U.S. patent application number 12/414497 was filed with the patent office on 2009-10-08 for novel proteosome-liposaccharide vaccine adjuvant.
This patent application is currently assigned to ID BIOMEDICAL CORPORATION OF QUEBEC. Invention is credited to David S. Burt, David Jones, George H. Lowell, Clement Rioux, Gregory L. White.
Application Number | 20090252762 12/414497 |
Document ID | / |
Family ID | 26956684 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090252762 |
Kind Code |
A1 |
Burt; David S. ; et
al. |
October 8, 2009 |
NOVEL PROTEOSOME-LIPOSACCHARIDE VACCINE ADJUVANT
Abstract
An adjuvant complex composed of bacterial outer membrane protein
proteosomes complexed to bacterial liposaccharide is prepared to
contain the component parts under a variety of conditions. The
complex can be formulated with antigenic material to form
immunogenic compositions, vaccines and immunotherapeutics. An
induced immune response includes protective antibodies and/or type
1 cytokines is shown for a variety of protocols.
Inventors: |
Burt; David S.; (Dollard des
Ormeaux, CA) ; Lowell; George H.; (Hampstead, CA)
; White; Gregory L.; (Ile Perrot, CA) ; Jones;
David; (Mountain View, CA) ; Rioux; Clement;
(Ile Dizard, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
ID BIOMEDICAL CORPORATION OF
QUEBEC
Laval
CA
|
Family ID: |
26956684 |
Appl. No.: |
12/414497 |
Filed: |
March 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10094424 |
Mar 11, 2002 |
7524509 |
|
|
12414497 |
|
|
|
|
60274232 |
Mar 9, 2001 |
|
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60327297 |
Oct 9, 2001 |
|
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|
Current U.S.
Class: |
424/206.1 ;
424/184.1; 424/204.1; 424/234.1; 424/265.1; 424/275.1 |
Current CPC
Class: |
C12N 2760/16151
20130101; A61K 2039/55516 20130101; A61K 2039/70 20130101; A61K
39/145 20130101; A61K 39/12 20130101; A61K 39/39 20130101; C12N
7/00 20130101; A61P 43/00 20180101; A61P 37/02 20180101; A61K 39/36
20130101; A61P 37/04 20180101; C12N 2760/16134 20130101; A61K
2039/55572 20130101; A61K 2039/543 20130101 |
Class at
Publication: |
424/206.1 ;
424/184.1; 424/275.1; 424/204.1; 424/234.1; 424/265.1 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61K 39/00 20060101 A61K039/00; A61K 39/35 20060101
A61K039/35; A61K 39/12 20060101 A61K039/12; A61K 39/02 20060101
A61K039/02; A61K 39/36 20060101 A61K039/36; A61P 37/04 20060101
A61P037/04 |
Claims
1.-33. (canceled)
34. A process for preparing an immunogenic composition comprising
mixing a proteosome-lipopolysaccharide adjuvant with an antigen to
form the composition, wherein the proteosome-lipopolysaccharide
adjuvant is formed from an outer membrane proteosome complexed with
a lipopolysaccharide preparation, wherein both the proteosome and
the lipopolysaccharide preparation are from gram-negative bacteria,
which proteosome-lipopolysaccharide adjuvant has a final
lipopolysaccharide content by weight as a percentage of the total
proteosome protein of at least 13%.
35. The process of claim 34 wherein the antigen is selected from a
peptide, a protein, a toxoid, a glycoprotein, a glycolipid, a
lipid, a carbohydrate, and a polysaccharide.
36. The process of claim 34 wherein the antigen is derived from a
biologic or infectious organism of the animal or plant kingdom, is
an allergen or a chemically or biologically modified allergen, or
is a chemical material.
37. The process of claim 34 wherein the antigen is whole or
disrupted microorganisms selected from viruses, bacteria and
parasites, attenuated and/or inactivated.
38. The process of claim 34 wherein the antigen is produced by a
synthetic or a recombinant molecular procedures.
39. The process of claim 34 wherein the antigen is Bet v 1a.
40. The process of claim 34 wherein the antigen is rBet v 1a.
41. The process of claim 34 wherein the antigen is recombinant
influenza antigen.
42. The process of claim 34 wherein the antigen is influenza split
antigen.
43. The process of claim 34 wherein the antigen is birch pollen
extract.
44. The process of claim 34 wherein the antigen is an immunogen
extract.
45.-54. (canceled)
55. The process according to claim 34 wherein the
proteosome-lipopolysaccharide adjuvant is prepared by a process
comprising mixing an outer membrane protein proteosome preparation
prepared from a first gram-negative bacteria and a
lipopolysaccharide preparation derived from a second gram-negative
bacteria to effect complexing of the outer membrane protein
proteosome and the lipopolysaccharide to form the
proteosome-lipopolysaccharide adjuvant.
56. The process of claim 55 wherein the first gram-negative
bacteria and the second gram-negative bacteria are the same.
57. The process of claim 55 wherein the first gram-negative
bacteria and the second gram-negative bacteria are the
different.
58. The process of claim 55 wherein the first gram-negative
bacteria is selected from genus Neisseria.
59. The process of claim 58 wherein the Neisseria is Neisseria
meningitidis.
60. The process of claim 55 wherein the second gram-negative
bacteria is selected from the genera Escherichia, Shigella,
Plesiomonas, and Salmonella.
61. The process of claim 60 wherein the second gram-negative
bacteria is selected from E. coli, S. flexneri, P. shigelloides,
and S. essens.
62. The process of claim 55 wherein the outer membrane protein
proteosome preparation and the lipopolysaccharide preparation are
mixed in a detergent solution.
63. The process of claim 62 wherein the detergent solution is
EMPIGEN.RTM. BB, TRITON X-100, MEGA-10.
64. The process of claim 62 further comprising removing detergent
by a dialysis, a diafiltration, or an ultrafiltration methodology
or combinations thereof.
65. The process of 62 further comprising removing detergent by a
diafiltration or an ultrafiltration methodology or a combination
thereof.
66. The process of claim 55 wherein the mixing includes
co-precipitation and/or lyophilization of both preparations.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/094,424, filed Mar. 11, 2002, now allowed,
which claims the benefit of U.S. Provisional Patent Application No.
60/274,232, filed Mar. 9, 2001 and U.S. Provisional Application No.
60/327,297, filed Oct. 9, 2001, all of which applications (three)
are incorporated herein by reference in their entireties.
FIELD OF INVENTION
[0002] This invention relates to adjuvants for enhancing the
immunogenicity and improvement of the immune response of antigens
and to methods and compositions for preparing and using them.
BACKGROUND OF THE INVENTION
[0003] The ability of antigens to induce protective immune
responses in a host can be enhanced by combining the antigen with
immunostimulants or adjuvants. Alum-based adjuvants are almost
exclusively used for licensed injectable human vaccines, however,
while alum enhances certain types of serum antibody responses (Type
2), it is poor at enhancing other types of antibody responses (Type
1) and is a poor activator of cellular immune responses that are
important for protection against intracellular pathogens and for
therapeutic vaccines for cancer and allergy. Furthermore, alum
enhances allergic reactions due to production of IgE. Although
numerous substances have been tested and shown to be potent
adjuvants for antibody and cellular (Type 1) immune responses in
animal models, very few have proved to be suitable for use in
humans due to unacceptable levels of reactogenicity and/or
disappointing immuno-enhancing abilities. Furthermore, there are
currently no licensed adjuvants capable of enhancing immune
responses at mucosal surfaces where the majority of infectious
agents enter the host. Indeed, development of the most promising
nasally delivered mucosal adjuvants, the bacterial enterotoxins
(e.g. mutated cholera and heat-labile toxins), have been halted in
North America due to their ability to be transported to, and cause
inflammation in the olfactory bulb region of the CNS of rodents.
There is a need for potent adjuvants that are safe in humans and
capable of inducing protective systemic and mucosal humoral and
cellular immune responses.
[0004] Lipopolysaccharides (LPS) from gram negative bacteria are
potent adjuvants. LPS activates the innate immune system causing
production of inflammatory cytokines such as IL-1, TNF-.alpha.,
IL-10 and IL-12 from macrophages and dendritic cells; IL-1, IL-6
and IL-8 from endothelial cells and IL-8 from epithelial cells. In
addition, LPS is a B cell activator in mice and, to a certain
extent in humans, as evidenced by B cell mitogenicity and
stimulation of polyclonal antibody secretion. LPS mediates its
effects by binding to CD14 molecules and activation of toll like
receptors (TLR) on the surface of antigen presenting cells leading
to the initiation of a transcriptional cascade, gene expression and
secretion of pro-inflammatory molecules.
