U.S. patent application number 09/935466 was filed with the patent office on 2002-02-28 for use of microparticles combined with submicron oil-in-water emulsions.
This patent application is currently assigned to Chiron Corporation. Invention is credited to O'Hagan, Derek, Ott, Gary S., Singh, Manmohan, Van Nest, Gary.
Application Number | 20020025329 09/935466 |
Document ID | / |
Family ID | 22090819 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025329 |
Kind Code |
A1 |
O'Hagan, Derek ; et
al. |
February 28, 2002 |
Use of microparticles combined with submicron oil-in-water
emulsions
Abstract
Compositions are provided which include biodegradable
microparticles with entrapped or adsorbed antigens, in combination
with submicron oil-in-water emulsions. Also provided are methods of
immunization which comprise administering to a vertebrate subject
(a) a submicron oil-in-water emulsion, and (b) a therapeutically
effective amount of a selected antigen entrapped in a
microparticle.
Inventors: |
O'Hagan, Derek; (Berkeley,
CA) ; Van Nest, Gary; (El Sobrante, CA) ; Ott,
Gary S.; (Oakland, CA) ; Singh, Manmohan;
(Hercules, CA) |
Correspondence
Address: |
Alisa A. Harbin
CHIRON CORPORATION
Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
|
Family ID: |
22090819 |
Appl. No.: |
09/935466 |
Filed: |
August 20, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09935466 |
Aug 20, 2001 |
|
|
|
09564416 |
May 2, 2000 |
|
|
|
6306405 |
|
|
|
|
09564416 |
May 2, 2000 |
|
|
|
09015736 |
Jan 29, 1998 |
|
|
|
6086901 |
|
|
|
|
60069724 |
Dec 16, 1997 |
|
|
|
Current U.S.
Class: |
424/278.1 ;
424/204.1; 424/228.1; 424/280.1; 424/283.1; 424/497; 424/70.11;
424/70.19; 424/70.9 |
Current CPC
Class: |
A61K 39/21 20130101;
A61P 31/12 20180101; Y10S 977/802 20130101; A61P 31/00 20180101;
A61P 31/14 20180101; A61K 2039/55566 20130101; A61K 2039/575
20130101; A61P 37/04 20180101; A61K 39/29 20130101; A61P 1/16
20180101; Y10S 977/918 20130101; A61K 39/39 20130101; A61K 2039/545
20130101; A61K 2039/54 20130101; Y02A 50/412 20180101; A61K 9/1075
20130101; A61K 9/1647 20130101; A61K 39/12 20130101; A61K
2039/55555 20130101; C12N 2770/24234 20130101; Y02A 50/401
20180101; Y10S 977/915 20130101; Y02A 50/30 20180101; C12N
2740/16134 20130101 |
Class at
Publication: |
424/278.1 ;
424/497; 424/70.9; 424/70.19; 424/70.11; 424/228.1; 424/204.1;
424/283.1; 424/280.1 |
International
Class: |
A61K 009/16; A61K
009/50; A61K 007/06; A61K 007/075; A61K 039/12; A61K 045/00; A61K
047/00 |
Claims
We claim:
1. A composition comprising a submicron oil-in-water emulsion, and
a selected antigen entrapped in, or adsorbed to, a biodegradable
microparticle.
2. The composition of claim 1, wherein the microparticle is formed
from a poly(.alpha.-hydroxy acid) selected from the group
consisting of poly(L-lactide), poly(D,L-lactide) and
poly(D,L-lactide-co-glycolide).
3. The composition of claim 2, wherein the microparticle is formed
from poly(D,L-lactide-co-glycolide.
4. The composition of claim 1, wherein the submicron oil-in-water
emulsion comprises 4-5% w/v squalene, 0.25-0.5% w/v Tween 80.RTM.,
and 0.5% w/v Span 85.RTM., and optionally,
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L--
alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamin-
e.
5. The composition of claim 1, wherein the selected antigen is a
viral antigen.
6. The composition of claim 5, wherein the selected antigen is
gp120.
7. The composition of claim 5, wherein the selected antigen is
p24gag.
8. The composition of claim 5, wherein the selected antigen is
hepatitis C virus E2.
9. The composition of claim 1, wherein the selected antigen is
entrapped in the microparticle.
10. The composition of claim 1, wherein the selected antigen is
adsorbed to the microparticle.
11. A composition comprising (a) a submicron oil-in-water emulsion
which comprises 4-5% w/v squalene, 0.25-0.5% w/v Tween 80.RTM., and
0.5% w/v Span 85.RTM., and optionally,
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L--
alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamin-
e, and (b) a selected antigen entrapped in, or adsorbed to, a
poly(D,L-lactide-co-glycolide microparticle.
12. The composition of claim 11, wherein the selected antigen is
entrapped in the microparticle.
13. The composition of claim 11, wherein the selected antigen is
adsorbed to the microparticle.
14. A method of immunization which comprises administering to a
vertebrate subject (a) a submicron oil-in-water emulsion, and (b) a
therapeutically effective amount of a selected antigen entrapped
in, or adsorbed to, a biodegradable microparticle.
15. The method of claim 14, wherein the microparticle is formed
from a poly(.alpha.-hydroxy acid) selected from the group
consisting of poly(L-lactide), poly(D,L-lactide) and
poly(D,L-lactide-co-glycolide).
16. The method of claim 15, wherein the microparticle is formed
from poly(D,L-lactide-co-glycolide.
17. The method of claim 14, wherein the submicron oil-in-water
emulsion comprises 4-5% w/v squalene, 0.25-0.5% w/v Tween 80.RTM.,
and 0.5% w/v Span 85.RTM., and optionally,
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L--
alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamin-
e.
18. The method of claim 14, wherein the selected antigen is a viral
antigen.
19. The method of claim 18, wherein the selected antigen is
gp120.
20. The method of claim 18, wherein the selected antigen is
p24gag.
21. The method of claim 18, wherein the selected antigen is
hepatitis C virus E2.
22. The method of claim 14, wherein the selected antigen is
entrapped in the microparticle.
23. The method of claim 14, wherein the selected antigen is
adsorbed to the microparticle.
24. The method of claim 14, wherein the submicron oil-in-water
emulsion is administered prior to the microparticle.
25. The method of claim 14, wherein the submicron oil-in-water
emulsion is administered subsequent to the microparticle.
26. The method of claim 14, wherein the submicron oil-in-water
emulsion is administered substantially concurrently with the
microparticle.
27. A method of immunization which comprises administering to a
vertebrate subject the composition of claim 11.
28. A method of making a composition comprising combining a
submicron oil-in-water emulsion with a selected antigen entrapped
in, or adsorbed to, a biodegradable microparticle.
29. The method of claim 28, wherein the selected antigen is
entrapped in the microparticle.
30. The method of claim 28, wherein the selected antigen is
adsorbed to the microparticle.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to provisional patent
application Ser. No. 60/069,724, filed Dec. 16, 1997, from which
priority is claimed under 35 USC .sctn.119(e)(1) and which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to vaccine
compositions. In particular, the invention relates to the use of
biodegradable microparticles including entrapped or adsorbed
antigens, in combination with submicron oil-in-water emulsions.
BACKGROUND OF THE INVENTION
[0003] Numerous vaccine formulations which include attenuated
pathogens or subunit protein antigens, have been developed.
