Note: Descriptions are shown in the official language in which they were submitted.
WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Vaccines
The present invention relates to a novel adjuvant system comprising a
polyoxyethylene sorbitan ester surfactant in combination with an octoxynol.
The
present invention provides said novel adjuvants, vaccines comprising them, and
methods for their manufacture and for their formulation into vaccines. The use
of the
adjuvants or vaccines of the present invention in the prophylaxis or therapy
of disease
is also provided. The adjuvants are particularly useful as mucosal adjuvants,
but are
also effective systemically. The adjuvants are especially useful in the
context of
influenza vaccines.
Apart from bypassing the requirement for painful injections and the associated
negative affect on patient compliance because of "needle fear", mucosal
vaccination
is attractive since it has been shown in animals that mucosal administration
of
antigens has a greater efficiency in inducing protective responses at mucosal
surfaces,
which is the route of entry of many pathogens. In addition, it has been
suggested that
mucosal vaccination, such as intranasal vaccination, may induce mucosal
immunity
not only in the nasal mucosa, but also in distant mucosal sites such as the
genital
mucosa (Mestecky, 1987, Journal of Clinical Immunology, 7, 265-276; McGhee and
Kiyono, Infectious Agents and Disease, 1993, 2, 55-73). Despite much research
in the
field, safe and effective adjuvants which are suitable for use in humans,
remain to be
identified. The present invention provides a solution to this problem.
Medical uses of certain non-ionic surfactants have been described. For
example,
intranasal administration of polyoxyethylene sorbitan esters, polyoxyethylene
ethers,
bile salts, and other permeation enhancers, for the enhancement of insulin
uptake in
the nasal cavity has been described (Hirai et al. 1981, International Journal
of
Pharmaceutics, 9, 165-172; Hirai et al. 1981, International Journal of
Pharmaceutics,
9, 173-184).
Other non-ionic surfactant formulations have been utilised. For example,
vaccine
preparations comprising an admixture of either polyoxyethylene castor oil or
caprylic/capric acid glycerides, with polyoxyethylene sorbitan monoesters, and
an
antigen, are capable of inducing systemic immune responses after topical
WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
administration to a mucosal membrane (WO 94/17827). This patent application
discloses the combination of the non-ionic surfactant TWEEN20TM
(polyoxyethylene
sorbitan monoester) and Imwitor742TM (caprylic/capric acid glycerides), or a
combination of TWEEN20TM and polyoxyethylene castor oil is able to enhance the
systemic immune response following intranasal immunisation. Details of the
effect of
this formulation on the enhancement of the immune response towards
intranasally
administered antigens have also been described in the literature (Gizurarson
et al.
1996. Vaccine Research, 5, 69-75; Aggerbeck et al. 1997, Vaccine, 15, 307-316;
Tebbey et al.,Virallmmunol 1999;12(1):41-5). In the examples shown in WO
94/17827, (in particular example 4) the concentration of TWEEN20TM that is
required
to enhance the immune response is very high (36%), whereas at 28% even in the
presence of the caprylic/capric acid glyceride no enhancement of the immune
response occurs.
Non-ionic surfactants have also been formulated in such a way as to form non-
ionic
surfactant vesicles (commonly known as NISV; US 5,679,355). Such formulations
of
non-ionic surfactants, often in the presence of cholesterol, form lipid-
bilayer vesicles
which entrap antigen within the inner aqueous phase or within the bilayer
itself.
WO 96/36352 and US 5,653,987 describe a liquid pharmaceutical agent comprising
at
least two absorption enhancers and water, primarily for oral insulin delivery,
wherein
the amount of each absorption enhancing agent is present in a concentration of
from 1
to 10 % w/w of the total formulation.
Surfactants are commonly used in the formulation of oil emulsion adjuvants for
systemic administration, and function to stabilise the oil droplets. For
example,
polyoxyethylene sorbitan esters (TWEENTM) and sorbitan fatty acid esters
(SPANTM)
are used to stabilise oil in water emulsions (EP 0 399 843, WO 95/17210).
Influenza virus vaccines have been prepared in the past by the use of Triton X-
100 or
a mixture of Tween and ether to split influenza virus. A clinical comparison
of the
systemic immunogenicity of the two splits shows that they are comparable
(Gross et
al. 1981. J. Clin Microbiol 14, 534-8). Other surfactants have also been
investigated
for their effect on the immunogenicity of the resulting split vaccine. In a
comparative
WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
study of parenteral administration Mukhlis et al. (1984 Vaccine 2, 199-203)
showed
that whole virus was more immunogenic than detergent disrupted virus, but that
between different detergents Triton X-100 and cetyl trimethyl ammonium bromide
(CTAB) gave marginally more immunogenic splits than the detergent empigen.
The applicant presents here the surprising finding that polyoxyethylene
sorbitan esters
in combination with an octoxynol together act as a potent adjuvant for
vaccines.
Advantageously, such compositions may be administered systemically, but are
sufficient to induce systemic immune responses when administered mucosally.
The
immune responses induced by mucosal administration of vaccines of the present
invention may be at least as high as or at least comparable to those observed
after a
systemic injection of conventional vaccine.
The present invention provides safe and potent adjuvants which are easy to
manufacture, which may be administered either through mucosal or systemic
routes.
In a first aspect the invention provides an adjuvant which comprises a
polyoxyethylene sorbitan ester and an octoxynol.
