 ~ 2 3 ~CT~4/004~
compared to the immunogenicity of the monovalent non-
adjuvanted formula and to the adjuvant 3D-MPL without
antigen in a lethal influenza challenge in mice.
For each vaccine, CB6Fl mice (30 per group) were
immunized subcutaneously with 2 injections given 3 weeks
apart with a preparation that contains 5 ~g of indicated
strain + 5 ~g 3D-MPL. Seven weeks after the first
injection, mice were intranasally challenged under
metofane anaesthesia with 5 LD50 of A/PR~8. Survival and
clinical signs of illness were monitored for 15 mice up
to 3 weeks after challenge. Lung virus titers were
determined by MDCK microassay [A. L. Frank et al, ~
Cl;n. M;croh;ol., 12:426-432 (1980)] on the remaining
animals (5 mice per group).
The results are reported in Table 15 below.
Table 15
Mice Lethal Challenge: Survival Rate
and Lung Virus Titers
Virus titer*
Vaccine Survival
Group (%) day 3 day 5 day 7
Alum + 3D-MPL 13 8.90+0.108.44+0.55 8.69+0.32
Singapore 80 8.10+0.177.15+0.23 3.09+0.10
Singapore 100# <1 <1 <1
+ 3D-MPL
A/PR/8 100# 8.01+0.029 3.93+0.70 <1
A/PR/8 + 3D-MPL 100# <1 <1 <1
* log TCID/lung (geometric mean + S.E.)
# no visible clinical symptoms
The two strains (A/PR/8 and Singapore) induce a
certain level of protection. The survival rate for both
groups was markedly higher than that of the control
- 63 -
WO94/1901~ 15 6 3 2 a PCT~W4/00448
preparation devoid of antigen (Alum + 3D-MPL), and lung
virus clearance was faster. Not surprisingly, A/PR/8-
vaccinated group (homologous with respect to challenge)
performed better than the Singapore-vaccinated group
theterologous with respect to challenge). Furthermore,
both strains supplemented with 3D-MPL induced 100%
survival, practically no lung viral titer, and no
clinical symptoms. Clearly the ~3D-MPL adjuvanted vaccine
of the invention induced superior protection against
lower respiratory track infection and therefore serious
clinical manifestations such as viral pneumonia. From
this experiment it therefore appears that 3D-MPL
adjuvantation improves homosubtypic and heterosubtypic
activity of the split vaccine and is therefore
anticipated to provide broader protection against
antigenic shifts and drifts than unadjuvanted vaccine.
F.x~m~l e 28 - Non-Teth~l Ch~llen~e in M;ce
The protocol was the same as Example 25 above,
except that mice were not anesthesized before intranasal
challenge. Virus titration in the respiratory tract,
virus neutralization assays and scanning electron
microscopy of trachea were performed.
Viral neutralization titration against A/PR/8 virus
was carried out on days 1, 5 and 9 post-challenge (Table
16).
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WO94/19013 PCT~4/00448
Table l6
Viral Neutralization Assay
Neutralizing titer*
Vaccine Group day l day 5 day 9
Alum + 3D-MPL <l <l <l
Singapore <l <l <l
Singapore <l <l <l
+3D-MPL
A/PR/8 l.6 2.3 2.2
A/PR/8 + 3D-MPL 3.5 3.6 3.3
* mean of log10 (5 mice)
Both A/PR/8 groups produced neutralizing antibodies,
with a titer consistently higher in the presence of 3D-
MPL. In contrast, the vaccination with Singapore did notinduce neutralization antibodies, even with the addition
of 3D-MPL. This indicated that the heterosubtypic
activity observed previously with Singapore and 3D-MPL
was not due to antibody and provides strong evidence that
cell-mediated immunity was responsible.
Scanning Electron Micrographs (SEM) of trachea were
performed on samples harvested on days 3 and 5. The
tissues were evaluated for histophathological changes in
the serious and cilliated epithelial cells lining the
trachea particularly desqumation. These changes are
indicative of the severity of an ongoing influenza
infection or the recovery from infection (see Table 17
below).
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W094/19013 2 ~ 2 5 PCT~W4/00
~Table 17
., ~, .
Non Lethal Challenge in Mice: SEM of Trachea
S
Mean severity score*
Vaccine Grpday l day 5Overall Score**
l0 Alum + 3D-MPL l.7 l.0 0
Singapore 2.0 l.7 2+
Singapore+MPL 2.4 2.4 3+
A/PR/8 2.0 2.0 2+
A/PR/8+3D-MPL2.0 2.4 3+
* semi quantitative, done blind.
Regenerating: l: severe lesion
2: partial lesion
3: normal tissue
** overall score, from 0 (no protection) to
4+ (complete protection)
The results of SEM suggest that vaccination with
either A/PR/8 or Singapore strain alone gave protection;
in both cases this positive effect is enhanced by 3D-MPL
addition, thereby indicating that adjuvanted split
vaccine may alter the progression of influenza disease.
Virus titers were determined in the nose, trachea
and lungs by MDCK microassay on days l, 3, 5, 7 and 9
post-challenge (5 mice per group). Results are shown in
Figs. 6A through 6F below. Not surprisingly, nasal
titers were higher than tracheal or pulmonary titers.
However, all the treatments assayed did not result in
clear differences in nasal or tracheal titers. In
contrast, lung titers were differentiated: A~PR/8 + 3D-
MPL and Singapore + 3D-MPL titers were always lower or
equal to the values of the antigen alone, or of 3D-MPL
alone (Figs. 6A through 6F). This indicates that 3D-MPL
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WO94/19013 21~ ~ 5 2 ~ PCT~W4/00~
adjuvantation confers a better lung protection in mice.
