U.S. patent application number 11/719646 was filed with the patent office on 2008-11-06 for vaccines and methods for using the same.
Invention is credited to Michele Kutzler, David B Weiner.
Application Number | 20080274140 11/719646 |
Document ID | / |
Family ID | 37968247 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274140 |
Kind Code |
A1 |
Weiner; David B ; et
al. |
November 6, 2008 |
Vaccines and Methods for Using the Same
Abstract
Compositions comprising one or more isolated nucleic acid
molecules that encode an immunogen in combination with one or more
of CTACK protein, TECK protein, MEC protein and functional
fragments thereof and/or an isolated nucleic acid molecule that
encodes an protein selected from the group consisting of: CTACK,
TECK, MEC and functional fragments thereof are disclosed. Methods
of inducing an immune response, including methods of inducing
mucosal immune responses, in an individual against an immunogen,
using such compositions are disclosed.
Inventors: |
Weiner; David B; (Merion,
PA) ; Kutzler; Michele; (Souderton, PA) |
Correspondence
Address: |
Pepper Hamilton LLP
400 Berwyn Park, 899 Cassatt Road
Berwyn
PA
19312-1183
US
|
Family ID: |
37968247 |
Appl. No.: |
11/719646 |
Filed: |
November 18, 2005 |
PCT Filed: |
November 18, 2005 |
PCT NO: |
PCT/US05/42231 |
371 Date: |
June 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60629737 |
Nov 19, 2004 |
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60647617 |
Jan 27, 2005 |
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Current U.S.
Class: |
424/208.1 ;
424/232.1; 424/277.1; 536/23.5; 536/23.72 |
Current CPC
Class: |
A61P 37/02 20180101;
A61K 39/21 20130101; A61K 2039/55516 20130101; C12N 2740/16234
20130101; A61K 39/39 20130101; C07K 14/005 20130101; A61K 39/12
20130101; C12N 2740/16222 20130101; Y02A 50/30 20180101; A61K
2039/541 20130101; A61K 2039/53 20130101; Y02A 50/394 20180101 |
Class at
Publication: |
424/208.1 ;
536/23.72; 536/23.5; 424/277.1; 424/232.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07H 21/04 20060101 C07H021/04; A61K 39/285 20060101
A61K039/285; A61K 39/21 20060101 A61K039/21 |
Claims
1. A composition comprising: an isolated nucleic acid molecule that
encodes an immunogen; and an isolated nucleic acid molecule that
encodes one or more proteins of selected from the group consisting
of: CTACK, TECK, MEC and functional fragments thereof.
2. The composition of claim 1 wherein said nucleic acid molecules
are plasmids.
3. The composition of claim 1 wherein said immunogen is a pathogen
antigen, a cancer-associated antigen or an antigen linked to cells
associated with autoimmune diseases.
4. The composition of claim 3 wherein said immunogen is a pathogen
antigen.
5. The composition of claim 4 wherein said immunogen is an HIV
antigen.
6. The composition of claim 5 wherein said HIV antigen is gag.
7. A composition comprising an isolated nucleic acid molecule
comprising a nucleotide sequence that encodes an immunogen; and a
nucleotide sequence that encodes one or more proteins of selected
from the group consisting of: CTACK, TECK, MEC and functional
fragments thereof.
8. The composition of claim 7 wherein said nucleic acid molecule is
a plasmid.
9. The composition of claim 7 wherein said immunogen is a pathogen
antigen, a cancer-associated antigen or an antigen linked to cells
associated with autoimmune diseases.
10. The composition of claim 9 wherein said immunogen is a pathogen
antigen.
11. The composition of claim 10 wherein said immunogen is an HIV
antigen.
12. The composition of claim 11 wherein said HIV antigen is
gag.
13. An injectable pharmaceutical composition comprising the
composition of claim 1.
14. A method of inducing an immune response in an individual
against an immunogen comprising administering to said individual a
composition of claim 1.
15. The method of claim 14, wherein said immune response is a
mucosal immune response.
16. A recombinant vaccine comprising a nucleotide sequence that
encodes an immunogen operably linked to regulatory elements, a
nucleotide sequence that encodes one or more proteins of selected
from the group consisting of: CTACK, TECK, MEC and functional
fragments thereof.
17. The recombinant vaccine of claim 16 wherein said immunogen is a
pathogen antigen, a cancer-associated antigen or an antigen linked
to cells associated with autoimmune diseases.
18. The recombinant vaccine of claim 17 wherein said immunogen is a
pathogen antigen.
19. The recombinant vaccine of claim 18 wherein said recombinant
vaccine is a recombinant vaccinia vaccine.
20. A method of inducing an immune response in an individual
against an immunogen comprising administering to said individual a
recombinant vaccine of claim 17.
21. The method of claim 20 wherein said immune response is a
mucosal immune response.
22. A live attenuated pathogen comprising a nucleotide sequence
that encodes one or more proteins of selected from the group
consisting of: CTACK, TECK, MEC and functional fragments
thereof.
23. A method of immunizing an individual against a pathogen
comprising administering to said individual the live attenuated
pathogen of claim 22.
24. The method of claim 23 wherein said immune response is a
mucosal immune response.
25. A method of inducing an immune response in an individual
against an immunogen comprising administering to said individual:
one or more of CTACK protein, TECK protein, MEC protein and
functional fragments thereof in combination with an isolated
nucleic acid molecule that encodes an immunogen; and/or a
recombinant vaccine that encodes an immunogen and/or a subunit
vaccine that comprises an immunogen and/or a live attenuated
vaccine and/or a killed vaccine.
26. The method of claim 25 wherein an isolated nucleic acid
molecule that encodes one or more proteins of selected from the
group consisting of: CTACK, TECK, MEC and functional fragments
thereof is administered to the individual.
27. The method of claim 25 wherein said immunogen is a pathogen
antigen, a cancer-associated antigen or an antigen linked to cells
associated with autoimmune diseases.
28. The method of claim 27 wherein said immunogen is a pathogen
antigen.
29. The method of claim 28 wherein said immunogen is an HIV
antigen.
30. The method of claim 29 wherein said HIV antigen is gag.
31. The method of any one of claim 24 wherein the immune response
is a mucosal immune response.
32. A composition comprising: one or more of CTACK protein, TECK
protein, MEC protein and functional fragments thereof in
combination with one or more of an isolated nucleic acid molecule
that encodes an immunogen; a recombinant vaccine comprising a
nucleotide sequence that encodes an immunogen operably linked to
regulatory elements; a live attenuated pathogen; and a killed
pathogen.
33. The composition of claim 32 comprising an isolated nucleic acid
molecule that encodes an immunogen and/or a recombinant vaccine
comprising a nucleotide sequence that encodes an immunogen operably
linked to regulatory elements; wherein said immunogen is a pathogen
antigen, a cancer-associated antigen or an antigen linked to cells
associated with autoimmune diseases.
34. The composition of claim 33 wherein said immunogen is a
pathogen antigen.
35. The composition of claim 34 wherein said immunogen is an HIV
antigen.
36. The composition of claim 35 wherein said HIV antigen is
gag.
37. The composition of claim 32 further comprising nucleic acid
molecule that encodes one or more proteins of selected from the
group consisting of: CTACK, TECK, MEC and functional fragments
thereof is administered to the individual.
38. An injectable pharmaceutical composition comprising the
composition of claim 32.
39. A method of inducing an immune response in an individual
against an immunogen comprising administering to said individual a
composition of claim 32.
40. The method of claim 39, wherein said immune response is a
mucosal immune response.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to improved vaccines, improved
methods for inducing immune responses, including mucosal immune
responses, and for prophylactically and/or therapeutically
immunizing individuals against immunogens.
BACKGROUND OF THE INVENTION
[0002] Infectious agents commonly enter the host across a mucosal
tissue such as the oral mucosa and other mucosa of the alimentary
canal, the respiratory tract including olfactory and conjunctival
mucosa, the mammary glands, and the genitourinary tract. The
mucosal immune system provides a secretory immunoglobulin response
to prevent infectious agents at these points of entry.
[0003] The secretory immune response includes clonal proliferation
of antigen-specific B cells and progressive isotype switching by
the B cell progeny to all subclasses of IgG- and IgA-secreting
cells. Antigens such as microorganisms, proteins, polysaccharides,
etc., that are encountered at a mucosal site can elicit local
production of antibodies into the secretions that bathe the mucosal
surface at the site, as well as other mucosal sites.
[0004] Secretory and circulating IgA production often exceeds that
of other immunoglobulin isotypes. Secretory IgA as well as IgM and
all subclasses of IgG have been found in virtually all external
secretions, including tears, saliva, colostrum and milk, and in the
mucous secretions of the respiratory, intestinal and genitourinary
tracts.
[0005] The secretory IgA performs a protective role in the
prevention of infectious diseases and for the inhibition of
allergic reactions at mucosal surfaces. Secreted IgA neutralizes
biologically active antigens, prevents uptake of antigens from the
intestinal tract, and inhibits adherence of bacteria to epithelial
surfaces.
[0006] Once antigen penetrates the mucosal epithelial cells,
antigen-presenting cell-dependent activation of paracortical T
cells and germinal center B cells within the Peyer's Patches is
observed. However, the inductive stimuli required for
differentiation of IgA-committed B cells is deferred until B cells
have migrated through efferent lymphatics into the mesenteric lymph
nodes after departure from the Peyer's Patches. Ultimately,
IgA-committed, antigen-sensitized B cells enter the circulation
through the lymph to populate various exocrine glands and mucosal
epithelia throughout the body. Under local influences which include
information provided by helper T cells, by the antigen and other
biochemical mediators, terminal differentiation into IgA-secreting
plasma cells occurs.
