U.S. patent application number 12/410083 was filed with the patent office on 2011-02-03 for nucleotide vector, composition containing such vector, and vaccine for immunization against hepatitis.
This patent application is currently assigned to Institut Pasteur. Invention is credited to Maryline Mancini, Marie-Louise Michel.
Application Number | 20110027318 12/410083 |
Document ID | / |
Family ID | 27252809 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110027318 |
Kind Code |
A1 |
Michel; Marie-Louise ; et
al. |
February 3, 2011 |
NUCLEOTIDE VECTOR, COMPOSITION CONTAINING SUCH VECTOR, AND VACCINE
FOR IMMUNIZATION AGAINST HEPATITIS
Abstract
Nucleotide vector composition containing such vector and vaccine
for immunization against hepatitis. Nucleotide vector comprising at
least one gene or one complementary DNA coding for at least a
portion of a virus, and a promoter providing for the expression of
such gene in muscle cells. The gene may be the S gene of the
hepatitis B virus. A nucleotide vector composition when
administered to even chronic HBV carriers is capable of breaking T
cell tolerance to the surface antigens of hepatitis B virus. A
vaccine preparation containing said bare DNA is injected into the
host previously treated with a substance capable of inducing a
coagulating necrosis of the muscle fibers.
Inventors: |
Michel; Marie-Louise;
(Paris, FR) ; Mancini; Maryline; (Paris,
FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Institut Pasteur
Paris
FR
Institut National de la Sante et de la Recherche
Medicale
Paris
FR
|
Family ID: |
27252809 |
Appl. No.: |
12/410083 |
Filed: |
March 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10142358 |
May 10, 2002 |
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12410083 |
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09570546 |
May 12, 2000 |
6429201 |
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10142358 |
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08799569 |
Feb 12, 1997 |
6133244 |
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09570546 |
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08706337 |
Aug 30, 1996 |
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08799569 |
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08633821 |
Aug 2, 1996 |
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PCT/FR94/00483 |
Apr 27, 1994 |
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08706337 |
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Current U.S.
Class: |
424/227.1 ;
424/184.1; 435/320.1; 514/44R |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2730/10134 20130101; A61P 31/20 20180101; A61K 2039/545
20130101; A61K 39/292 20130101; A61K 2039/575 20130101; A61P 37/04
20180101; A61K 39/12 20130101; A61K 48/00 20130101; A61K 39/00
20130101; A61K 2039/55566 20130101; A61K 2039/53 20130101; C12N
2730/10122 20130101 |
Class at
Publication: |
424/227.1 ;
435/320.1; 424/184.1; 514/44.R |
International
Class: |
A61K 39/29 20060101
A61K039/29; C12N 15/63 20060101 C12N015/63; A61K 39/00 20060101
A61K039/00; A61K 31/7088 20060101 A61K031/7088; A61P 37/04 20060101
A61P037/04; A61P 31/20 20060101 A61P031/20 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 22, 1993 |
FR |
93 12659 |
Claims
1. A nucleotide sequence comprising a promoter homologous to the
host and another regulatory sequence for the expression of a gene
or a DNA coding complement for S, preS.sub.2-S, or
preS.sub.1-preS.sub.2-S.
2. A vaccine comprising the nucleotide sequence according to claim
1.
3. A composition capable of inducing a cytotoxic response
comprising a gene or complementary DNA coding for at least a
portion of a virus protein, which is expressed in the muscle cells,
and an internal promoter.
4. The composition of claim 3, wherein said gene is the S gene.
5. The composition of claim 3, wherein said protein is the S,
preS.sub.2-S, or preS.sub.1-preS.sub.2-S protein.
6. A non-lipid pharmaceutical composition comprising at least one
substance capable of inducing a coagulating necrosis of the muscle
fibers and either a vector comprising a gene or complementary DNA
coding for at least a portion of a virus protein, and a promoter
allowing the expression of the gene in the muscle cells or a
complete or partial nucleotide sequence comprising a promoter
homologous to the host and another regulatory sequence for the
expression of a gene or a DNA coding complement for S,
preS.sub.2-S, or preS.sub.1-preS.sub.2-S.
7. The composition of claim 6, wherein said gene is the S gene.
8. The composition of claim 6, wherein said protein is the S,
S-preS.sub.2, or S-preS.sub.1-preS.sub.2 protein.
9. The composition of any one of claims 6 to 8, wherein said
composition is administered to a chronic HBV carrier.
10. A composition for inducing a B and/or T cell response in
chronic HBV carriers comprising a vector, wherein said vector
comprises a gene or complementary DNA coding for at least a portion
of a hepatitis B protein and a promoter.
11. The composition of claim 10, wherein the B cell response is
able to clear the circulating HBsAg.
12. The composition of claim 10, wherein said gene or complementary
DNA encodes a HBV protein selected from group consisting of pre-S2,
pre-S1, S protein, and a part of pre-S2, pre-S1, or S protein.
13. The composition of claim 10, wherein said gene or complementary
DNA encodes a pre-S2 or pre-S1 protein of HBV and an S protein of
HBV or a part of pre-S2 or pre-S1 protein and a part of S
protein.
14. A composition according to any one of claims 10, 11, 12, or 13
for inducing a T cell response.
15. A process of treating chronic HBV carriers comprising
administering to said chronic HBV carriers a therapeutically
effective amount of the composition according to any one of claims
10 to 13.
16. A recombinant plasmid containing a nucleotide sequence
according to any one of claims 10 to 13, wherein said sequence
encodes for the small and/or the middle forms of HBV surface
protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 08/706,337, filed Aug. 30, 1996, which is a
continuation-in-part of application Ser. No. 08/633,821, filed Apr.
22, 1996, which is based on International Application
PCT/FR94/00483, filed Apr. 27, 1994. The entire disclosure of each
of these applications is relied upon and incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to compositions for inducing
protective antibodies against hepatitis. It also relates to a
vector comprising a nucleotide sequence coding for at least a
portion of a virus protein, which is capable of being expressed in
muscle cells. In addition, the invention relates to compositions
capable of inducing a T cell response in chronic HBV carriers.
[0003] Hepatitis B is a widespread and serious international health
problem. In addition to causing acute hepatitis and liver damage,
the hepatitis B virus (HBV) can cause cirrhosis and hepatocellular
carcinoma (Davis, Hum. Molec. Genet. 2:1847-1851 (1993)).
[0004] The HBV is a 42-nm particle (Dane particle) consisting of a
lipoprotein envelope enclosing a core protein (capsid) and the
viral genome, which contains only four genes (S, C, P, X). The
major (or small) envelope protein, which includes the surface
antigen of HBV (HBsAG) is encoded by the S gene and is organized
into dimers of one glycosylated and one unglycosylated polypeptide
(Petersen, J. Biol. Chem. 256:6975-6983 (1981)). Present in smaller
amounts are the middle and large envelope proteins, which are
encoded by the pre-S2 and S or pre-S1, pre-S2 and S genes,
respectively. The predominant form of HBsAg secreted by infected
cells is not the Dane particle, however, but 22-nm particles or
filaments, which are empty viral envelopes composed solely or
predominantly of major (small) envelope protein and sometimes small
amounts of middle and large proteins (Maupas, Lancet 1:1367-1370
(1976)). The 22-nm particles are seen to persist in the plasma of
chronic carriers (Davis, 1993).
[0005] In 1976, the first vaccine against HBV comprising 22-nm
HBsAg particles was applied to humans (Maupas, 1976). The particles
were purified from the plasma of chronic carriers and treated to
eliminate possible co-purified infectious HBV or other pathogens.
While this vaccine was effective, mass immunization was not
feasible due to the long and expensive purification procedure, the
need to assay each batch on chimpanzees for safety, and the limited
supply of chronically infected human plasma (Maugh, Science
210:760-762 (1980); Stephenne, Vaccine 6:299-303 (1988)).
[0006] The present vaccines are produced employing genetic
engineering techniques to create HBsAg-producing cell lines. One
frequently used vaccine is a second generation vaccine based on
recombinant yeast cells containing the S gene for HBsAg
(Valenzuela, Nature 298:347-350 (1982)). Another vaccine commonly
used in France is a third generation vaccine based on a line of
Chinese hamster ovary cells containing both the S and pre-S2 genes
(Michel, Proc. Natl. Acad. Sci. USA 81:7708-7712 (1984)). While the
present protein vaccines are highly effective and safe, the
production and maintenance of these vaccines is time-consuming and
expensive (Davis, 1993). On the other hand, the production of a
viral vaccine is not feasible due to safety considerations.
