U.S. patent application number 09/486996 was filed with the patent office on 2002-05-16 for vaccines.
Invention is credited to GARCON, NATHALIE, MOMIN, PATRICIA MARIE CHRISTINE ALINE FRANCOISE.
Application Number | 20020058047 09/486996 |
Document ID | / |
Family ID | 10818632 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058047 |
Kind Code |
A1 |
GARCON, NATHALIE ; et
al. |
May 16, 2002 |
VACCINES
Abstract
The present invention relates to an oil in water emulsion
compositions, their use in medicine, in particular to their use in
augmenting immune responses to a wide range of antigens, and to
methods of their manufacture; the oil in water emulsion comprising
a metabolisable oil, a saponin and a sterol.
Inventors: |
GARCON, NATHALIE; (WAVRE,
BE) ; MOMIN, PATRICIA MARIE CHRISTINE ALINE FRANCOISE;
(BRUSSELLS, BE) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION
CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Family ID: |
10818632 |
Appl. No.: |
09/486996 |
Filed: |
April 24, 2000 |
PCT Filed: |
September 2, 1998 |
PCT NO: |
PCT/EP98/05714 |
Current U.S.
Class: |
424/283.1 |
Current CPC
Class: |
A61K 2039/55566
20130101; A61K 9/0019 20130101; Y02A 50/30 20180101; Y10S 514/938
20130101; A61K 47/28 20130101; Y10S 514/937 20130101; A61K
2039/55577 20130101; A61K 39/39 20130101; A61P 31/22 20180101; Y10S
514/943 20130101; A61P 37/04 20180101; A61K 2039/55511 20130101;
Y02A 50/412 20180101; A61P 33/06 20180101 |
Class at
Publication: |
424/283.1 |
International
Class: |
A61K 039/12; A61K
045/00; A61K 047/44 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 1997 |
GB |
9718901.3 |
Claims
1. A composition comprising an oil in water emulsion and a saponin,
wherein the oil droplets of said oil in water emulsion comprises a
metabolisable oil and a sterol.
2. A composition as claimed in claim 1, where the sterol is
cholesterol.
3. A composition as claimed in either of claims 1 or 2, wherein
said metabolisable oil is squalene.
4. A composition as claimed in any of claims 1 to 3, wherein said
saponin is a derivative of QuilA, such as QS21.
5. A composition as claimed in any of claims 1 to 4, wherein the
ratio of QS21:cholesterol is in the range of 1:1 to 1:10 (w/w).
6. A composition as claimed in any of claims 3 to 5, wherein the
ratio of squalene:QS21 is in the range from 1:1 to 250:1 (w/w).
7. A composition as claimed in any of claims 3 to 6, wherein the
ratio of squalene:QS21 is substantially 48:1 (w/w).
8. A composition as claimed in any one of claims 1 to 7, further
containing one or more other inmmunomodulators.
9. A composition as claimed any in any one of claims 1 to 7,
further containing one or more other immunomodulators, which
immunomodulator is selected from the group comprising: 3D-MPL and
.alpha.-tocopherol.
10. A vaccine composition comprising a composition as claimed in
any one of claims 1 to 9, further comprising an antigen or
antigenic preparation.
11. A vaccine composition as claimed in claim 10, where the antigen
or antigenic preparation is prepared from the group comprising:
Human Immunodeficiency Virus; Herpes Simplex Virus type 1; Herpes
Simplex Virus type 2; Human Cytomegalovirus; Hepatitis A, B, C or
E; Respiratory Syncitial Virus, Human Papilloma Virus; Influenza
Virus; Salmonella; Neisseria; Borrelia; Chlamydia; Bordetella;
Plasmodium, Toxoplasma, tuberculosis, and EBV.
12. A vaccine composition as claimed in claim 10, wherein the
antigen or antigenic preparation is a combination of the Malaria
antigens RTS,S and TRAP.
13. A vaccine composition as claimed in claim 10, wherein the
antigen or antigenic preparation is, or is derived from, a tumour
or host derived antigen.
14. A composition as claimed in any one of claims 1 to 9, wherein
the oil in water emulsion comprises oil droplets which have a
diameter which is less that 1 micron.
15. A composition as claimed in any one of claims 1 to 9, wherein
the oil in water emulsion comprises oil droplets which are in the
range of 120 to 750 nm in diameter.
16. A composition as claimed in any one of claims 1 to 9, wherein
the oil in water emulsion comprises oil droplets which are in the
range of 120 to 600 nm in diameter.
17. A vaccine adjuvant composition as claimed in any one of claims
1 to 9 for use as a medicament.
18. A method for manufacturing a vaccine as claimed in claims 11,
12 and 13 comprising admixing (a) an oil in water emulsion wherein
the oil droplets comprise a sterol, such as cholesterol; (b) QS21;
and (c) an antigen or antigenic preparation.
19. Use of a composition as claimed in any one of claims 1 to 9, in
the manufacture of a medicament for the treatment of a human
susceptible to or suffering from a disease.
20. Treatment of an individual susceptible to or suffering from a
disease by the administration of a vaccine composition as claimed
in any one of claims 10 to 13.
21. Treatment of an individual susceptible to or suffering from a
disease by the administration of a composition as claimed in any
one of claims 1 to 9.
22. A method of stabilising a saponin present in the aqueous phase
of an oil in water emulsion, comprising the addition of a sterol
into the oil phase of said oil in water emulsion.
23. A method as claimed in claim 22, wherein the saponin is
QS21.
24. A method as claimed in claims 22 or 23, wherein the sterol is
cholesterol.
25. A method as claimed in claim 22, characterised in the oil phase
of said oil in water emulsion comprises squalene, said saponin is
QS21, and wherein the ratio of squalene:QS21 is substantially 48:1
(w/w).
26. A method of quenching the reactogenicity of an oil in water
emulsion composition, which composition comprises a saponin, said
method comprising the addition of a sterol into the oil droplets of
said oil in water emulsion.
Description
[0001] The present invention relates to an oil in water emulsion
compositions, their use in medicine, in particular to their use in
augmenting immune responses to a wide range of antigens, and to
methods of their manufacture; the oil in water emulsion comprising
a metabolisable oil, a saponin and a sterol.
[0002] Induction of cytotoxic T-cell (CTL) responses occurs
naturally during infection of a target cell, or uncontrolled
synthesis of a tumour antigen, wherein enzymatic degradation of the
target antigen takes place in the cell cytoplasm. This phenomenon
allows cytoplasmic peptides derived from the pathogen, or tumour
specific antigen, to enter the Th1 (endogenous antigen processing)
pathway and be presented on the surface of the cell associated with
an MHC class 1 molecule. If a vaccine antigen does not enter into
the cytoplasm of the host cell, then it might be taken up by the
cell and enter the exogenous antigen processing pathway and
ultimately be presented on the surface of the cell associated with
a MHC class II molecule. This alternative route generally results
in T-helper responses and antigen specific antibody responses.
[0003] After conventional vaccination with subunit or non-living
vaccines, antigen generally does not enter the cytoplasm of a host
cell, and therefore will not enter the endogenous antigen
processing pathway and ultimately will not induce a CTL response.
CTL induction is believed to correlate with Th-1 cytokine profile
responses, specifically with IFN-.gamma. and IL-2 secretion.
IFN-.gamma. secretion is associated with protective responses
against intracellular pathogens, including parasites, bacteria and
viruses. Activation of leucocytes by IFN-.gamma. enhances killing
of intracellular pathogens and increases expression of Fc
receptors. Direct cytotoxicity may also occur, especially in
synergy with lymphotoxin (another product of TH1 cells).
IFN-.gamma. is also both an inducer and a product of NK cells,
which are major innate effectors of protection. TH1 type responses,
either through IFN-.gamma. or other mechanisms, provide
preferential help for murine IgG2a and human IgG1 immunoglobulin
isotypes.
[0004] International patent application No. WO 95/17210 discloses
an adjuvant emulsion system based on squalene, .alpha.-tocopherol,
and polyoxyethylene sorbitan monooleate (TWEEN 80), optionally
formulated with the immunostimulants QS21 and/or 3D-MPL. This
adjuvant formulation is a very potent inducer of a wide range of
immune responses.
[0005] These oil in water emulsions, when formulated with 3
De-O-acylated monophosphoryl lipid A (3D-MPL) and QS21 are potent
inducers of Th1 type immune responses. Accordingly, this system
when associated with antigen preferentially stimulate the
sub-isotype of IgG associated with Th1 responses (for example,
murine IgG2a and human IgG1) and also will induce significant
levels of IFN-.gamma. production and antigen specific CTL
responses. The observation that the basic oil in water/QS21/3D-MPL
formulation can induce strong CTL responses is significant, as
these responses in certain animal models have been shown to induce
protection against disease.
[0006] Immunologically active saponin fractions (e.g. Quil A)
having adjuvant activity derived from the bark of the South
American tree Quillaja Saponaria Molina are known in the art.
Derivatives of Quil A, for example QS21 (an HPLC purified fraction
derivative of Quil A), and the method of its production is
disclosed in U.S. Pat. No. 5,057,540. Amongst QS21 (known as QA21)
other fractions such as QA17 are also disclosed. The use of such
saponins in isolation is accompanied with disadvantage in that
local necrosis, that is to say, localised tissue death, occurs at
the injection site, thereby leading to pain.
[0007] 3 De-O-acylated monophosphoryl lipid A is a well known
adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is
often supplied as a mixture of 3 De-O-acylated monophosphoryl lipid
A with either 4, 5, or 6 acylated chains. It can be prepared by the
methods taught in GB 2122204B. A preferred form of 3 De-O-acylated
monophosphoryl lipid A is in in the form of an emulsion having a
small particle size less than 0.2 .mu.m in diameter, and its method
of manufacture is disclosed in European Patent No. EP0 671 948
B1.