In another aspect the invention provides a vaccine comprising an adjuvant
according
to the invention, together with an antigen.
Particularly preferred is a vaccine composition comprising an adjuvant
according to
the invention together with influenza virus antigen for administration to a
mucosal
surface, in particularly to the nasal mucosa. However, there are alternative
routes of
administration and other possible antigens for use in a vaccine according to
the
invention which will be described below.
Octoxynols and polyoxyethylene sorbitan esters are described in "Surfactant
systems"
Eds: Attwood and Florence ( 1983, Chapman and Hall). The octoxynol series,
including t-octylphenoxypolyethoxyethanol (TRITON X-100TM) is also described
in
Merck Index Entry 6858 (Page 1162, 12'h Edition, Merck & Co. Inc., Whitehouse
Station, N.J., USA; ISBN 0911910-12-3). The polyoxyethylene sorbitan esters,
including polyoxyethylene sorbitan monooleate (TWEEN80TM) are described in
WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Merck Index Entry 7742 (Page 1308, 12'h Edition, Merck & Co. Inc., Whitehouse
Station, N.J., USA; ISBN 0911910-12-3). Both may be manufactured using methods
described therein, or purchased from commercial sources such as Sigma Inc
Preferred octoxynols for use in the adjuvants according to the invention
include other
non-ionic surfactants from the Triton series, such as Triton X-45, Triton X-
102,
Triton X-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57, Triton N-
101
and Triton N-128, but t-octylphenoxypolyethoxyethanol (Triton X-100) is
particularly
preferred.
The adjuvants of the present invention comprise a polyoxyethylene sorbitan
ester and
an octoxynol. Preferably the octoxynol is t-octylphenoxypolyethoxyethanol
(TRITON
-X-100TM). Preferably the polyoxyethylene sorbitan ester is polyoxyethylene
sorbitan
monooleate (TWEEN80TM).
The adjuvant according to the invention may advantageously further comprise a
bile
salt or a cholic acid derivative.
Accordingly the adjuvant may comprise a polyoxyethylene sorbitan ester such as
polyoxyethylene sorbitan monooleate (Tween 80), an octoxynol such as t-
octylphenoxy polyethoxyethanol (Triton X-100) and a bile salt or cho'lic acid
derivative such as sodium deoxycholate or taurodeoxycholate. In a preferred
embodiment, the invention provides an adjuvant formulation comprising
polyoxyethylene sorbitan monooleate (Tween 80), t-octylphenoxy
polyethoxyethanol
(Triton X-100) and sodium deoxycholate.
Preferably, the total concentration of non-ionic surfactants present in the
adjuvant
formulation is less than 40%, more preferably up to about 20%. A preferred
range is
between about 0.001% to 20%, more preferably 0.01 to 10% and most preferably
up
to about 2% (w/v).
The individual non-ionic surfactants have preferred concentrations in the
final vaccine
composition as follows: octyl-or nonylphenoxy polyethoxyethanols such as
Triton X-
100 or other detergents in the Triton series: from 0.001 % to 20%, preferably
0.001
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WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
to 10%, more preferably from 0.001 to 1% and most preferably 0.005 to 0.1%
(w/v);
polyoxyethylene sorbitan esters such as Tween 80: 0.01 to 1 %, most preferably
about
0.0% (w/v).
Particularly prefered ranges for the concentrations of the non-ionic
surfactants are:
Tween 80TM: 0.01 to 1%, most preferably about 0.1% (v/v);
Triton X-100TM: 0.001 to 0.1, most preferably 0.005 to 0.02 % (w/v).
One aspect of the present invention is a vaccine formulation comprising a
polyoxyethylene sorbitan ester surfactant in combination with an octoxynol,
wherein
the antigen present in the vaccine is not entrapped within a non-ionic
surfactant
vesicle.
Inflluenza virus antigens for use in the vaccine according to the invention
may be any
form of influenza antigens suitable for raising an immune response, including
live or
inactivated whole virus, split virus, or subunit antigens prepared from whole
virus or
by recombinant means. Influenza virus for production of the antigen may be
grown in
embryonated eggs in a conventional process or the virus may be grown in tissue
culture. Suitable cell substrates for tissue culture of influenza include for
example
dog kidney cells such as MDCK cells, cells from a clone of MDCK, or MDCK-like
cells, monkey kidney cells such as AGMK cells including Vero cells, or any
other cell
type suitable for the production of influenza virus for vaccine purposes.
Suitable cell
substrates also include human cells e.g. MRC-5 cells. Suitable cell substrates
are not
limited to cell lines; for example primary cells such as chicken embryo
fibroblasts are
also included.
Preferred is an influenza virus antigen preparation which comprises split
virus which
has undergone a series of purification steps. Thus the antigen preparation may
be
produced by a number of different commercially applicable processes, for
example
the split flu process described in patent no. DD 300 833 and DD 211 444,
incorporated herein by reference. Commercially available split influenza
vaccine
includes FluarixTM which is sold by SmithKline Beecham.
VVO ~l/212~7 CA 02383413 2002-03-22 PCT/EP00/09366
Accordingly a preferred vaccine formulation according to the invention
comprises egg
or tissue culture derived influenza antigen, preferably split influenza
antigen, together
with a polyoxyethylene sorbitan ester and an octoxynol, optionally further
comprising
a bile salt or derivative of cholic acid. Most preferably such a formulation
comprises
split influenza virus antigen, polyoxyethylene sorbitan monooleate (Tween 80),
t-
octylphenoxypolyethoxyethanol (Triton X-100) and sodium deoxycholate.
