U.S. patent application number 12/992512 was filed with the patent office on 2011-03-24 for compositions comprising liposomes, an antigen, a polynucleotide and a carrier comprising a continuous phase of a hydrophobic substance.
This patent application is currently assigned to Immunovaccine Technologies Inc.. Invention is credited to Antar Fuentes-Ortega, Mohan Karkada, Lisa Diana MacDonald, Marc Mansour, Leeladhar Sammatur, Genevieve Mary Weir.
Application Number | 20110070298 12/992512 |
Document ID | / |
Family ID | 41397660 |
Filed Date | 2011-03-24 |
United States Patent
Application |
20110070298 |
Kind Code |
A1 |
Mansour; Marc ; et
al. |
March 24, 2011 |
Compositions Comprising Liposomes, An Antigen, A Polynucleotide and
A Carrier Comprising a Continuous Phase of a Hydrophobic
Substance
Abstract
The invention provides a composition comprising: an antigen;
liposomes; a polyI:C polynucleotide; and a carrier comprising a
continuous phase of a hydrophobic substance. Methods for making and
using the compositions are also provided.
Inventors: |
Mansour; Marc; (Halifax,
CA) ; Sammatur; Leeladhar; (Halifax, CA) ;
MacDonald; Lisa Diana; (Halifax, CA) ; Karkada;
Mohan; (Beechville, CA) ; Weir; Genevieve Mary;
(Dartmouth, CA) ; Fuentes-Ortega; Antar;
(Dartmouth, CA) |
Assignee: |
Immunovaccine Technologies
Inc.
Halifax
CA
|
Family ID: |
41397660 |
Appl. No.: |
12/992512 |
Filed: |
May 22, 2009 |
PCT Filed: |
May 22, 2009 |
PCT NO: |
PCT/CA2009/000692 |
371 Date: |
November 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61059043 |
Jun 5, 2008 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/184.1; 424/186.1; 424/210.1 |
Current CPC
Class: |
A61K 39/0007 20130101;
A61K 2039/55505 20130101; A61K 39/12 20130101; A61K 2039/55566
20130101; A61P 35/00 20180101; A61K 9/127 20130101; C12N 2710/20034
20130101; Y02A 50/464 20180101; A61P 31/16 20180101; A61K 47/06
20130101; Y02A 50/386 20180101; A61K 31/785 20130101; C12N
2760/16134 20130101; A61K 2039/55544 20130101; A61P 25/28 20180101;
Y02A 50/394 20180101; A61K 2039/55555 20130101; A61K 2039/575
20130101; A61K 9/19 20130101; A61K 39/00 20130101; Y02A 50/39
20180101; Y02A 50/388 20180101; A61K 9/10 20130101; Y02A 50/30
20180101; A61K 2039/55561 20130101; A61K 9/107 20130101; A61P 31/20
20180101; Y02A 50/466 20180101; A61K 39/39 20130101 |
Class at
Publication: |
424/450 ;
424/210.1; 424/184.1; 424/186.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61P 31/04 20060101 A61P031/04; A61P 31/10 20060101
A61P031/10; A61P 31/12 20060101 A61P031/12; A61P 33/00 20060101
A61P033/00; A61P 35/00 20060101 A61P035/00; A61P 31/16 20060101
A61P031/16; A61P 25/28 20060101 A61P025/28; A61K 39/145 20060101
A61K039/145; A61K 39/00 20060101 A61K039/00; A61K 39/12 20060101
A61K039/12 |
Claims
1. A composition comprising: (a) an antigen; (b) liposomes; (c) a
polyI:C polynucleotide; and (d) a carrier comprising a continuous
phase of a hydrophobic substance.
2. The composition according to claim 1, wherein the polyI:C
polynucleotide comprises RNA or DNA.
3. The composition according to claim 1, wherein the polyI:C
polynucleotide comprises RNA and DNA.
4. The composition according to claim 1, wherein the polyI:C
polynucleotide is a homopolymer or a heteropolymer.
5. The composition according to claim 1, wherein the polyI:C
polynucleotide comprises a homopolymeric polyI:C polynucleotide and
a heteropolymeric polyI:C polynucleotide.
6. A method for making a composition, said method comprising
combining, in any order: a) an antigen; (b) liposomes; (c) a
polyI:C polynucleotide; and (d) a carrier comprising a continuous
phase of a hydrophobic substance.
7. The method according to claim 6, wherein said antigen is
encapsulated in said liposomes.
8. The method according to claim 6, wherein said polyI:C
polynucleotide is encapsulated in said liposomes.
9. The method according to claim 6, wherein said polyI:C
polynucleotide is added outside said liposomes.
10. A composition prepared according to the method of claim 6.
11. A method comprising administering the composition according to
claim 1 to a subject in need thereof.
12. The method according to claim 11, which is a method for
inducing an antibody response and/or cell-mediated immune response
to said antigen in said subject.
13. The method according to claim 11, which is a method for the
treatment and/or prevention of a disease caused by a bacteria, a
virus, a fungus, a parasite, an allergen or a tumor cell that
expresses the antigen.
14. The method according to claim 13, wherein the treatment and/or
prevention comprises inducing an antibody and/or a cell mediated
immune response to the antigen in the subject, wherein the subject
has or is at risk of developing a viral infection.
15. The method according to claim 14, wherein the viral infection
is an influenza virus infection.
16. The method according to claim 13, wherein the treatment and/or
prevention comprises inducing an antibody and/or a cell mediated
immune response to the antigen in the subject, wherein the subject
has or is at risk of developing cancer.
17. The method according to claim 11, which is a method for the
treatment and/or prevention of a neurodegenerative disease, wherein
the neurodegenerative disease is associated with expression of the
antigen.
18. The method according to claim 17, wherein the neurodegenerative
disease is Alzheimer's disease.
19. The method according to claim 11, wherein the composition
induces an immune response in the subject that is at least
1.5.times. higher relative to a response induced by a control
composition.
20. The method according to claim 19, wherein the composition
induces an immune response in the subject that is at least 5.times.
higher relative to a response induced by a control composition.
21. The method according to claim 11, wherein the composition is
administered via a route that is nasal, oropharyngeal, ocular,
oral, rectal, sublingual, genitourinary mucosa, intranasal,
oropharyngeal, intratracheal, intrapulmonary, transdermal,
transpulmonary, intraarterial, intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous or submucosal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority from
U.S. Provisional Patent Application No. 61/059,043, filed Jun. 5,
2008.
FIELD OF THE INVENTION
[0002] The present application relates compositions comprising
liposomes, an antigen, a polyI:C polynucleotide and a carrier
comprising a continuous phase of a hydrophobic substance, and their
use.
BACKGROUND OF THE INVENTION
[0003] Conventional vaccines may comprise an antigen, an adjuvant
and a pharmaceutically acceptable carrier. It is known that a
polyI:C polynucleotide may be useful as an adjuvant. It is also
known that liposomes may be useful in vaccine compositions (see
Applicants' issued U.S. Pat. No. 6,793,923). However, to
Applicants' knowledge, the art does not teach or suggest combining
an antigen, a polyI:C polynucleotide, liposomes and a hydrophobic
carrier in a vaccine composition.
SUMMARY OF THE INVENTION
[0004] Applicants have now discovered that a composition comprising
an antigen, a polyI:C polynucleotide, liposomes and a carrier
comprising a continuous phase of a hydrophobic substance may
provide surprisingly higher antibody titers and a higher percentage
of activated or memory CD8+ T cells than either conventional
vaccine compositions containing polyI:C polynucleotides in an
aqueous carrier, or compositions comprising liposomes, a
hydrophobic carrier and an alum adjuvant.
[0005] Accordingly, in one aspect, the invention provides a
composition comprising: (a) an antigen; (b) liposomes; (c) a
polyI:C polynucleotide; and (d) a carrier comprising a continuous
phase of a hydrophobic substance.
[0006] In another aspect, the invention provides a method for
making a composition, said method comprising combining, in any
order: (a) an antigen; (b) liposomes; (c) a polyI:C polynucleotide;
and (d) a carrier comprising a continuous phase of a hydrophobic
substance. In an embodiment, the antigen is encapsulated in the
liposomes. In an embodiment, the polyI:C polynucleotide is
encapsulated in the liposomes.
[0007] In another aspect, the invention provides a composition
prepared according to the methods described above.
[0008] In another aspect, the invention provides a method
comprising administering a composition as described above to a
subject. In an embodiment, the method is a method for inducing an
antibody response or cell-mediated immune response to the antigen
in the subject.
[0009] Other aspects and features of the present invention will
become apparent to those of ordinary skill in the art upon review
of the following description of specific embodiments of the
invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0010] In the figures, which illustrate embodiments of the
invention by way of example only:
[0011] FIG. 1 is a graph showing the results of vaccination of
three groups of mice (n=9 or 10) as follows: Group 1 mice were
vaccinated with 1 microgram rHA and 4 micrograms polyI:C in a 30
microliter dose formulated as a liposome/polyI:C/hydrophobic
carrier vaccine (Vaccine B, the invention). Group 2 mice were
treated with Vaccine A comprising 1 microgram rHA and 60 micrograms
alum in a 30 microliter dose of liposome/alum/hydrophobic carrier
formulation. Group 3 mice were vaccinated with 1 microgram rHA and
60 micrograms alum per 30 microliter dose of control alum vaccine.
Humoral immune responses were measured by ELISA as described
herein. For each treatment group, the log 10 values of the endpoint
antibody titers were averaged and standard deviations calculated
for each time point. P values were calculated using the student T
test.
[0012] FIG. 2 is a graph showing the results of vaccination of two
groups of mice (n=9 or 10) as follows: Group 1 mice were vaccinated
with 1 microgram rHA and 4 micrograms polyI:C in a 30 microliter
dose formulated as a liposome/polyI:C/hydrophobic carrier vaccine
(Vaccine B, the invention). Group 2 mice were treated with 1
microgram rHA and 4 micrograms polyI:C per 30 microliter dose of
control polyI:C vaccine. Humoral immune responses were measured by
ELISA as described herein. For each treatment group, the log 10
values of the endpoint antibody titers were averaged and standard
deviations calculated for each time point. P values were calculated
using the student T test.
[0013] FIG. 3 is a graph showing the results of vaccination of two
groups of mice (n=8 or 9) as follows: Group 1 mice were vaccinated
with a single dose of 1 microgram rHA and 10 micrograms polyI:C in
a 50 microliter dose formulated as a lyophilized
liposome/polyI:C/hydrophobic carrier vaccine (Vaccine C, the
invention). Group 2 mice were treated with 1 microgram rHA and 100
micrograms alum per 50 microliter dose of control alum vaccine;
mice were boosted 21 days post-vaccination. Humoral immune
responses were measured by ELISA as described herein. For each
treatment group, the log 10 values of the endpoint antibody titers
were averaged and standard deviations calculated for each time
point.
[0014] FIG. 4. Enhanced anti-rHA antibody responses following
vaccination with rHA antigen formulated in a liposome/polyI:C/oil
carrier vaccine. Two groups of mice (n=9 or 10) were vaccinated as
follows: Group 1 mice were vaccinated with 1 microgram rHA and 4
micrograms polyI:C in a 30 microliter dose formulated as a
liposome/polyI:C/hydrophobic carrier vaccine (Vaccine B, the
invention). Group 2 mice were treated with Vaccine A, 1 microgram
rHA and 60 micrograms alum in a 30 microliter dose of
liposome/alum/hydrophobic carrier formulation. Humoral immune
responses were measured by ELISA as described herein. For each
treatment group, the log 10 values of the endpoint antibody titers
were averaged and standard deviations calculated for each time
point. P values were calculated using the student T test.
[0015] FIG. 5. Enhanced anti-rHA antibody responses following
vaccination with rHA antigen formulated in a liposome/polyI:C/oil
carrier vaccine. Two groups of mice (n=9 or 10) were vaccinated as
follows: Group 1 mice were vaccinated with 1 microgram rHA and 4
micrograms polyI:C in a 30 microliter dose formulated as a
liposome/polyI:C/hydrophobic carrier vaccine (Vaccine B, the
invention). Group 2 mice were treated with 1 microgram rHA and 4
micrograms polyI:C per 30 microliter dose of control polyI:C
vaccine. Humoral immune responses were measured by ELISA as
described herein. For each treatment group, the log 10 values of
the endpoint antibody titers were averaged and standard deviations
calculated for each time point. P values were calculated using the
student T test.
[0016] FIG. 6. Enhanced anti-rHA antibody responses following
vaccination with rHA antigen formulated in a lyophilized
liposome/polyI:C/oil carrier vaccine. Two groups of mice (n=9 or
10) were immunized as follows: Group 1 mice were vaccinated with a
single dose of 1.5 micrograms rHA and 12.5 micrograms polyI:C in a
50 microliter dose formulated as a lyophilized
liposome/polyI:C/hydrophobic carrier vaccine (Vaccine D, the
invention). Group 2 mice were treated with 1.5 micrograms rHA and
100 micrograms alum per 50 microliter dose of control alum vaccine;
mice were boosted 28 days (week 4) post-vaccination. Humoral immune
responses were measured by ELISA as described herein. For each
treatment group, the log 10 values of the endpoint antibody titers
were averaged and standard deviations calculated for each time
point. P values were calculated using the Student T test.
[0017] FIG. 7. Number of antigen-specific CD8 cells within a
CD8-positive T cell population following vaccination. Three groups
of BALB/c mice (n=4) were vaccinated as follows: Group 1 mice were
vaccinated with 1.5 micrograms of rHA and 12.5 micrograms of
RNA-based polyI:C adjuvant in a 50 microliter dose formulated as
lyophilized liposome/polyI:C/hydrophobic carrier vaccine (Vaccine
D, invention) intramuscularly. Group 2 mice were vaccinated with 50
microliters of Vaccine D subcutaneously. Group 3 mice were
vaccinated with 1.5 micrograms of rHA and 100 micrograms of Imject
Alum adjuvant in 50 microliters of 50 millimolar phosphate buffer
(pH 7.0) intramuscularly. All vaccines were given once without
boosting. Antigen-specific CD8+ T cells were detected twenty-two
days after vaccination in the splenocytes of animals using
tri-colour flow cytometric analysis. Cells were stained with
anti-CD8.beta.-APC, anti-CD19-FITC and a PE-pentamer specific for
H2-Dd bearing the immunodominant epitope of rHA, I9L. Results are
expressed as average percentage of pentamer positive cells in a
population of CD8.beta.-positive/CD19-negative cell population,
+/-standard deviation. The background staining detected in the
splenocytes isolated from naive cells was subtracted. *p=<0.025,
**p=<0.005, as compared to Group 3.
[0018] FIG. 8. Hemagglutination inhibition (HAI) titers following a
single vaccination against rHA formulated in the invention. One
group of mice and one group of rabbits (n=5) were vaccinated as
follows: The group of mice were vaccinated with 0.5 micrograms rHA
and 12 micrograms polyI:C in a 50 microliter dose formulated as a
lyophilized liposome/polyI:C/hydrophobic carrier vaccine (Vaccine
E, the invention). The group of rabbits were treated with Vaccine F
(the invention), 2 microgram rHA and 50 micrograms polyI:C in a 200
microliter dose of lyophilized liposome/polyI:C/hydrophobic carrier
formulation. Humoral immune responses were measured by
hemagglutination inhibition assay, as described herein; before
vaccination (pre-vaccination) and at 4 (rabbits) or 5 (mice) weeks
afterwards. For each animal group, the log 10 values of the HAI
titers were averaged and standard deviation calculated.
[0019] FIG. 9. Enhanced anti-.beta.-amyloid antibody responses
following vaccination with a mixture of .beta.-amyloid and F21E
peptides formulated in a liposome/polyI:C/oil carrier vaccine. Two
groups of mice (n=9) were vaccinated as follows: Group 1 mice were
vaccinated with 10 micrograms .beta.-amyloid, 20 micrograms F21E
and 200 micrograms alum in a 100 microliter dose formulated as a
liposome/alum/hydrophobic carrier vaccine (Vaccine G). Group 2 mice
were treated with 10 micrograms .beta.-amyloid, 20 micrograms F21E
and 10 micrograms polyI:C per 100 microliter dose formulated as
liposome/poly:IC/hydrophobic carrier (Vaccine H, the invention).
Humoral immune responses were measured by ELISA as described
herein. For each treatment group, the log 10 values of the endpoint
antibody titers were averaged and standard deviation calculated for
each time point. P values were calculated using the student T
test.
[0020] FIG. 10. Vaccines formulated in a
liposome/polyI:C/hydrophobic carrier formulation are capable of
raising cellular and humoral immune responses. Two groups of mice
(n=5) were vaccinated as follows: Group 1 mice were vaccinated with
0.5 micrograms rHA and 12 micrograms polyI:C in a 50 microliter
dose formulated as a lyophilized liposome/polyI:C
(high)/hydrophobic carrier vaccine (Vaccine E, the invention).
Group 2 mice were treated with 0.5 micrograms rHA and 2.5
micrograms polyI:C per 50 microliter dose formulated as lyophilized
liposome/polyI:C (low)/hydrophobic carrier (Vaccine I, the
invention). Indicators of humoral (IgG1) and cellular (IgG2A)
immune responses were measured by ELISA as described herein. For
each treatment group, the log 10 values of the endpoint antibody
titers were averaged and standard deviations calculated for each
time point.
[0021] FIG. 11 is a graph showing the average tumor volume of
C57BL/6 mice implanted with HPV16 E7 expressing C3 cells and
vaccinated eight days later as follows: Group 1 mice were
vaccinated with 100 microliters containing 15 micrograms of FP
antigen and 150 micrograms of RNA-based polyI:C formulated in an
emulsion with hydrophobic carrier (Control Emulsion vaccine). Group
2 mice were vaccinated with 100 microliters containing 15
micrograms of FP antigen and 150 micrograms of polyI:C formulated
in liposome/PolyI:C/hydrophobic carrier (Vaccine K, invention).
