U.S. patent application number 12/144130 was filed with the patent office on 2008-10-16 for method for inducing a cell-mediated immune response and improved parenteral vaccine formulations thereof.
This patent application is currently assigned to MERRION RESEARCH III LIMITED. Invention is credited to David J. Brayden.
Application Number | 20080254134 12/144130 |
Document ID | / |
Family ID | 36758535 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080254134 |
Kind Code |
A1 |
Brayden; David J. |
October 16, 2008 |
METHOD FOR INDUCING A CELL-MEDIATED IMMUNE RESPONSE AND IMPROVED
PARENTERAL VACCINE FORMULATIONS THEREOF
Abstract
A method of inducing either a T.sub.H1 polarised immune
response, a T.sub.H2 polarised immune response or a combined
T.sub.H1 and T.sub.H2 response to an antigen and associated vaccine
formulations are disclosed. A method is provided for inducing a
polarised T.sub.H1 response by parenteral administration of
microparticles sized such that at least 50% of the microparticles
are less than 5 .mu.m, the microparticles containing antigen
entrapped or encapsulated by a biodegradable polymer. Additionally,
a method is provided for inducing a polarised T.sub.H2 response by
parenteral administration of nanoparticles sized such that at least
50% of the nanoparticles are less than 600 nm, the nanoparticles
containing antigen entrapped or encapsulated by a biodegradable
polymer. Vaccine formulations containing the B. pertussis antigens
PTd, FHA or a combination of PTd and FHA are provided.
Inventors: |
Brayden; David J.; (Co.
Dublin, IE) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Assignee: |
MERRION RESEARCH III
LIMITED
|
Family ID: |
36758535 |
Appl. No.: |
12/144130 |
Filed: |
June 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11463194 |
Aug 8, 2006 |
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12144130 |
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09386266 |
Aug 31, 1999 |
7087236 |
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11463194 |
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60098760 |
Sep 1, 1998 |
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Current U.S.
Class: |
424/501 ;
424/184.1; 424/254.1; 424/489; 977/917 |
Current CPC
Class: |
A61K 9/0024 20130101;
A61K 2039/57 20130101; A61K 2039/55555 20130101; Y10S 530/825
20130101; A61K 9/0019 20130101; A61K 9/1647 20130101; A61K 39/099
20130101 |
Class at
Publication: |
424/501 ;
424/489; 424/184.1; 424/254.1; 977/917 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 39/00 20060101 A61K039/00; A61K 39/10 20060101
A61K039/10 |
Claims
1. A method of inducing a T.sub.H2 polarised immune response to an
antigen, comprising parenterally administering to a subject
nanoparticles sized such that at least 50% of the nanoparticles are
less than 600 nm, the nanoparticles comprising the antigen
entrapped or encapsulated by a biodegradable polymer.
2. The method of claim 1, wherein the nanoparticles are sized such
that at least 50% of the nanoparticles are less than 500 nm.
3. The method of claim 1, wherein the biodegradable polymer
comprises a copolymer of lactic acid and glycolic acid or
enantiomers thereof.
4. The method of claim 1, wherein the nanoparticles are formed
using a coacervation method.
5. The method of claim 1, wherein the antigen comprises a B.
pertussis antigen.
6. The method of claim 1, wherein the parenteral administration is
selected from the group consisting of intraperitoneal
administration, subcutaneous administration and intramuscular
administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/463,194, which is a divisional of U.S.
patent application Ser. No. 09/386,266, filed Aug. 31, 1999, now
U.S. Pat. No. 7,087,236, which claims priority from U.S.
Provisional Patent Application Ser. No. 60/098,760 filed Sep. 1,
1998, the disclosures of all of which are to be incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to vaccine formulations and to
methods for inducing an immune response that is polarised in favour
of either a cell-mediated T.sub.H1 immune response, a humoral
T.sub.H2 immune response or a combined T.sub.H1 and T.sub.H2
response. In particular, the present invention relates to
parenteral microparticulate and nanoparticulate vaccine
formulations comprising antigens entrapped or encapsulated within
polymer particles.
DESCRIPTION OF THE PRIOR ART
[0003] Controlled release antigen delivery systems have attracted
considerable interest in the continuing search for vaccine
carriers. The effectiveness of polymer matrices in the sustained
release of antigen was first demonstrated in 1979 with the
entrapment of bovine serum albumin in a non-degradable
ethylene-vinyl acetate copolymer pellet for subcutaneous
implantation [Preis et al, J. Immunol. Methods 28, 193-197 (1979)].
This composition induced an antibody response for six months after
administration and gave antibody levels similar to two injections
of the same total amount of antigen in complete Freund's
adjuvant.
[0004] More recently aluminium salts (see for example WO 94/15636
(CSL Ltd.)) and biodegradable polymers such as poly (L-lactide)
(hereinafter PLA) and poly (DL-lactide-co-glycolide) (hereafter
PLGA) have been used as carriers for vaccine antigens. WO 95/11008
(Genentech Inc.) discloses the use of PLGA microspheres for
encapsulating an antigen in which the ratio of lactide to glycolide
in the microspheres ranges from 100:0 to 0:100 weight percent, the
inherent viscosity of the PLGA polymers ranges from 0.1-1.2 dl/g
and the median diameter of the microspheres ranges from 20-100
.mu.M. The antigen can be continuously released from the
microspheres over an extended period in a triphasic pattern. A
method for encapsulating antigens in microspheres is also
disclosed.
[0005] Eldridge et al., J. Controlled Release 11, 205-214 (1990)
report that the oral administration of biodegradable PLA or PLGA
microspheres containing the staphylococcal enterotoxin B (SEB)
vaccine are absorbed into the Peyer's patches of the small
intestine. Uptake is restricted to particles .ltoreq.10 .mu.m in
diameter. The majority of microspheres <5 .mu.m were observed to
be transported to systemic lymphoid tissue (such as the spleen)
where the released antigen stimulated a serum antibody response.
The majority of those particles >5 .mu.m were found to be
retained in the Peyer's patches. EP 0 266 119 (The UAB Research
Foundation & Southern Research Institute) teaches an oral
composition comprising a bioactive agent, such as an antigen,
encapsulated in a biodegradable polymer excipient to form a
microcapsule less than or equal to 10 .mu.m that is capable of
being taken up selectively by the Peyer's patch. Similarly, EP 0
333 523 (The UAB Research Foundation & Southern Research
Institute) and EP 0 706 792 divided therefrom, teach compositions
for delivery of a bioactive agent, such as an antigen, to the
mucosally associated lymph tissue (MALT), comprising microcapsules
having sizes between 1-5 and 5-10 .mu.m for selective absorption
and retention in MALT.
[0006] EP 0 686 030 (Gesellschaft zur Forderung der
Industrieorientierten Forschung) teaches a method of potentiating
an immune response by embedding a model antigen in a biodegradable
biopolymer and injecting it in the form of a dispersion in order to
trigger a humoral and cellular response. In this instance PLGA
entrapped antigens were shown to elicit long lasting T helper,
antibody and cytotoxic T cell responses.
[0007] Moore et al., Vaccine 18 1741-1749 (1995) discloses that HIV
gp120 entrapped in PLGA solvent evaporated microspheres can induce
cytolytic activity (CTL) in mice splenic T cells upon nasal, s.c.
or i.p. administration. For this antigen, anti-HIV specific CD4+
and CD8+ T cells were induced leading to induction of T.sub.H1
cells and CTL respectively. In the case of i.p. ovalbumen (OVA)
immunisation, Maloy et al., Immunology 81, 661-667 (1994) disclose
that a single s.c. immunization with OVA-PLGA microspheres primed
significant OVA-specific responses and strong OVA-specific CTL
responses were found after i.p immunisation in mice. Newman et al.,
J. Controlled Release, 54, 49-59 (1998) disclose the use of OVA
peptide encapsulated in PLGA microspheres for inducing a T.sub.H1
type immune response in mice after s.c. delivery.
[0008] Distinction between the types of immune response in terms of
T.sub.H1 (cell-mediated) and T.sub.H2 (humoral/antibody) type
responses is important for protection against infectious diseases
induced by intracellular pathogens or extracellular toxins
respectively. The division of CD4+ lymphocytes into T.sub.H1 and
T.sub.H2 according to antibody sub-class and cytokine profile has
led to attempts to classify adjuvants accordingly. For instance,
aluminium hydroxide (also referred to as alum) is considered to
have a higher capacity for inducing T.sub.H2 rather than T.sub.H1
immunity [see, e.g., Men et al., Vaccine 13, 683-689 (1995)]. U.S.
Pat. No. 5,417,986 (US Army) describes the loading of PLGA
microspheres with antigens such as CFA (complete Freund's adjuvant)
and HepB sAg (hepatitis B surface antigen) and injected to give
both antibodies and T cell proliferation in animals.
