Title of Invention

A METHOD OF PRODUCING A MICROPARTICLE HAVING AN ADSORBENT SURFACE

Abstract A method of producing a microparticle having an adsorbent surface to which a vector construct capable of expressing a selected nucleic acid sequence is adsorbed, said method comprising the steps of: (a) emulsifying a mixture of a polymer solution and a detergent to form an emulsion, wherein the polymer solution comprises a polymer selected from the group consisting of a poly(α- hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and a polycyanoacrylate, wherein the polymer is present at a concentration of about 1% to about 30% in an organic solvent, and wherein the detergent is present in the mixture at a weight to weight detergent to polymer ratio of from about 0.00001:1 to about 0.5:1; (b) removing the organic solvent from the emulsion, to form said microparticle; and (c) adsorbing the vector construct to the surface of the microparticle, wherein said vector construct is selected from the group consisting of an ELVIS vector and an RNA vector construct.
Full Text MICROP ARTICLES FOR DELIVERY OF HETEROLOGOUS NUCLEIC ACIDS
Technical Field
The present invention relates generally to pharmaceutical compositions. In particular, the
invention relates to microparticles of polymers or submicron emulsions having adsorbent surfaces
wherein biologically active agents, particularly nucleic acids, such as plasrrdd DNA, Eukaryotic
Layered Vector Initiation Systems (ELVIS vectors) or RNA vector constructs, are adsorbed
thereto, methods for preparing such microparticles and submicron emulsions, and uses theere of
including induction of immune responses, vaccines, and delivery of heterologous nucleotide
sequences to eukaryotic cells and animals.
Background
Particulate carriers have been used in order to achieve controlled, parenteral delivery of
therapeutic compounds. Such carriers are designed to maintain the active agent in the delivery
system for an extended period of time Exarnples of particulate carriers include those derived from
polymethyl metnacrylate polymers, as well as microparticles derived frompoly(lactides) (see, e.g.,
U.S. Patent No. 3,773,919), poly (lactide-co-grycolides), known as PLG (see, e.g., U.S. Patent No.
4,767,628) and polyethylene glycol, known as PEG (see, e.g., U.S. Patent No. 5,648,095).
polymethyl metnacrylate polymers are nondegradable while PLG particles biodegrade by random
nonenzymatic hydrolysis of ester bonds to lactic and grycolic acids which are excreted along
normal metabolic pathways.
For example, U.S. Patent No. 5,648,095 describes the use of microspheres with
encapsulated pharmaceuticals as drug delivery systems for nasal, oral, pulmonary and oral delivery.
Slow-release formulations containing various polypeptide growth factors have also been
described. See, e.g., Intemarional Publication No. WO 94/12158, U.S. Patent No. 5,134,122 and
International Publication No. WO 96/37216.

Fattal et al, Journal of Controlled Release 53:137-143 (1998) describes nanoparticles
prepared from polyalkylcyanoacrylates (PACA) having adsorbed oligonucleotides.
Particulate carriers, such as micraparticles, have also been used with adsorbed or entrapped
antigens in attempts to elicit adequate immine responses. Such carriers present multiple copies of
a selected antigen to the immune system and promote trapping and retention of antigens in local
lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen
presentation through cytokine release. For example, commonly owned, co-pending Application
No. 09/015,652, filed January 29,1998, describes the use of antigen-adsorbed and antigen-
encapsulated rmcroparticles to stimulate cell-mediated immunological responses, as well as
methods of making the rmcroparticles.
In commonly owned Patent Application Serial No. 09/015,652, for example, a method of
forming rmcroparticles is disclosed which comprises combining a polymer with an organic solvent,
then adding an emulsion stabilizer, such as polyvinyl alcohol (PVA), then evaporating the organic
solvent, thereby forming rmcroparticles. The surface of the rmcroparticles comprises the polymer
and the stabilizer. Macromolecules such as ELVIS vectors, other nucleotides (DNA or RNA),
polypeptides, and antigens may then be adsorbed on those surfaces.
Adjuvants are compounds which are capable of potentiating an immune response to
antigens. Adjuvants can potentiate both humoral and cellular immunity. However, it is preferable
for certain pathogens to stimulate cellular immunity and, particularly, Thl cells. In many instances,
presently used adjuvants do not adequately induce Thl cell responses, and/or have deleterious side
effects.
Currently, the onry advants approved for human use in the United States are aluminum
These adjuvants hare been useful for some vaccines including hepatitis B, diphtheria,
polio, rabies, and influenza, but may not be useful for others. For example, reports indicate that
alum failed to improve the effectiveness of whooping cough and typhoid vaccines and provided
only a slight effect with adenovirus vaccines. Additionally, problems such as, induction of
granulomas at the ection site and lot-to-lot variation of alum preparations have been
experienced.
Microparticles prepared from biodegradable and biocompatible polymers, known as the
poly(lactide-co-glycolides) (PLG), have been demonstrated to be effective vehicles for a number of

antigens. In addition, PLG microparricles can control the rate of release of entrapped antigens and,
thus, offer potential for single-dose vaccines. Moreover, administration of biodegradable polymers
with entrapped antigens has been demonstrated in a range of animal models to induce potent
immune responses. O'HaganefaL, AdvancedDrug Deliv.Rev., 1998, 32,225-246 and Singh et
aL, AdvancedDrug Dettv. Rev., 1998, .34,285-304, the disclosures of which are incorporated
herein by reference in their entirety.
An emulsion comprising squalene, sorbrtaa trioleate (Span85™), and polysorbate 80
(Tween 80™) microfluidized to provide unirbrmry sized microdroplets, i e. MF59, has also been
shown to induce potent immune responses. MF59 formulations have been shown to induce
antibody titers from 5 to >100 times greater than those obtained with aluminum salt adjuvants.
MF59 has been demonstrated to enhance the immune response to antigens from numerous sources
including, for example, herpes simplex vims (HSV), human immunodeficiency virus (HIV),
mfliifin7a virus, hepatitis C vims (HCV), cytomegalovirus (CMV), hepatitis B virus (HBV), human
papillomavirus (HPV), and materia. Ott etal, Vaccine Design: The Subunit And Adjuvant
Approach, 1995, MR Powell and MJ. Newman, Eds., Plenum Press, New York, p. 277-296;
Singh et aL, Vaccine, 1998,16,1822-1827; Ott et aL, Vaccine, 1995,13,1557-1562; O'Hagan et
al,Mol. Medicine Today, 1997, February, 69-75; and Traquinafif al/.j Infect. Dis., 1996,174,
1168-75, the disclosures of winch are incorporated herein by reference in their entirety. MF59
adjuvant improves the fanmunogenicity of snbunit antigens while maintaining the safety and
tolerahflity profile of alum adjuvant Van Nest etal., Vaccines 92, 1992, Cold Spring Harbor
Laboratory Press, 57-62 and Valeria etal, J. Immunol, 1994,153, 4029-39, the disclosures of
which are incorporated herein by reference in their entirety. MF59 is further described in co-
pending U.S. application Serial No. 08/434,512, filed May 4,1995, which is assigned to the
assignee of the present invention, the disclosure of which is incorporated herein by reference in its
entirety. In animal studies, MF59 has not been found to be genotoxic, teratogenic, nor does it
cause sensitization. The mechanism of action of MF59 appears to be dependent upon the
generation of a strong CD4+ T cefl, ie., a Th2 cell response. MF59 adjuvants, however, elicit
little, if any, Thl responses, or cytotoxic T lymphocyte (CTL) responses.
Oligonucleotides comprising CpG motifs mixed with antigens have been demonstrated to
induce strong Thl immune responses. Roman etal., Nat. Med, 1997,3, 849-854; Weiner etal..

Proc. Natl. Acad. Sci. USA, 1997,94,10833-10837; Davis et al., J. Immunol., 1998,160, 870-
876; Chu etal, J. Exp. Med., 1997, 186,1623-1631; Lipfordef al, Eur. J. Immunol, 1997, 27,
2340-2344; and Moldoveanu et al. Vaccine, 1988,16,1216-1224, the disclosures of which are
incorporated herein by reference in their entirety. Unrnetbylated CpG dinucleotides are relatively
common in bacterial DNA, but are underrepresented and methylated in vertebrate DNA Bird,
Trends Genet, 1987,3,342-347. Bacterial DNA or synthetic oligonucleotides containing
unmethylated CpG motifs are also known to induce immune responses including, for example, B
cell proliferation, interleukin-6 and immunoglobulin secretion, and apoptosis resistance. Krieg et
al, Nature, 1995, 374,546-549; Khnman etal.,Proc Natl. Acad Sci. USA, 1996,93,2879-2883;
BaOasetal,J. Immunol, 1996,157,1840-1845; Cowdery etal, J. Immunol, 1996,156, 4570-
4575; Halpem etal, Cell Immunol, 1996,167,72-78; Yamatnoto etal, Jpn. J Cancer Res.,
1988, 79, 866-873; Stacey etal, J. Immunol, 1996,157,2116-2122; Messina et al,J. Immunol,
1991,147,1759-1764; Yi etal,J. Immunol, 1996, 757,4918-4925; Yief al, J. Immunol, 1996,
157, 5394-5402; Yi et al, J. Immunol, 1998,160, 4755-4761; and Yi etal, J. Immunol, 1998,
160, 5898-5906; PCT Publication WO 96/02555; PCT Publication WO 98/16247; PCT Publication
WO 98/18810; PCT Publication WO 98/40100; PCT Publication WO 98/55495; PCT Publication
WO 98/37919; and PCT Publication WO 98/52581, the disclosures of which are incorporated
herein by reference in their entirety.
It has also been shown that cationic tepid-based emulsions may be used as gene carriers.
See, e.g., Yi et al, Proc. Int'L Symp. Control. ReL Bioact. Mater., 24:653-654 (1997); Kim et al,
Proc. Int'L Symp. Control. ReL Bioact. Mater., 25:344-345 (1998); Kim et aL, Proc. Int'L Symp
ControL ReL Bioact Mater., 26, #5438 (1999). Cationic submicron emulsions, a somewhat recent
approach to pharmaceutical delivery, were first shown to have carrying capacity for small
molecule drugs (Elbaz et al 1993 Int. J. Pharm. 96 R1-R6). Use of the charged surface to stably
bind and protect oligonucleotides in serum has been demonstrated for both small oligomers
(Teixera et al (1999) Pharm Res 16 30-36) and plasmid DNA (Yi et al (2000) Pharm Res 17 314-
320.) DOTAP-based emulsions have been shown to enhance transfection in vitro and in vivo.
(Kim et al, supra).
An adjuvant which results in the increase of a Thl cell response which can be used for
prophylactic and therapeutic treatment is thus desirable. Such a response would be helpful in


U.S. Patents 5,814,482 and 6,015,686 disclose Eukaryotic Layered Vector Initiation
Systems (ELVIS vectors), particularly those derived and constructed from alphavirus genomes
(such as Sindbis vinis), for use in stitnulatmg an immune response to an antigen, in methods of
inhibiting pathogenic agents, and in delivery of heterologous nucleotide sequences to eukaryotic
cells and animals, among others.
Commonly owned International patent application PCT/US99/17308 and U.S. patent
application 09/715,902 disclose methods of making microparticles having adsorbed
macromolecules, such as a pharmaceutical, a polynucleotide, a polypeptide, a protein, a hormone,
an enzyme, a transcription or translation mediator, an intermediate in a metabolic pathway, an
immunomodulator, an antigen, an adjuvant, or combinations thereof, and the like.
Commonly owned Intemational patent application PCT/US00/03331 discloses methods of
making submicron emulsions having adsorbed macromolecules, such as a pharmaceutical, a
polymicleotide, a polypeptide, a protein, a hormone, an enzyme, a transcription or translation
mediator, an intermediate in a metabolic pathway, an mimunomodulator, an antigen, an adjuvant,
or combinations thereof, and the like.
Summary of the Invention
The inventors herein have discovered that the effectiveness of the various uses of nucleic
acids, particularly vector constructs capable of expressing a nucleic acid sequence, and more
particularly vector constructs comprising a heterologous nucleic acid sequence encoding an
antigen, such as such pCMV vectors, ELVIS vectors or RNA vector constructs may be enhanced
by adsorbing the vector constructs to polymer microparticles or submicron emulsions with
adsorbent surfaces, which facilitates introduction of the vector constructs, and of heterologous
nucleic acid sequences comprised in the vector constructs, into the cells of an animal
As disclosed in above described Intemational Patent Application PCT/US99/17308, a
method of forming microparticles with adsorbent surfaces capable of adsorbing a wide variety of
macromolecules has been invented. In one embodiment, the microparticles are comprised of both a

polymer and a detergent. The microparticles of the present invention adsorb such macromolecules
more efficiently than other microparticles currently available.
Several embodiments of the present invention utilize microparticles that are derived from a
polymer, such as a poly(α-hydrosy acid), apolyhydrocy butyric acid, apolycaprolactone, a
polyorthoester, a polyanhydride, apolyalky lcyanoacrylate, a polycyanoaaylate, and the like, and
are formed with detergents, such as canonic, anionic, or nomonic detergents, which detergents may
be used in combination. The present inventors have discovered that these microparticles yield
improved adsorption of vector constructs (e.g., ELVIS vectors, RNA vector constructs), as well as
viral antigens, and provide for superior immune responses.
As disclosed in above-described International Patent Application PCT/USOO/03331, a '
microparticle preparation comprising oil droplet submicron emulsions with ionic surfactants has
been invented. Such compositions readily adsorb macromolecules such as DNA, protein, and other
antigenic molecules. Several embodiments of the present invention utilize microparticles that are
derived from an oil droplet emulsion mat preferably comprises a metabolizable oil and an
emulsifying agent which are preferably present in the form of an oil-in-water emulsion having oil
droplets substantially all of which are less than 1 micron in diameter, preferably smaller than 250
run. Preferably, the composition exists in the absence of any polyoxypropylene-polyoxyemylene
block copolymer. The oil is preferably an animal oil, an unsaturated hydrocarbon, a terpenoid such
as, for example, squalene, or a vegetable oil The composition preferably comprises 0.5 to 20 %
by volume of the oil in an aqueous medium The emulsifying agent preferably comprises a non-
ionic detergent such as a polyoxyethylene sorbitan mono-, di-, or triester or a sorbitan mono-, di-,
or triether. Preferably, the composition comprises about 0.01 to about 5 % by weight of the
emulsifying agent.
Hence, in some embodiments, the particulate portion of the invention's composition is a
microparticle with an adsorbent surface, wherein the microparticle comprises a polymer selected
from the group consisting of a poly (α-hydroxy acid), a polyhy droxy butyric acid, a
polycaprolactone, a polyorthoester, a polyanhydride, and a polycyanoacrylate.
In another embodiments, the particulate portion of the invention's composition is a
submicron emulsion which comprise an oil droplet emulsion formulated with an ionic detergent.

In other embodiments, the microparticle further comprises vector constructs capable of
expressing a nucleic acid sequence, such as a selected ELVIS vector or RNA vector construct
adsorbed on the microparticle's surface, with the vector construct comprising a heterologous
nucleotide sequence encoding a polypeptide, a protein, a hormone, an enzyme, a transcription or
translation mediator, an intermediate in a metabolic pathway, an immunomodulator, an antigen, an
adjuvant, or combinations thereof, and the like.
In other embodiments, the invention is directed to a microparticle composition comprising a
nucleic acid, preferably vector constructs capable of expressing a nucleic acid sequence, such as a
selected pCMV vector, ELVIS vector or RNA vector construct, adsorbed to a microparticle of the
invention, and a pharmaceutically acceptable excipient.
In other embodiments, the invention is directed to a method of producing a microparticle
with an adsorbed nucleic acid, preferably vector constructs capable of expressing a nucleic acid
sequence, such as an ELVIS vector or RNA vector construct, the method comprising:
(a) combining a polymer solution comprising a polymer selected from the group
consisting of a poly(a-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a
polyorthoester, a polyanhydride, and a polycyanoacrylate, wherein the polymer is present at a
concentration of about 1% to about 30% in an organic solvent;
and an anionic, canonic, or nonionic detergent to the polymer solution, wherein the
detergent is present at a ratio of 0.001 to 10 (w/w) detergent to polymer, to form a
polymer/detergent mixture;
(b) dispersing the polymer/detergent mixture;
(c) removing the organic solvent;
(d) recovering the microparticle; and
(r) adsorbing an ELVIS vector or RNA vector construct to the surface of the
microparticle, wherein the ELVIS vector or RNA vector construct comprises a heterologous
nucleotide sequence encoding a polypeptide, a protein, a hormone, an enzyme, a
transcription or translation mediator, an intermediate in a metabolic pathway, an
immunomodulator, an antigen, an adjuvant, or combinations thereof, and the like.
Preferably, the polymer/detergent mixture is emulsified to form an emulsion prior to
removing the organic solvent.