Group 3 mice received 100 microliters of PBS only. All groups
contained eight mice. Tumor size was measured once a week for five
weeks after implantation. FIG. 11 shows the average tumor volume
calculated for each group +/-SEM. P values were calculated for
Group 1 and Group 2 using Students' T test, *p=<0.1,
**p=<0.05.
[0022] FIG. 12 is a graph showing the average tumor volume of
C57BL/6 mice implanted with HPV16 E7 expressing C3 cells and
vaccinated five days later as follows: Group 1 mice received 100
microliters containing 10 micrograms of FP antigen and 20
micrograms of DNA based polyI:C formulated in
liposome/PolyI:C/hydrophobic carrier (Vaccine L, invention). Group
2 mice received 50 microliters containing 10 micrograms of FP
antigen and 20 micrograms of DNA based polyI:C formulated in
lyophilized liposome/PolyI:C/hydrophobic carrier (Vaccine M,
invention). Group 3 mice received 50 microliters containing 10
micrograms of FP antigen formulated in lyophilized
liposome/hydrophobic carrier (Adjuvant control). Group 4 mice
received 100 microliters of PBS only. All groups contained ten (10)
mice. Tumor size was measured once a week for five weeks after
implantation. FIG. 12 shows the average tumor volume calculated for
each group +/-SEM. P values were calculated for Group 2 and Group 3
using Students' T test, *p=<0.05.
[0023] FIG. 13. Enhanced anti-rHA cellular response following
vaccination with rHA antigen formulated in a lyophilized
liposome/polyI:C/oil carrier vaccine. Two groups of mice (n=9 or
10) were immunized as follows: Group 1 mice were vaccinated with a
single dose of 1.5 micrograms rHA and 12.5 micrograms polyI:C in a
50 microliter dose formulated as a lyophilized
liposome/polyI:C/hydrophobic carrier vaccine (Vaccine D, the
invention). Group 2 mice were treated with 1.5 micrograms rHA and
100 micrograms alum per 50 microliter dose of control alum vaccine;
mice were boosted 28 days (week 4) post-vaccination. Antigen
specific cellular responses were measured by pentamer staining of
CD8+ T cells specific for the H2-Kd epitope IYSTVASSL and flow
cytometry. Mice vaccinated with the invention as described
generated an antigen-specific long-lasting cellular response. P
values were calculated using the Student T test.
DETAILED DESCRIPTION
[0024] The present application relates to compositions comprising
liposomes, an antigen, a polyI:C polynucleotide and a carrier
comprising a continuous phase of a hydrophobic substance and their
use.
[0025] Compositions of the invention, combining an antigen, a
polyI:C polynucleotide, liposomes and a carrier comprising a
continuous phase of a hydrophobic substance provided surprisingly
higher antibody titers than either conventional vaccine
compositions containing polyI:C polynucleotides in an aqueous
carrier, or compositions comprising liposomes, a hydrophobic
carrier and an alum adjuvant.
[0026] The data described in Examples 1 and 2 herein are summarized
in Table 1:
TABLE-US-00001 TABLE 1 Composition antibody titer (log10) antibody
titer (non-logged) (1) rHA antigen 5.41 256,000 alum adjuvant
liposomes hydrophobic carrier (2) rHA antigen 6.01 1,024,000
polyl:C PBS carrier (3) rHA antigen 6.91 8,192,000 polyl:C
liposomes hydrophobic carrier rHA = recombinant H5N1 influenza
hemagglutinin glycoprotein PBS = phosphate buffered saline
carrier
[0027] It will be seen from the above table (Table 1) that the
compositions of the invention (3) provided antibody titers that
were more than the additive effect of either the combination of
liposomes plus hydrophobic carrier (1), or the use of polyI:C (2).
The additive effect of (1) and (2) would be a non-logged antibody
titer of 256,000+1,024,000=1,280,000. Instead, replacing the alum
adjuvant in (3) with polyI:C gave an unexpectedly high non-logged
antibody titer of 8,192,000, 6.4 times the expected additive
effect. Furthermore, the antibody response generated with
composition (3) was long lasting and the effect observed at the
earlier time point (week 4 post-vaccination) described above was
maintained at week 16 post-vaccination (Examples 4 and 5). The data
described in Examples 4 and 5 herein are summarized in Table 2:
TABLE-US-00002 TABLE 2 Average antibody Average antibody titer
Composition titer (log10) (non-logged) (1) rHA antigen 5.11 128,824
alum adjuvant liposomes hydrophobic carrier (2) rHA antigen 5.23
169,824 polyl:C PBS carrier (3) rHA antigen 6.21 1,621,810 polyl:C
liposomes hydrophobic carrier
[0028] The additive effect of (1) and (2) would be a non-logged
average antibody titer of 128,824+169,824=298,648. Instead,
replacing the alum adjuvant in (3) with polyI:C gave an
unexpectedly high non-logged average antibody titer of 1,612,810,
5.4 times the expected additive effect.
[0029] The results observed with composition (3) described above
were duplicated in a separate study that used a composition
consisting of antigen (rHA), polyI:C, lyophilized liposomes, and a
hydrophobic carrier and described in Example 3. The average
antibody titer observed with this composition at week 8 post
vaccination was 2,884,031 (non logged) compared to 147,910
(non-logged) average titer observed with a standard alum-adjuvanted
vaccine delivered twice to enhance its activity. This 19.4 fold
average increase in titer was observed with one immunization of the
composition described.
[0030] Vaccine compositions containing polyI:C, liposomes, and a
hydrophobic carrier have the potential to generate antibody
responses and/or cellular responses against a broad range of
antigens. Examples 1 through 6 and Examples 8 and 9 demonstrate the
ability to raise a significantly higher antibody response when
combining all components of the composition against a recombinant
protein (rHA) or a short peptide (.beta.-amyloid). These
surprisingly high antibody titers were not observed without the use
of a polyI:C polynucleotide specifically in the vaccine composition
(Examples 1, 4, and 9), nor were they observed in the absence of
liposomes and a hydrophobic carrier despite the use of polyI:C
alone with an antigen (Examples 2 and 5). Similarly, the
combination of all components of the composition generated a
significantly more efficacious and longer-lasting cellular immune
response as illustrated in Example 7 and Examples 11 through 13
against a recombinant protein or a short peptide containing a known
CTL epitope. Significant antigen-specific immune responses were
detected when immunizing with the composition by at least two
immunization routes (Example 7). The unusual efficacy in
controlling tumor growth with the described invention were not
observed without the use of a polyI:C polynucleotide specifically
in the composition (Example 12) and were not observed without the
use of liposomes and despite the use of a polyI:C polynucleotide
and a hydrophobic carrier with the antigen (Example 11). The
ability to raise robust and long lasting humoral and cellular
responses simultaneously with at least one immunization using all
components of the described composition (Examples 6, 7, 10, and 13)
illustrates the particular usefulness of the composition in a wide
range of medical applications including infectious diseases and
cancers.
[0031] It is clear from the collection of examples described herein
that vaccine compositions consisting of an antigen, liposomes, a
hydrophobic carrier and ribo- or deoxyribo-polynucleotides
containing inosine and cytosine residues in more than one chemical
configuration are capable of inducing unusually strong immune
responses. The examples also describe more than one method to make
the desired composition.
Antigens
[0032] The compositions of the invention comprise one or more
antigens. As used herein, the term "antigen" refers to a substance
that can bind specifically to an antibody or to a T-cell
receptor.
[0033] Antigens useful in the compositions of the invention
include, without limitation, polypeptides, a microorganism or a
part thereof, such as a live, attenuated, inactivated or killed
bacterium, virus or protozoan, or part thereof.
[0034] As used herein and in the claims, the term "antigen" also
includes a polynucleotide that encodes the polypeptide that
functions as an antigen. Nucleic acid-based vaccination strategies
are known, wherein a vaccine composition that contains a
polynucleotide is administered to a subject. The antigenic
polypeptide encoded by the polynucleotide is expressed in the
subject, such that the antigenic polypeptide is ultimately present
in the subject, just as if the vaccine composition itself had
contained the polypeptide. For the purposes of the present
invention, the term "antigen", where the context dictates,
encompasses such polynucleotides that encode the polypeptide which
functions as the antigen.
[0035] Polypeptides or fragments thereof that may be useful as
antigens in the invention include, without limitation, those
derived from Cholera toxoid, tetanus toxoid, diphtheria toxoid,
hepatitis B surface antigen, hemagglutinin, neuraminidase,
influenza M protein, PfHRP2, pLDH, aldolase, MSP1, MSP2, AMA1,
Der-p-1, Der-f-1, Adipophilin, AFP, AIM-2, ART-4, BAGE,
.alpha.-fetoprotein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin
D1Ep-CAM, EphA2, EphA3, ELF-2, FGF-5, G250, Gonadotropin Releasing
Hormone, HER-2, intestinal carboxyl esterase (ICE), IL13R.alpha.2,
MAGE-1, MAGE-2, MAGE-3, MART-1, MART-2, M-CSF, MDM-2, MMP-2, MUC-1,
NY-EOS-1, MUM-1, MUM-2, MUM-3, p53, PBF, PRAME, PSA, PSMA, RAGE-1,
RNF43, RU1, RU2AS, SART-1, SART-2, SART-3, SAGE-1, SCRN 1, SOX2,
SOX10, STEAP1, surviving, Telomerase, TGF.beta.RII, TRAG-3, TRP-1,
TRP-2, TERT and WT1.
[0036] Viruses, or parts thereof, useful as antigens in the
invention include, without limitation, Cowpoxvirus, Vaccinia virus,
Pseudocowpox virus, Human herpesvirus 1, Human herpesvirus 2,
Cytomegalovirus, Human adenovirus A-F, Polyomavirus, Human
papillomavirus, Parvovirus, Hepatitis A virus, Hepatitis B virus,
Hepatitis C virus, Human immunodeficiency virus, Orthoreovirus,
Rotavirus, Ebolavirus, parainfluenza virus, influenza A virus,
influenza B virus, influenza C virus, Measles virus, Mumps virus,
Rubella virus, Pneumovirus, Human respiratory syncytial virus,
Rabies virus, California encephalitis virus, Japanese encephalitis
virus, Hantaan virus, Lymphocytic choriomeningitis virus,
Coronavirus, Enterovirus, Rhinovirus, Poliovirus, Norovirus,
Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and
varicella.
[0037] Bacteria or parts of thereof useful as antigens in the
invention include, without limitation, Anthrax, Brucella, Candida,
Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium
botulinum, Coccidioides immitis, Cryptococcus, Diphtheria,
Escherichia coli 0157: H7, Enterohemorrhagic Escherichia coli,
Enterotoxigenic Escherichia coli, Haemophilus influenzae,
Helicobacter pylori, Legionella, Leptospira, Listeria,
Meningococcus, Mycoplasma pneumoniae, Mycobacterium, Pertussis,
Pneumonia, Salmonella, Shigella, Staphylococcus, Streptococcus
pneumoniae and Yersinia enterocolitica.
[0038] The antigen may alternatively be of protozoan origin, e.g.
Plasmodium falciparum, which causes malaria.
[0039] The term "polypeptide" encompasses any chain of amino acids,
regardless of length (e.g., at least 6, 8, 10, 12, 14, 16, 18, or
20 amino acids) or post-translational modification (e.g.,
glycosylation or phosphorylation), and includes, for example,
natural proteins, synthetic or recombinant polypeptides and
peptides, denatured polypeptides and peptides, epitopes, hybrid
molecules, variants, homologs, analogs, peptoids, peptidomimetics,
etc. A variant or derivative therefore includes deletions,
including truncations and fragments; insertions and additions, for
example conservative substitutions, site-directed mutants and
allelic variants; and modifications, including peptoids having one
or more non-amino acyl groups (for example, sugar, lipid, etc.)
covalently linked to the peptide and post-translational
modifications. As used herein, the term "conserved amino acid
substitutions" or "conservative substitutions" refers to the
substitution of one amino acid for another at a given location in
the peptide, where the substitution can be made without substantial
loss of the relevant function. In making such changes,
substitutions of like amino acid residues can be made on the basis
of relative similarity of side-chain substituents, for example,
their size, charge, hydrophobicity, hydrophilicity, and the like,
and such substitutions may be assayed for their effect on the
function of the peptide by routine testing. Specific, non-limiting
examples of a conservative substitution include the following
examples:
TABLE-US-00003 Original Conservative Residue Substitutions Ala Ser
Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln
Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met;
Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu
[0040] Polypeptides or peptides that have substantial identity to a
preferred antigen sequence may be used. Two sequences are
considered to have substantial identity if, when optimally aligned
(with gaps permitted), they share at least approximately 50%
sequence identity, or if the sequences share defined functional
motifs. In alternative embodiments, optimally aligned sequences may
be considered to be substantially identical (i.e., to have
substantial identity) if they share at least 60%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% identity over a specified region.
The term "identity" refers to sequence similarity between two
polypeptides molecules. Identity can be determined by comparing
each position in the aligned sequences. A degree of identity
between amino acid sequences is a function of the number of
identical or matching amino acids at positions shared by the
sequences, for example, over a specified region. Optimal alignment
of sequences for comparisons of identity may be conducted using a
variety of algorithms, as are known in the art, including the
ClustalW program, available at http://clustalw.genome.ad.ip, the
local homology algorithm of Smith and Waterman, 1981, Adv. Appl.
Math 2: 482, the homology alignment algorithm of Needleman and
Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity
method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA
85:2444, and the computerised implementations of these algorithms
(such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, Madison, Wis., U.S.A.).
Sequence identity may also be determined using the BLAST algorithm,
described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using
the published default settings). For example, the "BLAST 2
Sequences" tool, available through the National Center for
Biotechnology Information (through the internet at
http://www.ncbi.nlm.nih.gov/BLAST/bl2seg/wblast2.cgi) may be used,
selecting the "blastp" program at the following default settings:
expect threshold 10; word size 3; matrix BLOSUM 62; gap costs
existence 11, extension 1. In another embodiment, the person
skilled in the art can readily and properly align any given
sequence and deduce sequence identity and/or homology by mere
visual inspection.
[0041] Polypeptides and peptides used to practice the invention can
be isolated from natural sources, be synthetic, or be recombinantly
generated polypeptides. Peptides and proteins can be recombinantly
expressed in vitro or in vivo. The peptides and polypeptides used
to practice the invention can be made and isolated using any method
known in the art. Polypeptide and peptides used to practice the
invention can also be synthesized, whole or in part, using chemical
methods well known in the art. See e.g., Caruthers (1980) Nucleic
Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp.
Ser. 225-232; Banga, A. K, Therapeutic Peptides and Proteins,
Formulation, Processing and Delivery Systems (1995) Technomic
Publishing Co., Lancaster, Pa. For example, peptide synthesis can
be performed using various solid-phase techniques (see e.g.,
Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol.
289:3-13) and automated synthesis may be achieved, e.g., using the
ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the
instructions provided by the manufacturer.
[0042] In some embodiments, the antigen may be a purified antigen,
e.g., from about 25% to 50% pure, from about 50% to about 75% pure,
from about 75% to about 85% pure, from about 85% to about 90% pure,
from about 90% to about 95% pure, from about 95% to about 98% pure,
from about 98% to about 99% pure, or greater than 99% pure.
[0043] As noted above, the term "antigen" also includes a
polynucleotide that encodes the polypeptide that functions as an
antigen. Nucleic acid-based vaccination strategies are known,
wherein a vaccine composition that contains a polynucleotide is
administered to a subject. The antigenic polypeptide encoded by the
polynucleotide is expressed in the subject, such that the antigenic
polypeptide is ultimately present in the subject, just as if the
vaccine composition itself had contained the polypeptide. For the
purposes of the present invention, the term "antigen", where the
context dictates, encompasses such polynucleotides that encode the
polypeptide which functions as the antigen.
[0044] As used herein and in the claims, the term "polynucleotide"
encompasses a chain of nucleotides of any length (e.g. 9, 12, 18,
24, 30, 60, 150, 300, 600, 1500 or more nucleotides) or number of
strands (e.g. single-stranded or double-stranded). Polynucleotides
may be DNA (e.g. genomic DNA or cDNA) or RNA (e.g. mRNA) or
combinations thereof. They may be naturally occurring or synthetic
(e.g. chemically synthesized). It is contemplated that the
polynucleotide may contain modifications of one or more nitrogenous
bases, pentose sugars or phosphate groups in the nucleotide chain.
Such modifications are well-known in the art and may be for the
purpose of e.g. improving stability of the polynucleotide.
[0045] The polynucleotide may be delivered in various forms. In
some embodiments, a naked polynucleotide may be used, either in
linear form, or inserted into a plasmid, such as an expression
plasmid. In other embodiments, a live vector such as a viral or
bacterial vector may be used.
[0046] One or more regulatory sequences that aid in transcription
of DNA into RNA and/or translation of RNA into a polypeptide may be
present. In some instances, such as in the case of a polynucleotide
that is a messenger RNA (mRNA) molecule, regulatory sequences
relating to the transcription process (e.g. a promoter) are not
required, and protein expression may be effected in the absence of
a promoter. The skilled artisan can include suitable regulatory
sequences as the circumstances require.
[0047] In some embodiments, the polynucleotide is present in an
expression cassette, in which it is operably linked to regulatory
sequences that will permit the polynucleotide to be expressed in
the subject to which the composition of the invention is
administered. The choice of expression cassette depends on the
subject to which the composition is administered as well as the
features desired for the expressed polypeptide.
[0048] Typically, an expression cassette includes a promoter that
is functional in the subject and can be constitutive or inducible;
a ribosome binding site; a start codon (ATG) if necessary; the
polynucleotide encoding the polypeptide of interest; a stop codon;
and optionally a 3' terminal region (translation and/or
transcription terminator). Additional sequences such as a region
encoding a signal peptide may be included. The polynucleotide
encoding the polypeptide of interest may be homologous or
heterologous to any of the other regulatory sequences in the
expression cassette. Sequences to be expressed together with the
polypeptide of interest, such as a signal peptide encoding region,
are typically located adjacent to the polynucleotide encoding the
protein to be expressed and placed in proper reading frame. The
open reading frame constituted by the polynucleotide encoding the
protein to be expressed solely or together with any other sequence
to be expressed (e.g. the signal peptide), is placed under the
control of the promoter so that transcription and translation occur
in the subject to which the composition is administered.
