Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ADJUVANT COMPOSITIONS
The present invention relates to an adjuvant composition comprising a
polyoxyethylene ether or polyoxyethylene ester, in combination with a
pharmaceutically acceptable excipient, and to a vaccine comprising such
adjuvant
compositions and antigen. In addition, the present invention relates to the
use of
polyoxyethylene ethers or esters in the manufacture of an adjuvant
formulations, and
vaccine formulations, and their use as medicaments.
Mucosal vaccination, for example intranasal and oral, may represent an easy
and more
convenient way of vaccination than traditional vaccination through systemic
injection.
The use of an injection to administer a vaccine dose is associated with a
number of
disadvantages, namely pain and irritation at the injection site following
injection.
These factors may lead to "needle-fear" which has been known to result in poor
patient compliance for vaccination regimes. Furthermore, conventional systemic
injections can be a source of infection in the region of the skin puncture.
Apart from bypassing the requirement for injection, mucosal vaccination is
attractive
since it has been shown in animals that mucosal administration of antigens has
a
greater efficiency of 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).
In order for mucosal immunisation to be a viable replacement for, or
alternative to,
immunisation through injection, this vaccination route will have to be able to
induce
systemic immunological responses at least as efficiently as those induced by
injection.
While it has been reported that certain antigens when administered via this
route are
able to induce systemic responses (Cahill et al.,1993, FEMS Microbiology
Letters,
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WO 99/52549 PCT/EP99l02278
107, 211-216}, most soluble antigens given intranasally by themselves induce
little or
no immune response.
A number of authors have investigated potential mucosal adjuvants to overcome
this
problem, which exert their adjuvant activity through various mechanisms
including:
encapsulation of the antigen (e.g. liposomes and microparticles); or via
direct
interaction with, and subsequent release of immunostimulatory cytokines from,
target
cells (e.g. cholera toxin and E.coli heat-labile toxin); or by enhancing the
uptake of
antigen across the epithelium (e.g. cholera toxin).
The applicant presents here the surprising finding that polyoxyethylene ethers
and
polyoxyethylene esters act as a potent adjuvants for vaccines. The adjuvants
of the
present invention are safe, easily steriiisable, and simple to administer.
Advantageously, such compositions are sufficient to induce systemic immune
responses when administered mucosally, which are at least as high as those
observed
after conventional systemic injection of the vaccine.
Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in
the
Merck index ( 12~' ed: entry 7717), where therapeutic uses are stated to
include: topical
anesthetic; anti-pruritic; and sclerosing agent activities. As a class, such
polyoxyethylene ethers, or esters, are non-ionic surfactants.
Intranasal administration of polyoxyethylene ethers and esters have been
described for
the enhancement of insulin uptake in the nasal cavity (Hirai et al. 1981,
International
Journal of Pharmaceutics, 9, I65-172; Hirai et al. 1981, International Journal
of
Pharmaceutics, 9, 173-184).
Other non-ionic surfactants have been utilised in vaccine formulations. It has
been
reported that 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
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WO 99152549 PCT/EP99102278
topical administration to a mucosal membrane (WO 9417827). This patent
application
discloses the combination of 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).
Novasomes (US 5,147,725) are paucilamenar vesicular structures comprising
Polyoxyethylene ethers and cholesterol encapsulate the antigen and are capable
of
adjuvanting the immune response to antigens after systemic administration.
Surfactants have also been formulated in such a way as to form non-ionic
surfactant
vesicles (commonly known as neosomes, WO 95/09651 ). Such vesicles, in the
presence of cholesterol form lipid-bilayer vesicles which are capable of
entrapping
antigen within the inner aqueous phase or within the bilayer itself.
We present here the surprising finding that relatively low concentrations of
polyoxyethylene ethers or esters are able to significantly enhance the
systemic
immune response towards co-administered antigens. Furthermore, when used in
mucosal vaccine formulations, the boosting effect of these adjuvants raises
the
systemic immunological responses to a level equal or superior to that achieved
by
conventional systemic injection of the antigen. These molecules represent a
class of
adjuvants suitable for application in humans either for conventional systemic
vaccine
purposes, or to replace systemic vaccination by mucosal vaccination.
As many available vaccine adjuvants function because of antigen encapsulation,
surprisingly the present invention functions as a potent vaccine adjuvants in
the form
of a non-vesicular solution or suspension. Thus, one embodiment of the present
invention provides for an adjuvant formulation comprising a surfactant of
formula (I),
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which is present in the form of a non-vesicular solution or suspension.
Another
embodiment of the present invention takes the form of a vaccine adjuvant
comprising
a surfactant of formula (I), formulated in the absence of cholesterol.
Vaccines and adjuvant formulations of the present invention comprise molecules
of
general formula (I):
HO(CHZCH20)o A-R
wherein, n is 1-SO, A is a bond or -C(O)-, R is C,_3° alkyl or Phenyl
C,_,o alkyl.
One embodiment of the present invention consists of a vaccine formulation
comprising a polyoxyethyIene ether of general formula (I), wherein n is
between 1
and 50, preferably 4-24, most preferably 9; the R component is C,_so,
preferably C4-CZo
alkyl and most preferably C,2 alkyl, and A is a bond. The concentration of the
polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-
10%, and
most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are
selected
from the following group: polyoxyethylene-9-Iauryl ether, polyoxyethylene-9-
steoryl
ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,
polyoxyethylene-35-Iauryi ether, and polyoxyethylene-23-lauryl ether.
A further embodiment of the present invention consists of a vaccine
composition
comprising a polyoxyethylene ester of general formula (I), wherein n is
between 1
and 50, preferably 4-24, most preferably 9; R is C,.so, preferably C4 to CZO
alkyl and
most preferably C,~ alkyl, and A is -C(O)-. The concentration of the
polyoxyethylene
ester should be in the range 0.1-20%, preferably from 0.1-10%, and most
preferably in
the range 0.1-1%. Preferred polyoxyethylene esters are selected from the
following
group: polyoxyethylene-9-lauryl esters, polyoxyethylene-9-steoryl esters,
polyoxyethylene-8-steoryl esters, polyoxyethylene-4-lauryl esters,
polyoxyethylene-
35-lauryl esters, and polyoxyethylene-23-Iauryl esters.