[0007] Immunization by DNA-based vaccines has been the object of
several studies since the beginning of the 1990s. A DNA-based
vaccine involves the transfer of a gene or at least a portion of a
gene, by direct or indirect means, such that the protein
subsequently produced acts as an antigen and induces a humoral-
and/or cellular-mediated immunological response.
[0008] Ulmer et al. (Science, 259:1745-1749 (1993)) obtained
protection against the influenza virus by induction of the
cytotoxic T lymphocytes through injection of a plasmid coding for
the influenza A nucleoprotein into the quadriceps of mice. The
plasmid used carries either the Rous sarcoma virus promoter or the
cytomegalo virus promoter.
[0009] Raz et al. (Proc. Natl. Acad. Sci. USA 90:4523-4527, (1993))
injected vectors comprising the Rous sarcoma virus promoter and a
gene coding for interleukin-2, interleukin-4, or the .beta.1-type
transforming growth factor (TGF-.beta.1). The humoral and
cell-mediated immune responses of the mice to which these plasmids
have been intramuscularly administered are improved.
[0010] Wang et al. (Proc. Natl. Acad. Sci. USA, 90:4156-4160,
(1993)) injected a plasmid carrying a gene coding for the envelope
protein of the HIV-1 virus into mice muscles. The plasmid injection
was preceded by treatment with bupivacaine in the same area of the
muscle. The authors demonstrated the presence of antibodies capable
of neutralizing the HIV-1 virus infection. However, the DNA was
injected twice a week for a total of four injections.
[0011] Davis et al. (Compte-Rendu du 28 eme Congres Europeen sur le
muscle, Bielefeld, Germany, 21-25 Sep. 1992) injected plasmids
carrying a luciferase or .beta.-galactosidase gene by pretreating
the muscles with sucrose or a cardiotoxin. The authors observed the
expression of luciferase or .beta.-galactosidase.
[0012] More recently, an article published in Science et Avenir
(September 1993, pages 22-25) indicates that Whalen and Davis
succeeded in immunizing mice against the hepatitis B virus by
injecting pure DNA from the virus into their muscles. An initial
injection of snake venom toxin, followed 5 to 10 days later by a
DNA injection, is generally described. However, the authors specify
that this method is not practical.
[0013] These studies were preceded by other experiments in which
various DNAs were injected, in particular into muscle tissues. For
example, the International application, PCT/US90/01515 (published
under No. WO-90/11 092), discloses various plasmid constructions,
which can be injected in particular into muscle tissues for the
treatment of muscular dystrophy. However, this document specifies
that DNA is preferentially injected in liposomes.
[0014] Additionally, Canadian patent CA-362.966 30 (published under
No. 1,169,793) discloses the intramuscular injection of liposomes
containing DNA coding, in particular, for HBs and HBc antigens. The
results described in this patent mention the HBs antigen
expression. The presence of anti-HBs antibodies was not
investigated.
[0015] International application PCT/FR92/00898 (published under
No. WO-93/06223) discloses viral vectors, which can be conveyed to
target cells by blood. These vectors are recognized by the cell
receptors, such as the muscle cells, and can be used in the
treatment of muscular dystrophy or of thrombosis.
[0016] The DNA-based vaccines suggested by the prior art have not
been capable of practical uses. For example, some bare DNA used to
vaccinate the mice was pure DNA from the virus. This type of
treatment can not be considered for human vaccination due to the
safety risks involved for the patients. Additionally, earlier
experiments in which the injected DNA is contained in liposomes did
not exhibit an immune response.
[0017] The present inventors have succeeded in developing effective
DNA-based immunizing compositions capable of inducing immune
responses against infectious viruses without the detrimental
effects on human health.
SUMMARY OF THE INVENTION
[0018] The present invention relates to a composition capable of
inducing T cell response, and more particularly, a cytotoxic
response comprising a nucleotide sequence expressed in muscle
cells. The nucleotide sequence comprises a gene or complementary
DNA coding for at least a portion of a virus protein and a promoter
allowing for the expression of the gene or complementary DNA in the
muscle cells.
[0019] The invention further relates to the vector, which serves as
a vehicle for the gene or complementary DNA coding for at least a
portion of a virus protein and a promoter allowing for the
expression of the gene or cDNA, which is administered to an
individual to be immunized.
[0020] In addition, the inventors have developed a non-lipid
pharmaceutical composition comprising at least, on the one hand, a
substance capable of inducing a coagulating necrosis of the muscle
fibers, such as bupivacaine, and, on the other hand, vector
including the gene or complementary DNA coding for at least a
portion of a virus protein, which is expressed in muscle cells, and
the promoter. Preferably, the substance capable of inducing a
coagulating necrosis of the muscle fibers is first administered
into the muscle of an individual to be immunized. Then, at least
five days later, the vector is administered into substantially the
same location of the individual's muscle.
[0021] The inventors discovered that the compositions of the
instant invention are capable of breaking T-cell tolerance to HBsAg
in a mouse model for chronic HBV carriers. Thus, the present
invention is further directed to the treatment of chronic HBV
carriers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] This invention will be described in greater detail with
reference to the drawings in which:
[0023] FIG. 1 is a schematic representation of pRC/CMV-HBs
plasmid.
[0024] FIGS. 2A to 2D are schematic representations of pCMVHB-S,
pCMVHB-S2.S., pCMVHB-S1.S2.S and pHBV-S1.S2.S plasmids,
respectively.
[0025] FIGS. 3, 4, and 5 are schematic restriction maps for
pCMVHB-S2.S, pCMVHB-S1.S2.S, and pRSV-HBS plasmids,
respectively:
[0026] FIG. 6 illustrates the secretion of antigenic HBs particles
(HBs Ag) in ng/ml (ordinates) as a function of the number of days
(abscissa) for cells carrying the pCMVHB-S, pCMVHB-S1.S2.S,
pHBV-S1.S2.S, pSVS, or pCMVHB-S2.S plasmids.
[0027] FIGS. 7A and 7B illustrate the determination on some
particles in FIG. 6 of the presence of the preS.sub.1 and
PreS.sub.2 antigens using, respectively, anti-preS.sub.1 and
anti-preS.sub.2 antibodies. The formation of antibody-antigen
complexes is shown by the optical density (ordinates), as a
function of antigen concentration.
[0028] FIGS. 8A to 8D represent the anti-HBS responses (HBS Ab as
ordinate, expressed as mUI/ml) and anti-preS2 (preS2 Ab as
ordinate, expressed in O.D.) of mice vaccinated by pCMVHB-S (8A),
pCMVHB-S2.S (8B), pCMVHB-S1.S2.S (8C), and pHBV-S1.S2.S (8D),
respectively.
[0029] FIG. 8E depicts the kinetics of appearance of anti-pre-S2
and anti-pre-S1 antibodies in sera from groups of mice injected
with HBV envelope-expressing plasmids (pSVS and pCMVS1S2S).
[0030] FIGS. 8F and 8G illustrate the kinetics of IgG and IgM
anti-HBs in mice immunized with HBV envelope-expressing plasmids.
The fine specificity of the antibodies was determined using
S-containing HBsAg of a heterologous subtype (ad; circles) of a
homologous subtype (ay; triangles), as well as HBsAg containing
approximately 30% of the middle (pre-S2 and S) protein of the ay
subtype (squares). The bound anti-mouse IgG antibodies are depicted
as a continuous line and bound anti-mouse IgM antibodies are
depicted as a dotted line.
[0031] FIG. 8H depicts the anti-HBs immune response in mice
injected with the two expression vectors pSVS (squares) and
pCMV-S1.S2.S (circles).
[0032] FIG. 9 illustrates the antibody response, IgG and IgM
immunoglobulins (titre as ordinates), of a mouse vaccinated by
pCMVHB-S2.S as a function of the number of weeks (abscissa).
[0033] FIGS. 10A to 10C represent the anti-group and anti-subtype
ay responses induced by DNA from pCMV-S (DNA) or from the HBS
antigen (prot), respectively, in mice B10 (10A), B10S (10B), and
B10M (10C).
[0034] FIGS. 10D to 10F represent the antigroup responses induced
by DNA from pCMV-S (DNA) or from the HBS antigens (prot),
respectively in mice B10 (10D), B10S (10E), and B10M (10F).
[0035] FIG. 11 represents a linear restriction map for the
pBS-SKT-S1.S2.S plasmid.
