Note: Descriptions are shown in the official language in which they were submitted.
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USE OF SUBMICRON OIL-IN-WATER
EMULSIONS WITH DNA VACCINES
Technical Field
The present invention relates generally to
vaccine compositions. In particular, the invention
relates to the use of submicron oil-in-water emulsions
with nucleic acid vaccines.
Backaround of the Invention
Numerous vaccine formulations which include
attenuated pathogens or subunit protein antigens, have
been developed. Conventional vaccine compositions
often include immunological adjuvants to enhance
immune responses. For example, depot adjuvants are
frequently used which adsorb and/or precipitate
administered antigens and which can retain the antigen
at the injection site. Typical depot adjuvants
include aluminum compounds and water-in-oil emulsions.
However, depot adjuvants, although increasing
antigenicity, often provoke severe persistent local
reactions, such as granulomas, abscesses and scarring,
when injected subcutaneously or intramuscularly.
Other adjuvants, such as lipopolysacharrides and
muramyl dipeptides, can elicit pyrogenic responses
upon injection and/or Reiter's symptoms (influenza-
like symptoms, generalized joint discomfort and
sometimes anterior uveitis, arthritis and urethritis).
Saponins, such as Quillaja saponaria, have also been
used as immunological adjuvants in vaccine
compositions against a variety of diseases. Recently,
MF59, a safe, highly immunogenic, submicron oil-in-
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water emulsion, has been developed for use in vaccine
compositions. See, e.g., Ott et al., "MF59 -- Design
and Evaluation of a Safe and Potent Adjuvant for Human
Vaccines" in Vaccine Design: The Subunit and Adjuvant
Approach (Powell, M.F. and Newman, M.J. eds.) Plenum
Press, New York, 1995, pp. 277-296.
Despite the presence of such adjuvants,
conventional vaccines often fail to provide adequate
protection against the targeted pathogen. In this
regard, there is growing evidence that vaccination
against intracellular pathogens, such as a number of
viruses, should target both the cellular and humoral
arms of the immune system.
Cytotoxic T-lymphocytes (CTLs) play an
important role in cell-mediated immune defense against
intracellular pathogens such as viruses and tumor-
specific antigens produced by malignant cells. CTLs
mediate cytotoxicity of virally infected cells by
recognizing viral determinants in conjunction with
class I MHC molecules displayed by the infected cells.
Cytoplasmic expression of proteins is a prerequisite
for class I MHC processing and presentation of
antigenic peptides to CTLs. However, immunization
with killed or attenuated viruses often fails to
produce the CTLs necessary to curb intracellular
infection. Furthermore, conventional vaccination
techniques against viruses displaying marked genetic
heterogeneity and/or rapid mutation rates that
facilitate selection of immune escape variants, such
as HIV or influenza, are problematic. Accordingly,
alternative techniques for vaccination have been
developed.
Direct injection of DNA and mRNA into
mammalian tissue for the purpose of eliciting an
immune response has been described. See, e.g., U.S.
Patent No. 5,589,466. The method, termed "nucleic
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acid immunization" herein, has been shown to elicit
both humoral and cell-mediated immune responses. For
example, sera from mice immunized with a human
immunodeficiency virus type 1 (HIV-1) DNA construct
encoding the envelope glycoprotein, gp160, were shown
to react with recombinant gp160 in immunoassays and
lymphocytes from the injected mice were shown to
proliferate in response to recombinant gp120. Wang et
al., Proc. Natl. Acad. Sci. USA (1993) 90:4156-4160.
Similarly, mice immunized with a plasmid containing a
genomic copy of the human growth hormone (hGH) gene,
demonstrated an antibody-based immune response. Tang
et al., Nature (1992) 356:152-154. Intramuscular
injection of DNA encoding influenza nucleoprotein
driven by a mammalian promoter has been shown to
elicit a CD8+ CTL response that can protect mice
against subsequent lethal challenge with virus. Ulmer
et al., Science (1993) 259:1745-1749.
Immunohistochemical studies of the injection site
revealed that the DNA was taken up by myeloblasts, and
cytoplasmic production of viral protein could be
demonstrated for at least six months.
Conventional uses of adjuvants have involved
codelivery of the adjuvants with antigens, in order to
invoke immune responses against the antigens.
Recently, the saponin adjuvant, QS-21, has been
reported to increase the cell-mediated immune response
to a naked DNA vaccine directed against HIV. Genetic
Engineering News, June 1, 1997, p. 33.
However, the use of other adjuvants,
including submicron oil-in-water emulsions, to enhance
immunogenicity of nucleic acid vaccines has not
heretofore been described. Furthermore, the temporal
dissociation between delivery of adjuvant and delivery
of nucleic acid vaccines has not previously been
described.
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Disclosure of the Invention
The present invention is based on the
surprising and unexpected discovery that the use of a
submicron oil-in-water emulsion serves to enhance the
immunogenicity of nucleic acid vaccines. The use of
such emulsions provides a safe and effective approach
for enhancing the immunogenicity of nucleic acid
vaccines against a wide variety of pathogens. The
submicron oil-in-water emulsion need not be
administered at the same time as the gene of interest,
but may be administered prior or subsequent to
delivery of the gene. Indeed, surprisingly good
results are seen when the emulsion is administered
prior to delivery of the gene.
Accordingly, in one embodiment, the
invention is directed to a method of immunization
which comprises administering a submicron oil-in-water
emulsion to a vertebrate subject, and transfecting
cells of said subject with a recombinant vector
comprising a nucleic acid molecule encoding an antigen
of interest, under conditions that permit the
expression of said antigen, thereby eliciting an
immunological response to said antigen of interest.
In additional embodiments, the recombinant
vector is a nonviral vector, or a viral vector, such
as a retroviral, vaccinia or canarypox vector.
In still another embodiment, the invention
is directed to a method of immunization which
comprises administering MF59 to a mammalian subject
and immunizing said subject with a recombinant vector
comprising a nucleic acid molecule encoding a viral
antigen of interest, under conditions that permit the
expression of said antigen, thereby eliciting an
immunological response to said antigen of interest.
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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 Figures
Figures lA and IH show the results of a SICr
release assay performed on splenocytes from C3H mice
given the specified adjuvant two days prior to
retroviral vector delivery, as described in Example
2a. Figure lA depicts results from mice administered
undiluted retrovirus vector 6A3. Figure 1B depicts
results from mice administered retrovirus vector 6A3,
diluted 1:10.
Figure 2 shows the average IgGl response to
gp120 in mice pretreated with the specified adjuvant
two days prior to retroviral vector delivery, as
described in Example 3.
Detailed Description of the Invention
The practice of the present invention will
employ, unless otherwise indicated, conventional
methods of virology, microbiology, molecular biology,
recombinant DNA techniques and immunology within the
skill of the art. Such techniques are explained fully
in the literature. See, e.g., Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (2nd Edition,
1989); DNA Cloning: A Practical Approach, vol. I & II
(D. Glover, ed.); 0ligonucleotide Synthesis (N. Gait,
ed., 1984); A Practical Guide to Molecular Cloning
(1984); Fundamental Virology, 2nd Edition, vol. I & II
(B.N. Fields and D.M. Knipe, eds.); Methods In
Enzymology {S. Colowick and N. Kaplan, eds., Academic
Press, Inc.); and Handbook of Experimental Immunology,
Vols. I-IV (D. M. Weir and C.C. Blackwell, eds., 1986,
Blackwell Scientific Publications)
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As used in this specification and the _
appended claims, the singular forms "a," "an" and
"the" include plural references unless the content
clearly dictates otherwise.