Conventional vaccine compositions often include immunological
adjuvants to enhance immune responses. For example, depot adjuvants
are frequently used which adsorb and/or precipitate administered
antigens and which can retain the antigen at the injection site.
Typical depot adjuvants include aluminum compounds and water-in-oil
emulsions. However, depot adjuvants, although increasing
antigenicity, often provoke severe persistent local reactions, such
as granulomas, abscesses and scarring, when injected subcutaneously
or intramuscularly. Other adjuvants, such as lipopolysacharrides,
can elicit pyrogenic responses upon injection and/or Reiter's
symptoms (influenza-like symptoms, generalized joint discomfort and
sometimes anterior uveitis, arthritis and urethritis). Saponins,
such as Quillaja saponaria, have also been used as immunological
adjuvants in vaccine compositions against a variety of
diseases.
[0004] More particularly, Complete Freund's adjuvant (CFA) is a
powerful immunostimulatory agent that has been successfully used
with many antigens on an experimental basis. CFA includes three
components: a mineral oil, an emulsifying agent, and killed
mycobacteria, such as Mycobacterium tuberculosis. Aqueous antigen
solutions are mixed with these components to create a water-in-oil
emulsion. Although effective as an adjuvant, CFA causes severe side
effects primarily due to the presence of the mycobacterial
component, including pain, abscess formation and fever. CFA,
therefore, is not used in human and veterinary vaccines.
[0005] Incomplete Freund's adjuvant (IFA) is similar to CFA but
does not include the bacterial component. IFA, while not approved
for use in the United States, has been used elsewhere in human
vaccines for influenza and polio and in veterinary vaccines for
rabies, canine distemper and foot-and-mouth disease. However,
evidence indicates that both the oil and emulsifier used in IFA can
cause tumors in mice.
[0006] Muramyl dipeptide (MDP) has been found to be the minimal
unit of the mycobacterial cell wall complex that generates the
adjuvant activity observed with CFA. See, e.g., Ellouz et al.,
Biochem. Biophys. Res. Commun. (1974) 59:1317. Several synthetic
analogs of MDP have been generated that exhibit a wide range of
adjuvant potency and side effects. For a review of these analogs,
see, Chedid et al., Prog. Allergy (1978) 25:63. Representative
analogs of MDP include threonyl derivatives of MDP (Byars et al.,
Vaccine (1987) 5:223), n-butyl derivatives of MDP (Chedid et al.,
Infect. Immun. 35:417), and a lipophilic derivative of a muramyl
tripeptide (Gisler et al., in Immunomodulations of Microbial
Products and Related Synthetic Compounds (1981) Y. Yamamura and S.
Kotani, eds., Excerpta Medica, Amsterdam, p. 167).
[0007] One lipophilic derivative of MDP is
N-acetylmuramyl-L-alanyl-D-isog-
luatminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3-huydroxyphosphorylox-
y)-ethylamine (MTP-PE). This muramyl tripeptide includes
phospholipid tails that allow association of the hydrophobic
portion of the molecule with a lipid environment while the muramyl
peptide portion associates with the aqueous environment. Thus, the
MTP-PE itself is able to act as an emulsifying agent to generate
stable oil-in-water emulsions. MTP-PE has been used in an emulsion
of 4% squalene with 0.008% Tween.RTM. 80, termed MTP-PE-LO (low
oil), to deliver the herpes simplex virus gD antigen with effective
results (Sanchez-Pescador et al., J. Immunol. (1988)
141:1720-1727), albeit poor physical stability. Recently, MF59, a
safe, highly immunogenic, submicron oil-in-water emulsion which
contains 4-5% w/v squalene, 0.5% w/v Tween.RTM. 80, 0.5% Span.RTM.
85, and optionally, varying amounts of MTP-PE, has been developed
for use in vaccine compositions. See, e.g., Ott et al.,
"MF59--Design and Evaluation of a Safe and Potent Adjuvant for
Human Vaccines" in Vaccine Design: The Subunit and Adjuvant
Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New
York, 1995, pp. 277-296.
[0008] Despite the presence of such adjuvants, conventional
vaccines often fail to provide adequate protection against the
targeted pathogen. In this regard, there is growing evidence that
vaccination against intracellular pathogens, such as a number of
viruses, should target both the cellular and humoral arms of the
immune system.
[0009] More particularly, cytotoxic T-lymphocytes (CTLs) play an
important role in cell-mediated immune defense against
intracellular pathogens such as viruses and tumor-specific antigens
produced by malignant cells. CTLs mediate cytotoxicity of virally
infected cells by recognizing viral determinants in conjunction
with class I MHC molecules displayed by the infected cells.
Cytoplasmic expression of proteins is a prerequisite for class I
MHC processing and presentation of antigenic peptides to CTLs.
However, immunization with killed or is attenuated viruses often
fails to produce the CTLs necessary to curb intracellular
infection. Furthermore, conventional vaccination techniques against
viruses displaying marked genetic heterogeneity and/or rapid
mutation rates that facilitate selection of immune escape variants,
such as HIV or influenza, are problematic. Accordingly, alternative
techniques for vaccination have been developed.
[0010] Particulate carriers with adsorbed or entrapped antigens
have been used in an attempt to elicit adequate immune 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.
Examples of particulate carriers include those derived from
polymethyl methacrylate polymers, as well as microparticles derived
from poly(lactides) and poly(lactide-co-glycolides- ), known as
PLG. Polymethyl methacrylate polymers are nondegradable while PLG
particles biodegrade by random nonenzymatic hydrolysis of ester
bonds to lactic and glycolic acids which are excreted along normal
metabolic pathways.
[0011] Recent studies have shown that PLG microparticles with
entrapped antigens are able to elicit cell-mediated immunity. For
example, microencapsulated human immunodeficiency virus (HIV) gp120
has been shown to induce HIV-specific CD4+ and CD8+ T-cell
responses in mice (Moore et al., Vaccine (1995) 13:1741-1749).
Similarly, microparticle-encapsulated ovalbumin has been shown to
be capable of priming cellular immune responses in vivo and can
induce mucosal IgA responses when administered orally (O'Hagan et
al., Vaccine (1993) 11:149-154). Additionally, both antibody and
T-cell responses have been induced in mice vaccinated with a
PLG-entrapped Mycobacterium tuberculosis antigen (Vordermeier et
al., Vaccine (1995) 13:1576-1582). Antigen-specific CTL responses
have also been induced in mice using a microencapsulated short
synthetic peptide from the circumsporozoite protein of Plasmodium
berghei.
[0012] However, the use of microparticles with entrapped or
adsorbed antigen, in combination with submicron oil-in-water
emulsions, has not heretofore been described.
DISCLOSURE OF THE INVENTION
[0013] The present invention is based on the surprising and
unexpected discovery that the use of biodegradable microparticles,
such as those derived from a poly(.alpha.-hydroxy acid), and
including entrapped or adsorbed antigen, in combination with
submicron oil-in-water emulsions, serves to enhance the
immunogenicity of the antigen. The use of such combinations
provides a safe and effective approach for enhancing the
immunogenicity of a wide variety of antigens.
[0014] Accordingly, in one embodiment, the invention is directed to
a composition comprising a submicron oil-in-water emulsion, and a
selected antigen entrapped in, or adsorbed to, a biodegradable
microparticle.
