Note: Descriptions are shown in the official language in which they were submitted.
WO 94/21292 PCT/EP94/00818
VACCINE COMPOSITIONS CONTAINING 3-0 DEACYLATED MONOPHOSPHORYL LIPID A.
The present invention relates to novel vaccine formulations, methods for
preparing them and to their use in therapy.
3-O-deacylated monophosphoryl lipid A (or 3 De-O-acylated
monophosphoryl lipid A) has formerly been termed 3D-MPL or d3-MPL to indicate
that position 3 of the reducing end glucosamine is de-O-acylated. For
preparation,
see GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryl
lipid A with 4, 5 or 6 acylated chains. Herein the term 3D-MPL (or d3-MPL) is
abbreviated to MPL since 'MPL' is a Registered Trademark of Ribi
Immunochem.,Montana which is used by Ribi to denote unambiguously their 3-O-
deacylated monophosphoryl lipid A product.
GB 2 220 211A mentions that the endotoxicity of the previously used
enterobacterial lipopolysacharides (LPS) is reduced while the immunogenie
properties are conserved. However GB 2 220 211 cited these findings merely in
connection with bacterial (Gram negative) systems. No mention of the particle
size
of the MPL was made. In fact the particle size of the known 3-O-deacylated
monophosphoryl lipid A has particles in excess of SOOnm.
In WO 92/16231 a vaccine formulation comprising a Herpes Simplex Virus
glycoprotein gD or immunological fragments thereof in conjunction with 3
deacylated monophosphoryl lipid A was disclosed. Again, no mention of the
particle
size of the 3 deacylated monophosphoryl lipid A was made.
In WO 92/06113 a vaccine formulation comprising HIV gp 160 or a
derivative thereof such as gp 120 in conjunction with 3 deacylated
monophosphoryl
lipid A was disclosed. No mention of particle size of the MPL was made.
The present invention provides a vaccine composition comprising.an antigen
in conjunction with 3-O-deacylated monophosphoryl lipid A (abbreviated herein
to
MPL) and a suitable carrier wherein the particle size of the MPL is 'small'
and in
general do not exceed 120nm when prepared.
Such a formulation is suitable for a broad range of monovalent or polyvalent
vaccines.
Surprisingly, it has been found that vaccine compositions according to the
invention have particularly advantageous properties as described herein. In
particular such formulations are highly immunogenic. Additionally sterility of
the
adjuvant formulation can be assured as the product is susceptible to sterile
filtration.
A further advantage of 'small' MPL arises when formulated with aluminium
hydroxide, as the MPL interacts with the aluminium hydroxide and the antigen
to
form a single entity.
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WO 94/21292 PCT/EP94/00818
In an embodiment of the invention, the antigen is a viral antigen, for example
an antigen against hepatitis infection (Hepatitis A, B; C, D, or E) or herpes
(HSV-1 or
HSV-2) infection as described hereinbelow. A review on modern hepatitis
vaccines,
including a number of key references, may be found in the Lancet, May 12th
1990 at
page 1142 ff (Prof A.L.W.F. Eddlesten). See also 'Viral Hepatitis and Liver
Disease'
(Vyas, B.N., Dienstag, J.L., and Hoofnagle, J.H., eds, Grune and Stratton,
Inc. (/984)
and 'Viral Hepatitis and Liver Disease' (Proceedings of the 1990 International
Symposium, eds F.B. Hollinger, S.M. Lemon and H. Margolis, published by
Williams and Wilkins). References to HSV-1 and HSV-2 may be found in WO
92/ 16231.
Infection with hepatitis A virus (HAV) is a widespread problem but vaccines
which can be used for mass immunisation are available, for example the product
'Havrix' (SmithKline Beecham Biologicals) which is a killed attenuated vaccine
obtained from the HM-175 strain of HAV [see 'Inactivated Candidate Vaccines
for
Hepatitis A' by F.E. Andre, A Hepburn and E.D'Hondt, Prog Med. Virol. Vol 37,
pages 72-95 (1990) and the product monograph 'Havrix' published by SmithKline
Beecham Biologicals (1991)].
Flehmig et al (loc eit., pages 56-71) have reviewed the clinical aspects,
virology, immunology and epidemiology of Hepatitis A and discussed approaches
to
the development of vaccines against this common viral infection.
As used herein the expression 'HAV antigen' refers to any antigen capable of
stimulating neutralising antibody to HAV in humans. The HAV antigen may
comprise live attenuated virus particles or inactivated attenuated virus
particles or
may be, for example an HAV capsid or HAV viral protein, which may conveniently
be obtained by recombinant DNA technology.
Infection with hepatitis B virus (HBV) is a widespread problem but vaccines
which can be used for mass immunisation are now available, for example the
product
'Engerix-B' (SmithKline Beecham plc) which is obtained by genetic engineering
techniques.
The preparation of Hepatitis B surface antigen (HBsAg) is well documented.
See. for example, Harford et al in Develop. Biol. Standard 54, page 125
(1983),
Gregg et al in Biotechnology9 5, page 479 (1987), EP-A- 0 226 846, EP-A-0 299
108
and references therein.
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 S 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
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WO 94/21292 ~ ~ PCT/EP94/00818
,, ,.
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 preS 1-preS2 -S
polypeptide
described in EP 0 198 474 (Endotronics) or analogues thereof such as those
described
in EP 0 304 578 (Mc Cormick and Jones). HBsAg as herein described can also
refer
to mutants, for example the 'escape mutant' described in WO 91/14703 or
European
Patent Application Publication Number 0 511 855 A l, 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.
Herpes Simplex virus Glycoprotein D is located on the viral envelope, 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 60kD. Of all the HSV envelope glycoproteins
this is probably the best characterized (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 elict neutralizing antibodies in
vivo
(Sing et als J. Med Virology 127: 59-65). However, latent HSV2 virus can still
be
reactivated and induce recurrence of the disease despite the presence of high
neutralizing antibodies titre in the patients sera. It is therefore apparent
that the
ability to induce neutralizing antibody alone is insufficient to adequately
control the
disease.
The mature recombinant truncated glycoprotein D (rgD2t) or equivalent
proteins secreted from mammalian cells, is preferably used in the vaccine
formulations of the present invention. Equivalent proteins include
glycoprotein gD
from HSV-1.
In a preferred aspect the rgD2t is HSV-2 glycoprotein D of 308 amino acids
which comprises amino acids 1 though 306 of the naturally occurring
glycoprotein
with the addition of Asparagine and Glutamine at the C terminal end of the
truncated
protein. 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 and Science ~ p524, and Biotechnology p527 June 1984. Such a vaccine when
formulated with small MPL according to the present invention has a superior
therapeutic potential as compared to known rgD2t formulations.
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WO 94/21292 PCT/EP94/00818
Whilst certain experimental and commercially available vaccines afford
excellent results it is an accepted fact that an optimal vaccine needs to
stimulate not
only neutralising antibody but also needs to stimulate as effectively as
possible
cellular immunity mediated through T-cells.
A particular advantage is that the vaccine formulations of the in~rention are
very effective in inducing protective immunity, even with very low doses of
antigen.
They provide excellent protection against primary and recurrent infection and
stimulate advantageously both specific humoral (neutralising antibodies) and
also
effector cell mediated (DTH) immune responses.
