U.S. patent application number 10/292136 was filed with the patent office on 2003-04-17 for method to enhance an immune response of nucleic acid vaccination.
This patent application is currently assigned to SmithKline Beecham Biologicals, s.a.. Invention is credited to Bruck, Claudine, Dalemans, Wilfried, Friede, Martin, Mechelen, Marcelle Van.
Application Number | 20030072768 10/292136 |
Document ID | / |
Family ID | 10823687 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030072768 |
Kind Code |
A1 |
Dalemans, Wilfried ; et
al. |
April 17, 2003 |
Method to enhance an immune response of nucleic acid
vaccination
Abstract
This invention provides a method to enhance an immune response
of nucleic acid vaccination by simultaneous administration of a
polynucleotide and a polypeptide of interest.
Inventors: |
Dalemans, Wilfried;
(Hoegaarden, BE) ; Mechelen, Marcelle Van;
(Wagnelee, BE) ; Bruck, Claudine; (Rixensart,
BE) ; Friede, Martin; (Farnham, GB) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham Biologicals,
s.a.
|
Family ID: |
10823687 |
Appl. No.: |
10/292136 |
Filed: |
November 12, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10292136 |
Nov 12, 2002 |
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09581368 |
Jun 12, 2000 |
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6500432 |
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09581368 |
Jun 12, 2000 |
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PCT/EP98/08152 |
Dec 11, 1998 |
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Current U.S.
Class: |
424/185.1 ;
424/486; 514/44R |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 9/1647 20130101; A61K 2039/57 20130101; A61K 2039/70 20130101;
A61K 2039/53 20130101; A61K 39/39 20130101; A61K 2039/55505
20130101; A61P 43/00 20180101; C12N 2760/18534 20130101; A61K
39/245 20130101; A61K 39/12 20130101; A61K 2039/55555 20130101;
A61K 39/155 20130101; A61K 39/21 20130101; A61K 2039/545 20130101;
A61P 37/04 20180101; C12N 2710/16634 20130101; C12N 2740/16134
20130101 |
Class at
Publication: |
424/185.1 ;
514/44; 424/486 |
International
Class: |
A61K 048/00; A61K
039/38; A61K 039/00; A61K 009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 1997 |
GB |
9726555.7 |
Claims
What is claimed is:
1. A method to enhance an immune response of nucleic acid
vaccination by simultaneous administration of: (i) a polynucleotide
encoding for a polypeptide of interest; and (ii) the polypeptide of
interest.
2. The method of claim 1 wherein the nucleic acid is DNA.
3. The method of claim 1 wherein the nucleic acid is RNA.
4. The method of claim 2 wherein the DNA and protein are
admixed.
5. The method of claim 1 wherein the polypeptide is administered
0-10 days after the polynucleotide.
6. The method of claim 5 wherein the polypeptide is administered
within 3-7 days after the polynucleotide.
7. The method of claim 1 wherein the polypeptide is presented in a
delayed release formulation and administered at the same time as
the polynucleotide.
8. A method to enhance an immune response of polypeptide
vaccination by simultaneous administration of: (i) a nucleic acid
encoding for a polypeptide of interest; and (ii) the polypeptide of
interest.
9. The method of claim 8 wherein the nucleic acid is DNA.
10. The method of claim 8 wherein the nucleic acid is RNA.
11. The method of claim 9 wherein the DNA and protein are
admixed.
12. The method of claim 8 wherein the polypeptide is presented in a
delayed release formulation and administered at the same time as
the polynucleotide.
13. The method of claim 8 wherein the immune response is a Th1
response.
14. A pharmaceutical composition comprising DNA+polypeptide,
wherein the DNA encodes the polypeptide of interest and wherein the
ratio of DNA:Polypeptide is from 1000:1 to 1:1 (w/w).
15. The pharmaceutical composition of claim 14 wherein the
polypeptide is presented in a delayed release formulation.
16. The method of claim 15 wherein the polypeptide is coated with a
biodegradable polymer comprising poly(capro-lactone) or
poly(lactide-co-glycolide).
17. A method to prepare a pharmaceutical formulation according to
claim 14, which method comprises: purifying a polynucleotide
encoding for a polypeptide of interest; purifying a polypeptide of
interest; and admixing the combination thereof.
18. A method to prepare a pharmaceutical formulation according to
claim 15, which method comprises: purifying a polynucleotide
encoding for a polypeptide of interest; purifying a polypeptide of
interest; encapsulating the polypeptide of interest in a delayed
release formulation; and admixing the combination thereof.
19. A vaccine comprising DNA+polypeptide, wherein the DNA encodes
the polypeptide of interest, and wherein the ratio of
DNA:Polypeptide is from 1000:1 to 1:1 (w/w).
20. The vaccine of claim 19 wherein the polypeptide is presented in
a delayed release formulation.
21. The vaccine according to claim 19 comprising DNA+polypeptide
and a suitable adjuvant.
22. The vaccine according to claim 20 comprising DNA+polypeptide
presented in a delayed release formulation and a suitable
adjuvant.
23. The use of DNA+polypeptide admixed together in the manufacture
of a composition for use in enhancing the immune response of a
mammal.
24. The use of DNA+polypeptide wherein the polypeptide is presented
in a delayed release formulation admixed together in the
manufacture of a composition for use in enhancing the immune
response of a mammal.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method to enhance an immune
response of nucleic acid vaccination by simultaneous administration
of a polynucleotide and a polypeptide of interest.
BACKGROUND OF THE INVENTION
[0002] In 1990 Wolff and colleagues (Wolff et al., Science
247:1465-1468 (1990)) reported that nonreplicating plasmid DNA
encoding reporter genes could be internalized by muscle cells,
without the use of any transfection vehicle, and that the encoded
proteins were expressed following injection of the plasmid DNA. The
subsequent finding that the expressed protein of "naked" (i.e.,
devoid of agents which promote transfection) plasmid DNA was
immunogenic upon intramuscular inoculation has opened a new area of
research with significant clinical impact.
[0003] As such, vaccination with nucleic acids (NAVAC) has become a
relatively new approach for vaccinating against a multitude of
diseases. It involves the in vivo production of a polypeptide
within a host cell rather than the more conventional method of
administering a polypeptide (or an attenuated, or killed
microorganism) that was first produced (or cultured) in vitro.
NAVAC also differs from more conventional vaccines in that the
compound administered consists of nucleic acids, i.e., DNA or RNA,
encoding selected antigen(s). Upon injection of these nucleic
acids, they are taken up by the recipient cells of a mammalian host
and the antigen encoded by such nucleic acid(s) is subsequently
expressed. Thereafter, the presence of a foreign antigen within a
host can elicit a specific immune response directed against the
antigen. It has been shown that NAVAC induces humoral, i.e.,
antibodies, as well as cell mediated immunity, i.e., T helper cell
responses and cytotoxic lymphocytes (see, e.g., Corr et al., J.
Exp. Med. 184:1555-1560 (1996); Doe et al., Proc. Natl. Acad. Sci.
93:8578-8583 (1996)).