Also forming an embodiment of the present invention are vaccine compositions
comprising polyoxyethylene phenyl ethers of general formula (I), wherein n is
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WO 99/52549 PCT/EP99/02278
between 1 and 50 but preferably 4-24 and most preferably 9, R is C,.,°
phenyl alkyl,
preferably C, - C~ phenyl alkyl, and most preferably C,~ phenyl alkyl, and A
is a
bond. The concentration of the polyoxyethylene ethers should preferably be in
the
range 0.1-10%, and most preferably in the range 0.25-1%.
The vaccine preparations of the present invention may be 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/intestinaUvaginaUrectal 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, characterised in that the
adjuvants are
not in the form of a vesicle.
In a preferred embodiment of the present invention provides for an adjuvant
for use in
mucosal vaccine formulations. Such adjuvants are well tolerated in humans and
are
potent in their induction of systemic immune responses. The adjuvants of the
present
invention may take the form of a solution, or non-vesicular solution or
suspension,
and as such do not have any of the problems associated with the manufacture,
stability, uniformity, and quality control of particulate adjuvant systems.
These
formulations are potent adjuvants and also exhibit low reactogenicity and are
well
tolerated by patients.
Preferably, the polyoxyethylene ethers of the present invention have
haemolytic
activity. The haemolytic activity of a polyoxyethylene ether may be measured
in vitro,
with reference to the following assay, and is as expressed as the highest
concentration
of the detergent which fails to cause lysis of the red blood cells:
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1. Fresh blood from guinea pigs is washed with phosphate buffered saline (PBS)
3 times in a desk-top centrifuge. After resuspension to the original volume
the blood
is further diluted 10 fold in PBS.
2. 50 ~1 of this blood suspension is added to 800 ~,1 of PBS containing two-
fold
dilutions of detergent.
3. After 8 hours the haemolysis is assessed visually or by measuring the
optical
density of the supernatant. The presence of a red supernatant, which absorbs
light at
570 nm indicates the presence of haemolysis.
4. The results are expressed as the concentration of the first detergent
dilution at
which hemolysis no longer occurs.
Within the inherent experimental variability of such a biological assay, the
polyoxyethylene ethers, or surfactants of general formula {I), of the present
invention
I 5 preferably have a haemolytic activity, of approximately between 0.5-0.0001
%, more
preferably between 0.05-0.0001%, even more preferably between 0.005-0.0001%,
and
most preferably between 0.003-0.0004%. Ideally, said polyoxyethylene ethers or
esters should have a haemolytic activity similar (i.e. within a ten-fold
difference) to
that of either polyoxyethylene-9 lauryl ether or polyoxyethylene-8 steoryl
ether.
The ratio of the length of the polyoxyethylene section to the length of the
alkyl chain
in the surfactant (i. e. the ratio of n: alkyl chain length), affects the
solubility of this
class of detergent in an aqueous medium. Thus, the adjuvants of the present
invention
may be in solution or may form particulate structures such as micelles. The
adjuvants
of the present invention are because of their non-vesicular nature are clear
and not
cloudy or opaque, stable and are easily sterilisable by filtration through a
220 nm
membrane, and are manufactured in a easy and controlled fashion.
Vaccines of the present invention may take the form of a non-vesicular
solution or
suspension of polyoxyethyiene ether or ester of general formula (I) in a
pharmaceutically acceptable excipient, such as PBS or water, and an antigen or
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antigenic preparation. Such a vaccine formulation may then be applied to a
mucosal
surface of a mammal in either a priming or boosting vaccination regime; or
alternatively be administered systemically, for example via the transdermal,
subcutaneous or intramuscular routes.
Other adjuvants which are known to enhance both mucosal and systemic
immunological responses include the bacterial enterotoxins derived from Vibrio
Cholerae and Eschericia Coli (namely cholera toxin (CT), and heat-labile
enterotoxin
(LT) respectively). CT and LT are heterodimers consisting of a pentameric ring
of (3-
subunits, cradling a toxic A subunit. Their structure and biological activity
are
disclosed in Clements and Finklestein, 1979, Infection and Immunity, 24:760-
769;
Clements et al., 1980, Infection and Immunity, 24,91-97. Recently a non-toxic
derivative of LT has been developed which lacks the proteolytic site required
to
enable the non-toxic form of LT to be " switched on" into its toxic form, once
released from the cell. This form of LT (termed mLT(R192G)) is rendered
insuceptible to proteolytic cleavage by a substitution of the amino acid
arginine with
glycine at position 192, and has been shown to have a greatly reduced toxicity
whilst
retaining its potent adjuvant activity. mLT(R.192G) is, therefore, termed a
proteolytic
site mutant. Methods for the manufacture of mLT(RI92G) are disclosed in the
patent
application WO 96/06627. Other mutant forms of LT include the active site
mutants
such as mLT(A69G) which contain a substitution of an glycine for an alanine in
position 69 of the LTA sequence. The use of mLT(R192G) as a mucosal vaccine is
described in patent application WO 96/06627. Such adjuvants may be
advantageously
combined with the non-ionic surfactants of the present invention.
