U.S. patent number 5,593,972 [Application Number 08/125,012] was granted by the patent office on 1997-01-14 for genetic immunization.
This patent grant is currently assigned to The Trustees of the University of Pennsylvania, The Wistar Institute. Invention is credited to Bin Wang, David B. Weiner, William V. Williams.
United States Patent |
5,593,972 |
Weiner , et al. |
January 14, 1997 |
Genetic immunization
Abstract
Methods of prophylactic and therapeutic immunization of an
individual against pathogen infection, diseases associated with
hyperproliferative cells and autoimmune diseases are disclosed. The
methods comprise the steps of administering to cells of an
individual, a nucleic acid molecule that comprises a nucleotide
sequence that encodes a protein which comprises at least one
epitope that is identical or substantially similar to an epitope of
a pathogen antigen, a hyperproliferative cell associated protein or
a protein associated with autoimmune disease respectively. In each
case, nucleotide sequence is operably linked to regulatory
sequences to enable expression in the cells. The nucleic acid
molecule is free of viral particles and capable of being expressed
in said cells. The cells may be contacted cells with a cell
stimulating agent. Methods of prophylactically and therapeutically
immunizing an individual against HIV are disclosed. Pharmaceutical
compositions and kits for practicing methods of the present
invention are disclosed.
Inventors: |
Weiner; David B. (Merion,
PA), Williams; William V. (Havertown, PA), Wang; Bin
(Havertown, PA) |
Assignee: |
The Wistar Institute
(Philadelphia, PA)
The Trustees of the University of Pennsylvania
(Philadelphia, PA)
|
Family
ID: |
26678092 |
Appl.
No.: |
08/125,012 |
Filed: |
September 21, 1993 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
29336 |
Mar 11, 1993 |
|
|
|
|
08342 |
Jan 26, 1993 |
|
|
|
|
Current U.S.
Class: |
514/44R;
424/278.1; 514/818; 514/615 |
Current CPC
Class: |
A61K
39/00 (20130101); A61K 39/21 (20130101); C07K
14/005 (20130101); C12N 15/87 (20130101); A61K
39/12 (20130101); C12N 2740/16134 (20130101); C12N
2740/16222 (20130101); C12N 2740/16334 (20130101); Y10S
514/818 (20130101); C12N 2740/16322 (20130101); C12N
2740/16034 (20130101); C12N 2740/16234 (20130101); Y02A
50/464 (20180101); Y10S 435/975 (20130101); Y02A
50/30 (20180101); C12N 2740/16122 (20130101); Y02A
90/26 (20180101); Y02A 90/10 (20180101); A61K
2039/53 (20130101) |
Current International
Class: |
A61K
39/12 (20060101); A61K 39/00 (20060101); A61K
39/21 (20060101); C07K 14/005 (20060101); C07K
14/16 (20060101); C12N 15/87 (20060101); A61K
045/05 (); A61K 048/00 (); A61K 031/00 () |
Field of
Search: |
;435/320.1
;424/93.1,93.2,93.21,278.1 ;514/44,615,818 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO90/11092 |
|
Oct 1990 |
|
WO |
|
WO91/12329 |
|
Aug 1991 |
|
WO |
|
WO93/17706 |
|
Sep 1993 |
|
WO |
|
WO93/23552 |
|
Nov 1993 |
|
WO |
|
Other References
F D. Ledley (1991) Human Gene Therapy 2:77-83. .
B. F. Haynes (1993) Science 260: 1279-1286. .
A. Hoffenbach et al. (1989) The Journal of Immunology 142: 452-462.
.
D. Torpey et al. (1993) Clinical Immunology and Immunopathology
68(3): 263-272. .
L. Butini et al. (1994) J. Cell. Biochem. Suppl. 18B: 147, Abstract
J306. .
A. Knuth et al (1991) Current Opinion in Immunology 3:659-664.
.
B. Wang et al (1993) Proc. Natl. Acad. Sci. USA 90:4156-4160. .
D. J. Wells (1993) Febs Letters 332 (1,2):179-182. .
R. F. Garry et al. (1990) Science 250: 1127-1129. .
A. M. Schultz et al., AIDS 7(suppl. 1):S161-S170 (1993). .
V. Glaser, Genetic Engineering News 16(1):6 (1996). .
H. N. Eisen, "Introduction to Immune Responses," in Microbiology,
Bernard D. Davis et al., eds. Hagerstown: Harper & Row,
Publishers, 1980, p. 294. .
Aldovini et al., "Mutations of RNA and Protein Sequences Involved
in Human Immunodeficiency Virus Type 1 Packaging Result in
Production of Noninfectious Virus," J. of Virology, 64: 1920-1926,
1990. .
Benvenisty et al., "Direct introduction of genes into rats and
expression of the genes," Proc. Natl. Acad. Sci. USA, 83:9551-9555,
1986. .
Brandsma et al., "Use of a rapid, efficient inoculation method to
induce papillomas by conttontail rabbit papillomavirus DNA shows
that the E7 gene is required," Proc. Natl. Acad. Sci. USA,
88:4816-4820, 1991. .
Desrosiers, "HIV with Multiple Gene Deletions as a Live Attenuated
Vaccine for AIDS," AIDS Research and Human Retroviruses, 8:411-421,
1992. .
Friedmann et al., "Progress Toward Human Gene Therapy," Science,
244:1275-1281, 1989. .
Kaneda et al., "Increased Expression of DNA Cointroduced with
Nuclear Protein in Adult Rat Liver," Science, 243:375-378, 1989.
.
Nicolau et al., "In vivo expression of rat insulin after
intravenous administration of the liposome-entrapped gene for rat
insulin I," Proc. Natl. Acad. Sci. USA, 80:1068-1072, 1983. .
Ronen et al., "Expession of wild-type and mutant p53 proteins by
recombinant vaccinia viruses," Nucleic Acids Research,
20:3435-3441, 1992. .
Schauer et al., "The N-Terminal Region of HIV-1 Integrase is
Required for Integration Activity, but not for DNA-Binding,"
Biochem. and Biophys. Res. Commun., 185:874-880, 1992. .
Seeger et al., "The cloned genome of ground squirrel hepatitis
virus is infectious in the animal," Proc. Natl. Acad. Sci. USA,
81:5849-5852, 1984. .
Wu et al., "Receptor-mediated Gene Delivery and expression in
Vivo," J. of Biological Chemistry, 263:14621-14624, 1988. .
Yang et al., "In vivo and in vitro gene transfer to mammalian
somatic cells by particle bombardment," Proc. Natl. Acad. Sci. USA,
87:9568-9572, 1990. .
Zelenin et al., "High-velocity mechanical DNA transfer of the
chloramphenicolacetyl transferase gene into rodent liver, kidney
and mammary gland cells in organ explants and in vivo," FEBS
Letts., 280:94-96, 1991. .
Acsadi et al., "Human dystrophin expression in mdx mice after
intramuscular injection of DNA constructs," Nature 352:815-818,
1991. .
Anderson, W. French, "Prospects for Human Gene Therapy," Science
226:401-409, 1984. .
Anilionis et al., "Structure of the glycoprotein gene in rabies
virus," Nature 294:275278, 1981. .
Benoit et al., "Destruction and regeneration of skeletal muscle
after treatment with a local anaesthetic, bupivacaine
(Marcaine.RTM.)," J. Anat. 107:547-556, 1970. .
Berman et al., "Protection of chimpanzees from infection by HIV-1
after vaccination with recombinant glycoprotein gp120 but not
gp160," Nature, 345:622-625, 1990. .
Brigham et al., "Rapid Communication: In Vivo Transfection of
Murine Lungs with a Functioning Prokaryotic Gene Using A Liposome
Vehicle," American Journal of the Medical Sciences, 298:278-281,
1989. .
Chaudhary et al., "A rapid method of cloning functional
variable-region antibody genes in Escherichia coli as single-chain
immunotoxins," Proc. Natl. Acad. Sci. USA, 87:1066-1070, 1990.
.
Chen et al., "HIV-1 gp41 contains two sites for interaction with
several proteins on the helper T-lymphoid cell line, H9," AIDS,
6:533-539, 1992. .
Cheng-Mayer et al., "Human Immunodeficiency virus can productively
infect cultured human glial cells," Proc. Natl. Acad. Sci. USA,
84:3526-3530, 1987. .
Crowe et al., "Improved cloning efficiency of polymerase chain
reaction (PCR) products after proteinase K digestion", Nucleic
Acids Research, 19:184, 1991. .
Desquenne-Clark et al., "T-cell receptor peptide immunization leads
to enhanced and chronic experimental allergic encephalomyelitis,"
Proc. Natl. Acad. Sci. USA, 88:7219-7223, 1991. .
Di Fiore et al., "erbB-2 Is a Potent Oncogene When Overexpressed in
NIH/3T3 Cells," Science, 237:178-182, 1987. .
Dubensky et al., "Direct transfection of viral and plasmid DNA into
the liver of spleen of mice," Proc. Natl. Acad. Sci. USA,
81:7529-7533, 1984. .
Felgner et al., "Gene Therapeutics," Nature, 349:351-352, 1991.
.
Fisher et al., "A molecular clone of HTLV-III with biological
activity," Nature, 316:262-265, 1985. .
Fisher et al., "HIV infection is blocked in vitro by recombinant
soluble CD4," Nature, 331:76-78, 1988. .
Goudsmit et al., "Human antibody response to a strain-specific
HIV-1 gp120 epitope associated with cell fusion inhibition," AIDS,
2:157-164, 1988. .
Hahn et al., "Suppression of Murine Lupus Nephritis By
Administration of an Anti-Idiotypic Antibody to Anti-DNA," J. of
Immunology, 132:187-190, 1984. .
Hall-Craggs, E. C. B., "Rapid Degeneration and Regeneration of a
Whole Skeletal Muscle Following Treatment with Bupivacain
(Marcain)," Experimental Neurology, 43:349-358, 1974. .
Howley, Peter M., "Papillomavirinae and Their Replication,"
Virology, Chapter 58:1625-1650, 1990. .
Howell et al., "Limited T-cell receptor Beta-chain heterogeneity
among interleukin 2 receptor-positive synovial T cells suggests a
role for superantigen in rheumatoid arthritis," Proc. Natl. Acad.
Sci. USA, 88:10921-10925, 1991. .
Israel et al., "Biological Activity of Polyoma Viral DNA in Mice
and Hamsters," J. of Virology, 29:990-996, 1979. .
Klein et al., "Transformation of Microbes, Plants and Animals by
Particle Bombardment," Bio/Technology, 10:286-291, 1992. .
Koenig et al., "Detection of AIDS Virus in Macrophages in Brian
Tissue from AIDS Patients with Encephalopathy," Science,
233:1089-1093, 1986. .
Kowalski et al., "Functional Regions of the Envelope Glycoprotein
of Human Immunodeficiency Virus Type 1," Science, 237:1351-1355,
1987. .
Langlois et al., "The Ability of Certain HIV Vaccines to Provoke
Reactions Against Normal Cells," Science, 255:292-293, 1992. .
Lasky et al., "Neutralization of the AIDS Retrovirus by Antibodies
to a Recombinant Envelope Glycoprotein," Science, 233:209-212,
1986. .
Lasky et al., "Delineation of a Region of the Human
Immunodeficiency Virus Type 1 gp120 Glycoprotein Critical for
Interaction with the CD4 Receptor," Cell, 50:975-985, 1987. .
Letvin et al., "Risks of Handling HIV," Nature, 349:573, 1991.
.
Maddon et al., "The T4 Gene Encodes the AIDS Virus Receptor and Is
Expressed in the Immune System and the Brain," Cell, 47:333-348,
1986. .
Montefiori et al., "Evaluation of Antiviral Drugs and Neutralizing
Antibodies to Human Immunodeficiency Virus by a Rapid and Sensitive
Microtiter Infection Assay," J. of Clinical Microbiology,
26:231-235, 1988. .
Morgenstern et al., "Advanced mammalian gene transfer: high titre
retroviral vectors with multiple drug selection markers and a
complementary helper-free packaging cell line," Nucl. Acids Res.,
18:3587-3596, 1990. .
Nabel et al., "Site-Specific Gene Expression in Vivo by Direct Gene
Transfer into the Arterial Wall," Science, 249:1285-1288, 1990.
.
Nara, Peter L., "Quantitative Infectivity Syncytium-Forming
Microassay," Basic Virologic Techniques, 77-86. .
Oksenberg et al., "Limited heterogeneity of rearranged T-cell
receptor V alpha transcripts in brains of multiple sclerosis
patients," Nature, 345:344-348, 1990. .
Osther et al., "Protective Humoral Immune Responses to the Human
Immunodeficiency Virus Induced in Immunized Pigs: A Possible Source
of Therapeutic Immunoglobulin Preparations," Hybridoma, 10:673-683,
1991. .
Osther et al., "The Quick Western Blot, A Novel Transportable
50-Minute HIV-1 Antibody Test," Transplantation, 47:834-838, 1989.
.
Paliard et al., "Evidence for the Effects of a Superantigen in
Rheumatoid Arthritis," Science, 253:325-329, 1991. .
Putney et al., "Development of an HIV Subunit Vaccine," V
International Conference on AIDS, Quebec, Canada; Jun. 4-9, 1989.
.
Schrier et al., "B-and T-Lymphocyte Responses to an Immunodominant
Epitope of Human Immunodeficiency Virus," J. of Virology,
62:2531-2536, 1988. .
Sun et al., "Generation and Characterization of Monoclonal
Antibodies to the Putative CD4-Binding Domain of Human
Immunodeficiency Virus Type 1 gp120," J. of Virology, 63:3579-3585,
1989. .
Shah et al., "Papillomaviruses," Virology, Chapter 59:1651-1676,
1990. .
Seed et al., "Molecular cloning of the CD2 antigen, the T-cell
erythrocyte receptor, by a rapid immunoselection procedure," Proc.
Natl. Acad. Sci. USA, 84:3365-3369, 1987. .
Szala et al., "Molecular cloning of cDNA for the
carcinoma-associated antigen GA733-2," Proc. Natl. Acad. Sci. USA,
87:3542-3546, 1990. .
Tang et al., "Genetic immunization is a simple method for eliciting
an immune response", Nature 356:152-154, 1992. .
Teitelbaum et al., "In Vivo Effects of Antibodies Against a High
Frequency Idiotype of Anti-DNA Antibodies in MRL Mice,"
132:1282-1285, 1984. .
Thomason et al., "Stable incorporation of a bacterial gene into
adult rat skeletal muscle in vivo," Cell Physiol., 27:C578-581,
1990. .
Ugen et al., (1992) Generation of Monoclonal Antibodies Against the
Amino Region of gp120 Which Elicits Antibody Dependent Cellular
Cytotoxicity, Cold Spring Harbor Laboratory, 1992. .
Ulmer et al., "Heterologous Protection Against Influenza by
Injection of DNA Encoding a Viral Protein," Science, 259:1745-1749,
1993. .
Vandenbark et al., "Immunization with a synthetic T-cell receptor
V-region peptide protects against experimental autoimmune
encephalomyelitis," Nature, 341:541-544, 1989. .
Vitadello et al., "Gene Transfer in Regenerating Muscle," J. of
Cellular Biochemistry, Suppl. 17E:252, Mar. 29-Apr. 25, 1993. .
Weiner et al., "Non-CD4 Molecules on Human Cells Important in HIV-1
Cell Entry," Vaccines, 115-120, 1989. .
Will et al., "Cloned HBV DNA causes hepatitis in chimpanzees,"
Nature, 299:740-742, 1982. .
Williams et al., "Molecular Diagnosis of Borrelia burgdorferi
Infection (Lyme Disease)," DNA and Cell Biology, 11:207-213, 1992.
.
Williams et al., "Restricted Heterogeneity of T Cell Receptor
Transcripts in Rheumatoid Synovium," J. Clin. Invest., 90:326-333,
1992. .
Wolff et al., "Direct Gene Transfer into Mouse Muscle in Vivo,"
Science, 247:1465-1468, 1990. .
Wolff et al., "Conditions Affecting Direct Gene Transfer into
Rodent Muscle In Vivo," BioTechniques, 11:474-485, 1991. .
Wucherpfennig et al., "Shared Human T Cell Receptor V Beta Usage to
Immunodominant Regions of Myelin Basic Protein," Science,
248:1016-1019, 1990. .
Fleckenstein et al., "Tumour induction with DNA of oncogenic
primate herpesviruses," Nature, 274:57-59, 1978. .
Boiron et al., "A Biological Property of Deoxyribonucleic Acid,"
Discussion and Preliminary Reports, 150-153, 1965. .
McCoy et al., "Human Colon Carcinoma Ki-ras2 Oncogene and Its
Correspondence Proto-Oncogene," Mol. and Cellular Biology,
4:1577-1582, 1984. .
Rowe, et al., "Studies of Mouse Polyoma Virus Infection," U.S.
Department of Health, Education and Welfare, 379-391, 1958. .
Orth et al., "Infectious and Oncogenic Effect of DNA Extracted from
Cells Infected with Polyoma Virus," P.S.E.B.M., 115:1090-1095,
1964. .
McCutchan et al., "Enhancement of the Infectivity of Simian Virus
40 Deoxyribonucleic Acid with Diethylaminoethyl-Dextran," J. of the
Nat. Cancer Institute, 41:351-356, 1968. .
Mayne et al., "Tumour Induction by Simian Adenovirus SA7 DNA
Fragments," Nature New Biology, 232:182-183, 1971. .
Sol et al., "Oncogenicity of SV40 DNA in the Syrian Hamster," J.
gen. Vir. 37:635-638, 1977. .
Rowe et al., "B. Ecology of a Mouse Tumor Virus," Perspectives in
Virology, 177-190..
|
Primary Examiner: Fleisher; Mindy
Assistant Examiner: Railey, II; Johnny F.
Attorney, Agent or Firm: Woodcock Washburn Kurtz Mackiewicz
& Norris
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-In-Part application of U.S.
patent application Ser. No. 08/029,336 filed Mar. 11, 1993, now
abandoned; which is a Continuation-In-Part application of U.S.
patent application Ser. No. 08/008,342 filed Jan. 26, 1993,
abandoned; both of which are incorporated herein by reference.
Claims
We claim:
1. A method of immunizing an individual comprising:
injecting into skeletal muscle tissue of said individual at a site
on said individual's body, bupivacaine and a DNA molecule that
comprises a DNA sequence that encodes an antigen from a pathogen,
said DNA sequence operatively linked to regulatory sequences which
control the expression of said DNA sequence;
wherein said DNA molecule is taken up by cells in said skeletal
muscle tissue, said DNA sequence is expressed in said cells and an
immune response is generated against said antigen.
2. The method of claim 1 wherein said pathogen is an intracellular
pathogen.
3. The method of claim 1 wherein said pathogen is a virus selected
from the group consisting of: human immunodeficiency virus, HIV;
human T cell leukemia virus, HTLV; influenza virus; hepatitis A
virus; hepatitis B virus; hepatitis C virus; human papilloma virus,
HPV; Herpes simplex 1 virus, HSV1; Herpes simplex 2 virus, HSV2;
Cytomegalovirus, CMV; Epstein-Barr virus, EBR; rhinovirus; and,
coronavirus.
4. The method of claim 1 wherein said pathogen is HIV and said DNA
molecule comprises a DNA sequence that encodes an HIV antigen.
5. The method of claim 1 wherein at least two non-identical DNA
molecules are injected into skeletal muscle tissue of said
individual at different sites on said individual's body, said
bupivacaine being injected into each of the different sites of an
individual; said non-identical DNA molecules each comprising DNA
sequences encoding one or more pathogen antigens of the same
pathogen.
6. A method of immunizing an individual comprising:
injecting into skeletal muscle tissue of said individual at a site
on said individual's body, bupivacaine and a DNA molecule that
comprises a DNA sequence that encodes a hyperproliferative
disease-associated protein operatively linked to regulatory
sequences;
wherein said DNA molecule is taken up by cells in said skeletal
muscle tissue, said DNA sequence is expressed in said cells, and an
immune response is generated against said hyperproliferative
disease-associated protein.
7. The method of claim 6 wherein said DNA molecule comprises a DNA
sequence encoding a target protein selected from the group
consisting of: protein products of oncogenes myb, myc, fyn, ras,
src, neu and trk; protein products of translocation gene bcr/abl;
P53; variable regions of antibodies made by B cell lymphomas; and
variable regions of T cell receptors of T cell lymphomas.
8. A method of immunizing an individual comprising:
injecting into skeletal muscle tissue of said individual,
bupivacaine and a DNA molecule that comprises a DNA sequence that
encodes an autoimmune disease-associated protein operatively linked
to regulatory sequences;
wherein said DNA molecule is taken up by cells in said skeletal
muscle tissue, said DNA sequence is expressed in said cells and an
immune response is generated against said autoimmune
disease-associated protein.
9. The method of claim 8 wherein said DNA molecule comprises a DNA
sequence encoding a target protein selected from the group
consisting of: variable regions of antibodies involved in B cell
mediated autoimmune disease; and variable regions of T cell
receptors involved in T cell mediated autoimmune disease.
Description
FIELD OF THE INVENTION
The present invention relates to use of genetic material as
immunizing agents. In particular, the present invention relates to
the introduction of DNA molecules into an individual's tissues or
cells that then can produce proteins capable of eliciting an immune
response.
BACKGROUND OF THE INVENTION
Vaccination and immunization generally refer to the introduction of
a non-virulent agent against which an individual's immune system
can initiate an immune response which will then be available to
defend against challenge by a pathogen. The immune system
identifies invading "foreign" compositions and agents primarily by
identifying proteins and other large molecules which are not
normally present in the individual. The foreign protein represents
a target against which the immune response is made.
The immune system can provide multiple means for eliminating
targets that are identified as foreign. These means include humoral
and cellular responses which participate in antigen recognition and
elimination. Briefly, the humoral response involves B cells which
produce antibodies that specifically bind to antigens. There are
two arms of the cellular immune response. The first involves helper
T cells which produce cytokines and elicit participation of
additional immune cells in the immune response. The second involves
killer T cells, also known as cytotoxic T lymphocytes (CTLs), which
are cells capable of recognizing antigens and attacking the antigen
including the cell or particle it is attached to.
Vaccination has been singularly responsible for conferring immune
protection against several human pathogens. In the search for safe
and effective vaccines for immunizing individuals against infective
pathogenic agents such as viruses, bacteria, and infective
eukaryotic organisms, several strategies have been employed thus
far. Each strategy aims to achieve the goal of protecting the
individual against pathogen infection by administering to the
individual, a target protein associated with the pathogen which can
elicit an immune response. Thus, when the individual is challenged
by an infective pathogen, the individual's immune system can
recognize the protein and mount an effective defense against
infection. There are several vaccine strategies for presenting
pathogen proteins which include presenting the protein as part of a
non-infective or less infective agent or as a discreet protein
composition.
One strategy for immunizing against infection uses killed or
inactivated vaccines to present pathogen proteins to an
individual's immune system. In such vaccines, the pathogen is
either killed or otherwise inactivated using means such as, for
example, heat or chemicals. The administration of killed or
inactivated pathogen into an individual presents the pathogen to
the individual's immune system in a noninfective form and the
individual can thereby mount an immune response against it. Killed
or inactivated pathogen vaccines provide protection by directly
generating T-helper and humoral immune responses against the
pathogenic immunogens. Because the pathogen is killed or otherwise
inactivated, there is little threat of infection.
Another method of vaccinating against pathogens is to provide an
attenuated vaccine. Attenuated vaccines are essentially live
vaccines which exhibit a reduced infectivity. Attenuated vaccines
are often produced by passaging several generations of the pathogen
through a permissive host until the progeny agents are no longer
virulent. By using an attenuated vaccine, an agent that displays
limited infectivity may be employed to elicit an immune response
against the pathogen. By maintaining a certain level of
infectivity, the attenuated vaccine produces a low level infection
and elicits a stronger immune response than killed or inactivated
vaccines. For example, live attenuated vaccines, such as the
poliovirus and smallpox vaccines, stimulate protective T-helper,
T-cytotoxic, and humoral immunities during their nonpathogenic
infection of the host.
