U.S. patent application number 11/717766 was filed with the patent office on 2007-10-11 for dna-vaccines based on constructs derived from the genomes of human and animal pathogens.
This patent application is currently assigned to POWDER JECT VACCINES, INC.. Invention is credited to Ralph P. Braun, Lendon G. Payne, Lee K. Roberts, William F. Swain.
Application Number | 20070237789 11/717766 |
Document ID | / |
Family ID | 37886040 |
Filed Date | 2007-10-11 |
United States Patent
Application |
20070237789 |
Kind Code |
A1 |
Swain; William F. ; et
al. |
October 11, 2007 |
DNA-vaccines based on constructs derived from the genomes of human
and animal pathogens
Abstract
Methods of eliciting an immune response in a subject by
administering one or more large genomic DNA fragments are provided.
Also provided are methods of identifying sequences encoding
antigenic polypeptides. Also provided are vaccine compositions
comprising one or more large genomic DNA fragments.
Inventors: |
Swain; William F.; (Madison,
WI) ; Roberts; Lee K.; (Madison, WI) ; Payne;
Lendon G.; (Madison, WI) ; Braun; Ralph P.;
(Madison, WI) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
POWDER JECT VACCINES, INC.
|
Family ID: |
37886040 |
Appl. No.: |
11/717766 |
Filed: |
March 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09705149 |
Nov 1, 2000 |
7196066 |
|
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11717766 |
Mar 14, 2007 |
|
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60163297 |
Nov 3, 1999 |
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Current U.S.
Class: |
424/230.1 ;
424/204.1; 424/229.1; 424/231.1 |
Current CPC
Class: |
A61K 39/12 20130101;
A61K 2039/545 20130101; C12N 2710/16634 20130101; A61K 39/245
20130101; A61P 31/22 20180101; A61K 2039/53 20130101 |
Class at
Publication: |
424/230.1 ;
424/204.1; 424/229.1; 424/231.1 |
International
Class: |
A61K 39/245 20060101
A61K039/245; A61K 39/25 20060101 A61K039/25; A61P 31/22 20060101
A61P031/22 |
Claims
1. A vaccine, comprising a construct that comprises one or more
pathogenic DNA genomic fragments, wherein (a) the collective size
of the fragment(s) is about 5-50 Kb and (b) at least one fragment
comprises at least one antigen coding sequence.
2. The vaccine of claim 1, wherein the pathogenic DNA fragments are
from a virus pathogen.
3. The vaccine of claim 2, wherein the virus is a herpes virus.
4. The vaccine of claim 3, wherein the herpes virus is selected
from the group consisting of a Herpes Simplex Virus (HSV),
Varicella Zoster Virus (VZV), Epstein Barr Virus (EBV) and a
Cytomegalovirus (CMV).
5. The vaccine of claim 4, wherein the HSV virus is herpes simplex
virus-2 (HSV-2).
6. The vaccine of claim 4, wherein the virus is a Herpes Simplex
Virus and the HSV genomic DNA fragment(s) express HSV antigens
consisting essentially of those encoded by the HSV immediate early
genes ICP 27, ICP 0, ICP 4 and ICP 22.
7. The vaccine of claim 6, wherein (i) the HSV genomic DNA
fragment(s) are from HSV-2 and (ii) correspond to the sequence
extending from the 7th to 10th EcoR1 sites shown in FIG. 1.
8. The vaccine of claim 1, wherein the vaccine comprises multiple
different constructs.
9. The vaccine of claim 8, wherein the constructs are coated onto
core carriers.
10. The vaccine of claim 9, wherein the core carriers have an
average diameter of about 0.5 to about 5 .mu.m and a density
sufficient to allow delivery into a subject.
11. The vaccine of claim 10, wherein the core carrier is made of
metal.
12. The vaccine of claim 11, wherein the metal is gold.
13. The vaccine of claim 1, wherein the construct is either (A) (i)
a plasmid and (ii) the collective size of the fragment(s) is about
5-25 Kb, or (B) (i) a cosmid and (ii) the collective size of the
fragment(s) is about 25-50 Kb.
14. The vaccine of claim 1, wherein the antigen coding sequence(s)
are not expressed by heterologous promoters.
15. A method for eliciting an immune response in a vertebrate
subject, comprising administering to a vertebrate subject the
vaccine of claim 1, whereby expression of the antigen coding
sequence(s) of the construct elicits an immune response against the
expressed antigen.
16. The method of claim 15, wherein (i) the constructs of the
vaccine are coated onto core carriers and (ii) the vaccine is
administered to the vertebrate subject using a particle-mediated
transdermal delivery technique.
17. The method of claim 15, further comprising repeating the
administration to provide a prime and a boost.
18. The method of claim 15, wherein the vaccine is in a formulation
that comprises an adjuvant
19. The method of claim 15 further comprising administering an
adjuvant.
20. A method of eliciting an immune response in an individual,
comprising administering constructs to an individual, wherein the
constructs comprise pathogenic genomic fragments that (i) comprise
an array of antigen-encoding sequence, but which (ii) do not
comprise sequences that encode immunodominant antigens or genes
whose products inhibit immune responses.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to U.S. provisional application
Ser. No. 60/163,297, filed 3 Nov. 1999, from which priority is
claimed pursuant to 35 U.S.C. .sctn.119(e)(1) and which application
is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to vaccine
compositions and methods of use thereof. More particularly, the
invention pertains to eliciting an immune response in a subject by
administering one or more large genomic DNA fragments to the
subject.
BACKGROUND
[0003] Vaccines which induce a cell-mediated immune response are
emerging as important strategies in combating parasites, autoimmune
disorders, allergic diseases and cancers. Conventional vaccination
strategies generally involve administration of either "live" or
"dead" vaccines. Ertl et al. (1996) J. Immunol. 156:3579-3582. The
so-called live vaccines include attenuated microbes and recombinant
molecules based on a living vector. The dead vaccines include those
based on killed whole pathogens, and subunit vaccines, e.g.,
soluble pathogen subunits or protein subunits. Live vaccines are
generally successful in providing an effective immune response in
immunized subjects; however, such vaccines can be dangerous in
immunocompromised or pregnant subjects, can revert to pathogenic
organisms, or can be contaminated with other pathogens. Hassett et
al. (1996) Trends in Microbiol. 8:307-312. Dead vaccines avoid the
safety problems associated with live vaccines; however such
vaccines often fail to provide an appropriate and/or effective
immune response in immunized subjects.
[0004] More recently, direct injection of plasmid DNA by
intramuscular (Wolff et al. (1990) Science 247:1465:1468) or
intradermal injection with a needle and syringe (Raz et al. (1994)
PNAS USA 91:9519-9523) has been described. Another approach
referred to as ballistic or particle-mediated DNA delivery employs
a needless particle delivery device to administer DNA-coated
microscopic gold beads directly into the cells of the epidermis.
(Yang et al. (1990) PNAS USA 87:9568-9572). Thus, a number of
delivery techniques can be used to deliver nucleic acids for
immunizations, including particle-mediated techniques which deliver
nucleic acid-coated microparticles into target tissue (see, e.g.,
co-owned U.S. Pat. No. 5,865,796, issued Feb. 2, 1999).
Particle-mediated nucleic acid immunization techniques have been
shown to elicit both humoral and cytotoxic T lymphocyte immune
responses following epidermal delivery of nanogram quantities of
DNA. Pertmer et al. (1995) Vaccine 13:1427-1430. Such
particle-mediated delivery techniques have been compared to other
types of nucleic acid inoculation, and found markedly superior.
Fynan et al. (1995) Int. J. Immunopharmacology 17:79-83, Fynan et
al. (1993) Proc. Natl. Acad. Sci. USA 90:11478-11482, and Raz et
al. (1994) Proc. Natl. Acad. Sci. USA 91:9519-9523.
[0005] A novel transdermal drug delivery system that entails the
use of a needleless syringe to deliver solid drug-containing
particles in controlled doses into and through intact skin has also
been described. In particular, commonly owned U.S. Pat. No.
5,630,796 to Bellhouse et al., describes a particle delivery device
(e.g., a needleless syringe) that delivers pharmaceutical particles
entrained in a supersonic gas flow. The particle delivery device is
used for transdermal delivery of powdered drug compounds and
compositions, for delivery of genetic material into living cells
(e.g., gene therapy) and for the delivery of biopharmaceuticals to
skin, muscle, blood or lymph. The device can also be used in
conjunction with surgery to deliver drugs and biologics to organ
surfaces, solid tumors and/or to surgical cavities (e.g., tumor
beds or cavities after tumor resection). Pharmaceutical agents that
can be suitably prepared in a substantially solid, particulate form
can be safely and easily delivered using such a device.
[0006] One particular particle delivery device generally comprises
an elongate tubular nozzle having a rupturable membrane initially
closing the passage through the nozzle and arranged substantially
adjacent to the upstream end of the nozzle. Particles of a
therapeutic agent to be delivered are disposed adjacent to the
rupturable membrane and are delivered using an energizing means
which applies a gaseous pressure to the upstream side of the
membrane sufficient to burst the membrane and produce a supersonic
gas flow (containing the pharmaceutical particles) through the
nozzle for delivery from the downstream end thereof. The particles
can thus be delivered from the needleless syringe at delivery
velocities of between Mach 1 and Mach 8 which are readily
obtainable upon the bursting of the rupturable membrane.
[0007] Another particle delivery device configuration generally
includes the same elements as described above, except that instead
of having the pharmaceutical particles entrained within a
supersonic gas flow, the downstream end of the nozzle is provided
with a bistable diaphragm which is moveable between a resting
"inverted" position (in which the diaphragm presents a concavity on
the downstream face to contain the pharmaceutical particles) and an
active "everted" position (in which the diaphragm is outwardly
convex on the downstream face as a result of a supersonic shockwave
having been applied to the upstream face of the diaphragm). In this
manner, the pharmaceutical particles contained within the concavity
of the diaphragm are expelled at a high initial velocity from the
device for transdermal delivery thereof to a targeted skin or
mucosal surface.
[0008] Transdermal delivery using the above-described device
configurations is generally carried out with particles having an
approximate size that generally ranges between 0.1 and 250 .mu.m.
Particles larger than about 250 .mu.m can also be delivered from
the device, with the upper limitation being the point at which the
size of the particles would cause untoward damage to the skin
cells. The actual distance which the delivered particles will
penetrate depends upon particle size (e.g., the nominal particle
diameter assuming a roughly spherical particle geometry), particle
density, the initial velocity at which the particle impacts the
skin surface, and the density and kinematic viscosity of the skin.
Target particle densities for use in needleless particle injection
generally range between about 0.1 and 25 g/cm.sup.3, and injection
velocities generally range between about 150 and 3,000 m/sec.
[0009] The level of effective protection achieved with DNA-vaccines
is similar to that elicited by traditional protein subunit vaccines
and killed or attenuated viral vaccines; but is traditionally less
than that observed in convalescent animals following recovery from
a natural infection. Manickan et al. (1997) Critical Review
Immunol. 17:139-154. Herpes infections are extremely prevalent and
are caused by two viruses, herpes simplex virus type 1 (HSV-1) and
herpes simplex virus type 2 (HSV-2). HSV-1 is usually acquired in
childhood and is the predominant cause of oral infections. HSV-2
infections are usually associated with sexually transmitted genital
infections. However, up to 25% of genital herpes is caused by
HSV-1. Natural infection appears to impart cross-protection between
the two HSV strains, in that, individuals infected with one strain
(e.g., HSV-1) have a low incidence of infection with the other
strain (i.e., HSV-2) despite exposure. Mertz et al. (1992) Ann.
