U.S. patent application number 11/629610 was filed with the patent office on 2007-08-16 for plasmid having three complete transcriptional units and immunogenic compositions for inducing an immune response to hiv.
Invention is credited to Michael Egan, John H. Eldridge, Zimra Israel, Maninder K. Sidhu.
Application Number | 20070190031 11/629610 |
Document ID | / |
Family ID | 35266872 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070190031 |
Kind Code |
A1 |
Sidhu; Maninder K. ; et
al. |
August 16, 2007 |
Plasmid having three complete transcriptional units and immunogenic
compositions for inducing an immune response to hiv
Abstract
The invention provides a DNA plasmid comprising: (a) a first
transcriptional unit comprising a nucleotide sequence that encodes
a first polypeptide operably linked to regulatory elements
including a first promoter and a first polyadenylation signal; (b)
a second transcriptional unit comprising a nucleotide sequence that
encodes a second polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal;
(c) a third transcriptional unit comprising a nucleotide sequence
that encodes a third polypeptide operably linked to regulatory
elements including a third promoter and a third polyadenylation
signal; and wherein said first, said second and said third
promoters are each derived from different transcriptional units;
and wherein said first, said second and said third polyadenylation
signals are each derived from different transcriptional units. The
invention further relates to immunogenic compositions for inducing
an immune response to HIV comprising combinations of two, three, or
four plasmids, where each plasmid is expressing a defined antigen,
which may be a single antigen or a fusion of two or three
antigens.
Inventors: |
Sidhu; Maninder K.; (New
City, NY) ; Eldridge; John H.; (Somers, NY) ;
Egan; Michael; (Washingtonville, NY) ; Israel;
Zimra; (New York, NY) |
Correspondence
Address: |
WYETH;PATENT LAW GROUP
5 GIRALDA FARMS
MADISON
NJ
07940
US
|
Family ID: |
35266872 |
Appl. No.: |
11/629610 |
Filed: |
June 15, 2005 |
PCT Filed: |
June 15, 2005 |
PCT NO: |
PCT/US05/21168 |
371 Date: |
December 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60580438 |
Jun 17, 2004 |
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60624983 |
Nov 3, 2004 |
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60662275 |
Mar 16, 2005 |
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Current U.S.
Class: |
424/93.2 ;
424/208.1; 435/456 |
Current CPC
Class: |
A61K 2039/53 20130101;
C12N 15/85 20130101; C12N 2740/16322 20130101; C12N 2830/00
20130101; A61K 39/12 20130101; C12N 2800/107 20130101; C12N
2740/16122 20130101; A61K 39/21 20130101; C12N 2740/16134 20130101;
A61K 2039/545 20130101; A61P 37/04 20180101; A61K 2039/55538
20130101; C12N 2840/20 20130101; C12N 2840/60 20130101; A61K
2039/54 20130101; C12N 2740/16222 20130101; C07K 14/005 20130101;
C12N 2740/16334 20130101; C12N 2740/16234 20130101; A61P 31/18
20180101 |
Class at
Publication: |
424/093.2 ;
435/456; 424/208.1 |
International
Class: |
A61K 39/21 20060101
A61K039/21; A61K 48/00 20060101 A61K048/00; C12N 15/867 20060101
C12N015/867 |
Claims
1. A DNA plasmid comprising: (a) a first transcriptional unit
comprising a nucleotide sequence that encodes a first polypeptide
operably linked to regulatory elements including a first promoter
and a first polyadenylation signal; (b) a second transcriptional
unit comprising a nucleotide sequence that encodes a second
polypeptide operably linked to regulatory elements including a
second promoter and a second polyadenylation signal; (c) a third
transcriptional unit comprising a nucleotide sequence that encodes
a third polypeptide operably linked to regulatory elements
including a third promoter and a third polyadenylation signal;
wherein said first, said second and said third promoters are each
derived from different transcriptional units; wherein said first,
said second and said third polyadenylation signals are each derived
from different transcriptional units; wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
second transcriptional unit, or wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
third transcriptional unit, or wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
second transcriptional unit and in the opposite direction from the
direction of transcription of said third transcriptional unit.
2-3. (canceled)
4. The plasmid of claim 1, wherein said first, second and third
promoters are selected from the group consisting of human
cytomegalovirus (HCMV) immediate early promoter, the simian
cytomegalovirus (SCMV) promoter, the murine cytomegalovirus (MCMV)
promoter, the herpes simplex virus (HSV) latency-associated
promoter-1 (LAP1), Simian virus 40 promoter, human elongation
factor 1 alpha promoter, and the human muscle cell specific desmin
promoter.
5. The plasmid of claim 1, wherein said first, second and third
polyadenylation signals are selected from the group consisting of
rabbit beta-globin poly(A) signal, synthetic polyA, HSV Thymidine
kinase poly A, Human alpha globin poly A, SV40 poly A, human beta
globin poly A, polyomavirus poly A, and Bovine growth hormone poly
A.
6-25. (canceled)
26. The plasmid of claim 1, wherein said plasmid is less than about
15 kilobase pairs in total size.
27-29. (canceled)
30. An immunogenic composition for inducing an immune response to
selected antigens in a vertebrate host, said immunogenic
composition comprising: (a) a DNA plasmid comprising a (i) a first
transcriptional unit comprising a nucleotide sequence that encodes
a first polypeptide operably linked to regulatory elements
including a first promoter and a first polyadenylation signal; (ii)
a second transcriptional unit comprising a nucleotide sequence that
encodes a second polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal;
(iii) a third transcriptional unit comprising a nucleotide sequence
that encodes a third polypeptide operably linked to regulatory
elements including a third promoter and a third polyadenylation
signal; wherein said first, second and third promoters are each
derived from different transcriptional units; wherein said first,
second and third polyadenylation signals are each derived from
different transcriptional units; wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
second transcriptional unit, or wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
third transcriptional unit, or wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
second transcriptional unit and in the opposite direction from the
direction of transcription of said third transcriptional unit and
(b) at least one of a pharmaceutically acceptable diluent,
adjuvant, carrier or transfection facilitating agent.
31. The immunogenic composition of claim 30, wherein said
transfection facilitating agent is bupivacaine.
32-36. (canceled)
37. A method of immunizing a vertebrate host against selected
antigens comprising administering to said vertebrate host an
immunogenic composition comprising: (a) a DNA plasmid comprising a
(i) a first transcriptional unit comprising a nucleotide sequence
that encodes a first polypeptide operably linked to regulatory
elements including a first promoter and a first polyadenylation
signal; (ii) a second transcriptional unit comprising a nucleotide
sequence that encodes a second polypeptide operably linked to
regulatory elements including a second promoter and a second
polyadenylation signal; (iii) a third transcriptional unit
comprising a nucleotide sequence that encodes a third polypeptide
operably linked to regulatory elements including a third promoter
and a third polyadenylation signal; wherein said first, second and
third promoters are each derived from different transcriptional
units; wherein said first, second and third polyadenylation signals
are each derived from different transcriptional units; wherein the
direction of transcription for said first transcriptional unit is
in the opposite direction from the direction of transcription of
said second transcriptional unit, or wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
third transcriptional unit, or wherein the direction of
transcription for said first transcriptional unit is in the
opposite direction from the direction of transcription of said
second transcriptional unit and in the opposite direction from the
direction of transcription of said third transcriptional unit and
(b) at least one of a pharmaceutically acceptable diluent,
adjuvant, carrier or transfection facilitating agent.
38. The method of claim 7, wherein said immunogenic composition is
administered to a mammal using in vivo electroporation.
39. The method of claim 8, wherein said electroporation involves
electrically stimulating the muscle with an electrical current
having a field strength in the range of from about 25 V/cm to about
800 V/cm.
40-42. (canceled)
43. An immunogenic composition for inducing an immune response to
human immunodeficiency virus (HIV) in a vertebrate host, said
immunogenic composition comprising: (a) a first DNA plasmid
comprising a single transcriptional unit comprising a nucleotide
sequence that encodes an HIV gag-pol fusion polypeptide, wherein
said single transcriptional unit is operably linked to regulatory
elements including a promoter and a polyadenylation signal; (b) a
second DNA plasmid comprising (i) a first transcriptional unit
comprising a nucleotide sequence that encodes an HIV nef-tat-vif
fusion polypeptide operably linked to regulatory elements including
a first promoter and a first polyadenylation signal; (ii) a second
transcriptional unit comprising a nucleotide sequence that encodes
an HIV envelope polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal;
wherein said first and second promoters are each derived from
different transcriptional units; and wherein said first and second
polyadenylation signals are each derived from different
transcriptional units; and wherein the direction of transcription
for said first transcriptional unit is in the opposite direction
from the direction of transcription of said second transcriptional
unit; or wherein the direction of transcription for said first
transcriptional unit is in the same direction from the direction of
transcription of said second transcriptional unit and said first
and second transcriptional units are separated by a spacer region
of at least one kilobase pairs; and (c) at least one of a
pharmaceutically acceptable diluent, carrier or transfection
facilitating agent.
44. The immunogenic composition of claim 10, wherein said
transfection facilitating agent is bupivacaine.
45. The immunogenic composition of claim 10, wherein said promoters
are selected from the group consisting of human cytomegalovirus
(HCMV) immediate early promoter, the simian cytomegalovirus (SCMV)
promoter, the murine cytomegalovirus (MCMV) promoter, the herpes
simplex virus (HSV) latency-associated promoter-1 (LAP1), Simian
virus 40 promoter, human elongation factor 1 alpha promoter, and
the human muscle cell specific desmin promoter.
46. The immunogenic composition of claim 10, wherein said
polyadenylation signals are selected from the group consisting of
rabbit beta-globin poly(A) signal, synthetic polyA, HSV Thymidine
kinase poly A, Human alpha globin poly A, SV40 poly A, human beta
globin poly A, polyomavirus poly A, and Bovine growth hormone poly
A.
47. The immunogenic composition of claim 12, wherein said promoter
on said first plasmid is the human cytomegalovirus (HCMV) immediate
early promoter.
48. The immunogenic composition of claim 13, wherein said
polyadenylation signal on said first plasmid is the Bovine growth
hormone poly A polyadenylation signal.
49. The immunogenic composition of claim 10, wherein said first DNA
plasmid encodes an HIV gag-pol fusion polypeptide, wherein said
fusion of the gag and pol genes of HIV or gag-pol gene is derived
from the HXB2 isolate of HIV.
50. The immunogenic composition of claim 12, wherein said first
promoter on said second plasmid is the human cytomegalovirus (HCMV)
immediate early promoter.
51. The immunogenic composition of claim 13, wherein said first
polyadenylation signal on said second plasmid is the SV40 poly A
polyadenylation signal.
52. The immunogenic composition of claim 10, wherein said HIV
nef-tat-vif fusion polypeptide is a nef, tat, and vif (NTV) fusion
protein expressed from a fusion of the nef, tat, and vif (ntv)
genes of HIV.
53. The immunogenic composition of claim 19, wherein said fusion of
the nef, tat, and vif genes of HIV or ntv gene is derived from the
NL4-3 isolate of HIV.
54. The immunogenic composition of claim 12, wherein said second
promoter on said second plasmid is the simian cytomegalovirus
(SCMV) promoter.
55. The immunogenic composition of claim 13, wherein said second
polyadenylation signal on said second plasmid is the Bovine growth
hormone (BGH) polyadenylation signal.
56. The immunogenic composition of claim 10, wherein said HIV
envelope polypeptide is derived from the primary isolate 6101 of
HIV.
57. A method of immunizing a vertebrate host against selected
antigens comprising administering to said vertebrate host an
immunogenic composition comprising: (a) a first DNA plasmid
comprising a single transcriptional unit comprising a nucleotide
sequence that encodes an HIV gag-pol fusion polypeptide, wherein
said single transcriptional unit is operably linked to regulatory
elements including a promoter and a polyadenylation signal; (b) a
second DNA plasmid comprising (i) a first transcriptional unit
comprising a nucleotide sequence that encodes an HIV nef-tat-vif
fusion polypeptide operably linked to regulatory elements including
a first promoter and a first polyadenylation signal; (ii) a second
transcriptional unit comprising a nucleotide sequence that encodes
an HIV envelope polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal;
wherein said first and second promoters are each derived from
different transcriptional units; and wherein said first and second
polyadenylation signals are each derived from different
transcriptional units; and wherein the direction of transcription
for said first transcriptional unit is in the opposite direction
from the direction of transcription of said second transcriptional
unit; or wherein the direction of transcription for said first
transcriptional unit is in the same direction from the direction of
transcription of said second transcriptional unit and said first
and second transcriptional units are separated by a spacer region
of at least one kilobase pairs; and (c) at least one of a
pharmaceutically acceptable diluent, adjuvant, carrier or
transfection facilitating agent.
58. The method of claim 24, wherein said immunogenic composition is
administered to a mammal using in vivo electroporation.
59. The method of claim 25, wherein said electroporation involves
electrically stimulating the muscle with an electrical current
having a field strength in the range of from about 25 V/cm to about
800 V/cm.
60. The method of claim 24, wherein said transfection facilitating
agent is bupivacaine.
61. The method of claim 24, wherein said promoters are selected
from the group consisting of human cytomegalovirus (HCMV) immediate
early promoter, the simian cytomegalovirus (SCMV) promoter, the
murine cytomegalovirus (MCMV) promoter, the herpes simplex virus
(HSV) latency-associated promoter-1 (LAP1), Simian virus 40
promoter, human elongation factor 1 alpha promoter, and the human
muscle cell specific desmin promoter.
62. The method of claim 24, wherein said polyadenylation signals
are selected from the group consisting of rabbit beta-globin
poly(A) signal, synthetic polyA, HSV Thymidine kinase poly A, Human
alpha-globin poly A, SV40 poly A, human beta globin poly A,
polyomavirus poly A, and Bovine growth hormone poly A.
63. The method of claim 28, wherein said promoter on said first
plasmid is the human cytomegalovirus (HCMV) immediate early
promoter.
64. The method of claim 29, wherein said polyadenylation signal on
said first plasmid is the Bovine growth hormone poly A
polyadenylation signal.
65. The method of claim 24, wherein said first DNA plasmid encodes
an HIV gag-pol fusion polypeptide, wherein said fusion of the gag
and pol genes of HIV or gag-pol gene is derived from the HXB2
isolate of HIV.
66. The method of claim 28, wherein said first promoter on said
second plasmid is the human cytomegalovirus (HCMV) immediate early
promoter.
67. The method of claim 29, wherein said first polyadenylation
signal on said second plasmid is the SV40 poly A polyadenylation
signal.
68. The method of claim 24, wherein said HIV nef-tat-vif fusion
polypeptide is a nef, tat, and vif (NTV) fusion protein expressed
from a fusion of the nef, tat, and vif (ntv) genes of HIV.
69. The method of claim 35, wherein said fusion of the nef, tat,
and vif genes of HIV or ntv gene is derived from the NL4-3 isolate
of HIV.
70. The method of claim 28, wherein said second promoter on said
second plasmid is the simian cytomegalovirus (SCMV) promoter.
71. The method of claim 29, wherein said second polyadenylation
signal is the Bovine growth hormone (BGH) polyadenylation
signal.
72. The method of claim 24, wherein said HIV envelope polypeptide
is derived from the primary isolate 6101 of HIV.
73-87. (canceled)
88. Use of an immunogenic composition as defined in any one of
claims 30 to 31, or 43 to 56 in the manufacture of a medicament for
immunizing a vertebrate host against selected antigens.
Description
FIELD OF THE INVENTION
[0001] This invention relates to plasmids, immunogenic compositions
and methods to improve prophylactic and therapeutic immune
responses to antigens.
BACKGROUND OF THE INVENTION
[0002] Immunization using plasmid DNA-based immunogenic
compositions is a powerful tool that is useful for developing
approaches to prevent or treat infectious diseases or in the
treatment of ongoing disease processes. Plasmid DNA immunization
has been extensively tested in animal models where it has been
found to be effective in inducing both cellular and humoral immune
responses against a wide variety of infectious agents and tumor
antigens. See Donnelly J J, et al., Ann. Rev. Immunol.; 15: 617-48
(1997); Iwasaki A, et al., J Immunol 158 (10): 4591-601 (1997);
Wayne, C. L. and Bennett M., Crit. Rev. Immunol., 18: 449-484
(1998).
[0003] An important advantage of plasmid DNA immunization is that
genes can be cloned, modified and positioned into a potential
plasmid DNA expression vector in such a way as to allow for
relevant post-transcriptional modifications, expression levels,
appropriate intracellular trafficking and antigen presentation.
Plasmid DNA vectors useful for DNA immunization are similar to
those employed for delivery of reporter or therapeutic genes.
Plasmid DNA-based immunization uses the subject's cellular
machinery to generate the foreign protein and stimulates the
subject's immune system to mount an immune response to the protein
antigen. Such plasmid DNA vectors generally contain eukaryotic
transcriptional regulatory elements that are strong viral
promoter/enhancer elements to direct high levels of gene expression
in a wide host cell range and a polyadenylation sequence to ensure
appropriate termination of the expressed mRNA. While, viral
regulatory elements are advantageous for use in plasmid DNA
vectors, the use of unmodified viral vectors to express the
relevant genes may raise safety and technical issues not
encountered with plasmid DNA.
[0004] Current plasmid DNA designs, however, limit the expression
of multiple genes from one vector backbone in a single target cell.
Therefore, to transfer and express multiple genes, co-transfection
of the target cells with separate plasmids is required. When cells
must be co-transfected with multiple plasmids, it is difficult to
achieve optimal expression of all encoded genes, especially when
the plasmid is being used in vivo. Previous attempts to overcome
these limitations and express two or more genes include the use of
the following: viral vectors, multiple alternatively spliced
transcripts from proviral DNA, fusion of genes, bicistronic vectors
containing IRES sequences (Internal ribosome entry site) from
viruses and dual expression plasmids. See Conry R. M. et al., Gene
Therapy. 3(1):67-74, (1996); Chen T T. et al., Journal of
Immunology. 153(10):4775-87, (1994); Ayyavoo V. et al., AIDS.
14(1):1-9, (2000); Amara R. R. et al., Vaccine. 20(15):1949-55,
(2002); Singh G, et al.,. Vaccine 20: 1400-1411 (2002).
[0005] None of the existing plasmid designs have solved the problem
of providing a DNA plasmid suitable for expressing more than two
independent open reading frames in human immunogenic compositions.
In the case of bicistronic vectors, in many instances, only the
first gene transcribed upstream of the IRES is expressed strongly
from either a plasmid or a retroviral vector. See Sugimoto Y., et
al., Hum. Gen. Ther. 6: 905-915 (1995); Mizoguchi H, et al., Mol.
Ther. 1:376-382 (2000). Dual expression cassettes on the other hand
have performed better. For example, it was found that co-delivery
of cDNA for B7-1 and human carcinoembryonic antigen (CEA) with a
single plasmid having two independent cassettes resulted in far
superior immune responses, when compared to separate plasmids. See
Conry R. M. et al., Gene Therapy. 3(1):67-74, (1996). However, in
this case the two independent cassettes involved both consisted of
homologous HCMV promoter and bovine growth hormone (BGH)
poly-adenylation sequences. The presence of homologous sequences
within a plasmid renders that plasmid unsuitable for use in DNA
immunogenic compositions, because the presence of homologous
sequences within the plasmid backbone increases the possibility of
recombination between the repeated sequences and results in vector
instability.
[0006] Another constraint one confronts when designing a plasmid
DNA vector for use in a human immunogenic composition involves size
and organization of the plasmid. As transcriptional units are added
to a plasmid, interference between transcriptional units can arise,
for example in the form of steric hindrance. The cell's RNA
transcription complex must be able to bind to the multiple sites on
a polytranscriptional unit plasmid, uncoil the DNA and effectively
transcribe the genes. Simply making the plasmid bigger is not
necessarily the best solution for several reasons including plasmid
instability, difficulty in plasmid manufacture and, most
importantly, dosing considerations. To design an improved plasmid
DNA multiple transcriptional unit vector, one must consider
placement of genes, spacing and direction of transcription of open
reading frames, level of expression, ease of manufacture, safety
and the ultimate dose of the vector necessary to immunize the
subject.
[0007] Therefore, there remains a need for innovative plasmid DNA,
non-viral vector designs for use in expressing multiple proteins
from complex pathogens like HIV, where a broad immune response to
many proteins is required. In addition, a need exists for
polyvalent DNA-based immunogenic compositions that can direct
expression of high levels of multiple HIV genes within a single
cell.
SUMMARY OF THE INVENTION
[0008] In one embodiment, the present invention provides a DNA
plasmid comprising: (a) a first transcriptional unit comprising a
nucleotide sequence that encodes a first polypeptide operably
linked to regulatory elements including a first promoter and a
first polyadenylation signal; (b) a second transcriptional unit
comprising a nucleotide sequence that encodes a second polypeptide
operably linked to regulatory elements including a second promoter
and a second polyadenylation signal; (c) a third transcriptional
unit comprising a nucleotide sequence that encodes a third
polypeptide operably linked to regulatory elements including a
third promoter and a third polyadenylation signal; wherein said
first, said second and said third promoters are each derived from
different transcriptional units; and wherein said first, said
second and said third polyadenylation signals are each derived from
different transcriptional units. In another embodiment of the
invention, the first, second and third polypeptides are expressed
in a eukaryotic cell.
[0009] In another embodiment, the present invention provides an
immunogenic composition for inducing an immune response to selected
antigens in a vertebrate host, the immunogenic composition
comprising: (a) a DNA plasmid comprising a (i) a first
transcriptional unit comprising a nucleotide sequence that encodes
a first polypeptide operably linked to regulatory elements
including a first promoter and a first polyadenylation signal; (ii)
a second transcriptional unit comprising a nucleotide sequence that
encodes a second polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal;
(iii) a third transcriptional unit comprising a nucleotide sequence
that encodes a third polypeptide operably linked to regulatory
elements including a third promoter and a third polyadenylation
signal; wherein the first, second and third promoters are each
derived from different transcriptional units; wherein said first,
second and third polyadenylation signals are each derived from
different transcriptional units; and (b) at least one of a
pharmaceutically acceptable diluent, adjuvant, carrier or
transfection facilitating agent. In a particular embodiment of the
invention, the transfection facilitating agent is bupivacaine. In
another embodiment of the invention, the first, second and third
polypeptides are expressed in a eukaryotic cell.
[0010] In certain embodiments of the invention, the immunogenic
composition is administered to a mammal using in vivo
electroporation. In a particular embodiment, electroporation
involves electrically stimulating the muscle with an electrical
current having a field strength in the range of from about 25 V/cm
to about 800 V/cm.
[0011] In still another embodiment, the present invention provides
a method of immunizing a vertebrate host against selected antigens
comprising administering to the vertebrate host an immunogenic
composition comprising: (a) a DNA plasmid comprising a (i) a first
transcriptional unit comprising a nucleotide sequence that encodes
a first polypeptide operably linked to regulatory elements
including a first promoter and a first polyadenylation signal; (ii)
a second transcriptional unit comprising a nucleotide sequence that
encodes a second polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal;
(iii) a third transcriptional unit comprising a nucleotide sequence
that encodes a third polypeptide operably linked to regulatory
elements including a third promoter and a third polyadenylation
signal; wherein said first, second and third promoters are each
derived from different transcriptional units; wherein the first,
second and third polyadenylation signals are each derived from
different transcriptional units; and (b) at least one of a
pharmaceutically acceptable diluent, adjuvant, carrier or
transfection facilitating agent. In another embodiment of the
invention, the first, second and third polypeptides are expressed
in a eukaryotic cell.
[0012] In another embodiment of the invention, the selected
antigens are derived from the group consisting of a bacterium, a
virus, an allergen and a tumor. In a particular embodiment, the
selected antigens are viral antigens derived from a virus selected
from the group consisting of Human immunodeficiency virus, Simian
immunodeficiency virus, Respiratory syncytial virus, Parainfluenza
virus type 1, Parainfluenza virus type 2, Parainfluenza virus type
3, Influenza virus, Herpes simplex virus, Human cytomegalovirus,
Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human
papillomavirus, Poliovirus, rotavirus and coronavirus (SARS).
[0013] In still another embodiment of the invention, the selected
antigens are bacterial antigens derived from a bacterium selected
from the group consisting of Haemophilus influenzae (both typable
and nontypable), Haemophilus somnus, Moraxella catarrhalis,
Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus
agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria
meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis,
Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis,
Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium,
Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio
cholerae, Corynebacterium diphtheriae, Mycobacterium tuberculosis,
Mycobacterium avium-Mycobacterium intracellulare complex, Proteus
mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus
epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia
burgdorferi, Pasteurella haemolytica, Pasteurella multocida,
Actinobacillus pleuropneumoniae and Mycoplasma gallisepticum.
[0014] In one embodiment of the invention, the vertebrate host is
selected from the group consisting of mammals, birds and fish. In a
certain embodiment of the invention, the vertebrate host is a
mammal selected from the group consisting human, bovine, ovine,
porcine, equine, canine and feline species.
[0015] In one embodiment of the invention, the first, second and
third promoters are active in eukaryotic cells. In other
embodiments of the invention, the first, second and third promoters
are selected from the group consisting of human cytomegalovirus
(HCMV) immediate early promoter, the simian cytomegalovirus (SCMV)
promoter, the murine cytomegalovirus (MCMV) promoter, the herpes
simplex virus (HSV) latency-associated promoter-1 (LAP1), Simian
virus 40 promoter, human elongation factor 1 alpha promoter, and
the human muscle cell specific desmin promoter.
[0016] In certain embodiments of the invention, the first, second
and third polyadenylation signals are selected from the group
consisting of rabbit beta-globin poly(A) signal, synthetic polyA,
HSV Thymidine kinase poly A, Human alpha globin poly A, SV40 poly
A, human beta globin poly A, polyomavirus poly A, and Bovine growth
hormone poly A.
[0017] In a particular embodiment of the invention, the first
transcriptional unit expresses a gag-pol fusion protein from a
fusion of the gag and pol genes of HIV. In one embodiment of the
invention, the fusion of the gag and pol genes of HIV or gag-pol
gene is derived from the HXB2 isolate of HIV.
[0018] In a certain embodiment of the invention, the second
transcriptional unit expresses an envelope protein from the
envelope gene of HIV. In a particular embodiment of the invention,
the envelope gene is derived from a primary isolate 6101 of
HIV.
[0019] In a specific embodiment of the invention, the third
transcriptional unit expresses a nef, tat, and vif (NTV) fusion
protein from a fusion of the nef, tat, and vif (ntv) genes of HIV.
In a particular embodiment of the invention, the fusion of the nef,
tat, and vif genes of HIV or ntv gene is derived from the NL4-3
isolate of HIV.
[0020] In a specific embodiment of the invention, in a three
transcriptional unit plasmid, the direction of transcription for
the first transcriptional unit is in the opposite direction from
the direction of transcription of the second transcriptional unit.
In another embodiment of the invention, the direction of
transcription for first transcriptional unit is in the opposite
direction from the direction of transcription of the third
transcriptional unit.
[0021] In a certain embodiment of the invention, the invention
provides a three transcriptional unit plasmid, which further
comprises a nucleotide sequence that encodes a selectable marker
operably linked to regulatory elements including a promoter and a
polyadenylation signal. In one embodiment, the selectable marker is
selected from the group consisting of kanamycin resistance gene,
ampicillin resistance gene, tetracycline resistance gene,
hygromycin resistance gene and chloroamphenicol resistance gene. In
another embodiment, the location of the selectable marker is
selected from the group consisting of spacer region 1, spacer
region 2 and spacer region 3. In a specific embodiment, the
location of the selectable marker is spacer region 2.
[0022] In another embodiment of the invention, the invention
provides a three transcriptional unit plasmid, which further
comprises a bacterial origin of replication. In another embodiment,
the location of the origin of replication is selected from the
group consisting of spacer region 1, spacer region 2 and spacer
region 3. In a specific embodiment, the location of the selectable
marker is spacer region 3. In a particular embodiment, the origin
of replication is the pUC origin of replication.
[0023] In one embodiment of the invention, the invention provides a
three transcriptional unit plasmid, wherein the plasmid is less
than about 15 kilobase pairs in total size. In another embodiment
of the invention, spacer region 1 is less than about 400 base
pairs, spacer region 2 is less than about 1100 base pairs and
spacer region 3 is less than about 1100 base pairs.
[0024] In one embodiment, the invention provides an immunogenic
composition for inducing an immune response to human
immunodeficiency virus (HIV) in a vertebrate host, said immunogenic
composition comprising: (a) a first DNA plasmid comprising a single
transcriptional unit comprising a nucleotide sequence that encodes
an HIV gag-pol fusion polypeptide, wherein said single
transcriptional unit is operably linked to regulatory elements
including a promoter and a polyadenylation signal; (b) a second DNA
plasmid comprising (i) a first transcriptional unit comprising a
nucleotide sequence that encodes an HIV nef-tat-vif fusion
polypeptide operably linked to regulatory elements including a
first promoter and a first polyadenylation signal; (ii) a second
transcriptional unit comprising a nucleotide sequence that encodes
an HIV envelope polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal,
wherein said first and second promoters are each derived from
different transcriptional units; and wherein said first and second
polyadenylation signals are each derived from different
transcriptional units; and wherein the direction of transcription
for said first transcriptional unit is in the opposite direction
from the direction of transcription of said second transcriptional
unit; or wherein the direction of transcription for said first
transcriptional unit is in the same direction from the direction of
transcription of said second transcriptional unit and said first
and second transcriptional units are separated by a spacer region
of at least one kilobase pairs; and (c) at least one of a
pharmaceutically acceptable diluent, carrier or transfection
facilitating agent. In a particular embodiment of the invention,
the transfection facilitating agent is bupivacaine. In a particular
embodiment, the promoter on the first plasmid is the human
cytomegalovirus (HCMV) immediate early promoter, the
polyadenylation signal on the first plasmid is the Bovine growth
hormone poly A polyadenylation signal and the first DNA plasmid
encodes an HIV gag-pol fusion polypeptide, wherein the fusion of
the gag and pol genes of HIV or gag-pol gene is derived from the
HXB2 isolate of HIV. In a certain embodiment, the first promoter on
the second plasmid is the human cytomegalovirus (HCMV) immediate
early promoter and the first polyadenylation signal on the second
plasmid is the SV40 poly A polyadenylation signal and the
polypeptide is a nef, tat, and vif (NTV) fusion protein expressed
from a fusion of the nef, tat, and vif (ntv) genes derived from the
NL4-3 isolate of HIV. In a particular embodiment, the second
promoter on the second plasmid is the simian cytomegalovirus (SCMV)
promoter, the second polyadenylation signal on the second plasmid
is the Bovine growth hormone (BGH) polyadenylation signal encoded
envelope polypeptide is derived from the primary isolate 6101 of
HIV.