Another means of immunizing against pathogens is provided by
recombinant vaccines. There are two types of recombinant vaccines:
one is a pathogen in which specific genes are deleted in order to
render the resulting agent non-virulent. Essentially, this type of
recombinant vaccine is attenuated by design and requires the
administration of an active, non-virulent infective agent which,
upon establishing itself in a host, produces or causes to be
produced antigens used to elicit the immune response. The second
type of recombinant vaccine employs infective non-virulent vectors
into which genetic material that encode target antigens is
inserted. This type of recombinant vaccine similarly requires the
administration of an active infective non-virulent agent which,
upon establishing itself in a host, produces or causes to be
produced, the antigen used to elicit the immune response. Such
vaccines essentially employ infective non-virulent agents to
present pathogen antigens that can then serve as targets for an
anti-pathogen immune response. For example, the development of
vaccinia as an expression system for vaccination has theoretically
simplified the safety and development of infectious vaccination
strategies with broader T-cell immune responses.
Another method of immunizing against infection uses subunit
vaccines. Subunit vaccines generally consist of one or more
isolated proteins derived from the pathogen. These proteins act as
target antigens against which an immune response may be mounted by
an individual. The proteins selected for subunit vaccine are
displayed by the pathogen so that upon infection of an individual
by the pathogen, the individuals immune system recognizes the
pathogen and mounts a defense against it. Because subunit vaccines
are not whole infective agents, they are incapable of becoming
infective. Thus, they present no risk of undesirable virulent
infectivity that is associated with other types of vaccines. It has
been reported that recombinant subunit vaccines such as the
hepatitis B surface antigen vaccine (HBsAg) stimulate a more
specific protective T-helper and humoral immune response against a
single antigen. However, the use of this technology to stimulate
board protection against diverse pathogens remains to be
confirmed.
Each of these types of vaccines carry severe drawbacks which render
them less than optimally desirable for immunizing individuals
against a particular pathogen.
It has been observed that absent an active infection, a complete
immune response is not elicited. Killed and inactivated vaccines,
because they do not reproduce or otherwise undergo an infective
cycle, do not elicit the CTL arm of the cellular immune response in
most cases. Additionally, killed and inactivated vaccines are
sometimes altered by the means used to render them inactivated.
These changes can affect the immunogenicity of the antigens.
Subunit vaccines, which are merely discreet components of a
pathogen, do not undergo any sort of infective cycle and often do
not elicit the CTL arm of the cellular immune response. Absent the
CTL arm, the immune response elicited by either vaccine is often
insufficient to adequately protect an individual. In addition,
subunit vaccines have the additional drawback of being both
expensive to produce and purify.
Attenuated vaccines, on the other hand, often make very effective
vaccines because they are capable of a limited, non-virulent
infection and result in immune responses involving a humoral
response and both arms of the cellular immune response. However,
there are several problems associated with attenuated vaccines.
First, it is difficult to test attenuated vaccines to determine
when they are no longer pathogenic. The risk of the vaccine being
virulent is often too great to properly test for effective
attenuation. For example, it is not practically possible to test an
attenuated form of Human Immunodeficiency virus (HIV) to determine
if it is sufficiently attenuated to be a safe vaccine. Secondly,
attenuated vaccines carry the risk of reverting into a virulent
form of the pathogen. There is a risk of infecting individuals with
a virulent form of the pathogen when using an attenuated
vaccine.
Recombinant vaccines require the introduction of an active
infective agent which, in many cases, is undesirable. Furthermore,
in cases where the recombinant vaccine is the result of deletion of
genes essential for virulence, such genes must exist and be
identified. In vaccines in which pathogen genes are inserted into
infective non-virulent vectors, many problems exist related to the
immune response elicited against the vector antigens. These
problems negatively impact the immune response elicited against the
target antigen. First, the recombinant vaccine introduces a great
number of vector antigens against which the immune system also
responds. Secondly, the vector can be used only once per individual
since, after the first exposure, the individual will develop
immunity to the vector. These problems are both present, for
example, in recombinant vaccines that employ vaccinia vectors such
as those disclosed in U.S. Pat. No. 5,017,487 issued May 21, 1991
to Stunnenberg et al. This technology has not been universally
successful against diverse pathogenic organisms. It is also
complicated by the large amount of excess vaccinia antigens
presented in the vaccinee. Once vaccinated with the vaccinia
vector, the vaccinee cannot be effectively vaccinated again using
the vaccinia vector.
Accordingly, the most effective vaccines for invoking a strong and
complete immune response carry the most risk of harming the
individual while the safer alternatives induce an incomplete, and
therefore, less effective immune response. Furthermore, many
subunit vaccines and recombinant vaccines using non-virulent
vectors to produce target proteins are most useful if a single
antigenic component can be identified which is singularly
protective against live challenge by a pathogen. However, both
technologies require that the protective component be identified.
Such identification is often both laborious and time-consuming.
A distinct advantage would exist if there were a rapid system for
directly testing subunit vaccination strategies without tissue
culture and in the absence of excess vector antigens. Furthermore,
it would be particularly advantageous if such a system could
deliver an antigen that could be presented for development of both
T cell immune arms.
There is a need for a means to immunize individuals against
pathogen infection which can elicit a broad, biologically active
protective immune response without risk of infecting the
individual.
HIV infection represents a great threat to the human population
today. Despite the intense resources expended and efforts made to
develop an effective vaccine, the problem remains intractable. No
vaccine is currently available that protects an individual against
HIV infection. There is a great need for a method of immunizing an
individual against HIV infection. There is a great need for an
effective immunotherapy method to combat the development of AIDS in
HIV infected individuals.
In addition to immunizing against pathogens, work has recently been
undertaken to develop vaccines against cancer. Cancer vaccines
currently being studied are essentially analogous to anti-pathogen
subunit vaccines. Anti-cancer subunit vaccines essentially
introduces a cancer-associated target protein into an individual.
An immune response is elicited against the target protein in the
same manner an immune response is elicited against a pathogen
protein in the individual. The target protein is a protein that is
specific to cancer cells. Subsequent appearance of the target
protein when cancer occurs provides an immunogenic target for an
immune response. Thus, the cancer vaccine immunizes an individual
against cancer cells, an "endogenous pathogen", by immunizing
against a target antigen specifically associated with the cancer.
Specific proteins are administered which represent targets for an
immunological response. As in the case of anti-pathogen subunit
vaccines, the immune response elicited is often incomplete and
insufficient to protect the individual. In particular,
administration of a protein or peptide does not elicit a CTL
response.
There is a need for an effective means to immunize individuals
against hyperproliferative disease such as cancer in order to
provide individuals with broad, biologically active protective
immunity against specifically targeted hyperproliferating
cells.
Many autoimmune diseases are mediated by specific antigen
receptors. Autoimmune diseases generally refer to those diseases
involving a self-directed immune response. Autoimmune diseases are
referred to as being B cell mediated or T cell mediated. For
example, Systemic Lupus Erythematosus (SLE) is considered a B cell
mediated autoimmune disease. Many of the clinical manifestations of
SLE are believed to be due to the presence of anti-DNA antibodies
in the patients' serum, which combine with the antigen to form
immune complexes. These immune complexes are deposited in tissues,
setting off the inflammatory cascade. Rheumatoid Arthritis (RA) is
an example of T cell mediated autoimmune disease. RA is believed to
be mediated by autoreactive T cells present in the synovium (joint
tissue), where they respond to an unknown antigen in the context of
class II major histocompatibility complex (MHC II) molecules, such
as HLA-DR4 which is genetically linked to RA. These T cells
recognize a specific antigen associated with MHC II via their T
cell antigen receptors (TCRs). Thus, autoreactive antigen
receptors, such as antibodies or T cell antigen receptors are
responsible for the initial recognition event in a series of
pathogenic, inflammatory events which culminates in the clinical
manifestations of autoimmune diseases such as SLE and RA.
Several studies have been performed in experimental systems where
such autoreactive antigen receptors have been targeted or deleted.
Animal model systems for autoimmune disease include a murine lupus
model which occurs in a strain of NZB/NZW mice, and an experimental
allergic encephalomyelitis (EAE) model which can be produced in
susceptible mouse and rat strains following inoculation with myelin
basic protein (MBP). In murine SLE, anti-idiotypic antibodies have
been used therapeutically in an attempt to delete the autoreactive
B cells which produce the autoreactive antibodies. In some cases,
these anti-idiotypic antibodies have improved clinical
manifestations of the disease (Hahn, B. H. and F. M. Ebling, 1984
J. Immunol. 132(1):187-190), while in others they have worsened
disease (Teitelbaum, D. et al., 1984 J. Immunol. 132(3):1282-1285).
Similarly, in EAE, antibodies to autoreactive T cell antigen
receptors have been utilized, as has been immunization with T cell
antigen receptor-derived peptides. Again, in some instances this
improves the disease Vandenbark, A., et al., 1989 Nature
341:541-544, while in other worsening of the disease occurs
(Desquenne-Clark, L., et al., 1990 Proc. Natl. Acad. Sci. USA
88:7219-7223).
Thus, while it is possible to vaccinate against autoimmune disease
in some cases, the nature of the immune response elicited affects
the clinical outcome of such therapies. For example, if the
vaccination results in development of an antibody response, with
subsequent anti-idiotype development, these anti-idiotypic
antibodies could target the autoreactive B cells or T cells for
complement-mediated lysis, with resulting clinical improvement.
Alternatively, if the immunization results in production of
non-complement fixing anti-idiotypic antibodies, these would bind
to the autoreactive B cells or T cells and cross-link their antigen
receptors. Typically, this leads to activation of the cells and
subsequent increased production of the autoreactive antibodies or T
cells, with worsening of the clinical condition. Alternatively, if
a predominant T cell response is elicited by vaccination, this
could result in either a helper T cell response which would be
expected to worsen disease or a killer/suppressor cell response
which should improve the disease.
There is a need for an effective means to immunize individuals
against and treat individuals suffering from autoimmune diseases
which would elicit a CTL response capable of targeting either B
cells that produce the antibodies involved in the disease (in the
case of B cell mediated autoimmune disease) or the T cells that
produce the specific T cell antigen receptor which are involved int
he disease (in the case of T cell mediated autoimmune disease).
The direct introduction of a normal, functional gene into a living
animal has been studied as a means for replacing defective genetic
information. In such studies, DNA is introduced directly into cells
of a living animal. Nabel, E. G., et al., (1990) Science
249:1285-1288, disclose site-specific gene expression in vivo of a
beta-galactosidase gene that was transferred directly into the
arterial wall in mice. Wolfe, J. A. et al., (1990) Science
247:1465-1468, disclose expression of various reporter genes that
were directly transferred into mouse muscle in vivo. The use of
direct gene transfer as an alternative anti-pathogen vaccination
method is suggested. Acsadi G., et al., (1991) Nature 352:815-818,
disclose expression of human dystrophin gene in mice after
intramuscular injection of DNA constructs. Wolfe, J. A., et al.,
1991 BioTechniques 11(4):474-485, which is incorporated herein by
reference, refers to conditions affecting direct gene transfer into
rodent muscle in vivo. Multiple injections of plasmid DNA are
reported to result in higher levels of protein production but not
to the extent that the levels of protein production are
proportional to additional plasmid DNA added. Felgner, P. L. and G.
Rhodes, (1991) Nature 349:351-352, disclose direct delivery of
purified genes in vivo as drugs without the use of retroviruses.
Use of direct gene transfer by single injection are suggested as a
possible vaccination strategy and a cellular immune response to HIV
gp120 resulting from introduction of plasmid DNA encoding the same
into cells is reported to have been observed. PCT International
Application Number PCT/US90/01515 published Oct. 4, 1990 discloses
methods of immunizing an individual against pathogen infection by
directly injecting naked polynucleotides into the individual's
cells in a single step procedure. The use of transfecting agents
other than lipofectins is specifically excluded from the disclosed
methods. The stimulation of inoculated cells is neither disclosed
nor suggested. An HIV vaccine is disclosed which consists of the
introduction of polynucleotides that encode the viral protein
gp120. The operability of this vaccine is not evidenced. Thomason,
D. B. et al., (1990) Cell Physiol. 27:C578-581 and PCT patent
application Ser. No. WO 91/12329 disclose administering bupivacaine
to muscle cells in order to induce satellite cell proliferation. In
particular, Thomason, D. B. et al., (1990) Cell Physiol.
27:C578-581 and PCT patent application Ser. No. WO 91/12329
disclose retroviral-mediated transfer of genes into adult tissue in
which a mitotically-active state of satellite cells is induced. The
retroviruses contain recombinant retroviral RNA that includes a
foreign reporter gene incorporated within the viral particle.
SUMMARY OF THE INVENTION
The present invention relates to a method of immunizing an
individual against a pathogen. The method comprises the steps of
contacting cells of said individual with an agent that facilitates
the uptake of DNA by the cells, the agent preferably being a cell
stimulating agent, and administering to the cells, a DNA molecule
that comprises a DNA sequence that encodes a peptide which
comprises at least an epitope identical or substantially similar to
an epitope displayed on a pathogen antigen operatively linked to
regulatory sequences. The DNA molecule is capable of being
expressed in the cells of the individual.
The present invention relates to a method of immunizing a human
against HIV. The method comprises the steps of administering to a
human a DNA molecule that comprises a DNA sequence that encodes at
least one peptide that comprises at least one epitope identical or
substantially similar to an epitope displayed on an HIV protein
operatively linked to regulatory sequences.
The present invention relates to a method of immunizing a human
against HIV. The method comprises the steps of administering two
different DNA molecules to different cells of the human. Each DNA
molecule comprises a DNA sequence that encodes at least one peptide
which comprises at least one epitope identical or substantially
similar to an epitope displayed on an HIV protein operatively
linked to regulatory sequences. The different DNA molecules are
each capable of being expressed in human cells. The different DNA
molecules comprise different DNA sequences that encode at least one
different peptide from the other.
The present invention related to a method of immunizing an
individual against a hyperproliferative disease. The method
comprises the steps of administering to cells of an individual, a
DNA molecule that comprises a DNA sequence that encodes a peptide
that comprises at least an epitope identical or substantially
similar to an epitope displayed on a hyperproliferative
disease-associated protein operatively linked to regulatory
sequences; the DNA molecule being capable of being expressed in the
cells.
The present invention relates to a method of immunizing an
individual against an autoimmune disease. The method comprises the
steps of administering to cells of an individual, a DNA molecule
that comprises a DNA sequence that encodes a peptide that comprises
at least an epitope identical or substantially similar to an
epitope displayed on an autoimmune disease-associated protein
operatively linked to regulatory sequences; the DNA molecule being
capable of being expressed in the cells.
The present invention relates to an HIV vaccine comprising a
pharmaceutically acceptable carrier or diluent and a DNA molecule
that encodes one or more peptides that each comprises at least an
epitope identical or substantially similar to an epitope displayed
on at least one HIV protein operatively linked to regulatory
sequences; the DNA molecule being capable of being expressed in
human cells.
The present invention relates to an HIV vaccine comprising two
inoculants. The first inoculant comprises a pharmaceutically
acceptable carrier or diluent and a first DNA molecule. The first
DNA molecule comprises a DNA sequence that encodes one or more
peptides that each comprises at least an epitope identical or
substantially similar to an epitope displayed on at least one HIV
protein operatively linked to regulatory sequences; the DNA
molecule being capable of being expressed in human cells. The
second inoculant comprises a pharmaceutically acceptable carrier or
diluent and a second DNA molecule. The second DNA molecule
comprises a DNA sequence that encodes one or more peptides that
each comprises at least an epitope identical or substantially
similar to an epitope displayed on at least one HIV protein
operatively linked to regulatory sequences; the DNA molecule being
capable of being expressed in human cells. The first and second DNA
molecules are different and encode different peptides.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a diagram depicting the construction of plasmid pM160
which was produced by inserting a PCR-generated fragment that
encodes the HIV-HXB2 glycoprotein gp160 into plasmid pMAMneoBlue
(Clonetech).
FIG. 1B is a photograph of an autoradiogram of a Western blot of
whole cell lysates of cells transfected with the pM160 plasmid (3G7
cells) versus vector-alone transfected cells (TE671 cells) showing
production of gp120 and gp41 in 3G7 cells and not in TE671
cells.
FIG. 2 is a photograph of an autoradiogram showing
immunoprecipitations of serum antibodies binding to .sup.125
I-gp160.
FIGS. 3A-3E are graphs showing ELISA results binding different sera
to various proteins immobilized on microtiter plates.
FIGS. 4A and 4B are photographs of MT-2 cells infected with
TCID.sub.50 HIV-1/III.sub.B cell-free virus that was preincubated
with serial dilutions of antisera.
FIG. 4C is a graph illustrating the neutralization values (V.sub.n
/V.sub.o) versus the dilution factors from results using control
serum (x=pMAMneoBlue vector-immunized mice) and test sera
(O=pM160-immunized mice).
FIGS. 4D-4G are photographs of H9/III.sub.B cells used in
experiments to examine syncytial inhibition using sera from
immunized and control animals.
FIG. 5 is a chart depicting the survival of immunized and
non-immunized mice challenged with HIV gp160-labelled and
unlabelled tumor cells. Mice were immunized with recombinant gp160
protein, vector DNA only or recombinant vector comprising DNA
encoding gp160. SP2/0 tumor cells or SP2/0-gp160 (SP2/0 cells
transfected with DNA encoding gp160 and expressing gp160) tumor
cells were introduced into the mice.
FIG. 6 is a plasmid map of pGAGPOL.rev.
FIG. 7 is a plasmid map of pENV.
FIG. 8 is shows four backbones, A, B, C and D, used to prepare
genetic construct.
FIG. 9 shows four inserts, 1, 2, 3 and 4 which are inserted into
backbones to produce genetic constructs.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of eliciting immune
responses in an individual which can protect an individual from
pathogen infection or combat diseases and disorders involving cells
that produce specific proteins. According to the present invention,
genetic material that encodes an immunogenic peptide or protein is
directly administered to an individual either in vivo or to the
cells of an individual ex vivo. The genetic material encodes a
peptide or protein that shares at least an epitope with an
immunogenic protein to be targeted. The genetic material is
expressed by the individual's cells to form immunogenic target
proteins that elicit an immune response. The resulting immune
response is broad based: in addition to a humoral immune response,
both arms of the cellular immune response are elicited. Thus, the
immune responses elicited by vaccination methods of the present
invention are particularly effective to protect against pathogen
infection or combat cells associated with hyperproliferative
diseases or autoimmune diseases.
The immune response elicited by the target protein that is produced
by vaccinated cells in an individual is a broad-based immune
response which involves B cell and T cell responses including
cytotoxic T cell (CTL) responses. The target antigens produced
within the cells of the host are processed intracellularly: broken
down into small peptides, bound by Class I MHC molecules, and
expressed on the cell surface. The Class I MHC-target antigen
complexes are capable of stimulating CD8.sup.+ T-cells, which are
phenotypically the killer/suppressor cells. Genetic immunization
according to the present invention is thus capable of eliciting
cytotoxic T-cell (CTL) responses (killer cell responses). It has
been observed that genetic immunization according to the present
invention is more likely to elicit CTL responses than other methods
of immunization.
The present invention is useful to elicit broad immune responses
against a target protein. Target proteins may be proteins
specifically associated with pathogens or the individual's own
"abnormal" cells. The present invention is useful to immunize
individuals against pathogenic agents and organisms such that an
immune response against a pathogen protein provides protective
immunity against the pathogen. The present invention is
particularly useful to protect an individual against infection by
non-encapsulated intracellular pathogens which produce proteins
within the host cells. The immune response generated against such
proteins is capable of eliminating infected cells using CTLs which
specifically recognize and eliminate such infected cells. The CTL
response is crucial in protection against pathogens such as viruses
and other intracellular pathogens which produce proteins within
infected cells. The present invention is useful to combat
hyperproliferative diseases and disorders such as cancer by
eliciting an immune response against a target protein that is
specifically associated with the hyperproliferative cells. In such
cases, a cytotoxic immune response against the hyperproliferating
cells which produce the target protein is elicited. The present
invention is useful to combat autoimmune diseases and disorders by
eliciting an immune response against a target protein that is
specifically associated with cells involved in the autoimmune
condition. In such cases, the cytotoxic immune response against
cells that produce the target protein is provided. The CTL response
can be utilized for the specific elimination of deleterious cell
types which, during their production of proteins, display antigens
bound by Class I MHC molecules. Therefore, genetic immunization
according to the present invention is more likely to result in
anti-pathogen protection and therapy than standard immunization
using killed, inactivated or protein--or peptide-based subunit
vaccines and furthermore, may be used in immunization procedures to
protect against and treat individuals suffering from cancer and
autoimmune diseases.
Genetic immunization according to the present invention elicits an
effective immune response without the use of infective agents or
infective vectors. Vaccination techniques which usually do produce
a CTL response do so through the use of an infective agent. A
complete, broad based immune response is not generally exhibited in
individuals immunized with killed, inactivated or subunit vaccines.
The present invention achieves the full complement of immune
responses in a safe manner without the risks and problems
associated with vaccinations that use infectious agents.
According to some embodiments of the present invention, cells are
treated with compounds that facilitate uptake of genetic constructs
by the cells. According to some embodiments of the present
invention, cells are treated with compounds that stimulate cell
division and facilitate uptake of genetic constructs.
Administration of compounds that facilitate uptake of genetic
constructs by the cells including cell stimulating compounds
results in a more effective immune response against the target
protein encoded by the genetic construct.
According to some embodiments of the present invention, the genetic
construct is administered to an individual using a needleless
injection device. According to some embodiments of the present
invention, the genetic construct is simultaneously administered to
an individual intradermally, subcutaneously and intramuscularly
using a needleless injection device. Administration of genetic
constructs using needleless injection devices is disclosed in U.S.
patent application Ser. No. 08/093,235 filed Jul. 15, 1993, which
is incorporated herein by reference.
According to the present invention, DNA or RNA that encodes a
target protein is introduced into the cells of an individual where
it is expressed, thus producing the target protein. The DNA or RNA
is linked to regulatory elements necessary for expression in the
cells of the individual. Regulatory elements for DNA include a
promoter and a polyadenylation signal. In addition, other elements,
such as a Kozak region, may also be included in the genetic
construct.
As used herein, the term "genetic construct" refers to the DNA or
RNA molecule that comprises a nucleotide sequence which encodes the
target protein and which includes initiation and termination
signals operably linked to regulatory elements including a promoter
and polyadenylation signal capable of directing expression in the
cells of the vaccinated individual. As used herein, the term
"expressible form" refers to gene constructs which contain the
necessary regulatory elements operable linked to a coding sequence
that encodes a target protein, such that when present in the cell
of the individual, the coding sequence will be expressed. As used
herein, the term "genetic vaccine" refers to a pharmaceutical
preparation that comprises a genetic construct.
As used herein, the term "target protein" refers to a protein
against which an immune response can be elicited. The target
protein is an immunogenic protein which shares at least an epitope
with a protein from the pathogen or undesirable cell-type such as a
cancer cell or a cell involved in autoimmune disease against which
immunization is required. The target protein is an immunogenic
protein derived from the pathogen or undesirable cell-type such as
a cancer cell or a cell involved in autoimmune disease. Target
proteins share epitopes with either pathogen-associated proteins,
proteins associated with hyperproliferating cells, or proteins
associated with autoimmune disorders, depending upon the type of
genetic vaccine. The immune response directed against the target
protein will protect the individual against the specific infection
or disease with which the target protein is associated. For
example, a genetic vaccine with a DNA or RNA molecule that encodes
a pathogen-associated target protein is used to elicit an immune
response that will protect the individual from infection by the
pathogen. Likewise, a genetic vaccine with a DNA or RNA molecule
that encodes a target protein associated with a hyperproliferative
disease such as, for example, a tumor-associated protein, is used
to elicit an immune response directed at hyperproliferating cells.