I. Definitions
In describing the present invention, the
following terms will be employed, and are intended to
be defined as indicated below.
By "nucleic acid immunization" is meant the
introduction of a nucleic acid molecule encoding one
or more selected antigens into a host cell, for the in
vivo expression of the antigen or antigens. The
nucleic acid molecule can be introduced into the
recipient subject, using nonviral vectors, viral
vectors or bacterial vectors (as described further
below) such as by injection, inhalation, oral,
intranasal and mucosal administration, or the like, or
can be introduced ex vivo, into cells which have been
removed from the host. In the latter case, the
transformed cells are reintroduced into the subject
where an immune response can be mounted against the
antigen encoded by the nucleic acid molecule.
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 when the antigen is
presented, or a humoral antibody response. Normally,
an epitope will include between about 3-15, generally
about 5-15, amino acids. For purposes of the present
invention, antigens can be derived from any of several
known viruses, bacteria, parasites and fungi. The
term also intends any of the various tumor antigens.
Furthermore, for purposes of the present invention, an
"antigen" refers to a protein which includes
modifications, such as deletions, additions and
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substitutions (generally conservative in nature), to _
the native sequence, so long as the protein maintains
the ability to elicit an immunological response.
These modifications may be deliberate, as through
site-directed mutagenesis, or may be accidental, such
as through mutations of hosts which produce the
antigens.
An "immunological response" to an antigen or
composition is the development in a subject of a
humoral and/or a cellular immune response to molecules
present in the composition of interest. For purposes
of the present invention, a "humoral immune response"
refers to an immune response mediated by antibody
molecules, while a "cellular immune response" is one
mediated by T-lymphocytes and/or other white blood
cells. One important aspect of cellular immunity
involves an antigen-specific response by cytolytic T-
cells ("CTL"s). CTLs have specificity for peptide
antigens that are presented in association with
proteins encoded by the major histocompatibility
complex (MHC) and expressed on the surfaces of cells.
CTLs help induce and promote the intracellular
destruction of intracellular microbes, or the lysis of
cells infected with such microbes. Another aspect of
cellular immunity involves an antigen-specific
response by helper T-cells. Helper T-cells act to
help stimulate the function, and focus the activity
of, nonspecific effector cells against cells
displaying peptide antigens in association with MHC
molecules on their surface. A "cellular immune
response" also refers to the production of cytokines,
chemokines and other such molecules produced by
activated T-cells and/or other white blood cells,
including those derived from CD4+ and CD8+ T-cells.
A composition or vaccine that elicits a
cellular immune response may serve to sensitize a
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vertebrate subject by the presentation of antigen in
association with MHC molecules at the cell surface.
The cell-mediated immune response is directed at, or
near, cells presenting antigen at their surface. In
addition, antigen-specific T-lymphocytes can be
generated to allow for the future protection of an
immunized host.
The ability of a particular antigen or
composition to stimulate a cell-mediated immunological
response may be determined by a number of assays, such
as by lymphoproliferation (lymphocyte activation)
assays, CTL cytotoxic cell assays, or by assaying for
T-lymphocytes specific for the antigen in a sensitized
subject. Such assays are well known in the art. See,
e.g., Erickson et al., J. Immunol. (1993) 151:4189-
4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-
2376; and the examples below.
Thus, an immunological response as used
herein may be one which stimulates the production of
CTLs, and/or the production or activation of helper T-
cells. The antigen of interest may also elicit an
antibody-mediated immune response. Hence, an
immunological response may include one or more of the
following effects: the production of antibodies by B-
cells; and/or the activation of suppresser T-cells
and/or 'y8 T-cells directed specifically to an antigen
or antigens present in the composition or vaccine of
interest. These responses may serve to neutralize
infectivity, and/or mediate antibody-complement, or
antibody dependent cell cytotoxicity (ADCC) to provide
protection to an immunized host. Such responses can
be determined using standard immunoassays and
neutralization assays, well known in the art.
A "coding sequence" or a sequence which
"encodes" a selected antigen, is a nucleic acid
molecule which is transcribed (in the case of DNA) and
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translated (in the case of mRNA) into a polypeptide in _
vivo when placed under the control of appropriate
regulatory sequences. 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
eucaryotie 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.
A "nucleic acid" molecule can include, but
is not limited to, procaryotic sequences, eucaryotie
mRNA, cDNA from eucaryotie mRNA, genomic DNA sequences
from eucaryotie (e. g., mammalian) DNA, and even
synthetic DNA sequences. The term also captures
sequences that include any of the known base analogs
of DNA and RNA.
By "vector" is meant any genetic element,
such as a plasmid, phage, transposon, cosmid,
chromosome, virus, virion, recombinant virus, etc.,
which can deliver gene sequences to a desired cell or
tissue. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
"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 operably linked to a coding
sequence is capable of effecting the expression of the
coding sequence when the proper enzymes are present.
The promoter need not be contiguous with the coding
sequence, so long as it functions to direct the
expression thereof. Thus, for example, intervening
untranslated yet transcribed sequences can be present
between the promoter sequence and the coding sequence
_g_
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and the promoter sequence can still be considered
"operably linked" to the coding sequence.
"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 "re-
combinant" as used with respect to a protein or
polypeptide means a polypeptide produced by expression
of a recombinant polynucleotide.
Two nucleic acid or polypeptide sequences
are "substantially homologous" when at least about
70%, preferably at least about 80-900, and most
preferably at least about 95%, of the nucleotides or
amino acids match over a defined length of the
molecule. As used herein, substantially homologous
also refers to sequences showing identity to the
specified nucleic acid or polypeptide sequence.
Nucleic acid sequences that are substantially
homologous can be identified in a Southern
hybridization experiment under, for example, stringent
conditions, as defined for that particular system.
Defining appropriate hybridization conditions is
within the skill of the art. See, e.g., Sambrook et
al . , supra; DNA Cloning, vols I & II, supra; Nucleic
Acid Hybridization, supra. Such sequences can also be
confirmed and further characterized by direct
sequencing of PCR products.
The terms "effective amount" or
"pharmaceutically effective amount" of an agent, as
provided herein, refer to a nontoxic but sufficient
amount of the agent to provide the desired
immunological response and corresponding therapeutic
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effect. As will be pointed out below, the exact _
amount required will vary from subject to subject,
depending on the species, age, and general condition
of the subject, the severity of the condition being
treated, and the particular antigen of interest, mode
of administration, and the like. An appropriate
"effective" amount in any individual case may be
determined by one of ordinary skill in the art using
routine experimentation.
As used herein, "treatment " refers to any of
(i) the prevention of infection or reinfection, as in
a traditional vaccine, (ii) the reduction or
elimination of symptoms, and (iii) the substantial or
complete elimination of the pathogen in question from
the patient. Treatment may be effected
prophylactically (prior to infection) or
therapeutically (following infection).
By "vertebrate subject" is meant any member
of the subphylum cordata, including, without
limitation, humans and other primates, including non-
human primates such as chimpanzees and other apes and
monkey species; farm animals such as cattle, sheep,
pigs, goats and horses; domestic mammals such as dogs
and cats; laboratory animals including rodents such as
mice, rats and guinea pigs; birds, including domestic,
wild and game birds such as chickens, turkeys and
other gallinaceous birds, ducks, geese, and the like.
The term does not denote a particular age. Thus, both
adult and newborn individuals are intended to be
covered. The system described above is intended for
use in any of the above vertebrate species, since the
immune systems of all of these vertebrates operate
similarly.
