DNA vaccination for treatment of multiple sclerosis and insulin-dependent
United States Patent: 7,579,328
Issued: August 25, 2009
Lawrence (Palo Alto, CA), Ruiz; Pedro (Menlo Park, CA), Garren; Hideki
(Palo Alto, CA)
Assignee: The Board of
Trustees of the Leland Stanford Junior University (Palo Alto, CA)
Appl. No.: 11/289,226
Filed: November 28, 2005
A pro-inflammatory T cell response is
specifically suppressed by the injection into a recipient of DNA encoding
an autoantigen associated with autoimmune disease. The recipient may be
further treating by co-vaccination with a DNA encoding a Th2 cytokine,
particularly encoding IL4. In response to the vaccination, the
proliferation of autoantigen-reactive T cells and the secretion of Th1
cytokines, including IL-2, IFN-.gamma. and IL-15, are reduced.
Description of the
SUMMARY OF THE INVENTION
Methods are provided for the suppression of pro-inflammatory T cell
responses in autoimmune disease. A mammalian host is vaccinated with a DNA
expression vector encoding an autoantigen fragment. In response to the
vaccination, pathogenic T cell proliferation is inhibited and production
of Th1 cytokines, including IL-2, IL-10, IFN-.gamma. and IL-15 is reduced.
In one embodiment of the invention, a nucleic acid encoding a Th2 cytokine
is co-administered with the autoantigen coding sequence. The use of IL-4
coding sequences is of particular interest. Suppressive vaccination
diminishes T cell pro-inflammatory responses in a specific, targeted
manner. Conditions that benefit from this treatment include autoimmune
diseases, tissue transplantation and other diseases associated with
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The subject methods provide a means for therapeutic treatment and
investigation of inflammation, through the suppression of pathogenic
antigen-specific T-cell responses. A DNA expression cassette is injected
into host tissue, for example muscle or skin. The vector comprises a DNA
sequence encoding at least a portion of an autoantigen. The vaccination
may also include DNA sequences encoding a Th2 cytokine, e.g. IL-4. In
response to this vaccination, a suppressive response is evoked.
Antigen-specific T cell proliferation is inhibited and Th1 cytokine
production is reduced.
Without limiting the scope of the invention, it is believed that the
methods described herein are a novel method of protective immunity, which
combines the effects of DNA vaccination and local gene delivery. After DNA
vaccination with a autoantigen epitope alone, T cells are anergic. This
may be in part due to the biological effects of DNA motifs like
unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs)
(Krieg et al. (1998) Trends in Microbiol. 6:23-27). The addition of IL4 as
a DNA co-vaccine rescues the anergy imposed by the autoantigen DNA
vaccine, and drives the response to a Th2 phenotype. STAT6 is activated in
draining lymph node cells by the IL4 DNA vaccine. It is believed that IL4
is produced from the DNA vaccine administered and that it interacts with
IL4 receptor on lymph node cells, which in turn causes the activation of
STAT6 downstream of the receptor. Immunization against the antigens that
trigger those autoimmune diseases caused by Th1 autoreactive cells,
diseases such as multiple sclerosis, juvenile diabetes and rheumatoid
arthritis, would be conditions where co-vaccination with DNA encoding IL-4
might prove beneficial
Autbantigens, as used herein, are endogenous proteins or fragments thereof
that elicit a pathogenic immune response. Of particular interest are
autoantigens that induce a T cell mediated inflammatory pathogenic
response. Suppressive vaccination with the relevant target autoantigen
finds use in the treatment of autoimmune diseases characterized by the
involvement of pro-inflammatory T cells, such as multiple sclerosis,
experimental autoimmune encephalitis, rheumatoid arthritis and insulin
dependent diabetes mellitus. Animal models, particularly small mammals,
e.g. murine, lagomorpha, etc. are of interest for experimental
The subject methods of suppressive immunization are used for prophylactic
or therapeutic purposes. Use used herein, the term "treating" is used to
refer to both prevention of disease, and treatment of pre-existing
conditions. The prevention of autoimmune disease involving the vaccine
autoantigen (VA), is accomplished by administration of the vaccine prior
to development of overt disease. The treatment of ongoing disease, where
the suppressive vaccination stabilizes or improves the clinical symptoms
of the patient, is of particular interest. Such treatment is desirably
performed prior to complete loss of function in the affected tissues.