[0009] Review of the above cited references and other literature in
the area shows that there is no general method for predicting or
anticipating the nature of the immune response induced by an
antigen in combination with a given adjuvant.
[0010] With respect to Bordetella pertussis, the fimbrae,
filmentous hemaglutinin (FHA), inactivated pertussis toxin (PTd)
and pertactin antigens have all been entrapped in PLGA
microspheres, administered individually by a variety of routes and
have been shown to protect against infection in response to
challenge in a mouse model of pertussis (see, e.g., Shahin et al.,
Infection and Immunity 63, 1195-1200 (1995); Jones et al.,
Infection and Immunity 64, 489-494 (1996); Cahill et al., Vaccine
13,455-462 (1995)). WO 93/21950 (Roberts and Dougan) teaches that
the antigens FHA and pertactin are immunogenic as a mixture or when
entrapped in PLGA and delivered to mucosal sites. Singh et al.,
Vaccine 16, 346-352 (1998) describe that two antigens entrapped
simultaneously in the same polymer particles can induce antibody
responses to each agent in rats after parenteral delivery. There is
evidence that the mouse model of aerolised pertussis infection
correlates with pertussis vaccine efficacy in children [Mills et
al., Infection and Immunity 66, 594-602 (1998)] and that T.sub.H1
cells play an important role in bacterial clearance [Mills et al.,
Infection and Immunity 61, 399-410 (1993)]. Further work by Ryan et
al., Immunology 93, 1-10 (1998) indicates that the long-term
protective immunity of a potent whole cell pertussis vaccine in
children is largely mediated by T.sub.H1 cells. A cellular
pertussis vaccines appear to involve a mixed population of T.sub.H1
and T.sub.H2 cells and their long term efficacy is unknown.
[0011] Despite the abovementioned prior art, the ability to predict
and control the type of immune response produced by a given vaccine
formulation remains a goal central to immunology research. This is
particularly true given the variation from disease to disease of
the relative importance of T.sub.H1 and T.sub.H2 components of the
immune response. For example, T.sub.H1 response can assist in
cytotoxic T cell activity which is important in clearances of
viruses, intracellular pathogens and some cancers.
[0012] Therefore, it is an object of the present invention to
provide a vaccine formulation which will elicit a significant and
reproducible polarised T.sub.H1 immune response in vivo. It is also
an object of this invention to provide methods to enhance the
T.sub.H1 T cell response compared to the T.sub.H2 response.
Additionally, it is an object of the present invention to provide a
vaccine formulation which will elicit a significant and
reproducible polarised T.sub.H2 immune response in vivo and methods
to enhance the T.sub.H2 T cell response compared to the T.sub.H1
response.
[0013] It is an another object of the present invention to provide
a parenteral vaccine formulation directed at a particular agent
such as an infectious agent which, after administration to the
subject, is capable of providing protective immunity against the
agent.
[0014] Further objects of the present invention include an improved
composition for use in the preparation of a B. pertussis vaccine
and a method for the vaccination against B. pertussis.
SUMMARY OF THE INVENTION
[0015] It has now been surprisingly found that polarisation of the
T.sub.H1 immune response over the T.sub.H2 immune response or that
polarisation of the T.sub.H2 immune response over the T.sub.H1
immune response can be induced by the choice of parenteral
administration of microparticles or nanoparticles comprising
antigen entrapped or encapsulated in a biodegradable polymer using
a suitable combination of polymer type, loading method, morphology
and size.
[0016] Furthermore, it has been found that a vaccine formulation
designed for a particular agent such as an infectious agent and
containing microparticles or nanoparticles comprising antigen
entrapped or encapsulated in a biodegradable polymer can, in
addition to inducing T cell proliferation, yield protective
immunity against the infectious agent.
[0017] Accordingly, the present invention provides a method of
inducing a T.sub.H1 polarised immune response to an antigen(s),
comprising parenterally administering to a subject, such as a
mammal and preferably a human, microparticles sized such that at
least 50% of the microparticles are less than 5 .mu.m, preferably
less than 3 .mu.m, the microparticles comprising the antigen(s)
entrapped or encapsulated by a biodegradable polymer. A vaccine
formulation for parenteral administration comprising microparticles
sized such that at least 50% of the microparticles are less than 5
.mu.m, preferably less than 3 .mu.m, the microparticles comprising
antigen entrapped or encapsulated by a biodegradable polymer is
also provided.
[0018] Additionally, the present invention provides a method of
inducing a T.sub.H2 polarised immune response to an antigen(s),
comprising parenterally administering to a subject, such as a
mammal and preferably a human, nanoparticles such that at least 50%
of the nanoparticles are less than 600 nm, preferably less than 500
nm, the nanoparticles comprising the antigen(s) entrapped or
encapsulated by a biodegradable polymer. A vaccine formulation for
parenteral administration comprising microparticles sized such that
at least 50% of the nanoparticles are less than 600 nm, preferably
less than 500 nm, the nanoparticles comprising antigen entrapped or
encapsulated by a biodegradable polymer is also provided.
[0019] The present invention also provides a method of inducing
both a potent T.sub.H1 and T.sub.H2 immune response to an
antigen(s), comprising parenterally administering to a subject,
such as a mammal and preferably a human, (1) microparticles sized
such that at least 50% of the microparticles are less than 5 .mu.m,
preferably less than 3 .mu.m, the microparticles comprising the
antigen(s) entrapped or encapsulated by a biodegradable polymer in
combination with (2) antigen(s) presented so as to produce an
immune response polarised in favor of a T.sub.H2 response. To
produce an immune response polarised in favor of a T.sub.H2
response, the antigen(s) can be presented as nanoparticles sized
such that at least 50% of the nanoparticles are less than 600 nm,
preferably less than 500 nm, the nanoparticles comprising the
antigen(s) entrapped or encapsulated by a biodegradable polymer; as
soluble antigen(s); and/or as antigen(s) adsorbed or presented at
least in part on the surface of a particle. The administration of
the antigen-containing microparticles in combination with the
antigen presented so as to produce an immune response polarised in
favor of a T.sub.H2 response can be simultaneous, separate, or
sequential. A vaccine formulation for parenteral administration
comprising antigen entrapped or encapsulated microparticles in
combination with antigen presented so as to produce an immune
response polarised in favor of a T.sub.H2 response such as antigen
entrapped or encapsulated nanoparticles is also provided.
[0020] The present invention also provides a method of providing
protective immunity against B. pertussis, comprising parenterally
administering to a subject microparticles sized such that at least
50% of the microparticles are less than 5 .mu.m, preferably less
than 3 .mu.m, the microparticles comprising at least one B.
pertussis antigen entrapped or encapsulated by a biodegradable
polymer. The present invention also provides a method of providing
protective immunity against B. pertussis, comprising parenterally
administering to a subject nanoparticles sized such that at least
50% of the nanoparticles are less the 600 nm, preferably less than
500 nm, the nanoparticles comprising at least one B. pertussis
antigen entrapped or encapsulated by a biodegradable polymer.
Additionally, the present invention provides a method of providing
protective immunity against B. pertussis, comprising parenterally
administering to a subject microparticles sized such that at least
50% of the microparticles are less than 5 .mu.m, preferably less
than 3 .mu.m, the microparticles comprising at least one B.
pertussis antigen entrapped or encapsulated by a biodegradable
polymer in combination with at least one B. pertussis antigen
presented so as to produce an immune response polarised in favor of
a T.sub.H2 response, such as at least one B. pertussis antigen
presented as nanoparticles sized such that at least 50% of the
nanoparticles are less than 600 nm, preferably less than 500 nm,
the nanoparticles comprising the at least one B. pertussis antigen
entrapped or encapsulated by a biodegradable polymer; as soluble B.
pertussis antigen; and/or as B. pertussis antigen absorbed or
presented at least in part on the surface of a particle.
[0021] Preferably, the antigen is capable of eliciting an immune
response upon administration, the antigen being entrapped and/or
encapsulated within a biocompatible, biodegradable polymer carrier
material. Routes for parenteral administration include
intraperitoneal (i.p.), subcutaneous (s.c.) and intramuscular
(i.m.) routes of administration. Preferably, the method for
entrapping and/or encapsulating the antigen within the polymer
carrier material is a solvent evaporation based process for
formation of antigen entrapped or encapsulated in microparticles or
a coacervation based process for formation of antigen entrapped or
encapsulated in nanoparticles.
[0022] The present invention further relates to a method for the
prevention of B. pertussis which method comprises eliciting a
T.sub.H1 immune response by the administration of a composition
comprising inactivated B. pertussis toxin and/or FHA encapsulated
in poly (DL-lactide-co-glycolide) microparticles, wherein
encapsulation of the inactivated B. pertussis toxin and/or FHA in
poly (DL-lactide-co-glycolide) particles is carried out by solvent
evaporation and wherein administration is by way of parenteral
injection.