[0007] Tissue-selective trafficking of memory and effector T and B
lymphocytes is mediated by a unique combination of adhesion
molecules and chemokines. Chemokines contribute to both lymphocytes
exit from circulation and localization and retention within
tissues. Memory T lymphocytes selectively re-circulate back through
tissues including skin and intestines and other mucosal tissues.
Chemokines and their receptors help control the movement of memory
lymphocytes subsets through skin and gut. Effector T cells homing
to the intestine and GALT express high levels of a4b7, whose
ligand,
[0008] MAdCAM-1 is expressed in the intestinal lamina propria and
Peyers Patches. These mucosal T cells also express CCR9, whose
ligand, TECK (CCL25) is selectively expressed by small intestinal
epithelial cells. DC's which home to the mucosae express CCR10
which is the receptor for the chemokines Mec or c-TACK. (Campbell
et al., J. Exp Med. 2002, 195:(1) 135-141. Johansson-Lindbom et
al., J. Exp. Med. 2003. 198(6), 963-969, which is incorporated
herein by reference.)
[0009] Cutaneous lymphocyte-associated antigen (CLA+) memory T
cells are preferentially Targeted by CTACK and MEC. (Morales et
al., 1999 PNAS, 96(25) 14470-14475, Jarmin et al., 2000 J. Immunol.
164:3460-3464). Therefore, CTACK, MEC and their receptors control
movement of memory lymphocyte subsets in skin and gut.
[0010] Subset of circulating a4b7+ integrin lymphocytes from the
small intestine co-express CCR9 and respond to TECK, and all T
lymphocytes in the small intestinal epithelium express CCR9 (Zabel
et al., 1999 J. Exp. Med 190:1241-1256). Therefore, TECK and CCR9
play a critical role in lymphocyte biology in the mucosae
[0011] Mucosal epithelia are major site of secretory IgA by
resident plasma cells. B cells secreting IgA also migrate
preferentially to mucosae and express a4b7. B cells from spleen,
MLN, and Peyer's patches express CCR9 (Butcher et al., 1999 Adv.
Immunol 72: 209-253, Kunkel et al., 2000 J. Exp. Med. 192:761-768).
Therefore, TECK and CCR9 participate in localization of B cells
that secrete IgA to mucosal sites.
[0012] Vaccine protocols can be improved by the delivery of agents
that modulate a person's immune responses to induce an improved
immune response. In some vaccination protocols in which the
individual is administered a vaccine that exposes the individual to
an immunogen against which the individual generates an immune
response, an agent is provided that increases the immune response
and/or selectively enhances a portion of the immune response (such
as the cellular arm or the humoral arm) which is desirable to treat
or prevent the particular condition, infection or disease.
[0013] Vaccines are useful to immunize individuals against target
antigens such as allergens, pathogen antigens or antigens
associated with cells involved in human diseases. Antigens
associated with cells involved in human diseases include
cancer-associated tumor antigens and antigens associated with cells
involved in autoimmune diseases.
[0014] In designing such vaccines, it has been recognized that
vaccines that produce the target antigen in cells of the vaccinated
individual are effective in inducing the cellular arm of the immune
system. Specifically, live attenuated vaccines, recombinant
vaccines which use avirulent vectors, and DNA vaccines each lead to
the production of antigens in the cell of the vaccinated individual
which results in induction of the cellular arm of the immune
system. On the other hand, killed or inactivated vaccines, and
sub-unit vaccines which comprise only proteins do not induce good
cellular immune responses although they do induce a humoral
response.
[0015] A cellular immune response is often necessary to provide
protection against pathogen infection and to provide effective
immune-mediated therapy for treatment of pathogen infection, cancer
or autoimmune diseases. Accordingly, vaccines that produce the
target antigen in cells of the vaccinated individual such as live
attenuated vaccines, recombinant vaccines that use avirulent
vectors and DNA vaccines are often preferred.
[0016] While such vaccines are often effective to immunize
individuals prophylactically or therapeutically against pathogen
infection or human diseases, there is a need for improved vaccines.
There is a need for compositions and methods that produce an
enhanced immune response.
[0017] Likewise, while some immunotherapeutics are useful to
modulate immune response in a patient there remains a need for
improved immunotherapeutic compositions and methods.
SUMMARY OF THE INVENTION
[0018] The present invention relates to a composition an isolated
nucleic acid molecule that encodes an immunogen in combination with
an isolated nucleic acid molecule that encodes one or more
chemokines selected from the group consisting of: CTACK, TECK, MEC
and functional fragments thereof.
[0019] The present invention further relates to a composition an
isolated nucleic acid molecule that encodes both an immunogen and
one or more chemokines selected from the group consisting of:
CTACK, TECK, MEC and functional fragments thereof.
[0020] The present invention relates to injectable pharmaceutical
compositions comprising an isolated nucleic acid molecule that
encodes an immunogen in combination with an isolated nucleic acid
molecule that encodes one or more chemokine selected from the group
consisting of: CTACK, TECK, MEC and functional fragments
thereof.
[0021] The present invention relates to injectable pharmaceutical
compositions comprising an isolated nucleic acid molecule that
encodes both an immunogen and one or more chemokines selected from
the group consisting of: CTACK, TECK, MEC and functional fragments
thereof.
[0022] The present invention further relates to methods of inducing
an immune response in an individual against an immunogen,
comprising administering to the individual a composition an
isolated nucleic acid molecule that encodes an immunogen in
combination with an isolated nucleic acid molecule that encodes one
or more chemokines selected from the group consisting of: CTACK,
TECK, MEC and functional fragments thereof.
[0023] The present invention further relates to methods of inducing
an immune response in an individual against an immunogen,
comprising administering to the individual a nucleic acid molecule
that encodes an immunogen and one or more chemokines selected from
the group consisting of: CTACK, TECK, MEC and functional fragments
thereof.
[0024] The present invention further relates to methods of inducing
a mucosal immune response in an individual against an immunogen,
comprising administering to the individual a composition an
isolated nucleic acid molecule that encodes an immunogen in
combination with an isolated nucleic acid molecule that encodes one
or more chemokines selected from the group consisting of: CTACK,
TECK, MEC and functional fragments thereof.
[0025] The present invention further relates to methods of inducing
a mucosal immune response in an individual against an immunogen,
comprising administering to the individual a nucleic acid molecule
that encodes an immunogen and one or more chemokines selected from
the group consisting of: CTACK, TECK, MEC and functional fragments
thereof.
[0026] The present invention further relates to recombinant
vaccines comprising a nucleotide sequence that encodes an immunogen
operably linked to regulatory elements, a nucleotide sequences that
encode one or more chemokines selected from the group consisting
of: CTACK, TECK, MEC and functional fragments thereof, and to
methods of inducing an immune response, including methods of
inducing a mucosal immune response, in an individual against an
immunogen comprising administering such a recombinant vaccine to an
individual.
[0027] The present invention further relates to a live attenuated
pathogen comprising a nucleotide sequence that encodes one or more
chemokines selected from the group consisting of: CTACK, TECK, MEC
and functional fragments thereof, and to methods of inducing an
immune response, including methods of inducing a mucosal immune
response, in an individual against a pathogen comprising
administering the live attenuated pathogen to an individual.
[0028] The present invention further relates to methods of inducing
an immune response in an individual against an immunogen comprising
administering to said individual one or more of CTACK protein, TECK
protein, MEC protein and functional fragments thereof in
combination with an isolated nucleic acid molecule that encodes an
immunogen; and/or a recombinant vaccine that encodes an immunogen
and/or a subunit vaccine that comprises an immunogen and/or a live
attenuated vaccine and/or a killed vaccine.
[0029] A method of inducing an immune response in an individual
against an immunogen comprising administering to said individual
one or more of CTACK protein, TECK protein, MEC protein and
functional fragments thereof in combination with an isolated
nucleic acid molecule that encodes an immunogen; and/or a
recombinant vaccine that encodes an immunogen and/or a subunit
vaccine that comprises an immunogen and/or a live attenuated
vaccine and/or a killed vaccine
[0030] The present invention further relates to composition
comprising: one or more of CTACK protein, TECK protein, MEC protein
and functional fragments thereof in combination with one or more of
an isolated nucleic acid molecule that encodes an immunogen; a
recombinant vaccine comprising a nucleotide sequence that encodes
an immunogen operably linked to regulatory elements; a live
attenuated pathogen; and a killed pathogen.
[0031] The present invention further relates to methods of inducing
an immune response in an individual against an immunogen comprising
administering to said individual a composition comprising one or
more of CTACK protein, TECK protein, MEC protein and functional
fragments thereof in combination with an isolated nucleic acid
molecule that encodes an immunogen; and/or a recombinant vaccine
that encodes an immunogen and/or a subunit vaccine that comprises
an immunogen and/or a live attenuated vaccine and/or a killed
vaccine
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 depicts a proposed mechanism of plasmid induced
cellular immune response in secondary lymphoid organs.
[0033] FIG. 2 shows construction of plasmids used in the Example
and data confirming the identity and expression of the chemokine
coding sequences.
[0034] FIG. 3 shows a depiction of the order of methodology
employed.
[0035] FIG. 4 shows IFN.gamma. ELISpot and Flow cytometry data
comparing vector only, pHIV-1 gag only, and pHIV-1+pMEC.
[0036] FIG. 5 shows IFN.gamma. ELISpot and Flow cytometry data
comparing vector only, pHIV-1 gag only, pHIV-1+pCTACK and
pHIV-1+pTECK.
[0037] FIG. 6 shows IFN.gamma. ELISpot data comparing vector only,
pHIV-1 gag only, pHIV-1+pCTACK and pHIV-1+pTECK when stimulated
with different HIV-1 Gag peptide pools.