Autoantigens known to be associated with disease include myelin proteins
with demyelinating diseases, e.g. multiple sclerosis and experimental
autoimmune myelitis; collagens and rheumatoid arthritis; insulin,
proinsulin, glutamic acid decarboxylase 65 (GAD65); islet cell antigen
(ICA512; ICA12) with insulin dependent diabetes. An association of GAD
epitopes with diabetes is described in a number of publications, including
U.S. Pat. No. 5,212,447; and European patent application no. 94.927940.0.
An association of insulin epitopes with autoimmune insulitis is described
in Griffin et al. (1995) Am. J. Pathol. 147:845-857. Rudy et al. (1995)
Mol. Med. 1:625-633 disclose an epitope that is similar in GAD and
The protein components of myelin proteins, including myelin basic protein
(MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and
myelin oligodendrocyte glycoprotein (MOG), are of particular interest for
use as immunogens of the invention. The suppression of T cell
responsiveness to these antigens is used to prevent or treat demyelinating
In one embodiment of the invention, the vaccine autoantigen is proteolipid.
For convenience, a reference sequence of human PLP is provided as SEQ ID
NO:1; and human myelin basic protein as SEQ ID NO:3. Proteolipid is a
major constituent of myelin, and is known to be involved in demyelinating
diseases (see, for example Greer et al. (1992) J. Immunol. 149:783-788 and
Nicholson (1997) Proc. Natl. Acad. Sci. USA 94:9279-9284).
The integral membrane protein PLP is a dominant autoantigen of myelin.
Determinants of PLP antigenicity have been identified in several mouse
strains, and include residues 139-151 (Tuohy et al. (1989) J. Immunol.
142:1523-1527), 103-116 (Tuohy et al., (1988) J. Immunol. 141:1126-1130],
215-232 (Endoh et al. (1990) Int. Arch. Allergy Appl. Immunol.
92:433-438), 43-64 (Whitham et al., (1991) J. Immunol. 147:3803-3808) and
178-191 (Greer, et al. (1992) J. Immunol. 149:783-788). Immunization with
native PLP or with synthetic peptides corresponding to PLP epitopes
induces EAE. Analogues of PLP peptides generated by amino acid
substitution can prevent EAE induction and progression (Kuchroo et al.
(1994) J. Immunol. 153:3326-3336, Nicholson et al. (1997) Proc. Natl.
Acad. Sci. USA 94:9279-9284).
MBP is an extrinsic myelin protein that has been studied extensively. At
least 26 MBP epitopes have been reported (Meinl et al. (1993) J. Clin.
Invest. 92:2633-2643). Of particular interest for use in the present
invention are residues 1-11, 59-76 and 87-99. Analogues of MBP peptides
generated by truncation have been shown to reverse EAE (Karin et al.,
(1998) J. Immunol. 160:5188-5194). DNA encoding polypeptide fragments may
comprise coding sequences for immunogenic epitopes, e.g. myelin basic
protein p84-102, more particularly myelin basic protein p87-99, (SEQ ID
NO:11) VHFFKNIVTPRTP (p87-99), or even the truncated 7-mer peptide (SEQ ID
NO:12) FKNIVTP. The sequences of myelin basic protein exon 2, including
the immunodominant epitope bordered by amino acids 59-85, are also of
interest. For examples, see Sakai et al. (1988) J Neuroimmunol 19:21-32;
Baxevanis et al (1989) J Neuroimmunol 22:23-30; Ota et al (1990) Nature
346:183-187; Martin et al (1992) J. Immunol. 148:1350-1366, Valli et al
(1993) J Clin Inv 91:616. The immunodominant MBP(84-102) peptide has been
found to bind with high affinity to DRB1*1501 and DRB5*0101 molecules of
the disease-associated DR2 haplotype. Overlapping but distinct peptide
segments were important for binding to these molecules; hydrophobic
residues (Val189 and Phe92) in the MBP (88-95) segment for peptide binding
to DRB1*1501 molecules; hydrophobic and charged residues (Phe92, Lys93) in
the MBP (89-101/102) sequence contributed to DRB5*0101 binding.