Intern. Med. 116:197-202. The reasons for these observations have
not been fully elucidated. Although near complete protection
against infection with herpes simplex virus (HSV) can be achieved
in mice and/or guinea pigs by vaccination with killed or modified
virus, the degree of protection in convalescent animals sets the
target for improvement of vaccine performance.
[0010] HSV is a double-stranded DNA virus having a genome of about
150-160 kbp. HSV-1 and HSV-2 genomes are colinear and share greater
than 50% homology over the entire genome. For some genes, the amino
acid identity between the two virus types is as much as 80 to 90%.
As a result of this similarity, many HSV-specific antibodies are
cross-reactive for both virus types.
[0011] The viral genome is packaged within an icosahedral
nucleocapsid which is enveloped in a membrane. The membrane (or
envelope) includes at least 10 virus-encoded glycoproteins, the
most abundant of which are gB, gC, gD, and gE. The viral
glycoproteins are involved in the processes of virus attachment to
cellular receptors and in fusion of the viral and host cell
membranes to permit virus entry into the cell. The glycoproteins
are located on the surface of the virion. Consequently, they are
targets of neutralizing antibody and antibody dependent cell
cytotoxicity (ADCC). Within a virus type, there is a limited (1 to
2%) strain-to-strain sequence variability of the glycoprotein
genes. The viral genome also encodes over 70 other proteins,
including virion proteins, such as VP16 and VP22 which are
associated with the virion tegument, located between the capsid and
the envelope. In addition, a group of approximately five ICPs are
encoded by the virus. (See, e.g., WO/9516779 regarding
ICP4-containing vaccines). These early proteins are synthesized
early in the viral replication cycle, in contrast to the envelope
glycoproteins which are only made late in the life cycle of the
virus.
[0012] For a review of the molecular structure and organization of
HSV, see, for example, Roizman and Sears (1996) "Herpes simplex
viruses and their replication" in Fields Virology, 3rd ed., Fields
et al. eds., Lippincott-Raven Publishers, Philadelphia, Pa.
[0013] One approach to HSV vaccine development has been the use of
isolated glycoproteins which have been shown to be both protective
and therapeutic. See, e.g., Burke et al., Virology (1991)
181:793-797; Burke et al., Rev. Infect. Dis. (1991) 13(Suppl
11):S906-S911; Straus et al., Lancet (1994) 343:1460-1463; Ho et
al., J. Virol. (1989) 63:2951-2958; Stanberry et al., J. Infect.
Dis. (1988) 157:156-163; and Stanberry et al., (1987) J. Infect.
Dis. 155:914-920; Stanberry, L. R. "Subunit Viral Vaccines:
prophylactic and therapeutic use." In: Aurelian L (ed.)
Herpesviruses, the Immune Systems and Aids. Kluwer, Boston, pp.
309-341. However, clinical trials have failed to demonstrate
significant protective immunity in humans vaccinated with a subunit
vaccine consisting of the gB and gD glycoproteins.
[0014] One method of identifying sequences encoding immunogenic
epitopes involves the use of expression libraries, and is known as
expression library immunization (ELI). International Publication WO
96/31613 reports that introducing cloned expression libraries from
cDNA or fragmented genomic DNA into a subject induces an immune
response which can be quantified to select libraries including
sequences with immunogenic epitopes. Selected pools are then
identified and further characterized. The genomic fragments used
are relatively small, between about 10 and 100 base pairs in
length. Moreover, in the ELI method, genomic fragments are spliced
into a vector having an exogenous (e.g., heterologous) promoter.
The genomic clones of ELI remain unidentified sequences and,
accordingly, any antigenic sequences must be extensively purified
and characterized before a specific vaccine can be developed.
[0015] Despite these reports, there remains a need for methods of
eliciting an immune response that more closely mimics an animal's
natural response to antigens. The present invention provides a
solution to this and other problems.
SUMMARY OF THE INVENTION
[0016] The present invention provides methods and compositions for
eliciting immune responses in a subject. In one aspect, the
invention includes a method for eliciting an immune response in a
vertebrate subject comprising administering constructs carrying
genomic DNA fragments obtained or derived from one or more
pathogens. Once the genomic DNA fragments have been administered to
the subject, antigen encoded by coding sequences present in the
genomic DNA fragments is expressed at an amount sufficient to
elicit an immune response. The genomic fragments are preferably
greater than 5 kilobases (kb) in size. In certain embodiments, the
construct is a plasmid carrying genomic DNA fragments between about
5 kb and 25 kb size. In yet other embodiments the construct is a
cosmid carrying genomic DNA fragments between about 25 kb and about
50 kb in size. In certain embodiments, expression of coding
sequences (i.e, antigen coding sequences) contained within the
genomic DNA fragments is not driven by a heterologous promoter. The
pathogens may be, for example, one or more types of bacteria or one
or more types of viruses (e.g., a herpes simplex virus or "HSV").
The constructs (e.g., plasmids or cosmids) may be administered, for
example, by transdermal administration.
[0017] In another aspect, the invention includes a method for
eliciting an immune response in a vertebrate subject. The method
includes the steps of: (a) providing a core carrier coated with
constructs carrying genomic DNA fragments obtained or derived from
one or more pathogens, wherein the genomic DNA fragments contain an
antigen coding sequence; and (b) administering the coated core
carrier to the subject using a particle-mediated transdermal
delivery technique, whereby antigen encoded by a coding sequence
present in the genomic DNA fragments is expressed in the subject in
an amount sufficient to elicit an immune response. The genomic DNA
fragments are greater than about 5 kilobases in size. In certain
embodiments, the construct is a plasmid and carries genomic DNA
fragments ranging from about 5 to about 25 kilobases in size. In
other embodiments, the construct is a cosmid and carries genomic
DNA fragments from about 25 to about 50 kilobases in size. In
certain embodiments, expression of coding sequences contained
within the genomic DNA fragments of the construct (e.g., plasmid or
cosmid) is not driven by a heterologous promoter. For any of the
plasmid or cosmid constructs described herein, the core carrier
typically has an average diameter of about 0.5 to about 5 .mu.m and
a density sufficient to allow transdermal delivery into the
subject. The core carrier can be comprised of a metal, for example,
gold. The pathogens can be, for example, one or more types of
bacteria, one or more viruses, or can be derived from two or more
different pathogens. In other embodiments, the methods involve
repeating step (b) to provide a prime and a booster
administration.
[0018] In another aspect of the invention, a method is provided for
identifying a sequence encoding an antigenic polypeptide. The
method entails the steps of: (a) administering constructs carrying
genomic DNA fragments obtained or derived from one or more
pathogens, wherein the genomic DNA fragments contain or are
suspected of containing a coding sequence for the antigenic
polypeptide and, upon delivery to the subject, the antigenic
polypeptide is expressed from the coding sequence in an amount
sufficient to elicit an immune response; and (b) identifying the
sequence on the construct encoding the antigenic polypeptide. In
one embodiment, step (b) comprises administering one or more
fragments of the constructs of step (a) and identifying which
fragment encodes the antigenic polypeptide. In another embodiment,
step (b) comprises sequencing the construct.
[0019] In yet another aspect, the invention includes a vaccine
composition comprising one or more constructs carrying genomic DNA
fragments obtained or derived from one or more pathogens, for
example, a vaccine composition comprising one or more constructs
(e.g., clone #68) carrying genomic DNA fragments from herpes
simplex virus-2 (HSV-2). In certain embodiments, the construct is a
plasmid carrying genomic DNA fragments ranging from about 5 kb to
25 kb in size. In yet other embodiments the construct is a cosmid
carrying genomic DNA fragments ranging from about 25 kb to about 50
kb in size. In any of the methods or compositions described herein,
the genomic fragments can include the immediate early regions
(e.g., ICP27, ICP0, ICP4, ICP22, etc.) of a virus such as HSV-2,
for example the region spanning from approximately nucleotide
114589 to 134980 of the HSV-2 genome, or an EcoRI fragment that
spans nucleotides 110931 to 139697 of the HSV-2 genome. The
sequence of the HSV-2 genome is available form published sources,
for example the sequence deposited with GenBank under Accession
Number NC.sub.--001798. In certain embodiments, expression of
coding sequences contained within the genomic DNA fragments of the
construct (e.g., plasmid or cosmid) is not driven by a heterologous
promoter. The vaccine compositions may also comprise one or more
adjuvants.
[0020] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic representation of various HSV-2
cosmids used in the present invention. The numbers represent the
designations for the cosmid clones that have been used to immunize
animals. Each cosmid contains approximately 30,000-40,000 base
pairs of DNA from HSV-2.
[0022] FIG. 2 depicts serum antibody levels in mice immunized with
HSV-2 cosmids.
[0023] FIG. 3 depicts antigen-specific spleen cell proliferation in
animals immunized with either an HSV-2 gD plasmid or an HSV-2
cosmid.
MODES FOR CARRYING OUT THE INVENTION
[0024] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
antigens or to antigen-coding nucleotide sequences. It is also to
be understood that different applications of the disclosed methods
may be tailored to the specific needs in the art. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments of the invention only, and is not
intended to be limiting.
[0025] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0026] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an", and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "an antigen" includes a mixture of
two or more such agents, reference to "a particle" includes
reference to mixtures of two or more particles, reference to "a
recipient cell" includes two or more such cells, and the like.
Definitions
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. The
following terms are intended to be defined as indicated below.
[0028] The term "vaccine composition" intends any pharmaceutical
composition containing an antigen (e.g., polynucleotide encoding an
antigen), which composition can be used to prevent or treat a
disease or condition in a subject. The term thus encompasses both
subunit vaccines, i.e., vaccine compositions containing antigens
which are separate and discrete from a whole organism with which
the antigen is associated in nature, as well as compositions
containing whole killed, attenuated or inactivated bacteria,
viruses, parasites or other microbes.
[0029] The term "transdermal" delivery intends intradermal (e.g.,
into the dermis or epidermis), transdermal (e.g., "percutaneous")
and transmucosal administration, i.e., delivery by passage of an
agent into or through skin or mucosal tissue. See, e.g.,
Transdermal Drug Delivery: Developmental Issues and Research
Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989);
Controlled Drug Delivery: Fundamentals and Applications, Robinson
and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal
Delivery of Drugs, Vols. 1-3, Kydonieus and Bemer (eds.), CRC
Press, (1987). Thus, the term encompasses delivery from a particle
delivery device (e.g., needleless syringe) as described in U.S.
Pat. No. 5,630,796, as well as particle-mediated delivery as
described in U.S. Pat. No. 5,865,796.
[0030] By "core carrier" is meant a carrier particle on which a
nucleic acid (e.g., DNA) is coated in order to impart a defined
particle size as well as a sufficiently high density to achieve the
momentum required for cell membrane penetration, such that the DNA
can be delivered using particle-mediated delivery techniques, for
example those described in U.S. Pat. No. 5,100,792. Core carriers
typically include materials such as tungsten, gold, platinum,
ferrite, polystyrene and latex. See e.g., Particle Bombardment
Technology for Gene Transfer, (1994) Yang, N. ed., Oxford
University Press, New York, N.Y. pages 10-11.