[0015] In another embodiment, the invention is directed to a
composition comprising (a) a submicron oil-in-water emulsion which
comprises 4-5% w/v squalene, 0.25-0.5% w/v Tween 80.RTM., and 0.5%
w/v Span 85.RTM., and optionally,
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(l'-2'-d-
ipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine, and (b)
a selected antigen entrapped in, or adsorbed to, a biodegradable
microparticle.
[0016] In yet another embodiment, the subject invention is directed
to a method of immunization which comprises administering to a
vertebrate subject (a) a submicron oil-in-water emulsion, and (b) a
therapeutically effective amount of a selected antigen entrapped
in, or adsorbed to, a biodegradable microparticle.
[0017] In still further embodiments, the invention is directed to a
method of making a composition comprising combining a submicron
oil-in-water emulsion with a selected antigen entrapped in, or
adsorbed to, a biodegradable microparticle.
[0018] In particularly preferred embodiments, the microparticle is
derived from a poly(.alpha.-hydroxy acid), preferably
poly(L-lactide), poly(D,L-lactide) or
poly(D,L-lactide-co-glycolide).
[0019] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows total IgG titers at 2, 6 and 10 weeks following
initial vaccination in mice immunized with gp120; gp120+MF59; PLG
with entrapped gp120; and PLG with entrapped gp120+MF59.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of 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 (Easton, Pa.: Mack Publishing Company, 1990); Methods
In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press,
Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific
Publications); and Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989).
[0022] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0023] 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.
[0024] I. Definitions
[0025] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0026] The term "microparticle" as used herein, refers to a
particle of about 100 nm to about 150 .mu.m in diameter, more
preferably about 200 nm to about 30 .mu.m in diameter, and most
preferably about 500 nm to about 10 .mu.m in diameter. Preferably,
the microparticle will be of a diameter that permits parenteral
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. Microparticles
for use herein will be formed from materials that are sterilizable,
non-toxic and biodegradable. Such materials include, without
limitation, poly(.alpha.-hydroxy acid), polyhydroxybutyric acid,
polycaprolactone, polyorthoester, polyanhydride. Preferably,
microparticles for use with the present invention are derived from
a poly(.alpha.-hydroxy acid), in particular, from a poly(lactide)
("PLA") or a copolymer of D,L-lactide and glycolide or glycolic
acid, such as a poly(D,L-lactide-co-glycolide) ("PLG" or "PLGA"),
or a copolymer of D,L-lactide and caprolactone. The 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 antigen. These parameters are discussed
more fully below.
[0027] By "antigen" is meant a molecule which contains one or more
epitopes that will stimulate a host's immune system to make a
cellular antigen-specific immune response when the antigen is
presented, or a humoral antibody response. Normally, an epitope
will include between about 3-15, generally about 5-15, amino acids.
For purposes of the present invention, antigens can be derived from
any of several known viruses, bacteria, parasites and fungi. The
term also intends any of the various tumor antigens. Furthermore,
for purposes of the present invention, an "antigen" refers to a
protein which includes modifications, such as deletions, additions
and substitutions (generally conservative in nature), to the native
sequence, so long as the protein maintains the ability to elicit an
immunological response. These modifications may be deliberate, as
through site-directed mutagenesis, or may be accidental, such as
through mutations of hosts which produce the antigens.
[0028] An "immunological response" to an antigen or composition is
the development in a subject of a humoral and/or a cellular immune
response to molecules present in the composition of interest. For
purposes of the present invention, a "humoral immune response"
refers to an immune response mediated by antibody molecules, while
a "cellular immune response" is one mediated by T-lymphocytes
and/or other white blood cells. One important aspect of cellular
immunity involves an antigen-specific response by cytolytic T-cells
("CTL"s). CTLs have specificity for peptide antigens that are
presented in association with proteins encoded by the major
histocompatibility complex (MHC) and expressed on the surfaces of
cells. CTLs help induce and promote the intracellular destruction
of intracellular microbes, or the lysis of cells infected with such
microbes. Another aspect of cellular immunity involves an
antigen-specific response by helper T-cells. Helper T-cells act to
help stimulate the function, and focus the activity of, nonspecific
effector cells against cells displaying peptide antigens in
association with MHC molecules on their surface. A "cellular immune
response" also refers to the production of cytokines, chemokines
and other such molecules produced by activated T-cells and/or other
white blood cells, including those derived from CD4+ and CD8+
T-cells.
[0029] A 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. The cell-mediated immune response is directed at, or
near, cells presenting antigen at their surface. In addition,
antigen-specific T-lymphocytes can be generated to allow for the
future protection of an immunized host.
[0030] The ability of a particular antigen or composition to
stimulate a cell-mediated immunological response may be determined
by a number of assays, such as by lymphoproliferation (lymphocyte
activation) assays, CTL cytotoxic cell assays, or by assaying for
T-lymphocytes specific for the antigen in a sensitized subject.
Such assays are well known in the art. See, e.g., Erickson et al.,
J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol.
(1994) 24:2369-2376; and the examples below.
[0031] Thus, an immunological response as used herein may be one
which stimulates the production of CTLs, and/or the production or
activation of helper T-cells. The antigen of interest may also
elicit an antibody-mediated immune response. Hence, an
immunological response may include one or more of the following
effects: the production of antibodies by B-cells; and/or the
activation of suppressor T-cells and/or .gamma..delta. T-cells
directed specifically to an antigen or antigens present in the
composition or vaccine of interest. These responses may serve to
neutralize infectivity, and/or mediate antibody-complement, or
antibody dependent cell cytotoxicity (ADCC) to provide protection
to an immunized host. Such responses can be determined using
standard immunoassays and neutralization assays, well known in the
art.
[0032] A vaccine composition which contains a selected antigen
entrapped or adsorbed with a microparticle, along with a submicron
oil-in-water emulsion adjuvant, or a vaccine composition containing
an antigen entrapped or adsorbed with a microparticle which is
coadministered with the subject submicron oil-in-water emulsion
adjuvant, displays "enhanced immunogenicity" when it possesses a
greater capacity to elicit an immune response than the immune
response elicited by an equivalent amount of the
microparticle/antigen without the submicron oil-in-water emulsion
adjuvant. Thus, a vaccine composition may display "enhanced
immunogenicity" because the antigen is more strongly immunogenic or
because a lower dose of antigen is necessary to achieve an immune
response in the subject to which it is administered. Such enhanced
immunogenicity can be determined by administering the
microparticle/antigen composition and submicron oil-in-water
emulsion, and microparticle/antigen controls to animals and
comparing antibody titers against the two using standard assays
such as radioimmunoassay and ELISAs, well known in the art.
[0033] The terms "effective amount" or "pharmaceutically effective
amount" of an agent, as provided herein, refer to a nontoxic but
sufficient amount of the agent to provide the desired immunological
response and corresponding therapeutic effect. As will be pointed
out below, the exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of the condition being treated, and the
particular antigen of interest, mode of administration, and the
like. An appropriate "effective" amount in-any individual case may
be determined by one of ordinary skill in the art using routine
experimentation.
[0034] As used herein, "treatment" refers to any of (i) the
prevention of infection or reinfection, as in a traditional
vaccine, (ii) the reduction or elimination of symptoms, and (iii)
the substantial or complete elimination of the pathogen in
question. Treatment may be effected prophylactically (prior to
infection) or therapeutically (following infection).