The transmembrane glycoprotein MOG is a minor component of myelin that has
been shown to induce EAE. Immunodominant MOG epitopes that have been
identified in several mouse strains include residues 1-22, 35-55, 64-96 (deRosbo
et al. (1998) J. Autoimmunity 11:287-299, deRosbo et al. (1995) Eur J.
Immunol. 25:985-993) and 41-60 (Leadbetter et al. (1998) J Immunol
For the treatment of diabetes, immunogens of interest include IA-2;
IA-2beta; GAD; insulin; proinsulin; HSP; glima 38; ICA69; and p52. For
example, insulin (which sequence is publicly available, for example from
Sures et al. (1980) Science 208:57-59; Bell et al. (1979) Nature
282:525-527; and Bell et al. (1980) Nature 284:26-32) has been found to
have immunodominant epitopes in the B chain, e.g. residues 9-23; as well
as in the pre-proinsulin leader sequence. Other autoantigens associated
with diabetes include glutamic acid decarboxylase 65 (GAD65), e.g.
residues 206-220; 221-235, 286-300; 456-470; and peptides including
residues p247, p509; p524 (Kauffman et al. (1993) Nature 366:69-72).
A DNA expression cassette encoding at least a portion of an autoantigen,
usually as part of a vector, is introduced into tissue of the vaccine
recipient. The minigene is expressed in the tissue, and the encoded
polypeptide acts as an immunogen, or antigen. The autoantigen sequence may
be from any mammalian or avian species, e.g. primate sp., particularly
humans; rodents, including mice, rats and hamsters; rabbits; equines,
bovines, canines, felines; etc. Of particular interest are the human and
mouse autoantigen segments. Generally, the sequence will have the same
species of origin as the animal host, preferably it will be autologous
The subject DNA expression cassette will comprise most or all of the
sequence encoding an autoantigen fragment, as defined by Kabat et al.,
supra. The coding sequence may be truncated at the 5' or 3' terminus and
may be a fragment of the complete polypeptide sequence. In one embodiment
of the invention, the sequence encodes a peptide fragment that is known to
be presented to pathogenic T cells, for example peptides presented by
Class II MHC molecules of the host. Such peptides have been described in
the literature, and are typically of about 8 to about 30 amino acids in
The vaccine may be formulated with one or a cocktail of autoantigen
sequences. While it has been found that a single sequence is capable of
suppressing a response to multiple epitopes, it may be desirable in some
cases to include multiple sequences, where each encodes a different
epitope. For example, see Leadbetter et al. (1998) J. Immunol.
161:504-512. A formulation comprised of multiple coding sequences of
distinct PLP epitopes may be used to induce a more potent and/or sustained
suppressive response. By specifically targeting multiple autoreactive T
cell populations, such a formulation may slow or prevent the development
of autoantigen resistance. The use of PLP sequences in combination with
other myelin protein epitopes may effectively suppress the repertoire of
myelin-reactive T cells. Similar autoantigen combinations to suppress
autoimmune response, e.g., glutamic acid decarboxylase (GAD) and
pancreatic islet cell autoantigen for the treatment of insulin dependent
diabetes, are contemplated.
In addition to the specific epitopes and polypeptides of autoantigens, the
immune response may be enhanced by the inclusion of CpG sequences, as
described by Krieg et al. (1998) Trends Microbiol. 6:23-27, and helper
sequence, King et al. (1998) Nat. Med. 4:1281-1286. Biological effects of
DNA motifs like unmethylated CpG dinucleotides in particular base contexts
(CPG-S motifs) may modulate innate immune responses when injected to
animals. Low numbers of CpG motifs, or the presence of imperfect motifs,
may act in the development of anergy by immunization with autoantigens.
The polypeptide coding sequence, which may be autoantigen or cytokine,
sequences are inserted into an appropriate expression cassette. The
expression construct is prepared in conventional ways. The cassette will
have the appropriate transcriptional and translational regulatory
sequences for expression of the sequence in the vaccine recipient cells.
The cassette will generally be a part of a vector, which contains a
suitable origin of replication, and such genes encoding selectable markers
as may be required for growth, amplification and manipulation of the
vector, prior to its introduction into the recipient. Suitable vectors
include plasmids, YACs, BACs, bacteriophage, retrovirus, and the like.