[0004] The induced immune responses have been shown to protect
against a subsequent challenge with the infectious organism from
which the antigen was originally obtained in a certain number of
animal models (e.g. Ulmer et al., Science 59:1745-1749 (1993);
Sedegah et al., Proc Natl Acad Sci USA 91:9866-9870 (1994);
Manickan et al., J. Immunol. 155:259-265 (1995)). NAVAC has been
shown to be much less efficient in larger animal species (when
compared to administration of adjuvanted proteins), and large
amounts of DNA have to be administered to achieve immunisation
levels comparable to that found in smaller animal species, e.g.,
mice (Gramzinski et al., Vaccine Research 5:173-183 (1996); Lu et
al., J. Virol. 70:3978-3991 (1996); Shiver et al.; Journal of
Pharmaceutical Sciences 85:1317-1324 (1996)). There is thus a need
to improve the efficacy of NAVAC, for example, by developing new
delivery and targeting mechanisms, new adjuvantation techniques,
other routes of administration, etc. It is the object of this
invention to provide another improvement to the efficacy of DNA
vaccination.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides a method for
enhancing the immune response of a nucleic acid vaccination by
administration of (i) a polynucleotide encoding a gene of interest,
which encodes a polypeptide and (ii) by also administering the
polypeptide simultaneously, that is, during the same ongoing immune
response. Preferably the administration of polynucleotide and
polypeptide occur within 0-10 days of each other. One preferred
embodiment is administration of polypeptide 3-7 days prior to
administration of polynucleotide. The polypeptide may be further
prepared prior to administration such that it is presented to the
immune system in such a way to provide a delayed release (e.g.,
encapsulated). In such a case, the polypeptide is preferably
administered at the same time (concurrently) as the
polynucleotide.
[0006] In another aspect, the present invention provides a method
for enhancing the immune response of a polypeptide vaccination by
administration of a nucleotide encoding a gene of interest, which
encodes a polypeptide of interest, and by also administering the
polypeptide simultaneously, that is, during the same ongoing immune
response.
[0007] In further related aspects, the present invention relates to
pharmaceutical compositions, vaccines, and methods to prepare such
compositions and vaccines, comprising a polynucleotide encoding a
gene of interest, and the corresponding polypeptide of interest,
where the ratio of polynucleotide to polypeptide is from 1000:1 to
1:1 (w/w). Optionally, the polypeptide is prepared prior to
administration such that it is presented to the immune system in
such a way to provide a delayed release.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 (Ig 14 post 2) shows the serological responses in the
immunised animals 2 weeks after the booster injection. The
combination of the two vaccine substrates strongly enhanced the
amount of RSV-FG specific antibodies compared to those obtained by
using the respective substrates separately ((DNA-F or FG protein)
2.times.).
[0009] FIG. 2 (IgG isotype 14 post 2) shows the isotype
distribution in the different vaccine groups. All the groups who
did receive a DNA vaccine at the first injection have a strong
content of IgG2a, indicative for a T helper response of type 1
(Th1), whereas the protein groups have a complete bias for the
opposite, that is the IgG1 isotype typical for a Th2 type
response.
[0010] FIG. 3 (Lymphoproliferation) shows the lymphoproliferation
of splenocytes from the different vaccine groups upon in vitro
restimulation with the FG antigen.
[0011] FIG. 4 shows the presence of CTL's in the different vaccine
groups.
[0012] FIG. 5 shows the total anti-gp120 IgG titers 14 days after
the second injection. IgG titers are strongly increased in the
group which received the two antigen forms (polynucleotide and
polypeptide). The polypeptide (protein) administration was delayed
for four days.
[0013] FIG. 6 shows the isotype distribution of anti-gp120 IgG
titers. Delayed protein delivery maintains considerable IgG2a
titers as observed for the DNA group, which is in contrast to the
protein-only group.
DETAILED DESCRIPTION OF THE INVENTION OF THE INVENTION
[0014] The present invention relates to a method for enhancing the
immune response of a mammal (e.g., mice, rabbits, primates, man) to
DNA vaccines by admixing two different compounds prior to
simultaneous administration. The first compound comprises a
polynucleotide (nucleic acid) such as DNA or RNA which encodes a
selected polypeptide that can stimulate protective immunity. The
second compound comprises a polypeptide, which preferably is the
same polypeptide (or substantially the same, i.e., having the same
immunodominant epitope(s)) encoded by the nucleic acid of this
invention. The enhancement relates to the total increase in immune
response, as determined by both humoral and cell mediated immune
responses. That is, there are measurable increases in one or more
of the following: total antibody titer (to the selected
polypeptide); in lymphoproliferation; and in CTL (cytotoxic T
lymphocyte) levels. Moreover, when nucleic acid such as DNA
encoding the gene of interest is admixed with the corresponding
polypeptide and (simultaneously) administered to a mammal, a
synergistic effect is observed. That is, not only is the DNA
vaccine capable of inducing an immune response in the presence of
protein (polypeptide), but the presence of such protein
(polypeptide) has been found to actually enhance the efficacy of
the DNA vaccine. Thus, one aspect of the present invention is a
composition comprising a polynucleotide and polypeptide for
enhancing an immune response. Optionally, the polypeptide may be
adjuvanted as well.
[0015] Conversely, the presence of specific nucleic acid (e.g., DNA
encoding the whole or substantial parts of the polypeptide) to a
polypeptide vaccine was found to increase that immune response as
well. That is, the interaction of a DNA/polypeptide mixture
enhances the efficacy of either individual component. This
enhancement is specific to the polynucleotide added, as a similar
polynucleotide not encoding the specific polypeptide was unable to
enhance the immune responses. One surprising aspect of the instant
invention was the fact that the DNA component and the polypeptide
component did not compete with each other in eliciting an immune
response, and that, to the contrary, a synergistic effect was
observed when DNA and protein were administered simultaneously.
[0016] The present invention differs from the "prime boost"
approach (Barnett et al., Vaccine 15:869-873 (1997)) where two
distinct vaccine preparations (one DNA, one protein) are prepared
and administered separately, at different times, and in a specific
order. That is, under the prime boost approach, a DNA molecule is
administered to "prime" the immune system and at some subsequent
time (weeks or months later) after the primary immune response is
established a protein is administered to boost the then
pre-existing immune response.
[0017] Another benefit of the present invention is that in certain
cases, where there is a predisposition to a strong immune response
(e.g., high percentage of responders to initial vaccination, high
level of antibody titers, CTL response, etc.), the combination of
both compounds (DNA+protein) can obviate the need for
immunostimulants.
[0018] In a further embodiment of this invention are included
formulations wherein the nucleic acid and protein antigen are
co-administered (preferably simultaneously), but the protein
antigen is encapsulated within a delayed-release vehicle which
liberates the protein antigen several days after administration.
Such formulations enhance the magnitude of the induced immune
response compared to nucleic acid alone, without biasing the immune
response.
[0019] Nucleic Acid
[0020] The polynucleotide materials used according to the present
invention comprise DNA or RNA sequences coding for polypeptides
that have useful therapeutic application, e.g., prophylactic or
therapeutic vaccines. According to the methods of the invention,
both expressible DNA and RNA can be delivered to cells to form
therein a polypeptide translation product. If the nucleic acids
contain the proper control sequences, they will direct the
synthesis of relatively large amounts of the encoded protein.