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II. Modes of Carrying Out the Invention _
The present invention is based on the
discovery that the use of submicron oil-in-water
emulsions in combination with nucleic acid
immunization, can provide a vigorous immune response,
even when the gene delivered encodes for a protein
which is by itself weakly immunogenic.
In particular, the method of the invention
provides for cell-mediated immunity, and/or humoral
antibody responses. Thus, in addition to a
conventional antibody response, the system herein
described can provide for, e.g., the association of
the expressed antigens with class I MHC molecules such
that an in vivo cellular immune response to the
antigen of interest can be mounted which stimulates
the production of CTLs to allow for future recognition
of the antigen. Furthermore, the method may elicit an
antigen-specific response by helper T-cells.
Accordingly, the methods of the present invention will
find use with any antigen for which cellular and/or
humoral immune responses are desired, including
antigens derived from viral, bacterial, fungal and
parasitic pathogens that may induce antibodies, T-cell
helper epitopes and T-cell cytotoxic epitopes. Such
antigens include, but are not limited to, those
encoded by human and animal viruses and can correspond
to either structural or non-structural proteins.
The technique is particularly useful for
immunization against intracellular viruses and tumor
cell antigens which normally elicit poor immune
responses. For example, the present invention will
find use for stimulating an immune response against a
wide variety of proteins from the herpesvirus family,
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 derived from
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varicella zoster virus (VZV), Epstein-Barr virus (EBV)
and cytomegalovirus (CMV) including CMV gB and gH; and
antigens derived from other human herpesviruses such
as HHV6 and HHV7. (See, e.g. Chee et al.,
Cytomegaloviruses (J. K. McDougall, ed., Springer-
Verlag 1990) pp. 125-169, for a review of the protein
coding content of cytomegalovirus; McGeoch et al., J.
Gen. Virol. (1988) 69:1531-1574, for a discussion of
the various HSV-1 encoded proteins; U.S. Patent No.
5,171,568 for a discussion of HSV-1 and HSV-2 gB and
gD proteins and the genes encoding therefor; Baer et
al., Nature (1984) 310:207-211, for the identification
of protein coding sequences in an EBV genome; and
Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816,
for a review of VZV.)
Polynucleotide sequences encoding antigens
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), can also be conveniently used in the techniques
described herein. By way of example, the viral
genomic sequence of HCV is known, as are methods for
obtaining the sequence. 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 (also known as E) and E2 (also
known as E2/NSI) and an N-terminal nucleocapsid
protein (termed "core") (see, Houghton et al.,
Hepatology (1991) 14:381-388, for a discussion of HCV
proteins, including E1 and E2). The sequences
encoding each of these proteins, as well as antigenic
fragments thereof, will find use in the present
methods. Similarly, the coding sequence for the b-
antigen from HDV is known (see, e.g., U.S. Patent No.
5,378,814) and this sequence can also be conveniently
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used in the present methods. Additionally, antigens
derived from HBV, such as the core antigen, the
surface antigen, sAg, as well as the presurface
sequences, pre-S1 and pre-S2 (formerly called pre-S),
as well as combinations of the above, such as sAg/pre-
S1, sAg/pre-S2, sAg/pre-S1/pre-S2, and pre-S1/pre-S2,
will find use herein. See, e.g., "HBV Vaccines - from
the laboratory to license: a case study" in Mackett,
M. and Williamson, J.D., Human Vaccines and
Vaccination, pp. 159-176, for a discussion of HBV
structure; and U.S. Patent Nos. 4,722,840, 5,098,704,
5,324,513; Beames et al., J. Virol. (1995) 69:6833-
6838, Birnbaum et al., J. Virol. (1990) 64:3319-3330;
and Zhou et al., J. Virol. (1991) 65:5457-5464.
Polynucleotide sequences encoding antigens
derived from other viruses will also find use in the
claimed methods, such as without limitation, proteins
from members of the families Picornaviridae (e. g.,
polioviruses, etc.); Caliciviridae; Togaviridae (e. g.,
rubella virus, dengue virus, etc.); Flaviviridae;
Coronaviridae; Reoviridae; Birnaviridae;
Rhabodoviridae (e. g., rabies virus, etc.);
Filoviridae; Paramyxoviridae (e. g., mumps virus,
measles virus, respiratory syncytial virus, etc.);
Orthomyxoviridae (e.g., influenza virus types A, B and
C, 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 HIVIIIb, HIVsF2, HIVI,p,~, HIV~"I,
HIV,,Q,~) ; HIV-1CM235~ HIV-lUS4% HIV-2; simian
immunodeficiency virus (SIV) among others.
Additionally, antigens may also be derived from human
papillomavirus (HPV) and the tick-borne encephalitis
viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik
ed. 1988); Fundamental Virology, 2nd Edition (B. N.
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Fields and D.M. Knipe, eds. 1991), for a description
of these and other viruses.
More particularly, genes encoding the gp120
envelope protein from any of the above HIV 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, New Mexico (1992); Myers et al., Human
Retroviruses and Aids, 1990, Los Alamos, New Mexico:
Los Alamos National Laboratory; and Modrow et al., J.
Virol. (1987) 61:570-578, for a comparison of the
envelope gene sequences of a variety of HIV isolates)
and sequences derived from any of these isolates will
find use in the present methods. Furthermore, the
invention is equally applicable to other immunogenic
proteins derived from any of the various HIV isolates,
including any of the various envelope proteins such as
gp160 and gp4l, gag antigens such as p24gag and
p55gag, as well as proteins derived from the pol
region.
As explained above, influenza virus is
another example of a virus for which the present
invention will be particularly useful. Specifically,
the envelope glycoproteins HA and NA of influenza A
are of particular interest for generating an immune
response. Numerous HA subtypes of influenza A have
been identified (Kawaoka et al., Virology (1990)
179:759-767; Webster et al., "Antigenic variation
among type A influenza viruses," p. 127-168. In: P.
Palese and D.W. Kingsbury (ed.), Genetics of influenza
viruses. Springer-Verlag, New York). Thus, the gene
sequences encoding proteins derived from any of these
isolates can also be used in the nucleic acid
immunization techniques described herein.
The techniques can be used for the delivery
of discrete antigens, larger portions of the genome in
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question and, for example, a proviral DNA which
includes nearly all of the viral genome.
The methods described herein will also find
use with DNA sequences encoding numerous bacterial
antigens, such as those derived from organisms that
cause diphtheria, cholera, tuberculosis, tetanus,
pertussis, meningitis, and other pathogenic states,
including, without limitation, Meningococcus A, B and
C, Hemophilus influenza type B (HIB), and Helicohacter
pylori. Examples of parasitic antigens include those
derived from organisms causing malaria and Lyme
disease.
Furthermore, the methods described herein
provide a means for treating a variety of malignant
cancers. For example, the system of the present
invention can be used to mount both humoral and cell-
mediated immune responses to particular proteins
specific to the cancer in question, such as an
activated oncogene, a fetal antigen, or an activation
marker. Such tumor antigens include any of the
various MAGEs {melanoma associated antigen E),
including MAGE 1, 2, 3, 4, etc. {Boon, T. Scientific
American (March 1993):82-89); any of the various
tyrosinases; MART 1 (melanoma antigen recognized by T
cells), mutant ras; mutant p53; p97 melanoma antigen;
CEA (carcinoembryonic antigen), among others.
It is readily apparent that the subject
invention can be used to prevent or treat a wide
variety of diseases.