[0038] FIG. 7 shows MIP1B expression data comparing vector only,
pHIV-1 gag only, pHIV-1+pMEC, pHIV-1+pCTACK and pHIV-1+pTECK when
stimulated with R10 or HIV-1 Gag.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0039] As used herein, "functional fragment" is meant to refer to a
fragment of an immunomodulating protein that, when delivered in
conjunction with an immunogen, provides an increased immune
response compared to the immune that is induced when the immunogen
is delivered without the fragment. Fragments are generally 10 or
more amino acids in length.
[0040] As used herein the term "target protein" is meant to refer
to peptides and protein encoded by gene constructs of the present
invention that act as target proteins for a target protein-specific
immune response. The terms "target protein" and "immunogen" are
used interchangeably and refer to a protein against which an immune
response can be elicited. The target protein is an immunogenic
protein that shares at least an epitope with a protein from the
pathogen or undesirable cell-type such as a cancer cell or a cell
involved in autoimmune disease against which an immune response is
desired. The immune response directed against the target protein
will protect the individual against and/or treat the individual for
the specific infection or disease with which the target protein is
associated.
[0041] As used herein, the term "genetic construct" refers to the
DNA or RNA molecules that comprise a nucleotide sequence which
encodes a target protein or immunomodulating protein. The coding
sequence includes initiation and termination signals operably
linked to regulatory elements including a promoter and
polyadenylation signal capable of directing expression in the cells
of the individual to whom the nucleic acid molecule is
administered.
[0042] As used herein, the term "expressible form" refers to gene
constructs that contain the necessary regulatory elements operable
linked to a coding sequence that encodes a target protein or an
immunomodulating protein, such that when present in the cell of the
individual, the coding sequence will be expressed.
[0043] As used herein, the term "sharing an epitope" refers to
proteins that comprise at least one epitope that is identical to or
substantially similar to an epitope of another protein.
[0044] As used herein, the term "substantially similar epitope" is
meant to refer to an epitope that has a structure that is not
identical to an epitope of a protein but nonetheless invokes a
cellular or humoral immune response that cross-reacts to that
protein.
[0045] As used herein, the term "intracellular pathogen" is meant
to refer to a virus or pathogenic organism that, at least part of
its reproductive or life cycle, exists within a host cell and
therein produces or causes to be produced, pathogen proteins.
[0046] As used herein, the term "hyperproliferative diseases" is
meant to refer to those diseases and disorders characterized by
hyperproliferation of cells.
[0047] As used herein, the tam "hyperproliferative-associated
protein" is meant to refer to proteins that are associated with a
hyperproliferative disease.
[0048] The invention arises from the discovery that when delivered
in combination with an immunogen, each of the chemokines CTACK,
TECK, MEC and functional fragments thereof, and combinations
thereof modulates immune responses. Accordingly, a combination of
these proteins may be delivered as components of a vaccine in order
to induce a therapeutic or prophylactic immune response or in
compositions useful to induce an immune response. In some
embodiments, the means to deliver the immunogen is a DNA vaccine, a
recombinant vaccine, a protein subunit vaccine, an attenuated
vaccine or a killed vaccine. In some embodiments, the means to
deliver one or more of CTACK, TECK, MEC and functional fragments
thereof is by expression of coding sequences included in a DNA
vaccine, a recombinant vaccine or an attenuated vaccine. In some
embodiments, the means to deliver one or more of CTACK, TECK, MEC
and functional fragments thereof is simply to administer the
protein directly or to incorporate the protein as part of a
recombinant vaccine, an attenuated vaccine or killed vaccine. In
some embodiments, the means to deliver the immunogen is a DNA
vaccine, a recombinant vaccine, a protein subunit vaccine, an
attenuated vaccine or a killed vaccine and the one or more of
CTACK, TECK, MEC and functional fragments thereof is by expression
of coding sequences included in a DNA vaccine, a recombinant
vaccine or an attenuated vaccine and/or by administering the
protein directly and/or incorporated as part of a recombinant
vaccine, an attenuated vaccine or killed vaccine.
[0049] Immune responses result in the production of antigen
specific antibodies and/or antigen specific T- and B-cells. Antigen
specific antibodies and/or cells provide the means to protect
against infection, to reduce or to clear existing infection. They
can also be isolated from the individual and used in other
applications such as passive immunity protocols, immunocolumns or
as reagents.
[0050] In some embodiments, CTACK, TECK and MEC are useful to
induce mucosal immune responses, even in protocols where the
composition is delivered systemically. In such embodiments, the
presence of one or more of CTACK, TECK and MEC recruit mucosal
immunity cells, such as Dendritic cells from mucosae tissue, such
as those expressing CCR10, or T cells from mucosae tissue, such as
those expressing CCR9. These mucosal immunity cells become engaged
in the immune response being generated against the co-expressed
immunogen and result in the presence of primed effector T cells and
activated dendritic cells at sites of mucosal tissue in the
individual. Thus, systemic administration of vaccines that comprise
one or more of the chemokines CTACK, TECK and MEC may be used to
induce broad mucosal immunity.
[0051] CTACK, MEC and TECK play an important role in the
recruitment of T and B lymphocytes and dendritic cells from the
mucosae. CTACK, TECK and/or MEC are delivered as part of or in
combination with various types of vaccines. The CTACK, TECK and/or
MEC may be delivered as proteins as part or, as a single
combination with and/or in combination with a DNA vaccine,
recombinant viral vaccine, live, attenuated vaccine or killed
vaccine. The CTACK, TECK and/or MEC may be delivered by delivering
nucleic acid molecules which encode the proteins. These nucleic
acid molecules may be incorporated within and/or delivered in a
single composition with and/or delivered separately but in
combination with a DNA vaccine, recombinant viral vaccine, live,
attenuated vaccine or killed vaccine.
[0052] Co-immunization with an immunogen such as by DNA vaccine or
other means plasmid plus one or more of these chemokines, such as
in a DNA vaccine or part of the coding sequence of another type of
vaccine, will provide a unique adjuvanting property by bringing
mucosal relevant cells to the site of vaccination thus bringing
mucosal relevant cells to the systemic immune system where the
mucosal relevant cells can be primed by systemic DNA vaccine. FIG.
1 depicts a proposed mechanism of plasmid induced cellular immune
response in secondary lymphoid organs. The plasmid or plasmids
containing coding sequences for an immunogen and one or more of
CTACK, TECK and MEC are delivered systemically to the individual
such as by intramuscular injection in addition to the antigenic
plasmid. The coding sequences are expressed by the plasmids in the
myocyte and Dendritic cells that take up the plasmids. The secreted
chemokine(s) recruit T cells and/or Dendritic cells including those
from mucosal tissues which express the receptor for the chemokine.
The recruited cells, which become antigen specific Effector T cells
and activated Dendritic cells specific for the immunogen, migrate
to the lymph system and mucosal sites, thereby providing a mucosal
immune response and mucosal protection.
[0053] The GENBANK Accession number for the nucleotide sequence for
murine (mouse) form of CTACK (CCL27) is accession number
NM.sub.--011336, which is incorporated herein by reference. The
GENBANK Accession number for the nucleotide sequence for human form
of CTACK is accession number AF082393, which also references
Morales, J., et al., CTACK, a skin-associated chemokine that
preferentially attracts skin-homing memory T cells, Proc. Natl.
Acad. Sci. U.S.A. 96 (25), 14470-14475 (1999) which are each
incorporated herein by reference.
[0054] The nucleic acid sequence for the CTACK mRNA set forth in
Genbank is:
TABLE-US-00001 1 atgaaggggc ccccaacctt ctgcagcctc ctgctgctgt
cattgctcct gagcccagac 61 cctacagcag cattcctact gccacccagc
actgcctgct gtactcagct ctaccgaaag 121 ccactctcag acaagctact
gaggaaggtc atccaggtgg aactgcagga ggctgacggg 181 gactgtcacc
tccaggcttt cgtgcttcac ctggctcaac gcagcatctg catccacccc 241
cagaacccca gcctgtcaca gtggtttgag caccaagaga gaaagctcca tgggactctg
301 cccaagctga attttgggat gctaaggaaa atgggctgaa gcccccaata
gccaaataat 361 aaagcagcat tggataa
[0055] The translation product set forth in Genbank is:
TABLE-US-00002 MKGPPTFCSLLLLSLLLSPDPTAAFLLPPSTACCTQLYRKPLSDKLLRKV
IQVELQEADGDCHLQAFVLHLAQRSICIHPQNPSLSQWFEHQERKLHGTL PKLNFGMLRKMG
[0056] CTACK is normally secreted from skin and interacts with the
CCR10 which is normally produced in immature mucosal Dendretic
cells.
[0057] The GENBANK Accession number for the nucleotide sequence for
murine (mouse) form of TECK (CCL25) is accession number
NM.sub.--009138, which is incorporated herein by reference. The
GENBANK Accession number for the nucleotide sequence for human form
of TECK is accession number HSU86358, which also references Vicari,
A. P., et al., TECK: a novel cc chemokine specifically expressed by
thymic dendritic cells and potentially involved in T cell
development, Immunology 7, 291-301 (1997), which are each
incorporated herein by reference.