[0005] Despite the adjuvant potential of LPS, its use in humans has
been restricted due to the associated endotoxicity mediated by the
lipid A portion of the molecule. Chemical modification of the lipid
A region of LPS was shown to substantially detoxify lipid A (e.g.,
monophosphoryl lipid A or MPL-A or e.g., alkali-detoxification to
remove certain fatty acids) while maintaining certain adjuvant
properties (see Qureshi et. al., J. Biol. Chem. 1982;
257:11808-15). While MPL-A exhibited potent adjuvant activity in
animals, the experience in humans has been inconsistent, showing
poor adjuvant activity with some antigens and unacceptable
reactogenicity overall in many situations.
[0006] Non-covalent proteosome-LPS complexes, containing
proteosomes from Neisseria meningitidis and purified LPS from
Shigella flexneri or Plesiomonas shigelloides, have been
administered to humans intranasally and orally in phase 1 and phase
2 clinical trials in the context of stand-alone vaccines. These
vaccines induce protective immune responses against Shigella
flexneri or S. sonnei infection, respectively, in animals (Mallet
et. al., Infect. Immun. 1995; 63:2382-86) and humans (Fries et.
al., Infect. Immun. 2001; 69:4545-53) when given via the intranasal
route. Further, these complexes were well-tolerated via the nasal
or oral routes in humans at very high doses (up to 1.5 mg of
proteosomes along with comparable amounts of LPS given intranasally
and up to 2 mg of each of the proteosome and LPS components given
orally) (Fries et. al., 2000) and showed no olfactory bulb or other
CNS associated toxicity in small animal toxicity studies.
Proteosomes consist predominantly of porin proteins and other outer
membrane proteins. Evidence suggests that proteosome porins may
also induce IL-12 from dendritic cells and induction of CD8+ T
cells (Jeannin et. al., Nature Immunology 2000; 1:502-509) and
activation of Hela cells to produce IL-8 (Pridmore et. al., J.
Infect. Dis. 2000; 10:183). Proteosome porins also upregulate B7.2
(CD28) co-stimulatory molecules on antigen presenting cells via the
activation of the toll-like receptor 2 (Massari et. al., J.
Immunol. 2002, 168:1533-1537).
[0007] Dalseg et. al. (in Vaccines 96 pp. 177-182 (Cold Spring
Harbor laboratory Press, 1996)) report the use of meningococcal
outer membrane vesicles (OMV's) as a mucosal adjuvant for
inactivated whole influenza virus. Dalseg and his associates and
collaborators have reported that the OMV's they prepare employ a
process that retains 6% to 9% of endogenous lipooligosaccharide
(LOS) remaining compared to the amount of total OMV protein by
weight. These OMV preparations have also been reported to
specifically retain 16% of detergent (deoxycholate) in their OMV's,
an amount that may be unhealthy or toxic in toxicity studies or in
humans.
BRIEF DESCRIPTION OF INVENTION
[0008] The instant invention (IVX-908) describes compositions of
and processes for production of novel formulations that are
adjuvants for antigens and result in adjuvanted vaccines or
immunotherapeutics when the invention and antigen(s) are combined
by simple mixing and the adjuvanted vaccines or immunotherapeutics
are delivered by a parenteral or mucosal route. The adjuvant
consists of two major components. The first component is an outer
membrane protein preparation of proteosomes prepared from
gram-negative bacteria including, but not limited to Neisseria
meningitidis. The second component is a preparation of
liposaccharide. Liposaccharide includes native or modified
lipopolysaccharide (LPS) and lipooligosaccharide derived from S.
flexneri or Plesiomonas shigelloides or other gram-negative
bacteria including, but not limited to, Shigella, Plesiomonas,
Escherichia or Salmonella species. The two components may be
formulated at specific initial ratios by processes described, so as
to optimize interaction between the components resulting in stable
non-covalent complexes of the components to each other. The
processes generally involve the mixing of the components in a
selected detergent solution (e.g., EMPIGEN BB, TRITON X-100, and/or
MEGA-10) and then effecting complexing of the components while
removing detergent by dialysis or, preferably, by
diafiltration/ultrafiltration methodologies. Mixing,
co-precipitation and/or lyophilization of the two components may
also be used to effect adequate complexing or association.
[0009] The end result of the process is the production of an
adjuvant that when administered together with antigens forms an
adjuvanted vaccine or immunotherapeutic that can be delivered by a
mucosal route (such as nasal, oral, oropharyngeal, ocular,
geniturinary mucosal including vaginal, sublingual, intrapulmonary,
intratracheal or rectal) or a parenteral route (such as
intramuscular, subcutaneous, intravenous, intraperitoneal,
submucosal, intradermal) or a transdermal, topical or transmucosal
route to induce enhanced levels of serum and/or mucosal antibodies
and/or type 1 cellular immune responses against the antigen
compared with the antigen alone given by the same routes. In the
following examples, mixtures containing proteosome-LPS (using LPS
from either Shigella or Plesiomonas or Escherichia or Salmonella)
and a mono or multivalent split or purified recombinant influenza
antigen and delivered by liquid or spray or by injection as an
adjuvanted influenza vaccine induced specific anti-influenza immune
responses including, for example one or more of the following: a)
serum IgG antibodies or serum antibodies measured in functional
assays including, but not limited to, hemagglutination inhibition
(HAI) antibodies; it is noted that HAI responses are significant
since their induction is known to correlate with protection against
influenza in humans; b) mucosal antibodies including IgA in mucosal
secretions collected from the respiratory, gastrointestinal or
genitourinary tracts including, but not limited to the nasopharynx,
lungs and vagina and c) correlates of cell-mediated immunity (CMI)
including the switch or decrease from higher or predominant type 2
responses to result in mixed, balanced, increased or predominant
type 1 responses, for example, as measured by the induction of
cytokines such as IFN-.gamma. without comparable increases in
induction of certain type 2 cytokines such as IL-5 whose levels
may, for example, be maintained, decreased, or absent. Such Type 1
responses are predictive of the induction of other CMI associated
responses such as development of cytotoxic T cells (CTLs)
indicative of Th1 immunity. The ability of the adjuvant given
nasally or intramuscularly to elicit these three types of responses
against the antigen indicate that the vaccine can provide immunity
against infectious diseases since functional serum antibodies
(including HAI antibodies) and virus specific lung antibodies are
generated. Also, the induction of vaginal IgA for mucosally
administered adjuvanted vaccines using the adjuvant of the instant
invention supports utilization against mucosal infections or
allergies distal from the site of immunization such as at the
gastrointestinal or genitourinary tracts. Furthermore, the
induction of type 1 of responses assists the elimination of
residual or intracellular virus, parasite or certain bacterial
pathogens. In addition the ability of the adjuvant to produce type
1 immune responses against the antigen will be beneficial for
producing effective therapeutic vaccines for example against
cancer, autoimmune diseases and allergy where CTL and Th1 cytokine
responses are important.
[0010] For example, allergic rhinitis can often be effectively
controlled by immunotherapy--a series of injections with increasing
doses of the substance against which the individual is allergic.
Allergic rhinitis can be cured in approximately 50% of individuals
who undergo classic immunotherapy. Successful immunotherapy is
associated with one or more of the following: a switch from T cell
responses that result in the production of type 2 cytokines (e.g.,
IL-5 and IL-4) to those that produce type 1 cytokines (e.g.,
IFN-.gamma.) and/or an increase in IgG and/or reduction in IgE
specific for the allergen. However, in order to achieve these
results, up to three years of repeated immunizations are required.
The use of allergens together with adjuvants that promote type 1
immune responses may enhance the effectiveness of such
immunotherapy and reduce the number of immunizations required.
[0011] In the following example we show the results of studies in
mice immunized intranasally with IVX-908 together with rBet v 1a as
a recombinant protein representing the major allergen of Birch tree
pollen or Birch tree pollen extract. The results for both the
recombinant protein and allergen extract demonstrate that IVX-908
converts T cell cytokine production against Bet v 1a from a type 2
to a predominately type 1 phenotype. Furthermore, the type 1
response is associated with the increased production of
allergen-specific serum IgG compared with the allergen alone, and a
reduction in Bet v 1a-specific serum IgE compared with allergen
administered with aluminum phosphate, a depot and Type 2 adjuvant
known to sensitize mice for allergic responses against an allergen.
Importantly, the increase in the type 1 cytokine, IFN .gamma. was
also observed following the immunization of allergic mice with the
same allergen given with IVX-908. The pre-allergic state of the
mice mimics the situation in allergic humans, suggesting that
IVX-908/allergen formulations may be candidates for therapeutic
allergy vaccines.
