U.S. patent application number 15/694023 was filed with the patent office on 2018-04-05 for thermostable vaccine compositions and methods of preparing same.
The applicant listed for this patent is The Regents of the University of Colorado, a body, Soligenix, Inc.. Invention is credited to Robert N. Brey, John Carpenter, Kimberly Hassett, Pradyot Nandi, Theodore Randolph.
Application Number | 20180092851 15/694023 |
Document ID | / |
Family ID | 49581482 |
Filed Date | 2018-04-05 |
United States Patent
Application |
20180092851 |
Kind Code |
A1 |
Hassett; Kimberly ; et
al. |
April 5, 2018 |
Thermostable Vaccine Compositions and Methods of Preparing Same
Abstract
Compositions relating to thermostable vaccines and methods of
preparing same. Specifically, methods of preparing thermostable
vaccines based on a recombinant ricin neurotoxin protein and uses
of co-adjuvants to develop a composition capable of eliciting an
immune response in a subject.
Inventors: |
Hassett; Kimberly; (Boulder,
CO) ; Nandi; Pradyot; (Denver, CO) ; Brey;
Robert N.; (Lakeside, IL) ; Carpenter; John;
(Littleton, CO) ; Randolph; Theodore; (Niwot,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of Colorado, a body
Soligenix, Inc. |
Denver
Princeton |
CO
NJ |
US
US |
|
|
Family ID: |
49581482 |
Appl. No.: |
15/694023 |
Filed: |
September 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13474661 |
May 17, 2012 |
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15694023 |
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61487206 |
May 17, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/19 20130101; A61K
39/39 20130101; Y02A 50/464 20180101; A61K 2039/55572 20130101;
A61K 47/26 20130101; Y02A 50/30 20180101; A61K 2039/55505 20130101;
A61K 39/00 20130101 |
International
Class: |
A61K 9/19 20060101
A61K009/19; A61K 47/26 20060101 A61K047/26; A61K 39/39 20060101
A61K039/39; A61K 39/00 20060101 A61K039/00 |
Goverment Interests
GOVERNMENT SUPPORT STATEMENT
[0002] This invention was made with government support under grant
UO1-A1-08-2210 from the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of preparing an immunologically-active adjuvant-bound
dried vaccine composition, the method comprising: (a) providing at
least one aluminum-salt adjuvant, at least one buffer system
containing at least one volatile salt, at least one glass-forming
agent and at least one antigen; (b) combining (a) together to
create a liquid vaccine formulation; (c) freezing the liquid
vaccine formulation in (b) to create a frozen vaccine formulation;
and (d) lyophilizing the frozen vaccine formulation in (c) to
create a dried vaccine composition, further wherein the dried
vaccine composition is capable of eliciting an immune response in a
subject.
2. The method of claim 1, wherein the immune response developed by
the subject may be humoral immunity and/or cell-mediated immunity
specific to the antigen.
3. The method of claim 1, wherein the at least one aluminum-salt
adjuvant is selected from the group consisting of aluminum
hydroxide, aluminum phosphate and aluminum sulfate.
4. The method of claim 1, wherein the at least one buffer system is
selected from the group consisting of acetate, succinate, citrate,
prolamine, arginine, glycine, histidine, borate, carbonate and
phosphate.
5. The method of claim 1, wherein the at least one buffer system is
selected from the group consisting of ammonium acetate, ammonium
formate, ammonium carbonate, ammonium bicarbonate, triethylammonium
acetate, triethylammonium formate, triethylammonium carbonate,
trimethylamine acetate trimethylamine formate, trimethylamine
carbonate, pyridinal acetate and pyridinal formate.
6. The method of claim 1, wherein the at least one glass-forming
agent is selected from the group consisting of trehalose, sucrose,
ficoll, dextran, sucrose, maltotriose, lactose, mannitol,
hydroxyethyl starch, glycine, cyclodextrin, and povidone.
7. The method of claim 1, wherein the at least one glass-forming
agent is trehalose and the trehalose is present in a
weight-to-volume concentration of from about 5% to about 15% in the
liquid vaccine formulation prior to freeze drying.
8. The method of claim 1, wherein the at least one glass-forming
agent is trehalose and the trehalose is present in a
weight-to-volume concentration of from about 8% to about 20% in the
liquid vaccine formulation.
9. The method of claim 1, wherein at least one
immunologically-active co-adjuvant is added in step (a).
10. The method of claim 9, wherein the at least one
immunologically-active co-adjuvant is selected from the group
consisting of lipid A, lipid A derivatives, monophosphoryl lipid A,
chemical analogues of monophosphoryl Lipid A, CpG containing
oligonucleotides, TLR-4 agonists, flagellin, flagellins derived
from gram negative bacteria, TLR-5 agonists, fragments of
flagellins capable of binding to TLR-5 receptors, saponins,
analogues of saponins, QS-21, purified saponin fractions, ISCOMS
and saponin combinations with sterols and lipids.
11. The method of claim 1, wherein the freezing step comprises one
of tray freezing, shelf freezing, spray-freezing and
shell-freezing.
12. The method of claim 1, wherein the freezing step includes use
of a pre-cooled tray to initiate the freezing step.
13. The method of claim 1, wherein the antigen is selected from or
derived from the group consisting of rotavirus, foot and mouth
disease virus, influenza A virus, influenza B virus, influenza C
virus, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7,
human parainfluenza type 2, herpes simplex virus, Epstein-Barr
virus, varicella virus, porcine herpesvirus 1, cytomegalovirus,
lyssavirus, Bacillus anthracis, anthrax PA and derivatives,
poliovirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E,
distemper virus, venezuelan equine encephalomyelitis, feline
leukemia virus, reovirus, respiratory syncytial virus, Lassa fever
virus, polyoma tumor virus, canine parvovirus, papilloma virus,
tick borne encephalitis virus, rinderpest virus, human rhinovirus
species, Enterovirus species, Mengovirus, paramyxovirus, avian
infectious bronchitis virus, human T-cell leukemia-lymphoma virus
1, human immunodeficiency virus-1, human immunodeficiency virus-2,
lymphocytic choriomeningitis virus, parvovirus B19, adenovirus,
rubella virus, yellow fever virus, dengue virus, bovine respiratory
syncitial virus, corona virus, Bordetella pertussis, Bordetella
bronchiseptica, Bordetella parapertussis, Brucella abortis,
Brucella melitensis, Brucella suis, Brucella ovis, Brucella
species, Escherichia coli, Salmonella species, Salmonella typhi,
Streptococci, Vibrio cholera, Vibrio parahaemolyticus, Shigella,
Pseudomonas, tuberculosis, avium, Bacille Calmette Guerin,
Mycobacterium leprae, Pneumococci, Staphlylococci, Enterobacter
species, Rochalimaia henselae, Pasteurella haemolytica, Pasteurella
multocida, Chlamydia trachomatis, Chlamydia psittaci,
Lymphogranuloma venereum, Treponema pallidum, Haemophilus species,
Mycoplasma bovigenitalium, Mycoplasma pulmonis, Mycoplasma species,
Borrelia burgdorferi, Legionalla pneumophila, Colstridium
botulinum, Corynebacterium diphtheriae, Yersinia entercolitica,
Rickettsia rickettsii, Rickettsia typhi, Rickettsia prowsaekii,
Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes,
trypanosomes, Leishmania species, Filarial nematodes,
trichomoniasis, sarcosporidiasis, Taenia saginata, Taenia solium,
Leishmania, Toxoplasma gondii, Trichinella spiralis, coccidiosis,
Eimeria tenella, Cryptococcus neoformans, Candida albican,
Apergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae,
malaria circumsporozoite protein, malaria merozoite protein,
trypanosome surface antigen protein, pertussis, alphaviruses,
adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer
membrane protein, streptococcal M protein, Influenza hemagglutinin,
cancer antigen, tumor antigens, toxins, clostridium perfringens
epsilon toxin, ricin toxin, pseudomonas exotoxin, exotoxins,
neurotoxins, cytokines, cytokine receptors, monokines, monokine
receptors, plant pollens, animal dander, and dust mites.
14. The method of claim 1, wherein dried vaccine composition is
reconstituted with an aqueous diluent to form a reconstituted
vaccine composition.
15. The method of claim 13, wherein the reconstituted vaccine
composition contains a mean particle diameter of less than 100
micrometers.
16. The method of claim 1, wherein the liquid vaccine formulation
is first prepared as a hypertonic mixture prior to freezing and
then adjusted to isotonic levels upon dilution of the dried vaccine
composition with an aqueous diluent.
17. A vaccine composition, comprising: (a) at least one
aluminum-salt adjuvant; (b) at least one buffering agent, wherein
the at least one buffering agent comprises a volatile salt; (c) at
least one glass forming agent; and (d) at least one antigen,
wherein the composition is lyophilized to create a dried vaccine
composition and further wherein the dried vaccine composition is
capable of eliciting an immune response in a subject.
18. The vaccine composition of claim 17, wherein the at least one
aluminum-salt adjuvant is selected from the group consisting of
aluminum hydroxide, aluminum phosphate and aluminum sulfate.
19. The vaccine composition of claim 17, wherein the at least one
buffering agent is selected from the group consisting of acetate,
succinate, citrate, prolamine, arginine, glycine, histidine,
borate, carbonate and phosphate.
20. The vaccine composition of claim 17, wherein the at least one
buffering agent is selected from the group consisting of ammonium
acetate, ammonium formate, ammonium carbonate, ammonium
bicarbonate, triethylammonium acetate, triethylammonium formate,
triethylammonium carbonate, trimethylamine acetate trimethylamine
formate, trimethylamine carbonate, pyridinal acetate and pyridinal
formate.
21. The vaccine composition of claim 17, wherein the at least one
glass-forming agent is selected from the group consisting of
trehalose, sucrose, ficoll, dextran, sucrose, maltotriose, lactose,
mannitol, hydroxyethyl starch, glycine, cyclodextrin, and
povidone.
22. The vaccine composition of claim 17, wherein the composition
includes at least one immunologically-active co-adjuvant.
23. The vaccine composition of claim 22, wherein the at least one
immunologically-active co-adjuvant is selected from the group
consisting of lipid A, lipid A derivatives, monophosphoryl lipid A,
chemical analogues of monophosphoryl Lipid A, CpG containing
oligonucleotides, TLR-4 agonists, flagellin, flagellins derived
from gram negative bacteria, TLR-5 agonists, fragments of
flagellins capable of binding to TLR-5 receptors, saponins,
analogues of saponins, QS-21, purified saponin fractions, ISCOMS
and saponin combinations with sterols and lipids.
24. The vaccine composition of claim 17, wherein the antigen is
selected from or derived from the group consisting of rotavirus,
foot and mouth disease virus, influenza A virus, influenza B virus,
influenza C virus, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2,
H7N3, H10N7, human parainfluenza type 2, herpes simplex virus,
Epstein-Barr virus, varicella virus, porcine herpesvirus 1,
cytomegalovirus, lyssavirus, Bacillus anthracis, anthrax PA and
derivatives, poliovirus, Hepatitis A, Hepatitis B, Hepatitis C,
Hepatitis E, distemper virus, venezuelan equine encephalomyelitis,
feline leukemia virus, reovirus, respiratory syncytial virus, Lassa
fever virus, polyoma tumor virus, canine parvovirus, papilloma
virus, tick borne encephalitis virus, rinderpest virus, human
rhinovirus species, Enterovirus species, Mengovirus, paramyxovirus,
avian infectious bronchitis virus, human T-cell leukemia-lymphoma
virus 1, human immunodeficiency virus-1, human immunodeficiency
virus-2, lymphocytic choriomeningitis virus, parvovirus B19,
adenovirus, rubella virus, yellow fever virus, dengue virus, bovine
respiratory syncitial virus, corona virus, Bordetella pertussis,
Bordetella bronchiseptica, Bordetella parapertussis, Brucella
abortis, Brucella melitensis, Brucella suis, Brucella ovis,
Brucella species, Escherichia coli, Salmonella species, Salmonella
typhi, Streptococci, Vibrio cholera, Vibrio parahaemolyticus,
Shigella, Pseudomonas, tuberculosis, avium, Bacille Calmette
Guerin, Mycobacterium leprae, Pneumococci, Staphlylococci,
Enterobacter species, Rochalimaia henselae, Pasteurella
haemolytica, Pasteurella multocida, Chlamydia trachomatis,
Chlamydia psittaci, Lymphogranuloma venereum, Treponema pallidum,
Haemophilus species, Mycoplasma bovigenitalium, Mycoplasma
pulmonis, Mycoplasma species, Borrelia burgdorferi, Legionalla
pneumophila, Colstridium botulinum, Corynebacterium diphtheriae,
Yersinia entercolitica, Rickettsia rickettsii, Rickettsia typhi,
Rickettsia prowsaekii, Ehrlichia chaffeensis, Anaplasma
phagocytophilum, Plasmodium falciparum, Plasmodium vivax,
Plasmodium malariae, Schistosomes, trypanosomes, Leishmania
species, Filarial nematodes, trichomoniasis, sarcosporidiasis,
Taenia saginata, Taenia solium, Leishmania, Toxoplasma gondii,
Trichinella spiralis, coccidiosis, Eimeria tenella, Cryptococcus
neoformans, Candida albican, Apergillus fumigatus,
coccidioidomycosis, Neisseria gonorrhoeae, malaria circumsporozoite
protein, malaria merozoite protein, trypanosome surface antigen
protein, pertussis, alphaviruses, adenovirus, diphtheria toxoid,
tetanus toxoid, meningococcal outer membrane protein, streptococcal
M protein, Influenza hemagglutinin, cancer antigen, tumor antigens,
toxins, clostridium perfringens epsilon toxin, ricin toxin,
pseudomonas exotoxin, exotoxins, neurotoxins, cytokines, cytokine
receptors, monokines, monokine receptors, plant pollens, animal
dander, and dust mites.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/474,661, filed May 17, 2012, which claims
the benefit of priority from U.S. Provisional Application No.