Preferably they encode antigens for organisms associated with
infectious diseases caused by viruses (including but not limited to
Hepatitis (all forms), HSV, HIV, CMV, EBV, RSV, VZV, HPV, polio,
influenza), parasites (e.g., from the genus Plasmodium), and
pathogenic bacteria (including but not limited to M. tuberculosis,
M. leprae, Chlamydia, Shigella, B. burgdorferi, enterotoxigenic E.
coli, S. typhosa, H. pylori, V. cholerae, B. pertussis, etc.).
Other antigens, such as found on cancerous cells (examples of known
targets for immune responses to tumors are e.g., E7 for HPV-induced
cervical carcinoma, Her-2/neu for breast and other cancers, MAGE-3
for melanoma and other cancers, tyrosinase, melanA/MART, or gp100,
a melanocyte-specific differentiation antigen) are also
contemplated within the scope of this invention.
[0021] The polynucleotides of the present invention may be in the
form of linear fragments, or non-linear fragments integrated into
plasmids or viral DNA. The polynucleotides (or nucleic acids) may
be associated with other materials which promote transfection such
as liposomal formulations, charged lipids (cationic or anionic:
e.g., Lipofectin.TM., Transfectam.TM., DOTAP.TM., cochleates),
polycations (e.g., polylysine, polyethylenimine, etc.), viral
proteins (e.g., histones), peptides that bind to nucleic acids
(e.g., condensing agents), dendrimers, PLPG microparticles,
etc.
[0022] Where the nucleic acid is DNA, promoters suitable for use in
various mammalian systems are well known. For example, suitable
promoters include CMV IE, RSV LTR, SV40, Adeno MLP,
metallothionein, etc.
[0023] The exact cellular and immunological mechanisms underlying
the induction of the immune response by NAVAC are not yet
completely understood. A hallmark of NAVAC is the induction of
cytotoxic responses, which is a part of the immune response that is
difficult to induce by protein (vaccination) approaches. In order
to induce a CTL response, the antigen must be presented in the
context of MHC-I molecules present within the cell. Since in NAVAC
the protein is expressed intracellularly, such MHC-I presentation
is readily achieved. Thus, nucleic acid vaccination is particularly
well suited for stimulating cellular immunity, in particular,
cytotoxic T cell responses to an antigen of interest.
[0024] Protein
[0025] The protein can be any polypeptide that has a useful
therapeutic application, e.g., as a prophylactic or therapeutic
vaccine. As noted above, the present invention is not limited to a
particular polypeptide, however, it would be desirable to target
proteins that may not intrinsically stimulate a strong cell
mediated immunity response, such as polypeptides found in
intracellular pathogens (for example, but not limited to M.
tuberculosis, M. leprae, Chlamydia), certain tumor-specific
antigens (e.g., found on breast or colon cancer), and weakly
immunogenic viral proteins (e.g., from Herpes, EBV, CMV, HPV,
etc.).
[0026] It is noted that a potential drawback of recombinant
proteins and subunit vaccines is the fact that glycoprotein
antigens may need to be presented in a specific conformation (i.e.,
identical to the native protein) to elicit an immunoprotective
response. When purified from recombinant expression systems, it is
sometimes difficult to insure that the antigens are
conformationally correct. However, the introduction of a gene
encoding an antigen in vivo and its subsequent intracellular
expression will insure that the protein product is synthesized,
modified and processed very similar to the native pathogen protein.
Thus, for example, the expression of a gene for a human viral
glycoprotein will likely result in the correct conformation of the
antigen. As a consequence, the native antigenic epitopes will be
presented to the immune system and can result, for example, in the
generation of neutralizing antibodies specific to the native viral
glycoprotein.
[0027] DNA+Protein
[0028] Normally a DNA vaccination elicits a predominantely Th1
helper type response. Conversely, most soluble non-particulate
antigen (i.e., protein based) vaccines elicit a predominately Th2
response. Once "primed", the immune system response remains
predominately Th1 or Th2, depending on the composition (i.e.,
nucleic acid or polypeptide) used for the initial vaccination.
Quite unexpectedly the combination of DNA+protein, when
administered simultaneously, exhibits a more balanced Th1+Th2
response. That is, both responses are elicited and the combination
of polynucleotide/polypeptide appears to act synergistically.
Moreover, the induction of CTL's is not hampered by the presence of
the extracellular protein.
[0029] Based on the polynucleotide/polypeptide immune response,
another advantage of the instant invention is that this approach
provides a method to treat latent viral infections. Several viruses
(for example members of the Herpes virus group, etc.) can establish
latent infections in which the virus is maintained intracellularly
in an inactive or partially active form. There are few ways of
treating such infections. However, by inducing a cytolytic immunity
against a latent viral protein, the latently infected cells can
eventually be targeted and eliminated. Thus, by administering a
polynucleotide/polypeptide composition, this effect should be
enhanced over pure NAVAC (i.e., nucleotide only) approach since a
stronger immune response, including cytolytic activity, can be
established.
[0030] A related application of this approach is to the treatment
of chronic pathogen infections. There are numerous examples of
pathogens which replicate slowly and spread directly from cell to
cell. These infections are chronic, in some cases lasting years or
decades. Examples of these are the slow viruses (e.g. Visna),
Hepatitis B, the Scrapie agent and HIV. Other examples of pathogens
include organisms of the genus Plasmodium, Mycobacterium,
Helicobacter, Borrelia, and Toxoplasm and well as others as noted
above.
[0031] Finally, this approach may also be applicable to the
treatment of malignant disease. For example, vaccination to mount a
strong immune response including a T-cell mediated component to a
protein expressed by the tumor cell, will potentially result in the
elimination of these cells.
[0032] As used above, "simultaneous" administration refers to the
same ongoing immune response. Preferably both compositions are
administered at the same time (concurrent administration of both
DNA+protein), however, one compound could be administered within a
few week's time (preferably 1-10 days) of the other (initial)
administration and still be considered as "simultaneous" since they
both act during the same ongoing immune response. Normally, when a
polypeptide is administered, the immune response is considered
immediate in that an immune response will initiate as soon as the
antigen is exposed to the immune system. In contrast, when nucleic
acid is administered, peak antigen expression (in vivo) is observed
3-7 days after administration, and thus antigen exposure to the
immune system is considered "delayed" when compared to the kinetics
of protein vaccination. Regardless of this difference in kinetics,
co-administration of nucleic acid and polypeptide can be considered
"simultaneous" by understanding that they are both functionally
present during the process of an ongoing immune response. In order
to present both antigenic forms (i.e. polypeptide as such and
polypeptide expressed by the administered polynucleotide),
virtually simultaneously to the immune system, formulations can be
conceived wherein the polypeptide is contained in such a way that
its release from the formulation is delayed after the
administration. This allows the expression of polypeptide from the
polynucleotide to occur first, which is then subsequently
complemented by the delayed released polypeptide from the
formulation.
[0033] Thus another aspect of the present invention is the
concurrent administration of both DNA+protein where the protein
(polypeptide) is present or administered in the form of
delayed-release particles intended to hide the antigen from the
immune system for a short period of time. Preferably it is 1-10
days.
[0034] Regardless of the different modes or possibilities of
"simultaneous" administration as described above, it is key to the
invention that both antigenic substrates are present during the
induction phase of an ongoing immune response. In comparison to
this, the prime boost concept refers to 2 separate immune
responses: (i) an initial priming of the immune system with a
polynucleotide followed by (ii) a secondary or boosting of the
immune system with a polypeptide many weeks or months after the
primary immune response has been established.