[0031] By "particle delivery device," or "needleless syringe," is
meant an instrument which delivers a particulate composition
transdermally, without a conventional needle that pierces the skin.
Particle delivery devices for use with the present invention are
discussed throughout this document.
[0032] By "antigen" is meant a molecule which contains one or more
epitopes that will stimulate a host's immune system to make a
cellular antigen-specific immune response, or a humoral antibody
response. Thus, antigens include proteins, polypeptides, antigenic
protein fragments, oligosaccharides, polysaccharides, and the like.
Furthermore, the antigen can be derived from any known virus,
bacterium, parasite, plants, protozoans, or fungus, and can be a
whole organism. The term also includes tumor antigens. Similarly,
an oligonucleotide or polynucleotide which expresses an antigen,
such as in DNA immunization applications, is also included in the
definition of antigen. Synthetic antigens are also included, for
example, polyepitopes, flanking epitopes, and other recombinant or
synthetically derived antigens (Bergmann et al. (1993) Eur. J.
Immunol. 23:2777-2781; Bergmann et al. (1996) J. Immunol.
157:3242-3249; Suhrbier, A. (1997) Immunol. and Cell Biol.
75:402-408; Gardner et al. (1998) 12th World AIDS Conference,
Geneva, Switzerland, Jun. 28-Jul. 3, 1998).
[0033] By "suitable immune response" is meant that the methods of
the invention can bring about in an immunized subject an immune
response characterized by the production of B and/or T lymphocytes
specific for an antigen or antigens.
[0034] The term "peptide" is used in it broadest sense to refer to
a compound of two or more subunit amino acids, amino acid analogs,
or other peptidomimetics. The subunits may be linked by peptide
bonds or by other bonds, for example ester, ether, etc. As used
herein, the term "amino acid" refers to either natural and/or
unnatural or synthetic amino acids, including glycine and both the
D or L optical isomers, and amino acid analogs and peptidomimetics.
A peptide of three or more amino acids is commonly called an
oligopeptide if the peptide chain is short. If the peptide chain is
long, the peptide is typically called a polypeptide or a
protein.
[0035] The term "pathogen" is used in a broad sense to refer to the
source of any molecule that elicits an immune response. Thus,
pathogens include, but are not limited to, virulent or attenuated
viruses, bacteria, fingi, protozoa, parasites, cancer cells and the
like. Typically, the immune response is elicited by one or more
peptides produced by these pathogens. As described in detail below,
genomic DNA encoding the antigenic peptides from these and other
pathogens is used to generate an immune response that mimics the
response to natural infection. It will also be apparent in view of
the teachings herein, that the methods include the use of genomic
DNA obtained from more than one pathogen.
[0036] The terms "nucleic acid molecule" and "polynucleotide" are
used interchangeably to and refer to a polymeric form of
nucleotides of any length, either deoxyribonucleotides or
ribonucleotides, or analogs thereof. Polynucleotides may have any
three-dimensional structure, and may perform any function, known or
unknown. Non-limiting examples of polynucleotides include a gene, a
gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA,
ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, isolated RNA of any sequence, nucleic acid probes, and
primers.
[0037] A polynucleotide is typically composed of a specific
sequence of four nucleotide bases: adenine (A); cytosine (C);
guanine (G); and thymine (T) (uracil (U) for thymine (T) when the
polynucleotide is RNA). Thus, the term polynucleotide sequence is
the linear representation of a polynucleotide molecule. This linear
representation can be input into databases in a computer having a
central processing unit and used for bioinformatics applications
such as functional genomics and homology searching.
[0038] A "vector" is any moiety capable of transferring gene
sequences to target cells (e.g., viral vectors, non-viral vectors,
particulate carriers, and liposomes). A "plasmid" vector is an
extrachromosomal genetic element which is capable of
self-replication in a host cell. A "cosmid" vector is a special
type of plasmid vector that uses the cos sequences of bacteriophage
lambda (.lamda.). The term "cos ends" or "cos sites" refers to the
single stranded 12 base pair complementary extensions of .lamda.
DNA. Cosmids can carry large inserts, for example up to around 50
kb in size, while typical plasmids carry fragments under about 10
kb in size. Because of their capacity to carry large fragments,
cosmids are useful for the construction of genomic libraries.
Typically, "vector construct," "expression vector," and "gene
transfer vector," mean any nucleic acid construct capable of
directing the expression of a gene of interest and which can
transfer gene sequences to target cells. Thus, the term includes
cloning and expression vehicles, as well as viral vectors. A
"genomic library" is a collection of recombinant nucleic acid
molecules which together represent the entire genome of an
organism.
[0039] A "coding sequence" or a sequence which "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in
the case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences (or "control elements"). The boundaries of the
coding sequence are determined by a start codon at the 5' (amino)
terminus and a translation stop codon at the 3' (carboxy) terminus.
A coding sequence can include, but is not limited to, cDNA from
viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from
viral or procaryotic DNA, and even synthetic DNA sequences. A
transcription termination sequence may be located 3' to the coding
sequence. Transcription and translation of coding sequences are
typically regulated by "control elements," including, but not
limited to, transcription promoters, transcription enhancer
elements, Shine and Delagarno sequences, transcription termination
signals, polyadenylation sequences (located 3' to the translation
stop codon), sequences for optimization of initiation of
translation (located 5' to the coding sequence), and translation
termination sequences.
[0040] A "promoter" is a nucleotide sequence which directs
transcription of a polypeptide-encoding polynucleotide. Promoters
can include inducible promoters (where expression of a
polynucleotide sequence operably linked to the promoter is induced
by an analyte, cofactor, regulatory protein, etc.), repressible
promoters (where expression of a polynucleotide sequence operably
linked to the promoter is repressed by an analyte, cofactor,
regulatory protein, etc.), and constitutive promoters. It is
intended that the term "promoter" or "control element" includes
full-length promoter regions and functional (e.g., controls
transcription or translation) segments of these regions.
[0041] An "isolated polynucleotide" molecule is a nucleic acid
molecule separate and discrete from the whole organism with which
the molecule is found in nature; or a nucleic acid molecule devoid,
in whole or part, of sequences normally associated with it in
nature; or a sequence, as it exists in nature, but having
heterologous sequences (as defined below) in association therewith.
A sequence is "derived or obtained from" a molecule if it has the
same or substantially the same basepair sequence as a region of the
source molecule, its cDNA, complements thereof, or if it displays
sequence identity as described below.
[0042] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, a given promoter that is operably
linked to a coding sequence (e.g., encoding an antigen of interest)
is capable of effecting the expression of the coding sequence when
the proper enzymes are present. The promoter or other control
elements need not be contiguous with the coding sequence, so long
as they function to direct the expression thereof. For example,
intervening untranslated yet transcribed sequences can be present
between the promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence.
[0043] "Recombinant" as used herein to describe a nucleic acid
molecule means a polynucleotide of genomic, cDNA, semisynthetic, or
synthetic origin which, by virtue of its origin or manipulation:
(1) is not associated with all or a portion of the polynucleotide
with which it is associated in nature; and/or (2) is linked to a
polynucleotide other than that to which it is linked in nature. The
term "recombinant" as used with respect to a protein or polypeptide
means a polypeptide produced by expression of a recombinant
polynucleotide.
[0044] Techniques for determining nucleic acid and amino acid
"sequence identity" also are known in the art. Typically, such
techniques include determining the nucleotide sequence of the mRNA
for a gene and/or determining the amino acid sequence encoded
thereby, and comparing these sequences to a second nucleotide or
amino acid sequence. In general, "identity" refers to an exact
nucleotide-to-nucleotide or amino acid-to-amino acid correspondence
of two polynucleotides or polypeptide sequences, respectively. Two
or more sequences (polynucleotide or amino acid) can be compared by
determining their "percent identity." The percent identity of two
sequences, whether nucleic acid or amino acid sequences, is the
number of exact matches between two aligned sequences divided by
the length of the shorter sequences and multiplied by 100. An
approximate alignment for nucleic acid sequences is provided by the
local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981). This algorithm can be applied to
amino acid sequences by using the scoring matrix developed by
Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff
ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.
14(6):6745-6763 (1986). An exemplary implementation of this
algorithm to determine percent identity of a sequence is provided
by the Genetics Computer Group (Madison, Wis.) in the "BestFit"
utility application. The default parameters for this method are
described in the Wisconsin Sequence Analysis Package Program
Manual, Version 8 (1995) (available from Genetics Computer Group,
Madison, Wis.). A preferred method of establishing percent identity
in the context of the present invention is to use the MPSRCH
package of programs copyrighted by the University of Edinburgh,
developed by John F. Collins and Shane S. Sturrok, and distributed
by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite
of packages the Smith-Waterman algorithm can be employed where
default parameters are used for the scoring table (for example, gap
open penalty of 12, gap extension penalty of one, and a gap of
six). From the data generated the "Match" value reflects "sequence
identity." Other suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art, for example, another alignment program is BLAST, used with
default parameters. For example, BLASTN and BLASTP can be used
using the following default parameters: genetic code=standard;
filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;
Descriptions=50 sequences; sort by .dbd.HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+Swiss protein+Spupdate+PIR. Details of these programs
can be found at the following internet address:
http://www.ncbi.nlm.gov/cgi-bin/BLAST.
[0045] Alternatively, homology can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. Two DNA, or two polypeptide sequences are
"substantially homologous" to each other when the sequences exhibit
at least about 80%-85%, preferably at least about 90%, and most
preferably at least about 95%-98% sequence identity over a defined
length of the molecules, as determined using the methods above. As
used herein, substantially homologous also refers to sequences
showing complete identity to the specified DNA or polypeptide
sequence. DNA sequences that are substantially homologous can be
identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. For example, stringent hybridization conditions can include
50% formamide, 5.times. Denhardt's Solution, 5.times.SSC, 0.1% SDS
and 100 .mu.g/ml denatured salmon sperm DNA and the washing
conditions can include 2.times.SSC, 0.1% SDS at 37.degree. C.
followed by 1.times.SSC, 0.1% SDS at 68.degree. C. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic
Acid Hybridization, supra.
[0046] As used herein the term "adjuvant" refers to any material
that enhances the action of a drug, antigen, polynucleotide, vector
or the like. It is intended, although not always explicitly stated,
that molecules having similar biological activity as wild-type or
purified peptide adjuvants (e.g., recombinantly produced or muteins
thereof) and nucleic acid encoding these molecules are intended to
be used within the spirit and scope of the invention.
[0047] As used herein, the term "treatment" includes any of
following: the prevention of infection or reinfection; the
reduction or elimination of symptoms; and the reduction or complete
elimination of a pathogen. Treatment may be effected
prophylactically (prior to infection) or therapeutically (following
infection).
[0048] By "vertebrate subject" is meant any member of the subphylum
cordata, particularly mammals, including, without limitation,
humans and other primates. The term does not denote a particular
age. Thus, both adult and newborn individuals are intended to be
covered.