In other embodiments, the invention is directed to a micropartacle produced by the above-
described methods. More preferably, a microparticle composition is produced, which also
comprises a pharmaceuticauy acceptable excipient.
In still other embodiments, the invention is directed to a method of delivering a
heterologous nucleotide sequence to a vertebrate subject, which comprises administering to a
vertebrate subject any of the compositions described above.
In additional embodiments, the invention is directed to a method for eliciting a cellular
immune response in a vertebrate subject comprising administering to the vertebrate subject a
therapeutically effective amount of a selected heterologous nucleotide sequence adsorbed to a
microparticle of the invention.
In other embodiments, the invention is directed to a method of immunization which
comprises administering to a vertebrate subject a therapeutically effective amount of any of the
microparticle compositions above. The composition may optionally contain unbound
macromolecules, and also may optionally contain adjuvants, including aluminum salts such as
aluminum phosphate, or an oligonucleotide comprising at least one CpG motif
In several preferred embodiments, the microparticles are formed from a poly(a-hydroxy
acid); more preferably, a poly(D,L-lactide-co-glycolide); and most preferably, a pofy(D,L-lactide-
co-gjycohde).
Each of the nonexhaustive previously described adsorbent microparticles may optionally
also have macromolecules entrapped within them, or in free solution. Thus, the invention
encompasses a variety of combinations wherein nucleic acid molecules are adsorbed on
microparticles and other nucleic acid molecules are entrapped or adsorbed. Moreover, the
microparticles of the invention may have more than one species of nucleic acid adsorbed thereon,
as well as other antigenic macromolecules adsorbed thereon Additionally, the microparticles may
have several species of nucleic acid and/or other antigenic macromolecules entrapped within
In other preferred embodiments, the microparticles are prepared in the form of submicron
emulsions as described above.
The present invention is also directed to immunogenic compositions comprising an
irnmunostimulating amount of a nucleic acid (e.g., a vector construct capable of expressing a
nucleic acid sequence, such as a selected ELVIS vector or RNA vector construct, where the

heterologous nucleotide sequence portion of the ELVIS vector or RNA vector construct may
encode an antigen), and an innnunostimulating amount of an adjuvant composition described
herein. In some embodiments of the invention, the immunogenic composition comprises a CpG
oligonucleotide in combination with the nucleic acid- adsorbed microparticles. The adsorbed
macromolecule itself is preferably an ELVIS vector or RNA vector construct encoding an
antigenic polypeptide.
In some preferred embodiments of the invention, the antigenic polypeptide is from a virus
such as, for example, hepatitis C virus (HCV), hepatitis B virus (HBV), herpes simplex virus
(HSV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), influenza virus (flu), and
rabies virus. Preferably, the antigenic polypeptide is selected from the group consisting of HSV
glycoprotein gD, HIV glycoprotein gpl20, HIV glycoprotein gpl 40, HIV p55 gag, and
polypeptides from the pol and tat regions. In other preferred embodiments of the invention, the
antigenic polypeptide is from a bacterium such as, for example, cholera, diphtheria, tetanus,
streptococcus (e.g., streptococcus B), pertussis, Neisseria meningitidis (e.g., meningitis B),
Neisseria gonorrhoeae, Helicobacter pylori, and Haemophilus influenza. In still other preferred
embodiments of the invention, the antigenic polypeptide is from a parasite such as, for example, a
malaria parasite.
Adjuvant compositions may comprise, for example, aluminum salts. Alternatively, adjuvant
compositions may comprise an oligonucleotide comprising at least one CpG motif The adjuvant
composition can also comprise an optional component which results in a positively charged
emulsion The oligonucleotide preferably comprises at least one phosphorothioate bond or peptide
nucleic acid bond. In preferred embodiments of the invention, the oligonucleotide comprises a
nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-28. In other preferred
embodiments of the invention, the oligonucleotide comprises a CpG motif flanked by two purines
immediately 5' to the motif and two pyrknidines immediately 3' to the motif In other preferred
embodiments of the invention, the oligonucleotide comprises a nucleotide sequence selected from
the group consisting of SBQ ID NOs: 19-28. Most preferred is SEQ ID NO:28. In some
preferred embodiments of the invention, the adjuvant composition further comprises a separate
rnirnvmostimulating agent which is preferably selected from the group consisting of alum, a
bacterial cell wall component, and muramyl peptide. The adjuvant composition itself may be in the

form of a second microparticle. The second microparticle may have adsorbed and/or entrapped
within a variety of nucleic acids and/or antigenic polypeptides, or other antigenic macromolecules.
Additionally, the immunogenic compositions may include the presence of free nucleic acid in
solutioa
The present invention is also AmctpA tn nwmnHg nf stimulating an fmrmmp raspnnsR in a
host ATP"1^ comprising administering to the animal an immunogenic composition described herein
in an amount effective to induce an imrmme response. The host animal is preferably a mammal,
more preferably a Rhesus macaque, and sol more preferably a human
viral, bacterial, or parasitic infection comprising administering to the animal an immunogenic
composition described herein in an amount effective to induce a protective response. The host
animal is preferably a mammal, more preferably a Rhesus macaque, and still more preferably a
human.
The present invention is also directed to methods of increasing a Thl immune response, or
a CTL response, or ryphopronferation, or cytokine production in a host animal comprising
administering to the animal an immunogenic composition described herein in an amount effective
to induce the Thl immune response, or the CTL response, or ryphoproliferation, or cytokine
production. The host anirrial is preferably a marntr^ more pi^^
more preferably a human
The present invention is also directed to methods of raising an immune response in a host
animal in which a microparticle-adsorbed macromolecule comprising a heterologous nucleic acid
sequence encoding a first antigen (e.g., plasmid DMA, such as pCMV or an ELVIS vector, or an
RNA vector construct) is first administered to the animal in an amount effective to elicit an
immunological response. Subsequently, a second antigen is administered to the animaL
The first antigen and the second antigen in these etnbodimeats can be the same or different,
and are preferably the same. Preferred antigens include bacterial and viral antigens, suchasHTV
antigens (e.g., gpl20, gpl40, gpl 60, p24gag and p55gag), hepatitis C virus antigens, influenza A
virus antigens, meningitis B bacterial antigens, and streptococcus B bacterial antigens. The second
antigen is preferably adsorbed to the microparticles described herein, or is coadministered with an
adjuvant, such as MF59. The macromolecule can also be coadministered with an adjuvant, if

desired. In some preferred embodiments, the macromolecule is administered two or more tunes
before the second antigen, which can also be administered two or more times.
According to one specific embodiment: (1) the macromolecule is administered (a) at a time
of initial administration, (b) at a time period ranging 1-8 weeks from the initial administration, and
(c) at a time period ranging 4-32 weeks from the initial administration, and (2) the second antigen
is administered (a) at a time period ranging from 8-50 weeks from the initial administration and (b)
at a time period ranging from 8-100 weeks from the initial administration
Delivery of the nricroparticle compositions of the invention may be performed by any
known method, mcmding direct injection (e.g., subcutaneousry, intraperitoneaUy, intravenously or
intramuscularly), and such delivery may also be enhanced by the use of electroporation (see, e.g.,
U.S. patent application no. 09/499,023, the disclosure of which is hereby incorporated by reference
in its entirety). Electroporation is the application of short electrical pulses to cells to increase
permeability of the cell membranes, thus facilitating DNA uptake by cells. Recently it has been
found that applying an electric field to tissues in vivo significantly increases DNA uptake and gene
expression (Matmesen, I., 1999, Gene Therapy 6:508). Among the tissues targeted for in vivo
electroporation have been skin, liver, tumors, and muscle. For DNA vaccine application, Widera et
al have shown mat in vivo electroporation substantially enhances DNA vaccine potency in mice,
guinea pigs, and rabbits (Widera, G., et al, 2000, J. Immunol 164:4635).
The ELVIS vectors of the above-described embodiments are generaBy DNA molecules
comprising a promoter that functions in a eukaryotic cell, a cDNA sequence for which the
transcription product is an RNA vector construct (e.g., alphavirus RNA vector replicon), and a 3'
termination region The RNA vector constructs preferabry comprise an RNA genome from a
picomavirus, togavirus, flavivirus, coronavirus, paramyxovirus, yellow fever virus, or alphavirus
(e.g., Sindbis virus, Semliki Forest virus, Venezuelan equine encephalitis virus, or Ross River
virus), and more preferably an alphavirus genome, which has been modified by the replacement of
one or more structural protein genes with a selected heterologous nucleic acid sequence encoding a
gene product of interest. The RNA vector constructs of the present invention generally are
obtained by transcription in vitro from a DNA template.
These and other aspects and 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
Fig. 1 provides a DNA sequence (SEQ ID NO:63) encoding a modified HTV-1 p55gag
polypeptide.
Fig. 2 provides a DNA sequence (SEQ ID NO:64) encoding a modified HTV-1 p55gag
polypeptide.
Fig. 3 provides a DNA sequence (SEQ ID NO:65) encoding a modified HTV-1 envelope
polypeptide.
Fig. 4 provides a DNA sequence (SEQ ID NO:66) encoding a modified HTV-1 envelope
polypeptide.
Fig. 5 provides a DNA sequence (SEQ ID NO: 67) encoding a modified HTV-1 p55gag
polypeptide.
Fig. 6 provides a DNA sequence (SEQ ID NO:68) encoding a modified HTV-1 p55gag
polypeptide.
Detailed Description of the Invention
The present invention is based upon the surprising discovery that rmcroparticles with
adsorbed nucleic acid molecules, preferably vector constructs capable of expressing a nucleic acid
sequence, and more preferably vector constructs comprising a heterologous nucleic acid sequence
encoding an antigen, such as pCMV vectors, ELVIS vectors or RNA vector constructs, elicit
enhanced immune responses. Additionally, the combination of microparticles with adsorbed
nucleic acid molecules (for example, microparticles with adsorbed pCMV vectors, ELVIS vectors
or RNA vector constructs) and adjuvants is useful for eliciting enhanced immune responses.
The invention is also based upon the surprising discovery that vector constructs comprising
antigen-encoding nucleic acid sequences, such as pCMV vectors, ELVIS vectors or RNA vector
constructs, in association with subsequent administration of antigen elicit enhanced
responses.
The practice of the present invention will employ, unless otherwise indicated, conventional
methods of chemistry, polymer chemistry, biochemistry, molecular biology, immunology and
pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See,

e.g., Remington's Pharmaceutical Sciences, 18th Edition (Eastern, Pennsylvania; Mack Publishing
Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press,
Inc.); Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. BlackweH, eds.,
1986, Blackwell Scientific Publications); Sambrook, et aL, Molecular Cloning: A Laboratory
Manual (2nd Edition, 1989); Handbook of Surface and Colloidal Chemistry (Birdi, K.S., ed, CRC
Press, 1997) and Seymour/Carraheri Polymer Chemistry (4th edition, Marcel Dekker Inc., 1996).
AD publications, patents and patent applications cited herein, whether supra or infra, are
hereby incorporated by reference in their entirety.
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. Thus, for example, the term
"microparticle" refers to one or more microparticles, and the like.
A Defffinrtions
In describing the present invention, the following terms will be employed, and are intended
to be defined as indicated below.
Unless stated otherwise, all percentages and ratios herein are given on a weight basis.
The term "microparticle" as used herein, refers to a particle of about 10 nm to about 150
pmin diameter, more preferably about 200 nmto about 30 urn in diameter, and most preferably
about 500 nmto about 10 umin diameter. Preferably, the microparticle will be of a diameter that
permits parenteral or mucosal administration without occluding needles and capillaries.
Microparticle size is readily determined by techniques well known in the art, such as photon
correlation spectroscopy, laser diffractometry and/or scanning electron microscopy. The term
"particle" may also be used to denote a microparticle as defined herein. Microparticle may
alternatively refer to a submicron emulsion composition as described herein
Polymer microparticles for use herein are formed from materials that are stenlizable, non-
toxic and biodegradable. Such materials include, without limitation, poly(a-hydroxy acid),
polyhydroxybutyric acid, polycaprolactone, polyorthoester, polyanhydride, PACA, and
polycyanoacrylate. Preferably, microparticles for use with the present invention are polymer
microparticles derived from a poly(α-hydroxy acid), in particular, from a poly(lactide) ("PLA") or a
copolymer of D,L-lactide and glycolide or gly colic acid, such as a poly(D,L-lacnde-co-glycolide)

("PLG" or "PLGA"), or a copolymer of D,L-lactide and caprolactone. The polymer microparticles
may be derived from any of various polymeric starting materials which have a variety of molecular
weights and, in the case of the copolymers such as PLG, a variety of lactide:glycolide ratios, the
selection of which will be largely a matter of choice, depending in part on the coadministered
macromolecule. These parameters are discussed more fully below. Alternatively, microparticles of
the invention are comprised in a submicron emulsion.
As used herein, the phrase "oil droplet emulsion" refers to an emulsion comprising a
metabolizable oil and an emulsifying agent The term "submicron emulsion" as used herein refers
to an oil droplet emulsion of the invention comprising droplets ranging in size from about 10 nm to
about 1000 nm
As used herein, the term "microparticle" may refer to a polymer microparticle as described
herein or a submicron emulsion composition as described herein.
The term "detergent" as used herein includes surfactants, dispersing agents, suspending
agents, and emulsion stabilizers. Anionic detergents include, but are not limited to, SDS (sodium
dodecyl sulfate), SLS (sodium lauryi sulfite), DSS (disulfosuccinate), sulphated fatty alcohols, and
the like. Catiomc detergents include, but are not limited to, cetrimide (cetyl trimethyl ammonium
bromide, or "CTAB"), benzalkonium chloride, DDA (dimethyl dioctodecyl ammonium bromide),
IX)TAP(dioleoyl-3-trimethylaluminimum-propane), and the like. Nomonic detergents include, but
are not limited to, PVA, povidone (also known as polyvinylpyrrolidone or PVP), sorbitan esters,
polysorbates, polyoxyethylated glycol monoethers, polyoxyethylated alkyl phenols, poloxamers,
and the like.
The term "zeta potential" as used herein, refers to the electrical potential that exists across
the interface of all solids and liquids, ie., the potential across the diffuse layer of ions surrounding
a charged colloidal particle. Zeta potential can be calculated from electrophoretic mobilities, i e.,
the rates at which colloidal particles travel between charged electrodes placed in contact with the
substance to be measured, using techniques well known in the art.
The term "macromolecule" as used herein refers to, without limitation, a pharmaceutical, a
polynucleotide, a polypeptide, a hormone, an enzyme, a transcription or translation mediator, an
intermediate in a metabolic pathway, an immunomodulator, an antigen, an adjuvant, or

combinations thereof Particular macromolecules for use with the present invention are described
in more detail below.
The term "pharmaceutical" refers to biologically active compounds such as antibiotics,
antiviral agents, growth factors, hormones, and the like, discussed in more detail below.
The term "adjuvant" refers to any substance that assists or modifies the action of a
pharmaceutical, including but not limited to immunological adjuvants, which increase or diversify
the immune response to an antigen.
A "polynucleotide" is a nucleic acid polymer, which typically encodes a biologically active
(e.g., immunogenic or therapeutic) protein or polypeptide. Depending on the nature of the
polypeptide encoded by the polynucleotide, a polynucleotide can include as little as 10 nucleotides,
e.g., where the polynucleotide encodes an antigen. Furthermore, a "polynucleotide" can include
both double- and single-stranded sequences and refers to, but is not limited to, cDNA from viral,
procaryotic or eucaryotic mRNA, genomic RNA and DNA sequences from viral (e.g. RNA and
DNA viruses and retroviruses) or procaryotic DNA, and especially synthetic DNA sequences. The
term also captures sequences mat include any of the known base analogs of DNA and RNA The
term further includes modifications, such as deletions, additions and substitutions (generally
conservative in nature), to a native sequence, preferably such that the nucleic acid molecule
encodes a therapeutic or antigenic protein. These modifications may be deliberate, as through site-
directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the
antigens.
The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are
not limited to a rninimum length of the product Thus, peptides, oligopeptides, dimers, multimers,
and the like, are included within the definition. Both full-length proteins and fragments thereof are
encompassed by the definition. The terms also include modifications, such as deletions, additions
and substitutions (generafly conservative in nature), to a native sequence, preferably such that the
protein maintains the ability to elicit an immunological response or have a therapeutic effect on a
subject to which the protein is administered.
By "antigen" is meant a molecule which contains one or more epitopes capable of
stimulating a host' s immune system to make a cellular antigen-specific immune, response when the
antigen is presented in accordance with the present invention, or a humoral antibody response. An

antigen may be capable of eliciting a cellular or humoral response by itself or when present in
combination with another molecule. Normally, an epitope will include between about 3-15,
preferably about 5-15, and more preferably about 7-15 amino acids. Epitopes of a given protein
can be identified using any number of epitope mapping techniques, well known in the art. See,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology, VoL 66 (Glenn B. Morris, Ed,
1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by
e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides
corresponding to portions of the protein molecule, and reacting the peptides with antibodies while
the peptides are still attached to the supports. Such techniques are known in the art and described
in, e.g., U.S. Patent No. 4,708,871; Geysenet aL (1984) Proc. Natl. Acad. Sci. USA 81:3998-
4002; Geysen et al (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in
their entireties. Similarly, conformational epitopes are readily identified by determining spatial
conformation of amino acids such as by, e.g., x-ray crystallography and 2-dirnensional nuclear
magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.
The term "antigen' as used hererind aotes bom subunit antigens, Le., antigens which are
separate and discrete from a whole organism with which the antigen is associated in nature, as well
as killed, attenuated or inactivated bacteria, viruses, parasites or other microbes. Antibodies such
as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can
mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used
herein Similarly, an oligonucleotide or polynucleotide which expresses a therapeutic or
immunogenic protein, or antigenic determinant in vivo, such as in gene therapy and nucleic acid
immunization applications, is also included m the definition of antigen herein
Further, tor purposes of the present invention, antigens can be derived from any of several
known viruses, bacteria, parasites and fungi, as well as 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 substitutions (generally conservative in nature), to
the native sequence, so long as the protein maintian the ability to elicit an immunological
response. These modifications may be deliberate, as through she-directed mutagenesis, or may be
accidental, such as through mutations of hosts which produce the antigens.

An "immunological response" or 'immune 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-rymphocytes and/or other white blood cells. One important aspect of cellular
immunity involves an antigen-specific response by cytolytic T-cells ("CTLs"). CTLs have
specificity for peptide antigens that are presented in association with proteins encoded by the major
histocompatibility complex (MHC) and expressed on the surfeces 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, chemolrines 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, such as an immunogenic composition, or vaccine that elicits a cellular
immune response may serve to sensitize a vertebrate subject by the presentation of antigen in
association with MHC molecules at the cell surface. Tte ceD-rnediated immune response is
directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-
rymphocytes can be generated to allow for the future protection of an minimized 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 h/mphoproliferation
(lymphocyte activation) assays, CTL cytotoxic cell assays, by assaying for T-tymphocytes specific
for the antigen m. a sensitized subject, or by measurement ofcytokine production by T cells in
resporjse to restimulation with antigen Such assays are well known in the art See, e.g., Erickson
et al, J. ImmmoL (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 whi& 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 Mowing effects: the production of antibodies by B-cefls; and/or the
activation of suppressor T-ceHs and/or yS T-cefls directed specificaUy to an antigen or antigens
present in the composition or vaccine of interest These responses may serve to neutralize
infectivity, and/or mediate antibody-complernent, or antibody dependent cell cytotoxicity (ADCC)
to jffovkte protection to an rmrmnrized host Such responses on be determined using standard
immunoassays and neutralization assays, well known in the art
A composition which contains a selected antigen adsorbed to a microparticie, displays
"enhanced immunogenicity" when it possesses a greater capacity to eKch an hnrmriM response than
the immune response elicited by an equivalent amount of the antigen when delivered without
assoriation wife the microparticie. Thus, a composition may display "airimced mniunogenicity"
because the antigen is more strongly immunogenic by virtue of adsorption to the microparticle, or
because a lower dose of antigen is necessary to achieve an immune response in the subject to which
it is administered. Such enhanced imrnunogenicity can be determined by administering the
rrricroparticle/aaitigen composition, and antigen controls to animals and comparing antibody titers
against the two using standard assays such as radiomknunoassay and ELISAs, well known in the
art.
The terms "effective amount1' or "pharmaceuticauy effective amount" of a given
composition, as provided herein, refer to a nontoxic but sufficient amount of the composition to
provide a desired response, such as an immunological response, and corresponding therapeutic
effect, or in the case of delivery of a therapeutic protein, an amount sufficient to effect treatment of
the subject, as denned below. As will be pointed out below, the exact amount required win 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 macromolecule of interest, mode of
administration, and the like. An appropriate "effective" amount many individual case may be
determined by one of ordinary skill in the art using routine experimentation.
By "vertebrate subject" is meant any member of the subphylum cordata, including, without
limitation, mammals such as cattle, sheep, pigs, goats, horses, and humans; domestic animals such
as dogs and cats; and birds, including domestic, wild and game birds such as cocks and hens
including chickens, turkeys and other gallinaceous birds. The term does not denote a particular
age. Thus, both adult and newborn animals are intended to be covered.