[0036] FIG. 12 represents the DNA-based immunization of transgenic
mice. Groups of 6 female transgenic mice were immunized once by
intramuscular injection of 100 .mu.g of the DNA plasmid pCMV-S2.S
(- -) or pCMV-LacZ (-.quadrature.-) five days after cardiotoxin
treatment. A group of 8 transgenic mice were injected with PBS
instead of DNA (non-immunized controls (-.DELTA.-). Mice were bled
at weekly intervals and the sera were analyzed for HBsAg (expressed
as ng/ml). Each point represents the mean titre for the group and
error bars represent the standard errors of the mean.
[0037] FIG. 13 describes the kinetics of appearance of anti-HBs
antibodies in mice following injection of pCMV-S2.S DNA. Sera were
taken as in FIG. 12 and the fine specificity of the antibodies was
determined using HBsAg particles containing either the S (open
symbols) or the S plus preS2 proteins (filled symbols). Anti-HBs
antibodies (Ig) were expressed as 1/log.sub.10 of the antibody
titre (determined by serial end-point dilution analysis). Circles
represent the immunized transgenic mice shown in FIG. 1, diamonds
are non-transgenic immunized mice, and squares are transgenic mice
injected with pCMV-LacZ. Symbols in the grey area correspond to
mice which gave no detectable seroconversion (titre <100).
[0038] FIG. 14 provides an anti-HBs IgG isotype profile in the sera
of six individual transgenic mice (solid columns) and three
non-transgenic mice (open columns) at 12 weeks after immunization
with pCMV-S2.S DNA. HBsAg-specific IgG1, IgG2a, IgG2b, and IgG3
antibodies were detected by ELISA with specific secondary
antibodies. Antibody titres are expressed as a serial end-point
dilutions, which was defined as the highest serum dilution that
resulted in an absorbance value two times greater than that of
non-immune or control serum, with a cutoff value of 0.05.
[0039] FIG. 15 describes the expression of HBV sequences in the
livers of transgenic and non-transgenic mice. Northern blot
analysis of 50 .mu.g of total RNA isolated from the livers of
transgenic mice (+) and their non-transgenic littermates (-) after
direct injection of DNA (A) or 26 days after adoptive transfer of
primed spleen cells (B). .sup.32P-labelled DNA probes specific for
HBV and .beta.-actin were used. The molecular weights (Kb) of the
two mRNAs encoded by the transgene are indicated.
A. Lane 1: non-immunized transgenic mouse.
[0040] Lanes 2-4: pCMV-LacZ immunized transgenic mice.
[0041] Lanes 5-9: transgenic mice immunized with pCMV-S2.S DNA.
B. Transgenic mice receiving primed spleen cells harvested from
non-transgenic mice 3-6 weeks after immunization with pCMV-LacZ
(lanes 2-3) or with pCMV-S2.S (lanes 4-8). Lane 9: transgenic mouse
receiving unprimed spleen T cells. Lane 1: RNA from the liver of
non-transgenic mouse is shown as a negative control. The
transferred spleen cell population is indicated on the top. HBsAg
titres (ng/ml) and the present (++) or the absence (-) of HBs
antibody at the time of sacrifice are indicated.
[0042] FIG. 16 describes adoptive transfer of primed spleen cells
into transgenic mice. Non-transgenic mice were immunized by
intramuscular injection of pCMV-S2.S or pCMV-LacZ DNA in order to
produce primed spleen cells for adoptive transfer into their
transgenic littermates. The mean titre of antibodies in the serum
of donor mice at the time of spleen harvest was 1.times.10.sup.5.
Eleven pCMV-S2.S recipients were bled at 2 or 3 days intervals and
their sera were analyzed for HBsAg (ng/ml) (-.box-solid.-) and
antibodies to HBsAg (-.quadrature.-), (ELISA, end-point dilution
titres). Results are shown as mean titres +/-SEM.
(- -): Mean titres of serum HBsAg in five control transgenic
recipient mice receiving either unprimed pCMV-LacZ-primed spleen
cells.
[0043] FIG. 17 describes the HBV mRNA content in the livers of
transgenic mice taken at various times after adoptive transfer of
HBsAg primed spleen cells. Northern blots were performed as in FIG.
15 and the quantitative determination of the HBV mRNA was done by
phosphoimager analysis after correction for mRNA loading and
variations in transfer efficiency as assessed by .beta.-actin
expression. The results are expressed as arbitrary units and
represented by grey columns. Background level of hybridization is
shown for RNA extracted from a non-transgenic mouse liver (open
column on right). Serum-HBsAg concentrations (ng/ml) at the time of
liver harvest are shown (-.smallcircle.-).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0044] The present invention concerns a composition capable of
inducing a cytotoxic response against viruses, such as hepatitis B.
The composition comprises a nucleotide sequence comprising a gene
or complementary DNA coding for at least a portion of a virus
protein, wherein said gene or complementary DNA is capable of being
expressed in muscle cells, and a promoter. The gene or
complementary DNA and the promoter is preferably carried by a
vector for administration into an individual to be immunized or
treated for infection. The composition is further preferably
capable of inducing protective antibodies.
[0045] The nucleotide sequence of a gene or complementary DNA may
code for at least a portion of a viral protein. Said "at least a
portion" of a viral protein signifies, in the context of the
invention, an antigenic portion of a protein with which to induce a
humoral or cell-mediated immunogenic response, such as a cytotoxic
response.
[0046] Moreover, the protein can be either a structure protein or a
regulatory protein. Preferably, the protein is a structure
protein.
[0047] In addition, the viral protein may be derived from any
infectious virus against which a humoral or cell-mediated
immunogenic response is desired. For example, the virus may be a
hepatitis virus, such as hepatitis A, hepatitis B, or a non-A,
non-B hepatitis virus, such as hepatitis C, E, or delta.
Alternatively, the virus may be a non-hepatitis virus, such as
HIV-1.
[0048] In a preferred embodiment of the invention, the gene or
complementary DNA codes for at least a portion of the surface
antigen of hepatitis B (HBsAg), particularly, in the S, preS2-S, or
preS1-preS2-S form of HBsAg, and where the gene encodes envelope
proteins.
[0049] Alternatively, the gene made code for HBsAg/ayw.
[0050] The gene or protein sequences for hepatitis A, hepatitis B,
and non-A, non-B hepatitis viruses, such as hepatitis C, E, or
delta, are described by the following documents, which are relied
upon and incorporated by reference herein:
[0051] French Patent FR 79 21 811;
[0052] French Patent FR 80 09 039;
[0053] European Patent EP 81 400 634;
[0054] French Patent FR 84 03 564;
[0055] European Patent EP 91 830 479; and
[0056] Najarian et al., Proc. Natl. Acad. Sci. USA, 82:2627-2631
(1985).
[0057] Alternatively, the gene or complementary DNA may code for at
least a portion of the gp160 protein of HIV-1 virus associated with
the p25 protein and/or the p55 protein and/or the p18 protein
and/or the Rev protein of HIV-1 virus.
[0058] In yet another alternative embodiment, the gene or
complementary DNA codes for a protein from a pathogenic
microorganism such as the bacterium causing diphtheria, whooping
cough, listeriosis, the tetanus toxin, etc.
[0059] The promoter is selected for its ability to allow the
efficient expression of the gene or complementary DNA in the muscle
cells. It may be heterologous, not naturally found in the host, or
preferably, homologous, while being originally active in a tissue
other than the muscle tissue. The promoter may be an internal or
endogenic promoter, i.e., a promoter of the virus from which the
gene or cDNA is taken. Such a promoter may be completed by a
regulatory element of the muscle or another tissue, in particular,
an activating element. Alternatively, the promoter may be from a
gene of a cytoskeleton protein, such as that described by Bolmon
(J. Submicros. Cytol. and Patholog., 22:117-122 (1990)) and Zehnlin
(Gene 78:243-254 (1989)). The promoter may alternatively be the
promoter from the virus HBV surface genes.
[0060] In a preferred embodiment, the promoter is advantageously
the promoter for cytomegalovirus (CMV).
[0061] The vector of the present invention comprises nucleotide
sequence as described above. In particular, the vector comprises
the DNA or complementary DNA coding for at least a portion of a
virus as defined above and a promoter allowing the expression of
the nucleotide sequence in muscle cells.
[0062] The vector must be capable of gene transfer of the
nucleotide sequence into the muscle cells. In addition, the vector
is selected in order to avoid its integration into the cell's DNA,
since such integrations are known to activate the oncogens and
induce cell canceration. Thus, the vector may be
non-replicative.
[0063] In an alternative embodiment, the vector may be replicative,
which would allow a high number of copies per cell to be obtained
and the immune response to be enhanced.
[0064] Suitable vectors include but are not limited to plasmids,
adenoviral vectors, retroviral vectors, and shuttle vectors.