[0058] The nucleic acid sequence for the TECK mRNA set forth in
Genbank is:
TABLE-US-00003 1 atgaacctgt ggctcctggc ctgcctggtg gccggcttcc
tgggagcctg ggcccccgct 61 gtccacaccc aaggtgtctt tgaggactgc
tgcctggcct accactaccc cattgggtgg 121 gctgtgctcc ggcgcgcctg
gacttaccgg atccaggagg tgagcgggag ctgcaatctg 181 cctgctgcga
tattctacct ccccaagaga cacaggaagg tgtgtgggaa ccccaaaagc 241
agggaggtgc agagagccat gaagctcctg gatgctcgaa ataaggtttt tgcaaagctc
301 caccacaaca tgcagacctt ccaagcaggc cctcatgctg taaagaagtt
gagttctgga 361 aactccaagt tatcatcatc caagtttagc aatcccatca
gcagcagcaa gaggaatgtc 421 tccctcctga tatcagctaa ttcaggactg
tgagccggct catttctggg ctccatcggc 481 acaggagggg ccggatcttt
ctccgataaa accgtcgccc tacagaccca gctgtcccca 541 cgcctctgtc
ttttgggtca agtcttaatc cctgcacctg agttggtcct ccctctgcac 601
ccccaccacc tcctgcccgt ctggcaactg gaaagaagga gttggcctga ttttaacctt
661 ttgccgctcc ggggaacagc acaatcctgg gcagccagtg gctcttgtag
agaaaactta 721 ggatacctct ctcactttct gtttcttgcc gtccaccccg
ggccatgcca gtgtgtcctc 781 tgggtcccct ccaaaaatct ggtcattcaa
ggatcccctc ccaaggctat gcttttctat 841 aacttttaaa taaaccttgg
ggggtgaatg gaataaaaa
[0059] The translation product set forth in Genbank is:
TABLE-US-00004 MNLWLLACLVAGFLGAWAPAVHTQGVFEDCCLAYHYPIGWAVLRRAWTYR
IQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAMKLLDARNKVFAKL
HHNMQTFQAGPHAVKKLSSGNSKLSSSKFSNPISSSKRNVSLLISANSGL
[0060] TECK is normally secreted in the colon, salivary gland,
bronchi and mammary gland, and interacts with the CCR10 which is
normally produced in immature mucosal Dendritic cells.
[0061] The GENBANK Accession number for the nucleotide sequence for
murine (mouse) form of MEC(CCL28) is accession number
NM.sub.--020279, which is incorporated herein by reference. The
GENBANK Accession number for the nucleotide sequence for human form
of MEC is accession number AF266504, which also references Pan, J.
et al., A novel chemokine ligand for CCR10 and CCR3 expressed by
epithelial cells in mucosal tissues, J. Immunol. 165 (6), 2943-2949
(2000), which are each incorporated herein by reference.
[0062] The nucleic acid sequence for the MEC mRNA set forth in
Genbank is:
TABLE-US-00005 1 tgatcgaaca gcctcacttg tgttgctgtc agtgccagta
gggcaggcag gaatgcagca 61 gagaggactc gccatcgtgg ccttggctgt
ctgtgcggcc ctacatgcct cagaagccat 121 acttcccatt gcctccagct
gttgcacgga ggtttcacat catatttcca gaaggctcct 181 ggaaagagtg
aatatgtgtc gcatccagag agctgatggg gattgtgact tggctgctgt 241
catccttcat gtcaagcgca gaagaatctg tgtcagcccg cacaaccata ctgttaagca
301 gtggatgaaa gtgcaagctg ccaagaaaaa tggtaaagga aatgtttgcc
acaggaagaa 361 acaccatggc aagaggaaca gtaacagggc acatcagggg
aaacacgaaa catacggcca 421 taaaactcct tattagagag tctacagata
aatctacaga gacaattcct caagtggact 481 tggccatgat tggttgtcct
gcatactgat gaaactactg atgtcagctg gtctgaagga 541 ccctaccaga
agctaaatca tcaaagaatg caatttccat atcctaatga ttcaatctcc 601
cttaccctga ccaatcagtg gcccaaattt tccagcccct tgcctcccag aaccccagcc
661 cagaactctt cagagattta agaatctcct cctacctcct gactcagcac
catgtaatca 721 ttaaactctc tgctgcaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaa
[0063] The translation product set forth in Genbank is:
TABLE-US-00006 MQQRGLAIVALAVCAALHASEAILPIASSCCTEVSHHISRRLLERVNMCR
IQRADGDCDLAAVILHVKRRRICVSPHNHTVKQWMKVQAAKKNGKGNVCH
RKKHHGKRNSNRAHQGKHETYGHKTPY
[0064] MEC is normally secreted by epithelial cells in the small
intestine and interacts with the CCR9 which is normally produced in
mucosal Th1 and Th2 cells.
[0065] CTACK protein, TECK protein and MEC protein may each be
isolated or produced routinely by those skilled in the art using
well known techniques and readily available starting materials.
[0066] In some embodiments for which protein is used, for example,
one having ordinary skill in the art can, using well known
techniques, isolates any of CTACK protein, TECK protein and MEC
protein from natural sources using, for example, immuno columns
which contain antibodies that specifically bind to the protein.
Alternatively, the protein may be separated using electrophoresis,
isolated from the electrophoresis matrix and purified by for
example dialysis to yield essentially pure protein. Other well
known protein purification technologies can be employed to produce
isolated, essentially pure protein.
[0067] In some embodiments for which protein is used, for example,
one having ordinary skill in the art can, using well known
techniques, inserts DNA molecules that encode any of CTACK protein,
TECK protein, MEC protein and functional fragments thereof into a
commercially available expression vector for use in well known
expression systems. For example, the commercially available plasmid
pSE420 (Invitrogen, San Diego, Calif.) may be used for production
of protein in E. coli. The commercially available plasmid pYES2
(Invitrogen, San Diego, Calif.) may, for example, be used for
production in S. cerevisiae strains of yeast. The commercially
available MAXBAC.TM. complete baculovirus expression system
(Invitrogen, San Diego, Calif.) may, for example, be used for
production in insect cells. The commercially available plasmid
pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) may, for example,
be used for production in mammalian cells such as Chinese Hamster
Ovary cells. One having ordinary skill in the art can use these
commercial expression vectors and systems or others to produce
protein by routine techniques and readily available starting
materials. (See e.g., Sambrook et al., Molecular Cloning a
Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989) which
is incorporated herein by reference.) Thus, the desired proteins
can be prepared in both prokaryotic and eukaryotic systems,
resulting in a spectrum of processed forms of the protein.
[0068] One having ordinary skill in the art may use other
commercially available expression vectors and systems or produce
vectors using well known methods and readily available starting
materials. Expression systems containing the requisite control
sequences, such as promoters and polyadenylation signals, and
preferably enhancers are readily available and known in the art for
a variety of hosts. See e.g., Sambrook et al., Molecular Cloning a
Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989).
Genetic constructs include the protein coding sequence operably
linked to a promoter that is functional in the cell line into which
the constructs are transfected. Examples of constitutive promoters
include promoters from cytomegalovirus or SV40. Examples of
inducible promoters include mouse mammary leukemia virus or
metallothionein promoters. Those having ordinary skill in the art
can readily produce genetic constructs useful for transfecting with
cells with DNA that encodes protein from readily available starting
materials. Such gene constructs are useful for the production of
CTACK, TECK and MEC and functional fragments thereof. The
expression vector including the DNA that encodes and of CTACK
protein, TECK protein, MEC protein and functional fragments is used
to transform the compatible host which is then cultured and
maintained under conditions wherein expression of the foreign DNA
takes place.
[0069] The protein produced is recovered from the culture, either
by lysing the cells or from the culture medium as appropriate and
known to those in the art. One having ordinary skill in the art
can, using well known techniques, isolate protein that is produced
using such expression systems. The methods of purifying protein
from natural sources using antibodies which specifically bind to a
specific protein as described above may be equally applied to
purifying protein produced by recombinant DNA methodology.
[0070] In addition to isolating proteins from natural sources or
producing proteins by recombinant techniques, automated peptide
synthesizers may also be employed to produce isolated, essentially
pure protein. Such techniques are well known to those having
ordinary skill in the art and are useful if derivatives which have
substitutions not provided for in DNA-encoded protein production.
According to some embodiments of the invention, the combination of
an immunogen and one or more chemokines selected from the group
consisting of: CTACK, TECK, MEC and functional fragments thereof is
delivered to an individual to modulate the activity of the
individual's immune system and thereby enhance the immune response
against the immunogen. When the nucleic acid molecules that encode
the chemokine(s) is taken up by cells of the individual the
nucleotide sequences that encode the chemokine(s) is expressed in
the cells and the proteins are thereby delivered to the individual.
Aspects of the invention provide methods of delivering the coding
sequences of the proteins on a single nucleic acid molecule, in
compositions comprising different nucleic acid molecules that
encodes one or more of the various chemokines, as part of
recombinant vaccines and as part of attenuated vaccines.
[0071] According to some aspects of the present invention,
compositions and methods are provided which prophylactically and/or
therapeutically immunize an individual against an immunogen such as
an allergen, a pathogen or abnormal, disease-related cells. The
vaccine may be any type of vaccine such as, a live attenuated
vaccine, a cell vaccine, a recombinant vaccine or a nucleic, acid
or DNA vaccine. By delivering nucleic acid molecules that encode an
immunogen and one or more chemokines selected from the group
consisting of: CTACK, TECK, MEC and functional fragments thereof
the immune response induced by the vaccine may be modulated. In
particular, mucosal immune responses may be induced even if the
composition is delivered to the individual by a non-mucosal route
of delivery.
[0072] Isolated cDNA that encodes the chemokine proteins are useful
as a starting material in the construction of constructs that can
produce that chemokine protein. Using standard techniques and
readily available starting materials, a nucleic acid molecule that
encodes an chemokine protein may be prepared.