To make 3 deacylated monophosphoryl lipid A with a small particle size, in
general not exceeding 120nm the procedure described in GB 2 220 211 may be
followed to obtain known 3D-MPL, (or commercial MPL of larger particle size
may
be purchased from Ribi Immunochem.) and the product may then be sonicated
until
the suspension is clear. The size of the particles may be estimatedvsing
dynamic
light scattering as described hereinbelow. In order to maintain the MPL size
in the
100 nm range after being formulated with aluminium hydroxide, the antigen and
the
buffer, tween 80 or sorbitol can be added. Under these conditions, it has been
established that MPL does not aggregate in the presence of phosphate buffer,
as may
happen during formulation without them. By doing so, the final formulation is
further defined and characterized. It has also been established that under
these
conditions, MPL still interacts with aluminium hydroxide and the antigen
forming
one single entity.
A clear solution of MPL forms an aspect of the invention. This solution may
be sterilised by passing through a filter.
Preferably the size of the particles is in the range 60-120nm.
Most advantageously the particle size is below 100nm.
The MPL as defined above will normally be present in the range of 10-200p.g,
preferably 25-SOp.g per dose wherein the antigen will be typically present in
a range
of 2-SOp.g per dose or more. The vaccine formulation of the present invention
may
additionally contain further immunostimulants, in a preferred embodiment the
vaccines of the present invention may include QS21 (sometimes referred to as
QA21).
This is an 13PLC fraction of a saponin extract derived from the bark of a
tree, Quillaja
Saponaria Molina and a method for its production is disclosed in US Patent
5,057,540.
The carrier may optionally be an oil in water emulsion, a lipid vehicle, or
aluminium hydroxide (aluminium hydroxide salt).
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.
squalene and an emulsifier such as Tween 80, in an aqueous carrier. The
aqueous
carrier may be, for example, phosphate buffered saline.
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CA 02157376 2004-06-29
Preferably the vaccine formulations will contain an antigen or antigenic
composition capable of eliciting an immune response against a human or animal
pathogen, which antigen or antigenic composition is derived from HIV-1, (such
as
gp120 or gp160; see WO 92/06113 and references therein), herpes virus such as
gD
or derivatives thereof or Immediate Early protein such as ICP27 from HSV-1 or
HSV-2, gB (or derivatives thereof) from Human cytomegalovirus, or gpI, a or
III
from Varicella Zoster Virus, or from a hepatitis virus such as hepatitis B
virus or
from other viral pathogens, such as Respiratory Syncytial virus, human
papilloma
virus or Influenza virus, or against bacterial pathogens such as SalrnoneUa,
Neisseria,
Borrelia (for example OspA or OspB or derivatives thereof), or C~lamydia, or
Bordetella for example P.69, PT and FHA, or against parasites such as
plasmodium or
Toxoplasma. The vaccine formulations of the present invention may contain a
tumour antigen, and be useful as an anticancer vaccine.
One embodiment of the invention is HAV antigen (for example as in Havrix)
in admixture with MPL and aluminium hydroxide as described hereinbelow.
A further embodiment of the invention is HB Virus Surface (HBsAg) antigen
(for example as in Engeiix-B) in admixture with MPL and aluminium hydroxide as
described hereinbelow.
A further specific embodiment of the invention is HBsAg antigen as (L*,S)
particles, defined hereinabove, in admixture with MPL and aluminium hydroxide.
Hepatitis A plus Hepatitis B combination vaccines may also be prepared in
accordance with the invention.
A further embodiment is a formulation according to the invention comrising
mature truncated glycoprotein D (rgD2t) or equivalent proteins as described
hereinabove. Yet a further embodiment is a formulation of the invention
comprising
an OspA antigen or derivative thet~eof from Borrelia burgdorferi. For example,
antigens, particularly OSpA antigens from the ZS7 or B31 strains may be used.
Yet a
further embodiment is a formulation of the invention comprising a flu antigen.
This
provides an improved influenza vaccine, especially if a 'split' virus is used.
The formulation may also be useful for utilising with herpetic light particles
such as described in International Patent Publication No. WO 92/19748A1 and,
International Patent Publication No. WO 92/13943A1.
Advantagously the vaccine composition of the invention contains other
antigens so that it is effective in the treatment or prophylaxis of one or
more other
bacterial, viral or fungal infections.
For example, hepatitis vaccine formulations according to the invention
preferably contain at least one other component selected from non-hepatitis
antigens
which are known in the art to afford protection against one or more of the
following:
diphtheria, tetanus, pertussis, Haemophilus influenzae b (Hib), and polio.
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WO 94/21292 PCT/EP94/00818
.
Preferably the vaccine according to the invention includes HBsAg as
hereinabove defined.
Particular combination vaccines within the scope of the invention include a
DTP (diphtheria-tetanus-pertussis) -hepatitis B combination vaccine
formulation, an
Hib-Hepatitis B vaccine formulation, a DTP-Hib-Hepatitis B vaccine formulation
and
an IPV (inactivated polio vaccine) -DTP-Hib-Hepatitis B vaccine formulation.
The above combinations may advantageously include a component which is
protective against Hepatitis A, especially the killed attenuated strain
derived from the
HM-175 strain as is present in Havrix.
Suitable components for use in such vaccines are already commercially
available and details may be obtained from the World Health Organisation. For
example the IPV component may be the Salk inactivated polio vaccine. The
pertussis
vaccine may comprise whole cell or acellular product.
Advantageously the hepatitis or combination vaccine according to the
invention is a paediatric vaccine.
The invention in a further aspect provides a vaccine composition according to
the invention for use in medical therapy, particularly for use in the
treatment or
prophylaxis of infections include viral and bacterial infections or for immuno
therapeutic treatment of cancer. In a preferred aspect the vaccine according
to the
invention is a therapeutic vaccine useful for the treatment of ongoing
infections, for
example hepatitis B or herpetic infections in humans suffering therefrom.
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, US Patent 4,235,877. Conjugation of proteins to macromoloecules is
disclosed, for example, by Likhite, US Patent 4,372,945 and by Arn~or et al,
US
Patent 4,474,757.
The amount of antigen 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 on which specific
immunogens
are employed. Generally it is expected that each dose will comprise 1-1000pg
of
total immunogen, preferably 2-100ug, most preferably 4-40pg. An optimal amount
for a particular vaccine can be ascertained by standard studies involving
observation
of antibody titres and other responses in subjects. Following an initial
vaccination,
subjects may receive a boost in about 4 weeks.
In a further aspect of the present invention there is provided a method of
manufacture of a vaccine effective in preventing or treating infection,
wherein the
method comprises mixing the antigen with a carrier and MPL wherein the
particle
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WO 94/21292 ~K~ PCT/EP94/00818
size of the MPL is no greater than 120nm, normally 6b-120nm, preferably about
or
less than 100nm.
The following examples illustrate the invention and its advantages.
Example 1: Preparation of MPL with a particle size of 60 - 120 nm
S Water for injection is injected in vials containing lyophilised 3-de-O-
acylated
monophosphoryl lipid A (MPL) from Ribi Immunochem, Montana using a syringe to
reach a concentration of 1 to 2 mg per ml. A preliminary suspension is
obtained by
mixing using a vortex. The content of the vials is then transferred into 25 ml
Corex
tubes with round bottoms (10 ml suspension per tube) and the suspension is
sonicated
using a water bath sonicator. When the suspension has become clear, the size
of the
particles is estimated using dynamic light scattering (Malvern Zetasizer 3).