61/487,206, filed on May 17, 2011, the entire contents of which are
herein incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of
dried vaccine compositions. More specifically, to methods of
producing dried vaccine compositions bound to adjuvant and
containing immunostimulatory molecules.
BACKGROUND OF THE INVENTION
[0004] Vaccines containing recombinant proteins benefit from or
absolutely require an adjuvant to elicit an immune response.
(Callahan et al., 1991, The importance of surface charge in the
optimization of antigen-adjuvant interactions, Pharm. Res.
8(7):851-858; Singh and O'Hagan 1999, Advances in vaccine
adjuvants, Nat Biotechnol 17(11): 1075-81; and O'Hagan et al.,
2001, Recent developments in adjuvants for vaccines against
infectious diseases, Biomol Eng 18(3): 69-85). Aluminum-salt
adjuvants are currently the most widely used adjuvants for general
use in humans because of the extensive history of safe use in
vaccines administered to children and adults. The only adjuvants
currently appearing in FDA-approved vaccines are the aluminum salt
adjuvants, aluminum hydroxide and aluminum phosphate. Aluminum-salt
adjuvants enhance the immunogenicity of vaccines and cause
significant improvements in the outcomes of vaccination by reducing
the dose level of protein in vaccine, elevating the titers of
protective antibodies, and reducing the need for annual vaccination
after a primary series of vaccination has been completed.
Nonetheless, there are significant limitations in the use of
aluminum-salt adjuvants in many subunit vaccines based on
recombinant proteins, peptides, and chemically synthesized
vaccines. These limitations include the general aspects of vaccine
storage and stability, since vaccine containing aluminum adjuvants
can be stored only within narrow temperature ranges, and cannot be
frozen. Further limitations include the generally accepted view
that aluminum adjuvants are relatively weak, do not foster the
development of cellular immunity, and may favor the development of
antibodies that are non-neutralizing in cases where neutralizing
antibodies are necessary to block viral infections or impede the
activity of biological toxins.
[0005] In the case of aluminum adjuvants, it has been suggested
that to provide adequate immunogenicity, antigens must be adsorbed
on the surface of the adjuvant. (Gupta et al., 1995, Adjuvant
Properties of Aluminum and Calcium Compounds. Pharmaceutical
Biotechnology. 6: 229-248; and White and Hem, 2000,
Characterization of aluminium-containing adjuvants, Dev Biol
(Basel) 103: 217-28). This adsorption is typically facilitated
through electrostatic interactions between the antigen and
adjuvant, and the formulation pH is usually chosen so that the
antigen and adjuvant are oppositely charged (Callahan et al. 1991).
The surface charge on the adjuvant also can be modified by surface
exchange reactions with buffer salts such as phosphate, succinate,
and citrate (Hem and White, 1984, Characterization of aluminum
hydroxide for use as an adjuvant in parenteral vaccines. J Parenter
Sci Technol, 38(1): p. 2-10; Chang et al., 1997, Role of the
electrostatic attractive force in the adsorption of proteins by
aluminum hydroxide adjuvant. PDA J Pharm Sci Technol, 51(1): p.
25-9; and Rinella et al., 1996, Treatment of aluminium hydroxide
adjuvant to optimize the adsorption of basic proteins. Vaccine,
14(4): p. 298-300.) The mechanisms of action of aluminum-salt
adjuvants are poorly understood, but likely due to several
different mechanisms. (Lindblad 2004. "Aluminium compounds for use
in vaccines" Immunol. Cell. Biol. 82(5):497-505; Gupta and Siber,
1995, Adjuvants for Human Vaccines--Current Status, Problems and
Future-Prospects. Vaccine 13(14):1263-1276; Gupta and Rost, 2000,
Aluminum Compounds as Vaccine Adjuvants, In O'Hagan D, editor
Vaccine Adjuvants: Preparation Methods and Research Protocols, ed.,
Totowa, N.J.: Humana Press Inc. p 65-89; Cox and Coulter, 1997,
Adjuvants--a classification and review of their modes of action,
Vaccine 15(3):248-256). Common proposed mechanisms are that the
adjuvant acts as a depot at the site of injection, wherein the
antigen is slowly released after administration. (Cox and Coulter,
1997). Another proposed mechanism is that the adjuvant aids in
delivery of the antigen to antigen-presenting cells (Lindblad
2004). A further proposed mechanism is that adjuvant serves as an
immunostimulator and elicits Th2 cytokines (Grun and Maurer 1989,
Different T helper cell subsets elicited in mice utilizing two
different adjuvant vehicles: the role of endogenous interleukin 1
in proliferative responses. Cell Immunol 121(1):134-145). Yet
another proposed mechanism is that adjuvant destabilizes protein
antigens on the surface of the adjuvant making them more
susceptible to proteolytic degradation (Jones et al., 2005, Effects
of adsorption to aluminum salt adjuvants on the structure and
stability of model protein antigens. J Biol Chem
280(14):13406-13414; and That et al., 2004. "Antigen stability
controls antigen presentation" J. Biol. Chem.
279(48):50257-50266).
[0006] Although the mechanism of action is not fully understood, it
is likely that surface area, surface charge, and morphology of the
adjuvant are important factors dictating the immune response to
antigens adsorbed onto these adjuvants (Hem and White 1984). It is
generally theorized that the smaller the particle size of the
vaccine adjuvant, the more immunogenic the vaccine preparation,
especially when particle size is approximately 1 micron, a size
best suited for uptake into professional antigen presenting cells
(Maa et al., 2003. Stabilization of alum-adjuvanted vaccine dry
powder formulations: mechanism and application. J Pharm Sci
92(2):319-332, Diminsky et al., 1999. Physical, chemical and
immunological stability of CHO-derived hepatitis B surface antigen
(HBsAg) particles. Vaccine 18(1-2):3-17).
[0007] Lyophilization (freeze drying) is a process frequently
utilized to improve long term stability of various protein
preparations. However, when vaccines formulated with aluminum-salt
adjuvants are processed in an attempt to improve stability through
freezing and lyophilization, a loss of potency occurs, where
potency is a summation of the quality of the vaccine measurable by
a series of tests that can include immunogenicity in animals,
chemical degradation of protein antigen, denaturation of protein
antigen, or loss of substituent immunogenic epitopes. Loss of
potency is associated with loss of efficacy in humans. Previous
studies have suggested that a freeze-dried vaccine product
containing adjuvant cannot be produced due to aggregation of the
adjuvant particles. (Diminsky et al., 1999; Maa et al., 2003). A
number of theories have been set forth to explain possible
mechanisms responsible for the loss of potency following
lyophilization of vaccines formulated with aluminum-salt adjuvants.
Particle aggregation may account for significant losses. For
example, the aggregation of aluminum hydroxycarbonate and magnesium
hydroxide gels after freezing and thawing has been attributed to
ice crystal formation which forces particles together, resulting in
irreversible aggregation. (Zapata et al., 1984, Mechanism of
freeze-thaw instability of aluminum hydroxycarbonate and magnesium
hydroxide gels. J Pharm Sci 73(1):3-8). This explanation has been
proposed by Maa et al., 2003 suggesting further that faster cooling
rates result in a greater rate of ice nucleation and the formation
of smaller ice crystals, which would not force aluminum particles
into an aggregate. Particle aggregation can thus account for losses
of potency, but other factors, such as loss of protein
configuration (tertiary structure), loss of protein secondary
structure, and modifications of primary structure through
deamidation or oxidation of amino acid side chains.
[0008] The capacity of particles to increase allergic sensitization
is predicted by particle number and surface area, not by particle
mass. Moorefield et al. showed that the degree of antigen
internalization of adjuvant particles is inversely related to the
particle size of the adjuvant aggregates (Moorefield et al., 2005.
"Role of aluminum-containing adjuvants in antigen internalization
by dendritic cells in vitro" Vaccine 23(13):1588-1595). Nygaard et
al. showed that the particle diameter, and thus surface area and
number of particles, and not mass or volume, is the dominant
property in the immunological response of polystyrene particles in
mice (Nygaard et al., 2004). While it is likely that the particle
size is an important characteristic parameter for immunogenicity,
there has yet to be a comprehensive study examining the particle
size distribution (PSD) as a function of formulation and cooling
rates along with other physical properties of the products
produced.
[0009] There is some consensus view that the more effective
vaccines with aluminum adjuvants are ones in which antigen is bound
to the aluminum surface, rather than free in solution (Lindblad,
2004, Aluminium adjuvants--in retrospect and prospect, Vaccine,
22:3658-68). For reproducibility of formulations and stability, it
is desirable to define conditions for optimal binding of antigen to
crystal surfaces, and conditions in which antigen does not desorb
over time or under elevated stress conditions. To construct
aluminum vaccines, it is necessary to carry out studies to optimize
binding and desorption. Aluminum adjuvants have a point of zero
charge (PZC) at a certain solution pH, but are charged at pHs above
or below this value (White and Hem, 2000, Characterization of
aluminium-containing adjuvants, Dev Biol (Basel), 103:217-28).
Selecting an optimal formulation pH is further complicated for a
recombinant protein vaccine for which binding to aluminum salt
adjuvants is generally required to obtain the desired immune
response (McInerney, Brennan et al., 1999, Analysis of the ability
of five adjuvants to enhance immune responses to a chimeric plant
virus displaying an HIV-1 peptide, Vaccine, 17:1359-68). To
facilitate protein binding to adjuvant, a solution pH is selected
in which the protein and adjuvant have opposite charges. However, a
solution pH that provides optimal protein stability, may not allow
for appropriate binding of the vaccine to adjuvants. In such a
scenario, a vaccine protein may have to be prepared at pH that is
suboptimal for stability and lyophilized with appropriate
stabilizing excipients to minimize degradation during long-term
storage.
[0010] Lyophilization of proteins to stabilize structure and
activity for storage and reconstitution has been commonly applied
to recombinant protein therapeutic proteins. This has been usually
accomplished by freeze drying in the presence of disaccharides such
as trehalose and other excipients that promote a glass state during
process and storage. Proteins can be stored for long term as long
as the product is stored below its glass transition temperature
(T.sub.g) above which the material transitions into a rubbery
state. Excipients are thought to stabilize protein in the amorphous
state through interactions of the stabilizer with specific sites
substituting for water during drying and by simultaneously
suppressing translational and rotational motions of the protein
molecule (.alpha.-relaxations) or portions of the molecule
(.beta.-relaxations). Drying technology has been less frequently
applied to long term storage of vaccines, especially in the case of
vaccines adsorbed to aluminum phosphate or aluminum hydroxide
adjuvants. Very little data is available on the storage of dried
vaccines under elevated temperature conditions, as most of the
attempts to generate dried vaccines have been to obtain inhalable
powders or preparations able to survive moderate excursions in
temperature. For example, because the yellow fever vaccine is use
primarily in tropical climates, lyophilization in the presence of
stabilizers (lactose, sorbitol) has been used to preserve viability
of the live virus vaccine (Monath, 1996, Stability of yellow fever
vaccine, Dev Biol Stand, 87:219-25). Without excipients during
lyophilization and storage, activity is rapidly lost above
-20.degree. C., but the stabilized vaccine can withstand more than
two weeks at 37.degree. C. A lyophilized dried vaccine for the
cattle disease rinderpest has also been developed and can be
employed for up to a month after leaving the cold chain in African
field conditions (House and Mariner, 1996, Stabilization of
rinderpest vaccine by modification of the lyophilization process,
Dev Biol Stand, 87:235-44). Similar attempts to use variations on
process and drying have been recently applied to measles vaccine
development (Burger, Cape et al., 2008, Stabilizing formulations
for inhalable powders of live-attenuated measles virus vaccine, J
Aerosol Med Pulm Drug Deliv, 21:25-34; Burger, Cape et al., 2008,
Stabilizing Formulations for Inhalable Powders of Live-Attenuated
Measles Virus Vaccine, J Aerosol Med) and for dried vaccine powders
for influenza where it is likely very important to devise
conditions that permit retention of the structure of the immunogen
(Amorij, Meulenaar et al., 2007, Rational design of an influenza
subunit vaccine powder with sugar glass technology: preventing
conformational changes of haemagglutinin during freezing and
freeze-drying, Vaccine, 25:6447-57; Amorij, Huckriede et al., 2008,
Development of Stable Influenza Vaccine Powder Formulations:
Challenges and Possibilities, Pharm Res). As a stabilizer, small
amounts of formaldehyde are occasionally added to vaccines,
including the current AVA anthrax vaccine (Biothrax.RTM.), and may
act by cross-linking proteins forming more immunogenic protein
aggregates on the surface of aluminum crystals (Little, Ivins et
al., 2007, Effect of aluminum hydroxide adjuvant and formaldehyde
in the formulation of rPA anthrax vaccine, Vaccine, 25:2771-7).
Formaldehyde had been used historically as the stabilizer of choice
in the older vaccines derived from culture supernatants, such as
tetanus toxoid, botulinum toxoids, and others. The current AVA
vaccine is labeled for 3 year stability, where stability is a
function of a number of biochemical evaluations and potency.