[0025] In still a further embodiment, the invention provides a
method of immunizing a vertebrate host against selected antigens
comprising administering to said vertebrate host an immunogenic
composition comprising: (a) a first DNA plasmid comprising a single
transcriptional unit comprising a nucleotide sequence that encodes
an HIV gag-pol fusion polypeptide, wherein said single
transcriptional unit is operably linked to regulatory elements
including a promoter and a polyadenylation signal; (b) a second DNA
plasmid comprising (i) a first transcriptional unit comprising a
nucleotide sequence that encodes an HIV nef-tat-vif fusion
polypeptide operably linked to regulatory elements including a
first promoter and a first polyadenylation signal; (ii) a second
transcriptional unit comprising a nucleotide sequence that encodes
an HIV envelope polypeptide operably linked to regulatory elements
including a second promoter and a second polyadenylation signal;
wherein said first and second promoters are each derived from
different transcriptional units; and wherein said first and second
polyadenylation signals are each derived from different
transcriptional units; and wherein the direction of transcription
for said first transcriptional unit is in the opposite direction
from the direction of transcription of said second transcriptional
unit; or wherein the direction of transcription for said first
transcriptional unit is in the same direction from the direction of
transcription of said second transcriptional unit and said first
and second transcriptional units are separated by a spacer region
of at least one kilobase pairs; and (c) at least one of a
pharmaceutically acceptable diluent, carrier or transfection
facilitating agent. In a particular embodiment of the invention,
the transfection facilitating agent is bupivacaine. In a particular
embodiment, the promoter on the first plasmid is the human
cytomegalovirus (HCMV) immediate early promoter, the
polyadenylation signal on the first plasmid is the Bovine growth
hormone poly A polyadenylation signal and the first DNA plasmid
encodes an HIV gag-pol fusion polypeptide, wherein the fusion of
the gag and pol genes of HIV or gag-pol gene is derived from the
HXB2 isolate of HIV. In a certain embodiment, the first promoter on
the second plasmid is the human cytomegalovirus (HCMV) immediate
early promoter and the first polyadenylation signal on the second
plasmid is the SV40 poly A polyadenylation signal and the
polypeptide is a nef, tat, and vif (NTV) fusion protein expressed
from a fusion of the nef, tat, and vif (ntv) genes derived from the
NL4-3 isolate of HIV. In a particular embodiment, the second
promoter on the second plasmid is the simian cytomegalovirus (SCMV)
promoter, the second polyadenylation signal on the second plasmid
is the Bovine growth hormone (BGH) polyadenylation signal encoded
envelope polypeptide is derived from the primary isolate 6101 of
HIV. In one embodiment, the immunogenic composition is administered
to a mammal using in vivo electroporation. In a particular
embodiment, the electroporation involves electrically stimulating
the muscle with an electrical current having a field strength in
the range of from about 25 V/cm to about 800 V/cm.
[0026] In one embodiment, the transfection facilitating agent is
bupivacaine. In one embodiment, the invention provides an
immunogenic composition for inducing an immune response to human
immunodeficiency virus (HIV) in a vertebrate host, the immunogenic
composition comprising: (a) a first DNA plasmid comprising a single
transcriptional unit comprising a nucleotide sequence that encodes
an HIV gag polypeptide, wherein said single transcriptional unit is
operably linked to regulatory elements including a promoter and a
polyadenylation signal; (b) a second DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV pol polypeptide, wherein said single transcriptional
unit is operably linked to regulatory elements including a promoter
and a polyadenylation signal; (c) a third DNA plasmid comprising
(i) a first transcriptional unit comprising a nucleotide sequence
that encodes an HIV nef-tat-vif fusion polypeptide operably linked
to regulatory elements including a first promoter and a first
polyadenylation signal; (ii) a second transcriptional unit
comprising a nucleotide sequence that encodes an HIV envelope
polypeptide operably linked to regulatory elements including a
second promoter and a second polyadenylation signal; wherein said
first and second promoters are each derived from different
transcriptional units; and wherein said first and second
polyadenylation signals are each derived from different
transcriptional units; and wherein the direction of transcription
for said first transcriptional unit is in the opposite direction
from the direction of transcription of said second transcriptional
unit; or wherein the direction of transcription for said first
transcriptional unit is in the same direction from the direction of
transcription of said second transcriptional unit and said first
and second transcriptional units are separated by a spacer region
of at least one kilobase pairs; and (d) a fourth DNA plasmid
comprising a nucleotide sequence that encodes an adjuvant
polypeptide, wherein said nucleotide sequence is operably linked to
regulatory elements including a promoter and a polyadenylation
signal; and (e) at least one of a pharmaceutically acceptable
diluent, carrier or transfection facilitating agent.
[0027] In another embodiment, the invention provides a method of
immunizing a vertebrate host against selected antigens comprising
administering to said vertebrate host an immunogenic composition
comprising: (a) a first DNA plasmid comprising a single
transcriptional unit comprising a nucleotide sequence that encodes
an HIV gag polypeptide, wherein said single transcriptional unit is
operably linked to regulatory elements including a promoter and a
polyadenylation signal; (b) a second DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV pol polypeptide, wherein said single transcriptional
unit is operably linked to regulatory elements including a promoter
and a polyadenylation signal; (c) a third DNA plasmid comprising
(i) a first transcriptional unit comprising a nucleotide sequence
that encodes an HIV nef-tat-vif fusion polypeptide operably linked
to regulatory elements including a first promoter and a first
polyadenylation signal; (ii) a second transcriptional unit
comprising a nucleotide sequence that encodes an HIV envelope
polypeptide operably linked to regulatory elements including a
second promoter and a second polyadenylation signal; wherein said
first and second promoters are each derived from different
transcriptional units; and wherein said first and second
polyadenylation signals are each derived from different
transcriptional units; and wherein the direction of transcription
for said first transcriptional unit is in the opposite direction
from the direction of transcription of said second transcriptional
unit; or wherein the direction of transcription for said first
transcriptional unit is in the same direction from the direction of
transcription of said second transcriptional unit and said first
and second transcriptional units are separated by a spacer region
of at least one kilobase pairs; and (d) a fourth DNA plasmid
comprising a nucleotide sequence that encodes an adjuvant
polypeptide, wherein said nucleotide sequence is operably linked to
regulatory elements including a promoter and a polyadenylation
signal; and (e) at least one of a pharmaceutically acceptable
diluent, carrier or transfection facilitating agent. In a
particular embodiment, the electroporation involves electrically
stimulating the muscle with an electrical current having a field
strength in the range of from about 25 V/cm to about 800 V/cm. In
one embodiment, the transfection facilitating agent is
bupivacaine.
[0028] In one embodiment the present invention provides an
immunogenic composition for inducing an immune response to HIV in a
vertebrate host, where the immunogenic composition comprises: a) a
first DNA plasmid comprising a single transcriptional unit
comprising a nucleotide sequence that encodes an HIV envelope
polypeptide, wherein the single transcriptional unit is operably
linked to regulatory elements including a promoter and a
polyadenylation signal; (b) a second DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV gag-pol fusion polypeptide, wherein the single
transcriptional unit is operably linked to regulatory elements
including a promoter and a polyadenylation signal; (c) a third DNA
plasmid comprising a single transcriptional unit comprising a
nucleotide sequence that encodes an HIV nef-tat-vif fusion
polypeptide, wherein the single transcriptional unit is operably
linked to regulatory elements including a promoter and a
polyadenylation signal; (d) a fourth DNA plasmid comprising a
nucleotide sequence that encodes an adjuvant polypeptide, wherein
the nucleotide sequence is operably linked to regulatory elements
including a promoter and a polyadenylation signal; and (e) at least
one of a pharmaceutically acceptable diluent, carrier or
transfection facilitating agent. In a particular embodiment, the
transfection facilitating agent is bupivacaine. In another
embodiment, the immunogenic composition containing bupivacaine is
administered in conjunction with electroporation. In a specific
embodiment, the HIV envelope, gag-pol, nef-tat-vif and adjuvant
polypeptides are expressed in a eukaryotic cell. In one embodiment,
the first, second, third and fourth plasmids contain promoters that
are active in eukaryotic cells.
[0029] In one embodiment the present invention provides a method of
immunizing a vertebrate host against selected antigens comprising
administering to the vertebrate host an immunogenic composition,
wherein the immunogenic composition comprises: a) a first DNA
plasmid comprising a single transcriptional unit comprising a
nucleotide sequence that encodes an HIV envelope polypeptide,
wherein the single transcriptional unit is operably linked to
regulatory elements including a promoter and a polyadenylation
signal; (b) a second DNA plasmid comprising a single
transcriptional unit comprising a nucleotide sequence that encodes
an HIV gag-pol fusion polypeptide, wherein the single
transcriptional unit is operably linked to regulatory elements
including a promoter and a polyadenylation signal; (c) a third DNA
plasmid comprising a single transcriptional unit comprising a
nucleotide sequence that encodes an HIV nef-tat-vif fusion
polypeptide, wherein the single transcriptional unit is operably
linked to regulatory elements including a promoter and a
polyadenylation signal; (d) a fourth DNA plasmid comprising a
nucleotide sequence that encodes an adjuvant polypeptide, wherein
the nucleotide sequence is operably linked to regulatory elements
including a promoter and a polyadenylation signal; and (e) at least
one of a pharmaceutically acceptable diluent, carrier or
transfection facilitating agent. In a particular embodiment, the
transfection facilitating agent is bupivacaine. In another
embodiment, the immunogenic composition containing bupivacaine is
administered in conjunction with electroporation. In a specific
embodiment, the HIV envelope, gag-pol, nef-tat-vif and adjuvant
polypeptides are expressed in a eukaryotic cell. In one embodiment,
the first, second, third and fourth plasmids contain promoters that
are active in eukaryotic cells.
[0030] In certain embodiments of the invention, the first, second,
third and fourth plasmids contain promoters that are selected from
the group consisting of human cytomegalovirus (HCMV) immediate
early promoter, the simian cytomegalovirus (SCMV) promoter, the
murine cytomegalovirus (MCMV) promoter, the herpes simplex virus
(HSV) latency-associated promoter-1 (LAP1), Simian virus 40
promoter, human elongation factor 1 alpha promoter, and the human
muscle cell specific desmin promoter. In certain embodiments of the
invention, the first, second, third and fourth plasmids contain
polyadenylation signals that are selected from the group consisting
of rabbit beta-globin poly(A) signal, synthetic polyA, HSV
Thymidine kinase poly A, Human alpha globin poly A, SV40 poly A,
human beta globin poly A, polyomavirus poly A, and Bovine growth
hormone poly A.
[0031] In a particular embodiment, the present invention provides
an immunogenic composition for inducing an immune response to HIV
in a vertebrate host, where the immunogenic composition comprises
four plasmids as described above, and where each plasmid further
comprises a selectable marker selected from the group consisting of
kanamycin resistance gene, ampicillin resistance gene, tetracycline
resistance gene, hygromycin resistance gene and chloroamphenicol
resistance gene. In another embodiment, each plasmid further
comprises a bacterial origin of replication. In still another
embodiment, the origin of replication is the pUC origin of
replication.
[0032] The invention also provides an immunogenic composition, and
wherein the fourth DNA plasmid comprises a primary transcriptional
unit and a secondary transcriptional unit comprising two nucleotide
sequences that encode two adjuvant polypeptides operably linked to
regulatory elements. In one embodiment, the primary transcriptional
unit comprises a nucleotide sequence that encodes an IL-12 p35
polypeptide operably linked to regulatory elements including a
promoter and a polyadenylation signal. In another embodiment, the
secondary transcriptional unit comprises a nucleotide sequence that
encodes an IL-12 p40 polypeptide operably linked to regulatory
elements including a promoter and a polyadenylation signal.
[0033] In another embodiment the present invention provides an
immunogenic composition for inducing an immune response to HIV in a
vertebrate host, where the immunogenic composition comprises: (a) a
first DNA plasmid comprising a single transcriptional unit
comprising a nucleotide sequence that encodes an HIV envelope
polypeptide, wherein the single transcriptional unit is operably
linked to regulatory elements including a promoter and a
polyadenylation signal; (b) a second DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV gag polypeptide, wherein the single transcriptional
unit is operably linked to regulatory elements including a promoter
and a polyadenylation signal; (c) a third DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV pol polypeptide, wherein the single transcriptional
unit is operably linked to regulatory elements including a promoter
and a polyadenylation signal; (d) a fourth DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV nef-tat-vif fusion polypeptide, wherein said single
transcriptional unit is operably linked to regulatory elements
including a promoter and a polyadenylation signal; (e) a fifth DNA
plasmid comprising a nucleotide sequence that encodes an adjuvant
polypeptide, wherein said nucleotide sequence is operably linked to
regulatory elements including a promoter and a polyadenylation
signal; and (f at least one of a pharmaceutically acceptable
diluent, carrier or transfection facilitating agent. In a specific
embodiment, the transfection facilitating agent is bupivacaine. In
another embodiment, the immunogenic composition containing
bupivacaine is administered in conjunction with electroporation. In
one embodiment, the HIV envelope, gag, pol, nef-tat-vif and
adjuvant polypeptides are expressed in a eukaryotic cell.
[0034] In another embodiment the present invention provides a
method of immunizing a vertebrate host against selected antigens
comprising administering to said vertebrate host an immunogenic
composition where the immunogenic composition comprises: (a) a
first DNA plasmid comprising a single transcriptional unit
comprising a nucleotide sequence that encodes an HIV envelope
polypeptide, wherein the single transcriptional unit is operably
linked to regulatory elements including a promoter and a
polyadenylation signal; (b) a second DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV gag polypeptide, wherein the single transcriptional
unit is operably linked to regulatory elements including a promoter
and a polyadenylation signal; (c) a third DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV pol polypeptide, wherein the single transcriptional
unit is operably linked to regulatory elements including a promoter
and a polyadenylation signal; (d) a fourth DNA plasmid comprising a
single transcriptional unit comprising a nucleotide sequence that
encodes an HIV nef-tat-vif fusion polypeptide, wherein said single
transcriptional unit is operably linked to regulatory elements
including a promoter and a polyadenylation signal; (e) a fifth DNA
plasmid comprising a nucleotide sequence that encodes an adjuvant
polypeptide, wherein said nucleotide sequence is operably linked to
regulatory elements including a promoter and a polyadenylation
signal; and (f) at least one of a pharmaceutically acceptable
diluent, carrier or transfection facilitating agent. In a specific
embodiment, the transfection facilitating agent is bupivacaine. In
another embodiment, the immunogenic composition containing
bupivacaine is administered in conjunction with
electroporation.
[0035] In one embodiment of the invention the first, second, third,
fourth and fifth plasmids contain promoters that are active in
eukaryotic cells. In certain embodiments, the first, second, third,
fourth and fifth plasmids contain promoters that are selected from
the group consisting of human cytomegalovirus (HCMV) immediate
early promoter, the simian cytomegalovirus (SCMV) promoter, the
murine cytomegalovirus (MCMV) promoter, and the herpes simplex
virus (HSV) latency-associated promoter-1 (LAP1), Simian virus 40
promoter, human elongation factor 1 alpha promoter, and the human
muscle cell specific desmin promoter. In other embodiments of the
invention, the first, second, third and fourth plasmids contain
polyadenylation signals that are selected from the group consisting
of rabbit beta-globin poly(A) signal, synthetic polyA, HSV
Thymidine kinase poly A, Human alpha globin poly A, SV40 poly A,
human beta globin poly A, polyomavirus poly A, and Bovine growth
hormone poly A.
[0036] In a particular embodiment, the present invention provides
an immunogenic composition for inducing an immune response to HIV
in a vertebrate host, where the immunogenic composition comprises
five plasmids as described above, and where each plasmid further
comprises a selectable marker selected from the group consisting of
kanamycin resistance gene, ampicillin resistance gene, tetracycline
resistance gene, hygromycin resistance gene and chloroamphenicol
resistance gene. In another embodiment, each plasmid further
comprises a bacterial origin of replication and wherein the origin
of replication is the pUC origin of replication.
[0037] The invention also provides an immunogenic composition, and
wherein the fifth DNA plasmid comprises a primary transcriptional
unit and a secondary transcriptional unit comprising two nucleotide
sequences that encode two adjuvant polypeptides operably linked to
regulatory elements. In one embodiment, the primary transcriptional
unit comprises a nucleotide sequence that encodes an IL-12 p35
polypeptide operably linked to regulatory elements including a
promoter and a polyadenylation signal. In another embodiment, the
secondary transcriptional unit comprises a nucleotide sequence that
encodes an IL-12 p40 polypeptide operably linked to regulatory
elements including a promoter and a polyadenylation signal.
[0038] Other aspects and embodiment of the present invention are
disclosed in the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows a circular schematic diagram of an illustrative
triple transcriptional unit DNA plasmid set up to express six HIV
genes or gene constructs in eukaryotic cells from three separate
open reading frames. FIG. 1 shows a linear but more detailed
schematic diagram of the same plasmid. The following abbreviations
are used: SCMV: Simian cytomegalavirus promoter, HCMV: Human
cytomegalovirus promoter, BGHpolyA: Bovine growth hormone poly
adenylation signal, kan: Kanamycin marker gene for resistance,
HSVIap1: Herpes simplex virus latency-associated promoter 1, SV40
polyA: Simian virus 40 poly adenylation signal SV40sd/sa: Simian
virus 40 splice donor and acceptor, gag-pol: HIV gag-pol fusion,
ntv: HIV nef-tat-vif fusion, env: HIV envelope.
[0040] FIG. 2 shows HIV gag expression in 293 cells. 293 cells were
transfected with 2 .mu.g of indicated plasmid DNA expression
vector. Forty-eight hours after transfection, cell associated HIV
proteins were visualized by Western blot. The promoters and open
reading frames for a particular plasmid are shown below:
[0041] Plasmids Transfected
[0042] 102: HCMV-gag
[0043] 201: HCMV-pol, SCMV-gag
[0044] 203: HCMV-gag/pol/nef/tat/vif, SCMV-env
[0045] 302: SCMV-gag/pol, HCMV-, Lap1-nef/tat/vif
[0046] 204: HCMV-gag/pol, SCMV-env
[0047] 303: SCMV-gag/pol, HCMV-env, Lap1-nef/tat/vif
[0048] 001: control plasmid without insert
[0049] FIG. 3 shows HIV pol expression in 293 cells. 293 cells were
transfected with 2 .mu.g of indicated plasmid DNA expression
vector. Forty-eight hours after transfections, cell associated HIV
proteins were visualized by Western blot. The promoters and open
reading frames for a particular plasmid are shown below:
[0050] Plasmids Transfected
[0051] 103: HCMV-pol
[0052] 201: HCMV-pol, SCMV-gag
[0053] 302: SCMV-gag/pol, HCMV-, Lap1-nef/tat/vif
[0054] 203: HCMV-gag/pol/nef/tat/vif, SCMV-env
[0055] 204: HCMV-gag/pol, SCMV-env
[0056] 303: SCMV-gag/pol, HCMV-env, Lap1-nef/tat/vif
[0057] 001: control plasmid without insert
[0058] FIG. 4 shows HIV nef/tat/vif (ntv) expression in 293 cells.
293 cells were transfected with 2 .mu.g of indicated plasmid DNA
expression vector. Forty-eight hours after transfections, cell
associated HIV proteins were visualized by Western blot. The
promoters and open reading frames for a particular plasmid are
shown below:
[0059] Plasmids Transfected
[0060] 104: HCMV-ntv
[0061] 105: Lap1-ntv
[0062] 202: HCMV-ntv, SCMV-env
[0063] 203: HCMV-gag/pol/nef/tat/vif, SCMV-env
[0064] 302: SCMV-gag/pol, HCMV-, Lap1-nef/tat/vif
[0065] 303: SCMV-gag/pol, HCMV-env, Lap1-nef/tat/vif
[0066] 001: control plasmid without insert
[0067] FIG. 5 shows HIV env expression in 293 cells. 293 cells were
transfected with 2 .mu.g of indicated plasmid DNA expression
vector. Forty-eight hours after transfections, cell associated HIV
proteins were visualized by Western blot. The promoters and open
reading frames for a particular plasmid are shown below:
[0068] Plasmids Transfected
[0069] 101: HCMV-env
[0070] 202: HCMV-ntv, SCMV-env
[0071] 203: HCMV-gag/pol/nef/tat/vif, SCMV-env
[0072] 204: HCMV-gag/pol, SCMV-env
[0073] 303: SCMV-gag/pol, HCMV-env, Lap1-nef/tat/vif
[0074] 001: control plasmid without insert
[0075] FIG. 6 shows HIV gag expression in 293 cells. 293 cells were
transfected with 1 .mu.g indicated plasmid DNA combination.
Forty-eight hours after transfections, cell associated HIV proteins
were visualized by Western blot. The promoters and open reading
frames for a particular plasmid are shown below: TABLE-US-00001
Lane Plasmid Combinations Transfected 1 301 (gag/pol) + 101(env) +
104(ntv) 2 201(gag, pol) + 202 (env, ntv) 3 203 (gag/pol/ntv, env)
4 303 (gag/pol, env, ntv) 5 101(env) + 102(gag) + 103(pol) +
104(ntv) 6 001 (control)
[0076] FIG. 7 shows HIV env expression in 293 cells. 293 cells were
transfected with 1 .mu.g of indicated plasmid DNA combination.
Forty-eight hours after transfections, cell associated HIV proteins
were visualized by Western blot. The promoters and open reading
frames for a particular plasmid are shown below: TABLE-US-00002
Lane Plasmid Combinations Transfected 1 152 (gag/pol) + 101(env) +
104(ntv) 2 201(gag, pol) + 202(env, ntv) 3 203(gag/pol/ntv, env) 4
303(gag/pol, env, env) 5 101(env) + 102(gag) + 103(pol) + 104(ntv)
6 001 (control)
[0077] FIG. 8 shows HIV ntv expression in 293 cells. 293 cells were
transfected with 1 .mu.g of indicated plasmid DNA combination.
Forty-eight hours after transfections, cell associated HIV proteins
were visualized by Western blot. The promoters and open reading
frames for a particular plasmid are shown below: TABLE-US-00003
Lane Plasmid Combinations Transfected 1 152 (gag/pol) + 101(env) +
104(ntv) 2 201(gag, pol) + 202(env, ntv) 3 203(gag/pol/ntv, env) 4
303(gag/pol, env, env) 5 101(env) + 102(gag) + 103(pol) + 104(ntv)
6 001 (control)
[0078] FIG. 9 shows HIV pol expression in 293 cells. 293 cells were
transfected with the indicated plasmid DNA concentration and
combination. Forty-eight hours after transfections, cell associated
HIV proteins were visualized by Western blot. The promoters and
open reading frames for a particular plasmid are shown below:
TABLE-US-00004 Plasmid concentration Lane Plasmid Combinations
Transfected Transfected (micrograms) 1 001 (control) 2 2 201(gag,
pol) + 202(ntv, env) 1 + 1 3 204(gag/pol, env) + 104(ntv) 1 + 1 4
203(gag/pol/ntv, env) 2 5 302(gag/pol, ntv) + 101(env) 1 + 1 6
303((gag/pol, env, ntv) 2
[0079] FIG. 10 shows HIV gag expression in 293 cells. 293 cells
were transfected with the indicated plasmid DNA concentration and
combination. Forty-eight hours after transfections, cell associated
HIV proteins were visualized by Western blot. The promoters and
open reading frames for a particular plasmid are shown below:
TABLE-US-00005 Plasmid concentration Lane Plasmid Combinations
Transfected Transfected (micrograms) 1 001 (control) 2 2 201(gag,
pol) + 202(ntv, env) 1 + 1 3 204(gag/pol, env) + 104(ntv) 1 + 1 4
203(gag/pol/ntv, env) 2 5 302(gag/pol, ntv) + 101(env) 1 + 1 6
303((gag/pol, env, ntv) 2
[0080] FIG. 11 shows HIV env Expression in 293 Cells. 293 cells
were transfected with the indicated plasmid DNA concentration and
combination. Forty-eight hours after transfections, cell associated
HIV proteins were visualized by Western blot. The promoters and
open reading frames for a particular plasmid are shown below:
TABLE-US-00006 Plasmid concentration Lane Plasmid Combinations
Transfected Transfected (micrograms) 1 001 (control) 2 2 201(gag,
pol) + 202(ntv, env) 1 + 1 3 204(gag/pol, env) + 104(ntv) 1 + 1 4
203(gag/pol/ntv, env) 2 5 302(gag/pol, ntv) + 101(env) 1 + 1 6
303((gag/pol, env, ntv) 2
[0081] FIG. 12 shows HIV ntv expression in 293 cells. 293 cells
were transfected with the indicated plasmid DNA concentration and
combination. Forty-eight hours after transfections, cell associated
HIV proteins were visualized by Western blot. The promoters and
open reading frames for a particular plasmid are shown below:
TABLE-US-00007 Plasmid concentration Lane Plasmid Combinations
Transfected Transfected (micrograms) 1 001 (control) 2 2 201(gag,
pol) + 202(ntv, env) 1 + 1 3 204(gag/pol, env) + 104(ntv) 1 + 1 4
203(gag/pol/ntv, env) 2 5 302(gag/pol, ntv) + 101(env) 1 + 1 6
303((gag/pol, env, ntv) 2
DETAILED DESCRIPTION OF THE INVENTION
[0082] DNA based immunogenic compositions provide an alternative to
traditional immunogenic compositions comprising administration of
protein antigens and an adjuvant. Instead, DNA based immunogenic
compositions involve the introduction of DNA, which encodes the
antigen or antigens, into tissues of a subject, where the antigens
are expressed by the cells of the subject's tissue. As used herein,
such immunogenic compositions are termed "DNA based immunogenic
compositions" or "nucleic acid-based immunogenic compositions." One
problem has been that when multiple genes are required for
generation of a protective immune response, multiple plasmids have
had to be used to individually express the genes. This imposes
manufacturing and regulatory burdens. Embodiments of the present
invention provide solutions to this problem with a plasmid design
capable of expressing three independent open reading frames in the
same cell. In certain embodiments of the invention, genes are fused
to make polyproteins and, in this way, many more proteins can be
can be expressed from a single plasmid. In one embodiment, six
proteins are expressed from the single plasmid.
[0083] A large number of factors can influence the expression of
antigen genes and/or the immunogenicity of DNA based immunogenic
compositions. Examples of such factors include the construction of
the plasmid vector, size of the plasmid vector, choice of the
promoter used to drive antigen gene expression, the number and size
of transcriptional units on the plasmid, stability of the RNA
transcripts, orientation of the transcriptional units within the
plasmid, reproducibility of immunization and stability of the
inserted gene in the plasmid. Embodiments of the present invention
provide plasmid designs that optimize many of these key
parameters.
[0084] The design and optimization of plasmid DNA vectors having
multiple transcriptional units is critical. To improve the actual
dose of antigen received by an immunized subject, the size of the
plasmid must be minimized, while the number of protein products and
quantity of protein produced should be maximized. To balance these
considerations, one must consider placement of genes; spacing of
transcriptional units; direction of transcription of the open
reading frames; levels of expression; promoter size, orientation
and strength; enhancer size, placement, orientation and strength;
open reading frame size and organization; ease of manufacture;
plasmid stability; safety; and the ultimate dose of the vector
necessary to immunize the subject.
[0085] An important consideration with the use of DNA plasmids for
immunization is manufacture of the plasmid. Due to potential safety
concerns, the manufacturing process and the final products must
undergo intense scrutiny and be subject to extensive quality
control. The result is reflected in high costs for such procedures.
As a result, any DNA immunization, which requires multiple
plasmids, will be proportionately more expensive and less likely to
be effective. Therefore, in certain embodiments of the present
invention, where manufacturing costs need to be controlled,
immunogenic compositions are provided comprising a single plasmid
per application suitable to induce immune responses in virtually
any disease process.
[0086] In some situations, in spite of higher manufacturing costs,
the use of combinations of plasmids each containing a single
transcriptional unit or two transcriptional units may lead to a
more effective immunogenic composition. In such cases, it is
important to design the immunogenic composition to have the optimal
number of plasmids encoding all of the genes necessary for inducing
an effective immune response. The use of a plasmid containing three
transcriptional units expressing all of the necessary genes instead
of multiple plasmids each containing a single transcriptional unit
must be balanced with the immunogenicity of particular antigens.
One advantage of the combination of single transcriptional unit
plasmids approach is that the individual genes may each be driven
by the same strong promoter. For example, the HCMV promoter can be
used in each plasmid, rather than only once per plasmid, as is the
case in a three transcriptional unit plasmid. In contrast, when
using a three transcriptional plasmid, the HCMV promoter can only
be used once to prevent the possibility of internal homologous
recombination and plasmid instability. For example, in a
composition having two antigen expressing plasmids where one
plasmid has one transcriptional unit and the second has two
transcriptional units. In such a composition, HCMV promoter may be
used to drive expression of the single antigen or fusion protein in
the plasmid with one transcriptional unit and it may also be used
to drive expression of one of the proteins or fusion proteins in
the plasmid having two transcriptional units.
[0087] In the case where the pathogen is human immunodeficiency
virus (HIV), immunogenic compositions are described with four
single transcriptional unit plasmids which contain nucleotide
sequences encoding, respectively, an HIV envelope polypeptide, an
HIV gag-pol fusion polypeptide, an HIV nef-tat-vif fusion
polypeptide, and an adjuvant polypeptide. If desired, two single
transcriptional unit plasmids may be used which contain nucleotide
sequences encoding, respectively, an HIV gag polypeptide and an HIV
pol fusion polypeptide, instead of the single transcriptional unit
plasmid containing a nucleotide sequence encoding an HIV gag-pol
fusion polypeptide (thus, in this aspect, five plasmids are
used).
[0088] In general, depending on their origin, promoters differ in
tissue specificity and efficiency in initiating mRNA synthesis
[Xiang et al., Virology, 209:564-579 (1994); Chapman et al., Nucle.
Acids. Res., 19:3979-3986 (1991)]. To date, most DNA based
immunogenic compositions in mammalian systems have relied upon
viral promoters derived from cytomegalovirus (CMV). The CMV may be
human or simian in origin. These have had good efficiency in both
muscle and skin immunization in a number of mammalian species.