Plasmids are the preferred vector according to the invention.
[0065] In a further preferred embodiment, the plasmid is partly
bacterial in origin and notably carries a bacterial replication
origin. Further preferred is a plasmid carrying a gene allowing for
its selection, as is known in the art, such as a gene for
resistance to an antibiotic.
[0066] The vector may also be provided with a replication origin
allowing it to replicate in the muscle cells of its host, as is
known in the art, such as the replication origin of the bovine
papilloma virus.
[0067] In addition, the vector may include a terminal transcription
sequence situated downstream of the gene.
[0068] The vectors may be obtained by methods known by those having
ordinary skill in the art. For example, methods of obtaining these
vectors include those by synthesis or by genetic engineering
methods. Such methods are described, for example, in the technical
manual Maniatis T. et al., Molecular Cloning, A Laboratory Manual,
Cold Spring Harbour: New York (1982).
[0069] Other suitable vectors are described. The pCMV/HBS or
pRCCMV-HBS plasmid, having the SEQ ID No. 1 sequence, was filed
under No. I-1370 with the Collection Nationale des Cultures des
Micro-organismes de l'Institut Pasteur (CNCM) on 21 Oct. 1993.
[0070] Additionally, the pRSV/HBS plasmid filed under. No. I-1371
with the CNCM on 21 Oct. 1993 is suitable for the instant
invention. This plasmid has a similar structure to pCMV/HBS, but
includes the Rous sarcoma virus (RSV) promoter instead of the
cytomegalovirus (CMV) promoter.
Other plasmids may be: [0071] pCMVHB-S1.S2.S constructed by
inserting the fragment BglII-BglII of the S gene, obtained from
pCP10, into a pBlueScript vector modified to contain supplementary
cloning sites in the "polylinker" portion. The fragment containing
the S gene was then removed by KpnI-BssHII digestion then cloned
into the corresponding sites of pcDNA 3 (In vitrogen, Rad Systems
Europe Ltd, Abingdon UK) so as to obtain pCMVHB-S1.S2.S. This
plasmid was filed under No. I-1411 with the CNCM; [0072]
pCMVHB-S2.S was obtained by eliminating the pre-S1 part of the HBs
gene from pCMVHB-S1.S2.S by KpnI/MstII digestion, then by bonding
the two extremities after treatment with 51 nuclease. pCMVHB-S2.S
was filed with the CNCM under No. I-1410; [0073] pHBV-S1.S2.S,
filed with the CNCM under No. I-1409, was obtained by inserting the
S gene BglII-BglII fragment, obtained from pCP10, into a
pBlueScript vector modified to contain supplementary cloning sites
in the "polylinker" portion; [0074] pBS-SKT-S1.S2.S codes for the
three envelope proteins S, S-preS.sub.1 and S-preS.sub.1-preS.sub.2
of the HBV virus; and [0075] pSVS codes for the three envelope
proteins, S, preS.sub.2-S, and preS.sub.1-preS.sub.2-S of the HBV
virus. The construction of the pSVS plasmid is described in EP 0
156 712 B1, which is incorporated herein by reference. Moreover,
pSVS has been deposited in the C.N.C.M. under No. I-1840 on Jan.
30, 1997.
[0076] The plasmid DNA may be administered in naked form or in a
liposome formulation.
[0077] The present invention further relates to nucleotide
sequences comprising a promoter homologous to the host and another
regulatory sequence for the expression of a gene or complementary
DNA coding for one of the above-mentioned proteins.
[0078] The invention is also directed to a vaccine or medicine
containing at least one vector, or a nucleotide sequence, such as
defined above. The vaccine or medicine according to the invention
is capable of inducing-protective antibodies against viruses such
as hepatitis B.
[0079] Alternatively, the vaccine or medicine according to the
invention is capable of inducing a T cell response in chronic HBV
carriers. Thus, the invention relates to the treatment of chronic
HBV carriers, wherein a composition of the invention is
administered into the muscle of the carrier in a therapeutically
effective amount.
[0080] "Chronic HBV carriers" are defined as individuals,
particularly mammals, initially infected by HBV, who fail to
resolve their infection. These individuals may remain persistently
infected by HBV and do not appear to be capable of eliciting a
multispecific polyclonal immune response to several HBV antigens,
as compared to those individuals capable of clearing the virus
following acute infection. In actively infected chronic patients,
the virus replicates in the liver and the disease is mostly
mediated by the immune response. Such viral replication is not
detected in other individuals.
[0081] Large amounts of empty viral particles carrying the HBsAg
are produced and secreted by the hepatocytes of chronic HBV
carriers. These particles persist in the serum and the
corresponding HBsAg-specific antibodies (anti-HBs) are not induced
or remain undetectable by the conventional techniques due to the
presence of immune complexes. Thus, tolerance to HBsAg is
characteristic of chronic HBV carriers.
[0082] The present invention relates to breaking the tolerance to
HBsAg in order to control the infection. The inventors discovered
that the lack of T cell response in a mouse model of chronic HBV
carriers may be responsible for this tolerance. Thus, the
"treatment" of said chronic HBV carriers relates to the inducement
of an immune response to break the T-cell tolerance to HBsAg and a
"therapeutically effective amount" is said to be that amount, which
induces the immune response to break the T-cell tolerance.
[0083] In a preferred embodiment, the composition administered to
chronic HBV carriers comprises a plasmid carrying the S2.S form of
HBsAg and particularly, is pCMV-S2.S.
[0084] The present invention further relates to a composition
capable of inducing a cytotoxic response comprised of at least one
nucleotide sequence expressed in the muscle cells and a promoter
such as defined above.
[0085] The present invention further relates to a non-lipid
pharmaceutical composition for immunizing an individual against a
viral infection, such as a hepatitis, including, on the one hand, a
substance capable of inducing a coagulating necrosis of the muscle
fibers, such as bupivacaine, and, on the other hand, vector such as
described above or including one of the nucleotide sequences coding
for at least a portion of a virus protein, complete or partial,
capable of being expressed in muscle cells. The "partial sequence"
is a sequence coding for at least six (6) amino acids.
[0086] The substance capable of inducing a coagulating necrosis of
the muscle fibers is preferably bupivacaine.
[0087] The substance capable of inducing a coagulating necrosis of
the muscle fibers is administered into the muscle of an individual
to be immunized, followed at least five (5) days later by
administration of the vector into the muscle of an individual to be
immunized. Preferably, the administration of the substance and the
vector is substantially in the same location in the individual's
muscle. In another preferred embodiment, the vector is administered
ten (10) days after administration of bupivacaine and substantially
in the same location of the individual's muscle.
[0088] The prior administration of bupivacaine has demonstrated an
unexpected increase in the effectiveness of the vector
administration as well as in the immunization of the individual.
Thus, the invention further relates to a method of increasing the
effectiveness of DNA-based vaccines, such as those described
above.
[0089] The compositions of the present invention may contain
additives, which are compatible and pharmaceutically
acceptable.
[0090] Moreover, the compositions of the present invention may be
administered by means known in the art and preferentially by
intramuscular or intradermal injection. The injection can be
carried out using a syringe designed for such use or a liquid jet
gun as described by Furth (Anal. Biochem., 205:365-368 (1992)).
[0091] The effective amount of bupivacaine used is that which
obtains sufficient degeneration of the muscle tissue in order to
achieve optimal immunization. An injection dosage of about 0.1 mg
to about 10 mg per injected composition is usually suitable.
[0092] The effective amount of the vector to be injected is that
amount effective to achieve optimal immunization or
immunotherapeutic treatment of the individual against the virus of
interest according to the protein coded by the gene carried by the
vector. An injection dosage of about 0.1 to about 1000 .mu.g of
vectors per individual is usually suitable.
[0093] The present invention is illustrated by, without in any way
being limited to, the following examples.
Example 1
Induction of Antibodies Against a Hepatitis B Surface Antigen by
Sequential Injection of Bupivacaine and of a Plasmid Carrying a
Gene Coding for the Antigen
[0094] (1) Materials and Methods
[0095] 1.1 Bupivacaine Pretreatment
[0096] All experiments were made on the muscles of the anterior
tibia (AT) of mice C57BL/6 aged between 5 to 7 weeks.
[0097] A single degeneration-regeneration cycle of the muscle
fibers is induced in the muscles of the anterior tibia of
non-anaesthetized mice, by intramuscular injection of 50 .mu.l
marcaine (bupivacaine 0.5%, DMSO 1%) sold by Laboratoires Astra,
France. The solution is injected using a tuberculosis syringe with
a needle fitted into a polyethylene sleeve in order to limit the
penetration depth to 2 mm.