The influenza vaccine according to the invention is preferably a multivalent
influenza
vaccine comprising two or more strains of influenza. Most preferably it is a
trivalent
vaccine comprising three strains. Conventional influenza vaccines comprise
three
strains of influenza, two A strains and one B strain. However, monovalent
vaccines,
which may be useful for example in a pandemic situation, are not excluded from
the
invention. A monovalent, pandemic flu vaccine will most likely contain
influenza
antigen from a single A strain.
The vaccine preparations of the present invention are preferably used to
protect or
treat a mammal susceptible to, or suffering from disease, by means of
administering
said vaccine via a mucosal route, such as the
oral/bucal/intestinal/vaginal/rectal or
nasal route. Such administration may be in a droplet, spray, or dry powdered
form.
Nebulised or aerosolised vaccine formulations also form part of this
invention.
Enteric formulations such as gastro resistant capsules and granules for oral
administration, suppositories for rectal or vaginal administration also form
part of this
invention. The present invention may also be used to enhance the
immunogenicity of
antigens applied to the skin (transdermal or transcutaneous delivery). In
addition, the
adjuvants of the present invention may be parentally delivered, for example
intramuscular, or subcutaneous administration. When used for intranasal
vaccination,
the vaccines of the present invention are preferably haemolytic in nature.
Depending on the route of administration, a variety of administration devices
may be
used. For example, for the preferred, intranasal route of administration, a
spray
device such as the commercially available AccusprayTM (Becton Dickinson) may
be
used.
W~ 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Preferred spray devices for intranasal use are devices for which the
performance of
the device is not dependent upon the pressure applied by the user. These
devices are
known as pressure threshold devices. Liquid is released from the nozzle only
when a
threshold pressure is attained. These devices make it easier to achieve a
consistent
spray with a regular droplet size. Pressure threshold devices suitable for use
with the
present invention are known in the art and are described for example in WO
91/13281, EP 311 863 B and EP 516 636 B, incorporated herein by reference.
Such
devices are commercially available from Pfeiffer GmbH.
Preferred intranasal devices produce droplets (measured using water as the
liquid) in
the range 1 to 200~m, preferably 10 to 120~m. Below 10~m there is a risk of
inhalation, therefore it is desirable to have no more than about 5% of
droplets below
lOpm. Droplets above 120~.m do not spread as well as smaller droplets, so it
is
desirable to have no more than about 5% of droplets exceeding 120~m.
Bi-dose delivery is a further preferred feature of an intranasal delivery
device for use
with the vaccines according to the invention. Bi-dose devices contain two
subdoses
of a single vaccine dose, one sub-dose for administration to each nostril.
Generally
the two sub-doses are in a single chamber and the construction of the device
allows
the efficient delivery of a single sub-dose at a time.
The invention provides in a further aspect a kit comprising an intranasal
administration device as described herein containing a vaccine formulation
according
to the invention. In a preferred embodiment of this aspect of the invention,
the
intranasal administration device is filled with an influenza vaccine.
For certain vaccine formulations, other vaccine components may be included in
the
formulation. As such the adjuvant formulations of the present invention may
also
comprise a bile acid or a derivative thereof, particular in the form of a
salt. These
include derivatives of cholic acid and salts thereof, in particular sodium
salts of cholic
acid or cholic acid derivatives. Examples of bile acids and derivatives
thereof include
cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid,
ursodeoxycholic acid, hyodeoxycholic acid and derivatives such as glyco-,
tauro-,
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WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
amidopropyl-1-propanesulfonic-, amidopropyl-2-hydroxy-1-propanesulfonic
derivatives of the aforementioned bile acids, or N,N-bis
(3Dgluconoamidopropyl)
deoxycholamide. A particularly preferred example is sodium deoxycholate
(NaDOC)
which may be present in the final vaccine dose.
Preferably, the adjuvant formulations of the present invention are
advantageous when
in the form of an aqueous solution or a suspension of non-vesicular forms.
Such
formulations are easy to manufacture reproducibly, and also to sterilise
(terminal
filtration through a 450 or 220 nm pore membrane) and are easy to administer
to the
nasal mucosa in the form of a spray without degradation of the complex
physical
structure of the adjuvant.
In a further aspect of the present invention, there is provided a method of
preparing a
vaccine which method comprises admixing an adjuvant according to the invention
with an antigen.
In a still further aspect, there is provided a method of inducing or enhancing
an
immune response in a subject, comprising admixing the antigen and the adjuvant
according to the invention, and administering said admixture to the subject
Preferably, the route of administration to the subject is via a mucosal
surface and
more preferably via the nasal mucosa. When the vaccine is administered via the
nasal
mucosa, the vaccine is preferably administered as a spray. In a preferred
method of
inducing or enhancing an immune response, a systemic response is induced by a
nasal
administration of the vaccine. Thus, a mucosal vaccine according to the
invention is
preferably capable of inducing a systemic immune response when administered
via a
mucosal route.
The present invention further provides the use of a polyoxyethylene sorbitan
ester,
and an octoxynol in the manufacture of an adjuvant formulation, in particular
an
adjuvant formulation for application to the mucosa of a patient. The present
invention
also relates to the use of a polyoxyethylene sorbitan ester, an octoxynol and
an
antigen, in the manufacture of a vaccine formulation, especially a vaccine
formulation
for application to the mucosa. Preferably the antigen is influenza virus
antigen.
