U.S. patent application number 10/834616 was filed with the patent office on 2005-01-06 for adjuvant for vaccine composition.
This patent application is currently assigned to Aventis Pasteur SA. Invention is credited to Haensler, Jean, Ronco, Jorge, Trannoy, Emmanuelle.
Application Number | 20050002952 10/834616 |
Document ID | / |
Family ID | 9468771 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050002952 |
Kind Code |
A1 |
Haensler, Jean ; et
al. |
January 6, 2005 |
Adjuvant for vaccine composition
Abstract
An amphipathic compound including a sterol-derived lipophilic
grouping bound to a cationic grouping for use as an adjuvant in the
delivery of a vaccine composition. In a particular embodiment, the
lipophilic grouping is a cholesterol derivative and the cationic
grouping is a quaternary ammonium or a protonatable amine. A
vaccine composition including one or more antigens with at least
one amphipathic compound having a sterol-derived lipophilic
grouping bound to a cationic grouping, is also disclosed.
Inventors: |
Haensler, Jean; (St.
Genis-les-Ollieres, FR) ; Trannoy, Emmanuelle; (Lyon,
FR) ; Ronco, Jorge; (Lyon, FR) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Aventis Pasteur SA
|
Family ID: |
9468771 |
Appl. No.: |
10/834616 |
Filed: |
April 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10834616 |
Apr 29, 2004 |
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08836576 |
Sep 9, 1997 |
|
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6780421 |
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08836576 |
Sep 9, 1997 |
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PCT/FR95/01495 |
Nov 14, 1995 |
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Current U.S.
Class: |
424/185.1 ;
424/450; 514/171 |
Current CPC
Class: |
A61K 2039/55555
20130101; A61K 39/145 20130101; A61K 39/12 20130101; A61K 9/0043
20130101; C12N 7/00 20130101; A61K 2039/545 20130101; A61P 31/16
20180101; A61P 31/12 20180101; A61K 9/1272 20130101; A61K 47/186
20130101; A61K 2039/543 20130101; A61K 39/39 20130101; A61K 9/0019
20130101; C12N 2760/16134 20130101 |
Class at
Publication: |
424/185.1 ;
424/450; 514/171 |
International
Class: |
A61K 039/00; A61K
009/127; A61K 031/56 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 14, 1994 |
FR |
94/13606 |
Claims
We claim:
1. A composition comprising at least one proteinaceous antigen and
an adjuvanting amount of
3-.beta.-(N-(N'-N'-dimethylaminoethane)carbamoy1) cholesterol.
2. The vaccine composition of claim 1 further comprising a neutral
lipid.
3. The composition of claim 2, wherein the ratio of the said
neutral lipid to f 3-B-(N-(N'-N'-dimethylaminoethane)carbamoy1)
cholesterol is greater than 1:4.
4. The composition of claim 2, wherein said neutral lipid is
ioleolyphosphatidylethanolamine or dioleoylphosphatidylcholine.
5. The composition of claim 1, wherein said
3-.beta.-(N-(N'-N'-dimethylami- noethane)carbamoy1) cholesterol is
dispersed in an aqueous environment in the form of liposomes.
6. The composition of claim 1, wherein said
3-.beta.-(N-(N'-N'-dimethlamin- oethane)carbamoy1) cholesterol
takes the form of liposomes including at least one antigen.
7. A method of inducing an immune response in a mammal comprising
administering the vaccine composition of claim1 to a mammal.
8. The method of claim 7 wherein said immune response is a humoral
immune response.
9. The method of claim 7, wherein said immune response is a
cytotoxic T cell response.
10. The method of claim 7, wherein said immune response is a
T.sub.1-type immune response.
11. The method of claim 7, wherein said antigen is an influenza
virus haemagglutinin.
12. The method of claim 7 wherein said composition is administered
by the subcutaneous route.
13. The method of claim 7, wherein said composition is administered
by the mucosal route.
14. The method of claim 7, wherein said composition is administered
by the intranasal route.
15. A method of inducing an immune response in a mammal comprising
administering the vaccine composition of. claim 2 to a mammal.