[0023] Additionally, the present invention further relates to a
method for the prevention of B. pertussis which method comprises
eliciting a T.sub.H2 immune response by the administration of a
composition comprising inactivated B. pertussis toxin and/or FHA
encapsulated in poly (DL-lactide-co-glycolide) nanoparticles,
wherein encapsulation of the inactivated B. pertussis toxin and/or
FHA in poly (DL-lactide-co-glycolide) particles is carried out by
coacervation and wherein administration is by way of parenteral
injection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the T.sub.H1/T.sub.H2 responses following
parenteral immunisation with KLH entrapped in PLGA. Four groups of
4 mice received i.p. inoculations with 5.0 .mu.g of KLH
encapsulated in PLGA microparticles (KLH-PLGA), adsorbed to alum
(KLH-alum) or in solution combined with empty PLGA microparticles
(KLH+PLGA). Mice were immunised twice (0 and 4 weeks) and
sacrificed two weeks later. Spleen cells from individual mice were
stimulated with 0.03-20 .mu.g/ml of KLH or with medium alone. After
3 days culture supernatants were tested for IL-5 and IFN-.gamma. by
specific immunoassays. Each bar represents the mean response for 4
mice in each group. Note the difference in the scale for IL-5
(pg/ml) and IFN-.gamma. (ng/ml);
[0025] FIG. 2 shows the T.sub.H1/T.sub.H2 responses following
parenteral immunisation with PTd-PLGA microparticles (batch PTd-1
of Example 2) prepared by solvent evaporation. Three groups of mice
received a single dose of 5 .mu.g PTd-PLGA, PTd with alum or in
solution in PBS. The levels of IFN-.gamma. and IL-5 were determined
by specific immunoassays in cultured spleen cells three days after
stimulation with PT. Medium=negative control; iPT=inactivated PT; B
pertussis=active pertussis bacteria and anti-CD3/PMA=the positive
control anti-CD3 antibody/phorbol 12-myristate-13 acetate;
[0026] FIG. 3 shows a plot of Log.sub.10 CFU counts per lung versus
Days after challenge for the control group (immunised with empty
PLGA microparticles), PTd+FHA+alum group (immunised with 5 .mu.g
each of PTd and FHA adsorbed onto alum) and PTd+FHA in PLG group
(immunised with 5 .mu.g each of PTd and FHA entrapped in PLGA
microparticles according to Examples 2 and 3) for the challenge
study in balb/c mice described in Example 7;
[0027] FIG. 4 shows the serum antibody titres to PTd following i.p.
administration as described in Example 7 of PTd+FHA in PLGA (5
.mu.g each of PTd and FHA entrapped in PLGA microparticles
according to Example 2 and 3); PTd+FHA+alum (5 .mu.g each of PTd
and FHA adsorbed onto alum; and PLGA (i.p.) (empty PLGA
microparticles) to balb/c mice;
[0028] FIG. 5 compares the T.sub.H1/T.sub.H2 responses following
parenteral immunisation of balb/c mice with PTd+FHA in PLGA (5
.mu.g each of PTd and FHA entrapped in PLGA microparticles
according to Example 2 and 3; 4 animals) and PTd+FHA+alum (5 .mu.g
each of PTd and FHA adsorbed onto alum; 4 animals). The levels of
IFN-.gamma. and IL-5 were determined by specific immunoassays in
cultured spleen cells three days after stimulation with PT.
iPT=inactivated PT; FHA=filamentous haemagglutinin; B
pertussis=active pertussis bacteria and anti-CD3/PMA=the positive
control anti-CD3 antibody/phorbol 12-myristate-13 acetate;
[0029] FIG. 6 shows the T.sub.H1/T.sub.H2 responses following i.p.
administration of low dose (1 .mu.g) FHA encapsulated in PLGA.
Spleen cells from individual mice were stimulated with medium alone
(0), inactivated PT (PT), filamentous haemagglutinin (FHA), active
pertussis bacteria (BP) and the positive control anti-CD3
antibody/phorbol 12-myristate-13 acetate (PMA/CD3);
[0030] FIG. 7 shows a plot of Log.sub.10 CFU counts per lung versus
Days after challenge for the control group (immunised with empty
PLGA microparticles), PTd-PLG group (immunised with 1 .mu.g of PTd
entrapped in PLGA microparticles), PTd/FHA-alum group (immunised
with 1 .mu.g each of PTd and FHA adsorbed onto alum), FHA-PLG
(immunised with 1 .mu.g of FHA entrapped in PLGA microparticles)
and PTd/FHA-PLG group (immunised with 1 .mu.g each of PTd and FHA
entrapped in PLGA microparticles) for the low dose challenge study
in balb/c mice described in Example 8;
[0031] FIG. 8 shows a plot of Log.sub.10 CFU counts per lung versus
days post challenge for the control group (immunised with empty
PLGA microparticles), PTd/FHA-alum group (immunised with 5 .mu.g
each of PTd and FHA adsorbed onto alum), and PTd/FHA-PLG group
(immunised with 5 .mu.g each of PTd and FHA entrapped in PLGA
microparticles) for the delayed challenge study in balb/c mice
described in Example 9;
[0032] FIG. 9 shows the T.sub.H1 response (IFN-.gamma.) and the
T.sub.H2 response (IL-5) following i.m. immunisation with Treatment
F of Example 10. The levels of IFN-.gamma. and IL-5 were determined
by specific immunoassays in cultured spleen cells from 5 animals
(Mouse 1 through Mouse 5) three days after stimulation with PT.
BG=negative control; PT=inactivated PT; B. pert=active pertussis
bacteria and PMA/a-CD3=the positive control anti-CD3
antibody/phorbol 12-myristate-13 acetate; and
[0033] FIG. 10 shows the T.sub.H1/T.sub.H2 responses following
parenteral immunisation with coacervated nanoparticulate Treatments
A-F of Example 11. The levels of IFN-.gamma. and IL-5 were
determined by specific immunoassays in cultured spleen cells three
days after stimulation with PT. iPT-1=inactivated PT (1.0
.mu.g/ml); iPT-5=inactivated PT (5.0 .mu.g/ml); FHA-1=FHA (1.0
.mu.g/ml); FHA-5=FHA (5.0 .mu.g/ml); BP=active pertussis bacteria
and PMA/CD3=the positive control anti-CD3 antibody/phorbol
12-myristate-13 acetate.
DETAILED DESCRIPTION OF THE INVENTION
[0034] While vaccine formulations which comprise antigens loaded
onto polymer particles are known in the prior art it has now been
found that the choice of biocompatible carrier material, the method
of loading of the biologically active agent (i.e., the method for
adsorbing and/or encapsulating the biologically active agent onto
and/or within the biocompatible, biodegradable polymer material),
the size of the particles and/or the route of administration are
all contributing factors in determining the nature of the immune
response produced. By a suitable combination of the above listed
determinants a composition may be prepared which elicits a
particular polarised immune response. Polarisation of the immune
response may be characterised by determination of the relative
proportions of T.sub.H1 and T.sub.H2 indicators, typically
cytokines such as IFN-.gamma., TNF, IL-2 or IL-12 and IL-5, IL-4,
IL-6 or IL-10 specific to T.sub.H1 and T.sub.H2 responses,
respectively.
[0035] It has now been found that polarisation of the T.sub.H1
immune response over the T.sub.H2 immune response can be induced by
parenteral administration of appropriately sized antigen-loaded
microparticles and that polarisation of the T.sub.H2 immune
response over the T.sub.H1 immune response can be induced by
parenteral administration of appropriately sized antigen-loaded
nanoparticles. While not wishing to be limited by any theory of the
mechanism behind this differentiation, it is possible that the
different polarization obtained from administration of antigen
entrapped microparticles and antigen entrapped nanoparticles may
relate to the physical positioning of the antigen. For instance,
some antigen may be presented on the exterior of the nanoparticles
compared to relatively more antigen entrapped within the
microparticles. Support of a relationship between T.sub.H2
polarisation and externally associated antigen can be found from
data showing a T.sub.H2 polarisation following presentation of
soluble antigen, such as that presented in Example 5 below
(administration of soluble antigen in combination with empty
microparticles).
[0036] Preferable routes for parenteral administration include i.p,
i.m., and s.c., most preferably i.p. and i.m.