[0049] In a related embodiment, the antigen may be an allergen and
may be derived from, without limitation, cells, cell extracts,
proteins, polypeptides, peptides, polysaccharides, polysaccharide
conjugates, peptide and non-peptide mimics of polysaccharides and
other molecules, small molecules, lipids, glycolipids, and
carbohydrates of plants, animals, fungi, insects, food, drugs,
dust, and mites. Allergens include but are not limited to
environmental aeroallergens; plant pollens (e.g. ragweed/hayfever);
weed pollen allergens; grass pollen allergens; Johnson grass; tree
pollen allergens; ryegrass; arachnid allergens (e.g. house dust
mite allergens); storage mite allergens; Japanese cedar pollen/hay
fever; mold/fungal spore allergens; animal allergens (e.g., dog,
guinea pig, hamster, gerbil, rat, mouse, etc., allergens); food
allergens (e.g. crustaceans; nuts; citrus fruits; flour; coffee);
insect allergens (e.g. fleas, cockroach); venoms: (Hymenoptera,
yellow jacket, honey bee, wasp, hornet, fire ant); bacterial
allergens (e.g. streptococcal antigens; parasite allergens such as
Ascaris antigen); viral antigens; drug allergens (e.g. penicillin);
hormones (e.g. insulin); enzymes (e.g. streptokinase); and drugs or
chemicals capable of acting as incomplete antigens or haptens (e.g.
the acid anhydrides and the isocyanates).
PolyI:C Polynucleotides
[0050] PolyI:C polynucleotides are double stranded polynucleotide
molecules (either RNA or DNA or a combination of DNA and RNA)
containing inosinic acid residues (I) and cytidylic acid residues
(C), and which induce the production of inflammatory cytokines,
such as interferon. They are typically composed of one strand
consisting entirely of cytosine-containing nucleotides and one
strand consisting entirely of inosine-containing nucleotides
although other configurations are possible. For instance, each
strand may contain both cytosine-containing and inosine-containing
nucleotides. In some instances, either or both strand may
additionally contain one or more non-cytosine or non-inosine
nucleotides.
[0051] It has been reported that polyI:C can be segmented every 16
residues without an effect on its interferon activating potential
(Bobst, 1981). Furthermore, the interferon inducing potential of a
polyI:C molecule mismatched by introducing a uridine residue every
12 repeating cytidylic acid residues (Hendrix, 1993), suggests that
a minimal double stranded polyI:C molecule of 12 residues is
sufficient to promote interferon production. Others have also
suggested that regions as small as 6-12 residues, which correspond
to 0.5-1 helical turn of the double stranded polynucleotide, are
capable of triggering the induction process (Greene, 1978). If
synthetically made, polyI:C polynucleotides are typically about 20
or more residues in length (commonly 22, 24, 26, 28 or 30 residues
in length). If semisynthetically made (e.g. using an enzyme), the
length of the strand may be 500, 1000 or more residues.
[0052] PolyI:C act as mimics of viral genomes and are particularly
useful for modulating the immune system in vivo. Synthetic poly
I:poly C homopolymers for example has been reported to enhance
innate immunity by inducing interferon gamma non-specifically when
delivered systemically in vivo by intravenous or intramuscular
injection (Krown 1985, Zhu 2007). Several variants of poly inosinic
and cytidylic acid polymers have been described over the years (de
Clercq 1978, Bobst 1981, De Clercq 1975, Guschlbauer 1977, Fukui
1977, Johnston 1975, U.S. Pat. No. 3,906,092 1971, Kamath 2008,
Ichinohe 2007), some of which included the use of covalently
modified residues, the use of ribo and deoxy-ribo inosinic and
cytidylic residues, the use of homopolymers and alternating
co-polymers that contain inosinic and cytidylic acid residues, and
the introduction of specific residues to create mismatched
polymers.
[0053] The use of double stranded polynucleotides containing
inosinic and cytidylic acids has been reported for the treatment of
a number of viral diseases (Kende 1987, Poast 2002, U.S. Pat. No.
6,468,558 2002, Sarma 1969, Stephen 1977, Levy 1978), cancer (Durie
1985, Salazar 1996, Theriault 1986, Nakamura 1982, Talmadge 1985,
Droller 1987), autoimmune disease like multiple sclerosis (Bever
1986), and other infectious diseases such as malaria (Awasthi 1997,
Puri 1996). The efficacy of polyI:C molecules has been further
enhanced in some cases by complexing the molecule with positively
charged poly-lysine and carboxymethyl-cellulose, effectively
protecting the polynucleotide from nuclease degradation in vivo
(Stephen 1977, Levy 1985), or by complexing polyI:C with positively
charged synthetic peptides (Schellack 2006).
[0054] In addition to its uses as a non-specific enhancer of innate
immunity, polyI:C is also useful as adjuvant in vaccine
compositions. The enhancement of innate immunity can lead to an
enhanced antigen specific adaptive immunity, possibly through a
mechanism that involves, at least in part, NK cells, macrophages
and/or dendritic cells (Chirigos 1985, Salem 2006, Alexopoulou
2001, Trumpfheller 2008). Evidence for the use of polyI:C molecules
in this context originates from various vaccine studies for
controlling infectious diseases (Houston 1976, Stephen 1977,
Ichinohe 2007, Sloat 2008, Agger 2006, Padalko 2004) and the
prevention or treatment of cancer by a variety of vaccine
modalities (Zhu 2007, Cui 2006, Salem 2005, Fujimura 2006, Llopiz
2008). These studies demonstrate that polyI:C enhances humoral
responses as evident from enhanced antibody responses against
specific infectious disease antigens. PolyI:C is also a potentiator
of antigen-specific cellular responses (Zhu 2007, Zaks 2006, Cui
2006, Riedl 2008). The adjuvanting effects of PolyI:C molecules are
believed to occur, at least partially, by inducing interferon-gamma
through their interaction with toll like receptors (TLR) such as
TLR3, TLR4, TLR7, TLR8 and TLR9 (Alexopoulou 2001, Trumpfheller
2008, Schellack 2006, Riedl 2008), with TLR3 being particularly
relevant for most polyI:C molecules. Evidence also suggests that
polyI:C molecules may exert their effect, at least in part, by
interacting with receptors other than TLRs, such as the RNA
helicase retinoic acid induced protein I (RIG-I)/melanoma
differentiation associated gene 5 (MDA5) (Alexopoulou 2001,
Yoneyama 2004, Gowen 2007, Dong 2008). The mechanism of action of
polyI:C molecules remains to be fully understood.
[0055] Accordingly, as used herein, a "polyI:C" or "polyI:C
polynucleotide" is a double-stranded polynucleotide molecule (RNA
or DNA or a combination of DNA and RNA), each strand of which
contains at least 6 contiguous inosinic or cytidylic acid residues,
or 6 contiguous residues selected from inosinic acid and cytidylic
acid in any order (e.g. IICIIC or ICICIC), and which is capable of
inducing or enhancing the production of at least one inflammatory
cytokine, such as interferon, in a mammalian subject. PolyI:C
polynucleotides will typically have a length of about 8, 10, 12,
14, 16, 18, 20, 22, 24, 25, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000 or more
residues. The upper limit is not believed to be essential.
Preferred polyI:C polynucleotides may have a minimum length of
about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30
nucleotides and a maximum length of about 1000, 500, 300, 200, 100,
90, 80, 70, 60, 50, 45 or 40 nucleotides.
[0056] Each strand of a polyI:C polynucleotide may be a homopolymer
of inosinic or cytidylic acid residues, or each strand may be a
heteropolymer containing both inosinic and cytidylic acid residues.
In either case, the polymer may be interrupted by one or more
non-inosinic or non-cytidylic acid residues (e.g. uridine),
provided there is at least one contiguous region of 6 I, 6 C or 6
I/C residues as described above. Typically, each strand of a
polyI:C polynucleotide will contain no more than 1 non-I/C residue
per 6 I/C residues, more preferably, no more than 1 non-I/C residue
per every 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 I/C
residues.
[0057] The inosinic acid or cytidylic acid (or other) residues in
the polyI:C polynucleotide may be derivatized or modified as is
known in the art, provided the ability of the polyI:C
polynucleotide to promote the production of an inflammatory
cytokine, such as interferon, is retained. Non-limiting examples of
derivatives or modifications include e.g. azido modifications,
fluoro modifications, or the use of thioester (or similar) linkages
instead of natural phosphodiester linkages to enhance stability in
vivo. The polyI:C polynucleotide may also be modified to e.g.
enhance its resistance to degradation in vivo by e.g. complexing
the molecule with positively charged poly-lysine and
carboxymethylcellulose, or with a positively charged synthetic
peptide.
[0058] The polyI:C polynucleotide will typically be included in the
compositions of the invention in an amount from about 0.001 mg to 1
mg per unit dose of the composition.
Liposomes
[0059] Liposomes are completely closed lipid bilayer membranes
containing an entrapped aqueous volume. Liposomes may be
unilamellar vesicles (possessing a single bilayer membrane) or
multilamellar vesicles characterized by multimembrane bilayers,
each bilayer may or may not be separated from the next by an
aqueous layer. A general discussion of liposomes can be found in
Gregoriadis G. Immunol. Today, 11:89-97, 1990; and Frezard, F.,
Braz. J. Med. Bio. Res., 32:181-189, 1999. As used herein and in
the claims, the term "liposomes" is intended to encompass all such
vesicular structures as described above, including, without
limitation, those described in the art as "niosomes",
"transfersomes" and "virosomes".
[0060] Although any liposomes may be used in this invention,
including liposomes made from archaebacterial lipids, particularly
useful liposomes use phospholipids and unesterified cholesterol in
the liposome formulation. The cholesterol is used to stabilize the
liposomes and any other compound that stabilizes liposomes may
replace the cholesterol. Other liposome stabilizing compounds are
known to those skilled in the art. For example, saturated
phospholipids produce liposomes with higher transition temperatures
indicating increased stability.
[0061] Phospholipids that are preferably used in the preparation of
liposomes are those with at least one head group selected from the
group consisting of phosphoglycerol, phosphoethanolamine,
phosphoserine, phosphocholine and phosphoinositol. More preferred
are liposomes that comprise lipids which are 94-100%
phosphatidylcholine. Such lipids are available commercially in the
lecithin Phospholipon.RTM. 90 G. When unesterified cholesterol is
also used in liposome formulation, the cholesterol is used in an
amount equivalent to about 10% of the amount of phospholipid. If a
compound other than cholesterol is used to stabilize the liposomes,
one skilled in the art can readily determine the amount needed in
the composition.
[0062] Liposome compositions may be obtained, for example, by using
natural lipids, synthetic lipids, sphingolipids, ether lipids,
sterols, cardiolipin, cationic lipids and lipids modified with poly
(ethylene glycol) and other polymers. Synthetic lipids may include
the following fatty acid constituents; lauroyl, myristoyl,
palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or
combinations of these fatty acids.
Carriers
[0063] The carrier of the composition comprises a continuous phase
of a hydrophobic substance, preferably a liquid hydrophobic
substance. The continuous phase may be an essentially pure
hydrophobic substance or a mixture of hydrophobic substances. In
addition, the carrier may be an emulsion of water in a hydrophobic
substance or an emulsion of water in a mixture of hydrophobic
substances, provided the hydrophobic substance constitutes the
continuous phase. Further, in another embodiment, the carrier may
function as an adjuvant.
[0064] Hydrophobic substances that are useful in the compositions
as described herein are those that are pharmaceutically and/or
immunologically acceptable. The carrier is preferably a liquid but
certain hydrophobic substances that are not liquids at atmospheric
temperature may be liquefied, for example by warming, and are also
useful in this invention. In one embodiment, the hydrophobic
carrier may be a Phosphate Buffered Saline/Freund's Incomplete
Adjuvant (PBS/FIA) emulsion.
[0065] Oil or water-in-oil emulsions are particularly suitable
carriers for use in the present invention. Oils should be
pharmaceutically and/or immunologically acceptable. Suitable oils
include, for example, mineral oils (especially light or low
viscosity mineral oil such as Drakeol.RTM. 6VR), vegetable oils
(e.g., soybean oil), nut oils (e.g., peanut oil), or mixtures
thereof. In an embodiment, the oil is a mannide oleate in mineral
oil solution, commercially available as Montanide.RTM. ISA 51.
Animal fats and artificial hydrophobic polymeric materials,
particularly those that are liquid at atmospheric temperature or
that can be liquefied relatively easily, may also be used.
Other Components
[0066] The composition may further comprise one or more
pharmaceutically acceptable adjuvants, excipients, etc., as are
known in the art: See, for example, Remington's Pharmaceutical
Sciences (Remington's Pharmaceutical Sciences, Mack Publishing
Company, Easton, Pa., USA 1985) and The United States
Pharmacopoeia: The National Formulary (USP 24 NF19) published in
1999.
[0067] The term "adjuvant" refers to a compound or mixture that
enhances the immune response to an antigen. An adjuvant can serve
as a tissue depot that slowly releases the antigen and also as a
lymphoid system activator that non-specifically enhances the immune
response (Hood et al, Immunology, 2d ed., Benjamin/Cummings: Menlo
Park, Calif., 1984; see Wood and Williams, In: Nicholson, Webster
and May (eds.), Textbook of Influenza, Chapter 23, pp. 317-323).
Often, a primary challenge with an antigen alone, in the absence of
an adjuvant, will fail to elicit a humoral immune response.
[0068] Suitable adjuvants include, but are not limited to, alum,
other compounds of aluminum, Bacillus of Calmette and Guerin (BCG),
TiterMax.RTM., Ribi.RTM., incomplete Freund's adjuvant (IFA),
saponin, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, Corynebacteriumparvum, QS-21,
Freund's Complete Adjuvant (FCA), adjuvants of the TLR agonist
family such as CpG, falgellin, lipopeptides, peptidoglycans,
imidazoquinolines, single stranded RNA, lipopolysaccharides (LPS),
heat shock proteins (HSP), and ceramides and derivatives such as
.alpha.Gal-cer. Suitable adjuvants also include cytokines or
chemokines in their polypeptide or DNA coding forms such as, but
not limited to, GM-CSF, TNF-.alpha., IFN-.gamma., IL-2, IL-12,
IL-15, IL-21. A suitable alum adjuvant is sold under the trade name
Imject Alum.RTM. (Pierce, Rockford, Ill.), that consists of an
aqueous solution of aluminum hydroxide (45 mg/ml) and magnesium
hydroxide (40 mg/ml) plus inactive stabilizers.
[0069] The amount of adjuvant used depends on the amount of antigen
and on the type of adjuvant. One skilled in the art can readily
determine the amount of adjuvant needed in a particular
application.
[0070] An immune response elicited in subjects administered a
composition of the invention may be formulated to bias the immune
response towards an antibody or a cell mediated immune response.
This may be achieved by using agents, such as adjuvants, that
predominantly induce a Th1 or Th2 response. For example, a
CpG-containing oligonucleotides (in which the CpG dinucleotide is
unmethylated) may be used to induce a predominantly Th1 response,
thus favoring a cell mediated response.
Compositions
[0071] Methods for making liposomes are well known in the art. See
e.g. Gregoriadis (1990) and Frezard (1999) both cited previously.
Any suitable method for making liposomes may be used in the
practice of the invention, or liposomes may be obtained from a
commercial source. Liposomes are typically prepared by hydrating
the liposome components that will form the lipid bilayer (e.g.
phospholipids and cholesterol) with an aqueous solution, which may
be pure water or a solution of one or more components dissolved in
water, e.g. phosphate-buffered saline (PBS), phosphate-free saline,
or any other physiologically compatible aqueous solution.
[0072] In an embodiment, a liposome component or mixture of
liposome components, such as a phospholipid (e.g. Phospholipon.RTM.
90G) and cholesterol, may be solubilized in an organic solvent,
such as a mixture of chloroform and methanol, followed by filtering
(e.g. a PTFE 0.2 .mu.m filter) and drying, e.g. by rotary
evaporation, to remove the solvents.
[0073] Hydration of the resulting lipid mixture may be effected by
e.g. injecting the lipid mixture into an aqueous solution or
sonicating the lipid mixture and an aqueous solution. During
formation of liposomes, the liposome components form single
bilayers (unilamellar) or multiple bilayers (multilamellar)
surrounding a volume of the aqueous solution with which the
liposome components are hydrated.
[0074] In some embodiments, the liposomes are then dehydrated, such
as by freeze-drying or lyophilization.
[0075] The liposomes are combined with the carrier comprising a
continuous hydrophobic phase. This can be done in a variety of
ways.
[0076] If the carrier is composed solely of a hydrophobic substance
or a mixture of hydrophobic substances (e.g. use of a 100% mineral
oil carrier), the liposomes may simply be mixed with the
hydrophobic substance, or if there are multiple hydrophobic
substances, mixed with any one or a combination of them.
[0077] If instead the carrier comprising a continuous phase of a
hydrophobic substance contains a discontinuous aqueous phase, the
carrier will typically take the form of an emulsion of the aqueous
phase in the hydrophobic phase, such as a water-in-oil emulsion.
Such compositions may contain an emulsifier to stabilize the
emulsion and to promote an even distribution of the liposomes. In
this regard, emulsifiers may be useful even if a water-free carrier
is used, for the purpose of promoting an even distribution of the
liposomes in the carrier. Typical emulsifiers include mannide
oleate (Arlacel.TM. A), lecithin, Tween.TM. 80, and Spans.TM. 20,
80, 83 and 85. Typically, the volume ratio (v/v) of hydrophobic
substance to emulsifier is in the range of about 5:1 to about 15:1
with a ratio of about 10:1 being preferred.
[0078] The liposomes may be added to the finished emulsion, or they
may be present in either the aqueous phase or the hydrophobic phase
prior to emulsification.
[0079] The antigen may be introduced at various different stages of
the formulation process. More than one type of antigen may be
incorporated into the composition (e.g. an inactivated virus,
attenuated live virus, protein or polypeptide).
[0080] In some embodiments, the antigen is present in the aqueous
solution used to hydrate the components that are used to form the
lipid bilayers of the liposomes (e.g. phospholipid(s) and
cholesterol). In this case, the antigen will be encapsulated in the
liposome, present in its aqueous interior. If the resulting
liposomes are not washed or dried, such that there is residual
aqueous solution present that is ultimately mixed with the carrier
comprising a continuous phase of a hydrophobic substance, it is
possible that additional antigen may be present outside the
liposomes in the final product. In a related technique, the antigen
may be mixed with the components used to form the lipid bilayers of
the liposomes, prior to hydration with the aqueous solution.