Another factor known to affect the immune response elicited by DNA
immunization is the method of DNA delivery; parenteral routes can
yield low rates of gene transfer and produce considerable
variability of gene expression. See Montgomery et al., DNA Cell
Bio., 12:777-783 (1993). High-velocity inoculation of plasmids,
using a gene-gun, enhanced the immune responses of mice, presumably
because of a greater efficiency of DNA transfection and more
effective antigen presentation by dendritic cells. See Fynan et
al., Proc. Natl. Acad. Sci., 90:11478-11482 (1993B); Eisenbraun et
al., DNA Cell Biol., 12: 791-797 (1993). Vectors containing the
nucleic acid-based immunogenic composition of the invention may
also be introduced into the desired host by other methods known in
the art, e.g., transfection, electroporation, microinjection,
transduction, cell fusion, DEAE dextran, calcium phosphate
precipitation, lipofection (lysosome fusion), or a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-967
(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut
et al., Canadian Patent Application No. 2,012,311, filed Mar. 15,
1990.
[0089] Accordingly, the present invention relates to plasmids,
immunogenic compositions and methods for the genetic immunization
of vertebrates such as mammals, birds and fish. The plasmids,
immunogenic compositions and methods of the present invention can
be particularly useful for mammalian subjects including human,
bovine, ovine, porcine, equine, canine and feline species. The
plasmids, immunogenic compositions and methods are described in
detail below and with reference to the cited documents that are
incorporated by reference to provide detail known to one of skill
in the art.
A. DNA Plasmids, Vectors, Constructs, Immunogenic Compositions
[0090] The terms plasmid, construct and vector are used throughout
the specification. As used herein, the term "plasmid" refers to a
circular, supercoiled DNA molecule into which various nucleic acid
molecules coding for regulatory sequences, open reading frames,
cloning sites, stop codons, spacer regions or other sequences
selected for structural or functional regions are assembled and
used as a vector to express genes in a vertebrate host. Further, as
used herein, "plasmids" are capable of replicating in a bacterial
strain. As used herein, the term "construct" refers to a particular
vector or plasmid having a specified arrangement of genes and
regulatory elements. A nucleic acid sequence can be "exogenous,"
which means that it is foreign to the cell into which the vector is
being introduced, "heterologous" which means that it is derived
from a different genetic source or "homologous", which means that
the sequence is structurally related to a sequence in the cell but
in a position within the host cell nucleic acid in which the
sequence is ordinarily not found. One of skill in the art would be
well equipped to construct a vector or modify a plasmid of the
invention through standard recombinant techniques, which are
described in See, e.g., Sambrook et al, Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor Laboratory, New York, (1989)
and references cited therein at, for example, pages 3.18-3.26 and
16.17-16.27 and Ausubel et al., Current Protocols in Molecular
Biology, Wiley Interscience Publishers, New York (1995) both
incorporated herein by reference.
[0091] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a designated nucleic acid molecule encoding an
antigen or antigens can be inserted for introduction into a cell
where it can be expressed. Vectors include plasmids, cosmids,
viruses (bacteriophage, animal viruses, and plant viruses), and
artificial chromosomes (e.g., YACs). The term "expression vector"
refers to a vector containing a nucleic acid sequence coding for at
least part of a gene product capable of being transcribed. In some
cases, RNA molecules are then translated into a protein,
polypeptide, or peptide. In other cases, these sequences are not
translated, for example, in the production of expressed interfering
RNA (eiRNA), short interfering RNA (siRNA), antisense molecules or
ribozymes. Expression vectors can contain a variety of "control
sequences," which refer to nucleic acid sequences necessary for the
transcription and possibly translation of an operably linked coding
sequence in a particular host organism. In addition to control
sequences that govern transcription and translation, vectors and
expression vectors may contain nucleic acid sequences that serve
other functions as well and are described below.
[0092] The terms "nucleic acid" and "oligonucleotide" are used
interchangeably to mean multiple nucleotides (i.e. molecules
comprising a sugar (e.g. ribose or deoxyribose) linked to a
phosphate group and to an exchangeable organic base, which is
either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or
uracil (U)) or a substituted purine (e.g. adenine (A) or guanine
(G)). As used herein, the terms refer to oligoribonucleotides as
well as oligodeoxyribonucleotides. The terms shall also include
polynucleosides (i.e. a polynucleotide minus the phosphate) and any
other organic base containing polymer. Nucleic acid molecules can
be obtained from existing nucleic acid sources (e.g. genomic or
cDNA), but may be synthetically produced (e.g. produced by
oligonucleotide synthesis).
[0093] The phrase "each derived from different transcriptional
units", as used herein means that each of the regulatory control
elements of a similar function, such as the promoters, are all of
different origin and are not homologous to each other to such a
level that genetic instability through recombination may arise in
the plasmid. See Herrera et al., Biochem. Biophys. Res. Commun.
279:548-551 (2000).
[0094] Immunogenic compositions of this invention include a triple
transcriptional unit DNA plasmid comprising a DNA sequence encoding
at least three selected antigens to which an immune response is
desired. In the plasmid, the selected antigens are under the
control of regulatory sequences directing expression thereof in a
mammalian or vertebrate cell. Immunogenic compositions of this
invention also include combinations of plasmids encoding selected
antigens. Such combinations may be comprised of two, three or four
plasmids encoding additional selected antigens. There may be one,
two, or three transcriptional units on any particular plasmid
within the combination. Furthermore, additional plasmids encoding
adjuvant polypeptides may be included in the immunogenic
compositions of the invention.
[0095] Non-viral, plasmid vectors useful in this invention contain
isolated and purified DNA sequences comprising DNA sequences that
encode the selected immunogen and antigens. The DNA molecule
encoding the target antigens may be derived from viral or non-viral
sources such as bacterial species or tumor antigens that have been
designed to encode an exogenous or heterologous nucleic acid
sequence. Such plasmids or vectors can include sequences from
viruses or phages. A variety of non-viral vectors are known in the
art and may include, without limitation, plasmids, bacterial
vectors, bacteriophage vectors, "naked" DNA, DNA condensed with
cationic lipids or polymers, as well as DNA formulated with other
transfection facilitating agents, for example the local anesthetic
such as bupivacaine, discussed below.
[0096] Components of the plasmids of this invention may be obtained
from existing vectors. Examples of bacterial vectors include, but
are not limited to, sequences derived from bacille Calmette Guerin
(BCG), Salmonella, Shigella, E. coli, and Listeria, among others.
Suitable plasmid vectors for obtaining components include, for
example, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18,
pUC19, pLG339, pR290, pK37, pKC101, pAC105, pVA51, pKH47, pUB110,
pMB9, pBR325, Col E1, pSC101, pBR313, pML21, RSF2124, pCR1, RP4,
pBAD18, and pBR328.
[0097] Other components may be obtained from inducible expression
vectors. Examples of suitable inducible Escherichia coli expression
vectors include pTrc (Amann et al., Gene, 69:301-315 (1988)), the
arabinose expression vectors (e.g., pBAD18, Guzman et al, J.
Bacteriol., 177:4121-4130 (1995)), and pETIId (Studier et al.,
Methods in Enzymology, 185:60-89 (1990)). Target gene expression
from the pTrc vector relies on host RNA polymerase transcription
from a hybrid trp-lac fusion promoter. Target gene expression from
the pETIId vector relies on transcription from a T7 gn10-lac fusion
promoter mediated by a coexpressed viral RNA polymerase T7 gn I.
This viral polymerase is supplied by host strains BL21 (DE3) or HMS
I 74(DE3) from a resident prophage harboring a T7 gn1 gene under
the transcriptional control of the lacUV5 promoter. The pBAD system
relies on the inducible arabinose promoter that is regulated by the
araC gene. The promoter is induced in the presence of
arabinose.
[0098] Regulatory components may be obtained from inducible
promoters that are regulated by exogenously supplied compounds,
including, the zinc-inducible sheep metallothionine (MT) promoter,
the dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV)
promoter, the tetracycline inducible system (Gossen et al, Science
268:1766-1769 (1995) and the repamycin inducible system (Magari et
al, J din Invest, 100:2865-2872 (1997)).
[0099] Transcriptional control signals in eukaryotes are comprised
of promoter and enhancer elements. "Promoters" and "enhancers" as
used herein refer to DNA sequences that interact specifically with
proteins involved in transcription. See Maniatis, T., et al.,
Science 236:1237 (1987). As discussed above 5'-untranslated regions
may be combined with promoters and enhancers to enhance expression
of the selected antigens. The promoter, enhancers and other
regulatory sequences that drive expression of the antigen in the
desired mammalian or vertebrate subject may similarly be selected
from a wide list of promoters known to be useful for that purpose.
A variety of such promoters are disclosed below. In an embodiment
of the immunogenic DNA plasmid composition described below, useful
promoters are the human cytomegalovirus (HCMV) promoter/enhancer
(described in, e.g., U.S. Pat. Nos. 5,158,062 and 5,385,839,
incorporated herein by reference), the human herpes virus
latency-associated promoters 1 and 2 (HSVLap1 & HSVLap2:
sometimes referred to as "latency-active promoters 1 & 2") and
the simian cytomegalovirus (SCMV) promoter enhancer. See Goins W.
F. et al., J. Virology 68:2239-2252 (1994); Soares, K. J. et al.,
Virology 70:5384-5394; Goins W. F. et al., J. Virology 73:519-532
(1999). The murine cytomegalovirus (MCMV) promoter is also suitable
for use.
[0100] Other useful transcriptional control elements include
posttranscriptional control elements such as the constitutive
transport enhancers (CTE) or CTE-like elements such as RNA
transport elements (RTE), which aid in transport of unspliced or
partially spliced RNA to the cytoplasm. See U.S. Pat. No. 5,585,263
to Hammarskjold et al., and Zolotukhin et al., J. Virol.
68:944-7952 (1994)). CTE or RTE are desirable because they have
been shown to improve expression, and because many genes require
the presence of post-transcriptional control elements. There are
several types of CTE and CTE-like elements, which function using
different pathways. See Tabernero et al., J. Virol. 71:95-101
(1997). See also International application WO 99/61596, which
describes a new type of post-transcriptional control element that
is able to replace CTE.
[0101] Gene expression can also be enhanced by the inclusion of
polynucleotide sequences that function at the level of supporting
mRNA accumulation, increasing mRNA stability or through the
facilitation of ribosome entry all of which mechanisms produce
greater levels of translation. In particular embodiments of the
invention, certain 5' untranslated regions and introns can be
combined with promoters and enhancers to produce composite or
chimeric promoters capable of driving higher levels of gene
expression.
[0102] Examples of 5' untranslated regions useful for enhancing
gene expression include the adenovirus tripartite leader sequence
(Adtp) which can be inserted downstream of a promoter to increase
the expression of a of a gene or transgene by enhancing
translation, without modifying the specificity of the promoter. See
W. Sheay et al., Biotechniques 15(5):856-62 (1993). The 5'UTR of
the chimpanzee and mouse elongation factor 1 alpha (EF-1.alpha.)
mRNAs contains an intron known to enhance the gene expression
through increasing RNA transcription and/or RNA stability. See S.
Y. Kim et al., J Biotechnol. 14; 93(2):183-7 (1993). The 5'-UTR of
the mRNA encoding the eukaryotic initiation factor 4g (eIF4g) is
characterized by the presence of a putative internal ribosome entry
site (IRES) and displays a strong promoter activity. See B. Han B.
& J. T. Zhang Mol Cell Biol 22(21):7372-84 (2002). In addition,
the 5'UTR of human heat shock protein 70 (Hsp70) mRNA contains an
element that increases the efficiency of mRNA translation under
normal cell culture conditions by up to an order of magnitude. See
S. Vivinus et al.,. Eur J Biochem. 268(7):1908-17 (2001). The 5'UTR
of the NF-kappaB Repressing Factor acts as a potent IRES and also
functions as a translational enhancer in the context of
monocistronic mRNAs. See A. Oumard et al., Mol Cell Biol.
20(8):2755-9 (2000). When associated and added between the CAP and
the initiation codon, the SV40 5'UTR and the R region from human T
cell leukemia virus (HTLV) Type 1 Long Terminal Repeat (SUR)
increase translation efficiency possibly through mRNA
stabilization. See Y. Takebe et al., Mol Cell Biol. 8(1):466-472)
(1988).
[0103] In particular embodiments of the invention, regulatory
sequences for inclusion in a nucleic acid molecule, DNA plasmid or
vector of this invention include, without limitation, a promoter
sequence, an enhancer sequence, 5' untranslated region sequence,
intron, CTE, RTE, a polyadenylation sequence, a splice donor
sequence and a splice acceptor sequence, a site for transcription
initiation and termination positioned at the beginning and the end,
respectively, of the gene to be translated, a ribosome binding site
for translation in the transcribed region, an epitope tag, a
nuclear localization sequence, an internal ribosome entry site
(IRES) element, a Goldberg-Hogness "TATA" element, a restriction
enzyme cleavage site, a selectable marker and the like. Enhancer
sequences include, e.g., the 72 bp tandem repeat of SV40 DNA or the
retroviral long terminal repeats or LTRs, etc. and are employed to
increase transcriptional efficiency. See Wasylyk, et al., Nucleic
Acid Res. 12:5589-5608 (1984).
[0104] These other components useful in DNA plasmids, including,
e.g., origins of replication, polyadenylation sequences (e.g.,
bovine growth hormone (BGH) polyA, simian virus 40 (SV40) polyA),
drug resistance markers (e.g., kanamycin resistance), and the like,
may also be selected from among widely known sequences, including
those described in the examples and mentioned specifically
below.
[0105] Selection of individual promoters and other common plasmid
elements are conventional and many such sequences are available
with which to design the plasmids useful in this invention. See,
e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, (1989) and references cited
therein at, for example, pages 3.18-3.26 and 16.17-16.27 and
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, New York (1989). All components of the plasmids useful
in this invention may be readily selected by one of skill in the
art from among known materials in the art and available from the
pharmaceutical industry.
[0106] Examples of suitable genes, which express antigens or
polypeptides, are identified in the discussion below. In one
embodiment of the plasmids and immunogenic compositions herein, the
selected antigens are HIV-1 antigens, including those expressed by
the gag, pol, env, nef, vpr, vpu, vif and tat genes. In one
embodiment, the coding and noncoding sequence and other components
of the DNA plasmid are optimized, such as by codon selection
appropriate to the intended host and by removal of any inhibitory
sequences, also discussed below with regard to antigen
preparation.
[0107] According to embodiments of the present invention, a
composition contains one plasmid expressing at least three selected
antigens. Alternatively, the plasmid composition also comprises one
DNA plasmid comprising a DNA sequence encoding at least three
copies of the same selected antigen or polypeptide of interest. In
one embodiment of the present invention, a composition may contain
one plasmid expressing multiple selected antigens from multiple
open reading frames. In another embodiment, the plasmid composition
comprises one DNA plasmid comprising a DNA sequence encoding
multiple copies of similar open reading frames encoding multiple
selected antigens, for example multiple env genes from different
clades.
[0108] In a particular embodiment of the invention, the use of
combinations of plasmids, each expressing a single antigen, may
lead to a more effective immunogenic composition. For example, in
one embodiment, the present invention provides an immunogenic
composition where the immunogenic composition contains four
plasmids, each encoding an HIV immunogen or an adjuvant. One such
specific immunogenic composition contains the following combination
of four plasmids: (a) a first DNA plasmid that has a single
transcriptional unit with a nucleotide sequence that encodes an HIV
envelope polypeptide; (b) a second DNA plasmid that has a single
transcriptional unit with a nucleotide sequence that encodes an HIV
gag-pol fusion polypeptide; (c) a third DNA plasmid that has a
single transcriptional unit with a nucleotide sequence that encodes
an HIV nef-tat-vif fusion polypeptide; (d) a fourth DNA plasmid
that has a nucleotide sequence that encodes an adjuvant
polypeptide; and (e) at least one of a pharmaceutically acceptable
diluent, carrier or transfection facilitating agent. In a specific
embodiment, the promoter driving the expression of each of the HIV
genes is the HCMV promoter and the polyA sequence for each of the
HIV genes is the bovine growth hormone polyA.
[0109] In a specific embodiment of the invention, where the use of
combinations of plasmids each expressing a single antigen is
desired, it may be advantageous to use more plasmids containing
more individual genes encoding individual polypeptides and fewer
fusion genes encoding fusion polypeptides. For example, in one
embodiment the present invention provides an immunogenic
composition where the immunogenic composition contains five
plasmids each encoding and an HIV immunogen or an adjuvant. In this
embodiment, the immunogenic composition comprises: (a) a first DNA
plasmid that has a single transcriptional unit with a nucleotide
sequence that encodes an HIV envelope polypeptide; (b) a second DNA
plasmid that has a single transcriptional unit with a nucleotide
sequence that encodes an HIV gag polypeptide; (c) a third DNA
plasmid that has a single transcriptional unit with a nucleotide
sequence that encodes an HIV pol polypeptide; (d) a fourth DNA
plasmid that has a single transcriptional unit with a nucleotide
sequence that encodes an HIV nef-tat-vif fusion polypeptide; (e) a
fifth DNA plasmid that has a nucleotide sequence that encodes an
adjuvant polypeptide. In a specific embodiment, the promoter
driving the expression of each of the HIV genes is the HCMV
promoter and the polyA sequence for each of the HIV genes is the
bovine growth hormone polyA.
[0110] In still a further embodiment, the DNA plasmids and
immunogenic compositions may further contain, as an individual DNA
plasmid component or as part of the antigen-containing DNA plasmid,
a nucleotide sequence that encodes a desirable cytokine, lymphokine
or other genetic adjuvant. A description of such suitable adjuvants
for which nucleic acid sequences are available is provided below.
In the embodiments exemplified in this invention, a desirable
cytokine for administration with the DNA plasmid composition of
this invention is Interleukin-12.
[0111] The DNA plasmid composition may be administered in a
pharmaceutically acceptable diluent, excipient or carrier, such as
those discussed below. Although the composition may be administered
by any selected route of administration, in one embodiment a
desirable method of administration is coadministration
intramuscularly of a composition comprising the plasmid molecules
with bupivacaine as the transfection facilitating agent, described
below.
B. Physical Arrangement of Elements within the Plasmid
[0112] A practical consideration for designing a vertebrate
immunogenic composition is the amount of DNA that can be
effectively administered when immunizing subjects. When dose is
considered, limiting the total size of the plasmid, while
simultaneously maximizing the number of complete transcriptional
units within the plasmid provides a strategy for creating plasmid
DNA designs. The advantages of minimizing plasmid size and
maximizing the number of genes expressed are that dose of
immunogenic protein delivered per microgram of DNA injected is
enhanced. In addition, is known that as vector size increases, so
does the potential for vector instability. See Herrera et al.,
Biochem. Biophys. Res. Commun. 279:548-551 (2000). Therefore to
achieve this goal, the size of the individual regulatory control
elements, such as the promoters, should be considered and balanced
with the strength of the promoter required for a given expression
level. Similarly, the size of open reading frames contributes to
the overall size of the plasmid. As used herein, DNA regions in
between transcriptional units, which are occupied by DNA not having
a regulatory or selected antigen encoding role, are referred to
herein as "spacer regions". The size of the spacer regions is
important in determining the level of transcriptional interference
between transcriptional units, the level of steric hindrance and
the total plasmid size. Therefore, the size of each element,
whether it is protein coding, regulatory control or a spacer region
must be carefully considered and limited to the smallest effective
numbers of base pairs.
[0113] Embodiments of the present invention provide a triple
transcriptional unit DNA plasmid that is less than or equal to
about 18 kilo base pairs (kb) of DNA in total length. In an
alternate embodiment, the present invention provides a triple
transcriptional unit DNA plasmid that is less than or equal to
about 17 kb of DNA in total length. Another embodiment of the
present invention provides a triple transcriptional unit DNA
plasmid that is less than or equal to about 16 kb of DNA in total
length. A certain embodiment of the present invention provides a
triple transcriptional unit DNA plasmid that is less than or equal
to about 15 kb of DNA in total length. Still another embodiment of
the present invention provides a triple transcriptional unit DNA
plasmid that is less than or equal to about 14 kb of DNA in total
length. A specific embodiment of the present invention provides a
triple transcriptional unit DNA plasmid that is less than or equal
to about 13 kb of DNA in total length. A particular embodiment of
the present invention provides a triple transcriptional unit DNA
plasmid that is less than or equal to about 12 kb of DNA in total
length. Another embodiment of the present invention provides a
triple transcriptional unit DNA plasmid that is less than or equal
to about 11 kb of DNA in total length.
[0114] As used herein, "about" or "approximately" shall generally
mean within 20 percent of a given value or range.
[0115] As defined in FIG. 1, orientation of the direction of
transcription between the three transcriptional units is another
consideration for DNA plasmid design. One of skill in the art of
molecular biology would appreciate that in a circular DNA plasmid,
there are only two directions of transcription. Therefore, in a
plasmid with three transcriptional units, at least two of them will
be going in the same direction. In a certain embodiment of the
invention, the direction of transcription for the first
transcriptional unit is in the opposite direction from the
direction of expression of the second transcriptional unit. In
another embodiment of the invention, the direction of transcription
for the first transcriptional unit is in the opposite direction
from the direction of expression of the second transcriptional unit
and the direction of transcription of the third transcriptional
unit is in the same direction as the second transcriptional unit.
In still another embodiment of the invention, the direction of
transcription for the first transcriptional unit is in the opposite
direction from the direction of expression of the second
transcriptional unit and the direction of transcription of the
third transcriptional unit is in the same direction as the first
transcriptional unit.
[0116] One of skill in the art will appreciate that the numbering
of the transcriptional units as "first", "second" and "third" is
for convenience only. The three transcriptional units can be
arranged in any order around the plasmid.
[0117] In a plasmid with two transcriptional units, certain
constraints exist regarding the direction of transcription for the
two transcriptional units. If the directions of the transcription
for the two transcriptional units are in the opposite direction,
then the two transcriptional units may be separated by a spacer
region of as small as 200 bp from one another, alternatively by a
spacer region of small as 300 bp from one another, or alternatively
by a spacer region of small as 400 bp from one another.
[0118] In a plasmid with two transcriptional units, if the
directions of the transcription for the two transcriptional units
are in the same directions, then the two transcriptional units
should be separated by a spacer region of at least about 500 bp
from one another. In another embodiment, the two transcriptional
units should be separated by a spacer region of at least about 600
bp from one another. In still another embodiment, the two
transcriptional units should be separated by a spacer region of at
least about 700 bp from one another. In a certain embodiment, the
two transcriptional units should be separated by a spacer region of
at least about 800 bp from one another. In another embodiment, the
two transcriptional units should be separated by a spacer region of
at least about 900 bp from one another. In still another
embodiment, the two transcriptional units should be separated by a
spacer region of at least about 1000 bp from one another.
[0119] In another embodiment of the invention, the direction of
transcription for the first transcriptional unit is in the same
direction as the direction of expression of the second
transcriptional unit. In still another embodiment of the invention,
the direction of transcription for the first transcriptional unit
is in the same direction as the direction of expression of the
second transcriptional unit and the direction of transcription of
the third transcriptional unit is in the same direction as the
second transcriptional unit. In a particular embodiment of the
invention, the direction of transcription for the first
transcriptional unit is in the same direction as the direction of
expression of the second transcriptional unit and the direction of
transcription of the third transcriptional unit is in the opposite
direction as the first transcriptional unit.
[0120] The size of the spacer regions is one variable that can be
manipulated to relieve transcriptional interference between
transcriptional units, decrease steric hindrance and to control
overall plasmid size. In the embodiment shown in FIG. 1, there is a
spacer region separating transcriptional units 1 and 2 that is
located in between the SCMV and HCMV promoters. As used herein, the
spacer region separating transcriptional units 1 and 2 is known as
"spacer region 1." In the embodiment shown in FIG. 1, there is a
spacer region separating transcriptional units 2 and 3 that is
located in between the SV 40 poly A and HSV Lap 1 promoter. As used
herein, the spacer region separating transcriptional units 2 and 3
is known as "spacer region 2." In the embodiment shown in FIG. 1,
there is a third spacer region separating transcriptional units 3
and 1 that is located in between the BGH poly A and rabbit
betaglobin poly A. As used herein, the spacer region separating
transcriptional units 3 and 1 is known as "spacer region 3." See
FIG. 1.
[0121] Another feature of the invention is that overall plasmid
size may be minimized by using the spacer regions of the eukaryotic
plasmid to fulfill plasmid and or adjuvant functions. For example,
in the embodiment shown in FIG. 1, spacer region 3 also includes
the bacterial origin of replication. In addition, in the embodiment
shown in FIG. 1, spacer region 2 includes the kanamycin gene for
growth in bacteria. In other embodiments, the spacer regions
include CpG island sequences for stimulating the immune response.
In another embodiment, the spacer regions include CTE and or RTE
sequences for enhancing expression of antigens. In still another
embodiment of the invention, the spacer region can include enhancer
sequences. In another embodiment of the invention, the spacer
region can include untranslated sequences known to be useful in
enhancing expression.
[0122] In one embodiment of the invention, spacer region 1 is less
than about 5 kb, alternatively less than about 4 kb in size. In
another embodiment of the invention, spacer region 1 is less than
less than about 3 kb, alternatively less than about 2 kb in size.
In a certain embodiment of the invention, spacer region 1 is less
than about 1 kb in size. In a particular embodiment of the
invention, spacer region 1 is between about 800 base pairs (bp) and
about 1000 bp in size. In an alternate embodiment of the invention,
spacer region 1 is between about 600 bp and about 800 bp in size.
In a certain embodiment of the invention, spacer region 1 is
between about 400 bp and about 600 bp in size. In another
embodiment of the invention, spacer region 1 is between about 300
bp and about 400 bp in size. In another embodiment of the
invention, spacer region 1 is less than about 400 bp in size. In a
specific embodiment of the invention, spacer region 1 is between
about 200 bp and about 300 bp in size. In a particular embodiment
of the invention, spacer region 1 is between about 100 bp and about
200 bp in size. In another embodiment of the invention, spacer
region 1 is between about 10 bp and about 100 bp in size.
[0123] In one embodiment of the invention, spacer region 2 is less
than less than about 5 kb, alternatively less than about 4 kb in
size. In another embodiment of the invention, spacer region 2 is
less than less than about 3 kb, alternatively less than about 2 kb
in size. In a certain embodiment of the invention, spacer region 2
is less than about 1 kb in size. In another embodiment of the
invention, spacer region 2 is less than about 1100 bp in size. In a
particular embodiment of the invention, spacer region 2 is between
about 800 base pairs (bp) and about 1000 bp in size. In an
alternate embodiment of the invention, spacer region 2 is between
about 600 bp and about 800 bp in size. In a certain embodiment of
the invention, spacer region 2 is between about 400 bp and about
600 bp in size. In another embodiment of the invention, spacer
region 2 is between about 300 bp and about 400 bp in size. In a
specific embodiment of the invention, spacer region 2 is between
about 200 bp and about 300 bp in size. In a particular embodiment
of the invention, spacer region 2 is between about 100 bp and about
200 bp in size. In another embodiment of the invention, spacer
region 2 is between about 10 bp and about 100 bp in size.
[0124] In one embodiment of the invention, spacer region 3 is less
than less than about 5 kb, alternatively less than about 4 kb in
size. In another embodiment of the invention, spacer region 3 is
less than less than about 3 kb, alternatively less than about 2 kb
in size. In a certain embodiment of the invention, spacer region 3
is less than about 1 kb in size. In another embodiment of the
invention, spacer region 3 is less than about 1100 bp in size. In a
particular embodiment of the invention, spacer region 3 is between
about 800 bp and about 1000 bp in size. In an alternate embodiment
of the invention, spacer region 3 is between about 600 bp and about
800 bp in size. In a certain embodiment of the invention, spacer
region 3 is between about 400 bp and about 600 bp in size. In
another embodiment of the invention, spacer region 3 is between
about 300 bp and about 400 bp in size. In a specific embodiment of
the invention, spacer region 3 is between about 200 bp and about
300 bp in size. In a particular embodiment of the invention, spacer
region 3 is between about 100 bp and about 200 bp in size. In
another embodiment of the invention, spacer region 3 is between
about 10 bp and about 100 bp in size.
C. Antigens Expressed by Immunogenic Compositions of this
Invention
[0125] As used herein, "polypeptide" refers to selected protein,
glycoprotein, peptide or other modified protein antigens, which are
encoded by the plasmids and immunogenic compositions of this
invention. Embodiments of the invention provide plasmids and
immunogenic compositions, which induce an immune response to
"polypeptides" in a vertebrate host to a selected antigen. As used
herein, the term "selected antigen" refers to these polypeptides.
The selected antigens, which comprise the polypeptides, when
expressed by the plasmid DNA, may include a protein, polyprotein,
polypeptide, peptide, fragment or a fusion thereof derived from a
pathogenic virus, bacterium, fungus, parasite, prion or
combinations thereof. Alternatively, the selected antigens, may
include a protein, polyprotein, polypeptide, peptide, fragment or
fusion thereof derived from a cancer cell or tumor cell. In another
embodiment, the selected antigens may include a protein,
polyprotein, polypeptide, peptide, fragment or fusion thereof
derived from an allergen so as to interfere with the production of
IgE so as to moderate allergic responses to the allergen. In still
another embodiment, the selected antigens may include a protein,
polyprotein, polypeptide, peptide, fragment or fusion thereof
derived from a molecule or portion thereof which represents those
produced by a host (a self molecule) in an undesired manner, amount
or location, such as those from amyloid precursor protein, so as to
prevent or treat disease characterized by amyloid deposition in a
vertebrate host. In one embodiment of this invention, the selected
antigens may include a protein, polyprotein, polypeptide, peptide
or fragment derived from HIV-1.
[0126] Embodiments of the present invention are also directed to
immunogenic compositions comprising a plasmid encoding the selected
antigens (1) from a pathogenic virus, bacterium, fungus or parasite
to elicit the immune response in a vertebrate host, or (2) from a
cancer antigen or tumor-associated antigen from a cancer cell or
tumor cell to elicit a therapeutic or prophylactic anti-cancer
effect in a mammalian subject, or (3) from an allergen so as to
interfere with the production of IgE so as to moderate allergic
responses to the allergen, or (4) from a molecule or portion
thereof which represents those produced by a host (a self molecule)
in an undesired manner, amount or location, so as to reduce such an
undesired effect.
[0127] In another embodiment, a desirable immunogenic composition
may utilize a triple transcriptional unit plasmid of this
invention, which encodes selected antigens to induce an immune
response aimed at preventing or to treating one of the following
viral diseases: Human immunodeficiency virus, Simian
immunodeficiency virus, Respiratory syncytial virus, Parainfluenza
virus types 1-3, Influenza virus, Herpes simplex virus, Human
cytomegalovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C
virus, Human papillomavirus, Poliovirus, rotavirus, caliciviruses,
Measles virus, Mumps virus, Rubella virus, adenovirus, rabies
virus, canine distemper virus, rinderpest virus, Human
metapneumovirus, avian pneumovirus (formerly turkey rhinotracheitis
virus), Hendra virus, Nipah virus, coronavirus, parvovirus,
infectious rhinotracheitis viruses, feline leukemia virus, feline
infectious peritonitis virus, avian infectious bursal disease
virus, Newcastle disease virus, Marek's disease virus, porcine
respiratory and reproductive syndrome virus, equine arteritis virus
and various Encephalitis viruses, and Coronavirus, such as SARS
virus.