The
treatment is continued until the size of the MPL particles is in the range 60 -
120 nm.
Suspensions can in some cases be stored at 4 degrees C without significant
aggregation up to 5 months. Isotonic NaCI (O.15M) or isotonic NaCI plus lOmM
phosphate induces a rapid aggregation (size >3-Sp.m).
Example 2: Production of large scale sterile soluble MPL with a particle size
of
below IOOnm.
Lyophilised 3 de-O-acylated monophosphoryl lipid A was obtained from Ribi
Immunochem, and suspended in water for injection (WFI). The suspension was
continuously pumped through an ultrasound flow cell. The flow cell is
typically
made of glass or stainless steel with PTFE seals so as to comply with GMP
constraints. The Ultrasound is generated utilising an Ultrasound generator and
a
titanium sonic horn (sonotrode) acquired from Undatim Ultrasonics (Louvain-La-
Neuve, Belgium). A heat exchanger is incorporated into the loop to avoid
degradation of the product by heat. The temperature of the MPL between the
inlet
and the outlet of the flow cell is kept between + 4°C and 30°C,
and the difference of
the temperature between the inlet and outlet is kept below 20°C. It
will be
appreciated that heat is also removed as the material passes through the
apparatus.
The apparatus used is schematically depicted in figure 1.
2.1. Sonication
The MPL powder (50 to 500 mg) is suspensed in WFI at concentration
between 1 and 2 mg/ml.
The MPL suspension (under stirring conditions) is continuously pumped
through the sonication loop (see figure 1) at a flow rate between 50 and 100
ml per
minute in order to reach the equilibrium temperature of the system which is
between
+ 4 and + 15°C.
The own frequency spectrum of the sonic horn in the configuration of the
system (Power, Flow Cell, Liquid Flow rate, T°,) is set according to
suppliers
WO 94/21292 ~ PCT/EP94/00818
instructions for the equipment. Pre-established limits are fixed between 19000
Hertz
and 21000 Hertz for the 20,000 Hertz transducer.
The generator allows the control of the optimal efficiency of the sonication
(more transmission of energy with less heat) at a given time interval.
The temperature during the process is maintained below 30°C to
avoid MPL
degradation.
The process is complete when the particle size is reduced below 100nm and
the solution is clear by visual inspection. During the sonication process
samples are
taken for particle size evaluation by photocorrelation spectroscopy (dynamic
light
scattering) using a Malvern Zetasizer type 3 in the same manner as example 1.
The
total liquid residence time in the sonicator flow cell is calculated to be
between 2.5
and 3.5 minutes (see table 1), using a 20 ml. capacity flow cell and SOmI/min
recirculation flow rate. This flow rate gives an average residence time of 25
seconds
per cycle and normally less than 10 cycles are needed to obtain the desired-
effect of
small particle MPL.
2.2. Sterilization Process
The resulting "solubilized" MPL is sterilized by dead end filtration on a
hydrophilic PVDF 0.22mcm membrane. The observed pressure is usually below 1
bar. At least 25 mg of "solubilized" MPL are easily processed per square
centimetre
with a recovery above 85°l0.
2.3. Storage/Stability
Sterile "solubilized" MPL is stored at +2° to 8°C. Stability
data (Malvern) did
not show any significant difference of particle size after 6 months storage.
(See Table
2).
Example 3: Hepatitis B vaccine formulation
MPL (particle size less than 100 nm) was obtained as described in Example 1.
Aluminium hydroxide was obtained from Superfos (Alhydrogel).
The MPL was resuspended in water for injection at a concentration varying
from 0.2 to 1 mg/ml by sonication in a water bath until the particles reach a
size of
between 80 and S00 nm as measured by photo correlation light scattering.
1 to 20p.g of HBsAg (S- antigen as in Engerix B) in phosphate buffer solution
at lmg/ml) is adsorbed on 30 to 100p.g of aluminium hydroxide (solution at
10.38
Al3+ mg/ml) for one hour at room temperature under agitation. To the solution
is
then added 30 to SOp.g of MPL (solution 1 mg/ml). Volume and osmolarity are
adjusted to 600p.1 with water for injection and phosphate buffer Sx
concentrated.
Solution is incubated at room temperature for 1 hour and kept at 4oC until
use.
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WO 94/21292 ~ a~~.~~'~~~~ PCT/EP94/00818
Maturation of the formulation occurs during storage. This represents 10
injecting
doses for testing in mice.
Example 4: Hepatitis A vaccine formulation
r~LPL (particle size less than 100nm) was obtained as described in Example 1.
Aluminium hydroxide was obtained from Superfos (Alhydrogel).
HAV (360 to 22 EU per dose) is preadsorbed on 10°l0 of the
aluminium
hydroxide final concentration (O.Smg/ml). The MPL (12.5 to 100p.g per dose) is
added to the solution.
The remaining aluminium hydroxide is added to the solution and left for one
hour at room temperature. Volumes are adjusted with phosphate buffer
(phosphate
lOmM, NaCI 150mM) and the final formulation is then stored at 4oC until use.
Example 5 - Comparison Of Adjuvant Efficacy Of A Recombinant Herpes
Simplex Glycoprotein D Subunit Vaccine
5.1 In this study, the ability of various Al(OH)3 MPL formulations to improve
the
protective immunity of a truncated glycoprotein D from Herpes Simplex virus
type 2
(HSV2) (rgD2t) was evaluated in the prophylactic and therapeutic guinea pig
models.
Immunogenicity studies were also performed in primates. The aim of these
experiments was to investigate the impact of the size of 3-de-O-acylated
monophosphoryl lipid A (MPL) particles on the immunogenicity and protective
efficacy of rgD2t Al(OH)3 MPL formulations in rodents and in primates. Three
different Al(OH)3 MPL formulations of small size MPL were tested:
Al(OH)3 MPL 100nm (as described previously)
Al(OH)3 MPL 100nm with sorbitol
Al(OH)3 MPL 100nm with tween.
5.2 Antigen-adjuvant preparations and immunisation schedules
5.2.1. Antigen formulations
Aluminium hydroxide (Al(OH)3) was obtained from Superfos (Alhydrogel
Superfos, Denmark). MPL was obtained from Ribi Immunochem Research Inc.
5.2.1.1. rgD2t (AI(OH)3)/MPL TEA
MPL was resuspended by sonication in a water bath, to give sizes comprising
between 200 and 600nm. The preparation was prepared according to patent
application W092/16231, and was stored at 4°C until use.
A dose contained Sp.g rgD2t, 0.5 mg Al(OH)3 and SOp,g MPL.
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WO 94/21292 ' PCT/EP94/00818
5.2.1.2. rgD2t/AI(OH)3/MPL 100nm
MPL (particle size less than 100nm) was obtained as described in Example 1.
rgD2t was adsorbed on aluminium hydroxide and incubated lh room temperature.
MPL was added to the solution at the required concentration and incubated
further for
1 hour at room temperature.
The preparation was completed with PBS to reach a final concentration of
lOmM P04, 150mM NaCI. The final formulation was further incubated for 30
minutes at room temperature and stored at 4°C until use.
