Note: Descriptions are shown in the official language in which they were submitted.
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Vaccines
The present invention relates to novel vaccine formulations, to methods of
their production and to their use in medicine. In particular, the present
invention
relates to vaccines containing alum. an antigen, an immunologically active
fraction
derived from the bark of Quillaj a S aponaria Molina such as QS21, and a
sterol.
Immunologically active saponin fractions having adjuvant activity derived
from the bark of the South American tree Quillaja Saponaria Molina are known
in the
art. For example QS21, also known as QA21, an Hplc purified fraction from the
Quillaja Saponaria Molina tree and it's method of its production is disclosed
(as
QA21) in US patent No. 5,057,540. Quillaja saponin has also been disclosed as
an
adjuvant by Scott et al, Int. Archs. Allergy Appl. Immun., l985, 77. 409.
However,
the use of QS21 as an adjuvant is associated with certain disadvantages. For
example
when QS21 is injected into a mammal as a free molecule it has been observed
that
necrosis, that is to say, localised tissue death, occurs at the injection
site.
WO 96/33739 discloses vaccine formulations comprising an antigen, an
immunologically active fraction derived from the bark of Quillaja Saponaria
Molina
such as QS21, and a sterol.
It has now surprisingly been found that incorporation of alum in vaccine
formulations containing MPL, QS21 and SUV enhances both humoral and cellular
responses and that vaccine formulations containing MPL, QS21, SUV and alum are
non-toxic with a good reactogenicity profile and have enhanced adjuvant
activity. In
addition, the combined adjuvant appears to favour TH1 responses.
In a first aspect the invention provides a vaccine adjuvant comprising alum,
an
immunologically active saponin fraction, and a sterol. By the term 'alum' is
meant
aluminium hydroxide or aluminium phosphate.
Preferably the adjuvant compositions of the invention contain the
immunologically active saponin fraction in substantially pure form. Preferably
the
compositions of the invention contain QS21 in substantially pure form, that is
to say,
the QS21 is at least 90% pure, preferably at least 95% pure and most
preferably at
least 98% pure.
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Other immunologically active saponin fractions useful in compositions of the
invention include QA 17/QS 17. Compositions of the invention comprising QS21
and
cholesterol show decreased reactogenicity when compared to compositions in
which
the cholesterol is absent, while the adjuvant effect is maintained. In
addition it is
known that QS21 degrades under basic conditions where the pH is about 7 or
greater.
A further advantage of the present compositions is that the stability of QS21
to base-
mediated hydrolysis is enhanced in formulations containing cholesterol.
Preferred sterols include ~i-sitosterol, stigmasterol, ergosterol,
ergocalciferol
and cholesterol. These sterols are well known in the art, for example
cholesterol is
disclosed in the Merck Index, 1 lth Edn., page 341, as a naturally occurring
sterol
found in animal fat. Most preferably the sterol is cholesterol.
Preferred compositions of the invention are those forming a liposome
structure, that is to say small unilammelar vesicles (SUV). Compositions where
the
sterol/immunologically active saponin fraction forms an ISCOM structure also
form
an aspect of the invention.
The ratio of QS21 : sterol will typically be in the order of 1 : 100 to 1 : 1
weight to weight. Preferably excess sterol is present, the ratio of QS21 :
sterol being
at least 1 : 1 w/w. Typically for human administration QS21 and sterol will be
present
in a vaccine in the range of about 1 p.g to about l00 p.g, preferably about 10
p.g to
about 50 p.g per dose of QS21.
The liposomes preferably contain a neutral lipid, for example
phosphatidylcholine, which is preferably non-crystalline at room temperature,
for
example eggyolk phosphatidylcholine, dioleoyl phosphatidylchoiine or dilauryl
phosphatidylcholine. The liposomes may also contain a charged lipid which
increases
the stability of the Iipsome-QS21 structure for liposomes composed of
saturated
lipids. In these cases the amount of charged lipid is preferably 1-20% w/w,
most
preferably 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), most
preferably 20-25%.
