Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
ADJLTVANT COMPOSITIONS COMPRISING A M]NELZAL SALT AND
ANOTHER IMMUNOSTIMULATING COMPOUND
FIELD OF THE INVENTION
The present invention relates to the field of immunology and is particularly
concerned with adjuvants, i.e. materials which modulate immune response to an
antigen.
This application is a division of co-pending Canadian Patent Application
Serial No. 2,192,659, filed June 15, 1995.
BACKGROUND OF THE INVENTION
Vaccines have been used for many years to protect humans and animals
against a wide variety of infectious diseases. Such conventional vaccines
consist of
attenuated pathogens (for example, polio virus), killed pathogens (for
example,
Bordetella pertussis) or immunogenic components of the pathogen (for example,
diphtheria toxoid). Some antigens are highly immunogenic and are capable alone
of
eliciting protective immune responses. Other antigens, however, fail to induce
a
protective immune response or induce only a weak immune response.
In the development of some vaccines and immunogenic compositions, there is
a trend to use smaller and well defined immunogenic and protective materials.
Recent
advances in molecular genetics, protein biochemistry, peptide chemistry, and
immunobiology have provided economical and effiruient technologies to identify
and
produce large quantities of pure antigens from various pathogens. However,
some
such materials may not be sufficiently immunogenic, due to either their small
size
(especially synthetic peptides) or the lack of intrinsic immunostimu-latory
properties
thereof.
Immunogenicity can be significantly improved if the antigens are co-
administered with adjuvants. Adjuvants enhance the immunogenicity of an
antigell
but are not necessarily immunogenic themselves. Adjuvants may act by retaining
tile
antigen locally near the site of administration to produce a depot effect
facilitating a
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slow, sustained release of antigen to cells of the immune
system. Adjuvants can also attract cells of the immune
system to an antigen depot and stimulate such celis to
elicit immune responses.
Immunostimulatory agents or adjuvants have been used
for many years to improve the host immune response-s to,
for example, vaccines. intrinsic adjuvants, such as
lipopo7.ysaccharides, normally are the components of the
killed or attenuated bacteria used as vaccines.
Extrinsic adjuvants are immunomodulators whi-ch are
typically non-covalently linked to antigens and are
formul-at-ed to enhanee the host immuaE responses. Thus,
adjuvants have been identified that enhance the immune
response to antigens delivered parenterally. Some of
these adjuvants are toxic; however, and can cause
undesirable side-effects, making them unsuitable for use
in humans and many animals. Indeed, only aluminum
hydroxide and aluminum phosphate tco].lecti.vely commonly
referred to as alum) are routinely used as adjuvants in
human and veterinary vaccines. The efficacy of alum in
increasing antibody responses to diptheria and tetanus
toxoids is well established and, more recently, a HBsAg
vaccine has been adjuvanted with alum. While the
usefulness of alum is well established for some
applications, it has limitations. For example, alum is
ineffective for influenza vaccination and inconsistently
elicits a cell mediated immune response. The antibodies
elicited by alum-adjuvanted antigens are mainly of the
IgGI isotype in the mouse, which may not be optimal for
protection by some vaccinal agents.
A wide range of extrinsic adjuvants can provoke
potent immune responses to antigens. These include
saponins complexed to membrane protein antigens Ummune
stimulating complexes), pluronic polymers with mineral
oil, killed mycobacteria in mineral oil, Freund's
complete adjuvant, bacterial products, such as muramyl
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dipeptide (MDP) and lipopolysaccharide -(LPS), as well as
lipid A, and liposomes.
To efficiently induce humoral immune responses (HIR)
and cell-mediated immunity (-CMI), immunogens are
emulsified in adjuvants. Many adjuvants are toxic,
inducing granulomas, acute and chronic inflammations
(Freund's complete adjuvant, FCA), cytolysis (saponins
and Pluronic polymers) and pyrogenicity, arthritis and
anterior uveitis (LPS and MDP). Although FCA is an
excellent adjuvant and widely used in research, it is not
licensed for use in human or veterinary vaccines because
of its toxicity.
Desirable characteristi,ce of ideal adjuvants
include:
3.5 (1) lack of toxicity;
(2) ability to stimulate a long-lasting immune response;
(3) simplicity of manufacture and stability in long-term
storage;
(4) ability to elicit both 0+II and HIR to antigens
administered by various routes, if required;
(5) synergy with other adjuvants;
(6) capability of selectively interacting with
populations of antigen presenting cells (APC);
(7) ability to specifically elicit appropriate THl or Ts2
cell-specific immune responses; and
(8) ability to selectively increase appropriate antibody
isotype levels (for example, IgA) against antigens.
US Patent No. 4,855,283 granted to Zockhoff et al on
August 8, 1989 teaches glycolipid an,alogues including N-
glycosylamides,N-g3ycosylur.eas and N-glycosylcarbamates,
each of which is substituted in the sugar residue by an
amino acid, as immuno-modulators or adjuvants. Thus,
Lockhoff et al. (US Patent No. 4,855,283) reported that
N-glycolipid analogs displaying structural similarities
to the naturally-occurring glycolipids, such as
glycosphingolipids and glycoglycerolipids, are capable of
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eliciting strong immune responses in both herpes simplex
virus vaccine and pseudorabies virus vaccine. Some
glycolipids have been synthesized from long -chain-
alkylamines and fatty acids that are linked directly with
the sugars through the anomeric carbon atom, to mimic the.
functions of the naturally occurring lipid residues.
U.S. Patent No. 4,258,029 granted to Moloney,
assigned to the assignee hereof, teaches that octadecyl
tyrosine hydrochloride (OTH).functioned as an adjuvant
l0 =when complexed with tetanus toxoid and formalin
inactivated type I, II and III poliomyelitis virus
vaccine. Also, Nixon-George et al. (1990), J. Immunology
.144:4798-4802 reported that octadecyl esters of aromatic
amino acids complexed with a recombinant hepatitis B
surface antigen, enhanced the host immune responses
against hepatitis B virus. .
Lipidation of synthetic peptides has also been used
to increase their immunogenicity. Thus, Wiesmuller
((1989), Vaccine Z:29-33) describes a peptide with a.
sequence homologous to a foot-and-mouth' disease viral
protein coupled to an adjuvant tripalmityl-S-glyceryl-
cysteinylserylserine, being a synthetic analogue of the
N-terminal part of the lipoprotein from Gram negative
bacteria. Furthermozre, Deres et al. (1989, NaturB
342:561) reported j,n y,'Lvo. priming of virus-specific
cytotoxic T lymphocytes with synthetic lipopeptide
vaccine which comprised of modified synthetic peptides
derived from influenza virus nucleoprotein by linkage to
a lipopeptide, N-palmityl-S-[2,3-bis(palmitylxy)-(2RS)-
propyl-[R]-cysteine (TPC).
The adjuvants and immunostimulating compounds
described above may not provide for adjuvanticity for all
antigens delivered to a variety of hosts under many
conditions.
A~NpE~ SM0
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It would be desirable to provide adjuvant compositions that do not suffer from
the disadvantages and limitations of currently available adjuvants.
SUMMARY OF INVENTION
The present invention is directed towards the provision of improved adjuvant
compositions. In accordance with one aspect of the present invention, as clain-
ied in
the parent application Serial No. 2,192,659, there is provided an adjuvant
composition
for modulating an immune response to an antigen administered to a host, the
composition comprising:
(a) a mineral salt adjuvant; and
(b) at least one other adjuvant.
The multiple adjuvant compositions provided herein exhibit a surprisingly
unexpected adjuvanting effect on an antigen which is greater than the
adjuvanting
effect attainable by one of the adjuvants alone. The enhanced effect nlay be
additive
of the adjuvanting effect of the individual adjuvants and, in particular
embodiments, a
synergistic effect is attained.
The mineral salt adjuvant preferably comprises aluminum hydroxide or
aluminum phosphate, although other known mineral salt adjuvants, such as
calcium
phosphate, zinc hydroxide or calcium hydroxide, may be used. The at least one
other
adjuvant may be a glycolipid analog, an octadecyl ester of an amino acid (such
as an
aromatic amino acid) or a lipoprotein. The lipoprotein may be a synthetic
analogue of
an N-terminal portion of lipoprotein.