[0037] Biologically active agents suitable for the practice of the
present invention are typically antigens capable of eliciting a
polarised T.sub.H1 immune response (e.g., viral antigens, cancer
antigens, allergens etc.), a polarized T.sub.H2 response (e.g.,
toxoid antigens, parasite antigens etc.) or a mixed
T.sub.H1/T.sub.H2 response upon administration. Preferred antigens
include those selected from the list comprising of PTd, inactivated
pertussis toxin or pertactin; FHA, filamentous hemaglutinin; TT,
tetanus toxoid; HIV gp-120; hepatitis B surface antigen; DT,
diptheria toxoid; HSV, herpes simplex type 1; HPV, human papilloma
virus; polio; influenza epitopes; H. pylori; shigella; chlorea;
salmonella; rotavirus; RSV, respiratory virus; yellow fever;
hepatitis A and C; meningoccoccal types A-C; pneumococcal;
parasites such as leischmania; mycobacteria such as tuberculosis;
and cancer vaccine antigens.
[0038] As used herein, the term "protective immunity" refers to at
least 75% clearance, more preferably 90% clearance of the
challenging agent, such as an infectious agent, from the subject
preferably within 2 weeks after the introduction of the challenging
agent, more preferably within 1 week, most preferably within 3
days.
[0039] As used herein, the term "pharmaceutically effective amount"
refers to the amount of antigen required to elicit a protective
immunity response to that antigen. For instance, in mice,
protective immunity is achieved with an amount of a B. pertussis
antigen(s) in the 1-5 .mu.g range for each antigen given
parenterally in multiple doses, such as two doses, or in a single
dose.
[0040] As used herein, references to the sizes of microparticles
and/or nanoparticles refer to sizes as determined by visual
assessment of scanning electron micrographs and/or, where
indicated, laser light diffractometry.
[0041] It has been found that the choice of biocompatible,
biodegradable polymer material used as a carrier for entrapping or
encapsulating the antigen, the size of the resulting particles
and/or the method of loading the carrier with antigen are important
in defining the nature of immune response achieved. Preferably the
biocompatible, biodegradable polymer material is a copolymer of
lactic acid and glycolic acid, such as 50:50 poly
(D,L-lactide-co-glycolide), poly (lactide-co-glycolide), and
enantiomers thereof or a polymer of lactic acid, such as poly
(lactide) and enantiomers thereof. The antigen can be loaded by a
solvent evaporation type process, a coacervation process or a spray
drying process, preferably by a solvent evaporation type process or
a coacervation method. Further details of the loading processes are
given in the Examples below.
[0042] As will be further appreciated from the examples below the
nature of the immune response elicited by antigen loaded polymer
particles does not depend on a single factor, but is governed by a
combination of a number of factors.
EXAMPLES
[0043] All percentages are by weight (w/w) unless other wise
stated. The following abbreviation are used throughout the
examples: KLH, keyhole limpet hemacyanin; PTd, inactivated
pertussis toxin; FHA, filamentous hemaglutinin; PLA, poly lactide;
PLGA, poly lactide-co-glycolide; DCM, dichloromethane; PVA, poly
vinyl alcohol; PBS, phosphate buffer solution.
Example 1
Preparation of KLH-PLGA Microparticles Using a Solvent Evaporation
Method
[0044] A polymer solution of PLGA [poly (D,L-lactide-co-glycolide),
50:50; i.v.=0.94 dl/g; supplied by Boehringer Ingelheim] in
dichloromethane (10% PLGA in 10 ml DCM) was prepared two hours
prior to use and subsequently chilled 30 minutes prior to use. The
antigen, KLH (supplied by Calbiochem as a powder), was prepared as
an aqueous solution (5.1 mg KLH in 1 ml water) containing 2% PVA. A
first water-in-oil emulsion was prepared by adding the antigen
solution to the polymer solution and homogenising for 1 min. at
24,000 rpm on ice. This first emulsion was poured slowly into an
aqueous solution of PVA (40 ml, 3% PVA) forming a second
water-oil-water emulsion and homogenisation was continued for 2
min. with a 15 sec. break [1 min.; 15 sec. break; 1 min.]. The
resulting emulsion was stirred for 2 hours to evaporate the
dichloromethane. The antigen-loaded particles (75% yield) were
collected by centrifugation (10,000 rpm for 15 min).
[0045] The morphology and the particle size of the KLH-PLGA
particles were examined by scanning electron microscopy (SEM) using
a Leica Cambridge S360. Samples were mounted on stubs, gold coated
and scanned at magnifications of .times.3,000-10,000. Particle size
assessment by SEM was carried out by dividing the micrographs at
the 5,000 or 10,000 magnification into different fields and
counting the number of particles greater and less than 3 microns
and 5 microns. Particle size determination was also carried out by
laser diffractometry using a Malvern Mastersizer S Ver. 2.14. The
microparticles were suspended in filtered 0.1% Tween 20, sonicated
for 5 minutes and analysed with continuous stirring. KLH-PLGA
particles prepared as detailed above were found to have a smooth
spherical appearance and a D50% of 2.5 .mu.m by laser light
diffraction. By SEM, it could be seen that at least 50% of the
particles had a diameter less than 5 microns.
[0046] The loading of microparticles with antigen was determined by
digesting 10 mg of loaded microparticles in 3 ml of 5% SDS/0.1 M
NaOH for up to 60 hours with continuous shaking at room
temperature. The particles were completely digested during this
period. The pH of the solution was adjusted to pH 11.2 with 0.1 M
HCl and protein content was determined using a Bicinchoninic acid
(BCA) protein assay kit. Equivalent control particles containing no
antigen were also digested. The loading was calculated as
follows:
Actual loading ( g / mg ) = concentration in sample ( g / ml )
.times. total volume digested ( ml ) weight of particles ( mg )
##EQU00001## % entrapment efficiency = actual loading ( g / mg )
.times. 100 theoretical loading ( g / mg ) ##EQU00001.2##
where the theoretical loading is calculated from the amount of
antigen added to the formulation divided by the amount of polymer
used. KLH-PLGA particles prepared according to the present example
were found to have a loading of 3.1 .mu.g antigen/mg particles,
giving an entrapment efficiency of 94%.
[0047] The in vitro release of antigen from the loaded particles
was determined as follows: antigen loaded microparticles and
control microparticles (prepared in a similar manner, but
containing no antigen) were accurately weighed and dispersed in PBS
containing 0.02% sodium azide as a bacteriostatic agent. Samples
were immersed in a water bath at 37.degree. C. and shaken
continuously. At appropriate time intervals, 2.2 ml aliquots were
removed with a syringe, filtered and the protein content measured
in duplicate by BCA assay. KLH-PLGA particles prepared according to
the present example were found to release 80% of loaded antigen
after 1 hour and 100% of loaded antigen after 24 hours.
[0048] The procedure detailed above was repeated to form a second
batch of KLH-PLGA microparticles. This second batch of
microparticles appeared smooth and spherical under SEM with at
least 50% of the particles less than 5 microns, the D50% was
determined to be 2.2 .mu.m; the loading was found to be 3.5
.mu.g/mg representing 94% entrapment efficiency; and 76% of the
antigen was determined to be released after 1 hour, with 90% being
released after 24 hours.
[0049] Antigen loaded microparticles obtained from these two
batches were pooled together for an immunogenicity study in mice as
discussed in Example 5 below.
Example 2
Preparation of PTd-PLGA Microparticles Using a Solvent Evaporation
Method
[0050] Using a method substantially the same as that described in
Example 1 above, PTd (supplied by Katetsuken) loaded PLGA particles
were prepared. The polymer solution was 6.7% PLGA in 15 ml DCM and
the antigen solution was 744 .mu.g PTd in 2 ml water containing
0.9% PVA. The first water-in-oil emulsion was poured into 80 ml
aqueous PVA (3% PVA) to form the water-oil-water emulsion. The
emulsion was left over night to evaporate the DCM. After collection
(88% yield), the microparticles were washed with chilled autoclaved
water (30 ml).
[0051] Characterisation of these particles, identified as PTd-1 in
Table 1 below, showed that the microparticles formed were smooth
and spherical in appearance with at least 50% of the particles less
than 5 microns in diameter. Laser light diffractometry showed that
the particles had a D50% of 2.5 .mu.m. The microparticles were
loaded with antigen at 0.12 .mu.g/mg, representing an entrapment
efficiency of 15%.
[0052] The in vitro release of PTd loaded microparticles was
determined according to the following method: 30 mg of
microparticles were dispersed in PBS (4.0 ml) containing 0.02%
sodium azide. The sample was placed in a water bath at 37.degree.
C. and shaken continuously. At appropriate time intervals the
sample was removed from the water bath and centrifuged to pellet
the particles. The supernatant was removed and the protein content
was determined in duplicate. Three ml of fresh PBS was added to the
microparticles to maintain sink conditions and the incubation was
continued. PTd-PLGA particles prepared according to the present
example (PTd-1) were found to release 22% of loaded antigen after 1
hour and 56% of loaded antigen after 24 hours followed by biphasic
release over 20 days.