[0081] In an alternative approach, the antigen may instead be mixed
with the carrier comprising a continuous phase of a hydrophobic
substance, before, during, or after the carrier is combined with
the liposomes. If the carrier is an emulsion, the antigen may be
mixed with either or both of the aqueous phase or hydrophobic phase
prior to emulsification. Alternatively, the antigen may be mixed
with the carrier after emulsification.
[0082] The technique of combining the antigen with the carrier may
be used together with encapsulation of the antigen in the liposomes
as described above, such that antigen is present both within the
liposomes and in the carrier comprising a continuous phase of a
hydrophobic substance.
[0083] The above-described procedures for introducing the antigen
into the composition apply also to the polyI:C. That is, the
polyI:C may be introduced into e.g. any one or more of: (1) the
aqueous solution used to hydrate the components that are used to
form the lipid bilayers of the liposomes; (2) the components used
to form the lipid bilayers of the liposomes; or (3) the carrier
comprising a continuous phase of a hydrophobic substance, before,
during, or after the carrier is combined with the liposomes. If the
carrier is an emulsion, the polyI:C may be mixed with either or
both of the aqueous phase or hydrophobic phase prior to
emulsification. Alternatively, the polyI:C may be mixed with the
carrier after emulsification.
[0084] The technique of combining the polyI:C with the carrier may
be used together with encapsulation of the polyI:C in the
liposomes, such that polyI:C is present both within the liposomes
and in the carrier comprising a continuous phase of a hydrophobic
substance.
[0085] The polyI:C can be incorporated in the composition together
with the antigen at the same processing step, or separately, at a
different processing step. For instance, the antigen and the
polyI:C may both be present in the aqueous solution used to hydrate
the lipid bilayer-forming liposome components, such that both the
antigen and polyI:C become encapsulated in the liposomes.
Alternatively, the antigen may be encapsulated in the liposomes,
and the polyI:C mixed with the carrier comprising a continuous
phase of a hydrophobic substance. It will be appreciated that many
such combinations are possible.
[0086] If the composition contains one or more adjuvants, the
adjuvant can be incorporated in the composition together with the
antigen at the same processing step, or separately, at a different
processing step. For instance, the antigen and adjuvant may both be
present in the aqueous solution used to hydrate the lipid
bilayer-forming liposome components, such that both the antigen and
adjuvant become encapsulated in the liposomes. Alternatively, the
antigen may be encapsulated in the liposomes, and the adjuvant
mixed with the carrier comprising a continuous phase of a
hydrophobic substance.
[0087] Stabilizers such as sugars, anti-oxidants, or preservatives
that maintain the biological activity or improve chemical stability
to prolong the shelf life of antigen, adjuvant, the liposomes or
the continuous hydrophobic carrier, may be added to such
compositions.
[0088] In some embodiments, an antigen/polyI:C mixture may be used,
in which case the antigen and the polyI:C polynucleotide are
incorporated into the composition at the same time. An
"antigen/polyI:C mixture" refers to an embodiment in which the
antigen and polyI:C polynucleotide are in the same diluent at least
prior to incorporation into the composition. The antigen and
polyI:C polynucleotide in an antigen/polyI:C mixture may, but need
not necessarily be chemically linked, such as by covalent
bonding.
[0089] Similarly, in some embodiments, an antigen/adjuvant mixture
may be used, in which case the antigen and adjuvant are
incorporated into the composition at the same time. An
"antigen/adjuvant mixture" refers to an embodiment in which the
antigen and adjuvant are in the same diluent at least prior to
incorporation into the composition. The antigen and adjuvant in an
antigen/adjuvant mixture may, but need not necessarily be
chemically linked, such as by covalent bonding.
[0090] In some embodiments, the carrier comprising a continuous
phase of a hydrophobic substance may itself have
adjuvanting-activity. Incomplete Freund's adjuvant, is an example
of a hydrophobic carrier with adjuvanting effect. As used herein
and in the claims, when the term "adjuvant" is used, this is
intended to indicate the presence of an adjuvant in addition to any
adjuvanting activity provided by the carrier comprising a
continuous phase of a hydrophobic substance.
[0091] The compositions as described herein may be formulated in a
form that is suitable for oral, nasal, rectal or parenteral
administration. Parenteral administration includes intravenous,
intraperitoneal, intradermal, subcutaneous, intramuscular,
transepithelial, intrapulmonary, intrathecal, and topical modes of
administration. Preferred routes include intramuscular,
subcutaneous and intradermal administration to achieve a depot
effect. In embodiments where the composition of the invention is
for the treatment of cancer tumors, the composition may be
formulated for delivery by injection directly into the tumor, or
adjacent to the tumor. In some embodiments, the composition may be
delivered evenly over or throughout the tumor to enhance the
biodistribution and hence enhance the therapeutic benefit.
[0092] In further embodiments, a composition of the invention may
be formulated with DNA based polyI:C, RNA based polyI:C or a
mixture of RNA and DNA based polyI:C. In this context, a RNA and
DNA mixture may relate to nucleotides, such that each strand may
comprises DNA and RNA nucleotides; to the strands, such that each
double stranded polynucleotide has one DNA strand and one RNA
strand; to the polynucleotide, such that a composition contains
polyI:C polynucleotides, each of which are wholly composed of RNA
or wholly composed of DNA; or combinations thereof.
[0093] In other embodiments, the compositions of the invention may
be formulated for use in combination with a T cell epitope or a B
cell epitope. The T cell epitope may be a universal T cell epitope
and the B cell epitope may be a universal B cell epitope. As used
herein, a "universal epitope" may be any epitope that is broadly
recognized, for example, by T cells or B cells of multiple strains
of an animal. In one embodiment, the T cell epitope may be a
tetanus toxoid peptide such as F21E. In another embodiment, the T
cell epitope may be PADRE, a universal helper T cell epitope. Other
universal epitopes that may be suitable for use in the context of
the invention are known to the skilled person or may be readily
identified using routine techniques.
[0094] In related embodiments, a composition of the invention
comprises a polyI:C polynucleotide and an antigen, where the
presence of the polyI:C polynucleotide and the antigen in terms of
weight or number of molecules is in a ratio of less than 1 to
1,000, of less than 1 to 900, of less than 1 to 800, of less than 1
to 700, of less than 1 to 500, of less than 1 to 400, of less than
1 to 300, of less than 1 to 200, of less than 1 to 100, of less
than 1 to 50, of less than 1 to 10, of less than 1 to 5, of less
than 1 to 2, of about 1 to 1, of greater than 2 to 1, of greater
than 5 to 1, of greater than 10 to 1, of greater than 50 to 1, of
greater than 100 to 1, of greater than 200 to 1, of greater than
300 to 1, of greater than 400 to 1, of greater than 500 to 1, of
greater than 600 to 1, of greater than 700 to 1, of greater than
800 to 1, of greater than 900 to 1, of greater than 1,000 to 1.
[0095] The optimal amount of polyI:C polynucleotide to antigen to
elicit an optimal immune response may depend on a number of factors
including, without limitation, the composition, the disease, the
subject, and may be readily ascertained by the skilled person using
standard studies including, for example, observations of antibody
titers and other immunogenic responses in the host.
Kits and Reagents
[0096] The present invention is optionally provided to a user as a
kit. For example, a kit of the invention contains one or more of
the compositions of the invention. The kit can further comprise one
or more additional reagents, packaging material, containers for
holding the components of the kit, and an instruction set or user
manual detailing preferred methods of using the kit components.
Methods of Use
[0097] The invention finds application in any instance in which it
is desired to administer an antigen to a subject. The subject may
be a vertebrate, such as a fish, bird or mammal, preferably a
human.
[0098] In some embodiments, the compositions of the invention may
be administered to a subject in order to elicit and/or enhance an
antibody response to the antigen.
[0099] As used herein, to "elicit" an immune response is to induce
and/or potentiate an immune response. As used herein, to "enhance"
an immune response is to elevate, improve or strengthen the immune
response to the benefit of the host relative to the prior immune
response status, for example, before the administration of a
composition of the invention.
[0100] An "antibody" is a protein comprising one or more
polypeptides substantially or partially encoded by immunoglobulin
genes or fragments of immunoglobulin genes. The recognized
immunoglobulin genes include the .kappa., .lamda., .alpha.,
.gamma., .delta., .epsilon. and .mu. constant region genes, as well
as myriad immunoglobulin variable region genes. Light chains are
classified as either .kappa. or .lamda.. Heavy chains are
classified as .gamma., .mu., .alpha., .delta., or .epsilon., which
in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and
IgE, respectively. A typical immunoglobulin (antibody) structural
unit comprises a protein containing four polypeptides. Each
antibody structural unit is composed of two identical pairs of
polypeptide chains, each having one "light" and one "heavy" chain.
The N-terminus of each chain defines a variable region primarily
responsible for antigen recognition. Antibody structural units
(e.g. of the IgA and IgM classes) may also assemble into oligomeric
forms with each other and additional polypeptide chains, for
example as IgM pentamers in association with the J-chain
polypeptide.
[0101] Antibodies are the antigen-specific glycoprotein products of
a subset of white blood cells called B lymphocytes (B cells).
Engagement of antigen with antibody expressed on the surface of B
cells can induce an antibody response comprising stimulation of B
cells to become activated, to undergo mitosis and to terminally
differentiate into plasma cells, which are specialized for
synthesis and secretion of antigen-specific antibody.
[0102] As used herein, the term "antibody response" refers to an
increase in the amount of antigen-specific antibodies in the body
of a subject in response to introduction of the antigen into the
body of the subject.
[0103] One method of evaluating an antibody response is to measure
the titers of antibodies reactive with a particular antigen. This
may be performed using a variety of methods known in the art such
as enzyme-linked immunosorbent assay (ELISA) of antibody-containing
substances obtained from animals. For example, the titers of serum
antibodies which bind to a particular antigen may be determined in
a subject both before and after exposure to the antigen. A
statistically significant increase in the titer of antigen-specific
antibodies following exposure to the antigen would indicate the
subject had mounted an antibody response to the antigen.
[0104] Other assays that may be used to detect the presence of an
antigen-specific antibody include, without limitation,
immunological assays (e.g. radioimmunoassay (RIA)),
immunoprecipitation assays, and protein blot (e.g. Western blot)
assays; and neutralization assays (e.g., neutralization of viral
infectivity in an in vitro or in vivo assay).
[0105] In some embodiments, the compositions of the invention may
be administered to a subject in order to elicit and/or enhance a
cell-mediated immune response to the antigen. As used herein, the
term "cell-mediated immune response" refers to an increase in the
amount of antigen-specific cytotoxic T-lymphocytes, macrophages,
natural killer cells, or cytokines in the body of a subject in
response to introduction of the antigen into the body of the
subject.
[0106] Historically, the immune system was separated into two
branches: humoral immunity, for which the protective function of
immunization could be found in the humor (cell-free bodily fluid or
serum that contain antibodies) and cellular immunity, for which the
protective function of immunization was associated with cells.
Cell-mediated immunity is an immune response that involves the
activation of macrophages, natural killer cells (NK),
antigen-specific cytotoxic T-lymphocytes, and the release of
various cytokines in response to a `non-self` antigen. Cellular
immunity is an important component of adaptive immune response and
following recognition of antigen by cells through their interaction
with antigen-presenting cells such as dendritic cells, B
lymphocytes and to a lesser extent, macrophages, protects the body
by various mechanisms such as: [0107] 1. activating
antigen-specific cytotoxic T-lymphocytes that are able to induce
apoptosis in body cells displaying epitopes of foreign antigen on
their surface, such as virus-infected cells, cells with
intracellular bacteria, and cancer cells displaying tumor antigens;
[0108] 2. activating macrophages and natural killer cells, enabling
them to destroy intracellular pathogens; and [0109] 3. stimulating
cells to secrete a variety of cytokines that influence the function
of other cells involved in adaptive immune responses and innate
immune responses.
[0110] Cell-mediated immunity is most effective in removing
virus-infected cells, but also participates in defending against
fungi, protozoans, cancers, and intracellular bacteria. It also
plays a major role in transplant rejection.
[0111] Detection of Cell Mediated Immune Response Following
Vaccination
[0112] Since cell mediated immunity involves the participation of
various cell types and is mediated by different mechanisms, several
methods could be used to demonstrate the induction of immunity
following vaccination. These could be broadly classified into
detection of: i) specific antigen presenting cells; ii) specific
effector cells and their functions and iii) release of soluble
mediators such as cytokines.
i) Antigen presenting cells: Dendritic cells and B-cells (and to a
lesser extent macrophages) are equipped with special
immuno-stimulatory receptors that allow for enhanced activation of
T cells, and are termed professional antigen presenting cells
(APC). These immuno-stimulatory molecules (also called as
co-stimulatory molecules) are up-regulated on these cells following
infection or vaccination, during the process of antigen
presentation to effector cells such as CD4 and CD8 cytotoxic T
cells. Such co-stimulatory molecules (such as CD80, CD86, MHC class
I or MHC class II) can be detected by using flow cytometry with
fluorochrome-conjugated antibodies directed against these molecules
along with antibodies that specifically identify APC (such as CD11c
for dendritic cells). ii) Cytotoxic T cells: (also known as Tc,
killer T cell, or cytotoxic T-lymphocyte (CTL)) are a sub-group of
T cells which induce the death of cells that are infected with
viruses (and other pathogens), or expressing tumor antigens. These
CTLs directly attack other cells carrying certain foreign or
abnormal molecules on their surface. The ability of such cellular
cytotoxicity can be detected using in vitro cytolytic assays
(chromium release assay). Thus, induction of adaptive cellular
immunity can be demonstrated by the presence of such cytotoxic T
cells, wherein, when antigen loaded target cells are lysed by
specific CTLs that are generated in vivo following vaccination or
infection.
[0113] Naive cytotoxic T cells are activated when their T-cell
receptor (TCR) strongly interacts with a peptide-bound MHC class 1
molecule. This affinity depends on the type and orientation of the
antigen/MHC complex, and is what keeps the CTL and infected cell
bound together. Once activated the CTL undergoes a process called
clonal expansion in which it gains functionality, and divides
rapidly, to produce an army of "armed"-effector cells. Activated
CTL will then travel throughout the body in search of cells bearing
that unique MHC Class I+peptide. This could be used to identify
such CTLs in vitro by using peptide-MHC Class I tetramers in flow
cytometric assays.
[0114] When exposed to these infected or dysfunctional somatic
cells, effector CTL release perforin and granulysin: cytotoxins
which form pores in the target cell's plasma membrane, allowing
ions and water to flow into the infected cell, and causing it to
burst or lyse. CTL release granzyme, a serine protease that enters
cells via pores to induce apoptosis (cell death). Release of these
molecules from CTL can be used as a measure of successful induction
of cellular immune response following vaccination. This can be done
by enzyme linked immunosorbant assay (ELISA) or enzyme linked
immunospot assay (ELISPOT) where CTLs can be quantitatively
measured. Since CTLs are also capable of producing important
cytokines such as IFN-.gamma., quantitative measurement of
IFN-.gamma.-producing CD8 cells can be achieved by ELISPOT and by
flowcytometric measurement of intracellular IFN-.gamma. in these
cells.
CD4+ "helper" T-cells: CD4+ lymphocytes, or helper T cells, are
immune response mediators, and play an important role in
establishing and maximizing the capabilities of the adaptive immune
response. These cells have no cytotoxic or phagocytic activity; and
cannot kill infected cells or clear pathogens, but, in essence
"manage" the immune response, by directing other cells to perform
these tasks. Two types of effector CD4+ T helper cell responses can
be induced by a professional APC, designated Th1 and Th2, each
designed to eliminate different types of pathogens.
[0115] Helper T cells express T-cell receptors (TCR) that recognize
antigen bound to Class II MHC molecules. The activation of a naive
helper T-cell causes it to release cytokines, which influences the
activity of many cell types, including the APC that activated it.
Helper T-cells require a much milder activation stimulus than
cytotoxic T-cells. Helper T-cells can provide extra signals that
"help" activate cytotoxic cells. Two types of effector CD4+ T
helper cell responses can be induced by a professional APC,
designated Th1 and Th2, each designed to eliminate different types
of pathogens. The two Th cell populations differ in the pattern of
the effector proteins (cytokines) produced. In general, Th1 cells
assist the cellular immune response by activation of macrophages
and cytotoxic T-cells; whereas Th2 cells promote the humoral immune
response by stimulation of B-cells for conversion into plasma cells
and by formation of antibodies. For example, a response regulated
by Th1 cells may induce IgG2a and IgG2b in mouse (IgG1 and IgG3 in
humans) and favor a cell mediated immune response to an antigen. If
the IgG response to an antigen is regulated by Th2 type cells, it
may predominantly enhance the production of IgG1 in mouse (IgG2 in
humans). The measure of cytokines associated with Th1 or Th2
responses will give a measure of successful vaccination. This can
be achieved by specific ELISA designed for Th1-cytokines such as
IFN-.gamma., IL-2, IL-12, TNF-.alpha. and others, or Th2-cytokines
such as IL-4, IL-5, IL10 among others.
iii) Measurement of cytokines: released from regional lymph nodes
gives a good indication of successful immunization. As a result of
antigen presentation and maturation of APC and immune effector
cells such as CD4 and CD8 T cells, several cytokines are released
by lymph node cells. By culturing these LNC in vitro in the
presence of antigen, antigen-specific immune response can be
detected by measuring release if certain important cytokines such
as IFN-.gamma., IL-2, IL-12, TNF-.alpha. and GM-CSF. This could be
done by ELISA using culture supernatants and recombinant cytokines
as standards.
[0116] Successful immunization may be determined in a number of
ways known to the skilled person including, but not limited to,
hemagglutination inhibition (HAI) and serum neutralization
inhibition assays to detect functional antibodies; challenge
studies, in which vaccinated subjects are challenged with the
associated pathogen to determine the efficacy of the vaccination;
and the use of fluorescence activated cell sorting (FACS) to
determine the population of cells that express a specific cell
surface marker, e.g. in the identification of activated or memory
lymphocytes. A skilled person may also determine if immunization
with a composition of the invention elicited an antibody and/or
cell mediated immune response using other known methods. See, for
example, Current Protocols in Immunology Coligan et al., ed. (Wiley
Interscience, 2007).
[0117] In further embodiments, the compositions of the invention
may be administered to a subject to elicit and/or enhance an
antibody and a cell mediated immune response to the antigen.