16. The method of claim 5, wherein said antigen is an influenza
virus haemagglutinin.
17. A method of inducing an immune response in a mammal comprising
administering the vaccine composition of claim 3 to a mammal.
18. The method of claim 17, wherein said antigen is an influenza
virus haemagglutinin.
19. A method of inducing an immune response in a mammal comprising
administering the vaccine composition of claim 4 to a mammal.
20. The method of claim 19, wherein said antigen is an influenza
virus haemagglutinin.
Description
[0001] This is a continuation of application Ser. No. 08/836,576,
filed Sep. 9, 1997, which is a U.S. National Phase application of
international application PCT/FR95/01495, filed Nov. 14, 1995.
These applications are hereby incorporated by reference in their
entirety.
[0002] The present invention relates to the field of vaccine
compositions. More especially, the invention relates to new
adjuvants used for increasing the immunogenicity of vaccine
compositions.
[0003] There are a large number of antigens which, when injected
into animals, will cause a production of antibodies which are
specific to them. One of the principles of vaccination is to
stimulate antibody production by the body of a man or an animal by
administering chosen antigens thereto. The antibodies thus produced
will then enable the body to defend itself against a subsequent
infection. However, some antigens do not bring about sufficient
stimulation of the immune system when they are administered alone.
Hence an adjuvant which will enable the body's immune response to
be increased has to be added to them in order to obtain a
sufficient amount of antibody to be protective.
[0004] Among known adjuvants, aluminum hydroxide and aluminum
phosphate, which are customarily used in human vaccines, may be
mentioned. However, these compounds do not possess an adjuvant
property with respect to all antigens. In particular, they do not
enable the immunogenicity of influenza vaccine to be increased.
[0005] There is hence a need to be able to have adjuvants at one's
disposal which enable the immunogenicity of the antigens
administered in a vaccine composition to be increased, without any
risk of toxicity.
[0006] In addition, it is advantageous to have adjuvants at one's
disposal which are capable of inducing an immune response that
manifests itself in a production of secretory antibodies, such as
IgAs.
[0007] To this end, the invention provides for the use of an
amphipathic compound comprising a lipophilic group derived from a
sterol linked to a cationic group, for the production of a vaccine
composition.
[0008] A subject of the invention is also the use of such an
amphipathic compound as adjuvant in the administration of a
vaccine.
[0009] A subject of the invention is also a vaccine composition
comprising at least one antigen, characterized in that it
comprises, in addition, at least one amphipathic compound
possessing a lipophilic group derived from a sterol linked to a
cationic group.
[0010] A further subject of the invention is a product containing
at least one antigen and one amphipathic compound comprising a
lipophilic group derived from a sterol linked to a cationic group,
as a combination product for use simultaneously, separately or
staggered over time in vaccination.
[0011] Another subject of the invention is a method for inducing an
immune response in a mammal, consisting in administering at least
one antigen to the mammal, characterized in that it consists in
administering, in addition, at least one amphipathic compound
comprising a lipophilic group derived from a sterol linked to a
cationic group.
[0012] For the purposes of the present invention, the term
amphipathic denotes a compound which possesses both a hydrophobic
portion and a hydrophilic portion.
[0013] Among sterol derivatives capable of yielding the compounds
according to the invention, cholesterol, phytosterol and ergosterol
may be mentioned. Cholesterol derivatives are especially
suitable.
[0014] The cationic group of the amphipathic compounds according to
the invention can consist of a quaternary ammonium or an amine
which can be protonated.
[0015] The lipophilic group is attached to the cationic group by
means of an ester, ether, amide or carbamoyl link, among which
ester, amide and carbamoyl links have the advantage that they can
be hydrolysed in the cell.
[0016] The linkage between the 2 groups is preferably effected via
a spacer arm consisting of a branched or unbranched alkyl chain
comprising from 1 to 20 carbon atoms.
[0017] Among the compounds suitable for the purposes of the
invention, there may be mentioned those mentioned in Patent U.S.