In a particular embodiment, the glycolipid may be a glycosylamide and may
have the formula:
R$--- O-CH2
0 CO-X-R=
R'-0 N\
Ri
R'--O NH - W
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wherein Rl denotes hydrogen or saturated or singly or multiply unsaturated
alkyl
radical having up to 50 carbon atoms;
X represents -CH2-, -0- or -NH-;
R2 denotes hydrogen or a saturated or singly or multiply unsaturated alkyl
radical
having up to 50 carbon atoms,
R3, R4, and R5, independently of one another, denotes hydrogen, or SO42-, or
P042",
or other negatively charged moiety, or -CO-R6, R6 being an alkyl radical
having up to
carbon atoms;
R7 is L-alanyl, L-alpha-aminobutyryl, L-arginyl, L-asparginyl, L-aspartyl, L-
cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxypropyl, L-isoleucyl, L-
leucyl,
L-lysyl, L-methionyl, L-omithinyl, L-phenylalanyl, L-prolyl, L-seryl, L-
threonyl, L-
tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers;
and pharmaceutically acceptable salts thereof. In an embodiment, the
glycosylamide
may be N - (2-deoxy-2-L-leucylamino-(3-D-glucopyranosyl) - N -
octadecyldodecanamide acetate.
In accordance with a ftirther aspect of the present invention as claimed in
the
parent application Serial No. 2,192,659, there is provided an immunogenic
composition for eliciting an immune response in a host, including a human, the
composition comprising:
(a) at least one antigen;
(b) a mineral salt adjuvant; and
(c) at least one other adjuvant.
Convenient antigens which may be included in said immunogenic
compositions and in respect of which an immune response is modulated, include
microbial pathogens, bacteria, viruses, proteins, glycoproteins lipoproteins,
peptides,
glycopeptides, lipopeptides,
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toxoids, carbohydrates, and tumor-specific antigens. Mixtures of two or more
antigens may be employed.
Such peptides, glycopeptides or lipopeptides may include an amino acid
sequence corresponding to an antigenic determinant of HIV, Rubella virus,
Respiratory Syncytial Virus, Bordetella pertussis, Haemopliilus influenzae oi-
Streptocococcus pneumoniae, including those specific synthetic peptides shown
in
Table I below (SEQ ID NOS: 1 to 15) (The Tables appear at end of the
descriptive
text) or a functional analog thereof. The toxoid may be a pertussis toxoid
while the
protein may be influenza hemagglutinin or a parainfluenza virus subunit, such
as the
HN or F proteins of PIV-3.
In a particular aspect of the present invention, as claimed in the parent
application Serial No. 2,192,659, there is provided a kit for preparing an
immunogenic composition, comprising:
(a) means for containing a mineral salt adjuvant;
(b) means for containing at least one other adjuvant;
(c) means for containing at least one antigen; and
(d) means for combining the mineral salt adjuvant, at least one other adjuvant
and at
least one antigen to produce the immunogenic composition.
In a further aspect of the present invention as clainled herein, there is
provided
a compound comprising an antigen, including any of those referred to above,
covalently linked to a glycolipid analog, such as a glycosylamide, as well as
immunogenic compositions comprising the same for generating an immune response
in a host, including a human. The glycosylamide may have the formula I above.
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By covalently bonding an antigen to a glycolipid
analog, a discrete molecule is produced which exhibits a
surprisingly unexpected enhanced adjuvanting effect on
the antigen whi-ch is greater than the adjuvanting effect
attainable in the absence of such covalent bonding, as in
a mixture of the two components. A further enhanced
adjuvanting effect may be attained for such covalently-
bonded antigen by incorporating a mineral salt adjuvant
with such compounds.
The antigen may be .covalently linked to the
glycolipid analog at a carboxy or amino terminus or other
suitable site compatible with ..covalent linkage of the
antigen by, for example, a-cross-3.inker having a reactive
function, such as maleimidyl, succinimidyl, 2-
pyridyldithio, NI32, SH, and -CO-it8, where P.8 is -OH, N3, -
O-alkyl (C,-C,),-OCgFS, H, Br, or Cl.
Advantages of the present invention include:
(a) ease of fo-rmulation;
(b) effectiveness of adjuvanticity; and
(c) compatibility of antigens with the adjuvat3t
composition.
BRIEF DEHCRTPTION DE '{'HE DRAWrNGS
Figure 1 shows antibody responses to HIV peptides in
guinea pigs formulated with adjuvants according to one
embodiment of the invention;
Figure 2 shows antibody responses in guinea pigs to
a cocktail of HIV peptides formulated with adjuvants
according to an embodiment of the present invention;
Figure 3 shows haemagglutinin inhibition antibody
responses in mice to a Human parainfluenza virus subunit
vaccine formulated with adjuvants aecoxding to the
present invention;
Figure 4 shows virus neutralization antibody
responses to a FIuman parainfluenza virus subunit vapccine
formulated with adjuvants according to the present
invention;
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Figure 5 shows guinea pig immune responses t-o a
split influenza vaccine formulated with adjuvants
according to an embodiment of the present invention
against three influenza virus strains;
Figure 6 shows guinea pig immune responses
(determined by anti-PT ELISA) to pertussis toxoid
formulated with adjuvants according to an embodiment or
the present invention;
Figure 7 shows guinea pig immune responses
(determined by the CHO cell neutralizing assay)
(Grunstrom et al. 1985 J. Infec. Dis. 151:646-649) to
pertuairis toxoid ~ormulated with adjuvants according to
an embodiment of the present invention;
Figure 8 shows a guinea pig immune response to an
HIV peptide CLTB36 formulated with adjuvants or
conjugated to BAY R1005 according to an embodiment of the
present invention; and
Figure 9 shows guinea pig antibody responses to RIV
peptide (,CLTB 36) ~formulated with alum or conjugated to
BAY R3.005 according to an .embodiment of the present
invention.
MNE}2iP,I, DESCRIPTTON OF TRE INVBNTLON
It will be apparent to those 'skill.ed in the art,
that the various embodiments of the present invention
have many applications in the fields of medicine and, _n
particular, vaccination, diagnosis, generation of
immunological agents, and treatment of infections with
pathogens, including bacteria and viruses. A further
non-limiting discussion of such uses is further presented
below. As noted above, the present invention re?ates, in
one aspect, to adjuvant compositions useful for
modulating the immune response to an antigen.
Synthetic antigens, including vaccines, may be
prepared by chemically synthesizing peptides sharing
antigenic determinants with proteins, for example, of
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HIV-1, rubella virus, RSV, Haemophilus influenza.e type b,
Bordetella pertussis and Streptococcus pMgumoniae or
other antigens. These peptides, lipid derivatives of
such peptides as well as viral antigens or bacterial
5 antigens, may be used either individually or combined as
a cocktail, and formulated with synthetic adjuvants and
mineral salts to provide an immunogenic composition.
These compositions can be used to immunize mammals, for
example, by the intramuscular or parenteral routes, or by
10 delivery to mucosal surfaces using microparticles,
capsules, liposomes and targeting mol-ecules, such as
toxins and antibodies.
Reference will now be made in detail to the
presently preferred embodiments of the invention, which
together with the following Etamples, help to explain the
invention.
Antigen Selection
Several antigens were selected to exemplify the
present invention. Advances in biotechnology now,enabl.e
bacterial and viral antigens to be identified and
purified on a large scale. However, subunit or syathetic
vaccine candidates are sometimes of low immunogenicity,
due to their size (especially synthetic peptides) or the
lack of intrinsic immunostimulatory properties. Thus,
external additives are often required to enhance their
immunogenicity. Several antigens have been chosen which
are able to elicit strong TgG antibody responses in
adjuvants, such as CFA. The sel-ected antigens include
synthetic peptides (Table 1) sharing antigenic
determinants with the proteius of HiV-1, rubelia vi-xus
(RV), respiratory syncytial virus (RSV), . Haemophilus
influenzae type b (Hib), Bordetella pertussis and
Strentococcus pneumoniae, and the HN and F proteins f-rom =
parainfluenza virus 3(PfV3), pertussis toxoid ancl
chemically-disrupted influenza virus.