Polynucleotide sequences coding for the
above-described molecules can be obtained using
recombinant methods, such as by screening cDNA and
genomic libraries from cells expressing the gene, or
by deriving the gene from a vector known to include
the same. Furthermore, the desired gene can be
isolated directly from cells and tissues containing
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the same, using standard techniques, such as phenol _
extraction and PCR of cDNA or genomic DNA. See, e.g.,
Sambrook et al., supra, for a description of
techniques used to obtain and isolate DNA. The gene
of interest can also be produced synthetically, rather
than cloned. The nucleotide sequence can be designed
with the appropriate codons for the particular amino
acid sequence desired. In general, one will select
preferred codons for the intended host in which the
sequence will be expressed. The complete sequence is
assembled from overlapping oligonucleotides prepared
by standard methods and assembled into a complete
coding sequence. See, e.g., Edge, Nature (1981)
292:756; Nambair et al., Science (1984) 223:1299; Jay
et al., J. Biol. Chem. (1984) 259:6311.
Next, the gene sequence encoding the desired
antigen can be inserted into a vector which includes
control sequences operably linked to the desired
coding sequence, which allow for the expression of the
gene in vivo in the subject species. For example,
typical promoters for mammalian cell expression
include the SV40 early promoter, a CMV promoter such
as the CMV immediate early promoter (Chapman et al.,
Nucl. Acids Res. (1991) 19:3979-3986), the mouse
mammary tumor virus LTR promoter, the adenovirus major
late promoter (Ad MLP), and the herpes simplex virus
promoter, among others. Other nonviral promoters,
such as a promoter derived from the murine
metallothionein gene, will also find use for mammalian
expression. Typically, transcription termination and
polyadenylation sequences will also be present,
located 3' to the translation stop codon. Preferably,
a sequence for optimization of initiation of
translation, located 5' to the coding sequence, is
also present. Examples of transcription
terminator/polyadenylation signals include those
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derived from SV40, as described in Sambrook et al.,
supra, as well as a bovine growth hormone terminator
sequence. Introns, containing splice donor and
acceptor sites, may also be designed into the
constructs for use with the present invention.
Enhancer elements may also be used herein to
increase expression levels of the mammalian
constructs. Examples include the SV40 early gene
enhancer, as described in Dijkema et al., EMBO J.
(1985) 4:761, the enhancer/promoter derived from the
long terminal repeat (LTR) of the Rous Sarcoma Virus,
as described in Gorman et al., Proc. Natl. Acad. Sci.
USA (1982b) 79:6777 and elements derived from human
CMV, as described in Boshart et al., Cell (1985)
41:521, such as elements included in the CMV intron A
sequence.
Furthermore, plasmids can be constructed
which include a chimeric gene sequence, encoding e.g.,
multiple antigens of interest, for example derived
from more than one viral isolate. Additionally, genes
coding for immune modulating agents which can enhance
antigen presentation, attract lymphocytes to the site
of gene expression or promote expansion of the
population of lymphocytes to the site of gene
expression or promote expansion of the population of
lymphocytes which respond to the expressed antigen,
can also be present. Such agents include cytokines,
lymphokines, and chemokines, including but not limited
to IL-2, modified IL-2 (cys125~ser125), GM-CSF, IL-12,
y-interferon, IP-10, MIPlc~, MIPl~i and RANTES.
Additionally, immune molecules such as TAP
transporters, costimulatory molecules such as B7, ,~2M,
class I or II MHC genes (syngeneic or allogeneic), and
other genes coding for proteins that are required for
efficient immune responses but are not expressed due
to specific inhibition or deletion, will also find use
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in the constructs. This is particularly relevant in
tumor cells and in some infected cells where antigen
presentation is often reduced.
The above sequences can be administered
using separate vectors or can be present on the vector
bearing the gene encoding the antigen of interest. If
present on the same vector, the additional gene
sequences can either precede or follow the gene
encoding the antigen of interest in a dicistronic gene
configuration. Additional control elements can be
situated between the various genes for efficient
translation of RNA from the distal coding region.
Alternatively, a chimeric transcription unit having a
single open reading frame encoding both the gene of
interest and the modulator, can also be constructed.
Either a fusion can be made to allow for the synthesis
of a chimeric protein or alternatively, protein
processing signals can be engineered to provide
cleavage by a protease such as a signal peptidase,
thus allowing liberation of the two or more proteins
derived from translation of the template RNA. Such
signals for processing of a polyprotein exist in,
e.g., flaviviruses, pestiviruses such as HCV, and
picornaviruses, and can be engineered into the
constructs. The processing protease may also be
expressed in this system either independently or as
part of a chimera with the antigen and/or cytokine
coding region(s). The protease itself can be both a
processing enzyme and a vaccine antigen.
Once complete, the constructs are used for
nucleic acid immunization using standard gene delivery
protocols. Methods for gene delivery are known in the
art. See, e.g., U.S. Patent Nos. 5,399,346,
5,580,859, 5,589,466. Genes can be delivered either
directly to the vertebrate subject or, alternatively,
delivered ex vivo, to cells derived from the subject
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and the cells reimplanted in the subject. Genes can _
be delivered using nonviral vectors, as described
above, viral vectors or bacterial vectors.
A number of viral based systems have been
developed for gene transfer into mammalian cells. For
example, retroviruses provide a convenient platform
for gene delivery systems. A selected gene can be
inserted into a vector and packaged in retroviral
particles using techniques known in the art. The
recombinant virus can then be isolated and delivered
to cells of the subject either in vivo or ex vivo. A
number of retroviral systems have been described (see,
e.g., U.S. Patent No. 5,219,740; International
Publication Nos. WO 91/02805 and WO 93/15207; Miller
and Rosman, BioTechniques (1989) 7:980-990; Miller,
A.D., Human Gene Therapy (1990) 1:5-14; Scarpa et al.,
Virology (1991) 180:849-852; Burns et al., Proc. Natl.
Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie
and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-
109) .
A number of adenovirus vectors have also
been described. Unlike retroviruses which integrate
into the host genome, adenoviruses persist
extrachromosomally thus minimizing the risks
associated with insertional mutagenesis (Haj-Ahmad and
Graham, J. Virol. (1986) 57:267-274; Bett et al., J.
Virol. (1993) 67:5911-5921; Mittereder et al., Human
Gene Therapy (1994) 5:717-729; Seth et al., J. Virol.
(1994) 68:933-940; Barr et al., Gene Therapy (1994)
1:51-58; Berkner, K.L. BioTechniques (1988) 6:616-629;
and Rich et al., Human Gene Therapy (1993) 4:461-476).
Additionally, various adeno-associated virus
(AAV) vector systems have been developed for gene
delivery. AAV vectors can be readily constructed
using techniques well known in the art. See, e.g.,
U.S. Patent Nos. 5,173,414 and 5,139,941;
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WO 99/02132 PCT/US98/14310
International Publication Nos. WO 92/01070 (published
23 January 1992) and WO 93/03769 (published 4 March
1993); Lebkowski et al., Molec. Cell. Biol. (1988)
8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold
Spring Harbor Laboratory Press); Carter, B.J. Current
Opinion in Biotechnology (1992) 3:533-539; Muzyczka,
N. Current Topics in Microbiol. and Immunol. (1992)
158:97-129; Kotin, R.M. Human Gene Therapy (1994)
5:793-801; Shelling and Smith, Gene Therapy (1994)
1:165-169; and Zhou et al., J. Exp. Med. (1994)
179:1867-1875.
