U.S. patent number 6,506,386 [Application Number 09/744,800] was granted by the patent office on 2003-01-14 for vaccine comprising an iscom consisting of sterol and saponin which is free of additional detergent.
This patent grant is currently assigned to SmithKline Beecham Biologicals, S.A.. Invention is credited to Martin Friede, Nathalie Garcon.
United States Patent |
6,506,386 |
Friede , et al. |
January 14, 2003 |
Vaccine comprising an iscom consisting of sterol and saponin which
is free of additional detergent
Abstract
The present invention provides an improved adjuvant formulation
and a process for producing said adjuvant. The adjuvant comprises
an ISCOM structure comprising a saponin, said ISCOM structure being
devoid of additional detergent.
Inventors: |
Friede; Martin (Farnham,
GB), Garcon; Nathalie (Wavre, BE) |
Assignee: |
SmithKline Beecham Biologicals,
S.A. (Rixensart, BE)
|
Family
ID: |
10836763 |
Appl.
No.: |
09/744,800 |
Filed: |
June 4, 2001 |
PCT
Filed: |
August 03, 1999 |
PCT No.: |
PCT/EP99/05587 |
PCT
Pub. No.: |
WO00/07621 |
PCT
Pub. Date: |
February 17, 2000 |
Foreign Application Priority Data
Current U.S.
Class: |
424/184.1;
424/278.1; 424/283.1; 514/25 |
Current CPC
Class: |
A61K
39/39 (20130101); A61P 37/04 (20180101); A61P
37/08 (20180101); A61K 2039/55577 (20130101) |
Current International
Class: |
A61K
39/39 (20060101); A61K 039/39 () |
Field of
Search: |
;424/184.1,278.1,283.1,208.1 ;514/25 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Kersten, et al., "On the Structure of Immune-Stimulating
Saponin-Lipid Complexes (Iscoms)", Biochimica et Biophysica Acta,
1062(2): 165-71 (1991). .
Sjolander, et al., "Uptake and Adjuvant Activity of Orally
Delivered Saponin and ISCOM.TM. Vaccines", Advanced Drug Delivery
Reviews, 34: 321-338 (1998). .
Barr, et al., "ISCOMs (Immunostimulating Complexes): The First
Decade", Immunology and Cell Biology, 74: 8-25 (1996)..
|
Primary Examiner: Scheiner; Laurie
Attorney, Agent or Firm: Kinzig; Charles M. Gimmi; Edward
R.
Claims
What is claimed is:
1. An adjuvant composition comprising a sterol, a saponin, and a
phospholipid, characterised in that the adjuvant is in the form of
an ISCOM and that it is free of additional detergent, other than
the saponin.
2. An adjuvant composition as claimed in claim 1, wherein the ratio
of saponin:sterol (w/w) exceeds 1.
3. An adjuvant composition as claimed in claim 1, wherein the ratio
of saponin to sterol is in the range of 1:1 to 100:1 (w/w).
4. An adjuvant composition as claimed in claim 1, wherein the ratio
of saponin to sterol is 5:1.
5. An adjuvant composition as claimed in any one of claims 1 to 4,
wherein the saponin is Quil A or extract thereof.
6. An adjuvant composition as claimed in claim 5, wherein the
extract of Quil A is QS21.
7. An-adjuvant composition as claimed in claim 1, wherein the
sterol is cholesterol.
8. An adjuvant composition as claimed in claim 1, wherein the
phospholipid is phosphatidylcholine.
9. An adjuvant composition as claimed in claim 8, wherein
phosphatidylcholine is dioloeoylphosphatidylcholine or dilauryl
phosphatidylcholine.
10. An adjuvant composition as claimed in claim 7, wherein the
ratio of cholesterol to phospholipid is 50% (w/w).
11. An adjuvant composition as claimed in claim 10, wherein the
ratio of cholesterol to phospholipid is 20-25% (w/w).
12. A vaccine comprising an adjuvant composition as claimed in any
one of claims 1 to 11, further comprising an antigen.
13. A vaccine composition as claimed in claim 12, wherein the
antigen is an antigen or antigenic composition derived from any of
Human Immunodeficiency Virus, Feline Immunodeficiency Virus,
Varicella Zoster virus, Herpes Simplex Virus type 1, Herpes Simplex
virus type 2, Human cytomegalovirus, Hepatitis A, B, C, or E,
Respiratory Syncytial virus, human papilloma virus, Influenza
virus, Hib, Meningitis virus, Salmonella, Neisseria, Borrelia,
Chlamydia, Bordetella, Plasmodium, or Toxoplasma.