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8ynthetic Adjuvants
Synthetic adjuvants, such as glycolipid analogs
(Lockhoff et al., US Patent No. 4,855,283), lipopeptide
(Wiesmuller et al. (1989), Vaccine 7:29-33) and octadeyl
ester of aromatic amino acids (Moloney, et al., US Patent
No. 4,258,029) have been shown to act as adjuvants to
enhance the immunogenicity of viral and bacterial
antigens. Therefore, three adjuvants, namely N-palmityl-
S-(2,3-bis(palmityloxy)-(2RS)-propyl-[R]-cysteine (TPC,
Wiesmuller et al., Vaccine (1989) 8:29-33); N-(2-deoxy-2-
L-leucylamino-{3-D-glucopyranosyl)-N-octadecyldodecanamide
acetate (BAY R1005, O. Lockhoff, Angew. Chem. Int. Ed.
Engl. (1991) 30:1611-1620); and octadecyl-tyrosine_(OTH)
(Nixon-George et al. (1990), J. Immunology 144:4798-4802),
were selected as starting'molecules;for designing more
potent synthetic adjuvant compositions. These thr.ee
classes of synthetic adjuvants were synthesized and
characterized. The synthesis of the three classes of
adjuvants required less than ten steps of reaction. The
20. protocols for the adjuvant synthesis are well established
and reported in the literature. Scale-up production for
BAY R1005 and octadecyT-tyrosine would be' within ;the
skill of the art.
All three classes of synthetic adjuvant are
insoluble in water or aqueous buffer, such as phosphate
buffered saline (PBS)., They form a milky solution when
mixed with water or aqueous buffer. They are non-tox3c,
as judged by a lack of adverse reactions in mice injected
with 1 to 2 mg, and non-pyrogenic in a rabbit pyrogen
test. All three classes of synthetic adjuvant are very
stable in powdered form at -20*C and can be suspended
into aqueous buffer for long term storage at 4-C.
To analyse the effectiveness of the synthetic
adjuvants, 100 g of. synthetic peptides (Table 1)
containing known functional T-helper and B-cell epitopes
were used as antigens. Fifteen peptides, including
AMENDf,p SWET
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epitopes from HIV-1, RSV, Rubella virus, H. influenzae,
B. nertussis, and S. pneumoniae, were individually
injected intramuscularly into guinea pigs, either in the
presence of FCA, or alum (aluminium phosphate), or TPC,
or SAY R1005, or OTH or PBS. The effect of adjuvants on
antibody responses were evaluated using peptide-specific
ELISAs. As shown in Table 2 below, all peptides
emulsified in FCA elicited strong antibody responses,
whereas peptides in PBS =either elicited very low anti-
peptide titer or failed to elicit any detectable antibody
response. After three immunizations, the fifteen
peptides absorbed onto alum were capable of inducing
paptide-specific IgG antibody responses. Only two
peptides (RV-EP27 and PSP-AA) in the presence of BAY
R1005, failed to elicit significant antibody responses
after three immunizations. One of the peptides (RV-EP27)
that failed to elicit significant antibody responses in
BAY R1005, was highly immunogenic and induced neptide-
specific antibody response when TpC was used as adjuvant.
In most cases, both synthetic adjuvants, TPC and OTFI,
enhanced the immunogenicity of the peptide, but the
reactive titers were much lower than those obtained from
alum or CPA. These results are consistent with published
data reported by other workers, and demonstrate the
adjuvant potential of the synthetic adjuvants and -the
capacity of these pepti.des to be adjuvanted.
In one aspect, the present invention provides an
adjuvant composition for triodulating an immune response to
an antigen adminietered to a host, the -composition
comprising a mineral salt adjuvant and at least one other
adjuvant. To exemplify this aspect of the inveuti-on,
several HIV-1 peptides were used in guinea pig
immunogenicity studies. Pept3des were absorbed onto alum
first and then emulsified with the synthetic adjuvant
before injecting into different groups of guinea pigs.
Guinea pigs were also immunized eithAr with pepti,des
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absorbed onto alum, or peptides emulsified with synthetic
adjuvant, as controls. The antibody resporise results
obtained with the synthetic adjuvant BAY R1O05 are shown
in Fig. a.. No adverse reactions were seen in any of the
guinea pigs. It was surprisingly found, according to the
present invention, that alum (3 mg/mL) combined with a
synthetic adjuvant (BAY R1.005 (I mg/mL) )resulted in
increased anti-peptide antibody -responees after two
immunizations in comparison to those titers produced by
the same peptide using either synthetic adjuvant or
mineral salts alone as adjuvant. These studies thus
indicate that the immune r-eesponse to an antigen that has
already been enhanced by adsorption to alum under
standard condit ions can unexpectedly be further enhanced
by another adjuvant, according to the present invention.
The unexpected immune response enhancing capacity of
adjuvant compositions of the present invention was
further demonstrated for syntheti~c peptides RSV-P and RV-
EP27 (Table 1) . When these peptides were adjuvanted with
alum alone, antibody titres of 32000 JRSV-F) and 12800
(RV-EP27) were obtained (Table 3). Wb-en the aame
peptides were adjuvanted with BAY R1005, they were poorly
immunogenic. However, when these peptides were
adjuvanted with an adjuvant compo,sit3on comprising alum
and BAY R1005, antibody titres of 128, 000 (RSV-F) and
64000 (RV-EP27) were obtained. These results thus show
the capaCity of this adjuvant composition to enhance the
immune response to an antigen beyond that obtainable by
either individual adjuvant. In fact, an ammunrie response
was obtained that was more than the =sum of the immune
response to each of the adjuvants individually, i.e. a
synergistic effect was obtained.
To further aseess the adjuvant effect of the present
invention, the HIJ and F gly.coproteins from parainfl iuenza
virus 3(PZV3) were absorbed first onto alum and then
emulsified with BAY RI0o5 before injecting
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intraperitoneally {i.p.) to -CD1 mice. As.controls, the
same antigens were either absorbed onto alum, or
emulsified with BAY R:1005, or mixed with P$S and then
used to immunize i.p. groups of CD1 mice. 'No adverse
=
reactions were seen in any of the mice. lt was
surprisingly found that the primary antibody responses to =
HIN and F, were best with the alum/BAY R1005 adjuvant
composition. The functional antibody responses as
measured by haemagglutinin inhibition and virus
neutralization assays, revealed that mice immunized with
antigen formulated with an adjuvant composition of the
present invention generated higher antibody levels than
obtained with a single adjuvant (Figs. 3 and 4).
To determine whether the steps of formulation would
affect the adjuvant effects of the compositions of the
present invention, conanercially-available split flu
vaccine was first emulsified with BAY R1005 (1 mg/mL) and
then absorbed onto alum (3 mg/mL) before injecting
intraperitoneally (i.p.) into mice. As -contrals, the
same preparation was= either absorbed onto alum, or
emulsified with BAY R1005, or mixed with PBS and then was
used to immunize different groups of mice. Again, no
adverse reactions were: seen in any of the groups of mice.
The BAY R1005/alum coaibination adjuvant formulation.gav.e
the best anti-HA antibody responses (Fig. 5) against
various influenza strains.
The results obtained with the split flu vaccine
indicate that the sequence of steps used to formulate the
antigen with the different adjuvants is not material to
the adjuvanting effect obtained.
The adjuvant compositions of the present invention
were also able to modulate the immune response to
gluteraldehyde-inactivated pertussis toxoid. Thus,
guinea pigs were immunized with pertussis toxoid in alum
alone, BAY R1005 or BAY R1005 anti-PT and toxin
neutralizing antibody titres determined (Figures S and
CA 02625156 2008-04-10
I.5
7). The results presented indicate that the highest ;and
a synergistic) immurie response was obtain=ed when the
pertussis toxoid was for_--~lated with alum and BAY R1-005
according to the present invention.
Immuaogenicitiy of Peptides Covalently Liaked to Synthetic
Adjuvants
The task of providing a synthetic peptide as a self-
sufficient immunogen capable of eliciting both humoral
and cell-mediated inunune r.esponaes, is very challenging.
