Note: Descriptions are shown in the official language in which they were submitted.
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Method of Screening for Therapeutics for Infectious Diseases
Background of the Invention
Field of the Invention
The present invention relates to methods of identifying therapeutics useful
for infectious diseases. More specifically, the present invention relates to
methods of identifying antigens which are produced by infected cells, and the
use
of such antigens in immunogenic compositions or vaccines to treat or prevent
infection.
Related Art
The immune system is the primary biological defense of the host (self)
against potentially pernicious agents (non-self): These agents may be
pathogens,
such as bacteria or viruses, as well as modified self cells, including virus-
infected
cells, tumor cells or other abnormal cells of the host. Collectively, these
targets
of the immune system are referred to as antigens. The recognition of antigen
by
the immune system rapidly mobilizes immune mechanisms to destroy that
antigen, thus preserving the sanctity of the host environment.
Antigens may provoke antibody-mediated responses andlor cell-mediated
responses. Cells of the immune system termed B lymphocytes, or B cells,
produce
antibodies that specifically recognize and bind to the foreign substance.
Other
lymphocytes termed T lymphocytes, or T cells, both effect and regulate the
cell-
mediated response resulting eventually in the elimination of the antigen.
A variety of T cells are involved in the cell-mediated response. Some
induce particular B cell clones to proliferate and to produce antibodies
specific
for the antigen. Others recognize and destroy cells that present foreign
antigens
on their surfaces. Certain T cells regulate the response by either stimulating
or
suppressing other cells.
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Prospects for development of broadly effective tumor vaccines have been
advanced by evidence that several self-proteins can be recognized as tumor
antigens by immune T cells (Van den Eynde et al., J. Exp. Med.173:1373 (1991);
Bloom et al., J. Exp. Med. 185:453 (1997); Van Der Bruggen et al., Science
254:1643 (1991); Gaugler et al., J. Exp. Med. 179:921 (1994); Boel et al.,
Immutaity 2:167 (1995); Van Den Eynde et al., J. Exp. Med. 182:689 (1995);
Kawakaxni et al., Proc. Natl. Acad. Sci. U.S.A. 91:3515 (1994); Kawakaxni et
al.,
Proc. Natl. Acad. Sci. U.S.A. 91:6458 (1994); Brichard et al., J. Exp. Med.
178:489 (1993)). Several genes and gene families that are expressed in
melanoma and a fraction of tumors of other types but are silent in normal
adult
tissues except testis have been identified: MAGE-1 (van Der Bruggen, P., C.
Traversari, P. Chomez, C. Lurguin, E. De Plaen, B. Van Den Eynde, A. Knuth,
and T. Boon. 1991. Science 254: 1643-1647); MAGE-3 (Gaugler, B., B. Van
den Eynde, P. van der Bruggen, P. Romero, J.J. Gaforio, E. De Plaen, B. Lethe,
F. Brasseur, and T. Boon. 1994. J. Exp. Med. 179:921-930); GAGE (Boel, P.,
C. Wildman, M.L. Sensi, R. Brausseur, J.C. Renauld, P. Coulie, T. Boon and P.
van der Bruggen. 1995. Immunity 2: 167-175); and GAGE (Van den Eynde, B.,
O. Peeters, O. De Backer, B.Gaugler, S.Lucas, and T. Boon. 1995. J. Exp. Med.
182: 689-698). In each case these gene products were identified by isolation
of
melanoma specific cytotoxic T cells from patients, and demonstration that the
corresponding gene products are immunogenic.
Infected cells sometimes express self-proteins that are not expressed in
uninfected cells. Geiss et al., Virology 266:8-16 (2000); Scheuring et al.,
AIDS
12:563-570 (1998).
Summary of the Invention
The present invention provides a method of screening for therapeutics for
infectious diseases, comprising identifying host cell gene products which are
differentially expressed in infected cells, screening the differentially
expressed
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gene products for immunogenicity, and determining which gene products are
irnmunogenic.
The present invention also provides a method comprising identifying host
cell gene products which are differentially expressed in infected cells,
identifying
which of the differentially expressed gene products are expressed
embryonically,
screening the differentially- and embryonically-expressed gene products fox
immunogenicity, and determining which gene products are immunogenic.
The differentially expressed gene products may be identified using
subtractive hybridization, representational difference analysis, differential
display, or ordered microarrays of nucleic acids.
Immunogenicity includes cytotoxic T lymphocyte responses, T helper
responses, and B cell responses, such as antibody production.
Brief Description of the Figures
Figure 1 shows the results of hybridization to an array of 24 cDNA clones
selected following subtractive hybridization of cDNA from HIV-infected THP-1
monocytic cell line minus uninfected THP-1 cDNA + HIV DNA.
Figure 2 shows hybridization to Northern blots of poly-A RNA from
uninfected and HIV-infected cells.
Detailed Description of the Preferred Embodiments
Altered features of an infected cell which are recognized by the immune
system as non-self may be the basis for development of treatments or vaccines
against infectious diseases. Since many pathogens elude immune surveillance
by frequent reproduction and mutation, it is of considerable value to develop
a
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vaccine that targets host gene products that are not likely to be subject to
mutation. Thus, the present invention relates to a method of identifying
potential
therapeutics useful for the treatment or prevention of infectious diseases. By
"treatment" is meant reduction in symptoms, reduction in pathogen load,
reduction in the rate of pathogen replication, and/or no worsening of
symptoms,
pathogen load, or pathogen replication over a specified period of time.
Host gene products that are overexpressed in infected cells are identified.
Those that are shown to be overexpressed by a factor of 9 or greater in
infected
cells as compared to uninfected cells axe the most likely to be immunogenics.
Optionally, relative gene expression is then determined in a broad panel of
normal tissues. It is expected that immune tolerance will be induced to gene
products expressed at relatively high levels in any normal tissue. Such gene
products are excluded from further analysis. Immunogenicity is then directly
assayed.
Thus, in one embodiment, a method is provided comprising identifying
host cell genes which are differentially expressed in infected cells,
screening the
gene products of the differentially expressed host cell genes for
immunogenicity,
and determining which differentially expressed host cell gene products are
immunogenic.
In another embodiment, a method is provided comprising identifying host
cell genes which are differentially expressed in infected 'cells, identifying
which
of the differentially expressed genes are expressed in embryonic tissue,
screening
the gene products of said differentially- and embryonically-expressed genes
for
immunogenicity, and determining which differentially expressed host cell gene
. products are immunogenic. Developmentally regulated gene products are a very
important pool of potential neoantigens since, once gene expression is turned
off,
it is no longer part of the definition of immunological "selp' and tolerance
is not
maintained.
In another embodiment, a method is provided comprising identifying host
cell genes which are differentially expressed in infected cells, identifying
which
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of the differentially expressed genes are expressed in embryonic tissue,
identifying which of the differentially and embryonically-expressed genes are
not
expressed in other adult tissues, screening the gene products of said
differentially-
and embryonically-expressed genes which are not expressed in adult tissue for
immunogenicity, and determining which differentially expressed host cell gene
products are immunogenic.
In another embodiment, a method is provided comprising identifying host
cell genes which are differentially expressed in infected cells, identifying
which
which of the differentially-expressed genes are not expressed in other adult
tissues, screening the gene products of said differentially-expressed genes
which
are not expressed in adult tissue for immunogenicity, and determining which
differentially expressed host cell gene products are immunogenic.
Any cell type that is capable of being infected can be used in the method
of the invention. Suitable cells include, but are not limited to, mammalian
cells,
including animal (rodents, including mice, rats, hamsters and gerbils),
primates,
and human cells, including cells of all types, including breast, skin, lung,
cervix,
colorectal, leukemia, brain, etc.
Cells include dividing cells, non dividing cells, terminally differentiated
cells, pluripotent stem cells, committed progenitor cells and uncommitted stem
cells.
Cells and cell types also include muscle cells such as cardiac muscle cells,
skeletal muscle cells and smooth muscle cells, myofibrils, intrafusal fibers
and
extrafusal fibers; skeletal system cells such as osteoblasts, osteocytes,
osteoclasts
and their progenitor cells.; and epithelial cells such as squamous epithelial
cells,
including endothelial cells, cuboid epithelial cells and columnar epithelial
cells.
