Us 9603799

US0096.
03799B2
(12) United States Patent (10) Patent No.: US 9,603,799 B2

Sorayya et al. (45) Date of Patent: Mar. 28, 2017
(54) LIPOSOMAL VACCINE ADJUVANTS AND FOREIGN PATENT DOCUMENTS
METHODS OF MAKING AND USING SAME
WO WO9515762 A1 * 6, 1995
(71) Applicant: HTD Biosystems Inc., Pleasanton, CA
(US)
OTHER PUBLICATIONS
(72) Inventors: Aryo Sorayya, Danville, CA (US);
Mitra Mosharraf, Danville, CA (US); Watson DS, Endsley AN, Huang L. Design considerations for
Rajiv Nayar, Danville, CA (US) liposomal vaccines: influence of formulation parameters on anti
body and cell-mediated immune responses to liposome associated
(*) Notice: Subject to any disclaimer, the term of this antigens. Vaccine. Mar. 16, 2012:30(13):2256-72. doi: 10.1016/j.
patent is extended or adjusted under 35 vaccine.2012.01.070. Epub Feb. 2, 2012. Review. Erratum in:
U.S.C. 154(b) by 0 days. Vaccine. Aug. 24, 2012:30(39):5799.*
Lutwyche P. Cordeiro C. Wiseman DJ, St-Louis M. Uh M. Hope
(21) Appl. No.: 14/216,605 MJ. Webb MS. Finlay BB. Intracellular delivery and antibacterial
activity of gentamicin encapsulated in pH-sensitive liposomes.
(22) Filed: Mar. 17, 2014 Antimicrob Agents Chemother. Oct. 1998:42(10):2511-20.*
Ewert KK. Ahmad A. Bouxsein NF, Evans HM, Safinya CR.
(65) Prior Publication Data Non-viral gene delivery with cationic liposome-DNA complexes.
Methods Mol Biol. 2008:433:159-75.*
US 2014/0341974 A1 Nov. 20, 2014 Sorayya, A. “Designing a Novel Freeze-Stable Tetanus Vaccine.”
Intel International Science and Engineering Fair (Intel ISEF), 2013.
Fraga et al. Influence of phospholipid composition on cationic
Related U.S. Application Data emulsions/DNA complexes: physiochemical properties, cytotoxic
ity, and transfection on Hep G2 cells. Int J. Nanomedicine (2011)
(63) Continuation-in-part of application No. 13/837,637, 6:2213-20. Epub Oct. 7, 2011.
filed on Mar. 15, 2013, now abandoned. Chen et al. “An overview of liposome lyophilization and its future
potential.” J. Control Release (2010) 142:299-311. Epub Oct. 27.
2009.
(51) Int. Cl. O'Hagan, DT. Recent advances in immunological adjuvants: the
A6K 9/27 (2006.01) development of particulate antigen delivery systems. Expert Opin
A6 IK 39/08 (2006.01) Investig Drugs. (1998) 7(3):349-59.
A6 IK 9/00 (2006.01) Sorayya, A. (2012) Contra Costa County Science & Engineering
A6 IK 39/00 (2006.01) Fair. “Development of a lyophilizable vaccine for delivering a
A6 IK 47/24 (2006.01) model protein antigen.” 1st Place. (Also presented at CA State
A 6LX 39/39 (2006.01) Science Fair where same presentation won "Project of the Year” in
(52) U.S. Cl. 2012).
CPC ............ A61K 9/127 (2013.01); A61K 9/0019 Sorayya A. “Overcoming the Cold Chain: Designing a Novel
(2013.01); A61K 39/0005 (2013.01); A61 K Freeze-Stable Vaccine.” CA State Fair Project 2012 Project Sum
mary. Apr. 30, 2012.
39/08 (2013.01); A61K 39/39 (2013.01); A61 K Sorayya et al. Designing a novel freeze-stable vaccine as an
47/24 (2013.01); A61 K 2039/545 (2013.01); alternative to aluminum-based vaccines. Abstract. 2012 AAPA
A61 K 2039/55555 (2013.01) Annual Meeting and Exposition Oct. 14-17, 2012.
(58) Field of Classification Search Nayar and Mosharraf. (2010) “Effective approaches to formulation
CPC ... A61K 2300/00; A61K 39/39; A61K 9/0019; development and biopharmaceuticals, in Formulation and Process
A61K 9/127; A61K 2039/55511; A61 K Development Strategies for Manufacturing Biopharmaceuticals'
2039/55555; A61K 39/12: A61K 9/1075; (eds F. Jameel and S. Hershenson), John Wiley & Sons, Inc.
A61 K9/19: A61K 9/51; A61K 31/00 Hoboken, NJ USA.
See application file for complete search history. Altin and Parish. (2006) "Liposomal vaccines—targeting the deliv
ery of antigen.” Methods 40:39-52.
(56) References Cited (Continued)
U.S. PATENT DOCUMENTS
Primary Examiner—Rachel B Gill
5,290,563 A 3, 1994 Millet-Genin et al. (74) Attorney, Agent, or Firm — Lewis Kohn & Walker
6,045,828 A * 4/2000 Bystrom .............. A61K 9.0075
424/404 LLP; David M. Kohn: Kari Moyer-Henry
2005/O142114 A1 6, 2005 Gieseler et al.
2008.0089927 A1* 4/2008 Malinin ........................ 424/450
2008. O145413 A1 6, 2008 Panzner et al. (57) ABSTRACT
2008/0268028 A1 10/2008 Zurbriggen et al.
2010. 0233251 A1 9/2010 Andrian et al. A vaccine adjuvant composition comprising: a lipid selected
2011 0002983 A1 1/2011 Hipler et al. from the group consisting of dipalmitoyl phosphatidlcho
2011/0092739 A1 4/2011 Chen et al. line (DPPC), dipalmitoyl phosphatidylglycerol (DPPG),
2012, OO15865 A1 1/2012 Zelphati et al. dioleoyl phosphatidylcholine (DOPC), and cholesterol and
2013/0177629 A1 7, 2013 Martin et al.
2013/0287.857 A1 10/2013 von Andrian et al. containing a positively or negatively charged lipid with
2013,0323298 A1 12/2013 Goodwin et al. associated/entrapped protein antigen.
2014, OO17279 A1 1/2014 Brito et al.
2014/OO79774 A1 3/2014 Brinker et al. 7 Claims, 28 Drawing Sheets
US 9,603,799 B2
Page 2
(56) References Cited
OTHER PUBLICATIONS
Gregoriadis et al. (1999) “Vaccine Entrapment in Liposomes.”
Methods 19:156-162.
Milicic et al. (2012) Small Cationic DDA:TBD Liposomes as
Protein Vaccine Ajvants Obviate the Need for TLR Agonists in
Inducing Cellular and Humoral Responses. PLoS One 7(3): e34255.
doi:10.1371journal.pone.0034255.
Mishra et al. (2007) "Liposomes as adjuvant for combination
vaccines.” Indian Journal of Experimental Biology 45:237-241.
* cited by examiner
U.S. Patent US 9,603,799 B2
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US 9,603,799 B2
1. 2
LPOSOMAL VACCNE ADJUVANTS AND Currently, aluminum-based adjuvants, such as aluminum
METHODS OF MAKING AND USING SAME phosphate and aluminum hydroxide, dominate their field
and, prior to 2009, were the only licensed adjuvants in the
CROSS REFERENCE TO RELATED U.S. Unfortunately, while the use of adjuvants such as alum
APPLICATIONS and MF59 (oil-in-water, proprietary adjuvant owned by
Novartis) can augment certain response to specific Ags. Such
This application is a continuation-in-part of U.S. patent agents can sometimes lead to induction of undesired, inap
application Ser. No. 13/837,637, filed Mar. 15, 2013, the propriate responses (ie. generation of a humoral response
contents of which are incorporated by reference herein. 10
rather than a cell mediated response) (Roberts et al., “Phase
2 study of the g209-2M melanoma peptide vaccine and
FIELD OF THE INVENTION low-dose interleukin-2 in advanced melanoma, J. Immu
nother, 29(1):95-101 (2006)).
This application relates generally to the field of vaccine Additionally, these inorganic adjuvants face numerous
compositions and methods of making or using same. Spe 15 other problems as they are frost sensitive and not readily
cifically, this application relates to liposomal vaccine com lyophilizable. The limitations placed on vaccines by adju
positions for use in promoting a specific immune response, vants that are not freeze-compatible severely restrict the use
and methods of processing Such compositions in a manner of Such vaccines and make them unavailable in many areas
which promotes degrees of protection of the composition in the world. In fact, liquid formulations of Al-based vac
from external environmental factors. cines against diphtheria, pertussis, tetanus, hepatitis B and
BACKGROUND OF THE INVENTION
influenza (type B) should not be frozen (Kartoglu et al.,
“Validation of the shake test for detecting freeze damage to
adsorbed vaccines’, Bull World Health Organ., 88:624-631
The majority of vaccines currently in development belong (2010)). Unfortunately, practices that expose a wide variety
to a specific class of subunit vaccine compositions, which 25 of vaccines to Sub-Zero temperatures are widespread, in both
consist of recombinant or purified pathogen-specific pro developed and developing countries, across all levels of the
teins or encoded antigens (from DNA) that will be expressed respective health systems (Matthias et al., “Freezing tem
and presented in vivo in order to accomplish or elicit the peratures in the vaccine cold chain: a systematic literature
desired immunogenic response. This type of vaccine pres review'', Vaccine, 25:691-697 (2007)).
ents an antigen to the immune system without introducing 30 When a vaccine is damaged by freezing, the potency lost
viral particles, whole or otherwise. While evidence suggests can never be restored, resulting in permanent damage to the
that live, attenuated pathogens and viral vectors can induce underlying composition itself. As a result, freeze-damaged
protective effects, they often cause unwanted side effects or vaccines have lower immunogenicity and are more likely to
raise safety concerns, which is one reason why subunit cause local reactions. Such as sterile abscesses (Dimayuga et
vaccines have risen to prominence in the field (Arvin et al., 35 al., “Effects of freezing on DTP and DTP-IPV vaccines,
“New viral vaccines”, Virology, 344:240-249 (2006): Yang adsorbed, Can. Commun. Dis. Rep., 21:101-103 (1995);
et al., “A novel peptide isolated from phage library to Mansoor et al., “Vaccine adverse events reported in New
Substitute a complex system for a vaccine against Staphylo Zealand, NZ Med. J., 110:270-272 (1997)). Accordingly,
cocci infection, Vaccine, 24:1117-1123 (2006)) much has been devoted to overcoming the inherent problems
One weakness of this technique is that isolated proteins 40 within the cold chain relative to vaccine compositions.
can be denatured and thus will be associated with antibodies The cold-chain, a Supply chain for pharmaceutical drugs
that are distinct from the desired antibodies. Another method based on temperature control, is a laborious process that
of making Subunit vaccine involves extracting an antigen's attempts to keep vaccines at the Suggested 2-8°C. range, and
gene from the targeted virus (or bacterium) and inserting this thus costs companies and organizations (ie. UNICEF) mil
gene into another virus or attenuated bacterium to make a 45 lions of dollars every year. Freeze-sensitive vaccines repre
recombinant virus or bacteria. sent over 30% of the S439 million UNICEF spent on all
Researchers have also inserted an antigen's gene derived vaccines in 2005 and the S757 million spent in 2010.
from a targeted virus into yeast. Examples of Such resultant Carrying-containers using ice (prominent in developing
vaccines are well known in the art, including Subunit viral countries), defective refrigerators, and extreme cold cli
vaccines derived from hepatitis B surface antigen (HBSAG) 50 mates can impel these vaccines to freeze and render them
produced in yeast cells (Recombivax HB from Merck). ineffective. Rate of exposure to freezing temperatures in
Such subunit vaccines, when administered alone, have developed and developing countries is 13.5% and 21.9%,
relatively low efficacy for immune system activation, gen respectively—making this a global concern. Freezing is a
erally exhibiting poor immunogenicity (Toes et al., “Peptide risk at any level of the cold chain, and serves as a major
vaccination can lead to enhanced tumor growth through 55 problem for many salient vaccines.
specific T-cell tolerance induction, Proc. Natl. Acad. Sci., In the face of the above mentioned limitations with
93:7855-7860 (1996)) and thus require the addition of respect to aluminum-based adjuvants in vaccine composi
adjuvants in order to elicit the appropriate level of immune tions, it has been shown that liposomes may be a viable
system response to a particular antigen, initially through the alternative as an adjuvant providing similar immunogenic
innate, then Subsequently the adaptive immune system 60 ity, without the problems associated with freeze sensitivity
(Grasso, Pet al., Essentials of Pathology for Toxicologists, and lyophilization. Furthermore, these freeze sensitive adju
CRC Press (2002)). vants found in the prior art have also failed to elicit adequate
Preferably, the adjuvant should be able to improve or immune responses in many cases and, often times, do not
facilitate antigen uptake by antigen presenting cells (APCs) bind effectively to all protein antigens. This has spurred
and, ideally, then induce an Ag-specific immune response 65 interest in other forms of adjuvants which may be more
while simultaneously eliciting minimal toxicity to the indi versatile and without the encumbrances identified in Al
vidual. based adjuvants.