[0035] The DNA+protein complex can thus be administered as two
separate events or combined (admixed) to permit one administration.
Preferably, the DNA+protein are admixed. Admixing can occur just
prior to use, or when the two components are manufactured (and
formulated), or any time in between.
[0036] The compositions and vaccines of the invention comprise
DNA+protein (polypeptide) in a ratio of 1000:1 (i.e., 1 mg DNA/1 ug
protein) to 1:1 to 1:100 (w/w). Preferably it is in the range of
100:1 to 1:1. More preferably it is in the range of 20:1 to 1:1.
One preferred ratio is 5:1
[0037] Adjuvants
[0038] The polynucleotides, polypeptides and
polynucleotide+polypeptide mixture (complex) of the present
invention, when adjuvanted, are preferably adjuvanted in the
vaccine formulation of the invention. Vaccine preparation is
generally described in New Trends and Developments in Vaccines,
Voller et al. (eds.), University Park Press, Baltimore, Md., 1978.
Fullerton describes encapsulation within liposomes, U.S. Pat. No.
4,235,877. Suitable adjuvants include an aluminum salt such as
aluminum hydroxide gel (alum), aluminum phosphate, or algammulin,
but may also be a salt of calcium, iron or zinc, or may be an
insoluble suspension of acylated tyrosine, or acylated sugars,
cationically or anionically derivatised polysaccharides, or
polyphosphazenes.
[0039] Suitable adjuvant systems include, for example, a
combination of monophosphoryl lipid A, preferably 3-de-O-acylated
monophosphoryl lipid A (3D-MPL) together with an aluminum salt. An
enhanced system involves the combination of a monophosphoryl lipid
A and a saponin derivative particularly the combination of QS21 and
3D-MPL as disclosed in WO 94/00153, or a less reactogenic
composition where the QS21 is quenched with cholesterol as
disclosed in WO 96/33739. A particularly potent adjuvant
formulation involving QS21 3D-MPL & tocopherol in an oil in
water emulsion is described in WO 95/17210 and is a preferred
formulation. In addition, the DNA could serve as an adjuvant as
well by encoding the gene of interest and CpG sequences. Such CpG
sequences, or motifs, are known in the art.
[0040] Formulations
[0041] Administration of pharmaceutically acceptable salts of the
polynucleotides and proteins described herein is included within
the scope of the invention. Such salts may be prepared from
pharmaceutically acceptable non-toxic bases including organic bases
and inorganic bases. Salts derived from inorganic bases include
sodium, potassium, lithium, ammonium, calcium, magnesium, and the
like. Salts derived from pharmaceutically acceptable organic
nontoxic bases include salts of primary, secondary, and tertiary
amines, basic amino acids, and the like. For a helpful discussion
of pharmaceutical salts, see S. M. Berge et al., J Pharm Sci
66:1-19 (1977), the disclosure of which is hereby incorporated by
reference.
[0042] The protein component of the formulation may be administered
in the form of delayed-release particles intended to hide the
antigen from the immune system for several days, and to then
liberate the antigen. In doing so the protein antigen is liberated
once the DNA has initiated the priming of the immune response. The
concept of using delayed-release formulations for achieving both
priming and boosting of the immune response with protein antigens
has been discussed in the literature, for example see Coombes, A.
et al. (Vaccine, 14:1429-1438 (1996,)). Most published attempts at
achieving delayed-release formulations of protein antigens have
used microparticles composed of poly(lactide-co-glycolide) in which
the protein antigen is entrapped. Other formulations have been
prepared with polyacrylate, latex, starch, cellulose and dextran.
Another type of carrier that can be used as a delayed delivery
vehicles (i.e., for delayed release formulations) are
supramolecular biovectors (SMBVs). SMBVs comprise a non-liquid
hydrophillic core, such as a cross-linked polysaccharide or a
cross-linked oligosaccharide and, optionally, an external layer
comprising an amphiphillic compound, such as a phospholipid. See,
for example, U.S. Pat. No. 5,151,264, PCT applications WO94/20078,
WO94/23701 and WO96/06638.
[0043] In a preferred embodiment of the invention the protein
antigen is adsorbed to alum, and the alum particles are then coated
with a bio-degradeable polymer such as poly(capro-lactone) or
poly(lactide-co-glycolide). Unlike published methods for
encapsulating protein antigen, such formulations provide a hermetic
layer of polymer around the antigen, preventing antigen liberation
for a period of time ranging from a day to several weeks.
[0044] Polynucleotides and proteins for injection, a preferred
route of delivery, may be prepared in unit dosage form in ampoules,
or in multidose containers. The polynucleotides and proteins may be
present in such forms as suspensions, solutions, or emulsions in
oily or preferably aqueous vehicles. Alternatively, the
polynucleotide salt may be in lyophilized form for reconstitution,
at the time of delivery, with a suitable vehicle, such as sterile
pyrogen-free water. Both liquid as well as lyophilized forms that
are to be reconstituted will comprise agents, preferably buffers,
in amounts necessary to suitably adjust the pH of the injected
solution. For any use, particularly if the formulation is to be
administered intravenously, the total concentration of solutes
should be controlled to make the preparation isotonic, hypotonic,
or weakly hypertonic. Nonionic materials, such as sugars, are
preferred for adjusting tonicity, and sucrose is particularly
preferred. Any of these forms may further comprise suitable
formulatory agents, such as starch or sugar, glycerol or saline.
The compositions per unit dosage, whether liquid or solid, may
contain from 0.1% to 99% of polynucleotide and proteinaceous
material.
[0045] The unit dosage ampoules or multidose containers, in which
the polynucleotides and polypeptides are packaged prior to use, may
comprise an hermetically sealed container enclosing an amount of
polynucleotide (and/or polypeptide) or solution containing a
polynucleotide (and/or polypeptide) suitable for a pharmaceutically
effective dose thereof, or multiples of an effective dose. The
polynucleotide and polypeptide are packaged as a sterile
formulation, and the hermetically sealed container is designed to
preserve sterility of the formulation until use.
[0046] Dosage and Route of Administration of DNA/Protein
Complex
[0047] The polynucleotides may be delivered to various cells of the
animal body, including for example, muscle, skin, brain, lung,
liver, spleen, or to the cells of the blood. Administration of the
polynucleotide/protein complex is not limited to a particular route
or site. For example, the route could be intramuscular,
intradermal, epidermal (using for example a gene gun), intra pinna,
oral, vaginal, nasal, etc. Preferably the route is intramuscular,
intradermal or epidermal.
[0048] Preferred tissues to target are muscle, skin and mucous
membranes. Skin and mucous membranes are the physiological sites
where most infectious antigens are normally encountered. Skin
associated lymphoid tissues contain specialized cells like
keratinocytes, Langerhans' cells, and other dendritic cells which
are involved in the initiation and further regulation of immune
responses.
[0049] The dosage to be administered depends to a large extent on
the condition and size of the subject being treated as well as the
frequency of treatment and the route of administration. Regimens
for continuing therapy, including dose and frequency may be guided
by the initial response and clinical judgment. The parenteral route
of injection into the interstitial space of tissues is preferred a
preferred route, although other parenteral routes, such as
inhalation of an aerosol formulation, may be required in specific
administration, as for example to the mucous membranes for the
nose, throat, bronchial tissues or lungs. In addition, oral
administration may be used for vaccination against infectious
diseases of mucosal surfaces such as the nose, lungs and sinuses.