[0128] In a particular embodiment, immunogenic compositions
comprising the triple transcriptional unit plasmids of this
invention include those encoding selected antigens from pathogens
causing emerging diseases such as severe acute respiratory virus
(SARS), human herpes virus 8 (HHV-8), Hantaanvirus, Vibrio cholera
0139, Helicobacter pylori and Borrelia burgdorferi.
[0129] In another embodiment, immunogenic compositions comprising
the plasmids of this invention include those directed to the
prevention and/or treatment of bacterial diseases caused by,
without limitation, Haemophilus influenzae (both typable and
nontypable), Haemophilus somnus, Moraxella catarrhalis,
Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus
agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria
meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis,
Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis,
Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium,
Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio
cholerae, Corynebacterium diphtheriae, Mycobacterium tuberculosis,
Mycobacterium avium-Mycobacterium intracellulare complex, Proteus
mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus
epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia
burgdorferi, Pasteurella haemolytica, Pasteurella multocida,
Actinobacillus pleuropneumoniae and Mycoplasma gallisepticum.
[0130] Embodiments of the present invention are also directed to
immunogenic compositions comprising a plasmid encoding selected
antigens from, without limitation, Aspergillis, Blastomyces,
Candida, Coccidiodes, Cryptococcus and Histoplasma. In certain
embodiments, such immunogenic compositions comprising a plasmid
encoding selected antigens from fungi are used for the prevention
and/or treatment of fungal disease.
[0131] In another embodiment, of the present invention are also
directed to immunogenic compositions comprising a plasmid encoding
selected antigens from, without limitation, Leishmania major,
Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium,
Trichomonas, Toxoplasma gondii and Pneumocystis carinii. In
particular embodiments, such immunogenic compositions comprising a
plasmid encoding selected antigens of parasites are used for the
prevention and/or treatment of parasitic disease.
[0132] In a particular embodiment, this invention provides
immunogenic compositions for eliciting a therapeutic or
prophylactic anti-cancer effect in a vertebrate host, which
comprise a plasmid encoding a selected antigen such as a cancer
antigen or tumor-associated antigen, including, without limitation,
prostate specific antigen, carcino-embryonic antigen, MUC-1, Her2,
CA-125 and MAGE-3. In some embodiments, the same antigen or
variants of the antigen may be placed in multiple transcriptional
units to enhance transcription and ultimate dose of a particular
target antigen.
[0133] Embodiments of the invention, also provide immunogenic
compositions comprising plasmids encoding selected antigens that
are allergens for use in moderating responses to allergens in a
vertebrate host, include those containing an allergen or fragment
thereof. Examples of such allergens are described in U.S. Pat. No.
5,830,877 and International Patent Publication No. WO99/51259,
which are hereby incorporated by reference. Such allergens include,
without limitation, pollen, insect venoms, animal dander, fungal
spores and drugs. The immunogenic compositions of the invention may
be used to interfere with the production of IgE antibodies, a known
cause of allergic reactions.
[0134] Embodiments of the present invention are also directed to
immunogenic compositions comprising a plasmid encoding selected
antigens for moderating responses to self molecules in a vertebrate
host. The selected antigens include those containing a self
molecule or a fragment thereof. Examples of such self molecules
include the e-chain of insulin that is involved in diabetes, the
G17 molecule involved in gastroesophageal reflux disease, and
antigens which down regulate autoimmune responses in diseases such
as multiple sclerosis, lupus and rheumatoid arthritis. Also
included is the .beta.-amyloid peptide (also referred to as A.beta.
peptide), which is an internal, 39-43 amino acid fragment of
amyloid precursor protein (APP), which is generated by processing
of APP by the .beta. and .gamma. secretase enzymes. The A.beta.1-42
peptide has the following sequence: Asp Ala Glu Phe Arg His Asp Ser
Gly Tyr Glu Val H is H is Gln Lys Leu Val Phe Phe Ala Glu Asp Val
Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile
Ala (SEQ ID NO:1).
[0135] It is also desirable in the selection and use of the
sequences encoding the selected antigens for design of the DNA
plasmids of this invention to alter codon usage of the selected
antigens encoding gene sequences, as well as the DNA plasmids into
which they are inserted, in order to increase the expression of the
antigens and/or to remove inhibitory sequences therein. The removal
of inhibitory sequences can be accomplished by using the technology
discussed in detail in U.S. Pat. Nos. 5,965,726; 5,972,596;
6,174,666; 6,291,664; and 6,414,132; and in International Patent
Publication No. WO01/46408, incorporated by reference herein.
Briefly described, this technology involves mutating identified
inhibitor/instability sequences in the selected gene, preferably
with multiple point mutations.
[0136] As one specific embodiment exemplified below, the DNA
plasmid and immunogenic compositions of this invention desirably
employ one or more sequences optimized for HIV-1 genes, such as the
gag, pol, env nef, tat, and vif.
[0137] The triple transcriptional unit plasmid of this invention is
also suitable for use to transfect, transform or infect a host cell
to express three or more proteins of polypeptides in vitro.
D. Promoters Useful in the Transcriptional Units
[0138] The DNA plasmids of the invention comprise one, two or three
transcriptional units. Each transcriptional unit comprises at least
one promoter. Therefore, in certain embodiments of the invention,
the nucleic acid encoding a selected antigen is under
transcriptional control of a promoter. A "promoter" refers to a DNA
sequence recognized by the synthetic machinery of the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a gene. The phrase "under transcriptional control"
means that the promoter is in the correct location and orientation
in relation to the nucleic acid to control RNA polymerase
initiation and transcription of the gene.
[0139] The term promoter is used herein to refer to a group of
transcriptional control modules that are clustered around the
initiation site for the RNA polymerase. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0140] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0141] Suitable promoters for use in any of the transcriptional
units include all promoters active in eukaryotic cells. Examples of
suitable eukaryotic promoters include human cytomegalovirus (HCMV)
immediate early promoter (optionally with the HCMV enhancer) (see,
e.g., Boshart et al, Cell, 41:521-530 (1985)), the simian
cytomegalovirus (SCMV) promoter, the murine cytomegalovirus (MCMV)
promoter, the herpes simplex virus (HSV) LAP1 promoter, the simian
virus 40 (SV40) promoter, the Human elongation factor 1 alpha
promoter, the retroviral long terminal repeats (LTRs), the muscle
cell specific desmin promoter, or any other promoter active in an
antigen presenting cell.
[0142] In addition, suitable eukaryotic promoters may be
characterized as being selected from among constitutive promoters,
inducible promoters, tissue-specific promoters and others. Examples
of constitutive promoters that are non-specific in activity and
employed in the DNA plasmids encoding selected antigens include,
without limitation, the retroviral Rous sarcoma virus (RSV)
promoter, the retroviral LTR promoter (optionally with the RSV
enhancer), the SV40 promoter, the dihydrofolate reductase promoter,
the .beta.-actin promoter, the phosphoglycerol kinase (PGK)
promoter, and the EF1.alpha. promoter (Invitrogen). Inducible
promoters that are regulated by exogenously supplied compounds,
include, without limitation, the arabinose promoter, the
zinc-inducible sheep metallothionine (MT) promoter, the
dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV)
promoter, the T7 polymerase promoter system (WO 98/10088); the
ecodysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA,
93:3346-3351(1996)), the tetracycline-repressible system (Gossen et
al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the
tetracycline-inducible system (Gossen et al, Science,
268:1766-1769, (1995) see also Harvey et al., Curr. Opin. Chem.
Biol., 2:512-518, (1998)), the RU486-inducible system (Wang et al,
Nat. Biotech., 15:239-243, (1997) and Wang et al, Gene Ther.,
4:432-441, (1997)) and the rapamycin-inducible system (Magari et
al., J. Clin. Invest., 100: 2865-2872, (1997)).
[0143] Other types of inducible promoters that may be useful in DNA
plasmids of the invention are those regulated by a specific
physiological state, e.g., temperature or acute phase or in
replicating cells only. Useful tissue-specific promoters include
the promoters from genes encoding skeletal .beta.-actin, myosin
light chain 2A, dystrophin, muscle creatine kinase, as well as
synthetic muscle promoters with activities higher than
naturally-occurring promoters (see Li et al., Nat. Biotech.,
17:241-245, (1999)). Examples of promoters that are tissue-specific
are known for the liver (albumin, Miyatake et a. J. Virol.,
71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al.,
Gene Ther., 3: 1002-9, (1996); alpha-fetoprotein (AFP), Arbuthnot
et al., Hum. Gene Ther., 7:1503-14, (1996)), bone (osteocalcin,
Stein et al., Mol. Biol. Rep., 24:185-96, (1997); bone
sialoprotein, Chen et al., J. Bone Miner. Res., 11:654-64, (1996)),
lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8, (1988);
immunoglobulin heavy chain; T cell receptor .alpha. chain),
neuronal (neuron-specific enolase (NSE) promoter, Andersen et al.
Cell. Mol. Neurobiol., 13:503-15, (1993); neurofilament light-chain
gene, Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5,
(1991); the neuron-specific ngf gene, Piccioli et al., Neuron,
15:373-84, (1995)); among others. See, e.g., International Patent
Publication No. WO00/55335 for additional lists of known promoters
useful in this context.
E. Polyadenylation Signals Useful in the Transcription Units
[0144] The DNA plasmids of the invention comprise three
transcriptional units and each transcriptional unit comprises at
least one polyadenylation signal. A "polyadenylation signal", as
defined herein refers to a stop sequence (or stop site) that
terminates transcription of a particular transcriptional unit and
ensures that the nucleic acid sequence encoding a polypeptide is
transcribed and translated properly. The stop site can be synthetic
or of natural origin. Examples of stop sites include, but are not
limited to, a polyadenylation signal and a synthetic bi-directional
transcriptional stop site. Typically, the polyadenylation signal
arrests transcription of DNA sequences.
[0145] Suitable polyadenylation signals for use in any of the
transcriptional units include all polyadenylation signals active in
eukaryotic cells. Examples of eukaryotic polyadenylation signals
include rabbit beta-globin poly(A) signal, a signal that has been
characterized in the literature as strong (Gil and Proudfoot, Cell
49: 399-406 (1987); Gil and Proudfoot, Nature 312: 473-474 (1984)).
One of its key features is the structure of its downstream element,
which contains both UG- and U-rich domains. Other poly A signals
include synthetic polyA, HSV Thymidine kinase poly A, (see Cole, C.
N. and T. P. Stacy, Mol. Cell. Biol. 5:2104-2113 (1985)); Human
alpha globin poly A SV40 poly A (See Schek, N, Cooke, C., and J. C.
Alwine, Mol. Cell Biol. 12:5386-5393 (1992)); human beta globin
poly A (See Gil, A., and N. J. Proudfoot, Cell 49:399-406 (1987));
polyomavirus poly A (See Batt, D. B and G. G. Carmichael Mol. Cell.
Biol. 15:4783-4790 (1995); Bovine growth hormone poly A, (Gimmi, E.
R., Reff, M. E., and I. C. Deckman, Nucleic Acid Res. (1989)). Many
other polyadenylation signals are known in the art, and will also
be useful in embodiments of the invention.
[0146] Both the early and late polyadenylation signals of SV40 are
useful in the various embodiments of the invention. See Schek, et
al., Mol. Cell Biol. 12:5386-5393 (1992). These sequences are
encoded within the 237-base pair fragment between the BamnHI site
at nucleotide 2533 and the BclI site at nucleotide 2770 of the SV40
genome (Carswell and Alwine, Mol. Cell. Biol. 9:4248; 1989).
Carswell and Alwine concluded that, of the two SV40 polyadenylation
signals, the late signal was more efficient, most likely because it
comprises both downstream and upstream sequence elements that
facilitate efficient cleavage and polyadenylation.
[0147] Additional polyadenylation sites can be identified or
constructed using methods that are known in the art. A minimal
polyadenylation site is composed of AAUAAA and a second recognition
sequence, generally a G/U rich sequence, found about 30 nucleotides
downstream. As used herein, the sequences are presented as DNA,
rather than RNA, to facilitate preparation of suitable DNAs for
incorporation into expression vectors. When presented as DNA, the
polyadenylation site is composed of AATAAA, with, for example, a
G/T rich region downstream. Both sequences must be present to form
an efficient polyadenylation site. The purpose of these sites is to
recruit specific RNA binding proteins to the RNA. The AAUAAA binds
cleavage polyadenylation specificity factor (CPSF; Murthy K. G.,
and Manley J. L. (1995), Genes Dev 9:2672-2683), and second site,
frequently a G/U sequence, binds to Cleavage stimulatory factor
(CstF; Takagaki Y. and Manley J. L. (1997) Mol Cell Biol
17:3907-3914). CstF is composed of several proteins, but the
protein responsible for RNA binding is CstF-64, a member of the
ribonucleoprotein domain family of proteins (Takagaki et al. (1992)
Proc Natl Acad Sci USA 89:1403-1407).
F. Carriers, Diluents, Facilitating Agents, Adjuvants and
Formulations Useful for the Immunogenic Compositions of this
Invention
[0148] The DNA plasmids and immunogenic compositions useful in this
invention, further comprise an pharmaceutically acceptable diluent,
excipient or a pharmaceutically acceptable carrier. In one
embodiment, said pharmaceutically acceptable diluent is sterile
water, sterile isotonic saline or a biological buffer. The
antigenic compositions may also be mixed with such diluents or
carriers in a conventional manner. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with administration to humans or other vertebrate
hosts. The appropriate carrier is evident to those skilled in the
art and will depend in large part upon the route of
administration.
[0149] Still additional excipients that may be present in the
immunogenic compositions of this invention are adjuvants,
facilitating agents, preservatives, surface active agents, and
chemical stabilizers, suspending or dispersing agents. Typically,
stabilizers, adjuvants, and preservatives are optimized to
determine the best formulation for efficacy in the human or
veterinary subjects.
1. Adjuvants
[0150] An adjuvant is a substance that enhances the immune response
when administered together with an immunogen or antigen. A number
of cytokines or lymphokines have been shown to have immune
modulating activity, and thus may be used as adjuvants, including,
but not limited to, the interleukins 1-.alpha., 1-.beta., 2, 4, 5,
6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15,
16, 17 and 18 (and its mutant forms), the interferons-.alpha.,
.beta. and .gamma., granulocyte-macrophage colony stimulating
factor (see, e.g., U.S. Pat. No. 5,078,996 and ATCC Accession
Number 39900), macrophage colony stimulating factor (MCSF),
granulocyte colony stimulating factor (GCSF), and the tumor
necrosis factors .alpha. and .beta. (TNF). Still other adjuvants
useful in this invention include a chemokine, including without
limitation, MCP-1, MIP-1.alpha., MIP-1.beta., and RANTES. Adhesion
molecules, such as a selectin, e.g., L-selectin, P-selectin and
E-selectin may also be useful as adjuvants. Still other useful
adjuvants include, without limitation, a mucin-like molecule, e.g.,
CD34, GlyCAM-1 and MadCAM-1, a member of the integrin family such
as LFA-1, VLA-1, Mac-1 and p150.95, a member of the immunoglobulin
superfamily such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3,
CD2 and LFA-3, co-stimulatory molecules such as CD40 and CD40L,
growth factors including vascular growth factor, nerve growth
factor, fibroblast growth factor, epidermal growth factor, B7.1,
B7.2, PDGF, BL-1, and vascular endothelial growth factor, receptor
molecules including Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3,
TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2,
and DR6. Still another adjuvant molecule includes Caspase (ICE).
See, also International Patent Publication Nos. WO98/17799 and
WO99/43839, incorporated herein by reference.
[0151] In one embodiment, the desired adjuvant is IL-12 protein,
which is expressed from a plasmid. See, e.g., U.S. Pat. Nos.
5,457,038; 5,648,467; 5,723,127 and 6,168,923, incorporated by
reference herein. In one embodiment, the cytokine may be
administered as a protein. In a certain embodiment, IL-12 is
expressed from one or two of the three transcriptional units of the
DNA plasmid of the invention. Alternatively, Il-12 is expressed
independently from a separate plasmid. In another embodiment, a
plasmid encoding and expressing IL-15 is administered instead of a
plasmid encoding and expressing IL-12.
[0152] Suitable adjuvants used to enhance an immune response
include, without limitation, MPL.TM. (3-O-deacylated monophosphoryl
lipid A; Corixa, Hamilton, Mont.), which is described in U.S. Pat.
No. 4,912,094, which is hereby incorporated by reference. Also
suitable for use as adjuvants are synthetic lipid A analogs or
aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or
analogs thereof, which are available from Corixa (Hamilton, Mont.),
and which are described in U.S. Pat. No. 6,113,918, which is hereby
incorporated by reference. One such AGP is
2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl
2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-t-
etradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is
also known as 529 (formerly known as RC529). This 529 adjuvant is
formulated as an aqueous form or as a stable emulsion.
[0153] Still other adjuvants include mineral oil and water
emulsions, aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, etc., Amphigen, Avridine, L121/squalene,
D-lactide-polylactide/glycoside, pluronic polyols, muramyl
dipeptide, killed Bordetella, saponins, such as Stimulon.TM. QS-21
(Antigenics, Framingham, Mass.), described in U.S. Pat. No.
5,057,540, which is hereby incorporated by reference, and particles
generated therefrom such as ISCOMS (immunostimulating complexes),
Mycobacterium tuberculosis, bacterial lipopolysaccharides,
synthetic polynucleotides such as oligonucleotides containing a CpG
motif (U.S. Pat. No. 6,207,646, which is hereby incorporated by
reference), a pertussis toxin (PT), or an E. coli heat-labile toxin
(LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g.,
International Patent Publication Nos. WO 93/13302 and WO 92/19265,
incorporated herein by reference.
[0154] Also useful as adjuvants are cholera toxins and mutants
thereof, including those described in published International
Patent Application number WO 00/18434 (wherein the glutamic acid at
amino acid position 29 is replaced by another amino acid (other
than aspartic acid), preferably a histidine). Similar CT toxins or
mutants are described in published International Patent Application
number WO 02/098368 (wherein the isoleucine at amino acid position
16 is replaced by another amino acid, either alone or in
combination with the replacement of the serine at amino acid
position 68 by another amino acid; and/or wherein the valine at
amino acid position 72 is replaced by another amino acid). Other CT
toxins are described in published International Patent Application
number WO 02/098369 (wherein the arginine at amino acid position 25
is replaced by another amino acid; and/or an amino acid is inserted
at amino acid position 49; and/or two amino acids are inserted at
amino acid positions 35 and 36).
[0155] In some embodiments, plasmid DNA that encodes an adjuvant
may be administered in an immunogenic composition. In such cases,
an adjuvant whose DNA is inserted into a plasmid for inclusion in
the immunogenic compositions of the invention includes, but are not
limited to, interleukin-1 (IL-1), IL-5, IL-10, IL-12, IL-15, IL-18,
TNF-.alpha., TNF-.beta. and BL-1 (as described in published
International Patent Application WO 98/17799); B7.2 (as described
in published International Patent Application WO 00/51432); IL-8,
RANTES, G-CSF, IL-4, mutant IL-18, IL-7, TNF-R (as described in
published International Patent Application WO 99/43839); and mutant
CD80 (as described in published International Patent Application WO
00/66162). As used herein, the term "IL-12 protein" is meant to
refer to one or both human IL-12 subunits including single chain
IL-12 proteins in which the two subunits are encoded by a single
coding sequence and expressed as a single protein having a linker
sequences connecting the two subunits.
[0156] In a particular embodiment, the cytokine is administered as
a nucleic acid composition comprising a DNA sequence encoding the
cytokine under the control of regulatory sequences directing
expression thereof in a mammalian cell. In still another
embodiment, the cytokine-expressing plasmid is administered with
the DNA plasmid encoding selected antigens in an immunogenic
composition. In still another embodiment, the cytokine is
administered between the administrations of a priming immunogenic
composition and a boosting immunogenic composition. In yet another
embodiment, the cytokine is administered with the boosting step. In
still another embodiment, the cytokine is administered with both
priming and boosting compositions.
[0157] In certain embodiments of the invention, CpG DNA may be
included in the plasmid as an adjuvant. As used herein, CpG DNA
refers to an oligonucleotide containing at least one unmethylated
CpG dinucleotide nucleic acid molecule which contains an
unmethylated cytosine-guanine dinucleotide sequence (i.e. "CpG
DNA") or DNA containing a 5' cytosine followed by 3' guanosine and
linked by a phosphate bond) and activates the immune system. See
U.S. Pat. No. 6,406,705 to Davis et al., and U.S. Pat. No.
6,207,646 to Krieg et al., which are hereby incorporated by
reference in their entirety. CpG DNA from bacterial DNA, but not
vertebrate DNA, has direct immunostimulatory effects on peripheral
blood mononuclear cells (PBMC) in vitro. This lymphocyte activation
is due to unmethylated CpG dinucleotides, which are present at the
expected frequency in bacterial DNA (1/16), but are
under-represented (CpG suppression, 1/50 to 1/60) and methylated in
vertebrate DNA. It is has been suggested that the rapid immune
activation in response to CpG DNA may have evolved as one component
of the innate immune defense mechanisms that recognize structural
patterns specific to microbial molecules. See U.S. Pat. No.
6,406,705 to Davis et al., and U.S. Pat. No. 6,207,646 to Krieg et
al., which are hereby incorporated by reference in their
entirety.
[0158] In certain embodiments, the subject is administered a
combination of adjuvants, wherein the combination of adjuvants
includes at least one oligonucleotide containing at least one
unmethylated CpG DNA dinucleotide and at least one non-nucleic acid
adjuvant such as IL-12.
2. Facilitating Agents or Co-Agents
[0159] Immunogenic compositions composed of polynucleotide
molecules desirably contain optional excipients such as
polynucleotide transfection facilitating agents or "co-agents",
such as a local anesthetic, a peptide, a lipid including cationic
lipids, a liposome or lipidic particle, a polycation such as
polylysine, a branched, three-dimensional polycation such as a
dendrimer, a carbohydrate, a cationic amphiphile, a detergent, a
benzylammonium surfactant, or another compound that facilitates
polynucleotide transfer to cells. Such a facilitating agent
includes the local anesthetic bupivacaine or tetracaine (see U.S.
Pat. Nos. 5,593,972; 5,817,637; 5,380,876; 5,981,505 and 6,383,512
and International Patent Publication No. WO98/17799, which are
hereby incorporated by reference). Other non-exclusive examples of
such facilitating agents or co-agents useful in this invention are
described in U.S. Pat. Nos. 5,703,055; 5,739,118; 5,837,533;
International Patent Publication No. WO96/10038, published Apr. 4,
1996; and International Patent Publication No WO94/16737, published
Aug. 8, 1994, which are each incorporated herein by reference.
[0160] Most preferably, the transfection facilitating agent is
present in an amount that forms one or more complexes with the
nucleic acid molecules. When the transfection facilitating agent is
mixed with nucleic acid molecules or plasmids of this invention, it
forms a variety of small complexes or particles that pack the DNA
and are homogeneous. Thus, in one embodiment of the immunogenic
compositions of this invention, the complexes are formed by mixing
the transfection facilitating agent and at least one plasmid of
this invention.
[0161] In a particular embodiment, an immunogenic composition of
the invention may be comprised of more than one type of plasmid.
Alternatively, in another embodiment of the compositions of the
invention, the transfection facilitating agent may be pre-mixed
with each plasmid separately. The separate mixtures are then
combined in a single composition to ensure the desired ratio of the
plasmids is present in a single immunogenic composition, if all
plasmids are to be administered in a single bolus administration.
Alternatively, the transfection facilitating agent and each plasmid
may be mixed separately and administered separately to obtain the
desired ratio.
[0162] Where, hereafter, the term "complex" or "one or more
complexes" or "complexes" is used to define this embodiment of the
immunogenic composition, it is understood that the term encompasses
one or more complexes. Each complex contains a plasmid. Preferably,
the complexes are between about 50 to about 150 nm in diameter.
When the facilitating agent used is a local anesthetic, preferably
bupivacaine, an amount from about 0.1 weight percent to about 1.0
weight percent based on the total weight of the polynucleotide
composition is preferred. See, also, International Patent
Publication No. WO99/21591, which is hereby incorporated by
reference, and which teaches the incorporation of benzylammonium
surfactants as co-agents, preferably administered in an amount
between about 0.001-0.03 weight %. According to the present
invention, the amount of local anesthetic is present in a ratio to
said nucleic acid molecules of about 0.01-2.5% w/v local anesthetic
to about 1-10 .mu.g/ml nucleic acid. Another such range is about
0.05-1.25% w/v local anesthetic to about 100 .mu.g/ml to 1 mg/ml
nucleic acid.
3. Other Additives to the Immunogenic Compositions
[0163] Other excipients can be included in the immunogenic
compositions of this invention, including preservatives,
stabilizing ingredients, surface active agents, and the like.
[0164] Suitable exemplary preservatives include chlorobutanol,
potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the
parabens, ethyl vanillin, glycerin, phenol, and
parachlorophenol.
[0165] Suitable stabilizing ingredients that may be used include,
for example, casamino acids, sucrose, gelatin, phenol red, N-Z
amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate,
and dried milk.
[0166] Suitable surface active substances include, without
limitation, Freunds incomplete adjuvant, quinone analogs,
hexadecylamine, octadecylamine, octadecyl amino acid esters,
lysolecithin, dimethyl-dioctadecylammonium bromide),
methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g.,
pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl
peptide and dipeptide, dimethylglycine, tuftsin; oil emulsions; and
mineral gels, e.g., aluminum phosphate, etc. and immune stimulating
complexes (ISCOMS). The plasmids may also be incorporated into
liposomes for use as an immunogenic composition. The immunogenic
compositions may also contain other additives suitable for the
selected mode of administration of the immunogenic composition. The
immunogenic composition of the invention may also involve
lyophilized polynucleotides, which can be used with other
pharmaceutically acceptable excipients for developing powder,
liquid or suspension dosage forms. See, e.g., Remington: The
Science and Practice of Pharmacy, Vol. 2, 19.sup.th edition (1995),
e.g., Chapter 95 Aerosols; and International Patent Publication No.
WO99/45966, the teachings of which are hereby incorporated by
reference.
[0167] These immunogenic compositions can contain additives
suitable for administration via any conventional route of
administration. In some embodiments, the immunogenic composition of
the invention is prepared for administration to human subjects in
the form of, for example, liquids, powders, aerosols, tablets,
capsules, enteric-coated tablets or capsules, or suppositories.
Thus, the immunogenic compositions may also include, but are not
limited to, suspensions, solutions, emulsions in oily or aqueous
vehicles, pastes, and implantable sustained-release or
biodegradable formulations. In one embodiment of the invention, the
immunogenic compositions are prepared as a formulation for
parenteral administration, the active ingredient is provided in dry
(i.e., powder or granular) form for reconstitution with a suitable
vehicle (e.g., sterile pyrogen-free water) prior to parenteral
administration of the reconstituted composition. Other useful
parenterally-administrable formulations include those which
comprise the active ingredient in microcrystalline form, in a
liposomal preparation, or as a component of a biodegradable polymer
system. Compositions for sustained release or implantation may
comprise pharmaceutically acceptable polymeric or hydrophobic
materials such as an emulsion, an ion exchange resin, a sparingly
soluble polymer, or a sparingly soluble salt.
[0168] The immunogenic compositions of the present invention, are
not limited by the selection of the conventional, physiologically
acceptable carriers, diluents and excipients such as solvents,
buffers, adjuvants, facilitating agents or other ingredients useful
in pharmaceutical preparations of the types described above. The
preparation of these pharmaceutically acceptable compositions, from
the above-described components, having appropriate pH isotonicity,
stability and other conventional characteristics is within the
skill of the art.
F. Dosages and Routes of Administration, Electroporation for
Immunogenic Compositions
[0169] In general, selection of the appropriate "effective amount"
or dosage for the components of the immunogenic composition(s) of
the present invention will also be based upon the identity of the
selected antigens in the immunogenic composition(s) employed, as
well as the physical condition of the subject, most especially
including the general health, age and weight of the immunized
subject. The method and routes of administration and the presence
of additional components in the immunogenic compositions may also
affect the dosages and amounts of the DNA plasmid compositions.
Such selection and upward or downward adjustment of the effective
dose is within the skill of the art. The amount of plasmid required
to induce an immune response, preferably a protective response, or
produce an exogenous effect in the patient without significant
adverse side effects varies depending upon these factors. Suitable
doses are readily determined by persons skilled in the art.
[0170] The immunogenic compositions of this invention are
administered to a human or to a non-human vertebrate by a variety
of routes including, but not limited to, intranasal, oral, vaginal,
rectal, parenteral, intradermal, transdermal (see, e.g.,
International patent publication No. WO 98/20734, which is hereby
incorporated by reference), intramuscular, intraperitoneal,
subcutaneous, intravenous and intraarterial. The appropriate route
is selected depending on the nature of the immunogenic composition
used, and an evaluation of the age, weight, sex and general health
of the patient and the antigens present in the immunogenic
composition, and similar factors by an attending physician.
[0171] The order of immunogenic composition administration and the
time periods between individual administrations may be selected by
the attending physician or one of skill in the art based upon the
physical characteristics and precise responses of the host to the
application of the method. Such optimization is expected to be well
within the skill of the art.
[0172] In another embodiment, a method is provided for
co-expressing in a single cell, in vivo, one, two or three open
reading frames of discrete gene products, which comprises
introducing between about 0.1 .mu.g and about 100 mg of a
polynucleotide into the tissue of the mammal.
[0173] The immunogenic compositions may be administered and the
uptake of the plasmids enhanced by the use of electroporation at
the time of administration. To perform electroporation, electrodes
are placed about 1-4 mm apart, near the area where the
polynucleotide is injected. The exact position or design of the
electrodes can be varied so long as current is permitted to pass
through the muscle fibers perpendicular to their direction in the
area of the injected polynucleotide. See U.S. Pat. No. 5,273,525 to
G. A. Hofmann; U.S. Pat. No. 5,869,326 to G. A. Hofmann; U.S. Pat.
No. 5,993,434 to S. B. Dev, et al.; U.S. Pat. No. 6,014,584 to G.
A. Hofmann, et al.; U.S. Pat. No. 6,068,650 to G. A. Hofmann, et
al.; U.S. Pat. No. 6,096,020 to G. A. Hofmann; U.S. Pat. No.
6,233,482 to G. A. Hofmann, et al.; U.S. Pat. No. 6,241,701 to G.
