Note: Descriptions are shown in the official language in which they were submitted.
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VACCINES
The present invention relates to a novel vaccine formulations and their use in
medicine, particularly in the prevention of malaria infections. In particular
the
present invention is concerned with a CpG oligonucleotide and a malarial
antigen.
Malaria, is one of the world's major health problems with 2 to 4 million
people
dying from the disease each year. One of the most acute forms of the disease
is
caused by the protozoan parasite, Plasmodium falciparum which is responsible
for
most of the mortality attributable to Malaria.
The life cycle of P. falciparum is complex, requiring two hosts, man and
mosquito
for completion. The infection of man is initiated by the inoculation of
sporozoites
in the saliva of an infected mosquito. The sporozoites migrate to the liver
and there
infect hepatocytes where they differentiate, via the exoerythrocytic
intracellular
stage, into the merozoite stage which infects red blood cells (RBC) to
initiate
cyclical replication in the asexual blood stage. The cycle is completed by the
differentiation of a number of merozoites in the RBC into sexual stage
gametocytes
which are ingested by the mosquito, where they develop through a series of
stages
in the midgut to produce sporozoites which migrate to the salivary gland.
The sporozoite stage of P. falciparum has been identified as a potential
target of a
malaria vaccine. The major surface protein of the sporozoite is known as
circumsporozoite protein (CS Protein). This protein from strain 7G8 has been
cloned, expressed and sequenced (Dame et al Science 225 (1984) p593). The
protein from strain 7G8 is characterised by having a central immunodominant
repeat
region comprising a tetrapeptide Asn-Ala-Asn-Pro repeated 37 times but
interspersed with four minor repeats Asn-Val-Asp-Pro. In other strains the
number
of major and minor repeats vary as well as their relative position. This
central
portion is flanked by an N and C terminal portion composed of non-repetitive
amino
acid sequences designated as the repeatless portion of the CS protein.
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It has been shown that irradiated sporozoites can provide significant
protection
against experimental human malaria (Am. J. Trop. Med. Hyg. 24: 297-402, 1975).
However, production difficulties makes the use of irradiated sporozoite
impractical
from the point of view of producing a vaccine.
Several groups have proposed subunit vaccines based on the circumsporozoite
protein. Two of these vaccines have undergone clinical testing; one is a
synthetic
peptide, the other is a recombinant protein (Ballou et al Lancet: i 1277
(1987) and
Herrington et al Nature 328:257 (1987).
These vaccines were successful in stimulating an anti-sporozoite response.
Nonetheless, the magnitude of the response was disappointing, with some
vaccinees
not making a response at all. Furthermore, the absence of "boosting" of
antibody
levels on subsequent injections and results of in vitro lymphocyte
proliferation
assays suggested that T-cells of most of these volunteers did not recognise
the
immuno-dominant repeat. Nonetheless, one vaccinee in each study did not
develop
parasitemia.
The present invention provides a new, improved malaria vaccines which not only
produces a humoral response, but also a cellular immune response. Preferably
the
antigen induces the production of neutralising antibodies against the
immunodominant repeat. Most preferably, the antigen should also elicit
effector T
cell mediated immune responses of the CD4+ and CD8+ cytotoxic T lymphocyte
(CTL) type and of the delayed type hypersensitivity type and also, preferably
be
able to induce T helper (TH) memory cells.
International patent application No. WO 93 / 10152 (SmithKline Beecham
Biologicals s.a) provides a hybrid protein comprising substantially all the
C-terminal portion of the CS protein, four or more tandom repeats of the
immunodominant region, and the Surface antigen from Hepatitis B virus (HBsAg).
Preferably the hybrid protein comprises a sequence which contains at least 160
amino acids which is substantially homologous to the C-terminal portion of the
CS
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protein. The CS protein may be aevoid of the last 12 amino-acids from the C
terminal.
In particular there is provided a protein which comprises a portion of the CS
protein
of P. falciparum substantially as corresponding to amino acids 210-398 of P.
falciparum 7G8 fused in frame via a linear linker to the N-terminal of HBsAg.