By "prarrnaceuticauy acceptable" or "pharmacologically acceptable" is meant a material
which is not biologically or otherwise undesirable, Le., the material may be administered to an
individual along with the mkroparticle formulation without causing any undesirable biological
effects in the individual or interacting in a deleterious marmerwimauyofthecomrwrienteofthe
composition in which it is contained.
The term "excpenf refers to substances that are rortmvnly provided wimm finished
dosage forms, and include vehicles, binders, disintegrants, fillers (diluents), robricants, gndants
(now enhancers), compression aids, colors, sweeteners, preservatives, suspensing/dispersing
agents, firm formers/coatings, flavors and panting inks.
By "physiological pH" or a "pH in the physiological range" is meant a pH in the range of
approximately 7.2 to 8.0 inclusive, more typically in the range of approximately 7.2 to 7.6
inclusive.
As used herein, "treatment" (including variations thereof) for example, "treat" or "treated")
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 or disorder in question. Treatment may be effected prophylacticauy (prior to infection)
or therapeutically (following infection).
As used hereto, the phrase "nucleic acid" refers to DNA, RNA, or chimeras formed
therefrom
As used herein, the phrase "oligonucleotide comprising at least one CpG motif" refers to a
polynucleotide comprising at least one CpG dinucleotide. Oligonucleotides comprising at least one
CpG motif can comprise multiple CpG motifs. These oligonucleotides are also known as "CpG
oligonucleotides" in the art As used herein, the phrase "CpG motif' refers u a dinucleotide
portion of an oligonucleotide which comprises a cytosine nucleotide followed by a guanosine
nucleotide. 5-methylcytosine can also be used in place of cytosine.
As used herein, "arphavirus RNA vector replicon," "KNA vector replicon," "RNA vector
construct," and "rephcon" refer to an RNA molecule which is capable of directing its own
amplification or self-replication in vivo, within a target cefi. An alphavirus-derived RNA vector
replicon should contain the following ordered elements: 5' viral sequences required in cis for
replication (also referred to as 5' CSE), sequences which, when expressed, code for biologically

active alphavirus nonstructural protons (e.g., nsPl, nsP2, nsP3, nsP4), 3' viral sequences required
in cis for replication (also referred to as 3' CSE), and a polyaderrylate tract. An alplmvirus-derived
RN A vector repHcon also may contain a viral subgenomic "junction region" promoter, sequences
from one or more structural protein genes or portions thereof; extraneous nucleic acid molecules)
which are of a size sufficient to allow production of viable virus, as well as heterologous
sequences) to be expressed.
As used herein, "Eukaryotic Layered Vector Initiation System," "ELVIS, "or "ELVIS
vector" refers to an assembly which is capable of directing the expression of a sequence^) or
gene(s) of interest The eukaryotic layered vector initiation system should contain a 5' promoter
which is capable of initiating in vivo (i.e., within a cell) the synthesis of RNA from cDNA, and a
viral vector sequence which is capable of directing its own replication in a eukaryotic cell and also
expressing a heterologous sequence. In preferred embodiments, the nucleic acid vector sequence is
an alphavirus-derived sequence and is comprised of a 5' sequence which is capable of initiating
transcription of an alphavirus SNA (also referred to as 5' CSE), as well as sequences which, when
expressed, code for bdologicalry active alphavirus nonstructural proteins (eg., nsPl, nsP2, nsP3,
nsP4), and an alphavirus RNA polymerase recognition sequence (also referred to as 3' CSE). In
addition, the vector sequence may include a viral subgenomic "junction region" promoter,
sequences from one or more structural protein genes or portions thereof, extraneous nucleic acid
molecule^) which are of a size sufficient to allow optimal amplification, a heterologous sequence
to be expressed, one or more restriction sites for insertion of heterologous sequences, as well as a
polyadenyktion sequence. The eukaryotic layered vector initiation system may also contain splice
recognition sequences, a catalytic ribozyme processing sequence, a nuclear export signal, and a
transcription termination sequence.
"Atphavirus vector construct" refers to an assembly which is capable of directing the
expression of a sequence or gene of interest Such vector constructs are generally comprised of a
5' sequence which is capable of irritiating transcrnitkin ofanarphavirm RNA (also inferred to as S'
CSE), as well as sequences which, when expressed, code for biologically active atphavirus
nonstructural proteins (e.g., nsPl, nsP2, nsP3, nsP4), an arphavirus RNA polymerase recognition
sequence (also referred to as 3' CSE), and a polyaderrylate tract In addition, the vector construct
may include a viral subgenomic "junction region" promoter, sequences from one or more structural

protein genes or portions thereof extraneous nucleic acid molecule(s) which are of a size sufficient
to allow production of-viable virus, a 5' promoter which is capable of initiating the synthesis of
viral RNA fiom cDNA in vitro or in vivo, aheterologous sequence to be expressed, and one or
more restriction sites for insertion of heterologous sequences.
As used herein, the phrase "vector construct" generally refers to any assembly which is
capable of directing the expression of a nucleic acid sequence^) or gene(s) of interest Hie vector
construct typically includes transcriptional promoter/enhancer or locus defining elements), or
other elements which control gene expression by other means such as alternate splicing, nuclear
RNA export, post-translational modification of messenger, or post-transcriptianal modification of
protein In addition, the vector construct typically includes a sequence which, when transcribed, is
operabry linked to the sequences) or gene(s) of interest and acts as a translation initiation
sequence. The vector construct may also optionally include a signal which directs pbryadenylation,
a selectable marker, as well as one or more restriction sites and a translation termination sequence.
In addition, if the vector construct is placed into a retrovirus, the vector construct may include a
packaging signal, long terminal repeats (LTRs), and positive and negative strand primer binding
sites appropriate to the retrovirus used (if these arc not already present). Examples of vector
constructs include ELVIS vectors, which comprise the cDNA complement of RNA vector
constructs, RNA vector constructs themselves, alphavirus vector constructs, CMV vector
constructs and the like.
One specific type of vector construct is a "plasmid", which refers to a circular double
stranded DNA, loop into which additional DNA segments can be ligated. Specific plasmids
described below include pCMV and pSINCP.
According to some embodiments of the present inv tition, compositions and methods are
provided which treat, mrJiiHing prophylacticaDy and/or therapeutically immunize, a host animal
against viral, fungal, mycoplasma, bacteria], or protozoan infections, as well as against tumors.
The methods of the present invention are useful for conferring prophylactic and/or therapeutic
immunity to a mammal, preferably a human. The methods of the present invention can also be
practiced on mammals, other than humans, "ic^nd"^ mammals in biomedical research settings.

B. General Methods
1. Polymer Microparticles with Adsorbed Macromolecules
Polymer microparticles, including FLA and PLG microparticles, efficiently adsorb
biologically active macromolecules. Further, these microparticles adsorb a great variety of
molecules, including charged and/or bulky macromolecules. Thus the
macromolecule/microparticles used in connection with the present invention can be used as a
delivery system to deliver me biologically active components in order to treat, prevent and/or
diagnose a wide variety of diseases.
A wide variety of macromolecules can be delivered in association with the microparticles
including, but not limited to, pharmaceuticals such as antibiotics and antiviral agents, nonsteroidal
antiinflammatory drugs, analgesics, vasodilators, cardiovascular drugs, psychotropics, neuroleptics,
antidepressants, antiparkmson drugs, beta blockers, calcium channel blockers, bradykinin
inhibitors, ACE-inhibitors, vasodilators, prolactin inhibitors, steroids, hormone antagonists,
antihistamines, serotonin antagonists, heparin, chemotherapeutic agents, antineoplastics and
growth Actors, including but not limited to PDGF, EGF, KGF, IGF-1 and IGF-2, FGF,
polynucleotides which encode therapeutic or immunogenic proteins, immunogenic proteins and
epitopes thereof for use in vaccines, hormones including peptide hormones such as insulin,
proinsulin, growth hormone, GHRH, LHRH, EOF, somatostatin, SNX-111, BNP, insulinotropin,
ANP, FSH, LH, PSH and hCG, gonadal steroid hormones (androgens, estrogens and
progesterone), thyroid-stimulating hormone, inMbin, cholecystokirrin, ACTH, CKF, dynorphins,
endorphins, mrlothfllin, fibronectin fragments, galanin, gastrin, insulinotropin, glucagon, GTP-
binding protein fragments, guanyhn, the leukokinins, magainin, mastoparans, dermaseptin,
systemin, neuromedins, neurotensin, pancreastatin, pancreatic polypeptide, substance P, secretin,
thymosin, and the Eke, enzymes, transcription or translation mediators, intermediates in metabolic
pathways, irnrnunornodulators, such as any of the various cytokines including interleukin-1,
interleukm-2, interleukin-3, interleukin-4, and gamma-interferon, antigens, and adjuvants.
In some preferred embodiments of the invention, the macromolecule is nucleic acid, more
preferably a vector construct such as an ELVIS vector, or RNA vector construct. One particular
advantage of the present invention is the ability of the microparticles with adsorbed ELVIS vector
to generate cell-mediated immune responses in a vertebrate subject The ability of the antigen/

microparticles of the present invention to elicit a cell-mediated immmifs response against a selected
antigen provides a powerful tool against infection by a wide variety of pathogens. Accordingly, the
antigen/ microparticles of the present invention can be incorporated into vaccine compositions.
Thus, in addition to a conventional antibody response, the systems herein described can
provide far, e.g., the association of the expressed antigens with class IMHC 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 methods may
elicit an antigen-specific response by helper T-celk Accordingly, the methods of the present
invention will find use with any macromolecule for which cellular and/or humoral immune
responses are desired, preferably antigens derived from viral pathogens mat 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 microparticles of the present invention are particularly useful for immunization against
intraceltalar viruses 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 famuy, 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 varicella zoster
virus (VZV), Epstedn-Barr virus (EBV) and cytomegalovirus (CMV) jnrJndTng 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 McDougaH, ed., Springer-Verlag 1990) pp. 125-169, for a review
of the protein coding content of cytomegalovirus; McGeoch et aL, J. Gen. Virol. (1988) 6j>:1531-
1574, for a dist jssion 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.)
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. Hie HCV genome encodes several viral proteins, including El (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 El
and E2). Each of these proteins, as well as antigenic fragments thereof, will find use in the present
composition and methods.
Similarly, the sequence for the 5-antigen from HDV is known (see, e.g., U.S. Patent No.
5,378,814) and this antigen can also be conveniently used in the present composition and methods.
Additionally, antigens derived from HBV, such as the core antigen, the surface antigen, SAg, as
well as the presurface sequences, pre-Sl and pre-S2 (formerly called pre-S), as well as
amibinations of the above, such as SAg/pre-Sl, SAg/pre-S2, SAg/pre-Sl/pre-S2, and pre-Sl/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, incorporated
herein by reference in their entireties; BeamesetaL,^ Virol (1995) 62:6833-6838, Bimbaumet
al,/. Virol (1990) 64:3319-3330; and Zhou etaL,/. Virol (1991)65:5457-5464.
Antigens derived from other viruses will also find use in the c^mmvi compositions and
methods, such as without Imitation, proteins from members of the families Picomaviridae (e.g.,
pohoviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, eta); Flavrviridae;
Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae;
Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc.);
Orthomyxoviridae (e.g., jnthienza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;
Retroviradae (e.g., HTLV-I; HTLV-H; HTV-1 (also known as HTLV-IH, LAV, ARV, hTLR,
etc.)), including but not limited to antigens from the isolates HTVn>, HTVSR, HTVLAV, HTVLAI,
HTVMH), HTV-IOMCIS, HTV-IUM; HTV-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. Fields and D.M. Knipe, eds. 1991), for a description of these and
other viruses.

More particularly, the gpl20 or gpl40 envelope proteins from any of the above HTV
isolates, including members of the various genetic subtypes of HTV, 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 Retrotfruses 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 sequences of a variety of HTV isolates) and antigens 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 HTV isolates, including any of the
various envelope proteins such as gpl60 and gp41, gag antigens such as p24gag and p55gag, as
well as proteins derived from the pol and tat regions. Any of these proteins and antigens may also
be modified for use in the present invention. For example, Figures 1,2, 5, and 6 provide DNA
sequences encoding modified gag antigens (SEQ ID NOs: 63, 64, 67, and 68), and Figures 3 and 4
provide DNA sequences encoding modified envelope antigens (SEQ ID NOs: 65 and 66).
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 mflnm?* viruses," p. 127-168. In: P. Palese and D.W. Kingsbury (ed), Genetics of
influenza viruses. Springer-Verlag, New York). Thus, proteins derived from any of these isolates
can also be used in the compositions and methods described herein.
The compositions and methods described herein will also find use with numerous bacterial
antigens, such as those derived from organisms that cause diphtheria, cholera, tuberculosis,
tet uus, pertussis, meningitis, and other pathogenic states, mrhiHing without limitation, Bordetella
pertussis, Neisseria meningt tides (A, B, C, Y), Neisseria gonorrhoeae, Helicobacter pylori, and
Haemophilus influenza. Hemophilus influenza type B (HT£), Helicobacter pylori, and
combinations thereof Examples of antigens fromAfeisserto meningitiaes B are disclosed in the
following co-owned patent applications: PCT/US99/09346; PCTIB98/01665; and PCT
IB99/00103. Examples of parasitic antigens include those derived from organisms causing malaria
and Lyme disease.
Additional antigens for use with the invention, some of which are also listed elsewhere in

this application, include the following (references are listed immediately below):
- A protein antigen from N. meningitidis serogroup B, such as those in Reft. 1 to 7 below.
- an outer-membrane vesicle (OMV) preparation fromN. meningitidis serogroup B,
such as those disclosed in Reft. 8, 9,10,11 etc. below.
- a saccharide antigen fromN. meningitidis serogroup A, C, W135 and/or Y, such as
the oligosaccharide disclosed in Ref 12 below from serogroup C (see also Ref 13).
. a saccharide antigen from Streptococcus pneumoniae [e.g. Refs. 14, IS, 16].
- an antigen from A', gonorrhoeae [e.g., Reft. 1,2,3],
- an antigen from Chlamydia pneumoniae [e.g., Reds. 17,18,19,20,21,22,23].
- an antigen from Chlamydia trachomatis [e.g. 24].
- an antigen from hepatitis A virus, such as inactivated virus [e.g., Reft. 25,26].
- an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g., Reft. 26,27].
- an antigen from hepatitis C virus [e.g. Ref 28].
- an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous
haemaglutinin (FHA) from 5. pertussis, optionally also in combination with pertactin and/or
agglutinogens 2 and 3 [e.g., Reft. 29 & 30].
- a diphtherk antigen, such as diritherk toxoid [e.g, chapter 3 of Ref 31] e.g. the
CRM197 mutant [e.g., Ref 32].
- a tetanus antigen, such as a tetanus toxoid [e.g, chapter 4 of Ref 31].
- a protein antigen from Helicobacter pylori such as CagA [e.g. Ref. 33], VacA [e.g. Ref 33],
NAP [e.g. Ref 34], HopX [eg. Ref 35], HopY [e.g. Ref. 35] and/or urease.
- a saccharide antigen framHaemophllus influenzae B [e.g. Ref 13].
- an antigen from Porphyramonas gingivalis [e.g. Ref 36].
- polio antigenfs) [e.g. Reft. 37,38] such as IPV or OPV.
- rabies antigen(s) [e.g. Ref 39] such as ryopUlized inactivated virus [e.g. Ref 40, Rabavert™).
- measles, mumps and/or rubella antigens [e.g., chapters 9,10 and 11 of Ref 31].
- influenza antigeniY) [e.g. chapter 19 of Ref 31], such as the haemagglutinui and/or
neuraminidase surface proteins.
- an antigen fromMoraxella catarrhalis [e.g, time 41].

- an antigen from Streptococcus agalactiae (Group B streptococcus) [e.g. Refs. 42, 43]
-an antigen from Streptococcus pyogenes (Group A streptococcus) [e.g. Refs. 43,44,45].
- an antigen from Staphylococcus aureus [e.g. Re£ 46].
- Compositions comprising one or more of these antigens.
Where a saccharide or carbohydrate antigen is used, it is preferably conjugated to a carrier
protein in order to enhance nnmunogemcity [e.g. Refs. 47 to 56]. Preferred carrier proteins are
bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRM^ diphtheria toxoid is
particularly preferred. Other suitable carrier proteins include N. meningitidis outer membrane
protein [eg. Ref 57], synthetic peptides [e.g. Reft. 58, 59], heat shock proteins [eg. Ref 60],
pertussis proteins [e.g. Refs. 61,62], protein D fcomH. Influenzae [e.g. Ref 63], toxin A or B
fromC. difficile [e.g. Ref 64], etc. Where a mixture comprises capsular saccharides from both
serogroups A and C, it is preferred that the ratio (w/w) of MenA saccharide. MenC saccharide is
greaterthan 1 (e.g. 2:1, 3:1,4:1,5:1,10:1 or higher). Saccharides from different serogroups ofN.
meningitidis may be conjugated to the same or different carrier proteins.
Any suitable conjugation reaction can be used, with any suitable linker where necessary.
Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis
toxin by chemical and/or means [Ref 30].
Where diphtheria antigen is included in the composition it is preferred also to include
tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred
also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it
is preferred also to include diphtheria and tetanus antigens.
It is readily apparent that the subject invention can be used to deliver a wide variety of
macromolecules and hence to treat, prevent and/or diagnose a large number of diseases. In some
embodiments, the rnacromolecule/microparticle compositions of the present invention can be used
for site-specific targeted delivery. For example, intravenous administration of the
rnacromolecule/microparticle compositions can be used for targeting the lung, liver, spleen, blood
circulation, or bone marrow.
The adsorption of macromolecules to the surface of the adsorbent microparticles (or to
submicron emulsions of the present invention) occurs via any bonding-interaction mechanism,
including, but not limited to, ionic bonding, hydrogen bonding, covalent bonding, Van der Waals

bonding, physical entrapment, and bonding through hydrophflic/hydrophobic interactions. Those
of ordinary skill in the art may readily select detergents appropriate for the type of maoromolecule
to be adsorbed.
For example, microparticles manufactured in the presence of charged detergents, such as
anionic or catiooic detergents, may yield microparticles with a surface having a net negative or a
net positive charge, which can adsorb a wide variety of molecules. For example, microparticles
manufactured with anionic detergents, such as sodium dodecyl sulfate (SDS), ie. SDS-PLG
microparticles, adsorb positively charged antigens, such as proteins. Similarly, microparticles
manufactured with canonic detergents, such as hexadecylhirnethylanxnooium bromide (CTAB),
ie. CTAB-PLGmicroparticles, adsorb negatively charged rnacromolecules, such as DNA. Where
the rnacromolecules to be adsorbed have regions of positive and negative charge, canonic, anionic,
nonionic or zwitterioinic detergents may be appropriate.
Biodegradable polymers for manufacturing microparticles for use with the present invention
are readily commercially available from, e.g., Boehringer Ingelheim, Germany and Birmingham
Polymers, Inc., Birrnmgharn, AL. For example, useful polymers for forming the microparticles
herein include homopolymers, copolymers and polymer blends derived from the following:
pofyhydroxybutyric acid (also known as polyhydroxyburyrate); polyhydroxy valeric acid (also
known as polyhydroxyvalerate); polygrycoKc add (PGA) (also known as polygtycoKde): poly lactic
acid (PLA) (also known as polylactide); polydioxanone; polycaprolactone; polyormoester, and
polyanhydride. More preferred are poly(a-hydroxy acids), such as poly(L-lactide), pofy(D,L-
Iactide) (both known as "PLA" herein), poly(hydoxybutyrate), copolymers of D,L-lactide and
glycolide, such as poly(D,L-lactide-co-glycolide) (designated as "PLG" or "PLGA" herein) or a
copolymer of D,L-lactide and caprolactone. Particularly preferred polymers for use hei ein are
PLA and PLG polymers. These polymers are available in a variety of molecular weights, and the
appropriate molecular weight for a given use is readily determined by one of skill in the art. Thus,
e.g., for PLA, a suitable molecular weight wffl be on the order of about 2000 to 5000. For PLG,
suitable molecular weights will generally range from about 10,000 to about 200,000, preferably
about 15,000 to about 150,000.
If a copolymer such as PLG is used to form the microparticles, a variety of lactide: glycolide
ratios will find use herein and the ratio is largely a matter of choice, depending in part on the