[0008] In order for any oil in water composition to be suitable for
human administration, the oil phase of the emulsion system has to
comprise a metabolisable oil. The meaning of the term metabolisable
oil is well known in the art. Metabolisable can be defined as
"being capable of being transformed by metabolism" (Dorland's
Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition
(1974)). The oil may be any vegetable oil, fish oil, animal oil or
synthetic oil, which is not toxic to the recipient and is capable
of being transformed by metabolism. Nuts, seeds, and grains are
common sources of vegetable oils. Synthetic oils are also part of
this invention and can include commercially available oils such as
NEOBEE.RTM. and others. Squalene
(2,6,10,15,19,23-Hexamethyl-2,6,10,14- ,18,22-tetracosahexaene) is
an unsaturated oil which is found in large quantities in
shark-liver oil, and in lower quantities in olive oil, wheat germ
oil, rice bran oil, and yeast, and is a particularly preferred oil
for use in this invention. Squalene is a metabolisable oil virtue
of the fact that it is an intermediate in the biosynthesis of
cholesterol (Merck index, 10th Edition, entry no.8619).
[0009] Oil in water emulsions per se are well known in the art, and
have been suggested to be useful as adjuvant compositions (EPO
399843).
[0010] The oil in water emulsions described in International patent
application No. WO 95/17210 obviously hold great advantages over
conventional non-Th1 inducing adjuvants. However, the inclusion of
QS21 has so far made this potent adjuvant reactogenic, leading to
pain at the site of injection.
[0011] Formulations comprising QS21 with a sterol are known from
International Patent Application No. PCT/EP96/01464, No oil in
water emulsions disclosed in this document. Sterols are well known
in the art, for example cholesterol is well known and is, for
example, disclosed in the Merck Index, 11th Edn., page 341, as a
naturally occurring sterol found in animal fat.
[0012] The present inventors have found that oil in water
formulations containing a sterol, and QS21, have reduced local
reactogenicity after injection into a host with respect to
comparable emulsions formulated without a sterol. This is
surprising since sterols, being oil soluble, would be expected to
dissolve into the heart of the oil droplet, whilst on the other
hand QS21 is primarily expected to be associated with the aqueous
phase. Therefore, it would be expected that the sterol would be
physically distinct from the QS2 1. Nonetheless, these formulations
are surprisingly less reactogenic than those oil in water emulsions
not containing a sterol.
[0013] Accordingly, one preferred embodiment of the present
invention provides a composition comprising a saponin, an oil in
water emulsion, and a sterol. Especially preferred embodiments of
this include compositions wherein the saponin is the non-toxic
fraction of Quil A known as QS21, the oil in water emulsion
comprises a metabolisible oil, such as squalene, and wherein the
sterol is cholesterol. Such a composition may further comprise
other immunomodulators including: .alpha.-tocopherol and
polyoxyethylene sorbitan monooleate (TWEEN 80), and 3D-MPL. The
inclusion of cholesterol in the formulation much reduces the local
reactogenicity of the composition once injected into a recipient.
Other sterols that can easily act as alternatives for cholesterol
include .beta.-sitosterol, stigmasterol, ergosterol, and
ergocalciferol.
[0014] Such embodiments of the present invention are used as
vaccine adjuvant systems, and once combined with antigen form
potent vaccines. Advantageously they preferentially induce a Th1
response.
[0015] Embodiments of the present invention include composition
comprising an oil in water emulsion, a saponin and a sterol,
characterised in that a reduced reatogenicity profile is induced
upon administration to a host in comparison to the reactogenicity
profile observed after administration of the same composition from
which the sterol has been omitted.
[0016] Previous examples of oil in water adjuvant emulsions as
disclosed in International patent application No. WO 95/17210
involved large quantities of squalene. The ratio of
squalene:saponin (w/w) in such vaccine preparations was in the
region of 240:1. An additional benefit that the addition of
cholesterol bestows is the opportunity to reduce the total level of
oil in the emulsion. This leads to a reduced cost of manufacture,
improvement of the overall comfort of the vaccination, and also
qualitative and quantitative improvements of the resultant immune
responses, such as improved IFN-.gamma. production. Accordingly,
the adjuvant system of the present invention typically comprises a
ratio of squalene:saponin (w/w) in the range of 200:1 to 300:1,
also the present invention can be used in a "low oil" form the
preferred range of which is 1:1 to 200:1, preferably 20:1 to 100:1,
and most preferably substantially 48:1, this vaccine retains the
beneficial adjuvant properties of all of the components, with a
much reduced reactogenicity profile. Accordingly, the particularly
preferred embodiments have a ratio of squalene:QS21 (w/w) in the
range of 1:1 to 250: 1, also a preferred range is 20:1 to 200:1,
preferably 20:1 to 100:1, and most preferably substantially
48:1.
[0017] The emulsion systems of the present invention have a small
oil droplet size in the sub-micron range. Preferably the oil
droplet sizes will be in the range 120 to 750 nm, and most
preferably from 120-600 nm in diameter.
[0018] The formulations of the invention are suitable for a broad
range of monovalent or polyvalent vaccines, once combined with an
antigen or antigenic composition/combination. Additionally the oil
in water emulsion may contain 3 de-O-acylated monophosphoryl lipid
A (3D-MPL) and/or polyoxyethylene sorbitan trioleate (such as SPAN
85). Additionally the preferred form of 3 De-O-acylated
monophosphoryl lipid A is disclosed in International patent
application published under No. 92116556-SmithKline Beecham
Biologicals s.a.
[0019] 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,
gp120 or gp160), human herpes viruses, such as gD or derivatives
thereof or Immediate Early protein such as ICP27 from HSV1 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, 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 binding proteins, PilC,
adhesins); Streptococcus spp, including S. pneumoniae (for example
capsular polysaccharides and conjugates thereof, PsaA, PspA,
streptolysin, choline-binding proteins), S. pyogenes (for example M
proteins or fragments thereof, C5A protease, lipoteichoic acids),
S. agalactiae, S. mutans; Haemophilus spp, including H. influenzae
type B (for example PRP and conjugates thereof), non typeable H.
influenzae (for example OMP26, high molecular weight adhesins, P5,
P6, lipoprotein D), 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 thereof); Bacillus spp., including B.
anthracis (for example botulinum toxin and derivatives thereon,;
Corynebacterium spp., including C. diphtheriae (for exarnple
diphtheria toxin and derivatives thereof); Borrelia spp., including
B. burgdorferi (for exarnple 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 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.
[0020] Derivatives of Hepatitis B Surface antigen are well known in
the art and include, inter alia, those PreS1, 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.
[0021] In a preferred embodiment of the present invention vaccines
containing the claimed adjuvant comprise the HPV viruses considered
to be responsible for genital warts, (HPV 6 or HPV 11 and others),
and the HPV viruses responsible for cervical cancer (HPV16, HPV18
and others). Particularly preferred forms of vaccine comprise L1
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 GB 95 15478.7, and proteinD(1/3)-E7 disclosed in GB
9717953.5.
[0022] Vaccines of the present invention further 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. It's 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, RAP 1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP,
SALSA, PfEXP1, Pfs25, Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and
their analogues in Plasmodium spp.
[0023] 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, 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.
[0024] 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.
[0025] 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 the stanworth decapeptide).
[0026] The ratio of the QS21 to cholesterol (w/w), present in a
preferred embodiment of the present invention, is envisaged to be
in the range of 1:1 to 1:20, substantially 1 :10.
[0027] The ratio of QS21:3D-MPL (w/w) will typically be in the
order of 1:10 to 10:1; preferably 1:5 to 5:1 and often
substantially 1:1. The preferred range for optimal synergy is 2.5:1
to 1:1 3D MPL: QS21. Typically for human administration QS21 and 3D
MPL will be present in a vaccine in the range 1 .mu.g-100 .mu.g,
preferably 10 .mu.g-50 .mu.g per dose. Typically the oil in water
will comprise from 2 to 10% squalene, from 2 to 10%
.alpha.-tocopherol and from 0.4 to 2% polyoxyethylene sorbitan
monooleate (TWEEN 80). Preferably the ratio of squalene:
.alpha.-tocopherol is equal or less than 1 as this provides a more
stable emulsion. Sorbitan trioleate (SPAN 85) may also be present
at a level of 0.5 to 1%. In some cases it may be advantageous that
the vaccines of the present invention will further contain a
stabiliser, for example other emulsifyers/surfactants, including
Caprylic acid (merck index 10th Edition, entry no.1739), of which
Tricaprylin is a particularly preferred embodiment.
[0028] Vaccine preparation is generally described in New Trends and
Developments in Vaccines, edited by Voller et al., University Park
Press, Baltimore, Md., U.S.A. 1978. Conjugation of proteins to
macromolecules is disclosed by Likhite, U.S. Pat. No. 4,372,945 and
by Armor et al., U.S. Pat. No. 4,474,757.
[0029] 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 .mu.g of protein, preferably 1-500 .mu.g,
preferably 1-100 .mu.g, of which 1 to 50 .mu.g is the most
preferable range. 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
immunisations adequately spaced.
[0030] The compositions of the present invention can 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 the histamine releasing
decapeptide of IgE (known as the Stanworth decapeptide),
recombinantly produced protein and peptides, and chimeric fusion
proteins.
[0031] In a further aspect of the present invention there is
provided a vaccine as herein described for use in medicine.
[0032] Also provided by the present invention is a method of
quenching the reactogenicity of a saponin, preferably QS21,
containing oil in water emulsion, comprising the addition of a
sterol, preferably cholesterol, into the oil phase of the oil in
water emulsion.
[0033] QS21 in aqueous solution is known to degenerate over time
into an adjuvant-inactive form, QS21-H, which degeneration is
mediated by OH hydrolysis by the aqueous medium. Such degeneration
may occur when the QS21 is present in the aqueous phase of an oil
in water adjuvant. Surprisingly it has been found that the addition
of cholesterol to the oil phase of the oil in water emulsion has
the effect of maintaining the QS21 in its active form, with obvious
benefits to the shelf-life of the adjuvant/vaccine formulation. The
present invention provides a method of stablilising a preparation
of a saponin, preferably QS21, in its non-hydrolysed
adjuvant-active form, when the QS21 is present in an oil in water
emulsion based adjuvant. This method comprises the addition of a
sterol, preferably cholesterol, into the oil phase of an oil in
water emulsion.