Particularly preferred and advantageous compositions of the invention contain
MPL (~-deacylated mono-phosphoryl lipid A, also known as 3D-MPL). 3D-MPL is
known from GB 2 220 211 (Ribi) as a mixture of 3 types of De-O-acylated
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monophosphoryl lipid A with 4, ~ or 6 acylated chains and is manufactured by
Ribi
Immunochem, Montana. A preferred form is disclosed in International Patent
Application 94!2129Z.
Suitable compositions of the invention are those wherein liposomes are
S initially prepared without MPL, and MPL is then added, preferably as 100 nm
particles. The MPL is therefore not contained within the vesicle membrane
(known as
MPL out). Compositions where the MPL is contained within the vesicle membrane
(known as MPL in) also form an aspect of the invention. The antigen can be
contained within the vesicle membrane or contained outside the vesicle
membrane.
Preferably soluble antigens are outside and hydrophobic or lipidated antigens
are
either contained within or outside the membrane.
In a preferred aspect of the invention, liposomes/SUV are first added to the
QS21 and then mixed with alum which results in a significant proportion of the
QS21
binding to the alum (via interaction through the liposomes). Such a
formulation, when
injected, is expected to result in a slower release of QS21 to the body, due
to a depot
effect of the alum, than if the QS21 was free or in un-fixed liposomes. The
formulation containing MPL, QS21, SUV and alum are particularly advantageous
as
they are non-toxic and highly immunogenic.
Preferably the vaccine formulations will contain an antigen or antigenic
composition capable of eliciting an immune response against a human or animal
pathogen. In a first aspect the present invention therefore provides a vaccine
composition comprising alum, an antigen, an immunologically active saponin
fraction
and a sterol.
Antigen or antigenic compositions known in the art can be used in the
compositions of the invention, including polysaccharide antigens. antigen or
antigenic
compositions derived from HIV-1, (such as gp120 or gp160), any of Feline
Immunodeficiency virus, human or animal herpes viruses, such as gD or
derivatives
thereof or Immediate Early protein such as ICP27 from HSV 1 or HSV2,
cytomegalovirus (especially human) (such as gB or derivatives thereot~,
Varicella
Zoster Virus (such as gpI, II or III), or from a hepatitis virus such as
hepatitis B virus
for example Hepatitis B Surface antigen or a derivative thereof, hepatitis A
virus,
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hepatitis C virus and hepatitis E virus, or from other viral pathogens, such
as
Respiratory Syncytial virus (for example HSRV F and G proteins or immunogenic
fragments thereof disclosed in US Patent 5,149,650 or chimeric polypeptides
containing immunogenic fragments from HSRV proteins F and G. eg FG
glycoprotein
disclosed in US Patent 5,194,595), antigens derived from meningitis strains
such as
meningitis A. B and C, Streptococcus Pneumonia, human papilloma virus,
Influenza
virus, Haemophilus Influenza B (Hib), Epstein Barr Virus (EBV), or derived
from
bacterial pathogens such as Salmonella, Neisseria, Borrelia (for example OspA
or
OspB or derivatives thereof), or Chlamydia, or Bordetella for example P.69, PT
and
FHA, or derived from parasites such as plasmodium or toxoplasma.
HSV GIycoprotein D (gD) or derivatives thereof is a preferred vaccine
antigen. It is located on the viral membrane, and is also found in the
cytoplasm of
infected cells (Eisenberg R.J. et al; J of Virol 1980 35 428-435). It
comprises 393
amino acids including a signal peptide and has a molecular weight of
approximately
60 kD. Of all the HSV envelope glycoproteins this is probably the best
characterised
(Cohen et al J. Virology 60 157-166). In vivo it is known to play a central
role in
viral attachment to cell membranes. Moreover, glycoprotein D has been shown to
be
able to elicit neutralising antibodies in vivo and protect animals from lethal
challenge. A truncated form of the gD molecule is devoid of the C terminal
anchor
region and can be produced in mammalian cells as a soluble protein which is
exported
into the cell culture supernatant. Such soluble forms of gD are preferred. The
production of truncated forms of gD is described in EP 0 139 417. Preferably
the gD
is derived from HSV-2. An embodiment of the invention 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 allow for
the
mature soluble 283 amino acid protein to be secreted from a host cell.