[0012] It is noted that the instant invention can readily adjuvant
vaccines containing single, monovalent or multi-component antigens
such as peptides, proteins, toxoids, glycoproteins, glycolipids,
carbohydrates and/or polysaccharides, isolated from biologic
organisms of the animal or plant kingdom that may be infectious
organisms, such as parasites, viruses and bacteria, or may be
extracts or purified or chemically modified extracts of allergens
derived from unicellular or multicellular organisms or may be
chemical material. It is also envisioned that whole or disrupted
microorganisms including viruses, bacteria or parasites, attenuated
or inactivated could be used as antigen. These materials may also
be produced by synthetic or recombinant molecular procedures to
induce immunity to and protect against several strains of a
particular organism or multiple organisms or disease-causing agents
or against allergies, cancer or auto-immune diseases. The utility
in human and veterinary fields is proposed. Furthermore, the
invention can be used to enhance immunity when given together with
the antigen as an adjuvanted vaccine or immunotherapeutic as
priming or boosting immunizations prior to or subsequent to
administering the antigen (by mucosal or parenteral routes) without
the instant invention.
[0013] For parenteral, nasal, oral or suppository use, the adjuvant
may be given together with amounts of a variety of excipients or
other adjuvants including oils, emulsions, nano-emulsions, fats,
waxes, buffers, or sugars, as diluents or vehicles customary in the
art to provide stable delivery of the product in the desired
delivery format.
[0014] Of particular note, it is emphasised that using the instant
invention as an adjuvant is particularly novel since it may, in a
preferred embodiment, combine the adjuvant effect of proteosomes
together with the immunostimulatory potential of LPS. This complex
would not have been predicted to be effective from prior art since
it contains full-length LPS that is normally toxic when given
alone. As a stable proteosome complex LPS is non-toxic by the nasal
and parenteral routes in the given examples as verified by both
pre-clinical safety, immunogenicity and toxicity as well as in
clinical studies in FDA-approved phase I and phase II clinical
trials.
[0015] The instant invention may be composed of purified or
recombinant bacterial outer-membrane proteins from gram-negative
bacteria species including but not limited to Neisseria
meningitidis strains. The LPS can be derived from gram negative
bacteria such as, but not limited to Shigella or Plesiomonas or
Escherichia or a Salmonella species and can be from the same or
different species of the bacteria used to provide the outer
membrane protein proteosomes. In the preferred embodiment the final
liposaccharide or LPS content by weight as a percentage of the
total proteosome protein can be between about 13% and 300% and,
depending on the specificity of the application and route of
administration may be effective and practical for use at
liposaccharide or LPS percentages of 20% to 200%, or may be further
distinguished in a particular application at a liposaccharide
percentage of between 30% to 150%. The instant invention together
with antigen is designed to deliver adjuvanted vaccines by mucosal
(nasal, sub-lingual, oral or rectal) or parenteral (intramuscular,
subcutaneous, intradermal or transdermal) routes for use in the
prevention or treatment of cancer, autoimmune, viral or microbial
diseases or against certain toxins or biologic threat agents or
allergies whether acquired by mucosal routes such as and specially
by inhalation, or by ingestion or sexual transmission, or by
parenteral routes such as transdermal, intradermal or subcutaneous
or intramuscular.
[0016] An embodiment of the instant invention is a process for
preparing proteosomes with endogenous lipooligosaccharide (LOS)
content of between 0.5% up to about 5% of total protein. Another
embodiment of the instant invention specifies a process for
preparing proteosomes with endogenous liposaccharide of between
about 12% to about 25%, and in a preferred embodiment, between 15%
and 20% of total protein.
[0017] The instant invention specifies a composition containing
liposaccharide derived from any gram negative bacterial species
which may preferably be naturally or recombinantly different from
or the same as the gram negative bacterial species which is the
source of the proteins in the invention. The composition of the
present invention may be optimised, specifically specified by the
formulators and varied at will to contain amounts of proteosomes
and liposaccharide such that the resultant composition of the
instant invention contains liposaccharide to an amount that is at
least about 13% by weight of the weight of total proteosome protein
and in a preferred embodiment, may be from 15% to 300% and may be
further preferred, depending on the application, to be between 20%
to 200% of the total protein on a weight:weight basis or even
between 30% and 150% of the total protein.
[0018] A most preferred embodiment of the instant invention is the
adjuvant composition wherein the proteosomes are prepared from
Neisseria meningitidis and the liposaccharide is prepared from
Shigella flexneri or Plesiomonas shigelloides and the final
liposaccharide content is between 50% to 150% of the total
proteosome protein by weight.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIGS. 1A and B show, respectively two embodiments for the
manufacture of proteosome bulk material (Flow Chart 1A and Flow
Chart 1B).
[0020] FIG. 2 shows a scheme for the manufacture of S. flexneri 2a
LPS (Flow Chart 2).
[0021] FIG. 3 shows a scheme for the manufacture of IVX-908
proteosome-LPS adjuvant (Flow Chart 3).
[0022] FIGS. 4 A and 4B show the levels of specific serum IgG (A)
and lung lavage IgA (B) elicited when a constant amount of HA was
mixed with different amounts of IVX-908 and used to immunize mice
intranasally. FIGS. 4C and 4D show the levels of specific serum IgG
(C) and lung lavage IgA (D) elicited when a constant amount of
IVX-908 (either 1 or 0.3 .mu.g) was mixed with different amounts of
HA and used to immunize mice intranasally.
[0023] FIG. 5A shows the numbers of immunized (n=10) or control
(n=9) mice surviving challenge with a live, mouse-adapted,
homotypic variant influenza virus. FIG. 5B shows mean weight loss
(a measure of morbidity associated with infection by influenza
virus) in the survivors in each group. Error bars indicate standard
errors on the mean.
[0024] FIG. 6 shows specific antibody responses in serum of mice
immunized i.n. or i.m. with Ovalbumin with or without IVX-908.
Titers are expressed as geometric mean concentrations of specific
IgG (.mu.g/ml) with 95% confidence limits indicated by error
bars.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Results show the following activities of IVX-908 adjuvant
when mixed with recombinant and split antigens from influenza
virus:
A. By the Injectable Route:
[0026] 1. Induces up to eight-fold increases in serum HAI and IgG
compared with injectable split flu influenza vaccine alone [0027]
2. Shifts elicited immune responses to Type 1 (CMI) responses
compared to split flu influenza vaccine alone
B. By the Nasal Route:
[0027] [0028] 1. Induces >100-fold increases in serum HAI and
IgG responses, compared with split flu influenza antigen alone
given by the nasal route [0029] 2. Induces up to 10-fold higher
specific serum HAI and IgG compared with split flu given by
injection [0030] 3. Induces >100-1000 fold higher specific IgA
in lung and/or nose compared with split flu influenza antigen alone
given nasally or by injection [0031] 4. Induces up to 160-fold
higher specific IgA in genital tract compared with split flu
influenza antigen alone given nasally or by injection [0032] 5.
Shift to Type 1 (CMI) responses compared to split flu alone [0033]
6. Amounts of IVX-908 as little as 0.3 .mu.g to 1 .mu.g are
sufficient to achieve optimal enhancement of serum IgG responses
against split-flu HA [0034] 7. Recombinant influenza HA
co-administered with IVX-908, induces responses which are
protective against mortality and morbidity, and superior to those
induced by injection or i.n. administration of the antigen alone
[0035] 8. IVX-908 prepared at protein:LPS ratios of 3:1 to 1:3
using LPS from Shigella, Escherichia and Salmonella species were
effective.
[0036] The results show that respiratory or parenteral immunization
with the instant invention and influenza split flu antigen induces
enhanced specific anti-influenza HA antibody formation in each of
the serum and mucosal bio-samples compared to immunizing with the
influenza split product without adjuvant.
[0037] Results show the following activities of IVX-908 adjuvant
when mixed with rBet v 1a, the major allergen from Birch pollen as
either recombinant allergen or Birch pollen allergen extract and
administered via the nasal route. [0038] 1. The nasal IVX-908 and
rBet v 1a mixture enhanced induction of the type 1 cytokine,
IFN-.gamma. by 50- and 74-fold compared with Bet v 1a alone and Bet
v 1a formulated in aluminium phosphate respectively. The nasal
IVX-908 and Birch pollen extract (BPEx) mixture enhanced induction
of the type 1 cytokine, IFN-.gamma. by >44- and 3-fold compared
with Bet v 1a alone and Bet v 1a formulated in aluminium phosphate
respectively. [0039] 2. The increases in IFN-production by the
IVX-908/Bet v 1a and IVX-908/BPEx mixtures were not associated with
an increase in IL-5 secretion, indicating that IVX-908 directed the
immune response against Bet v 1a towards a type 1-biased T cell
response. [0040] 3. Serum IgE induced by the IVX-908 Bet v 1a and
IVX-908/BPEx mixtures were approximately 37- and 44-fold lower than
that induced by the allergens given with aluminium phosphate,
respectively. [0041] 4. Allergen-specific serum IgG was increased
by >400-fold and 22-fold for mice immunized with the IVX-908/Bet
v 1a and IVX-908/BPEx mixtures compared with Bet v 1a and BPEx
alone, respectively. [0042] 5. In mice sensitized with Bet v 1a
plus alum, the production of the type 1 cytokine, IFN-.gamma. was
increased by 4.7- and 33-fold following immunization with
IVX-908/rBet v 1a and IVX-908/BPEx, respectively, compared with the
corresponding allergens alone. In these mice, the levels of the
type 2 cytokine, IL-5 were reduced compared to the corresponding
allergens alone. [0043] 6. In mice immunized nasally with
IVX-908/allergen mixtures and subsequently given a sensitizing
injection with Bet v 1a plus alum the type 1 cytokine, IFN-.gamma.
increased by 10-fold compared with birch pollen extract alone. In
these mice, the levels of the type 2 cytokine, IL-5, were not
similarly elevated and indeed were somewhat reduced compared to
birch pollen extract alone.