Although a moderate amount of stability can be achieved with liquid
suspension vaccines, it is not likely that all stability parameters
can be met for longer storage periods that are required for
vaccines to be stockpiled and distributed.
[0011] Successful drying of therapeutic proteins, while retaining
structure and function, is dependent on the knowledge of the
degradation pathways that occur in solution, which can be retarded
or eliminated by appropriate drying and excipients for
stabilization. For vaccines, function is largely determined by
immunogenicity and protection studies, rather than enzymatic
activity. In the case of protein immunogens that are adsorbed to
aluminum adjuvants crystals, the measurement of function and other
parameters in vitro is correspondingly more difficult, since
protein may be sequestered and difficult to remove for analysis.
Thus, function can only be tested by immunogenicity and protection
studies. The tertiary conformation of proteins can obviously affect
enzymatic functions, if present, but also can affect immunogenicity
of B cell epitopes dependent on conformation. Linear B and T cell
epitopes contained therein can be also affected by oxidation (of
methionine and cysteine residues) and deamidation (especially of
asparagine residues). pH is one of the most critical formulation
variables governing stability of therapeutic proteins. (Carpenter,
Chang et al., 2002, Rational design of stable lyophilized protein
formulations: theory and practice, Pharm Biotechnol, 13:109-33;
Chi, Krishnan et al., 2003, Physical stability of proteins in
aqueous solution: mechanism and driving forces in nonnative protein
aggregation, Pharm Res, 20:1325-36) By affecting the conformational
and colloidal stability of proteins in solution, pH can greatly
modulate their aggregation rates (Chi, Krishnan et al., 2003,
Physical stability of proteins in aqueous solution: mechanism and
driving forces in nonnative protein aggregation, Pharm Res,
20:1325-36). In addition, rates of deamidation are strongly
dependent on pH (Manning, Patel et al., 1989, Stability of protein
pharmaceuticals, Pharm Res, 6:903-18). There can be different
optimal pH values for physical and chemical stability for a given
protein (Kolvenbach, Narhi et al., 1997, Granulocyte-colony
stimulating factor maintains a thermally stable, compact, partially
folded structure at pH2, J Pept Res, 50:310-8). For example,
physical stability may be optimal at a pH where deamidation is
unacceptably rapid (Chang, Reeder et al., 1996, Development of a
stable freeze-dried formulation of recombinant human interleukin-1
receptor antagonist, Pharm Res, 13:243-9). In such cases,
development of a lyophilized formulation where the rates of these
reactions are minimized may provide a viable strategy to obtain a
stable product. The few published studies examining effects of
pre-lyophilization solution pH on the stability of therapeutic
proteins during lyophilization and storage in dried formulations
demonstrated the importance of this parameter (Prestrelski, Pikal
et al., 1995, Optimization of lyophilization conditions for
recombinant human interleukin-2 by dried-state conformational
analysis using Fourier-transform infrared spectroscopy, Pharm Res,
12:1250-9; Chang, Reeder et al., 1996, Development of a stable
freeze-dried formulation of recombinant human interleukin-1
receptor antagonist, Pharm Res, 13:243-9; Katayama, Kirchhoff et
al., 2004, Retrospective statistical analysis of lyophilized
protein formulations of progenipoietin using PLS: determination of
the critical parameters for long-term storage stability, J Pharm
Sci, 93:2609-23). These studies demonstrated the difficulty in
identifying a pre-lyophilization solution pH that confers adequate
physical and chemical stability to the proteins studied during
lyophilization and storage. However, degradation of proteins could
be minimized if sufficient amounts of stabilizing excipients are
included in the formulation. For example, when recombinant human
interleukin-1-receptor antagonist (rhIL-1ra) was formulated prior
to lyophilization in a solution containing suboptimal sucrose at
levels less than 0.3 mass ratio of sucrose/protein and at pH less
than 6.5, severe protein aggregation occurred after lyophilization,
during storage and reconstitution (Chang, Reeder et al., 1996,
Development of a stable freeze-dried formulation of recombinant
human interleukin-1 receptor antagonist, Pharm Res, 13:243-9).
Protein aggregation was minimized following lyophilization from a
solution at pH greater than 6, although, deamidation occurred at an
unacceptably high rate. Following lyophilization from a solution
containing amounts of sucrose greater than 0.3 sucrose/protein mass
ratio at pH 6.5, both destabilization pathways could be inhibited.
In another example, interleukin-2 (IL-2) had significantly greater
structural perturbation during freeze-drying at pH 7, which
resulted in higher levels of aggregation after storage and
rehydration than samples lyophilized from solutions at pH 5
(Prestrelski, Pikal et al., 1995, Optimization of lyophilization
conditions for recombinant human interleukin-2 by dried-state
conformational analysis using Fourier-transform infrared
spectroscopy, Pharm Res, 12:1250-9). The addition of sucrose to the
pre-lyophilization solution formulation at pH 7 improved the
stability of IL-2 during storage following lyophilization. More
recently, this approach to pre-formulation has been taken with
anthrax rPA to create a dried powder vaccine candidate for nasal
administration (Jiang, Joshi et al., 2006, Anthrax vaccine powder
formulations for nasal mucosal delivery, J Pharm Sci, 95:80-96). In
this case, conditions for optimizing pH and excipient stabilizers
were established for rPA in solution prior to lyophilization. As
trehalose was one of the excipients determined to stabilize soluble
rPA to thermal stress, there was evidence of at least 30 days
stability at 40.degree. C. for the dried trehalose-containing
vaccines in terms of the total content of rPA in comparison to
liquid samples in which rPA quickly disappeared. In an effort to
obtain a dried powder composition for epidermal delivery using a
gas-driven injection device, it was found that rapid freezing of
aluminum-adsorbed hepatitis B vaccine (HBsAg) in the presence of a
mixture of mannitol, glycine, and dextran (not more than .about.6%
w/v of total excipients) resulted in vaccines that retained
particle size and relative immunogenicity in mice after a rapid
freezing (spray freeze drying) that involved injection of the
sprayed vaccine into liquid nitrogen prior to drying (Maa, Zhao et
al., 2003, Stabilization of alum-adjuvanted vaccine dry powder
formulations: mechanism and application, J Pharm Sci, 92:319-32).
The behavior of the spray-freeze dried vaccines under thermal
stress conditions was not determined, although normally lyophilized
vaccine aggregated after processing and was minimally immunogenic.
Diminished immunogenicity was associated with aluminum particle
aggregation after reconstitution.
[0012] More lately, Roser et al. have suggested lyophilization
methods that will prevent aggregation of aluminum particles. Roser
et al., U.S. Pat. No. 6,890,512 disclose a method of preventing
gross aggregation during dehydration and rehydration of
particulates in suspension by adding to a particulate suspension of
aluminum hydroxide in excess of 15% (w/v) of trehalose. Trehalose,
alpha.-D-glucopyranosyl-alpha-D-glucopyranoside, is a naturally
occurring disaccharide responsible for protection of plant cells
from desiccation. Trehalose has been shown to prevent denaturation
of proteins during desiccation by forming sugar glasses that
immobilize protein structure. However, Roser et al., while
disclosing prevention of gross particle aggregation, do not
disclose the importance of freezing rate of a particulate
suspension or other factors critical to control and maintain
pre-lyophilization particle size and protein structure in an
aluminum-salts containing vaccine in the presence of trehalose.
Maintenance of particle size is a critical parameter in controlling
the degree of adsorption of protein immunogens to the surface of
aluminum particles, and is influenced by several factors during
lyophilization cycle in addition to the content of trehalose or
other glassifying excipients. These factors influence the
immunogenicity and generation of protective immune responses.
[0013] Aluminum-salt adjuvants provide a well explored means to
augment the immunogenicity of protein or peptide subunit vaccines.
However, a variety of exploratory formulations to enhance vaccines
have been developed as more potent alternative to aluminum-salts
adjuvants, but are not currently available in FDA-licensed human
vaccines. Formulations designed to enhance immune responses include
a variety of compositions based on water-in-oil emulsions,
oil-in-water emulsions, self-assembling macrostructures, cytokines,
saponins, toll-like receptor agonists (TLR-4, TLR-5, and TLR-9),
immunostimulatory double stranded RNA species, unmethylated DNA
oligonucleotides, and polymeric microparticles and nanostructures.
Many of these compositions are directed towards improving the
immunogenicity of injected vaccines, and some variations can be
applied to altering routes of delivery for intranasal or oral
vaccination. As an example of one class of immunostimulatory
molecules that can be used to enhance vaccine immunogenicity,
bacterial DNA, but not vertebrate DNA, can be used because of
direct immunostimulatory effects that activate lymphocytes. This is
due to unmethylated CpG dinucleotides are present at the expected
frequency in bacterial DNA but are under-represented and methylated
in vertebrate DNA (Krieg et al., 1995). Activation may also be
triggered by addition of synthetic oligodeoxynucleotides (ODN) that
contain an unmethylated CpG dinucleotide in a particular sequence
context. CpG DNA induces proliferation of almost all (>95%) B
cells and increases immunoglobulin (Ig) secretion. This B cell
activation by CpG DNA is T cell independent and antigen
non-specific. However, B cell activation by low concentrations of
CpG DNA has strong synergy with signals delivered through the B
cell antigen receptor for both B cell proliferation and Ig
secretion (Krieg et al., 1995). This strong synergy between the B
cell signaling pathways triggered through the B cell antigen
receptor and by CpG DNA promotes antigen specific immune responses.
In addition to its direct effects on B cells, CpG DNA also directly
activates monocytes, macrophages, and dendritic cells to secrete a
variety of cytokines, including high levels of IL-12 (Klinman et
al., 1996; Halpern et al., 1996; Cowdery et al., 1996). These
cytokines stimulate natural killer (NK) cells to secrete
gamma-interferon (IFN-.gamma.-) and have increased lytic activity
(Klinman et al., 1996, supra; Cowdery et al., 1996, supra; Yamamoto
et al., 1992; Ballas et al., 1996). Overall, CpG DNA induces a Thl
like pattern of cytokine production dominated by IL-12 and
IFN-gamma with little secretion of Th2 cytokines (Klinman et al.,
1996). Other molecules stimulate toll like receptors. One example
is flagellin, the protein subunit comprising numerous bacterial
flagella. Flagellin is a TLR-5 ligand and triggers at least one of
the biological functions of antigen presenting cells upon such
binding. Flagella are found on the surface of rod and spiral shaped
bacteria, including members of the genera Escherichia, Salmonella,
Proteus, Pseudomonas, Bacillus, Campylobacter, Vibrio, Treponema,
Legionella, Clostridia, and Caulobacter. The conserved regions of
flagellins are important for TLRS binding, while the polymorphic
central region can be deleted without affecting binding to TLRS.
Flagellin sequences from numerous bacteria are available in the
art, such as Genbank accession numbers D13689, YP.sub.-275549,
YP.sub.-275550, AAU18718, AAU18717, ZP.sub.-00743095, EA052626,
YP.sub.-315348, AAT28337, AAT28336, AAT28335, AAT28334, AAT28333,
AAZ36356, AAZ33167, AAZ94424, AAZ91670, NP.sub.-414908, BAD18052,
and BAD18051. As a third example of purified adjuvant immune
stimulants, non toxic chemically synthesized or enzymatically
modified derivatives of gram negative lipopolysaccharides are
potent adjuvants and act by stimulating lymphocytes through TLR-4
binding and activation. For example, monophosphoryl lipid A (MPL)
is a derivative of the lipid A component of lipopolysaccharide and
is a potent activator of pro-inflammatory cytokines. Although
native lipid A and its parent LPS have powerful pyrogenic
properties and in humans induce febrile responses (Greisman and
Homick, J Immunol, 109:1210-1215 (1972); Greisman and Homick, J
Infect Dis, 128:257-263 (1973); Abemathy and Spink, J Clin Invest,
37:219-225 (1958); Rietschel et al, supra; and Raetz, supra
(1993)), MPL and its chemically synthesized analogues are not toxic
but induce a compendium of host proinflammatory cytokines including
IL-1, IL-6, and TNF-alpha.
[0014] In addition, to enhance the immune response to subunits
adsorbed to aluminum salts, it is likely that co-adjuvants will be
required in order to generate effective antibody responses in
humans after one or two doses. A number of adjuvant compounds that
are compatible with aluminum salts have been evaluated as adjuvants
in recent years. Primarily these adjuvants include Monophosphoryl
Lipid A (MPL) and QS-21, and CpG sequences. Recent data with
anthrax vaccine indicates in human studies that AVA, an A1OH
adsorbed vaccine, can be significantly enhanced by adding CpG 7909
to the adjuvant formulations in non-human primates and humans, in
terms of total anti-rPA antibodies and anthrax toxin neutralizing
antibodies, although no data describe the long term thermal
stability of CpG-containing vaccines (Klinman, 2006, CpG
oligonucleotides accelerate and boost the immune response elicited
by AVA, the licensed anthrax vaccine, Expert Rev Vaccines,
5:365-9). MPL and QS-21 have been also used with aluminum salts as
well as in proprietary oil emulsion formulations being developed by
Glaxo Smith Kline Biologics. QS-21 has been evaluated in AlOH
vaccines in humans and animal models with good evidence of
tolerability and systemic safety. QS-21 is thought to bind to
aluminum salts through ionic and hydrophobic interactions, as well
as some part of it remaining in solution (in aqueous vaccines) in a
micellar form. QS-21 is a saponin purified from tree bark with
broad adjuvant effects to induce both antibody and cell mediated
immunity. Though the mechanism is not understood, dose levels
effective in conjunction with human vaccines have been evaluated.