[0053] Additional batches of PTd-PLGA microparticles were made
following substantially the same procedure as given above using
quantities of the various components as summarised in Table 1
below. In batches PTd-2 through PTd-6, no PVA was added to the
initial antigen solution and 40 ml chilled autoclaved water was
used to wash the recovered microparticles. In each case the
resulting antigen loaded microparticles were found to be smooth and
spherical in appearance with at least 50% of the particles less
than 5 microns in diameter.
TABLE-US-00001 TABLE 1 Batch PTd Aq. Vol % DCM 3% PVA Load % D50% 1
hr 24 hr No. (.sup..mu.g) (ml) PLGA (ml) (ml) (.sup..mu.g/mg) EE
(.sup..mu.m) (%) (%) PTd-1 744 2 6.7 15 80 0.12 15 2.5 22 56 PTd-2
1536 1.0 4 10 40 1.2 31 3.0 30 PTd-3 2760 1.5 4 20 80 1.3 33 3.3 *
* PTd-4 3130 1.5 4 20 80 1.3 34 2.4 * * PTd-5 2140 2.0 4 20 80 1.1
42 3.2 * * PTd-6* -- -- -- -- -- -- -- -- 17 21 3% PVA is the
volume of PVA solution to which the antigen/PLGA water-in-oil
emulsion is added; Load is the antigen loading of the
microparticles; % EE is the % entrapment efficiency; D50% is the
average diameter of the microparticles; 1 hr is the antigen
released after 1 hour; 24 hr is the antigen released after 24
hours. *The loaded microparticles obtained from PTd3, PTd-4 and
PTd-5 were pooled for antigen release assay and i.p. protection
study (see Example 7 below).
Example 3
Preparation of FHA-PLGA Microparticles Using a Solvent Evaporation
Method
[0054] A procedure substantially similar to that used in Example 2
was employed for the preparation of FHA-loaded PLGA microparticles.
Two batches of FHA-PLGA microparticles were prepared. For these two
batches (FHA-1 and FHA-2 in Table 2 below) the polymer solution was
4% PLGA in 20 ml DCM and the antigen solution was 0.87 .mu.g FHA in
2 ml water containing no PVA. The first water-in-oil emulsion was
poured into 80 ml aqueous PVA (3% PVA) to form the water-oil-water
emulsion. The characteristics of these two batches are given in
Table 2 below. FHA-1 and FHA-2 were pooled (the pooled
microparticles are labelled FHA-3 in Table 2) for antigen release
determination and i.p. protection studies (see Example 7 below).
SEM analysis showed the FHA-1 and FHA-2 microparticles to be smooth
and spherical in nature with at least 50% of the particles less
than 5 microns in diameter.
TABLE-US-00002 TABLE 2 Example Loading D50% No. (mg/mg) % EE
(.sup..mu.m) 1 hr (%) 24 hr (%) FHA-1 0.94 87 3.0 * * FHA-2 1.09
100 4.3 * * FHA-3* -- -- -- 25 49
Example 4
Preparation of Antigen Entrapped or Encapsulated Nanoparticles
[0055] An aqueous solution (A) of a polymer, surface active agent,
surface stabilising or modifying agent or salt, or surfactant
preferably a polyvinyl alcohol (PVA) or derivative with a %
hydrolysis 50-100% and a molecular weight range 500-500,000, most
preferably 80-100% hydrolysis and 10,000-150,000 molecular weight,
is introduced into a vessel. The mixture (A) is stirred under low
shear conditions at 10-2000 rpm, preferably 100-600 rpm. The pH
and/or ionic strength of this solution may be modified using salts,
buffers or other modifying agents. The viscosity of this solution
may be modified using polymers, salts, or other viscosity enhancing
or modifying agents.
[0056] A polymer, preferably poly(lactide-co-glycolide),
polylactide, polyglycolide or a combination thereof or in any
enantiomeric form is dissolved in water miscible organic solvents
to form organic phase (B). Most preferably, a combination of
acetone and ethanol is used in a range of ratios from 0:100
acetone: ethanol to 100:0 acetone: ethanol depending upon the
polymer used. Additional polymer(s), peptide(s) sugars, salts,
natural/biological polymers or other agents may also be added to
the organic phase (B) to modify the physical and chemical
properties of the resultant particle product.
[0057] An antigen or bioactive substance may be introduced into
either the aqueous phase (A) or the organic phase (B). The organic
phase (B) is added into the stirred aqueous phase (A) at a
continuous rate. The solvent is evaporated, preferably by a rise in
temperature over ambient and/or the use of a vacuum pump. The
particles are now present as a suspension (C).
[0058] The particles (D) are then separated from the suspension (C)
using standard colloidal separation techniques, preferably by
centrifugation at high `g` force, filtration, gel permeation
chromatography, affinity chromatography or charge separation
techniques. The supernatant is discarded and the particles (D)
re-suspended in a washing solution (E) preferably water, salt
solution, buffer or organic solvent(s). The particles (D) are
separated from the washing liquid in a similar manner as previously
described and re-washed, commonly twice.
[0059] The particles may then be dried. Particles may then be
further processed for example, tabletted, encapsulated or spray
dried.
[0060] The release profile of the particles formed above may be
varied from immediate to controlled or delayed release dependent
upon the formulation used and/or desired.
[0061] Antigen loading may be in the range 0-90% w/w.
[0062] Specific examples include the following:
[0063] A PTd (168 .mu.g/ml) or FHA (264 .mu.g/ml) solution was
first dispersed in a PVA (mwt=13000-23000; 98% hydrolysis) solution
while stirring at 400 rpm with the temperature set at 25.degree. C.
A polymer solution (prepared by dissolving PLGA; 50:50; either
RG504 or RG504H supplied by Boehringer Ingelheim into the organic
phase) was added slowly into the aqueous phase to form coacervates
that hardened following evaporation of the organic solvent. The
nanoparticles were then recovered by centrifugation at 15,000 rpm
for 30 minutes and washed three times with autoclaved deionised
water. The wet pellet was allowed to dry at ambient temperature
under a vacuum. Batches having a theoretical loading of 0.3% PTd
(RG504H and RG504 polymer), 0.2% FHA (RG504H and RG504 polymer)
were prepared according to Table 3.
TABLE-US-00003 TABLE 3 PLGA 5% w/v polymer Acetone Ethanol antigen
Batch PVA soln. (g) (ml) (ml) (ml) 0.3% PTd 546.4 2.991 67.5 7.5
53.6 0.2% FHA 577.3 2.994 67.5 7.5 22.7
[0064] Scanning microscopy was employed to assess the nanoparticle
morphology and size. The nanoparticles were mounted onto SEM stubs,
sputter coated using an Emitech K550 sputter coater set at 25 mA
for 3 minutes and scanned using a Leica Cambridge S360. Micrographs
were taken at magnifications of 500-20,000.times.. Particle size
assessment by SEM was carried out by dividing the micrographs at
the 15,000 magnification into different fields and counting the
number of particles greater and less than 600 nm and 500 nm. The
SEM analysis showed that for both the PTd-PLGA and FHA-PLGA
nanoparticles were approximately spherical in shape with smooth
surfaces. At least 50% of the particles were less than 600 nm at
the 15 k magnification, although there was some evidence of
aggregation.
[0065] Antigen loading was determined by measuring the total
protein content of the nanoparticles using a BCA protein assay as
described in Example 1. The nanoparticles prepared according to the
present example were found to have the potencies and encapsulation
efficiencies as given in Table 5.
TABLE-US-00004 TABLE 4 Potency Encapsulation Batch (.sup..mu.g/ml)
efficiency (%) 0.3% PTd -RG504H 1.3 43.3 0.2% FHA - RG504H 0.9 45.0
0.3% PTd -RG504 1.3 43.3 0.2% FHA - RG504 1.0 50.0
[0066] The in vitro release of antigen from the loaded particles
was determined by suspending 50 mg of nanoparticles in 10 ml PBS,
pH 7.4, containing 0.02% w/v sodium azide in glass tubes and
incubating at 37.degree. C. At predetermined time intervals, a 3 ml
sample was removed and the total protein released was determined by
the BCA protein assay described above. PTd-PLGA formulations showed
a large burst effect of approximately 45% in the first hour
followed by a very gradual release up to 55% at 24 hours. In
comparison, FHA-PLGA formulations showed a much lower burst release
of 14% at 1 hours up to 18% after 24 hours.
Example 5
Immune Response Upon ip. Administration of KLH-PLGA Microparticles
to Balb/c Mice
[0067] The immunogenicity of KLH entrapped in biodegradable
microparticles was assessed in mice following parenteral (i.p.)
administration and compared with the same antigen in solution
(phosphate buffered saline: PBS) or adsorbed to alum. Further
control groups included KLH in solution with empty PLGA
microparticles, empty PLGA microparticles alone or PBS alone.