[0098] As marcaine is an anesthetic, injections into the right and
left legs were performed at 10 to 30 minute intervals to prevent an
overdose.
[0099] 1.2 DNA Preparation
[0100] The plasmid used was constructed by cloning into a modified
pBlueScript vector of the XhoI-BglII restriction fragment of the
pCP10 plasmid, which contains the gene coding for the HBs surface
antigen and the non-translated sequences, both upstream and
downstream, including the polyadenylation signal.
[0101] The S gene was then recovered by digestion using KpnI-BssHII
enzymes and the fragment was cloned into the site of the pRC/CMV
vector sold by In Vitrogen. The final plasmid construction was
called pCMV-HBS and was filed under No. 1-1370 with the CNCM.
[0102] This plasmid is represented schematically in FIG. 1. The CMV
promoter is situated between the 288 nucleotide, which is the
cleavage position of MluI, and the 896 nucleotide, which is the
cleavage position of KpnI. The DNA fragment including the
structural gene of the HBs antigen structure was cloned between the
896 and 2852 nucleotides (position of BssH III)
[0103] The HBs gene spreads between the 911 (XhoI position) and
2768 nucleotides (BglII position), respectively.
[0104] The complete sequence for this plasmid is sequence SEQ ID
No. 1.
[0105] The purified plasmid DNA was prepared by standard methods
then redissolved in PBS buffer and stored at -20.degree. C. until
the injection was performed.
[0106] 1.3 DNA Injection
[0107] One to five days after the marcaine injection, DNA was
injected into the same area, the mouse being anaesthetized using
sodium pentobarbital (75 mg/kg interperitonal path).
[0108] The DNA solution, which contains 50 .mu.g of plasmid DNA and
50 .mu.l of PBS buffer, was injected by a single intramuscular
injection through the skin into the anterior tibia muscles
undergoing regeneration.
[0109] The injections were performed bilaterally into the two legs
of the mice, each animal thus receiving a total of 100 .mu.g of
recombinant plasmid DNA. As for the marcaine injection, the DNA
solution was injected using the tuberculosis syringe with the
needle described previously.
[0110] A single intramuscular DNA injection was performed in each
leg.
[0111] 2. Results
[0112] The results obtained are summarized in Table 1 below.
[0113] They show very clearly that a DNA injection after treatment
with marcaine allows a large number of seric antibodies to be
obtained against the hepatitis B surface antigen.
[0114] These results are surprising. From the analysis of the state
of the art it was not inferred that a plasmid would allow the
induction of anti-HBs antibodies, which could be found in the serum
and thus allow an effective vaccination.
[0115] The ease of application of the plasmid vaccination, and the
fact that boosters would not be necessary, allows the consideration
of a large scale vaccination.
Example 2
Comparison of the Efficiency of a Plasmid Injection in the Presence
and Absence of Lipids
[0116] A dose of 10 .mu.g plasmid DNA from the SV40-luciferase
vector available commercially ("pGL2-Control Vector" from Promega,
reference E1 11) in 50 .mu.l of physiological solution was injected
into the sucrose pretreated muscle following the method of Davis et
al. (Hum. Gene Ther. 4:151-159 (1993)). The injected DNA is mixed
earlier with lipids such as dioctadecylamidoglycyl spermine (DOGS)
or the following mixtures: DOGS+spermidine, and
DOGS+polyethyleneglycol (PEG). The luciferase activity was
determined 5 days after the injection.
[0117] These results are shown in table II below.
[0118] They show that the presence of lipids (DOGS) very
significantly reduces the efficiency of the plasmid injection with
respect to a composition with no lipids (Control).
Example 3
Comparison of the Responses of Mice and Rabbits to Plasmids
Carrying Different Promoters and Envelope Genes for the HBV
Virus
[0119] Five plasmids were constructed allowing the expression of
one, two, or three envelope proteins for the HBV virus. In three of
the constructions (pCMVHB-S, pCMVHB-S2.S, pCMVHB-S1.S2.S) the genes
coding for the HBV virus envelope proteins are put under
transcriptional control of the promoter of the CMV virus precursor
genes (FIG. 1, FIG. 2A to 2C, FIGS. 3 and 4). The fourth plasmid
(pHBV-S1.S2.S) uses the promoter for the HBV virus surface genes
contained in the pre-S1 region of this virus (Cattaneo et al.
(1983) Nature, 305, 336) (FIG. 2D) as a transcriptional controlling
element. In another plasmid, pSVS, the three envelope proteins were
placed under control of the SV40 promoter (pSVS) (Michel et al.,
Proc. Natl. Acad. Sci. USA, 81:7708-7712 (1984)). The construction
of the pSVS plasmid is further described in EP0 156 712 B1. In the
five constructions, the polyadenylation signal used is contained in
the HBV sequences present in 3' of the S gene.
[0120] 1. In Vitro Control of the Vector Efficiency.
[0121] To control the efficiency of these vectors in vitro in
eukaryotic cells, mouse fibroblasts or myoblasts were transfected.
FIG. 6 illustrates secretion kinetics of HBs particles in the
culture supernatants. The low antigen levels produced by
transfection of the pCMVHB-S1.S2.S vector are compatible with a
large degree of synthesis of the large envelope protein starting
from the CMV promoter. This protein being myristilated in its amino
terminal region is retained in the endoplasmic reticulum (Ganem,
Current Topics in Microbiology and Immunology, 168:61-83 (1991)).
Retention in the cell of proteins carrying the pre-S1 determinants
was confirmed by immunofluorescence.
[0122] The composition of the secreted particles was analyzed in an
ELISA sandwich system using as capture antibodies a monoclonal
mouse antibody specific to the pre-S1 (FIG. 7A) or pre-S2 (FIG. 7B)
determinants and rabbit anti-HBs polyclonal serum as second
antibodies. These experiments show that the HBsAg particles
produced starting from pCMVHB-S1.S2.S vector carry pre-S1 and
pre-S2 determinants showing the presence of the large and medium
envelope proteins of the HBV virus. Particles secreted after the
transfection of the pCMVHB-S2.S and pHBV-S1.S2.S vectors carry, in
addition to the HBs determinants, pre-S2 determinants
characteristic of the medium envelope proteins. It was observed
that the proportion of pre-S1 and pre-S2 epitopes on the pSVS
particles was twice the amount found on pCMV-S1.S2.S HBsAg
particles.
[0123] 2) DNA Inoculation
[0124] DNA purified on a Quiagen column was injected by an
intramuscular path in a single injection of 100 .mu.g (50
.mu.g/leg) in the anterior tibia muscle of mice C57BL/6 (8 mice per
group). Five days prior to the injection, the muscle was pretreated
with cardiotoxin in order to induce degeneration followed by
regeneration of muscles cells, thus favoring DNA capture by these
cells.
[0125] The DNA injection experiments were also carried out for
rabbits. In this case, pCMVHB-S DNA was administered into normal
muscle without degeneration, either by using an injection gun
without needle called Biojector.RTM., or by conventional syringes
fitted with needles.
[0126] 3) Anti-Hbs Responses for Mice Vaccinated with DNA
[0127] An anti-HBs antibody response is induced by a single
injection of any one of the four plasmids used.
[0128] The antibody response was analyzed using a commercial
anti-HBs antibody detection kit (Monolisa anti-HBs, Diagnostic
Pasteur). Anti-preS2 antibodies are detected by an ELISA system
using, on the solid phase, a peptide from the pre-S2 (AA 120-145)
region corresponding to a B major epitope carried by this area
(Neuarth et al., 315:154 (1985)).
[0129] FIGS. 8A to 8D illustrate the anti-HBs (HBs-Ab) response
kinetics expressed in milli-international units/ml and the
anti-pre-S2 response (preS2Ab) determined as optical density (492
nm) for 1/100 diluted serums. Detection was carried out using a
mouse anti-immunoglobulin antibody (IgG) labeled with
peroxidase.
[0130] The injection of the pCMVHB-S plasmid (FIG. 8A) induces a
constant anti-HBs antibody synthesis. Seroconversion was observed
in 100% of mice from one week after the injection with an antibody
average level of 48 mUI/ml (from 12 to 84 mUI/ml, standard
deviation (SD)=28), which is 4 to 5 times superior to the threshold
required in man to provide protection (10 mUI/ml)
[0131] The induced response for a single injection of pCMVHB-S2.S
plasmid (FIG. 8B) is characterized by the very early appearance of
anti-HBs antibodies. These antibodies reach an average level of 439
mUI/ml (from 104 to 835 mUI/ml; SD=227) at one week then decline
before increasing again to reach the initial level at 13 weeks. The
significance of this antibody peak will be discussed later. A peak
for anti-pre-S2 IgG antibodies is observed at two weeks.