coadministered macromolecule and the rate of degradation desired. For example, a 50:50 PLG
polymer, containing 50% D,L-lactide and 50% glycolide, will provide a fast resorbing copolymer
while 75:25 PLG degrades more slowly, and 85:15 and 90:10, even more slowly, due to the
increased lactide component. It is readily apparent that a suitable ratio of lacude:grycoEde is easily
determined by one of skill in the art based, for example, on the nature of the antigen aid disorder in
question. Moreover, in embodiments of the present invention wherein antigen or adjuvants are
entrapped within microparticles, mixtures of microparticles with varying lactide: glycolide ratios
will find use herein in order to achieve the desired release kinetics for a given macromolecule and
to provide for both a primary and secondary immune response. Degradation rate of the
microparticles of the present invention can also be controlled by such factors as polymer molecular
weight and polymer crystalliniry. PLG copolymers with varying lactide:grycolide ratios and
molecular weights are readily available commercially from a number of sources including from
Boehringer Ingfllhftim, Germany and Birmingham Polymers, Inc., Birmingham, AL. These
polymers can also be synthesized by simple pofycondensation of the lactic acid component using
techniques well known in the art, such as described in Tabata et aL, J. Biomed Mater. Res. (1988)
22:837-858.
'Where used, preferred poly(D,L-lactide-co-grycolide) polymers are those having a
lactide/grycoMe molar ratio ranging from 30:70 to 70:30, more preferably 40:60 to 60:40, and
having a molecular weight ranging from 10,000 to 100,000 Daltons, more preferably from 30,000
Daltons to 70,000 Daltons.
The polymer microparticles are prepared using any of several methods well known in the
art. For example, in some embodiments, double emulsion/solvent evaporation techniques, such as
those described in U.S. Patent No. 3,523,907 and Ogawa et aL, Chem. Pharm. Bull (1988)
36:1095-1103, can be used herein to make the rmcroparticles. These techniques involve the
formation of a primary emulsion consisting of droplets of polymer solution, which is subsequently
mixed with a continuous aqueous phase containing a particle stabilizer/ surfactant.
Alternatively, a water-in-oil-in-water (w/o/w) solvent evaporation system can be used to
form the microparticles, as described by OHagan et aL, Vaccine (1993) 11:965-969,
PCT/US99/17308 (WO 00/06123) to O'Hagan et aL and Jeffery et aL, Pharm Res. (1993)
10:362. In mis technique, the particular polymer is typically combined with an organic solvent,

such as ethyl acetate, dimethylchloride (also called methylene chloride and dichloromethane),
acetonitrile, acetone, chloroform, and the Hce. The polymer will be provided in about a 1-30%,
preferably about a 2-15%, more preferably about a 3-10% and most preferably, about a 4-6%
solution, in organic solvent The polymer solution is men combined with an aqueous solution and
emulsified to form an o/w emulsion. The aqueous solution can be, for example, deionized water,
normal saline, or a buffered solution such as phosphate-buffered saline (PBS) or a sodium
citrate/etirylenediammetetraacetic acid (sodium ritrate/ETDA) buffer solution. Preferably, the
volume ratio of polymer solution to aqueous liquid ranges from about 5:1 to about 20:1, more
preferably about 10:1. Emulsification is conducted using any equipment appropriate for this task,
and is typically a high-shear device such as, e.g., an homogenizes
A volume of the o/w emulsion is then optionally preferably combined with a larger volume
of an aqueous solution, which preferably contains a canonic, anionic, or nonionic detergent. The
volume ratio of aqueous solution to o/w emulsion typically ranges from about 2:1 to 10:1, more
typically about 4:1. Examples of anionic, canonic and nonionic detergents "appropriate for the .
practice of the invention are listed above and include SDS, CTAB and PVA, respectively. Certain
macromolecules may adsorb more readily to microparticles having a combination of stabilizers
and/or detergents, for example, a combination of PVA and DOTAP. Moreover, in some instances,
it may be desirable to add detergent to the above organic solution. Where a nonionic detergent
such as PVA an emulsion, stabilizer is used, it is typically provided in about a 2-15% solution, more
typicalry about a 4-10% solution Where a cationic or anionic detergent is used, it is typically
provided in about a 0.05-5% solution, more typically about a 0.25-1% solution Generally, a
weight to weight detergent to polymer ratio in me range of fromabout 0.00001:1 to about 0.5:1
will be used, more preferabry from about 0.0001:1 to about 0.5:1, more preferably fromabout
0.001:1 to about 0.5:1, and even more preferably from about 0.005:1 to about 0.5:1.
The mixture is then homogenized to produce a stable w/o/w double emulsion Organic
solvents are then evaporated. The formulation parameters can be manipulated to allow the
preparation of small microparticles on the order of 0.05 urn (50 nm) to larger microparticles 50 urn
or even larger. See, e.g., Jefiery et al, Pharm. Res. (1993) 10:362-368; McGee et al, J.
Microencap. (1996). For example, reduced agitation results in larger microparticles, as does an

increase in internal phase volume. Small particles are produced by low aqueous phase volumes
with high concentrations of emulsion stabilizers.
Additional information can be found in U.S. Application Serial No. , Attorney Docket
Nos. PP 16502.002, entitled "Mcroparticles with Adsorbed Macromolecules" filed September 28,
20O1.
The formulation parameters can be manipulated to allow the preparation of small
microparticles on the order of 0.05 um (50 iim) to larger microparticles 50 urn or even larger. See,
e.g., Jeffery et aL,Pharm. Res. (1993) 10:362-368; McGee et al, J. Microencap. (1996). For
example, reduced agitation results in larger microparticles, as does an increase in internal phase
volume. Small particles are produced by low aqueous phase volumes with high concentrations of
emulsion stabilizers.
Microparticles can also be formed using spray-drying and coacervarion as described in, e.g.,
Thomasin et aL, J. Controlled Release (1996) 41:131; U.S. Patent No. 2,800,457; Masters, K.
(1976) Spray Drying 2nd Ed. Wiley, New York; air-suspension coating techniques, such as pan
coating and WurSter coating, as described by Hall et aL, (1980) The "Wurster Process" in
Controlled Release Technologies: Methods, Theory, and Applications (A.F. Kydonieus, ed), Vol
2, pp. 133-154 CRC Press, Boca Raton, Florida and Deasy, P.B., Crit. Rev. Ther. Drug Carrier
Syst. (1988) S(2):99-139; and ionic gelation as described by, e.g., Lim et aL, Science (1980)
210:908-910.
Particle size can be determined by, e.g., laser light scattering, using for example, a
spectrometer incorporating a helium-neon laser. Generally, particle size is determined at room
temperature and involves multiple analyses of the sample in question (e.g., 5-10 times) to yield an
average value for toe particle diameter. Particle size is also readily determined using scanning
electron microscopy (SEM).
Alternative embodiments of the present invention utilize nricroparticle preparations
comprising a submicron emulsion, which preferably includes an ionic surfactant For instance,
MF59 or others may be used as the base oil-containing submicron emulsion, while ionic surfactants
may include, but are not limited to, ttokoyl-3-Trimethylammonium-Propane (DOTAP), Dioleoyl-
sn-Grycerch3-Bthylphosphocholine(DEPC) and dioleoyl-phosphatidic acid (DPA), each of which
are soluble in squalene. Prototypic ionic emulsions may be formulated by dissolving each of the

detergents in squalene/10% Span 85 at concentrations ranging from 4-52 mg/ml squalene. Tie
squalene/surfactant mixrures may be emulsified with 0.5% Tween 80/H2O at 5ml squalene/100 ml
H2O. A pre-emulsion may be formed by bomogenization with a Silverson homogenizer (5
minutes, 5000 RPM) and final emulsions may be made by rnicrofhridization (~10,000psi, 5 passes,
MiCTofluidizer 110S). Additional discussion concerning submicron emulsions can be found infra.
Following preparation, micropartides can be stored as is or freeze-dried for future use.
Typically, in order to adsorb macromolecules to the micropartides, the microparticle preparation is
simply mixed with the macromolecule of interest and the resulting formulation can again be
ryopbilized prior to use. Generally, macromolecules are added to the microparticles to yield
microparticles with adsorbed macromolecules having a weight to weight ratio of from about
0.0001:1 to 0.25:1 macromolecules to microparticles, preferably, 0.001:1 to 0.1, more preferably
0.01 to 0.05. Macromolecule content of the microparticles can be determined using standard
techniques.
As noted above, macromolecules for use in connection with the present invention include
proteins, preferably antigen molecules, and nucleic acids, preferably vector constructs capable of
expressing a nucleic acid sequence, such as CMV-based vectors, ELVIS vectors or RNA vector
constructs.
The polymer microparticles of the present invention may have macromolecules entrapped
or encapsulated within them, as weD as having macromolecules adsorbed thereon Thus, for
example, one of skill in the art may prepare n accordance with the invention microparticles having
encapsulated adjuvants with ELVIS vector adsorbed thereon, or microparticles having
encapsulated antigen with RNA vector construct adsorbed thereon The invention contemplates a
variety of combinations of nucleic acid macromolecules adsorbed on and entrapped within
microparticles, along with other nucleic acids as well as other antigenic molecules. In some
preferred embodiments, the microparticles of the invention have ELVIS vectors or RNA vector
constructs adsorbed thereon.
Additionally, any of the embodiments of the microparticles of the invention may be
delivered in conjunction with electroporation.
Once the macromolecule-adsorbed microparticles and/or submicron emulsion
microparticles are produced, they are formulated, along with any desired adjuvants, into

pharmaceutical compositions mrinrling vaccines, to treat, prevent and/or diagnose a wide variety of
disorders, as described above. The compositions will generally include one or more
phannaceutically acceptable exdpients. For example, vehicles such as water, saline, glycerol,
pofyethylene-grycol, hyaluronic acid, ethanol, etc. may be used. Other exdpients such as wetting
or emulsifying agents, biological buffering substances, and the like, may be present in such vehicles.
A biological buffer can be virtually any solution which is pharmacologically acceptable and which
provides the formulation with the desired pH, ie., a pH in the physiological range. Examples of
buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered
saline, and the like. Other excipients known in the art can also be introduced into me finaldosage
form, including binders, dismf egrants, fillers (diluents), lubricants, gKdanis (flow enhancers),
compression aids, colors, sweeteners, preservatives, suspensing/dispersing agents, film
formers/coatings, flavors and printing inks.
The compositions of the invention will comprise a therapeutically effective amount of one
or more macromolecules of interest That is, an amount of rnacromolecule/microparticle will be
included in the compositions, which will cause the subject to produce a sufficient response, in order
to prevent, reduce, eliminate or diagnose symptoms. The exact amount necessary will vary,
depending on the subject being treated; the age and general condition of the subject to be treated;
the severity of the condition being treated; in the case of an immunological response, the capacity
of the subject's immune system to synthesize antibodies; the degree of protection desired and 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, where the macromolecule is : polynucleotide,
an effective dose will typically range from about 1 ng to about 10 rug, more preferably from about
10 ng to about 1 mg, and most preferably about 100 ug to about 1 rag of the macromolecule
delivered per dose; where the macromolecule is an «"tigfn. an effective dose will typically range
from about 1 ugto about 100 rng, more preferably from about 10 ug to about 1 mg, and most
preferably about 50 ug to about 1 mg of the macromolecule delivered per dose.
Once formulated, the compositions of the invention can be administered parenteralfy, e.g.,
by injection. The con^osftions can be injected either subcutaneousry, intraperitoneaDy,

intravenously or intramuscularly. Other modes of administration include nasal, mucosal, rectal,
vaginal, oral and pulmonary administration, suppositories, and transdermal or transcutaneous
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 administration may be with 1-10
separate doses, followed by other doses given at subsequent time intervals, chosen to maintain
and/or reinforce the therapeutic response, for example at 1-4 months for a second dose, and if
needed, a subsequent dose(s) after several months.
In certain embodiments of the invention, a series of one or more injections of a vector
construct (which comprises a heterologous nucleic acid sequence encoding an antigen) is followed
by a series of one or more injections of antigen (also referred to herein as "boosts"). As a specific'
example, the vector construct may be administered in three injections: (a) at a time of initial
administration, (b) at a time period ranging 1-8 weeks from the initial administration, and (c) at a
time period ranging 4-32 weeks from the initial administration, while the antigen may be
administered in two injections: (a) at a time period ranging from 8-50 weeks from the initial
administration and (b) at a time period ranging from 8-100 weeks from the initial administration.
The dosage regimen will also, at least in part, be determined by the need of the subject and
be dependent on the judgment of the practitioner.
Furthermore, if prevention of disease is desired, the microparticles with adsorbed vector
constructs are generally administered prior to primary infection with the pathogen of interest If
treatment of disease (other than prevention) is desired, e.g., the reduction of symptoms or
recurrences, the microparticles with adsorbed vector constructs are generally administered
subsequent to primary infection.
2 Oil Droplet Krniiljamiy
In other embodiments of the present invention, an oil droplet emulsion (particularly, a
subnxcron emulsion) is prepared comprising a metabolizable oil and an emulsifying agent.
Molecules such as an oligonucleotide comprising at least one CpG motif may be combined with the
oil droplet emulsion to form an adjuvant
The oil droplet emulsion preferably comprises a metabolizable oil and an emulsifying agent,
wherein the oil and the emulsifying agent are present in the form of an oil-in-water emulsion having

oil droplets substantially all of which are less than one micron in diameter. Suhrrdcron emulsions,
with droplets in this preferred size range, show a surprising superiority over other emulsions
containing oil and emulsifying agents in which the oil droplets are significantly larger than those
provided by the present invention. In preferred embodiments, the emulsion is positively charged as
a result of a cationic detergent being used as the emulsifying agent or, alternatively, contains a
canonic detergent in addition to the emulsifying agent This allows for the adsorption of
nucleotide antigenic molecules, such as CpG oligonucleotides or vector constructs.. Alternatively,
the use of an anionic detergent allows for the adsorption of molecules such as proteins.
Although individual components of the submicron emulsion compositions of the present
invention are generally known, such compositions have not been combined in the same mariner.
Accordingly, the individual components, although described below both generally and in some
detail for preferred embodiments, are well known in the art, and the terms used herein, such as
metabolizable oil, emulsifying agent, imrnunostimulating agent, muranryl peptide, and lipophilic
muramyl peptide, are sufficiently well known to describe these compounds to one skilled in the art
without further description.
One component of these compositions is a metabolizable, non-tone oil, preferably one of
about 6 to about 30 carbon atoms including, but not limited to, alkanes, alkenes, alkynes, and their
corresponding acids and alcohols, the ethers and esters thereof, and mixtures thereof The oil can
be any vegetable oil, fish oil, animal oil or synthetically prepared oil which can be metabolized by
the body of the host animal to which the adjuvant will be administered and which is not toxic to the
subject The host animal is typically a mammal, and preferabry a human. Mineral oil and similar
toxic petroleum distillate oils are expressly excluded from this invention.
The oil component of this invention c -in also be any long chain alkane, alkene or alkyne, or
an acid or alcohol derivative thereof either as the free acid, its salt or an ester such as a mono-, or
di- or triester, such as the triglycerides and esters of 1,2-propanediol or similar poly-hydroxy
alcohols. Alcohols can be acylated employing amino- or poly-functional acid, for example acetic
acid, propanoic acid, citric acid or the like. Ethers derived from long chain alcohols which are oils
and meet the other criteria set forth herein can also be used.
The individual alkane, alkene or alkyne moiety and its acid or alcohol derivatives will
generally have about 6 to about 30 carbon atoms. The moiety can have a straight or branched

chain structure. It can be fully saturated or have one or more double or triple bonds. Where mono
or poly ester- or ether-based oils are employed, the limitation of about 6 to about 30 carbons
applies to the individual fatty acid or fatty alcohol moieties, not the total carbon count
Any metaboKzable oil, particularly from an animal, fish or vegetable source, can be used
herein. It is essential that the oil be metabolized by the host to winch it is administered, otherwise
the oil component can cause abscesses, granulomas or even carcinomas, or (when used in
veterinary practice) can make the meat of vaccinated birds and animals unacceptable for human
consumption due to the deleterious effect the unmetabolized oil can have on the consumer.
For a detailed description of such submicron emulsions, see International Publication No.
WO 90/14837, and commonly owned Intemational Patent Application PCT/USOO/03331.
The oil component of these adjuvants and immunogenic compositions will be present in an
amount from about 0.5% to about 20% by volume but preferably no more than about 15%,
especially in an amount of about 1% to about 12%. It is most preferred to use from about 1% to
about 4% ofl.
The aqueous portion of these submicron emulsion compositions is preferably buffered
saline or, more preferably, unadulterated water. Because these compositions are intended for
parenteral administration, it is preferable to make up final buffered solutions used as immunogenic
compositions so that the tonicity, Le., osmolality, is essentially the same as normal physiological
fluids in order to prevent post-administration swelling or rapid absorption of the composition
because of differential ion concentrations between the composition and physiological fluids It is
also preferable to buffer the saline in order to maintain pH compatible with normal physiological
conditions. Also, in certain instances, it can be necessary to maintain the pH at a particular level in
order to ensure the stability of certain composition components such as the grycopeptides.
Any physiologically acceptable buffer can be used herein, but phosphate buffers are
preferred. Other acceptable buffers such acetate, tris, bicarbonate, carbonate, or the like can bs
used as substitutes for phosphate buffers. The pH of the aqueous component will preferably be
between about 6.0-8.0.
When the submicron emulsion is initially prepared, however, unadulterated water is
preferred as the aqueous component of the emulsion. Increasing the salt concentration makes it
more difficult to achieve the desired small droplet size. When the final immunogenic compositions

is prepared from the adjuvant, the antigenic material can be added in a buffer at an appropriate
osmolality to provide the desired immunogenic compositioa
The quantity of the aqueous component employed in these compositions will be that
amount necessary to bring the value of the composition to unity. That is, a quantity of aqueous
component sufficient to make 100% will be mixed, with the other components listed above, in
order to faring the compositions to volume.
A substantial number of emulsifying and suspending agents are generally used in the
pharmaceutical sciences. These include naturally derived materials such as gums from trees,
vegetable protein, sugar-based polymers such as alginates and cellulose, and the hke. Certain.
oxypolymers or polymers having a hydroxide or other hydropmhc substituent on the carbon
backbone have surfactant activity, for example, povidone, polyvinyl alcohol, and glycol ether-based
mono- and poly-functional compounds. Long chain fatry-acid-derived compounds form a third
substantial group of emulsifying and suspending agents which could be used in this inventioa Any
of the foregoing surfactants are useful so long as they are non-toxic.
Specific examples of suitable emulsifying agents (also referred to as surfactants or
detergents) which can be used in accordance with the present invention are disclosed in commonly
owned International patent application PCT/US00/0331. Surfactants are generally divided into
four basic types: anionic, canonic, zwitterionic, and nonionic. Examples of anionic detergents
include, but are not limited to, alginic acid, capryUc acid, choke acid, 1-decanesulfonic acid,
deoxycholic acid, 1-dodecanesulfonic acid, N-lauroylsarcosme, and taurocholic acid, and the like.
Canonic detergents include, but are not limited to, cerrimide (hexadecytairfletbylaramnium
bromide, or CTAB), benzalkomum chloride, dimethyl dioctodecyl ammonium (DDA) bromide,
DCT AP, dodecyhriinemylamrnonhim bromide, benzyldimethylhexadecyl ammonium chloride,
cetylpyridinium chloride, methylbenzethonium chloride, and 4-picoline dodecyl sulfate, and the
like. Examples of zwitterionic detergents include, but are not limited to, 3-[(3-cholamidopropyl)-
dnnemylammonio]-I-propanesulfonate (commonly abbreviated CHAPS), 3-[(choIamidopropyi)--
dimiemylaminomo]-2-hydroxy-l-propanesulfonate (generafly abbreviated CHAPSO) N-dodecyl-
N.N-diniethyl-3-ammonio-l-propanesulfonate, and ryso-a-phosphaudylcholine, and the like.
Examples of nonionic detergents include, but are not limited to, decanoyl-N-methylglucamide,
diethylene glycol monopenryl ether, n-dodecyl p^D-ghicopyranoside, ethylene oxide condensates of

