U.S. patent application number 12/573903 was filed with the patent office on 2010-01-28 for use of microparticles with adsorbed antigen to stimulate immune responses.
This patent application is currently assigned to NOVARTIS VACCINES AND DIAGNOSTICS, INC.. Invention is credited to John Barackman, Jina Kazzaz, Derek O'Hagan, Gary S. Ott, Gary Van Nest.
Application Number | 20100021548 12/573903 |
Document ID | / |
Family ID | 26713053 |
Filed Date | 2010-01-28 |
United States Patent
Application |
20100021548 |
Kind Code |
A1 |
O'Hagan; Derek ; et
al. |
January 28, 2010 |
USE OF MICROPARTICLES WITH ADSORBED ANTIGEN TO STIMULATE IMMUNE
RESPONSES
Abstract
The use of poly(lactide) or poly(lactide-co-glycolide)
microparticles with adsorbed antigen is disclosed. The
microparticles are useful for enhancing CTL responses to a selected
antigen.
Inventors: |
O'Hagan; Derek; (Winchester,
MA) ; Van Nest; Gary; (El Sobrante, CA) ; Ott;
Gary S.; (Oakland, CA) ; Barackman; John; (San
Leandro, CA) ; Kazzaz; Jina; (San Rafael,
CA) |
Correspondence
Address: |
NOVARTIS VACCINES AND DIAGNOSTICS INC.
INTELLECTUAL PROPERTY- X100B, P.O. BOX 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
NOVARTIS VACCINES AND DIAGNOSTICS,
INC.
EMERYVILLE
CA
|
Family ID: |
26713053 |
Appl. No.: |
12/573903 |
Filed: |
October 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10189104 |
Jul 3, 2002 |
7597908 |
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12573903 |
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09015652 |
Jan 29, 1998 |
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10189104 |
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60036316 |
Jan 30, 1997 |
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60069749 |
Dec 16, 1997 |
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Current U.S.
Class: |
424/489 ;
424/208.1; 424/209.1; 424/227.1; 424/228.1 |
Current CPC
Class: |
A61P 31/16 20180101;
A61K 2039/54 20130101; C12N 2760/16134 20130101; A61K 2039/545
20130101; A61K 39/39 20130101; A61K 39/12 20130101; A61K 39/145
20130101; A61K 2039/55555 20130101; Y02A 50/30 20180101; A61K
39/245 20130101; A61P 37/04 20180101; A61P 31/12 20180101; A61K
9/1647 20130101; C12N 2710/16634 20130101; C12N 2740/16234
20130101; Y02A 50/396 20180101; A61K 39/21 20130101; A61K 9/167
20130101 |
Class at
Publication: |
424/489 ;
424/208.1; 424/228.1; 424/227.1; 424/209.1 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 39/21 20060101 A61K039/21; A61K 39/29 20060101
A61K039/29; A61K 39/145 20060101 A61K039/145 |
Claims
1. A composition comprising an antigen derived from a pathogen
adsorbed to a microparticle that comprises a poly(.alpha.-hydroxy
acid); and a pharmaceutically acceptable excipient.
2. The composition of claim 1, wherein said antigen is derived from
a viral pathogen.
3. The composition of claim 2, wherein the microparticle comprises
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).
4. The composition of claim 3, wherein the microparticle comprises
poly(D,L-lactide-co-glycolide).
5. The composition of claim 2, wherein the antigen comprises HIV
gp120.
6. The composition of claim 2, wherein the antigen comprises HIV
p24gag.
7. The composition of claim 2, wherein the antigen comprises
Influenza A hemagglutinin antigen.
8. The composition of claim 2, wherein said antigen comprises a
hepatitis B viral antigen.
9. The composition of claim 2, wherein said antigen comprises a
hepatitis C viral antigen.
10. The composition of claim 2, wherein said antigen comprises an
influenza A viral antigen.
11. The composition of claim 2, wherein said antigen comprises an
HIV antigen.
12. The composition of claim 2, wherein said composition is an
injectable composition.