[0118] The invention finds broad application in the prevention and
treatment of any disease susceptible to prevention and/or treatment
by way of administration of an antigen. Representative applications
of the invention include cancer treatment and prevention, gene
therapy, adjuvant therapy, infectious disease treatment and
prevention, allergy treatment and prevention, autoimmune disease
treatment and prevention, neuron-degenerative disease treatment,
and atherosclerosis treatment, drug dependence treatment and
prevention, hormone control for disease treatment and prevention,
control of a biological process for the purpose of
contraception.
[0119] Prevention or treatment of disease includes obtaining
beneficial or desired results, including clinical results.
Beneficial or desired clinical results can include, but are not
limited to, alleviation or amelioration of one or more symptoms or
conditions, diminishment of extent of disease, stabilisation of the
state of disease, prevention of development of disease, prevention
of spread of disease, delay or slowing of disease progression,
delay or slowing of disease onset, conferring protective immunity
against a disease-causing agent and amelioration or palliation of
the disease state. Prevention or treatment can also mean prolonging
survival of a patient beyond that expected in the absence of
treatment and can also mean inhibiting the progression of disease
temporarily, although more preferably, it involves preventing the
occurrence of disease such as by preventing infection in a
subject.
[0120] The skilled artisan can determine suitable treatment
regimes, routes of administration, dosages, etc., for any
particular application in order to achieve the desired result.
Factors that may be taken into account include, e.g.: the nature of
the antigen; the disease state to be prevented or treated; the age,
physical condition, body weight, sex and diet of the subject; and
other clinical factors. See, for example, "Vaccine Handbook",
edited by the Researcher's Associates (Gaku-yuu-kai) of The
National Institute of Health (1994); "Manual of Prophylactic
Inoculation, 8th edition", edited by Mikio Kimura, Munehiro
Hirayama, and Harumi Sakai, Kindai Shuppan (2000); "Minimum
Requirements for Biological Products", edited by the Association of
Biologicals Manufacturers of Japan (1993).
Immune Responses
[0121] A composition of the invention may be used to induce an
antibody response and/or cell-mediated immune response to the
antigen that is formulated in the composition in a subject in need
thereof. An immune response may be elicited and/or enhanced in a
subject in need thereof to any antigen and/or to the cell that
expresses it. Thus, in embodiments of the invention, a composition
may comprise an antigen derived from a bacteria, a virus, a fungus,
a parasite, an allergen or a tumor cell, and may be formulated for
use in the treatment and/or prevention of a disease caused by a
bacteria, a virus, a fungus, a parasite, an allergen or a tumor
cell, respectively.
[0122] A composition of the invention may be suitable for use in
the treatment and/or prevention of cancer in a subject in need
thereof. The subject may have cancer or may be at risk of
developing cancer. Cancers that may be treated and/or prevented by
the use or administration of a composition of the invention
include, without limitation, carcinoma, adenocarcinoma, lymphoma,
leukemia, sarcoma, blastoma, myeloma, and germ cell tumors. In one
embodiment, the cancer may be caused by a pathogen, such as a
virus. Viruses linked to the development of cancer are known to the
skilled person and include, but are not limited to, human
papillomaviruses (HPV), John Cunningham virus (JCV), Human herpes
virus 8, Epstein Barr Virus (EBV), Merkel cell polyomavirus,
Hepatitis C Virus and Human T cell leukaemia virus-1. A composition
of the invention may be used for either the treatment or
prophylaxis of cancer, for example, in the reduction of the
severity of cancer or the prevention of cancer recurrences. Cancers
that may benefit from the compositions of the invention include any
malignant cell that expresses one or more tumor specific
antigen.
[0123] A composition of the invention may be suitable for use in
the treatment and/or prevention of a viral infection in a subject
in need thereof. The subject may be infected with a virus or may be
at risk of developing a viral infection. Viral infections that may
be treated and/or prevented by the use or administration of a
composition of the invention include, without limitation,
Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, Human herpesvirus
1, Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F,
Polyomavirus, Human papillomavirus, Parvovirus, Hepatitis A virus,
Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus,
Orthoreovirus, Rotavirus, Ebolavirus, parainfluenza virus,
influenza A virus, influenza B virus, influenza C virus, Measles
virus, Mumps virus, Rubella virus, Pneumovirus, Human respiratory
syncytial virus, Rabies virus, California encephalitis virus,
Japanese encephalitis virus, Hantaan virus, Lymphocytic
choriomeningitis virus, Coronavirus, Enterovirus, Rhinovirus,
Poliovirus, Norovirus, Flavivirus, Dengue virus, West Nile virus,
Yellow fever virus and varicella.
[0124] In one embodiment, a composition of the invention may be
used to treat and/or prevent an influenza virus infection in a
subject in need thereof. Influenza is a single-stranded RNA virus
of the family Orthomyxoviridae and is often characterized based on
two large glycoproteins on the outside of the viral particle,
hemagglutinin (HA) and neuraminidase (NA). Numerous HA subtypes of
influenza A have been identified (Kawaoka et al., Virology (1990)
179:759-767; Webster et al., "Antigenic variation among type A
influenza viruses," p. 127-168. In: P. Palese and D. W. Kingsbury
(ed.), Genetics of influenza viruses. Springer-Verlag, New
York).
[0125] A composition of the invention may be suitable for use in
the treatment and/or prevention of a neurodegenerative disease in a
subject in need thereof, wherein the neurodegenerative disease is
associated with the expression of an antigen. The subject may have
a neurodegenerative disease or may be at risk of developing a
neurodegenerative disease. Neurodegenerative diseases that may be
treated and/or prevented by the use or administration of a
composition of the invention include, without limitation,
Alzheimer's disease, Parkinson's disease, Huntington's disease, and
amyotrophic lateral sclerosis (ALS).
[0126] In one embodiment, a composition of the invention may be
used to treat and/or prevent Alzheimer's disease in a subject in
need thereof. Alzheimer's disease is characterized by the
association of .beta.-amyloid plaques and/or tau proteins in the
brains of patients with Alzheimer's disease (see, for example,
Goedert and Spillantini, Science, 314: 777-781, 2006). Herpes
simplex virus type 1 has also been proposed to play a causative
role in people carrying the susceptible versions of the apoE gene
(Itzhaki and Wozniak, J Alzheimers Dis 13: 393-405, 2008).
[0127] A subject administered or treated with a composition of the
invention may result in the increase of an antibody and/or cell
mediated immune response to the antigen relative to a subject
treated with a control composition. As used herein, a "control
composition" may refer to any composition that does not contain at
least one component of the claimed composition. Thus a control
composition does not contain at least one of 1) an antigen, 2)
liposome, 3) polyI:C or 4) a hydrophobic carrier. In one
embodiment, a control composition does not contain polyI:C. In
other embodiments, a control composition may contain alum instead
of polyI:C.
[0128] A subject administered or treated with a composition of the
invention may elicit an antibody immune response that is at least
1.50.times., at least 1.75.times., at least 2.times., at least
2.5.times., at least 3.times., at least 3.5.times., at least
4.times., at least 4.5.times., or at least 5.times. higher relative
to a subject treated with a control composition. In one embodiment,
the antibody titre (expressed in terms of log 10 value) from the
serum of a subject treated with a composition of the invention is
at least 0.05, at least 0.10, at least 0.15, at least 0.20, at
least 0.25 or at least 0.30 higher than that of a subject treated
with a control composition.
[0129] A subject administered or treated with a composition of the
invention may elicit a cell mediated immune response that is at
least 1.50.times., at least 1.75.times., at least 2.times., at
least 2.5.times., at least 3.times., at least 3.5.times., at least
4.times., at least 4.5.times., or at least 5.times. higher relative
to a subject treated with a control composition.
[0130] A subject administered or treated with a composition of the
invention may elicit a memory T cell population that is at least
1.50.times., at least 1.75.times., at least 2.times., at least
2.5.times., at least 3.times., at least 3.5.times., at least
4.times., at least 4.5.times., or at least 5.times. higher relative
to a subject treated with a control composition.
[0131] A subject administered or treated with a composition of the
invention may prevent the development and/or delay the onset of a
tumor in a subject, relative to a subject treated with a control
composition.
[0132] The invention is further illustrated by the following
non-limiting examples.
Example 1
[0133] Pathogen free, female CD1 mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0134] The H5N1 recombinant hemagglutinin protein, was purchased
from Protein Sciences (Meridien, Conn., USA). This recombinant
protein has an approximate molecular weight of 72,000 daltons and
corresponds to the hemagglutinin glycoprotein, an antigenic protein
present on the surface of the H5N1 influenza virus. This
recombinant protein, hereafter designated rHA, was used as a model
antigen to test the efficacy of vaccine formulations. rHA was used
at 1 microgram per 30 microliter dose.
[0135] Vaccine efficacy was assessed by enzyme-linked immunosorbent
assay (ELISA), a method that allows the detection of
antigen-specific antibody levels in the serum of immunized animals.
Performing the ELISA on sera collected from immunized mice on a
regular interval (every four weeks for example), is useful for
monitoring the antibody responses to a given vaccine formulation.
Briefly, a 96-well microtiter plate is coated with antigen (rHA, 1
microgram/milliliter) overnight at 4 degrees Celsius, blocked with
3% gelatin for 30 minutes, then incubated overnight at 4 degrees
Celsius with serial dilutions of sera, typically starting at a
dilution of 1/2000. A secondary reagent (protein G conjugated to
alkaline phosphatase, EMD chemicals, Gibbstown, N.J., USA) is then
added to each well at a 1/500 dilution for one hour at 37 degrees
Celsius. Following a 60 minute incubation with a solution
containing 1 milligram/milliliter 4-nitrophenyl phosphate disodium
salt hexahydrate (Sigma-Aldrich Chemie GmbH, Switzerland), the 405
nanometer absorbance of each well is measured using a microtiter
plate reader (ASYS Hitech GmbH, Austria). Endpoint titers are
calculated as described in Frey A. et al (Journal of Immunological
Methods, 1998, 221:35-41). Calculated titers represent the highest
dilution at which a statistically significant increase in
absorbance is observed in serum samples from immunized mice versus
serum samples from naive, non-immunized control mice. Titers are
presented as log 10 values of the endpoint dilution.
[0136] To formulate vaccine described herein, a 10:1 w:w homogenous
mixture of S100 lecithin and cholesterol (Lipoid GmbH, Germany) was
hydrated in the presence of a rHA solution in phosphate buffered
saline (pH 7.4) to form liposomes with encapsulated rHA. In brief,
33 micrograms of rHA were first suspended in 300 microliters of
phosphate buffered saline (pH 7.4) then added to 132 milligrams of
the S100 lecithin/cholesterol mixture to form approximately 450
microliters of a liposome suspension encapsulating the rHA antigen.
The liposome preparation was extruded by passing the material
through a manual mini-extruder (Avanti, Alabaster, Ala., USA)
fitted with a 200 nanometer polycarbonate membrane. For every 450
microliters of liposome suspension containing rHA, two milligrams
of Imject Alum adjuvant (Pierce, Rockford, Ill., USA) was added.
For every 500 microliters of a liposome/antigen/adjuvant
suspension, an equal volume of a mineral oil carrier equivalent to
Freund's incomplete adjuvant (known as Montanide.TM. ISA 51,
supplied by Seppic, France) was added to form a water-in-oil
emulsion with the liposome suspension contained within the water
phase of the emulsion and the oil forming a continuous hydrophobic
phase. Each vaccine dose consisted of 30 microliters of the
above-described emulsion containing liposomes, rHA antigen, alum
adjuvant, and the mineral oil carrier. This vaccine formulation
will be referred to as liposome/alum/hydrophobic carrier.
[0137] To formulate the vaccine corresponding to the invention, the
same procedures described above were used with the following
exception: following the formation of liposomes encapsulating rHA,
and after extruding the liposome suspension through a 200 nanometer
polycarbonate membrane, 133 micrograms of polyI:C adjuvant (Pierce,
Rockford, Ill., USA) were added to every 450 microliters of
liposomes. For every 500 microliters of a liposome/antigen/adjuvant
suspension, an equal volume of a mineral oil carrier (Montanide.TM.
ISA 51, Seppic, France) was added to form a water-in-oil emulsion
with the liposome suspension contained in the water phase of the
emulsion and the oil forming the continuous phase. Each vaccine
dose consisted of 30 microliters of the above described emulsion
containing liposomes, rHA antigen, polyI:C adjuvant, and the
mineral oil carrier. This particular formulation will be referred
to as liposome/polyI:C/hydrophobic carrier.
[0138] The efficacy of the two emulsion formulations described
above was compared to the efficacy of a control vaccine consisting
of 1 microgram of rHA and 60 micrograms of alum adjuvant in 30
microliters of phosphate buffered saline (pH 7.4). Two groups of
mice (9 or 10 mice per group) were injected once (no boosting) with
liposome vaccine formulations, intramuscularly, as follows: Group 1
mice were vaccinated with Vaccine B comprising 1 microgram of rHA
antigen formulated in 30 microliters of
liposome/polyI:C/hydrophobic carrier as described above. Each
vaccine dose effectively contained 4 micrograms of polyI:C. Group 2
mice were vaccinated with Vaccine A comprising 1 microgram of rHA
formulated in 30 microliters of liposome/alum/hydrophobic carrier
as described above. Each vaccine dose effectively contained 60
micrograms of alum. The control group of mice (Group 3, n=10) was
injected intramuscularly with the control alum vaccine consisting
of 1 microgram of rHA and 60 micrograms of alum suspended in
phosphate buffered saline. Serum samples were collected from all
mice at 18 days and 28 days post-immunization. Antibody titers in
these sera were examined by ELISA as described above.
[0139] Group 3 mice generated a detectable antigen-specific
antibody response as was expected following the administration of
an alum-adjuvanted control vaccine. Not surprisingly, Group 2 mice
vaccinated with a liposome/alum/hydrophobic carrier formulation
generated a considerably higher antibody response. While these
results were expected, the use of polyI:C adjuvant instead of alum
adjuvant in a liposome/polyI:C/hydrophobic carrier formulation
(Group 1 mice), yielded some unexpected result; antibody titers
were significantly higher than those generated by the
liposome/alum/hydrophobic carrier formulation (Group 2).
[0140] Group 3 mice, vaccinated with the aqueous control
formulation described above, generated endpoint titers up to
1/32,000 and 1/64,000 at 18 and 28 days post-vaccination (log 10
values of 4.51 and 4.81 respectively). The endpoint titers at 18
and 28 days post-vaccination in Group 2 were up to 1/256,000 (log
10 value of 5.41). The presence of such antibody responses at 18
and 28 days (4 weeks) post-vaccination confirms that a genuine
immune response was generated as a result of vaccination. Group 1
mice that were injected with the formulation corresponding to the
invention were able to generate an enhanced immune response with
endpoint titers reaching up to 1/1,024,000 (log 10 value of 6.01)
at 18 days post-vaccination and 1/8,192,000 (a log 10 value of
6.91) at four weeks post-immunization. These results indicate that
liposome/hydrophobic carrier formulations containing a polyI:C
adjuvant are capable of generating a significantly enhanced in vivo
immune response compared to liposome/alum/hydrophobic carrier and
aqueous/alum control vaccinations.
Example 2
[0141] Pathogen free, female CD1 mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0142] As in example 1, H5N1 recombinant hemagglutinin protein,
corresponding to the hemagglutinin glycoprotein on the surface of
the H5N1 influenza virus, was purchased from Protein Sciences
(Meridien, Conn., USA). This recombinant protein, hereafter
designated rHA, was used as a model antigen to test the efficacy of
vaccine formulations. rHA was used at 1 microgram per 30 microliter
dose.
[0143] To formulate the vaccine corresponding to the invention, the
same procedures as described in example one were used. In summary,
33 micrograms of rHA were suspended in 300 microliters of phosphate
buffered saline (pH 7.4) then added to 132 milligrams of a S100
lecithin/cholesterol mixture (Lipoid GmbH, Germany) to form
approximately 450 microliters of a liposome suspension
encapsulating the rHA antigen. The liposome preparation was
extruded by passing the material through a manual mini-extruder
(Avanti, Alabaster, Ala., USA) fitted with a 200 nanometer
polycarbonate membrane. For every 450 microliters of liposome
suspension containing rHA, 133 micrograms of polyI:C adjuvant
(Pierce, Rockford, Ill., USA) was added. For every 500 microliters
of a liposome/antigen/adjuvant suspension, an equal volume of a
mineral oil carrier (Montanide.TM. ISA 51, Seppic, France) was
added to form a water-in-oil emulsion with the liposome suspension
contained within the water phase of the emulsion and the oil
forming a continuous hydrophobic phase. Each vaccine dose consisted
of 30 microliters of the above described emulsion containing
liposomes, rHA antigen, polyI:C adjuvant, and the mineral oil
carrier. This particular formulation will be referred to as
liposome/polyI:C/hydrophobic carrier.
[0144] The efficacy of the liposome/polyI:C/hydrophobic carrier
vaccine described above was compared to the efficacy of an aqueous
control vaccine containing polyI:C adjuvant. Two groups of mice (9
or 10 mice per group) were injected once, intramuscularly, with 30
microliters per dose. Group 1 mice were vaccinated with Vaccine B
comprising 1 microgram of rHA and 4 micrograms of polyI:C
formulated in 30 microliters of liposome/polyI:C/hydrophobic
carrier as described above. Group 2 mice were injected with 30
microliters of the control polyI:C vaccine comprising 1 microgram
rHA and 4 micrograms polyI:C formulated in phosphate buffered
saline (pH 7.4). Serum samples were collected from all mice at 18
and 28 days post-immunization. rHA antibody titers of the sera
samples were examined by ELISA as described in example 1.
[0145] Group 2 mice generated a detectable antigen-specific
antibody response following the administration of a
polyI:C-adjuvanted control vaccine. Group 1 mice, vaccinated with
the liposome/polyI:C/hydrophobic carrier formulation, yielded
significantly enhanced endpoint titers compared to those of Group
2. Group 2 mice generated titers up to 1/128,000 (log 10 value of
5.11) at 18 days post-vaccination and up to 1/1,024,000 (log 10
equal to 6.01) at 28 days (4 weeks) post-vaccination. As noted in
example 1, the presence of such antibody responses confirms a
genuine immune response generated as a result of the vaccination.