General Overview of the Invention
[0049] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0050] The present invention provides novel methods of eliciting an
immune response in a subject. Briefly, vector libraries carrying
large polynucleotide (e.g., DNA) fragments representing the genomes
of one or more pathogens (e.g., cells, tissues, viruses, etc.) are
generated. The vectors are typically in the form of plasmids or
cosmids, depending upon the size of the genomic fragments. The
libraries may contain overlapping or non-overlapping inserts. One
or more selected clones of the genomic library are then
administered to a subject in an amount effective to elicit an
immune response. In this way, a large number of antigens (and their
corresponding epitopes) can be administered to the subject in a
single immunization step. In addition, because the large genomic
fragments carried by the constructs (e.g., cosmids or plasmids)
include their endogenous expression control elements, further
molecular manipulations (for example insertion of heterologous
promoter sequences to drive expression from coding sequences
present in the genomic fragments) are not required. However,
heterologous control may be present in the constructs, for example
when the genomic sequences are derived from procaryotes such as
bacteria. Further, because the endogenous control elements (e.g.,
cis and/or trans-acting regulatory elements) drive antigen
expression in the host subject and the immunogenic gene-products
are thus produced at levels similar to that of a natural infection,
the overall immune response elicited by the constructs more closely
mimics the response due to natural infection. In contrast, the use
of heterologous promoters in previously described expression
library immunization would result in non-selective expression of
the pathogen's genes.
[0051] The invention also includes an efficient method for
identifying sequences encoding antigenic fragments. In particular,
use of constructs having a known genetic composition allows for a
method of identifying sequences that encode antigenic polypeptides.
For example, a cosmid or plasmid clone that induces an immune
response in a subject is identified. The precise sequence(s)
involved in the immune response can then be determined by methods
known in the art, for example sequencing of the clone, or by
further fragmenting of the cosmid or plasmid insert and testing
these smaller fragments for their immunogenicity.
[0052] The polynucleotides of the present invention may be
introduced into cells in vitro or in vivo, for example by
conventional transfection techniques or by coating the
polynucleotides onto particles and then administering the coated
particles to the cells using particle-mediated transfection.
Alternatively, the polynucleotides may be provided in a particulate
(e.g., powder) form, discussed more fully below and in the
disclosure of International Publication Number WO 98/10750, which
is incorporated by reference herein.
[0053] Advantages of the present invention include, but are not
limited to, (i) expanding the number of antigens used to elicit an
immune response; (ii) providing an array of antigens (e.g.,
epitopes) which more closely mimics that of a natural infection;
(iii) achieving co-delivery of the antigens and their associated
wild-type regulatory elements into the same cell to achieve
coordinated expression of multiple antigens; (iv) eliciting an
immune response similar to that elicited by natural infection; (v)
eliciting an immune response that is more protective than that
elicited by natural infection, e.g., by elimination of an
immunodominant antigen or elimination of genes that inhibit immune
responses; (vi) identifying the most effective combination of
antigens for subsequent production of plasmids for use in eliciting
an immune response; and (vii) triggering the antigen processing and
presentation pathways that are normally involved in the clearance
of intracellular infections.
Antigens
[0054] The methods described herein are useful in eliciting an
immune response against a wide variety of cells, tissues and human
or animal pathogens. These pathogens contain one or more antigens.
Non-limiting examples of sources for genomic DNA for cosmid or
plasmid libraries include viruses, bacterial cells, fungal cells,
and other pathogenic organisms.
[0055] Suitable viral antigens include, but are not limited to,
those obtained or derived from the hepatitis family of viruses,
including hepatitis A virus (HAV), hepatitis B virus (HBV),
hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis
E virus (HEV) and hepatitis G virus (HGV). See, e.g., International
Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV
genome encodes several viral proteins, including E1 and E2. See,
e.g., Houghton et al. (1991) Hepatology 14:381-388. Genomic
fragments containing sequences encoding these proteins, as well as
antigenic fragments thereof, will find use in the present methods.
Similarly, the coding sequence for the .delta.-antigen from HDV is
known (see, e.g., U.S. Pat. No. 5,378,814).
[0056] In like manner, a wide variety of proteins from the
herpesvirus family can be used as antigens in the present
invention, including proteins derived from herpes simplex virus
(HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD
and gH; antigens from varicella zoster virus (VZV), Epstein-Barr
virus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; and
antigens from other human herpesviruses such as HHV6 and HHV7.
(See, e.g. Chee et al. (1990) Cytomegaloviruses (J. K. McDougall,
ed., Springer-Verlag, pp. 125-169; McGeoch et al. (1988) J. Gen.
Virol. 69:1531-1574; U.S. Pat. No. 5,171,568; Baer et al. (1984)
Nature 310:207-211; and Davison et al. (1986) J. Gen. Virol.
67:1759-1816.)
[0057] Human immunodeficiency virus (HIV) antigens, such as gp120
molecules for a multitude of HIV-1 and HIV-2 isolates, including
members of the various genetic subtypes of HIV, are known and
reported (see, e.g., Myers et al., Los Alamos Database, Los Alamos
National Laboratory, Los Alamos, N. Mex. (1992); and Modrow et al.
(1987) J. Virol. 61:570-578) and antigen-containing genomic
fragments derived or obtained from any of these isolates will find
use in the present invention. Furthermore, other immunogenic
proteins derived or obtained from any of the various HIV isolates
will find use herein, including fragments containing one or more of
the various envelope proteins such as gp160 and gp41, gag antigens
such as p24gag and p55gag, as well as proteins derived from the
pol, env, tat, vif, rev, nef, vpr, vpu and LTR regions of HIV.
[0058] Antigens derived or obtained from other viruses will also
find use herein, such as without limitation, antigens from members
of the families Picornaviridae (e.g., polioviruses, rhinoviruses,
etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue
virus, etc.); Flaviviridae; Coronaviridae; Reoviridae (e.g.,
rotavirus, etc.); Bimaviridae; Rhabodoviridae (e.g., rabies virus,
etc.); Orthomyxoviridae (e.g., influenza virus types A, B and C,
etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles
virus, respiratory syncytial virus, parainfluenza virus, etc.);
Bunyaviridae; Arenaviridae; Retroviradae (e.g., HTLV-I; HTLV-II;
HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.)), including
but not limited to antigens from the isolates HIV.sub.IIIb,
HIV.sub.SF2, HIV.sub.LAV, HIV.sub.LAI, HIV.sub.MN);
HIV-1.sub.CM235, HIV-1.sub.US4; HIV-2, among others; simian
immunodeficiency virus (SIV); Papillomavirus, the tick-bourne
encephalitis viruses; and the like. See, e.g. Virology, 3rd Edition
(W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N.
Fields and D. M. Knipe, eds. 1991), for a description of these and
other viruses.
[0059] In some contexts, it may be preferable that the selected
viral antigens are obtained or derived from a viral pathogen that
typically enters the body via a mucosal surface and is known to
cause or is associated with human disease, such as, but not limited
to, HIV (AIDS), influenza viruses (Flu), herpes simplex viruses
(genital infection, cold sores, STDs), rotaviruses (diarrhea),
parainfluenza viruses (respiratory infections), poliovirus
(poliomyelitis), respiratory syncytial virus (respiratory
infections), measles and mumps viruses (measles, mumps), rubella
virus (rubella), and rhinoviruses (common cold).
[0060] Genomic fragments containing bacterial and parasitic
antigens can be obtained or derived from known causative agents
responsible for diseases including, but not limited to, Diptheria,
Pertussis, Tetanus, Tuberculosis, Bacterial or Fungal Pneumonia,
Otitis Media, Gonnorhea, Cholera, Typhoid, Meningitis,
Mononucleosis, Plague, Shigellosis or Salmonellosis, Legionaire's
Disease, Lyme Disease, Leprosy, Malaria, Hookworm, Onchocerciasis,
Schistosomiasis, Trypamasomialsis, Lesmaniasis, Giardia,
Amoebiasis, Filariasis, Borelia, and Trichinosis. Still further
antigens can be obtained or derived from unconventional viruses
such as the causative agents of kuru, Creutzfeldt-Jakob disease
(CJD), scrapie, transmissible mink encephalopathy, and chronic
wasting diseases, or from proteinaceous infectious particles such
as prions that are associated with mad cow disease.
[0061] Specific pathogens can include M. tuberculosis, Chlamydia,
N. gonorrhoeae, Shigella, Salmonella, Vibrio Cholera, Treponema
pallidua, Pseudomonas, Bordetella pertussis, Brucella, Franciscella
tulorensis, Helicobacter pylori, Leptospria interrogaus, Legionella
pneumophila, Yersinia pestis, Streptococcus (types A and B),
Pneumococcus, Meningococcus, Hemophilus influenza (type b),
Toxoplasma gondic, Complylobacteriosis, Moraxella catarrhalis,
Donovanosis, and Actinomycosis; fungal pathogens including
Candidiasis and Aspergillosis; parasitic pathogens including
Taenia, Flukes, Roundworms, Amebiasis, Giardiasis, Cryptosporidium,
Schistosoma, Pneumocystis carinii, Trichomoniasis and Trichinosis.
Thus, the present invention can also be used to provide a suitable
immune response against numerous veterinary diseases, such as Foot
and Mouth diseases, Coronavirus, Pasteurella multocida,
Helicobacter, Strongylus vulgaris, Actinobacillus pleuropneumonia,
Bovine viral diarrhea virus (BVDV), Klebsiella pneumoniae, E. coli,
Bordetella pertussis, Bordetella parapertussis and
brochiseptica.
Adjuvants
[0062] In some embodiments, the present invention may effectively
be used with any suitable adjuvant or combination of adjuvants. For
example, suitable adjuvants include, without limitation, adjuvants
formed from aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, aluminum sulfate, etc; oil-in-water and
water-in-oil emulsion formulations, such as Complete Freunds
Adjuvants (CFA) and Incomplete Freunds Adjuvant (IFA); adjuvants
formed from bacterial cell wall components such as adjuvants
including lipopolysaccharides (e.g., lipid A or monophosphoryl
lipid A (MPL), Imoto et al. (1985) Tet. Lett. 26:1545-1548),
trehalose dimycolate (TDM), and cell wall skeleton (CWS); heat
shock protein or derivatives thereof; adjuvants derived from
ADP-ribosylating bacterial toxins, including diphtheria toxin (DT),
pertussis toxin (PT), cholera toxin (CT), the E. coli heat-labile
toxins (LT1 and LT2), Pseudomonas endotoxin A, Pseudomonas exotoxin
S, B. cereus exoenzyme, B. sphaericus toxin, C. botulinum C2 and C3
toxins, C. limosum exoenzyme, as well as toxins from C.
perfringens, C. spiriforma and C. difficile, Staphylococcus aureus
EDIN, and ADP-ribosylating bacterial toxin mutants such as
CRM.sub.197, a non-toxic diphtheria toxin mutant (see, e.g., Bixler
et al. (1989) Adv. Exp. Med. Biol. 251:175; and Constantino et al.
(1992) Vaccine); saponin adjuvants such as Quil A (U.S. Pat. No.
5,057,540), or particles generated from saponins such as ISCOMs
(immunostimulating complexes); chemokines and cytokines, such as
interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-12, etc.), interferons (e.g., gama interferon), macrophage
colony stimulating factor (M-CSF), tumor necrosis factor (TNF),
defensins 1 or 2, RANTES, MIP1-.alpha. and MIP-2, etc; muramyl
peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine
(thr-MDP), N-acetyl-nornuramyl-.sup.L-alanyl-.sup.D-isoglUtamine
(nor-MDP),
N-acetylmuramyl-.sup.L-alanyl-.sup.D-isoglutaminyl-.sup.L-alanine-2-(1'-2-
'-dipalmitoyl-sn-glycero-3 huydroxyphosphoryloxy)-ethylamine
(MTP-PE) etc.; adjuvants derived from the CpG family of molecules,
CpG dinucleotides and synthetic oligonucleotides which comprise CpG
motifs (see, e.g., Krieg et al. Nature (1995) 374:546, Medzhitov et
al. (1997) Curr. Opin. Immunol. 9:4-9, and Davis et al. J. Immunol.