Cells that can be used in the method of the present invention also include
nervous system cells such as neurons, including cortical neurons, inter
neurons,
central effector neurons, peripheral effector neurons and bipolar neurons; and
neuroglia, including Schwann cells, oligodendrocytes, astrocytes, microglia
and
ependyma.
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Additionally, endocrine and endocrine-associated cells may also be used
such cells as pituitary gland cells including epithelial cells, pituicytes,
neuroglia,
agranular chromophobes, granular chromophils (acidophils and basophils);
adrenal gland cells including epinephrine-secreting cells, non-epinephrine-
secreting cells, medullary cells, cortical cells (cells of the glomerulosa,
fasciculata
and reticularis); thyroid gland cells including epithelial cells (principal
and
parafollicular); parathyroid gland cells including epithelial cells (chief
cells and
oxyphils); pancreas cells including cells of the islets of Langerhans (alpha,
beta
and delta cells); pineal gland cells including parenchymal cells and
neuroglial
cells; thymus cells including parafollulicular cells; cells of the testes
including
seminiferous tubule cells, interstitial cells ("Leydig cells"), spermatogonia,
spermatocytes (primary and secondary), spermatids, spermatozoa, Sertoli cells
and myoid cells; cells of the ovary including ova, oogonia, oocytes, granulosa
cells, theca cells (internal and external), germinal epithelial cells and
follicle cells
(primordial, vesicular, mature and atretic).
Circulatory system cells are also included such cells as heart cells
(myocardial cells); cells of the blood and lymph including erythropoietin-
sensitive stem cells, erythrocytes, leukocytes (such as eosinophils, basophils
and
neutrophils (granular cells) and lymphocytes and monocytes (agranular cells)),
thrombocytes, tissue macrophages (histiocytes), organ-specific phagocytes
(such
as I~upffer cells, alveolar macrophages and microglia), B-lymphocytes, T-
lymphocytes (such as cytotoxic T cells, helper T cells and suppressor T
cells),
megaloblasts, monoblasts, myeloblasts, lymphoblasts, proerythroblasts,
megakaryoblasts, promonocytes, promyelocytes, prolymphocytes, early
normoblasts, megakaryocytes, intermediate normoblasts, metamyelocytes (such
as juvenile metamyelocytes, segmented metamyelocytes and polymorphonuclear
granulocytes), late normoblasts, reticulocytes and bone marrow cells.
Respiratory system cells are also included such as capillary endothelial
cells and alveolar cells; as are urinary system cells such as nephrons,
capillary
endothelial cells, granular cells, tubule endothelial cells and podocytes;
digestive
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system such as simple columnar epithelial cells, mucosal cells, acinar cells,
parietal cells, chief cells, zymogen cells, peptic cells, enterochromaffin
cells,
goblet cells, Argentaffen cells and G cells; and sensory cells such as
auditory
system cells (hair cells); olfactory system cells such as olfactory receptor
cells and
columnar epithelial cells; equilibrium/vestibular apparatus cells including
hair
cells and supporting cells; visual system cells including pigment cells,
epithelial
cells, photoreceptor neurons (rods and cones), ganglion cells, amacrine cells,
bipolar cells and horizontal cells are also included.
Additionally, mesenchymal cells, stromal cells, hair cells/follicles,
adipose (fat) cells, cells of simple epithelial tissues (squamous epithelium,
cuboidal epithelium, columnar epithelium, ciliated columnar epithelium and
pseudostratified ciliated columnar epithelium), cells of stratified epithelial
tissues
(stratified squamous epithelium (keratinized and non-keratinized), stratified
cuboidal epithelium and transitional epithelium), 'goblet cells, endothelial
cells
of the mesentery, endothelial cells of the small intestine, endothelial cells
of the
large intestine, endothelial cells of the vasculature capillaries, endothelial
cells
of the microvasculature, endothelial cells of the arteries, endothelial cells
of the
arterioles, endothelial cells of the veins, endothelial cells of the venules,
etc.;cells
of the connective tissue include chondrocytes, adipose cells, periosteal
cells,
endosteal cells, odontoblasts, osteoblasts, osteoclasts and osteocytes;
endothelial
cells, hepatocytes, keratinocytes andbasal keratinocytes, muscle cells, cells
of the
central and peripheral nervous systems, prostate cells, and lung cells, cells
in the
lung, breast, pancreas, stomach, small intestine, and large intestine;
epithelial
cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes,
mucin-
producing goblet cells, and other epithelial cells and their progenitors of
the skin,
lung, liver, and gastrointestinal tract may be used in the methods of the
present
invention, preferably the selection and screening methods.
The method of the present invention can be used to screen for antigens
which are differentially expressed in cells infected with any infectious
agent,
including viruses, fungal agents, mycobacteria, bacteria or parasitic agents.
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In one embodiment, the cells are infected with human immunodeficiency
virus (HIV). This method of vaccine development is broadly applicable to any
infectious agent but especially to infectious agents that, like HIV, replicate
or
mutate rapidly, for example, hepatitis C virus and many RNA viruses (because
they depend on RNA polymerases which are more error prone since they do not
have a "proof reading" function).
In other embodiments, the cells are infected with infectious agents causing
chickenpox, shingles, rubella, influenza, rubeola, mumps, yellow fever,
mononucleosis, rabies, acute viral gastroenteritis, poliomyelitis, subacute
sclerosing panencephalitis, encephalitis, Colorado tick fever, pharyngitis,
croup,
bronchiolitis, viral pneumonia, pleurodynia, aseptic meningitis, keratitis,
conjunctivitis, viral leukemias, rabies, polio, myocarditis, hepatitis A,
hepatitis
B, hepatitis C, hepatitis D, hepatitis E; and any infections caused by
adenoviruses,
coxsackieviruses, parainfluenza viruses, respiratory syncytial virus,
reovirus,
cytomegalovirus, Epstein-Barn virus, herpes simplex viruses, herpes-zoster-
varicella virus, rhinoviruses, rotaviruses, papolomaviruses, enteroviruses,
paramyxoviruses, parvoviruses, apthoviruses, Ebola virus, Marburg virus,
vesicular stomatitis virus, coronaviruses, Lassa virus, lymphocytic
choriomeningitis virus, Machupo virus, Junin virus, human papillomavirus, or
poxviruses.
Further examples of viruses, include, but are not limited to the following
DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae,
Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,
Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such
as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,
Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,
Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as
Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I,
HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within
these families can cause a variety of diseases or symptoms, including, but not
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limited to: arthritis, bronchiollitis, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E,
Chronic
Active, Delta), meningitis, opportunistic infections (e.g., A>DS), pneumonia,
Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually
transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
Moreover, parasitic agents include, but not limited to, the following
families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
These parasites can cause a variety of diseases or symptoms, including, but
not
limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g.,
dysentery, giardiasis), liver disease, lung disease, opportunistic infections
(e.g.,
A)DS related), Malaria, pregnancy complications, and toxoplasmosis.
Fungal pathogens include, but are not limited to Candida albicans and
pneumocystis carnii. Mycobacterial pathogens include, but are not limited to,
M.
tuberculosis, M. avium.
Differetztial expression
Host cell gene products which are "differentially expressed" in infected
cells include gene products which are upregulated during infection, i.e.,
expressed
in a cell during both infection and non-infection but at higher levels during
infection; and those which are expressed in a cell only during infection.
In one embodiment, differential expression is determined by subtractive
hybridization. Methods of subtractive hybridization are known in the art. See,
for example, U.S. Patent Nos. 5,827,658; 5,700,644; and 5,525,471.
In another embodiment, differential expression is determined by
representational difference analysis (RDA). RDA is a subtractive hybridization
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based method applied to "representations" of total cellular DNA (Lisitsyn, N.
and
N., M. Wigler. 1993. Science 259: 946-951). The differential display methods
of Liang and Pardee (1992, Science 257:967-971) employ an arbitrary 10
nucleotide primer and anchored oligo-dT to PCR amplify an arbitrary subset of
fragments from a more complex set of DNA molecules.