[0073] The present invention relates to compositions for delivering
immunogens and chemokines. Aspects of the present invention relate
to nucleic acid molecules that comprise a nucleotide sequence that
encodes one or more of CTACK, TECK, MEC and functional fragments
thereof operably linked to regulatory elements in combination with
a nucleotide sequence that encodes an immunogen operably linked to
regulatory elements. Aspects of the present invention relate to
compositions which comprise a nucleic acid molecule that comprises
a nucleotide sequence that encodes one or more of CTACK, TECK, MEC
and functional fragments thereof operably linked to regulatory
elements in combination with a nucleic acid molecule that comprises
a nucleotide sequence that encodes an immunogen operably linked to
regulatory elements. Aspects of the present invention relate to
compositions comprising one or more of CTACK protein, TECK protein,
MEC protein and functional fragments thereof in combination with
one or more of an isolated nucleic acid molecule that encodes an
immunogen; a recombinant vaccine comprising a nucleotide sequence
that encodes an immunogen operably linked to regulatory elements; a
live attenuated pathogen; and a killed pathogen. The present
invention further relates to injectable pharmaceutical compositions
that comprise such nucleic acid molecules and compositions.
[0074] The nucleic acid molecules may be delivered using any of
several well known technologies including DNA injection (also
referred to as DNA vaccination), recombinant vectors such as
recombinant adenovirus, recombinant adenovirus associated virus and
recombinant vaccinia.
[0075] DNA vaccines are described in U.S. Pat. Nos. 5,593,972,
5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859,
5,703,055, 5,676,594, and the priority applications cited therein,
which are each incorporated herein by reference. In addition to the
delivery protocols described in those applications, alternative
methods of delivering DNA are described in U.S. Pat. Nos. 4,945,050
and 5,036,006, which are both incorporated herein by reference.
[0076] Routes of administration include, but are not limited to,
intramuscular, intranasally, intraperitoneal, intradermal,
subcutaneous, intravenous, intraarterially, intraocularly and oral
as well as topically, transdermally, by inhalation or suppository
or to mucosal tissue such as by lavage to vaginal, rectal,
urethral, buccal and sublingual tissue. Preferred routes of
administration include intramuscular, intraperitoneal, intradermal
and subcutaneous injection. Genetic constructs may be administered
by means including, but not limited to, traditional syringes,
needleless injection devices, or "microprojectile bombardment gone
guns".
[0077] When taken up by a cell, the genetic construct(s) may remain
present in the cell as a. functioning extrachromosomal molecule
and/or integrate into the cell's chromosomal DNA. DNA may be
introduced into cells where it remains as separate genetic material
in the form of a plasmid or plasmids. Alternatively, linear DNA
that can integrate into the chromosome may be introduced into the
cell. When introducing DNA into the cell, reagents that promote DNA
integration into chromosomes may be added. DNA sequences that are
useful to promote integration may also be included in the DNA
molecule. Alternatively, RNA may be administered to the cell. It is
also contemplated to provide the genetic construct as a linear
minichromosome including a centromere, telomeres and an origin of
replication. Gene constructs may remain part of the genetic
material in attenuated live microorganisms or recombinant microbial
vectors which live in cells. Gene constructs may be part of genomes
of recombinant viral vaccines where the genetic material either
integrates into the chromosome of the cell or remains
extrachromosomal. Genetic constructs include regulatory elements
necessary for gene expression of a nucleic acid molecule. The
elements include: a promoter, an initiation codon, a stop codon,
and a polyadenylation signal. In addition, enhancers are often
required for gene expression of the sequence that encodes the
target protein or the immunomodulating protein. It is necessary
that these elements be operable linked to the sequence that encodes
the desired proteins and that the regulatory elements are operably
in the individual to whom they are administered.
[0078] Initiation codons and stop codon are generally considered to
be part of a nucleotide sequence that encodes the desired protein.
However, it is necessary that these elements are functional in the
individual to whom the gene construct is administered. The
initiation and termination codons must be in frame with the coding
sequence.
[0079] Promoters and polyadenylation signals used must be
functional within the cells of the individual.
[0080] Examples of promoters useful to practice the present
invention, especially in the production of a genetic vaccine for
humans, include but are not limited to promoters from Simian Virus
40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human
Immunodeficiency Virus (MV) such as the BIV Long Terminal Repeat
(LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as
the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous
Sarcoma Virus (RSV) as well as promoters from human genes such as
human Actin, human Myosin, human Hemoglobin, human muscle creatine
and human metalothionein.
[0081] Examples of polyadenylation signals useful to practice the
present invention, especially in the production of a genetic
vaccine for humans, include but are not limited to SV40
polyadenylation signals and LTR polyadenylation signals. In
particular, the SV40 polyadenylation signal that is in pCEP4
plasmid (Invitrogen, San Diego Calif.), referred to as the SV40
polyadenylation signal, is used.
[0082] In addition to the regulatory elements required for DNA
expression, other elements may also be included in the DNA
molecule. Such additional elements include enhancers. The enhancer
may be selected from the group including but not limited to: human
Actin, human Myosin, human Hemoglobin, human muscle creative and
viral enhancers such as those from CMV, RSV and EBV.
[0083] Genetic constructs can be provided with mammalian origin of
replication in order to maintain the construct extrachromosomally
and produce multiple copies of the construct in the cell. Plasmids
pVAX1, pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain
the Epstein Barr virus origin of replication and nuclear antigen
EBNA-1 coding region which produces high copy episomal replication
without integration.
[0084] In some preferred embodiments related to immunization
applications, nucleic acid molecule(s) are delivered which include
nucleotide sequences that encode a target protein, the
immunomodulating protein and, additionally, genes for proteins
which further enhance the immune response against such target
proteins. Examples of such genes are those which encode other
cytokines and lymphokines such as alpha-interferon,
gamma-interferon, platelet derived growth factor (PDGF),
TNF.alpha., TNF.beta., GM-CSF, epidermal growth factor (EGF), IL-1,
IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86 and
IL-15 including IL-15 having the signal sequence deleted and
optionally including the signal peptide from IgE. Other genes which
may be useful include those encoding: MCP-1, MIP-1.alpha., MIP-1p,
IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1,
MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2,
ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18,
CD40, CD40L, vascular growth factor, IL-7, nerve growth factor,
vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1,
p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER,
TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2,
p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,
JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,
TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, O.times.40,
O.times.40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E,
NKG2F, TAP1, TAP2 and functional fragments thereof
[0085] An additional element may be added which serves as a target
for cell destruction if it is desirable to eliminate cells
receiving the genetic construct for any reason. A herpes thymidine
kinase (tk) gene in an expressible form can be included in the
genetic construct. The drug gangcyclovir can be administered to the
individual and that drug will cause the selective killing of any
cell producing tk, thus, providing the means for the selective
destruction of cells with the genetic construct.
[0086] In order to maximize protein production, regulatory
sequences may be selected which are well suited for gene expression
in the cells the construct is administered into. Moreover, codons
may be selected which are most efficiently transcribed in the cell.
One having ordinary skill in the art can produce DNA constructs
that are functional in the cells.
[0087] In some embodiments, gene constructs may be provided to in
order to produce coding sequences for the immunomodulatory proteins
described herein linked to IgE signal peptide.
[0088] One method of the present invention comprises the steps of
administering nucleic acid molecules intramuscularly, intranasally,
intraperitoneally, subcutaneously, intradermally, or topically or
by lavage to mucosal tissue selected from the group consisting of
inhalation, vaginal, rectal, urethral, buccal and sublingual.
[0089] In some embodiments, the nucleic acid molecule is delivered
to the cells in conjunction with administration of a polynucleotide
function enhancer or a genetic vaccine facilitator agent.
Polynucleotide function enhancers are described in U.S. Pat. Nos.
5,593,972, 5,962,428 and International Application Serial Number
PCT/US94/00899 filed Jan. 26, 1994, which are each incorporated
herein by reference. Genetic vaccine facilitator agents are
described in U.S. Ser. No. 021,579 filed Apr. 1, 1994, which is
incorporated herein by reference. The co-agents that are
administered in conjunction with nucleic acid molecules may be
administered as a mixture with the nucleic acid molecule or
administered separately simultaneously, before or after
administration of nucleic acid molecules. In addition, other agents
which may function transfecting agents and/or replicating agents
and/or inflammatory agents and which may be co-administered with a
GVF include growth factors, cytokines and lymphokines such as
a-interferon, gamma-interferon, GM-CSF, platelet derived growth
factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2,
IL-4, IL-6, IL-10, IL-12 and IL-15 as well as fibroblast growth
factor, surface active agents such as immune-stimulating complexes
(ISCOMS), Freunds incomplete adjuvant, LPS analog including
monophosphoryl Lipid A (WL), muramyl peptides, quinone analogs and
vesicles such as squalene and squalene, and hyaluronic acid may
also be used administered in conjunction with the genetic construct
In some embodiments, an immunomodulating protein may be used as a
GVF. In some embodiments, the nucleic acid molecule is provided in
association with PLG to enhance delivery/uptake.
[0090] The pharmaceutical compositions according to the present
invention comprise about 1 nanogram to about 2000 micrograms of
DNA. In some preferred embodiments, pharmaceutical compositions
according to the present invention comprise about 5 nanogram to
about 1000 micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 10 nanograms to about 800
micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 0.1 to about 500
micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 1 to about 350 micrograms
of DNA. In some preferred embodiments, the pharmaceutical
compositions contain about 25 to about 250 micrograms of DNA. In
some preferred embodiments, the pharmaceutical compositions contain
about 100 to about 200 microgram DNA.
[0091] The pharmaceutical compositions according to the present
invention are formulated according to the mode of administration to
be used. In cases where pharmaceutical compositions are injectable
pharmaceutical compositions, they are sterile, pyrogen free and
particulate free. An isotonic formulation is preferably used.
Generally, additives for isotonicity can include sodium chloride,
dextrose, mannitol, sorbitol and lactose. In some cases, isotonic
solutions such as phosphate buffered saline are preferred.
Stabilizers include gelatin and albumin. In some embodiments, a
vasoconstriction agent is added to the formulation.