[0034] Also provided by the present invention is the process for
the production of an adjuvant or vaccine preparation comprising the
addition of cholesterol to a metabolisable oil, followed by
emulsification of the oil phase; into which emulsion is added QS21,
and optionally 3D-MPL, .alpha.-tocopherol, and antigen.
[0035] The vaccine preparation of the present invention may be used
to protect or treat a mammal susceptible to, or suffering from a
disease, by means of administering said vaccine via systemic or
mucosal route. These administrations may include injection via the
intramuscular, intraperitoneal, intradermal or subcutaneous routes;
or via mucosal administration to the oral/alimentary, respiratory,
genitourinary tracts.
EXAMPLE 1
Preparation of the Oil in Water Emulsion Adjuvants
[0036] The oil in water emulsion adjuvant formulations used in the
subsequent examples were each made comprising the following oil in
water emulsion component: 5% Squalene, 5% .alpha.-tocopherol, 2.0%
polyoxyethylene sorbitan monooleate (TWEEN 80).
[0037] The emulsion was prepared as follows as a 2 fold
concentrate. All examples used in the immunological experiments are
diluted with the addition of extra components and diluents to give
either a 1.times.concentration (equating to a squalene:QS21 ratio
(w/w) of 240:1) or further dilutions thereof.
[0038] Briefly, the TWEEN 80 is dissolved in phosphate buffered
saline (PBS) to give a 2% solution in the PBS. To provide 100 ml of
a two fold concentrate emulsion, 5 ml of DL alpha tocopherol and 5
ml of squalene are vortexed to mix thoroughly. 95 ml of PBS/Tween
solution is added to the oil and are mixed thoroughly. The
resulting emulsion is then passed through a syringe needle and
finally microfluidised by using an M110S Microfluidics machine. The
resulting oil droplets have a size of approximately 145-180 nm
(expressed as z av. measured by PCS) and is termed "full dose"
SB62.
[0039] These formulations can be sterile filtered through a 0.2
.mu.m filter. The other adjuvant/vaccine components (QS21, 3D-MPL
or antigen) are added to the emulsion in simple admixture.
[0040] The antigen containing vaccines used herein are formulated
either with full dose SB62 adjuvant to give a high squalene:QS21
ratio (240:1) or with a lower amount of emulsion to give a low
ratio formulation (48:1), these adjuvant formulations are called
SB62 and SB62' respectively. Other vaccines were formulated with
the addition of cholesterol to the oil phase of the emulsion prior
to the emulsifying process, wherein the QS21:cholesterol ratio of
1:10 (denoted by the addition of the letter "c").
EXAMPLE 2
Reactogenicity Studies with Vaccines Comprising Oil in Water
Emulsions and QS21 with the Optional Addition of Cholesterol
[0041] A study was conducted to examine the local and systemic
reactogenicity of various vaccine formulations containing the
Herpes Simplex Virus (HSV) glycoprotein gD2t. Oil in water vaccine
(o/w) adjuvants containing QS21 are known to produce moderate
adverse effects upon administration to a host. This study compared
the reactogenic profile resulting from vaccination with a gD2t/o/w
vaccine, with that from the same vaccine formulations which further
contained cholesterol.
[0042] Reactogenicity Study Experimental Procedure
[0043] Groups of 5 SPF bred New Zealand White albino rabbits were
inoculated by intramuscular injection into the right hind leg
muscle (gastrocnemius), with 0.5 ml of the adjuvant preparations
(for details of production see example 1). Samples were taken
before and after vaccination to assay the percentage blood
polymorpho-neutrophils (as a measure of inflammation, % PMN), and
Creatine phosphokinase (as a measure of muscle damage, CPK). The
animals were sacrificed 3 days after vaccination for histological
examination of the injection site.
1TABLE 1 Groups of animals and formulations used in example 2.
Adjuvant formulation Antigen MPL QS21 SB62 Chol PBS o/w Group
gD2t(.mu.g) (.mu.g) (.mu.g) (.mu.l) (.mu.g) (.mu.l) dose 1 20 50 50
250 -- 250 1/1 2 20 50 50 125 -- 375 1/2 3 20 50 50 83.3 -- 416.7
1/3 4 20 50 50 62.5 -- 437.5 1/4 5 20 50 50 50 -- 450 1/5 6 -- --
-- -- -- 500 -- 7 20 50 50 250 500 250 1/1 8 20 50 50 125 500 375
1/2 9 20 50 50 83.3 500 416.7 1/3 10 20 50 50 62.5 500 437.5 1/4 11
20 50 50 50 500 450 1/5 12 -- -- -- -- -- 500 -- footnotes: SB62 =
full dose oil in water emulsion PBS = Phosphate Buffered Saline
[0044] In this experiment the vaccine preparation in group 1 the
SB62 stock preparation is diluted with the addition of extra
components and diluents to give a 1.times.concentration (1/1). In
other groups the SB62 final dilution varies between 1/2 to 1/5.
Groups 1 to 5 have a squalene:QS21 ratio (w/w) of 240:1, 120;1,
80:1, 60:1 and 48:1 respectively.
[0045] The antigen used in this study is a truncated HSV-2
glycoprotein D of 308 amino acids, which comprises amino acids 1
through 306 naturally occurring glycoprotein with the addition
Asparagine and Glutamine at the C terminal end of the truncated
protein devoid of its membrane anchor region. This form of the
protein includes the signal peptide which is cleaved to yield a
mature 283 amino acid protein. The production of such a protein in
Chinese Hamster ovary cells has been described in Genentech's
European patent EP-B-139 417. The antigen is used in the vaccine
formulations of the present invention as is designated
gD.sub.2t.
[0046] CPK levels, in units per litre (U/L), were determined from
serum at various time points throughout the experiment, using
commercially available reagents (Abbot) and a Abbot Vision System
analyser. Levels of PMN in blood samples were determined
concurrently using a Sysmex K-1000 Haematology analyser (Toa
Medical Electronics Co.).
[0047] Reactogenicity Results
[0048] CPK Levels
2TABLE 2 CPK concentrations pre and post injection. Mean CPK (U/L)
Group Day 0 (SD) Day 1 (SD) Day 3 (SD) 1 1093(202) 3308(2013)
1995(1047) 2 818(215) 3701(1430) 1842(915) 3 784(228) 3346(1434)
2321(780) 4 946(228) 2963(1246) 2316(593) 5 808(686) 3976(1311)
1963(1177) 6 726(163) 769(107) 1208(388) 7 1687(527) 994(541)
1667(249) 8 1006(309) 836(469) 1408(1113) 9 1367(536) 1012(462)
1171(503) 10 899(373) 1083(737) 731(282) 11 1137(310) 952(257)
1610(441) 12 1086(713) 1078(321) 1475(1642)
[0049] From table 2, it is clear that when cholesterol was added to
these formulations, no muscle damage was observed in terms of CPK.
CPK levels were substantially the same as, or lower than, those
seen before vaccination or after vaccination with PBS. Vaccine
preparations not containing cholesterol induced a significant
increases in blood CPK levels on day 1. These CPK levels were
independent of SB62 dilution.
[0050] % PMN Results
[0051] The results relating to the % PMN observed after vaccination
can be summarised by the following. A transient PMN burst was
observed on day 1 in all animals injected, independent of SB62
dilution and presence of cholesterol.
[0052] Conclusions
[0053] The addition of cholesterol suppresses the reactogenicity in
terms of muscle damage of QS21/o/w emulsion adjuvant
formulations.
[0054] The addition of cholesterol did not influence the induction
of the desirable inflammatory response. The effect on the CPK
levels and PMN burst effect was independent of the amount of o/w
emulsion present in the vaccine formulation.
EXAMPLE 3
Immunogenicity Studies in Mice with the Glycoprotein gD2t from
HSV
[0055] A study was conducted in Balb/C mice with oil in water
emulsion vaccine formulations using the Herpes Simplex Virus
glycoprotein gD2t as the antigen. The study investigated the
induction of gD2t specific humoral and cellular immune responses
(cytokine production and cellular proliferation), and investigated
the consequences of the addition of cholesterol to the
formulation.
[0056] Groups of 10 Balb/C mice were immunised in the rear footpads
(50 .mu.l per footpad) with the following formulations, at days 0
and 28:
3TABLE 3 Groups of mice and vaccine formulations used in example 3.
Group Vaccine formulation 13 gD2t (2 .mu.g)/3D-MPL(5 .mu.g)/QS21(5
.mu.g)/SB62(25 .mu.l) 14 gD2t (2 .mu.g)/3D-MPL(5 .mu.g)/QS21(5
.mu.g)/SB62(25 .mu.l)/ Cholesterol(50 .mu.g) 15 gD2t (2
.mu.g)/3D-MPL(5 .mu.g)/Alum(50 .mu.g)
[0057] The vaccines were prepared using the SB62 oil in water
emulsion adjuvants as described in example 1. The antigen used in
this study is a truncated HSV-2 glycoprotein D of 308 amino acids,
which comprises amino acids 1 through 306 naturally occurring
glycoprotein with the addition Asparagine and Glutamine at the C
terminal end of the truncated protein devoid of its membrane anchor
region. This form of the protein includes the signal peptide which
is cleaved to yield a mature 283 amino acid protein. The production
of such a protein in Chinese Hamster ovary cells has been described
in Genentech's European patent EP-B-139 417. The antigen is used in
the vaccine formulations of the present invention as is designated
gD2t.
[0058] Serology
[0059] Sera was obtained from 5 mice from each group at 14 days
after the second immunization, and again from the 5 remaining mice
28 days after the second immunization. Each serum sample was tested
in ELISA for anti-gD2t Ig titers and the isotype distribution using
pooled sera was measured.
[0060] Cytokine Production
[0061] Spleen and lymph node cells were also isolated 14 and 28
days (n=5) after the second immunisation. Pooled samples were
analysed for both gD2t-specific proliferation and cytokine
(IFN-.gamma. and IL-5) secretion.
[0062] Results from the Mouse Studies
[0063] The results from the mice immunised with the gD2t vaccines
are summarised in the following table. The magnitude of the
response with respect to each parameter measured is indicated by
the number of "+" signs.