The
linker may comprise a portion of preS2 from HBsAg.
A particularly preferred embodiment is the hybrid protein designated RTS (or
RTS,S). This hybrid consists of:
~ A methionine-residue, encoded by nucleotides 1059 to 1061, derived from the
Saccharomyces cerevisiae TDH3 gene sequence. (Musti A.M. et al Gene 1983
25 133-143).
~ Three amino acids, Met Ala Pro, derived from a nucleotide sequence (1062 to
1070) created by the cloning procedure used to construct the hybrid gene.
~ A stretch of 189 amino acids, encoded by nucleotides 1071 to 1637
representing
amino acids 210 to 398 of the circumsporozoite protein (CSP) of Plasmodium
falciparum strain 7G8 (Dame et al su ra).
~ An amino acid (Arg) encoded by nucleotides 1638 to 1640, created by the
cloning procedure used to construct the hybrid gene.
~ Four amino acids, Pro Val Thr Asn, encoded by nucleotides 1641 to 1652, and
representing the four carboxy terminal residues of the hepatitis B virus (adw
serotype) preS2 protein (9).
~ A stretch of 226 amino acids, encoded by nucleotides 1653 to 2330, and
specifying the S protein of hepatitis B virus (adw serotype).
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In an alternative embodiment there is provided a hybrid protein designated
RTS*
(or RTS*,S), which was generated using the CSP gene sequence from P.
falciparum
NF54 (Mol. Biochem Parisitol. 35 : 185-190, 1989) and comprises substantially
all
of the region 207 to 395 of the CS protein from P falciparum NF54.
In particular RTS* comprises:
~ A Methionine, encoded by nucleotides 1059 to 1061, derived from the
TDH3 gene sequence (see Musti et al, loc cit)..
~ Three amino acids, Met Ala Pro, derived from a nucleotide sequence (1062
to 1070) created by the cloning procedure used to construct the hybrid gene.
~ A stretch of 189 amino acids, encoded by nucleotides 1071 to 1637
representing amino acids 207 to 395 of the circumsporozoite protein (CSP)
of Plasmodium falciparum strain NF54 (Mol.Biochem.Parasitol, 35:185-
190, 1989).
~ An amino acid (Gly) encoded by nucleotides 1638 to 1640, created by the
cloning procedure used to construct the hybrid gene.
~ Four amino acids, Pro Val Thr Asn, encoded by nucleotides 1641 to 1652,
and representing the four carboxy terminal residues of the hepatitis B virus
(adw serotype) preS2 protein (Nature 280:815-819, 1979).
~ A stretch of 226 amino acids, encoded by nucleotides 1653 to 2330, and
specifying the S protein of hepatitis B virus (adw serotype) (Nature 280:815-
819,1979)
International patent application no. WO 90/01496 describe an antigen known as
Trap (or TRAP) from P. falciparum. An apparent homologue of Trap is described
in W092/11868 and relates to an antigen called SSP2 from P. yeolii.
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International patent application WO 98/ 05355 describes, inter alia, a malaria
vaccine based on a combination of Trap and RTS,S.
Immunomodulatory oligonucleotides contain unmethylated CpG dinucleotides
("CpG") and are known (WO 96/02555, EP 468520). CpG is an abbreviation for
cytosine-guanosine dinucleotide motifs present in DNA. Historically, it was
observed that the DNA fraction of BCG could exert an anti-tumour effect. In
further studies, synthetic oligonucleotides derived from BCG gene sequences
were
shown to be capable of inducing immunostimulatory effects (both in vitro and
in
vivo). The authors of these studies concluded that certain palindromic
sequences,
including a central CG motif, carried this activity. The central role of the
CG motif
in immunostimulation was later elucidated in a publication by Krieg, Nature
374,
p546 1995. Detailed analysis has shown that the CG motif has to be in a
certain
sequence context, and that such sequences are common in bacterial DNA but are
rare in vertebrate DNA.