fatty alcohols (e.g., sold under the trade name Lubrol), polyoxyethylene ethers of fatty acids
(particularly CM-CM fatty acids), polyoxyethylene sorbitan fatty acid ethers (e.g., sold under the
trade name Tween), and sorbitan fatty acid ethers (e.g., sold under the trade name Span), and the
like.
A particularly useful group of surfactants are the sorbitan-based non-ionic surfactants, such
as the commercialr/ available SPAN® or ARLACEL®, usually with a letter or number designation
which distinguishes between the various mono-, di- and triester substituted sorbrtans. A related
group of surfactants comprises polyoxyethylene sorbitan monoesters and polyoxyethylene sorbitan
triesters, commercialry available under the mark TWEEN®. The TWEEN® surfactants can be
combined with a related sorbitan monoester or triester surfactants to promote emulsion stability.
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 hnmunostnrnilating agent (adding such agents decreases droplet size). Actual droplet
size will vary with the particular detergent, oil, and immtmosthnulatizig 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 0.8 microns in
diameter, and most preferably less than 0.5 microns in diameter. By substantially all is meant at
least about 80% (by number), preferably at least about 90%, more preferabry at least about 95%,
and most preferabry at feast about 98%. The particle size distribution is typically Gaussian, so that
the average diameter is smaller than the stated limits.
A preferred oil droplet emulsion is MF59. MF59 can be made according to the procedures
described in, for example, (Metal, Vaccine Design: The Submit And Adjuvant Approach, 1995,
MR Powell and M.J. Newman, Eds., Plenum Press, New York, p. 277-296; Singh et al, Vaccine,
1998,16,1822-1827; Ott etal, Vaccine, 1995,13,1557-1562; and Valensi etal, J. Immunol,
1994,153,4029-39, the disclosures of which are incorporated herein by reference in their entirety.
Other oil droplet emulsions include, for example, SAF, comaining 10% Squalane, 0.4%
Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a

submicron emulsion 01 vortexed to generate a larger particle size emulsion, and Ribi® adjuvant
system (RAS), (Ribi Imrnunochem, Hamton, 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 dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS
(DetoxJ) (for a further discussion of suitable submicron oil-in-water emulsions for use herein, see
commonly owned, patent application no. 09/013,736, filed on January 29,1998).
After preparing the microparticles of the invention, whether of the polymer type or the
submicron emulsion type, macromolecules such as polypeptides and vector constructs may be
adsorbed thereto as previously discussed. The submicron emulsion microparticles of the present
invention may also have macromolecules entrapped or encapsulated within them, as well as having
macromolecules adsorbed thereon. Thus, for example, one of skill in the art may prepare in
accordance with the invention microparticles having encapsulated adjuvants with ELVIS vector
adsorbed thereon,, or microparticles having encapsulated antigen with RNA vector construct
adsorbed thereon. The invention contemplates a variety of combinations of nucleic acid
macromolecules adsorbed on and entrapped within microparticles, along with other nucleic acids as
well as other antigenic molecules. Preferably, the microparticles of the invention have ELVIS
vectors or RNA vector constructs adsorbed thereon Additionally, any of the embodiments of the
microparticles of the invention may be delivered in conjunction with electroporaaon.
3. ELVIS vectors
ELVIS vectors are Eukaryotic Layered Vector Initiation Systems, which are generally
described in U.S. Patents 5,814,482 and 6,015,686, cited above, as well as in International Patent
Applications WO 97/38087 and WO 99/18226. In one embodiment, an ELVIS vector is derived
from the genome of an abphavirus, more preferably from Sindbis virus (SIN), SemlM Forest virus
(SFV), Venezuelan equine encephalitis virus (VEE), or Ross River virus (RRV). The alphavirus is
an RNA virus of approximately 11-12 kb in length, which contains a 5' cap and a 3' polyadenylate
taiL The mature infectious virus is composed of the genomic RNA enveloped by the nucleocapsid
and envelope proteins. Alphavirus infection of host cefls occurs by a receptor specific event and
culminates in release of genomic RNA into the cytoplasm During viral replication, the viral-

encoded envelope glycoproteins El and E2 are synthesized and embedded in the host cell
membrane, through which progeny virions bud and release to the outside of the host celL
Replication of the viral genome begins with the genomic RNA strand serving as the
template for synmesis of a con^ementary negative RNA strand. The negative RNA strand then
serves as a template for full-length genomic RNA, and for an intenialfyininsrted positive-strand
subgenotmc RNA Tte nonstructural proteins are translated from the genomic strand, while lie
structural proteins are translated from the subgaaomic strand. All the viral genes are expressed
first as polyproteins, then post-translalionauy processed into individual proteins by proteolytic
cleavage.
An alphavirus vector replicon may be buQt by replacing certain portions of the viral genome
(e.g., structural protein genes) with a selected heterologous nucleic acid sequence. Thus, in certain
embodiments, an alphavirus replicon vector may comprise a 5' sequence capable of initiating
transcription of an alphavirus, a nucleotide sequence encoding the alphavirus nonstructural
proteins, an alphaviral junction region promoter, an alphavirus RNA polymerase recognition site,
and a 3' polyadenylate tract Additionally, the alphavirus vector replicon may be contained as a
cDNA copy within an alphavirus vector construct Such vector constructs typically comprise a 5'
promoter capable of initiating synthesis of RNA from cDNA positioned upstream and operabty
associated with the vector cDNA, such mat transcription produces the vector replicon RNA The
vector construct also may contain and a 3' sequence controlling transcription termination. A
heterologous nucleic acid sequence may be present upstream or downstream of the viral junction
regkm.
The ELVIS vector capitalizes on the mechanism of RNA virus replication to achieve
delivery of a heterologous nucleotide sequence of interest by using a double-layered approach (for
example, based on the above-described alphavirus vector construct). In gsueral, an ELVIS vector
provides a layered expression system capable of amplifying the amount of RNA encoding the gene
product of interest because the first layer initiates transcription of a second layer. Thus, a typical
ELVIS vector comprises a 5' promoter capable of initiating synthesis of RNA from cDNA, a
cDNA complement of a construct capable of autonomous replication in a cell, and which construct
is also capable of expressing a heterologous nucleic acid sequence, and a 3' sequence controlling
transcription termination. The construct capable of autonomous replication and expression of the

selected nucleic acid sequence may be an alphavirus vector construct Thus, the first layer of the
DNA ELVIS vector transcribes the RNA atyhavirus vector construct, from which expression of
the selected heterologous nucleic acid sequence is achieved.
An alphavirus-based ELVIS vector may be constructed by first preparing a cDNA
cornplernentary to an alphavirus genome. ThecDNA corresponding to the genomic RNA is then
. . deleted of sequences encoding one or more viral structuralr/roteins which men may be replaced
with heterologous DNA encoding the gene-product of iiterest, thereby preventing packaging of
mature virus and —«Hinj> amplification of the heterologous sequence. The modified cDNA
containing the heterologous sequence is then inserted within the first layer of the ELVIS vector.
Upon entry into the cell and nucleus, the ELVIS vector will be transcribed and the resulting mRNA
molecules, which are RNA vectors capable of self-replication, will begin to replicate and translate
polypeptides, including the heterologous gene of interest
While atypical Sindbis-derived alphavirus vector construct is preferred, other alphavirus
species may be readily used according to the teachings provided herein. Alternatively, vectors
derived from any RNA virus may be utilized, particularly those from positive-stranded viruses.
The construction of an ELVIS vector, in general, is described in U.S. Patents 5,814,482
and 6,015,686. Briefly, RNA is obtained from an SNA virus, then cDNA is synthesized by PCR
amplification using appropriate primers for particular genes or portions of the RNA virus, which
primers may also contain additional restriction sites as necessary. The cDNA fragments are then
cloned into a plasmid and transformed into an appropriate host such as E. coli. Positive colonies
are grown for plasmid purification, and then plasmids are assembled into the desired ELVIS vector
with a portion having heterologous DNA such as a reporter gene (e.g., GFP) or a desired gene
coding for an antigen. Example 3 below describes a pjticular preferred ELVIS vector (pSINCP)
used in accordance with the instant invention.
4. RNA and pCMV vector constructs
In other embodiments of the present invention, an RNA vector construct or RNA replicon
vector is used directly, without file requirement for introduction of DNA into a cell and transport
to the nucleus where transcription would occur. By using the RNA vector for direct delivery into
the cytoplasm of the host cell, autonomously replication and translation of the heterologous nucleic

acid sequence occurs efficiently. In this embodiment, the RNA vector construct or RNA rephcon
vector is obtained by in vitro transcription from a DNA-based vector construct Preferably, Hie
RNA vector construct or RNA replicon vector is derived from the genome of an alphavirus, more
preferably from Sindbis virus (SIN), Semtilri Forest virus (SFV), Venezuelan equine encephalitis
virus (VEE), or Ross River virus (RRV). In other embodimeDts, me RNA vector construct is
derived from a vinis other than an alphavirus. Preferably, such other viruses used for the
derivation of RNA vector constructs are positive-stranded RNA viruses, and more preferably they
are picomaviruses, fkvrvimses, rotaviruses, or coronaviruses. Conyositioris and methods for in
vitro transcription of alphavmis-based RNA vectors is provided in detail elsewhere (see U.S.
Patent 5,842,723 and Roto et aL, 1999, PNAS 96:4598-603). The RNA vector is men adsorbed to
a nicroparticle of the invention for delivery as detailed herein 'While a typical alphavirus RNA
vector from SIN, SFV, VEE or RRV is preferred, similar vectors derived from other alphavirus
species may be readily substituted.
In other embodiments of the present invention, pCMV vector constructs are used. Such
vector constructs are well known in the art A particularly preferred pCMV vector contains the
jmrnftrtintft-mirly anhanrar/pmmntBr of P.MV and a hnvfne growth hnrmrmfi terminatnr It is
described in detail in Chapman, B. S., et aL 1991. "Effect of nitron A from human cytomegalovirus
(Towne) immediate-early gene on heterologous expression in nwrnrratian cells." Nucleic Adds
Res. 19:3979-86.
S Adjuvants
Adjuvants may optionally be used to enhance the effectiveness of the pharmaceutical
compositions, wnh Thl stimulating adjuvants being particularly preferred. The adjuvants may be
administered concurrently with the microparticles of the present invention, e.g., in the same
composition or in separate compositions. Alternatively, an adjuvant may be administered prior or
subsequent to the micropartide compositions of the present invention In another embodiment, the
adjuvant, such as an immunological adjuvant, may be encapsulated in the nicroparticle. Adjuvants,
just as any macromolecules, may be encapsulated within the microparticles using any of the several
methods known in the art See, e.g., U.S. Patent No. 3,523,907; Ogawa et aL, Chan Pharm. Bull.
(1988) 36:1095-1103; OTHagan et aL, Vaccine (1993) Jl:965-969 and Jefferey et aL, Pharm. Res.

(1993) 10:362. Alternatively, adjuvants may be adsorbed on the micropaiticle as described above
for any macromolecule. Alternatively, adjuvants may comprise the oil droplet emulsions of the
present invention.
Immunological adjuvants include, but are not limited to: (1) aluminum salts (alum), such as
aluminum hydroxide, ahmanum phosphate, atoninum sulfate, etc.; (2) other oil-in water emulsion
formulations (with or without other specmcinminnostuiailating agents such as muramyl peptides
(see below) or bacterial cell wall components), such as for example (a) MFS9 (International
Publication No. WO90/14837; Chapter 10 in Vaccine design: the subunit an adjuvant approach,
eds. Powell & Newman, PlenumPress 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5%
Span 85 (optionally containing various amounts of MTP-PE (see below), although not required)
formulated into subrmcron particles using a microfluidizer such as Model HOY microfluidizer
(Miaofluidics, Newton, MA), (b) SAF, containing 10% Squakne, 0.4% Tween 80, 5% pluronic-
blocked polymer L121, and thr-MDP (see below) either rmcroitanzed into a submicron emulsion
or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi
Immuuochem, 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
dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™) (fbT a further
discussion of suitable submicron oil-in-water emulsions for use herein, see commonly owned,
patent application no. 09/015,736, filed on January 29,1998); (3) saponin adjuvants, such as Quil
A, or QS21 (e.g, Sthnulon™ (Cambridge Bioscience, Worcester, MA)) may be used or particle
generated therefrom such as ISCOMs (irnmunostimulating complexes), which ICOMS may be
devoid of additional detergent e.g., WO00/07621; (4) Complete Freunds Adjuvant (CFA) and
Incomplete Freunds .■' Jjuvant (IFA); (5) cytokines, such as interleukms (e.g. JL-1, IL-2, IL-4, IL-
5, BL-6, EL-7, IL-12 (W099/44636), etc.), interferons (e.g. gamma interferon), macrophage colony
stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) monophosphoryi lipid A (MPL)
or 3-O-deacyiated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantial
absence of alum when used with pneumococcal saccharides e.g. WOOO/56358; (7) combinations of
3dMPL with, for example, QS21 and/or oil-in-water emulsions, e.g., EP-A-0835318, BP-A-
0735898, BP-A-0761231; (8) oligonucleotides comprising CpGmotifs (Romanet el, Nat. Med,
1997,3,849-854, Weiner et aL, PNAS USA, 1997, 94,10833-10837; Davis et al, J. Immunol.

1988,160, 870-876; Chu et al, J. Exp. Med., 1997,186,1623-1631; Lipford et al., Eur. J.
Immunol. 1997, 27, 2340-2344; Moldoveanu et aL, Vaccine, 1988,16,1216-1224, Krieg et aL,
Nature, 1995, 374, 546-549; Klinman et aL, PNAS USA. 1996, 93,2879-2883: Ballas et aL, J.
Immunol., 1996,157,1840-1845; Cowdeiy et aL, J. Immunol, 1996,156,4570-4575; Halpern et
aL, Cell Immunol, 1996,167,72-78; Yamamoto etd.,Jpn.J. Cancer Res., 1988,79, 866-873;
Stacey et aL, J. Immunol 1996,157,2116-2122; Messina et aL, J. Immunol, 1991,147,1759-
1764; Yi et aL, J. Immunol, 1996,157,4918-4925; Yi et aL, J. Immunol, 1996,157,5394-5402;
Yiet al,./. Immunol,.1998,160,4755-4761; and Yiet aL,/. Immunol, 1998,160, 5898-5906;
International patent applications WO96/02555, W098/16247, WO98/18810, WO98/40100,
W098/55495, W098/37919 and W098/52581) La containing at least one CG dinucleotide, with 5
mcthylcytosine optionally being used in place of cytosme; (9) a polyoxyethylene ether or a
polyoxyetbylene ester e.g. W099/52549; (10) a polyoxyethylene sorbitan ester surfactant in
combination with an octoxynol (WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant
in combination with at least one additional non-ionic surfactant such as an octoxynol
(WOO 1/21152); (11) a saponin and an immunostimulatory oligonucleotide (e.g., a CpG
oligonucleotide) (WO00/62800); (12 ) an imtnfflostimulant and a particle of metal sah e.g.
WO00/23105; (13) a saponin and an oil-in-water emulsion e.g. W099/1I241; (14) a saponin (e.g.,
QS21) + 3dMPL + EH2 (optionally + a sterol) eg. W098/57659; (15) detoxified mutants of a
bacterial ADF-rJbosylating toxin such as a cholera torn (CT), a pertussis toxin (FT), or an E. coli
heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type ammo
acid at position 63) LT-R72 (where argmine is substituted for the wild-type amino acid at position
72), CT-S109 (where serine is substituted for me 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 (16) other substances that act as mmunostimulating agents to enhance the
effectiveness of the composition.. Alum (especially aluminum phosphate and/or hydroxide) and
MF59 are preferred.
Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-
isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), N-

acetyhmrarnyl-L-alanyl-D-isogruatniiryl-L-^^
huydroxyphosphoiyloxy)-ethylamiiie (MTP-PE), etc.
For additional examples of adjuvants, see Vaccine Design, The Submit and the Adjuvant
Approach, Powell, M.F. and Newman, MJ, eds., Plenum. Press, 1995)
Thus, an optional additional component of the compositions of the present invention
preferably is an adjuvant such, as aluminum salts or an oligonucleotide which comprises at least one
CpG motif As used herein, the phrase "CpG motif refers to a dinucleotide portion of an
oligonucleotide which comprises a cytosine nucleotide followed by a guanosine nucleotide. Such
oligonucleotides can be prepared using conventional oligonucleotide synthesis well known to the
skilled artisan. Preferably, the oligonucleotides of the invention comprise a modified backbone,
such as a phosphorotbioate or peptide nucleic acid, so as to confer nuclease resistance to the
oligonucleotide. Modified backbones are well known to those skilled in the ait. Preferred peptide
nucleic acids are described in detail in U.S. Patent Numbers 5,821,060,5,789,573, 5,736,392, and
5,721,102, Japanese Patent No. 10231290, European Patent No. 839,828, and PCT Publication
Numbers WO 98/42735, WO 98/42876, WO 98/36098, WO 98/27105, WO 98/20162, WO
98/16550, WO 98/15648, WO 98/04571, WO 97/41150, WO 97/39024, and WO 97/38013, the
disclosures of which are incorporated herein by reference in their entirety.
The oligonucleotide preferably comprises between about 6 and about 100 nucleotides,
more preferably between about 8 and about 50 nucleotides, most preferably between about 10 and
about 40 nucleotides. In addition, the oligonucleotides of the invention can comprise substitutions
of the sugar moieties and nitrogenous base moieties. Preferred oligonucleotides are disclosed in,
for example, Krieg etal, Proc. Natl. Acad, Set USA, 1998,95,12631-12636, Khnmane/ai,
Proc. 7 ;tl. Acad. Sci. USA, 1996,93,2879-2883, Werner et al, Proa Natl. Acad Sci. USA,
1997,94,10833-10837, Chu etal, J. Exp.Med, 1997,186,1623-1631, Brazolot-MuTanera/.,
Proc. Natl. Acad Sci. USA, 1998,95,15553-15558, BaOas et aL, J. Immunol, 1996,157,1840-
1845, Cowdery et al, J. Immunol., 1996,156,4570-4575, Halpem et al, Cell Immunol, 1996,
167, 72-78, Yamamoto et al.,Jpn. J. Cancer Res., 1988, 79, 866-873, Stacey et al, J. Immunol,
1996,157,2116-2122, Messina et al, J. Immunol, 1991,147,1759-1764, Yi etal, J. Immunol,
1996,157,4918-4925, Yi etal., J. Immunol, 1996,157, 5394-5402, Yi etal, J. Immunol, 1998,
160,4755-4761, Roman etal, Nat. Med, 1997,3, 849-854, Davis etal, J. Immunol, 1998,160,