(1998) 160:870-876) such as TCC ATG ACG TTC CTG ATG CT (SEQ ID
NO:1) and ATC GAC TCT CGA GCG TTC TC (SEQ ID NO: 2); and synthetic
adjuvants such as PCPP (Poly[di(carboxylatophenoxy)phosphazene)
(Payne et al. Vaccines (1998) 16:92-98). Such adjuvants are
commercially available from a number of distributors such as
Accurate Chemicals; Ribi Immunechemicals, Hamilton, Mont.; GIBCO;
Sigma, St. Louis, Mo.
[0063] The adjuvant may delivered individually or delivered in a
combination of two or more adjuvants. In this regard, combined
adjuvants may have an additive or a synergistic effect in promoting
an immune response. A synergistic effect is one where the result
achieved by combining two or more adjuvants is greater than one
would expect than by merely adding the result achieved with each
adjuvant when administered individually.
Preparation of Genomic Libraries
[0064] Genomic libraries can be produced by any method known in the
art. A variety of sources can be used for the genomic DNA. Genomic
DNA may be commercially available, for example, from sources such
as Advanced Biotechnologies Inc (ABI) and Clonetech, Inc. Another
standard source is, of course, genomic DNA isolated from an
organism (e.g., virus, bacteria, parasite, or other pathogen), a
cell (e.g., a cancer cell), or selected tissue. Alternatively, DNA
can be prepared from RNA (e.g., RNA viruses) by techniques
available in the art. Genomic DNA from the selected source can be
isolated by standard procedures, which typically include successive
phenol and phenol/chloroform extractions followed by ethanol
precipitation. After precipitation, the DNA from an organism, cell
or tissue of interest can be treated with a restriction
endonuclease which either completely or partially digests the DNA
to produce the appropriate fragments. DNA fragments of a selected
size can be separated by a number of techniques, including agarose
or polyacrylamide gel electrophoresis or pulse field gel
electrophoresis (Carle et al. (1984)Nuc. Acid Res. 12:5647-5664;
Chu et al. (1986) Science 234:1582; Smith et al. (1987) Methods in
Enzymology 151:461), to provide an appropriate size starting
material for cloning.
[0065] Another method for obtaining nucleic acid sequences for use
herein is by recombinant means. Thus, a desired nucleotide sequence
can be excised from a vector carrying the same using standard
molecular biology procedures. Site specific DNA cleavage can be
performed by treating with the suitable restriction enzyme (or
enzymes) under conditions which are generally understood in the
art, and the particulars of which are specified by manufacturers of
commercially available restriction enzymes. If desired, size
separation of the cleaved fragments may be performed by
polyacrylamide gel or agarose gel electrophoresis using standard
techniques.
[0066] Restriction cleaved fragments may be blunt ended, if
desired, by treating with the large fragment of E. coli DNA
polymerase I (Klenow) in the presence of the four deoxynucleotide
triphosphates (dNTPs) using standard techniques. The Klenow
fragment fills in at 5' single-stranded overhangs but digests
protruding 3' single strands, even though the four dNTPs are
present. If desired, selective repair can be performed by supplying
only one, or several, selected dNTPs within the limitations
dictated by the nature of the overhang. After Klenow treatment, the
mixture can be extracted with e.g. phenol/chloroform, and ethanol
precipitated. Treatment under appropriate conditions with S1
nuclease or BAL-31 results in hydrolysis of any single-stranded
portion.
[0067] Once genomic fragments have been prepared or isolated, such
sequences can be cloned into any suitable vector construct or
replicon. Numerous cloning vectors are known to those of skill in
the art, and the selection of an appropriate cloning vector is a
matter of choice. In one preferred embodiment, the genomic
fragments are cloned into cosmids to generate cosmid libraries.
When using cosmid cloning vectors, the fragments are large,
preferably between about 20,000 bp (20 kb) and 50,000 base pairs
(50 kb) in size (or any integer there between), preferably between
about 25 kb and 50 kb, more preferably between about 30-35 kb and
50 kb, and even more preferably between about 35 kb and about 50
kb. Suitable cosmid vectors are commercially available, for example
the SuperCos 1 Cosmid Vector Kit (Stratagene, La Jolla, Calif.).
Ligation of the DNA into the cosmid is performed as instructed by
the manufacturer or may be empirically determined using methods
known in the art in view of the teachings of this
specification.
[0068] In another preferred embodiment, the genomic fragments are
cloned into plasmids to generate plasmid libraries. When using
plasmid cloning vectors, the fragments are typically between about
5,000 bp (5 kb) and 25,000 base pairs (25 kb) in size (or any
integer there between), preferably between about 10 kb and 25 kb,
more preferably between about 10-15 kb and 25 kb, and even more
preferably between about 15 kb and 20 kb. Suitable plasmid vectors
are commercially available. Ligation of the DNA into the plasmid is
performed using methods well known in the art in view of the
teachings of this specification.
[0069] As described above, one advantage of the present invention
is that the large genomic fragments include endogenous
transcription and translation regulatory elements. Such regulatory
control sequences include, for example, promoters (a sequence
associated with initiating transcription), enhancers (a cis-acting
sequence that enhances transcription) and other elements including
those which cause the expression of a coding sequence to be turned
on or off in response to a chemical or physical stimulus, including
the presence of a regulatory compound. Thus, in a preferred
embodiment, the number of molecular modifications to the vectors
and/or inserted polynucleotides is minimal.
[0070] It is also possible that selected nucleotide sequences
within the vector constructs can be placed under the control of
heterologous regulatory sequences such as a heterologous promoter,
for example when the genomic fragments are derived from bacteria or
other prokaryotes, and expression is desired in a eukaryotic
subject. Modification of the sequences encoding the particular
protein of interest may be desirable to achieve this end. For
example, in some cases it may be necessary to modify the sequence
so that it is attached to the control sequences with the
appropriate orientation; i.e., to maintain the reading frame. The
control sequences and other regulatory sequences may be ligated to
the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector which already contains the control sequences and
an appropriate restriction site.
Administration of Polynucleotides
[0071] The genomic fragments and ancillary substances described
herein may be administered by any suitable method. In a preferred
embodiment, described below, the polynucleotide fragments are
administered by coating a suitable construct (e.g., cosmids or
plasmids) containing the fragments onto core carrier particles and
then administering the coated particles to the subject or cells.
However, the genomic fragments may also be delivered using a viral
vector or using non-viral systems, e.g., naked nucleic acid
delivery.
[0072] Viral Vectors
[0073] A number of viral based systems have been used for gene
delivery. For example, retroviral systems are known and generally
employ packaging lines which have an integrated defective provirus
(the "helper") that expresses all of the genes of the virus but
cannot package its own genome due to a deletion of the packaging
signal, known as the psi sequence. Thus, the cell line produces
empty viral shells. Producer lines can be derived from the
packaging lines which, in addition to the helper, contain a viral
vector which includes sequences required in cis for replication and
packaging of the virus, known as the long terminal repeats (LTRs).
The genomic fragment(s) of interest can be inserted into the vector
and packaged in the viral shells synthesized by the retroviral
helper. The recombinant virus can then be isolated and delivered to
a subject. (See, e.g., U.S. Pat. No. 5,219,740.) Representative
retroviral vectors include but are not limited to vectors such as
the LHL, N2, LNSAL, LSHL and LHL2 vectors described in e.g., U.S.
Pat. No. 5,219,740, incorporated herein by reference in its
entirety, as well as derivatives of these vectors, such as the
modified N2 vector described herein. Retroviral vectors can be
constructed using techniques well known in the art. See, e.g., U.S.
Pat. No. 5,219,740; Mann et al. (1983) Cell 33:153-159.
[0074] Adenovirus based systems have been developed for gene
delivery and are suitable for delivering the genomic fragments
described herein. Human adenoviruses are double-stranded DNA
viruses which enter cells by receptor-mediated endocytosis. These
viruses are particularly well suited for genetic transfer because
they are easy to grow and manipulate and they exhibit a broad host
range in vivo and in vitro. For example, adenoviruses can infect
human cells of hematopoietic, lymphoid and myeloid origin.
Furthermore, adenoviruses infect quiescent as well as replicating
target cells. Unlike retroviruses which integrate into the host
genome, adenoviruses persist extrachromosomally thus minimizing the
risks associated with insertional mutagenesis. The virus is easily
produced at high titers and is stable so that it can be purified
and stored. Even in the replication-competent form, adenoviruses
cause only low level morbidity and are not associated with human
malignancies. Accordingly, adenovirus vectors have been developed
which make use of these advantages. For a description of adenovirus
vectors and their uses see, e.g., Haj-Ahmad and Graham (1986) J.
Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;
Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.
(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy
1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; Rich et al.
(1993) Human Gene Therapy 4:461-476.
[0075] Adeno-associated viral vectors (AAV) can also be used to
administer certain of the smaller genomic fragments (e.g., 5 kb)
described herein. AAV vectors can be derived from any AAV serotype,
including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,
AAVX7, etc. AAV vectors can have one or more of the AAV wild-type
genes deleted in whole or in part, preferably the rep and/or cap
genes, but retain one or more functional flanking inverted terminal
repeat (ITR) sequences. A functional ITR sequence is generally
deemed necessary for the rescue, replication and packaging of the
AAV virion. Thus, an AAV vector includes at least those sequences
required in cis for replication and packaging (e.g., a functional
ITR) of the virus. The ITR need not be the wild-type nucleotide
sequence, and may be altered, e.g., by the insertion, deletion or
substitution of nucleotides, so long as the sequence provides for
functional rescue, replication and packaging.
[0076] AAV expression vectors are constructed using known
techniques to at least provide as operatively linked components in
the direction of transcription, control elements including a
transcriptional initiation region, the DNA of interest and a
transcriptional termination region. The control elements are
selected to be functional in a mammalian cell. The resulting
construct which contains the operatively linked components is
bounded (5' and 3') with functional AAV ITR sequences. Suitable AAV
constructs can be designed using techniques well known in the art.
See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International
Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO
93/03769 (published 4 Mar. 1993); Lebkowski et al. (1988) Molec.
Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold
Spring Harbor Laboratory Press); Carter, B. J. (1992) Current
Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current
Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994)
Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene
Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med.
179:1867-1875.
[0077] Conventional Pharmaceutical Preparations
[0078] Formulation of a preparation comprising the genomic DNA
fragments of the present invention, with or without addition of an
adjuvant composition, can be carried out using standard
pharmaceutical formulation chemistries and methodologies all of
which are readily available to the ordinarily skilled artisan. For
example, compositions containing one or more genomic fragments
(e.g., present in a plasmid or cosmid) can be combined with one or
more pharmaceutically acceptable excipients or vehicles to provide
a liquid preparation.