Pat. No. 5,283,185 and, in particular:
[0018] cholesteryl-3.beta.-carboxamidoethylenetrimethyl-ammonium
iodide,
[0019] cholesteryl-3.beta.-carboxamidoethylenamine,
[0020] cholesteryl-3.beta.-oxysuccinamidoethylene-trimethylammonium
iodide,
[0021]
3.beta.-[N-(N',N'-dimethylaminoethane)carbamoy1]cholesterol,
[0022] 3.beta.-[N-(polyethylenimine)carbamoyl]cholesterol among
which 3.beta.-[N-(N',N'-dimethylaminoethane)carbamoy1]cholesterol,
cholesterol is especially advantageous.
[0023] The amphipathic compounds according to the invention may be
obtained by condensation between a sterol derivative and a compound
containing a cationic group, according to one of the methods
described in "Advanced Organic Chemistry" Part B: Reactions and
Synthesis (F. A. Carey and R. J. Sundberg--Plenum Publishing
Corp.). More especially, some of the compounds according to the
invention may be prepared according to the methods described in
Patent U.S. Pat. No. 5,283,185.
[0024] The amphipathic compounds obtained in alcoholic solution can
then be dispersed in water or in an aqueous buffer, and can yield a
suspension of micelles or of liposomes. Advantageously, the
amphipathic compounds of the invention are combined with a neutral
lipid such as a phospholipid, for example
dioleoylphosphatidylethanolamine (DOPE) or
dioleoylphosphatidylcholine (DOPC). This combination causes the
amphipathic compounds according to the invention to organize
themselves in the form of liposomes rather than micelles during the
phase of dispersion in an aqueous environment. The molar proportion
of neutral lipid combined with the amphipathic compounds is
preferably greater than 20%.
[0025] The products obtained according to the invention did not
give rise to any acute toxicity reaction when they were inoculated
into mice.
[0026] The antigen used to induce a protective immune response
consists of any antigen customarily used in a vaccine composition,
either alone or in combination with another antigen.
[0027] In particular, the amphipathic compounds according to the
invention prove to be good immunoadjuvants when they are combined
with the influenza virus vaccine comprising, in particular: the HA
protein which is a haemagglutinin located at the surface of the
influenza virus envelope, the NP protein which is a capsid
nucleoprotein linked to the viral RNA and an M protein or protein
"matrix" of the envelope.
[0028] Combination between the antigen whose immunogenicity it is
desired to increase and the micellar or liposomal suspension of
amphipathic compounds takes place spontaneously by hydrophobic and
electrostatic interaction on mixing the constituents.
[0029] The vaccine compositions obtained possess good stability.
However, the liposomal suspension appears preferable to the
micellar suspension.
[0030] In addition, the liposomal suspension can be sterilized by
filtration and lyophilized.
[0031] It is obvious that it is possible to add ingredients
traditionally used in vaccines, such as water, physiological saline
or a buffer substance, to the vaccine compositions obtained.
[0032] Administration of the vaccine compositions obtained
according to the invention may be performed by all the routes
customarily used for the administration of vaccines, and in
particular by the subcutaneous or intranasal route. It is also
possible to choose a different route for the primary immunization
and the booster immunization.
[0033] It is possible to administer separately the composition
comprising the antigen and the composition containing the
amphipathic compounds according to the invention; however, the
administration of a liposomal composition of amphipathic compounds
according to the invention combined with the antigen makes it
possible not only to increase the humoral type immune response, but
also to induce specific cytotoxic T lymphocytes.
[0034] A better understanding of the invention will be gained on
reading the non-limiting examples which follow, reference being
made to the figures.
[0035] FIG. 1 illustrates the reaction scheme for the production of
DC chol. FIGS. 2 to 6 depict the results of tests of induction of
cytotoxic T lymphocytes for each group of mice mentioned in Example
8.
[0036] FIG. 7 depicts the results mentioned in Example 11.
[0037] FIGS. 8 and 9 depict the results mentioned in Example
12.
[0038] FIGS. 10 and 11 depict the results mentioned in Example
13.
[0039] FIGS. 12 and 13 depict the results mentioned in Example
14.
[0040] FIGS. 14 and 15 depict the results mentioned in Example
15.
[0041] FIG. 16 depicts the results mentioned in Example 16.