13. The composition of claim 2, wherein said composition further
comprises an immunological adjuvant.
14. The composition of claim 13, wherein the immunological adjuvant
comprises a monophosphorylipid A compound.
15. The composition of claim 2, wherein the microparticle has a
diameter between 500 nanometers and 10 microns.
16. The composition of claim 2, wherein the microparticle comprises
a poly(lactide-co-glycolide).
17. The composition of claim 16, wherein said
poly(lactide-co-glycolide) contains 50% D,L-lactide and 50%
glycolide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/189,104, filed Jul. 3, 2002, entitled "Use Of
Microparticles With Adsorbed Antigen To Stimulate Immune
Responses", which is a continuation of U.S. patent application Ser.
No. 09/015,652, filed Jan. 29, 1998, now abandoned, which claims
the benefit of U.S. Provisional Patent Application Ser. Nos.
60/036,316, filed Jan. 30, 1997 and 60/069,749, filed Dec. 16,
1997. Each of the prior applications 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
microparticles with adsorbed antigen for stimulating immunological
responses, as well as to methods for making the microparticles.
BACKGROUND
[0003] Many pharmaceutical compositions include adjuvants in order
to increase activity, antigenic potency and to enhance stability of
the formulation. In this regard, vaccine compositions often include
immunological adjuvants to enhance cell-mediated and humoral immune
responses. For example, depot adjuvants are frequently used which
adsorb) and/or precipitate administered antigens and which serve to
retain the antigen at the injection site. Typical depot adjuvants
include aluminum compounds and water-in-oil emulsions. However,
depot adjuvants, although increasing antigenicity, often provoke
severe persistent local reactions, such as granulomas, abscesses
and scarring, when injected subcutaneously or intramuscularly.
Other adjuvants, such as lipopolysacharrides and muramyl
dipeptides, can elicit pyrogenic responses upon injection and/or
Reiter's symptoms (influenza-like symptoms, generalized joint
discomfort and sometimes anterior uveitis, arthritis and
urethritis).
[0004] 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.
[0005] 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 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.
[0006] 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.
[0007] 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).
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).
[0008] While offering significant advantages over other more toxic
systems, antigen-entrapped PLG microparticles suffer from some
drawbacks. For example, the production of microparticles is
difficult and involves the use of harsh chemicals that can denature
the antigen and destroy the immunogenicity thereof. Furthermore,
antigen instability can occur due to the high shear forces used to
prepare small microparticles and due to interfacial effects within
the emulsions used.
[0009] The use of antigens adsorbed to microparticles avoids these
drawbacks. However, reports on the immunogenicity of microparticles
with adsorbed antigen have been mixed. In fact, experimenters have
postulated that antigens must be entrapped in microparticles in
order to achieve an adequate adjuvant effect. See, e.g., Eldridge
et al., Infect. Immun. (1991) 59:2978-2986; Eldridge et al.,
Seminars in Hematology (1993) 30:16-25; Nakaoka et al., J.
Controlled Release (1995) 37:215-224; Sah et al., J. Controlled
Release (1995) 35:137-144; and Duncan et al.,
"Poly(lactide-co-glycolide Microencapsulation of Vaccines for
Mucosal Immunization" in Mucosal Vaccines (Academic Press, Inc.,
1996).
[0010] More particularly, microparticle-encapsulated and -adsorbed
ovalbumin have been shown to prime cellular immune responses in
vivo and induce mucosal IgA responses when administered orally.
However, entrapped antigen elicited better responses than adsorbed
antigen (O'Hagan et al., Vaccine (1993) 11:149-154). Coombes et
al., Vaccine (1996) 14:1429-1438 also describes experiments using
both ovalbumin-encapsulated and -adsorbed microparticles. Antibody
responses to the adsorbed antigen were significantly lower than
those elicited by administration of entrapped ovalbumin. Finally,
antigen-specific CTL responses have been reported in mice using a
short synthetic peptide from the circumsporozoite protein of
Plasmodium berghei microencapsulated in biodegradable microspheres
or adsorbed on empty microspheres (Men et al., Vaccine (1997)
15:1405-1312).