A genetic vaccine with a DNA or RNA molecule that encodes a target
protein that is associated with T cell receptors or antibodies
involved in autoimmune diseases is used to elicit an immune
response that will combat the autoimmune disease by eliminating
cells in which the natural form of target protein is being
produced. Target proteins may be either pathogen-associated
proteins, proteins associated with hyperproliferating cells, or
proteins associated with auto-immune disorders, depending upon the
type of genetic vaccine.
As used herein, the term "sharing an epitope" refers to proteins
which comprise at least one epitope that is identical to or
substantially similar to an epitope of another protein.
As used herein, the term "substantially similar epitope" is meant
to refer to an epitope that has a structure which is not identical
to an epitope of a protein but nonetheless invokes an cellular or
humoral immune response which cross reacts to that protein.
The genetic construct of genetic vaccines comprise a nucleotide
sequence that encodes a target protein operably linked to
regulatory elements needed for gene expression. Accordingly,
incorporation of the DNA or RNA molecule into a living cell results
in the expression of the DNA or RNA encoding the target protein and
thus, production of the target protein.
When taken up by a cell, the genetic construct which includes the
nucleotide sequence encoding the target protein operably linked to
the regulatory elements may remain present in the cell as a
functioning extrachromosomal molecule or it may integrate into the
cell's chromosomal DNA. DNA may be introduced into cells where it
remains as separate genetic material in the form of a plasmid.
Alternatively, linear DNA which can integrate into the chromosome
may be introduced into the cell. When introducing DNA into the
cell, reagents which promote DNA integration into chromosomes may
be added. DNA sequences which are useful to promote integration may
also be included in the DNA molecule. Since integration into the
chromosomal DNA necessarily requires manipulation of the
chromosome, it is preferred to maintain the DNA construct as a
replicating or non-replicating extrachromosomal molecule. This
reduces the risk of damaging the cell by splicing into the
chromosome without affecting the effectiveness of the vaccine.
Alternatively, RNA may be administered to the cell. It is also
contemplated to provide the genetic construct as a linear
minichromosome including a centromere, telomeres and an origin of
replication.
The necessary elements of a genetic construct of a genetic vaccine
include a nucleotide sequence that encodes a target protein and the
regulatory elements necessary for expression of that sequence in
the cells of the vaccinated individual. The regulatory elements are
operably linked to the DNA sequence that encodes the target protein
to enable expression.
The molecule that encodes a target protein is a protein-encoding
molecule which is translated into protein. Such molecules include
DNA or RNA which comprise a nucleotide sequence that encodes the
target protein. These molecules may be cDNA, genomic DNA,
synthesized DNA or a hybrid thereof or an RNA molecule such as
mRNA. Accordingly, as used herein, the terms "DNA construct",
"genetic construct" and "nucleotide sequence" are meant to refer to
both DNA and RNA molecules.
The regulatory elements necessary for gene expression of a DNA
molecule include: a promoter, an initiation codon, a stop codon,
and a polyadenylation signal. In addition, enhancers are often
required for gene expression. It is necessary that these elements
be operable in the vaccinated individual. Moreover, it is necessary
that these elements be operably linked to the nucleotide sequence
that encodes the target protein such that the nucleotide sequence
can be expressed in the cells of a vaccinated individual and thus
the target protein can be produced.
Initiation codons and stop codon are generally considered to be
part of a nucleotide sequence that encodes the target protein.
However, it is necessary that these elements are functional in the
vaccinated individual.
Similarly, promoters and polyadenylation signals used must be
functional within the cells of the vaccinated individual.
Examples of promoters useful to practice the present invention,
especially in the production of a genetic vaccine for humans,
include but are not limited to promoters from Simian Virus 40
(SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human
Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat
(LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as
the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous
Sarcoma Virus (RSV) as well as promoters from human genes such as
human Actin, human Myosin, human Hemoglobin, human muscle creatine
and human metalothionein.
Examples of polyadenylation signals useful to practice the present
invention, especially in the production of a genetic vaccine for
humans, include but are not limited to SV40 polyadenylation signals
and LTR polyadenylation signals. In particular, the SV40
polyadenylation signal which is in pCEP4 plasmid (Invitrogen, San
Diego Calif.), referred to as the SV40 polyadenylation signal, is
used.
In addition to the regulatory elements required for DNA expression,
other elements may also be included in the DNA molecule. Such
additional elements include enhancers. The enhancer may be selected
from the group including but not limited to: human Actin, human
Myosin, human Hemoglobin, human muscle creatine and viral enhancers
such as those from CMV, RSV and EBV.
Genetic constructs can be provided with mammalian origin of
replication in order to maintain the construct extrachromosomally
and produce multiple copies of the construct in the cell. Plasmids
pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the
Epstein Barr virus origin of replication and nuclear antigen EBNA-1
coding region which produces high copy episomal replication without
integration.
An additional element may be added which serves as a target for
cell destruction if it is desirable to eliminate cells receiving
the genetic construct for any reason. A herpes thymidine kinase
(tk) gene in an expressible form can be included in the genetic
construct. When the construct is introduced into the cell, tk will
be produced. The drug gangcyclovir can be administered to the
individual and that drug will cause the selective killing of any
cell producing tk. Thus, a system can be provided which allows for
the selective destruction of vaccinated cells.
In order to be a functional genetic construct, the regulatory
elements must be operably linked to the nucleotide sequence that
encodes the target protein. Accordingly, it is necessary for the
initiation and termination codons to be in frame with the coding
sequence.
In order to maximize protein production, regulatory sequences may
be selected which are well suited for gene expression in the
vaccinated cells. Moreover, codons may be selected which are most
efficiently transcribed in the vaccinated cell. One having ordinary
skill in the art can produce DNA constructs which are functional in
vaccinated cells.
In order to test expression, genetic constructs can be tested for
expression levels in vitro using tissue culture of cells of the
same type as those to be vaccinated. For example, if the genetic
vaccine is to be administered into human muscle cells, muscle cells
grown in culture such as solid muscle tumors cells of
rhabdomyosarcoma may be used as an in vitro model to measure
expression level.
The present invention provides methods of conferring a broad based
protective immune response against pathogen infection,
hperproliferative diseases and autoimmune diseases without the use
of infectious agents. The genetic constructs used in the present
invention are not incorporated with retroviral particles. The
genetic constructs are taken up by the cell without viral
particle-mediated insertion such as that which occurs when
retorvirus particles with retroviral RNA that is incorporated in
retroviral particles infects a cell. As used herein, the term "free
from viral particles" is meant to refer to genetic constructs that
are not incorporated within viral particles. In some embodiments,
the genetic constructs constitute less than a complete,
replicatable viral genome such that upon introduction into the
cell, the genetic construct possesses insufficient genetic
information to direct production of infectious vital particles. As
used herein, the term "incomplete viral genome" is meant to refer
to a genetic construct which contains less than a complete genome
such that incorporation of such a genetic construct into a cell
does not constitute introduction of sufficient genetic information
for the production of infectious virus.
One aspect of the present invention provides a method of conferring
a broad based protective immune response against pathogen
infection, diseases associated with hyperproliferative cells or
autoimmune diseases by administering genetic constructs to cells
contacted with an agent that facilitates the uptake of genetic
material, particularly cell stimulating agents. The genetic
construct may be administered with or without the use
microprojectiles.
The present invention may be used to immunize an individual against
all pathogens such as viruses, prokaryote and pathogenic eukaryotic
organisms such as unicellular pathogenic organisms and
multicellular parasites. The present invention is particularly
useful to immunize an individual against those pathogens which
infect cells and which are not encapsulated such as viruses, and
prokaryote such as gonorrhoea, listeria and shigella. In addition,
the present invention is also useful to immunize an individual
against protozoan pathogens which include a stage in the life cycle
where they are intracellular pathogens. As used herein, the term
"intracellular pathogen" is meant to refer to a virus or pathogenic
organism that, at least part of its reproductive or life cycle,
exists within a host cell and therein produces or causes to be
produced, pathogen proteins.
Table 1 provides a listing of some of the viral families and genera
for which vaccines according to the present invention can be made.
DNA constructs that comprise DNA sequences which encode the
peptides that comprise at least an epitope identical or
substantially similar to an epitope displayed on a pathogen antigen
such as those antigens listed on the tables are useful in
vaccines.
In addition to being particularly effective against pathogens which
infect the cells of an individual, the present invention is also
useful to immunize an individual against other pathogens including
prokaryotic and eukaryotic protozoan pathogens as well as
multicellular parasites. Table 2 contains a list of bacterial and
eukaryotic pathogens for which vaccines according to the present
invention may be made.
In order to produce a genetic vaccine to protect against pathogen
infection, genetic material which encodes immunogenic proteins
against which a protective immune response can be mounted must be
included in the genetic construct. Whether the pathogen infects
intracellularly, for which the present invention is particularly
useful, or extracellularly, it is unlikely that all pathogen
antigens will elicit a protective response. Because DNA and RNA are
both relatively small and can be produced relatively easily, the
present invention provides the additional advantage of allowing for
vaccination with multiple pathogen antigens. The genetic construct
used in the genetic vaccine can include genetic material which
encodes many pathogen antigens. For example, several viral genes
may be included in a single construct thereby providing multiple
targets. In addition, multiple inoculants which can be delivered to
different cells in an individual can be prepared to collectively
include, in some cases, a complete or, more preferably, an
incomplete such as a near complete set of genes in the vaccine. For
example, a complete set of viral genes may be administered using
two constructs which each contain a different half of the genome
which are administered at different sites. Thus, an immune response
may be invoked against each antigen without the risk of an
infectious virus being assembled. This allows for the introduction
of more than a single antigen target and can eliminate the
requirement that protective antigens be identified.
The ease of handling and inexpensive nature of DNA and RNA further
allow for more efficient means of screening for protective
antigens. Genes can be sorted and systematically tested much more
easily than proteins. The pathogenic agents and organism for which
the vaccine is being produced to protect against is selected and an
immunogenic protein is identified. Tables 1 and 2 include lists of
some of the pathogenic agents and organisms for which genetic
vaccines can be prepared to protect an individual from infection by
them.
Another aspect of the present invention provides a method of
conferring a broad based protective immune response against
hyperproliferating cells that are characteristic in
hyperproliferative diseases and to a method of treating individuals
suffering from hyperproliferative diseases. As used herein, the
term "hyperproliferative diseases" is meant to refer to those
diseases and disorders characterized by hyperproliferation of
cells. Examples of hyperproliferative diseases include all forms of
cancer and psoriasis.
It has been discovered that introduction of a genetic construct
that includes a nucleotide sequence which encodes an immunogenic
"hyperproliferating cell"-associated protein into the cells of an
individual results in the production of those proteins in the
vaccinated cells of an individual. As used herein, the term
"hyperproliferative-associated protein" is meant to refer to
proteins that are associated with a hyperproliferative disease.
These proteins can elicit a broad biologically active immune
response in the individual including CTLs that can effectively
combat and eliminate hyperproliferating cells in the individual.
Thus, to immunize against hyperproliferative diseases, a genetic
construct that includes a nucleotide sequence which encodes a
protein that is associated with a hyperproliferative disease is
administered to an individual. When expressed, the protein produced
elicits an immune response directed at cells that produce the
protein.
In order for the hyperproliferative-associated protein to be an
effective immunogenic target, it must be a protein that is produced
exclusively or at higher levels in hyperproliferative cells as
compared to normal cells. Target antigens include such proteins,
fragments thereof and peptides which comprise at least an epitope
found on such proteins. In some cases, a
hyperproliferative-associated protein is the product of a mutation
of a gene that encodes a portein. The mutated gene encodes a
protein which is nearly identical to the normal protein except it
has a slightly different amino acid sequence which results in a
different epitope not found on the normal protein.
Such target proteins include those which are proteins encoded by
oncogenes. Generally, oncogenes can be divided into three groups
depending upon the portion of the cell where their gene products
are found. Oncogenes such as myb, myc, fyn, and the translocation
gene bcr/abl encode products that remain in the nucleus and are
involved in transcription and cell cycle events. Gene products of
oncogenes such as ras, src and P53 are generally found in the
cytoplasm. Membrane bound products of oncogenes include neu, trk
and EGRF. While protein products of these genes are often found in
normal cells, they exist at greater levels in cancer cells. Thus,
cancer cells can be expected to be more likely to have these
proteins bound to Class I MHC molecules at the cell surface.
Accordingly, CTLs which specifically recognize the target
protein/MHC I complex will be more effective against cancer
cells.
In addition to oncogene products as target antigens, variable
regions of antibodies made by B cell lymphomas and variable regions
of T cell receptors of T cell lymphomas can also be used as target
antigens. These antigens are discussed and described in greater
detail below in the section referring to autoimmune disease.
However, it is contemplated that similar vaccination strategies can
be used for treating and preventing these types of cancer.
Additionally, other tumor-associated proteins can be used as target
proteins. Such proteins are generally those which are found at
higher levels in tumor cells. Examples include the protein
recognized by monoclonal antibody 17-1A and folate binding
proteins.
While the present invention may be used to immunize an individual
against one or more of several forms of cancer, the present
invention is particularly useful to immunize an individual who is
predisposed to develop a particular cancer or who has had cancer
and is therefore susceptible to a relapse.
Developments in genetics and technology as well as epidemiology
allow for the determination of probability and risk assessment for
the development of cancer in individual. Using genetic screening
and/or family health histories, it is possible to predict the
probability a particular individual has for developing any one of
several types of cancer. Those individuals identified as being
predisposed to developing a particular form of cancer can, by using
the methods of the present invention, take prophylactic steps
towards reducing the risk of cancer. According to the present
invention, high-risk individuals can be immunized against the form
of cancer that they have a predisposition to develop.
Similarly, those individuals who have already developed cancer and
who have been treated to remove the cancer or are otherwise in
remission are particularly susceptible to relapse and reoccurrence.
As part of a treatment regimen, such individuals can be immunized
against the cancer that they have been diagnosed as having had in
order to combat a recurrence. Thus, once it is known that an
individual has had a type of cancer and is at risk of a relapse,
they can be immunized in order to prepare their immune system to
combat any future appearance of the cancer.
The present invention provides a method of treating individuals
suffering from hyperproliferative diseases. In such methods, the
introduction of genetic constructs serves as an immunotherapeutic,
directing and promoting the immune system of the individual to
combat hyperproliferative cells that produce the target
protein.
The present invention provides a method of treating individuals
suffering from autoimmune diseases and disorders by conferring a
broad based protective immune response against targets that are
associated with autoimmunity including cell receptors and cells
which produce "self"-directed antibodies.
T cell mediated autoimmune diseases include Rheumatoid arthritis
(RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis,
insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis,
reactive arthritis, ankylosing spondylitis, scleroderma,
polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's
granulomatosis, Crohn's disease and ulcerative colitis. Each of
these diseases is characterized by T cell receptors that bind to
endogenous antigens and initiate the inflammatory cascade
associated with autoimmune diseases. Vaccination against the
variable region of the T cells would elicit an immune response
including CTLs to eliminate those T cells.
In RA, several specific variable regions of T cell receptors (TCRs)
which are involved in the disease have been characterized. These
TCRs include V.beta.-3, V.beta.-14, V.beta.-17 and V.alpha.-17.
Thus, vaccination with a DNA construct that encodes at least one of
these proteins will elicit an immune response that will target T
cells involved in RA. See: Howell, M.D., et al., 1991 Proc. Natl.
Acad. Sci. USA 88:10921-10925; Paliard, X., et al., 1991 Science
253:325-329; Williams, W. V., et al., 1992 J. Clin. Invest.
90:326-333; each of which is incorporated herein by reference.
In MS, several specific variable regions of TCRs which are involved
in the disease have been characterized. These TCRs include
V.beta.-7 and V.alpha.-10. Thus, vaccination with a DNA construct
that encodes at least one of these proteins will elicit an immune
response that will target T cells involved in MS. See:
Wucherpfennig, K. W., et al., 1990 Science 248:1016-1019;
Oksenberg, J. R., et al., 1990 Nature 345:344-346; each of which is
incorporated herein by reference.
In scleroderma, several specific variable regions of TCRs which are
involved in the disease have been characterized. These TCRs include
V.beta.-6, V.beta.-8, V.beta.-14 and V.alpha.-16, V.alpha.-3C,
V.alpha.-7, V.alpha.-14, V.alpha.-15, V.alpha.-16, V.alpha.-28 and
V.alpha.-12. Thus, vaccination with a DNA construct that encodes at
least one of these proteins will elicit an immune response that
will target T cells involved in scleroderma.
In order to treat patients suffering from a T cell mediated
autoimmune disease, particularly those for which the variable
region of the TCR has yet to be characterized, a synovial biopsy
can be performed. Samples of the T cells present can be taken and
the variable region of those TCRs identified using standard
techniques. Genetic vaccines can be prepared using this
information.
B cell mediated autoimmune diseases include Lupus (SLE), Grave's
disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune
thrombocytopenia, asthma, cryoglobulinemia, primary biliary
sclerosis and pernicious anemia. Each of these diseases is
characterized by antibodies which bind to endogenous antigens and
initiate the inflammatory cascade associated with autoimmune
diseases. Vaccination against the variable region of antibodies
would elicit an immune response including CTLs to eliminate those B
cells that produce the antibody.
In order to treat patients suffering from a B cell mediated
autoimmune disease, the variable region of the antibodies involved
in the autoimmune activity must be identified. A biopsy can be
performed and samples of the antibodies present at a site of
inflammation can be taken. The variable region of those antibodies
can be identified using standard techniques. Genetic vaccines can
be prepared using this information.
In the case of SLE, one antigen is believed to be DNA. Thus, in
patients to be immunized against SLE, their sera can be screened
for anti-DNA antibodies and a vaccine can be prepared which
includes DNA constructs that encode the variable region of such
anti-DNA antibodies found in the sera.
Common structural features among the variable regions of both TCRs
and antibodies are well known. The DNA sequence encoding a
particular TCR or antibody can generally be found following well
known methods such as those described in Kabat, et al. 1987
Sequence of Proteins of Immunological Interest U.S. Department of
Health and Human Services, Bethesda Md., which is incorporated
herein by reference. In addition, a general method for cloning
functional variable regions from antibodies can be found in
Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci. USA 87:1066,
which is incorporated herein by reference.
According to the invention, the genetic vaccine may be administered
directly into the individual to be immunized or ex vivo into
removed cells of the individual which are reimplanted after
administration. By either route, the genetic material is introduced
into cells which are present in the body of the individual. Routes
of administration include, but are not limited to, intramuscular,
intraperitoneal, intradermal, subcutaneous, intravenous,
intraarterially, intraoccularly and oral as well as transdermally
or by inhalation or suppository. Preferred routes of administration
include intramuscular, intraperitoneal, intradermal and
subcutaneous injection. Genetic constructs may be administered by
means including, but not limited to, traditional syringes,
needleless injection devices, or "microprojectile bombardment gene
guns". Alternatively, the genetic vaccine may be introduced by
various means into cells that are removed from the individual. Such
means include, for example, ex vivo transfection, electroporation,
microinjection and microprojectile bombardment. After the genetic
construct is taken up by the cells, they are reimplanted into the
individual. It is contemplated that otherwise non-immunogenic cells
that have genetic constructs incorporated therein can be implanted
into the individual even if the vaccinated cells were originally
taken from another individual.
The genetic vaccines according to the present invention comprise
about 1 nanogram to about 1000 micrograms of DNA. In some preferred
embodiments, the vaccines contain about 10 nanograms to about 800
micrograms of DNA. In some preferred embodiments, the vaccines
contain about 0.1 to about 500 micrograms of DNA. In some preferred
embodiments, the vaccines contain about 1 to about 350 micrograms
of DNA. In some preferred embodiments, the vaccines contain about
25 to about 250 micrograms of DNA. In some preferred embodiments,
the vaccines contain about 100 micrograms DNA.
The genetic vaccines according to the present invention are
formulated according to the mode of administration to be used. One
having ordinary skill in the art can readily formulate a genetic
vaccine that comprises a genetic construct. In cases where
intramuscular injection is the chosen mode of administration, an
isotonic formulation is preferably used. Generally, additives for
isotonicity can include sodium chloride, dextrose, mannitol,
sorbitol and lactose. In some cases, isotonic solutions such as
phosphate buffered saline are preferred. Stabilizers include
gelatin and albumin. In some embodiments, a vaso-constriction agent
is added to the formulation. The pharmaceutical preparations
according to the present invention are provided sterile and pyrogen
free.
Genetic constructs may optionally be formulated with one or more
response enhancing agents such as: compounds which enhance
transfection, i.e. transfecting agents; compounds which stimulate
cell division, i.e. replication agents; compounds which stimulate
immune cell migration to the site of administration, i.e.
inflammatory agents; compounds which enhance an immune response,
i.e. adjuvants or compounds having two or more of these
activities.
As used herein, the term "transfection agent" is meant refer to an
agent that promotes and facilitates the uptake of genetic material
by the cells. According to the present invention, cells are
contacted with one or more transfection agents prior to,
simultaneously with or subsequent to administration of the genetic
construct.
As used herein, the term "replicating agent" is meant to refer to
an agent that stimulates cell division and replication. According
to the present invention, cells are contacted with one or more
replicating agents prior to simultaneously with, or subsequent to
administration of the genetic construct.
As used herein, the term "inflammatory agent" is meant to refer to
an agent that induces migration and chemotaxis of cells involved in
an immune response to the site in an individual where it is
administered. According to the present invention, cells are
contacted with one or more inflammatory agents prior to,
simultaneously with, or subsequent to administration of the genetic
construct. An inflammatory agent can be an irritant which disrupts
or damages tissue. Thus, in addition to the cells that are normally
present at the site of administration, the migrating immune cells
can come into contact with and take up the administered genetic
construct.
As used herein, the term "cell stimulating agent" refers to a
compound that is both a transfection agent in that it facilitates
DNA and RNA uptake by cells and a replicating agent in that is
stimulates cell division and replication. As used herein, the terms
"cell stimulating agent" or "cell proliferative agent" are used
interchangeably and refer to compounds which are transfecting
agents and replicating agents. Cell stimulating agents facilitate
DNA and RNA uptake, and stimulate cell division.
In some embodiments, the transfecting agent used is preferably a
cell stimulating agent. In some embodiments, a transfecting agent
is used which is also an inflammatory agent. In some embodiments, a
transfecting agent is used which is also an adjuvant. In some
embodiments, a transfecting agent is used which is also an
inflammatory agent and an adjuvant. In some embodiments, a cell
stimulating agent is used which is also an inflammatory agent. In
some embodiments, a cell stimulating agent is used which is also an
adjuvant. In some embodiments, a cell stimulating agent is used
which is also an inflammatory agent and an adjuvant. In some
embodiments, a replicating agent is used which is also an
inflammatory agent. In some embodiments, a replicating agent is
used which is also an adjuvant. In some embodiments, a replicating
agent is used which is also an inflammatory agent and an adjuvant.
In some embodiments, an inflammatory agent is used which is also an
adjuvant.
In a preferred embodiment, bupivacaine, a well known and
commercially available pharmaceutical compound, is administered
prior to, simultaneously with or subsequent to the genetic
construct. Bupivacaine and the genetic construct may be formulated
in the same composition. Bupivacaine is particularly useful as a
cell stimulating agent in view of its many properties and
activities when administered to tissue. Bupivacaine promotes and
facilitates the uptake of genetic material by the cell. As such, it
is a transfecting agent. Administration of genetic constructs in
conjunction with bupivacaine facilitates entry of the genetic
constructs into cells. Bupivacaine is believed to disrupt or
otherwise render the cell membrane more permeable. Cell division
and replication is stimulated by bupivacaine. Accordingly,
bupivacaine acts as a replicating agent. Administration of
bupivacaine also irritates and damages the tissue. As such, it acts
as an inflammatory agent which elicits migration and chemotaxis of
immune cells to the site of administration. In addition to the
cells normally present at the site of administration, the cells of
the immune system which migrate to the site in response to the
inflammatory agent can come into contact with the administered
genetic material and the bupivacaine. Bupivacaine, acting as a
transfection agent, is available to promote uptake of genetic
material by such cells of the immune system as well.