Microparticles from the two batches of Example 1 were pooled and
suspended in PBS at a concentration equivalent to 100 .mu.g of
antigen per ml or diluted accordingly for lower doses. Each mouse
was immunised with 0.3 ml once or twice at a four week interval and
immune responses were assessed 2 weeks after the last
immunisation.
[0068] Serum and mucosal secretions (lung homogenates) were tested
for anti-KLH IgG and IgA antibody levels by ELISA. Systemic
cellular immune responses were assessed using spleen from immunised
mice. The spleen cells from 4 to 6 individual mice in each
experimental group were cultured in triplicate wells of duplicate
96-well microtitre plates with a range of concentrations of antigen
(0.16 to 100 .mu.g/ml). The mitogens Concanavlin A or PMA and
anti-CD3-antibody or medium alone were included as positive and
negative controls respectively. After 24 and 72 hours supernatants
were removed from one plate and stored at -70.degree. C. for
cytokine analysis. The levels of interferon gamma. (IFN-.gamma.)
and interleukin-5 (IL-5) were determined by immunoassay as
quantifiable markers of induction of antigen-specific T.sub.H1 and
T.sub.H2 subpopulations respectively. Additionally, the
proliferation of T cell cultures were assessed in four day
cultures, by [.sup.3H]-thymidine incorporation.
[0069] A single i.p. immunisation with 20 .mu.g KLH entrapped in
PLGA microparticles induced potent cellular immune responses with
high nanogram levels of IFN-.gamma. produced by spleen cells
following in vitro stimulation with KLH over a wide dose range.
Picogram levels of IL-5 were also detected in antigen-stimulated
spleen cell supernatants but the levels were comparatively lower
than that observed with spleen cells from animals immunised with
KLH adsorbed to alum. Overall the responses were polarised to
T.sub.H2 with antigen adsorbed onto alum and to T.sub.H1 with
microencapsulated antigen. FIG. 1 shows the immune response
following the second immunisation at four weeks. The levels of IL-5
for the microencapsulated KLH were comparable with those observed
with alum, but the production of IFN-.gamma. was significantly
higher. Furthermore, potent antigen-specific proliferation was
observed in spleen cells from mice immunised with KLH entrapped in
PLGA microparticles. The stimulation indices (derived by dividing
the response to antigen by the response to medium alone) were
significantly higher than those observed with spleen cells from
mice immunised with KLH adsorbed to alum at all antigen doses
tested in vitro.
[0070] Following parenteral immunisation with microencapsulated
KLH, the levels of KLH-specific IgG in serum were significantly
higher than those generated with the soluble antigen and were equal
to or greater than that induced with alum absorbed antigen.
Co-injection with empty PLGA also appeared to significantly boost
the antibody responses to soluble antigen. Although each of the
animals immunised with 20 .mu.g of soluble KLH by the i.p. route
generated detectable antibody response, the titres were more than
10 fold higher when the antigen was combined with empty PLGA
microparticles prior to immunisation. These findings suggest that
the T.sub.H2 and antibody, but not the T.sub.H1 response was
significantly boosted following co-injection of empty
microparticles with soluble antigen, whereas T.sub.H1 responses are
enhanced with the microencapsulated antigen.
[0071] These results demonstrate that the entrapment of soluble
antigen KLH in PLG microparticles significantly enhanced T cell
proliferative responses over that observed with soluble antigen and
was comparable to that observed with alum adsorbed antigen by the
i.p. route. Moreover, encapsulation of the antigen in PLGA appears
to favour the induction of T.sub.H1 cells.
Example 6
Immune Response Upon ip. Administration of PTd-PLGA Microparticles
to Balb/c Mice
[0072] Similarly to the immunisation regimen of Example 5, batch
PTd-1 of Example 2 was used in a preliminary parenteral
immunisation study in which mice were immunised with two i.p.
inoculations of 5 .mu.g PTd entrapped in PLGA microparticles,
adsorbed to alum or in solution combined with empty PLGA
microparticles. Control mice were immunised with PBS alone or with
empty PLGA microparticles alone. Antibody responses were detected
by ELISA two weeks after administration of the second dose. Potent
anti PTd-IgG titres were observed after each of the two
immunisations, with PTd-PLGA titres on the order of
1.times.10.sup.6, comparable to those obtained with alum, being
obtained after the second immunisation. No responses were seen in
mice exposed to empty PLGA microparticles. Surprisingly, PTd in
solution gave responses in 3/5 mice; these responses are unlikely
to be sustained over time compared to PTd-PLGA or PTd-alum and they
were not seen in the first immunisation. The effects of PTd in
solution were enhanced in the presence of empty PLGA.
[0073] FIG. 2 shows the cytokine analysis after the first i.p.
immunisation, demonstrating a dominant T.sub.H1 cell-mediated
immune response to PTd-PLGA. Upon re-exposure to pertussis, a high
level of IFN-.gamma. (T.sub.H1) and only modest levels of IL-5
(T.sub.H2) were seen in the spleen cell cultures form animals
previously immunised with PTd-PLGA. Cells re-exposed to pertussis
following a PTd-alum immunisation gave relatively strong
IFN-.gamma. and IL-5 production.
Example 7
Pertussis Challenge Study Following ip. Immunisation of Balb/c Mice
With the Antigen Combination PTd+FHA
[0074] Groups of 20 balb/c mice were immunised i.p. with 5 .mu.g
each of PTd and FHA entrapped in PLGA microparticles (using PTd-6
of Example 2 and FHA-3 of Example 3) or adsorbed to alum. The
control group received empty PLGA microparticles. The ability of
PLGA-entrapped antigen to protect against B. pertussis was examined
in a respiratory challenge model. Briefly, following two doses of
antigen, four weeks apart, a respiratory B. pertussis infection was
initiated in 16 mice per experimental group by aerosol challenge of
approximately 2.times.10.sup.10 cfu/ml (approximately
10.sup.410.sup.5 cfu per mouse lung) two weeks after the second
immunisation. The mice were sacrificed at different time points
over a two-week period and lung homogenates were cultured and
examined after 5 days culture for the number of colony forming
units (CFU). Four mice from each group were sacrificed prior to
challenge to test immune responses on the day of challenge.
[0075] The results from the CFU counts 2 hours and 3, 7, 10 and 14
days after challenge, which are shown in FIG. 3, reveal a high
level of protection with both PLGA microparticle entrapped and alum
adsorbed antigens. These treatments provided clearance of B.
pertussis by the third day post challenge following challenge 6
weeks after immunisation. The potency index which compares the
counts for the test "vaccine" with the un-immunised control group
were 88.8 and 88.4 for the PTd+FHA in PLGA and PTd+FHA adsorbed to
alum respectively. These potency indices equal or exceed the levels
of potency for commercial a cellular vaccines, including 3 and 5
component vaccines.
[0076] The immune responses on the day of challenge revealed very
strong anti-PT antibody responses with the PLG entrapped antigens
as shown in FIG. 4. The end point titres were log.sub.10 5.8 for
the PLGA group and 5.0 for the alum group. The anti-FHA antibody
titres were of the order of 5.0 for both alum and PLGA groups.
[0077] Results of the CMI studies revealed positive T cell
proliferative responses against inactivated PT and FHA in all mice
immunised with PTd and FHA microencapsulated in PLGA or adsorbed to
alum. The T cell responses against PT were stronger in the PLGA
group, whereas the responses to FHA were stronger in the alum
group. FIG. 5 shows the corresponding cytokine analysis from
splenic T cells following parenteral immunisation with 5 .mu.g each
of PTd and FHA entrapped in PLGA microparticles (T.sub.H1
polarisation) and 5 .mu.g each of PTd and FHA adsorbed onto alum
(mixed T.sub.H1/T.sub.H2 responses). These results confirm that as
in the case of KLH entrapped in PLGA, PTd entrapped in PLGA and FHA
entrapped in PLGA, entrapment of PTd and FHA together in PLGA
polarises the immune response towards T.sub.H1 after i.p. delivery.
In the case of alum, a mucosal or exclusive T.sub.H2 result is
found.
Example 8
Pertussis Challenge Study Following ip. Immunisation of Balb/c Mice
With the Antigen Combination PTd+FHA (Reduced Dose)
[0078] Groups of 20 balb/c mice were immunised parenterally (i.p.)
at week 0 and at week 4 with 1 .mu.g each of PTd and FHA entrapped
in PLGA microparticles; 1 .mu.g each of PTd and FHA adsorbed to
alum; 1 .mu.g of PTd entrapped in PLGA microparticles or 1 .mu.g
FHA entrapped in PLGA microparticles. The control group received
empty PLGA microparticles. The ability of PLGA-entrapped antigen
(low dose) to protect against B. pertussis was examined in the
respiratory challenge model as described in Example 7. The mice
were sacrificed at different time points over a two-week period
after the aerosol challenge and lung homogenates were cultured and
examined after 5 days culture for the number of colony forming
units (CFU). Four mice from each group were sacrificed prior to
challenge to test immune responses on the day of challenge.