[0035] By "pharmaceutically acceptable" or "pharmacologically
acceptable" is meant a material which is not biologically or
otherwise undesirable, i.e., the material may be administered to an
individual along with the microparticle adjuvant formulations
without causing any undesirable biological effects or interacting
in a deleterious manner with any of the components of the
composition in which it is contained.
[0036] 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.
[0037] By "vertebrate subject" is meant any member of the subphylum
cordata, including, without limitation, humans and other primates,
including non-human primates such as chimpanzees and other apes and
monkey species; farm animals such as cattle, sheep, pigs, goats and
horses; domestic mammals such as dogs and cats; laboratory animals
including rodents such as mice, rats and guinea pigs; birds,
including domestic, wild and game birds such as chickens, turkeys
and other gallinaceous birds, ducks, geese, and the like. The term
does not denote a particular age. Thus, both adult and newborn
individuals are intended to be covered. The system described above
is intended for use in any of the above vertebrate species, since
the immune systems of all of these vertebrates operate
similarly.
[0038] II. Modes of Carrying Out the Invention
[0039] The present invention is based on the discovery that the use
of microparticles with entrapped or adsorbed antigen, in
combination with submicron oil-in-water emulsions, provide a
vigorous immune response, even when the antigen is by itself weakly
immunogenic. The submicron oil-in-water adjuvants of the present
invention can be incorporated into vaccine compositions containing
the desired microparticle/antigen, or can be administered
separately, either simultaneously with, just prior to, or
subsequent to, a microparticle/antigen-containing composition.
Furthermore, the formulations of the invention may be used to
enhance the activity of antigens produced in vivo, i.e., in
conjunction with DNA immunization.
[0040] Although the individual components of the vaccine
compositions and methods described herein were known, it was
unexpected and surprising that such combinations would enhance the
efficiency of antigens beyond levels achieved when the components
were used separately.
[0041] The method of the invention provides for cell-mediated
immunity, and/or humoral antibody responses. Thus, in addition to a
conventional antibody response, the system herein described can
provide for, e.g., the association of the expressed antigens with
class I MHC molecules such that an in vivo cellular immune response
to the antigen of interest can be mounted which stimulates the
production of CTLs to allow for future recognition of the antigen.
Furthermore, the methods may elicit an antigen-specific response by
helper T-cells. Accordingly, the methods of the present invention
will find use with any antigen for which cellular and/or humoral
immune responses are desired, including antigens derived from
viral, bacterial, fungal and parasitic pathogens that may induce
antibodies, T-cell helper epitopes and T-cell cytotoxic epitopes.
Such antigens include, but are not limited to, those encoded by
human and animal viruses and can correspond to either structural or
non-structural proteins.
[0042] The technique is particularly useful for immunization
against intracellular viruses and tumor cell antigens which
normally elicit poor immune responses. For example, the present
invention will find use for stimulating an immune response against
a wide variety of proteins from the herpesvirus family, including
proteins derived from herpes simplex virus (HSV) types 1 and 2,
such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens
derived from varicella zoster virus (VZV), Epstein-Barr virus (EBV)
and cytomegalovirus (CMV) including CMV gB and gH; and antigens
derived from other human herpesviruses such as HHV6 and HHV7. (See,
e.g. Chee et al., Cytomegaloviruses (J. K. McDougall, ed.,
Springer-Verlag 1990) pp. 125-169, for a review of the protein
coding content of cytomegalovirus; McGeoch et al., J. Gen. Virol.
(1988) 69:1531-1574, for a discussion of the various HSV-1 encoded
proteins; U.S. Pat. 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.)
[0043] Antigens from the hepatitis family of viruses, including
hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus
(HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and
hepatitis G virus (HGV), can also be conveniently used in the
techniques described herein. By way of example, the viral genomic
sequence of HCV is known, as are methods for obtaining the
sequence. See, e.g., International Publication Nos. WO 89/04669; WO
90/11089; and WO 90/14436. The HCV genome encodes several viral
proteins, including E1 (also known as E) and E2 (also known as
E2/NSI) and an N-terminal nucleocapsid protein (termed "core")
(see, Houghton et al., Hepatology (1991) 14:381-388, for a
discussion of HCV proteins, including E1 and E2). Each of these
proteins, as well as antigenic fragments thereof, will find use in
the present methods. Similarly, the sequence for the
.delta.-antigen from HDV is known (see, e.g., U.S. Pat. No.
5,378,814) and this antigen can also be conveniently used in the
present methods. Additionally, antigens derived from HBV, such as
the core antigen, the surface antigen, sAg, as well as the
presurface sequences, pre-S1 and pre-S2 (formerly called pre-S), as
well as combinations of the above, such as sAg/pre-S1, sAg/pre-S2,
sAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See,
e.g., "HBV Vaccines--from the laboratory to license: a case study"
in Mackett, M. and Williamson, J. D., Human Vaccines and
Vaccination, pp. 159-176, for a discussion of HBV structure; and
U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513, incorporated herein
by reference in their entireties; Beames et al., J. Virol. (1995)
69:6833-6838, Birnbaum et al., J. Virol. (1990) 64:3319-3330; and
Zhou et al., J. Virol. (1991) 65:5457-5464.
[0044] Antigens derived from other viruses will also find use in
the claimed methods, such as without limitation, proteins from
members of the families Picornaviridae (e.g., polioviruses, etc.);
Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus,
etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae;
Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae;
Paramyxoviridae (e.g., mumps virus, measles virus, respiratory
syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus
types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae
(e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV,
hTLR, etc.)), including but not limited to antigens from the
isolates HIV.sub.IIIb, HIV.sub.SF2, HIV.sub.LAV, HIV.sub.LAI,
HIV.sub.MN); HIV-1.sub.CM235, HIV-1.sub.US4; HIV-2; 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.
[0045] More particularly, the gp120 envelope proteins from any of
the above HIV isolates, including members of the various genetic
subtypes of HIV, are known and reported (see, e.g., Myers et al.,
Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N.
Mex. (1992); Myers et al., Human Retroviruses and Aids, 1990, Los
Alamos, New Mexico: Los Alamos National Laboratory; and Modrow et
al., J. Virol. (1987) 61:570-578, for a comparison of the envelope
sequences of a variety of HIV 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 HIV isolates,
including any of the various envelope proteins such as gp160 and
gp41, gag antigens such as p24gag and p55gag, as well as proteins
derived from the pol region.
[0046] As explained above, influenza virus is another example of a
virus for which the present invention will be particularly useful.
Specifically, the envelope glycoproteins HA and NA of influenza A
are of particular interest for generating an immune response.
Numerous HA subtypes of influenza A have been identified (Kawaoka
et al., Virology (1990) 179:759-767; Webster et al., "Antigenic
variation among type A influenza viruses," p. 127-168. In: P.
Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses.
Springer-Verlag, New York). Thus, proteins derived from any of
these isolates can also be used in the immunization techniques
described herein.
[0047] The methods described herein will also find use with
numerous bacterial antigens, such as those derived from organisms
that cause diphtheria, cholera, tuberculosis, tetanus, pertussis,
meningitis, and other pathogenic states, including, without
limitation, Meningococcus A, B and C, Hemophilus influenza type B
(HIB), and Helicobacter pylori. Examples of parasitic antigens
include those derived from organisms causing malaria and Lyme
disease.