Bupivacaine is related chemically and pharmacologically to the
aminoacyl local anesthetics. It is a homologue of mepivacaine and
related to lidocaine. Bupivacaine renders muscle tissue voltage
sensitive to sodium challenge and effects ion concentration within
the cells. A complete description of bupivacaine's pharmacological
activities can be found in Ritchie, J. M. and N. M. Greene, The
Pharmacological Basis of Therapeutics, Eds.: Gilman, A. G. et al,
8th Edition, Chapter 15:3111, which is incorporated herein by
reference. Bupivacaine and compounds that display a functional
similarity to bupivacaine are preferred in the method of the
present invention.
Bupivacaine-HCl is chemically designated as
2-piperidinecarboxamide,
1-butyl-N-(2,6-dimethylphenyl)monohydrochloride, monohydrate and is
widely available commercially for pharmaceutical uses from many
sources including from Astra Pharmaceutical Products Inc.
(Westboro, Mass.) and Sanofi Winthrop Pharmaceuticals (New York,
N.Y.), Eastman Kodak (Rochester, N.Y.). Bupivacaine is commercially
formulated with and without methylparaben and with or without
epinephrine. Any such formulation may be used. It is commercially
available for pharmaceutical use in concentration of 0.25%, 0.5%
and 0.75% which may be used on the invention. Alternative
concentrations which elicit desirable effects may be prepared if
desired. According to the present invention, about 250 .mu.g to
about 10 mg of bupivacaine is administered. In some embodiments,
about 250 .mu.g to about 7.5 mg is administered. In some
embodiments, about 0.50 mg to about 5.0 mg is administered. In some
embodiments, about 1.0 mg to about 3.0 mg is administered. In some
embodiments about 5.0 mg is administered. For example, in some
embodiments about 50 .mu.l to about 2 ml, preferably 50 .mu.l to
about 1500 .mu.l and more preferably about 1 ml of 0.5%
bupivacaine-HCl and 0.1% methylparaben in an isotonic
pharmaceutical carrier is administered at the same site as the
vaccine before, simultaneously with or after the vaccine is
administered. Similarly, in some embodiments, about 50 .mu.l to
about 2 ml, preferably 50 .mu.l to about 1500 .mu.l and more
preferably about 1 ml of 0.5% bupivacaine-HCl in an isotonic
pharmaceutical carrier is administered at the same site as the
vaccine before, simultaneously with or after the vaccine is
administered. Bupivacaine and any other similarly acting compounds,
particularly those of the related family of local anesthetics may
be administered at concentrations which provide the desired
facilitation of uptake of genetic constructs by cells.
In some embodiments of the invention, the individual is first
subject to bupivacaine injection prior to genetic vaccination by
intramuscular injection. That is, up to, for example, up to a about
a week to ten days prior to vaccination, the individual is first
injected with bupivacaine. In some embodiments, prior to
vaccination, the individual is injected with bupivacaine about 1 to
5 days before administration of the genetic construct. In some
embodiments, prior to vaccination, the individual is injected with
bupivacaine about 24 hrs before administration of the genetic
construct. Alternatively, bupivacaine can be injected
simultaneously, minutes before or after vaccination. Accordingly,
bupivacaine and the genetic construct may be combined and injected
simultaneously as a mixture. In some embodiments, the bupivacaine
is administered after administration of the genetic construct. For
example, up to about a week to ten days after administration of the
genetic construct, the individual is injected with bupivacaine. In
some embodiments, the individual is injected with bupivacaine about
24 hrs after vaccination. In some embodiments, the individual is
injected with bupivacaine about 1 to 5 days after vaccination. In
some embodiments, the individual is administered bupivacaine up to
about a week to ten days after vaccination.
In addition to bupivacaine, mepivacaine, lidocaine, procains,
carbocaine and methyl bupivacaine, other similarly acting compounds
may be used as response enhancing agents. Such agents acts a cell
stiumulating agents which promote the uptake of genetic constructs
into the cell and stimulate cell replication as well as initiate an
inflammatory response at the site of administration.
Other contemplated response enhancing agents which may function
transfecting agents and/or replicating agents and/or inflammatory
agents and which may be co-adminstered with bupivacaine and similar
acting compounds include lectins, growth factors, cytokines and
lymphokines such as .alpha.-interferon, gamma-interferon, platelet
derived growth factor (PDGF), gCSF, gMCSF, TNF, epidermal growth
factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12 as well
as collagenase, fibroblast growth factor, estrogen, dexamethasone,
saponins, surface active agents such as immune-stimulating
complexes (ISCOMS), Freund's incomplete adjuvant, LPS analog
including monophosphoryl Lipid A (MPL), muramyl peptides, quinone
analogs and vesicles such as squalene and squalane, hyaluronic acid
and hyaluronidase may also be used administered in conjunction with
the genetic construct. In some embodiments, combinations of these
agents are administered in conjunction with bupivicaine and the
genetic construct. For example, bupivacaine and either hyaluronic
acid or hyaluronidase are co-administered with a genetic
construct.
The genetic construct may be combined with collagen as an emulsion
and delivered parenterally. The collagen emulsion provides a means
for sustained release of DNA. 50 .mu.l to 2 ml of collagen are
used. About 100 .mu.g DNA are combined with 1 ml of collagen in a
preferred embodiment using this formulation.
In some embodiments of the invention, the genetic construct is
injected with a needleless injection device. The needleless
injection devices are particularly useful for simultaneous
administration of the material intramuscularly, intradermally and
subcutaneously.
In some embodiments of the invention, the genetic construct is
administered with a response enhancing agent by means of a
microprojectile particle bombardment procedure as taught by Sanford
et al. in U.S. Pat. No. 4,945,050 issued Jul. 31, 1990, which is
incorporated herein by reference.
In some embodiments of the invention, the genetic construct is
administered as part of a liposome complex with a response
enhancing agent.
In some embodiments of the invention, the individual is subject to
a single vaccination to produce a full, broad immune response. In
some embodiments of the invention, the individual is subject to a
series of vaccinations to produce a full, broad immune response.
According to some embodiments of the invention, at least two and
preferably four to five injections are given over a period of time.
The period of time between injections may include from 24 hours
apart to two weeks or longer between injections, preferably one
week apart. Alternatively, at least two and up to four separate
injections are given simultaneously at different sites.
In some embodiments of the invention, a complete vaccination
includes injection of a single inoculant which contains a genetic
construct including sequences encoding one or more targeted
epitopes.
In some embodiments of the invention, a complete vaccination
includes injection of two or more different inoculants into
different sites. For example, in an HIV vaccine according to the
invention, the vaccine comprises two inoculants in which each one
comprises genetic material encoding different viral proteins. This
method of vaccination allows the introduction of as much as a
complete set of viral genes into the individual without the risk of
assembling an infectious viral particle. Thus, an immune response
against most or all of the virus can be invoked in the vaccinated
individual. Injection of each inoculant is performed at different
sites, preferably at a distance to ensure no cells receive both
genetic constructs. As a further safety precaution, some genes may
be deleted or altered to further prevent the capability of
infectious viral assembly. As used herein, the term "pharmaceutical
kit" is meant to collectively refer to multiple inoculant used in
the present invention. Such kits include separate containers
containing different inoculants and/or cell stimulating agents. It
is intended that these kits be provided to include a set of
inoculants used in an immunizing method.
While the disclosure herein primarily relates to uses of the
methods of the present invention to immunize humans, the methods of
the present invention can be applied to veterinary medical uses
too. It is within the scope of the present invention to provide
methods of immunizing non-human as well as human individuals
against pathogens and protein specific disorders and diseases.
Accordingly, the present invention relates to genetic immunization
of mammals, birds and fish. The methods of the present invention
can be particularly useful for mammalian species including human,
bovine, ovine, porcine, equine, canine and feline species.
While this disclosure generally discusses immunization in the
context of prophylactic methods of protection, the term
"immunizing" is meant to refer to both prophylactic and therapeutic
methods. Thus, a method of immunizing includes both methods of
protecting an individual from pathogen challenge or occurrence or
proliferation of specific cells as well as methods of treating an
individual suffering from pathogen infection, hyperproliferative
disease or autoimmune disease. Accordingly, the present invention
may be used as a vaccine for prophylactic protection or in a
therapeutic manner; that is, as immunotherapeutic methods and
preparations.
Other aspects of the invention include the use of bupivacaine and
related, similarly acting cell stimulating agents in methods of
introducing therapeutic genes into cells of an individual. Thus,
one aspect of the present invention relates gene therpy; that is,
to methods of introducing nucleic acid molecules that encode
therapeutic proteins into the cells of an individual. The
administration protocols and genetic constructs useful in gene
therapy applications are the same as those described above for
genetic immunization except the genetic constructs include
nucleotide sequences that encode proteins whose presence in the
individual will eliminate a deficiency in the individual and/or
whose presence will provide a therapeutic effect on the
individual.
The Examples set out below include representative examples of
aspects of the present invention. The Examples are not meant to
limit the scope of the invention but rather serve exemplary
purposes. In addition, various aspects of the invention can be
summarized by the following description. However, this description
is not meant to limit the scope of the invention but rather to
highlight various aspects of the invention. One having ordinary
skill in the art can readily appreciate additional aspects and
embodiments of the invention.
EXAMPLES
Example 1
According to the present invention, an effective vaccine has been
produced which can invoke a protective immune response against HIV
infected cells as well as cell free virus. As the awareness of AIDS
and HIV infection has grown, repeated attempts and vast
expenditures of resources and efforts have been made to produce an
HIV vaccine. Despite enormous efforts, little progress has been
made thus far and the long felt need for an HIV vaccine has gone
unabated.
The present invention provides an HIV vaccine using direct genetic
immunization. Genetic constructs are provided which, when delivered
into the cells of an individual, are expressed to produce HIV
proteins. According to some embodiments, the production of all
viral structural proteins in the cells of the individual elicit a
protective immune response which protects against HIV infection.
The HIV vaccine of the present invention may be used to immunize
uninfected individuals from HIV infection or serve as an
immunotherapeutic for those individuals already infected. The HIV
vaccine of the present invention invokes an immune response
including CTLs which recognize and attack HIV infected cells and
recognize the widest contingent of HIV protein. Thus, uninfected
individuals are protected from HIV infection.
In some embodiments, the present invention relates to a method of
immunizing an individual against HIV by administering two
inoculants. These two inoculants comprise at least two and
preferably more than two, a plurality or all of the genes of the
HIV virus. However, the inoculants are not delivered together.
Accordingly, an inoculated cell will not be administered a complete
complement of genes. The vaccinated individual will receive at
least two different and preferably more than two, more preferably a
plurality or all of the viral genes. Immune responses can then be
directed at the total complement of HIV protein target.
This strategy serves two purposes. First, it is unknown which
target protein is most effective as an immunizing antigen to
protect an individual against infection. Thus, immunizing with two
or more provides a greater probability that the vaccinated
individual will be provided with sufficient immunogenic target
proteins for eliciting a protective immune response. Secondly, HIV
proteins are known to undergo structural changes due to mutation.
By providing multiple antigenic targets, the probability that a
viral particle will escape detection by the immune response is
reduced despite structural changes in one or more viral proteins.
Accordingly, it is desirable to vaccinate an individual with
multiple and preferably a nearly complete or complete complement of
genes encoding viral proteins.
If a single cell is provided with a complete complement of viral
genes, it is possible that a complete infectious virus can be
assembled within the cell. Accordingly, a genetic construct
according to the present invention is not provided with such a full
complement of genes. Furthermore, two or more inoculants, each
having an incomplete set of genes and combined having up to a full
complement of viral genes, are administered to different cells,
preferably at a distant site from each other to ensure that no
vaccinated cell will inadvertently be exposed to a full set of
genes. For example, a portion of the HIV genome may be inserted
into a first construct and the remaining portion of the HIV genome
is inserted in a second construct. The first construct is
administered to an individual as a genetic vaccine in the muscle
tissue of one arm while the second construct is administered to an
individual as a genetic vaccine in the muscle tissue of the
individual's other arm. The individual may be exposed to a full set
of viral genes; thus essentially vaccinating against the whole
virus but with no risk that an infectious viral particle will be
assembled.
As an additional safety precaution, even when genetic material is
delivered by two or more inoculants at distant parts of the
individual's body, one or more essential genes can be deleted or
intentionally altered to further ensure that an infectious viral
particle cannot be formed. In such embodiments, the individual is
not administered a complete functional set of viral genes.
A further safety precaution provides non-overlapping portions of
the viral genome on the separate genetic constructs that make up
the separate inoculants respectively. Accordingly, recombination
between the two genetic constructs is prevented.
In some embodiments of the present invention, a full complement of
structural genes are provided. The structural genes of HIV consist
of gag, pol and env. These three genes are provided on two
different DNA or RNA constructs. Accordingly, in one preferred
embodiment, gag and pol are on one DNA or RNA construct and env is
on another. In another preferred embodiment, gag is on one DNA or
RNA construct and pol and env is on the other. In another preferred
embodiment, gag and env are on one DNA or RNA construct and pol is
on the other. Optionally, in any of these combinations, HIV
regulatory genes may also be present. The HIV regulatory genes are:
vpr, vif, vpu, nef, tat and rev.
The DNA construct in a preferred embodiment consists of a promoter,
an enhancer and a polyadenylation signal. The promoter may be
selected from the group consisting of: HIV LTR, human Actin, human
Myosin, CMV, RSV, Moloney, MMTV, human Hemoglobin, human muscle
creatine and EBV. The enhancer may be selected from the group
consisting of: human Actin, human Myosin, CMV, RSV, human
Hemoglobin, human muscle creatine and EBV. The polyadenylation
signal may be selected from the group consisting of: LTR
polyadenylation signal and SV40 polyadenylation signal,
particularly the SV40 minor polyadenylation signal among
others.
In some embodiments, the two inoculant vaccine is administered
intramuscularly at spatially segregated tissue of the individual,
preferably in different appendages, such as for example in the
right and left arms. Each inoculant of the present invention may
contain from about 0.1 to about 1000 micrograms of DNA. Preferably,
each inoculant contains about 1 to about 500 micrograms of DNA.
More preferably, each inoculant contains about 25 to about 250
micrograms of DNA. Most preferably, each inoculant contains about
100 micrograms DNA.
The inoculant in a preferred embodiment is in a sterile isotonic
carrier, preferably phosphate buffered saline or saline
solution.
In some embodiments, prior to vaccine administration, the tissue to
be vaccinated is injected with a cell proliferating agent,
preferably bupivacaine. Bupivacaine injections may be performed up
to about 24 hours prior to vaccination. It is contemplated that
bupivacaine injection will occur immediately before vaccination.
About 50 .mu.l to about 2 ml of 0.5% bupivacaine-HCl and 0.1%
methylparaben in isotonic NaCl is administered to the site where
the vaccine is to be administered, preferably, 50 .mu.l to about
1500 .mu.l, more preferably about 1 ml.
In other embodiments, a cell proliferating agent, preferably
bupivacaine is included in the formulation together with the
genetic construct. About 50 .mu.l to about 2 ml of 0.5%
bupivacaine-HCl and 0.1% methylparaben in isotonic NaCl is
administered to the site where the vaccine is to be administered,
preferably, 50 .mu.l to about 1500 .mu.l, more preferably about 1
ml.
Accordingly, some embodiments comprise a two inoculant vaccine: one
inoculant comprising a DNA or RNA construct having two HIV
structural genes, the other inoculant comprising a DNA or RNA
construct having the third, remaining HIV structural gene such that
the combined inoculants contain a full complement of HIV structural
genes. The structural genes on each DNA construct are operably
linked to a promoter, an enhancer and a polyadenylation signal. The
same or different regulatory elements may control expression of the
viral genes. When vaccinating an individual, the two inoculants are
administered intramuscularly to different sites, preferably on
different arms. In some embodiments of the invention, bupivacaine
is first administered at the site where inoculant is to be
administered. In some embodiments of the invention, bupivacaine is
included in the formulations together with the genetic
constructs.
In some embodiments, the vaccination procedure is repeated at least
once and preferably two or three times. Each vaccination procedure
is performed from 24 hours to two months apart.
In some embodiments, the vaccine is administered using a needleless
injection device. In some embodiments, the vaccine is administered
hypodermically using a needleless injection device thus providing
intramuscular, intradermal, subcutaneous administration
simultaneously while also administering the material
interstitially.
Preferred genetic constructs include the following. Plasmids and
Cloning Strategies:
Two plasmids were constructed: one which contains HIV gag/pol and
the other which contains HIV env.
The HIV-1 genomic clone pNL43 was obtained through the NIH AIDS
Research and Reference Reagent Program (ARRRP), Division of AIDS,
NIAID, NIH, from Dr. Malcom Martin, and can be used as the starting
material for HIV-1 viral genes for genetic constructs.
Alternatively, any HIV molecular clone of infected cell can,
through use of the polymerase chain technology, be modified
sufficiently for construction including the HXB2 clone the MN clone
as well as the SF or BAL-1 clone. The pNL43 clone is a construct
that consists of HIV-1 proviral DNA plus 3 kb of host sequence from
the site of integration cloned into pUClS.
Construction of pNL-puro-env plasmid:
This plasmid was constructed for expression of gag pol. The StuI
site within the non-HIV 5' flanking human DNA of pNL43 was
destroyed by partial digestion with StuI followed by digestion of
the free ends with E. coli polymerase 1. The linear plasmid was
filled and then self ligated, leaving a unique StuI site within the
HIV genome. This plasmid, pNLDstu, was then digested with the
blunting enzymes StuI and BsaBI which eliminated a large section of
the coding sequence for gp120. The SV40 promoter and puromycin
resistance coding region (puromycin acetyl transferase (PAC)) were
isolated from pBABE-puro (Morgenstern and Land, 1990 Nucl. Acids
Res. 18(12):3587-3596, which is incorporated herein by reference,
kindly provided by Dr. Hartmut Land of the Imperial Cancer Research
Fund) using EcoRI and ClaI. This fragment was blunted, then cloned
into the StuI/BsaBI-digested pNLDstu. A clone was selected with the
SV40-puro fragment in the correct orientation so that the 3' LTR of
HIV could provide poly A functions for the PAC message. This
plasmid was designated pNLpuro.
Cloning strategy for deletion of vpr regulatory gene from the HIV
gag pol vector:
A region from just upstream of the unique PflMI site to just after
the vif termination codon was amplified via PCR using primers that
introduced a non-conservative amino acid change (glu.fwdarw.val) at
aminoacid 22 of vpr, a stop codon in the vpr reading frame
immediately after amino acid 22, and an EcoRI site immediately
following the new stop codon. This PCR fragment was substituted for
the PflMI-EcoR I fragment of pNLpuro or pNL43. This substitution
resulted in the deletion of 122 nucleotides of the open reading
frame of vpr, thus eliminating the possibility of reversion that a
point mutation strategy entails. The resulting plasmids,
pNLpuroAvpr, encode the first 21 natural amino acids of vpr plus a
valine plus all other remaining HIV-1 genes and splice junctions in
their native form. Such deletion strategy would also be applicable
to nef, vif, and vpu and allow for structural gene expression but
protect from the generation of a live recombinant virus.
Plasmid construction for envelope expression:
The DNA segment encoding the envelope gene of HIV-1 HXB2 was cloned
by the polymerase chain reaction (PCR) amplification technique
utilizing the lambda cloned DNA obtained from the AIDS Research and
Reference Reagent Program. The sequences of the 5' and 3' primers
are 5'-AGGCGTCTCGAGACAGAGGAGAGCAAGAAATG-3' (SEQ ID NO:1) with
incorporation of XhoI site and 5'-TTTCCCTCTAGATAAGCCATCCAATCACAC-3'
(SEQ ID NO: 2) with incorporation of XbaI site, respectively, which
encompass gp160, tat and rev coding region. Gene specific
amplification was performed using Taq DNA polymerase according to
the manufacturer's instructions (Perkin-Elmer Cetus Corp.). The PCR
reaction products were treated with 0.5 ug/ml proteinase K at
37.degree. C. for thirty minutes followed by a phenol/chloroform
extraction and ethanol precipitation. Recovered DNA was then
digested with Xhol and Xbal for two hours at 37.degree. C. and
subjected to agarose gel electrophoresis. The isolated and purified
Xhol-Xbal PCR fragment was cloned into Bluescript plasmid
(Stratagene Inc., La Jolla, Calif.) and then subcloned into the
eukaryotic expression vector pMAMneoBlue (Clontech Laboratories,
Inc., Palo Alto, Calif.). The resulting construct was designated as
pM160. The plasmid DNA was purified with CsCl gradient
ultracentrifugation.
An alternative envelope expression plasmid construction called
HIV-1 env-rev plasmid:
The region encoding the two exons of rev and the vpu and envelope
open reading frames of HIV-1 HXB2 was amplified via PCR and cloned
into the expression vector pCNDA/neo (Invitrogen). This plasmid
drives envelope production through the CMV promoter.
Production and Purification:
The plasmid in E. coli (DH5 alpha) is grown up as follows: An LB
plus ampicillin agar plate is streaked with the desired plasmid
culture from frozen stock. The plate is incubated overnight (14-15
hours) at 37.degree. C. A single colony is taken from the plate and
inoculated into 15 ml of LB medium with a peptone preparation and
50 .mu.g/ml ampicillin. This culture is grown at 37.degree. C.
while being shaken (ca. 175 rpm) for 8-10 hours. OD.sub.600
readings should be at least 1.0. 1 liter of LB medium with peptone
and 50 .mu.g/ml ampicillin is inoculated with 1.0 OD of culture.
These 1-2 liter cultures are grown overnight at 37.degree. C. while
being shaken (175 rpm).
Plasmid grown in E. coli (strain DH5 alpha) are harvested and
purified by the following methods. General procedures for the lysis
of cells and purification of plasmid can be found in "Molecular
Cloning: A Laboratory Manual", 2nd Edition, J. Sambrook, E. F.
Fritsch, and T. Maniatis, Cold Spring Harbor Press, 1989. The cells
are concentrated and washed with glucose-tris-EDTA pH 8.0 buffer.
The concentrated cells are lysed by treatment with lysozyme and
briefly treated with 0.2N KOH, the pH is then adjusted 5.5 with
potassium acetate/acetic acid buffer. Insoluble material is removed
by centrifugation. To the supernatant is added 2-propanol to
precipitate the plasmid. The plasmid is redissolved in tris-EDTA
buffer and further purified by phenol/chloroform extraction and an
additional precipitation with 2-propanol.
Endotoxin can optionally be removed by a variety of methods
including the following: specific adsorption by immobilized
materials such as polymyxin ("Endotoxin removed from hemoglobin
solution using polymyxin B-immobilized fibre (PMX-F) . . . ", Tani
et al , Biomater Artif Cells Immobilization Biotechnol.
20(2-4):457-62 (1992); "Efficient endotoxin removal with a new
sanitizable affinity column: Affi-Prep Polymyxin", Issekutz J.
Immunol Methods 61(3):275-81 (1983)); anti-endotoxin monoclonal
antibodies, such as 8A1 and HA-1A.TM. (Centocor, Malvern, Pa.;
"Human Monoclonal Antibody HA-1A Binds to Endotoxin via an Epitope
in the Lipid A Domain of Lipopolysaccharide" Bogard et al. J.
Immunol. 150(10):4438-4449 (1993); Rietschel et al., Infect.