[0011] However, none of the above studies describe the use of
antigen-adsorbed microparticles, using viral antigens, to stimulate
cell-mediated immune responses. Accordingly, there is a continued
need for effective and safe adjuvants for use in a variety of
pharmaceutical compositions and vaccines.
SUMMARY OF THE INVENTION
[0012] The inventors herein have found, surprisingly, that
adsorbing selected viral antigens to microparticles derived from a
poly(.alpha.-hydroxy acid), provides for superior immune responses.
Accordingly, then, the invention is primarily directed to methods
and compositions which include such microparticles, as well as to
processes for producing the same. The use of microparticles with
adsorbed antigens provides a safe and effective approach for
enhancing the immunogenicity of a wide variety of antigens.
[0013] Accordingly, in one embodiment, the invention is directed to
a composition comprising a selected viral antigen adsorbed to a
poly(.alpha.-hydroxy acid) microparticle and a pharmaceutically
acceptable excipient.
[0014] In an additional embodiment, the invention is directed to a
method of immunization which comprises administering to a
vertebrate subject a therapeutically effective amount of the
microparticle composition above.
[0015] In yet an additional embodiment, the invention is directed
to a method for eliciting a cellular immune response in a
vertebrate subject comprising administering to a vertebrate subject
a therapeutically effective amount of a selected viral antigen
adsorbed to a poly(.alpha.-hydroxy acid) microparticle.
[0016] In yet a further embodiment, the invention is directed to a
method of producing a composition comprising:
[0017] (a) providing a viral antigen;
[0018] (b) adsorbing the viral antigen to a poly(.alpha.-hydroxy
acid) microparticle; and
[0019] (c) combining the microparticle with the adsorbed antigen
with a pharmaceutically acceptable excipient.
[0020] In particularly preferred embodiments, the microparticles
above are formed from poly(D,L-lactide-co-glycolide.
[0021] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0022] 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).
[0023] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0024] 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.
A. 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. 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
adsorbed to a microparticle, 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
antigen when delivered without association with the microparticle.
Thus, a vaccine composition may display "enhanced immunogenicity"
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 immunogenicity can be determined
by administering the microparticle/antigen composition, and 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 antigen/microparticle, as provided herein, refer to a
nontoxic but sufficient amount of the antigen/microparticle 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] By "vertebrate subject" is meant any member of the subphylum
cordata, including, without limitation, mammals such as cattle,
sheep, pigs, goats, horses, and man; 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
"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 formulation 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.
[0035] 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.
[0036] 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).
B. General Methods
[0037] Central to the present invention is the discovery that PLA
and PLG microparticles with adsorbed viral antigens can generate
cell-mediated immune responses in a vertebrate subject. The ability
of the antigen/microparticles of the present invention to elicit a
cell-mediated immune response against a selected antigen provides a
powerful tool against infection by a wide variety of viruses. The
antigen/microparticles of the present invention can be incorporated
into vaccine compositions. Furthermore, the adjuvant formulations
of the invention may be used to enhance the activity of antigens
produced in vivo, i.e., in conjunction with DNA immunization.
[0038] Although the individual components of the vaccine
compositions and methods described herein were known, it was
unexpected and surprising that such combinations would produce
potent cell-mediated immune responses beyond levels achieved when
the components were used separately. 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
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.
[0039] The technique is particularly useful for immunization
against intracellular 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 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).
[0040] 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.
[0041] 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.
[0042] 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, N. Mex.: 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.
[0043] 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.
[0044] It is readily apparent that the subject invention can be
used to mount an immune response to a wide variety of antigens and
hence to treat or prevent a large number of diseases.
[0045] The selected antigen is adsorbed to a microparticle for
subsequent delivery. 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., Birmingham, Ala. 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(hydroxybutyrate), 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 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 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, and most preferably about 50,000 to
about 100,000.
[0046] 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.