[0048] Furthermore, the methods described herein provide a means
for treating a variety of malignant cancers. For example, the
system of the present invention can be used to mount both humoral
and cell-mediated immune responses to particular proteins specific
to the cancer in question, such as an activated oncogene, a fetal
antigen, or an activation marker. Such tumor antigens include any
of the various MAGEs (melanoma associated antigen E), including
MAGE 1, 2, 3, 4, etc. (Boon, T. Scientific American (March
1993):82-89); any of the various tyrosinases; MART 1 (melanoma
antigen recognized by T cells), mutant ras; mutant p53; p97
melanoma antigen; CEA (carcinoembryonic antigen), among others.
[0049] It is readily apparent that the subject invention can be
used to prevent or treat a wide variety of diseases.
[0050] The selected antigen is-entrapped in, or adsorbed to, a
microparticle for subsequent delivery. Biodegradable polymers for
manufacturing microparticles useful in the present invention are
readily commercially available from, e.g., Boehringer Ingelheim,
Germany and Birmingham Polymers, Inc., Birmingham, AL. For example,
useful polymers for forming the microparticles herein include those
derived from polyhydroxybutyric acid; polycaprolactone;
polyorthoester; polyanhydride; as well as a poly(.alpha.-hydroxy
acid), such as poly(L-lactide), poly(D,L-lactide) (both known as
"PLA" herein), poly(hydoxybutyrate), copolymers of D,L-lactide and
glycolide, such as poly(D,L-lactide-co-glyc- olide) (designated as
"PLG" or "PLGA" herein) or a copolymer of D,L-lactide and
caprolactone. Particularly preferred polymers for use herein are
PLA and PLG polymers. These polymers are available in a variety of
molecular weights, and the appropriate molecular weight for a given
antigen is readily determined by one of skill in the art. Thus,
e.g., for PLA, a suitable molecular weight will be on the order of
about 2000 to 250,000. For PLG, suitable molecular weights will
generally range from about 10,000 to about 200,000, preferably
about 15,000 to about 150,000, and most preferably about 50,000 to
about 100,000.
[0051] 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 antigen 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 lactide:glycolide is
easily determined by one of skill in the art based on the nature of
the antigen and disorder in question. Moreover, mixtures of
microparticles with varying lactide:glycolide ratios will find use
in the formulations in order to achieve the desired release
kinetics for a given antigen 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 crystallinity. PLG copolymers
with varying lactide:glycolide ratios and molecular weights are
readily available commercially from a number of sources including
from Boehringer Ingelheim, Germany and Birmingham Polymers, Inc.,
Birmingham, Ala. These polymers can also be synthesized by simple
polycondensation 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.
[0052] The antigen/microparticles are prepared using any of several
methods well known in the art. For example, double emulsion/solvent
evaporation techniques, such as described in U.S. Pat. No.
3,523,907 and Ogawa et al., Chem. Pharm. Bull. (1988) 36:1095-1103,
can be used herein to form the microparticles. These techniques
involve the formation of a primary emulsion consisting of droplets
of polymer solution containing the antigen (if antigen is to be
entrapped in the microparticle), which is subsequently mixed with a
continuous aqueous phase containing a particle
stabilizer/surfactant.
[0053] More particularly, a water-in-oil-in-water (w/o/w) solvent
evaporation system can be used to form the microparticles, as
described by O'Hagan et al., Vaccine (1993) 11:965-969 and Jeffery
et al., Pharm. Res. (1993) 10:362. In this technique, the
particular polymer is combined with an organic solvent, such as
ethyl acetate, dimethylchloride (also called methylene chloride and
dichloromethane), acetonitrile, acetone, chloroform, and the like.
The polymer will be provided in about a 2-15%, more preferably
about a 4-10% and most preferably, a 6% solution, in organic
solvent. An approximately equal amount of an antigen solution,
e.g., in water, is added and the polymer/antigen solution
emulsified using e.g, an homogenizer. The emulsion is then combined
with a larger volume of an aqueous solution of an emulsion
stabilizer such as polyvinyl alcohol (PVA) or polyvinyl
pyrrolidone. The emulsion stabilizer is typically provided in about
a 2-15% solution, more typically about a 4-10% solution. The
mixture is then homogenized to produce a stable w/o/w double
emulsion. Organic solvents are then evaporated.
[0054] The formulation parameters can be manipulated to allow the
preparation of small (<5 .mu.m) and large (>30 .mu.m)
microparticles. 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 PVA.
[0055] Microparticles can also be formed using spray-drying and
coacervation as described in, e.g., Thomasin et al., J. Controlled
Release (1996) 41:131; U.S. Pat. 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, Fla. 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.
[0056] The above techniques are also applicable to the production
of microparticles with adsorbed antigens. In this embodiment,
microparticles are formed as described above, however, antigens are
mixed with the microparticles following formation.
[0057] 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 the
particle diameter. Particle size is also readily determined using
scanning electron microscopy (SEM).
[0058] Prior to use of the microparticles, antigen content is
generally determined so that an appropriate amount of the
microparticles may be delivered to the subject in order to elicit
an adequate immune response. Antigen content of the microparticles
can be determined according to methods known in the art, such as by
disrupting the microparticles and extracting the entrapped antigen.
For example, microparticles can be dissolved in dimethylchloride
and the protein extracted into distilled water, as described in,
e.g., Cohen et al., Pharm. Res. (1991) 8:713; Eldridge et al.,
Infect. Immun. (1991) 59:2978; and Eldridge et al., J. Controlled
Release (1990)11:205. Alternatively, microparticles can be
dispersed in 0.1 M NaOH containing 5% (w/v) SDS. The sample is
agitated, centrifuged and the supernatant assayed for the antigen
of interest using an appropriate assay. See, e.g., O'Hagan et al.,
Int. J. Pharm. (1994) 103:37-45.
[0059] As explained above, a submicron oil-in-water emulsion
formulation will also be administered to the vertebrate subject,
either prior to, concurrent with, or subsequent to, delivery of the
antigen/microparticle.
[0060] Submicron oil-in water emulsions for use herein include
nontoxic, metabolizable oils and commercial emulsifiers. Examples
of nontoxic, metabolizable oils include, without limitation,
vegetable oils, fish oils, animal oils or synthetically prepared
oils. Fish oils, such as cod liver oil, shark liver oils and whale
oils, are preferred, with squalene,
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, found
in shark liver oil, particularly preferred. The oil component will
be present in an amount of from about 0.5% to about 20% by volume,
preferably in an amount up to about 15%, more preferably in an
amount of from about 1% to about 12% and most preferably from 1% to
about 4% oil.
[0061] The aqueous portion of the adjuvant can be buffered saline
or unadulterated water. Since the compositions are intended for
parenteral administration, it is preferable to make up the final
solutions so that the tonicity, i.e., osmolality, is essentially
the same as normal physiological fluids, in order to prevent
post-administration swelling or rapid absorption of the composition
due to differential ion concentrations between the composition and
physiological fluids. If saline is used rather than water, it is
preferable to buffer the saline in order to maintain a pH
compatible with normal physiological conditions. Also, in certain
instances, it may be necessary to maintain the pH at a particular
level in order to insure the stability of certain composition
components. Thus, the pH of the compositions will generally be pH
6-8 and pH can be maintained using any physiologically acceptable
buffer, such as phosphate, acetate, tris, bicarbonate or carbonate
buffers, or the like. The quantity of the aqueous agent present
will generally be the amount necessary to bring the composition to
the desired final volume.