To determine whether a peptide antigen coval.ently linked
to an adjuvant can e:Licit both humoxal and cell-mediated
immune responses, peptide =CI,TB-36 was synthesized with
synthetic adjuvant BAY R1005 covalently linked at the N-
terminus. During the preparation of the CLTB-36
covalently linked wi'th BAY R1005, it may be desirable to
use Fmoc peptide synthesis chemistry and temporarily
protect reactive functional groups, for example, a].cohols
by t-butyl and acids by ester groups. Suitable
protection-deprotection conditions and protocols -are
described in Examp].es her-ein. The peptide-adjuvant
conjugate was purified by RP-HPLC and used to immunize
guinea pigs. These immunogenicity studies revealed that
CLTB-36 covalently linked to BAY R100=5 was as immunogeni-C
as CLTB-36 formulateci with (BAY RIOOS + alum) (Figures 8,
9 and Table 4). The anti-CLTB-36 antibody titer obtained
was about 3-fold and 20-fold higher than those elici:ed
either in the presence of BAY R1005 or alum, respectively
(Fig. S) . In addition, the peptide-adjuvant conjugate
(BAY-CI,T13-36) required fewer immunizations and less
antigen to elicit the same degree of anti-peptide
antibody responses as shown in Figure 9. These results
show that an antigen with a built-in immunomodulator can
induce strong immune responses.
Vaccine nrenaration and use
As indicated above, the present invention, in one
embodiment, provides adjuvant mixtures useful for
CA 02625156 2008-04-10
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formulating immunogenic compositions, suitable to be used as, for example,
vaccines.
The immunogenic composition elicits an immune response by the host to wllich
it is
administered including the production of antibodies by the host. The
immunogenic
compositions include at least one antigen in one embodiment. This antigen may
be an
inactivated pathogen or an antigenic fraction of a pathogen. The pathogen may
be, for
example, a virus, a bacterium or a parasite. The pathogen may be inactivated
by a
chemical agent, such as formaldehyde, glutaraldehyde, (3-propiolactone,
ethyleneimine and derivatives, or other compotulds. The pathogen may also be
inactivated by a physical agent, su.ch as UV radiation, gamma radiation, "heat
sliock"
and X-ray radiation.
An antigenic fraction of a;pathogen can be produced by means of chemical or
physical decomposition methods, followed, if desired, by separation of a
fraction by
means of chromatography, centrifugation and similar techniques. In general,
low
molecular components are then obtained which, although purified, may have low
immunogenicity. Alternatively, atitigens or haptens can be prepared by llleans
ol'
organic synthetic methods, or, in the case of, for example, polypeptides and
proteins,
by means of recombinant DNA methods.
Vaccines containing peptides are generally well known in the art, as
exemplified by U.S. Patents 4,601,903; 4,599,231; 4,599,230; and 4,596,792.
The use of peptides in vivc- may first require their chemical modification
since
the peptides themselves may not have a sufficiently long seruln and/or tissue
half-life
and/or sufficient immunogenicity.
For this purpose, the molecule of the invention may optionally be linked to a
carrier molecule. Many suitable linkages are known, e.g., using the side
chains of the
Tyr residues. Suitable carriers include, e.g.,
CA 02625156 2008-04-10
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keyhole limpet hemocyanin iKLH), serum albumin, purified
protein derivative of tube.culin (PPD), ovalumin, non-
protein carriers and many others.
in addition, it may be advantageous to modify the
peptides in order to impose a conformational restraint
upon them. This might be useful, for example, -to mimic
a naturally-occurring =conformation of the peptide in the
context of the native protein in order to opti-mize the
effector immune responses that are elicited.
Modified peptides are referred to herein as "peptide
analogs". The term "peptide analog" extends to any
functional chemical equivalent of a peptide characterized
by its increased atability and/or efficacy and
immunogenicity in vivo or in vitro in respect of the
practice of the invention. The term "peptide analog" is
also used herein to extend to any amino acid derivative
of the peptides as described herein. Repti-de -a.na?=ogs
contemplated herein are produced by procedures that
include, but are not limited to, modifications to side
chains, incorporation of unnatural amino acids and/or
their derivatives during peptide synthesis and the use of
cross-linkers and other methods which impose
conformational -constraint on the peptides or -their
analogs.
it will be apparent that the peptide9 etcoloyed
herein as antigens can be modified in a variety of
different ways without significantly affecting the
functionally important immunogenic behaviour thereof.
Possible modifi>cations to the peptide sequence may
include the following:
one or more individual amino acids can be
substituted by amino acids having comparable or similar
properties, thus:
V may be substituted by I;
T may be substituted by S;
K may be substitut.ed by R; or
CA 02625156 2008-04-10
. =õ
- = = . .
. . . = ,
. . . =~
18
L may be substituted by I, V or M.
One or more of the amino acids of peptides of the
invention can be replaced by a"retro-inverso" amino
acid; i.e., a bifunctional amine having a functionad,
group corresponding to an amino acid, as discursed in
published International application WO 91/13909.
one or more amino acids can be deleted.
Structural analogs mimicking the 3-dimensional
structure of the peptide can be ueed in place of the
lo peptide.
= Examples of side chain modifications contemplated by
the present invention include modification of amino
groups, such as by reductive alkylation by 'reaction with
an aldehyde followed by reduction with NaBH4; amida:ion
with methylacetimidate; acetylation , with acetic
anhydride; carbamylation of amino groups with 2, 4, 6,
trinitrobenzene sulfonic acid (TNBS); alkylation of amino
groups with succinic anhydride and tetrahydrophthalic
anhydride; and pyridoxylation of lysine with pyz=i=doxal-
5'-phosphate followed by reduction with NaBH4.
The guanidino group of arginine x.esidu+es may be
modified by the formation of heterocyclic condensation
products with reagents, such a's 2, 3-butanedione,
phenylglyoxal and glyoxal.
The carboxyl group may be modified by casbodiimide
activation via o-acylisourea formation followed by
subsequent derivatisation, for example, to a
corresponding amide.
Sulfhydryl groups may be modified by methods, such
as carboxymethylation with iodoacetic acid or
iodoacetamide; performic acid oxidation to cystei-- acid;
formation of mixed disulphides with other thiol
compounds; reaction with maleimide; maleic anhydri~de or
other substituted maleimide; formation of mercurial
derivatives using 4-chlorome-rcuribenZoat.e, 4-
chlor6mercuriphenylsulfonic acid, phenylmercury chlo+ri,de,
CA 02625156 2008-04-10
19
2-chloromercuri=c-4-nitrophenol and other mercurials;
carbamylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example,
oxidation with N-bromosu=inimide or alkylation of the
indole ring with 2-hydroxy-5-nitrobenxyl bromide or
sulphonyl halides. Tryosine residues may be altred by
nitration with tetranitromethane to form a 3-
nitrotyrosine derivative.
Modification of the imidazole ring of a histidine
residue may be accomplished by alkylation with iodoacetic
acid derivatives or N-carbethoxylation with
diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not
limited to, use of norleucine, 4-amino butyric acid, 4-
amino-3-hydroxy-5-phenylpentanoic acid, 6-arcinohexanoic
acid, t-butylglycine, norvaline, phenyiglycine,
ornithine, sarcosine, 4-amino-3-hydroxy-6-mehty3heptanoic
acid, 2-thienyl alanine and/or D-isomers of amino acids.
The immunogenic compositions may be prepared as
injectables, as liquid solutions or emuls3ons. The
antigens and immunogenic compositiions may be mixed with
physiologically acceptable carriers which are coyppatible
therewith. These may include water, saline, dextrose,
glycerol, ethanol and-combinations Lhereof. The vaccine
may further contain auxiliary substances, such as wetting
or emulsifying agents or pH buffering agents, to further
enhance their effectiveness. Vaccines may be
administered by injection subcutaneously or
intramuscularly.