4TABLE 4 Summary table showing the anti-gD2t immune responses in
mice (example 3). Cytokine Serology production Group IgG IgG 2a and
b IL-5 IFN-.gamma. 13 + + + + + + + + + 14 + + + + + + + + + + 15 +
+ + + + +
[0064] The addition of cholesterol did not, therefore, effect the
magnitude or quality of the anti-gD2t immune responses in the
murine model.
EXAMPLE 4
Immunogenicity Studies in Rhesus Monkeys with the Glycoprotein gD2t
from HSV
[0065] Groups of 5 rhesus monkeys were immunuised intramuscularly
in the posterior part of the right leg (500 .mu.l) with the
following vaccine formulations:
5TABLE 5 Vaccine formulations used in the Rhesus monkey model
(example 4). Group Vaccine formulation 16 gD2t (20 .mu.g)/3D-MPL(50
.mu.g)/QS21(50 .mu.g)/SB62(250 .mu.l) 17 gD2t (20 .mu.g)/3D- MPL(50
.mu.g)/QS21(50 .mu.g)/SB62(250 .mu.l)/Cholesterol(500 .mu.g) 18
gD2t (20 .mu.g)/3D-MPL(50 .mu.g)/Alum(500 .mu.g)
[0066] The vaccines were prepared using the SB62 emulsion adjuvants
as described in example 1. The antigen used in this study is a
truncated HSV-2 glycoprotein D of 308 amino acids, which comprises
amino acids 1 through 306 naturally occurring glycoprotein with the
addition Asparagine and Glutamine at the C terminal end of the
truncated protein devoid of its membrane anchor region. This form
of the protein includes the signal peptide which is cleaved to
yield a mature 283 amino acid protein. The production of such a
protein in Chinese Hamster ovary cells has been described in
Genentech's European patent EP-B-139 417. The antigen is used in
the vaccine formulations of the present invention as is designated
gD2t.
[0067] The monkeys were vaccinated on days 0, 28 and 84. Serum was
taken from each monkey at 14, 28, and 42 days after the third
vaccination. Each serum was tested in ELISA for anti-gD2t Ig
titres. Results were expressed as ELISA units (EU). Neutralisation
assays were also performed, which evaluated the ability of serial
dilutions of the sera to neutralise in vitro the infectivity of
HSV-2 (strain HG-52). Results were expressed as mid-point titres
after regression analysis.
[0068] A DTH test was performed at 42 days after the third
vaccination. 28 .mu.g gD2t diluted in PBS (total volume of 100
.mu.l) was injected intradermally in duplicate. Controls consisted
of PBS alone. Skin thickness was measured prior to, and 24 hours
after, injection. Data was expressed as specific increase of skin
thickness (difference between the site injected with gD2t and the
site injected with PBS).
[0069] Results from the Rhesus Monkey Studies
[0070] The results from the Rhesus monkeys immunised with the gD2t
vaccines are summarised in the following table (data shown is the
mean from the 5 monkeys).
6TABLE 6 Summary table showing the anti gD2t immune responses
induced in Rhesus monkeys. Immune parameter Serum anti-gD2t
Neutralisation titre Group titre (14d post VII)* (28d post VIII)*
DTH (mm)** 16 14,000 900 0.9 17 16,000 800 1 18 4000 400 1.1
*geometric mean titre of the group **average of the group
[0071] Conclusions
[0072] Both of the SB62 based vaccine formulations induced very
high titres of anti-gD2t antibodies in the monkeys. Furthermore,
these vaccines also induced neutralising antibody and stimulated
DTH responses. The inclusion of cholesterol had no effect on the
performance of the vaccines.
EXAMPLE 5
Reactogenicity Studies with Oil in Water Emulsion Adjuvants with
QS21 with the Optional Addition of Cholesterol
[0073] A study was conducted to examine the local and systemic
reactogenicity of various adjuvant formulations. Oil in water
vaccine adjuvants containing QS21 are known to produce moderate
adverse clinical symptoms upon administration to a host. This study
compared the resultant reactogenic profile with that resulting from
the same adjuvant formulations which further contained
cholesterol.
[0074] Experimental Procedure
[0075] The oil in water emulsions tested were produced using
techniques described in example 1. Groups of 5 SPF bred New Zealand
White albino rabbits were inoculated by intramuscular injection
into the right hind leg muscle (gastrocnemius), with 0.5 ml of the
adjuvant preparations. Samples were taken before and after
vaccination to assay the percentage blood polymorpho-neutrophils
(as a measure of inflammation, % PMN), and Creatine phosphokinase
(as a measure of muscle damage, CPK). The animals were sacrificed 3
days after vaccination for histological examination of the
injection site.
7TABLE 7 Groups of rabbits used in example 5.: Group Vaccine
preparation 19 SB62', QS21(50 .mu.g), MPL(50 .mu.g) 20 SB62'c,
Q521(50 .mu.g), MPL(50 .mu.g) 21 QS21(50 .mu.g) 22 QS21(50 .mu.g)
23 SB62 24 SB62c 25 PBS footnotes: SB62 = full dose oil in water
emulsion SB62' = 1/5th dose SB62 SB62'c, = SB62' containing
cholesterol in the oil phase PBS = Phosphate Buffered Saline
[0076] CPK levels, in units per litre (U/L), were determined from
serum at various time points throughout the experiment, using
commercially available reagents (Abbot) and a Abbot Vision Systems
analyser. % PMN in blood samples were determined concurrently using
a Sysmex K-1000 Haematology analyser (Toa Medical Electronics
Co.).
[0077] Three days after injection post-mortem inspection of the
rabbits determined lesion size at injection site, and local
histopathology.
[0078] Results
[0079] CPK Levels Pre and Post Vaccination
8TABLE 8 Mean CPK concentrations in Rabbits Mean CPK (U/L) Group
Day 0 (SD) Day 1 (SD) Day 3 (SD) 19 764 (545) 2868 (1284) 1539
(487) 20 1364 (1842) 871 (543) 1360 (309) 21 400 (191) 2860 (1405)
1364 (552) 22 962 (783) 1650 (343) 1370 (475) 23 863 (762) 719
(306) 1164 (426) 24 606 (274) 599 (172) 1336 (779) 25 401 (107) 778
(176) 666 (164)
[0080] From Table 8, above, it can be seen that adjuvant
formulations containing QS21 show a marked increase in plasma CPK
levels 1 day after injection, indicating a significant level of
muscle damage at the site of injection (groups 19, 21, and 22).
[0081] The addition of cholesterol to the adjuvant formulation
quenches this effect and as such no increases in CPK are seen after
vaccination (group 20). The results attained using this adjuvant
are very similar to those obtained with PBS or SB62 given alone
(see groups 23, 24, and 25).
9TABLE 9 Percentage PMN changes in blood % PMLN % PMN Day-5 Day-1
Day 0 Group (SD) (SD) (SD) Day 1 (SD) Day 3 (SD) 19 14.1 (2.1) 18.9
(2.1) 15.2 (1.8) 34.0 (4.6) 12.9 (1) 20 17.3 (1.1) 20.7 (3.2) 18.0
(2.8) 40.2 (8.4) 16.7 (3.9) 21 15.8 (1.9) 18.4 (1.6) 15.6 (1.3)
25.7 (5.6) 14.3 (2.2) 22 14.3 (2.3) 16.3 (2.2) 14.7 (1.9) 14.6
(3.2) 15.9 (2.7) 23 15.5 (1.2) 16.5 (1.4) 15.7 (2.1) 31.9 (7.1)
14.0 (3.2) 24 16.5 (1.7) 20.0 (2.7) 13.6 (2.6) 32.3 (2.7) 14.9
(2.5) 25 15.0 (2.9) 18.2 (2.9) 16.4 (2.2) 16.1 (5.0) 12.4 (2.2)
[0082] The percentage of blood PMN is taken to be a readout of the
magnitude of the local inflammation reaction in response to
vaccination. As can be seen from table 9, the addition of
cholesterol to the QS21 containing adjuvant formulation does not
significantly affect the inflammatory process at day 1
post-vaccination, despite the absence of muscle damage as indicated
in table 8 . The addition of cholesterol does not affect the
inflammatory process induced by SB62.
10TABLE 10 Histological examination Site of injection
Histopathology Group Rabbit les size (mm) necro rhabdo infiltr
oedem haemo Remark 19 1 + 3 .times. 3 .times. 2 1 1 3 2 2 2 + 30
.times. 10 .times. 6 4 2 3 2 2 3 + 25 .times. 18 .times. 6 4 3 3 2
3 4 + 28 .times. 10 .times. 5 4 2 3 2 3 5 + 27 .times. 14 .times. 4
4 2 3 2 2 20 6 + 3 .times. 2 .times. 2 1 2 3 1 2 7 s 0 0 0 1 0 0 8
- 0 1 0 1 0 0 9 - 0 1 2 2 1 1 10 + 4 .times. 2 .times. 2 1 2 3 1 2
21 11 + 24 .times. 13 .times. 5 4 2 3 2 3 12 + 25 .times. 12
.times. 7 4 3 3 2 3 13 + 22 .times. 12 .times. 4 4 2 3 2 3 14 + 15
.times. 10 .times. 4 4 2 3 2 3 15 + 22 .times. 12 .times. 7 4 3 3 2
3 22 16 + 14 .times. 7 .times. S 3 3 3 2 3 17 + 8 .times. 4 .times.
2 3 2 3 2 4 18 + 10 .times. 8 .times. 3 3 1 3 2 1 19 + 4 .times. 3
.times. 2 2 2 3 2 4 20 + 12 .times. 7 .times. 2 2 3 3 2 3 23 21 - 0
0 2 2 1 1 22 s 0 0 1 2 1 1 23 - 0 0 0 1 0 0 24 - 0 0 0 0 0 0 25 - 0
0 0 0 0 0 24 26 - 0 0 1 2 0 0 27 s 0 0 1 2 0 0 28 - 0 0 1 2 0 0 29
- 0 0 0 2 0 0 30 s 0 1 2 2 1 1 25 31 s 0 0 0 1 0 0 32 s 0 0 0 0 0 0
33 s 0 0 0 2 1 1 34 - 0 0 0 0 0 0 35 - 0 0 0 0 0 0 footnotes: les =
lesion necro = necrosis infiltr = lymphocytic infiltration oedem =
oedema haemo = haemorrhage rhabdo = rhabdomyolosis Gradation: 0 =
no sign 1 = very slight 2 = slight 3 = moderate 4 = severe + =
present s = spot - = no sign
[0083] Histological examination at the site of injection confirms
the earlier CPK data shown in table 8, in that local damage was
reduced significantly by the addition of cholesterol into the
adjuvant formulation.