US 9,603,799 B2
3 4
In the approximately 1,400 publications about liposomal phobic antigens) or adsorbed into the liposomal Surface
vaccines since 1974, over 25% have been published in the through covalent or charge-dependent, electrostatic interac
past three years, propelling the creation of multiple vaccines tion (Taneichi et al., “Induction of differential T-cell epitope
using liposomal adjuvants against influenza (InflexalRV) by plain- and liposome-coupled antigen'. Bioconjug. Chem.,
and hepatitis (Epaxal(R). Both of these vaccines must be 17:899-904 (2006)). More recently, strong evidence has
stored at 2-8°C. and should not be frozen. Other liposomal indicated great potential for enhancing immunogenicity of
vaccines that are currently moving toward regulatory cationic liposomes through addition of toll-like receptor
approval are based on synthetic, cationic lipids which are (TLR) agonists (Bal et al., “Co-encapsulation of antigen and
insufficiently immunogenic, and are thus often combined toll-like receipt ligand in cationic liposomes affects the
with immunostimulators such as lipid A. Similar studies 10 quality of the immune response in mice after intradermal
have also proven an effective composition in other alterna vaccination, Vaccine, 29:1045-1052 (2011)). Similarly,
tive, bioactive lipid based vaccine compositions using Lipid liposomal encapsulation of CpG oligonucleotides has been
A (Coler et al., “Development and Characterization of shown to enhance and prolong innate system stimulation and
Synthetic Glucopyranosyl Lipid Adjuvant System as a Vac advanced the CpG-induced immune protection against List
cine Adjuvant”. PLoS One, 6:e 16333 (2011)). 15 eria (Gursel et al., “Sterically stabilized cationic liposomes
Liposomes are lipid-bilayer, vesicular structures within improve the uptake and immunostimulatory activity of CpG
which a variety of Substances may be entrapped and deliv oligonucleotides”, J. Immunol., 167:3324-3328 (2001)).
ered in vivo in a safe and effective manner. Liposomes are Methods of manufacturing the antigen loaded liposomes
composed largely of natural or synthetic phospholipids of the prior art consist primarily of embodiments described
which, over the last several decades, have been utilized for in the literature involving reverse phase evaporation and
effective delivery of therapeutic agents ranging from related techniques (Sazoka et al., “Rapid separation of low
enzyme replacement therapy (Jain et al., “Muco-adhesive molecular weight solute from liposomes without dilution',
multivesicular liposomes as an effective carrier for trans Proc. Natl. Acad. Sci., 75:4194 (1978)). With respect to
mucosal insulin delivery”. J. Drug Target, 15:417-427 liposomal compositions consisting of glucopyranosyl lipid
(2007)), to intracellular delivery of chelating agents in cases 25 adjuvant (GLA), the methods used to develop Such adju
of heavy metal poisoning (Rahman et al., “Preparation and vants are formulated as aqueous Suspensions and have been
prolonged tissue retention of liposome-encapsulated chelat used in the past to characterize the usefulness of synthetic
ing agents”. J. Lab. Clin. Med., 83:640-647 (1974)), to even TLR4 agonists (Anderson et al., “Physicochemical charac
possible treatments for certain cancers (Gregoriadis et al., terization and biological activity of synthetic TLR4 agonist
"Drug-carrier potential of liposomes in cancer chemo 30 formulations”. Colloids Surf. B. Biointerfaces, 75:123-132
therapy”, Lancet, 1:1313-1316 (1974)). (2010)). However, many of the known methods of making
More recently, liposomes have been found to be suitable liposomal vaccines in the prior art have encountered several
vaccine adjuvants in having the ability to prevent antigen difficulties, ranging from error in antigen binding to non
degradation while enhancing its uptake by APCs (Gregori specific effects relating to the aqueous Suspension. Most
adis et al., “The immunological adjuvant and vaccine carrier 35 importantly, such vaccine formulations still suffer from the
properties of liposomes”. J. Drug Target, 2:351-356 (1994); general defect found in the Al-based vaccines relating to
Brunel et al., “Cationic lipid DC-Chol induces an improved freeze sensitivity. Thus, there remains a great need in the art
and balanced immunity able to overcome the unresponsive to solve the problem of generating a vaccine composition
ness to the hepatitis B vaccine, Vaccine, 17:2192-2203 capable of eliciting a consistent immunogenic response,
(1999)). 40 with a high degree of selectivity and efficacy, with such
Liposomes have been considered as useful vehicles for activity not being diminished in the presence of Sub-Zero
the containment of particular antigens, though the choice of temperatures.
lipid used in the synthesis of liposomes greatly impacts their
physico-chemical and immunogenic properties. Much SUMMARY OF THE INVENTION
research has been devoted to the use of many diverse lipids 45
with the aim of refining the adjuvanting effect of liposome The present invention provides methods and composi
delivered vaccines (Gluck, R., "Liposomal presentation of tions comprising injectable, liposomal vaccines further com
antigens for human vaccines. Vaccine Design. The Subunit prised of members selected from the group consisting of
and Adjuvant Approach., 347-361 (1995)). Phospholipid natural lipids, dipalmitoyl phosphatidylcholine (DPPC), dio
molecules, in particular, have been examined for their dis 50 leoyl phosphatidylcholine (DOPC) and cholesterol with
tinctive regions of non-polar (comprised of one of more fatty either a negatively charged lipid comprising dipalmitoyl
acid chains or cholesterol) and polar (consisting of a phos phosphatidylglycerol (DPPG) or a positively charged lipid
phate group linked to tertiary or quarternary ammonium comprising Stearylamine (SA).
salts). The polar region can have a net negative (anionic), In one aspect, the present invention provides for a com
neutral or positive (cationic) Surface charge, which directly 55 position comprising at least one of DPPC, DOPC, choles
impacts the specific behavior and function of the specific terol and a charged lipid with an entrapped or adsorbed
liposome (Milicic et al., “Small cationic DDA:TDB lipo protein antigen capable of inducing an antibody response
somes as protein vaccine adjuvants obviate the need for TLR against an antigen in a Subject. An alternative embodiment
agonists in inducing cellular and humoral responses”. PLOS provides for a cationic liposomal vaccine composition com
ONE, 7(3):1-10 (2012)). 60 prising DPPC:DOPC:cholesterol:SA in a molar ratio of
While the potential of antigens entrapped in liposomes for approximately 40:20-30:20:10-20 and wherein the cationic
use as potential vaccines have shown promising results, liposomal vaccine is formulated as a lyophilized composi
there are a great many alternatives to preferred liposomal tion. Preferably, the molar ratio of DPPC:DOPC:cholesterol:
compositions, particularly with respect to entrapment of the SA is 40:25:20:15. Optionally, the cationic liposomal vac
antigens. For instance, antigens to be delivered may either 65 cine is formulated as an aqueous solution.
be entrapped within the aqueous compartment of the lipo Another alternative embodiment in the compositions of
Somes, incorporated into the lipid bilayer membrane (hydro the present invention provides for an anionic liposomal
US 9,603,799 B2
5 6
vaccine composition comprising DPPC:DOPC:cholesterol: In another aspect, the protein antigen can be selected from
DPPG in a molar ratio of approximately 40:20-30:20:10-20 or derived from the group consisting of rotavirus, foot and
and wherein the anionic liposomal vaccine is formulated as mouth disease virus, influenza A virus, influenza B virus,
a lyophilized composition. Preferably, the molar ratio of influenza C virus, H1N1, H2N2, H3N2, H5N1, H7N7,
DPPC:DOPC:cholesterol:DPPG is 40:25:20:15. Optionally, 5 H1N2, H9N2, H7N2, H7N3, H10N7, human parainfluenza
the anionic liposomal vaccine is formulated as an aqueous type 2, herpes simplex virus, Epstein-Barr virus, varicella
Solution. virus, porcine herpesvirus 1, cytomegalovirus, lyssavirus,
In another aspect, the positively charged lipid is at least Bacillus anthracis, anthrax PA and derivatives, poliovirus,
one selected from the group consisting of stearylamine (SA), hepatitis A, hepatitis B, hepatitis C, hepatitis E, distemper
dioleoyl trimethylammoniumpropane (DOTAP), dimethyl 10 virus, Venezuelan equine encephalomyelitis, feline leukemia
dioctadecylammonium (DDAB), ethylphosphocholine virus, reovirus, respiratory syncytial virus, Lassa fever virus,
(Ethyl PC), dipalmitoyl trimethylammoniumpropane polyoma tumor virus, canine parvovirus, papilloma virus,
(DPTAP) and dipalmitoyl trimethylammonium (DPTMA), tick borne encephalitis virus, rinderpest virus, human rhi
as well as variants thereof. In another aspect, the negatively novirus species, Enterovirus species, Mengovirus,
charged lipid is at least one selected from the group con 15 paramyxovirus, avian infectious bronchitis virus, human
sisting of dipalmitoyl phosphate (DPPA), dipalmitoyl phos T-cell leukemia-lymphoma virus 1, human immunodefi
phatidylglycerol (DPPG), dihexadecanoyl phosphoserine ciency virus-1, human immunodeficiency virus-2, lympho
(DPPS), and ditetradecyl phosphoglycerol (Diether PG), as cytic choriomeningitis virus, parvovirus B19, adenovirus,
well as variants thereof. rubella virus, yellow fever virus, dengue virus, bovine
In yet another aspect, the present invention provides for respiratory syncitial virus, corona virus, Bordetella pertus
methods of manufacturing a liposomal vaccine composition sis, Bordetella bronchiseptica, Bordetella parapertussis,
comprising: (a) providing a lipid blend, further comprising Brucella abortis, Brucella melitensis, Brucella Suis, Bru
at least a pair of natural lipids consisting of at least one cella ovis, Brucella species, Escherichia coli, Salmonella
saturated natural lipid and at least one unsaturated natural species, Salmonella typhi, Streptococci, Vibrio cholera,
lipid, and cholesterol: (h) combining the lipid blend together 25 Vibrio parahaemolyticus, Shigella, Pseudomonas, tubercu
with the cholesterol; (c) dissolving (b) in a cosolvent; (d) losis, avium, Bacille Calmette Guerin, Mycobacterium lep
drying (c) in a water bath under nitrogen gas at a temperature rae, Pneumococci, Staphylococci, Enterobacter species,
above the gel to liquid crystalline temperature of the lipids Rochalimaia henselae, Pasteurella haemolytica, Pasteurella
(about 50° C.) to form a homogenous lipid-blend film (e) multocida, Chlamydia trachomatis, Chlamydia psittaci,
placing (d) under vacuum to remove the residual solvent, (f) 30 Lymphogranuloma venereum, Treponema pallidum, Haemo
hydrating (e) with an effective amount of at least one antigen philus species, Mycoplasma bovigenitalium, Mycoplasma
solution; and (g) agitating (f) to form multilamellar vesicles pulmonis, Mycoplasma species, Borrelia burgdorferi,
(MLVs) with entrapped or adsorbed antigen, wherein the Legionalla pneumophila, Colstridium botulinum, Coryne
liposomal vaccine composition is freeze stable. bacterium diphtheriae, Yersinia entercolitica, Rickettsia
Implementations of the above aspects can include one or 35 rickettsii, Rickettsia typhi, Rickettsia prowsaekii, Ehrlichia
more of the following: Liposomes consisting of a vaccine chafeensis, Anaplasma phagocytophilum, Plasmodium fall
composition comprising of DPPC, DOPC, cholesterol and a ciparum, Plasmodium vivax, Plasmodium malariae, Schis
charged lipid, with an entrapped or adsorbed protein antigen tosomes, trypanosomes, Leishmania species, Filarial nema
capable of inducing an antibody response against the antigen todes, trichomoniasis, sarcosporidiasis, Taenia saginata,
in a subject. Preferably, liposomes can be used as an 40 Taenia solium, Leishmania, Toxoplasma gondii, Trichinella
adjuvant. Additionally, the liposomes can be used as a means spiralis, coccidiosis, Eimeria tenella, Cryptococcus neofor
for entrapment of an antigen in a vaccine composition. mans, Candida albican, Apergillus fumigatus, coccid
Liposomes of the present invention do not lose their immu ioidomycosis, Neisseria gonorrhoeae, malaria circumsporo
nogenicity after being exposed to freezing temperatures Zoite protein, malaria merozoite protein, trypanosome
during multiple freeze-thaws. Preferably, the adjuvants of 45 Surface antigen protein, pertussis, alphaviruses, adenovirus,
the present invention are freeze-stable adjuvants. diphtheria toxoid, tetanus toxoid, meningococcal outer
In another aspect, the composition of the present inven membrane protein, Streptococcal M protein, Influenza
tion is preferably in a molar ratio of 40:25:20 of DPPC: hemagglutinin, cancer antigen, tumor antigens, toxins,
DOPC:cholesterol and 15 for a negatively charged lipid. Clostridium perfiringens epsilon toxin, ricin toxin,
Preferably, the negatively charged lipid is DPPG. The com 50 pseudomonas exotoxin, exotoxins, neurotoxins, cytokines,
position is preferably in a molar ratio of 40:25:20 of DPPC: cytokine receptors, monokines, monokine receptors, plant
DOPC:cholesterol and 15 for a positively charged lipid. pollens, animal dander, and dust mites. The particles can be
Preferably, the positively charged lipid is SA. Preferably, the used in formulation of vaccines against diphtheria, tetanus,
composition is capable of being stable in freezing tempera pertussis (whooping cough), influenza, hepatitis B, botu
tures and retaining immunogenic activity after more than 55 linium toxin, anthrax, or combination vaccines Such as,
one freeze-thaw cycles, further wherein the composition is PedvaxHIB (Haemophilus b Conjugate and Meninococcal
finally freeze-dried at a temperature of about -45° C. Protein Conjugate), Comvax(R) (Meningococcal Protein
The mean hydrodynamic particle diameter of the lipo Conjugate and Hepatitis B, recombinant antigens), Tripe
Somes of the present invention is in the size range of dia(R) (Diphtheria and Tetanus Toxoids and Acellular Pertus
300-1000 nm. Preferably, the composition comprising the 60 sis antigens). Infanix(R) (Diphtheria and Tetanus Toxoids
liposomes maintains immunogenicity after lyophilization at and Acellular Pertussis antigens), or any other vaccines that
a freezing temperature range between about -30°C. to about loses its potency upon freezing.