It is currently believed that this evolutionary difference allows the
vertebrate
immune system to detect the presence of bacterial DNA (as occurring during an
infection) leading consequently to the stimulation of the immune system. The
immunostimulatory sequence as defined by Krieg is:
Purine Purine CG pyrimidine pyrimidine and where the CG motif is not
methylated.
In certain combinations of the six nucleotides a palindromic sequence is
present.
Several of these motifs, either as repeats of one motif or a combination of
different
motifs, can be present in the same oligonucleotide. The presence of one or
more of
these immunostimulatory sequence containing oligonucleotides can activate
various
immune subsets, including natural killer cells (which produce interferon y and
have
cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977).
Although other unmethylated CpG containing sequences not having this consensus
sequence have now been shown to be immunomodulatory.
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The present invention provides an improved vaccine formulation comprising a
CpG
oiigonucleotide and a malaria antigen. In particular, RTS,S or RTS,S* or Trap
or
immunologically equivalent derivatives thereof.
Vaccine preparation is generally described in Vaccine Design - The subunit and
adjuvant approach (Ed. Powell and Newman) Pharmaceutical Biotechnology Vol. 6
Plenum Press 1995. Encapsulation within liposomes is described by Fullerton,
US
Patent 4,235,877.
The preferred oligonucleotides preferably contain two or more CpG motifs
separated by six or more nucleotides. The oligonucleotides of the present
invention
are typically deoxynucleotides. In a preferred embodiment the internucleotide
in
the oligonucleotide is phosphorodithioate, or more preferably a
phosphorodithioate
bond, although phosphodiester and other internucleotide bonds are within the
scope
of the invention including oligonucleotides with mixed internucleotide
linkages.
The sequences preferably contain all phosphorodithioate modified
internucleotide
linkages. Preferred oligonucleotides have the following sequences:
Oligo (internal5'-SEQUENCE-3' CpG Thi
designation*)
WD1001 TCC ATG ACG TTC CTG ACG TT + +
WD1002 TCT CCC AGC GTG CGC CAT + +
WD 1003 ACC GAT AAC GTT GCC GGT GAC G + -
WD1004 G*G*G GTC AAC GTT GAG* G*G*G* G*G + Mix
WD 1005 TCC ATG AGC TTC CTG AGC TT - +
WD1006 TCC ATG ACG TTC CTG ACG TT + -
WD1007 ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG + +
TCG TCG TTT TGT CGT TTT GTC GTT + +
* alternatively referred to as WD001-WD007
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In the above table a + in the Thio column indicates the presence of a thioate
modification. 'Mix' indicates a mixture of thioate modification and sequence
without
thioate modification (the asterisks indicate the linkages with a thioate
modification).
A - in the Thio column indicates absence of a thioate modification. A + in the
CpG
column indicates a the presence of a CpG motif and a - in the CpG column
indicates
absence of a CpG motif. For example WD1005 contains a GpC rather than a CpG
motif, thus it is marked with a - in the CpG column of the table. WD 1007
contains a
palindromic motif (GACGTC) as well as other non-palindromic CpG sequences.
This
is also within the scope of a CpG oligonucleotide as the term is used in the
present
application.
The oligonucleotides utilised in the present invention may be synthesized by
any
method known in the art (eg EP 0 468 520). Conveniently, such oligonucleotides
may be synthesized utilising an automated synthesizer. Methods for producing
phosphorothioate oligonucleotides or phosphorodithioate are described in US
patent
5,666,153, US patent 5,278,302 and W095/26204.
The amount of protein in each vaccine does 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 ~g of protein, preferably 2-100 p,g, most preferably 5-50 fig.
An
optimal amount for a particular vaccine can be ascertained by standard studies
involving observation of appropriate immune responses in subjects. Following
an
initial vaccination, subjects may receive one or several booster immunisations
adequately spaced.