[0044] The results demonstrate that IVX-908/allergen formulations
induce strong type 1 cytokine responses in allergen naive and
sensitized mice, suggesting that these formulations prepared with
purified or recombinant proteins or extracts of allergens may be
used as vaccines or therapeutics for specific immunotherapy for
allergic diseases.
[0045] Results show the following activities of IVX-908 adjuvant
when mixed with ovalbumin (OVA), a known poor immunogen and given
by the nasal or injectable route. [0046] 1. Enhances OVA-specific
serum IgG titers by greater than 60- and 75-fold via the nasal and
injectable routes respectively compared with antigen alone, [0047]
2. Enhances the secretion of OVA-specific IFN-.gamma. and IL-5 from
re-stimulated splenocytes compared with antigen alone resulting in
a balanced type of immune response.
EXAMPLES
Example 1
Production of Proteosomes
[0048] Two examples of outer membrane protein proteosome
preparations are shown. These preparations were purified from type
2 Neisseria meningitidis by extraction of phenol-killed bacterial
paste with a solution of 6% EMPIGEN BB (EBB) (Albright and Wilson,
Whithaven, UK) in 1 M calcium chloride followed by precipitation
with ethanol, solubilization in 1% EBB-Tris/EDTA-saline and then
precipitation with ammonium sulphate. The precipitates were
re-solubilized in the 1% EBB buffer, diafiltered and stored in an
EBB buffer at -70.degree. C. A flow chart of this process, which
resulted in proteosomes having a liposaccharide content of between
0.5% and 5%, is shown in Flowchart 1A (FIG. 1A) on the following
pages. Proteosomes may also be prepared by omitting the ammonium
sulphate precipitation step to shorten the process as desired with
resultant proteosomes having a liposaccharide content of between
12% and 25%, and may, depending upon the materials, be between 15%
and 20% as shown in Flowchart 1B (FIG. 1B).
Example 2
Production of Liposaccharides
[0049] The example in Flowchart 2 (FIG. 2) shows the process for
the isolation and purification of LPS from S. flexneri or P.
shigelloides bacteria. This process can similarly be used for
preparing LPS from other gram-negative bacteria including, but not
restricted to Shigella, Plesiomonas, Escherichia and Salmonella
species. Following growth of the bacteria by fermentation, the cell
paste was re-hydrated with 3 mL 0.9M NaCl, 0.005 M EDTA/g paste.
Ten mg lysozyme/g paste was also added. Lysozyme digestion was
allowed to proceed for 1 hour at room temperature. Fifty U/mL
Benzonase (DNase) was then added with 0.025M MgCl.sub.2. DNase
digestion was allowed for 30 minutes to proceed at room
temperature. The suspension was then cracked by passage through a
microfluidizer at 14,000 to 19,000 psi. Fresh DNase (50 U/mL) was
added and the suspension was digested for a further 30 min at room
temperature. The digested cell suspension was heated to 68.degree.
C. in a water bath. An equal volume of 90% phenol (at the same
temperature) was added and the mixture was incubated with shaking
at 68.degree. C. for 30 min. The mixture was centrifuged at
4.degree. C. to separate the aqueous and organic phases. The
aqueous phase was harvested and the organic phase was re-extracted
with WFI (water for injection) at 68.degree. C. for 30 min. The
mixture was centrifuged at 4.degree. C. to separate the aqueous and
organic phases and the aqueous phases were combined. Twenty percent
ethanol and 10 mM CaCl.sub.2 were added to the combined aqueous
phase to precipitate nucleic acids. The mixture was stirred at
4.degree. C. overnight. Precipitated nucleic acids were then
pelleted by centrifugation at 10,000.times.G for 30 minutes and the
supernatant was collected.
[0050] The supernatant was concentrated and diafiltered using a
30,000 MW hollow fiber cartridge into 0.15M NaCl, 0.05M Tris, 0.01M
EDTA and 0.1% EMPIGEN BB, pH 8.0. Finally, the LPS was
sterile-filtered using a 0.22 .mu.m Millipak 60 filter unit
aliquoted into sterile storage containers and frozen at -80.degree.
C.
Example 3
Preparation and Characterisation of a Proteosome-Liposaccharide
Adjuvant Complex
[0051] The adjuvant is manufactured by non-covalently complexing
Proteosomes to LPS. The LPS can be derived from any of a number of
gram negative bacteria including, but not limited to Shigella or
Plesiomonas or Escherichia or Salmonella species as described in
Flowchart 3. Briefly, Proteosomes and LPS were thawed overnight at
4.degree. C. and adjusted to 1% EMPIGEN BB in TEEN buffer.
Proteosomes were thawed overnight and adjusted to 1% EMPIGEN BB in
TEEN buffer. The two components were mixed at quantities resulting
in a final Proteosome:LPS wt/wt ratio of between 10:1 and 1:3 and
stirred for 15 minutes at room temperature. The LPS-Proteosome
mixture was diafiltered on an appropriately sized (e.g., Size 9)
10,000 MWCO hollow fiber cartridge into TNS buffer (0.05 M Tris,
150 mM NaCl pH 8.0). The diafiltration was stopped when EMPIGEN
content in the permeate was <50 ppm (by EMPIGEN Turbidity Assay
or by a Bradford Reagent Assay). The bulk adjuvant IVX-908 was
concentrated and adjusted to 5 mg/mL protein (by Lowry assay).
Finally, the adjuvant was sterile-filtered using a 0.22 .mu.m
Millipak 20 filter unit. The bulk adjuvant was aliquoted into
sterile storage containers and frozen.
[0052] The Proteosome-LPS complex was tested for EMPIGEN (400 ppm)
using reverse-phase HPLC; protein content by Lowry, LPS content by
measurement of 2-keto-3-deoxyoctonate (KDO) assay. The said
invention was further characterized for particle size distributions
as determined by quantitative number weighted analysis using a
particle sizer (Brookhaven Instruments model 90 plus or similar
machine) (10-100 nm). However, the particle size for the complex
will increase with a higher proteosome to LPS ratio. Stability of
the complex formulations should be consistent with the previously
demonstrated S. flexneri LPS vaccine. These data demonstrate
complex stability at both refrigerated and accelerated temperature
(25 and 37.degree. C.).
Example 4
Preparation of IVX-908 Influenza Antigen or Birch Pollen Allergen
or Ovalbumin Mixtures
[0053] The current invention was prepared by mixing the IVX-908
Proteosome-LPS adjuvant (Example 3) with antigen in proportions
that promote optimal formulations for stability and immunological
outcomes.
Example 5
Mouse Immunization Protocols for Influenza Antigen Studies
[0054] BALB/c mice were immunized intranasally or intramuscularly
on days 1 and 21 with antigens in volumes of 25 or 100 l
respectively containing between 0.3 and 3 .mu.g influenza
hemagglutinin (HA) as A/Beijing/262/95 or an A/Beijing/262/95 plus
A/Sydney/5/97 bivalent detergent split antigen (GMP commercial
licensed antigen) either alone or mixed with 0.3-3 .mu.g IVX-908
adjuvant (containing LPS at various Proteosome:LPS wt/wt ratio).
Control mice were given intranasal immunizations with phosphate
buffered saline. Animals were bled on day 35 via the saphenous vein
or by cardiac puncture. Nasal or lung lavage or vaginal washes were
taken on day 35. The lungs of each mouse were surgically exposed
and a canula inserted in the trachea. Using a syringe containing
phosphate buffered saline supplemented with 0.1% bovine serum
albumin and protease inhibitors (0.2 mM AEBSF, 1 g/ml Aprotinin,
3.25 .mu.M Bestatin and 10 .mu.M Leupeptin), 1 nasal lavage sample
(approximately 1 ml) and 2 lung lavage samples (2.times.1 ml) were
collected. The lung lavage fluids from individual animals were
combined, vortexed and centrifuged to remove cell debris, and
supernatants stored at -70.degree. C. until assayed by ELISA.