[0062] Emulsifying agents suitable for use in the oil-in-water
formulations include, without limitation, sorbitan-based non-ionic
surfactants such as those commercially available under the name of
Span.RTM. or Arlacel.RTM.; polyoxyethylene sorbitan monoesters and
polyoxyethylene sorbitan triesters, commercially known by the name
Tween.RTM.; polyoxyethylene fatty acids available under the name
Myrj.RTM.; polyoxyethylene fatty acid ethers derived from lauryl,
acetyl, stearyl and oleyl alcohols, such as those known by the name
of Brij.RTM.; and the like. These substances are readily available
from a number of commercial sources, including ICI America's Inc.,
Wilmington, DE. These emulsifying agents may be used alone or in
combination. The emulsifying agent will usually be present in an
amount of 0.02% to about 2.5% by weight (w/w), preferably 0.05% to
about 1%, and most preferably 0.01% to about 0.5. The amount
present will generally be about 20-30% of the weight of the oil
used.
[0063] The emulsions can also contain other immunostimulating
agents, such as muramyl peptides, including, but not limited to,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(l'-2'-dipalmitoyl-s-
n-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
Immunostimulating bacterial cell wall components, such as
monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), may also be present.
[0064] For a description of various suitable submicron oil-in-water
emulsion formulations for use with the present invention, see,
e.g., International Publication No. WO 90/14837; Remington: The
Science and Practice of Pharmacy, Mack Publishing Company, Easton,
Pa., 19th edition, 1995; Van Nest et al., "Advanced adjuvant
formulations for use with recombinant subunit vaccines," In
Vaccines 92, Modern Approaches to New Vaccines (Brown et al., ed.)
Cold Spring Harbor Laboratory Press, pp. 57-62 (1992); and Ott et
al., "MF59--Design and Evaluation of a Safe and Potent Adjuvant for
Human Vaccines" in Vaccine Design: The Subunit and Adjuvant
Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New
York (1995) pp. 277-296.
[0065] In order to produce submicron particles, i.e., particles
less than 1 micron in diameter and in the nanometer size range, a
number of techniques can be used. For example, commercial
emulsifiers can be used that operate by the principle of high shear
forces developed by forcing fluids through small apertures under
high pressure. Examples of commercial emulsifiers include, without
limitation, Model 110Y microfluidizer (Microfluidics, Newton,
Mass.), Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.), and
Rainnie Minilab Type 8.30H (Miro Atomizer Food and Dairy, Inc.,
Hudson, Wis.). The appropriate pressure for use with an individual
emulsifier is readily determined by one of skill in the art. For
example, when the Model 110Y microfluidizer is used, operation at
5000 to 30,000 psi produces oil droplets with diameters of about
100 to 750 nm.
[0066] The size of the oil droplets can be varied by changing the
ratio of detergent to oil (increasing the ratio decreases droplet
size), operating pressure (increasing operating pressure reduces
droplet size), temperature (increasing temperature decreases
droplet size), and adding an amphipathic immunostimulating agent
(adding such agents decreases droplet size). Actual droplet size
will vary with the particular detergent, oil and immunostimulating
agent (if any) and with the particular operating conditions
selected. Droplet size can be verified by use of sizing
instruments, such as the commercial Sub-Micron Particle Analyzer
(Model N4MD) manufactured by the Coulter Corporation, and the
parameters can be varied using the guidelines set forth above until
substantially all droplets are less than 1 micron in diameter,
preferably less than about 0.8 microns in diameter, and most
preferably less than about 0.5 microns in diameter. By
substantially all is meant at least about 80% (by number),
preferably at least about 90%, more preferably at least about 95%,
and most preferably at least about 98%. The particle size
distribution is typically Gaussian, so that the average diameter is
smaller than the stated limits.
[0067] Particularly preferred submicron oil-in-water emulsions for
use herein are squalene/water emulsions optionally containing
varying amounts of MTP-PE, such as the submicron oil-in-water
emulsion known as "MF59" (International Publication No. WO
90/14837; Ott et al., "MF59--Design and Evaluation of a Safe and
Potent Adjuvant for Human Vaccines" in Vaccine Design: The Subunit
and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum
Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v
Squalene (e.g., 4.3%), 0.25-0.5% w/v Tween 80.RTM., and 0.5% w/v
Span 85.RTM. and optionally contains various amounts of MTP-PE,
formulated into submicron particles using a microfluidizer such as
Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For
example, MTP-PE may be present in an amount of about 0-500
.mu.g/dose, more preferably 0-250 .mu.g/dose and most preferably,
0-100 .mu.g/dose. MF59-0, therefore, refers to the above submicron
oil-in-water emulsion lacking MTP-PE, while MF59-100 contains 100
.mu.g MTP-PE per dose. MF69, another submicron oil-in-water
emulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v
Tween 80.RTM., and 0.75% w/v Span 85.RTM. an optionally MTP-PE. Yet
another submicron oil-in-water emulsion is SAF, containing 10%
squalene, 0.4% Tween 80.RTM., 5% pluronic-blocked polymer L121, and
thr-MDP, also microfluidized into a submicron emulsion.
[0068] Once the submicron oil-in-water emulsion is formulated it
can be administered to the vertebrate subject, either prior to,
concurrent with, or subsequent to, delivery of the microparticle.
If administered prior to immunization with the microparticle, the
adjuvant formulations can be administered as early as 5-10 days
prior to immunization, preferably 3-5 days prior to immunization
and most preferably 1-3 or 2 days prior to immunization with the
antigens of interest. If administered separately, the submicron
oil-in-water formulation can be delivered either to the same site
of delivery as the microparticle compositions or to a different
delivery site.
[0069] If simultaneous delivery is desired, the submicron
oil-in-water formulation can be included with the microparticle
compositions. Generally, the microparticles and submicron
oil-in-water emulsion can be combined by simple mixing, stirring,
or shaking. Other techniques, such as passing a mixture of the two
components rapidly through a small opening (such as a hypodermic
needle) can also be used to provide the vaccine compositions.
[0070] If combined, the various components of the composition can
be present in a wide range of ratios. For example, the
microparticle and emulsion components are typically used in a
volume ratio of 1:50 to 50:1, preferably 1:10 to 10:1, more
preferably from about 1:3 to 3:1, and most preferably about 1:1.
However, other ratios may be more appropriate for specific
purposes, such as when a particular antigen is both difficult to
incorporate into a microparticle and has a low immungenicity, in
which case a higher relative amount of the antigen component is
required.
[0071] Once formulated, the compositions of the invention are
administered parenterally, generally by injection. The compositions
can be injected either subcutaneously, intraperitoneally,
intravenously or intramuscularly. Dosage treatment may be a single
dose schedule or a multiple dose schedule. A multiple dose schedule
is one in which a primary course of vaccination may be with 1-10
separate doses, followed by other doses given at subsequent time
intervals, chosen to maintain and/or reinforce the immune response,
for example at 1-4 months for a second dose, and if needed, a
subsequent dose(s) after several months. The boost may be with a
microparticle/submicron oil-water-emulsion given for the primary
immune response, or may be with a different formulation that
contains the antigen. 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 vaccines are generally administered prior
to primary infection with the pathogen of interest. If treatment is
desired, e.g., the reduction of symptoms or recurrences, the
vaccines are generally administered subsequent to primary
infection.