In another aspect of the invention, Hepatitis B surface antigen is a preferred
vaccine antigen.
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As used herein the expression 'Hepatitis B surface antigen' or 'HBsAg'
includes any HBsAg antigen or fragment thereof displaying the antigenicity of
HBV
surface antigen. It will be understood that in addition to the 226 amino acid
sequence
of the HBsAg antigen (see Tiollais et al, Nature, 317, 489 ( 1985) and
references
therein) HBsAg as herein described may, if desired, contain all or part of a
pre-S
sequence as described in the above references and in EP-A- 0 278 940. In
particular
the HBsAg may comprise a polypeptide comprising an amino acid sequence
comprising residues 12-52 followed by residues 133-145 followed by residues
175-
400 of the L-protein of HBsAg relative to the open reading frame on a
Hepatitis B
virus of ad serotype (this polypeptide is referred to as L*; see EP 0 414
374). HBsAg
within the scope of the invention may also include the pre-S1-preS2-S
polypeptide
described in EP 0 198 474 (Endotronics) or close analogues thereof such as
those
described in EP 0 304 578 (Nlc Cormick and Jones). HBsAg as herein described
can
also refer to mutants, for example the 'escape mutant' described in WO
9l/14703 or
European Patent Application Number 0 511 855A1, especially HBsAg wherein the
amino acid substitution at position 145 is to arginine from glycine.
Normally the HBsAg will be in particle form. The particles may comprise for
example S protein alone or may be composite particles, for example (L*,S)
where L*
is as defined above and S denotes the S-protein of HBsAg. The said particle is
advantageously in the form in which it is expressed in yeast.
The preparation of hepatitis B surface antigen S-protein is well documented.
See for example, Harford et al (I983) in Develop. Biol. Standard 54, page 125,
Gregg
et a1 (1987) in Biotechnology, 5, page 479, EP 0 226 846, EP 0 299 108 and
references therein.
In another embodiment, the vaccine antigen is an RSV antigen. In particular
an F/G antigen. US patent 5l94595 (Upjohn) describes chimeric glycoproteins
containing immunogenic segments of the F and G glycoproteins of RSV and
suggests
that such proteins can be expressed from a variety of systems including
bacterial,
yeast, mammalian (eg CHO cells) and insect cells (using for example a
baculovirus).
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Wathen et al (J. Gen. Virol. l989, 70, 2625-2635) describes a particular RSV
FG chimeric glycoprotein expressed using a baculovirus vector consisting of
amino
acids i -489 of the F protein linked to amino acids 97-279 of the G protein.
The fornnulations within the scope of the invention rnay also contain an anti-
s tumour antigen and be useful for immunotherapeutically treating cancers.
Vaccine preparation is generally described in New Trends and Developments
in Vaccines, edited by Voller et al., University Park Press, Baltimore,
Maryland,
U.S.A. 1978. Encapsulation within liposomes is described, for example, by
Fullerton,
U.S. Patent 4.235,877. Conjugation of proteins to macromolecules is disclosed,
for
example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent
4,474,757.
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 vaccines. 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 mcg of protein, preferably 2-100 mcg, most preferably 4-40
mcg.
An optimal amount for a particular vaccine can be ascertained by standard
studies
involving observation of appropriate immune responses in subjects. Following
an
initial vaccination, subjects may receive one or several booster immunisation
adequately spaced.
The formulations of the present invention may be used for both prophylactic
and therapeutic purposes.
Accordingly in a further aspect, the invention therefore provides use of a
vaccine composition of the invention for the treatment of human patients. The
invention provides a method of treatment comprising administering an effective
amount of a vaccine of the present invention to a patient. In particular, the
invention
provides a method of treating viral, bacterial, parasitic infections or cancer
which
comprises administering an effective amount of a vaccine of the present
invention to a
patient.