A. Hofmann; U.S. Pat. No. 6,418,341 to G. A. Hofmann, et al.; U.S.
Pat. No. 6,451,002 to S. B. Dev, et al.; U.S. Pat. No. 6,516,223 to
G. A. Hofmann; U.S. Pat. No. 6,763,264 to G. A. Hofmann; U.S. Pat.
No. 6,110,161 to I. Mathiesen, et al.; all of which are
incorporated by reference in their entirety.
[0174] Once the electrodes are in position, the muscle is
electroporated or electrically stimulated. The stimulation is
delivered as a pulse having a predetermined amplitude and duration.
In order to optimize the transfection efficiencies, the parameters
of pulse duration, voltage, capacitance, field strength, number,
wave type may be varied and transfection efficiencies compared.
Electrical pulses are pulsed electric fields applied via
electroporation. The pulse can be unipolar, bipolar, exponential or
square wave form. Voltages have ranged from approximately 0 to 1000
volts; the pulse durations have ranged from 5 microseconds to 5
milliseconds; the number of pulses have ranged from a single pulse
to 30,000 pulses; and the pulse frequency within trains have ranged
from 0.5 Hz to 1000 Hz. Useful ranges for field strength are in the
range of from about 25 V/cm to about 800 V/cm. Electric pulses
contemplated for use in the practice of the present invention
include those pulses of sufficient voltage and duration to cause
electroporation. See Hofmann, G. A. Cells in electric fields. In E.
Neumann, A. E. Sowers, & C. A. Jordan (Eds.), Electroporation
and electrofusion in cell biology (pp. 389-407). Plenum Publishing
Corporation (1989).
G. Kit Components
[0175] In still another embodiment, the present invention provides
a pharmaceutical kit for ready administration of an immunogenic,
prophylactic, or therapeutic regimen for treatment of any of the
above-noted diseases or conditions for which an immune response to
a selected antigen is desired. This kit is designed for use in a
method of inducing a high level of antigen-specific immune response
in a mammalian or vertebrate subject. The kit contains at least one
immunogenic composition comprising a DNA plasmid comprising three
transcriptional units encoding a set of selected antigens or
peptides. Multiple prepackaged dosages of the immunogenic
compositions can be provided in the kit for multiple
administrations.
[0176] Where the above-described immunogenic compositions
comprising a DNA plasmid does not also express a cytokine or other
adjuvant, such as IL-12, the kit also optionally contains a
separate cytokine/adjuvant composition or multiple prepackaged
dosages of the cytokine/adjuvant composition for multiple
administrations. These cytokine compositions are generally nucleic
acid compositions comprising a DNA sequence encoding the selected
cytokine under the control of regulatory sequences directing
expression thereof in a mammalian or vertebrate cell. Other
adjuvants may optionally be provided in a prepackaged vial either
as a solution, liquid or solid.
[0177] The kit also contains instructions for using the immunogenic
compositions in a prime/boost method. The kits may also include
instructions for performing certain assays, various carriers,
excipients, diluents, adjuvants and the like above-described, as
well as apparatus for administration of the compositions, such as
syringes, spray devices, etc. Other components may include
disposable gloves, decontamination instructions, applicator sticks
or containers, among other compositions.
[0178] In order that this invention may be better understood, the
following examples are set forth. The examples are for the purpose
of illustration only and are not to be construed as limiting the
scope of the invention. All documents, publications and patents
cited in the following examples are incorporated by reference
herein.
EXAMPLES
Example 1
Selection and Modification of HIV Genes
[0179] One of skill in the art would appreciate that sequence
information from many viruses and bacteria is available in the art.
More particularly, sequence information can be used to clone genes
for use in expressing polypeptides in plasmids of the invention.
Information on many sequences from HIV and other pathogens is
available from the HIV sequence database at the Los Alamos National
Laboratory and the National Center for Biotechnology Information at
the United States National Library of Medicine, (8600 Rockville
Pike, Bethesda, Md. 20894).
[0180] In one embodiment of the invention, the following HIV genes
were selected for inclusion into a single exemplary DNA plasmid
expressing most of the HIV genome: gag gene from the HXB2 isolate
and the pol gene from the HXB2 isolate. The complete HXB2 sequence
is listed in the GenBank computer database under the accession
number K03455. The nef, tat and vif genes were derived from the
NL4-3 isolate. The complete NL4-3 sequence is listed in the GenBank
computer database under the accession number M19921. The HIV
envelope gene was derived from a primary isolate 6101 obtained from
Dr. David Montefiore. The complete HIV envelope sequence is listed
in the GenBank computer database under the accession numbers
AY612855 and bankit625244.
[0181] To allow for the inclusion of most of the HIV genome into a
single expression plasmid, gene fusions were prepared using full
length gag-pol genes and nearly full length nef-tat-vif genes. In
addition, the protease cleavage site between the gag and pol genes
was removed. All HIV genes used in the embodiments of this
invention were RNA optimized (sequence modified) for high-level
protein expression. See U.S. Pat. Nos. 5,965,726; 5,972,596;
6,174,666; 6,291,664; and 6,414,132.
[0182] Alternatively, the HIV genes may be optimized in accordance
with the methods provided in U.S. Application No. 60/576,819, filed
on Jun. 4, 2004. According to this method, the expression of genes
is enhanced by replacing certain wild type codons with "surrogate"
codons. The enhanced sequence of the polynucleotide is determined
by selecting suitable surrogate codons. Surrogate codons are
selected in order to alter the A and T (or A and U in the case of
RNA) content of the naturally-occurring (wild-type) gene. The
surrogate codons are those that encode the amino acids alanine,
arginine, glutamic acid, glycine, isoleucine, leucine, proline,
serine, threonine, and valine. Therefore, the modified nucleic acid
sequence has surrogate codons for each of these amino acids
throughout the sequence. For the remaining 11 amino acids, no
alterations are made, thereby leaving the corresponding
naturally-occurring codons in place.
[0183] Standard techniques were employed to modify the above HIV
genes to improve their safety and to optimize their expression. See
Sambrook J, Fritsch E F and Maniatis T. Molecular cloning: A
laboratory manual, 2.sup.nd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor N.Y. (1989). For example, the following
genetic modifications were used to enhance safety (i.e., by
inactivating viral enzymes) and maximize the breadth of HIV genes
included in a subsequent vector:
[0184] 1) Fusion polyproteins of HIV-1 gag-pol were created in a
single open reading frame by removing the gag terminator and pol
initiator from the respective genes and mutations were introduced
in the wild type frameshift region to eliminate the formation of
two individual proteins. In this example of a fusion construct the
frameshift "slippery" sequence TTTTTT U (SEQ ID NO:2) in wild type
gagpol has been changed to cTTcTg (SEQ ID NO:3). For information on
constructing a gag-pol fusion gene, see Megede, J. Z. et al. J.
Virology 77:6197-6207 (2003), the disclosure of which is hereby
incorporated by reference in its entirety. The wild type gag-pol
fusion protein contains a 56 amino acid open reading frame
polypeptide with no function, which separates the gag and pol
genes. In order to minimize the overall size of the present
construct, the gag polyprotein, which has the final four residues
of the (Lys-Gly-Arg-Pro) (SEQ ID NO:4), was modified so as to be
followed by a reduced ten amino acid intergenic region
(Asp-Arg-Gln-Gly-Thr-Val-Ser-Phe-Asn-Phe) (SEQ ID NO:5). The first
four residues of the pol polyprotein remain (Pro-Gln-Ile-Thr) (SEQ
ID NO:6). No deviations from the wild-type coding regions of gag
and pol genes were made to facilitate expression within the triple
transcriptional unit plasmid.
[0185] 2) All proteolytic activity of HIV-1 protease was
inactivated by deleting the nucleotides that code for three active
site amino acids (Asp-Thr-Gly from 25-27). See Loeb et al. Nature,
340:397 (1989); Wu et al. J Virol, 70: 3378 (1996).
[0186] 3) Reverse transcriptase (RT) was inactivated by deleting
nucleotides that code for the following four amino acids: Tyr 183,
Met 184, Asp 185, Asp 186. See Larder et al., Nature, 327: 716-717
(1987); Larder et al. PNAS, 86: 4803-4807 (1989).
[0187] 4) RNAse activity was abolished by deleting the nucleotides
that code for a single amino acid: glu 478. See Davies et al.,
Science, 252:88-95 (1991); Schatz et al. 1989, FEBS lett.
257:311-314 (1989).
[0188] 5) Integrase function was abolished by deleting the
nucleotides that code for the following three amino acids: Asp 626,
Asp 678 and Glu 714. See Wiskerchen et al. J. Virol, 69: 376-386
(1995); Leavitt et al. J. Biol. Chem., 268: 2113-2119 (1993).
[0189] 6) A single open reading frame was created for the HIV-1
nef, tat and vif genes by fusing the following coding regions in
frame (nef amino acid residues 4-206; tat amino acid residues 2-80;
vif amino acid residues 2-192) to encode a single polyprotein. This
polyprotein is referred to as nef-tat-vif or ntv.
[0190] 7) As a safety precaution the nef and tat proteins were
inactivated by removal of the myristylation signal (residues 1-3,
MGG) of nef and deletion of two cysteines (C30 & C34) from
tat.
Example 2
Construction of Single, Double and Triple Transcriptional Unit
Plasmids
[0191] The plasmids discussed in these examples are set forth in
Tables 1 and 2.
[0192] A triple transcriptional unit expression cassette was
constructed by using a variety of components in a circular double
stranded DNA plasmid. See FIG. 1. The first component was a first
transcriptional unit for expressing polypeptides in eukaryotic
cells, composed of the simian cytomegalovirus (SCMV) promoter, a
cloning site and bovine growth hormone (BGH) poly-A signal. The
second component is a second transcriptional unit for expressing
polypeptides in eukaryotic cells, which consists of human
cytomegalovirus (HCMV) immediate early promoter, a cloning site and
the SV40 polyadenylation (polyA) signal. Separating the first and
second transcriptional units is spacer region 1. The third
component is a third transcriptional unit for expressing
polypeptides in eukaryotic cells and is composed of the Herpes
simplex virus Lap1 promoter, the SV40 splice donor/acceptor, a
cloning site, and a rabbit beta globin poly-A signal. See Goins W.
F. et al., J. Virology 68:2239-2252 (1994); Soares, K. J. et al.,
Virology 70:5384-5394; Goins W. F. et al., J. Virology 73:519-532
(1999). Separating the second and third transcriptional units is
spacer region 2. Also included with spacer region 2 is a chimeric
bacterial kanamycin resistance (km.sup.r) gene, adenylyl
4'-nucleotidyl transferase type 1a. See Shaw K J, et al.,.
Microbiol. Reviews 57: 138-163 (1993) and Sadale, Y, et al., J.
Bacteriol. 141: 1178-1182 (1980). This gene has been devised to
confer resistance to a limited number of aminoglycosides while it
enables selection of bacteria containing the plasmid. Separating
the third and first transcriptional units is spacer region 3.
Spacer region 3 includes a pUC bacterial origin of replication that
is required for propagation of the plasmid in bacteria.
Example 3
Triple Transcriptional Unit Plasmid Containing Six HIV Genes
[0193] As a demonstration of the use of the three transcriptional
unit plasmid DNA vectors, a plasmid vector capable of co-expressing
three eukaryotic open reading frames was created. The three
transcriptional unit plasmid DNA vector was created by inserting
the following selected genes encoding HIV-1 antigens into the
triple transcriptional unit expression cassette described in
Example 2. All cloning techniques were performed following
conventional procedures (Sambrook et al. 1989).
[0194] First, an HIV-1 gag-pol fusion gene was inserted into the
PmeI-XhoI cloning site between the SCMV and BGH poly-A sites of the
first transcriptional unit. The gag gene was derived from the HXB2
isolate, and, similarly, the pol gene was also derived from the
HXB2 isolate. The complete HXB2 sequence is listed in the GenBank
computer database under the accession number K03455. One of skill
in the art would understand that other HIV-1 gag and pol genes from
other clades or other viral or bacterial genes could be inserted in
a similar fashion. Sequence information on HIV and other pathogens
is available from the HIV sequence database at the Los Alamos
National Laboratory and the National Center for Biotechnology
Information at the United States National Library of Medicine, 8600
Rockville Pike, Bethesda, Md. 20894.
[0195] Next, a full-length envelope gene (gp160) derived from a
primary isolate (6101) of HIV-1 was inserted into the MluI cloning
site between the HCMV and SV40 poly-A sites of the second
eukaryotic transcriptional unit. The 6101 envelope sequence can be
obtained in the GenBank computer database under the accession
numbers AY612855 and bankit625244.
[0196] Finally, a gene construct coding for an HIV nef-tat-vif
(NTV) fusion protein, which included nef residues 4-206 fused to
tat residues 2-80 and fused to vif residues 2-192 was inserted into
the KpnI-EcoRV cloning site between the HSVLap1 promoter and rabbit
beta-globin poly-A signals. The nef, tat, and vif genes were
derived from the NL4-3 isolate of HIV-1. The complete HIV-1 NL4-3
sequence is listed in the GenBank computer database under the
accession number M19921.
[0197] Therefore, as constructed, the gag-pol open reading frame
was placed under the control of SCMV promoter and BGH poly-A sites
in the first transcriptional unit; the envelope open reading frame
was placed under the control of HCMV promoter and SV40 poly-A
signals in the second eukaryotic transcriptional unit; and the
nef-tat-vif fusion open reading frame was placed under the control
of HSV Lap1/SV40 intron and rabbit beta-globin poly-A signals in
the third eukaryotic transcriptional unit.
Example 4
Expression of HIV Genes from Single, Double, Triple Transcriptional
Unit Plasmids
Materials and Methods: Cells and Transfection
[0198] The plasmid expressing six HIV genes described in Example 3
was evaluated in vitro for the ability to express the encoded
proteins. The cells used for all in vitro expression studies were
293 cells and RD cells that were obtained from the American Type
Culture Collection (ATCC). The procedure for expressing HIV
proteins in these cells was as follows: Cells were plated 24 hrs
prior to transfection at a density of 2.times.10.sup.5 cells per 35
mm diameter well and transfected with purified plasmid DNA. For
transfection 2 .mu.g of plasmid was mixed with Fugene transfection
reagent (Roche Diagnostics, Indianapolis, Ind.) and layered over
cells in a total volume of 100 .mu.l. Next, the cells were
incubated with 2 ml of DMEM media (BRL) with 10% FBS for 48 hrs.
Finally, cell lysates were harvested for further analysis
Detection of Expressed Proteins
[0199] Specific detection of HIV proteins was accomplished using a
western blot assay. For example, a western blot assay for each of
gag, pol, envelope and vif proteins was done by separating the
protein mixture using SDS polyacrylamide gel electroproresis. Next,
the separated proteins were then transferred onto PVDF membranes
(Invitrogen, Carlsbad, Calif.). Prestained molecular weight markers
and recombinant HIV-1 p24 (gag), p66 (pol), gp160 (env) and vif
proteins (Invitrogen) were used as size standards and positive
controls, respectively. Detection of gag, pol, env and vif
expression was accomplished by immunostaining. The PVDF membranes
having the bound and separated proteins were incubated with
antibodies specific to the respective proteins. Secondary
antibodies conjugated to alkaline phosphatase (Invitrogen) were
used and color detection was performed by using the chromogenic
detection kit (Invitrogen)
Expression of HIV Genes from Single, Double and Triple
Transcriptional Unit Plasmids
[0200] Expression of HIV genes from the triple transcriptional unit
plasmid was evaluated and compared to expression of the same genes
from each of a single transcriptional unit plasmid and a double
transcriptional unit plasmid. The single transcriptional unit
plasmid had a single eukaryotic transcriptional unit that contained
an HCMV promoter and BGH poly-A signal as expression regulatory
elements. The single transcriptional unit plasmids are numbered
from 101 through 105, plus 110 and 111 as shown in Table 1. For
example, plasmid 101 contained the HIV env gene as the open reading
frame in the single transcriptional unit. Similarly, plasmid 102
contained the HIV gag gene as the open reading frame in the single
transcriptional unit. In addition, plasmid 103 contained the HIV
pol gene as the open reading frame in the single transcriptional
unit and plasmid 104 contained the HIV nef-tat-vif (ntv) gene
fusion as the open reading frame in the single transcriptional
unit. Plasmid 101 also contained the HIV nef-tat-vif (ntv) gene
fusion as the open reading frame in the single transcriptional
unit, except it was driven by the Lap1 promoter rather than HCMV as
in plasmid 104. Finally, plasmid 110 contained the HIV
gag-pol-nef-tat-vif gene fusion as the open reading frame in the
single transcriptional unit and plasmid 111 contained the HIV
gag-pol gene fusion as the open reading frame in the single
transcriptional unit.
[0201] The double transcriptional unit plasmids had two complete
eukaryotic transcriptional units. The double transcriptional unit
plasmids were numbered from 201 to 204 and 212 as shown in Table 1.
The expression regulatory elements for the double transcriptional
unit plasmids were comprised of an HCMV promoter coupled with an
SV40 polyA in the first transcriptional unit and a SCMV promoter
coupled with a BGH poly-A signal in the second transcriptional
unit. In this embodiment, Plasmid 201 contained the HIV pol gene in
the first transcriptional unit and HIV gag gene in the second
transcriptional unit. Plasmid 202 contained the HIV nef-tat-vif
gene fusion gene in the first transcriptional unit and HIV env gene
in the second transcriptional unit. Plasmid 203 contained a HIV
gag-pol-nef-tat-vif gene fusion gene in the first transcriptional
unit and HIV env gene in the second transcriptional unit. Plasmid
204 contained the HIV gag-pol gene fusion gene in the first
transcriptional unit and HIV env gene in the second transcriptional
unit.
[0202] In some embodiments an adjuvant is provided by having it
expressed from a plasmid. In such cases, the plasmid must contain
the appropriate number of transcriptional units. For the sake of
clarity, and in order to distinguish from antigen plasmids, the
primary, secondary and tertiary terminology will be used to refer
to adjuvant plasmids having one or two or three transcriptional
units. For example, IL-12 is an adjuvant that is made up of two
polypeptides. An appropriate plasmid is plasmid 212, which
contained the IL-12 p35 subunit expressed under control of the HCMV
immediate early promoter and SV40 polyadenylation signal in the
primary transcriptional unit, and the IL-12 p40 subunit is
expressed under control of the simian CMV promoter (SCMV) and BGH
polyadenylation signal in the secondary transcriptional unit.
[0203] The triple transcriptional unit plasmids had three complete
eukaryotic transcriptional units and were numbered 301, 302 and
303. See Table 2. The difference between the three plasmids was in
the number of HIV open reading frames that were inserted. The
expression regulatory elements for the triple transcriptional unit
plasmids were comprised of an SCMV promoter coupled with a BGH
poly-A signal in the first transcriptional unit, an HCMV promoter
coupled with an SV40 polyA in the second transcriptional unit and
an HSVLap1 promoter coupled with a rabbit betaglobin poly-A signal
in the third transcriptional unit. As shown in Table 2, plasmid
number 301 is a triple transcriptional unit plasmid, but with only
one transcriptional unit having an inserted open reading frame.
Specifically, plasmid 301 contained the gag-pol fusion gene open
reading frame in the first transcriptional unit. Plasmid number 302
is the triple transcriptional unit plasmid having two
transcriptional units with inserted open reading frames, the
gag-pol in the first transcriptional unit and an HIV nef-tat-vif
fusion gene open reading frame in the third transcriptional unit
(no genes were inserted in the second transcriptional unit).
Finally, plasmid number 303 is the triple transcriptional unit
plasmid having all three transcriptional units with inserted open
reading frames, the gag-pol gene fusion open reading frame in the
first transcriptional unit, env gene open reading frame in the
second transcriptional unit and nef-tat-vif fusion gene open
reading frame in the third transcriptional unit. TABLE-US-00008
TABLE 1 Single and Double Transcriptional Unit Plasmids* Plasmid
No. HIV Construct Type 001 Empty vector control Control/No TUs 101
HCMV-env-BGH polyA Single 102 HCMV-gag-BGH polyA Single 103
HCMV-pol-BGH polyA Single 104 HCMV-ntv-BGH polyA Single 105
Lap1-ntv-Rabbit beta globin polyA single 110 HCMV-gag-pol-ntv-BGH
polyA Single/fusion 111 HCMV-gag-pol-BGH polyA Single/fusion 201
HCMV-pol-SV40 polyA, SCMV-gag-BGH Double polyA 202 HCMV-ntv-SV40
polyA, SCMV-env-BGH Double polyA 203 HCMV-gag-pol-ntv-SV40 polyA,
SCMV- Double env-BGH polyA 204 HCMV-gag-pol-SV40 polyA, SCMV-env-
Double BGH polyA 212 **HCMV-mIL-12 p35-SV 40 polyA, Adjuvant
SCMV-mIL-12 p40-BGH polyA *The following abbreviations are used:
SCMV: Simian cytomegalavirus promoter, HCMV: Human cytomegalovirus
promoter, HSVlap1: Herpes simplex virus latency-associated promoter
1, gag-pol: HIV gag-pol fusion, ntv: HIV nef-tat-vif fusion, env:
HIV envelope, mIL-12: murine interleukin-12.
[0204] TABLE-US-00009 TABLE 2 Triple Transcriptional Unit Plasmids*
Plasmid No. HIV Construct No. ORFs 301 SCMV-gag-pol-BGH polyA,
HCMV-[none], one Lap1-[none] 302 SCMV-gag-pol-BGH polyA,
HCMV-[none], two Lap1: ntv-Rabbit beta globin polyA 303 SCMV:
gag-pol-BGH polyA, HCMV-env-SV40 three polyA, Lap1: ntv-Rabbit beta
globin polyA *The following abbreviations are used: SCMV: Simian
cytomegalavirus promoter, HCMV: Human cytomegalovirus promoter,
HSVlap1: Herpes simplex virus latency-associated promoter 1,
gag-pol: HIV gag-pol fusion, ntv: HIV nef-tat-vif fusion, env: HIV
envelope, HCMV-[none], Lap1-[none] indicates the transcriptional
units did not contain an open reading frame (see plasmid 301);
**Il-12 can be either murine or rhesus macaque or human
[0205] As discussed above, multiple single and double
transcriptional unit plasmids were constructed for use in comparing
with the expression of the triple transcriptional unit plasmids.
See Tables 1 and 2. The expression patterns of these gag, pol, env,
nef-tat-vif, gag-pol and gag-pol-nef-tat-vif containing constructs
were evaluated by transiently transfecting 293 and/or RD cells with
the single, double, and triple transcriptional unit plasmids and
analyzing cell lysates by western blots using appropriate
antibodies.
[0206] The in vitro expression of gag in cell lysates from various
constructs was performed and the results were detected using
Western blots. See FIG. 2 and Table 1. Gag and pol proteins were
detected with mouse anti gag monoclonal and human polyclonal sera
respectively. Molecular weight markers and HIV p24 were included in
the first two lanes as standards. The single transcriptional unit
plasmid 102, which expressed gag, was run in the first sample lane.
The plasmids having two transcriptional units and two
transcriptional units with an inserted open reading frame were
plasmids 201, 203 and 204 all produced significant amounts of gag,
or gag-containing polyproteins such as gag-pol-nef-tat-vif, or
gag-pol. In the gag-pol fusion constructs, frameshift sequences
between gag and pol were mutated to allow gag and pol expression
from the same reading frame. The two transcriptional unit plasmids
201, 203 and 204 produced less gag than the single transcriptional
unit plasmid 102. The double or triple transcriptional unit
plasmids, which encoded gag-pol fusions, expressed equivalent
amounts of gag-pol polyprotein which migrated with an expected size
of .about.180 kd. Expression of gag from plasmid 203 that encodes a
large gag-pol-ntv polyprotein was also detected in cell lysates of
transfected cells and the protein migrated at an expected size of
.about.220 kD. Expression from this large fusion (plasmid 203),
however, was lower than that of plasmids 302 and 303 encoding
gag-pol. The three transcriptional unit plasmid 303 also produced
significant amounts of gag in the form of gag-pol polyprotein but
less gag than the single and about equivalent to the level produced
from double transcriptional unit plasmids. The three
transcriptional unit plasmid 302, which had two open reading frames
inserted and one transcriptional unit without an open reading frame
produced gag at approximately the same level as the two
transcriptional unit plasmids. See FIG. 2.
[0207] The in vitro expression profile of pol in cell lysates from
various constructs was performed and the results as detected using
Western blots followed a similar pattern as observed in the case of
gag. See FIG. 3 and Table 1. In this case, pol proteins were
detected with human polyclonal sera. Molecular weight markers and
HIV reverse transcriptase were included in the first two lanes as
standards. The single transcriptional unit plasmid 103, which
expressed pol, was run in the first sample lane. Next, plasmids
201, 203 and 204 having two transcriptional units and two
transcriptional units with an inserted open reading frame all
produced significant amounts of pol, or pol-containing polyproteins
such as gag-pol-nef-tat-vif, or gag-pol. In contrast to the
situation with gag, the two transcriptional unit plasmids 201, 203
and 204 produced about the same level of pol as the single
transcriptional unit plasmid 103. The pol, and gag-pol fusions
expressed pol polyprotein which migrated with expected sizes of
approximately 110 kd for pol, approximately 180 kd for gag-pol and
approximately 250 kd for gag-pol-nef-tat-vif. The three
transcriptional unit plasmid 303 also produced pol in the form of
gag-pol polyprotein but less pol than the single and double
transcriptional unit plasmids. Again, the three transcriptional
unit plasmid 302, which had two open reading frames inserted and
one transcriptional unit without an open reading frame expressed
pol in the form of a gag-pol polyprotein at approximately the same
level as the two transcriptional unit plasmids 201 and 203. See
FIG. 3. In this example, plasmid 204 expressed greater levels of
pol than the other two transcriptional unit plasmids 201 and 203.
See FIG. 3.
[0208] A similar analysis was performed for the in vitro expression
in cell lysates of the fusion of HIV regulatory proteins known as
nef-tat-vif or NTV. See FIG. 4 and Table 1. NTV protein was
detected with mouse anti-vif monoclonal antibody. Molecular weight
markers and recombinant HIV vif p23 were included in the first two
lanes, respectively, as standards. Two single transcriptional unit
plasmids 104 and 105, which expressed NTV from either the HCMV or
Lap 1 promoters respectively, were run in the first two sample
lanes. See FIG. 4. The level of nef-tat-vif expression was about
the same from both plasmids. Next, two plasmids having two compete
transcriptional units with an inserted open reading frame (plasmids
202 and 203) both produced significant amounts of nef-tat-vif
polyprotein. The level of nef-tat-vif protein expression appeared
less for plasmid 203, but this was expected because the polyprotein
being expressed was so large (gag-pol-nef-tat-vif .about.220 kD).
The three transcriptional unit plasmid 302, which had two open
reading frames inserted, and one transcriptional unit without an
open reading frame, produced nef-tat-vif at approximately the same
level as the single transcriptional unit plasmid. See FIG. 4. The
three transcriptional unit plasmid 303, which had three open
reading frames inserted, also produced significant amounts of
nef-tat-vif polyprotein. Specifically, the three transcriptional
unit plasmid 303 produced less nef-tat-vif than the single
transcriptional unit plasmids (104 and 105) and about equivalent to
or better than the level of nef-tat-vif polyprotein produced from
the double transcriptional unit plasmids (202 and 203). See FIG.
4.
[0209] The ability of various single, double and triple
transcriptional unit plasmids to express the HIV-envelope gene in
cell lysates was assessed. See FIG. 5 and Table 1. Envelope protein
was detected with mouse anti-env monoclonal antibody. Molecular
weight markers and recombinant HIV gp120 were included in the first
two lanes, respectively, as standards. The first sample lane
contains the protein expressed from a single transcriptional unit
plasmid 101, which expressed env from the HCMV promoter. See FIG.
5. Significant amounts of envelope glycoprotein were expressed.
Next, three plasmids having two compete transcriptional units with
two inserted open reading frames (plasmids 202, 203 and 204)
produced significant amounts of envelope glycoprotein. In each
case, envelope gene was controlled by the SCMV promoter. The three
transcriptional unit plasmid 303 also produced significant amounts
of env glycoprotein, but the level of expression was reduced by 2-3
fold, when compared to single and double transcriptional unit
plasmids (101, 202, 203 and 204). See FIG. 5.
Conclusion
[0210] Based upon semi-quantitative in vitro expression analysis,
the data indicate that all the inserted HIV genes, including
gag-pol, env and ntv, were expressed at significant levels from the
triple promoter plasmid carrying three independent transcriptional
units.
Example 5
Expression of Multiple Genes Via Multiple Plasmids or by a Single
Plasmid at Constant DNA Concentration Per Plasmid
[0211] Next, the expression from a single triple transcriptional
unit plasmid encoding multiple genes was compared to multiple
plasmids, each expressing a single gene from the same array of
genes, where the DNA per plasmid was held constant at 1 .mu.g. In
each case, the total amount of DNA was also held constant at 4
.mu.g by supplementing with plasmid DNA without an open reading
frame insert. HIV gag expression was evaluated using cultured cells
that were transiently transfected with 1 .mu.g of each plasmid, and
cell lysates were analyzed by western blot. As shown in FIG. 6, HIV
gag expression was readily detected in lane 2 (two plasmids), lane
3 (one plasmid), lane 4 (one plasmid), and lane 5 (4 plasmids). HIV
gag expression was low in lane 1 (three plasmids). The three
transcriptional unit plasmid 303 again produced significant amounts
of gag protein, although less than the combinations containing more
plasmids.
[0212] HIV env expression from single or multiple plasmids was
evaluated and the results are shown in FIG. 7. Again, 1 .mu.g of
each plasmid was transiently transfected into cultured cells and
cell lysates were analyzed by western blot. The results demonstrate
that HIV env expression was readily detected in lane 1 (3
plasmids), lane 2 (two plasmids), lane 3 (one plasmid), lane 4 (one
plasmid), and lane 5 (4 plasmids). In each case the total amount of
DNA was held constant at 4 .mu.g by supplementing with plasmid DNA
without an open reading frame insert to make the total amount of
DNA equal to 4 .mu.g. The three transcriptional unit plasmid 303
again produced significant amounts of env glycoprotein. See FIG. 7.
In this case, the single three transcriptional unit plasmid 303
produced comparable amounts of env glycoprotein to that produced in
lane 5 where 4 plasmids were used.
[0213] As shown in FIG. 8, HIV nef-tat-vif expression from single
or multiple plasmids was evaluated using 1 .mu.g of each plasmid
transiently transfected into cultured cells and cell lysates were
analyzed by western blot. See FIG. 8. The results demonstrate that
HIV nef-tat-vif expression was detected in lane 1 (3 plasmids),
lane 2 (2 plasmids), lane 3 (one plasmid), lane 4 (one three
transcriptional unit plasmid), and lane 5 (4 plasmids). See FIG. 8.