Accordingly, in an alternative embodiment of the present invention the
polyoxyethylene ether, or ester, will further be combined with other adjuvants
or
immunostimulants including Cholera toxin and its B subunit, Monophospharyl
Lipid
A and its non-toxic derivative 3-de-O-acylated monophosphoryl lipid A (as
described
in UK patent no. GB 2,220,211 ), saponins such as Quil A (derived from the
bark of
the South American tree Quillaja Saponaria Molina), and fractions thereof,
including
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QS21 and QS17 (US 5,057,540; Kensil, C. R., Crit Rev Ther Drug Carrier Syst,
1996, 12 (1-2):I-55; EP 0 362 279 BI; Kensil et al. (1991. J. Immunology vol
146,
431-437; WO 99/10008) and the oligonucleotide adjuvant system containing an
unmethylated CpG dinucleotide (as described in WO 96/02555). A particularly
preferred immunostimulant used in conjunction with POE is CpG
immunostimulatory
oligonucleotide, which formulations are potent in the induction and boosting
of
immune responses in larger animals. Preferred oligonucleotides have the
following
sequences: The sequences preferably contain all phosphorothioate modified
internucleotide linkages.
OLIGO 1: TCC ATG ACG TTC CTG ACG TT
OLIGO 2: TCT CCC AGC GTG CGC CAT
OLIGO 3: ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG
The CpG oligonucleotides utilised in the present invention may be synthesized
by any
method known in the art (eg EP 468520). Conveniently, such oligonucleotides
may
be synthesized utilising an automated synthesizer.
Alternatively polyoxyethylene ethers or esters may be combined with vaccine
vehicles
composed of chitosan or other polycationic polymers, polylactide and
polylactide-co-
glycolide particles, particles composed of polysaccharides or chemically
modified
polysaccharides, cholesterol-free liposomes and lipid-based particles, oil in
water
emulsions (WO 95/17210), particles composed of glycerol monoesters, etc. The
polyoxyethylene ethers or esters may also be admixed with powdered excipients
such
as lactose containing antigen which can be administered as a dry powder.
Adjuvants of the present invention comprise the surfactants: polyoxyethylene
ethers
or esters wherein the polyoxyethylene ethers or esters are not present in the
form of
vesicles. Accordingly, the present invention includes the use of
polyoxyethylene
ethers and esters of general formula (I) in the manufacture of adjuvant
compositions
and vaccines, wherein the surfactant of general formula (I) is not present in
a vesicular
form.
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w0 99/52549 PC'T/EP99102278
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-I, (such
as tat,
nef, gp120 or gp160), 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 Barn virus {such as gp350 or derivatives thereof), Varicella
Zoster
Virus (such as gpl, II and IE63), or from a hepatitis virus such as hepatitis
B virus (for
example Hepatitis B Surface antigen or a derivative thereofj, 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, meningitides (for example
capsular
polysaccharides and conjugates thereof, transfenin-binding proteins, Iactofen-
in
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 thereofj, enterohemorragic E. coli,
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W4 99152549 PCT/EP99/02278
enteropathogenic E. toll (for example shiga toxin-like toxin or derivatives
thereofj;
Yibrio spp, including Y. 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 toll; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis,
S.
enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp,
including H.
pylori (for example unease, catatase, 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 thereofj, C. botulinum (for
example
botulinurn toxin and derivative thereof), C. docile (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. afielii (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.
trachomatBs (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
proteins), T. denticola, T. hyodysenteriae; or derived from parasites such as
Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii
(for
example SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica; Babesia
spp.,
including B. microti; 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.
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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
90106951;
WO 99/03884). Other preferred bacterial vaccines comprise antigens derived
from
Haemophilus spp., including H. inJluenzae type B (for example PRP and
conjugates
thereof), non typeable H. inJluenzae, 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 Morexella
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 PreSl, 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, gp120, especially
when
expressed in CHO cells. In a further embodiment, the vaccine formulation of
the
invention comprises gD2t as hereinabove defined.
In a preferred embodiment of the present invention vaccines containing the
claimed
adjuvant comprise antigen derived from the Human Papilloma Virus (HPV)
considered to be responsible for genital warts, (HPV 6 or HPV 1 I 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, L 1, and L2.
I1
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WO 99152549 PCT/EP99/02278
The most preferred forms of fusion protein are: L2E7 as disclosed in WO
96/26277,
and proteinD(1/3)-E7 disclosed in GB 9717953.5 (PCT/EP98/05285).
S 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 L1E7 fusion proteins (WO 96J11272).
Particularly preferred HPV 16 antigens comprise the early proteins E6 or E7 in
fusion
with a protein D carrier to form Protein D - E6 or E7 fusions from HPV 16, or
combinations thereof; or combinations of E6 or E7 with L2 (WO 96126277).
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.
Vaccines of the present invention further comprise antigens derived from
parasites
that cause Malaria. For example, preferred antigens from Plasmodia,~alciparum
include RTS,S and TRAP. RTS is a hybrid protein comprising substantially all
the C-
tenninal portion of the circumsporozoite (CS) protein of P.falciparum linked
via four
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amino acids of the preS2 portion of Hepatitis B surface antigen to the surface
(S)
antigen of hepatitis B virus. It's full structure is disclosed in the
International Patent
Application No. PCT/EP92/02591, published under Number WO 93/10152 claiming
priority from LTK 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. PCTlGB89/00895, 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, LSA1, LSA3, STARP, SALSA, PfEXPI,
Pfs25, Pfs28, PFS27/25, Pfsl6, Pfs48/45, Pfs230 and their analogues in
Plasmodium
sPP.
The formulations may also contain an anti-tumour antigen and be useful for the
immunotherapeutic treatment 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,
BAGS 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 a1. (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 99/52549 PCT/EP991p2Z78
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.CoIi) termed (Lipo-OspA) or a non-lipidated
derivative.
Such non-Iipidated derivatives include the non-Iipidated NS I-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.
IS
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 B 1
)).
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 I-1000 p,g of protein, preferably 1-500 p,g, preferably I-IOO~cg,
most
preferably 1 to 50pg. Art 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.
I4
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WO 99/52549 PCT/EP99/02278
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-
s derived antigens, including GnRH and IgE peptides, recombinantly produced
protein
or peptides, and chimeric fusion proteins.