[0084] Severe necrosis, accompanied with moderate rhabdomyolosis
oedema and haemorrhage was observed with all vaccine adjuvants
containing "unquenched" QS21 (groups 19, 21, and 22) These signs
were associated with very large lesions at the site of
injection.
[0085] The inclusion of cholesterol (group 20) reduced the
macroscopical appearance of lesions (in only 2 of the 5 rabbits)
when compared to those observed in group 19, and significantly
reduced the severity of other histopathological signs when
present.
[0086] Conclusions
[0087] It is clear from the results described above that the use of
the adjuvant formulations comprising QS21, or indeed QS21 alone,
cause a significant amount of local damage at the site of
injection. This deleterious effect can be successfully abrogated by
the inclusion of cholesterol to the adjuvant formulation.
EXAMPLE 6
[0088] Immunogenicity Studies with Malaria Antigens TRAP and
RTS,S
[0089] Imnmunisation experiments using the Plasmodium falciparum
Malaria antigens TRAP and RTS,S in combination with various
adjuvants, each based on an oil in water emulsion system. 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 preS.sub.2 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 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.
[0090] TRAP antigens are described in the International Patent
Application No. PCT/GB89/00895, published under WO 90/01496. TRAP
antigens are polypeptides, so called Thrombospondin Related
Anonymous Proteins, which share homology with various P. falciparum
proteins.
[0091] Various formulations with two different squalene:QS21
ratios, optionally with cholesterol at a QS21 cholesterol ratio
(w/w) of 1:10, were combined with the malaria antigens and compared
in their ability to induce humoral and cell mediated immune
responses (T-cell proliferation and cytokine production). These
formulations were produced using the techniques described in
example 1.
[0092] Groups of 5 mice (six weeks old female mice, strain
C57/BL6.times.CBA/J[H-2k]) were immunised twice (in 2.times.50
.mu.l volumes) in the hind foot-pad, 14 days apart, with either 10
.mu.g RTS,S or 4 .mu.g TRAP combined with various oil in water
emulsion systems (SB62). 14 days following the second immunisation
the production of cytokines (IL5 and IFN-.gamma.) and T-cell
proliferation was analysed after in vitro restimulation of spleen
and lymph nodes cells with the malaria antigens. Antibody response
to RTS,S and TRAP and the isotypic profile that was induced was
investigated by ELISA.
11TABLE 11 Animal Groups Group No. Antigen Adjuvant 26 RTS,S
SB62/QS21/3D-MPL 27 TRAP SB62/QS21/3D-MPL 28 RTS,S/TRAP
SB62/QS21/3D-MPL 29 RTS,S AlOH/QS21/3D-MPL 30 RTS,S/TRAP
AlOH/QS21/3D-MPL 31 RTS,S SB62c/QS21/3D-MPL 32 RTS,S/TRAP
SB62c/QS21/3D-MPL 33 RTS,S SB62'/QS21/3D-MPL 34 RTS,S/TRAP
SB62'/QS21/3D-MPL 35 -- SB62/QS21/3D-MPL 36 Vac. Vir. 3D7
Footnotes: SB62-oil in water emulsion full dose SB62'-oil in water
emulsion exemplified in the figures as SB62 1/5th dose SB62c or
SB62'c-oil in water emulsion (either dose) plus cholesterol in the
oil phase. Vac. Vir. 3D7 = a recombinant vaccinia virus construct
expressing CS protein and administered at 10.sup.6PFU per
mouse.
[0093] Methodology
[0094] T-cell Proliferation
[0095] Spleen or popliteal lymph node cells were aseptically
removed and washed. 100 .mu.l of cells in RPMI medium (1%
heat-inactivated normal mouse serum, NMS) containing
2.times.10.sup.6/ml of cells were cultured in round bottomed plates
in the presence of RTS,S or TRAP antigens. Following stimulation
for 96 hours with 0.1, 0.5, and 2.5 .mu.g of antigen, or 48 hours
with 2 .mu.g/ml ConA, the cells were labelled with
.sup.3H-Thymidine (1 .mu.Ci/well) for 16 hours before harvesting
and counting in a .beta.-counter.
[0096] RPMI Medium
[0097] RPMI 1640 without L-glutamine (Life technologies
No.31870025), 2 mM L-glutamine (Life technologies No.25030024), 50
.mu.M 2-Mercaptoethanol (Life technologies No.11360039), 1 mM
Sodium Pyruvate (Life technologies No.11360039), 1.times.MEM non
essential amino acids (10.times.stock, Life technologies No.11
140035), 100 IU/ml penicillin-100 .mu.g/ml streptomycin (Life
technologies No.15140114).
[0098] Cytokine Detection
[0099] Spleen or popliteal lymph node cells were aseptically
removed and. 1000 .mu.l of cells in RPMI medium (5%
heat-inactivated fetal calf serum, FCS) containing
5.times.10.sup.6/ml of cells were cultured in 24 well flat bottomed
plates in the presence of RTS,S or TRAP antigens. The plates were
then incubated (37.degree. C., 5% CO.sub.2) for a number of hours
with 0.5, and 2.5 .mu.g of antigen, or 4 .mu.g/ml final of
ConA.
[0100] The length of time that the cells were incubated depended on
the particular cytokine to be detected, IL-2 was stimulated for 72
hours, IL-5 was 72 or 96 hours, and IFN-.gamma. was 96 hours. Each
cytokine was detected using commercially available ELISA kits (IL-2
and IFN-.gamma., Duoset Genzyme No.80-3573-00 and 80-3931-00
respectively; IL-5 was detected using the Pharmingen kit).
[0101] Serology
[0102] Antibodies directed against TRAP were analysed using a
sandwich ELISA. A sheep anti-TRAP antiserum was coated onto ELISA
plates which was used to capture TRAP antigen added at 0.5
.mu.g/ml. Titrations of pooled serum from the experimental groups
were added and incubated. Finally, biotinylated anti-mouse
isotype-specific antibodies followed by streptavidin-peroxidase,
were used to detect bound TRAP-specific antibodies.
[0103] Anti HBV humoral responses were analysed by a direct ELISA,
HBsAg was coated onto the ELISA plate at 1 .mu.g/ml. Pooled serum
from the different experimental groups were titrated and bound
antibodies were detected as described above.
[0104] Results
[0105] Proliferation of Lymphoid Cells in Response to Antigen
[0106] The proliferative responses in response to antigen can be
seen in the following figures. All vaccine preparations stimulated
cells in the local popliteal lymph node which were capable of
proliferating in vitro in response to antigen, the magnitude of
which was independent of the addition of cholesterol.
[0107] All vaccine preparations were capable of stimulating splenic
cells which proliferated in vitro in response to antigen. When
considering the stimulation indices, the preparations which
elicited the highest responses in the spleen were the ones
containing cholesterol and those having the low ratio squalene:QS21
(1/5th dose SB62).
[0108] FIG. 1, shows the proliferative responses of popliteal lymph
node cells (in raw counts per minute (CPM) form) derived from the
experimental groups after stimulation with TRAP and RTS,S
antigens.
[0109] FIG. 2, shows the proliferative responses of splenic cells
(in raw counts per minute (CPM) form) derived from the experimental
groups after stimulation with TRAP and RTS,S antigens.
[0110] FIG. 3, shows the proliferative responses of popliteal lymph
node cells (Stimulation index) derived from the experimental groups
after stimulation with TRAP and RTS,S antigens.
[0111] FIG. 4, shows the proliferative responses of splenic cells
(Stimulation index) derived from the experimental groups after
stimulation with TRAP and RTS,S antigens.
[0112] Discussion of Proliferation Results
[0113] FIGS. 1 and 2, clearly show that all of the vaccine
formulations stimulate lymphoid cells which are capable of
proliferating in vitro in the presence of antigen in a dose
dependent manner. The raw cpm data suggests that the inclusion of
cholesterol in the adjuvant formulations has no effect on the
magnitude of the proliferative responses (for example a comparison
between groups 26 and 31, termed RTS,SIMPL/QS21/SB62 and
RTS,S/MPL/QS21/SB62c respectively).
[0114] Examination of the cpm together with the stimulation index
results (FIGS. 3 and 4, obtained by dividing the raw cpm for
antigen specific proliferation by that derived from non-antigen
specific proliferation (medium alone)) shows that the vaccine
formulation which generates the highest proliferative responses
depends on the origin of the lymphocyte measured. The adjuvant
formulations containing the low ratio of squalene:QS21 generate the
highest proliferative responses in the spleen. Whereas in the local
lymph node whole dose SB62 formulations, with or without
cholesterol, generate the highest proliferation responses.
[0115] In Vitro Cytokine Production Upon Stimulation with
Antigen
[0116] Cytokine production, measured in vitro in response to
antigen, can be both a quantitative and qualitative measure of the
induction of immune responses in vivo. In general high levels of
IFN-.gamma. and IL-2 are taken to be a measure of Th1-type immune
responses and IL-5 is considered to be a Th2-type cytokine. The
following figures demonstrate evidence that the addition of
cholesterol has no qualitative or quantitative effects on the
cytokine profile produced in vitro in response to antigen. The use
of SB62' containing a reduced ratio of squalene:QS21 (termed SB62
1/5th dose) had a marked effect in enhancing the production of
IFN-.gamma. (FIG. 6).
[0117] FIG. 5, shows the IL-2 production of spleen cells after
stimulation with TRAP or RTS,S antigen 14 days after VII.
[0118] FIG. 6, shows the IFN-.gamma. production by spleen cells
after stimulation with TRAP or RTS,S antigen 14 days after VII.
[0119] FIG. 7, shows the IL-5 production by spleen cells after
stimulation with TRAP or RTS,S antigen 14 days after VII.