Additional viral vectors which will find use
for delivering the nucleic acid molecules encoding the
antigens of interest include those derived from the
pox family of viruses, including vaccinia virus and
avian poxvirus. By way of example, vaccinia virus
recombinants expressing the genes can be constructed
as follows. The DNA encoding the particular antigen
is first inserted into an appropriate vector so that
it is adjacent to a vaccinia promoter and flanking
vaccinia DNA sequences, such as the sequence encoding
thymidine kinase (TK). This vector is then used to
transfect cells which are simultaneously infected with
vaccinia. Homologous recombination serves to insert
the vaccinia promoter plus the gene encoding the
antigen of interest into the viral genome. The
resulting TK-recombinant can be selected by culturing
the cells in the presence of 5-bromodeoxyuridine and
picking viral plaques resistant thereto.
Alternatively, avipoxviruses, such as the
fowlpox and canarypox viruses, can also be used to
deliver the genes. Recombinant avipox viruses,
expressing immunogens from mammalian pathogens, are
known to confer protective immunity when administered
to non-avian species. The use of an avipox vector is
particularly desirable in human and other mammalian
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species since members of the avipox genus can only -
productively replicate in susceptible avian species
and therefore are not infective in mammalian cells.
Methods for producing recombinant avipoxviruses are
known in the art and employ genetic recombination, as
described above with respect to the production of
vaccinia viruses. See, e.g., WO 91/12882; WO
89/03429; and WO 92/03545.
Molecular conjugate vectors, such as the
adenovirus chimeric vectors described in Michael et
al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et
al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103,
can also be used for gene delivery. Additionally, the
gene of interest can be delivered using pseudovirions,
such as a noninfectious retrovirus-like particle,
described in e.g., International Publication No. WO
91/05864, published 2 May 1991.
Members of the Alphavirus genus, such as but
not limited to vectors derived from the Sindbis and
Semliki Forest viruses, will also find use as viral
vectors for delivering the gene of interest. For a
description of Sinbus-virus derived vectors useful for
the practice of the instant methods, see,
International Publication No. WO 95/07995.
A vaccinia based infection/transfection
system can be conveniently used to provide for
inducible, transient expression of the gene of
interest in a host cell. In this system,,cells are
first infected in vitro with a vaccinia virus
recombinant that encodes the bacteriophage T7 RNA
polymerase. This polymerase displays exquisite
specificity in that it only transcribes templates
bearing T7 promoters. Following infection, cells are
transfected with the polynucleotide of interest,
driven by a T7 promoter. The polymerase expressed in
the cytoplasm from the vaccinia virus recombinant
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transcribes the transfected DNA into RNA which is then
translated into protein by the host translational
machinery. The method provides for high level,
transient, cytoplasmic production of large quantities
of RNA and its translation products. See, e.g.,
Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA
(1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad.
Sci. USA (1986) 83:8122-8126.
As an alternative approach to infection with
vaccinia or avipox virus recombinants, or to the
delivery of genes using other viral vectors, an
amplification system can be used that will lead to
high level expression following introduction into host
cells. Specifically, a T7 RNA polymerase promoter
preceding the coding region for T7 RNA polymerase can
be engineered. Translation of RNA derived from this
template will generate T7 RNA polymerase which in turn
will transcribe more template. Concomitantly, there
will be a cDNA whose expression is under the control
of the T7 promoter. Thus, some of the T7 RNA
polymerase generated from translation of the
amplification template RNA will lead to transcription
of the desired gene. Because some T7 RNA polymerase
is required to initiate the amplification, T7 RNA
polymerase can be introduced into cells along with the
templates) to prime the transcription reaction. The
polymerase can be introduced as a protein or on a
plasmid encoding the RNA polymerase. For a further
discussion of T7 systems and their use for
transforming cells, see, e.g., International
Publication No. WO 94/26911; Studier and Moffatt, J.
Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene
(1994) 143:245-249; Gao et al., Biochem. Biophys. Res.
Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.
Acids Res. (1993) 21:2867-2872; Chen et al., Nuc.
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Acids Res. (1994) 22:2114-2120; and U.S. Patent No. -
5,135,855.
Bacterial vectors may also be used to
deliver the gene of interest, such as but not limited
to vectors derived from Mycobacteria, such as M.
smegmatis and M. bovis bacillus Calmette-Guerin (BCG)
(see, e.g., Stover et al., Nature (1991) 351:456 and
Aldovini and Young, Nature (1991) 351:479);
Salmonella-derived vectors, such as attenuated mutants
of S. typhimurium, S. sobrinus and S. dublin (see,
e.g., Cardenas and Clements, Vaccine (1993) 11:126 and
Schodel et al., Vaccine (1993) 11:143); as well as
vectors derived from E. coli; Listeria, such as L.
monocytogenes (see, e.g., Frankel et al., J. Immunol.
(1995) 155:4775); Shigella, and the like.
The gene of interest can also be packaged in
liposomes prior to delivery to the subject or to cells
derived therefrom, with or without the accompanying
antigen. Lipid encapsulation is generally
accomplished using liposomes which are able to stably
bind or entrap and retain nucleic acid. The ratio of
condensed DNA to lipid preparation can vary but will
generally be around 1:1 (mg DNA:micromoles lipid), or
more of lipid. For a review of the use of liposomes
as carriers for delivery of nucleic acids, see, Hug
and Sleight, Biochim. Biophys. Acta. (1991) 1097:1-17;
Straubinger et al., in Methods of Enzymology (1983),
Vol. 101, pp. 512-527.
Liposomal preparations for use in the
instant invention include cationic (positively
charged), anionic (negatively charged) and neutral
preparations, with cationic liposomes particularly
preferred. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner
et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-
7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA
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(1989) 86:6077-6081); and purified transcription _
factors (Debs et al., J. Biol. Chem. (1990) 265:10189-
10192), in functional form.
Cationic liposomes are readily available.
For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-
triethylammonium (DOTMA) liposomes are available under
the trademark Lipofectin, from GIBCO BRL, Grand
Island, NY. (See, also, Felgner et al., Proc. Natl.
Acad. Sc.i. USA (1987) 84:7413-7416) . Other
commercially available lipids include transfectace
(DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other
cationic liposomes can be prepared from readily
available materials using techniques well known in the
art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci.
USA (1978) 75:4194-4198; PCT Publication No. WO
90/11092 for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)
liposomes.
Similarly, anionic and neutral liposomes are
readily available, such as from Avanti Polar Lipids
(Birmingham, AL), or can be easily prepared using
readily available materials. Such materials include
phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG),
dioleoylphoshatidyl ethanolamine (DOPE), among others.
These materials can also be mixed with the DOTMA and
DOTAP starting materials in appropriate ratios.
Methods for making liposomes using these materials are
well known in the art.
The liposomes can comprise multilammelar
vesicles (MLVs), small unilamellar vesicles (SUVs), or
large unilamellar vesicles (LUVs). The various
liposome-nucleic acid complexes are prepared using
methods known in the art. See, e.g., Straubinger et
al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp.
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512-527; Szoka et al., Proc. Natl. Acad. Sci. USA _
(1978) 75:4194-4198; Papahadjopoulos et al., Biochim.
Biophys. Acta (19'75) 394 :483; Wilson et al. , Cell
(1979) 17:77); Deamer and Bangham, Biochim. Biophys.
Acta (1976) 443:629; Ostro et al., Biochem. Biophys.
Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl.
Acad. Sci. USA (1979) 76:3348); Enoch and
Strittmatter, Proc. Natl. Acad. Sci. USA (1979)
76:145); Fraley et al., J. Biol. Chem. (1980)
255:10431; Szoka and Papahadjopoulos, Proc. Natl.
Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et
al., Science (1982) 215:166.
The DNA and/or protein antigens) can also
be delivered in cochleate lipid compositions similar
to those described by Papahadjopoulos et al., Biochem.