The vaccines of the present invention may also be administered via the oral
route. In
such cases the pharmaceutically acceptible excipient may also include alkaline
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 stabIilisers of vaginal creams and suppositories. The vaccines
of the
present invention may also be administered by the rectal mute. 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 maybe 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 autoimmune disease. In a further aspect of the present invention
there is
provided a vaccine as herein described for use in medicine. Vaccine
preparation is
generally described in New Trends and Developments in Vaccines, edited by
Volley et
al., University Park Press, Baltimore, Maryland, U.S.A. 1978.
The present invention relates to the use of polyoxyethylene ethers or esters
of general
formula (I) in the manufacture of an adjuvant formulation, comprising a
surfactant of
formula (I) and a pharmaceutically acceptable excipient. The present invention
relates
to the use of polyoxyethylene ethers or esters of general formula (I) in the
manufacture of vaccine formulation, comprising a surfactant of formula (I) and
a
pharmaceutically acceptable excipient and an antigen. The present invention
also
I5
CA 02325939 2000-09-25
WO 99/52549 PCT/EP99/02278
relates to the use of polyoxyethylene ethers or esters of general formula (I)
in the
manufacture of an adjuvant formulation or vaccine, as described above, wherein
the
formulation does not contain cholesterol. The present invention further
provides the
use of polyoxyethylene ethers or esters of general formula (I) in the
manufacture of an
adjuvant formulation or vaccine, as described above, wherein the formulation
is a
non-vesicular solution or suspension.
Examples of suitable pharmaceutically acceptable excipients include water,
phosphate
buffered saline, isotonic buffer solutions.
Alternative terms or names for polyoxyethylene lauryl ether are disclosed in
the CAS
registry. The CAS registry number of polyoxyethylene lauryi ether is:
CAS REGISTRY NUMBER: 9002 92-0
The present invention is illustrated by, but not restricted to, the following
examples.
Ezample 1, Techniques used to measure antigen specific antibody (Ab) responses
.
ELISA for the measurement of OspA-specific serum IgG
Maxisorp Nunc immunoplates are coated overnight at 4°C with SO p,Uwell
of 1 p,g/ml
of antigen OspA diluted in PBS (in rows B to H of plate), or with 50 p.l of 5
pg/ml
purified goat anti-mouse Ig (Boerhinger), in PBS (row A). Free sites on the
plates
were blocked (1 hour, 37°C) using saturation buffer : PBS comtaining
1%BSA, 0.1%
polyoxyethylene sorbitan monolaurate (TWEEN 20), and 4% Normal Bovine Serum
(NBS). Then, serial 2-fold dilutions (in saturation buffer, 50 p.Uwell) of IgG
isotype
mixture, diluted in saturation buffer (50 ~1 per well), was added as a
standard curve
(mixture of mouse monoclonal antibodies IgGI, IgG2a and IgG2b from Sigma,
starting at 200 ng/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) vrrith washing buffer (PBS, 0.1% polyoxyethylene sorbitan
monolaurate (TWEEN 20)). Then, biotinylated goat anti-mouse IgG (Amersham)
16
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WO 99/52549 PCT/EP99/02278
diluted 1/5000 in saturation buffer are incubated (50 p,l/well) for lhr
30mins, at 37°C.
After 3 washings, and subsequent addition of streptavidin-horseradish
pemxidase
conjugate (Amersham), plates are washed 5 times and incubated for 20 min at
room
temperature with 50 pUwell of revelation buffer (OPDA 0.4 mg/ml (Sigma) and
HzO~
0.03% in SOmM pH 4.5 citrate buffer). Revelation is stopped by adding 50
pUwell
HZSO, 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.
Anti-TT, anti-FHA and anti-influenza IgG titres were measured using a similar
technique, by replacing the OspA coating antigen with either TT, FHA, or whole
influenza antigen. TT was supplied by a commercially available source
(Behring).
FHA was produced and purified by methods described in EP 0 427 462 B. Whole
influenza virus, inactivated with b-propriolactone (BPL), was supplied by SSD
GmBH (Dresden Germany).
ELISA for the measurement of S. Pneumoniae polysaccharide (PS 14 and PS 19~
specific serum IgG in mice: .
Maxisorp Nunc immunoplates are coated for 2 hours at 37°C with 100
pl/well of 5
pg/m1 (PS 14) or 20 ~,g/ml (PS 19) antigen diluted in PBS. The plates are then
washed
(x3) with washing buffer (PBS, O.I% palyoxyethylene sorbitan monolaurate
(TWEEN 20)). Then, serial 2-fold dilutions (in PBS TWEEN 20,100 p,l per well)
of
PS14 or PS19-specific monoclonal Ab (mAb) IgGI added as a standard curve
(starting at 785 ng/ml for PS 14 or 2040 ng/ml for PS 19, and put in row A)
and serum
samples (starting at a 1/20 dilution and put in rows B to H) are incubated for
30mins
at 20°C under agitation. Before to be added and diluted on the plate,
both mAb
standards and serum samples are pre-incubated with Common Polysaccharides
(CPS)
for 1 hour at 37°C, in order to eliminate aspecific reactions. The
plates are then
washed (x3) with washing buffer (PBS TWEEN 20). 'Then, peroxydase-conjugated
goat anti-mouse IgG (Jackson) diluted 1/5000 in PBS TWEEN 20 are incubated
(100
~Uwell) for 30 min at 20°C under agitation. After 3 washings, plates
are incubated for
17
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WO 99/52549 PCT/Ep99/02278
15 min at room temperature with I00 ~Uwell 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 uUwell HCl 1N. Optical densities are read at 492 and 630 ram by
using
Biorad 3550 immunoreader. Antibody titre are calculated by the 4 parameter
mathematical method using SoftMaxPro software.