[0079] The results from the CFU counts 2 hours and 3, 7, 10 and 14
days after challenge reveal a high level of protection with 1 .mu.g
of FHA and PTd either microencapsulated in PLGA or adsorbed to alum
as shown in FIG. 7, Both of these treatments provide substantial
clearance of B. pertussis by the third day post challenge following
challenge 6 weeks after immunisation.
[0080] The cytokine data demonstrates significant polarisation to
T.sub.H1 for the groups treated with FHA and PTd entrapped in PLGA,
with PTd entrapped in PLGA and with FHA entrapped in PLGA while a
mixed T.sub.H1/T.sub.H2 response if seen for the group treated with
FHA and PTd adsorbed to alum. FIG. 6 shows the cytokine analysis
for FHA entrapped in PLGA microparticles, demonstrating the
T.sub.H1 polarisation of this formulation. The control group
produced little response in the T cells.
[0081] ELISA antibody titres for the mice in the five treatment
groups were evaluated 2 weeks after the second immunisation, which
occurred at week 4. Table 3 presents the mean (SD) serum IgG titres
for each group of 4 mice. The results show that the anti-PT
antibody titres are not significantly different between the
different groups that received formulations that included PTd.
However, the anti-FHA antibody levels are significantly stronger in
the mice that received alum adsorbed antigens. The levels were
about 10 fold lower in the mice that received FHA entrapped in PLGA
or FHA and PLGA entrapped in PLGA microparticles.
TABLE-US-00005 TABLE 5 Immunogen Anti-PT Anti-FHA PTd-PLGA 5.05
(0.48) <1.00 PTd/FHA-PLGA 4.97 (0.32) 4.54 (0.89) PTd/FHA-alum
5.20 (0.24) 5.44 (0.16) FHA-PLGA <1.00 4.47 (0.94) Empty-PLGA
<1.00 <1.00
Example 9
Pertussis Challenge Study Following ip. Immunisation of Balb/c Mice
With the Antigen Combination PTd+FHA (Delayed Challenge)
[0082] Three groups of 20 balb/c mice were immunised parenterally
(i.p.) at week 0 and at week 4 with 5 .mu.g each of PTd and FHA
entrapped in PLGA microparticles and 5 .mu.g each of PTd and FHA
adsorbed to alum and a control group (empty PLGA microparticles).
The ability of PLGA-entrapped antigen to protect against B.
pertussis was examined in the respiratory challenge model.
Following two doses of antigen, four weeks apart, a respiratory B.
pertussis infection was initiated in 16 mice per experimental group
by aerosol challenge at week 12. The mice were sacrificed at
different time points over a two-week period after the aerosol
challenge and lung homogenates were cultured and examined after 5
days culture for the number of colony forming units (CFU). Four
mice from each group were sacrificed prior to challenge to test
immune responses on the day of challenge.
[0083] The results of the cytokine analysis at the 12 week time
point are consistent with those reported above for analysis at the
6 week time point, showing a polarisation of the T cell response to
type 1 with PTd and FHA entrapped in microparticles and to type 2
with alum adsorbed antigens. Overall, these results reveal
persistence, and perhaps even further polarisation, of the T.sub.H1
response after immunisation with antigen entrapped in PLGA
microparticles versus alum.
[0084] As shown in FIG. 8, the results from the CFU counts 2 hours
and 3, 7, 10 and 14 days after challenge reveal a high level of
protection with 5 .mu.g of FHA and PTd either microencapsulated in
PLGA or adsorbed to alum. Both of these treatments provide
clearance of B. pertussis by the third day post challenge following
challenge 12 weeks after immunisation.
Example 10
Immune Response Upon Parenteral Administration (ip., s.c., i.m) of
PTd-PLGA and FHA-PLGA Microparticles to Balb/c Mice
[0085] Seven groups of 5 balb/c mice were immunized by three
different parenteral routes at week 0 and at week 4 with 1 .mu.g
each of PTd and FHA in either saline solution or entrapped in PLGA
microparticles manufactured similarly to those of Examples 2 and 3
(PTd loading=1.42 .mu.g/mg; FHA loading=1.22 .mu.g/mg; 1.52 mg
particles per dose) as follows:
[0086] Treatment A: PTd+FHA in solution intraperitoneal (i.p.)
[0087] Treatment B: PTd+FHA in solution subcutaneous (s.c.)
[0088] Treatment C: PTd+FHA in solution intramuscular (i.m.)
[0089] Treatment D: PTd+FHA entrapped in PLGA intraperitoneal
(i.p.)
[0090] Treatment E: PTd+FHA entrapped in PLGA subcutaneous
(s.c.)
[0091] Treatment F: PTd+FHA entrapped in PLGA intramuscular
(i.m.)
[0092] Treatment E: Saline only (control)
[0093] The i.p. immunisations were administered in 0.3 ml, the s.c.
immunisations (on the back) in 0.2 ml and the i.m. immunisation at
two sites in 0.1 ml.
[0094] Immune responses to these treatments were assessed two weeks
subsequent to the second immunisation at week 6. Individual spleen
cell preparations from mice per experimental group were tested for
antigen-induced proliferation and cytokine production. Serum
samples (week 6) from individual mice were assessed over an 8-fold
dilution range for anti-PT and anti-FHA IgG.
[0095] Analysis of the serum IgG responses revealed clear effects
due to the route of administration on the antibody titres, with
striking differences between soluble and PLGA entrapped antigen.
The s.c. route generated slightly weaker anti-PT antibody response
with both soluble and PLGA entrapped antigens (end-point log titres
of .about.3.5 and 3.7, respectively). The anti-FHA titres were also
lowest when soluble and PLGA-entrapped antigens were given by the
s.c. route (end-point log titres of 1.8 and 0.4, respectively).
However, very strong anti-PT and anti-FHA responses induced with
the PLGA microparticle entrapped antigens were observed after i.p.
immunisation (end-point log titres of 4.5 and 2.7, respectively).
In contrast, immunisation by the i.m. route induced the strongest
IgG responses with the antigens in solution (end-point anti-PT and
anti-FHA log titres were 4.3 and 3.8 for i.m. solution).
[0096] Overall, the anti-PT responses were strongest with the PLGA
entrapped antigens by the i.p. route, whereas the strongest
anti-FHA responses were observed with antigens in solution by the
i.m. route. These results are consistent with the increased
immunogenicity of particulate antigens in the peritoneal cavity, a
site rich in phagocytic APC and the slower clearance of soluble
antigen administered by the i.m. route.
[0097] T cell proliferative responses against inactivated PT and
FHA shows that the strongest responses are observed in mice that
received microencapsulated or soluble PTd and FHA by the s.c.
routes.
[0098] Analysis of the cytokine production from spleen cells showed
that all of the immunogens induced potent antigen-specific T cell
responses but also revealed striking differences between
administration of antigens entrapped in PLGA or in solution by
different parenteral routes. Overall, the antigens entrapped in
PLGA induced a T cell response which was polarised to the T.sub.H1
subtype following any route of parenteral immunisation, whereas the
response was more polarised to T.sub.H2 with the antigen in
solution.
[0099] The best T cell priming with the soluble antigens was
observed with the i.m. route followed by s.c., with the poorest
results observed for the i.p. route, especially for IL-5 producing
cells. Potent T.sub.H2 cytokine production was induced with the
soluble antigens given by the i.m. route; the levels of FHA-induced
IL-5 exceed 2000 pg/ml for spleen cells in 4 of 5 mice. In
contrast, as shown in FIG. 9 for i.m. administration, analysis of
the cytokine production following parenteral administration of PLGA
microencapsulated antigen to mice revealed that the i.m. route
induced IFN-.gamma. production by each of the mice in response to
each of the antigen preparations tested whereas IL-5 production was
weak or undetectable. This was exactly the pattern seen for the
i.p. route as described in FIGS. 1, 5 and 6. However, the responses
were less polarised to T.sub.H1 with PLGA microencapsulated
antigens administered by the s.c. route; the IFN-.gamma. levels
were lower and more inconsistent and significant IL-5 responses
were detected in 3 of 5 mice. The finding that the i.m route
results in potent T.sub.H2 priming with soluble antigen but
T.sub.H1 priming with PLGA entrapped antigen is new and highly
significant.
[0100] Similarly to the immunisation regimen given above, a repeat
of the above parenteral route study was undertaken in which groups
of balb/c mice were immunized at week 0 and week 4 with
inoculations of 1 .mu.g of each of the antigens according to the
following treatments:
[0101] Treatment 1: PTd+FHA in solution subcutaneous (s.c.)
[0102] Treatment 2: PTd+FHA in solution intramuscular (i.m.)