Alternatively, the immunogenic compositions formed
according to the present invention, may be formulated and
delivered in a manner to evoke an immune response at
mucosal surfaces. Thus, the imm-tinogenia composition may
be administered to mucosal surfaces by, for example, the
nasal or oral (intragastric) routes. Aldternatively,
CA 02625156 2008-04-10
2,0
other modes of administration including suppositories may
be desirable. gor suppositories, binders and carriers
may include, for example, polyalkylem glycols and
triglycerides. Oral formulations may include normally
employed incipients, such as pharmaceutical grades of
saccharine, cellulose and magnasium =carbonar.e.
These -compositions may take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 1 to 9S4; of the
immunogenic compositions of the present invention.
The immunogenic compositions are administered in a
manner compatible with the dosage formulation, and in
such amount as to be therapeutically effective,
protective and immunogenic. The qua_ntity to h.e
administered depends on the subject to the immunized,
including, for example, the capaci-ty of the -subject's
immune system to synthesize antibodies, and if needed, to
produce a cell-mediabed immune response. P-recise amounts
of antigen and immunogenic composition to be administered
depend on the judgement of the practitioner. However,
suitable dosage ranges are readily determinabL.e by athose
skilled in the art and may be of the order of micrograms
to milligrams. Suitable regimes for initial
administration and booster doses ar.e al-so variable, but
may include an initial administration followed by
subsequent administrations. The dosage of the vaccine
may also depend on the route of administration and will
vary according to the size of the host.
The concentration of antigen in an immunogenic
composition according to the invention is in general i to
95%-. A vaccine which contains antligpnic material of only
one pathogen is a monovalent vaccine. Vaccines which
contain antigenic mat.erial of several pathogens are
combined vaccines and also belong to the pre$e:.t
invention. Such combined vaccines contain, for example,
material from various pathogens or from various strains
CA 02625156 2008-04-10
21
of the same pathogen, or from combinations of various
pathogens.
Immunoassays
in one embodiment, the adjuvant mixtures of the
present invention a-re useful for the production of
immunogenic compositions that can be used to generate
antigen-specific antibodies that are useful in the
specific identification of that antigen in an immunoassay
according to a diagnostic Embodiment. Such immunoassays
include enzyme-linked immunoaorbent assays I;sLISA), RiAs
and other non-enzyme linked antibody binding assays or
procedur-es known in the art. In Ei,I-SA assays, the
antigen-specific antibodies are immobilized onto a
selected surface; for =exampl.e, the wells of a polystyrene
microtiter plate. After washing to remove incompletely
adsorbed antibodies, a nonspecifiC protein, such as a
solution of bovine serum albumin (BSA) or casei.n, that is
known to be antigenically neutral with regard to the test
sample may be bound to the selected surface. This allows
for blocking of nonspeaific adsorption sites on the
immobilizing surface and thus reduces the background
caused by nonspecific bindings of antigens onto the
surface. The immobiaizing surface is then conta.:t-ed with
a sample, such as clinical or biologi=cal materials, to be
tested in a manner conducive to immune 7co=.plex
(antigen/antibody) formation. This may include -diluting
the sample with diluents, such as BSA, bovine gamma
globulin (Bt3G) and/or phosphate buffered saline
(PBS) /Tween. The sample in then allowed to incubate for
from about 2 to 4 hours, at temperatures such as of the
order of about 25' to 37~-C_ Following incubation, the
sample-contacted surface is washed to remove non-
imnunocomplexed material. The washing procedure may
include washing with aAsolution such as PBS/Tween, or a
borate buffer.
CA 02625156 2008-04-10
22
Following formation of specific immunocomplexes
between the antigen in the test sample and the bound
antigen-specific antibodies, and subsequent washing, the
occurrence, and even amount, of immunocomplex formation
may be determined by subjectingthe immunocompl,ex to a
second antibody having specificity for the antigen. To
provide detecting means, the second antibody may have an
associated activity, such as an enzymatic activity, that
will generate, for example, a ~col our development upon
1o incubating with an appropriate chromogenic substrate.
Quantification may then achieved by measu=ring -the degree
of colour generation using, for example, a visible
spectra spectrophotometer. 1n an additional embodimeat,
the present invention includes a diagnostic kit
comprising antigen-specific antibodies generated by
immunization of a host with immunogenic compositions
produced according to the present invention.
EXAMPLES
The above disclosure generally describesthe.present
invention. A more complete understanding .can be obtained
by reference to the following specific Examples. These
Examples are described solely for puxposes of
illustration and are not intended to limit the scope of
the invention. Changes in form and substitution of
equivalents are contemplated ascircumstances may suggest
or render expedient.. Although snecific terms have been
employed herein, such terms are intended in a descriptive
sense and not for purposes of limitations.
Example 1
This Example describes the preparation of synthetic
peptides.
Peptides (Table 1) were synthesi=z.ed using an ABk
430A peptide synthesizer and optimized t-Boc chemistry as
described by the manufacturer, then cleaved frcm the
resin by hydroflouric acid {HF} . The peptides were
purified by reversed-phase high pe,rforTnance liquid
CA 02625156 2008-04-10
23
chromatography (RP-HPLC) on a Vydac 4C4 semi-preparativ.e
column (1 x 30 cm) using a 10 to 5a% acetonitrile
gradient in 0.1k trifluoryl acetic acid ITFA) developed
over 40 minutes at a f3ow rate of 2 mL/min. Al1
synthetic peptides (Table 1) were >95% pure as judged by
analyti=cal HPLC. Am3.no acid composition analyses of
these peptides performed on a Waters Pico-Tag system were
in good agreement with their theoretical compositions.
F=xample 2
This Example describes the synthesis of adjuvants.
N-alkyl-N-g].ycosyl-.carboxamides (N-(2-L-l.eucine-
amino-2-deoxy-~-D-Slucopyranosyl)-I3-octadecyl-dodecanoic
acid amide, BAY R30,05 and its analogs) were prepared
following the process disclosed in Lockhoff et al., U.S.
Patent No. 4,855,283. BAY R14005 and its analogs can be
further purified by ion-exchange chromatography. The
octadeyl ester of an aromatic amino acid y(o3"H) was
prepared according to Moloney et al., US Patent No.
4,258,029. Fmoc-,S-(2,3-bis(palmitoy2oxy)-{2R]-propyl)-
[R]-cysteine using the ~coupling procedure of rmoc
chemistry into a growing peptide ch-ain and then acylated
at the N-terminus by a palmityl .group. TPC derivatives
can be either tripa?mityl-Cys-Ber-Ser-Asn-Ala JAd) or
tripalmityl-=Cys-Ser-Glu-Glu-G1u-,Glu (Ad-4), or
tripalmityi-Cys-Ser-Lye-Lys-Lys-Lys (Ad-2).
Example 3
This Exantple describes the formulation of adjuvantc.
with an antigen.
The adjuvant BAY RI005, OTH or TpC derivatives were
mixed with sterile distilled water or saline buffer using
for example a sonicator for two to thirty minutes. The
adjuvants were prepared on a laboratory sca3.e as 40tock
solutions of 10 mg/mL for BAY R1005, 3. mg/mL for OTA and
TPC derivatives. The synthetic adjuvant stock solution
was added to an antigen to produce 1 mg/mL BAY R1O05 or
200 g/mL OTH or TPC derivatives. The antigen may be
CA 02625156 2008-04-10
24
obtained by, for example, methods of peptide oynthesis
and recombinant techniques that are not explicitly
described in this disclosure but are amply reported in
the scientific literature and are within the scope of
those skilled in the art.
Example 4
This Bxample describes the preparation of adjuva=at
compositions and immunogenic compositions containing a
mineral salt adjuvant and at least one other adjuvant.
' A synthetic adjuvant may be prepared as described in
Example 2 and the mineral salt adjuvant may be clinical
grade aluminum phosphate obtainable from, for example,
Connaught Laboratories Limited, Toronto Canada.