[0072] The compositions will generally include one or more
"pharmaceutically acceptable excipients or vehicles" such as water,
saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol,
etc. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the like, may be
present in such vehicles.
[0073] The compositions will comprise a "therapeutically effective
amount" of the antigen of interest. That is, an amount of antigen
will be included in the compositions which, when in combination
with the submicron-oil-in water emulsion, will cause the subject to
produce a sufficient immunological response in order to prevent,
reduce or eliminate 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 capacity of the
subject's immune system to synthesize antibodies; the degree of
protection desired; the severity of the condition being treated;
the particular antigen selected and its mode of administration,
among other factors. An appropriate effective amount can be readily
determined by one of skill in the art. Thus, a "therapeutically
effective amount" will fall in a relatively broad range that can be
determined through routine trials. For example, for purposes of the
present invention, an effective dose will typically range from
about 1 .mu.g to about 100 mg, more preferably from about 10 .mu.g
to about 1 mg, and most preferably about 50 .mu.g to about 500
.mu.g of the antigen delivered per dose.
[0074] III. Experimental
[0075] 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.
[0076] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
EXAMPLE 1
Preparation of p24 Antigen-Entrapped Microparticles
[0077] Materials used to formulate the microparticles were as
follows:
[0078] (1) poly(D,L-lactide-co-glycolide), 50:50 mol ratio lactide
to glycolide, MW average=70-100 kDa (50:50 PLGA high viscosity
polymer) (Medisorb Technologies International, Cincinnati,
Ohio);
[0079] (2) 8% polyvinyl alcohol (PVA) 3-83 (Mowiol, Frankfurt,
Germany) in water saturated 10% with ethyl acetate by adding 6 ml
of the latter and stirring in a screw cap glass bottle for 10
minutes; and
[0080] (3) p24gag/sf2, in 30 mM Tris, pH 7.5, at a concentration of
6.1 mg of antigen/ml with 20:1 sucrose:protein.
[0081] P24gag microparticles were prepared by a solvent extraction
technique as follows. To make the microparticles, 2.58 g of the
polymer solution was sonicated with 0.8 ml of the antigen solution
for 30 seconds. The primary emulsion was homogenized with 60 grams
of the saturated PVA solution using a benchtop homogenizer with a
20 mm probe at 10 K rpm for 1 minute. The resulting emulsion was
immediately added to 2.5 L of water, stirred for two hours for
solvent extraction, filtered through a 38 .mu. mesh, washed by
centrifugation 3 times, and a portion sonicated for one minute in a
water bath sonicator, then sized by laser diffraction measurement.
The mean diameter size of the microparticles was 10 .mu.m. The
microspheres were lyophilized and stored at -20.degree. C.
EXAMPLE 2
Immungenicity of p24gag/sf2 Antigen-Entrapped Microparticles with
MF59
[0082] 10 Baboons were divided into two groups (five baboons per
group) and administered the formulations specified in Table 1. For
Group 1, equal parts of the adjuvant MF59-0, and p24gag/sf2 (in a
citrate/Tris buffer) were combined to yield a total of 0.7 ml. The
composition was gently mixed and 500 l (to yield 100 .mu.g
p24gag/sf2/dose) of vaccine was injected intramuscularly (IM) in
the thigh muscle. For Group 2, 1.5 ml of 2.times. phosphate
buffered saline (PBS) was added to 46.8 mg of the PLG-entrapped
p24gag/sf2 formulation produced in Example 1. The material was
vortexed for about 30 seconds until all beads were in suspension.
1.5 ml of MF59-0 was added to the resuspended beads to yield a
total of 3 ml. The composition was gently mixed and 500 .mu.l (to
yield 100 .mu.g p24gag/sf2/dose) of vaccine was injected IM in the
thigh muscle.
[0083] Both groups of animals were boosted twice at 4 week
intervals following the initial injection, with 500 .mu.l of the
vaccine composition. Two weeks following the second boost (10 weeks
after the initial immunization) serum was collected and IgG titers
evaluated by a standard ELISA, essentially as described below.
[0084] As shown in Table 1, entrapped p24gag/sf2+MF59 elicited a
significantly greater antibody response than nonentrapped
p24gag/sf2+MF59.
1 TABLE 1 Mean IgG Group/Formulation titers Group 1 19,976
p24gag/sf2 100 .mu.g + MF59-0 Group 2 85,725 p24gag/sf2 100 .mu.g
in PLG Microparticles + MF59-0
EXAMPLE 3
Preparation of qp120/sf2 Antigen-Entrapped Microparticles Materials
used to formulate the microparticles were as follows:
[0085] (1) 3.0 g of the polymer poly(D,L-lactide-co-glycolide)
composed of a 50:50 mol ratio of lactide to glycolide with a
molecular weight average of 80 Kdal, (Boehringer Ingelheim Resomer
RG505), was dissolved in 50 ml of dichloromethane (DCM, HPLC grade,
obtained from Aldrich);
[0086] (2) 16 g polyvinyl alcohol (13-23 Kdal molecular weight
average, ICN Biomedicals, Aurora, OH) was dissolved in 200 ml
deionized water; and
[0087] (3) HIV gp120sf2 antigen (Chiron, clinical grade) was used,
at a concentration of 7.2 mg antigen/ml in 30 mM sodium citrate, pH
6.0, buffer.
[0088] Microparticles were prepared as follows. 1.67 ml of the HIV
gp120sf2 antigen were added to 16.7 ml of the
poly(D,L-lactide-co-glycoli- de) solution in a 30 ml glass
heavy-walled test tube. The solution was homogenized 3 minutes at
23,000 RPM using a small, hand-held homogenizer equipped with 10 mm
diameter generator. The homogenate was then slowly poured into 66.8
ml of the polyvinyl alcohol solution in a 150 ml glass beaker while
homogenizing at 12,000 RPM using a bench scale homogenizer equipped
with a 20 mm diameter generator for a total homogenization time of
3 minutes. The beaker containing the resulting double emulsion was
equipped with a small magnetic stir bar. This was then allowed to
sit overnight at ambient temperature under moderate (approximately
1000 RPM) stirring rate to evaporate the DCM solvent. The resulting
microparticles prepared in this way were washed to remove excess
PVA and un-entrapped antigen. Washing was accomplished by
repeatedly (3 times total) diluting the microparticle preparation
in approximately 450 ml deionized water, centrifuging to pellet
microparticles, decanting off supernatant, and resuspending the
microparticles in approximately 30 ml deionized water. After the
final resuspension step, the microparticles were lyophilized and
stored at -20.degree. C.
[0089] Small samples (10-30 mg) of the lyophilized microparticles
were utilized to measure particle size distribution and antigen
content. The size distribution of the microparticles thus prepared
was measured by dynamic laser light scatter using a Malvern
Mastersizer instrument and determined to have a median size of 0.6
.mu.m. The antigen content (% load) was measured by dissolving
samples of the microparticles in a 0.1 M sodium hydroxide, 1%
sodium dodecyl sulfate solution, then measuring protein content
using a standard bicinchoninic acid (BCA) assay (Pierce, Rockford,
IL). The % load of the microparticles was measured in this manner
and determined to contain 0.7% protein by weight.