In another embodiment, differential expression is determined by the
modified differential display described below.
In another embodiment, differential expression is determined using
microarrays. Preferably, differential expression is determined using ordered
microarrays of nucleic acids. Two color differential hybridization may be
used.
Methods of making and using microarrays are known in the art. See, e.g., Eisen
and Brown, metlaods in Ehzymol. 303:179-205 (1999); Bowtell, Nature Genet.
Suppl. 21:25-32 (1999); Cheung et al., Nature Geuet. Suppl. 21:15-19 (1999);
Duggan et al., Nature GefZet. Suppl. 21: 10-14 (1999); Lipshutz et al., Nature
Genet. Suppl. 21:20-24 (1999); and U.S. Patent Nos. 6,060,288; 6,060,240;
6,045,996; 6,033,860 and 6,004,755.
Gene expression in embryonic tissues is known to be more complex than
in adult tissues. Many of these genes are downregulated in the adult and
would,
therefore, not be expected to induce tolerance in newly arising lymphocytes of
the
adult. If expression of any of these gene products is again upregulated in
infected
cells, as is known to happen for some such genes in cells that undergo tumor
transformation, then these would encode antigens that could be targeted for
immunotherapy. An ordered library of cDNA clones expressed during early
embryonic development can be made. See, e.g., Tanaka et al., PNAS
97:9127-9132 (2000). Microarrays representing the ordered library can be
produced and be employed to efficiently identify developmentally regulated
genes
that are overexpressed in infected cells. This is a powerful tool that greatly
accelerates the process of identifying specific host genes that encode novel
antigens in infected cells infected.
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Identification of genes which are expressed in embryonic tissue and/or are
not expressed in adult tissue can be done by any method known in the art. In
one
embodiment, Discovery LineTM RNA and Gene PoolTM cDNA (Invitrogen), which
is either total RNA or first strand cDNA prepared from over 30 different human
normal adult or fetal tissues, is screened for expression of the
differentially
expressed gene. Those sequences that are consistently expressed in infected
cells
but which have low or undetectable expression in diverse normal tissues
especially thymus are more likely to be immunogenic.
Immunogenicity
"Immune response" encompasses humoral and cell-mediated immune
responses, including, but not limited to, antibody response, cytotoxic T
lymphocyte response, T helper response, inflammation, cytokine production, and
complement. A gene product is immunogenic if it induces one or more of these
immune responses.
The ability of a differentially expressed gene product to modulate an
immune response can be readily determined by an in vitro assay. T cells for
use
in the assays include transformed T cell lines, such as T cell hybridomas, or
T
cells which are isolated from a mammal, e.g., from a human or from a rodent
such as a mouse. T cells can be isolated from a mammal by known methods. See,
for example, Shimonkevitz et al., J. Exp. Med. 158:303 (1983).
One way to demonstrate immunogenicity in humans is by stimulation of
a primary T cell response in vitro. A suitable assay to determine if a gene
product
is capable of modulating the activity of T cells is conducted by coculturing T
cells
and antigen presenting cells. The most effective antigen presenting cells for
stimulation of a primary immune response are dendritic cells (DC). In order to
efficiently introduce antigen into DC, recombinants of vaccinia, retroviral,
or
adenoviral vectors are generated for the same gene product and employed DC
infected with these vectors to alternately stimulate autologous human T cells.
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Alternate cycles of stimulation with different vectors minimize selection of a
vector specific response and focus immune reactivity on the recombinant gene.
Alternatively, the differentially expresssed gene product may be added to the
culture medium. Production of IL-2 is measured. An increase in IL-2 production
over a standard indicates the compound can stimulate an immune response and
is immunogenic.
Thus, in one embodiment, immunogenicity is determined by cultivating
T cells with antigen-presenting cells, adding a differentially expressed gene
product to the cell culture, and measuring IL-2 production. In another
embociment, immunogenicity is determined by cultivating T cells with antigen-
presenting cells, transfecting the antigen-presenting cells with a vector
expressing
the differentially expressed gene product, and measuring 1L-2 production.
Other in vitro assays of T cell activation include secretion of other
cytokines (IFN-'y, TNF-a, GM-CSF) measured by ELISA, ELISpot, or flow
cytometric detection (Luminex bead system). Many of these methods are
described in Current Protocols in hnmunology (John Wiley & Sons, New York).
Alternatively, rather than measurement of an expressed protein such as
IL-2, modulation of T cell activation can be suitably determined by changes in
antigen-dependent T cell proliferation as measured by radiolabelling
techniques
or colorimetric MTT assay as are recognized in the art. For example, a labeled
(e.g., tritiated) nucleotide may be introduced to an assay culture medium.
Incorporation of such a tagged nucleotide into DNA serves as a measure of T
cell
proliferation. This assay is not suitable for T cells that do not require
antigen
presentation for growth, e.g., T cell hybridomas. A difference in the level of
T
cell proliferation following contact with the compound of the invention
indicates
the complex modulates activity of the T cells. An increase in T cell
proliferation
indicates the compound can stimulate an immune response.
Additionally, cytotoxic T lymphocyte (CTL) activity can be detected using
a standard 4 hour SICr release assay, as well known in the art.
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In vivo assays also may be suitably employed to determine the ability of
a compound of interest to activate T cells. For example, a compound of
interest
can be assayed for its ability to inhibit immunoglobulin class switching (i.e.
IgM
to IgG). See, e.g., Linsley et al., Science 257:792-795 (1992)).
In vivo assays may also be suitably employed to determine the ability of
a compound to induce antibody production. A compound of interest can be
administered to a mammal such as a mouse, blood samples obtained from the
mammal at the time of initial administration and several times periodically
thereafter (e.g. at 2, 5 and 8 weeks after administration). Serum is collected
from
the blood samples and assayed for the presence of antibodies raised by the
immunization. Antibody concentrations may be determined.
Alternatively, the differentially expressed genes may be screened for
complement-dependent cytotoxicity (CDC) or antibody-dependent cellular
cytotoxicity (ADCC). See U.S. Pat. No. 5,500,362 for ADCC and CDC assays.
In any complex mixture of potential immunogens, as would be present in
an infected cell, some antigens will induce a strong response and others a
weak
response. This has important practical implications. A defect in antigen
processing is not readily corrected with present methods, but a deficiency in
T
cell clonal expansion can be overcome by the most fundamental of all
immunological manipulations - vaccination. Rather than examine what is
immunogenic in an infected cell, it may be more profitable to evaluate what
can
become immunogenic if the representation of specific T cells is first
augmented
by vaccination. The most promising candidates for this purpose are the
products
of genes which are differentially expressed during infection.
Thus, in one embodiment, a method is provided comprising identifying
genes which are differentially expressed in infected cells, followed by
immunization with a the differentially expressed gene product or a recombinant
expression vector comprising the differentially expressed gene, and isolation
of
CTL specific for one or more of these gene products. The immunogenicity of
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these peptide targets in man can be established by highly efficient in vitro
stimulation of human T cells with autologous peptide-pulsed dendritic cells.
Cellular peptides derived by degradation of endogenously synthesized
proteins are translocated into a pre-Golgi compartment where they bind to
class
I MHC molecules for transport to the cell surface. These class I MHC:peptide
complexes are the target antigens for specific CD8+ cytotoxic T cells. Since
all
endogenous proteins turn over, peptides derived from any cytoplasmic or
nuclear
protein may bind to an MHC molecule and be transported for presentation at the
cell surface. This allows T cells to survey a much larger representation of
cellular
proteins than antibodies which are restricted to recognize conformational
determinants of only those proteins that are either secreted or integrated at
the cell
membrane. Class I-bound peptides are generally 8-10 residues in length and
accommodate amino acids side chains of restricted diversity at certain key
positions that match pockets iri the MHC peptide binding site. These key
features
of peptides that bind to a particular MHC molecule constitute a peptide
binding
motif.