Conveniently, the expression vector will be a plasmid. Prior to
vaccination, the cassette may be isolated from vector sequences by
cleavage, amplification, etc. as known in the art. For injection, the DNA
may be supercoiled or linear, preferably supercoiled. The cassette may be
maintained in the host cell for extended periods of time, or may be
transient, generally transient. Stable maintenance is achieved by the
inclusion of sequences that provide for integration and/or maintenance,
e.g. retroviral vectors, EBV vectors and the like.
The expression cassette will generally employ an exogenous transcriptional
initiation region, i.e. a promoter other than the promoter which is
associated with the T cell receptor in the normally occurring chromosome.
The promoter is functional in host cells, particularly host cells targeted
by the cassette. The promoter may be introduced by recombinant methods in
vitro, or as the result of homologous integration of the sequence by a
suitable host cell. The promoter is operably linked to the coding sequence
of the autoantigen to produce a translatable mRNA transcript. Expression
vectors conveniently will have restriction sites located near the promoter
sequence to facilitate the insertion of autoantigen sequences.
Expression cassettes are prepared comprising a transcription initiation
region, which may be constitutive or inducible, the gene encoding the
autoantigen sequence, and a transcriptional termination region. The
expression cassettes may be introduced into a variety of vectors.
Promoters of interest may be inducible or constitutive, usually
constitutive, and will provide for high levels of transcription in the
vaccine recipient cells. The promoter may be active only in the recipient
cell type, or may be broadly active in many different cell types. Many
strong promoters for mammalian cells are known in the art, including the
.beta.-actin promoter, SV40 early and late promoters, immunoglobulin
promoter, human cytomegalovirus promoter, retroviral LTRs, etc. The
promoters may or may not be associated with enhancers, where the enhancers
may be naturally associated with the particular promoter or associated
with a different promoter.
A termination region is provided 3' to the coding region, where the
termination region may be naturally associated with the variable region
domain or may be derived from a different source. A wide variety of
termination regions may be employed without adversely affecting
The various manipulations may be carried out in vitro or may be performed
in an appropriate host, e.g. E. coli. After each manipulation, the
resulting construct may be cloned, the vector isolated, and the DNA
screened or sequenced to ensure the correctness of the construct. The
sequence may be screened by restriction analysis, sequencing, or the like.
A small number of nucleotides may be inserted at the terminus of the
autoantigen sequence, usually not more than 20, more usually not more than
15. The deletion or insertion of nucleotides will usually be as a result
of the needs of the construction, providing for convenient restriction
sites, addition of processing signals, ease of manipulation, improvement
in levels of expression, or the like. In addition, one may wish to
substitute one or more amino acids with a different amino acid for similar
reasons, usually not substituting more than about five amino acids in the
In one embodiment of the invention the autoantigen is co-vaccinated with
DNA sequences encoding a Th2 cytokine, which group includes IL-4, IL-10,
TGF-.beta., etc. IL4 is of particular interest. The lymphokine IL-4 has
T-cell and mast cell growth factor activities. Human IL4 is an 18-kD
glycoprotein. For convenience the amino acid sequence is provided herein
as SEQ ID NO:13, and the DNA sequence as SEQ ID NO:14 (Yokota et al.
(1986) P.N.A.S. 83:5894-5898). This sequence is the preferred sequence of
the invention. However, the invention is not limited to the use of this
sequence in constructs of the invention. Also of use are closely related
variant sequences that have the same biological activity, or substantially
similar biological activity. A specific STAT6 DNA-binding target site is
found in the promoter of the IL4 receptor gene; and STAT6 activates IL4
gene expression via this site. Interferons inhibit IL4-induced activation
of STAT6 and STAT6-dependent gene expression, at least in part, by
inducing expression of SOCS1 (see Kotanides et al. (1996) J. Biol. Chem.
Variant sequences encode protein subunits which, when present in a DNA
construct of the invention, give the protein one or more of the biological
properties of IL-4 as described above. DNA sequences of the invention may
differ from a native IL-4 sequence by the deletion, insertion or
substitution of one or more nucleotides, provided that they encode a
protein with the appropriate biological activity as described above.
Similarly, they may be truncated or extended by one or more nucleotides.