It is also possible to pre-administer the CpG oligonucleotide as a 'priming
formulation' shortly prior to vaccination with the malaria antigen, for
example 1
day before.
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Accordingly, according to another aspect of the invention, there is provided a
method for the prevention or amelioration of plasmodium infection in a
patient,
comprising administering an effective amount of either a malaria antigen and a
CpG
oligonucleotide (as hereinabove defined) or an effective amount of the CpG
oligonucleotide followed after a suitable time by an effective amount of a
malaria
antigen.
There is also provided a kit comprising effective amounts of a CpG
oligonucleotide-
containing formulation for use as a priming formulation for pre-administration
to
human patients and a malaria antigen for injection at some suitable time
later, as
described hereinabove.
Preferred CpG oligonucleotides are those indicated in the table hereinabove.
Suitably the CpG will be present in the range 10 pg per dose to 1000 ~.g,
preferably
10-100~,g, especially 25-75 pg, for example 50 pg per dose.
Suitably the vaccine used in the present invention may comprise a carrier such
as an
aluminium salt, eg aluminium hydroxide [A1(OH)3], aluminium phosphate or
aluminium phosphate sulfate (alum), or a non-toxic oil in water emulsion or a
mixture thereof.
If an aluminium salt (preferably aluminium hydroxide) is used as a carrier it
is
generally present in the range of 50 to 100 pg, preferably 100 to 500 p,g per
dose.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, eg
squalene and
an emulsifier such as (polysorbitan monoleate) Tween 80, in an aqueous carrier
such as phosphate buffered saline.
If desired the vaccine used in the present invention may comprise an
additional
adjuvant, preferably a saponin adjuvant such as QS21 as described for example
in
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WO 9517210, optionally in the presence of a sterol, such as cholesterol as
described
for example in PCT/EP96/01464. The vaccine of the invention may also comprise
monophosphoryl lipid A and derivatives thereof known in the art. A preferred
derivative is 3 de-O-acylated monophosphoryl lipid A, described in British
Patent
No.2220211.
Accordingly vaccine formulations of the present invention may additionally
comprise other pharmaceutical excipients or immunostimulants. In a preferred
embodiment the vaccine formulation additionally comprises an aluminium salt,
preferably aluminium hydroxide.
The present invention will now be described with reference to the following
examples:
IM1VIUNOGENICITY STUDIES USING RTS,S FORMULATED WITH CPG
OR CPG/ALUM
Evaluation of CpG and CpG/alum in mice
Experiment outline
An immunogenicity study was conducted to evaluate the ability of CpG to serve
as
an adjuvant for cytotoxic T lymphocyte (CTL) induction. Groups of mice were
immunized with RTS,S formulated with CpG oligonucleotide alone or in
combination with aluminum hydroxide. After two immunizations spleen cells were
examined for the presence of HbsAg-specific effector cells.
Table 1: Groups of mice
Group antigen adjuvant
1 RTS,S CpG/alum
2 RTS,S CpG
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Formulation
Component batches used.
COMPONENT BRAND BATCH CONCENTRATION BUFFER
NUMBER (MG/ML)
RTSS 24851 0.664 P/N 6.8
Al(OH)3 Superfos97A0027 10.380 H20
CpG (WD1001) Eurogen 32425581 5 Hz0
etech
Formulation process:
Formulations were prepared 3 days before each injection. All incubations were
carried out at room temperature with agitation.
CpGlalum group 1 (SOO~cIldose)
RTS, S (8.7~.g) and gp120 (8.7~,g) were adsorbed on 100~,g of Al(OH)3 or A1P04
for 1 hour. The formulation was buffered with a 10-fold concentrated P04/NaCI
pH
6.8 solution before addition of 100~,g of CpG (WD1001). After 15 min, 50
~cg/ml
of thiomersal was added as preservative.
CpG group 2 (SOO~,Ildose)
RTS,S (8.7~cg) and Gp120(8.7~,g) were diluted in PBS pH 6.8 before addition of
100~,g of CpG (WD1001). After 5 min, 50 ~.g/ml of thiomersal was added as
preservative.