A major concern for the development of broadly effective human vaccines
is the extreme polymorphism of HLA class I molecules. Extensive
characterization of peptide binding motifs of different human class I MHC
molecules has suggested that there are four major subtypes of HLA-A and HLA-
B alleles such that many peptides will bind to multiple members of a single
subtype. One attractive strategy, therefore, is to target representative
members
of these four subtypes: HLA-A2, HLA-A3, HLA-B7 and HLA-B44. Each
subtype has an average representation across ethnic populations of between 40%
and 50%. It is estimated that the combination of all four subtypes would cover
95% of the population.
Mice which are transgenic for human CD8 and a human HLA antigen can
be used to determine whether a particular differentially expressed gene
product
is immunogenic in humans.
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The invention will be better understood by reference to the specific
embodiments detailed in the examples which follow.
Examples
Example 1. Representational Difference Atzalysis (RDA)
The PCR SELECTTM variation of RDA is marketed by Clontech (Palo
Alto, CA). The following general protocol is a summary of the manufacturer's
recommendations. cDNA is synthesized from both a tracer (represented by
infected cell mRNA) and a driver (represented by parental, non-infected cell
mRNA). "Representations" of both tracer and driver cDNA are created by
digestion with RsaI which cuts the four-base recognition sequence GTAC to
yield
blunt end fragments. Adaptors, which eventually serve as primer sites for PCR,
are ligated to the 5' ends of only the tracer cDNA fragments. Two aliquots of
tracer representation are separately ligated with~two different adapters. A
series
of two hybridizations are carried out. In the first set of hybridizations,
each
adapter ligated tracer sample is denatured and hybridized with a ten fold
excess
of the denatured representation of driver cDNA for 8 hours. Under these
conditions re-annealing of all molecules is incomplete and some of both the
high
and Iow copy molecules remain single stranded. Since re-annealing rates are
faster for more abundant species, this leads to normalization of the
distribution
through relative enrichment of low copy number single stranded molecules. The
two hybridization reactions with each of the different adapter ligated tracer
cDNA
representations are then combined without fractionation or further
denaturation
but with addition of more freshly denatured driver in a second hybridization
reaction that is allowed to proceed further to completion, approximately 20
hours.
An aliquot of the products from the second hybridization is used as a
template for a high stringency PCR reaction, using the known sequences at the
5' ends of the ligated adapters as primers. The key here is that only tracer
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sequences that 1) remain single stranded through the first hybridization and
2)
hybridize to a complementary tracer sequence ligated to the alternate adapter
in
the second hybridization can be exponentially amplified during PCR. This
excludes both tracer and driver species that either remain single stranded or
that
have hybridized to excess driver (since they have a complementary primer at
only
one or neither end of the molecule), as well as tracer sequences that
hybridize to
a molecule with the same adapter (because the adapters are longer than the
primers and hybridize to their own complement with higher affinity when it is
present on the opposite end of a denatured single stranded molecule - a
reaction
termed "Suppression PCR" by Clontech). Finally, a second high stringency PCR
is performed using nested primers built into the adapters so as to further
reduce
background and enrich for differentially expressed, sequences. The products of
the
second PCR are electrophoresed and visualized on an agarose gel. Individual
bands are excised and subcloned for further analysis.
Example 2. Modified Differential Display of Gettes Eucodittg Potential
Immuttoge>zs
In the following example, the differential display methods of Liang and
Pardee (1992, Science 257:967-971) were modified to improve resolution of
DNA fragments and reduce the frequency of false positives.
The differential display method as originally described by Liang and
Pardee (1992, Science 257:967-971) employs an arbitrary 10 nucleotide primer
and anchored oligo-dT to PCR amplify an arbitrary subset of fragments from a
more complex set of DNA molecules. In principle, differences among the
fragments generated from normal and infected cells should reflect differences
in
gene expression in the two cell types. In practice, this method sometimes
works
well but often gives rise to numerous false positives. That is, bands which
appear
to be differentially displayed are, upon further characterization, found not
to be
differentially expressed. This is presumably due to variable PCR amplification
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of individual species in complex populations and a relatively high background
that can obscure less prominent bands. Since considerable effort is required
to
establish differential expression, these endemic false positives are costly in
terms
of efficiency and productivity.
A single arbitrary primer may also be used for differential display, as
described by Welch et al.. Use of single primers does, however, require
synthesis
of a much larger set of independent primers to achieve the same coverage of a
complex cDNA population.
In order to improve the resolution of fragments and reduce the frequency
of false positives, a second arbitrary primer is substituted for the anchored
oligo-dT employed in PCR amplification. This results in fewer DNA products
in each PCR reaction so that individual DNA fragments can be more reliably
resolved on sequencing gels.
Because each subset of fragments generated in this modified differential
display protocol is a smaller representation of total cDNA, more primer pairs
are
required for adequate sampling. Employing the negative binomial distribution,
it can be predicted that if 12 independent primers are utilized in all 66
possible
primer pair combinations there is a greater than 85% probability that for an
average size eukaryotic cDNA at least one primer pair will amplify a
representative PCR fragment of size >_70bp.
Following is a lists of sequences of 12 arbitrary decamers from which
primer pairs are selected for modified differential display. The specific
primers
were chosen on the basis of their sequence diversity, 3' hybridization
affinity, and
minimal pair-wise hybridization.
TAC AAC GAG G MR_1 TCG GTC ACA G ~ 9
GTC AGA GCA T ~ 2 ATC TGG TAG A M12_ 10
GGA CCA AGT C ~ 5 CTT ATC CAC G MR_ 1l
TCA GAC TTC A ~ 7 CAT GTC TCA A MR_ l2
TAC CTA TGG C ~ 8 GAT CAA GTC T MR_ 14
TGT CAC ATA C MR _15 CTG ATC CAT G Lddl
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A separate cDNA synthesis reaction with 0.1 ,ug polyA-RNA and
Superscript IIReverse Transcriptase (GibcoBRL) is carried out with each
primer.
Five percent of the cDNA product made with each member of a primer pair is
mixed together with that primer pair for amplification in 30 PCR cycles using
I~len Taq Polymerase Mix (Clontech). The PCR primers are used for cDNA
synthesis to avoid the 3' bias imposed by oligo-dT primed cDNA synthesis. The
relative orientation of the two primers in cDNA is randomized by carrying out
a
separate synthesis with each primer. These cDNA can be mixed in the same
combinations as the primers chosen for PCR amplification. PCR amplified
cDNA fragments are resolved on 6% acrylamide gels and dried for
autoradiography. Those bands which are differentially displayed in at least 2
infected cells samples samples and not in the .parental cells are cut out and
rehydrated. An aliquot (1/5) of the DNA recovered is reamplified using the
same
primer set and the same PCR conditions but without addition of isotope. This
second PCR product is resolved on 1% agarose and individual bands are
recovered by incubation with 13 agarase I (GibcoBRL). Each DNA fragment
recovered is cloned by blunt end ligation into the pcDNA3.1/Zeo (+) phagemid ,
vector (Invitrogen). Since it is possible that a single band may include more
than
one molecular species, at least 4 different transformants with an insert of
appropriate size are picked for further characterization. Northern analysis,
RNase protection assays and semi-quantitative PCR are employed to confirm
differential expression.
Example 3. Selectiott of Full Length cDNA Encoding Potential
Immurtogens
This section presents methods for facilitating selection of corresponding
full length cDNAs from fragments of differentially expressed genes identified
by
representational difference analysis or by modified differential display. .A
single
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stranded biotinylated probe is synthesized from isolated cDNA fragments and is
used to select the longer cDNA that contain a complementary sequence by
solution hybridization to single stranded circles rescued from a phagemid
infected
cell cDNA library. This method is especially well-suited to the use of DNA
fragments isolated by the modified differential display method employing two
arbitrary primers. The same arbitrary primers employed for PCR amplification
of a given fragment in differential display can be modified to generate a
single
stranded hybridization probe from that fragment. This avoids the need to
sequence, select and synthesize a new pair of fragment specific primers for
each
new fragment of interest.
i) The two oligonucleotides of a pair of PCR primers employed in
differential display are modified: (biotin-dT)-dT- (biotin-dT) is incorporated
at
the 5' end of one primer and a phosphate is incorporated at the 5' end of the
second primer. These modified primers are incorporated by PCR into the two
strands of a differential display fragment that was selected following the
original
PCR amplification with the same unmodified arbitrary primers. From this double
stranded PCR product, the strand labelled with a 5' phosphate is digested with
~,
exonuclease to generate a single stranded biotin-labeled probe.
ii) Single stranded (ss) DNA circles are rescued from a phagemid
cDNA library using the M13K07 packaging defective phage as helper virus. This
library is constructed in the pcDNA3.IlZeo(+) phagemid (Invitrogen, Carlsbad,
CA) with insertion of (ApaI)oligo-dT primed cDNA between the Apa I and Eco
RV restriction sites. A key manipulation to achieve the efficient ligation
necessary for construction of a high titer cDNA library is to insure that cDNA
inserts are 5' phosphorylated by treating with T4 polynucleotide kinase prior
to
ligation. The biotin-labeled single stranded probe generated from the
differential
display fragment is hybridized in solution to the ssDNA circles of the
phagemid
library. The biotin-labeled hybridization complexes can then be separated from
unrelated ssDNA on streptavidin magnetic beads and the ss circles eluted for
further analysis.