Immunity page 3863 (1993)); positively charged depth filters
("Depyrogenation by endotoxin removal with positively charged depth
filter cartridge", Hou et al., J. Parenter Sci Technol. 44(4):204-9
(July-August 1990)); poly(gamma-methyl L-glutamate) ("Removal of
endotoxin from culture supernatant of Bortedella pertussis with
aminated poly (gamma-methyl L-glutamate) spherical beads", Hirayama
et al., Chem. Pharm. Bull. (Tokyo) 40(8):2106-9 (1992)); histidine
("Specific removal of endotoxin from protein solutions by
immobilized histidine", Matsumae et al., Biotechnol. Appl. Biochem.
12:(2):129-40 (1990)); hydrophobic interaction columns and
membranes (e.g., "Removal of endotoxin from protein solutions by
phase separation using Triton X-114", Aida et al., J. Immunol
Methods 132(2):191-5 (1990); "Novel endotoxin adsorbing materials,
polymyxin-sepharose and polyporous polyethylene membrane for
removal of endotoxin from dialysis systems", Umeda et al., Biomater
Artif Cells Artif Organs 18(4):491-7 (1990); "The effect of
hydrophobic interaction on endotoxin adsorption by polymeric
affinity matrix", Hou et al., Biochem. Biophys. Acta 1073(1):149-54
(1991); "Endotoxin removal from water using microporous
polyethylene chopped fibres as a new adsorbent", Sawada et al., J.
Hyg. (London) 97(1):103-14 (1986)); specific hydrophobic resins
useful for removing endotoxin including hydrophobic
polystyrene/divinylbenzene or divinylbenzene resins such as
Brownlee Polypore Resin (Applied Biosystems, Palo Alto, Calif.);
XUS 40323.00 (Dow Chemical, Midland, Mich.); HP20, CHP20P
(Mitsubishi Kasei, U.S.); Hamilton PRP-1, PRP-infinity (Hamilton,
Reno, Nev.); Jordi Reversed-Phase DVB, Jordi Gel DVB, Polymer Labs
PLgel.TM. (Alltech, Deerfield, Ill.); Vydac PLx.TM. (Separations
Group, Hesperia, Calif.); other endotoxin removing materials and
methods include Detoxi-Gel.TM. Endotoxin Removing Gel (Pierce
Chemical, Rockford, Ill.); Application Note 206, "Chromatographic
removal of endotoxins and/or ethanol from albumin", (Pharmacia
Biotech Inc, Piscataway, N.J.) See also generally, "Endotoxin
Detection and Elimination in Biotechnology", Sharma, Biotech. App.
Biochem. 8:5-22 (1986).
Preferred anti-endotoxin monoclonal antibodies bind to the
conserved domains of endotoxin, preferably antibodies to lipid A,
the most structurally conserved portion of the endotoxin molecule.
Such anti-lipid A monoclonal antibodies include the high affinity
murine IgG monoclonal antibody 8A1 and the human anti-lipid A
IgM(k) monoclonal antibody HA-1A.TM.. HA-1A.TM. was derived from a
human B E. coli J5 vaccine. HA-1A.TM.. HA-1A.TM. is reported to be
broadly cross-reactive with a variety of bacterial endotoxins
(lipopolysaccharides).
Example 2
In experiments designed to compare the immunogenic response
elicited by genetic vaccination and protein vaccination, animal
models were designed using tumor cells that specifically express a
foreign target protein. Three immune competent mouse models have
been developed which express foreign antigens. Three clonal tumor
cell lines which are derived from the Balb/c mouse strain are used.
The cell lines are: 1) a lymphoid cell line which does not
metastasize significantly to other tissues but forms large palpable
tumors which appear to kill the animal within an 8-12 week period;
2) a murine melanoma cell line with some ability to metastasize,
mostly to the lung, and in which, following inoculation with 1
million cells, results in the development in the mice of large
palpable tumors which similarly kill the animal within 10-12 weeks;
and 3) a murine lung adenocarcinoma cell line which metastasizes to
multiple tissues and kills the animal within 12 or more weeks.
Subclones have been selected which can display foreign antigens in
an unrecognized form. When transfected tumors are implanted into a
parent mouse strain, unlike the majority of similar murine tumor
lines, the animals do not make a protective immune response to the
foreign antigens displayed and the tumors are accepted. These
tumors then kill the animal with the same phenotype in the same
time frame as the original untransfected tumor. Using these models,
the immune response elicited by genetic vaccination against an
antigen can be measured.
It was observed that mice vaccinated with a genetic vaccine
comprising a genetic construct that resulted in production of the
target protein by the cell's of the mouse elicited an immune
response including a strong cytotoxic that completely eliminated
tumors displaying the target protein but with no effect on tumors
that did not. In mice inoculated with the target protein itself,
the immune response elicited thereby was much less effective. The
tumors were reduced in size but, due to an absence of a cytotoxic
response, they were not eliminated. As controls, untransfected
tumors were used in experiments comparing the immune response of
animals vaccinated with the genetic vaccine, subunit vaccine and
unvaccinated animals. These experiments clearly demonstrate that
the genetic vaccine produced a broader, more effective immune
response which was capable, by virtue of CTL's, of completely
eliminating tumors. By contrast, immunization using intact target
protein produced a more limited, less effective immune
response.
Example 3
In some embodiments of the invention, the infectious virus, HIV,
which is responsible for AIDS is the pathogenic agent against which
a genetic vaccine has been designed. The viral protein gp160, which
is processed into gp120 and gp41, is the target protein against
which a genetic vaccine is produced. The genetic vaccine contains a
DNA construct that comprises a DNA sequence encoding gp160 operably
linked regulatory elements. When administered to an individual, the
DNA construct of the genetic vaccine is incorporated into the cells
of the individual and gp160 is produced. The immune response that
is elicited by the protein is broad based and includes the humoral
and both arms of the cellular immune response. The broad biological
response provides superior protection to that achieved when the
protein itself is administered.
The following is a description of the use of genetic immunization
for elicitation of an anti-human immunodeficiency virus type 1
(HIV-1) immune response in mice by administering a DNA construct
that contains a DNA sequence which encodes the HIV envelope
glycoprotein gp160. The gp160 construct (pM160) expresses
biologically active HIV-1 envelope proteins in vivo.
Mice were injected intramuscularly with pM160 and subsequently
analyzed for anti-HIV immune responses. The antisera from animals
immunized in this manner produce anti-HIV envelope glycoprotein
immune responses as measured by enzyme linked immunosorbent assay
(ELISA) and immunoprecipitation assays. The antisera neutralizes
HIV-1 infection and inhibits HIV-1 induced syncytium formation.
The observed neutralization and anti-syncytial activity may be the
result of reactivity of the elicited antibodies to functionally
important regions of the HIV-1 envelope protein, such as the V3
loop of gp120, CD4 binding site and the N-terminal "immunodominant"
region of gp41, among others.
The DNA construct (pM160) encoding the HIV-1/HXB2 (Fisher, A. G.,
et al., (1985) Nature 316:262-265) gp160 membrane bound
glycoprotein under control of a RSV enhancer element with the MMTV
LTR as a promoter (FIG. 1A) was tested to determine whether this
membrane-bound protein, when expressed by endogenous cells, can
generate an anti-pathogen immune responses. The construct was
generated as follows. The DNA segment encoding the envelope gene of
HIV-1 HXB2 was cloned by the polymerase chain reaction (PCR)
technique amplification utilizing the lambda cloned DNA obtained
from eh AIDS repository. The sequences of the 5' and 3' primers are
5'-AGGCGTCTCGAGACAGAGGAGAGCAAGAAATG-3' (SEQ ID NO:11) with
incorporation of XhoI site and 5'-TTTCCCTCTAGATAAGCCATCCAATCACAC-3'
(SEQ ID NO:12) with incorporation of XbaI site, respectively, which
encompass gp160, tat and rev coding region. Gene specific
amplification was performed using Taq DNA polymerase according to
manufacturer's instruction (Perkin-Elmer Cetus Corp.) The PCR
reaction products were treated with 0.5 .mu.g/ml proteinase K at
37.degree. C. for thirty minutes followed by a phenol/chloroform
extraction and ethanol precipitation (Crowe, J. S., et al., (1991)
Nucl. Acids Res. 19:184). Recovered DNA was then digested with XhoI
and XbaI for two hours at 37.degree. C. and subjected to agarose
gel electrophoresis. The isolated and purified XhoI-XbaI PCR
fragment was cloned into Bluescript plasmid (Stratagene Inc., La
Jolla, Calif.) and then subcloned into the eukaryotic expression
vector pMAMneoBlue (Clontech, Inc.). The resulting construct was
designated as pM160. The plasmid DNA was purified with CsCl
purification (Sambrook, J. et al., (1989 Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
The pM160 construct, which contains a DNA sequence that encodes
gp160, was transfected into a human rhabdomyosarcoma cell line,
TE671 (Stratton, M. R., et al., (1989) Carcinogenesis 10:899-905),
to evaluate its expression before introduction into living animals.
Transfection of pM160 construct into TE671 cells was performed
according to Wang, B., et al., (1992) AIDS Human Retr., in press.
Briefly, 2 .mu.g of purified pM160 was added to 2.times.10.sup.6
TE671 cells (Stratton, M. R., et al., (1989) Carcinogenesis
10:899-905) and subject to electroporation. Following
electroporation, the cells were grown in fresh medium for forty
eight hours prior to the addition of 500 .mu.g/ml neomycin for
selection. Individual cells expressing gp160 envelope protein were
isolated by binding to M450 magnetic beads (Dynal) which was coated
with mixture of monoclonal anti-gp120 antibodies, namely ID6, AD3
and AC4 (Ugen, K. E. et al., (1992) Generation of Monoclonal
Antibodies Against the Amino Region of gp120 Which Elicits Antibody
Dependent Cellular Cytotoxicity, Cold Spring Harbor Laboratory,
1992). Clones were isolated by limiting dilution of the gp160
expressing cells. One of such clone was designated as the 3G7 cell
line. Expression of gp120 and gp41 was determined by Western blot
analysis of whole cell lysates of 3G7 cells versus vector-alone
transfected TE671 cells, performed as previously described (Osther,
K., et al., (1989) Transplantation 47:834-8; and Weiner, D. B., et
al., (1989) Vaccines, Cold Spring Harbor Press, 115-120).
Typically, the mature HIV envelope glycoprotein gp160 is processed
into gp120 and gp41 (Kowalski, M., et al., (1987) Science
237:1351-135). The expression of HIV gp120 and gp41 by the pM160
transfected cell line 3G7 were observed in Western blot analysis
with anti-gp160 specific serum (Osther, K., et al., (1991)
Hybridoma 10:673-683) (FIG. 1B). Functional expression of gp160 by
this cell line was further demonstrated by the ability of 3G7 but
not TE671 cells to fuse with several CD4.sup.+ T-cell cell
lines.
In the genetic immunization procedure described herein, the
quadriceps muscles of BALB/c mice were injected with 100 .mu.l of
0.5% bupivacaine-HCl and 0.1% methylparaben in isotonic NaCl using
a 27-gauge needle to stimulate muscle cell regeneration and
facilitate uptake of the genetic construct. Twenty-four hours
later, the same injection sites were then injected with either 100
.mu.g of pM160 or with 100 .mu.g of pMAMneoBlue as a control
plasmid (FIG. 1A). The mice were boosted by injecting the same
amount of DNA construct three times at two week intervals in the
same manner but without pre-treatment with bupivacaine-HCl.
For the recombinant gp160 immunization, BALB/C mice were initially
immunized with 1 g of glycosylated recombinant (HIV-1/III.sub.B)
gp160 (MicroGeneSys Inc.) in complete Freund's adjuvant followed by
three boosters of 1 .mu.g of gp160 each in incomplete Freund's
adjuvant at two week intervals. The production of antibody against
HIV-1 gp160 was determined by testing the mouse sera for their
ability to immunoprecipitate gp160. Immunoprecipitation was
performed using 1.times.10.sup.6 cpm of .sup.125 I labeled rgp160,
mouse sera and protein-G agarose beads (GIBCO, Inc.) as previously
described by Osther, K., et al., (1991) Hybridoma 10:673-683, which
is incorporated herein by reference. The specific precipitations
were analyzed by 10% SDS-PAGE. Lane 1 is 1 .mu.l of preimmune mouse
serum reacted with the .sup.125 I-gp160. Lane 2 is 1 .mu.l of mouse
serum immunized from the pM160 immunized mice. Lane 3 is 1 .mu.l of
1:100 dilution of ID6 monoclonal anti-gp120 antibody (Ugen, K. E.,
et al., (1992) Generation of Monoclonal Antibodies Against the
Amino Region of gp120 Which Elicits Antibody Dependent Cellular
Cytotoxicity, Cold Spring Harbor Laboratory) as a positive control.
The arrow indicates the specifically immunoprecipitated .sup.125
I-gp160 envelope glycoprotein.
.sup.125 I-labelled gp160 was specifically immunoprecipitated with
antisera derived from the pM160-immunized animals (FIG. 2, lane 2)
as well as the positive control anti-gp120 monoclonal antibody, ID6
(FIG. 2, lane 3). In contrast, the preimmune sera (FIG. 2, lane 1)
only showed minimal activity in the same assay.
Eight of ten mice immunized with the pM160 construct were positive
for reactivity against gp160 as determined by ELISA and the immune
responses from the animal with the highest anti-gp160 titer was
analyzed in detail. Four mice immunized with the control vector all
showed a similar negative reactivity to gp160 in ELISA and one of
these sera was used as the control for subsequent experiments.
It has been shown that HIV neutralizing antibodies are specifically
targeted to several epitopes in gp120 and gp41, which include the
V3 loop in gp120 (Goudsmit, J. et al., (1988) AIDS 2:157-164; and
Putney, S. D., et al., (1989) Development Of An HIV Subunit
Vaccine, Montreal), the CD4 binding site near the carboxy terminus
of gp120 (Lasky, L. A., et al., (1987) Cell 50:975-985) as well as
the immunodominant loop of gp41 just downstream of the N-terminal
fusion region (Schrier, R. D., et al., (1988) J. Virol.
62:2531-2536).
To determine whether the anti-gp160 antibodies elicited in these
mice are reactive to these important regions of the envelope
glycoproteins, peptides for the BRU/V3 loop, peptides for the MN/V3
loop, peptides for the HXB2/gp41 N-terminus or peptides for
HXB2/CD4 binding site were absorbed to microtiter plates and
specific reactivities of the mouse antisera determined in ELISA
assays. One .mu.g/ml of gp160 or 10 .mu.g/ml of each peptide was
coated to microtiter plates in 0.1M bicarbonate buffer (pH 9.5)
overnight at 4.degree. C., blocked with 2% bovine serum albumin in
PBS, and reacted with goat anti-mouse IgG conjugated with HRPO
(Fisher) for one hour at 37.degree. C. and developed with TMB
substrate (Sigma) for 10-30 minutes at room temperature in the
dark. Results are reported in FIG. 3. Antisera were as follows:
(-+-) is preimmune sera, (-.times.-) is the pMAMneoBlue vector
immunized sera, (-.largecircle.-) is the pM160 immunized sera,
(-.DELTA.-) is from mice immunized with the rgp160 protein. FIG. 3A
shows results using a rgp160 protein coated plate. FIG. 3B shows
results using a BRU/V3 loop peptides (CNTRKRIRIQRGPGRAFVTIGK (SEQ
ID NO:13)) coated plate. FIG. 3C shows results using a plate coated
with MN/V loop peptides (YNKRKRIHIQRGPGRAFYTTKNIIC (SEQ ID NO:14))
with the QR sequence from HIV-1/III.sub.B in bold-faced type. FIG.
3D shows the results using a HXB2/CD4 binding site peptides
(CRIKQFINMWQEVGKAMYAPPISGIRC (SEQ ID NO:15)) coated plate. FIG. 3E
shows the results using a BRU/gp41 immunodominant region peptides
(RILAVERYIKDQQLLGIWGCSGKLIC (SEQ ID NO:16)) coated plate.
For the recombinant gp160 immunization. BALB/C mice were initially
immunized with 1 .mu.g of glycosylated recombinant
(HIV-1/III.sub.B) gp160 (MicroGeneSys Inc.) in complete Freund's
adjuvant followed by three boosters of 1 .mu.g of gp160 each in
incomplete Freund's adjuvant at two week intervals.
FIG. 3 shows that antiserum from the pM160 construct immunized
mouse has significantly higher reactivity to the BRU and MN/V3 loop
peptides, the CD4 binding site peptide and the immunodominant gp41
peptide than the recombinant gp160 protein (rgp160) immunized
serum. Interestingly, the antiserum from the rgp160 immunized mouse
had much higher titer against the rgp160 than the pM160 immunized
antiserum, but lower activity against the three specific
neutralization epitopes of gp160 tested (FIG. 3a-d).
To determine whether the antisera generated by DNA immunization
possessed antiviral activity, the ability of the antisera to
neutralize HIV-1 infection was examined. Cell-free HIV-1/III.sub.B
virus at 100 TCID.sub.50 was incubated with serial dilutions of the
antisera before being used to infect MT-2 target cells (Montefiori,
D.C., (1988) J. Clin. Microbio. 26:231-235).
One hundred TCID.sub.50 HIV-1/III.sub.B cell-free virus was
preincubated with serial dilutions of antisera for one hour at
37.degree. C. Following incubation the pretreated virus was then
plated on the 4.times.10.sup.4 of target cell line, MT-2 for one
hour at 37.degree. C., following infection the MT-2 cells were
washed three times and then incubated at 37.degree. C. at 5%
CO.sub.2. Fusion was evaluated three days later quantitatively by
visually counting the number of syncytia per well in triplicate
experiments under a phase contrast microscope.
The results are reported in FIG. 4. FIG. 4A shows the results using
vector-immunized mouse sera compared with FIG. 4B which shows the
results using pM160 immunized sera. Neutralization values (V.sub.n
/V.sub.o) versus the dilution factors (Nara, P., (1989) Techniques
In HIV Research eds. Aldovini, A. & Walkter, B. D., 77-86M
Stockton Press) are illustrated in FIG. 4C. The control serum
(-.times.) was from pMAMneoBlue vector immunized mice. The test
sera (.largecircle.) were from pM160 immunized mice.
Syncytia inhibition was performed as described by Osther, K., et
al., (1991) Hybridoma 10:673-683. The H9/III.sub.B cell line was
pre-incubated with serial dilutions (1:100, 1:200, and 1:400) of
antisera were made in 96 well plates in a total volume of 50 .mu.l
for thirty minutes at 37.degree. C. at 5% CO.sub.2. Fusion was
evaluated three days later quantitatively by visually counting the
number of syncytia per well under a phase construct microscope.
FIG. 4D is the target cells co-cultivated with HIV-1/III.sub.B cell
line treated with preimmune serum. FIG. 4E is the same as FIG. 4D
but treated with vector control immunized serum. FIG. 4F is the
same as FIG. 4D but treated with rgp160 immunized serum. FIG. 4G is
the same as FIG. 4D but treated with pM160 immunized serum. FIGS.
4D to 4G show that inhibition of syncytia was apparent at dilution
at 1:200 in these assays. MT-2 cells were infected with cell-free
HIV-1/III.sub.B which had been preincubated with vector-immunized
antiserum readily formed syncytia (FIG. 4A). In comparison,
preincubation with pM160 immunized mouse serum prevented syncytium
formation (FIG. 4B). The neutralization kinetics were determined by
V.sub.n /V.sub.o versus serial dilutions of antisera (Nara, P.,
(1989) Techniques In HIV Research, eds. Aldovini, A. & Walker,
B. D., 77-86, M Stockton Press) (FIG. 4C). The serum from the pM160
immunized mouse had biologically active neutralizing activity at
dilutions of up to 1:320 while control antisera did not show
similar activity.
To determine if the antiserum from the pM160 immunized mouse could
inhibit envelope-mediated virus spread through direct cell-to-cell
fusion, syncytium inhibition assays were performed. Antiserum from
the pM160 immunized mouse inhibits HIV-1 induced syncytium
formation at 1:200 dilutions (FIG. 4G). In contrast, the preimmune
sera (FIG. 4D), antisera from the rgp160 immunized mice (FIG. 4F)
and antisera from the control vector-immunized animals (FIG. 4E)
failed to inhibit syncytium formation at the same dilutions.
Observations from the neutralization (FIGS. 4A-C) and syncytium
inhibition assays (FIGS D-G) of these sera correlates with the
observed ELISA reactivities (FIG. 3). The antiserum from the pM160
immunized mouse which showed a high level of binding to
neutralizing epitopes likewise demonstrated high level anti-viral
activities; conversely, sera with little binding to these epitopes
including the antiserum from rgp160 immunized mice have low
anti-viral activity.
Low level neutralizing activity has been observed by other groups
when using rgp160 immunization (Lasky, L. A. et al., (1986) Science
233:209-212; and Berman P. W., et al., (1990) Nature 345:622-625.
The reasons for the more effective generation of anti-viral
activities by the genetic immunization than by recombinant protein
immunization are not clear. However, the differences in the
generated immune responses may be due to the introduction of the
gp160 gene directly into the mouse muscle cells and expression of
this gene in vivo which may correctly process the products and lead
to more effective processing of the target antigen.
HIV enters cells binding to the CD4 molecule found predominantly on
human helper T-cells, macrophages, and possibly glial cells
(Maddon, P. J., et al., (1986) Cell 47:333-348; Koenig, S., et al.,
(1986) Science 233:1089-1093; and Cheng-Mayer, C., et al., (1987)
Proc. Natl. Acad. Sci. USA 84:3526-3530). Interruption of this
binding has been shown to prevent HIV infection in vivo (Fisher, R.
A., et al., (1988) Nature 331:76-78; and Sun, H. C., et al., 1989
J. Virol. 63:3579-85).
To test whether the antisera from pM160 immunized mice can inhibit
gp120 binding to CD4-bearing T-cells, a direct inhibition assay
monitored by fluorocytometry was employed (Chen, Y. H., et al.,
(1992) AIDS 6:533-539. It was observed that serum from the pM160
construct-immunized mouse was able to block the binding of gp120 to
CD4-bearing T-cells: a 1:15 dilution of immune serum inhibited
FITC-gp120 binding to CD4.sup.+ SupT1 cells by 22%.+-.2% in
replicate experiments as evaluated by flow cytometry. This
indicates that this region for HIV entry into target cells can also
be functionally inhibited by this antiserum. These data are
consistent with observed ELISA reactivity of the antiserum to the
CD4 binding site peptides (FIG. 3c).
Immunoglobulin isotyping studies were performed by using a
commercial murine monoclonal antibody isotyping kit (Sigma). Of the
anti-gp160 specific antibodies elicited by pM160 immunization, 19%
are IgG1, 51% are IgG2, 16% are IgG3, 10% are IgM and 5% are IgA.
The predominance of IgG isotypes indicates that a secondary immune
response has taken place, and further suggests that helper T-cells
can be elicited by genetic immunization.
To determine whether immunization with the DNA construct can lead
to the generation of anti-DNA antibodies in these experimental
animals, pM160 and pMAMneoBlue DNAs were coated onto microtiter
plates and specific binding was determined by ELISA using sera all
immunized animals. No significant binding to plasmid DNA was
observed. Thus, using genetic material for inoculation into muscle
tissue appears unlikely to produce an anti-plasmid DNA
response.
Introducing construct DNA into mouse muscle by needle injection may
cause inconsistent results, as this technique does not provide a
means to control DNA uptake by muscle cells. Injection of construct
DNA alone (n.apprxeq.4) with bupivacaine pretreated animals
(n.apprxeq.4) was compared. The immune responses observed in the
two groups were dissimilar, with 25% and 75% animals responding in
ELISA assays respectively. Increased efficiency may be achieved by
use of a direct DNA delivery system such as particle bombardment
(Klein, T. M. et al., (1992) Bio/technology 10:286-291.
Evidence of neutralization, syncytia inhibition, inhibition of
CD4-gp120 binding, and specific binding to several important
regions on the gp160 demonstrate that introduction of a DNA
construct encoding HIV gp160 membrane-bound glycoprotein directly
into muscle cells of living animals can elicit specific humoral
responses, and generate biologically relevant anti-viral
antibodies.