Biophys. Acta. (1975) 394:483-491. See, also, U.S.
Patent Nos. 4,663,161 and 4,871,488.
Particulate systems and polymers can be used
for the in vivo or ex vivo delivery of the gene of
interest. For example, polymers such as polylysine,
polyarginine, polyornithine, spermine, spermidine, as
well as conjugates of these molecules, are useful for
transferring a nucleic acid of interest. Similarly,
DEAE dextran-mediated transfection, calcium phosphate
precipitation or precipitation using other insoluble
inorganic salts, such as strontium phosphate, aluminum
silicates including bentonite and kaolin, chromic
oxide, magnesium silicate, talc, and the like, will
find use with the present methods. See, e.g.,
Felgner, P.L., Advanced Drug Delivery Reviews (1990)
5:163-187, for a review of delivery systems useful for
gene transfer.
Additionally, biolistic delivery systems
employing particulate carriers such as gold and
tungsten, are especially useful for delivering genes
of interest. The particles are coated with the gene
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to be delivered and accelerated to high velocity, _
generally under a reduced atmosphere, using a gun
powder discharge from a "gene gun." For a description
of such techniques, and apparatuses useful therefore,
see, e.g., U.S. Patent Nos. 4,945,050; 5,036,006;
5,100,792; 5,179,022; 5,371,015; and 5,478,744.
The recombinant vectors (with or without
associated lipids or carriers) are formulated into
compositions for delivery to the vertebrate subject.
These compositions may either be prophylactic (to
prevent infection) or therapeutic (to treat disease
after infection). The compositions will comprise a
"therapeutically effective amount" of the gene of
interest such that an amount of the antigen can be
produced in vivo so that an immune response is
generated in the individual to which it is
administered. The exact amount necessary will vary
depending on the subject being treated; the age and
general condition of the subject to be treated; the
capacity of the subject's immune system to synthesize
antibodies; the degree of protection desired; the
severity of the condition being treated; the
particular antigen selected and its mode of
administration, among other factors. An appropriate
effective amount can be readily determined by one of
skill in the art. Thus, a "therapeutically effective
amount" will fall in a relatively broad range that can
be determined through routine trials. For example,
for purposes of the present invention, an effective
dose will typically range from about 1 ~.g to about 100
mg, more preferably from about 10 ~,g to about 1 mg, of
the DNA constructs.
The compositions will generally include one
or more "pharmaceutically acceptable excipients or
vehicles" such as water, saline, glycerol,
polyethyleneglycol, hyaluronic acid, ethanol, etc.
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Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the
like, may be present in such vehicles. Certain
facilitators of nucleic acid uptake and/or expression
can also be included in the compositions or
coadministered, such as, but not limited to,
bupivacaine, cardiotoxin and sucrose.
Once formulated, the compositions of the
invention can be administered directly to the subject
or, alternatively, delivered ex vivo, to cells derived
from the subject, using methods such as those
described above. For example, methods for the ex vivo
delivery and reimplantation of transformed cells into
a subject are known in the art and will include e.g.,
dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection,
lipofectamine and LT-1 mediated transfection,
protoplast fusion, electroporation, encapsulation of
the polynucleotide(s) (with or without the
corresponding antigen) in liposomes, and direct
microinjection of the DNA into nuclei.
Direct delivery of the compositions in vivo
will generally be accomplished with or without viral
vectors, as described above, by injection using either
a conventional syringe or a gene gun, such as the
Accell° gene delivery system (Agracetus, Inc.,
Middleton, WI). The constructs can be injected either
subcutaneously, epidermally, intradermally,
intramucosally such as nasally, rectally and
vaginally, intraperitoneally, intravenously, orally or
intramuscularly. For example, delivery of DNA into
cells of the epidermis provides access to skin-
associated lymphoid cells and provides for a transient
presence of DNA in the vaccine recipient. Other modes
of administration include oral and pulmonary
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administration, suppositories, and transdermal _
applications.
Dosage treatment may be a single dose
schedule or a multiple dose schedule. A multiple dose
schedule is one in which a primary course of
vaccination may be with 1-10 separate doses, followed
by other doses given at subsequent time intervals,
chosen to maintain and/or reinforce the immune
response, for example at 1-4 months for a second dose,
and if needed, a subsequent doses) after several
months. The boost may be with the nucleic acid
vaccines or may comprise subunit antigen compositions
including the antigen encoded by the delivered nucleic
acid constructs. The dosage regimen will, at least in
part, be determined by the need of the subject and be
dependent on the judgment of the practitioner.
If prevention of disease is desired, the
vaccines are generally administered prior to primary
infection with the pathogen of interest. If treatment
is desired, e.g., the reduction of symptoms or
recurrences, the vaccines are generally administered
subsequent to primary infection.
As explained above, a submicron oil-in-water
emulsion formulation will also be administered to the
vertebrate subject, either prior to, concurrent with,
or subsequent to, delivery of the gene. If
simultaneous delivery is desired, the submicron oil-
in-water formulation can be included in the nucleic
acid compositions. Alternatively, and preferably, the
oil-in-water emulsions are administered separately,
prior to delivery of the gene, either to the same site
of delivery as the nucleic acid compositions or to a
different delivery site. If administered prior to
nucleic acid immunization, the formulations can be
administered as early as 5-10 days prior to nucleic
acid immunization, preferably 3-5 days prior to
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nucleic acid immunization and most preferably 1-3 or 2 _
days prior to immunization with the nucleic acids of
interest.
Examples of suitable submicron oil-in-water
formulations for use with the present invention will
include nontoxic, metabolizable oils, such as
vegetable oils, fish oils, animal oils or
synthetically prepared oils. Fish oils, such as cod
liver oil, shark liver oils and whale oils, are
preferred, with squalene, 2,6,10,15,19,23-hexamethyl-
2,6,10,14,18,22-tetracosahexaene, found in shark liver
oil, particularly preferred. The oil component will
be present in an amount of from about 0.5% to about
20o by volume, preferably in an amount up to about
15%, more preferably in an amount of from about 1% to
about 12% and most preferably from 1% to about 4% oil.
The aqueous portion of the adjuvant can be
buffered saline or unadulterated water. If the
compositions are to be administered parenterally, it
is preferable to make up the final solutions so that
the tonicity, i.e., osmolality, is essentially the
same as normal physiological fluids, in order to
prevent post-administration swelling or rapid
absorption of the composition due to differential ion
concentrations between the composition and
physiological fluids. If saline is used rather than
water, it is preferable to buffer the saline in order
to maintain a pH compatible with normal physiological
conditions. Also, in certain instances, it may be
necessary to maintain the pH at a particular level in
order to insure the stability of certain composition
components. Thus, the pH of the compositions will
generally be pH 6-8 and pH can be maintained using any
physiologically acceptable buffer, such as phosphate,
acetate, tris, bicarbonate or carbonate buffers, or
the like. The quantity of the aqueous agent present
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will generally be the amount necessary to bring the _
composition to the desired final volume.