QS-21 with aluminum has been evaluated in clinical studies and
independent safety studies of QS-21 formulated with antigens have
been studied. QS-21 has been associated with stinging at the site
of injection (that resolves), with very little evidence of systemic
side effects (Waite, Jacobson et al., 2001, Three double-blind,
randomized trials evaluating the safety and tolerance of different
formulations of the saponin adjuvant QS-21, Vaccine, 19:3957-67).
Several studies in humans have shown that QS-21 enhances responses
to antigens that are adsorbed to aluminum. These include several
trials in malaria vaccine candidates (Nardin, Oliveira et al.,
2000, Synthetic malaria peptide vaccine elicits high levels of
antibodies in vaccinees of defined HLA genotypes, J Infect Dis,
182:1486-96; Kashala, Amador et al., 2002, Safety, tolerability and
immunogenicity of new formulations of the Plasmodium falciparum
malaria peptide vaccine SPf66 combined with the immunological
adjuvant QS-21, Vaccine, 20:2263-77), HIV gp120 (Evans, McElrath et
al., 2001, QS-21 promotes an adjuvant effect allowing for reduced
antigen dose during HIV-1 envelope subunit immunization in humans,
Vaccine, 19:2080-91) and more recently Rhesus macaque trials of
Dengue virus subunits in which neutralizing titers and protection
were enhanced by QS-21 (Putnak, Coller et al., 2005, An evaluation
of dengue type-2 inactivated, recombinant subunit, and
live-attenuated vaccine candidates in the rhesus macaque model,
Vaccine, 23:4442-52). The solution stability of QS-21 has been well
studied under long term stability studies, and has shown that
adjuvant active QS-21 (which actually consists of two isomeric
forms) is highly stable in slightly acidic buffers for over 4
years, whereas less than 10 days at 40.degree. C. (Kensil and
Kammer, 1998, QS-21: a water-soluble triterpene glycoside adjuvant,
Expert Opin Investig Drugs, 7:1475-82). QS-21 is stored as a dried
powder and in that form is stable indefinitely.
[0015] Ricin toxin is a 64 kDa protein produced by castor beans
(Ricinus communis) (Doan L G. Ricin: mechanism of toxicity,
clinical manifestations, and vaccine development. A review. Journal
of Toxicology--Clinical Toxicology 2004;42(2):201-8; Audi J, Belson
M, Patel M, Schier J, Osterloh J. Ricin poisoning: a comprehensive
review. JAMA 2005;294(18):2342-51). The holotoxin consists of two
polypeptide chains (A and B) joined by a disulfide bond. The A
chain (RTA) is a ribosome inactivating protein (RIP) that inhibits
protein synthesis in mammalian cells. The B chain (RTB) is a lectin
that binds to galactose residues on the surface of cells. Once
internalized by a cell, RTA translocates into the cytosol where it
enzymatically inactivates 60S ribosomes (Smallshaw, J E and
Vitetta, E S, A lyophilized formulation of RiVax, a recombinant
ricin subunit vaccine, retains immunogenicity. Vaccine 2010 Mar.
11; 28(12): 2428-2435). A single molecule of RTA in the cytoplasm
of a cell completely inhibits protein synthesis. The reported
estimated lethal dose of ricin in humans is 1-25 .mu.g/kg when
inhaled, injected, or ingested (Audi et al). Because of its wide
availability and extraordinary toxicity, ricin represents a
potential agent for use in bioterrorism and is therefore classified
by the Centers for Disease Control, Atlanta Ga. (CDC) as a level B
biothreat. Ricin intoxication can be prevented in experimental
animals by vaccination with toxoid or deglycosylated ricin A chain
(dgRTA), or by passive immunization with anti-ricin antibodies.
However the toxoid is considered to be too toxic for routine use in
humans and dgRTA is difficult and expensive to produce, and also
retains both active sites and could induce toxic side effects in
humans. Passive immunization with anti-ricin antibodies is only
effective if the ricin dose is relatively low and the antibody is
administered within a few hours after exposure (Hewetson J F,
Rivera V R, Creasia D A, Lemley P V, Rippy M K, Poli Mass.
Protection of mice from inhaled ricin by vaccination with ricin or
by passive treatment with heterologous antibody. Vaccine
1993;11(7):743-6).
[0016] In order to avoid these limitations, a recombinant RTA
vaccine (RiVax) was developed (Smallshaw et al.). RiVax
incorporated two point mutations, Y80A and V76M, to greatly reduce
or eliminate both of its known toxicities, i.e. ribotoxicity and
vascular leak-inducing ability. In the absence of adjuvant, RiVax
is non-toxic and immunogenic in mice, rabbits and humans (Smallshaw
J E, Firan A, Fulmer J R, Ruback S L, Ghetie V, Vitetta E S. A
novel recombinant vaccine which protects mice against ricin
intoxication. Vaccine 2002;20(27-28):3422-7). Such a model has been
showed to yield positive results without much of the toxicity
implicated with other vaccine models.
[0017] Ricin is currently listed by NIAID and the Centers for
Disease Control and Prevention (CDC) as a level B Biothreat agent
(Rotz, Khan et al., 2002, Public health assessment of potential
biological terrorism agents, Emerging Infectious Diseases,
8:225-30.). The vaccine candidate is based on a recombinant subunit
vaccine against ricin toxin obtained by genetic inactivation of
residues in the ricin toxin A chain (RTA) that are involved in well
characterized activities of the molecule. This modified molecule is
immunogenic in mice, rabbits, and humans and induces antibodies
that neutralize the toxin or are involved with clearance of the
toxin systemically or mucosally in each species. In the case of
smallpox or anthrax, terrorist induced epidemics could be
controlled by mass vaccination, selective vaccination, or ring
vaccination after evidence of an outbreak (Halloran, Longini et
al., 2002, Containing bioterrorist smallpox, Science, 298:1428-32;
Kaplan, Craft et al., 2002, Emergency response to a smallpox
attack: the case for mass vaccination, Proc Natl Acad Sci U S A,
99:10935-40; Bozzette, Boer et al., 2003, A model for a
smallpox-vaccination policy, N Engl J Med, 348:416-25; Kretzschmar,
van den Hof et al., 2004, Ring vaccination and smallpox control,
Emerg Infect Dis, 10:832-41). On the other hand, vaccination
against bioterrorist exposure to biological toxins, because the
agents do not replicate, is more likely to be used in select
populations, such as the military or first responders, rather than
mass vaccination. The isolated A and B subunit of ricin can be
produced in E. coli and other recombinant hosts. The B chain is
also a candidate for inclusion in a vaccine, but is considered less
immunogenic and less protective than the A chain (Maddaloni, Cooke
et al., 2004, Immunological characteristics associated with the
protective efficacy of antibodies to ricin, Journal of Immunology,
172:6221-8). Although ricin A chain is at least 1000-fold less
toxic than the native ricin, it still retains enzymatic activity
that may result in toxicity when used as a vaccine (Thorpe, Detre
et al., 1985, Modification of the carbohydrate in ricin with
metaperiodate-cyanoborohydride mixtures. Effects on toxicity and in
vivo distribution, European Journal of Biochemistry, 147:197-206.).
Several key amino acid residues in ricin A chain, Y80, Y123, E177,
R180, N209, and W211, constitute its enzymatically-active site
(Olson, 1997, Ricin A-chain structural determinant for binding
substrate analogues: a molecular dynamics simulation analysis,
Proteins, 27:80-95.; Lebeda and Olson, 1999, Prediction of a
conserved, neutralizing epitope in ribosome-inactivating proteins,
International Journal of Biological Macromolecules, 24:19-26).
Mutations in some of these amino acid residues have yielded ricin A
chains with negligible toxicity as determined by the inhibition of
protein synthesis (Kim and Robertus, 1992, Analysis of several key
active site residues of ricin A chain by mutagenesis and X-ray
crystallography, Protein Engineering, 5:775-9.). An additional site
and residues involved in the binding of RTA to endothelial cells
have also been identified, which occur extracellularly and do not
require toxin entry into host cells (Baluna and Vitetta, 1999, An
in vivo model to study immunotoxin-induced vascular leak in human
tissue, J Immunother, 22:41-7; Baluna, Coleman et al., 2000, The
effect of a monoclonal antibody coupled to ricin A chain-derived
peptides on endothelial cells in vitro: insights into
toxin-mediated vascular damage, Exp Cell Res, 258:417-24;
Smallshaw, Ghetie et al., 2003, Genetic engineering of an
immunotoxin to eliminate pulmonary vascular leak in mice, Nature
Biotechnology, 21:387-91). The endothelial binding site on RTA is
implicated in the damage to isolated HUVEC cells and to the
induction of vascular leak syndrome (VLS), which has been
determined to be a dose limiting toxicity in the use of
RTA-containing immunotoxins (Smallshaw, Ghetie et al., 2003,
Genetic engineering of an immunotoxin to eliminate pulmonary
vascular leak in mice, Nature Biotechnology, 21:387-91). The
portion of RTA involved in both pulmonary vascular leak, vascular
leak in human skin xenografts in SCID mice, and HUVEC cells appears
to involve amino acid residues L74, D75, and V76 (Baluna, Rizo et
al., 1999, Evidence for a structural motif in toxins and
interleukin-2 that may be responsible for binding to endothelial
cells and initiating vascular leak syndrome, Proceedings of the
National Academy of Sciences of the United States of America,
96:3957-62.). Therefore, an A chain vaccine taking into account
those toxic sites has been constructed and comprises a double
mutant of ricin A chain. Tyrosine 80 is mutated to alanine in the
enzymatic site and valine 76 is mutated to methionine in the
vascular leak site to minimize any possible in endothelial cell
damage. It is known now based on crystal structure data that this
protein is identical in structure to native ricin A chain (RTA) ,
indicating that the point mutations contained in the molecule do
not disrupt any potential tertiary structure (or potential
conformationally-dependent epitopes). When administered
intramuscularly (i.m.) to mice in the absence of adjuvant, the
mutant A chain elicited antibodies that recognized ricin and
animals were protected from a 10.times.LD.sub.50 dose of ricin
(Smallshaw, Firan et al., 2002, A novel recombinant vaccine which
protects mice against ricin intoxication, Vaccine, 20:3422-7.).
[0018] Native PA is the dominant immunogen in AVA (anthrax vaccine
adsorbed) and target for protective immunity in pre-exposure and
post exposure prophylaxis as a single component vaccine. Several
aluminum adsorbed recombinant PA vaccines (2.sup.nd generation) ,
based on expression of native PA in avirulent B. anthracis, have
been advanced and tested in recent Phase I trials, and have been
shown to be immunogenic in relationship to AVA (Gorse, Keitel et
al., 2006, Immunogenicity and tolerance of ascending doses of a
recombinant protective antigen (rPA102) anthrax vaccine: a
randomized, double-blinded, controlled, multicenter trial, Vaccine,
24:5950-9; Campbell, Clement et al., 2007, Safety, reactogenicity
and immunogenicity of a recombinant protective antigen anthrax
vaccine given to healthy adults, Hum Vaccin, 3:205-11).
[0019] The major correlates of immunity have been well
characterized in rabbit aerosol spore challenge studies (total
ELISA-reactive antibodies and toxin neutralizing activity
(TNA))(Little, Ivins et al., 2004, Defining a serological correlate
of protection in rabbits for a recombinant anthrax vaccine,
Vaccine, 22:422-30). It is well known that anthrax toxin is a
tripartite toxin consisting of a set of three plasmid-encoded
proteins expressed by B. anthracis: Protective Antigen (PA; 83
kDa), Lethal Factor (LF; 90 kDa) and Edema Factor (EF; 89 kDa). LF
and EF are transported from the extracellular surface into the
cytoplasm by the heptamerized PA where they act by enzymatically
modifying molecular targets of mammalian cells. LF is a
metalloprotease that cleaves and activates several
mitogen-activated protein kinases (MAPK kinases) and EF is a
calmodulin-dependent adenylate cyclase that causes a rapid increase
in intracellular cAMP levels. (Young and Collier, 2007, Anthrax
toxin: receptor binding, internalization, pore formation, and
translocation, Annu Rev Biochem, 76:243-65). These proteins are
nontoxic individually, but when administered together are a potent
toxin, causing rapid cell death.
[0020] The AVA vaccine is still the only vaccine for anthrax and
the major move to improve on it is based on several perceived
shortcomings: requirement for a 6-dose regimen in order to achieve
solid immunity and the perception that it is unsafe and
reactogenic, and that the preparative processing of AVA is crude
and lacks consistency. Although PA is the major immunogen in AVA,
it is not clear whether the small amounts of LF and EF that may be
present in some lots contribute to the vaccine's effectiveness. A
major factor in loss of immunogenicity of anthrax rPA involves
accelerated deamidation on the adjuvant surface. This could be
prevented by the presence of small amounts of phosphate to lower
the surface pH. There are other particular rPA vaccine candidates
that are being developed. AVA or PA-based vaccines in general
induce toxin neutralizing antibodies (Pitt, Little et al., 1999, In
vitro correlate of immunity in an animal model of inhalational
anthrax, J Appl Microbiol, 87:304; Reuveny, White et al., 2001,
Search for correlates of protective immunity conferred by anthrax
vaccine, Infect Immun, 69:2888-93; Little, Ivins et al., 2004,
Defining a serological correlate of protection in rabbits for a
recombinant anthrax vaccine, Vaccine, 22:422-30). The mechanism
underlying the protective action of PA-based vaccines is
attributable to anti-PA antibodies that protect the host from
intoxication and thus allow the immune system to deal with the
organism, though PA vaccine is not designed to limit the onset of
infection.