[0120] Serology
[0121] Another measure of immunity that can correlate to a
Th1-type, or alternatively a Th2-type, immune response is the IgG
sub-isotype which is elicited. A preferential stimulation of the
IgG1 sub-isotype is generally taken to be a measure of the
induction of a Th2-type immune response, and conversely IgG2a and
IgG2b is taken to be a measure of a Th1 type immune response.
[0122] ELISA studies were performed on pooled mouse serum and the
mid-point titres for both the HBsAg and TRAP specific antibodies
were ascertained. From these figures, the ratio of the antigen
specific IgG1 and IgG2a mid-point titres was calculated and taken
to be a measure of the Th1/Th2 balance of the humoral immune
response.
12TABLE 12 The ratio of IgG1:IgG2a, representing the Th1/Th2
balance. A ratio <1 represents a Th1-type immune response, a
ratio of >1 indicating a Th2-type response. Ratio of mid-point
titres IgG1:IgG2a Group HBsAg TRAP 26 0.44 27 0.36 28 1.46 1.68 29
0.37 30 0.39 11.83 31 0.28 32 0.2 7.21 33 0.66 34 0.3 0.77
[0123] Discussion of Serological Results
[0124] Pools of mouse serum were analysed from each group and were
found to have successfully stimulated HBsAg and TRAP specific
antibodies. In general, antibody mid-point titres against HBsAg
were higher than those found against TRAP. The isotype distribution
differed between the two antigens. RTS,S in all formulations
elicited a clear Th1 pattern, as indicated by an IgG1:IgG2a ratio
below 1.
[0125] In contrast, TRAP-specific antibodies exhibited a Th2-type
isotype pattern. The only exceptions to this observation were
groups 2, who received TRA-P alone, and group 9, who received
TRAP/RTS,S in a SB62' formulation (containing a low ratio of
squalene:QS21, termed SB621/5th dose).
EXAMPLE 7
Immunogenicity Studies with Recombinant Antigen S,L*
[0126] A study was conducted in Balb/C mice in order to compare the
immunogenicity of various S,L* containing formulations. S,L* is a
composite antigen comprising a modified surface antigen L protein
(L*) and an S-protein both derived from the Hepatitis B virus (HB.
This composite antigen is the subject of European Patent
application No. EP 0 414 374.
[0127] Various formulations with differing ratios of squalene:QS21,
optionally with cholesterol at a QS21:cholesterol ratio of 1:10,
were combined with S,L* and compared in their ability to induce
humoral and cell mediated immune responses (cytokine production and
CTL). These oil in water adjuvant emulsions were produced using
methods described in example 1. S,L* formulated on Aluminium
hydroxide (AlOH.sub.3) was used as a Th2 inducing control.
[0128] Briefly, groups of 10 mice were immunised intramuscularly 4
times at 3 weeks interval with 2 .mu.g lyophilised S,L* combined
with various oil in water emulsion systems (SB62). 14 days
following the fourth immunisation the production of cytokines (IL5
and IFN-.gamma.) and CTL activity was analysed after in vitro
restimulation of spleen and lymph nodes cells with S,L* antigen.
Antibody response to S,L* and the isotypic profile induced were
monitored by ELISA at 21 days post II and 14 days post IV.
[0129] Groups of Mice
[0130] Groups of 10 Balb/C mice were immunised intramuscularly with
formulations described below. SB62 was formulated together with the
antigen at a normal (240:1, SB62) or low (48:1, SB62') ratio of
squalene:QS21, optionally with the addition of cholesterol (c).
13TABLE 13 Groups of mice described in example 7: Antigen Adjuvant
Group S,L* name Composition of adjuvant formulation GR 1 2 .mu.g
SB62 25 .mu.l SB62/5 .mu.g QS21/5 .mu.g 3D-MPL GR 2 2 .mu.g SB62c
25 .mu.l SB62c/5 .mu.g QS21/5 .mu.g 3D-MPL GR 3 2 .mu.g SB62' 5
.mu.l SB62/5 .mu.g QS21/5 .mu.g 3D-MPL GR 4 2 .mu.g SB62'c 5 .mu.l
SB62c/5 .mu.g QS21/5 .mu.g 3D-MPL GR 5 2 .mu.g Alum 50 .mu.g
AlOH.sub.3
[0131] Immunisation Scheme
[0132] Animals were immnunised intramuscularly in the leg (50 .mu.l
for all groups except for group 5 where 100 .mu.l was injected) at
days 0, 21, 42 and 63. Blood was taken from the retroorbital sinus
at various time points post immunisations. On day 77, animals from
each group were sacrificed, spleens and lymph nodes draining the
site of injection (iliac lymph nodes) were taken out for in vitro
restimulation. Pools of 3 or 4 spleens and 1 pool of 10 LN were
obtained for each group and treated separately in the in vitro
assays.
[0133] Mouse Serology
[0134] Quantitation of anti-HBs antibody was performed by Elisa
using HB surface antigen as coating antigen. Antigen and antibody
solutions were used at 50 .mu.l per well. Antigen was diluted at a
final concentration of 1 .mu.g/ml in PBS and was adsorbed overnight
at 4.degree. C. to the wells of 96 wells microtiter plates
(Maxisorb Immuno-plate, Nunc, Denmark). The plates were then
incubated for 1 hr at 37.degree. C. with PBS containing 1% bovine
serum albumin and 0.1% Tween 20 (saturation buffer). Two-fold
dilutions of sera (starting at {fraction (1/100)} dilution) in the
saturation buffer were added to the HBs-coated plates and incubated
for 1 hr 30 min at 37.degree. C. The plates were washed four times
with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse IgG1,
IgG2a, IgG2b or Ig (Amersham, UK) diluted {fraction (1/1000)} in
saturation buffer was added to each well and incubated for 1 hr 30
min at 37.degree. C. After a washing step,
streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted
{fraction (1/5000)} in saturation buffer was added for an
additional 30 min at 37.degree. C. Plates were washed as above and
incubated for 20 min with a solution of o-phenylenediamine (Sigma)
0.04% H.sub.2O.sub.2 0.03% in 0.1% TWEEN 20, 0.05M citrate buffer
pH4.5. The reaction was stopped with H.sub.2SO.sub.4 2N and read at
492/620 nm. ELISA titers were calculated from a reference by
SoftmaxPro (using a four parameters equation ) and expressed in
EU/ml.
[0135] T Cell Proliferation
[0136] 2 weeks after the second immunisation, mice were killed,
spleen and lymph nodes were removed aseptically in pools (3 or 4
organs per pool for splenic cells, 1 pool of 10 organs for LNC).
Cell suspensions were prepared in RPMI 1640 medium (GIBCO)
containing 2 mM L-glutamine, antibiotics, 5.times.10.sup.-5 M
2-mercaptoethanol, and 1% syngeneic normal mouse serum. Cells were
cultured at a final concentration of 2.times.10.sup.6 cells/mi (for
LNC or SPC) in 200 .mu.l in round-bottomed 96 well-plates with
different concentrations (10-0.03 .mu.g/ml) of S,L* antigen
(25D84). Each test was carried out in quadriplicate. After 96 hr of
culture at 37.degree. C. under 5% CO.sub.2, the cells were pulsed
for 18hr with 3H-Thymidine (Amersham, UK, 5 Ci/mmol) at 0.5
.mu.Ci/well and then harvested on fibre glass filters with a cell
harvester. Incorporated radioactivity was measured in a liquid
scintillation counter. Results are expressed in cpm (mean cpm in
quadriplicate wells) or as stimulation indices (mean cpm in
cultures of cells with antigen/mean cpm in cultures of cells
without antigen).
[0137] Cytokine Production
[0138] 2 weeks after the second immunisation, mice were killed,
spleen and lymph nodes were removed aseptically in pools (3 or 4
organs per pool for splenic cells, 1 pool of 10 organs for LNC).
Cell suspensions were prepared in RPMI 1640 medium (GIBCO)
containing 2 mM L-glutamine, antibiotics, 5.times.10.sup.-5 M
2-mercaptoethanol, and 5% foetal calf serum. Cells were cultured at
a final concentration of 2.5 to 5.times.10.sup.6 cells/ml
(respectively for LNC or SPC) in 1 ml, in flat-bottomed 24 well-
with different concentrations (1-0.01 .mu.g/ml) of S,L* (25D84).
Supernatants were harvested 96 hrs later and frozen until tested
for the presence of IFNg and IL-5 by Elisa.
[0139] IFN-.gamma. Production
[0140] Quantitation of IFN.gamma. was performed by Elisa using
reagents from Genzyme. Samples and antibody solutions were used at
50 .mu.l per well. 96-well microtiter plates (Maxisorb
Immnuno-plate, Nunc, Denmark) were coated overnight at 4.degree. C.
with 50 .mu.l of hamster anti-mouse IFNg diluted at 1.5 .mu.g/ml in
carbonate buffer pH 9.5. Plates were then incubated for 1 hr at
37.degree. C. with 100 .mu.l of PBS containing 1% bovine serum
albumin and 0.1% Tween 20 (saturation buffer). Two-fold dilutions
of supernatant from in vitro stimulation (starting at 1/2) in
saturation buffer were added to the anti-IFNg-coated plates and
incubated for 1 hr 30 at 37.degree. C. The plates were washed 4
times with PBS Tween 0.1% (wash buffer) and biotin-conjugated goat
anti-mouse IFNg diluted in saturation buffer at a final
concentration of 0.5 .mu.g/ml was added to each well and incubated
for 1 hr at 37.degree. C. After a washing step, AMDEX conjugate
(Amersham) diluted {fraction (1/10000)} in saturation buffer was
added for 30 min at 37.degree. C. Plates were washed as above and
incubated with 50 .mu.l of TMB (Biorad) for 10 min. The reaction
was stopped with H.sub.2SO.sub.4 0.4N and read at 450 nm.
Concentrations were calculated using a standard curve (mouse
IFN.gamma. standard) by SoftmaxPro (four parameters equation) and
expressed in pg/ml.