[0132] The appearance of anti-HBs antibodies induced by injection
of pCMVHBV-S1.S2.S plasmids (FIG. 8C) and pHBV-S1.S2.S (FIG. 8D) is
slightly delayed as the mice only seroconvert to 100% after two
weeks. The seroconversion profile is identical; it is characterized
by an initial response, which is specific to the pre-S2 antigen
followed by an anti-HBs response, which gradually increases to
reach a level of 488 mUI/ml (from 91 to 1034 mUI/ml; SD=552)
(pCMVHBS1.S2.S) and 1725 mUI/ml (from 143 to 6037 mUI/ml; SD=1808)
(pHBV-S1.S2.S) at 13 weeks.
[0133] Anti-HBs antibodies were not detected earlier than 2 weeks
after pSVS injection. However, at its peak level (12 weeks),
HBs-specific antibodies induced were two times greater than that
with pCMV-S1.S2.S. The sera from mice injected with either pSVS or
pCMV-S1.S2.S indicated that they contained both group-specific (a)
and subtype-specific (y) antibodies. However, antibodies in
pCMV-S1.S2.S-treated mice appeared to show a stronger affinity to
pre-S2 than those treated with pSVS.
[0134] 4) Anti-HBS Response of Rabbits Injected with DNA
[0135] Results presented in tables III and IV show that the
antibody levels detected at 8 weeks in rabbits immunized using the
Biojector are significantly higher than those obtained by a DNA
injection by needle.
[0136] 5) Specificity of Clinically Defined Antibodies
[0137] Synthetic peptides were use to determine the relative
amounts of pre-S2 and pre-S1-specific antibodies for mice injected
with pSVS or pCMV-S1.S2.S. The binding to a synthetic peptide
pre-S2 peptide (residues 120-145) or pre-S1 peptide (residues
12-49) was measured and depicted in FIG. 8E. IgM and IgG titers
were present after two weeks. While the total titers declined over
the next two week, IgG titers continued to increase and was
maintained for at least 6 months after DNA injection mice injected
with each vector. FIGS. 8F and 8G demonstrate that antibodies to
other pre-S2 determinants of the middle protein are induced after
pCMV-S1.S2.S injection, or alternatively, that the response to
pre-S2 is overwhelmed by anti-pre-S1 antibodies in pSVS injected
mice.
[0138] The peptide encompassing residues 12-49 had previously been
shown to bind human antibodies specific to pre-S1 on native HBsAg
particles (Milich et al., J. Immunol. 137:315-344 (1986)) and
peptide 94-117 to be a dominant antibody binding site for murine
antibodies (Milich et al., 1986). Antibodies to pre-S1 peptide
12-49, but not to peptide 94-114, were detected in the sera of mice
injected with pSVS only. The specificity of the antibody induced
suggests that the particles produced after muscle transfection are
closely related to particles produced in vivo during infection in
humans.
[0139] FIG. 8H depicts the antibody levels in mice injected with
expression vectors pSVS (squares) and pCMV-S1.S2.S (circles). After
two weeks of DNA injection, 100% of the injected mice had
seroconverted to a titer of at least 10 mIU/ml, which is recognized
as a level to be protective in humans. At 12 weeks, these levels
were 50 to 100 times higher.
Example 4
Humoral Responses of Mice to Genetic Vaccine
[0140] 1) Qualitative Analysis of the Humoral Response
[0141] ELISA systems applied to the solid phase of the HBs antigens
of varying composition with respect to the determinants presented
on the solid phase and using mouse antibodies specific to IgM or
IgG as second antibodies gave a qualitative analysis of the
antibody response that was achieved.
[0142] In all cases, the single injection of DNA in mice is
characterized by the early appearance of HBsAg specific to IgM
followed immediately by conversion to IgG isotype antibodies, which
is characteristic of the memory response induced by the auxiliary T
cells. The antibody response to the DNA injection is characterized
by its prematurity. Indeed, seroconversion is achieved 8 to 15 days
after the injection depending on the DNA type used and in all cases
the plateau is achieved in four weeks and maintained constantly
over a period of 12 weeks.
[0143] The use of the heterologous subtype HBs antigens (ad) fixed
on ELISA plates allows the formation/detection of the presence, in
the serum of immunized mice, of antibodies specific to the anti-a
group, and by difference in reactivity with respect to HBsAg of the
same subtype (ay), of antibodies specific to the anti-y subtype.
The presence of antibodies specific to determinants of the HBsAg
group is very important as the former are capable of giving
protection against the heterologous subtype virus during virulent
tests in chimpanzees (Szmuness et al., N. Engl. J. Med.
307:1481-1486 (1982)).
[0144] Analysis of the response induced by the pCMV-S2.S vector
shows that it has a remarkable similarity with the one, which can
be observed in man during infection. It is characterized by an
extremely early (8 days) peak for IgM which is specific to the
pre-S2 region immediately followed by conversion to anti-pre-S2 IgG
(FIG. 9). This response is followed by the appearance of IgM then
IgG anti-HBs antibodies. The anti-HBs antibody production is
constant and reaches a maximum after 4 weeks. At 13 weeks IgG
anti-HBs and anti-pre-S2 remain at a constant level.
[0145] The anti-subtype (y) response precedes that of the
anti-group response (a) in the same way as that described for the
vaccination with the recombinant vaccine (Tron et al., J. Infect.
Dis. 160:199-204).
[0146] Thus, injection of a vector encoding the small and the
middle forms of HBV envelope protein (pCMV-S2.S) into normal mice
induced a strong and long-lasting humoral response to the preS2
domain of the middle protein and to subtype- and group-specific
HBsAg determinants.
[0147] The response obtained with the three other DNA vaccines
illustrates the commutation of class IgM-IgG, which is
characteristic of the secondary response.
[0148] The response being first of all directed against the subtype
before being against the HBsAg group determinants.
[0149] The long term response, which was studied for pCMVHB-S DNA,
shows that the antibody peak is reached within 3 months and this
remains at a constant level 6 months later (Table V) (Davis et al.,
Vaccine, 14 (9):910-915 (1996)).
[0150] 2. Immunization of Mice with Genetic Vaccine and
Non-Response
[0151] The high number of non-responders to the classical vaccine
(2.5 to 5%) remains a major problem for vaccination against
hepatitis B. It has been possible to correlate the non-response in
man to certain HLA types (Krustall et al., J. Exp. Med. 175:
495-502 (1992)) and to a defect in the antigen presentation or
stimulation of the auxiliary T cells.
[0152] To study the possible impact of the genetic vaccination on
the HBsAg non-response, a range of mice strains that were used for
which the response to various HBV virus envelope proteins is
controlled genetically and has been well characterized by Millich
et al. (J. Immunol. 137:315 (1986)). The pCMVHB-S construction
previously described was injected into B10 (H-2.sup.f), B10.S
(H-2.sup.s), and B10.M (H-2.sup.f) mice muscles.
[0153] The B10 strain responds to the three virus envelope
proteins. The B10.S strain does not respond to HBsAg, however this
non-response can be overcome by immunization with HBsAg antigens
which are carrying pre-S2 determinants. The B10M strain is totally
non-responsive to both HBs and pre-S2 antigens. A response for the
latter strain can be achieved by immunization using HBsAg carrying
pre-S1 determinants.
[0154] The mice immunized by the DNA received a single injection
(100 .mu.g) in the regenerating muscle. Control mice were injected
with two intraperitoneal injections of protein at an interval of
one month, the first of 2 .mu.g HBsAg to which the complete Freund
additive (CFA) was added and the second of 2 .mu.g HBsAg to which
the incomplete Freund additive (IFA) was added.
[0155] The results obtained for pCMVHB-S are illustrated by FIGS.
10A to 10F.
[0156] In the B10 strain (good responder), the DNA-induced response
is earlier than that induced by the protein after a single
injection.
[0157] The appearance of anti-HBs antibodies subtype specific then
group specific after immunization with pCMVHB-S DNA was observed in
the B10S strain (nonresponder to HBsAg in the absence of pre-S2).
Group specific anti-HBs antibodies are observed in HBs protein
immunized mice only after the second injection.
[0158] A group and subtype specific anti-HBs response is obtained
for DNA immunization of strain B10M (nonresponder to HBsAg in
absence of pre-S1), whereas only a subtype specific response is
induced by the protein with two injections being required.
[0159] The response induced by the three vector types is compared
in the three mice strains.