[0079] Auxiliary substances, such as wetting or emulsifying agents,
pH buffering substances and the like, may be present in the
excipient or vehicle. These excipients, vehicles and auxiliary
substances are generally pharmaceutical agents that do not induce
an immune response in the individual receiving the composition, and
which may be administered without undue toxicity. Pharmaceutically
acceptable excipients include, but are not limited to, liquids such
as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and
ethanol. Pharmaceutically acceptable salts can also be included
therein, for example, mineral acid salts such as hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of
organic acids such as acetates, propionates, malonates, benzoates,
and the like. It is also preferred, although not required, that the
preparation will contain a pharmaceutically acceptable excipient
that serves as a stabilizer, particularly for peptide, protein or
other like molecules if they are to be included in the vaccine
composition. Examples of suitable carriers that also act as
stabilizers for peptides include, without limitation,
pharmaceutical grades of dextrose, sucrose, lactose, trehalose,
mannitol, sorbitol, inositol, dextran, and the like. Other suitable
carriers include, again without limitation, starch, cellulose,
sodium or calcium phosphates, citric acid, tartaric acid, glycine,
high molecular weight polyethylene glycols (PEGs), and combination
thereof. A thorough discussion of pharmaceutically acceptable
excipients, vehicles and auxiliary substances is available in
REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991),
incorporated herein by reference.
[0080] Certain facilitators of nucleic acid uptake and/or
expression ("transfection facilitating agents") can also be
included in the compositions, for example, facilitators such as
bupivacaine, cardiotoxin and sucrose, and transfection facilitating
vehicles such as liposomal or lipid preparations that are routinely
used to deliver nucleic acid molecules. Anionic and neutral
liposomes are widely available and well known for delivering
nucleic acid molecules (see, e.g., Liposomes: A Practical Approach,
(1990) RPC New Ed., IRL Press). Cationic lipid preparations are
also well known vehicles for use in delivery of nucleic acid
molecules. Suitable lipid preparations include DOTMA
(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride),
available under the tradename Lipofectin.TM., and DOTAP
(1,2-bis(oleyloxy)-3-(trimethylammonio)propane), see, e.g., Felgner
et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416; Malone et
al. (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081; U.S. Pat. Nos.
5,283,185 and 5,527,928, and International Publication Nos WO
90/11092, WO 91/15501 and WO 95/26356. These cationic lipids may
preferably be used in association with a neutral lipid, for example
DOPE (dioleyl phosphatidylethanolamine). Still further
transfection-facilitating compositions that can be added to the
above lipid or liposome preparations include spermine derivatives
(see, e.g., International Publication No. WO 93/18759) and
membrane-permeabilizing compounds such as GAL4, Gramicidine S and
cationic bile salts (see, e.g., International Publication No. WO
93/19768).
[0081] Alternatively, the nucleic acid molecules of the present
invention may be encapsulated, adsorbed to, or associated with,
particulate carriers. Suitable particulate carriers include those
derived from polymethyl methacrylate polymers, as well as PLG
microparticles derived from poly(lactides) and
poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993)
Pharm. Res. 10:362-368. Other particulate systems and polymers can
also be used, for example, polymers such as polylysine,
polyarginine, polyomithine, spermine, spermidine, as well as
conjugates of these molecules.
[0082] The formulated vaccine compositions will thus typically
include a polynucleotide (e.g., a plasmid or cosmid) containing at
least one genomic fragment from a selected pathogen in an amount
sufficient to mount an immunological response. An appropriate
effective amount can be readily determined by one of skill in the
art. Such an amount will fall in a relatively broad range that can
be determined through routine trials. For example, immune responses
have been obtained using as little as 1 .mu.g of DNA, while in
other administrations, up to 2 mg of DNA has been used. It is
generally expected that an effective dose of polynucleotides
containing the genomic fragments will fall within a range of about
10 .mu.g to 1000 .mu.g, however, doses above and below this range
may also be found effective. The compositions may thus contain from
about 0.1% to about 99.9% of the polynucleotide molecules.
[0083] Administration of Conventional Pharmaceutical
Preparations
[0084] Administration of the above-described pharmaceutical
preparations can be effected in one dose, continuously or
intermittently throughout the course of treatment. Delivery will
most typically be via conventional needle and syringe for the
liquid compositions and for liquid suspensions containing
particulate compositions. In addition, various liquid jet injectors
are known in the art and may be employed to administer the present
compositions. Methods of determining the most effective means and
dosages of administration are well known to those of skill in the
art and will vary with the delivery vehicle, the composition of the
therapy, the target cells, and the subject being treated. Single
and multiple administrations can be carried out with the dose level
and pattern being selected by the attending physician. It should be
understood that more than one genomic fragment can be carried by
the delivered polynucleotide vector construct. Alternatively,
separate vectors (e.g., cosmids or plasmids), each expressing one
or more antigens derived from any pathogen can also be delivered to
a subject as described herein.
[0085] Furthermore, it is also intended that the polynucleotides
delivered by the methods of the present invention be combined with
other suitable compositions and therapies. For instance, in order
to augment an immune response in a subject, the compositions and
methods described herein can further include ancillary substances
(e.g., adjuvants), such as pharmacological agents, cytokines, or
the like. Ancillary substances may be administered, for example, as
proteins or other macromolecules at the same time, prior to, or
subsequent to, administration of the DNA vaccines (e.g., cosmids or
plasmids) described herein. The nucleic acid molecule compositions
may also be administered directly to the subject or, alternatively,
delivered ex vivo, to cells derived from the subject, using methods
known to those skilled in the art.
[0086] Coated Particles
[0087] In one embodiment, constructs containing the genomic
fragments (e.g, plasmids or cosmids), and other ancillary
components such as adjuvants are delivered using carrier particles.
Particle-mediated delivery methods for administering such nucleic
acid preparations are known in the art. Thus, once prepared and
suitably purified, the above-described plasmid and cosmid
constructs can be coated onto carrier particles (e.g., core
carriers) using a variety of techniques known in the art. Carrier
particles are selected from materials which have a suitable density
in the range of particle sizes typically used for intracellular
delivery from an appropriate particle delivery device. The optimum
carrier particle size will, of course, depend upon the diameter of
the target cells.
[0088] For the purposes of the present invention, tungsten, gold,
platinum and iridium core carrier particles can be used. Tungsten
and gold particles are preferred. Tungsten particles are readily
available in average sizes of 0.5 to 2.0.mu./m in diameter.
Although such particles have optimal density for use in particle
delivery methods, and allow highly efficient coating with DNA,
tungsten may potentially be toxic to certain cell types.
Accordingly, gold particles or microcrystalline gold (e.g., gold
powder A1570, available from Engelhard Corp., East Newark, N.J.)
will also find use with the present methods. Gold particles provide
uniformity in size (available from Alpha Chemicals in particle
sizes of 1-3 .mu.m, or available from Degussa, South Plainfield,
N.J. in a range of particle sizes including 0.95 .mu.m) and reduced
toxicity.
[0089] A number of methods are known and have been described for
coating or precipitating DNA or RNA onto gold or tungsten
particles. Most such methods generally combine a predetermined
amount of gold or tungsten with plasmid DNA, CaCl.sub.2 and
spermidine. The resulting solution is vortexed continually during
the coating procedure to ensure uniformity of the reaction mixture.
After precipitation of the nucleic acid, the coated particles can
be transferred to suitable membranes and allowed to dry prior to
use, coated onto surfaces of a sample module or cassette, or loaded
into a delivery cassette for use in a suitable particle delivery
device.
[0090] Peptide adjuvants (e.g., cytokines and bacterial toxins),
can also be coated onto the same or similar core carrier particles.
For example, peptides can be attached to a carrier particle by
simply mixing the two components in an empirically determined
ratio, by ammonium sulfate precipitation or other solvent
precipitation methods familiar to those skilled in the art, or by
chemical coupling of the peptide to the carrier particle. The
coupling of L-cysteine residues to gold has been previously
described (Brown et al., Chemical Society Reviews 9:271-311
(1980)). Other methods would include, for example, dissolving the
peptide adjuvant in absolute ethanol, water, or an alcohol/water
mixture, adding the solution to a quantity of carrier particles,
and then drying the mixture under a stream of air or nitrogen gas
while vortexing. Alternatively, the adjuvant can be dried onto
carrier particles by centrifugation under vacuum. Once dried, the
coated particles can be resuspended in a suitable solvent (e.g.,
ethyl acetate or acetone), and triturated (e.g., by sonication) to
provide a substantially uniform suspension. The core carrier
particles coated with the adjuvant can then be combined with core
carrier particles carrying the genomic fragment constructs and
administered in a single particle injection step, or administered
separately from the genomic fragment compositions.
[0091] Administration of Coated Particles
[0092] Following their formation, core carrier particles coated
with the nucleic acid preparations of the present invention, alone
or in combination with e.g., adjuvant preparations, are delivered
to a subject using particle-mediated delivery techniques.
[0093] Various particle delivery devices suitable for
particle-mediated delivery techniques are known in the art, and are
all suited for use in the practice of the invention. Current device
designs employ an explosive, electric or gaseous discharge to
propel the coated core carrier particles toward target cells. The
coated particles can themselves be releasably attached to a movable
carrier sheet, or removably attached to a surface along which a gas
stream passes, lifting the particles from the surface and
accelerating them toward the target. An example of a gaseous
discharge device is described in U.S. Pat. No. 5,204,253. An
explosive-type device is described in U.S. Pat. No. 4,945,050. One
example of an electric discharge-type particle acceleration
apparatus is described in U.S. Pat. No. 5,120,657. Another electric
discharge apparatus suitable for use herein is described in U.S.
Pat. No. 5,149,655. The disclosure of all of these patents is
incorporated herein by reference in their entireties.
[0094] The coated particles are administered to the subject to be
treated in a manner compatible with the dosage formulation, and in
an amount that will be effective to bring about a desired immune
response. The amount of the composition to be delivered which, in
the case of nucleic acid molecules is generally in the range of
from 0.001 to 100.0 .mu.g, more typically 0.01 to 10.0 .mu.g of
nucleic acid molecule per dose, and in the case of peptide or
protein molecules is 1 .mu.g to 5 mg, more typically 1 to 50 .mu.g
of peptide, depends on the subject to be treated. The exact amount
necessary will vary depending on the age and general condition of
the individual being immunized and the particular nucleotide
sequence or peptide selected, as well as other factors. An
appropriate effective amount can be readily determined by one of
skill in the art upon reading the instant specification.
[0095] Thus, an effective amount of the genomic fragments herein
described will be sufficient to bring about a suitable immune
response in an immunized subject, and will fall in a relatively
broad range that can be determined through routine trials.
Preferably, the coated core particles are delivered to suitable
recipient cells in order to bring about an immune response (e.g.,
T-cell activation) in the treated subject.
[0096] Particulate Compositions
[0097] Alternatively, the genomic fragments of the present
invention (e.g. plasmid or cosmid constructs carrying the genomic
fragments), as well as one or more selected adjuvants, can be
formulated as a particulate composition. More particularly,
formulation of particles comprising a genomic fragment of interest
can be carried out using standard pharmaceutical formulation
chemistries and methodologies all of which are readily available to
the reasonably skilled artisan. For example, one or more vector
construct and/or adjuvants can be combined with one or more
pharmaceutically acceptable excipients or vehicles to provide a
vaccine composition. Auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the like, may be
present in the excipient or vehicle. These excipients, vehicles and
auxiliary substances are generally pharmaceutical agents that do
not themselves induce an immune response in the individual
receiving the composition, and which may be administered without
undue toxicity. Pharmaceutically acceptable excipients include, but
are not limited to, liquids such as water, saline,
polyethyleneglycol, hyaluronic acid, glycerol and ethanol.