[0141] IL-5 Production
[0142] Quantitation of IL5 was performed by Elisa using reagents
from Pharmingen. Samples and antibody solutions were used at 50
.mu.l per well. 96-well microtiter plates (Maxisorb Irnmuno-plate,
Nunc, Denmark) were coated overnight at 4.degree. C. with 50 .mu.l
of rat anti-mouse IL5 diluted at 1 .mu.g/ml in carbonate buffer pH
9.5. Plates were then incubated for 1 hr at 37.degree. C. with 100
.mu.l PBS containing 1% bovine serum albumin and 0.1% TWEEN 20
(saturation buffer). Two-fold dilutions of supernatant from in
vitro stimulation (starting at 1/2) in saturation buffer were added
to the anti-IL5-coated plates and incubated for 1 hr 30 at
37.degree. C. The plates were washed 4 times with PBS Tween 0.1%
(wash buffer) and biotin-conjugated rat anti-mouse IL5 diluted in
saturation buffer at a final concentration of 1 .mu.g/ml was added
to each well and incubated for 1 hr at 37.degree. C. After a
washing step, AMDEX conjugate (Amersham) diluted {fraction
(1/10000)} in saturation buffer was added for 30 min at 37.degree.
C. Plates were washed as above and incubated with 50 .mu.l of TMB
(Biorad) for 15 min. The reaction was stopped with H.sub.2SO.sub.4
0.4N and read at 450 nm. Concentrations were calculated using a
standard curve (recombinant mouse IL5) by SoftmaxPro (four
parameters equation) and expressed in pg/ml.
[0143] CTL Induction
[0144] 2 weeks after the second immunisation, mice were killed,
spleens were removed aseptically in pools of 3 or 4 mice (2 pools
of 3 and one pool of 4 mice per group). Cell suspensions were
prepared in RPMI 1640 medium (GIBCO) containing 2 mM L-glutamine,
antibiotics, 5.times.10.sup.-5 M 2-mercaptoethanol, and 5% foetal
calf serum. Cells were cultured at a final concentration of
2.times.10.sup.6 cells/ml in 10 ml medium containing 2 .mu.g/ml
S,L* and 1.25% ConA sup (25 cm.sup.2 Falcon flasks) and incubated
for 8 days at 37.degree. C. under 5% CO.sub.2.
[0145] CTL Assay
[0146] The day before the CTL assay (d7), target cells were
prepared by incubation of P815 cells (10.sup.6 cells/ml) with S,L*
or peptide S.sub.28-39 at 10 .mu.g/ml. Following 1 hr incubation in
15 ml Falcon tubes in a small volume, cells are transferred to 24
well plates and incubated ON at 37.degree. C.
[0147] The day of the assay, 2.times.10.sup.6 S,L* and S.sub.28-39
pulsed P815 cells and P815-S are centrifugated, resuspended in 50
.mu.l FCS and incubated with 75 .mu.l .sup.51Cr(375 .mu.Ci) for 1
hr at 37.degree. C. (shaking every 15'). Cells are then washed 4
times with 10 ml complete medium and incubated for 30' at 4.degree.
C. following the 4th wash. Cells are then centrifuigated and
resuspended at a concentration of 2.times.10.sup.4 cells/ml.
[0148] Effector cells are then centrifugated, counted and
resuspended at 2.times.10.sup.6 cells/ml. Three fold serial
dilutions of effector cells are done in 96 V-bottomed plates,
starting at a concentration of 2.times.10.sup.5 cells /well/100
.mu.l.
[0149] 2.times.10.sup.3 target cells in 100 .mu.l are added to
effector cells in triplicate. Spontaneous and maximum release are
assessed by incubating target cells respectively with medium or
Triton X100 3%.
[0150] Plates are centrifugated 3' at 700 rpm and incubated for 4
hrs at 37.degree. C. Following the incubation time, 50 .mu.l of
supernatant is transfered from each well to Luma-plates and dryed
overnight before counting in Top-count scintillation counter.
Results are expressed as specific lysis and calculated as
follows:
% SR=(mean cpm sample-mean cpm medium/mean cpm max-mean cpm
medium).times.100
[0151] Results
[0152] Serology
[0153] Humoral responses (Ig and isotypes) were measured by Elisa
using HB surface antigen as coating antigen. Only the time point:
21 days post II was analysed. The results are shown in FIG. 8 and
9.
[0154] FIG. 8, Shows the titres of anti-Hepatitis B virus antibody
responses (Ig) expressed as both individual mouse sera and average
(21 days post II).
[0155] FIG. 9, Shows the sub-isotype distribution of Hbs specific
IgG in the serum the vaccinated mice.
[0156] As can be seen in FIG. 8, SB62 related formulations induce
much higher antibody titers than the S,L* Alum formulation.
[0157] Analysis of mean titres from individual sera suggest that
higher antibody titers are obtained with SB62c and SB62'c
formulations ( roughly 2 fold higher antibody titers than SB62 and
SB62' respectively).
[0158] Statistical analysis on individual sera (Anoval test Newman
Keuls) show no significant difference in antibody titers induced by
SB62c and SB62'c or equally between the antibody titers induced by
SB62 and SB62'c.
[0159] The sub-isotypic distribution profile (as shown in FIG. 9)
is comparable for all SB62 related formulations (25-30% IgG2a)
whereas Alum induce only 4% IgG2a.
[0160] Cell-Mediated Immune Responses
[0161] Cell-mediated immune responses (lymphoproliferation,
IFN.gamma./IL5 production and CTL) were measured at 14 days post IV
after in vitro restimulation of splenic and iliac lymph nodes cells
with S,L* antigen.
[0162] Cytokine Production
[0163] Cytokine production (IFN-.gamma. and IL-5) has been measured
following 96 h of in vitro restimulation of splenic cells and iliac
lymph node cells with S,L*. The results are shown in FIGS. 10 to
13.
[0164] FIG. 10, Shows the results of analysis of IFN-.gamma.
production by splenic cells (mean of data obtained with three
pools/group).
[0165] FIG. 11, Shows the results of analysis of IL-5 production by
splenic cells (mean of data obtained with three pools/group).
[0166] FIG. 12, Shows the results of analysis of IFN-.gamma.
production by Iliac lymph node cells (mean of data obtained with
three pools/group).
[0167] FIG. 13, Shows the results of analysis of IL-5 production by
Iliac lymph node cells (mean of data obtained with three
pools/group).
14TABLE 14 Ratio of IFN-.gamma.: IL-5 producing cells detected in
splenic cells Groups Restimulation GR1 GR2 GR3 GR4 GR5 S,L* 10
.mu.g/ml 22.9 10.7 51.7 17.0 0.9
[0168] Discussion
[0169] A IFN-.gamma.: IL-5 ratio>1 clearly suggests that a pro
TH1 response is induced by SB62 related formulations (calculated at
10 .mu.g/ml S,L*) (see table 14).
[0170] The strongest IFN-.gamma. production is obtained after
restimulation of splenic cells from animals immunised with S,L*
SB62' and SB62'c. SB62c formulations induce stronger IFN-.gamma.
production than the corresponding SB62 formulations (splenic
cells).
[0171] Higher levels of IL-5 are produced by animals immunised with
S,L* SB62c formulations than S,L* SB62 formulations not containing
cholesterol. S,L* Alum immunised animals produce the highest levels
of IL-5.
[0172] No significant difference is observed in ileac lymph node
cell IFN-.gamma. production between SB62 and SB62c
formulations.
[0173] The strongest IFN-.gamma. production is obtained after
restimulation of splenic cells from animals immunised with S,L*
SB62'c.
[0174] Cytotoxic T Cell Responses
[0175] S,L* specific CTL responses observed in the spleen cells of
mice two weeks after the second immunisation are shown in FIG.
14.
[0176] FIG. 14, Shows the CTL activity of splenic T-cells
stimulated in vitro for 7 days with S,L* antigen (mean % specific
lysis of three pools).
[0177] Discussion
[0178] S,L* specific CTL is stimulated by vaccination with all oil
in water emulsion formulations.
[0179] A stronger CTL response is observed with formulations
containing SB62' emulsions when looking at limiting E/T ratio such
as 3/1.
[0180] Conclusions
[0181] 1. The TH1 type profile of the immune response induced by
all SB62 related formulations is further confirmed by the
IFN-.gamma./IL-5 ratio.
[0182] 2. A comparable isotypic profile (25-30% IgG2a) is obtained
with all SB62 related formulations suggesting the induction of a
TH1 type HBs specific immune response.
[0183] 3. All SB62 related formulations induce specific CTL, with a
slight improvement seen by administration of SB62'.
[0184] 4. No significant difference is observed between antibody
titers induced following immunisation with SB62c and SB62'.
[0185] 5. The strongest IFN-.gamma. production is observed
following immunisation with SB62'c.
15TABLE 15 Summary table of the immune parameters induced by the
vaccine formulation described in example 7. Immune Formulations
containing S,L* parameter SB62 SB62c SB62' SB62'c Alum Ab titers
+++ +++ ++ +++ + TH type TH1 (29) TH1 (26) TH1 (29) TH1 (30) TH2
(4) (% IgG2a) IFN-.gamma. + ++ +++ ++++ + IL-5 - + + ++ +++ CTL + +
++ ++ -
EXAMPLE 8
Reactogenicity Study in Rabbits Using L2E7 Antigen Formulated in
Various SB62 Adjuvants
[0186] This study investigated the reactogenicity of various
vaccine formulations after administration into rabbits.
Formulations were given intramuscularly (IM, in a 500 .mu.l volume)
as a single administration on day 0, in male white New Zealand
rabbits weighing between 2 and 2.5 kg (5 animals in each group).
Blood was collected on day-5, +1, +2, +3 and +4 for PMN and CPK
determinations. Additional bleedings were performed on day -7 and
in order to get the PMN background of the animals prior injection.
On day 4, animals were sacrificed and necropsied in order to
examine the injection site macroscopically. The injection site was
collected and preserved in formaldehyde for histopathological
examinations.
[0187] The antigen used in this study is a fusion protein
comprising the L2 and E7 proteins from Human Papilloma virus. L2E7
fusion proteins are disclosed in GB 95/15478.7.