The immunogenic composition was prepared by first
1.''s mixing BAY R1005 with sterile distilled water or saline
buffer using a sonicator for two to thirty minutes. Ten
mL of the desired amount (1 to 200 g per mL) of antigen
absorbed onto 3.1 mg per mL of A1P04 suspension for 1 to
24 hr, was added to 1 mL BAY R1005 stock solution, and
the solution was mixed gently for 30 minutss at room
temperature.
reparation of an HIV synthetic Aeptid.e wmmungcr.e-tlic
composition
A BAY R1005 stock solution of 10 mg/mL was prepared
as described above. Ten mL of the desired amount (20 ,to
200 g per mL) of HIV synthetic peptides (Table 1)
absorbed onto 3.1 mg per mL of A1B04 suspension for i to
24 hr, were then added to 1 mi, BAY R1a05 stock solution,
and the solution mixed gently for 30 minutes at room
temperature. The HIV synthetic peptide vaccine
formulation was stored at 4'C ftr long term storage. The
vaccine formulation was gently mixed for 10 minutes at
room temperature before injecting into hosts.
p eparation of a flu vaccin-e ,formu tion
A BAY R1005 stock solution of 20 mg/mL was .pr=epared
as described above. Ten mL solution containing twenty
CA 02625156 2008-04-10
human doses of flu split vaccine obtained comrnercially
from Pasteur Merieux SV, Lyon, France, were added into 1
mL BAY R1005 stock solution. The mixture was vortexed
for 10 minutes at room temperature. The emulsified flu
5 vaccine was then mixed with 3.1 mg per mL of AIPDe
suspension for 1 to 24 hr, and then the vaccine
formulation stored at 4'C.
Prenaration of RSV neptide vaccine formulation
A HAY R1.005 stock solution .of 1-0 mg/mL was prepared
10 as described above. Ten mL of the desired arnount t10D gg
per mL) of RSV synthetic peptides (Table 1) absorbed onto
3.1 mg per mL of AIPO, suspension f-or I to 24 hr, were
added to 1 mL BAY R1005 stock solution, and the solution
mixed gently for 30 minutes at room temperature. The RSV
15 synthetic peptide vaccine formulation was stored at 4 c.
Preparation of PIV3 vaccine formulaLion
A BAY Ri005 stock solution of 10 mg/mL was prepared
as described above. Ten mL of the desired amount (0.3 to
3 pg per mL) of PIV3 subunit vaccine .cont+ainin.g IM and F
20 proteins absorbed onto 3.1 mg per mL of AlPQ4 suspension
for 1 to 24 hr. The PXV3 subunit vaccine was prepared
according to published International paten&- application
WO 91/00104, assigned to the assignee he+eof. The alum-
absorbed PIV3 antigens were added to 1 mL BAY it1o85 -st-ock
25 solution, and the solution mixed gently for 3-0 minutes at
room temperature. The PIV3 vaccine formulation was
stored at 4'C.
Preparation of flu BRA vaccine forcnulation
A BAY R1005 stock solution of 10 mg/mL was prepared
as described above. Ten mL of the desired amount (0.5 to
10 g per mL) of flu BHA subunit vaccine were absorbed
onto 3.1 mg per mL of AlPo4 suspension for 1to 24 hr.
The flu HHA subunit vaccine prepared aecording to=Brand
and Skehel (Nature New Biol. 1972, 238:3-45-147), w*as
provided by Dr. D. Burt of Connaught Laboratories
Limited, Willowdale, Ont. Canada. The alum-absorbed flu
CA 02625156 2008-04-10
26
BHA was added to 1 mL BAY R1.005 stock solution, and the
solution mixed gently for 30 minutes at room temperature.
The flu BHA vaccine formulation was stored at 4'C.
Pre.o ration of pertu$sis toxoid vaccine formulation
A BAY R1005 stock solution of 10 mg/mL was prepared
as described above. Ten mL of the desired amount (1 to
20 pg per mL) of pertussis toxoid, prepared according to
Tan et al. U.S. Patent No. 4,997,915, assigned to the
assignee hereof, were absorbed onto 3.1 mg per mi, of
AIPOs suspension for 1 to 24 hr. The alum-absorbed
pertussis toxoid was added to 1 mL of BAY R10D5 stock
solution, and the solution mixed gently for 30 minutes at
room temperature. The pertussis toxoid vaccine
formulation was stored at 4',C.
Example 5
This Example describes the general immunization
protocol used for testing adjuvant compositions and
immunogenic compositions.
Guln-ea pic immunizations
Three guinea pigs were used. The animals were pre-bled
at day 0 and then injected IM with S00 AL of the
formulation containing the desired amount of antigen, 1.5
mg of A1PO4, and 5U0 g of immunodulators BAY R3.005 or
its derivatives on day 1, 14 and 29. Blood samples were
obtained on day 28, and animals were bled out on day 42.
The antibody titr.es were assayed for peptide-specific IgG
antibodies using an antigen specific enzyme-linked
immunosorbent assay tF.L'~*SA). The functional antibody
responses were measured using virus neutralization and/or
haemagglutination inhibition (I3AI) assays and, ~-or
pertussis toxoid, the ability of antisera to inhibit
induced CHO ce1.1 c3.ust.ering was determined.
Mouse imrnunizations
Three to five mice were used. The animals were pra-
bled at day 0 and then injected IP with 200 L of the
formulatiion containing the desired amount of antigen,
CA 02625156 2008-04-10
27
0.6 mg of A1PO4, and 200 g of adjuvant BAY R1005 or its derivatives on day 1,
14
and 29. Blood samples were obtained on day 28, and animals were bled out on
day
42. The antibody titres were assayed for peptide-specific IgG antibodies using
an
antigen specific enzyme-linked immunosorbent assay (ELISA). The functional
antibody responses were measured using virus neutralization and/or
haemagglutination inhibition (HAI) assays.
Example 6
This Example describes an analysis of the immune response to immunogenic
compositions.
Antigen-specific ELISAs
Microtiter wells (Nunc-Immunoplate, Nunc, Denmark) were coated with 200
ng of purified antigen (PT, HA, PIV3, HN and F) or 500 ng of individual
peptides in
50 L of coating buffer (15mM Na2CO3, 35 niM NaHCO3, pH 9.6) for 16 hr at room
temperature. The plates were then blocked with 0.1% (w/v) BSA in phosphate
buffer
saline (PBS) for 30 minutes at room temperature. Serially diluted antisera
were added
to the wells and incubated for 1 hr at room temperature. After removal of the
antisera,
the plates were washed five times with PBS containing 0.1 %(w/v) Tween*-20 and
0.1 % (w/v) BSA. F(ab')2 fragments from goat anti-rabbit, guinea pig, mouse,
or
human IgG antibodies conjugated to horseradish peroxidase (Jackson
ImmunoResearch Labs Inc., PA) were diluted (1/8,000) with wasliing bufrer, and
added onto the microtiter plates. After 1 hr incubation at room temperature,
the plates
were washed five times with the washing buffer. The plates were then developed
using the substrate tetramethylbenzidine (TMB) in H202 (ADI, Toronto). The
reaction
was stopped with 1N H2SO4 and the optical density was measured at 450 nm using
a
Titretek Multiskan II (Flow Labs., Virginia). Two irrelevant pertussis toxin
peptides
NAD-S1 (19 residues) and S3(123-154) (32 residues) were included as negative
controls in the
*Trade-mark
CA 02625156 2008-04-10
28
peptide-specific SLISAs. Assays were performed in
triplicate, and the reactive titer of each antisserum was
defined as the dilution consistently showing 2-fold
increase absorbance value over those obtained from the
negative controls.
HaeMagglutination inhibition (HAI) assays
The assay is based on the capacity of antisPra
capable of neutralizing either influenza virus or PIV3 to
agglutinate red blood cells of guinea pigs JPIV3 ) or
chicken (flu virus) . Red blood cells are added to t:he
wells of a 96-well microtitration plate containing serial
dilutions of antisera and a constant amount of virus.
Following incubation, HAI titer is read by determining
the degree of haemagglutinin inhibition. The lowest
dilution of antisera blocking haemag.glutination is the
endpoint titer. A reference satnple with a kno%m ?izLI is
run in parallel.
Virus neutralizatian assavs
The assay is based on the capacity of antiaera to
inhibit PIV3 growth in Vero cells. To Vero cells grown
on wells of a 96-well microtitration plate, seri.al
dilutions of antisera and a constant amount=of virus are
added. Following incubation, VN titer of each antisera
is read by the determination of 50%- inhibition of
cytopathic effect caused by PIV3. The lowAst dilution of
antisera blocking the virus cytopathic effect is the
endpoint titer. A reference sample with a known VN titer
is run in parallel.