[0160] 3. Genetic Vaccine in HBsAg Transgenic Mice
[0161] Transgenic mice of the C57BL/6 strain is used as a model of
chronic HBV carriers since they constitutively express HBsAg. A
single injection of the pCMV-S2.S DNA into HBsAg-transgenic mice
(H-2.sup.b) provoked a decrease in titres of circulating HBsAg
(FIG. 12) and the concomitant appearance of anti-HBs antibodies,
which increased over time (FIG. 13). In some of the mice, antigen
was eliminated from the serum as early as four weeks after
injection of the DNA and remained undetectable for at least twelve
weeks without further injections of DNA. In the remaining mice,
antigen levels also fell and were maintained at low levels. These
effects were not due to non-specific immune stimulation induced by
the injection procedure or the presence of DNA per se, since HBsAg
levels were unaffected (FIG. 12) and no anti-HBs were detected
(FIG. 13) in control transgenic mice injected with PBS alone or a
DNA vector expressing beta-galactosidase (.beta.-gal; pCMV-LacZ)
(Davis, 1993), even though the latter procedure induced high levels
of anti-B-gal antibodies (ELISA titres>10.sup.5 by 12 weeks post
immunization).
[0162] Free antibodies were first detectable in the plasma of
transgenic mice 2-4 weeks following a single injection of pCMV-S2.S
DNA (FIG. 13). The first antibodies detected were preS2-specific
since they reacted only with particles carrying this epitope but
not with particles devoid of it. Anti-HBs antibodies were not
observed until 8 weeks, at which time there was a complete
clearance of circulating HBsAg (see FIG. 12). It is remarkable
that, even though the transgenic mice had been tolerant to high
levels of circulating HBsAg, DNA-based immunization was able to
induce titres of anti-HBs comparable to those induced in
non-transgenic controls, and that these antibodies were able to
completely neutralize the circulating HBsAg. The isotype profile of
the anti-HBs antibodies was identical in transgenic and in
non-transgenic mice and included IgG2 as well as IgG1 with some
IgG3 (FIG. 14). Such isotype switching of autoreactive B cells
strongly suggests that the DNA-mediated immunization triggered CD4+
T helper cells.
[0163] 3.a. Cytokine Production from Spleen Cells of Transgenic and
Non-Transgenic Mice
[0164] To further characterize the T-helper subset, the cytokine
production from spleen cells in culture was analyzed. Spleens were
removed from transgenic mice 20 weeks after DNA injection and cell
suspensions were specifically stimulated in vitro with HBsAg. These
cultures produced .gamma.-interferon (IFN-.gamma.), low levels of
IL-2 and TNF-.alpha., but no IL-4 as depicted in Table VII. The
secretion of IgG2a and .gamma.-interferon is consistent with a
predominant Th1 response, however, detection of IgG1 suggests that
the Th2 response was also induced.
[0165] 3.b. Regulation of Transgene Expression in pCMV-S2.S
Vaccinated Transgenic Mice
[0166] The rapid clearance of circulating antigen from immunized
mice did not appear to result from a significant or persistent
HBsAg-specific cytopathic effect on the liver since levels of
transaminase activity in the plasma remained normal subsequent to
injection of DNA and histological examination of the liver at the
time of HBsAg clearance showed not evidence of necrosis or
inflammation (not shown).
[0167] Since the inventors could not correlate the persistent
clearance of the transgene product with any apparent destruction of
the transgene-expressing liver cells, the HBV mRNA content in the
livers of transgenic B10 mice was evaluated. At 12 weeks after
immunization with pCMB-S2.S, the mRNA was decreased in the livers
of those mice which had partially cleared the antigen, and was
undetectable in those which had completely eliminated HBsAg from
their sera (FIG. 15A, lanes 5 and 6-7 respectively). This effect is
persistent since HBV mRNA remained undetectable in the livers of
mice analyzed 20 weeks after DNA injection (FIG. 15A, lanes 8-9).
In contrast, HBV mRNA was not diminished in livers taken from
untreated transgenic mice or control transgenic mice, which had
been injected with pCMV-LacZ DNA (FIG. 15A, lane 1 and lanes 2-4
respectively). This indicates that the inhibition of viral gene
expression in transgenic mice injected with pCMV-S2.S was not due
to a non-specific effect such as the release of cytokines with
injection-induced inflammation and/or with an immune response
against transfected muscle cells expressing a foreign antigen
(i.e., .beta.-gal). Thus, the HBsAg-specific immune response
induced by immunization with plasmid DNA appears to be responsible
for controlling hepatic transgene expression by some non-cytopathic
mechanism.
[0168] 3.c. Injection of DNA Induced B and T-Cell Response to
HbsAg
[0169] To determine which component of the immune response is
implicated in the down-regulation of HBV-specific mRNA and in the
observed decrease or elimination of the circulating antigen,
adoptive transfer experiments were performed. Fully immunocompetent
non-transgenic mice were immunized once with the pCMV-S2.S DNA
vector and when ELISA titres of serum antibodies to HBsAg had
reached at least 10.sup.4, both the serum and the primed spleen
cells were harvested from the mice for transfer into their
transgenic littermates.
[0170] Passive transfer of serum-derived antibodies on a single
occasion into transgenic mice induced a rapid but transient
decrease in circulating HBsAg levels (mice 2.21 and 4.26, Table
VI). In other transgenic mice, circulating antigen was maintained
at undetectable levels for a longer period by intra-peritoneal
injection of hyperimmune sera every 2-3 days over a period of 17
days (mice 1.3.5 and 1.3.6, Table 2). Neither single nor chronic
administration of antibodies resulted in decreased HBV-specific
mRNA in the liver (not shown) indicating that the humoral response
after DNA-based immunization was not responsible for the
down-regulation of transgene expression or the long-term
elimination of the transgene product.
[0171] Injection of primed spleen cell suspensions obtained from
pCMV-S2.S immunized non-transgenic donor mice into transgenic
littermate recipients resulted in a rapid clearance of circulating
HBsAg (by 7 days) and a concomitant appearance of anti-HBs
antibodies, which were sustained (FIG. 16). This indicates that the
transferred T and B cells were functional within the environment of
the transgenic mice and that the B cells were activated, probably
by the circulating antigen. Adoptive transfer of HBsAg-primed
spleen cells was also able to induce a complete disappearance of
HBV mRNA in the liver by 17 days (FIG. 17 and FIG. 15B, lanes 4,
5). The inability to detect serum HBsAg occurred 10 days prior to
the disappearance of HBV mRNA suggesting that two separate
mechanisms may be responsible for the observed clearance of the
antigen: an initial transient elimination due to formation of
immune complexes and a subsequent more permanent control of
transgene transcription. Serum transaminase activity and
histological examination of liver sections were monitored every 2-3
days after adoptive transfer and found to be normal for up to 17
days, after which some histological sections of liver exhibited a
few small necrotic foci. These were sometimes accompanied by the
presence of inflammatory cells, but in no case did the necrotic
regions involve more that 5% of the hepatic cells. In addition, a
few apoptotic hepatocytes were detected in centrilobular areas
within some randomly distributed lobules (not shown). The changes
induced by adoptive transfer with HBsAg-primed spleen cells were
HBsAg-specific since injection of .beta.-gal-primed spleen cells
into transgenic recipients had no effect on the levels of serum
HBsAg (FIG. 16) or liver HBV mRNA (FIG. 14B, lanes 2, 3).
[0172] 3.d. T Cells are Able to Control Transgene Expression in the
Absence of Antibody Production.
[0173] To determine the spleen cell population, which was involved
in the decrease or disappearance of circulating HBsAg and liver HBV
mRNA, adoptive transfer was carried out with fractionated B- or
T-spleen cells obtained from non-transgenic donor mice immunized
with pCMV-S2.S. After depletion of T-cells, the transfer of
HBsAg-primed spleen cells into transgenic mice did not induce
anti-HBs and had no effect on levels of circulating HBsAg or liver
HBV mRNA (FIG. 15B, lane 6). This indicates that antibody
production in DNA-immunized transgenic mice is T-cell dependent. In
contrast, transfer of B cell-depleted spleen cells resulted in
clearance of circulating HBsAg within 14-17 days although antibody
to HBsAg was not detected in these recipient mice at these or later
times (not shown). Thus, antigen-antibody complex formation is not
required for, but has a synergistic effect on the elimination of
circulating antigen (FIG. 16). Furthermore, it appears that
HBsAg-specific T cells are also responsible for down regulation of
transgene expression since only transfer of T--but not B--or
unprimed T-cells was able to reduce HBV mRNA in the liver to
undetectable levels (FIG. 15B, lane 6-9).