[0092] Aspects of the present invention relate to compositions
comprising one or more of CTACK protein, TECK protein, MEC protein
and functional fragments thereof in combination with one or more of
an isolated nucleic acid molecule that encodes an immunogen; a
recombinant vaccine comprising a nucleotide sequence that encodes
an immunogen operably linked to regulatory elements; a live
attenuated pathogen; and a killed pathogen. In some embodiments,
the composition comprises an isolated nucleic acid molecule that
encodes an immunogen and/or a recombinant vaccine comprising a
nucleotide sequence that encodes an immunogen operably linked to
regulatory elements; wherein said immunogen is a pathogen antigen,
a cancer-associated antigen or an antigen linked to cells
associated with autoimmune diseases. In some embodiments, the
immunogen is an HIV antigen such as HIV gag. In some embodiments,
the composition further comprises a nucleic acid molecule that
encodes one or more proteins of selected from the group consisting
of: CTACK, TECK, MEC and functional fragments thereof is
administered to the individual. In some embodiments, the
composition is an injectable pharmaceutical composition.
[0093] According to some embodiments of the invention, methods of
inducing immune responses, including methods of inducing mucosal
immune responses, against an immunogen are provided by delivering a
combination of the immunogen and one or more of CTACK, TECK, MEC
and functional fragments thereof to an individual. The vaccine may
be a live attenuated vaccine, a cell vaccine, a recombinant vaccine
or a nucleic acid or DNA vaccine. In some embodiments, methods of
inducing an immune response in individuals against an immunogen,
including methods of inducing mucosal immune responses, comprise
administering to the individual one or more of CTACK protein, TECK
protein, MEC protein and functional fragments thereof in
combination with an isolated nucleic acid molecule that encodes an
immunogen; and/or a recombinant vaccine that encodes an immunogen
and/or a subunit vaccine that comprises an immunogen and/or a live
attenuated vaccine and/or a killed vaccine. The one or more of
CTACK protein, TECK protein, MEC protein and functional fragments
thereof may be administered prior to, simultaneously with or after
administration of the isolated nucleic acid molecule that encodes
an immunogen; and/or recombinant vaccine that encodes an immunogen
and/or subunit vaccine that comprises an immunogen and/or live
attenuated vaccine and/or killed vaccine. In some embodiments, an
isolated nucleic acid molecule that encodes one or more proteins of
selected from the group consisting of: CTACK, TECK, MEC and
functional fragments thereof is administered to the individual.
[0094] In some embodiments, the immunogen is a pathogen antigen, a
cancer-associated antigen or an antigen linked to cells associated
with autoimmune diseases. In some embodiments, the immunogen is an
HIV antigen such as HIV gag.
[0095] The present invention is useful to elicit enhanced immune
responses against a target protein, i.e. proteins specifically
associated with pathogens, allergens or the individual's own
"abnormal" cells. The present invention is useful to immunize
individuals against pathogenic agents and organisms such that an
immune response against a pathogen protein provides protective
immunity against the pathogen. The present invention is useful to
combat hyperproliferative diseases and disorders such as cancer by
eliciting an immune response against a target protein that is
specifically associated with the hyperproliferative cells. The
present invention is useful to combat autoimmune diseases and
disorders by eliciting an immune response against a target protein
that is specifically associated with cells involved in the
autoimmune condition.
[0096] According to some aspects of the present invention, DNA or
RNA that encodes a target protein and immunomodulating proteins is
introduced into the cells of tissue of an individual where it is
expressed, thus producing the encoded proteins. The DNA or RNA
sequences encoding the target protein and one or both
immunomodulating proteins are linked to regulatory elements
necessary for expression in the cells of the individual. Regulatory
elements for DNA expression include a promoter and a
polyadenylation signal. In addition, other elements, such as a
Kozak region, may also be included in the genetic construct.
[0097] In some embodiments, expressible forms of sequences that
encode the target protein and expressible forms of sequences that
encode both immunomodulating proteins are found on the same nucleic
acid molecule that is delivered to the individual.
[0098] In some embodiments, expressible forms of sequences that
encode the target protein occur on a separate nucleic acid molecule
from the nucleic acid molecules that contain expressible forms of
sequences that encode one or more immunomodulatory proteins. In.
some embodiments, expressible forms of sequences that encode the
target protein and expressible forms of sequences that encode one
or more of the immunomodulatory proteins occur on a one nucleic
acid molecule that is separate from the nucleic acid molecule that
contain expressible forms of sequences that encode one or more of
the immunomodulating proteins. Multiple different nucleic acid
molecules can be produced and delivered according to the present
invention and delivered to the individual. For example, in some
embodiments, expressible forms of sequences that encode the target
protein occur on separate nucleic acid molecule from the nucleic
acid molecules that contain expressible forms of sequences that
encode one or more of the two immunomodulating proteins which occur
on separate nucleic acid molecule from the nucleic acid molecules
that contain expressible forms of sequences that encode one or more
immunomodulating proteins. In such cases, all three molecules are
delivered to the individual.
[0099] The nucleic acid molecule(s) may be provided as plasmid DNA,
the nucleic acid molecules of recombinant vectors or as part of the
genetic material provided in an attenuated vaccine or cell vaccine.
Alternatively, in some embodiments, the target protein and/or
wither or both immunomodulating proteins maybe delivered as a
protein in addition to the nucleic acid molecules that encode them
or instead of the nucleic acid molecules which encode them.
[0100] Genetic constructs may comprise a nucleotide sequence that
encodes a target protein or an immunomodulating protein operably
linked to regulatory elements needed for gene expression. According
to the invention, combinations of gone constructs that include one
that comprises an expressible form of the nucleotide sequence that
encodes a target protein and one that includes an expressible form
of the nucleotide sequence that encodes an immunomodulating protein
are provided. Incorporation into a living cell of the DNA or RNA
molecule(s) that include the combination of gene constructs results
in the expression of the DNA or RNA and production of the target
protein and one or more immunomodulating proteins. An enhanced
immune response against the target protein results.
[0101] The present invention may be used to immunize an individual
against all pathogens such as viruses, prokaryote and pathogenic
eukaryotic organisms such as unicellular pathogenic organisms and
multicellular parasites. The present invention is particularly
useful to immunize an individual against those pathogens which
infect cells and which are not encapsulated such as viruses, and
prokaryote such as gonorrhea, listeria and shigella. In addition,
the present invention is also useful to immunize an individual
against protozoan pathogens that include a stage in the life cycle
where they are intracellular pathogens. Table 1 provides a listing
of some of the viral families and genera for which vaccines
according to the present invention can be made. DNA constructs that
comprise DNA sequences that encode the peptides that comprise at
least an epitope identical or substantially similar to an epitope
displayed on a pathogen antigen such as those antigens listed on
the tables are useful in vaccines. Moreover, the present invention
is also useful to immunize an individual against other pathogens
including prokaryotic and eukaryotic protozoan pathogens as well as
multicellular parasites such as those listed on Table 2.
[0102] In order to produce a genetic vaccine to protect against
pathogen infection, genetic material that encodes immunogenic
proteins against which a protective immune response can be mounted
must be included in a genetic construct as the coding sequence for
the target. Whether the pathogen infects intracellularly, for which
the present invention is particularly useful, or extracellularly,
it is unlikely that all pathogen antigens will elicit a protective
response. Because DNA and RNA are both relatively small and can be
produced relatively easily, the present invention provides the
additional advantage of allowing for vaccination with multiple
pathogen antigens. The genetic construct used in the genetic
vaccine can include genetic material that encodes many pathogen
antigens. For example, several viral genes may be included in a
single construct thereby providing multiple targets.
[0103] Tables 1 and 2 include lists of some of the pathogenic
agents and organisms for which genetic vaccines can be prepared to
protect an individual from infection by them. In some preferred
embodiments, the methods of immunizing an individual against a
pathogen are directed against HIV, HSV, HCV, WNV or HBV.
[0104] Another aspect of the present invention provides a method of
conferring a protective immune response against hyperproliferating
cells that are characteristic in hyperproliferative diseases and to
a method of treating individuals suffering from hyperproliferative
diseases. Examples of hyperproliferative diseases include all forms
of cancer and psoriasis.
[0105] It has been discovered that introduction of a genetic
construct that includes a nucleotide sequence which encodes--an
immunogenic "hyperproliferating cell"--associated protein into the
cells of an individual results in the production of those proteins
in the vaccinated cells of an individual. To immunize against
hyperproliferative diseases, a genetic construct that includes a
nucleotide sequence that encodes a protein that is associated with
a hyperproliferative disease is administered to an individual.
[0106] In order for the hyperproliferative-associated protein to be
an effective immunogenic target, it must be a protein that is
produced exclusively or at higher levels in hyperproliferative
cells as compared to normal cells. Target antigens include such
proteins, fragments thereof and peptides; which comprise at least
an epitope found on such proteins. In some cases, a
hyperproliferative-associated protein is the product of a mutation
of a gene that encodes a protein. The mutated gene encodes a
protein that is nearly identical to the normal protein except it
has a slightly different amino acid sequence which results in a
different epitope not found on the normal protein. Such target
proteins include those which are proteins encoded by oncogenes such
as myb, myc, fyn, and the translocation gene bcr/abl, ras, src,
P53, neu, trk and EGRF. In addition to oncogene products as target
antigens, target proteins for anti-cancer treatments and protective
regimens include variable regions of antibodies made by B cell
lymphomas and variable regions of T cell receptors of T cell
lymphomas which, in some embodiments, are also used target antigens
for autoimmune disease. Other tumor-associated proteins can be used
as target proteins such as proteins that are found at higher levels
in tumor cells including the protein recognized by monoclonal
antibody 17-IA and folate binding proteins or PSA.