14. A process for the manufacture of an adjuvant composition,
comprising the following steps: (a) the formation of cholesterol
containing small unilamellar liposomes (SUL) in the absence of
detergent; and (b) admixing the preformed liposomes with saponin at
a ratio of saponin:cholesterol (w/w) exceeding 1.
15. A process for the manufacture of a vaccine composition,
comprising the following steps: (a) taking an adjuvant composition
produced according to the process of claim 14; and (b) adding an
antigen or an antigenic composition.
Description
The present invention provides an improved adjuvant formulation and
a process for producing said adjuvant. The adjuvant comprises an
ISCOM structure comprising a saponin, said ISCOM structure being
devoid of additional detergent. Also provided is an improved method
of producing an adjuvant, and vaccines comprising the adjuvant of
the present invention.
For many vaccines it is generally accepted that in order to
generate significant levels of antigen specific immune responses,
it is necessary to help the immune system by the inclusion of an
adjuvant. The term adjuvant comes from the Latin of the verb "to
Help" which is adjuvare. A number of adjuvants which help the
immune response to a co-administered antigen to achieve greater
magnitude than that observed if the antigen was given alone are
known in the art. These include metallic salts, such as aluminium
hydroxide or phosphate; liposomes, the bacterially derived
monophosphoryl lipid A, Cholera toxin, and numerous others.
Adjuvants may be classed as immunostimulants which have a direct
stimulatory effect on the cells of the immune system, or may be
classed as "vehicles" which function as carriers which present
antigen to the immune system more efficiently than when the antigen
is given alone. Alternatively, adjuvants may function in a
combination of these mechanisms.
Specific adjuvants may also be used to drive the immune response
into a particular desired characteristic. In theory, any given
immune response may characterised into two mutually exclusive
extremes of immune effector mechanisms. One extreme being
predominantly a humoral response (characterised by the generation
of Th2-type cytokines and immunoglobulin production) and a second
extreme of a predominantly cell-mediated immune response
(characterised by the generation of Th1-type cytokines and
cytotoxic T cells). Generally speaking what is actually observed in
real life is a balance of these two extremes, with any given
response being described as being predominantly humoral (Th2-type)
or predominantly cell-mediated (Th1-type). Thus for any particular
pathogen, if it is desired that a vaccine should induce a
predominantly Th1-type immune response, then the vaccine should be
formulated with a known Th1-type inducing adjuvant.
One such adjuvant which is known to induce a balance of humoral and
cell mediated immune response, which may include strong
cell-mediated and also strong humoral responses, are the
Immune-stimulating complexes (so called ISCOMs).
ISCOMs are three dimensional `cage-like` structures which have been
shown to form upon detergent removal from mixtures of saponins,
detergents and cholesterol. ISCOMs and their use in vaccines are
disclosed in EP 0 109 942 "Immunogenic protein or peptide complex,
method of producing said complexes and use thereof as an immune
stimulant and as a vaccine" . This patent discloses ISCOMs
comprising antigen with hydrophobic regions and a glycoside
(saponin), characterised in that the complex has an open spherical
structure consisting of circular subunits or parts of the spheric
structure. ISCOMs are thus open structures of around 30 nm in
diameter with a morphology which is different from liposomal
structures. The ISCOMs and parts thereof also usually have a lower
sedimentation constant than corresponding micelles and a higher
sedimentation constant than the corresponding monomeric form of
protein or peptide, and a higher sedimentation constant than the
corresponding liposome. The classical "cage-like" structure of
ISCOMs can be seen in the electron microscopy studies of EP 0 242
380 B1 and EP 0 180 564 B1.
During their manufacture, phospholipids or additional protein
antigens may be included in the structure. These ISCOM-protein
complexes have been used as very potent vaccines (EP 0 109 942 B1).
Alternatively, preformed ISCOMs without any additional antigen may
be mixed with extraneous antigen to form a vaccine wherein the
antigen is not in a complex with the ISCOM (EP 0 436 620 B1). These
vaccine formulations have also been shown to induce high levels of
immune responses.
ISCOM/protein complexes have also been formed by the covalent
conjugation of the protein antigen onto the surface of the ISCOM
(EP 0 180 564 B1). The use of ISCOMs for mucosal vaccination has
also been described Mowat et al. Immunology, 72, 317-322 (1991).