[0174] 4. Genetic Vaccine in Clinical Trials
[0175] H. A. Thoma, Progress in Hepatitis B Immunization, P.
Coursaget and M. J. Thong (Eds.), Colloque INSERM: Paris, France,
194:35-42 (May 3-5, 1990), the entire contents of which are
incorporated by reference herein, reports that a third generation
vaccine containing portions of pre-S1, pre-S2, and S proteins
demonstrates fast and high immune response in clinical trials in
man as compared to other available vaccines.
[0176] It is generally thought that the humoral response to HBs
antigens is sufficient by itself to give protection. The presence
of antibodies directed against other determinants (pre-S1 and
pre-S2) carried by the virus envelope proteins, themselves
protectors, could improve the response quality. The experiments
reported here as a whole illustrates that the humoral response
induced by the genetic anti-hepatitis B vaccination is greater in
several fields than that which can be achieved for the classical
vaccination.
[0177] In terms of seroconversion levels, the 100% level is
obtained, after only one injection, from day 8 for mice immunized
with pCMV-HBS DNA and pCMVHB-S2.S.
[0178] In terms of response level, the 10 mUI/ml threshold level,
considered sufficient to give protection in man, is always greatly
exceeded.
[0179] In terms of the speed of response, in 8 days a very high
level of anti-pre-S2 antibodies is obtained for the pCMVHB-S2.S
vector and it is known that the former are capable of giving
protection by themselves (Itoh et al., (1986) Proc. Natl. Acad.
Sci. USA 83, 9174-9178).
[0180] In terms of response stability, anti-HBs antibodies remain
constant at a high level for more than 6 months.
[0181] In terms of response quality, type IgG antibodies
characteristic of a response, which is dependent on the auxiliary T
cells and therefore on a memory response, are obtained. Moreover,
the single injection of DNA encoding the HBV protein is sufficient
to break T-cell tolerance in transgenic mice expressing the same
envelope sequences in the liver.
[0182] In terms of anti-viral activity, the antibodies are specific
to the viral subtype, but especially group-specific and therefore
susceptible to giving a cross protection and to clear HBsAg
particles in HBsAg transgenic mice.
[0183] In terms of biological significance, the response profile
obtained by pCMVHB-S2.S immunization mimes totally that which is
observed in man after a resolved viral infection.
[0184] The immune response resulting from plasmid expression of the
HBsAg leads to both clearance of circulating antigen as well as the
induction of T-cell responses capable of suppressing HBV mRNA
accumulation in the liver in a transgenic mouse model. Despite the
high concentration of transgene product in the circulation,
auto-antibodies are induced soon after DNA injection, although
initially these are directed only against the preS2 epitope.
[0185] In terms of treating HBV chronic carriers, the murine model
shown here demonstrates that T-cell non-responsiveness can be
overcome by using DNA-mediated immunization. The induced response
mimics in some aspects that required to clear a viral infection,
namely an adequate cellular immune response to regulate viral gene
expression without killing infected cells and an adequate humoral
response to prevent the spread of free virus to uninfected cells.
Thus, the inventors have arrived at the first demonstration of an
immunotherapeutic application of DNA-mediated immunization against
an infectious disease and particularly, provides a treatment for
HBV chronic carriers.
TABLE-US-00001 TABLE I Induction of antibodies against the
hepatitis B surface antigen Level of antibodies against hepatitis B
surface antigen in Number of the serum (mIU/ml) 15 days after 35
days after Description mice Before DNA injection DNA injection DNA
injection DNA injected 1 day 5 0 average: 56 from average: 59 after
marcaine 5 to >140 treatment DNA injected 5 days 5 0 average: 71
from average: 47 after marcaine 21 to >100 treatment
TABLE-US-00002 TABLE II Luciferase RLU/sec/muscle (Average .+-.
SEM) RLU = Percentage relative Group Relative Light Unit to the
control Control 43 082 .+-. 5 419 100% 4X DOGS 20 .+-. 7 0.06% DOGS
- Spermidine 50 .+-. 23 0.12% PEG-DOGS 0 .+-. 0 0.00%
TABLE-US-00003 TABLE III Immunization with the Biojector.sup.R
pCMV-HB.S N.degree. 0 weeks 2 weeks 8 weeks 2.1 0 517 380 2.2 0 374
322 3.1 0 250 418 4.1 0 400 4045 4.2 0 88 86 4.3 0 314 420 6.1 0
415 1001 6.2 0 1543 3517 6.3 0 1181 141 Average 0 566 mUI/ml 1148
mUI/ml SD 0 476 1521 SEM 0 159 507 N 9 9 9 CV 84% 133%
TABLE-US-00004 TABLE IV Immunization by injection using a needle
pCMV-HB.S N.degree. 0 weeks 2 weeks 8 weeks 1.1 1 0 1 5.1 0 287 186
5.2 0 162 798 5.3 0 305 203 7.1 0 86 175 7.2 0 1100 dead Average 0
325 mUI/ml 273 mUI/ml SD 0 401 305 SEM 0 164 136 N 6 6 5 CV 245%
124% 112%
TABLE-US-00005 TABLE V Long term response of a mouse vaccinated
with pCMVHB-S 1 2 3 6 month months months months * a-HBs titre 227
662 1299 1082 in mUI/ml * a-HBs 3.5 .times. 10.sup.-4 5 .times.
10.sup.-4 0.5 .times. 10.sup.-4 9 .times. 10.sup.-4 ELISA titre
Table VI
[0186] Serum titres of HBsAg in Tg mice passively transferred with
antibodies to HBsAg. Results are shown for seven Tg mice injected
intraperitonealy once (mice 4-23, 2-21, 6-11 and 4-26) or every 2-3
days (mice 1-3-16, 1-3-5, 1-3-6) with either anti-HBs immune sera
(anti-HBs Ab) or normal mouse sera (NMS). HBsAg titers (ng/ml) were
determined in the sera collected at the indicated time. Mice were
killed (.dagger.) 26 hours or 17 clays after the transfer and their
livers were harvested for extraction of mRNA and Northern blot
analysis. ND: Not Done
TABLE-US-00006 Injected Bleeding (days) Mouse n.sup.o serum 0 0.25
1 2 3 6 10 15 17 4-23 NMS 429 404 477 420 440 452 ND 252 502
.dagger. 2-21 anti-HBs Ab 1321 0 13 61 373 725 ND 1028 1356
.dagger. 6-11 NMS 696 548 442 .dagger. 4-26 anti-HBs Ab 1080 0 22
.dagger. 1-3-16 NMS 565 ND 542 ND 326 562 328 647 693 .dagger.
1-3-5 anti-HBs Ab 721 ND 0 ND 0 0 0 3 0 .dagger. 1-3-6 anti-HBS Ab
548 ND 3 ND 0 0 3 6 0 .dagger.
Table VII
[0187] Secretion of cytokines by spleen cells in culture.
Splenocytes of pCMV-S2.S-immunized Tg and non-Tg mice were
incubated with medium or stimulated with concanavalin A (ConA, 2.5
.mu.g/ml), preS2 peptide (10 .mu.g/ml) or HBsAg particles (3
.mu.g/ml) for 72 hr. Antigen-specific culture supernatants were
harvested for determination of cytokine levels (pg/ml) at 24 hr for
TNF-.alpha. and IL-2 determinations and at 48 hr for IFN-.gamma.
and IL-4. Data are as the arithmetic mean.+-.SEM of 5 to 6 spleens
independently tested in two experiments.
TABLE-US-00007 Mice Cytokines Medium ConA preS2 peptide HBsAg
Non-Tg IFN-.gamma. 2 .+-. 1 1,611 .+-. 377 92 .+-. 42 60 .+-. 28
TNF-.alpha. 28 .+-. 13 846 .+-. 87 267 .+-. 23 58 .+-. 17 IL-2 2
.+-. 1 2,584 .+-. 233 3 .+-. 2 4 .+-. 2 IL-4 6 .+-. 4 62 .+-. 15 4
.+-. 4 8 .+-. 6 Tg IFN-.gamma. 2 .+-. 2 1,709 .+-. 12 329 .+-. 227
39 .+-. 25 TNF-.alpha. 28 .+-. 12 871 .+-. 13 405 .+-. 137 83 .+-.
46 IL-2 1 .+-. 1 4,136 .+-. 578 1 .+-. 1 1 .+-. 1 IL-4 11 .+-. 8 49
.+-. 3 12 .+-. 6 11 .+-. 9
Sequence CWU 1
1
* * * * *