A dose contained Sp.g rgD2t, 0.5 mg Al(OH)3 and SOp.g MPL.
5.2.1.3. rgD2t/AI(OH)3/MPL 100nm sorbitol
MPL was prepared as described in Example 1 . rgD2t was adsorbed on
aluminium hydroxide and incubated 1 h at room temperature. A 50% sorbitol
solution was then added to reach a final concentration of S%. Tris- lOmM
solution
was then added to make up the final desired volume, and the solution was
incubated
lh at room temperature under agitation.
The Formulation was stored at 4°C until use.
A dose contained Sp.g rgD2t, 0.5 mg Al(OH)3 and SOltg MPL.
5.2.1.4. rgD2t/AI(OH)3/MPL 100nm tween
MPL was prepared as described in Example 1 . In order to maintain the MPL
size to 100 nm, tween 80 was added to the solution at a concentration such
that it will
be equal to 0.01 % in the final formulation. The formulation is then prepared
as
described in formulation 5.2.1.3 above.
A dose contained Sp.g rgD2t, 0.5 mg Al(OH)3 and SOp.g MPL.
5.3. Guinea pig prophylactic experiment
In these experiments, groups of guinea pigs were vaccinated at days 0 and 28
with Sp.g rgD2t in two different Aluminium hydroxide MPL formulations.
Immunisations were given subcutaneously in an O.SmI dose. One month after the
second vaccination, guinea pigs were challenged intravaginally with 105 pfu of
HSV2
strain MS. They were monitored daily for the development of primary and
recurrent
HSV2 disease (days 4 to 39 post challenge).
5.3.1. Guinea pig therapeutic experiments
In these experiments, guinea pigs were challenged at day 0 with I05 pfu
HSV2 strain MS. After recovery from primary infection, they were evaluated
daily
for recurrent herpetic disease (days 13 to 21 ). Guinea pigs were vaccinated
at days 21
and 42 with rgD2t Al(OH)3 MPL vaccine. Vaccines were administrated
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WO 94/21292 ~ ~ ~ PCT/EP94I00818
subcutaneously in a 0.5 ml dose. Animals were monitored daily for herpetic
lesions
until day ~ 60 or ~ 84.
5.3.2. Primate Immunogenicity studies
The immunogenicity of rgD2t Al(OH)3 combined with MPL in sorbitol was
evaluated in African Green Monkeys. Groups of monkeys were vaccinated at days
0
and 28 with 20p,g rgD2t and O.Smg AI(OH)3 combined with 50,20 or Sp.g MPL in
sorbitol. Specific humoral (ELISA and neutralising titers) and effector cell
mediated
(delayed type hypersensitivity response :DTH) immune responses were evaluated.
Each monkey group contained 5 animals. The formulations were administered
intramuscularly in a 1 ml dose. Preparation of the formulations was done as
described above. Animals were bled ~ every two weeks for antibody
determination.
DTH response was tested 14 days after the second vaccination. A description
of the skin test is given below.
5.4. Read-outs
Assays were set up to evaluate the specific antibody response induced by
vaccination with rgD2t AI(OH)3 MPL formulations (determination anti-rgD2t
ELISA
titers and anti-HSV2 neutralising titers). The protective efficacy of these
gD2
formulations was assessed in the prophylactic and therapeutical guinea pig
models.
Immunogenicity studies were also conducted in monkeys. Specific humoral and
DTH responses were evaluated.
5.4.1. ELISA and neutralising titers
Anri-rgD2t antibody titers and anti-HSV2 neutralising activity were
determined according the methods described in patent application No.
W092/16231
5.4.2. Delayed type hypersensitivity (DTH)
The rgD2t formulations were also tested for their ability to induce a T cell
specific immune response in monkeys as measured by the induction of delayed -
type
hypersensitivity (DTH) responses.
African Green monkeys were vaccinated at days 0 and 28 with 20p.g gD2
vaccine formulation administered intramuscularly. They were skin tested 14
days
after the second vaccination by intradermal injection on the belly of 15 or
Sp.g of
rgD2t in saline. They were also skin tested with saline as control. The site
of
injection was inspected 24 and 48 hours later for erythema and induration and
the size
of these local reactions was measured.
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WO 94!21292 PCT/EP94/00818
5.4.3. Guinea pig intravaginal challenge model
The guinea pig model for HSV genital infection has been described by L.
Stanberry et al (J. of Infectious Diseases 1982, 146: 397-403; Intervirology
1985, 24:
226-231). Briefly, in prophylactic experiments, the guinea pigs were
challenged
intravaginally with 105 pfu of HSV2 strain MS, one month after the last
vaccination.
The clinical course of primary disease was monitored by daily observation of
the '
incidence and severity of genital skin lesions during the 4-12 day post
challenge
period. Animals were then examined daily for evidence of recurrent herpetic
lesions , '
from days 13 to 39. In therapeutic experiments, guinea pigs were challenged at
day 0
with 105 pfu HSV2 strain MS. After recovery from primary infection, they were
evaluated daily for recurrent herpetic disease (days 13 to 21) and were then
randomized according to their primary and recurrent scores (providing an
equivalent
distribution of animals with mild to severe infection in each group) to
receive either
no treatment or vaccination. Vaccines were administered on days 20 and 4.1
after
challenge. The pattern of recurrent disease was generally observed until ~70
post
challenge.
The herpetic lesions were quantitated by using a lesion score scale ranging
from 0 to 32.
Scoring system
Lesion type Score
None 0
Vaginal lesions -
- Bleeding 0.5
- Redness for one or two days without0.5
bleeding
- Redness and bleeding for a day 1
- Redness without bleeding for at 1
least 3 days
External herpetic vesicles
- < 4 small vesicles 2
- >_ 4 small vesicles or only one 4
big vesicle
- >_ 4 large lesions 8
- Fusing large lesions 16
- Fusing large lesions on all external32 .
genital area
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Clinical read-outs
Primary infection
- Lesion severity = sum of the daily scores for the days 4 to 12 post
infection.
The lesion severity is expressed as arithmetic mean ~ SD as well as median
(more
appropriate for non-parametric test).
- Primary infection incidences = % of animals having experienced a maximum
lesion score of 0, 0.5, 1, 2, 4, 8 or 16 (rarely 32).
Primary infection index = Ei (max.score i) x (incidence %) with i = 0, 0.5, 2,
4,8or16.
Recurrent disease
- Recurrence day number = number of recurrence days for the days 13 to 39
post-infection. One recurrence is preceded and followed by a day without
lesion and
characterized by at least two days with erythema or one day with vesicle(s).
Recurrence day numbers are expressed as arithmetic means ~ SD and medians.
- Recurrence severity = sum of the daily scores for the days 13 to 39 post-
infection. Results are expressed as arithmetic means ~ SD and medians.
5.5. Results
The protective efficacy of different rgD2t Al(OH)3 MPL formulations was
compared in prophylactic and therapeutic experiments in guinea pigs.
Immunogenicity studies were also conducted in primates. The aim of these
experiments was to compare the immunogenicity and protective efficacy of rgD2t
Al(OH)3 combined with different MPL particles size.