8
w0 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Particularly preferred are adjuvants and vaccines for administration to the
nasal
mucosa.
Preferably the administering of a vaccine according to the invention comprises
the
administration of a priming or a boosting dose of the vaccine, such as a
priming or a
boosting dose of influenza vaccine comprising an influenza antigen
preparation.
It is foreseen that compositions of the present invention will be used to
formulate
vaccines containing antigens derived from a wide variety of sources. For
example,
antigens may include human, bacterial, or viral nucleic acid, pathogen derived
antigen
or antigenic preparations, tumour derived antigen or antigenic preparations,
host-
derived antigens, including GnRH and IgE peptides, recombinantly produced
protein
or peptides, and chimeric fusion proteins.
Preferably the vaccine formulations of the present invention contain an
antigen or
antigenic composition capable of eliciting an immune response against a human
pathogen, which antigen or antigenic composition is derived from HIV-1, (such
as tat,
nef, gp 120 or gp 160), human herpes viruses, such as gD or derivatives
thereof or
Immediate Early protein such as ICP27 from HSV 1 or HSV2, cytomegalovirus
((esp
Human)(such as gB or derivatives thereof), Rotavirus (including live-
attenuated
viruses), Epstein Barr virus (such as gp350 or derivatives thereof), Varicella
Zoster
Virus (such as gpI, II and IE63), or from a hepatitis virus such as hepatitis
B virus (for
example Hepatitis B Surface antigen or a derivative thereof), hepatitis A
virus,
hepatitis C virus and hepatitis E virus, or from other viral pathogens, such
as
paramyxoviruses: Respiratory Syncytial virus (such as F and G proteins or
derivatives
thereof), parainfluenza virus, measles virus, mumps virus, human papilloma
viruses
(for example HPV6, 11, 16, 18, ..), flaviviruses (e.g. Yellow Fever Virus,
Dengue
Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) or
Influenza virus
(whole live or inactivated virus, split influenza virus, grown in eggs or MDCK
cells,
or Vero cells or whole flu virosomes (as described by R. Gluck, Vaccine, 1992,
10,
915-920) or purified or recombinant proteins thereof, such as HA, NP, NA, or M
proteins, or combinations thereof), or derived from bacterial pathogens such
as
Neisseria spp, including N. gonorrhea and N. meningitidis (for example
capsular
polysaccharides and conjugates thereof, transferrin-binding proteins,
lactoferrin
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WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
binding proteins, PiIC, adhesins); S. pyogenes (for example M proteins or
fragments
thereof, CSA protease, lipoteichoic acids), S. agalactiae, S. mutans; H.
ducreyi;
Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis
(for
example high and low molecular weight adhesins and invasins); Bordetella spp,
including B. pertussis (for example pertactin, pertussis toxin or derivatives
thereof,
filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and
B.
bronchiseptica; Mycobacterium spp., including M. tuberculosis (for example
ESAT6,
Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M.
smegmatis; Legionella spp, including L. pneumophila; Escherichia spp,
including
enterotoxic E. coli (for example colonization factors, heat-labile toxin or
derivatives
thereof, heat-stable toxin or derivatives thereof), enterohemorragic E. coli,
enteropathogenic E. coli (for example shiga toxin-like toxin or derivatives
thereof);
Vibrio spp, including V. cholera (for example cholera toxin or derivatives
thereof);
Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp,
including
Y. enterocolitica (for example a Yop protein) , Y. pestis, Y.
pseudotuberculosis;
Campylobacter spp, including C. jejuni (for example toxins, adhesins and
invasins)
and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S.
choleraesuis, S.
enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp,
including H.
pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp,
including
P. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;
Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,
including C.
tetani (for example tetanus toxin and derivative thereof), C. botulinum (for
example
botulinum toxin and derivative thereof), C. difficile (for example clostridium
toxins A
or B and derivatives thereofj; Bacillus spp., including B. anthracis (for
example
botulinum toxin and derivatives thereof); Corynebacterium spp., including C.
diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia
spp.,
including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for
example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA,
DbpB), B. andersonii (for example OspA, OspC, DbpA, DbpB), B. hermsii;
Ehrlichia
spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis;
Rickettsia spp, including R. rickettsii; Chlamydia spp., including C.
trachomatis (for
example MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP,
heparin-binding proteins), C. psittaci; Leptospira spp., including L.
interrogans;
Treponema spp., including T. pallidum (for example the rare outer membrane
W~ 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
proteins), T. denticola, T. hyodysenteriae; or derived from parasites such as
Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii
(for
example SAG2, SAGS, Tg34); Entamoeba spp., including E. histolytica; Babesia
spp.,
including B. micron; Trypanosoma spp., including T. cruzi; Giardia spp.,
including
G. lamblia; Leshmania spp., including L. major; Pneumocystis spp., including
P.
carinii; Trichomonas spp., including T. vaginalis; Schisostoma spp., including
S.
mansoni, or derived from yeast such as Candida spp., including C. albicans;
Cryptococcus spp., including C. neoformans.