HTV-1 virus neutralization assavs
The ability. of antisera to neutralize HIV-i was
determined in a syncytia (multi-nuclei giant ,cel3.$)
iahibition assay. Ten uL of serially diluted antiserum
was added to the wells of a 95-well tissue culture plate.
5- 10 X 103 HIV-1 infected -CEM cells (in 50 L) were
then added to each well. 7 x 10' uninfected MOLT-4 celXe
(in 50 ;tL) were then added to each well. The plates were
CA 02625156 2008-04-10
29
then incubated in a COz incubator overnight. 3n sampl-es
where no syncytia were formed 4i.e. functional, virus-
neutralizing antibodies were present in the antiserum),
these plates were incubated for a further 24 hours and
s then reexamined for syncytia formation. The number of
syncytia was then scored under an inverted microscope.
The lowest dilution of antisera blocking 90t of virus
syncytia formation is the endpoint titer. A reference
sample with a known virus neutralising titer was run in
parallel.
Example 7
This Example describes the protocol that can be used.
for the generation of antigen-specific T-ye3.Z lines by
immunogenic preparations of the present invention.
BALB/c (H-2a) mice purchased #rom Charles River
Animal F'a.rm (Montreal, Canada) are individually primed
subcutaneously with desired amount of amount of antigen
(1 to 100 jug) emulsified with either BAY R1005 or its
derivatives. The animals are boosted twice with the same
dose of immunogen at 3 week intervals. Ten days after
the last boost, spleens of immunized mice are removed.
Splenocytes are cultured at 6 x 3.05 cells per well in a
final volume of 200 L of RPMI 1640 medium (Flow Lab.)
supplemented with 10W heat-inactivated fetal calf serum
((3ibco), 2 mM L-glutamine (FZow Lab.), IU0 U/mL)
penicillin (Flow Lab.) and 5 x20-5 M 2-merca,ptoetbanol
(Sigma) in the presence of varying concentrations (1, 10
and 100 pg per mL) of antigen in 96-well plates (Nunc,
Denmark). Cultures are kept in a humidif ied incubator in
the presence of 5t- O0./air. Triplicate cultures are
performed for each concentration of antigen. Five days
later, 150 L of l0& rat concanavalin A culture
supernatant diluted in culture medium is added to the
microtiter plate wells as a source of Interleukin-2 {IL-
2) to expand antigen-specific T-cells. Six days later,
150 L of supernatant is removed from each mi.c-roculture,
CA 02625156 2008-04-10
and 150 EcL of fresh IL-2 containing culture supernatant
added to further expand and maintain the viability of the
antigen-specific T-cells. After a further 6 day-
incubation, the cells are washed three times, each time
5 with 200 L of culture medium.
Bach set of cultures is then stimulated with the
corresponding concentrations (1, 10 and 100 jcg per mL) of
the antigen in the presence of 2 x 105 irradiated (1,500
rad) BALB/c spleen cells in a final volume of 200 AL of
10 culture medium. Sixty EcL of supernatant are then removed
from each microculture. The supernatants from each
triplicate cultures set are pooled. All supematants are
assayed for IL-2, Interleukin-4 and Interfexon-gamma
(IFN-7), using murine IL-2 and IL-4 ELISA kits purchased
15 from Endogen Inc. (MA, USA) respectively. Assay of ZPN--y
can be done using a mouse IFN-7 ELISA kit supplied by
Genzyme Corporation (MA, USA). Test culture supernatants
can be assayed at 1 in 5 dilution according to the
manufacturers' instructions.
20 Example 8
This Example describes the covalent linkage of BAY
R1005 to a peptide.
N- (glutarylacylamido-2-deoxy-P-D-giucopyranasyl) -N-
alkyl-carboxamide which can be used to covalently link
25 BAY Ra.005 derivatives to either a peptide or protein was
synthesized as follows. To a solution of N-(2-amino-2-
deoxy-p-D-glucopyranosyl ) -N-alkyl-carboxamide =( 2 mmol ) in
dioxane (28 mL) was added di-isopropylethyl amine 18
mmol) and glutaric anhydride (12 mmol). The reaction
30 mixture was stirred overnight at room temperature under
argon. Ammonium hydroxide (28 mL) was added to the
mixture, and then it was stirred for an additional 18
hours. The product was formed as a precipitate which was
filtered, and then redissolved in a solution containing
water and t-butanol (2 : 1). The solution was then
acidified by the gradual addition of acetic acid (4 mL).
CA 02625156 2008-04-10
31
The solution was then lypholized to afford the product in
73 t yield, mass spectroscopic analyses {FAB-HRMS) of N-
(glutarylacylamido-2--deoxy-g-D-glucopyranosyl)-N-a?kyl-
carboxamide, C43 HB3 N2 08, calculated 755.6149; and found
755.6129.
Resin-bound side-chain protected CLTB-36 peptide was
synthesized using P-moc chemistry as follows. Two
hundred to 500 mg of resirn carrying the N-Fmoc protected
first amino acid residue was placed in a reaction vessel.
The resin was washed 4 times with DAIIr, then prewashed
with a 5 ot solution of piperidine in DMF (5 mL for 1
mixzute) and deprotected with a 5tt$ solution of piperidine
in DMF (10 mL for 9 minutes). The resin was then washed
with DMF (S times, 10 mL each). The peptide resin was
then coupled with 5 et_ruivalent of the desired Fcnoc-
protected ' amino acids activated with phosphonium
hexafluorophosphate esr.er (pfp.e) in the presence of DIEA
at room temperature for 2 to 3 hr and washed with DW 45
tim.es, 10 mL each). After the final DMF washing step, an
aliquot was taken for a ninhydrine test. if the test was
negative, one goes to step 1 for coupling of the next
amino acid. 1f the test was positiv.e or -sZightly
positive, the coupling and DMF washing steps v,werA
repeated.
The N-(gZutarylacylamido-2-deor.y-f3-D-
glucopyranosyl)-N-alkyl-:carboxamide was conjugated to
synthetic peptide CL1B-3-6 as folZows: N-
Hydroxysuccinimide 1335 ug, 1.12 eq.) was added. to a
solution containing the glycolipid prepared as -descri3aed
above (2.0 mg) and dicyclohexylcarbodiamide di,ssolved in
dichloromethane (2 mY,). The resulting mixture was then
stirred for 4 hours at room temperature. Tre
dicyclohexylurea was filtered and the resulting filtrate
was evaporated to dryness. The dried active ester was
redissolved in DMF (0.1 mL) and was added into a pepticle-
resin containing side-chain protected CL,TS-36 at molar
CA 02625156 2008-04-10
32
ratio of 1.1 to 1. The reaction mixture was stirred
overnight at room temperature. The excess reagents were
filtered and the peptide-resin was washed with 4 x 10 mL
of dichloromethane. After drying, the peptide-resin was
treated with TPA to release lipidated CLTB-36 from resin.
The crude lipidated peptide was further purified by i3PLC
using a C4 Vydac column U x 35 cm) with a acetonitrile
gradient from 20 to 60% developed within 40 minutes at a
flow rate 2 mL/minute. Amino acid composition analyses
of lipidated CLTB-36 performed on a Waters Pico-Tag
system were in good agreement with their theoretical
compositions, and the presence of fatty acids were
confirmed by t3C analyses of the acid hydrolysate.
The N-(glutarylacylamido-2-deoxy-P-D-glucopyranosyl-
i5 N-alkyl-carboxamide was conjugatedto flu recombinant NP
protein as follows:
N-I;ydroxysuccinimide (335 ug, 1.12 eq.) was added to a
solution containing the glycolipid prepared as described
above (2.0 mg) and dicyclohexylcarbodiamide dissolved in
dichloromethane ( 2 mL). The resuiting mixture was then
stirred for 4 hours at room temperature. The
dicyclohexylurea was filtered and the resulting filtrate
was evaporated to dryness. The dried active ester 52.25
mg) was redissolved in DMF (0.1 mI,) and was added irnto a
protein solution (2 mg rNPI protein dissolved in 2 mD of
25 mM phosphate bu#fer, pE 7.8) at a molar ratio of 20 :
1. The reaction mixture was stirred overnight at 4'C,
and then was dialysed against 4 x 4i, of the phosphate
buffer. The incorporation of N-(glutarylacylamido-2-
deoxy-B-D-glu.copyranosyl)-N-alkyl-,carboxamide to flu
recombinant NP was determined and confirmed using gas
chromatography (GC) for lipid analyses of the acid
hydrolysate (6 N of HCI for 2 hr at 110*C) of the
modified protein.