In preferred protocols, a formulation comprising the
polynucleotide/protein complex in an aqueous carrier is injected
into tissue in amounts of from 10 .mu.l per site to about 5 ml per
site. The concentration of polynucleotide/protein complex in the
formulation is from about 0.1 .mu.g/ml to about 20 mg/ml, and
preferably in the range of 100-1000 ug/ml.
EXAMPLES
[0050] The examples below are carried out using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. The examples
are meant to illustrate, but not limit the invention.
Example 1
[0051] Vaccine Preparation
[0052] A. Plasmid DNA
[0053] The coding sequence of the RSV-F protein (Collins et al.,
Proc. Natl. Acad. Sci. 81:7683-7687 (1984)) (574 amino acids) was
cloned into the eukaryotic expression vector JA4304 (Lu et al., J.
Virol 70:3978-3991 (1996)) using cloning techniques well known by
persons skilled in the art. Briefly, the RSV-F insert was cloned as
a HindIII-EcoRV fragment into linearised JA4304 at the HindIII and
blunt-ended BglII restriction sites. Recombinant plasmids were
analyzed by restriction digests and were sequenced at the insertion
sites. Plasmid DNA for injection was purified by Qiagen columns
(Maxi-Prep), followed by two phenol/chloroform extractions, ether
extraction, and ethanol precipitation. The plasmid DNA was
resuspended into sterile water and stored at -20.degree. C.
Similarly, the coding sequence used to express the HSV gD protein
(McGeoch et al., J. Gen. Virol 68: 13-38 (1987)) was contained in a
HindIII-Bam HI-BgIII double restriction fragment, cloned into
JA4304 HindIII-BgIII.
[0054] B. Protein
[0055] The coding sequence of the RSV F and G proteins (Huang et
al., Virus Res. 2:157-173 (1985)) was cloned and a chimeric F/G
protein was made. Very briefly, plasmid pEE14-FG containing a
chimeric construct comprising of a fusion between amino acid
sequences of F (1-526) and G (69-298) was received from a
collaboration with A. BOLLEN (ULB/CRI, Belgium). This FG fusion
protein contains a total of 755 amino acids. It starts at the
N-terminal signal sequence of F and lacks the C-terminal
transmembrane domain (526-574) of the F glycoprotein. Then, fused
to the 3' end of the coding region is the extracellular region of G
glycoprotein, lacking an amino-terminal region and a carboxy
terminal region. The pEE14-FG expression plasmid was generated by
the insertion of the FG coding sequence from pNIV2857 (A. Bollen,
ULB/CRI, Belgium) as an Asp7181 (blunt) 5'-HindIII (blunt) 3'
restriction fragment (2188 bp) into the Smal site of pEE14
(Celltech). It will be appreciated that there are many of
expression plasmids known in the art that can be used.
[0056] CHO K1 cells derived were transfected with 20 ug of pEE14-FG
plasmid DNA twice CsCl purified using a Ca-phosphate/glycerol
transfection procedure. Cell clones were selected according to the
procedure of the GS (glutamine synthetase) expression system
(Crocett et al., BioTechniques, 8:662 (1990)) and amplified in the
presence of 25 micro molar methionine sulphoximine (MSX) in G MEM
medium containing no glutamine and supplemented with 10% dialyzed
FBS (Foetal Bovine Serum). Following transfection, 39 MSX resistant
clones were screened in 24-well plates and their supernatants were
tested for secretion of the FG fusion protein. All transfectants
proved to be positive for F antigen expression using a specific
`Sandwich` ELISA assay (i.e., rabbit polyclonal anti FG serum/FG
Antigen/mAb19). Monoclonal antibody 19 recognizes a conformational
F1 epitope and is neutralizing.
[0057] The 3 best FG-producer clones (n.sup.o 7, 13 and 37) were
single-cell subcloned in a limiting dilution assay using 0.07 cells
per well in a 96-well plates. A total of 59 positive subclones were
obtained and the 16 best FG-producers were further characterized by
western blot and ELISA. Again, the 8 best FG-subclones were further
amplified and their FG expression was evaluated in presence and
absence of sodium butyrate (2 mM) or DMSO (1 or 2%). Six subclones
were amplified and cell vials were made and stored at -80.degree.
C. or in liquid N.sub.2. Finally, the 3 best FG subclones were
selected. These are CHOK1 FG .sup.o 7.18, .sup.o 13.1, and .sup.o
37.2.
[0058] Western blot analyses (non-reducing conditions) with
monoclonal mAb19 indicated a major band of FG at about 135 kDa.
[0059] The addition of Sodium butyrate in CHO-FG cell culture
medium increased the expression level of FG 3 to 12 fold depending
on the subclone and cell culture growth conditions. In particular,
subclone CHO-FG 13.1 expressed FG protein 8-10 fold higher in the
presence of butyrate.
[0060] Frozen cell culture supernatant is was thawed in the cold
room (4.degree. C.-7.degree. C.). Protease inhibitors (for example,
1/1000 Aprotinin, 0.5 mg/litre leupeptin, 0.5 mM Pefabloc) are
added. The thawed supernatant is loaded onto a column packed with
Pharmacia SP Sepharose HP.TM. or fast flow resin. The SP Sepharose
HP.TM. or fast flow resin is equilibrated with 20 mM MES pH 6.5, 1%
thesit (buffer D). After loading, the column is washed with buffer
D until the absorbance at 280 nm returns to baseline. The column is
eluted successively with buffer D containing 150 mM NaCl, than 300
mM NaCl and finally 1 M NaCl.
[0061] FG is eluted with buffer D containing 300 mM NaCl. The
eluate could be, if necessary, concentrated on a Filtron OMEGA 30
or 50 kDa membrane. Eluate, whether concentrated or not, is loaded
onto a Pharmacia Superdex 200.TM. 16/60 column equilibrated and
eluted in PBS, 1% Tween 80. 1-3 mg of total protein (Lowry) are
injected per run.
[0062] As noted, a truncated version of recombinant glycoprotein
Herpes Simplex Virus (HSV) type 2 (rgD.sub.2t) was also prepared.
Such protein and DNA coding sequences are known in the art, see,
e.g., EP 139 417B, U.S. Pat. No. 5,656,457, WO 92/16231.
[0063] Recombinant Human Immunodeficiency Virus type 1 (HIV-1)
envelope glycoprotein gp120 was prepared. Gp120 expression and the
DNA coding sequence is known in the art as well, see, e.g., U.S.
Pat. No. 5,653,985, U.S. Pat. No. 5,614,612, EP 290 261B.
Example 2
[0064] Immunisation of Mice
[0065] A. Plasmid DNA Vaccination
[0066] Plasmid DNA (encoding the RSV-F protein "DNA F") was
resuspended at a concentration of 10 .mu.g/50 .mu.l of sterile PBS
just prior to injection. Anaesthetized mice were injected twice
with 50 .mu.l of this DNA vaccine into the tibialis anterior
muscle; the booster injection was given four weeks after the first.
In certain groups, the DNA vaccine was only given at the first
injection.