Preferred bacterial vaccines comprise antigens derived from Streptococcus spp,
including S. pneumoniae (for example capsular polysaccharides and conjugates
thereof, PsaA, PspA, streptolysin, choline-binding proteins) and the protein
antigen
Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial
Pathogenesis, 25, 337-342), and mutant detoxified derivatives thereof (WO
90/06951;
WO 99/03884). Other preferred bacterial vaccines comprise antigens derived
from
Haemophilus spp., including H. influenzae type B (for example PRP and
conjugates
thereof), non typeable H. injZuenzae, for example OMP26, high molecular weight
adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived
peptides (US 5,843,464) or multiple copy varients or fusion proteins thereof.
Other
preferred bacterial vaccines comprise antigens derived from Moraxella
Catarrhalis
(including outer membrane vesicles thereof, and OMP106 (W097/41731)) and from
Neisseria mengitidis B (including outer membrane vesicles thereof), and NspA
(WO
96/29412).
Derivatives of Hepatitis B Surface antigen are well known in the art and
include, inter
alia, those PreS 1, PreS2 S antigens set forth described in European Patent
applications
EP-A-414 374; EP-A-0304 578, and EP 198-474. In one preferred aspect the
vaccine
formulation of the invention comprises the HIV-1 antigen, gp 120, especially
when
expressed in CHO cells. In a further embodiment, the vaccine formulation of
the
invention comprises gD2t as hereinabove defined.
In a particular embodiment of the present invention vaccines containing the
claimed
adjuvant comprise antigen derived from the Human Papilloma Virus (HPV)
VVD 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
considered to be responsible for genital warts, (HPV 6 or HPV 11 and others),
and the
HPV viruses responsible for cervical cancer (HPV 16, HPV 18 and others).
Particularly preferred forms of genital wart prophylactic, or therapeutic,
vaccine
comprise L 1 particles or capsomers, and fusion proteins comprising one or
more
antigens selected from the HPV 6 and HPV 11 proteins E6, E7, L1, and L2.
The most preferred forms of fusion protein are: L2E7 as disclosed in WO
96/26277,
and protein D(1/3)-E7 disclosed in GB 9717953.5 (PCT/EP98/05285).
A preferred HPV cervical infection or cancer, prophylaxis or therapeutic
vaccine
composition may comprise HPV 16 or 18 antigens. For example, L 1 or L2 antigen
monomers, or L 1 or L2 antigens presented together as a virus like particle
(VLP) or
the L 1 alone protein presented alone in a VLP or capsomer structure. Such
antigens,
virus like particles and capsomer are per se known. See for example
W094/00152,
W094/20137, W094/05792, and W093/02184.
Additional early proteins may be included alone or as fusion proteins such as
preferably E7, E2 or ES for example; particularly preferred embodiments of
this
includes a VLP comprising LlE7 fusion proteins (WO 96/11272).
Particularly preferred HPV 16 antigens comprise the early proteins E6 or E7 in
fusion
with a protein D Garner to form Protein D - E6 or E7 fusions from HPV 16, or
combinations thereof; or combinations of E6 or E7 with L2 (WO 96/26277).
Alternatively the HPV 16 or 18 early proteins E6 and E7, may be presented in a
single
molecule, preferably a Protein D- E6/E7 fusion. Such vaccine may optionally
contain
either or both E6 and E7 proteins from HPV 18, preferably in the form of a
Protein D
- E6 or Protein D - E7 fusion protein or Protein D E6/E7 fusion protein.
The vaccine of the present invention may additionally comprise antigens from
other
HPV strains, preferably from strains HPV 6, 11, 31, 33, or 45.
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WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Vaccines of the present invention may comprise antigens derived from parasites
that
cause Malaria. For example, preferred antigens from Plasmodia falciparum
include
RTS,S and TRAP. RTS is a hybrid protein comprising substantially all the C-
terminal
portion of the circumsporozoite (CS) protein of P. falciparum linked via four
amino
acids of the preS2 portion of Hepatitis B surface antigen to the surface (S)
antigen of
hepatitis B virus. Its full structure is disclosed in the International Patent
Application
No. PCT/EP92/02591, published under Number WO 93/10152 claiming priority from
UK patent application No.9124390.7. When expressed in yeast RTS is produced as
a
lipoprotein particle, and when it is co-expressed with the S antigen from HBV
it
produces a mixed particle known as RTS,S. TRAP antigens are described in the
International Patent Application No. PCT/GB89/00895, published under WO
90/01496. A preferred embodiment of the present invention is a Malaria vaccine
wherein the antigenic preparation comprises a combination of the RTS,S and
TRAP
antigens. Other plasmodia antigens that are likely candidates to be components
of a
multistage Malaria vaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP,
RAP1, RAP2, Sequestrin, PfEMPI, Pf332, LSAI, LSA3, STARP, SALSA, PfEXPl,
Pfs25, Pfs28, PFS27/25, Pfsl6, Pfs48/45, Pfs230 and their analogues in
Plasmodium
spp.
The formulations may alternatively contain an anti-tumour antigen and be
useful for
the immunotherapeutic treatment of cancers. For example, the adjuvant
formulation
finds utility with tumour rejection antigens such as those for prostrate,
breast,
colorectal, lung, pancreatic, renal or melanoma cancers. Exemplary antigens
include
MAGE 1 and MAGE 3 or other MAGE antigens for the treatment of melanoma,
PRAME, BAGE or GAGE (Robbins and Kawakami, 1996, Current Opinions in
Immunology 8, pps 628-636; Van den Eynde et al., International Journal of
Clinical &
Laboratory Research (submitted 1997); Correale et al. ( 1997), Journal of the
National
Cancer Institute 89, p293. Indeed these antigens are expressed in a wide range
of
tumour types such as melanoma, lung carcinoma, sarcoma and bladder carcinoma.