[0021] There are 72 vaccines presently approved by FDA in the
United States (U.S. Food and Drug Administration. Complete List of
Vaccines Licensed for Immunization and Distribution in the US. FDA.
Jun. 3, 2010). Of these 72 vaccines, 36% contain an aluminum
adjuvant and 30% are freeze dried. None of the 72 vaccines contain
both an aluminum adjuvant and freeze dried component.
[0022] There is a need in the art to develop methods of producing
thermostable, immunologically-active freeze dried vaccine
preparations which incorporates recombinant antigens to promote
rapid onset of protective immunity.
SUMMARY OF THE INVENTION
[0023] The disclosure provides a method of production of
thermostable, freeze dried vaccine adjuvant-containing
preparations. The disclosure further provides a method of
production of thermostable, freeze dried vaccine preparations in
which the vaccine antigens are recombinant antigens.
[0024] In one embodiment, the disclosure provides a method of
preparing an immunologically-active adjuvant-bound dried vaccine
composition, the method comprising: combining at least one
aluminum-salt adjuvant, at least one buffer system containing at
least one volatile salt, at least one glass-forming agent, at least
one immunologically active co-adjuvant and at least one antigen to
create a liquid vaccine formulation; freezing the liquid vaccine
formulation to create a frozen vaccine formulation; and
lyophilizing the frozen vaccine formulation to create a dried
vaccine composition, where the composition is capable of eliciting
an immune response in a subject. The immune response developed by
the subject may be humoral immunity and/or cell-mediated immunity
specific to the antigen. In one aspect, the at least one
aluminum-salt adjuvant is selected from the group consisting of
aluminum hydroxide, aluminum phosphate and aluminum sulfate. In
another aspect, the aluminum-salt adjuvant is aluminum hydroxide.
In a further aspect, the at least one buffer system is selected
from the group consisting of acetate, succinate, citrate,
prolamine, arginine, glycine, histidine, borate, carbonate and
phosphate buffer systems. In yet another aspect, the at least one
buffer system is selected from the group consisting of ammonium
acetate, ammonium formate, ammonium carbonate, ammonium
bicarbonate, triethylammonium acetate, triethylammonium formate,
triethylammonium carbonate, trimethylamine acetate trimethylamine
formate, trimethylamine carbonate, pyridinal acetate and pyridinal
formate. In another aspect, the at least one glass-forming agent is
selected from the group consisting of trehalose, sucrose, ficoll,
dextran, sucrose, maltotriose, lactose, mannitol, hydroxyethyl
starch, glycine, cyclodextrin, and povidone. In a further aspect,
the glass-forming agent is trehalose. In one aspect, the
glass-forming agent trehalose is present in a weight-to-volume
concentration of from about 5% to about 15% in the liquid vaccine
formulation prior to freeze drying. In another aspect, the
glass-forming agent trehalose is present in a weight-to-volume
concentration from about 8% to about 20% in the liquid vaccine
formulation. In another embodiment, at least one
immunologically-active co-adjuvant is added to the method steps. In
this aspect, the at least one immunologically-active co-adjuvant is
selected from the group consisting of lipid A, lipid A derivatives,
monophosphoryl lipid A, chemical analogues of monophosphoryl Lipid
A, CpG containing oligonucleotides, TLR-4 agonists, flagellin,
flagellins derived from gram negative bacteria, TLR-5 agonists,
fragments of flagellins capable of binding to TLR-5 receptors,
saponins, analogues of saponins, QS-21, purified saponin fractions,
ISCOMS, saponin combinations with sterols and lipids. In a further
aspect, the co-adjuvant compound is QS-21. In a further aspect, the
freezing step comprises one of tray freezing, shelf freezing,
spray-freezing and shell-freezing. In preferred embodiment, the
freezing step includes use of a pre-cooled tray to initiate the
freezing step.
[0025] In another aspect, the antigen is selected from or derived
from the group consisting of rotavirus, foot and mouth disease
virus, influenza A virus, influenza B virus, influenza C virus,
H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human
parainfluenza type 2, herpes simplex virus, Epstein-Barr virus,
varicella virus, porcine herpesvirus 1, cytomegalovirus,
lyssavirus, Bacillus anthracis, anthrax PA and derivatives,
poliovirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E,
distemper virus, venezuelan equine encephalomyelitis, feline
leukemia virus, reovirus, respiratory syncytial virus, Lassa fever
virus, polyoma tumor virus, canine parvovirus, papilloma virus,
tick borne encephalitis virus, rinderpest virus, human rhinovirus
species, Enterovirus species, Mengovirus, paramyxovirus, avian
infectious bronchitis virus, human T-cell leukemia-lymphoma virus
1, human immunodeficiency virus-1, human immunodeficiency virus-2,
lymphocytic choriomeningitis virus, parvovirus B19, adenovirus,
rubella virus, yellow fever virus, dengue virus, bovine respiratory
syncitial virus, corona virus, Bordetella pertussis, Bordetella
bronchiseptica, Bordetella parapertussis, Brucella abortis,
Brucella melitensis, Brucella suis, Brucella ovis, Brucella
species, Escherichia coli, Salmonella species, Salmonella typhi,
Streptococci, Vibrio cholera, Vibrio parahaemolyticus, Shigella,
Pseudomonas, tuberculosis, avium, Bacille Calmette Guerin,
Mycobacterium leprae, Pneumococci, Staphlylococci, Enterobacter
species, Rochalimaia henselae, Pasteurella haemolytica, Pasteurella
multocida, Chlamydia trachomatis, Chlamydia psittaci,
Lymphogranuloma venereum, Treponema pallidum, Haemophilus species,
Mycoplasma bovigenitalium, Mycoplasma pulmonis, Mycoplasma species,
Borrelia burgdorferi, Legionalla pneumophila, Colstridium
botulinum, Corynebacterium diphtheriae, Yersinia entercolitica,
Rickettsia rickettsii, Rickettsia typhi, Rickettsia prowsaekii,
Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes,
trypanosomes, Leishmania species, Filarial nematodes,
trichomoniasis, sarcosporidiasis, Taenia saginata, Taenia solium,
Leishmania, Toxoplasma gondii, Trichinella spiralis, coccidiosis,
Eimeria tenella, Cryptococcus neoformans, Candida albican,
Apergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae,
malaria circumsporozoite protein, malaria merozoite protein,
trypanosome surface antigen protein, pertussis, alphaviruses,
adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer
membrane protein, streptococcal M protein, Influenza hemagglutinin,
cancer antigen, tumor antigens, toxins, clostridium perfringens
epsilon toxin, ricin toxin, pseudomonas exotoxin, exotoxins,
neurotoxins, cytokines, cytokine receptors, monokines, monokine
receptors, plant pollens, animal dander, and dust mites.
[0026] In another embodiment, the disclosure provides a vaccine
composition, comprising: at least one aluminum-salt adjuvant; at
least one buffering agent, wherein the at least one buffering agent
comprises a volatile salt; at least one glass forming agent; and at
least one antigen, wherein the composition is lyophilized to create
a dried vaccine composition and further wherein the dried vaccine
composition is capable of eliciting an immune response in a
subject. In one aspect, the at least one aluminum-salt adjuvant is
selected from the group consisting of aluminum hydroxide, aluminum
phosphate and aluminum sulfate. In another aspect, the at least one
buffering agent is selected from the group consisting of acetate,
succinate, citrate, prolamine, arginine, glycine, histidine,
borate, carbonate and phosphate. In an alternative aspect, the at
least one buffering agent is selected from the group consisting of
ammonium acetate, ammonium formate, ammonium carbonate, ammonium
bicarbonate, triethylammonium acetate, triethylammonium formate,
triethylammonium carbonate, trimethylamine acetate trimethylamine
formate, trimethylamine carbonate, pyridinal acetate and pyridinal
formate. In yet another aspect, the at least one glass-forming
agent is selected from the group consisting of trehalose, sucrose,
ficoll, dextran, sucrose, maltotriose, lactose, mannitol,
hydroxyethyl starch, glycine, cyclodextrin, and povidone. In a
further aspect, the antigen is selected from or derived from the
group consisting of rotavirus, foot and mouth disease virus,
influenza A virus, influenza B virus, influenza C virus, H1N1,
H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human
parainfluenza type 2, herpes simplex virus, Epstein-Barr virus,
varicella virus, porcine herpesvirus 1, cytomegalovirus,
lyssavirus, Bacillus anthracis, anthrax PA and derivatives,
poliovirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E,
distemper virus, venezuelan equine encephalomyelitis, feline
leukemia virus, reovirus, respiratory syncytial virus, Lassa fever
virus, polyoma tumor virus, canine parvovirus, papilloma virus,
tick borne encephalitis virus, rinderpest virus, human rhinovirus
species, Enterovirus species, Mengovirus, paramyxovirus, avian
infectious bronchitis virus, human T-cell leukemia-lymphoma virus
1, human immunodeficiency virus-1, human immunodeficiency virus-2,
lymphocytic choriomeningitis virus, parvovirus B19, adenovirus,
rubella virus, yellow fever virus, dengue virus, bovine respiratory
syncitial virus, corona virus, Bordetella pertussis, Bordetella
bronchiseptica, Bordetella parapertussis, Brucella abortis,
Brucella melitensis, Brucella suis, Brucella ovis, Brucella
species, Escherichia coli, Salmonella species, Salmonella typhi,
Streptococci, Vibrio cholera, Vibrio parahaemolyticus, Shigella,
Pseudomonas, tuberculosis, avium, Bacille Calmette Guerin,
Mycobacterium leprae, Pneumococci, Staphlylococci, Enterobacter
species, Rochalimaia henselae, Pasteurella haemolytica, Pasteurella
multocida, Chlamydia trachomatis, Chlamydia psittaci,
Lymphogranuloma venereum, Treponema pallidum, Haemophilus species,
Mycoplasma bovigenitalium, Mycoplasma pulmonis, Mycoplasma species,
Borrelia burgdorferi, Legionalla pneumophila, Colstridium
botulinum, Corynebacterium diphtheriae, Yersinia entercolitica,
Rickettsia rickettsii, Rickettsia typhi, Rickettsia prowsaekii,
Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes,
trypanosomes, Leishmania species, Filarial nematodes,
trichomoniasis, sarcosporidiasis, Taenia saginata, Taenia solium,
Leishmania, Toxoplasma gondii, Trichinella spiralis, coccidiosis,
Eimeria tenella, Cryptococcus neoformans, Candida albican,
Apergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae,
malaria circumsporozoite protein, malaria merozoite protein,
trypanosome surface antigen protein, pertussis, alphaviruses,
adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer
membrane protein, streptococcal M protein, Influenza hemagglutinin,
cancer antigen, tumor antigens, toxins, clostridium perfringens
epsilon toxin, ricin toxin, pseudomonas exotoxin, exotoxins,
neurotoxins, cytokines, cytokine receptors, monokines, monokine
receptors, plant pollens, animal dander, and dust mites.
[0027] In an alternative embodiment, the vaccine composition
further includes at least one immunologically-active co-adjuvant.
In one aspect, the at least one immunologically-active co-adjuvant
is selected from the group consisting of lipid A, lipid A
derivatives, monophosphoryl lipid A, chemical analogues of
monophosphoryl Lipid A, CpG containing oligonucleotides, TLR-4
agonists, flagellin, flagellins derived from gram negative
bacteria, TLR-5 agonists, fragments of flagellins capable of
binding to TLR-5 receptors, saponins, analogues of saponins, QS-21,
purified saponin fractions, ISCOMS and saponin combinations with
sterols and lipids.
[0028] In yet another embodiment, the disclosure provides a method
of controlling particle size in an adjuvant-bound dried vaccine
composition, the method comprising: combining at least one
aluminum-salt adjuvant, at least one buffer system, at least one
glass-forming agent, and at least one antigen to create a liquid
vaccine formulation; freezing the liquid vaccine formulation to
create a frozen vaccine formulation; and lyophilizing the frozen
vaccine formulation to create a dried vaccine composition, wherein
following dilution of the dried vaccine composition with an aqueous
diluent to form a reconstituted vaccine composition the mean
particle diameter of the reconstituted vaccine composition is less
than 100 micrometers. In another aspect, the at least one
aluminum-salt adjuvant is selected from the group consisting of
aluminum hydroxide, aluminum phosphate and aluminum sulfate. In a
further aspect, the aluminum-salt adjuvant is aluminum hydroxide.
In another aspect, the at least one buffer system is selected from
the group consisting of acetate, succinate, citrate, prolamine,
histidine, borate, carbonate and phosphate buffer systems. In a
further aspect, the at least one buffer system is selected from
succinate and phosphate buffer systems. In one aspect, the at least
one glass-forming agent is selected from the group consisting of
trehelose, sucrose, ficoll, dextran, sucrose, maltotriose, lactose,
mannitol, hydroxyethyl starch, glycine, cyclodextrin, povidone, and
potassium salts. In a specific aspect, the glass-forming agent is
trehalose. In a further specific aspect, the glass-forming agent
trehalose is present in a weight to volume concentration of from
about 5% to about 20% in the liquid vaccine formulation. In another
aspect, the glass-forming agent trehalose is present in a weight to
volume concentration of from about 7% to about 15% in the liquid
vaccine formulation. In one aspect, the freezing step comprises one
of tray freezing, shelf freezing, spray-freezing and
shell-freezing. In another aspect, the freezing step comprises
spray-freezing. In a further aspect, the mean particle diameter of
the reconstituted vaccine composition is less than 6 micrometers.