The present invention has been exemplified by
reference to particular examples and embodiments.
CA 02625156 2008-04-10
33
Numerous adaptations, variations and modifications may be
made to the particular examples and embodiments without
departing from the essence of the invention which is
defined in the claims.
S24MARY OF M DISCL(7SURE
In summary of this disclosure, the present invention
provides novel adjuvant compositions which are able to
elicit an enhanced immune response against antigens and
novel compounds comprising antigen covalently bonded to
:L0 a glycolipid analog. Modifications are possible within
the scope of the invention.
CA 02625156 2008-04-10
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Table 1
SYNTSETIC PEPTIDES USED IN TSE STUDIES
PEPTIDES ANlINO ACID SEQDBNCE SBQ ID NO:
HIV peptidea =
CLTS-36 GP1tEP GPGRAFYTTlCN 1
CLTB-70 GPKB IY'IGFGAFRTTGR 2
CLTB-72 GPKBP IR143tGGRAPUTIGK 3
CLTB-74 GPEF=TPpRDB'VDRFYiCN3'RKSITICGPGbtVIYATGQ 4
CLTS-76 GPXEPBRDYVDRF'YIQ]TRQSTPIGLGQALY3'TRG 5
P24E-GP41C GPKSPFRD1fVDRF]CKSLISESQNQQEXNEQELLELDiCWAfi 6
RSV peptides
RSV-F QSYSI4S1I1CE8VLAYAVQLPLPLYGViI)TP 7
RSV-FIIDa PIVHICQSCSISIQIETVIEFQQ 8
8SV-Gl TKQRQNIC8PS1MaTDFHFSYlaIdFVPG 9
RSV-GZ CSDIlQPTI.'WAIClGtIPNlXPG1tT 10
peztnssia peptide
69K-P1 PQPGPQPPQPPQPQPEAPAPQPPC 11
Hib peptides
HIBPl-5E NQITSALSTQQEFRDL~.'Yi+PSXDtG;WSLQD32F.C 12
TBP2-3 TV('IaCTYQVEAC'C6NLSYVlCFGM 13
Rubella virus peptide
RV-SP27 PDPGDI,ZT$YINIIIYTGNQQSRWGi-GSPD]MPDWiASP'V:cQRESP 14
S. pneuaoaiae pepticle
PSP-AA IKBIDEBESED7lA10rGFRAP I5
CA 02625156 2008-04-10
~ 4)
~ a o d o o d o d~, a 47
= ~ o o O o p o 0 0 o Q Q Q w sl' m V~ t0 ~T OD Ci d~ ~i b T+ ~'~i 'l W
10 N w tfl 1D N R1 %D 1n
~ ~ rt N .4 N
co + y
~
iu
is ~ -~ ~
~
aCi 43 a o o 0 0 0 0 o p p o 0 0
N N V~ N V~ N m O O O O O O O Q Q E
-.q
W U r~1 10 r~1 ~0 th N tD N+D ~' ~N 10 W 7r '~i O
ri ~i ++! ri to 10 . i ~
H W
m. . . .
+
3.41 ~ L 34
~
0
r'1 w 01 o Nf o O f+l f9 O O O o O O d d O -e~i
)?3C1 nl + Ip p O m O O e9 +++ tf1 O O O O O O O O i1
V~'.7 p m f9 OD IA m m W O N 7N m Uf 1t1 Y) aM 94
1
O W N M V1 N N Y1 Pf O 44 N (4N to LL
w rA 41 [~ m v N V V' rl tl frl M (q O W
0! b + > 1]~ 0
TI ~~1 + :11 4)
41 N
O 0
i," , 'lJ
r 14
+ a~ 0)
t) ~ m V W
E y ~ O tD O 1R w 1D p d p d d 1+1 O m O ,.~f ~, m df
41 + O tD O Uf 1D ~D O O d O O N+ O IO o s7 '~' ~O
.C + O 1D iq O rl rl O m U7 O m f'~+ If+ M tD +1
r r w r w = r r r r r b d 1~
d~o r 1h ~- 01 N (~ rl m cV m N e+f ~-1 ~ LI p~
ro rt ~ Pf a rV t9 N1 ~ rt rn d~ ~ m a
+ m to
y ~ o r+ w
ms o
b+
~
o 0 0 o p o 0 0 0 0 0 0 o p d C+ Cai x
O O O O O O O O O O O O O O -r1 0
O ~ O 1t1 tfI O O p V~ t0 O O O 11 O uf tn CL iJ N
Q' l0 N 1!1 tf1 1D N m tA m N O N N (i G
1Q N N l 'C7
a
41 W M . - I ri f'C C / N rl N rI N .-1 rl ri C) U Q)
N ri cn w +c %o r1 Nr ,t m V X m
~ -H N a
O C! +i
O O nl O P'S O O O O O O O d O O
~ d O Nl O P7 O O O O O O O O O O i1 iA.17
m ~ a V~ Y1 M Iff W tA 1q !'I 10 V N ED V N w -~
bf tV N CO rl CI .{ ri 0
~ GI
i
W 5+
i 0 i y~.7 y
Rt t Ci
a1 1 -r+ a
4.1 asIG Ls f..
a o
' i N w a . ' ~-r i o
0 tn ~ o N a io w c~ v+ ri at 2
V o M P r l~ [r L7 N rl N 4 ei f+1
-rl l, f il, w U{J ca r- (L :
J.7 al 10 m W 1 1 1 1 W Ck i N 3
sL E~ E4 F= ~ ~=r > > 9 > ~ cn ae a a
n~ ~+ ~ t~ u r.'~i u a a a~ a tt x o H a .. õ ,
CA 02625156 2008-04-10
36
TABLE 3
Guinea pigs antibody responses to synthetic pepti-des
formulated with different adjuvants
Peptide-8pecaific Aatibody Titrel
---------------------------------------
Adjuvants used in the itamunizata.on
Peptide BAY R30,05
Immunogensl alum BAY R100S + alum
RSV-F 32,000 3,850 128,000
RSV-FND2 7,800 10,000 25"600
RV-EP27 12,800 80o 64,~O00
Mean titer of three guinea pige received three
inj ections .
All peptide immunogens are used l00 jig of peptide
per dose formulated with different adjuvants.
CA 02625156 2008-04-10
37
T.A83,B 4
]119TIBODY RSSPONSB TO CIaTB-36 pOBad=T8D II$ DIFF888ZTT I1DJtTVABTS
Average t3.tQre against
------------------------ --------------------
CLTS-36 [O;T13-56' Virus
Syncytin-
Antiaera3 Dose (20 g) formuetion
Cattiaea piga alum 6,400 1,833 <10
B71Y R1005 62,500 4,333 35
alvm + BAY &2005 36,S00 6,250 24
SAY ft1005 conjugat=d 20,833 S,833 23
100 g in a1mn~ 32,000 12,500 32
Groups of guinea pigs (n = 3) were iatmunized with 20 }g of
CLTB-36 emulsified in BAY R1005 and/or absorbed onto alnm at
day 1. Booster immunizationa were at 2, 4 and 6 weeks with the
same antigens. Blood samples were collected every 2 Weeke after
the third injection. Sera were aaalyzed by peptide-specific
ELISAs, virus neutralization and syncytia=~ozmation inhibition
assays.
s Peptide CLTB-56 is the B-cell epitope of 4=TB-36 and has amino
acid sequence of 1Q1QlKRSBIt',tpGRhrr1TKN (S$Q 3D NO: 16) .
A 100 jeg of CLTB-36 absorbed onto alum was used.