The following examples and data illustrates the invention.
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Example 1 Preparation of vaccine containing alum, SUV MPL and QS21
1.1 Method of preparation of SUV
A mixture of lipid (such as phosphatidylcholine either from egg-yolk or
synthetic) and cholesterol in organic solvent, is dried down under vacuum (or
alternatively under a stream of inert gas). An aqueous solution (such as
phosphate
buffered saline) is then added, and the vessel agitated until all the lipid is
in
suspension. This suspension is then microfluidised until the liposome size is
reduced
to 100 nm, and then sterile filtered through a 0.2 p,m filter. Extrusion or
sonication
could replace this step. Typically the cholesterol: phosphatidycholine ratio
is 1:4
(w/w), and the aqueous solution is added to give a final cholesterol
concentration of 5
to 50 mg/mi.
1.2 Antigen ( 1-500 p.g, preferably 10-100 pg) is added to alum eg (aluminium
hydroxide or aluminium phosphate) ( 100-500 fig) in water. The volume of water
is
chosen so that the volume of the final formulation is S00 p.l. After
incubating for 15-
30 minutes, 50 wg of MPL is added in the form of small-particle MPL
(W094/21292).
The MPL is left to adsorb onto the alum for 15-30 minutes at room temperature.
10
times concentrated phosphate buffered saline ( 1.5 M sodium chloride, 0.5M
sodium
phosphate pH 7.5) is then added in such a volume so as to render the final
formulation
isotonic. This formulation is incubated at room temperature for 15-30 minutes.
QS21 (50 pg) is then added to SUV (containing between 50 and 250 pg
cholesterol). This mixture is added to the above alum/antigen/MPL/buffer
mixture. If
required a bacteriostatic such as thiomersal is added (50 p.g).
Example 2
Table 1 shows the binding of QS21 to alum in the presence and absence of
liposomes containing 25% (w/w) in dioleoyl phosphatidylcholine, and using a
five-
fold excess of cholesterol over QS21.
Formulation SUV ~ p.g QS21
bound
500 ug Alum+50 pg QS21 0 < i 0
500 ug Alum+50 ~g QS21 250 pg chol + 1 mg DOPC > 40
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In order to increase the binding of QS21 to alum, the quantity of Iiposomes
can be decreased. This decreases the cholesterol:QS2I ratio, however it has
been
shown that the QS21 remains non-toxic for cholesterol:QS21 ratios of 1:1 and
greater.
Table 2 shows that if the quantity of alum is decreased (from 500 p.g to 100
pg) the
quantity of QS2I that is bound decreases significantly, and the quantity of
MPL that is
bound also decreases. By adding less liposomes, yet maintaining a
cholesterol:QS21
ratio of 1:1 or greater, increased quantities of QS21 and MPL can be bound to
the
alum.
Formulation Chol/QS21 ~g QS21 boundpg MPL bound
500 p.g alum + ~0 pg QS21 5l1 42 >48
+ 50 pg
MPL
l00 pg alum + 50 pg QS21 ~/1 17 >40
+ 50 pg
MPL
100 p.g alum + 50 pg QS21 2/1 30 >45
+ 50 pg
MPL
100 pg alum + 50 p.g QS21 1/1 40 >45
+ 50 pg
MPL
Example 3
The adjuvant effect of a combination of antigen (gD2t from Herpes Simple:c
Virus-2 - expressed in CHO cells and comprises 283 amino acid from the mature
N-
terminal of the mature glycoprotein) with MPL and QS21 in combination with
liposomes was tested with and without alum. The formulations were tested in
African
Green Monkeys.
African Green Monkeys were immunised twice (0, 28 days) with 20 ~g gD2t
plus 50 ~g MPL plus 50 qg QS21 with or without liposomes (250 p.g cholesterol
plus
1 mg DOPC) and with or without 500 pg alum. On day 42 the immune response was
analysed.
The results are outlined below in figures 1 to 4.