[0107] While the present invention may be used to immunize an
individual against one or more of several forms of cancer, the
present invention is particularly useful to prophylactically
immunize an individual who is predisposed to develop a particular
cancer or who has had cancer and is therefore susceptible to a
relapse. Developments in genetics and technology as well as
epidemiology allow for the determination of probability and risk
assessment for the development of cancer in individual. Using
genetic screening and/or family health histories, it is possible to
predict the probability a particular individual has for developing
any one of several types of cancer.
[0108] Similarly, those individuals who have already developed
cancer and who have been treated to remove the cancer or are
otherwise in remission are particularly susceptible to relapse and
reoccurrence. As part of a treatment regimen, such individuals can
be immunized against the cancer that they have been diagnosed as
having had in order to combat a recurrence. Thus, once it is known
that an individual has had a type of cancer and is at risk of a
relapse, they can be immunized in order to prepare their immune
system to combat any future appearance of the cancer.
[0109] The present invention provides a method of treating
individuals suffering from hyperproliferative diseases. In such
methods, the introduction of genetic constructs serves as an
immunotherapeutic, directing and promoting the immune system of the
individual to combat hyperproliferative cells that produce the
target protein.
[0110] The present invention provides a method of treating
individuals suffering from autoimmune diseases and disorders by
conferring a broad based protective immune response against targets
that are associated with autoimmunity including cell receptors and
cells which produce "self"-directed antibodies.
[0111] T cell mediated autoimmune diseases include Rheumatoid
arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome,
sarcoidosis, insulin dependent diabetes mellitus (1DDM), autoimmune
thyroiditis, reactive arthritis, ankylosing spondylitis,
scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis,
Wegener's granulomatosis, Crohn's disease and ulcerative colitis.
Each of these diseases is characterized by T cell receptors that
bind to endogenous antigens and initiate the inflammatory cascade
associated with autoimmune diseases. Vaccination against the
variable region of the T cells would elicit an immune response
including CTLs to eliminate those T cells.
[0112] In RA, several specific variable regions of T cell receptors
(TCRs) that are involved in the disease have been characterized.
These TCRs include V.beta.-3, V.beta.-14, 20 V.beta.-17 and
V.alpha.-17. Thus, vaccination with a DNA construct that encodes at
least one of these proteins will elicit an immune response that
will target T cells involved in RA. See: Howell, M. D., et al.,
1991 Proc. Nat. Acad. Sci. USA 88:10921-10925; Piliard, X., et al,
1991 Science 253:325-329; Williams, W. V., et al., 1992 J Clin.
Invest. 90:326-333; each of which is incorporated herein by
reference. In MS, several specific variable regions of TCRs that
are involved in the disease have been characterized. These TCRs
include VfP and V.alpha.-10. Thus, vaccination with a DNA construct
that encodes at least one of these proteins will elicit an immune
response that will target T cells involved in MS. See:
Wucherpfennig, K. W., et al., 1990 Science 248:1016-1019;
Oksenberg, J. R., et al, 1990 Nature 345:344-346; each of which is
incorporated herein by reference.
[0113] In scleroderma, several specific variable regions of TCRs
that are involved in the disease have been characterized. These
TCRs include V.beta.-6, V.beta.-g, V.beta.-14 and V.alpha.-16,
V.alpha.-3C, V.alpha.-7, V.alpha.-14, V.alpha.-15, V.alpha.-16,
V.alpha.-28 and V.alpha.-12. Thus, vaccination with a DNA construct
that encodes at least one of these proteins will elicit an immune
response that will target T cells involved in scleroderma.
[0114] In order to treat patients suffering from a T cell mediated
autoimmune disease, particularly those for which the variable
region of the TCR has yet to be characterized, a synovial biopsy
can be performed. Samples of the T cells present can be taken and
the variable region of those TCRs identified using standard
techniques. Genetic vaccines can be prepared using this
information.
[0115] B cell mediated autoimmune diseases include Lupus (SLE),
Grave's disease, myasthenia gravis, autoimmune hemolytic anemia,
autoimmune thrombocytopenia, asthma, cryoglobulineinia, primary
biliary sclerosis and pernicious anemia. Each of these diseases is
characterized by antibodies that bind to endogenous antigens and
initiate the inflammatory cascade associated with autoimmune
diseases. Vaccination against the variable region of antibodies
would elicit an immune response including CTLs to eliminate those B
cells that produce the antibody.
[0116] In order to treat patients suffering from a B cell mediated
autoimmune disease, the variable region of the antibodies involved
in the autoimmune activity must be identified. A biopsy can be
performed and samples of the antibodies present at a site of
inflammation can be taken. The variable region of those antibodies
can be identified using standard techniques. Genetic vaccines can
be prepared using this information.
[0117] In the case of SLE, one antigen is believed to be DNA. Thus,
in patients to be immunized against SLE, their sera can be screened
for anti-DNA antibodies and a vaccine can be prepared which
includes DNA constructs that encode the variable region of such
anti-DNA antibodies found in the sera.
[0118] Common structural features among the variable regions of
both TCRs and antibodies are well known. The DNA sequence encoding
a particular TCR or antibody can generally be found following well
known methods such as those described in Kabat, et al 1987 Sequence
of Proteins of Immunological Interest U.S. Department of Health and
Human Services, Bethesda Md., which is incorporated herein by
reference. In addition, a general method for cloning functional
variable regions from antibodies can be found in Chaudhary, V. K.,
et al, 1990 Proc. Natl. Acad Sci. USA 87:1066, which is
incorporated herein by reference.
[0119] In addition to using expressible forms of immunomodulating
protein coding sequence to improve genetic vaccines, the present
invention relates to improved attenuated live vaccines, improved
killed vaccines and improved vaccines that use recombinant vectors
to deliver foreign genes that encode antigens and well as subunit
and glycoprotein vaccines. Examples of attenuated live vaccines,
those using recombinant vectors to deliver foreign antigens,
subunit vaccines and glycoprotein vaccines are described in U.S.
Pat. Nos. 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209;
5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424;
5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668;
5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734;
5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309;
5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and
6,589,529, which are each incorporated herein by reference. Gene
constructs are provided which include the nucleotide sequence that
encodes an immunomodulating protein is operably linked to
regulatory sequences that can function in the vaccine to effect
expression. The gene constructs are incorporated in the attenuated
live vaccines and recombinant vaccines to produce improved vaccines
according to the invention.
[0120] The present invention provides an improved method of
immunizing individuals that comprises the step of delivering gene
constructs to the cells of individuals as part of vaccine
compositions which include are provided which include DNA vaccines,
attenuated live vaccines and recombinant vaccines. The gene
constructs comprise a nucleotide sequence that encodes an
immunomodulating protein and that is operably linked to regulatory
sequences that can function in the vaccine to effect expression.
The improved vaccines result in an enhanced cellular immune
response. In some embodiments, the CTAC, TECK, MEC or functional
fragment thereof may be included as a protein in combination with
or part of the viral composition or delivered separately. In some
embodiments, one or more of the CTAC, TECK, MEC or functional
fragment thereof is part of the structure of the vaccine protein,
vaccine glycoprotein vaccine vector or attenuated or killed
pathogen. For example, fusion proteins comprising the CTAC, TECK,
MEC or functional fragment may be part of the structural proteins
that make up the virus, organism or part of a fusion protein that
may include the glycoprotein or protein subunit. In other examples,
the CTAC, TECK, MEC or functional fragment is an intact protein
which is complexed with DNA vaccine, glycoprotein or protein
subunit or part of the viral vector or organism. In some
embodiments, compositions comprise one or more of the CTAC, TECK,
MEC or functional fragment thereof is a protein which is part of
the structure of the vaccine vector or attenuated or killed
pathogen. In some embodiments, compositions are provided in which
the one or more CTAC, TECK, MEC or functional fragment thereof is
combined with a DNA vaccine, glycoprotein or protein subunit or
part of the viral vector or organism. In some embodiments, nucleic
acid molecules encoding the one or more CTAC, TECK, MEC or
functional fragment thereof is combined with a DNA vaccine,
glycoprotein or protein subunit or part of the viral vector or
organism. In some embodiments, nucleic acid molecules encoding the
one or more CTAC, TECK, MEC or functional fragment thereof is a
portion of a DNA vaccine, or part of the genome of a viral vector
or organism.
EXAMPLE
[0121] Plasmids were constructed using the pVax1 (Invitrogen)
backbone, inserting coding sequences for murine CTACK, murine TECK
or murine MEC into the multiple cloning region of pVax1, placing
the coding sequences under the regulatory control of the
cytomegalovirus promoter and bovine growth hormone polyadenylation
signal. (FIG. 2) Inserts were confirmed by restriction digest
conformation (FIG. 2). RD cells were transfected with vector or
construct (pCTACK or pTECK) to confirm that the coding sequences
would be expressed. Testing was not done to confirm pMEC expression
as antibodies against murine MEC were not available. The data shown
in FIG. 2 confirms that CTACK and TECK was expressed.
[0122] Balb/C mice (N=4 per group) were immunized with vector only,
plasmid pHIV-1 gag only (pVax-1 with HIV-1 gag inserted at multiple
cloning site), plasmid pHIV-1 gag+pCTACK, plasmid pHIV-1 gag+pTECK
and plasmid pHIV-1 gag+pMEC on days 0 and 14. Mice were sacrificed
at day 21 and spleens were removed for immune analysis (FIG. 3).
Plates containing 2.times.10.sup.5 splenocytes in triplicate were
incubated with medium only, medium+concavalin A or medium+pools of
Gag peptides. Pools of Gag peptide contained 15 amino acid
fragments of Gag each having 11 amino acid overlap with another
peptide in the pool. Pools were divided into three groups spanning
the 122 amino acid length of gag.