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Example 4. The ilse of Ordered Microarrays to Identz; fy Getae Expression
A powerful recent development for analysis of differential gene
expression is the use of ordered microarrays of cDNA in two color differential
hybridization. Schena et al., Science 270:467-470 (1995); Schena et al., PNAS
93:10614-10619 (1996); and DeRisi et al., Nature Genetics 14:457-460 (1996).
The microarrays maybe used to determine differential gene expression in
infected and uninfected cells. The microarrays may also be used to determine
expression patterns of genes in adult and embryonic tissue.
The probe for the microarrays is prepared as follows:
Amount Company Final Concentration
5 X First strand 80 ~,1 Gibco 1X
buffer
pd(T)12-18, lmg/ml10 ~,l Pharmacia 25 ,ug/ml
5mM dA,T,GTP 40 ~,1 Pharmacia 0.5 mM
O.1M DTT 40 p,1 10 mM
Rnase Inhibitor, 20 ~,1 Boehringer 2 units/~,1
40
units/ul
250 uCi alpha-33P 25 ~,1 Amersham
dCTP
150 ug RNA 150 ~,l
water 15 ,u1
final volume 380 ~,1
One reaction is made for two sets of membranes (one set contains 7 blots,
A-G) or two reactions for 3 sets of membranes.
190 ~,1 is aliquoted per 0.5 ml tube. Tubes are put in thermocycler:
65°C
for 10 minutes; 1 °C for 75 seconds; repeat for 23 cycles; and cooled
down to
42 ° C. 10 ~,l Superscipt II Reverse transcriptase/tube (Gibco) is
added. The tubes
are incubated at 42°C for 45 minutes. Another 10 ~,l Superscipt II
Reverse
transcriptase/tube (Gibco) is added. The tubes are incubated another 45
minutes
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at 42°C. Added to each tube are: 25 ~,l Superscript II Reverse
transcriptase; 25
~,l 0.5M EDTA; and 50 ~,1 1M NaOH; the tubes are incubated at 65°C for
20
minutes. 12.5 ~ul 1 M Tris-HCI, pH 7.5 is added. 1 ~,l is removed for "Total
cpm", and added to 3 mL ECO-scinct (Bio-Rad).
The remaining probe is purified on Bio-spin 6 columns, using 8 columns
per probe. The caps are snapped from bottom, then the top of column is
removed.
The column is drained by gravity, and the flowthrough is discarded. The column
is spun at 1000xg, for 2 min. The column is transferred to a fresh collection
tube,
add 70 ~,l/ column and spun 1000xg, 2 minutes. The flow through is pooled from
all columns, and 1 ~,l is counted in 3 ml ECO-stint.
Mycrohybe (Research Genetics) is warmed to hybridization temperature.
For two sets: 43 ml Microhybe, 0.5 ml denatured salmon sperm DNA 5 mg/ml
stock. Membranes are wet in 2X SSC for 5 minutes. Membranes are placed in
ml Falcon screw cap conical with 3 ml/tube and incubated 3-4 hours at 55
15 65°C forprehybridization.
CoT DNA, yeast tRNA, and probe is denatured for 5 minutes at 99°C,
then placed on ice. For two sets, 0.5 ml 50 mg/ml Poly A(Pharmacia) is mixed
to a final concentration of 1 mg/ml; 0.5 ml 1 mg/ml human CoT1 DNA (Gibco),
to a final concentration of 17 ~,g/ml; 0.5 ml 50 mg/ml yeast tRNA (Sigma), to
a
final concentration of 1 mg/ml; 6 ml 50% dextran sulfate, to a final
concentration
of 10%; 22.5 ml Microhybe; and 0.6 ml probe. Pre-hybe is discarded, and 2 mL
hybe solution/tube is added and incubated at 55-65°C overnight.
The membranes are rinsed 1x at room temperature with 250 ml 2X
SSC/0.5% SDS altogether in one container, followed by incubation 2X for 25
minutes at room temperature with 250 ml 2X SSC/0.5% SDS altogether in one
container. The membranes are then incubated 2X for 30 minutes at 65 °C
with
2X SSC/0.5% SDS, 3 ml/tube (one membxane/tube). The membranes are
incubated 2X for 30 minutes at 65°C in O.1XSSC/0.5% SDS 3 ml/tube (one
membrane/tube).
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The membranes are wrapped in plastic wrap and exposed to
phosphorimager for 10 days.
Example 5: In vitro Assay forDetermination of CTL Response to Differentially
Expressed Gene Products
To determine whether the products of differentially expressed genes are
immunogenic, groups of three (HLA-A2.1 x huCDB)Fl transgenic mice are
immunized intravenously with 5 x 106 pfu of each recombinant vaccinia virus
which express the differentially expressed gene of interest (Bennink and
Yewdell,
1990, Current Topics in Microbiol. and Immunol. 163: 153-178). After at least
two weeks, mice are sacrificed and CD8+ splenic T cells are enriched on
anti-CD8 coated magnetic beads. CD8+ cytolytic precursors are restimulated in
vitro with parental SV-HUC cells that are transfected with the recombinant
differentially expressed gene previously isolated in the pcDNA3.1/Zeo(+)
plasmid expression vector. Substitution of the plasmid recombinant in place of
the vaccinia vector for restimulation in vitro is necessary to avoid a large
vaccinia
vector specific response. After five days in vitro culture, cytolytic activity
is
determined by SICr release from SV-HUC target cells transfected with either
the
specific recombinant plasmid or a control ovalbumin gene recombinant.
Exarnple 6. In Vitro Assay for the Ifnmunogenicity of Differentially Expressed
Gene Products Using Dendritic Cells
Dendritic cells (DC) are the most potent stimulators of T cell responses
identified to date. To test immunogenicity of differentially expressed gene
products, DC are incubated with the relevant gene products and assayed for the
ability to activate human autologous T lymphocytes.
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Immature dendritic cells are prepared from healthy donors according to
the method of Bhardwaj and colleagues (Reddy, A. et al., . Blood 90:3640-3646
(1997)). Briefly, PBMC are incubated with neuraminidase-treated sheep
erythrocytes and separated into rosetted T cell (ER+) and non-T cell (ER-)
fractions. The ER+ fraction is cryopreserved for later use. The ER- fraction
(2x106 cells per well) is cultured in serum-free RPMI medium containing
1000U/xnl rhGM-CSF, 1000 U/ml rhIL-4 and 1 % autologous plasma. This
medium is replenished every other day. At day 7, the non-adherent immature DC
are harvested from the culture and re-plated in maturation conditions (1000
U/ml
GM-CSF, 1000 U/ml IL-4, 1% autologous plasma and 12.5-50% monocyte-
conditioned medium) for 2-4 days. Cells manipulated in this manner have
morphological and surface characteristics (CD83''~) of mature DC.