To test whether the vaccine is capable of eliciting a protective
immune response, the animal model described above was used. Tumor
cells were transfected with DNA encoding p160, confirmed to express
the protein and implanted into the animal. Controls included
untransfected tumor lines.
Genetically immunized animals were vaccinated with plasmid pm160.
Controls included unvaccinated animals, animals vaccinated with
vector DNA only and animals administered the gp160 protein.
Results demonstrate that the immune response of genetically
vaccinated mice was sufficient to completely eliminate the
transfected tumors while having no effect on untranslated tumors.
gp160 protein vaccination led to some reduction in tumor size in
transfected tumors as compared to untransfected tumors but had no
effect on mortality. Unvaccinated animals showed similar mortality
for both transfected and untransfected tumors.
Example 4
The following is a list of constructs which may be used in the
methods of the present invention. The vector pBabe.puro, which is
used as a starting material to produce many of the below listed
constructs, was originally constructed and reported by Morgenstern,
J. P. and H. Land, 1990 Nucl. Acids Res. 18(12):3587-3596, which is
incorporated herein by reference. The pBabe.puro plasmid is
particularly useful for expression of exogenous genes in mammalian
cells. DNA sequences to be expressed are inserted at cloning sites
under the control of the Moloney murine leukemia virus (Mo MuLV)
long terminal repeat (LTR) promoter. The plasmid contains the
selectable marker for puromycin resistance.
Example 5
Plasmid pBa.V.alpha.3 is a 7.8 kb plasmid that contains a 2.7 kb
EcoRI genomic fragment encoding the T cell receptor Va3 region
containing the L, V and J segments cloned into the EcoRI site of
pBabe.puro. The T cell receptor-derived target protein is useful in
the immunization against and treatment of T cell mediated
autoimmune disease and clonotypic T cell lymphoma and leukemia.
Example 6
Plasmid pBa.gagpol-vpr is a 9.88 kb plasmid that contains the
gag/pol and vif genes from HIV/MN cloned into pBabe.puro. The vpr
gene is deleted. The plasmid which contains these HIV viral genes,
which encode HIV target proteins, is useful in the immunization
against and treatment of HIV infection and AIDS. The HIV DNA
sequence is published in Reiz, M. S., 1992 AIDS Res. Human Retro.
8:1549, which is incorporated herein by reference. The sequence is
accessible from Genbank No.: M17449, which is incorporated herein
by reference.
Example 7
Plasmid pM160 is an 11.0 kb plasmid that contains the 2.3 kb PCR
fragment encoding the HIV-I/3B envelope protein and rev/tat genes
cloned into pMAMneoBlue. The nef region is deleted. The plasmid
which contains these HIV viral genes, which encode HIV target
proteins, is useful in the immunization against and treatment of
HIV infection and AIDS. The DNA sequence of HIV-1/3B is published
in Fisher, A., 1985 Nature 316:262, which is incorporated herein by
reference. The sequence is accessible from Genbank No.: K03455,
which is incorporated herein by reference.
Example 8
Plasmid pBa.VL is a 5.4 kb plasmid that contains PCR fragment
encoding the VL region of an anti-DNA antibody cloned into
pBabe.puro at the XbaI and EcoRI sites. The antibody-derived target
protein is an example of a target protein useful in the
immunization against and treatment of B cell mediated autoimmune
disease and clonotypic B cell lymphoma and leukemia. A general
method for cloning functional variable regions from antibodies can
be found in Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci.
USA 87:1066, which is incorporated herein by reference.
Example 9
Plasmid pOspA.B is a 6.84 kb plasmid which contains the coding
regions encoding the OspA and OspB antigens of the Borrelia
burgdorferi, the spirochete responsible for Lyme's disease cloned
into pBabe.puro at the BamHI and SalI sites. The PCR primers used
to generate the OspA and OspB fragments are
5'-GAAGGATCCATGAAAAAATATTTATTGGG-3' (SEQ ID NO:3) and
5'-ACTGTCGACTTATTTTAAAGCGTTTTTAAG-3' (SEQ ID NO: 4). See: Williams,
W. V., et al. 1992 DNA and Cell. Biol. 11(3):207, which is
incorporated herein by reference. The plasmid which contains these
pathogen genes, which encode target proteins, is useful in the
immunization against Lyme's disease.
Example 10
Plasmid pBa. Rb-G is a 7.10 kb plasmid which contains a PCR
generated fragment encoding the rabies G protein cloned into
pBabe.puro at the BamHI site. The plasmid which contains this
pathogen gene, which encodes the rabies G protein, is useful in the
immunization against Rabies. The DNA sequence is disclosed in
Genebank No.:M32751, which is incorporated herein by reference. See
also: Anilionis, A., et al., 1981 Nature 294:275, which is
incorporated herein by reference.
Example 11
Plasmid pBa. HPV-L1 is a 6.80 kb plasmid which contains a PCR
generated fragment encoding the L1 capsid protein of the human
papillomavirus (HPV) including HPV strains 16, 18, 31 and 33 cloned
into pBabe.puro at the BamHI and EcoRI sites. The plasmid is useful
in the immunization against HPV infection and the cancer caused
thereby. The DNA sequence is disclosed in Genebank No.:M15781,
which is incorporated herein by reference. See also: Howley, P.,
1990 Fields Virology, Volume 2, Eds.: Channock, R. M. et al.
Chapter 58:1625; and Shah, K. and P. Howley, 1990 Fields Virology,
Volume 2, Eds.: Channock, R. M. et al. Chapter 59; both of which
are incorporated herein by reference.
Example 12
Plasmid pBa.HPV-L2 is a 6.80 kb plasmid which contains a PCR
generated fragment encoding the L2 capsid protein of the human
papillomavirus (HPV) including HPV strains 16, 18, 31 and 33 cloned
into pBabe.puro at the BamHI and EcoRI sites. The plasmid is useful
in the immunization against HPV infection and the cancer caused
thereby. The DNA sequence is disclosed in Genebank No.:M15781,
which is incorporated herein by reference. See also: Howley, P.,
1990 Fields Virology, Volume 2, Eds.: Channock, R. M. et al.
Chapter 58:1625; and Shah, K. and P. Howley, 1990 Fields Virology,
Volume 2, Eds.: Channock, R. M. et al. Chapter 59; both of which
are incorporated herein by reference.
Example 13
Plasmid pBa.MNp7 is a 5.24 kb plasmid which contains a PCR
generated fragment encoding the p7 coding region including the HIV
MN ag (core protein) sequence cloned into pBabe.puro at the Bam HI
site. The plasmid which contains these HIV viral genes, which
encode HIV target proteins, is useful in the immunization against
and treatment of HIV infection and AIDS. Reiz, M. S., 1992 AIDS
Res. Human Retro. 8:1549, which is incorporated herein by
reference. The sequence is accessible from Genbank No.:M17449,
which is incorporated herein by reference.
Example 14
Plasmid pGA733-2 is a 6.3 kb plasmid that contains the GA733-2
tumor surface antigen cloned from the colorectal carcinoma cell
line SW948 into pCDM8 vector (Seed, B. and A. Aruffo, 1987 Proc.
Natl. Acad. Sci. USA 84:3365, which is incorporated herein by
reference) at BstXI site. The tumor-associated target protein is an
example of a target protein useful in the immunization against and
treatment of hyperproliferative disease such as cancer. The GA733-2
antigen is a useful. target antigen against colon cancer. The GA733
antigen is reported in Szala, S. et al., 1990 Proc. Natl. Acad.
Sci. USA 87:3542-3546, which is incorporated herein by
reference.
Example 15
Plasmid pT4-pMV7 is a 11.15 kb plasmid that contains cDNA which
encodes human CD4 receptor cloned into pMV7 vector at the EcoRI
site. The CD4 target protein is useful in the immunization against
and treatment of T cell lymphoma. Plasmid pT4-pMV7 is available
from the AIDS Repository, Catalog No. 158.
Example 16
Plasmid pDJGA733 is a 5.1 kb plasmid that contains the GA733 tumor
surface antigen cloned into pBabe.puro at the BamHI site. The
tumor-associated target protein is an example of a target protein
useful in the immunization against and treatment of
hyperproliferative disease such as cancer. The GA733 antigen is a
useful target antigen against colon cancer.
Example 17
Plasmid pBa.RAS is a 6.8 kb plasmid that contains the ras coding
region that was first subcloned from pZIPneoRAS and cloned into
pBabe.puro at the BamHI site. The ras target protein is an example
of a cytoplasmic signalling molecule. The method of cloning ras is
reported in Weinberg 1984 Mol. Cell. Biol. 4:1577, which is
incorporated herein by reference. Ras encoding plasmid are useful
for the immunization against and treatment of hyperproliferative
disease such as cancer; in particular, ras related cancer such as
bladder, muscle, lung, brain and bone cancer.
Example 18
Plasmid pBa.MNp55 is a 6.38 kb plasmid which contains a PCR
generated fragment encoding the p55 coding region including the HIV
MN gag precursor (core protein) sequence cloned into pBabe.puro at
the BamHI site. The plasmid which contains these HIV viral genes,
which encode HIV target proteins, is useful in the immunization
against and treatment of HIV infection and AIDS. Reiz, M. S., 1992
AIDS Res. Human Retro. 8:1549, which is incorporated herein by
reference. The sequence is accessible from Genbank No.:M17449,
which is incorporated herein by reference.
Example 19
Plasmid pBa.MNp24 is a 5.78 kb plasmid which contains a PCR
generated fragment from the pMN-SF1 template encoding the p24
coding region including the whole HIV MN gag coding region cloned
into pBabe.puro at the BamHI and EcoRI sites. The plasmid which
contains these HIV viral genes, which encode HIV target proteins,
is useful in the immunization against and treatment of HIV
infection and AIDS. Reiz, M. S., 1992 AIDS Res. Human Retro.
8:1549, which is incorporated herein by reference. The sequence is
accessible from Genbank No.: M17449, which is incorporated herein
by reference.
Example 20
Plasmid pBa.MNp17 is a 5.5 kb plasmid which contains a PCR
generated fragment encoding the p17 coding region including the HIV
MN gag (core protein) sequence cloned into pBabe.puro at the BamHI
and EcoRI sites. The plasmid which contains these HIV viral genes,
which encode HIV target proteins, is useful in the immunization
against and treatment of HIV infection and AIDS. Reiz, M. S., 1992
AIDS Res. Human Retro. 8:1549, which is incorporated herein by
reference. The sequence is accessible from Genbank No.: M17449,
which is incorporated herein by reference.
Example 21
Plasmid pBa.SIVenv is a 7.8 kb plasmid which contains a 2.71 PCR
generated fragment amplified from a construct containing SIV 239 in
pBR322 cloned into pBabe.puro at the BamHI and EcoRI sites. The
primers used are 5'-GCCAGTTTTGGATCCTTAAAAAAGGCTTGG-3' (SEQ ID NO:5)
and 5'-TTGTGAGGGACAGAATTCCAATCAGGG-3' (SEQ ID NO:6). The plasmid is
available from the AIDS Research and Reference Reagent Program;
Catalog No. 210.
Example 22
Plasmid pcTSP/ATK.env is a 8.92 kb plasmid which contains a PCR
generated fragment encoding the complete HTLV envelope coding
region from HTLV-1/TSP and /ATK isolates subcloned into the
pcDNA1/neo vector. The primers used are
5'-CAGTGATATCCCGGGAGACTCCTC-3' (SEQ ID NO:7) and
5'-GAATAGAAGAACTCCTCTAGAATTC-3' (SEQ ID NO:8). Plasmid
pcTSP/ATK.env is reported in 1988 Proc. Natl. Acad. Sci. USA
85:3599, which is incorporated herein by reference. The HTLV env
target protein is useful in the immunization against and treatment
of infection by HTLV and T cell lymphoma.
Example 23
Plasmid pBa.MNgp160 is a 7.9 kb plasmid which contains a 2.8 kb PCR
generated fragment amplified from a construct containing MNenv in
pSP72 and cloned into pBabe.puro at the BamHI and EcoRI sites. The
primers used are 5'-GCCTTAGGCGGATCCTATGGCAGGAAG-3' (SEQ ID NO:9)
and 5'-TAAGATGGGTGGCCATGGTGAATT-3' (SEQ ID NO:10). Reiz, M. S.,
1992 AIDS Res. Human Retro. 8:1549, which is incorporated herein by
reference. The sequence is accessible from Genbank No.:M17449,
which is incorporated herein by reference. The plasmid which
contains these HIV viral genes, which encode HIV target proteins,
is useful in the immunization against and treatment of HIV
infection and AIDS.
Example 24
Plasmid pC.MNp55 is a 11.8 kb plasmid which contains a 1.4 kb PCR
generated fragment amplified from the gag region of MN isolate and
cloned into the pCEP4 vector. The plasmid which contains these HIV
viral genes, which encode HIV target proteins, is useful in the
immunization against and treatment of HIV infection and AIDS. Reiz,
M. S., 1992 AIDS Res. Human Retro. 8:1549, which is incorporated
herein by reference. The sequence is accessible from Genbank No.:
M17449, which is incorporated herein by reference.
Example 25
Plasmid pC.Neu is a 14.2 kb plasmid that contains a 3.8 kb DNA
fragment containing the human neu oncogene coding region that was
cut out from the LTR-2/erbB-2 construct and subcloned into the
pCEP4 vector. The pC.Neu plasmid is reported in DiFiore 1987
Science 237:178, which is incorporated herein by reference. The neu
oncogene target protein is an example of a growth factor receptor
useful as a target protein for the immunization against and
treatment of hyperproliferative disease such as cancer; in
particular, colon, breast, lung and brain cancer.
Example 26
Plasmid pC.RAS is a 11.7 kb plasmid that contains a 1.4 kb DNA
fragment containing the ras oncogene coding region that was first
subcloned from pZIPneoRAS and subcloned into pCEP4 at the BamHI
site. The pC.RAS plasmid is reported in Weinberg 1984 Mol. Cell.
Biol. 4:1577, which is incorporated herein by reference. The ras
target protein is an example of a cytoplasmic signalling molecule.
Ras encoding plasmid are useful for the immunization against and
treatment of hyperproliferative disease such as cancer; in
particular, ras related cancer such as bladder, muscle, lung, brain
and bone cancer.
Example 27
Plasmid pNLpuro is a 15 kb plasmid which contains HIV gag/pol and
SV40-puro insertion. The plasmid which contains these HIV viral
genes, which encode HIV target proteins, is useful in the
immunization against and treatment of HIV infection and AIDS.
Example 28
A DNA construct was designed to test the effectiveness of a genetic
vaccine against human CD4 in mice. These experiments were designed
to test the ability of a vaccine to protect against a T lymphoma
antigen. In T cell lymphoma, CD4 is a tumor specific antigen.
Accordingly, this model demonstrates the ability of the genetic
vaccine to protect against T lymphoma. Further, these experiments
tested the effectiveness against a member of the immunoglobulin
superfamily of molecules. CD4 is highly conserved between human and
murine species.
The animal model used was described above. Tumor cells were
transfected with DNA encoding CD4, confirmed to express the protein
and implanted into the animal. Controls included untransfected
tumor lines. Although the animals were immunocompetent, an immune
response was not directed against the implanted, CD4-labelled
tumors in unvaccinated animals.
Genetically immunized animals were vaccinated with plasmid
pT4-pMVT, a 11.15 kb plasmid that contains cDNA which encodes human
CD4 receptor cloned into pMV7 vector at the EcoRI site. Plasmid
pT4-pMV7 is available from the AIDS Repository, Catalog No. 158.
Controls included unvaccinated animals and animals administered the
CD4 protein.
In the genetic immunization procedure described herein, the
quadriceps muscles of BALB/c mice were injected with 100 .mu.l of
0.5% bupivacaine-HCl and 0.1% methylparaben in isotonic NaCl using
a 27-gauge needle to stimulate muscle cell regeneration to
facilitate uptake of the genetic construct. Twenty-four hours
later, the same injection sites were then injected with either 100
.mu.g of pT4-pMV7 or with 100 .mu.g of pMV7 as a control plasmid.
The mice were boosted by injecting the same amount of DNA construct
three times at two week intervals in the same manner but without
pre-treatment with bupivacaine-HCl.
Animals received 1,000,000 CD4-labelled tumor cells. In
non-vaccinated animals, large tumors formed and death resulted
after about 7-10 weeks. Vaccinated animals did not develop similar
deadly tumors.
Results demonstrate that the immune response of genetically
vaccinated mice was sufficient to completely eliminate the
transfected tumors while having no effect on untransfected tumors.
CD4 protein vaccination led to some reduction in tumor size in
transfected tumors as compared to untransfected tumors but had no
effect on mortality. Unvaccinated animals showed similar mortality
for both transfected and untransfected tumors.
Example 29
A DNA construct was designed to test the effectiveness of a genetic
vaccine against human GA733 in mice. These experiments were
designed to test the ability of a vaccine to protect against GA733
associated cancer such as colon cancer. The animal model used was
described above. Tumor cells were transfected with DNA encoding
GA733, confirmed to express the protein and implanted into the
animal. Controls included untransfected tumor lines.
Genetically immunized animals were vaccinated with plasmid
pGA733-2, a 6.3 kb plasmid that contains the GA733-2 tumor surface
antigen cloned from the colorectal carcinoma cell line SW948 into
pCDM8 vector at BstXI site following the method described above.
Controls included unvaccinated animals and animals administered the
GA733 protein.
Results demonstrate that the immune response of genetically
vaccinated mice was sufficient to completely eliminate the
transfected tumors while having no effect on untranslated tumors.
GA733 protein vaccination led to some reduction in tumor size in
transfected tumors as compared to untransfected tumors but had no
effect on mortality. Unvaccinated animals showed similar mortality
for both transfected and untransfected tumors.
Example 30
A DNA construct was designed to test the effectiveness of a genetic
vaccine against human p185neu in mice. These experiments were
designed to test the ability of a vaccine to protect against
p185neu associated cancer such as breast, lung and brain cancer.
The animal model used was described above. Tumor cells were
transfected with DNA encoding neu, confirmed to express the protein
and implanted into the animal. Controls included untransfected
tumor lines.
Genetically immunized animals were vaccinated with plasmid
pLTR-2/erbB-2, a 14.3 kb plasmid that contains the human neu
oncogene coding region cloned into the LTR-2 vector at the XhoI
site following the method described above. The 5'LTR and 3'LTR are
from Moloney-MuLV LTR. Controls included unvaccinated animals and
animals administered the p185neu protein.
Results demonstrate that the immune response of genetically
vaccinated mice was sufficient to completely eliminate the
transfected tumors while having no effect on untranslated tumors.
p185 protein vaccination led to some reduction in tumor size in
transfected tumors as compared to untransfected tumors but had no
effect on mortality. Unvaccinated animals showed similar mortality
for both transfected and untransfected tumors.
Example 31
A DNA construct was designed to test the effectiveness of a genetic
vaccine against human Ras in mice. These experiments were designed
to test the ability of a vaccine to protect against Ras associated
cancer such as bladder, muscle, lung, brain and bone cancer. The
animal model used was described above. Tumor cells were transfected
with DNA encoding Ras, confirmed to express the protein and
implanted into the animal. Controls included untransfected tumor
lines.
Genetically immunized animals were vaccinated with plasmid pBa.RAS
is a 6.8 kb plasmid that contains the ras coding region that was
first subcloned from pZIPneoRAS and cloned into pBabe.puro at the
BamHI site following the vaccination method described above. The
ras target protein is an example of a cytoplasmic signalling
molecule. The method of cloning ras is reported in Weinberg 1984
Mol. Cell. Biol. 4:1577, which is incorporated herein by reference.
Controls included unvaccinated animals and animals administered the
Ras protein.
Example 32
A DNA construct was designed to test the effectiveness of a genetic
vaccine against human rabies G protein antigen in mice. The animal
model used was described above. Tumor cells were transfected with
DNA encoding rabies G protein, confirmed to express the protein and
implanted into the animal. Controls included untransfected tumor
lines.
Genetically immunized animals were vaccinated with plasmid pBa.
Rb-G is a 7.10 kb plasmid which contains a PCR generated fragment
encoding the rabies G protein cloned into pBabe.puro at the BamHI
site, following the vaccination method described above. The rabies
G target protein is an example of a pathogen antigen. The DNA
sequence is disclosed in Genebank No.:M32751. Controls included
unvaccinated animals and animals administered the G protein.
Example 33
A DNA construct was designed to test the effectiveness of a genetic
vaccine against Lyme's disease antigen in mice. The animal model
used was described above. Tumor cells were transfected with DNA
encoding OspA and Osp B, confirmed to express the protein and
implanted into the animal. Controls included untransfected tumor
lines.
Genetically immunized animals were vaccinated with plasmid pOspA.B
is a 6.84 kb plasmid which contains the coding regions encoding the
OspA and Osp. B antigens of the Borrelia burgdorferi, the
spirochete responsible for Lyme's disease cloned into pBabe.puro at
the BamHI and SalI sites, following the vaccination method
described above. The OspA and OspB target proteins are examples of
pathogen antigens. The PCR primers used to generate the OspA and
OspB fragments are 5'-GAAGGATCCATGAAAAAATATTTATTGGG-3' (SEQ ID
NO:3) and 5'-ACTGTCGACTTATTTTAAAGCGTTTTTAAG-3' (SEQ ID NO: 4). See:
Williams, W. V., et al. 1992 DNA and Cell. Biol. 11(3):207, which
is incorporated herein by reference. Controls included unvaccinated
animals and animals administered OspA and OspB proteins.
Example 34
A DNA construct was designed to test the effectiveness of a genetic
vaccine against a human T cell receptor variable region in mice.
These experiments were designed to test the ability of a vaccine to
protect against a T cell receptor derived protein associated cancer
such as T cell lymphoma and T cell mediated autoimmune disease. The
animal model used was described above. Tumor cells were transfected
with DNA encoding Ras, confirmed to express the protein and
implanted into the animal. Controls included untransfected tumor
lines.
Genetically immunized animals were vaccinated with plasmid
pBa.V.alpha.3 is a 7.8 kb plasmid that contains a 2.7 kb EcoRI
genomic fragment encoding the T cell receptor V.alpha.3 region
containing the L, V and J segments cloned into the EcoRI site of
pBabe.puro following the vaccination method described above.
Example 35
The plasmid pM160 can be used as a starting material for several
plasmids useful to express one or more genes from the env portion
of HIV. As described above, the DNA segment encoding the envelope
gene of HIV-1 HXB2 was cloned by the polymerase chain reaction
(PCR) amplification technique utilizing the lambda cloned DNA
obtained from the AIDS Research and Reference Reagent Program. The
sequences of the 5' and 3' primers are
5'-AGGCGTCTCGAGACAGAGGAGAGCAAGAAATG-3' (SEQ ID NO:1) with
incorporation of XhoI site and 5'-TTTCCCTCTAGATAAGCCATCCAATCACAC-3'
(SEQ ID NO: 2) with incorporation of XbaI site, respectively, which
encompass gp160, tat and rev coding region. The nef gene is absent.
Gene specific amplification was performed using Taq DNA polymerase
according to the manufacturer's instructions (Perkin-Elmer Cetus
Corp.). The PCR reaction products were treated with 0.5 ug/ml
proteinase K at 37.degree. C. for thirty minutes followed by a
phenol/chloroform extraction and ethanol precipitation. Recovered
DNA was then digested with Xhol and Xbal for two hours at
37.degree. C. and subjected to agarose gel electrophoresis. The
isolated and purified Xhol-Xbal PCR fragment was cloned into
Bluescript plasmid (Stratagene Inc., La Jolla, Calif.) and then
subcloned into the eukaryotic expression vector pMAMneoBlue
(Clontech Laboratories, Inc., Palo Alto, Calif.). The resulting
construct was designated as pM160. The plasmid DNA was purified
with CsCl gradient ultracentrifugation. The restriction enzyme map
for pMAMneoBlue plasmid is available from the manufacturer and may
be used by those having ordinary skill in the art to engineer, that
is to change, delete and add various elements using standard
molecular biology techniques and widely available starting
material.