[0067] B. Protein Vaccination
[0068] FG protein vaccine. The chimeric F/G protein ("FG") was
prepared at a dose of 2 .mu.g/50 .mu.l by resuspending purified FG
into 150 mM NaCl, 10 mM PO4 (pH 7.4). Phenoxyethanol was added as
preservative at a concentration of 0.5%. The protein vaccine was
prepared 7 days before the primary injection, and stored at
4.degree. C.
[0069] C. Mixed DNA/Protein Vaccination
[0070] Seven days prior to the primary injection, 2 .mu.g of FG per
dose was mixed with 26.95 .mu.l H.sub.2O and 4.25 .mu.l of 1.5M
NaCl, 100 mM PO4 (pH 7.4); in addition, 0.25 .mu.l of
phenoxyethanol was added as preservative. This preparation was
stored at 4.degree. C. Just prior to the respective injections
(first or secondary), 5.65 .mu.l of DNA (corresponding to 10 .mu.g)
encoding F was added to this protein preparation.
[0071] The immunisation scheme is presented below:
1 First injection Second (booster) injection DNA F 10 .mu.g PBS DNA
F 10 .mu.g DNA F 10 .mu.g DNA F 10 .mu.g FG 2 .mu.g PBS FG 2 .mu.g
FG 2 .mu.g FG 2 .mu.g DNA F 10 .mu.g + FG 2 .mu.g DNA F 10 .mu.g +
FG 2 .mu.g
Example 3
[0072] Serological Responses upon Mixed Vaccine Administration
[0073] Mice were immunised twice with the mixed DNA/protein vaccine
composition and the relevant controls, and their serological
response was analysed using the following protocol.
[0074] 3.a. Protocol: Enzyme-Linked ImmunoSorbent Assays
(ELISA)
[0075] Maxisorp Nunc immunoplates were coated overnight at
4.degree. C. with 50 .mu.l/well of 1 .mu.g/ml FG antigen diluted in
PBS (rows B to H of the plate) or with 50 .mu.l of 5 .mu.g/ml
purified goat anti-mouse immunoglobulin antiserum (Boehringer)
diluted in PBS (row A--IgG standard curve). Saturation of the
plates was done for 1 Hr at 37.degree. C. with 100 .mu.l/well of
PBS BSA 1% Tween 20 0.1% NBCS 4% (saturation buffer). Then, serial,
2-fold dilutions of IgG (mouse polyclonal antiserum, SIGMA) in
saturation buffer (50 .mu.l per well) were put in rowA as standard
starting at 200 ng/ml; serum samples were applied in rows B to H
starting at a 1/50 dilution further serially 2-fold diluted;
incubation was for 1 Hr 30 at 37.degree. C. Washing was done 3
times with PBS Tween 20 (0.1%). Then, biotinylated goat anti-mouse
IgG antiserum (Amersham) diluted 5000.times. in saturation buffer
was added (50 .mu.g/well) and incubated for 1 H 30 at 37.degree. C.
After 3 washings as above and subsequent addition for 30 min at
37.degree. C. of streptavidin biotinylated horseradish peroxydase
complex (Amersham) diluted 1000.times. in saturation buffer (50
.mu.l/well), plates were washed 5 times and incubated for 15 min at
room temperature (in darkness) with 50 .mu.l/well of substrate
solution (OPDA 0.4 mg/ml and H.sub.2O.sub.2 0.03% in 50 mM pH4.5
citrate buffer). The reaction was stopped by adding H.sub.2SO.sub.4
2N (50 .mu.l/well). The color reaction was read on a multiscan
ELISA reader at wavelength 492/620 nm. Antibody titers were
calculated by the 4 parameter mathematical method using SoftMaxPro
software.
[0076] The same protocol was used to determine the specific isotype
distribution of the antisera using mouse monoclonals (SIGMA) as
standard and isotype-specific biotinylated goat anti-mouse IgG1,
IgG2a and IgG2b antisera (Amersham) for the samples. The isotype
distribution was expressed as the percentage of the total IgG
specific immune response.
[0077] 3.b. Results
[0078] FIG. 1 (Ig 14 post 2) shows the serological responses in the
immunised animals 2 weeks after the booster injection. The
combination of the two vaccine substrates strongly enhanced the
amount of RSV-FG specific antibodies compared to those obtained by
using the respective substrates separately ((DNAF or FG) 2.times.).
Mixing the two vaccine compounds ((DNA+FG) 2.times.) was as
effective as administering them in two separate injections by first
priming with DNA and subsequent boosting with the protein alone
(DNAF/FG). The total IgG titer of the mixed DNA/protein vaccine was
much higher than the sum of the respective titers of the separate
substrates indicating synergy upon mixing of the two vaccine
substrates. FIG. 2 (IgG isotype 14 post 2) shows the isotype
distribution in the different vaccine groups. All the groups who
did receive a DNA vaccine at the first injection have a strong
content of IgG2a, indicative for a T helper response of type 1
(Th1), whereas the protein groups have a complete bias for the
opposite, that is the IgG1 isotype typical for a Th2 type response.
Surprisingly, the mixed DNA+protein group also had a high level of
the IgG2a isotype. This is unexpected since the immediate presence
of the protein is supposed to direct the system towards a Th2 type
response and would therefore impact on the DNA vaccine, which
antigen is produced in vivo later on. The fact that increased
antibody levels were obtained (FIG. 1) excludes the possibility
that (i) the protein was not immunoreactive in this formulation and
(ii) that the observed response was solely due to the DNA
vaccine.
Example 4
[0079] Lymphoproliferative Responses upon Mixed Vaccine
Administration
[0080] Induction of T helper cell activity and determination of the
type of T helper cells were assessed as follows.
[0081] 4.a. Protocols: In vitro Detection of FG-Specific
Lymphoproliferation and Cytokine Production by Mice Spleen and
Lymph Node Cells
[0082] In vitro Stimulation
[0083] Spleens and iliac lymph nodes were taken on day 15 after
second immunization. Spleens and lymph nodes of animals belonging
to the same group were pooled, gently triturated in medium 1 (RPMI
1640 completed with 2 mM L glutamine+5 10.sup.-2 mM
2-mercaptoethanol+1 mM sodium pyruvate+1.times.non essential MEM
amino acids+100 IU/ml penicillin+100 .mu.g/ml streptomycin) (5
ml/spleen cells, 1 ml/lymph node cells) and centrifuged at 1200 rpm
at RT for 10 minutes. Cell pellets were then resuspended in medium
1 (3 ml/spleen cells, 1 ml/lymph node cells), cell suspensions were
counted on a multisizer counter and adjusted to 2.times.10.sup.6
cells/ml in medium 2 (medium 1+1% heat inactivated normal mouse
serum). 100 .mu.l of these cell suspensions were added per well of
U bottom 96 well plates. For specific restimulation, 100 .mu.l of a
serial, 2-fold dilution of purified FG (diluted in medium 2,
ranging from 1.5 to 0.023 .mu.g/per well) was used. Controls
received 100 .mu.l of medium only. Total stimulation was assessed
by the addition of 100 .mu.l ConA (Boehringer) at a final
concentration of 5 .mu.g/ml. All stimulation conditions were
performed in triplicate. Plates were then incubated at 37.degree.