The total amount of DNA was held constant at 4 .mu.g. The three
transcriptional unit plasmid 303 produced significant amounts of
nef-tat-vif protein, although less than the combination containing
two plasmids.
Conclusion
[0214] As shown in FIGS. 6, 7 and 8, using the three
transcriptional unit plasmid (303), all three open reading frames
coding for gag-pol, env and ntv proteins were expressed
simultaneously at similar levels, thus confirming the functionality
of this plasmid.
Example 6
Expression of Multiple Genes Via Two Plasmids or by a Single
Plasmid at Constant Total DNA Concentration
[0215] The expression of HIV genes gag, pol, env and nef-tat-vif
was compared between the triple transcriptional unit plasmid at 2
.mu.g concentration and combinations of two plasmids each at 1
.mu.g DNA. The total DNA concentration was held constant at 2 .mu.g
as indicated in FIGS. 9, 10, 11 and 12.
[0216] FIG. 9 shows that pol protein expression was similar from
either of the two plasmid combinations or from the triple
transcriptional unit plasmid. Lane 2 shows western blots of pol
protein expressed from the combination of plasmids 201 and 202, two
double transcriptional unit plasmids constructed to express the
entire array of HIV genes, gag, pol, nef-tat-vif and env. Next,
expression of pol protein from two combinations of a double
transcriptional unit plasmid and a single transcriptional unit
plasmid, which were expressing gag, pol, env and nef-tat-vif in
various configurations, was evaluated using western blots of pol
protein. See FIG. 9, lane 3 (plasmids 204 and 104) and lane 5
(plasmids 302 and 101). In each case there is detectable pol
expression. Lane 4 contains western blots of pol protein expressed
from plasmid 203, which is a double transcriptional unit plasmid
expressing the entire array of HIV genes, gag-pol-nef-tat-vif and
env. See FIG. 9. Lane 6 contains western blots of pol protein
expressed from plasmid 303, which is an example of a triple
transcriptional unit plasmid expressing the entire array of HIV
genes, gag-pol env and nef-tat-vif, as described in Examples 2 and
3. See FIG. 9.
[0217] FIGS. 10 and 11 compare gag and envelope protein expression
from the two plasmid combinations with protein expression from the
triple transcriptional unit plasmid. Lane 2 shows western blots of
gag and env proteins expressed from the combination of plasmids 201
and 202, which were two double transcriptional unit plasmids
constructed to express the entire array of HIV genes, gag, pol,
nef-tat-vif and env. Next, expression of gag and env proteins from
combinations of a double transcriptional unit plasmid and a single
transcriptional unit plasmid was evaluated using western blots. See
FIGS. 10 and 11: lane 3 (plasmids 204 and 104) and lane 5 (plasmids
302 and 101). Plasmid 302 is a three transcriptional unit plasmid
functioning as a two transcriptional unit plasmid because it has
only two inserted open reading frames. See Table 2. There was
detectable gag and env expression in each case. See FIG. 10. Lane 4
exemplifies western blots of gag and env proteins expressed from
plasmid 203, which was a double transcriptional unit plasmid
expressing the entire array of HIV genes, gag-pol-nef-tat-vif and
env. See FIGS. 10 and 11. Lane 6 contains western blots of gag and
env proteins expressed from the triple transcriptional unit plasmid
303 described in Examples 2 and 3. See FIGS. 10 and 11. Expression
of gag and env proteins from the triple transcriptional unit
plasmid 303 was comparable to that of the combinations of
plasmids.
[0218] FIG. 12 compares nef-tat-vif polyprotein expression from
various plasmid combinations with protein expression from the
triple transcriptional unit plasmid using western blot detection.
Lane 2 shows western blots of nef-tat-vif polyprotein expressed
from the combination of plasmids 201 and 202, two double
transcriptional unit plasmids designed to express HIV genes, gag,
pol, nef-tat-vif and env. Lanes 3 and 5 show expression, as
detected using western blots, of nef-tat-vif polyprotein from two
different combinations of double transcriptional unit plasmids and
a single transcriptional unit plasmid. See FIG. 12: lane 3
(plasmids 204 and 104) and lane 5 (plasmids 302 and 101). As
discussed above, plasmid 302 is a three transcriptional unit
plasmid functioning as a two transcriptional unit plasmid because
it has only two inserted open reading frames. See Table 2. In this
case, the nef-tat-vif protein expression from plasmid 302 seen in
lane 5 was of a lower level than from plasmid combinations of 201
and 202 (lane 2) or 204 and 104 (lane 3). See FIG. 12. Lane 4
depicts nef-tat-vif polyprotein expressed from plasmid 203, which
was a double transcriptional unit plasmid expressing the entire
array of HIV proteins, gag-pol-nef-tat-vif and env. See FIG. 12.
Lane 6 depicts nef-tat-vif polyprotein expressed from the triple
transcriptional unit plasmid 303. See FIG. 12. Expression from 303
of nef-tat-vif was significantly higher than from plasmid 302.
Noticeably, the expression from a two transcriptional unit plasmid
(203) expressing a large gag-pol-nef-tat-vif polyprotein from one
promoter and env protein from the other was substantially lower
than that of plasmid 303 encoding the same genes from three
independent transcriptional units.
[0219] In summary, using the triple transcriptional unit plasmid,
three open reading frames could be expressed simultaneously at
approximately equivalent levels and overall levels were comparable
to both single and dual promoter constructs encoding those genes.
The in vitro gene expression data suggests a lack of significant
promoter interference when multiple HIV genes are expressed from a
triple transcriptional unit plasmid. Therefore, the individual
transcriptional units are placed appropriately in the vector.
Example 7
Expression of Multiple Genes Via Multiple Plasmids or by a Single
Plasmid without Holding the Total DNA Concentration Constant
[0220] The expression from a single triple transcriptional unit
plasmid encoding multiple genes was compared to multiple plasmids,
expressing the same array of genes, where the DNA per plasmid was
held constant at 1 .mu.g. In contrast to Example 5, the total
amount of DNA was not supplemented with plasmid DNA without an open
reading frame insert to make up for the total amount of DNA. The
data are not shown, but are summarized below.
[0221] In this example, HIV gag, pol, env and ntv expression was
evaluated using cultured 293 cells that were transiently
transfected with 1 .mu.g of each plasmid and cell lysates were
analyzed by western blot. HIV gag expression was detected from
transfections with combinations with three plasmids (101, 104,
301), two plasmids (201 and 202), one plasmid (203), one plasmid
(303), and four plasmids (101, 102, 103, 104). The three
transcriptional unit plasmid 303 produced significant amounts of
gag protein as compared to combinations requiring more plasmids.
Specifically, the three transcriptional unit plasmid 303 produced
more gag polyprotein than the two transcriptional unit plasmid 203
having all six HIV genes and slightly less than the combination of
two transcriptional unit plasmids 201 and 202 having all six HIV
genes. The expression of gag in from the combination of three
plasmids (101, 104, 301) was weak where gag was expressed as a
gag-pol fusion driven by the SCMV promoter.
[0222] HIV env expression from single or multiple plasmids was also
evaluated. The results demonstrated that HIV env expression was
easily detected from combinations with three plasmids (301, 101 and
104), two plasmids (201 and 202), one plasmid (203), one plasmid
(303), and four plasmids (101, 102, 103 and 104). The total amount
of DNA depended on the number of plasmids being used, with 1 .mu.g
of DNA transfected per plasmid. In this case the three
transcriptional unit plasmid 303 produced more env glycoprotein
than any other plasmid or plasmid combination.
[0223] HIV nef-tat-vif expression from single or multiple plasmids
was evaluated using 1 .mu.g of each plasmid transiently transfected
into cultured cells and cell lysates were analyzed by western blot.
HIV nef-tat-vif expression was detected from combinations with
three plasmids (301, 101 and 104), two plasmids (201 and 202), one
plasmid (203), one plasmid (303), and four plasmids (101, 102, 103
and 104). The three transcriptional unit plasmid 303 produced
significant amounts of nef-tat-vif protein.
Conclusion
[0224] A triple transcriptional unit plasmid encoding multiple HIV
genes that express high levels of specific proteins in a
rev-independent manner was designed and constructed, which
confirmed that a single plasmid construct expressed three
transcripts independently and efficiently. In this example,
expression of HIV genes from the triple transcriptional unit
plasmid was compared to the expression of the same genes from
either single or double transcriptional unit constructs. The data
indicate that gene expression from a triple transcriptional unit
plasmid was lower when compared to those being expressed by single
or dual expression cassettes. However, in the above example it was
found that HCMV promoter-driven gene expression was higher than
SCMV promoter, followed by HSV-lap1 promoter. This difference in
strength of the promoters in the triple transcriptional unit
construct should be considered when positioning genes for
expressing antigens of higher versus lower immunogenicity in the
plasmid.
Example 8
Murine Immunization Studies with Plasmid Vectors Containing One,
Two or Three Complete Transcriptional Units
[0225] Murine studies were performed to establish and compare
immunogenic functionality of the three transcriptional unit plasmid
vector expressing proteins from six HIV-1 genes including gag, pot,
env, nef, tat and vif. Specifically, the relative ability of
various single, double and triple plasmid DNA-based immunogenic
compositions to elicit multi-antigen-specific cell-mediated immune
responses in Balb/c mice was compared.
[0226] Balb/c mice were immunized intramuscularly with 100 total
.mu.g doses of DNA as outlined in Table 3. In all cases,
immunogenic compositions were formulated with 0.25% bupivacaine and
injected into the quadricep muscles in a 100 .mu.l volume. Ten days
after the second immunization, animals were sacrificed and the
serum and spleens were isolated for immune assays. Sera of
immunized mice were analyzed for anti-gag, and anti-env specific
antibody titers. Spleens were used to measure antigen-specific
IFN-gamma secreting cells using ELISPOT assays as described
below.
Animals
[0227] For these studies, 4-6 week old female Balb/c mice were
used. Mice were maintained in accordance with the Guide for the
Care and Use of Laboratory Animals (National Research Council,
National Academic Press, Washington, D.C., 1996). In addition,
procedures for the use and care of the mice were approved by Wyeth
Research's Institutional Animal Care and Use Committee.
Immunogenic Compositions and Immunization
[0228] Various plasmid DNA expression vectors encoding HIVenv
gp160, gag p55, pol, or a nef-tat-vif fusion protein were used as
the experimental immunogenic compositions, and the empty expression
vector backbone was used as a control immunogenic composition
vector. See Table 3 below for study design. HIV gene expression by
the various expression vectors was confirmed by Western blot after
transient transfection of human rhabdosarcoma (RD) cells. See
Examples 4-7.
[0229] The adjuvant used for these studies was also delivered via a
DNA plasmid. In this example, all animals were co-injected with 25
.mu.g of plasmid no. 212 expressing Il-12. This adjuvant plasmid is
a two-transcriptional unit expression plasmid (plasmid no. 212 in
Table 1) encoding murine IL-12 p35 and p40 genes. See Table 1. The
IL-12 p35 subunit was expressed under control of the HCMV immediate
early promoter and SV40 polyadenylation signal, while the IL-12 p40
subunit was expressed under control of the simian CMV promoter
(SCMV) and BGH polyadenylation signal. Production of murine IL-12
was confirmed after transient transfection of RD cells by screening
cell supernatants using an anti-mouse IL-12 p70 capture ELISA
(Endogen, Woburn, Mass.) (data not shown). TABLE-US-00010 TABLE 3
Mouse Study Design - Two Immunizations Immunization Group Plasmid
Total No. Schedule No. No. Plasmid description DNA.(ug) mice (week)
1 303 HCMV-env; SCMV-gag/pol; 100 9 0-3 lap-ntv 1a 203
HCMV-gag/pol; SCMV-env; 100 9 0-3 2b 101 + 110 HCMV-env 50 9 0-3
HCMV-gag-pol-ntv 50 2c 104 + 204 HCMV-ntv 50 9 0-3 HCMV-gag-pol,
SCMV-env 50 2d 111 + 202 HCMV-gag-pol 50 9 0-3 HCMV-ntv, SCMV-env
50 2e 201 + 202 HCMV-pol, SCMV-gag 50 9 0-3 HCMV-ntv, SCMV-env 50
3a 111 HCMV-gag/pol 33 9 0-3 101 HCMV-env 33 104 HCMV-ntv 33 3b 101
HCMV-env 33 9 0-3 104 HCMV-ntv 33 201 HCMV-pol, SCMV-gag 33 3c 102
HCMV-gag 33 9 0-3 103 HCMV-pol 33 202 HCMV-ntv, SCMV-env 33 4 001
Vector control 100 6 0-3
[0230] Expression plasmids for immunization were produced by
Puresyn, Inc. (Malvern, Pa.). Plasmids were propagated in E. coli,
isolated from cells by alkaline lysis, purified by column
chromatography and were formulated individually at a concentration
of 2.5 mg/mL in isotonic citrate buffer (29.3 mM sodium citrate,
0.67 mM citric acid, 150 mM NaCl, 0.34 mM EDTA, pH=6.4-6.7)
containing 0.25% bupivacaine as a facilitating agent to allow for
the formation of DNA:bupivacaine complexes. For all groups, the
adjuvant plasmid was mixed with the antigen expressing plasmids as
part of the immunogenic composition. Final plasmid preparations
were shown to consist of >90% supercoiled plasmid DNA and
residual endotoxin was shown to be <30 EU/mg DNA (data not
shown). Immediately prior to immunization, the immunogenic
compositions were prepared by mixing the appropriate plasmid
expression vector formulations. The resulting immunogenic
compositions were administered by intramuscular injection into both
quadriceps muscles (0.1 cc total injection volume, with 0.05 cc per
site) using an 18 gauge needle and 0.3 mL syringe.
Murine IFN-.gamma. ELISPOT Assay
[0231] ELISPOT (or ElisaSpot, short for Enzyme-linked ImmunoSpot
Assay) originally was developed as a method to detect
antibody-secreting B-cells. The method has now been adapted to
determine T-cell reactions to a specific antigen, usually
represented as number of activated cells per million. In the
present example, Interferon gamma (IFN-gamma) production was used
as a read-out for activation of single cells.
[0232] In this analysis, ELISPOT served to determine cytotoxic
T-cell activity elicited by immunogenic compositions expressing
specific HIV antigens. For the determination of IFN-.gamma. ELISPOT
responses, a Mouse IFN-.gamma. ELISPOT kit (material number 551083,
BD Biosciences, San Diego Calif.) was used. ELISPOT Assays were
performed in ninety-six-well micotiter plates with a membrane
bottom to each well. Specifically, ninety-six-well flat-bottom
ELISPOT plates (ImmunoSpot, Cellular Technology Limited, Cleveland
Ohio) were coated overnight with a purified anti-mouse
.gamma.-interferon (mIFN-.gamma.) monoclonal antibody (Material No.
51-2525KC, BD-Biosciences, San Diego Calif.) at a concentration of
10 mcg/mL, after which the plates were washed three times with
sterile 1.times. phosphate buffered saline (1.times.PBS) and then
blocked for 2 hours with R10 complete culture medium (RPMI-1640
containing 10% heat inactivated (HI) fetal bovine serum (FBS) and 2
mM L-glutamine, 100 units/mL penicillin, 100 mcg/mL streptomycin
sulfate, 1 mM sodium pyruvate, 1 mM HEPES, 100 mcM non-essential
amino acids). Mouse spleens were first processed by grinding the
spleens between the frosted end of two sterile microscope slides.
The resulting homogenate was resuspended in 10 mls of in complete
R05 culture medium (RPMI 1640 medium supplemented with 5% FBS, 2 mM
L-glutamine, 100 units/mL penicillin, 100 mcg/mL streptomycin
sulfate, 1 mM sodium pyruvate, 1 mM HEPES, 100 mcM non-essential
amino acids) and splenocytes were subsequently isolated by
Ficoll-Hypaque density gradient centrifugation and resuspended in
complete R10 culture medium containing either 2 mcg/mL Con-A
(Sigma), peptide pools (15 mers overlapping by 11 amino acids; 2.5
mcM each final peptide concentration) spanning HIV gag p55, HIV-1
6101 env gp160, pol, nef, tat, vif, or medium alone. Input cell
numbers were 4.times.10.sup.5 splenocytes per well
(4.times.10.sup.6 splenocytes/mL) and assayed in duplicate wells.
Splenocytes were incubated for 22-24 hours at 37.degree. C. and
then removed from the ELISPOT plate by first washing 3 times with
deionized water and incubating on ice for 10-20 minutes. Then
plates were washed 6 times with 1.times.PBS containing 0.1%
Tween-20. Thereafter, plates were treated with an anti-mouse
IFN-.gamma. biotinylated detection antibody (5.0 mcg/ml, Material
No. 51-1818KZ, BD-Biosciences, San Diego Calif.) diluted with R10
and incubated overnight at 4.degree. C. ELISPOT plates were then
washed 10 times with 1.times.PBS containing 0.1% Tween-20 and
treated with 100 mcL per well of streptavidin-horseradish
peroxidase conjugate (Catalog No. 51-9000209, BD-Biosciences, San
Diego Calif.)) diluted 1:100 with R10 and incubated an additional 1
hour at room temperature. The unbound streptavidin-horseradish
peroxidase conjugate was removed by rinsing the plate 6 times with
1.times.PBS containing 0.1% Tween-20 and 3 times with 1.times.PBS.
Next, the peroxidase substrate was prepared by diluting 20 mcL/mL
of AEC Chromogen in AEC substrate solution (Catalog No. 551951,
BD-Biosciences, San Diego Calif.). Color development was initiated
by adding 100 mcL/well of substrate solution for 3-5 minutes.
Finally, the plates were rinsed with water and were air-dried. The
results were determined using an ELISPOT analyzer or imaging device
that takes a picture of a single well of the ELISPOT plate and then
the spots were enumerated. In this case, the resulting spots were
counted using an Immunospot Reader (CTL Inc., Cleveland, Ohio).
Peptide-specific IFN-.gamma. ELISPOT responses were considered
positive if the response (minus media background) was .gtoreq.3
fold above the media response and >50 spot forming cells
excreting interferon gamma per 10.sup.6 splenocytes (#SFC/10.sup.6
splenocytes).
[0233] As shown in Table 4, individual HIV-1 antigen and total
HIV-specific IFN-gamma ELISPOT responses in mice after
multi-plasmid DNA immunizations were measured after two
immunizations with immunogenic compositions made up of the plasmids
shown in Table 3. TABLE-US-00011 TABLE 4 Murine Immune Responses
Following Two Immunizations gag- pol- env- ntv#- Total HIV-
specific specific specific specific specific Group ID response*
response response response response Control 2 0 3 0 5 1a 46 43 238
4 331 2e 29 138 181 12 360 2c 102 118 203 44 467 1 20 39 468 2 529
3b 16 109 404 20 548 2d 188 185 251 8 632 2b 43 65 548 6 662 3a 139
105 802 18 1064 3c 174 378 616 11 1179 *antigen-specific IFN-gamma
ELISPOT responses were reported as the spot forming cells
(#SFC/10.sup.6 splenocytes) excreting interferon gamma per 10.sup.6
splenocytes. #ntv, nef-tat-vif fusion protein.
[0234] In all cases, the nef-tat-vif specific responses were
relatively low. It was lowest in group 1 mice where nef-tat-vif was
under the control of the lap1 promoter. However, in the above
examples 4-7 it was found that HCMV promoter-driven gene expression
was higher than with the SCMV promoter, and SCMV-promoter driven
gene expression was higher than with the HSV-lap1 promoter. This
difference in strength of the promoters being utilized in the
triple promoter construct may be responsible for the lower induced
immune responses observed when this construct was used in an
immunogenic composition.
[0235] Regarding the use of fusion proteins, comparing the ELISPOT
response to HIV pol in 3a and 3c, it appears that there is some
reduced immunogenicity when fusion polypeptides are used rather
than single polypeptides.
[0236] Another consideration is the relative immunogenicity of the
protein being examined. For example, by examining 3b versus 3c
(where HCMV promoter-driven gene expression drives each of the
genes, env, gag, pol and nef-tat-vif, on a single plasmid
containing a single transcription unit), there still remains a
hierarchy of immunogenicity that is approximately
env>pol>gag>nef-tat-vif. As discussed above, promoter
strength and relative immunogenicity should both be considered in
the design of individual plasmids and combinations of plasmids for
use in immunogenic compositions.
[0237] Next, another study was performed to evaluate the effect on
immune responses when three immunizations using one, two and three
plasmid immunogenic compositions. See Table 5. Groups of six mice
were immunized as described above, except that they were immunized
three times at three-week intervals rather than two times at
three-week intervals. See Table 5. Groups 1, 2e and 3a utilize the
same immunogenic compositions as in Table 3. In addition, in the
study using three immunizations a new plasmid, designated 301, was
constructed to directly compare HCMV promoter-driven gene
expression of a gag/pol fusion protein with SCMV promoter-driven
gene expression of a gag/pol fusion protein. Compare groups 3a and
4b in Tables 5 and 6. This plasmid also allowed the comparison of
the immunogenic potential of gag-pol fusion being expressed from a
triple transcriptional unit plasmid with the gag-pol fusion and env
genes being expressed from three single transcriptional unit
plasmids driven by similar promoters. Compare groups 1 and 4b in
Tables 5 and 6. Spleen tissue was harvested 17 days after the final
boost and analyzed for antigen specific ELISPOT responses to the
individual HIV proteins. TABLE-US-00012 TABLE 5 Murine Study Design
- Three Immunizations Immunization .sup.1Group Plasmid Total DNA
No. Schedule No. No. Plasmid description (ug) mice (week) 1 303
HCMV-env; SCMV-gag/pol; 100 9 0-3-6 lap-ntv 2e 201 + 202 HCMV-pol,
SCMV-gag 50 9 0-3-6 HCMV-ntv, SCMV-env 50 3a 111 HCMV-gag/pol 33 9
0-3-6 101 HCMV-env 33 104 HCMV-ntv 33 4b 101 HCMV-env 33 9 0-3-6
104 HCMV-ntv 33 301 SCMV-gag/pol, HCMV- 33 [none], Lap1-[none]
control 001 Vector control 100 6 0-3-6 .sup.1Groups 1, 2e and 3a
utilize the same immunogenic compositions as in Table 3, except
that three immunizations were carried out.
[0238] The total induced cellular immune responses from the three
transcriptional until plasmid were approximately the same or higher
than cellular immune responses induced by immunogenic compositions
containing single and double transcriptional unit plasmids. See
Table 6. TABLE-US-00013 TABLE 6 Murine Cellular Immune Responses -
Three Immunizations gag- pol- env- ntv#- Total HIV- specific
specific specific specific specific Group ID response* response
response response response 1 34 58 986 1 1077 2e 32 363 431 69 895
3a 174 162 713 82 1131 4b 47 35 722 79 883 control 0 0 3 2 5
*antigen-specific IFN-gamma ELISPOT responses were reported as the
#SFC/10.sup.6 splenocytes. #ntv, nef-tat-vif fusion protein.
[0239] The ELISPOT results of the following three immunizations of
the immunogenic compositions indicated that HIV cellular immune
responses after three immunizations with the three transcriptional
unit plasmid-based immunogenic composition were increased by 100%
following the third immunization. However, the balance of the
response can still vary depending on the strength of the promoters
involved and the relative immunogenicity of the antigens. Clearly,
for some situations where a manufacturing advantage is necessary,
the tripe transcriptional unit plasmid will be a good vehicle for
administering three or more genes in an immunogenic
composition.
[0240] All plasmid designs tested thus far in immunogenic
compositions have been found to correctly express the antigens and
to be immunogenic, activating cellular immune responses after three
immunizations. However, nef, tat and vif specific responses were
undetectable when placed under the control of HSV Lap1 promoter in
the triple promoter construct.
[0241] Under some scenarios, immunogenic compositions which induce
broad, and balanced cellular immune responses to a range of
antigens would be preferable. In this case, two and three pDNA
immunogenic composition designs (2d, 3a and 3c) as shown in Tables
3 and 4 appear capable of eliciting potent (>600 SFC/10.sup.6
cells), balanced, HIV-specific ELISPOT responses and were selected
for further testing in non-human Primates. See Example 9.
Example 9
Macaque Immunization Studies with Plasmid Vectors Containing One or
Two Complete Transcriptional Units
[0242] In Example 8, Tables 3 and 4, three pDNA immunogenic
compositions, particularly the immunogenic compositions used in
groups 2d, 3a and 3c, appeared capable of eliciting potent (>600
SFC/10.sup.6 cells), balanced, HIV-specific ELISPOT responses to
all six HIV proteins and were selected for further testing in
non-human primates.
Experimental Design
[0243] For this study, a total of 30 Mamu-A*01 negative,
captive-bred, male rhesus macaques (Macaca mulatta) of Indian
origin were used. Macaques were housed at the New Iberia Research
Center (New Iberia, La.) and maintained in accordance with the
Guide for the Care and Use of Laboratory Animals (National Research
Council, National Academic Press, Washington, D.C., 1996). In
addition, procedures for the use and care of the macaques were
approved by Wyeth Research's Institutional Animal Care and Use
Committee.
Immunizations:
[0244] Expression plasmids for immunization were produced by
Puresyn, Inc. (Malvern, Pa.). Plasmids were propagated in E. coli,
isolated from cells by alkaline lysis, and purified by column
chromatography. The plasmids were then individually formulated at a
concentration of 2.5 mg/mL in isotonic citrate buffer (29.3 mM
sodium citrate, 0.67 mM citric acid, 150 mM NaCl, 0.34 mM EDTA,
pH=6.4-6.7) containing 0.25% bupivacaine to allow for the formation
of DNA:bupivacaine complexes. Final plasmid preparations were shown
to consist of >90% supercoiled plasmid DNA and residual
endotoxin was shown to be <30 EU/mg DNA (data not shown).
[0245] The adjuvant used for the rhesus macaque studies was a DNA
plasmid that was delivered as part of the immunogenic composition.
This adjuvant plasmid is a two-trancriptional unit expression
plasmid (plasmid no. 212 in Table 1) encoding rhesus IL-12 p35 and
p40 genes. See Table 7. The IL-12 p35 subunit was expressed under
control of the HCMV immediate early promoter and SV40
polyadenylation signal, while the IL-12 p40 subunit was expressed
while under control of the simian CMV promoter (SCMV) and BGH
polyadenylation signal. Bioactivity of the plasmid-expressed rhesus
IL-12 was confirmed by assaying supernatants from transiently
transfected RD cells for their capacity to induce IFN-.gamma.
secretion in resting rhesus peripheral blood lymphocytes (PBLs;
data not shown). TABLE-US-00014 TABLE 7 Macaque Study Design Group
Plasmid Total DNA No. No. No. .sup.1Plasmid description (ug) animal
2d 111 + 202 HCMV-gag-pol 4.25 6 HCMV-ntv, SCMV-env 4.25 212
HCMV-IL-12 p35, 1.5 SCMV-IL-12 p40 3a 111 HCMV-gag/pol 2.8 6 101
HCMV-env 2.8 104 HCMV-ntv 2.8 212 HCMV-IL-12 p35, 1.5 SCMV-IL-12
p40 3c 102 HCMV-gag 2.8 6 103 HCMV-pol 2.8 202 HCMV-ntv, SCMV-env
2.8 212 HCMV-IL-12 p35, 1.5 SCMV-IL-12 p40 3cE.sup.2 102 HCMV-gag
0.56 6 103 HCMV-pol 0.56 202 HCMV-ntv, SCMV-env 0.56 212 HCMV-IL-12
p35, 0.30 SCMV-IL-12 p40 4a.sup.3 102 HCMV-gag 2.1 6 101 HCMV-env
2.1 103 HCMV-pol 2.1 104 HCMV-ntv 2.1 212 HCMV-IL-12 p35, 1.5
SCMV-IL-12 p40 4 - 001 Vector control 8.5 6 control 212 HCMV-IL-12
p35, 1.5 SCMV-IL-12 p40 .sup.1All groups received 1.5 mg of plasmid
no. 212 (HCMC-IL-12 p35, SCMV-IL-12 p40) encoding rhesus macaque
IL-12 (rIL-12) as adjuvant. .sup.2A second Group 3c was included
where electroporation was added to the administration protocol.
.sup.3An additional group (4a) was added to the macaque study at a
later time to determine the immunogenicity of the indicated 4
vector vaccine design.
[0246] All macaques were immunized on a schedule of 0, 4, and 8
weeks. Immediately prior to immunization, the appropriate plasmid
expression vector formulations were mixed to create immunogenic
compositions and administered by intramuscular injection (groups
2d, 3a, 3c and controls) into both deltoid muscles and both
quadriceps muscles (1 ml injection volume, 2.5 mg DNA per site)
using an 18 gauge needle and 3 mL syringe.
[0247] Group 3cE macaques were immunized with pDNA by intramuscular
injection into both deltoid muscles and both quadriceps muscles
using standard 1 mL syringes with 21 gauge needles (Braun)
positioned 8.0 mm apart and, followed immediately by
electrostimulation (i.e., electroporation). The injection volume
was 0.2 ml providing 0.5 mg plasmid DNA per site per injection for
a total of 2 mg total DNA. Therefore, the electroporation group
(3cE) received 1/5 the total DNA administered to the other
groups.
[0248] In this example, the electroporation conditions were as
follows: six 20 ms unipolar pulses at 250 mA and about 100 V/cm.
There was a 250 ms pause between each pulse.
[0249] In the absence of electroporation, the results shown in
Table 8 indicated that immunogenic compositions based on a
combination of plasmids having a single transcriptional unit (group
3a) produced the highest total cellular immune responses after ten
or sixteen weeks as compared to immunogenic compositions based on a
combination of plasmids containing at least one plasmid with more
than one transcriptional unit. Compare 3a with 2d and 3c.
TABLE-US-00015 TABLE 8 Total HIV-Specific IFN-Gamma ELISPOT
Responses Over Time After Multi-Plasmid DNA Vaccination Total
HIV-specific IFN-gamma ELISpot response* Group Base- Week Week ID
line Week 2 Week 4 Week 6 Week 8 10 16 2d 43.8 .+-. 10.5 286.5 .+-.
234.9 278.7 .+-. 104.5 403.1 .+-. 89.9 348.3 .+-. 108.8 769.9 .+-.
340.4 407.5 .+-. 82.2 3a 29.5 .+-. 12.8 61.5 .+-. 23.2 204.8 .+-.
26.4 635.0 .+-. 230.5 365.8 .+-. 47.1 1652.5 .+-. 563.3 1015.3 .+-.
584.8 3c 35.5 .+-. 9.0 56.5 .+-. 12.3 138.3 .+-. 32.5 892.5 .+-.