The ISCOM structure has been improved for use in mucosal
applications by the incorporation of membrane targeting proteins
(WO 97/30728).
The saponins are plant derived glycosides, a number of which have
been studied extensively for their biological properties (The Plant
Glycosides, McIlroy, R. J., Edward Arnold and co., London, 1951).
The saponins used most predominantly in the art for the production
of ISCOMs are those derived from the plants Quillaja saponaria
molina, Aesculus hippocastanum or Gyophilla struthium. Extracts of
the bark of Quillaja saponaria molina which are known to have
adjuvant activity are known in the art, for example Quil A
(Dalsgaard, K., 1974, Saponin adjuvants III, Archiv.fur dis Gesamte
Virusforschung, 44, 243-254). Also pure fractions of Quil A have
been described which retain adjuvant activity whilst being less
toxic than Quil A, for example QS21 (EP 0 362 279 B1, and U.S. Pat.
No. 5,057,540). QS21 is also described in Kensil et al. (1991. J.
Immunology vol 146, 431-437).
ISCOMs comprising other purified less toxic fractions of Quil A
have been used in the manufacture of vaccines. These structures
have been reported to have adjuvant activity (WO 96/11711).
Alternative particulate structures containing a saponin and a
sterol, other than ISCOMs which are also less toxic than the
saponin alone, have also been described (WO 96/33739).
Other saponins which have been described in the literature include
Escin, which has been described in the Merck index (12.sup.th ed:
entry 3737) as a mixture of saponins occuring in the seed of the
horse chestnut tree, Lat: Aesculus hippocastanum. Its isolation is
described by chromatography and purification (Fiedler,
Arzneimittel-Forsch. 4, 213 (1953)), and by ion-exchange resins
(Erbring et al., U.S. Pat. No. 3,238,190). Fractions of escin have
been purified and shown to be biologically active (Yoshikawa M, et
al. (Chem Pharm Bull (Tokyo) 1996 August;44(8):1454-1464)).
Sapoalbin from Gypsophilla struthium (R. Vochten et al., 1968, J.
Pharm.Belg., 42, 213-226) has also been described in relation to
ISCOM production.
ISCOMs are conventionally formed through two steps (e.g. as
described in EP 0 109 942 ). 1, solubilisation of membrane and
membrane proteins with detergent; 2, removal of solubilising agent
by several means whilst at the same time contacting the membrane
components with the saponin whose concentration is at least equal
to the critical micellular concentration of the saponin, or
removing the solubilising agent and directly transferring the
antigen to the solution of saponin. U.S. Pat. No. Patent No.
4,578,269 teaches particular methods of separating the antigen from
the solubilising agent. These methods include, amongst others:
centrifugation through a gradient of solubilisation agent into an
inverse gradient of saponin; or alternatively the solubilised
antigen can be mixed with saponin followed by centrifugation of the
mixture and dialysis to remove excess detergent.
EP 0 242 380 teaches of an improvement in this manufacturing
process. This patent tells how the addition of lipids to the
process prevents the formation of antigen/glycoside micelles, and
ensures that the antigen/glycoside structures are all ISCOM-like.
The specification states that the lipids may be added at any stage,
at a molar ratio of at least 0.1:1 of lipids to antigen, and
preferably 1:1. Examples of lipids include cholesterol and
phosphatidyl choline. Thus, a method for producing an immunogenic
complex between an antigen and a polar triterpensaponin, associated
by the attraction between the hydrophobic regions of the
triterpensaponin, lipid and antigen,
said complex must be formed by: (i) mixing antigen and lipid with a
solubilising agent, thus forming complexes between the antigen,
lipid and the solubilising agent, and (ii) removing the
solubilising agent from the mixture in the presence of the saponin
whose concentration is at least equal to the critical micellular
concentration of the saponin, or removing the solubilising agent
and directly transferring the mixture to the solution of saponin;
wherein the solubilisation agent is selected from a group
comprising ionic or non-ionic detergents, Zwitterionic detergents,
or detergents based on gallic acid; wherein the complex has a
higher sedimentation constant that the monomeric antigen, and a
lower sedimentation constant than the corresponding micelles; and
wherein the complex has an open spherical structure, the
improvement comprising adding to the solubilised antigen at least
one lipid selected from the group comprising of fats, glycerol
ethers, waxes, phospholipids, glycolipids, isoprenoids, steroids,
and mixtures thereof, prior to the contact of the solubilised
antigen with the glycoside-containing solution. Alternatively,
ISCOMs have been produced using the solubilising agent to
solubilise the antigen and/or lipids, followed by the spontaneous
formation of the ISCOM structure without the removal of the
solubilising agent. U.S. Pat. No. 4,981,684, EP 0 415 794 A1, and
EP 0 766 967 A1 all teach a process for the production of ISCOMs
with water insoluble antigens. This process comprises the
solubilising of the antigen in a solubilising agent, admixing the
solubilised antigen, a glycoside and a sterol and forming ISCOMs
without the removal of the solubilising agent.