Alternatively, DNA sequences suitable for the practice of the invention
may be degenerate sequences that encode the naturally occurring IL-4
protein. Typically, DNA sequences of the invention have at least 70%, at
least 80%, at least 90%, at least 95% or at least 99% sequence identity to
a native IL-4 coding sequence. They may originate from any species, though
DNAs encoding human proteins are preferred. Variant sequences may be
prepared by any suitable means known in the art.
With respect of substitutions, conservative substitutions are preferred.
Typically, conservative substitutions are substitutions in which the
substituted amino acid is of a similar nature to the one present in the
naturally occurring protein, for example in terms of charge and/or size
and/or polarity and/or hydrophobicity. Similarly, conservative
substitutions typically have little or no effect on the activity of the
protein. Proteins of the invention that differ in sequence from naturally
occurring IL-4 may be engineered to differ in activity from naturally
occurring IL-4. Such manipulations will typically be carried out at the
nucleic acid level using recombinant techniques, as known in the art.
The vaccine may be formulated with one or a cocktail of autoantigen
sequences, which may be on the same or different vectors. The DNA vectors
are suspended in a physiologically acceptable buffer, generally an aqueous
solution e.g. normal saline, phosphate buffered saline, water, etc.
Stabilizing agents, wetting and emulsifying agents, salts for varying the
osmotic pressure or buffers for securing an adequate pH value, and skin
penetration enhancers can be used as auxiliary agents. The DNA will
usually be present at a concentration of at least about 1 ng/ml and not
more than about 10 mg/ml, usually at about from 100 .mu.g to 1 mg/ml.
In some embodiments of the the present invention, the patient is
administered both an autoantigen encoding sequence and a Th2 cytokine
encoding sequence. The cytokine and autoantigen can be delivered
simultaneously, or within a short period of time, by the same or by
different routes. In one embodiment of the invention, the two sequences
are co-formulated, meaning that they are delivered together as part of a
single composition. The coding sequences may be associated with one
another by covalent linkage in a single nucleic acid molecule, where they
may be present as two distinct coding sequences separated by a
translational stop, or may be be present as a single fusion protein. The
two sequences may also by joined by non-covalent interaction such as
hydrophobic interaction, hydrogen bonding, ionic interaction, van der
Waals interaction, magnetic interaction, or combinations thereof.
Alternatively, the two constructs may simply be mixed in a common
suspension, or encapsulated together in some form of delivery device such
as, for example, an alginate device, a liposome, chitosan vesicle, etc.
(see, for example, WO 98/33520, incorporated herein by reference).
The vaccine may be fractionated into two or more doses, of at least about
1 .mu.g, more usually at least about 100 .mu.g, and preferably at least
about 1 mg per dose, administered from about 4 days to one week apart. In
some embodiments of the invention, the individual is subject to a series
of vaccinations to produce a full, broad immune response. According to
this method, at least two and preferably four injections are given over a
period of time. The period of time between injections may include from 24
hours apart to two weeks or longer between injections, preferably one week
apart. Alternatively, at least two and up to four separate injections are
given simultaneously at different parts of the body.
The DNA vaccine is injected into muscle or other tissue subcutaneously,
intradermally, intravenously, orally or directly into the spinal fluid. Of
particular interest is injection into skeletal muscle. The genetic vaccine
may be administered directly into the individual to be immunized or ex
vivo into removed cells of the individual which are reimplanted after
administration. By either route, the genetic material is introduced into
cells which are present in the body of the individual. Alternatively, the
genetic vaccine may be introduced by various means into cells that are
removed from the individual. Such means include, for example, transfection,
electroporation and microprojectile bombardment. After the genetic
construct is taken up by the cells, they are reimplanted into the
individual. Otherwise non-immunogenic cells that have genetic constructs
incorporated therein can betaken from one individual and implanted into
An example of intramuscular injection may be found in Wolff et al. (1990)
Science 247:1465-1468. Jet injection may also be used for intramuscular
administration, as described by Furth et al. (1992) Anal Biochem
205:365-368. The DNA may be coated onto gold microparticles, and delivered
intradermally by a particle bombardment device, or "gene gun".