Emulsifying agents suitable for use in the
oil-in-water formulations include, without limitation,
sorbitan-based non-ionic surfactants such as those
commercially available under the name of Span° or
Arlacel°; polyoxyethylene sorbitan monoesters and
polyoxyethylene sorbitan triesters, commercially known
by the name Tween°; polyoxyethylene fatty acids
available under the name Myrj°; polyoxyethylene fatty
acid ethers derived from lauryl, acetyl, stearyl and
oleyl alcohols, such as those known by the name of
Brij°; and the like. These substances are readily
available from a number of commercial sources,
including ICI America's Inc., Wilmington, DE. These
emulsifying agents may be used alone or in
combination. The emulsifying agent will usually be
present in an amount of 0.02% to about 2.5% by weight
(w/w), preferably 0.05% to about 1%, and most
preferably 0.01% to about 0.5. The amount present
will generally be about 20-30% of the weight of the
oil used. The emulsions can also contain other
immunostimulating agents, such as muramyl peptides,
including, but not limited to, N-acetyl-muramyl-L-
threonyl-D-isoglutamine (thr-MDP), N-acteyl-
normuramyl-L-alanyl-D-isogluatme (nor-MDP), N-
acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-
(1'-2'-dipalmitoyl-sn-glycero-3-
huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
Immunostimulating bacterial cell wall components, such
as monophosphorylipid A (MPL), trehalose dimycolate
(TDM), and cell wall skeleton (CWS), may also be
present. In order to produce submicron particles,
i.e., particles less than 1 micron in diameter and in
the nanometer size range, a number of techniques can
be used. For example, commercial emulsifiers can be
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used that operate by the principle of high shear
forces developed by forcing fluids through small
apertures under high pressure. Examples of commercial
emulsifiers include, without limitation, Model 110Y
microfluidizer (Microfluidics, Newton, MA), Gaulin
Model 30CD (Gaulin, Inc., Everett, MA), and Rainnie
Minilab Type 8.30H (Miro Atomizer Food and Dairy,
Inc., Hudson, WI). The appropriate pressure for use
with an individual emulsifier is readily determined by
one of skill in the art. For example, when the Model
110Y microfluidizer is used, operation at 5000 to
30,000 psi produces oil droplets with diameters of
about 100 to 750 nm.
The size of the oil droplets can be varied
by changing the ratio of detergent to oil (increasing
the ratio decreases droplet size), operating pressure
(increasing operating pressure reduces droplet size),
temperature (increasing temperature decreases droplet
size), and adding an amphipathic immunostimulating
agent (adding such agents decreases droplet size).
Actual droplet size will vary with the particular
detergent, oil and immunostimulating agent (if any)
and with the particular operating conditions selected.
Droplet size can be verified by use of sizing
instruments, such as the commercial Sub-Micron
Particle Analyzer (Model N4MD) manufactured by the
Coulter Corporation, and the parameters can be varied
using the guidelines set forth above until
substantially all droplets are less than 1 micron in
diameter, preferably less than about 0.8 microns in
diameter, and most preferably less than about 0.5
microns in diameter. By substantially all is meant at
least about 80% (by number), preferably at least about
90%, more preferably at least about 95%, and most
preferably at least about 980. The particle size
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distribution is typically Gaussian, so that the
average diameter is smaller than the stated limits.
Particularly preferred submicron oil-in-
water emulsions for use herein are squalene/water
emulsions optionally containing varying amounts of
MTP-PE, such as the submicron oil-in-water emulsion
known as "MF59" (International Publication No. WO
90/14837; Ott et al., "MF59 -- Design and Evaluation
of a Safe and Potent Adjuvant for Human Vaccines" in
Vaccine Design: The Subunit and Adjuvant Approach
(Powell, M.F. and Newman, M.J. eds.) Plenum Press, New
York, 1995, pp. 277-296). MF59 contains 4-5% w/v
Squalene (e.g., 4.3%), 0.25-0.5% w/v Tween 80~, and
0.5% w/v Span 85° and optionally contains various
amounts of MTP-PE, formulated into submicron particles
using a microfluidizer such as Model 110Y
microfluidizer (Microfluidics, Newton, MA). For
example, MTP-PE may be present in an amount of about
0-500 ~.g/dose, more preferably 0-250 ~g/dose and most
preferably, 0-100 ~g/dose. MF59-0, therefore, refers
to the above submicron oil-in-water emulsion lacking
MTP-PE, while MF59-100 contains 100 ~.g MTP-PE per
dose.
MF69, another submicron oil-in-water
emulsion for use herein, contains 4.3% w/v squalene,
0.25% w/v Tween 80°, and 0.75% w/v Span 85~ an
optionally MTP-PE. Yet another submicron oil-in-water
emulsion is SAF, containing 10% squalene, 0.4% Tween
80°, 5% pluronic-blocked polymer L121, and thr-MDP,
also microfluidized into a submicron emulsion. Also
useful with the present invention is the RibiT""
adjuvant system {RAS), (Ribi Immunochem, Hamilton, MT)
containing 2% Squalene, 0.2% Tween 80, and one or more
bacterial cell wall components from the group
consisting of monophosphorylipid A {MPL), trehalose
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dimycolate (TDM), and cell wall skeleton (CWS), _
preferably MPL + CWS (Detoxt"") .
For a description of various submicron oil-
in-water emulsion formulations for use with the
present invention, see, e.g., International
Publication No. WO 90/14837; Remington: The Science
and Practice of Pharmacy, Mack Publishing Company,
Easton, Pennsylvania, 19th edition, 1995; Van Nest et
al., "Advanced adjuvant formulations for use with
recombinant subunit vaccines," In Vaccines 92, Modern
Approaches to New Vaccines (Brown et al., ed.) Cold
Spring Harbor Laboratory Press, pp. 57-62 (1992); and
Ott et al., "MF59 -- Design and Evaluation of a Safe
and Potent Adjuvant for Human Vaccines" in Vaccine
Design: The Subunit and Adjuvant Approach (Powell,
M.F. and Newman, M.J. eds.) Plenum Press, New York
(1995) pp. 277-296.
It has also surprisingly been found that
other adjuvants, in addition to submicron oil-in-water
emulsions, administered prior to delivery of the gene
of interest, also serve to enhance the immunogenicity
of the antigen encoded by the gene. Such adjuvants
include, but are not limited to: (1) aluminum salts
(alum), such as aluminum hydroxide, aluminum
phosphate, aluminum sulfate, etc.; (2) saponin
adjuvants, such as StimulonT'" (Cambridge Bioscience,
Worcester, MA), or particles generated therefrom such
as ISCOMs (immunostimulating complexes); (3) Complete
Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant
(IFA); (4) cytokines, such as interleukins (IL-l, IL-
2, etc.), macrophage colony stimulating factor (M-
CSF), tumor necrosis factor (TNF), etc.; (5)
detoxified mutants of a bacterial ADP-ribosylating
toxin such as a cholera toxin (CT), a pertussis toxin
(PT), or an E. coli heat-labile toxin (LT),
particularly LT-K63 {where lysine is substituted for
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the wild-type amino acid at position 63) LT-R72 (where
arginine is substituted for the wild-type amino acid
at position 72}, CT-S109 (where serine is substituted
for the wild-type amino acid at position 109), and PT-
K9/G129 (where lysine is substituted for the wild-type
amino acid at position 9 and glycine substituted at
position 129) (see, e.g., International Publication
Nos. W093/13202 and W092/19265); and (6) other
substances that act as immunostimulating agents to
enhance the immunogenicity of the antigen.
III. Experimental
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.
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
CTL Responses in Mice Immunized with Adiuvant
Formulations Prior to Administration of HIV Plasmid
DNA
BALB/c mice were divided into four treatment
groups. One group, which received no adjuvant, served
as a control. Group 2 was injected bilaterally with
50 ~.l of alum, mixed 1:1 with saline, in the tibialis
anterior (TA) muscles. Group 3 was injected as above
with MF59-0 (4.3% w/v squalene, 0.5% w/v Tween 80~,
0.5% w/v Span 85), mixed 1:1 with saline, and group 4
with MF59-100 (homogenization of a modified MF59
formulation containing 100 ~,g/dose MTP-PE), mixed 1:1
with saline. (See Van Nest et al., "Advanced adjuvant
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formulations for use with recombinant subunit
vaccines," in Vaccines 92, Modern Approaches to New
Vaccines (Brown et al., ed.) Cold Spring Harbor
Laboratory Press, pp. 57-62 (1992); and Ott et al.,
~~MF59 -- Design and Evaluation of a Safe and Potent
Adjuvant for Human Vaccines" in Vaccine Design: The
Subunit and Adjuvant Approach (Powell, M.F. and
Newman, M.J. eds.) Plenum Press, New York (1995) pp.