[0123] ELISpot testing was done to measure IFN.gamma. production
from cells from naive, vector only, plasmid pHIV-1 gag only (pVax-1
with HIV-1 gag inserted at multiple cloning site), plasmid pHIV-1
gag+pCTACK, plasmid pHIV-1 gag+pTECK and plasmid pHIV-1 gag+pMEC
that were exposed to HIV-1 Gag peptide pool. Production of
IFN.gamma. in response to Gag indicates that the cells activated by
Gag in an antigen specific manner.
[0124] Flow cytometry was performed on cells from naive, vector
only, plasmid pHIV-1 gag only (pVax-1 with HIV-1 gag inserted at
multiple cloning site), plasmid pHIV-1 gag+pCTACK, plasmid pHIV-1
gag+pTECK and plasmid pHIV-1 gag+pMEC that were exposed to HIV-1
Gag peptide pool staining for the markers CD3+, CD8+, CD4+ and
CD107+. CD3+ selects for T cells. Most CTLs are CD8+ although some
may be CD4+. CD107+ (also referred to as Lamp-1) is a marker for
antigen specific activated CTLs.
[0125] FIG. 4 shows data for MEC. IFN.gamma. ELISpot data shows a
small increase in IFN.gamma. spots. No increase was observed in
CD3+/CD8+/CD107+ and CD3+/CD4+/CD107+ although it is not clear
whether this was due to a lack of expression of MEC, insufficient
expression or a lack of activity.
[0126] FIG. 5 shows data for CTACK and TECK. IFN.gamma. ELISpot
data shows a significant increase in IFN.gamma. spots in groups
which included pCTACK or pTECK. Results from CD3+/CD8+/CD107+ show
a significant increase in percent positive populations in groups
which included pCTACK or pTECK. No increase was seen in
CD3+/CD4+/CD107+ data.
[0127] TECK, which is a chemokine that attracts T cells from the
mucosal environment, is the most potent driver of CD8 CTL responses
among the selected set of mucosal relevant chemokines tested.
However CTACK, a chemokine which should attract DC of mucosal
origin, is very interesting as well. As shown in FIG. 6, IFN.gamma.
ELISpot data using different pools show that C-TACK is stimulated
by one or more peptides in pool 3 as well as pool 2. TECK shows
stimulation by one or more Gag peptides in pool 2 which is
consistent with data from pHIV-1 gag only as well as observations
seen elsewhere. The presence of CTACK appeared to render the cells
stimulated by an additional, different epitope.
[0128] MIP1B ELISA were done to determine if MIP1B would be
produced when cells from the various groups are stimulated by R10
or HIV-1 gag p55. FIG. 7 shows that in response to stimulation by
Gag p55 MIP1B is produced by cells from each of the groups pHIV-1
gag+pCTACK, pHIV-1 gag+pTECK, and pHIV-1 gag+pMEC relative to that
produced by the control groups pHIV-1 gag only or pVax1 only. The
amount of MIP1B produced in by cells from the pHIV-1 gag+pTECK
group was significantly greater than the amount produced by either
the pHIV-1 gag+pCTACK group or the pHIV-1 gag+pMEC group.
TABLE-US-00007 TABLE 1 Picornavirus Family Genera: Rhinoviruses:
(Medical) responsible for - 50% cases of the common cold.
Etheroviruses: (Medical) includes polioviruses, coxsackieviruses,
echoviruses, and human enteroviruses such as hepatitis A virus.
Apthoviruses: (Veterinary) these are the foot and mouth disease
viruses. Target antigens: VP1, VP2, VP3, VP4, VPG Calcivirus Family
Genera: Norwalk Group of Viruses: (Medical) these viruses are an
important causative agent of epidemic gastroenteritis. Togavirus
Family Genera: Alphaviruses: (Medical and Veterinary) examples
include Senilis viruses, RossRiver virus and Eastern & Western,
Equine encephalitis. Reovirus: (Medical) Rubella virus.
Flariviridue Family Examples include: (Medical) dengue, yellow
fever, Japanese encephalitis, St. Louis encephalitis and tick borne
encephalitis viruses. West Nile virus (Genbank NC001563, AF533540,
AF404757, AF404756, AF404755, AF404754, AF404753, AF481864, M12294,
AF317203, AF196835, AF260969, AF260968, AF260967, AF206518 and
AF202541) Representative Target antigens: E NS5 C Hepatitis C
Virus: (Medical) these viruses are not placed in a family yet but
are believed to be either a togavirus or a flavivirus. Most
similarity is with togavirus family. Coronavirus Family: Infectious
bronchitis virus (poultry) (Medical and Porcine transmissible
gastroenteric virus (pig) Veterinary) Porcine hemaglutinating
encephalomyelitis virus (pig) Feline infectious peritonitis virus
(cats) Feline enteric coronavirus (cat) Canine coronavirus (dog)
SARS associated coronavirus The human respiratory coronaviruses
cause ~40 cases of common cold. EX. 224E, OC43 Note - coronaviruses
may cause non-A, B or C hepatitis Target antigens: E1 - also called
M or matrix protein E2 - also called S or Spike protein E3 - also
called BE or hemagglutin- elterose glycoprotein (not present in all
coronaviruses) N - nucleocapsid Rhabdovirus Family Genera:
Vesiliovirus Lyssavirus: (medical and veterinary) rabies Target
antigen: G protein N protein Filoviridue Family: Hemorrhagic fever
viruses such as (Medical) Marburg and Ebola virus Paramyxovirus
Genera: Paramyxovirus: (Medical and Veterinary) Family: Mumps
virus, New Castle disease virus (important pathogen in chickens)
Morbillivirus: (Medical and Veterinary) Measles, canine distemper
Pneuminvirus: (Medical and Veterinary) Respiratory syncytial virus
Orthomyxovirus The Influenza virus Family (Medical) Bungavirus
Family Genera: Bungavirus: (Medical) California encephalitis, LA
Crosse Phlebovirus: (Medical) Rift Valley Fever Hantavirus:
Puremala is a hemahagin fever virus Nairvirus (Veterinary) Nairobi
sheep disease Also many unassigned bungaviruses Arenavirus Family
LCM, Lassi fever virus (Medical) Reovirus Family Genera: Reovirus:
a possible human pathogen Rotavirus: acute gastroenteritis in
children Orbiviruses: (Medical and Veterinary) Colorado Tick fever,
Lebombo (humans) equine encephalosis, blue tongue Retrovirus Family
Sub-Family: Oncorivirinal: (Veterinary) (Medical) feline leukemia
virus, HTLVI and HTLVII Lentivirinal: (Medical and Veterinary) HIV,
feline immunodeficiency virus, equine infections, anemia virus
Spumavirinal Papovavirus Family Sub-Family: Polyomaviruses:
(Medical) BKU and JCU viruses Sub-Family: Papillomavirus: (Medical)
many viral types associated with cancers or malignant progression
of papilloma. Adenovirus (Medical) EX AD7, ARD., O.B. - cause
respiratory disease - some adenoviruses such as 275 cause enteritis
Parvovirus Family Feline parvovirus: causes feline enteritis
(Veterinary) Feline panleucopeniavirus Canine parvovirus Porcine
parvovirus Herpesvirus Family Sub-Family: alpha- Genera:
Simplexvirus (Medical) herpesviridue HSVI (Genbank X14112,
NC001806), HSVII (NC001798) Varicellovinis: (Medical Veterinary)
pseudorabies - varicella zoster Sub-Family - beta- Genera:
Cytomegalovirus (Medical) herpesviridue HCMV Muromegalovirus
Sub-Family. Genera: Lymphocryptovirus (Medical) Gamma- EBV -
(Burkitts lympho) herpesviridue Rhadinovirus Poxvirus Family
Sub-Family: Genera: Variola. (Smallpox) Chordopoxviridue Vaccinia
(Cowpox) (Medical - Parapoxivirus - Veterinary Veterinary)
Auipoxvirus - Veterinary Capripoxvirus Leporipoxvirus Suipoxviru's
Sub-Family: Entemopoxviridue Hepadnavirus Hepatitis B virus Family
Unclassified Hepatitis delta virus
TABLE-US-00008 TABLE 2 Bacterial pathogens Pathogenic gram-positive
cocci include: pneurnococcal; staphylococcal; and streptococcal.
Pathogenic gram-negative cocci include: meningococcal; and
gonococcal. Pathogenic enteric gram-negative bacilli include:
enterobacteriaceae; pseudomonas, acinetobacteria and eikenella,
melioidosis;, sahnonella; shigellosis; hemophilus; chancroid;
brucellosis; tularemia; yersinia (pasteurella); streptobacillus
mortiliformis and spirillum; listeria monocytogenes; erysipelothrix
rhusiopathiae; diphtheria, cholera, anthrax; donovanosis (granuloma
inguinale); and bartonellosis. Pathogenic anaerobic bacteria
include: tetanus; botulism; other clostridia; tuberculosis;
leprosy; and other mycobacteria. Pathogenic spirochetal diseases
include: syphilis; - treponematoses: yaws, pinta and endemic
syphilis; and leptospirosis. Other infections caused by higher
pathogen bacteria and pathogenic fungi include: actinomycosis;
.nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and
coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis;
sporotrichosis; paracoccidiodomycosis, petriellidiosis,
torulopsosis, mycetoma, and chromomycosis; and dermatophytosis.
Rickettsial infections include rickettsial and rickettsioses.
Examples of mycoplasma and chlarnydial infections include:
mycoplasma pneurnoniae; lymphogranuloma venereum; psittacosis; and
perinatal chlamydial infections. Pathogenic eukaryotes Pathogenic
protozoans and helminths and infections thereby include: amebiasis;
malaria; leishmaniasis; trypanosomiasis; toxoplasmosis;
pneurnocystis carinii; babesiosis; giardiasis; trichinosis;
filariasis; schistosomiasis; nematodes; trematodes or flukes; and
cestode (tapeworm) infections.
* * * * *