Microparticle DNA vaccination has been described in the literature (see,
for example, Tang et al. (1992) Nature 356:152-154). Gold microprojectiles
are coated with the vaccine cassette, then bombarded into skin cells.
The genetic vaccines are formulated according to the mode of
administration to be used. One having ordinary skill in the art can
readily formulate a genetic vaccine that comprises a genetic construct. In
cases where intramuscular injection is the chosen mode of administration,
an isotonic formulation is used. Generally, additives for isotonicity can
include sodium chloride, dextrose, mannitol, sorbitol and lactose.
Isotonic solutions such as phosphate buffered saline are preferred.
Stabilizers include gelatin and albumin.
According to the present invention, prior to or contemporaneously with
administration of the genetic construct, cells may be administered a cell
stimulating or cell proliferative agent, which terms are used
interchangeably and refer to compounds that stimulate cell division and
facilitate DNA and RNA uptake.
Bupivacaine or compounds having a functional similarity may be
administered prior to or contemporaneously with the vaccine. Bupivacaine
is a homologue of mepivacaine and related to lidocaine. It renders muscle
tissue voltage sensitive to sodium challenge and effects ion concentration
within the cells. In addition to bupivacaine, mepivacaine, lidocaine and
other similarly acting compounds, other contemplated cell stimulating
agents include lectins, growth factors, cytokines and lymphokines such as
platelet derived growth factor (PDGF), gCSF, gMCSF, epidermal growth
factor (EGF) and IL-4. About 50 .mu.l to about 2 ml of 0.5%
bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceutical
carrier may be administered to the site where the vaccine is to be
administered, preferably, 50 .mu.l to about 1500 .mu.l, more preferably
about 1 ml. The genetic vaccine may also be combined with collagen as an
emulsion and delivered intraperatonally. The collagen emulsion provides a
means for sustained release of DNA. 50 .mu.l to 2 ml of collagen are used.
The efficiency of DNA vaccination may be improved by injection of
cardiotoxin into the tissue about one week prior to the vaccination, as
described by Davis et al. (1993) FEBS Lett. 333:146-150, and in the
examples. The cardiotoxin stimulates muscle degeneration and regeneration.
The muscle is injected with from about 0.1 to 10 .mu.M of cardiotoxin
dissolved in a pharmacologically acceptable vehicle.
The condition that is being treated, and the host immune status will
determine the choice of autoantigen sequence(s). The host may be assessed
for immune responsiveness to a candidate vaccine autoantigen by various
methods known in the art.
The diagnosis may determine the level of reactivity, e.g. based on the
number of reactive T cells found in a sample, as compared to a negative
control from a naive host, or standardized to a data curve obtained from
one or more patients. In addition to detecting the qualitative and
quantitative presence of auto-antigen reactive T cells, the T cells may be
typed as to the expression of cytokines known to increase or suppress
inflammatory responses. It may also be desirable to type the epitopic
specificity of the reactive T cells.
T cells may be isolated from patient peripheral blood, lymph nodes, or
preferably from the site inflammation. Reactivity assays may be performed
on primary T cells, or the cells may be fused to generate hybridomas. Such
reactive T cells may also be used for further analysis of disease
progression, by monitoring their in situ location, T cell receptor
utilization, etc. Assays for monitoring T cell responsiveness are known in
the art, and include proliferation assays and cytokine release assays.
Proliferation assays measure the level of T cell proliferation in response
to a specific antigen, and are widely used in the art. In an exemplary
assay, patient lymph node, blood or spleen cells are obtained. A
suspension of from about 10.sup.4 to 10.sup.7 cells, usually from about
10.sup.5 to 10.sup.6 cells is prepared and washed, then cultured in the
presence of a control antigen, and test antigens. The test antigens may be
peptides of any autologous antigens suspected of inducing an inflammatory
T cell response. The cells are usually cultured for several days.
Antigen-induced proliferation is assessed by the monitoring the synthesis
of DNA by the cultures, e.g. incorporation of .sup.3H-thymidine during the
last 18 H of culture.
Enzyme linked immunosorbent assay (ELISA) assays are used to determine the
cytokine profile of reactive T cells, and may be used to monitor for the
expression of such cytokines as IL-2, IL-4, IL-5, .gamma.IFN, etc. The
capture antibodies may be any antibody specific for a cytokine of
interest, where supernatants from the T cell proliferation assays, as
described above, are conveniently used as a source of antigen. After
blocking and washing, labeled detector antibodies are added, and the
concentrations of protein present determined as a function of the label
that is bound.