C. in a 5% CO.sub.2 incubator for respectively 48 hrs (ConA total
stimulation) or 72 h for FG specific restimulation. Thereafter, 100
.mu.l of supernatant was withdrawn out of each well and replaced by
fresh medium 1 supplemented with .sup.3H-thymidine, 1 .mu.Ci/well
(Amersham). Plates were incubated for another 18 to 24 hr at
37.degree. C. in a 5% CO.sub.2 incubator before being harvested.
Thymidine incorporation was determined in a beta counter after cell
lysis on nitrocellulose filters. The results are expressed in cpm
(average of triplicates) for each restimulation condition.
[0084] Cytokine Detection
[0085] Spleen cells were processed in the same way as described
here above except that they were adjusted to 5.10.sup.6 cells/ml in
medium 3 (medium 1+10% heat inactivated FCS). 1 ml of these cell
suspensions were added per well of a 24 well plate. Restimulation
was done by adding purified FG antigen at 5 .mu.g/ml (25
.mu.l/well) to cells (medium was used as control). Plates were
incubated at 37.degree. C. in a 5% CO.sub.2 incubator for 72 hr.
Supernatants were withdrawn and tested for the presence of
cytokines (IFN.gamma. and IL-5) by Elisa. Maxisorp Nunc
immunoplates were coated overnight at 4.degree. C. with 50
.mu.l/well of respectively 1.5 .mu.g/ml rat monoclonal anti-mouse
INFg (Genzyme) or 1 .mu.g/ml rat monoclonal anti-mouse IL5
(Pharmingen) diluted in 0.1M bicarbonate buffer (pH 9.5).
Saturation of plates was done for 1 H at 37.degree. C. with 100
.mu.l/well of PBS BSA 1% Tween 20 (0.1%) NBCS 4% (saturation
buffer). Then, standards were applied in row A by putting serial,
2-fold dilutions of either IFNg (Genzyme) or IL5 (Pharmingen) in
saturation buffer (50 .mu.l per well) starting at 2430 pg/ml or
2000 pg/ml, respectively. Samples were also serially, 2-fold
diluted and added to the other rows of the plate. Plates were
incubated for 1 H 30 at 37.degree. C. Washing was done 3 times with
PBS Tween 20 0.1%. Then, biotinylated goat anti-mouse INFg or IL5
antiserum (Genzyme and Pharmingen, respectively) diluted at a
concentration of 0.5 or 1.0 .mu.g/ml (respectively) in saturation
buffer was incubated (50 .mu.l/well) for 1 H 30 at 37.degree. C.
After 3 washings as above and subsequent addition for 30 min at
37.degree. C. of AMDEX.TM. (Amersham) diluted 10000.times. in
saturation buffer (50 .mu.l/well), plates were washed 5 times and
incubated for 10 min at room temperature (in darkness) with 50
.mu.l/well of TMB substrate solution (Biorad). The reaction was
stopped by adding 50 .mu.l/well of H.sub.2SO.sub.4 (0.4N). Color
intensity was read on a multiscan ELISA reader at wavelength
450/630 nm. The results are expressed in pg/ml IFNg or IL5
referring to the corresponding calibration curves using the 4
parameters method.
[0086] 4.b. Results
[0087] FIG. 3 (Lymphoproliferation) shows the lymphoproliferation
of splenocytes from the different vaccine groups upon in vitro
restimulation with the FG antigen. Combining of the two vaccine
compounds into one composition strongly increases this cellular
response compared to monovalent (DNA or protein) vaccines or the
DNA prime+protein boost immunisation. Table 1 (IFNg and IL5) shows
the secretion of specific cytokines, IFN gamma and IL5
respectively, from the proliferating cells. The secretion of the
Th2 cytokine, IL5, is low despite the strong lymphoproliferation of
the mixed vaccine group. The IFNg/IL-5 ratio (Table 2), being a
measure of the relative ratio of Th2 to Th1 type of T helper
response, is consistent with the isotype distribution of this group
(see FIG. 2) and further confirms the impact of the DNA in the
mixed vaccine composition on the induction of a Th1 type of immune
response.
Example 5
[0088] Cytotoxic T Cell Responses upon Mixed Vaccine
Administration
[0089] The induction of cytotoxic T cells (CTL's) was determined as
follows.
[0090] Splenocytes were prepared as described in the previous
section and 2.10.sup.7 spleen cells (effector cells) were grown in
a 25 cm.sup.2 flask with 10 ml of medium 3 (see example 4). These
cells were stimulated for seven days (at 37.degree. C., 7%
CO.sub.2) by the addition of RSV infected naive spleen cells
(stimulator cells) at a ratio of 10/1 (E/S). These latter
stimulator cells were obtained by infecting 2.10.sup.6 haemolyzed
normal spleen cells with RSV (MOI 0.5) for 1 hr 30 at 37.degree. C.
In preparation of the cytotoxicity assay, EMT6 syngeneic target
cells (H2d mammary adenocarcinoma cells provided by Dr B. Rouse,
Knoxville) were prepared as follows. 3..times.10.sup.6 EMT6 were
collected by centrifugation, washed once with medium 1 and
resuspended in 200 .mu.l medium 1. Cells were infected with control
vaccinia virus (PSC11, negative control) or a recombinant vaccinia
virus expressing RSV-F at an MOI of 10 for 1 hr at 37.degree. C.
(vaccinia viruses were obtained from Dr. A. Bollen, Brussels).
Infected cells were then adjusted to 1 ml with medium 3 are
incubated overnight at 37.degree. C. and 7% CO.sub.2.
[0091] Infected cells were then washed, collected by
centrifugation, and resuspended in 50 .mu.l of FCS. 50 .mu.l of
radiolabeled sodium chromate (DuPont) at 370 MBq/ml was added for 1
hr. Target cells were subsequently washed three times in medium 1,
counted, and adjusted to a final concentration of 2..times.10.sup.4
target cells/ml. For the cytotoxicity assay, Cr51 loaded EMT6
target cells were placed in the wells of a 96 wells microplate in
the presence of different ratios of restimulated effector cells
(E/T ratios of 100/1-30/1-10/1-3/1-1/1). All samples were done in
duplicate at 100 .mu.l/well. Microplates containing effectors and
targets cells were then centrifuged for 5 minutes at 800 rpm and
incubated for 4 hrs at 37.degree. C. with CO.sub.2. Thereafter, 50
.mu.l of supernatants were transferred on a lumaplate (Packard),
dried overnight and counted. Percentage of lysis was calculated as
follows: (cpm sample-cpm spontaneous release)/(cpm max (triton3%
lysis)-cpm spontaneous release).times.100.
[0092] Results
[0093] FIG. 4 shows the presence of CTL's in the different vaccine
groups. As expected, DNA vaccination induces CTL whereas
non-adjuvanted FG protein has a CTL value which is the same as the
background lysis towards control target cells. A single
administration of the DNA vaccine did prime for CTL induction and
further boosting with either protein (alone) or DNA (alone) did not
substantially increase this response. Mixing of the two vaccine
compounds (DNA+protein) did not impact on the induction of the CTL
response by the DNA component, as CTL's could be detected. It is
observed that admixing a DNA vaccine to a non-adjuvanted protein
vaccine allows an extra dimension to its immune response, namely
the induction of a CTL response.