The humoral response was measured as IgG against the gD protein. Figure 1
shows that the combination of MPL+QS21+SUV+alum induced higher titres than in
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the absence of alum, Figure 2 shows that the formulation of the invention
provided
the superior antigen specific proliferation.
The data shows that incorporation of aluminium hydroxide in vaccine
formulations containing MPL and QS21 and SUV enhances both the humoral and
cellular responses. This is an unexpected finding since it is generally
accepted that
aluminium as an adjuvant tends to favour Th2 type responses, yet the results
presented
here demonstrate that the response contains a significant Th 1 component which
is not
depressed by the addition of alum.
The formulation containing MPL and QS21 and SUV and alum is non-toxic
and highly immunogenic.
Example 4 Production of RSV FG CHO cell derived proteins
The plasmid pEE 14-FG contains a chimeric construct comprising of a fusion
between amino acid sequences of F (1-525) and G (69-298) and was received from
a
collaboration with A. BOLLEN (ULB/CRI, Belgium). This FG fusion protein
contains a total of 755 amino acids. It starts at the N-terminal signal
sequence of F and
lacks the C-terminal transmembrane domain (525-574) -anchor domain- of F
glycoprotein. Then, followed the extracellular region of G glycoprotein,
without the
amino-terminal region that contains the Signal/Anchor domain of G, a typical
class II
glycoprotein.
The pEE 14-FG expression plasmid was generated by the insertion of the FG
coding sequence from pNIV2857 (A. Bollen, ULB/CRI, Belgium) as an Asp7181
(blunt) 5' - HindIII (blunt) 3' restriction fragment (2188 bp) into the SmaI
site of
pEE 14 (Celltech). A Kozak sequence in lieu of the FG start ATG was generated
into
the pNIV 2857 construction as follows:
pEE 14---ccc gtacc ATG GAG -----x-----CAG TAG aagct ggg ---pEE 14
(SmaI) Metl Gln(298)Stop
Asp718I(klenow) HindIII(klenow)
3 0 5' F( 1-525)-------x--------G(69-298)3'
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The F sequence in pEEl4-FG is from SS2 RSV strain, and was kindly made
available by Dr. PRINGLE as a cDNA construct in a Vaccinia vector (Baybutt and
Pringle, J.Gen.ViroL, 1987, 2789-2796). The G sequence is from A2 RSV strain
and
was generated from a recombinant G Vaccinia virus obtained from Dr. G. WERTZ
5 (Alabama, USA).
CHO Kl transfection and stable FG protein expression.
CHO Kl cells derived from MCB 024M (Celltech) were transfected with 20
ug of pEE 14-FG plasmid DNA twice CsCI purified using the Ca-phosphate -
glycerol
10 transfection procedure. Cell clones were selected according to the
procedure of the GS
(glutamine synthetase ) expression system (Crocett et al BioTechn., 1990,
VolB, 662)
and amplified in the presence of 25 micro molar methionine sulphoximine (MSX)
in
G MEM medium containing no glutamine and supplemented with 10% dialyzed FBS
(Foetal Bovine Serum). Following transfection, 39 MSX resistant clones were
screened in 24-well plates and their supernatants were tested for secretion of
the FG
fusion protein. A11 transfectants proved to be positive for F antigen
expression using a
specific 'Sandwich' ELISA assay (i.e. rabbit polyclonal anti FG serum /
Antigen /
mAB 19). Monoclonal antibody 19 recognises a conformational F 1 - epitope and
is
neutralising.
The 3 best FG-producer clones (n~ 7, 13 and 37) were single-cell subcloned in
a limiting dilution assay using 0.07 cells per well in a 96-well plates. A
total of ~ 9
positive subclones were obtained and the 16 best FG-producers were further
characterised by western blot and ELISA. Again, the 8 best FG-subclones were
further amplified and their FG expression was evaluated in presence and
absence of
sodiumbutyrate (2mM) or DMSO ( 1 or 2%). Six subclones were amplified and cell
vials were made and stored at -80~C and liquid N2. Finally, the 3 best FG-
subclones
were selected. These are CHOK1 FG ~ 7.18, ~ 13.1 , and ~ 37.2.