[0047] The antigen-containing 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 make the microparticles. 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.
[0048] 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. The polymer solution is 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.
[0049] 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.
[0050] 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.
[0051] 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).
[0052] Following preparation, microparticles can be stored as is or
freeze-dried for further use. In order to adsorb antigen to the
microparticles, the microparticle preparation is simply mixed with
the antigen of interest and the resulting formulation can again be
lyophilized prior to use. Protein content of the microparticles can
be determined using standard techniques.
[0053] A particularly preferred method for adsorbing antigen onto
prepared microparticles is as follows. Microparticles are
rehydrated and dispersed to an essentially monomeric suspension of
microparticles using dialyzable detergents. Useful detergents
include, but are not limited to, any of the various
N-methylglucamides (known as MEGAs), such as
heptanoyl-N-methylglucamide (MEGA-7), octanoyl-N-methylglucamide
(MEGA-8), nonanoyl-N-methylglucamide (MEGA-9), and
decanoyl-N-methylglucamide (MEGA-10); cholic acid; sodium cholate;
deoxycholic acid; sodium deoxycholate; taurocholic acid; sodium
taurocholate; taurodeoxycholic acid; sodium taurodeoxycholate;
3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS);
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate
(CHAPSO); N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate
(ZWITTERGENT 3-12); N,N-bis-(3-D-gluconeamidopropyl)-deoxycholamide
(DEOXY-BIGCHAP); N-octylglucoside; sucrose monolaurate; glycocholic
acid/sodium glycocholate; laurosarcosine (sodium salt);
glycodeoxycholic acid/sodium glycodeoxycholate. The above
detergents are commercially available from e.g., Sigma chemical
Co., St. Louis, Mo. Generally, a ratio of about 0.0156:1 detergent
to microparticle (w:w) will be used, more preferably about 0.625:1,
even more preferably about 0.25:1 and most preferably about 1:1 to
2:1, detergent to microparticle (w:w).
[0054] The microparticle/detergent mixture is then physically
ground, e.g., using a ceramic mortar and pestle, until a smooth
slurry is formed. An appropriate aqueous buffer, such as phosphate
buffered saline (PBS) or Tris buffered saline, is then added and
the resulting mixture sonicated or homogenized until the
microparticles are fully suspended. The antigen of interest is then
added to the microparticle suspension and the system dialyzed to
remove detergent. The polymer microparticles and detergent system
are preferably chosen such that the antigen of interest will adsorb
to the microparticle surface while still maintaining activity of
the antigen. The resulting microparticles containing surface
adsorbed antigens may be washed free of unbound antigen and stored
as a suspension in an appropriate buffer formulation, or
lyophilized with the appropriate excipients, as described further
below.
[0055] Once the antigen/microparticles are produced, they are
formulated into vaccine compositions to treat and/or prevent a wide
variety of viral disorders, as described above. 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,
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, i.e., 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.
[0056] Adjuvants may be used to enhance the effectiveness of the
pharmaceutical compositions. 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 microparticle compositions of the present invention. Such
adjuvants include, but are not limited to: (1) aluminum salts
(alum), such as aluminum hydroxide, aluminum phosphate, aluminum
sulfate, etc.; (2) oil-in-water emulsion formulations (with or
without other specific immunostimulating agents such as muramyl
peptides (see below) or bacterial cell wall components), such as
for example (a) MF59 (International Publication No. WO 90/14837),
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 submicron particles using a
microfluidizer such as Model 110Y microfluidizer (Microfluidics,
Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%
pluronic-blocked polymer L121, and thr-MDP (see below) either
microfluidized into a submicron emulsion or vortexed to generate a
larger particle size emulsion, and (c) Ribi.TM. adjuvant system
(RAS), (Ribi Immunochem, Hamilton, Mont.) 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.TM.) (for a further discussion of suitable submicron
oil-in-water emulsions for use herein, see commonly owned, patent
application attorney docket no. 2300-1397, filed on even date
herewith); (3) saponin adjuvants, such as Stimulon.TM. (Cambridge
Bioscience, Worcester, Mass.) may be used or particle generated
therefrom such as ISCOMs (immunostimulating complexes); (4)
Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant
(IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.),
macrophage colony stimulating factor (M-CSF), tumor necrosis factor
(TNF), etc.; and (6) other substances that act as immunostimulating
agents to enhance the effectiveness of the composition. Alum and
MF59 are preferred.