Mature (or immature) DC are pulsed with the gene products of the interest
for a short period followed by cocultivation with autologous T cells in 24-
well
plates for a period of 7-I4 days. In some experiments, these may be total T
lymphocytes, but it may also be desirable to fractionate CD4 and CD8 cells
using
magnetic separation systems (Miltenyi Biotech). Total T lymphocytes are
incubated with the appropriate antibody-magnetic bead conjugates to isolate
total
CD4, CDB, naive CD4+CD45RA+, - naive CD8+CD45RA+, memory
CD4+CD45R0+ or memory CD8+CD45R0+ lymphocytes. For naive CD4 and
CD8 lymphocytes, a cytokine cocktail consisting of IL-2 (20 U/ml), IL-I2 (20
U/ml), IL-18 (10 ng/ml), IFN-gamma (1 ng/ml) and a monoclonal antibody
specific for IL-4 (50 ug/ml) is especially potent in enhancing DC activation
of
cytotoxic T cells in vitro, At the end of the incubation period, the DC are
washed and cultured in maturation conditions (1000 U/ml GM-CSF, 1000 U/ml
IL-4, 1% autologous plasma and 12.5% monocyte-condition medium) for 2-4
days. Cells manipulated in this manner are viable and have morphological and
surface characteristics (CD83+) of mature DC. Following the activation period,
CTL activity is assessed in a 4 hour SICr release assay.
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An efficient means to express a specific gene product for presentation by
dendritic cells is through infection with a recombinant viral vector. Human DC
infected with either retroviral, vaccinia, or adenoviral vectors recombinant
for the
same foreign. gene are employed to alternately stimulate autologous human T
cells. Cycling T cell stimulation with different vector recombinants
significantly
reduces the strong anti-vector response and promotes outgrowth of CTL specific
for the recombinant gene product of interest (Chaux, P. et al.,. J. Irnmunol.
163:2928-2936 (1999)).
Dendritic cells from a normal donor can be transduced with a retroviral
rector containing a gene differentially expressed in an infected cell. These
infected DC are employed to stimulate autologous T cells in vitro. After 12
days,
T cells are restimulated in the presence of IL-2 (20U/ml), IL,-12(20U/ml), and
IL-18(l0ng/ml) with DC from the same donor infected with a vaccinia virus
recombinant of the differentially expressed gene (MOI=1, 16 hours). A third
cycle of stimulation is subsequently carried out with DC infected with an
adenoviral recombinant of the differentially expressed gene. Specific lysis of
infected target cells by the T cells stimulated in vitro with DC infected by
recombinant vectors is measured.
Exanzple 7. Differential expression of hostgene products in hTIV 1 infected
monocytic cells.
In this example, deregulated gene expression in HIV-1 infected cells is
identified that might give rise to novel antigens encoded by the host rather
than
the virus. In contrast to highly mutable viral antigens, host encoded antigens
are
expected to be a relatively stable target for protective immune responses and
would not have any of the risks associated with immunization with attenuated
virus.
Subtractive hybridization was employed to identify genes differentially
expressed in HIV-1 infected vs. uninfected cells. HIV-1 genes are naturally
differentially expressed in HIV-1 infected cells. To eliminate HIV genes from
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consideration and control for subtraction efficiency, the subtraction driver
was
spiked with 1 % HIV sequences.
Figure 1 shows the results of hybridization to an array of 24 cDNA clones
selected following subtractive hybridization of cDNA from infected THP-1
monocytic cell line minus uninfected THP-1 cDNA + HIV DNA. Three different
probes were employed to test for differential gene expression: HIV genomic DNA
from a plasmid vector, cDNA from the normal uninfected THP-1 monocytic cell
line, and cDNA from HIV-1 infected THP-1. Only one clone in this set, B 1,
hybridized to the HIV probe (top panel). Seven clones gave detectable
hybridization to the uninfected cDNA probe (middle panel). These included one,
G3, which appeared to be downregulated in HIV-1 infected cells (compare
bottom panel). As can be seen by comparing bottom and middle panels, a larger
number of clones selected by subtractive hybridization demonstrate the
expected
preferential hybridization to a cDNA probe from infected cells.
Individual clones from this and other groups were tested by hybridization
to Northern blots of poly-A RNA from uninfected and infected cells. The
results
for several representative clones are shown in Figure 2 (these derive from a
different set than those illustrated in Figure 1, but illustrate the same
patterns of
expression). Clone 296 is, like clone B1 in Figure 1, an unsubtracted HIV
sequence. Clones 85a and 49 represent known genes, CTP synthetase (bands at
approximately 7 kb and 4kb) and tricarboxylate carrier (approximately 5 kb),
that
are significantly overexpressed in the infected cells. Clone 89 (2.5 kb) is a
novel
sequence of unknown function, but, by normalizing the RNA loaded in each lane
to the relative expression of housekeeping genes (actin and G3PDIi), clone 89
was found to be overexpressed in infected cells by only a factor of three
relative
to uninfected cells.
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Example 8. Imfnutzogenicity of Gene Products Differentially Expressed in
HIV infected Cells
There would be three major advantages to an ALDS vaccine based on
alterations in host gene expression during HIV-1 infection rather than on gene
products of the HIV virus itself. 1) Since the fidelity of host gene
replication is
far greater than that of HIV-1 and, especially, since host genes do not
replicate
with anything like the frequency of the HIV-1 genome, antigens encoded by host
genes would represent a relatively stationary target much less prone to immune
evasion through mutation. 2) Since key regulatory genes of HIV-1, rev, tat,
and
nef, are expressed early in the infectious cycle and may also be expressed in
some
persistently infected cells, host cell antigens induced through expression of
these
regulatory genes might enable the immune system to also target these
reservoirs
of latent infection. This has taken on increased importance since, as noted
above,
it is now known that important reservoirs of infection are resistant to triple
drug
therapy. 3) A vaccine that targets deregulated host gene products that are not
expressed in normal uninfected cells would not have any of the risks
associated
with use of an attenuated viral vaccine (for example, that its activity might
be
reconstituted by recombination with another viral genome).
Evidence for transactivation of cellular genes by human retroviruses has
been reported for both HIV-1 and HTLV-1. Rosenblatt, et al., 1995, Curr.
Topics in Micro. T_m_m__unol. 193:25-49. Two early viral gene products, tat
and
rev, are central to transactivation. Tat stimulates HIV-1 gene expression
during
transcription initiation and elongation. Tat functions primarily through
specific
interactions with TAR, the transactivation response element downstream of the
transcriptional start site, and several cellular cofactors to increase the
processivity
of RNA polymerase If complexes during HIV-1 transcription elongation.
Suggestive evidence for potential regulatory interactions of tat with host
genes
include at least two human mRNA that have been reported to contain TAR-like
sequences as well as the existence of a cellular protein, TRP-2, which has
been
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shown to bind to the functional tat-binding trinucleotide bulge on TAR. A
number of different cellular proteins, including transcription factors Sp-1
and
TFIIF, have been found to bind directly to tat protein. Other cellular
proteins
r
have been shown to bind to either the Rev Response Element, RRE, or directly
to the rev protein. Such interactions may lead directly or indirectly to
deregulated
expression of host genes and that some of these host gene products may give
rise
to imrriunogenic targets in H1V-1 infected cells that could be the basis for
immunotherapeutic intervention.
For optimal sensitivity, it is necessary that a large fraction of the cell
population be infected. Initial experiments focus on altered host gene
expression
following HIV-1 infection of GHOST clone 3 and U87MG derived cell lines that
express high levels of both CD4 and CCR5 co-receptors. Observations of altered
host gene expression can then be confirmed or extended in monocytic and T cell
lines. In order to obtain the s~.rne high frequency of infection in these
latter cell
lines, the HIV-1 env(-) mutant is pseudotyped with the broad-host-range
vesicular
stomatitis virus envelope glycoprotein G (VSV-G). Host gene expression is be
characterized following infection with both rev(+) and rev(-) virus. The rev(-
)
mutants express early regulatory genes of HIV-1 that have been shown to also
be
expressed in some latently infected cell lines. If immunogenic molecules can
be
identified among genes expressed early in infection, then this might make it
possible to target the reservoir of latently infected cells that appears to
escape
other forms of therapy. The rev(+) virus expresses, in addition to rev, late
HIV-1
accessory genes, vpu, vif, vpr and gag that may induce further quantitative or
qualitative changes in host gene expression.