The promoter controlling gp160/rev/tat gene expression is MMTV LTR.
The promoter may be deleted and replaced with Actin promoter,
myosin promoter, HIV LTR promoter and CMV promoter.
The gene conferring ampicillin resistance may be deleted or
otherwise inactivated. The gene conferring neomycin resistance may
be placed under the control of a bacterial promoter.
The Rous sarcoma virus enhancer may be deleted from the plasmid.
The RSV enhancer may be replaced with the muscle creatine
enhancer.
The gp160/rev/tat genes overlap and share the same nucleotide
sequences in different reading frames. The rev gene may be deleted
by changing its initiation codon to a different codon. Similarly,
the tat gene may be eliminated by the same means. In each plasmid
except those using the HIV LTR promoter to control gp160/rev/tat,
either rev, tat, or both rev and tat may be eliminated. In plasmids
using the HIV LTR promoter, tat must be present.
The following Table lists pM160-modified plasmids. Each plasmid has
an inactivated ampicillin gene. Each has deleted the RSV enhancer.
Some have no enhancer (no); some have creatine muscle enhancer
(CME). Some have the HIV rev gene (yes) while it is deleted in
others (no). Some have the HIV tat gene (yes) while it is deleted
in others (no).
______________________________________ Construct Promoter enhancer
rev tat ______________________________________ RA-1 Actin no yes
yes RA-2 Actin no yes no RA-3 Actin no no yes RA-4 Actin CME yes
yes RA-5 Actin CME yes no RA-6 Actin CME no yes RA-7 CMV no yes yes
RA-8 CMV no yes no RA-9 CMV no no yes RA-10 CMV CME yes yes RA-11
CMV CME yes no RA-12 CMV CME no yes RA-13 MMTV no yes yes RA-14
MMTV no yes no RA-15 MMTV no no yes RA-16 MMTV CME yes yes RA-17
MMTV CME yes no RA-18 MMTV CME no yes RA-19 Myosin no yes yes RA-20
Myosin no yes no RA-21 Myosin no no yes RA-22 Myosin CME yes yes
RA-23 Myosin CME yes no RA-24 Myosin CME no yes RA-25 HIV-1 LTR no
yes yes RA-26 HIV-1 LTR no no yes RA-27 HIV-1 LTR CME yes yes RA-28
HIV-1 LTR CME no yes ______________________________________
Constructions RA-29 to RA-56 are identical to RA-1 to RA-32
respectively except in each case the promoter controlling the
neomycin gene is a bacterial promoter.
Example 36
The plasmid pNLpuro may be used as a starting material to produce
several different plasmids which express the HIV gag/pol genes. As
described above, pNLpuro was constructed for expression of gag pol.
The HIV-1 genomic clone pNL43 was obtained through the NIH AIDS
Research and Reference Reagent Program (ARRRP), Division of AIDS,
NIAID, NIH, from Dr. Malcom Martin. The pNL43 clone is a construct
that consists of HIV-1 proviral DNA plus 3 kb of host (i.e. human)
sequence from the site of integration (5' and 3' of the HIV
sequence) cloned into pUC18. The StuI site within the non-HIV 5'
flanking human DNA of pNL43 was destroyed by partial digestion with
StuI followed by digestion of the free ends with E. coli polymerase
1. The linear plasmid was filled and then self ligated, leaving a
unique StuI site within the HIV genome. This plasmid, pNLDstu, was
then digested with the blunting enzymes StuI and BsaBI which
eliminated a large section of the coding sequence for gp120. The
SV40 promoter and puromycin resistance coding region (puromycin
acetyl transferase (PAC))were isolated from pBABE-puro (Morgenstern
and Land, 1990 Nucl. Acids Res. 18(12):3587-3596, which is
incorporated herein by reference, kindly provided by Dr. Hartmut
Land of the Imperial Cancer Research Fund) using EcoRI and ClaI.
This fragment was blunted, then cloned into the StuI/BsaBI-digested
pNLDstu. A clone was selected with the SV40-puro fragment in the
correct orientation so that the 3' LTR of HIV could provide poly A
functions for the PAC message. This plasmid was designated
pNLpuro.
The vpr regulatory gene is deleted from the HIV gag pol vector in
order to eliminate a necessary regulatory protein from the set of
genes to be introduced by vaccination. A region from just upstream
of the unique PflMI site to just after the vif termination codon
was amplified via PCR using primers that introduced a
non-conservative amino acid change (glu.fwdarw.val) at amino acid
22 of vpr, a stop codon in the vpr reading frame immediately after
amino acid 22, and an EcoRI site immediately following the new stop
codon. This PCR fragment was substituted for the PflMI-EcoR I
fragment of pNLpuro or pNL43. This substitution resulted in the
deletion of 122 nucleotides of the open reading frame of vpr, thus
eliminating the possibility of reversion that a point mutation
strategy entails. The resulting plasmids, pNLpuro.DELTA.vpr, encode
the first 21 natural amino acids of vpr plus a valine plus all
other remaining HIV-1 genes and splice junctions in their native
form. Such deletion strategy would also be applicable to nef, vif,
and vpu and allow for structural gene expression but protect from
the generation of a live recombinant virus.
In addition to vpr, other changes may be made by those having
ordinary skill in the art to plasmid pNL43puro using standard
molecular biology techniques and widely available starting
material.
The human flanking sequences 5' and 3' of the HIV sequences can be
removed by several methods. For example, using PCR, only HIV,
SV40-puro, and pUC18 sequences can be amplified and
reconstructed.
The psi region of HIV, which is important in the packaging of the
virus, can be deleted from pNL43puro-based plasmids. In order to
delete the psi region, the pNLpuro plasmid is cut with SacI and
SpeI. This digestion removes the psi region as well as the 5' LTR
which is upstream and portion of the gag/pol region which is
downstream of psi. In order to reinsert the deleted non-psi
sequences, PCR amplification is performed to regenerate those
sequences. Primers are designed which regenerate the portions of
the HIV sequence 5' and 3' to psi without regenerating psi. The
primers reform the SacI site at the portion of the plasmid 5' of
the 5' LTR. Primers go downstream from a site upstream of the SacI
site to a site just 3' of the 5' end of the psi region, generating
an AatI site at the 3' end. Primers starting just 5' of the psi
region also generate an AatI site and, starting 3' of the SpeI
site, regenerate that site. The PCR generated fragments are
digested with SacI, AatI and SpeI and ligated together with the
SacI/SpeI digested pHLpuro-psi-fragment. The HIV 5'LTR promoter can
be deleted and replaced with Moloney virus promoter, MMTV LTR,
Actin promoter, myosin promoter and CMV promoter.
The HIV 3'LTR polyadenylation site can be deleted and replaced with
SV40 polyadenylation site.
The gene conferring ampicillin resistance may be deleted or
otherwise inactivated.
The following is a list of pNLpuro-based constructions in which HIV
psi and vpr regions are deleted and human flanking regions 5' and
3' of the HIV sequences are deleted.
______________________________________ Construct Promoter poly(A)
Amp.sup.r ______________________________________ LA-1 Moloney HIV
3' LTR yes LA-2 Moloney SV40 yes LA-3 Moloney HIV 3' LTR no LA-4
Moloney SV40 no LA-5 CMV HIV 3' LTR yes LA-6 CMV SV40 yes LA-7 CMV
HIV 3' LTR no LA-8 CMV SV40 no LA-9 MMTV HIV 3' LTR yes LA-10 MMTV
SV40 yes LA-11 MMTV HIV 3' LTR no LA-12 MMTV SV40 no LA-13 HIV 5'
LTR HIV 3' LTR yes LA-14 HIV 5' LTR SV40 yes LA-15 HIV 5' LTR HIV
3' LTR no LA-16 HIV 5' LTR SV40 no
______________________________________
Constructions LA-17 to LA-32 are identical to LA-1 to LA-16
respectively except in each case at least one of the human flanking
sequence remains.
Example 37
In another construction for expressing the env gene, that region of
HIV may be inserted into the commercially available plasmid pCEP4
(Invitrogen). The pCEP4 plasmid is particularly useful since it
contains the Epstein Barr virus origin of replication and nuclear
antigen EBNA-1 coding region which produces high copy episomal
replication without integration. pCEP4 also contains the hygromycin
marker under the regulatory control of the thymidine kinase
promoter and polyadenylation site. The HIV env coding region is
placed under the regulatory control of the CMV promoter and SV40
polyadenylation site. The HIV env coding region was obtained as a
2.3 kb PCR fragment form HIV/3B, Genebank sequence K03455. The
resulting pCEP4-based plasmid, pRA-100, is maintained
extrachromosomally and produces gp160 protein.
Example 38
In another construction for expressing the env gene, that region of
HIV may be inserted into the commercially available plasmid pREP4
(Invitrogen). The pREP4 plasmid is particularly useful since it
contains the Epstein Barr virus origin of replication and nuclear
antigen EBNA-1 coding region which produces high copy episomal
replication without integration. pREP4 also contains the hygromycin
marker under the regulatory control of the thymidine kinase
promoter and polyadenylation site. The HIV env coding region is
placed under the regulatory control of the RSV promoter and SV40
polyadenylation site. The HIV env coding region was obtained as a
2.3 kb PeR fragment form HIV/3B, Genebank sequence K03455. The
resulting pCEP4-based plasmid, pRA-101, is maintained
extrachromosomally and produces gp160 protein.
Example 39
In another construction for expressing the gag/pol genes, that
region of HIV may be inserted into the commercially available
plasmid pCEP4 (Invitrogen). The pCEP4 plasmid is particularly
useful since it contains the Epstein Barr virus origin of
replication and nuclear antigen EBNA-1 coding region which produces
high copy episomal replication without integration. pCEP4 also
contains the hygromycin marker under the regulatory control of the
thymidine kinase promoter and polyadenylation site. The HIV gag/pol
coding region is placed under the regulatory control of the CMV
promoter and SV40 polyadenylation site. The HIV gag/pol coding
region was obtained from HIV MN, Genebank sequence MI7449, and
includes the vif gene. The vpr gene is not included. The resulting
pCEP4-based plasmid, pLA-100, is maintained extrachromosomally and
produces GAG55, reverse transcriptase, protease and integrase
proteins.
Example 40
In another construction for expressing the gag/pol genes, that
region of HIV may be inserted into the commercially available
plasmid pREP4 (Invitrogen). The pREP4 plasmid is particularly
useful since it contains the Epstein Barr virus origin of
replication and nuclear antigen EBNA-1 coding region which produces
high copy episomal replication without integration. pREP4 also
contains the hygromycin marker under the regulatory control of the
thymidine kinase promoter and polyadenylation site. The HIV gag/pol
coding region is placed under the regulatory control of the CMV
promoter and SV40 polyadenylation site. The HIV gag/pol coding
region was obtained from HIV MN, Genebank sequence MI7449, and
includes the vif gene. The vpr gene is not included. The resulting
pREP4-based plasmid, pLA-101, is maintained extrachromosomally and
produces GAG55, reverse transcriptase, protease and integrase
proteins.
Example 41
The following construction, referred to herein as pGAGPOL.rev, is
useful to express HIV gag/pol genes.
The plasmid includes a Kanamycin resistance gene and a pBR322
origin of DNA replication. The sequences provided for transcription
regulation include: a cytomegalovirus promoter; a Rous sarcoma
virus enhancer; and an SV40 polyadenylation signal. The HIV-1
sequences included in pGAGPOL.rev include a sequence that encodes
gag; a sequence that encodes pol; a sequence that encodes reverse
transcriptase which contains a small deletion; a sequence that
encodes the inactive amino terminus of Int; and a sequence that
encodes rev. Each of the HIV sequences are derived from HIV-1
strain HXB2.
Several safety features are included in pGAGPOL.rev. These include
use of the CMV promoter and a non-retroviral poly(A) site.
Furthermore, deletion of the .psi. sequence limits the ability to
package viral RNA. In addition, multiple mutations of the reverse
transcriptase yield an enzymatically inactive product. Moreover, a
large deletion of integrase yields an inactive product and a
Kanamycin resistance marker is used for stabilizing bacterial
transformants.
Plasmid pGAGPOL.rev is constructed as follows.
Step 1. A subclone of part of the HIV-1 (HXB2) genome that is
cloned into Bluescript (Stratagene) is used. The subclone of HIV-1
contains the complete 5'LTR and the rest of the HIV-1 genome to
nucleotide 5795 (Genebank numbering). The HIV-1 sequences are
obtained from the HXB2D plasmid (AIDS Repository).
Step 2. PCR part of gag from the open reading frame HXB2D plasmid
(AIDS Repository). Cut PCR fragment with NotI and SpeI and ligate
with HIV-1 subclone described above restricted with NotI and
SpeI.
Step 3. PCR gag/pol junction and part of pol-encoding sequences
from the HXB2D plasmid (AIDS Repository) with primers SEQ ID NO.:17
and SEQ ID NO.:18. Cut PCR product with ClaI and ligate together.
Cut ligated fragments with BcII and SalI and ligate with plasmid
from Step 2 digested with BcII and SalI.
Step 4. Cut plasmid from Step 3 with BspMI and EcoRI and religate
with adapters formed by annealing linkers SEQ ID NO.:19 and SEQ ID
NO.:20.
Step 5. Cut plasmid from Step 4 with NotI and SalI and ligate with
plasmid from either 4a or 4b in description written for pENV
(below). Cut also with NotI and SalI.
Step 6. Restrict plasmid from Step 5 with SalI and MluI and ligate
with PCR product obtained by PCR of rev with primers SEQ ID NO.:21
and SEQ ID NO.:22.
Step 7. Cut plasmid from Step 6 with NotI and ligate with product
obtained by PCR of the rev responsive element in the HXB2D plasmid
(AIDS Repository) with primers SEQ ID NO.:23 and SEQ ID NO.:24.
Steps 6 and 7 are optional.
Example 42
The following construction, referred to herein as pENV, is useful
to express HIV env genes.
The plasmid includes a Kanamycin resistance gene and a pBR322
origin of DNA replication. The sequences provided for transcription
regulation include: a cytomegalovirus promoter; a Rous sarcoma
virus enhancer; and an SV40 polyadenylation signal. The HIV-1
sequences included in pENV include a sequence that encodes vpu; a
sequence that encodes rev; a sequence that encodes gp160; a
sequence that encodes 50% of nef; a sequence that encodes vif; and,
a sequence that encodes vpr with a 13 amino acid carboxy-end
deletion. The vpu, rev, gp160 and nef sequences are derived from
HIV-1 strain MN. The vif and vpr sequences are derived from HIV-1
strain HXB2.
Several safety features are included in pGAGPOL.rev. These include
use of the CMV promoter and a non-retroviral poly(A) site.
Furthermore, tat has been deleted and a 50% deletion of nef yields
an "inactive" nef product. In addition, vif and vpr are placed out
of normal sequence and a partial deletion of vpr further ensures an
inactive vpr product.
Plasmid pENV is constructed as follows.
Step 1. Start with pUC18 digested with HindIII and EcoRI. The
resulting fragment that contains the ColE1 origin of replication
and the laci gene should be ligated with the EcoRI/HindIII fragment
from pMAMneoBlue that contains the our sarcoma virus enhancer. The
resulting plasmid or pMAMneo-Blue from Clontech (Palo Alto, Calif.)
can then be digested with HindIII and BgII. Using standard
techniques, ligate with fragment containing kn gene obtained by PCR
of geneblock plasmid (Pharmacia).
Step 2. If pMAMneo-Blue used as starting plasmid, digest with MluI
and EcoRI, fill in the ends with Klenow fragment of Polymerase I
and religate.
Step 3. Them, with either pMAMneo-Blue or pUC18-derived plasmid,
digest with HindIII and ligate with the SV40 polyA site and early
splicing region obtained by PCR of pCEP4 (Invitrogen, San Diego
Calif.) with primers SEQ ID NO.:25 and SEQ ID N0.:26.
Step 4a. Digest with BamHI and ligate with the CMV promoter
obtained by PCR of pCEP4 (Invitrogen, San Diego Calif.) with
primers SEQ ID NO.:27 and SEQ ID NO.:28.
Step 4b. Digest with BamHI and ligate with the MoMLV LTR obtained
by PCR with primers SEQ ID NO.:29 and SEQ ID NO.:30.
Step 5. Digest with NotI and MluI and ligate with GP160 coding
region obtained by PCR of pMN-ST1 with primers SEQ ID NO.:31 and
SEQ ID NO.:32.
Step 6. Digest with MluI and ligate with sequences that encode vif
in its entirety and vpr with a 13aa carboxy-end deletion by CPR of
HXB2D plasmid (AIDS Repository) with primers SEQ ID NO.:33 and SEQ
ID NO.:34.
Example 43
An immunization system is provided which comprises:
a pharmaceutical composition comprising about 100 .mu.g of pGAGPOL.
rev in an isotonic, pharmaceutically acceptable solution; and,
a pharmaceutical preparation comprising 100 .mu.g of pENVin an
isotonic, pharmaceutically acceptable solution. In addition, the
immunization system preferably comprises a pharmaceutical
composition comprising about 1 ml of 0.5% bupivacaine-HCl and 0.1%
methylparaben in an isotonic pharmaceutical carrier.
In such a preferred immunization system, a first set of
administrations is performed in which bupivacaine and one of the
two pharmaceutical compositions are administered intramuscularly to
an individual, preferably into a muscle of an arm or buttock.
Bupivacaine and the other of the two pharmaceutical compositions
are administered intramuscularly to the individual at a different
site, preferably remote from the site of the administration of the
one pharmaceutical composition, preferably into a muscle of the
other arm or buttock. Subsequence sets of administrations may be
performed later in time, preferably 48 hours to two weeks or more
later.
The immunization system may be used to vaccinate an individual in
order to protect that individual from HIV infection or to treat an
HIV infected individual with an immunotherapeutic.
Example 44
In some embodiments, the present invention relates to a method of
immunizing an individual against HIV by administering a single
inoculant. This inoculant includes a genetic construct that
comprises at least one, preferably two, more preferably more than
two or a plurality of the genes of the HIV virus or all of the
structural genes. However, the inoculant does not contain a
complete complement of all HIV genes. If a single cell is provided
with a complete complement of viral genes, it is possible that a
complete infectious virus can be assembled within the cell.
Accordingly, a genetic construct according to the present invention
is not provided with such a full complement of genes. As a safety
precaution, one or more essential genes can be deleted or
intentionally altered to further ensure that an infectious viral
particle cannot be formed.
In some embodiments of the present invention, at least portions of
one, two or all HIV structural genes are provided. The structural
genes of HIV consist of gag, pol and env. Portions of at least one
of these three genes are provided on a genetic construct.
Accordingly, in some embodiments, at least a portion of each of gag
and pol are provided on a genetic construct; in some embodiments,
at least a portion of env is provided on a genetic construct; in
some embodiments, at least a portion of gag is provided on a
genetic construct; in some embodiments at least a portion of each
of pol and env are provided on a genetic construct; in some
embodiments, at least a portion of each of gag and env are provided
on a genetic construct; in some embodiments at least a portion of
pol is provided on a genetic construct. Optionally, the entire gene
is provided. Optionally, in any of these constructs, HIV regulatory
genes may also be present. The HIV regulatory genes are: vpr, vif,
vpu, nef, tat and rev.
Example 45
As used herein, the term "expression unit" is meant to refer to a
nucleic acid sequence which comprises a promoter operably linked to
a coding sequence operably linked to a polyadenylation signal. The
coding sequence may encode one or more proteins or fragments
thereof. In preferred embodiments, a expression unit is within a
plasmid.
As used herein, the term "HIV expression unit" is meant to refer to
a nucleic acid sequence which comprises a promoter operably linked
to a coding sequence operably linked to a polyadenylation signal in
which the coding sequence encodes a peptide that comprises an
epitope that is identical or substantially similar to an epitope
found on an HIV protein. "Substantially similar epitope" is meant
to refer to an epitope that has a structure which is not identical
to an epitope of an HIV protein but nonetheless invokes an cellular
or humoral immune response which cross reacts to an HIV protein. In
preferred embodiments, the HIV expression unit comprises a coding
sequence which encodes one or more HIV proteins or fragments
thereof. In preferred embodiments, an HIV expression unit is within
a plasmid.
In some embodiments of the present invention, a single genetic
construct is provided that has a single HIV expression unit which
contains DNA sequences that encode one or more HIV proteins or
fragments thereof. As used herein, the term "single HIV expression
unit construct" is meant to refer to a single genetic construct
that contains a single HIV expression unit. In preferred
embodiments, a single HIV expression unit construct is in the form
of a plasmid.
In some embodiments of the present invention, a single genetic
construct is provided that has more than one HIV expression units
in which each contain DNA sequences that encode one or more HIV
proteins or fragments thereof. As used herein, the term "multiple
HIV expression unit genetic construct" is meant to refer to a
single plasmid that contains more than one HIV expression units. In
preferred embodiments, a multiple HIV expression unit construct is
in the form of a plasmid.
In some embodiments of the present invention, a single genetic
construct is provided that has two HIV expression units in which
each contain DNA sequences that encode one or more HIV proteins or
fragments thereof. As used herein, the term "two HIV expression
unit genetic construct" is meant to refer to a single plasmid that
contains two HIV expression units, i.e a multiple HIV expression
unit genetic construct that contains two HIV expression unit
genetic expression units. In a two HIV expression unit genetic
construct, it is preferred that one HIV expression unit operates in
the opposite direction of the other HIV expression unit. In
preferred embodiments, a two HIV expression unit construct is in
the form of a plasmid.
In some embodiments of the present invention, an HIV genetic
vaccine is provided which contains a single genetic construct. The
single genetic construct may be a single HIV expression unit
genetic construct, a two HIV expression unit genetic construct or a
multiple HIV expression unit genetic construct which contains more
than two HIV expression units.
In some embodiments of the present invention, an HIV genetic
vaccine is provided which contains more than one genetic construct
in a single inoculant.
In some embodiments of the present invention, an HIV genetic
vaccine is provided which contains more than one genetic construct
in more than one inoculant. As used herein, the term "multiple
inoculant" is meant to refer to a genetic vaccine which comprises
more than one genetic construct, each of which is administered
separately. In some embodiments of the present invention, an HIV
genetic vaccine is provided which contains two genetic constructs.
Each genetic construct may be, independently, a single HIV
expression unit genetic construct, a two HIV expression unit
genetic construct or a multiple HIV expression unit genetic
construct which contains more than two HIV expression units. In
some embodiments, both genetic constructs are single HIV expression
unit genetic constructs. In some embodiments, both genetic
constructs are two HIV expression unit genetic constructs. In some
embodiments, both genetic constructs are multiple HIV expression
unit genetic constructs. In some embodiments, one genetic construct
is a single HIV expression unit genetic construct and the other is
a two HIV expression unit genetic construct. One having ordinary
skill in the art can readily recognize and appreciate the many
variations depending upon the number of genetic constructs used in
a genetic vaccine and the number of HIV expression units that may
be present on each genetic construct.
It is preferred that the genetic constructs of the present
invention do not contain certain HIV sequences, particularly, those
which play a role in the HIV genome integrating into the
chromosomal material of the cell into which it is introduced. It is
preferred that the genetic constructs of the present invention do
not contain LTRs from HIV. Similarly, it is preferred that the
genetic constructs of the present invention do not contain a psi
site from HIV. Further, it is preferred that the reverse
transcriptase gene is deleted and the integrase gene is deleted.
Deletions include deletion of only some of the codons or replacing
some of the codons in order to essentially delete the gene. For
example, the initiation codon may be deleted or changed or shifted
out of frame to result in a nucleotide sequence that encodes an
incomplete and non-functioning.