Vaginal washes were performed by inserting a tampon wetted with 25
.mu.l of supplemented phosphate buffered saline (as above), into
the vagina of the mouse for 30 seconds. After removing the tampon,
the procedure was repeated with the opposite end of the tampon. The
tampon was stored frozen at -70.degree. C. and reconstituted in
ELISA blocking buffer (see Example 8) at time of assay.
Example 6
Serum Hemagglutination Inhibition Assay (HAI)
[0055] Prior to determination of HAI activity, mouse sera were
heated at 56.degree. C. to inactivate complement. Elimination of
non-specific agglutination was achieved by treating mouse sera with
receptor destroying enzyme (RDE). To 0.1 ml of serum was added 0.4
ml of RDE (100 units/ml) for 12 to 18 hr at 37.degree. C. 0.3 ml of
sodium citrate (2.5%) was added for 30 min at 56.degree. C. to
inactivate the RDE. The sample volume was made up to 1 ml with PBS
(to give final sample dilution of 1:10). Two-fold serial dilutions
of each sample were tested for their ability to inhibit the
agglutination of 0.5% chick red blood cells by whole influenza
virus in a standard HAI assay.
Example 7
Measurement of Specific Anti-Flu Antibodies in Sera, in Lung, Nasal
and Vaginal Fluids
[0056] Sera were collected after each immunization; lung and nasal
cavity lavage fluids and vaginal washes were collected after the
last immunization. ELISA was performed using whole virus or
detergent split antigen as the detecting antigen. Briefly, 96 well
round bottom microtiter plates (e.g., Costar EIA/RIA 96 well
Easywash Plates, Corning, N.Y.) were coated with antigen and
incubated overnight. After aspiration of the antigen using a plate
washer, plates were washed once with PBS containing 0.1% Tween
(PBS-T) and incubated with blocking buffer containing PBS-T plus 2%
powdered milk. After aspirating the blocking buffer and washing
with PBS-T, samples of sera, lung or nasal cavity lavage fluids, or
vaginal washes serially diluted 2-fold in blocking solution, were
added and the plates were incubated for two hours at 37.degree. C.
After washing with PBS-T, affinity purified horseradish peroxidase
(HRP)-labelled goat anti-mouse IgG or IgA was added and plates were
incubated at 37.degree. C. for 30 min. After aspirating and washing
twice with PBS-T, developing solution was added and plates were
incubated for 30 min at r.t. and stopped by addition of
H.sub.2SO.sub.4 prior to determining the absorbance values using a
microtiter ELISA plate reader (Molecular Devices, Menlo Park,
Calif.). Antibody titers in the Tables are expressed as ng/ml of
specific IgG or IgA determined from a standard curve produced using
an ELISA capture assay using affinity purified mouse IgG and IgA
standards (Sigma).
Example 8
Enhanced Immunogenicity and Immunity Elicited by IVX-908 Adjuvanted
Influenza Vaccines
[0057] This example shows the serum and mucosal antibody responses
induced following nasal immunization with monovalent
(A/Beijing/262/95) or nasal or intramuscular immunization with
bivalent (A/Beijing/262/95 plus A/Sydney/5/97) antigens given with
or without IVX-908 adjuvants. Mice received 2 doses of antigen
containing 0.3 .mu.g HA and IVX-908 (which IVX-908 consists of a
1:1 wt/wt proteosome to LPS ratio with 1.2 .mu.g of proteosome
protein for every 0.3 .mu.g of HA) per strain of influenza antigen
used. Anti-influenza IgG antibodies in sera were analysed by HAI;
IgG in sera and IgA antibodies in lung and nasal cavity fluids were
analysed by ELISA. Results are shown and summarised in Tables 1-3.
Briefly:
IVX-908 Adjuvanted Influenza Vaccine Given Nasally:
[0058] 1. elicited between 50 to 250-fold higher serum IgG
responses than the split Flu influenza antigen alone given nasally
and up to 10-fold greater than the influenza vaccine given by
injection (i.m.) (Tables 1-3), [0059] 2. elicited 16 to 100-fold
higher serum HAI responses than split Flu alone given nasally and
up to 8-fold higher than elicited by giving the split product
influenza vaccine alone by injection (Tables 1-3), [0060] 3.
elicited between 20 to 120-fold higher IgA responses in the nasal
cavity than the split Flu influenza vaccine alone given nasally or
by injection (i.m.) (Table 1), [0061] 4. elicited 50 to
>600-fold higher specific IgA responses in the lung than split
Flu influenza vaccine alone given nasally or by injection (i.m.)
(Tables 1-3), [0062] 5. induced 30 to >160-fold increases in
specific vaginal IgA compared with split Flu influenza vaccine
alone given nasally or by injection (Table 2).
IVX-908 Adjuvanted Influenza Vaccine Given Intramuscularly:
[0062] [0063] 1. induces up to 5-fold increases in specific serum
IgG and up to 8-fold increase in serum HAI compare to the split Flu
influenza vaccine alone given by injection (table 3)
[0064] The data demonstrate that IVX-908 prepared with LPS from
either P. shigelloides (Tables 1 and 3) or S. flexneri (Table 2)
when mixed with influenza split antigens, enhances both the serum
and mucosal antigen-specific immune responses. Furthermore, IVX-908
adjuvanted the HA-specific immune responses against each of the
individual monovalent HA antigens when given as a multivalent
preparation (Tables 2 and 3).
TABLE-US-00001 TABLE 1 Adjuvant effect of IVX-908 via the
intranasal route with monovalent antigen. Murine serum HAI, IgG and
mucosal IgA induced by split flu antigen (A/Beijing/262/95) mixed
with IVX-908 adjuvant (3 .mu.g HA per dose at 4:1 IVX-908:HA ratio)
following nasal immunization. Split Flu + IVX-908 nasal Split nasal
Split IM PBS Serum IgG (ng/mL)* 3,205,360 24,774 290,844 250 HAI
(GMT)** 640 .ltoreq.10 160 .ltoreq.10 Lung IgA (ng/mL)* 6,168 32 10
10 Nasal IgA (ng/mL)* 1,531 85 13 10 All samples taken 14 days post
2.sup.nd immunization. IVX-908 prepared with P. shigelloides LPS.
*are Geometric Means for 10 mice/group **HAI for sera pooled from
10 mice/group
TABLE-US-00002 TABLE 2 Adjuvant effect via the nasal route with
bivalent antigen. Murine anti-A/Beijing/262/95 (H1) serum HAI, IgG
and mucosal IgA induced by bivalent split flu antigen
(A/Beijing/262/95 H1 and A/Sydney/5/97 (H3) mixed with IVX-908
adjuvant (0.3 .mu.g HA/strain per dose at 4:1 IVX-908:HA ratio)
given nasally Split nasal Split IM PBS A. Anti-A/Beijing/262/95
(H1) response Split Flu + IVX-908 nasal Serum IgG (ng/mL)* 427,600
1,682 97,810 2000 HAI (GMT)** 160 .ltoreq.10 20 .ltoreq.10 Lung IgA
(ng/mL)* 1,276 5 10 4 Vaginal IgA (ng/mL)* 833 8 5 4 B.
Anti-A/Sydney/5/97 (H3) response Split Flu + IVX-908 Serum IgG
(ng/mL)* 32,835 643 84,712 2000 HAI (GMT)** 80 .ltoreq.10 320
.ltoreq.10 Lung IgA (ng/mL)* 358 4 4 4 Vaginal IgA (ng/mL)* 141 5 4
4 All samples taken 14 days post 2.sup.nd immunization IVX-908
prepared with S. flexneri LPS. are Geometric Means for 10
mice/group **HAI for sera pooled from 10 mice/group
TABLE-US-00003 TABLE 3 Adjuvant effect via the nasal or
intramuscular route. Murine anti-A/Beijing/262/95 (H1) serum HAI,
IgG and mucosal IgA induced by bivalent split flu antigen
(A/Beijing/262/95 H1 and A/Sydney/5/97 (H3) mixed with IVX-908
adjuvant (0.3 .mu.g HA/strain per dose at 4:1 IVX-908:HA ratio)
given nasally or intramuscularly Nasal Muscular Immunization
Immunization Split Flu + Split Split Flu + Split IVX-908 Flu
IVX-908 Flu PBS A. Anti-A/Beijing/262/95 (H1) response Serum IgG
313,369 1,682 488,665 97,810 2000 (ng/mL)* HAI (GMT)** 160
.ltoreq.10 160 20 .ltoreq.10 Lung IgA (ng/mL)* 1,006 5 16 10 4 B.