870-876, Lipford et al, Eur. J. Immunol, 1997,27,2340-2344, Moldoveanu et al, Vaccine,
1988,16,1216-1224, Yi etal, J. Immunol, 1998,160,5898-5906, PCT Publication WO
96/02555, PCT Publication WO 98/16247, PCT Publication WO 98/18810, PCT Publication WO
98/40100, PCT Publication WO 98/55495, PCT Publication WO 98/37919, and PCT Publication
WO 98/52581, the disdosures of wbidi are incorporated hOTOT It is
to be understood that the oligonucleotides of the invention comprise at least one CpG motif but
can contain a plurality of CpG motifs.
Preferred oligonucleotides comprise nucleotide sequences such as, for example,
tccatgacgttcctgacgtt (SEQ ID NO:l), ataatcgacgttcaagcaag (SEQ ID NO:2),
ggggtcaacgttgagggggg (SEQ ID NO:3), tctcccagcgtgcgccat (SEQ ID NO:4),
gagaacgctcgaccttcgat (SEQ ID NO:5), tccatgtcgttcctgatgct (SEQ ID NO:6), tccatgacgttcctgatgct
(SEQ ID NO:7), gctagacgttagcgt (SEQ ID NO:8), atcgactctcgagcgttctc (SEQ ID NO:9),
gaaccttccatgctgttccg (SEQ ED NO: 10), gctagatgttagcgt (SEQ ID NO:l 1), tcaacgtt (SEQ ID
NO:12), gcaacgtt (SEQ ID NO:13), tcgacgtc (SEQ ID NO:14), tcagcgct (SEQ ID NO: 15),
tcaacgct (SEQ ED NO:16), tcatcgat (SEQ ED N0.17), tcttegaa (SEQ ID NO:18),
tgactgtgaacgttcgagatga (SEQ ED NO: 19), tgactgtgaacgttagcgatga (SEQ ED NO:20),
tgactgtgaacgttagagcgga (SEQ ED NO:21), gtttgcgcaacgttgttgccat (SEQ ED NO:22),
atggcaacaacgttgcgcaaac (SEQ ED NO:23), cattggaaaacgttcttcgggg (SEQ ED NO:24),
ccccgaagaacgttttccaatg (SEQ ED NO:25), attgacgtcaat (SEQ ED NO:26), ctttccattgacgtcaatgggt
(SEQ ED NO:27), and tccatacgttcctgacgtt (SEQ ED NO:28). En preferred embodiments of the
invention, the oligonucleotide comprises a CpG motif flanked by two purines at the 5' side of the
motif and two pyrirnidines at the 3' side of the motif It is to be understood, however, that any
oligonucleotide comprising a CpG motif can be used in the present invention as long as the
oligonucleotide induces an increase in Thl lymphocyte stimulation when combined with the
microparticle compositions described herein.
6. Antigens
The present invention is also directed to immunogenic compositions comprising the
microparticles described above with adsorbed macromolecules, preferably vector constructs
encoding antigens and/or antigen per se. Generally, an antigen stimulates the proliferation of T-

lymphocytes, preferably Thl rymphocytes, with receptors for the antigen and can react with die
lymphocytes to initiate the series of responses designated {^-mediated immunity. An antigen may
thus induce a CTL response, and/or a humoral response, and may induce cytokine production.
An epitope is within the scope of mis definition of antigen. An epitope is that portion of an
antigenic molecule or antigenic complex that determines its immunological specificity. Commonly,
an epitope is a peptide or polysaccharide in naturally occurring antigens. In artificial antigens it can
be a low molecular weight substance such as anarsauflic acid derivative. An epitope win react
specifically in vivo or in vitro with homologous antibodies or T rymphocytes. Alternative
descriptors are antigenic determinant, antigenic structural grouping and haptenic grouping.
In preferred embodiments of the invention, the antigenic substance is derived from a virus
such as, for example, human irnmunodeficiency virus (HTV), hepatitis B virus (HBV), hepatitis C
virus (HCV), herpes simplex virus (HSV), cytomegalovirus (CMV), influenza virus (flu), and
rabies virus. Preferably, the antigenic substance is selected from the group consisting of HSV
grycoprotehi gD , HTV glycoprotein gp!20, HTV p55 gag, and polypeptides fromThe pol and tat
regions. In other preferred embodiments of the invention, the antigenic substance is derived from a
bacterium such as, for example, Helicobacter pylori, Haemophilus influenza, cholera, diphtheria,
tetanus, Neisseria meningitidis, and pertussis, m other preferred embodiments of the invention,
thp antigmjr. siihstanpe V from a parasite such as, for example, a malaria parasite In another
preferred ernlwdnnent of the present invention, the antigen is adsorbed to the surface of a
microparticle of the present invention.
Antigens can be produced by methods known in the art or can be purchased from
commercial sources. Antigens within the scope of this invention include whole inactivated virus
particles, isolated virus proteins and protein subunits, whole cells and bacteria, cell membrane and
cell wall proteins, and the like. Some preferred antigens are described below.
Herpes simplex virus (HSV) rgD2 is a recombinant protein produced in genetically
engineered Chinese hamster ovary cells. This protein has the normal anchor region truncated,
resulting in a glycosylated protein secreted into tissue culture medium The gD2 can be purified in
the CHO medium to greater than 90% purity. Human immunodeficiency virus (HTV) env-2-3 is a
recombinant form of the HTV enveloped protein produced in genetically engineered
Saccharomyces cerevisae. This protein represents the entire protein region of HTV gpl20 but is

non-grycosylated and denatured as purified from the yeast HTV gpl20 is a fully glycosylated,
secreted form of gpl20 produced in CHO cells in a fashion similar to the gD2 above. Additional
HSV antigens suitable for use in immunogenic compositions are described in PCT Publications WO
85/04587 and WO 88/02634, the disclosures of which are incorporated herein by reference in their
entirety. Mixtures of gB and gD antigens, which are truncated surface antigens lacking the anchor
regions, are particularly preferred
Additional HTV antigens suitable for use in immunogenic compositions are described in
U.S. applications serial no. 490,858, filed March 9,1990, and published European application
number 181150 (May 14,1986), as well as U.S. applications serial nos. 60/168,471; 09/475,515;
09/475,504; and 09/610,313, the disclosures of which are incorporated herein by reference in their
entirety.
Cytomegalovirus antigens suitable for use in immunogenic compositions are described in
U.S, Patent No. 4,689,225, U.S. application serial number 367,363, filed June 16,1989 and PCT
Publication WO 89/07143, the disclosures of which are incorporated herein by reference in their
entirety.
Hepatitis C antigens suitable for use in immunogenic compositions are described in
PCT/US88/04125, published European application number 318216 (May 31,1989), published
Japanese application number 1-500565 filed November 18,1988, Canadian application 583,561,
and EPO 388,232, disclosures of which are incorporated herein by reference in their entirety. A
different set of HCV antigens is described in European patent application 90/302866.0, filed March
16,1990, and U.S. application serial number 456,637, filed December 21,1989, and
PCTVUS90/01348, the disclosures of which are incorporated herein by reference in their entirety.
Immunogenic compositions of the invention can be used to imrmmi7f! birds and mammals
against diseases and infection, mrrnHing without limitation cholera, diphtheria, tetanus, pertussis,
influenza, measles, meningitis, mumps, plague, poliomyelitis, rabies, Rocky Mountain spotted
fever, nibella, smallpox, typhoid, typhus, feline leukemia virus, and yellow fever.
Certain immunogenic compositions of the invention wul employ an effective amount of an
antigen. For example, there miry be included an amount of antigen which, in combination with an
adjuvant, will cause the subject to produce a specific and sufficient immunological response, so as

to impart protection to the subject from the subsequent exposure to a virus, bacterium, fungus,
mycoplasma, or parasite.
In other embodiments, a composition comprising an antigen will be used to boost the
immunological response of a previously administered vector construct, which preferably comprises
a heterologous nucleic acid sequence mat encodes the antigen. More preferably, the antigen is
associated with (e.g., adsorbed to) the miaoparticles described herein and/or the antigen is
coadministered with an adjuvant.
No single dose designation can be assigned which will provide specific guidance for each
and every antigen which can be employed in this invention. The effective amount of antigen will be
a function of its inherent activity and purity and is empirically determined by those of ordinary skill
in the art via routine experimentation. It is contemplated that the adjuvant compositions of this
invention can be used in conjunction with whole cell or viral immunogenic compositions as well as
with purified antigens or protein submit or peptide immunogenic compositions prepared by
recombinant DNA techniques or synthesis.
Where the antigen is provided in connection with an emulsion, because the adjuvant
compositions of the invention are stable, the antigen and emulsion can typically be mixed by simple
shaking. Other techniques, such as passing a mixture of the adjuvant and solution or suspension of
the antigen rapidly through a small opening (such as a hypodermic needle), readily provide a useful
immunogenic composition.
The immunogenic compositions according to the present invention comprise about 1
nanogram to about 1000 micrograms of nucleic acid, preferably DNA such as, for example, CpG
oligonucleotides. In some preferred embodiments, the immunogenic compositions contain about
10 nanograms to about 800 micrograms of nucleic acid. In some preferred embodiments, the
immunogenic compositions contain about 0.1 to about 500 micrograms of nucleic acid. In some
preferred embodiments, the immunogenic compositions contain about 1 microgram to about 10
milligrams of nucleic acid. In some preferred embodiments, the immunogenic compositions
contain about 250 micrograms to about 1 miHifiram of nucleic acid. In some preferred
embodiments, the immunogenic compositions contain about 500 micrograms to about 1 milligram
of nucleic acid. One skilled in the art can readily formulate an immunogenic composition
comprising any desired amount of nucleic acid. The immunogenic compositions according to the

present invention are provided sterile and pyrogen free. The immunogenic compositions can be
conveniently administered in unit dosage form and can be prepared by any of the methods well
known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical
Sciences (Mack Pub. Co., Easton, PA, 1980), the disclosure of which is incorporated herein by
reference in its entirety.
Trie present invention is also directfd to i»wrtiiMk "f £*""»Vftiog "> immnnft Taspunsa in a
host animal comprising administering to the animal one or more immunogenic compositions
described above in an amount effective to induce an immune response. The host animal is
preferably a mammal, more preferably a human. Preferred routes of administration include, but axe
not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial,
inrxaoccular and oral as well as transdermal or by inhalation or suppository. Most preferred routes
of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection.
According to some embodiments of the present invention, the immunogenic compositions are
administered to a host animal using a needleless injection device, which are well known and widely
available. One having ordinary stall in the art can, following the teachings herein, use needleless
injection devices to deliver immunogenic compositions to cells of an individual. Additionally, the
embodiments of the invention may be delivered together with electroporation.
The present invention is also directed to methods of immunizing a host animal against a
viral, bacterial, or parasitic infection comprising administering to the animal one or more
immunogenic compositions described above in an amount effective to induce a protective response.
The host animal is preferably a manuring more preferably a human. Preferred routes of
administration are described above. WWe prophylactic or therapeutic treatment of the host animal
can be directed to any pathogen, preferred pathogens, including, 1 t not limited to, the viral,
bacterial and parasitic pathogens described above.
The present mvention is also directed to methods of inducing an immune response in a host
animal comprising administering to the animal one or more immunogenic compositions described
above in an amount effective to induce m immune response. The host animal is preferably a
marrmal, more preferably a human. Preferred routes of administration are described above. One
skilled in the art is readily familiar with immune responses and measurements thereof

The present invention contemplates the use of polymer rmcroparticles or submicron
emulsion rmcroparticles with adsorbed macromolecule to elicit an immune response alone, or in
combination with another macromolecule. That is, the invention encompasses rmcroparticles with
adsorbed nucleic acid, submicron emulsions with adsorbed nucleic acid or Jmnnnostimiilating
molecule, and the combination of rmcroparticles with adsorbed nucleic acid together with
subrmcron emulsions with adsorbed nucleic acid or rmmmostminlating molecule. Electroporation
may also be used to improve delivery of the nucleic arid.
As demonstrated by the following Examples, the present invention's polymer rmcroparticles
with adsorbed macromolecules elicit strong immune, responses. Additionally, the present
invention's submicron emulsion rmcroparticles also elicit strong immune responses. The
combination of the present invention's rmcroparticles with adsorbed macromolecules is therefore a
powerful tool for eliciting immune responses.
All references cited herein are hereby incorporated by reference in their entirety.
C. 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. Those skilled in the art win recognize modifications that are within
the spirit and scope of the invention
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts,
temperatures, eta), but some experimental error and deviation should, of course, be allowed for.
Rvamplf 1
Preparation of Pnhrmnr Mifroparticles with AA^r}m\ fJivlsir. Arid
PLG-CTAB rmcroparticles were prepared using a modified solvent evaporation process.
Briefly, the micioparricles were prepared by emulsifying 10 ml of a 5% w/v polymer solution in
methylene chloride with 1 ml of T.E. buffer at high speed using an EKA homogenizer. The primary
emulsion was then added to 50ml of distilled water containing cetyl trimethyl ammonium bromide
(CTAB) (0.5% w/v). This resulted in the formation of a w/o/w emulsion which was stirred at 6000

rpm for 12 hours at room temperature, allowing the methylene chloride to evaporate. The resulting
microparticles were washed twice in distilled water by centrifugation at 10,000 g and freeze dried.
For a typical batch of 100 mg of DNA adsorbed microparticles, 100 mg of PLG-CTAB
catkmic microparticles were weighed into a glass vial and resuspended with 5 ml volume of
200ug/ml of DNA solution (Le., meplasmid pCMV or pSINCP containing gpl40 orp55gag) in
T.E. Buffer. The suspension was vortexed for a one minute to uniformly disperse the
microparticles in the DNA solution. The vial was set an a shaker (slow speed) at 4 C for overnight
adsorption. The next day the microparticles were centrifuged down at 8000 rpm on a Beckman
centrifuge for 10 minutes and the supernatant was collected for DNA quantitation. The pellet was
washed once with IX TE buffer by resuspending the pellet in 1 X TE buffer, dispersing with a
spatula and centrifugnig at 8,000 rpm for 10 minutes. The final pellet was resuspended in a
minimum amount of de-ionized water (about 2 ml) by dispersing the pellet with a spatula, and
freeze dried on a bench top ryophflizer (Labconco) for 24 hours.
The supernatant was assayed for DNA content by reading the absorbance at 260 run
Amount of DNA adsorbed on the microparticles was calculated by subtracting the amount in the
supernatant from the total DNA input (1 mg per 100 mg of microparticles. The total load was
estimated by dissolving 5 mg of final formulation in 0.S M NaOH/1% SDS solution and reading the
clear solution after hydrolysis at 260 ma
Example 2
Preparation of Submtcrnri ernnlsim) Microparticles with Adsorbed Nucleic Acid
Asubmicron emulsion formed from MF59 and DOTAP was prepared by providing DOTAP
(in chloroform) in a beaker and alio vmg it to evaporate down to 200uL Tween (0.5% w/w),
Squalene (5.0% w/w) and Span (0.5% w/w) were added and homogenized for 1 minute using an
Omni homogenizer with a 1 Oram probe at 10K revs/mm in order to provide a homogeneous
feedstock for final ernulsmcation This was passed 5 times through a Microfluidizer Mil OS
homogenizer (Microfhidics Co., Newton, MA) at -800 psi. The zeta potential of the emulsion,
which is a measure of net surface charge, was measured on a DELS A 440 SX Zetasizer from
Coulter and found to be approximately + 55 mV.

DNA (either 1 mg HIV-1 gpl40 DNA or 0.5 mg of p55 gag DNA, present in pCMV or
pSINCP) was adsorbed by incubation with the subnricron emulsion overnight at 4°C.
Rxamplft 3
Preparation of ELVIS Vectors and Other Vector Constructs for Adsorption to Microparticles
Construction of alphavirus-based ELVIS and replicon vectors was performed using Sindbis
virus as a representative example. As will be appreciated, the following may be readily applied to
the derivation of vectors from any alphaviros by one of skill in the art. Approximately 107 BHK-21
cells were infected with the SINDCchiron strain of Sindbis virus (ATCC deposit VR-2643, April
13,1999) at a MOI of 1 PFU/cefl. At 24 hours post-infection, after development of CPE, total
RNA was isolated from the cells using the TRIzol Reagent (GIBCO/BRL) according to the
manufacturer's instructions. After purification, viral RNA was dissolved in nuclease-free water,
aliquoted, and stored at -80°C for subsequent use in cDNA cloning.
Synthesis of cDNA was accomplished by PCR amplification, using the primer sets shown
below (Sindbis nucleotide numbering indicated for each primer):



Primer pairs 1-5 were used for doming of the virus structural protein genes, while pairs 6-
14 were for the virus nonstructural protein genes. Oligonucleotides in pairs 1-5 contained
additional sequences representing restriction eozyae sites for £coJQ and ifi«flll,whidi are not
present in subgenomic RNA of Sindfais virus. Oligonucleotides 6-14 contained sites for Sacl and
Xhol, which are not present in the whole genome of previously sequenced strains of Sindbis virus
(these sites are underlined).
Each reverse transcription (RT) reaction was performed in a 50 ul volume using the
SuperscriptO enzyme (GIBCO/BRL), according to the manufacturer's instructions. Reaction
mixtures contained the amount of RNA equivalent to 10s cells and 50 prnoles of each primer
shown below.
Maturel: primersl, 3 and 5
Mixture2: primers 2 and 4
Mixture3: primers 6,9 and 12
Mixture4: primers 8,11 and 14

RT reactions were frozen and then used subsequently for PCR amplification. PCR
reactions were performed using Vent DNA polymerase (NEB) as recommended by the
manufacturer. Each 50 ul PCR reaction contained 3 pi of RT mixtures described above and 50
pmoles of primers. A total of 14 reactions were performed (Table 1).