[0188] CPK (creatin phosphate kinase) is a marker of muscle lesion
and can be used as a read-out to assess the local reactogenicity
during muscle damage. PMN (polymorphonuclear neutrophils) is used
to assess both local and inflammatory process induced by the
injection and, in a lesser extent, systemic side-effects related to
this local inflammation (flu-like symptoms, fever, headache).
16TABLE 16 Groups of animals used in example 8 Group Antigen
Diluant A L2E7 (300 .mu.g) SB62 B L2E7 (300 .mu.g) SB62c C L2E7
(300 .mu.g) PBS pH6.8 D -- SB62 E -- SB62c F -- PBS pH6.8
[0189] Results
[0190] PMN
[0191] During the course of the experiment (excluding day 1 post
vaccination), all rabbits at had an average baseline of PMNs at
25.7% of total cells (SD 7.6).
[0192] In the control group that received PBS alone, the level of
PMNs remained at baseline throughout the course of the experiment,
that is 24.2% from day -7 to day +4, (SD 5.8). The results can be
summarized as follows:
17 % PMNs average before and % PMNs after injection average after
(days -7, -5, +3 and injection % PMNs % of Group +4) day 1
difference ncrease A 22.8% (+/-5.6%) 50.6% (+/-5.3%) 27.8% 122% B
24.1% (+/-7.2%) 56% (+/-6.9%) 31.9% 132% C 24.9% (+/-7.6%) 35.2%
(+/-8.7%) 10.3% 41% D 23.5% (+/-5.2%) 56.7% (+/-9.3%) 33.2% 141% E
25.1% (+/-6.4%) 59.3% (+/-7.1%) 34.2% 136% F 23.1% (+/-6.1%) 27.4%
(+/-5.8%) 4.3% 19%
[0193] Discussion
[0194] Injection of SB62 or SB62c alone or in combination with
antigen induced strong responses in PMN levels on day 1 post
vaccination. The addition of cholesterol therefore had no effect on
inflammatory responses post vaccination.
[0195] Injection of L2E7 in PBS induces an only slight increase in
PMNs percentage (41% increase) despite the high concentration of
antigen used (300 .mu.g).
[0196] No significant difference in PMNs percentage is observed in
the control group during the whole experiment.
[0197] CPK
[0198] Individual CPK(U/L) results measured on day -5, 1, 2, 3 and
4 are shown in FIG. 15. The results can be summarised as
follows:
[0199] SB62 formulations with or without antigen induce a
significant increase in CPK on day 1.
[0200] SB62c formulations with or without antigen do not induce
significant CPK release on day 1.
[0201] FIG. 15, Summary figure shows the mean CPK results for each
group.
[0202] Histopathological Analyses
[0203] Individual data from histopathological analyses are shown in
Table 17, and can be summarized as follows:
[0204] Macroscopical examinations at necropsy on day 4 revealed
abnormalities at the site of injection in animals that had received
SB62 based formulations with or without antigen. Most animals
treated with SB62 based formulations with or without antigen showed
a wide local muscle lesion.
[0205] Local muscle damage was significantly reduced when SB62c was
used (small spots in all rabbits except for 2 animals that
developed lesions that are smaller than lesions induced by
SB62).
[0206] No abnormalities were detected in PBS or antigen vaccinated
animals (besides slight lymphocyte infiltrate in 2 animals treated
with antigen and 1 animal treated with PBS).
[0207] Microscopic examinations showed some histological changes at
the injection site related to muscle damage (necrosis,
rhabdomyolysis, haemorrhage) and local inflammatory process
(lymphocyte and monocyte infiltrates). These signs were observed in
all rabbits injected with SB62 based formulations with or without
antigen, and were much more severe than those observed in animals
injected with SB62c containing formulations.
[0208] Histological examination of the site of injection confirms
the CPK release data showing a significant reduction in local
damage with SB62c formulations.
18TABLE 17 Histological examination result with SB62 based
formulations with L2E7 antigen. Site of injection Histopathology
Group Rabbit les size (mm) necro rhabdo infiltr haemo infl apone
Remark A 4 + 30 .times. 15 .times. 5 3 2 3 2 2 15 + 24 .times. 7
.times. 4 3 2 2 2 2 17 + 25 .times. 10 .times. 3 3 2 2 2 2 27 + 27
.times. 14 .times. 8 3 2 3 2 2 28 + 25 .times. 8 .times. 5 3 2 3 3
2 B 2 + 10 .times. 2 .times. l 1 2 2 1 1 1 11 s 0 0 0 0 0 21 + 15
.times. 8 .times. 2 0 0 2 1 2 infl diffus 22 - 0 0 0 1 0 2 infl
diffus 29 s 0 0 2 2 1 2 C 1 s s 0 0 - 0 0 3 - - 0 0 - 0 1 18 - - 0
0 - 0 1 23 - - 0 0 1 0 1 24 - - 0 0 2 0 1 D 5 + 10 .times. 3
.times. 2 2 2 3 1 1 7 s s 0 0 0 0 1 2 9 + 18 .times. 10 .times. 4 3
3 3 1 14 + 16 .times. 2 .times. 5 3 3 3 1 2 33 s s 0 1 2 0 2 E 8 s
s 0 1 2 1 2 10 s s 0 1 2 0 2 20 s s 0 1 2 0 2 25 s s 1 1 1 0 2 infl
diffus 30 s s 0 1 1 0 2 F 6 - - 0 0 2 0 2 13 - - 0 0 0 0 0 16 - - 0
0 0 0 0 26 - - 0 1 2 0 0 34 - - 0 0 0 0 0 footnotes: les = lesion
necro = necrosis infiltr = lymphocytic infiltration inf apone =
lymphocytic infiltration in muscle aponevrose oedem = oedema haemo
= haemorrhage rhabdo = rhabdomyolosis infl diffus = infiltration
diffuse Gradation: 0 = no sign 1 = very slight 2 = slight 3 =
moderate 4 = severe + = present s = spot - = no sign
EXAMPLE 9
Stabilization of QS21 by Addition of Cholesterol
[0209] It has previously been described that QS21-H is hydrolysis
product of QS21, that is no longer active as adjuvant. It is formed
by cleavage of the QS21 molecule by OH from the aqueous solution.
This reaction occurs where the pH of the aqueous medium is above a
value of 6.5, and is accelerated by higher temperature. The
oil-in-water emulsions described in this patent application (for
example SB62) are known to exhibit a stabilising effect such that
the hydrolysis of QS21 into QS21-H is inhibited. Upon dilution of
the oil in water emulsion in the presence of constant QS21, they
lose this stabilising property and the QS21 degenerates into the
inactive QS21-H form. Surprisingly, emulsions containing additional
Cholesterol, who at 1/1 ratio do not show an improved QS21
stability, maintain the stabilising effect even at a 1/5
dilution.
[0210] QS21 and QS21-H are assayed directly into the emulsion. This
is achieved by chemically derivatising the complete formulation,
and by performing a selective extraction step that dissolves the
QS21, but leaves most interfering matrix compounds behind. The
assay is HPLC based, and the compounds are dansylated. The
dansylation is performed by drying down a sample of the emulsion,
and adding 100 .mu.l of 3.5 mg Dansyl hydrazine/ml C/M 2/1 and 100
.mu.l of 1:4 Acetic acid: C/M 2/1 in that order. The mixture is
well vortexed and incubated at 60.degree. C. for 2 hours. The
reaction mixture is dried in the Speedvac. It is reconstituted in
500 .mu.l 30% ACN in H2O, and centrifugated twice at 14000 rpm for
two minutes. The supernatants are then collected in an autosampler
tube. A standard curve is obtained by preparing QS21 and QS21-H in
a mixture that contains the same compounds as the emulsion under
study.
[0211] The HPLC assay is ran on a Vydac 218TP54 5.mu. particle size
C18 RP column, 250*4.6 mm. Solvents are A:H20+0,05%
TFA(trifluoracetic acid) and B:Acetonitrile+0,05% TFA. The gradient
table is:
19 Time (min) % A % B 0 70 30 2 70 30 15 50 50 17 50 50 17.1 10 90
19 10 90 21 70 30 25 70 30
[0212] The Flow rate is 1 ml/min. Detection is in fluorescence,
with excitation at 345 nm and emission at 515 nm. 50 .mu.l is
injected of both the sample and the standards. The column heater is
set to 37.degree. C. for this separation. Peaks for QS21, QS21-iso
and QS21-H are distinguished on the chromatogram.
[0213] A series of samples with the following composition were
analysed:
20 Composition SB62 SB62c MPL QS21 SB62 250 .mu.l -- 50 .mu.g 50
.mu.g SB62' 50 .mu.l -- 50 .mu.g 50 .mu.g SB62c -- 250 .mu.g 50
.mu.g 50 .mu.g SB62'c -- 50 .mu.g 50 .mu.g 50 .mu.g
[0214] Assay of QS21/QS21-H was performed after incubation of the
samples at various time intervals and temperatures (4.degree. C.
and 37.degree. C.). The data for 1 month at 37.degree. C. in this
model correlate well with stability of QS21 after prolonged storage
at 4.degree. C. (eg 2 years).
21TABLE 18 HPLC QS2J assay: % of QS21-H generated over time 3
months (4.degree. C.) + Composition 3 months (4.degree. C.) 6
months (4.degree. C.) 7 days (37.degree. C.) 1 month (37.degree.
C.) SB62 1% 2% 3% 15% SB62' 1% 1% 9% 31% SB62c 2% 2% 3% 17% SB62'c
2% 2% 3% 21%
[0215] This results shown in the table above shows clearly (both
for 7 days and 1 m) the effect of adding a sterol, in this case
cholesterol, to SB62' in maintaining the stability of QS21.
Summary to the Invention
[0216] It is clear from the examples above that the present
invention encompasses an oil in water emulsion which preferentially
induces a strong Th1-type immune responses. Embodiments of the
present invention, as described in the examples, include
composition comprising an oil in water emulsion, a saponin and a
sterol, characterised in that a reduced reatogenicity profile is
induced upon administration to a host in comparison to the
reactogenicity profile observed after administration of the same
composition from which the sterol has been omitted. The addition of
cholesterol, however, does not adversly affect quantitatively or
qualitatively the immune responses thus induced.
* * * * *