Pharmaceutically acceptable salts can be included therein, for
example, mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
It is also preferred, although not required, that the nucleic acid
composition will contain a pharmaceutically acceptable carrier that
serves as a stabilizer, particularly for peptide, protein or other
like adjuvants or ancillary materials. Examples of suitable
carriers that also act as stabilizers for peptides include, without
limitation, pharmaceutical grades of dextrose, sucrose, lactose,
trehalose, mannitol, sorbitol, inositol, dextran, and the like.
Other suitable carriers include, again without limitation, starch,
cellulose, sodium or calcium phosphates, citric acid, tartaric
acid, glycine, high molecular weight polyethylene glycols (PEGs),
and combination thereof. A thorough discussion of pharmaceutically
acceptable excipients, carriers, stabilizers and other auxiliary
substances is available in REMINGTONS PHARMACEUTICAL SCIENCES (Mack
Pub. Co., N.J. 1991), incorporated herein by reference.
[0098] The formulated compositions will include an amount of the
genomic fragment of interest which is sufficient to mount an
immunological response, as defined above. An appropriate effective
amount can be readily determined by one of skill in the art. Such
an amount will fall in a relatively broad range, generally within
the range of about 0.1 .mu.g to 25 mg or more of the nucleic acid
construct of interest, and specific suitable amounts can be
determined through routine trials. The compositions may contain
from about 0.1% to about 99.9% of the nucleic acid molecule. If an
adjuvant is included in the composition, or the methods are used to
provide a particulate adjuvant composition, the adjuvant will be
present in a suitable amount as described above. The compositions
are then prepared as particles using standard techniques, such as
by simple evaporation (air drying), vacuum drying, spray drying,
freeze drying (lyophilization), spray-freeze drying, spray coating,
precipitation, supercritical fluid particle formation, and the
like. If desired, the resultant particles can be densified using
the techniques described in commonly owned International
Publication No. WO 97/48485, incorporated herein by reference.
[0099] Single unit dosages or multidose containers, in which the
particles may be packaged prior to use, can comprise a hermetically
sealed container enclosing a suitable amount of the particles
comprising a suitable nucleic acid construct (e.g., a plasmid or
cosmid) and/or the selected adjuvant (e.g., to provide a vaccine
composition). The particulate compositions can be packaged as a
sterile formulation, and the hermetically sealed container can thus
be designed to preserve sterility of the formulation until use in
the methods of the invention. If desired, the containers can be
adapted for direct use in a particle delivery device. Such
containers can take the form of capsules, foil pouches, sachets,
cassettes, and the like. Appropriate particle delivery devices
(e.g., needleless syringes) are described herein.
[0100] The container in which the particles are packaged can
further be labelled to identify the composition and provide
relevant dosage information. In addition, the container can be
labelled with a notice in the form prescribed by a governmental
agency, for example the Food and Drug Administration, wherein the
notice indicates approval by the agency under Federal law of the
manufacture, use or sale of the antigen, adjuvant (or vaccine
composition) contained therein for human administration.
[0101] The particulate compositions (comprising one or more genomic
fragments of interest alone, or in combination with a selected
adjuvant) can then be administered using a transdermal delivery
technique. Preferably, the particulate compositions will be
delivered via a powder injection method, e.g., delivered from a
needleless syringe system such as those described in commonly owned
International Publication Nos. WO 94/24263, WO 96/04947, WO
96/12513, and WO 96/20022, all of which are incorporated herein by
reference. Delivery of particles from such needleless syringe
systems is typically practised with particles having an approximate
size generally ranging from 0.1 to 250 .mu.m, preferably ranging
from about 10-70 .mu.m. Particles larger than about 250 .mu.m can
also be delivered from the devices, with the upper limitation being
the point at which the size of the particles would cause untoward
damage to the skin cells. The actual distance which the delivered
particles will penetrate a target surface depends upon particle
size (e.g., the nominal particle diameter assuming a roughly
spherical particle geometry), particle density, the initial
velocity at which the particle impacts the surface, and the density
and kinematic viscosity of the targeted skin tissue. In this
regard, optimal particle densities for use in needleless injection
generally range between about 0.1 and 25 g/cm.sup.3, preferably
between about 0.9 and 1.5 g/cm.sup.3, and injection velocities
generally range between about 100 and 3,000 m/sec, or greater. With
appropriate gas pressure, particles having an average diameter of
10-70 .mu.m can be accelerated through the nozzle at velocities
approaching the supersonic speeds of a driving gas flow.
[0102] If desired, these needleless syringe systems can be provided
in a preloaded condition containing a suitable dosage of the
particles comprising the genomic fragments and/or the selected
adjuvant. The loaded syringe can be packaged in a hermetically
sealed container, which may further be labelled as described
above.
[0103] Thus, the method can be used to obtain nucleic acid
particles having a size ranging from about 10 to about 250 .mu.m,
preferably about 10 to about 150 .mu.m, and most preferably about
20 to about 60 .mu.m; and a particle density ranging from about 0.1
to about 25 g/cm.sup.3, and a bulk density of about 0.5 to about
3.0 g/cm.sup.3, or greater.
[0104] Similarly, particles of selected adjuvants having a size
ranging from about 0.1 to about 250 .mu.m, preferably about 0.1 to
about 150 .mu.m, and most preferably about 20 to about 60 .mu.m; a
particle density ranging from about 0.1 to about 25 g/cm.sup.3, and
a bulk density of preferably about 0.5 to about 3.0 g/cm.sup.3, and
most preferably about 0.8 to about 1.5 g/cm.sup.3 can be
obtained.
[0105] Administration of Particulate Compositions
[0106] Following their formation, the particulate compositions
(e.g., powder) can be delivered transdermally to the tissue of a
vertebrate subject using a suitable transdermal delivery technique.
Various particle delivery devices suitable for administering the
substance of interest are known in the art, and will find use in
the practice of the invention. A particularly preferred transdermal
particle delivery system employs a needleless syringe to fire solid
particles in controlled doses into and through intact skin and
tissue. See, e.g., U.S. Pat. No. 5,630,796 to Bellhouse et al.
which describes a needleless syringe (also known as "the
PowderJect.RTM. particle delivery device"). Other needleless
syringe configurations are known in the art and are described
herein.
[0107] Compositions containing a therapeutically effective amount
of the powdered molecules described herein can be delivered to any
suitable target tissue via the above-described particle delivery
devices. For example, the compositions can be delivered to muscle,
skin, brain, lung, liver, spleen, bone marrow, thymus, heart,
lymph, blood, bone cartilage, pancreas, kidney, gall bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system,
eye, gland and connective tissues. For nucleic acid molecules,
delivery is preferably to, and the molecules expressed in,
terminally differentiated cells; however, the molecules can also be
delivered to non-differentiated, or partially differentiated cells
such as stem cells of blood and skin fibroblasts.
[0108] The powdered compositions are administered to the subject to
be treated in a manner compatible with the dosage formulation, and
in an amount that will be prophylactically and/or therapeutically
effective. The amount of the composition to be delivered, generally
in the range of from 0.5 .mu.g/kg to 100 .mu.g/kg of nucleic acid
molecule per dose, depends on the subject to be treated. Doses for
other pharmaceuticals, such as physiological active peptides and
proteins, generally range from about 0.1 .mu.g to about 20 mg,
preferably 10 .mu.g to about 3 mg. The exact amount necessary will
vary depending on the age and general condition of the individual
to be treated, the severity of the condition being treated, the
particular preparation delivered, the site of administration, as
well as other factors. An appropriate effective amount can be
readily determined by one of skill in the art.
[0109] Thus, a "therapeutically effective amount" of the present
particulate compositions will be sufficient to bring about
treatment or prevention of disease or condition symptoms, and will
fall in a relatively broad range that can be determined through
routine trials.
[0110] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
EXAMPLES
[0111] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
Example 1
Nucleic Acid Immunization Using HSV-2 Cosmids
[0112] In order to assess the specificity and effectiveness of
nucleic acid immunization using DNA vaccine cosmids containing
HSV-2 genomic DNA, the following studies were carried out.
A. Cosmid Preparation
[0113] HSV-2 Cosmids:
[0114] HSV-2 genomic DNA was obtained by infecting Vero cells (ATCC
# CCL-81) with HSV-2 strain MS (ATCC # VR-54T) and isolating
genomic DNA 10' from the infected cells. Cosmids containing
fragments of the HSV-2 genome were generated by digesting HSV-2
genomic DNA, as described above, with the restriction enzyme EcoRI
(New England Biolabs). As shown in FIG. 1, the HSV genome has been
mapped and contains at least 10 EcoR1 sites, indicated in the
figure with arrows. The digested DNA was ligated into cosmids using
the SuperCos 1 Cosmid Vector Kit from Stratagene following the
manufacturers instructions. Positive cosmids were first identified
from the fragment sizes generated by an EcoRI digest of purified
cosmid DNA. The digested DNA was loaded onto a 1% agarose gel (15
hour pulse field electrolysis, run time at 6V/cm; 120.degree.
angle, 14.degree. C. in 0.5.times.TBE buffer). Further
identification was based on restriction maps generated with other
restriction enzymes. This preliminary analysis was used to
determine fragment locations relative to the HSV-2 genome shown in
FIG. 1. For example, cosmid #68 contains a fragment of the HSV-2
genome extending from the 7th to 110th EcoR1 sites shown, while
clone #74 contains an HSV-2 fragment extending from the 1 st
through 4th depicted EcoR1 sites.
B. Preparation of Coated Microparticles
[0115] Cosmid DNA was coated onto 1-3 .mu.m gold particles (Degussa
Corp., South Plainfield, N.J.) using techniques described by
Eisenbraun et al. (1993) DNA Cell Biol. 12:791-797. Briefly, cosmid
DNA was affixed to gold particles by adding 13.26 mg of 1-3 micron
gold powder (Degussa) and an appropriate amount of cosmid DNA (2
.mu.g per mg of gold powder) to a 1.5 ml centrifuge tube containing
500 .mu.l of 0.05 M spermidine (Sigma). Cosmid DNA and gold were
coprecipitated by the addition of 50011 of 10% CaCl.sub.2
(Fujisawa) dropwise while vortexing, after which the precipitate
was allowed to settle for several minutes. The gold/DNA precipitate
was concentrated by centrifugation in a microcentrifuge for 15
seconds, washed three time in absolute ethanol (Spectrum) and
resuspended in an appropriate amount of ethanol (8.84 mg gold
powder/ml of ethanol) and polyvinyl pyrrilodine (PVP) (Spectrum)
0.05 mg/ml of ethanol. The suspension was then transferred to a
glass vial which was capped and immersed in a sonicating water bath
for 2-5 seconds to resolve clumps. The ethanol solution was then
injected into Tefzel tubing (McMaster-Carr) and the gold/DNA
adhered to the side of the tubing using centrifugal force. The
remaining ethanol was then ejected using a small, controlled flow
of nitrogen gas. The tube was then dried for one hour with a flow
of nitrogen gas, and then cut into half inch cartridges. The
cartridges were stored in, a tightly capped glass scintillation
vials with dessicant at 4.degree. C. until use.