5.5.1. Prophylactic experiments
Two experiments were performed to evaluate the potential of different rgD2t
Al(OH)3 MPL vaccines to provide protection against primary and recurrent HSV2
disease, when administered to guinea pigs prior to intravaginal viral
inoculation.
Experiment 1: Comparison of MPL 100 nm sorbitol versus MPL TEA
Group of female hartley guinea pigs (200-250g) were immunized at days 0
and 28 with Sp.g rgD2t Al(OH)3 combined with small size MPL particles (100nm;
MPL in sorbitol), or with larger MPL particles (MPL in TEA). Control animals
were
injected according to the same protocol with adjuvant alone or were untreated.
The
animals were bled 14 and 28 days after the second vaccination for antibody
determinations by ELISA and neutralization assays. They were challenged 29
days
after the second vaccination with 105 pfu of HSV2 strain MS intravaginally.
After
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WO 94/21292 PCT/EP94/00818
challenge, the guinea pigs were monitored daily for clinical signs of acute
infection
(days 4 to 12 post challenge) and for evidence of recurrent herpetic disease
(days 13
to 39 post challenge).
a) Induction of humoral immunity
As shown in Table 3, higher ELISA and neutralizing titers were induced when
the small size MPL particles were used in the rgL2t Al(OH)3 formulation.
b) Effect of vaccination on primary HSV2 infection (Table 3)
As compared to the control group that became infected and experienced acute
primary infection, both vaccinated groups showed significantly reduced lesion
severity (p < 0.00005). A significantly lower skin lesion incidence was
observed in
the rgD2t Al(OH)3 MPL 100 nm vaccinated group (p<0.06).
c) Effect of vaccination on recurrent HSV2 disease
Results are given in Table 4. As compared to the controls, both vaccines were
able to alter the development of recurrent herpetic disease, as measured
by~reduction
in the number of recurrent episodes (p<0.02 for rgD2t Al(OH)3 MPL 100 nm)
d) Conclusions
Both formulations were able to provide significant protection against primary
infection and to reduce recurrent disease. These results show that the rgD2t
Al(OH)3
formulation containing small size MPL particles has a very potent prophylactic
efficacy.
Experiment 2: Efficacy of AI(OH)3 MPL 100 nm
Hartley guinea pigs (200-250g) were immunized at days 0 and 28 with Spg
gD2 formulated in Al(OH)3 MPL 100 nm. Immunizations were given
subcutaneously in a 0.5 ml dose. A dose of SOp.g MPL was used in the Al(OH)3
MPL formulation. Control animals were injected according the same protocol
with
adjuvant alone or were untreated. The animals were bled 14 and 28 days after
the
second vaccination for antibody determination by ELISA and neutralization
assays.
The guinea pigs were challenged 29 days after the last immunization with 105
pfu of
HSV2 strain MS intravaginally.
a) Induction of humoral immunity
As shown in Table 3, the vaccinated group produced good ELISA and
neutralizing titers. Control group did not develop any detectable antibody
response.
b) Effect of vaccination on primary HSV2 infection (Table 3)
As compared to the control group that became infected and experienced acute
primary infection, the vaccinated group showed significant reduced lesion
severity (p
< 0.00005) and incidence (p<0.002). There was no evidence of external skin
lesions
in any of the vaccinated guinea pigs.
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c) Effect of vaccination on recurrent HSV2 disease (Table 4)
As compared to the controls, the rgD2t AI(OH)3 MPL vaccine was able to
alter the development of recurrent herpetic disease, as measured by
significant
reduction in the severity of recurrent episodes (p<0.00005), and in the
incidence of
recurrent episodes (p<0.01).
d) Conclusions
rgD2t Al(OH)3 combined with small size MPL particles is very potent in
providing protection against primary and recurrent HS V2 infection in guinea
pigs.
From the experiments described above one could conclude that small size
MPL Al(OH)3 formulations obtained through two different strategies induce at
least
as potent prophylactic response as does large size MPL Al(OH)3 formulation. In
addition small size MPL has the advantage of being sterilised easily before
use.
5.5.2. Therapeutic experiments
The aim of these experiments was to compare the therapeutical potential of
different rgD2t Al(OH)3 MPL formulations on the course of the recurrent
herpetic
disease in guinea pigs with established HS V2 infection.
Guinea pigs were inoculated intravaginally at day 0 with 105 pfu HSV2 strain
MS. They were monitored daily for clinical signs of acute infection (days 4 to
12) as
well as for evidence of recurrent herpetic disease (days 13 to 20). Animals
were
randomized into different experimental groups according to their primary and
recurrent scores, providing an equivalent distribution of animals with mild to
severe
infection in each group. Guinea pigs without evidence of clinical signs of
infection
were not enrolled in the protocol. Vaccines were administrated subcutaneously
at
days 21 and 42 after challenge.
The therapeutic efficacy of rgD2t Al(OH)3 MPL formulations was evaluated
in three different experiments.
Experiment 1: Efficacy of rgD2t AI(OH)3 combined with large size MPL
particles (MPL in TEA)
Guinea pigs experiencing recurrent disease were randomized to receive either
20p.g rgD2t Al(OH)3 combined with large size MPL particles (MPL TEA) or
adjuvant alone. Vaccines were administered at days 21 and 42 post challenge.
The
pattern of recurrent disease was observed until day 84.
As shown in Table 3, the rgD2t Al(OH)3 MPL TEA formulation was not
effective in reducing the ongoing recurrent disease.
Experiment 2: Efficacy of rgD2t AI(OH)3 MPL 100nm
Two groups of guinea pigs were either vaccinated with 20p.g rgD2t Al(OH)3
combined with small size MPL particles (MPL 100nm) or untreated.
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Vaccinations were given at days 20 and 41 post challenge. The recurrent
disease was followed until day 69.
As shown in Table 5 in contrast with data in Experiment 1 where large size
MPL was used, vaccination with the rgD2t Al(OH)3 MPL 100nm vaccine altered the
recurrences of established HSV2 disease, as compared to the control group, by
significantly decreasing the recurrence severity (-39%, p<0.05) and the
recurrence
day number (-28%, p<0.1).
Experiment 3: Comparative efficacy of AI(OH)3 combined with small size MPL
particles
In this experiment, a third strategy for obtaining small size MPL was used:
addition of tween, eg Tween 80.
The experimental groups were as follows:
Group 1 (n=15) : 20 p.g rgD2t/Al(OH)3 MPL 100nm with tween
Group 2 (n=15) : 20 pg rgD2t/Al(OH)3 MPL 100nm with sorbitol
Group 3 (n=16) : controls
Controls were either untreated or vaccinated with Al(OH)3 MPL alone.
Vaccines were administered at days 21 and 42 post challenge. The pattern of
recurrent disease was observed until day 60 post challenge.
The results are shown in Table 5. A clear significant therapeutical effect was
observed in animals vaccinated with the two rgD2t/Al(OH)3 MPL formulations.
Both formulations significantly reduced the recurrence severity, the
recurrence day
number and the number of recurrent episodes.
Conclusions
A very potent therapeutic effect against established recurrent HSV2 genital
disease was observed with rgD2t Al(OH)3 WPL formulations containing small size
MPL particles (ca. 100nin). In contrast, this therapeutic effect was not
observed
when large MPL size particles (MPL in TEA) were added to the rgD2t Al(OH)3
vaccine.