-50° C. in the presence of a lyoprotectant (e.g. Sucrose or In yet another aspect, the present invention provides for
trehalose). The adjuvant liposomes of the present invention using a novel lipid composition as an adjuvant, wherein the
can be used as an alternative to freeze-sensitive aluminum 65 liposomal vaccine is prepared with at least one entrapped
salt adjuvants in all vaccine formulations containing these antigen having an immunogenic response in a Subject. In a
adjuvants. preferred embodiment, the lipid composition maintains its
US 9,603,799 B2
7 8
immunogenic activity after freezing and lyophilization. liposome (reconstituted) after 28 months at room tempera
Optionally, the at least one entrapped antigen may be ture; and (e) freeze-dried liposome (reconstituted) after 13
selected from the group consisting of polynucleotides, poly months at 5° C.).
peptides, recombinant proteins, synthetic peptides, protein FIG. 5 depicts an exemplary average amount of mouse
extract, cells (including tumor cells), tissues, polysaccha 5
anti-tetanus toxoid antibody for different formulations
rides and lipids, including fragments thereof. (Absaso).
In another preferred aspect, the liposomal compositions of
the present invention do not require the presence of a FIG. 6 shows results from a mouse immunogenicity study,
co-adjuvant such that the methods and compositions of the wherein the average titers of mouse anti-tetanus toxoid IgG
present invention do not include the following: Lipid A. 10 from 5 mice are compared against each formulation. These
Lipid A derivatives, monophosphoryl lipid A, monophos are the same data from FIG. 8, but without correcting for
phoryl lipid A derivatives, lipopolysaccharide, muramyl 100x dilution factors and background (naive mouse).
dipeptide, CpG containing oligonucleotides, TLR-4 ago FIG. 7 depicts the particle size distribution of liposomal
nists, flagellin, flagellins derived from gram negative bac 15 vaccines with 0.1 ug/ml TLC by DLS (before and after
teria, TLR-5 agonists, fragments of flagellins capable of freeze-drying, Smoothness 20 applied). These results are
binding to TLR-5 receptors, saponins, analogues of from Study 1. (a) Liquid anionic liposomal vaccine -0.1
saponins, QS-21, purified Saponin fractions, immune stimu ug/ml TLC. (Formulation 2; SP-318-2); (b) Lyophilized
lating complexes (ISCOMS) and saponin combinations with anionic liposomal vaccine -0.1 g/ml TLC. (Formulation 3;
sterols. In a preferred embodiment, the methods and com SP-318-3); (c) Liquid cationic liposomal vaccine -0.1 ug/ml
positions of the present invention provide compositions TLC. (Formulation 4: SP-318-4); (d) Lyophilized cationic
having at least one antigen and lacking a co-adjuvant, liposomal vaccine -0.1 ug/ml TLC. (Formulation 5; SP-318
wherein the compositions retain their immunogenicity after 5).
exposure to freezing temperatures, after multiple freeze FIG. 8 shows the average amount of mouse anti-tetanus
thaws, and after being freeze-dried or lyophilized. 25
antibody for cationic liposomes with 0.1 g/ml TLC. These
Such liposomal vaccine products offer numerous advan results are from Study 1.
tages over Al-based vaccines in regards to safety, freeze FIG. 9 shows the particle size distribution of liposomal
stability, tolerability, biodegradability, and versatility.
Hence, this unique liposomal based adjuvant can be vaccines, before and after freeze-drying, with smoothness 20
employed instead of Aladjuvants in current freeze sensitive 30 applied. These results are from Study 2. (a) Liquid anionic
vaccines against for example diphtheria, tetanus, pertussis liposomal vaccine -0.4 ug/ml TLC. (Formulation 2; SP-329
(whooping cough), influenza and anthrax. The novel vaccine 2); (b) Lyophilized anionic liposomal vaccine -0.4 ug/ml
adjuvant designed thus provides a technological platform for TLC. (Formulation 3: SP-329-3); (c) Liquid cationic lipo
development of immunogenic, freeze-stable vaccines, pre somal vaccine -0.4 g/ml TLC. (Formulation 4: SP-329-4);
venting product damage during accidental freezing in the 35 and (d) Lyophilized cationic liposomal vaccine -0.4 ug/ml
cold chain. TLC. (Formulation 5; SP-329-5).
Although liposomes have been used before as vaccines FIG. 10 shows a standard curve of mouse anti-tetanus
(Powell et al., Vaccine Design, The subunit and Adjuvant antibodies obtained from Study 2.
Approach, Pharm. Biotech, 6:159 (1995), the liposomal
vaccine described herein as a preferred embodiment of the 40 FIG. 11 shows the average amount of mouse anti-tetanus
present invention is composed of natural or synthetic lipids antibody for each formulation (U/ml) in Study 2. The bars
and does not require the addition of immune adjuvants such show the standard deviations.
as Lipid A derivatives. Hence, the rigid liposomal vaccine FIG. 12 shows the average amount of mouse anti-tetanus
described in the present invention is of a novel composition antibody for anionic liposome with TLC at 0.4 ug/ml as
of a specific size range that can act as a replacement for 45
compared to TLC without adjuvant (Study 2). The bars show
Al-based vaccines. These liposomes, because of their lipid the standard deviations.
composition and size, may also have targeting properties to
specific antigen presenting cells or other immunomodula FIG.13a shows the dose dependence and overall levels of
tory cell types and may induce both humoral and cell anti-tetanus antibodies raised for the different formulations
mediated immune response. 50 of TLC (TLC alone, anionic liposomes with TLC and
cationic liposomes with TLC). FIG. 13b indicates the dose
BRIEF DESCRIPTION OF THE DRAWINGS dependence of immune response for anionic liposomes as
compared to TLC only. The anionic liposome formulations
FIG. 1 shows an exemplary immune response (450 nm at higher lipid content were prepared at pH 4, whereas those
absorbance) of the various formulations at different dilutions 55 at lower lipid content were prepared at pH 7.
of the sera 80,000, 160,000, and 320,000-fold. The amount FIG. 14a shows a comparison of the immunogenicity in
of absorbance at 450 nm reflects the amount of antibody mice of different formulations as a function of TLC con
present in the sera.
FIG. 2 shows an exemplary standard curve of mouse centration. FIG. 14b indicates a similar comparison across
anti-lysozyme antibody. 60 the immunogenicity levels in mice of different formulations
FIG. 3 shows an exemplary average amount of mouse as a function of TLC concentration on a logarithmic scale.
anti-lysozyme antibody for different formulations (mg/ml). FIG. 15a shows comparison between anionic lipid com
FIG. 4 shows the particle size distribution of liposomal positions after 2 shots and 28 days after the 1 shot, except
vaccines across a variety of composition types, processing results for freeze-dried anionic liposomes (yellow symbols)
variations, time points and temperature variations. (a) Liquid 65 which are based on 1 shot and 14 days after the shot. The
liposome at t0; (b) liquid liposome at 13 months; (c) dashed symbols are expected values after 28 days read from
freeze-dried liposome (reconstituted) at t0; (d) freeze-dried the linear regression lines from the actual values after 28
US 9,603,799 B2
10
days and 2 shots. FIG. 15b presents the additional cationic which is classified as a lipid. It is insoluble in water but
lipid composition data points. soluble in a number of organic solvents.
As used herein, the term “immune response' of a subject
DETAILED DESCRIPTION OF THE shall mean the development of a humoral immune response,
INVENTION a cellular immune response, or a humoral and a cellular
immune response to an antigen.
Definitions As used herein, the term “immunogenic' shall mean
capable of evoking an immune or antigenic response in a
As used herein, the term “amino acid refers to naturally 10
Subject.
occurring and synthetic amino acids, as well as amino acid As used herein, the term “immunologically protective
analogs and amino acid mimetics that function in a manner amount’ or “immunologically effective amount” refers to
similar to the naturally occurring amino acids. Naturally the quantity or amount Sufficient to induce an immunogenic
occurring amino acids are those encoded by the genetic response in a Subject. The immunogenic response may be
code, as well as those amino acids that are later modified, 15 Sufficient for diagnostic purposes or other testing, or may be
e.g., hydroxyproline, gamma-carboxyglutamate, and adequate to prevent signs or symptoms of disease, including
O-phosphoserine. Amino acid analogs refers to compounds adverse health effects or complications thereof, caused by
infection with a disease agent. Either humoral immunity or
that have the same basic chemical structure as a naturally cell-mediated immunity or both may be induced. The immu
occurring amino acid, i.e., an a carbon that is bound to a nogenic response of an animal to an immunogenic compo
hydrogen, a carboxyl group, an amino group, and an R sition may be evaluated, e.g., indirectly through measure
group, e.g., homoserine, norleucine, methionine Sulfoxide, ment of antibody titers, lymphocyte proliferation assays, or
methionine methyl sulfonium. Such analogs have modified directly through monitoring signs and symptoms after chal
R groups (e.g., norleucine) or modified peptide backbones, lenge with wild type strain, whereas the protective immunity
but retain the same basic chemical structure as a naturally 25 conferred by a vaccine can be evaluated by measuring, e.g.,
occurring amino acid. Amino acid mimetics refers to chemi reduction in clinical signs such as mortality, morbidity,
cal compounds that have a structure that is different from the temperature number, overall physical condition, and overall
general chemical structure of an amino acid, but that func health and performance of the Subject. The immune response
tions in a manner similar to a naturally occurring amino acid. 30 may comprise, without limitation, induction of cellular
As used herein, the term “adjuvant” refers to a pharma and/or humoral immunity.
cological or immunological agent that, when added to vac As used herein, the term “lipids’ refers to any of a group
cines, have the ability to stimulate a subjects immune of organic compounds, including the fats, oils, waxes, Ste
system's response to a target antigen, but do not, individu rols, and triglycerides, that are insoluble in water but soluble
ally, confer immunity. Adjuvants may act in a variety of
35 in nonpolar organic solvents, are oily to the touch, and
together with carbohydrates and proteins constitute the
ways in their presentation of an antigen to the immune principal structural material of living cells.
system, including but not limited to, acting as a depot or a As used herein, the term “liposome' shall mean a micro
housing for the antigen (Such as liposomes), wherein the scopic spherical particle formed by a lipid bilayer enclosing
40
antigen is presented over an extended period of time, there an aqueous compartment and capable of entrapping or
fore maximizing the immune response prior to the body’s housing a drug, antigen, vaccine, enzyme or another Sub
clearance of Such antigen. stance capable of being targeted to cells in the body.