PCR reactions for fragments 1-5 were performed using the following conditions: 12 cycles
of 95°C for 30 seconds, 56°C for 30 seconds and 74°C for 90 seconds. For fragments 6-14, the
number of cycles was changed from 12 to 15. A small aliquot of each reaction mixture was
analyzed by agarose gel electrophoresis to confirm the presence of the fragmentsof the expected
size. The remaining reaction mixture was extracted with phenol-chloroform and DNA fragments
were precipitated using ethanoL

For cloning, fragments 1-5 were digested with HindHl and EcoBl, and then ligated with
plasmid pRS2 (pUCl 9 with additional restriction sites in polyKnker) treated with the same
enzymes. Fragments 6-14 were digested with Sadl sndXhol and hgated with the same pRS2
plasmid treated with Sad. and Xho\. AH recombinant plasmids were transformed into the £. coli
XL-1 Blue strain (Stratagene, La Jolla, CA).
In addition, cDNA clones representing the subgenomic promoter region and 3'-end
nontranslated regions also were generated using the following primer pairs:

Positive colonies for each transformation were grown for plasmid purification using a
QIAGEN kit according to the manufacturer's instructions. The fragments, designated pl-p!4
correspondingry, were then assembled into the appropriate vector configurations.
The construction of a Eukaryotic Layered Vector Initiation System (ELVIS) and an
alphavirus vector construct for in vitro transcription of rephcon vector RNA was accomplished
using the Sindbis virus cDNA clones pl-pl4, plus the subgenomic and 3'-end region fragments as
follows. AaApal-MscI fragment, containing the promoter for SP6 SNA polymerase and start of
the Sindbis virus genomic RNA, was hgated with Ha&Msc\-Xho\ fragment of cloned fragment 14 in
Apd-Xhol digested plasmid pRS2. The resulting plasmid was named pl5. Next, the Sacl-EcoW.
fragment of p8, the EcdSl-Nsil fragment of p7 and the Nsil-Xhol fragment of p6 were Hgated into
Sacl-Xhol digested pRS2. The resulting plasmid was named pi6. Next, the Sacl-Murft fragment
of pl2, theMunl-Nhel fragment of pi 1 and the Nhel-Xhol fragment of plO were ligated into Sacl-
Xkol digested pRS2 plasmid. The resulting plasmid was named pl7. The Apal-ApdLlbagaxaL of
plS sndlheApaU-Xhol fragment of pi 3 then were ligated into Apal-Xhol treated pRS2, resulting
in the plasmid named pl8. Next, IheApal-NsH fragment of pl8 and the JVM-A7u>I fragment of
pl7 were ligated together iaApd-JOiol treated pRS2. The resulting plasmid was named pl9.
Finally, me^paWvrll fragment of pi 9, HieAwlI-SalGl fragment of p9 and the SalQl-Barrim

fragment of pl6 were ligated together into a previously constructed Sindbis replicon vector
expressing the GFP reporter (see Dubensky et aL, J. Virol 70:508-519,1996; Polo et al, 1999,
ibid; and U.S. Patent 5,843,723), that had been digested with^/pal-BamHI to remove the existing
nonstructural protein genes. The resulting Sindbis vector construct, which contains sequences
derived from the SINDCchiron virus strain and also encodes a GFP reporter, was designated
SINCR-GFP (also known as DCSP6SINgfp). Preparation of replicon RNA from mis reporter
construct, as well as Sindbis vector constructs expressing various other heterologous sequences
(e.g., antigens, described in the specification and below) was performed by linearization of the
DNA using Pme\ followed by in vitro transcription using bacteriophage SP6 polymerase as
described previously (Polo et aL, ibid; Dubensky et aL, ibid).
Similarly, the same Sindbis sequences were used for assembly into an alphavirus-based
Eukaryotic Layered Vector Initiation System (see U.S. 5,814,482 and 6,015,686), in which the
transcription of self-amplifying vector RNA takes place directly within ELVIS plasmid DNA-
transfected eukaryotic cells via a eukaryotic promoter (e.g., RNA polymerase 0 promoter). An
ELVIS plasmid DNA, which also expressed GFP reporter, was constructed by replacing Sindbis
virus derived sequences in an existing ELVIS vector with the corresponding SINCR-GFP
sequences from above. Starring with the previously described ELVIS vector pSINl .5 (Hariharan
et aL, J. Virol. 72:950-958,1998), the plasmid backbone first was modified by substituting the
plasmid backbone whh that from pCMVLink (zur Megede et aL, J. Virol. 74:2628-2635,2000)
using two Sad sites found in each plasmid, to generate the intermediate construct known as
ELVIS1.5CB. Next, one of the two Sad sites of ELVIS1.5CB (located adjacent to the SIN 3'-
end) was eliminated by partially digesting with Sad, blunt-ending using T4 DNA polymerase, and
iien Hgating into the modified she, &Pmel linker 5'-GTTTAAAC-3\ The correct plasmid without
the targeted Sad site was designated ELVIS 1.5CBdlSac. This intermediate plasmid then was
prepared for insertion of the new SIN nonstructural protein genes by digestion with Sad and Xhol.
The corresponding nonstructural genes were obtained by PCR amplification from SINCR-GFP
using the oligonucleotide primers
5'CCTATGAGCTCGTTTAGTGAACCGTATTGACGGCGTAGTACACAC (SEQ ED NO:61)
and 5'CCTATCTCGACKJGTGGTGTTGTAGTATTAGTC (SEQ ID NO:62), followed by
digestion with Sad soAXhol, and ligation, to produce the intermediate construct SINCP-Not

Finally, one of the two Noll sites present in this construct was eliminated by partial digest and
Klenow fill-in, to leave only one Nod. site in the polylinker. This newly constructed ELVIS vector
was designated SINCP (or pSINCP).
Insertion of heterologous sequences (e.g., antigen-encoding genes) into the SINCR or SINCP
alphavirus vectors is performed primarily by digestion withXhol/Notl oiXhoVXbcA, followed by
ligation with a desired DNA fragment that also has Xhol/NoS. or XhoUXbcAtenmsL Alternatively,
these sites may be blunt-ended or other polylinker sites may be used (or other heterologous
sequences may be replaced) to allow cloning of a greater number of inserts. For example, the
HTV-1 p55gag (SF2 strain) and gpl40 env (SF162 strain) encoding genes were inserted into these
vectors. Specifically, the codon-optimized HTV p55gagmod sequence (see commonly owned U.S.
Patent Application 09/475,515; zur Megede et al, ibid) was inserted by digesting the vectors with
XholiXbdl and figuring in the p55gagmod fragment obtained by digestion with SaWXbaS.. The
resulting vectors were designated SINCR-p55gag and SINCP-p55gag. Similarly, codon-optimized
HTV gpI40 sequences (described in Example 10 and Bamett et aL, 2001. J. ViroL 75:5526-40),
were inserted into both the SINCR and SINCP plasmids to generate the constructs SINCR-gpl40
and SINCP-gpl40. Formulation of ELVIS plasmid DNA (pSINCP) and RNA vector replicons
transcribed in vitro from the SINCR plasmids is performed as described elsewhere in the
Examples.
Example 4
Tmmiifiy.afjnn r>f RpesUS Macaques with
Antigen with pCMV or pSINCP Plasmids
using Aficroparticles or frihmicrnn amnkmns
PLG potymer microparticles and MF59 submicron emulsions were formed as described
above in previous Examples 1 and 2. Groups of microparticles and submicron emulsions were
made in order to analyze the different effects of immunizing rhesus macaques with a plasmid vector
construct, pCMV- gpl40 or pCMV-p55gag (see commonly owned U.S. Patent Application
09/475,515), on microparticles or in a submicron emulsion, as well as comparing the effect of using

an ELVIS plasmid, pSINCP-gpl40 or pSINCP-p55gag, constructed as described above. Six
groups of animals were immunized with different formulations as follows:
Group 1 used pCMV- gpl40 and pCMV - p55 gag without microparticles OT submicron
emulsions.
Group 2 used pCMV- gpl40 and pCMV - p55 gag adsorbed on PLG/CTAB
microparticles.
Group 3 used pCMV- gpl40 and pCMV - p55 gag adsorbed to an MF59-DOTAP
submicron emulsion.
Group 4 used pSINCP- gpl40 and pSINCP - p55 gag without microparticles or submicron
emulsions.
Group 5 used pSINCP- gpl40 and pSINCP - p55 gag adsorbed on PLG/CTAB
microparticles.
Group 6, a control, used no antigen, no microparticles, and no submicron emulsions.
For each group of animals, 5 rhesus macaques (only 4 for group 6) were immunized with
sufficient quantities of material such mat the dosage of vector with gpl40 DNA was 1.0 mg each,
and vector containing p55 gag DNA was 03 mg each, except for the control which had none. The
animals were immunized a second time four weeks after the first irnmunizatiori, and a third time 14
weeks after the first immunization. Serum was analyzed at weeks 2 (2wpl), 6 (2wp2), 11-12
weeks (7wp2), and 16 (2wp3). The route of irnmunization was IM TA. FoHowing immunizations,
plasma anri-p55gag and anti-gpl40 IgG titers were measured, the results of which appear below in
Tables 2 and 3 as geometric mean titers.




The same animals were also analyzed for lymptoproliferatioa This assay measures specific
proliferation of T cells in vitro in response to restimuMon with antigen. Rhesus macaque
peripheral blood mononuclear cells (PBMC) were purified from heparinized whole blood by
centrifugation on Ficofl-Hypaque gradients. PBMC were cultured at the number of 2 x 10s per
well in flat bottom nricrotiter plates in the presence or absence of 3 rnicrogramsM of purified
recombinant p55gag protein. Six replicate cultures per condition were initiated. After 4 days of
culture tritiated thymidine fl3H]TdR) was added (1 microcurie per well). Cultures were continued
overnight and harvested the following day. Cells were deposited onto glass microfiber filter sheets.
Filter sheets were exposed to scintillation fluid and counted in liquid scintillation counter. For
each condition fHJTdR incorporation, measured as the mean counts per mm (cpm) for the 6

replicates was calculated. The results appear below in Table 6. Geometric Mean Stimulation
Index (GMSI) is calculated as counts per minute (cpm) of p55gag stimulated cells divided by cpm
of unstimulated cells, thus, the larger the GMSI, the more positive the result.

The same animals were also analyzed for induction of intracellular cytokine production.
This assay measures specific production of cytokines by T ceDs in vitro in response to brief
restirnulationwrth antigen. Rhesus mataque peripheral bteodrnononuclear cells (PBMC) were
purified fromheparumed whole blood by ceotrimgation on Ficoll-Hypaque gradients. Aliquots of
1 x 10* PBMC were stimulated with a pool of synthetic overlapping peptides that span the gag (or
env) protein sequence in the presence of a co-stimulatory anti-CD28 monoclonal antibody.
Brefeldm A was added to allow the accumulation of newly synthesized cytokines within cells.
After overnight incubation PBMC were stained wim commercially available, fhiorescenuy labeled
nonoclonal antibodies for the presence of iraracelodar interferon^ (IFN-TO and tumor necrosis
factor-a (INF-a) and for cell surface CD4 and CD8 markers. Stained cell samples were analyzed
on a flow cytometer and data were acquired tor approximately 50,000 - 100,000 PBMC. The
frequency of cytoldne-posmve cells was determined for each sample using commercially available
software. The results for gpl40 and pSSgag are shown in Tables 7 and 8, respectively. The Tables
show the number of responding animals, where a responder is defined as an animal scoring greater
than 100 CD4 cells per 100,000 expressing TNF-a and EFN- r as measured by intracellular
staining.


RNA Vector Constructs
RNA vector constructs (e.g., replicons) may be adsorbed to micropartides for delivery of
heterologous nudedc acid sequmces to me cells of animals. The RNA vector construct generaDy
comprises a viral RNA which has had a region of the genomic RNA (e.g., structural protein gene)
replaced with the selected heterologous sequence, derived from the DNA coding sequence tor the
gene-product of interest Representative examples of RNA vector constructs include, but are not
finited to, alphavirus RNA vectors (see for example, US Patent 5843723, PCT publication WO
99A 8226, and Polo et aL, 1999, PNAS 96:4598-4603), picomavirus RNA vectors (see for

example, US Patent 6156538, and Vignuzzi et ai, 2001, J Gen ViroL 82:1737-47), fkvivirus RNA
vectors (see for example, Varnavski et al 1999, Virology 255:366-75), and rabivirus RNA vectors
(see for example, Pugachev et ai, 2000, J ViroL 74:10811-5).RNA vector constructs for use in the
present invention generally may be obtained from plasmid cDNA constructs as a source of starting
mntgrifl^ by the standard process of in vitro transcription (see references above). Similarly to
plasmid DNA, these RNA vector constructs then may be adsorbed to microparticles of the
invention as described elsewhere in the examples. For example, the RNA vector constructs are
adsorbed onto the microparticles by incubating 100 mg of canonic microparticles in a 1 mg/ml
solution of DNA at 4°C for 6 hr. The microparticles are then separated by centrifugation, the
pellet washed with TE buffer, and the microparticles freeze-dried. Reconsutution and delivery of
the PLG-formulated RNA vector constructs is similar to that described for DNA, using for
example at least 1 ug, 10 ug, lOOug, or 1000 ug of formulated RNA vector construct for delivery.
Ryrpple 6
An experiment with mice was performed to analyze the effect ofthe adjuvant aluminum
phosphate (arum) in mice. Polymer mtcroparticles were prepared as above described with or
without pCMV-p55gag adsorbed thereon. 10 micrograms of DNA, whether naked or adsorbed to
the PLG microparticles was injected in groups of 6 CB6 Fl mice on weeks 0 and 6, without or
without alum The results, as geometric mean titers of antibody, are shown below in Table 9.


Rxflmpl«7
Electropoiation with Microparticles and ELVIS Vectors and KNA Vector Constructs
Electroporation may be used in combination with polymer microparticles or subrnicron
emulsion microparticles made with any of the nucleic acids described above, such as plasmid DNA]
ELVIS vectors, and SNA vector constructs.
tft"npple8
Induction of Immune Response in Rhesus with Prime and B""s* TmrniirV^QTiy
An experiment was performed to determine the effect of priming with DNA and boosting
with protein adsorbed to PLG rdaoparticles. Particularly, PLG-SDS microparticles were
prepared as described above and in commonly owned International patent application
PCT7US99/17308, and purified recombinant pSSgag protein was adsorbed thereto. A group of
animals was immunized with 1 mg pCMV-p55gag. The animals were immunized again at 4 weeks,
then again at 8 weeks. The animals were boosted with p55gag protein adsorbed to PLG
microparticles at 41 weeks. The results are shown in Table 10 below, which shows antibody titer
for responders, induction of helper T cell lymphoproliferation (mean stimulation index of
responders), and induction of CTL (number of responders, based on greater than 10% lysis at two
or more consecutive B:T ratios). Results were measured at 14 weeks post 3rd prime for the prime
columns, and 2 weeks post boost for the boost columns. Numbers in parentheses indicate the
number of responders out of a total of 4 animals.


Ryampla Q
Induction of Nf"trali7infr AnfibpHiey
Hie sera from the riiesus macaques in Example 5 above were tested for inhibition of two
different HTV-1 strains (SF2 and SF162) using PMBC-grown virus stocks and a
CCR5+/CXCR.4+/CD4+T cell line based assay. Sera were used at a dilution of 1:20. Inhibitory
activity was measured and expressed as a percentage inhibition. The inhibitory activity of the
unimnk' sera prior to immunizations were subtracted from the results for each animal Hie results
for each ofthe five animals in each group are shown in Table 11 below.


FyfflnplelO
Preparation of Plasmids
Plasmids encoding HIV-1 p55gag and gpl40env driven by the human cytomegalovirus
(CMV) promoter were grown ia Escherichia coli strain DH5a, purified using a Qiagen Eadofree
Plasmid Giga kit (Qiagen, Inc.), and resuspended in 0.9% sodium chloride (Abbott Laboratories,
North Chicago, ID). The pCMV vector used contains the immediate-early enhancer/promoter of
CMV and a bovine growth hormone terminator and is described ia detail elsewhere (Chapman, B.
S., et aL 1991. "Effect of nitron A from human cytomegalovirus (Towne) immediate-early gene on
heterologous expression in mammalian cells." Nucleic Acids Res. 19:3979-86). The HTV gag
plasmid DNA vaccine (pCMVgag) contains a synthetically constructed p55gag gene, with codons
reflecting mammalian usage, derived from the HIV-1 SF2 strain as previously described (zur
Megede, ]., et aL 2000. "Increased expression and immunogenicity of sequence-modified human
immunodeficiency virus type 1 gag gene." J ViroL 74:2628-35). The HTV env plasmid DNA
vaccine (pCMVgpl40) consisted of a human tissue plasminogen activator (tPA) signal sequence
and the gpl40 from HIV-1 SF162 strain, codan optimized for high level expression in mammalian
cells (Bamett, S. W., et 2001. Tfceahfliry of anofigom^humaniniiiunodeficie^ virus type 1
(HIV-1) envelope antigen to elicit neutralizing antibodies against primary HTV-1 isolates is
improved following partial deletion of the second hypervariable region." J ViroL 75:5526-40). The
SINCP plasmid vector with either HTV-1 p55gag or gpl40env has been described in Example 3
above.
Example 11
Preparation of PrOfrflH
The protein and cDNA sequences for the gpl 60env.SFl 62 have been published in Cheng-
Mayer, C, M. Quiroga, J. W. Tung, D. Dina, and J. A Levy. 1990. "Viral determinants of human
immunodeficiency virus type 1 T-cefl or macrophage tropism, cytoparhogenicity, and CD4 antigen
modulation." J Virol 64:4390-8. These sequences can be found under Genbank accession number
M65024. Recombinant HTV-1 gl40.SF162(dV2) protein was expressed in Chinese hamster ovary
cells and purified as previously described (Bamett, S. W., ET. 2001. J ViroL 75:5526-40).

Recombinant HTV-1.SF2 p55 gag protein was expressed in yeast and purified by cation exchange
chromatography (Chiron Corporation, Emeryville, CA). The p55gag cDNA sequence from the
SF2 strain of HIV-1 (Genbank accession number K02007) was cloned into aubiquain expression
vector, resulting in the addition of glycine and arghune to the N-terminus of the wild-type
sequence. The recombinant pS5gagproteh was extracted from Ae yeast cell pellet usirjg 50 mM
phosphate, 6M area, pH7.9, followed by S-fractogel (canonic) ion exchange chromatography.
Ehrtion of the p55gag was obtained with a linear NaCl gradient (peak at 0.4m NaCl). The
estimated purity was 90% by SDS-P AGE
DNA-adsorbed polvflactide-co-glvcolidel PLG rrricropartides
PLG polymer (RG505) was obtained from Boehringer Ingelheim. Catiomc rnicroparticles
were prepared using a modified solvent evaporation process. Briefly, the micro-particles were
prepared by emulsifying 10 ml of a 5% (wt/vol) polymer solution in methylene chloride wrthl ml of
plwsrihate-buffere was then added to 50 ml of distilled water containing (^tyrtarnethylamtoomum bromide (CTAB)
(0.5% wt/vol), resulting in the formation of a. water-m-off-m-water emulsion, which was stirred at
6,000 rpm fori 2 h at room temperature, allowing the methylene chloride to evaporate. The
resulting micropartides were washed twice in distilled water by centrifugation at 10,000g and
freeze-dried. Plasmid DNA from Example 11 was adsorbed onto the rnicroparticles by incubating
100 mg of canonic rnicroparticles 5ml of a 200 rmcrogranvml solution of DNA at 4°C for 63b. The
micropartides were then separated by centrifugation, the pellet was washed with TE (Tris-EDTA)
buffer, and the micropartides were freeze-dried.
Example 13
Protein-adsorbed PI.O micrnptirncles
Blank rnicroparticles were prepared by a solvent evaporation technique. Briefly,
rnicroparticles were prepared by homogenizing 10ml 6% w/v polymer solution in methylene
chloride, with 40 ml of distilled water containing SDS (1% w/v) at high speed using a 10mm
probe. This resulted in an oil in water emulsion, which was stirred at 1000 rpm for 12 hours at

room temperature, and the methylene chloride was allowed to evaporate. The resulting
microparticles were filtered through a 38ummesh, washed 3 times in distilled water, and freeze-
dried. The size distribution of the microparticles was determined using a particles size analyzer
(Master sirer, Malvern Instruments, UK).
SOmg lyophfluad SDS blank particles were incubated with 0.5mg of p55 gag protein &om
Example 12 in 1 Oml 25mM Borate buffer pH9 wim 6M Urea Particles were left on a lab rocker,
(Aliquot mixer, Miles labs) at room temperature for 5 hours. The microparticles were separated
from the incubation medium by centrifugation, and the SDS pellet was washed once with Borate
buffer with 6M Urea then three times with distilled water, and fyophilized.
The loading level of protein adsorbed to microparticles was determined by dissolving 1 Omg
of the microparticles in 2ml of 5% SDS-0.2M sodium hydroxide solution at room temperature.
Protein concentration was measured by BCA protein assay (Pierce, Rockford, Illinois). The Zeta
potential for bom blank and adsorbed microparticles was measured using a Malvern Zeta analyzer
(Malvern Instruments, UK).
Rrampte 14
Preparation of Protein wim MF59 Adjuvant
Recombinant HTV-1 gl40.SF162(dV2) protein from Example 12 was combined with MF59
adjuvant as previousry described (Bamett, S. W., et 2001. J Virol 75:3526-40).
BzampJeJl
lpmunizanon
Male and female rhesus macaques were housed at Southern Research Institute (Frederick,
MD).
PlasrrM DNA imrnunization was performed at weeks 0,4, and 14. Rhesus were given
intramuscular injections of 0.5 mg of pCMVgag from Example 11 (in saline or formulated wim
PLG/CTAB microparticles as described in Example 13 or formulated with MF59/DOTAP as
described in Example 2) and 1.0 mg of pCMVenv from Example 11 (in saline or formulated with
PLG/CTAB microparticles as described in Example 13) at 4 separate sites per animal (0.25 mg

pCMVgagm upper right arm and upper right leg; 0.5 mg pCMVanv in upper left arm and upper
left leg). Alternatively, rhesus were given intramuscular injections of 0.5 mg of pSINCPgag from
Example 11 (in saline or formulated with PLG/CTAB microparticles as described in Example 13)
and 1.0 mg of pSINCPenv from Example 11 (m saline or formulated with PLG/CTAB
microparticles as described in Example 13) at 4 separate sites per animal
Rhesus were boosted by intramuscular injection of 0.2 mg recombinant p55gag pxotein/PLG
microparticles from Example 14 at week 29 and with 0.1 mg recombinant gpl40env(dV2)
protein/MF59 adjuvant from Example IS at week 38.
Eyqmplnlfi
Antibody responses
At various times following immunization, heparinized blood was collected from anesthetized
animals and plasma was recovered by centnfugation Anti-HTV Gag and Env antibodies were
measured by enzyme-linked immunosorbent assay (ELISA) as follows. Wells of micnroter plates
were coated with recombinant HTV-1.SF2 pS5gag protein or recombinant HTV-1.SF162 gpl40env
protein at 5 microgram/ml in PBS, SO microliters per wen, and incubated at 4°C overnight The
plates were washed six times with wash buffer (PBS, 0.3% Tween 20) and blocked at 37°C for 1 h
with 200 microliters per well of blocking buffer (PBS, 0.3% Tween 20,5% goat serum). Test
samples were diluted 1:25 and then serially diluted teee&W in blocldng b was aspirated, and then the plates were incubated at room temperature for 1 h with 70 microliters
per well of each plasma dilution After being washed six times, the plates were incubated for 1 h at
37°C with horseradish peroxidase-conjugated anti-IgG (1:8,000 dilution). Following six washes,
the plates were developed with TMB substrate for 15 minutes. The reaction was stopped with 2N
HCl and the optical densities (OD) measured at a wavelength of 450 nm. The titer was calculated
to be the reciprocal of the dilution at which an OD450 „ of 0.5 was achieved.