In conclusion, results obtained in guinea pigs clearly demonstrate the
prophylactic efficacy of rgD2t Al(OH)3 MPL formulations prepared with small
size
MPL particles. These formulations have an improved therapeutic potential as
compared to rgD2t Al(OH)3 combined with large size MPL particles.
5.5.3 Immunogenicity studies of rgD2t AI(OH)3 combined with small size MPL
particles in primates
The immunogenicity of rgD2t Al(OH)3 combined with small size MPL
particles (MPL 100nm sorbitol) was evaluated in primates (African Green
Monkeys).
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Doses of 50, 20 or Sp.g of MPL 1(?0nm were combined with 20p.g of rgD2t and
Al(OH)3 (O.Smg). Two vaccinations were given at 0 and 1 months. Specific
humoral (ELISA and neutralizing titers) and effector cell mediated (DTH)
immune
responses were measured.
a) Experimental procedure
Three groups of 5 African Green Monkeys were vaccinated at days 0 and 28
with 201tg of gD2t Aluminium hydroxide formulation containing 50, 20 or Sp.g
of
MPL. Immunisations were given intramuscularly in a lml dose. Animals were bled
every two weeks for antibody determination by ELISA (anti-gD2 titers) and
neutralization assays. The three vaccine formulations were also compared for
their
ability to induce a T cell mediated immunity in vivo, as measured by the
induction of
a specific delayed-type hypersensitivity (DTH) response. Three monkeys in each
group were skin tested 14 days after the second vaccination with 15 or Sp.g of
gD2t,
given in saline on the belly. They were also skin tested with saline alone as
control.
Erythema and induration at the site of the intradern~al inoculation were
monitored 24
hours and 48 hours later.
b) Results
Serological and DTH responses are shown in Table 6. Groups of monkeys
vaccinated with the gD2t Al(OH)3 formulation containing either 50 or 20~tg of
MPL
produced significantly more neutralizing antibodies than the group receiving
the Sp.g
MPL dose (p<0.003 and p<0.008, respectively). There was no significant
difference
in the ELISA or neutralizing titers measured in the 50 or 20p.g MPL groups. A
correlation between the MPL dose and the effect on the effector cell mediated
immune response was observed. A strong DTH response was detected in the
majority
of the monkeys (3/4) vaccinated with the 50 or 20p.g MPL formulation. In
contrast,
only one monkey from the S~.g MPL group developed a skin test response.
c) Conclusions
The data described above demonstrate that the adjuvant effect of Al(OH)3
combined with small size MPL particles is also effective in primates and is
not
restricted to small animal species. A correlation between the MPL dose and the
immunogenicity of the rgD2t Aluminium hydroxide MPL formulation could be
observed in monkeys, with 20 and SO~tg giving the best serological and DTH
responses.
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WO 94/21292 PCT/EP94/00818
Example 6: CLINICAL STUDIES with Lyme and Hepatitis B vaccines and
small MPL
6.1. Lyme disease vaccine comprising a fusion protein of NS1(1-81) from
influenza virus and OspA derived from B.burgdorferi ZS7.
S Preparations of formulations
6.1.1. NS 1-OspA/aluminium hydroxide
NS 1-OspA prepared according to the procedures described in WO 93/04175
was adsorbed on aluminium hydroxide and incubated 1 hr at room temperature.
Final volume was adjusted with phosphate buffer (P04 10 mM, NaCI 150 mM).
Formulation was stored at 4°C until use.
A dose contains lOltg NS1-OspA/SOOp.g aluminium hydroxide
6.1.2. NS1-OspA/aluminium hydroxide/MPL
NS 1-OspA was adsorbed on aluminium hydroxide and incubated 1 hr at room
temperature. MPL prepared as described previously was then added to the
formulation and incubated again 1 hr at room temperature. The formulation was
then
adjusted to the final volume with phosphate buffer (lOmM P04, 150 mM NaCI).
Formulation was stored at 4°C until use.
A dose contains lOp.g OspA/500p.g Al(OH)3/SO~tg MPL
6.1.3. Immunization schedule
Human volunteers were injected three times intramuscularly with lml of a
given formulation at days 0, 31 and 62. Sera were taken 30 days post I, II and
III.
They were then analysed by ELISA for total IgG anti OspA and for LA-2 like
antibody response in an inhibition test (LA-2 Mab was shown to be protective
antibody against infection in mice).
6.2. HBsAg/MPL formulations in humans
6.2.1. Preparation of Formulations
HBsAg 20p.g/Aluminium hydroxide SOOp.g
HBsAg was adsorbed on the total final amount of aluminium hydroxide and
final volume was adjusted with phosphate buffer saline (P04 10 mM, NaCI 150
mM)
at lml per dose. Formulation wa stored at 4°C until use. -
6.2.2. HBsAg 20~tg/Atuminium hydroxide 100~tg
HBsAg was formulated as described previously but adsorbed only on l~ftg
of Al(OH)3. The final volume was (lml) dose
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PCTlEP94/00818
6.2.3. HBsAg 20~tg/Aluminium hydroxide 100p.g/MPL SOp.g
HBsAg was adsorbed on 100p.g aluminium hydroxide and incubated lh at
mom temperature. MPL was then added at the acquired concentration and
incubated
lh at room temperature. The formulation was then adjusted to the final volume
(lml
per dose) with appropriate buffer (as above) and stored at 4°C until
use.
6.2.4. Immunization schedule
Human volunteers (20 per group) were injected LM. with lml of one of the
given formulations. Sera were collected on months 0, l, 3 and 6. They were
analysed for neutralizing antibodies with the commercially available Abbot
test.
6.3. RESULTS
Table 8 shows that MPL used in combination with aluminium hydroxide and
NS 1-OspA in a form of particles of 100nm is efficient at producing
higher.antibody
titers of inhibitory nature than the antigen on aluminium hydroxide and that
the
kinetics of seroconversion are faster.
This established that for a soluble antigen, like NS 1-OspA, in humans, MPL
formulated as small particle keeps the adjuvant properties that it already
exhibited in
animals with other soluble antigens.
Table 7 shows that the adjuvant effect lost by reducing the amount of
aluminium hydroxide formulation present in Hepatitis B formulations can be
recovered by adding MPL in the form described in this patent. The MPL also
improves the seroconversion rate.
Example 7: Combination vaccine formulation - Hepatitis B +Hepatitis A
HBsAg is adsorbed on 90% of the final amount of aluminium hydroxide
(O.Smg/ml) and incubated overnight at room temperature. The pH is adjusted to
6.2
and the preparation is left 14 days at room temperature for maturation.
Hepatitis A antigen at 360 to 22EU per dose, in the form of an inactivated
derivative of the HM-175 strain (as in Havrix) is preadsorbed on 10% of the
aluminium hydroxide final concentration (O.Smg/ml). The remaining aluminium
hydroxide is then added to the solution and left for one hour at room
temperature
under agitation.
The HAV adsorbed on aluminium hydroxide is then added to the HBsAg
formulation.
MPL (particle size less than 100 nm) is added to the HAV/HBsAg solution at
a final concentration of 12.5 to 100ug per 1 ml dose, the volume is adjusted
to the
final dose volume, and the formulation is stored at 4oC until used.
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Example 8: Combination vaccines containing additional antigens
Combination vaccines may be prepared by adding one or more of the desired
antigens to the formulations described in Example 2 or Example 3 or Example 4
above.