As used herein, the term “antigen” refers to any Substance As used herein, the term “lyoprotectant” refers to stabi
which provokes an adaptive immune response including, but 45 lizers used to prevent denaturation of proteins during freeze
not limited to, killed, inactivated, attenuated, or modified drying and Subsequent storage. In order to be effective,
live bacteria, viruses, or parasites. The term may also lyoprotectants must be retained amorphous. Lyoprotectants
include polynucleotides, polypeptides, recombinant pro of the present invention include, but are not limited to,
teins, synthetic peptides, protein extract, cells (including 50
trehalose. Sucrose and mannitol.
tumor cells), tissues, polysaccharides, or lipids, or fragments As used herein, the term “subject” refers to any animal,
thereof, individually or in any combination thereof. The including humans, for which the administration of an adju
term may also include antibodies, such as anti-idiotype vant composition is desired. It includes mammals and non
antibodies or fragments thereof, and to synthetic peptide mammals, including primates, livestock, companion ani
mimotopes that can mimic an antigen or antigenic determi 55 mals, laboratory test animals, captive wild animals, ayes
nant. (including in ova), reptiles, and fish.
As used herein, the term “antibody” refers to an immu As used herein, the term “vaccine' shall mean any com
noglobulin molecule that can bind to a specific antigen as the position that includes an antigen, the administration of
result of an immune response to that antigen. Immunoglobu which resulting in an immune response in a Subject having
60
lins are serum proteins composed of “light' and “heavy' received such administration.
polypeptide chains having “constant” and “variable' regions The present invention describes methods and composi
and are divided into classes (e.g., IgA, Ig), IgE, IgG, and tions related to freeze stable vaccines in order to overcome
IgM) based on the composition of the constant regions. the inherent problems identified in the vaccines of the state
As used herein, the term “cholesterol refers to a white 65 of the art. Presently, for instance, there are numerous vac
crystalline substance with a chemical formula of cines on the market against tetanus toxoid, a sampling of
C. sub.27H. Sub.45OH. It is a cyclic hydrocarbon alcohol, which is noted at Table 1.
US 9,603,799 B2
11 12
TABLE 1.
List of Tetanus Vaccines currently in the market
Vaccine Description Manufacturer Storage Condition
PEDIAEDX Suspension for Intramuscular Injection GSK 2-8° C. Do NOT
(Diphtheria and Tetanus Toxoids and FREEZE, discard if
Acellular Pertussis Adsorbed, Hepatitis B OZel
(Recombinant) and Inactivated Poliovirus
Vaccine)
Adacel (Tetanus Toxoid, Reduced Single-dose vials and prefilled Syringes Sanofi Store between 2-8 C.
Diphtheria Toxoid and Acellular Pertussis containing a 0.5-mL suspension for im (35°-46°F). DO NOT
Vaccine Adsorbed) injection. FREEZE. Discard
product if exposed to
reezing.
BOOSTRIX Single-dose vials and prefilled Syringes GSK 2-8° C. Do NOT
(Tetanus Toxoid, Reduced Diphtheria containing a 0.5-mL suspension for im FREEZE, discard if
Toxoid and Acellular Pertussis Vaccine, injection OZel
Adsorbed)
DAPTACEL (Diphtheria and Tetanus Suspension for im injection, Supplied in single Sanofi 2-8° C. Do NOT
Toxoids and Acellular Pertussis Vaccine dose (0.5 mL) vials FREEZE, discard if
Adsorbed) OZel
Pentacel (Diphtheria and Tetanus Toxoids Pentacel consists of a liquid vaccine Sanofi Store the DTaP-IPV
and Acellular Pertussis Adsorbed, component (DTaP-IPV component) and a vial and the Hib vial in
nactivated Poliovirus and Haemophilus b lyophilized vaccine component (ActHIB he original box in the
Conjugate (Tetanus Toxoid Conjugate) vaccine). Reconstitute the refrigerator at 35°-46 F.
Vaccine Suspension for Intramuscular ActHIB vaccine component with the DTaP- (2-8° C.). Protect from
njection IPV component immediately before ight. Do not freeze
administration.
NFANRIX (Diphtheria and Tetanus Single-dose vials and prefilled Syringes GSK Store refrigerated
ds and Acellular Pertussis containing a 0.5-mL suspension for injection. between 2° and 8° C.
ne. Adsorbed) Suspension for Intramuscular Injection (36° and 46 F.). Do not
reeze. Discard if the
vaccine has been
OZel.
KINRIX (Diphtheria and Tetanus A single intramuscular injection (0.5 mL). GSK Store refrigerated
Toxoids and Acellular Pertussis Adsorbed Single-dose vials and prefilled Syringes between 2° and 8° C.
and Inactivated Poliovirus Vaccine) containing a 0.5-mL suspension for injection. (36° and 46°F). Do not
Suspension for Intramuscular Injection reeze. Discard if the
vaccine has been
OZel.
ActHIB (R), Haemophilus b Conjugate ActEHIB WACCINE RECONSTITUTED Sanofi Store lyophilized
Vaccine (Tetanus Toxoid Conjugate), WITH O.4% SODIUM CHLORIDE vaccine packaged with
produced by Sanofi Pasteur SA, is a sterile, DILUENT Saline diluent,
lyophilized powder which is reconstituted at Vial, 1 Dose, lyophilized vaccine (5 x 1 Dose Diphtheria and Tetanus
the time of use with either saline diluent vials per package), packaged with 0.6 mL vial Toxoids and Pertussis
(0.4% Sodium Chloride) containing diluent (5 x 0.6 mL vials per or Tripedia vaccine at
package) (contains NO preservative). 2° to 8° C. (35° to 46°F).
DO NOT FREEZE.
TENIVAC (Tetanus and Diphtheria Suspension for injection Supplied in 0.5 mL. Sanofi Should be stored at 2
Toxoids Adsorbed) single-dose vials or syringes o 8° C. (35° to 46°F).
Suspension for Intramuscular Injection DO NOT FREEZE.
Tod (generic) Asterile vaccine for intramuscular injection. MassBiologics Store at 2 C.-8° C.
Tetanus and Diphtheria Toxoids Adsorbed Includes a traceamount of thimerosal mercury (36° F-46°F). DO NOT
(Td) derivative, (so.3 mcg mercury dose) (not as FREEZE. Discard
a preservative) from the manufacturing product if exposed to
process. The tetanus and diphtheria toxoids reezing
induce at least 2 units and 1 unit of antitoxin
per ml of serum, respectively, in the guinea
pig potency test.
As evidenced by Table 1, all of the tetanus vaccines Example 2. In these embodiments the molar ratio of DPPC:
currently out in the market employ an adjuvant system that DOPC:cholesterol was 40:25:20 and that of the charged
requires a storage environment at Some temperature range lipid was 15. Chicken egg lysozyme and tetanus light chain
above Zero (ie. 2°C.-8°C.) but no more than 10°C. for any (TLC) were used as model protein antigens, respectively.
period of time. More importantly, for the vast majority of The charged liposomal adjuvants with the associated protein
these vaccines, instruction is provided that, should the antigen were immunogenic and did not lose their immuno
vaccines be exposed to freezing (ie. Sub-Zero) temperatures, genic activity after multiple freeze-thaw cycles and also
the product should be discarded. 60 additional freezing to -40° C. during lyophilization. Thus,
The immunogenicity of a new liposomal adjuvant con they can be used in vaccine formulations as an alternative to
sisting of a lipid blend composition comprising the follow Aluminum salt adjuvants.
ing lipids: Dipalmitoyl phosphatidylcholine (DPPC), Dio The vaccine adjuvant designed provides a technological
leoyl phosphatidylcholine (DOPC), Cholesterol was tested platform for development of immunogenic, freeze-stable
with either a negative charge lipid: Dipalmitoyl phosphati 65 vaccines, preventing product damage during accidental
dylglycerol (DPPG) in Example 1 or with a positively freezing in the cold chain. The liposomal vaccine adjuvant
charged lipid: Octadecylamine (Stearylamine, SA) in can be used to develop vaccines that are stable against
US 9,603,799 B2
13 14
freezing. The novel freeze-dried liposomal vaccine demon Preparation of Liquid and Lyophilized Liposomal Vaccines
strated efficacy similar to that of a liquid aluminum-phos (Formulations 1 & 2)
phate based vaccine as measured by the antibody response Specific amounts of DPPC, DOPC, cholesterol and DPPG
to an antigen, preferably lysozyme, in mice. The results in a molar ratio of 40:25:20:15 were dissolved in a co
showed that the liposomal vaccine was freeze-stable and did
not lose its immunogenic activity upon freezing/freeze Solvent containing chloroform, methanol, and water. The
drying. lipid blend was dried in a 45° C. water bath under a stream
Such a liposomal vaccine product offers numerous advan of nitrogen gas, and any remaining residual solvent was
tages over aluminum-based vaccines in regards to safety, removed under vacuum overnight to ensure complete des
freeze-stability, tolerability, biodegradability, and versatility. 10 iccation.
It is recommended to use this adjuvant instead of aluminum The dried lipid-blend (50mg) was hydrated in 1 ml of an
based adjuvants in current freeze sensitive vaccines against
for example diphtheria, tetanus, pertussis (whooping cough), 8.9 mg/ml lysozyme stock solution and vortexed to form
influenza and anthrax. The novel vaccine adjuvant designed MLVs. In order to improve the entrapping efficacy, the
thus provides a technological platform for development of 15
liposome solution was freeze-thawed five times using an
immunogenic, freeze-stable vaccines, preventing product acetone-ice bath and a warm-water bath. The liposome
damage during accidental freezing in the cold chain. mixture was centrifuged at 14,000 rpm for 20 min to
EXAMPLES separate the liposomes from the unbound lysozyme solution.
The unbound lysozyme in Supernatant was removed and the
I. Vaccine Preparation Using Lysozyme as the amount of unbound lysozyme in the Supernatant was deter
Antigen mined by UV spectroscopy (n=3). The average unbound
concentration was subtracted from the initial lysozyme
Vaccine Preparation concentration to acquire the concentration of bound/en
The immunogenicity of a lysozyme-liposomal vaccine 25 trapped lysozyme.
(Lipo-LyZ) was investigated before and after freeze-drying
(Formulations 1 and 2, respectively). Lysozyme adsorbed to The liposome pellet containing entrapped lysozyme was
Adju-PhoSR (Adju-LyZ) was used as an aluminum salt resuspended in 1 ml of a sterile filtered 10% w/w sucrose
based vaccine reference. Also a solution of lysozyme with solution and vortexed. To reduce the size of liposomes and
out adjuvant was used as a reference. The four different 30
to obtain a more homogenous particle distribution, the
formulations are summarized in Table 2. All formulations liposome solution was extruded five times through two 800
were prepared in a laminar flow hood using depyrogenated nm polycarbonate filters in a 10 ml extruder using nitrogen
vials and utensils, as it is described below. gas at pressures around 50-100 psi. Lysozyme concentration
in the concentrated liposome solution was determined and
TABLE 2 adjusted to 200 ug/ml by diluting the solution in 10%
35
Formulation descriptions Sucrose. The lysozyme concentration in the final Solution
was determined by UV spectroscopy.
Formula- Lyopro- Dosage
tion Adjuvant Antigen tectant form Eight vials were filled with 1 ml of liposome solution
each. Four of the vials were freeze-dried in a lyophilizer by
1 DPPCDPPG? Chicken Sucrose Liquid 40
first freezing the solution at -45° C. at 1°C./min and holding
DOPCCholesterol Egg
(Lipo-Lyz (Aq)) Lysozyme it at this temperature for 2 hours. Primary drying was
2 DPPCDPPG? Chicken Sucrose Lyophilized performed at -20° C. shelf temperature for 20 hours at a
DOPCCholesterol Egg chamber pressure of 150 mTorr. After primary drying, the
(Lipo-LyZ (FD)) Lysozyme
3 Aluminum Chicken Sucrose Liquid 45
shelf temperature was increased to 25° C. at a rate of 0.2°
phosphate Egg C./min. Secondary drying was performed at 25° C. shelf
(Adju-Phos (R) Lysozyme temperature for 10 hours at a chamber pressure of 100
(Adju-Lyz (Aq)) mTorr.