Anti-A/Sydney/5/97 (H3) response Serum IgG 62,064 643 253,860
84,712 2,000 (ng/mL)* HAI (GMT)** 160 .ltoreq.10 320 320 20 Lung
IgA (ng/mL)* 200 4 10 4 4 All samples taken 14 days post 2.sup.nd
immunization. Adjuvant prepared with P. Shigelloides LPS. are
Geometric Means for 10 mice/group **HAI for sera pooled from 10
mice/group
Example 9
The Shift of Immune Responses from Type 2 to Type 1 by Nasal
Proteosome Influenza Vaccines
[0065] Spleen cell cultures from mice immunized with Proteosome-LPS
adjuvanted and non-adjuvanted influenza split antigens were
analyzed for their production of T cell cytokines interferon gamma
(IFN-.gamma.) and IL-5 as an indicator of induction of Th1 or Th2
type immune responses, respectively. Briefly, Balb/c mice were
immunized either intranasally or intramuscularly as described in
Example 6 with a bivalent formulation containing 3 .mu.g influenza
HA from with A/Beijing/262/95 plus A/Sydney/5/97 with or without 24
.mu.g IVX-908 Proteosome-LPS. Mice were euthanized 14 days after
the second immunization and the spleens from 5 mice from each group
were harvested and cells teased into a single cell suspension using
a 100-.mu.m nylon cell strainer (Becton Dickinson, N.J.). Spleen
cells were cultured at 2.0.times.10.sup.6 cells/ml (200 .mu.l/well)
in RPMI 1640 medium (Gibco BRL, Life technologies, Burlington, ON)
containing 8% fetal bovine serum (heat-inactivated for 1 hr at
56.degree. C.; Gibco BRL), 2 mM glutamine (Gibco BRL), 50 .mu.M
2-mercaptoethanol (Sigma Chemical Co., St-Louis, Mo.) and 50
.mu.g/ml gentamycin (Gibco BRL) with or without UV-inactivated,
purified A/Beijing/265/95 (H1N1) and IVR-108 reassortant (H3N2)
influenza viruses (NIBSC, Hertfordshire, UK) in 96-well cell
culture cluster (Corning, N.Y.). Cells were incubated for 72 hrs at
37.degree. C. and supernatants harvested and frozen at -80.degree.
C. Murine cytokines levels were measured using sandwich ELISA kits
(OptEIA.RTM. set, purchased from Pharmingen, San Diego, Calif.)
according to manufacturer's instructions. Recombinant cytokines
were used as standards.
[0066] Briefly, results in Table 4 demonstrate that IVX-908 given
together with a multivalent bivalent split flu antigen to form an
adjuvanted influenza vaccine given either nasally or
intramuscularly induces uniquely the type 1 cytokine, INF , without
detectable IL-5, a type 2 cytokine. Conversely, bivalent influenza
antigen alone given nasally or intramuscularly induces a mixed type
1 and type 2 immune response as evidenced by the production of both
INF-.gamma. and IL-5. These results indicate that IVX-908 induces
enhanced antigen-specific serological responses and biases T cell
responses against antigens towards the type 1 of immunity. Type 1
immune responses are important for the clearance of intracellular
pathogens, for the development of anti-tumor responses and in the
control of allergic responses.
TABLE-US-00004 TABLE 4 Murine cytokine induction from spleen cells.
Mice were immunized with bivalent split flu antigen
(A/Beijing/262/95 H1 and A/Sydney/5/97 H3) and IVX-908 adjuvant (3
.mu.g HA/strain per dose at 4:1 IVX-908:HA ratio) given nasally or
intramuscularly. IVX-908 adjuvant was prepared with P. shigelloides
LPS. Spleen cells were re-stimulated with whole inactivated
A/Beijing/262/95 (H1) or a Sydney (H3) reassortant. Nasal Muscular
Immunization Immunization Cytokine Split Flu + Split Flu + (pg/mL)
IVX-908 Split Flu IVX-908 Split Flu A. A/Beijing/262/95 (H1)
immunization and re-stimulation INF-.gamma. 6934 272 171 834 IL-5 0
173 0 277 B. A/Sydney/5/97 (H3) immunization and re-stimulation
INF-.gamma. 9,690 0 2,657 4111 IL-5 0 635 0 820
[0067] INF-.gamma. and IL-5 were determined in supernatants of
mouse spleen cells re-stimulated as described in Example 10 with
whole inactivated virus (1.25 .mu.g/mL) and are expressed in
.mu.g/mL of culture supernatant. Results are the means of
triplicate cultures, and have had the values obtained for
IFN-.gamma. and IL-5 (pg/mL) from spleen cells of PBS immunized
mice already subtracted.
Example 10
Defining Optimal Amounts and Ratios of IVX-908 and Hemagglutinin
Antigen to Maximise Adjuvantation
[0068] Mice were immunized i.n. on days 0 and 14 with 1 .mu.g of HA
(H3N2 strain, A/Sydney/5/97) mixed with IVX-908 (proteosome
protein:S. flex LPS, 1:1) in decreasing amounts from 10 .mu.g to
0.03 .mu.g. A subsequent study varied the amount of HA from 3 to
0.3 .mu.g while keeping the amount of IVX-908 constant at 1 or 0.3
.mu.g. In both studies, blood, lung lavage, nasal wash fluid and
spleens were collected at euthanasia on day 21 and analyzed for IgG
or IgA content, or used to prepare splenocytes for in vitro
stimulation as appropriate (as described in Example 9 above).
Significance of the data was assessed by ANOVA analysis using
Tukey-Kramer pair-wise comparisons.
[0069] FIGS. 4A and 4B show that above a threshold at 0.3-1 .mu.g
of IVX-908, the elicited immune responses leveled-off, and below
this threshold, the elicited responses diminished significantly.
Keeping the amount of IVX-908 constant at this threshold, a second
study was performed varying the amount of HA between 3 .mu.g and
0.3 .mu.g. The results in FIGS. 4C and 4D show that maximal
systemic and mucosal immune responses were obtained when HA was
mixed with IVX-908 above a threshold of 1-3 g of HA (administered
i.n. with either 0.3 .mu.g or 1 .mu.g of IVX-908). The results
indicate that in order to elicit optimal immune responses in mice,
1-3 .mu.g of HA should be mixed with 0.3-1 .mu.g of IVX-908.
[0070] As in other studies, analysis of the cytokines released from
in vitro stimulated splenocytes showed that i.n. administration of
HA with IVX-908 elicited responses primarily of type 1
phenotype.
Example 11
Enhancement of Systemic and Mucosal Immune Responses, and
Protection Against Live Virus Challenge, Elicited by Intranasal
Administration of Recombinant Hemagglutinin Mixed with IVX-908
[0071] Baculovirus-derived recombinant influenza hemagglutinin
(rHA; H1N1 strain A/Texas/36/91), supplied as a full-length
uncleaved protein (HA0), was purchased from a commercial source.
The immunogenicity of the rHA was assessed by immunization of
groups of 15, 6-8 week old female BALB/c mice. For intranasal
(i.n.) immunizations, mice were lightly anesthetized, 25 .mu.l of
vaccine containing 2 g of rHA with or without IVX-908 (8 .mu.g
proteosome protein and 8 g S. flex LPS), or PBS was applied to the
nares (12.5 l per nostril) and the mice allowed to inhale the
droplets. Intramuscular (i.m.) immunization was achieved by
injection of 25 .mu.l (2 .mu.g rHA) into the hind limbs. All mice
were immunized on days 0 and 21. Ten animals from each group were
challenged on day 48 by i.n. instillation of 8 LD.sub.50 of
mouse-adapted homotypic variant influenza virus (A/Taiwan/1/86) to
assess protection. Any deaths were recorded, and weight loss was
used as a surrogate for morbidity; mice were weighed immediately
before and every 2 days after challenge. Mice losing .gtoreq.30% of
their pre-challenge body weight or showing a lesser weight loss
(.gtoreq.20%) in conjunction with other clinical signs of distress
and/or morbidity (e.g., pilo-erection, hunched posture, reduced
mobility) were deemed to have met the experimental endpoint
criteria and were euthanized. The 5 non-challenged mice from each
group were euthanized on day 51 and exsanguinated by cardiac
puncture. Serum was separated from clotted blood and stored at
-70.degree. C. until assay. Spleens were removed aseptically and
processed for in vitro re-stimulation (as described in Example 9
above). Nasal washes and lung lavage were performed as previously
described.
[0072] Table 5a shows the systemic and mucosal responses in samples
collected on day 51, and Table 5b shows the amounts of IFN-.gamma.
and IL-5 released from splenocytes following specific in vitro
stimulation. FIG. 5 a) shows the protection against mortality, and
b) protection against morbidity, in immunized or control mice
following challenge with live, homotypic variant, mouse-adapted
virus.