It is also preferred that the genetic constructs of the present
invention do not contain a transcribable tat gene from HIV. The tat
gene, which overlaps the rev gene may be completely deleted by
substituting the codons that encode rev with other codons that
encode the same amino acid for rev but which does not encode the
required tat amino acid in the reading frame in which tat is
encoded. Alternatively, only some of the codons are switched to
either change, i.e. essentially delete, the initiation codon for
tat and/or change, i.e. essentially delete, sufficient codons to
result in a nucleotide sequence that encodes an incomplete and
non-functioning tat.
It is preferred that a genetic construct comprises coding sequences
that encode peptides which have at least an epitope identical to or
substantially similar to an epitope from HIV gag, pol, env or rev
proteins. It is more preferred that a genetic construct comprises
coding sequences that encode at least one of HIV gag, pol, env or
rev proteins or fragments thereof. It is preferred that a genetic
construct comprises coding sequences that encode peptides which
have more than one epitopes identical to or substantially similar
to an epitope from HIV gag, pol, env or rev proteins. It is more
preferred that a genetic construct comprises coding sequences that
encode more than one of HIV gag, pol, env or rev proteins or
fragments thereof.
In some embodiments, a genetic construct comprises coding sequences
that encode peptides which have at least an epitope identical to or
substantially similar to an epitope from HIV vif, vpr, vpu or nef
proteins. In some embodiments, a genetic construct comprises coding
sequences that encode at least one of HIV vif, vpr, vpu or nef
proteins or fragments thereof.
A single HIV expression unit genetic construct may comprise coding
regions for one or more peptides which share at least one epitope
with an HIV protein or fragment thereof in a single expression unit
under the regulatory control of single promoter and polyadenylation
signal. It is preferred that genetic constructs encode more than
one HIV protein or fragment thereof. The promoter may be any
promoter functional in a human cell. It is preferred that the
promoter is an SV40 promoter or a CMV promoter, preferably a CMV
immediate early promoter. The polyadenylation signal may be any
polyadenylation signal functional in a human cell. It is preferred
that the polyadenylation signal is an SV40 polyadenylation signal,
preferably the SV40 minor polyadenylation signal. If more than one
coding region is provided in a single expression unit, they may be
immediately adjacent to each other or. separated by non-coding
regions. In order to be properly expressed, a coding region must
have an initiation codon and a termination codon.
A two HIV expression unit genetic construct may comprise coding
regions for one or more peptides which share at least one epitope
with an HIV protein or fragment thereof on each of the two
expression units. Each expression unit is under the regulatory
control of single promoter and polyadenylation signal. In some
embodiments, it is preferred that genetic constructs encode more
than one HIV protein or fragment thereof. In some embodiments, it
is preferred that nucleotide sequences encoding gag and pol are
present on one expression unit and nucleotide sequences encoding
env and rev are present on the other. The promoter may be any
promoter functional in a human cell. It is preferred that the
promoter is an SV40 promoter or a CMV promoter, preferably a
immediate early CMV promoter. The polyadenylation signal may be any
polyadenylation signal functional in a human cell. It is preferred
that the polyadenylation signal is an SV40 polyadenylation signal,
preferably the SV40 minor polyadenylation signal. If more than one
coding region is provided in a expression unit, they may be
immediately adjacent to each other or separated by non-coding
regions. In order to be properly expressed, a coding region must
have an initiation codon and a termination codon.
According to some embodiments of the present invention, the MHC
Class II crossreactive epitope in env is deleted and replaced with
the analogous region from HIV II.
When a genetic construct contains gag and/or pol, it is generally
important that rev is also present. In addition to rev, a rev
response element may be provided with gag and pol for increased
expression of those genes.
When genetic constructs are produced that it is preferred that the
env gene used in plasmid 1 is derived from MN or MN-like isolates
including clinical isolates resembling MN, preferably non-syncytial
inducing clinical isolates, preferably those that are macrophage
tropic from early stage clinical isolates.
Multiple proteins may be produced from a single expression unit by
alternative splicing. Splicing signals are provided tp allow
alternative splicing which produces different messages encoding
different proteins.
Example 46
FIG. 8 shows four backbones, A, B, C and D. FIG. 9 shows 4 inserts,
1, 2, 3 and 4. Insert 1 supports expression of gag and pol; the rev
response element was cloned in a manner to conserve the HIV splice
acceptor. Insert 2 is similar to insert 1 as it too supports
expression of gag and pol except the rev reponse element was cloned
without conserving the HIV splice acceptor Insert 3 supports
expression of gag and pol, includes a deletion of the integrase
gene and does not include the presence of the cis acting rev
response element. Insert 4 supports expression of rev, vpu and env.
The env may have the MHC class II cross reactive epitope altered to
eliminate crossreactivity and the V3 loop may be alterered to
eliminate the possibility of syncytia formation.
In some embodiments, backbone A is used with insert 1. Such
constructs optionally contain the SV40 origin of replication.
Plasmid pA1ori+ is backbone A with insert 1 and the SV40 origin of
replication. Plasmid pA1ori- is backbone A with insert 1 without
the SV40 origin of replication. Additionally, either pA1ori+ or
pA1ori- may include integrase yielding pA1ori+int+ and pA1ori-int+,
respectively. Plasmids pA1ori+, pA1ori-, pA1ori+int+ and
pA1ori-int+ may be further modified by functionally deleing the
reverse transcriptase (RT) gene yielding pA1ori+RT-, pA1ori-RT-,
pA1ori+int+RT- and pA1ori-int+RT-, respectively.
In some embodiments, backbone A is used with insert 2. Such
constructs optionally the SV40 origin of replication. Plasmid
pA2ori+ is backbone A with insert 2 and the SV40 origin of
replication. Plasmid pA2ori- is backbone A with insert 1 without
the SV40 origin of replication. Additionally, either pA2ori+ or
pA2ori- may include integrase yeilding pA2ori+int+ and pA2ori-int+,
respectively. Plasmids pA2ori+, pA2ori-, pA2ori+int+ and
pA2ori-int+ may be further modified by functionally deleing the
reverse transcriptase (RT) gene yielding pA2ori+RT-, pA2ori-RT-,
pA2ori+int+RT- and pA2ori-int+RT-, respectively.
In some embodiments, backbone B is used with insert 1. Such
constructs optionally the SV40 origin of replication. Plasmid
pB1ori+ is backbone B with insert 1 and the SV40 origin of
replication. Plasmid pB1ori- is backbone B with insert 1 without
the SV40 origin of replication. Additionally, either pB1ori+ or
pB1ori- may include integrase yeilding pB1ori+int+ and pB1ori-int+,
respectively. Plasmids pB1ori+, pB1ori-, pB1ori+int+ and
pB1ori-int+ may be further modified by functionally deleting the
reverse transcriptase (RT) gene yielding pB1ori+RT-, pB1ori-RT-,
pB1ori+int+RT- and pB1ori-int+RT-, respectively.
In some embodiments, backbone B is used with insert 2. Such
constructs optionally the SV40 origin of replication. Plasmid
pB2ori+ is backbone B with insert 2 and the SV40 origin of
replication. Plasmid pB2ori- is backbone B with insert 1 without
the SV40 origin of replication. Additionally, either pB2ori+ or
pB2ori- may include integrase yeilding pB2ori+int+ and pB2ori-int+,
respectively. Plasmids pB2ori+, pB2ori-, pB2ori+int+ and
pB2ori-int+ may be further modified by functionally deleing the
reverse transcriptase (RT) gene yielding pB2ori+RT-, pB2ori-RT-,
pB2ori+int+RT- and pB2ori-int+RT-, respectively.
In some embodiments, backbone A minus rev is used with insert 3.
Such constructs optionally the SV40 origin of replication. Plasmid
pA/r-3ori+ is backbone A with insert 2 and the SV40 origin of
replication. Plasmid pA/r-3ori- is backbone A minus rev with insert
3 without the SV40 origin of replication. Additionally, either
pA/r-3ori+ or pA/r-3ori- may include integrase yeilding
pA/r-3ori+int+ and pA/r-3ori-int+, respectively. Plasmids
pA/r-3ori+, pA/r-3ori-, pA/r-3ori+int+ and pA/r-3ori-int+ may be
further modified by functionally deleing the reverse transcriptase
(RT) gene yielding pA/r-3ori+RT-, pA/r-3ori-RT-, pA/r-3ori+int+RT-
and pA/r-3ori-int+RT-, respectively.
In some embodiments, backbone C is used with insert 1. Such
constructs optionally the SV40 origin of replication. Plasmid
pC1ori+ is backbone C with insert 1 and the SV40 origin of
replication. Plasmid pC1ori- is backbone C with insert 1 without
the SV40 origin of replication. Additionally, either pC1ori+ or
pC1ori- may include integrase yeilding pC1ori+int+ and pC1ori-int+,
respectively. Plasmids pC1ori+, pC1ori-, pC1ori+int+ and
pC1ori-int+ may be further modified by functionally deleing the
reverse transcriptase (RT) gene yielding pC1ori+RT-, pC1ori-RT-,
pC1ori+int+RT- and pC1ori-int+RT-, respectively.
In some embodiments, backbone C is used with insert 2. Such
constructs optionally the SV40 origin of replication. Plasmid
pC2ori+ is backbone C with insert 2 and the SV40 origin of
replication. Plasmid pC2ori- is backbone C with insert 2 without
the SV40 origin of replication. Additionally, either pC2ori+ or
pC2ori- may include integrase yeilding pC2ori+int+ and pC2ori-int+,
respectively. Plasmids pC2ori+, pC2ori-, pC2ori+int+ and
pC2ori-int+ may be further modified by functionally deleing the
reverse transcriptase (RT) gene yielding pC2ori+RT-, pC2ori-RT-,
pC2ori+int+RT- and pC2ori-int+RT-, respectively.
In some embodiments, backbone C is used with insert 3. Such
constructs optionally the SV40 origin of replication. Plasmid
pC3ori+ is backbone C with insert 3 and the SV40 origin of
replication. Plasmid pC3ori- is backbone C with insert 3 without
the SV40 origin of replication. Additionally, either pC3ori+ or
pC3ori- may include integrase yeilding pC3ori+int+ and pC3ori-int+,
respectively. Plasmids pC3ori+, pC3ori-, pC3ori+int+ and
pC3ori-int+ may be further modified by functionally deleing the
reverse transcriptase (RT) gene yielding pC3ori+RT-, pC3ori-RT-,
pC3ori+int+RT- and pC3ori-int+RT-, respectively.
In some embodiments, backbone D is used with insert 4. Such
constructs optionally the SV40 origin of replication. Plasmid
pD4ori+ is backbone D-with insert 4 and the SV40 origin of
replication. Plasmid pD4ori- is backbone D with insert 4 without
the SV40 origin of replication.
Example 47
In some embodiments, a single expression unit/single inoculant
genetic vaccine is provided which comprises a genetic construct
that includes a coding sequence which encodes a peptide that has at
least one epitope which is an identical to or substantially similar
to epitopes of HIV proteins. The coding sequence is under the
regulatory control of the CMV immediate early promoter and the SV40
minor polyadenylation signal.
In some embodiments, a single expression unit/single inoculant
genetic vaccine is provided which comprises a genetic construct
that includes a coding sequence which encodes at least one HIV
protein or a fragment thereof. The coding sequence is under the
regulatory control of the CMV immediate early promoter and the SV40
minor polyadenylation signal. The HIV protein is selected from the
group consisting of gag, pol, env and rev. In some embodiments it
is preferred that the genetic vaccine is provided which comprises a
genetic construct that includes a coding sequence which encodes at
least two HIV proteins or a fragments thereof selected from the
group consisting of gag, pol, env and rev or fragments thereof. In
some embodiments, it is preferred that the genetic vaccine is
provided which comprises a genetic construct that includes a coding
sequence which encodes at least three HIV proteins or a fragments
thereof selected from the group consisting of gag, pol, env and rev
or fragments thereof. In some embodiments, it is preferred that the
genetic vaccine is provided which comprises a genetic construct
that includes a coding sequence which encodes gag, pol, env and rev
or fragments thereof.
In some embodiments, a dual expression unit/single inoculant
genetic vaccine is provided which comprises a genetic construct
that includes two expression units each of which comprises a coding
sequence which encodes a peptide that has at least one epitope
which is an identical to or substantially similar to epitopes of
HIV proteins. The coding sequence is under the regulatory control
of the CMV immediate early promoter and the SV40 minor
polyadenylation signal. The two expression units are encoded in
opposite directions of each other.
In some embodiments, a dual expression unit/single inoculant
genetic vaccine is provided which comprises a genetic construct
that includes two expression units each of which comprises a coding
sequence which encodes at least one HIV protein or a fragment
thereof. Each expression unit comprises a coding sequence that is
under the regulatory control of the CMV immediate early promoter
and the SV40 minor polyadenylation signal. The HIV protein is
selected from the group consisting of gag, pol, env and rev. In
some embodiments it is preferred that the genetic vaccine is
provided which comprises a genetic construct that includes two
expression units, at least one of which comprises a coding which
encodes at least two HIV proteins or a fragments thereof selected
from the group consisting of gag, pol, env and rev or fragments
thereof and the other comprises at least one HIV proteins or a
fragments thereof selected from the group consisting of gag, pol,
env and rev or fragments thereof. In some embodiments, it is
preferred that the genetic vaccine is provided which comprises a
genetic construct that includes two expression units, at least one
of which comprises a coding sequence which encodes at least three
HIV proteins or a fragments thereof selected from the group
consisting of gag, pol, env and rev or fragments thereof and the
other comprises at least one HIV proteins or a fragments thereof
selected from the group consisting of gag, pol, env and rev or
fragments thereof. In some embodiments, it is preferred that the
genetic vaccine is provided which comprises a genetic construct
that comprises two expression units and includes a coding sequence
which encodes gag, pol, env and rev or fragments thereof.
Table 1
Picornavirus Family
Genera:
Rhinoviruses: (Medical) responsible for .about.50% cases of the
common cold.
Ehteroviruses: (Medical) includes polioviruses, coxsackieviruses,
echoviruses, and human enteroviruses such as hepatitis A virus.
Apthoviruses: (Veterinary) these are the foot and mouth disease
viruses.
Target antigens: VP1, VP2, VP3, VP4, VPG
Calcivirus Family
Genera:
Norwalk Group of Viruses: (Medical) these viruses are an important
causative agent of epidemic gastroenteritis.
Togavirus Family
Genera:
Alphaviruses: (Medical and Veterinary) examples include Senilis
viruses, RossRiver virus and Eastern & Western Equine***
encephalitis.
Reovirus: (Medical) Rubella virus.
Flariviridue Family
Examples include: (Medical) dengue, yellow fever, Japanese
encephalitis, St. Louis encephalitis and tick borne encephalitis
viruses.
Hepatitis C Virus: (Medical) these viruses are not placed in a
family yet but are believed to be either a togavirus or a
flavivirus. Most similarity is with togavirus family.
Coronavirus
Family: (Medical and Veterinary) Infectious bronchitis virus
(poultry) Porcine transmissible gastroenteric virus (pig)
Porcine hemagglutinatiny encephalomyelitis virus (pig)
Feline infectious peritonitis virus (cats)
Feline enteric coronavirus (cat)
Canine coronavirus (dog)
The human respiratory coronaviruses cause .about.40 cases of common
cold. EX. 224E, 0C43 Note--coronaviruses may cause non-A, B or C
hepatitis
Target antigens:
E1--also called M or matrix protein
E2--also called S or Spike protein
E3--also called HE or hemagglutinelterose glycoprotein (not present
in all coronaviruses)
N--nucleocapsid
Rhabdovirus Family
Genera: Vesiliovirus
Lyssavirus: (medical and veterinary) rabies
Target antigen:G protein N protein
Filoviridue Family: (Medical)
Hemorrhagic fever viruses such as Marburg and Ebola virus
Paramyxovirus Family:
Genera:
Paramyxovirus: (Medical and Veterinary)
Mumps virus, New Castle disease virus (important pathogen in
chickens)
Morbillivirus: (Medical and Veterinary) Measles, canine
distemper
Pneuminvirus: (Medical and Veterinary)
Respiratory syncytial virus
Orthomyxovirus Family (Medical)
The Influenza virus
Bungavirus Family
Genera:
Bungavirus: (Medical) California encephalitis, LA Crosse
Phlebovirus: (Medical) Rift Valley Fever
Hantavirus: Puremala is a hemahagin fever virus
Nairvirus (Veterinary) Nairobi sheep disease
Also many unassigned bungaviruses
Arenavirus Family (Medical)
LCM, Lassa fever virus
Reovirus Family
Genera:
Reovirus: a possible human pathogen
Rotavirus: acute gastroenteritis in children
Orbiviruses: (Medical and Veterinary) Colorado Tick fever, Lebombo
(humans) equine encephalosis, blue tongue
Retrovirus Family
Sub-Family:
Oncorivirinal: (Veterinary) (Medical) feline leukemia virus, HTLVI
and HTLVII
Lentivirinal: (Medical and Veterinary) HIV, feline immunodeficiency
virus, equine infections, anemia virus Spumavirinal
Papovavirus Family
Sub-Family:
Polyomaviruses: (Medical) BKU and JCU viruses
Sub-Family:
Papillomavirus: (Medical) many viral types associated with cancers
or malignant progression of papilloma
Adenovirus (Medical)
EX AD7, ARD., O.B.--cause respiratory disease--some adenoviruses
such as 275 cause enteritis
Parvovirus Family (Veterinary)
Feline parvovirus: causes feline enteritis
Feline panleucopeniavirus
Canine parvovirus
Porcine parvovirus
Herpesvirus Family
Sub-Family:
alphaherpesviridue
Genera:
Simplexvirus (Medical) HSVI, HSVII Varicellovirus:
(Medical--Veterinary) pseudorabies--varicella zoster
Sub-Family--betaherpesviridue
Genera:
Cytomegalovirus (Medical)
HCMV
Muromegalovirus
Sub-Family:
Gammaherpesviridue
Genera:
Lymphocryptovirus (Medical)
EBV--(Burkitts lympho)
Rhadinovirus
Poxvirus Family
Sub-Family:
Chordopoxviridue (Medical--Veterinary)
Genera:
Variola (Smallpox)
Vaccinia (Cowpox)
Parapoxivirus--Veterinary
Auipoxvirus--Veterinary
Capripoxvirus
Leporipoxvirus
Suipoxvirus
Sub-Family:
Entemopoxviridue
Hepadnavirus Family
Hepatitis B virus
Unclassified
Hepatitis delta virus
Table 2
Bacterial pathogens
Pathogenic gram-positive cocci include:
pneumococcal; staphylococcal; and streptococcal.
Pathogenic gram-negative cocci include:
meningococcal; and gonococcal.
Pathogenic enteric gram-negative bacilli include:
enterobacteriaceae; pseudomonas, acinetobacteria and eikenella;
melioidosis; salmonella; shigellosis; hemophilus; chancroid;
brucellosis; tularemia; yersinia (pasteurella); streptobacillus
moniliformis and spirillum ; listeria monocytogenes; erysipelothrix
rhusiopathiae; diphtheria; cholera; anthrax; donovanosis (granuloma
inguinale); and bartonellosis.
Pathogenic anaerobic bacteria include: tetanus; botulism; other
clostridia; tuberculosis; leprosy; and other mycobacteria.
Pathogenic spirochetal diseases include: syphilis; treponematoses:
yaws, pinta and endemic syphilis; and leptospirosis. Other
infections caused by higher pathogen bacteria and pathogenic fungi
include: actinomycosis; nocardiosis; cryptococcosis, blastomycosis,
histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis,
and mucormycosis; sporotrichosis; paracoccidiodomycosis,
petriellidiosis, torulopsosis, mycetoma and chromomycosis; and
dermatophytosis.
Rickettsial infections include rickettsial and rickettsioses.
Examples of mycoplasma and chlamydial infections include:
mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and
perinatal chlamydial infections.
Pathogenic eukaryotes
Pathogenic protozoans and helminths and infections thereby include:
amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis;
pneumocystis carinii; babesiosis; giardiasis; trichinosis;
filariasis; schistosomiasis; nematodes; trematodes or flukes; and
cestode (tapeworm) infections.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 34 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AGGCGTCTCGAGACAGAGGAGAGCAAGAAATG32 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TTTCCCTCTAGATAAGCCATCCAATCACAC30 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAAGGATCCATGAAAAAATATTTATTGGG29 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ACTGTCGACTTATTTTAAAGCGTTTTTAAG30 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCCAGTTTTGGATCCTTAAAAAAGGCTTGG30 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TTGTGAGGGACAGAATTCCAATCAGGG27 (2) INFORMATION FOR SEQ ID NO:7: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CAGTGATATCCCGGGAGACTCCTC24 (2) INFORMATION FOR SEQ ID NO:8: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GAATAGAAGAACTCCTCTAGAATTC25 (2) INFORMATION FOR SEQ ID NO:9: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCCTTAGGCGGATCCTATGGCAGGAAG27 (2) INFORMATION FOR SEQ ID NO:10: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
TAAGATGGGTGGCCATGGTGAATT24 (2) INFORMATION FOR SEQ ID NO:11: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AGGCGTCTCGAGACAGAGGAGAGCAAGAAATG32 (2) INFORMATION FOR SEQ ID
NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TTTCCCTCTAGATAAGCCATCCAATCACAC30 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:13:
CysAsnThrArgLysArgIleArgIleGlnArgGlyProGlyArgAla 151015
PheValThrIleGlyLys 20 (2) INFORMATION FOR SEQ ID NO:14: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:14:
TyrAsnLysArgLysArgIleHisIleGlnArgGlyProGlyArgAla 151015
PheTyrThrThrLysAsnIleIleCys 2025 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:15:
CysArgIleLysGlnPheIleAsnMetTrpGlnGluValGlyLysAla 151015
MetThrAlaProProIleSerGlyIleArgCys 2025 (2) INFORMATION FOR SEQ ID
NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ArgIleLeuAlaValGluArgTyrIleLysAspGlnGlnLeuLeuGlyIle 151015
TrpGlyCysSerGlyLysLeuIleCys 2025 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TTGTTTAACTTTTGATCGATCCATTCC27 (2) INFORMATION FOR SEQ ID NO:18: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GATTTGTATCGATGATCTGAC21 (2) INFORMATION FOR SEQ ID NO:19: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
TGTAGTAGCAAAAGAAATAGTTAAG25 (2) INFORMATION FOR SEQ ID NO:20: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AATTCTTAACTATTTCTTTTGCTAC25 (2) INFORMATION FOR SEQ ID NO:21: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
ATTTGTCGACTGGTTTCAGCCTGCCATGGCAGGAAGAAGC40 (2) INFORMATION FOR SEQ
ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:22: ACGACGCGTATTCTTTAGCTCCTGACTCC29 (2) INFORMATION FOR SEQ ID
NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GCTGACGGTAGCGGCCGCACAATT24 (2) INFORMATION FOR SEQ ID NO:24: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GTATTAAGCGGCCGCAATTGTT22 (2) INFORMATION FOR SEQ ID NO:25: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 78 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
AAAAAGCTTCGCGGATCCGCGTTGCGGCCGCAACCGGTCACCGGCGACGCGTCGGTCGAc60
CGGTCATGGCTGGGCCCC78 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CCCAAGCTTAGACATGATAAGATACATTG29 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
CTAGCAGCTGGATCCCAGCTTC22 (2) INFORMATION FOR SEQ ID NO:28: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
GGATTTCTGGGGATCCAAGCTAGT24 (2) INFORMATION FOR SEQ ID NO:29: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
TATAGGATCCGCGCAATGAAAGACCCCACCT31 (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
ATATGGATCCGCAATGAAAGACCCCCGCTGA31 (2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
TAAAGCGGCCGCTCCTATGGCAGGAAGACG30 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
ATTACGCGTCTTATGCTTCTAGCCAGGCACAATG34 (2) INFORMATION FOR SEQ ID
NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
ATTACGCGTTTATTACAGAATGGAAAACAGATGGCAGGTG40 (2) INFORMATION FOR SEQ
ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:34: ATTACGCGTTATTGCAGAATTCTTATTATGGC32
__________________________________________________________________________
* * * * *