EXAMPLE 1
Synthesis of 3.beta.-[N-(N',N'-dimethylaminoethane)-
carbamoy1]cholesterol (DC chol)
[0042] DC chol is synthesized by reacting cholesteryl chloroformate
and N,N-dimethylethylenediamine according to the scheme in FIG. 1,
as described in the paper by X. Gao and L. Huang (BBRC 179 (1):
280-285).
[0043] A solution of cholesteryl chloroformate (2.25 g, 5 mmol in 5
ml of dry chloroform) is added dropwise to an excess of a solution
of N,N-dimethylethylenediamine (2 ml, 18.2 mmol, in 3 ml of dry
chloroform) at 0.degree. C. After extraction of the solvent by
evaporation, the residue is purified by 2 successive
recrystallizations in absolute ethanol at 4.degree. C., and dried
under vacuum. 0.545 g of DC chol is thereby obtained in the form of
a white powder. The structure of the compound was verified by NMR
and mass spectrometry. The results obtained are in agreement with
the data published in the paper cited above.
EXAMPLE 2
Preparation of a Vaccine Composition against Influenza Virus from a
Micellar Suspension of DC chol 2.3 mg
[0044] 30 mg of DC chol obtained according to Example 1 are
dissolved in 100 .mu.l of ethanol. 75 .mu.l of the solution thereby
obtained are injected via a Hamilton syringe into 3 ml of water
kept stirring at 45.degree. C. After a further 5 minutes of
stirring at 45.degree. C., the micellar suspension obtained is
mixed with 200 .mu.l of monovalent vaccine against influenza virus
(Strain A/Singapore) comprising, in particular, as antigens: the
haemagglutinin HA, nucleoprotein NP and M protein.
[0045] The mixture obtained is divided into vaccine doses of 0.3
ml. Each dose comprises 5 .mu.g of HA and 2.3 mg of DC chol.
EXAMPLE 3
Preparation of a Vaccine Composition against Influenza Virus from a
Micellar Suspension of DC chol 0.45 mg.
[0046] The procedure is as in Example 2, starting from 6 mg of DC
chol obtained according to Example 1.
EXAMPLE 4
Preparation of a Suspension of Liposomes Consisting of DC chol
Cmbined with dioleoylphosphatidylethanolamine (DOPE)
[0047] 18 mg of dioleoylphosphatidylethanolamine (DOPE) and 4.5 mg
of DC chol obtained according to EXAMPLE 1, which is dissolved in 3
ml of chloroform, are mixed.
[0048] The chloroform is evaporated off under vacuum to form a
lipid film, which is subjected to dessication under vacuum and then
resuspended in 3 ml of water.
[0049] After hydration for 24 hours at 4.degree. C., the dispersion
is subjected to sonication for 5 to 10 minutes in an ultrasound
bath (Laboratory Supplies - Hicksville - N.Y.) to form
liposomes.
[0050] This suspension is stable for at least 6 months at 4.degree.
C.
EXAMPLE 5
Preparation of a Suspension of Liposomes Consisting of DC chol
Combined with dioleoylphosphatidylcholine (DOPC)
[0051] The procedure is as in Example 4, replacing the 18 mg of
DOPE by 18 mg of dioleoylphosphatidylcholine (DOPC).
[0052] A liposomal suspension which is stable for at least 6 months
at 4.degree. C. is obtained.
EXAMPLE 6
Preparation of a Vaccine Composition Against Influenza Virus from a
DC chol/DOPE Liposomal Suspension
[0053] 3 ml of a lipsomal suspension obtained according to Example
4 are mixed with 0.2 ml of strain A/Singapore monovalent influenza
vaccine containing the equivalent of 50.mu.g of the antigen
consisting of the haemagglutinin HA.
[0054] The mixture obtained is then divided into 10 vaccine doses
of 0.3 ml, each containing 5 .mu.g of HA and 0.45 mg of DC
chol.
EXAMPLE 7
Preparation of a Vaccine Composition Against Influenza Virus from a
DC chol/DOPC Liposomal Suspension
[0055] 3 ml of a liposomal suspension obtained according to Example
5 are mixed with 0.2 ml of strain A/Singapore monovalent influenza
vaccine.