ELISA for the measurement of OspA-specific serum Ig Abs in monke~rs~
Maxisorp Nunc irnmunoplates are coated overnight at 4°C with 50 ~Uwell
of I pg/ml
OspAdiluted 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 ~.1
per well) of a reference serum added as a standard curare (serum having a mid-
point
titer of 60000 ELISA Unit/ml, starting at 12 EU/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
pUwell)
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 uUwell of revelation buffer
(OPDA
0.4 mg/ml (Sigma) and HZOz 0.03% in SOmM pH 4.5 citrate buffer). Revelation is
stopped by adding 50 pUwell HZSO, 2N. Optical densities are read at 492 and
630 ram
by using Biorad 3550 immunoreader. Antibody titre are calculated by the 4
parameter
mathematical method using SoftMaxPro software,
Anti-influenza immunoglobulin titres were measured using a similar technique,
by
replacing the OspA coating antigen with whole influenza virus antigen,
inactivated
with (i-propiolactone (BPL), supplied by SSD GmBH manufacturer (Dresden,
Germany).
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WO 99/52549 PCT/EP99/02278
ELISA for the measurement of OspA-specific nasal IgA Abs in monkeys'
Maxisorp Nunc immunoplates are coated overnight at 4°C with 50 ~Uwell
of 1 ~g/ml
antigen OspA diluted in PBS (in rows B to H of plate), or with 50 ~l of 5
~g/ml
purified goat anti-human IgA (Sigma), in PBS (row A). Free sites on the plates
are
blocked (1 hour, 37°C) using saturation buffer : PBS comtaining 1%BSA,
0.1%
polyoxyethylene sorbitan monolaurate (TWEEN 20), and 4% Normal Bovine Serum
(NBS). Then, serial 2-fold dilutions (in saturation buffer, 50 ~.l per well)
of a
reference secretion added as a standard curve (secretion having a mid-point
titer of
3000 ELISA Unitlml, starting at 30 EUlml and put in row A ) and nasal swabs
(starting at a 1/5 dilution and put in rows B to H) are incubated for 2hr at
22°C. The
plates are then washed (x3) with washing buffer (PBS, 0.1% polyoxyethylene
sorbitan
monolaurate (TWEEN 20)). Then, biotinylated goat anti-human IgA (ICN) at 0.2
~.g/ml in saturation buffer are incubated (50 pUwell) 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 10 min at room
temperature
with 50 pUwell of revelation buffer.(TMB, Biorad). Revelation is stopped by
adding
50 pUwell H2S0, 0.4N. Optical densities are read at 450 and 630 nm by using
Biorad
3550 immunoreader. Antibody titre are calculated by the 4 parameter
mathematical
method using SoftMaxPro software. Samples are considered to be positive when
their
IgA titre exceed the cut-off of the assay (0.3 EU/ml).
Inhibition assa for the measurement of serum LA2 like Antibody titres to lipo
OspA
Antibody titres in the vaccinees were studied with respect to their LA2-like
specificity. LA2 is a marine monoclonal antibody which recognizes a
conformational
OspA epitoge at the surface of the bacteria and has been shown to be able to
kill B.
burgdorferi in vitro, as well as to protect mice against a challenge with
laboratory-
grown spirochete (Schaible UE et al. 1990. Proc Natl Acad Sci USA 87:3768-
3772).
Moreover, LA-2 mab has been shown to correlate with bactericidal antibodies,
and
studies on human sera showed also a good correlation between the total anti-
OspA
IgG titers and the LA-2 titers (as measured by ELISA).
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WO 99/52549 PCT/EP99102278
Maxisorp Nunc inununoplates are coated overnight at 4°C with 50
p.Uwell of
O.Spg/ml lipo OspA diluted in PBS. Free sites were blocked with saturation
buffer for
lhr at 37°C with (100 pUwell of saturation buffer: PBS/ BSA 1%/ Tween
20 0.1%/
NBS 4%). Serial 2-fold diIutions of LA2 monoclonal Ab (mAb) starting at 4
~g/ml
were diluted in saturation buffer (50 p,l per well) to form a standard curve.
Dilutions
of serum samples from the vaccinees (starting at a 1/10 dilution) were also
added and
the plates incubated for 2hrs at 37°C. The plates were washed after
incubation 3 times
with PBS/ TWEEN 20 (O.i%). LA2 mAb-peroxidase conjugate (1/10,000) diluted in
saturation buffer was added to each well (50 ~1/well) and incubated for lhr at
37°C.
After 5 washings, plates are incubated for 20 min at room temperature (in
darkness)
with 50 pUwell of revelation buffer (OPDA 0.4 mg/ml and H202 0.03% in SOmM pH
4.5 citrate buffer). The reaction and colour formation was stopped with HZSO,
2N.
Optical densities are read at 492 and 630 nm by using Biorad 3550
immunoreader.
1 S LA2-like Ab titers are calculated by the 4 parameter mathematical method
using
SoftMaxPro software. LA2-like antibody titres were determined by comparison
with
the standard curve.
Example 2 Intranasal boosting of mice with OspA antigen
Female Balb/c mice (8 animals per group) aged 8 weeks were immunised
intramuscularly with I pg of the antigen lipo-OspA on 50 ~,g alum. After 3
months
the mice were boosted intranasall
y (under anesthesia) with 10 pl of solution (5 ~1 per
nostril, delivered as droplets by pipette) containing either A: 5 ~Cg lipo-
OspA; B: 5 pg
Iipo-OspA in 36 % tween-20, 10% Imwitor 742 ; C: 5 ug lipo-OspA in 36 % tween
20; D: 5 ~cg lipo-OspA in 18% polyoxyethylene-9 lauryl ether.
14 days after the boost the sera were assayed for Abs against lipo-OspA by IgG
and
LA2 anti-OspA ELISA (see example I). The results, see figure 1, indicate that
Iipo-
OspA administered intranasally is able to boost the systemic lipo-OspA
specific IgG
titres. This boost is only marginally increased by the presence of tween-20
plus
Imwitor 742 or tween-20 alone. Polyoxyethylene-9 lauryl ether, on the other
hand,
CA 02325939 2000-09-25
WO 99152549 PCT/EP99/02Z78
induces a very significant boost. A similar pattern is observed for the LA2
response
(see figure 2).