ISCOMs produced without antigen, so called Iscomatrix, have also
been described WO 96/11711.
Thus, several process for the manufacture of ISCOMs have been
described. All of these processes require the presence of an
additional detergent other than the saponin itself. The ISCOMs are
then either removed from the vaccine by dialysis or centrifugation,
or are left in the vaccine formulation.
Despite the attempt at detergent removal in some of these methods,
all of the resultant ISCOMs adjuvants or vaccines will contain some
additional detergent, other than the saponin itself. Detergents by
their very nature associate with lipid membranes, and so will never
be totally removed. Furthermore, the dialysis method of removing
substances works on a principle of equilibrium, thus, it is
physically impossible to remove all traces of the detergent. Such
formulations containing residual detergent may be less stable and
more toxic than the adjuvants of the present invention without the
detergent being present.
Examples of detergents that have been used in the production of
ISCOMs include, sodium cholate, n-Octyl glucopyranoside,
polyoxyethylene ethers or phenyl ethers, TritonX-100
(octylphenolether of polyethylene oxide), acylpolyoxyethylene
esters, acyl polyoxyethylene sorbitan esters (the Tween series),
the SPAN series, ionic detergents such as the gallic acid
detergents (bile salts).
The present invention provides for adjuvant formulations comprising
a saponin and a sterol, characterised in that the adjuvant is in
the form of an ISCOM, and that said ISCOM is free of additional
detergent, other than the saponin.
Also, provided is a process for the production of an ISCOM
comprising a saponin and a sterol, characterised in that the
process is free of additional detergents, other than the
saponin.
Vaccines are also provided by the present invention comprising an
adjuvant formulation comprising a saponin and a sterol,
characterised in that the adjuvant is in the form of an ISCOM, and
that said ISCOM is free of additional detergent, other than the
saponin, and an antigen.
Preferably, the process of the present invention comprises two
steps: 1. The formation of cholesterol containing small unilamellar
liposomes (SUL) in the absence of detergent, and; 2. Admixing the
preformed liposomes with saponin at a ratio of saponin:cholesterol
(w/w) exceeding 1.
Adjuvant formulations thus formed are in the form of an ISCOM, said
ISCOMs being free of detergent.
Optionally the SUL may be formed in the presence of antigen such
that SUL are formed in association with antigen. This may be
particularly preferable when using hydrophobic antigen. Also, the
SUL may be formed in the presence of reactive phospholipids such as
phosphatidyl ethanolamine or phosphatidyl serine or chemically
derivatised forms thereof such that antigen may be conjugated to
the ISCOM using commonly known heterobifunctional cross-linkers,
such as SPDP. Alternatively a vaccine may be formed by simple
admixing of the ISCOMs formed by the process of the present
invention, or ISCOMs which are free of additional detergent, with
antigen.
In order for a vaccine or adjuvant to be suitable for
administration into a human, it has to comply with rigorous safety
and quality control checks. Currently, there is no detergent
available which is generally recognised as safe for injection. All
detergents used to date in the production of ISCOMs are liable to
be reactogenic, and induce cell lysis and necrosis at the site of
injection. Thus, any ISCOM based vaccines containing even trace
amounts of detergents must be supported by extensive reactogenicity
and safety studies before gaining regulatory approval.
The adjuvants and vaccines of the present invention do not by
definition have this requirement as they do not contain any
detergent. The reactogenicity studies required for such vaccines
and adjuvants only have to focus on the role of the saponin. It is,
therefore, much easier to gain approval for these "clean" ISCOM
products, than it is for vaccines containing additional reactogenic
material.
A vaccine manufacturer must produce large quantities of a product
in a manner which is reproducible and susceptible to quality
control (QC) and Good Manufacturing Processing conditions (GMP).