[0073] The results demonstrate that: [0074] 1 Serum responses
elicited by IVX-908+rHA were 4.times. and 100.times. higher
respectively than the responses induced by rHA alone given i.m. or
i.n. [0075] 2 Only i.n. rHA administered with IVX-908 elicited
detectable mucosal IgA responses. [0076] 3 I.n. immunization with
IVX-908+rHA induced responses of type 1 phenotype in contrast to
i.m. rHA alone which induced responses of type 2 phenotype. [0077]
4 In contrast to rHA immunized or control mice, all mice ( 10/10)
immunized i.n. with IVX-908+rHA survived live virus challenge. 8/10
and 1/10 mice immunized i.m. or i.n. with rHA alone survived whilst
no control mice survived. [0078] 5 Mice immunized i.n. with
IVX-908+rHA suffered no weight loss following challenge. The
surviving mice immunized with rHA alone by either i.n. or i.m.
routes, all lost significant amounts of weight, indicating
morbidity as a result of infection following challenge. Thus i.n.
IVX-908+rHA protected mice against morbidity as well as mortality
following challenge.
[0079] Table 5a shows the systemic and mucosal responses elicited
by immunization of mice with 2 .mu.g of rHA, with or without
IVX-908, as described in example 11. HI titer is the reciprocal of
the maximum dilution of serum which will inhibit hemagglutination,
and immunoglobulin levels (IgG or IgA) are expressed as Geometric
Mean Concentrations with 95% confidence limits in parentheses.
ND=not detected.
TABLE-US-00005 IVX-908 + rHA (IN) rHA (IM) rHA (IN) PBS HI titer
1280 320 10 10 Serum 109.3 (51.5-232.3) 25.0 (12.1-51.4) 1.1
(0.9-1.4) 1.0 IgG (ug/ml) Nasal IgA 77 (30-196) ND ND ND (ng/ml)
Lung IgA 265 (112-629) ND ND ND (ng/ml)
[0080] Table 5b shows the amounts (pg/ml; determinations performed
in triplicate) of IFN-.gamma. and IL-5 released into culture
supernatants following in vitro stimulation of splenic T cells from
mice immunized with 2 .mu.g of vaccine or control material.
Splenocytes were restimulated with inactivated mouse-adapted
A/Taiwan influenza virus.
TABLE-US-00006 IVX-908 + rHA rHA (IM) rHA (IN) PBS IFN-.gamma.
(pg/ml) 12960 2918 3081 3266 IL-5 (pg/ml) 3 34 3 3
Example 12
Induction of Serum and Mucosal Antibodies and Shift of Immune
Responses from Type 2 Towards Type 1 by Nasal IVX-908/Bet v 1a
Allergen Formulation
[0081] Recombinant Bet v 1a protein was expressed in E. coli with a
His-Tag (His) added at the amino terminus and purified by affinity
chromatography on nickel columns. BALB/c mice were immunized
intranasally (in volumes of 28 .mu.l (Table 6) or 36 .mu.l (Table
7) three times at two (Table 7) or three (Table 6) weeks apart with
either 10 .mu.g Bet v 1a as purified recombinant protein (rBet v
1a) or birch pollen extract (BPEx) (Greer Labs. Inc.) alone or as a
mixture of 10 .mu.g rBet v 1a or BPEx plus 10 .mu.g of IVX-908
(Tables 6 and 7). Control mice were given intranasal immunizations
with phosphate buffered saline (PBS). Other mice were given 10
.mu.g Bet v 1a in 2 mg aluminum phosphate intraperitoneally in a
volume of 150 .mu.l on days 0 and 21 (Table 6), or 3 .mu.g birch
pollen extract (BPEx) (Greer Labs. Inc.) in 1 mg aluminum phosphate
on day 0 (Table 7). One (Table 6) or three (Table 7) weeks after
the final immunization, animals were bled by cardiac puncture
subsequent to obtaining lung lavage fluids. Bet v 1a-specific IgE
(OptEIA.RTM. Mouse IgE Set; BD Pharmingen, Mississauga, Ontario),
IgG, IgG1 and IgG2a in serum, and IgA and total IgA in
broncho-alveolar lavages were measured by ELISA. The levels of
secreted IFN-.gamma. and IL-5 were determined in the supernatants
from spleen cell cultures (10.times.10.sup.6 splenocytes/mL) after
two and three days respectively following re-stimulation in vitro
with 10 .mu.g/ml Bet v 1a. Cytokines were detected by ELISA (BD
Pharmingen; Mississauga, Ontario). In table 8, an example is shown
for cytokine induction in mice injected intraperitoneally on day 71
with a single dose of 10 .mu.g rBet v 1a in 2 mg aluminum phosphate
following 3 nasal immunizations on days 0, 17 and 29 with 10 .mu.g
birch pollen extract (BPEx) (Greer Labs. Inc.) alone or as a
mixture with 10 .mu.g of IVX-908. In Table 9, an example is shown
for cytokine induction following 3 immunizations of rBet v 1a or
BPEx with or without IVX-908 in mice previously sensitized
intraperitoneally with a single dose of 10 .mu.g Bet v 1a in 2 mg
aluminum phosphate.
[0082] Results for T cell cytokine and serum and mucosal
immunoglobulin responses following intranasal immunization with an
IVX-908/rBet v 1a or an IVX-908/BPEx mixture are shown in Tables 6,
7, 8 and 9.
IVX-908 Adjuvanted rBet v 1a or BPEx Given Nasally to Naive Mice
(Tables 6 and 7): [0083] 1. directed the T cell response induced by
Bet v 1a allergen from a type-2 biased to a higher or predominantly
type-1 phenotype. This was due to the enhanced production of
IFN-.gamma. by spleen cells from mice given IVX-908 formulated
allergen compared to rBet v 1a or BPEx alone or with aluminum
phosphate with a lowering (for IVX-908/BPEx) or maintenance (for
IVX-908/rBet v 1a) of the production of IL-5, [0084] 2. enhanced
production of Bet v 1a-specific serum IgG compared with rBet v 1a
or BPEx given alone, and, [0085] 3. produced a 37-43 fold reduction
in levels of serum IgE levels compared with that induced by rBet v
1a in aluminium phosphate, an immunizing regime known to sensitize
animals for allergic responses on subsequent challenge with
antigen.
TABLE-US-00007 [0085] TABLE 6 Induction of murine cytokines and
serum and mucosal antibodies by 10 ug rBet v 1a alone or formulated
with IVX-908 (10 ug 1:1 protein:LPS) via the nasal route, or with 2
mg aluminium phosphate by the intraperitoneal route as described in
Example 10. rBet v 1a + rBet v rBet v 1a IVX-908 1a + Alum PBS
IFN-.gamma. (pg/mL) 53 2,598 35 0 IL-5 (pg/mL) 965 905 1,746 0
IL-5/IFN-.gamma. ratio 18 0.4 50 0 Serum IgE (ng/mL) 8 77 2,832 8
Serum IgG (ng/mL) 27 11,111 901,497 3.8 Lung IgA/total IgA (%) 1.3
0.4 1.7 0.4
Results for IFN-.gamma. and IL-5 are expressed as the mean pg/mL
for triplicate cultures from spleens pooled from 5 mice/group.
Serum IgG is expressed as the sum of IgG1 and IgG2a titers. Lung
IgA is shown as specific IgA expressed as % total IgA. All
immunoglobulin titers were calculated using geometric mean titers
for samples from 7 to 10 (IgG and IgE) or 5 (IgA) mice/group.
IVX-908 was prepared with S. flexneri LPS.
TABLE-US-00008 TABLE 7 Induction of murine cytokines and serum IgG
by 10 ug birch pollen extract (BPEx) alone or formulated with 10 ug
IVX-908 via the nasal route as described in Example 12. For BPEx +
alum, mice were given a single i.p. immunization of 3 ug birch
pollen extract together with 1 mg aluminum phosphate. BPEx BPEx +
IVX-908 BPEx + Alum PBS IFN-.gamma. (pg/mL) <10 435 142 0 IL-5
(pg/mL) 431 143 290 0 IL-5/IFN-.gamma. ratio >43.1 0.33 2 0
Serum IgE (ng/mL) 16 19 829 16 Serum IgG (ng/mL) 105 2,300 nd
7.5
Results for IFN-.gamma. and IL-5 are expressed as the mean pg/mL
for triplicate cultures from spleens pooled from 4-5 mice/group.
Serum IgG is for sera pooled from 15 mice except for the
BPEx+IVX-908 group where the geometric mean of results from 15
individual mice was calculated. Serum IgE for the BPEx+IVX-908
group represents the geometric means from sera from 15 individual
mice while BPEx+Alum results are geometric means for 86 individual
mice. Serum IgE levels for BPEx and PBS were measured in sera
pooled from 15 animals. IVX-908 was prepared with S. flexneri LPS.