Group 1 mice, vaccinated with the vaccine corresponding to the
invention, were able to generate endpoint titers reaching up to
1/1,024,000 (log 10 value of 6.01) at 18 days post-vaccination and
1/8,192,000 (a log 10 value of 6.91) at four weeks
post-immunization. These results indicate that liposome/hydrophobic
carrier formulations containing a polyI:C adjuvant are capable of
generating a significantly enhanced in vivo immune response
compared to an aqueous/polyI:C control vaccination.
Example 3
[0146] Pathogen free, female CD1 mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0147] As in examples 1 and 2, H5N1 recombinant hemagglutinin
protein, corresponding to the hemagglutinin glycoprotein on the
surface of the H5N1 influenza virus, was purchased from Protein
Sciences (Meridien, Conn., USA). This recombinant protein,
hereafter designated rHA, was used as a model antigen to test the
efficacy of vaccine formulations. rHA was used at 1 microgram per
50 microliter dose.
[0148] To formulate vaccine corresponding to the invention, a 10:1
w:w homogenous mixture of S100 lecithin and cholesterol (Lipoid
GmbH, Germany) was hydrated in the presence of a rHA and polyI:C
adjuvant (Pierce, Rockford, Ill., USA) solution in phosphate buffer
to form liposomes with encapsulated rHA and adjuvant. In brief, 20
micrograms of rHA and 200 micrograms polyI:C were first suspended
in 250 microliters of 50 millimolar phosphate buffer (pH 7.4) then
added to 132 milligrams of the S100 lecithin/cholesterol mixture to
form approximately 400 microliters of a liposome suspension
encapsulating the rHA antigen and polyI:C adjuvant. The liposome
preparation was diluted in half using 50 millimolar phosphate
buffer (pH 7.4) and then extruded by passing the material through a
manual mini-extruder (Avanti, Alabaster, Ala., USA) fitted with a
200 nanometer polycarbonate membrane. Sized liposomes were then
lyophilized using the Virtis Advantage freeze dryer (SP Industries,
Warminister, Pa., USA). For every 800 microliters of original
liposome suspension containing rHA and polyI:C, one milliliter of a
mineral oil carrier equivalent to Freund's incomplete adjuvant
(known as Montanide.TM. ISA 51, supplied by Seppic, France) was
used to reconstitute the lyophilized liposomes. Each vaccine dose
consisted of 50 microliters of the above described formulation
combining liposomes, rHA antigen, polyI:C adjuvant, and the mineral
oil carrier. This vaccine formulation will be referred to as
lyophilized liposome/polyI:C/hydrophobic carrier.
[0149] The efficacy of the lyophilized liposome formulation
described above was compared to the efficacy of a control vaccine
consisting of 1 microgram of rHA and 100 micrograms of Imject Alum
adjuvant (Pierce, Rockford, Ill., USA) in 50 microliters of 50
millimolar phosphate buffer (pH 7.4). Group 1 mice (N=8) were
injected once (no boosting) with Vaccine C comprising 1 microgram
of rHA antigen and 10 micrograms of polyI:C adjuvant formulated in
50 microliters of lyophilized liposome/polyI:C/hydrophobic carrier
as described above. Group 2 mice (N=9) were vaccinated twice (day 0
and day 21) with the control alum vaccine comprising 1 microgram of
rHA and 100 micrograms of alum adjuvant suspended in 50 millimolar
phosphate buffer. Serum samples were collected from all mice at 3,
4, and 8 weeks post-immunization. rHA antibody titers in these sera
were examined by ELISA as described in example 1.
[0150] Group 2 mice generated a detectable antigen-specific
antibody response following the administration of an
alum-adjuvanted control vaccine. Group 1 mice, vaccinated with a
single dose of the lyophilized liposome/polyI:C/hydrophobic carrier
formulation, yielded significantly enhanced endpoint titers
compared to those of Group 2, despite that Group 2 animals were
vaccinated twice (primary immunization plus boost). Group 2 mice
generated titers up to 1/128,000 (log 10 value of 5.11) at three
weeks post-vaccination (before boost) and up to 1/1,024,000 (log 10
equal to 6.01) and 1/512,000 (log 10 equal to 5.71) at four and
eight weeks respectively (after boost). As noted in example 1, the
presence of such antibody responses confirms a genuine immune
response generated as a result of the vaccination. Group 1 mice,
vaccinated with the vaccine corresponding to the invention, were
able to generate endpoint titers reaching up to 1/2,048,000 (log 10
value of 6.31) at three weeks post-vaccination and 1/8,192,000 (a
log 10 value of 6.91) at four and eight weeks post-immunization.
These results indicate that single dose lyophilized
liposome/hydrophobic carrier formulations containing a polyI:C
adjuvant are capable of generating a significantly enhanced in vivo
immune response compared to a boosted, aqueous alum control
vaccination. The immune responses generated in this example are
equivalent to the immune responses generated by a vaccine of the
invention presented in Examples 1 and 2.
Example 4
[0151] Pathogen free, female CD1 mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0152] As in the previous examples, H5N1 recombinant hemagglutinin
protein (Protein Sciences, Meridien, Conn., USA) corresponding to
the hemagglutinin glycoprotein present on the surface of the H5N1
influenza virus, hereafter designated rHA, was used as a model
antigen to test the efficacy of vaccine formulations. rHA was used
at 1 microgram per 30 microliter dose.
[0153] Vaccines described herein were formulated as described in
Example 1. Briefly, 33 micrograms of rHA were suspended in 300
microliters of phosphate buffered saline (pH 7.4) then added to 132
milligrams of a homogeneous (10:1, w:w) S100 lecithin/cholesterol
mixture (Lipoid GmbH, Germany) to form approximately 450
microliters of a liposome suspension encapsulating the rHA antigen.
The liposome preparation was extruded by passing the material
through a manual mini-extruder (Avanti, Alabaster, Ala., USA)
fitted with a 200 nanometer polycarbonate membrane. For every 450
microliters of liposome suspension containing rHA, two milligrams
of Imject Alum adjuvant (Pierce, Rockford, Ill., USA) was added.
For every 500 microliters of a liposome/antigen/adjuvant
suspension, an equal volume of a mineral oil carrier (Montanide.TM.
ISA 51, supplied by Seppic, France) was added to form a
water-in-oil emulsion with the liposome suspension contained within
the water phase of the emulsion and the oil forming a continuous
hydrophobic phase. Each vaccine dose consisted of 30 microliters of
the above described emulsion containing liposomes, rHA antigen,
alum adjuvant, and the mineral oil carrier. This vaccine
formulation will be referred to as liposome/alum/hydrophobic
carrier.
[0154] To formulate the vaccine corresponding to the invention, the
same procedures as described above were used with the following
exception: following the formation of liposomes encapsulating rHA,
and after extruding the liposome suspension through a 200 nanometer
polycarbonate membrane, 133 micrograms of RNA-based polyI:C
adjuvant (Pierce, Rockford, Ill., USA) were added to every 450
microliters of liposomes. For every 500 microliters of a
liposome/antigen/adjuvant suspension, an equal volume of a mineral
oil carrier (Montanide.TM. ISA 51, Seppic, France) was added to
form a water-in-oil emulsion with the liposome suspension contained
in the water phase of the emulsion and the oil forming the
continuous phase. Each vaccine dose consisted of 30 microliters of
the above described emulsion containing liposomes, rHA antigen,
polyI:C adjuvant, and the mineral oil carrier. This particular
formulation will be referred to as liposome/polyI:C/hydrophobic
carrier.
[0155] The efficacy of the two emulsion formulations described
above was compared as described in Example 1. Two groups of mice (9
or 10 mice per group) were injected once (no boosting) with
liposome vaccine formulations, intramuscularly, as follows: Group 1
mice were vaccinated with Vaccine B comprising 1 microgram of rHA
antigen and 4 micrograms of polyI:C adjuvant formulated in 30
microliters of liposome/polyI:C/hydrophobic carrier (the
invention). Group 2 mice were vaccinated with 1 microgram of rHA
and 60 micrograms of alum adjuvant formulated in 30 microliters of
liposome/alum/hydrophobic carrier. Group 2 vaccine was a control
formulation (Vaccine A) containing the generic adjuvant alum. Serum
samples were collected from all mice at 18 and 28 days
post-immunization and then every four weeks for a total of 16
weeks. Antibody titers in these sera were examined by ELISA as
described in Example 1.
[0156] The endpoint titers in Group 2 were up to 1/256,000 at 8 and
12 weeks and 1/512,000 at 16 weeks post-immunization (log 10 values
of 5.41 and 5.71 respectively). Group 1 mice that were injected
with the formulation corresponding to the invention were able to
generate an enhanced immune response with endpoint titers reaching
up to 1/4,096,000 (log 10 value of 6.61) at 8, 12 and 16 weeks
post-vaccination. These results confirm that liposome/hydrophobic
carrier formulations containing a polyI:C adjuvant are capable of
generating a significantly enhanced in vivo immune response that is
on average 10 times greater than what is achieved using a control
vaccine lacking polyI:C (P values<than 0.01 at all time points
between weeks 4 and 16 post-vaccination). The dramatic improvement
in the immune response generated was a result of using the polyI:C
adjuvant specifically instead of alum in the
antigen/liposome/adjuvant/mineral oil carrier composition. The
stronger immune response generated with the vaccine of this
invention was robust, as it persisted at significantly superior
levels compared to the alum containing vaccine for a minimum of 16
weeks.
Example 5
[0157] Pathogen free, female CD1 mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0158] As in the previous examples, H5N1 recombinant hemagglutinin
protein, corresponding to the hemagglutinin glycoprotein on the
surface of the H5N1 influenza virus, was purchased from Protein
Sciences (Meridien, Conn., USA). This recombinant protein,
hereafter designated rHA, was used as a model antigen to test the
efficacy of vaccine formulations. rHA was used at 1 microgram per
30 microliter dose.
[0159] To formulate the vaccine corresponding to the invention, the
same procedures as described in Example 2 were used. In summary, 33
micrograms of rHA were suspended in 300 microliters of phosphate
buffered saline (pH 7.4) then added to 132 milligrams of a S100
lecithin/cholesterol mixture (Lipoid GmbH, Germany) to form
approximately 450 microliters of a liposome suspension
encapsulating the rHA antigen. The liposome preparation was
extruded by passing the material through a 200 nanometer
polycarbonate membrane. For every 450 microliters of liposome
suspension containing rHA, 133 micrograms of RNA-based polyI:C
adjuvant (Pierce, Rockford, Ill., USA) was added. For every 500
microliters of a liposome/antigen/adjuvant suspension, an equal
volume of a mineral oil carrier (Montanide.TM. ISA 51, Seppic,
France) was added to form a water-in-oil emulsion with the liposome
suspension contained within the water phase of the emulsion and the
oil forming the continuous phase. Each vaccine dose consisted of 30
microliters of the above described emulsion containing liposomes,
rHA antigen, polyI:C adjuvant, and the mineral oil carrier. This
particular formulation will be referred to as
liposome/polyI:C/hydrophobic carrier.
[0160] The efficacy of the liposome/polyI:C/hydrophobic carrier
vaccine described above was compared to the efficacy of an aqueous
control vaccine containing rHA antigen and RNA-based polyI:C
adjuvant. Two groups of mice (9 or 10 mice per group) were injected
once, intramuscularly, with 30 microliters per dose. Group 1 mice
were vaccinated with Vaccine B comprising 1 microgram of rHA and 4
micrograms of polyI:C formulated as liposome/polyI:C/hydrophobic
carrier as described above. Group 2 mice were injected with the
control polyI:C vaccine comprising 1 microgram rHA and 4 micrograms
polyI:C formulated in phosphate buffered saline (pH 7.4). Serum
samples were collected from all mice at 18 and 28 days
post-immunization and then every four weeks for a total of 16
weeks. rHA antibody titers of the sera samples were examined by
ELISA as described in Example 1.
[0161] Group 2 mice generated a detectable, antigen-specific
antibody response following the administration of a
polyI:C-adjuvanted control vaccine. Group 1 mice, vaccinated with
the liposome/polyI:C/hydrophobic carrier formulation, yielded
significantly enhanced endpoint titers compared to those of Group
2. Group 2 mice generated titers up to 1/512,000 (log 10 value of
5.71) at 8 weeks and up to 1/2,048,000 (log 10 equal to 6.31) at 12
and 16 weeks post-vaccination. As noted previously, the presence of
such antibody responses confirms a genuine immune response
generated as a result of the vaccination. Group 1 mice, vaccinated
with the vaccine corresponding to the invention, were able to
generate endpoint titers reaching up to 1/4,096,000 (log 10 value
of 6.61) at 8, 12 and 16 weeks post-immunization. These results
confirm that liposome/hydrophobic carrier formulations containing a
polyI:C adjuvant are capable of generating a durable and
substantially higher in vivo immune response compared to an
aqueous/polyI:C control vaccination (P value<0.02 at week 4 and
week 16 post-vaccination). Antibody titers that were 7 times higher
on average at early (week 4 post vaccination) and 9 times higher on
average at late (week 16 post-vaccination) time points were
achieved in the presence of liposomes and a hydrophobic carrier in
the vaccine. This suggests that the liposome and hydrophobic
carrier components are important for generating the strong immune
responses observed.
Example 6
[0162] Pathogen free, female BALB/c mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0163] As in Examples 1 through 5, H5N1 recombinant hemagglutinin
protein, corresponding to the hemagglutinin glycoprotein on the
surface of the H5N1 influenza virus, was purchased from Protein
Sciences (Meridien, Conn., USA). This recombinant protein,
hereafter designated rHA, was used as a model antigen to test the
efficacy of vaccine formulations. rHA was used at 1.5 micrograms
per 50 microliter dose.
[0164] To formulate vaccine corresponding to the invention, a 10:1
(w:w) homogenous mixture of S100 lecithin and cholesterol (Lipoid
GmbH, Germany) was hydrated in the presence of rHA in phosphate
buffer to form liposomes with encapsulated rHA and followed by the
addition of polyI:C (Pierce, Rockford, Ill., USA). In brief, 30
micrograms of rHA were suspended in 750 microliters of 50
millimolar phosphate buffer (pH 7.0) then added to 132 milligrams
of the S100 lecithin/cholesterol mixture to form approximately 900
microliters of a liposome suspension encapsulating the rHA antigen.
The liposome preparation was extruded by passing the material
through a semi-automatic extruder (Avestin, Ottawa, ON, Canada)
fitted with a 200 nanometer polycarbonate membrane at a flow rate
of 100 milliliters per minute. 250 micrograms of RNA-based polyI:C
adjuvant in 50 millimolar phosphate buffer (pH 7.0) was added to
sized liposomes to dilute the preparation to 1 milliliter.
Liposomes were then lyophilized using the Virtis Advantage freeze
dryer (SP Industries, Warminister, Pa., USA). For every 1
milliliter of original liposome suspension containing rHA and
polyI:C, 800 microliters of a mineral oil carrier (Montanide.TM.
ISA 51, Seppic, France) was used to reconstitute the lyophilized
liposomes. Each vaccine dose consisted of 50 microliters of the
above described formulation containing liposomes, rHA antigen,
polyI:C adjuvant, and the mineral oil carrier. This vaccine
formulation will be referred to as lyophilized
liposome/polyI:C/hydrophobic carrier.
[0165] The efficacy of the lyophilized liposome formulation
described above was compared to the efficacy of a control vaccine
consisting of 1.5 micrograms of rHA and 100 micrograms of Imject
Alum adjuvant (Pierce, Rockford, Ill., USA) in 50 microliters of 50
millimolar phosphate buffer (pH 7.0). Group 1 mice (N=10) were
injected intramuscularly, once (no boosting), with Vaccine D
comprising 1.5 micrograms of rHA antigen and 12.5 micrograms of
RNA-based polyI:C adjuvant formulated in 50 microliters of
lyophilized liposome/polyI:C/hydrophobic carrier as described
above. Group 2 mice (N=10 at weeks 3 and 4 reduced to N=9 at weeks
6 and 9 due to unplanned non-vaccine related termination of one
animal) were vaccinated twice (day 0 and day 28) with a control
alum vaccine comprising 1.5 micrograms of rHA and 100 micrograms of
alum adjuvant suspended in 50 millimolar phosphate buffer. Serum
samples were collected from all mice at 3, 4, 6 and 9 weeks
post-immunization. rHA antibody titers in these sera were examined
by ELISA as described in Example 1.
[0166] Group 2 mice generated an antigen-specific antibody response
only after the administration of 2 doses (primary immunization and
boost) of an alum-adjuvanted control vaccine. Group 1 mice,
vaccinated with a single dose of the lyophilized
liposome/polyI:C/hydrophobic carrier formulation, yielded
significantly enhanced endpoint titers compared to those of Group 2
at all time points tested despite that Group 2 animals were
vaccinated twice. Group 2 mice recorded background titers 3 weeks
after the primary vaccination and one individual generated a
maximum titer of 1/8,000 (log 10 equal to 3.39) at 4 weeks. After
the boost, Group 2 mice generated titers up to 1/64,000 (log 10
value of 4.81) at 6 and 9 weeks post-immunization. Group 1 mice,
vaccinated with the vaccine corresponding to the invention, were
able to generate endpoint titers up to 1/128,000 (log 10 of 5.11)
at 3 and 4 weeks post-vaccination and 1/512,000 (a log 10 value of
5.71) at 6 and 9 weeks post-immunization. These results confirm,
using a different mouse species than the one used in Example 3,
that a single dose of lyophilized liposome/hydrophobic carrier
formulations containing a polyI:C adjuvant is capable of generating
a significantly enhanced in vivo humoral immune response compared
to even a boosted, aqueous/alum control vaccination. Antibody
levels were 24 times higher than a single dose of the control
vaccine at week 4 post-vaccination (P value<0.001) and 9 times
higher than two doses of the control vaccine at the later time
point of 9 weeks post-vaccination (P value<0.01). Furthermore,
results from Examples 3 and 6 indicate that the polyI:C adjuvant
can be incorporated into the lyophilized liposome/hydrophobic
carrier formulation either before or after liposome extrusion.