The methods previously used for the removal of detergent from
ISCOMs are difficult to control under these conditions. For
example, the scaling up of dialysis process is limited by the size
of the dialysis equipment, and is also inherently variable
depending on many factors including ambient temperature and media
osmolarity. Equally, the scale of ISCOM production using the
centrifuge method was previously dependent of the size of your
centrifuge. Thus, it is difficult to produce a product using the
previous methods in a large scale, controlled and reproducible
manner. The process of the present invention is not limited by the
size of any equipment, it is also susceptible to QC and GMP control
throughout the process. For example a batch of liposomes may be
produced and released for sterility and size, also a batch of
saponin may be released for sterility and purity, all before the
liposomes and saponin are admixed.
The presence or absence of additional detergent in the final
preparations of ISCOMs formed by the classical methods can be
determined by gas chromatography, or HPLC.
The process of the present invention does not require this variable
detergent removal step and is therefore much easier to control.
Each intermediate used in the process of the present invention may
be produced and released from a QC point of view before the final
step of admixing the pre-formed liposomes with the saponin.
Additionally, the process of the present invention is not limited
in the quantity of the final product.
The adjuvants of the present invention are suitable for
administration to the recipiant via any route, including systemic
routes such as intramuscular or subcutaneous or transdermal, or via
a mucosal route such as intranasal or oral. Saponin based adjuvant
formulations which are not haemolytic are known (WO 96/33739).
However, in certain circumstances the adjuvant of the present
invention may beneficially retain significant haemolytic activity
of the saponin, for example when used as an intranasal vaccine, or
when some reactogenicity may be tolerated.
The SUL formed during the process of the present invention may be
manufactured using well known techniques of the art. Such processes
which do not involve additional detergent include sonication,
microfluidisation, or membrane extrusion. For example phosphatidyl
choline (PC) dissolved in ethanol may be added to a flask and dried
under vacuum or inert gas. PBS or other pharmaceutically acceptable
excipient may then be added and the contents of the flask
sonicated. Optionally the lipid suspension may be microfluidised to
attain a uniform preparation of SUL of around 100 nm in diameter.
The SUL comprise cholesterol and also include one or more
phospholipids. The ratio of cholesterol to phospholipid is at most
50% and preferably 20-25% (w/w). The phospholipid is preferably
phosphatidylcholine and is most preferably chosen so as to have a
low transition temperature e.g. Dioloeoylphosphatidylcholine or
dilauryl phosphatidylcholine. Optionally a charged phospholipid
(e.g. phosphatidylglycerol or phosphatidyl serine) may be
added.
The saponins for use in the present invention include saponins
derived from Quillaja Saponaria Molina, Aesculus hippocastanum or
Gyophilla struthium. Particularly preferred saponins are QuilA or
extracts therefrom from Quillaja Saponaria Molina. Particularly
preferred extracts from Quil A include QS21. Typically for human
administration the saponin will be present in a vaccine in the
range 1 .mu.g-100 .mu.g, preferably 10 .mu.g-50 .mu.g per dose.
The adjuvants of the present invention comprise a sterol. Ratios of
saponin:sterol in adjuvants of the present invention is
substantially in the range between 1:1 to 100:1 (w/w), preferably
between 1:1 to 10:1 (w/w), and most preferably 5:1 (w/w). The
sterol is preferably cholesterol.
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 thereof);
Corynebacterium spp., including C. diphtheriae (for example
diphtheria toxin and derivatives thereof); Borrelia spp., including
B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii
(for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA,
OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA,
DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agent
of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including
R. rickettsii; Chlamydia spp., including C. trachomatis (for
example MOMP, heparin-binding proteins), C. pneumoniae (for example
MOMP, heparin-binding proteins), C. psittaci; Leptospira spp.,
including L. interrogans; Treponema spp., including T. pallidum
(for example the rare outer membrane proteins), T. denticola, T.
hyodysenteriae; or derived from parasites such as Plasmodium spp.,
including P. falciparum; Toxoplasma spp., including T. gondii (or
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.
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.
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.
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, RAP1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP,
SALSA, PfEXP1, Pfs25, Pfs28, PFS27/25, Pfs16, 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, 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 (GA377), 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.