4 None Chicken Sucrose Liquid
(Lyz) Egg Preparation of 200 ug/ml Lysozyme/Adju-Phos(R Vaccines
Lysozyme 50 (Formulation 3)
Preparation of a 9 Mg/ml Stock Lysozyme Solution in PBS In order to prepare an aluminum salt based vaccine, 0.5 ml
In order to prepare 1 L phosphate buffer saline (PBS), 1 of 8.9 mg/ml lysozyme stock solution and 1 ml 2% (20
pouch PBS powder was dissolved in 1 L water for injection mg/ml) Adju-Phos(R suspension were mixed in a 1.5 ml
(WFI). The mixture was then stirred on a magnetic stirrer for 55
centrifuge tube and stored for 42 minutes at room tempera
5 minutes. The solution was then filtered through a 0.22 um ture to allow for lysozyme to be adsorbed to Adju-Phos. The
Polyvinylidene Difluoride (PVDF) filter, using a Corning 1 mixture was then centrifuged for 5 min at 5,000 rpm to pellet
L Filter System and a vacuum pump. the adsorbed lysozyme-Adju-Phos(R complex. The superna
50.1 mg lysozyme was weighed using an analytical bal tant was removed and measured by UV spectroscopy to
ance. 5 ml of PBS was measured using a sterile, 5 ml pipette 60 determine the amount of unbound lysozyme. The concen
and added to the lysozyme powder. The solution was stirred tration of bound lysozyme was determined by Subtracting
on a magnetic stirrer for 3 minutes until the solution was this value from the initial added amount. Based on the
dissolved. The solution was then filtered through a 0.22 um amount of bound lysozyme, 10%. Sucrose solution was
sterile filter. The actual concentration of lysozyme in the added to the Adju-Phos(R/lysozyme pellet to acquire a total
filtered solution was determined by UV Spectrophotometry. 65 weight of 28 g. This would give an adjuvant:antigen ratio of
This stock solution was used in preparation of different 24 w/w, i.e. similar to that used in Lipo-formulations (For
vaccine formulations. mulations 1 and 2), further described in Table 3.
US 9,603,799 B2
15 16
TABLE 3 For the liposomal formulations, the mean particle diam
eter was also determined after one year storage of the
Final Formulation Target Concentration (mg/ml) and Ratios samples at 2-8° C. The lyophilized liposome sample was
Target Target Target also stored at room temperature more than two years. This
Adjuvant LyZoSyme ratio sample was reconstituted and analyzed by DLS (Table 6b).
Formula- Concentration Concentration (Adjuvant
tion Name (mg/mL) (mg/mL) LyZ) Mice Immunization
1 Lipo-LyZ (Aq) 5 O.2O 25 100 ul (2x50 ul) of each formulation was injected intra
2 Lipo-Lyz(Lyo) 5 O.2O 25 muscularly (IM) in the shoulder of four female CD-1 mice
3 Adu-LyZ (Aq) O.71 O.O3 24 10 (Charles River, Hollister Calif.). A booster shot was admin
4 Lyz O O.2O O
istered on day 14. Twenty mice were tested in groups of four.
16 were used for vaccine testing and 4 were naive mice as
Preparation of Lysozyme with No Adjuvant in 10% Sucrose negative control. All of the animals were observed imme
(Formulation 4) diately after dosing and daily thereafter. On Day 28, serum
In order to prepare a lysozyme solution with no adjuvant, 15 was collected from the immunized mice as well as the
10 ml of 200 g/ml lysozyme solution was prepared by control group and the antibody response to each vaccine was
diluting 226 ul lysozyme stock solution (8.9 mg/ml) with determined by Indirect Enzyme-Linked Immunosorbent
10% sucrose until a total weight of 10 g was obtained. Assay (Indirect ELISA) to chicken egg lysozyme.
Concentration of lysozyme in the final formulation was
determined by UV spectroscopy. Statistical analysis of the induced immune responses was
UV Spectroscopy performed, including a two-tail t-test. Differences were
50 ul of each sample was diluted in PBS as needed and considered significant if p <0.05.
analyzed in an Agilent 8453 UV spectrophotometer (Agilent The study design and group designation are Summarized
Technologies, USA) equipped with a Peltier cell holder at in Table 4.
TABLE 4
Study Group Designations
Group? Study Event
Test No. mice, Dose Day 28
Article if group Volume Sex Day 0 - Dose Day 14 - Dose Terminal Bleed
1 4 100 ul in F Intramuscular Intramuscular Cardiac Puncture
two sites
two sites
two sites
two sites
5 (naive 4 NA F None - Naive Animals Cardiac Puncture
animals)
20° C. and a 1 cm path length quartz cell was used. Data was Prior to dosing, animals will be arbitrarily assigned to
collected in a wavelength range of 200-500 nm, with an treatment groups. On Day 0, each mouse will be injected IM
integration time of 10 seconds at 1 nm intervals. The 45 with 100 ul of test article (50 ul in each shoulder) using an
concentration of lysozyme was determined by Eqn. 1. appropriate size Syringe and beveled needle (i.e. 1 cc insulin
C-(A2so-Abstsaso), e. (Eqn. 1) Syringe w/26 gauge (or Smaller) needle).
Group 1 was dosed with Formulation 1 (Lipo-LyZ (Aq));
Where Abs, so is the light scattering interference at 280 Group 2 was dosed with Formulation 2 (Lipo-LyZ (Lyo));
nm determined by logarithmic regression extrapolation 50
Group 3 was dosed with Formulation 3 (Adju-LyZ (Aq));
through absorbencies at 320 and 350 nm, and Aso is the Group 4 was dosed with Formulation 4 (Lyz). Group 5
absorbance at 280 nm. An extinction coefficient (e) of 2.63 animals were naive animals and did not receive any test
ml/(mgXcm) was used to calculate the lysozyme concentra article. On Day 14, each animal received a booster injection
tion (C) in mg/ml according to Eqn 1. The average concen of the appropriate test article. On Day 28, animals were
tration of lysozyme was calculated based on three readings. 55
exsanguinated via cardiac puncture.
Particle Size Characterization Using Dynamic Light Scat
tering (DLS) The blood was collected into tubes containing no antico
All samples were analyzed on a Precision Detector DLS agulant. The tubes were centrifuged at -2800 rpm for at least
instrument PD2000DLSPP' and PDDLS/CoolBatch 90T 10 minutes. Sera were placed into appropriately labeled
using quartz cuvettes (Precision Detectors). Measurements 60 tubes and stored at -16° C. to -22° C. until analysis by
were done at 20° C. using a refractive index of 1.3330 and ELISA for chicken egg lysozyme IgG antibodies.
a viscosity of 0.01002 Poise. Sample time was 15 usec and Test Article Preparation
3 sec run duration with a total of 60 accumulations per Group 1: SP-255a: Formulation 1 (lysozyme--liposomes):
measurement. Data was analyzed using Precision Decon One vial was used per scheduled dosing time point for all
volve software. Each sample was analyzed in triplicate. The 65 animals in the dose group. Prior to injection, the vial was
mean hydrodynamic diameter by intensity and standard gently inverted at least 10 times and then gently swirled to
deviation (SD) were calculated (n=3). obtain a homogenous mixture.
US 9,603,799 B2
17 18
Group 2: SP-255b: Formulation 2 (lyophilized lysozyme-- before and after freeze-drying of liposomes. When these
liposomes): One lyophilized vial and one diluent vial was samples were characterized after one year storage at 5° C.
used per scheduled dosing time point for all animals in the the mean particle diameter were relatively unchanged with
dose group. Prior to injection, the lyophilized cake was the corresponding values of 639 nm (+6 nm) and 647 nm
reconstituted with 1 ml of diluent. The reconstituted vial was 5 (t14 nm) for the liquid and lyophilized liposomes, respec
Swirled to obtain a homogenous mixture. tively (Table 7a, b).
Group 3: SP-256a, Formulation 3 (lysozyme+Adju The mean particle diameter for the lyophilized liposomal
Phos): One vial was used per scheduled dosing time point
for all animals in the dose group. The vial was vigorously vaccine sample that was stored at room temperature for 28
shaken to obtain good homogeneity. 10 months (Table 7b) was 691 nmit58 nm. Surprisingly, after 2
Group 4: SP-256b, Formulation 4 (lysozyme in 10% years of storage of the lyophilized vaccine at room tempera
Sucrose): One vial was used per scheduled dosing time point ture, the particle size of liposomes is unchanged and no
for all animals in the dose group. Prior to injection, the vial aggregation is observed.
was gently inverted at least 10 times and then gently Swirled The average particle size in Adju-LyZ formulation was
to obtain a homogenous mixture. 15 >2000 nm and thus outside the range of the DLS instrument.
Group 5: Naive animals; no test article was administered. According to literature, the particle size of Adju-Phos(R) is in
Dosing Procedure the range of 1-10 um. The mean hydrodynamic diameter of
Four groups of 4 CD-1 female mice were dosed intra lysozyme in solution was 8.4+1.4 nm (Table 6).
muscularly with 100 ul of test article on Day 0 and Day 14.
Serum was collected from each animal on Day 28. Addi- 20 TABLE 6
tionally, serum was collected from 4 naive female CD-1
mice on Day 28 as a control group. The serum was frozen Characterization of Final Vaccine Formulations, Mean Particle
and stored at -80° C. until analysis by ELISA for chicken Diameters and Antibody Titers of the Vaccines
egg lysozyme IgG antibodies.
Determination of Amount Bound and Unbound Lysozyme 25 Measured Particle Antibody
The concentration of lysozyme stock solution, unbound Lysozyme (mg/ml) diameter (nm) titer (mg/ml)
and bound lysozyme, was determined by UV spectroscopy. Vaccine Mean (SD) Mean (SD) Mean (SD)
The results are listed in Table 5. According to these results,
the concentration of lysozyme stock solution was 8.9 mg/ml Lipo-LyZ (Aq) 0.26 (+0.01) 707 (+5) 177 (+68)
(+0.1), the concentration of free lysozyme in the Supernatant 30 Lipo-LyZ (Lyo) 0.27 (+0.03) 667 (+113) 306 (+207)
was determined to be 5.9 mg/ml (+0.0) out of 8.9 in the Adju-Lyz (Aq) 0.03 (+0.00) >2000 478 (+136)
liposome-lysozyme solution supernatant and 2.2 (+0.0) LyZ (Aq) 0.22 (+0.00) 8.4 (+1.4) 50 (+50)
mg/ml (out of 3.0 mg/ml) in the Adju-Phos(R)-lysozyme
Supernatant. The amounts of bound/entrapped lysozyme to Particles were outside the upper size limit of the DLS instrument and could not be
Adju-Phos(R/liposomes were thus 26% w/w and 34% w/w, 35 measured accurately
respectively. These values are in the expected range.
TABLE 5
Measurements of Bound and Unbound Lysozyme
Adjuvant Lysozyme Unbound
Concentration Concentration Initial free Bound Entrapped
No Name (mg/mL) (mg/mL) Adjuvant LyZ (mg/mL) (mg/mL) (%)
1 Lipo- 50 8.9 0.1 S.6 5.9 O.O 3.0 - O.O 34
LyZ(Aq)
2 Lipo-Lyz 50 8.9 0.1 S.6 5.9 O.O 3.0 - O.O 34
(Lyo)
3 Adju-Lyz 2O 3.0 - 0.1 6.7 2.2 O.O O.8 O.O 26
(Aq)
4 Lyz O 8.9 0.1 O
Lysozyme Concentration in Final Vaccine Formulations TABLE 7a

As shown in Table 6, the average lysozyme concentra
tions in liquid and lyophilized liposomal vaccines were 0.26 Stability of Liquid Liposomal Vaccine
mg/ml (+0.01 mg/ml) and 0.27 mg/ml (+0.03 mg/ml), 55
respectively. Formulation of Lysozyme (Lipo-LyZ (Aq))
The concentration of lysozyme in the liquid Adju-Phos(R)
formulation and in the lysozyme solution with no adjuvant Measured Particle Antibody
were 0.03 (+0.00 mg/ml) and 0.22 mg/ml (+0.00 mg/ml), Lysozyme (mg/ml) diameter (nm) titer (mg/ml)
respectively and as expected. 60 Vaccine Mean (SD) Mean (SD) Mean (SD)
tering (DLS) tO 0.26 (+0.01) 707 (+5) 177 (+68)
The particle size data are shown in Tables 6 and 7. The 13 months ND 639 (+6) ND
mean hydrodynamic particle diameters of liposomes from
distributions by intensity, before and after lyophilization 65
were 707 nm (+5 nm) and 667 nm (113 nm), respectively. NTD = Not Determined
No significant change in particle diameter was thus observed
US 9,603,799 B2
19 20
TABLE 7b Freeze-Stability of Vaccines
It is well-known that aluminum-based vaccines are
Stability of Lyophilized Liposomal Vaccine freeze-sensitive (Wolff et al., “Development of a formula
Formulation of LYSOZYme (Lipo-LYZ (Lyo tion protecting aluminum hydroxide adjuvanted vaccines
Measured Antibody during lyophilization”, Proc. 6" World Meeting Pharma.