Example 7
[0167] Pathogen free, female BALB/c mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0168] As in Examples 1 through 6, H5N1 recombinant hemagglutinin
protein, corresponding to the hemagglutinin glycoprotein on the
surface of the H5N1 influenza virus, was purchased from Protein
Sciences (Meridien, Conn., USA). This recombinant protein,
hereafter designated rHA, was used as a model antigen to test the
efficacy of vaccine formulations. rHA was used at 1.5 micrograms
per 50 microliter dose.
[0169] In this example, the lyophilized
liposome/polyI:C/hydrophobic carrier was administered
intramuscularly once (no boosting) or subcutaneously once (no
boosting) to evaluate the generation of antigen-specific cytotoxic
lymphocyte response.
[0170] To formulate the vaccine corresponding to the invention, the
same procedures as described in Example 6 were used. In summary,
liposomes were formulated by hydrating a 10:1 (w:w) homogeneous
mixture of S100 lecithin and cholesterol (Lipoid GmbH, Germany) in
the presence of rHA in phosphate buffer followed by the addition of
RNA-based polyI:C (Pierce, Rockford, Ill., USA). The liposome
suspension was lyophilized and resuspended in a mineral oil carrier
(Montanide ISA 51.TM., SEPPIC, France). Each vaccine dose (Vaccine
D) consisted of 50 microliters of the above described formulation
containing liposomes (6.6 mg of S100/cholestrol lipids), rHA
antigen (1.5 micrograms), polyI:C adjuvant (12.5 micrograms), and
the mineral oil carrier. This vaccine formulation will be referred
to as lyophilized liposome/polyI:C/hydrophobic carrier. Mice in
Group 1 (n=4) received this formulation intramuscularly as in
Example 6. Group 2 mice (n=4) received this vaccine
subcutaneously.
[0171] Mice in Group 3 (n=4) were vaccinated with the control alum
vaccine consisting of 1.5 micrograms of rHA and 100 micrograms of
Imject Alum adjuvant (Pierce, Rockford, Ill., USA) in 50
microliters of 50 millimolar phosphate buffer (pH 7.0). Mice were
injected intramuscularly once (no boost). Group 4 mice (n=2) were
naive and did not receive any immunization.
[0172] Twenty-two days after vaccination, animals were euthanized
by carbon dioxide induced asphyxiation. The spleens were collected
and individual single cell suspensions prepared using standard
procedures. Red blood cells were lysed using ACK lysis buffer (0.15
M NH4Cl, 10 mM KHC03, 0.1 mM Na2EDTA in distilled H20). To augment
the antigen specific T cells, splenocytes were cultured at
1.times.10 6 cells per millilitre in RPMI 1640 (Invitrogen,
Burlington, ON, Canada) complete media containing 1%
Penicillin/Streptomycin/Glutamine, 0.1% 2-mercaptoethanol
(Sigma-Aldrich, St. Louis, Mo., USA), and 10% fetal bovine serum
(Hyclone, Logan, Utah, USA) supplemented with 20 units per
millilitre of recombinant human IL-2 (Sigma-Aldrich) and 10
micrograms per millilitre rHA for 4 days at 37.degree. C., 5%
carbon dioxide. Tri-colour flow cytometric analysis was performed
on splenocytes to detect antigen-specific CD8+ T cells. Cells were
blocked with a 10 minute treatment at room temperature of FC-block
(eBioscience, San Diego, Calif., USA). Cells were then stained with
phycoerythrin (PE)-labeled IYSTVASSL (I9L)/H2-Kd pentamer obtained
from Proimmune (Bradenton, Fla., USA) for 20 minutes at 4.degree.
C. I9L is the H2-Kd immunodominant epitope of rHA (518-528), and
the pentamer reagent detects MHC I presentation of this epitope by
the mouse. Cells were then stained with anti-CD19-fluorescein
isothiocyanate (FITC) (eBioscience) and
anti-CD8.beta.-Allophycocyanin (APC) (eBioscience) for 30 minutes
at 4.degree. C. protected from light, washed and fixed in 50
millimolar phosphate buffer (pH 7.0) with 0.1% paraformaldehyde.
5.times.10 5 cells were acquired on a FACSCalibur.TM. flow
cytometer (BD Bioscience, Missisauga, ON, Canada) and analysed
using WinList 6.0 software (Verity Inc, Topsham, Me., USA). Results
were gated based on forward and side scatter, and antigen-specific
CD8 T cells were defined as pentamer positive, CD8.beta. positive
and CD19 negative. Statistical analysis was performed using
two-tailed Students' T test.
[0173] Mice vaccinated with the control alum-based formulation
generated a small population of antigen-specific CD8 T cells
(0.045%). Mice vaccinated with the lyophilized
liposome/polyI:C/hydrophobic carrier formulation of the present
invention, delivered by the intramuscular or subcutaneous route,
generated a significantly higher population of antigen-specific CD8
T cells (0.23% and 0.17% respectively; p=<0.025 for both
compared to alum control). These results demonstrate that rHA
formulated in the invention can be delivered intramuscularly or
subcutaneously and generate a significantly higher antigen-specific
CD8+ T cell population representative of a cellular immune response
compared to a conventional vaccine formulation using alum.
Example 8
[0174] Pathogen free, female CD-1 mice, 6-8 weeks of age, and
female New Zealand White rabbits, 2-3 kilograms in weight, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filtered air circulation.
[0175] As in Examples 1 through 7, H5N1 recombinant hemagglutinin
protein, corresponding to the hemagglutinin glycoprotein on the
surface of the H5N1 influenza virus, was purchased from Protein
Sciences (Meridien, Conn., USA). This recombinant protein,
hereafter designated rHA, was used as a model antigen to test the
efficacy of vaccine formulations. rHA was used at 0.5 micrograms
per 50 microliter dose in mice and 2 micrograms per 200 microliter
dose in rabbits.
[0176] Vaccine efficacy was assessed by hemagglutination inhibition
assays (HAI) conducted by Benchmark Biolabs (Lincoln, Nebr., USA).
Briefly, serum samples were pre-treated with a receptor destroying
enzyme and pre-absorbed to chicken red blood cells to avoid any
non-specific hemagglutination inhibition reactions. Serial
dilutions of sera were then incubated with 0.7% equine red blood
cells, 0.5% bovine serum albumin and 8 HA units of
A/Vietnam/1203/2004[H5N1] influenza virus. Calculated titers
represent the highest dilution at which the serum sample can
completely inhibit hemagglutination of the red blood cells.
[0177] To formulate the first vaccine corresponding to the
invention, a 10:1 (w:w) homogenous mixture of S100 lecithin and
cholesterol (Lipoid GmbH, Germany) was hydrated in the presence of
rHA in phosphate buffer to form liposomes with encapsulated rHA and
followed by the addition of RNA-based polyI:C (Pierce, Rockford,
Ill., USA). Briefly, 10 micrograms of rHA were first suspended in
650 microliters of 50 millimolar phosphate buffer (pH 7.0) then
added to 132 milligrams of the S100 lecithin/cholesterol mixture to
form approximately 800 microliters of a liposome suspension
encapsulating the rHA antigen. The liposome preparation was then
extruded by passing the material through a manual mini-extruder
(Avanti, Alabaster, Ala., USA) fitted with a 200 nanometer
polycarbonate membrane. 240 micrograms of polyI:C adjuvant in 50
millimolar phosphate buffer (pH 7.0) were added to sized liposomes.
Liposomes were then lyophilized using the Virtis Advantage freeze
dryer (SP Industries, Warminister, Pa., USA). The lyophilized
material was reconstituted with a mineral oil carrier
(Montanide.TM. ISA 51, supplied by Seppic, France) up to the
original 1 milliliter volume of solublized liposomes. Each vaccine
dose as delivered to mice, consisted of 50 microliters of the above
described formulation combining liposomes, rHA antigen, polyI:C
adjuvant, and the mineral oil carrier. These vaccine formulations
will be referred to as lyophilized liposome/polyI:C/hydrophobic
carrier.
[0178] To formulate the second vaccine, also corresponding to the
invention, the same procedures described above were used with the
following exceptions: following the formation of liposomes
encapsulating rHA antigen, the liposome preparation was extruded by
passing the material through a manual mini-extruder fitted with two
400 nanometer polycarbonate membranes. 250 micrograms of the
RNA-based polyI:C adjuvant in 50 millimolar phosphate buffer (pH
7.0) was added to sized liposomes to dilute the preparation to 1
milliliter. Liposomes were then lyophilized using the Virtis
Advantage freeze dryer and the lyophilized material reconstituted
to the original 1 milliliter using a mineral oil carrier
(Montanide.TM. ISA 51, Seppic, France). Each vaccine dose delivered
to rabbits consisted of 200 microliters of the above described
formulation containing liposomes, rHA antigen, polyI:C adjuvant,
and the mineral oil carrier. This vaccine formulation will also be
referred to as lyophilized liposome/polyI:C/hydrophobic
carrier.
[0179] The efficacy of the lyophilized liposome formulations
described above was tested using two different animal models.
Animals were vaccinated with comparable formulations; the injection
volume was adjusted as appropriate for the size of the animals. One
group of mice (N=5) were injected intramuscularly with Vaccine F
comprising 0.5 micrograms of rHA antigen and 12 micrograms of
polyI:C adjuvant formulated in 50 microliters of lyophilized
liposome/polyI:C/hydrophobic carrier as described above. One group
of rabbits (N=5) were injected subcutaneously with Vaccine E
comprising 2 micrograms of rHA antigen and 50 micrograms of polyI:C
adjuvant formulated in 200 microliters of lyophilized
liposome/polyI:C/hydrophobic carrier as described above. All
animals were bled before injection and then again at either 4 or 5
weeks post-immunization. HAI titers in these sera were examined by
the H5N1 hemagglutination inhibition assay described above.
[0180] By 4 or 5 weeks post-vaccination with lyophilized
liposome/polyI:C/hydrophobic carrier formulations both the mice and
rabbits generated HAI titers that indicate protection against
influenza H5N1. A HAI serum titer of 40 (log 10 equal to 1.60) is
typically accepted to mean an individual has a protective level of
antibodies targeting a specific strain of influenza. At 5 weeks
post-vaccination mice generated titers ranging from 128 (log 10 of
2.11) to 512 (log 10 of 2.71). At 4 weeks post-immunization rabbits
generated HAI titers ranging from 64 (log 10 equal to 1.81) up to
1024 (log 10 of 3.01). It is generally accepted that a single
vaccination of rHA used at the dosages described above is incapable
of inducing the high HAI titers achieved in all vaccinated subject.
Titers of this magnitude, generated in two different animal models,
show that the lyophilized liposome/polyI:C/hydrophobic carrier
formulations is particularly effective in generating strong
antibody levels in the protective range (HAI>20 or log
value>1.3) in all vaccinated subject in as little as 4 weeks
following vaccination.
Example 9
[0181] Pathogen free, female CD1 mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0182] The amyloid .beta. protein fragment (1-43) was purchased
from Anaspec (San Jose, Calif., USA) and used as a model antigen to
test the efficacy of vaccine formulations. This peptide, hereafter
referred to as .beta.-amyloid, has a molecular weight of
approximately 4,600 daltons and is associated the formation of
plaques in the brains of Alzheimer's patients. .beta.-amyloid was
used at 10 micrograms per 100 microliter dose.
[0183] The 21 amino acid peptide FNNFTVSFWLRVPKVSASHLE, hereafter
referred to as F21 E, was purchased from NeoMPS (San Diego, Calif.,
USA). This tetanus toxoid peptide (amino acids 947-967) is
identified as being a T-helper epitope. F21 E was used as a model
T-helper epitope to test the efficacy of vaccine formulations; it
was used at 20 micrograms per 100 microliter dose.
[0184] As in Examples 1 through 6, vaccine efficacy was assessed by
enzyme-linked immunosorbent assay (ELISA). The same procedures as
described in Example 1 were used with changes to allow for the
detection of .beta.-amyloid specific antibodies. Briefly, a 96-well
microtiter plate is coated with antigen (.beta.-amyloid, 1
microgram/milliliter) overnight at 4 degrees Celsius, blocked with
3% gelatin for 30 minutes, then incubated overnight at 4 degrees
Celsius with serial dilutions of sera, typically starting at a
dilution of 1/1000. A secondary reagent (protein G conjugated to
alkaline phosphatase, EMD chemicals, Gibbstown, N.J., USA) is then
added to each well at a 1/500 dilution for one hour at 37 degrees
Celsius. Following a 60 minute incubation with a solution
containing 1 milligram/milliliter 4-nitrophenyl phosphate disodium
salt hexahydrate (Sigma-Aldrich Chemie GmbH, Switzerland), the 405
nanometer absorbance of each well is measured using a microtiter
plate reader (ASYS Hitech GmbH, Austria). Endpoint titers are
calculated as described in Frey A. et al (Journal of Immunological
Methods, 1998, 221:35-41). Calculated titers represent the highest
dilution at which a statistically significant increase in
absorbance is observed in serum samples from immunized mice versus
serum samples from naive, non-immunized control mice. Titers are
presented as log 10 values of the endpoint dilution.
[0185] To formulate vaccine described herein, a 10:1 w:w homogenous
mixture of S100 lecithin and cholesterol (Lipoid GmbH, Germany) was
hydrated in the presence of a .beta.-amyloid and F21E solution in
phosphate buffered saline (pH 7.4) to form liposomes with
encapsulated antigen and T-helper. In brief, 100 micrograms of
.beta.-amyloid and 200 micrograms of F21E were first suspended in
300 microliters of phosphate buffered saline (pH 7.4) then added to
132 milligrams of the S100 lecithin/cholesterol mixture to form
approximately 450 microliters of a liposome suspension
encapsulating the .beta.-amyloid antigen and F21E T-helper. The
liposome preparation was extruded by passing the material through a
manual mini-extruder (Avanti, Alabaster, Ala., USA) fitted with a
400 nanometer polycarbonate membrane. For every 450 microliters of
liposome suspension containing .beta.-amyloid and F21E, 2
milligrams of Imject Alum adjuvant (Pierce, Rockford, Ill., USA)
was added. For every 500 microliters of a
liposome/antigen/T-helper/adjuvant suspension, an equal volume of a
mineral oil carrier (known as Montanide.TM. ISA 51, supplied by
Seppic, France) was added to form a water-in-oil emulsion with the
liposome suspension contained within the water phase of the
emulsion and the oil forming a continuous hydrophobic phase. Each
vaccine dose consisted of 100 microliters of the above-described
emulsion containing liposomes, .beta.-amyloid antigen, F21E
T-helper, alum adjuvant, and the mineral oil carrier. This vaccine
formulation will be referred to as liposome/alum/hydrophobic
carrier.
[0186] To formulate the vaccine corresponding to the invention, the
same procedures described above were used with the following
exception: following the formation of liposomes encapsulating
.beta.-amyloid and F21E, and after extruding the liposome
suspension through a 400 nanometer polycarbonate membrane, 100
micrograms of RNA-based polyI:C adjuvant (Pierce, Rockford, Ill.,
USA) were added to every 450 microliters of liposomes. For every
500 microliters of a liposome/antigen/T-helper/adjuvant suspension,
an equal volume of a mineral oil carrier (Montanide.TM. ISA 51,
Seppic, France) was added to form a water-in-oil emulsion with the
liposome suspension contained in the water phase of the emulsion
and the oil forming the continuous phase. Each vaccine dose
consisted of 100 microliters of the above described emulsion
containing liposomes, .beta.-amyloid antigen, F21E T-helper,
polyI:C adjuvant, and the mineral oil carrier. This particular
formulation will be referred to as liposome/polyI:C/hydrophobic
carrier.
[0187] The efficacy of the two emulsion formulations described
above was compared. Two groups of mice (9 mice per group) were
injected intraperitoneally with liposome vaccine formulations as
follows: Group 2 mice were vaccinated with Vaccine G comprising 10
micrograms of .beta.-amyloid and 20 micrograms of F21E formulated
in 100 microliters of liposome/alum/hydrophobic carrier as
described above. Each vaccine dose effectively contained 200
micrograms of alum. Group 1 mice were vaccinated with Vaccine H
comprising 10 micrograms of .beta.-amyloid antigen and 20
micrograms F21 E formulated in 100 microliters of
liposome/polyI:C/hydrophobic carrier as described above. Each
vaccine dose effectively contained 10 micrograms of polyI:C. Serum
samples were collected from all mice at 4, 8 and 12 weeks
post-immunization. Antibody titers in these sera were examined by
ELISA as described above.
[0188] Group 2 mice, vaccinated with a single dose of a
liposome/alum/hydrophobic carrier formulation, generated a
detectable antigen-specific antibody response as was expected. The
endpoint titers at 4 and 8 weeks post-vaccination were up to
1/32,000 (log 10 value of 4.51) and at 12 weeks they were up to
1/64,000 (log 10 of 4.81). The presence of such antibody responses
confirms that a genuine immune response was generated as a result
of vaccination. Group 1 mice that were injected once with the
formulation corresponding to the invention were able to generate an
enhanced immune response with endpoint titers reaching up to
1/256,000 (log 10 value of 5.41) at 4, 8 and 12 weeks
post-vaccination. The titers generated with the invention were 7
times higher on average at every time point relative to titers
generated by the control formulation containing the generic
adjuvant alum. The increase in titers achieved with the invention
was statistically significant (P value<0.01 at weeks 8 and 12
post-vaccination). These results confirm through the use of a
different antigen model that liposome/hydrophobic carrier
formulations containing a polyI:C adjuvant are capable of
generating a significantly enhanced in vivo immune response
compared to a liposome/alum/hydrophobic vaccination.
Example 10
[0189] Pathogen free, female CD1 mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0190] The H5N1 recombinant hemagglutinin protein was purchased
from Protein Sciences (Meridien, Conn., USA). This recombinant
protein has an approximate molecular weight of 72,000 daltons and
corresponds to the hemagglutinin glycoprotein, an antigenic protein
present on the surface of the H5N1 influenza virus. This
recombinant protein, hereafter designated rHA, was used as a model
antigen to test the efficacy of vaccine formulations. rHA was used
at 0.5 micrograms per 50 microliter dose.