[0116] The DNA-coated gold particles were then loaded into
Tefzel.RTM. tubing as described in U.S. Pat. No. 5,584,807 to
McCabe, and the tubing was cut into 1.27 cm lengths to serve as
cartridges in a particle delivery device. The helium-pulse
PowderJect.RTM. XR particle delivery device has been previously
described (see, U.S. Pat. No. 5,584,807) and was obtained from
PowderJect Vaccines, Madison, Wis. In the vaccinations, each 1.27
cm cartridge nominally contained 0.5 mg gold particles coated with
1 .mu.g of DNA.
C. Immunizations
[0117] Balb/c mice were immunized by particle-mediated delivery
using a single shot of gold beads coated with cosmid (approximately
1 .mu.g cosmid/mg gold, 0.5 mg gold delivered per shot). Animals
were boosted 4 weeks after priming. Serum was collected at the time
of boosting (4 week) and two weeks later (6 week).
[0118] Serum antibody levels in mice at 6 weeks were detected using
an ELISA. Briefly, HSV-2 antigen was prepared at a concentration of
10 .mu.g/ml in PBS. One hundred microliters of the HSV-2 antigen
preparation was added to each well of a 96 well plates (Costar),
for a concentration of approximate 1 .mu.g of protein/well. The
plates were incubated overnight at 4.degree. C. and then washed 3
times with PBS/0.05% Tween 20 solution. The plates were blocked
with PBS/Tween with 5% dry milk for 1 hour at room temperature.
Dilute rabbit anti-HSV-2 (control) antibody or mouse serum
(unknowns) at 1:100 in PBS/Tween was added to appropriate wells.
The plates were washed 3 times with PBS/0.05% Tween 20. A 1:8000
dilution of biotin conjugated goat anti-rabbit in PBS/Tween was
prepared and 100 .mu.l added to each well. The plates were washed 3
times with PBS/0.05% Tween 20. A 1:8000 dilution of
streptavidin-HRP was prepared, 100 .mu.l added to each well for a 1
hour incubation at room temperature. The plates were washed 6 times
with PBS/0.05% Tween 20. TMB was prepared just prior to use, 100
.mu.l added to each well and incubated for 15 at room temperature.
Reactions were stopped by the addition of 100 .mu.l 1 N
H.sub.2SO.sub.4 and optical density values read on an automated
plate reader at 450 nm. Values from the 1:160 dilution were
averaged for the individual animals (4 per group) and the optical
density of the control group (animals vaccinated with empty vector)
was subtracted. Results for particular cosmids are shown in FIG. 2.
The numbers above the bars are the names of the different cosmid
clones.
D. Cell Mediated Immune Response
[0119] Spleen cell proliferation assays were done to further assess
the ability of an HSV-2 genome-derived cosmid to induce a
cell-mediated immune (CMI) response. This assay measures the
capacity of spleen-derived lymphocytes cultured in vitro to
proliferate, i.e., grow and divide thus increasing in number, in
response to HSV-2 antigens. The greater the amount of
proliferation, as assessed by a higher stimulation index score, the
stronger the immune response against the antigens.
[0120] BALB/c mice were immunized with either a plasmid containing
the gene for expression of the HSV-2 gD-antigen PCR amplified from
HSV-2 genomic DNA cloned into a pTarget vector (Promega), or with a
HSV-2 genomic-derived cosmid (designated No. 68 as described
above). A PowderJect.RTM. XR particle delivery device was used to
vaccinate the mice by delivery of plasmid/cosmid DNA-coated
gold-particles into the epidermis. Mice were immunized with a prime
and two boosts (each given 4 weeks apart). Each immunization
consisted of a single injection that delivered 1 .mu.g of DNA/0.5
mg gold. These mice had also been injected in their ear pinna with
protein extracts (100 .mu.g in PBS), 1-2 weeks after the third
boost immunization, to assess their ability to mount a delayed-type
hypersensitivity response. The right and left ears of these animals
were injected with protein extracts derived from HSV-2 infected and
uninfected cultured VERO cells (ATCC #CCL-81), respectively.
[0121] Three weeks after protein injection two mice were sacrificed
from each immunization group (designated in the figure legend-box
as "gD Plasmid and Cosmid #68"), along with two non-immune naive
mice. The spleen cells from these animals were isolated and
cultured in vitro with either pure gD protein (0.1 .mu.g/well), or
HSV-2 infected VERO cell protein extract (25 or 10 .mu.g/well), or
no added antigen. After 3 days in culture the antigen induced
proliferation responses of the different spleen cell populations
were quantified by the amount of radioactively labeled thymidine
that was incorporated into the dividing cells (measured as counts
per minute in a scintillation counter). Stimulation indices were
calculated by dividing the amount of radioactivity, i.e., counts
per minute (cpm), incorporated within antigen-stimulated spleen
cells by the cpm of their non-antigen-stimulated spleen cell
counterparts. In FIG. 3, the stimulation indices of the
antigen-stimulated non-immune naive spleen cells were subtracted
from that of the spleen cells derived from the immunized mice to
give a true representation of the level of antigen-specific
cell-mediated immunity elicited by the gD-antigen expressing
plasmid compared to that elicited by the HSV-2 genomic-derived
cosmid.
[0122] These results clearly demonstrate that the HSV-2
genomic-derived cosmid #68 is capable of eliciting a strong CMI
response against HSV-2 antigens in mice. The CMI response to gD
protein was slightly higher for spleen cells obtained from gD
plasmid immunized mice compared to that of spleen cells obtained
from mice immunized with cosmid #68. In contrast, the CMI response
to protein extract from HSV-2 infected VERO cells, which contains a
multiple array of HSV-2 antigens, was significantly higher for the
spleens cells obtained from the mice immunized with cosmid #68
compared to that of the spleen cells obtained from the mice
immunized with the single gD-antigen expressing plasmid. Thus,
these data demonstrate the utility of using a cosmid-based
DNA-vaccine to produce a stronger CMI response.
[0123] Similarly experiments were carried out with above-described
HSV-2 genomic-derived cosmids in a guinea pig animal model system.
The cosmids also elicited a strong CMI response in these animals.
In subsequent challenge studies in the immunized guinea pigs,
protective immunity was not observed; however, these results were
deemed inconclusive.
Example 2
Nucleic Acid Immunization Using HSV-2 Plasmids
A. Plasmid Construction
[0124] HSV-2 genomic DNA was obtained by infecting VERO cells (ATCC
#CCL-81) with HSV-2 strain MS (ATCC #VR-54T) and isolating genomic
DNA from purified viral particles. PCR was performed using the
genomic DNA to amplify segments for cloning into a plasmid
vector.
[0125] The primer gB2, having the following sequence: CGC GTC TAG
AAA CGT TCG CGA CCA CGG GTG AC (SEQ ID NO: 3) and primer gB3,
having the following sequence: CGC GTC TAG ATG ATG GGG TCC CGC TAA
CTC GC (SEQ ID NO: 4), which correspond to sequences at 58160 and
52930 of the HSV-2 genome, respectively, were used to amplify a
product of approximately 5200 bp by PCR. A second PCR product was
generated with using primer gB2 (SEQ ID NO: 3) and primer gB4,
having the following sequence: CGC GTC TAG ACC TTC ATG ACC GCG CTG
GTC CT (SEQ ID NO: 5) which correspond to sequences at 58160 and
49670, respectively, of the HSV-2 genome. This produced a product
of approximately 8500 bp. Both products contain the genomic
sequences coding for the glycoprotein B protein.
[0126] The PCR cycle specifications were: 98.degree. C. 30 seconds,
70.degree. C. 10 minutes, for 35 cycles. PCR reactions were carried
out using the Expand Long template PCR system (Boehringer
Mannheim). Reaction conditions were 20 pmoles of each primer, 1
.mu.g of HSV-2 genomic DNA, 1.times. buffer #1 (5 mM Tris-HCl, pH
9.2, 1.6 mM (NH.sub.4).sub.2SO.sub.4, 1.75 mM MgCl.sub.2), 200
.mu.M deoxynucleotides (dNTPs, Sigma), 1% DMSO, 10 mM MgSO.sub.4,
H.sub.2O to 50 .mu.L and 1U of Expand DNA polymerase. PCR products
were "A-tailed" by incubating the products with 1U Taq DNA
polymerase (Promega) for 10 minutes at 72.degree. C. after the
addition of an additional 100 .mu.M dNTPs. The PCR fragments were
purified from agarose gels and ligated into pGEM-T Easy vector
system (Promega) using manufacturers instructions. TOP 10 E. coli
competent bacteria (Stratagene) were transformed with the ligation
mixtures by electroporation and bacteria retaining plasmid were
isolated on agar plates prepared with ampicillin. DNA minipreps
were made from overnight cultures of selected bacterial colonies
and purified plasmids were tested for the desired inserts. Mobility
on agarose gels and an appropriate restriction pattern was used to
identify positive plasmids.
B. Preparation of Coated Microparticles
[0127] Plasmid DNA was coated onto 1-3 .mu.m gold particles
(Degussa Corp., South Plainfield, N.J.) using techniques described
by Eisenbraun et al. (1993) DNA Cell Biol. 12:791-797. Briefly,
plasmid DNA was affixed to gold particles by adding gold powder and
an appropriate amount of plasmid DNA to a centrifuge tube
containing spermidine. Plasmid DNA and gold were coprecipitated by
the addition of CaCl.sub.2 (Fujisawa) dropwise while vortexing,
after which the precipitate was allowed to settle for several
minutes. The gold/DNA precipitate was concentrated by
centrifugation in a microcentrifuge, washed three time in absolute
ethanol and resuspended in an appropriate amount of ethanol and
polyvinyl pyrrilodine (PVP). The suspension was then transferred to
a glass vial which was capped and immersed in a sonicating water
bath for 2-5 seconds to resolve clumps. The ethanol solution was
then injected into Tefzel.RTM. tubing (McMaster-Carr) and the
gold/DNA adhered to the side of the tubing using centrifugal force.
The remaining ethanol was then removed using a small, controlled
flow of nitrogen gas. The tube was then dried for with a flow of
nitrogen gas, and then cut into cartridges. The cartridges were
stored in a tightly capped glass scintillation vials with dessicant
at 4.degree. C. until use.
C. Immunizations
[0128] C57/black mice were immunized by particle-mediated delivery
using a single shot of gold beads coated with plasmid
(approximately 2 .mu.g plasmid/mg gold, 0.5 mg gold delivered per
shot). Animals are boosted 4 weeks after priming. Serum is
collected at the time of boosting (4 week) and is also collected
two weeks later (6 week).
[0129] Serum antibody levels in mice at 6 weeks is detected using
an ELISA substantially identical to that described in Example 1
above. Spleen cell proliferation assays are also carried out to
further assess the ability of an HSV-2 genome-derived plasmid to
induce a cell-mediated immune (CMI) response.
[0130] Thus, this study demonstrates the utility of using a
plasmid-based DNA-vaccine to elicit an immune response.
[0131] Accordingly, novel compositions for eliciting an immune
response have been described. Methods of using these compositions
have also been described. Although preferred embodiments of the
subject invention have been described in some detail, it is
understood that obvious variations can be made without departing
from the spirit and the scope of the invention as defined by the
appended claims.
Sequence CWU 1
1
5 1 20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 tccatgacgt tcctgatgct 20 2 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 2 atcgactctc gagcgttctc 20 3 32 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 3
cgcgtctaga aacgttcgcg accacgggtg ac 32 4 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 4 cgcgtctaga
tgatggggtc ccgctaactc gc 32 5 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 5 cgcgtctaga
ccttcatgac cgcgctggtc ct 32
* * * * *
References