Other Tumor-Specific antigens are suitable for use with adjuvant of the
present
invention and include, but are not restricted to Prostate specific antigen
(PSA) or Her-
2/neu, KSA (GA733), MUC-1 and carcinoembryonic antigen (CEA). Accordingly in
one aspect of the present invention there is provided a vaccine comprising an
adjuvant
composition according to the invention and a tumour rejection antigen.
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WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Additionally said antigen may be a self peptide hormone such as whole length
Gonadotrophin hormone releasing hormone (GnRH, WO 95/20600), a short 10 amino
acid long peptide, in the treatment of many cancers, or in immunocastration
It is foreseen that compositions of the present invention will be used to
formulate
vaccines containing antigens derived from Borrelia sp.. For example, antigens
may
include nucleic acid, pathogen derived antigen or antigenic preparations,
recombinantly produced protein or peptides, and chimeric fusion proteins. In
particular the antigen is OspA. The OspA may be a full mature protein in a
lipidated
form virtue of the host cell (E.Coli) termed (Lipo-OspA) or a non-lipidated
derivative.
Such non-lipidated derivatives include the non-lipidated NS1-OspA fusion
protein
which has the first 81 N-terminal amino acids of the non-structural protein
(NS1) of
the influenza virus, and the complete OspA protein, and another, MDP-OspA is a
non-lipidated form of OspA carrying 3 additional N-terminal amino acids.
Vaccines of the present invention may be used for the prophylaxis or therapy
of
allergy. Such vaccines would comprise allergen specific (for example Der p1)
and
allergen non-specific antigens (for example peptides derived from human IgE,
including but not restricted to the stanworth decapeptide (EP 0 477 231 B1)).
The amount of protein in each vaccine dose is selected as an amount which
induces an
immunoprotective response without significant, adverse side effects in typical
vaccinees. Such amount will vary depending upon which specific immunogen is
employed and how it is presented. Generally, it is expected that each dose
will
comprise 1-1000 ~.g of protein, preferably 1-500 fig, preferably 1-100~g, most
preferably 1 to SOp.g. An optimal amount for a particular vaccine can be
ascertained
by standard studies involving observation of appropriate immune responses in
subjects. Following an initial vaccination, subjects may receive one or
several booster
immunisation adequately spaced.
The vaccines of the present invention may also be administered via the oral
route. In
such cases the pharmaceutically acceptable excipient may also include antacid
14
WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
buffers, or enteric capsules or microgranules. The vaccines of the present
invention
may also be administered by the vaginal route. In such cases, the
pharmaceutically
acceptable excipients may also include emulsifiers, polymers such as
CARBOPOL~,
and other known stablilisers of vaginal creams and suppositories. The vaccines
of the
present invention may also be administered by the rectal route. In such cases
the
excipients may also include waxes and polymers known in the art for forming
rectal
suppositories.
The formulations of the present invention may be used for both prophylactic
and
therapeutic purposes. Accordingly, the present invention provides for a method
of
treating a mammal susceptible to or suffering from an infectious disease or
cancer, or
allergy, or auto-immune disease. In a further aspect of the present invention
there is
provided an adjuvant combination and a vaccine as herein described for use in
medicine. Vaccine preparation is generally described in New Trends and
Developments in Vaccines, edited by Voller et al., University Park Press,
Baltimore,
Maryland, U.S.A. 1978.
In an alternative, related embodiment of the present invention the adjuvant of
the
present invention may further be combined with other adjuvants including
Cholera
toxin and its B subunit, Monophosphoryl Lipid A and its non-toxic derivative 3-
de-O-
acylated monophosphoryl lipid A (as described in UK patent no. GB 2,220,211 ),
immunologically active saponin fractions e.g. Quil A derived from the bark of
the
South American tree Quillaja Saponaria Molina and derivatives thereof (for
example
QS21, US Patent No.5,057,540), and the oligonucleotide adjuvant system CpG (as
described in WO 96/02555), especially S'TCG TCG TTT TGT CGT TTT GTC GTT3'
(SEQ ID NO. 1 ).
The present invention is illustrated by, but not limited to, the following
examples.
In the examples below we used whole egg-grown flu virus inactivated with
formaldehyde, or TWEEN-ether split virus or NaDOC split egg-grown virus
supplemented with Triton X-100. The concentrations of the Tween-80 and Triton
X-
100 are shown in the examples.
VV~ 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Example 1, Methods used to measure antibody (Ab) responses in sera
ELISA for the measurement of influenza-specific serum Ig Abs:
Maxisorp Nunc immunoplates are coated overnight at 4°C with 50 pl/well
of 1 p.g/ml
HA of ~i-propiolactone (BPL) inactivated influenza virus (supplied by SSD GmBH
manufacturer, Dresden, Germany) diluted in PBS. Free sites on the plates are
blocked
(1 hour, 37°C) using saturation buffer: PBS containing 1%BSA, 0.1%
polyoxyethylene sorbitan monolaurate (TWEEN 20). Then, serial 2-fold dilutions
(in
saturation buffer, 50 ~l per well) of a reference serum added as a standard
curve
(serum having a mid-point titer expressed as ELISA Unit/ml, and put in row A )
and
serum samples (starting at a 1/100 dilution and put in rows B to H) are
incubated for
lhr 30mins at 37°C. The plates are then washed (x3) with washing buffer
(PBS, 0.1%
polyoxyethylene sorbitan monolaurate (TWEEN 20)). Then, biotinylated goat anti-
human Ig (Amersham) diluted 1/3000 in saturation buffer are incubated (50
pl/well)
for lhr 30mins, at 37°C. After 3 washings, and subsequent addition of
streptavidin-
horseradish peroxidase conjugate (Amersham), plates are washed 5 times and
incubated for 20 min at room temperature with 50 p.l/well of revelation buffer
(OPDA
0.4 mg/ml (Sigma) and HZOZ 0.03% in 50mM pH 4.5 citrate buffer). Revelation is
stopped by adding 50 ~l/well HZS04 2N. Optical densities are read at 492 and
630 nm
by using Biorad 3550 immunoreader. Antibody titre are calculated by the 4
parameter
mathematical method using SoftMaxPro software.