EXAMPLE 4
Immungenicity of gp120/sf2 Antigen-Entrapped Microparticles with
MF59 in Baboons
[0090] A similar experiment was run as-described in Example 2,
using gp120/sf2 in place of p24gag/sf2. In particular, gp120/sf2
was combined with MF59-0 and 50 .mu.g administered to Group 1
baboons, as described above. Additionally, the PLG-entrapped
gp120/sf2 from Example 1 was combined with MF59-0 as described and
50 .mu.g administered to the Group 2 animals.
[0091] Both groups of animals were boosted at 4 weeks following the
initial injection, with 500 .mu.l of the vaccine composition. Serum
samples were collected four weeks after the initial dose (4wp1), as
well as four weeks following the second dose (4wp2) and 8 weeks
following the second dose (8wp2) and IgG titers evaluated by ELISA
as follows. 96-well ELISA plates (Nunc U96, cat# 449824) were
coated with 100 Al per well of 2 .mu.g/ml gp120/sf2 antigen in 50
mM sodium borate buffer, pH 9.0. The plates were incubated
overnight at 4.degree. C. Baboon serum samples, initially diluted
1:50 to 1:1000 in 100 mM sodium phosphate, 1 mM EDTA, 0.5 M sodium
chloride buffer, pH 7.5 (dilution solution), were serially diluted
with dilution solution 1:2 from top to bottom of the ELISA plate
(one column per serum sample) such that samples were diluted by a
factor of 1-, 2-, 4-, 8-, 16-, 32-, 64- and 128-fold greater than
the initial dilution, with a final volume of 100 .mu.l sample per
well. A column containing dilution solution only (blank), and a
standard serum (standard) were included on each plate for
comparison purposes. ELISA plates were incubated 1 hour at
37.degree. C. After washing plates extensively with 0.05%
Triton-X100 solution, 100 Al per well of a 1:5000 diluted Goat
anti-Monkey IgG-HRP conjugate solution (Organon Teknike Corp., West
Chester, Pa., cat# 55432) was added. Plates were incubated 1 hour
at 37.degree. C. Plates were again washed extensively with 0.05%
Triton-X100. 100 .mu.l TMB peroxide developer solution (Kirkegaard
& Perry labs, Gaithersburg, Md.) were added to each well. Color
reaction was allowed to develop for approximately 3 minutes before
stopping by adding 50 .mu.l per well 2 M HCl. Plates were read
using an ELISA reader at 450 nm. Resulting OD values for each plate
were subtracted from baseline OD using average values from a blank
column. Titers for each serum sample were expressed as the dilution
required to achieve an OD of 0.5 as determined by fitting resulting
data to a log-logit function.
[0092] As shown in Table 2, entrapped gp120/sf2+MF59 elicited a
greater antibody response than nonentrapped gp120/sf2+MF59 in all
groups with the response seen at four weeks after the first dose
being significantly higher.
2TABLE 2 Mean IgG Mean IgG Mean IgG titers titers titers
Group/Formulation 4wp1 4wp2 8wp2 Group 1 10 3297 1118 gp120/sf2 50
.mu.g + MF59-0 Group 2 637 5120 1733 gp120/sf2 50 .mu.g entrapped
in PLG Microparticles + MF59-0
EXAMPLE 5
Immungenicity of qp120/sf2 Antigen-Entrapped Microparticles with
MF59 in Mice
[0093] The ability of HIV gp120 to stimulate an immune response
when entrapped or adsorbed to PLG microparticles and coadministered
with MF59 was also tested in mice as follows. Balb/C mice, 6-7
weeks in age, were divided into four groups and administered
intramuscularly 50 .mu.l of a vaccine composition containing 10
.mu.g of HIV gp120, and adjuvant as specified in Table 3. The
various compositions were prepared as described in Example 4
above.
3TABLE 3 Animal Adjuvant Antigen Volume per Injection Group numbers
Name Dose Name Dose Site Animal Route 1 (10) PBS HIV 10 .mu.g 50
.mu.l 50 .mu.l IM gP120 (soluble) 2 (30) MF59-0 25 .mu.l HIV 10
.mu.g 50 .mu.l 50 .mu.l IM gP120 (soluble) 3 (30) PLG/ 1.3 mg HIV
10 .mu.g 50 .mu.l 50 .mu.l IM gp120 gP120 (entrapped) 4 (30)
PLG/gp120 1.3 mg HIV 10 .mu.g 50 .mu.l 50 .mu.l IM in MF59-0 25
.mu.l gP120 (entrapped)
[0094] Animals were boosted at 4 and 8 weeks following the initial
injection. Serum was collected at 2, 6 and 10 weeks following
injection and IgG titers evaluated by a standard ELISA, as
described in Example 4.
[0095] The results are shown in Table 4 and FIG. 1. In all cases,
IgG titers were higher in the group administered PLG-entrapped
gp120+MF59 than IgG titers in the other groups, and significantly
higher than the group administered MF59 alone. At 10 weeks
following injection, IgG titers were significantly higher in the
group adminstered PLG-entrapped gp120+MF59 as compared to all other
groups.
4 TABLE 4 Total IgG Formulation 2 weeks 6 weeks 10 weeks gp120 9 9
19 gp120 + MF59 9 65 851 PLG/gp120 54 40728 62167 PLG/gp120 + MF59
82 70672 113172
EXAMPLE 6
Immungenicity of HCV E2 Antigen-Entrapped Microparticles with MF59
in Mice
[0096] The ability of the hepatitis C virus (HCV) E2 antigen to
stimulate an immune response when entrapped or adsorbed to PLG
microparticles and coadministered with MF59 was tested as follows.
Mice were divided into six groups and administered intramuscularly
50 .mu.l of a vaccine composition containing 5 .mu.g of HCV E2
antigen and adjuvant as specified in Table 5. The compositions were
prepared essentially as described above.
[0097] Animals were boosted at 4 and 8 weeks following the initial
injection. Serum was collected at 2, 6, 10 and 12 weeks following
injection and IgG titers evaluated by a standard ELISA, essentially
as described above.
[0098] As shown in Table 6, antibody titers for HCV E2, either
adsorbed or entrapped in PLG microparticles, and coadministered
with MF59, were higher than those seen when PLG or MF59 were
administered alone.
5 TABLE 5 Adjuvant E2 Group # Name Dose Dose 1 MF59 50 .mu.l -- 2
PLG mixed 500 .mu.g 5 .mu.g 3 PLG adsorbed 500 .mu.g 5 .mu.g 4 PLG
entrapped 500 .mu.g 5 .mu.g 5 PLG adsorbed + MF59 500 .mu.g 5 .mu.g
6 PLG entrapped + MF59 500 .mu.g 5 .mu.g
[0099]
6TABLE 6 PLG PLG PLG PLG ads. + entr. + Weeks MF59 PLG adsorbed
entrapped MF59 MF59 0 0.43 0.39 0.42 0.5 0.47 0.52 2 0.37 0.21 0.19
0.28 2.26 0.54 6 31.48 4.71 5.67 49.96 98.77 175.69 10 155.04 6.74
31.35 176 418 425 12 141 1.13 20.33 21.66 123 188
[0100] Accordingly, the use of submicron oil-in-water emulsions
with antigen-entrapped and -adsorbed microparticles is disclosed.
Although preferred embodiments of the subject invention have been
described in some detail, it is understood that obvious variations
can be made without departing from the spirit and the scope of the
invention as defined by the appended claims.
* * * * *