The above diagnostic assays may be performed with various peptides derived
from the autologous protein of interest. A series of peptides having the
sequence of an auto-antigen, e.g. PLP, MBP, etc. may be used. Possible
peptides may be screened to determine which are immunodominant in the
context of autoimmune disease.
The immunodominant peptides may be defined by screening with a panel of
peptides derived from the test protein. The peptides have the amino acid
sequence of a portion of the protein, usually at least about 8 and not
more than about 30 amino acids, more usually not more than about 20 amino
acids in length. The panel of peptides will represent the length of the
protein sequence, i.e. all residues are present in at least one peptide.
Preferably overlapping peptides are generated, where each peptide is
frameshifted from 1 to 5 amino acids, thereby generating a more complete
set of epitopes. The peptides may be initially screened in pools, and
later screened for the exact epitope to which the T cell will respond, as
previously described. Immunodominant peptides are recognized by a
significant fraction of the HLA restricted, responsive hybridomas, usually
at least about 10%, more usually at least about 25%, and may be as much as
The subject therapy will desirably be administered during the
presymptomatic or preclinical stage of the disease, and in some cases
during the symptomatic stage of the disease. Early treatment is
preferable, in order to prevent the loss of function associated with
inflammatory tissue damage. The presymptomatic, or preclinical stage will
be defined as that period not later than when there is T cell involvement
at the site of disease, e.g. islets of Langerhans, synovial tissue,
thyroid gland, etc., but the loss of function is not yet severe enough to
produce the clinical symptoms indicative of overt disease. T cell
involvement may be evidenced by the presence of elevated numbers of T
cells at the site of disease, the presence of T cells specific for
autoantigens, the release of performs and granzymes at the site of
disease, response to immunosuppressive therapy, etc.
Degenerative joint diseases may be inflammatory, as with seronegative
spondylarthropathies, e.g. ankylosing spondylitis and reactive arthritis;
rheumatoid arthritis; gout; and systemic lupus erythematosus. The
degenerative joint diseases have a common feature, in that the cartilage
of the joint is eroded, eventually exposing the bone surface. Destruction
of cartilage begins with the degradation of proteoglycan, mediated by
enzymes such as stromelysin and collagenase, resulting in the loss of the
ability to resist compressive stress. Alterations in the expression of
adhesion molecules, such as CD44 (Swissprot P22511), ICAM-1 (Swissprot
P05362), and extracellular matrix protein, such as fibronectin and
tenascin, follow. Eventually fibrous collagens are attacked by
metalloproteases, and when the collagenous microskeleton is lost, repair
by regeneration is impossible.
There is significant immunological activity within the synovium during the
course of inflammatory arthritis. While treatment during early stages is
desirable, the adverse symptoms of the disease may be at least partially
alleviated by treatment during later stages. Clinical indices for the
severity of arthritis include pain, swelling, fatigue and morning
stiffness, and may be quantitatively monitored by Pannus criteria. Disease
progression in animal models may be followed by measurement of affected
joint inflammation. Therapy for inflammatory arthritis may combine the
subject treatment with conventional NSAID treatment. Generally, the
subject treatment will not be combined with such disease modifying drugs
as cyclosporin A, methotrexate, and the like.
A quantitative increase in myelin autoreactive T cells with the capacity
to secrete IFN-gamma is associated with the pathogenesis of MS and EAE,
suggesting that autoimmune inducer/helper T lymphocytes in the peripheral
blood of MS patients may initiate and/or regulate the demyelination
process in patients with MS. The overt disease is associated with muscle
weakness, loss of abdominal reflexes, visual defects and paresthesias.
During the presymptomatic period there is infiltration of leukocytes into
the cerebrospinal fluid, inflammation and demyelination. Family histories
and the presence of the HLA haplotype DRB1*1501, DQA1*0102, DQB1*0602 are
indicative of a susceptibility to the disease. Markers that may be
monitored for disease progression are the presence of antibodies in the
cerebrospinal fluid, "evoked potentials" seen by electroencephalography in
the visual cortex and brainstem, and the presence of spinal cord defects
by MRI or computerized tomography. Treatment during the early stages of
the disease will slow down or arrest the further loss of neural function.