HemaQglutination Inhibition (HAI2 activity of Flu-specific serum Abs in mice
Sera (25 p.1) are first treated for 20 minutes at room temperature (RT) with
100 p.1
borate 0.5M buffer (pH 9) and 125 p1 Dade Behring-purchased kaolin. After
centrifugation (30 minutes, 3000 RPM or 860 g), 100 p1 supernatant
(corresponding
to a 1/10 dilution of the serum) are taken and incubated for 1 hour at
4°C with 0.5%
chicken red blood cells. Supernatant is collected after centrifugation for 10
minutes at
3200 RPM (970 g). Both operations are done for eliminating the natural
hemagglutinating factors contained in the sera. Then, 25 ~l treated-sera are
diluted in
25 u1 PBS (serial 2-fold dilutions starting at 1/20) in 96 well Greiner
plates. BPL
inactivated whole virus is added (25 u1 / well) at a concentration of 4
Hemagglutination Units (i.e. at a dilution which is 4-fold lower than the last
one
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WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
provoking an agglutination of red blood cells) for 30 minutes at RT under
agitation.
Chicken red blood cells are then added (25 ~.1 / well) for 1 hour at RT.
Plates are
finally kept overnight at 4°C before to be read. The HAI titer
corresponds to the
inverse of the last serum dilution inhibiting the virus-induced
hemagglutination.
Example2, Effect of TWEEN80 and Triton on the intranasal immunogenicity of
inactivated whole influenza virus in mice
In the past, the pre-clinical evaluations of alternative influenza vaccines
(e.g.
adjuvanted parenteral vaccines, DNA-based vaccines or mucosally delivered
vaccines) have mainly been performed in naive animals. In general, the
promising
results obtained from these studies were not confirmed in humans. This was
probably
due to the fact that the majority of adults have been immunologically "primed"
through natural infections before vaccination, unlike the naive animals.
Therefore, the best way to evaluate intranasal influenza vaccines in animal
models
would be to test their ability to boost pre-established immune responses in
nasally
primed animals. We assess in the present example the effect of TWEEN-80 and
Triton X-100 on such responses.
The priming was done in female Balb/c mice (8 weeks old) at day 0 by
administering
with a pipette (under anesthesia) in each nostril 2.5 ~g HA of BPL-inactivated
A/Beijing/262/95 influenza virus contained in 10 ~,l PBS. After 28 days, mice
(6
animals/group) were boosted intranasally (under anesthesia) with 20 p1 of
solution ( 10
u1 per nostril, delivered as droplets by pipette) containing 5 p.g HA of BPL-
inactivated A/Beijing/262/95 influenza virus in either A: PBS; B: TWEEN80
(0.11%)
plus Triton X-100 (0.074%) ; or by C: intramuscular injection of 1.5 ~,g HA of
influenza vaccine. Antigens were supplied by SSD GmBH manufacturer (Dresden,
Germany). HAI Ab responses were measured in sera as described in Example 1.
As shown in the Figure 1, when formulated with TWEEN80 and Triton, inactivated
influenza virus delivered intranasally.is capable of boosting pre-established
systemic
HAI Ab responses as efficiently as the classical parenteral influenza vaccine.
However, the same antigen given intranasally in the absence of TWEEN80 and
Triton
is significantly less immunogenic.
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WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Example 3: A comparison of the immunogenicity of an intranasal split influenza
vaccine with TWEEN80 & TRITONX 100 compared to a licensed conventional
parenteral vaccine (FluarixTM) in healthy adult subjects.
Formulations used in the study
Two formulations (A,B) of egg-derived split influenza antigens have been
evaluated.
A is an intranasal formulation and B is the FluarixT""/a-Rix~ given
intramuscularly.
The formulations contain three inactivated split virion antigens prepared from
the
WHO recommended strains of the 1998/1999 season.
The device used for intranasal delivery is the AccusprayT"' intranasal
syringes from
Becton Dickinson. 100p.1 of the A formulation is sprayed in each nostril.
Composition of the formulations.
The intranasal formulation (A) contains the following inactivated split
virions:
1. 30~g HA A/Beijing/262/95 (H1N1)
2. 30p,g HA A/Sydney/5/97 (H3N2)
3. 30p,g HA of B/Harbin/7/94
and phosphate buffered saline pH 7.4~ 0.1, Tween 80 0.1%, Triton X-100 0.015%,
Na
deoxycholate 0.0045% and thiomersal below 35~g/ml.
The volume of one dose is 200p.1 (100p.1 sub-doses for each nostril).