In one aspect, the concentration of the glass forming agent in the
selecting step is decreased as the rate of cooling the liquid
vaccine formulation to a frozen state in the cooling step is
increased.
[0029] In an alternative embodiment, the disclosure provides an
adjuvant composition for use in a dried vaccine composition, the
adjuvant composition comprising: an aluminum-salt adjuvant, a
glass-forming agent, and a buffer salt. In one aspect, the
aluminum-salt adjuvant is selected from aluminum hydroxide and
aluminum phosphate. In another aspect, the glass-forming agent is
trehalose. In a further aspect, the buffer salt is selected from
one or more of the group consisting of sodium succinate, potassium
succinate, sodium phosphate and potassium phosphate.
[0030] In yet another embodiment, the disclosure provides an
adjuvant-bound dried vaccine composition having limited mean
particle diameter, the composition produced by a method comprising:
blending at least one adjuvant, at least one glass forming agent,
and at least one antigen in a buffer system to create a liquid
vaccine formulation; cooling the liquid vaccine formulation rapidly
to a frozen state to create a frozen vaccine formulation; and
lyophilizing the frozen vaccine formulation to create a dried
vaccine composition, wherein following dilution of the dried
vaccine composition with an aqueous diluent to form a reconstituted
vaccine composition, the mean particle diameter of the
reconstituted vaccine composition is less than 100 micrometers.
[0031] In another embodiment, the disclosure provides a method of
controlling particle size in a frozen vaccine formulation, the
method comprising: combining at least one aluminum-salt adjuvant,
at least one buffer system, at least one glass-forming agent, and
at least one antigen to create a liquid vaccine formulation;
freezing the liquid vaccine formulation to create a frozen vaccine
formulation; and lyophilizing the frozen vaccine formulation to
create a dried vaccine composition, wherein following thawing and
dilution of the dried vaccine composition with an aqueous diluent
to form a reconstituted vaccine composition the mean particle
diameter of the reconstituted vaccine composition is less than 100
micrometers. In one aspect, the at least one aluminum-salt adjuvant
is selected from the group consisting of aluminum hydroxide,
aluminum phosphate and aluminum sulfate. In another aspect, the
aluminum-salt adjuvant is aluminum hydroxide. In a further aspect,
the at least one buffer system is selected from the group
consisting of acetate, succinate, citrate, prolamine, histidine,
borate, carbonate and phosphate buffer systems. In one aspect, the
at least one buffer system is selected from succinate and phosphate
buffer systems. In another aspect, the at least one glass-forming
agent is selected from the group consisting of trehalose, sucrose,
ficoll, dextran, sucrose, maltotriose, lactose, mannitol,
hydroxyethyl starch, glycine, cyclodextrin, povidone, and potassium
salts. In a specific aspect, the glass-forming agent is trehalose.
In a further specific aspect, the glass-forming agent trehalose is
present in a weight to volume concentration of from about 5% to
about 20% in the liquid vaccine formulation. In another specific
aspect, the glass-forming agent trehalose is present in a weight to
volume concentration of from about 7% to about 15% in the liquid
vaccine formulation. In one aspect, the freezing step comprises one
of tray freezing, shelf freezing, spray-freezing and
shell-freezing. In another aspect, the freezing step comprises
spray-freezing. In a further aspect, the mean particle diameter of
the reconstituted vaccine composition is less than 6
micrometers.
[0032] In another aspect, the liquid vaccine formulation is
prepared as a hypertonic mixture prior to freezing, wherein upon
dilution of the dried vaccine composition with an aqueous diluent
to form a reconstituted vaccine composition, the tonicity of the
reconstituted vaccine composition is adjusted to isotonic levels.
In yet another aspect, a formulation is prepared wherein the
volatile salt is removed by lyophilization, yielding, upon
reconstitution, a vaccine preparation with tonicity reduced
relative to the starting formulation while retaining the same
concentration of antigens and adjuvants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 depicts particle size distributions before and after
freeze drying and reconstitutions based on % surface area.
[0034] FIG. 2 depicts particle size distributions of histidine
formulations based on % surface area before and after freeze
drying.
[0035] FIG. 3 depicts particle size distributions of arginine
formulations based on % surface area before and after freeze
drying.
[0036] FIG. 4 depicts particle size distributions of glycine
formulations based on % surface area before and after freeze
drying.
[0037] FIGS. 5(a)-5-(c) show Alhydrogel particles settling over
time in 10 mM histidine buffer pH 6. (a) No settling; (b) After 30
minutes of settling; and (c) After 3 hours of settling.
[0038] FIG. 6 depicts particle size distributions based on surface
area after allowing particles to settle for varying amounts of time
before freeze drying with -10.degree. C. pre-cooled shelves.
Samples contained 1 mg/mL Al in 10 mM histidine buffer at pH 6.
[0039] FIG. 7 depicts particle size distributions based on surface
area after allowing particles to settle for varying amounts of time
before freeze drying with -10.degree. C. pre-cooled shelves.
Samples contained 1 mg/mL Al and 8 w/v % trehalose in 10 mM
histidine buffer at pH 6.
[0040] FIG. 8 shows a comparison of mean particle size between
formulations of 1 mg/mL Al in 10 mM histidine with and without
trehalose while varying the settling time before freeze drying.
[0041] FIG. 9 demonstrates the steps during lyophilization in which
1 ml vaccine samples contained in 3 ml glass vials were treated to
varying freezing rates with an FTS System LyoStar Freeze Drying
System before primary and secondary freezing. After freeze drying,
vials were purged with nitrogen gas sealed and stored at
-80.degree. C. before further analysis.
[0042] FIGS. 10(A)-10(D) show particle size distributions before
and after freeze drying cycles under four conditions and increasing
concentrations of trehalose. Faster rates of freezing before
primary and secondary drying and higher concentrations of trehalose
result in particle size distributions after freeze drying most
similar to the initial particle distribution. From slowest to
fastest: room temperature tray A (A), -10.degree. C. Pre-cooled
Tray (B), Liquid Nitrogen dip (C), Liquid Nitrogen spray Freeze
Drying (D). Formulation consisted of 1 mg/ml as Alhydrogel, 10 mM
histidine, pH 6.0, with 0-12% trehalose.
[0043] FIG. 11 shows the dependence of particle size distribution
after freeze drying of room temperature freeze drying. Room
temperature freeze drying was carried out as in FIG. 10(A). With
room temperature incubation on trays prior to the freezing cycle,
particle size distribution shifted from <1 micron to >than 20
microns, and the presence of 8% trehalose reduce the magnitude of
the particle size shift.
[0044] FIG. 12 shows SDS PAGE of RTA dissolved in 10 mM histidine,
pH 6.0, 144 mM NaCl with 50% w/v glycerol in comparison to RTA
dialyzed, concentrated and stored at -20 degree C. (Top
panel--silver stain, bottom panel--Coomassie stain. The same set of
samples was used to perform both studies).
[0045] FIG. 13 shows adsorption of RTA to Alhydrogel prior to
lyophilizaiont. The concentration of RTA was varied keeping the
concentration of Al at 1 mg/mL. At least 95% of the RTA protein was
adsorbed to the surface of Alhydrogel at pH 6.0.
[0046] FIG. 14 shows the results of vaccination of a liquid RTA
vaccine adsorbed to Alhydrogel in which various dose of RTA were
used to vaccinate groups of 8 Swiss Webster mice. When the vaccine
was stored at 40.degree. C. for 1 month prior to vaccination, none
of the animals exposed to ricin toxin survived and a significant
loss of immunogenicity was observed. The vaccine stored at
4.degree. C. for one month induced total protection at the highest
doses and partial protection in mice at lower doses.
[0047] FIG. 15 shows rRTA antibody titers after one injection (week
3) and after two injections (week 5) for each vaccine after no
incubation, 1 week and 1 month incubation at 40.degree. C. Average
titers are shown as the average of only the mice that responded
with the standard deviation of those mice.
[0048] FIG. 16 shows endpoint titer data from immunized mice.
Endpoint Titers =reciprocal endpoint anti-RTA titers. Neutralizing
IC50 Titers =the dilution of sera required to protect 50% of the
cells in a well from ricin cytotoxicity Not shown here, but none of
the sham immunized mice (#1-10 (except 5) had any anti-RTA titers
in their sera. #1 sham immunized mouse sera was also tested for
neutralizing capacity in vitro, and did not protect cells.
[0049] FIG. 17 shows total and neutralizing titers obtained from
individual sera post 2nd vaccination of Swiss Webster mice with
adsorbed liquid vaccine prior to lyophilization.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0050] Trehalose dehydrate (high purity, low endotoxin) was
obtained from Ferro Pfanstiehl (Cleveland, Ohio). Arginine,
glycine, histidine, sodium citrate, and ammonium acetate were
purchased from Sigma Chemical Company (St. Louis, Mo.).
Alhydrogel.TM. 2.0% (aluminum hydroxide adjuvant), made by Brenntag
Biosector, was purchased through E.M. Sergeant Pulp & Chemical
Co, Inc (Clifton, N.J.). 3-ml and 5-ml lyophilization vials and
caps were obtained from West Pharmaceutical Services.
Sample Preparation
[0051] Aqueous solutions were prepared containing different
concentrations of trehalose (0-15 w/v %). Unless otherwise noted,
samples were prepared in 10 mM buffer (as indicated) at pH 6.0 and
contained 1 mg/ml Al (as Alhydrogel.TM.). Samples were processed as
one-ml aliquots. With the exception of the adjuvant, all aqueous
solutions were passed through a 0.2 .mu.m filter prior to
formulation.
Surface Charge Zeta Potential
[0052] Zeta potentials were measured for suspensions of aluminum
hydroxide (Alhydrogel) in various formulations to probe
electrostatic interactions. Formulations without antigen were then
prepared to determine if aggregation of particles occurred during
freeze drying. Alhydrogel at a concentration of 1 mg Al/mL was
combined in 10mM buffer (glycine, arginine, histidine, ammonium
acetate, sodium citrate) at pH 6 with the stabilizer trehalose
ranging from 0-12w/v %. To determine if the rate of freezing
affects particle aggregation, formulations were freeze dried using
four methods of freezing: Room Temperature Tray Freezing,
-10.degree. C. Pre-cooled Tray Freezing, Liquid Nitrogen Dip
Freezing, and Liquid Nitrogen Spray Freezing before primary and
secondary drying. Protein was also added to formulations to see its
effect on particle size after freeze drying and reconstitution.
Particle size distributions in the range of 0.04-2000 .mu.m were
characterized by laser diffraction for each formulation.
Lyophilization
[0053] An FTS Systems Lyostar lyophilizer was used for the
freeze-drying of samples. Samples were frozen at various cooling
rates as follows from slowest to fastest: (i) Vials prepared at
room temperature were placed on the lyophilizer trays and kept at
room temperature for 1 hour prior to inaiting the (ii.) Frozen by
placing the samples in lyophilizer, equilibrating 1 hr at a shelf
temperature of -10.degree. C., then cooling the shelves at
0.5.degree. C./min to -40.degree. C. ("-10 pre-cooled
tray-freezing"); (ii.) Frozen by immersion of bottom of vial into
liquid N2 (LN2 Dip Freeze Drying); and (iii.) Spray-freezing by
dropping by -20 .mu.l droplets into liquid N.sub.2. (LN2 spray
freeze drying). Tray-frozen and liquid N2-immersed samples were
processed in 3-ml lyophilization vials, while the spray-frozen
samples were processed in 5-ml lyophilization vials. Vials
containing samples frozen using liquid N2 were quickly transferred
to the lyophilizer placed on lyophilizer shelves pre-cooled to
-40.degree. C. Samples were spaced in the lyophilizer so that they
were each separated from one another and were encircled with a row
of vials containing water.
[0054] Primary drying of the samples was achieved by setting the
shelf temperature to -20.degree. C. and applying vacuum at 60 mTorr
for 20 hours, and was followed by secondary drying, in which shelf
temperatures were ramped from -20.degree. C. to 0.degree. C. at
0.2.degree. C./min, to 30.degree. C. at 0.5.degree. C./min and
finally held at 30.degree. C. for 5 hours. Samples were sealed
under vacuum and reconstituted with DI water prior to analysis. The
variations of the freezing and drying cycles are depicted in (FIGS.
10(a)-10(D)).
Particle Size Distributions
[0055] Particle size distributions (PSD) were measured using a
Beckman-Coulter LS230 laser diffraction particle size analyzer.
Three one-ml samples were required for each run, and three
replicates of each run were completed per formulation. Reported
PSD's are surface area weighted and are composites of three
runs.
Examples
[0056] I. -10.degree. C. Pre-Cooled Tray Freeze Drying with Varying
Settling Time of Particles 10 mM histidine buffer at pH 6.0, 1
mg/mL Al from Alhydrogel, and 0, 4, 8 or 12 w/v % trehalose was
combined and rotated end over end for 30 minutes at 4.degree. C. 1
mL of the solution was placed in each 3 mL glass freeze drying
vial. The formulations were placed on -10.degree. C. pre-cooled
shelves in the freeze drier and freeze dried as follows in the
table below. Following freeze drying, the chamber was backfilled
with dry nitrogen gas and the vials were sealed.