BssakJuL
CvtoMc T Lymphocyte fCTD responses
A pool of 51 synthetic peptides 20 ammo acids (aa) long, overlapping by 10 aa, and spanning
p55 gag, and a pool of 66 synthetic peptides 20 aa long, overlapping by 10 aa, and spanning gpl40
were prepared. Rhesus macaque peripheral blood mononuclear cells (PBMC) were separated from
heparinized blood by centrifugation on Ficofl-Paque (Pharmacia Biotech, Piscataway, N.J.)
gradients. PBMC were cultured for 8 days in 24-weH plates at 3 x 106 per well in 1.5 ml of AIM-
V/RPMI1640 (50:50) culture medium ( fetal bovine serum Gag-specific CTL were stimulated by the addition of the gag peptide pool and
env-specrSc CTL were stimulated by the addition of the env peptide pool Cultures were
supplemented with recombinant human intedeukin-7 (IL-7; 15 ng/ml; R&D Systems, Minneapolis,
Mmn.). Human recombinant IL-2 (20IUM; Proleukin;Chiron) was added on days 1,3, and 6.
Stable rhesus B-rymphoblastoid cell lines (B-LCL) were derived by exposing PBMC to herpesvirus
papio-contairjmg culture supernatant from the S594 cell line (Falk, L, et aL 1976. Properties of a
baboon fymphotropic herpesvirus related to Epstein-Barr virus. Int J Cancer. 18:798-807. Rabin,
H., et aL 1976. Virological studies of baboon (Papio hamadryas) lymphoma; isolation and
characterization of foamyviruses. J Med PrimatoL 5:13-22.) in the presence of 0.5 microgram/ml
cyclosporin A (Sigma, St Louis, MO). Autologous B-LCL were infected with recombinant
vaccinia virus (rW) encoding HTV-1.SF2 gag-pol (rWgag-poI) or HTV-1.SF162 gpl60env
(rWgpl60env) (PFU:ceIl ratio of 10) and concurrentry labeled with NaI31Cr]20« (NEN, Boston,
MA) at 25 mdcrocurie per 1 x 10s B-LCL. After overnight culture at 37°C, rW-infected, 51Cr-
labded B-LCL were washed and then added (2,500 per round-bottomed well) to duplicate wells
containing threefold serial dilutions of cultured PBMC. Then 10s unlabeled, uninfected B-LCL

were added per well to inhibit nonspecific cytolysis. After 4h incubation at 37°C, 50 microliters of
culture supernatauts were harvested and added to LumaPlates (Packard, Meriden, CT), and
radioactivity was counted (counts per minute (cpm)) with a Microbeta 1450 liquid scintillation
counter (Wallac, Gaithersburg, MD). slCr released from rysed targets was normalized by using the
formula: % Specific MCr Release = 100% x (mean experimental cpm - SR)/(MR - SR), where SR
= mean cpm from targets alone and MR = mean cpm from targets exposed to Triton X-100. An
animal was determined to have a positive, p55gag-specific response if at two consecutive dilutions
of the gag peptide pool-stimulated PBMC the lysis of rWgag-pol-infected B-LCL exceeded lysis
of rWgpl60env-infected B-LCL by at least 10% and if at two consecutive dilutions of cultured
PBMC the lysis of rWgag-pol-infected B-LCL by the gag peptide pool-stimulated PBMC exceed
lysis of rWgag-pol-infected B-LCL by env peptide pool-stimulated B-LCL by at least 10%. An
animal was determined to have a positive, gpl60-specific response if at two consecutive dilutions
of the env peptide pool-stimulated PBMC the lysis of rWgpl60env-infected B-LCL exceeded
lysis of rWgag-pol-infected B-LCL by at least 10% and if at two consecutive dilutions of cultured
PBMC the lysis of rWgpl60env-infected B-LCL by the env peptide pool-stimulated PBMC
exceed lysis of rWgpl60env-infected B-LCL by gag peptide pool-stimulated B-LCL by at least
10%.







TframplR 19
TnlraceHnlBT rytnlrma imTTrnnnflynT'fiiCence and flow cytometry
Rhesus PBMC (1 x 106 per well) were cultured overnight in the presence of Brefeldin A
(Pharmingen, San Diego, CA) and anti-CD28 monoclonal antibody (mAb) (Phanningen) and in the
presence or absence of the gag or env peptide pools. Duplicate wells were prepared for each
condition of stimulation. The next day ceDs were stained with peridinin chlorophyll protein
(PerCP)-conjugated anti-CD8 mAb and aHophycocyanin (APC)-conjugated anti-CD4 mAb
(Becton Dickinson, San Jose, CA), fixed and permeaMized (Cytofrx/Cytoperm, Pharmingen), and
stained with fluorescein isotfnocyanate (FJTC)-conjugated anti-tumor necrosis factor-a (TNF-a)
mAb and phycoerythrin (PE)-conjugated anti-interferon-y (IFN-y) mAb (Pharmingen). Stained cell
samples were analyzed using a FACSCalibur™ flow cytometer and CeDQuest™ software (Becton
Dickinson). The fraction of cells positively stained for IFN-y and TNF-a was calculated for the
CD4+8- and CD8+4- T cell subsets. The number of gag" or env-specific cells was calculated by
subtraction of the average IPN-^/TNF-a fraction found in the unstimulated control wells from the
average IFN-y^ENF-a fraction found in the gag- or env-stimulated wells.
For a given T cell subset (CD4+8- or CD8+4-) and antigen (gag or env) a response was designated
as positive if the fraction of antigen-specific cells was at least 0.1%.





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WE CLAIM:
1. A method of producing a microparticle having an adsorbent surface to
which a vector construct capable of expressing a selected nucleic acid
sequence is adsorbed, said method comprising the steps of:
(a) emulsifying a mixture of a polymer solution and a detergent to form
an emulsion, wherein the polymer solution comprises a polymer
selected from the group consisting of a poly(a-hydroxy acid), a
polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a
polyanhydride, and a polycyanoacrylate, wherein the polymer is
present at a concentration of about 1% to about 30% in an organic
solvent, and wherein the detergent is present in the mixture at a
weight to weight detergent to polymer ratio of from about 0.00001:1
to about 0.5:1;
(b) removing the organic solvent from the emulsion, to form said
microparticle; and
(c) adsorbing the vector construct to the surface of the microparticle,
wherein said vector construct is selected from the group consisting
of an ELVIS vector and an RNA vector construct.

2. The method as claimed in claim 1, wherein the vector construct comprises
a heterologous nucleic acid sequence encoding a member selected from
the group consisting of a pharmaceutical, a polypeptide, a hormone, an
enzyme, a transcription or translation mediator, an intermediate in a
metabolic pathway, an immunomodulator, an antigen, and an adjuvant.
3. The method as claimed in claim 2, wherein the heterologous nucleic acid
sequence encodes an antigen selected from the group consisting of HIV
gp120, HIV gp140, HTV p24gag, HIV p55gag, and Influenza A
hemagglutinin antigen.
4. The method as claimed in any one of the previous claims, wherein said
heterologous nucleic acid sequence encodes an HIV gag polypeptide and
comprises a sequence having at least 90% identity to a sequence
selected from the group consisting of nucleotides 844-903 of SEQ ID
NO:63, nucleotides 841-900 of SEQ ID NO:64, nucleotides 1213-1353 of
SEQ ID NO: 67, and nucleotides 82-1512 of SEQ ID NO:68.
5. The method as claimed in any one of claims 1 to 3, wherein said
heterologous nucleic acid sequence encodes an HIV envelope
polypeptide and comprises a sequence having at least 90% identity to a
sequence selected from the group consisting of nucleotides 1513-2547 of
SEQ ID NO:65 and nucleotides 1210-1353 of SEQ ID NO:66.

6. A microparticle made as claimed in the method of any one of the previous
claims.
7. The microparticle with an adsorbent surface to which a biologically active
macromolecule has been adsorbed comprising: a microparticle selected
from the group consisting of (a) a polymer microparticle comprising (i) a
polymer selected from the group consisting of a poly(a-hydroxy acid), a
polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a
polyanhydride and a polycyanoacrylate; and a detergent; and (b) a
submicron emulsion comprising:
(i) a metabolizable oil; and (ii) one or more emulsifying agents; and
the biologically active macromolecule, wherein the biologically active
macromolecule is a nucleic acid molecule comprising at least one vector
construct selected from the group consisting of an ELVIS vector and an
RNA vector construct.
8. The microparticle as claimed in claim 7, wherein the selected microparticle
is the submicron emulsion, and (a) the oil is a terpenoid and (b) the one or
more emulsifying agents comprise one or more non-ionic detergents and
one or more cationic detergents.

9. The microparticle as claimed in claim 8, wherein the oil is squalene and
the one or more emulsifying agents comprise: a polyoxyethylene sorbitan
fatty acid ester, a sorbitan fatty acid ester, and DOTAP.
10. The microparticle as claimed in claim 7, wherein said polymer
microparticle is selected as said microparticle.
11. The microparticle as claimed in claim 10, wherein said polymer
microparticle comprises a poly(α-hydroxy acid) selected from the group
consisting of poly(L-lactide), poly(D,L-lactide) and poly(D,L-lactide-co-
glycolide).
12. The microparticle as claimed in claim 10 or 11, further comprising a
second biologically active macromolecule entrapped within the
microparticle, wherein the second biologically active macromolecule is a
member selected from the group consisting of a polynucleotide, a
polynucleoside, a pharmaceutical, a polypeptide, a hormone, an enzyme,
a transcription or translation mediator, an intermediate in a metabolic
pathway, an immunomodulator, an antigen, and an adjuvant.
13. The microparticle as claimed in any one of claims 10 to 12, wherein said
vector construct is an ELVIS vector.

14.The microparticle as claimed in claim 13, wherein said ELVIS vector
comprises a cDNA complement of an RNA vector construct derived from a
member selected from the group consisting of alphavirus, picomavirus,
togavirus, flavivirus, coronavirus, paramyxovirus, and yellow fever virus,
and wherein said RNA vector construct further comprises a selected
heterologous nucleotide sequence.
15. The microparticle as claimed in any one of claims 10 to 12, wherein said
vector construct is an RNA vector construct derived from a member
selected from the group consisting of alphavirus, picomavirus, togavirus,
flavivirus, coronavirus, paramyxovirus, and yellow fever virus, and wherein
said RNA vector construct comprises a selected heterologous nucleotide
sequence.
16.The microparticle as claimed in claim 14 or 15, wherein said vector
construct is derived from an alphavirus selected from the group consisting
of Sindbis virus, Semliki Forest virus, Venezuelan equine encephalitis
virus, or Ross River virus.
17. The microparticle as claimed in claim 13 or 14, wherein said vector
construct comprises a heterologous nucleic acid sequence encoding a
member selected from the group consisting of a pharmaceutical, a

polypeptide, a hormone, an enzyme, a transcription or translation
mediator, an intermediate in a metabolic pathway, an immunomodulator,
an antigen, and an adjuvant.
18. The microparticle as claimed in claim 17, wherein said heterologous
nucleic acid sequence encodes an antigen selected from the group
consisting of HIV gp120, HIV gp140, HIV p24gag, HIV p55gag, and
influenza A hemagglutinin antigen.
19. The microparticle as claimed in claim 18, wherein said heterologous
nucleic acid sequence encodes an HIV gag polypeptide and comprises a
sequence having at least 90% identity to a sequence selected from the
group consisting of nucleotides 844-903 of SEQ ID NOs:63, nucleotides
841-900 of SEQ ID NO: 64, nucleotides 1213-1353 of SEQ ID NO: 67,
and nucleotides 82-1512 of SEQ ID NO:68.
20. The microparticle as claimed in claim 18, wherein said heterologous
nucleic acid sequence encodes an HIV envelope polypeptide and
comprises a sequence having at least 90% identity to a sequence
selected from the group consisting of nucleotides 1513-2547 of SEQ ID
NO: 65 and nucleotides 1210-1353 of SEQ ID NO:66.

21. The microparticle as claimed in claim 13, wherein said vector construct is
a vector selected from the group consisting of the ELVIS vectors pSINCP-
gp140 and pSINCP-p55gag.
22.The microparticle as claimed in any one of claims 10 to 21 further
comprising at least one second biologically active macromolecule
adsorbed on the surface thereof, wherein the second biologically active
macromolecule is at least one member selected from the group consisting
of a polypeptide, a polynucleotide, a polynucleoside, an antigen, a
pharmaceutical, a hormone, an enzyme, a transcription or translation
mediator, an intermediate in a metabolic pathway, an immunomodulator,
and an adjuvant.
23.The microparticle as claimed in claim 22, wherein the second biologically
active macromolecule is an antigen selected from the group consisting of
HIV gp120, HIV gp140, HIV p24gag, HIV p55gag, and influenza A
hemagglutinin antigen, a polynucleotide which encodes HIV gp140, or is
an adjuvant.
24. The microparticle as claimed in claim 23, wherein the second biologically
active macromolecule is an adjuvant that is an aluminum salt.

25. A microparticle composition comprising a microparticle as claimed in any
one of claims 7 to 24 and a pharmaceutically acceptable excipient.
26. The microparticle composition as claimed in claim 25, further comprising
an adjuvant.
27. The microparticle composition as claimed in claim 26, wherein the
adjuvant is (a) a member selected from the group consisting of a CpG
oligonucleotide or (b) an aluminum salt which is aluminum phosphate.
28. The microparticle composition as claimed in any one of claims 25 to 27 for
use in inducing or raising an immune response in a host animal.
29. The microparticle composition as claimed in any one of claims 25 to 27 for
use in delivering a therapeutically effective amount of a macromolecule to
a host animal wherein the host animal is a vertebrate.
30. The microparticle composition as claimed in any one of claims 25 to 27 for
use in treatment of a disease or as a vaccine.

31. The microparticle composition as claimed in claims 28 to 29, wherein said
animal is a human.


A method of producing a microparticle having an adsorbent surface to which a
vector construct capable of expressing a selected nucleic acid sequence is
adsorbed, said method comprising the steps of: (a) emulsifying a mixture of a
polymer solution and a detergent to form an emulsion, wherein the polymer
solution comprises a polymer selected from the group consisting of a poly(α-
hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a
polyanhydride, and a polycyanoacrylate, wherein the polymer is present at a
concentration of about 1% to about 30% in an organic solvent, and wherein the
detergent is present in the mixture at a weight to weight detergent to polymer
ratio of from about 0.00001:1 to about 0.5:1; (b) removing the organic solvent
from the emulsion, to form said microparticle; and (c) adsorbing the vector
construct to the surface of the microparticle, wherein said vector construct is
selected from the group consisting of an ELVIS vector and an RNA vector
construct.

Documents:

270-KOLNP-2003-ASSIGNMENT.pdf

270-KOLNP-2003-CORRESPONDENCE.pdf

270-KOLNP-2003-EXAMINATION REPORT.pdf

270-KOLNP-2003-FORM 13.pdf

270-KOLNP-2003-FORM 18.pdf

270-KOLNP-2003-FORM 26.pdf

270-KOLNP-2003-FORM 3.pdf

270-KOLNP-2003-FORM 5.pdf

270-KOLNP-2003-GRANTED-ABSTRACT.pdf

270-KOLNP-2003-GRANTED-CLAIMS.pdf

270-KOLNP-2003-GRANTED-DESCRIPTION (COMPLETE).pdf

270-KOLNP-2003-GRANTED-DRAWINGS.pdf

270-KOLNP-2003-GRANTED-FORM 1.pdf

270-KOLNP-2003-GRANTED-FORM 2.pdf

270-KOLNP-2003-GRANTED-SPECIFICATION.pdf

270-KOLNP-2003-OTHERS.pdf

270-KOLNP-2003-REPLY TO EXAMINATION REPORT.pdf


Patent Number 250654
Indian Patent Application Number 270/KOLNP/2003
PG Journal Number 03/2012
Publication Date 20-Jan-2012
Grant Date 16-Jan-2012
Date of Filing 03-Mar-2003
Name of Patentee CHIRON CORPORATION
Applicant Address 4560 HORTON STREET, EMERYVILLE, CA 94608, USA
Inventors:
# Inventor's Name Inventor's Address
1 OTTEN GILLIS 35 WILLIAMS LANE, FOSTER CITY, CA 94404, USA
2 DONNELLY JOHN JAMES 46 FIELDBROOK PLACE, MORAGA, CA 94556, USA
3 ULMER JEFFREY 160 SUMMERFIELD STREET, DANVILLE, CA 94506, USA
4 POLO JOHN M. 28034 EI PORTAL DRIVE, HAYWARD, CA 94542, USA
5 BARNETT SUSAN 4240 21ST STREET, SAN FRANCISCO, CA 94114, USA
6 SINGH MANMOHAN 127 PEPPERWOOD STREET, HERCULES, CA 94547, USA
7 DUBENSKY THOMAS W JR. 6 PACIFIC AVENUE, PIEDMONT, CA 94611, USA
8 O'HAGAN DEREK 2373 WOOLSEY STREET, BERKELEY, CA 94705, USA
PCT International Classification Number A61K 9/00
PCT International Application Number PCT/US2001/30540
PCT International Filing date 2001-09-28
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/236,105 2000-09-28 U.S.A.
2 60/315,905 2001-08-30 U.S.A.