Example 9: Increase Of Humoral Immunity And Induction Of Cell Mediated
Immunity By Immunization Of Mice With HBsAg formulated With Aluminium
Hydroxide And MPL
9.1. Effect Of AD(OH)3 + Mpl On Induction Of Anti-HBs Antibodies
Balb/c mice were immunized by the subcutaneous route or by the intradermal
route with recombinant HBsAg adsorbed on Al(OH)3 with MPL as adjuvant. Mice
were immunized twice with HBsAg/Al/MPL formulations and the antibody response
was measured after the first and the second doses. Total Ig were measured by
ELISA
or AUSAB kit (Abbott Lab, Ill.) and a particular attention was given to the
induction
of antibodies of the IgG2a isotype since this isotype is mainly induced by
secretion of
g-Interferon. The induction of this isotype thus indirectly reflects the
activation of
cell mediated immunity, namely the activation of Thl.
The ratio HBsAg/MPL has been investigated as well as the size of MPL
particles.
9.1.1. EXPERIMENT I - Effect of MPL (> 500 nm) dose on immunogenicity of
rec.HBsAg adsorbed on AI(OH)3
Groups of 10 female Balblc mice were injected by the subcutaneous route
with 2.5 mcg of recHBsAg adsorbed on SO mcg of Al+++ (as Al(OH)3) and
increasing amounts of MPL (3.1 to 50 mcg) with a particle size of > S00 nm.
The
mice were injected twice in a volume of 100 mcl and at 2 weeks internal.-They
were
bled 2 weeks after the first injection (partial bleeding) and one week after
the booster.
Total anti-HBs IgG and specific IgG2a were measured by ELISA using recHBsAg as
capture antigen. The titers were expressed as the reciprocal of the dilution
corresponding to 50% of the maximal value (mid-point dilution). The results
indicate
an increase of both specific IgG and IgG2a with increasing doses of MPL,
particularly for doses of 12.5 to 50 mcg. The effect is seen for both primary
and
secondary responses and is particularly obvious for IgG2a (up to 20 fold
increase)
indirectly indicating a secretion of g-interferon induced by the immunization
with
MPL.
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9.1.2. EXPERIMENT II - Comparison of clinical lots of adsorbed recHBsAg
containing or not containing MPL (> 500 nm)
3 clinical lots of recHBsAg adsorbed on Al(OH)3 were prepared : lot
DSAH16 contained no MPL and served as control. Lots DSAR501 and 502 were
prepared in a similar way (20 mcg of recHBsAg adsorbed on 0.5 mg Al+++ as
Al(OH)3) but contained 50 mcg of MPL (> 500 nm).
The 3 lots were injected subcutaneously to groups of 10 mice (200 mcl
containing 2.5 mcg HBsAg, 100 mcg Al+++ and 6.25 mcg MPL), twice at 2 weeks
interval. The mice were bled at day 14 and 1 week after the booster. Anti-HBs
antibodies were measured using AUSAB kit or an in-house ELISA for either IgG
or
IgG2a. The results are given in table 2. They indicate that, 2 weeks after the
first
injection, the 2 lots containing MPL induce a very significant anti-HBs
response (12.4
and 41.9 mIU/ml) while the lot which does not contain MPL only induces a
marginal
response (0.75 mIU/ml). The number of responders is also higher.with MEL (9/IO
and 9/10 versus 1/10 in absence of MPL). The effect of MPL is confirmed after
the
booster since the titers obtained for lots DSAR501 and 502 are about 6 fold
higher
than that observed without MPL.
This indicates that, at least in mice, MPL (> 500 nm) can improve both the
kinetics of the anti-HBs response and the level of the anti-HBs response.
These results were confirmed when specific IgG and IgG2a are measured after
immunization with lots DSAH16 (without MPL) and DSAR502 (with MPL): the anti-
HBs IgG titer is 5 (primary response) and 3 (secondary response) times higher
when
MPL is present.
For the IgG2a response, the effect of MPL is even more striking, at least
after
the second dose, indicating a preferential induction of IgG2a. This indirectly
reflects
activation of cell-mediated immunity (secretion of gamma-interferon) by the
preparation containing MPL.
9.1.3. Experiment III: Effect of MPL (< 100 nm) dose on immunogenicity of
recombinant HBsAg adsorbed on AI(OH)3
Groups of 10 mice (Balb/c, female, 7 weeks old) were injected subcutaneously
with 1 mcg of recombinant HBsAg adsorbed on 50 mcg of Al+++ (as Al(OH)3) and
in presence of increasing amounts (3.1 to 25 mcg) of MPL(< 100 nm). The mice
were injected twice at 2 weeks interval with a volume of 200 mcl. They were
bled 2
weeks after the first injection and 1 week after the booster. The anti-HBs
response
was evaluated by ELISA (total Ig, IgG, IgG2a) on pooled sera. The titers were
expressed as mid-point dilutions (reciprocal of the dilution giving 50 % of
the highest
values). The results indicate that as few as 3.1 mcg of MPL induce a strong
increase
of the antibody response both for primary and secondary responses. The
response
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WO 94/21292 PCT/EP94100818
culminates for 6.25 mcg and decreases afterwards to become similar to that
found
without MPL when high doses of MPL (25 mcg) are used. The pattern of the
antibody response is similar for IgG, IgG2a and total Ig. It contrasts with
results
obtained for MPL of higher size (> 500 nm) and shows that small size (< 100
nm)
S particles of MPL are more effective than larger size (> 500 nm) particles
(at least for
humoral immunity), since less MPL is needed to obtain the maximal effect. The
highest activity of small size MPL was confirmed in several experiments.
As shown for larger size MPL (> 500 nm), the adjuvant effect of MPL is
higher for IgG2a than for total IgG or Ig. At the maximal effect of the
secondary
response (6.25 mcg of MPL), there is a 25 fold increase for IgG2a while the
increase for IgG or total Ig was 7.6 and 4.3 respectively.
9.2. Induction of Celt-Mediated Immunity by recHBsAg adsorbed on
AI(OH)3 - effect of MPL
If humoral immunity is sufficient to protect against Hepatitis B, the
induction
of cell-mediated immunity (CTL, Th 1 ) could be of particular importance for
the
treatment of the disease.
New formulations are required however for therapeutic vaccines since
Al(OH)3 is capable of improving humoral immunity but not cell mediated
immunity.
We have investigated the effect of MPL on the induction of Thl cells capable
of secreting IL-2 and g-(i.e. gamma) interferon in Balb/c mice immunized with
recHBsAg adsorbed on Al(OH)3.
9.2.1. EXPERIMENT I - Effect of MPL (> 500 nm) on induction of Thl cells
after immunization of Balb/c mice with AI(OH)3 adsorbed HBsAg
A group of 10 Balb/c mice (female, 5 weeks old) were immunized by
injection in each footpad of 30 mcl containing 10 mcg of HBsAg, 15 mcg of
Al+++
(as Al(OH)3) and 15 mcg of MPL. Control mice were injected similarly with the
same amount of recHBsAg either mixed with FCA (positive control) or adsorbed
on
Al(OH)3 without MPL (negative control).