[0057] 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-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
[0058] The compositions will comprise a "therapeutically effective
amount" of the antigen of interest. That is, an amount of
antigen/microparticle will be included in the compositions which
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.
[0059] Once formulated, the compositions of the invention can be
administered parenterally, e.g., by injection. The compositions can
be injected either subcutaneously, intraperitoneally, intravenously
or intramuscularly. Other modes of administration include oral and
pulmonary administration, suppositories, and transdermal
applications. Dosage treatment may be a single dose schedule or a
multiple dose schedule. A multiple dose schedule is one in which a
primary course of vaccination may be with 1-10 separate doses,
followed by other doses given at subsequent time intervals, chosen
to maintain and/or reinforce the immune response, for example at
1-4 months for a second dose, and if needed, a subsequent dose(s)
after several months. 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.
C. Experimental
[0060] 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.
[0061] 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 Ha-Entrapped Microspheres Using a Solvent
Evaporation Technique
[0062] In a 15 ml glass test tube was placed 0.5 ml 5 mg/ml
Influenza A/Beijing93 hemagglutinin antigen (HA) and 5 ml 6% w:w
PLG (poly D,L-lactide-co-glycolide) in dichloromethane, 50:50 mol
ratio lactide to glycolide, MW average=70-100 kDa, (Medisorb
Technologies International). The solution was homogenized for 2
minutes at high rpm using a hand held homogenizer. The homogenate
was added to 20 ml 8% polyvinyl alcohol (PVA) (12-23 kDa) in a 100
ml glass beaker. This was homogenized for two minutes at a 10,000
rpm using a bench scale homogenizer equipped with a 20 mm diameter
generator. The solution was stirred at room temperature at a
moderate rate using a magnetic stir bar until the solvents were
evaporated. Microspheres were resuspended in water and washed
several times with water, using centrifugation to pellet
microspheres between washes. Microspheres were dried in the
presence of desiccant (Dririte CaSO.sub.4) under vacuum. Mean
volume size was determined to be 0.9 .mu.m by laser diffraction
measurement. Protein content of the microspheres was determined to
be 0.5% w:w by amino acid compositional analysis.
Example 2
Preparation of Ha-Adsorbed Microspheres Using a Solvent Evaporation
Technique
[0063] In a 100 ml glass beaker was placed 10 ml water and 100 ml
4% w:w PLG in dichloromethane, 50:50 mol ratio lactide to
glycolide, MW average=80 kDa (Boehringer Ingelheim). The solution
was homogenized for three minutes at 10,000 rpm using a bench scale
homogenizer equipped with a 35 mm diameter generator. 400 ml 10%
PVA (12-23 kDa) was added while continuing to homogenize for an
additional three minutes. The solution was stirred at room
temperature overnight, at a moderate rate using a magnetic stir
bar, until the dichloromethane evaporated. Microspheres were washed
several times with water using centrifugation to pellet
microspheres between washes and the microspheres freeze-dried. 123
mg of freeze-dried microspheres were added to 2.4 ml 1 mg/ml
Influenza A/Beijing93 HA antigen in a glass vial and freeze-dried
after overnight incubation at 4.degree. C. Mean volume size was
determined to be 0.34 um by laser diffraction measurement. Protein
content was approximately 2% w:w after freeze-drying.