[0103] Treatment 3: PTd+FHA entrapped in PLGA subcutaneous
(s.c.)
[0104] Treatment 4: PTd+FHA entrapped in PLGA intramuscular
(i.m.)
[0105] Treatment 5: empty PLGA subcutaneous (s.c.) (control)
[0106] The ability of these s.c. and i.m. treatments to protect
against B. pertussis was examined in the respiratory challenge
model as described in Example 7. The results from the CFU counts
reveal a high level of protection from Treatments 3 and 4
(microparticles injected s.c. and i.m). Both of these treatments,
as well as that of Treatment 2 (antigens in solution, i.m.),
provide substantial clearance of B. pertussis by the third day post
challenge following challenge 2 weeks after the second
immunisation. In contrast, neither Treatment 5 (empty PLGA, s.c.)
or Treatment 1 (antigens in solution, s.c.) show clearance at the
third day post challenge.
Example 1
Immune Response Upon Parenteral Administration of PTd-PLGA and
FHA-PLGA Nanoparticles to Balb/c Mice
[0107] The immunogenicity of coacervate formulations of PTd, FHA
and the combination of PTd and FHA entrapped in biodegradable PLGA
nanoparticles was assessed in mice following parenteral (i.p.)
administration and compared to the administration of PTd and FHA in
solution and empty PLGA nanoparticles. 6 groups of mice were
immunised i.p. with FHA and/or PTd formulations according to
Example 4 (5 .mu.g FHA and/or PTd in 0.3 ml deionised water) or
with empty PLGA nanoparticles (control) as follows:
[0108] Treatment A: empty PLGA nanoparticles
[0109] Treatment B: PTd and FHA in solution
[0110] Treatment C: PTd-PLGA (RG504) nanoparticles
[0111] Treatment D: FHA-PLGA (RG504) nanoparticles
[0112] Treatment E: PTd-PLGA (RG504)+FHA-PLGA (RG504)
nanoparticles
[0113] Treatment F: PTd-PLGA (RG504H)+FHA-PLGA (RG504H)
nanoparticles Each mouse was dosed with two i.p. inoculations at
weeks 0 and 4; immune responses were tested 2 weeks after the
second immunisation (week 6).
[0114] Overall, very potent antibody responses were generated with
antigens entrapped in these PLGA coacervate nanoparticles, with
mean anti-PT serum IgG endpoint log titres of 4.3 and mean anti-FHA
serum endpoint log IgG titres of 4.6. The anti-FHA antibody titres
were modestly stronger with entrapped FHA compared to FHA in
solution; however, the anti-PT titres for the entrapped and
solution formulations were not significantly different.
Formulations containing both PTd and FHA entrapped nanoparticles
did not raise significantly different antibody responses compared
to those generated by either entrapped antigen alone. The responses
are almost identical using the two different PLGA polymers.
[0115] As shown in FIG. 10, in contrast to the T.sub.H1
polarisation found upon i.p. immunisation with microparticulate
entrapped antigens discussed above, the most striking feature of
the antigen-specific spleen cell cytokine production was the strong
polarisation of the response to T.sub.H2 for all these
nanoparticulate formulations. High levels of IL-5 were produced by
spleen cells stimulated in vitro with FHA (1 or 5 .mu.g/ml) or
inactivated PT (1 or 5 .mu.g/ml) or killed B. pertussis. The only
significant antigen-specific IFN-.gamma. production observed was
against PT and killed B. pertussis in mice given PTd entrapped in
PLGA.
[0116] A repeat experiment in which groups of 5 mice were immunised
i.p. with PTd+FHA in solution, PTd+FHA in PLGA nanoparticles
(formulations as outlined in Example 4) and empty PLGA
nanoparticles (control) was undertaken following the same protocol
as given above in this example. Spleen cell preparations from 5
mice per leg were tested individually for antigen-induced
proliferation and cytokine production and serum samples from
individual mice were assessed over an 8-fold dilution range for
anti-PT and anti-FHA IgG.
[0117] Again, antibody responses induced with the pertussis
antigens entrapped in PLGA nanoparticles were very strong, with end
point titres in the range 4.0 to 5.0. The response to PT was almost
one log stronger with the PLGA entrapped antigens when compared
with the soluble antigens. The responses to FHA were stronger
overall that to PT and there was not a significant difference in
the end point titres in sera from mice immunised with soluble or
PLGA entrapped antigens.
[0118] Strong proliferative T cell responses to FHA and killed
bacteria were observed in individual spleen cell preparations from
all mice immunised with PTd and FHA either in solution or entrapped
in PLGA. There was no significant difference between the two
immunogens. Analysis of cytokine production by spleen cells
revealed very high levels of IL-5 (in the region of 1500 to 3000
pg/ml) and very modest levels of IFN-.gamma. in response to FHA in
all mice immunised with PTd and FHA, either in solution or
entrapped in PLGA. FIG. 10 shows the cytokine data from the
coacervated nanoparticles. The responses to inactivated B.
pertussis were somewhat lower but were still in the region of 500
pg/ml compared with levels less than 15 pg/ml in control mice
immunised with empty PLGA nanoparticles. Therefore, the overall
pattern is a polarisation towards a T.sub.H2 response by
administration of soluble antigens or antigens entrapped in
coacervated nanoparticles.
Example 12
Preparation of KLH-PLA Microparticles by a Spray Drying Method
[0119] A PLA (poly D, L lactide; molecular weight 16,000 solution;
i.v.=0.27 dl/g; supplied by Boehringer Ingelheim, R203) solution in
ethylacetate (5% PLA in 150 ml ethylacetate) was prepared two hours
prior to use. The polymer solution was homogenised (at 24,000 rpm)
using an IKA Ultra Turrax T25 homogeniser with an S1 head while the
KLH antigen solution (62 mg KLH in MES (2-[n-morphlino]
ethanesulfonoic acid)) was added slowly. The emulsion was cooled on
ice and homogenisation was continued for 1 min. The single emulsion
thus prepared was spray dried using a Buchi 191 mini spray drier
with continuous stirring using a magnetic stirrer. The following
parameters were used in the spray drying step:
TABLE-US-00006 TABLE 6 Parameter Value Parameter Value Inlet 60
Aspirator (%) 100 temperature (.degree. C.) Outlet 45 Pump rate*
(ml/min.) 5 temperature (.degree. C.) Flow rate 700-800 Pressure
(mbar) -40 *Pump rate was set at 25%, the actual rate of solvent
pumped through varied from 5 to 6 ml/min.
[0120] The particles were collected immediately from both the
collection vessel and the cyclone. The antigen-loaded
microparticles were characterised according to the procedures
outlined above for the previous examples. The microparticles formed
according to the present invention (yield 33%) were found to be
smooth and spherical in nature. The loading (.mu.g/mg) was 7.4 with
an entrapment efficiency of 91% and a D50% by laser light
diffractometry of 4.6 .mu.m. The in vitro release of KLH was found
to be 10% within one hour and 49% on day 23.
[0121] Similar to the Examples above, mice were immunised with two
i.p. inoculations of 20 .mu.g KLH either entrapped in PLA
microparticles or adsorbed to alum. Titres were obtained when the
particles were administered to mice systemically (1.times.10.sup.5)
which were comparable with those obtained in the KLH-alum group.
Furthermore, the KLH-PLA particles resulted in a dominant T.sub.H2
response when administered i.p. which is in contrast to that
observed in the above examples for KLH-PLGA microparticles in which
polarisation was towards the T.sub.H1 type.
Example 13
Preparation of PTd-PLA Microparticles by a Spray Drying Method
[0122] PTd-PLA microparticles were formed by a spray drying method
similar to that of Example 12 with the exceptions that, to prevent
phase separation during spray drying, the homogenisation speed was
increased to 24,000 rpm, the polymer viscosity was increased by
using 5% R203 and the w/o emulsion was stirred during spraying. The
release profile of the resultant particles was characterised by a
marked burst of 50-60% in the first hour followed by a very slow
release phase over a three month period of time.
[0123] Serum anti-PTd IgG levels were determined in mice immunised
i.p. with 5 .mu.g PTd in spray dried PLA microparticles and
compared to immunisation with PTd-alum, empty PLA particles mixed
with soluble PTd, or PTd in solution. However, no antibody or
T-cell responses were obtained in mice immunised systemically with
PTd-PLA.
[0124] The integrity of PTd, FHA and KLH following either the
solvent evaporation (see Examples above) or spray drying processes
were examined semi-quantitatively by PAGE gel analysis. More PTd
remains intact when extracted from particles prepared by the
solvent evaporation method relative to spray dried particles. Thus,
the PTd may have been partially degraded during the spray drying
process. The data suggest that while spray-drying is problematic
for maintaining antigenic structure in the case of PTd, this cannot
be assumed for less labile antigens and it will therefore need to
be assessed on a case by case basis
* * * * *