277-296).
Three days later, mice received a retroviral
vector, HIV-IT (International Publication No. WO
91/02805; Ziegner, et al., AIDS (1995) 9:43-50). HIV-
IT encodes the entire HIV-IIIIB env gene, preceded by
the first exon of the rev gene to facilitate HIV-I
protein expression. 21 days following injection of
the adjuvant, primed splenocytes were harvested, CTL
were restimulated in vitro, and CTL activity assays
were conducted.
Quantitative determination of CTLs was made
using a limiting dilution assay, also known as CTL
precursor frequency assay (CTLp), based on the single
hit Poisson model as described by Taswell, J. Immunol.
(1981) 126:1614-1619. The assays were conducted using
methods well known in the art. Briefly, for limiting
dilution assays, the in vitro restimulation step is
done clonally, rather than in bulk splenocyte cultures
(see Example 2, below). Sixty wells of a microculture
plate were filled with a fixed number of primed
splenocytes. Another set of 60 wells received a
different number of primed cells; a third set of wells
received a third number of cells; and so on. All
wells also received irradiated target cells to
stimulate any CTL in the well specific for the target
antigen. After incubation for 7-10 days, aliquots of
cells were transferred to wells containing
radiolabelled target cells and SlCr release was
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measured. Individual wells were scored either "+" or
"-" for CTL activity, compared to the baseline release
from wells containing labelled targets, but no
effectors.
After tallying the numbers of wells
containing CTL activity at each concentration of input
primed splenocytes, the frequency of CTL in the input
population can be calculated according to formulae set
forth in Taswell, J. Immunol. (1981) 126:1614-1619.
(Data presented here were calculated by the "minimum
x2" method; calculations using the "maximum likelihood"
method yielded nearly identical results.)
Tab~~e ~~ 1
Pretreatment CTL Precursor Frequency
None 55,615
(45,968 - 70,388)
Alum 40,903
(35,333 - 48,557)
MF59-0 17,528
(14,810 - 21,468)
MF59-100 48,079
(40,009 - 60,227)
These numbers represent the inverse frequency of CTL
among the primed splenocytes. In other words, in the
spleens of animals preprimed with MF59-0, then
immunized with HIV-IT, roughly 1 in 17,000 splenocytes
are CTL specific for the antigens encoded by HIV-IT;
whereas in animals which received no adjuvant, fewer
than 1 in 55,000 splenocytes are HIV-IT specific CTL.
Given in parentheses is the range of the 95~
confidence interval for the frequency calculation,
based upon the input data. The enhancement of CTL
activity by MF59-0 pretreatment in this experiment is
statistically significant; alum pretreatment shows a
weaker enhancement of CTL activity; and MF59-100 does
not appear to have any effect on this response.
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Example 2
CTL Responses in Mice Immunized with Adj,uvant
Formulations Prior to Administration of
HBV Retroviral Vector
C3H mice were divided into six treatment
groups of three mice/group and injected with adjuvant
(1:1 mixed with 140 mM NaCl) in the TA muscles, as
described above. Two days later, mice received
retroviral vector 6A3, either undiluted or diluted, in
the same muscle sites. The following groups were
included:
Control--undiluted vector
*Alum pretreatment; undiluted vector
*MF59-0 pretreatment; undiluted vector
Control--diluted vector (1:10)
Alum pretreatment; diluted vector (1:10)
MF59-0 pretreatment; diluted vector (1:10)
* due to sample contamination during restimulation,
only 2 samples in each of these groups
Vector 6A3 is a retroviral vector that
encodes a chimeric protein which is a fusion between
the hepatitis B core protein, and the neon protein.
See, e.g., International Publication No WO 93/15207.
Previous work has shown that this vector induces weak
CTL responses in C3H mice.
21 days following injection of the adjuvant,
spleens were harvested individually, and standard CTL
activity assays were performed. Briefly, spleen cells
from immunized BALB/c mice were cultured,
restimulated, and assayed for CTL activity against
slCr-labelled target cells which expressed the HIV
env/rev antigens and were thus susceptible to lysis by
vector-induced CTL. Using known methods, target cells
(T) were cultured with effector {E) cells at various
E:T ratios for 4 hours. Aliquots of culture
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supernatants were harvested, and the release of SlCr _
into the supernatants was quantitated by scintillation
counting. 5lCr in the supernatants of experimental
wells was compared with release from targets incubated
without effectors ("spontaneous release"), and with
targets incubated with detergent ("maximal release").
The "o specific release", a measure of CTL activity,
is calculated using the formula:
% specific - (cpm released - spontaneous release) x 100
release (maximal release - spontaneous release)
The % specific release at varying E:T ratios is
plotted in Figures lA (undiluted vector) and 1B
(diluted vector). As can be seen, MF59-0
significantly enhanced CTL induction, even when the
vector was diluted such that little CTL activity was
seen without the adjuvant pretreatment. It is
interesting to note that two of three animals
receiving diluted vector and MF59-0 pretreatment
showed CTL responses stronger than animals receiving
undiluted vector without pretreatment, suggesting that
vector activity is enhanced 10-fold or more by MF59-0.
Alum also showed an enhancing effect in this study;
however, this effect was weaker than that seen with
MF59-0.
b. A second study was performed in parallel
with the above study. Here, C3H mice were given MF59-
0 either one day prior or two days prior to injection
with retroviral vector 5A3. CTL precursor frequencies
were determined, as described in Example 1. Results
are shown in Table 2.
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Table 2 . _
Pretreatment CTL Precursor Frequency
None 135,918
(119,508 - 157,552)
MF59-0 on day (-1) 46,667
(40
773 - 54
555)
,
,
MF59-0 on day (-2) 14,761
(11,586 - 20,337)
As in Table 1, results are presented as
inverse frequency of CTL, with 95% confidence limits
in parentheses. MF59-0 pretreatment significantly
enhances the induction of CTL specific for 6A3
antigens; here, the effect is more pronounced when
adjuvant is given 2 days prior to vector.
Example 3
IgG Induction in Mice Immunized with Ad~uvant
Formulations Prior to Administration of HIV-IT
Balb/c mice were divided into four treatment
groups as described in Example 1 and administered
alum, MF59-0 or MF59-100, each combined 1:1 with 140
mM NaCl, into the TA muscles, as described. Two days
after adjuvant administration, mice were given the
HIV-IT retrovirus vector as described in Example 1.
At week nine, mice received a boost of HIV-IT vector
without adjuvant.
Serum samples were collected prior to the
first treatment, and at regular intervals thereafter,
and levels of IgGl specific for HIV gp120 determined
using standard ELISAs. See, e.g., Fuller et al., AIDS
Res. Hum. Retroviruses (1994) 10:1433-1441. The
results are shown in Figure 2. Data are presented as
the average of O.D. 450 for 1:100 serum dilutions. As
can be seen, both alum and MF59-100 pretreatment
enhanced Ig induction.
Accordingly, the use of submicron oil-in
water emulsions with nucleic acid immunization
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techniques is disclosed. 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.
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