Stored
at Temp
Lysozyme
(mg/ml)
Particle
diameter (nm)
titer
(mg/ml)
Biopharm. Pharma Tech., Barcelona, ES (2008)) and as
Time (° C.) Mean (SD) Mean (SD) Mean (SD)
further summarized herein (Table 1). According to aspects of
the present invention, the liposomal vaccine compositions
tO NA 0.26 (+0.01) 667 (+113) 306 (+207) described herein were freeze-stable; they did not lose their
13 months 5+3 0.27 (+0.03) 647 (+14) ND 10 immunogenicity despite multiple freeze-thaw cycles and, in
28 months 22 + 2 0.32 (+0.00) 691 (+58) 1,918 (+3.406) the case of the freeze-dried formulation, freezing at -45° C.
Assumed to be unchanged after lyophilization, during lyophilization also failed to erode the immunogenic
ity of the claimed composition. Similarly, after freeze
Characterization of Antibody Response to Each Formulation drying, no negative attributes Such as large aggregates or
by Indirect ELISA 15 increase in liposome sizes were observed, as discussed
FIG. 1 shows an exemplary immune response (450 nm below.
absorbance) of the various formulations as compared at The Effect of Particle Size on Immune Response
different dilutions of the sera (80,000, 160,000, and 320, Liposome mean particle diameters before and after
000-fold). The amount of absorbance at 450 nm reflects the lyophilization remained relatively identical (Table 6) and
amount of antibody present in the sera. representative particle size distribution by intensity were
FIG. 2 shows an exemplary standard curve of mouse shown (FIG. 4a-e) at varying composition features and
anti-lysozyme antibody, while FIG. 3 depicts an exemplary across different time points, including liquid liposome at t0
average amount of mouse anti-lysozyme antibody for a (FIG. 4a), liquid liposome at 13 months (FIG. 4b), freeze
given formulation (mg/ml). dried liposomes (reconstituted) at t0 (FIG. 4c), freeze-dried
The amount of mouse anti-lysozyme antibody from the 25 liposome (reconstituted) after 28 months at room tempera
immune response of each of the four formulations was ture (FIG. 4d) and freeze-dried liposome (reconstituted)
determined using a standard curve for purified mouse anti after 13 months at 5° C. (FIG. 4e).
lysozyme antibody (Raybiotech). A representative standard It is further evidence of the value of the compositions of
curve is shown (FIG. 2), with the standard curves being the present invention, particularly in view of the longevity of
similar for all of the four ELISA plates. 30 the liposomal structural integrity under room temperature
The average quantified immune response to the lysozyme conditions over many months. Indeed, this Suggests that the
vaccines from each group of mice (n=4) and the correspond slight difference in immune response induced between the
ing standard deviations are shown (FIG. 3). The group of two formulations was not due to a change in particle size.
naive mice did not give any immune response as expected This is also corroborated by observing that despite the large
(negative control) and lysozyme without adjuvant had the 35 difference in particle size between liposomal formulations
lowest amount of antibodies induced. and the Adju-PhoSR) formulation, similar immune responses
The liquid liposomal vaccine induced approximately were induced. In fact, it has been shown that anti-lysozyme
three times the amount of antibodies as the lysozyme titers were independent of the particle size for vaccines
without adjuvant. The lyophilized liposomes showed a six adjuvanted with either aluminum hydroxide or aluminum
fold increase in antibodies. The Adju-Phos(R (Adju-LyZ) 40 phosphate and also unaffected by the level of antigen bind
vaccine had the highest immune response, a nine-fold ing to the adjuvant.
increase from the lysozyme alone. Characteristics of the Stability of Lyophilized Liposomes at Room Temperature
various vaccines are summarized in Table 6. According to the methods and compositions of the present
A two-tail t-test performed to evaluate the significance invention, as stated expressly herein at Table 7b, there were
between the lyophilized liposomal formulation and Adju 45 no changes in mean particle diameter of liposomes after 2
Phos(R formulation, as well as the liquid and lyophilized years in storage at room temperature. Similarly, there was no
liposomal formulations, gave p-values of 0.06 and 0.11, change noted in the average lysozyme concentration in the
respectively. This showed that there was no significant lyophilized liposomal lysozyme vaccine that was reconsti
difference between lyophilized liposomal formulation and tuted after more than 2 years storage at room temperature.
Adju-Phos(R formulation, as well as the liquid and 50 These results suggest that the formulation was stable at room
lyophilized liposomal formulations at the 95% confidence temperature in regards to particle size and antigen concen
limit. tration.
The formulations, in order of increasing immunogenic
response, are as follows: II. Liposomal Vaccine Composition with Tetanus
55 Light Chain Antigen
Lysozyme formulations liquid liposomal
formulations lyophilized liposomal formulation Lipids used in this formulation include the following:
as Adju-Phos(R formulation. dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phospha
The results show that all the vaccine formulations (Lipo tidylcholine (DOPC), cholesterol, and a negatively charged
LyZ (Aq), Lipo-LyZ (Lyo) and Adju-LyZ (Aq)) were better 60 lipid dipalmitoyl phosphatidylglycerol (DPPG). These lipids
than the control lysozyme (LyZ) solution, inducing a sig were supplied from Avanti Lipids. Octadecylamine, which is
nificant immune response in mice. The findings of this study the equivalent of Stearylamine (SA), a positively charged
clearly demonstrate that the freeze-stable liposomal vaccine lipid, was supplied from Fluka.
can be as immunogenic as an aluminum-based vaccine. In Recombinant light chain from tetanus toxin (TLC) 10 ug
addition, the freeze stable liposomal vaccine can be 65 was supplied by List Biological Laboratory Inc, Cat. No.
lyophilized into a stable, dry product that has the potential 650 A, lot 6503A1 and was used as the model antigen. TLC
to become a room temperature stable vaccine. has a molecular weight of 50,000 Da, a pl of 5.06 and is a
US 9,603,799 B2
21 22
non-toxic protein. TLC is the smaller polypeptide chain of sample diluent was made by mixing 2.5 ml sample diluent
tetanus toxoid and retains the enzymatic activity encoded by with 47.5 ml Milli-QR) water.
the holotoxin. Results from the mouse immunogenicity study are shown
Tween-20 (10% w/w Solution), Trehalose, lactose, (FIGS. 5 and 6), where the average titers of mouse anti
HEPES, PBS, sodium citrate, citric acid, sodium hydroxide tetanus toxoid IgG from 5 mice are compared among the
and Milli-QR) water comprised the remainder of the com formulations (FIG. 6) against the standard curve (FIG. 5).
position in Solution. The results clearly show that the cationic liposomes
A positively charged liposomal adjuvant system was containing TLC in both the liquid formulation that had been
made in a similar fashion as described in Example I, except freeze-thawed three times and the lyophilized formulation
with the lipid blend molar ratio of 40:25:20:15 containing 10
that had been frozen to -40° C. and lyophilized gave a
DPPC:DOPC:cholesterol:SA. Recombinant tetanus light significant immune response and generated IgG antibodies
chain (TLC) was entrapped and/or associated with the lipid to tetanus toxoid as measured by ELISA (Table 12). The
blend by hydration in the protein solution. The solution was naive and TLC solution without adjuvants did not give a
subjected to three freeze-thaw cycles and extruded through
800 nm pore Nucleopore membranes. The free unassociated 15 significant immune response against the tetanus toxoid.
TLC was removed by dialysis and the liposomal TLC Particle size was analyzed and specific sizes described in
complex was diluted to around 120 ng/ml protein. Table 11.
As a control, a non-adjuvant solution of just the TLC
Solution was injected into mice. All the Solutions were III. Lipid Blend Composition with Tetanus Light
totally dosed at 10 ng TLC per mouse (Table 8). One IM Chain as Antigen
injection of 50 ul/mouse was administered into 5 mice per
group and serum was collected following two weeks after In order to experimentally deduce preferred compositions
the second booster injection of the test articles. and preparatory methods of different lipid blends, and their
Immunogenicity to the tetanus toxoid was evaluated using resultant immunogenicity, liposomal vaccine formulations
the mouse anti-tetanus toxoid IgG ELISAkit that detects and were prepared and described, as indicated in Table 8.
TABLE 8
TLC Dosing and Total Lipid Content in Liposomal Vaccine Formulations
Total
Antigen
Ratio injected Total lipid
Antigen Adjuvant (Adjuvant after 2 injected
Booster COC COC. Vol. in Antigen Adjuvant protein) doses after 2
Sample ID shot Injftime point (ug/mL) (mg/mL) (ul) dose dose (ugug) (ng) doses (ug)
Anionic liposomes SP-318-2 Y 50 O.1 6 50 5 O.3 60,000 10 600
(study 1) Aq
Anionic liposomes SP-318-3 Y 50 O.1 6 50 5 O.3 60,000 10 600
(study 1) FD
Cationic liposomes SP-318-4 Y 50 O.1 6 50 5 O.3 60,000 10 600
(study 1) Aq
Catonic liposomes 5P-318-5 Y 50 O.1 6 50 5 O.3 60,000 10 600
(study 1) FD
Anionic liposomes 5P-329-2 y 100 ul (2 x 50) 0.4 25 100 40 2.5 31,250 8O SOOO
(study 2) Aq
Anionic liposomes 5P-329-3 y 100 ul (2 x 50) 0.4 25 100 40 2.5 31,250 8O SOOO
(study 2) FD
Cationic liposomes SP-329-4 y 100 ul (2 x 50) 0.4 25 100 40 2.5 31,250 8O SOOO
(study 2) AQ
Cationic liposomes SP-329-5 y 100 ul (2 x 50) 0.4 25 100 40 2.5 31,250 8O SOOO
(Study 2) FD
50
quantifies tetanus toxoid-specific IgG in mouse serum of Preparation of Lipid Blend
vaccinated or immunized animals (ELISA Kit Cat No. In order to prepare the cationic liposomes, two vials were
930-130-TMG, Alpha Diagnostic International, San Anto prepared, each with about 6.6 mg SA, 48.6 mg DPPC, and
nio, Tex., USA). 12.7 mg cholesterol added to 1.6 ml of 20 mg/ml DOPC/
The material for the ELISA consisted of the following: 55 chloroform (32 mg). The total amount of lipid in each vial
mouse anti-tetanus toxoid IgG ELISA was used for quanti was 100 mg.
tative determination of anti-tetanus toxoid IgG in serum. Similarly, to prepare the anionic liposomes, two vials
This kit included tetanus toxoid coated strip plate (Part were prepared, each with about 43.5 mg DPPC, 16.6 mg
930-111, lot 11045P6), anti-tetanus toxoid IgG calibrators DPPG, and 11.5 mg cholesterol in 1.45 ml DOPC/chloro
(10 U/ml, 20 U/ml. 40 U/ml and 80 U/ml), mouse X-tetanus 60 form (29 mg). The total amount of lipid in each vial was 100
toxoid IgG (as positive control, part 930-132), low NSB ng.
sample diluent (Part TBTm). TMB substrate, and stop Since all the lipids failed to dissolve in chloroform, an
solution (part 80101, dilute sulfuric acid). Wash solution 83% methanol/water solution was added to obtain a clear
concentrate (Cat. WB-100), sample diluent concentrate (Cat. solution. The lipid blends were first dried in a 45° C. water
No. SD-20T) and anti-mouse IgG-HRP-conjugate concen 65 bath under nitrogen gas, and then the residual solvent was
trate (Part H-MsC-112b) were also utilized. The wash solu removed under vacuum overnight, to ensure complete des
tion was diluted 100x in milliO water prior to use. A working iccation. Two lipid blend vials were used per study.