Example 3
Immunogenicity of HA-Entrapped and -Adsorbed Microspheres
[0064] The HA-entrapped and adsorbed microspheres, produced as
described above, were administered to mice and the mice were
boosted after 28 days, as shown in Table 1. A total dose of 4 .mu.g
of HA-adsorbed microparticles was administered. A total dose of
HA-entrapped microparticles was administered. Serum was collected
at day 42 and evaluated for total HIA and total Ig. The results are
shown in Table 1. As can be seen, the HA-adsorbed microparticles
were more immunogenic than the HA-entrapped formulation. 1
TABLE-US-00001 TABLE 1 Serum Anti-HA .mu.g HA Response prime/boost
at Day 42 Animal Group day 0/day 28 Total Ig HIA HA-adsorbed 2/2
7.00E+05 1280 HA-encapsulated 1/4 1.50E+05 160
Example 4
Preparation of Ha-Adsorbed Micro Spheres Using a Spray Drying
Technique
[0065] 2% (w:w) poly(d,l-lactide-co-glycolide) (Medisorb
Technologies, 50:50 mol ratio lactide to glycolide, 70-100 Kdal MW
or equivalent) in dichloromethane was spray dried using a Buchi
mini spray-dryer (model B-191) at an inlet temperature of
67-68.degree. C., an outlet temperature of 55.degree. C., a spray
pressure of 80 PSI, and a spray flow of 800 L/hr. Resulting
microparticles were determined to be 1-5 .mu.m in diameter by light
microscopy examination against size standards.
[0066] 450 mg of the spray dried microparticles and nine ml 10%
MEGA-10 detergent (2:1 w:w ratio MEGA-10 to microparticles) were
placed in a ceramic mortar. The mixture was ground using a ceramic
pestle until a smooth slurry formed. 22.5 ml of phosphate buffered
saline (PBS) were added and the mixture was homogenized three
minutes using a bench scale homogenizer at 25,000 RPM with a 10 mm
diameter generator, until microparticles were fully
resuspended.
[0067] A/Beijing HA bulk antigen, containing 1 mg/ml protein
content as assayed by a bicinchoninic acid (BCA) protein assay
(Pierce, Rockford, Ill.), and approximately 0.2 mg/ml HA activity,
as assayed by single radial immunodiffusion (SRID) was adsorbed to
the microparticles as follows. 6 ml A/Beijing HA bulk antigen was
diluted with 9.6 ml PBS and then added to 8.4 ml of the
microparticle slurry (final composition: 0.25 mg/ml protein, 120 mg
microparticles, 1% w:v MEGA-10, 5% w:w protein:particle ratio). The
mixture was dialyzed extensively using a 50,000 molecular weight
cutoff cellulose dialysis membrane against PBS until MEGA-10 was
removed, as measured by colorimetric assay. The dialysate was
removed from the dialysis bag and centrifuged to pellet
microparticles. Supernatant was removed and discarded and the
microparticles washed with two changes of PBS, with centrifuging
between washes. 30 ml PBS were used per wash. Protein load was
measured by standard methods, using BCA at approximately 1.4%
protein content by weight microparticles.
Example 5
Immunogenicity of HA-Adsorbed Microspheres Produced by Spray
Drying
[0068] In order to test the immunogenicity of the microparticles
produced in Example 4, groups of Balb/C mice (n=10) were immunized
intramuscularly according to the schedule shown in Table 2. Priming
and boosting were performed one month apart. Dosing was done with
A/Beijing antigen based on HA activity (SRID) either as a soluble
antigen in PBS alone, or surface adsorbed to microparticles. Serum
samples were taken two weeks and four weeks post boost immunization
and assayed for A/Beijing specific total Ig titers by a
calorimetric based ELISA. Serum samples were further evaluated for
hemagglutination inhibition activity (HI). Results of the ELISA and
HI assays are summarized in Table 2. As indicated, intramuscular
immunization with HA-adsorbed microparticles resulted in equivalent
or measurably higher 1 g and HI titers than immunization with HA
alone.
[0069] A/Beijing HA encapsulated into PLG microparticles using a
standard microencapsulation technique were shown to elicit poor HI
responses after intramuscular administration indicating that
denaturation of HA occurred during the encapsulation process.