IVX-908 Adjuvanted BPEx Given Nasally to Mice and Subsequently
Injected with rBet v 1a Plus Alum (Table 8): [0086] 1. increased
the production of the type 1 cytokine, IFN-.gamma. by 10-fold
compared with BPEx given alone [0087] 2. and slightly lowered the
levels of the type 2 cytokine, IL-5.
TABLE-US-00009 [0087] TABLE 8 Induction of cytokines in mice
injected intraperitoneally with rBet v1a plus alum following 3
nasal immunizations with 10 .mu.g birch pollen extract alone or
formulated 1:1 with IVX-908 (10 .mu.g protein:LPS) as described in
Example 12. BPEx + BPEx IVX-908 IFN-.gamma. (pg/mL) 31 330 IL-5
(pg/mL) 384 276 IL-5/IFN-.gamma. ratio 13 0.8
Results for IFN-.gamma. and IL-5 are expressed as the geometric
means (pg/mL) from spleen cultures from 8-10 individual mice/group.
IVX-908 was prepared with S. flexneri LPS. IVX-908 Adjuvanted rBet
v 1a or BPEx Given Nasally to rBet v 1a Sensitized Mice (Table 9):
[0088] 1. increased the production of the type 1 cytokine,
IFN-.gamma. by 4.7- and 33-fold for IVX-908/rBet v 1a and
IVX-908/BPEx respectively compared with the corresponding allergens
alone and [0089] 2. lowered the levels of the type 2 cytokine,
IL-5
TABLE-US-00010 [0089] TABLE 9 Induction of murine cytokines by 10
.mu.g rBet v 1a or birch pollen extract given nasally alone or with
10 .mu.g IVX-908 in rBet v 1a-sensitized mice as described in
Example 12. rBet v 1a + BPEx + rBet v 1a IVX-908 BPEx IVX-908 PBS
IFN-.gamma. (pg/mL) 126 593 295 9790 55 IL-5 (pg/mL) 2353 1747 8160
6270 460 IL-5/IFN-.gamma. ratio 19 3 28 0.6 8
Results for IFN-.gamma. and IL-5 are expressed as the geometric
means (pg/mL) from spleen cultures from 4-5 mice/group. IVX-908 was
prepared with S. flexneri LPS.
[0090] The data in Tables 6, 7, 8 and 9 demonstrate that allergens
(purified recombinant proteins or extracts) formulated with IVX-908
induce type 1 immune responses in mice. These formulations
maintained the production of type 1 cytokines in mice subsequently
injected intraperitoneally with a sensitizing injection of rBet v
1a plus alum. Importantly, these formulations also enhanced the
production of type 1 cytokines in mice that had previously been
sensitized or made allergic to the allergen. These results suggest
the potential utility of IVX-908/allergen formulations as
therapeutic vaccines for allergic diseases.
Example 13
Enhancement of Immune Responses Against a Poor Immunogen
[0091] Mice were immunized as above by either the i.n. or i.m.
routes, with Ovalbumin (OVA--a poorly immunogenic, soluble protein)
in decreasing amounts from 100 .mu.g to 5 .mu.g, with or without 1
.mu.g of IVX-908 (proteosome protein:LPS 1:1, using P. shig LPS).
Following immunization on days 0 and 14, mice were euthanized on
day 21 and serum, lung lavage fluids and spleens collected for
analysis. Serum GMCs are shown in FIG. 6.
[0092] The data confirms that unadjuvanted OVA is poorly
immunogenic and elicited barely detectable serum IgG titers even
when mice were immunized with 100 .mu.g of OVA by either i.n. or
i.m. routes. However when mixed with IVX-908, over 60-fold rises in
titers were observed by both routes of immunization, albeit at
higher concentrations (.gtoreq.25 .mu.g) of OVA. No mucosal
responses were detected in any of the immunized mice. Analysis of
the cytokine profiles elicited by OVA or OVA+IVX-908 showed that
when immunized i.n., co-administration of IVX-908 induced the
secretion of elevated levels of IFN-.gamma., IL-2, IL-4 and IL-5
from splenocytes. Thus unlike HA which induced release of cytokines
indicative of a type 2 phenotype response which switched to a type
1 phenotype when HA was administered with IVX-908, adjuvanting of
the poorly immunogenic, soluble OVA appeared to be associated with
induction of a balanced type 1/type 2 phenotype response.
Example 14
Effect of Varying the Amount of LPS in IVX-908 on Elicited
Immunity
[0093] To determine the effects of varying the ratio of proteosome
to LPS in IVX-908 on elicited immunity, a study was performed in
which mice were immunized i.n. as above with 3 .mu.g of HA (H3N2
strain A/Sydney) mixed with 1 .mu.g (as LPS) of IVX-908 (1:1 or 1:2
complex of proteosomes and P. shigelloides LPS). At euthanasia,
blood and lung washes were collected and analyzed by ELISA for
specific IgG or IgA respectively. The results are shown in table 9,
and indicate that although both IVX-908s elicit virtually identical
levels of specific serum IgG, there is a highly significant
difference (P.ltoreq.0.001) between the mucosal IgAs elicited by
the different IVX-908s. Clearly the IVX-908 comprising proteosomes
complexed 1:1 with P. shigelloides LPS elicited higher titers of
specific mucosal IgA in lung lavage fluids and therefore possesses
more mucosal adjuvant activity than the 1:2 proteosome protein:LPS
complex.
Table 9 shows immunoglobulin levels (IgG or IgA) expressed as
geometric mean concentrations with 95% confidence limits in
parentheses, in serum and lung washes from mice immunized i.n. with
HA+IVX-908 (Pr:LPS 1:1 or 1:2).
TABLE-US-00011 IVX908 (Proteosome IVX908 (Proteosome protein:LPS,
1:2) protein:LPS, 1:1) Serum IgG (.mu.g/ml) 158.8 (105.4-239.2)
166.8 (108.5-256.3) Lung IgA (ng/ml) 393 (157-981) 2026
(1230-3335)
Example 15
Adjuvant Effects of IVX-908 Prepared with LPS from Different
Organisms
[0094] To determine the adjuvanticity of IVX-908 made by complexing
proteosomes to LPS from novel organisms, IVX-908 preparations were
made using LPS from a non-pathogenic E. coli (017) and from
Salmonella essen. IVX-908 preparations were made by mixing
proteosomes and the LPS in 3:1, 1:1 and 1:3 (w/w) ratios in the
presence of EMPIGEN, and removal of detergent by dialysis in
dialyzing cassettes. Mice were immunized i.n. on day 0 and 14 with
3 .mu.g of HA (B/Guangdong) mixed with 3 .mu.g or 0.3 .mu.g (as
LPS) of IVX-908. Control mice received 3 .mu.g HA i.n. At
euthanasia on day 21, blood was collected and analyzed by ELISA for
specific IgG. The results are shown in Table 10, and indicate that
IVX-908 preparations made with LPS from pathogens other than
Shigella species are capable of enhancing immune responses to a
vaccine antigen. For IVX-908 prepared with E. coli LPS, the 1:1 and
1:3 ratios of proteosomes to LPS at a dose of 0.3 .mu.g LPS gave
significant (P.ltoreq.0.001) enhancement of the anti-HA serum IgG
response compared with HA alone given i.n. All ratios of Pr:LPS (S.
essen) at both doses tested elicited significant (P.ltoreq.0.001)
enhancement of serum anti-HA responses over HA alone given i.n. The
results for IVX-908 made with S. essen were comparable to those
obtained for IVX-908 made with LPS from Shigella species.
Table 10 shows serum anti-HA IgG titers expressed as geometric mean
concentrations (.mu.g/ml) with 95% confidence limits in parentheses
for 8 mice per group immunized i.n. with HA+IVX-908 preparations
containing LPS from different gram negative bacteria and at
different Pr:LPS ratios.
TABLE-US-00012 Serum IgG (.mu.g/ml) Pr:LPS ratio Immunogen 3:1 1:1
1:3 HA + Pr:E. coli 0.83 (0.79-0.87) 4.75 (2.53-8.91) 38.93
(28.19-53.75) LPS (0.3 .mu.g LPS) HA + Pr:S. essen LPS 19.89
(12.12-32.63) 28.24 (18.14-43.98) 22.91 (13.43-39.09) (0.3 .mu.g
LPS) HA + Pr:S. essen LPS (3 .mu.g 76.41 (43.62-133.86) 38.52
(20.64-71.9) 69.05 (31.15-153.04) LPS) HA + Pr:P. shig LPS (3 .mu.g
38.97 (16.53-91388 LPS) HA + Pr:S. flex LPS (3 .mu.g 19.19
(7.39-49.8) LPS) HA 0.83 (0.77-0.89)
[0095] To the extent necessary to understand or complete the
disclosure of the present invention, all publications, patents, and
patent applications mentioned herein are expressly incorporated by
reference therein to the same extent as though each were
individually so incorporated.
* * * * *