US 9,603,799 B2
23 24
Preparation of 10 ug/ml TLC Stock Solution at pH 7.4 or pH shelf temperature to remove any residual water from the
4.0 in 0.05% Tween cake. A Summary of drying conditions are described in Table
A 0.05% Tween-20 solution was made by diluting 25 ul 9.
Tween-20 in 5 ml water. In Study 1, each vial of lyophilized
recombinant Tetanus light chain (10 ug) was reconstituted 5 TABLE 9
with 1 ml 0.5% Tween-20 solution to obtain a 10 ug/ml Freeze-Drying Conditions
tetanus solution in 20 mM HEPES (pH 7.4) and 1.25%
lactose. Freeze-drying Time Rampf
In Study 2, 0.05% Tween-20 solutions were made either steps Temperature (Minutes) Hold Pressure
10
in citrate buffer (pH 3.7) or in 20 mM HEPES (pH 7.4) with Freezing +5 15 H 1 ATM
1.25% lactose. 1 ml of each solution was used for reconsti -45 60 (120) R 1 ATM
tution of one TLC vial. -45 60 (240) H 1 ATM
Primary -45 60 H 150 (100) mTorr
Hydration of Lipid Blend Drying -30 (-40) 30 R 150 (100) mTorr
Cationic and anionic lipid blends were then hydrated with 15 -30 (-40) 1200 H 150 (100) mTorr
0.9 ml of the TLC stock solutions. In Study 1, both cationic Secondary +25 240 R 150 (100) mTorr
Drying +25 600 H 150 (100) mTorr
and anionic liposomes were made at pH 7.4, whereas in
Study 2, the liposomes were made at pH 7.4 and 4.0, The parenthetical values are parameters used in Study 2.
respectively. The mixtures were stirred on a magnetic stirrer
for 15 minutes and sonicated for 10 seconds until the lipids Preparation of Diluents
were totally hydrated into a milky homogenous Solution. Diluents (reconstitution solutions for the freeze-dried
Multiple Freeze-Thaw Cycles vaccines) were also prepared for the lyophilized samples by
In order to improve the entrapping efficacy, the liposome filling 1 ml of WFI into five vials.
Solutions were freeze-thawed three times using an acetone Preparation of TLC without Adjuvant
ice bath and a warm-water bath. The solutions were then 25 In Study 2, 0.15 ml of TLC solution (10 ul in 0.05%
diluted to a concentration of 6 ug/ml TLC in Study 1 and 5 Tween-20) was diluted in 2.5 ml HEPES, 10% trehalose,
ug/ml in Study 2. 0.05% Tween-20 (pH 7.4) to obtain a 0.6 g/ml TLC
Extrusion Solution.
To reduce the size of liposomes and to obtain a more Mice Immunization
homogenous particle distribution, each liposome solution 30 50 and 100 ul (2x50 ul) of each formulation was injected
was extruded ten times through two 800 nm polycarbonate IM in the shoulder of five female CD-1 mice (Charles River,
filters (Nucleopore) in a 10 ml Extruder (Northern Lipids) at Hollister Calif.) in Study 1 and 2, respectively. A booster
50 psi using nitrogen gas. shot was administered on day 14. Mice were tested in groups
Dialysis of five. 5 naive mice were used as negative control and did
In order to separate the free TLC from the incorporated 35
not receive any injections. All of the animals were observed
TLC, the liposome solutions were dialyzed through Float immediately after dosing and daily thereafter. On Day 28,
A-Lyzer G2 (Spectrum labs) with 300,000 Da (Study 1) or serum was collected from the immunized mice as well as the
1,000 kDa (Study 2) molecular weight cut off (MWCO) control group. All animal testing was conducted according
membranes against PBS buffer (pH 7.4) overnight (Study 1). to an approved Animal Care and Use Protocol (ACUP). The
In Study 2, 1.5 ml of liposomes were dialyzed against 40
either 1.25% lactose, 20 mM citrate buffer, pH 4.0 (anionic animal studies were carried out in an animal facility that is
liposomes) or 500 ml of 1.25% lactose, 20 mM HEPES fully accredited by the Association for Assessment and
(cationic liposomes) overnight (18 hrs). Accreditation of Laboratory Animal Care International.
Indirect ELISA Methods and Results
300,000 Da MWCO membranes were used because lipo
somes would be unable to pass, but the free TLC, which is 45 Immunogenicity to the tetanus toxoid was evaluated using
smaller (50,000 Daltons), would be able to pass through the a mouse anti-tetanus toxoid IgG ELISA kit that detects and
membrane. quantifies tetanus toxoid-specific IgG in mouse serum of
Filling vaccinated or immunized animals (ELISA Kit Cat No.
After dialysis each solution was transferred to a tube in a 930-130-TMG, Alpha Diagnostic International, San Anto
sterile hood. 50 nio, Tex., USA).
A solution containing 100 ml 10 wt.% trehalose in 10 mM The mouse sera was diluted (100:900 ul) in the working
Hepes buffer was prepared by dissolving 238.3 mg HEPES sample diluent (10x). Then each sera was diluted from 1:10
and 10 g trehalose in 90 g water until final weight of solution to 1:100 in LNSB buffer (10x).
reached 100 g (pH of the buffer was measured to be 7.05) Each well of a 96-well polystyrene microtiter plate (Alpha
and sterile filtered using a 0.22 micron filtration flask. 55 Diagnostics) was incubated with 200 ul wash buffer for 5
4.5 ml 10% trehalose in a 10 mM Hepes solution was min. The wells were then rinsed and 100 ul of each diluted
added to 0.5 ml of each liposome solution. Assuming a 20% sample was added. The plate then incubated for one hour on
entrapment efficiency for TLC in liposomes, the concentra an orbital shaker (VWR) at 150 rpm. The plate was removed
tion of TLC should be about 0.1 ug/ml for cationic lipo and washed four times using washing buffer. 100 ul HRP
Somes and anionic liposomes in the Study 1, and 0.4 ug/ml 60 conjugated IgG Anti-Mouse antibody was diluted in 9900 ul
in the Study 2. working solution diluent. 100 ul of the diluted solution was
Freeze-Drying subsequently added to each well and incubated for 30 min.
For each formulation, five of the vials were freeze-dried The wells were then washed again five times and 100 ul
in a Vertis Freeze-dryer (Vertis Genesis 12XL) by first TMB substrate was added to each well. The wells were
freezing the solution at -45° C. and then subliming the ice 65 incubated in the dark for 15 minto allow for the reaction and
at -30° C. and -40°C. under high vacuum in Study 1 and color change to take place. When the color in the wells
2, respectively. Secondary drying was performed at 25°C. turned blue, 100 ul stop solution was added to each well.
US 9,603,799 B2
25 26
Absorbance of each well was measured at 450 nm (and Characterization of Antibody Response by Indirect ELISA
also at 630 nm to normalize for well background), using a for Liposomes with 0.1 ug/ml TLC (Study 1)
Spectromax(R) 190 microplate reader (Molecular Devices). The amount of mouse anti-tetanus antibody from the
Two duplicate readings were obtained per sample at each immune response of each of the formulations was deter
wavelength. The absorbance at 630 nm were subtracted from 5 mined using a standard curve (FIG. 5) generated from a
the corresponding values at 450 nm to reflect the amount of purified preparation of mouse anti-tetanus antibody.
antibody in the serum at each dilution. The mean of two The average quantified immune response to the tetanus
duplicate readings were calculated for each sample. vaccines from each group of mice and the standard devia
A standard curve was plotted using the absorbance values 10
tions are shown in Table 11. The average immune response
for the standards with known mouse anti-tetanus toxoid for cationic liposomes-TLC (0.1 ug/ml) was also examined
antibodies at 10 U/ml, 20 U/ml, 40 U/ml and 80 U/ml. (FIG. 8) compared to a no adjuvant control.
Mouse anti-tetanus toxoid antibody titers were calculated Anionic liposomes did not show a significant immune
from absorbance readings that fell on the standard curve for response with tetanus at the dose used (50 ul of 0.12 lug/ml
the mouse anti-tetanus toxoid standards and corrected for at t=0 and 14 days, i.e. 6 ng TLC and 0.3 mg lipid per
15
dilution factors (the mouse sera was diluted one hundred injection). Cationic liposomes induced 4-6 times more anti
fold in all cases, except in cases of mice immunized with body titers against tetanus toxoid than TLC without adjuvant
cationic liposomes. The sera of mice immunized with liquid (Table 11).
and lyophilized cationic liposomes (TLC 0.4 g/ml (Study
2)) were diluted totally one thousand fold and five hundred TABLE 11
fold, respectively).
The mean and standard deviations of the antibody titers Average antibody response formulation
for each formulation were calculated. The mean values of calculated from standard curve (FIG. 5
antibody titers for 5 mice were calculated for all samples. Antibody Particle
The mean values obtained for each sample was subtracted 25 response' Diameter (nm)
from those obtained for naive mice. Two-sample, one-tailed Sample ID Formulation (U/mL) Mean SD
t-Tests were performed for the mean antibody titers of the
liquid and lyophilized anionic and cationic liposomes (Study SP-318-6 No adjuvant 871 - 1451
SP-318-2 Anionic liposome (liquid) 82 - 271 S21 37
2) to see whether lyophilization induced a significant dif SP-318-3 Anionic liposome (FD) 1,183 + 1,655 357 12
ference in immunogenicity of the formulations. An alpha SP-318-4 Cationic liposome (liquid) 5,605 + 6,399 313 8
level of 0.05 was used. A p-value was calculated. If the 30 SP-318-5 Cationic liposome (FD) 3,358 + 3,395 349 25
p-alpha, the difference in the immunogenicity of the for
mulations before and after lyophilization would be signifi These values are already corrected for the amount obtained for naive mice (73 126).
Cant.
These data are obtained from particle size distribution by intensity using smoothness 20
(n = 3).
tering (DLS) 35 Results from Study 2
50 ul of each liposomal vaccine was diluted 40-60 times The particle size analysis results for Study 2 are presented
and analyzed on a Precision Detector DLS instrument (FIG. 9a-d) and Table 12, with no significant change in the
(PD2000DLSplus and PDDLS/CoolBatch90T) using quartz mean particle diameter of liposomes observed by DLS
cuvettes (Precision Detectors). Measurements were done at before and after freeze-drying. The results show that the
20° C. using a refractive index of 1.3330 and a viscosity of mean particle diameter of anionic liposomes were more or
0.01002 Poise. Sample time was 15 usec and 3 sec run less unchanged after freeze drying.
duration with a total of 60 accumulations per measurement. The mean particle diameter of cationic liposomes had
Data was analyzed using Precision Deconvolve software.
Each sample was analyzed in triplicate. A Smoothing param increased slightly from 462 (+37) to 512 (+106) nm after
eter of 20 was applied. The mean hydrodynamic diameters freeze drying. This increase in particle size did not have any
by intensity and standard deviations (SD) were calculated 45 significant impact on the immune response obtained in mice
(n-3). (Tables 12 and 13).
Results from Study 1
In Study 1, the particle size characterization of liposomal TABLE 12
vaccines with 0.1 g/rat TLC was analyzed. Representative
particle size distribution by intensity were shown (FIG. 50 Data Summary for Immune Responses and Particle
Sizes of the Liposomal Formulations with 0.4
7a-d), with mean particle diameters (n=3) and standard mg/ml TLC (Study 2) (n = 5/formulation
deviations listed in Table 10.
Mouse Anti
Particle Tetanus Toxoid
TABLE 10
55 Sample
Diameter (nm) IgG (U/mL)
Vaccine Mean SD Mean SD
Particle Size Data for Liposomal Vaccine
with 0.1 Lig/ml TLC (Study l SP-329-2 Anionic liposomes- 311 8 7,080 + 8,916
Particle
TLC (Aq)
SP-329-3 Anionic liposomes- 315 - 24 3,621 + 5,525
Diameter (nm) TLC (FD)
Samples ID Formulation Mean SD 60 SP-329-4 Cationic liposomes- 462 - 37 30,633 + 40,697
SP-318-2 Liquid anionic liposomes S21 37 TLC (Aq)
SP-318-3 Lyophilized anionic liposomes 357 12 SP-329-5 Cationic liposomes- 512 106 34,140 + 44,228
SP-318-4 Liquid cationic liposomes 313 8 TLC (FD)
SP-318-5 Lyophilized cationic liposomes 349 25 SP-329-6 TLC ND 1,316 + 1,741
These data are obtained from particle size distribution by intensity using smoothness 20 65 In these values, the response obtained for naive mice has already been subtracted.