Human insulin-dependent diabetes mellitus (IDDM) is a disease
characterized by autoimmune destruction of the .beta. cells in the
pancreatic islets of Langerhans. An animal model for the disease is the
non-obese diabetic (NOD) mouse, which develops autoimmunity. NOD mice
spontaneously develop inflammation of the islets and destruction of the
.beta. cells, which leads to hyperglycemia and overt diabetes. Both
CD4.sup.+ and CD8.sup.+ T cells are required for diabetes to develop:
CD4.sup.+ T cells appear to be required for initiation of insulitis,
cytokine-mediated destruction of .beta. cells, and probably for activation
of CD8.sup.+ T cells. The CD8.sup.+ T cells in turn mediate .beta. cell
destruction by cytotoxic effects such as release of granzymes, perforin,
TNF.alpha. and IFN.gamma.. Reactivities to several candidate autoantigens,
including epitopes of insulin and glutamic acid decarboxylase (GAD), have
In one embodiment of the invention, the coding sequence used for
vaccination provides for an immunogenic insulin epitope. Immunodominant
epitopes include the B chain, in particular residues 9-23, which have been
implicated in both human disease and in animal models. Epitopes of the
pre-proinsulin have also been implicated as immunodominant epitopes.
Protection from diabetes is associated with down regulation of IFN-.gamma.
and IL-10 in pancreatic lymph node cells in response to the insulin
peptide encoded in the vaccine. It has been found that T cells immunized
with an immunodominant insulin epitope express substantially lower levels
of IFN-.gamma. in response to activation.
The depletion of .beta. cells results in an inability to regulate levels
of glucose in the blood. Overt diabetes occurs when the level of glucose
in the blood rises above a specific level, usually about 250 mg/dl. In
humans a long presymptomatic period precedes the onset of diabetes. During
this period there is a gradual loss of pancreatic .beta. cell function.
The disease progression may be monitored in individuals diagnosed by
family history and genetic analysis as being susceptible. The most
important genetic effect is seen with genes of the major
histocompatibility locus (IDDM1), although other loci, including the
insulin gene region (IDDM2) also show linkage to the disease (see Davies
et al, supra and Kennedy et al. (1995) Nature Genetics 9:293-298).
Markers that may be evaluated during the presymptomatic stage are the
presence of insulitis in the pancreas, the level and frequency of islet
cell antibodies, islet cell surface antibodies, aberrant expression of
Class II MHC molecules on pancreatic .beta. cells, glucose concentration
in the blood, and the plasma concentration of insulin. An increase in the
number of T lymphocytes in the pancreas, islet cell antibodies and blood
glucose is indicative of the disease, as is a decrease in insulin
concentration. After the onset of overt diabetes, patients with residual b
cell function, evidenced by the plasma persistence of insulin C-peptide,
may also benefit from the subject treatment, to prevent further loss of
Mammalian species susceptible to inflammatory conditions include canines
and felines; equines; bovines; ovines; etc. and primates, particularly
humans. Animal models, particularly small mammals, e.g. murine, lagomorpha,
etc. may be used for experimental investigations. Animal models of
interest include those involved with the production of antibodies having
isotypes associated with IL-4 production, e.g. IgE, IgG1 and IgG4. Other
uses include investigations where it is desirable to investigate a
specific effect in the absence of T cell mediated inflammation.
It is to be understood that this invention is not limited to the
particular methodology, protocols, formulations and reagents described, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
Claim 1 of 10 Claims
1. A method of reducing destruction of
.beta. cells in a mammal in need thereof, the method comprising
administering intramuscularly to the mammal a modified plasmid DNA vector
comprising an expression cassette, the expression cassette comprising a
promoter active in mammalian cells operably linked to a DNA encoding an
insulin autoantigen targeted in IDDM, wherein the insulin autoantigen is
selected from the group consisting of insulin B chain, pre-proinsulin, and
proinsulin, wherein the modified plasmid DNA vector has a low number of
CpG motifs compared to an unmodified plasmid DNA vector, so as to thereby
reduce the destruction of .beta. cells in the mammal.
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