The most efficient and reliable way to determine immunogenicity, is to
employ human dendritic cells pulsed with immunodominant peptides for
stimulation of autologous human T cells in vitro. However, in order to
identify
immunodominant peptides, it is necessary to first induce specific T cells. It
is
first determined whether a gene product is potentially immunogenic by the
ability
to induce specific CTL in HLA-A2 and human CD8 double transgenic mice. The
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murine T cells selected are then be tested for crossreactivity on HIV-1
infected
and uninfected targets. If differential reactivity is confirmed, then the same
T
cells can be employed to identify which of the peptide sequences that express
an
HLA binding motif in that gene product are immunodominant. It is then be
possible to determine whether human T cells are capable of responding to these
identified peptides presented by mature autologous dendritic cells ox whether
they
may have been rendered tolerant perhaps due to expression of related gene
products in other normal tissues.
Genes which are differentially expressed in HIV-infected cells are
identified using microarrays, the PCR SELECTTM cDNA subtraction method
(Clontech Laboratories), or the modified version of differential display
method
described above may be used to identify differentially expressed genes.
Microarrays have been used to monitor host cell gene expressioin in HIV
infected
cells. Geiss et al., Virol. 266:x-16 (2000).
In the present example, immunogenic peptides which bind HLA-A2. I are
identified. However, the experiments used in this example can also be used to
identify immunogenic peptides which bind to the A3, B7 and B44 subtypes.
To facilitate identification of genetic interactions between virus and host,
cells from which RNA is readily and reproducibly available for cDNA synthesis
and Northern analysis are employed, such as the human THP-1 monocytic cell
line (ATCC, TIB 202). This cell line shares more phenotypic and functional
markers with normal mature monocytes than most other available monocytic cell
lines. THP-1 is Fc receptor positive and phagocytic and provides costimulator
activity for T cell responses to Con A. THP-I expresses several other
histologic
and enzymatic markers of monocytes, most notably HLA-DR, and treatment with
160 nM phorbol diester (TPA) induces THP-1 to differentiate into cells with
the
functional characteristics of mature macrophage. HIV-1 infection of THP-1 has
been previously reported. Shattock, et al. , J. Virol. 67:3569-3575 (1993). It
is
especially useful that THP-1 expresses HLA-A2 (haplotype: HLA-A2, A9, B5,
DRW1, and DRW2).
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Gene expression in HIV-1 infected and uninfected THP-1 that are
untreated or TPA induced is compared. If specific gene deregulation associated
with HIV-1 infection is identified in this monocytic cell line, it will be of
great
interest to determine whether similar alterations of gene expression are
induced
in HIV-1 infected T cell lines. Sup-T1 and CEM are suitable T cell lines in
which to investigate these effects. Both lines are available from the AIDS
Research Reagent Repository.
To enhance the efficiency of HIV-1 infection HIV-1 env(-) mutants that
have been pseudotyped with the vesicular stomatitis virus envelope
glycoprotein
I O G (VS V-G) are employed. Use of the VS V pseudotype has the advantage that
the
interaction of viral and host genes can be studied independently of membrane
CD4 and chemokine receptor expression in the target cell. There is, however, a
concern that some effects on gene expression may be mediated by VS V envelope
interactions. Two controls are incorporated to identify possible effects of
the
VSV envelope alone or in concert with the HIV genome.
THP-1 cells are also infected with a VSV-G pseudotyped defective MuLV
based vector that expresses Thy-1.2 under the Moloney murine sarcoma virus
LTR. This VS V-G pseudotyped defective MuLV vector is packaged by triple co-
transfection of COS cells with the defective MuLV based plasmid (pSRLthy),
together with the packaging and env(-) deficient MuLV gag and pol expression
construct pSV(-)env(-)MLV, and with the VSV-G expression construct pHCMV-
G. An et al., J. Virol. 71:1397-1404 (1997). This control should identify
changes
in host cell gene expression that can be attributed to the VSV envelope alone.
A number of cell lines have been modified to express high levels of CD4
and CCR5 co-receptor so that a high frequency of HIV-1 infection is feasible.
Changes in gene expression induced by infection of CCR5 transfected GHOST
clone 3 cells are compared with the HIV-1 NIL-3 strain and with VSV-G
pseudotyped NL4-3 genome. This control should identify changes in host cell
gene expression that can be attributed to interaction between the VSV envelope
and the HIV-1 genome.
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The temporal pattern of HIV-1 RNA expression in the course of infection
has been well characterized. The transition from multiply spliced to singly
spliced
and unspliced transcripts is a key event that has been shown to be blocked in
some chronically infected cell lines. Butera et al., J. Virol. 68:2726-2730
(1994); Cannon et al., J. Virol. 68:1993-1997 (1994). This form of blocked
early-stage latency can be overcome by cellular activation with phorbol ester
or
cytokines and appears to be a result of either limited transcription at the
site of
proviral integration or an inherent deficiency in cellular regulatory factors.
It has
been suggested that a similar state of blocked early-stage latency may occur
in
some cells during the course of natural infection i>2 vivo. Since viral
mutants in
rev give rise to the same pattern of RNA transcription characteristic of
chronically infected cell lines, it will be interesting to investigate
patterns of
differential gene expression in cells infected with rev(-) mutants. In
comparison
to the study of chronically infected cell lines (e.g. Ul/HIV-1) this has the
advantage that specific effects on gene expression can be compared in
different
target cell populations infected with the same mutant virus and, importantly,
that
the immediately relevant uninfected cell controls are directly available for
comparison.
Viral trtutants:
HIV-1~_3env(-): An envelope-defective molecular clone of HIV-1~_3
was constructed by deletion of the envelope gene sequences between two bgllI
restriction endonuclease sites. Planelles et al., J. Virol. 69:5883-5889
(1995).
A related clone, HIV-1~_3 Thy-l.2env(-), has the murine thymocyte Thy-1.2
surface antigen introduced into the nef open reading frame by deletion of the
nef
gene sequences between XhoI and I~pnI sites. This clone is especially useful
to
determine the frequency of infected cells in a population.
HIV-1~_3env(-)rev(-): The Rev open reading frame (orf) was disrupted
by introducing an oligonucleotide encoding double stop codons in all 3 reading
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frames into the Rev gene. The insertion of this oligonucleotide would be into
a
BamHI site present in approximately the middle of the Rev orf. The BamHI site
is at nucleotide position 7886 in the HIV provirus clone (DHIV-3nef).
Unfortunately, this HIV provirus clone contained an additional BamHI site in
the
, vector (not in the HIV DNA), at position 9143 (the HIV sequence in this
vector
is from position 1 to 9129). This extra BamHI site was removed by digesting
DHIV-3nef with ~naI (position 9149) and XbaI (position 9131), blunt ending
with pfu polymerase, and religating the DNA. The ligated DNA was transformed
into XL-10 Gold bacteria and clones were identified that contained a unique
BamHI site in the Rev gene. This clone is designated DHIV-3nef BamHI(-).
In order to disrupt the Rev orf two single stranded, complementary
oligonucleotides were synthesized and annealed together. This double stranded
oligonucleotide encoded 5' BamHI sticky end / double stop codons (TAA) in all
3 reading frames / Eco RI site / Bam HI sticky end. This oligo was ligated
into
the Bam HI site of DHIV-3nef BamHI(-). Following transformation, clones that
contained the oligonucleotide were identified by restriction digest analysis
using
EcoRI. Sequence analysis confirmed that these clones contained the Rev
knockout oligonucleotide. These clones were designated DHIV3-nef Rev(-).
VSV-Gpseudotypedenv-deficientHIV-1 areproducedbyelectroporation
of COS-7 cells with p HIV-l~_3 env(-) (or p HIV-1~_3 env(-)rev(-)) and
pHCMV-G, which expresses the VSV-G gene under the control of the CMV
promoter (39). In the case of the rev(-) plasmid, triple cotransfection with
pcRev
provides the necessary rev functions in trans. Viral supernatants are
harvested at
48 and 72 hrs and are titred by activation of the b-Galactosidase gene in the
MAGI cell assay (40). It is expected, on the basis of prior experience with
HeLa
cells, that THP-1 infection with VSV-G pseudotyped HIV-1~_3env(-) at m.o.i.=3
will result in greater than 75 ~lo infected cells. Control experiments will be
carried
out by infection of untreated and TPA induced THP-1 monocytic cells with VSV
G pseudotyped HIV-1~_3 Thy-l.2env(-) to allow simple scoring of the
frequency of infected cells by FACS analysis of Thy-1.2 expression.