[0056] The mixture obtained is then divided into 10 vaccine doses
of 0.3 ml, each containing 5 .mu.g of HA and 0.45 mg of DC
chol.
[0057] EXAMPLE 8:
Immunization
[0058] 5 groups of 4 Balb/c mice are immunized by 3 subcutaneous
injections performed on DO, D21 and D36 with the following vaccine
compositions:
[0059] Group A: 0.3 ml of diluted strain A/Singapore monovalent
influenza vaccine containing 5 .mu.g of HA in 0.3 ml of PBS,
[0060] Group B: 0.3 ml of vaccine composition obtained according to
Example 2,
[0061] Group C: 0.3 ml of vaccine composition obtained according to
Example 3,
[0062] Group D: 0.3 ml of vaccine composition obtained according to
Example 6,
[0063] Group E: 0.3 ml of vaccine composition obtained according to
Example 7.
EXAMPLE 9
Assay for Anti-HA Antibodies
[0064] In order to perform the assays for neutralizing antibodies,
the sera of mice are sampled on D21, D36 and D51, and the titration
of anti-HA antibodies is performed by means of the technique of
inhibition of influenza virus-induced haemagglutination.
[0065] Table 1 below illustrates the results obtained for each
group of mice after one injection, and Table 2 the results after 2
injections.
1TABLE 1 1
[0066]
2TABLE 2 2
[0067] The titres of neutralizing antibodies in the mouse sera are
presented in the form of log2 of the highest dilution inducing
haemagglutination inhibition.
[0068] These results show clearly the adjuvant role played by DC
chol. In effect, the level of anti-HA antibodies is markedly higher
for the groups of mice which have received DC chol in comparison
with mice which have received the vaccine without adjuvant (Group
A).
[0069] It is important to note that the levels of neutralizing
antibodies in Groups B, C, D and E are greater than the level of
neutralizing antibodies in Group A, even after a single injection
of the different vaccine compositions. These titres increase
slightly more and stabilize after the second injection. The results
obtained after the 3.sub.rd injection are substantially equal to
those for the 2.sub.nd injection (not shown).
[0070] Tests carried out with a trivalent influenza vaccine
comprising strain A/Texas, strain B/Panama and strain A/Beijing
also demonstrated the adjuvant power of DC chol.
EXAMPLE 10
Demonstration of the Induction of Cytotoxic T Cells
[0071] The spleen cells of the mice of each of the groups mentioned
in Example 8 are removed on D51.
[0072] These effector cells are restimulated in vitro in the
presence of syngeneic stimulatory cells infected with the strain
A/Singapore virus corresponding to the vaccine tested. The specific
cytotoxic function of these stimulated cells is demonstrated using
as target cells the P815 mastocytoma line sensitized by incubation
with a peptide which is a CTL epitope of the haemagglutinin of the
virus (specific response against the HA) or with a peptide which is
a CTL epitope of the nucleoprotein of the virus (specific response
against the NP). Non-specific lysis (background) is measured on
P815 cells which are unsensitized or sensitized with a peptide
which is a CTL epitope of the HIV virus (V3MN).
[0073] Lysis of the target cells is measured by a radioactive
technique based on loading of the target cells with Cr-51 and on
release of this radioelement during cell lysis.
[0074] The results presented in FIGS. 2 to 6, which illustrate the
percentage cytotoxicity in terms of the ratio of effector cells to
target cells for each of the groups of mice tested, show that it is
especially advantageous to use a liposomal composition of DC chol
especially in combination with a neutral lipid, since this
composition makes it possible to induce specific cytotoxic T
lymphocytes in addition to the humoral type immune response
obtained as a result of the adjuvant action of the DC chol.