Six days after the immunization, the mice were killed and the popliteal lymph
nodes were removed. The lymph node cells (LNC 2.105/ml) were cultivated for
different periods of time (24 hrs to 74 hrs) in RPMI medium supplemented with
1 %
of negative mouse serum and containing 5 mcg/ml of recHBsAg. After termination
of the culture, the amount of 1L-2, INF-g and IL-4 secreted in the medium was
measured. IL-2 was estimated by its ability to stimulate the proliferation
(evaluated
by incorporation of 3H-Thymidine) of an IL-2-dependent CTL line (VDA2 cells)
and
the titer was expressed as stimulation index (SI = amount of 3H-Thymidine
incorporated in stimulated cells/amount of 3H-Thymidine incorporated in non
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stimulated cells). The amount of IL-4 and INF-g was measured using commercial
ELISA kits (Holland Biotechnology for IFN-g and Endogen for IL-4). The titers
were expressed in pg of IFN-g/ml.
The results indicate that no significant amount of IL-2, IL-4 or INF-g is
secreted by LNC from mice immunized with HBsAg adsorbed on Al(OH)3. On the
contrary, high levels of IL-2 (LS. = 38 at 48 hrs) and a significant amount of
INF-g
are secreted by LNC from mice immunized with HBsAg adsorbed on Al(OH)3 +
MPL. This secretion is similar (INF-g) or higher (IL-2) to that observed for
mice
immunized with HBsAg + FCA and the in vitro secretion occurs earlier.
No IL-4 was detected after immunization with HBsAg adsorbed on Al(OH)3
even in presence of MPL.
This secretion profile indicates that specific Th 1 cells (IL-2, INF-g) have
been
induced by immunization with adsorbed HBsAg in presence of MPL but not in
absence of MPL. However, no Th2 (IL-4) were detected in these conditions of
immunization.
9.2.2. EXPERIMENT II - Effect of the dose of MPL (< 100 nm) on the
induction of Thl cells after immunization of Balb/c mice with Al(OH)3 adsorbed
recHBsAg
Groups of 5 Balb/c mice were immunized in each of the 2 footpads with30
mcl containing 10 mcg of recHBsAg adsorbed on 15 mcg of Al+++ (as Al(OH)3) and
with increasing amounts of MPL ( 100 nm, 0 to 15 mcg).
Six days after the injection, the mice were killed and the popliteal lymph
node
cells (LNC) were cultivated at 2.106 cells/ml in RPMI supplemented with 1
°!o
negative mouse serum for different periods of time (24 hrs to 96/25) in
presence of 5
mcg/ml of recHBsAg.
The secretion of IL-2 was measured by stimulation of the proliferation of
VDA2 cells and concentration of IL-2 is expressed as Stimulation Index (SI);
the
secretion of INF-g was measured using a commercial kit and expressed in pg/ml.
It was found that the secretion of IL-2 is dramatically increased by the lower
dose of MPL (7.5 mcg) and a maximal effect is obtained for 15 mcg of MPL.
The secretion of IL-2 is generally more important at 24 hrs than at 48 or 72
hrs.
The secretion of INF-g is absent when HBsAg is adsorbed on Al(OH)3 in
absence of MPL. A small dose (7.5 mcg) of MPL induces a secretion of INF-g and
again, the maximal effect is obtained for 15 mcg of MPL. Contrary to what is
observed for IL-2, the secretion of INF-g is delayed in the culture and
increases with
time up to 96 hours.
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Taken together these data indicate that MPL (less than 100 nm) is a potent
inducer of Th 1 when combined with HBsAg adsorbed on Al(OH)3.
The effect of a formulation containing HBsAg adsorbed on Al(OH)3 and MPL on
the
induction of both humoral and cell-mediate immunity in Balb/c mice has
been investigated. The results indicate that MPL clearly improves the kinetics
of the
anti-HBs response since much more anti-HBs antibodies are found after both the
primary and secondary immunizations. The quality of the anti-HBs is also
modified
and a preferential induction of IgG2a has been observed, reflecting indirectly
secretion of INF-g and thus induction of a cell-mediated immunity.
Direct evaluation of the induction of Th 1 cells by formulations containing
HBsAg, Al(OH)3 and MPL clearly indicates that MPL is a potent inducer of Thl
cells secreting both IL-2 and INF-g. This kind of formulation is thus
important in the
development of therapeutic vaccines.
Best results were obtained using MPL of less than 100nm particle size.
For Tables showing the results of experiments described above, see Tables 9-
14 below.
13. Conclusions
Overall the data suggests that small MPL is an improved immunostimulant in
primates including man, over large size MPL. This combined with the ability to
make large scale sterile Iots renders small MPL as a suitable immunostimulant
for a
range of human or animal health vaccines.
-24-
WO 94/21292 PCT/EP94/00818
Table 1:
MPL particle and filtration recovery using different sonication parameters
Tria ConcentrationTotal residenceParticle sizeRecovery
n after
(mg/ml) time in the before filtrationfiltration
flow (%)
cell (min) (nm)
16 1 2,5 92 104
17 1 3 79 78,5
18 1 3,5 95 86,4
19 2 2,8 77 N.A.
20 1 2,8 98 N.A.
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TABLE 6
IMMUNOGENICITY OF gD2t ALUMINIUM HYDROXIDE MPL 100nm (sorbitol)
IN PRIMATES
SEROLOGICAL AND DTH RESULTS
VACCINE MONKEY ANTIBODY DTH RESPONSE **
No. RESPONSE* (Induration)
ELISA NEUTRA gD2 gD2
TTTER TITER PBS Sp.g l5~tg
KQ 100 18630 800 - + ++
20p.g gD2tKQ 101 5554 1600 - -
Aluminium KQ 102 14870 800 - ++ +-+-+-
hydroxide
SOitg MPL KQ 103 5486 1600 - _++ _ +++
KQ 104 16270 1600 ND ND ND
GMT 10655 1213
KQ 105 16170 800 - + ++
20p.g gD2 KQ 106 4389 800 - - -
Aluminium KQ 107 20440 1600 - ++ +++
hydroxide
20~tg MPL KQ 108 5613 800 - + +
KQ 109 6765 1600 ND ND ND
GMT 8876 1056
KQ 110 2486 200 - - -
20p.g gD2tKQ 111 9918 800 - ++ +++
Aluminium KQ I12 2526 400 - - -
hydroxide
Sp.g MPL KQ 1 I3 7137 400 - - -
KQ 114 8396 400 ND ND ND
GMT 5181 400
*Measured 14 days post II/GMT = geometric mean titer
ELISA titer= midpoint titer
NEUTRA titer = reciprocal of the highest serum dilution giving 100% protection
against the cytopathogen effect
**skin test on day 14 post II
Induration 24 hrs reading
+ = 1 mm
++ = 1-Smm
+++=>Smm
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Table 8
Immunogenicity of clinical batches of OspA In Humans
Anti-OspA in the LA-2 inhibition assay
(ng equiv LA-2/ml) (GMT)
Vaccine Pre Post I Post II Post III 30
30 30
Do D28 D56 D84
NS 1-OspA 118 233 409 768
on Aluminium
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SC (%) 2.6 77.2 86.5 100
NS 1-OspA+MPL134 269 865 2424
on Aluminium
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SC (lo) l 2.6 -.' g8.6 _[ ~7.2 100
N=80
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