Immunological methods
Nine Balb/C mice per group received into the hind footpads 100 ~,l vaccine
twice at
a two-week-interval. Two weeks later spleen cells were harvested and used to
determine the induction of HBsAg-specific CTL.
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For CTL analysis cells were cultured for 7 days in 6-well plates in the
presence of
~,g per ml of synthetic peptide pCMI003 corresponding to an HBsAg CTL
epitope (Schirmbeck et al., 1995). At the end of the culture period cells were
assessed in duplicate for HBsAg-specific cytolytic activity in standard [5'Cr]-
release
5 assays using control and S-transfected P815 cells. Minimum and maximum
release
were determined with target cells without effector cells and by the addition
of 3 %o
(v/v) Triton X-100, respectively. Results are expressed as % [SlCr]-release
(cpm of
exp. culture - cpm of spont. release / cpm of max. release - cpm of spont.
release).
10 Results
Spleen cells from both groups of mice exhibited HBsAg-specific effector cell
function in SICr release assays (Figure 1). Untransfected P815 target cells
were
lysed to a much lesser degree than similar target cells expressing the HBV s
gene.
Lysis of the target cells diminished with decreasing effector to target cell
ratios.
Conclusions
Immunization with RTS,S in combination with CpG or CpG/alum induces HBsAg-
specific CTL in mice.
EVALUATION OF CPG AND CPG/ALUM IN RHESUS MONKEYS
Experiment outline
An immunogenicity study was conducted to evaluate the adjuvant effect of CpG
in
non-human primates. Groups of five monkeys were immunized twice with RTS,S in
combination with CpG or CpG/alum. After the second immunization the immune
response of the animals was assessed. Antibodies to HBsAg and
lymphoproliferative
as well as cytokine responses were evaluated.
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Table 2. Groups of monkeys
Group antigen adjuvant
1 RTS,S CpG/alum
2 RTS,S CpG
Formulation
Component batches used.
COMPONENT BRAND BATCH CONCENTRATION Buffer
NUMBER (MG/ML)
RTS,S ERTS1X058 1.372 P/N 6.8
Al(OH)3 Superfos 96A0089 10.380 H20
CpG WD 1001 5 H20
Formulation process:
Formulations were prepared one day before each injection. All incubations were
carried out at room temperature with agitation.
CpGlalum group 1 (SOO~,Ildose)
RTS,S (SO~cg) was adsorbed on SOO~sg of Al(OH)3 for 1 hour. The formulation
was
buffered with a 10-fold concentrated P04/NaCI pH 6.8 solution before addition
of
SOO~,g of CpG (WD1001). After 15 min, SO ~,g/ml of thiomersal was added as
preservative.
H20+Al(OH)3+RTS,S-1H-lOxPN-15m-CpG-15m-Thio
CpG group 2 (500~c1/dose)
RTS,S (SO~,g) was diluted in P04/NaCI buffer pH 6.8 before addition of SOO~.g
of
CpG WD1001. After 15 min, 50 ~,g/ml of thiomersal was added as preservative.
HZO +RTSS + lOxPN-lSm-CpG-15m-Thio
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Immunological methods
Five rhesus monkeys (Macaca mulatta) per group were immunized twice
intramuscularly with 500 td of vaccine at a four-week-interval. Sera and
peripheral
blood mononuclear cells (PBMC) were taken at several occasions.
HBsAg-specific antibodies in monkey sera were determined in a radio immuno
assay (RIA, Abbott) according to the manufacturer's instructions.
Lymphoproliferation was assessed by using density gradient-purified PBMC from
immunized rhesus monkeys. Cells were seeded in quadruplicates at 1x105 in 100
~,l
RPMI/S % FCS per well in round bottom 96 well plates. Then another 100 ~,l of
medium alone or containing soluble RTS,S (10 tcg/ml) were added and parallel
cultures were incubated for 48 hrs. Thereafter, 100 tcl culture supernatant
were
replaced by fresh medium containing 1 ~,Ci [3H]-thymidine. After 16 hrs cells
were
harvested onto filter plates and incorporated radioactivity was determined in
a (3-
counter. Results are expressed in cpm and in stimulation indices (SI, = cpm
antigen-containing cultures/cpm medium alone cultures), SI greater than 3 are
considered as a positive response.