277.5 300.0 .+-. 95.9 786.7 .+-. 213.1 816.3 .+-. 330.6 3cE 41.5
.+-. 13.6 1405.0 .+-. 422.0 346.3 .+-. 72.7 1287.9 .+-. 365.6
3349.6 .+-. 1575.9 3637.8 .+-. 863.7 8140.8 .+-. 1819.0 4a 18.8
.+-. 8.2 52.1 .+-. 13.3 43.3 .+-. 16.6 272.9 .+-. 60.0 230.0 .+-.
40.5 190.6 .+-. 38.9 nd.sup.1 control 32.0 .+-. 12.5 10.2 .+-. 2.7
33.2 .+-. 12.0 24.2 .+-. 9.3 16.7 .+-. 4.0 12.1 .+-. 4.1 47.1 .+-.
13.7 *Total HIV-specific IFN-gamma ELISpot responses are reported
as the mean #SFC/10.sup.6 PBLs .+-. standard error. .sup.1nd, not
done
[0250] A surprising result was that electroporation enhanced the
total cellular immune responses by more than 450% at ten weeks and
by more that 990% at sixteen weeks. Compare 3cE with 3c. The
results shown in Table 8 indicated that immunogenic compositions
based on a combination of plasmids containing at least one plasmid
with more than one transcriptional unit when combined with
electroporation produced the highest total cellular immune
responses after ten or sixteen weeks as compared to immunogenic
compositions based on a combination of plasmids having a single
transcriptional unit. Compare group 3c and group 3a.
[0251] In the macaque study, excluding the use of electroporation,
group 3a developed the highest ten or sixteen week total HIV
antigen-specific ELISPOT responses (1,652 and 1015 SFC/10.sup.6
cells). This response was not statistically different relative to
group 2d (770 SFC/10.sup.6 cells) or group 3c (787 SFC/10.sup.6
cells). See Table 8. However, the highest ELISPOT response was
achieved with the use of electroporation. See group 3cE in Table
8.
[0252] Interestingly, the peak immune response following booster
immunizations where electroporation was used was later than for the
non-electroporation groups. For example, the total HIV specific
IFN-gamma ELIspot response for group 3a animals peaked around week
6 following the week 4 immunization or boost. See Table 8. In
contrast, for the electroporation group, the peak was closer to
week 10. See Table 8.
[0253] The cellular immune response was further analyzed as
IFN-gamma ELISPOT responses to the six HIV proteins. Table 9 shows
IFN-gamma ELISPOT responses to the HIV env, gag, pol and a fusion
protein of nef-tat-vif proteins. In the macaque study, again
excluding the use of electroporation, group 3a developed the
highest ten-week HIV antigen-specific ELISPOT responses to env and
nef-tat-vif. See Table 9. Group 3c animals developed the highest
ELISPOT response to gag and group 2d developed the highest ELISPOT
response to pol protein. Compare 3a with 2d and 3c in Table 9. By
far the highest ELISPOT response was achieved with the use of
electroporation. See group 3cE in Table 9. TABLE-US-00016 TABLE 9
Individual HIV Antigen-Specific IFN-Gamma ELISPOT Responses At Week
10 After Multi-Plasmid DNA Vaccination Group Antigen-specific
IFN-gamma ELISPOT response* ID Env Gag Pol ntv total 2d 360.4 .+-.
111.8 107.9 .+-. 45.2 204.0 .+-. 182.6 97.6 .+-. 67.6 769.9 .+-.
340.4 3a 1170.4.sup.1 .+-. 427.0 .sup. 43.8 .+-. 17.5 173.8 .+-.
97.7 264.6.sup.3 .+-. 113.8.sup. 1652.5 .+-. 563.3 3c 412.1 .+-.
131.7 246.3.sup.2 .+-. 59.7 .sup. 106.7 .+-. 60.5 21.7 .+-. 8.9
786.7 .+-. 213.1 3cE 861.1 .+-. 292.5 1147.9 .+-. 356.9 1023.1 .+-.
384.0 605.7 .+-. 159.3 3637.8 .+-. 863.7 4a 132.9 .+-. 33.9 29.4
.+-. 6.5 9.1 .+-. 5.4 19.2 .+-. 7.9 190.6 .+-. 38.9 control 7.1
.+-. 3.4 1.7 .+-. 0.8 2.5 .+-. 1.1 0.8 .+-. 0.5 12.1 .+-. 4.1
*individual HIV antigen-specific IFN-gamma ELISPOT responses are
reported as the mean #SFC/10.sup.6 PBls .+-. standard error.
.sup.1Statistically higher env-specific ELISPOT response relative
to group 2d (p < 0.05). .sup.2Statistically higher gag-specific
ELISPOT response relative to group 3a (p < 0.05).
.sup.3Statistically higher ntv-specific ELISPOT response relative
to group 3c (p < 0.05).
[0254] Table 10 shows IFN-gamma ELISPOT responses to the HIV env,
gag, pol and a fusion protein of nef-tat-vif proteins at week
sixteen, 8 weeks after the last immunization. Excluding the use of
electroporation, group 3a developed the highest sixteen-week HIV
antigen-specific ELISPOT responses to env and nef-tat-vif, while
group 3c developed the highest ten-week HIV antigen-specific
ELISPOT responses to gag and pol. The highest ELISPOT response was
achieved with the use of electroporation. See group 3cE in Table
10.
[0255] Tables 9 and 10 show that increasing the number of antigen
expressing plasmids from 3 to 4 in the immunogenic composition
decreased immune response to all of the HIV proteins. See Tables 9
and 10.
[0256] Tables 9 and 10 also show that the plasmids in group 2d with
two antigen expressing plasmids in the immunogenic composition,
where one plasmid has two transcriptional units, induced the
broadest and most balanced immune response to all of the HIV
proteins. See Tables 9 and 10. TABLE-US-00017 TABLE 10 Individual
HIV antigen-specific IFN-gamma ELISpot responses at week 16 after
multi-plasmid DNA vaccination. Group Antigen-specific IFN-gamma
ELISPOT response* ID Env Gag Pol ntv total 2d 217.5 .+-. 33.3 76.3
.+-. 25.8 81.3 .+-. 32.2 32.5 .+-. 14.3 407.5 .+-. 82.2 3a 831.0
.+-. 457.8 39.7 .+-. 35.6 80.2 .+-. 68.7 64.3 .+-. 25.6 1015.3 .+-.
584.8 3c 437.5 .+-. 187.9 250.0 .+-. 88.2 96.3 .+-. 68.0 32.5 .+-.
10.7 816.3 .+-. 330.6 3cE 1984.7 .+-. 698.1 1975.3 .+-. 567.2
2305.6 .+-. 786.2 1875.3 .+-. 624.4 8140.8 .+-. 1819.0 4a nd.sup.1
nd nd nd nd control 22.5 .+-. 7.2 5.0 .+-. 2.3 9.2 .+-. 3.6 10.4
.+-. 4.4 47.1 .+-. 13.7 *individual HIV antigen-specific IFN-gamma
ELISpot responses are reported as the mean #SFC/10.sup.6 PBLs .+-.
standard error. .sup.1nd, not done
[0257] Table 11 shows IFN-gamma ELISPOT responses to the HIV env,
gag, pol and a fusion protein of nef-tat-vif proteins at thirty
weeks, 22 weeks after the last immunization. In the macaque study,
again excluding the use of electroporation, group 3a developed the
highest HIV antigen-specific ELISPOT responses to env, pol and
nef-tat-vif. See Table 11. Group 3c animals developed the highest
ELISPOT response to gag. Compare 3a with 2d and 3c in Table 11. The
highest ELISPOT response was achieved with the use of
electroporation. See group 3cE in Table 11.
[0258] In both the mouse and macaque studies, antigen-specific
ELISPOT responses were generally highest in groups receiving each
individual gene by itself under control of the HCMV promoter. In
the macaque study, electroporation was a more important factor in
producing immune responses than whether the immunogenic composition
contained plasmids having one versus two complete transcriptional
units or whether fusion proteins were used. TABLE-US-00018 TABLE 11
Individual HIV antigen-specific IFN-gamma ELISpot responses at week
30 after multi-plasmid DNA vaccination. Group Antigen-specific
IFN-gamma ELISpot response* ID Env Gag Pol ntv total 2d 44.2 .+-.
11.6 6.7 .+-. 3.1 8.8 .+-. 6.3 4.6 .+-. 3.6 64.2 .+-. 16.0 3a 184.0
.+-. 105.4 5.6 .+-. 3.7 14.0 .+-. 6.9 10.2 .+-. 4.7 213.9 .+-.
119.1 3c 52.5 .+-. 11.7 25.4 .+-. 6.6 2.9 .+-. 2.0 0.8 .+-. 0.8
81.7 .+-. 19.6 3cE 831.3 .+-. 339.1 768.9 .+-. 216.7 907.4 .+-.
476.5 886.4 .+-. 371.8 3,393.9 .+-. 920.4 4a.sup.1 nd nd nd nd nd
control 9.6 .+-. 4.8 0.0 .+-. 0.0 1.6 .+-. 1.2 0.0 .+-. 0.0 11.3
.+-. 5.8 *individual HIV antigen-specific IFN-gamma ELISpot
responses were reported as the mean #SFC/10.sup.6 PBLs .+-.
standard error. .sup.1Not done
[0259] Cellular Immune Response to Individual HIV Proteins Over
Time
[0260] IFN-gamma ELISPOT responses were measured at weeks 2, 4, 6,
8, 10 and 16 to individual HIV proteins env, gag, pol, nef, tat,
and vif following immunization with the plasmids described in Table
7. The results are presented in Tables 12-17. TABLE-US-00019 TABLE
12 HIV env-specific IFN-gamma ELISpot responses over time after
multi-plasmid DNA vaccination. HIV env-specific IFN-gamma ELISpot
response* Group Base- Week Week ID line Week 2 Week 4 Week 6 Week 8
10 16 2d 17.7 .+-. 4.5 204.0 .+-. 162.8 182.3 .+-. 64.8 295.1 .+-.
60.9 209.6 .+-. 66.1 360.4 .+-. 111.8 217.5 .+-. 33.3 3a 5.3 .+-.
2.2 43.8 .+-. 19.6 165.3 .+-. 20.6 577.9 .+-. 224.5 308.8 .+-. 38.6
1170.4 .+-. 427.0 831.0 .+-. 457.8 3c 21.0 .+-. 8.4 26.3 .+-. 7.1
84.8 .+-. 20.2 538.3 .+-. 174.2 192.1 .+-. 71.1 412.1 .+-. 131.7
437.5 .+-. 187.9 3cE 23.2 .+-. 9.5 598.3 .+-. 203.9 144.2 .+-. 30.9
382.9 .+-. 87.2 1165.8 .+-. 647.7 861.1 .+-. 292.5 1984.7 .+-.
698.1 4a 14.6 .+-. 8.7 24.2 .+-. 10.1 22.1 .+-. 9.6 254.2 .+-. 57.5
169.2 .+-. 33.5 132.9 .+-. 33.9 nd.sup.1 control 13.7 .+-. 5.4 3.0
.+-. 1.6 17.2 .+-. 9.0 17.1 .+-. 6.0 9.2 .+-. 2.6 7.1 .+-. 3.4 22.5
.+-. 7.2 *HIV env-specific IFN-gamma ELISpot responses were
reported as the mean #SFC/10.sup.6 PBLs .+-. standard rror.
.sup.1nd, not done
[0261] TABLE-US-00020 TABLE 13 HIV gag-specific IFN-gamma ELISpot
responses over time after multi-plasmid DNA vaccination. HIV
gag-specific IFN-gamma ELISpot response* Group Base- Week Week ID
line Week 2 Week 4 Week 6 Week 8 10 16 2d 6.8 .+-. 1.5 23.5 .+-.
16.7 36.0 .+-. 18.3 28.1 .+-. 5.7 59.6 .+-. 31.2 107.9 .+-. 45.2
76.3 .+-. 25.8 3a 2.2 .+-. 1.0 9.0 .+-. 3.4 21.5 .+-. 4.9 17.5 .+-.
11.5 10.0 .+-. 2.7 43.8 .+-. 17.5 39.7 .+-. 35.6 3c 4.5 .+-. 2.1
19.0 .+-. 6.7 51.7 .+-. 15.6 229.6 .+-. 67.0 86.7 .+-. 21.8 246.3
.+-. 59.7 250.0 .+-. 88.2 3cE 4.8 .+-. 2.9 709.6 .+-. 244.1 161.3
.+-. 38.3 381.7 .+-. 78.5 1169.6 .+-. 551.6 1147.9 .+-. 356.9
1975.3 .+-. 567.2 4a 2.1 .+-. 8.7 12.4 .+-. 3.7 5.4 .+-. 2.4 10.0
.+-. 4.0 27.5 .+-. 6.2 29.4 .+-. 6.5 nd.sup.1 control 3.2 .+-. 2.2
1.0 .+-. 0.6 7.7 .+-. 4.5 1.7 .+-. 0.8 2.1 .+-. 1.2 1.7 .+-. 0.8
5.0 .+-. 2.3 *HIV gag-specific IFN-gamma ELISpot responses are
reported as the mean #SFC/10.sup.6 PBLs .+-. standard error.
.sup.1nd, not done
[0262] TABLE-US-00021 TABLE 14 HIV pol-specific IFN-gamma ELISpot
responses over time after multi-plasmid DNA vaccination. HIV
pol-specific IFN-gamma ELISpot response* Group Base- Week Week ID
line Week 2 Week 4 Week 6 Week 8 10 16 2d 12.2 .+-. 4.3 33.8 .+-.
31.3 27.7 .+-. 7.6 53.3 .+-. 32.3 41.7 .+-. 25.1 204.0 .+-. 182.6
81.3 .+-. 32.2 3a 7.3 .+-. 4.1 1.8 .+-. 0.9 7.3 .+-. 2.9 17.5 .+-.
7.9 15.0 .+-. 4.5 173.8 .+-. 97.7 80.2 .+-. 68.7 3c 6.5 .+-. 3.4
3.5 .+-. 2.1 1.8 .+-. 1.3 102.1 .+-. 42.3 17.1 .+-. 6.8 106.7 .+-.
60.5 96.3 .+-. 68.0 3cE 3.7 .+-. 2.4 54.6 .+-. 30.5 22.1 .+-. 9.1
316.3 .+-. 215.8 497.9 .+-. 179.7 1023.1 .+-. 384.0 2305.6 .+-.
786.2 4a 1.7 .+-. 1.1 9.3 .+-. 6.8 2.5 .+-. 1.3 5.4 .+-. 2.0 13.8
.+-. 4.8 9.1 .+-. 5.4 nd.sup.1 control 10.7 .+-. 4.4 3.2 .+-. 2.8
4.7 .+-. 3.0 2.1 .+-. 1.6 4.2 .+-. 2.7 2.5 .+-. 1.1 9.2 .+-. 3.6
*HIV pol-specific IFN-gamma ELISpot responses are reported as the
mean #SFC/10.sup.6 PBLs .+-. standard error. .sup.1nd, not done
[0263] TABLE-US-00022 TABLE 15 HIV nef-specific IFN-gamma ELISpot
responses over time after multi-plasmid DNA vaccination. HIV
nef-specific IFN-gamma ELISpot response* Group Base- Week Week ID
line Week 2 Week 4 Week 6 Week 8 10 16 2d 4.8 .+-. 3.2 16.3 .+-.
16.3 22.5 .+-. 16.7 12.4 .+-. 6.0 32.9 .+-. 14.4 43.7 .+-. 27.6
24.6 .+-. 12.3 3a 20.1 .+-. 9.8 2.5 .+-. 2.0 7.9 .+-. 3.6 13.8 .+-.
5.2 22.5 .+-. 9.8 192.1 .+-. 76.7 54.8 .+-. 25.4 3c 4.2 .+-. 4.2
0.4 .+-. 0.4 0.0 .+-. 0.0 10.4 .+-. 7.5 3.3 .+-. 2.5 10.0 .+-. 8.1
18.3 .+-. 9.6 3cE 5.1 .+-. 3.4 11.9 .+-. 7.2 11.7 .+-. 7.7 67.1
.+-. 56.6 281.7 .+-. 207.0 403.2 .+-. 158.3 1276.2 .+-. 516.3 4a
0.4 .+-. 0.4 1.7 .+-. 1.4 5.4 .+-. 3.1 2.1 .+-. 2.1 10.4 .+-. 5.0
8.3 .+-. 4.4 nd.sup.1 control 3.6 .+-. 2.8 0.8 .+-. 0.8 0.8 .+-.
0.8 0.0 .+-. 0.0 0.0 .+-. 0.0 0.0 .+-. 0.0 2.9 .+-. 1.5 *HIV
nef-specific IFN-gamma ELISpot responses are reported as the mean
#SFC/10.sup.6 PBLs .+-. standard error. .sup.1nd, not done
[0264] TABLE-US-00023 TABLE 16 HIV tat-specific IFN-gamma ELISpot
responses over time after multi-plasmid DNA vaccination. HIV
tat-specific IFN-gamma ELISpot response* Group Base- Week Week ID
line Week 2 Week 4 Week 6 Week 8 10 16 2d 7.1 .+-. 3.3 0.8 .+-. 0.5
7.1 .+-. 4.2 8.5 .+-. 3.3 2.9 .+-. 2.1 4.6 .+-. 2.1 3.8 .+-. 1.4 3a
10.0 .+-. 5.3 3.8 .+-. 2.3 2.9 .+-. 1.2 4.2 .+-. 2.0 8.8 .+-. 7.3
14.6 .+-. 8.2 1.7 .+-. 1.2 3c 6.2 .+-. 4.5 6.3 .+-. 2.9 0.4 .+-.
0.4 8.3 .+-. 3.5 0.4 .+-. 0.4 1.3 .+-. 1.3 2.9 .+-. 1.2 3cE 7.6
.+-. 5.2 22.4 .+-. 13.8 2.1 .+-. 1.0 25.0 .+-. 17.8 75.0 .+-. 42.4
29.3 .+-. 19.9 190.0 .+-. 88.4 4a 0.0 .+-. 0.0 1.8 .+-. 1.5 5.8
.+-. 2.9 1.3 .+-. 1.3 5.8 .+-. 3.7 10.3 .+-. 6.1 nd.sup.1 control
5.1 .+-. 4.5 0.8 .+-. 0.5 2.1 .+-. 1.6 3.3 .+-. 1.5 0.0 .+-. 0.0
0.0 .+-. 0.0 2.1 .+-. 1.2 *HIV tat-specific IFN-gamma ELISpot
responses are reported as the mean #SFC/10.sup.6 PBLs .+-. standard
error. .sup.1nd, not done
[0265] TABLE-US-00024 TABLE 17 HIV vif-specific IFN-gamma ELISpot
responses over time after multi-plasmid DNA vaccination. HIV
vif-specific IFN-gamma ELISpot response* Group Base- Week Week ID
line Week 2 Week 4 Week 6 Week 8 10 16 2d 9.4 .+-. 3.9 7.9 .+-. 7.9
3.3 .+-. 2.9 5.8 .+-. 2.7 1.7 .+-. 1.2 8.7 .+-. 8.1 4.2 .+-. 1.9 3a
12.9 .+-. 8.5 0.4 .+-. 0.4 0.4 .+-. 0.4 4.2 .+-. 2.3 0.8 .+-. 0.8
12.1 .+-. 12.1 7.8 .+-. 2.7 3c 6.4 .+-. 4.8 0.8 .+-. 0.5 0.0 .+-.
0.0 3.8 .+-. 2.0 0.4 .+-. 0.4 2.5 .+-. 2.5 11.3 .+-. 3.3 3cE 8.9
.+-. 5.9 8.2 .+-. 5.1 5.0 .+-. 2.6 115.0 .+-. 51.6 159.6 .+-. 64.8
173.2 .+-. 103.6 409.1 .+-. 129.9 4a 0.0 .+-. 0.0 2.8 .+-. 1.8 2.1
.+-. 0.8 0.0 .+-. 0.0 3.3 .+-. 1.1 0.6 .+-. 0.2 nd.sup.1 control
6.8 .+-. 2.2 1.2 .+-. 0.8 0.8 .+-. 0.8 0.0 .+-. 0.0 1.3 .+-. 1.3
0.0 .+-. 0.0 5.4 .+-. 3.1 *HIV vif-specific IFN-gamma ELISpot
responses are reported as the mean #SFC/10.sup.6 PBLs .+-. standard
error. .sup.1nd, not done
[0266] Tables 12-17, which show immune responses to individual
proteins over time indicate that increasing the number of antigen
expressing plasmids from 3 to 4 in the immunogenic composition,
resulted in decreased immune response to all of the HIV proteins at
this given concentration of DNA administered. See Tables 12-17.
Example 10
Estimation of the Percentage of HIV Specific CTL and Helper
Cells
[0267] The relative amounts of HIV specific CTL and helper cells
were estimated by first depleting unfractionated peripheral blood
lymphocytes (PBLs) of CD4+ or CD8+ cells prior to measuring total
HIV-specific IFN-gamma ELISpot responses at weeks 10 and 16.
[0268] Preparation of Bead Depleted PBLs
[0269] CD4+ or CD8+ cells were depleted from unfractionated PBLs
using magnetic polystyrene beads coated with anti-human CD4- or
CD8-specific mouse monoclonal antibodies, as per the manufacturer's
instructions (Dynal Biotech, Oslo, Norway). Briefly, freshly
isolated rhesus PBLs were washed and resuspended to a final
concentration of 2.times.10.sup.6 cells/mL in ice cold 1.times.PBS
containing 2% FBS. Dynal microbeads coated with either anti-human
CD4- or anti-CD8-specific mouse monoclonal antibodies were washed
three times with 1.times.PBS containing 2% FCS then added to
unfractionated PBLs at a 5:1 bead to cell ratio, and incubated for
one hour at 4.degree. C. on a rotating/tilting apparatus. After
incubation, the bead/cell suspension was placed in a magnetic
column, and the flow through containing either CD4+ or CD8+ cell
depleted PBLs was collected. The cells were washed once with
complete culture medium supplemented with 5% FBS, and resuspended
to the original volume with complete culture medium supplemented
with 5% FBS. Equal volumes of unfractionated, and bead depleted
PBLs, were used directly in the ELISpot assay.
[0270] The efficiency of CD4+ and CD8+ cell subset depletion and
the precise numbers of CD4+ and CD8+ cells added to the ELISpot
plate were subsequently quantified by flow cytometry. Briefly, bead
depleted PBLs were washed once with 1.times.PBS containing 2% FBS
and stained for 15 minutes at room temperature with the following
monoclonal antibodies: anti-rhesus macaque CD3-fluorescein
isothiocyanate (FITC, clone SP34; BD Pharmingen, San Jose, Calif.);
anti-human CD4-phycoerythrin (PE, clone M-T477; BD Pharmingen, San
Jose, Calif.); anti-human CD8-peridinin chlorophyll protein (PerCP;
clone SK1; BD Pharmingen, San Jose, Calif.); and anti-human
CD20-allophycocyanin (APC, clone L27; BD Pharmingen, San Jose,
Calif.). Cells were then washed once with 1.times.PBS containing 2%
FBS, 0.02% azide and resuspended in 1.times.PBS containing 1%
paraformaldehyde. FACS analysis was performed on a FACSCalibur Flow
Cytometer (Becton Dickinson, Franklin Lakes, N.J.) and analyzed
using CellQuest Software. The percent CD4+ or CD8+ cell depletion
was routinely >95% (data not shown). TABLE-US-00025 TABLE 18
Total HIV-specific IFN-gamma ELISpot responses at week 10 and 16 in
unfractionated and CD4+ or CD8+ cell depleted PBLs. Week 10 Week 16
Group CD4 CD8 CD4 CD8 ID Unfrac depleted depleted Unfrac depleted
depleted 2d 1,501 .+-. 632 1,494 .+-. 801 364 .+-. 77 902 .+-. 173
758 .+-. 141 431 .+-. 93 3a 2,524 .+-. 789 1,239 .+-. 662 997 .+-.
222 1,821 .+-. 906 1,059 .+-. 689 539 .+-. 175 3c 1,484 .+-. 359
908 .+-. 268 536 .+-. 147 1,532 .+-. 556 856 .+-. 308 607 .+-. 203
3cE 6,651 .+-. 1,326 10,563 .+-. 3,388 1,921 .+-. 274 13,361 .+-.
2,770 21,051 .+-. 7,067 2,754 .+-. 543 4a 1,591 .+-. 281 688 .+-.
119 954 .+-. 248 nd.sup.1 nd nd control 6 .+-. 2 31 .+-. 10 34 .+-.
15 187 .+-. 12 107 .+-. 31 118 .+-. 24 *Total HIV-specific
IFN-gamma ELISpot responses are reported as the mean #SFC/10.sup.6
unfractionated, CD4+ or CD8+ depleted PBLs .+-. standard error.
.sup.1nd, not done
[0271] The results shown in Table 18 provide an estimate of the
relative percentage of HIV specific CTL cells versus helper cells
participating in a particular induced immune response. A few
general observations may be drawn from the data. First groups 2d,
3a and 3c elicit similar magnitudes of cellular immune response to
HIV. Group 3a appears to induce a higher level of immune response,
but the amount of variation in the assay is also greater with that
group. Where electroporation was used in conjunction with
immunization, the magnitude of the immune response to the plasmids
in group 3c was enhanced by about 5 fold to about 10 fold. See
Table 18, compare 3cE and 3c. It is also worthy of note that many
more cells were participating in the immune response as a result of
the use of electroporation with the immunization.
Example 11
HIV Specific Antibody Titers Induced by Multi-Plasmid
Immunization
[0272] An immunogenic composition (IC) containing plasmid DNA
provides several advantages over other types of immunogenic
composition technologies currently in use. For example, DNA based
ICs, in contrast to conventional protein based subunit ICs, allow
for the encoded antigen to be efficiently processed and presented
by the major histocompatability complex (MHC) Class I antigen
processing pathway. The class I antigen processing pathway is
critical for the induction of CD8+ T-cell mediated immune
responses. However, conventional protein based subunit ICs
typically outperform DNA based ICs in terms of their ability to
elicit antigen-specific antibody responses.
[0273] For the determination of HIV viral lysate-specific antibody
titers, ELISA plates were coated for 18 hours at 4.degree. C. with
detergent disrupted HIV-1.sub.MN at 20 ng/well, (Advanced
Biotechnologies, Columbia, Md.). The detergent disrupted
HIV-1.sub.MN was diluted in carbonate/bicarbonate buffer (15
mMNa.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.6). For the
determination of HIV env-specific antibody titers, ELISA plates
were coated with purified HIV-1 6101 gp120 (kindly provided by
Larry Liao, Duke University, 20 ng/well) diluted in 1.times.PBS.
Following the 18 hour incubation with HIV proteins, the ELISA
plates were then washed five times with 1.times.PBS containing 0.1%
Tween 20 and blocked for 2 hours at room temperature with
1.times.PBS containing 0.1% Tween 20 and 3% BSA. Serum samples from
immunized and control animals were diluted with 1.times.PBS
containing 1% BSA and 0.1% Tween-20, added to the ELISA plates at a
starting dilution of 1:100 and further diluted 3-fold across the
plates. The diluted serum samples were incubated overnight at
4.degree. C. with the protein coated plates. Detection of
antigen-specific immunoglobulin was accomplished by incubating a
biotin conjugated primary antibody specific for primate IgG for 2
hours ar room temp. This antibody was diluted 1:30,000 with
1.times.PBS supplemented with 0.1% Tween-20, 1% BSA, Accurate
Scientific, Westbury, N.Y. Next, the primary antibody was washed
away and followed with a 1 hour room temperature incubation of
streptavidin-horseradish peroxidase conjugated anti-biotin
secondary antibody (500 units/ml stock, diluted 1:10,000 with
1.times.PBS supplemented with 0.1% Tween-20, 1% BSA, Roche
Immunochemical, Indianapolis, Ind.). Finally, color was developed
by the addition of 100 mcL/well of TMB (3,3',5,5'-tetramethyl
benzidine, Sigma). Antigen-specific antibody titers were defined as
the reciprocal of the last serum dilution giving an O.D..sub.450
greater than the same animal's naive serum (i.e. week 0)+3 standard
deviations.
[0274] HIV envelope titers for certain time points over the first
16 weeks of multi-plasmid DNA immunizations were determined and are
shown in Table 19. HIV-1 6101 env gp120 ELISA titers were
calculated as the reciprocal of the last serum dilution giving an
O.D..sub.450 greater than the same animal's naive serum (i.e. week
0)+3 standard deviations. The data in Table 19 (as well as in Table
20 below) were presented as the mean log.sub.10 titer.+-.standard
error of the mean. In this case, HIV-1 env titers .ltoreq.2.00
represent an endpoint titer of less than 1:100 and were below the
limit of detection. TABLE-US-00026 TABLE 19 HIV-1 6101 env gp120
specific ELISA antibody titers over time after multi-plasmid DNA
Immunization. Group.sup.1 HIV-1 env ELISA titer* ID Week 2 Week 4
Week 6 Week 8 Week 10 Week 16 2d 2.00 .+-. 0.00 2.00 .+-. 0.00 2.08
.+-. .035 2.43 .+-. 0.21 2.73 .+-. 0.27 2.59 .+-. 0.20 3a 2.00 .+-.
0.00 2.00 .+-. 0.00 2.16 .+-. 0.10 2.64 .+-. 0.32 2.95 .+-. 0.28
2.56 .+-. 0.29 3c 2.00 .+-. 0.00 2.00 .+-. 0.00 2.16 .+-. 0.16 2.48
.+-. 0.21 2.80 .+-. 0.32 2.95 .+-. 0.37 3cE 2.16 .+-. 0.16 2.72
.+-. 0.16 4.39 .+-. 0.49 3.67 .+-. 0.44 5.18 .+-. 0.20 4.78 .+-.
0.23 4a nd.sup.1 nd nd nd nd nd control 2.00 .+-. 0.00 2.08 .+-.
0.08 2.00 .+-. 0.00 2.16 .+-. 0.10 2.16 .+-. 0.16 2.32 .+-. 0.16
*Data were reported as the mean log.sub.10 titer .+-. standard
error of the mean. HIV-1 env titers .ltoreq.2.00 represent an
endpoint titer of less than 1:100 and were below the limit of
detection. .sup.1nd indicates not done
[0275] As shown in Table 19, group 3c animals immunized with
immunogenic compositions based on a combination of plasmids
containing at least one plasmid with more than one transcriptional
unit achieved the highest non-electroporation titers at week 16.
However, the results for groups 2d and 3a were somewhat similar,
but with groups 3a animals showing the highest titers at weeks 8
and 10. See Table 19, compare 3a with 2d and 3c. An immunogenic
composition based on a combination of plasmids containing at least
one plasmid with more than one transcriptional unit and receiving
electroporation-electrostimulation with immunization developed by
far the highest titers to the HIV envelope protein. See Table 19,
Compare 3c with 3cE.