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Fresh primary monocytes are resistant to HIV-1 infection iv vitro.
Susceptibility to infection, however, increases rapidly during the first 24
hrs of
in vitro culture. This appears to be related to differentiation induced by
adherence to plastic. Viral yield from monocytic cultures can be greatly
enhanced
by addition of GM-CSF and M-CSF to promote differentiation. In order to
compare host gene deregulation in HIV-1 infected THP-1 and normal monocytes,
Ficoll/Hypaque isolated PBMC are resuspended at 5x106 cells/ml in RPMf with
lOmM HEPES and no serum in plastic tissue culture dishes for 2 hrs incubation
at 37°C. Monolayers are carefully washed to remove all non-adherent
cells
(recovery is 10 to 20%, with >90% CD14+ CD3- cells). Cells are maintained in
complete medium for 7 days in vitro prior to infection with VSV-G pseudotyped
HIV-1~_3 env(-) (or HIV-1~_3 env(-)rev(-)) at m.o.i.=3. Expression of specific
genes previously identified as differentially expressed in HIV-1 infected THP-
1
cells are determined by Northern blot and RNAse protection assays with RNA
extracted from infected and uninfected primary monocyte cultures. Since THP-1
cells can be induced by GM-CSF as well as by TPA, an interesting variation on
this experiment is to compare HIV-1 infected, GM-CSF induced THP-1 and GM-
CSF activated primary monocytes (monocyte derived macrophage).
To determine the expression pattern in other normal adult and fetal
tissues of any gene which is differentially expressed in HIV-1 infected
monocytic
cells, Discovery LineTM RNA and Gene PoolTM cDNA (Invitrogen) is used. Those
sequences that are consistently expressed in HIV-1 infected monocytic cells
but
which have low or undetectable expression in diverse normal tissues especially
thymus are more likely to be immunogenic.
.
Construction of Vaccihia Virus Recombifzants
The ease of cloning and propagation in a variety of host cells has led to
the widespread use of poxvirus vectors for expression of foreign proteins and
as
delivery vehicles for vaccine antigens. Recently, two laboratories have
reported
on a direct ligation protocol obviating the need for homologous recombination
to
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generate poxvirus chimeric genomes. Merchlinsky et al., Virology 190: 522-526
(1992); Scheiflingler et al., Proc. Natl. Acad. Sci. USA 89: 9977-9981 (1992).
In order to make this method more generally useful, a new direct ligation
vector
was constructed, vEL/tk, that incorporates unique NotI and ApaI restriction
sites
downstream of the early/late 7.5k vaccinia promoter. This vector gives higher
levels of expression of the recombinant gene, permits directional cloning of
DNA, and largely eliminates the background of non-recombinant virus following
ligation. Merchlinsky et al., 1997. Virology, 238: 444-451 (1997).
HLA-restricted respofzse of murine T cells ire HLA-A2/Kb afzd humaf2 CD8
transgenic mice
The avidity of interaction between the cytolytic T cell receptor and
MHC:peptide complex on the target cell must, in general, be enhanced by a
parallel interaction between the CD8 molecule on the T cell membrane and MHC
class I of the target. Since murine CD8 does not interact efficiently with
human
HLA class I, induction of HLA-restricted T cell responses in HLA-transgenic
mice requires that either a second transgene for human CD8 be introduced or
that
the HLA molecule be modified to permit interaction with murine CD 8. For HLA-
A2.1, the latter can be accomplished by construction of a chimeric HLA
molecule, HLA-A2/Kb, with the al and a2 domains of HLA-A2.1 and the a3
domain of murine H-2Kb. Co-expression of human CD8 in the HLA transgenic
is desirable because CTL induced in these mice for crossreactivity on human
HLA-2+, HIV-1-infected cells is tested. If the T cells did not express human
CDB, then it is necessary to transfect the chimeric HLA-A2lKb gene even into
target populations that express native HLA-A2.1. Double transgenic (HLA-
A2/Kb x huCDB) Fl hybrid mice are therefore used for induction of HLA-A2.1
restricted murine T cell responses.
HLA transgenic mice have been previously employed to characterize
peptide epitopes of HTLV-1 in association with HLA-B35 (Schonbach et al.,
Virology. 226: 102-12 (1996)), and epitopes of Hepatitis C Virus ( Shirai et
al.,
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J. Immunol. 154: 2733-42 (1995)), Human Papilloma Virus type 16 (Ressing et
al., J. Immunol. 154:5934-43 (1995)), and circumsporozoite protein of
Plasmodium falciparum (Blum-Tirouvanziam et al., J. Immunol. 154: 3922-31)
in association with HLA-A2.1. In these experiments as well as in a broad
survey
of the immune response of HLA-A2.1 transgenic mice to HLA-A2.1 binding
peptides (Wentworth et al., Eur. J, of Immunol. 26: 97-101 (1996)), it was
concluded that there is an extensive overlap between the T cell repertoire of
mouse and man.
vaccireatiore with Yaccircia Virus recombinafat of differentially expressed
genes
To determine whether the products of differentially expressed genes are
immunogenic, groups of three (HLA-A2/Kb x huCDB)Fl transgenic mice are
immunized intravenously with 5 x 106 pfu of vaccinia virus recombinant for a
differentially expressed gene. Bennink et al., Current Topics in Microbiol.
and
Immunol. 163: 153-178 (1990). After at least two weeks, mice are sacrificed
and
CD8+ splenic T cells are enriched on anti-CD8 coated magnetic beads. CD8+
cytolytic precursors are restimulated ire vitro with THP-1 monocytic cells
that are
transfected With the recombinant differentially expressed gene previously
isolated
in the pcDNA3.l/Zeo(+) plasmid expression vector. Substitution of the plasmid
recombinant in place of the vaccinia vector for restimulation in vitro is
necessary
to avoid a large vaccinia vector specific response. After five days in vitro
culture,
cytolytic activity is determined by SiCr release from THP-1 target cells
transfected
with either the specific recombinant plasmid or a control ovalbumin gene
recombinant.
This same cytolytic assay can be readily applied to determine whether
CTL epitopes are also presented by other HLA compatible HIV-1 infected cells.
For T cells induced in (HLA-A2/Kb x huCDB)Fl transgenic mice, HLA
compatible targets include cells that either express native HLA-A2.1 or that
have
been transfected with HLA-A2.1 (or HLA-A2/Kb).
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In order to demonstrate immunogenicity, human dendritic cells pulsed
with immunodominant peptides for presentation to autologous T cells in vitro
are
used. However, in order to identify immunodorninant peptides, it is necessary
to
first induce specific T cells. A two-phase strategy can be used in which it is
first
determined whether a gene product is immunogenic by the ability to induce
specific CTL in HLA-A2 and human CD8 double transgenic mice. The T cells
selected will then be tested for crossreactivity on HIV-1 infected, HLA
compatible tumors that express the corresponding mRNA and, if tumor reactivity
is confirmed, will be used to identify which of the peptide sequences that
express
an HLA binding motif in that gene product are immunodominant. It will then be
possible to determine whether human T cells are capable of responding to these
identified peptides or whether they may have been rendered tolerant.
There are publicly available programs for identification of peptides in a
given sequence that express a human or murine MHC binding motif. Parker et
al., Journal of Immunology 152:163 (1994). Specific T cells can then be used
to identify the immunodominant peptides. In the absence of tolerance,
presentation of these peptides by mature dendritic cells (DC) is a very
efficient
means of stimulating primary, peptide-specific T cell responses i~z vitro.
It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are
possible in light of the above teachings and, therefore, are within the scope
of the
appended claims.
The entire disclosure of all publications (including patents, patent
applications, journal articles, laboratory manuals, books, or other documents)
cited herein are hereby incorporated by reference.