The comparator FluarixT""/a-Rix~ is the SmithKline Beecham Biologicals'
commercial inactivated trivalent split influenza vaccine. The dose of 500p.1
is
administered intramuscularly.
This dose contains;
l5p.g HA of the three strains mentioned above, Tween 80 between 500 and 1000
~g
per ml (0.05%-0.1%), Triton X-100 between 50 and 170p.g/ml (0.005%-0.017%),
sodium deoxycholate maximum 100pg/ml, thiomersal 100ug/ml and
phosphate buffered saline pH between 6.8 and 7.5.
18
WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Immunogenic~ Study
An open, controlled and randomised study evaluated the immunogenicity of an
intranasal split influenza vaccine formulated with Tween 80 & Triton X-100
compared to the conventional parenteral vaccine (i.e. FluarixTM). Twenty
healthy
adult subjects (aged 18-40 years) received one dose of FluarixTM and ten
subjects
received one dose of the intranasal influenza vaccine. The intranasal
formulation
(200.1) contained the following inactivated virions: 30ug of haemagglutinin
A/Beijing/262/95 (H1N1), 30pg of haemagglutinin A/Sydney/5/97 (H3N2), 30p,g of
haemagglutinin B/Harbin/7/94 with phosphate buffered saline (pH 7.4 ~ 0.1),
Tween
80 (0.1%), Triton X-100 (0.015%), sodium deoxycholate (0.0045%) and thiomersal
(<35~.g/ml).
There was an eight-day follow-up period for solicited local and general
symptoms and
both vaccines were well-tolerated regarding safety and reactogenicity. No
serious
adverse events related to vaccination were reported.
The immunogenicity of the vaccines was examined by assessing the serum
haemagglutination inhibition (HI) titres to determine the seroconversion rate
(defined
as the percentage of vaccinees who have at least a 4-fold increase in serum HI
titres
on day 21 compared to day 0, for each vaccine strain), conversion factor
(defined as
the fold increase in serum HI Geometric Mean Titres (GMTs) on day 21 compared
to
day 0, for each vaccine strain) and seroprotection rate (defined as the
percentage of
vaccinees with a serum HI titre >_40 after vaccination (for each vaccine
strain) that is
accepted as indicating protection). Generally, an influenza vaccine needs to
have > or
equal to 40% seroconversion rate, > or equal to 70% seroprotection rate and a
conversion factor of > or equal to 2.5, for each strain, in order to meet the
international regulatory requirements. This applies for adults between 18-60
years;
different criteria apply for the elderly.
In addition, the mucosal IgA antibody response was assessed by Enzyme Linked
Immunosorbent Assay (ELISA).
19
W~ 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
HI seropositivity, serconversion and seroprotection rates twenty-one days
after one
dose of FluarixTM or the intranasal formulation can be seen in Table 1.
Table 1:
HI seropositivity, seronversion and seroprotection rates at 21 days post dose
1
SeropositivitySeroprotectionSeroconversion
Strain Group Timing N
n % n % n
A/BeijingIntranasal vaccineDay 20 4 20.0 0 0.0
plus 0
Tween 80 & TitronDay 20 17 85.0 15 75.0 15 75.0
X100 21
FluarixTM Day 19 4 21.1 3 15.8
0
Day 19 19 100.0 18 94.7 19 100.0
21
A/SydneyIntranasal vaccineDay 20 13 65.0 3 15.0
plus 0
Tween 80 & TitronDay 20 20 100.0 19 95.0 15 75.0
X100 21
FluarixTM Day 19 14 73.7 1 5.3
0
Day 19 19 100.0 18 94.7 16 84.2
21
B/HarbinIntranasal vaccineDay 20 10 50.0 7 35.0
plus 0
Tween 80 & TitronDay 20 20 100.0 18 90.0 14 70.0
X100 21
FluarixTM Day 19 17 89.5 11 57.9
0
Day 19 19 100.0 19 100.0 15 78.9
21
Seropositivity (n,%) : number and percentage of subjects with titer _> 10
Seroprotection (n,%) : number and percentage of subjects with titer >_ 40
Seroconversion (n,%) : number and percentage of subjects with at least a 4-
fold increase in titres
from day 0 to day 21
In all cases, the conversion factor (fold increase in serum HI GMTs after
vaccination)
was greater than 2.5, the level required for a successful influenza vaccine.
The percentage of subjects with a two-fold or a four-fold increase in the
specific/total
mucosal IgA antibody ratio between day 21 and day 0 ( 1 dose) can be seen in
Table 2.
WO 01/21207 CA 02383413 2002-03-22 PCT/EP00/09366
Table 2:
Percentages of subiects with a two-fold or a four-fold increase in the
specific/total
IQA ratio between day 21 and day 0 ( 1 dose).
2 fold increase4 fold increase
Strain Group N ("/o) (%)
A/BeijingTween & Triton20 55.0 30.0
FluarixTM 19 52.6 26.3
A/SydneyTween & Triton20 65.0 45.0
FluarixTM 19 47.4 5.3
B/HarbinTween & Triton20 40.0 30.0
FluarixTM 19 26.3 5.3
Summary
The immunogenicity results tabulated above show that the intranasal
formulation
produced similar levels of seropositivity, seroconversion and seroprotection
to those
produced by the conventional parenteral vaccine (FluarixTM) twenty-one days
after
one dose. The intranasal formulation produced a better mucosal IgA response
after
one dose than the conventional parenteral vaccine (FluarixTM).
21