It is foreseen that compositions of the present invention will be
used to formulate vaccines containing antigens derived from
Borrelia sp. For example, antigens may include nucleic acid,
pathogen derived antigen or antigenic preparations, recombinantly
produced protein or peptides, and chimeric fusion proteins. In
particular the antigen is OspA. The OspA may be a full mature
protein in a lipidated form virtue of the host cell (E.Coli) termed
(Lipo-OspA) or a non-lipidated derivative. Such non-lipidated
derivatives include the non-lipidated NS1-OspA fusion protein which
has the first 81 N-terminal amino acids of the non-structural
protein (NS1) of the influenza virus, and the complete OspA
protein, and another, MDP-OspA is a non-lipidated form of OspA
carrying 3 additional N-terminal amino acids.
Vaccines of the present invention may be used for the prophylaxis
or therapy of allergy. Such vaccines would comprise allergen
specific (for example Der p1) and allergen non-specific antigens
(for example the stanworth decapeptide).
Preferably, antigens to be used in the present invention are
provided in aqueous solution or aggregates in aqueous suspension.
Also forming part of the present invention are antigens with are
present in a detergent containing solution or suspension. Thus
vaccines of this type comprise ISCOM structure which are free of
detergent, in the presence of external antigen in a solution or
suspension of detergent.
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, most preferably 1 to 50 .mu.g. An optimal amount for a
particular vaccine can be ascertained by standard studies involving
observation of appropriate immune responses in subjects. Following
an initial vaccination, subjects may receive one or several booster
immunisation adequately spaced.
Also provided by the present invention is a process for the
manufacture of an vaccine composition comprising the following
steps: (1) the formation of cholesterol containing small
unilamellar liposomes (SUL) in the absence of detergent; (2)
admixing the preformed liposomes with saponin at a ratio of
saponin:cholesterol (w/w) exceeding 1; (3) admixing an antigen with
the product of step 2.
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. Furthermore, use of an vaccine in the manufacture of a
medicament for the immunoprophylaxis or therapy of disease or
infection or cancer is also provided. 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.
The present invention is illustrated but not restricted by the
following examples.
EXAMPLE 1
Preparation of ISCOMS by Addition of QS21 to Small Unilamellar
Liposomes (SUL).
Dioloeoyl phosphatidylcholine (1 g) in ethanol was mixed with
cholesterol (250 mg) in ethanol (solubilised by warming) and the
ethanol removed under vacuum. Phosphate buffered saline (25 ml) was
added and the flask agitated to suspend the lipids. The resulting
multilamellar liposomes were microfluidised until the particle size
was 100 nm as determined by photon correlation spectroscopy. The
liposomes were filter sterilised through 0.22 .mu.m filters and
stored at 4.degree. C.
QS21 (obtained from Aquila Biopharmaceuticals, Mass.) was dissolved
in water at 2 mg/ml, the pH adjusted to 7 with HCl, and the
solution filter sterilised through sterile 0.22 .mu.m filters. To
100 .mu.l of this solution (200 .mu.g QS21) was added 20 .mu.l SUL
(containing 40 .mu.g cholesterol).
The original SUL and the QS21-SUL mixture were examined by
transmission electron microscopy of frozen hydrated material
(cryo-electron microscopy). FIG. 1 shows that the SUL are
predominantly spherical unilamelar vesicles with size ranging from
40 to 200 nm. FIG. 2 shows that when the QS21 is added at a
five-fold excess over cholesterol the vesicular structures
disappear and are replaced by spherical cage-like structures. These
closely resemble the open spherical ISCOM structure. In FIG. 2
there is also evidence of ISCOMs in the process of being formed by
budding off from a vesicle. In the micrographs the bar represents
100 nm. Arrow heads point towards clearly discernible open
spherical structures resembling ISCOMs.
EXAMPLE 2
Comparison of Density of SUL and ISCOMs Prepared by Adding QS21 to
SUL.
SUL were prepared as in example 1 except that a trace of
radioactive (.sup.3 H) cholesterol was included and the SUL were
prepared by sonication. ISCOMs were prepared by adding QS21
directly to these SUL at a QS21:cholesterol ratio of 5:1.
The samples were layered onto a 10-60% sucrose gradient and
centrifuged in an SW-40 rotor 34000 rpm and fractions collected and
analysed for radioactivity. The radioactivity associated with the
SUL was found only at the top of the gradient, whereas most of the
radioactivity associated with the ISCOMs formed by adding QS21 to
the SUL was found further down the tube indicating the ISCOMs have
a higher density than the liposomes.
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