Both sets of mice experiments (lethal and non lethal
challenge) indicate that influenza vaccination with two
subtypes of HlNl strain (Singapore or A/PR/8) and
subsequent homotypic challenge is improved by the
incorporation of 3D-MPL as an adjuvant according to this
invention.
F.x~m~l e ~9 - I~mllnogen; C; ty of the V~cc;ne
For each vaccine, CB6Fl mice (15 per group) were
immunized subcutaneously with 2 injections given 3 weeks
apart of a preparation that contains one tenth of a human
dose of conventional unadjuvanted Singapore monovalent
split vaccine or a vaccine composition of this invention
containing the Singapore strain with 3D-MPL. A human
dose is defined as a 0.5 ml injection containing 15 ~g HA
of each viral strain. The formulation used herein
contained a hemaglutinin (HA) to 3D-MæL ratio of 1.5 ~g
HA per 5 ~g 3D-MPL to 5 ~g HA per 5 ~g 3D-MPL. A control
group received only 3D-MPL (5 ~g). Three weeks after the
second injection, mice were bled and sera antibodies were
individually assayed by inhibition of haemagglutination.
Titers were calculated against a calibrated reference.
Mice were considered responders if they have antibody
titers greater than the cut-off value.
The results are reported in Table 18. Both types of
formulations induce anti-haemagglutination antibodies in
all animals, thus the seroresponse rate is maximal.
However, the immune response is significantly enhanced by
the vaccine formulation of the present invention
containing 3D-MPL, as indicated by a geometric mean titer
more than 5 times higher than that of the vaccine
containing the Singapore strain alone.
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WO94/19013 21~ 2 ~ PCT~4/00
Table 18
Vaccine Geometric
5 Group Individual titers* mean titer Seroresponse
3D-MPL <25; <25; <25; <25; <25;
(5 ~g) <25; <25; <25; <25;`<25; <25 0/15
<25; <25; <25; <25; <25;
Singapore 100; 200; 200 75; 200,
(1.5 ~g HA) 100 25 800 300 50 150 15/15
200 200 600 200 50
Singapore 1200 600 400 400 1600
ll(1.5 ~g HA)I 800 200 1600 1600 400l 828 ! 15/15 !!
¦¦+ 3D-MPL ¦ 400 3200 400 3200 1200¦
115 ~g
* cut-off value is lower than 25
This experiment indicates that antibody response in
mice is improved by the use of the 3D-MPL adjuvanted
vaccine of this invention.
Fx~le 30 - Hypersensit;v;ty Stu~y ;n Gl~;ne~ P;g
A hypersensitivity study was conducted in guinea
pigs to determine whether the addition of 3D-MPL
(particle size < 100 nm, Example 26) to a trivalent split
vaccine modifies hypersensitivity. The trivalent
vaccine, designated Trivalent Influsplit, was prepared as
described in Example 25 and contained an HlN1 strain
Singapore/6/86, an H3N2 strain Beijing/353/84 and a Type
B strain, B/Yamaghta/16/88.
The sensitizing agent [allantoic fluid 0.5 or 2.5
mg + 3D-MPL 50 ~g; one human dose (0.5 ml injection
containing 15 ~g HA for each of these strains of
influenza virus) Trivalent Influsplit + 3D-MPL 50 ~g] was
given intraperitoneally by six injections, at days 0, 3,
5, 7, 10, and 12. Guinea pigs were allowed to rest for 4
weeks and then challenged intravenously, under
anesthesia, with the challenging agent (allantoic fluid
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WO94/19013 215 ~ S 2 S PCT~P94/004~
O.45 mg; one human dose Trivalent Influsplit ~ 3D-MPL 50
~g). The animals were observed 30 minutes after
challenge, and again after 2 or 3 hours. The observed
symptoms (scratching, breathing problems, convulsions,
death) were recorded. Where no symptoms arose from the
first challenge, the animals were re-challenged with
allantoic fluid (1.3 mg) 24 hours later.
The results of this assay are shown in Tables l9A
and 19B below.
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WO 94/19013 215 6 :3 2 ~ PCT/EP94/00448
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-71 -
WO94/19013 21~ 6 S 2 S PCT~4/00448 ~
The absence of effect for groups l to 3 (negative
controls) indicates that hypersensitivity requires prior
intraperitoneal sensitization. The absence of
hypersensitization in group 5 indicates that Influsplit
cannot sensitize to allantoic fluid. A similar
conclusion can be drawn from group 6 (sensitization to
allantoic fluid and challenge with Influsplit). In fact,
results from groups 5 and 6 also~i~dicate that the
vaccine does not contain residues of allantoic fluid
proteins. The addition of 3D-MæL either to the
sensitizing agent (groups 7 and 8) or to the challenging
agent (group 9) did not change the response (compare
group 7 vs 4; group 8 vs 5; group 9 vs 6).
This experiment demonstrated that the trivalent
Influsplit vaccine given as sensitizing agent with or
without 3D-MæL is not able to induce hypersensitivity.
The trivalent Influsplit vaccine given as challenging
agent with or without 3D-MæL is not able to trigger any
hypersensivity reaction in ~n;m~ls previously
hypersensitized with allantoic fluid; and within the
experimental conditions, 3D-MPL does not have any
noticeable effect on hypersensitivity reactions. Thus,
the vaccine compositions of this invention are safe for
administration to mammals.
Numerous modifications and variations of the present
invention are included in the above-identified
specification and are expected to be obvious to one of
skill in the art. Such modifications and alterations to
the compositions and processes of the present invention
are believed to be encompassed in the scope of the claims
appended hereto.
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