These data are obtained from particle size distribution by intensity using smoothness 20
(n = 3). (n = 3).
US 9,603,799 B2
27 28
Characterization of Antibody Response by Indirect ELISA As evidenced in FIG. 13a, there was a considerable
for Liposomes with 0.4 ug/ml TLC (Study 2) distinction across the dose dependent range of immune
The amount of mouse anti-tetanus antibody from the response in aggregate levels of anti-tetanus antibody IgG
immune response of each of the formulations was deter raised between the anionic and cationic variants of the lipid
mined using a standard curve (FIG. 10) generated from a 5 adjuvants used in both studies. Specifically, the cationic
purified preparation of mouse anti-tetanus antibody. The liposomes, whether liquid or freeze-dried, raised at least five
average quantified immune response to the tetanus vaccines times the antibody levels when compared with the anionic
from each group of mice and the standard deviations were liposomes.
calculated (FIG. 11) and summarized (Table 12). Average A deeper analysis of the data from the dose dependent
amounts of mouse anti-tetanus antibody for cationic lipo 10 ranges of immune response across all TLC formulations
somes with TLC (0.4 ug/ml) in study 2 are compared to TLC found a benefit of anionic liposomes as compared to TLC
without adjuvant in FIG. 8.
As indicated, TLC alone had a very low amount of alone (FIG. 13b). The anionic liposome formulations were
antibodies induced (FIG. 11; Table 12). The liquid and prepared at two different pH concentrations, depending on
lyophilized anionic liposomes induced approximately five lipid content (higher lipid content formulations were pre
and three times as many antibodies, respectively, as the TLC 15 pared at pH 4 and lower lipid content formulations were
alone. The liquid and lyophilized cationic liposomes showed prepared at pH 7).
almost a forty- and fifty-fold increase in antibodies, respec Additional examination of comparisons of the immuno
tively. genicity in mice of different formulations as a function of
The results reveal that there is a significant difference TLC concentration was also undertaken (FIG. 14a). Spe
between the cationic and anionic liposome formulations, cifically, when analyzing immunogenicity as a function of
with the cationic liposomes inducing a much stronger total TLC injected (ng), the data consistently indicates the
immune response. The liposomal vaccines retained their benefit of the cationic formulations (both liquid and freeze
immunogenic activity after multiple freeze-thaws and also dried) when compared with the anionic formulations. Such
after freeze-drying. benefit remains even when the data are viewed in a log scale
The results reveal that there is a significant difference 25 format (FIG. 14b), indicating the remarkable improvement
between the cationic and anionic liposome formulations, attained with respect to immunogenicity when altering the
with the cationic liposomes inducing a much stronger charged lipid in the liposomal vaccine adjuvant from DPPG
immune response. Two-sample, one-tailed t-Tests were per (anionic) to SA (cationic). This additionally would enable
formed for the immune response obtained for liquid and the development of vaccine requiring a lower dose of TLC,
lyophilized anionic and cationic liposomes to see whether 30 therefore reducing costs as well as potential negative reac
lyophilization induced a significant difference in immuno
genicity of the formulations. An alpha level of 0.05 was tions to an individual’s immune system.
used. For the anionic formulations, a p-value of 0.243 was V. Stability Analysis from Studies
obtained, and for the cationic formulations a p-value of
p=0.450 was obtained, with all results summarized at Table
13. Since alpha<p in both cases, it cannot be concluded that 35 Lyophilized liposomal tetanus vaccines (SP-318-3,
there was a significant difference in the formulation after SP-318-5, SP-329-3 and SP-329-5) were stored at 5° C.i.2°
lyophilization in either case. C. for about 1 year. At the end of this time they were also
stored at room temperature (22° C.2° C.) for 2 days and
TABLE 13 then tested in mice for immunogenicity. In these studies the
40 dose was increased from 50 ul to 80 ul for anionic liposomes
Parameters for the T-Test (Study 2 with 0.1 g/ml TLC (SP-318-3 and SP-318-5 from Tetanus
study 1).
TLC-Anionic liposomes TLC-Cationic liposomes Since the lyophilized liposomal vaccine vials from Study
Freeze- Freeze 2 of tetanus were limited, the mice received only 1 dose in
Liquid dried (FD) Liquid dried (FD) 45 the groups receiving anionic and cationic liposomes con
X = 7,080 X = 3,621 X = 30,633 X = 34,140
taining 0.4 ug/ml TLC and 2.5 mg/ml adjuvant (lipid)
Sx = 8,916 Sx = 5,525 Sx = 40,697 Sx = 44,228 (SP-329-3 and SP-329-5). The sera of mice were analyzed 2
n =5 n=5 n=5 n=5 weeks after the 1st dose by ELISA (as previously detailed)
Ha: Liq > FD, t = 0.7374, Ha: Liq < FD, t = -0.1305, and at the end of study, i.e. 28 days after the 1st dose.
df = 6.68, p = 0.243 df = 7.945 p = 0.450 50 1 Year Stability Study of TLC Liposomes at 2-8° C.
The IgG response against tetanus toxoid in mice obtained
The mean particle size for cationic and anionic liposomes after the first shot, 14 days after the shot, showed that in
were relatively constant before and after freeze-drying average for both anionic liposomes (SP-318-3 and SP-329
(Table 12). There was a slight increase in mean particle size 3), similar IgG response was obtained in mice (490+76 U/ml
for the cationic liposomes after freeze-drying. According to 55 and 470+48, respectively). This showed that increase of the
statistical analysis this increase in particle size did not amount of TLC injected from 10 ng to 40 ng in each case,
significantly affect the immune response of the vaccine or increase in the amount of adjuvant (lipid) injected from
formulation. Average amounts of mouse anti-tetanus anti 0.48 mg to 2.5 mg in each case, did not have any effect on
body for anionic liposomes with TLC (0.4 ug/ml), both the immune response obtained in mice after Such a short
liquid and freeze-dried, were compared against TLC without 60 time as 14 days after the 1st shot. Similar results were
adjuvant (FIG. 12). obtained for cationic liposomes at lower dose of TLC (10
ng) and lipid (0.48 mg), i.e 445+76 U/ml.
IV. Comparison of Studies 1 and 2 However, the cationic liposomes at higher dose (40 ng
TLC and 2.5 mg lipid) gave a higher immune response
The results obtained for the studies above are plotted as a 65 (1,268+237 U/ml), 14 days after the 1st shot was received by
function of lipid adjuvant or TLC in the dose received by mice, as detailed and compared with specific stability ele
mice (FIGS. 13-15). ments from each composition (Table 14).
US 9,603,799 B2
29 30
TABLE 1.4
Stability Results for Cationic and Anionic Lyophilized Liposomes Associated with TLC
Immune
Wol. Total Immune response (28
inji lipid response days after 1st
TLC time Conc Ratio (14 days shot or 14
Booster TLC in point Lipid after 1st (Lipid TLC) after 1st days after
ID Antigen shot (ug/mL) (ng) (ul) (ug/mL) shot (ug) (ugug) shot) 2nd shot)
SP-318-3 TLC Y O.12 10 8O 6OOO 480 50,000 490 76 TBD
(1 Year at (expecting
5° C.) 1,300)
SP-318-5 TLC Y O.12 10 8O 6OOO 480 50,000 445 76 TBD
5° C.) 4,000)
SP-329-3 TLC N O4O 40 100 25,000 2,500 62,500 470 - 48 TBD
5° C.) 2,000)
SP-329-5° TLC N O4O 40 100 25,000 2,500 62,500 1,268 + 237 TBD
5° C.) 6,000)
SP-318-3 is the anionic liposomal vaccine with 0.12 ugml TLC and 6,000 ugml lipid
SP-318-5 is the cationic liposomal vaccine with 0.12 lug/ml TLC and 6,000 ugml lipid
SP-329-3 is the anionic liposomal vaccine with 0.40 ugml TLC and 25,000 ugml lipid
SP-329-5 is the cationic liposomal vaccine with 0.12 lug/ml TLC and 25,000 ugml lipid
Immunogenicity was also assessed (FIG. 15a-b) as com 25 ings falling within the scope of the generic disclosure also
pared across lipid charge (anionic versus cationic) and time form part of these inventions. This includes the generic
after first shot. Final prophetic amounts are plotted based on description of each invention with a proviso or negative
expected values after 28 days as read from linear regression limitation removing any subject matter from the genus,
lines from actual values attained after 28 days and 2 shots. regardless of whether or not the excised materials specifi
30
It is also clear that all liposomal vaccines described in the cally resided therein. In addition, where features or aspects
present invention provide a better immune response, with of an invention are described in terms of the Markush group,
Sustained greater immunogenicity, and a lower antigen dose, those schooled in the art will recognize that the invention is
as compared to TLC adsorbed to the Adju-Phos(R)-like also thereby described in terms of any individual member or
systems of the prior art. subgroup of members of the Markush group. It is also to be
35
All of the features disclosed in this specification may be understood that the above description is intended to be
combined in any combination. Each feature disclosed in this illustrative and not restrictive. Many embodiments will be
specification may be replaced by an alternative feature apparent to those of in the art upon reviewing the above
serving the same, equivalent, or similar purpose. Thus, description. The scope of the invention should therefore, be
determined not with reference to the above description, but
unless expressly stated otherwise, each feature disclosed is 40
should instead be determined with reference to the appended
only an example of a generic series of equivalent or similar claims, along with the full scope of equivalents to which
features. As used in this specification and in the appended such claims are entitled. Those skilled in the art will
claims, the singular forms include the plural forms. For recognize, or will be able to ascertain using no more than
example the terms “a,” “an,” and “the include plural routine experimentation, many equivalents to the specific
references unless the content clearly dictates otherwise. 45 embodiments of the invention described. Such equivalents
Additionally, the term “at least preceding a series of are intended to be encompassed by the following claims.
elements is to be understood as referring to every element in
the series. The inventions illustratively described herein can What is claimed is:
suitably be practiced in the absence of any element or 1. An injectable immunogenic composition comprised of
elements, limitation or limitations, not specifically disclosed 50 a liposome-associated antigen, wherein said liposome com
herein. Thus, for example, the terms “comprising,” “includ prises: dipalmitoyl phosphatidylcholine (DPPC), dioleoyl
ing.” “containing.” etc. shall be read expansively and with phosphatidylcholine (DOPC), cholesterol and stearylamine
out limitation. Additionally, the terms and expressions (SA) wherein said antigen is entrapped within the aqueous
employed herein have been used as terms of description and compartment of said liposome, entrapped within the lipid
not of limitation, and there is no intention in the use of Such 55 bilayer, and/or adsorbed to the liposomal surface wherein
terms and expressions of excluding any equivalents of the the DPPC:DOPC:cholesterol:positively charged lipid molar
future shown and described or any portion thereof, and it is ratio is 40:20-30:20:10-20; wherein said antigen is at least
recognized that various modifications are possible within the one member selected from the group consisting of poly
scope of the invention claimed. Thus, it should be under nucleotides, polypeptides, recombinant proteins, synthetic
stood that although the present invention has been specifi 60 peptides and protein extract isolated from pathogens
cally disclosed by preferred embodiments and optional selected from the group consisting Corynebacterium diph
features, modification and variation of the inventions herein theria, Bordetella pertussis, influenza virus, hepatitis B
disclosed can be resorted by those skilled in the art, and that virus, Clostridium botulinum and Bacillus anthracis and
Such modifications and variations are considered to be further wherein the composition includes lysozyme.
within the scope of the inventions disclosed herein. The 65 2. The immunogenic composition of claim 1, wherein the
inventions have been described broadly and generically injectable, liposomal vaccine is formulated as a lyophilized
herein. Each of the narrower species and Subgeneric group composition.
US 9,603,799 B2
31 32
3. The immunogenic composition of claim 1, further
wherein the liposomes maintain a mean hydrodynamic par
ticle diameter of between about 300 nm to about 1000 nm.
4. The immunogenic composition of claim 1, wherein the
liposomal vaccine maintains immunogenicity after
lyophilization and in the presence of at least one lyopro
tectant.
at least one lyoprotectant is selected from the group con
sisting of Sucrose, trehalose and mannitol. 10
liposomal composition is stable after storage at Sub-Zero
temperatures and room temperatures for a period of time
greater than six months.
7. The immunogenic composition of claim 1, wherein the 15
vaccine is formulated in the absence of a co-adjuvant.
k k k k k

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(12) United States Patent (10) Patent No.: US 9,603,799 B2

(56) References Cited

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