Example 3 Intranasal boosting of mice with OspA antigen
Groups of mice were primed as described in example 2. The nice were then
boosted
(using the method described in example 2) with 5 pg Iipo-OspA alone (group A
and
C) or in the presence of B: 1 % sodium taurocholic acid; D: 1 % dodecyl-
maltoside; E:
36% tween 20 or F: 18% polyoxyethylene-9 lauryl ether. Since the experiment
with
I O groups A and B was performed at a different moment to that with groups
C,D,E and F
they are separated on the figures below (see figure 3). It is clear that 1 %
sodium
taurocholate does not significantly adjuvant the boost above that obtained
with the
antigen alone. Dodecyl-maltoside at I %, or tween-20 at 36% provide a slight
adjuvant
effect, but only polyoxyethylene-9 lauryl ether provides a very significant
15 enhancement of the IgG response. A similar effect is observed for the LA2
response
(see figure 4).
Example 4 Intranasal boosting of mice - Dose range study
20 In order to assess the concentration of polyoxyethylene-9 Iaury1 ether
required to
provide the nasal adjuvanticity observed in the previous examples, we
performed a
dose-range assay, and in order to show that this effect can be achieved using
other
polyoxyethylene ethers we investigated the use of polyoxyethylene-23 lauryl
ether.
Mice primed as in example 1 were boosted intranasally with 10 pl containing 5
pg of
25 lipo-OspA in either A: PBS; B: 1 % polyoxyethylene-9 lauryl ether; C: 2%
polyoxyethylene-9 Iauryl ether; D: S% poIyoxyethylene-9 lauryl ether; E: 1
polyoxyethylene-23 lauryl ether or; F: 10 % polyoxyethylene-23 lauryl ether.
14 days
after the boost the sera were analysed as in example 2.
30 Figures S and 6, below, show that concentrations of polyoxyethylene-9
Iauryl ether as
low as I % show a very significant enhancement of the immune response.
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WO 99/52549 PCT/EP99/02278
Polyoxyethylene-23-Iauryl ether also significantly enhances the intranasal
boost
response.
Example 5 Combination vaccine - intranasal boosting
In order to asses the applicability of polyoxyethylene ethers to the
enhancement of
systemic immune responses after intranasal boosting, female baib/c mice were
primed
infra-muscularly with the commercial DTPa vaccine (Diptheria, Tetanus,
accetular
Pertussis vaccine: IIVFANRIXTM SmithKline Beecham, Belgium). The mice were
primed once intramuscularly with 2 X 50 pI injections corresponding to 20% of
the
human dose. Three months later the mice were boosted (as in example 2)
intranasally
with either tetanus toxoid (TT: 5 ~.g) or filamentous haemagglutinin (FHA: 5
~.g) in
A: PBS; B: I% polyoxyethylene-9 lauryl ether, or; C: by intramuscular
injection of
the DTPa vaccine (2x50p,I). 14 days after the boosting the sera were analysed
for their
TT and FHA specific IgG. The titres are shown in figures 7 and 8.
It is clear that for TT the protein by itself does not induce a significant
boost, but
polyoxyethylene-9 lauryl ether is able to significantly boost the immune
response.
Surprisingly, the response obtained by intranasai boosting in the presence of
this
adjuvants is greater than that obtained following intramuscuIar boosting of
the
immune response.The administration of FHA by itself, induces an immune
response
which is further significantly enhanced by addition of the polyoxyethylene-9
lauryl
ether as an adjuvant.
Example 6 Intranasal boosting ofAGMs
Many adjuvants have been shown to work in small rodents, but to have no effect
when
tested in larger mammals. In order to asses whether polyoxyethylene ethers
were able
to exert an adjuvant effect on intranasal boosting when this was performed in
larger
species, African Green monkeys (AGMs: 4 animals per group) were primed
intramuscularly with lipo-OspA (10 fig) on ~~ (500 pg) by intramuscuIar
injection.
IO months later the animals were boosted intranasally with 200 ~l (I00 pl per
nostril
22
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WO 99/52549 PCT/EP99/02278
administered under anesthesia with a bidose spray device from Pfeiffer GmBH,
Germany) containing 60 ~g lipo-OspA in either A: PBS; or B: 1% polyoxyethylene-
9
Iauryl ether. After 14 days the sera were tested for anti-OspA immunoglobulin,
and
LA2 titres.Figures 9 and 10, show the geometric mean titres titres for each of
the
groups. Group C consisting of 10 AGMs that had received both the priming and
the
boost by intramuscular injection of lipo-OspA on alum were assayed for anti-
OspA
immunoglobulin responses (geometric mean titres shown for LA2 titres only,
figure
I 0).
Lipo-OspA alone was able to boost the systemic response when administered
intranasally to monkeys, but this boost is very significantly enhanced by the
addition
of 1 % polyoxyethylene 9 Iauryl ether. Surprisingly, the titres obtained
following
intranasal boosting in the presence of polyoxyethylene 9 lauryl ether are also
greater
than those obtained following an intramuscular injection (group C).
EaampIe 7 Intranasal priming and boosting of.4GMs
In the previous examples we demonstrated that polyoxyethylene ethers could
adjuvant
an intranasal boosting of the systemic response. In this example we examine
whether
naive animals can be primed and boosted by the nasal route to induce a
systemic
immune response. In addition, in order to investigate the applicability of
these
adjuvants to larger animals, this experiment was performed in African Green
Monkeys (AGMs).