[0276] Total HIV titers to whole virus lysate was determined for
weeks 2, 4, 6, 8, 10, and 16 weeks of multi-plasmid DNA
immunizations are shown in Table 20. HIV-1.sub.MN viral
lysate-specific ELISA titers were determined as the reciprocal of
the last serum dilution giving an O.D..sub.450 greater than the
same macaque's naive serum (i.e. pre-immune)+3 standard deviations.
In this table, the data were reported as the mean log.sub.10
titer.+-.standard error of the mean. Note that antibody titers
.ltoreq.1.70 represent an endpoint titer of less than 1:50 and were
below the limit of detection. The results in Table 20 at week 16
were similar to these presented in Table 19. TABLE-US-00027 TABLE
20 Total HIV-1-specific ELISA antibody titers over time after
multi-plasmid DNA vaccination. Total HIV-1 ELISA titer* Group Week
Week ID Week 2 Week 4 Week 6 Week 8 10 16 2d 1.70 .+-. 0.00 1.70
.+-. 0.00 1.75 .+-. 0.05 1.75 .+-. 0.05 2.04 .+-. 0.28 1.70 .+-.
0.00 3a 1.75 .+-. 0.05 1.75 .+-. 0.05 1.70 .+-. 0.00 1.70 .+-. 0.00
1.70 .+-. 0.00 1.70 .+-. 0.00 3c 2.06 .+-. 0.19 2.11 .+-. 0.18 1.75
.+-. 0.05 1.88 .+-. 0.13 1.85 .+-. 0.07 1.90 .+-. 0.06 3cE 1.88
.+-. 0.13 1.88 .+-. 0.13 3.46 .+-. 0.53 2.38 .+-. 0.34 4.36 .+-.
0.16 3.75 .+-. 0.29 4a nd.sup.1 nd nd nd nd nd control 1.70 .+-.
0.00 1.75 .+-. 0.05 1.70 .+-. 0.00 1.70 .+-. 0.00 1.91 .+-. 0.21
1.91 .+-. 0.21 *Data were reported as the mean log.sub.10 titer
.+-. standard error of the mean. Antibody titers .ltoreq.1.70
represent an endpoint titer of less than 1:50 and were below the
limit of detection. .sup.1nd indicates not done
Example 12
Effect of Multi-Plasmid Immunization on Various Serological
Parameters and Body Weight in Macaques
[0277] The peripheral blood white blood cell counts (WBC) in
macaques used in the study were determined over time by complete
blood count analysis and reported as the mean WBC
(.times.1000/ml).+-.standard error. See Table 21. TABLE-US-00028
TABLE 21 Total WBC counts (.times.1000) in macaques immunized with
plasmid DNA vaccines with and without electroporation Week Group ID
-2 0 2 4 6 8 10 16 2d 10.3 .+-. 1.1 8.8 .+-. 1.4 8.1 .+-. 1.0 7.2
.+-. 0.7 7.1 .+-. 1.0 8.6 .+-. 0.7 6.9 .+-. 0.9 6.6 .+-. 0.4 3a 8.6
.+-. 1.4 5.5 .+-. 0.8 7.9 .+-. 1.3 6.0 .+-. 0.9 6.3 .+-. 1.0 7.3
.+-. 1.1 7.8 .+-. 1.1 8.0 .+-. 1.6 3c 9.4 .+-. 1.4 6.3 .+-. 0.6 8.0
.+-. 0.8 7.0 .+-. 0.8 7.3 .+-. 0.9 9.9 .+-. 0.9 8.4 .+-. 1.4 7.8
.+-. 1.2 3cE 11.0 .+-. 1.7 12.1 .+-. 1.5 8.2 .+-. 1.1 18.4 .+-. 2.0
11.0 .+-. 1.3 13.1 .+-. 1.3 9.3 .+-. 0.9 7.9 .+-. 0.5 4a 11.6 .+-.
0.8 10.3 .+-. 1.4 8.9 .+-. 0.8 8.0 .+-. 0.8 8.2 .+-. 0.5 7.9 .+-.
0.5 8.3 .+-. 0.7 nd.sup.1 control 7.6 .+-. 0.9 5.6 .+-. 0.7 7.1
.+-. 0.9 5.7 .+-. 0.6 5.9 .+-. 0.7 7.6 .+-. 1.3 5.6 .+-. 0.5 6.6
.+-. 0.7 *Peripheral blood white blood cell counts (WBC) as
determined by complete blood count analysis are reported as the
mean WBC (.times.1000/ml) .+-. standard error. .sup.1nd, not
done
[0278] Peripheral blood red blood cell counts (RBC) in animals used
in the study were determined over time by complete blood count
analysis and reported as the mean RBC
(.times.10.sup.6/ml).+-.standard error. See Table 22.
[0279] The peripheral blood hemoglobin levels (g/dL) in animals
used in the study were determined over time by complete blood count
analysis and reported as the mean hemoglobin level.+-.standard
error. See Table 23.
[0280] Multi-plasmid immunization with the plasmids and immunogenic
compositions described in Table 7 did not produce any adverse
effects on the WBCs, RBCs and hemoglobin levels in animals used in
this study. See Tables 21-23. One clear positive effect was
detected when electroporation was used with the immunogenic
composition used to immunize group 3cE. In this group, the number
of WBC was significantly elevated throughout the time course of the
study. See Table 21. TABLE-US-00029 TABLE 22 Total RBC counts
(.times.10.sup.6) in macaques immunized with plasmid DNA vaccines
with and without electroporation. Week Group ID -2 0 2 4 6 8 10 16
2d 5.60 .+-. 0.12 5.64 .+-. 0.03 5.62 .+-. 0.08 5.69 .+-. 0.09 5.70
.+-. 0.11 5.67 .+-. 0.11 5.74 .+-. 0.06 5.91 .+-. 0.08 3a 5.61 .+-.
0.19 5.36 .+-. 0.17 5.39 .+-. 0.17 5.40 .+-. 0.13 5.39 .+-. 0.15
5.53 .+-. 0.18 5.32 .+-. 0.14 5.70 .+-. 0.16 3c 5.39 .+-. 0.13 5.32
.+-. 0.14 5.43 .+-. 0.09 5.46 .+-. 0.13 5.38 .+-. 0.14 5.45 .+-.
0.10 5.52 .+-. 0.13 5.69 .+-. 0.09 3cE 5.63 .+-. 0.15 5.91 .+-.
0.09 5.80 .+-. 0.07 5.60 .+-. 0.21 5.87 .+-. 0.10 5.57 .+-. 0.13
5.70 .+-. 0.07 5.75 .+-. 0.11 4a 5.99 .+-. 0.11 5.68 .+-. 0.09 5.97
.+-. 0.08 5.77 .+-. 0.11 5.84 .+-. 0.07 5.79 .+-. 0.12 5.54 .+-.
0.10 nd.sup.1 control 5.69 .+-. 0.18 5.49 .+-. 0.13 5.57 .+-. 0.09
5.63 .+-. 0.09 5.61 .+-. 0.08 5.66 .+-. 0.09 5.73 .+-. 0.12 5.94
.+-. 0.13 *Peripheral blood red blood cell counts (RBC) were
determined by complete blood count analysis and reported as the
mean RBC (.times.10.sup.6/ml) .+-. standard error.
[0281] TABLE-US-00030 TABLE 23 Total hemaglobin levels in macaques
immunized with plasmid DNA vaccines with and without
electroporation. Week Group ID -2 0 2 4 6 8 10 16 2d 12.5 .+-. 0.3
12.7 .+-. 0.2 12.5 .+-. 0.2 12.6 .+-. 0.2 12.5 .+-. 0.1 12.6 .+-.
0.2 12.9 .+-. 0.2 13.1 .+-. 0.2 3a 13.1 .+-. 0.3 12.6 .+-. 0.3 12.6
.+-. 0.3 12.5 .+-. 0.3 12.6 .+-. 0.3 13.0 .+-. 0.3 12.8 .+-. 0.4
13.4 .+-. 0.2 3c 12.7 .+-. 0.3 12.6 .+-. 0.2 12.7 .+-. 0.2 12.7
.+-. 0.2 12.6 .+-. 0.4 13.0 .+-. 0.3 13.2 .+-. 0.3 13.5 .+-. 0.3
3cE 12.8 .+-. 0.3 13.4 .+-. 0.2 13.0 .+-. 0.2 13.1 .+-. 0.3 13.4
.+-. 0.2 12.9 .+-. 0.2 13.0 .+-. 0.1 13.3 .+-. 0.2 4a 13.5 .+-. 0.3
13.1 .+-. 0.2 13.5 .+-. 0.2 13.1 .+-. 0.2 13.2 .+-. 0.2 13.1 .+-.
0.2 12.5 .+-. 0.2 nd.sup.1 control 13.3 .+-. 0.3 12.8 .+-. 0.3 13.0
.+-. 0.2 12.9 .+-. 0.2 13.0 .+-. 0.2 13.2 .+-. 0.1 13.6 .+-. 0.3
13.9 .+-. 0.3 *Peripheral blood hemoglobin levels (g/dL) as
determined by complete blood count analysis are reported as the
mean hemoglobin level .+-. standard error. .sup.1nd, not done
[0282] Peripheral blood platelet levels as determined in animals
used in the study were determined over time by complete blood count
analysis and reported as the mean platelet level
(.times.1000).+-.standard error. See Table 24.
[0283] Percent hematocrit levels in animals used in the study were
determined over time by complete blood count analysis and reported
as the mean percent hematocrit level.+-.standard error. See Table
25.
[0284] Peripheral blood total lymphocyte numbers as determined in
animals used in the study were determined over time by complete
blood count analysis and reported as the mean total lymphocyte
number.+-.standard error. See Table 26.
[0285] Peripheral blood total CD3.sup.+ T-lymphocyte numbers in
animals used in the study were determined over time by complete
blood count analysis and reported as the mean total CD3.sup.+
T-lymphocyte number.+-.standard error. See Table 27.
[0286] Peripheral blood total CD3.sup.+CD4.sup.+ Th-lymphocyte
numbers in animals used in the study were determined over time by
complete blood count analysis and reported as the mean total
CD3.sup.+CD4.sup.+ Th-lymphocyte number.+-.standard error. See
Table 28.
[0287] Peripheral blood total CD3.sup.+CD8.sup.+ T-lymphocyte
numbers in animals used in the study were determined over time by
complete blood count analysis and reported as the mean total
CD3.sup.+CD8.sup.+ T-lymphocyte number.+-.standard error. See Table
29.
[0288] Peripheral blood total CD20.sup.+ lymphocyte numbers in
animals used in the study were determined over time by complete
blood count analysis and reported as the mean total CD20.sup.+
lymphocyte number.+-.standard error. See Table 30. TABLE-US-00031
TABLE 24 Total platelet counts (.times.1000) in macaques immunized
with plasmid DNA vaccines with and without electroporation. Week
Group ID -2 0 2 4 6 8 10 16 2d 404 .+-. 42 433 .+-. 19 399 .+-. 21
411 .+-. 16 392 .+-. 30 420 .+-. 13 448 .+-. 17 394 .+-. 21 3a 419
.+-. 41 418 .+-. 31 399 .+-. 28 441 .+-. 25 402 .+-. 30 411 .+-. 20
450 .+-. 35 380 .+-. 17 3c 454 .+-. 19 404 .+-. 13 418 .+-. 21 405
.+-. 19 391 .+-. 13 423 .+-. 41 381 .+-. 23 381 .+-. 27 3cE 384
.+-. 29 389 .+-. 30 414 .+-. 31 389 .+-. 33 431 .+-. 33 315 .+-. 33
400 .+-. 24 347 .+-. 24 4a 364 .+-. 21 373 .+-. 9 339 .+-. 16 368
.+-. 15 355 .+-. 16 357 .+-. 16 360 .+-. 19 nd.sup.1 control 458
.+-. 39 412 .+-. 33 386 .+-. 47 383 .+-. 14 386 .+-. 43 414 .+-. 35
409 .+-. 27 378 .+-. 34 *Peripheral blood platelet levels as
determined by complete blood count analysis are reported as the
mean platelet level (.times.1000) .+-. standard error. .sup.1nd,
not done
[0289] TABLE-US-00032 TABLE 25 Percent hematocrit in macaques
immunized with plasmid DNA vaccines with and without
electroporation. Week Group ID -2 0 2 4 6 8 10 16 2d 38.3 .+-. 0.9
38.5 .+-. 0.5 37.8 .+-. 0.6 38.6 .+-. 0.4 38.4 .+-. 0.4 38.6 .+-.
0.5 38.9 .+-. 0.4 40.2 .+-. 0.5 3a 39.9 .+-. 1.0 37.7 .+-. 0.8 38.4
.+-. 1.1 38.3 .+-. 0.9 38.0 .+-. 0.8 39.7 .+-. 1.1 38.3 .+-. 1.1
40.7 .+-. 0.7 3c 38.9 .+-. 0.8 37.8 .+-. 0.8 38.7 .+-. 0.5 38.8
.+-. 1.1 38.4 .+-. 1.1 39.3 .+-. 0.9 39.7 .+-. 0.9 40.6 .+-. 0.8
3cE 39.1 .+-. 0.9 40.6 .+-. 0.5 39.7 .+-. 0.6 39.6 .+-. 0.9 40.9
.+-. 0.5 39.0 .+-. 0.6 40.0 .+-. 0.3 39.9 .+-. 0.5 4a 41.3 .+-. 0.8
38.8 .+-. 0.6 40.8 .+-. 0.5 39.6 .+-. 0.4 40.1 .+-. 0.6 39.8 .+-.
0.6 37.9 .+-. 0.5 nd.sup.1 control 40.3 .+-. 1.0 38.5 .+-. 0.6 39.3
.+-. 0.4 39.4 .+-. 0.4 39.6 .+-. 0.5 40.3 .+-. 0.5 40.8 .+-. 0.7
41.8 .+-. 0.7 *Percent hematocrit levels as determined by complete
blood count analysis are reported as the mean percent hematocrit
level .+-. standard error. .sup.1nd, not done
[0290] TABLE-US-00033 TABLE 26 Total lymphocyte numbers in macaques
immunized with plasmid DNA vaccines with and without
electroporation. Week Group ID -2 0 2 4 6 8 10 16 2d 3444 .+-. 554
4399 .+-. 521 3952 .+-. 578 4038 .+-. 462 3646 .+-. 677 4631 .+-.
574 3600 .+-. 581 3018 .+-. 422 3a 2955 .+-. 613 2901 .+-. 452 2706
.+-. 405 2910 .+-. 434 2804 .+-. 459 3631 .+-. 714 3186 .+-. 775
3814 .+-. 736 3c 3213 .+-. 448 3097 .+-. 369 3192 .+-. 407 3343
.+-. 559 3417 .+-. 699 4268 .+-. 667 3098 .+-. 678 3925 .+-. 805
3cE 3157 .+-. 331 3737 .+-. 718 4441 .+-. 608 2737 .+-. 383 4835
.+-. 822 5286 .+-. 987 4927 .+-. 575 4385 .+-. 612 4a 4850 .+-. 348
3763 .+-. 381 4268 .+-. 339 3471 .+-. 149 4544 .+-. 363 3494 .+-.
248 3408 .+-. 248 nd.sup.1 control 2638 .+-. 230 3685 .+-. 784 3280
.+-. 349 3037 .+-. 334 3828 .+-. 456 4392 .+-. 465 3451 .+-. 358
3470 .+-. 220 *Peripheral blood total lymphocyte numbers as
determined by complete blood count analysis are reported as the
mean total lymphocyte number .+-. standard error. .sup.1nd, not
done
[0291] TABLE-US-00034 TABLE 27 Total CD3.sup.+ T-lymphocyte numbers
in macaques immunized with plasmid DNA vaccines with and without
electroporation. Week Group ID -2 0 2 4 6 8 10 16 2d 1778 .+-. 356
2469 .+-. 265 2167 .+-. 306 2299 .+-. 257 2051 .+-. 356 2917 .+-.
313 2261 .+-. 318 1852 .+-. 218 3a 1697 .+-. 291 1796 .+-. 269 1681
.+-. 255 1910 .+-. 327 1822 .+-. 322 2536 .+-. 450 2344 .+-. 619
2772 .+-. 523 3c 1862 .+-. 215 1815 .+-. 175 1862 .+-. 187 1949
.+-. 279 2080 .+-. 341 2679 .+-. 313 2019 .+-. 385 2458 .+-. 426
3cE 1716 .+-. 223 1926 .+-. 421 2718 .+-. 427 1417 .+-. 241 3139
.+-. 560 3437 .+-. 680 3229 .+-. 360 2928 .+-. 457 4a 2848 .+-. 240
2141 .+-. 263 2481 .+-. 265 1881 .+-. 95 2851 .+-. 328 2153 .+-.
212 2141 .+-. 224 nd.sup.1 control 1455 .+-. 85 2188 .+-. 484 1883
.+-. 218 1749 .+-. 258 2334 .+-. 382 2789 .+-. 334 2352 .+-. 341
2291 .+-. 197 *Peripheral blood
[0292] TABLE-US-00035 TABLE 28 Total CD3.sup.+CD4.sup.+
Th-lymphocyte numbers in macaques immunized with plasmid DNA
vaccines with and without electroporation. Week Group ID -2 0 2 4 6
8 10 16 2d 1117 .+-. 226 1463 .+-. 197 1348 .+-. 219 1371 .+-. 190
1317 .+-. 225 1770 .+-. 208 1435 .+-. 225 1457 .+-. 266 3a 934 .+-.
143 1007 .+-. 158 986 .+-. 156 1084 .+-. 191 1078 .+-. 198 1425
.+-. 242 1291 .+-. 322 1535 .+-. 287 3c 1132 .+-. 167 1108 .+-. 130
1178 .+-. 129 1195 .+-. 176 1283 .+-. 209 1598 .+-. 208 1229 .+-.
224 1480 .+-. 256 3cE 1034 .+-. 155 1115 .+-. 194 1622 .+-. 267 827
.+-. 124 1752 .+-. 271 1917 .+-. 347 1673 .+-. 165 1628 .+-. 165 4a
1774 .+-. 220 1362 .+-. 202 1528 .+-. 202 1171 .+-. 91 1743 .+-.
247 1363 .+-. 163 1360 .+-. 174 nd.sup.1 control 877 .+-. 79 1292
.+-. 259 1162 .+-. 117 1109 .+-. 155 1430 .+-. 239 1659 .+-. 226
1437 .+-. 178 1353 .+-. 139 *Peripheral blood total
CD3.sup.+CD4.sup.+ Th-lymphocyte numbers as determined by complete
blood count analysis are reported as the mean total
CD3.sup.+CD4.sup.+ Th-lymphocyte number .+-. standard error.
.sup.1nd, not done
[0293] TABLE-US-00036 TABLE 29 Total CD3.sup.+CD8.sup.+
T-lymphocyte numbers in macaques immunized with plasmid DNA
vaccines with and without electroporation. Week Group ID -2 0 2 4 6
8 10 16 2d 627 .+-. 141 1008 .+-. 105 807 .+-. 96 908 .+-. 87 729
.+-. 147 1137 .+-. 131 811 .+-. 103 678 .+-. 92 3a 729 .+-. 159 778
.+-. 118 691 .+-. 94 823 .+-. 139 729 .+-. 120 1111 .+-. 224 1041
.+-. 285 1254 .+-. 251 3c 663 .+-. 61 661 .+-. 69 635 .+-. 61 709
.+-. 102 744 .+-. 122 1023 .+-. 111 712 .+-. 151 884 .+-. 149 3cE
626 .+-. 78 774 .+-. 229 1067 .+-. 169 542 .+-. 114 1409 .+-. 334
1431 .+-. 348 1528 .+-. 206 1270 .+-. 294 4a 1005 .+-. 47 721 .+-.
70 901 .+-. 74 628 .+-. 53 994 .+-. 95 699 .+-. 64 718 .+-. 58
nd.sup.1 control 540 .+-. 92 876 .+-. 252 695 .+-. 151 625 .+-. 141
870 .+-. 172 1104 .+-. 184 872 .+-. 215 880 .+-. 131 *Peripheral
blood total CD3.sup.+CD8.sup.+ T-lymphocyte numbers as determined
by complete blood count analysis are reported as the mean total
CD3.sup.+CD8.sup.+ T-lymphocyte number .+-. standard error.
.sup.1nd, not done
[0294] TABLE-US-00037 TABLE 30 Total CD20.sup.+ lymphocyte numbers
in macaques immunized with multi- plasmid DNA vaccines with and
without electroporation. Week Group ID -2 0 2 4 6 8 10 16 2d 1468
.+-. 309 1287 .+-. 347 1369 .+-. 403 1131 .+-. 328 1337 .+-. 391
1300 .+-. 331 993 .+-. 301 918 .+-. 275 3a 1071 .+-. 296 857 .+-.
204 859 .+-. 218 767 .+-. 175 782 .+-. 195 799 .+-. 229 575 .+-.
115 746 .+-. 189 3c 1143 .+-. 269 994 .+-. 205 1155 .+-. 264 1089
.+-. 283 1083 .+-. 340 1322 .+-. 380 902 .+-. 295 1175 .+-. 356 3cE
1081 .+-. 140 968 .+-. 139 1221 .+-. 156 923 .+-. 125 1147 .+-. 201
1080 .+-. 173 1006 .+-. 138 966 .+-. 118 4a 1332 .+-. 186 1127 .+-.
162 1247 .+-. 113 1255 .+-. 148 1051 .+-. 104 938 .+-. 100 987 .+-.
91 nd.sup.1 control 984 .+-. 161 1134 .+-. 296 1171 .+-. 169 1027
.+-. 183 1206 .+-. 221 1223 .+-. 204 912 .+-. 144 945 .+-. 164
*Peripheral blood total CD20.sup.+ lymphocyte numbers as determined
by complete blood count analysis are reported as the mean total
CD20.sup.+ lymphocyte number .+-. standard error. .sup.1nd, not
done
[0295] Multi-plasmid immunization with the plasmids and immunogenic
compositions described in Table 7 also did not produce any adverse
effects on the platelet counts (Table 24), percent hematocrit
(Table 25), total lymphocyte numbers (Table 26), total CD3+
T-lymphocyte numbers (Table 27), total CD3+CD4+ Th-lymphocyte
numbers (Table 28), total CD3+CD8+ T-lymphocyte numbers (Table 29),
and total CD20+ T-lymphocyte numbers (Table 30), in animals used in
this study. Again, in these analyses a positive effect on total
lymphocyte numbers (Table 26), total CD3+ T-lymphocyte numbers
(Table 27), total CD3+CD4+ Th-lymphocyte numbers (Table 28), total
CD3+CD8+ T-lymphocyte numbers (Table 29), was detected when
electroporation was used in conjunction with the bupivacaine
formulated immunogenic composition to immunize group 3cE. In this
group, the number of lymphocytes in each of these categories was
significantly elevated at times during the course of the study.
[0296] The body weights of animals used in the study were monitored
on a weekly basis. Body weights (kg) were reported as the mean body
weight.+-.standard error. See Table 31. TABLE-US-00038 TABLE 31
Body weight (kg) of macaques immunized with multi-plasmid DNA
vaccines with and without electroporation. Week Group ID -2 0 2 4 6
8 10 16 2d 3.74 .+-. 0.27 3.63 .+-. 0.27 3.84 .+-. 0.29 3.93 .+-.
0.28 3.98 .+-. 0.29 4.16 .+-. 0.29 4.00 .+-. 0.28 4.05 .+-. 0.28 3a
3.63 .+-. 0.19 3.56 .+-. 0.19 3.74 .+-. 0.22 3.75 .+-. 0.22 3.83
.+-. 0.25 3.98 .+-. 0.23 3.85 .+-. 0.25 3.96 .+-. 0.25 3c 3.70 .+-.
0.23 3.65 .+-. 0.20 3.87 .+-. 0.24 3.97 .+-. 0.25 4.16 .+-. 0.25
4.26 .+-. 0.29 4.14 .+-. 0.26 4.28 .+-. 0.30 3cE 3.67 .+-. 0.23
3.91 .+-. 0.23 4.03 .+-. 0.28 3.99 .+-. 0.26 4.04 .+-. 0.28 4.12
.+-. 0.25 4.06 .+-. 0.27 4.14 .+-. 0.30 4a 3.67 .+-. 0.19 3.72 .+-.
0.21 3.83 .+-. 0.22 3.77 .+-. 0.19 3.85 .+-. 0.18 3.71 .+-. 0.18
3.72 .+-. 0.14 nd.sup.1 control 3.61 .+-. 0.23 3.66 .+-. 0.20 3.91
.+-. 0.18 4.03 .+-. 0.19 4.15 .+-. 0.18 4.24 .+-. 0.19 4.21 .+-.
0.20 4.29 .+-. 0.21 *Body weights (kg) are reported as the mean
body weight .+-. standard error. .sup.1nd, not done
[0297] Finally, this analysis indicates that multi-plasmid
immunization with the plasmids and immunogenic compositions
described in Table 7 also did not produce any adverse effects on
the body weights (Table 31) of animals used in this study.
Example 13
Murine Immunization Studies Using Immunogenic Compositions
Comprising Four Plasmids Each Having a Single Transcriptional
Unit
[0298] Previous examples suggested that in situations where the
total immune response must be maximized then it may be advantageous
to use an immunogenic composition based on a combination of
plasmids having a single transcriptional unit expressing a single
antigen per plasmid. In this example, murine immunization studies
were performed to compare immunogenic functionality of immunogenic
compositions based on four plasmids with immunogenic compositions
based on three plasmids. More particularly, the immunogenic
functionality of an immunogenic composition based on four
individual plasmids directing the expression of six HIV-1 genes
including gag, pol, env, and only one fusion of nef-tat-vif genes
was compared to immunogenic compositions based on three individual
plasmids directing the expression of six HIV-1 genes including env,
a fusion of gag-pol genes and a second fusion of nef-tat-vif genes.
Immunogenic functionality was evaluated as relative ability of
various three and four plasmid DNA-based immunogenic compositions
to elicit multi-antigen-specific cell-mediated immune responses in
Balb/c mice. The HIV genes and sequences were described in Example
1. The three plasmid immunogenic compositions from groups 3a and 3c
were the same as described in Examples 8 and 9. See Tables 1 and
32.
[0299] Immunogenic Compositions and Immunization
[0300] Plasmid DNA expression vectors encoding HIVenv gp160, gag
p55, pol (or a gag-pol fusion), or a nef-tat-vif fusion protein
were used as the experimental immunogenic compositions, and the
empty expression vector backbone was used as a control immunogenic
composition vector. See Table 32 below for study design. HIV gene
expression by the various expression vectors was confirmed by
Western blot after transient transfection of human rhabdosarcoma
(RD) cells. See Examples 4-7.
[0301] Group 3a has three plasmids with a single transcriptional
unit plasmid each, but where two of the antigens are fusion
proteins (gag-pol and nef-tat-vif). Group 3c also has three
plasmids but where two of the plasmids have a single
transcriptional unit and the third plasmid has two complete
transcriptional units. See Table 32. Only one of the antigens is
expressed as a fusion protein (nef-tat-vif). Group 4a has four
plasmids with a single transcriptional unit plasmid each, but where
only one of the antigens was a fusion protein (nef-tat-vif).
[0302] The adjuvant used for these studies was also delivered via a
DNA plasmid. In this example, all animals were co-injected with 25
.mu.g of plasmid no. 212 encoding murine IL-12 p35 and p40 genes
and expressing murine Il-12. See Table 1.
[0303] Balb/c mice were immunized intramuscularly with 100 total
.mu.g doses of DNA as outlined in Table 32. In all cases,
immunogenic compositions were formulated with 0.25% bupivacaine and
injected into the quadricep muscles in a 100 .mu.l volume. Ten days
after the second immunization, animals were sacrificed and the
serum and spleens were isolated for immune assays. Spleens were
used to measure antigen-specific IFN-gamma secreting cells using
ELISPOT assays as described below.
Animals
[0304] For these studies, 4-6 week old female Balb/c mice were
used. Mice were maintained in accordance with the Guide for the
Care and Use of Laboratory Animals (National Research Council,
National Academic Press, Washington, D.C., 1996). In addition,
procedures for the use and care of the mice were approved by Wyeth
Research's Institutional Animal Care and Use Committee.
TABLE-US-00039 TABLE 32 Murine Study Design - Two Immunizations
Total Immunization .sup.1Group Plasmid Plasmid DNA No. Schedule No.
No. description (ug) mice (week) 3a 111 HCMV-gag/pol 33 8 0-3 104
HCMV-ntv 33 101 HCMV-env 33 3c 102 HCMV-gag 33 8 0-3 103 HCMV-pol
33 202 HCMV-ntv, 33 SCMV-env 4a 101 HCMV-env 25 8 0-3 102 HCMV-gag
25 103 HCMV-pol 25 104 HCMV-ntv 25 5 001 Vector control 100 4 0-3
.sup.1Groups 3a and 3c utilize the same immunogenic compositions as
in Table 3.
[0305] The data shown in Table 33 indicates that increasing the
number of antigen expressing plasmids from 3 to 4 in the
immunogenic composition did not produce any dramatic increase in
immune response to HIV proteins. See Table 33. TABLE-US-00040 TABLE
33 Murine Immune Responses Following Two Immunizations gag- pol-
ntv#- Total HIV- Group specific specific env-specific specific
specific ID response* response response response response Control 3
0 9 1 13 3a 163 247 1564 116 2090 3c 436 1155 671 83 2345 4a 294
662 1150 123 2229 *antigen-specific IFN-gamma ELISPOT responses are
reported as the spot forming cells (#SFC/10.sup.6 splenocytes)
excreting interferon gamma per 10.sup.6 splenocytes. #ntv,
nef-tat-vif fusion protein
[0306] All documents cited herein are incorporated by reference.
Various modifications and minor alterations in the method and
components are believed to be clear to those of skill in the art.
Sequence CWU 1
1
6 1 42 PRT Homo sapiens 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val
Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly
Val Val Ile Ala 35 40 2 6 DNA Human immunodeficiency virus type 1 2
tttttt 6 3 6 DNA Human immunodeficiency virus type 1
misc_difference (1)..(1) mutation to allow read through
misc_difference (4)..(4) mutation to allow read through
misc_difference (6)..(6) mutation to allow read through 3 cttctg 6
4 4 PRT Human immunodeficiency virus type 1 4 Lys Gly Arg Pro 1 5
10 PRT Human immunodeficiency virus type 1 5 Asp Arg Gln Gly Thr
Val Ser Phe Asn Phe 1 5 10 6 4 PRT Human immunodeficiency virus
type 1 6 Pro Gln Ile Thr 1
* * * * *