EXAMPLE 11
Study of the Immune Response as a Function of the Dose of DC chol
Used
[0075] Vaccine compositions of 300 .mu.l are prepared, each
containing strain A/Singapore monovalent influenza vaccine and
containing either 15 or 5 .mu.g of HA, in combination with DC
chol/DOPC liposomes at a variable concentration. The preparation of
liposomal suspension is carried out in a manner similar to that of
Example 4, replacing the 18 mg of dioleoylphosphatidylethanolamine
(DOPE) by 13.5 mg of dioleoylphosphatidylcholine (DOPC) and taking
up the lipid film obtained with an amount of water which varies
according to the desired DC chol concentration.
[0076] The vaccine compositions obtained are injected into groups
of 5 8- to 10-week-old female Balb/c mice on D0 and D28. The sera
are sampled on D42 and the anti-HA antibodies are assayed by the
agglutination inhibition (HAI) technique.
[0077] The following compositions are tested in this way:
[0078] 15 .mu.g of HA+0 .mu.g of DC chol
[0079] 15 .mu.g of HA+400 .mu.g of DC chol
[0080] 5 .mu.g of HA+0 .mu.g of DC chol
[0081] 5 .mu.g of HA+25 .mu.g of DC chol.
[0082] 5 .mu.g of HA+50 .mu.g of DC chol
[0083] 5 .mu.g of HA+100 .mu.g of DC chol
[0084] 5 .mu.g of HA+200 .mu.g of DC chol
[0085] 5 .mu.g of HA+300 .mu.g of DC chol
[0086] 5 .mu.g of HA+400 .mu.g of DC chol
[0087] The results expressed in the form of log base 2 of the
highest dilution inducing haemagglutination inhibition are depicted
in FIG. 7, and show that only above 100 .mu.g of DC chol/dose is
the maximum benefit of the adjuvant effect of DC chol obtained.
EXAMPLE 12
Demonstration of the Induction of Different Antibody Isotypes
[0088] A comparative study is carried out of the antibodies induced
in 3 groups of 5 8- to 10-week-old female BALB/c adult mice which
have received 2 subcutaneous injections performed on D0 and D28
with the following vaccine compositions:
[0089] 1st group: 0.3 ml of diluted strain A/Singapore monovalent
influenza vaccine containing 5 .mu.g of HA in 0.3 ml of PBS,
[0090] 2nd group: 0.3 ml of diluted strain A/Singapore monovalent
influenza vaccine containing 5 .mu.g of HA with 0.1mg of aluminum
hydroxide as adjuvant,
[0091] 3rd group: 0.3 ml of vaccine composition obtained according
to Example 11 and containing 5 .mu.g of HA and 400 .mu.g of DC chol
in combination with DOPC.
[0092] The sera of the mice are sampled on D28 and on D42 and the
IgGl and IgG2 produced are assayed by the ELISA technique.
[0093] The results obtained in the primary response are depicted in
FIG. 8 and those obtained in the secondary response are depicted in
FIG. 9.
[0094] These results illustrate the adjuvant effect of DC chol,
both in the primary response and in the secondary response, in
comparison to aluminum hydroxide which is an adjuvant of the prior
art. It will be noted, in addition, that there is a large increase
in the IgG2a produced, signifying that DC chol acts by promoting
the development of TH1 type lymphocytes.
EXAMPLE 13
Demonstration of the Action of DC chol in Elderly Mice
[0095] The same experiment is carried out exactly as in Example 12
but, in this instance, the mice tested are 20 to 22-month-old mice
which are more difficult to stimulate.
[0096] The results obtained in the primary response are depicted in
FIG. 10 and those for the secondary response in FIG. 11.
[0097] Here too, DC chol has an adjuvant effect both for the
primary response and for the secondary response, and in particular
with respect to the induction of IgG2a antibodies.
EXAMPLE 14
HAI Levels in Adult Mice and in Elderly Mice
[0098] In the tests carried out in Examples 12 and 13, the
neutralizing antibodies are determined in the immunized mice by the
haemagglutination inhibition (HAI) test.
[0099] The results obtained in the primary response are illustrated
in FIG. 12 and those for the secondary response in FIG. 13. The
titres obtained, expressed in the form of log base 2 of the highest
dilution inhibiting haemagglutination, shows clearly the adjuvant
role of the DC chol/DOPC liposomes both in adult mice and in
elderly mice.