African Green Monkeys (3 animals per group) were primed and boasted
intranasally
with 60 ug of Iipo-OspA delivered in 200 ~1 (100 ~tl per nostril delivered
with a
bidose spray-device from Pfeiffer GmBH, Germany) of A: PBS; B: I%
polyoxyethylene-9 lauryl ether. 14 days after the boosting the sera were
assayed for
their Osp-A specific immunoglobulin. Figure l I, shows that when Lipo-OspA is
not
adjuvanted, no systemic immune response can be detected following intranasal
23
CA 02325939 2000-09-25
WO 99/52549 PCT/EP99102278
priming and boosting. Whea polyoxyethylene-9 lauryl ether is used as an
adjuvant,
this vaccination schedule induced significant anti-OspA titres.
Ezample 8, Intranasal adjuvant effect of CpG on the induction of systemic and
nasal
S humoral immune responses to lipo OspA antigen in primates
This model was designed to investigate the priming and boosting effect of
polyoxyethylene-9 lauryl ether (POE-9LE), with and without additional
immunostimulants, in a primate priming and boosting model. Serum and nasal
immunoglobulin responses were measured. The inununostimulant used in this
study
was the CpG 1001 as described in example 9.
Experimental procedure
African Green monkeys were primed and boosted intranasally at days 0 (pI) and
14
1 S (pIn. Vaccines were given using a bi-dose spray delivery system from the
Pfeiffer
company (100 p,l in each nostril, under anesthesia). Formulations tested were:
Group Antigen Ad, juvant n= Route
1 LipoOspA (60p.g)None 2 i.n.
2 lipoOspA (60~tg)CpG (100pg) 3 i.n.
3 lipoOspA (b0pg)CpG (100~g), POE-9 LE 3 i.n.
(0.25%)
4 lipoOspA (60pg)POE (0.25%) 4 i.n.
S lipoOspA {60~cg)POE (O.S%) 4 i.n.
Tg Ab titers to lipo OspA were measured in sera collected at day 14 post-pII.
Antigen-
specific nasal IgA were measured using a very sensitive ELISA in nasal swabs
collected at the same time, animals were considered positive when their igA
titres
exceeded a pre-determined Ievel which was significantly above background
levels.
Results
2S Serum OspA-speck immunoglobulin
24
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WO 99/52549 PCT/EP99/02278
Figure 12 shows the levels of serum anti-lipo-OspA immunoglobuiin responses
observed at day i 4 post-pII. Lipo-OspA given as a priming and boosting
formulation
alone did not induce any detectable serum immunoglobulin. This response was
not
improved in the presence of CpG. A dose of 0.25 % and 0.5 % of POE-9 LE
elicited
greater immune responses that those observed after vaccination with CpG alone,
although the 0.5% dose is much more efficient in this respect. However, when
combined with CpG, the 0.25 % dose induces an Ab response similar in magnitude
to
that obtained with 0.5 % dose, indicating a synergistic effect of the CpG and
POE
components.
Nasal OspA-speck IgA
As observed for the serum Ig response, vaccines containing lipo OspA alone or
combined with CpG are unable to elicit detectable nasal IgA Abs (see figure 13
for a
summary of ali nasal responses). Only 25% animals given lipo OspA in
combination
with 0.25% polyoxyethylene lauryl ether were found to be "nasal IgA" positive
(versus 50% in the 0.5% POE-9 LE). When CpG is added to this 0.25% POE
formulation, 100% animals develop an IgA response. Therefore, a synergy
between
CpG and polyoxyethylene lauryl ether is also obtained for the induction of
mucosal
antibodies.
Thus, a synergy between polyoxyethylene lauryl ether and CpG is obtained in
monkeys for the induction of antigen specific serum immunoglobulins and nasal
IgA.
Ezample 9, Intranasal adjuvant ej, j''ect of CpG on the boosting of systemic
humoral
immune responses to lipo OspA antigen
The following example was designed to investigate the effect of the addition
of other
immunostimulants into the polyoxyethylene ether (POE-9 LE) adjuvant system in
a
murie booster model. CpG is a known immunomodulatory oligonucleotide described
in PCT WO 96/02555. The immune response boosted by these vaccine formulations
CA 02325939 2000-09-25
WO 99/52549 PC'T/EP99/02278
were at least as high as those induced by conventional i.m. boosting
vaccinations. The
formulations were further compared to a well known intranasal adjuvant, the
heat-
labile enterotoxin from E.Coli (mLT).
The CpG sequences used in this experiment were CpG 1001 (TCC ATG AGC TTC
CTG ACG TT), CpG 1002 (TCT CCC AGC GTG CGC CAT), and the negative
control the non-immunostimulatory sequence CpG1005 (TCC ATG AGC TTC CTG
AGC TT).
IO Experimental procedure
Balb/c mice were primed at day 0 by intramuscular administration of 100 ~1
vaccine
containing 1 pg lipo OspA adsorbed on 50 ~g aluminium hydroxyde. At day 107,
intranasal booster was given in I O pl (5 p,l in each nostril), by nasal drop
administration with a micropipette under anesthesia. Groups of 6 mice were
boosted
either intranasally (i.n.) or intramuscularly (i.m.) with the following
vaccine
formulations:
Group Antigen Adjuvant Route
1 LipoOspA (Spg)AIOH, (SO~g) i.m.
2 LipoOspA (Sp,g)CpG1005 (20p,g), POE-9 LE i.n.
{1%)
3 LipoOspA {Sp,g)CpG1002 (20~,g), POE-9 LE i.n.
(1%)
LipoOspA (SPg)CpG1001 (20~,g), POE-g LE i.n.
LipoOspA (S~g)(1%) i.n.
CpG1005 (201tg)
6 LipoOspA (Sp,g)CpG1002 (20p,g) i.n.
7 LipoOspA (Spg)CpG1001 (20pg) i.n.
LipoOspA (S~g)POE-9 LE (I%) i.n.
9 LipoOspA {S~cB)mLT (S~eg) i.n.
10 LipoOspA (S~g)None i.n.
II Unboosted
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Bleedings were performed the day of boosting, and 14 days after the boost
(pII).
Specific serum IgG titers to OspA and LA2 titers were determined by ELISA on
individual sera.