[0191] Both the humoral (TH1) and cellular (TH2) immune responses
were assessed by enzyme-linked immunosorbent assay (ELISA), a
method that allows the detection of antigen-specific antibody
levels in the serum of immunized animals. Briefly, a 96-well
microtiter plate is coated with antigen (rHA, 1
microgram/milliliter) overnight at 4 degrees Celsius, blocked with
3% gelatin for 30 minutes, then incubated overnight at 4 degrees
Celsius with serial dilutions of sera, typically starting at a
dilution of 1/2000. A secondary antibody, anti-IgG, is then added
to each well at a 1/2000 dilution for one hour at 37 degrees
Celsius. For the detection of IgG2A antibodies, indicative of a TH1
cellular response, goat anti-mouse IgG2A (SouthernBiotech,
Birmingham, Ala., USA) was used. For the detection of a TH2 humoral
response a goat anti-mouse IgG1 (SouthernBiotech, Birmingham, Ala.,
USA) secondary reagent was used. Following a 60 minute incubation
with a solution containing 1 milligram/milliliter 4-nitrophenyl
phosphate disodium salt hexahydrate (Sigma-Aldrich Chemie GmbH,
Switzerland), the 405 nanometer absorbance of each well is measured
using a microtiter plate reader (ASYS Hitech GmbH, Austria).
Endpoint titers are calculated as described in Frey A. et al
(Journal of Immunological Methods, 1998, 221:35-41). Calculated
titers represent the highest dilution at which a statistically
significant increase in absorbance is observed in serum samples
from immunized mice versus serum samples from naive, non-immunized
control mice. Titers are presented as log 10 values of the endpoint
dilution.
[0192] To formulate vaccines corresponding to the invention, a 10:1
w:w homogenous mixture of S100 lecithin and cholesterol (Lipoid
GmbH, Germany) was hydrated in the presence of a rHA solution in
phosphate buffer to form liposomes with encapsulated rHA and
followed by the addition of RNA-based polyI:C (Pierce, Rockford,
Ill., USA) as described in Example 8. In brief, 10 micrograms of
rHA were first suspended in 650 microliters of 50 millimolar
phosphate buffer (pH 7.0) then added to 132 milligrams of the S100
lecithin/cholesterol mixture to form approximately 800 microliters
of a liposome suspension encapsulating the rHA antigen. The
liposome preparation was then extruded by passing the material
through a manual mini-extruder (Avanti, Alabaster, Ala., USA)
fitted with a 200 nanometer polycarbonate membrane. PolyI:C
adjuvant in 50 millimolar phosphate buffer (pH7.0) was added to
sized liposomes to dilute the preparation to 1 milliliter. For the
"high dose" polyI:C formulation, 240 micrograms of polyI:C in
phosphate buffer was added and for the "low dose" polyI:C
formulation 50 micrograms of polyI:C were added. Liposomes were
then lyophilized using the Virtis Advantage freeze dryer (SP
Industries, Warminister, Pa., USA). The lyophilized material was
reconstituted with a mineral oil carrier (Montanide.TM. ISA 51,
supplied by Seppic, France) up to the original 1 milliliter volume
of solublized liposomes. Each vaccine dose consisted of 50
microliters of the above described formulation combining liposomes,
rHA antigen, polyI:C adjuvant, and the mineral oil carrier. These
vaccine formulations will be referred to as lyophilized
liposome/polyI:C (high)/hydrophobic carrier and lyophilized
liposome/polyI:C (low)/hydrophobic carrier.
[0193] The TH1 and TH2 responses generated, as a result of
vaccination with the lyophilized liposome formulations containing
polyI:C adjuvant, were compared. Two groups of mice (N=5 per
groups) were injected intramuscularly with 50 microliters of either
Vaccine E comprising 0.5 micrograms rHA and 12 micrograms polyI:C
formulated as lyophilized liposomes/polyI:C (high)/hydrophobic
carrier (Group 1) or Vaccine I comprising 0.5 micrograms rHA and
2.5 micrograms polyI:C formulated as lyophilized liposomes/polyI:C
(low)/hydrophobic carrier (Group 2). Serum samples were collected
at 5 weeks post-immunization and IgG1 and IgG2A antibody titers
examined as described above.
[0194] Group 1 mice generated IgG1 titres up to 2,048,000 (log 10
value of 6.31) at 5 weeks post-immunization which is comparable to
the humoral response results of the similar lyophilized
liposomes/polyI:C/hydrophobic carrier formulation used in Example
3. The IgG2A titers, indicative of a cellular response, were up to
4,096,000 (log 10 equal to 6.61) at 5 weeks post-vaccination. Group
2 mice, vaccinated with a lower dose of polyI:C, generated at 5
weeks post-vaccination IgG1 titers up to 4,096,000 (log 10 of 6.61)
and IgG2A titers also up to 4,096,000. Results show that polyI:C
adjuvant formulated at various concentrations in a lyophilized
liposome/hydrophobic carrier formulation is able to generate both
humoral (TH2) and cellular (TH1) immune responses. These results
suggest that the formulations described above are capable of
generating cellular and humoral immune responses in vaccinated
subjects.
Example 11
[0195] Pathogen free, female C57BL6 mice, 4-6 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0196] The antigen used in vaccine formulations was a fusion
protein consisting of the H2-Db immunodominant epitope of HPV16 E7
(49-57; RAHYNIVTF) fused to the universal T helper epitope PADRE.
This antigen, hereafter referred to as FP, was synthesized by
Anaspec Inc. (San Jose, Calif.). The adjuvant was a RNA-based poly
inosine-cytosine RNA molecule provided by Sigma-Genosys (St. Louis,
Mo.).
[0197] The efficacy of the invention comprising liposomes, an
RNA-based poly I:C molecule, and a hydrophobic carrier was tested
in vivo using a C3 tumor challenge model. C3 cells contain the
human papilloma virus 16 (HPV16) genome and as a result, present on
their surface the HPV16 E7 epitope (amino acids 49-57; RAHYNIVTF)
which can be targeted by vaccination. C3 cells grow into measurable
solid tumors when injected subcutaneously. Three groups of mice
(n=8 per group) were implanted subcutaneously in the flank with the
HPV16 E7 expressing tumor cell line C3 (5.times.10 5 cells/mouse)
on day 0. On day 8, mice in Groups 1 and 2 were vaccinated
subcutaneously in the opposing flank with 100 microliters of
vaccine. Group 3 mice received PBS only and served as the tumor
growth control. Tumor volume was measured once a week using
callipers to record the shortest diameter and longest diameter for
5 weeks post implantation. Tumor volume was calculated using the
following formula: longest measurement.times.(shortest measurement)
2 divided by 2.
[0198] The control vaccine (conventional emulsion) used to immunize
Group 1 was formulated by mixing 300 micrograms of FP antigen and 3
milligrams of PolyI:C adjuvant in 1 millilitre of PBS. For every
500 microliters of antigen/adjuvant suspension, an equal volume of
a mineral oil carrier (Montanide.TM. ISA 51, supplied by Seppic,
France) was added to form a water-in-oil emulsion. Each vaccine
dose consisted of 100 microliters of the described emulsion
containing FP antigen (15 micrograms) and polyI:C adjuvant (150
micrograms) and the mineral oil carrier. This vaccine formulation
will be referred to as polyI:C/hydrophobic carrier.
[0199] To formulate vaccine (Vaccine K) corresponding to the
invention for Group 2, the same procedures as described in Example
1 were used. Briefly, 150 micrograms of FP antigen was mixed with a
DOPC lecithin/cholesterol mixture (10:1, w:w; Lipoid GmbH, Germany)
dissolved in tert-butanol and lyophilized. Liposomes were
formulated by adding 1 millilitre of 50 millimolar phosphate buffer
(pH 7.0) containing 1.5 milligrams of polyI:C. The liposome
preparation was extruded by passing the material through a manual
mini-extruder (Avanti, Alabaster, Ala., USA) fitted with a 200
nanometer polycarbonate membrane. Liposome size was confirmed at
200 nanometers using a Malvern Particle Size Analyzer
(Worchestershire, United Kingdom). For every 500 microliters of a
liposome/antigen/adjuvant suspension, an equal volume of a mineral
oil carrier (Montanide.TM. ISA 51, supplied by Seppic, France) was
added to form a water-in-oil emulsion with the liposome suspension
contained within the water phase of the emulsion and the oil
forming a continuous hydrophobic phase. Each vaccine dose consisted
of 100 microliters of the described emulsion containing liposomes
(13.2 milligrams of DOPC/cholesterol), FP antigen (15 micrograms),
polyI:C adjuvant (150 micrograms), and the mineral oil carrier.
This vaccine formulation will be referred to as
liposome/polyI:C/hydrophobic carrier.
[0200] The results of this experiment are shown in FIG. 11. Group 1
mice had partial protection from tumor growth and started to
develop measurable tumors by week 4 post implantation. The mice in
Group 2, vaccinated with the invention, developed significantly
smaller tumors that were only detectable by week 5 (p<0.1). The
mice in the control group developed tumors with expected kinetics,
starting at week 3 post implantation.
[0201] These results indicate that tumor-specific antigens
formulated in the liposome/polyI:C/hydrophobic carrier formulation
was more effective at therapeutically treating an established tumor
in mice than when formulated with polyI:C/hydrophobic carrier. The
optimal therapeutic effect could only be achieved when liposomes
were present in the formulation, clearly indicating that liposomes
are a critical component of the invention.
Example 12
[0202] Pathogen free, female C57BL6 mice, 4-6 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0203] As in Example 11, the antigen used in vaccine formulations
was a fusion protein consisting of the H2-Db immunodominant epitope
of HPV16 E7 (49-57; RAHYNIVTF) fused to the universal T helper
epitope PADRE. This antigen, hereafter referred to as FP, was
synthesized by Anaspec Inc. (San Jose, Calif.). The adjuvant was a
DNA-based poly inosine-cytosine DNA molecule consisting of 13 (IC)
repeats and synthesized by Operon MWG (Huntsville, Ala., USA).
[0204] The efficacy of the invention comprising liposomes, a
DNA-based polyI:C and a hydrophobic carrier was tested in vivo
using the C3 tumor challenge model described earlier. Four groups
of mice (n=8 per group) were implanted subcutaneously in the flank
with the HPV16 E7 expressing tumor cell line C3 (5.times.10 5
cells/mouse) on day 0. On day 5, mice in Groups 1 to 3 were
vaccinated subcutaneously in the opposing flank with vaccine. Group
4 mice received PBS only and served as the tumor growth control.
Tumor volume was measured once a week using callipers to record the
shortest diameter and longest diameter for 5 weeks post
implantation. Tumor volume was calculated using the following
formula: longest measurement.times.(shortest measurement{circumflex
over (0)}2 divided by 2.
[0205] Mice in Group 1 were vaccinated with Vaccine L comprising a
liposome/antigen/poly IC/hydrophobic carrier. The vaccine was
formulated as in Example 11. Each dose volume was 100 microliters
and contained liposomes, FP (10 micrograms), poly IC (20
micrograms) and was emulsified with the mineral oil carrier. Mice
in Group 2 were vaccinated with Vaccine M comprising a lyophilized
liposome/antigen/poly IC/hydrophobic carrier. Briefly, a 10:1 (w:w)
homogenous mixture of DOPC lecithin and cholesterol (Lipoid GmbH,
Germany) was hydrated in the presence of 200 micrograms of FP and
400 micrograms of poly IC in 0.5% PEG/water to form 1 milliliter of
liposomes with encapsulated antigen and adjuvant. The liposome
preparation was extruded by passing the material 20 times through a
manual extruder (Avanti, Alabaster, Ala., USA) fitted with two 400
nanometer polycarbonate membranes. Liposome size was confirmed at
200 nanometers using a Malvern Particle Size Analyzer
(Worchestershire, United Kingdom). Liposomes containing antigen and
adjuvant were lyophilized using the Virtis Advantage freeze dryer
(SP Industries, Warminister, Pa., USA). The lyophilized material
was reconstituted in oil up to the original volume of solublized
liposomes with a mineral oil carrier (Montanide.TM. ISA 51, Seppic,
France). Each dose volume was 50 microliters and contained
liposomes (6.6 mg of DOPC/cholesterol), FP (10 micrograms), polyI:C
(20 micrograms) and the mineral oil carrier. Mice in Group 3 were
vaccinated with a lyophilized liposome/antigen/hydrophobic carrier
formulated as for Group 2, except without the poly IC adjuvant
(adjuvant control).
[0206] Results of this experiment are shown in FIG. 12. Group 1 and
group 2 mice did not develop measurable tumors throughout the
length of the study. Mice in Group 3, which were vaccinated with
the lyophilized liposome formulation with FP but no adjuvant,
started to develop tumors at week 3 post implantation. Mice in the
PBS control group developed tumors with expected kinetics, starting
at week 3 post implantation.
[0207] These results indicate that vaccine formulations of the
present invention require a poly IC adjuvant to be efficacious in a
tumor challenge model. In this example, a DNA-based polyI:C
adjuvant formulated in a liposome/hydrophobic carrier or in a
lyophilized liposome/hydrophobic carrier formulation generated an
effective immune response with therapeutic effect with as little as
one immunization.
Example 13
[0208] Pathogen free, female BALB/c mice, 6-8 weeks of age, were
obtained from Charles River Laboratories (St Constant, QC, Canada)
and were housed according to institutional guidelines with water
and food ad libitum, under filter controlled air circulation.
[0209] As in previous examples, H5N1 recombinant hemagglutinin
protein, corresponding to the hemagglutinin glycoprotein on the
surface of the H5N1 influenza virus, was purchased from Protein
Sciences (Meridien, Conn., USA). This recombinant protein,
hereafter designated rHA, was used as a model antigen to test the
efficacy of vaccine formulations. rHA was used at 1.5 micrograms
per 50 microliter dose.
[0210] Vaccine efficacy was assessed by immunofluorescence staining
of memory CD8 cells, similar to the method described in Example 7.
Syngenic splenocytes from BALB/c mice were activated for 48 hours
at 37 degrees Celsius with 10 micrograms/milliliter of
lipopolysaccharide and the resulting blasts were treated with 50
micrograms/milliliter mitomycin C for 20 minutes at room
temperature. Following repeated washes, the activated blast cells
were used as antigen presenting stimulator cells for expanding
flu-specific CD8 memory cells from vaccinated mice. Spleen cells
from naive or immunized mice were cultured with blast cells at a
ratio of 5:1 and cultures were stimulated with rHA at 0.1
micrograms/milliliter for 6 days at 37 degrees Celsius, 5 percent
carbon dioxide. Harvested cells were used for immunofluorescence
staining with anti-CD8-fluorescein isothiocyanate (FITC)
(eBioscience, San Diego, Calif., USA) antibodies and phycoerythrin
(PE)-conjugated Pro5 Flu-pentamer reagent (H2-Kd, IYSTVASSL,
Proimmune, Oxford, UK). Anti-CD19-allophycocyanin (APC)
(eBioscience) was also used to exclude any non-specific binding of
pentamer to B cells. Stained cells were collected on a FACSCalibur
flow-cytometer (BD Bioscience, Mississauga, ON, Canada) and data
analysis was done using WinList 6.0 software (Verity Software
House, Topsham, Me., USA). Results were gated based on forward and
side scatter, and antigen-specific CD8 T cells were defined as
pentamer positive, CD8 .beta. positive and CD19 negative.
Statistical analysis was performed using Students' T-test.
[0211] To formulate the vaccine corresponding to the invention, the
same procedures as described in Examples 6 and 7 were used. In
summary, a 10:1 (w:w) homogeneous mixture of S100 lecithin and
cholesterol (Lipoid GmbH, Germany) was hydrated in the presence of
rHA in phosphate buffer (pH 7.0), to form liposomes encapsulating
rHA, and followed by the addition of RNA-based polyI:C (Pierce,
Rockford, Ill., USA). The liposome suspension was extruded through
a semi-automatic extruder (Avestin, Ottawa, ON, Canada) and the
sized liposomes lyophilized (Virtis Advantage freeze dryer, SP
Industries, Warminister, Pa., USA) and reconstituted in a mineral
oil carrier (Montanide ISA 51.TM., SEPPIC, France). Each vaccine
dose consisted of 50 microliters of the above described formulation
containing liposomes, rHA antigen, polyI:C adjuvant, and the
mineral oil carrier. This vaccine formulation will be referred to
as lyophilized liposome/polyI:C/hydrophobic carrier.
[0212] The efficacy of the lyophilized liposome formulation
described above was compared to the efficacy of a control vaccine
consisting of 1.5 micrograms of rHA and 100 micrograms of Imject
Alum adjuvant (Pierce, Rockford, Ill., USA) in 50 microliters of 50
millimolar phosphate buffer (pH 7.0). Group 1 mice (N=5) were
injected intramuscularly, once (no boosting), with 1.5 micrograms
of rHA antigen and 12.5 micrograms of polyI:C adjuvant formulated
in 50 microliters of lyophilized liposome/polyI:C/hydrophobic
carrier as described above. This vaccine corresponds to the same
vaccine used in Examples 6 and 7 (vaccine D, the invention). Group
2 mice (N=5) were vaccinated twice (day 0 and day 28) with a
control vaccine consisting of 1.5 micrograms of rHA and 100
micrograms of alum adjuvant suspended in 50 millimolar phosphate
buffer. Twenty-one weeks post-vaccination, animals were euthanized
by carbon dioxide induced asphyxiation, the spleens were collected
and individual single cell suspensions prepared using standard
procedures. The presence of flu-specific CD8 memory T cells was
then assessed using the flu pentamer immunofluorescence staining
described above.
[0213] Mice vaccinated with the control alum-based formulation
generated a small population of antigen-specific CD8 memory T
cells, mean population size of 0.02 percent and considered
background (standard deviation 0.02 percent). Mice vaccinated with
the lyophilized liposome/polyI:C/hydrophobic carrier formulation
corresponding to the invention on the other generated a
significantly higher population (P<0.02) of antigen-specific CD8
memory T cells, mean population size of 0.51 percent (standard
deviation 0.10 percent). These results are significant as they
demonstrate that single dose lyophilized liposome/hydrophobic
carrier formulations containing polyI:C adjuvant generate a large,
long-lasting, antigen-specific CD8 memory T cell population whereas
an aqueous/alum control vaccine could not generate any significant
and lasting cellular response even after two immunizations.
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[0261] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0262] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural reference unless
the context clearly dictates otherwise. Unless defined otherwise
all technical and scientific terms used herein have the same
meaning as commonly understood to one of ordinary skill in the art
to which this invention belongs.
[0263] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
* * * * *
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