Example 6
[0094] Enhanced Humoral Responses upon Mixed Vaccine Administration
Are Due to the Presence of a Specific Polynucleotide Encoding the
Polypeptide of Interest
[0095] It has been shown that in certain cases plasmid DNA can
exert an adjuvant effect when co-administered with a heterologous
polypeptide (Sato et al., Science 273:352-354 (1996)). To exclude
that the observed enhancement of the humoral response as
illustrated in Example 3 was due to an aspecific adjuvant effect of
the plasmid DNA, following experiment was performed. Mice were
immunised as described in Example 3 with the following
preparations: 10 .mu.g DNA-F, 10 .mu.g DNA-F plus 2 .mu.g FG (mixed
antigens), 2 .mu.g FG, or 10 .mu.g pJA4304 plus 2 .mu.g FG. This
latter group resembles the mixed antigen group but with the
difference that the plasmid DNA JA4304, having the same vector
backbone as DNA-F, is not expressing the F polypeptide. Serological
responses were analysed at 13 days after the second injection.
2 ANTIGEN Average IgG (.mu.g/ml) Standard Deviation DNA-F 10 .mu.g
5.8 8.2 DNA-F 10 .mu.g + FG 2 .mu.g 60.8 20.5 FG 2 .mu.g 5.1 5.5
DNA-JA4304 + FG 2 .mu.g 4.4 3.9
[0096] From these results it is very clear that mixing the two
specific antigen compositions (i.e. polypeptide FG and DNA-F) is
strongly enhancing the humoral responses, and that a synergistic
effect occurs. This enhancement is depending on the presence of a
plasmid encoding the antigen since a similar, non-coding plasmid is
not capable of enhancing the immune response.
Example 7
[0097] A Short Delayed Administration of a Polypeptide Antigen Is
Enhancing the Humoral Response of the Respective Polynucleotide
Vaccine
[0098] Polypeptide antigens have a more rapid presentation to the
immune system than polynucleotide vaccines, since the encoded
polypeptide has first to be expressed after uptake of the
polynucleotide by the cell. A short delayed delivery of the
polypeptide antigen presents the two immunogenic compositions
(virtually) simultaneously to the immune system.
[0099] Mice were injected twice with the following immunogens: 30
.mu.g DNA-gp120, 10 .mu.g gp120 protein, 30 .mu.g DNA-gp120
followed four days later by 10 .mu.g gp120 protein (delayed protein
delivery). Humoral responses and isotype distribution were
determined essentially as described in Example 3 using gp120
protein to capture the anti-gp120 specific antibodies.
[0100] FIG. 5 shows the total anti-gp120 IgG titers 14 days after
the second injection. IgG titers are strongly increased in the
group which received the two antigen forms (polynucleotide and
polypeptide) where the protein administration was delayed for four
days.
[0101] FIG. 6 shows the isotype distribution. As can be seen,
delayed protein delivery maintains considerable IgG2a titers as
observed for the DNA group, and this in strong contrast to the
protein-only group.
Example 8
[0102] Preparation of Delayed-release Formulations
[0103] 20 .mu.g of the antigen gD2t was adsorbed onto 500 .mu.g
alum (aluminium hydroxide) in H.sub.2O (final volume 90 .mu.l). A
solution of poly(capro-lactone)diol (average Mn 2000, softening
temperature 50.degree. C.) in ethanol (5 mg/ml: at 70.degree. C.)
was added to a ratio of 10:1 (polycaprolactone:alum) and
temperature allowed to equilibrate to 25.degree. C. This results in
co-acervation of the polycaprolactone onto the particles of alum.
The resulting coated particles were collected by centrifugation and
washed with water.
[0104] For in vitro release kinetics, samples were prepared using
radio-iodinated gD2t. The gD2t was iodinated using lodogen (Pierce)
by following the manufacturer's instructions. In vitro release
kinetics were performed in 250 mM sodium phosphate. This medium
causes instantaneous release of gD2t from alum.
Polycaprolactone-coated alum was incubated in this medium at
37.degree. C. for 8 days, and the release of antigen into the
medium assayed by radioactivity measurements. For uncoated alum,
the gD2t was released into the medium within 10 minutes. For
polycaprolactone coated alum there was a slight (5-25%) release
within the first 24 hours, followed by slow release over 8
days.
[0105] For in vivo studies the polycaprolactone-coated alum was
mixed with DNA coding for gD at a ratio of 2 .mu.g protein for 10
.mu.g DNA.
Example 9
[0106] Increased Induction of Humoral Responses with Delayed
Release Formulations
[0107] In order to allow simultaneous administration of both DNA
and the corresponding protein antigen, delayed release formulations
prepared as described above were tested. Mice were injected with
the following antigen compositions (ALOH-PLC refers to
polycaprolactone-coated alum containing gD2t):
3 GROUP DAY 0 DAY 4 1 10 .mu.g DNA-gD 2 10 .mu.g DNA-gD 2 .mu.g gD
/ A1OH 3 2 .mu.g gD / A1OH 4 10 .mu.g DNA-gD + 2 .mu.g gD / A1OH-
PLC 5 2 .mu.g gD / A1OH-PLC
[0108] Humoral responses were determined 13 days after the first
injection and are represented in the following Table:
4 Average titer GROUP (.mu.g/ml) Standard deviation GMT 1 5.7 3.8
4.4 2 17.3 19.3 11.1 3 1 1.1 0.7 4 16 8.3 13.9 5 0.9 0.5 0.9
[0109] As can be seen from these results, combined protein and DNA
immunisation leads to a stronger induction of the humoral response.
The fact that titers of group 2 (protein administered 4 days after
the DNA), and group 4 (delayed release formulation with
simultaneous injection of DNA and formulated protein) are very
similar, strongly indicates that delayed release of the protein has
taken place and that the concomitant presence of the protein at the
time of polynucleotide directed expression strongly enhances the
induction of the humoral response induced by the DNA vaccine. As
can be seen, virtually simultaneous presentation of the polypeptide
from both vaccine compositions to the immune system can be achieved
by a short delayed delivery of the polypeptide after the nucleic
acid vaccination.
[0110] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
[0111] While the preferred embodiments of the invention are
illustrated by the above, it is to be understood that the invention
is not limited to the precise instructions herein disclosed and
that the right to all modifications coming within the scope of the
following claims is reserved.
5TABLE 1 Prime Boost RSV 14 Post 2 IgG IgG1 IgG2a IgG2b Prolif IL5
INFg CTL DNAF/ 1 19 51 29 1387 300 2406 16 PBS DNAF/ 2 13 77 10
1272 300 1069 22 DNAF PBS/ 0 100 0 0 807 300 391 6 FG FG 2X 2 100 0
0 861 367 360 5 DNAF/ 120 57 39 4 1837 2195 3272 15 FG (DNAF + 149
32 67 1 4190 300 1553 20 FG) 2X .mu.g/ml % % % CPM pg/ml pg/ml %
Cr51 re- lease
[0112]
6TABLE 2 RSV 14 Post 2 INFg IL5 Ratio INFg/IL5 DNAF/PBS 2406 300
8.02 DNAF/DNAF 1069 300 3.56 PBS/FG 391 300 1.30 FG 2.times. 360
367 0.98 DNAF/FG 3272 2195 1.49 (DNAF + FG) 2.times. 1553 300 5.18
pg/ml pg/ml
* * * * *