TABLE-US-00001 TABLE 1 Initial Final Temp Temp Stage Time for Step
(.degree. C.) (.degree. C.) Pressure Rate Freezing 0.25 hours -10
-10 Atmospheric Constant temp -10 1 hours -10 -40 Atmospheric
-0.5.degree. C./min 1 hour -40 -40 Atmospheric Constant temp -40
Primary 0.5 hours -40 -40 60 mTorr Constant Drying temp -40 0.5
hour -40 -20 60 mTorr Increase temp 20 hours -20 -20 60 mTorr
Constant temp -20 Secondary 1 hour 40 min -20 0 60 mTorr
0.2.degree. C./min Drying 1 hour 0 30 60 mTorr 0.5.degree. C./min 5
hours 30 30 60 mTorr Constant temp 30
Particle Size Analysis
[0057] Particle size analysis was done on the solutions before they
were freeze dried as well as on the freeze dried samples
reconstituted in 1 mL of DI water. Laser diffraction particle size
analysis was done using a LS 230 instrument made by Beckman. For
the analysis no sonication was done on the sample chamber. The
model used for calculating particle size distributions used a
solution refractive index of 1.33 and a sample refractive index of
1.57. Approximately 6 mL of sample was required to be added to
filtered DI water in the analyzer before the reading was taken. For
each run three ninety second averaged particle size distributions
were taken. For each formulation three runs were taken.
Results
[0058] When formulations contained higher concentrations of
trehalose (8-12 w/v %) the initial particle size distribution was
able to be maintained as seen in FIG. 1.
II. -10.degree. C. Pre-Cooled Tray Freeze Drying 10 mM buffer, 1
mg/mL Al from alhydrogel, 10w/v % trehalose with and without 0.26
mg/mL rRTA was combined and rotated end over end for 30 minutes at
4.degree. C. 1 mL of the solution was placed in each 3 mL glass
freeze drying vial. The formulations were placed on -10.degree. C.
pre-cooled shelves in the freeze drier and freeze dried as follows
in the table below. Following freeze drying, the chamber was
backfilled with dry nitrogen gas and the vials were sealed.
TABLE-US-00002 TABLE 2 Initial Final Temp Temp Stage Time for Step
(.degree. C.) (.degree. C.) Pressure Rate Freezing 0.25 hours -10
-10 Atmospheric Const. temp 5 1 hours -10 -40 Atmospheric
-0.5.degree. C./min 1 hour -40 -40 Atmospheric Const. temp -40
Primary 0.5 hours -40 -40 60 mTorr Const. Drying temp -40 0.5 hour
-40 -20 60 mTorr Increase temp 20 hours -20 -20 60 mTorr Const.
temp -20 Secondary 1 hour 40 min -20 0 60 mTorr 0.2.degree. C./min
Drying 1 hour 0 30 60 mTorr 0.5.degree. C./min 5 hours 30 30 60
mTorr Const. temp 30
Particle Size Analysis
[0059] Particle size analysis was done on the solutions before they
were freeze dried as well as on the freeze dried samples
reconstituted in 1 mL of DI water. Laser diffraction particle size
analysis was done using a LS 230 instrument made by Beckman. For
the analysis no sonication was done on the sample chamber. The
model used for calculating particle size distributions used a
solution refractive index of 1.33 and a sample refractive index of
1.57. Approximately 6-7 mL of sample was required to be added to
filtered DI water in the analyzer before the reading was taken. For
each run three ninety second averaged particle size distributions
were taken. For each formulation three runs were taken.
Results
[0060] In arginine, histidine, and glycine buffers containing 10
w/v % trehalose, both with and without rRTA protein present, the
particle size distribution was able to be maintained before freeze
drying and after using -10.degree. C. pre-cooled shelves before
freeze drying. Particle size distributions can be seen in FIGS.
1-3. Particle size distributions could possibly be maintain better
with pre-cooled shelves before freeze drying than tray freeze
drying because the adjuvant particles have less time to settle
before the formulation freezes when pre-cooled shelves are
used.
III. -10.degree. C. Pre-Cooled Tray Freeze Drying with Varying
Settling Time of Particles at 3 Hour, 30 Minute and 0 Timepoints
before Freeze Drying
[0061] 10 mM histidine buffer at pH 6, 1 mg/mL Al from alhydrogel,
and 0 or 8 w/v % trehalose was combined and rotated end over end
for 30 minutes at 4.degree. C. 1 mL of the solution was placed in
each 3 mL glass freeze drying vial. The vials were divided into
three groups that were allowed to rest for 3 hours, 30 minutes and
0 minutes before being placed in the freeze drier. Once vials were
filled they were allowed to sit at 4.degree. C. until it was time
to be loaded in the freeze drier. The formulations were placed on
-10.degree. C. pre-cooled shelves in the freeze drier and freeze
dried as follows in the table below. Following freeze drying, the
chamber was backfilled with dry nitrogen gas and the vials were
sealed.
TABLE-US-00003 TABLE 3 Initial Final Temp Temp Stage Time for Step
(.degree. C.) (.degree. C.) Pressure Rate Freezing 0.25 hours -10
-10 Atmospheric Constant temp 5 1 hours -10 -40 Atmospheric
-0.5.degree. C./min 1 hour -40 -40 Atmospheric Constant temp -40
Primary 0.5 hours -40 -40 60 mTorr Constant Drying temp -40 0.5
hour -40 -20 60 mTorr Increase temp 20 hours -20 -20 60 mTorr
Constant temp -20 Secondary 1 hour 40 min -20 0 60 mTorr
0.2.degree. C./min Drying 1 hour 0 30 60 mTorr 0.5.degree. C./min 5
hours 30 30 60 mTorr Constant temp 30
Particle Size Analysis
[0062] Particle size analysis was done on the solutions before they
were freeze dried as well as on the freeze dried samples
reconstituted in 1 mL of DI water. Laser diffraction particle size
analysis was done using a LS 230 instrument made by Beckman. For
the analysis no sonication was done on the sample chamber. The
model used for calculating particle size distributions used a
solution refractive index of 1.33 and a sample refractive index of
1.57. Approximately 6 mL of sample was required to be added to
filtered DI water in the analyzer before the reading was taken. For
each run three ninety second averaged particle size distributions
were taken. For each formulation two runs were taken.
Results
[0063] Before samples were placed in the freeze drier they were
allowed to settle for 0 minutes, 30 minutes or 3 hours. In FIGS.
5(a)-5(c), a vial containing 1 mg/mL Al from Alhydrogel in 10 mM
histidine is shown at various time points during settling. Without
settling, the formulation appears to be cloudy throughout the
solution (FIG. 5a). After 30 minutes of settling the majority of
the alhydrogel particles appear to be close to the bottom of the
vial with a slightly cloudy solution above (FIG. 5b). After 3 hours
of settling the alhydrogel particles have settled closer to the
bottom of the vial and leave a clear solution above the alhydrogel
layer (FIG. 5c).
[0064] When the formulation contained alhydrogel and histidine
without trehalose, the particle size distribution was shifted
towards larger particles from the initial particle size
distribution (FIG. 6). Formulations that were allowed to settle for
less time produced slightly smaller particles than those allowed to
settle for longer periods of time.
[0065] When formulations contained 8 w/v % trehalose, the amount of
time the samples were allowed to settle before being placed in the
freeze drier effected the particle size distribution (FIG. 7). When
the formulation was not allowed to settle before being placed in
the freeze drier, the particle size distribution was very similar
to the initial particle size distribution before freeze drying.
After 30 minutes of settling the particle size distribution starts
to shift to larger particle sizes and at 3 hours of settling the
particles are significantly larger than the initial particle size
distribution.
[0066] When comparing the formulations with trehalose in comparison
the ones without trehalose, trehalose presence in the formulation
in maintains the particle size distribution after the freeze drying
process. Although the initial mean particle size before freeze
drying is the same with and without trehalose present in the
formulation, the mean particle size after freeze drying is smaller
when trehalose is present in the formulation at each amount of
settling before freeze drying as can be seen in FIG. 8. From these
experiments we can also see the importance of not allowing the
samples to settle before loading in the freeze drier if it is
desired to maintain the initial particle size.
IV. Immunogenicty of Ricin Vaccine Subunit in Experimental
Animals.
[0067] As an example, a thermostable lyophilized ricin subunit
vaccine was constructed and tested. Ricin A chain vaccine was used
because it is subject to aggregation and denaturation in aqueous
buffers and is prone to losses in structural integrity that affect
immunogenicity and the induction of neutralizing antibodies
involved in protection against ricin toxin exposure. A lyophilized
ricin vaccine was prepared as follows. RTA dissolved in glycerol
was dialyzed against 10 mM histidine buffer, pH 6.0 to remove
glycerol (FIG. 11). The liquid suspension vaccine was placed into
vials and subjected to lyophilization as described in FIGS.
10(A)-10(D) to compare precooled freeze drying at -10 degrees C.
with vaccine at room temperature prior to initiating of the primary
freeze drying cycle at -40.degree. C. The dried vaccines were
stored either at refrigeration temperature (4-8.degree. C.) or at
elevated temperature (40-60.degree. C.). Samples from the stored
lyophilized vaccine were withdrawn periodically and tested for
structural integrity by assessment of binding of a diagnostic
monoclonal antibody termed R70 (Neal, O'Hara et al., 2010, A
monoclonal immunoglobulin G antibody directed against an
immunodominant linear epitope on the ricin A chain confers systemic
and mucosal immunity to ricin, Infect Immun, 78:552-61). In
addition, vaccines were subjected to additional biophysical tests
including the determination of intrinsic fluorescence diagnostic of
tertiary structure of protein bound to aluminum, determination of
residual water, and immunogenicity/potency in mice. Immunogenicity
was determined by injecting Swiss Webster mice as below and
determining total antibodies against the vaccine by ELISA and
determination of ricin neutralizing antibodies. Mice were exposed
to ricin toxin at day 35 by injection of 10.times.LD50 dose of
toxin and lethality was determined in the exposed animals. In
addition, peptide scans were performed in which serum from
vaccinated and control mice were assessed for response to
overlapping peptides encompassing the RTA molecule. This was done
to determine the immunodominant regions and their preservation
during high and low temperature storage conditions. When control
liquid vaccine was used to vaccinate mice 3.times. by intramuscular
injection, incubation of the vaccine at 40.degree. C. for one month
resulted in loss of immunogenicity and the ability to induce
protective immunity (FIG. 14).
[0068] During the study, each Swiss Webster mouse was bled three
times and injected with a vaccine formulation twice. Before the
initial injection mice were bled and then on day 0 injected with a
vaccine formulation. The initial bleeding was necessary so that
each mouse could be its own baseline. 21 days later the mice were
bled and injected with a booster vaccine formulation. 35 days after
the initial injection the mice were bled one last time. Before
bleeding procedures the mice were anesthetized using is isofluorane
inhalant. Blood was drawn from the retro-orbital venous sinus of
the mice. A drop of proparacaine was put on the eye from which
blood was drawn and then blood was collected using 504, capillary
tubes. Approximately 100-2004, of blood was drawn during each
bleeding.
TABLE-US-00004 TABLE 4 Group Contents Negative Control Freeze dried
Alhydrogel in histidine buffer Negative Control Freeze dried
Alhydrogel in ammonium acetate buffer Positive Control Liquid
formulation of rRTA and Alhydrogel Experimental Group 1 Freeze
dried (Room temp shelves) rRTA and Alhydrogel in histidine buffer
Experimental Group 2 Freeze dried (Room temp shelves) rRTA and
Alhydrogel in ammonium acetate buffer Experimental Group 3 Freeze
dried (Pre-cooled shelves) rRTA and Alhydrogel in histidine buffer
Experimental Group 4 Freeze dried (Pre-cooled shelves) rRTA and
Alhydrogel in ammonium acetate buffer
[0069] To create variations in the formulation particle size,
different buffers such as histidine and ammonium acetate and the
variation of freezing rate before freeze drying (such as room
temperature shelves or pre-cooled shelves before freeze drying)
were used. All samples contained the disaccharide trehalose up to
15% (w/V) and Alhydrogel is an aluminum hydroxide vaccine adjuvant
used at 0.85 -1 mg/ml total aluminum.
V. Controlled Lyophilization of Adsorbed Ricin Vaccine.
[0070] The central objective of this invention is to make subunit
vaccines by employing controlled lyophilization of protein,
aluminum adjuvant, and immunostimulant components for
reconstitution with water at the point of use. Using aluminum
adjuvant, it has not been feasible or possible up to this point to
adequately combine these components together without loss of
vaccine effectiveness on the one hand and gross clumping and
inability to rehydrate adequately. A number of different conditions
for precisely controlling points in the lyophilization cycle
examining a spectrum of buffer conditions, salt conditions, and
lyophilization cycle conditions and have reported that we had been
able to define conditions for retaining gross integrity including
protein structure pre and post lyophilization.
[0071] VI. Generation of Prototype Freeze Dried Vaccines. A series
of freeze dried formulation was made according to the general
lyophilization schemes presented in Table 1. Freeze dried
formulations with RTA protein and placebo formulations without
protein were created containing 1.0 mg Al/mL, 8 w/v % trehalose and
0.2 or 0 mg/mL rRTA in 10 mM histidine or ammonium acetate buffer
pH 6, with either pre-cooling (PC) prior to lyophilization or room
temperature incubation prior to lyophilization. Formulations were
prepared by mixing with a stir bar at 4-8.degree. C. for 1 hour to
allow protein to adsorb to Alhydrogel adjuvant. 1 mL of formulation
was placed in a 3 mL glass vial and freeze dried as described in
Table 4. Samples from each process condition were incubated at
40.degree. C. and withdrawn for analysis and vaccination studies at
1 week, one month (and continuing through month 6). Pre- and
post-lyophilization samples were also obtained.