Westernblot analyses (non-reducing conditions) with monoclonal mAB 19
indicated a major band of FG at about I35 kDa. The purified FG protein from
recombinant Baculovirus FG infected cells (UPJOHN) appeared as major broad
bands
at +/-1 OOkDa and other bands at +/-70kDa under similar blot conditions.
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The addition of Sodium butyrate in CHO-FG cell culture medium increased
the expression level of FG 3 to 12 fold depending on the subclone and cell
culture
growth conditions. In particular, subclone CHO-FG 13.1 expressed 8-10 fold
more FG
protein in the presence of butyrate (WB/ELISA).
Expression level determination was performed by ELISA (mAB 19 or MoAb
AK13) using purified FG baculo protein as standard, as well as by western blot
analysis using serial dilution.
Depending of the ELISA assay and cell culture conditions, the expression
level of CHO-FG 13.1 is 5-12 ug of FG/ml after treatment with butyrate. Under
accumulation conditions and medium replacements (3 to S days ) yields of 16 to
28 ug
of secreted FG protein /ml were obtained.
CHOK1 FG l3.1 cell line was adapted to grow in suspension and serumfree
(S/SF) conditions using a proprietary growth medium. Cell line CHO-FG 13.1
S/SF
grown in a medium without butyrate expressed similar yields as the parental
adherent
cell line grown in medium with butyrate. The addition of butyrate to CHO-FG
13.l
5/SF media has little effect on production of FG ( 1.5 to 2 fold increase).
Long term expression evaluation and preliminary genetic characterisation
showed that CHO-FG 13.1 and the S/SF adapted 13.1 cell line were stable,
contained
intact FG expression cassettes giving rise to one single~mRNA band of about
3000
nucleotides long (Southern and Northern analyses). The CHO-FG clone 13.1 S/SF
was further used for production of FG antigen.
The use of alum/MPL/QS21 /SUV for the enhancement of the immune
response in African Green Monkeys towards the FG protein from RS V
(Respiratory
Syncytial Virus).
The FG protein (fusion protein containing the F- and G- proteins from RSV)
was expressed in CHO cells and purified. 20 ~g of the purified protein was
adsorbed
on alum (S00 pg) to which monophosphoryl lipid A (MPL: 50 pg) was added. After
incubating 30 minutes at room temperature, phosphate buffered saline was
added.
Then either SUV alone or a mixture of SUV and QS21 (50 ~g QS21, SUV containing
250 ~g cholesterol and 1 mg DOPC) were added. African green monkeys were
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12
injected three times with these formulations, or with FG on alum alone or FG
mixed
with MPL, SUV and QS21 in the absence of alum.
Figure S below show the RSV neutralising titres and the FG-ELISA titres
obtained for each formulation. It is clear that the group alum/MPL/QS21 /SUV
induces the highest titres.
Example ~ Comparison of QS2I / SUV containing formulations with Alum
formulation of Hepatitis B vaccine containing SL* antigen
Introduction
SL* was produced in accordance with the procedure set out in European
Patent application No. 4l4374.
An immunogenicity study was conducted in Balb/c mice to compare the
humoral responses induced by QS2I-SUV containing formulations in presence or
not
of Al(OH)3. MPL dose was 5pg, QS21 5pg, SUV contained 25p8 cholesterol and
100pg DOPC.
The experimental protocol is described in Material and Methods.
Briefly, mice were immunised intramuscularly in the leg twice at 4 weeks
interval
with SL* vaccines containing vehicle, immunostimulants or combinations of
both.
Anti-HBs humoral responses (IgG and isotypes) were analysed.
The following groups were included in the study:
1. SL* (2ug) Al(OH)3 (50 ug)
2. SL* (2ug) Al(OH)3 (50 ug) / MPL / QS21-SUV
3. SL* (2ug) Al(OH)3 (50 ug) i QS21-SUV
4. SL* (2ug) MPL / QS21-SUV
Results
Humoral responses were measured by Elisa as described in Material and
Methods. Two time points were analyses: 28 days after the first injection (28
post I)
and 14 days following the booster injection {14 post II).