Flat bottom 96 well plates were prepared by coating an IFN-y-specific capture
antibody in 50 ~,l PBS for 4 hrs at 37 °C. The plates were washed three
times and
PBMC were seeded similar to lymphoproliferation assays. After 48 hrs of
culture
the plates were washed thrice with PBS/0.05 % Tween 20 and 50 ~cl of
biotinylated
secondary IFN-y-specific antibody diluted in PBS/Tween/1 % FCS were added for
2 hrs. The plates were washed again and a gold-conjugated a-biotin antibody
was
incubated for 1 hr. After additional washings the ELIspots were visualized by
using
a silver enhancing kit (50 ~cl per well). The reaction was stopped after
approx. 30
min by adding deionized water. Cytokine-secreting cells were enumerated by
microscopic examination.
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Results
Analysis of HBsAg-specific antibodies in sera of the monkeys revealed that all
animals in the two groups had developed specific immune responses (Figure 2).
Some responses were detectable already after one immunization. Interestingly,
these
responses were boosted by the second immunization only in group 1, while
titers in
group 2 remained more or less constant.
Induction of specific lymphoproliferation by immunization with RTS,S in
combination with CpG or CpG/alum was evaluated before immunization and 6 days
post secondary immunization. All 10 animals did not exhibit any specific
lymphoproliferation (SI > 3) at the study start (data not shown). In contrast,
all
animals in group 1 possessed strong lymphoproliferative responses 6 days post
boost immunization (Figure 3). All animals from group 2 did, however, remain
negative in this analysis.
The presence of RTS,S-specific IFN-y-secreting cells was investigated in all
monkeys before immunization and 6 days after the second dose. IFN-y-secreting
cells could not be evaluated from pre-immunization samples due to technical
difficulties. However, such cells were detectable after secondary immunization
(Figure 4). All animals in group 1 exhibited a positive response, while only
one
animal in group 2 was positive.
Conclusions
Immunization with RTS,S in combination with CpG induces immune responses in
non-human primates. After two immunizations CpG alone induces low level HB
sAg-specific antibodies, while CpG combined with alum induces high titer
antibodies as well as vigorous lymphoproliferative and IFN-y responses.
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Figure legends
Figure 1: CTL activity of spleen cells from immunized mice. Effector cell
activity
was assessed by examining 5'Cr release of P815 cells (open circles) or s-
transfected
P815 cells (closed circles).
Figure 2: HBsAg-specific antibody responses in immunized rhesus monkeys.
Specific antibodies were evaluated using a commercially available RIA.
Individual
values from multiple time points for each animal are shown in the table, and
group
averages are shown in the table and as a graphic.
Figure 3: RTS,S-specific lymphoproliferation in immunized rhesus monkeys 6
days
post second immunization. PBMC were stimulated with RTS,S antigen and
lymphoproliferative responses were measured by 'H-thymidine incorporation.
Results are expressed in cpm and as SI.
Figure 4: RTS,S-specific IFN-y-secreting cells from immunized rhesus monkeys.
IFN-y-secreting cells were visualized by the ELIspot method. Cytokine-
secreting
cells resulting in a colored spot were enumerated by microscopic examination
and
results are expressed semi-quantitatively (- = 0-5, + = 5-15, + + = 15-35,
+ + + = 35-50, + + + + _ > 50).
References
Schirmbeck, R., Boehm, W., Melber, K., Reimann, J. (1995). Processing of
exogenous heat-aggregated (denatured) and particulate (native) Hepatitis B
surface
antigen for class I-restricted epitope presentation. J. Immunol. 155:4676-
4684.
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