TABLE-US-00005 TABLE 5 Freeze Drying Cycle Initial Final Temp Temp
Stage Time for Step (.degree. C.) (.degree. C.) Pressure Rate
-10.degree. C. 0.25 hour -10 -10 Atmospheric Constant Pre- temp -10
Cooled Tray Freezing 1 hour -10 -40 Atmospheric -0.5.degree. C./min
1 hour -40 -40 Atmospheric Constant temp -40 Primary 0.5 hours -40
-40 60 mTorr Constant Drying temp -40 0.5 hour -40 -20 60 mTorr
Increase temp 20 hours -20 -20 60 mTorr Constant temp -20 Secondary
1 hour 40 min -20 0 60 mTorr 0.2.degree. C./min Drying 1 hour 0 30
60 mTorr 0.5.degree. C./min 5 hours 30 30 60 mTorr Constant temp
30
VII. Particle Size Analysis of Reconstituted Dried Vaccines.
[0072] Particle size analysis was done on the solutions before they
were freeze dried as well as on the freeze dried samples
reconstituted in 1 mL of deionized water. Laser diffraction
particle size analysis was conducted using a LS 230 instrument made
by Beckman. For the analysis no sonication was done on the sample
chamber. The model used for calculating particle size distributions
used a solution refractive index of 1.33 and a sample refractive
index of 1.57. Approximately 6 mL of sample was required to be
added to filtered DI water in the analyzer before the reading was
taken. For each run three ninety second averaged particle size
distributions were taken. For each formulation three runs were
taken. The particle size distribution of the placebo stability
study samples is being monitored over with using laser diffraction.
The initial Time 0 liquid formulations all had similar particle
size distributions and mean particle sizes based on surface area as
can be seen in Table 5. When formulations were Tray Freeze Dried
from Room Temperature, an increase in particle size was seen. When
formulations were Tray Freeze Dried from -10.degree. C. Pre-Cooled
Shelves, the particle size distribution stayed very similar to the
initial particle size distribution.
TABLE-US-00006 TABLE 6 Mean particle size .+-. standard deviation
based on surface area Vaccine Time Point RT His RT AA PC His PC AA
Time 0 - 0.35 .+-. 0.01 0.34 .+-. 0.01 0.35 .+-. 0.01 0.35 .+-.
0.01 Liquid Time 0 - 9.43 .+-. 0.31 8.11 .+-. 0.74 0.38 .+-. 0.06
0.49 .+-. 0.05 FD Time 1 10.69 .+-. 0.41 8.96 .+-. 0.16 0.44 .+-.
0.09 0.42 .+-. 0.05 Week - FD Time 1 10.31 .+-. 0.63 9.09 .+-. 0.12
0.46 .+-. 0.12 0.55 .+-. 0.07 Month - FD
VIII. Vaccination of Animals.
[0073] Female Swiss Webster mice 5-6 weeks old were vaccinated with
50 .mu.L of the indicated formulations containing 10 microgram of
RTA protein subcutaneously on Day 0 and 20. Mice under anesthesia
by isoflurane were bled through the retro orbital cavity collecting
approximately 2004, of blood on Day 0, 20 and 34. In each group 10
mice were used. Mice were housed 5 per cage and were allowed food
and water all the time. Serum was separated from blood by
centrifugation at 10,000 rpm for 14 minutes at 4.degree. C.
[0074] Total antibody to RTA in individual sera from vaccinated
Swiss Webster mice was determined by ELISA and for determination of
neutralizing antibodies (FIGS. 6 and 7). Nunc flat bottom MaxiSorb
96 well plates were coated with 50 .mu.L/well of stock protein
diluted in PBS to 1 .mu.g rRTA/mL and incubated at 2-6.degree. C.
overnight. Plates were washed 4 times with 300 .mu.L/well of PBS
with 0.05% Tween 20. Plates were blocked with 300 .mu.L/well of PBS
with 1% BSA and incubated at room temperature for 2 hours. Plates
were washed as previously described. 40 .mu.L of PBS with 1% BSA
and 0.05% Tween 20 was added to each well. Serum was initially
diluted in a dilution buffer of PBS with 1% BSA and 0.05% Tween 20.
70 .mu.L of sample was added to the starting well and then a seven
in-plate 2.33-fold dilution was created for each sample. The plate
was then incubated for 2 hours at room temperature. Plates were
washed again. 40 .mu.L of HRP-conjugated donkey anti-mouse antibody
diluted 10,000 times was added to each well and incubated for 2
hours at room temperature. Plates were washed again. TMB was added
to each well at 40 .mu.L and incubated for 30 minutes. Stop
solution of 2N sulfuric acid was added at 40 .mu.L to each well.
The plate was read at 450 nm. Endpoint dilution analysis of
individual serum samples from vaccinated mice is shown in FIG. 15.
The vaccines tested are abbreviated as follows:
RT His--Negative control (room temperature tray freeze dried in
histidine with no protein) RT AA--Negative control (room
temperature tray freeze dried in ammonium acetate with no protein)
His+rRTA Liquid--Positive control (liquid formulation in histidine
with protein) RT His+rRTA--Experimental 1 (room temperature tray
freeze dried in histidine with protein) RT AA +rRTA--Experimental 2
(room temperature tray freeze dried in ammonium acetate with
protein) RPC His+rRTA--Experimental 3 (Pre-Cooled tray freeze dried
in histidine with protein) PC AA+rRTA--Experimental 4 (Pre-Cooled
tray freeze dried in ammonium acetate with protein)
[0075] When vaccines were stored for one or one month at 40.degree.
C., there was no significant difference in the capacity of the
vaccines to generate antibodies against RTA (by ELISA) after one
injection of 10 microgram (week 3 titers) or 2 injections (week 5)
in relationship to vaccine prepared without storage at 40.degree.
C. (time 0 in FIG. 15). At week three, 90-100% of mice in each
experimental and positive control group responded and by week five
all experimental and positive control responded (Table 3). More
important, serum obtained from post 2 (week 5) contained antibodies
that neutralized ricin (in vitro) where the titers and the
proportion of mice with such titers were not obviously different
from time 0 vaccines (FIG. 16) or the liquid vaccines (FIG. 17).
Further, neutralizing titers decreased after storage of lyophilized
RTA vaccine at 40.degree. C. for 1 month, in sera from mice that
were given vaccine placed on a room temperature tray before freeze
drying. In contrast, vaccines made by precooling prior to freeze
drying had better total and neutralizing anti-RTA titers than those
immunized with liquid vaccine.
TABLE-US-00007 TABLE 7 Number of mice responding to the vaccine
after week 3 and week 5 # of with Antibody Titer Vaccine Week 3
Week 5 Positive Control - 9/10 10/10 Liquid His + rRTA Time 0 - RT
His + rRTA 10/10 10/10 Time 0 - RT AA + rRTA 10/10 10/10 Time 0 -
PC His + rRTA 9/10 10/10 Time 0 - PC AA + rRTA 10/10 10/10 Time 1
Week - RT His + rRTA 10/10 10/10 Time 1 Week - RT AA + rRTA 9/9
10/10 Time 1 Week - PC His + rRTA 10/10 10/10 Time 1 Week - PC AA +
rRTA 9/10 10/10 Time 1 Month - RT His + rRTA 9/10 10/10 Time 1
Month - RT AA + rRTA 10/10 10/10 Time 1 Month - PC His + rRTA 10/10
10/10 Time 1 Month - PC AA + rRTA 10/10 10/10
IX. Vaccination of Animals with Vaccines Containing Secondary
Co-Adjuvants.
[0076] A series of freeze dried formulation was made according to
the general lyophilization schemes presented in Table 1. Freeze
dried formulations with RTA protein and placebo formulations
without protein were created containing 1.0 mg Al/mL, 8 w/v %
trehalose and 0.2 or 0 mg/mL rRTA and 60 micrograms of TLR-4
agonist, a synthetic derivative of monophosphoryl Lipid A (MPL)
termed PHAD, obtained from Avanti Polar Lipids (Alabaster, AL).
Vaccines were made in several different manners. In method (1), RTA
protein was adsorbed (bound) to aluminum hydroxide in 10 mM
histidine or ammonium acetate buffer pH 6 in the presence of 8%
trehalose, followed by addition of PHAD agonist to the aqueous
suspension. For this method, RTA stored in stabilizer buffer
consisting of 10 mM histidine, pH6.0, and 144 mM NaCl, was
subjected to dialysis into glycerol- and salt-free buffer prior to
adsorption to aluminum adjuvant. In method (2), RTA stored in
stabilizing glycerol buffer was diluted 10 fold in 10 mM histidine,
pH 6.0, 144 mM NaCl prior to the addition of aluminum to the
diluted stabilizing buffer. For this method adsorption was allowed
to occur at 4C.degree. for more than 5 hours so that greater than
95% of the RTA became bound to aluminum gel particles.
Subsequently, the aluminum particles were allowed to settle to the
bottom of the adsorption vessel or the mixture was subjected to
centrifugation to separate the particles from the aqueous buffer.
To this Aluminum mixture was added a buffer system (ammonium
acetate or histidine) containing 8% trehalose. In this manner the
isotonicity of the system could be maintained. For method 1 and
method 2, subsequent lyophilization proceeded with either
pre-cooling (PC) prior to lyophilization or room temperature
incubation prior to lyophilization.
[0077] Samples from each process condition were incubated at
4.degree. C. and 40.degree. C. and withdrawn for analysis and
vaccination studies at 1 week, one month, 2 months, 3 months, 6
months, 9 months, 12 months, 18 months, and 24 months. For potency
analysis, Swiss Webster mice were vaccinated with a concentration
range that kept the ajduvant components (aluminum and PHAD)
constant while varying the dose of RTA immunogen. For control
studies, mice were vaccinated with vaccine that did not contain
co-adjuvant PHAD, using the same dose range as the PHAD containing
lyophilized vaccines. Two vaccination protocols were used.
[0078] One set of mice were vaccinated with one dose of vaccine on
study day 1, and another set of mice was vaccinated with vaccine on
study days 1 and 21. Serum was obtained from animals at the time of
each vaccination and two weeks thereafter. For final analysis, mice
were exposed to 10.times.LD 50 of ricin toxin on day 35 and
survivors were recorded. The animals that were vaccinated with the
PHAD-containing dried reconstituted vaccine samples demonstrated a
significant shift of the dose response curve toward lower doses of
RTA immunogen for the serological endpoints (total RTA reactive
antibodies and ricin neutralizing antibodies) and also demonstrated
protective immunity at the lower dose range when subjected to ricin
exposure in comparison to the vaccine without the co-adjuvant.
Equally significant, the vaccine samples that were incubated at the
higher temperature also demonstrated enhanced immune response,
indicating that all of the components of the vaccine were
stabilized. Furthermore, the PHAD vaccines induced a broader immune
response reflected by a higher titer of neutralizing antibodies and
broader response to neutralizing epitopes.
X. Glass Transition Temperature.
[0079] Glass transition temperature (Tg) is an indicator of
stability of the vaccine product. Below or near the Tg the vaccine
behaves as a glass and all components of the vaccines are
stabilized within the glass. Above the Tg, the sample becomes is
less stable, and the components within the matrix also become less
stable. The Tg is measured by differential scanning calorimetry in
the following manner. The Tg of the sample is determined by
subjecting the sample to a controlled temperature program from
0.degree. C. to 150.degree. C. at a rate of 10.degree. C./min. The
heat flow to and from the sample is measured and expressed as a
shift in the baseline. The Tg is expressed as the temperature at
the midpoint of this baseline shift.
[0080] Lyophilized RTA vaccines subjected to DSC analysis
demonstrate a high glass transition temperature in excess of
100.degree. C. and lower than 0.5% water content (Karl Fischer
analysis).
[0081] As used in this specification and in the appended claims,
the singular forms include the plural forms. For example the terms
"a," "an," and "the" include plural references unless the content
clearly dictates otherwise. Additionally, the term "at least"
preceding a series of elements is to be understood as referring to
every element in the series. The inventions illustratively
described herein can suitably be practiced in the absence of any
element or elements, limitation or limitations, not specifically
disclosed herein. Thus, for example, the terms "comprising,"
"including," "containing," etc. shall be read expansively and
without limitation. Additionally, the terms and expressions
employed herein have been used as terms of description and not of
limitation, and there is no intention in the use of such terms and
expressions of excluding any equivalents of the future shown and
described or any portion thereof, and it is recognized that various
modifications are possible within the scope of the invention
claimed. Thus, it should be understood that although the present
invention has been specifically disclosed by preferred embodiments
and optional features, modification and variation of the inventions
herein disclosed can be resorted by those skilled in the art, and
that such modifications and variations are considered to be within
the scope of the inventions disclosed herein. The inventions have
been described broadly and generically herein. Each of the narrower
species and subgeneric groupings falling within the scope of the
generic disclosure also form part of these inventions. This
includes the generic description of each invention with a proviso
or negative limitation removing any subject matter from the genus,
regardless of whether or not the excised materials specifically
resided therein. In addition, where features or aspects of an
invention are described in terms of the Markush group, those
schooled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group. It is also to be understood that the
above description is intended to be illustrative and not
restrictive. Many embodiments will be apparent to those of in the
art upon reviewing the above description. The scope of the
invention should therefore, be determined not with reference to the
above description, but should instead be determined with reference
to the appended claims, along with the full scope of equivalents to
which such claims are entitled. Those skilled in the art will
recognize, or will be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the invention described. Such equivalents are intended to be
encompassed by the following claims.
* * * * *