S Results
As shown in figure I4 (showing OspA specific serum IgG as measured by antigen
specific ELISA), and figure 15 (showing bacteriocidal LA2 titres in serum), no
improvement of the serum OspA-specific Ab responses was imparted by CpG alone.
The formulation of OspA with polyoxyethylene lauryl ether enhanced the
resultant
IgG and LA2 titers. The best responses were observed when lipo-OspA was
formulated with both polyoxyethylene lauryl ether and CpG.
Ezample 10 Dose study
As described in the example 4, concentrations of polyoxyethylene-9 lauryl
ether as
low as I% show a very significant enhancement of the immune response. In order
to
assess the concentration of polyoxyethylene-9 lauryl ether required to provide
the
nasal adjuvanticity observed in the previous examples, a dose-range assay with
lower
doses was performed.
Balb/c mice primed as in example 2 were boosted intranasally with 10 ~,1
containing 5
~tg of lipo-OspA in either A: PBS; B: 1 % polyoxyethylene-9 lauryl ether; C:
0.5%
golyoxyethylene-9 lauryl ether; D: 0.25% polyoxyethylene-9 lauryl ether; or;
E: by
intramuscular injection of 1 pg lipo-OspA adsorbed on 50 ~g Alum. I4 days
after the
boost the sera were analyzed as in example I .
Results
Figures 16 and 17, below, show that concentrations of polyoxyethylene-9 lauryl
ether
as low as 0.25% show a very significant enhancement of the immune response.
Even
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with such a low dose of adjuvant, the Ab response reached is similar to that
elicited by
the parenteral vaccine.
Ezampte 11, Anti-in, fluenza vaccination in mice
In order to assess the applicability of polyoxyethylene ethers to the
enhancement of
systemic anti-influenza immune responses after intranasal boosting, female
Balb/c
nuce were primed infra-muscularly with classical monovalent split influenza
vaccine.
a
The mice were primed twice intramuscularly at days 0 and 14 with 100 ~1
injections
containing 1.5 pg equivalent hemagglutinin A (HA) of A/Singapore/6/86 split
monobulk. Three months later the mice were boosted (as in example 2)
intranasally
with 1.5 ~g equivalent HA of inactivated whole A/Singapore/6/86 virus in A:
PBS; B:
1% polyoxyethylene-9 lauryl ether, or; C: by intramuscular injection of the
split
A/Singapore/6/86 vaccine (1.5 ~g equivalent HA). 14 days after the boosting
the sera
were analyzed for their A/Singapore/6/86 virus-specific IgG.
Results
The titres are shown in figure 18. It is clear that the plain antigen by
itself does not
induce a significant boost, but polyoxyethylene-9 lauryl ether is able to
significantly
boost the immune response. The Ab titres reached in the presence of this
adjuvant are
not significantly lower than those elicited by the patenteral vaccine.
Eiample 12 Anti-influenza vaccination in monkeys
In the example 11, we demonstrated that polyoxyethylene-9 lauryl ether
enhanced the
immunogenicity of influenza antigen in mice. In order to assess whether this
surfactant was able to exert a similar adjuvant effect in larger species,
African Green
monkeys (AGMs: 2 animals per group and per blood collection day) were primed
and
boosted intranasally (as in example 6) with 50 ug equivalent HA of inactivated
whole
AIBeijing/262/95 virus in 200 ~1 of A: PBS; B: 0.5% polyoxyethylene-9 lauryl
ether.
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At days 2, 7 and 14 after the boosting the sera were assayed for their
A/Beijing/262/95
virus-specific Ig Abs. Figure 19 shows clearly that when polyoxyethylene-9
lauryl
ether is used as an adjuvant, the immune response to influenza antigen is
improved.
Ezample 13 Vaccination studies with polysaccharide antigens
The preceding examples demonstrate the ability of polyoxyethylene-9 Iauryl
ether to
adjuvant the immune responses elicited to protein-type antigens. In this
example, we
examine whether this adjuvant is able to enhance the boosting effect of
nasally-
delivered polysaccharide antigens in mice primed parenteraIly. The mice were
primed
once subcutaneously with 100 ~,I injections containing S. pneumoniae PS14 and
PS19
polysaccharides {1 p,g each one) conjugated to the protein D carrier. Two
months
later, the mice were boosted intranasatly (under anesthesia) with 40 pl of
solution ( 10
pl per nostril at time 0 followed 30 minutes later by 10 p.l per nostril
again, delivered
as droplets by pipette) containing 1 p,g PS 14 and 1 g.g pS 19 conjugates in
either A:
NaCI 150 mM pH 6.1; B: 1% polyoxyethylene-9 lauryl ether. 14 days after the
boost
the sera were assayed for their PS 14 and PS 19-specific IgG Abs.
Results
As shown in figures 20 and 21, the administration of PS 14 or PS 19 by itself
induces a
boosting response which is further enhanced by addition of polyoxyethylene-9
lauryl
ether as an adjuvant.
Ezample 14 Polyoxyethylene-8 stearyl ether
In order to show that the adjuvant effect of polyoxyethylene-9 lauryl ether
can be
achicved using other polyoxyethylene ethers we investigated the use of
polyoxyethylene-8 stearyl ether.
Balblc mice primed as in example 2 were boosted intranasally with 10 ~,l
containing 5
ug of lipo-OspA in either A: PBS; B: 1 % polyoxyethylene-9 lauryl ether; C: 1
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polyoxyethylene-8 stearyl ether; or, D: by intramuscular injection of 1 p,g
lipo-OspA
adsorbed on SO pg Alum. 14 days after the boost the sera were analyzed as in
example
1.
Results
Figures 22 and 23 show that polyoxyethylene-8 stearyl ether is as potent as
polyoxyethylene-9 lauryl ether for enhancing the boosting response to the
antigen. Ab
titres reached with both polyoxyethylene ethers are similar to those elicited
by the
parenterat vaccine.