EXAMPLE 15
Intranasal Immunization
[0100] Vaccine compositions are prepared as described in Example
11, in this instance of 50 .mu.l comprising 5 .mu.g of HA (in the
form of strain A/Singapore monovalent influenza vaccine) combined
with 200 .mu.g of DC chol present in the form of DC chol/DOPC (in a
mass ratio of 1 to 4) liposomes.
[0101] Two groups of 4 BALB/c mice are immunized intranasally twice
with an interval of 4weeks.
[0102] The 1st group of mice (G.sub.1) receives 50 .mu.l of the
vaccine composition containing 200 .mu.g of DC chol, whereas the
second group (G.sub.2) receives 50 .mu.l of the same monovalent
influenza vaccine but without adjuvant. The immune responses of
each group are analysed by ELISA assay of the sera sampled 3 weeks
after the booster immunization.
[0103] The results, expressed in the form of log base 10 of the
ELISA titre, are depicted in FIG. 14 as regards the serum IgG and
in FIG. 15 as regards the serum IgA.
[0104] It will thus be noted that, in a protocol of strict mucosal
administration, the DC chol/DOPC liposomes enable the local and
general immune responses to be increased by a factor of at least
2.
[0105] The adjuvant role performed by the DC chol/DOPC liposomes in
the general immune responses was also observed when, instead of
measuring the IgG and IgA titres by ELISA, the anti-HA antibody
titres were measured by HAI; the increase in the local immune
response was also observed in an ELISPOT count of the number of
cells secreting IgG and the number of cells secreting IgA in the
lungs of the immunized mice, and also in the determination of the
ratio of the level of specific IgG (or of IgA) to the level of
total IgG (or of IgA, respectively) measured by ELISA in the
nasopharyngeal lavage fluids of the immunized mice.
EXAMPLE 16
Immunization by Combined Subcutaneous and Intranasal
Administration
[0106] Two groups of 4 BALB/c mice are immunized twice with an
interval of 4 weeks with, on each occasion, the following vaccine
compositions:
[0107] Group 3 (G.sub.3): a vaccine composition of 300 .mu.l
injected subcutaneously comprising 4 .mu.g of HA (in the form of
strain A/Singapore monovalent influenza vaccine) combined with 6
.mu.g of DC chol (in the form of DC chol/DOPC liposomes), and
[0108] a vaccine composition of 50 .mu.l administered intranasally
comprising 0.25 .mu.g of HA (in the form of strain A/Singapore
monovalent influenza vaccine) combined with 6 .mu.g of DC chol (in
the form of DC chol/DOPC liposomes),
[0109] Group 4 (G.sub.4):--a vaccine composition of 300 .mu.l
injected subcutaneously comprising 4 .mu.g of HA combined with 200
.mu.g of DC chol, and
[0110] a vaccine composition of 50 .mu.l administered intranasally
comprising 0.25 .mu.g of HA combined with 200 .mu.g of DC chol.
[0111] The sera of the immunized mice are sampled 3 weeks after the
booster administration and, for each mouse, the level of
neutralizing antibodies is assayed by the HAI haemagglutination
inhibition technique.
[0112] The results obtained, expressed in the form of log base 2 of
the titre, are depicted in FIG. 16, and show that, in a protocol of
mixed administration combining the subcutaneous and intranasal
routes, DC chol has an adjuvant effect, and that the increase in
the does of DC chol from 6 to 200 .mu.g enables the immune response
to be increased.
[0113] An identical dose effect could be observed when, instead of
measuring the level of anti-HA antibodies by the HAI technique, the
serum IgG was measured by the ELISA technique of the number of
cells secreting IgG and the number of cells secreting IgA was
measured in the lungs of the immunized mice (by the ELISPOT
technique), or the ratio of the amounts of specific IgG (or IgA) to
the amounts of total IgG (or IgA, respectively) was measured in the
nasopharyngeal lavage fluids of the immunized mice.
[0114] Results similar to those described in Examples 11 to 16 were
obtained by replacing DOPC by DOPE, as well as by varying the DC
chol/neutral lipid ratio, maintaining the molar proportion of
neutral lipid combined with DC chol at not less than 20%.
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