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13
Post I and post II anti-HBs response analysed on pooled sera are presented in
Figure
6.
These data show that in primary response, comparable antibody titers are
induced by all formulations containing QS2I-SUV while a weaker response is
observed when Al(OH)3 alone is used.
In secondary response, the lowest antibody response was also induced by
AI(OH)3 containing vaccine. However, all formulations containing QS21-SUV did
not behave the same way.
The two formulations containing Al(OH)3 QS21-SUV (+/- MPL) induced the
strongest antibody response (2x higher than MPL / QS21-SUV).
Although no statistical analysis has been performed, results on individual
sera
confirm this observation.
The combination of AI(OH)3 and QS21-SUV (+/- MPL) also qualitatively
affects the immune response as shown by the isotypic profile of the humoral
response
1 ~ (Figure 7).
Al(OH)3 induces a clear TH2 type of immune response (only 3 % IgG2a)
whereas Al(OH)3 / QS21-SUV (+/- MPL) formulations induce up to 46% IgG2a.
Conclusion
The combination of Alum with QS21-SUV (+i-MPL) induces higher antibody
titers than formulations containing vehicle or immunostimulants alone.
Material and Methods
Immunisations
10 groups of ~ female Balb/c mice (6-8 weeks) were immunised
intramuscularly in the leg (gastrocnemien) twice at 4 weeks interval with SOpI
vaccine
containing 2 ~g SL* formulated in Al(OH)3(~0 ug equivalent A13+), Al(OH)3 /
QS21-SUV, Al(OH)3 / MPL / QS21-SUV, MPL / QS21-SUV. A dose of ~ ug of
immunostimulants was used.
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Animals were bled on day 28 (28 post I) and 42 (14 post II) for antibody
determination by Elisa.
Formulations
Components batches used.
Formulation process
SL* (2ug) is adsorbed or not for 15 min on 50 ug of water diluted Al(OH)3.
If needed, 5 ug of MPL is added to the preparation as a suspension of 100 nm
particles (MPL out) for 15 min. If needed, ten fold concentrated buffer is
added
before adding 5 ug of QS21 mixed with liposomes in a weight ratio QS21 /
Cholesterol of 1/5.
Thiomersal is added to the formulations 15 min after QS21/SUV addition.
Formulations containing QS21-SUV are buffered with PBS pH 7.4 and the
others are prepared in PBS pH 6.8
Serology
Quantitation of anti-HBs antibody was performed by Elisa using HBs
(Hep286) as coating antigen. Antigen and antibody solutions were used at 50 ul
per
well. Antigen was diluted at a final concentration of 1 ug/ml in PBS and was
adsorbed
overnight at 4~c to the wells of 96 wells microtiter plates (Maxisorb Immuno-
plate,
Nunc, Denmark). The plates were then incubated for 1 hr at 3 7~c with PB S
containing
1 % bovine serum albumin and 0.1 % Tween 20 (saturation buffer). Two-fold
dilutions
of sera (starting at 1/100 dilution) in the saturation buffer were added to
the HBs-
coated plates and incubated for 1 hr 30 min at 37~c. The plates were washed
four
times with PB S 0. l % Tween 20 and biotin-conjugated anti-mouse IgG 1, IgG2a,
IgG2b or a mix of the three antibodies (Amersham, UK) diluted 1/l000 in
saturation
buffer was added to each well and incubated for 1 hr 30 min at 37~c. After a
washing
step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted
l/5000
in saturation buffer was added for an additional 30 min at 37~c. Plates were
washed as
above and incubated for 20 min with a solution of o-phenylenediamine (Sigma)
0.04% H202 0.03% in 0.1 % tween 20 0.05M citrate buffer pH4.5. The reaction
was
CA 02267191 1999-04-O1
WO 98/15287 PCT/EP97/05578
stopped with H2S04 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.