Therefore, presentation of antigen on the surface of microparticles
presents advantages over microencapsulation of the antigen and
surprisingly, shows an adjuvant effect. 2
TABLE-US-00002 TABLE 2 Immunization Serum Titers Schedule Two Weeks
Four Weeks Group Prime Boost Post Boost Post Boost # (Day 0) (Day
28) Total Ig HI Total Ig HI 1 14 .mu.g HA 52,000 150 207,000 160 2
14 .mu.g HA 236,000 320 415,000 320 (HA .mu.- particles) 3 1 .mu.g
14 .mu.g HA 1,160,000 1,280 911,000 1,280 HA 4 1 .mu.g 14 .mu.g HA
1,310,000 2,560 1,360,000 1,280 HA (HA .mu.- particles)
Example 6
Preparation of PLG-Entrapped HSVgD2 Microspheres
[0070] HSVgD2-entrapped PLG microparticles were prepared by a
solvent evaporation technique, generally as described above.
Briefly, the microparticles with a 1% w/w antigen loading level
were prepared by adding 2 ml of antigen solution and emulsifying at
high speed using a silverson homogenizer, with 10 ml of a 5% w/v
PLG polymer solution in methylene chloride. The primary emulsion
was then added to 50 ml of distilled water containing PVA (10%
w/v). This resulted in the formation of a w/o/w emulsion which was
again homogenized at high speed for 4 minutes. The resulting
emulsion was stirred at 1000 rpm for 12 hours at room temperature
and the methylene chloride was allowed to evaporate. The
microparticles were filtered, washed twice in distilled water and
lyophilized.
Example 7
Preparation of PLG-Adsorbed HSVgD2 Microspheres
[0071] Blank microparticles were prepared by a solvent evaporation
technique. Briefly, the microparticles with a 0% w/w protein
loading level (Blank or Placebo) were prepared by adding 2 ml of
normal saline solution and emulsifying at high speed using a
silverson homogenizer, with 10 ml of a 10% w/v PLG polymer solution
in methylene chloride. The primary emulsion was then added to 50 ml
of distilled water containing polyvinyl alcohol (10% w/v). This
resulted in the formation of a w/o/w emulsion which was stirred
again at high speed for 4 minutes. The resulting emulsion was
stirred at 1000 rpm for 12 hours at room temperature and the
methylene chloride was allowed to evaporate. The microparticles
were filtered, washed twice in distilled water and lyophilized. The
Blank PLG Microparticles were added to a HSVgD2 Protein solution
and mixed well by shaking the suspension on a test tube shaker at
room temperature for two hours. The suspension was then frozen at
-80 C. The frozen suspension was lyophilized for use as an
associated HSVgD2 formulation.
Example 8
Immunogenicity of HSVgD2-Entrapped and Adsorbed Microspheres
[0072] The HSVgD2-entrapped and adsorbed microspheres, produced as
described above, were intramuscularly administered to mice and the
mice were boosted after 28 days. A total dose of 10 .mu.g of the
microparticles was administered. Serum was collected at 4 and 8
weeks and IgG and neutralization titers evaluated. The results are
shown in Table 3. As can be seen, HSVgD2 adsorbed with
microparticles gave higher neutralization titers than the
HSVgD2-entrapped microparticles. 3
TABLE-US-00003 TABLE 3 4 weeks post 2 8 weeks post 2 Neutralization
Ratio IgG Neutralization Ratio Formulation IgG titers Titers
Neut./IgG Titers Titers Neut./igG HSVgD2 1 5.4 .times. 10.sup.-5 58
1.04 .times. 10.sup.-4 2.26 .times. 10.sup.5 68 3.01 .times.
10.sup.-4 .nu.m entrapped HSVgD2 8.54 .times. 10.sup.5 192 2.26
.times. 10.sup.-4 2.23 .times. 10.sup.5 136 6.10 .times. 10.sup.-4
400 nm adsorbed
Example 9
Preparation of Gag-Adsorbed and Entrapped Microspheres
[0073] Solutions used to make Gag-adsorbed 0.4 .mu.m microparticle
formulations were as follows:
(1) 4% RG 503 PLG (Boehringer Ingelheim) in dimethyl chloride. (2)
10% PVA (ICN) in water.
(3) PBS
[0074] In particular, the internal emulsion was made by adding 1.25
ml of PBS to 12.5 ml of polymer solution and homogenizing for 2.5
minutes at 23 k, using a hand-held IKA homogenizer with a small
probe. The second emulsion was made by adding the internal emulsion
to 50 ml of the PVA solution and homogenizing for 3 minutes using a
benchtop homogenizer with a 20 mm probe at 10 K rpm. The emulsion
was left stirring overnight for solvent evaporation. The formed
microspheres were then filtered through a 38.mu. mesh, sized in the
Malvern Master sizer, then washed with water by centrifugation 3
times, and lyophilized.
[0075] P24 gag was adsorbed to the microspheres as follows.
A. 5% Adsorbed Microspheres
[0076] 200 mg of the lyophilized placebo microspheres were
incubated with rocking overnight at room temperature, with 80 ml
0.25 mg/ml P24 gag protein in PBS. The next day, the microspheres
were centrifuged and the supernatant assayed by BCA for gag
concentration to determine the amount adsorbed. The microspheres
were washed once with PBS and lyophilized. The lyophilized
microspheres were incubated with another 40 ml 0.25 mg/ml P24 gag
in PBS with rocking at room temperature overnight. Microspheres
were centrifuged the next day and the supernatant was assayed for
protein by BCA. The microspheres are washed once with PBS and
lyophilized. The lyophilized microspheres were analyzed for total
protein adsorbed by base hydrolysis.
B. 1% Adsorbed Microspheres
[0077] 100 mg 0.4 .mu.m placebo microspheres were incubated by
rocking at room temperature overnight with 10 ml 0.2 mg/ml P24 gag
in PBS. The next day the microspheres were centrifuged and the
supernatant assayed for protein by BCA. The microspheres were
washed once with PBS, lyophilized, then assayed for adsorbed
protein by base hydrolysis.
Example 10
Immunogenicity of Gag-Adsorbed Microspheres
[0078] The gag-adsorbed microspheres, produced as described in
Example 9, as well as gag-encapsulated microspheres and blank
microspheres as controls, were administered to mice, as described
above, and CTL activity assayed two weeks following the final
immunization. As shown in Tables 4 and 5, microparticles with
surface presented gag (1%) induced CTL activity, while the same
amount of gag-encapsulated in biodegradable particles did not. 5%
surface-adsorbed gag was also better than incorporated protein for
induction of CTL activity.
TABLE-US-00004 TABLE 4 Percent specific Lysis of targets Deb-assay
1 Effector E:T Ratio SV/O SV/p7g MC/p7g PLG 60:1 3 23 1 Surface 1%
12:1 2 10 2 2.4:1 1 3 1 PLG 60:1 0 1 -1 encapsulated 1% 12:1 0 1 1
2.4:1 0 1 -1 gag alone 60:1 0 6 1 12:1 1 5 1 2.4:1 1 2 2 Vaccinia
gag 60:1 2 27 0 12:1 1 10 2 2.4:1 1 4 2
TABLE-US-00005 TABLE 5 Percent specific Lysis of targets Deb-assay
1 Effector E:T Ratio SV/O SV/p7g MC/p7g PLG 60:1 3 32 2 Surface 5%
12:1 2 13 0 2.4:1 1 5 1 PLG 60:1 13 18 12 encapsulated 5% 12:1 5 8
4 2.4:1 1 2 0 gag alone 60:1 5 9 4 12:1 2 4 3 2.4:1 2 1 3 Vaccinia
gag 60:1 9 32 11 12:1 1 14 4 2.4:1 0 6 1
[0079] Thus, the use of antigen-adsorbed microparticles to
stimulate cell-mediated immunological responses, as well as methods
of making the microparticles, are 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.
* * * * *